Chieki Wada

p53 Mutations and Prognosis in Bladder Tumors

The incidence of loss of heterozygosity on chromosome 17p and p53 gene mutations was assessed in 43 bladder tumor patients. Histological findings, cigarette smoking and prognosis were examined for possible correlation with the presence or absence of loss of heterozygosity on 17p and p53 mutations. Of 20 informative cases 10 (50.0%) showed loss of heterozygosity of 17p13, including 9 (90.0%) with disease beyond stage pT2. The p53 mutations were detected in 20 of 43 patients (46.5%), including 9 (95.0%) with disease beyond grade 2 and 17 (85.0%) with cancer beyond stage pT2. The incidence of p53 gene mutations was not significantly influenced by habitual smoking but G:C to T:A substitutions, often observed in lung cancers, were detected only in mutations from smokers (5 of 10 or 50%, p < 0.05). Groups with and without loss of heterozygosity showed essentially the same results, while significant differences were found for groups with grades 1 and 2 to 3 (p < 0.05) cancer, stages pT1 and pT2 to 4 (p < 0.01) disease, and with and without p53 gene mutations (p < 0.01, Cox-Mantel test). Genetic alternation in chromosome 17p and p53 mutations would, thus, appear to occur more frequently in high grade and invasive bladder tumors. Cigarette smoking may possibly be a determining factor of mutations of the p53 gene in bladder tumors. Our results indicate that an unfavorable prognostic factor may possibly be linked not only to histopathological findings but the presence of a p53 mutation in bladder tumors as well. Accordingly, mutations of the p53 gene may be deeply involved in late events of tumorigenesis and possibly useful as ideal molecular markers for prognosis in bladder tumors.

Infrequent involvement of p53 mutations and loss of heterozygosity of 17p in the tumorigenesis of renal cell carcinoma

Restriction fragment length polymorphism (RFLP) analysis and the polymerase chain reaction of the single-strand conformation polymorphism (PCR-SSCP) method were conducted to assess the loss of heterozygosity of chromosome 17p and mutations of the p53 gene in 30 surgical specimens of human renal cell carcinoma. Six of 29 tumors (20.6%) showed loss of heterozygosity on chromosome 17p in RFLP analysis, and in none of 21 tumors could a mutation be found on exons 5 to 8 of the p53 gene in PCR-SSCP analysis. We conclude that the p53 gene mutation does not play a role in the development of the majority of cases of renal cell carcinoma and that there may be another tumor suppressor gene on 17p.

Molecular cloning of rat cerebellin-like protein cDNA which encodes a novel membrane-associated glycoprotein

Rat cerebellin and an apparent metabolite des-Ser1-cerebellin have been shown to be located in cerebellar Purkinje cells and the dorsal cochlear nucleus. A cDNA clone was isolated by screening a rat brain cDNA library using an oligonucleotide corresponding to rat cerebellins. The clone encodes 224 amino acid residues of a glycoprotein a part of whose sequence is virtually the same as that of cerebellins and which contains one putative transmembrane spanning domain. Neither an N-terminal cleavable signal peptide nor dibasic pair specific endopeptidase directly precedes or follows the cerebellin-like sequence. Expression of the cerebellin-like protein gene is developmentally regulated in rat cerebellum and shows tissue specific alternative splicing in the brain, adrenal gland and spleen of the rat. The results of Southern blot indicated the gene to possibly be a member of some gene family. Rat cerebellin-like protein, possibly a precursor of cerebellins, is a novel membrane-associated glycoprotein expressed in nervous, adrenal and immune systems and appears to synaptic functions in the central nervous system.

MICROSATELLITE INSTABILITY IN TRANSITIONAL CELL CARCINOMA OF THE URINARY TRACT AND ITS RELATIONSHIP TO CLINICOPATHOLOGICAL VARIABLES AND SMOKING

To determine whether microsatellite instability is involved in the development of transitional cell carcinoma (TCC) of the urinary tract, a microsatellite instability assay was carried out using PCR with 9 microsatellite loci. Thirty‐eight TCC samples (30 patients with bladder cancer, 5 with renal pelvic tumors and 3 with ureteral tumors) and 1 lymph node with metastasis were examined. Microsatellite instability was found in 8 of 38 tumors examined, and 3 showed alterations in more than 2 microsatellite loci. All 8 tumors were beyond grade 2 and stage pT2 advanced tumors. Stages pT1‐2 and pT3‐4 patients differed significantly. Microsatellite instability was greater in smokers than non‐smokers, but the differences were not significant. Microsatellite instability in TCC of the urinary tract is rare in superficial tumors but more common in invasive tumors. Microsatellite alterations would thus appear to occur, and possibly be importantly involved, in the tumorigenesis of urinary tract TCC.

Infrequent involvement of mutations on neurofibromatosis type 1, H-ras, K-ras and N-ras in urothelial tumors

The neurofibromatosis type 1 (NF1) gene is considered a tumor-suppressor gene whose product acts upstream of ras. The ras gene is an oncogene very commonly detected in human cancers and consists of three families, H-ras, K-ras and N-ras. These genes are converted to active oncogenes by point mutations in codon 12, 13, or 61. Examination was made of the mutations of these genes in 39 urothelial malignant tumors (31 bladder cancer, 6 renal pelvic tumor, and 2 ureter tumors) using polymerase chain reaction single-stranded conformation polymorphism and direct sequencing methods. Three of 39 (7.7%) cases showed mobility shifts in the ras family gene but no point mutations in NF1 and N-ras genes could be detected. Mutations were found in 1 case in H-ras at codon 13 (GGT-GTT/GGT) and K-ras at codon 12 in 2 cases (GGT-GCT/GGT, GGT-GTT/GGT). All 3 cases had progressed far beyond grade 2 and stage pT2. It follows from the above that NF1 and ras gene mutations are infrequent in the pathogenesis of urothelial tumors.

Development ally regulated expression of the calcitonin gene related peptide (CGRP) in rat lung endocrine cells

SummaryCalcitonin (CT) and the calcitonin generelated peptide (CGRP) are generated by alternative RNA processing from a single CT/CGRP gene. Recently, we reported the existence of CGRP-immunoreactivity and CGRP mRNA in endocrine cells or Kulchitsky (K) cells of human and rat lung [Wada et al. 1987b]. In this report, an examination was made of developmental changes in the expression of the CGRP gene in rat lungs by immunohistochemistry, radioimmunoassay (RIA) and Northern hybridization. CGRP-positive K-cells in lung tissue appeared on the 18th day of gestation. Their number was greatest on the 20th day of gestation and then decreased postnatally. The level of CGRP in rat lung was found to be highest in a 1-day-old neonate by RIA. In the Northern hybridization of rat lung using the CGRP 3′ non-coding region (exon 6) of the first human CT/CGRP gene as the probe, 1.0 kilobase (kb) CGRP mRNA was found to be abundant on the 20th day of gestation and in a 1 day-old neonate. It thus appears that CGRP in rat lung is essential for pulmonary adaptation at birth and/or from the last intrauterine stage to the early neonatal period.

Relationships among expression, transcription and rearrangement of T-cell receptor Β gene in T-cell lymphomas

The expression of T-cell receptors (TCR) in malignant lymphomas was examined immunohistochemically by monoclonal antibodies which react with the TCRΒ or TCRδ chain. TCRΒ was expressed in 16 out of 47 non-Hodgkins lymphomas. These included 15 T-cell lymphomas and 1 Ki-1 lymphoma. The anti-TCRΒ chain antibody,ΒF1, did not react with 26 B-cell lymphomas, 1 Ki-1 lymphoma or 6 Hodgkins disease. The anti-TCRδ chain antibody, TCRδ1, did not react with any type of malignant lymphoma. Although TCRΒ and CD3 were co-expressed in normal lymphoid tissues and most T-cell lymphomas, 3 cases of CD3+CD4+ CD8−T-cell lymphoma failed to express TCR0. TCRΒ and Ig JH gene configurations in malignant lymphomas were examined by Southern hybridization. Although each of 9 T-cell lymphomas had a rearranged TCRΒ locus, TCRΒ gene rearrangement in the 3 cases ofΒF1−CD3+T-cell lymphomas was demonstrated by Southern blot. No transcripts of the TCRΒ gene could be found in 2 out of the 3ΒF1−CD3+T-cell lymphomas by Northern blot, indicating the lack of TCRΒ protein expression to be due to non-transcription of the TCR gene. Loss of TCRΒ proteins in these T-cell lymphomas is thus quite likely to be associated with T-cell tumour activation and progression, since 3ΒF1−CD3+T-cell lymphomas expressed CD25 (interleukin-2 receptor) to a high degree.

A catalytic antibody that accelerates the hydrolysis of carbonate esters. Prediction of the binding-site structure of the substrate.

Monoclonal antibodies catalyzing the hydrolysis of p-nitrophenyl alkyl carbonate were obtained using p-nitrophenyl phosphonate as hapten. One of the antibodies, 4A1, has a relatively high activity for the substrate having a bulky group. To determine the amino acid residues related to the binding of the bulky group, we determined the amino acid sequences of VL and VH regions of 4A1 by the cycle sequencing method, built the three-dimensional structure of the V regions, labeled 4A1 with [14C]phenyl glyoxal in the presence and absence of I-1 or I-13, and analyzed the labeled incubation mixture with SDS–PAGE. From these results, the possibility that Arg-H28 of the heavy chain is involved in binding the bulky group of the substrate is discussed.