Summary Recently, we have found a high frequency of p53 gene mutations in human functional adrenal tumours. As the tumorigenesis is a multigene defect, we believe that other oncogenes may also be involved in the initiation or progression of adrenal tumours. Using the singlestrand conformational polymorphism (SSCP) method, we chose the ras oncogenes as the target in this screening procedure because their high mutation rates were detected in thyroid tumours. For the ras oncogenes analysed, exon 1 to exon 2 of H-ras and K-ras genes in the tumour tissues of 13 Conn's syndrome, two adrenal Cushing's syndrome, two non-functional adrenal tumours, one adrenocortical hyperplasia and eight phaeochromocytomas and its paired adjacent normal adrenal tissues were amplified and sequenced. No mutations were detected in the H-ras gene. But mutations of the K-ras gene were detected in 46% (6 of 13) of Conn's syndrome; the hot spots were located at codon 15, 16, 18 and 31, which were different from those previously found in other tumours (codon 12, 13 and 61). Northern blot analysis with 1.1 kb K-ras cDNA revealed that K-ras mRNA was more than tenfold over-expressed in four of Conn's syndrome, one case of Cushing's syndrome and one case of adrenocortical hyperplasia. The mutation sites and mutation type were not found in other tissues, which confered that this was highly related to adrenocortical tumours. Yet, the correlation between K-ras oncogene and adrenocortical tumours needs to be clarified by further studies.
Four new cembrane-type diterpenes; numerosol A–D (1–4); along with a known steroid; gibberoketosterol (5); were isolated from the Taiwanese soft coral Sinularia numerosa. The structures of these metabolites were determined by extensive analysis of spectroscopic data. Gibberoketosterol (5) exhibited cytotoxicity against P-388 (mouse lymphocytic leukemia) cell line with an ED50 of 6.9 μM.
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.
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