Differential regulation of IL-6 promoter activity in a human ovarian-tumor cell line transfected with various p53 mutants: involvement of AP-1.
In human ovarian carcinomas, the p53 tumor-suppressor gene is frequently mutated. Interleukin-6 (IL-6) in these tumors is known to stimulate tumor-cell proliferation. In order to evaluate the effect of several p53 phenotypes on the IL-6 promoter activity, the human ovarian wild-type (wt)-p53 cell line A2780 was stably transfected with an empty plasmid (CMV) or (m)-175-, m-248- or m-273-p53. Electrophoretic mobility-shift assays revealed differences in activator protein-1 (AP-1) DNA-binding activity in the various clones. The CMV and m-273 clone had comparable amounts of AP-1. The m-175 clone displayed the least and m-248 the most pronounced AP-1 binding. Supershift analysis of AP-1/DNA complexes with antibodies against the AP-1 sub-units, c-Fos, FosB, Fra-1, Fra-2, c-Jun, JunB, and JunD, revealed that the AP-1/DNA complexes in the various clones had different compositions. Fra proteins were basically present only in m-175 and m-248 AP-1. IL-6-promoter activity was evaluated in the presence and absence of the AP-1 binding site which showed that the m-175-transfected clone has a transcriptional suppressing AP-1, whereas the CMV and the m-273 clones have an activating AP-1. Exposure of the p53 clones to tumor-necrosis factor-alpha (TNF-alpha) clearly altered the AP-1/DNA complex composition. IL-6-promoter activity was enhanced by TNF-alpha irrespective of the presence of an AP-1 binding site, while the degree of activation differed in the various clones, being most pronounced in the m-175 and m-248 clones. The results demonstrate that the basic and activated IL-6-promoter activity is differently regulated in the various p53 clones, possibly due to alterations in the AP-1 composition.
Asschert
J G
JG
Department of Internal Medicine, University Hospital, Groningen, The Netherlands.
De Vries
E G
EG
De Jong
S
S
Withoff
S
S
Vellenga
E
E
D018076:DNA, Complementary / Q000235:genetics; D004268:DNA-Binding Proteins / Q000378:metabolism; D005260:Female; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D016158:Genes, p53; D005815:Genetic Code; D006801:Humans; D015850:Interleukin-6 / Q000235:genetics*; D010051:Ovarian Neoplasms / Q000235:genetics* / Q000473:pathology; D010641:Phenotype; D011401:Promoter Regions, Genetic; D018808:Transcription Factor AP-1 / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D015850:Interleukin-6; D018808:Transcription Factor AP-1; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha