ATR, a human phosphatidylinositol 3-kinase-related kinase, is an important component of the cellular response to DNA damage. In the present study, we evaluated the role of ATR in modulating the response of cells to S phase-associated DNA double-stranded breaks induced by topoisomerase poisons. Prolonged exposure to low doses of the topoisomerase I poison topotecan (TPT) resulted in S phase slowing because of diminished DNA synthesis at late-firing replicons. In contrast, brief TPT exposure, as well as prolonged exposure to the topoisomerase II poison etoposide, resulted in subsequent G 2 arrest. These responses were associated with phosphorylation of the checkpoint kinase Chk1. The cell cycle responses and phosphorylation of Chk1 were markedly diminished by forced overexpression of a dominant negative, kinase-inactive allele of ATR. In contrast, deficiency of the related kinase ATM had no effect on these events. The loss of ATR-dependent checkpoint function sensitized GM847 human fibroblasts to the cytotoxic effects of the topoisomerase I poisons TPT and 7-ethyl-10-hydroxycamptothecin, as assessed by inhibition of colony formation, increased trypan blue uptake, and development of apoptotic morphological changes. Expression of kdATR also sensitized GM847 cells to the cytotoxic effects of prolonged low dose etoposide and doxorubicin, albeit to a smaller extent. Collectively, these results not only suggest that ATR is important in responding to the replication-associated DNA damage from topoisomerase poisons, but also support the view that ATM and ATR have unique roles in activating the downstream kinases that participate in cell cycle checkpoints. ATR1 has been identified as one of the protein kinases that transduces signals to the cell cycle machinery during normal DNA replication (1) and after DNA damage (2-4). Like the structurally related kinases human ATM, Schizosaccharomyces pombe Rad3 (Rad3 SP ), and Saccharomyces cerevisiae Mec1 (Mec1 Sc ), ATR contains a conserved C-terminal kinase domain that phosphorylates downstream substrates (5). The nature of the DNA damage that activates ATR, the identity of its substrates, and the impact of ATR on cell cycle progression are currently the subject of extensive investigation.Previous studies have suggested that ATR and ATM might have distinct but overlapping functions. In response to IR, ATR has been observed to phosphorylate and activate the checkpoint kinase Chk1 (4), which in turn phosphorylates Cdc25c, inactivating its phosphatase activity and contributing to the ensuing G 2 arrest (6 -8). In contrast, ATM, which appears to play the more critical role in response to IR, phosphorylates Chk2 (1, 9, 10). Despite these differences, Chen et al. (11) observed that Chk1 overexpression can complement the G 2 /M checkpoint defect in AT cells and restore IR resistance. These results suggest redundancy and overlap in the specific roles of ATM and ATR.Several observations indicate that ATM and ATR are also important in the intra-S checkpoint, a series of biochemical reaction...

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Synthetic kappa-opioid receptor (KOR) agonists induce dysphoric and pro-depressive effects, and variations in the KOR (OPRK1) and prodynorphin (PDYN) genes have been shown to be associated with alcohol dependence. We genotyped 23 single nucleotide polymorphisms (SNPs) in the PDYN and OPRK1 genes in 816 alcohol dependent subjects and investigated their association with (1) negative craving measured by a subscale of the Inventory of Drug Taking Situations (IDTS); (2), a self-reported history of depression; and, (3) the intensity of depressive symptoms measured by the Beck Depression Inventory-II (BDI). In addition, 13 of the 23 PDYN and OPRK1 SNPs, which were previously genotyped in a set of 1248 controls, were used to evaluate association with alcohol dependence. SNP and haplotype tests of association were performed. Analysis of a haplotype spanning the PDYN gene (rs6045784, rs910080, rs2235751, rs2281285) revealed significant association with alcohol dependence (p=0.00079) and with negative craving (p=0.0499). A candidate haplotype containing the PDYN rs2281285-rs1997794 SNPs that was previously associated with alcohol dependence was also associated with negative craving (p=0.024) and alcohol dependence (p=0.0008) in this study. A trend for association between depression severity and PDYN variation was detected. No associations of OPRK1 gene variation with alcohol dependence or other studied phenotypes were found. These findings support the hypothesis that sequence variation in the PDYN gene contributes to both alcohol dependence and the induction of negative craving in alcohol dependent subjects.

Limonene and related monoterpenes have been shown to impair the incorporation of mevalonic acid-derived isoprene compounds, that is farnesyl pyrophosphate, into RAS and RAS-related proteins. As farnesylation is critical for RAS's membrane localization and function, the isoprenylation pathways have received attention as potential targets of anti-RAS pharmacologic maneuvers. We have expanded on these prior studies and demonstrate that one of limonene's metabolic derivatives, perillyl alcohol, decreases the levels of antigenic RAS in the human-derived myeloid THP-1 and lymphoid RPMI-8402 cell lines. Both limonene and perillyl alcohol decrease levels of [35S]methionine-labeled RAS proteins in cells that have been pulsed with radiolabeled methionine for 4 h. In contrast, lovastatin, which inhibits hydroxymethylglutaryl coenzyme A reductase and thus depletes cells of farnesyl pyrophosphate, does not diminish levels of total antigenic RAS but rather results in a shift in the RAS protein; levels of farnesylated RAS decrease whereas levels of unmodified/unfarnesylated RAS increase. As limonene and perillyl alcohol do not induce such a shift, we conclude that these monoterpenes decrease farnesylated RAS protein levels by a mechanism that is clearly distinct from that of either depleting cells of farnesyl pyrophosphate or inhibiting the enzyme farnesyl protein transferase that catalyzes the post-translational farnesylation of RAS. Perillyl alcohol decreases antigenic RAS levels but does not decrease levels of another membrane-tethered protein, the alpha subunit of the heterotrimeric G protein. Furthermore, perillyl alcohol decreases the levels of radiolabeled methionine incorporated into immunoprecipitable RAS to a greater extent than it decreases radiolabeled methionine incorporated into total cellular protein. Thus there is some degree of specificity for the activity of perillyl alcohol to depress RAS levels.