Validating the Prognostic Utility of the ABCD-GENE Score in Asian Patients with Acute Coronary Syndrome Patients on Clopidogrel.

Background: The ABCD-GENE score, which links cytochrome P450 2C19 (CYP2C19) phenotype and high platelet reactivity (HPR) to the risk of major adverse cardiovascular events (MACE) in clopidogrel users, has been validated in white and Japanese populations. The prognostic implications of the score in other Asian cohorts, however, have been largely unchartered. The aim of this study was to validate the prognostic utility of the ABCD-GENE score in a heterogeneous Asian acute coronary syndrome (ACS) cohort. Methods and Results: In this single-centre, retrospective cohort evaluation of 423 ACS patients, the objectives were to characterise the best cut-off score for MACE prognostication by comparing the adjusted 1-year risk of MACE between groups above and below the candidate cut-off scores using Cox regression; and for on-clopidogrel HPR prediction using receiver operating characteristic (ROC) analysis and Youden's index. In the adjusted Cox model, an ABCD-GENE score cut-off at 10 points significantly predicts the 1-year risk of MACE (adjusted HR 3.771; 95% CI [1.041-13.661]). Female sex, baseline LDL, history of ACS and angiotensin receptor blocker use were additional independent predictors of MACE. On ROC analysis the ideal cut-off for HPR prediction was 7 points. However, that did not independently predict the 1-year risk of MACE (adjusted HR 1.595; 95% CI [0.425-5.989]). Conclusion: The original ABCD-GENE score 10-point cut-off moderately predicts MACE in a heterogeneous, Asian ACS population at 1 year. Additional predictors of MACE were also identified in the present cohort, and these findings should be prospectively validated in larger ACS cohorts.

Identification of delta helicase as the bovine homolog of HUPF1: demonstration of an interaction with the third subunit of DNA polymerase delta.

Delta helicase is a 5' to 3' DNA helicase that partially co-purifies with DNA polymerase delta (pol delta) from fetal bovine thymus tissue. We describe the resolution of delta helicase from pol delta on heparin-agarose chromatography and its purification to apparent homogeneity by affinity purification on single-stranded DNA-cellulose chromatography, unique-sequence RNA-agarose chromatography, and ceramic hydroxyapatite chromatography. Delta helicase isolated from fetal bovine thymus had an apparent M(r) of 115 kDa in SDS-PAGE, and photo-crosslinked to [alpha-32P]ATP. Tandem mass spectrometry peptide mass data derived from the bovine polypeptide matched to human UPF1 (HUPF1), a 5' to 3' RNA and DNA helicase, and a requisite component of the mRNA surveillance complex. Antisera against HUPF1 cross-reacted with delta helicase on western analysis, and delta helicase activity was immunoinactivated by pre-incubation with antibodies to HUPF1, suggesting that delta helicase is the bovine homolog of HUPF1. Immunoprecipitation experiments demonstrated that HUPF1 interacts with the 66-kDa third subunit of pol delta in vivo.

p27 phosphorylation by Src regulates inhibition of cyclin E-Cdk2.

The kinase inhibitor p27Kip1 regulates the G1 cell cycle phase. Here, we present data indicating that the oncogenic kinase Src regulates p27 stability through phosphorylation of p27 at tyrosine 74 and tyrosine 88. Src inhibitors increase cellular p27 stability, and Src overexpression accelerates p27 proteolysis. Src-phosphorylated p27 is shown to inhibit cyclin E-Cdk2 poorly in vitro, and Src transfection reduces p27-cyclin E-Cdk2 complexes. Our data indicate that phosphorylation by Src impairs the Cdk2 inhibitory action of p27 and reduces its steady-state binding to cyclin E-Cdk2 to facilitate cyclin E-Cdk2-dependent p27 proteolysis. Furthermore, we find that Src-activated breast cancer lines show reduced p27 and observe a correlation between Src activation and reduced nuclear p27 in 482 primary human breast cancers. Importantly, we report that in tamoxifen-resistant breast cancer cell lines, Src inhibition can increase p27 levels and restore tamoxifen sensitivity. These data provide a new rationale for Src inhibitors in cancer therapy.

Direct interaction of proliferating cell nuclear antigen with the small subunit of DNA polymerase delta.

The interaction between proliferating cell nuclear antigen (PCNA) and DNA polymerase delta is essential for processive DNA synthesis during DNA replication/repair; however, the identity of the subunit of DNA polymerase delta that directly interacts with PCNA has not been resolved until now. In the present study we have used reciprocal co-immunoprecipitation experiments to determine which of the two subunits of core DNA polymerase delta, the 125-kDa catalytic subunit or the 50-kDa small subunit, directly interacts with PCNA. We found that PCNA co-immunoprecipitated with human p50, as well as calf thymus DNA polymerase delta heterodimer, but not with p125 alone, suggesting that PCNA directly interacts with p50 but not with p125. A PCNA-binding motif, similar to the sliding clamp-binding motif of bacteriophage RB69 DNA polymerase, was identified in the N terminus of p50. A 22-amino acid oligopeptide containing this sequence (MRPFL) was shown to bind PCNA by far Western analysis and to compete with p50 for binding to PCNA in co-immunoprecipitation experiments. The binding of p50 to PCNA was inhibited by p21, suggesting that the two proteins compete for the same binding site on PCNA. These results establish that the interaction of PCNA with DNA polymerase delta is mediated through the small subunit of the enzyme.