Requirements for attachment and subsequent growth of canine prostatic epithelial cells in culture

Abstract

The attachment and spreading of canine prostatic epithelial cells in primary monolayers and their subsequent proliferation were studied in Primaria and in polystyrene dishes either uncoated or coated with collagen type I, fibronectin, laminin, poly-D-lysine, or natural extracellular matrices (nECM) produced by canine prostatic epithelial or fibroblastic cells. Cells were inoculated in serum-free medium or in medium supplemented with either dialyzed fetal bovine serum (dFBS) or charcoal-treated dog serum at 10%. Dihydrotestosterone (DHT) and 5 alpha-androstane 3 alpha, 17 beta-diol (3 alpha, 17 beta-diol) at 10(-6) M or a mixture of steroids (androstenedione, testosterone, DHT, 3 alpha, 17 beta-diol, 5 alpha-androstane 3 beta, 17 beta-diol, estrone, and estradiol) were also added. Of all components and dishes tested, only dFBS and the nECM produced by prostatic epithelial cells increased cell attachment (850% and 450%, respectively). When the latter preparations were used in combination, an additive effect (1,500%) was observed, and the subsequent addition of dog serum after the attachment period yielded the highest number of growing prostatic epithelial cells. When prostatic epithelial cells were inoculated either in dishes coated with a nECM derived from prostatic fibroblasts or in presence of dog serum, a 50% inhibition in their plating efficiency was observed. The presence of collagen type I, fibronectin, laminin, or an nECM in the cultures, together with various steroids, had no effect on the cell unresponsiveness to steroids nor did it alter the mitogenic effect of dog serum. Thus, the attachment and spreading of canine prostatic epithelial cells in monolayers are mediated by nonsteroidal factors present in dFBS and in their nECM. Their presence, together with steroids, does not elicit a proliferative response to steroids nor did it increase the effect of growth-promoting factors in dog serum.

Serum and prostatic growth‐promoting factors for steroid‐independent epithelial cells of adult dog prostate

“…3); consequently, all further experiments on their growth regulation were performed with low-density epithelial cell cultures. It is noteworthy that when attached in serum-free medium, these cells remain serum-responsive [9]. or medium 199 ( U ) supplemented with 10% dFBS and antibiotics.…”

“…The total volume (V,) was calculated from the dimensions of the column. static epithelial cells in primary cultures and reinforce our earlier conclusion of their steroid unresponsiveness [8,9]. Indeed, inhibitors of sex steroid action, namely the antiandrogens Anandron and Cyproterone Acetate which are very potent competitors of the specific binding of Methyltrienolone to these prostatic epithelial cells [ 101, were unable to prevent their growth.…”

“…It has been reported in vitro that the growth of epithelial cells isolated from adult human and canine prostates is not enhanced by exogenous sex steroids [6-81. In the canine model, the presence of an extracellular matrix does not induce a proliferative response to sex steroids and does not alter the mitogenic effect of dog serum [9] in spite of the fact that these cells bind androgens specifically [ 101. These findings have led to the proposal that in the mature prostate, the growth of basal epithelial cells is androgen independent.…”

Models for studying benign prostatic hyperplasia

“…The presence of these factors, together with steroids, did not elicit a proliferative response to steroids nor did they increase the effect of growth-promoting factors in dog serum. 35,36 Recently, Lehr et al 37 reported the isolation of canine prostate epithelial cells from a sexually intact, adult beagle. Epithelial clones were immortalized by tranfection with the Simian Virus 40 large T-antigen cDNA.…”

Involvement of vitronectin and of a natural extracellular matrix in prostatic cell to cell contact and interaction with the substratum in culture

“…This study also demonstrates that the nECM ensures cell spreading through a passive process (not altered by the presence of cycloheximide) as early as the 1st day of culture in serum-free medium. Its presence did not result in any increase in cell division or in the responsiveness of prostatic epithelial cells to steroids and/or to growth-promoting factors present in dog serum [16] but growth could only be evidenced when proper attachment and spreading had occurred. These events are therefore a prerequisite to their growth and the presence of a prostatic matrix may be a determinant.…”