Overexpression and gene amplification of BAG-1L in hormone-refractory prostate cancer.

BAG-1L (Bcl-2-associated anthanogene 1) has been found to interact with androgen receptor (AR), and has been suggested to be involved in the development of prostate cancer. In order to determine the presence of genetic and/or expression alterations of BAG-1L in prostate cancer, we analysed human prostate cancer cell lines and xenografts as well as patient samples of untreated, hormone-naïve, and hormone-refractory prostate carcinomas for sequence variations using denaturing high-performance liquid chromatography (DHPLC), for gene copy number using fluorescence in situ hybridization (FISH), and for expression using both quantitative RT-PCR and immunostaining. Only one sequence variation was found in all 37 cell lines and xenografts analysed. BAG-1 gene amplification was detected in two xenografts. In addition, gene amplification was found in 6 of 81 (7.4%) hormone-refractory clinical tumours, whereas no amplification was found in any of the 130 untreated tumours analysed. Additionally, gain of the BAG-1 gene was observed in 27.2% of the hormone-refractory tumours and in 18.5% of the untreated carcinomas. In a set of 263 patient samples, BAG-1L protein expression was significantly higher in hormone-refractory tumours than in primary tumours (p = 0.002). Altogether, these data suggest that amplification and overexpression of BAG-1L may be involved in the progression of prostate cancer.

Oxidative/nitrosative stress and peroxiredoxin 2 are associated with grade and prognosis of human renal carcinoma.

Peroxiredoxins (Prxs) 1-6 were assessed in 138 renal cell carcinomas (RCC) using immunohistochemistry and selected samples by Western blotting analysis. Oxidative/nitrosative damage was evaluated using nitrotyrosine immunoreactivity. The expressions of Prxs were correlated with tumor grade and survival and nitrotyrosine reactivity. Non-malignant kidney tubular cells showed positivity with variable intensity for all six Prxs. In RCCs, most cases were positive for Prxs 1 and 2, while only 15-20% of tumors showed expression for Prxs 3 and 4. Prx 2 was associated with tumors of a lower grade (p=0.009) and with a lower frequency of distant metastases (p=0.046). Patients with tumors expressing Prx2 had better prognosis (p=0.027). Instead, nitrotyrosine was significantly associated with high grade tumors (p=0.001). Compared with the non-malignant kidney tubular cells, low Prx expression in the tumor cells can make them more susceptible to oxidative damage. Prx 2 was more abundantly expressed in low grade tumors, suggesting that this protein could play a role in preventing the development of oxidative damage, which in turn can lead to the activation of pathways leading to aggressive tumors.

Altered levels of Smad2 and Smad4 are associated with human prostate carcinogenesis.

Alterations have been demonstrated in ligand and cognate receptor system of the transforming growth factor beta (TGF-beta) pathway in prostate cancer (PC). Still, little is known about changes in the activity of the intracellular Smad cascade of TGF-beta signaling during prostate carcinogenesis. We used immunohistochemistry to analyze phosphorylated Smad2 (p-Smad2), nuclear Smad4 and inhibitory-Smad7 in epithelial cells of normal, hyperplastic and malignant prostate. Specimens comprised 49 tissue cores of PC, 10 benign prostate hypertrophies and three normal prostates. Nuclear p-Smad2 (P<0.001) and nuclear Smad4 (P=0.023) were significantly decreased in PC with remarkable variations in cytoplasmic Smad7 levels. Substantial decreases in p-Smad2 and Smad4 levels were found in specimens with primary Gleason grades 3 and 4, whereas in grade 5, levels were markedly higher. Our results provide the first evidence for changes and reversible attenuation in the Smad system of the TGF-beta pathway during prostate carcinogenesis.

Antioxidant enzymes in renal cell carcinoma.

The aim of the study was to estimate the significance of oxidative/nitrosative damage and expression of antioxidant enzymes in renal cell carcinomas (RCC). For this we investigated immunohistochemically six antioxidant enzymes (AOEs) including MnSOD, ECSOD, thioredoxin, thioredoxin reductase, and gammaglutamyl cysteine synthetase heavy and light chain in 138 RCCs. As an indicator of oxidative/nitrosative damage, sections were stained with an antibody to nitrotyrosine. The extent of apoptosis was evaluated by TUNEL method and proliferation by immunohistochemistry to Ki67. Variable expression of all AOEs could be seen in RCC with expression of MnSOD being strongest. Nitrotyrosine was significantly associated with high grade tumors. MnSOD was associated with tumors of a lower stage. Cases showing ECSOD reactivity had higher and cases expressing thioredoxin lower apoptotic index than other tumors. No association with patient prognosis was observed. According to the results renal cell carcinomas show oxidative/nitrosative damage which, according to nitrotyrosine staining, was higher in high grade tumors. Of AOEs, MnSOD was more abundantly expressed in low stage tumors suggesting that its antioxidant function could play a main role to prevent development of oxidative damage leading to more aggressive tumors.

A novel organotypic culture model for normal human endometrium: regulation of epithelial cell proliferation by estradiol and medroxyprogesterone acetate.

BACKGROUND: A novel organotypic culture system was established for modelling the hormonal responses of the normal human endometrium in vitro. METHODS: Endometrial epithelial cells were cultured as glandular organoids within reconstituted extracellular matrix (Matrigel) in tissue culture inserts and stromal cells on plastic below the epithelial compartment. The effects of estradiol (E2) and E2 together with medroxyprogesterone acetate (MPA) on cell proliferation and the expression of estrogen receptor alpha (ERalpha) and progesterone receptor (PR) were studied in 10 epithelial-stromal co-cultures and in three parallel monocultures of epithelial organoids. RESULTS: In co-cultures, E2 was shown to increase the percentage of Ki67-positive cells by approximately 2-fold relative to untreated controls. In the presence of MPA, a significant decrease in cell proliferation was detected. Similar results were obtained when the corresponding percentages of Ki67-positive organoids were calculated instead of individual cells. In the absence of stromal fibroblasts, Ki67 epithelial labelling remained below the control value after both hormonal treatments. Epithelial organoids retained their capacity to express estrogen and progesterone receptors in culture. E2 was shown to markedly increase and MPA to down-regulate the expression of PR. The expression of ERalpha was only slightly affected by either hormonal treatment. CONCLUSIONS: The present organotypic model provides a novel in vitro system in which to study the effects of steroids in the normal human endometrium both in terms of cell proliferation and gene expression. The culture system holds promise as a useful method to screen novel steroid compounds and may help to circumvent problems related to the use of animal models.

Membranous location of EGFR immunostaining is associated with good prognosis in renal cell carcinoma.

Epidermal growth factor receptor (EGFR) is a key factor in tumorigenesis. The association between EGFR expression and prognosis in renal cell carcinoma (RCC) is not clear. In our study of 134 RCCs, the cellular location of immunostaining was evaluated and patients with EGFR-positive tumours with prominent membranous staining had a good prognosis. Their overall survival was significantly longer (P=0.004) than that of patients with either EGFR-negative tumours or with mainly cytoplasmic staining. However, further studies on the different EGFR expression patterns in RCC are needed to clarify their role in the progression of the disease.

Consistency of prostate cancer grading results in screened populations across Europe.

OBJECTIVE: To assess the consistency of grading outcome among seven of eight participating centres of the European Randomised Screening Program of Prostate Cancer (ERSPC), a multicentre randomized trial intended to detect a difference in prostate cancer-related mortality between screened participants and a control group. Currently, tumour stage and grade in prostatectomy specimens represent the most predictive variables for biological behaviour. In prostate needle biopsies the tumour grade is a strong factor for deciding therapy. PATIENTS AND METHODS: Within the ERSPC all prostate cancers detected in needle biopsies were graded according to the Gleason score system. Gleason scores were compressed in three categories of < or = 6, 7 and 8-10. Data for grading outcome were obtained from the databases from seven individual centres; in one centre the slide sets with cancer were separately reviewed. RESULTS: Combining the data of seven ERSPC centres 66% of cancers detected in the screening arm were Gleason score < or = 6 and 92% were < or = 7. Gleason score 8-10 cancers varied from 2 to 11%. This variation in Gleason scores may be attributed to differences in the population characteristics and biopsy indications. CONCLUSIONS: These data indicate that in the seven ERSPC centres most screen-detected cancers have favourable characteristics on biopsy. Men with these cancers are amenable for treatment with curative intent. The observed differences in Gleason score distribution in different centres may partly be attributed to geographical differences and differences in the age range of the screened populations.

Increased expression of FGF-8 isoforms and FGF receptors in human premalignant prostatic intraepithelial neoplasia lesions and prostate cancer.

SUMMARY: Fibroblast growth factor 8 (FGF-8) is implicated in growth of prostate cancer. Alternative splicing of the human FGF-8 gene potentially allows coding for four protein isoforms (a, b, e, and f). These isoforms differ in their binding to FGF receptors (FGFR) and in their mitogenic and transforming capacity in transfection assays. Here, we used RT-PCR and immunohistochemistry to study the expression of FGF-8 and FGFR isoforms in human prostate cancer (n = 31). Nonmalignant prostate specimens from cystoprostatectomies (n = 24) were examined as controls. Most prostate cancer samples and some control prostates also contained prostatic intraepithelial neoplasia (PIN) lesions. FGF-8a and e were expressed at significantly higher frequencies in prostate cancer (FGF-8a, 55%; FGF-8e, 45%) than in control samples (FGF-8a, 17%, p = 0.0052; FGF-8e, 8%, p = 0.0031). On the contrary, FGF-8b was found at an equal frequency in prostate cancer (55%) and in control prostates (50%). Furthermore, a combination of two or three FGF-8 isoforms (a, b, and/or e) was also expressed at a higher frequency in prostate cancer than in control samples (45% and 8%, respectively, p = 0.0031). Immunohistochemistry with an antibody recognizing all FGF-8 isoforms was more strongly immunoreactive in prostate cancer cells and PIN lesions than in normal-type epithelium. The receptor splicing variants FGFR1IIIc and FGFR2IIIc, which are activated by FGF-8, were found both in prostate cancer and control samples. Interestingly, immunoreactivity for FGFR1 and FGFR2 was much stronger in prostate cancer cells and PIN than in normal epithelium. These results demonstrate, for the first time, that FGF-8 isoforms and their receptors FGFR1IIIc and FGFR2IIIc are expressed at an increased level not only in prostate cancer but also in premalignant PIN lesions. These data suggest that FGF-8 may have an important autocrine role in the development of human prostate cancer. In addition to FGF-8b, the FGF-8 isoforms a and e may be involved in this process.

Prolactin is a survival factor for androgen-deprived rat dorsal and lateral prostate epithelium in organ culture.

PRL is one of several polypeptide factors that regulate growth and differentiation of prostate epithelium besides steroid hormones. This hormone may also participate in the development of pathologic changes of the prostate, as evidenced by marked prostate hyperplasia in hyperprolactinemic mice. We have previously demonstrated expression of PRL receptors and androgen-dependent local production of PRL in rat and human prostate epithelium, suggesting the existence of an autocrine loop. We now show that PRL acts as a survival factor for epithelial cells of rat dorsal and lateral prostate but not ventral prostate, using long-term organ cultures as an in vitro model. Culture of prostate explants in androgen-free medium was associated with a transient surge of apoptosis during the first 2-4 days of culture in rat ventral, dorsal, and lateral prostate tissues, as quantified by either nuclear morphology or in situ DNA fragmentation analysis. PRL significantly inhibited apoptosis in androgen-deprived dorsal and lateral prostate cultures, by 40-60%, as determined by the two methods. The present study has established conditions and methodology for analysis of apoptosis in organ cultures of rat prostate and suggests a physiological role for PRL as a survival factor for prostate epithelium.

Prolactin and prolactin receptors are expressed and functioning in human prostate.

Prolactin is widely expressed in different tissues, and it is presumed to have both local and systemic actions. In males it is known to influence reproductive functions but the significance and mechanisms of prolactin action in male accessory reproductive tissues are poorly understood. Here we show that prolactin acts as a direct growth and differentiation factor for human prostate, as measured by changes in DNA synthesis and epithelial morphology of organ cultures. Furthermore, we report the expression in human prostate of a short prolactin receptor form in addition to the long form, based upon ligand cross-linking studies and RT-PCR analysis of mRNA expression. The highest density of prolactin receptors was detected in the secretory epithelial cells by immunohistochemistry. Finally, we report that prolactin is locally produced in human prostate epithelium, as evidenced by marked prolactin immunoreactivity in a significant portion of prostate epithelial cells, with parallel expression of prolactin mRNA in human prostate. Collectively, these data provide significant support for the existence of an autocrine/paracrine loop of prolactin in the human prostate and may shed new light on the involvement of prolactin in the etiology and progression of neoplastic growth of the prostate.

Multilocular renal cyst. Report of two cases and review of the literature.

Multilocular renal cyst (MRC), or cystic nephroma (CN), is a rare tumour of the kidney. Approximately one hundred cases have been reported, half of these in children. Normally these lesions have been confirmed to be benign cystic lesions, but it is possible for them to transform into malignancy. In children this type of tumour may simulate Wilms' tumour. We report two cases of MRC disease with clinical findings, pathology, treatment and survival.

Hormone regulation of human prostate in organ culture.

We have established organ cultures of human prostate for in vitro analysis of the hormone responsiveness of prostatic carcinoma. Tissue samples were obtained from total prostatectomies for localized cancer. Normal prostate tissues with age-related hyperplastic changes were obtained from cystoprostatectomies of bladder cancer patients representing the same age group, and they wer cultivated as controls. The explants of prostates were cultured for 7 days in basal medium containing 5% dextran charcoal-treated fetal calf serum, insulin (0.08 IU/ml), and dexamethasone (10(-7) M) with or without dihydrotestosterone (DHT) (10(-7) M) or estradiol (10(-9) M). Control prostates showed involutive changes of morphology when cultured in basal medium. These changes were prevented by DHT, which also maintained a strong epithelial immunostaining for PSA (prostate specific antigen), which was used as a marker for tissue-specific functions. The concentration of PSA in the medium was high. The rate of [3H]thymidine incorporation into DNA was stimulated by DHT in some cultures of control prostates, but no increase was seen in the others. Androgen stimulation of [3H]thymidine incorporation was consistently inhibited by the antihormone cyproterone acetate. The main morphological response of cultured control prostates to estradiol was induction of squamous metaplasia. This was associated with increased incorporation of [3H]thymidine, which was radioautographically localized to the basal layer of epithelium. Estradiol effects were counteracted by the antihormone toremifene. The expression of androgen receptor mRNA and protein in cultured control prostate was demonstrated by Northern blotting and immunohistochemistry, respectively. Also, the expression of estrogen receptor was demonstrated by the polymerase chain reaction analysis of total mRNA from cultured control and cancer prostate. The cultured explants of prostate cancer maintained the overall morphology of the original carcinoma. However, the presence of DHT improved the morphology of cancerous acini in all better differentiated carcinomas (3 grade I and 5 grade II), and corresponding responses to DHT were observed in the rate of DNA labeling with [3H]thymidine. In 2 of 3 grade I carcinomas, DHT increased DNA synthesis, but in grade II cancers the patterns of hormone responses were more variable. The poorly differentiated grade III prostatic carcinomas did not respond to either hormone as measured by [3H]thymidine uptake, and no hormone effects could be seen in morphology. Immunostaining for PSA differed from that in control prostates: besides cancerous acini, the surrounding stroma was also intensively stained, which suggests unpolarized and impaired secretion of PSA by the cancer cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Apoptosis in toremifene-induced growth inhibition of human breast cancer cells in vivo and in vitro.

BACKGROUND: Antiestrogens inhibit the stimulative effects of estrogens on breast cancer growth, but the mechanism(s) by which they trigger tumor regression are not completely understood. Growth retardation and tumor regression can be achieved by enhanced cell death and/or arrested cell proliferation. PURPOSE: Our aim was to investigate the effect of a new antiestrogen, toremifene, on human breast cancer cells grown either in culture or as tumors in nude mice. METHODS: The growth and morphology of in vitro cultured cells of the human breast cancer cell line MCF-7 were monitored by time-lapse video. MCF-7 cells and ZR-75-1 human breast cancer cells were grown as tumors in nude mice and subsequently examined by electron microscopy. The integrity of DNA isolated from these cells was determined by standard gel electrophoretic techniques. Northern blot hybridization analysis was used to determine the steady-state levels of the mRNAs for testosterone-repressed prostatic message-2 (TRPM-2), tumor growth factor beta-1 (TGF beta 1), and pS2 (a small, cysteine-rich protein of unknown function). RESULTS: Time-lapse video microscopy of the cell cultures indicated that treatment with 7.5 microM toremifene for 3 days caused approximately 60% of the cells to exhibit morphologic characteristics typical of cells undergoing programmed death, or apoptosis. The number of mitoses gradually decreased to zero over a 3- to 4-day period. Estrogen withdrawal for the same length of time resulted in an approximately equal number of apoptoses and mitoses. These changes were not associated with the pattern of DNA fragmentation, detectable as ladders in agarose gels, that is characteristic of the DNA of cells undergoing apoptosis. Elevated levels of TRPM-2 and TGF beta 1 mRNAs were observed in in vitro or in vivo grown tumor cells treated with 5-10 microM toremifene. Elevated levels of TRPM-2, but not TGF beta 1, mRNA were observed in the tumor cells after estrogen withdrawal. The steady-state level of pS2 mRNA in the tumor cells dropped in response to either toremifene treatment or estrogen withdrawal. CONCLUSION: Toremifene causes growth inhibition of estrogen-sensitive breast cancer cells by inducing some cells to undergo apoptosis and by inhibiting other cells from entering mitosis. The higher than normal amounts of TRPM-2 and TGF beta 1 protein that would likely result from the elevated levels of TRPM-2 and TGF beta 1 mRNAs measured in these cells after toremifene treatment may have an important role in the growth inhibition process. IMPLICATION: Apoptosis as an active, targeted process provides a potential new therapeutic approach for treating breast cancer.

Interaction of male and female sex hormones in cultured rat prostate.

The organ culture of the rat ventral prostate was chosen as a model to determine whether any of the estrogen effects in vivo on the prostate are direct and expressed at the hormone concentrations normally found in the male. During 2 weeks of culture, estradiol at the high concentration of 10(-5) M blocked the androgenic activation of [3H]thymidine incorporation into DNA. The inhibition was localized in epithelium. Protein content of testosterone-treated explants and the accumulation of prostatein in the medium were considerably decreased, indicating inhibition of secretion. Antiandrogenic effects were not seen in morphology of estrogen-treated explants. The lower concentrations (from 10(-9) M to 10(-6) M) of estradiol increased the volume density of epithelium from day 7 onwards. The height of epithelium was concomitantly increased. The volume density of epithelium as well as the percentage of acini with metaplastic changes were significantly increased. These epithelial changes were less pronounced in the presence of androgen, suggesting that physiological concentrations of androgen prevent the expression of estrogen action in the morphology of the prostate. A change in staining with peanut (PNA)- and wheat germ agglutinin (WGA)-lectins indicated defective secretory capacity in metaplastic epithelium. In spite of the increased protein content in the explants, no constant pattern of the changes in prostatein accumulation could be recorded. Although the concentrations of estrogen required to induce squamous metaplasia were still unphysiological, the occurrence of this abnormal differentiation of the prostatic epithelium suggests that the cooperative action of estrogen is involved in androgen-dependent normal epithelial growth and possibly also in promoting growth of prostatic neoplasia.

Effect of glucose on the major testosterone-maintained protein in the cultured rat ventral prostate.

The effect of glucose on the androgen-maintained protein synthesis was studied in the cultured rat ventral prostate. The explants were cultivated for 5 days in the glucose-free medium containing 10% fetal calf serum with or without 10 mM glucose and 10(-7) M testosterone. In some experiments tunicamycin, a specific inhibitor of protein glycosylation was added to the glucose-containing medium. The morphological integrity of the tissue was maintained in all the mediums used. At the end of the culture, the explants were incubated with [35S]methionine. Soluble radioactive proteins were separated by the SDS-polyacrylamide gel electrophoresis and analyzed further by the fluorography. Glucose was necessary for the testosterone-maintained accumulation of three components (Mr less than 14,000) of the major prostatic secretory protein. The electrophoretic migration, glycosylation pattern and immunological data (not shown) indicated that it was the well-known prostatic binding protein. On the other hand, two prominent polypeptides (Mr 70,000 and 100,000) appeared in the absence of glucose. Glucose starvation and the inhibition of glycosylation with tunicamycin caused similar effects on the labelling of the newly-synthesized soluble proteins. The mechanisms of glucose maintenance of the major prostatic protein and suppression of two high molecular weight proteins seemed to be different, although glycosylation was probably involved in both glucose effects.