Nobuo Tsuchida

Promoter hypermethylation profile of tumor‐associated genes p16, p15, hMLH1, MGMT and E‐cadherin in oral squamous cell carcinoma

Aberrant promoter hypermethylation of tumor‐associated genes leading to their inactivation is a common event in many cancer types. Using a sensitive restriction‐multiplex PCR method, we studied the promoter hypermethylation profile of the p16, p15, hMLH1, MGMT and E‐cad genes in oral squamous cell carcinoma (OSCC) of Indians. We analyzed a total of 51 samples for the p15 tumor‐suppressor gene and 99 samples for each of the remaining genes. Our studies indicate an incidence of promoter hypermethylation of 23% each for p16 and p15, 8% for hMLH1, 41% for MGMT and 35% for E‐cad. We observed aberrant hypermethylation of the promoter region of at least 1 of these genes in 74.5% of cases (n = 51) for which all the 5 genes were studied. Abnormal methylation was detected in tumors irrespective of stage and location in the oral cavity, whereas no abnormal methylation was detectable in normal oral squamous tissues obtained from 25 OSCC patients. Detection of aberrant hypermethylation patterns of cancer‐associated genes listed above is therefore suitable for diagnosis of OSCC in individuals at high risk for this disease.

Uniform nomenclature for the mitochondrial contact site and cristae organizing system

The mitochondrial inner membrane contains a large protein complex that functions in inner membrane organization and formation of membrane contact sites. The complex was variably named the mitochondrial contact site complex, mitochondrial inner membrane organizing system, mitochondrial organizing structure, or Mitofilin/Fcj1 complex. To facilitate future studies, we propose to unify the nomenclature and term the complex “mitochondrial contact site and cristae organizing system” and its subunits Mic10 to Mic60.

Cytoplasmic retention of the p53 tumor suppressor gene product is observed in the hepatitis B virus X gene-transfected cells

It has been suggested that hepatitis B virus (HBV) X gene activates X gene expression by disrupting the function of p53 tumor suppressor gene (Takada et al., 1996). To find out their connection, effect of X protein expression on the nuclear localization of p53 protein in human hepatoma cells was examined by the immunofluorescent double-staining technique. The location of transiently-expressed p53 protein was examined in X gene-transfected cells, where X protein was detected in the cytoplasm. The nuclear location of transiently-expressed p53 protein was changed to the cytoplasm by X protein co-expression. Endogenous p53 protein was also observed in the cytoplasm by X protein expression. The transcriptional activation domain of X protein and the carboxy-terminal region of p53 protein were found mutually responsible for the cytoplasmic retention of p53 protein in X gene-transfected cells. Therefore, the cytoplasmic retention of p53 protein may be closely correlated to the function of X protein expressed in transfected cells.

Aberrant expression and function of p53 in T-cells immortalized by HTLV-I Tax1

The expression and function of p53 tumor suppressor protein was investigated in T‐cells immortalized by the Tax1 protein of HTLV‐I. Conformationally wild‐type p53 was expressed at elevated levels in Tax1‐immortalized T‐cells by post‐transcriptional mechanisms when compared with normal T‐cells. Luciferase assays with a reporter plasmid containing p53‐binding sites revealed an impairment in the transactivating function of p53 in Tax1‐immortalized T‐cells. Our results suggest an important role for Tax1 in the aberrant expression and function of p53 observed in many HTLV‐I transformed cells.

Transcriptional activation of the human stress-inducible transcriptional repressor ATF3 gene promoter by p53.

Activating transcription factor 3 (ATF3) is an immediate early response gene that is induced in cells exposed to a variety of stress stimuli. In this report, upon exposure of cells to ultraviolet (UV) or proteasome inhibitor MG132, ATF3 protein was induced more efficiently in cells with intact p53 allele than in those with null mutant p53 allele. In Saos-2 cells harboring the temperature-sensitive mutant p53(Val-138), the expression of ATF3 gene was more significant at permissive temperature of 32.5 degrees C than at non-permissive 37.5 degrees C. Reporter assay of the human ATF3 gene promoter identified two p53-responsive elements at -379 to -370 and -351 to -342 from the transcriptional start site. These elements were capable of conferring p53 responsiveness to a heterologous promoter and specifically bound p53 protein in electrophoretic mobility shift assay. Furthermore, ATF3 gene promoter was more significantly activated by UV in cells with wild p53 allele. These results clearly show that the human ATF3 gene is one of the target genes directly activated by p53 and may suggest a functional link between stress-inducible transcriptional repressor ATF3 and p53.

Ras oncogenes in oral cancer: The past 20 years

Oral squamous cell carcinoma (OSCC) of head and neck is associated with high morbidity and mortality in both Western and Asian countries. Several risk factors for the development of oral cancer are very well established, including tobacco chewing, betel quid, smoking, alcohol drinking and human papilloma virus (HPV) infection. Apart from these risk factors, many genetic factors such as oncogenes, tumor suppressor genes and regulatory genes are identified to involve in oral carcinogenesis with these risk factors dependent and independent manner. Ras is one of the most frequently genetically deregulated oncogene in oral cancer. In this review, we analyze the past 22years of literature on genetic alterations such as mutations and amplifications of the isoforms of the ras oncogene in oral cancer. Further, we addressed the isoform-specific role of the ras in oral carcinogenesis. We also discussed how targeting the Akt and MEK, downstream effectors of the PI3K/Akt and MAPK pathways, respectively, would probably pave the possible molecular therapeutic target for the ras driven tumorigenesis in oral cancer. Analysis of these ras isoforms may critically enlighten specific role of a particular ras isoform in oral carcinogenesis, enhance prognosis and pave the way for isoform-specific molecular targeted therapy in OSCC.

Aberrant expression of the p53 tumor suppressor gene in adult T-cell leukemia and HTLV-I-infected cells.

By immunoprecipitation analysis, enhanced p53 expression was detected in 3 of 4 adult T‐cell leukemia (ATL) cell lines, 1 of 3 HTLV‐I‐infected cell lines and 1 of 5 fresh ATL samples, compared with phytohemagglutinin‐stimulated peripheral blood lymphocytes. Among these 5 high expressers, p53 missense mutations were indicated in 2 ATL cell lines and 1 fresh ATL sample by extensive p53 cDNA and genomic DNA polymerase chain reaction single‐strand conformation polymorphism analysis. No mutation was found throughout the entire coding region of the remaining 2 high expressers (1 ATL and 1 HTLV‐I‐infected cell lines) and low expressers of p53 (2 HTLV‐I‐infected cell lines). Tax oncoprotein expression was found in these 2 high p53 expressers in which p53 mutation was not present, but not in low p53 expressers or cells carrying this mutation. The levels of p53 mRNA were similar among the samples regardless of p53 levels. Posttranscriptional mechanisms other than missense mutation would thus appear to increase p53 in the Tax‐expressing cells hut not in cells containing undetectablc levels of Tax. No complex formation between p53 and Tax was observed.

Novel Apoptosis-Inducing Activity in Bacteroides forsythus: a Comparative Study with Three Serotypes of Actinobacillus actinomycetemcomitans

ABSTRACT Bacteroides forsythus, which has been reported to be associated with periodontitis but has not been recognized as a key pathogen, was found to induce cytolytic activity against HL-60 and other human leukemic cells. This cytolytic activity was demonstrated according to three different criteria: (i) loss of both mitochondrial membrane potential and membrane integrity in cells treated with bacterial extracts and then with Rh123 and propidium iodide, respectively, as demonstrated by flow cytometry; (ii) damage to cytoplasmic membrane, as revealed by scanning electron microscopy (SEM); and (iii) DNA ladder formation and activation of caspase-3. These results indicate that B. forsythus produced an apoptosis-inducing factor(s) found to be composed of protein as judged by heat and trypsin sensitivity. In addition to extracts from B. forsythus, the culture supernatant of this bacterium has the ability to induce a cytolytic effect against peripheral white blood cells, especially lymphocytes. For comparison with B. forsythus, the same analyses were applied to two strains with different serotypes ofActinobacillus actinomycetemcomitans, serotypes a (ATCC 43717) and c (ATCC 43719), in addition to previously reported apoptosis-inducing serotype b (ATCC 43718), which was used as a positive control. The strains of A. actinomycetemcomitansserotypes a and b induced apoptosis in HL-60 cells as judged by the above three criteria but to a slightly lesser extent than did B. forsythus, while the serotype c strain produced apoptosis to a negligible extent. Detailed SEM images showed that the A. actinomycetemcomitans serotype a strain induced large-pore formation and the serotype b strain produced small pores with typical blebbing, while B. forsythus induced severe membrane ruffling. Further DNA ladder formation and caspase-3 activation were observed in the serotype a and b strains but not in the serotype c strain. The present paper is the first report of a protein factor(s) from B. forsythus and the A. actinomycetemcomitans serotype a strain which induces apoptotic cell death.

p53 gene mutations in oral carcinomas from India

In this study, we analyzed 53 oral squamous‐cell carcinomas among Indians for the presence of alterations in the tumor‐suppressor gene p53 by PCR‐SSCP and sequencing methods. Our results showed that 21% (II/53) of oral carcinomas analyzed carried mutations within the exons 5–8 of the p53 gene. We have identified II single‐base pair substitutions consisting of 10 mis‐sense mutations and one at the splice acceptor site, and one deletion mutation involving 4 consecutive bases. The majority of the base substitutions were transitions (5 TA to CG and 5 GC to AT), while only one transversion (TA to GC) was observed. Probable hot‐spots for the mutation induction were identified at codons 149 and 274, which have not been observed before in head‐and‐neck cancers. The mutational spectrum might have originated from base alkylations at guanine and thymine residues, caused by some alkylating agents. The present results are thus consistent with the involvement of tobacco‐related nitrosoamines in the etiology of oral squamous‐cell carcinoma.

Interaction between hsp70 and hsp40, eukaryotic homologues of DnaK and DnaJ, in human cells expressing mutant‐type p53

We have recently identified a novel 40‐kDa heatshock protein hsp40 as a mammalian homologue of bacterial DnaJ protein. Here we demonstrate the physical interaction between hsp70 (DnaK homologue) and hsp40 in human cells as determined by immunoprecipitation methods. Co‐immunoprecipitation of hsp70 with hsp40 was dependent on the presence of ATP or unfolded protein (reduced carboxymethylated α‐lactalbumin). A mutant type of tumor suppressor gene product, mtp53, was co‐immunoprecipitated not only with hsp70 but also with hsp40. These results suggest the existence of a hsp70(DnaK)/ hsp40(DnaJ) chaperone system in mammalian cells.