Cancer cells differ from normal cells in many characteristics including loss of differentiation and uninhibited cell proliferation. Recent studies have focused on the identification of factors contributing to cell growth and differentiation. Gut-enriched Krüppel-like factor (GKLF or KLF4) is a newly identified eukaryotic transcription factor and has been shown to play a role in regulating growth arrest. We have previously shown that GKLF mRNA levels were significantly decreased in colon cancer tissues, and that over-expression of GKLF in colonic adenocarcinoma cells (HT-29) resulted in reduction of cyclin D1 (CD1) mRNA and protein levels. The current study was undertaken to determine the mechanisms by which GKLF inhibited CD1 expression. In a transient transfection system, GKLF suppressed CD1 promoter activity by 55%. Sequential deletion and site-directed mutation analysis of the CD1 promoter have identified the sequence between -141 and -66, a region containing an Sp1 response element, to be essential for GKLF function. By electrophoretic mobility gel shift assay, recombinant GKLF and nuclear extracts from HT-29 cells were found to bind to the Sp1 motif on the CD1 promoter. The inhibitory effect of GKLF on the CD1 promoter activity was completely abolished by excessive amount of Sp1 DNA and GKLF significantly reduced the stimulatory function of Sp1 suggesting that GKLF and Sp1 may compete for the same binding site on the CD1 promoter. These results indicate that GKLF is a transcriptional repressor of the CD1 gene and that the inhibitory effect of GKLF is, in part, mediated by interaction with the Sp1 binding domain on its promoter.
We report here that des-methyl, des-amino pateamine A (DMDA-PatA), a structurally simplified analogue of the marine natural product pateamine A, has potent antiproliferative activity against a wide variety of human cancer cell lines while showing relatively low cytotoxicity against nonproliferating, quiescent human fibroblasts. DMDAPatA retains almost full in vitro potency in P-glycoprotein-overexpressing MES-SA/Dx5-Rx1 human uterine sarcoma cells that are significantly resistant to paclitaxel, suggesting that DMDA-PatA is not a substrate for P-glycoprotein-mediated drug efflux. Treatment of proliferating cells with DMDA-PatA leads to rapid shutdown of DNA synthesis in the S phase of the cell cycle. Cell-free studies show that DMDA-PatA directly inhibits DNA polymerases α and γ in vitro albeit at concentrations considerably higher than those that inhibit cell proliferation. DMDA-PatA shows potent anticancer activity in several human cancer xenograft models in nude mice, including significant regressions observed in the LOX and MDA-MB-435 melanoma models. DMDA-PatA thus represents a promising natural product-based anticancer agent that warrants further investigation.
Cancer cells differ from normal cells in many aspects, including hyperproliferation and loss of differentiation. Recent research has focused on the role of transcription factors in regulating abnormal cell growth. Gut-enriched Krüppel-like factor (GKLF) is a newly identified eukaryotic zinc finger protein expressed extensively in the gastrointestinal tract. In the current study, we demonstrated that GKLF mRNA levels were significantly decreased in the dysplastic epithelium of the colon, including adenomatous polyp and cancer. GKLF immunostains in the normal colon were higher at the surface epithelium and gradually decreased toward the crypt, but this gradient was not present in the adenomatous and cancerous mucosa. Constitutive overexpression of GKLF DNA in a human colonic adenocarcinoma cell line (HT-29) decreased [(3)H]thymidine incorporation, whereas suppression of GKLF gene increased DNA synthesis, indicating that downregulation of the GKLF gene might contribute to cellular hyperproliferation. Cyclin D1 (CD1) protein level and CD1-associated kinase activity were decreased in HT-29 cell overexpressed GKLF cDNA, and CD1 promoter activity was profoundly suppressed by GKLF. When HT-29 cells were cultured in the presence of sodium butyrate, GKLF mRNA levels increased as cells acquired more differentiated phenotypes. These results suggest that GKLF plays an important role in regulating cell growth and differentiation in the colonic epithelium and that downregulation of GKLF expression may cause colonic cells to become hyperproliferative. Furthermore, GKLF appears to be a transcriptional repressor of the CD1 gene.
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