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Expression and function of HOXA genes in the normal ovary and ovarian carcinoma Ota, Takayo 2006

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EXPRESSION AND FUNCTION OF HOXA GENES IN THE NORMAL OVARY AND OVARIAN CARCINOMA by TAKAYO OTA M . D . , K I N K I UNIVERSITY, 1996 M . S c , THE U N I V E R S I T Y OF BRITISH C O L U M B I A , 2000 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (REPRODUCTIVE & D E V E L O P M E N T A L SCIENCES) THE U N I V E R S I T Y OF BRITISH C O L U M B I A November, 2006 © Takayo Ota, 2006 11 Abstract Homeobox genes, which code for families of transcription factors, act at the top of genetic hierarchies. H O X genes specify positional identity during development, and in adult tissues, regulate differentiation and proliferation. Most ovarian carcinomas are derived from the ovarian surface epithelium (OSE). In contrast to other carcinomas, OSE differentiates further with neoplastic progression and acquires Mtillerian duct-derived epithelial properties. I tested the hypothesis that growth and differentiation in ovarian carcinogenesis are regulated by H O X genes. I studied three genes: H O X A 4 , A7 and A9. H O X A 7 has been reported to play a role in the differentiation of ovarian cancers, while H O X A 9 is expressed in normal oviductal epithelium which resembles ovarian serous adenocarcinomas. Furthermore, microarray studies showed that H O X A 4 and H O X A 7 , but not H O X A 9 , were highly expressed in ovarian carcinomas. In this study, I produced a well-characterized polyclonal anti-HOXA7 antibody and investigated its distribution in ovarian carcinomas. H O X A 7 expression was not related to grade. H O X A 7 was expressed mainly in the cytoplasm, however, nuclear staining correlated with prognosis, suggesting that H O X A 7 in different subcellular locations has different functions. H O X A 4 expression in normal OSE was not consistently related to proliferation but increased at low cell density, low [Ca ] and with EGF stimulation, and H O X A 4 s iRNA enhanced motility. In ovarian cancer cultures, H O X A 4 s iRNA enhanced motility, increased proliferation in three-dimensional cultures and reduced CA125 secretion. These results imply that H O X A 4 regulates differentiation and cell-cell interrelationships, and that increased H O X A 4 expression may reflect a response to maintain homeostasis during neoplastic progression. I l l The study was expanded to investigate H 0 X A 7 distribution in human ovarian folliculogenesis. Granulosa cells were negative in primordial, but positive in primary follicles. As they matured, H O X A 7 relocated to the cytoplasm. H O X A 7 expression in granulosa cells increased with proliferation, and in culture it was regulated by GDF-9. These results indicate that, in folliculogenesis, H O X A 7 expression undergoes cell type-and stage-specific changes, which are regulated, in part, by GDF-9. In summary, H O X genes contribute to the development of ovarian follicles and carcinomas. Controlling H O X genes may be one of the critical methods to overcome diseases associated with ovarian folliculogenesis and carcinogenesis. Table of Contents Abstract i i Table of Contents iv List of Tables vi i i List of Figures ix List of Abbreviations x i i Acknowledgements xv 1. Introduction 1 1.1. H O X genes 5 1.1.1. H O X genes 5 1.1.2. Regulation of H O X genes 9 1.1.3. Targets of H O X genes 11 1.2. Homeobox genes in the female reproductive system 12 1.2.1. Non-clustered homeobox genes in the development of the female reproductive system 12 1.2.2. Expression of clustered homeobox (HOX) genes in the reproductive system 14 1.2.3. Mammalian ovary 14 1.2.4. Miillerian ducts 17 1.2.5. Function of H O X genes in the female reproductive system 20 1.2.6. Regulation of the expression of H O X genes in the female reproductive system 21 1.2.7. The expression of AbdB-type genes in the pathophysiological conditions other than tumors 23 1.3. Folliculogenesis 24 1.3.1. GDF-9 25 1.3.2. TGF-p i 28 1.3.3. Homeobox genes in folliculogenesis 29 V 1.4. Ovarian cancer 31 1.4.1. Epidemiology of epithelial ovarian cancer 31 1.4.2. Ovarian surface epithelium (OSE) ...32 1.4.3. EGF receptor (EGFR) in ovarian surface epithelium and ovarian cancer 33 1.4.4. C a 2 + 35 1.4.5. Homeobox genes and carcinogenesis 36 1.4.6. H O X in epithelial ovarian carcinoma 37 1.5. Three H O X A genes 39 1.5.1. H O X A 4 39 1.5.2. H O X A 7 42 1.5.3. H O X A 9 • 44 1.6. Hypothesis and aims 45 2. Materials and Methods 47 2.1. Tissues 47 2.2. Tissue microarray 49 2.3. Cell culture 50 2.4. Antibody production 51 2.4.1. Immunization 51 2.4.2. ELISA 52 2.4.3. Antibody-affinity purification .53 2.4.4. G S T / H O X A 7 fusion protein 53 2.4.5. Site-directed mutagenesis 54 2.5. Treatments of cultured cells 55 2.6. Proliferative index 57 2.7. RT-PCR 58 2.8. Southern blot analysis 61 2.9. Western blot analysis 61 2.10. Immunofluorescence 63 2.11. Immunohistochemistry 64 2.12. Scoring and statistics for tumor arrays 64 2.13. s iRNA transfection 65 2.14. Scratch assay for cell motility 66 2.15. Matrigel cell culture 67 2.16. CA125 measurement 67 2.17. Statistics 68 3. Results... 69 3.1. The production and antibody characterization of anti-HOXA7 antibody 69 3.2. The expression and regulation of H O X A 7 in human ovarian follicles..79 3.2.1. H O X A 7 expression in human ovarian follicles 79 3.2.2. H O X A 7 protein expression in the cell cycle 85 3.2.3. GDF-9 and TGF-(31 regulate H O X A 7 protein expression and proliferation in cultured S V O G 87 3.3. H O X A 7 expression in ovarian surface epithelium (OSE), granulosa cell tumors and tissue microarray 90 3.3.1. H O X gene expression in c D N A array and the confirmation by RT-PCR 90 3.3.2. H O X A 7 expression in OSE-z'w vivo 91 3.3.3. Tissue microarrays in epithelial ovarian carcinomas 95 3.3.4. Granulosa cell tumors 109 3.4. Expression of H O X A 4 , H O X A 7 and H O X A 9 in normal OSE and ovarian cancer in culture 109 3.5. Effects of EGF on H O X A 4 expression and proliferation in normal OSE 116 3.6. Effects of extracellular [Ca 2 +] on H O X A 4 expression in IOSE cells 119 3.7. H O X A 4 expression and proliferation in ovarian cancer cell lines 124 3.8. Estrogen effects on H O X A 4 expression in IOSE, O V C A R 3 and SKOV3 cells 124 3.9. Effects of H O X A 4 on the growth rates in IOSE cells 126 3.10. Downregulation of H O X A 4 in normal OSE, O V C A R 3 and O V C A R 8 132 3.11. Effects of H O X A 4 modulation on cellular motility 132 3.12. Effects of downregulation of H O X A 4 on ovarian cancer cell lines 140 3.12.1. E-cadherin and collagen type III expression ....140 3.12.2. Effects of H O X A 4 on CA125 140 3.13. Effects of H O X A 4 in three-dimensional Matrigel culture 142 3.14. Proliferative effects of downregulation of H O X A 4 in normal OSE with EGF treatment 145 3.15. Proliferative effects of downregulation of H O X A 4 in O V C A R 3 in 2 dimensional culture 145 4. Discussion 149 4.1. The expression of H O X A 7 in ovarian follicles, normal OSE and ovarian cancer 149 4.2. H O X A 4 in normal ovarian surface epithelium and epithelial ovarian carcinomas • 160 4.3. Future studies 166 4.3.1. Folliculogenesis 166 4.3.2. H O X A 7 and ovarian carcinomas 167 4.3.3. H O X A 4 and ovarian carcinomas 167 5. References 169 List of Tables Table 1 O ld and new nomenclature of H O X genes 10 Table 2 H O X expression in human normal ovary 16 Table 3 H O X expression in epithelial ovarian cancer 40 Table 4 Cl inical and pathological features of benign, low malignant potential tumors and serous ovarian carcinomas 48 Table 5 List of primers 60 Table 6 Staining intensity of H O X A 7 in ovarian follicles during follicular development 84 Table 7 Tumor characteristics and the intensity of H O X A 7 overall expression 96 Table 8 Tumor characteristics and the intensity of H O X A 7 nuclear expression 97 Table 9 Normal O S E cases in culture I l l Table 10 Effects of H O X A 4 on CA125 secretion in O V C A R 3 143 ix List of Figures Figure 1. c D N A microarray, unsupervised hierarchical clustering for homeobox genes in benign ovarian tumors, ovarian tumors of low malignant potentials and ovarian carcinoma 2 Figure 2. Conservation of genomic organization and expression patterns of H O X genes 7 Figure 3. The spatial domains of expression of H O X A 9 , H O X A 1 0 , H O X A 1 1 , and H O X A 1 3 in the developing human paramesonephric duct 18 Figure 4. Antibody titers in serum by E L I S A 70 Figure 5. Expression of H O X A 7 m R N A and protein in ovarian cancer cells O V C A R 3 (positive control) and the leukemia lines K562 (negative control) 71 Figure 6. Affinity purified H O X A 7 antibody titers by E L I S A 73 Figure 7. Expression of H O X A 7 protein in ovarian cancer cells O V C A R 3 (positive control) and the leukemia lines K562 (negative control) by affinity purified antibodies 74 Figure 8. Expression of H O X A 7 in a mouse embryo 76 Figure 9. Immunofluorescent detection of H O X A 7 in O V C A R 3 77 Figure 10. A point mutation in H O X A 7 sequence 78 Figure 11. S D S - P A G E analysis of G S T / H O X A 7 fusion protein 80 Figure 12. Immunohistochemical staining for H O X A 7 in paraffin-embedded cell Lines 81 Figure 13. Immunohistochemical staining for H O X A 7 in human ovaries 82 Figure 14. Immunofluorescence staining by an t i -HOXA7 and anti-Ki-67 antibody in cultured immortalized granulosa cells ( S V O G ) 86 Figure 15. Effects of G D F - 9 on S V O G cells over 24hr 88 6   T G p l on S V O G cells over 8hr 9X Figure 17. Confirmation of c D N A microarray by R T - P C R 92 Figure 18. H O X A 7 expression in ovarian surface epithelium 94 Figure 19. Expression of H O X A 7 in tumor microarray-1 98 Figure 19B. Expression of H O X A 7 in tumor microarray-1 (nuclei) 100 Figure 20. Expression of H O X A 7 in tumor microarray-2 102 Figure 20B. Expression of H O X A 7 in tumor microarray-2 (nuclei) 104 Figure 21. Relationship between H O X A 7 overall expression and cumulative survival patients with epithelial ovarian tumors 107 Figure 22. Relationship between H O X A 7 nuclear expression and cumulative survival patients with epithelial ovarian tumors 108 Figure 23. Immunohistochemical staining for H O X A 7 in granulosa cell tumors.. .110 Figure 24. Morphology of normal O S E 113 Figure 25. H O X A expression in normal O S E in culture 114 Figure 26. Expression of H O X A 4 , A 7 and A 9 in epithelial ovarian cell lines by R T - P C R 115 Figure 27. Expression of H O X A 4 in normal O S E , immortalized O S E (IOSE) and ovarian cancer lines 117 Figure 28. Effects of 20ng/ml E G F on H O X A 4 in normal O S E for 0-60 min 118 Figure 29. Effects of 20ng/ml E G F on H O X A 4 in normal O S E over 5-24 hr 120 Figure 30. Effects of extracellular [Ca 2 + ] on H O X A 4 expression in IOSE cells 122 Figure 31. H O X A 4 expression, cell morphology and proliferation in ovarian cancer cell lines 125 Figure 32. Estradiol effects on three H O X A genes in IOSE cells 127 Figure 33. Effects of H O X A 4 down-regulation in IOSE cells 129 Figure 34. Effects of s iRNAs on IOSE cells 131 X I Figure 35. Effects of control s iRNAs #1 and #2 on the growth rate of IOSE cells 133 Figure 36. Downregulation of H O X A 4 by s i R N A 134 Figure 37. Effects of H O X A 4 downregulation on cellular motility in normal O S E cells 136 Figure 38. Effects of downregulation of H O X A 4 on cellular motility in O V C A R 3 and O V C A R 8 cells 138 Figure 39. E-cadherin expression in O V C A R 3 141 Figure 40. Effects of H O X A 4 downregulation of morphology of O V C A R 3 cells in the three dimensional culture 144 Figure 41. Effects of H O X A 4 downregulation of H O X A 4 by s i R N A on proliferation in normal O S E cells 146 Figure 42. Effects of H O X A 4 downregulation of H O X A 4 by s i R N A on proliferation in O V C A R 3 147 Figure 43. Nuclear localization signals (NLSs) on H O X A 7 153 List of Abbreviations 2-D two-dimensional 3-D three-dimensional Abd-A Abdominal-A Abd-B Abdominal-B A B T S 2,2'-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid A N O V A analysis of Variance Ant-C Antennapedia complex Arg arginine A T C C American Type Culture Collection B C C A B C Cancer Agency BMP-15 bone morphogenetic protein-15 Bx-C Bithorax complex B L A S T Basic Local Alignment Search Tool bp base pair C cervical C a 2 + calcium CaR calcium sensing receptor c D N A complementary deoxyribonucleic acid cpm counts per minute D N A deoxy ribonucleic acid E embryonic day E C M extracellular matrix EGF epidermal growth factor E G F R epidermal growth factor receptor EHS Engelbreth-Holm-Swarm ELISA Enzyme-Linked Immunosorbent Assay E M S A electrophoretic mobility shift assay Emx empty spiracles homolog ES embryonic stem EST expressed sequence tag FIGO International Federation of Gynecology and Obstetrics FBS fetal bovine serum F S H follicle-stimulating hormone G A P D H glyceraldehyde-3 -phosphate dehydrogenase GDF-9 growth differentiation factor-9 GI gastrointestinal GST glutathione S-transferase H O X homeobox hr hour IOSE SV40 Tag immortalized normal OSE IPTG isopropyl-beta-D-thiogalactopyranoside IVF in-vitro fertilization Jak/Stat Janus kinases / signal transducers and activa transcription K L H keyhole limpet hemocyanin L H luteinizing hormone L M P ovarian tumors of low malignant potential Lys lysine M A P K mitogen-activated protein kinase Meis Myeloid ecotropic viral integration site 1 homologues M R K H syndrome Mayer Rokitansky Kiister Hauser Syndrome m R N A messenger ribonucleic acid NES nuclear exporting signals N L S nuclear localization signal Nobox newborn-ovary homeobox-encoding gene Obox oocyte specific homeobox gene Oct-4 octamer-binding transcription factor-4 OSE ovarian surface epithelium PAGE PolyAcrylamide Gel Electrophoresis Pax paired box gene Pbx pre-B-cell leukemia transcription factor PBS phosphate buffer solution p.c. post coital Pem reproductive homeobox on X chromosome, 5 PI3K Phosphoinositide-3 kinase Psx2 reproductive homeobox on X chromosome, 9 R A retinoic acid R A R E retinoic acid response elements Rhox reproductive homeobox genes on the X chromosome R N A ribonucleic acid xiv RT-PCR reverse transcription polymerase chain reaction Sebox skin-embryo-brain-oocyte-specific homeobox gene SDS sodium dodecyl sulphate s iRNA small interfering R N A S V O G immortalized human granulosa cells T thoracic TBS tris buffered saline TGF-P transforming growth factor-P TGM1 Transglutaminase type I TIMP-1 tissue inhibitors of metalloproteinases-1 Tox reproductive homeobox on X chromosome, 8 TPA 12-O-tetradecanoyl phorbol-13 -acetate TTBS TBS/tween-20 U C ulcerative colitis U V ultraviolet w/v weight per volume X V Acknowledgements Most of all I would like to thank my research supervisor, Dr. Ne l ly Auersperg. Her scientific enthusiasm inspired me and I am glad to work with her. I sincerely thank my committee members, Dr. Peter C K . Leung, Dr. Blake Gi lks , Dr. Keith Humphries, Dr. Basi l Ho Yuen and Dr. Dianne Mi l l e r for giving me thoughtful advice. M y best regards I want to give to Dr. Michael R. Hayden for helping to produce anti-H O X A 7 antibodies and Dr. Wilfred A . Jefferies and Kyung-Bok Choi for helping to make a G S T - H O X A 7 construct. I am also very grateful to Dr. A . J . W . Hsueh for his gift o f G D F - 9 , Dr. I. Clark-Lewis for giving me H O X A 7 peptides, Dr. E . M . Simpson for giving me mouse embryos. I also want to thank all people in our research department, especially in our lab, Michelle Woo, Arezoo Astanehe, Sarah Maines-Bandiera and Clara Salamanca for being kind and patient all the time. M y special thanks to Roshni Nair for helping me to go through my PhD program. I would like to thank all of my friends, especially, Takako Okuyama, M i k i k o Shimabukuro, Chikako Nishida, Chisato Mizutani and Chiharu Kameyama for their great support from Japan. I also want to thank Mehran Goreishi, Maggie Lee, her all family members and friends, Sun-Ji Park, Marisol , Edgar, Emiliano, Priya, Ilesh, Viera and Smita who supported me in Canada to make my life more "normal" outside the lab. Finally, I would like to thank my parents, my grandmother, my aunts, my brother and sister who encourage me to pursue my dream. This study is supported by B C Foundation for Non-Animal Research and Ovarian Cancer Canada. 1 1. Introduction Rationale Epithelial ovarian carcinomas are derived from the ovarian surface epithelium (OSE). In contrast to other carcinomas, OSE differentiates further with neoplastic progression and acquires properties of Mullerian duct-derived epithelia, such as oviduct or endometrial epithelium. Homeobox (HOX) genes play a major role in tissue and organ differentiation in development, and regulate differentiation in adult tissues. Therefore, I wished to test the hypothesis that H O X genes may also influence differentiation of ovarian cancers. I chose to study three H O X A genes, H O X A 4 , A7 and A9 , since c D N A microarray studies of ovarian benign tumors, low malignant potential tumors and serous adenocarcinomas, in collaboration with Dr. Gilks (in collaboration with Department of Pathology, U B C ; Fig. l) , showed H O X A 4 and H O X A 7 to be overexpressed in ovarian carcinomas, and because H O X A 9 is particularly expressed in normal adult mouse and human oviductal epithelium (Taylor et al., 1997). H O X A 7 was chosen initially for further study because of the findings by Naora, which suggested that H O X A 7 plays a role in the aberrant epithelial differentiation of the ovarian surface epithelium (OSE) in the early development of ovarian carcinoma (Naora et al., 2001a). There were no antibodies available to the H O X A 7 , and I therefore initiated my project 2 Fig.l. c D N A microarray, unsupervised hierarchical clustering for homeobox genes in benign ovarian tumors, ovarian tumors of low malignant potential and ovarian carcinomas. The c D N A microarray slide was scanned in red and green channels (Gilks et al., 2005). Red color shows upregulation, green color shows downregulation and black color shows the middle points of upregulation and downregulation of the genes. rt rt rt « , . « V (U « « H fl j 11 Jj Jj Jj Jj u II 1 1 U i S i W W n I t ' l l ' ' ietf a U kMd W o o o o o o o o o o o o o o o o o o o o o o « « m b y y L U 15 O I i I l • i i r ( n « i e MSlfSSflflffllilff 109546 EPHIS3 E»KB3 Hs.2913 M4 55591 221495 HOXftlO hrareo box A10 Hs. 110637 11953229 185291 HOXftlO homeo box 111 Hs. 110637 ftft284178 331037 HOX A 9 homeo box 19 Hs.127428 AI676154 331144 HQX17 bateo box ft7 Hs.355540 11762187 102536 10?7 acniaaorin 7 Hs.25475 H27752 98406 HOXD9 homeo box D9 Hs.236646 11424747 118489 1050X2 mesenchyme homeo box 2 (movrth arrest-specific homeo box) Hs.7 120132 C0L211 collaaen. tvoe II. alpha 1 (primary osteoarthritis, spondyloe] 31233S ESTs Hs.145939 11368019 117570 GPC4 CTlVTJicaJi 4 Hs.58367 ¥95635 111189 H0Xft4 homeo box 14 Hs.77637 11448557 114048 ESTs Hs.226923 R84679 4 by producing a well characterized polyclonal antibody to H O X A 7 protein and used this antibody to define the distribution of H O X A 7 in ovarian carcinomas. Since little is known about the role of H O X genes in the normal ovary, the project was expanded to include a study of H O X A 7 protein in normal ovarian folliculogenesis. Finally, because H O X A 4 was the most highly overexpressed H O X gene in carcinomas in Dr. Gilks ' expression array, I investigated its possible roles in the growth and differentiation of normal and neoplastic OSE in culture. In summary, the purpose of this project was to investigate the distribution and the mechanisms regulated by H O X genes in ovarian folliculogenesis and ovarian carcinomas. This thesis is separated into four parts: (i) the production of anti-human H O X A 7 antibody, (ii) the distribution and regulation of H O X A 7 in human ovarian folliculogenesis, (iii) the histochemical distribution of H O X A 7 in ovarian carcinomas and possible relationships to clinical parameters and (iv) the regulation and function of three H O X A genes, mainly H O X A 4 , in normal ovarian surface epithelium and in ovarian cell lines in culture. 5 1.1. HOX genes 1.1.1. H O X genes Homeobox genes are regulatory genes, which code for families of transcription factors and act at the top of genetic hierarchies (Veraksa et al., 2000). In the mammalian genome, 200 homeobox genes are present (Del Bene and Wittbrodt, 2005). Homeobox genes are divided into two groups; clustered (HOX) and non-clustered. H O X genes specify positional identity during development, along the anterior-posterior axis. In adult tissues, H O X genes regulate differentiation and proliferation. Hox genes are highly conserved from yeast to human beings (Acampora et al., 1989). Hox genes were first identified in the fruit fly Drosophila melanogaster (Lewis, 1978). In Drosophila, eight homeotic genes are clustered into two loci in the third chromosome, known as the Antennapedia complex (Ant-C) and the Bithorax complex (Bx-C). Thus far, 39 H O X genes, homologues of Anntenapedia-like genes, have been identified in mammals (Daftary and Taylor, 2006; Veraksa et al., 2000). Four groups of human H O X genes (A, B , C and D) are clustured on different chromosomes (7pl5.3, 17p21.3, 12ql3.3 and 2q31; Apiou et al., 1996). Each homeobox gene contains a sequence coding for 61 amino acids, which comprises the homeodomain (Gehring et 6 al., 1990), and binds to specific D N A sequences (Grier et al., 2005). On the basis of peptide sequence identity of the several domains, and the relative positions of genes, 13 different sets of genes are separated (Fig.2; Veraksa et al., 2000). Gene duplication and divergence are thought to form the four clusters in vertebrates (Favier and Dolle, 1997). Genes which are duplicated, for example, H O X A 1 and H O X B 1 , are referred to as paralogous and are located to the same relative positions. The H O X genes on the chromosome are arranged. along the anterior-posterior body axis. In. development, genes at the 3' end clusters are expressed earliest and the anterior boundary of the region is well defined, but the posterior boundary is indefinite (Favier and Dolle, 1997). Interestingly, in adult tissues, the embryonically restricted H O X gene expression is conserved along the anterior-posterior axis (Morgan, 2006). For example, H O X 7 (HOXA7, HOXB7) expression is limited to the middle abdominal region in the mesoderm in the embryo. In the adult, H O X A 7 is expressed in most tissues, such as heart, lung, liver, kidney, pancreas and colon (Kim et al., 2000), but the expression is higher in the kidney rather than the brain. Hence, overall, the spatial expression of homeobox genes is unchanged over the course of development. 7 Fig.2. Conservation of genomic organization and expression patterns of HOX genes, (modified from Veraksa et al. 2000). The lower half of the figure shows that four clusters of HOX genes in mammals. The anterior expression boundaries between the nervous system and the segmented mesoderm are shifted. The upper half of the figure shows Drosophila Hox genes, aligned with their mammalian orthologs. Drosophila Hox cluster Ancestral Hox cluster 3 X HOXA HOX8 HOXC lab pb fen D M S c r te U b x a b d ' A A M ' e A1 A2 A3 A4 AS AB A7 A9 A10 A11 A13 O - C H O O 81 82 83 84 B5 B6 B7 88 89 813 n J r ~ C4 CS C6 CB CS CIO C11 C\2 C13 01 L HOXD - Q -D3 04 08 09 D10 DU 012 013 3' + Anterior Posterior 9 Table 1 shows old and currently used names of H O X genes (Scott, 1992). Capitalized H O X mean genes for homo sapiens and non-capitalized Hox mean genes for mice. 1.1.2. Regulation of H O X genes Few regulators of H O X genes have been identified. Retinoic acid (RA) is a well-characterized regulator of H O X genes in development (Daftary and Taylor, 2006). R A acts via the retinoic acid receptor complex, which regulates target gene binding to enhancers, retinoic acid response elements (RAREs). Thus far, RAREs have been found in four H O X genes (Hoxal, Hoxbl , Hoxa4, Hoxd4), which indicates that the expression of those H O X genes is directly regulated by R A (Conlon, 1995; Doerksen et al., 1996). R A treatment and alteration of H O X genes expression lead to the similar transformation of the axial patterning. Excess R A and overexpression of H O X genes cause the vertebrae to change into posterior identities, e.g., C7 transforms T l (Kessel and Gruss, 1991; Krumlauf, 1994). The effects of R A are stronger at the 3' ends of the H O X clusters, which are expressed most anteriorly. R A receptor expression and its target H O X genes determine the location of a definitive segmental boundary. Estrogen and progesterone also regulate H O X genes, which is discussed in section 1.2.6. Table 1. O ld and new nomenclature of H O X genes (modified from Scott 1993). Groups: H , Human, M , mouse. Anterior Drosophila Hox A Hox new old H old M new 1 lab A1 1F 1.6 B1 2 pb A2 1K 1.11 B2 3 A3 1E 1.5 B3 4 Dfd A4 1D 1.4 B4 5 Scr A5 1C 1.3 B5 6 Antp A6 1B 1.2 B6 7 Ubx A7 1A 1.1 B7 8 Abd-A B8 9 Abd-B A9 1G 1.7 B9 10 A10 1H 1.8 11 A11 11 1.9 12 13 A13 1J 1.10 Posterior (Evx1) HoxC HoxD old old new old old new old old H M H M H M 21 2.9 D1 4G 4.9 2H 2.8 2G 2.7 D3 4A 4.1 2F 2.6 C4 3E D4 4B 4.2 2A 2.1 C5 3D 3.4 2B 2.2 C6 3C 3.3 2C 2.3 2D 2.4 C8 3A 3.1 D8 4E 4.3 2E 2.5 C9 3B 3.2 D9 4C 4.4 C10 31 3.6 D10 4D 4.5 C11 3H 3.7 D11 4F 4.6 C12 3F D12 4H 4.7 C13 3G D13 41 4.8 (Evx2) 11 1.1.3. Targets of Hox genes The homeodomain, which is encoded for by a homeobox, recognizes and binds to specific D N A sequences, indicating homeobox genes as transcription factors (Svingen and Tonissen, 2006; Del Bene and Wittbrodt, 2005). The homeodomain is highly conserved and consists of 60 aminoacids, forms a helix-turn-helix motif and an additional domain close to the first helix. A core D N A consensus sequence is recognized by most of the homeoproteins, raising the question how transcriptional specificity can be achieved. The specificity is partially obtained by the H O X cofactors, i.e., Pbx/Meis. Most H O X proteins interact with Pbx (Chang et al., 1997), but only groups 9, 10 and 13 have been shown to interact with Meis (Chang et al., 1996, Williams et al., 2005). H O X proteins bind directly to cofactors and the specificities may be regulated in the different cofactors peptide domains used by different H O X genes (Williams et al., 2005). Recent review papers have shown that homeoboxes are likely to regulate proliferation, differentiation, migration and cell adhesion (Pearson et al., 2005). A number of target genes for H O X proteins have been found, however, the list of direct target genes is still 12 incomplete. By promoter studies, more direct target genes should be identified to understand the function of H O X proteins more clearly. 1.2. Homeobox genes in the female reproductive system 1.2.1. Non-clustered homeobox genes in the development of the female reproductive system In mammals, the urogenital system originates from the intermediate mesoderm. In mice, the male reproductive tract develops from the mesonephric duct (the Wolffian duct) by embryonic day 9.5 (E9.5) and most of the female reproductive system arises from the paramesonephric duct (the Miillerian duct) in the parallel to the Wolffian duct by day E l 1.5 (Bard and Kaufman, 1999). At this stage, embryos have both male and female reproductive systems and their sex is indistinguishable. Later, in female embryos, the Wolffian duct degenerates due to the absence of male hormones, and the Miillerian duct differentiates into fallopian tubes, uterus, cervix and upper third of vagina. Several genes have been identified to be necessary to the development of female reproductive system (Yin and Ma, 2005). Two homeobox genes, Pax2 and Emx2 are essential at the early stage of Miillerian duct development. 13 The intermediate mesoderm is divided into two different components, the ductal epithelium and the nephrogenic mesenchyme. The epithelial part develops into the genital tracts, ureters and collecting duct system in the kidney, whereas the mesenchymal part undergoes epithelial transformation to form nephrons. Pax2, a member of the paired homeobox gene family, is expressed in both epithelial and mesenchymal parts (Dressier et al., 1990). Pax2 null mice lack the entire genital tracts, except gonads (Torres et al., 1995). Both the Wolffian duct and the Miillerian duct initially form, but later degenerate. Emx2, a mouse homologue of Drosophila empty spiracles, is expressed in the epithelial components of the urogenital system during development (Miyamoto et al., 1997). Emx2 null mice lack the urogenital system, kidneys, ureters, gonads and genital tracts. The Wolffian duct develops, later degenerates. Before degeneration, Pax2 is expressed normally in the mutant. In these mutants, the Miillerian duct never forms, the thickening of the coelomic epithelium is not clear and the invaginations of the coelomic epithelium are also poor. These results indicate that Pax2 and Emx2 are required in a specific time period during mesonephric development (Yin and Ma, 2005). The induction of the Wolffian duct, the Miillerian duct and the development of gonads requires different signals. 14 1.2.2. Expression of clustered homeobox (HOX) genes in the reproductive system The genital expression in Drosophila (Estrada and Sanchez-Herrero, 2001; Foronda et al., 2005) and in mammals is determined by the H O X Abdominal-A (Abd-A) and Abdominal-B (Abd-B) genes (Fig.2). The Abd-A gene directs H O X 8 genes and the Abd-B genes direct H O X 9 to HOX13 genes in mammals. Abd-A and Abd-B genes are Bx-C genes, one of the two loci of H O X clusters, and most posteriorly expressed. Abd-A genes have been shown to be required for gonad development, the internal female genitalia, including the uterus, oviducts and seminal receptacle (a receptacle in the reproductive tracts of certain female invertebrates, especially in insects, in which spermatozoa are received and stored until needed to fertilize ova; Karch et al., 1985; Foronda et al., 2005). The absence of Abd-B genes modifies genital structures to legs or antenna in the genital disc (Estrada and Sanchez-Herrero, 2001). 1.2.3. Mammalian ovary Naora et al. reported that H O X A 7 is expressed in inclusion cysts in the normal ovary, in serous cystadenomas and in differentiated serous adenocarcinomas, but not in normal adult OSE and undifferentiated ovarian carcinoma (Naora et al., 2001a). 15 Epithelial ovarian carcinomas are thought to frequently originate from the inclusion cysts (Scully et al., 1995). Besides H O X A 7 , as a non-Abd-A and -B type gene, which indicates that the gene is thought to be not required for reproductive system development, H O X B 7 was studied in the reproductive system (Naora et al., 2001b; Vogels et al., 1990; Table 2). Hoxb7 is not found in the mouse embryonic ovary. Hoxb7 is expressed in the adult mouse ovary, and more specifically, H O X B 7 is expressed in normal human adult OSE. The H O X B 7 expression is higher in ovarian carcinomas, which is not related to the grade of tumor (Naora et al., 2001b). Among Abd-B-type H O X genes, the normal human adult ovary expresses H O X A 9 and H O X C 9 , but not HOXD10 (Redline et al., 1994). Unfortunately, the authors did not define the location of the expressions in the ovary. The same group has shown that HoxdlO expression is positive in mouse adult ovary, which is negative in human (Redline et al., 1992), using the same methods. These results indicate that the contradictory results maybe due to the difference of species in spite of the conservation of H O X genes among species. Table 2. H O X expression in human normal ovary 1 2 3 4 5 6 7 8 9 10 11 12 13 H O X A N D N D N D N D N D N D (OSE) + + N D N D H O X B N D N D N D N D N D N D + (OSE) N D N D N D H O X C N D N D N D N D + N D N D N D N D H O X D N D N D N D N D N D - N D N D N D N D - not determined Ref. Redline et al. 1994, Naora et al. 2001a, Pando and Taylor 2002 17 1.2.4. Mullerian ducts The expression of the H O X A cluster has a role in the differentiation of the Mullerian duct in the female reproductive system of mice and human (Taylor et al., 1997; Fig.3). The Abd-B type genes in the Hoxa cluster, i.e., Hoxa9, HoxalO, Hoxal 1 and Hoxal3, are expressed diffusely and homogeneously at El5.5 in mice. The dispersed expression pattern is lost at 2 weeks of age when the expression of Hoxa9 is restricted to the fallopian tubes and the uterus expresses HoxalO and Hoxal 1. The HoxalO expression is higher in uterine epithelial cells than in the stroma. In contrast, Hoxal 1 expression is higher in uterine stroma than in the epithelium. Hoxal 1 is also expressed in the cervix, where Hoxal3 is expressed as well. Hoxal3 expression is localized in the vaginal epithelium. Hence, the Hoxa expression pattern is spatially narrowed along the anterior posterior axis and the pattern persists in adult mice. Moreover, the expression pattern in adult mice corresponds the expression in human tissues. H O X A 9 is expressed in the fallopian tubes. H O X A 1 0 is expressed in the uterus. HOXA11 is expressed in the uterus and the cervix. H O X A 13 is expressed in the cervix and the upper vagina. 18 Fig.3. The spatial domains of expression of H O X A 9 , H O X A 1 0 , H O X A 1 1 , and H O X A 13 in the developing human paramesonephric duct. In keeping with the property of colinearity, H O X A 9 is expressed in the developing oviduct, H O X A 10 is expressed in the uterus, H O X A 1 1 is expressed in both the uterine and the vagina and H O X A 13 is expressed in the developing vagina. (modified from Taylor et al. 2000) 20 Another set of Abd-B type genes in the H O X D cluster has a similar expression pattern to those in the H O X A cluster (Dolle et al., 1991). The spatially restricted expression of H O X D genes is earlier than H O X A in the development. The normal female reproductive system expresses non-Abd-B type genes as well as Abd-B type genes. H O X A 7 is expressed in the fallopian tubes and endometrium (Naora et al., 2001a). The Mullerian ducts express Hoxb7 in the 12.5-day mouse embryo (Vogels, 1990). H O X B 7 m R N A expression is higher in ovarian carcinomas compared to normal ovarian surface epithelium in vivo (Naora et al., 2001b). Overexpression of H O X B 7 in immortalized normal ovarian surface epithelial cells enhances cellular proliferation and secretion of basic fibroblast growth factors (Naora et al. 2001b). These findings indicate that H O X genes have other functions in addition to mediating the formation of segment-specific structures. 1.2.5. Function of H O X genes in the female reproductive system H O X A 10 and H O X A 11 in the uterus control implantation. Mutation of HoxalO or Hoxal 1 results in sterility. In HoxalO-mutant female mice, the uterotubal junction is absent and the proximal uterus to the oviduct is narrowed (Benson et al., 1996). 21 Hoxal 1-deficient female mice have intact ovaries (Hsieh-Li et al., 1995), but their uteri are smaller and there is little stromal tissue development and less glandular formation (Gendron et al., 1997). However, both HoxalO- or Hoxal 1-mutated mice are sterile, which implies that the sterility might be due to the factors for implantation other than structural changes (Benson et al., 1996). Mutant mice ovulate normally and produce embryos, which survive when transferred to wild-type mice. 1.2.6. Regulation of the expression of H O X genes in the female reproductive system Throughout the menstrual cycle, the expressions of both H O X A 1 0 and H O X A 11 are present, but they are significantly increased in the mid-secretory phase. Expression of H O X A 10 and H O X A 11 was upregulated by estrogen and progesterone application in cultured primary endometrial stromal cells (Taylor et al., 1998; Taylor et al., 1999). This indicates that sex steroid hormones regulate the expression of H O X A 10 and H O X A 1 1 . H O X A 10 and H O X A 11 expression in endometrium oscillates with the menstrual cycle and the expression patterns of H O X A 10 and H O X A 11 are similar to each other (Gendron et al., 1997; Taylor et al., 1998, 1999a). 22 In two studies, hormonal application has paradoxical effects on H O X expression in the endometrium. Estrogen or estrogen with progesterone increased (Taylor et al., 1998, 1999) or decreased (Ma et al., 1998) HOXAlO/HoxalO and H O X A l l / H o x a l l expression. In contrast, progesterone enhanced HOXAlO/HoxalO expression in both studies (Ma et al., 1998; Taylor et al., 1998). The differences might be because Taylor et al. (1998) used only endometrium, whereas M a et al. (1998) used whole uterine tissue to extract R N A . The whole uterine tissue contains more myometrium than endometrium and Cermik et al. (2001) has shown that estrogen with progesterone decreases the H O X A 10 expression in the myometrium. However, even taking into account the myometrial tissues in the study by M a et al. (1998), the opposite effects of estrogen are still difficult to explain. Estrogen increases the H O X A 10 expression in myometrium (Cermik et al., 2001). One possibility is that steroid hormones cause different effects on H O X expression in different species. Although no human females with H O X A 10 and H O X A 11 mutations have been described, females with lower implantation rates have lower H O X A 10 and H O X A 11 expression in the secretory phase (Taylor et al., 1999a). To distinguish developmental effects from maternal effects on infertility caused by H O X A 1 0 expression, HoxalO 23 expression was changed by transfection of antisense in adult mice (Bagot et al., 2000). Blocking HoxalO expression by antisense transfection inhibited implantation, whereas HoxalO overexpression results in larger litter size. In ectopic pregnancy, H O X A 1 0 expression is increased at the site of implantation in the fallopian tubes (Salih and Taylor, 2004). These experiments demonstrate that expression of H O X A 1 0 is associated with implantation and has therapeutic potential as a fertility drug or contraceptive (Bagot et al., 2000). 1.2.7. The expression of AbdB-tvpe H O X genes in pathophysiological conditions other than tumors Mutations of more posterior genes, H O X A 13 and HOXD13, contribute to the malformation in the caudal part of reproductive system. In addition, these genes are essential for limb development. A nonsense mutation of H O X A 13 results in the hand-foot-genital syndrome (Mortlock and Innis, 1997). The first digits in both hands and feet are hypoplastic and the carpal and tarsal ossification are delayed, causing their shortenings (Goodman and Scambler, 2001). Such women have uterine malformations, resulting from Miillerian duct fusion defects. A mutation in HOXD13 has been found in syndactyly and Polydactyly (Goodman and Scambler, 2001). There is no literature to 24 demonstrate human genital defects in H O X D 13 mutation alone. Hoxdl3 mutant female mice are fertile (Dolle et al., 1993), but not Hoxal3 + / 7Hoxdl3 _ / " because of the failure in developing the caudal part of Mullerian ducts (Warot et al., 1997), indicating that the H O X A cluster may have stronger effects on the urogenital development than the H O X D cluster. However, in human, in various forms of congenital absence of the uterus and vagina (Mayer-Rokitansky-Kiister-Hauser; M R K H syndrome), with is lack of limb anomalies, sequence analysis of coding regions of H O X A 7 to H O X A 1 3 genes and their co-factor, PBX1 did not show any mutations (Burel et al., 2006). Mutations of H O X genes are not involved and genes other than H O X genes might be involved in the M R K H syndrome (Bruel et al., 2006). 1.3. Folliculogenesis Folliculogenesis is the process of the growth and the differentiation of ovarian follicles from the primordial stage into preovulatory follicles (Barnett et al., 2006). Primordial follicles are considered the resting pool, which is gradually depleted during reproductive life. Initiation of follicular growth from the primordial to primary stage is characterized both by changes of granulosa cell shape and number and an increase in oocyte size. 25 The process of ovarian folliculogenesis is dependent on both endocrine factors and paracrine factors. The main paracrine factors secreted by oocytes belong to the TGF-P superfamily, in particular, growth differentiation factor-9 (GDF-9), bone morphogenetic protein-15 (BMP-15; also called GDF-9B), TGF-P and activin. In the middle of my study, I found that H O X A 7 was not expressed in granulosa cells in primordial follicles and started being expressing in primary follicles. One of the TGF-P superfamily members, GDF-9, which is essential for ovarian follicular development is expressed in the oocytes, however, the expression pattern also starts in primary follicles like H O X A 7 (see below). Therefore, I chose to study GDF-9 to see the regulation of H O X A 7 . I also studied TGF-p i as a representative of TGF-P subfamily to show whether the GDF-9 effects are specific to H O X A 7 . Here is a short summary for GDF-9 and TGF-P 1. 1.3.1. GDF-9 GDF-9 is essential for normal folliculogenesis (Juengel and McNatty, 2005). It has been shown in rats and mice that GDF-9 is one of the principal factors which regulate granulosa cell proliferation and differentiation in early folliculogenesis, where granulosa cells are not receptive to gonadotropin hormones. In rats, mice and human, 26 the expression of m R N A and protein was observed in oocytes in primary follicles (Laitinen et al., 1998; Aaltonen et al., 1999; Jaatinen et al., 1999). Female mice lacking functional GDF-9 are infertile and follicle growth is arrested at the primary stage (Dong et al., 1996). Although both recombinant GDF-9 and F S H treatment in immature female rats increase ovarian weight, GDF-9 particularly increases the number of primary and small preantral follicles (Vitt et al., 2000a). On the other hand, FSH does not influence the number of primary follicles and increases the number of preantral follicles. In vitro, GDF-9 enhances the survival and progression of human follicles to the secondary stage (Hreinsson et al., 2002). GDF-9 prevents apoptosis in preantral follicles through phosphatidylinositol 3-kinase (PI3K) /Akt pathway (Orisaka et al. 2006). Granulosa cells are targets of GDF-9. Conflicting GDF-9 effects have been shown between species and experiments by several researchers. In general, GDF-9 is mitogenic for granulosa cells from small antral and even preovulatory follicles, which is observed in rats (Vitt et al., 2000b; Nilsson and Skinner, 2002; McNatty et al., 2005a). In addition, in human organ culture, GDF-9 increases the diameters of follicles, without changing the diameters of oocytes (Hreisson et al., 2002), which indicates 27 proliferation of granulosa cells. GDF-9 stimulates estradiol production in undifferentiated and differentiated granulosa cells and progesterone production in differentiated granulosa cells (Elvin et al., 1999; Vitt et al., 2000). GDF-9 inhibits FSH-stimulated estradiol and progesterone production and L H receptor expression in rats (Vitt et al., 2000a), but the effect on progesterone have been inconsistent in mice (Elvin et al., 1999). GDF-9 itself increases inhibin production in rats and human (Hayashi et al., 1999; Kaivo-oja et al., 2003) and GDF-9 enhances FSH-induced inhibin a and inhibin P in rats (Roh et al., 2003), which implies that GDF-9 regulates FSH secretion. Again, the stimulatory effects for inhibin a are not observed in mice (Varani et al., 2002). These discrepancies could be explained by species differences and experimental procedures (McNatty et al., 2005a,b). GDF-9 has a role not only in smaller follicles but also in peri-ovulatory stages of follicles, because it facilitates cumulus expansion to help the release of the oocyte to the pelvic cavity (Elvin et a l , 1999; Vanderhyden et al. 2003). Injection of s iRNA to GDF-9 limits oocyte cumulus expansion (Gui and Joyce, 2005). Dragovic et al. showed that GDF-9 is not the only cumulus expansion factor (Dragovic et al., 2005), but this study used a neutralizing antibody against human GDF-9 and partially purified 28 mouse GDF-9 to mice. The neutralizing antibody only partially inhibited granulosa cell proliferation in another study (Gilchrist et al., 2004), hence, based on their result, we cannot conclude that GDF-9 has no effects on cumulus expansion (Pangas and Matzuk, 2005). 1.3.2. TGF-p l TGF-P 1 is one of the TGF-P subfamilies comprised of three proteins; p i , P2 and P3 in mammals. In humans, TGF-p 1 is present in primary oocytes (Chegini and Flanders, 1992). Granulosa cells and theca cell layers are also positive for T G F - p l and the intensity of the staining was stronger as follicles matured. Similar to GDF-9, the effects of TGF-P 1 on granulosa cells vary by species. In rodents, TGF-P 1 stimulates cellular proliferation (Dorrington et al., 1988; Saragueta et al., 2002), however, in other species, such as pigs and sheep, the effects are mild stimulatory or inhibitory (May et a l , 1988; Juengel et al., 2004). In rodents, TGF-p i stimulates FSH-induced progesterone production (Dodson and Schomberg, 1987; Knecht et al., 1987), whereas TGF-p i inhibits basal and FSH-stimulated progesterone production in pigs and sheep (Mondschein et al., 1988; Juengel et al., 2004). TGF-p i stimulates the expression of inhibin/activin PB, not inhibin/activin PA or inhibin a in human granulosa luteal cells 29 (Eramaa and Ritvos, 1996). Together with GDF-9, ovarian TGF-P 1 promotes folliculogenesis: they both can stimulate granulosa cell proliferation, stimulate progesterone production and increase inhibin production (Juengel and McNatty, 2005). However, the basis for the different action of GFD-9 and TGF-P 1 in the regulation of H O X A 7 expression through the same signaling pathways needs to be elucidated (Mazerbourg and Hsueh, 2006). 1.3.3. Homeobox genes in ovarian folliculogenesis Several homeobox genes have been found to be expressed in the ovary and to regulate folliculogenesis (Pangas and Rajkovic, 2006). Some homeobox genes are particularly expressed in oocytes. Using expressed sequence tags (ESTs), Nobox (newborn-ovary homeobox-encoding gene) is found in mouse new born ovary (Suzumori et al., 2002). The gene is expressed in the ovary as well as testis and pancreas (Huntriss et al., 2006). Nobox m R N A is expressed in oocytes from primordial through antral follicles, but not in granulosa cells, theca cells and corpus luteum (Suzumori et al., 2002). In Nobox null mice, follicles do not grow beyond the primordial stage and several oocyte-specific genes, including GdjV are downregulated, suggesting that the Nobox gene controls early folliculogenesis (Rajkovic et al., 2004). However, the mutation of N O B O X was 30 absent in women with premature ovarian failure, suggesting that other than Nobox genes may cause ovarian failure (Zhao et al., 2005). Obox family transcripts are found in mouse oocytes and their expression starts from primary follicles (Rajkovic et al., 2002). Sebox, A skin-embtyo-brain-oocyte-specific homeobox gene, is a paired-like homeobox gene, expressed in brain, skin, two cell embryos and oocytes (Cinquanta et al., 2000). In oocytes, the Sebox m R N A expression is high in early antral stage follicles and becomes low when follicle matures in late stages of follicle maturation. The role of the Obox and Sebox genes are unknown. Rhox is one of newly identified homeobox genes clusters on the mouse X chromosome (Maclean et al., 2005). Rhox5 (previously called Pern) protein is expressed in periovulatory mural granulosa cells, not in cumulus granulosa cells (Pitman et al., 1998). Rhox5 is absent from less mature follicles, which suggest that Rhox5 could have ovulation specific function (Pitman et al. 1998). Rhox8 (Tox; Kang et al., 2004) is expressed in theca cells in the preantral follicles and whereas in mural granulosa cells and in the theca cells in the antral follicles. Rhox9 (Psx2; Takasaki et al., 2000) was discovered by sequencing c D N A libraries from E12-13 urogenital ridges. Psx2 is 31 expressed in both male and female embryos, but not in the adult tissues. The specific function of these genes in the female reproductive system requires future studies. 1.4. Ovarian cancer 1.4.1. Epidemiology of epithelial ovarian cancer Ovarian carcinoma is the fourth (Canadian Cancer Statistics, 2006) and the fifth (Center for Diseases Control and Prevention, 2004/2005) leading cause of cancer death in Canada and North America, respectively. Currently, only 25 % patients are diagnosed in Stage 1, which can be cured in 90% of patients (Bast et al., 2005). The high fatality rate is due to a lack of early symptoms and appropriate diagnostic markers, as well as frequent recurrences of tumors that are resistant to therapy. Hence, detection at earlier stages might improve survival and outcome of ovarian cancer patients. About 85% to 90% of ovarian cancers are epithelial ovarian carcinomas. Epithelial ovarian tumors consist of many types, each with subtypes, including serous, mucinous, endometrioid, clear cell and transitional types (International Federation of Gynecology and Obstetrics: FIGO). The serous type was by far the most common. 32 Epidemiologic studies indicate that incessant ovulation might be one of the factors to cause epithelial ovarian carcinomas (Auersperg et al., 1998). Decreases in ovulation, such as in response to oral contraceptives or multiparity, reduce risk of ovarian carcinomas (Ozols et al., 2000). 1.4.2. Ovarian surface epithelium (OSE) Epithelial ovarian carcinomas are believed to be derived from the ovarian surface epithelium (OSE). OSE is a simple squamous-to-cuboidal mesothelial epithelium which covers the ovary. OSE originates from part of the coelomic epithelium. In the embryo, the coelomic epithelium gives rise to granulosa cells, Mullerian duct epithelia and the peritoneum (Auersperg et al., 2001). These epithelia have a common origin with OSE. However, OSE is relatively uncommitted; it is able to differentiate into the Mullerian duct type epithelia, i.e., the epithelia of fallopian tubes, uterus and upper vagina. OSE is responsive to the environment. Normal adult OSE expresses both epithelial and mesenchymal markers in primary culture (Auersperg et al., 1994). With time and passages, OSE undergoes epithelial-mesenchymal transition, in which epithelial 33 characters are lost (Auersperg et al., 1994). This characteristic leads to increase cell motility during the ovulation repair, and conversion to ovarian stroma when trapped within the ovary (Auersperg et al., 2001). In contrast, borderline and low grade ovarian carcinoma cells are committed to an epithelial phenotype. The epithelial phenotype might have an advantage for cell survival to promote neoplastic progression and modify responsiveness to hormone or growth factors. In the middle of my experiments, I found that H O X genes expression is related to proliferative activities of OSE. Using known mitogens to OSE, such as epidermal growth factor (EGF) and high extracellular concentration C a 2 + ([Ca 2 +]), I studied the regulation of H O X A 4 in normal OSE and in SV40 Tag immortalized normal OSE (IOSE) cells. Below, I summarize these mitogens briefly. 1.4.3. EGF receptor (EGFR) in ovarian surface epithelium and ovarian cancer EGF receptor (EGFR) is expressed in normal OSE and ovarian carcinomas (Berchuck et al., 1991; Rodriguez et al., 1991). E G F R is expressed in a wide range (13% to 77%) of ovarian cancers (Bauknecht et al., 1991; Meden et al., 1995). E G F R belong to the type I receptor tyrosine kinase class. The activation of E G F R triggers multiple signal 34 transduction pathways, such as phosphatidylinositol-34anase/Akt, Jak/STAT and ras/raf/MAPK (Rubin and Yarden, 2001). The activation results in cellular proliferation (Rodriguez et al., 1991; Salamanca et al., 2004), increased cell motility (Ellerbroek et al., 1998; Ahmed et al., 2005) and stimulated protease activities (Ellerbroek et al., 1998; Henic et al., 2006), which may play a important role in the progression of ovarian carcinomas. E G F R is a target for ovarian cancer treatment. Various E G F R targeting agents are currently in clinical trials (Dupont et al., 2005). There are two types; tyrosine kinase inhibitors and antibodies against EGF receptors. Tyrosine kinase inhibitors are gefitinib (ZD1839, Iressa) and erlotinib (OSI-774, Tarceva). Gefitinib and erlotinib are highly specific to E G F R receptors and have no effect on other tyrosine kinases, and competitively inhibit E G F R autophosphorylation (Albanell and Gascon, 2005). These drugs only have a low response rate as single agents (Cohen et al., 2004; Gordon et al., 2005). Monoclonal antibodies against EGFR, such as cetuximab, are currently being tested. Thus far, the clinical efficacy of the E G F R specific agents is poor. 35 In lung cancer, i f there are no E G F R mutations, the response to E G F R inhibitors is poor (Lynch et al., 2004). Thus, E G F R mutations increase the responsiveness to the E G F R inhibitors. Schilder's group stresses the importance of the mutation to the sensitivity of gefitinib, however, even ovarian carcinomas and cancer cell lines which do not have mutations respond to gefitinib (Schilder et al., 2005). In addition, thus far, only 2 of 57 advanced ovarian adenocarcinomas were found to have mutations (Schilder et al., 2005). In another study, no E G F R kinase domain mutations were found in advanced epithelia ovarian cancer (Lacroix et al., 2006). Other than mutations may be associated with the response to E G F R targeted drugs. 1.4.4 Caz+ Extracellular calcium sensing receptor (CaR) is a transmembrane G-protein coupled receptor and regulates cellular proliferation of ovarian surface epithelium (McNeil et al., 1998). Functional CaR is expressed in normal OSE and ovarian cancer cell lines (Wright et al., 2003). The responses to changes in extracellular calcium are cell-type-dependent. Fibroblasts and many epithelial cell types require high calcium (1.8-2.0 m M Ca 2 + ) for proliferation, whereas keratinocytes proliferate in low calcium and differentiate in high calcium concentrations more than 1.0 m M . Immortalized SV40 36 Tag OSE (IOSE) are epithelial cells, and IOSE proliferates more extracellular [Ca ] is increased from 0.8 m M to 1.8 m M (McNeil et al., 1998). This result indicates that OSE has mesenchymal characteristics. The proliferative response is PI3 K-dependent (Bilderback et al., 2002). In contrast, ovarian cancer cell lines also express CaR (McNeil et al., 1998), however, these cancer cells are less sensitive to extracellular [Ca 2 +] and show constant proliferative effects regardless of calcium concentration (Rodland, 2004). Thus, CaR controls proliferation and differentiation by responding to extracellular [Ca 2 + ], and with malignant transformation, cells lose the responsiveness to this environment factor. 1.4.5. Homeobox genes and carcinogenesis There is direct evidence which demonstrates that H O X genes have an effect on carcinogenesis in solid tumors when H O X genes are expressed at the wrong time and at wrong levels (Abate-Shen, 2003). One example is Oct-4, which controls the differentiation status of embryonic stem (ES) cells. In humans, Oct-4 overexpression is observed specifically in testicular germ cell tumors and the strongest expression is observed at intratubular stage (Gidekel et al., 2003). Increasing Oct-4 protein levels in ES cells is associated with increasing the possibility of tumor formation in mice 37 (Gidekel et al., 2003). These findings imply that Oct-4 deregulation is tissue specific and Oct-4 acts as an oncogene when overexpressed in a dose-dependent manner. Oct-4 may be an appropriate target for gene therapy for testicular germ cell tumors (Gidekel et al., 2003). 1.4.6. H O X in epithelial ovarian carcinomas Neoplastic changes influence the expression of H O X genes in tissues originated from Miillerian ducts. A recent study by Naora suggested that one of the H O X genes has a role in the epithelial differentiation of OSE (Naora et al., 2001a). B y immuno-screen lambda Z A P ovarian carcinomas c D N A expression library, anti-HOXA7 antibody was identified and found in serous ovarian carcinomas antigens in patient serum (Naora et al., 2001a). RT-PCR and immunohistochemical staining confirmed H O X A 7 expression in differentiated carcinomas. The H O X A 7 was also expressed in benign cystadenomas, while there was no or little expression in normal scraped OSE and in poorly differentiated carcinomas. These results suggested that H O X A 7 , as well as other H O X genes might have a role in the epithelial differentiation of OSE that takes place during neoplastic progression. 38 U s i n g t h e s a m e i m m u n e - s c r e e n i n g , N a o r a ' s g r o u p f o u n d t h a t a n o t h e r H O X g e n e , H O X B 7 , i s e x p r e s s e d a l i t t l e i n n o r m a l s c r a p e d O S E , w h e r e a s i t i s u p r e g u l a t e d i n o v a r i a n c a r c i n o m a s ( 2 0 0 1 b ) . I n c o n t r a s t t o H O X A 7 , H O X B 7 e x p r e s s i o n w a s n o t a s s o c i a t e d w i t h d i f f e r e n t i a t i o n o f o v a r i a n c a n c e r s . I n s t e a d , H O X B 7 i n c r e a s e d b a s i c f i b r o b l a s t g r o w t h f a c t o r s e c r e t i o n , w h i c h i n d u c e s c e l l u l a r p r o l i f e r a t i o n o f O S E . T w o g r o u p s h a v e p u b l i s h e d c D N A m i c r o a r r a y a n a l y s i s f o r d i f f e r e n t H O X g e n e s e x p r e s s i o n i n n o r m a l , b e n i g n , m a l i g n a n t o v a r i a n t i s s u e s a n d o v a r i a n c a n c e r c e l l l i n e s ( Y a m a s h i t a e t a l . , 2 0 0 2 ; T a i t e t a l . , 2 0 0 5 ) . T h e w e a k p o i n t o f t h e s e s t u d i e s i s t h a t t h e y u s e d R N A f r o m w h o l e o v a r i e s , n o t o n l y f r o m O S E a s a n o r m a l c o n t r o l . M o s t l y u p r e g u l a t e d g e n e s i n m a l i g n a n t t u m o r s c o n c e n t r a t e d i n c l u s t e r s A a n d B g e n e s . A l t h o u g h i n G i l k s ' s t u d y ( F i g . 1) , t h e c o n t r o l i s b e n i g n t u m o r s o v e r m a l i g n a n t t u m o r s , s i m i l a r t o G i l k s ' s t u d y , H O X A 4 u p r e g u l a t i o n i n m a l i g n a n t t u m o r s o v e r n o r m a l o v a r y w a s o b s e r v e d i n T a i t ' s s t u d y ( T a i t et a l . , 2 0 0 5 ) . H O X A 7 u p r e g u l a t i o n i n m a l i g n a n t t u m o r s w a s n o t s e e n b y c D N A m i c r o a r r a y a n a l y s i s , b u t w a s o b s e r v e d b y r e a l t i m e R T -P C R i n t u m o r s a m p l e s a n d o n e o v a r i a n c a n c e r c e l l l i n e ( Y a m a s h i t a e t a l . 2 0 0 2 ; T a i t e t a l . 2 0 0 5 ) . 39 Yamashita's group chose to study H O X B 7 and HOXB13, which are most upregulated in five different ovarian cancer cell lines compared to normal ovary. Downregulation of these two genes by antisense in S K O V 3 , decreased invasion of matrigel coated filters (Yamashita et al., 2006). Thus, H O X B 7 has at least two roles in OSE; regulating proliferation and invasion. Regulation of cell motility by H O X genes was also observed in keratinocytes by HOXD3 (Hansen et al., 2003) and in endometrial cancer cells by HOXD13 (Zhao et al., 2005). The expression of H O X genes in epithelial ovarian carcinomas by c D N A array is summarized in Table 3. 1.5. Three HOXA genes 1.5.1. H O X A 4 HoxA4 m R N A is not expressed in the mouse ovary (Wolgemuth et al., 1986). The expression and function of H O X A 4 is not known in female reproductive system. H O X A 4 m R N A is present in human unfertilized oocytes and cleaving embryos (Kuliev et al., 1996). In developing mice, Hoxa4 is seen in a variety of tissues, including the spinal cord, ganglia, and the mesodermal layer of the gut (Wolgemuth et al., 1986; Behringer et al., 1993), but there are no descriptions about the reproductive system. In the adult mouse, the expression is limited to the testis and germ cells that Table 3. HOX expression in epithelial ovarian cancer Paralog 1 2 3 4 5 6 7 8 9 10 11 12 13 HOXA A2+ A3+ A4+, A4+ A5+ A7+ A9+ A10+ HOXB B2+ B3+ B4 + B5+ B6+ B7+ B8+ B9+ B13+ HOXC C6- C13+ HOXD D13+ Ref. Tait et al. 2005, Yamashita et al. 2006 In red, in vivo, in black, in vitro; +, upregulation, -, downregulation. HOXA4 is the only gene which is upregulated both in vivo and in cell lines. 41 have reached meiotic stages (Behringer et al., 1993). The restricted expression of Hoxa4 in the testis led to the assumption that Hoxa4 has a function during spermatogenesis. Surprisingly, Hoxa4 null mice are viable and fertile (Horan et al., 1994). Hoxa4 paralogs might compensate the function. The Hoxa4 null mice have homeotic transformations, in which one structure replaces another, but other than homeo tic transformation, no abnormalities are observed in various organs. Homeosis can be caused by environmental factors leading to developmental anomalies, or by mutation. In Hoxa4 null mice, in the vertebral column, anterior (C3 to C2) and posterior (C7 to TI) transformations are observed (Horan et al., 1994). In contrast, overexpression of Hoxa4 in transgenic mice leads to congenital megacolon (Wolgemuth et al., 1989). The terminal colon is hypoganglionic, has abnormal enteric ganglia and thickened smooth muscles (Tennyson et al., 1993;1998). Megacolon is characteristic of a congenital condition known as Hirschsprung's disease, which is caused by a deficiency of myenteric ganglion cells. In case of Hoxa4 overexpressed transgenic mice, the migration of pelvic and enteric neural precursors are abnormal, which may be due to the overexpression of a signaling molecule produced by the enteric mesenchyme (Tennyson et al., 1998). H O X A 4 expression has not been studied 42 in human Hirschsprung's disease, hence I cannot conclude whether H O X A4 is related to this disease. A recent study has shown that H O X A 4 m R N A expression is downregulated in ulcerative colitis (UC) and colorectal carcinomas compared to normal control (Toiyama et al., 2005). Thus, H O X A 4 may play a role in the development of the colon and the pathogenesis of U C and UC-related colorectal carcinomas. At the cellular level, HoxA4 transcripts are upregulated in dissociated chicken embryo brain cells (Mauro et al., 1994). From these in vitro experiments, adhesion molecules have been proposed as targets of Hox genes. However, the expression of N-cadherin, a cell adhesion molecule, is not changed anywhere either in Hoxa4 null mice or in Hoxa4 overexpressed mice (Packer et al., 1997). Other Hox4 genes might take over Hoxa4-dependent missing functions. 1.5.2. H O X A 7 H O X A 7 is present in human unfertilized oocytes and cleaving embryos (Kuliev et al., 1996). The distribution of H O X A 7 in the adult human ovary was unknown prior to this 43 study, except that in normal ovarian surface epithelial cells, H O X A 7 is not expressed (Naora et al., 2001a). In transgenic mice, overexpression of Hoxa7 leads to craniofacial abnormalities and to death shortly after birth (Balling et al., 1989). On the other hand, Hoxa7 null mice are healthy, have no apparent abnormalities, including their reproductive systems, and both sexes are fertile (Chen et al., 1998), suggesting that redundant factors can substitute for H O X A 7 in its absence. In Hoxb7 null mice, the first two rib fuse, which implies that Hoxa7 is expressed at the level of C7, the cervical/thoracic border (Min et al., 1998). Therefore, Hoxa7 might have a function for upper thoracic regions (Chen et al., 1998). At the cellular level, during differentiation of F9 stem cells, the expression of murine Ffoxa7 is associated with chromatin (Schulze et al., 1987). In proliferating human neonatal keratinocytes, H O X A 7 binds directly to the differentiation-specific gene promoters of transglutaminase type I, and downregulates the cells' differentiation (La Celle and Polakowska, 2001). These results imply that Hoxa7 expression is associated with differentiation as well as proliferation. The regulation of transglutaminase by H O X A 7 was also investigated in hematopoietic cells (Leroy et al., 2004). Here, the 44 transglutaminase gene is involved in adhesion and migratory properties of monocytes, which suggests that H O X A 7 controls interactions between cells and extracellular matrix as well. 1.5.3. H O X A 9 Hoxa9 is found in mouse ovary (Villaescusa et al., 2004). The expression of H O X A 9 in normal human ovary is unknown. Hoxa9 knockout mice are healthy and fertile. Their white blood cells counts are reduced and they have smaller spleen and thymuses. Their committed erythroid and myeloid progenitor cells are reduced, but not primitive progenitor cells (Lawrence et al., 1997), indicating that the Hoxa9 has a function in the process of differentiation of hematopoietic cells. Mammary glands in mice deficient for Hoxa9, Hoxb9 and Hoxd9 are normal before pregnancy, but do not develop during pregnancy and after parturition. In wild type mice, the expression of these three Hox9 genes is reduced by pregnancy and lactation (Chen and Capecchi, 1999). The results indicate that the Hox9 genes have a role in the 45 development and the differentiation of the mammary gland in response to pregnancy. H O X A 9 expression persists in the adult differentiated oviduct of mice and humans but its role is unknown. 1.6. Hypothesis and Aims The hypotheses of this study were: (i) H O X proteins are expressed during ovarian folliculogenesis. (ii) The aberrant expression of H O X genes in ovarian carcinomas has a role in the differentiation of ovarian carcinomas. The specific aims of this study were: (1) Produce a well-characterized an anti-human H O X A 7 antibody. (2) (i) Investigate the distribution of H O X A 7 protein in normal human ovaries by immunohistochemistry and evaluate its expression in different stages of follicular development, (ii) Examine the possible roles of H O X A 7 in granulosa cells. (3) Study H O X A 7 expression in an ovarian tumor array and examine possible correlations with differentiation and prognosis. (4) Compare the H O X A 4 expression in normal OSE and ovarian cancer cell lines. 46 (5) Investigate the possible function of H O X A 4 in normal OSE and ovarian cancer cell lines. 47 2. Materials and Methods 2.1. Tissues Paraffin sections were obtained from grossly and histologically normal ovaries of 11 premenopausal women, with appropriate consent. Seven out of 11 tissues included ovarian follicles. Eight out of 11 tissues retained normal ovarian surface epithelial cells, invaginations or inclusion cysts. Mouse embryos for characterization of the antibody to H O X A 7 were kindly donated by Dr. Elizabeth M . Simpson (UBC). For confirmation of the c D N A microarray (Fig. 1), eight snap-frozen ovarian tumor samples were obtained from National Ovarian Cancer Association ovarian tumor bank. Ethical approval was granted by U B C for collection of these tumors. These were two ovarian tumors of Tow malignant potential (LMP), three grade 2 and three grade 3 ovarian carcinomas. Their Federation of Gynecology and Obstetrics (FIGO) stages and grades are presented at Table 4. Out of 22 tumors used in the c D N A microarray, I obtained eight tumors. I extracted R N A from them and did RT-PCR followed by Southern blot analysis. The methods for R N A extraction, RT-PCR and Southern blot analysis are described as below. 48 Table 4. Clinical and pathological features of benign, low malignant potential tumors and serous ovarian carcinomas Case# Age (years) FIGO stage Grade OvCal 75 III 3 OvCa3 74 Benign OvCa4* 37 III 1 OvCa5' 37 III LMP OvCa7 48 III 3 OvCa8 62 III 2 OvCalO 22 I LMP OvCall 50 III 2 OvCal2 • 72 Benign OvCal4 ' 50 III LMP OvCal6 87 Benign OvCal7 61 Benign OvCal9 34 I LMP OvCa22 27 I LMP OvCa23 52 III 2 OvCa24 80 Benign OvCa25 not known Benign OvCa27 77 I LMP OvCa30 52 IV 2 OvCa32 65 II 3 OvCa41 31 III LMP OvCa44 57 I LMP VOA85 83 III 3 VOA86 61 III 3 VOA95 39 III 3 LMP = low malignant potential * OvCa4 and OvCa5 are from right and left ovarian tumors, respectively, from a single patient. 49 2.2. Tissue microarray Tissue microarrays were prepared by members of the Department of Pathology, U B C . Tumor samples were collected from several hospitals in British Columbia, Canada, between 1984 and 2000. Histopathology, tumor grade and stage were reviewed by pathologists according to FIGO and Silverberg criteria, respectively. Clinical data included initial diagnosis, treatment, toxicity of chemotherapy and clinical outcomes. The samples were selected corresponding to the following criteria: referred to B C Cancer Agency for treatment, paraffin embedded tissue remaining, classified as moderate or high risk of recurrence More than 95 % of the tumors were chemotherapy treated. The tissue samples were fixed in formalin and embedded in paraffin. A hematoxylin-eosin stained slide allowed the selection of 2 representative cores of 0.6 mm in diameter. The tissue array was composed of one serous borderline, 211 serous, 131 endometrioid, 34 mucinous, 143 clear cell carcinoma, 7 transitional, 1 squamous, 7 undifferentiated, 4 unclassified adenocarcinomas, 1 metastatic carcinoma from GI tract. Another tissue microarray of ovarian neoplasms contained 6 specimens of granulosa cell tumors. The 6 granulosa cell tumors were all of the typical adult type, consisting of 50 sheets of uniform small cells that resembled normal granulosa cells. For immunohistochemistry, the tissue array was cut into 3.5-4 um sections. 2.3. Cell culture Normal human OSE cells were obtained at laparoscopic procedures for nonmalignant gynecological conditions from women 26 to 46 years old. Ovarian cancer cell lines, A2780, O V C A R 2 , O V C A R 4 , O V C A R 5 , O V C A R 8 , and O V C A R 1 0 were kindly provided by Dr. T.C. Hamilton at Fox Chase Cancer Center, Philadelphia. BG-1 was provided by Dr. Korach (NIH). CaOV3, O V C A R 3 , SKOV3, and an erythroleukemic cells line, K562 were obtained from American Type Culture Collection (ATCC). Immortalized OSE (IOSE) cell lines were generated by transfection with the immortalizing simian virus 40 Tag/tag into normal OSE (Auersperg et al. 1999). Immortalized human granulosa cells (SVOG) were obtained as described previously (Lie et al., 1996), by transfection of SV40 early genes Tag/tag into human granulosa cells obtained from IVF procedures. Normal OSE and IOSE were maintained in a 1:1 mixture of M-199/MCDB105 medium (Sigma) supplemented with 10% FBS (Hyclone Laboratories Ltd., Logan, UT) or in E G M medium (Clonetics, San Diego, C A ) which is E B M basal medium with 10 ng/ml epidermal growth factor, 1 u.g /ml hydrocortisone, 12 51 u.g/ml bovine brain extract and 2 % FBS. A l l cells were grown in a 5% CCVair atmosphere, and passaged using 0.06% trypsin/0.01% EDTA in Ca 2 +-,Mg 2 +-free Hanks' BSS. OSE cells were grown in E G M medium only in passage 0 and then switched to M-199/MCDB105/10%) FBS. They were used in passages 0 to 8. Ovarian cancer cell lines, except BG-1 , were maintained in a 1:1 mixture of 199/MCDB 105 with 5% FBS. BG-1 was maintained in phenol red-free DMEM/F12 medium (Sigma, St. Lois, MO) supplemented with 10% FBS. S V O G cells were maintained in 199/MCDB 105/10%) FBS with 0.4 u.g/ml hydrocortisone (BD Biosciences Clontech, Mountain View, CA) . K562 cells were maintained in RPMI 1640 medium (Invitrogen, Burlington, ON) with 15%> FBS. For normal OSE and O V C A R 3 , gentamicin (Invitrogen) was sometimes added to the culture medium. 2.4. Ant ibody Product ion 2.4.1. Immunizat ion Polyclonal antisera to H O X A 7 were generated by immunization of three New Zealand white rabbits (Rabbit T l , T2 and T3) with a human synthetic peptide conjugated to keyhole limpet hemocyanin ( K L H ; by Dr. I. Clark-Lewis, U B C ) . The peptide sequence was D K T D E G A L H G A A E A , 102-115, found to be specific for H O X A 7 in the uniprot 52 database at N C B I using the blast homology search engine, provided by Covance Research Products (Denver, PA). The peptide was mixed with Titermax gold adjuvant (Sigma) and a total of 500 ug was injected subcutaneously. Subsequent 100 ug boosts were injected after two weeks and every month thereafter for 12 months. The rabbit sera were obtained two-three weeks after the boost injection every month. 2.4.2. E L I S A Ninety-six well plates (Becton Dickinson Labware, Franklin, NJ) were coated overnight at 4 °C, with 50 ul, 50 ug/ml the synthetic peptide without K L H in PBS. Plates were blocked with 0.5% skim milk/PBS (blocking buffer) and coated with 50 ul serially diluted duplicate samples in blocking buffer for 2 hr at 37 °C. After incubation with horseradish peroxidase-linked anti-rabbit secondary antibody (Cal Biochem, La Jolla, CA) , plates were developed with A B T S substrate and read at 405 nm minus 490 nm (background), using a microplate photoreader (Bio-Tek Instruments Inc.). 53 2.4.3. Antibody-affinity purification H O X A 7 synthetic peptide without K L H dissolved in distilled water (8 mg) was coupled to 0.6g thiol activated-Sepharose 4B beads (Sigma) in 50 ml PBS at 25 °C for 4 hr. Filtered and diluted polyclonal antisera 1:1 with PBS were incubated in the above affinity column at 25 °C, overnight. The flowthrough was collected and kept at -20 °C. After the beads were washed by PBS, eluted slowly with 0.1 mol/1 glycine, pH 2.5. The eluates were collected at 500 ul each, neutralized to pH 7.0 -8.0 by 2 mol/1 Tris-HCl, pH 9.0 immediately and kept at -20 °C. 2.4.4. G S T / H O X A 7 fusion protein The full-length H O X A 7 c D N A (forward primer 5' - G G A T C C A T G A G T T C T T C G T A T T A T G T G A A C G C - 3 ' and reverse primer 5 ' - C T C G A G T C A T T C C T C C T C G T C T T C C T C T T - 3 ' ) were PCR amplified by Taq polymerase, subcloned into the vector pCRII (Invitrogen), then moved to p G E X 4T-1 vector (Amersham) at BamHI and Xhol sites. The construct was transformed into INVaF ' cells, confirmed by sequencing compared to Genbank (AF026397). G S T / H O X A 7 fusion protein was induced by adding 54 isopropylthio-P-o-galacto-pyranoside (IPTG) to a 0.08 m M final concentration for 4h at 30 C. After sonication, the fusion protein was affinity purified by incubation with glutathione sepharose 4B beads (Amersham), and eluting with 20 m M glutathione in 120 m M NaCl and 50 m M Tris-HCl, pH 8.0. 2.4.5. Site-directed mutagenesis A point-mutation in the GST-HOXA7 recombinant protein, which was outside the peptide sequence used raising the anti-HOXA7 antibody was corrected by site-directed mutagenesis. The p G E X 4T-1 expression vector carrying mutated H O X A 7 c D N A was used as the template and fixed from Gly-99 to Ser-99 by site-directed mutagenesis using QuickChange II X L Site-Directed Mutagenesis Kit (Stratagene, L a Jolla, C A ) . Internal complementary PCR primers were 5 ' - G A C C T C G C C A A A G G C G C C T G C G A C A A G - 3 ' and 5' - C T T G T C G C A G G C G C C T T T G G C G A G G T C - 3 ' . The PCR reaction consisted of 18 cycles of 95 °C for 15 sec, 60 °C for 50 sec, 68 °C for 1 min, and PfuUltra HF D N A polymerase was used for the PCR reaction. The correction was confirmed by sequencing the inserted H O X A 7 fragment. 55 2.5. Treatments for cultured cells For EGF (Sigma) treatment, normal OSE cells were plated at a density of 1.0 * 105 cells in 35 mm culture dishes or 2 x 104 cells/well in 24 well plates for [3H]-thymidine incorporation assay. After 24 hr, the media were changed from 10 % FBS to 2 % FBS for 24 hr before treatments with 20 ng/ml EGF. Parallel cultures were used for RT-PCR and [3H]-thymidine incorporation. For GDF-9 (growth differentiation factor-9, received as conditioned medium from Dr. A . Hsueh, Stanford Univ.) or TGF-p i ( R & D Systems Inc., Minneapolis, M N ) treatment, S V O G were plated at a density of 1.5 x 105 cells in 35 mm culture dishes or 2 x 104 cells/well in 24 well plates for [3H]-thymidine incorporation assay. The cells were serum starved for 24 hr before treatments with GDF-9 or TGF-P 1, then incubated with 200-300 ng/ml GDF-9 for 24 hr or 1-10 ng/ml TGF-p i for 24-48 hr. These concentrations were chosen based on Dr. Hsueh's recommendation, who carried out E L I S A assays of conditioned medium in his laboratory for GDF-9, and the company's instruction for TGF-P 1. For controls, GDF-9 and T G F p l were not added. Parallel S V O G culture were used for Western blot analysis and [3H]-thymidine incorporation. 56 To study the effects of 17-B-estradiol (E2; Sigma), 2 x 105 IOSE or O V C A R 3 cells were plated in 35 mm culture dishes. After a preincubation for 24 hr, the cells were treated with E2 at concentrations of 10"8 to 10"6 M in phenol-red free medium 199 (Sigma) with 2% charcoal/dextran treated FBS (HyClone) for 24 hr. Control cultures were treated with vehicle, 0.1 % absolute ethyl alcohol. To investigate the effects of changes in extracellular C a 2 + concentration ([Ca 2 +]), IOSE cells were seeded at 2 x 105 cells in 35 mm dishes or 2 x 104 cells/well in 24 well plates for [3H]-thymidine incorporation assay in 199/MCDB105/10%FBS (1.8 m M Ca 2 + ) and allowed to attach for 24 hr. The cells were then made quiescent by culture in serum-free Ham's F12 (0.3 m M C a 2 + ; Invitrogen) for 24 hr. The desired final calcium concentration was achieved by addition of calcium to calcium-free Dubelcco's modified Eagle's medium (Invitrogen): 0.05 m M to maintain the quiescent state and 2.0 m M to induce proliferation. Cells were lysed for R N A extraction after 24 hr treatment. For the [3H]-thymidine incorporation assay they were treated for 20 hr and 24 hr with either 0.05 m M or 2.0 m M calcium (Bilderback et al. 2002). 57 2.6. Proliferative Index Cellular proliferation was measured in three ways. (1) mitotic figures Mitotic figures were counted by visual examination in 2 microscopic fields with over 500 cells each at 4 * objective per culture. (2) DNA determination Cells were plated at 2 xl0 3 /wel l in 96-well strip plates (Corning Inc., Corning, N Y ) in 200 ul M-199/MCDB105/10%FBS (IOSE) or M-199/MCDB/5%FBS (ovarian cancer cell lines) and were harvested from dayl to day 6. Cells were washed with PBS and fixed with -20°C methanol for at least 20 min. After washing, cells were stained with 5.0 ug/ml Hoechst 33258 (Sigma), which binds specifically to non-denatured D N A , and analyzed on a fluorescence plate reader at wavelength filters 360/40 and 460/40. (3) [3H]-thymidine incorporation assay Cells were plated in 2 x 104 in 24-well plates in 0.5 ml medium. Initially 199/MCDB 105 were used for all cultures with 10% FBS (normal OSE, IOSE or SVOG) were incubated for 24 hr. One microcurie [3H]-thymidine (5.0 Ci/mmol; Amersham 58 Pharmacia Biotech.) was added 4 hr before the cell harvest. Then, the thymidine was precipitated with 0.5 ml 10% trichloroacetic acid for 20 min or more at 4 °C. The precipitate was washed in methanol and solubilized in 0.5 ml 0.1 N NaOH. In case of Matrigel cultures (see section 2.15), the precipitate.was not washed by methanol and solubilized in 1.0 ml 0.1 N NaOH. The incorporated radioactivity was measured in a Tri-Carb liquid scintillation analyzer (model 2100TR; Packard Instrument Co., Meriden, CT). Data are presented as the mean ± SD of quadruplicate measurements and are representative of three individual experiments. 2.7. RT-PCR Total RNA was extracted from cultured cells using trizol Reagent (Life Technologies, USA) or RNeasy kit (Qiagen Inc., Mississauga, ON) according to the manufacturer's procedure. The total RNA concentration and purity was determined by spectrophotometric analysis A260/280- Complementary DNA (cDNA) was transcribed from 1.5-3.0 ug total RNA using a First Strand cDNA synthesis kit (Amersham, Oakville, Ont., Canada). The cDNA was used as a template for polymerase chain reaction (PCR). Primers were designed through Primer3 (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3.cgi) or SGD 59 (http://seq.yeastgenoine.org/cgi-bin/web-priiner), or used modified published condition. PCR primers were listed in Table 5. A l l PCR primers span the introns to detect specific m R N A sequences. With these primers, the amplimers for H 0 X A 4 , H 0 X A 7 , H 0 X A 9 were 226-bp (GenBank N M 002141), 414-bp (GenBank AF032095) and 331-bp (GenBank U41813), respectively. The amplification reaction was carried in the linear range of the logarithmic phase unless it was mentioned. PCR was sometimes performed by touchdown. Each cycle consisted of denaturation at 94 °C for 30 s, primer annealing at 55 °C for 30 s, extension at 72°C for 60 s. This was followed by a final extension at 72°C for 5 min in a D N A thermal cycler (Mini cyclerTM, PTC-100 T M , MJ-Research, Bio-Rad). P-actin or G A P D H amplimers were included to normalize the relative amount of c D N A in each reaction, and a 510-bp or a 3 73-bp fragment was amplified under similar condition. To amplify the cDNA, 1 ul aliquots of the reverse-transcribed c D N A were subjected in 25 ul of reaction mixture containing; 1 U Taq polymerase (Invitrogen) and its buffer, 1 m M M g C h , 1 m M deoxy-NTP and 1 pmol each of specific oligonucleotide primers. Eight ul of PCR products was analyzed by agarose (1%) gel electrophoresis and visualized by ethidium bromide or S Y B R Safe™ D N A gel stain (Molecular Probes, Eugene, OR). The intensity of the PCR products was measured by Scion Image (Scion Corporation, Frederick, M A ) . The PCR products were purified by 60 Table 5. L i s t o f p r i m e r s H O X A 4 5' - C C C T G G A T G A A G A A G A T C C A - 3 ' a n d 5' - T C C A C T T C A T T C T C C G G T T C - 3 ' H O X A 7 5' - C T T A T A C A A T G T C A A C A G C C - 3 ' a n d 5 ' - T C C T T A T G C T C T T T C T T C C A - 3 ' H O X A 9 5' - C T G T T G A T G G T A G G C T G T A T - 3 ' 5' - A G G T G G A G A A A A T G A T G A A T - 3 ' p - a c t i n 5' - G G A C C T G A C T G A C T A C C T C A T G A A - 3 ' a n d 5' - T G A T C C A C A T C T G C T G G A A G G T G G - 3 ' G A P D H 5 ' - A T G T T C G T C A T G G G T G T G A A C C A - 3 ' a n d 5 ' - T G G C A G G T T T T T C T A G A C G G C A G - 3 ' 61 PCR purification kit (Qiagen) and verified by sequence analysis, compared to the published sequence in GenBank (NIH, www.ncbi.nlm.nih.gov). 2.8. Southern blot analysis After electrophoresis, PCR products were transferred to membranes (Schliecher & Schuell Biosciences Inc. Keene, NH) and fixed by U V irradiation. The blotted membranes were pre-hybridized for 1 hr at 42 °C in hybridization solution containing 50% formamide, 5 x SSC (75 m M sodium citrate, 0.75 M NaCl , pH 7.0), 0.1% w/v N-lauroylsarcosine, 0.02% w/v SDS and 2% w/v blocking reagent. The pre-hybridized membranes were hybridized overnight at 42 °C with digoxigenin-Iabeled probes. PCR products of H O X A 4 , H O X A 7 , H O X A 9 and P-actin were isolated from O V C A R 3 and cloned into PCRII vector using TA Cloning Kit (Invitrogen). The c D N A probes were labeled with digoxigenin c D N A labeling kit (Roche Molecular Biochemical, Laval, Canada). The hybridized membranes were detected by the alkaline phosphatase method. 2.9. Western blot analysis Cells were washed with ice-cold PBS and lysed by cold lysis buffer (1% Triton-X, 0.5%> sodium deoxycholate, 0.1% SDS in PBS, pH 7.4), including freshly added protease 62 inhibitor (Sigma). The extracts were placed on ice for 10 min, vortexed briefly and centrifuged for 15 min at 4 °C to remove cell debris. Total protein concentration was determined using a Bradford assay (Bio-Rad Laboratories, Hercules, CA) . The samples were boiled for 10 min before running the gels. SDS polyacrylmide gel electophoresis (SDS-PAGE) was performed using a 10-12 % separating gel. Ten % gels were used for E-cadherin and 12 % gels were used for H O X A 7 . Seventeen - 30 ug of proteins were then electrotransferred to a nitrocellulose or a PVDF membrane (Bio-Rad Laboratories). The membrane was blocked with 5-10% skim milk in PBS. H O X A 7 , GST protein, E-cadherin (Transduction Lab, B D bioscience, Mississauga, ON), p-actin, G A P D H were detected by primary antibody diluted 1:800-1,600 for H O X A 7 , 1:500 for GST, 1:3000 for p-actin, 1:100 for G A P D H (Santa Cruz Biotechnology Inc., Santa Cruz, C A ) in 10 % skim milk/PBS (HOXA7, GST), or in 5% skim milk/TBS (E-cadherin), or in 5% skim milk/TTBS (p-actin, GAPDH) for 1 hr at 25 °C. Subsequently, i f necessary, the signals were detected with secondary antibodies conjugated with horse radish peroxidase and visualized by E C L (Pierce, Rockford, IL). G A P D H were conjugated with horse radish peroxidase, hence, directly visualized. 6 3 2.10. Immunofluorescence M o u s e e m b r y o s at d a y 1 1 . 5 p . c . w e r e f i x e d i n 4 % p a r a f o r m a l d e h y d e / P B S , e q u i l i b r a t e d w i t h 3 0 % s u c r o s e / P B S , c o v e r e d w i t h O T C a n d f r o z e n i n l i q u i d n i t r o g e n . T e n u m f r o z e n s e c t i o n s w e r e t r a n s f e r r e d t o p o s i t i v e l y c h a r g e d s l i d e s , p e r m e a b i l i z e d i n 0 . 1 % T r i t o n X - 1 0 0 / P B S a n d b l o c k e d w i t h P r o t e i n B l o c k ( D A K O , M i s s i s s a u g a , O N ) f o r 1 h r . T h e s e c t i o n s w e r e i n c u b a t e d o v e r n i g h t a t 4 ° C w i t h p r i m a r y a n t i - H O X A 7 a n t i b o d y at 1 : 4 0 0 d i l u t i o n i n P r o t e i n B l o c k ( D A K O ) , f o l l o w e d b y T e x a s R e d - l a b e l e d g o a t - a n t i r a b b i t s e c o n d a r y a n t i b o d y f o r 1 h r at 2 5 ° C ( J a c k s o n I r n m u n o R e s e a r c h L a b o r a t o r i e s , I n c . , W e s t G r o v e , P A ) . C e l l s w e r e g r o w n o n g l a s s c o v e r s l i p s , f i x e d i n f r e s h l y p r e p a r e d 4 % p a r a f o r m a l d e h y d e i n P B S f o r 1 0 m i n at 4 ° C , p e r m e a b i l i z e d b y 0 . 1 % T r i t o n X - 1 0 0 i n P B S f o r 1 0 m i n a t 2 5 ° C , t h e n , p r o c e s s e d a s f o r m o u s e e m b r y o s t a i n i n g . O t h e r t h a n a n t i - H O X A 7 a n t i b o d y , a s p r i m a r y a n t i b o d i e s , a n t i - E - c a d h e r i n a n t i b o d y w a s u s e d a t 1 : 5 0 0 , o r a n t i - c o l l a g e n t y p e III a n t i b o d y at 1 :10 ( S o u t h e r n B i o t e c h n o l o g y A s s o c i a t e s , B i r m i n g h a m , A L ) , f o l l o w e d b y g o a t a n t i - m o u s e a n t i b o d y ( f o r a n t i - E - c a d h e r i n a n t i b o d y , A l e x a F l u o r 5 9 4 , M o l e c u l a r P r o b e s , E u g e n e , O r e g o n ) o r r a b b i t a n t i - g o a t a n t i b o d y ( f o r a n t i - c o l l a g e n t y p e III a n t i b o d y ; A l e x a F l u o r 4 8 8 , M o l e c u l a r P r o b e s ) . P r o l i f e r a t i v e a c t i v i t y o f c u l t u r e d c e l l s w a s d e t e r m i n e d b y d o u b l e s t a i n i n g w i t h a n t i - K i - 6 7 a n t i b o d y ( D A K O ) a t a 1:25 d i l u t i o n i n P r o t e i n B l o c k . K i - 6 7 i s p r e s e n t i n t h e n u c l e i o f c y c l i n g 64 cells. 2.11. Immunohistochemistry O V C A R 3 and K562 cell pellets were solidified by collagen gel, fixed in 4% paraformaldehyde, and embedded in paraffin. Sections of these cells, of the ovaries and of tissue arrays were deparaffinized, rehydrated, submitted to antigen retrieval by a steamer for 25-30 min in Target Retrieval Solution (DAKO) and then treated with 3% H2O2 for 30 min. Slides were blocked in 5% normal goat serum in PBS for 30 min followed by incubation with H O X A 7 primary antibody (1:400) overnight at 4 °C. The sections were then incubated with Envision Labelled Polymer/HRP (DAKO) for 30 min at RT and developed in diaminobenzine (DAKO) or in NovaRED substrate (Vector Laboratories, Inc., Burlington, ON), and counterstained with Mayers hematoxylin. As controls, we used rabbit IgG instead of H O X A 7 antibody. 2.12. Scoring and statistics for tumor arrays For tissue arrays, after immunohistochemistry, H O X A 7 protein expression was scored microscopically according to the intensity (0 for absence, 1 for low and 2 for high intensity) of staining. Nuclear staining was also scored according to the existence of the 65 staining in the epithelium (0 for absence, 1 for 5-95% of nuclei positive and 2 for 95-100%) of nuclei positive). A l l slides were analyzed in a blinded study for total protein by Dr. Gilks (UBC), and for nuclear expression by myself. The association between immunohistochemical staining intensity or nuclear positivity and grades was analyzed by Spearman's Rho Correlation Coefficient. Disease-specific survival was defined as the interval between diagnosis and death due to ovarian carcinomas. Significance of markers to predict survival was performed using Kaplan-Meier survival curve and log-rank statistic. Statistical significance was set at p<0.05. Statistical analysis was performed using JMP v.6 (SAS Institute, Cary, NC) . The data were binarised for H O X A 7 nuclear staining (0 and 1 classified as negative [0] and 2 classified as positive [1]). A l l statistics were carried out by Kalloger Consulting (Vancouver, Canada). 2.13. siRNA transfection siRNAs were purchased from Dharmacon, Inc. (Chicago, IL). Cells were seeded at 10 x lO 4 in 35 mm culture dishes for normal OSE, 12-16 x 104 cells/ well for O V C A R 3 and O V C A R 8 , and 8 x lO 4 cells/well for SKOV3 in 12 well plates and allowed to adhere for 66 24hr. siRNAs were transiently transfected into cells by using lipofectamine 2000 reagent (Invitrogen) in opt iMEM (Invitrogen) according to manufacturer's guidelines. For D N A determination experiments (see section 2.6), IOSE cells were plated as described above, allowed to attach for 24 hr, then, siRNAs were transfected. For [3H]-thymidine incorporation assay experiments (see section 2.6), normal OSE and O V C A R 3 were trypsinized after 24 hr of s iRNA transfection, then seeded in M-199/MCDB105/2% FBS (normal OSE) or M-199/MCDB105/5% FBS (OVCAR3) as described above. In case of normal OSE, after 24 hr, the cells were treated with E G F (See section 2.5). 2.14. Scratch assay for cell motility Cells were grown to near confluence within 72 hr after siRNAs transfection. Monolayers were wounded with the tip of a sterile 200 ul pipette. Points along the wound were marked and migration of cells into the wound was measured using an ocular micrometer. Six representative fields per culture were measured at time 0 and again after 5 hr (OSE and SKOV3), 24 hr (OVCAR3 and O V C A R 8 ) or 50 hrs (OVCAR3). Data were analyzed by A N O V A . 67 2.15. Matrigel cell culture Three-dimensional cultures were prepared using a modified procedure described by Carrio et al. (Carrio et al., 2005). O V C A R 3 were seeded at 12 x i o 4 cells/ well in 12 well plates. After 24 hr, s iRNA control scramble #2 or s iRNA to H O X A 4 was transfected for 24 hr. Then, cells were trypsinized and plated (3 x 104 cells in 24 well plates) on top of 200 ul of polymerized Matrigel (10.75 mg/ml; B D Biosciences, Bedford, M A ) in 0.5 ml M-199/MCDB 105 medium without serum. After 30 minutes of incubation at 37 °C, 250 ul, medium containing 10% Matrigel was added on top of the cells. More than 4 hr later, 250 ul M-199/MCDB 105 with 5 % FBS were added on the top of the 10%) Matrigel medium. After 4 days of cultivation, the morphology of the three-dimensional cultures was assessed by visual examination. 2.16. CA125 measurement O V C A R 3 was seeded at 16 x 104 cells/well in 12 well plates. After 24hr, s iRNA control scramble #2 or s iRNA H O X A 4 was transfected for 48hr. Then, media were changed and 24 hr later, conditioned media were collected and kept at - 70 °C until they were analyzed. The tests were performed on the Abbott A x S Y M analyzer, with a normal range of <35 kU/L (Tumor Marker Lab, PHSA Laboratories, Vancouver, BC) . 6 8 2.17. Statistics Data are presented as the mean ± SD and are representative of three individual experiments. Statistical analysis was performed using the Prism statistical software package (GraphPad Software, San Diego, CA) . Data were analyzed by Student's t-Test or A N O V A (Tukey i f necessary). Statistical significance was set at p<0.05. 69 3. Results The main purpose of this study was to examine the role of H O X genes in ovarian carcinogenesis. Since published data suggested that H O X A 7 may play a role in ovarian carcinogenesis (Naora et al., 2001a), and no antibodies to H O X A 7 protein were available, I started my study by producing such an antibody. However, before using this antibody to investigate ovarian cancer, I examined possible interrelationships between H O X A 7 and normal ovarian folliculogenesis, which represents one of the most complex differentiation processes in the adult woman. The remainder of the study deals with relationships of H O X A genes with ovarian carcinogenesis. 3.1. The production and antibody characterization of anti-HOXA7 antibody ELISA, RT-PCR, and Western blot analysis: B y E L I S A , the serum from two of three rabbits reacted significantly with the synthetic peptide (Fig.4). The sera were tested further using the ovarian cancer line O V C A R 3 and the erythroleukemic line K562 as positive and negative controls, respectively. O V C A R 3 was positive for H O X A 7 by R T - P C R (Fig.5) and, by Western blot analysis, serum from one rabbit (rabbit T I ) detected a single protein band in O V C A R 3 (Fig.5). The molecular weight was consistent with the published data for H O X A 7 (Swiss-Prot, accession number P31268). 70 T1 • 1st bleed A 2nd bleed • 3rd bleed • 4th bleed -5 -4 -3 -2 -1 0 log10(dilution) 12 0.75-1 o log10(dilution) Fig.4. Antibody titers in serum by ELISA. Rabbit TI and T 2 responded by boosting, but not rabbit T 3 . (a) o K HOXA7 actin (b) Fig.5. Expression of H O X A 7 m R N A and protein in ovarian cancer cells O V C A R 3 (positive control) and the leukemia lines K 5 6 2 (negative control), (a) A predicted 414 bp P C R product of H O X A 7 was obtained in O V C A R 3 , but not in K 5 6 2 . The P C R product was confirmed by sequence analysis (data not shown), (b) The arrow indicates a predicted 2 6 kDa single band in O V C A R 3 , but not in K 5 6 2 . o, O V C A R 3 ; k, K 5 6 2 . 72 The single band was observed in O V C A R 3 without reducing agents in sample buffer (data not shown). This serum was chosen for all subsequent experiments. K562, known as H O X A 7 negative by Northern blot analysis (Magli et al., 1991) and RT-PCR (Vieille-Grosjean et al., 1992), was negative both by Western blot analysis and RT-PCR (Fig.5). Affinity purification: Although rabbit T l polyclonal antibody showed the single band by Western blot analysis, I used an affinity chromatography by using the H O X A 7 synthetic peptide affinity column to purify the antibody more. As affinity-purifed antibodies, I chose to use fractions, which was the higher concentration of protein by Bradford assay. Fraction 4 was the highest and fraction 7 and 8 were lower than fraction 4, but they contained protein (data not shown). By ELISA, affinity purified sera and the sera before affinity purification reacted to the peptide H O X A 7 (Fig.6). As predicted, the flow-through before elution had no response. However, by western blot analysis, there were no bands by affinity-purified sera (Fig. 7) On the other hand, the sera before affinity purification and the flow-through sera before elution had bands in O V C A R 3 . This experiment was repeated twice, and I got the same result as above. Hence, non-affinity purified sera were used for the rest of the experiments. 73 • before purification log10(dilution) Fig.6. Affinity purified H O X A 7 antibody titers by E L I S A . F4, fraction number 4; F7, 8, fraction number 7 and 8, combined. Affinity purified sera and sera before affinity purification reacted by E L I S A . The flow-through before elution did not react. 74 o k o k o k o k 4 Fig .7 . Expression of H O X A 7 protein in ovarian cancer cells O V C A R 3 (positive control) and the leukemia lines K562 (negative control) by affinity purified antibodies. The black line indicates a predicted 26 kDa. 1 . before purification, 2. fraction 4, 3. fraction 7 and 8 combined, 4. flow-through. There were no bands by affinity purified antibodies. There were bands by non-purified and the flow-through sera before elution. O V C A R 3 ; k, K562. 75 Immunofluorescence microscopy: (I) Mouse embryos: I evaluated the specificity of our antibody in mouse embryos at 11.5 day p.c. At this stage, the central and peripheral nervous system has differentiated (Mahon et al., 1988). H O X A 7 protein was apparent in dorsal root ganglia between T3 and L I (Fig.8). In addition, it was present in the neural tube, but, as expected, was absent in the metanephros. Similar to Hox 2.1, where the polyclonal antibody bound to all tissues expressing m R N A (Wall et al., 1992), my H O X A 7 protein staining was consistent with its gene transcript localization by in situ hybridization (Mahon et al, 1988). In preimmune serum controls, the staining was negative (data not shown). (2) Cultured cells: . O V C A R 3 cells showed H O X A 7 expression in the nuclei (Fig.9), which was consistent with its function as a transcription factor, and with the studies in differentiated F9 and NIH3T3 cells with anti-Hoxl.l (mouse Hoxa7) monoclonal antibodies (Shulze et al., 1987). With pre-immune serum, H O X A 7 staining was non-specific (Fig.9). GST fusion protein: To further confirm the specificity of our anti-HOXA7 antibody, I created 2 lots of GST/HOXA7 fusion protein: one with a perfect sequence and one with one point mutation which was outside the peptide sequence used raising the anti-HOXA7 antibody (Fig. 10). Both fusion proteins produced the expected 54 kDa Fig.8. Expression of H O X A 7 in a mouse embryo. Parassagital section of a 11.5 day pc embryo stained with anti-HOXA7 antibody, (a) hematoxylin stain, (b) immunofluorescence. G, dorsal root ganglion (scale bars = 140 um). 7 7 Fig.9. Immunoflurescent detection of H O X A 7 in O V C A R 3 . (a) pre-immune serum, nuclei negative, (b) anti-HOXA7 antibody, nuclei positive (scale bars = 35 um). gtgctgcggc gagctccgtc caaaagaaaa tggggtttgg tgtaaatctg ggggtgtaat gttatcatat atcactctac ctcgtaaaac cgacactgaa agctgccgga caacaaatca caggtcaaaa ttatgagttc ttcgtattat gtgaacgcgc tttttagcaa atatacggcg gggacttctc tgttccaaaa tgccgagccg acttcttgct cctttgcgcc caactcacag agaagcggct acggggcggg cgccggcgcc ttcgcctcga ccgttccggg cttatacaat gtcaacagcc ccctttatca gagccccttt gcgtccggct acggcctggg cgccgacgcc tacggcaacc tgccctgcgc ctcctacgac caaaacatcc ccgggctctg cagtgacctc gccaaa ggc (Gly) g cctgcgacaa gacggacgag ggcgcgctgc age (Ser) atggcgcggc tgaggccaat ttccgcatct acccctggat geggtcttea ggacctgaca ggaagcgggg ccgccagacc tacacgcgct accagacgct ggagctggag aaggagttcc acttcaaccg ctacctgacg cggcgccgcc gcattgaaat cgcccacgcg ctctgcctca ccgagcgcca gattaagatc tggttccaga accgccgcat gaagtggaag aaagagcata aggacgaagg tccgactgcc gccgcagctc ccgagggcgc cgtgccctct gccgccgcca ctgctgccgc ggacaaggee gacgaggagg acgatgatga agaagaggaa gacgaggagg aatgaggggc egatcegggg ccctctctgc aceggacagt eggaaaageg tctttaagag actcactggt tttacttaca aaaatgggaa aaataaaaga aaatgtaaaa aacaaaaaca aaaacaaaaa agee Fig.10. A point mutation in the H O X A 7 sequence. The sequence in blue is the coding region. The sequence in red was used to raise polyclonal antibodies against H O X A 7 . The sequence in pink is the point-mutated region. The sequence from GenBank (AF026397)is ggc (Gly). The point-mutated sequence was age (Ser). The point-mutated sequence is outside of the sequence which was used to raise an t i -HOXA7 antibody. 79 bands by Western blot with anti-H0XA7 antibody and with anti-GST antibody (Fig. 11) , based on the molecular weight for H O X A 7 protein of 26 kDa and for GST protein of around 28 kDa. 3.2. The expression and regulation of HOXA7 in human ovarian follicles 3.2.1. HOXA7 expression in human ovarian follicles Immunohistochemisty: I used anti-HOXA7 antibody on paraffin sections of seven premenopausal ovaries, O V C A R 3 and K562 cells. H O X A 7 expression in O V C A R 3 was mainly but not entirely nuclear, while IgG controls and K562 were negative (Fig. 12) . The seven ovarian sections contained a total of 43 primordial, 28 primary and 6 secondary/preovulatory follicles. Oocyte nuclei and cytoplasm were weakly positive at all stages (Figs. 13A-E). In 42% of primordial follicles, granulosa cells were not stained and in 51%>, nuclei were weakly stained for H O X A 7 (Fig. 13A; Table 6). In contrast, granulosa cell nuclei in 93 % of primary follicles were either partially or completely positive (Fig.l3B). From the primary to the preovulatory stages, all follicles were strongly positive compared to their surrounding stroma (Fig.l3F). In secondary multilayered and preovulatory follicles, the staining of granulosa cells was variable, both within and between follicles, (Figs. 13D,E), however, when they stained, the Fig.ll. S D S - P A G E a n a l y s i s o f G S T / H O X A 7 f u s i o n p r o t e i n , ( a ) a n t i - H O X A 7 a n t i b o d y . ( b ) a n t i - G S T a n t i b o d y . T h e a r r o w i n d i c a t e s t h e G S T / H O X A 7 f u s i o n p r o t e i n ( a b o u t 54 k D a ) . T h e s a m e b a n d s w e r e o b s e r v e d w i t h b o t h a n t i b o d i e s , r, r e c o m b i n a n t p r o t e i n ; o, O V C A R 3 . Fig.12. Immunohistochemical staining for H O X A 7 in paraffin-embedded cell lines, (a) O V C A R 3 , IgG control, (b) O V C A R 3 stained with H O X A 7 antibody, and (c) K562 cells stained with H O X A 7 antibody (scale bars = 30 urn). 82 Fig.13. Immunohistochemical staining for H O X A 7 in human ovaries. (A) primordial follicles, H O X A 7 negative; (B,C) primary follicles, H O X A 7 positive; (D,E) secondary or multilayered follicles and (F,G) graafian follicles. (G) is close up of (F) (scale bars: A , B = 18 um; C = 40 um, D , E = 75 mn F = 500 urn, G = 95 u.m). Arrowheads in C and D show primodial follicles. (F) (i) follicles stain for H O X A 7 , in contrast to the surrounding stroma (ii) granulosa cells (G) stain heterogeneously, (iii) nuclei and cytoplasm stain in the theca interna (TI) and (iv) theca externa (TE) is completely negative, FF ; follicular fluid. (H) stromal cells vary but in general, stroma in the ovarian cortex stains more intensely than medullary stroma (scale bar = 350 (j,m). 84 Table 6. Staining intensity of H 0 X A 7 in ovarian follicles during follicular development Signal Follicle stages primordial primary secondary none weak strong 18 (42%) 22 (51 %>) 3 (7%) 2 9 (32%) 17 (61%) 1 (17%) 2 (33%) 3 (50%) Total follicles 43 28 85 intensity of the signal was high. 50% of granulosa cells in the secondary follicles were strongly positive. Similar to primary follicles, H O X A 7 was localized in the nuclei of secondary follicles, but in preovulatory follicles H O X A 7 had mainly translocated to the cytoplasm. There was no consistent difference between the cumulus and the mural granulosa cells. The theca interna was strongly positive, and the staining was cytoplasmic (Figs. 13F,G). In contrast, the theca externa was completely negative, strikingly different from other stromal areas. In general, the stroma in the ovarian cortex was more positive than in the medulla (Fig. 13H). 3.2.2. H O X A 7 protein expression in the cell cycle Proliferating cultured S V O G cells were double-stained with anti-HOXA7 antibody and anti-Ki-67 antibody. Ki-67 antigen is present in all active phases of cell division, but not in the quiescent or Go phase (Scholzen and Gerdes, 2000). H O X A 7 expression increased during mitosis: In prophase, the accumulation of H O X A 7 began. In metaphase, H O X A 7 expression was the highest, whereas during anaphase, the expression began to decrease and in telophase, staining was similar to that of interphase cells (Fig. 14). H O X A 7 was dispersed in the cytoplasm, in contrast to mouse cells where Hoxa7 protein co-distributed with chromatin (Schulze et al., 1987). 86 B c m •* >;s. F ^ • Fig.14. Immunofluorescence staining by anti-HOXA7 and anti-Ki-67 antibody in cultured immortalized granulosa cells (SVOG). SVOG were prepared from 60% confluent plates containing proliferating cells. (A, D) phase microscopy, (B, E) HOXA7, (C, F) Ki-67. HOXA7 staining is increased in mitotic cells (arrow -heads). Cells were double-stained for HOXA7 and Ki-67. A,B ,C and D,E,F, respectively, each represent the same field, (scale bars = 25 um). 87 3.2.3. GDF-9 and TGF-pi regulate HOXA7 protein expression and proliferation in cultured SVOG The presence of H O X A 7 in primary, but not in primordial follicles suggested that H O X A 7 expression might be induced by GDF-9 which is one of the oocyte-derived factors known to be produced at that stage of folliculogenesis, and to induce granulosa cell proliferation in other species. As a control member of the TGFp family, known not to have this function (Juengel and McNatty, 2005), I used TGFP-1. Since human primordial and primary follicles were not available, I examined the effect of GDF-9 on the proliferation of cultured S V O G cells. 24 hr treatment with 200-300 ng/ml GDF-9 significantly upregulated H O X A 7 protein expression (Fig. 15A). In contrast, 48hr treatment with 1-10 ng/ml TGF-p 1 downregulated H O X A 7 protein expression (Fig. 16A). To evaluate the effects of GDF-9 or TGF-pi on proliferation of S V O G cells, I treated them with GDF-9 for 24hr or TGF-P 1 for 24-48 hr, followed by [3H]thymidine incorporation. Similar to rat granulosa cells from antral and preovulatory follicles (Vitt et al., 2000), treatment with 200-300 ng/ml GDF-9 induced a significant increase in the thymidine uptake in S V O G cells (Fig.l5B). On the other hand, treatment with 1-10 ng/ml TGF-p i for 48 hr had no significant effect on thymidine uptake (Fig.l6B). GDF-9 (ng/ml) 0 200 300 (b) 200 300 GDF-9 (ng/ml) Fig. 15. Effects of GDF-9 on S V O G cells over 24 hr. (a) H O X A 7 expression by SDS-PAGE, (b) proliferation by [^thymidine incorporation assay. A7 , H O X A 7 . Parallel S V O G cultures were used for western blot analysis and [3H]thymidine incorporation.* indicates p < 0.05. 8 9 (a) A7 G A P D H TGF (ng/ml) 0 3 10 (b) - 1 5 0 o X S 100 SL 4) I 50 0 "io" T G F - p 1 (ng/ml) Fig.16. E f f e c t s o f T G F - B l o n S V O G c e l l s o v e r 4 8 h r . ( a ) H O X A 7 e x p r e s s i o n b y S D S - P A G E , ( b ) p r o l i f e r a t i o n b y [ 3 H ] t h y m i d i n e i n c o r p o r a t i o n a s s a y . A 7 , H O X A 7 ; T G F , T G F - 1 3 1 . P a r a l l e l S V O G c u l t u r e s w e r e u s e d f o r w e s t e r n b l o t a n a l y s i s a n d [ 3 H ] t h y m i d i n e i n c o r p o r a t i o n . 90 3.3. HOXA7 expression in ovarian surface epithelium (OSE), granulosa cell tumors and tissue microarray 3.3.1. HOX gene expression in cDNA array and the confirmation by RT-PCR In collaboration with Dr. Gilks (Department of Pathology, U B C ) , I received the results of a c D N A microarray, which showed the different expression of H O X genes between benign ovarian tumors, serous ovarian tumors of low malignant potential (LMP) and serous ovarian carcinoma (Fig. l ; Table 4). This result is a part of his published study (Gilks et al., 2005). The results showed that H O X A 4 , H O X A 7 and H O X A 10 were upregulated in the serous type of ovarian malignant tumors compared to L M P and benign tumors (Fig.l). HOXA10 has a transcript variant and one of them (GenBank AA953229) was upregulated in ovarian malignant tumors, but not the other (GenBank AA284178). H O X A 4 was most upregulated in ovarian tumors. Therefore, I chose to study this gene. Naora's study identified H O X A 7 to be associated with differentiation (Naora et al., 2001a), and she showed that H O X A 7 was upregulated in benign ovarian tumors and low grade tumors compared to high grade tumors. However, in our c D N A array, the expression was higher in high grade tumors than in L M P or benign tumors. H O X A 9 is 91 expressed in fallopian tubes, and the morphology of serous ovarian tumors resembles fallopian tubes, hence I expected the upregulation of H O X A 9 in malignant tumors, however, there was no change between benign, L M P and malignant tumors. H O X D 9 was downregulated in ovarian carcinoma compared to benign tumors and LMP. By RT-PCR, the different expressions in the c D N A microarray for three H O X A genes, H O X A 4 , H O X A 7 and H O X A 9 , was confirmed using five of the same tumors as were used for the c D N A microarray, and in three different tumors (Fig. 17). 3.3.2. H O X A 7 expression in O S E in vivo Eleven normal ovaries from premenopausal and postmenopausal normal ovaries were stained with anti-HOXA7 antibody. Only eight ovaries had OSE, either in invaginations or cysts. As Naora found (Naora et al., 2001a), most of OSE, which consisted of flat cells, did not express H O X A 7 (Fig.l8A). Positive nuclei and cytoplasmic staining was observed in cuboidal OSE (Fig.l8B), and increased staining was observed in columnar cells in OSE (Fig. 18C). The black material on the top of the OSE in this figure was due to painting of the sample with India ink during tissue handling in pathology. Invaginations may create inclusion cysts, which are considered to be the site of origin of epithelial ovarian tumors (Freeley and Wells, 2001). In OSE lining invaginations, 92 Fig.17. Confirmation of c D N A microarray by R T - P C R . 1 .0vCal4 , 2. O v C a l 9 , 3. OvCalO, 4. O v C a l 1, 5. OvCa23, 6. V O A 86, 7. V O A 89, 8. V O A 95. Five tumors were the same tumors as were used in c D N A microarray. The square colors were copied from F i g . l . neg, water control; L M P , low malignant potential tumor; grade 2 and grade 3, carcinomas. oo CO CD "D CO 0)1 CN 0 "D 0)1 CD L O CL co CN C I • i • i • • I • ml o O (J> O Fig. 18. H0XA7 expression in ovarian surface epithelium. (a,b,c) ovarian surface epithelial cells, (d,e) inclusion cysts. 95 both negative and positive staining was also observed (data not shown). Naora claimed that the epithelium lining inclusion cysts in all specimens was H O X A 7 positive, however, both positive and negative staining was observed in this study even when the cells were columnar in inclusion cysts (Fig.l8D,E; Naora et al., 2001a). 3.3.3. Tissue microarray - epithelial ovarian carcinomas To study the implication of H O X A 7 in biology of ovarian cancer, I performed immunohistochemistry using a tissue microarray composed of 542 ovarian epithelial tumors. The intensity of the staining was monitored by Dr. Gilks (UBC). In this study, 513 cases (1 serous borderline, 200 serous, 126 endometrioid, 34 mucinous, 138 clear cell carcinoma, 7 transitional, 7 undifferentiated, atypical) were used for the assessment. A n image of a representative core of each grade of tumors (1-3) is presented in Figs. 19, 19B, 20 and 20B. The staining pattern is summarized in Table 7 and Table 8. H O X A 7 staining showed cytoplasmic staining in all tumors, varying from very weak to intense, whereas 76% of epithelial ovarian carcinomas showed nuclear staining as well (Figs. 19B, 20B; Table 8). Negative staining of the stroma was observed in some tumors even when the epithelial cells were strongly positive (Figs. 19c,h). The positivity of the stroma was not related to the pathology of the tumors and the tumor grade. 9 6 Table 7. Tumor characteristics and the intensity of HOXA7 overall expression Histopathologic subtype intensity (mean ± SD) Serous 200 grade 1 10 1.30 ± 0 . 4 8 grade 2 54 1.57 ± 0 . 5 0 grade 3 136 1.54 ± 0 . 5 3 Clear cell 138 grade 1 47 1.47 ± 0 . 5 5 grade 2 76 1.49 ± 0 . 5 0 grade 3 15 1.60 ± 0 . 5 1 Endometrioid 126 grade 1 82 1.50 ± 0 . 5 7 grade 2 37 1.43 ± 0 . 5 0 grade 3 7 1.57 ± 0 . 5 2 Mucinous 34 gradel 14 1.43 ± 0 . 5 1 grade2 18 1.28 ± 0 . 4 6 grade3 2 1. Transitional 7 grade2 2 1.5 grade3 5 1.80 ± 0 . 4 5 Undifferentiated 7 grade2 1 2 grade3 6 1.83 ± 0 . 4 1 Serous borderline tumor Total 1 513 1 97 Table 8. Tumor characteristics and the intensity of HOXA7 nuclear expression Histopathologic subtype HOXA7 (-/+)* (133/480) positivity (mean ± SD) Serous 200 grade 1 10 6/4 1.30 ± 0 . 6 7 grade 2 54 20/34 1.56 ± 0 . 6 3 grade 3 136 41/95 1.61 ± 0 . 6 5 Clear cell 138 grade 1 47 4/43 1.89 ± 0 . 3 7 grade 2 76 13/63 1.80 ± 0 . 4 6 grade 3 16 6/9 1.60 ± 0 . 5 1 Endometrioid 126 grade 1 82 15/67 1.78 ± 0 . 5 0 grade 2 37 12/25 1.68 ± 0 . 4 7 grade 3 7 2/5 1.57 ± 0 . 7 9 Mucinous 34 grade1 14 3/11 1.79 ± 0 . 4 3 grade2 18 7/11 1.61 ± 0 . 5 0 grade3 2 1/1 1.50 ± 0 . 7 1 Transitional 7 grade2 2 1/1 1.50 ± 0 . 7 1 grade3 5 1/4 1.80 ± 0 . 4 5 Undifferentiated 7 grade2 1 0/1 2 grade3 6 1/5 1.83 ± 0.40 Serous borderline tumor 1 1 Total 513 * score 0 and 1 was considered as negative and score 2 as positive 98 Fig.19. Expression of H O X A 7 in tumor microarray-1. (A) serous type, (B) endometrioid type. (a,d,g,j), grade 1 (GI); (b,e,h,k), grade 2 (G2); (c,f,i,l), grade 3 (G3). F i g . l 9 B are enlarged pictures of Fig.19. Scale bar = 320 pm. 99 100 Fig.l9B. Expression of H O X A 7 in tumor microarray-1 (nuclei). (A) serous type, (B) endometrioid type. (a,d,g,j), grade 1 (GI); (b,e,h,k), grade 2 (G2); (c,f,i,l), grade 3 (G3 (a,b,c,g,h,i), nuclei are positive in the epithelial cells; (d,e,f,j,k,l), nuclei are negative or mixed positive and negative in the epithelial cells. Scale bar = 80 pm. 102 Fig.20. Expression of H O X A 7 in rumor microarray-2. (A) mucinous type, (B) clear cell type. (a,d,g,j), grade 1 (GI); (b,e,h,k), grade 2 (G2); (c,f,i,l), grade 3 (G3). Fig.20B is enlarged pictures of Fig.20. Scale bar = 320 um. 103 104 Fig.20B. Expression of H O X A 7 in tumor microarray-2 (nuclei). (A) mucinous type, (B) clear cell type. (a,d,g,j), grade 1 (GI); (b,e,h,k), grade 2 (G2); (c,f,i,l), grade 3 (G3). (a,b,c,g,h,i), nuclei are positive in the epithelial cells; (d,e,f,j,k,l), nuclei are negative or mixed positive and negative in the epithelial cells. Scale bar = 80 pm. 106 Naora et al. showed that H O X A 7 expression is correlated with grade in serous and endometrial epithelial ovarian carcinoma (Naora et al., 2001a). In contrast, in my study, by statistical analysis, there was an association between tumor grade and the intensity of H O X A 7 total expression, however, the correlation coefficient was 0.05, which was considered as insignificant (Table 7). There was no correlation between H O X A 7 nuclear expression and tumor grade (p = 0.3819; Table 8). E-cadherin is required for epithelial differentiation of primary epithelial ovarian carcinomas (Auersperg et al. 1999). However, in our study, consistent with there being no correlation with H O X A 7 overall expression and tumor grade, there was no correlation between H O X A 7 expression and E-cadherin expression (p = 0.75), and, also no correlation between H O X A 7 nuclear staining and E-cadherin expression (p = 0.5075). Survival analyses were performed based on Kaplan-Meier curves coupled to a log-rank test and Wilxocon test (Figs. 21, 22). The H O X A 7 overall staining intensity was not associated with patient survival either by a log-rank test (p = 0.8455) or by a Wilcoxon test (p = 0.7334). On the other hand, positive H O X A 7 nuclear staining showed a 107 o,o-1000 2000 3008 400© Tims (Days) 5000 0' I Fig.21. Relationship between H O X A 7 overall expression and cumulative survival of patients with epithelial ovarian tumors. Kaplan-Meier survival curve demonstrates no correlation H O X A 7 expression intensity with survival. Log-rank and Wilcoxon tests were used to verify the significance of the difference in survival. The intensity was scored as [0] for absence, [1] for low and [2] for high intensity of staining. Fig.22. Relationship between H O X A 7 nuclear expression and cumulative survival of patients with epithelial ovarian tumors. Kaplan-Meier survival curve demonstrates H O X A 7 nuclear expression significantly improved disease-specific survival. Log-rank and Wilcoxon tests were used to verify the significance of the difference in survival. Score 0 (negative) and score 1 (partially positive) were combined and showed as [0] and score 2 (positive) was showed as [ 1 ] . 109 significantly improved prognosis for the disease-specific survival by a log-rank test (p=0.0104) and by a Wilcoxon test (p = 0.0059). The difference of life expectanices from average of 5 years to 10 years. 3.3.4. Granulosa cell tumors Granulosa cell tumors were intensely H O X A 7 positive, except for rare negative regions in some of the tumors. Similar to normal preovulatory granulosa cells, the staining of the granulosa cell tumors was mainly cytoplasmic (Fig.23). Only 2 of 6 tumors did show focal nuclear staining as well (Fig.23). 3.4. Expression of HOXA4, HOXA7 and HOXA9 in normal OSE and ovarian cancer in culture Expression of HOXA4, HOXA7 and HOXA9 in normal OSE in culture in different proliferative stages I examined cases of normal cultured OSE for the 3 H O X A genes at different proliferative stages: (i) sub-confluent; (ii) confluent; and (iii) senescent, using mitotic figures as the proliferative index (Table 9). These normal OSE were grown in E G M medium in passage 0 and from passage 1, in M-199/MCDB105/10% FBS. In E G M , Fig.23. Immunohistochemical staining for HOXA7 in granulosa cell tumors. (A) Tumor N o . l : staining was in the cytoplasm, (B) Tumor No.2: staining was predominantly nuclear, (scale bars = 45 pm). I l l Table 9. Normal OSE cases in culture O S E Passage Confluency (%) Mitiotic counts Age morphology sub-confluent 362 1 80-90 0.74 35 atypical 371 2 90 0.42/0.32 35 atypical 363 3 80-90 0.55/0.73 37 epithelial 370 3 80-90 0.38 36 atypical 373 4 70-80 0.41 35 epithelial 364 5 80 0.22 27 epithelial confluent 371 2 100 0 35 atypical 363 3 100 0 37 epithelial 368 3 100 0 26 epithelial 361 4 100 0 37 epithelial 373 4 100 0 35 epithelial senescent 367 3 70 0 30 flat epi 360 7 70 0 32 atypical 364 8 60 0 27 flat epi Two values for mitotic counts were from two different dishes of the same case. Morphology and passage numbers were defined at the time of lysis. 112 cells were atypical, however, when switched to M-199/MCDB 105/10% FBS, the cells acquired an epithelial morphology (Fig.24; Salamanca et al., 2004). A l l 3 H O X genes were expressed in normal OSE. Expression of all 3 genes was reduced in senescent cultures (p<0.05), and in addition, H O X A 9 and H O X A 4 were lower in stationary than in rapidly proliferating cells (p<0.05), indicating a possible role of these genes in the regulation of OSE proliferation or motility (Fig.25). Expression of HOXA4, HOXA 7 and HOXA9 in normal OSE and ovarian cancer cell lines To investigate whether the three H O X A genes, H O X A 4 , H O X A 7 and H O X A 9 are expressed in ovarian cancer cell lines, 10 epithelial ovarian cancer cell lines and 2 SV40 Tag immortalized normal OSE (IOSE) lines were examined (Fig.26). A n erythroleukemic cell line, K562 was used as a negative control. Three ug of R N A was used to make cDNAs, and I used 1 u.1 of c D N A in 25 ul total PCR-mixture and performed 30 cycles of PCR for H O X A 4 , H O X A 7 and H O X A 9 (not in linear range of the expression). H O X gene expression varied between ovarian cancer cell lines. A l l ovarian cancer cell lines demonstrated similar expression of H O X A 4 , H O X A 7 and H O X A 9 . O V C A R 1 0 and A2780 were completely negative for all three H O X A genes. Fig.24. Morphology of normal OSE. (A) epithelial, (B) atypical, scale bar - 50 um. 114 sub-confl. confl. senescent A4 A7 A9 actin B^RSJBIt 4MMMH ^pJMJt M U M WHWj? W 4Mfc <••> SPB* « • * ( M A «•» H ^ M t M M * MnK «(• • 1 H m 4(MMk WBWte S^BWBBfc SSBMS^ 4MMH^  <B8MBW ^ W i ^ ^BK(^  4NHNM^  ^ffK/K^ A 4 A 7 A 9 o to lsub-eontf, i confl. I senescent Fig.25. HOXA expression in normal OSE in culture. HOX genes expression were asessed in three different cultlure conditions: (i) sub-confluent (sub-confl.), (ii) confluent (confl.) and (iii) senescent. Confluent cases were kept at least for 2 days after they became confluent. HOX expression was normalized to beta-actin. a: p < 0.05, vs. sub-confluent. A4, HOXA4; A7, HOXA7; A9, HOXA9; actin, beta-actin. 115 A4 A7 A9 1 2 3 4 5 6 7 8 9 10 11 12 13 14 actin Will IteSi^ l Fig.26. Expression of H O X A 4 , A7 and A 9 in epithelial ovarian cell lines by RT-PCR. 1. water control, 2. O V C A R 2 , 3. O V C A R 4 , 4. O V C A R 5 , 5. O V C A R 8 , 6. O V C A R 1 0 , 7. A2780, 8. B G - 1 , 9. CaOV3, 10. O V C A R 3 , 11. S K O V 3 , 12. K562, 13. IOSE-80, 14. IOSE-398. A n erythroleukemic cell lines,K562 was used as a negative control for three H O X A genes. A4 , H O X A 4 ; A7 , H O X A 7 ; A 9 , H O X A 9 ; actin, beta-actin. A l l P C R products were sequenced. 116 Both IOSE cells had all three H O X A genes and the expression was stronger than in the epithelial ovarian cancer cell lines (Fig.26). To compare the expression of ovarian cancer cell lines and normal OSE for H O X A 4 , which was the most highly expressed in ovarian carcinoma cell lines, PCR was performed using the same cases from Fig.25 and Fig.26. Two normal OSE cells were sub-confluent cases. H O X A 4 expression in one of normal cases was higher than in IOSE cells and other case was similar intensity to IOSE (Fig.27). 3.5. Effects of EGF on HOXA4 expression and proliferation in normal OSE To see the effects of EGF, I treated normal OSE with 20 ng/ml E G F in a time-dependent manner. In these experiments, I sometimes combined 2-3 normal OSE cases to do one experiment. For short time treatments, I used 0-60 min. Treatment with 20 ng/ml EGF downregulated H O X A 4 expression around 30 % at 60 min, but before 60 min, there was no differences (Fig.28). In this figure, at 10 min, H O X A 4 seemed to be downregulated by E G F treatment, however, in other separate experiments, this downregulation was not observed. For longer time treatment, I used 5-48 hrs. 1 2 3 4 5 6 7 A4 actin Fig.27. Expression of H O X A 4 in normal OSE, immortalized normal OSE (IOSE) and ovarian cancer lines. Among ovarian cancer lines, O V C A R 8 has the highest, O V C A R 3 , moderate, and O V C A R 1 0 , no expression of H O X A 4 . 1. water control, 2. IOSE-80, 3 . 0 V C A R 8 , 4. O V C A R 3 , 5. O V C A R 1 0 , 6. OSE361 passage 2, 7. OSE362 passage 2. A4, H O X A 4 ; actin, beta-actin. 118 (A) A4 beta-actin 0 10 20 30 60 (min) C C E C E C E C E tpft!gj|| :mm0 iggpMg£ W fP®Sf HRIr PP» ***mf (B) 1.00n 0.75H > | 0.50-E 0.25-0.00-control 3EGF , • • , • • ,.• • , • -mm • t • • , • my. 0 10 10 20 20 30 30 60 60 time (min) Fig.28. Effects of 20 ng/ml EGF on H O X A 4 expression in normal OSE. for 0-60 min. The H O X A 4 expression level was analyzed by semi-quantative RT-PCR. H O X A 4 expression was normalized to G A P D H . The results are representative of three individual experiments. A4 , H O X A 4 . 119 Twentyfour hr treatment with 20 ng/ml EGF upregulated H O X A 4 expression (50 % increase), but not 5 hr (Fig.29A) or 13 hr (data not shown). The upregulation was not obvious at 48 hr (data not shown). To further evaluate the effects of EGF on proliferation of normal OSE cells, I treated them with 20 ng/ml EGF for 24 hr, followed by [3F£]-thymidine incorporation. Treatment with EGF induced a significant increase in the thymidine uptake in normal OSE cells (Fig. 29B). These results indicate that EGF may regulate H O X A 4 expression in normal OSE and that the expression may be associated with proliferative activities. 3.6. Effects of extracellular [Ca2+] on H O X A 4 expression in IOSE cells High concentrations of extracellular [Ca 2 +] induce cellular proliferation in IOSE cells (McNeil et al. 1998; Bilderback 2002). To see the effects of extracellular [Ca 2 +] on H O X A 4 expression, IOSE were treated with low (0.05 mM) and high (2.0 mM) concentrations of C a 2 + and performed semi-quantitative RT-PCR. IOSE demonstrated a drop in H O X A 4 m R N A levels by 24 hr at the high concentration of Ca compared to low concentration (Fig.30A). To evaluate the effects of changing extracellular [Ca ] on the proliferation of IOSE cells, I changed the concentration for 16-24 hr from 0.3 m M to either 0.05 m M or 2.0 m M , followed by [ H]-thymidine incorporation. Consistent with 120 Fig.29. Effects of 20 ng/ml E G F on H O X A 4 expression in normal O S E over 5 - 24 hr. (A) The H O X A 4 expression level was analyzed by semi-quantative R T - P C R . H O X A 4 expression level is normalized to G A P D H . C, control (no E G F ) ; E , E G F ; A 4 , H O X A 4 . (B) proliferation by [ 3H]-thymidine incorporation assay. Data were analyzed by Student's t-test. The results are the means ± SD and representative of four individual experiments. * indicates p<0.01. c, control (no E G F ) . (A) 5h 24h 2.5-2.0-> £ 1.5-z 1.0-£ 0.5-0.0-(B) I 1 control cm EGF • , i i r 24 24 time (hr) 122 Fig.30. Effects of extracellular [Ca ] on H O X A 4 expression in IOSE cells. IOSE cells were treated with 0.05 m M or 2.0 m M [Ca 2 + ] for 24 hr. (A) The H O X A 4 expression level was analyzed by semi-quantitative R T - P C R . H O X A 4 expression level is normalized to G A P D H . A 4 , H O X A 4 . (B) proliferation by [ 3H]-thymidine incorporation assay. Data were analyzed by Student's t-test. The results are the means ± SD and representative of four individual experiments. * indicates p<0.05. 123 (A) A4 G A P D H 0.05 2+ flBillP ^BBliP ispswP ^ ^ ^ P •••sisiPf ^isppp C a c o n c e n t r a t i o n (mM) 0.05 C a 2 * concentrat ion (mM) (B) 120000n 100000 0 80000 C 600004 1 40000 § ** 20000 i Ca 2* concentration (mM) 124 previous studies, the high concentration of extracellular C a 2 + induced a significant increase in the thymidine uptake in IOSE cells (Fig. 30B). These results showed an inverse relationship between proliferation and H O X A 4 expression, in contrast to the effects of cell density and EGF on OSE cells. 3.7. H O X A 4 expression and proliferation in ovarian cancer cell lines To examine relationship between H O X A 4 expression and proliferation, I chose 3 ovarian cancer cell lines, O V C A R 8 , O V C A R 3 and O V C A R 1 0 . O V C A R 8 had the highest expression, O V C A R 3 was moderate and O V C A R 1 0 was negative for H O X A 4 expression (Fig.26). Using Hoechst staining, the amount of D N A was quantitated on dayl, day3 and day 6 (Fig.31). Growth in O V C A R 8 and O V C A R 1 0 were similar and these two cell lines grew faster than O V C A R 3 . There was no relationship between H O X A 4 expression and cellular proliferation. 3.8. Estrogen effects on H O X A 4 expression in IOSE, O V C A R 3 and S K O V 3 cells Epidemiological studies have shown that endogenous and exogenous steroid hormones may influence the pathogenesis of ovarian carcinoma. The incidence of invasive ovarian carcinoma increases with the use of estrogen containing hormone replacement Fig.31. H 0 X A 4 expression, cell morphology and proliferation in ovarian cancer cell lines, (a) OVCAR8-the highest expression, OVCAR3-moderate and O V C A R l O - n o expression of H O X A 4 in culture. O V C A R 3 and O V C A R 8 characterized by compact cell growth, (b) Proliferation assay by D N A staining using Hoechst 33258 dye. Scale bar = 60 um. 126 therapy in peri- or post-menopausal women (Rodriguez et al., 1995; Garg et al., 1998). HOXA 10 and HOXC4 are regulated by steroid hormones in the endometrium (Taylor et al., 1998) and breast cancer cell lines (Frasor et al. 2003), respectively. There are contradictory reports on whether or not estrogen is mitogenic for OSE in culture (Karlan et al, 1995). I tested the effects of estrogen on HOXA4, HOXA7 and HOXA9 expression by RT-PCR in IOSE, OVCAR3 and SKOV3. In IOSE and OVCAR3, there was a trend to increase the expression of HOXA7 mRNA in a concentration dependent manner (10"8 to 10"6 M) at 24 hr (Fig.32, OVCAR3, data not shown). However, it was not statistically significant. SKOV3 did not express these three HOXA genes (Fig.26) and even after the application of estrogen, I could not detect HOX gene expression (data not shown). Therefore, estrogen appears to have no significant effect the HOXA 4,7,9 genes in these cells. 3.9. Effects of HOXA4 modulation on the growth rates of IOSE cells Thus far, I have shown that HOXA4 expression was not consistently associated with proliferation. I therefore evaluated the effects of HOXA4 on the growth properties on IOSE cells. IOSE was transfected with siRNAs targeting HOXA4 or with control siRNAs scramble #1 or #2 (scramble #1 and scramble #2; Dharmacon, Inc., Chicago, 127 (A) -7 0 10 10 10 A 4 A 7 A 9 actin E2 cone. (mM) (B) A 4 A 7 A9 i i.o> E 0.6' 0.0' X 10'! X 10-I •o.o-10-' I f 10"8 10"7 10 - 6 l— 0.0-^ -1—T E2 concentration (mM) X X 10-' 10" io-f Fig.32. Estradiol effects on three HOXA genes in IOSE cells. IOSE cells were treated with l(fMto ]<fM of estrogen for 24 hr. Control culture was treated with vehicle. The expression levels of HOXA4, HOXA7 and HOXA9 were analyzed by semi-quantative RT-PCR. Data were shown as the means +/- SD and representative of three separate experiments. E2, estradiol; A4, HOXA4; A7, HOXA7; A9, HOXA9; actin, beta-actin. 128 IL). Scrambles #1 and #2 are confirmed at least 4 mismatches with all known human, mouse and rat genes by B L A S T analysis. A scramble #1 target gene is not provided by the company, whereas scramble #2 targets firefly luciferase gene (non-mammal). H O X A 4 s iRNA was 4 targeted mixture (SMARTpool™). H O X A 4 expression was assessed by RT-PCR in IOSE following transfection of siRNAs. As a transfection reagent, I used lipofectamine 2000 (Invitrogen), and the volume used of lipofectamine 2000 was 60% less than the company's recommendation. Microscopically, 50 n M of scramble #1 showed obvious cellular damage to the IOSE. Consequently, I used 0.1 nM-20 n M of siRNAs for IOSE. By RT-PCR, H O X A 4 expression was decreased by H O X A 4 siRNAs, compared to scramble #1 at 10 n M or 20 n M at 48 hr (Fig.33). H O X A 4 expression in scramble #1 transfected cells was similar to mock cells (treated with lipofectamine 2000 only). I analyzed the effects of H O X A 4 depletion on the growth of IOSE. In vitro growth rates were assessed under routine culture conditions by Hoechst nuclear staining. Scramble #1 showed significant cellular toxicity compared to no transfection control especially at 10 n M or 20 n M (Fig.33) and the toxic effects were also observed under microscope (Fig.34). Therefore, using scramble #1, I could not assess appropriate effects of 129 Fig.33. Effects of H O X A 4 down-regulation in IOSE cells. (A) Effects of s i R N A transfection on H O X A 4 m R N A expression in IOSE cells. The cell lines were transfected with control scramble #1 or H O X A 4 s i R N A . 1. water control, 2. mock, 3. 20 n M scramble (20s), 4. 20 n M H O X A 4 (20A4), 5. 10 n M scramble (10s), 6.10nM A 4 (10A4), 7. 1 n M scramble (Is), 8. I n M A 4 (1A4), 9. O . l n M scramble (0.1 s), 10. O . l n M A 4 (0.1 A4) . (B) Effects of H O X A 4 down regulation on the growth rates on IOSE cells on day 3. Scramle, H O X A 4 s i R N A or mock transfected IOSE cells were plated into 96-well plates. The growth rate was monitored by D N A determination assay on day 3. A 4 , H O X A 4 s i R N A . See Fig.34 for morphology. (A) A4 G A P D H 1 2 3 4 5 6 7 8 9 10 mBmm^mmmm^mm^mft <•» (B) 1000«i £ o C (0 XI 1_ o » XX re 750 £111 scramble #1 CZDA4 X mock 20 20 10 10 1 1 0.1 0.1 concentration (nM) 131 mock control #1 A4 4 Fig.34. Effects of siRNAs on IOSE cells. At 20 n M on day 5. control #1, control s iRNA scramble #1; A 4 , H O X A 4 s iRNA. Scale bar = 80 um. (see Fig.33). 132 H 0 X A 4 downregulation, I compared the effects of scramble #1 and #2 on growth assays. In this experiment, I examined two different amounts of lipofectamine 2000, (20% or 40%) of company's recommended amounts). On day 3, scramble #1 has already shown the toxicity, but not #2 (Fig.35a,b). On day 5, scramble #2 showed less toxic effects (Fig.35c,d). Therefore, scramble #2 only was used for my rest of the experiments. 3.10. Downregulation of H O X A 4 in normal OSE, O V C A R 3 and O V C A R 8 H O X A 4 expressing normal OSE, O V C A R 3 and O V C A R 8 were transfected with scramble #2 or s iRNA targeting H O X A 4 . In normal OSE cells, 20 n M or 50 n M of siRNAs was transfected with 40% of lipofectamine 2000, similar to IOSE cells. After 48hr, s iRNA H O X A 4 downregulated H O X A 4 (Fig.36). In O V C A R 3 and O V C A R 8 cells, maximum recommended doses of lipofectamine 2000 were used. After 48 hr, although at 20 n M in O V C A R 3 , downregulation of H O X A 4 was observed, at either 50 n M or 100 n M of siRNAs, H O X A 4 expression was downregulated more (Fig.36). 3.11. Effects of H O X A 4 modulation on cellular motility One of the functions of H O X genes is the regulation of motility (Yamashita et al. 2006). 133 Fig.35. Effects of control siRNAs #1 and #2 on the growth rate of IOSE cells, (a) day 3, 20% lipofectamine 2000, (b) day 3, 40% lipofectamine 2000, (c) day 5,20% lipofectamine 2000, (d) day 5,40% lipofectamie 2000 for s iRNAs transfection. c, no transfection; control #1, control scramble s iRNAs #1; control #2, control scramble siRNAs #2. 134 (A) A4 G A P D H 1 2 3 4 5 ' M i l I i i mmm immm. m&mm mwwt? ^ W S B ^ G A P D H (Q 13 14 15 16 A 4 G A P D H m&mmk- wmmsm M i i t t i i M g i M t V W ^ W W lilPPBPPW Fig.36. Downregulation of H 0 X A 4 by s iRNA. s iRNAs were transfected into normal OSE, 0 V C A R 3 and 0 V C A R 8 and after 48 hr, the expression levels were assessed by semi-quantative RT-PCR. (A) normal OSE, (B) 0 V C A R 3 , (C) 0 V C A R 8 . (1,6) mock, (2,9,15) 50 n M scramble (50s), (3,10,16) 50 n M H O X A 4 (50A4), (4,11) 20 n M scramble (20s), (5,12) 20 n M A4 (20A4), (7,13) 100 n M scramble (100s), (8,14)100 n M A 4 (100A4). The results are representative of at least two experiments. 135 In a previous study in our lab, EGF enhanced migration of OSE (Ahmed et al. 2006) and I expected that downregulation of H O X A 4 would inhibit motility. Therefore, I observed H O X A 4 effects on OSE under E G F treatment. E G F treatment promoted migration (Fig.37). But, in contrast to my hypothesis, treatment with s iRNA to H O X A 4 enhanced EGF-induced migration after 4 hr (Fig.37). O V C A R 3 and O V C A R 8 migrated more after s iRNA H O X A 4 treatment, without EGF stimulation (Fig.38). Even after 24 hr, the differences between scramble and s iRNA H O X A 4 treatment were observed. O V C A R 8 cells migrated more than O V C A R 3 cells. In contrast to the results of the scratch assay of EGF and s iRNA treatment of OSE, where the cells moved singly, the ovarian cancer cell lines moved like a sheet. Toxicity of scramble #2 at 100 n M was not observed microscopically and there was no difference between mock and scramble transfection. SKOV3 has little or no H O X A 4 expression (Fig.26). To see the effect of H O X A 4 on cell motility, SKOV3 cells were treated under the same conditions (100 nM) as O V C A R 3 and O V C A R 8 . s iRNA H O X A 4 transfection had no effects on SKOV3 (data not shown). 136 Fig.37. Effects of H O X A 4 downregulation on cellular motility in normal O S E cells. O S E cells were transfected by s iRNAs . After 48 hr, the medium was changed to 0.1% F B S overnight. The cultured cells were scratched, then treated with or without 20 ng/ml E G F . (A) the pictures are taken after 6 hr. (B) migrated distance were measured after 4 hr. Results were presented as the means +/- SD and analyzed by Student's t-test (no E G F vs. E G F ) or A N O V A (mock, scramble and A4) . a, vs. control, p<0.05; b, vs. mock or scramble, p<0.05. The results are representative of three separate experiments. A 4 , H O X A 4 s i R N A . 1 3 7 138 Fig.38. Effects of downregulation of H O X A 4 on cellular motility in O V C A R 3 and O V C A R 8 cells. O V C A R 3 (A) and O V C A R 8 (B) cells were transfected with 100 n M s iRNAs and after 72 hr, scratched. The migrated distance was measured after 48 hr in O V C A R 3 and 24 hr in O V C A R 8 . (a,d) scramble (b,e) H O X A 4 s i R N A . The results are presented as the means ± SD and analyzed by A N O V A . * vs. mock or scramble, p<0.05. The results are representative of three separate experiments. A 4 , H O X A 4 s i R N A . (B) 300 JL X mock scramble "A4~ g 300 3, S200 CD i S100 mock scramble A4 140 3.12. Effects of downregulation of HOXA4 on ovarian cancer cells 3.12.1. E-cadherin and collagen type III expression E-cadherin is an epithelial differentiation marker which is absent in normal OSE but present in low grade ovarian carcinomas while collagen type III is a mesenchymal marker which is present in OSE (Auersperg et al., 2001). O V C A R 3 expresses E-cadherin, but not collagen type III (Maines-Bandiera et al., 2004). After s iRNA transfection, O V C A R 3 was double-stained with E-cadherin antibody and collagen type III antibody. E-cadherin was localized at the cell borders and its expression varied among areas within cultures, hence, it was hard to conclude on the basis of immunofluorescence whether overall E-cadherin expression was changed by H O X A 4 downregulation (Fig.39). Collagen type III expression was negative and there was no change with H O X A 4 downregulation (data not show). E-cadherin expression was quantitated by western blot analysis, after siRNAs transfection. The total amount of E-cadherin was not changed by H O X A 4 downregulation (data not shown). 3.12.2. Effects of HOXA4 on CA125 CA125 is another epithelial differentiation marker of ovarian carcinomas. I evaluated the role of H O X A 4 in O V C A R 3 , which is known to secrete CA125. After 48 hr of Fig.39. E-cadherin expression in O V C A R 3 . These cells were transfected with s iRNA H O X A 4 . The expression was not uniform. Both (a) and (b) were stained by E-cadherin primary antibody. Scale bar = 35 um. 142 scramble #2 or siRNAs H O X A 4 transfection, medium was changed, then, conditioned medium was collected after 24 hr. To see the amount of CA125 production per cell, the raw data were standardized by the amount of CA125 per cell protein. H O X A 4 downregulation decreased CA125 secretion (Table 10). If scramble and H O X A 4 s iRNA were compared by paired Student's t-test, there was a significant difference. 3.13. Effects of HOXA4 in three-dimensional Matrigel culture I analyzed the pattern of O V C A R 3 cell growth in three-dimensional (3-D) culture using Matrigel. Matrigel is a solubilized basement membrane extracted from EHS (Englebreth-Holm-Swarm) mouse sarcoma. Its major component is laminin, followed by collagen IV, heparan sulfate proteoglycans, and entactin. O V C A R 3 cells were treated with scramble #2 or s iRNA H O X A 4 at 50, 70, 100 n M . Mock or scramble #2 treated O V C A R 3 formed compact colonies. There were no differences between mock and scramble in the pattern of formation or the size of the aggregates. H O X A 4 downregulation caused bigger colonies compared to scramble #2 (Fig.40). There were no differences in the sizes of colonies between the concentrations of siRNAs (data not shown). Table 10. Effects of H O X A 4 on CA125 secretion in O V C A R 3 . CA125/protein concentration (kU/L/pg/pl) e x p l e x p 2 e x p 3 m e a n + / ~ S D m o c k 3 6 9 . 9 5 2 4 0 2 1 9 . 2 9 2 7 5 . 1 + / - 7 9 . 4 s i R N A c o n t r o l 3 5 4 . 0 4 1 8 8 . 3 2 7 3 . 5 8 2 7 2 . 0 + / - 8 2 . 9 s i R N A A 4 3 0 8 . 9 6 1 5 2 . 8 5 2 1 9 . 6 1 2 2 7 . 1 + / - 7 8 . 3 P<0.05, compared s i R N A control and s i R N A A 4 , paired t-test. Fig.40. E f f e c t s o f H O X A 4 d o w n r e g u l a t i o n o n m o r p h o l o g y o f O V C A R 3 c e l l s c u l t u r e d i n t h e t h r e e d i m e n s i o n a l c u l t u r e , ( a ) p h a s e - c o n t r a s t m i c r o g r a p h s o f O V C A R 3 c e l l s t r a n s f e c t e d b y s i R N A s a f t e r 4 d a y s o n t o p o f M a t r i g e l . ( b ) m e a s u r e d 1 0 b i g g e s t c o l o n i e s i n c u l t u r e . T h e r e s u l t s a r e m e a n s w i t h s t a n d a r d d e v i a t i o n . * vs. m o c k o r s c r a m b l e , p < 0 . 0 5 . S c a l e b a r = 2 0 0 u m . A 4 , H O X A 4 s i R N A s . 145 3.14. Proliferative effects of downregulation of HOXA4 in normal OSE with EGF treatment The [3H]-thymidine incorporation assay was used to assess the effects of H O X A 4 downregulation in normal OSE. EGF enhanced proliferation in all three conditions. Microscopically, scramble #2 did not show any toxicity, however, by [3H]-thymidine incorporation assay, scramble #2 at 20 n M and 50 n M showed significantly lower proliferative activity compared to mock transfection (Fig.41, 20 n M data not shown) and compared to s iRNA H O X A 4 . Because of this apparent toxic effect of the scramble, it was not possible to draw conclusions and these experiments wi l l have to be repeated. 3.15. Proliferative effects of downregulation of HOXA4 in OVCAR3 in 2 dimensional culture The [3H]-thymidine incorporation assay was used to assess the effects of H O X A 4 downregulation on O V C A R 3 proliferation in conventional two-dimensional (2-D) culture. Microscopically, scramble #2 did not show any toxicity, however, by [3H]-thymidine incorporation assay, scramble #2 at 20 n M , 50 n M or 70 n M , showed significantly lower proliferative activities compared to mock transfection in both cells (Fig. 42, 70 n M data not shown). When compared between scramble and s iRNA 146 Fig.41. Effects of donwnegulation of H O X A 4 by s iRNA on proliferation in normal OSE cells. OSE cells were transfected by 50 n M siRNAs and after 24 hr, re-plated in 2 % FBS. After 16 hr, medium with/without 20 ng/ml EGF was added. Twentyfour hr later, thymidine incorporation was measured. The results are presented as the means +/- SD and analyzed by Student's t-test (control vs. EGF) or A N O V A (mock, scramble and A4). a, EGF vs. no EGF, p<0.05. b, H O X A 4 s iRNA vs. mock or scramble, p<0.05. The results are representative of three separate experiments. 20000i E 15000-1 CL O c 10000H P 50004 Unscramble Z J A 4 mock 50 50 20 s iRNA concentrat ion (nM) T 20 Fig.42. E f f e c t s o f d o w n r e g u l a t i o n o f H O X A 4 o n p r o l i f e r a t i o n i n O V C A R 3 . i n 2 - D . O V C A . R 3 c e l l s w e r e t r a n s f e c t e d b y s i R N A s , p l a t e d a n d m e a s u r e d t h y m i d i n e i n c o r p o r a t i o n a f t e r 2 4 h r . T h e r e s u l t s a r e p r e s e n t e d a s t h e m e a n s + / - S D a n d a n a l y z e d b y A N O V A ( m o c k , 5 0 n M A 4 , 2 0 n M A 4 ) . a , H O X A 4 s i R N A v.y. s c r a m b l e , p < 0 . 0 5 . T h e r e s u l t s a r e r e p r e s e n t a t i v e o f t h r e e s e p a r a t e e x p e r i m e n t s . 148 H 0 X A 4 transfection, H O X A 4 transfected cells showed higher proliferative activities than scramble at 20, 50 and 70 n M . However, the toxicity of the scramble was again observed, hence, it is difficult to draw conclusions. 149 4. Discussion 4.1. The expression of HOXA7 in ovarian follicles, normal OSE and ovarian cancer In this study, I describe the generation of a human HOXA7-specific antibody, its use in studying in the role of HOXA7 in human ovarian follicular development, and its expression in normal OSE and ovarian carcinoma. The reactivity of the antibody to HOXA7 by ELISA, Western blot analysis of cell lysates and recombinant protein, as well as immunolocalization in mouse embryos and the nuclei of cultured cells that were HOXA7 positive by RT-PCR provide convincing evidence that our antibody reacts specifically with HOXA7. However, this HOXA7 antibody could not be affinity purified. Activated thiol sepharose 4B, which was used to make affinity column, reacts with solutes containing thiol groups to form mixed disulfides. The peptide sequence used to raise an antibody was added at the N-terminus of the peptide sequence CGG-; C is cysteine, and contains the thiol (-SH) group. The results showed that anti-HOXA7 antibody could bind to the peptide sequence by affinity chromatography, ELISA and Western blot analysis, in which the protein was denatured by sodium dodecyl sulfate. It is difficult to think that there were differences between the peptide used in affinity chromatography and non-denatured protein. Most likely, the sepharose 4B has a longer spacer arm, which may have caused the combination between the peptide of HOXA7 150 and the arm or even H O X A 7 peptide itself to construct a different tertiary structure, which prevented it from being recognized by anti-HOXA7 antibody. Histochemical examination of normal human ovaries demonstrated that the ovarian follicles showed striking changes in H O X A 7 staining in the course of their development. Primordial follicular cells were mostly negative but, once they reached the primary stage, they stood out against the less intensely stained stroma. Thus, the amount of H O X A 7 protein was more abundant in developing follicles, starting with primary follicles, than in primordial follicles and stroma. Its amounts and distribution varied among follicular components. Focusing on the negative staining of granulosa cells in primordial follicles, but positive staining in later follicular developmental stages, I demonstrated that, in cultured granulosa cells (SVOG, human SV40 Tag-immortalized granulosa cells, Lie et a l , 1996), H O X A 7 expression is increased during mitosis. Moreover, GDF-9, which enhances early follicular growth and.differentiation (Juengel and McNatty 2005), increased H O X A 7 protein expression in cultured granulosa cells. In my study, the H O X A 7 protein was predominantly nuclear in proliferating cells in vitro, and in granulosa cells of small follicles. However, it was mainly cytoplasmic in 151 mouse embryos, the theca interna, preovulatory granulosa cells and in a part of the granulosa cells. In ovarian cancer, most ovarian carcinomas showed cytoplasmic H O X A 7 expression, but nuclear expression was also observed. There is increasing evidence that H O X proteins can be located in both nuclei and cytoplasm and that they may have different functions in these different subcellular locations (Naora et al., 2001a; Kosaka et al., 2004). The subcellular localization of homeobox genes is regulated by external stimuli (Soubry et al., 2005). For example, thrombopoietin induces nuclear translocation of H O X A 9 (Kirito et al., 2004). The nuclear proteins are transported from the cytoplasm to the nucleus i f they have nuclear localization signals (NLS). Nuclear translocation is important in regulating the transcriptional activity of homeobox proteins. H O X A 9 has two N L S sequences (Kirito et al., 2004). Mutation analysis has shown that both NLSs in the homeodomain are functional. When in the cytoplasm, N L S might be inactive through phosphorylation, intra- or intermolecular interactions, or cytoplasmic anchoring (Vandromme et al., 1996). I investigated whether H O X A 7 contains a nuclear localization signal (NLS). First, although H O X A 7 is part of the ubx family in Drosophila, I recognized in the human gene the same sequence of amino acids at 129-133, N-terminal region of antp, in 152 which a highly conserved Arg/Lys-rich sequence exists (Kasahara and Izumo, 1999). Second, we searched for the putative N L S , using the Columbia University Bioinformatics Center program (Cokol et al., 2000). The program predicted that H O X A 7 contains one putative N L S site at amino acids 156 to 189, which was found to cover the second and the third helix of the homeodomain (Fig. 43). Compared to H O X A 9 , the locations of N L S of H O X A 7 are similar to those of H O X A 9 , but their size is more limited (Kirito et al., 2004). I also found that, similar to H O X A 9 arid as generalized in other homeoproteins, H O X A 7 has a putative NES (nuclear exporting signals; Maizel et al., 1999). The subcellular localization of H O X A 7 might be the balance between N L S and NES signaling. The nuclear and cytoplasmic homeobox protein might have distinctive functions (Kosaka et al., 2004). The shift from a nuclear to a cytoplasmic location of H O X A 7 , as ovarian follicles progress from the primary to the preovulatory stage, may be related to the change from a predominantly proliferative to a predominantly secretory state which takes place with follicular maturation. It may be significant, in this regard, that preovulatory granulosa cells and theca interna, where H O X A 7 is predominantly cytoplasmic, all secrete steroids. H o m e o d o m a i n H e l i x l H e l i x 2 H e l i x 3 R K R G R O T Y T R Y O T L E L E K E F H F N R Y L T R R R R I E I A H A L C L T E R Q I K I W F Q N R R M K W K K E H K N L S # 1 N L S # 2 Fig.43. N u c l e a r l o c a l i z a t i o n s i g n a l s ( N L S s ) o n H O X A 7 . T h e r e a r e t h r e e p u t a t i v e N L S s i n H O X A 7 . 154 Proliferation of granulosa cells is controlled by responses to gonadotropins, steroids and growth factors (Vanderhyden, 2002). In primordial follicles, a single layer of flat granulosa, or follicular, cells are at a quiescent stage. These cells start proliferating at a slow rate, and acquire responsiveness to FSH receptors in preantral follicles, which accelerates follicular development (McGee et al., 1997). In addition, oocytes secrete growth factors, such as GDF-9 and bone morphogenetic protein-15 which also stimulate granulosa cell mitosis (Juengel and McNatty, 2005). M y study suggests that nuclear H O X A 7 protein expression in ovarian follicles is associated with granulosa cell proliferative stages. First, in the stationary primordial follicles, the follicular cells were predominantly H O X A 7 negative, while in primary follicles, which have initiated growth, nuclei of the single cuboidal layer of granulosa cells were strongly positive. Second, my in vitro results confirmed that GDF-9, which is a member of the TGF-P superfamily, does indeed stimulate human granulosa cell proliferation. Importantly, we found that this stimulation paralleled an increase in H O X A 7 protein levels. In contrast, TGF-P 1 reduced H O X A 7 expression, suggesting that the effect of GDF-9 on H O X A 7 may be specific. GDF-9 and TGF-P 1 share the downstream of signaling pathways and target promoter (Mazerbourg et al., 2003). The different action of GDF-9 and TGF-p i might be due to different degrees of the activation of the receptors. Thirdly, we double-155 stained S V O G cells with antibodies to H O X A 7 and anti-Ki-67. The increased H O X A 7 staining in mitotic cells in the current study suggests that H O X A 7 positively regulates the mitotic phase of the cell cycle. H O X A 7 protein expression might be regulated by hormonal activities, and H O X A 7 may, in turn, regulate other cell functions. Expression of H O X A 1 0 changes with the menstrual cycle in human endometrium, and is upregulated by estrogen and progesterone in primary endometrial stromal cells or Ishikawa cells, a well differentiated endometrial adenocarcinoma cell line (Taylor et al., 1998). In contrast, H O XA10 was suppressed by testosterone in the Ishikawa cells (Cermik et al., 2003). Theca interna and externa originate from the same stroma and differentiate into either LH-/hCG-responsive androgen producing cells, the theca interna, or contractile myofibroblast-like cells, the theca externa. Normal granulosa cells produce hormones such as estradiol and inhibin (Schumer and Cannistra, 2003). Thus, H O X A 7 expression and its nucleo-cytoplasmic distribution might be regulated in the theca interna by androgen or in granulosa cells by estrogen. Conversely, H O X A 7 may regulate endocrine functions of ovarian cells at the transcriptional level. Furthermore, theca externa has less proliferative and secretory activity compared to granulosa cells and 156 theca interna (Feranil et al., 2004), which may explain the lack of H O X A 7 protein expression, and supports our hypothesis that H O X A 7 is associated with proliferative and secretory activity. H O X A 7 staining of the stroma was stronger near the ovarian surface than in the medulla. It has been reported that in the bovine ovary, which has basically the same morphology and physiology as the human ovary, the structure of the stroma also changes with distance from the ovarian surface (Vigne et al., 1994). These differences may imply possible functional differences, which have not been studied. H O X A 7 protein was mainly localized in the cytoplasm in most epithelial ovarian cancers. However, nuclear H O X A 7 was also observed in more than 75% cases in my study. Importantly, i f H O X A 7 localization was nuclear, the prognosis of the disease-specific survival was significantly improved (Fig.22). As discussed above, H O X A 9 protein can also be expressed both in the nucleus and in the cytoplasm. Cytoplasmic H O X A 9 expression is also observed in acute myelogenous leukemia (Lawrence et al., 1999). Similar to H O X A 7 , the presence of nuclear Akt has been correlated with better prognosis parameters in prostate cancer (Le Page et al., 2006), and in lung cancer and 157 endometrial cancer (Shah et al., 2005; Uegaki et al., 2005). Currently, no mechanism has been identified which induces nuclear-cytoplasmic translocation of H O X protein expression in cancer. H O X A 7 nuclear expression might demonstrate the "normal" ability for the epithelial cells related to cell proliferative activity. The results of my studies of H O X A 7 protein expression in ovarian cancer showed major differences from the results of a similar study by Naora, which showed that H O X A 7 is upregulated in benign and low grade serous and endometriod type of ovarian tumors and downregulated in normal OSE in vivo and undifferentiated ovarian carcinomas by using RT-PCR and immunohistochemistry, including tissue arrays (Naora et al., 2001a; Cheng et al., 2005). These authors concluded that H O X A 7 expression is increased in differentiated tumors. In my study, normal OSE in vivo also did not show H O X A 7 expression. However, H O X A 7 was not upregulated in benign serous types of tumors and in low malignant potential tumors, and upregulation of H O X A 7 was found in higher grade ovarian tumors in a c D N A array. Dr. Gilks ' microarray results were confirmed by myself, using RT-PCR. Moreover, in my study, the analysis of tissue arrays showed that there was no association between H O X A 7 overall intensity and tumor grades in all histopathological types of tumors. E-cadherin is associated with 158 differentiation of tumors, however, E-cadherin expression and H O X A 7 intensity were also not associated with each other. As a result, only two points gave the same results as Naora's study: (i) lack of H O X A 7 expression by OSE and (ii) no association between tumor grade and H O X A 7 expression in the mucinous type of tumors. There are several possible reasons for these discrepancies. Naora used anti-mouse polyclonal Hoxa7 antibody (Covance) in her earlier study (Naora et al., 2001a) and anti-human H O X A 7 polyclonal antibody (Cheng et al., 2005) in the later study. The anti-mouse antibody was raised against the comparable site of peptide sequences to mine. However, there are three amino acid differences out of 14 between the two species. Their anti-human antibody was raised against a different sequence from my antibody. The discrepancies between our results might also be because of (i) the definition of histological differences, (ii) the differences between chemo-naive tissues and tissues after chemotherapy - in her study, there was no detailed description of the choice of ovarian tumors in regard to treatment, (iii) scoring differences, (iv) anti-mouse antibody was raised against non-human species and not well characterized by the company, (v) the epitope differences between her anti-human antibody and my antibody (her anti-human antibody was not as well characterized as mine) and (vi) her 159 [ i immunohistochemical staining procedures were not perfect, so that the staining had high background. This study provides the first insight into the expression and regulation of H O X genes, and H O X A 7 in particular, in human ovarian follicular development, using a carefully characterized antibody to human H O X A 7 . The results indicate that H O X A 7 protein expression is closely related to proliferative activities of developing follicles, and that its expression is regulated, at least in part, by oocyte-derived factors. The increase in H O X A 7 protein in response to GDF-9 represents the first demonstration of a possible regulatory role of human oocytes in follicular H O X gene expression. The mechanisms underlying these changes and the precise interrelationships between H O X A 7 expression and granulosa cell proliferation and functions require further investigations. In regard to H O X A 7 protein expression in ovarian cancer, our results do support Naora's suggestion that H O X A 7 may become an early marker to detect ovarian carcinoma. In contrast to her results, my findings suggest that both overall and nuclear H O X A 7 protein expression was not related to tumor grades. Rather, H O X A 7 nuclear protein expression was associated with the prognosis of disease-specific survival. 160 Therefore, H O X A 7 may play a role in the biology of ovarian cancer progression and the nuclear expression of H O X A 7 can be a clinical marker to evaluate patient's prognosis. 4.2. HOXA4 in normal ovarian surface epithelium and epithelial ovarian carcinomas. I demonstrated in this study, that three H O X A genes were expressed in culture by normal OSE and several ovarian carcinoma cell lines: H O X A 4 , H O X A 7 and H O X A 9 . However, I focused on H O X A 4 , which was the most highly expressed in the microarrays of ovarian carcinomas in vivo. The expression of H O X A 4 was acquired in ovarian tumors with transition from benign and borderline to malignant tumors in c D N A microarray analysis (Fig. 1). From the results of the c D N A microarray analysis, I hypothesized that H O X A 4 may act as an oncogene and assumed that the downregulation of H O X A 4 in ovarian cancer cell lines would cause cells to behave similar to normal cells. However, it is also possible that the increased H O X A 4 expression in the stroma of carcinomas might be involved in the higher expression in malignant tumors compared to benign ones (see Fig. 19 or Fig.20). When H O X A 4 expression was downregulated in ovarian cancer cells in vitro using siRNAs, cell migration was enhanced and bigger colonies were formed in three-dimensional (3-D) 161 cultures (Fig.40). M y results suggest that H 0 X A 4 downregulation increases neoplastic characteristics, which indicates that H O X A 4 might be a tumor suppressor in ovarian epithelial carcinomas. These surprising results, opposite to my hypothesis, might indicate that the H O X A 4 expression increase in ovarian carcinomas reflects protective effects against neoplastic progression. A similar phenomenon has been observed in cervical cancer (O'Neill and McCluggage, 2006). I found that in normal OSE cells, H O X A 4 expression was highest in sparse, migrating and proliferating cells in culture, but was reduced in OSE in vivo and in culture under conditions of high density where proliferation and migration are reduced. Cell density has an effect on H O X gene expression. H O X A 1 expression was dependent on cell density in breast cancer cell lines and H O X A 1 was upregulated when cells were confluent dependent on E-cadherin (Zhang et al. 2006). Moreover, cell density regulates the expression of extracellular matrix (ECM), such as collagenase (Johnson-Wint, 1980), tissue inhibitors of metalloproteinases-1 (TIMP-1; Nabeshima et al., 1994) and fibronectins (Inoue et al. 1998). OSE cells secrete E C M components and proteases on plastic in culture (Kruk et al., 1994) These E C M components may have an influence on 162 H O X gene expression. On the basis of these inital observations, we hypothesized that H O X A 4 may regulate proliferation. In keratinocytes, cell density and extracellular C a 2 + concentration determine cell fate. Keratinocytes proliferate at low density, whereas they differentiate at confluency. Similar to H O X A 4 in OSE, H O X A 7 expression in keratinocytes is downregulated at high density (La Celle and Polakowska, 2001). On the other hand, in contrast to OSE, keratinocytes proliferate at low C a 2 + concentrations and differentiate at high C a 2 + 2"t" concentrations. However, H O X A 5 and H O X A 7 were downregulated by high Ca concentration, which was similar to H O X A 4 in OSE. Hence, my results did not indicate that OSE proliferative activities are proportional to H O X A 4 expression. H O X A 7 downregulation by high [Ca 2 +] in keratinocytes, is accompanied by transgultaminase type I (TGM1) induction, which is a differentiation marker for keratinocytes (La Celle and Polakowska 2001). 12-O-tetradecanoyl phorbol-13-acetate (TPA), induced differentiation of keratinocytes, similar to low C a 2 + , upregulated H O X A 7 expression and downregulated TGM1 expression. This modulation occurs downstream of P K C activation (La Celle and Polakowska, 2001). The authors 163 concluded that in keratinocytes, H O X A 7 expression is increased with proliferation and reduced with differentiation. In OSE, H O X A 7 expression also increased with proliferation. EGF and high extracellular [Ca 2 +] activate PI3-K and extracellular signal regulated kinase (ERK) signaling pathways, which may regulate H O X A 4 expression. In my study, short term treatment (< 60 min) with EGF had almost no effects on H O X A 4 expression in normal OSE, whereas H O X A 4 was upregulated at 24 hr, concomitantly with increased proliferation. In addition, H O X A 4 downregulation was observed by high extracellular [Ca 2 +] at 24 hr. Consequently, these effects were most likely not direct effects but involved transcriptional activation. To identify the precise regulation, H O X A 4 promoter studies wi l l be needed. One of direct target genes of H O X might be integrins. H O X gene expression was associated with specific integrin expression in human metastatic melanoma cell lines (Cillo et al., 1996). HOXD3 altered a5 and (33 integrin expression in endothelial cells (Boudreau et al., 1997; Boudreau and Varner, 2004) and HOXD10 changed a3 expressions in breast cancer cells (Carrio et al., 2005). In endometrial cells, 03 integrin 164 is a target of H O X A 10 and its direct binding to four out of seven H O X binding sites in P3 integrin was demonstrated by E M S A (Daftary et al., 2002). The aberrant expression of several members of the integrin family is associated with grades and a high invasive potential in ovarian carcinomas (Maubant et al., 2005). Intergrins are large family of extracellular matrix receptors, and integrin expression is modulated by E C M and changes between 2-D and 3-D conditions (Weaver et al. 1997; Yamada and Clark, 2002). Each member of integrins is known to play important roles for the interaction with different types of E C M . Further studies wi l l identify which integrins are regulated by H O X A 4 in ovarian carcinomas. Subtypes of Akt/ protein kinase B (PKB), might be involved in HOXA4-induced regulation of cell motility. Akts are a target of integrin-linked kinase (Persad et al., 2001) as well as PI3-K (Toker and Yoeli-Lerner, 2006). Activating A k t l inhibits migration and invasion of breast cancer cell lines (Yoeli-Lerner et al., 2005), whereas Akt2 enhances migration, invasion and epithelio-mesenchymal transition (Irie et al., 2005). Thus, Akt subtypes may act in their specific ways in regulating cell migration and invasion (Toker and Yoeli-Lerner, 2006). H O X A 4 downregulation might inhibit 165 Akt2 and enhance A k t l expression, which may control cellular motility in ovarian cancer cells. In this study, I initiated the assessment of H O X A 4 functions in 3-D culture. Boudreau's group has shown that H O X D 10 does not inhibit cellular proliferation in conventional 2-D cultures, however, in 3-D cultures and in vivo, H O X D 10 stably transfected breast cancer cells stop cellular growth (Carrio et al., 2005). In my study, it is difficult to conclude whether H O X A 4 downregulation promoted proliferation in 2-D cultures. It is crucial to optimize the condition for s iRNA controls. I observed bigger colonies in 3-D cultures, hence, there is a possibility that the proliferation rate might be different between in 2-D and 3-D. These results imply that overexpression of H O X A 4 in vivo may prevent cell growth. In this study, H O X A 4 donwregulation inhibited CA125 secretion, however, E-cadherin expression was not changed. For these studies, further evaluation wil l be needed, however, these results suggest that H O X A 4 might be able to enhance epithelial differentiation. The regulation of CA125 secretion and E-cadherin by H O X A 4 is different. 166 In summary, H 0 X A 4 downregulation increased three characteristics which accompany ovarian neoplastic progression: motility, proliferation and differentiation. H O X A 4 regulates cellular motility and differentiation in 2-D culture which provides a convenient model system to study underlying molecular mechanisms. The signaling pathways related to these H O X A 4 functions should be elucidated, which might lead to the development of useful cancer chemotherapeutic agents. 4.3. Future studies 4.3.1. Folliculogenesis M y study has shown that H O X A 7 started presenting from primary follicles during folliculogenesis. The H O X A 7 expression was regulated specifically by GDF-9. Future studies should clarify (i) how GDF-9 regulates H O X A 7 expression (ii) the function of H O X A 7 in granulosa cells. It is already known that the downstream pathway of GDF-9 is through Smads and their specific target sequence has to be activated. However, we don't know how H O X A 7 is involved in these pathways. L i et al. (2006) have shown that H O X transcriptional activities are regulated by Smads in mammalian cell lines, HEK293T and LNCaP (Li et al., 2006). It is possible that GDF-9 regulates H O X A 7 167 transcriptional activities in granulosa cells. The function of H O X A 7 in granulosa cells wi l l be investigated by using s iRNA to H O X A 7 , or overexpression of H O X A 7 . 4.3.2. HOXA7 and ovarian carcinomas In my study, I have shown that H O X A 7 expression was both in the nucleus and in the cytoplasm in ovarian tumors. The overall expression and nuclear expression of H O X A 7 were not related to tumor differentiation., but the nuclear expression of H O X A 7 was related to prognosis. Future studies should identify (i) the different functions in the nucleus and cytoplasm of H O X A 7 , (ii) the mechanism of nuclear-cytoplasmic translocation of H O X A 7 in ovarian carcinoma. These experiments may answer how H O X A 7 plays a role in ovarian carcinogenesis. 4.3.3. HOXA4 and ovarian carcinomas In my study, H O X A 4 downregulation increased three characteristics which are related to neoplastic progression: cellular motility, proliferative and differentiation. This raises the important possibility that H O X A 4 may be a differentiation marker. Future studies should clarify (i) what, or which signaling pathways regulate H O X A 4 expression, (ii) what are the targets for H O X A 4 . One possibility is that using c D N A microarray, to 168 compare control vs. H O X A 4 s iRNA transfected cells, or control vs. H O X A 4 overexpressing cells might identify H O X A 4 downstream targets. To make sure i f they are direct targets for H O X A 4 , E M S A wi l l be needed. If H O X genes can reverse characteristics of ovarian tumors, or even prevent only invasive characteristics, bearing in mind that H O X genes may act at a top of genetic hierarchies, H O X genes could be a great target using for gene therapy. To do this, we need to deliver genes in a way which is specific to the ovary, or ovarian carcinomas to reduce the side effects. 169 5. References Aaltonen J, Laitinen M P , Vuqjolainen K , Jaatinen R, Horelli-Kuitunen N , Seppa L , Louhio H , Tuuri T, Sjoberg J, Butzow R, Hovata O, Dale L , Ritvos O. Human growth differentiation factor 9 (GDF-9) and its novel homolog GDF-9B are expressed in oocytes during early folliculogenesis. J Cl in Endocrinol Metab. 1999;84(8):2744-50. Abate-Shen C. Deregulated homeobox gene expression in cancer; cause of consequence? Nature Rev Cancer 2002;2:777-85. Abate-Shen C. Homeobox genes and cancer: new OCTaves for an old tune. Cancer Cell. 2003;4(5):329-30. Acampora D, D'Esposito M , Faiella A , Pannese M , Migliaccio E, Morelli F, Stornaiuolo A , Nigro V , Simeone A , Boncinelli E. The human H O X gene family. Nucleic Acids Res. 1989; 17(24): 10385-402. Ahmed N , Maines-Bandiera S,Quinn M A , Unger W G , Dedhar S, Auersperg N . Molecular pathways regulating EGF-induced epithelio-mesenchymal transition in human ovarian surface epithelium. A m J Physiol Cell Physiol. 2006;290(6):C 1532-42. Albanell J, Gascon P. Small molecules with E G F R - T K inhibitor activity. Curr Drug Targets. 2005;6(3):259-74. Apiou F, Flagiello D, Cillo C, Malfoy B , Poupon M F , Dutrillaux B . Fine mapping of human H O X gene clusters. Cytogenet Cell Genet. 1996;73(1-2):114-5. Auersperg N , Maines-Bandiera SL, Dyck H G , Kruk PA. Characterization of cultured human ovarian surface epithelial cells: phenotypic plasticity and premalignant changes. Lab Invest. 1994;71(4):510-8. Auersperg N , Edelson MI , Mok SC, Johnson SW, Hamilton TC. The biology of ovarian cancer. Semin Oncol. 1998;25(3):281-304. Auersperg N , Pan J, Grove B D , Peterson T, Fisher J, Maines-Bandiera S, Somasiri A , Roskelley C D . E-cadherin induces mesenchymal-to-epithelial transition in human 170 ovarian surface epithelium. Proc Natl Acad Sci U S A . 1999;96(ll):6249-54. Auersperg N , Wong AS , Choi K C , Kang SK, Leung PC. Ovarian surface epithelium: biology, endocrinologym and pathology. Endocr Rev. 2001;22:255-88. Bagot C N , Troy PJ, Taylor HS. Alteration of maternal HoxalO expression by in vivo gene transfection affects implantation. Gene Ther. 2000;7(16): 1378-84. Balling R, Mutter G, Gruss P, Kessel M . Craniofacial abnormalities induced by ectopic expression of the homeobox gene Hox-1.1 in transgenic mice. Cell. 1989;58(2):337-47. Bard J B L and Kaufman M H . The Anatomical Basis of Mouse Development, Academic Press, New York 1999. Bast R C Jr, Badgwell D, Lu Z, Marquez R, Rosen D, L iu J, Baggerly K A , Atkinson E N , Skates S, Zhang Z, Lokshin A , Menon U , Jacobs I, Lu K . New tumor markers: CA125 and beyond. Int J Gynecol Cancer. 2005; 15 Suppl 3:274-81. Bauknecht T, Kommoss F, Birmelin G, von Kleist S, Kohler M , Pfleiderer A . Expression analysis of EGF-R and TGFa in human ovarian carcinomas. Anticancer Res. 1991;ll(4):1523-8. Barnett K R , Schilling C, Greenfeld CR, Tomic D, Flaws JA. Ovarian follicle development and transgenic mouse models. Hum Reprod Update. 2006;12(5):537-55. Behringer RR, Crotty D A , Tennyson V M , Brinster R L , Palmiter R D , Wolgemuth DJ. Sequences 5' of the homeobox of the Hox-1.4 gene direct tissue-specific expression of lacZ during mouse development. Development. 1993;117(3):823-33. Benson G V , L i m H , Paria B C , Satokata I, Dey SK, Maas R L . Mechanisms of reduced fertility in Hoxa-10 mutant mice: uterine homeosis and loss of maternal Hoxa-10 expression. Development. 1996;122(9):2687-96. 171 Berchuck A , Kohler M F , Boente M P , Rodriguez G C , Whitaker RS, Bast R C Jr. Growth regulation and transformation of ovarian epithelium. Cancer. 1993;71(2 Suppl):545-51. Bilderback TR, Lee F, Auersperg N , Rodland K D . Phosphatidylinositol 3-kinase-dependent, M E K - independent proliferation in response to CaR activation. A m J Physiol Cell Physiol. 2002;283(l):C282-8. Boudreau N , Andrews C, Srebrow A , Ravanpay A , Cheresh D A . nduction of the angiogenic phenotype by Hox D3. J Cell Biol . 1997;139(l):257-64. Boudreau NJ , Varner JA. The homeobox transcription factor Hox D3 promotes integrin alpha5betal expression and function during angiogenesis. J Bio l Chem. 2004;279(6):4862-8. Burel A , Mouchel T, Odent S, Tiker F, Knebelmann B , Pellerin I, Guerrier D. Role of H O X A 7 to H O X A 13 and PBX1 genes in various forms of M R K H syndrome (congenital absence of uterus and vagina). J Negat Results Biomed. 2006;5:4. Carrio M , Arderiu G, Myers C, Boudreau NJ . Homeobox D10 induces phenotypic reversion of breast tumor cells in a three-dimensional culture model. Cancer Res. 2005;65(16):7177-85. Cermik D, Karaca M , Taylor HS. HOXA10 expression is repressed by progesterone in the myometrium: differential tissue-specific regulation of H O X gene expression in the reproductive tract. J Clin Endocrinol Metab. 2001;86(7):3387-92. Cermik D, Selam B , Taylor HS. Regulation of HOXA-10 expression by testosterone in vitro and in the endometrium of patients with polycystic ovary syndrome. J Cl in Endocrinol Metab. 2003;88(l):238-43. Chang CP, Brocchieri L , Shen WF, Largman C, Cleary M L . Pbx modulation of Hox homeodomain amino-terminal arms establishes different DNA-binding specificities across the Hox locus. M o i Cell Biol . 1996; 16(4): 1734-45. Chang CP, Jacobs Y, Nakamura T, Jenkins N A , Copeland N Q Cleary M L . Meis proteins 172 are major in vivo D N A binding partners for wild-type but not chimeric Pbx proteins. M o i Cell Biol . 1997; 17(10):5679-87. Chegini N , Flanders K C . Presence of transforming growth factor-beta and their selective cellular localization in human ovarian tissue of various reproductive stages. Endocrinology. 1992; 130(3): 1707-15. Chen F, Greer J, Capecchi M R . Analysis of Hoxa7/Ffoxb7 mutants suggests periodicity in the generation of the different sets of vertebrae. Mech Dev. 1998;77(l):49-57. Chen F, Capecchi M R . Paralogous mouse Hox genes, Hoxa9, Hoxb9, and Hoxd9, function together to control development of the mammary gland in response to pregnancy. Proc Natl Acad Sci U S A . 1999;96(2):541-6. Cheng W, L iu J, Yoshida H , Rosen D, Naora H . Lineage infidelity of epithelial ovarian cancers is controlled by H O X genes that specify regional identity in the reproductive tract. Nat Med. 2005; 11(5):531-7. Choi Y, Rajkovic A . Genetics of early mammalian folliculogenesis. Cell M o i Life Sci. 2006;63(5):579-90. Cillo C, Cantile M , Mortarini R, Barba P, Parmiani G, Anichini A . Differential patterns of H O X gene expression are associated with specific integrin and I C A M profiles in clonal populations isolated from a single human melanoma metastasis. Int J Cancer. 1996 May 29;66(5):692-7. Cinquanta M , Rovescalli A C , Kozak C A , Nirenberg M . Mouse Sebox homeobox gene expression in skin, brain, oocytes, and two-cell embryos. Proc Natl Acad Sci U S A . 2000;97(l6):8904-9. Cohen M H , Williams G A , Sridhara R, Chen G, McGuinn W D Jr, Morse D, Abraham S, Rahman A , Liang C, Lostritto R, Baird A , Pazdur R.. United States Food and Drug Administration Drug Approval summary: Gefitinib (ZD 1839; Iressa) tablets. Clin Cancer Res. 2004; 10(4): 1212-8. 173 Cokol M , Nair R, Rost B . Finding nuclear localization signals. E M B O Rep. 2000 Nov;l(5):411-5. Conlon R A . Retinoic acid and pattern formation in vertebrates. Trends Genet. 1995; ll(8):314-9. Daftary GS, Troy PJ, Bagot C N , Young SL, Taylor HS. Direct regulation of beta3-integrin subunit gene expression by H O X A 10 in endometrial cells. M o i Endocrinol. 2002;16(3):571-9. Daftary GS, Taylor HS. Endocrine regulation of H O X genes. Endocr Rev. 2006;27(4):331-55. Del Bene F, Wittbrodt J. Cell cycle control by homeobox genes in development and disease. Semin Cell Dev Biol . 2005;16(3):449-60. Dodson W C and Schomberg DW. The effect of transforming growth factor-B on follicle-stimulating hormone-induced differentiation of cultured rat granulosa cells. Endocrinology. 1987;120:512-516. Doerksen L F , Bhattacharya A , Kannan P, Pratt D, Tainsky M A . Functional interaction between a R A R E and an AP-2 binding site in the regulation of the human H O X A4 gene promoter.Nucleic Acids Res. 1996;24(14):2849-56. Dolle P, Izpisua-Belmonte JC, Brown J M , Tickle C, Duboule D. HOX-4 genes and the morphogenesis of mammalian genitalia. Genes Dev. 1991;5(10): 1767-7. Dolle P, Izpisua-Belmonte JC, Brown J, Tickle C, Duboule D. Hox genes and the morphogenesis of the vertebrate limb. Prog Clin Biol Res. 1993;383A:11-20. Dong J, Albertini DF, Nishimori K , Kumar TR, Lu N , Matzuk M M . Growth differentiation factor-9 is required during early ovarian folliculogenesis. Nature. 1996;383(6600):531-5. 174 Dorrington J, Chuma A V , Bendell JJ. Transforming growth factor beta and follicle-stimulating hormone promote rat granulosa cell proliferation.Endocrinology. 1988;123(l):353-9. Dragovic R A , Ritter L J , Schulz SJ, Amato F, Armstrong DT, Gilchrist R B Role of oocyte-secreted growth differentiation factor 9 in the regulation of mouse cumulus expansion. Endocrinology. 2005;146(6):2798-806. Dressier GR, Deutsch U , Chowdhury K , Nornes HO, Gruss P. Pax2, a new murine paired-box-containing gene and its expression in the developing excretory system. Development. 1990;109(4):787-95. Dupont J, Aghajanian C, Sabbatini P, Spriggs DR. New agents for the treatment of ovarian cancer: the next generation. Int J Gynecol Cancer. 2005; 15 Suppl 3:252-7. Ellerbroek S M , Hudson L G , Stack MS. Proteinase requirements of epidermal growth factor-induced ovarian cancer cell invasion. Int J Cancer. 1998;78(3):331-7. Elvin JA, Clark A T , Wang P, Wolfman N M , Matzuk M M . Paracrine actions of growth differentiation factor-9 in the mammalian ovary. M o i Endocrinol. 1999;13(6): 1035-48. Eramaa M and Ritvos O. Transforming growth factor-B 1 and -B 2 induce inhibin and activin beta B-subunit messenger ribonucleic acid levels in cultured human granulosa-luteal cells. Fertil Steril. 1996;65:954-960. Estrada B , Sanchez-Herrero E. The Hox gene Abdominal-B antagonizes appendage development in the genital disc of Drosophila. Development. 2001;128(3):331-9 1 Favier B , Dolle P. Developmental functions of mammalian Hox genes. M o i Hum Reprod. 1997;3(2):115-31. Feranil JB, Isobe N , Nakao T. Cell proliferation in the atretic follicles of buffalo and cattle ovary. Reprod Domest Anim. 2004;39(6):405-9. Foronda D, Estrada B , de Navas L , Sanchez-Herrero E. Requirement of Abdominal-A 175 and Abdominal-B in the developing genitalia of Drosophila breaks the posterior downregulation rule. Development. 2006;133(1):117-27. Frasor J, Danes J M , Komm B, Chang K C , Lyttle CR, Katzenellenbogen BS. Profiling of estrogen up- and down-regulated gene expression in human breast cancer cells: insights into gene networks and pathways underlying estrogenic control of proliferation and cell phenotype. Endocrinology. 2003;144(10):4562-74. Garg PP, Kerlikowske K , Subak L, Grady D. Hormone replacement therapy and the risk of epithelial ovarian carcinoma: a meta-analysis. Obstet Gynecol. 1998;92(3):472-9. Gehring WJ, Muller M , Affolter M , Percival-Smith A , Billeter M , Qian Y Q , Otting G, Wuthrich K . The structure of the homeodomain and its functional implications. Trends Genet. 1990;6(10):323-9. Gendron R L , Paradis H , Hsieh-Li H M , Lee DW, Potter SS, Markoff E. Abnormal uterine stromal and glandular function associated with maternal reproductive defects in Hoxa-11 null mice. Bio l Reprod. 1997;56(5): 1097-105. Gidekel S, Pizov G, Bergman Y , Pikarsky E. Oct-3/4 is a dose-dependent oncogenic fate determinant. Cancer Cell . 2003;4(5):361-70. Gilchrist R B , Ritter L J , Cranfield M , Jeffery L A , Amato F, Scott SJ, Myllymaa S, Kaivo-Oja N , Lankinen H , Mottershead D G , Groome NP, Ritvos O. Immunoneutralization of growth differentiation factor 9 reveals it partially accounts for mouse oocyte mitogenic activity. Biol Reprod. 2004;71(3):732-9. Gilks C B , Vanderhyden B C , Zhu S, van de Rijn M , Longacre TA. Distinction between serous tumors of low malignant potential and serous carcinomas based on global m R N A expression profiling. Gynecol Oncol. 2005;96(3):684-94. Goodman FR, Scambler PJ. Human H O X gene mutations. Cl in Genet. 2001 ;59(1): 1-11. Gordon A N , Finkler N , Edwards RP, Garcia A A , Crozier M , Irwin D H , Barrett E. Efficacy and safety of erlotinib HC1, an epidermal growth factor receptor (HER1/EGFR) tyrosine kinase inhibitor, in patients with advanced ovarian carcinoma: 176 results from a phase II multicenter study. Int J Gynecol Cancer. 2005;15(5):785-92. Grier D G , Thompson A , Kwasniewska A , McGonigle GJ, Halliday H L , Lappin TR. The pathophysiology of H O X genes and their role in cancer. J Pathol. 2005;205(2):154-71. Gui L M , Joyce IM. R N A interference evidence that growth differentiation factor-9 mediates oocyte regulation of cumulus expansion in mice. Biol Reprod. 2005;72(1): 195-9. Hansen SL, Myers C A , Charboneau A , Young D M , Boudreau N . HoxD3 accelerates wound healing in diabetic mice. A m J Pathol. 2003; 163(6):2421-31. Hayashi M , McGee E A , M i n G, Klein C, Rose U M , van Duin M , Hsueh A J . Recombinant growth differentiation factor-9 (GDF-9) enhances growth and differentiation of cultured early ovarian follicles. Endocrinology. 1999;140(3): 1236-44. Henic E, Sixt M , Hansson S, Hoyer-Hansen G, Casslen B. EGF-stimulated migration in ovarian cancer cells is associated with decreased internalization, increased surface expression, and increased shedding of the urokinase plasminogen activator receptor. Gynecol Oncol. 2006;101(l):28-39. Horan GS, Wu K , Wolgemuth DJ, Behringer RR. Homeotic transformation of cervical vertebrae in Hoxa-4 mutant mice. Proc Natl Acad Sci U S A . 1994;91(26):12644-8. Hreinsson JG, Scott JE, Rasmussen C, Swahn M L , Hsueh A J , Hovatta O. Growth differentiation factor-9 promotes the growth, development, and survival of human ovarian follicles in organ culture. J Clin Endocrinol Metab. 2002;87(1):316-21. Hsieh-Li H M , Witte DP, Weinstein M , Branford W, L i H , Small K , Potter SS. Hoxa 11 structure, extensive antisense transcription, and function in male and female fertility. Development. 1995;121(5):1373-85. Huntriss J, Hinkins M , Picton H M . c D N A cloning and expression of the human N O B O X gene in oocytes and ovarian follicles. M o i Hum Reprod. 2006;12(5):283-9. 177 Inoue T, Nabeshima K , Shimao Y , Kataoka H , Koono M . Cell density-dependent regulation of fibronectin splicing at the E D A region in fibroblasts: cell density also modulates the responses of fibroblasts to TGF-beta and cancer cell-conditioned medium. Cancer Lett. 1998;129(l):45-54. Irie H Y , Pearline R V , Grueneberg D, Hsia M , Ravichandran P, Kothari N , Natesan S, Brugge JS. Distinct roles of A k t l and Akt2 in regulating cell migration and epithelial-mesenchymal transition. J Cell Biol . 2005;171(6): 1023-34. Jaatinen R, Laitinen M P , Vuojolainen K , Aaltonen J, Louhio H , Heikinheimo K , Lehtonen E, Ritvos O. Localization of growth differentiation factor-9 (GDF-9) m R N A and protein in rat ovaries and c D N A cloning of rat GDF-9 and its novel homolog GDF-9B. M o i Cell Endocrinol. 1999; 156(1-2): 189-93. Johnson-Wint B . Regulation of stromal cell collagenase production in adult rabbit cornea: in vitro stimulation arid inhibition by epithelial cell products. Proc Natl Acad Sci U S A . 1980;77(9):5331-5. Juengel JL, Bibby A H , Reader K L , Lun S, Quirke L D , Haydon L J and McNatty K P . The role of transforming growth factor-B (TGF-B) during ovarian follicular development in sheep. Reprod Biol Endocrinol 2004;2:78-88. Juengel JL, McNatty K P . The role of proteins of the transforming growth factor-beta superfamily in the intraovarian regulation of follicular development. Hum Reprod Update. 2005;11(2): 143-60. Kaivo-Oja N , Bondestam J, Kamarainen M , Koskimies J, Vitt U , Cranfield M , Vuojolainen K , Kallio JP, Olkkonen V M , Hayashi M , Moustakas A , Groome NP, ten Dijke P, Hsueh A J , Ritvos O. Growth differentiation factor-9 induces Smad2 activation and inhibin B production in cultured human granulosa-luteal cells. J Clin Endocrinol Metab. 2003;88(2):755-62. Kang Y L , L i H , Chen W H , Tzeng Y S , Lai Y L , Hsieh-Li H M . A novel PEPP homeobox gene, TOX, is highly glutamic acid rich and specifically expressed in murine testis and ovary. Biol Reprod. 2004;70(3):828-36. 178 Karen F, Weiffenbach B , Peifer M , Bender W, Duncan I, Celniker S, Crosby M , Lewis E B . The abdominal region of the bithorax complex. Cell. 1985;43(l):81-96. Karlan B Y , Jones J, Greenwald M , Lagasse L D . Steroid hormone effects on the proliferation of human ovarian surface epithelium in vitro. A m J Obstet Gynecol. 1995;173(1):97-104 Kasahara H , Izumo S. Identification of the in vivo casein kinase II phosphorylation site within the homeodomain of the cardiac tisue-specifying homeobox gene product Csx/Nkx2.5. M o i Cell Biol . 1999;19(l):526-36. Kessel M , Grass P. Homeotic transformations of murine vertebrae and concomitant alteration of Hox codes induced by retinoic acid. Cell . 1991 ;67(1):89-104. K i m M H , Jin H , Seol E Y , Yoo M , Park HW. Sequence analysis and tissue specific expression of human H O X A 7 . M o i Biotechnol. 2000;14(l):19-24. Kirito K , Fox N , Kaushansky K . Thrombopoietin induces H O X A 9 nuclear transport in immature hematopoietic cells: potential mechanism by which the hormone favorably affects hematopoietic stem cells. M o i Cell Biol . 2004;24(15):6751-62. Knecht M , Feng P and Catt K . Bifunctional role of transforming growth factor-B during granulosa cell development. Endocrinology. 1987;120:1243-1249. Kosaka Y , Akimoto Y , Yokozawa K , Obinata A , Hirano H . Localization of HB9 homeodomain protein and characterization of its nuclear localization signal during chick embryonic skin development. Histochem Cell Bio l . 2004;122(3):237-47. Kruk PA, Uitto V J , Firth JD, Dedhar S, Auersperg N . Reciprocal interactions between human ovarian surface epithelial cells and adjacent extracellular matrix. Exp Cell Res. 1994;215(1):97-108. Krumlauf R. Hox genes in vertebrate development. Cell . 1994;78(2): 191-201. Kuliev A , Kukharenko V , Morozov G, Freidine M , Rechitsky S,, Verlinsky O, 179 Ivakhnenko V , Gindilis V , Strom C, Verlinsky Y . Expression of homebox-containing genes in human preimplantation development and in embryos with chromosomal aneuploidies. J Assist Reprod Genet. 1996; 13(2): 177-81. La Celle PT, Polakowska RR. Human homeobox H O X A 7 regulates keratinocyte transglutaminase type 1 and inhibits differentiation. J Bio l Chem. 2001;276(35):32844-53. Lacroix L , Pautier P, Duvillard P, Motte N , Saulnier P, Bidart J M , Soria JC. Response of ovarian carcinomas to gefitinib-carboplatin-paclitaxel combination is not associated with E G F R kinase domain somatic mutations. Int J Cancer. 2006; 118(4): 1068-9. Laitinen M , Vuojolainen K , Jaatinen R, Ketola I, Aaltonen J, Lehtonen E, Heikinheimo M , Ritvos O. A novel growth differentiation factor-9 (GDF-9) related factor is co-expressed with GDF-9 in mouse oocytes during folliculogenesis. Mech Dev. 1998;78(l-2): 135-40. Lawrence HJ, Helgason C D , Sauvageau G, Fong S, Izon DJ, Humphries R K , Largman C. Mice bearing a targeted interruption of the homeobox gene H O X A 9 have defects in myeloid, erythroid, and lymphoid hematopoiesis. Blood. 1997;89(6): 1922-30. Lawrence HJ, Rozenfeld S, Cruz C, Matsukuma K , Kwong A , Komuves L , Buchberg A M , Largman C. Frequent co-expression of the H O X A 9 and MEIS1 homeobox genes in human myeloid leukemias. Leukemia. 1999;13(12): 1993-9. Le Page C, Koumakpayi IH, Alam-Fahmy M , Mes-Masson A M , Saad F. Expression and localisation of Akt-1, Akt-2 and Akt-3 correlate with clinical outcome of prostate cancer patients. Br J Cancer. 2006;94(12): 1906-12. Leroy P, Berto F, Bourget I, Rossi B . Down-regulation of Hox A7 is required for cell adhesion and migration on fibronectin during early HL-60 monocytic differentiation. J Leukoc Biol . 2004;75(4):680-8. Lewis E B . A gene complex controlling segmentation in Drosophila. Nature. 1978;276(5688):565-70. 180 L i X , Nie S, Chang C, Qiu T, Cao X . Smads oppose Hox transcriptional activities. Exp Cell Res. 2006;312(6):854-64. Lie B L , Leung E, Leung PC, Auersperg N . Long-term growth and steroidogenic potential of human granulosa-lutein cells immortalized with SV40 large T antigen. M o i Cell Endocrinol. 1996; 120(2): 169-76. Lynch TJ, Bell DW, Sordella R, Gurubhagavatula S, Okimoto R A , Brannigan B W , Harris PL, Haserlat S M , Supko JG, Haluska FG, Louis D N , Christiani DC, Settleman J, Haber D A . Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med. 2004;350(21):2129-39. M a L, Benson G V , L i m H , Dey SK, Maas RL. Abdominal B (AbdB) Hoxa genes: regulation in adult uterus by estrogen and progesterone and repression in mullerian duct by the synthetic estrogen diethylstilbestrol (DES). Dev Biol . 1998;197(2):141-54. Maclean JA 2nd, Chen M A , Wayne C M , Bruce SR, Rao M , Meistrich M L , Macleod C, Wilkinson M F . Rhox: a new homeobox gene cluster. Cell. 2005;120(3):369-82. Maines-Bandiera SL, Huntsman D, Lestou V S , Kuo W L , Leung PC, Horsman RD, Wong A S , Woo M M , Choi K K , Roskelley CD, Auersperg N . Epithelio-mesenchymal transition in a neoplastic ovarian epithelial hybrid cell line. Differentiation. 2004-72(4): 150-61. Maizel A , Bensaude O, Prochiantz A , Joliot A . A short region of its homeodomain is necessary for engrailed nuclear export and secretion. Development. 1999;126(14):3183-90 Maubant S, Cruet-Hennequart S, Dutoit S, Denoux Y , Crouet H , Henry-Amar M , Gauduchon P. Expression of alpha V-associated integrin beta subunits in epithelial ovarian cancer and its relation to prognosis in patients treated with platinum-based regimens. J M o i Histol. 2005;36(l-2): 119-29. 181 Mauro V P , Wood IC, Krushel L , Crossin K L , Edelman G M . Cell adhesion alters gene transcription in chicken embryo brain cells and mouse embryonal carcinoma cells. Proc Natl Acad Sci U S A . 1994 Mar 29;91(7):2868-72. May JV, Stephenson L A , Turzcynski CJ, Fong HW, Mau Y H and Davis JS. Transforming growth factor 13 expression in the porcine ovary: evidence that theca cells are the major secretory source during antral follicle development. Biol Reprod. 1996;54:485^196. Mazerbourg S, Klein C, Roh J, Kaivo-Oja N , Mottershead D G , Korchynskyi O, Ritvos O, Hsueh A J . Growth differentiation factor-9 signaling is mediated by the type I receptor, activin receptor-like kinase 5. M o i Endocrinol. 2004;18(3):653-65. Mazerbourg S, Hsueh AJ . Genomic analyses facilitate identification of receptors and signalling pathways for growth differentiation factor 9 and related orphan bone morphogenetic protein/growth differentiation factor ligands. Hum Reprod Update. 2006;12(4):373-83. McGee E A , Perlas E, LaPolt PS, Tsafriri A , Hsueh A J . Follicle-stimulating hormone enhances the development of preantral follicles in juvenile rats. Bio l Reprod. 1997;57(5):990-8. McNatty K P , Juengel JL, Reader K L , Lun S, Myllymaa S, Lawrence SB, Western A , Meerasahib M F , Mottershead D G , Groome NP, Ritvos O, Laitinen M P . Bone morphogenetic protein 15 and growth differentiation factor 9 co-operate to regulate granulosa cell function in ruminants. Reproduction. 2005a; 129(4):481-7. McNatty K P , Juengel JL, Reader K L , Lun S, Myllymaa S, Lawrence SB, Western A , Meerasahib M F , Mottershead D G , Groome NP, Ritvos O, Laitinen M P . Bone morphogenetic protein 15 and growth differentiation factor 9 co-operate to regulate granulosa cell function. Reproduction. 2005b; 129(4):473-80. McNei l L , Hobson S, Nipper V , Rodland K D . Functional calcium-sensing receptor expression in ovarian surface epithelial cells. A m J Obstet Gynecol. 1998;178(2):305-13. 182 Meden H , Marx D, Raab T, Kron M , Schauer A , Kuhn W. EGF-R and overexpression of the oncogene c-erbB-2 in ovarian cancer: immunohistochemical findings and prognostic value. J Obstet Gynaecol. 1995;21(2): 167-78. M i n W, Woo HJ, Lee CS, Lee K K , Yoon W K , Park HW, K i m M H . 307-bp fragment in H O X A 7 upstream sequence is sufficient for anterior boundary formation. D N A Cell Biol . 1998;17(3):293-9. Miyamoto N , Yoshida M , Kuratani S, Matsuo I, Aizawa S. Defects of urogenital development in mice lacking Emx2. Development. 1997;124(9): 1653-64. Mondschein JS, Canning SF and Hammond J M . Effects of transforming growth factor-6 on the production of immunoreactive insulin-like growth factor I and progesterone and on [3H]thymidine incorporation in porcine granulosa cell cultures. Endocrinology. 1988; 123:1970-1976. Morgan R. Hox genes: a continuation of embryonic patterning? Trends Genet. 2006;22(2):67-9. Mortlock DP, Innis JW. Mutation of H O X A 13 in hand-foot-genital syndrome. Nat Genet. 1997; 15(2): 179-80. Nabeshima K , Kishi J, Kurogi T, Komada N , Kataoka H , Okada Y , Koono M . Stimulation of TIMP-1 and metalloproteinase production in co-cultures of human tumor cells and human fibroblasts. Cancer Lett. 1994;78(l-3): 133-40. Naora H , Montz FJ, Chai C Y , Roden RB. Aberrant expression of homeobox gene H O X A 7 is associated with mullerian-like differentiation of epithelial ovarian tumors and the generation of a specific autologous antibody response. Proc Natl Acad Sci U S A . 2001a;98(26): 15209-14. Naora H , Yang Y Q , Montz FJ, Seidman JD, Kurman RJ, Roden R B . A serologically identified tumor antigen encoded by a homeobox gene promotes growth of ovarian epithelial cells. Proc Natl Acad Sci U S A . 2001b;98(7):4060-5. 183 Nilsson EE, Skinner M K . Growth and differentiation factor-9 stimulates progression of early primary but not primordial rat ovarian follicle development. Biol Reprod. 2002;67(3): 1018-24. O'Nei l l CJ, McCluggage W G . p l6 expression in the female genital tract and its value in diagnosis. Adv Anat Pathol 2006;13(1):8-15. Orisaka M , Orisaka S, Jiang JY, Craig J, Wang Y , Kotsuji F, Tsang B K . Growth Differentiation Factor-9 Is Anti-Apoptotic during Follicular Development from Preantral to Early Antral Stage. M o i Endocrinol. 2006; [Epub ahead of print] Ozols RF, Rubin SC. Thomas G M , Robboy SJ. Epithelial ovarian cancer. In: Hoskins WJ, Perez C A , Young RC. Eds. Principles and practice of gynecologic oncology. 3th ed. Lippincott Williams & Wilkins. Philadephia, PA. 2000:982-1057. Packer AI , Elwell V A , Parnass JD, Knudsen K A , Wolgemuth DJ. N-cadherin protein distribution in normal embryos and in embryos carrying mutations in the homeobox gene Hoxa-4. Int J Dev Biol . 1997;41(3):459-68. Pando S M , Taylor HS. Homeobox gene expression in ovarian cancer. Cancer Treat Res. 2002;107:231-45. Pangas SA, Matzuk M M . The art and artifact of GDF9 activity: cumulus expansion and the cumulus expansion-enabling factor. Bio l Reprod. 2005;73(4):582-5. Pangas SA, Rajkovic A . Transcriptional regulation of early oogenesis: in search of masters. Hum Reprod Update. 2006;12(l):65-76. Pearson JC, Lemons D, McGinnis W. Modulating Hox gene functions during animal body patterning. Nat Rev Genet. 2005 ;6( 12): 893-904. Persad S, Attwell S, Gray V , Mawji N , Deng JT, Leung D, Yan J, Sanghera J, Walsh M P , Dedhar S. Regulation of protein kinase B/Akt-serine 473 phosphorylation by integrin-linked kinase: critical roles for kinase activity and amino acids arginine 211 and serine 343. J Biol Chem. 2001;276(29):27462-9. 184 Pitman JL, L in TP, Kleeman JE, Erickson GF, MacLeod C L . Normal reproductive and macrophage function in Pern homeobox gene-deficient mice. Dev Biol . 1998;202(2): 196-214. Rajkovic A , Yan C, Yan W, Klysik M , Matzuk M M . Obox, a family of homeobox genes preferentially expressed in germ cells. Genomics 2002;79:711-7. Rajkovic A , Pangas SA, Ballow D, Suzumori N , Matzuk M M . N O B O X deficiency disrupts early folliculogenesis and oocyte-specific gene expression. Science. 2004;305(5687): 1157-9. Redline RW, Williams A J , Patterson P, Collins T. Human H O X 4 E : a gene strongly expressed in the adult male and female urogenital tracts. Genomics. 1992;13(2):425-30. Redline RW, Hudock P, MacFee M , Patterson P. Expression of AbdB-type homeobox genes in human tumors. Lab Invest. 1994;71(5):663-70. Rodland K D . The role of the calcium-sensing receptor in cancer. Cell Calcium. 2004;35(3):291-5. Rodriguez C, Calle EE , Coates RJ, Miracle-McMahill H L , Thun M J , Heath C W Jr. Estrogen replacement therapy and fatal ovarian cancer. A m J Epidemiol. 1995;141(9):828-35. Rodriguez GC, Berchuck A , Whitaker RS, Schlossman D, Clarke-Pearson D L , Bast R C Jr. Epidermal growth factor receptor expression in normal ovarian epithelium and ovarian cancer. II. Relationship between receptor expression and response to epidermal growth factor. A m J Obstet Gynecol. 1991;164(3):745-50. Roh JS, Bondestam J, Mazerbourg S, Kaivo-Oja N , Groome N , Ritvos O, Hsueh A J . Growth differentiation factor-9 stimulates inhibin production and activates Smad2 in cultured rat granulosa cells. Endocrinology. 2003;144(1): 172-8. Rubin I, Yarden Y . The basic biology of HER2. Ann Oncol. 2001;12 Suppl l:S3-8. 185 Salamanca C M , Maines-Bandiera SL, Leung PC, Hu Y L , Auersperg N . Effects of epidermal growth factor/hydrocortisone on the growth and differentiation of human ovarian surface epithelium. J Soc Gynecol Investig. 2004; 11(4):241-51. Salih S M , Taylor HS. H O X A 10 gene expression in human fallopian tube and ectopic pregnancy. A m J Obstet Gynecol. 2004; 190(5): 1404-6. Saragueta PE, Lanuza G M , Baranao JL. Autocrine role of transforming growth factor betal on rat granulosa cell proliferation. Biol Reprod. 2002;66(6): 1862-8. Schilder RJ, Sill M W , Chen X , Darcy K M , Decesare SL, Lewandowski G, Lee R B , Arciero C A , Wu H , Godwin A K . Phase II study of gefitinib in patients with relapsed or persistent ovarian or primary peritoneal carcinoma and evaluation of epidermal growth factor receptor mutations and immunohistochemical expression: a Gynecologic Oncology Group Study. Cl in Cancer Res. 2005 Aug l;ll(15):5539-48. Schulze F, Chowdhury K , Zimmer A , Drescher U , Gruss P. The murine homeo box gene product, Hox 1.1 protein, is growth-controlled and associated with chromatin. Differentiation. 1987;36(2): 130-7. Scott MP. Vertebrate homeobox gene nomenclature. Cell. 1992;71(4):551-3. Schumer ST, Cannistra SA. Granulosa cell tumor of the ovary. J Cl in Oncol. 2003;21(6):1180,9 Scully RE. Pathology of ovarian cancer precursors. J Cell Biochem Suppl. 1995;23:208-18. Shah A , Swain W A , Richardson D, Edwards J, Stewart DJ, Richardson C M , Swinson D E , Patel D, Jones JL, O'Byrne K J . Phospho-akt expression is associated with a favorable outcome in non-small cell lung cancer. Clin Cancer Res. 2005;ll(8):2930-6 Soubry A , van Hengel J, Parthoens E, Colpaert C, Van Marck E, Waltregny D, Reynolds A B , van Roy F. Expression and nuclear location of the transcriptional repressor Kaiso is regulated by the tumor microenvironment. Cancer Res. 2005;65(6):2224-33. 186 Svingen T, Tonissen K F . Hox transcription factors and their elusive mammalian gene targets. Heredity. 2006;97(2):88-96. Suzumori N , Yan C, Matzuk M M , Rajkovic A . Nobox is a homeobox-encoding gene preferentially expressed in primordial and growing oocytes. Mech Dev 2002;111: 137-41. Tait D L , Zhang J, Gurlov S, McKinney K , Wachris J, Mougeot JL, Wagstaff JE, Bahrani-Mostafavi Z. Differential expression of Hox genes in ovarian cancer. Proc Amer Assoc Cancer Res 2005:46(3586). Takasaki N , Mclsaac R, Dean J. Gpbox (Psx2), a homeobox gene preferentially expressed in female germ cells at the onset of sexual dimorphism in mice. Dev Biol . 2000;223(1): 181-93. Taylor HS, Vanden Heuvel G B , Igarashi P. A conserved Hox axis in the mouse and human female reproductive system: late establishment and persistent adult expression of the Hoxa cluster genes. Biol Reprod. 1997;57(6): 1338-45. Taylor HS, Aric i A , Olive D, Igarashi P. H O X A 1 0 is expressed in response to sex steroids at the time of implantation in the human endometrium.J Clin Invest. 1998;101(7):1379-84. Taylor HS, Igarashi P, Olive D L , Aric i A . Sex steroids mediate H O X A 11 expression in the human peri-implantation endometrium. J Cl in Endocrinol Metab. 1999a;84(3): 1129-35. Taylor HS. The role of H O X genes in human implantation. Hum Reprod Update. 2000;6(l):75-9. Tennyson V M , Gershon M D , Sherman D L , Behringer RR, Raz R, Crotty D A , Wolgemuth DJ. Structural abnormalities associated with congenital megacolon in transgenic mice that overexpress the Hoxa-4 gene. Dev Dyn. 1993;198(l):28-53. Tennyson V M , Gershon M D , Wade PR, Crotty D A , Wolgemuth DJ. Fetal development 187 of the enteric nervous system of transgenic mice that overexpress the Hoxa-4 gene. Dev Dyn. 1998;211 (3):269-91. Toiyama Y , Mizoguchi A , Kimura K , Araki T, Yoshiyama S, Sakaguchi K , M i k i C, Kusunoki M . Persistence of gene expression changes in noninflamed and inflamed colonic mucosa in ulcerative colitis and their presence in colonic carcinoma. World J Gastroenterol. 2005;ll(33):5151-5. Toker A , Yoeli-Lerner M . Akt signaling and cancer: surviving but not moving on. Cancer Res. 2006;66(8):3963-6. Torres M , Gomez-Pardo E, Dressier GR, Gruss P. Pax-2 controls multiple steps of urogenital development. Development. 1995;121(12):4057-65. Uegaki K , Kanamori Y , Kigawa J, Kawaguchi W, Kaneko R, Naniwa J, Takahashi M , Shimada M , Oishi T, Itamochi H , Terakawa N . PTEN-positive and phosphorylated-Akt-negative expression is a predictor of survival for patients with advanced endometrial carcinoma. Oncol Rep. 2005;14(2):389-92. Vanderhyden B. Molecular basis of ovarian development and function. Front Biosci. 2002;7:d2006-22. Vanderhyden B C , Macdonald E A , Nagyova E, Dhawan A . Evaluation of members of the TGFbeta superfamily as candidates for the oocyte factors that control mouse cumulus expansion and steroidogenesis. Reprod Suppl. 2003;61:55-70. Vandromme M , Gauthier-Rouviere C, Lamb N , Fernandez A . Regulation of transcription factor localization: fine-tuning of gene expression. Trends Biochem Sci. 1996;21(2):59-64. Varani S, Elvin JA, Yan C, DeMayo J, DeMayo FJ, Horton HF, Byrne M C , Matzuk M M . Knockout of pentraxin 3, a downstream target of growth differentiation factor-9, causes female subfertility. M o i Endocrinol. 2002;16(6): 1154-67. Veraksa A , Del Campo M , McGinnis W. Developmental patterning genes and their conserved functions: from model organisms to humans. M o i Genet Metab. 188 2000;69(2):85-100. Vigne JL, Halburnt L L , Skinner M K . Characterization of bovine ovarian surface epithelium and stromal cells: identification of secreted proteins. Biol Reprod. 1994;51(6):1213-21. Villaescusa JC, Verrotti A C , Ferretti E, Farookhi R, Blasi F. Expression of Hox cofactor genes during mouse ovarian follicular development and oocyte maturation. Gene. 2004;330:1-7. Vitt U A , Hayashi M , Klein C, Hsueh AJ . Growth differentiation factor-9 stimulates proliferation but suppresses the follicle-stimulating hormone-induced differentiation of cultured granulosa cells from small antral and preovulatory rat follicles. Biol Reprod 2000a;62(2):370-7. Vitt U A , McGee E A , Hayashi M , Hsueh A J . In vivo treatment with GDF-9 stimulates primordial and primary follicle progression and theca cell marker CYP17 in ovaries of immature rats.Endocrinology. 2000b;141(10):3814-20. Vogels R, de Graaff W, Deschamps J. Expression of the murine homeobox-containing gene Hox-2.3 suggests multiple time-dependent and tissue-specific roles during development. Development. 1990; 110(4): 1159-68. Wang F, Weaver V M , Petersen OW, Larabell C A , Dedhar S, Briand P, Lupu R, Bissell M J . Reciprocal interactions between beta 1-integrin and epidermal growth factor receptor in three-dimensional basement membrane breast cultures: a different perspective in epithelial biology. Proc Natl Acad Sci U S A . 1998;95(25): 14821-6. Warot X , Fromental-Ramain C, Fraulob V , Chambon P, Dolle P. Gene dosage-dependent effects of the Hoxa-13 and Hoxd-13 mutations on morphogenesis of the terminal parts of the digestive and urogenital tracts. Development. 1997; 124(23):4781-91. Weaver V M , Petersen OW, Wang F, Larabell C A , Briand P, Damsky C, Bissell M J . Reversion of the malignant phenotype of human breast cells in three-dimensional culture and in vivo by integrin blocking antibodies. J Cell Bio l . 1997;137(l):231-45. 189 Williams T M , Williams M E , Innis JW. Range of H O X / T A L E superclass associations and protein domain requirements for H 0 X A 1 3 : M E I S interaction. Dev Biol . 2005;277(2):457-71. Wolgemuth DJ, Engelmyer E, Duggal R N , Gizang-Ginsberg E, Mutter G L , Ponzetto C, Viviano C, Zakeri ZF. Isolation of a mouse c D N A coding for a developmentally regulated, testis-specific transcript containing homeo box homology. E M B O J. 1986; 5(6): 1229-35. Wolgemuth DJ, Behringer RR, Mostoller M P , Brinster R L , Palmiter RD. Transgenic mice overexpressing the mouse homoeobox-containing gene Hox-1.4 exhibit abnormal gut development. Nature. 1989;337(6206):464-7. Wright JW, Stouffer R L , Rodland K D . Estrogen inhibits cell cycle progression and retinoblastoma phosphorylation in rhesus ovarian surface epithelial cell culture. M o i Cell Endocrinol. 2003;208(l-2): 1-10. Yamada M K , Clark K . Survival in three dimensions. Nature. 2002;149:790-1. Yamashita T, Katayama H , Ishiya T, Kato Y, Ishikawa M . Abnormal gene expression profile of homeobox containing gene H O X in ovarian cancer. Jap J Cancer Res 2002; 93 Suppl:333(2260), in Japanese Yamashita T, Tazawa S, Yawei Z, Katayama H , Kato Y , Nishiwaki K , Yokohama Y , Ishikawa M . Suppression of invasive characteristics by antisense introduction of overexpressed H O X genes in ovarian cancer cells. Int J Oncol. 2006;28(4):931-8. Yin Y, Ma L . Development of the mammalian female reproductive tract. J Biochem 2005;137:677-83. Yoeli-Lerner M , Y i u G K , Rabinovitz I, Erhardt P, Jauliac S, Toker A . Akt blocks breast cancer cell motility and invasion through the transcription factor N F A T . M o i Cell. 2005;20(4):539-50. Zhang X , Emerald BS, Mukhina S, Mohankumar K M , Kraemer A , Yap A S , Gluckman PD, Lee K O , Lobie PE. H O X A 1 is required for E-cadherin-dependent 190 anchorage-independent survival of human mammary carcinoma cells. J Bio l Chem. 2006;281(10):6471-81. Zhao X X , Suzumori N , Yamaguchi M , Suzumori K . Mutational analysis of the homeobox region of the human N O B O X gene in Japanese women who exhibit premature ovarian failure. Fertil Steril. 2005;83(6): 1843-4. Zhao Y , Yamashita T, Ishikawa M . Regulation of tumor invasion by HOXB13 gene overexpressed in human endometrial cancer. Oncol Rep. 2005;13(4):721-6. 

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