Cyrus Vaziri

A p53-Dependent Checkpoint Pathway Prevents Rereplication

Eukaryotic cells control the initiation of DNA replication so that origins that have fired once in S phase do not fire a second time within the same cell cycle. Failure to exert this control leads to genetic instability. Here we investigate how rereplication is prevented in normal mammalian cells and how these mechanisms might be overcome during tumor progression. Overexpression of the replication initiation factors Cdt1 and Cdc6 along with cyclin A-cdk2 promotes rereplication in human cancer cells with inactive p53 but not in cells with functional p53. A subset of origins distributed throughout the genome refire within 2-4 hr of the first cycle of replication. Induction of rereplication activates p53 through the ATM/ATR/Chk2 DNA damage checkpoint pathways. p53 inhibits rereplication through the induction of the cdk2 inhibitor p21. Therefore, a p53-dependent checkpoint pathway is activated to suppress rereplication and promote genetic stability.

MicroRNAs as modulators of smoking-induced gene expression changes in human airway epithelium

We have shown that smoking impacts bronchial airway gene expression and that heterogeneity in this response associates with smoking-related disease risk. In this study, we sought to determine whether microRNAs (miRNAs) play a role in regulating the airway gene expression response to smoking. We examined whole-genome miRNA and mRNA expression in bronchial airway epithelium from current and never smokers (n = 20) and found 28 miRNAs to be differentially expressed (P < 0.05) with the majority being down-regulated in smokers. We further identified a number of mRNAs whose expression level is highly inversely correlated with miRNA expression in vivo. Many of these mRNAs contain potential binding sites for the differentially expressed miRNAs in their 3′-untranslated region (UTR) and are themselves affected by smoking. We found that either increasing or decreasing the levels of mir-218 (a miRNA that is strongly affected by smoking) in both primary bronchial epithelial cells and H1299 cells was sufficient to cause a corresponding decrease or increase in the expression of predicted mir-218 mRNA targets, respectively. Further, mir-218 expression is reduced in primary bronchial epithelium exposed to cigarette smoke condensate (CSC), and alteration of mir-218 levels in these cells diminishes the induction of the predicted mir-218 target MAFG in response to CSC. These data indicate that mir-218 levels modulate the airway epithelial gene expression response to cigarette smoke and support a role for miRNAs in regulating host response to environmental toxins.

ATR inhibition selectively sensitizes G1 checkpoint-deficient cells to lethal premature chromatin condensation.

Premature chromatin condensation (PCC) is a hallmark of mammalian cells that begin mitosis before completing DNA replication. This lethal event is prevented by a highly conserved checkpoint involving an unknown, caffeine-sensitive mediator. Here, we have examined the possible involvement of the caffeine-sensitive ATM and ATR protein kinases in this checkpoint. We show that caffeines ability to inhibit ATR (but not ATM) causes PCC, that ATR (but not ATM) prevents PCC, and that ATR prevents PCC via Chk-1 regulation. Moreover, mimicking cancer cell phenotypes by disrupting normal G1 checkpoints sensitizes cells to PCC by ATR inhibition plus low-dose DNA damage. Notably, loss of p53 function potently sensitizes cells to PCC caused by ATR inhibition by a small molecule. We present a molecular model for how ATR prevents PCC and suggest that ATR represents an attractive therapeutic target for selectively killing cancer cells by premature chromatin condensation.

Rad18 Regulates DNA Polymerase κ and Is Required for Recovery from S-Phase Checkpoint-Mediated Arrest

ABSTRACT We have investigated mechanisms that recruit the translesion synthesis (TLS) DNA polymerase Polκ to stalled replication forks. The DNA polymerase processivity factor PCNA is monoubiquitinated and interacts with Polκ in cells treated with the bulky adduct-forming genotoxin benzo[a]pyrene dihydrodiol epoxide (BPDE). A monoubiquitination-defective mutant form of PCNA fails to interact with Polκ. Small interfering RNA-mediated downregulation of the E3 ligase Rad18 inhibits BPDE-induced PCNA ubiquitination and association between PCNA and Polκ. Conversely, overexpressed Rad18 induces PCNA ubiquitination and association between PCNA and Polκ in a DNA damage-independent manner. Therefore, association of Polκ with PCNA is regulated by Rad18-mediated PCNA ubiquitination. Cells from Rad18−/− transgenic mice show defective recovery from BPDE-induced S-phase checkpoints. In Rad18−/− cells, BPDE induces elevated and persistent activation of checkpoint kinases, indicating persistently stalled forks due to defective TLS. Rad18-deficient cells show reduced viability after BPDE challenge compared with wild-type cells (but survival after hydroxyurea or ionizing radiation treatment is unaffected by Rad18 deficiency). Inhibition of RPA/ATR/Chk1-mediated S-phase checkpoint signaling partially inhibited BPDE-induced PCNA ubiquitination and prevented interactions between PCNA and Polκ. Taken together, our results indicate that ATR/Chk1 signaling is required for Rad18-mediated PCNA monoubiquitination. Recruitment of Polκ to ubiquitinated PCNA enables lesion bypass and eliminates stalled forks, thereby attenuating the S-phase checkpoint.

A Benzo[a]pyrene-induced Cell Cycle Checkpoint Resulting in p53-independent G1 Arrest in 3T3 Fibroblasts

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor of the basic helix-loop-helix family. Although physiological ligands for the AhR have not been identified, carcinogenic polycyclic aromatic hydrocarbons such as Benzo[a]pyrene (B[a]P) are high affinity AhR ligands that induce nuclear translocation and sequence-specific DNA binding of the AhR. AhR-regulated genes include members of the cytochrome P-450 family that are known to oxidize B[a]P to form genotoxic (DNA-damaging) metabolites. Murine Swiss 3T3 cells express high levels of AhR. Treatment of Swiss 3T3 cells with B[a]P during the G1 phase of the cell cycle resulted in growth arrest, as shown by inhibition of growth factor-stimulated DNA synthesis. By contrast, other murine 3T3 fibroblasts not expressing detectable levels of AhR did not undergo growth arrest in response to B[a]P. The AhR antagonist α-naphthoflavone prevented B[a]P-induced growth arrest, further demonstrating that cessation of cell growth was mediated by the activated AhR. A nongenotoxic AhR ligand (2,3,7,8-tetrachlorodibenzo-p-dioxin) did not elicit growth arrest, showing that ligand activation of the AhR alone was insufficient to block cell cycle progression. However, genomic DNA from B[a]P-treated Swiss 3T3 cells contained covalent adducts, whereas that from 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated cells did not, showing that G1 arrest correlated with DNA damage resulting from genotoxic B[a]P metabolites. B[a]P-induced DNA damage and growth arrest was coincident with elevated levels of nuclear p53 protein and induction of the p53-regulated mdm-2 proto-oncogene. However, Swiss 3T3 fibroblasts expressing “dominant negative” mutant p53, as well as primary fibroblasts from p53−/− “knockout” mice, also underwent growth arrest in response to B[a]P. Therefore, B[a]P-induced growth arrest occurs via p53-independent mechanisms.

The Chk1-mediated S-phase checkpoint targets initiation factor Cdc45 via a Cdc25A/Cdk2-independent mechanism.

DNA damage induced by the carcinogen benzo[a]pyrene dihydrodiol epoxide (BPDE) induces a Chk1-dependent S-phase checkpoint. Here, we have investigated the molecular basis of BPDE-induced S-phase arrest. Chk1-dependent inhibition of DNA synthesis in BPDE-treated cells occurred without detectable changes in Cdc25A levels, Cdk2 activity, or Cdc7/Dbf4 interaction. Overexpression studies showed that Cdc25A, cyclin A/Cdk2, and Cdc7/Dbf4 were not rate-limiting for DNA synthesis when the BPDE-induced S-phase checkpoint was active. To investigate other potential targets of the S-phase checkpoint, we tested the effects of BPDE on the chromatin association of DNA replication factors. The levels of chromatin-associated Cdc45 (but not soluble Cdc45) were reduced concomitantly with BPDE-induced Chk1 activation and inhibition of DNA synthesis. The chromatin association of Mcm7, Mcm10, and proliferating cell nuclear antigen was unaffected by BPDE treatment. However, the association between Mcm7 and Cdc45 in the chromatin fraction was inhibited in BPDE-treated cells. Chromatin immunoprecipitation analyses demonstrated reduced association of Cdc45 with the β-globin origin of replication in BPDE-treated cells. The inhibitory effects of BPDE on DNA synthesis, Cdc45/Mcm7 associations, and interactions between Cdc45 and the β-globin locus were abrogated by the Chk1 inhibitor UCN-01. Taken together, our results show that the association between Cdc45 and Mcm7 at origins of replication is negatively regulated by Chk1 in a Cdk2-independent manner. Therefore, Cdc45 is likely to be an important target of the Chk1-mediated S-phase checkpoint.

The novel SPARC family member SMOC-2 potentiates angiogenic growth factor activity

SMOC-2 is a novel member of the SPARC family of matricellular proteins. The purpose of this study was to determine whether SMOC-2 can modulate angiogenic growth factor activity and angiogenesis. SMOC-2 was localized in the extracellular periphery of cultured human umbilical vein endothelial cells (HUVECs). Ectopically expressed SMOC-2 was also secreted into the tissue culture medium. In microarray profiling experiments, a recombinant SMOC-2 adenovirus induced the expression of transcripts required for cell cycle progression in HUVECs. Consistent with a growth-stimulatory role for SMOC-2, its overexpression stimulated DNA synthesis in a dose-dependent manner. Overexpressed SMOC-2 also synergized with vascular endothelial growth factor or with basic fibroblast growth factor to stimulate DNA synthesis. Ectopically expressed SMOC-2 stimulated formation of network-like structures as determined by in vitro matrigel angiogenesis assays. Fetal calf serum enhanced the stimulatory effect of overexpressed SMOC-2 in this assay. Conversely, small interference RNA directed toward SMOC-2 inhibited network formation and proliferation. The angiogenic activity of SMOC-2 was also examined in experimental mice by subdermal implantation of Matrigel® plugs containing SMOC-2 adenovirus. SMOC-2 adenovirus induced a 3-fold increase in the number of cells invading Matrigel® plugs when compared with a control adenoviral vector. Basic fibroblast growth factor and SMOC-2 elicited a synergistic effect on cell invasion. Taken together, our results demonstrate that SMOC-2 is a novel angiogenic factor that potentiates angiogenic effects of growth factors.

Critical role for mouse Hus1 in an S-phase DNA damage cell cycle checkpoint.

ABSTRACT Mouse Hus1 encodes an evolutionarily conserved DNA damage response protein. In this study we examined how targeted deletion of Hus1 affects cell cycle checkpoint responses to genotoxic stress. Unlike hus1− fission yeast (Schizosaccharomyces pombe) cells, which are defective for the G2/M DNA damage checkpoint, Hus1-null mouse cells did not inappropriately enter mitosis following genotoxin treatment. However, Hus1-deficient cells displayed a striking S-phase DNA damage checkpoint defect. Whereas wild-type cells transiently repressed DNA replication in response to benzo(a)pyrene dihydrodiol epoxide (BPDE), a genotoxin that causes bulky DNA adducts, Hus1-null cells maintained relatively high levels of DNA synthesis following treatment with this agent. However, when treated with DNA strand break-inducing agents such as ionizing radiation (IR), Hus1-deficient cells showed intact S-phase checkpoint responses. Conversely, checkpoint-mediated inhibition of DNA synthesis in response to BPDE did not require NBS1, a component of the IR-responsive S-phase checkpoint pathway. Taken together, these results demonstrate that Hus1 is required specifically for one of two separable mammalian checkpoint pathways that respond to distinct forms of genome damage during S phase.

Identification of a novel REV1-interacting motif necessary for DNA polymerase κ function

When a replicative DNA polymerase (Pol) is stalled by damaged DNA, a “polymerase switch” recruits specialized translesion synthesis (TLS) DNA polymerase(s) to sites of damage. Mammalian cells have several TLS DNA polymerases, including the four Y‐family enzymes (Polη, Polι, Polκ and REV1) that share multiple primary sequence motifs, but show preferential bypass of different DNA lesions. REV1 interacts with Polη, Polι, and Polκ and therefore appears to play a central role during TLS in vivo. Here we have investigated the molecular basis for interactions between REV1 and Polκ. We have identified novel REV1‐interacting regions (RIRs) present in Polκ, Polι and Polη. Within the RIRs, the presence of two consecutive phenylalanines (FF) is essential for REV1‐binding. The consensus sequence for REV1‐binding is denoted by x‐x‐x‐F‐F‐y‐y‐y‐y (x, no specific residue and y, no specific residue but not proline). Our results identify structural requirements that are necessary for FF‐flanking residues to confer interactions with REV1. A Polκ mutant lacking REV1‐binding activity did not complement the genotoxin‐sensitivity of Polk‐null mouse embryonic fibroblast cells, thereby demonstrating that the REV1‐interaction is essential for Polκ function in vivo.

Chromatin unfolding by Cdt1 regulates MCM loading via opposing functions of HBO1 and HDAC11-geminin.

The efficiency of metazoan origins of DNA replication is known to be enhanced by histone acetylation near origins. Although this correlates with increased MCM recruitment, the mechanism by which such acetylation regulates MCM loading is unknown. We show here that Cdt1 induces large scale chromatin decondensation that is required for MCM recruitment. This process occurs in G1, is suppressed by Geminin, and requires HBO1 HAT activity and histone H4 modifications. HDAC11, which binds Cdt1 and replication origins during S-phase, potently inhibits Cdt1-induced chromatin unfolding and re-replication, suppresses MCM loading, and binds Cdt1 more efficiently in the presence of Geminin. We also demonstrate that chromatin at endogenous origins is more accessible in G1 relative to S-phase. These results provide evidence that histone acetylation promotes MCM loading via enhanced chromatin accessibility. This process is regulated positively by Cdt1 and HBO1 in G1 and repressed by Geminin-HDAC11 association with Cdt1 in S-phase, and represents a novel form of replication licensing control.