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Effect of all-trans-retinoic acid on bovine papillomavirus (BPV)-1 DNA-induced mouse C127 cell transformation… Li, Gang 1989

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EFFECT OF ALL-TRANS-RETINOIC ACID ON BOVINE PAPILLOMAVIRUS (BPV)-l DNA-INDUCED MOUSE C127 CELL TRANSFORMATION AND NUMBER OF BPV DNA COPIES By GANG LI B.M., Nanjing Medical College, People's Republic of China, 1984 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Zoology) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA January, 1989 © Gang L i , 1989 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Zoology The University of British Columbia Vancouver, Canada Date February 28, 1989 DE-6 (2/88) ABSTRACT Bovine papillomavirus (BPV)-l DNA-induced transformation of mouse C127 cells was used as a model for examining the action of all-trans -retinoic acid (RA), which is currently being tested as a chemopreventive and chemotherapeutic agent. Prior to studying the action of RA, the transformation frequency and the nature of transformed clones were studied. Transformed colonies formed two weeks after v i r a l DNA transfection. The transformation frequency was proportional to the amount of v i r a l DNA used for transfection. The 8 transformed colonies examined contained 20 to 100 copies of v i r a l genomes per c e l l on average. The BPV DNA copy number was stably inherited over 19 passages. The transformed cells had a heterogeneous number of BPV DNA copies. BPV DNA was present extrachromosomally in more than 95% of the subclones containing the v i r a l genomes. Transformed cells became elongated after the cells reached confluency. The effect of RA was examined on (1) transformation induced in C127 cells by transfection with plasmid pdBPV-1 (142-6), which contains DNA of bovine papillomavirus (BPV), (2) the capacity of transformed BPV DNA-containing clones to form colonies with transformed properties (i.e., p i l i n g up into multilayered colonies), and (3) the number of BPV DNA copies in transformed c e l l s . At non-toxic doses ranging from 10"^ to 1 0 M , RA reduced the frequency of transformed foci in a dose-dependent manner. The extent of inhibition depended on the length of RA treatment and on the time that elapsed between DNA transfection and RA treatment. RA exerted only a i i i slight inhibitory effect during the f i r s t days after transfection. Complete inhibition was observed when the cells were continuously exposed after transfection to RA at doses ranging from 0.5 to 1 x 10""' M. The inhibitory effect of RA on transformation was reversible. Transformed foci started to form after withdrawal of RA treatment. RA reduced the saturation density of the transformed cells without reducing their growth rate. RA specifically suppressed the piling-up phenotype of the transformed c e l l s . The doses for complete suppression of transformed phenotype is similar to those for complete inhibition of v i r a l DNA-induced transformation. The number of BPV DNA copies gradually decreased when the cells were grown over several generations in the presence of RA (5 x 10"^ M). After five weeks of treatment (approximately 30 c e l l generations), the c e l l cultures appeared to have less than one copy of BPV DNA. The number of BPV-1 DNA copies was examined after withdrawal of RA. The low number of BPV DNA copies in the RA-treated c e l l population did not increase for at least 10 passages when the cells were subcultured before reaching confluence. After 5 weeks treatment with RA, the transformed c e l l populations that contained less than one BPV DNA copy lost the transformed phenotype. However, a tiny fraction of cells (1 in 13,000) s t i l l had more than 10 BPV DNA copies and retained the capacity to develop into transformed colonies. The tiny fraction of cells were not resistant to RA. After the RA treatment was extended to 10 weeks, transformed cells completely lost BPV DNA. The BPV DNA free cells do not express a transformed phenotype. iv The elongated morphology of the transformed cells were also reversed to normal (star-shaped) after 10 week treatment with RA. The above results recommend the use of retinoids as chemopreventive and chemotherapeutic agents for papillomavirus-induced tumours. V TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS v LIST OF TABLES v i i LIST OF FIGURES v i i i ABBREVIATIONS x ACKNOWLEDGEMENTS xi INTRODUCTION 1 1. Papillomaviruses and Cancer 1 2. Genomic Organization of Papillomavirus DNA 2 3. Transformation Capacity of Papillomaviruses 3 4. Biological Functions of Retinoids 6 5. Retinoids and Cancer 9 6. Objectives 11 MATERIALS AND METHODS 13 1. Cell Cultures 13 2. Bovine Papillomavirus (BPV) 13 3. Transformation Assay 13 4. Establishment of Cell Lines from Transformed Foci 14 5. Treatment with all-trans-Retinoic Acid (RA) 14 6. Microphotography 14 7. DNA Extraction 15 8. Blot Hybridization 15 9. Estimation of BPV DNA Copy Number 16 v i RESULTS 17 1. BPV-1 DNA-Induced C127 Cell Transformation and Characterization of Transformed Clones 17 1.1. Transformation frequency 17 1.2. Selection and characterization of BPV DNA-transformed clones 17 2. Inhibition of Cell Transformation by RA 27 2.1. Dose-dependent inhibition of c e l l transformation by RA 27 2.2. Stage-dependent responses to RA 34 3. Effect of RA on Phenotype of Transformed Cells 37 3.1. Growth rate 37 3.2. Inhibition of focus-forming capacity of transformed cells by RA 37 4. Effect of RA on the Number of BPV DNA Copies 41 4.1. RA-induced reduction in the number of BPV DNA copies 41 4.2. Stability of low BPV DNA copy number 45 4.3. Focus - forming capacity of RA-treated B3 cells 45 5. Continuation of RA Treatment 50 DISCUSSION 62 1. BPV-1 DNA-Induced Mouse C127 Cell Transformation 62 2. Inhibition of Cell Transformation by Retinoids 63 3. Effect of RA on Transformed Cells 64 4. Response of HPV-Carrying Human Lesions to Retinoids 66 5. Mechanism of RA Action: An Unresolved Issue 68 REFERENCES 71 v i i LIST OF TABLES Page Table 1 Reduced capacity of transformed clones to form colonies with transformed properties in the presence of retinoic acid 40 Table 2 Estimates of the number of BPV DNA copies of B3 cells treated with RA for 5 weeks from densitometer scanning of autoradiographs 44 TABLE OF FIGURES Figure 1 Genomic organization of BPV-1 Figure 2 Structural configuration of retinoids Figure 3 Frequency of C127 c e l l transformation induced by BPV DNA Figure 4 BPV DNA copy numbers of 8 transformed clones Figure 5 Southern analysis of DNAs from 22 subclones of a BPV DNA-transformed clone (B3) Figure 6 Southern analysis of DNAs from 28 subclones of BS9 (a subclone of B3) Figure 7 Morphology of C127 cells and BPV-1 DNA-transformed B3 cells Figure 8 Inhibition of BPV-1 DNA-induced C127 c e l l transformation by RA Figure 9 Effect of RA on the clone-forming capacity of C127 cells and BPV-1 DNA-induced c e l l transformation Figure 10 Effect of length and period of RA treatment on c e l l transformation Figure 11 Effect of RA on the growth rate of transformed cells Figure 12 Reduction of BPV DNA copy numbers by RA Figure 13 Maintenance of low BPV DNA copy numbers following termination of RA treatment Figure 14 Focus - forming capacity of transformed cells treated with RA for 5 weeks Figure 15 Maintenance of transformed phenotype for 1 in 13,000 transformed cells treated with RA for 5 weeks Figure 16 BPV DNA copy numbers in 24 transformed colonie which developed after treatment with RA for 5 weeks Southern analysis of DNAs from 24 transformed colonies which developed after treatment with RA for 5 weeks Reversal of transformed phenotype and elimination of BPV DNA after 10-week RA treatment Morphology of transformed cells treated with RA for 10 weeks X ABBREVIATIONS BPV Bovine papillomavirus DMBA 7,12-dimethylbenz[a]anthracene DMEM Dulbecco's modified minimal essential medium DMSO dimethylsulfoxide DNA deoxyribonucleic acid EDTA ethylenediaminetetracetate EV epidermodysplasia verruciformis FBS fetal bovine serum HPV human papillomavirus MCA 3-methylcholanthrene MNNG N-methyl-N'-nitro-N-nitrosoguanidine ORF open reading frame PV papillomavirus RA all-trans-retinoic acid rpm revolutions per minute SDS sodium dodecyl sulphate Tris tris-(hydroxymethyl)-aminomethane x i ACKNOWLEDGEMENTS I would like to express my gratitude: to my supervisors, Dr. H.F. Stich and Dr. S.S. Tsang, for their guidance, technical instructions, suggestions, discussions, and encouragement throughout my project; to the members of my supervisory committee, Dr. J.D. Berger, Dr. H. Brock and Dr. T. G r i g l i a t t i , for their encouragement and help; and to Ms. R. Pritchard for c r i t i c a l reading of the manuscript. The financial support from the State Education Commission, People's Republic of China, and the National Cancer Institute of Canada research grant to Dr. S.S. Tsang is acknowledged. 1 INTRODUCTION 1. Papillomaviruses and Cancer Papillomaviruses (papilla=nipple; oma=tumour) are a group of species-specific viruses inf e c t i n g humans and a wide range of animals. The viruses produce i n th e i r host benign tumours (papillomas) which contain a variable amount of infectious v i r u s . Common hand warts and plantar warts are the most recognizable cutaneous l e s i o n s , but i t i s now clear that papillomaviruses (PV) are associated with a variety of lesions on many squamous e p i t h e l i a l surfaces, including the cervix, vaginal w a l l , vulva, penis, larynx, tongue, buccal mucosa and conjunctiva, as well as the skin (for review, see P f i s t e r , 1984; McCance, 1986). The tumours are usually s e l f - l i m i t i n g and regress. However, a number of papillomaviruses induce tumours that may eventually progress to carcinomas. The oncogenic potential of animal papillomaviruses i n natural conditions i s evidenced by the transformation of c o t t o n t a i l rabbit papillomas into invading, destroying, and frequently metastasizing, squamous c e l l carcinomas which may occur i n up to 25% of the rabbits within several months (Syverton, 1952). Furthermore, malignant transformation of alimentary tract papillomas of c a t t l e , mainly esophageal and nominal, induced by bovine papillomavirus (BPV) type 4, has been observed (Jarrett et a l . . 1978). Conversion of human papillomas into squamous c e l l carcinomas has also been noted for epidermodysplasia verruciformis (EV) lesions, condylomata acuminata, condylomata plana, and laryngeal papillomas. About one-third of EV patients develop cancer between 2 and 60 years 2 after the onset of verrucosis, on average after 24 years (Lutzner, 1978). Two human papillomaviruses (HPV), type 5 and type 8, are commonly found in these carcinomas, in a free extrachromosomal state (Orth et a l . , 1980). The carcinomas are found mainly at light-exposed sites, such as the face, hands and arms (Jablonska et a l . . 1972). This may point to other cofactors, here most probably ultraviolet l i g h t , during the malignant conversion of EV papillomas. Human papillomaviruses as agents playing a role in the etiology of genital carcinomas, especially cervical carcinomas, was suggested only a decade ago (zur Hausen, 1977). However, a great deal of work has been done to investigate whether the association of HPV infection and incidence of genital carcinomas is casual or causal. Over 80% of malignant genital carcinomas are associated with HPV infection (Durst et a l . , 1983; zur Hausen, 1987). More than ten HPV types are detected in the genital tract. Among this group, HPV-16 and HPV-18 have been consistently associated with premalignant and malignant lesions of the female genital tract (Gissmann and Schneider, 1986; Reid et a l . . 1987), and their DNAs have been found in an integrated state in invasive cancers (Boshart et a l . . 1984; Durst et a l . . 1985). Furthermore, i t was found that the integration site of HPV-18 into the cellular genome is near proto-oncogene myc, which suggests that malignant conversion of PV-induced papillomas may be due to trans-activation of cellular oncogenes (Durst et a l . . 1987). 2. Genomic Organization of Papillomavirus DNA The papillomavirus is a single piece of circular double stranded and superhelical DNA about 8,000 nucleotides in length, which is encased 3 i n a p r o t e i n c a p s i d c o n s i s t i n g o f v i r a l c oded p r e t e i n s ( P f i s t e r , 1 984 ) . The a p p r o x i m a t e s i z e o f t h e v i r i o n i s 54 nm. Many d i f f e r e n t v a r i a n t s have b e en i d e n t i f i e d ; t h e y a r e d i s t i n g u i s h e d by t h e d i v e r s i t y i n t h e i r n u c l e o t i d e s equence , as d e t e r m i n e d by s t a n d a r d DNA-DNA h y b r i d i z a t i o n e x p e r i m e n t s . More t h a n 50 t y p e s o f human p a p i l l o m a v i r u s e s have been i d e n t i f i e d . The c omp l e t e DNA sequences o f BPV-1 (Chen e t a l . . 1 9 8 2 ) , c o t t o n t a i l r a b b i t p a p i l l o m a v i r u s ( G i r i e t a l . , 1 985 ) , and q u i t e a few HPV genomes (Danos e t a l . . 1983; Dar tmann e t a l . . 1986; Fuchs e t a l . . 1986; Schwarz e t a l . . 1983 ; S e e d o r f e t a l . . 1985) have b e en d e t e r m i n e d . A l i g n m e n t o f t h e i r s equences r e v e a l s t h a t t h e y p o s s e s s s i m i l a r g e n e t i c o r g a n i z a t i o n o f p r o t e i n - c o d i n g p o t e n t i a l , r e c o g n i z e d as open r e a d i n g f r ames (ORFs) ( F i g u r e 1 ) . Two o f t h e s e , d e s i g n a t e d L l and L 2 , code f o r s t r u c t u r a l p r o t e i n s o f t h e v i r u s e s , and t h e o t h e r 8 ORFs ( E l t o E8) a r e i m p o r t a n t i n v i r a l r e p l i c a t i o n and c e l l u l a r t r a n s f o r m a t i o n (How ley e t a l . . 1986; P f i s t e r e t a l . , 1 9 8 6 ) . The E l gene i s b e l i e v e d t o be r e s p o n s i b l e f o r s t a b l e e x t r a c h r o m o s o m a l DNA ma i n t e nan c e o f t h e v i r u s ( S a r v e r e t a l . , 1984; L u s k y and B o t c h a n , 1 9 8 5 ) . T r a n s f o r m a t i o n f u n c t i o n s seem t o be encoded b y t h e E6/E7 and t h e E2 /E5 ORFs ( S a r v e r e t a l . , 1984; Yang e t a l . , 1985; Choo e t a l . . 1 9 8 7 ) . A l l t h e ORFs a r e encoded i n messenge r RNAs t r a n s c r i b e d f r om one o f t h e two DNA s t r a n d s . 3 . T r a n s f o r m a t i o n C a p a c i t y o f P a p i l l o m a v i r u s e s B o t h human and a n i m a l p a p i l l o m a v i r u s e s a r e e s s e n t i a l l y e p i t h e l i o t r o p i c . On l y t h e t op few l a y e r s o f c e l l s c o n t a i n i n f e c t i o u s v i r u s ( B r a un e t a l . . 1983; McCance e t a l . , 1 9 8 3 ) , a l t h o u g h v i r a l DNA can be d e t e c t e d i n t h e l o w e r l a y e r s ( O r t h e t a l . . 1 9 7 7 ) . T h i s c l o s e 4 Figure 1. Genomic organization of BPV-1. The genome is measured in kilobase pairs (kb). The open bars represent open reading frames, which are labelled "E" or "L" depending on their functions. Gene functions that have been mapped for BPV-1 are li s t e d below the genome. 5 I CD E l E2 T OD CD 3 I HU [ |E3l lE5| L l L2 I TJ 03 7945 k b t r a n s -f o r m a t i o n e p i s o m a l p e r s i s t e n c e i i t r a n s f o r m a t i o n m i n o r c a p s i d m a j o r c a p s i d e n h a n c e r s t i m u l a t i o n t-6 coincidence of papillomavirus replication with epithelial differentiation is most lik e l y the characteristic of this group of viruses that has hampered attempts to replicate them in in vitro c e l l cultures. However, a few papillomaviruses have been found to be able to transform fibroblasts in v i t r o . Among them, bovine papillomavirus type 1 is the best characterized. The oncogenic potential of the PV DNA has been repeatedly demonstrated in in vitro experiments. The v i r a l DNA can induce cellular transformation of several different tissue culture c e l l s . These experiments were f i r s t performed with animal PV DNA such as BPV DNA (Lowy et a l . . 1980; Sarver et a l . . 1982), and more recently with human papillomavirus DNA (Watts et a l . . 1984; Yasumoto et a l . . 1986; P i r i s i et a l . , 1987). Bovine papillomavirus is capable of transforming mouse c e l l s . Subgenomic segment equal to 69% of the BPV genome, the early region, contains a l l the necessary functions (Lowy et a l . , 1980). A l l the transcripts present in cells transformed by intact BPV are confined to this 69% region. It is believed that the BPV-1 DNA in transformed cells exists predominantly, i f not exclusively, as non-integrated episomal molecules (Lancaster, 1981; Binetruy et a l . . 1982; Sarver et a l . . 1982). 4. Biological Functions of Retinoids Retinoids are naturally occurring compounds and synthetic derivatives of retinol (Vitamin A) (Figure 2). Retinoids are essential in the control of epithelial c e l l growth, cellular differentiation, and in the inhibition of carcinogenesis (reviewed by Lotan, 1980; Peto, 1983; Sporn and Roberts, 1983; Lipmann et a l . . 1987). For example, 7 Figure 2. S t r u c t u r a l configuration of r e t i n o i d s . A: r e t i n o l (vitamin A). B: a l l - t r a n s - r e t i n o i c a c i d . C: 1 3 - c i s - r e t i n o i c a c i d . 9 retinoids have been shown to affect the differentiation of both epithelial cells and mesenchymal cells in cultures that have not been exposed to a transforming agent (Yuspa et a l . . 1981). Retinoids can induce terminal differentiation in mouse F9 teratocarcinoma cells (Strickland and Mahdavi, 1978), and in human promyelecytic leukemia cells (Breitman et a l . . 1980; Strickland et a l . , 1983; Yen et a l . . 1984). In addition, retinoic acid aslo has striking effects on pattern formation and may be the natural morphogen providing the positional information in the postulated morphogenetic gradient of the developing ckin limb bud (Thaller and Eichele, 1987), axolotl limb (Crawford and Stocum, 1988) and palatal shelves of embryonic mice (Abbott et a l . , 1988) . 5. Retinoids and Cancer Retinoids have been shown to antagonize carcinogenesis in numerous in vivo and in vitro models. Vitamin A or synthetic retinoids can inhibit carcinogenesis by chemical and physical carcinogens (Lotan, 1980; McCormick et a l . . 1981; Muto and Moriwaki, 1984; Zile et a l . . 1986). The results overwhelmingly indicate that retinoids are capable of preventing the development of cancer of the skin, respiratory tract, urinary tract and mammary gland. It was also found that retinoids inhibit the development and growth of PV-induced papillomas (McMichael, 1965). In v i t r o , retinoids were found to inhibit and reverse hyperplasia and squamous metaplasia induced by benzo(a)pyrene in organ cultures of hamster trachea (Crocker and Sander, 1970) and preneoplastic changes induced by methylcholanthrene or N-methyl-N'-nitro-N-nitrosoguanidine 10 (MNNG) in organ cultures of mouse prostate glands (Lasnitck and Goodman, 1974). In addition, retinoids inhibit the carcinogen- or v i r a l DNA-induced neoplastic transformation of rodent cells (Harisiadis et a l . , 1978; Dickens et a l . . 1979; Merriman and Bertram, 1979). Recently, retinoic acid was shown to inhibit cellular transformation induced by the ras-oncogene or myc-oncogene (Dotto et a l . . 1985; Roberts et a l . . 1985). Retinoids were also found to inhibit the appearance and development of skin papillomas during the croton oil-promotion phase of dimethybenzanthracene-initiated mouse skin carcinogenesis (Bollag, 1971) . Treatment of mouse skin with retinoic acid resulted in a large depression in the induction of ornithine decarboxylase (Verma et a l . , 1979; Loprinzi and Verma, 1985; Verma, 1988), which is the rate-limiting enzyme in the synthesis of polyamine, whose accumulation is believed to play a role in the mechanism of tumour promotion (O'Brien, 1976). Furthermore, retinol has been shown to inhibit the promoting activity of betel quid ingredients in BPV-1 DNA-induced C3H/10T1/2 c e l l transformation (Stich and Tsang, 1989). The administration of retinoids to humans has led to the inhibition of benign and malignant skin lesions (Bollag and Matter, 1981), oral leukoplakia (Koch, 1981), and cervical lesions (Meyskens et a l . , 1983). Using intermediate endpoints, such as the frequency of micronucleated c e l l s , the oral administration of beta-carotene and vitamin A has shown a protective effect in tobacco/betel nut chewers (Stich et a l . , 1984a,b; Stich e t _ a l . , 1988a,b). More relevant to this research are the intervention studies on precancerous lesions or carcinomas which contain HPV DNA and in which 11 HPV genes are expressed. Retinoids have been repeatedly and successfully applied in the treatment of common, plantar and f l a t warts, and preneoplastic lesions and cancers of patients with EV (Lutzner et a l . , 1981; Pfister, 1984; Mahrle, 1985). In some c l i n i c a l t r i a l s , retinoids induced the disappearance or a 100- to 1500-fold reduction of v i r a l DNA and/or v i r a l antigens in skin lesions (Jablonska et a l . . 1981; Lutzner, 1981; Gross et a l . . 1983; Lutzner et a l . . 1984). 6. Objectives During the past two years, we have established the in vitro v i r a l carcinogenesis model in which BPV-1 DNA was used to transform mouse C127 c e l l s . Although all-trans-retinoic acid (RA) has been successfully applied in the treatment of PV-containing warts, the mechanism of i t s effects remains unknown. The projects investigate whether the chemopreventive and chemotherapeutic actions of RA are due to the suppression of papillomavirus DNA replication, and therefore a reduction of BPV DNA copy number in transformed c e l l s , and whether the transformed cells s t i l l retain a transformed phenotype after withdrawal of RA. Effect of RA on the frequency of BPV DNA-induced transformation The objective of this project is to reveal whether RA can inhibit transformation induced by the entire genome of BPV, whether this inhibition depends on the dose of chemopreventive agents, and whether the degree of RA inhibition depends on a particular stage of transformation. Effect of RA on the transformed properties of BPV DNA-transformed cells 12 Since RA has been proven effective in the treatment of PV-containing warts and patients with EV, i t is of interest to examine the effect of RA on the phenotype of transformed C127 c e l l s . Does RA reduce the growth rate of the transformed cells? Does treatment with RA reverse the transformed phenotype to a "normal" (non-transformed) one? Does this reversion require higher doses of RA than the inhibition of BPV DNA-induced C127 c e l l transformation? Effect of RA on the number of copies of BPV DNA in transformed cells The replication of BPV DNA may be inhibited specifically by RA . Answers to the following questions w i l l be sought. Does treatment with RA reduce the number of extrachromosomal BPV DNA copies? Does the reduction of BPV DNA copies occur in a l l the cells? Does a loss of the BPV sequence lead to the reversion of cells with a transformed phenotype into cells with a "normal" phenotype? Upon withdrawal of RA, do the v i r a l gene copy numbers return to the pretreatment level? 13 MATERIALS AND METHODS 1. Cell Cultures Mouse C127 cells (American Type Culture Collection, Rockville, MD) were maintained in Dulbecco's modified minimal essential medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Gibco), 60 ng/ml p e n i c i l l i n (1,670 units/mg), and 100 /*g/ml streptomycin in CO2 incubators (5% CO2, 37°C). Medium was changed twice weekly. 2. Bovine Papillomavirus (BPV) Plasmid pdBPV-1 (142-6) (American Type Culture Collection) was used as the source of BPV DNA. This plasmid consists of the full-length BPV-1 genome inserted at it s unique Bam HI sites in the plasmid pML2d, which is a deletion derivative of pBR322 lacking the DNA sequence from bases 1,095 to 2,485 (Sarver et a l . . 1982). The a b i l i t y of the BPV genome in the plasmid to transform mouse C127 cells has been documented previously (Sarver et a l . . 1982; Schiller et a l . . 1984). 3. Transformation Assay Cells were seeded at a density of 1 x 10^ cells/ml per 100-mm tissue culture plate. BPV DNA with 20-40 ng of carrier calf thymus DNA was precipitated with calcium phosphate (Parker" and Stark, 1979), and added to the cells after 24 hr. The medium was removed, and 5 ml 15% glycerol in HBS buffer was added to the cells after 20 hr. The HBS buffer contained 137 mM NaCl, 5 mM KC1, 5.5 mM dextrose, 0.7 mM Na2HP04, and 21 mM Hepes at pH 7.05. The glycerol solution was removed after 2 min. The c e l l cultures were rinsed twice with 5 ml fresh medium, and re-14 fed with 10 ml fresh medium. After 24 hr, the cells were subcultured into culture dishes (100 mm diameter). The culture medium was changed twice weekly. The c e l l cultures were fixed and stained with 0.1% methylene blue dissolved in 50% methanol for 30 min after 14 to 21 days and transformed foci were scored. 4. Establishment of Cell Lines from Transformed Foci The centre portion of each transformed foci was transferred into a drop of 0.02% EDTA and 0.05% trypsin solution on a 25 mm diameter petri dish by means of a ste r i l i z e d Pasteur pipette. The extirpated clones were trypsinized for 5 min and mechanically dispersed and f i n a l l y 2 ml DMEM containing 10% FBS were added to the petri dish. The cells were passaged twice in tissue culture flasks with an area of 80 mm before they were stored frozen in liquid nitrogen. 5. Treatment with all-trans-Retinoic Acid (RA) RA (Sigma Chemical Co., St Louis, MO) was dissolved in o dimethylsulfoxide (DMSO) at a concentration of 3.3 x 10 M, and stored at -70°C. Since RA is light-sensitive, a l l manipulations involving RA were carried out in yellow light. In control plates, cells only receive DMSO. 6. Microphotography The methylene blue-stained cells were photographed with an Olympus photomicrographic system with a magnification of 400x. The Kodak 2415 films were developed in Kodak D-19 for 4 min. 15 7. DNA Extraction Culture medium was removed from the petri dish. The cells were rinsed twice with 5 ml ice-cold phosphate buffered saline (PBS), and scraped with a rubber policeman. Using a wide-mouthed pipette, the cells were collected in 10 ml PBS and the c e l l suspension was transferred into a Corex centrifuge tube. The cells were centrifuged for approximately 2 min at 700g (2000 rpm/min) un t i l the cells were pelleted. Cells were lysed and digested with SET (100 mM NaCl, 1 mM EDTA, 10 mM Tris-HCl) buffer containing 1 mg/ml proteinase K (Sigma Chemical Co., St Louis, MO) and 0.5% sodium dodecyl sulfate (SDS) at 37°C for 3 hr with rotation (Gross-Bellard et a l . . 1973). Cellular DNA was extracted with phenol, phenol/chloroform (1:1) and chloroform/isoamylalcohol (24:1), and by ethanol precipitation. RNA was removed by digestion with pancreatic RNase (Sigma Chemical Co., St Louis, MO). The DNA concentration of the samples was measured with a Lambda 3 UV/VIS spectrophotometer (Perkin-Elmer). 8. Blot Hybridization For Southern blotting, DNA was digested with Bam HI restriction enzyme, and subjected to electrophoresis in 0.8% agarose (Bio-Rad) gels. Gels were soaked once in 0.25 M HCl for 10 min to parti a l l y hydrolyse DNA, twice in 1.5 M NaCl, 0.5 M NaOH for 15 min each to denature double strand DNA, and twice in 1.5 M NaCl, 0.5 M Tris-Cl (pH 7.5) for 20 min each to neutralize. After capillary transfer of DNA onto nitrocellulose f i l t e r s (Schleicher & Schuell Inc.) in 20x SSC buffer (3 M NaCl, 0.3 M sodium citrate, pH 7.0) overnight, the f i l t e r s were dried in vacuo for 2 hr at 80°C (Maniatis et a l . . 1982). 16 For slot blotting, DNA was diluted with 200 fj.1 TE (10 mM Tris-HCl, 1 mM EDTA) buffer, denatured with a 0.1 volume of 3 M NaOH at 65°C for 30 min, neutralized with an equal volume of 2 M sodium acetate, and applied to nitrocellulose f i l t e r s (Maniatis et a l . . 1982). Filters were baked for 2 hr at 80°C under vacuum. Fil t e r s were prehybridized for 30 min at 42°C with 50% formamide, 5x SSC (lx SSC is 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0), lx Denhardt's (0.02% bovine serum albumin, 0.02% F i c o l l , 0.02% polyvinylpyrrolidone), 0.1% SDS, 50 mM Na2HP04 (pH 7.0), 1 mM EDTA, and 100 pg/ml yeast tRNA. Filters were then hybridized hybridized for 24 hr at 42°C in a similar solution containing with 2 x 10^ cpm (counts per 39 minute)/ml of J iP nick-translated pdBPV-1 (142-6) probe (specific Q activity >10 cpm/pg). Filters were washed three times for 5 min each in 2x SSC/0.1% SDS at room temperature, and then twice for 15 min each in O.lx SSC/0.1% SDS at 68°C. The f i l t e r s were then exposed to X-ray film (Kodak X-Omat AR5) with an intensifying screen at -70°C. 9. Estimation of BPV Copy Numbers The average number of BPV DNA copies per c e l l was estimated by comparing the extent of hybridization to a known quantity of BPV DNA. I have used 14 pg pdBPV-1 (142-6) as one gene copy equivalent of BPV DNA per 10 jug cellular DNA (Watts et a l . . 1984). The intensity of hybridization was quantitated by scanning the autoradiograph with a GS 300 densitometer (Hoefer S c i e n t i f i c ) , and integrating the peak areas with an SP4100 integrator (Spectra Physics). 17 RESULTS 1. BPV-1 DNA-Induced C127 Cell Transformation and Characterization of Transformed Clones 1.1. Transformation frequency Various amounts of BPV DNA were used to transfect 10^ C127 c e l l s . The cells were incubated in DMEM supplemented with either 10% fetal bovine serum or 10% calf serum. Figure 3 shows that the frequency of transformed colonies was directly proportional to the amount of BPV DNA used for transfection. However, the transformation frequency was higher when the cells were incubated in 10% fetal bovine serum. Therefore, a l l the following experiments were carried out in 10% fetal bovine serum. 1.2. Selection and characterization of BPV DNA-transformed clones Eight transformed foci were isolated and expanded into c e l l lines designated B3 to B10. The cellular DNA was extracted from 8 c e l l lines and slot blot analysis revealed that these transformed c e l l lines contain 20 to 100 copies of BPV DNA (Figure 4). To gain information on the heterogeneity of transformed clones with respect to the number of BPV DNA copies, B3 cells were subcloned. Approximately 100 B3 cells were seeded into a 100-mm petri dish and allowed to grow into individual colonies. Twenty-two colonies were picked with a st e r i l i z e d Pasteur pipette (as described in Material and Methods) and expanded into c e l l lines designated BS1 to BS22. Southern blot analysis showed that 22.7% (5/22) (lanes 5, 16, 17, 19 and 21) had no detectable BPV DNA, 18.2% (4/22) (lanes 1,2,4 and 11) contained 18 Figure 3. Frequency of C127 c e l l transformation induced by BPV DNA. Mouse C127 cells at passage 14 were seeded at a density of 1 x 10 cells per 100-mm tissue culture plates in Dulbecco's modified minimal essential medium (DMEM) supplemented with either 10% fetal bovine serum ( • ) or 10% calf serum ( • ). 24 hr later, the cells were transfected with variable amounts of BPV DNA and 25 fig calf thymus DNA. Two days later, the cells were subcultured into 100-mm culture dishes at a ratio of 1:4. The media were changed twice weekly. The cells cultured in 10% fetal bovine serum DMEM were stained after 2 weeks. The cells cultured in 10% calf serum medium were stained after 32 days. Regression lines were computed from the data. Fetal bovine serum, Y=l.17+0.745X (n=15). Calf serum, Y=-5+0.322X (n=14). Bars, S.E. 19 20 Figure 4. BPV DNA copy numbers of 8 transformed clones, measured by slot blot hybridization, as described in Materials and Methods. 5 ng DNA from each sample were used, a, BPV DNA genome equivalents, b, DNAs from C127 and transformed c e l l s . 400 c i 2 7 1 0 0 mm 4 m B 3 so *m 4 m B 4 20 mm € » B 5 10 — 4 k W B 6 5 — 4 • B 7 — 3 8 » B 9 V B I O 22 less than 60 copies, and the rest (59.1%, 13/22) contained 60 to 150 copies (Figure 5). 27.3% (6/22) of the subclones (lanes 4, 12, 13, 14, 15 and 22) lost the plasmid pML2 sequences. This phenomenon may be due to recombination of the plasmid pdBPV-1 (142-6) at Bam HI site during transfection, which was also observed by Law et a l . (1981). The molecular weight of BPV DNA in a l l 17 subclones which contained the v i r a l genomes is 8 kb, indicating that BPV DNA present episomally in transformed c e l l s . However, one subclone (lane 3) contained an additional band other than 8 kb, which suggest that some of the v i r a l DNA copies were integrated into cellular genome. To decide whether the presence of those clones which did not contain BPV DNA were due to contamination of non-transformed C127 cells during the cloning procedure or to the spontaneous loss of v i r a l genome, 28 re-subclones, designated BSS1 to BSS28, were isolated from the BS9, which contained about 60 copies of episomal BPV DNA. The DNAs were extracted from each re-subclone and subjected to Southern blot hybridization. Figure 6 shows 10.7% of the re-subclones (3/28) (lanes 4, 7 and 18) lost v i r a l genomes. The spontaneous loss of BPV DNA may be due to the stringent conditions during the subcloning procedure when only 100 cells were seeded into a culture dish. Figure 6 also shows that among 25 re-subclones containing BPV DNA, 22 had only episomal copies, 2 (lanes 3 and 6) contained both integrated and extrachromosomal BPV DNA, and 1 (lane 11) contained only integrated copies of BPV DNA. The BPV DNA was episomal in the majority of the re-subclones containing v i r a l genomes (96%, 24/25). The integration event happened only in a small percentage of the cells and the number of integrated BPV DNA copies is very low comparing to the episomal DNA copies. The significance of the 23 Figure 5. Southern blot analysis of DNAs from 22 subclones of B3. 2 fig DNA from each sample were used. A: BPV DNA copy equivalents were mixed with 2 fig of DNA from C127 c e l l s . B: DNAs from 22 subclones of B3 (BS1-22). Lambda Hind III size standards (kb) are shown at l e f t of panel. Arrow indicates integrated BPV DNA. 24 25 Figure 6. Southern blot analysis of DNAs from 28 subclones of BS9 (BSS1-28). 2 /ig DNA from each sample were used. Lambda Hind III size standards (kb) are shown at l e f t of panel. Arrows indicate integrated BPV DNA. 27 integrated BPV DNA with respect to the transformed phenotype is not clear. The morphological differences between "normal" C127 c e l l and transformed cells were examined under a microscope. Both C127 cells and B3 cells were seeded into a petri dish. The cultures were fixed and stained with 0.1% methylene blue at both subconfluent and confluent stages. The microphotographs (Figure 7) show that the morphology of C127 and transformed cells is star-shaped in both cases before they reach confluency. However, C127 cells stop dividing after they reach confluency, while B3 cells continue to divide and pile up. The piling-up cells are long, thin and stripe-shaped. 2. Inhibition of Cell Transformation by RA 2.1. Dose-dependent inhibition of c e l l transformation by RA Two days after BPV DNA transfection, various doses of RA were added to the culture medium for 3 weeks. Control petri dishes were not exposed to RA. Comparing the number of transformed foci of RA-treated dishes with that of controls (Figure 8), i t was found that BPV DNA-induced transformation was inhibited by RA in a dose-dependent manner (Figure - ft 9). At a concentration of 5 x 10" M, RA caused a complete inhibition of cellular transformation. In order to prove that the reduced number of transformed foci by RA is not due to the k i l l i n g of the c e l l s , the cloning capacity of C127 cells was tested in the absence and in the presence of RA. One hundred C127 cells were seeded into 100 mm diameter dishes and incubated in DMEM supplemented with 10% FBS containing various doses of RA. Cell cultures 28 Figure 7. Morphology of C127 cells and BPV-1 DNA-transformed B3 c e l l s . Both C127 and B3 cells were seeded into 60-mm petri dishes at a density of 5 x 10 cells per dish. Cell cultures were stained with 0.1% methylene blue after 24 hr (A and C) or 96 hr (B and D). A and B: C127 c e l l s . C and D: B3 c e l l s . A and C; subconfluent stage. B and D: confluent stage. 400x. 29 30 Figure 8. Inhibiti on of BPV-1 DNA-induced C127 c e l l transformation by RA. 0.84 fig of pdBPV-1 (142-6) was used to transfect 106 C127 c e l l s . Two days later the cells were subcultured at a ratio of 1:4. Then, the cells were cultured either in the absence (A) or in the presence (B) of RA (5 x 10" M). The c e l l cultures were stained in 14 days. 32 Figure 9. Effect of RA on the clone-forming capacity of C127 cells ( • ), and the development of transformed colonies following transfection with a BPV DNA-containing plasmid (pdBPV-1) (142-6). Two experiments were performed, in either tr i p l i c a t e ( • ) or duplicate ( A ) plates, for each dose of RA. The control plates without RA had an average of 64 foci per plate for the tri p l i c a t e experiment and 86 for the duplicate experiment. 33 34 were stained with 0.1% methylene blue 7 days later and colonies were counted. Figure 9 also shows that a dose of RA up to 1 x 10"^ M does not affect the cloning capacity of C127 c e l l s . 2.2. Stage-dependent responses to RA RA was added to C127 cultures at different intervals for different periods of time after DNA transfection. Inhibition of focus formation induced by RA is shown in Figure 10. RA was applied at a concentration of 5 x 10"^ M, which completely prevented the development of transformed colonies when administered for the entire study period (18 days). The results indicate that RA exerted a relatively small inhibitory effect during the f i r s t few days after subculture of the transfected c e l l s . A 3.5-day treatment period, starting on the day after the cells were subcultured (day 0), reduced the transformation frequency by only 10%. If RA treatment was initiated on the 4th day, the inhibition of focus formation was comparable to that of exposure to RA over the entire study period. The weak response of the early post-transfection period to the inhibitory action of RA was also evident upon comparison of the 7-day exposures. RA seems to exert i t s main inhibitory effect when cells reach confluency. The results also show that longer treatment produces stronger inhibition in any period during transformation (comparison of 3.5-day and 7-day treatment). 35 Figure 10. Effect of length and period of RA treatment on c e l l transformation. Mouse C127 cells at the 11th passage were transfected with BPV DNA. Two days later (day 0), the cells were subcultured at a ratio of 1:5 into medium containing 0.015% DMSO or medium containing DMSO plus 5 x 10" M RA. Media were changed twice weekly. Cells were fed with RA-containing medium for the indicated period of time. The average number of foci per plate was 27 in the absence of RA. 36 DAYS 8 10 12 14 16 18 INHIBITION RA J 1 1 1 1 1 1 1 1 °/o EXPOSURE COAYSJ 17 7 55 7 an 7 1 • 3-5 3Q 3-5 27 3-5 37 3. Effect of RA on Phenotype of Transformed Cells 3.1. Growth rate The effects of RA on the growth rate and saturation density were examined on BPV-1 DNA transformed B3 c e l l s . Cells were seeded into 60-mm petri dishes at a density of 1 x 10^ cells per dish, and incubated in 10% FBS DMEM containing 0.02% DMSO or RA dissolved in DMSO (RA, 5 x 10"6 M). Figure 11 shows that RA, at a concentration of 5 x 10"^ M, had l i t t l e effect on the growth rate of B3 c e l l s . However, the saturation density of transformed cells was reduced by 35% in the presence of RA. 3.2. Inhibition of foci forming capacity of transformed cells by RA The following experiments were designed to reveal whether RA can affect the behaviour of transformed c e l l s . Three c e l l lines of transformed C127 cells were used in this study (B3, B5, and B10). The experiment consisted of mixing approximately 100 cells from B3, B5 or B10 clones with 5 x 10^ normal non-transformed C127 c e l l s , seeding them into a petri dish, and exposing these mixed cultures to RA at doses ft ft ranging from 1 x 10" M to 5 x 10" M. In the absence of RA, the approximately 100 transformed cells mixed with normal cells formed multilayered, transformed foci (44 for B3, 42 for B5, and 76 for B10). The inhibitory effect of RA on the formation of transformed colonies by cells derived form B3, B5 and B10 clones is shown in Table 1. The results indicate that the phenotype of the transformed c e l l s , i.e.. the ab i l i t y to form foci of piled-up c e l l s , is suppressed by treatment with RA. 38 Figure 11. Effect of RA on the growth rate of transformed c e l l s . B3 cells at passage 16 were seeded into 60-mm petri dishes at a density of 1 x 10^ cells/dish, and incubated in 10% fetal bovine serum DMEM containing 0.02% DMSO or RA dissolved in DMSO (RA, 5 x 10"° M). Media were changed on the fourth day. Total c e l l counts for RA-treated and untreated cultures were carried out in a hemocytometer at specific time intervals. Data are derived from tri p l i c a t e dishes. Bars indicate standard error. 40 Table 1. Reduced capacity of cells from 3 transformed BPV DNA-containing clones (B3, B5 and BIO) to form colonies with transformed properties in the presence of retinoic acid Retinoic acid Inhibition of development of transformed colonies (%) (M) B3 B5 B10 0 0 0 0 2 x 10"7 29 24 45 5 x 10"7 56 57 55 1 x 10"6 70 64 79 5 x 10"6 100 100 100 41 4. Effect of RA on the Number of BPV DNA Copies 4.1. RA-induced reduction in the number of BPV DNA copies Experiments were designed to investigate the effect of RA on the number of extrachromosomal BPV DNA copies in transformed c e l l s . Transformed B3 c e l l s , which contained approximately 60 copies of BPV DNA per c e l l on average, were used. Throughout the entire study, the cells were subcultured at the time of confluency. Each time, 1 x 10^ cells were seeded into new 100-mm petri dishes. RA (5 x 10 M) was added to one series of subcultures, whereas a comparable series received no RA treatment. Total cellular DNA was extracted from untreated and RA-treated cells of each subculture, and cleaved with the restriction endonuclease Bam HI. Each episomal molecule of the pdBPV-1 (142-6) plasmid DNA was thus converted into 2 linear fragments. An 8-kb and 2.6-kb fragment corresponding to the cloned BPV-1 DNA and the pML2d plasmid DNA were detected by hybridization with 3 2P-labelled pdBPV-1 (142-6) plasmid DNA. In two separate experiments, continuous exposure to RA caused a gradual reduction in the number of BPV DNA copies. An example is shown in Figure 12. Estimates of BPV DNA copies following RA treatment are presented in Table 3. The average number of BPV DNA copies per c e l l was reduced to less than one after the cells had gone through 9 subcultures (about 27 population doublings). In untreated B3 c e l l cultures, the copy number of BPV DNA remained constant throughout a - 6 comparable number of passages. The B3 cells treated with RA (5 x 10" M) for 5 weeks were named B3-RA5. 42 Figure 12. Effect of RA on the copy numbers of BPV DNA. RA (5 x 10"° M) was applied to B3 cells for 9 passages (3 to 11). 1 /jg of DNA samples was digested with Bam HI and subjected to Southern hybridization. A: BPV DNA genome equivalents. B: DNA samples from untreated B3 c e l l s . C: DNA samples from c e l l cultures continuously exposed to RA (DNA bands in passage 11 are too faint to be detected in the photograph). Lambda Hind III size standards are shown at l e f t of panel. 8kb-G E N O M E C O P Y P A S S A G E N U M B E R E Q U I V A L E N T S U N T R E A T E D R A - T R E A T E D 100 50 20 10 5 2 3 4 5 6 7 8 9 10 4 5 6 7 8 9 10 11 ^ . ^ j - ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ 2-6 kb-A 44 Table 2. Estimates of the number of BPV DNA copies of B3 cells treated with RA for 5 weeks from densitometer scanning of autoradiography Treatment BPV DNA copy number in different c e l l passages 3 4 5 6 7 8 9 10 11 Untreated 58 50 70 72 73 72 55 48 ND* RA-treated ND 54 34 18 9 4 2 1 0.5 ND, not done. 45 4.2. Stability of low BPV DNA copy number The s t a b i l i t y of this low BPV DNA copy number was examined by subculturing the B3-RA5 cells for 10 passages in the absence of RA. The cells were subcultured before they reached confluency. BPV DNA copy numbers were estimated at each of the 10 passages with slot hybridization. The average BPV DNA copy number remained less than one copy per c e l l (Figure 13). It did not return to the original level of approximately 60 copies found in the original B3 c e l l l i n e . Figure 13 also shows that the BPV DNA copy number in transformed cells without RA treatment was constant over 19 passages examined. 4.3. Foci forming capacity of RA-treated B3 cells The capacity of the B3-RA5 cells to form transformed colonies was examined in two different experiments. B3-RA5 cells were mixed with "normal" (no RA treatment and no exposure to BPV DNA-containing plasmid) C127 c e l l s , and seeded into petri dishes. After 3 weeks, the number of transformed colonies was scored. This mixture of B3-RA5 and C127 cells was cultured in the absence of RA. The results show that the B3-RA5 c e l l population, which had an average of less than one BPV DNA copy per c e l l , did not form transformed foci (Figure 14). The experiment involved only 30, 60, 300, or 600 RA-treated c e l l s , respectively. Thus, the possibility could not be discarded that a tiny fraction of B3-RA5 c e l l population may have retained the capacity to form foci with a transformed phenotype. To resolve this issue, 1.5 x 10^ B3-RA5 cells were seeded in a 100-mm petri dish, and allowed to grow to confluency in the absence of RA. Three weeks thereafter, the number of transformed colonies was scored in 3 petri dishes, and was found to be 113, 120 and 46 Figure 13. Maintenance of low BPV DNA copy numbers following termination of retinoic acid treatment. • , no RA treatment; • , RA treatment (5 x 1 0 M ) . Arrow indicates termination of RA treatment. 47 48 Figure 14. Foci formation of B3 cells and RA-treated B3 c e l l s . • , B3 ce l l s . • , RA-treated B3 c e l l s . Approximately 50 100, 500, and 1000 B3 cells or RA-treated B3 cells were seeded with 10 C127 cells into 60-mm petri dishes. Cell cultures were stained after 20 days. For testing the actual surviving c e l l s , approximately 100 B3 cells or RA-treated B3 cells were seeded into 60-mm petri dishes. Cultures were stained after 8 days and colonies counted: B3 c e l l s , 38+2; RA-treated B3 c e l l s , 59+3. Data are derived from triplicate plates. 49 60-1 N U M B E R O F S U R V I V I N G C E L L S 50 123, respectively. The results indicate that approximately 1 in 13,000 B3 cells which received 5 x 1 0 M RA treatment for 5 weeks can form transformed colonies (Figure 15). From these transformed colonies, 24 were isolated and expanded into c e l l lines, designated RRC1 to RRC24 (After Removal of Retinoic acid, transformed Colonies formed). The number of BPV DNA copies was examined with slot blot hybridization (Figure 16): 12 colonies carried approximately 10-20 copies, 8 colonies had between 21 and 30 copies, and 4 colonies contained 31 to 40 copies. None of the colonies had less than 10 copies. The DNA samples were then subjected to Southern hybridization analysis in order to investigate whether integration of BPV DNA into the cellular genome was responsible for the phenomenon that 1 in 13,000 B3-RA5 cells s t i l l contained more than 10 copies of BPV DNA and retained a transformed phenotype. The results showed that BPV DNA remained episomal in a l l RR.C c e l l lines examined (Figure 17). Only 4 clones contained both episomal and integrated copies of BPV DNA (lanes 1, 9, 10 and 16), which also occurred in non-treated clones (Figures 5 and 6). The integrated BPV DNA presented in a very low number of copies. Therefore, integration events cannot account for the phenomenon that a tiny fraction of the cells escaped the RA-treatment. 5. Continuation of RA Treatment After 5-week treatment with RA, BPV DNA copy numbers in B3 cells decreased to less than one, and only 1 in 13,000 cells retained a transformed phenotype. The question must be raised whether the tiny fraction of cells which retained a transformed phenotype would be eliminated i f the transformed cells were exposed to RA for a longer 51 Figure 15. 1 in 13,000 B3 cells can form transformed foci after 5-week RA treatment. Transformed B3 cells were treated with RA (5 x 10"6 M) for 5 weeks. At the end of treatment, 1.5 x 10^ cells were seeded into a 100-mm petri dish and cultured in the absence of RA for 3 weeks. 52 53 Figure 16. BPV DNA copy numbers in 24 transformed colonies which developed after seeding 1.5 x 10 B3 cells treated with RA for 5 weeks, as measured by slot blot hybridization, as described in Materials and Methods. A and B: 5 fig of DNA from 24 different transformed colonies. C: 5 fig of DNA from C127 cells mixed with 0 to 100 copy equivalents of pdBPV-1 (142-6) DNA per c e l l . 54 100 50 20 10 5 0 55 Figure 17. Southern blot analysis of 24 transformed colonies which developed after seeding 1.5 x 10 B3 cells treated with RA for 5 weeks. 2 ng DNA from each sample were digested with restriction enzyme, and subjected to Southern blot hybridization, as described in Materials and Methods. Lambda Hind III size standards (kb) are shown at right of panel. 56 <? 3 C O O tN t> <5 ^ CN CN I I I I I I 57 period of time. B3-RA5 cells which had received RA treatment for 5 weeks were re-exposed to RA at 5 x 10" M for another 5 weeks. At the end of the treatment, 1 x 10^ cells were seeded in a 100-mm petri dish, and allowed to grow to confluency in the absence of RA. Three weeks later, the c e l l cultures were stained, and no transformed focus was observed (Figure 18a). DNA was extracted from the cells treated with RA for 10 weeks, and subjected to slot and Southern blot hybridization. The results showed that after 10-week treatment with RA the BPV DNA in the transformed cells was no longer detectable (Figure 18b,c). The morphology of the transformed cells treated with RA for 10 weeks was then compared to C127 cells and transformed cells to see whether a 10-week treatment with RA would reverse the morphology of the transformed c e l l s . Figure 19 shows that RA treatment for 10 weeks reversed the morphology of transformed cells from long, thin and stripe-shaped to normal. 58 Figure 18. Reversal of transformed phenotype and elimination of BPV DNA after 10-week RA treatment. Transformed B3 cells were treated with RA (5 x 10"^ M) for 10 weeks. After 10-week RA treatment, 1 x 10^ cells were seeded into a 100-mm petri dish, and cultured in the absence of RA for 3 weeks (A). DNA was extracted from the cells treated with RA for 10 weeks, and subjected to slot (B) and Southern (C) blot hybridization. 5 Hg DNA were used in either method. 100, 10, 0.5 are standard copies of BPV DNA. B3-RA, DNA from B3 cells treated with RA for 10 weeks. B3, DNA from B3 cells without RA treatment. 60 Figure 19. Morphology of transformed B3 cells treated with RA for 10 weeks. Cells were seeded into 60 mm petri dishes at a density of 5 x 10^ cells per plate. Cultures were stained with 0.1% methylene blue after 24 hr (A) or 96 hr (B). 400x. 61 62 DISCUSSION 1. BPV-1 DNA-Induced Mouse C127 Cell Transformation The results show that the transformation frequency is directly proportional to the amount of BPV DNA used in transfection (Figure 3). The results also reveal that BPV-1 DNA-transformed cells contain 20 to 100 copies of v i r a l genomes, which is comparable to 20-120 copies and 40-150 copies of BPV-1 DNA in transformed C127 cells reported by Law et a l . (1981) and Turek et a l . (1982), respectively, and 50-200 copies the numbers of 50-200 of HPV-1 v i r a l DNA in cultured epidermal keratinocytes observed by LaPorta and Taichman (1982). The copy number is stably inherited from generation to generation for at least 19 passages, or about 70 generations (Figure 13). However, the transformed B3 cells have a heterogeneous composition with respect to BPV DNA copy number (Figures 5 and 6). Some cells even spontaneously lost episomal BPV DNA molecules. Integrated copies of BPV DNA into cellular genomes were observed in a small percentage of subclones (Figures 5 and 6). The integrated BPV DNA present in a small number of copies and the integration sites appear to be random. The significance of the integrated copies in transformed cells remains unknown. The induction of papillomas of rabbit skin by Shope papillomavirus has been used as in vivo model to test the effect of retinoids on papillomavirus-induced carcinogenesis (McMichael, 1965; Ito, 1980). However, the use of BPV-1 DNA-induced mouse C127 c e l l transformation has the following advantages: (1) BPV-1 DNA-induced C127 c e l l transformation assay is highly reproducible, (2) The v i r a l DNA used for transfection can be easily propagated in the bacteria, (3) The time for 63 transformation assay is 2-3 weeks, while the induction of tumours by Shope papillomavirus in rabbit skin requires several months, and (4) The BPV DNA-transformed cells contain high copy number (usually 50-100) of v i r a l genomes, which ensures ease of detection of v i r a l DNA. Therefore, the system is eminently suitable for studying the effect of RA on maintenance of BPV DNA copy number. 2. Inhibition of Cell Transformation by Retinoids The results from this study reveal that RA is a potent inhibitor of transformation of mouse C127 cells induced by BPV-1 DNA. The degree of inhibition was dependent on the RA concentration, the time of i n i t i a t i o n of RA treatment and the duration of treatment. RA, at a concentration of - 6 j x 10"° M, caused complete inhibition of cellular transformation. Inhibition of transformation was most pronounced when RA treatment commenced during the second week after v i r a l DNA transfection (84%). RA seems to exert i t s main inhibitory effect when cells reach confluency. It appears that RA treatment of BPV-1 DNA-transfected C127 c e l l cultures resulted in a reversible inhibition of transformation, since earlier removal of RA resulted in a weaker inhibition of transformation (Figure 10, comparison of three 7-day RA treatments). The nature of the effects of retinoids on cellular transformation seems to be affected by many factors. The inhibition of RA on N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced rat tracheal epithelial c e l l transformation was irreversible (Mass et a l . , 1984; Fitzgerald et a l . , 1986), while the inhibition by retinyl acetate, retinol and retinaldehyde of 3-methylcholanthrene (MCA)- induced neoplastic transformation in C3H/10T1/2 cells was reversible (Merriman and Bertram, 64 1979). Retlnylidene dlmedone prevented mammary gland transformation caused by 7,12-dimethylbenz[a]anthracene (DMBA), benzo[a]pyrene and N-2-fluorenylacetamide (Dickens and Sorof, 1980). RA had an enhancing effect on the early stage and an inhibitory effect on the later stage of Syrian hamster embryo c e l l transformation induced by BP (Rivedal and Sanner, 1985) . RA enhanced mouse epidermal c e l l transformation by DMBA, MNNG and MCA (Kulesz-Martin et a l . . 1986). Therefore, i t should be concluded that the effect of retinoids on c e l l transformation depends on the c e l l system, the carcinogens or v i r a l DNA used for inducing transformation, and the stage of transformation on which the retinoid is applied. The differences may reflect alternative mechanisms responsible for the carcinogenic processes by different agents in various c e l l systems. 3. Effect of RA on Transformed Cells RA had l i t t l e effect on the growth rate of transformed B3 c e l l s . However the saturation density of B3 cells decreased by 35% in the presence of RA, at a concentration of 5 x 10"^ M. Density-dependent growth inhibition by retinoids was found to be accompanied by changes in glycolipid and glycoprotein synthesis, and i t has been suggested that retinoids may induce some membrane-mediated growth control involving glycolipids and glycoproteins (Linder et a l . , 1981) . In addition, the gene expression of proteins of cytoskeleton and extracellular matrix, such as keratins, collagen, collagenase, transglutaminase and laminin, have been found to be regulated by RA (Strickland and Mahdavi, 1979; Strickland et a l . . 1980; Brinckerhoff and Harris, 1981; Fuchs and Green, 1981; G i l f i x and Eckert, 1985; Oikarinen et a l . . 1985). 65 It must be noted that the morphology of transformed cells at subconfluency and confluency is different (Figure 7). RA had no inhibitory effect on the growth of transformed cells at the subconfluent stage (Figure 11), while specifically inhibiting transformed cells from pi l i n g up (Table 1). It appears that the genes responsible for the transformed phenotype, which enable the cells to pi l e up and which are suppressed by RA, are expressed only when the cells reach confluency. After a 5-week treatment with RA, the BPV DNA copy number decreased from 60 to less than 1 copy on average per c e l l (Figure 12, Table 2). It is most l i k e l y that the reduction in the number of BPV DNA copies is due to the inhibition by RA of the replication of BPV molecules rather than to a preferential k i l l i n g of cells harboring high copy numbers of BPV DNA, since about 70% of transformed cells contained more than 40 copies of BPV DNA (Figure 5), and RA had no effect on the growth rate of the transformed cells (Figure 11). The observation that RA reduced BPV DNA copy number in transformed cells is in agreement with the fact that RA inhibited polyomavirus replication (Russell and Blalock, 1985) and that RA decreased HPV-3 and HPV-5 v i r a l DNA by 100- to 1500-fold in the lesions of epidermodysplasia verruciformis (Lutzner et a l . . 1981; Gross et a l . . 1983; Lu, 1985). The reduction of BPV DNA copy number by RA is irreversible since the BPV DNA copy numbers of RA-treated cells do not return to the original level after 10 passages, or approximately 40 divisions (Figure 13), and the subclones of RA-treated B3 cells do not contain high copy numbers of BPV DNA. Similarly, a complete loss of BPV DNA copies from transformed mouse C127 cells and reversal of transformed properties was obtained by prolonged treatment with interferon (Turek et a l . , 1982). 66 However, the reduction of BPV DNA copies of RA-exposed cells starts earlier and progresses more quickly, as compared with the c e l l cultures treated with interferon. The majority of the transformed cells which had been treated with RA for 5 weeks and contained less than one copy of BPV DNA per c e l l cannot form transformed foci after removal of RA (Figure 14). However, after a 5-week treatment with RA, 1 in 13,000 cells s t i l l retained a transformed phenotype and a l l these transformed colonies contained 10-40 copies of BPV DNA genomes (Figure 16). Therefore, a certain minimum number of BPV DNA copies apparently is necessary to maintain a transformed phenotype. The factors that cause different responses of transformed cells to RA remain unclear. The possibility of the integration was eliminated by the fact that a l l these transformed colonies contained extrachromosomal BPV DNA while integrated BPV DNA only present in 12% of the clones and the number of the integrated BPV copies was low (Figure 17). The tiny fraction of c e l l population was not resistant to RA since after additional 5-week treatment with RA these cells were eliminated from the cultures (Figure 18). 4. Response of HPV-Carrying Human Lesions to Retinoids Retinoids have been successfully applied in the treatment of HPV-containing preneoplastic lesions. For example, i t has been reported that the patients with epidermodysplasia verruciformis induced by HPV-3, HPV-5, HPV-8 or HPV-17 improved markedly after treatment with Ro 10-9359 (Tigason) for 1 to 2 months (Edelson et a l . . 1981; Jablonska et a l . . 1981; Lutzner et_ a l . , 1981; Jablonska e t _ a l . , 1982; Lutzner et a l . , 1984; van Voorst Vader et a l . . 1987). Treatment with Tigason for 2 67 months caused a 100 fold reduction in HPV-5 v i r a l DNA in the lesions of epidermodysplasia verruciformis (Lutzner et a l . , 1981). Treatment of HPV-2-induced common warts with Ro 10-9359 also resulted rapid improvement (Gross et a l . , 1983). However, the reappearance of pre-neoplastic lesions on cessation of treatment with retinoids is an issue of considerable concern. Oral leukoplakias which contain human papilloma DNA (Maitland et a l . , 1987) regressed only temporarily during the treatment period (Koch, 1981; Hong et a l . , 1986). The inhibitory effect of etretinate (Tigason) on epidermodysplasia verruciformis, was also reversed on removal of the chemopreventive agent (Jablonska et a l . . 1981; Lutzner et a l . . 1984). Similarly, papillomas induced by Shope papillomavirus in rabbits showed atrophy following vitamin A injections, but grew again when treatment stopped (McMichael, 1965). Gross et a l . (1983) reported that the treatment of HPV-2-induced warts with aromatic retinoid Ro 10-9359 (Tigason) for 12 weeks improved the lesions and reduced v i r a l DNA to undetectable le v e l , and discontinuation of therapy led to a complete relapse of the cutaneous lesions and the same type of virus genomes were again detected 10 weeks after therapy. The question must be raised whether the reappearance of the lesions was due to too short intervention periods. A prolonged exposure to retinoids may result in a complete loss of v i r a l DNA in what would amount to a "cure". The BPV DNA transformation system may contribute to the interpretation of the recurrance phenomenon. Although a 5-week treatment with RA reduced BPV DNA copy number to less than one per c e l l on average, s t i l l 1 in 13,000 cells contain more than 10 copies of BPV 68 DNA and retain transformed phenotype. By analogy, in the c l i n i c a l t r i a l by Gross et a l . . (1983), there may have a small fraction of cells which contained high copy of HPV-2 genome after 12-week treatment with Tigason, and the presence of these cells caused a complete relapse of the cutaneous lesions. After the RA treatment of the BPV-1 DNA-containing cells is extended to 10 weeks, the BPV DNA appears to be completely eliminated from the c e l l s . The BPV DNA free cells do not express a transformed phenotype (Figure 18). These results indicate that a retinoid treatment should be maintained long enough to eliminate the v i r a l DNA in a l l the c e l l s . 5. Mechanism of RA Action: An Unresolved Issue RA inhibits BPV-1 DNA replication in C127 cells and inhibits the transformed phenotype. The questions concerning the mechanisms of action of RA are whether inhibition of transformed phenotype is due to inhibition of BPV DNA replication, and the importance of BPV-1 DNA copy number in maintaining the transformed phenotype. The results show that the reduction of BPV DNA copy number to less than one by RA required a 5-week treatment (Figure 12), while RA inhibited BPV-1 DNA-induced c e l l transformation in (Figure 10) and inhibited the transformed cells from expressing a transformed phenotype (Table 1) in two weeks. These results indicate that the reduction of BPV DNA copy number is not a prerequisite for the inhibition of the transformed phenotype. RA must have other mechanism(s) to suppress the transformed phenotype. As discussed in several recent reviews (Lotan, 1980; Sporn and Roberts, 1983; Goodman, 1984; Lippman et a l . . 1987), the major 69 biological effects include regulation of enzyme synthesis, membrane function, growth factors, binding proteins, genomic and postgenomic expression, extracellular effects, immunological ac t i v i t y , and the PK-C cascade system. The effect of RA on gene expression has been of great interest towards the mechanisms of i t s action in differentiation and carcinogenesis. Retinoids control the expression of many proteins which are either direct constituents of the cytoskeleton and extracellular matrix or participate in the formation of cytoskeleton and matrix. Retinoids also modulate the effects of many mitogens and transforming growth factors which affect the c e l l membrane, such as epidermal growth factor (EGF), platelet-derived growth factor (PDGF), colony-stimulating factor (CSF), sarcoma growth factor derived from the Moloney murine sarcoma virus - transformed 3T3 c e l l s , and other tumour promoters and hormones (reviewed by Lippman et a l . , 1987) . Retinoids may also antagonize the action of oncogenes or their products, which are normally genetically repressed polypeptides that change the regulated growth pattern of normal cells to a more malignant pattern, since i t has been shown that retinoic acid modifies c-myc gene transcription (Amatruda et a l . , 1985; Thiele et a l . . 1985; Griep and DeLuca, 1986; Dean et a l . . 1986) . However, this study focuses on the inhibitory effect of RA on the transformation of cultured cells and the number of BPV DNA copies, and on the reve r s i b i l i t y of the RA inhibition. This study did not tackle the question of expression of BPV DNA genes in transformed cells and the effect of RA on this phenomenon. It could be expected that the inhibition of the transformed phenotype by RA is due to the inhibition on BPV gene expression. During treatment with RA, the expression of BPV 70 genes is suppressed, and thus the transformed phenotype of the cells is inhibited. In the meantime, RA inhibits BPV DNA replication and reduces the copy number of the v i r a l genome. However, i f the treatment is not long enough to eliminate the v i r a l DNA, the v i r a l genes w i l l be expressed after withdrawal of RA, and lead to the expression of the transformed phenotype of the c e l l . In future studies, answers to the following questions should be sought. Will retinoids inhibit the expression of BPV sequences? How quickly does a change in gene expression occur after treatment with retinoids? Is the effect of retinoids reversible upon withdrawal of the treatment? 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