Prostate cancer cell-platelet bidirectional signaling promotes calcium mobilization, invasion and apoptotic resistance via distinct receptor-ligand pairs.

Platelets play a crucial role in cancer and thrombosis. However, the receptor-ligand repertoire mediating prostate cancer (PCa) cell-platelet interactions and ensuing consequences have not been fully elucidated. Microvilli emanating from the plasma membrane of PCa cell lines (RC77 T/E, MDA PCa 2b) directly contacted individual platelets and platelet aggregates. PCa cell-platelet interactions were associated with calcium mobilization in platelets, and translocation of P-selectin and integrin αIIbß3 onto the platelet surface. PCa cell-platelet interactions reciprocally promoted PCa cell invasion and apoptotic resistance, and these events were insensitive to androgen receptor blockade by bicalutamide. PCa cells were exceedingly sensitive to activation by platelets in vitro, occurring at a PCa cell:platelet coculture ratio as low as 1:10 (whereas PCa patient blood contains 1:2,000,000 per ml). Conditioned medium from cocultures stimulated PCa cell invasion but not apoptotic resistance nor platelet aggregation. Candidate transmembrane signaling proteins responsible for PCa cell-platelet oncogenic events were identified by RNA-Seq and broadly divided into 4 major categories: (1) integrin-ligand, (2) EPH receptor-ephrin, (3) immune checkpoint receptor-ligand, and (4) miscellaneous receptor-ligand interactions. Based on antibody neutralization and small molecule inhibitor assays, PCa cell-stimulated calcium mobilization in platelets was found to be mediated by a fibronectin1 (FN1)-αIIbß3 signaling axis. Platelet-stimulated PCa cell invasion was facilitated by a CD55-adhesion G protein coupled receptor E5 (ADGRE5) axis, with contribution from platelet cytokines CCL3L1 and IL32. Platelet-stimulated PCa cell apoptotic resistance relied on ephrin-EPH receptor and lysophosphatidic acid (LPA)-LPA receptor (LPAR) signaling. Of participating signaling partners, FN1 and LPAR3 overexpression was observed in PCa specimens compared to normal prostate, while high expression of CCR1 (CCL3L1 receptor), EPHA1 and LPAR5 in PCa was associated with poor patient survival. These findings emphasize that non-overlapping receptor-ligand pairs participate in oncogenesis and thrombosis, highlighting the complexity of any contemplated clinical intervention strategy.

Apixaban Concentrations in Routine Clinical Care of Older Adults With Nonvalvular Atrial Fibrillation.

BACKGROUND: Direct-acting oral anticoagulants are first-line agents for prevention of stroke in patients with nonvalvular atrial fibrillation (NVAF), but data are limited for the oldest patients, and with reduced dosing. OBJECTIVES: To determine steady-state apixaban peak and trough concentrations during routine care of older adults with NVAF, compare concentrations to clinical trial concentrations, and explore factors associated with concentrations. METHODS: A cross-sectional study of medically stable older adults with NVAF (≥75 years or ≥70 years if Black) receiving apixaban. Peak (2-4.4 hours post-dose) and trough (before next dose) concentrations were determined by anti-Xa activity calibrated chromogenic assay. Patient characteristics associated with concentrations were determined by multivariate modeling. RESULTS: The median age of patients (n = 115) was 80 (interquartile range: 77-84) years. The cohort comprised 46 women and 69 men; of which 98 are White, 11 Black, and 6 Asian. With 5 mg twice daily per labelling (n = 88), peak concentrations were higher in women: 248 ± 105 vs 174 ± 67 ng/mL in men (P < 0.001) and exceeded expected 95% range in 6 of 30 vs 0 of 55 men (P = 0.002). With 2.5 mg twice daily per label (n = 11), concentrations were <5 mg twice daily (peak: 136 ± 87 vs 201 ± 90 ng/mL, P = 0.026; trough: 65 ± 28 vs 109 ± 56 ng/mL, P < 0.001), but not different than 2.5 mg twice daily without reduction criteria (n = 13; peak: 132 ± 88; trough: 65 ± 31 ng/mL). Covariates associated with concentrations included sex, number of daily medications, and creatinine clearance. CONCLUSIONS: Older women had higher than expected peak apixaban concentrations, and 2.5 mg twice daily produced lower concentrations than standard dosing. Factors not currently included in dosing recommendations affected concentrations. The impact of apixaban concentrations on outcomes needs evaluation.

Race and Drug Toxicity: A Study of Three Cardiovascular Drugs with Strong Pharmacogenetic Recommendations.

The Clinical Pharmacogenetics Implementation Consortium (CPIC®) establishes evidence-based guidelines for utilizing pharmacogenetic information for certain priority drugs. Warfarin, clopidogrel and simvastatin are cardiovascular drugs that carry strong prescribing guidance by CPIC. The respective pharmacogenes for each of these drugs exhibit considerable variability amongst different ethnic/ancestral/racial populations. Race and ethnicity are commonly employed as surrogate biomarkers in clinical practice and can be found in many prescribing guidelines. This is controversial due to the large variability that exists amongst different racial/ethnic groups, lack of detailed ethnic information and the broad geographic categorization of racial groups. Using a retrospective analysis of electronic health records (EHR), we sought to determine the degree to which self-reported race/ethnicity contributed to the probability of adverse drug reactions for these drugs. All models used individuals self-reporting as White as the comparison group. The majority of apparent associations between different racial groups and drug toxicity observed in the "race only" model failed to remain significant when we corrected for covariates. We did observe self-identified Asian race as a significant predictor (p = 0.016) for warfarin hemorrhagic events in all models. In addition, patients identifying as either Black/African-American (p = 0.001) or Other/Multiple race (p = 0.019) had a lower probability of reporting an adverse reaction than White individuals while on simvastatin even after correcting for other covariates. In both instances where race/ethnicity was predictive of drug toxicity (i.e., warfarin, simvastatin), the findings are consistent with the known global variability in the pharmacogenes described in the CPIC guidelines for these medications. These results confirm that the reliability of using self-identified race/ethnic information extracted from EHRs as a predictor of adverse drug reactions is likely limited to situations where the genes influencing drug toxicity display large, distinct ethnogeographic variability.

Differences in the Platelet mRNA Landscape Portend Racial Disparities in Platelet Function and Suggest Novel Therapeutic Targets.

The African American (AA) population displays a 1.6 to 3-fold higher incidence of thrombosis and stroke mortality compared with European Americans (EAs). Current antiplatelet therapies target the ADP-mediated signaling pathway, which displays significant pharmacogenetic variation for platelet reactivity. The focus of this study was to define underlying population differences in platelet function in an effort to identify novel molecular targets for future antiplatelet therapy. We performed deep coverage RNA-Seq to compare gene expression levels in platelets derived from a cohort of healthy volunteers defined by ancestry determination. We identified > 13,000 expressed platelet genes of which 480 were significantly differentially expressed genes (DEGs) between AAs and EAs. DEGs encoding proteins known or predicted to modulate platelet aggregation, morphology, or platelet count were upregulated in AA platelets. Numerous G-protein coupled receptors, ion channels, and pro-inflammatory cytokines not previously associated with platelet function were likewise differentially expressed. Many of the signaling proteins represent potential pharmacologic targets of intervention. Notably, we confirmed the differential expression of cytokines IL32 and PROK2 in an independent cohort by quantitative real-time polymerase chain reaction, and provide functional validation of the opposing actions of these two cytokines on collagen-induced AA platelet aggregation. Using Genotype-Tissue Expression whole blood data, we identified 516 expression quantitative trait locuses with Fst values > 0.25, suggesting that population-differentiated alleles may contribute to differences in gene expression. This study identifies gene expression differences at the population level that may affect platelet function and serve as potential biomarkers to identify cardiovascular disease risk. Additionally, our analysis uncovers candidate novel druggable targets for future antiplatelet therapies.

The ACCOuNT Consortium: A Model for the Discovery, Translation, and Implementation of Precision Medicine in African Americans.

The majority of pharmacogenomic (PGx) studies have been conducted on European ancestry populations, thereby excluding minority populations and impeding the discovery and translation of African American-specific genetic variation into precision medicine. Without accounting for variants found in African Americans, clinical recommendations based solely on genetic biomarkers found in European populations could result in misclassification of drug response in African American patients. To address these challenges, we formed the Transdisciplinary Collaborative Center (TCC), African American Cardiovascular Pharmacogenetic Consortium (ACCOuNT), to discover novel genetic variants in African Americans related to clinically actionable cardiovascular phenotypes and to incorporate African American-specific sequence variations into clinical recommendations at the point of care. The TCC consists of two research projects focused on discovery and translation of genetic findings and four cores that support the projects. In addition, the largest repository of PGx information on African Americans is being established as well as lasting infrastructure that can be utilized to spur continued research in this understudied population.

Teaching students in clinical programs about pharmacogenomics: do they understand drug-drug interactions?

Teaching the clinical implementation of pharmacogenomics to students in clinical programs first requires careful consideration of their aptitude in basic clinical pharmacologic concepts. Prior to developing training exercises on drug-gene interactions, educators must first assess student competency in identifying and managing drug-drug interactions given the similarities in identifying and managing these sources of medication error.

Impact of a personal CYP2D6 testing workshop on physician assistant student attitudes toward pharmacogenetics.

AIM: We assessed the impact of personal CYP2D6 testing on physician assistant student competency in, and attitudes toward, pharmacogenetics (PGx). MATERIALS & METHODS: Buccal samples were genotyped for CYP2D6 polymorphisms. Results were discussed during a 3-h PGx workshop. PGx knowledge was assessed by pre- and post-tests. Focus groups assessed the impact of the workshop on attitudes toward the clinical utility of PGx. RESULTS: Both student knowledge of PGx, and its perceived clinical utility, increased immediately following the workshop. However, exposure to PGx on clinical rotations following the workshop seemed to influence student attitudes toward PGx utility. CONCLUSION: Personal CYP2D6 testing improves both knowledge and comfort with PGx. Continued exposure to PGx concepts is important for transfer of learning.

Novel genetic predictors of venous thromboembolism risk in African Americans.

Venous thromboembolism (VTE) is the third most common life-threatening cardiovascular condition in the United States, with African Americans (AAs) having a 30% to 60% higher incidence compared with other ethnicities. The mechanisms underlying population differences in the risk of VTE are poorly understood. We conducted the first genome-wide association study in AAs, comprising 578 subjects, followed by replication of highly significant findings in an independent cohort of 159 AA subjects. Logistic regression was used to estimate the association between genetic variants and VTE risk. Through bioinformatics analysis of the top signals, we identified expression quantitative trait loci (eQTLs) in whole blood and investigated the messenger RNA expression differences in VTE cases and controls. We identified and replicated single-nucleotide polymorphisms on chromosome 20 (rs2144940, rs2567617, and rs1998081) that increased risk of VTE by 2.3-fold (P< 6 × 10(-7)). These risk variants were found in higher frequency among populations of African descent (>20%) compared with other ethnic groups (<10%). We demonstrate that SNPs on chromosome 20 are cis-eQTLs for thrombomodulin (THBD), and the expression of THBD is lower among VTE cases compared with controls (P= 9.87 × 10(-6)). We have identified novel polymorphisms associated with increased risk of VTE in AAs. These polymorphisms are predominantly found among populations of African descent and are associated with THBD gene expression. Our findings provide new molecular insight into a mechanism regulating VTE susceptibility and identify common genetic variants that increase the risk of VTE in AAs, a population disproportionately affected by this disease.

Improving nurse practitioners' competence with genetics: Effectiveness of an online course.

PURPOSE: The purpose of this study was to assess the effectiveness of an online genetics course for improving nurse practitioners' knowledge, competence, and comfort with genetic principles and their application to clinical practice. DATA SOURCES: A genetics knowledge test and survey were administered to 232 nurse practitioner students, between 2011 and 2013, before and after completing a 15-week online genetics course taught by a multidisciplinary team of instructors at a private east coast U.S. university. The 65-item survey allowed participants to rate competence regarding genetic principles, diseases, and terminology, as well as comfort performing various clinical tasks related to genetics. The 21-item knowledge test contained multiple choice questions regarding core competencies in genetics. Paired t-tests were used to compare mean pre- and postscores. CONCLUSIONS: Participants significantly increased postcourse knowledge (p < .001) and comfort with genetic core competencies and clinical skills related to genetics (p < .001). This study demonstrates the effectiveness of an online genetics course for increasing nurse practitioners' knowledge, competence, and confidence with genetics and identifies specific topics educators should consider when designing curricula for nurse practitioners. IMPLICATIONS FOR PRACTICE: Findings from this study can improve genetics education for nurse practitioners, which will in turn improve patient health.

Targeted single molecule sequencing methodology for ovarian hyperstimulation syndrome.

BACKGROUND: One of the most significant issues surrounding next generation sequencing is the cost and the difficulty assembling short read lengths. Targeted capture enrichment of longer fragments using single molecule sequencing (SMS) is expected to improve both sequence assembly and base-call accuracy but, at present, there are very few examples of successful application of these technologic advances in translational research and clinical testing. We developed a targeted single molecule sequencing (T-SMS) panel for genes implicated in ovarian response to controlled ovarian hyperstimulation (COH) for infertility. RESULTS: Target enrichment was carried out using droplet-base multiplex polymerase chain reaction (PCR) technology (RainDance®) designed to yield amplicons averaging 1 kb fragment size from candidate 44 loci (99.8% unique base-pair coverage). The total targeted sequence was 3.18 Mb per sample. SMS was carried out using single molecule, real-time DNA sequencing (SMRT® Pacific Biosciences®), average raw read length = 1178 nucleotides, 5% of the amplicons >6000 nucleotides). After filtering with circular consensus (CCS) reads, the mean read length was 3200 nucleotides (97% CCS accuracy). Primary data analyses, alignment and filtering utilized the Pacific Biosciences® SMRT portal. Secondary analysis was conducted using the Genome Analysis Toolkit for SNP discovery l and wANNOVAR for functional analysis of variants. Filtered functional variants 18 of 19 (94.7%) were further confirmed using conventional Sanger sequencing. CCS reads were able to accurately detect zygosity. Coverage within GC rich regions (i.e.VEGFR; 72% GC rich) was achieved by capturing long genomic DNA (gDNA) fragments and reading into regions that flank the capture regions. As proof of concept, a non-synonymous LHCGR variant captured in two severe OHSS cases, and verified by conventional sequencing. CONCLUSIONS: Combining emulsion PCR-generated 1 kb amplicons and SMRT DNA sequencing permitted greater depth of coverage for T-SMS and facilitated easier sequence assembly. To the best of our knowledge, this is the first report combining emulsion PCR and T-SMS for long reads using human DNA samples, and NGS panel designed for biomarker discovery in OHSS.

Kinase insert domain receptor/vascular endothelial growth factor receptor 2 (KDR) genetic variation is associated with ovarian hyperstimulation syndrome.

BACKGROUND: The objective of this investigation was to determine if kinase insert domain/vascular endothelial growth factor receptor 2 (KDR/VEGFR2) genetic variation was associated with the development of ovarian hyperstimulation syndrome (OHSS) in patients undergoing controlled ovarian hyperstimulation (COH). METHODS: This was a case-control study of 174 patients who underwent controlled ovarian stimulation. Patient blood samples were genotyped for single nucleotide polymorphisms (SNPs) spanning the KDR locus. OHSS development, clinical outcome variables, SNP and haplotype frequencies were compared between control (n = 155) and OHSS (n = 19) groups. RESULTS: Patients who developed OHSS had significantly higher response markers (estradiol levels of the day of hCG administration, number of follicles developed, number of eggs retrieved) than control patients. When adjusted for age and self-identified race, the rs2305945 G/T genotype was associated (P = 0.027) with a decreased risk (OR = 0.30; 95% CI = 0.10, 0.93) of developing OHSS using an overdominant model. The rs2305945 G/T variant was also associated with decreased COH response (number of follicles, number of eggs retrieved) in an overdominant model. The rs2305948, rs1870378, rs2305945 (C-T-G) haplotype was associated with both decreased COH response and OHSS risk (unadjusted OR = 0.10; 95% CI = 0.01, 0.80, P = 0.031). CONCLUSIONS: The KDR receptor is believed to play a central role OHSS development and is a target for pharmacological prevention of OHSS. These results indicate that genetic variation in the KDR gene may impact individual risk of developing OHSS from COH. In addition, the rs2305948 SNP and C-T-G haplotype might serve as potential biomarkers for poor ovarian response to COH.

Engaging the next generation of healthcare professionals in genomics: planning for the future.

There is broad agreement that healthcare professionals require fundamental training in genomics to keep pace with scientific advancement. Strong models that promote effective genomic education, however, are lacking. Furthermore, curricula at many institutions are now straining to adapt to the integration of additional material on next-generation sequencing and the bioethical and legal issues that will accompany clinical genomic testing. This article advocates for core competencies focused on job function, which will best prepare providers to be end-users of healthcare information. In addition, it argues in favor of online and blended learning models that incorporate student genotyping and specific training in the ethical, legal and social issues raised by genomic testing.

Association between the luteinizing hormone/chorionic gonadotropin receptor (LHCGR) rs4073366 polymorphism and ovarian hyperstimulation syndrome during controlled ovarian hyperstimulation.

BACKGROUND: The aim of this study was to determine the relationship between a purported luteinizing hormone/chorionic gonadotropin (LHCGR) high function polymorphism (rs4539842/insLQ) and outcome to controlled ovarian hyperstimulation (COH). METHODS: This was a prospective study of 172 patients undergoing COH at the Fertility and IVF Center at GWU. DNA was isolated from blood samples and a region encompassing the insLQ polymorphism was sequenced. We also investigated a polymorphism (rs4073366 G > C) that was 142 bp from insLQ. The association of the insLQ and rs4073366 alleles and outcome to COH (number of mature follicles, estradiol level on day of human chorionic gonadotropin (hCG) administration, the number of eggs retrieved and ovarian hyperstimulation syndrome (OHSS)) was determined. RESULTS: Increasing age and higher day 3 (basal) FSH levels were significantly associated with poorer response to COH. We found that both insLQ and rs4073366 were in linkage disequilibrium (LD) and no patients were homozygous for both recessive alleles (insLQ/insLQ; C/C). The insLQ variant was not significantly associated with any of the main outcomes to COH. Carrier status for the rs4073366 C variant was associated (P = 0.033) with an increased risk (OR 2.95, 95% CI = 1.09-7.96) of developing OHSS. CONCLUSIONS: While age and day 3 FSH levels were predictive of outcome, we found no association between insLQ and patient response to COH. Interestingly, rs4073366 C variant carrier status was associated with OHSS risk. To the best of our knowledge, this is the first report suggesting that LHCGR genetic variation might function in patient risk for OHSS.

First report of warfarin dose requirements in patients possessing the CYP2C9*12 allele.

BACKGROUND: Warfarin is the most frequently prescribed anticoagulant in North America and Europe. It is administered as a racemate, but S-warfarin is principally responsible for its anticoagulant activity. Cytochrome P450 (CYP) 2C9 is the enzyme primarily responsible for the metabolism of S-warfarin. Numerous variant alleles of CYP2C9 have been identified. The CYP2C9*12 (rs9332239) allele harbors a P489S substitution in CYP2C9 which has been shown to result in a 40% decline in catalytic activity in vitro. CASES: Four Caucasian patients with a low mean weekly warfarin dose (MWWD) were genotyped for CYP2C9, VKORC1 and APOE variant alleles. None of the four patients carried the common CYP2C9 variant alleles (*2, *3, *5, *6, *7, *8, *9, *11, *13) despite a relatively low MWWD (23.4±7.94 mg) compared to 208 patients carrying the CYP29C9*1 genotype (32.2±12.65 mg). Given that CYP2C9*12 confers decreased in vitro activity to the enzyme, we investigated whether these patients carried this allele. All four patients were CYP2C9*12 CT heterozygotes. Individual comparisons with patients possessing the same VKORC1 and APOE genotypes also demonstrated lower dose requirements in the patients that possessed CYP2C9*12 allele. CONCLUSIONS: There are no reports of the clinical impact of rs9332239 on CYP2C9 substrates. This is the first report of patients with the rare CYP2C9*12 genotype and lower warfarin dose requirements.

Assessment of K-Ras mutant frequency and micronucleus incidence in the mouse duodenum following 90-days of exposure to Cr(VI) in drinking water.

Chronic exposure to high concentrations of hexavalent chromium [Cr(VI)] as sodium dichromate dihydrate (SDD) in drinking water induces duodenal tumors in mice, but the mode of action (MOA) for these tumors has been a subject of scientific debate. To evaluate the tumor-site-specific genotoxicity and cytotoxicity of SDD in the mouse small intestine, tissue pathology and cytogenetic damage were evaluated in duodenal crypt and villus enterocytes from B6C3F1 mice exposed to 0.3-520mg/L SDD in drinking water for 7 and 90 days. Allele-competitive blocker PCR (ACB-PCR) was used to investigate the induction of a sensitive, tumor-relevant mutation, specifically in vivo K-Ras codon 12 GAT mutation, in scraped duodenal epithelium following 90 days of drinking water exposure. Cytotoxicity was evident in the villus as disruption of cellular arrangement, desquamation, nuclear atypia and blunting. Following 90 days of treatment, aberrant nuclei, occurring primarily at villi tips, were significantly increased at ≥60mg/L SDD. However, in the crypt compartment, there were no dose-related effects on mitotic and apoptotic indices or the formation of aberrant nuclei indicating that Cr(VI)-induced cytotoxicity was limited to the villi. Cr(VI) caused a dose-dependent proliferative response in the duodenal crypt as evidenced by an increase in crypt area and increased number of crypt enterocytes. Spontaneous K-Ras codon 12 GAT mutations in untreated mice were higher than expected, in the range of 10(-2) to 10(-3); however no treatment-related trend in the K-Ras codon 12 GAT mutation was observed. The high spontaneous background K-Ras mutant frequency and Cr(VI) dose-related increases in crypt enterocyte proliferation, without dose-related increase in K-Ras mutant frequency, micronuclei formation, or change in mitotic or apoptotic indices, are consistent with a lack of genotoxicity in the crypt compartment, and a MOA involving accumulation of mutations late in carcinogenesis as a consequence of sustained regenerative proliferation.

The current and future state of pharmacogenomics medical education in the USA.

Healthcare professionals (e.g., physicians, physician assistants, pharmacists, nurses and genetic counselors) believe pharmacogenomics (PGx) is essential to personalized medicine; however, they still lack confidence prescribing, dosing, interacting with other healthcare professionals and counseling patients with regard to PGx. This is due to the inadequate incorporation of PGx content into professional curricula. Compared with other health professions, Doctor of Pharmacy programs have integrated more PGx content. Unlike other healthcare professionals, pharmacists have extensive training in pharmacology, drug selection, drug dosage, drug-drug interactions and are uniquely accessible to patients. We suggest pharmacists are the best poised to facilitate incorporating PGx into therapeutic decision-making. Based on our experience as undergraduate and pharmacy PGx educators, we further reflect on our experience educating future healthcare professionals on PGx.

Pharmacogenomics instruction in US and Canadian medical schools: implications for personalized medicine.

AIMS: To determine the extent of pharmacogenomics instruction at US and Canadian medical schools, characterize perceptions of curricular coverage, identify curricular resources and compare responses with similar studies conducted in US pharmacy schools and British medical schools. MATERIALS & METHODS: A survey was sent to the pharmacology department chairs of US and Canadian medical schools accredited by the Liaison Committee on Medical Education or the American Osteopathic Association's Commission on Osteopathic College Accreditation. Data were collected from July 2009 to February 2010. RESULTS: A total of 56% of eligible medical schools responded (90 out of 160). Of these schools, 82% (74 out of 90) incorporated pharmacogenomics into their curriculum. However, only 28% (21 out of 74) had more than 4 h of the required didactic pharmacogenomic coursework, and only 29% (22 out of 75) were planning to increase the number of pharmacogenomic coursework hours in the next 3 years. Pharmacogenomics coursework was most often contained within a required pharmacology course (66%; 49 out of 74) taught in the second professional year (72%; 53 out of 74). A total of 57% (44 out of 77) considered pharmacogenomics instruction at their own school as 'poor' or 'not at all adequate' while 76% (54 out of 71) considered it 'poor' or 'not at all adequate' at most medical schools. CONCLUSION: Most US and Canadian medical schools have begun to incorporate pharmacogenomics material into their curriculum; however, the extent of instruction is less than that of US pharmacy schools. To adequately prepare physicians to practice in the era of personalized medicine, medical schools should be encouraged to incorporate greater pharmacogenomic material in their curriculum.

Polo-like kinase 1 enhances survival and mutagenesis after genotoxic stress in normal cells through cell cycle checkpoint bypass.

Polo-like kinase 1 (Plk1) is a key regulator of mitosis. Aberrant Plk1 activity is found in tumors, but little is known regarding its role in the DNA damage response of normal cells and its potential contribution to the early stages of carcinogenesis. Inappropriate survival signaling after DNA damage may facilitate clonal expansion of genetically compromised cells, and it is known that protein tyrosine phosphatase (PTP) inhibitors activate key survival pathways. In this study, we employed hexavalent chromium [Cr(VI)], a well-documented genotoxicant, to investigate the mechanism by which survival pathway activation could lead to loss of checkpoint control via a mechanism involving Plk1. We recently reported that PTP inhibition enhances clonogenic survival and mutagenesis after Cr(VI) exposure by overriding Cr-induced growth arrest. Here, we report that checkpoint bypass, facilitated by PTP inhibition, was associated with decreased Cdk1 Tyr15 phosphorylation, as well as increased Plk1 activity and nuclear localization. Plk1 was necessary for increased survival after PTP inhibition and Cr(VI) exposure in normal human fibroblasts via enhanced mitotic progression. In addition, pharmacological inhibition of Plk1 abolished the PTP inhibitor-induced bypass of the G(2)/M checkpoint. Notably, Plk1 overexpression increased survival and mutagenesis after Cr(VI) exposure in wild-type Saccharomyces cerevisiae. Taken together, our data indicate that Plk1 activation and nuclear localization are necessary for PTP-regulated mitotic progression after DNA damage. Our studies highlight a role for Plk1 in the loss of checkpoint control, increased survival and mutagenesis after genotoxic exposure in normal cells, which in turn may lead to genomic instability and carcinogenesis.

Development of an undergraduate pharmacogenomics curriculum.

Pharmacogenomic biomarkers are becoming increasingly common in medicine and drug development. However, there is a genuine concern that the healthcare workforce will be ill-equipped to translate this information to clinical practice. As a result, a major effort is underway to educate future healthcare professionals on pharmacogenomics. This paper describes the development of a year-long course that aims to instill the fundamental concepts of this rapidly growing field into the minds of undergraduate students. This course offers the advantage of exposing students to the concepts of pharmacogenomics prior to their enrollment in PhD, PharmD or MD/DO graduate programs.

DNA polymerase zeta is essential for hexavalent chromium-induced mutagenesis.

Translesion synthesis (TLS) is a unique DNA damage tolerance mechanism involved in the replicative bypass of genetic lesions in favor of uninterrupted DNA replication. TLS is critical for the generation of mutations by many different chemical and physical agents, however, there is no information available regarding the role of TLS in carcinogenic metal-induced mutagenesis. Hexavalent chromium (Cr(VI))-containing compounds are highly complex genotoxins possessing both mutagenic and clastogenic activities. The focus of this work was to determine the impact that TLS has on Cr(VI)-induced mutagenesis in Saccharomyces cerevisiae. Wild-type yeast and strains deficient in TLS polymerases (i.e. Polzeta (rev3), Poleta (rad30)) were exposed to Cr(VI) and monitored for cell survival and forward mutagenesis at the CAN1 locus. In general, TLS deficiency had little impact on Cr(VI)-induced clonogenic lethality or cell growth. rad30 yeast exhibited higher levels of basal and induced mutagenesis compared to Wt and rev3 yeast. In contrast, rev3 yeast displayed attenuated Cr(VI)-induced mutagenesis. Moreover, deletion of REV3 in rad30 yeast (rad30 rev3) resulted in a significant decrease in basal and Cr(VI) mutagenesis relative to Wt and rad30 single mutants indicating that mutagenesis primarily depended upon Polzeta. Interestingly, rev3 yeast were similar to Wt yeast in susceptibility to Cr(VI)-induced frameshift mutations. Mutational analysis of the CAN1 gene revealed that Cr(VI)-induced base substitution mutations accounted for 83.9% and 100.0% of the total mutations in Wt and rev3 yeast, respectively. Insertions and deletions comprised 16.1% of the total mutations in Cr(VI) treated Wt yeast but were not observed rev3 yeast. This work provides novel information regarding the molecular mechanisms of Cr(VI)-induced mutagenesis and is the first report demonstrating a role for TLS in the fixation of mutations induced by a carcinogenic metal.