Protein kinase C (PKC) plays a crucial role(s) in regulation of growth and differentiation of cells. In the present study, we examined possible roles of the ␣, ␦, , and isoforms of PKC in squamous differentiation by overexpressing these genes in normal human keratinocytes. Because of the difficulty of introducing foreign genes into keratinocytes, we used an adenovirus vector system, Ax, which allows expression of these genes at a

hic-5 was originally isolated as an H2O2-inducible cDNA clone whose product was normally found at focal adhesions. In this study, we found that Hic-5 accumulated in the nucleus in response to oxidants such as H2O2. Other focal adhesion proteins including paxillin, the most homologous to Hic-5, remained in the cytoplasm. Mutation analyses revealed that the C- and N-terminal halves of Hic-5 contributed to its nuclear localization in a positive and negative manner, respectively. After the finding that leptomycin B (LMB), an inhibitor of nuclear export signal (NES), caused Hic-5 to be retained in the nucleus, Hic-5 was demonstrated to harbor NES in the N-terminal, which was sensitive to oxidants, thereby regulating the nuclear accumulation of Hic-5. NES consisted of a leucine-rich stretch and two cysteines with a limited similarity to Yap/Pap-type NES. In the nucleus, Hic-5 was suggested to participate in the gene expression of c-fos. Using dominant negative mutants, we found that Hic-5 was actually involved in endogenous c-fos gene expression upon H2O2 treatment. Hic-5 was thus proposed as a focal adhesion protein with the novel aspect of shuttling between focal adhesions and the nucleus through an oxidant-sensitive NES, mediating the redox signaling directly to the nucleus.

Hic-5 is a paxillin homologue with four LIM domains in its C-terminal region, localized mainly in focal adhesions in normal fibroblasts. Hic-5 is also known to associate with focal adhesion kinase (FAK) or the related CAKbeta, and with vinculin. In the present study, we examined changes in Hic-5 and paxillin protein levels in primary mouse embryo fibroblasts (MEF) during mortal and immortal stages. The Hic-5 level was markedly decreased when cells became immortalized, whereas that of paxillin was increased. The vinculin level was not changed significantly. Hic-5 was mainly localized in focal adhesion plaques of mortal MEF but was localized in the nuclear periphery in the immortalized MEF; the number of focal adhesion plaques was decreased in these cells. Mouse Hic-5 contains three LD domains in its N-terminal half, and the first LD domain (LD1) appears to be involved in interaction with FAK. However, this interaction was not essential for recruitment of Hic-5 to focal adhesions, since its subcellular localization was similar in FAK(-/-) cells. Forced expression of Hic-5 decreased colony forming ability of MEF from FAK(+/+) mice, but not of FAK(-/-) cells. These observations suggested the involvement of Hic-5 in determination of cellular proliferative capacity in collaboration with other cytoskeletal components.

DNA adducts are a major cause of DNA mutation and DNA mutation-related diseases, but the simultaneous identification of multiple DNA adducts has been a challenge for a decade. An adductome approach using consecutive liquid chromatography and double mass spectrometry after micrococcal nuclease treatment has paved the way to demonstrations of numerous DNA adducts in a single experiment and is expected to contribute to the comprehensive understanding of overall environmental and endogenous exposures to possible mutagens in individuals. In this report, we applied an adductome approach to gastric mucosa samples taken at the time of a gastrectomy for gastric cancer in Lujiang, China, and in Hamamatsu, Japan. Seven lipid peroxidation-related DNA adducts [1,N6-etheno-2'-deoxyadenosine, butanone-etheno-2'-deoxycytidine (BεdC), butanone-etheno-2'-deoxy-5-methylcytidine, butanone-etheno-2'-deoxyadenosine (BεdA), heptanone-etheno-2'-deoxycytidine, heptanone-etheno-2'-deoxyadenosine (HεdA) and heptanone-etheno- 2'-deoxyguanosine] were identified in a total of 22 gastric mucosa samples. The levels of these adducts ranged from 0 to 30,000 per 10(9) bases. Although the presence of Helicobacter pylori DNA in the mucosa was not related to these adducts level, the levels of BεdC, BεdA and HεdA were higher in the Japanese gastric mucosa samples. The profiles of these 7 adduct levels among the 21 cases were capable of discriminating between the possible origins (China or Japan) of the gastric mucosa samples. Our report is the first demonstration of lipid peroxidation-related DNA adducts in the human stomach, and these observations warrant further investigation in the context of the significance of DNA adducts in human gastric carcinogenesis.

PKC is activated on the cell membrane by phospholipids, thereby transducing signals to intracellular pathways. We provide here another function of PKC, namely, regulating cell cycle by interaction with the cyclin E/ cdk2/p21 complex. Among the 10 isoforms of PKC, PKCZ is predominantly expressed in squamous cell epithelia and induces terminal di erentiation of keratinocytes. PKCZ that is endogenously expressed or overexpressed was found to associate with the cyclin E/cdk2/p21 complex in keratinocytes of mice and humans. Requirement of a possible adaptor protein to the binding was suggested by the reconstitution of PKCZ and the cyclin E/cdk2/p21 complex which were prepared from human keratinocytes or Sf9 insect cells. Colocalization of PKCZ with cdk2 and cyclin E was observed in the cytoplasm, particularly in the perinuclear region. p21 was phosphorylated in the complex in a PKC-activator dependent manner. Association of PKCZ with cdk2 resulted in marked inhibition of cdk2-kinase activity when measured by phosphorylation of Rb. Dominant negative PKCZ associated with the cyclin E/cdk2/p21 complex, but caused a little inhibition of cdk2 kinase activity. Among the known regulatory mechanisms of cdk2 activity, dephosphorylation of Thr160 was demonstrated. Oncogene (2000) 19, 6334 ± 6341.

We found that 12-O-tetradecanoylphorbol-13-acetate (TPA) promoted anchorage-independent growth but did not affect anchorage-dependent growth of MIA PaCa-2 human pancreatic carcinoma cells. TPA markedly activated mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase in an anchorage-independent manner. Two protein kinase C (PKC) isoforms, conventional PKC (cPKC) and novel PKC (nPKC), but not apical PKC, translocated from the cytosolic to the particulate fraction upon TPA treatment. To identify the PKC isoforms involved in the regulation of anchorage-independent growth, four PKC isoforms (alpha, delta, epsilon, and zeta) were forced to be expressed in MIA PaCa-2 cells with an adenovirus vector. Overexpression of nPKCdelta or nPKC epsilon activated MAPK and promoted anchorage-independent growth. Overexpression of cPKCalpha alone did not influence anchorage-independent growth but lowered the concentration of TPA that was required to enhance such growth. Expression of constitutively active MAPK kinase-1 (MEK1) also promoted anchorage-independent growth. Furthermore, PKC inhibitors or an MEK inhibitor completely suppressed both TPA-induced activation of MAPK and promotion of anchorage-independent growth, but a cPKC-selective inhibitor partially suppressed TPA-induced promotion of the growth. Based on these results, we suggest that MAPK activation, mediated by certain isoforms of PKC, plays a part in oncogenic growth of MIA PaCa-2 cells. In summary, our data indicated that specific inhibitors of the cPKC and nPKC signaling pathway might be selective anti-oncogenic growth agents for some types of human pancreatic cancer.

No abstract

The dopaminergic brain pathway is involved in many addictive behaviours, hence represents a good candidate in the study of smoking behaviour and nicotine addiction. Dopamine beta hydroxylase (DBH) is an enzyme that catalyses the conversion of dopamine into noradrenaline. This study, the first of its kind, was done to investigate the role of DBH rs5320 polymorphism in smoking behaviour of elderly Japanese. This was done by collecting blood samples from 2521 subjects with various smoking habits to genotype the DBH rs5320 polymorphism. Participants also had to fill out a questionnaire containing questions regarding their lifestyles. Some of the questions were from the Fagerströ m Test for Nicotine Dependence (FTND) and the Tobacco Dependence Screener (TDS). It was found that male ever-smokers with AA genotype smoked less cigarettes per day than those with GG and AG genotypes. FTND scores were also lowest in male ever-smokers with AA genotype and in female ever-smokers with AG genotype. There was no correlation detected between the TDS scores and any of the genotypes. This study shows that DBH rs5320 polymorphism influences nicotine dependence.