[CANCER RESEARCH 49, 5889-5894. November I. 1989] Gene Activity during the Early Phase of Androgen-stimulated Rat Prostate Regrowth1 Aaron E. Katz, Mitchell C. Benson, Gilbert J. Wise, Carl A. Olsson, Mark G. Bandyk, Ihor S. Sawczuk, Philip Tomashefsky, and Ralph Buttyan Department of Urology, Maimonides Medical Center, Brooklyn, New York 11219 [A. E. K., G. J. W.], and Department of Urology, Columbia University, New York, New York 10032 [M. C. B., C. A. O., M. G. B., l. S. S., P. T., R. B.] ABSTRACT Androgenic steroids regulate the proliferation rate of normal and malignant prostate cells. In order to investigate the molecular basis of this control, we utilized Northern and Western blot techniques to measure changes in the expression of individual genes during the early phase of prostate regrowth. Vestigial ventral prostate glands of 7-day castrated rats showed increased numbers of replicating cells within 12 h of contin uous pharmacological testosterone replacement as demonstrated by flow cytometric procedures. The period prior to the onset of proliferative enhancement was accompanied by the sequential induction of a variety of transcripts encoding gene products often associated with cell growth. Within l h of treatment, mature mRNA transcripts for c-fos were induced 6-fold, returning to control levels by 2 h. Other genes showed transiently elevated transcript levels after 2 h (c-Ha-ro?, c-Ki-ras) or after 8h (c-myc, c-myb, 0-actin, and M, 70,000 heat shock protein) of testosterone replacement. Expression of the polypeptide encoded by c-I la-ra.v was coordinately enhanced (2-fold) during this period, but not to the levels of the transcript (20-fold induction). Transcripts encoding basic fibroblast growth factor were increased 16 h and later, subsequent to the earlier enhancement in the proliferation rate. By placing these genes in a defined temporal order with regard to their expression in response to testosterone, we have constructed a map of gene activity during early prostate regrowth. This map shows a number of genes, the products of which might partic ipate in the mechanism by which androgens induce mitogenesis of pros tate cells. INTRODUCTION Androgenic steroids are characterized by their ability to reg ulate the gene activity of androgen-sensitive cells. Like other steroid hormones, androgens mediate their effect through an intracellular receptor protein which may stimulate (or repress) the expression of specific gene products, depending on the nature of the target cell. For some cells, androgens also have a mitogenic influence. As exemplified by the developing mam malian prostate gland, testosterone will induce the rapid growth of this organ by increasing the rate of cellular proliferation. Currently, the relationship between the androgen stimulus and the mitogenic response is unclear. Studies on other systems of inducible cell growth have demonstrated that cell proliferation is a complex process requiring the coordinated participation of multiple gene products (1, 2). Thus, in the prostate gland, the mitogenic effect of androgenic steroids is likely to be mediated by the action of a battery of gene products expressed, either directly or indirectly, under the influence of androgens. This investigation was designed to increase our understand ing of the molecular mechanism by which androgens regulate prostate growth. We have begun to catalogue the acute changes in gene activity that accompany androgen-stimulated prostate cell proliferation in a regrowing rat ventral prostate model. Using probes for gene products known to participate in cellular Received 4/3/89; revised 7/21/89; accepted 8/4/89. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This work was partially supported by a grant from the Maimonides Research and Development Foundation and from the NIH (CA47848) (R. B.). growth, we were able to quantitate the expression of a large number of genes as a function of time following androgen stimulation. The genes that we assayed for included a variety of protooncogenes (c-fos, c-myc, Ha-ras, Ki-ras, and c-myb), genes required for cellular maintenance and homeostasis (actin, tubulin, protein kinase C, and M, 70,000 heat shock protein), and genes encoding specific growth factors (c-sis and basic fibroblast growth factor). By placing these genes in a defined temporal order in terms of their acute response to testosterone, we have been able to construct a preliminary map of gene activity during androgen-stimulated proliferation of prostate cells. Although the expression of many different gene products increased during acute prostate regrowth, the intense response of a few particular genes implies the possibility that their products may drive the mitogenic response to androgens. MATERIALS AND METHODS Androgenic Manipulation of Rats. Mature (350-375 g) male Sprague- Dawley rats (Camm Industries, Wayne, NJ) were surgically castrated under sodium pentobarbitol anesthesia (35 mg/kg i.p.). Castrated rats were maintained under standard laboratory conditions for 7 days to allow regression of the ventral prostate gland. At this time, testosterone was returned by an injection of testosterone propionate (2 mg/kg i.m. in sesame oil). At various times following testosterone treatment, groups of rats were sacrificed with sodium pentobarbitol (100 mg/kg). Ventral prostate tissue was removed and frozen in liquid nitrogen for later RNA and protein extraction. Rats maintained for longer than 12 h were given repeated injections at 12-h intervals to maintain pharma cological testosterone levels during the length of the experiment (up to 60 h). Flow Cytometric Analysis of Cell Cycle Components during Testoster one Stimulation of Prostate Regrowth. Ventral prostate glands were collected from groups of rats at 7 days after castration and at specified hourly intervals after testosterone stimulation of 7-day castrated rats. Individual glands were mechanically minced into 1-mm3 pieces and the cells were dispersed with gentle stirring in a trypsin-EDTA solution (Sigma Chemical Co., St. Louis, MO). Following digestion, samples were passed through cotton gauze and were centrifuged at 3000 rpm for 10 min. The cell pellet was washed with Dulbecco's modified Eagle's medium containing 10% fetal calf serum and then incubated in a propidium iodide solution (50 ^g/ml) containing 4 HIMsodium citrate (pH 7.8), 30 units/ml DNase-free RNase, and 0.1% Triton X-100. Flow cytometric measurements were performed using a Coulter EPICS 752 flow cytometer (argon laser, 488 nm) equipped with a dedicated M DADS computer. For each analysis 50,000 nuclei were counted. PARA 1 analysis of cell cycle components was used. By this means, the percentage of nuclei in the G0 + d, S, and G2 + M phase of each sample was determined. The coefficient of variation for every sample analyzed remained below 5.0. Extraction of Poly(A)+2 mRNA and Quantitation of Transcript Levels. Pooled groups of frozen ventral prostate glands from 5 rats/time point were pulverized in liquid nitrogen and the powder was homogenized in a solution of 5 M guanidine ¡sothiocyanate-5% 2-mercaptoethanol. As described previously (3), RNA was precipitated from the homogenate 2The abbreviations used are: poly(A)* mRNA, polyadenylated mRNA; SDS, sodium dodecyl sulfate; cDNA, complementary DNA; bFGF, basic fibroblast growth factor; p21, M, 21.000 protein. 5889 on June 6, 2016. © 1989 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from