characterization of rat ventral prostatic epithelial cells in collagen gel culture

The Prostate 7:305-319 (1985)

Characterization of Rat Ventral Prostatic Epithelial Cells in Collagen Gel Culture Terence O’Connor and Dilip K. Sinha

Department of Breast Surgery and Breast Cancer Research Unit, Roswell Park Memorial Institute, Buffalo, New York

The characterization of rat ventral prostatic epithelial cells grown in collagen gel culture was undertaken to determine the usefulness of this culture technique for the study of prostatic growth and differentiation in vitro. The results of these studies demonstrate that embedding prostatic epithelial cells in a matrix of collagen gel permitted rapid proliferation and maintenance of the cells for a prolonged pericd of time in a state that showed remarkable structural resemblance and physiological responsiveness to that found in vivo. Three- dimensional outgrowths from dissociated single epithelial cells and/or small aggregates of epithelial cells were obtained which resembled structures actually found within the prostate gland, as determined by histological and visual examination of the cellular growths present within the gels. In addition, the responsiveness of the cells in the collagen gel cultures to various hormonal additives, including insulin, testosterone, prolactin, and estradiol corre- lated closely to the manner in which these cells have been shown to respond in vivo. The general characteristics of growth of the cells in the collagen gel cultures, the effect of various sera and different pH levels upon growth, and the ability of the cells to produce acid phosphatase were also investigated.

Key words: ventral prostate, epithelial cells, primary cell cultures, collagen gel matrix, hormones, sera

INTRODUCTION

In an effort to understand the many factors involved in the growth and differentiation of the prostate gland in vivo, a variety of in vitro techniques have been utilized. Studies involving organ cultures of the prostate have been used mainly to define the various substances necessary for maintaining the anatomical and functional integrity of the gland in vitro. Organ-type culture, although it maintains the structural integrity between the epithelium and the mesenchyme, which has been reported to be of critical importance for the functional integrity of the prostate gland in vivo, suffers from several drawbacks. This type of culture is of relatively short duration and cannot be used to determine the effects of specific substances upon single cellular elements of the gland. Cell culture studies have overcome many of the deficiencies inherent in the organ culture system, but most cell cultures, including those of prostate gland cells, have cultivated the cells in monolayer type cultures with the cells growing on an “unnatural” substratum, such as plastic or glass. The physiological significance of such studies may thus be questioned, since the response of the prostate cells under these conditions may not necessarily resemble that found in the in vivo state.

In order to overcome this objection, the use of the recently developed collagen gel matrix culture system was investigated, since this system appears to more closely

Received March 22, 1985, accepted May 15, 1985.

Address reprint requests to Dilip K. Sinha, PhD, Department of Breast Surgery, Roswell Park Memorial Institute, 666 Elm Street, Buffalo, NY 14263.

0 1985 Alan R. Liss, Inc.

306 O’Connor and S i a

approximate physiologic conditions for the growth of the cells. This result is achieved by embedding prostatic cells in a matrix of collagen gel. Previous studies [l-51 utilizing the collagen substrate culture system have reported an increased maintenance of the differentiated state and an enhanced degree of tissue organization, which is greater than that observed with other substrate surfaces such as plastic or glass. Furthermore, it has been shown that many types of cells maintained in vitro in collagenous matrix are able to respond to trophic factors in a fashion reminiscent of that observed in vivo [ 1,641. In this investigation, primary cultures of prostate cells were maintained for prolonged periods of time, during which the cells were examined for responsiveness to a variety of substances, such as sera and hormones.

MATERIALS AND METHODS Cell Dissociation

The ventral prostate glands were aseptically removed from two to four sexually mature Sprague-Dawley rats and gently sliced in a parallel fashion with surgical razor blades. The minced glands were placed in a screw-capped microfernbach flask containing a 0.45 % collagenase solution (collagenase type III, 162 units/mg, Milli- pore Corporation, Freehold, NJ) in Earle’s Balanced Salt Solution (EBSS, Ca++ and Mg++ free) and placed in a Dubnoff water bath at 37°C for approximately two hours, during which time the tissue was allowed to dissociate.

Upon dissociation the cell suspension was passed through nylon meshes of pore sizes 400 and 100 pm (Nitex mesh, Tetko Products, Depew, NY) to remove any undissociated elements. The cells were then washed twice with EBSS and resuspended in RPMI 1640 medium containing 10% porcine serum. The dissociated cells were enriched for prostatic epithelial cells by layering the cells upon a 2-8 % ficoll gradient containing a 20% ficoll cushion at its base. After centrifugation at 600 rpm for ten minutes, the bottom 10-20-ml fraction of the gradient just above the 20% cushion was consistently found to contain predominantly epithelial elements. The cells from this fraction were collected and washed twice with RPMI 1640 medium containing 10% porcine serum. The number of cells thus obtained was determined by staining an aliquot of the cell suspension with crystal violet and counting in a hemacytometer [9]. Viability was assessed using the trypan blue exclusion test. The cells were plated at a density of 105 viable cells per well in a 24-well plastic dish (Falcon Plastics, Cockeyville, MD), unless otherwise indicated.

Collagen Gel Preparation Stock solutions of collagen were prepared as originally described by Michalo-

poulus and Pitot [8]. Briefly, lg. rat-tail collagen fibers were dissolved in 300 ml of a 1:1,OOO acetic acid solution at 4°C. Two to three days later, the collagen solution was centrifuged at 10,800g for one hour, in order to remove the undissolved fibers. Four parts of this stock solution were mixed with a 2: 1 mixture of 10 x Eagle’s Minimum Essential Medium and 0.34 N NaOH to form the gel. Placement of the above mixture on ice delayed gelation for several minutes. Into each well of Falcon Multiwell plastic petri plates, 0.3 ml of gel mixture was added to form a base layer. Upon this gelled base was layered 0.5 ml of gel containing the desired number of cells to be plated. After that layer had gelled, 0.5 ml of the desired supplemented media was added.

Prostate Epithelial Cell Culture 307

Medium and Additives The basic medium for the culture consisted of RPMI 1640 supplemented with

various concentrations of sera and/or hormones. Pennstrep (GIBCO, Grand Island, NY) and fungizone (lyophilized amphotericin B, GIBCO, Grand Island, NY) were added at concentrations of 50 units/ml and 1 pg/ml, respectively, to maintain sterility. A small amount of a 0.63 M sodium bicarbonate solution was also added to maintain the pH of the media at approximately 7.4. Sera was heat inactivated in a dry oven at 60°C for 30 minutes. For charcoal adsorption, 1 g activated charcoal was added to 100 ml serum and mixed, using a magnetic stirrer, for 15 minutes. Charcoal was then removed by centrifugation at 9,000g for 15 minutes at 4°C. This process was carried out twice. These standard techniques (heat inactivation and charcoal adsorption) have been shown to effectively remove all peptide and steroid hormones from the sera. Prolactin and insulin, the two peptide hormones used in these studies, were dissolved fresh in solutions of 0.01 N NaOH and 0.01 N HCl, respectively, and stored at 4°C for periods of not more than seven days. The steroid hormones-namely, testosterone and estradiol-were stored indefinitely at 4 "C in absolute ethanol solutions. Additions of the above steroid hormones to the media never raised the alcohol concentration of the media above 0.2%. Media for all cultures were changed every third day. DNA assay

Cells were removed from inside the gels by digestion with 0.5 ml 25% acetic acid solution in 10 X 75 mm siliconized culture tubes (Dispo Culture Test Tube, Coming Glass Works, Coming, NY) and placed in a Dubnoff gyratory water bath at 37°C for approximately 30 minutes. Upon digestion of the gel, the cells were collected by centrifugation at 1,400 rpm for 15 minutes. The cell pellet was stored at -20°C until the time of DNA assay. The amount of DNA in each tube was determined by a fluorometric assay [lo]. For determination of cell number from total DNA, a standard curve was used. This standard curve was generated by using epithelial cells from 7,12-dimethylbenz[a]anthracene (DMBA)-induced rat mammary tumor counted in a hemacytometer. Autoradiography

Cultures were labeled for one hour with 2 pCi of 3H-thymidine (40-60 Ci/mM, New England Nuclear, Boston, MA) per ml of media. After labeling, the gels containing the growing cells were rinsed briefly in EBSS and dissolved in 25 % acetic acid solution. The released cells were collected by centrifugation and resuspended in 70% ethanol for several hours. The cells were then affixed to microscope slides by centrifugation in cytology buckets (Cytophysics, Nashville, TN) for 30 minutes at 2,000 rpm. The slides were processed for autoradiography using a high-speed scintillation autoradiography method [ 111. The slides were dip coated with NTB-3 emulsion (Eastman Kodak, Rochester, NY) and upon drying, the slides were briefly immersed in scintillation cocktail fluid. After dark exposure at -70°C for 24 hours, the slides were developed, fuced, washed with water, and stained with hematoxylin and eosin. The labeling index was determined by counting at least 2,000 cells from each slide and the labeling index (LI) was expressed as percent labeled cells.

RESULTS

Freshly dissociated cells were embedded in collagen gel and the general pattern of growth was monitored by viewing the cultures in an inverted phase-contrast

microscope. When the prostatic epithelial cells were cultured in medium supplemented with 20% porcine serum and hormonal additives (insulin, prolactin, and testosterone), three basic types of growth were observed. Some cells grew three- dimensionally into masses of cells containing radiating projections resembling ductlike structures (Fig. 1). Other epithelial cells grew into an oval mass of cells (Fig. 2). These oval masses were in reality hollow, saclike structures, a fact which was later confmed by histological sections of the gels. Either single or multiple layers of cells surrounding the sac had a remarkable resemblance to the normal histological structure of the prostate (Fig. 3). The third pattern observed was a monolayer-type growth, which apparently occurred when the cells reached the top of the collagen gel and spread as a monolayer sheet across the surface of the collagen gel.

The presence of growth in the cultures was apparent by light microscopy and was further confirmed by measurements of the total DNA content of the cells growing within the gels and also by autoradiography as a function of time. An insight was gained into the requirements for growth of prostatic epithelial cells in collagen gel by culturing these cells in one of four basic media types, including RPMI 1640 media supplemented as follows: (1) with 20% porcine serum plus a combination of the hormones insulin, testosterone, and prolactin; (2) with 20% porcine serum only; (3)

Fig. I . Prostate epithelial cell mass growing in collagen gel. Note the ductlike growth spreading from the cell mass. Phase contrast. ~ 2 8 0 .


Fig. 2. Spherical growth of prostate epithelial cells in collagen gel. Three-dimensional saclike growth is evident. Phase contrast. X400.

with a combination of the aforementioned hormones only; or (4) with no serum or hormones.

The results of this study can be seen in Figure 4 and Table I. A substantial four- to sixfold increase in cell number could be achieved within a relatively short period of time (15 days) in collagen gel cultures of prostatic epithelial cells containing media supplemented with 20% porcine serum with or without hormones. While only a small increase in cell number was observed during the first six days in culture, a steady increase in cell number occurred from day 6 to day 15. The addition of insulin (5.0 pg/ml of media), testosterone (1.0 pglml), and prolactin (5.0 pg/ml) to media containing 20% porcine serum resulted in an even greater stimulus to growth, compared to media containing 20 % porcine serum only, thus indicating the hormonal responsiveness of the cells in the collagen gel culture.

A large increase in labeling indices (from 5% to 25% labeled cells) was observed to occur from day 3 to day 6 in cells cultured in the presence of media containing 20% porcine serum plus hormones. A lesser increase in labeling indices (from 6% to 17% labeled cells) was seen in cells cultured in the presence of media containing 20% porcine serum but no hormonal additives. The increase in labeling indices observed in the above cultures tended to precede the increases in cell numbers in the cultures by approximately three days. After the sixth day of culture, a steady decline in labeling indices was observed and by day 12 labeling indices had decreased to levels similar to those observed on day 3 of culture. Cells cultivated in media containing 20% porcine serum plus hormones had higher labeling indices on day 9

310 O'Connor and Sinha

-

-

Fig. 3. Histological section of outgrowths of prostate cells in the collagen gel. Note the formation of prostatelike lobular structures in the gel. H & E. X480.

X

20 g q- -'5" I

Z L 10

W

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7 r I

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DAYS IN CULTURE

Fig. 4. Pattern of growth of ventral prostrate epithelial cells in collagen gel during 15 days of culture. Solid lines represent total cell number and broken lines indicate labeling index. 0, Serum and hormones; A serum only; , no hormones, no serum; W , hormones only.


TABLE 1. Effect of Serum and Hormones on Growth of Prostate Epithelial Cells in Culture?

DAYS MEDONLY MED + PS MED + HORM MED + HORM + PS 3 1.71 f 0.01 0.99 f 0.06 1.85 f 0.11 1.33 f 0.18 6 1.77 f 0.13 0.87 f 0.24 1.42 f 0.12 1.69 f 0.09 9 1.62 f 0.08 2.12 f 0.35 1.51 f 0.04 2.50 f 0.44

12 1.32 f 0.04 2.50 f 0.70* 1.53 f 0.16 6.08 f 0.48* 15 1.51 f 0.04 4.04 f 0.84** 1.50 f 0.14 6.69 f 0.45**

?Figures represent number of cells X I d f standard error. MED, medium; HORM, hormones: insulin 5 pg/ml, prolactin 5 pg/ml, testosterone 1 pg/ml; PS, heat inactivated, charcoal adsorbed 20% porcine serum. *P = < .01. P = < .05. **

and especially on day 6, as compared to cells cultivated in the presence of media containing 20% porcine serum only. The decline in the levels of labeling indices which occurred after day 6 may be possibly have been due to an inhibition of growth caused by crowding of the cells in the limited space available for growth in the multiwell plates.

Very low labeling indices (< 1 % labeled cells) and only small increases in cell number were achieved in cultures containing media which lacked serum. Thus the presence of serum in the media appeared to be a necessary requirement for growth in these cultures. The addition of hormones to media lacking serum produced the same effect that was observed in cultures lacking both serum and hormones. This would indicate that the stimulatory effect of these hormones on the cells requires the presence of serum. Once the general pattern of growth of the cells and the requirements for growth were determined, the role of each individual hormone and the effect of different types of sera were investigated.

Types of sera Various types of sera, including fetal calf, horse, and porcine serum, were

examined in order to determine which would be most effective in promoting the growth of the ventral prostate epithelial cells in collagen gel cultures. It was observed that raising the concentration of each of the different types of sera in the media, from the lowest concentration tested ( 5 % ) to the highest (20%), led to an increased stimulation of epithelial growth, as determined by increases in cell numbers in the cultures. In subsequent studies, porcine serum at a concentration of 20% was used, because this was found to consistently promote excellent growth of the cultured cells.

Effect of pH Variations in pH levels ranging from 7.2 to 7.8 were examined in order to

determine the effect of this parameter on the growth of prostate epithelial cells in collagen gel culture (Fig. 5) . A fully supplemented media containing 20% porcine serum plus a combination of hormones was adjusted with 1 N HCl and various amounts of a sodium bicarbonate solution, in order to attain the desired pH in the media, which was placed inside the 5 % C 0 2 incubator. The results demonstrated that a pH level of 7.5 in the culture media provided the conditions most conducive to growth, as determined by increased DNA content and labeling indices. Greater cell numbers and higher labeling indices were achieved in cultures maintained at pH levels

312 O’Connor and Sinha

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Fig. 5. Growth of prostate epithelial cells at different pH levels of the medium, maintained at 37°C under 95% air and 5% C 0 2 atmosphere. Solid line represents total cell number; broken line indicates the labeling index.

of 7.5 and above (pH 7.6-7.8), as compared to cultures maintained at a pH level below 7.5 (pH 7.2-7.4).

Hormones The effect of various peptide and steroid hormones on the proliferation of rat

ventral prostate epithelial cells in collagen gel culture was also examined. Each hormone was tested in combination with a basic media containing RPMI 1640 plus 20% porcine serum (heat inactivated and charcoal adsorbed). The effect of these hormones on cellular growth was assessed by increases in the amount of total DNA and also by increases in 3H-thymidine incorporation (labeling indices), measured usually two to three weeks after initiation of the culture.

The effect of insulin on prostatic epithelial cell growth was examined at concentrations ranging from 0 to 50 pg/ml media. Figure 6 demonstrates that insulin significantly stimulated prostatic epithelial cell growth at doses ranging from 0.05 pg/ml to 50 pg/ml, as compared to control cultures containing no insulin. Insulin at a dose level of 0.005 pglml produced a slight, although not significant, increase in both cell number and cell labeling indices in the cultures. The greatest cell number and highest labeling index was obtained when insulin was administered at a dose of 0.5 pg/ml. Dose levels of 0.05 pg/ml, 5.0 pg/ml, and 50 pg/ml of media were only slightly less stimulatory.

Prolactin was also shown to have a significant stimulatory effect on cell growth in these cultures (Fig. 7). Each increment in the dosage of prolactin from 0 to 5.0 pg/ml of media resulted in a progressively greater number of cells and higher labeling indices. Every dose level of prolactin tested in this culture, with the exception of 10 pglml, was found to significantly stimulate growth as compared to control cultures containing no prolactin. Prolactin at a dose level of 5 pglml of media was determined to be the most stimulatory, and this concentration was thus used in all subsequent studies involving prolactin.


INSULIN CONCENTRATION (pg/ml )

Fig. 6. Effect of different concentrations of insulin on the growth of prostate cells in collagen culture. Solid line represents total number of cells; the broken line indicates the labeling index.

0 0005 0.050 0.50 5.0 10

PROLACTIN CONCENTRATION ()rg/ml)

Fig. 7. Effect of different doses of prolactin on ventral prostate epithelial cells.

Testosterone was also observed to have a stimulatory effect on prostatic epithelial cell growth in these cultures (Fig. 8). A dosage range of 0-10 pg/ml of media was tested, and it was found that increasing the dosage of testosterone from 0 to 0.1 pg/ml resulted in a progressively greater number of cells. A further increase in the dosage of testosterone resulted in less stimulation than was observed at the 0.1 pg/ml dose level, and the highest dose tested (10 pg/ml) was found to exert a slight inhibitory effect upon cellular DNA production, when compared to control cultures containing no testosterone. The effect of increasing the dosage of testosterone upon labeling

L Y n s z

TESTOSTERONE CONCENTRATION bg/ml)

Fig. 8. Effect of different concentrations of testosterone on cell number and labeling index of ventral prostate cells in collagen gel culture.

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ESTRAOIOL CONCENTRATION (pg/ml)

Fig. 9. Total cell number and labeling index of prostate epithelial cells in culture, as influenced by different concentrations of estradiol.

indices tended to parallel the effect of testosterone upon cell number, with a greater labeling index obtained with each increment in the dose of testosterone from 0 to 1.0 pg/ml. It was found that a testosterone dose of 10 pg/ml was less stimulatory than other doses tested, but there was still an observable increase in 3H-thymidine incorporation (labeling indices) compared to cells cultured in media lacking testosterone.

The effect on cell growth of estradiol at concentrations ranging from 0 to 10 pg/ml was also examined (Fig. 9). Interestingly, estradiol was found to have little or no sthulatory effect on the cultured cells and, in fact, when used in the higher concentrations of 1.0 pg/ml and 10 pg/ml of media, estradiol exerted an inhibitory effect on both total DNA and thymidine incorporation.


After the optimal stimulatory level of each individual hormone had been identi- fied, various combinations of these hormones were then tested, in order to determine the most effective level of hormonal supplementation in the culture for cell growth. Thus, insulin (0.5 pg/ml media), prolactin (5.0 pg/ml), testosterone (0.1 pg/ml), and estradiol (0.1 pg/ml) were evaluated in various combinations, in order to determine their ability to stimulate prostatic epithelial cell growth in collagen gel culture (Fig. 10, Table II). Both insulin and testosterone, when added singularly to a medium containing 5 9% porcine serum, produced cultures containing a significantly greater number of cells, as compared to control cultures containing media supplemented with 5 % porcine serum only. Insulin was found to be more stimulatory in this regard than

f ie combination of testosterone and prolactin produced a significantly testosterone.

2.0 0 Q x I .5

(L w

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2 0.5 V

S I I*T I+T+P I*T+P+E* T TIP

Fig. 10. Effect of individual and combinations of hormones on growth of prostate epithelial cells. Open bars represent cell number; hatched bars indicate labeling index. The first group contained serum only (S); all other groups contained serum, in addition to the indicated hormones: I (insulin); T (testosterone); P (prolactin); & (estradiol).

TABLE II. Effect of Different Hormones on Cell Number and Labeling Index of Prostate Epithelial Cellst

Cell No. Labeling Index Hormones ( X 16) (%I None Testosterone

None Estradiol

None Prolactin

None Insulin

(0.1 agln-4

(10 pglml)

(5 p g w

(0.5 pglml)

4.89 f 0.51 6.36 f 0.84

1.90 f 0.44 0.52 f 0.02***

2.74 f 0.18 6.52 f 1.37

4.38 f 0.28 6.13 f 0.75

2.73 f 0.06 11.50 f 1.54***

4.84 f 0.59 0.64 f 0.14**

7.22 f 0.69 12.73 f 0.53**

8.32 5 1.10 17.08 f 0.98**

?Only the most effective dose of hormones is depicted in this table. *p = < .05. **

t.. p = < .01. p = c .02.

greater increase in the number of cells, when compared to cultures containing testosterone only. Insulin administered in combination with testosterone resulted in a greater increase in cell growth than did the addition of testosterone alone, but this combination was no more effective in stimulating growth than media supplemented with insulin alone. The greatest number of cells and highest labeling indices were obtained in cultures containing insulin in combination with both prolactin and testosterone. This hormonal combination was therefore defined as the optimal supplement of hormones and was subsequently used in all further cultures when a maximum level of stimulation was desired. The addition of estradiol to the full hormonal supplement of insulin, prolactin, and testosterone resulted in lower cell numbers and low labeling indices, compared to cultures containing insulin, prolactin, and testosterone, without estradiol . Acid Phosphatase

The ability of rat ventral prostate epithelial cells to produce the enzyme acid phosphatase was examined in collagen gel culture. Using a modification of the method for detecting acid phosphatase developed by Gomori [ 121, prostatic epithelial cells obtained from the collagen gel cultures were demonstrated to possess acid phosphatase activity (Fig. 11). Even after immersion in a 10% neutral formalin solution overnight, which has been shown to be inhibitory to the lysosomal form of acid phosphatase, but

Fig. 11. Acid phosphatase staining of prostate epithelial cells from culture. Note the intense reaction of the sphere of cells in the center. x 120.


not inhibitory to acid phosphatase of prostatic epithelial cell origin [ 131, an intense reaction was observed to occur in cells removed from the collagen gel cultures.

DISCUSSION

The main objective of this investigation was to identify a culture system which could be used to maintain prostatic epithelial cells in a state resembling that found in vivo, in order to study the various factors involved in prostatic growth and function. This investigation was precipitated by the suspicion that culturing cells on a surface such as glass or plastic would likely cause the cells to respond in a fashion uncharac- teristic of their natural state. Normally, epithelial cells from the prostate line the acini within tubuloalveolar structures and are surrounded by a matrix, consisting of stromal collagen and various mesenchymal elements. By embedding the cells in a matrix of collagen gel, it is possible to mimic partially the natural environment of these cells, since the collagen gel system allows the cells to grow in a three-dimensional fashion similar to that found in vivo. Previous studies using a variety of cells have shown that culturing cells in collagenous matrices maintains them in a viable state for longer periods of time than would be experienced if the same cells had been cultured on a matrix of glass or plastic [3,5,8,14]. Purified collagen substrates and collagenous matrices have also been shown to maintain differentiated functions, as well as to induce differentiation, in various types of cultured cells [ 1,2,5,15].

In this study, we found that rat ventral prostatic cells grown in a collagen gel culture develop into structures strongly resembling those found within the intact gland, a pattern of growth distinctly different from that observed when the cells are plated upon glass or plastic substrates. The importance of this distinctly different growth pattern cannot be overstated, since it has been reported that cellular shape may play an important role in the growth of epithelial cells and the responsiveness of these cells to serum growth factors [7,16].

In addition to the similarity in morphology achieved with this culture system, it was also possible to demonstrate that the ventral prostatic epithelial cells grown in a matrix of collagen responded to hormonal factors in a manner quite similar to that which occurs in the intact organ. Thus, in vivo, the rat ventral prostate has been shown to respond to a variety of hormones and trophic factors. The major hormone involved in prostatic growth would appear to be testosterone or, more importantly, its metabolite, dihydroxy-testosterone [ 171. Depleting the plasma of testosterone indirectly via hypophysectomy has been shown to result in severe atrophy and cessation of function in the rat prostate. Restoration of growth and function can be achieved in these rats by exogenous administration of testosterone, thus demonstrating the re- quired presence of testosterone for prostatic function [ 181.

Prolactin has been shown to have at least a synergistic effect with androgens on prostatic growth, since in the above hypophysectomized rats, testosterone was unable to fully restore prostatic function and growth, unless prolactin was also present [ 191. The presence of receptors for prolactin also suggests a not yet totally defined role of prolactin in the regulation of the prostate. Similarly, insulin has been reported to have synergistic or permissive effects of prostatic growth, since the presence of insulin appears to be necessary for the full restorative effect of testosterone upon prostatic growth in castrated rats [20]. The exact role played by estrogens on prostatic function has not yet been determined, despite the fact that estrogen receptors have been found

318 O’COMOr and Sinha

in the prostate [21]. The major inhibitory effects exerted by estrogens on the prostate appear to be exerted indirectly through the hypothalamic-pituitary-testicular axis by blocking the synthesis of testosterone and thus lowering plasma testosterone levels [22,23]. Whenever a direct effect of estrogens upon prostatic growth is noted, it is usually exerted at the level of the fibromuscular stroma [24-261. However, it has been shown that estrogens can indeed exert some inhibitory effects upon the prostatic epithelium, if administered in high pharmacological doses [24].

The results obtained in this investigation closely agree with those of the aforementioned in vivo experiments. Epithelial cells from the rat ventral prostate cultured in collagen gel responded to various hormonal manipulations in a manner quite reminiscent of that observed in vivo. While cells in these cultures were not totally dependent upon testosterone for growth, a definite stimulatory effect of testosterone on prostatic growth was observed. Also, epithelial cell growth could be altered by varying the doses of prolactin and insulin in the cultures. These two hormones have been shown to affect prostatic growth in vivo. A slight inhibitory effect on growth was achieved when estrogen was administered. This was especially evident at the higher concentrations of estrogen tested and is again similar to results obtained in vivo.

CONCLUSIONS

Based on these investigations, collagen gel matrix would appear to be an ideal culture system for the elucidation of the various factors involved in prostatic growth and function, since this culture system allows cells to grow and to respond to hormonal stimulation in a manner similar to that observed in vivo. The collagen gel culture system thus offers distinct advantages over both monolayer or organ-type culture systems, and would seem to have great potential for further studies on the delineation of both normal and abnormal prostate physiology.

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