Hot and not-so-hot females: reproductive state and thermal preferences of female Arizona Bark Scorpions (Centruroides sculpturatus).

For ectotherms, environmental temperatures influence numerous life history characteristics, and the body temperatures (Tb ) selected by individuals can affect offspring fitness and parental survival. Reproductive trade-offs may therefore ensue for gravid females, because temperatures conducive to embryonic development may compromise females' body condition. We tested whether reproduction influenced thermoregulation in female Arizona Bark Scorpions (Centruroides sculpturatus). We predicted that gravid females select higher Tb and thermoregulate more precisely than nonreproductive females. Gravid C. sculpturatus gain body mass throughout gestation, which exposes larger portions of their pleural membrane, possibly increasing their rates of transcuticular water loss in arid environments. Accordingly, we tested whether gravid C. sculpturatus lose water faster than nonreproductive females. We determined the preferred Tb of female scorpions in a thermal gradient and measured water loss rates using flow-through respirometry. Gravid females preferred significantly higher Tb than nonreproductive females, suggesting that gravid C. sculpturatus alter their thermoregulatory behaviour to promote offspring fitness. However, all scorpions thermoregulated with equal precision, perhaps because arid conditions create selective pressure on all females to thermoregulate effectively. Gravid females lost water faster than nonreproductive animals, indicating that greater exposure of the pleural membrane during gestation enhances the desiccation risk of reproductive females. Our findings suggest that gravid C. sculpturatus experience a trade-off, whereby selection of higher Tb and increased mass during gestation increase females' susceptibility to water loss, and thus their mortality risk. Elucidating the mechanisms that influence thermal preferences may reveal how reproductive trade-offs shape the life history of ectotherms in arid environments.

Evaluation of the chemopreventive potential of retinoids using a novel in vitro human prostate carcinogenesis model.

The prevalence of prostatic intraepithelial neoplasia (PIN) and latent prostatic carcinoma, representing multiple steps in carcinogenesis and progression to invasive carcinoma, makes them relevant targets for prevention. A unique family of human prostate epithelial cell lines, which mimic steps in prostate carcinogenesis and progression, were used to evaluate the chemopreventive potential of all-trans-retinoic acid (RA) and N-(4-hydroxyphenyl)retinamide (4-HPR). The effects of RA and 4-HPR on anchorage-dependent growth of an immortalized, non-tumorigenic cell line RWPE-1 and two tumorigenic cell lines, WPE1-NB14 and WPE1-NB11, derived from RWPE-1 by exposure to N-methyl-N-nitrosourea (MNU), were examined. Both tumorigenic cell lines grow more rapidly than the parent RWPE-1 cell line in monolayer culture. Further, while RWPE-1 cells do not form colonies in agar, both tumorigenic cell lines do, with a colony forming efficiency (CFE) of 1.85 and 2.04% for WPE1-NB14 and WPE1-NB11 cells, respectively. Both RA and 4-HPR inhibited anchorage-dependent growth of all cell lines and anchorage-independent growth of WPE1-NB14 and WPE1-NB11 cells, in a dose-dependent manner, however, 10 times more RA than 4-HPR was required to produce the same effect. RWPE-1 cells are not invasive but WPE1-NB11 cells are significantly more invasive than WPE1-NB14 cells. Both RA and 4-HPR inhibited invasion in vitro by WPE1-NB11 and WPE1-NB14 cells where the more malignant WPE1-NB11 cells showed greater inhibition of invasion by 4-HPR than by RA. Overall, 4-HPR was more effective than RA in inhibiting growth and invasion but the response varied amongst the cell lines. These three cell lines mimic progressive steps in carcinogenesis and progression, from immortalized, non-tumorigenic RWPE-1 cells, to the less malignant WPE1-NB14 to the more malignant WPE1-NB11 cells, and provide powerful models for studies on secondary and tertiary prevention, i.e. promotion and progression stages, respectively, of prostate cancer.

N-(4-hydroxyphenyl)retinamide (4-HPR) decreases neoplastic properties of human prostate cells: an agent for prevention.

The development of prostate cancer through a multistep process of carcinogenesis may have a long latent period of 20-30 years. It is possible that progression to a malignant state could be blocked or reversed during this time. This study focuses on the ability of the synthetic retinoid, N-(4-hydroxyphenyl)-retinamide (4-HPR), to reverse changes associated with malignant transformation and tumor progression, towards a normal phenotype. To examine the responsiveness of cells at different steps of prostate carcinogenesis, three immortalized, but non-tumorigenic (RWPE-1, WPE1-7 and WPE1-10), and one human prostate carcinoma cell line (DU-145), were used. The effects of 4-HPR on cell proliferation, expression of intermediate filament proteins cytokeratin 18 and vimentin, and tumor suppressor proteins p53 and pRb were examined by immunostaining and compared. Results show that 4-HPR caused inhibition of growth in all cell lines in a dose-dependent manner. 4-HPR induced an increase in staining for cytokeratin 18, a marker of differentiation for prostate epithelial cells. While all cell lines showed strong immunostaining for vimentin, treatment with 4-HPR for 8 days caused a marked decrease in staining for vimentin in all cell lines. In an in vitro assay, 4-HPR also caused inhibition of invasion by DU-145 cells in a dose-dependent manner. Furthermore, 4-HPR treatment was effective in significantly decreasing the abnormal nuclear staining for the tumor suppressor proteins p53 and pRb. Because 4-HPR decreased invasion-associated vimentin expression, inhibited invasion, and normalized p53 and pRb immunostaining, we propose that 4-HPR may be an effective agent for secondary and tertiary prevention, i.e. promotion and progression stages, respectively, of prostate cancer.

The role of alpha 6 beta 1 integrin and EGF in normal and malignant acinar morphogenesis of human prostatic epithelial cells.

Complex multiple interactions between cells and extracellular matrix occur during acinar morphogenesis involving integrin receptors and growth factors. Changes in these interactions occur during carcinogenesis as cells progress from a normal to a malignant, invasive phenotype. We have developed human prostatic epithelial cell lines of the same lineage, which represent multiple steps in carcinogenesis, similar to prostatic intraepithelial neoplasia and subsequent tumor progression. The non-tumorigenic, RWPE-1 and the tumorigenic WPE1-NB27 and WPE1-NB26 cell lines were used to examine their ability to undergo acinar morphogenesis in a 3-D cell culture model and its relationship to invasion, integrin expression and EGF presence. An inverse relationship between the degree of acinar formation and invasive ability was observed. The non-tumorigenic, non-invasive RWPE-1 and the low tumorigenic, low invasive, WPE1-NB27 cells show high and decreased acinar forming ability, respectively, while the more invasive WPE1-NB26 cells show a loss of acinar formation. While RWPE-1 acini show basal expression of alpha 6 beta 1 integrin, which correlates with their ability to polarize and form acini, WPE1-NB27 cells lack alpha 6 but show basal, but weaker expression of beta 1 integrin. WPE1-NB26 cells show loss alpha 6 and abnormal, diffused beta 1 integrin expression. A dose-dependent decrease in acinar formation was observed in RWPE-1 cells when cell proliferation was induced by EGF. Anti-functional antibody to EGF caused an increase in acinar formation in RWPE-1 cells. These results suggest that malignant cells lose the ability to undergo acinar morphogenesis and that the degree of this loss appears to be related to invasive ability, EGF levels and alterations in laminin-specific integrin expression. This model system mimics different steps in prostate carcinogenesis and has applications in the secondary and tertiary prevention of prostate cancer.

Human cell lines as an in vitro/in vivo model for prostate carcinogenesis and progression.

BACKGROUND: The study of prostate carcinogenesis and tumor progression is made difficult by the lack of appropriate in vitro and in vivo models. High prevalence of prostatic intra-epithelial neoplasia and latent prostatic carcinoma, representing multiple steps in carcinogenesis to invasive carcinoma, are relevant targets for cancer prevention. From the RWPE-1, immortalized, non-tumorigenic, human prostate epithelial cell line, we have derived four tumorigenic cell lines with progressive malignant characteristics. METHODS: Cell lines were derived by exposure of RWPE-1 to N-methyl-N-nitrosourea (MNU), selected and cloned in vivo and in vitro, and characterized by prostatic epithelial and differentiation markers, karyotype analysis, anchorage-independent growth, invasiveness, tumorigenicity, and pathology of the derived tumors. RESULTS: Cytokeratins 8 and 18, androgen receptor, and prostate-specific antigen expression in response to androgen, confirm prostatic epithelial origin. RWPE-1 cells do not grow in agar and are not tumorigenic in mice, but the growth, tumorigenicity, and tumor pathology of the MNU cell lines correlate with their invasive ability. The WPE1-NA22 (least malignant) form small, well-differentiated, and WPE1-NB26 cells (most malignant) form large, poorly differentiated, invasive tumors. Overall, loss of heterozygosity for chromosomes 7q, 13q, 18q, and 22, and gain of 5, 9q, 11q, and 20, was observed. The MNU cell lines, in order of increasing malignancy are; WPE1-NA22, WPE1-NB14, WPE1-NB11, and WPE1-NB26. CONCLUSIONS: This family of cell lines with a common lineage represents a unique and relevant model which mimics stages in prostatic intra-epithelial neoplasia (PIN) and progression to invasive cancer, and can be used to study carcinogenesis, progression, intervention, and chemoprevention.

Cadmium-induced malignant transformation of human prostate epithelial cells.

Prostate cancer has become epidemic, and environmental factors such as cadmium may be partly responsible. This study reports malignant transformation of the nontumorigenic human prostatic epithelial cell line RWPE-1 by in vitro cadmium exposure. The cadmium-transformed cells exhibited a loss of contact inhibition in vitro and rapidly formed highly invasive and occasionally metastatic adenocarcinomas upon inoculation into mice. The transformed cells also showed increased secretion of MMP-2 and MMP-9, a phenomenon observed in human prostate tumors and linked to aggressive behavior. Cadmium-induced malignant transformation of human prostate epithelial cells strongly fortifies the evidence for a potential role of cadmium in prostate cancer.

Laminin-1 and alpha6beta1 integrin regulate acinar morphogenesis of normal and malignant human prostate epithelial cells.

BACKGROUND: Cell-matrix interactions via integrin receptors are critical for acinar morphogenesis. The non-tumorigenic, human prostate epithelial cell line RWPE-1 was used in a three-dimensional (3D) cell culture model to identify the matrix protein and its integrin receptor required for acinar morphogenesis. METHODS: 3D cultures, immunostaining, confocal microscopy, and Western blot analysis were used to examine acinar formation on matrix proteins and to determine integrin receptor expression. RESULTS: RWPE-1 cells differentiate into acini of polarized cells with a distinct lumen in 3D Matrigel culture. In contrast, the malignant WPE1-NB26 prostate epithelial cells form solid cell masses. In 3D gels of laminin-1, type IV collagen, or fibronectin, RWPE-1 cells form acini only in laminin-1. Anti-laminin-1 antibody reduces acinar formation in a dose-dependent manner. Polarized RWPE-1 cells showed basal expression of alpha6 and beta1 integrin subunits. Blocking antibodies to alpha6 or beta1 reduced acinar formation to 9 and 6 percent of control, respectively. The beta1 integrin colocalized with focal adhesion kinase (FAK). Inhibition of extracellular signal-regulated kinase kinase activity significantly reduced acinar formation to 38 percent of control, suggesting that beta1 integrin-mediated signal transduction may be regulated through a FAK pathway. CONCLUSIONS: While basal expression of alpha6beta1 integrin in RWPE-1 cells correlates with their ability to polarize and form acini, a decrease or loss of alpha6, and diffused beta1 expression in WPE1-NB26 cells correlates with loss of acinar-forming ability. Results show that laminin-1 and a functional alpha6beta1 integrin receptor are required for acinar morphogenesis. This novel 3D cell culture model is useful for elucidating regulation of acinar morphogenesis and its loss during prostate carcinogenesis.

Cadmium induces c-myc, p53, and c-jun expression in normal human prostate epithelial cells as a prelude to apoptosis.

Cadmium is a suspected human prostatic carcinogen shown to induce prostatic tumors and proliferative lesions in rats. The carcinogenic mechanism of cadmium is unknown, but its poor mutagenicity points toward an epigenetic mechanism. Here we studied the effect of cadmium on genes involved in growth regulation of prostate epithelial cell using the human prostate epithelial cell line RWPE-1, which is immortalized but not transformed and is androgen-responsive. Treatment with 10 microM cadmium resulted in transient increases in c-myc and p53 mRNA levels that peaked at 2-fold and 1.4-fold, respectively, compared to control after 2 h. In contrast, c-jun mRNA levels were increased >3-fold after 2, 4, and 6 h and 20-fold after 24 h. DNA synthesis decreased after 24 h of cadmium exposure. Further study revealed a significant increase in apoptosis after 48 h of cadmium exposure. However, approximately 35% of the cells were still viable and appeared normal, indicating this subpopulation was more resistant to cadmium. Furthermore, these resistant cells had 2.5-fold more metallothionein than untreated control cells. This suggests that cadmium could act to select for apoptotic-defective cells in vivo, thereby increasing the likelihood of tumor formation. This work represents the first description of cadmium affecting oncogene expression in a human cell model of a potential in vivo target site of cadmium carcinogenesis.

Modulation of the malignant phenotype of human prostate cancer cells by N-(4-hydroxyphenyl)retinamide (4-HPR).

A long latent period of 20 to 30 years may be involved in the multistep process of carcinogenesis represented by prostatic intraepithelial neoplasia (PIN) in the prostate. It is, therefore, possible that progression to a malignant state could be blocked or reversed during this time. Retinoids not only have the ability to block steps in the process of carcinogenesis but they may also modulate or reverse some malignant characteristics of cancer cells. This study focuses on the ability of N-(4-hydroxyphenyl)-retinamide (4-HPR), a synthetic retinoid, to reverse malignant characteristics towards a normal phenotype, using the human prostate carcinoma cell line DU-145. These malignant characteristics include abnormal cell proliferation, intermediate filament expression, motility, invasion, and cell survival. Results show that 1 microM and 10 microM 4-HPR caused 31% and 96% inhibition of growth, while all-trains retinoic acid (ATRA) produced similar effects at 10 and 100 microM, making 4-HPR ten times more effective than ATRA. While DU-145 cells show strong immunostaining for vimentin, treatment with 1 microM 4-HPR for eight days caused a marked decrease in vimentin staining. This was accompanied by a change from an elongated to an epithelial cell morphology. Densitometric analysis of Western blots for vimentin showed a 53% decrease in vimentin expression in 1 microM 4-HPR treated cells. Concomitant with the decrease in vimentin expression, cell motility and invasive ability also decreased by 32% and 52%, respectively. Growth inhibition was accompanied by DNA fragmentation and apoptosis. Exposure of cells to 1 microM 4-HPR caused a marked upregulation of nuclear retinoid receptors RARalpha and a detectable expression of RARgamma. These results suggest that inhibition of growth and vimentin expression, and induction of apoptosis by 4-HPR in prostate cancer cells may occur via a receptor-mediated mechanism involving transrepression of AP-1 by retinoid receptors. We propose that vimentin may serve as a useful intermediate marker for early detection of prostate cancer in biopsy specimens and that 4-HPR may be effective in blocking several steps in prostate carcinogenesis as well as the progression of PIN to invasive carcinoma.

A human prostatic stromal myofibroblast cell line WPMY-1: a model for stromal-epithelial interactions in prostatic neoplasia.

Here we report the characterization of an SV40 large-T antigen-immortalized stromal cell line, WPMY-1, derived from the same prostate as our previously described epithelial cell lines. The WPMY-1 cells were determined to be myofibroblasts on the basis of co-expression of smooth muscle alpha-actin and vimentin. They also show positive staining for androgen receptor, large-T antigen, and positive but heterogeneous staining for p53 and pRb. Their growth is stimulated by the synthetic androgen mibolerone to 145% of control (100%). Platelet-derived growth factor BB, epidermal growth factor and basic fibroblast growth factor, at 10 ng/ml, stimulated growth to 138, 143 and 146% of control, respectively. Transforming growth factor-beta, at 10 ng/ml, inhibited serum-induced growth to 65% of control in the presence of 1% serum, and bFGF-induced growth to 30% of control. A serum-free medium was developed for optimal growth of WPMY-1 cells. They show anchorage-independent growth in soft agar. Studies on paracrine interactions show that myofibroblast-conditioned medium causes a marked inhibition of growth in WPE1-10 cells, while conditioned medium from WPE1-10 prostatic epithelial cells caused only a small increase in the growth of WPMY-1 cells. WPMY-1 cells secrete very low levels of MMP-9 but high levels of MMP-2, markedly higher than the epithelial cells. These epithelial and myofibroblast cell lines, derived from the same prostate, provide novel and useful models for studies on paracrine stromal-epithelial interactions in carcinogenesis, tumor progression, prevention and treatment of prostate cancer and benign prostatic hyperplasia.

Laser scanning analysis of cell-cell communication in cultured human prostate tumor cells.

OBJECTIVE: To investigate gap-junctional intercellular communication (GJIC) in LNCaP and DU145 human prostate cancer cells. STUDY DESIGN: Normal rat liver F344 (WB1) cells were used as positive controls. Functional GJIC was inspected using either the scrape-loading/dye transfer (SL/DT) method or fluorescence recovery after photobleaching (FRAP) analysis. In the former, GJIC activity was expressed as a measure of the extent of diffusion of Lucifer Yellow after cell monolayers were scraped using a surgical blade and exposed to dye for a few minutes at room temperature. In the latter, cells were incubated for 15 minutes at 37 degrees C with 5,6-carboxyfluorescein diacetate dye and the dye transfer visualized by photobleaching individual cells with a 488-nm laser and monitoring the recovery of fluorescence using a laser cytometer. RESULTS: The preliminary results obtained indicate that neither LNCaP nor DU145 cells have functional GJIC, while, as expected, WB1 cells show unimpaired GJIC activity. Equivalent results were consistently obtained using either SL/DT or the FRAP approach. However, using FRAP analysis, DU145 cells only showed weak recovery of fluorescence after a total observation interval of 15 minutes. CONCLUSION: The present data, though preliminary, suggest that disruption of GJIC may play a role in development of malignancy in the human prostate.

Interleukin-6 and epidermal growth factor promote anchorage-independent growth of immortalized human prostatic epithelial cells treated with N-methyl-N-nitrosourea.

BACKGROUND: Epidermal growth factor (EGF) and interleukin (IL)-6 are implicated in the growth of benign and malignant prostatic epithelial cells. We investigated the role of EGF and IL-6 during the process of prostate carcinogenesis. METHODS: Using growth in soft agar as an index of transformation, we examined the effect of EGF and IL-6 on the enhancement of N-methyl-N-nitrosourea (MNU)-initiated transformation of immortalized, nontumorigenic prostatic epithelial cell lines (PWR-1E and RWPE-1) developed in our laboratory. The effect of EGF and IL-6 on the growth of MNU-induced transformants isolated from soft agar was assessed both in monolayer culture and in a soft agar. RESULTS: After a 1 hr exposure to N-methyl-N-nitrosourea (50 microg/ml), cells (5 x 10(4)) were grown in soft agar in the presence of EGF (5 ng/ml) or IL-6 (10 or 100 ng/ml). Addition of EGF or IL-6 significantly increased colony formation in soft agar of both immortalized prostatic epithelial cell lines initiated with MNU (P < 0.001-0.05). Only a very small number of colonies was observed with the parental cell lines PWR-1E and RWPE-1 not exposed to MNU, and their numbers increased by the addition of EGF or IL-6. All of the transformants, derived by exposure to MNU and isolated from soft agar, exhibited a higher cell growth potential in monolayer cultures than did their parental cell lines. Furthermore, as compared to the parental cell lines, growth response of MNU-transformants to 5alpha-dihydrotestosterone (5alpha-DHT), EGF, or IL-6 in monolayer culture was better in 5 of 8, 6 of 8, and 7 of 8 cell lines, respectively. All of the MNU-transformants exhibited a far higher colony-forming efficiency in soft agar than did the parental cell lines. However, the degree of responsiveness to EGF or IL-6 in soft agar varied among the MNU-transformants. CONCLUSIONS: The results of the present study suggest that IL-6 and EGF may enhance prostate carcinogenesis in vitro by preferentially stimulating the growth of transformed cells.

Long-term quantitative results following fundoplication and antroplasty for gastroesophageal reflux and delayed gastric emptying in children.

BACKGROUND: The operative management of children with combined gastroesophageal reflux and delayed gastric emptying is controversial. This study measures the long-term follow-up of gastric emptying in children who have undergone gastroesophageal fundoplication combined with antroplasty. METHODS: Fifteen randomly selected children with gastroesophageal reflux and scintigraphically demonstrated delayed gastric emptying underwent fundoplication and antroplasty. Each patient had another gastric emptying scintigraphic study performed an average of 3.6 years postoperation. RESULTS: All patients reported improvement of their symptoms compared with before the operation, and none required further medical therapy for gastroesophageal reflux or experienced dumping syndrome. Eleven of the 15 patients had significant long-term improvement of their gastric emptying postoperatively. The mean percent of isotope meal remaining in the stomach at 90 minutes improved from 72% preoperatively to 40% postoperatively (P = 0.0005). CONCLUSIONS: Gastric emptying in children with gastroesophageal reflux and delayed gastric emptying is significantly improved for several years in three-fourths of patients after fundoplication and antroplasty. Fundoplication and concomitant antroplasty are recommended for symptomatic children with documented gastroesophageal reflux and delayed gastric emptying.

Effects of 'crack' cocaine on pulmonary alveolar permeability.

BACKGROUND: Lung clearance of 99mTc-labeled diethylenetriamine pentaacetate (DTPA) is a sensitive test of altered alveolar epithelial permeability that has been found to be increased in smokers of tobacco, as well as a small number of healthy smokers of crack cocaine, suggesting the possibility of subclinical crack-related lung injury. STUDY OBJECTIVE: To evaluate further whether habitual smoking of cocaine alone alters alveolar permeability, whether crack smoking adds to or potentiates the effects of tobacco and/or marijuana, and whether experimental cocaine smoking acutely alters DTPA lung clearance. DESIGN: Observational cohort study (habitual cocaine smoking) and single-blind crossover study (experimental cocaine administration). SUBJECTS: Fourteen habitual smokers of cocaine alone (CS), 19 smokers of cocaine and tobacco (CTS), 3 smokers of cocaine and marijuana, 12 smokers of cocaine, tobacco, and marijuana (CMTS), and 5 smokers of marijuana plus tobacco (MTS). Results obtained in the crack-smoking subjects were compared with data previously obtained in 10 nonsmokers (NS), 9 smokers of tobacco alone (TS), 10 smokers of marijuana alone (MS), and 4 additional MTS. METHODS: Subjects underwent measurements of DTPA radioaerosol lung clearance after refraining from marijuana and/or cocaine for > 12 h and from tobacco for >2 h. Ten of the 48 crack users were tested on two days 1 to 2 weeks apart within 2 h of experimental smoking of three physiologically active or inactive doses (total 98.8+/-15.5 or 8.5+/-2.5 mg, respectively) of cocaine base. Lung clearance half-times (T1/2) were computed from time-activity curves for each lung. RESULTS: T1/2 values for each lung in CS and MS were comparable to those of NS, while TS, MTS, CTS, and CMTS had significantly shorter clearance rates than NS (p<0.01; three-way analysis of variance). No additive or interactive effects on T1/2 were noted among tobacco, cocaine, and/or marijuana. No acute effect of experimental cocaine smoking on T1/2 was noted. CONCLUSION: Whereas regular smoking of tobacco alone or with other substances increases alveolar epithelial permeability, habitual smoking of cocaine and/or marijuana has no measurable effect on alveolar permeability in the absence of tobacco nor any additive effect to that of tobacco alone.

Androgen responsive adult human prostatic epithelial cell lines immortalized by human papillomavirus 18.

Prostate cancer and benign tumors of the prostate are the two most common neoplastic diseases in men in the United States, however, research on their causes and treatment has been slow because of the difficulty in obtaining fresh samples of human tissue and a lack of well characterized cell lines which exhibit growth and differentiation characteristics of normal prostatic epithelium. Non-neoplastic adult human prostatic epithelial cells from a white male donor were immortalized with human papillomavirus 18 which resulted in the establishment of the RWPE-1 cell line. Cells from the RWPE-1 cell line were further transformed by v-Ki-ras to establish the RWPE-2 cell line. The objectives of this study were to: (1) establish the prostatic epithelial origin and androgen responsiveness of RWPE-1 and RWPE-2 cell lines; (2) examine their response to growth factors; and (3) establish the malignant characteristics of the RWPE-2 cell line. Immunoperoxidase staining showed that both RWPE-1 and RWPE-2 cells express cytokeratins 8 and 18, which are characteristic of luminal prostatic epithelial cells, but they also coexpress basal cell cytokeratins. These cell lines show growth stimulation and prostate specific antigen (PSA) and androgen receptor (AR) expression in response to the synthetic androgen mibolerone, which establishes their prostatic epithelial origin. Both cell lines also show a dose-dependent growth stimulation by EGF and bFGF and growth inhibition when exposed to TGF-beta, however, the transformed RWPE-2 cells are less responsive. RWPE-1 cells neither grow in agar nor form tumors when injected into nude mice with or without Matrigel. However, RWPE-2 cells form colonies in agar and tumors in nude mice. In the in vitro invasion assay, RWPE-1 cells are not invasive whereas RWPE-2 cells are invasive. Nuclear expression of p53 and Rb proteins was heterogeneous but detectable by immunostaining in both cell lines. The RWPE-1 cells, which show many normal cell characteristics, and the malignant RWPE-2 cells, provide useful cell culture models for studies on prostate growth regulation and carcinogenesis.

Acinar differentiation by non-malignant immortalized human prostatic epithelial cells and its loss by malignant cells.

Invasive prostatic carcinomas and prostatic intraepithelial neoplasia (PIN) are characterized by a loss of normal cell organization, cell polarity, and cell:cell and cell:basement membrane adhesion. The objective of this study was to establish in vitro three-dimensional (3-D) cell models which can be used to investigate mechanisms involved in acinar morphogenesis and differentiation in normal prostatic epithelium and their abnormalities in cancer cells. The process of acinar morphogenesis, including structural and functional differentiation, was investigated by culture on basement membrane gels (Matrigel). The human papillomavirus 18 immortalized, non-tumorigenic cell line RWPE-1, the v-Ki-ras transformed, tumorigenic RWPE-2 cell line derived from RWPE-1 cells (see previous paper pp. 1221-1229) and the human prostatic carcinoma cell line DU-145 were used. When cultured on Matrigel, RWPE-2 cells remain as single cells or form small aggregates and DU-145 cells form large amorphous cell aggregates without any organization or lumen. In contrast, RWPE-1 cells form acini of polarized epithelium with a distinct lumen, show a distinct laminin basement membrane, and express alpha6beta1 integrins at their basal end. Exposure to conditioned medium from NIH 3T3 cultures accelerates glandular morphogenesis. Parallel cultures maintained as monolayers on plastic remain as monolayers. In the presence of the synthetic androgen mibolerone, acinar cells express prostate specific antigen (PSA) as determined by immunostaining. We conclude that normal prostate cells can undergo acinar morphogenesis while tumorigenic cells have lost this ability. The 3-D cultures provide physiologically relevant in vitro models for elucidating regulation of growth, morphogenesis and differentiation in the normal human prostate, for defining heterotypic interactions in benign prostatic hyperplasia and for establishing the basis for the loss of normal cell organization in early neoplastic lesions such as PIN as well as during tumor progression in prostate cancer.

ras Activation of human prostate epithelial cells induces overexpression of parathyroid hormone-related peptide.

Immortalized adult and fetal prostate cell lines grown in serum-free conditions produce low levels of parathyroid hormone-related peptide (PTHRP) in the presence of growth factors as assessed by mRNA analysis, PTHRP immunoreactivity, and immunohistochemistry. Subsequent infection of these cells with Kirsten murine sarcoma virus containing an activated Ki-ras oncogene induces at least a 10-20-fold increase in PTHRP expression and production of both adult and fetal immortalized cell lines in the presence of the same growth factors. These results provide the first evidence of direct activation of PTHRP by the ras oncogene in human prostate cells and suggest its potential usefulness as a tumor marker in prostate malignancies.

Malignant transformation of human prostate epithelial cells by N-nitroso-N-methylurea.

We report the malignant transformation of adult human prostate epithelial cells after multiple exposures to the chemical carcinogen N-nitroso-N-methylurea. Such transformants showed morphological alterations and anchorage-independent growth in soft agar and induced carcinomas when transplanted into nude mice. No p53 or ras mutations were observed. Stepwise chromosomal changes in the progression to tumorigenicity were observed. Loss of the p arms of chromosome 8 (p10>pter) and chromosome 10 (p10>pter) and gain of the q arm of chromosome 8 (q10>qter) were only observed in the tumor outgrows. These findings provide the first evidence of malignant transformation of human prostate epithelial cells exposed to a chemical carcinogen.