The Prostate 13:119-130 (1988)

Calcium Channel Antagonists Delay Regression of Androgen-Dependent Tissues and Suppress Gene Activity Associated With Cell Death John Connor, lhor S. Sawczuk, Mitchell C. Benson, Philip Tomashefsky, Kathleen M. OToole, Carl A. Olsson, and Ralph Buttyan

Departments of Urology (J.C., I.S.S., M.C.B., P. T., C.A.O., R.B.) and Pathology (i? T., K. M.O.), Columbia University, New York, NY

Androgen deprivation subsequent to castration of an adult male rat results in the regression of sexual accessory tissues. Regression of these tissues involves the massive death of androgen-dependent cells. Using the rat ventral prostate gland as a model to study androgen-programed cell death, we have characterized a series of molecular events that accompany its regression. This analysis has shown that there was a sequential induction of specific gene transcripts in the ventral prostate gland following castration. The first event in this cascade was an abrupt induction of transcripts encoding clfos. Since clfos expression has been linked to perturbations in intracellular Ca2+ levels, we investigated whether membrane-mediated Ca2+ flux might be an early physiological step involved in the death of prostatic cells. To test this, rats were treated simultaneously upon castration with either verapamil or nifedipine, two different calcium channel antagonist drugs. Compared to the ventral prostate glands of untreated castrated rats, the glands of the calcium channel antagonist-treated rats showed a significant delay in all parameters associated with regression (loss of wet weight and DNA content and delay in histological changes associated with prostatic regression). The seminal vesicle glands of treated rats also showed signs of delayed regression. Furthermore, calcium channel antagonists suppressed the induction of transcripts encoding both c-fos and testosterone-repressed prostate message-2 (TRPM-2), a gene expressed exclusively by dying cells, during the first 62 hr following castration. These findings further support a role for calcium ion influx in the pathway leading to hormonally programed prostate cell death, and suggest the intriguing possibility that modulation of this activity can alter the process by which cells die.

Key words: prostate gland, tissue regression, c-fos, TRPM-2, programed cell death, calcium

INTRODUCTION

Shortly following castration of an adult male rat, sexual accessory organs undergo drastic changes in secretory activity, cellular composition, and structural architecture. This process, generally referred to as glandular regression or involution,

Received for publication April 12, 1988; accepted May 17, 1988.

Address reprint requests to Ralph Buttyan, Department of Urology, Columbia University, 630 W. 168th Street, New York, NY 10032.

0 1988 Alan R. Liss, Inc.

120 Connor et al.

reflects the extensive death of androgen-dependent cells in these organs. Studies of the effects of androgen withdrawal on the rat ventral prostate gland have shown that this organ provides a particularly suitable model of androgen-dependent programed cell death. Within the first 10 days after androgen deprivation, up to 80% of the cells of the ventral prostate die [l-41. By replenishing low levels of testosterone in a castrated rat, however, prostate cells can be maintained in a vital state despite losing the ability to proliferate [ 5 ] . This observation indicates that androgens act as an antagonist of cell death in ventral prostate cells.

Molecular analysis of regressing prostate tissue has already shown that novel RNA transcripts and proteins appear abruptly during this period [6-81. Other studies which show that protein and RNA synthesis inhibitors can delay the death of androgen-dependent prostate cells [2,9] support the concept that prostate cells are actively recruited into the process of death, perhaps by the novel products synthesized in the absence of androgens. As one approach towards deciphering the mechanism by which androgen deprivation leads to prostate cell death, we have begun to catalog the acute molecular changes that occur in these cells following androgen ablation.

The earliest notable change in ventral prostate tissue after castration is a dramatic decline in nuclear androgen receptor content. Nuclear androgen receptors are reduced to undetectable levels within the first 12 hr after castration [lo]. Following this, mRNA transcript levels for genes characteristically associated with cell proliferation are induced in a sequential cascade. Transcripts encoding c-fus are transiently induced within 36 hr after castration, followed by the induction of c-myc transcripts after 48 hr and heat-shock 70K transcripts after 72 hr [ 111. Since this cascade pattern of gene activity is associated with at least three different cellular processes (proliferation, differentiation, and programed death), we have termed it a “reactive cascade.”

The expression of c-fos, the first gene induced during the reactive cascade, has been linked to increases in intracellular calcium ion levels, degradation of phospha- tidy1 inositol, and protein kinase C activation [12-141. Based on this association, we have begun to study these specific parameters to determine whether our gene expression studies allow us to predict early cellular physiological changes that accompany prostate cell death and regression. Further indication that calcium ion flux might play a role in hormonally regulated prostate cell death can be inferred from the observation that treatment of castrated rats with chloroquin can significantly delay regression of the ventral prostate gland [2]. While this phenomenon has been linked to stabilization of lysosomal membranes and a delay in autophagous events, chloroquin and related drugs are also suspected of lowering intracellular Ca2+ levels by either suppressing membrane Ca2+ flux or altering the ability of a cell to release Ca2+ from sequestered stores [ 15-17]. Therefore, in this report, we analyzed whether other drugs whose action is specifically linked to the blockade of cellular membrane Ca2+ transport can delay or prevent regression of the ventral prostate gland in the rat.

The two different types of calcium channel antagonists utilized in these experiments, nifedipine and verapamil, act by blocking calcium flux at the slow or “L”-type calcium channel [18]. When these drugs were added to the medium of qviescent cultured cells, the expression of c-fos, normally inducible by mitogens, was suppressed [ 191. Calcium channel antagonists have already shown significant value in vivo in preventing damage to renal, cardiac, and brain cells following experimentally

Calcium Channel Antagonists and Cell Death 121

induced ischemia [20-221. Our results here indicate that these types of drugs are also effective in delaying the death of cells of hormone-dependent tissues following hormonal ablation and suggest that various pathways of programed cell death share a common mechanism.

MATERIALS AND METHODS Animals and Tissue

Mature male Sprague-Dawley rats weighing 400 to 425 g (Camm Industries, Wayne, NJ) were castrated by scrota1 incision under sodium pentobarbitol anesthesia. Groups of rats were treated simultaneously upon castration with either verapamil (100 mg) or nifedipine (SO mg) in subcutaneous pellet implants designed and specified by the manufacturer (Innovative Research, Inc., Toledo, OH) to release a constant dose of drug over a 21-day period. At indicated time intervals after treatment, rats from treated and untreated groups were sacrificed with a lethal dose of sodium pentobar- bitol, and ventral prostate tissue was dissected out. Following wet weight determi- nation, specimens of ventral prostate used for histological preparations were fixed in Bouin’s solution, while specimens used for RNA and DNA extraction were frozen in liquid nitrogen and maintained at - 85°C until processing. Fixed tissues were dehydrated and embedded in paraffin before sectioning. Thin sections were rehydra- ted, then stained with hematoxylin and eosin.

DNA and RNA Extraction and Analysis

For quantitation of glandular DNA content, whole frozen ventral prostate glands were homogenized in ice-cold 0.2 M perchloric acid (PCA). The precipitate was washed twice in cold 0.2 M PCA and twice in cold acetone before being solubilized in 0.3 M NaOH at 37°C for 1 hr. DNA was precipitated following the addition of an equal volume of 0.3 M PCA and incubation on ice, then the DNA pellet was resolubilizing in 0.5 M PCA at 60°C. Aliquots of the acid extract were assayed for DNA content by the diphenylamine color assay as described by Burton [23].

RNA was extracted from frozen ventral prostate glands as has been described previously [ 111. Polyadenylated mRNA was selected from total RNA by oligo dt-cellulose chromatography, then 1 0-Fg aliquots were electrophoresed on denaturing agarose gels and transferred to charge-modified nylon filter paper as has been described [ 1 I]. The Northern blot was initially hybridized to a mixture of denatured [32P]-labeled probes for c-fos [24] and testosterone-repressed prostate message-2 (TRPM-2) [8] (at a 1O:l concentration), which were prepared by nick translation. Following autoradiography , the Northern blot was stripped of labeled probe by immersion in boiling water, then rehybridized to a probe for p-actin [ 2 S ] . Relative quantitation of band densities on Northern blot autoradiograms was performed using a Joyce-Loebel scanning densitometer with integration of absorption peaks.

RESULTS

Groups of mature male Sprague-Dawley rats (400-42s g) were treated simul- taneously at the time of castration with either verapamil (100 mg) or nifedipine

122 Connor et al.

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Fig. 1. Changes in rat ventral prostate wet weight following castration or castration with calcium channel antagonist treatment. Sprague-Dawley rats (400 g) were surgically castrated under sodium pentobarbital anesthesia. Select groups of castrated rats received simultaneous treatment with calcium channel antagonist drugs. Time-released pellets of 100 mg verapamil (B) or 50 mg nifedipine (a) were implanted subcutaneously. These pellets are designed to release a constant dose of drug over 21 days. At the indicated day after castration, rats were sacrificed and ventral prostate tissue was dissected out and weighed. Bars show the mean wet weight of ventral prostate tissue for each group of animals; error bars indicate the standard error calculated for each group, P values, as shown, were determined by the “ t” test. Parentheses above the bars indicate the number of animals in each group. Wet weights of drug-treated animals were compared to the weights of ventral prostate tissue from intact rats (m) and from untreated castrated rats (0) at equivalent time intervals.

(50 mg). Treatment was administered by subcutaneous implant of pellets designed to release an equivalent dose of drug over a 21-day period. Animals from each group were sacrificed on days 3-6 after treatment; the ventral prostate glands were removed and scored for wet weight and total DNA content. These parameters were compared with data obtained at equivalent time points from untreated castrated rats or from control intact rats. Neither drug regimen completely blocked regression. As shown in Figure 1, however, rats receiving either calcium channel antagonist drug demon- strated a statistically significant elevation (P < .05-.005 for most groups) in glandular wet weight at each of the time points compared to castrated control rats. In fact, at the first time point analyzed (3-day castrates), drug-treated animals showed no significant difference in ventral prostate wet weight compared to intact rat ventral prostate glands, while untreated castrated rats already showed a decrease to approximately half-normal glandular weight. The drug dosages chosen for this first experiment were slightly higher than the highest dosages recommended for human usage. At this dose, nifedipine appears to be a more potent inhibitor of regression compared to verapamil. Figure 2 shows a plot of ventral prostate wet weight over time for the castrated control and castrated nifedipine-treated rats. The rate of regression

Calcium Channel Antagonists and Cell Death 123

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DAYS AFTER CASTRATION

Fig. 2. Rate of regression of ventral prostate glands of untreated castrated rats or castrated rats treated with calcium channel antagonist drugs. Mean wet weights of ventral prostates from untreated castrated rats (0) or from castrated nifedipine-treated rats (O), as shown in Figure 1, were plotted over time. The relative rate of regression, as shown, was calculated from these curves.

calculated from these curves shows that prostatic regression in nifedipine-treated rats (t l12 = 7.65 days) occurred at approximately half the rate of control castrated rats (tl12 = 3.25 days).

To determine whether this apparent delay in the regression rate was due to a delay in the loss of cells rather than to drug-induced water uptake or retention, ventral prostate tissues derived from the initial experiment were subject to microscopic histological examination and were analyzed for total DNA content. Thin sections of fixed ventral prostate tissue from an intact rat show characteristic patterns of ducts dilated from the abundance of prostatic secretions (Fig. 3A). As the gland undergoes involution following castration, the secretory ducts shrink and acquire heterogeneous morphological characteristics. Those ducts near the periphery of the gland are the most sensitive to androgen withdrawal and show the first signs of involution [26]. A photomicrograph of thin sections through a peripheral region of the ventral prostate gland of a 3-day castrated rat is shown in Figure 3B. This gland shows contracted ducts with a disordered epithelial cell layer and interstitial lymphocytic infiltration typical of regressing prostate tissue [4,27]. In the peripheral ducts of the calcium antagonist-treated 3-day castrated rats (Fig. 3C,D), however, the cellular organiza- tion remains remarkably similar to that of ducts of the gland from an intact rat, although epithelial cells appear to be slightly smaller. Histological sections through regressing ventral prostate glands of the 5-day treated castrated rats showed a regression pattern similar to that of the 3-day untreated castrated rats (not shown). Thus, histological examination of regressing ventral prostate tissue from calcium channel antagonist-treated rats confirms, at the cellular level, a delay in glandular involution resulting from drug treatment.

This is also supported by maintenance of DNA levels in glands of castrated drug-treated rats. Representative glands from each group were processed to quantitate total glandular DNA content. As shown in Figure 4, the loss of DNA from the ventral prostate gland during regression was significantly retarded by the simultaneous

124 Connor et al.

Fig. 3. Histology of regressing rat ventral prostate glands. Rat ventral prostate glands dissected from intact (A), untreated 3-day castrated (B), or 3-day castrated rats treated with nifedipine (C) or verapamil (D) were fixed, dehydrated, and embedded in paraffin. Thin sections were stained with hematoxylin and eosin. X 125.

Calcium Channel Antagonists and Cell Death 125

I 0 1 2 3 4 5 6

DAYS AFTER CASTRATION

Fig. 4. Loss of DNA from rat ventral prostate tissue at various times after castration. Individual ventral prostate glands from intact or calcium channel antagonist-treated (verapamil and nifedipine) (0) or -untreated rats (0) were extracted for DNA and assayed as indicated. Curves indicate mean DNA content of ventral prostate glands during regression. Error bars indicate standard error at each point.

treatment of castrated rats with calcium channel-blocking drugs. Combined, our results clearly suggest that the delay in prostatic regression subsequent to treatment with calcium channel-blocking drugs is related to stabilization in the cellular population of the gland rather than to water uptake or retention. It is of further interest that concomitant analysis of another androgen-dependent gland of the rat, the seminal vesicle, from these same animals, also indicated that calcium channel antagonist treatment inhibited its regression. Although wet weight determinations for the seminal vesicle glands in this experiment were not performed due to the inability to control the secretory fluid content, Figure 5 contrasts the size of a fixed representative seminal vesicle gland from a 3-day untreated castrated rat to glands from 3-day castrates treated with either nifedipine or verapamil. The glands of the calcium channel antagonist-treated castrates were markedly larger than the glands from comparable untreated control candidates.

Effects of Calcium Channel Antagonist Drugs on the Induction of mRNA Transcripts Encoding c-fos and TRPM-2 During Regression of the Rat Ventral Prostate Gland

We initiated this project based on the finding that the induction of transcripts for c-fos, a gene whose expression is linked to abrupt changes in intracellular Ca2+ levels, was an early event during regression of the rat ventral prostate gland [ 1 11. Since both calcium channel antagonist drugs showed a significant effect in delaying the regression of the ventral prostate gland, we also analyzed mRNA extracted from regressing ventral prostate tissue of treated and untreated rats to determine whether these drugs also down-modulated the expression of c-fos, or other genes whose expression is linked to prostate cell death.

In the previous study, it was found that c-fos activity fluctuated during regression in rough approximation of a diurnal cycle (transcript levels were elevated at morning time points and depressed at evening time points) [ 1 13. Therefore, in this experiment, rats were castrated in the evening, and selected groups received

126 Connor et al.

Fig. 5. Comparison of fixed unilateral lobes of a seminal vesicle gland from a mature intact rat (1) an age-matched 3-day untreated castrated rat (U), or a 3-day castrated rat treated with either verapamil (V) or nifedipine (N).

simultaneous treatment with nifedipine or verapamil, as indicated. At 36 and 62 hr following castration, peak times for c-fos expression, groups of rats were sacrificed, then ventral prostate tissue was removed, pooled, and frozen. This tissue and ventral prostate tissue from untreated castrated rats at comparable time points were processed to extract polyadenylated mRNA. The mRNA was electrophoresed and transferred to a nylon filter, and the Northern blot was simultaneously hybridized to [32P]-labeled probes for c-fos and TRPM-2. TRPM-2 is a gene that is expressed at low to undetectable levels in the normal prostate gland, but is induced to very high levels in regressing prostate tissue [8]. In situ hybridization analysis has shown that this gene is expressed only by dying acinar epithelial cells of the regressing rat ventral prostate and by other rat tissues subject to degenerative atrophy and death [28]. As has been found previously, both c-fus and TRPM-2 transcript levels were much higher in 36- and 62-hr castrated prostate tissues than in control prostate tissue from intact rats (Fig. 6A). While visual inspection of the autoradiograms shows that treatment with calcium channel antagonist drugs significantly decreased the levels of both c-fos and TRPM-2 transcripts in regressing prostate tissue (Fig. 6A), we performed densitometric analysis of the autoradiogram to quantitate transcript levels. By this method, we determined that calcium channel antagonist treatment suppressed c-fos transcripts to 30.9% (V) and 13.9% (N) of the levels of untreated castrates at 36 hr and to 50% (V) and 16% (N) of the levels of untreated castrates at 62 hr. While TRPM-2 activity was also suppressed by these drugs, the level of suppression was less than for c-fos. TRPM-2 transcript expression was 52% (V) and 72% (N) of the levels of untreated castrates at 36 hr and 70% (V) and 56% (N) of the levels of the untreated castrate rats at 62 hr.

Figure 6 also shows that chloroquin, a drug that has previously been shown to delay regression, also suppressed the expression of these genes, although, at the dosage used, not to the extent of the specific calcium channel-blocking drugs. c-fos transcript levels were 85% and TRPM-2 transcript levels were 72% of the levels of untreated castrated rats at 62 hr. Subsequent rehybridization of the same Northern

Calcium Channel Antagonists and Cell Death 127

Fig. 6. Northern blot analysis of C-~OS, TRPM-2, and p-actin transcript levels in mRNA extracted from ventral prostate tissues of intact rats, castrated rats, or castrated rats treated with either verapamil (V), nifedipine (N), or chloroquin ( C ) for the times indicated. Aliquots of 10 pg polyadenylated mRNA were electrophoresed on denaturing agarose gels and transferred to nylon filters. The Northern blot was initially hybridized to combined [32P]-labeled probes for c-fos and TRPM-2 at a 1O:l ratio of probe concentration (A). Following autoradiography, the blot was stripped of probe and rehybridized with denatured [32P]-labeled probe for p-actin (B).

blot with a probe for p-actin showed a rough equivalence in each lane (Fig. 6B) so that the depressed levels of c-fos and TRPM-2 transcripts in RNA from drug-treated rats were not the result of unequal loading of RNA samples.

DISCUSSION

Hormones often provide the stimulus for cell growth and synthesis in multicel- lular organisms. However, similar to the regression of the prostate gland resulting from androgen depletion, hormonal signals can also initiate a genetically programed series of events inevitably resulting in cell death. This process is frequently invoked as embryos acquire the adult form during development. In addition, many adult tissues retain sensitivity to specific hormones; withdrawal or supplementation of the hormone stimulus will cause glandular atrophy and regression. Although a wide variety of hormones can initiate this process, depending on the cell type, common physiological changes are shared by the different types of cells that undergo programed cell death [29]. Among these changes is an abrupt increase in cytoplasmic Ca2+ levels. It is of special interest that several types of cytotoxic chemicals or deleterious environmental conditions (heat shock) can also cause drastic increases in intracellular Ca2+ [30,3 11. Experimental evidence from one model of programed cell

128 Connor et al.

death has already indicated that an increase in calcium ion levels may be one of the more important events in the process leading to cell death. Mammalian thymocytes cultured in low calcium or calcium-free media are resistant to glucocorticoid toxicity

Our analysis of gene activity during death of prostatic cells has led us to propose that acute disturbances of intracellular Ca2+ also accompany prostatic regression. This was implied from our finding that the first gene induced during prostatic repression was cfos. Recent studies suggest a role for c-fos in coupling short-term events elicited by extracellular stimuli to long-term alterations in gene activity [33]. This gene is one of the first to be expressed by a cell following exposure to biochemical agents that activate calcium ion influx [12-14,191. As shown in this report, our ability to delay the regression of the rat ventral prostate gland with the use of calcium channel antagonist drugs supports our hypothesis and further indicates the importance of calcium ion flux in the mechanism by which prostatic cells are driven to their death in the absence of androgens. It is of interest that if all the models of programed cell death involve sudden accumulation of intracellular Ca2+, our results also indicate that this phenomenon may be mediated by the activity of specific ion channels and may not be the result of random membrane leakage.

Living cells normally maintain a relatively steep Ca2+ gradient across the cell membrane by means of distinct membrane-associated calcium ion-pumping mecha- nisms. The activity of these Ca2+ channels is tightly regulated, in conjunction with other ion channels, by surface membrane receptors for hormones or growth factors. Androgenic steroids are classically believed to act, by means of a cytoplasmic receptor protein, to alter the transcriptional activity of specific nuclear genes. While androgen-associated changes in gene activity could secondarily influence the activity of calcium channels, androgens and their receptors might also act directly at the cell membrane to regulate cellular Ca2+ flux. Alternatively, membrane-associated androgen-binding proteins distinct from the cytoplasmic androgen receptor have been detected, but remain uncharacterized [34]. These proteins might also play a role in regulating intracellular Ca2 + levels.

Regardless of its source, toxicity of a sudden surge in intracellular Ca2+ could result from a variety of disruptive physical changes in the cell. Mitochondria1 membrane function and cellular metabolism can be drastically affected by high levels of Ca2+ [20]. More pertinent to our studies on prostate regression, Kyprianou and Isaacs [ 101 have demonstrated that prostatic nuclear DNA is fragmented following castration, This fragmentation is thought to be related to activation of a nuclear Ca2+-Mg2+-dependent endonuclease. By blocking the entry of Ca2+ into prostate cells with the calcium channel antagonist drugs, perhaps this enzyme is maintained in an inactive form, thus preventing the fragmentation of DNA.

The results presented here emphasize the potential usefulness of gene expres- sion patterns as an aid in the interpretation of complex cellular processes. To date, the concept of programed cell death remains more poorly understood than that of cellular proliferation or differentiation. Since cell death contributes equally to the homeostatic maintenance of tissue as proliferative processes, and since the hormonal induction of cell death remains an important mainstay in the treatment of metastatic prostate cancer, the role of Ca2+ influx in hormonally programed prostate cell death requires further exploration. Although the calcium channel antagonist drugs at the dosages used in these experiments did not completely block cell death and regression, it is

[321.

Calcium Channel Antagonists and Cell Death 129

possible that higher doses may have accomplished this. Unfortunately, the potential cardiotoxicity of these drugs prevents us from attempting these experiments in the rat. As an alternate approach to this problem, it might be possible to enhance the rate of regression of androgen-dependent tissues by simultaneous treatment with calcium channel agonists.

ACKNOWLEDGMENTS

The authors gratefully acknowledge the technical assistance of Po-Ying Ng . This work was supported by grants R23-CA39010 (M.C.B.) and (R.B.) from the U.S. Public Health Service. I.S.S. is an American Urological Association Scholar and is the recipient of a National Kidney Foundation Young Investigator Award.

REFERENCES

1. Bruchovsky N, Lesser B, van Doorne E, Craven S: Hormonal effects on cell proliferation in the rat

2. Lee C: Physiology of castration-induced regression in the rat prostate. Prog Clin Biol Res

3. Kerr JFR, Searle J : Deletion of cells by apoptosis during castration-induced involution of the rat

4. Sanford ML, Searle JW, Kerr JFR: Successive waves of apoptosis in the rat prostate after repeated

5 . Isaacs JT: Antagonistic effect of androgens on prostate cell death. Prostate 5:545-557, 1984. 6. Anderson KM, Baranowski J , Economous SG, Rubenstein M: A qualitative analysis of acidic

proteins associated with regressing, growing, or dividing rat ventral prostate cells. Prostate

7. Lee C, Tsai Y, Harrison H, Sensibar J: Proteins of the rat prostate. I. Preliminary characterization by two-dimensional electrophoresis. Prostate 7: 171-182, 1985.

8. Montpetit ML, Lawless KR, Tenniswood MR: Androgen-repressed messages in the rat ventral prostate. Prostate 8 9 - 3 6 , 1986.

9. Stanisic T, Sadlowsky R, Lee C, Grayhack JT: Partial inhibition of castration-induced ventral prostate regression with actinomycin D and cycloheximide. Invest Urol 16: 19-22, 1978.

10. Kyprianou N, Isaacs JT: Activation of programmed cell death in the rat ventral prostate after castration. Endocrinology 122552-562, 1988.

11. Buttyan R, Zakeri Z, Lockshin R, Wolgemuth D: Cascade induction of c-fos, c-myc and heat shock 70K transcripts during regression of the rat ventral prostate gland. Mol Endocrinol2:650-657, 1988.

12. Bravo R, Burckhardt J, Curran T, Muller R: Stimulation and inhibition of growth by EGF in different A43 1 cell clones are accompanied by the rapid induction of c-fos and c-myc proto-oncogenes. EMBO

13. Morgan J , Curran T: Role of ion flux in the control of clfos expression. Nature 322552-555, 1986. 14. Bravo R, Neuberg M, Burckhardt J, Almendral J, Wallich R, Muller R: Involvement of common and

cell type-specific pathways in clfos gene control. Stable induction by CAMP in macrophages. Cell 49: 25 1-260, 1987.

15. Nayler WG: An effect of quinidine sulfate on the lipid-facilitated transport of calcium ions in cardiac muscle. Am Heart J 71:363-367, 1966.

16. Huddart H, Saad KHM: Quinine and lanthanium effects on contractility and calcium movements of rat ileal smooth muscle. Gen Pharmacol 8:341-354, 1977.

17. Ikhinmwin MK, Sofola OA, Elebute 0: The effects of calcium ions on the depression of cardiac contractility by chloroquinine and quinine. Eur J Pharmacol 9507-510, 1981.

18. Greenberg DA: Calcium channels and calcium channel antagonists. Ann Neurol 21:317-330, 1987. 19. Curran T, Morgan JI: Barium modulates c&s expression and post-translational modification. Proc

prostate. Vit Horm 33:61-102, 1975.

75A1145-159, 1981.

prostate. Virchows Arch B 13:87-94, 1973.

withdrawal of testosterone stimulation. Pathology 16:406-410, 1984.

41151-166, 1983.

J 4~1193-1197, 1985.

Natl Acad Sci USA 83:8521-8524, 1986.

130 Connor et al.

20. Schrier RW, Arnold PE, Van Putten VJ, Burke TJ: Cellular calcium in ischemic acute renal failure:

21. Cheung JY, Bonventre JV, Mallis CD, Leaf A: Calcium and ischemic injury. New Engl J Med

22. White BC, Winegar CD, Wilson RF, Krause GS: Early amelioration of neurologic deficit by lidoflazine after fifteen minutes of cardiopulmonary arrest in dogs. Ann Emerg Med 122-9, 1983.

23. Burton K: Measurement of DNA with the diphenylamine assay. Biochem J 62:315-323, 1956. 24. Sambucetti LC, Schaber M, Kramer R, Crome R, Curran T: Thefos gene product undergoes

extensive post-translation modification in eucaryotic but not in procaryotic cells. Gene 43:69-77, 1986.

25. Cleveland DW, Lopata MA, McDonald RJ, Cowan NJ, Rutter WJ, Kirshner MW: Number and evolutionary conservation of a- and P-tubulin and cytoplasmic P- and y-actin genes using specific cloned cDNA probes. Cell 20:95-105, 1980.

26. Surimura Y, Cunha GR, Donjacour AA: Morphological and histological study of castration-induced degeneration and androgen-induced regeneration in the mouse prostate. Biol Reprod 34:973 -983, 1986.

27. English HF, Drago JR, Santen RJ: Cellular response to androgen depletion and repletion in the rat ventral prostate: Autoradiography and morphometric analysis. Prostate 7:41-5 1, 1985.

28. Buttyan R: Unpublished observations. 29. Trump BF, Berezesky IK, Osornio-Vargas AR: Cell death and the disease process. The role of

calcium. In Bowen ID, Lockshin RA (eds): “Cell Death in Biology and Pathology.” London: Chapman & Hale, 1981, pp 209-223.

30. Schanne RAX, Kane AB, Young EE: Calcium dependence of toxic cell death: A final common pathway. Science 206:701-702, 1979.

3 1 . Stevenson MA, Caldenvood SK, Hahn GM: Rapid increases in inositol triphosphate and intracellular calcium after heat shock. Biochem Biophys Res Commun 137:826-833, 1986.

32. Kaiser N, Edelman IS: Further studies on the role of calcium in glucocorticoid-induced lymphocy- tolysis. Endocrinology 103:936-942, 1978.

33. Marx JL: Thefos gene as a “master switch”. Science 237:854, 1987. 34. Szego CM, Pietras RJ: Membrane recognition and effector sites in steroid hormone action. In

Litwack G (ed): “Biochemical Actions of Hormones,” Vol 8. New York: Academic Press, 1981, pp 307-463.

Role of calcium entry blockers. Kidney Intl 32:313-331, 1987.

31: 1670-1676, 1984.