Saturated fat intake and prostate cancer aggressiveness: results from the population-based North Carolina-Louisiana Prostate Cancer Project.

BACKGROUND: Epidemiologic and laboratory evidence supports a role for cholesterol in prostate cancer (PC). Dietary saturated fat content impacts serum cholesterol levels. However, epidemiologic associations between saturated fat and PC aggressiveness are inconsistent. We hypothesized that high saturated fat intake would be associated with increased PC aggressiveness, and that statin use would modify this association. METHODS: Of 1854 PC cases in the North Carolina-Louisiana PC Project, 321 (17%) were classified as high aggressive (Gleason sum ⩾8, PSA>20 ng ml-1, or Gleason sum ⩾7 and clinical stage T3-4) or low/intermediate aggressive (all other cases). Using low/intermediate aggressive cases as the referent group, we examined the association between tertiles of total fat-adjusted saturated fat intake and high aggressive PC using logistic regression, overall and stratified by race and statin use. We examined total fat-adjusted polyunsaturated and monounsaturated fatty acids (PUFA and MUFA, respectively), trans fat and cholesterol intake in secondary analysis. RESULTS: High total fat-adjusted saturated fat intake was associated with an elevated odds ratio (OR) for aggressive PC (ORT3vsT1 1.51; 95% CI 1.10-2.06; P-trend=0.009), with an attenuated association in statin users (ORT3vsT1 1.16; 95% CI 0.67-2.01; P-trend=0.661) compared with non-users (ORT3vsT1 1.71; 95% CI 1.16-2.51; P-trend=0.053). High total fat-adjusted cholesterol intake was associated with aggressive PC in European Americans (ORT3vsT1 1.62; 95% CI 1.02-2.58; P-trend=0.056), but not African Americans (ORT3vsT1 0.92; 95% CI 0.60-1.42; P-trend=0.750). High total fat-adjusted PUFA was inversely associated with PC aggressiveness (ORT3vsT1 0.75; 95% CI 0.55-1.03), although this was not significant. No associations were found between total fat-adjusted MUFA or trans fat and PC aggressiveness. CONCLUSIONS: High total fat-adjusted saturated fat intake was associated with increased PC aggressiveness, with a suggestion of a stronger effect in men not using statins. The association between total fat-adjusted cholesterol intake and PC aggressiveness was most pronounced in European Americans.

Association of fatty-acid synthase polymorphisms and expression with outcomes after radical prostatectomy.

BACKGROUND: Fatty-acid synthase (FASN), selectively overexpressed in prostate cancer (PCa) cells, has been described as linked to the aggressiveness of PCa. Constitutional genetic variation of the FASN gene and the expression levels of FASN protein in cancer cells could thus be expected to predict outcome after radical prostatectomy (RP). This study evaluates the associations of malignant tissue status, neoadjuvant androgen deprivation therapy (NADT) and single-nucleotide polymorphisms (SNPs) of FASN with FASN protein expression in prostate tissue. The study then examines the associations of FASN SNPs and gene expression with three measures of post-prostatectomy outcome. METHODS: Seven tagging FASN SNPs were genotyped in 659 European American men who underwent RP at Roswell Park Cancer Institute between 1993 and 2005. FASN protein expression was assessed using immunohistochemistry. The patients were followed for an average of 6.9 years (range: 0.1-20.6 years). Outcome was assessed using three end points: biochemical failure, treatment failure and development of distant metastatic PCa. Cox proportional hazards analyses were used to evaluate the associations of the tagging SNPs and FASN expression with these end points. Bivariate associations with outcomes were considered; the associations also were controlled for known aggressiveness indicators. RESULTS: Overall, no SNPs were associated with any known aggressiveness indicators. FASN staining intensity was stronger in malignant than in benign tissue, and NADT was associated with decreased FASN staining in both benign and malignant tissue. The relationships of FASN SNPs and staining intensity with outcome were less clear. One SNP, rs4246444, showed a weak association with outcome. FASN staining intensity also showed a weak and seemingly contradictory relationship with outcome. CONCLUSIONS: Additional study with longer follow-up and populations that include more metastatic patients is warranted.

American College of Radiology Appropriateness Criteria(®)--locally advanced (high-risk) prostate cancer.

The American College of Radiology Appropriateness Criteria are evidence-based guidelines for specific clinical conditions that are reviewed every 2 years by a multidisciplinary expert panel. The guideline development and review include an extensive analysis of current medical literature from peer-reviewed journals and the application of a well-established consensus methodology (modified Delphi) to rate the appropriateness of imaging and treatment procedures by the panel. In those instances where evidence is lacking or not definitive, expert opinion may be used to recommend imaging or treatment. This review focuses on locally advanced prostate cancer and the evidence for treatment outcomes, both toxicity and efficacy, across the three major treatment modalities of external beam radiotherapy, brachytherapy and surgery. Only data that could pass contemporary quality metrics were used to form this report. This body of literature suffers from an absence of trials prospectively comparing therapies for efficacy and a lack of long-term prospective comparisons of toxicity. Upon review of these data, the authors concluded that there are several acceptable methods for the treatment of locally advanced prostate cancer that is highly dependent of the patient's clinical (both prostate cancer-specific and comorbidity-specific) parameters at diagnosis.

Comparison of clinical and pathological features in African-American and Caucasian patients with localized prostate cancer.

OBJECTIVE: To examine patient characteristics, prostate specific antigen (PSA) levels, and established preoperative and pathological prognostic factors to determine differences between Caucasian and African-American patients with localised prostate cancer, as it remains controversial whether African-American men present with more aggressive disease. PATIENTS AND METHODS: One hundred consecutive patients (aged 53-76 years) undergoing radical retropubic prostatectomy (RRP) at an equal-access tertiary-care centre were retrospectively reviewed. All patients had preoperative PSA levels, a physical examination (including clinical staging), and sextant biopsy. Insurance information was also collected. The same urological oncologist determined clinical staging and performed all the RRPs, and the same genitourinary pathologist determined the Gleason grade for biopsies and surgical specimens, pathological stage, percentage of tumour involvement, and specimen weight. African-American and Caucasian patients were compared for PSA, clinical stage, pathological stage, biopsy and pathological Gleason grade, organ confinement, margin status and specimen weight. Using preoperative and pathological data, both groups were also compared for over- and under-staging and -grading. The Wilcoxon rank test with P < 0.05 was used to determine statistically significant differences. RESULTS: African-American patients were more likely to be Medicaid or self-insured than Caucasian patients. Age, biopsy grade and clinical stage were not significantly different between the groups. African-American patients presented with a mean PSA level of 11.9 ng/mL and Caucasians with a mean of 8.5 ng/mL (P = 0.03). When clinical and biopsy data were compared with pathological data there were no differences between the groups in under/over-grading or under/over-staging. African-American patients had larger prostates per surgical specimen than their Caucasian counterparts (59.3 g vs 51.6 g, respectively; P = 0.04). CONCLUSIONS: In a referred, equal-access system, African-American patients presented with higher serum PSA levels and had larger prostates in the surgical specimen. However, African-American patients did not present at an earlier age or with higher Gleason grade or clinical stage, nor were pathological grade and stages higher. Other pathological features were no different. African-American patients were not under- or over-staged or under- or over-graded more than their Caucasian counterparts. This retrospective study does not suggest that African-American men present with more aggressive disease.

Establishment of short-term primary human prostate xenografts for the study of prostate biology and cancer.

Human tissue xenograft models are currently the only tool for conducting in vivo analyses of intact human tissue. The goal of the present study was to develop reliable methods for successful generation of short-term primary tissue xenografts from benign and tumor-derived human prostate tissue. Primary human prostate xenografts were established in athymic nu/nu mice from eight of eight benign and five of five prostate cancer tissues, collected from a total of 10 patients who underwent radical prostatectomy for the treatment of prostate cancer. An average of 13 xenografts was established per specimen. Two tissue specimens were cryopreserved for >1 month before successful generation of prostate xenografts. After 1 month in vivo, xenograft tissues were harvested and examined regarding: gross evidence of vascularization; tissue morphology; proliferation; apoptosis; and expression of androgen receptor, prostate-specific antigen, and high molecular weight cytokeratins specific for basal cells in the prostate. Direct comparison of the original tissue specimen and the 1-month xenografts revealed similar histology; similar apoptotic and proliferative fractions in most cases; and comparable expression levels and expression patterns of androgen receptor, prostate-specific antigen, and high molecular weight cytokeratins. These data demonstrate that primary human prostate xenografts, benign and malignant, can be established routinely from human prostate tissue surgical specimens, and that the xenografts maintain tissue architecture and expression of key prostatic markers. The development of this methodology, including the technique for cryopreservation of human tissue, will allow multiple (successive) analyses of human prostate tissue to be conducted throughout time using a tissue sample derived from a single patient; and simultaneous analysis of human prostate tissues derived from a cohort of patients.

Androgen receptor regulation of G1 cyclin and cyclin-dependent kinase function in the CWR22 human prostate cancer xenograft.

Human prostate cancer is initially dependent on androgens for growth, and androgen-dependent cells undergo apoptosis after castration. However, a subset of androgen-responsive cells survives and eventually proliferates in the absence of testicular androgen. The high levels of androgen receptor in both androgen-dependent and recurrent tumors led us to investigate androgen regulation of cell cycle proteins in human prostate cancer using the CWR22 xenograft. Cellular proliferation decreased dramatically in CWR22 tumors after castration. Testosterone propionate (TP) treatment of castrated mice restored cellular proliferation after 24-48 hours. Growth of CWR22 tumors in the absence of testicular androgen recurred several months after castration. CDK1 and CDK2, and cyclin A and cyclin B1 messenger RNAs were decreased 6 days after castration, increased 6-12 hours after TP treatment, and were expressed at high levels in recurrent CWR22 tumors. Coimmunoprecipitated cyclin B1/CDK1 and cyclin D1/CDK4 protein complexes decreased after castration and increased after TP treatment of castrated mice. In addition, CDK1 and CDK2 kinase activities were upregulated by androgen in parallel with hyperphosphorylation of retinoblastoma (Rb) protein. Despite the absence of testicular androgen in recurrent CWR22, the levels of these androgen-regulated cyclin/ CDK protein complexes and hyperphosphorylation of Rb were equal to or greater than in tumors from intact mice. The results indicate that androgen receptor regulates cellular proliferation by control of CDK and cyclins at the transcriptional level and by post-translational modifications that influence cell cycle protein activity.

A mechanism for androgen receptor-mediated prostate cancer recurrence after androgen deprivation therapy.

The development and growth of prostate cancer depends on the androgen receptor and its high-affinity binding of dihydrotestosterone, which derives from testosterone. Most prostate tumors regress after therapy to prevent testosterone production by the testes, but the tumors eventually recur and cause death. A critical question is whether the androgen receptor mediates recurrent tumor growth after androgen deprivation therapy. Here we report that a majority of recurrent prostate cancers express high levels of the androgen receptor and two nuclear receptor coactivators, transcriptional intermediary factor 2 and steroid receptor coactivator 1. Overexpression of these coactivators increases androgen receptor transactivation at physiological concentrations of adrenal androgen. Furthermore, we provide a molecular basis for this activation and suggest a general mechanism for recurrent prostate cancer growth.

Androgen receptor stabilization in recurrent prostate cancer is associated with hypersensitivity to low androgen.

The androgen receptor (AR) is highly expressed in androgen-dependent and recurrent prostate cancer (CaP) suggesting it has a role in the growth and progression of CaP. Previously proposed mechanisms for AR reactivation in recurrent CaP include altered growth factor signaling leading to protein phosphorylation and AR mutations that broaden ligand specificity. To further establish a role for AR in recurrent CaP, we compared several properties of AR in relation to the growth response to low levels of androgens in model systems of androgen-dependent and recurrent CaP. AR from all of the tumors and cell lines bound [3H]R1881 with similar high affinity (mean Kd, 0.12 nM). In the absence of androgen, AR in androgen-dependent LNCaP cells was unstable with a degradation half-time (t(1/2)) of 3 h at 37 degrees C. In contrast, AR was 2-4 times more stable in recurrent CWR22 tumors (t(1/2), >12 h) and CWR-R1 or LNCaP-C4-2 cell lines (t(1/2), 6-7 h) derived from recurrent prostate tumors. In the recurrent CWR22 tumor and its CWR-R1 cell line grown in the absence of androgen, AR immunostaining was entirely nuclear, whereas under the same conditions AR in LNCaP-C4-2 and LNCaP cells was predominantly nuclear but was also detected in the cytoplasm. High level expression, increased stability, and nuclear localization of AR in recurrent tumor cells were associated with an increased sensitivity to the growth-promoting effects of dihydrotestosterone in the femtomolar range. The concentration of dihydrotestosterone required for growth stimulation in CWR-R1 and LNCaP-C4-2 cells was four orders of magnitude lower than that required for androgen-dependent LNCaP cells. The results suggest that AR is transcriptionally active in recurrent CaP and can increase cell proliferation at the low circulating levels of androgen reported in castrated men.

HE2beta and HE2gamma, new members of an epididymis-specific family of androgen-regulated proteins in the human.

HE2 is an epididymis-specific sperm-binding secretory protein. We isolated a family of HE2-related complementary DNAs from a human caput/corpus library. The transcripts code for identical 71-amino acid N-termini and different C-termini, and 5'- and 3'-untranslated regions. Compared with the original HE2, HE2beta and HE2gamma proteins have a 25-amino acid deletion near the C-terminus, and HE2gamma isoforms have a second deletion. These frame-shifting deletions result in C-termini differing in length, amino acid sequence, including number of cysteines, and isoelectric point. Identical sequences and deletion start and stop points indicate the HE2 isoforms are derived from alternative splicing of 8 or more exons of a single gene. Northern hybridization revealed that the 0.9-kb messenger RNA (mRNA) is most abundant in human caput; there is much less of it (20%) in corpus and little (<5%) in cauda. In castrated Macaca mulatta, HE2 mRNA decreased to 10% of sham-operated levels. Testosterone replacement maintained HE2 mRNA 3- to 5-fold higher than castrate levels, indicating its androgen dependence. Immunohistochemical staining revealed that the beta1 form is highly expressed in principal cells of the initial segment and caput. It is secreted into the lumen and binds to the sperm surface in the postacrosomal and neck regions. The beta2 form is expressed in principal cells primarily in efferent ducts.

Immunohistochemical quantitation of androgen receptor expression using color video image analysis.

BACKGROUND: The immunostaining features of the androgen receptor (AR) have been studied in prostate cancer (CaP) to predict the outcome of androgen deprivation therapies. We have developed an automatic video color image analysis system for quantitation of AR expression in large samples of prostatic nuclei. METHODS: Essential criteria of immunostaining have been examined to establish a linear relationship between AR protein content and mean optical density (MOD) of the immunoperoxidase-substrate reaction product. Titration of monoclonal AR antibody, F39.4.1, and concentration and reaction time of substrate were optimized using color video image analysis. The methodology was tested twice. First, CWR22 human CaP xenograft specimens, harvested from testosterone (T)-stimulated, castrated and T-resupplemented mice, were immunostained to demonstrate the dependence of AR expression on serum androgen levels. Second, AR expression was measured in archived clinical specimens. RESULTS: In CWR22 tumor-bearing mice castrated for 6 days, AR MOD decreased to 57% of T-stimulated, intact mice. After 72 hrs of T treatment, AR MOD returned to the level measured in T-stimulated, intact mice. Sixteen radical prostatectomy specimens and 16 transurethral resection of prostate (TURP) specimens were double-labeled with F39.4.1 and anti-cytokeratin MAb (13betaE12) specific for basal epithelial cells. Benign epithelial cells exhibited lower AR MOD in prostatectomy compared to TURP specimens (P < 0.01). Differences in AR immunostaining intensity may have resulted from differences in tissue fixation of whole organ versus small tissue specimens. CONCLUSIONS: AR immunostaining can be quantitated accurately using optimized immunohistochemical criteria and video image analysis.

Prostate cancer risk and polymorphism in 17 hydroxylase (CYP17) and steroid reductase (SRD5A2).

Prostate cancer is the most common malignancy in males and is the second most common cause of cancer mortality in American men. Polymorphisms have been identified in two genes, the 17-hydroxylase cytochrome P450 gene (CYP17) and the steroid 5-reductase type II gene (SRD5A2) that are involved with androgen biosynthesis and metabolism. The CYP17 A2 allele contains a T-->C transition in the 5' promoter region that creates an additional Sp1-type (CCACC box) promoter site. The SRD5A2 valine to leucine (V89L) polymorphism has been correlated with lower dihydroxytestosterone levels. We tested genotypes in 108 prostate cases and 167 controls along with samples (n = 340) from several different ethnic groups. The CYP17 A2 allele (combined A1/A2 and A2/A2 genotypes) occurred at a higher frequency in Caucasian patients with prostate cancer (70%) than in Caucasian clinical control urology patients (57%), suggesting that the A2 allele may convey increased risk for prostate cancer [odds ratio (OR) = 1.7, 95% confidence interval (CI) = 1.0-3.0]. Blacks and Caucasians had a similar frequency of the A2 genotype (16 and 17%, respectively) while Taiwanese had the highest frequency (27%). The SRD5A2 leucine genotype was most frequent in Taiwanese (28%), intermediate in Caucasians (8.5%) and lowest in Blacks (2.5%). Genotypes having a SRD5A2 leucine allele were somewhat more common in prostate cancer cases (56%) than in controls (49%) (OR = 1.4, 95% CI = 0.8-2.2) but this difference was not significant. These results support the hypothesis that some allelic variants of genes involved in androgen biosynthesis and metabolism may be associated with prostate cancer risk.

Androgen receptor up-regulates insulin-like growth factor binding protein-5 (IGFBP-5) expression in a human prostate cancer xenograft.

The insulin-like growth factor (IGF) binding proteins (IGFBPs) are important modulators of IGF action in many tissues including human prostate. IGFBPs and the androgen receptor (AR) are expressed in CWR22, an androgen-dependent epithelial cell human CaP xenograft that retains biological characteristics of human CaPs, including regression following androgen withdrawal and recurrent growth of AR-containing cells in the absence of testicular androgens beginning several months after castration. Northern blot and in situ hybridization analyses demonstrated that IGFBP-5 is androgen-regulated in CWR22. IGFBP-5 messenger RNA (mRNA) decreased by 90% following castration of tumor-bearing mice compared with noncastrate androgen-stimulated mice. Testosterone treatment of CWR22 tumor-bearing mice 6 or 12 days after castration increased IGFBP-5 mRNA 10- to 12-fold. Levels of other IGFBP mRNAs did not change following androgen withdrawal and replacement. IGFBP-5 protein in tumor extracts bound 125I-labeled IGF-I in ligand blot assays and the amounts of IGFBP-5 measured by immunoblotting paralleled the levels of IGFBP-5 mRNA. Androgen-induced expression of IGFBP-5 was at a maximum level within 24 h after testosterone replacement, whereas the major increase in cell proliferation as measured by Ki-67 immunostaining occurred between 24-48 h. This time course suggested IGFBP-5 may be a mediator of androgen-induced growth of CWR22. In tumors that recurred several months following castration, IGFBP-5 mRNA and protein increased to levels that approached those in androgen-stimulated CWR22 tumors from noncastrate mice. IGFBP-5 immunohistochemical staining of prostate tissue specimens from patients was stronger in androgen-dependent and androgen-independent CaP than in areas of intraepithelial neoplasia (PIN) or benign prostatic hyperplasia (BPH). IGFBP-5 mRNA in these specimens was localized predominantly to stromal cells and IGFBP-5 protein to epithelial cell membranes.

Overexpression of cyclin D1 is rare in human prostate carcinoma.

BACKGROUND: Overexpression of cyclin D1 has been documented in a number of human cancers. Increased expression of cyclin D1 can contribute to cellular transformation and abnormal proliferation. METHODS: Quantitative RT-PCR and/or Western blot analysis were used to determine the level of cyclin D1 expression in 96 human prostate tumors, 15 benign prostate hyperplasias, 4 prostate cancer cell lines, and 3 xenografts. RESULTS: Our results demonstrate that 4.2% of the prostate tumors examined overexpressed cyclin D1 transcripts. In the cell lines, expression was normal, with the exception that reduced levels of cyclin D1 transcript and protein were observed in the DU145 cell line, as expected from cells with mutant RB. Normal levels of cyclin D1 were found in all xenograft tumors and BPH specimens examined. CONCLUSIONS: These data show that overexpression of cyclin D1 occurs rarely in human prostate tumors. However, when overexpression of cyclin D1 does occur, it may identify a subset of tumors with a different molecular biology.

Isolation and partial characterization of an epithelial cell line (RPE-F344) from the regenerating prostate of a normal adult male rat.

Normal prostate epithelial cells are difficult to propagate in vitro without experimental immortalization. The goal of this study was to isolate and characterize a propagable epithelial cell line from normal adult rat prostate. Enrichment of proliferation-competent cells was accomplished in vivo by initiating a single cycle of prostatic involution/regeneration. The RPE-F344 cell line was established from an androgen-deprived, involuted prostate four days after the initiation of regeneration by administration of testosterone. The cell line has been cultured in vitro for >50 passages, forms a uniform monolayer in culture, exhibits contact inhibition at confluence, and does not form colonies in soft agar. Immunocytochemical and RT-PCR analyses demonstrated that the RPE-F344 cells express anti-apoptotic genes associated with cell survival, and several growth factor receptors important in prostate development and homeostasis. RPE-F344 cells are p27kip1 negative, telomerase positive, and express high molecular weight cytokeratins specific for prostatic basal cells. They also express low levels of androgen receptor (AR) and prostatic acid phosphatase (PAP); features associated with secretory luminal epithelial cells. RPE-F344 cells are maintained in vitro without androgen supplementation, but addition of 15nM dihydrotesterone (DHT) to the culture media results in a significant but transient enhancement of cellular proliferation. Establishment of RPE-F344-like colonies from rat prostate is limited to the ventral and dorsal lobes of the prostate 2-4 days after initiation of regeneration, suggesting that RPE-F344 cells may originate from a stem cell-like compartment that is responsible for regenerative repopulation.

Androgen receptor expression in androgen-independent prostate cancer is associated with increased expression of androgen-regulated genes.

The human prostate cancer (CaP) xenograft, CWR22, mimics human CaP. CWR22 grows in testosterone-stimulated nude mice, regresses after castration, and recurs after 5-6 months in the absence of testicular androgen. Like human CaP that recurs during androgen deprivation therapy, the recurrent CWR22 expresses high levels of androgen receptor (AR). Immunohistochemical, Western, and Northern blot analyses demonstrated that AR expression in the androgen-independent CWR22 is similar to AR expression in the androgen-dependent CWR22 prior to castration. Expression of prostate-specific antigen and human kallikrein-2 mRNAs, two well-characterized androgen-regulated genes in human CaP, was androgen dependent in CWR22. Despite the absence of testicular androgen, prostate-specific antigen and human kallikrein-2 mRNA levels in recurrent CWR22 were higher than the levels in regressing CWR22 tumors from 12-day castrate mice and similar to those in the androgen-stimulated CWR22. Other AR-regulated genes followed a similar pattern of expression. Differential expression screening identified androgen regulation of alpha-enolase and alpha-tubulin as well as other unknown mRNAs. Insulin-like growth factor binding protein-5, the homeobox gene Nkx 3.1, the AR coactivator ARA-70, and cell cycle genes Cdk1 and Cdk2 were androgen regulated in CWR22. In recurrent CWR22, the steady-state levels of all these AR-dependent mRNAs were similar to those in the androgen-stimulated CWR22, despite the absence of testicular androgen. Expression of AR and AR-regulated genes in the androgen-deprived recurrent CWR22 at levels similar to the androgen-stimulated CWR22 suggests that AR is transcriptionally active in recurrent CWR22. Induction of these AR-regulated genes may enhance cellular proliferation in relative androgen absence but through an AR-dependent mechanism. Alternatively, in androgen-independent tumors, induced expression of the AR-regulated gene network might result from a non-AR transcription control mechanism common to these genes.

The role of N-acetylation polymorphisms in smoking-associated bladder cancer: evidence of a gene-gene-exposure three-way interaction.

Arylamines are known bladder carcinogens and are an important constituent of tobacco smoke. The handling of arylamines in the body is complex and includes metabolism by NAT1 and NAT2, enzymes that play a role in both activation and detoxification of arylamines and their congeners. Both NAT1 and NAT2 are polymorphic, with alleles that have been shown to correlate with higher or lower enzyme activity. To explore the combined role of these genes and exposure on bladder cancer risk, we examined the NAT1 and NAT2 genotype in a case-control study of bladder cancer in which detailed exposure histories were available on all 230 cases and 203 frequency-matched controls. Using PCR-RFLP genotyping, we determined NAT2 genotype for the five most common alleles, NAT2*4, NAT2*5, NAT2*6, NAT2*7, NAT2*14 (frequently referred to as WT, M1, M2, M3, and M4, respectively). Similarly, the NAT1 genotype was determined for the four most common alleles NAT1*3, NAT1*4, and NAT1*11, and the putative high-activity allele, NAT1*10. No association between NAT2 genotype and bladder cancer risk was found whether genotype was considered alone or in combination with smoking, in either stratified or logistic regression analysis that adjusted for age, sex, and race. Stratified and logistic regression analysis both demonstrated an increased risk for individuals carrying the NAT1*10 allele among smokers. There was evidence of a gene-dosage effect, such that those who were homozygous for the NAT1*10 allele had the highest risks. There was also evidence of a statistically significant gene-environment interaction, such that bladder cancer risk depends on both NAT1 genotype and smoking exposure. Interestingly, although NAT2 genotype did not influence risk either alone or in combination with smoking exposure, there was evidence of a statistically significant gene-gene-environment three-way interaction. Bladder cancer risk from smoking exposure is particularly high in those who inherit NAT2 slow alleles in combination with one or two copies of the NAT1*10 allele. A biological mechanism for this finding is suggested.

Patient positioning for radical retropubic prostatectomy.

Proper positioning of a surgical patient reduces morbidity and mortality. We describe a method of patient positioning involving elevation of the lower extremities and protection of the brachial plexus that reduces complication rates in radical retropubic prostatectomy.

Isolation and characterization of propagable cell lines (HUNC) from the androgen-sensitive Dunning R3327H rat prostatic adenocarcinoma.

The Dunning H rat prostate tumor (R3327H) is a widely used experimental model of human prostatic adenocarcinoma (CaP). The Dunning H tumor has been characterized as androgen-sensitive, androgen-receptor (AR) positive, prostate-specific antigen and prostatic acid phosphatase (PAP) positive. To date, the tumor has been maintained by serial passage in vivo because of the lack of an in vitro cell line that retains the characteristics of the in vivo tumor. The objective of the present study was to establish a propagable cell line from R3327H adenocarcinoma that maintained androgen sensitivity and expression of AR, PSA and PAP. Tissue harvested from an in vivo R3327H tumor was dissociated with collagenase and placed into Richter's improved media (with supplements). A cytokeratin-positive epithelial cell line (HUNC-E) and a vimentin-positive stromal cell line (HUNC-S) were generated from the primary culture, subcultured continuously for >300 days, and passaged >50 times. Survival of the HUNC-E cell line in vitro depended on several media supplements, including nicotinamide, insulin, transferrin, selenium and epidermal growth factor (EGF). HUNC-E cells expressed AR and produced PSA and PAP throughout the culture period, as confirmed by immunocytochemistry and Western blot analyses. Addition of 14 nM testosterone (T) or dihydrotestosterone (DHT) to HUNC-E cells, stimulated DNA synthesis as well as anchorage-independent growth and PSA production, which demonstrated the androgen-sensitive nature of the cells in vitro. When HUNC-E and HUNC-S cells were combined in a 3:1 ratio and introduced subcutaneously into syngeneic male hosts, tumors formed in 2/3 animals with an average latency of 7 months. RT-PCR and immunocytochemical characterization of the HUNC cell lines revealed that the cells expressed several growth factors and their cognate receptors, including HGF, TGF-alpha and the TGF-betas, indicating the establishment of potential autocrine loops in the neoplastic cells. The HUNC-E and HUNC-S CaP cell lines, which retain the characteristics of the epithelial and stromal components of the in vivo R3327H tumor, will allow a more thorough and informative molecular and biological analysis of prostatic adenocarcinoma.

Downregulation of the beta4 integrin subunit in prostatic carcinoma and prostatic intraepithelial neoplasia.

Integrins are adhesion receptors thought to be important in the process of cancer cell invasion and metastasis. Unlike other integrins, which attach a cell to extracellular matrix molecules, the alpha6beta4 integrin participates in the formation of hemidesmosomes, attaching epithelial cells to the basement membrane. Investigations of the alpha6beta4 integrin in human prostatic carcinoma have yielded conflicting results and have been primarily qualitative rather than quantitative. Expression of the beta4 integrin subunit was determined using rat monoclonal antibody 439-9B and image analysis in regions of benign prostatic epithelium (BPE), high-grade prostatic intraepithelial neoplasia (PIN), and prostatic carcinoma (CaP) in 38 patients treated by radical prostatectomy for clinically localized CaP. The beta4 integrin subunit was significantly downregulated in CaP compared with BPE; PIN stained intermediate in intensity between BPE and CaP. Thirty-four of 35 patients showed downregulation of the beta4 integrin subunit, and all 15 patients with PIN had downregulation of beta4 in PIN as compared with BPE. Degree of downregulation of the beta4 integrin subunit did not add prognostic significance to the information present at initial biopsy (age, clinical stage, clinical grade, and serum prostate-specific antigen level). There was no correlation between intensity of staining of CaP, absolute change in staining, or percent loss of beta4 integrin subunit staining with age, pathological stage, or Gleason's score. Downregulation of the beta4 integrin in CaP and PIN compared with BPE may be correlated with neoplastic transformation of the prostate and loss of hemidesmosomes or basal epithelial cells.