The Oct-4 gene encodes a transcription factor that is specifically expressed in embryonic stem cells and germ cells of the mouse embryo. Cells that differentiate into somatic tissues lose Oct-4 expression. Regulation of Oct-4 gene transcription involves a TATA-less minimal promoter and two upstream elements: the proximal (PE) and distal enhancers (DE). We report here the nucleotide sequence of the 5' upstream regulatory regions of the human and murine Oct-4 genes. A comparative alignment analysis between these regions and those of the bovine Oct-4 ortholog reveals four conserved regions of homology (CR 1 to 4) between these species (66-94% conservation). The 1A sequence within the mouse PE is located approximately half-way between CR 2 and CR 3. A putative Sp1/Sp3 binding site and the overlapping hormone responsive element (HRE) in CR1 are identical in all three species. A high number of CCC(A/T)CCC motifs exhibit various levels of homology in these upstream regions. We discuss the importance of these and other sequences and present candidate factors that may bind and regulate Oct-4 gene expression.
Nucleotide excision repair (NER) is a multi-enzyme DNA repair pathway in eukaryotes. Several NER genes in this pathway including XPB, XPD, XPA and ERCC-1 have been implicated in anticancer drug resistance in human tumor cells. In this study, we assessed the levels of the above-mentioned proteins in the NCI panel of 60 human tumor cell lines in relation to the cytotoxicity patterns of 170 compounds that constitute the standard agent (SA) database. The database consists of drugs used in the clinic for which a mechanism of action has been at least partially defined. The ERCC-1, XPD and XPB protein expression patterns yielded significant negative Pearson correlations with 13, 32 and 17 out of the 170 compounds, respectively (using p<0.05). XPA produced a random assortment of negative and positive correlations, and did not appear to confer an overall resistance or sensitivity to these drugs. Protein expression was also compared with a pre-defined categorization of the standard agents into six mechanism-of-action groups resulting in an inverse association between XPD and alkylating agent sensitivity. Our present data demonstrate that XPD protein levels correlate with resistance to alkylating agents in human tumor cell lines suggesting that XPD is implicated in the development of this resistance. NER activity, using the in vitro cell-free system repair assay, revealed no correlation between NER activity and the level of XPD protein in four cell lines with widely varying XPD protein levels. This lack of correlation may be due to the contribution of XPD to other functions including interactions with the Rad51 repair pathway.
Resistance to the nitrogen mustards in patients with chronic lymphocytic leukemia (CLL) correlates with an enhanced removal of melphalan-induced DNA interstrand cross-links. This finding suggests that DNA repair enzymes may be involved in this process. The activity of 3-methyladenine-DNA glycosylase, which can release altered bases, including adducts at the N-7 position of guanine, was increased significantly in lymphocytes from patients with resistant CLL compared with those from untreated CLL patients. Since glycosylase activity varies with cell proliferation, the amount of [3H]thymidine incorporated into DNA was determined and found to be elevated almost threefold in lymphocytes from patients with resistant CLL. The ratio of glycosylase activity to level of thymidine incorporation did not differ between these two groups of patients. Northern blot analysis of ERCC1 gene (a putative DNA repair enzyme involved in nucleotide excision repair) expression in lymphocytes from patients with CLL revealed multiple gene transcripts (1.1, 3.4, and 3.8 kilobases). In addition, analysis of two samples revealed the presence of a 2.6-kilobase transcript. The 2.6-kilobase transcript was recognized by specific RNA probes that hybridize to antisense ERCC1 transcripts. Levels of expression of the 1.1-kilobase protein encoding transcript in lymphocytes from patients with resistant CLL were increased twofold to threefold above those of untreated patients with CLL. These results indicate that increased expression of ERCC1 and increased activity of 3-methyladenine-DNA glycosylase occur with the development of resistance to the nitrogen mustards in patients with CLL, suggesting a role for enhanced DNA repair in this process.
SUMMARYWe examined the O 6 -methylguanine-DNA methyltransferase (MGMT) protein as well as MGMT activity levels and the excision repair cross-complementing rodent repair deficiency gene, ERCC2 (XPD), protein levels in 14 human tumor cell lines not selected for chloroethylnitrosourea (CENU) resistance. These results were compared with 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) cytotoxicity and UV light sensitivity. MGMT protein correlated significantly with MGMT activity (r ϭ 0.9497, p ϭ 0.0001). There was no significant linear correlation between BCNU cytotoxicity and MGMT content as determined by both Western analysis (r ϭ 0.139, p ϭ 0.6348) and activity assay (r ϭ 0.131, p ϭ 0.6515). However, MGMT-rich cell lines were found to be more resistant than MGMT-poor cell lines to BCNU (t ϭ 2.2375, p ϭ 0.0225) but not to UV (t ϭ 1.1734, p ϭ 0.1317). Furthermore, the most BCNU-sensitive cell lines were all MGMT-poor. UV sensitivity was significantly correlated to BCNU cytotoxicity (r ϭ 0.858, p ϭ 0.0001). Significant correlations were found between ERCC2 protein levels and BCNU cytotoxicity (r ϭ 0.786, p ϭ 0.0009) or UV sensitivity (r ϭ 0.874, p ϭ 0.0001). Our results confirm that MGMT plays an important role in CENU resistance, but not in UV resistance. The correlation of UV sensitivity with BCNU cytotoxicity suggests that nucleotide excision repair is an important modifying factor of MGMT-mediated innate CENU resistance in human tumor cell lines, especially in highly resistant cell lines. ERCC2 may be implicated in this process.
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