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Background: Using a functional analysis of prostate cancer cells, we found a CD24-dependent inactivation of mutant p53, but the clinical significance of this observation remained uncertain. Here, we validated these results with samples of human prostate cancer and explored the role of a CD24-p53 axis in racial disparities of prostate cancer., Methods: Samples of formalin-fixed, paraffin-embedded prostate cancer from 141 European Americans (EAs) and 147 African Americans (AAs) in two independent sample cohorts were assessed for protein expression of CD24, mutant p53, mouse double minute 2 human homolog (MDM2), and cyclin dependent kinase inhibitor 2A (ARF) using immunohistochemical analyses. All samples were analyzed for TP53 R175H and TP53 R273H ., Results: CD24, mutant p53, MDM2, and ARF proteins were expressed in 55%, 24%, 39%, and 68% of prostate cancer samples, respectively. CD24 and mutant p53 were present more frequently in late-stage and metastatic prostate cancer. The presence of CD24 was associated with a greater than fourfold risk of metastasis, which included lymph node and distant metastases. H score analysis showed positive correlations of CD24 expression with mutant p53 (r = .308, P < .001) and MDM2 (r = .227, P = .004). There was a negative correlation for CD24 with ARF (r = -.280, P < .001). A racial disparity was evident for CD24 (AAs/EAs: 64% vs 47%; P = .004) but not for mutant p53 (AA/EA: 28% vs 21%; P = .152). In 32 CD24 + /mutant p53 + cases, a TP53 R273H mutation was found in five cases, but no TP53 R175H mutation was found., Conclusion: The CD24-p53 axis may contribute to aggressive and metastatic prostate cancers, especially those of AAs. This observation enhances understanding of the pathogenesis of prostate cancer and its associated racial disparities., (© 2020 Wiley Periodicals, Inc.)

Background: African-American (AA) men tend to present with more aggressive prostate cancer (Gleason score >7) than European-American (EA) men. Vitamin D and its metabolites are implicated in prostate cancer biology with vitamin D deficiency, indicated by its metabolite levels in serum or plasma, usually observed in AA men., Objective: To determine if 1, 25-dihydroxy vitamin D3 [1,25(OH) 2 D] plasma levels in AA and EA prostate cancer patients alter the risk of having aggressive prostate cancer., Design: Research subjects from the North Carolina-Louisiana Prostate Cancer Project (AA n = 435 and EA n = 532) were included. Plasma metabolites 1,25(OH) 2 D and 25-hydroxyvitamin D3 [25(OH)D] were measured using liquid chromatography with tandem mass spectrophotometry. Research subjects were classified into low (Gleason sum < 7, stage T1-T2, and Prostate-specific antigen (PSA) < 9 ng/mL) or high (Gleason sum > 8 or Gleason sum = 7 with 4 + 3, or PSA > 20 ng/mL, or Gleason sum = 7 and stage T3-T4) aggressive disease., Results: Research subjects in the second and third tertiles of plasma levels of 1, 25(OH) 2 D had lower odds of high aggressive prostate cancer (AA [OR T2vsT1 : 0.66, 95%CI: 0.39-1.12; OR T3vsT1 : 0.83, 95%CI: 0.49-1.41] and EA [OR T2vsT1 : 0.68, 95%CI: 0.41-1.11; OR T3vsT1 : 0.67, 95%CI: 0.40-1.11]) compared with the first tertile, though confidence intervals included the null. Greater 1,25(OH) 2 D/25(OH)D molar ratios were associated with lower odds of high aggressive prostate cancer more evidently in AA (OR Q4vsQ1 : 0.45, CI: 0.24-0.82) than in EA (OR Q4vsQ1 : 0.64, CI: 0.35-1.17) research subjects., Conclusions: The 1,25(OH) 2 D/25(OH)D molar ratio was associated with decreased risk of high aggressive prostate cancer in AA men, and possibly in EA men. Further studies analyzing vitamin D polymorphisms, vitamin D binding protein levels, and prostatic levels of these metabolites may be useful. These studies may provide a better understanding of the vitamin D pathway and its biological role underlying health disparities in prostate cancer., (© 2019 The Authors. The Prostate Published by Wiley Periodicals, Inc.)

Background: Associations between carotenoid intake and prostate cancer (CaP) incidence have varied across studies. This may result from combining indolent with aggressive disease in most studies. This study examined whether carotenoid intake and adipose tissue carotenoid levels were inversely associated with CaP aggressiveness., Methods: Data on African-American (AA, n = 1,023) and European-American (EA, n = 1,079) men with incident CaP from North Carolina and Louisiana were analyzed. Dietary carotenoid intake was assessed using a detailed-food frequency questionnaire (FFQ), and abdominal adipose tissue samples were analyzed for carotenoid concentrations using high-performance liquid chromatography. Multivariable logistic regression was used in race-stratified analyses to calculate odds ratios (ORs) and 95% confidence intervals (95%CI) comparing high aggressive CaP with low/intermediate aggressive CaP., Results: Carotenoid intake differed significantly between AAs and EAs, which included higher intake of lycopene among EAs and higher β-cryptoxanthin intake among AAs. Comparing the highest and lowest tertiles, dietary lycopene was associated inversely with high aggressive CaP among EAs (OR = 0.55, 95%CI: 0.34-0.89, Ptrend  = 0.02), while an inverse association was observed between dietary β-cryptoxanthin intake and high aggressive CaP among AAs (OR = 0.56, 95%CI: 0.36-0.87, Ptrend  = 0.01). Adipose tissue α-carotene and lycopene (cis + trans) concentrations were higher among EAs than AAs, and marginally significant inverse linear trends were observed for adipose α-carotene (Ptrend  = 0.07) and lycopene (Ptrend  = 0.11), and CaP aggressiveness among EAs only., Conclusions: These results suggest that diets high in lycopene and β-cryptoxanthin may protect against aggressive CaP among EAs and AAs, respectively. Differences in dietary behaviors may explain the observed racial differences in associations. Prostate 76:1053-1066, 2016. © 2016 Wiley Periodicals, Inc., Competing Interests: Statement: The authors have no conflict of interest to disclose., (© 2016 Wiley Periodicals, Inc.)

Background: PSA is a biomarker for diagnosis and management of prostate cancer. PSA is known to have anti-tumorigenic activities, however, the physiological role of PSA in prostate tumor progression is not well understood., Methods: Five candidate peptides identified based upon computer modeling of the PSA crystal structure and hydrophobicity were synthesized at >95% purity. The peptides in a linear form, and a constrained form forced by a di-sulfide bond joining the two ends of the peptide, were investigated for anti-angiogenic activity in HUVEC., Results: None of the five PSA-mimetic peptides exhibited PSA-like serine protease activity. Two of the peptides demonstrated significant anti-angiogenic activity in HUVEC based on (i) inhibition of cell migration and invasion; (ii) inhibition of tube formation in Matrigel; (iii) anti-angiogenic activity in a sprouting assay; and (iv) altered expression of pro- and anti-angiogenic growth factors. Constrained PSA-mimetic peptides had greater anti-angiogenic activity than the corresponding linearized form. Complexing of PSA with ACT eliminated PSA enzymatic activity and reduced anti-angiogenic activity. In contrast, ACT had no effect on the anti-angiogenic effects of the linear or constrained PSA-mimetic peptides. Modeling of the ACT-PSA complex demonstrated ACT sterically blocks the anti-angiogenic activity of the two bioactive peptides., Conclusions: The interaction of a hydrophilic domain on the surface of the PSA molecule with a target on the cell membrane of prostate endothelial and epithelial cells was responsible for the anti-angiogenic or anti-tumorigenic activity of PSA: enzymatic activity was not associated with anti-angiogenic effects. Furthermore, since PSA and ACT are both expressed within the human prostate tissue microenvironment, the balance of their expression may represent a mechanism for endogenous regulation of tissue angiogenesis., (© 2015 Wiley Periodicals, Inc.)

Background: Controversies remain over the safety and efficacy of vitamin E (i.e., α-tocopherol) supplementation use for the prevention of prostate cancer (CaP); however, associations of different tocopherol forms and CaP aggressiveness have yet to be examined., Methods: This study examined whether food intake of tocopherols, vitamin E supplement use, and adipose tissue biomarkers of tocopherol were associated with CaP aggressiveness among African-American (AA, n = 1,023) and European-American (EA, n = 1,079) men diagnosed with incident CaP. Dietary tocopherols were estimated from a food frequency questionnaire, supplement use from questionnaire/inventory, and biomarkers from abdominal adipose samples measured using high-performance liquid chromatography. Odds ratios (ORs) and 95% confidence intervals (95%CIs) were estimated from logistic regression comparing high-aggressive CaP to low/intermediate aggressive CaP, adjusting for covariates., Results: Dietary intakes of α-and δ-tocopherol were related inversely to CaP aggressiveness among EAs [OR (95%CI), highest versus lowest quartile: α-tocopherol, 0.34 (0.17-0.69), P(trend)  = 0.006; δ-tocopherol, 0.45 (0.21-0.95) P(trend)  = 0.007]. Inverse associations between dietary and supplemental α-tocopherol and CaP aggressiveness were observed among AAs, though these did not reach statistical significance [OR (95%CI), highest versus lowest quartile: dietary α-tocopherol, 0.58 (0.28-1.19), P(trend)  = 0.20; supplemental α-tocopherol, 0.64 (0.31-1.21) P(trend)  = 0.15]. No significant association was observed between adipose tocopherol levels and CaP aggressiveness [OR (95%CI), highest versus lowest quartiles of α-tocopherol for EAs 1.43 (0.66-3.11) and AAs 0.66 (0.27-1.62)]., Conclusions: The inverse associations observed between dietary sources of tocopherols and CaP aggressiveness suggests a beneficial role of food sources of these tocopherols in CaP aggressiveness., Competing Interests: Statement: The authors have no conflict of interest to disclose., (© 2015 Wiley Periodicals, Inc.)

Background: Currently available 5α-reductase inhibitors are not completely effective for treatment of benign prostate enlargement, prevention of prostate cancer (CaP), or treatment of advanced castration-recurrent (CR) CaP. We tested the hypothesis that a novel 5α-reductase, 5α-reductase-3, contributes to residual androgen metabolism, especially in CR-CaP., Methods: A new protein with potential 5α-reducing activity was expressed in CHO-K1 cellsandTOP10 E. coli for characterization. Protein lysates and total mRNA were isolated from preclinical and clinical tissues. Androgen metabolism was assessed using androgen precursors and thin layer chromatography or liquid chromatography tandem mass spectrometry., Results: The relative mRNA expression for the three 5α-reductase enzymes in clinical samples of CR-CaP was 5α-reductase-3 ≫ 5α-reductase-1> 5α-reductase-2. Recombinant 5α-reductase-3 protein incubations converted testosterone, 4-androstene-3,17-dione (androstenedione) and 4-pregnene-3,20-dione (progesterone) to dihydrotestosterone, 5α-androstan-3,17-dione, and 5α-pregnan-3,20-dione, respectively. 5α-Reduced androgen metabolites were measurable in lysates from androgen-stimulated (AS) CWR22 and CR-CWR22 tumors and clinical specimens of AS-CaP and CR-CaP pre-incubated with dutasteride (a bi-specific inhibitor of 5α-reductase-1 and 2)., Conclusion: Human prostate tissues contain a third 5α-reductase that was inhibited poorly by dutasteride at high androgen substrate concentration in vitro, and it may promote DHT formation in vivo, through alternative androgen metabolism pathways when testosterone levels are low., (© 2013 Wiley Periodicals, Inc.)

Background: Few genetic risk factors have been uncovered that contribute specifically to the racial disparity in prostate cancer (CaP) observed in African Americans (AA). With the advent of ancestry informative marker (AIM) single nucleotide polymorphism (SNP) panels and powerful genetic strategies such as mapping by admixture linkage disequilibrium (MALD) it is possible to discover genes that underlie ethnic variation in disease risk., Methods: One thousand one hundred thirty AA CaP cases enrolled in the North Carolina-Louisiana Prostate Cancer Project (PCaP) were genotyped using a 1,509 AIM SNP panel. MALD was performed using ADMIXMAP to test for linkage between CaP risk and ancestry estimates at each AIM SNP., Results: The largest increase of African ancestry was observed at marker rs12543473 (P = 0.0011), located on chromosome 8q24.21, and the greatest excess of European ancestry was observed at marker rs10768140 (P = 0.0004) at chromosome 11p13., Conclusions: The study confirmed the 8q24 risk loci and identified a novel genomic region on 11p13 that is associated with CaP risk. These findings should be replicated in larger AA populations and combined with fine mapping data to further refine the novel 11p13 CaP risk loci., (© 2013 Wiley Periodicals, Inc.)

Background: Genome-wide association studies have established a number of replicated single nucleotide polymorphisms (SNPs) for susceptibility to prostate cancer (CaP), but it is unclear whether these susceptibility SNPs are also associated with disease aggressiveness. This study evaluates whether such replication SNPs or other candidate SNPs are associated with CaP aggressiveness in African-American (AA) and European-American (EA) men., Methods: A 1,536 SNP panel which included 34 genome-wide association study (GWAS) replication SNPs, 38 flanking SNPs, a set of ancestry informative markers, and SNPs in candidate genes and other areas was genotyped in 1,060 AA and 1,087 EA men with incident CaP from the North Carolina-Louisiana Prostate Cancer Project (PCaP). Tests for association were conducted using ordinal logistic regression with a log-additive genotype model and a 3-category CaP aggressiveness variable., Results: Four GWAS replication SNPs (rs2660753, rs13254738, rs10090154, rs2735839) and seven flanking SNPs were associated with CaP aggressiveness (P < 0.05) in three genomic regions: One at 3p12 (EA), seven at 8q24 (5 AA, 2 EA), and three at 19q13 at the kallilkrein-related peptidase 3 (KLK3) locus (two AA, one AA and EA). The KLK3 SNPs also were associated with serum prostate-specific antigen (PSA) levels in AA (P < 0.001) but not in EA. A number of the other SNPs showed some evidence of association but none met study-wide significance levels after adjusting for multiple comparisons., Conclusions: Some replicated GWAS susceptibility SNPs may play a role in CaP aggressiveness. However, like susceptibility, these associations are not consistent between racial groups., (Copyright © 2012 Wiley Periodicals, Inc.)

Background: Prostate specific antigen (PSA) is a well known biomarker for early diagnosis and management of prostate cancer. Furthermore, PSA has been documented to have anti-angiogenic and anti-tumorigenic activities in both in vitro and in vivo studies. However, little is known about the molecular mechanism(s) involved in regulation of these processes, in particular the role of the serine-protease enzymatic activity of PSA., Methods: Enzymatic activity of PSA isolated directly from seminal plasma was inhibited specifically (>95%) by incubation with zinc2+ . Human umbilical vein endothelial cells (HUVEC) were utilized to compare/contrast the physiological effects of enzymatically active versus inactive PSA., Results: Equimolar concentrations of enzymatically active PSA and PSA enzymatically inactivated by incubation with Zn2+ had similar physiological effects on HUVEC, including inhibiting the gene expression of pro-angiogenic growth factors, like VEGF and bFGF, and up-regulation of expression of the anti-angiogenic growth factor IFN-γ; suppression of mRNA expression for markers of blood vessel development, like FAK, FLT, KDR, TWIST-1; P-38; inhibition of endothelial tube formation in the in vitro Matrigel Tube Formation Assay; and inhibition of endothelial cell invasion and migration properties., Discussion: Our data provides compelling evidence that the transcriptional regulatory and the anti-angiogenic activities of human PSA are independent of the innate enzymatic activity., (Copyright © 2011 Wiley-Liss, Inc.)

Background: Genome-wide association studies (GWAS) have identified numerous common SNPs associated with prostate cancer (CaP) risk in men of European descent. This study evaluates GWAS SNPs associated with CaP in African Americans (AAs) and European Americans (EA)., Methods: Eight hundred SNPs were genotyped, including 32 from European-based GWAS and 35 flanking SNPs, in 417 AA and 455 EA cases from the NC-LA Prostate Cancer Project (PCaP) and compared to 925 AA and 1,687 EA controls from Illumina's iControlDB. The 32 GWAS SNPs were evaluated for their predictive power to discriminate between cases and controls using ROC curves., Results: Of the 32 GWAS SNPs, 13 were significant at P < 0.05 in EA and 4 in AA (rs6983267, rs7017300, rs1859962, rs6501455). Three of 35 flanking SNPs, all from chromosome 8q, reached study-wide significance (P < 3.5 × 10(-5)); 2 in AA (rs10505476 rs6985504) and 1 in EA (rs16901970). Among the remaining 656 SNPs, 2 were associated with CaP (P < 3.5 × 10(-5)): rs1472606 (OR: 1.43 in EA) and rs9351265 (OR: 1.48 in AA) both in intergenic regions. For the 32 GWAS SNPs, ROC plots yielded AUC estimates too low for clinical use (EA AUC = 0.60 and AA AUC = 0.56)., Conclusions: This study confirms a large proportion of CaP associated regions implicated by European-based GWAS and provides evidence that some regions may be important in AA CaP risk. Despite the identification of a large panel of GWAS replicated SNPs for CaP, this panel is not appropriate for clinical screening., (Copyright © 2010 Wiley-Liss, Inc.)

Background: Cancer stem cells are defined by their self-renewal and multi-potential capabilities and are hypothesized to be the source of primary and recurrent cancers. The stem cell properties of self-renewal and pluripotency in embryonic stem cells and germ cells are regulated by Oct4A, a splice variant of the POU5F1 (Oct3/4) gene, while the function of the alternative splice variant, Oct4B, is unknown. Rare cells that express Oct4 were identified in several somatic cancers, however, the differential contributions of the Oct4A and Oct4B variants were not determined., Methods: Oct4A expression and co-localization with lineage markers was performed with PCR and immunohistochemistry., Results: Rare Oct4A expressing cells are present in human benign and malignant prostate glands and the number of Oct4A expressing cells increases in prostate cancers with high Gleason scores. Oct4A expressing cells were non-proliferative, and did not co-express markers of basal epithelial cell or luminal epithelial cell differentiation, or AMACR, a marker of prostate cancer epithelial cells. A subpopulation of the Oct4A expressing cells co-expressed Sox2, an embryonic stem cell marker, but did not express other putative stem cell markers, such as ABCG2, NANOG or CD133. The majority of Oct4A expressing cells co-expressed chromogranin A, and a subset of Oct4A expressing cells co-expressed synaptophysin, both markers of neuroendocrine differentiation., Conclusion: The increased number of cells that expressed Oct4A in prostate cancer compared to benign prostate, and in cancers of increasing grade, suggests that Oct4A/Chromogranin A co-expressing cells represent neuroendocrine cells in prostate cancer., (2008 Wiley-Liss, Inc.)

Background: The North Carolina-Louisiana Prostate Cancer Project (PCaP) is a multidisciplinary study of social, individual, and tumor-level causes of racial differences in prostate cancer aggressiveness., Methods: A population-based sample of incident prostate cancer cases from North Carolina and Louisiana will include 1,000 African Americans and 1,000 Caucasian Americans. Study nurses administer structured questionnaires and collect blood, adipose tissue, urine, and toenail samples during an in-home visit. Clinical data are abstracted from medical records, diagnostic biopsies are reviewed and assayed, and tissue microarrays are constructed from prostatectomy samples. Prostate cancer aggressiveness is classified based on PSA, clinical stage, and Gleason grade., Results: Preliminary data demonstrate between- and within-group differences in patient characteristics, screening, and treatment by race and state. Participation exceeds 70% in all groups., Conclusions: Preliminary data support the feasibility of this comprehensive study to help determine the focus of public health efforts to reduce racial disparities in prostate cancer mortality., ((c) 2006 Wiley-Liss, Inc.)

Background: Neuroendocrine (NE) cell differentiation is proposed to facilitate prostate cancer (CaP) recurrence following androgen deprivation through secretion by NE cells of growth factors and neuropeptides that support survival and proliferation of CaP cells and vasculature., Methods: The effect of androgen deprivation on NE differentiation and tumor cell proliferation in the CWR22 model of human CaP was determined by immunohistochemical analysis of the NE cell marker synaptophysin and the cell proliferation marker Ki67., Results: A significant increase in the number of NE cells was observed in CWR22 tumors between 28 and 66 days after castration compared to intact mice, that preceded the increase in tumor cell proliferation that began 70 days after androgen deprivation heralding recurrence. There was a significant positive correlation between the number of tumor-associated NE cells and proliferating CaP cells in tumors from mice after 28-34 days of androgen withdrawal., Conclusion: In the CWR22 model, androgen deprivation induces an increase in tumor-associated NE cells prior to increased tumor cell proliferation, with NE cells possibly promoting tumor survival and recurrent disease., (Copyright 2004 Wiley-Liss, Inc.)

Introduction: The phenotypic plasticity of the human prostate stem cell within human prostate tissue was examined to determine the response of the stem cell to changes in the androgenic environment., Methods: Prostate xenografts were transplanted into athymic nu/nu mice implanted with testosterone pellets, allowed to establish for 1 month time point, the hosts were castrated and pellets removed, and following 1 month of androgen deprivation, the hosts were stimulated with androgen for 2 days to induce proliferation of the residual population of stem cells (2-month time point)., Results: Glands in benign xenografts harvested at the 1- and 2-month time points contained basal cell layers that expressed p63 and high molecular weight cytokeratin, and in which essentially all of the cellular proliferation was localized, consistent with the proposed localization of the prostate stem cell. Benign glandular structures in the xenografts were populated by basal, secretory epithelial, neuroendocrine (NE), or squamous cells overlaying the basal cell layer, whereas, adenocarcinoma glands in the xenografts resembled the original prostate cancer (CaP) tissue., Conclusions: In this human prostate primary xenograft model, the residual stem cell population that survives transplantation, or androgen deprivation, maintains significant pluripotentiality as demonstrated by the capacity to generate progeny that differentiate along multiple lineages in response to microenvironmental signals, particularly along the secretory epithelial lineage in response to androgen, and along the NE cell lineage in response to androgen deprivation., (Copyright 2004 Wiley-Liss, Inc.)

Due to the recent discovery of exercise-induced arterial hypoxemia (EIAH) in healthy active women with normal levels of peak oxygen uptake (V'(O(2)peak), this study examined the incidence of EIAH in prepubescent females. Nineteen healthy, active, prepubescent females (X +/- SD: age = 11.1 +/- 1.6 years; height = 145.8 +/- 9.1 cm; weight = 35.6 +/- 7.0 kg) performed a progressive maximal exercise test on an electronically braked cycle ergometer starting at 0 W and increasing power by 15 W. min(-1). During this test, expired gases, heart rate (HR), and percent arterial oxyhemoglobin saturation (%SaO(2)) were measured. Results for physiological variables at maximal exercise were as follows: V'(O(2)peak) = 43.7 +/- 7.0 ml x kg(-1) x min(-1); HR(max) = 199 +/- 5 beats x min(-1); %SaO(2) = 96.6 +/- 1.2%. For nearly all subjects, the %SaO(2) at maximal exercise was above levels that would reduce V'(O(2)peak). Therefore, in comparison to previous reports of EIAH in adult women with similar V'(O(2)peak), EIAH does not appear to occur in the prepubescent female population., (Copyright 2002 Wiley-Liss, Inc.)

Background: The uptake of nitrate by phytoplankton is a central issue in biological oceanography due to its importance to primary production and vertical flux of biogenic carbon. Nitrate reductase catalyzes the first step of nitrate assimilation, the reduction of NO(3) to NO(2). A cytometric protocol to detect and quantify relative changes in nitrate reductase (NR) protein content of the marine centric diatom Skeletonema costatum is presented., Methods: Immunolabeling of NR protein was achieved with polyclonal antibodies raised against S.costatum NR. Antisera specific to a NR protein subunit and to a NR polypeptide sequence were compared, and cytometric results of NR protein abundance were related to Western analyses. Changes in cellular NR abundance and activity were followed during an upwelling simulation experiment in which S. costatum was exposed to a shift from ammonia to nitrate as major nitrogen source., Results: NR protein could be detected in NO(3)-grown cells and at extremely low levels hardly discernible by Western Blot densiometry in NH(4)-grown cells. The protocol allowed observation of early stages of NR induction during an upwelling simulation. NR abundance increased after the nutrient shift to reach a new physiological "steady-state" 96 hrs later. NR activity exhibited diel variation with maxima at mid-day. NR abundance as estimated by both flow cytometry and Western analysis exhibited a hyperbolic relationship to NR activity. This pattern suggests post-translational activation of NR protein., Conclusions: The presented protocol allows the differentiation of NH(4)- versus NO(3)-grown algae as well as the monitoring of early stages in the induction of nitrate assimilatory capacities., (Copyright 2000 Wiley-Liss, Inc.)

Telomeres, which are specialized structures consisting of T2AG3 repeats and proteins at the ends of chromosomes, may be essential for genomic stability. To test whether telomere length maintenance preserves genomic stability in rats (Rattus rattus and Fischer 344), we assayed telomerase activity and telomere length in the rat hepatic epithelial stem-like cell line WB-F344 during aging in vitro and in tumor-derived lines. Telomerase activity in the parental WB-F344 line was repressed at low and intermediate passage levels in vitro and reexpressed at high passages. Southern blot hybridization and quantitative fluorescence in situ hybridization analyses demonstrated that telomeres were significantly eroded at intermediate passage levels, when telomerase was repressed, and at high passage levels, when telomerase was expressed. Fluorescence in situ hybridization analysis also revealed interstitial telomeric sequences in rat chromosomes. Tumor-derived WB-F344 cell lines that express telomerase had variably shortened telomeres. Cytogenetic analyses performed on WB-F344 cells at low, intermediate, and high passages demonstrated that chromosome instability was most severe in the intermediate passage cells. These data suggest that telomere shortening during aging of rat hepatic epithelial stem-like WB-F344 cells in vitro and during selection of tumorigenic lines in vivo may destabilize chromosomes. Expression of telomerase in high passage cells appeared to partially stabilize chromosomes.

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.

We previously demonstrated that a locus (or loci) linked to the D11S436 marker, which is within the approximately 6-Mb cen-p12 region of human chromosome 11, suppresses the tumorigenic potential of some rat liver epithelial tumor microcell hybrid (MCH) cell lines. To more precisely map this putative liver tumor suppressor locus, we examined 25 loci from human chromosome 11 in suppressed MCH cell lines. Detailed analysis of these markers revealed a minimal area of overlap among the suppressed MCH cell lines corresponding to the chromosomal region bounded by (but not including) microsatellite markers D11S1319 and D11S1958E and containing microsatellite markers D11S436, D11S554, and D11S1344. Direct examination of the kang ai 1 (KA/1) prostatic adenocarcinoma metastasis suppressor gene (which is closely linked to D11S1344) produced evidence suggesting that this locus was not responsible for tumor suppression in this model system. In addition, our data strongly suggested that the putative liver tumor suppressor locus was distinct from other known 11p tumor suppressor loci, including the multiple exotoses 2 locus (at 11p11.2-p12), Wilms' tumor 1 locus (at 11p13), and Wilms' tumor 2 locus (at 11p15.5). The results of this study significantly narrowed the chromosomal location of the putative liver tumor suppressor locus to a region of human 11p11.2-p12 that is approximately 950 kb. This advance forms the basis for positional cloning of candidate genes from this region and, in addition, identified a number of chromosomal markers that will be useful for determining the involvement of this locus in the pathogenesis of human liver cancer.

Comparative chromosomal mapping studies and investigations of tumor-associated chromosomal abnormalities suggest that the development of hepatic tumors in humans and rats may share a common molecular mechanism that involves inactivation of the same tumor suppressor genes or common genetic loci. We investigated the potential of human chromosomes 2 and 11 to suppress the tumorigenic phenotype of rat liver epithelial tumor cell lines. These tumor cell lines (GN6TF and GP7TB) display elevated saturation densities in culture, efficiently form colonies in soft agar, and produce subcutaneous tumors in 100% of syngeneic rat hosts with short latency periods. Introduction of human chromosome 11 by microcell fusion markedly altered the tumorigenicity and the transformed phenotype of GN6TF cells. In contrast, the tumorigenic potential and phenotype of GP7TB cells was unaffected by the introduction of human chromosome 11, indicating that not all rat liver tumor cell lines can be suppressed by loci carried on this chromosome. Introduction of human chromosome 2 had little or no effect on the tumorigenicity or cellular phenotype of either tumor cell line, suggesting the involvement of chromosome 11-specific loci in the suppression of the GN6TF tumor cell line. The GN6TF-11neo microcell hybrid cell lines displayed significantly reduced saturation densities in monolayer cultures, and their ability to grow in soft agar was completely inhibited. Although GN6TF-11neo cells ultimately formed tumors in 80-100% of syngeneic rat hosts, the latency period for tumor formation was much longer. Molecular characterization of GN6TF-11neo microcell hybrid cell lines indicated that some of the clonal lines had spontaneously lost significant portions of the introduced human chromosome, partially delineating the chromosomal location of the putative tumor suppressor locus to the region between the centromere and 11p12. Molecular examination of microcell hybrid-derived tumor cell lines further defined the minimal portion of human chromosome 11 capable of tumor suppression in this model system to the region 11p11.2-p12.

Spontaneous and chemically induced mutation was examined in the lambda light chain immunoglobulin gene in a human B-lymphoblastoid cell strain (T5-1). The hemizygous lambda gene is a unique mutational target gene which codes for a protein that is both expressed on the cell membrane and secreted. Mutations in the lambda gene were detected by analysis of western blots of isoelectric focusing gel electrophoresis of T5-1 cell conditioned culture medium. None of 5,841 individual clones established from vehicle-exposed populations had detectable variations in the isoelectric banding pattern of the constitutively secreted lambda immunoglobulin protein. In contrast, 113 of 6,128 clonal populations established from N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-exposed populations exhibited stable variations in expression of the lambda immunoglobulin: isoelectric variants (n = 3) and non-secretors (n = 110). MNNG-induced mutations in the lambda gene, which resulted in lambda immunoglobulin proteins with altered isoelectric points (pIs), occurred at a frequency of no less than 4.9 x 10(-4) mutations/cell, indicating the mature rearranged lambda immunoglobulin gene is comparably sensitive to carcinogen induced mutation as other human autosomal target genes. Approximately one-half of the MNNG-induced non-secretor mutant clones lacked lambda mRNA while one-half maintained constitutive transcription and expression of the lambda immunoglobulin on the cell surface, demonstrating that carcinogen damage interdicted gene function at multiple points.

Sodium butyrate acts as a differentiation-promoting agent for a wide variety of cell types, including some tumor cell lines. In this study, we examined the effects of sodium butyrate (SB) on the functional differentiation of cultured WB-F344 rat liver epithelial stemlike cells. Treatment of WB-F344 cells with 3.75 mM SB resulted in an inhibition of cellular proliferation, alterations to normal cellular morphology (increased cell size and decreased nuclear/cytoplasmic ratio), and significant increases in cellular protein synthesis. The SB-mediated changes in cell morphology, proliferative status, and protein catabolism were accompanied by development of dexamethasone-inducible tyrosine aminotransferase (TAT) enzyme activity. Culture of WB-F344 cells in growth medium containing SB and dexamethasone (DEX; 1 x 10(-6) M) resulted in greater than sevenfold increase in the basal TAT activity compared with control cultures. An additional sixfold increase in TAT activity was observed when cells cultured in medium containing SB and DEX were exposed to 1 x 10(-7) M DEX during the last 24 hours of culture. The DEX-inducible TAT activity developed by SB-treated WB-F344 cells responded to the modulating effects of insulin and L-tyrosine in a manner that closely resembled that reported for cultured hepatocytes and hepatoma cell lines. These studies show that treatment of WB-F344 rat liver epithelial stemlike cells with the differentiation-promoting agent SB in vitro leads to expression of the differentiation-specific hepatocyte enzyme TAT.

Flow cytometry offers great potential for the study of xenobiotic metabolism in intact cells. We explored this application by the use of ethoxyfluorescein ethyl ester (EFEE) and isolated rat hepatocytes, a classic system for studying such reactions. EFEE is only weakly fluorescent and it diffuses freely into viable cells, where it is metabolized to fluorescein by a process dependent upon mixed-function oxidase activity. In the current study, viable hepatocytes were first identified by flow cytometric assessment of fluorescein diacetate staining. The viable subpopulation was also identifiable on the basis of forward and right angle light scattering properties alone, and it was in this fraction that EFEE metabolism was measured. Metabolism of EFEE to fluorescein was quantified by flow cytometry. SKF 525A, alpha-naphthoflavone, and metyrapone, classic inhibitors of mixed-function oxidation, each inhibited the metabolism of EFEE. These results demonstrate the potential of EFEE for use in flow cytometric studies of drug metabolism, such as in multiparameter mechanistic assays of cellular xenobiotic metabolism and toxicity, and in the isolation by fluorescence-activated cell sorting of subpopulations which differ in this activity.

Fluorescence-activated cell sorting (FACS) was used to establish clonal populations of a human lymphoblastoid cell strain that contain spontaneously occurring and N-methyl-N'-nitro-N-nitrosoguanidine-induced mutations in the lambda immunoglobulin gene. Multiple rounds of FACS using a monoclonal antibody specific for the membrane-expressed human lambda immunoglobulin were used to enrich the population fraction of cells lacking a wild-type lambda immunoglobulin on the cell surface. Approximately 20% of the clonal populations established after five rounds of FACS-mediated enrichment did not express the lambda immunoglublin epitope recognized by the monoclonal antibody used for selection. However, evaluation of the FACS-selected mutant clonal populations with a polyclonal antilambda antibody, or a monoclonal antibody directed against a different epitope on the lambda immunoglobulin made by the T5-1 cells, indicated that the mutant clonal populations expressed lambda immunoglobulin on their cell surfaces. Additionally, the presence of lambda mRNA and of both secreted and cytoplasmic lambda protein confirmed the transcription and translation of the lambda immunoglobulin gene. These data suggest that FACS-mediated selection employing epitope-specific monoclonal antibodies provides a powerful technique for isolation of cell populations that express mutations within the coding region of the lambda immunoglobulin gene.

Several passage cycles of poorly metastatic malignant epithelial cells through immunosuppressed mice failed to induce enhanced metastasis-forming ability of cells derived from either the primary subcutaneous tumours or the resultant lung metastases. In vitro treatment of cultured malignant cells with dimethyl sulphoxide (DMSO) induced a reversible change in phenotype towards increased invasiveness but did not significantly increase metastasis formation. A cloned-cell line from a spontaneous in vitro transformant in the presence of DMSO was highly invasive and highly metastatic. In vitro treatment of cultured cells with 2% (v/v) DMSO produced alterations in morphology with decreased growth rate of all cell lines and decreased anchorage-independent colony formation in several malignant cell lines. All in vitro and in vivo effects were reversible following both short- and long-term (1 year) culture of cells in the presence of DMSO, suggesting epigenetic effects. These data support the concept of independent genetic controls for the invasiveness of tumours and the ability to form metastases.

An in vitro cell model of mouse lung alveologenic carcinoma consisting of preneoplastic nonmalignant cells, spontaneously transformed cells, and urethane-induced malignant cells was analyzed for phenotypic and genotypic changes associated with the transition to neoplasia. The polymerase chain reaction (PCR) was used to amplify the cDNA derived from the c-Ki-ras mRNA corresponding to exons 1 and 2 of the proto-oncogene. This approach allowed analysis of the gene transcription product rather than potentially unexpressed DNA. Direct sequencing of the PCR product identified a common single point mutation, alone or together with wild-type mRNA for c-Ki-ras, in all of the malignant clones. An A----G transition in the second base position of codon 61 was common to spontaneously malignant and chemical tumor-derived cell lines and to lines selected for lung metastatic behavior. The absence of the mutation in the nonmalignant cells suggests that the Ki-ras mutation may be a factor in onset or maintenance of the malignant phenotype and, at least in vitro, may be a late event in the transformation process.

Perturbation of DNA replication by chemical-DNA adducts produced by exposure to mutagenic/carcinogenic chemicals results in mutagenic or cytotoxic damage in the DNA. Demonstration of a correlation between cell cycle dependency of cytotoxicity and point mutation at the Na+/K+ ATPase gene could suggest that the two consequences of chemical exposure are caused by the same damage in the template DNA and that both are mediated through DNA replication-associated mechanisms. N-methyl-N'-nitro-N-nitrosoguanidine, N-ethyl-N'-nitro-N-nitrosoguanidine, 4-nitroquinoline-1-oxide, and benzo(a)pyrene-trans-7,8-dihydrodiol-9,10-epoxide demonstrated cell cycle-related patterns of cytotoxicity in 10T1/2 cells, with maximal cell killing produced by exposure in early S phase, and were highly efficient mutagens of the Na+/K+ ATPase gene relative to their cytotoxic potential. In contrast, methyl methanesulfonate and N-acetoxy-N-2-fluorenylacetamide were maximally cytotoxic in cell populations exposed in early G1 phase and were weak mutagens of the Na+/K+ ATPase gene at comparable levels of cytotoxicity. These data suggest that mutagenic/carcinogenic chemicals that are effective at producing mutations by misreplication kill cells by a related mechanism that may be associated with the perturbation of DNA replication.

The teratogenic potential of nicotine and a primary metabolite, cotinine, was examined with FETAX (Frog Embryo Teratogenesis Assay: Xenopus). Early embryos of Xenopus laevis were exposed for 96 hr to nicotine or cotinine in two separate static renewal tests of each compound without addition of the metabolic activation system (MAS). Two static renewal tests of nicotine with the MAS were also conducted. Addition of the MAS to nicotine reduced the LC50 from an average of 136 to 20 mg/L. However, the EC50 (malformation) was increased from 0.4 to 5.8 mg/L upon activation. The LC50 and EC50 values for cotinine averaged 4,340 and 720 mg/L, respectively. Based on mortality/malformation index values, growth end points, and the types and severity of the induced malformations, nicotine and cotinine scored as potential teratogens. Metabolism of nicotine to more polar metabolites increased the nicotine concentration required to induce terata. The results are indicative of the versatility of FETAX in developmental toxicity testing.