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| CELLULAR CONTROL OF HUMAN MULTIDRUG-RESISTANCE-1 (MDR-1) GENE-EXPRESSION IN ABSENCE AND PRESENCE OF GENE AMPLIFICATION IN HUMAN CANCER-CELLS |
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| Author(s): KOHNO K, TANIMURA H, SATO S, NAKAYAMA Y, MAKINO Y, WADA M, FOJO AT, KUWANO M |
| Source: JOURNAL OF BIOLOGICAL CHEMISTRY Volume: 269 Issue: 32 Pages: 20503-20508 Published: AUG 12 1994 |
| Times Cited: 40 References: 37 |
| Abstract: The Kst-6 cell line is a human KB carcinoma cell line that has a stably integrated chloramphenicol acetyltransferase (CAT) reporter gene under the control of the human mdr-1 promoter. Using Kst-6 cells as the parental cell line, five vincristine resistant sublines, designated Kst-V5, -V25, V50, -V75, and -V100, were isolated by exposure to increasing concentrations of drug. These sublines showed increased resistance to vincristine when compared with parental Kst-6 cells. Two sublines, V25-1 and V25-2, were further isolated from Kst-V25 after culture in the presence of vincristine, and V100-1 and V100-2 were also isolated from Kst-V100. Southern analysis demonstrated mdr-1 gene amplification in Kst-V50, Kst-V75, Kst-V100, V100-1, and V100-2 cells, respectively, but not in Kst-V5, Kst-V25, V25-1, and V25-2 cells. In contrast, increased mdr-1 expression was documented by Northern analysis in Kst-V25, V25-1, and V25-2 cells and five cell lines with mdr-1 amplification. Southern blot analysis utilizing a CAT probe demonstrated a stable copy number in all vincristine-resistant sublines. However, Northern analysis documented increased CAT expression in Kst-V5, Kst-V25, V25-1, and V25-2 cells but reduced mRNA levels in the cell line with amplification of the endogenous mdr-1 gene. Expression of the CAT gene was increased along with the endogenous mdr-1 gene in the early steps of the selection but decreased with the onset of gene amplification. There appeared almost similar mRNA levels of two trans-acting factor genes, SP-1 and MDR-NF1/YB-1, which are supposed to be involved in mdr-1 gene promoter activation, among all cell lines used in this study. These findings suggest that transcription of both the CAT gene fused to the mdr-1 promoter and the endogenous mdr-1 gene is enhanced through activation by trans-acting factors in the early steps of drug selection. However, the quantity of trans-activating factor is limiting, and with the onset of gene amplification, less is available for activation of the CAT gene, resulting in decreased expression. |
| Document Type: Article |
| Language: English |
| Reprint Address: KOHNO, K (reprint author), KYUSHU UNIV, SCH MED, DEPT BIOCHEM, FUKUOKA 812, JAPAN |
Addresses:
1. NCI, MED BRANCH, BETHESDA, MD 20892 USA |
| Publisher: AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 9650 ROCKVILLE PIKE, BETHESDA, MD 20814-3996 USA |
| Subject Category: Biochemistry & Molecular Biology |
| IDS Number: PB317 |
| ISSN: 0021-9258 |
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