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Biochemical studies of chloroleukemia in female Sprague-Dawley rats Lee, Lilian Miu Yee 1965

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BIOCHEMICAL STUDIES OF CHLOROLEUKEMIA IN FEMALE SPRAGUE-DAWLEY RATS by L i l i a n Miu Yee Lee B.Sc. M c G i l l University, 1963 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of Biochemistry We accept t h i s thesis as conforming to required standard THE UNIVERSITY OF BRITISH COLUMBIA September, 1965. In presenting th i s thes i s in p a r t i a l f u l f i lment of the requirements for an advanced degree at the Un ivers i ty of B r i t i s h Columbia, I agree that the L ibrary sha l l make i t f r ee l y a v a i l a b l e fo r reference and study. I fur ther agree that per -mission for extensive copying of th i s thes i s for scho la r l y purposes may be granted by the Head of my Department or by h i s representatives., It i s understood that copying or p u b l i -ca t ion of th i s thes i s for f i n a n c i a l gain sha l l not be allowed without my wr i t ten permiss ion. Department of The Un ivers i ty of B r i t i s h Columbia Vancouver 8, Canada Date ABSTRACT The chloroleukemia (chloroma) i n the Cancer Research Centre, U.B.C. could be transplanted into adult Sprague-Dawley rats with tumour homogenates, whole blood and a s c i t i c f l u i d containing i n t a c t c e l l s . C e l l - f r e e f i l t r a t e s of the a s c i t i c f l u i d did not give r i s e to tumours. I t was considered probable that i n t a c t c e l l s were required for the transmission of this leukemia. The present study showed that there was a 'latent period' of 10 to 12 days after which the chloroma grew r a p i d l y u n t i l the death of the animal (ca 27 days). The tumour contained substantial amounts of free protoporphyrin and had high myeloperoxidase a c t i v i t y . The protoporphyrin concentration increased markedly and continuously with the age of the tumour but i t s r a t e of synthesis (from glycine-2-C^) declined after the 19th day, i.e..protoporphyrin accumulated i n the tumour. The myeloperoxidase a c t i v i t y increased between the 13th and 15th day afte r transplantation and thereafter f e l l sharply. The e f f e c t of two drugs - Vinblastine (VLB) and 3,5-dicar-bethoxy-1,4-dihydrocollidine (DDC) on certain aspects of the biosynthetic a c t i v i t y of this p a r t i c u l a r adult chloroma was investigated. VLB i n h i b i t e d the incorporation of g l y c i n e - 2 - C ^ into both RNA and protein e a r l i e r and to a greater extent than into the DNA and protoporphyrin. This tumour, although of hematopoietic o r i g i n , did not therefore show the high s e n s i t i -v i t y of normal bone marrow to VLB. DDC markedly increased the A -aminolevulinic acid synthetase a c t i v i t y and protopor-phyrin concentration i n the l i v e r of rats but had no e f f e c t on the tumour protoporphyrin synthesis although there was more than a 20-fold increase i n A -aminolevulinic a c i d synthetase a c t i v i t y . I t was suggested therefore that the enzyme A-aminolevulinic acid synthetase may be r a t e - l i m i t i n g i n the l i v e r but not i n the tumour. In the course of the present work, an improved method for the separation of porphyrin esters based on p a r t i t i o n chromatography on alumina was developed. TABLE OF CONTENTS Page INTRODUCTION 1 Chloroleukemia 1 Properties and biosynthesis of porphyrins 3 Chemical porphyria 6 Present inves t i g a t i o n 8 EXPERIMENTAL 12 I Material 12 II Methods 14 (1) Tumour transplantation 14 (2) U l t r a v i o l e t spectroscopy 14 (3) Measurement of r a d i o a c t i v i t y 14 (4) Estimation of porphyrins 15 (5) Extraction of porphyrins from chloromas and l i v e r s 16 (6) I d e n t i f i c a t i o n of the porphyrins 19 A) I d e n t i f i c a t i o n of free porphyrins.... 19 (i) Paper chromatography 20 ( i i ) Paper electrophoresis 21 ( i i i ) Column chromatography on t a l c . . . 21 B) I d e n t i f i c a t i o n of the porphyrin esters 22 (i) Column chromatography on alumina 22 (7) Extraction of nucleic acids from the chloroma 26 (8) The incorporation of glycine-2-C-1-^ into chloroma protein 27 (9) Extraction and estimation of myeloperoxi-dase from the chloroma....... 28 (i) Extraction of myeloperoxidase from the tumour 28 ( i i ) Estimation of myeloperoxidase a c t i v i t y 29 v. Page (10) Estimation of urinary porphobilinogen.... 30 (11) Estimation of ^-aminolevulinic a c i d synthetase a c t i v i t y i n the l i v e r and the tumour 30 (a) Assay of ^ - a m i n o l e v u l i n i c a c i d synthetase 31 (b) Estimation of A -aminolevulinic acid 32 RESULTS . 34 I Transplantation..... 34 II The r e l a t i o n s h i p of tumour growth, protoporphyrin concentration and myeloperoxidase a c t i v i t y of the chloroma 37 III Incorporation of g l y c i n e - 2 - 0 ^ into the proto-porphyrin and the nucle i c acids of the chloroma 40 (1) I s o l a t i o n of radioactive protoporphyrin... 40 (2) E f f e c t of age of tumour on the rate of protoporphyrin and nucleic acid synthesis by the chloroma i n vivo 42 IV E f f e c t of v i n b l a s t i n e on the biosyntheis of protoporphyrin, n u c l e i c acids and protein of the chloroma 42 (1) Time course of g l y c i n e - 2 - C ^ incorporation into chloroma protoporphyrin, n u c l e i c acids and proteins 43 (2) E f f e c t of VLB on glycine-2-Cl^ incorporation into the protoporphyrin, n u c l e i c acids and pi?otein of the chloroma 44 V E f f e c t of 3,5-dicarbethoxy-l,4-dihydrocollidine (DDC) on the biosynthesis of porphyrin i n the l i v e r and the chloroma 45 (1) Conditions a f f e c t i n g the stimulation of porphyrin synthesis with DDC i n the l i v e r of tumour-free r a t s i n vivo 46 VI-Page (i) E f f e c t of route of administration of DDC '.. . 46 ( i i ) Protoporphyrin concentration and i t s rate of synthesis i n l i v e r s of tumour-free rat s receiving DDC by stomach tube 47 (2) E f f e c t of DDC on porphyrin synthesis i n the l i v e r and the tumour of chloroleukemic r a t s 47 (3) E f f e c t of DDC on the induction of ^ - a m i n o l e v u l i n i c a c i d synthetase i n r a t s 49 DISCUSSION . .. 52 SUMMARY 61 BIBLIOGRAPHY 63 VH TABLES Page l a E x t i n c t i o n c o e f f i c i e n t s of porphyrins i n hydrochloric acid solution (to face) 16 l b E x t i n c t i o n c o e f f i c i e n t s of porphyrin methyl esters i n chloroform...... ...(to face) 16 2 Development of chloroleukemia and s u r v i v a l time i n 10 generations of Sprague-Dawley r a t s inoculated i n t r a p e r i t o n e a l l y with 0.2 ml of chloroma homogenate (to face) 34 3 T r a n s p l a n t a b i l i t y of chloroleukemia (to face) 36 4 The tumour weight, protoporphyrin concentration and myeloperoxidase a c t i v i t y of the chloroma on d i f f e r e n t days afte r transplantation (to face) 37 5 E f f e c t of age of tumour on g l y c i n e - 2 - C ^ incorporation into the protoporphyrin and nucleic acids of r a t chloroma (to face) 42 6 Time course of glycine-2-G"^ incorporation into chloroma protoporphyrin, nucleic acids and protein...(to face) 43 7 The e f f e c t of v i n b l a s t i n e on the incorporation of glycine-2 -c!4 into the protoporphyrin, nucleic acids and protein of the chloroma (to face) 44 8 E f f e c t of route of DDC administration on protoporphyrin concentration i n the l i v e r s of tumour-free rats (to face) 46 9 E f f e c t of DDC on the concentration of protoporphyrin and rate of protoporphyrin synthesis from glycine-2-C-^ i n l i v e r s of tumour-free rat s (to face) 47 10 The e f f e c t of DDC on the urinary excretion of porphobilinogen (PBG) i n tumour-free and chloro-leukemic rat s (to face) 48 11 The e f f e c t of DDC on protoporphyrin accumulation i n tumour-free and chloroleukemic rats (to face) 49 12 The induction of A -aminolevulinic acid synthetase by DDC i n tumour-free and chloroleukemic r a t s . . ( t o face) 50 1/ i i i FIGURES Page 1 Structure of some common porphyrins and their precursors and the iron complexes of protoporphyrin (to face) 3 2 The biosynthetic pathways of porphyrin (to face) 4 3 Source of carbon and nitrogen atoms derived from glycine i n protoporphyrin and purine nucleus...(to face) 11 4a Absorption spectra of uro-, copro- and protoporphyrin i n HC1 (to face) 15 4b Absorption spectra of coproporphyrin tetramethyl ester and protoporphyrin dimethyl ester i n ethylene di c h l o r i d e : n-hexane mixture .(to face) 15 5 Extraction of porphyrins from the tumour (to face) 17 6 Chromatography of protoporphyrin dimethyl ester on A1 20 3 ..(to face) 24 7 Re-chromatography of fractions 35 to 38 on A1 20 3 (to face) 24 8 Extraction of nucleic acids and protein from the chloroma (to face) 26 9 Post-mortem photographs of tumour-free and chloroleu-kemic Sprague-Dawley rats 35 10 Tumour weight and the amount of protoporphyrin i n chloroleukemic r a t s on d i f f e r e n t days after transplantation (to face) 38 11 Myeloperoxidase a c t i v i t y i n chloromas of d i f f e r e n t age 38 12 Chromatography of chloroma protoporphyrin on alumina i n dim l i g h t (to face) 40 13 Chromatography of chloroma protoporphyrin on alumina i n normal l i g h t i n g of the laboratory...(to face) 40 Figures (cont'd) Page 14 Absorption spectrum of material i n the 'secondary' peak (Fraction 30, F i g 13) ..('to face) 41 15 E f f e c t of VLB on the incorporation of glycine-into the protoporphyrin, nucleic acids and protein of chloroma (to face) 45 X ACKNOWLEDGEMENTS The author wishes to express her sincere gratitude to Dr. C.T. Beer for his supervision, encouragement and c r i t i c i s m during t h i s work. The author i s also indebted to Dr. J.F. Richards for his h e l p f u l guidance i n some parts of thi s work and to Dr. R.L. Noble for the use of the Cancer Research Centre laboratory f a c i l i t i e s . The author i s gr a t e f u l for the technical assistance of Miss M. Pearce. The f i n a n c i a l assistance of the Medical Research Council of Canada i s g r a t e f u l l y acknowledged. -1-INTRODUCTION Chloroleukemia: Chloroma or chloroleukemia has been known for over 100 years i n man (1,2). I t i s characterized by a leukemic blood picture and l o c a l tumours of a yellowish-green colour i n various s i t e s within the body cavity. Under u l t r a v i o l e t l i g h t , the tumour masses of the chloroma show a red fluorescence. I t i s now known that chloroleukemia i s derived from hematopoietic tis s u e , probably the bone marrow. Chloroleukemia i n the Sprague-Dawley r a t was i n i t i a t e d by the intravenous i n j e c t i o n of actinium-227 by Cowden et a l (3), and i t s f i r s t successful transplantation was reported by Zipf et a l (4). The i n j e c t i o n of whole blood, a s c i t i c f l u i d or tissue homogenates from leukemic rat s a l l produced successful transplants, although of d i f f e r e n t e f f i c i e n c i e s , i n weanling r a t s . Ninety-five per cent of the ra t s showed p o s i t i v e 'takes' of the tumour and the average s u r v i v a l time of these animals was 42 days. Morphologically and anatomically, the tumour resembled the Shay chloroleukemia which developed i n the Wistar r a t afte r the g a s t r i c i n s t i l l a t i o n of 20-methylcholanthrene (5). The spontaneous occurrence of chloroleukemia i n Sprague-Dawley rats has not been reported although i t has been observed i n the Wistar s t r a i n (6). I t i s well established that chloroleukemia may be produced i n r a t s by the inoculation of c e l l - f r e e f i l t r a t -es obtained from animals with leukemia or even from tumours of -2-non-hematopoietic o r i g i n (7). This strongly suggests a v i r a l o r i g i n of the tumour. Chloroleukemia i n the r a t c l o s e l y resembles the human chloroleukemia (8). The biochemical properties of the human chloroma have been studied by several workers i n the l a s t two decades (1,2,9,10). Due to the l i m i t e d amounts of c l i n i c a l material a v a i l a b l e and the shortcomings of quantitative methods, c o n f l i c t i n g data have been reported. Agner (9) suggested that the green colour of the tumour was due to myeloperoxidase (formerly c a l l e d verdoperoxidase) while others i n s i s t e d that choleglobin was responsible for the colour (1,10). Thomas suggested a protoporphyrin-protein complex was responsible for both the green colour and the red fluorescence (2). However, Humble (1) could not f i n d porphyrins i n the tumour i n spite of i t s red fluorescence. Recently, Schultz and his associates have shown that the red fluorescence i n the chloroma of the r a t i s due, at least i n part, to a porphyrin that appears to be a dicarboxylic a c i d and which has the s o l u b i l i t y and spectrophotometric absorption c h a r a c t e r i s t i c of protoporphyrin (11,12). They claimed to have i s o l a t e d pure protoporphyrin from the tumour. A green pigment, s l i g h t l y soluble i n saline but insoluble i n 65% to 707o alcohol, has been i s o l a t e d from the chloroma and was shown to have a high peroxidase a c t i v i t y with an absorption spectrum c l o s e l y resembling that of myeloperoxidase (11,13,14,15). The prosthe-FIGURE 1 Structure of some common porphyrins and their precursors and the iron complexes of protoporphyrin. M COOH I C H , I 2 C H , I 2 CO I H 2 C M H 2 ^ - A m i n o l e v u l i n i c a c i d (b) (c) COOH C H , COOH I 2 I CH„ C H H P o r p h o b i l i n o g e n ? P U r o p o r p h y r i n o g e n H I t\ = M e t h y l ; V - V i n y l ; ? * P r o p i o n y l ', A *> -3-t i c group of this enzyme i s covalently bound and appears to contain an iron chelated porphyrin (9) or a porphyrin degra-dation product similar to that found i n choleglobin (16). The high protoporphyrin content of the chloroma may be the r e s u l t of a " l e s i o n " i n the malignant c e l l s of the "heme" biosynthetic pathway normally present i n the hematopoietic tissue from which the tumour originates. Properties and biosynthesis of porphyrins: The porphyrins are macroeyelie compounds formed by the linkage of four pyrrole rings through methene (=CH-) bridges. They are d i f f e r e n t i a t e d s t r u c t u r a l l y by the nature and the arrangement of the sidechains on the pyrrole r i n g . The p o s i -tions of the substituents r e l a t i v e to each other give r i s e to the d i f f e r e n t isomeric configurations, the chemical structures of which have been established lar g e l y by Fischer (17). The three free porphyrins commonly found i n nature are uroporphyrin I I I , coproporphyrin III and protoporphyrin IX ( f i g 1 d,e,f) a l l of which are r e l a t e d to the " I I I " s e r i e s . Although the alterna-ting single and double bonds of the porphyrin provide a resonating structure, i t i s important to point out that the v i n y l sidechains of protoporphyrin are e a s i l y oxidized by oxygen to formyl group i n the presence of l i g h t (18). Porphyrins r e a d i l y combine with various metals to form FIGURE 2 The biosynthetic pathways of porphyrin Glyc ine + S u c c i n y l CoA Pyr idoxa l phosphate (1) A - a m i n o l e v u l i n i c a c i d (A-ALA) (2) Porphobi l inogen (PBG) (3) Type I isomers The enzymes for c a t a l y z i n g the above reac t i ons a r e : -( 1 ) A - A m i n o l e v u l i n i c a c i d synthetase (A -ALA synthetase) (2) A - A m i n o l e v u l i n i c dehydrase (3) Porphobi l inogen deaminase (4) Uroporphyrinogen isomerase (5) Fe r roche la ta se J(3)(4) Uroporphyrinogen III i Uroporphyrin III Coproporphyrinogen III • Coproporphyrin I I I -» Prqtoporphyr inogen 1 / Protoporphyr in IX I Fe-"-(5) Heme - 4 -chelates i n which the metal i s co-ordinatively bound to the nitrogen atoms but only those with i r o n and magnesium have important b i o l o g i c a l r o l e s . Chelation occurs both with free porphyrins and with porphyrin esters. The metal complexes are soluble i n organic solvents. The iron complexes are known c o l l e c t i v e l y as "hemes" of which the commonest i s iron-proto-porphyrin i n which the i r o n may be i n the ferrous or f e r r i c state ( f i g 1 g , h j i ) . In common prac t i c e , the terms "heme" and "hematin" r e f e r to ferrous or f e r r i c protoporphyrin r e s p e c t i v e l y . Heme i s the prosthetic group of hemoglobin, the cytochromes and a number of enzymes such as catalase, peroxidase and tryptophan pyrrolase. A i r r e a d i l y oxidizes heme to hematin or, i f chloride ions are present, to hemin ( f e r r i c protoporphyrin chloride) ( f i g 1 h , i ) . Present knowledge concerning the biosynthesis of porphyrins i s outlined i n figure 2. Glycine combines with succinyl CoA to form es-amino-p-keto ad i p i c a c i d which undergoes decarboxylation to produce A-aminolevulinic a c i d (A-ALA) ( f i g 1 a ) . This reaction proceeds i n mitochondria which supply the succinyl CoA by way of the Krebs cycle, and i s catalysed by the enzyme ^- a m i n o l e v u l i n i c a c i d synthetase (A-ALA synthetase). The enzyme seems to be rather l a b i l e and i s protected to some extent by mercaptoethanol and chelators (19). The precise mechanism of the reaction has not yet been elucidated. I t appears l i k e l y -5-that the reaction takes place by a concerted condensation-decarboxylation reaction rather than v i a the formation of free g-amino-p-keto adipic a c i d as an intermediate (20). The next step i n porphyrin biosynthesis involves the condensation of two molecules of A-ALA to form the pyrrole intermediate porphobilinogen (PBG) ( f i g 1 b). This reaction i s catalysed by the enzyme / I -aminolevulinic a c i d dehydrase (also known as ^ - a m i n o l e v u l i n i c a c i d hydrolyase) and i t i s present i n large amounts i n the l i v e r , kidney and the bone marrow. The reaction involves the addition of one molecule of A-ALA to a second molecule, followed by dehydration and c y c l i z a t i o n . Four PBG molecules are then linked together by methylene (-CH2-) bridges to form the colourless tetrapyrrole uroporphyr-inogen ( f i g 1 c ) . The porphyrinogens are reduced porphyrins containing s i x extra hydrogen atoms as compared to the corres-ponding porphyrins. Bogorad (21) was able to separate two factors concerned i n the enzymic synthesis of uroporphyrinogen I I I . The f i r s t was a r e l a t i v e l y stable enzyme, porphobilinogen deaminase, which i f present alone converted PBG to uroporphyr-inogen I with the elimination of four molecules of ammonia. The second factor, uroporphyrinogen isomerase, had no action on either PBG or uroporphyrinogen I but i f present together with the deaminase, i t modified the action of the l a t t e r so that uroporphyrinogen III res u l t e d ( f i g 2). Decarboxylation of uroporphyrinogen III gives r i s e to copr©porphyrinogen I I I . The -6-uro- and coproporphyrins are formed when the methylene bridges of the corresponding porphyrinogens are oxidized. Between coproporphyrinogen and protoporphyrin, there occurs not only a decarboxylation but also a desaturation step, two vinyl groups ultimately taking the place of two propionic acid residues. The mechanism of this transformation has not been elucidated at present. The introduction of ferrous iron into the protoporphy-r i n to form heme is catalysed by the enzyme ferrochelatase. Chemical porphyria Free porphyrins have no known biological functions and they accumulate as by-products of the normal biosynthetic pathway leading to the hemes (fig 1 g). Under normal circum-stances only small amounts of free porphyrins occur in tissues. The porphyria diseases are characterized by the excretion of large amounts of porphyrins or precursors in the urine. In general there are two types of porphyria: (i) "Erythropoietic' porphyria", (so named because the bone marrow appears to be the site of metabolic error) in which there is a deposition of porphyrins in the skin, bones and other tissues, and ( i i ) the more common "hepatic" type ("acute porphyria" and "porphyria cutanea tarda") in which there is an abnormal and excessive production of porphyrins and their precursors in the l i v e r . "Acute porphyria" i s characterized by the excretion of large -7-quantities of porphobilinogen and ^ - a m i n o l e v u l i n i c acid i n the urine whereas i n "porphyria cutanea tarda", high concentra-tions of. uro- and coproporphyria are found i n both the urine and feces. . "Acute porphyria" may be inherited as a Mendelian t r a i t (22-25). The disease can also be induced a r t i f i c i a l l y by cer t a i n chemicals, and for t h i s reason, the term "chemical porphyria" i s assigned to the drug induced-porphyria producing symptoms which mimic those of hepatic porphyria. Chemically induced changes i n porphyrin metabolism were f i r s t described by Schmid and Schwartz (26) who fed "sedormid" (allyl-isopropyl-acetyl-urea) to r a b b i t s . Labbe (27) obtained similar e f f e c t s with the chemically r e l a t e d compound allylisopropylacetamide. Certain barbiturates (28), sulfonal (29), hexachlorobenzene (30) and 3,5-dicarbethpxy-l,4-dihydrocollidine (DDC) (31,32) have also been shown to cause experimental porphyria i n animals. The action of 3,5-carbethoxy-l,4-dihydrocollidine (DDC) on the l i v e r was f i r s t studied by Solomon and Figge (31,32) who noticed that the administration of t h i s drug to mice and guinea pigs caused an increase of l i v e r copro- and protoporphyrins and the appearance of uro- and protoporphyrin i n the urine. Granick (33) and Urata (34) found a s i g n i f i c a n t increase i n the a c t i v i t y of the l i v e r mitochondrial enzyme, ^ - a m i n o l e v u l i n i c a c i d synthetase, when guinea pigs were fed DDC. The concentra--8-t i o n of ^.-aminolevulinic a c i d was fo r t y f o l d that of the normal l i v e r . In a recent paper, Granick (35) suggested that DDC may act by tampering with a repressor control mechanism of the porphyrin biosynthetic chain i n the l i v e r : Regulator gene Operator Str u c t u r a l gene gene DDC Aporepressor Heme /\ -ALA synthetase The i n h i b i t i o n of the operator gene i s l i f t e d by porphyria-inducing drugs which act by in t e r a c t i n g with heme or prevent heme from combining with the aporepressor to form the repressor, Under th i s condition, the s t r u c t u r a l gene i s now avail a b l e for the production of /± -aminolevulinic a c i d synthetase. An a l t e r -native i n t e r p r e t a t i o n of the action of DDC was given by Labbe (36) who proposed that these drugs act by i n h i b i t i n g the formation of heme from Fe++ and protoporphyrin by s p e c i f i c a l l y i n h i b i t i n g the ferro-protoporphyrin chelatase. At present the explanation for the increase of £-aminolevulinic a c i d synthe-tase a c t i v i t y i s s t i l l open to question. Present i n v e s t i g a t i o n : The induction of a transplantable chloroleukemia i n the -9-Sprague-Dawley r a t s by Zipf et a l (4) i n weanling rat s made possible controlled studies of the chloroma. Although the chloroleukemic l i n e i n Sprague-Dawley rats i n our laboratory was obtained from Zipf, the tumour became changed (mutated) so t h a t - i t now can be r e a d i l y transplanted into ajdult_ Sprague-Dawley r a t s . I t gives r i s e to large tumour masses and grows much more r a p i d l y than the o r i g i n a l l i n e . Hence, i t appeared to be convenient for biochemical investigations of tumours r e l a t e d to the hematopoietic ti s s u e s . The anti-leukemic Vinca a l k a l o i d - Vinblastine (VLB) depresses the r a t bone marrow and causes a marked decrease i n the number of c i r c u l a t i n g leucocytes (37). Noble (38) reported that chloroleukemia i n the Sprague-Dawley r a t s i s very sens i t i v e to VLB treatment. I f rat s were treated with VLB over the period of 1-13 days after transplant of the tumour, t o t a l l y 'cured' rats could be r e a d i l y produced; i f VLB treatment was delayed to the 14th day, there were no i n d e f i n i t e survivors. The l a t e stage at which VLB can i n h i b i t the development of the tumour i s unique for the chloroleukemia as no other experimental tumour can be arrested t h i s l a t e with the a l k a l o i d . I t appeared therefore that the tumour might be p a r t i c u l a r l y suitable for studying the biochemical mode of action of t h i s class of com-pound. As a prerequisite to the biochemical studies i n the chlor--10-oma, i t was desirable to e s t a b l i s h the growth pattern, trans-p l a n t a b i l i t y and the r e p r o d u c i b i l i t y of the tumour. This would permit the selection of a period of steady tumour growth (free of necrosis and hemorrhage) i n which the biochemical studies could most conveniently be made. As a r a p i d l y growing tissue, the tumour w i l l be synthesiz-ing i t s n u c l e i c acids, proteins and other e s s e n t i a l c e l l u l a r components at a high r a t e . In addition, the tumour produces substances more or less peculiar to i t s e l f (or i t s tissue of origin) v i z . protoporphyrin and myeloperoxidase. The production of these substances r e l a t i v e to tumour growth may indicate i f they are e s s e n t i a l or only i n c i d e n t a l to the development of the leukemic condition. As mentioned above, free porphyrins accumu-l a t e as by-products of the biosynthetic pathway which leads to the synthesis of heme. Under normal conditions only small amounts of free porphyrins occur i n animal tissues. The presence of large amounts of protoporphyrins i n the tumour i s therefore rather an exceptional f i n d i n g . A f t e r the growth c h a r a c t e r i s t i c s and some of the biosynthe-t i c patterns of the tumour had been established, i t was possible to choose a s p e c i f i c period to study the e f f e c t of two drugs on c e r t a i n of the biochemical reactions - e s p e c i a l l y the biosynthesis of nucleic acids, proteins and protoporphyrin i n the tumour. One of the drugs studied - v i n b l a s t i n e - retards tumour growth and has been shown by Richards and Beer (39) to i n h i b i t the synthesis of DNA markedly i n suspensions of r a t FIGURE 3 , Source of carbon and nitrogen atoms derived from glycine in protoporphyrin and purine nucleus. (a) NH N • • • • N HN Protoporphyrin / M - methyl ; V = vinyl ; p = propionyl Cb) N H N HC N N \ U Purine nucleus -11-thymus c e l l s . I t was of i n t e r e s t to see i f t h i s observation made i n v i t r o with a non-malignant tissue could be extended to an i n vivo study with the chloroma and i n p a r t i c u l a r to compare the e f f e c t of vi n b l a s t i n e on the biosynthesis of the nucleic acids and the protoporphyrin:;. The other drug -3,5-Dicarbeth<n<xy-l,4-dihydrocollidine (DDC) - causes a very large increase i n the synthesis of protoporphyrin by the l i v e r s of rodents. Its e f f e c t on protoporphyrin synthesis i n both the l i v e r and the tumour of the ra t s with chloroleukemia has been studied. Glycine i s a common precursor for both the nucleic acids and the porphyrins. Those carbon and nitrogen atoms of the purine nucleus and the porphyrin r i n g which are derived from glycine are shown i n f i g 3 a and b. Since glycine i s also r e a d i l y incorporated into proteins, i t was advantageous there-fore i n the present work to use g l y c i n e - 2 - C ^ as a tracer i n studying the e f f e c t of time, drugs etc. on the r e l a t i v e u t i l i z a t i o n of the isotope i n these various biosynthetic pro-cesses . -12-EXPERIMENTAL I. Material: Animals: Adult female Sprague-Dawley rats (100 to 130 gms) from the stock at the Cancer Research Centre i n U.B.C. were used i n a l l experiments. A l l animals were maintained on Purina Laboratory Chow die t ad libitum. The chloroleukemic l i n e i n our laboratory was i n i t i a l l y transplanted into weanling Sprague-Dawley rats but i t has 'changed' so that i t now can be transplanted into adult r a t s . Protoporphyrin IX dimethyl ester ( c r y s t a l l i n e ; Grade I) and Hemin (Bovine; 2X c r y s t a l l i z e d ) were purchased from Sigma Chemical Company, St. Louis, Missouri, U.S.A. Coproporphyrin  tetramethyl ester ( c r y s t a l l i n e ; Grade I) and uroporphyrin  octamethy1 ester ( c r y s t a l l i n e ; Grade I) were obtained from Mann Research Laboratory Incorporation, New York, U.S.A. Squibb Ether, U.S.P. was obtained from E.R. Squibb and Sons Ltd. The ether was washed with a fresh solution of FeS04 i n d i l u t e H2SO4 and d i s t i l l e d water before use to remove peroxides. The absence of the l a t t e r was confirmed when a small sample of the ether f a i l e d to l i b e r a t e iodine when shaken with an equal volume of 2% potassium iodide solution a c i d i f i e d with a few drops of d i l u t e HCl. A-Aminolevulinic acid hydrochloride ('A1 grade) was obtained from C a l i f o r n i a Corporation for Biochemical Research, Los Angeles, U.S.A. Co-enzyme A (from yeast); pyridoxal-5'-phosphate (codecarboxylase) ( c r y s t a l l i n e , monohydrate) and adenosine-5'-triphosphate (disodium s a l t from equine muscle;crystalline) -13-were obtained from Sigma Chemical Company, St. Louis, Missouri, U.S.A. Dimethyl-amino-benzaldehyde (M.P. 73-75°); a c e t y l acetone (Practical) and N,N-dimethyl-p-phenylenediamine monohydrochloride were obtained from Matheson Coleman and B e l l Company. 3,5-Dicarbethoxy-l,4-dihydrocollidine (DDC) was obtained from A l d r i c h Chemical Company Inc., Milwaukee, Wisconsin. A suspen-sion of 500 mgs of DDC i n 10 mis of corn o i l i n a centrifuge tube was heated i n a water bath (100°C) u n t i l completely dissolved. The tube was then stoppered, shaken vigorously whilst cooling i n cold water to form a uniform viscous, creamy and stable suspension. One ml of t h i s suspension (equivalent to 50 mgs of DDC) was given to animals by means of either a stomach tube or by subcutaneous and intraperitoneal i n j e c t i o n s . Radioactive glycine: Glycine-2-C- L^ was obtained from Merck Sharp and Dohme of Canada Limited. 0.1 m i l l i c u r i e : (6.3 mgs) was dissolved i n 5.0 mis of s t e r i l e saline to give a solution containing 20 microcuries per ml. 0.5 ml of thi s solution was injected i n t r a p e r i t o n e a l l y into each r a t . Vinblastine: Vinblastine sulphate was supplied by the E l i L i l l y Company. 4.5 mgs were dissolved i n 15 mis of s t e r i l e s a l i n e . A dose of 1.5 mgs/kg was injected i n t r a p e r i t o n e a l l y . Talcum powder, (u.S .Pi) was supplied by A l l i e d Chemical Company, New York, New York, U.S.A. S i l i c i c a cid powder (100 mesh, S i 0 2 x H 20) and Alumina (Grade 1, Woehlm, Neutral) were obtained from Mallinckrodt Chemical Works -14-and Alupharm Chemicals r e s p e c t i v e l y . Ethylene d i c h l o r i d e ('purified'); n-hexane (Fischer c e r t i f i e d reagant) and chloroform (Reagent ACS) were obtained from J.T. Baker Chemical Company, Fischer S c i e n t i f i c Company and Nichols Chemical Corp. Ltd., r e s p e c t i v e l y . I I . Methods: (1) Tumour t r an splantation: The chloroleukemia was transplant-ed into Sprague-Dawley rats as follows:- donor chloroleukemic r a t s were k i l l e d by decapitation and the tumours dissected out from the peritoneal cavity with s t e r i l e precautions. About 0.25 gm of v i a b l e chloroleukemic tissue (free from hemorrhage and necrosis) was homogenized by a few strokes for about 1 min. at room temperature i n 10 mis of saline (0.9% NaCl) i n a glass homogenizer with a very l o o s e - f i t t i n g glass Ipestle. Tumours were induced by the i n t r a p e r i t o n e a l i n j e c t i o n of 0.2 ml of the suspension. (2) U l t r a v i o l e t Spectroscopy: The absorption spectra of porphyrins i n the v i s i b l e and U.V. were measured with a Cary 11 Recording Spectrophotometer against reference c e l l s containing the corresponding solvent. A l l colorimetric estimations were made with a Model 14 Coleman Universal Spectrophotometer. (3) Measurement of r a d i o a c t i v i t y : Aliquots of samples were plated on platinum or aluminum planchettes and dried under an i n f r a red lamp. The r a d i o a c t i v i t y was measured i n a Nuclear FIGURE 4 a Absorption spectra of uro-, copro- and protoporphyrin i n HC1 Uroporphyrin Coproporphyria Protoporphyrin o •H 00 o P. o 400 500 600 700 800 Wavelength (ir^i) d (0 C •a o 400 500 600 700 800 Wavelength (»ju) o" n a 400 500 600 700 800 Wavelength (nai) FIGURE 4 b Absorption spectra of coproporphyrin tetramethyl ester and protoporphyrin dimethyl ester i n ethylene dichloride:n-hexane mixture o •H m C O Coproporphyrin ester 400 500 600 700 800 Wavelength (nai) o •H S •a u •H •» Protoporphyrin ester 400 500 600 700 800 Wavelength (ip) -15-Chicago windowless gas-flow counter. The high voltage supply-to the Geiger counter was set at about the mid point of the plateau. The background count of the planchette was determined before use. (4) Estimation of porphyrins: In solutions of strong acids, porphyrins have a purple-red colour; i n organic solvents or i n a l k a l i n e solutions, they are red-brown i n colour. By spectro-photometrie analysis a series of f a i r l y sharp and c h a r a c t e r i s t i c absorption bands may be defined. A l l porphyrins have an intense absorption band at about 400 imi. This i s the 'Soret' band c h a r a c t e r i s t i c of a l l conjugated tetrapyrroles but lacking when the conjugation i s broken as i n b i l e pigments and porphyr-inogens. The p o s i t i o n and magnitude of the Soret band d i f f e r somewhat for each porphyrin. Solutions of porphyrins i n mineral acids have two bands i n the v i s i b l e region plus the Soret band. The spectra of uro-, copro- and protoporphyrin i n HC1 are shown i n f i g 4a. In contrast, the spectra of most common porphyrins i n organic solvents or weak a l k a l i consist of four r e l a t i v e l y sharp bands i n the v i s i b l e region i n addi-t i o n to the Soret band. The spectra of coproporphyrin t e t r a -methyl ester and protoporphyrin dimethyl esters are shown i n f i g 4b. The absorption spectra of. porphyrins are important for th e i r i d e n t i f i c a t i o n and estimation. The i n t e n s i t y of the Soret band can be measured e a s i l y . The e x t i n c t i o n c o e f f i c i e n t s TABLE 1 a Extinction c o e f f i c i e n t s of porphyrins i n hydrochloric acid solution* Porphyrin Cone. HC1 Soret max 17 E i / o E ug/ml (m|j.) 1 cm 1 cm Uroporphyrin III 0.5 N 405.5 6520 0.652 Coproporphyrin III 0.1 N 399.5 7470 0.747 Protoporphyrin 1.40 N 408 4890 0.489 '''Data quoted from Riming ton (40) TABLE 1 b Extinction c o e f f i c i e n t s of porphyrin methyl esters i n chloroform** 1 Porphyrin ester Soret max (mp,) E Y/o 1 cm £ |ig/ml 1 cm Uroporphyrin III octamethyl ester 405-406 2300 0.23 Coproporphyrin III tetramethyl ester 1 399.5 2530 0.253 j. (Protoporphyrin dimethyl ester 407.5 2890 0.289 **Data quoted from Rimington (40). -16-of uro-, copro- and protoporphyrins and their esters given i n table 1 a and b are quoted from Rimington (40). A 1% absorbance of a solution whose concentration i s 1 |j,g/ml. The porphyrin concentration of a solution (|j,g/ml) can therefore be In some parts of the work the porphyrins were measured i n solvent mixtures of ethylene d i c h l o r i d e and n-hexane. It was established that the extinc t i o n c o e f f i c i e n t at the Soret band region i n a 4:1 mixture of ethylene dic h l o r i d e and n-hexane was 987o of that i n pure chloroform. The HC1 extracts from the chloroma and the l i v e r s of animals treated with DDC when chromatographed on alumina, showed that 75-80?o was due to the protoporphyrin. In view of t h i s , occasionally porphyrin concentration i n these tissues was estimated d i r e c t l y from the HC1 extracts as sp e c i f i e d i n the text. (5) Extraction of porphyrins from chloromas and l i v e r s : The procedure used for the extraction of porphyrins from chloroma and l i v e r outlined i n figure 5 i s similar to that described by Schwartz and Wikoff for red blood c e l l s (41). Since the v i n y l side chains of protoporphyrin are extremely calculated by dividing the observed AMAX?1 i j _ c n i by the appropriate FIGURE 5 Extraction of porphyrins from the tumour Tumour (S.O-7.0 gn\) Homogenized with 20 mis EtoAc/HOAc (4:1) Suspension > 2 (1 ml) samples taken out f o r n u c l e i c a c i d ; estimation Centrifuged at 1100 x g ca. 10 mins 4°C Supernatant P r e c i p i t a t e Repeated e x t r a c t i o n with two 10 ml portions of EtoAc/HOAc & centrifugpri Combined supernatants Supernatants J ' P r e c i p i t a t e Washed with 3% NaOAc ( f i r s t washing contained 0.1 ml 12 solution) Discard Aqueous f r a c t i o n (uroporphyrin) Organic EtoAc/HOAc f r a c t i o n Repeated e x t r a c t i o n with 5 ml p o r t i o n s 3N HC1 3N HC1 e x t r a c t Organic f r a c t i o n Discard (1) D i l u t e d with d i s t i l l e d water to 1.40 N. (2) Adjusted to pH ca 4.0 with 10N NaOH and IN NaOH, (3) T r a n s f e r r e d to ether and d r i e d down. (4) Ester i f ied with 50 mis 5% (V) H^SO^MeOH. -17-sensi t i v e to l i g h t (18) a l l manipulations were carried out i n dim l i g h t . Five to seven gms of the tumour were homogen-ized i n 20 mis of a 4:1 mixture of ethyl acetate-acetic a c i d at high speed with a S e r v a l l blendor at 4°C. Two 1 ml samples of the brown ethyl acetate-acetic a c i d suspension were set aside for nucleic acid determinations. The r e s t of the suspension was centrifuged at 1100 xg at 4°C for 10 mins and the supernatant removed and preserved. The p r e c i p i t a t e was re-extracted twice with 10 mlsportions of the 4:1 mixture of ethyl acetate-acetic acid, the f i n a l supernatant being p r a c t i c a l l y colourless. The combined supernatants were washed once with 10 mis of 3% NaOAc containing 0.1 ml of d i l u t e iodine solution (100 mgs I 2 + 500 mgs KI + 10 mis of d i s t i l l e d water) and twice with 5 mis of 3% NaOAc. (The addition of the iodine solution f a c i l i t a t e s the oxidation of any porphyrihoro gen present i n the organic phase to porphyrins (42).) Since the s o l u b i l i t y of a porphyrin i n aqueous media i s proportional to the number of carboxyl groups i t has, uroporphyrin with 8 carboxyl groups i s r e a d i l y soluble i n water and i t i s found i n the aqueous washes. The l a t t e r were therefore preserved for the spectrophotometric measurement. The porphyrins remain ing i n the ethyl acetate-acetic a c i d solvent were then removed by repeated extractions with 5 ml portions of 3N HCl u n t i l the HCl extract no longer showed the presence of porphyrins (absence of the Soret band). Usually 5 or 6 extractions were -18-s u f f i c i e n t . The 3N HC1 extracts were d i l u t e d with water to a concentration of 1.40 N for spectrophotometry measure-ment of the porphyrins. The U.V. data showed that the combined 1st and 2nd HCl extracts contained 85-90% of the t o t a l porphyrin extractable from the tumour and these were used for the subsequent estimation of porphyrins i n the tumour. The porphyrin may be i d e n t i f i e d and estimated as the free porphyrin or after conversion to the ester. Free porphyrins are r e l a t i v e l y unstable and i n general practice, most workers prefer using the more stable esters. The e s t e r i f i c a t i o n procedure used i n the present work was essen-t i a l l y that of Falk (43). The HCl extracts were cooled for about 20 mins at 4°C and then adjusted with ION NaOH and 1 N NaOH to a pH of 3-4. (The i s o e l e c t r i c point of most porphy-r i n s araa found between pH 3 to 4.) I t was observed that the o r i g i n a l purple colour of the porphyrin solution changed to brown as the i s o e l e c t r i c point was approached. The porphy-r i n s i n the aqueous pH 4 solution jfca 20 mis) were then transferred to ether by extracting 4 times with 10 mis portions of peroxide-free ether. Spectroscopic examination of the r e s i d u a l aqueous f r a c t i o n showed that only 2 to 5% of the o r i g i n a l porphyrins remained unextracted. The combined ether extracts were washed 3 times with 5 mis of water and evaporated to dryness i n vacuo. (The aqueous washings were free of porphyrins.) The residue was then dissolved i n 50 mis -19-of cold 5% (V/V) I^SO^-methanol and the e s t e r i f i c a t i o n of the porphyrins was allowed to proceed at -10°C for 18 to 20 hours. F i f t y mis of i c e cold water were then added and the mixture was shaken with 25 mis of chloroform. The aqueous phase was then removed. The chloroform containing the porphyrin ester was washed 3 times with 50 ml portions of water, once with 50 mis of 2N NH^OH and three more times with 50 mis of water. There was no loss of the porphyrin ester from the chloroform on washing. Aliquots of the chloroform solution were removed i f required for r a d i o a c t i v i t y measurement, and the r e s t evapo-rated to dryness i n vacuo. The porphyrin ester was r e - d i s s o l v -ed i n a suitable solvent for chromatography as described below. (6) I d e n t i f i c a t i o n of the porphyrins: At present, there i s no procedure which permits accurate quantitative analysis of a l l porphyrins present i n b i o l o g i c a l material. In the l i t e r a t u r e , there i s no agreement by the various workers on the r e l a t i v e merits of the e x i s t i n g a n a l y t i c a l methods. A preliminary survey of a number of these was therefore made. A) I d e n t i f i c a t i o n of free porphyrins I d e n t i f i c a t i o n of free porphyrins by paper chromatography, paper electrophoresis and column chromatography on t a l c were attempted. Authentic porphyrins as the free compound were obtained by hydrolyzing a few mgs of the pure porphyrin esters -20-i n 1.0 ml of 7N HCl at room temperature for about 40 hours (43). The HCl solution was di l u t e d with water to 1.4 N and evaporated at room temperature i n vacuo i n a dessicator containing KOH p e l l e t s and concentrated H£S0^. The residue was dissolved i n a small volume (2 mis) of 2N NH^OH. (i) Paper chromatography Aliquots of authentic free porphyrins i n 2N NH^OH were spotted on Whatman #1 paper (25 cm x 35 cm) which was developed by ascending chromatography i n a mixture of 2:6 l u t i d i n e - ^ O i n presence of ammonia vapour according to the method of Eriksen (44). The paper was allowed to dry at room temperature and the porphyrins then located by thei r red fluorescence i n U.V. l i g h t . In every chromatographic run, a small non-fluor-escent zone was found above the protoporphyrin spot. This might be due to an impurity i n the 'authentic' protoporphyrin sample. Many attempts were made to elute the porphyrins from the paper using various concentrations of HCl, ammonia and ace t i c acid, but i n no case could more than 50% of the applied porphyrins be recovered. Brown stains were found on the paper after e l u t i o n . Since free porphyrins are unstable i n certai n solvents and e a s i l y oxidized on exposure to a i r i n the l i g h t these brown stains could be the decomposition products of the free porphyrins obtained as a r e s u l t of e l u t i o n . The paper chromatographic method, although useful i n i d e n t i f y i n g small amounts of free porphyrins, i s not a sa t i s f a c t o r y a n a l y t i c a l method for quantitative analysis i n the present -21-wor k. ( i i ) Paper electrophoresis I t has been claimed by S t e r l i n g and Redeker (45) that free porphyrins can be separated by paper electrophoresis on Whatman #3 paper with a pH 8.6 EDTA buffer (Disodium s a l t of ethylene diamine t e t r a a c e t i c a c i d dihydrate). Paper electrophoresis was therefore attempted. Authentic porphyr-ins were applied i n small volumes of 2N NH^OH. Electrophor-esis was c a r r i e d out for 4 hours at 200 v o l t s at 4°C and the porphyrins then located i n U.V. l i g h t . Although uroporphyrin moved 10-15 cm from the o r i g i n , both copro- and protoporphyrins stayed at the o r i g i n . ( S t e r l i n g et a l reported a separation of copro- from protoporphyrin by ca 0.5 cm from each other on paper.) The recovery of porphyrins from paper aft e r electrophoresis was i n the range of 40-50% of the t o t a l amount o r i g i n a l l y applied. This method i s therefore not adequate for the i s o l a t i o n of porphyrins from b i o l o g i c a l material i n which copro- and protoporphyrin are present together. ( i i i ) Column chromatography on t a l c A t a l c column was used for the p u r i f i c a t i o n of free porphyrins by Comfort (46) who employed varying strengths of aqueous HCl for e l u t i o n . This method, as i t stood, was unsatisfactory since large volumes of acid had to be used to achieve complete separation. I t was f e l t that i t might -22-be possible to develop i t into a serviceable a n a l y t i c a l procedure e.g. by using d i f f e r e n t e l u t i o n conditions. I t was found that the t a l c (5 gms i n a column 1.0 diameter) absorbed the porphyrins so strongly that i t was extremely d i f f i c u l t to elute them, not only with HCl of varying concentrations, but also with ammonia or solvents such as acetone-HCl, pyridine, dioxane, morpholine-^O, etc. The t a l c column therefore did not show s u f f i c i e n t promise as an a n a l y t i c a l t o o l for quantitative porphyrin assay to warrant further study i n the present work. B) I d e n t i f i c a t i o n of porphyrin esters Since the foregoing attempts to separate and recover the free porphyrins had not been s a t i s f a c t o r y , the separation of porphyrin esters by column chromatography was investigated. Column chromatography on alumina Nicholas (47) found that mixtures of chloroform and petroleum ether eluted the porphyrin esters from alumina i n the order of proto-, copro- and uroporphyrin. When the adsorptive power of the materials and the conditions of development were standardized reproducible separations of porphyrin esters were obtained. I t seems that the alumina column i s a p o t e n t i a l l y u s e f u l t o o l for the quantitative estimation of porphyrin esters. Twenty gms of alumina (Grade 1) i n an Erlenmeyer f l a s k were p a r t i a l l y deactivated by the addition of 2 mis of water. -23-The stoppered f l a s k was shaken vigorously and aft e r standing overnight, the dry free-flowing powder was suspended i n 20 mis of chlorof orm-n-hexane mixture (1:4) and then slowly poured into a glass column (1.8 cm x 30 cm) f i t t e d with a cotton plug at the bottom. The alumina was allowed to s e t t l e 6y gravity and the surface then protected by a layer of micro glass beads. Protoporphyrin dimethyl ester (20y) dissolved i n 1.0 ml of a mixture of chloroform-n-hexane (1:4) was applied to the column, which was then developed with 100 mis of chloroform-n-hexane (1:4.) . The protoporphyrin dimethyl ester was mobilized by this solvent quite e a s i l y but there was a s i g n i f i c a n t d i f f u s i o n of zone i n the lower portion of the column r e s u l t i n g i n unsatisfact-ory r e s o l u t i o n . Although the spectrum of the protoporphyrin dimethyl ester i n the effluents was not changed the chloroform was found to cause a discontinuity i n the e l u t i o n pattern thus giving r i s e to ambiguous r e s u l t s . In order to minimize the extent of d i f f u s i o n and t a i l i n g of zones a gradient e l u t i o n was developed. The following describes the method as ultimately adopted for the estimation of porphyrins from the tumour and the l i v e r . Protoporphyrin dimethyl ester (1 mg) i n 1 ml of ethylene dichloride was d i l u t e d with 4 mis of n-hexane and then applied to an alumina column (1.8 cm x 30 cm). Ethylene dic h l o r i d e was chosen i n place of chloroform because: i) I t i s a more stable solvent; i i ) I t does not need washing and drying before use; i i i ) I t does not develop a c i d i t y ; iv) I t does not contain phosgene and other impurities normally FIGURE 6 Chromatography of Protoporphyrin Dimethyl Ester on A 1 2 0 3 b 0 10 20 30 40 50 60 Fracti o n Number 1 FIGURE 7 Re-Chromatography of Fractions 35 to 38 on AI2O F r a c t i o n Number -24-found i n chloroform. The protoporphyrin dimethyl ester was eluted from the column with a linear gradient of ethylene dichloride. and n-hexane. (One hundred mis of a 1:4 mixture of ethylene d i c h l o r i d e and n-hexane i n the mixing chamber and 100 mis of a 4:1 mixture of ethylene d i c h l o r i d e and n-hexane i n the reservoir.) The e f f l u e n t was c o l l e c t e d i n 4.0 ml f r a c t i o n s . Spectra and o p t i c a l densities were then measured. The e l u t i o n pattern as shown i n figure 6 gave two peaks (peaks a and b). The spectra of both peaks were porphyrin type. Although effluents between f r a c t i o n 25 to 32 absorbed U.V. around 400 m\± they did not fluoresce. E f f l u e n t s between f r a c t i o n 33 to 48 had spectra resembling those of pure protoporphyrin dimethyl ester and a l l of them fluoresced i n U.V. l i g h t . This established that the main peak (peak b) was the protoporphyrin dimethyl ester. Sixty percent of the protoporphyrin dimethyl ester applied to the column was recovered i n t h i s major peak. Fractions 46 to 54 forming the shoulder of the major peak (peak b) also f l u o r -esced and absorbed around 400 m|_u An examination of the column i n U.V. l i g h t a f t e r chromatography revealed a wide zone of fluorescent material on top of the alumina. This and the non-fluorescent peak (peak a) may be due to impurities i n the commercial protoporphyrin dimethyl ester or to a decomposition of the protoporphyrin as a r e s u l t of exposure to a i r i n the l i g h t . The behaviour of the pure protoporphyrin dimethyl -25-ester (fractions 33-48) on re-chromatography was therefore examined. The t o t a l protoporphyrin dimethyl ester i n the combined fra c t i o n s 35 to 38 was calculated from the o p t i c a l density. The solvent was then removed i n a stream of N 2 and the residue was re-chromatographed on a new alumina column. The elu t i o n pattern ( f i g 7) s t i l l showed two peaks (peaks a' and b') but the shoulder between fr a c t i o n s 49 to 54 was absent. This indicated that the shoulder was probably due to impurities i n the commercial protoporphyrin ester. As i n the previous run, fractions 28 to 32 did not fluoresce and i t was found l a t e r that t h i s peak (peak a 1) was an a r t e f a c t r e s u l t i n g from the exposure of protoporphyrin to l i g h t (see section on glycine-2-C^ incorporation into the protoporphyrin of the chloroma) . The recovery of protoporphyrin dimethyl ester i n the e f f l u e n t comprising the major peak (peak b 1) enclosed by fractions 33 to 43 was 75-80% of the t o t a l amount applied to the column. Only a very small trace of red fluorescent material remained on the top of the alumina column. Since coproporphyrin might also be present i n the tumour, i t was necessary to check the chromatographic behaviour of a sample of authentic coproporphyrin ester on the alumina column. I t was found that coproporphyrin ester stayed at the top of the column i f a l i n e a r ethylene dic h l o r i d e n-hexane gradient was used. (Same gradient as for the elu t i o n of protoporphyrin.) figure 8 Extraction of nucleic acids and protein from the chloroma 7.0 gm tumour Homogenized with 20 ml EtoAc/HoAc (4:1) 1 ' < 1.0 ml suspension 1.0 ml suspension 18.0 ml suspension = 350 mg tissue «350 mg tissue • 6 .3 gm tissue PORPHYRIN EXTRACTION Duplicate samples for nucleic acids determination Centrifuged at b°C 1100 x g for ca_ 6 mins F Supernatant 1 Precipitate L i p i d extraction Centrifuged 1 Supernatant Precipitate Salt extraction with 10% NaCl at 100°C Supernatant EtOH precipitation I Centrifuged • 1 Centrifuged Precipitate PROTEIN DETERMINATION Supernatant Precipitate DA r \ l k a l i hydrolysis! with 0, overnight \ 2)Centrifuged 3NKDH 1 Precipitate Supernatant Extraction with 3N HCl 0°C| I Centri fuged ' | I | Discard Precipitate r Supernatant RNA . | Dissolved in 1 M NH^ OH DNA -26-However, i f the column was then eluted with 50 mis of chloro-form, the coproporphyria ester moved r a p i d l y down the column and could be recovered i n the effluents i n a y i e l d of 75%. The foregoing r e s u l t s indicate that the alumina column, as modified, i s very suitable for the i d e n t i f i c a t i o n of protoporphyrin. I t was easy to manipulate and gave consistent r e s u l t s i n both the high recovery and the p o s i t i o n of e l u t i o n . Also, i t was found that the presence of traces of acid or water i n the sample did not a f f e c t the e l u t i o n pattern. This method was adopted i n the estimation of protoporphyrin i s o l a t e d from the tumour and the l i v e r . (7) Extraction of Nucleic Acids from the Chloroma The n u c l e i c acids i n the tumour were extracted by a modification of the procedure of Hecht and Potter (48). Two 1 ml samples of the EtOAc/HOAc tumour homogenate were c e n t r i -fuged at 1100 x g at 4°C for ca 10 mins ( f i g 8). L i p i d s were extracted with the following solvent sequence: acetone, EtOH, EtOH:CHCl 3 (1:1); EtOH:ether (3:1) and f i n a l l y ether. The p r e c i p i t a t e was well s t i r r e d with each solvent before re-centrifuging. The f i n a l residue was allowed to dry at room temperature and was then extracted with 6 mis of 10% NaCl at 100°C for 30 mins; the pH was kept at 7.0 to 7.4 throughout the extraction by the addition of 0.1N NaOH using phenol red as i n t e r n a l i n d i c a t o r . The extraction was repeated with 4 mis -27-of 10%. NaCl at 100°C for 5 mins. The residue obtained during NaCl extraction was retained for the estimation of glycine-2-C ^ incorporation into the protein as described i n the following section. Two and a h a l f volumes of cold 100% EtOH were added to the supernatant to p r e c i p i t a t e the sodium nucleates. After standing at 4°C overnight, the sodium nucleates were centrifuged and the RNA degraded to the aci d soluble monoribonucleotides by incubating for 20 hours with l§0 ml 0.3N KOH at 37°C. The mixture was centrifuged to remove a small amount of insoluble material. The a l k a l i n e supernatant was c h i l l e d and the DNA prec i p i t a t e d by the addition of 1/5 volume of cold 3N HCl. The pr e c i p i t a t e was washed with 1.0 ml cold 0.1N HCl and the com-bined HCl fractio n s were made up to 5 mis with water. The r a d i o a c t i v i t y of the RNA was determined i n the acid soluble ribonucleotide f r a c t i o n by counting a dried a l i q u o t of the HCl solu t i o n . The DNA p r e c i p i t a t e was dissolved i n 5 mis of 1M NH4OH and the r a d i o a c t i v i t y determined on dried aliquots of this s o l u t i o n . The amount of RNA was determined by the ribose •fke. i n RNA us i n g j o r c i n o l reaction (49) and the RNA phosphorus (50). The amount of DNA was measured by the indole reaction (51) and the DNA phosphorus (50). The s p e c i f i c a c t i v i t i e s were calculated by div i d i n g the c.p.m. i n an aliquo t by the amount of RNA or DNA present i n the sample. (8) The incorporation of glycine-2-C-*-^ into chloroma protein The p r e c i p i t a t e remaining aft e r the nucleic acids had been extracted with NaCl ( f i g 8) was well s t i r r e d with 5 mis of cold -28-5% t r i c h l o r o a c e t i c a c i d and centrifuged at 1500 xg for 5 mins. The extraction was repeated once. The protein residue was washed three times with 5 ml portions of ether to remove any-r e s i d u a l l i p i d , and was allowed to dry overnight at room temperature. I t was then dissolved i n 5 mis of warm 987o formic acid and aliquots of the solution dried on platinum planchettes for counting i n a windowless gas-flow Geiger counter. The weight of the protein plated was obtained by weighing each of the dried platinum planchettes before and a f t e r p l a t i n g . Since none of the plated protein samples weighed more than 0.2 mg/sq. cm. the correction for self-absorption was less than 2%. (9) Extraction and estimation of myeloperoxidase from the chloroma (i) Extraction of myeloperoxidase from the tumour: Two gms of tumour was homogenized for 5 mins at 0°C. with 12.0 mis of sodium phosphate buffer (0.2 M; pH 6.2) i n a motor-driven glass homogenizer f i t t e d with a t e f l o n p e s t l e . The suspension was then centrifuged at 60', 000 x g for 15 mins i n a Spinco centrifuge (Model L Preparative Centrifuge). The extraction was repeated twice with 10 mis of phosphate buffer. The myeloperoxidase a c t i v i t y i n each supernatant was estimated by the method described below. The supernatant of the 4th extract had no a c t i v i t y . I t was found that the supernatant of the 1st, 2nd and the 3rd extraction constituted 93.5%, 6% and 0.5% r e s p e c t i v e l y of the t o t a l myeloperoxidase a c t i v i t y i n the 2.0 gm of the tumour. I t was f e l t therefore that the myelo--29-peroxidase a c t i v i t y i n the f i r s t extract was a r e l i a b l e representation of the t o t a l myeloperoxidase extractable from the tumour. ( i i ) Estimation of myeloperoxidase a c t i v i t y : Myeloperoxidase was estimated by a modification of the method of Straus (52) . In t h i s method, N,N dimethy1-p-phenyl-ene diamine i s used as a hydrogen donor and Ho0 o as H acceptor. ^ 2 Myeloperoxidase transforms the N,N dimethyl-p-phenylene diamine into a red pigment of semiquinone character. The amount of red colour formed by peroxidase i s proportional to the concentra-ti o n of the enzyme and to the time of incubation during the f i r s t 20 to 80 seconds. Myeloperoxidase a c t i v i t i e s are expres-sed as changes i n o p t i c a l density at 515 m\x/30 second/gm wet tis s u e . One ml of the clear supernatant from the f i r s t extract was d i l u t e d to 10 mis with the phosphate buffer. Two mis of thi s solution were then incubated with 2.0 mis of phosphate buffer and 6 mis of water at 20°C for 2 mins i n a colorimeter tube. 0.4 ml of 0.18% H202 solution and 0.3 ml of a fre s h l y prepared solution of N,N dimethyl-p-phenylene diamine hydro-chloride were then added. The contents were mixed and the o p t i c a l density at 515 mo. immediately read against d i s t i l l e d water at in t e r v a l s of 10 seconds for a t o t a l period of 100 seconds. Appropriate corrections were made for any colour developed by the reagents. -30-Preparation of hematin: A solution of hematin was prepared immediately before use by d i s s o l v i n g commercial bovine hemin (2x c r y s t a l l i z e d ) (Mann Research Laboratory) i n a mixture of 3 parts of 0.01 N NaOH and 7 parts of pH 6.2 sodium phosphate buffer. Varying amounts of the hematin solution were incuba-ted with the enzyme extracts i n an attempt to stimulate the a c t i v i t y of the enzyme. The r e s u l t s were corrected for the "pseudo" peroxidase a c t i v i t y of hematin i n and the colour reagent. (10) Estimation of urinary porphobilinogen The determination of porphobilinogen followed the method of Vahlquist (53), which i s based on the reaction of porpho-bilinogen with the Ehrlich-Hg reagent i n a c i d solution to form a red compound. Ehrlich-Hg reagent: 0.7 gm of Hg C l 2 and 4.0 gms of dimethyl-aminobenzaldehyde were dissolved i n 40 mis of 70% per c h l o r i c a c i d . 168 mis of g l a c i a l a c e t i c a c i d were then added and the solution d i l u t e d with d i s t i l l e d water to 220 mis. 0.1 ml of urine d i l u t e d to 5 mis with water was mixed with 5 mis of the Ehrlich-Hg reagent and the o p t i c a l density at 555 mo. read after 10 mins. . E h r l i c h reagent d i l u t e d with an equal volume of water was used i n the reference c e l l . The O.D. x 2.8 = micro moles of porphobilinogen/ml urine (53). (11) Estimation of A-aminolevulinic a c i d synthetase a c t i v i t y i n the l i v e r and the tumour: -31-(a) Assay of ^-aminolevulinic acid synthetase A -Aminolevulinic a c i d synthetase a c t i v i t y was determined by the formation of A -aminolevulinic acid according to the method of Granick (34). In this method, succinyl CoA i s generated from succinate, ATP and Co-enzyme A by the enzyme succinyl CoA thiokinase. The succinyl CoA then reacts with glycine under the influence of 4-aminolevulinic acid synthe-tase and co-factor pyridoxal phosphate to give ^-aminolevu-l i n i c a cid according to the reactions outlined i n - f i g u r e 2. 2.0 gms of f r e s h l y excised l i v e r s or tumours were homogen-ized i n 0.25 M sucrose (1 volume per gm of wet tissue) at 0°C with a motor-driven glass homogenizer. 0.2 ml of the homogenate was incubated with:-T r i s buffer, pH 7.4 100 u. moles Glycine 100 p, moles MgCl 2.6H 20 40 [i moles Sucrose 500 \i moles Succinate 100 u. moles Pyridoxal phosphate 2 u. moles Co-enzyme A 0.5 a. moles ATP 2 jj, moles D i s t i l l e d water to make 2.0 ml i n a 25 ml Erlenmeyer f l a s k at 37°C for 1 hour. The reaction was terminated by adding 2.0 ml of 0.3M t r i c h l o r o a c e t i c a c i d and the p r e c i p i t a t e d protein removed -32-by centrifugation. The ^ -aminolevulinic a c i d i n the supernatant was then determined as described below, (b) Estimation of A - a m i n o l e v u l i n i c a c i d A - A m i n o l e v u l i n i c acid was estimated by the method of Mauzerall and Granick (54). In t h i s determination, £ -aminolevulinic a c i d i s condens-ed with a c e t y l acetone to form a pyrrole, which having a free ,Q}1 p o s i t i o n , can react with the E h r l i c h reagent to form a coloured compound:-H / = \ xeH'3 H H ' H ^ — — y V 0 H 3 l y r r o l e E h r l i c h r e a g e n t C o l o u r e d Compound +, E^O Reagents: i ) Acetate buffer, pH 4.6 was made by adding 57 mis of g l a c i a l a c e t i c a c i d (1 mole) to 136 gms of sodium acetate trihydrate (1 mole) and d i l u t i n g to 1 l i t r e with d i s t i l l e d water. i i ) Ehrlich-Hg reagent: as described above i i i ) A c etyl acetone: p r a c t i c a l . A c e t y l acetone (0.2 ml) was added to 2.0 ml of the supernatant from the incubation mixture i n a test-tube and the mixture d i l u t e d to 10 ml with the pH 4.6 sodium acetate buffer. The tube was stoppered and heated at 100°C for 10 mins. After cooling to room temperature, 5 ml of the solution were mixed with 5 mis of E h r l i c h reagent. After 15 mins, the -33-o p t i c a l density was read at 555 m\i against a reference c e l l containing the E h r l i c h reagent d i l u t e d with an equal volume of water. TABLE 2 Development of chloroleukemia and su r v i v a l time i n 10 generations of Sprague-Dawley r a t s inoculated i n t r a p e r i toneally with 0.2 ml of chloroma homogenate. Generation number Number of rats injected Number of posi-t i v e transplant Average su r v i v a l time + S.D. (Days)* 34** 5 4 27 + 1 35 6 6 25 + 1 36 6 5 26 + 1 37 12 11 27 + 1 38 12 11 27 + 1 39 12 11 27 + 1 40 12 12 24 + 1 41 12 12 29 ± * 42 6 6 26 + 1 43 11 10 25 + 1 * Average su r v i v a l time calculated for animals which a c t u a l l y developed tumours ( i . e . 'No takes' were discarded) ** This series of experiment was started when 34 generations had already been observed i n this laboratory. -34-RESULTS I. Transplantation. The transplant l i n e of chloroleukemic Sprague-Dawley r a t s i n our laboratory was obtained from the Miami Valley Hospital i n the Spring of 1961. I n i t i a l l y , the tumour was transplanted into weanling r a t s . In September 1962, perhaps due to mutation, transplantation into adult Sprague-Dawley r a t s became possible. Adult female Sprague-Dawley rats (ca 2 months old) weighing 100-130 grams were used i n a l l the present experiments. As shown i n table 2, i n t r a p e r i t o n e a l i n j e c t i o n of the chloroma homogenates produced 92% p o s i t i v e transplants and an average s u r v i v a l time of 27 days with a range of 24 to 29 days. The high 'take' of the tumour and the consistent lifel.span of the animals suggest that this tumour may be very usef u l for biochemical studies. Inoculated animals showing signs of a 'take' were e a s i l y recognized by the abdominal enlargements r e s u l t i n g from the growth of chloromatous masses. A few days before death, the rear legs of the animals were paralysed. Post-mortem examinations revealed the presence of yellowish-green tumours i n f i l t r a t i n g the mesentery, thymus and the l i v e r s i n a l l animals. The tumour was also frequently found i n the spleen, kidneys and diaphragm. Post-mortem photographs of tumour-free and chloroleukemic rats are shown i n figure 9. On exposure to u l t r a - v i o l e t l i g h t a l l tumours fluoresced bright red. Although very l i t t l e or no a s c i t i c f l u i d was found i n - 3 5 -FIGURE 9 Post-mortem photographs of tumour-free and chloroleukemic Sprague-Dawley r a t s . Tumour-free Sprague-Dawley r a t Chloroleukemic Sprague-Dawley r a t C = Chloroma Age of the tumour = 20 days TABLE 3 Tr a n s p l a n t a b i l i t y of chloroleukemia Material injected I.P. (0.2 ml) No. of c e l l s injected No. of rats injected No. of pos i -t i v e transplants Average 'take' time (days) Chloroma homogenate 1 m i l l i o n 6 6 10-12 Whole blood 6 3 2 mos. A s c i t i c f l u i d 600,000 6 5 10-12 A s c i t i c f l u i d supernatant 12,000 6 3 10-12 C e l l - f r e e a s c i t i c f l u i d no c e l l s 4 0 No detec-table tum-our aft e r 6 months -36-chloroleukemic rat s 11 to 14 days old, as much as 10-15 mis was frequently found i n the peritoneal cavity around the 21st day after the transplantation. About t h i s time the tumours usually became rather necrotic and hemorrhagic. However, i n no case did a tumour .regress aft e r i t had developed. I t was mentioned i n the introduction that chloroleukemia may be produced i n rats by the inoculation of d i f f e r e n t c e l l - f r e e f i l t r a t e s obtained from animals with leukemia or even from tumours of non-hematopoietic o r i g i n (7). I t has been suggested that chloroleukemias are of v i r a l o r i g i n . For example, a chloroleu-kemia virus p a r t i c l e varying i n size from 50 to 150 mp, has been described by G r a f f i (55). In view of t h i s , the a b i l i t y of whole blood, a s c i t i c f l u i d and c e l l - f r e e a s c i t i c f l u i d from donor leukemic rats to induce chloroleukemias i n host r a t s was investigated. The a s c i t i c f l u i d from the peritoneal cavity of tumour-bearing r a t s was centrifuged at 1500 x g for 15 mins i n a c l i n i c a l centrifuge to sediment the c e l l s . I t was found by counting i n a haemocytometer that 60 c e l l s / c u mm were s t i l l i n the supernatant. 0.2 ml of the supernatant was used d i r e c t l y for inoculating each of the 6 host r a t s , and the r e s t was then drawn through a 0.45 p, pore size " M i l l i p o r e " f i l t e r (type HA) into a 5 ml syringe. Microscopic examination of the f i l t r a t e i n a haemocytometer showed that i t was now c e l l - f r e e . The development of tumours from these i n o c u l i are shown i n table 3. The data i l l u s t r a t e a peculiar feature of the chloroma i n that there was a substantial lapse of time ('take TABLE 4 The tumour weight, protoporphyrin concentration and myeloperoxidase a c t i v i t y of the chloroma on d i f f e r e n t days after transplantation. Days a f t e r transplantation Average wt of tumour + S.D. (gms) Protoporphyrin concentration* (p,gs/gm tumour) Myeloperoxidase activity**/gm tumour 9 No detectable tumour 11 3.2 + 1.8 0.57 + 0.04 Not determined 13 7.5 + 3.0 2.90 + 0.36 0.23 15 13.0 + 4.0 4.20 + 0.30 0.485 17 19.1 + 3.6 10.30 + 0.59 0.48 19 28.6 + 4.3 12.80 + 0.35 0.15 21 33.5 + 4.5 14.70 + 0.36 Not determined 23 47.0 + 6.2 18.90 + 0.40 0.12 25 51.9 +3.2 20.35 + 0.60 Not determined Number of rats used i n each experiment^ 6 ^Protoporphyrin was estimated as the free porphyrin i n HCl as described i n the experimental section ^^Myeloperoxidase a c t i v i t y i s expressed as the change i n o p t i c a l density i n 30 seconds at 515 imi per gm tumour as described i n the experimental section. -37-time') a f t e r the inoculation before the tumour was detectable. The appearance of tumour by either the a s c i t i c f l u i d or supernatant of the a s c i t i c f l u i d was the same as that observed with the chloroma homogenate i . e . there was a lag period of 10-12 days and thereafter followed a similar course until?, the death at 27-30 days. Although the a s c i t i c f l u i d and whole blood gave the same percentage of tumour takes, the 'take time' with the whole blood was delayed for 2 months - thereafter the rate of tumour growth was as rapid as i n the chloroma homogenate. The slow 'take' of the blood may be r e l a t e d to the number of tumour c e l l s i n the blood. There was no sign of a p o s i t i v e transplant using c e l l - f r e e a s c i t i c f l u i d even though observation was maintained for 6 months. I I . The r e l a t i o n s h i p of tumour growth, protoporphyrin concen-t r a t i o n and myeloperoxidase a c t i v i t y of the chloroma. The rate of growth of the tumour induced by the chloroma homogenate i s shown i n table 4 and figure 10. I t may be seen that there was no tumour detectable before the 9th day after transplantation. After the 11th day, the tumour began to develop and thereafter itgrew st e a d i l y i n an almost linear manner u n t i l the death of the animal around the 27-30 day. I t may also be noted that the tumour increased i n weight r a p i d l y as the tumour aged. I t would appear that the period of steady growth (days 15 to 19) would be most convenient for studying the e f f e c t of drugs on the metabolism of the tumour. FIGURE 10 Tumour weight and the amount of protoporphyrin i n chloroleukemic r a t s on d i f f e r e n t days a f t e r transplantation 5 10 15 20 25 Days a f t e r transplantation -38-FIGURE 11 Myeloperoxidase a c t i v i t y i n chloromas of d i f f e r e n t age 0 5 10 15 i 20 25 Days a f t e r transplantation 1 Note: 1 Myeloperoxidase a c t i v i t y i s expressed as the change i n o p t i c a l density i n 30 seconds at 515 mp. as described ^ i n Methods -39-The r e l a t i o n s h i p of protoporphyrin concentration and myelo-peroxidase a c t i v i t y to the growth of the chloroma i s shown i n table 4. The protoporphyrin concentration i n the tumour ([j,g protoporphyrin/gm tumour) increased markedly suggesting an accumulation of the protoporphyrin i n the tumour. The amount of protoporphyrin i n the tumour (jig protoporphyrin/tumour) was not d i r e c t l y proportional to the tumour weight but rather increased markedly even when the tumour was becoming necrotic and hemorrhagic i . e . days 23-25 ( f i g 10). The myeloperoxidase activity/gm tumour also increased quite markedly during the early period of rapid tumour growth reaching a maximum on the 17*th day a f t e r transplantation (table 4 and f i g 11). However, the myeloperoxidase a c t i v i t y then declined r a p i d l y to a value which i s less than 30% of the maximum around the 23rd day after the transplantation. Figure 11 also shows the t o t a l myeloperoxi-dase a c t i v i t y i n the tumour. The s l i g h t increase i n myeloperoxi-dase per tumour on day 23 i s r e l a t e d to the increase i n t o t a l tumour mass rather than increased enzyme concentration. In view of Greengard's work on the a c t i v a t i o n of the r a t l i v e r enzyme tryptophan pyrrolase with hematin (56), the e f f e c t of hematin on the myeloperoxidase a c t i v i t y of the tumour was studied, p a r t i c u l a r l y to see i f the 'low' peroxidase a c t i v i t y of the older tumours e.g. day 23, could be stimulated by the addition of hematin. I t was found that hematin, even at a concentration of 7.5 \M had no e f f e c t on the myeloperoxidase a c t i v i t y of the tumour extracts (Greengard found that hematin FIGURE 12 Chromatography of chloroma protoporphyrin on alumina i n dim l i g h t c o •H b O CJ u C cu u o O 10 20 30 J*o 50 Fraction Number FIGURE 13 Chromatography of chloroma protoporphyrin on alumina i n normal l i g h t i n g of the laboratory c o •H 60 CU T3 CJ tO 4-» CU U o » o F r a c t i o n Number -40-at concentrations as low as 0.5 \M stimulated the a c t i v i t y of r a t l i v e r tryptophan pyrrolase). The r e s u l t with hematin i s therefore rather inconclusive since i t i s not known i f hematin w i l l act as the prosthetic group of the myeloperoxidase. I I I . Incorporation of glycine-2-C^^ into the protoporphyrin  and the nucleic acids of the chloroma. Since glycine i s a common precursor of both protoporphyrin and nucleic acids, i t was used to study the rate of synthesis of these compounds i n the tumour and to see i f there was any c o r r e l a t i o n between their rate of synthesis and the age of the tumour. 14 6 Radioactive glycine-2-C (1.5 x 10 cpm) was injected i n t r a p e r i t o n e a l l y into chloroleukemic r a t s i n pairs on days 13, 15, 17, 19, 21 and 23 af t e r transplantation. Five hours afte r the i n j e c t i o n , the animals were k i l l e d and the incorpora-t i o n of glycine-2-C-1-4 into the protoporphyrin and the nucleic ac i d fractions then measured as described i n the experimental section. (1) I s o l a t i o n of radioactive protoporphyrin The porphyrins were extracted from the tumour and converted to the esters and t h e i r r a d i o a c t i v i t i e s determined. The crude esters were then chromatographed on alumina as described. The porphyrin ester from the tumour was eluted from the column i n a p o s i t i o n corresponding to authentic protoporphyrin dimethyl ester ( f i g 12). Its absorption spectrum was also i d e n t i c a l FIGURE 14 Absorpt ion Spectrum of material In the 'secondary' peak (Fraction 30, E i g 13) -41-with that of the pure dimethyl ester. There was only a very small amount of coproporphyrin i n the tumour (ca VL of the protoporphyrin). The r a d i o a c t i v i t y of the porphyrin ester fractions from the column coincided with the o p t i c a l density of the e f f l u e n t . A small radioactive peak (peak a) appeared between fr a c t i o n s 28 and 33. Although absorbing at 405 mo. these fractions did not fluoresce i n U.V. l i g h t . This secondary peak (peak a) was found i n every chromatographic run of the porphyrin ester. However, i t s height varied from run to run, and i t was suspected that i t might be due to a photo-oxidation product of the protoporphyrin ester (18). In support of t h i s suggestion, i t was found that i f the ester was prepared and chromatographed i n the normal l i g h t i n g of the laboratory, the peak (peak a') was much greater than i f the experiment was performed with another portion of the same sample i n near darkness ( f i g 13). The spectrum of the effluents comprising the non-fluorescent peak (peak a) d i f f e r e d from that of authentic protoporphyrin (fractions 34 to 43) i n having the Soret band displaced to 405 mjj, and two secondary peaks i n the v i s i b l e (instead of the normal Soret band at 407m(i, and four secondary peaks i n the v i s i b l e ) ( f i g 14). However, the r a d i o a c t i v i t y per uni t o p t i c a l density of the material i n peaks a and a' ( f i g 12 and 13) was the same irr e s p e c t i v e of whether th i s experiment was done i n bright or dim l i g h t . This indicated that the small peak a was derived from the protoporphyrin (or i t s ester) during manipulation and that i t s formation could be greatly reduced i f care i s taken TABLE 5 E f f e c t of age of tumour on g l y c i n e - 2 - C ^ incorporation* into the protoporphyrin and nucleic acids of r a t chloroma. Days a f t e r inoculation Average wt of tumour (gms) Protoporphyrin (cpm/gm tumour)+ RNA DNA (cpm/gm tumour x IO" 2) 13 5|J0 255 215 ' 90 15 15.8 340 100 40 , 17 17.6 370 15.8 70 19 29.0 425 113 72 21 33.5 200 77 27 23 49.4 200 57 18 Number of ra t s per group =2 *Glycine-2-C'1-^ was injected i n t r a p e r i t o n e a l l y into each animal on the days s p e c i f i e d and the animals were k i l l e d 5 hours aft e r the i n j e c t i o n . "^Protoporphyrin of the tumour was measured as the protoporphyrin ester. -42-to exclude l i g h t . I t i s therefore an a r t e f a c t of the method rather than of biochemical o r i g i n . (2) E f f e c t of age of tumour on the rate of protoporphyrin and  nucleic acids synthesis by the chloroma i n vivo. The l a b e l l i n g of the nucleic acids and porphyrins by g l y c i n e - 2 - G ^ i n the chloroma on days 13, 15, 17, 19, 21 and 23 aft e r transplantation i s shown i n table 5. The rate of proto-porphyrin synthesis (expressed as cpm protoporphyrin/gm tumour) i n the tumour increased s t e a d i l y over the period 13 to 19 days after transplantation and then f e l l very r a p i d l y to a constant l e v e l and much lower as the tumour aged. In contrast, i n the period immediately following tumour development i . e . at day 13, the rate of l a b e l l i n g of the nucleic acids was very high (probably coinciding with the r a p i d increase i n weight of the tumour). Apart from this i n i t i a l maximum,the rate of nucleic acidy synthesis reached a secondary maximum within the 17 to 19 day period a f t e r transplantation and thereafter r a p i d l y declined. I t would appear that t h i s early period of tumour growth (15 to 19 days) i s suitable for studying the biosynthesis of the porphyrins and the nucleic acids i n the chloroma. I t may be noted that t h i s period of 4-5 days, i n which both reproducible growth and high biosynthetic rates were obtained, i s less than o n e - f i f t h of the t o t a l tumour l i f e . IV. E f f e c t of v i n b l a s t i n e on the biosynthesis of protoporphyrin, nucleic acids and protein of the chloroma. TABLE 6 Time course of gl y c i n e - 2 - C ^ incorporation into chloroma protoporphyrin, nucleic acids and protein. Average wt of tumour (gms) Glycine-2-C 1 4 incorporation period (hrs) Pr o top orphyr i n * (cpm/gm tumour) RNA DNA (cpm/gm tumour) x 10-2 Protein (cpm/gm tumour x 10-3 9.6 1 340 51 11 96 13.1 2 1100 200 75 230 6.1 4 704 400 220 448 13.7 6 680 145 62 150 14.2 8 550 145 60 .150 Number of rats per group = 4 Age of tumour = 16 days ^Incorporation of glycine-2-C^ 4 into the protoporphyrin was measured as the protoporphyrin ester. -43-(1) Time course of glycine-2-C incorporation into chloroma  protoporphyrin, nucleic acids and protein;. The foregoing experiment established that the rate of synthesis of both the protoporphyrin and the nucleic acids i n the r a t chloroma reached a maximum between the 15 and 19 days after trans plantation. The l a b e l l i n g of these compounds afte r a 5 hour g l y c i n e - i n c o r p o r a t i o n period was ample for rapid counting. However, i n order to study the immediate e f f e c t of a drug (e.g. vinblastine) on the metabolism and biosynthetic reactions of the tumour, i t i s necessary to use an isotope incorporation period s u b s t a n t i a l l y shorter than the time over which the drug acts. At the same time the l a b e l l i n g of the products e.g. porphyrins, nucleic acids and protein; , has to be high enough for accurate counting. The degree of l a b e l l i n g of the protoporphyrin, nucleic acids and protein aft e r various periods of isotope incorporation was therefore measured. Sixteen days after inoculation, tumour-bearing rat s i n groups of four were given i n t r a p e r i t o n e a l i n j e c t i o n s of glycine-2-C^ (1.1 x lO^ cpm) . The tumours were removed at intervals of 1,2,4,6 and 8 hours after the i n j e c t i o n and the incorporation of g l y c i n e - 2 - C ^ into the protoporphyrin, nucleic acids and protein of the tumour were then determined. Table 6 shows that the incorporation of g l y c i n e - 2 - C ^ into the protoporphyrin was at a maximum 2 hours a f t e r the i n j e c t i o n by which time there were enough counts for accurate measurement TABLE 7 The e f f e c t of vi n b l a s t i n e * on the incorporation of g l y c i n e - i n t o the protoporphyrin, nucleic acids and protein of the chloroma. Group number Duration of VLB treatment (hrs) Average wt of tumour (gms) Pr o toporphyr i n * * (cpm/gm tumour) RNA DNA (cpm/gm tumour) x 10-2 Protein (cpm/gm tumour) x IO" 3 I None 10.4 577 172 43 218 (control) II 3 11.0 740 160 46 224 III 6 11.7 715 125 55 172 IV 12 11.2 572 100 33 135 V 24 11.2 329 65 17 115 . . . . Number of r a t s (wt = 125 + 5 gms) per group = 4 *Dose of VLB = 1.5 mg/kg body weight (given i n 0.7 ml saline) **Incorporation of glycine-2-C^4 into the protoporphyrin was measured i n the ester. -44-of r a d i o a c t i v i t y . The l a b e l l i n g of the nucleic acids and protein reached a maximum after 4 hours, but was high enough after 2 hours to permit accurate counting. This time i n t e r v a l was chosen therefore as the glycine incorporation time i n studying the short term e f f e c t s of v i n b l a s t i n e on the chloroma. In passing, i t may be suggested that the 4 hour period i n thi s experiment i s probably an anomaly. As seen i n table 6, the average weight of the tumour i n the 4 hour experiment was unexpectedly low. Since the same amount of radioactive glycine was injected into each animal, the a c t i v i t y of smaller tumours w i l l be extremely high and thi s may probably account, at lea s t i n part, for the much higher l a b e l l i n g i n the 4 hour period i n thi s experiment. (2) E f f e c t of VLB on g l y c i n e - 2 - C ^ incorporation into the protoporphyrin, nucleic acids and protein of the chloroma. I t was concluded from the foregoing experiment that a 2 14 hour incorporation period of glycine-2-C would be s u f f i c i e n t for accurate counting of the protoporphyrin, nucleic acids and protein fractions of the chloroma. The e f f e c t of VLB on the l a b e l l i n g of these tumour components was investigated. On the" 15th day after tumour transplantation, female Sprague-Dawley r a t s weighing 125 + 5 gms i n groups of four (Groups I I , I I I , IV, V and table 7) were given i n t r a p e r i t o n e a l i n j e c t i o n s of VLB sulphate (1.5 mg/kg body weight). At in t e r v a l s of 3,6,12 and 24 hours aft e r the i n j e c t i o n of VLB, g l y c i n e - 2 - C ^ FIGURE 15 E f f e c t of VLB on the incorporation of g l y c i n e - 2 - C 1 4 into the protoporphyrin, nucleic acids and protein of chloroma \ -45-(1.2 x 10^ cpm) was given i n t r a p e r i t o n e a l l y to each animal. A control group (group I) received 0.7 ml of d i s t i l l e d water, followed 6 hours l a t e r by the i n j e c t i o n of glycine-2-C-^ (1.2 x 10^ cpm). Two hours after the i n j e c t i o n of the radioactive glycine, the animals were k i l l e d and the tumours removed. The incorporation of the isotope into the protopor-phyrin, nucleic acids and protein fractions of the tumour was determined by the methods described i n the experimental section and the r e s u l t s are shown i n table 7. The incorporation of g l y c i n e - 2 - C ^ into the protoporphyrin and the DNA of the tumour was stimulated i n the i n i t i a l 6 hour:; period of VLB treatment aft e r which incorporation dropped below the control value. On the other hand, VLB s i g n i f i c a n t l y i n h i b i t e d the incorporation of the isotope into the RNA within 3 hours of the i n j e c t i o n of the a l k a l o i d and the incorporation continued to decline up to 24 hours of VLB treatment. The incorporation of g l y c i n e - 2 - C ^ into the protein was s l i g h t l y stimulated i n the f i r s t 3 hours of VLB treatment a f t e r which incorporation dropped below the control values. Figure 15 shows the e f f e c t of VLB on the l a b e l l i n g of the protoporphyrin, nucleic acids and protein expressed as % of the control values. V. E f f e c t of 3,5-dicarbethoxy-l,4-dihydrocollidine (DDC) on the biosynthesis of porphyrin i n the l i v e r and the chloroma. As mentioned i n the introduction, Solomon and Figge (32) TABLE 8 E f f e c t of route of DDC* administration on protoporphyrin concentration i n the l i v e r s of tumour-free r a t s . Route of administration Average wt of l i v e r s (gms) ug protoporphyrin"*" per gm l i v e r Stimulation 7o of control Control (corn o i l ) 8.4 1.4 -Intraperitoneal 6.8 6.2 340 Subcutaneous 7.2 10.0 610 Stomach tube 8.4 39.0 2700 Number of r a t s per group = 4 *Dose = 50 mg DDC i n 1 ml corn o i l given d a i l y for 5 days. "h?rotoporphyrin was estimated as the free porphyrin i n HCl. -46-found that 3,5-dicarbethpxy-l,4-dihydrocollidine (DDC) causes very marked increases i n the protoporphyrin concen-t r a t i o n i n the l i v e r s of normal (tumour-free) r a t s . I t would be of i n t e r e s t to see i f the biosynthesis of the protoporphy-r i n i n the chloroma can be s i m i l a r l y stimulated. The e f f e c t of DDC on both the l i v e r and the chloroma was therefore stu-died. (1) Conditions a f f e c t i n g the stimulation of porphyrin synthesis with DDC i n the l i v e r of tumour-free rat s  i n v i v o . i ) E f f e c t of route of administration of DDC 1.0 ml of DDC suspension (50 mg DDC/ml corn o i l ) was given d a i l y over a period of 5 days either subcutaneously, i n t r a p e r i t o n e a l l y or by feeding with a stomach tube to female tumour-free Sprague-Dawley r a t s (weight: 120 + 5 gms) i n groups of four. The s i t e of subcutaneous i n j e c t i o n of the suspension was varied d a i l y to avoid the formation of l o c a l i z e d cysts which might hinder the absorption of the drug. After 5 days of DDC treatment the r a t s were k i l l e d and the protoporphyrin i n the l i v e r s of each group determined. From the data i n table 8, i t may be noted that whereas intrap e r i t o n e a l and subcutaneous injections of DDC gave 4-fold and 7-fold increases i n l i v e r protoporphyrin concentration, the administration of DDC by stomach tube caused a 28-fold increase i n l i v e r protoporphyrin concentration compared to TABLE 9 E f f e c t of DDC on the concentration of protoporphyrin and rate of protoporphyrin synthesis from glycine-2-C^ 4 i n l i v e r s of tumour-free r a t s . Duration of DDC treatment (days) [xg protoporphyrin* per gm l i v e r R a dioactivity of protoporphyrin cpm per gm l i v e r Control (corn o i l ) 1.50 65 2 6.00 4340 ! 4 30.00 5400 i 7 i 38.50 930 1 ^Protoporphyrin was estimated as the free porphyrin i n HCl. -47-the group which had received only corn o i l . In the subse-quent experiments^ the DDC was therefore given by stomach tube, i i ) Protoporphyrin concentration and i t s rate of synthesis i n l i v e r s of tumour-free r a t s receiving DDC by stomach tube. Groups of female tumour-free Sprague-Dawley r a t s (wt: 120 + 5 gms) were given 1.0 ml of DDC corn o i l suspension (50 mg/ml corn o i l ) d a i l y for periods of 2,4 and 7 days by stomach tube. At the end of these periods glycine-2-C14 ^2.2 x 10^ cpm) was injected i n t r a p e r i t o n e a l l y into each r a t . Five hours l a t e r , the animals were k i l l e d and the protoporphyrin concentration i n the l i v e r was determined and the r a d i o a c t i v i t y of the protoporphyrin measured. As shown i n table 9, the concentration of protoporphyrin i n the l i v e r was increasing throughout the 7 day period of DDC treatment. The very marked increase between days 2-4 corresponds to the period during which the rate of protoporphy-14 r i n synthesis was very high as shown by the C data. However^ the rate of synthesis from glycine-2-C^ 4 had f a l l e n to less than 20% of the maximum during the l a t t e r part of this period (4-7 days) even though the concentration of protoporphyrin was s t i l l increasing. (2) E f f e c t of DDC on porphyrin synthesis i n the l i v e r and the  tumour of chloroleukemic r a t s . The fore-going experiments showed that DDC administration caused a marked increase i n the l i v e r protoporphyrin concentra-t i o n of non-tumour bearing r a t s , and established a suitable TABLE 10 The e f f e c t of DDC"*" on the urinary excretion of porphobilinogen (PBG) i n tumour-free and chloroleukemic r a t s . Duration of DDC treatment (Days) Tumour-free rats PBG u- mo les/24 hr urine Chloroleukemic r a t s * PBG (j, moles/24 hr urine 0 (Control)** 1 2 3 1.25 4.8 28.5 31.5 55.8 17.0 4.8 6.7 15.9 50.7 73.6 33.6 Number of rats per group = 4 +Dose = 50 mg DDC i n 1 ml corn o i l given d a i l y for 5 days by stomach tube. *Tumour was 18 days old at s t a r t of DDC administration. **Twenty-four hour urine was co l l e c t e d i n the control group and corn o i l was then given d a i l y for 5 days i n these animals which also serve as the control group for the estimation of protoporphyrin concentration (see table 11). -48-requirement for administering the compound. The e f f e c t of DDC on the protoporphyrin concentration i n the l i v e r and tumour of chloroleukemic rat s was investigated. Disturb-ances i n control mechanisms operating i n the tetrapyrrole synthesis have been found to r e s u l t i n an abnormally high excretion of porphobilinogen - a precursor i n the biosynthesis of porphyrins (32). The e f f e c t of DDC on the urinary excretion of porphobilinogen was therefore examined i n both tumour-free and tumour-bearing r a t s . On day 18 aft e r inoculation, tumour-bearing rats were given 50 mgs DDC i n 1.0 ml corn o i l for 5 days. A group of tumour-free rat s was also given the same amount of DDC d a i l y for 5 days. The d a i l y urinary excretion of porphobilinogen i n both groups was determined using the method of Vahlquist (53). At the end of the 5 day treatment, the protoporphyrin concentration i n the l i v e r s and the tumours of both groups was estimated. I t may be noted (table 10) that i n the absence of DDC treatment, the chloroleukemic rat s excreted nearly 4 times as much porphobilinogen i n the urine as the control animals. Following the administration of DDC, the urinary excretion of porphobilinogen i n both the tumour-free and tumour-bearing r a t s rose to a maximum on the 4th day of DDC treatment; thereafter, the amount of the compound i n the urine decreased markedly even though DDC was s t i l l being given. With the exception of the 2nd c o l l e c t i o n , the d a i l y urinary excretion TABLE 11 The e f f e c t of DDC* on protoporphyrin accumulation i n tumour-free and chloroleukemic r a t s . Treatment (5 days) Average wt of tissue Liver tumour Protoporphyrin** p,g per gm tissue Liver Tumour Tumour-free r a t s Corn o i l 7.3 - 1.5 -DDC 8.1 - 36.0 -Chloroleukemic r a t s * Corn o i l 6.9 29.4 1.3 13.4 DDC 7.6 32.0 20.0 12.0 Number of r a t s per group = 4 ^Dose = 50 mg DDC i n 1 ml corn o i l given d a i l y for 5 days by stomach tube. *Tumour was 18 days old at s t a r t of DDC administration. **Protoporphyrin i n the l i v e r and the tumour was estimated as the free porphyrin, j -49-of t h i s porphyrin precursor was s i g n i f i c a n t l y higher i n the chloroleukemic r a t s . The peak of urinary porphobilinogen excretion on the 4th day of DDC treatment probably correspond-ed to a period during which the c o n t r o l l i n g mechanism i n porphyrin biosynthesis was impaired to a large extent, allow-ing precursors (and porphyrins) to be formed continuously at a maximum r a t e . The decrease i n urinary excretion of porpho-bilinogen a f t e r the 4th day may be correlated with the f a l l i n the rate of l i v e r protoporphyrin synthesis which also occurred a f t e r DDC had been given for 4 days (table 9). Table 11 shows the protoporphyrin concentration i n the l i v e r s and tumours of both groups at the end of 5 days treatment with DDC. The protoporphyrin concentration i n the l i v e r s of the DDC treated tumour-free and chloroleukemic rats was increased 24-fold and 15-fold respectively over the corresponding groups which received corn o i l only. However, i n spite of the large increase i n the concentration of protoporphyrin i n the l i v e r of the tumour-bearing r a t s there was no concomitant increase i n the protoporphyrin of the tumour i t s e l f . (3) E f f e c t of DDC on the induction of A-aminolevulinic acid  synthetase i n r a t s . A - a m i n o l e v u l i n i c acid synthetase i s the f i r s t enzyme i n the biosynthetic chain leading to the A - a m i n o l e v u l i n i c acid and ultimately the porphyrins. Granick (34) has shown that TABLE 12 The induction of A-aminolevulinic a c i d synthetase by DDC i n tumour-free and chloroleukemic r a t s . Treatment Average wt of tissue (gms) Liver Tumour [X moles A -ALA produced i n enzyme assay* Liver Tumour ug protoporphyrin** per gm tissue Liver Tumour Tumour-free r a t s Corn o i l DDC Chloroleukemic rats+ Corn o i l DDC 8.6 7.8 7.5 23.3 8.2 26.4 0.1 100.0 0.1 0.5 76.0 11.4 1.5 1.5 1.4 .12.8 1.4 12.0 Number of r a t s per group = 3 Age of tumour = 18 days /\ -ALA = A-aminolevulinic acid * A -aminolevulinic a c i d synthetase a c t i v i t y was estimated by the A-ALA produced when homogenates of the tissues were incubated as described i n the experimental section. **Protoporphyrin was estimated as the free porphyrin i n HCl. -50-DDC markedly stimulates the a c t i v i t y of t h i s enzyme i n the l i v e r of guinea pigs. The e f f e c t of DDC on the induction of ^ - a m i n o l e v u l i n i c acid synthetase i n the tumours and l i v e r s of chloroleukemic and tumour-free r a t s has therefore been studied. Granick found that the increase i n enzyme a c t i v i t y could be detected within 24 hours i f f a i r l y large doses of DDC were given - e.g. 1.5 gm DDC/kg and t h i s dose has been used i n the present experiment. Tumour-free and chloroleukemic r a t s (wt = 130 + 5 gms) were each given 200 mgs DDC i n 1.0 ml corn o i l by stomach tube. Twenty-four hours a f t e r the administration of the drug, the animals were k i l l e d , and the £-aminolevulinic ac i d synthetase a c t i v i t y i n both the l i v e r s and the tumour were measured by the method of Mauzerall and Granick (54) as described i n the experimental section. Table 12 shows that the ^ - a m i n o l e v u l i n i c a c i d synthetase a c t i v i t i e s i n the l i v e r s of tumour-free and chloroleukemic r a t s were stimulated 1000 and 760-fold r e s p e c t i v e l y by the administration of DDC. In contrast to the marked increase i n the a c t i v i t y of the enzyme i n the l i v e r of chloroleukemic r a t s , the enzyme a c t i v i t y i n the tumour was only stimulated 23-fold. DDC therefore was far more e f f e c t i v e i n the l i v e r than i n the tumour for the induction of the enzyme. I t may be noted that i n spite of the increase of the synthetase a c t i v i t y i n the l i v e r s and tumours at 24 hours, there was at t h i s time no increase i n the protoporphyrin concentration -51-of either tissue ( c f . the s i g n i f i c a n t increase i n l i v e r protoporphyrin concentration i n 48 hours, table 9). -52-DISCUSSION The o r i g i n a l chloroleukemic l i n e i n the Sprague-Dawley r a t was transplanted into weanling r a t s and the average s u r v i v a l time was approximately 42 days (4). The chloro-leukemic l i n e i n our laboratory i s unique i n that, not only i s the average s u r v i v a l time r e l a t i v e l y short (27 days, table 2) but the tumour can also be r e a d i l y transplanted into adult r a t s . Successful transplant of the tumour was over 90% and there was consistent tumour development from group to group. These b i o l o g i c a l findings suggest that the tumour might be very u s e f u l for studying the metabolism and biology of malignancies r e l a t e d to the hematopoietic system (e.g. bone marrow). There i s evidence that some chloroleukemia are of v i r a l o r i g i n . G r a f f i , (55) for example, i s o l a t e d from tumour extracts by f i l t r a t i o n p a r t i c l e s 50 to 150 mu i n diameter with which he was able to transmit the disease. However, i n the present work (table 3), although tumour homogenates, whole blood and a s c i t i c f l u i d containing whole c e l l s a l l produced p o s i t i v e transplants, c e l l - f r e e f i l t r a t e s obtained by passing the a s c i t i c f l u i d through a ' M i l l i p o r e ' f i l t e r with a pore size of 450 mu did not give r i s e to tumours. Since p a r t i c l e s considerably larger than those described by G r a f f i would pass through th i s f i l t e r , i t appears that i n t a c t c e l l s are required for successful transmission of t h i s -53-chloroma. The tumour being used i n the present work there-fore resembles the chloroleukemia of Zipf et a l (4) which i n weanling Sprague-Dawley rats could only be transmitted by i n t a c t c e l l s and not by leukemic c e l l p a r t i c l e s or sub-c e l l u l a r f i l t r a t e s . The b i o l o g i c a l data on tumour growth (table 4) show that the chloroma has a 10 to 12 day period of apparent quiescence during which the injected malignant c e l l s may l i e dormant. Immediately a f t e r the "latent period' the tumour grew r a p i d l y u n t i l the death of the animal (ca day 27). Although the size of the tumour was small during the early period of growth, i t grew to about one-third of the t o t a l body weight by day 25 a f t e r the transplantation. The b i o l o g i c a l and biochemical state of the c e l l s during the 'latent period' i s not known nor has the nature of the stimulus which then leads to r a p i d c e l l p r o l i f e r a t i o n been elucidated. The present findings on tumour growth are s i g n i f i c a n t i n r e l a t i o n s h i p to the e f f e c t of v i n b l a s t i n e on the development of the tumour. Noble (38) found that chloroleukemia i n the Sprague-Dawley r a t s i s very sensitive to VLB treatment. I f r a t s were treated with VLB over the period of 1 to 13 days af t e r transplantation of the tumour, t o t a l l y 'cured' r a t s could be r e a d i l y produced; i f VLB treatment was delayed to the 14th day, there were no i n d e f i n i t e survivors. I t would now appear from the r e s u l t s (table 4 and f i g 10) that the -54-a l k a l o i d a f f e c t s the c e l l s only during the 'latent period' when there i s no detectable c e l l growth. Vinblastine i s a very powerful mitoti c poison - a r r e s t i n g d i v i d i n g c e l l s i n the metaphase (57) and i t s anti-tumour action has been ascribed to t h i s property. However, since the chloroma c e l l s are s e n s i t i v e to VLB when their rate of d i v i s i o n i s extremely low ('latent period') and almost i n s e n s i t i v e when they are r a p i d l y p r o l i f e r a t i n g , i t would appear u n l i k e l y that the a l k a l o i d i n h i b i t s tumour growth s o l e l y on the basis of i t s a n t i - m i t o t i c a c t i v i t y . From the point of view of studying VLB therapy on the chloroma i t i s disappointing since the tumour i s only s e n s i t i v e to the a l k a l o i d during the 'latent period'. The period of steady tumour growth (day 15 to 19, table 4 and f i g 10) appears to be very suitable for biochemical investigations and for studying the e f f e c t of drugs on the metabolism of the chloroma. During t h i s period the tumour i s metabolically active (table 5) and although of reasonable size i t s n u t r i t i o n a l requirements can apparently s t i l l be met by metabolites from external sources. Tumours over 20 days old tended to become necrotic and hemorrhagic and were probably outstripping the supply of metabolites; their biochemical a c t i v i t y (table 5) also declined. Chloromas c h a r a c t e r i s t i c a l l y contain s i g n i f i c a n t amounts of free porphyrin and have high myeloperoxidase a c t i v i t y . -55-Although the structure of the prosthetic group of the peroxidase has not been established with certainty, i t appears to be a porphyrin derivative (16) showing some s i m i l a r i t y to the prosthetic group of choleglobin. In addition, i n U.V. l i g h t p u r i f i e d myeloperoxidase shows the t y p i c a l bright red fluorescence of the porphyrins. There may be therefore more than a casual r e l a t i o n s h i p between the free protoporphyrin and the myeloperoxidase i n the chloroma. The concentration of protoporphyrin i n the chloroma ( i . e . |xg protoporphyrin/gm tumour) increased very markedly as the tumour aged and grew i n size (table 4). On the other hand, the rate of protoporphyrin synthesis declined somewhat afte r the 19th day (table 5). This indicates that the protoporphyrin concentration increases because i t s rate of u t i l i z a t i o n has diminished. In mammal-ian tissues the most important outlet for the protoporphyrin i s i n the biosynthesis of heme of which i t i s the immediate precursor ( f i g 2). A block i n t h i s ferro-chelatase mediated reaction might, therefore be expected to lead to an accumula-tio n of the protoporphyrin. In contrast to the continuous increase i n the protoporphyrin concentration throughout the l i f e span of the tumour, the myeloperoxidase a c t i v i t y per gm tumour only increased markedly between days 13 to 15 af t e r transplantation and thereafter f e l l very sharply (table 4 and f i g 11). Myeloperoxidase may e x i s t i n vivo i n both -56-active and ina c t i v e form e.g. Apoenzyme (inactive) +. Prosthetic group ^ Holoenzyme(Active) (Protein) (Probably a porphyrin deriva-tive) The decrease i n myeloperoxidase a c t i v i t y as the tumour ages may be r e l a t e d to a block i n the synthesis of the prosthetic group from protoporphyrin; a l t e r n a t i v e l y the synthesis of the apoenzyme may have been impaired. I t i s not possible i n the present evidence to make a choice between these alterna-tives . I t was found by Richards et al (39) and by Beer (58) that v i n b l a s t i n e i n h i b i t s very profoundly the synthesis of DNA i n the thymus and the bone marrow of rodents re c e i v i n g the drug. RNA synthesis was not affected to nearly the same •i i * degree and i n v i t r o studies indicated that the a l k a l o i d may in t e r f e r e with the synthesis or u t i l i z a t i o n of the deoxy-nucleotides. On the other hand, Creasey et a l (59,60) found that i n the E h r l i c h a s c i t e s carcinoma the a l k a l o i d i n h i b i t e d p a r t i c u l a r l y the synthesis of soluble RNA and had only a minor e f f e c t on the DNA synthesis. They assumed that the oncolytic a c t i v i t y of VLB was r e l a t e d to i t s a n t i - m i t o t i c properties and speculates that the lack of s-RNA might i n t e r f e r e e s p e c i a l l y with the synthesis of the 'spindle' protein required for the mitotic apparatus. The two groups used d i f f e r e n t types of tissues i . e . normal (non-malignant) and malignant and t h i s may i n part account for the apparent-l -57-l y c o n f l i c t i n g r e s u l t s . Since the bone marrow of normal animals i s very sensitive to VLB (37,58), the biochemical e f f e c t of the a l k a l o i d on a tumour c l o s e l y r e l a t e d to i t e.g. the chloroma had s p e c i a l i n t e r e s t . The r e s u l t s i n table 7 and figure 15 c l e a r l y indicate that DNA synthesis i n the chloroma i s comparatively in s e n s i -t i v e to VLB i n contrast to the extreme s e n s i t i v i t y shown by normal bone marrow (37) and thymus (39). In f a c t , the incorporation of glycine-2-C-'-4 into the tumour DNA was even stimulated somewhat i n i t i a l l y and only f e l l below the control values about 9 hours a f t e r the a l k a l o i d was i n j e c t e d . The synthesis of RNA, on the other hand,was s i g n i f i c a n t l y depressed within 2 or 3 hours. Although the r e s u l t s show that the a l k a l o i d i n h i b i t s RNA synthesis before and to a greater extent than the DNA, they do not indicate i f t h i s i s the r e s u l t of interference with the DNA-dependent RNA polymerase reaction or with s-RNA formation as found by Creasey on the E h r l i c h ascites carcinoma (59,60). An analysis of the RNA f r a c t i o n would be very h e l p f u l i n answering th i s question. Considering the biochemical r e l a t i o n s h i p of RNA and protein synthesis, i t i s probably quite s i g n i f i c a n t that the decline i n the rate of protein synthesis c l o s e l y follows the RNA curve (table 7 and f i g 15). I t would seem u n l i k e l y that the i n h i b i t i o n of RNA and protein synthesis i s due to an o v e r a l l (non-specific) depression of c e l l u l a r metabolic -58-a c t i v i t y since the biosynthesis of both the DNA and the protoporphyrin are not i n h i b i t e d u n t i l much l a t e r (table 7 ,and f i g 15) . Although hematopoietic systems have been shown to be very sen s i t i v e to VLB (37, 58) the r e s u l t s obtained i n the present inves t i g a t i o n indicate that biochemically the tumour does not resemble too c l o s e l y i t s tissue of o r i g i n ( i . e . the bone marrow) i n i t s response to the a l k a l o i d . The DDC work i s another attempt to disturb s e l e c t i v e l y a biochemical process c h a r a c t e r i s t i c of the tumour i . e . the protoporphyrin and to see i f the response of the tumour c e l l s i n any way resembles that of normal t i s s u e . The preliminary studies (table 8) showed that the administration of DDC to r a t s - p a r t i c u l a r l y with stomach tube - caused a very large increase (24-fold) i n l i v e r protoporphyrin concentration. A similar although not quite marked increase (15-fold) was found i n the l i v e r s of r a t s bearing the chloroma (table 11). On the other hand, although the tumour i s c l e a r l y already equipped with the biochemical machinery for synthesizing the protoporphyrin there was no increase i n the concentration of th i s compound i n the tumour when DDC was given (table 11). This r a i s e s i n t e r e s t i n g questions on the manner i n which the metabolism of the protoporphyrin i s influenced by drugs. Various suggestions have been put forward to explain the increase i n protoporphyrin concentration i n the l i v e r s of -59-DDC-treated animals. Thus Granick (34,35) found that there was a marked increase i n ^ - a m i n o l e v u l i n i c a c i d synthetase (^-ALA synthetase) a c t i v i t y ( f i g 2) of the l i v e r s of guinea pigs r e c e i v i n g the DDC and on the basis of this he proposed that the enzyme was r a t e - l i m i t i n g i n the biosyntheticypath-way leading to the protoporphyrin. The l a t t e r then accumulated as a r e s u l t of its^nore r a p i d synthesis. Granick advanced a speculative theory that DDC may act by disturbing a repressor control on porphyrin biosynthesis (as described i n the Introduction). Labbe (36) , on the other hand, suggest-ed that the DDC i n h i b i t e d the action of the enzyme ferr o -chelatase and that the protoporphyrin accumulation increased because i t s removal was impeded. Thus the ^-ALA synthetase a c t i v i t y of the i i v e r of rats was stimulated 1000-fold by DDC (table 12). In addition, the r e s u l t s i n table 9 provide d i r e c t experimental evidence for the proposal that t h i s leads i n vivo to a greatly increased r a t e of l i v e r protoporphyrin synthesis ( y 80-fold stimulation). Nearly as great a stimulation was found i n the l i v e r of tumour-bearing r a t s i n d i c a t i n g that the tumour had not impaired the response of the host l i v e r to the drug. The f a i l u r e of DDC to increase the protoporphyrin concen-t r a t i o n of the chloroma suggests either a difference i n the biosynthetic pathway leading to the compound or that the pathway i s less sensitive to regulation by the drug. The ava i l a b l e evidence - (synthesis of protoporphyrin from -60-g l y c i n e - 2 - C ^ , table 9; presence of ^-ALA synthetase, table 12.) and of increased amounts of porphobilinogen i n the urine of tumour-bearing animals, table 10) does not indicate major differences on the pathways i n the two t i s s u e s . Furthermore, DDC also caused a 23-fold stimulation of ^-ALA synthetase a c t i v i t y of the tumour (table 12) and although this increase and the f i n a l a c t i v i t y of the enzyme i n the tumour was much less than i n the l i v e r i t probably accounts for the extra porphobilinogen excreted i n the urine of the tumour-bearing r a t s (table 10). However, since there was no concurrent increase i n the protoporphyrin of the chloroma i t may be i n f e r r e d that the A-ALA synthetase was not r a t e - l i m i t i n g i n the tumour. The r e s u l t s with VLB and DDC have i n general demonstra-ted that r e l a t i v e to normal tissues e.g. l i v e r and bone marrow, the biochemical processes studied i n the chloroma are much less s e n s i t i v e to external interference e.g. drugs. This may be r e l a t e d to the r e j e c t i o n of external biochemical controls which i s c h a r a c t e r i s t i c of many malignant ti s s u e s . An understanding of the nature of t h i s phenomenon i s important i n the development of r a t i o n a l therapy. -61-SUMMARY 1. The t r a n s p l a n t a b i l i t y , growth c h a r a c t e r i s t i c s and certain aspects of the biosynthetic a c t i v i t y of the chloroleukemia (chloroma) i n the adult Sprague-Dawley rats have been i n v e s t i -gated. 2. Successful transplantation with the chloroma homogenate was over 90% and i n t a c t c e l l s appeared to be e s s e n t i a l for the transmission of the chloroma. There was a 'latent period' of 10 to 12 days during which time there was no detectable tumour development. Thereafter, the tumour grew r a p i d l y u n t i l the death of the animals at about 27 days after the transplantation. 3. The tumour had been shown to contain substantial amounts of protoporphyrin and have high myeloperoxidase a c t i v i t y . The rate of protoporphyrin synthesis from glycine-2-C^ 4 declined quite r a p i d l y a f t e r the 19th day although the protoporphyrin concentra-t i o n increased continuously as the tumour aged. I t i s suggested that the tumour accumulated the compound as i t aged. Myeloperoxi-dase a c t i v i t y increased markedly between the 13th to 15th day and then f e l l sharply as the tumour grew. 4. Vinblastine (VLB) i n h i b i t e d the incorporation of glycine-2-C^ 4 into the RNA and protein of the tumour within 3 hours of i t s i n j e c t i o n . On the other hand, protoporphyrin and DNA synthesis was not i n h i b i t e d u n t i l about 9-12 hours aft e r the i n j e c t i o n of the a l k a l o i d . -62-5. 3,5-Dicarbethoxy-l,4-dihydrocollidine (DDC) markedly stimulated the synthesis of protoporphyrin i n the l i v e r but had no e f f e c t i n the tumour. 6. I t was concluded that the chloroma was not p a r t i c u l a r l y sensitive to the action of VLB and DDC. 7 e A comparison of the e x i s t i n g methods for separating porphyrins was examined and an improved method for estimating porphyrin esters based on p a r t i t i o n chromatography on alumina was developed. -63-BIBLIOGRAPHY 1. Humble, J.G. Aetiology of chloroma and'the nature of the green pigment. Quart. J . Med. 39:299-312 (1946). 2. Thomas, J. B u l l . Soc. chim. b i o l . 20:1058 (1938). ,3. Cowden, R.N. and M i l l e r , M. B i o l o g i c a l Research Quarterly Progress Report, Mound Laboratory, Miamisburg, Ohio. Report No. MLM 989, June 1954. 4. 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