3,170 research outputs found

    Rapid Evolution of BRCA1 and BRCA2 in Humans and Other Primates

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    The maintenance of chromosomal integrity is an essential task of every living organism and cellular repair mechanisms exist to guard against insults to DNA. Given the importance of this process, it is expected that DNA repair proteins would be evolutionarily conserved, exhibiting very minimal sequence change over time. However, BRCA1, an essential gene involved in DNA repair, has been reported to be evolving rapidly despite the fact that many protein-altering mutations within this gene convey a significantly elevated risk for breast and ovarian cancers. Results: To obtain a deeper understanding of the evolutionary trajectory of BRCA1, we analyzed complete BRCA1 gene sequences from 23 primate species. We show that specific amino acid sites have experienced repeated selection for amino acid replacement over primate evolution. This selection has been focused specifically on humans and our closest living relatives, chimpanzees (Pan troglodytes) and bonobos (Pan paniscus). After examining BRCA1 polymorphisms in 7 bonobo, 44 chimpanzee, and 44 rhesus macaque (Macaca mulatta) individuals, we find considerable variation within each of these species and evidence for recent selection in chimpanzee populations. Finally, we also sequenced and analyzed BRCA2 from 24 primate species and find that this gene has also evolved under positive selection. Conclusions: While mutations leading to truncated forms of BRCA1 are clearly linked to cancer phenotypes in humans, there is also an underlying selective pressure in favor of amino acid-altering substitutions in this gene. A hypothesis where viruses are the drivers of this natural selection is discussed.National Institutes of Health R01-GM-093086, 8U42OD011197-13National Science Foundation BCS-07115972Burroughs Wellcome FundMolecular Bioscience

    Interferon, the Cell Cycle and Herpesvirus

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    An E2F1-Mediated DNA Damage Response Contributes to the Replication of Human Cytomegalovirus

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    DNA damage resulting from intrinsic or extrinsic sources activates DNA damage responses (DDRs) centered on protein kinase signaling cascades. The usual consequences of inducing DDRs include the activation of cell cycle checkpoints together with repair of the damaged DNA or induction of apoptosis. Many DNA viruses elicit host DDRs during infection and some viruses require the DDR for efficient replication. However, the mechanism by which DDRs are activated by viral infection is poorly understood. Human cytomegalovirus (HCMV) infection induces a DDR centered on the activation of ataxia telangiectasia mutated (ATM) protein kinase. Here we show that HCMV replication is compromised in cells with inactivated or depleted ATM and that ATM is essential for the host DDR early during infection. Likewise, a downstream target of ATM phosphorylation, H2AX, also contributes to viral replication. The ATM-dependent DDR is detected as discrete, nuclear γH2AX foci early in infection and can be activated by IE proteins. By 24 hpi, γH2AX is observed primarily in HCMV DNA replication compartments. We identified a role for the E2F1 transcription factor in mediating this DDR and viral replication. E2F1, but not E2F2 or E2F3, promotes the accumulation of γH2AX during HCMV infection or IE protein expression. Moreover, E2F1 expression, but not the expression of E2F2 or E2F3, is required for efficient HCMV replication. These results reveal a novel role for E2F1 in mediating an ATM-dependent DDR that contributes to viral replication. Given that E2F activity is often deregulated by infection with DNA viruses, these observations raise the possibility that an E2F1-mediated mechanism of DDR activation may be conserved among DNA viruses

    Elucidating the role of DNA damage and human cytomegalovirus in medulloblastoma and glioblastoma

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    The most common primary malignant brain tumor in children is Medulloblastoma, while Glioblastoma is the most common in adults. Treatment for both include some combination of surgery, radiation therapy, and chemotherapy. The evolution of most primary malignant brain tumors is unknown, although varying degree of genomic instability caused by defects in the DNA Damage Response (DDR) is suspected. Lately, even human cytomegalovirus (HCMV) has emerged as a suspected pathogen possibly implicated in malignant tumor evolution. Nevertheless, the causes of the chromosomal instability and its potential links with HCMV infection and/or resistance to genotoxic therapies (i.e. radiation and chemotherapy) remain largely unknown. Thus, the main aim of this PhD thesis is to investigate the role of HCMV in the context of DDR in human Medulloblastoma and Glioblastoma. In the 1st study, we turned our attention towards Glioblastoma (GBM). We examined the ability of HCMV to induce a more aggressive cancer stem cell (CSC)-like phenotype in primary GBM cell lines. HCMV infection induced a stem cell phenotype in primary GBM cell lines as determined by changes in the cellular gene expression profile and by the conferred ability of cells to grow as neurospheres in vitro, and this phenotype was prevented by treatment with the anti-viral drug ganciclovir. As CSCs are known to be resistant to chemotherapy, our results imply that HCMV may enhance the malignancy grade of the tumor, and possibly contribute to therapy resistance. In the 2nd study, we found pronounced endogenous DNA damage signaling and constitutive activation of DNA damage checkpoint kinase cascades across our medulloblastoma cohort. The bulk of the specimens also showed expression of HCMV immediate early and late proteins, in comparative analyses using three immunohistochemical protocols. Cell culture experiments validated the chronic endogenous replication stress in medulloblastoma cell lines and showed sharply differential, intriguing responses of normal cells and medulloblastoma cells to HCMV infection. Our results strongly indicate that in human medulloblastomas, the DDR checkpoint barrier is widely activated, at least in part due to replication stress. Furthermore, we propose that unorthodox the highly prevalent HCMV may impact the medulloblastoma host cell replication stress and DNA repair mechanisms. In the 3rd study, we examined cancer stem cell markers (CD133, CD15, VEGFR2) and HCMV protein expression in human medulloblastoma specimens and medulloblastoma cell lines, at the same time considering also the replication stress and DNA damage response, as cancer stem cells are often more resistant to standard-of-care radiation and chemotherapy treatments. Our immunohistochemistry analysis on clinical material identified widespread expression of the VEGFR2 receptor and CD15, yet more limited expression of CD133 compared to GBM. In addition, assessments of expression of HCMV early and late proteins have been carried out in parallel, along with cell culture experiments with HCMV infection and replication stress responses in medulloblastoma cell lines. Remarkably, we found that unlike the ‘non-stem cell’ medulloblastoma cell lines, the cell line that showed robust stemness phenotype featured a very distinct response to DNA replication stress and HCMV infection, both emerging hallmarks of brain cancers. In the 4th study, we show that HCMV infection induced replication stress (RS) and triggered host DNA damage response (DDR) in permissive and non-permissive human cells. Further, we show that undergoing standard-of-care genotoxic radiochemotherapy in patients with HCMV-positive glioblastomas correlated with elevated HCMV markers after tumor recurrence. We propose a model to explain oncomodulatory effects of HCMV, through RS induction, DDR subversion, cell death inhibition and host-cell’s genome destabilization. Our findings provide fresh insights into HCMV pathobiology and inspiration for future strategies to combine radio-chemotherapy with anti-viral drugs for cancer treatment

    Modulation of the DNA damage response during the life cycle of human papillomaviruses

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    Human papillomavirus (HPV) is the most common sexually transmitted viral infection. Infection with certain types of HPV pose a major public health risk as these types are associated with multiple human cancers, including cervical cancer, other anogenital malignancies and an increasing number of head and neck cancers. The HPV life cycle is closely tied to host cell differentiation with late viral events such as structural gene expression and viral genome amplification taking place in the upper layers of the stratified epithelium. The DNA damage response (DDR) is an elaborate signaling network of proteins that regulate the fidelity of replication by detecting, signaling and repairing DNA lesions. ATM and ATR are two kinases that are major regulators of DNA damage detection and repair. A multitude of studies indicate that activation of the ATM (Ataxia telangiectasia mutated) and ATR (Ataxia telangiectasia and Rad3-related) pathways are critical for HPV to productively replicate. This review outlines how HPV interfaces with the ATM- and ATR-dependent DNA damage responses throughout the viral life cycle to create an environment supportive of viral replication and how activation of these pathways could impact genomic stability

    Studies on early cellular responses during Epstein-Barr virus infection

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    The human gamma-herpesvirus Epstein-Barr virus (EBV) has been implicated in the pathogenesis of a broad spectrum of lymphoid and epithelial cell malignancies. A characteristic property of the virus is the capacity to establish a non-productive growthpromoting infection in B-lymphocytes. Although the induction of cell proliferation is a key feature in oncogenesis, it is not sufficient for full malignancy. In the work presented in this thesis my colleagues and I have asked whether the virus might contribute to oncogenesis by triggering additional events that are required for tumor progression. Replicative immortality is dependent on the activation of mechanisms that maintain the integrity of telomeres. Malignant cells achieve this by activating telomerase or a recombination-dependent pathway known as alternative lengthening of telomeres (ALT). We observed multiple signs of telomere dysfunction consistent with the activation of ALT in newly EBV infected Blymphocytes. These include accumulation of telomere-associated promyelocytic leukemia nuclear bodies (APBs), telomeric-sister chromatid exchange (T-SCE), and low expression of telomere associated proteins such as TRF1, TRF2, POT1, and ATRX, pointing to telomere de-protection as possible cause of telomere damage. The early phase of EBV induced B-cell immortalization is characterized by the accumulation of DNA damage and activation of a DNA damage response (DDR) that limits the efficiency of growth transformation. By comparing the response of B-lymphocytes infected with EBV or stimulated with a potent Bcell mitogen, we found that significant higher levels of damage occur in EBV infected blasts due to stronger and sustained accumulation of reactive oxygen species (ROS). Quenching of ROS did not affect the kinetics and magnitude of viral gene expression but dramatically decreased the efficiency of B-cell transformation, which correlated with selective downregulation of the viral LMP1 and the phosphorylated form of the cellular transcription factor STAT3. Analysis of the mechanism by which high levels of ROS support LMP1 expression revealed selective inhibition of viral microRNAs that target the LMP1 transcript. Viral products that are delivered to the infected cells by the incoming virions are likely to play important roles in regulating the cellular response to infection. One of such products, the large tegument protein BPLF1, is a cysteine protease with potent ubiquitin and NEDD8-specific deconjugase activities. We found that targeting of the deneddylase activity of BPLF1 to nucleus of productively infected cells requires processing of the catalytic N-terminus by caspase-1. Inhibition of caspase-1 severely impairs viral DNA synthesis and the release of infectious viruses. Collectively, the findings summarized in this thesis provide new insights on the capacity of EBV to contribute to tumor initiation and progression by triggering events, such as oxidative stress and ALT, that favor the acquisition of both genomic instability and replicative immortality. Regulation of viral functions by the cellular response to danger signals delivered by incoming virions may further contribute to the remodeling of the host cell environment allowing successful infection

    Vaccinia virus modulates the host cell cycle to promote infection

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    Vaccinia Virus (VACV) is well-known as the vaccine used for the eradication of smallpox. It serves as the model orthopox virus and has gained further clinical significance as an oncolytic virus. As a member of the poxvirus family, VACV is a double-stranded DNA virus that replicates exclusively in the cytoplasm of infected cells. Early research suggested that VACV alters the host cell cycle and inhibits cellular DNA synthesis. Later, VACV was described to modulate key cell cycle regulators during late timepoints of infection. However, the relevance of this cell cycle subversion to VACV replication and how it is achieved remains undefined. In this PhD project, I combined state of the art techniques with classical assays to determine the (viral) effector proteins, their mode of action, and the contribution of the host cell cycle to productive VACV infection. Using recombinant VACV strains, RNAi, biochemistry, and super-resolution microscopy, I demonstrate that VACV early gene expression inhibits cell proliferation after viral entry. Concurrently, the cellular CDK inhibitor p21 is upregulated, while the tumour suppressor p53 is targeted for degradation by the viral kinase B1 and/or its paralog pseudokinase B12. The second wave of viral gene expression shifts the cell cycle from G1 to S/G2/M, while still inhibiting cell proliferation. Additionally, the viral kinase F10 was shown to be necessary and sufficient to cause degradation of p21, and for activation of the cellular DNA damage response (DDR), a process known to be essential for viral DNA replication. By probing these cellular pathways with a small molecule inhibitor library I defined their requirement for the viral life cycle. Screening for defects in viral late gene expression, I found inhibition of Aurora Kinases, selected CDKs, ATR and Chk1/2 interferes with infection. Collectively, I demonstrate that VACV modulates cell cycle checkpoints and identify the viral kinases B1 and F10 as potential temporal controllers of the host cell cycle that serve to promote productive viral replication

    A systematic approach to cancer: evolution beyond selection.

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    Cancer is typically scrutinized as a pathological process characterized by chromosomal aberrations and clonal expansion subject to stochastic Darwinian selection within adaptive cellular ecosystems. Cognition based evolution is suggested as an alternative approach to cancer development and progression in which neoplastic cells of differing karyotypes and cellular lineages are assessed as self-referential agencies with purposive participation within tissue microenvironments. As distinct self-aware entities, neoplastic cells occupy unique participant/observer status within tissue ecologies. In consequence, neoplastic proliferation by clonal lineages is enhanced by the advantaged utilization of ecological resources through flexible re-connection with progenitor evolutionary stages

    HIV-1 Vpr-Mediated G2 Arrest Involves the DDB1-CUL4AVPRBP E3 Ubiquitin Ligase

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    Human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) has been shown to cause G2 cell cycle arrest in human cells by inducing ATR-mediated inactivation of p34cdc2, but factors directly engaged in this process remain unknown. We used tandem affinity purification to isolate native Vpr complexes. We found that damaged DNA binding protein 1 (DDB1), viral protein R binding protein (VPRBP), and cullin 4A (CUL4A)—components of a CUL4A E3 ubiquitin ligase complex, DDB1-CUL4AVPRBP—were able to associate with Vpr. Depletion of VPRBP by small interfering RNA impaired Vpr-mediated induction of G2 arrest. Importantly, VPRBP knockdown alone did not affect normal cell cycle progression or activation of ATR checkpoints, suggesting that the involvement of VPRBP in G2 arrest was specific to Vpr. Moreover, leucine/isoleucine-rich domain Vpr mutants impaired in their ability to interact with VPRBP and DDB1 also produced strongly attenuated G2 arrest. In contrast, G2 arrest–defective C-terminal Vpr mutants were found to maintain their ability to associate with these proteins, suggesting that the interaction of Vpr with the DDB1-VPRBP complex is necessary but not sufficient to block cell cycle progression. Overall, these results point toward a model in which Vpr could act as a connector between the DDB1-CUL4AVPRBP E3 ubiquitin ligase complex and an unknown cellular factor whose proteolysis or modulation of activity through ubiquitination would activate ATR-mediated checkpoint signaling and induce G2 arrest
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