Thirteen novel mutations of the replicated region of PKD1 in an Asian population.

BACKGROUND: Mutations of PKD1 are thought to account for approximately 85% of all mutations in autosomal dominant polycystic kidney disease (ADPKD). The search for PKD1 mutations has been hindered by both its large size and complicated genomic structure. To date, few mutations that affect the replicated segment of PKD1 have been described, and virtually all have been reported in Caucasian patients. METHODS: In the present study, we have used a long-range polymerase chain reaction (PCR)-based strategy previously developed by our laboratory to analyze exons in the replicated region of PKD1 in a population of 41 unrelated Thai and 6 unrelated Korean families with ADPKD. We have amplified approximately 3.5 and approximately 5 kb PKD1 gene-specific fragments (5'MR and 5'LR) containing exons 13 to 15 and 15 to 21 and performed single-stand conformation analysis (SSCA) on nested PCR products. RESULTS: Nine novel pathogenic mutations were detected, including six nonsense and three frameshift mutations. One of the deletions was shown to be a de novo mutation. Four potentially pathogenic variants, including one 3 bp insertion and three missense mutations, were also discovered. Two of the nonconservative amino acid substitutions were predicted to disrupt the three-dimensional structure of the PKD repeats. In addition, six polymorphisms, including two missense and four silent nucleotide substitutions, were identified. Approximately 25% of both the pathogenic and normal variants were found to be present in at least one of the homologous loci. CONCLUSION: To our knowledge, this is the first report of mutation analysis of the replicated region of PKD1 in a non-Caucasian population. The methods used in this study are widely applicable and can be used to characterize PKD1 in a number of ethnic groups using DNA samples prepared using standard techniques. Our data suggest that gene conversion may play a significant role in producing variability of the PKD1 sequence in this population. The identification of additional mutations will help guide the study of polycystin-1 and better help us to understand the pathophysiology of this common disease.

Mitomycin C induces apoptosis in a caspases-dependent and Fas/CD95-independent manner in human gastric adenocarcinoma cells.

We investigated the mechanism of mitomycin C (MMC)-induced apoptosis in SNU-16 human gastric adenocarcinoma cells. Caspase-8 and caspase-3 were activated in MMC-treated cells whereas caspase-1 was not activated, and cytochrome c was released from mitochondrial membrane to cytosol suggesting that caspase-9 was activated during the MMC-induced apoptotic process. Protein kinase C (PKC) delta was cleaved to its characteristic 40 kDa fragment in a caspase-3-dependent manner; on the other hand PKC zeta was cleaved to approximately 40 kDa independently of caspase-3 in the drug-induced apoptosis of the cells. Incubation with z-DEVD-fmk and benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk) almost completely abrogated MMC-induced DNA fragmentation, indicating that activation of these caspases was crucially involved in MMC-induced apoptosis. Activation of caspase-8 in response to Fas triggering by recruitment of caspase-8 to the Fas has also been found, however, MMC did not induce FasL and Fas expression, as evidenced by reverse transcriptase-polymerase chain reaction and Western blotting. Taken together, these findings indicate that MMC-induced apoptosis in SNU-16 cells was mediated by caspase-8, caspase-9, and caspase-3 activation independently of FasL/Fas interactions.

Alteration of signal-transducing molecules in tumor-infiltrating lymphocytes and peripheral blood T lymphocytes from human colorectal carcinoma patients.

Tumor development or growth is accompanied by impaired immune responses, such as a poor proliferative response or down-regulated cytolytic T lymphocyte activity. Although recent reports have suggested that modification of the signal-transducing molecule is responsible for impaired immune responses in tumor-bearing hosts, the causes of defective immune function are not yet completely understood. Furthermore, the clinical significance of the findings is not yet clear. In this study, we investigated the alteration of several signal-transducing molecules in peripheral blood T lymphocytes (T-PBL) as well as in tumor-infiltrating lymphocytes (TIL) from human colorectal carcinoma patients and their relationship with the impaired host immune responses. A greater reduction in CD3zeta chain level was observed in TIL than in T-PBL from tumor-bearing hosts. CD3zeta chain reduction in T-PBL correlated with the clinicopathological stage of a tumor, especially with the status of lymph node metastasis. The levels of p56lck and p59fyn protein tyrosine kinase in T-PBL were also compared between tumor-bearing hosts and normal healthy volunteers. In T-PBL from tumor-bearing hosts, expression of protein tyrosine kinase p59fyn was significantly lower than that of p56lck. However, the level of CD3zeta chain expression did not correlate with T lymphocyte functions such as T lymphocyte proliferative response or allogeneic target cell lysis.

Expression of a novel Bcl-2 related gene, Bfl-1, in various human cancers and cancer cell lines.

The expression of Bfl-1 gene, a novel Bcl-2 related gene, was determined by Northern blot analysis using a radiolabeled cDNA specific for Bfl-1 gene in 82 surgically resected tissue specimens of 28 gastric cancers, 15 colon cancers, nine breast cancers, eight bone and soft tissue sarcomas, five ovarian cancers, nine colon adenomas and eight gastric adenomas. A high rate of expression was observed in gastric and colon cancer, at 86 and 93%, respectively. In breast cancer, bone and soft tissue sarcoma and ovarian cancer, the expression rate was 33, 25 and 40%, respectively. In stomach cancer, the expression rate of Bfl-1 gene in metastatic lymph nodes was 82%, which was higher than 50% of the primary sites (p < 0.02). The intensity of RNA bands of the gastric cancer specimens was compared according to the stage, demonstrating that there was no difference in the expression levels of Bfl-1 gene between the stages in both primary sites and metastatic lymph nodes. Bfl-1 gene was expressed in three (33%) out of nine adenomas of the colon, while it was not detected in all eight gastric adenomas, We also examined the RNA expression of Bfl-1 gene in 22 human cancer cell lines consisting of five stomach cancer, four squamous cell carcinoma, three lung cancer, three cervical cancer, two colon cancer, two brain cancer, two leukemia and one osteosarcoma cell lines. Bfl-1 gene band was detected in one (5%) cervical cancer cell line, SiHa. The results of cancer tissue specimens indicate that Bfl-1 gene may play an important role in carcinogenesis of human cancers and may be involved in a relatively early phase of the adenoma-carcinoma sequence in colon cancer development. However, the mechanism responsible for the very low rate of expression in established cell lines is not clearly understood and further investigation is necessary to clarify the mechanism involved.