Tissue interactions and prostatic growth: II. Morphological and biochemical characterization of adult mouse prostatic hyperplasia induced by fetal urogenital sinus implants

Current hypotheses regarding the causes of human benign prostatic hyperplasia have implicated both steroid hormone imbalance and tissue interactions. To examine the role of the latter we have further investigated the phenomenon of urogenital sinus-induced hyperplasia of the adult mouse ventral prostate. Urogenital sinuses (UGS) or purified urogenital mesenchyme (UGM) from C3H mice were implanted into the ventral prostates or coagulating glands of 50- to 90-day-old BALB/c-nu/nu hosts. The animals were sacrificed at 15, 30, and 180 days postimplantation to establish time dependence. Wet weight and DNA content were used as measures of net growth. Glucose phosphate isomerase (GPI) isozyme analysis was used to determine the relative contributions of C3H and BALB/c cells to the enlarged chimeric ventral prostates. It was determined that the induced growth is time-dependent and that the UGS induces 2- to 4-fold more growth than UGM. GPI analysis shows that UGM-induced growth was composed primarily of host-derived cells whereas the UGS is composed nearly equally of host- and implant-derived cells. Histologic analysis reveals that the UGS implants induce marked epithelial proliferation. The proliferating glands occur in clusters, and the epithelium within the glands appears cribriform. Foci of postobstructive cystic atrophy are also found. Remnants of the implanted UGS are still present even at 180 days postimplantation. UGM-induced growth is of a more subtle nature and appears morphologically similar to the sham-operated controls. In view of the morphologic similarity with human disease, as well as the time and hormonal dependence of UGS-induced ventral prostatic hyperplasia, this model represents the basis for a unified hypothesis regarding the roles of tissue interaction and hormonal milieu in human benign prostatic hyperplasia.     

Prostatic growth effects of rat urogenital sinus and human prostatic tissue in the rat

It is known that fetal urogenital sinus (UGS) implanted into one ventral lobe of an adult mouse prostate produces a significantly increased DNA content in that lobe. In our study, we attempted to determine whether that growth was of host or of graft tissue. UGS from fetal rats and ventral prostate from adult rats were implanted in various sites in syngeneic Fischer rats, alone or as a recombinant tissue complex. Human benign prostatic hyperplasia (BPH) tissue was implanted into the ventral prostate of adult, male, hooded nude rats to determine whether it stimulated prostatic growth. All UGS-implanted in situ ventral lobes enlarged. Mean DNA content of UGS and ventral prostate implanted together in the flank was not different from the mean DNA content of UGS and ventral prostate implanted separately in the flank (P = 0.69). Mean DNA content of in situ control ventral lobe and of UGS grown singly in the flank was less than that of a UGS-implanted in situ ventral lobe (P less than 0.01). Thus overgrowth in the UGS-implanted in situ ventral prostate was location-dependent, but which tissue(s) enlarged remains unclear. Implanted human BPH tissue did not stimulate rat prostatic growth.     

Inhibition of prostatic epithelial cell proliferation by a factor secreted specifically by prostatic stromal cells

Stromal cells from the prostate were recently shown to inhibit clonal growth of the prostatic carcinoma cell lines PC-3 (hormone-independent) and LNCaP (hormone-sensitive) in coculture. Our study revealed that stromal cell-conditioned medium strongly inhibited proliferation of PC-3 and LNCaP cells when grown in monolayer culture. Antiproliferative activity was found to be reversible, and was produced specifically by prostatic stromal cells and not by stromal cells derived from skin, foreskin, uterus, kidney, and Wilms' tumor. Inhibition was not species-specific, since the cell lines AT-2.1 and MATLyLu, derived from the Dunning rat prostate tumor, were also sensitive. No inhibition, however, occurred on breast and renal carcinoma cell lines, suggesting a prostate-specific action. The putative inhibiting factor(s) could be concentrated and partially purified by ammonium sulfate precipitation. The possible role in stromal control of epithelial cell proliferation is discussed.     

Clonal growth of human prostatic epithelial cells is stimulated by fibroblasts

The interaction of fibroblasts with adult human prostatic epithelial cells was studied in vitro. Stimulation of epithelial cell proliferation, measured by clonal growth assay, was demonstrated when prostatic epithelial cells were grown in coculture with fibroblasts. Epithelial growth in cocultures with fibroblasts was greater than could be obtained in isolated culture in an "optimized," serum-free medium previously described. Fibroblasts were able to compensate for the deletion of several growth factors from this "optimized" medium, including epidermal growth factor and insulin, but were notably unable to replace bovine pituitary extract. Epithelial growth stimulation identical to that achieved in coculture was produced by medium conditioned by fibroblasts. Similar results were obtained by using fibroblasts of adult human prostate origin, adult human skin, human fetal lung fibroblasts (IMR-90), and mouse 3T3 cells. No difference in response was demonstrated between prostatic epithelial cells derived from normal or malignant tissues. Fixed fibroblast monolayers and extracellular matrix prepared from fibroblast cultures failed to stimulate prostatic epithelial growth. These results suggest that a soluble growth factor is secreted by prostatic fibroblasts and other human fibroblasts of nonprostatic origin, as well as by embryonic mesenchymal cells from nonhuman species, which is capable of producing a marked proliferative response in vitro from adult human prostatic epithelium. This proliferative response could not be reproduced by the addition of a variety of known growth factors to prostatic epithelial cell cultures.     

Regulation of prostatic growth and function by peptide growth factors

Polypeptide growth factors are positive and negative regulators of prostatic growth and function. Expression and biological effects of epidermal growth factor (EGF), transforming growth factors (TGFs) α and β, fibroblast growth factors (FGFs), and insulin-like growth factors (IGFs) in the prostate have been extensively studied. EGF and TGFα, which share the same receptor, are strong mitogens for prostatic epithelial and stromal cells. Their paracrine mode of action in normal tissue and early-stage tumors is apparently altered towards an autocrine stimulation in hormone-independent tumors, which gain the ability to produce TGFα by themselves. TGFβ has a dual role in the regulation of prostatic growth. It inhibits growth of prostatic epithelial cells in culture and mediates programmed cell death after androgen withdrawal. However, advanced prostatic carcinomas become insensitive to the inhibitory effect of TGFβ. Several members of the FGF family have been identified in the prostate. They are mainly or exclusively expressed in the stromal cells, and stimulate the epithelial cells. In the rat Dunning tumor model, progression is accompanied by distinct changes in the expression of FGFs and their receptors. In the hyperplastic tissue, basic FGF (bFGF) is accumulated. This growth factor is also a potent angiogenic inducer, expression of which may determine the metastatic capability of a tumor. IGFs are paracrine growth stimulators in the normal and hyperplastic prostate. It is still under consideration whether prostatic cancer cells gain the ability to produce IGF-I by themselves and thus shift to an autocrine mode of IGF-I stimulation. Growth factors also interact with the androgen-signaling pathway. IGF-I in particular, other growth factors as well, can activate the androgen receptor. © 1996 Wiley-Liss, Inc.     

Morphologic and biochemical alterations in rat prostatic tumors induced by fetal urogenital sinus mesenchyme

Because fetal urogenital sinus mesenchyme (UGM) has been found to be highly inductive when recombined with normal adult prostate tissues or normal and neoplastic bladder epithelium, we investigated whether fetal UGM also interacts with established hormone-responsive and unresponsive rat Dunning and Nb prostate tumors. Our results indicate that: 1) fetal UGM acts directly on selected rat prostatic tumors by inducing histomorphologic changes (e.g., inducing acinar ductal structures and secretory activity) in the tumors toward more differentiated forms resembling that of the adult prostate gland; 2) fetal UGM either increased the growth rate of or maintained the sizes of three of the four interacting rat prostatic tumors; and 3) fetal UGM markedly reduced the lactate dehydrogenase activity of Nb-autonomous tumor toward a level comparable to that of the normal rat prostate gland. Our data suggest that fetal UGM can directly affect the growth and differentiative functions of selected rat prostatic tumors.     

四黄前列片对尿生殖窦植入法小鼠前列腺增生模型的影响

目的 探讨阴黄前列片对小鼠前列腺增生模型的影响。方法 选用NIH系雄性小鼠，依文献方法，行前列腺腹叶内植入尿生殖实组织，制备BPH动物模型，给药30d后处死动物，取前列腺组织，称重测量体积，组织切片观察前列腺腺腔嘶积和腺上皮高度，用免疫组化法研究实验各组前列腺组织中PCNA抗原和Caspase-3蛋白的表达。结果 四黄前列片可缩小前列腺体积，降低腺腔的面积和腺上皮高度，并能抑制前列腺组织中PCNA的表达，增加Caspase-3的表达。结论 四黄前列片能抑制模型小鼠前列腺增生，其机理可能与抑制前列腺细胞的增殖和诱导凋亡有关。     

Regulation of urogenital smooth muscle patterning by testosterone and estrogen during prostatic induction

BACKGROUND: Smooth muscle (SM) has been proposed to play an important role in controlling prostate organogenesis by regulating signaling between inductive mesenchyme and developing epithelial prostatic buds. METHODS: We have examined the effects of testosterone and estrogen upon SM patterning in the embryonic rat urogenital tract (UGT) using in vitro organ cultures, immunohistochemistry, and Western blotting. RESULTS: We observed that testosterone elicited a sexually dimorphic difference in SM structure of embryonic UGTs, in cultures grown with testosterone. The addition of estrogen led to an increase in the rate of SM closure, in both males and females. To quantify the effects of steroids upon SM we used Western blotting of SM actin, which showed that estrogen stimulated SM content, while testosterone reduced SM content. Finally, we examined the expression of ERalpha, ERbeta, PR, and SM actin under different hormonal treatments of UGTs grown in vitro. The expression patterns of ERalpha and ERbeta were largely unchanged by hormonal treatment, while PR showed a much broader expression pattern in response to estradiol. CONCLUSIONS: Our results indicate that testosterone can directly regulate SM patterning and content in the UGT, and that SM is sensitive to both androgens and estrogens.     

The influence of mesenchyme of neonatal seminal vesicle and embryonic urogenital sinus on the morphologic and functional cytodifferentiation of dunning prostatic adenocarcinoma: Roles of growth factors and proto-oncogenes

Previous studies have demonstrated that male genital tract mesenchymes can induce morphogenetic changes in the rat prostatic Dunning tumor (DT). However, the mechanism of these cellular interactions remains unknown. The aims of this paper are: (I) to examine the influence of neonatal seminal vesicle mesenchyme (SVM) and embryonic urogenital sinus mesenchyme (UGM) on the growth, and morphologic and functional cytodifferentiation of DT; (2) to investigate the possible role of growth factors and receptors including epidermal growth factor and its receptor, basic fibroblast growth factor and transforming growth factor-beta, and two proto-oncogenes, c-fos and c-jun, in these tumor-mesenchymal interactions. Combination of mesenchymes (SVM or UGM) with DT enhanced the growth and induced an apparently more normal morphologic cytodifferentiation in vivo with formation of large tubules lined by highly differentiated columnar epithelial cells and reappearance of fibromuscular stroma. The SDS-PAGE analysis has shown that the DT + UGM enlarged and small tubules secreted proteins different from those of parental DT, demonstrating that mesenchymes can also modulate the functional expression of DT. Interestingly, our immunohistochemical data demonstrate that all the selected growth factors, receptors, and proto-oncogenes are upregulated in the mesenchyme-induced DT epithelial cells, suggesting that these cellular regulators may be closely associated with the mesenchymal induction on the DT phenotypic changes in vivo. This finding implicates the potential role of these growth factors, receptors, and proto-oncogenes in the epigenetic pathway of prostate carcinogenesis via tumor-stromal interactions.     

Regional differences in the inductive activity of the mesenchyme of the embryonic mouse urogenital sinus

Embryonic urogenital sinuses (UGS) of 16-day-old mice were divided into two or three zones (cranial half and caudal half; or cranial third, intermediate, and caudal thirds). Following tryptic digestion, these zones were separated into their mesenchymal (UGM) and epithelial (UGE) components. The cranial and caudal zones of the UGM were recombined separately with the different zones of the UGE or with adult mouse urinary bladder epithelium (BLE). Following four weeks of growth in syngeneic male hosts, tissue recombinants of cranial UGM + caudal UGE contained numerous prostatic ducts and urethral glands. Conversely, recombinants composed of caudal UGM + cranial UGE developed into fibromuscular tissue covered with urethral epithelium and did not contain prostate, but occasionally contained urethral glands. Urethral glands were usually present in grafts of cranial UGM + cranial UGE or in grafts of the intact intermediate third of the UGS. In heterotypic recombinants of the UGM + adult BLE, prostatic glands were induced only when cranial UGM was utilized. Urethral glands were not observed in tissue recombinants prepared with adult BLE. These data suggest that regional differences in mesenchymal inductive ability and epithelial responsiveness play a role in the harmonious morphogenesis of the male lower genitourinary tract.     

Regulation of prostate-specific antigen gene expression in LNCaP human prostatic carcinoma cells by growth,dihydrotestosterone, and extracellular matrix

We have examined the role of extracellular matrix (ECM), cell growth, and dihydrotestosterone on the expression of prostate-specific antigen (PSA) by human prostatic carcinoma cells LNCaP. ECM induced a transient decrease in PSA mRNA even in the presence of growth factors. PSA mRNA, but not actin mRNA, was down-regulated on ECM in a biphasic manner and was not detected up to 48 hr after culture, but was re-expressed after 3 days. Cycloheximide and actinomycin D pretreatment did not prevent ECM-induced down-regulation of PSA mRNA, while actinomycin D-treated cells on plastic maintained stable PSA mRNA levels. DNA synthesis and PSA glycoprotein secretion were also transiently suppressed on ECM. LNCaP growth inhibition correlated with decreased glyceraldehyde phosphate dehydrogenase mRNA levels. However, the transient growth suppression induced by ECM was not observed with primary endothelial cells on Matrigel. Down-regulation of PSA mRNA by culture on Matrigel was reversible upon transfer to a different matrix substrate. Re-expression was highest on heparan sulfate proteoglycan (4-fold) and fibronectin or collagen I (2-fold) compared to plastic or laminin. Our results indicate that the morphology and proliferation of LNCaP cells may be regulated by the ability of ECM to control cellular differentiation and proliferation. © 1994 Wiley-Liss, Inc.     

The induction of new ductal growth in adult prostatic epithelium in response to an embryonic prostatic inductor

Tissue recombinants were prepared with a single epithelial ductal tip from adult prostate and mesenchyme from either the embryonic urogenital sinus or adult urinary bladder. Recombinants were grown in vivo beneath the renal capsule of male hosts. After 4 weeks of in vivo growth, extensive growth of arborizing ducts was apparent in recombinants composed of urogenital sinus mesenchyme and a single adult prostatic ductal tip. One-dimensional polyacrylamide gel electrophoresis indicated that these recombinants contained many of the proteins of the mature prostate. Heterospecific recombinants (rat urogenital sinus mesenchyme and mouse prostatic epithelium) showed the ductal tissue to be derived solely from the prostatic epithelium. In recombinants of a prostatic ductal tip with mesenchyme from the urinary bladder, ductal growth was absent, the ductal tip was maintained as a single, discrete, epithelial structure, and the protein composition of these recombinants more closely resembled that of the bladder. The results demonstrate that the epithelial cells of the adult prostate can participate in new ductal growth in response to an embryonic prostatic inductor. These data provide experimental evidence to support the hypothesis that human benign prostatic hyperplasia may result from the anomalous reactivation of embryonic growth potential in the adult prostate.     

Effect of prostatic growth factor,basic fibroblast growth factor,epidermal growth factor,and steroids on the proliferation of human fetal prostatic fibroblasts

To study the relationship between androgen metabolism and the pathogenesis of benign prostatic hypertrophy, we purified a growth factor from benign hyperplastic tissue of human prostates and assayed the proliferative responses of human fetal prostatic fibroblasts to the purified growth factor (hPGF), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), dihydrotestosterone (DHT), and estradiol (E₂). Prostatic tissue extracts were fractionated using heparin-Sepharose chromatography. The fraction that eluted with 1.3–1.7 M NaCl contained the majority of mitogenic activity. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS/PAGE) of the lyophilyzed active fraction showed a band at 17,000 daltons. Human prostatic fibroblasts were isolated from fetal prostate and tested for their proliferative responses to hPGF, bFGF, EGF, DHT, and E₂. hPGF, as well as bFGF and EGF, did increase tritiated thymidine incorporation into the cultured fibroblasts. DHT(10⁻⁷ M) had a significant stimulatory effect on cell growth in serum-free media after 6 days of culture. E₂(10⁻⁷ M) had no effect on cell proliferation. The combination of DHT and E₂ showed no synergistic effect. We conclude that our purified hPGF, bFGF, and EGF promote cell growth directly, DHT indirectly, while E₂ does not. The effect of DHT appears to be mediated via the increased production and/or secretion of growth factor(s). Possibly, the bFGF-like hPGF purified from human benign hyperplastic prostatic tissue is such a mediator. © 1996 Wiley-Liss, Inc.     

Quantal relationship between prostatic dihydrotestosterone and prostatic cell content: critical threshold concept

The shape of the prostatic dihydrotestosterone (DHT) dose vs prostatic cell number response curve can be used to determine if the androgen-induced increase in prostatic cell number occurs as a continuous graded process increasing with all levels of prostatic DHT, or whether the process is quantal, beginning to occur only when a critical threshold of prostatic DHT is reached. To make this determination, castrated-adrenalectomized rats, castrated rats given no treatment, and castrated rats given testosterone-filled silastic implants of varying lengths were used to construct such a prostatic DHT dose/prostatic cell number response curve ranging from undetectable to pharmacologically high levels of prostatic DHT. The shape of this dose-response curve was found not to be continuously hyperbolic, but instead, to be discontinuously sigmoidal. This demonstrates that, in the rat, androgen-induced increase in prostatic cell number occurs as a quantal process which can only begin when the concentration of prostatic DHT is above a critical threshold value (ie, 0.4 ng/10(8) cells for the rat ventral prostate). Thus, in order to completely prevent this androgenic stimulation of prostatic cell number, the prostatic DHT level has to be lowered to below this critical threshold but does not have to be completely eliminated.     

2,3,7,8-tetrachlorodibenzo-p-dioxin inhibits prostatic epithelial bud formation by acting directly on the urogenital sinus

PURPOSE: In utero and lactational 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure causes lobe specific inhibition of prostate development in C57BL/6 mice due primarily to region specific inhibition of prostatic epithelial bud formation by the urogenital sinus (UGS). This inhibition requires that the receptor for TCDD, the aryl hydrocarbon receptor (AhR), must be present. We tested the hypothesis that TCDD inhibits prostatic epithelial bud formation by acting directly on the UGS. MATERIALS AND METHODS: UGSs were removed from WT and AhR null mutant (AhRKO) male C57BL/6 mice on gestation day 14 and incubated in vitro with vehicle, 10-8 M testosterone or 10-8 M testosterone plus 10-9 M TCDD for 5 days. Budding was evaluated by a newly developed technique, namely scanning electron microscopy of UGS epithelium after removal of UGS mesenchyme. RESULTS: Few buds were present in UGSs of either genotype in the absence of testosterone, while many were observed when testosterone was present. TCDD prevented prostatic epithelial buds from forming in UGSs from WT mice but it had no effect on UGSs from AhRKO mice. CONCLUSIONS: TCDD can act directly on the UGS to cause AhR dependent inhibition of prostatic epithelial bud formation. Because this inhibition occurred at a TCDD concentration similar to the estimated concentration at which TCDD inhibits bud formation in vivo, it appears that TCDD inhibits prostatic budding primarily via direct effects on the UGS rather than indirectly through effects on other organs.     

Role of mesenchymal-epithelial interactions in mammary gland development

The mammary gland is a hormone-target organ derived from epidermis and develops as a result of reciprocal mesenchymal-epithelial interactions. The induction of mammary differentiation from indifferent epidermal cells by mammary mesenchyme implies induction of the complement of hormone receptors characteristic of normal mammary epithelium in cells of the epidermis. Considering the facts that mammary epithelial differentiation is induced by mammary mesenchyme and that certain aspects of hormone response (androgen-induced mammary regression) are inextricably linked to mesenchymal-epithelial interactions, it is evident that the biology of the mammary gland arises from and is maintained via cell-cell interactions. As a corollary, perturbation of stromal-epithelial interactions in adulthood may play a role in mammary carcinogenesis and in turn may provide opportunities for differentiation therapy.