Faculty Publications

Replicative DNA polymerases possess 3' --> 5' exonuclease activity to reduce misincorporation of incorrect nucleotides by proofreading during replication. To examine if this proofreading activity modulates DNA synthesis of damaged templates, we constructed a series of recombinant human DNA polymerase delta (Pol delta) in which one or two of the three conserved Asp residues in the exonuclease domain are mutated, and compared their properties with that of the wild-type enzyme. While all the mutant enzymes lost more than 95% exonuclease activity and severely decreased the proofreading activity than the wild-type, the bypass efficiency of damaged templates was varied: two mutant enzymes, D515V and D402A/D515A, gave higher bypass efficiencies on templates containing an abasic site, but another mutant, D316N/D515A, showed a lower bypass efficiency than the wild-type. All the enzymes including the wild-type inserted an adenine opposite the abasic site, whereas these enzymes inserted cytosine and adenine opposite an 8-oxoguanine with a ratio of 6:4. These results indicate that the exonuclease activity of human Pol delta modulates its intrinsic bypass efficiency on the damaged template, but does not affect the choice of nucleotide to be inserted.

Long-term exposure to estrogens influences the development of breast cancer in women, but the precise mechanisms involved are not clearly defined. Our working hypothesis is that estrogen modulates this process by two separate processes. One involves the binding of estradiol to estrogen receptor (ER) alpha with stimulation of cell proliferation. Errors in DNA occurring during replication result in fixed mutations when not repaired. The other process results from the formation of genotoxic metabolites of estradiol, which can bind to DNA, cause depurination, and result in mutations. Our collaborative group, funded by a Department of Defense Center of Excellence grant, has examined this hypothesis using a variety of experimental methods. Estradiol and its catechol-estrogen metabolite 4-OH-estradiol causes mutations in cell culture systems and can transform benign MCF-10F cells, allowing them to cause tumors in SCID mice. We have demonstrated loss of heterozygosity and gains and losses of DNA segments by comparative genomic hybridization methodology. The depurinated estradiol-guanine and -adenine adducts are measurable in MCF-7 breast cancer cells in culture and in mouse mammary tissue. The double transgenic, alpha estrogen receptor knockout/Wnt-1 knockin mouse model allows us to dissect out the separate effects of ER-mediated and ER-independent actions of estradiol. Knock out of the ER alpha delays the onset of breast tumors in this model, demonstrating a role of receptor-mediated actions. Oophorectomy delays the onset of tumors and reduces overall incidence, providing evidence for an ER-independent effect. Taken together, these data suggest that both ER-dependent and genotoxic ER-independent effects of estradiol mediate breast cancer development.

Testicular germ cell tumors (TGCT) have been expected to have a strong underlying genetic component. We conducted a genome-wide scan among 277 TGCT cases and 919 controls and found that seven markers at 12p22 within KITLG (c-KIT ligand) reached genome-wide significance (P < 5.0 x 10 (-8) in discovery). In independent replication, TGCT risk was increased threefold per copy of the major allele at rs3782179 and rs4474514 (OR = 3.08, 95% CI = 2.29-4.13; OR = 3.07, 95% CI 2.29-4.13, respectively). We found associations with rs4324715 and rs6897876 at 5q31.3 near SPRY4 (sprouty 4; P < 5.0 x 10(-6) in discovery). In independent replication, risk of TGCT was increased nearly 40% per copy of the major allele (OR = 1.37, 95% CI = 1.14-1.64; OR = 1.39, 95% CI = 1.16-1.66, respectively). All of the genotypes were associated with both seminoma and nonseminoma TGCT subtypes. These results demonstrate that common genetic variants affect TGCT risk and implicate KITLG and SPRY4 as genes involved in TGCT susceptibility.

Background: Data protection is important for all information systems that deal with human-subjects data. Grid-based systems - such as the cancer Biomedical Informatics Grid (caBIG) - seek to develop new mechanisms to facilitate real-time federation of cancer-relevant data sources, including sources protected under a variety of regulatory laws, such as HIPAA and 21CFR11. These systems embody new models for data sharing, and hence pose new challenges to the regulatory community, and to those who would develop or adopt them. These challenges must be understood by both systems developers and system adopters. In this paper, we describe our work collecting policy statements, expectations, and requirements from regulatory decision makers at academic cancer centers in the United States. We use these statements to examine fundamental assumptions regarding data sharing using data federations and grid computing. Methods: An interview-based study of key stakeholders from a sample of US cancer centers. Interviews were structured, and used an instrument that was developed for the purpose of this study. The instrument included a set of problem scenarios - difficult policy situations that were derived during a full-day discussion of potentially problematic issues by a set of project participants with diverse expertise. Each problem scenario included a set of open-ended questions that were designed to elucidate stakeholder opinions and concerns. Interviews were transcribed verbatim and used for both qualitative and quantitative analysis. For quantitative analysis, data was aggregated at the individual or institutional unit of analysis, depending on the specific interview question. Results: Thirty-one (31) individuals at six cancer centers were contacted to participate. Twenty-four out of thirty-one (24/31) individuals responded to our request-yielding a total response rate of 77%. Respondents included IRB directors and policy-makers, privacy and security officers, directors of offices of research, information security officers and university legal counsel. Nineteen total interviews were conducted over a period of 16 weeks. Respondents provided answers for all four scenarios (a total of 87 questions). Results were grouped by broad themes, including among others: governance, legal and financial issues, partnership agreements, de-identification, institutional technical infrastructure for security and privacy protection, training, risk management, auditing, IRB issues, and patient/subject consent. Conclusion: The findings suggest that with additional work, large scale federated sharing of data within a regulated environment is possible. A key challenge is developing suitable models for authentication and authorization practices within a federated environment. Authentication - the recognition and validation of a person's identity - is in fact a global property of such systems, while authorization - the permission to access data or resources - mimics data sharing agreements in being best served at a local level. Nine specific recommendations result from the work and are discussed in detail. These include: (1) the necessity to construct separate legal or corporate entities for governance of federated sharing initiatives on this scale; (2) consensus on the treatment of foreign and commercial partnerships; (3) the development of risk models and risk management processes; (4) development of technical infrastructure to support the credentialing process associated with research including human subjects; (5) exploring the feasibility of developing large-scale, federated honest broker approaches; (6) the development of suitable, federated identity provisioning processes to support federated authentication and authorization; (7) community development of requisite HIPAA and research ethics training modules by federation members; (8) the recognition of the need for central auditing requirements and authority, and; (9) use of two-protocol data exchange models where possible in the federation.

BACKGROUND: Flow cytometry technology is widely used in both health care and research. The rapid expansion of flow cytometry applications has outpaced the development of data storage and analysis tools. Collaborative efforts being taken to eliminate this gap include building common vocabularies and ontologies, designing generic data models, and defining data exchange formats. The Minimum Information about a Flow Cytometry Experiment (MIFlowCyt) standard was recently adopted by the International Society for Advancement of Cytometry. This standard guides researchers on the information that should be included in peer reviewed publications, but it is insufficient for data exchange and integration between computational systems. The Functional Genomics Experiment (FuGE) formalizes common aspects of comprehensive and high throughput experiments across different biological technologies. We have extended FuGE object model to accommodate flow cytometry data and metadata. METHODS: We used the MagicDraw modelling tool to design a UML model (Flow-OM) according to the FuGE extension guidelines and the AndroMDA toolkit to transform the model to a markup language (Flow-ML). We mapped each MIFlowCyt term to either an existing FuGE class or to a new FuGEFlow class. The development environment was validated by comparing the official FuGE XSD to the schema we generated from the FuGE object model using our configuration. After the Flow-OM model was completed, the final version of the Flow-ML was generated and validated against an example MIFlowCyt compliant experiment description. RESULTS: The extension of FuGE for flow cytometry has resulted in a generic FuGE-compliant data model (FuGEFlow), which accommodates and links together all information required by MIFlowCyt. The FuGEFlow model can be used to build software and databases using FuGE software toolkits to facilitate automated exchange and manipulation of potentially large flow cytometry experimental data sets. Additional project documentation, including reusable design patterns and a guide for setting up a development environment, was contributed back to the FuGE project. CONCLUSION: We have shown that an extension of FuGE can be used to transform minimum information requirements in natural language to markup language in XML. Extending FuGE required significant effort, but in our experiences the benefits outweighed the costs. The FuGEFlow is expected to play a central role in describing flow cytometry experiments and ultimately facilitating data exchange including public flow cytometry repositories currently under development.

A core set of oncoproteins is overexpressed or functionally activated in many types of cancer, and members of this group have attracted significant interest as subjects for development of targeted therapeutics. For some oncoproteins such as EGFR/ErbB1, both small molecule and antibody agents have been developed and applied in the clinic for over a decade. Analysis of clinical outcomes has revealed an initially unexpected complexity in the response of patients to these agents. Diverse factors, including developmental lineage of the tumor progenitor cell, co-mutation or epigenetic modulation of genes encoding proteins in an extended EGFR signaling network or regulating core survival responses in individual tumors, and environmental factors including inflammatory agents and viral infection, all have been identified as modulating response to treatment with EGFR-targeted drugs. Second and third generation therapeutic strategies increasingly incorporate knowledge of cancer type-specific signaling environments, in a more personalized treatment approach. This review takes squamous cell carcinoma of the head and neck (SCCHN) as a specific example of an EGFR-involved cancer with idiosyncratic biological features that influence design of treatment modalities, with particular emphasis on commonalities and differences with other cancer types.

The tumor suppressor p53 induces apoptosis by altering the transcription of pro-apoptotic targets in the nucleus and by a direct, nontranscriptional role at the mitochondria. Although the post-translational modifications regulating nuclear apoptotic functions of p53 have been thoroughly characterized, little is known of how transcription-independent functions are controlled. We and others identified acetylation of the p53 DNA binding domain at lysine 120 as a critical event in apoptosis induction. Although initial studies showed that Lys-120 acetylation plays a role in p53 function in the nucleus, we report here a role for Lys-120 acetylation in transcription-independent apoptosis. We demonstrate that the Lys-120-acetylated isoform of p53 is enriched at mitochondria. The acetylation of Lys-120 does not appear to regulate the ability of p53 to interact with the pro-apoptotic proteins BCL-XL and BAK. However, displacement of the inhibitory MCL-1 protein from BAK is compromised when Lys-120 acetylation is blocked. Functional studies show that mutation of Lys-120 to a nonacetylated residue, as occurs in human cancer, inhibits transcription-independent apoptosis, and enforced acetylation of Lys-120 enhances transcription-independent apoptosis. These data support a model whereby Lys-120 acetylation contributes to both the transcription-dependent and -independent apoptotic pathways induced by p53.

Previous studies have identified interleukin 6 (IL-6) as an important cytokine with prognostic significance in ovarian cancer. Activation of the IL-6- Stat3 pathway contributes to tumor cell growth, survival and drug resistance in several cancers, including ovarian cancer. To explore potential therapeutic strategies for interrupting signaling through this pathway, we assessed the ability of CDDO-Me, a synthetic triterpenoid, to inhibit IL-6 secretion, Stat3 phosphorylation, Stat3 nuclear translocation and paclitaxel sensitivity in several cell line model systems. These studies demonstrated that CDDO-Me significantly inhibits IL-6 secretion in paclitaxel-resistant ovarian cancer cells and specifically suppresses IL-6- or oncostatin M-induced Stat3 nuclear translocation. Treatment with CDDO-Me significantly decreases the levels of Stat3, Jak2, and Src phosphorylation in ovarian and breast cancer cell lines with constitutively activated Stat3. This inhibition of the IL-6-Stat3 pathway correlated with suppression of the anti-apoptotic Stat3 target genes Bcl-XL, survivin, and Mcl-1, and with apoptosis induction as measured by monitoring PARP and its cleavage product, as well as by quantitative measurement of the apoptosis-associated CK18Asp396. Furthermore, CDDO-Me increases the cytotoxic effects of paclitaxel in the paclitaxel-resistant ovarian cancer cell line OVCAR8(TR) (2 to 5-fold) and of cisplatin in the cisplatin-resistant ovarian cancer cell line A2780cp70 (2 to 4-fold). Our data confirm that CDDO-Me interrupts the signaling of multiple kinases involved in the IL-6-Stat3 and Src signaling pathways. Inhibition is likely achieved through multiple points within these pathways. In a model system of established acquired drug resistance, CCDO- Me is effective at partially reversing the drug-resistance phenotype.

Objective. The primary objective of the study was to prospectively assess quality of life (QOL) among women at increased risk of ovarian cancer who are undergoing risk-reducing salpingo-oophorectomy (RRSO) or serial screening. Methods. Women at increased risk of ovarian cancer who were undergoing RRSO were recruited into the study. At-risk women undergoing serial screening for early detection of ovarian cancer served as a comparison group. Participants completed measures of QOL, sexual functioning, body image, depressive symptoms, and a symptom checklist at baseline (prior to surgery for women obtaining RRSO), and then at 1-month, 6-months, and 12-months post baseline. Results. Women who underwent surgery reported poorer physical functioning, more physical role limitations, greater pain, less vitality, poorer social functioning, and greater discomfort and less satisfaction with sexual activities at 1-month assessment compared to baseline. In contrast, women undergoing screening experienced no significant decrements in QOL or sexual functioning at 1-month assessment. Most QOL deficits observed in the surgical group were no longer apparent by 6-month assessment. Women in the surgery group were more likely to report hot flashes and vaginal dryness, but over time, symptoms of vaginal discomfort decreased to a greater extent in women who had RRSO compared to women undergoing screening. No differences in body image or depressive symptoms were observed between the two groups at any time point. Conclusions. Short-term deficits in physical functioning and other specific domains of QOL were observed following RRSO, but most women recovered baseline functioning by 6- and 12-month assessments. Issues regarding the potential impact of surgery on short-term sexual functioning should be considered and weighed carefully, particularly among younger women. (C) 2008 Elsevier Inc. All rights reserved.

BACKGROUND: The family of zinc finger-containing GATA transcription factors plays critical roles in cell lineage specification during early embryonic development and organ formation. GATA4 and GATA6 were found to be frequently lost in ovarian cancer, and the loss is proposed to account for dedifferentiation of the cancer cells. METHODOLOGY/PRINCIPAL FINDINGS: We further investigated the expression of GATA4 and GATA6 in ovarian surface epithelial lesions and histological subtypes of ovarian carcinomas by immunostaining. GATA4 and GATA6 were found to be absent in high percentages (80 to 90%) of serous, clear cell, and endometrioid ovarian cancer examined. In contrast, both were found positive in 11 out of 12 cases of mucinous carcinomas, suggesting the expression of the GATA factors can distinguish mucinous cancer from other histological subtypes. GATA4 was frequently lost in preneoplastic lesions such as morphologically normal inclusion cysts and epithelial hyperplasia adjacent to malignant cells. The loss of GATA6 correlates closely with neoplastic morphological transformation of ovarian surface epithelia. In culture, GATA4 expression was progressively reduced upon passaging primary ovarian surface epithelial cells, which correlated with changes in histone modification of the GATA4 locus. A reduced GATA6 gene dosage as in GATA6 (+/-) mice led to an increased pre-neoplastic changes and inclusion cysts in the ovaries, suggesting the loss of GATA6 contributes to ovarian cancer development. CONCLUSIONS/SIGNIFICANCE: This study suggests that the expression status of GATA4 and GATA6 may dictate distinct pathologic pathways leading to serous or mucinous ovarian carcinomas. The readily loss of GATA4 expression through changes in chromatin conformation suggests a potential non-phenotypic initiating event, leading to subsequent loss of GATA6, morphological transformation, and ultimate tumorigenesis.

"Race-specific" prostate-specific antigen (PSA) needs evaluation in men at high risk for prostate cancer for optimizing early detection. Baseline PSA and longitudinal prediction for prostate cancer were examined by self-reported race and genetic West African (WA) ancestry in the Prostate Cancer Risk Assessment Program, a prospective high-risk cohort. Eligibility criteria were age 35 to 69 years, family history of prostate cancer, African American race, or BRCA1/2 mutations. Biopsies were done at low PSA values (<4.0 ng/mL). WA ancestry was discerned by genotyping 100 ancestry informative markers. Cox proportional hazards models evaluated baseline PSA, self-reported race, and genetic WA ancestry. Cox models were used for 3-year predictions for prostate cancer. Six hundred forty-six men (63% African American) were analyzed. Individual WA ancestry estimates varied widely among self-reported African American men. Race-specific differences in baseline PSA were not found by self-reported race or genetic WA ancestry. Among men with >= 1 follow-up visit (405 total, 54% African American), 3-year prediction for prostate cancer with a PSA of 1.5 <4.0 ng/mL was higher in African American men with age in the model (P = 0.025) compared with European American men. Hazard ratios of PSA for prostate cancer were also higher by self-reported race (1.59 for African American versus 1.32 for European American, P = 0.04). There was a trend for increasing prediction for prostate cancer with increasing genetic WA ancestry. "Race-specific" PSA may need to be redefined as higher prediction for prostate cancer at any given PSA in African American men. Large-scale studies are needed to confirm if genetic WA ancestry explains these findings to make progress in personalizing prostate cancer early detection.