Anti-RPA2 monoclonal antibody, clone 23G4.4

We previously performed SEREX (serological identification of antigens by recombinant expression cloning) using the sera of patients with esophageal squamous cell carcinoma (SCC), and isolated a variant clone (AK093616) of ubiquitin-conjugating enzyme EN (UBE2I). This clone was tentatively designated as UBE21-v5 and analyzed for biological function by transient transfection of the cDNA into activated Ha-ras-transformed NIH3T3 (ras-NIH) mouse fibroblasts. Chemosiensitivity to 92 cytotoxic drugs was compared between UBE2I-v5-transfected cells and the parental ras-NIH cells. The UBEN-v5-transfected cells were more sensitive than the parental cells to anticancer drugs such as vincristine (VCR), mitoxantrone (MIT) and etoposide (VP-16). The regression analysis of the total chemosensitivity pattern of UBE2I-v5-transfected cells revealed that the function of UBE21-v5 was positively related to RPA2 (replication protein A2), Rho-GDI (Rho guanine nucleotide dissociation inhibitor a), FUS (putative tumor suppressor) and TKT (transketolase) but negatively related to Per-1 (period-1), Ran (nuclear Ras-related protein), PTEN (phosphatase and tensin homolog), C/EBP alpha (CCAAT/enhancer binding protein alpha) and the tumor suppressor p53. Thus, it is possible that UBE21-v5 plays a role in carcinogenesis by suppressing the function of C/EBP alpha and/or p53 via RPA2-like activity.

A novel karenitecin, BNP1350, is a topoisomerase I-targeting anticancer agent with significant antitumor activity in vitro and in vivo. A BNP1350-resistant human head and neck carcinoma A253 cell line, denoted A253/BNPR, was developed. The A253/BNPR cell line was approximately 9-fold resistant to BNP1350 and 4-fold cross-resistant to another topoisomerase I inhibitor SN-38, the active metabolite of irinotecan. After drug treatment with equimolar concentrations of BNPI 350 (0.7 muM) for 2 h, activation of the DNA double-strand break repair protein complexes was similar in the two cell lines, suggesting that DNA dsb repair is not attributable to resistance to BNP1350 in the A253/BNPR cells. Cell cycle analysis indicates that the A253 cell line accumulated primarily in S phase, but G(2) phase accumulation was observed in the A253/BNPR cell line at 48 h after drug removal. Elevated chk1 phosphorylation at Ser(345) following DNA damage induced by BNP1350 was accompanied by G2 accumulation in the A253/BNPR cell line, while exposure to equimolar concentrations of BNP1350 (0.7 muM) induced S-phase arrest and no increased phosphorylation of chk1 at Ser(345) in the A253 cell line. Under the same conditions, increased chk1 activity was observed in the A253/BNPR cell line, but not in the A253 cell line. Moreover, stimulated binding of 14-3-3 proteins to chk1 was observed in BNP1350-treated A253/BNPR cells. To confirm relationship between chk1 expression/phosphorylation and drug resistance to topo I poisons, we examined the effects of chk1 or chk2 antisense oligonucleotides on the cellular growth inhibition. Chk1 antisense oligonucleotide can sensitize the A253/BNPR cells to killing by topo I inhibitor BNP1350, but no significant sensitization of BNP1350-induced growth inhibition was observed in the drug-sensitive cell line. Chk2 antisense oligonucleotide has only a small sensitization effect on BNP1350-induced growth inhibition in both cell lines. The data indicate that the chk1 signaling pathways that mediate cell cycle checkpoint are associated with cellular resistance to BNP1350 in the A253/BNPR cell line. (C) 2002 Elsevier Science (USA). All rights reserved.