S. Chevalier

Enhancement of the detection of alkali-resistant phosphoproteins in polyacrylamide gels.

Following their separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, labeled proteins obtained from cultured canine prostatic epithelial cells incubated with [35S]-methionine and [32P]phosphate were subjected to alkali treatment, a method that is currently used to detect phosphotyrosine-containing proteins. Significant amounts of 35S-labeled material were lost during the alkali treatment. The crosslinking of proteins within the gels by glutaraldehyde treatment eliminated protein losses and did not alter the efficiency of phosphoester bond hydrolysis by alkali treatment. Consequently, the time required to detect alkali-resistant phosphoproteins by autoradiography was greatly reduced. Prostatic phosphoproteins were also shown to contain phosphotyrosine, indicating the presence of tyrosine protein kinase activity in these proliferating epithelial cells.

Proliferation and differentiation of canine prostatic epithelial cells in culture

When cultured in monolayers, non-secretory epithelial cells from canine prostates actively synthesize DNA, RNA and proteins; subsequently, mitotic figures and an increase in cell number are observed. During this culture period, cell size and acid phosphatase activity remain constant. As the culture proceeds, these cells mature into secretory cells as evidenced by a gradual shift in their density in Percoll gradients to the density of secretory cells and by an increase in the cellular content of acid phosphatase. During maturation, the size of the non-secretory cells increases and their morphology changes and becomes similar to that of the secretory cells. When a homogeneous population of secretory cells is cultured, DNA synthesis is minimal and few mitotic figures may be observed while cell number and cell density remain constant. Early in the culture period, their size increases and by 2 weeks their acid phosphatase activity is 2--3-fold higher than that of the non-secretory cells. Thus, upon culture, the non-secretory epithelial cells enter and proceed through the cell cycle with evidence of DNA synthesis and mitosis. Those cells leaving the cycle undergo maturation into secretory cells which further differentiate with the concomitant appearance of acid phosphatase activity. This model will be useful to study prostatic hyperplasia and hypertrophy and the control mechanisms involved in these phenomena.

Characterization of canine prostatic cells from normal and hyperplastic glands.

Secretory and non-secretory epithelial cells and fibroblasts obtained from normal and hyperplastic canine prostate glands and from prostates of 6-week castrated dogs are cultured in monolayers. Prostatic fibroblasts are grown in non-selective culture medium and found at densities of 1.040-1.045 g/ml in Percoll gradients. Enriched populations of each epithelial cell type area obtained by varying the duration of the culture combined with the use of selective MEM D-Val mixture. When separated by centrifugation in Percoll density gradients, the secretory cells (high A.P.) are found at densities of 1.02-1.03 g/ml whereas the non-secretory cells (low A.P.) have densities of 1.05-1.06 g/ml. Both epithelial cell types are present in the normal and hyperplastic glands at the time of explantation. There is no correlation between the prostatic weight and the proportion of each cell type present in the tissue. On the basis of cell density in Percoll gradients and A.P. activity, those prostates with a high percentage of non-secretory epithelial cells yield better attachment and overall cultures than glands consisting mainly of secretory cells. Our results strongly suggest that non-secretory cells are precursors of the secretory type. In addition, the cells involved in the aging process of the culture are the secretory epithelial cells.

Androgen binding as evidenced by a whole cell assay system using cultured canine prostatic epithelial cells

The androgen receptor content in the prostate has been usually evaluated using subcellular fractions without taking into account cellular and functional heterogeneity of the gland. Using enriched populations of immature canine prostatic epithelial cells cultured in primary monolayers, a whole cell assay system was developed to measure androgen receptors. Tritiated dihydrotestosterone (DHT) and/or methyltrienolone (R1881) in serum-free medium were used as ligands and Triamcinolone acetonide (0.5 microM) was added to prevent the binding of R1881 to other types of receptors. The amount of radiolabelled ligand specifically bound to the cells was determined at equilibrium. Specific binding was proportional to the number of cells seeded. Scatchard analysis revealed the presence of at least two types of binding sites. The Kd for the high affinity binding site was 2 x 10(-9) M. Competition studies indicated that this component was specific for androgens; Methyltrienolone, Mibolerone and the antiandrogen RU 23908 were the most efficient competitors. They were followed by DHT, 5 alpha-androstane-3 alpha, 17 beta-diol, testosterone, estradiol and estrone. Progesterone, 5 alpha-androstane-3 beta, 17 beta-diol and epitestosterone were not inhibitors. The level of specific binding was 11.0 +/- 7.6 fmol of bound R1881 per 10(6) cells (n = 34) or 2075 +/- 1434 fmol per mg of DNA; these values correspond to an average of 6624 +/- 4577 sites per cell. Thus, using this whole cell assay system, specific and androgen receptors were detected in immature prostatic epithelial cells in culture. This assay will therefore be useful to study the interrelationship between androgen binding activity and specific cell functions.

Induction of acid phosphatase synthesis in canine prostatic epithelial cells in vitro

The increase in acid phosphatase (AP) activity in cultured canine prostatic epithelial cells was investigated as a biochemical marker of in vitro cellular differentiation. The enzyme was studied in secretory and non-secretory epithelial cell populations obtained from control and cycloheximide-treated cultures over a period of 3 weeks and compared to the AP present in tissue and cellular extracts from normal canine prostates. The progressive increase in AP activity with the duration of culture was strongly inhibited by cycloheximide in both cell populations. The degree of inhibition was more pronounced late in the culture when AP activity increased at a faster rate in secretory cells. Cycloheximide inhibited protein biosynthesis by 70-80% as evidenced by a reduction in the incorporation of amino acids into acid-insoluble material. However, the specific activities of AP in the cellular extracts were similar in control and cycloheximide-treated cultures and increased sharply by 3-4-fold in the secretory cells after 12 days of culture. When extracts derived from control and cycloheximide-treated cells of various duration were submitted to electrophoresis in polyacrylamide gels (PAGE), a unique pattern of three bands of AP activity with Rf values of 0.18, 0.27 and 0.38 was obtained. In controls the AP activity in the band with an Rf of 0.18 increased preferentially during the culture period and was more important quantitatively in secretory cells. In cycloheximide-treated cultures the increase of AP activity associated with the band with an Rf of 0.18 was more strongly inhibited. The addition of tartrate to the staining mixture inhibited all three bands of AP activity. Similar results were obtained when extracts derived from freshly dispersed cells as well as from normal canine prostatic tissue were submitted to PAGE; the AP activity was resolved into 3 bands with Rf values of 0.15-0.18, 0.23-0.27 and 0.33-0.38; all three bands were inhibited by the addition of tartrate and the first band was predominant. Thus, the increase in AP activity in prostatic epithelial cells in a culture medium supplemented with serum and deprived of sex steroids is due to the de novo synthesis of a major form of the enzyme by the secretory cells.

Quantification of cytosolic steroid receptors in secretory and non-secretory epithelial cells of the canine prostate

Radiolabelled methyltrienolone, dihydrotestosterone and estradiol were used as ligands to identify and quantify androgen and estrogen receptors in freshly dispersed cells from the canine prostate. Soluble extracts (cytosols) were obtained from secretory and non-secretory epithelial cells separated on the basis of their density in Percoll gradients. For both cell types, as well as for the whole prostate, Scatchard plot analyses were linear and showed a single class of high affinity binding sites: Kd values of 3.6 +/- 2.2 X 10(-9) M and 3.0 +/- 1.2 X 10(-10) M were measured for the androgen and estrogen receptors, respectively. The number of binding sites for the cytosolic androgen receptor, expressed per mg of protein or per mg of DNA, was 2.4- to 6.7-fold higher in the non-secretory cells compared to the secretory cells. However, these two cell types contained a similar number of specific sites for the estrogens. The specificities of the androgen and estrogen receptors were shown to be identical for the two cell types: the binding of [3H]R1881 was strongly inhibited by unlabelled R1881, 5 alpha-androstane-3 alpha, 17 beta-diol and dihydrotestosterone, while 5 alpha-androstane-3 beta, 17 beta-diol, estradiol and estrone did not displace bound R1881. The addition of triamcinolone acetonide did not alter the binding of R1881 in extracts of either cell type or in the whole prostate. The binding of [3H]estradiol to the estrogen receptor was highly specific since a strong displacement was only observed with estradiol (83%).

5α-reductase and 3α-hydroxysteroid dehydrogenase activities in isolated canine prostatic epithelial cells

Kinetic parameters of two enzymes, 5 alpha-reductase and 3 alpha-hydroxysteroid dehydrogenase, have been calculated for freshly isolated canine prostatic epithelial cells separated into secretory and non-secretory cells on the basis of their density in Percoll gradients. For 5 alpha-reductase, a Km value of 3 X 10(-6) M was obtained in both epithelial cell types, and similar Vmax values of 1.7 X 10(-12) and 1.9 X 10(-12) moles of dihydrotestosterone formed/min/10(6) cells (P greater than 0.50) were calculated in secretory and non-secretory cells, respectively. The Km of 3 alpha-hydroxysteroid dehydrogenase (dihydrotestosterone----3 alpha-androstanediol) varied between 2.2 and 2.8 X 10(-6) M (P greater than 0.50) with respective Vmaxs of 9 and 24 X 10(-12) moles of 3 alpha-androstanediol formed/min/10(6) cells (P less than 0.005) in secretory and non-secretory cells. For the reverse reaction, that is the transformation of 3 alpha-androstanediol to dihydrotestosterone, the Kms obtained were 0.4 and 0.5 X 10(-6) M (P greater than 0.50) with Vmaxs of 14 and 19 X 10(-12) moles of dihydrotestosterone formed/min/10(6) cells (P less than 0.50) in secretory and non-secretory cells, respectively. Vmax values for 3 alpha-hydroxysteroid dehydrogenase are 10-fold higher than the ones for 5 alpha-reductase. Moreover, Km values for the reaction 3 alpha-androstanediol----DHT are 5-fold lower than those calculated for the reverse reaction and for 5 alpha-reductase. This could explain why the major metabolic pathway in the canine prostate gland is the conversion of 3 alpha-androstanediol to dihydrotestosterone.

Alkali-resistant protein phosphorylation and tyrosine kinase activity of epithelial cell types from normal and metaplastic canine prostates.

To determine how functional changes such as growth and differentiation in the prostatic epithelium would alter protein phosphorylation on tyrosyl residues, differentiated (secretory) and basal (non-secretory) prostatic epithelial cells from normal and from metaplastic canine prostates were labeled with [32P]phosphate and their alkali-resistant phosphoproteins were analyzed by autoradiography after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The tyrosine protein kinase activity of the cells was also assayed by phosphorylation of a synthetic substrate, poly(Glu80-NaTyr20), in the presence of [gamma-32P]ATP. The prostates were characterized by tissue morphology and the cells by their density on Percoll gradients and [3H]thymidine incorporation into DNA. In prostates from intact dogs, tall columnar secretory epithelial cells were predominant and the non-secretory or basal epithelial cells were quiescent and did not incorporate [3H]thymidine. In metaplastic glands obtained from estrogen-treated castrated dogs, most of the isolated cells were non-secretory and synthesized DNA. Alkali-resistant phosphoproteins were present in all cell types. Except for two common phosphoproteins, p44 and p82, the relative phosphoprotein distribution within the gel differed when normal prostatic cells were compared to the metaplastic cells. However, the phosphoprotein patterns were the same in secretory and non-secretory cells from the same prostate tissue. On the other hand, the relative labeling intensity of phosphoproteins was always higher in non-secretory cells compared to corresponding secretory cells. Accordingly, tyrosine protein kinase (TPK) activity, expressed on a cell basis, was also higher in non-secretory cells; the ratios of TPK activities, non-secretory over corresponding secretory cells, was always higher than unity for all preparations but overlapped when cells from metaplastic glands were compared to those isolated from normal prostates. Thus, non-secretory epithelial cells isolated from either normal or metaplastic glands have a higher ability than corresponding secretory cells to phosphorylate endogenous alkali-resistant phosphoproteins and their TPK activity, measured with the synthetic substrate poly(Glu80-NaTyr20), is also higher.

The major form of protein tyrosine kinase in the dog prostate is expressed by a 50 kDa polypeptide

We have already reported that the protein tyrosine kinase (PTK) activity in the dog prostate is distributed in cytosolic (75%) and particulate (Triton X‐100‐solubilized) fractions and that upon gel filtration, both PTKs migrate as entities of M r 44 000 [(1991) Biochem. Cell. Biol. 69, 146–153]. Herein we demonstrate by immunoprecipitation with anti‐phosphotyrosine antibodies that the soluble PTK has the ability to undergo self‐phosphorylation. In addition, the polypeptide responsible for that enzymatic activity has been identified by 2 approaches; (1) a two‐dimensional electrophoresis, in which the first dimension performed in non‐denaturing conditions allowed the localization of the native enzyme, while the second dimension (SDS‐PAGE) permitted the analysis of alkali‐resistant phosphoproteins corresponding to the activity; (2) protein renaturation after SDS‐PAGE followed by in situ phosphorylation (with [γ‐32P]ATP) of polyGT electrophoresed together with the enzyme preparation; the exclusive presence of the radiolabeled phosphotyrosine in the renatured protein confirmed its enzymatic nature. Using these methods, the major form of PTK in the dog prostate was shown to be expressed by a 50 kDa polypeptide which possesses autophosphorylation sites and which is present in the cytosol as an active monomer.

Multiple binding components for methyltrienolone in canine prostatic epithelial cells.

Using a whole cell assay system, the androgen binding capacity of canine prostatic epithelial cells was evaluated in relation to their function. Radiolabeled Methyltrienolone (R1881) was used as the ligand in the presence of an excess of Triamcinolone acetonide and the amount of [3H]R1881 bound to the cells at equilibrium was determined by either displacement or saturation studies. With immature cells in culture (3 days of attachment), displacement analysis revealed the presence of high affinity binding sites which were also present in cells cultured for 10 days. With freshly dispersed prostatic cells (mostly secretory epithelial cells) as well as with older cells in culture (17 and 24 days), only less specific binding sites were observed with both unlabeled R1881 and/or dihydrotesterone (DHT). In contrast, only the high affinity androgen receptor (AR) was present in cytosolic extracts prepared from normal glands. Displacement studies performed with cultured cells at different stages of growth also showed that the basal level as well as the degree of low affinity binding increased during the maturation of non-proliferating cells. The presence of multiple binding components was demonstrated by saturation studies performed with either cultured or freshly dispersed cells. The first component, that was saturated at 5 nM of [3H]R1881, was due to AR while the other two binding components, showing positive-cooperativity (Hill coefficients of 1.90 and 5.07, respectively), were saturated at concentrations of 15 and 30 nM of [3H]R1881. In contrast, the Hill coefficient for the AR was 0.88 indicating the presence of an independent component. It was calculated that only 11.4% of the total uptake of R1881 was attributed to AR binding, suggesting that the remainder may represent an intracellular pool of androgens. Thus, a whole cell binding assay represents a dynamic system for the detection, by saturation studies, of binding components that are not revealed using the conventional displacement studies or cell-free systems. It is proposed that these acceptor sites may play a role in differentiated prostatic function rather than in cell proliferation.