The effects of testosterone, 5α-dihydrotestosterone and adenosine 3′,5′-monophosphate on cell proliferation and differentiation in RAT prostate

Biocbimica et Biophysica Acta, 308 (1973) 426-437 ~C) Elsevier Scientific Publishing C o m p a n y , A m s t e r d a m - Printed in T h e N e t h e r l a n d s BBA 97657

T H E EFFECTS OF T E S T O S T E R O N E , 5 ~ - D i H Y D R O T E S T O S T E R O N E A N D A D E N O S I N E Y , 5 ' - M O N O P H O S P H A T E ON C E L L P R O L I F E R A T I O N A N D D I F F E R E N T I A T I O N IN RAT P R O S T A T E

BARRY LESSER and NICHOLAS BRUCHOVSKY

Department of Medicine, University of Alberta, Edmonton, Alberta (Canada) (Received D e c e m b e r 4th, 1972)

SUMMARY

The ability of testosterone, 5c~-dihydrotestosterone and adenosine 3',5'-monophosphate to stimulate the regeneration of prostate in castrated rats has been studied using D N A synthesis, nuclei per prostate and wet weight as parameters of tissue growth. Male rats castrated 7 days previously were treated daily with subcutaneous injections of either testosterone or 5c~-dihydrotestosterone. 24-36 h after the beginning of treatment with 5e-dihydrotestosterone, the rates of D N A synthesis and of cell production increased sharply above those observed in control animals. These elevated rates were maintained for 3-4 days, and by the 5th day the number of cells per prostate reached a normal level. After this time, despite continued administration of hormones, the rate of cell production fell markedly, and the rate of D N A synthesis continued to decline until Day 14 when it reached a negligible level. The growth response induced by testosterone was considerably slower than that by 5~-dihydrotestosterone, indicating that the former is a less potent androgen. Adenosine Y,5'-monophosphate does not appear to be involved in the effect of androgens on cell proliferation.

INTRODUCTION

The male sex accessory organs, whose growth and function are under the direct control of androgens, can potentially serve as an excellent model system for the study of the regulation of cell proliferation by steroid hormones. Burkhart 1 demonstrated that a single injection of 0.1 mg testosterone propionate to 40-day castrated rats produces a wave of mitotic activity in both prostate and seminal vesicles. Later studies by Cavazos and Melampy z confirmed these results in rats while Allen 3 obtained similar findings in target tissues of mice. More recently, in analyzing the effect of testosterone propionate on mouse prostate in terms of cell renewal rate and mitotic cycle, Tuohimaa and Niemi 4 observed that the cell cycle is greatly shortened after the administration of testosterone. Sheppard e t al. 5 were the first to describe the stimulatory action of androgens on D N A synthesis in target organs. Coffey e t al. 6 demonstrated that testosterone

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propionate administered daily to rats induces a large increase in DNA polymerase activity of soluble prostate extracts and in incorporation of radioactive thymidine into prostatic cells both in vivo and in vitro. This effect was found to be accompanied by an increase in prostatic weight and DNA content and to be transitory despite continued administration ot hormone. Doeg et al. 7 using rat prostate and seminal vesicle and Geigel et al. s studying hamster prostate, seminal vesicle and flank organ, have confirmed these findings. The finding by Bruchovsky and Wilson9 and Anderson and Liao ~° that 5ctdihydrotestosterone is selectively concentrated by rat prostatic nuclei after an intravenous injection of labelled testosterone has focused much attention on this metabolite as the androgen directly responsible at the cellular level for certain androgenic effects. Robel et al. 1 ~ have found that 5~-dihydrotestosterone is not only more potent than testosterone in maintaining epithelial height and secretory activity in prostatic explants in culture, but is also more effective in stimulating epithelial hyperplasia. Schmidt e t al. 12 have reported that 5~-dihydrotestosterone has a greater effect than testosterone on the wet weight and DNA content ot prostate and seminal vesicles of immature castrate rats. Despite the considerable body of information on the growth-promoting effects of androgens, knowledge of the sequential changes in different parameters of growth for a particular target organ is surprisingly incomplete. Consequently, we have undertaken studies to characterize the proliferative and functional responses of rat prostate in greater detail. In this work, incorporation of [Me-3H]thymidine into DNA and changes in number of nuclei per prostate have been used as measures of cell proliferation. These parameters have been correlated with wet weight of tissue on the assumption that the latter is an appropriate indicator of functional state. Further, the kinetics of the growth response have been studied with autoradiographic techniques in order to obtain an estimate of the fraction of cells involved in the regeneration of prostate. Finally this system was used to compare the potency of testosterone and 5c~-dihydrotestosterone, and to determine whether adenosine 3',5'-monophosphate is a mediator of the proliferative response induced by these agents. MATERIALS AND METHODS

Animals

Adult male Wistar rats (270-340 g) were purchased from Woodlyn Laboratories, Guelph, Ontario, and maintained on a diet of Rockland rat chow and water a d l i b i t u m . Orchiectomy was performed via the scrotal route under diethyl ether anesthesia. Animals were injected subcutaneously with various doses of testosterone or 5a-dihydrotestosterone (Steraloids, Pawling, N.Y.) in about 0.6 ml of an aqueous medium containing (v/v) 10 % ethanol+ 10 % polyoxyethylene sorbitan monopalmitate (Tween 40, Sigma Chemical Co., St. Louis, Mo., U.S.A.). Controls received equal volumes ot vehicle. In the adenosine 3',5'-monophosphate experiments, both adenosine 3',5'-monophosphate (Sigma) and theophylline (l,3-dimethylxanthine; Merck, Sharp and Dohme, Pointe Claire, Quebec, Canada) were injected intraperitoneally in a total volume of 2 ml 0.9 % NaC1. After varying periods of treatments the rats were killed by decapitation and prostates quickly removed, stripped of connective tissue, placed on ice and weighed. Three to seven prostates, depending on their size,

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were pooled for each experiment, and all points represent at least triplicate determinations.

Incorporation of [Me-3H]thymidine in vitro The prostates were chopped with a Sorvall TC-2 Tissue Sectioner (Sorvall, Norwalk, Conn., U.S.A.) and washed with ice cold phosphate-buffered saline 13. The tissue (250-900 rag) was resuspended in 5 ml Eagle's minimal essential medium (pH 7.4) (Grand Island Biological Co., Berkeley, Calif., U.S.A.; Catalogue No. F-11) at 37 °C and incubated with gentle shaking in an atmosphere of CO2-O2 (5:95, v/v). After 5 min of temperature equilibration, 5 ml of the above medium containing 10 #Ci [Me-3H]thymidine/ml was added to give a radioactivity concentration of 5 ttCi/ml and a thymidine concentration of 0.02 mM in a total volume of 10 ml. Following a 20-min incubation, incorporation was terminated by pouring the reaction mixture over crushed ice and diluting it with ice cold phosphate-buffered saline. The suspension was centrifuged in a Sorvall GLC-I centrifuge (HL-4 rotor, Ray 12.5 cm) at 4 0 0 × 9 for 5 min and the tissue washed once with ice cold phosphate-buffered saline. Incorporation was determined to p~oceed linearly for at least 40 min after a lag period of 5 min and maximal incorporation was achieved with a radioactivity concentration of 5/tCi/ml.

Incorporation of [Me-3 H]thymidine in vivo Animals were injected intraperitoneally with a dose of 50 pCi/ [Me-3H]thymi dine per 100 g body weight in a volume of about 0.3 ml distilled water 40 min prior to decapitation. The prostates were removed, placed on ice, weighed, chopped as above, and washed once with ice cold phosphate-buffered saline. Some prostates were sectioned and prepared for autoradiography as described below.

Preparation of tissue extracts All operations were carried out at 0-2 °C except where stated otherwise. The washed tissue was suspended in 20 ml of 0.01 M Tris-HC1 buffer (pH 7.5) containing 0.25 M sucrose, 1.5 m M CaCI 2, 0.05/~M EDTA, 5 mM MgCI z and 0.5 mM mercaptoethanol and manually homogenized in a Dounce homogenizer with 25 strokes of the loose fitting pestle. The suspension was next filtered through 3 layers oI gauze, homogenized with 10 strokes of the tight fitting pestle, and then centrifuged at 700 x g for 10 min. The supernatant was decanted and processed as the cytoplasmic fraction. The pellet was washed in 0.01 M Tris-HCl buffer (pH 7.5), containing 0.05 M NaCl, 0.05 #M EDTA, 5 mM MgCI z, 0.5 mM mercaptoethanol and resuspended in 5 ml of the same medium. The suspension was centrifuged at 40 × g for 2 rain to remove debris and the supernatant was decanted as the nuclear fraction. An aliquot of the nuclear fraction was stained with methylene blue and the number of nuclei counted using a haemocytometer. Another aliquot was prepared for autoradiography as described below. The rest of the suspension was sonicated using a Bronwill Biosonik lII (Bronwill Scientific, Rochester, N.Y., U.S.A.) with two 10-s pulses at a setting of 40. Aliquots of the cytoplasmic fraction and the nuclear sonicate were counted to determine total radioactivity present in these fractions. The rest of the fractions were brought to l0 ~ (w/v) with trichloroacetic acid. The precipitate was washed twice

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with 5ml 5°/ /o (w/v) trichloroacetic acid and digested with 1.5 ml 0.5 M NaOH at 37 °C for 90 min. A small volume (0.15 ml) of 70 ~o HC104 was added to the digest and the precipitate formed after 30 rain was extracted with 3 ml of 1.6 M HCIO4 at 70 °C for 20 rain. Aliquots of the resulting supernatant were assayed in duplicate for radioactivity with a Beckman LS-250 liquid scintillation system (Beckman Instruments Fullerton, Calif., U.S.A.) using as scintillant 6 g 2,5-diphenyloxazole (AmershamSearle Corp., Arlington Heights, II1., U.S.A.), 216 g Bio-solv (BBS-3; Beckman) and 75 ml water per 1 scintillation grade toluene (Fisher Scientific Co., Montreal, Quebec, Canada). Counting efficiency was about 30 ~ and external standardization was used to convert cpm to dpm. Backgrounds were 30-35 cpm. Aliquots were also assayed for DNA using the diphenylamine procedure of Burton 14 with calf thymus DNA (Sigma) as standard. A utoradiography Aliquots of nuclear suspension were centrifuged at 700 ×g for 5 min. The pellets were resuspended in 1 ml 10 ~ (v/v) phosphate-buffered saline for 5 rain to swell nuclei and then twice in 1 ml acetic acid-methanol (3:1, v/v). After fixation the nuclei were resuspended in 0.2 ml acetic acid-methanol, applied to microscope slides and air dried. The slides were dipped in Ilford L4 emulsion (llford, England), exposed for 3 weeks at 0 °C in total darkness, developed in Kodak D19 developer (Canadian Kodak Sales, Toronto, Ontario, Canada), and stained with haematoxylin and eosin. Alternatively, prostates were dehydrated, embedded in paraffin, and sectioned at 4 tzm thickness. "Ihe slices were applied to slides, rehydrated and dipped in Kodak NTB2 emulsion. After 4-6 weeks exposure at 0 °C, the slides were developed in Kodak Dektol and stained with haematoxylin and eosin. For determination of percentages of labelled nuclei, 500 or 1000 nuclei were counted at random 3 times, and the results averaged. Radioisotopes [Me. 3H]Thymidine at a specific activity of 50-55 Ci/mmole was purchased from New England Nuclear Corp. (Boston, Mass., U.S.A.). The purity of this material was checked monthly by chromatography on phosphoethyleneimine-cellulose plates (Brinkmann Instruments, Rexdale, Ontario, Canada) using water as the mobile phase and was considered acceptable only if it was 90 ~ or greater. RESULTS Effect of castration on the prostate The effects ot castration on prostatic weight, number of nuclei per prostate and rate of incorporation of labelled thymidine into DNA by prost3tic minces in vitro are shown in Fig 1. The earliest effect of castration is a rapid decline in incorporation of [Me-3H] thymidine to 25 ~ of normal within 24 h and this rate is maintained until Day 4 when a further decline occurs. The constant rate of incorporation between the 1st and 4th days suggests that small amounts of endogenous steroid persist during this time and are sufficient to support a low level of DNA synthesis in the prostate. By Day 6

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Fig. 1. Regression of the prostate following castration. Male rats were castrated at Day 0. At daily intervals for 10 days and then at 14 days following castration animals were sacrificed and 3-7 prostates were pooled, weighed and incubated in vitro with [Me-3H]thymidinefor 20 rain. Nuclei were then isolated and counted and incorporation of radioactivity into nuclear DNA determined. Results are expressed as mean ±S.E. for at least 3 determinations. Values obtained for normal animals are shown as the zero time points on the ordinate. G-C), incorporation of [Me-3H]thymidine;IS]---O, nuclei isolated per prostate; A - - - A wet weight of prostate. incorporation has dropped to 5 % of normal, after which this minimal level is maintained. In contrast to the rapid effects on D N A synthesis, both prostatic wei~ht and nuclear content do not drop significantly below the normal level until Day 4 and then decline to about 20 and 15 ~ of normal, respectively, by Day 7, after which very little further change occurs. Over the interval of the experiment little valiation of body weights of the rats was observed. This time course of events indicates that the primary effect of androgen deprivation on the prostate is an immediate arrest of cell proliferation (as manifested by the drop in D N A synthesis) with subsequent atrophy due to continuing cell loss flora the tissue. Light microscope observations of prostate slices indicate that the main cytological effects of castration are increased amounts of connective tissue between acini, decreased vascularity, decreased acinar size, and greatly reduced epithelial height. However, the cellular organization is maintained.

Effect o f 5ct dihydrotestosterone on the regeneration of prostate Regeneration of prostatic tissue was induced in rats castrated 7 days previously with daily injections of 400/~g of 5~-dihydrotestosterone per 100 g body weight. Small changes in body weights were observed during the course of the experiment but never exceeded 10 ~ of the weights at the initiation of treatment. No differences in any of the parameters were observed between control animals treated with vehicle and untreated castrates. Fig. 2 shows that there is a latent period of 24-36 h after beginning of treatment before the incorporation of labelled thymidine into nuclear D N A can be detected in vitro. The incorporation then increases rapidly to a maximum by the 3rd day of

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Fig. 2. T h e effect o f 5~-dihydrotestosterone on the g r o w t h o f prostate. Male rats were castrated 7 days prior to the beginning o f the experiment. Starting at D a y 0, a n i m a l s were injected s u b c u t a n e o u s l y with 400 t~g o f 5~-dihydrotestosterone per 100 g b o d y weight every 24 h. After various periods o f t r e a t m e n t , g r o u p s o f 3-5 a n i m a l s were killed a n d prostates pooled. T h e prostates were weighed, t h e n m i n c e d a n d i n c u b a t e d with [ M e - a H ] t h y m i d i n e in vitro for 20 rain. Nuclei were isolated a n d counted, a n d incorporation o f radioactivity into nuclear D N A w a s determined. Results are expressed as m e a n -LS.E. for at least 3 separate determinations. O - O , i n c o r p o r a t i o n o f [Me-3H]thymidine, [ ] - - - - I , n u m b e r o f nuclei recovered per prostate; A . . . ~ , wet weight o f prostate; 0 , II, • values o f [Me-3H] t h y m i d i n e incorporation, nuclear content a n d wet weight, respectively for prostates from n o r m a l rats.

about 200 times the rate for untreated controls and 10 times the normal rate. Two factors account for the increased rates of incorporation of thymidine into DNA. Firstly, higher amounts of precursor are taken up by nuclei and secondly, the percentage of nuclear label converted to acid-insoluble material increases from less than 10 % in controls to 100 % in hormone treated tissue. By Day 10 the rate has dropped to below the normal level and by Day 14 to the level observed in control untreated castrates despite continued administration of hormone. In all cases, negligible amounts of acid insoluble material appear in the cytoplasmic fraction. The percentage of labelled nuclei as determined by autoradiography correlates directly with levels ot incorporation of thymidine (correlation coefficient 0.78). Therefore the changes in rate of incorporation are due to participation by varying numbers of nuclei rather titan variation in rate of synthesis among individual nuclei. At the peak of incorporation at Day 3, about 3 % of the nuclei are labelled. Wet weights and the number of nuclei per prostate are significantly above the control values (P < 0.01) by 36 h after beginning of treatment. These parameters increase rapidly as D N A synthesis approaches a maximum and normal levels are attained by Day 5 and Day 7, respectively. The rate of increase in numbers of nuclei

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then decreases to 1/4 the formec rate, while weights continue to increase at the same rate. By Day 14, wet weights are about twice the normal level, while nuclear number is about 30 % greater than normal. DNA content was found to be 12.23~0.57 pg/nucleus (mean zkS.E.)and no significant variations were observed throughout the period of treatment. During the period of rapid cell proliferation, between the 2nd and 5th day, the doubling time tor the cell population is about 40 h. Tuohimaa and Niemi 4 have found a cell cycle time of 20 h and an S phase of 7 h in mouse prostate proliferating in response to testosterone. Assuming that these values are reasonable estimates of the same parameters in our system, it would appear that the fraction of cells proliferating in response to 5~-dihydrotestosterone is about 50 %. In order to ascertain that the effects on inco, poration of thymidine by minces in vitro in response to in vivo administration of hormone are not an artifact of the in vitro incubation, radioactive thymidine was administered in vivo after various periods of hormone treatment, and incorporation into DNA measured during a 40rain period. Fig. 3 demonstrates that the time course in vivo is the same as that observed in vitro. When the nuclei were examined by autoradiography it was found that, as in the in vitro assay, there is excellent correlation (correlation coefficient = 0.99) between incorporation of thymidine and the percentage of labelled nuclei. At Day 3, the peak of incorporation, 13 % of the nuclei are labelled after 40 rain of exposure to isotope. Again using the cell cycle parameters of Tuohimaa and Niemi 4, this labelling index would be consistent with a growth fraction of about 37 ~.

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Fig. 3. I n c o r p o r a t i o n o f t h y m i d i n e in vivo during prostatic growth induced by 5~-dihydrotestosterone. Male rats were castrated 7 days prior to the beginning o f the experiment. T r e a t m e n t with daily doses o f 400 fig o f 5~-dihydrotestosterone per 100 g body wt was c o m m e n c e d at D a y 0. At 1 2day intervals groups o f 3-4 a n i m a l s were injected intraperitoneally with 50 ffCi o f [ M e - 3 H ] t h y m i d i n e per 100 g b o d y weight, a n d i n c o r p o r a t i o n o f label into nuclear D N A determined following a 40-rain exposure to isotope.

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While the in vitro and in vivo assays for incorporation of thymidine show parallel changes in rate of D N A synthesis during hormone treatment, the two assays are o f course not strictly comparable. At Day 3, 13 % of the nuclei are labelled in vivo but only 3 ~ in vitro. These differences are probably due to loss of cell viability prior to and during the in vitro incubation. Thus the in vitro assay can be used to measure changes in the rate of D N A synthesis as a result of various treatments, and has been used routinely in the work to be reported subsequently. However, for measurement o f absolute rates of D N A synthesis, or for the true number of cells involved, the in vivo assay must be used. Examination of prostatic slices under the light microscope reveals that hormone induced regeneration results in restoration of the normal morphology, with decreased connective tissue, large acini and tall columnar epithelium. Autoradiographs of slices do not reveal any obvious distinguishing characteristics between labelled and unlabelled cells.

Effect of a single dose of 5~-dihydrotestosterone on prostatic regeneration In order to determine the length o f time a dose of a n d r o g e n r e m a i n s effective, prostates were examined at various intervals after a d m i n i s t r a t i o n of a single dose of 400 #g o f 5~-dihydrotestosterone per 100 g body weight to 7-day castrates. The results are shown in Fig. 4. It is evident that a single dose can induce a n increased rate of D N A synthesis with the same time course, a l t h o u g h a smaller magnitude, as observed d u r i n g repeated a d m i n i s t r a t i o n . Both prostatic weight a n d nuclear n u m b e r s are stimulated to increase for at least 7 days at a b o u t half the rate produced by multiple doses o f h o r m o n e , a n d neither parameter attains the n o r m a l level.

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Fig. 4. Effect of a single dose of 5~-dihydrotestosterone on prostatic growth. Male rats were castrated 7 days prior to the beginning of the experiment. On Day 0, the rats were given an injection of 400/~g of 5~-dihydrotestosterone per 100 g body weight. At various times thereafter, 3-7 prostates were pooled, weighed and incubated in vitro with [Me-3H]thymidine for 20 min. Nuclei were then isolated and counted, and incorporation of label into nuclear DNA determined. Results are expressed as mean ±S.E. for at least 3 separate experiments. O - O , incorporation of [Me-3H]thymidine; F1-- -~, number of nuclei isolated per prostate; + - - - A , wet weight of prostate.

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Effects of testosterone on regeneration of the prostate In order to compare the relative potency of testosterone and 5~-dihydrotestosterone, 7-day castrate rats were treated with daily doses of 400 #g of testosterone per 100 g body weight for varying periods of time. During the course of the experiment the increase in body weights of the rats was at most 10 ~. Fig. 5 shows that the DNA synthetic response induced by testosterone is very broad and variable unlike the sharp peak observed with 5~-dihydrotestosterone treatment. Furthermore, the rates of increase of wet weight and nuclear content are only about 25 ~ of those elicited by 5~dihydrotestosterone, and by Day 3 both parameters are significantly less for the testosterone treated animals compared to those treated with 5~-dihydrotestosterone. There appears to be a lag period of about 7 days during which growth is very slow and then more rapid growth between the 7th and 14th days. This growth is almost as rapid as the peak rate of growth induced by 5~-dihydrotestosterone. It takes 14 days for testosterone to return wet weight and nuclear content to their normal values, while 5~-dihydrotestosterone does so in 7 and 5 days, respectively. 30C

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Fig. 5. The effect of testosterone on the growth of prostate. Male rats were castrated 7 days prior to the beginning of the experiment. Starting at Day 0, animals were injected subcutaneously with 400 #g of testosterone per 100 g body weight every 24 h. After various periods of treatment, groups o f 3-5 animals were killed and prostates pooled. The prostates were weighed, then minced and incubated with [Me-3H]thymidine in vitro for 20 rain. Nuclei were isolated and counted and incorporation of radioactivity into nuclear D N A was determined. Results are expressed as mean ± S . E . for at least three separate determinations. O - - O , incorporation of [Me-3Hlthymidine; - - - [ ] , number of nuclei isolated per prostate; & - - - & , wet weight of prostate; O, II, A, values of [Me-aH]thymidine incorporation, nuclear content and wet weight, respectively, for prostates from normal rats.

Effects of dose of androgen on rates of DNA synthesis Rats were treated daily tor 2 days with various doses of testosterone or 5~dihydrotestosterone and rate of incorporation of [Me-3H]thymidine determined in

A N D R O G E N S A N D CELL P R O L I F E R A T I O N

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vitro. The results are shown in Fig. 6. At all doses except the lowest, the values for 5~dihydrotestosterone treatment are consistently greater than those for testosterone. However, due to considerable variability in the response after this short period of treatment, the differences are statistically significant only at the highest dose tested.

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Fig. 6. The effect o f dose o f testosterone and 5~-dihydrotestosterone on the incorporation o lMe-3H]thymidine. Male rats castrated 7 days previously were given a subcutaneous injection o various doses o f testosterone or 5~-dihydrotestosterone in 10 % ethanol + 10 % Tween 40 and the injection was repeated 24 h later. 48 h after the first injection, incorporation o f lMe-3H]thymidine by prostatic minces in vitro was determined. Results are shown as mean 5:S.E. for at least 3 separate determinations. U - m , 5ct-dihydrotestosterone-treated animals; O - O , testosterone-treated animals.

Effect of adenosine 3',5'-monophosphate on prostatic growth Rats were treated daily for 2 days with intraperitoneal injections of 10 mg adenosine 3',5'-monophosphate q-10 mg theophylline. Results are shown in Table I, along with the corresponding effects of a dose of 400/~g of 5~-dihydrotestosterone TABLE I E F F E C T OF A D E N O S I N E 3",5"-MONOPHOSPHATE ON P R O S T A T I C G R O W T H 7-day castrate rats were treated with 10 m g adenosine 3'5'-monophosphate + l0 mg theophylline or with 400/~g 5ct-dihydrotestosterone per 100 g body wt at 24-h intervals. Control animals received vehicle only. 48 h after beginning o f treatment, prostates from 4 rats were pooled and weighed. Nuclei were isolated and counted, and incorporation o f [Me-aH]thymidine by minces in vitro determined. Results are expressed as m e a n ~ S.E. for triplicate determinations, and significance levels refer to the control values.

Treatment

Weight of prostate (m#)

Control 49±5 Adenosine 3',5'-monophosphate 47d_3 5 ~-Dihydrotestosterone 87 i 11 (P < 0.05) * 20 min incubation

Nuclei per prostate ( × 10 -7) 0.42±0.04 0.31±0.04 0.77 ± 0.20 (P <7 0.2)

*Radioactivity incorporated per try DNA (dpm) 4.15±0.69 4.30±0.68 716.12 ± 11.54 (P < 0.001)

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per 100 g body weight for comparison. It is evident that adenosine 3',5'-monophosphate has no direct effect on proliferation of rat prostate, while of course 5~-dihydrotestosterone has very large effects even after this short period ot treatment. DISCUSSION

It is clear from the results presented in Figs 1,2, 4 and 5 that three fundamental types of cellular response can be easily distinguished in rat prostate: firstly, the atrophy of the prostate induced by the withdrawal of androgens; secondly, the regeneration of prostate in response to the administration of steroid to castrated animals; and thirdly, cellular differentiation accompanied by the gradual decline of cell division after an extended period of hormone treatment. The regression of prostate after castration results in pint from the immediate cessation of cell renewal as indicated by a sharp decline of DNA synthesis (Fig. 1). The number of cells per prostate, however, remains constant until the 3rd day after which it begins to fall rapidly. By the 4th day, 50 ~ of the cells have disappeared and only about 15 G remain after 6 days; this fraction is then relatively constant until at least 14 days after castration. It is not clear whether the surviving cells represent a population of stem cells with the capacity to withstand androgen deprivation or whether the cells are simply random survivors. From the 1st day to the 5th day after the beginning of treatment of castrated rats with 5~-dihydrotestosterone, the primary cellular response is proliferation (Fig. 2). The phase of cell division is preceded by a latent period of 24-36 h during which it has been shown by other workers that there is synthesis of RNA 6' 15 - 17 and of protein 6,18,, 9 but not of DNA. In effect it would seem that the synthetic processes result in the production of factors required for D N A synthesis and cell multiplication. Once DNA replication and cell proliferation have started, they are maintained at a very high level for 3-4 days. During this period we estimate that only about 37-50 ~o of the cell population is ploliferating at any one time. Approx. 3 doublings of the cell population are necessary in order to restore the cellular complement of the prostate to a normal level. The source and fate of the p~oliferating cells remains obscure; however, it is within the scope of current methodology to determine whether one or both of the daughter cells ol a dividing cell re-enter the proliferative cycle or alternatively whether both daughter cells differentiate after mitosis. In the latter case non-dividing cells would continuously replace the cycling cells which differentiate after a single division. By the 5th day of 5~-dihydrotestosterone treatment, the number of cells in the prostate has reached a normal level. After this time the rate of cell production declines to about 25 ~o of its level during the preceding 4 days. This decrease is accompanied by and actually slightly preceded by, a sharp drop in the rate of DNA synthesis. In addition, whereas during the first 5 days prostatic weight and nuclear number increase in parallel, after the 5th day wet weight increases much more rapidly than the number of nuclei so that prostatic mass per cell steadily increases, suggesting cellular enlargement and production of prostatic secretions, All three of these observations indicate that about 5 days after restoration of 5a-dihydrotestosterone the cellular response changes from proliferation to differentiation. This shift is so complete that by the 14th day the level of DNA synthesis is negligible.

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Both the relative effects of various doses of testosterone and 5c~-dihydrotestosterone on stimulation of the early DNA synthetic response in regenerating prostate (Fig. 6) and more significantly the relative rates at which equal doses induce prostatic regeneration (Figs 2 and 5) suggest that 5~-dihydrotestosterone is a more potent androgen than testosterone. This finding is in agreement with the work of Schmidt e t al. 12. If one assumes that these hormones share a common mechanism and site of action, then this finding is consistent with the hypothesis that conversion of testosterone to 5c~-dihydrotestosterone is necessary for expression of the proliferative effects of this hormone. The very slow growth observed during the first week of testosterone administration may represent the time required for induction of the enzyme that converts testosterone to 5c~-dihydrotestosterone. Singhal e t al. 2° have provided evidence for the involvement of adenosine 3',5'-monophosphate as a mediator for androgenic stimulation of certain prostatic enzymes. Using the same doses and method of administration, we have found that adenosine 3',5'-monophosphate has no effect on cell proliferation in the prostate and hence does not appear to be a direct mediator of the early proliferative effects of androgens. The effects measured by Singhal e t al. 2° are essentially differentiated responses and thus our results ale not necessarily incompatible with theirs. ACKNOWLEDGEMENTS

This research was supported by grants from the Medical Research Council of Canada (MA-3729) and the National Cancer Institute of Canada. One of us (B. L.) is a recipient of a Medical Research Council Studentship and one of us (N. B.) is a Scholar of the Medical Research Council of Canada. We thank Dr W. Weinstein for assistance in carrying out autoradiographic procedures. REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

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