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Carcinogenesis |
Departments of Urology [S. W. H., Y. K. H., G. D. G., G. R. C.], Anatomy [Y. W., M. C., B. Z., G. R. C.], and Pathology [D. S.], University of California-San Francisco, San Francisco, California 94143
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-estrogen (ER), or progesterone (PR) receptors by the parental BPH-1 cell line or by any of the tumor-derived cell strains. The cells uniformly coexpressed both basal and luminal cell-type cytokeratins and the basal cell marker p63. When grafted beneath the renal capsule of athymic mouse hosts, all of the tumor-derived cell strains consistently formed tumors. These were predominantly poorly or moderately differentiated squamous or adenosquamous tumors, similar in organization to the primary tumors from which the cell strains were derived. The cell strains continued to express both basal- and luminal-type cytokeratins in vivo. Some of the cell strains also coexpressed vimentin. E-cadherin expression was absent from many of the cells, although patches of cells expressing this marker were seen. The cells continued to express SV40T antigen. These cell strains, which are all derived from a common nontumorigenic progenitor, represent a useful resource for examining genetic and phenotypic changes during carcinogenesis. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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At present, there are a limited number of models of prostatic carcinogenesis (5) . Prostate cancer is a slowly developing disease of aging men and dogs and has an extremely low incidence of spontaneous occurrence in laboratory animals. A variety of methods, including hormonal induction and targeted expression of oncogenes have been used to induce prostate cancer in rats and mice (6, 7, 8, 9) . In a few cases, it has been possible to examine the range of tumor types derived from a common rodent precursor cell or tumor strain (10 , 11) .
There is a dearth of model systems to examine the induction of tumors in human prostatic epithelium. Although models that manipulate already malignant human prostatic cells and tissues are available (12) , there are few models in which cancer is induced in a previously benign human prostatic epithelium. Bae et al. and Jackson-Cook et al. (13 , 14) used repeated cycling of the generally nontumorigenic P69SV40T cell line through nude mouse hosts to produce tumorigenic sublines. Webber et al. and Bello et al. (5 , 15) have used both Ki-ras and the chemical carcinogen methylnitrosourea (MNU) to derive a series of tumorigenic sublines from the nontumorigenic RWPE-1 line.
SV40T3 was introduced into primary cultures of prostatic epithelial cells using the Zipneo viral construct containing a selectable neo cassette to generate the immortalized BPH-1 cell line (16) . SV40T interacts with and inactivates both p53 and pRb, thus eliminating these two important tumor suppressor pathways (17) . However many SV40T-expressing cells, including the BPH-1 cell line, are not tumorigenic (16 , 18) . The production of tumors in immuno-incompetent hosts is a clear marker of tumorigenicity and is easily demonstrated. The opposite of tumorigenicity (nontumorigenicity) implies an inability to form tumors. However the mere absence of tumors after transplantation into an immuno-incompetent host does not of itself prove that a cell line or graft is "nontumorigenic." This terminology requires that the cells or tissues, which were grafted or injected, have the ability to survive at the graft site and not to grow to form a tumor. We have previously demonstrated that parental BPH-1 cells grafted to athymic hosts can be recovered from the graft site for up to 1 year after grafting and that these cells do not form tumors at the site (0/125 attempts; Ref. 4 ). BPH-1 cells can however be considered to be genetically initiated, in that they have suffered a major genetic insult (attributable to the expression of SV40T), which renders them susceptible to further genetic damage and to progression along a pathway to malignancy.
Prostatic carcinogenesis involves genetic alterations to epithelial cells including activation of oncogenes (19, 20, 21, 22) and inactivation of tumor suppressor genes (23 , 24) . Alterations in tumor suppressor genes such as the RB3 and p53 genes have been suggested to play a role in the development of human prostate cancer (23 , 25, 26, 27) . The RB gene encodes a Mr 110,000 nuclear protein involved in cell cycle control (28) . RB gene mutations have been reported in 16.4% of primary human prostatic cancers, which suggests that inactivation of RB may play a role in carcinogenesis in at least a subset of prostatic carcinomas (29, 30, 31) . Estimates of the levels of p53 mutations in prostate cancer vary widely. Commonly reported figures suggest 20–50% of advanced stage tumors contain these mutations (32) . However estimates of p53 mutations as high as 56% in high-grade prostatic intraepithelial neoplasia and 72% in prostatic carcinoma have been made, based on immunohistochemical data (33) .
Androgens have long been known to elicit prostatic epithelial differentiation as a result of paracrine actions dependent on receptors located in the urogenital sinus mesenchyme (34) . In contrast, differentiated function of prostatic epithelium (expression of secretory markers) is apparently dependent on the presence of ARs located in the epithelial cells (35) . More recent data show that ERs in both stromal and epithelial cells play a role in mediating estrogenic effects in the prostate (36) . We have previously demonstrated that the nontumorigenic BPH-1 cell line can be induced to form tumors either by the influence of stromal cells that are derived from human prostate tumors (3) or by recombination with rUGM and stimulation with a combination of T+E2, presumably as a result of paracrine interactions (4) . This communication describes the isolation and characterization of a series of 10 tumorigenic sublines with various degrees of invasive potential derived from the nontumorigenic parental BPH-1 line. The data presented here demonstrate for the first time that genetically initiated but nontumorigenic human prostatic epithelial cells can undergo permanent malignant transformation as a result of their previous exposure to CAFs. We further demonstrate that cells derived from hormonally induced tumors are likewise tumorigenic. These cell lines will be a useful resource with which to investigate the genetic and phenotypic lesions induced by the in vivo process of carcinogenesis.
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MATERIALS AND METHODS |
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CAFs were prepared from human prostate tumors as described previously (3) . Briefly, tumors were identified using histopathological analysis of stained frozen sections. Five-mm3 tissue fragments immediately adjacent to identified carcinoma were used. Specimens were digested with collagenase and hyaluronidase (37) and placed into culture in RPMI 1640 containing penicillin, streptomycin, and Fungizone supplemented with 10% FBS. After 10 days of growth, the fibroblastic cells were separated from contaminating epithelial and endothelial cells by differential trypsinization. Subsequent immunocytochemical characterization confirmed their fibroblastic nature, as described previously (3) .
Pregnant rats were obtained from Simenson (Gilroy, CA). rUGM was prepared from 18-day embryonic fetuses (copulatory plug date denoted as day 0). Urogenital sinuses were dissected from fetuses and separated into epithelial and mesenchymal components by tryptic digestion, as described previously (38) . rUGM was then further reduced to single cells by a 90-min digestion at 37°C with 187 units/ml collagenase (Life Technologies, Inc., Grand Island, NY). After digestion, the cells were washed extensively with RPMI 1640 tissue culture medium. Viable cells were then counted using a hemacytometer, and viability was determined by trypan blue exclusion.
Cell recombinants were prepared by mixing 100,000 epithelial (BPH-1) cells with 250,000 rUGM cells or CAFs in suspension. For control grafts, 350,000 cells of each cell type (BPH-1, rUGM, or CAF) were used. Cells were pelleted and resuspended in 50 µl of neutralized rat tail collagen prepared as described previously (39) . The recombinants were allowed to set at 37°C for 15 min and were then covered with growth medium (RPMI 1640 + 5% FBS) containing testosterone (10-8 M) and were cultured overnight. Recombinants were then grafted beneath the renal capsule of adult male outbred athymic mice (Simenson). All of the animals were housed in the University of California-San Francisco laboratory animal resource center with food and drinking water ad libitum under controlled conditions (12 h light, 12 h dark, 20°C ± 2°C).
Mice carrying rUGM + BPH-1 recombinants were treated hormonally by surgical implantation of Silastic capsules containing E2 (17-ß-estradiol benzoate; Sigma Chemical Co.) and testosterone propionate (Sigma Chemical Co.) as described previously (4) . Control animals received empty Silastic tubing. Three months postgrafting, hosts were killed by anesthetic overdose followed by cervical dislocation. Kidneys were excised, and grafts were dissected free of the host kidney, weighed, and then processed for immunohistochemistry and for cell isolation.
Isolation of Cell Strains.
Grafts, which had formed tumors, were cut into small fragments using forceps and scalpel. Control grafts composed of BPH-1 + rUGM from untreated hosts were treated in the same manner. The resultant tissue fragments were plated overnight in tissue culture flasks in a small volume (1.2 ml in a 25-cm2 flask) of tissue culture medium (RPMI 1640 containing 5% FBS). The following day, the medium and any unattached fragments were aspirated and replaced with 5 ml of fresh medium. After 1 week of growth, the medium was additionally supplemented with 250 µg/ml G418 (Clontech, Palo Alto, CA). G418 selection was applied for 2 weeks, during which time all of the nonresistant cells died. The resulting cell populations were expanded in culture and characterized as described below.
Regrafting Experiments.
Epithelial cells (350,000 cells) of each of the derived cell strains were suspended in 50 µl of rat tail collagen gel, as described above, and grafted to the kidney capsules of male athymic mouse hosts. After 1 month of growth, the hosts were killed, and the grafts were removed from the kidney, weighed, fixed in formalin, and processed to paraffin. Before fixation, small pieces of the CAFTD grafts were removed and placed in culture as described above to isolate a "second generation" of TD cells. These were again grown in athymic mouse hosts for 1 month, excised and weighed, and fixed for immunohistochemical analysis. The lineage of these cell strains are shown in Fig. 1
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(clone 1D5) was purchased from Dako (Carpenteria, CA). Anti-Ki67 was purchased from Immunotech (Westbrook, ME). Purified rabbit and mouse IgGs were obtained from Zymed Corp. (South San Francisco, CA). Biotinylated antirabbit and antimouse IgG were obtained from Amersham International (Arlington Heights, IL). Biotinylated antigoat antibody was purchased from Sigma Chemical Co. Peroxidase-linked avidin/biotin complex reagents were obtained from Vector Laboratories (Burlingame, CA). After incubation with primary antibody, sections were washed with PBS and incubated with appropriate biotinylated secondary antimouse immunoglobulin diluted with PBS at 1:200 for 30 min at room temperature. After incubation with the secondary antibody, sections were washed in PBS (three 10-min washes) and then incubated with avidin-biotin complex (Vector Laboratories, Foster City, CA) for 30 min at room temperature. After a further 30-min of washing in PBS, immunoreactivity was visualized using 3', 3'-diaminobenzidine (DAB) in PBS and 0.03% H2O2. Sections were counterstained with hematoxylin and were dehydrated in graded alcohols. Control sections were processed in parallel with mouse or rabbit non-immune IgG (Dako) at the same concentration as the primary antibodies. For immunocytochemical analysis cells were grown on sialized microscope slides, fixed briefly in 100% ethanol, and washed in PBS. Cells were then blocked and processed as described above, except that epitope retrieval was not performed.
Proliferation Assays.
Proliferation assays were initiated by plating 25,000 BPH-1 or experimental cells in 0.5 ml of 5% FBS-containing medium into one well of a 24-well plate at 37°C and grown overnight to allow cells to attach. After attachment, serum-containing medium along with nonattached cells were aspirated. Experimental culture medium [RPMI 1640 (0.5 ml per well) with 2.5% dextran coated charcoal-stripped heat inactivated serum] was added, and cultures were continued another 5 days with one medium change.
Cultures were terminated for proliferation assays by aspirating culture medium and fixing cells with 0.2 ml of 11% glutaraldehyde per well on a rotary shaker for at least 20 min at room temperature. Glutaraldehyde was then removed, and plates were washed thoroughly with tap water and allowed to air-dry (
1 h). Cells were subsequently stained with 0.5 ml of 0.1% crystal violet per well in 200 nM boric acid (at pH 6.3) for 20 min on a rotary shaker. Crystal violet staining solution was aspirated from each well, and plates were again washed extensively with tap water and allowed to air-dry.
Proliferation measurement was started by adding 0.2 ml of 10% acetic acid per well for 20 min on a rotary shaker to solubilize the bound dye. Absorbance of the dye was measured at 590 nm with an Elx800UV automated microplate reader (BIO-TEK Instruments, Winooski, VT). Cell proliferation values were determined via extrapolation from a standard curve established with BPH-1 cells. Results are presented as the mean of at least three replicates with SE at 95% probability.
Preparation of Soft Agar Plates.
A bottom layer of 1 ml was prepared with a 1:1 mixture of 1% agar (50°C) and warm 2x DMEM and was poured into a well of a 6-well plate. This layer was allowed to solidify at 4°C for at least 15 min. A top layer was then constructed by mixing 500 µl of warm 2x DMEM with 200 µl of sterile dH2O and 300 µl of 1% agar (50°C) along with the cells at the appropriate density. Typically, 2 wells of a 6-well plate were each seeded with 103, 104, or 105 cells per well. The top layer with cells was poured on top of the solidified bottom layer and allowed to gel. After both layers had set, cultures were fed with 1 ml of RPMI 1640 supplemented with 5% FBS. Medium was gently removed and feedings delivered at least three times a week. After 4 weeks of culture, as much medium as possible was removed and cultures were stained with 10 µg/ml of tetrazolium violet (Sigma Chemical Co.) in medium overnight. Anchorage-independent growth was assessed by counting cell colonies of more than 200 µm in diameter in a single well of a 6-well plate. Data are presented as colonies present per 10,000 cells originally plated.
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RESULTS |
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. In addition to these cell lineages a strain of cells derived from BPH-1 + rUGM grafts to untreated hosts was also derived and designated BPH1UGM.
When grown on tissue culture plastic, the TD cell strains had a cobblestone or slightly elongated morphology. Although there were strain-to-strain differences, the TD cells were in general less tightly adherent to each other than the parental BPH-1 cells (Fig. 2)
. In vitro the cell strains uniformly expressed SV40T antigen, confirming their origin. The basal cell-specific markers, high-molecular weight cytokeratins and p63, were uniformly expressed as was the luminal cell-specific marker, cytokeratin 8 (Table 1)
. ARs, -ERs, and PRs were not detected by immunocytochemistry in the parental cell line or in any of the TD cell strains. Vimentin was not detected in the parental BPH-1 cell line but was weakly expressed in three of the BPH1CAFTD and both of the BPH1TETD strains (Table 1)
. Expression was slightly stronger in subconfluent than in confluent cultures. E-cadherin and ß-catenin expression was patchy in the tumorigenic sublines, with some cells showing no expression, others expressing poorly, and some demonstrating normal levels of protein. The expression of a range of markers by these cell strains is summarized in Table 1
. The BPH1UGM cells expressed the same markers, had the same morphology, and behaved in the same way, in all of the in vitro and in vivo assays, as the parental BPH-1 line.
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. In contrast the CAFTD sublines all exhibited some ability to form colonies in soft agar. The lone exception was BPH1CAFTD-04, which formed many fewer colonies than BPH1CAFTD-03, from which it was derived. This may reflect chance selection of a population of cells with low colony-forming ability during the selection of this strain. Results of the soft agar assay are summarized in Table 2
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but engulfed the host kidney given sufficient time (Fig. 4)
. Histologically, the tumors were squamous or adenosquamous (Fig. 5)
, similar to the tumors from which they were derived. Some tumors grew as noninvasive proliferative lesions on the surface of the host kidney. Others formed invasive lesions that engulfed and destroyed kidney tissue (Fig. 5)
. The tumors continue to express SV40T antigen. They coexpress markers characteristic of both basal and luminal epithelial cells (high-molecular weight cytokeratins, cytokeratin 14, p63, and cytokeratins 8 and 18). Ki67 labeling index (percentage of epithelial nuclei exhibiting positive staining with an anti-Ki67 antibody, based on counts of at least 2000 tumor epithelial cells) is comparable with, and in some cases elevated over, the 15% level previously described in BPH-1 epithelial cells recombined with rUGM in untreated nude mouse hosts (Ref. 4
; Table 3
). E-cadherin expression was patchy in the tumors, and in many places, the protein was not detectable. In some areas, apparently normal levels of membrane-localized protein were seen. In other areas, the protein was diffusely localized throughout the cell. Steroid hormone receptors (AR, PR, and ER) were not detected in the epithelial cells. ER was seen in cells of the kidney capsule and in some blood vessels within the tumor (Fig. 5)
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DISCUSSION |
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Malignant transformation in this model is most likely to be caused by genetic changes in the cells, a phenomenon described more fully in the article by Phillips et al. (41) . It is significant that there are characteristic patterns of genetic change that occur in four separate lineages of BPH-1 cells exposed to CAFs, although these were derived from separate experiments using different CAF populations. The BPH1CAFTD lines all share recurrent, and complex (harlequin) chromosomal rearrangements resulting in loss of 8p, 11p, and 20p, and high-level amplification of 1p, 11q, and 20q (41) . This type of specific genetic change is an important observation that underlines the concept that stromal microenvironment may play a crucial role in both promoting carcinogenesis and in eliciting genetic changes during malignant progression. The mechanism by which such common genetic changes could be induced by stromal environment is at present unclear, although the changes in cellular adhesion seen in these tumors may provide a clue to the cause of the genetic changes. We previously demonstrated that malignant changes are associated with a reduction or loss of membranous E-cadherin localization in the BPH-1 epithelial cells that form the tumors (3 , 4) . The cell lines described here likewise have a general tendency toward reduced expression and a loss of membrane localization of adherens junction proteins. Interactions between E-cadherin and p53 have been postulated to result in genomic instability (42) . In a broader context, cellular adhesion has been proposed to modulate neoplastic processes by altering the p53 pathways that control genomic stability (43) . However, in the present model, the p53 pathway is disrupted in both the benign and malignant phenotypes (because of the presence of SV40T), which suggests that this route may not be involved in progression from the nontumorigenic to the tumorigenic state here.
One model, which is in some ways similar, is a study showing that the human prostate cancer cell line LNCaP C4–2, when injected into nude mice, is capable of inducing genetic changes in host stromal cells around the injection site (44) . In this instance, genetically damaged and tumorigenic epithelial cells apparently induce "neoplastic transformation in stromal cells of the host organ by some, as yet unknown, epigenetic mechanism(s)" (44) . In the present model, the situation is somewhat different in that apparently genetically normal but phenotypically modified stromal cells are inducing permanent malignant transformation in genetically initiated but nonmalignant epithelial cells. It is, however, entirely possible that similar epigenetic mechanisms are involved in this process.
Our model of tumorigenesis in the BPH-1 cell line raises the issue of the relative importance and role of genetics versus epigenetic factors in tumorigenesis. CAF-induced tumorigenesis of BPH-1 cell is clearly an epigenetic effect that leads to further genetic alterations in the course of the tumorigenetic process. T+E2-induced carcinogenesis in BPH-1 cells requires interaction with rUGM as well as treatment with T+E2. Hormonal carcinogenesis induced by T+E2 in rUGM+BPH-1 recombinants, therefore, is also likely to involve epigenetic as well as genetic mechanisms. Thus, the pathway to hormonal carcinogenesis surely can involve both epigenetic and genetic mechanisms. The dominance of genetic versus epigenetic mechanisms in determining progression to tumorigenesis or the tumorigenetic state per se appears to vary on an individual case-by-case basis.
There are many instances in which the single addition of a dominant acting oncogene is sufficient to convert a nontumorigenetic cell to a fully tumorigenetic cell. Such observations emphasize the important genetic change as a key determinant of malignancy. On the other hand, studies with highly malignant teratocarcinoma cells emphasize the dominance of epigenetic factors in the expression of benign versus malignant growth. Small numbers of teratocarcinoma cells transplanted into a host will consistently form tumors that will lead to the demise of the host (45) . Such highly malignant cells, when microinjected into mouse blastocysts, will participate in normal development producing a range of benign tissues representing all germ layers. Indeed, because of contribution of teratocarcinoma cells to the germ line, it is possible to derive mouse strains from teratocarcinoma cells (46) . Thus, despite the genetic alterations within the parental teratocarcinoma cells, epigenetic factors can elicit reversion of highly malignant cells to benign cells.
The ability of T+E2 to induce malignancy in BPH-1 cells has been previously demonstrated (4) . The original description of these tumors emphasizes that stromal cells are required for sex hormone-induced carcinogenesis. We also demonstrated that transplantable tumors can be established from these T+E2-induced tumors. The present study demonstrates that epithelial cells that are isolated from these hormone-induced tumors retain their malignant potential. The BPH1TETD strains shared the 11q and 20q amplifications seen in BPH1CAFTD but also had high-level amplifications of 7p encompassing the v-erb (epidermal growth factor receptor) region. In addition to the unbalanced translocations seen in the CAFTD cells, TETD cells also had examples of reciprocal translocations, the exact genetic implication of which is currently unclear but may reflect the unique pathways to tumorigenesis induced by TE/rUGM (41) . Unlike the situation in CAF-induced carcinogenesis, there are well-established mechanisms by which sex steroid hormones, especially estrogens, can induce genetic change. Metabolism of natural and synthetic estrogens generates free radicals that are capable of DNA damage (47) . Both diethylstilbestrol and estradiol can induce sister chromatid exchange (48, 49, 50) . T+E2 treatment also increases DNA strand breakage as a result of free radical action (51) .
There are a number of signaling mechanisms that could act to promote carcinogenesis; these include the Wnt/frizzled pathway, which may act to regulate cellular adhesion through adherens junctions (for a recent summary see Ref. 52 ). Growth factors are also obvious potential mediators of paracrine interactions between epithelial and stromal cells. A number of growth factors, notably members of the IGF and the fibroblastic growth factor (FGF) families have been implicated as potentially playing a role in models of prostate cancer. As an example; attention has focused on IGFs because of data suggesting that they may be linked to human prostate cancer progression (53, 54, 55) . Mutations of IGF-1 or its receptor are known to delay prostatic development (56) , whereas overexpression of the ligand is suggested to induce prostatic carcinogenesis (57) . In murine models of prostate cancer, IGF-1 levels were also seen to rise in conjunction with progression of the disease (58) . Clearly this is a complex field that is not, at present, well understood.
A comparison of the in vivo and in vitro characteristics of the various cell lines reveals that behavior in the two environments is only loosely linked. Thus, for example, in the present study, only tumorigenic epithelial cells formed colonies in soft agar. However, the inability to form colonies under these conditions does not preclude a cell strain from being highly tumorigenic and invasive in vivo. These observations reinforce the importance of determining the true in vivo potential of cells to form tumors before extrapolating from data obtained in vitro.
The present communication describes a series of cell substrains with a variety of malignant phenotypes generated from a clonally derived nontumorigenic human prostatic epithelial cell line. These cells have the advantage that the sublines have a common genetic heritage, having all been originally derived from a single cell. This will allow a characterization of the effects of specific genetic insults. BPH-1 was immortalized with SV40 large T only, rather than the whole SV40 early region, resulting in a clonally derived cell line that, although immortalized, is not tumorigenic in nude mice. We have previously demonstrated that this cell line can undergo some aspects of prostatic differentiation and can be induced to form tumors both by sex steroid hormones and by its stromal microenvironment (3 , 4) . The lines described retain epithelial characteristics and are tumorigenic but nonmetastatic. Both invasive and noninvasive phenotypes were seen, although these did not correlate with other characteristics examined.
In summary, this report demonstrates that stromal microenvironment and hormonal carcinogens can elicit permanent malignant transformation in human prostatic epithelial cells. We further characterize a series of cell strains that will be useful tools to further examine the processes involved in carcinogenesis elicited by various mechanisms.
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FOOTNOTES |
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1 Supported by NIH Grants DK52721, DK52708, CA64872, DK47517, CA59831, and CA89520. 
2 To whom requests for reprints should be addressed, at Department of Urologic Surgery, A 1302 Medical Center North, Vanderbilt University Medical Center, Nashville, TN 37232-2765. Phone: (615) 322-5823; Fax: (615) 322-8990; E-mail: simon.hayward{at}mcmail.vanderbilt.edu
3 The abbreviations used are: SV40T, SV40 large T (antigen); RB, retinoblastoma; CAF, carcinoma-associated fibroblast; rUGM, rat urogenital sinus mesenchyme; FBS, fetal bovine serum; TD, tumor-derived; AR, androgen receptor; ER, estrogen receptor; E2, estradiol; T+E2, testosterone and E2; PR, progesterone receptor; IGF, insulin-like growth factor.
Received 6/ 4/01. Accepted 9/18/01.
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-glycerolphosphate dehydrogenase in benign prostatic hyperplasia cells in primary culture. J. Urol., 138: 648-653, 1987.[Medline]
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P. A. Watson, K. Ellwood-Yen, J. C. King, J. Wongvipat, M. M. LeBeau, and C. L. Sawyers Context-Dependent Hormone-Refractory Progression Revealed through Characterization of a Novel Murine Prostate Cancer Cell Line Cancer Res., December 15, 2005; 65(24): 11565 - 11571. [Abstract] [Full Text] [PDF]
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M. S. Joesting, S. Perrin, B. Elenbaas, S. E. Fawell, J. S. Rubin, O. E. Franco, S. W. Hayward, G. R. Cunha, and P. C. Marker Identification of SFRP1 as a Candidate Mediator of Stromal-to-Epithelial Signaling in Prostate Cancer Cancer Res., November 15, 2005; 65(22): 10423 - 10430. [Abstract] [Full Text] [PDF]
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 M. V. Maffini, J. M. Calabro, A. M. Soto, and C. Sonnenschein Stromal Regulation of Neoplastic Development: Age-Dependent Normalization of Neoplastic Mammary Cells by Mammary Stroma Am. J. Pathol., November 1, 2005; 167(5): 1405 - 1410. [Abstract] [Full Text] [PDF]
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 D M Peehl Primary cell cultures as models of prostate cancer development Endocr. Relat. Cancer, March 1, 2005; 12(1): 19 - 47. [Abstract] [Full Text] [PDF]
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R. Berger, P. G. Febbo, P. K. Majumder, J. J. Zhao, S. Mukherjee, S. Signoretti, K. T. Campbell, W. R. Sellers, T. M. Roberts, M. Loda, et al. Androgen-Induced Differentiation and Tumorigenicity of Human Prostate Epithelial Cells Cancer Res., December 15, 2004; 64(24): 8867 - 8875. [Abstract] [Full Text] [PDF]
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 E. L. Rosenthal, A. McCrory, M. Talbert, W. Carroll, J. S. Magnuson, and G. E. Peters Expression of Proteolytic Enzymes in Head and Neck Cancer-Associated Fibroblasts Arch Otolaryngol Head Neck Surg, August 1, 2004; 130(8): 943 - 947. [Abstract] [Full Text] [PDF]
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 M. D. Westfall and J. A. Pietenpol p63: molecular complexity in development and cancer Carcinogenesis, June 1, 2004; 25(6): 857 - 864. [Abstract] [Full Text] [PDF]
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 G. Castoria, M. Lombardi, M. V. Barone, A. Bilancio, M. Di Domenico, D. Bottero, F. Vitale, A. Migliaccio, and F. Auricchio Androgen-stimulated DNA synthesis and cytoskeletal changes in fibroblasts by a nontranscriptional receptor action J. Cell Biol., May 12, 2003; 161(3): 547 - 556. [Abstract] [Full Text] [PDF]
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 B.-e. Wang, J. Shou, S. Ross, H. Koeppen, F. J. de Sauvage, and W.-Q. Gao Inhibition of Epithelial Ductal Branching in the Prostate by Sonic Hedgehog Is Indirectly Mediated by Stromal Cells J. Biol. Chem., May 9, 2003; 278(20): 18506 - 18513. [Abstract] [Full Text] [PDF]
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K. C. Day, M. T. McCabe, X. Zhao, Y. Wang, J. N. Davis, J. Phillips, M. Von Geldern, T. Ried, M. A. KuKuruga, G. R. Cunha, et al. Rescue of Embryonic Epithelium Reveals That the Homozygous Deletion of the Retinoblastoma Gene Confers Growth Factor Independence and Immortality but Does Not Influence Epithelial Differentiation or Tissue Morphogenesis J. Biol. Chem., November 8, 2002; 277(46): 44475 - 44484. [Abstract] [Full Text] [PDF]
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J. Shou, R. Soriano, S. W. Hayward, G. R. Cunha, P. M. Williams, and W.-Q. Gao Expression profiling of a human cell line model of prostatic cancer reveals a direct involvement of interferon signaling in prostate tumor progression PNAS, March 5, 2002; 99(5): 2830 - 2835. [Abstract] [Full Text] [PDF]
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