Hideaki Oda

Increased insulin sensitivity and hypoglycaemia in mice lacking the p85α subunit of phosphoinositide 3-kinase

The hallmark of type 2 diabetes, the most common metabolic disorder, is a defect in insulin–stimulated glucose transport in peripheral tissues. Although a role for phosphoinositide–3–kinase (PI3K) activity in insulin–stimulated glucose transport and glucose transporter isoform 4 (Glut4) translocation has been suggested in vitro, its role in vivo and the molecular link between activation of PI3K and translocation has not yet been elucidated. To determine the role of PI3K in glucose homeostasis, we generated mice with a targeted disruption of the gene encoding the p85α regulatory subunit of PI3K (Pik3r1; refs 3, 4, 5). Pik3r1−/− mice showed increased insulin sensitivity and hypoglycaemia due to increased glucose transport in skeletal muscle and adipocytes. Insulin–stimulated PI3K activity associated with insulin receptor substrates (IRSs) was mediated via full–length p85α in wild–type mice, but via the p50α alternative splicing isoform of the same gene in Pik3r1−/− mice. This isoform switch was associated with an increase in insulin–induced generation of phosphatidylinositol(3,4,5)triphosphate (PtdIns(3,4,5)P 3) in Pik3r1−/− adipocytes and facilitation of Glut4 translocation from the low–density microsome (LDM) fraction to the plasma membrane (PM). This mechanism seems to be responsible for the phenotype of Pik3r1−/− mice, namely increased glucose transport and hypoglycaemia. Our work provides the first direct evidence that PI3K and its regulatory subunit have a role in glucose homeostasis in vivo.

Cardiovascular anomaly, impaired actin bundling and resistance to src-induced transformation in mice lacking p130Cas

p130Cas (Cas), the protein encoded by the Crkas gene (also known as Cas), is an adaptor molecule with a unique structure that contains a Src homology (SH)-3 domain followed by multiple YXXP motifs and a proline-rich region. Cas was originally cloned as a highly tyrosine-phosphorylated protein in cells transformed by v-Src (refs 2,3) or v-Crk (ref. 4) and has subsequently been implicated in a variety of biological processes including cell adhesion, cell migration, growth factor stimulation, cytokine receptor engagement and bacterial infection. To determine its role in vivo, we generated mice lacking Cas. Cas-deficient embryos died in utero showing marked systemic congestion and growth retardation. Histologically, the heart was poorly developed and blood vessels were prominently dilated. Electron microscopic analysis of the heart revealed disorganization of myofibrils and disruption of Z-disks. In addition, actin stress fiber formation was severely impaired in Cas-deficient primary fibroblasts. Moreover, expression of activated Src in Cas-deficient primary fibroblasts did not induce a fully transformed phenotype, possibly owing to insufficient accumulation of actin cytoskeleton in podosomes. These findings have defined Cas function in cardiovascular development, actin filament assembly and Src-induced transformation.

Acetylation of GATA‐3 affects T‐cell survival and homing to secondary lymphoid organs

Acetylation of a transcription factor has recently been shown to play a significant role in gene regulation. Here we show that GATA‐3 is acetylated in T cells and that a mutation introduced into amino acids 305–307 (KRR‐GATA3) creates local hypoacetylation in GATA‐3. Remarkably, KRR‐GATA3 possesses the most potent suppressive effect when compared with other mutants that are disrupted in putative acetylation targets. Expressing this mutant in peripheral T cells results in defective T‐cell homing to systemic lymphnodes, and prolonged T‐cell survival after activation. These findings have significant implications in that the acetylation state of GATA‐3 affects its physiological function in the immune system and, more importantly, provides evidence for the novel role of GATA‐3 in T‐cell survival and homing to secondary lymphoid organs.

Mutations of the Ki‐ras, p53 and APC genes in adenocarcinomas of the human small intestine

In contrast to the origins of colorectal carcinomas, the mechanisms of carcinogenesis in the small intestine remain unclear. We therefore analyzed the mutational status of the Ki‐ras, p53, and adenomatous polyposis coli (APC) genes in primary carcinomas of the small intestine and compared the mutation patterns with those established for colorectal cancers. DNA was extracted from 15 formalin‐fixed, paraffin‐embedded lesions. Codons 12, 13 and 61 of the Ki‐ras gene, exons 5–8 of the p53 gene, and codons 1268–1569, which contain the mutation cluster region (MCR) of the APC gene, were amplified by means of PCR, subcloned and sequenced. Mutations of the Ki‐ras and p53 genes were observed in 8 (53.3%) and 4 lesions (26.7%), respectively. The mutational frequency of the Ki‐ras gene in the present series of small intestinal carcinomas was similar, while that of the p53 gene was slightly lower than the reported frequencies for colorectal carcinomas. Only one case showed a mutation of the APC gene, involving an insertional mutation of an adenine at codons 1554–1556 with formation of a stop codon immediately downstream. Since the occurrence of an APC mutation is considered an early event in colorectal carcinogenesis, our findings indicating an extremely low frequency of such changes in and around the MCR suggest that carcinomas of the small intestine arise via a genetic pathway distinct from that involved in the development of carcinomas of the colorectum. Int. J. Cancer, 70:390–395, 1997.

Hepatitis B and C viruses infection, lifestyle and genetic polymorphisms as risk factors for hepatocellular carcinoma in Haimen, China.

A case‐control study was carried out to investigate the impact of factors including virus infection, aflatoxin B1, microcystins, smoking/drinking and dietary habits as well as genetic polymorphisms of aldehyde dehydrogenase 2 (ALDH2) and cytochrome P4502E1 (CYP2E1), on susceptibility to hepatocellular carcinoma (HCC) in Haimen, China. A total of 248 patients with HCC and 248 sex‐, age‐ and residence‐matched population‐based controls were recruited into the study. Virus infection, and ALDH2 and CYP2E1 gene polymorphisms were assessed in 134 paired cases and controls. By univariate analysis, hepatitis B virus (HBV) infection (odds ratio [OR]=9.75; 95% confidence interval [CI] =4.71–20.2), history of intravenous injection (OR=1.50; 95%CI=1.02–2.22), average income (OR=0.63; 95% CI=0.43–0.92), frequent intake of foods rich in protein, e.g., egg (OR=0.6; 95% CI=0.42–0.87), chicken (OR=0.53; 95% CI=0.35–0.79), pork (OR=0.67; 95% CI=0.46–0.98) and fresh fish (OR=0.58; 95% CI=0.39–0.87) significantly differed between cases and controls. However, peanut intake (OR=0.66; 95% CI=0.43–1.01), source of drinking water, including tap (OR=1.33; 95% CI=0.81–2.20), deep well (OR=0.94; 95% CI=0.56–1.55), shallow well (OR=0.85; 95% CI=0.55‐–1.30), river (OR=0.95; 95% CI=0.65–1.38), ditch (OR=1.09; 95% CI=0.76–1.55) and pond water (OR=1.0; 95% CI=0.14–7.10) were not significantly associated with risk. Univariate analysis also indicated that the 1–1 genotype of ALDH2 (OR=1.38; 95% CI=0.86–2.23) as well as the Pst1‐ and Rsa1‐digested c1/c1 genotype of CYP2E1 (OR=1.36; 95% CI=0.81–2.28), was slightly more frequent in the case group. On multivariate analysis, HBV infection (OR=13.9; 95% CI=5.78–33.6) and history of intravenous injection (OR=2.72; 95% CI=1.24–6.00) were still associated with significantly increased risk of HCC, while frequent intake of fresh fish (OR=0.32; 95% CI=0.12–0.86) decreased this risk. These findings suggest that whereas peanut intake, water sources as well as genetic polymorphisms in ALDH2 and CYP2E1 do not significantly correlate with the risk of HCC, HBV infection is a main risk factor, and dietary items rich in protein, especially fresh fish, might protect against the risk of HCC in Haimen, China.

Depletion of mitochondrial fission factor DRP1 causes increased apoptosis in human colon cancer cells

Mitochondria play a critical role in regulation of apoptosis, a form of programmed cell death, by releasing apoptogenic factors including cytochrome c. Growing evidence suggests that dynamic changes in mitochondrial morphology are involved in cellular apoptotic response. However, whether DRP1-mediated mitochondrial fission is required for induction of apoptosis remains speculative. Here, we show that siRNA-mediated DRP1 knockdown promoted accumulation of elongated mitochondria in HCT116 and SW480 human colon cancer cells. Surprisingly, DRP1 down-regulation led to decreased proliferation and increased apoptosis of these cells. A higher rate of cytochrome c release and reductions in mitochondrial membrane potential were also revealed in DRP1-depleted cells. Taken together, our present findings suggest that mitochondrial fission factor DRP1 inhibits colon cancer cell apoptosis through the regulation of cytochrome c release and mitochondrial membrane integrity.

Sarcomatoid renal cell carcinoma. A study of its proliferative activity

Background. Sarcomatoid renal cell carcinoma is an uncommon renal tumor consisting of carcinomatous and sarcomatous components. Although this tumor shows highly malignant biologic behavior, little is known about its proliferative activity.

Mutational Analysis of the p53 and K-ras Genes and Allelotype Study of the Rb-1 Gene for Investigating the Pathogenesis of Combined Hepatocellular-Cholangiocellular Carcinomas

Because combined hepatocellular‐cholangiocellular carcinoma is rare and its biological features and pathogenesis have not been well established, we investigated alterations of the p53, K‐ras and Rb‐1 genes, as well as expression patterns of carcinoembryonic antigen and keratin, in seven combined hepatocellular‐cholangiocarcinoinas out of 557 hepatocellular carcinomas autopsied at Tokyo University during 30 years. Mutations of the p53 gene were found in two cases, at codon 244 (GGC to TGC) in the cholangiocellular carcinoma component of case 1 (mixed type, showing an intimate intermingling of both elements) and at codon 234 (TAC to AAC) in both components of case 5 (combined type, consisting of contiguous but independent masses of both elements). Mutation of the K‐ras gene (codon 12, GGT to GAT) was seen only in the cholangiocellular carcinoma component of clinically apparent double cancer, case 6. Allelic alteration of the Rb‐1 gene was observed in two cases, deletion of both alleles in the hepatocellular carcinoma component of case 3 (combined type) and replication error of the same pattern in both components of case 4 (mixed type). Immunohistochemical analysis showed that the hepatocellular carcinoma components of five cases (cases 2, 3, 5, 6, 7) were immunoreactive for keratin, suggesting biliary epithelial transformation. In four of the five cases (cases 3 and 5 combined, case 7 mixed and case 6 double cancer), cholangiocellular carcinoma components were also positive for keratin. These results suggest that both components of combined hepatocellular‐cholangio‐carcinoma have the same genetic and phenotypic character and might have arisen from the same origin in some cases.

DNA repair and cancer: Lessons from mutant mouse models

DNA damage, if the repair process, especially nucleotide excision repair (NER), is compromised or the lesion is repaired by some other error‐prone mechanism, causes mutation and ultimately contributes to neoplastic transformation. Impairment of components of the DNA damage response pathway (e.g., p53) is also implicated in carcinogenesis. We currently have considerable knowledge of the role of DNA repair genes as tumor suppressors, both clinically and experimentally. The deleterious clinical consequences of inherited defects in DNA repair system are apparent from several human cancer predisposition syndromes (e.g., NER‐compromised xeroderma pigmentosum [XP] and p53‐deficient Li‐Fraumeni syndrome). However, experimental studies to support the clinical evidence are hampered by the lack of powerful animal models. Here, we review in vivo experimental data suggesting the protective function of DNA repair machinery in chemical carcinogenesis. We specifically focus on the three DNA repair genes, O6‐methylguanine‐DNA methyltransferase gene (MGMT), XP group A gene (XPA) and p53. First, mice overexpressing MGMT display substantial resistance to nitrosamine‐induced hepatocarcinogenesis. In addition, a reduction of spontaneous liver tumors and longer survival times were evident. However, there are no known mutations in the human MGMT and therefore no associated cancer syndrome. Secondly, XPA mutant mice are indeed prone to spontaneous and carcinogen‐induced tumorigenesis in internal organs (which are not exposed to sunlight). The concomitant loss of p53 resulted in accelerated onset of carcinogenesis. Finally, p53 null mice are predisposed to brain tumors upon transplacental exposure to a carcinogen. Accumulated evidence in these three mutant mouse models firmly supports the notion that the DNA repair system is vital for protection against cancer.

Different frequencies of p53 codon‐249 hot‐spot mutations in hepatocellular carcinomas in Jiang‐Su province of China

Environmental carcinogens often induce specific mutations in the p53 gene, apparent in tumors. The relation between aflatoxin B1(AFB1)‐related hepatocellular carcinomas (HCCs) and hot spot at codon 249 of the p53 gene has received a great deal of attention, but its significance is still controversial. To clarify this problem, we analyzed the p53‐mutational status of HCCs in Jiang‐su province in China, where AFB1 contamination of the staple food significantly differs between the northern and southern parts (prominent only in the latter), while other conditions are quite similar. Background liver status and mutations in exons 5 to 8 of p53 in a total of 31 cases were divided approximately equally between the 2 areas. In all, 15 tumors exhibited a total of 17 mutations in the p53 gene; 9 cases from the southern part of the province had the hot‐spot mutation at codon 249 (9/16, 56%), but only one case from the northern part (1/15, 8%). These results suggest that AFB1 contamination may correlate with codon‐249 mutations in HCC. Int. J. Cancer 82:187–190, 1999.