A Significant Decrease of the Transcriptional Activity of p53 Mutants Deriving from Human Functional Adrenal Tumors

Abstract

Recently, our laboratory has found a high incidence (77%) of p53 gene mutations in human functional adrenal tumors. Furthermore, the majority of mutant sites were assembled at codons 100, 102, and 249. These mutation sites are not common, and there have been no studies addressing whether or not these mutants points or mutant styles cause the p53 protein to lose function. It has been well known that p53 is a transcription factor. To examine the transcriptional activities of these mutant p53 genes from patients with functional adrenal tumors, we constructed p53 expression plasmids from tumors and paired adjacent normal adrenal gland tissues, using a transient co-transfection assay with a reporter gene in H358 cells. Wild-type p53 from normal adrenal gland tissues specifically trans-activates the expression of a chloramphenicol acetyltransferase (CAT) reporter gene in H358 cells. Three mutant p53 proteins (at codons 100, 102, and 249, respectively) from tumors showed a >90% loss of transcriptional activity. One mutant at codon 68, other than at hot spots, remained at approximately 65% transcriptional activity. An immunoprecipitation assay showed that the mutant proteins of codon 68 and codon 102 could respond to the three monoclonal antibodies (PAbDO-1, PAb1620, and PAb421), indicating that there were no obvious changes in the antigenicity of the proteins. However, the mutant protein of codon 249 could not respond to the carboxy-terminus-specific antibody PAb421 and conformation-specific antibody PAb1620, indicating that there were some obvious changes in the conformation of the mutant proteins. The mutant protein of codon 100 could not be detected by immunoprecipitation assay but could be analyzed by Western blot. In a further study using a DNA-binding assay, it was shown that the loss of transcriptional activity was caused by the loss of DNA-binding ability. These results show that the p53 mutants, derived from functional adrenal tumors, actually lost DNA-binding ability and decreased the transcriptional activity. However, the role of the mutant protein in the tumorigenesis of functional adrenal tumors requires further investigation.

Mutation of p53 in Recurrent Hepatocellular Carcinoma and Its Association with the Expression of ZBP-89

“…This is consistent with the report which indicated that a point mutation in codon 68 of exon 4 decreased approximately 35% transcriptional activity of the p53 gene. 28 The p53 nuclear import or retention is essential for its normal function in growth inhibition or induction of apoptosis. 23,29 A defect in the regulation of p53 nuclear import or export may result in tumorigenesis.…”

Mutant K-ras oncogene regulates steroidogenesis of normal human adrenocortical cells by the RAF-MEK-MAPK pathway

“…Our laboratory has been studying human functional adrenocortical tumours since 1993 (Lin et al, 1994(Lin et al, , 1996(Lin et al, , 1998a(Lin et al, ,b, 2000Shu et al, 2001;Wu et al, 2002). We have found that the K-ras gene enhanced the efficiency of cell proliferation and elevation of cortisol production in bovine adrenocortical cells.…”

Mutations of K-ras oncogene in human adrenal tumours in Taiwan

“…No electrophoretic mobility shift over the samples tested suggests normal conformations over these samples. P is the positive control of K-ras codon 12 mutant derived from SW480 human colon adenocarcinoma cell line (Lin et al, 1994;1996). As the tumorigenesis is a multigene defect (Knudson, 1989) British Journal of Cancer (1998) …”