Soichiro Torigoe

We analyzed 47 primary sporadic human renal cell carcinomas (39 clear cell and 8 non-clear cell) for mutations of the von Hippel-Lindau (VHL) tumor suppressor gene using the polymerase chain reaction and single strand conformational polymorphism analysis of DNA. All of the positive cases in single strand conformational polymorphism analyses were further characterized by direct sequencing. Somatic mutations were detected in 22 (56%) of 39 clear cell renal carcinomas including 15 deletions, 3 insertions, 3 missense mutations, and 1 nonsense mutation. Nineteen of these mutations predicted to produce truncation of the VHL protein. These mutations mainly occurred in the last one-third region of exons 1, 2, and 3. In addition, loss of heterozygosity of the VHL gene was observed in 16 (84%) of 19 informative clear cell renal carcinomas. No somatic mutations were detected in 8 non-clear cell carcinomas. These results show that the VHL tumor suppressor gene is one of the major tumor suppressor genes in human renal cell carcinomas, especially in the clear cell subtype renal cell carcinoma. Clear cell carcinoma might be distinguished from other pathological types of renal cell carcinomas by molecular genetic techniques.

Hemangioblastoma is one of the benign tumors in the central nervous system. It is often associated with the von Hippel-Lindau (VHL) disease, a well known hereditary tumor syndrome. It is believed that inactivation of both alleles of VHL tumor suppressor gene is essential in the tumorigenic processes in hemangioblastomas associated with VHL disease. The molecular basis for the development of sporadic hemangioblastomas is not known. Here, we analyzed 13 cases of primary sporadic hemangioblastomas for somatic mutations of VHL gene with single strand conformational polymorphism analyses of the tumor DNAs. We detected abnormal single strand conformational polymorphism pattern in 7 tumors (54%). Of these 7 possibly mutated tumors, we successfully characterized 3 tumors by direct sequencing. We were unable to sequence 4 tumors because of the poor quality of DNA obtained from paraffin blocks. Somatic mutations in the 3 tumors were 2 missense mutations and 1 microdeletion. These mutations were observed in 1 tumor in exon 1 and 2 tumors in exon 2. Our results suggest that mutations of VHL tumor suppressor gene are involved in the development of at least 20% of sporadic central nervous system hemangioblastomas.

DNA polymerase beta is a nuclear protein essential to DNA repair in mammalian cells. A high frequency of mutations in this gene has been reported in colorectal cancers. To clarify the tumorigenesis steps of human prostate cancers in the molecular basis, we examined the entire coding region of the human DNA polymerase beta gene in human prostate cancer tissues using polymerase chain reaction, single-strand conformational polymorphism analysis of RNA, and sequencing analysis. Consequently, we detected DNA polymerase beta gene mutations in 2 of 12 cases (17%). The first case is an A to G transition at nucleotide 893, resulting in a substitution of the amino acid from tyrosine to cysteine. In the second case, we found an A to G transition at nucleotide 305, a T deletion at nucleotide 569, and an A insertion into the 6 repeats of A from nucleotide 612 to 617. This T deletion shifted the subsequent reading frame and resulted in the premature termination at codon 163 instead of 336. The two cases were advanced grade and stage. Present results suggest that polymerase beta gene mutations, although they occurred at relatively low frequency, are involved in certain cases of human prostate carcinogenesis.

The genetic background underlying the growth and development of human prostatic cancer is not yet clear. Here we searched for possible mutations in the entire coding region of tumor suppressor gene p53 in primary human prostatic carcinomas, using polymerase chain reaction and single-strand conformational polymorphism analysis of RNA. We found p53 gene mutations in 4 of 21 cases (19%). DNA sequencing of the polymerase chain reaction products revealed missense point mutations that resulted in amino acid changes in exon 5 or 3 in three cases and single base deletions in exon 7 in two cases. One case contained both a missense point mutation and a single base deletion. Three of these four cases were pathologically diagnosed as poorly differentiated adenocarcinomas, and three of the four cases were clinically localized to stage C or D. None of seven noncancerous prostate tissues nor three well-differentiated adenocarcinoma tissues showed any mutations. The present results suggest that p53 gene mutation is involved in the late progression steps of human prostate carcinogenesis.