Todd A. Thompson

Cytochromes CYP1A1 and CYP1B1 in the rat mammary gland: Cell-specific expression and regulation by polycyclic aromatic hydrocarbons and hormones

Cultured rat mammary cells express both CYP1A1 and CYP1B1 in response to polycyclic aromatic hydrocarbons (PAH) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in a cell type-specific manner. The expression of each P450 was determined functionally (regioselective PAH metabolism), as apoprotein (immunoblots) and as mRNA (Northern hybridization). The epithelial rat mammary cells (RMEC) expressed CYP1A1, however only after PAH or TCDD treatment. CYP1B1 protein was scarcely detected in these induced RMEC but was surprisingly active as a participant in 7,12-dimethylbenz[a]anthracene (DMBA) metabolism shown through selective antibody inhibition (40% of total activity). CYP1B1 was selectively expressed in the stromal fibroblast population of rat mammary cells to the exclusion of CYP1A1. In the rat mammary fibroblasts (RMF), CYP1B1 protein and associated activity were each present at low levels constitutively and were highly induced by benz[a]anthracene (BA) to a greater extent than by TCDD (12- versus 6-fold). However, BA (10 microM) and TCDD (10 nM) stimulated the 5.2-kb CYP1B1-specific mRNA equally. These increases are consistent with the involvement of the aryl hydrocarbon (Ah) receptor in the transcription of the CYP1B1 gene and with the additional stabilization of CYP1B1 protein by BA, previously observed in embryo fibroblasts. Exactly this regulation of CYP1B1-dependent activity was seen in RMEC suggesting that this arises from exceptionally active CYP1B1 in a small proportion (5%) of residual RMF. The constitutive expression and PAH inducibility of CYP1B1 and CYP1A1 proteins in RMF and RMEC, respectively, were each substantially decreased (approximately 75%) by a hormonal mixture (17 beta-estradiol (0.2 microM) progesterone (1.5 microM) cortisol (1.5 microM) and prolactin (5 micrograms/ml)). Progesterone and cortisol, added singly to RMF suppressed CYP1B1 protein expression (approximately 80%) in both untreated and BA-induced cells, while cortisol also suppressed the 5.2-kb CYP1B1 mRNA. In contrast, 17 beta-estradiol stimulated constitutive expression of CYP1B1 protein (50-75%) and mRNA level (2- to 3-fold), but did not affect CYP1B1 expression in BA-treated RMF. The expression of CYP1A1 and CYP1B1 is therefore highly cell specific even though each is regulated through the Ah receptor. Each P450 exhibits a surprisingly similar pattern of hormonal regulation even though expressed in different cell types.

JunD Mediates Androgen-Induced Oxidative Stress in Androgen Dependent LNCaP Human Prostate Cancer Cells

Numerous and compelling evidence shows that high level of reactive oxygen species (ROS) plays a key role in prostate cancer occurrence, recurrence and progression. The molecular mechanism of ROS overproduction in the prostate gland, however, remains mostly unknown. Unique AP‐1 transcription factor JunD has been shown to inhibit cell proliferation, promote differentiation and mediate stress responses in a variety of eukaryotic cells. We previously reported that androgen–androgen receptor induced ROS production in androgen‐dependent LNCaP human prostate cancer cells is associated with increased JunD level/AP‐1 transcriptional activity.

The curcumin analog ca27 down-regulates androgen receptor through an oxidative stress mediated mechanism in human prostate cancer cells

The androgen receptor (AR) plays a critical role in prostate cancer development and progression. Therefore, the inhibition of AR function is an established therapeutic intervention. Since the expression of the AR is retained and often increased in progressive disease, AR protein down‐regulation is a promising therapeutic approach against prostate cancer. We show here that the curcumin analog 27 (ca27) down‐regulates AR expression in several prostate cancer cell lines.

Detection of Intracellular Granularity Induction in Prostate Cancer Cell Lines by Small Molecules Using the HyperCyt® High-Throughput Flow Cytometry System

Prostate cancer is a leading cause of death among men due to the limited number of treatment strategies available for advanced disease. Discovery of effective chemotherapeutics involves the identification of agents that inhibit cancer cell growth. Increases in intracellular granularity have been observed during physiological processes that include senescence, apoptosis, and autophagy, making this phenotypic change a useful marker for identifying small molecules that induce cellular growth arrest or death. In this regard, epithelial-derived cancer cell lines appear uniquely susceptible to increased intracellular granularity following exposure to chemotherapeutics. We have established a novel flow cytometry approach that detects increases in side light scatter in response to morphological changes associated with intracellular granularity in the androgen-sensitive LNCaP and androgen-independent PC3 human prostate cancer cell lines. A cell-based assay was developed to screen for small molecule inducers of intracellular granularity using the HyperCyt® high-throughput flow cytometry platform. Validation was performed using the Prestwick Chemical Library, where known modulators of LNCaP intracellular granularity, such as testosterone, were identified. Nonandrogenic inducers of granularity were also detected. A further screen of ~25,000 small molecules led to the identification of a class of aryl-oxazoles that increased intracellular granularity in both cell lines, often leading to cell death. The most potent agents exhibited submicromolar efficacy in LNCaP and PC3 cells. (Journal of Biomolecular Screening. 2009:596-609)

Ethanol-induced oxidative stress is associated with EGF receptor phosphorylation in MCF-10A cells overexpressing CYP2E1.

Breast cancer is the most common cancer and the second leading cause of cancer-related mortality worldwide. The etiology of breast cancer is very diverse and ethanol (EtOH) consumption is a well-established risk factor for breast cancer in women. However, the mechanism by which EtOH exerts its carcinogenic activity in breast tissue remains unknown. CYP2E1 is known to metabolize ethanol and produce reactive oxygen species (ROS), including superoxide in epithelial cells. Therefore, in the present studies, we investigated whether there is an increase in ROS following overexpression of CYP2E1 in MCF-10A cells. We found that 30 and 100 mM EtOH increased ROS levels after 2 h treatment in CYP2E1 overexpressing cells. Based on these results and our previous studies with ROS-producing chemicals, we also examined epidermal growth factor receptor (EGFR) activation following exposure to ethanol. We found that there was an increase in phosphorylation of pY1086 EGFR after 18 h EtOH treatment in CYP2E1 overexpressing cells. These studies support a hypothesis that EtOH might increase human mammary cell activation, via an EGFR-dependent signaling mechanism associated with oxidative stress.

A multifunctional androgen receptor screening assay using the high-throughput Hypercyt® flow cytometry system

The androgen receptor (AR) is a steroid hormone receptor which regulates transcription of androgen‐sensitive genes and is responsible for the development and maintenance of male secondary sexual characteristics. Chemicals that interfere with AR activity may lead to pathological conditions in androgen‐sensitive tissues. A variety of reporter systems have been developed, driven by androgen‐sensitive promoters, which screen for chemicals that modulate androgenic activity. We have developed a flexible, high‐throughput AR transcriptional activation assay, designated the Multifunctional Androgen Receptor Screening (MARS) assay, to facilitate the identification of novel modulators of AR transcriptional activity using flow cytometry. Androgen‐independent human prostate cancer‐derived PC3 cells were transiently cotransfected with an expression vector for the wild‐type human AR and an androgen‐sensitive promoter regulating the expression of destabilized enhanced GFP (dsEGFP). The transfected cells were stimulated with established androgenic and antiandrogenic compounds and assessed for increased or decreased dsEGFP expression. To screen for antagonists of AR transcription, the AR agonist R1881 was coadministered at submaximal concentrations with potential AR antagonists. The assay was formatted for high‐throughput screening using the HyperCyt® flow cytometry system. Agents with established androgenic and antiandrogenic activity were used for validation of the MARS assay. AR agonists were found to potently induce dsEGFP. Furthermore, AR agonists induced dsEGFP expression in a dose‐dependent manner. Alternatively, AR antagonists blocked dsEGFP expression when coadministered with low‐dose R1881, which also occurred in a dose‐dependent manner. Modulators of AR transcriptional activity can be successfully identified by the MARS assay, utilizing a rapid, flexible, sensitive, and high‐throughput format. Dose–response curves can be successfully generated for these compounds, allowing for an assessment of potency. Because of its simplicity and high‐throughput compatibility, the MARS assay and HyperCyt® system combined with flow cytometric analysis represents a valuable and novel addition to the current repertoire of AR transcriptional activation screening assays.

Decreased susceptibility to NMU-induced mammary carcinogenesis in transgenic rats carrying multiple copies of a rat ras gene driven by the rat Harvey ras promoter.

Ras protein over-expression has been observed in human breast cancers although the significance of Ras over-expression in the etiology of breast cancer is unknown and its contribution to breast cancer prognosis is still debated. In this study, the over-expression of both wild-type Harvey and Kirsten Ras proteins as contributors to rat mammary carcinogenesis were examined using a transgenic rat model. Three rat transgenic lines (designated HrHr transgenics) carrying three to six copies of wild-type rat Harvey ras driven by the wild-type rat Harvey ras promoter were produced. In addition, transgenic lines carrying either three or seven copies of the Kirsten ras gene under the same promoter (HrKr) were produced. No pathological changes in the mammary gland were observed in any of the HrHr or HrKr transgenic rat line heterozygotes. Two of the Ras transgenic lines, HrHr (R8) and HrKr (4334), had a significant reduction in NMU-induced rat mammary cancer when compared to their non-transgenic littermates. All five Ras transgenic lines developed fewer carcinomas than their non-transgenic littermates following NMU exposure. The percentage of NMU-induced G35 to A35 activating mutations in the endogenous Harvey ras gene in mammary carcinomas from the HrHr, HrKr transgenic rats and their non-transgenic littermates was similar (∼50%). In contrast, less than 1% of the NMU-induced carcinomas in these Ras transgenic rats had an activating ras mutation in their transgenes. These findings highlight the potential of Ras to function as a modifier gene in repressing mammary carcinogenesis.

1α,25-Dihydroxyvitamin D3 down-regulates expression of prostate specific membrane antigen in prostate cancer cells

Prostate specific membrane antigen (PSMA) expression correlates with prostate cancer grade and is increased in hormone‐refractory prostate cancer. The increased expression of PSMA following androgen deprivation therapy may be a consequence of the down‐regulation of PSMA expression by androgen. Moreover, 1α,25‐dihydroxyvitamin D3 (1,25‐VD) has been shown to suppress prostate cancer progression as well as cell motility and invasion. Since PSMA is positively correlated with both of these characteristics, we hypothesized that 1,25‐VD would regulate PSMA expression.

Health disparities in clinical practice patterns for prostate cancer screening by geographic regions in the United States: a multilevel modeling analysis.

Background:To our knowledge, no previous study has examined state-level geographic variability and its predictors in clinical practice patterns to screen for prostate cancer in the United States.Methods:We used the Behavioral Risk Factor Surveillance System 2010 data set to analyze geographic variability (by state) and its associated predictors in receiving a PSA test and/or a digital rectal examination (DRE). The study population consisted of men aged ⩾50 years who responded as yes/no when asked about having a PSA test or DRE performed during the last year. We build two multilevel logistic regression models, differing in dependent variables, that is, (1) any prostate cancer screening (PCS) (either PSA and/or DRE), and (2) PCS based on PSA testing (PSAT). Individual characteristics (age, education, employment, marriage, income, race/ethnicity, self-reported health status, obesity, alcohol consumption, smoking status, personal physician presence, and health insurance coverage) were treated as level-1 variables and state characteristics (number of doctors per 100 000 persons per state, US regions and metropolitan statistical area (MSA) codes) were treated as level-2 variables.Results:We found significant geographic variability in receiving PCS and PSAT screening in the United States. For PCS, MSA code was an independent predictor, with men living in urban areas having lower odds of screening (odds ratio (OR)=0.8, 95% confidence interval (CI)=0.7–0.9). In PSAT, the number of doctors per 100 000 persons per state was an independent predictor, with lowest quartile states (0–25% quartile) having lower odds of PSA-based screening (OR=0.78, 95% CI=063–0.94). In both models, all level-1 variables were independent predictors (P<0.05) of PCS, except self-reported health status.Conclusions:Men living in urban areas and states with lower prevalence of doctors have lower odds of screening for prostate cancer and PSAT, respectively, after adjusting for individual variables. Future studies should examine the reasons for these health disparities.

Variants in autophagy-related genes and clinical characteristics in melanoma: a population-based study

Autophagy has been linked with melanoma risk and survival, but no polymorphisms in autophagy‐related (ATG) genes have been investigated in relation to melanoma progression. We examined five single‐nucleotide polymorphisms (SNPs) in three ATG genes (ATG5; ATG10; and ATG16L) with known or suspected impact on autophagic flux in an international population‐based case–control study of melanoma. DNA from 911 melanoma patients was genotyped. An association was identified between (GG) (rs2241880) and earlier stage at diagnosis (OR 0.47; 95% Confidence Intervals (CI) = 0.27–0.81, P = 0.02) and a decrease in Breslow thickness (P = 0.03). The ATG16L heterozygous genotype (AG) (rs2241880) was associated with younger age at diagnosis (P = 0.02). Two SNPs in ATG5 were found to be associated with increased stage (rs2245214 CG, OR 1.47; 95% CI = 1.11–1.94, P = 0.03; rs510432 CC, OR 1.84; 95% CI = 1.12–3.02, P = 0.05). Finally, we identified inverse associations between ATG5 (GG rs2245214) and melanomas on the scalp or neck (OR 0.20, 95% CI = 0.05–0.86, P = 0.03); ATG10 (CC) (rs1864182) and brisk tumor infiltrating lymphocytes (TILs) (OR 0.42; 95% CI = 0.21–0.88, P = 0.02), and ATG5 (CC) (rs510432) with nonbrisk TILs (OR 0.55; 95% CI = 0.34–0.87, P = 0.01). Our data suggest that ATG SNPs might be differentially associated with specific host and tumor characteristics including age at diagnosis, TILs, and stage. These associations may be critical to understanding the role of autophagy in cancer, and further investigation will help characterize the contribution of these variants to melanoma progression.