Yukiharu Sawada

Detection of Human Papillomavirus DNA Sequences in Oral Squamous Cell Carcinomas and Their Relation to p53 and Proliferating Cell Nuclear Antigen Expression

Background. The etiology of oral squamous cell carcinoma (SCC) is still obscure. Since human papillomavirus (HPV) DNAs are associated with carcinoma of the uterine cervix, carcinomas of the oral cavity were investigated to ascertain if these viruses are present in squamous carcinomas of this anatomic site.

Structure and gene organization in the transforming Hind III-G fragment of Ad12

The nucleotide sequence of the transforming Hind III-G fragment of Ad12 DNA which encompasses the left 6.8% of the genome has been determined. The fragment was 2320 nucleotides long, and contained a GC cluster at positions 126-155 and a region extremely rich in AT at positions 1098-1142 (number from the leftmost end). Possible coding regions for the two transforming gene products were assigned. The predicted coding region for T antigen g is positions 502-1069 and positions 1144-1373, which are joined by splicing (266 amino acid residues, 30 kd), and that for T antigen f is positions 1845-2126 (94 amino acid residues, 10 kd). The sequence of the Hind III-G fragment was compared with that of the transforming DNA fragment of Ad5 which encompasses the left 8.0% of the genome (2809 nucleotides). There are several discrete regions with significant sequence homology. The comparison suggests that the regions in the left two thirds of the Ad5 and Ad12 transforming DNA fragments (map units 0-4.7% in Ad5 and 0-4.4% in Ad12) bear some resemblance in their gene organizations, and code for proteins containing structurally homologous regions.

Thes physical state of human papillomavirus 16 DNAxs in cervical carcinoma and cervical intraepithelial neoplasia

Cervical carcinomas and cervical intraepithelial neoplasias (CIN) were analyzed for the presence of human papillomavirus (HPV) DNA using Southern blot hybridization. of the five HPV types examined (HPV types 6, 11, 16, 18, and 33), HPV 16 DNA was detected most frequently. in most HPV 16‐positive carcinomas examined, HPV 16 DNA was present in an integrated state in cellular DNA with or without the coexistence of episomal species. in one case, however, only episomal species were detected. Among seven cases of HPV 16‐positive CIN, four contained HPV 16 DNA only in the episomal state and the rest contained HPV 16 DNA only in the integrated state, but the coexistence of both states was not found. These results suggest that the integration of HPV 16 DNA is not necessary for cells to become malignant, although it is frequently associated with malignant cells.

Characterization of adenovirus type 40 E1 region

The left-most 3.9 kb of adenovirus type 40 (Ad40) DNA has been sequenced using cloned viral DNA fragments. The Ad40 E1 region is deduced to code for at least four polypeptides, 221 and 249 amino acids as E1A products in addition to 166 and 475 amino acids as E1B products. E1B polypeptides share about 50% homology with well-defined adenovirus types, 2/5, 7, and 12, throughout the E1B sequences. E1A homology of Ad40 to these types is relatively lower than that of E1B, while highly conserved regions of E1A are retained to a certain level in Ad40 as well. Activity for morphological transformation of Ad40 E1A on 3Y1 cells is considerably lower when compared to that of Ad5 and Ad12 E1A genes. Transient chloramphenicol acetyltransferase (CAT) expression assay shows that Ad40 E1A has a trans-acting function, though lower than that of other E1A genes, on adenovirus early promoter. The Ad40 E1A promoter also holds only a little cis-acting activity in 3Y1 cells. Lower activities of both Ad40 E1A promoter and certain E1A functions may explain in part the difficulty in propagation of Ad40.

Three different classes of human adenovirus transforming DNA sequences: Highly oncogenic subgroup A-, weakly oncogenic subgroup B-, and subgroup C-specific transforming DNA sequences

Abstract DNA-DNA homology measurements showed the presence of three different classes of human adenovirus transforming gene sequences: one specific for highly oncogenic subgroup A (types 12, 18, and 31), the second specific for weakly oncogenic subgroup B (types 3, 7, and 16), and the third specific for transforming subgroup C (types 2, 5, and 6). Adenovirus transforming DNA sequences with molecular weights of 1.6–1.8 × 106 are in common or almost identical among members within the same subgroup. Less than one-third of the transforming DNA sequences are shared among members of different subgroups. All of the DNA fragments containing transforming DNA sequences investigated are located at the end of the molecule.

Cloning and characterization of rat p27Kip1, a cyclin-dependent kinase inhibitor

Cyclin-dependent kinase (Cdk) inhibitors play significant roles in the cell cycle control of various biological phenomena. To characterize the role of Cdk inhibitors in rat cells, we isolated a cDNA encoding rat p27Kip1, a 27-kDa Cdk inhibitor. The 1.04-kb cDNA of rat p27 contained an open reading frame of 197 amino acids that shared high homology with mammalian p27 and significant homology with mammalian p21Cip1 and p57Kip2. p27 mRNA was detected in most rat tissues and cell lines. The levels of p27 protein expression were similar in rat cell lines transformed by E1A and in normal cells. Rat p27 was able to interact with Cdk 2/4 and cyclin A/D in rat cells, but the amounts of rat p27 in Cdk2 complexes were different between transformed cells and normal cells. Thus, the formation of stable complexes of rat p27 may be modulated by E1A. Rat p27 protein could inhibit the increased Cdk2-associated kinase activity in transformed rat cells.

Adenovirus type 5 and adenovirus type 12 recombinant viruses containing heterologous E1 Genes are viable, transform rat cells, but are not tumorigenic in rats

Two sets of adenovirus type 5 (Ad5)-adenovirus type 12 (Ad12) recombinant viruses were constructed and analyzed. In one case the Ad12 E1A, E1B, or E1A plus E1B genes were substituted for the corresponding Ad5 genes in the Ad5 chromosome. The second set contained the Ad5 E1A, E1B, or E1A plus E1B genes in place of the cognate Ad12 genes in the Ad12 chromosome. The hybrid viruses were all viable and expressed the appropriate E1 antigens. They were able to transform secondary rat fibroblasts, but at reduced efficiency as compared to either parental virus. Fibroblasts transformed with the recombinant Ad5 virus carrying the Ad12 E1A plus E1B genes were tumorigenic in newborn, syngeneic rats. Some of the cell lines transformed with the Ad5 virus containing the Ad12 E1A gene were tumorigenic but none of the recombinants with the Ad12 E1B gene was able to induce tumors in this assay. Although Ad12 was tumorigenic, none of the Ad5 or Ad12 recombinant viruses induced tumors in newborn rats injected either intracerebrally or subcutaneously with virus particles.

Nucleotide sequence of the adenovirus type 40 inverted terminal repeat: Close relation to that of adenovirus type 5

Human adenovirus type 40 (Ad40) is a pathogen that causes acute infantile gastroenteritis. Ad40 has the distinct characteristic of being difficult to propagate in conventional cultured human cells. The nucleotide sequence of the inverted terminal repeat (ITR) of Ad40, which includes the origin of adenoviral DNA replication, was determined using recombinant plasmid DNA. By using our newly developed program to express the ITR homologies simply, we found that the ITR of Ad40, which is 163 nucleotides long, was related most closely to that of adenovirus type 5, which replicates efficiently.

Nuclear export of adenovirus E4orf6 protein is necessary for its ability to antagonize apoptotic activity of BH3-only proteins

The adenovirus E4orf6 is a viral oncoprotein known to cooperate with the E1A gene product in transforming primary murine cells. It has been shown to inhibit the apoptotic activities of p53 and p73 through direct binding to these proteins. Here, we demonstrate that the adenovirus E4orf6 protein inhibits apoptosis mediated by BNIP3 and Bik, which are BH3-only proteins of the Bcl-2 family. This activity was not mediated by p53 and p73 because E4orf6 had the same effect on the apoptosis in Saos-2 cells that do not express p53-related genes. It was also ascertained that E4orf6 could change the mitochondrial localization of BNIP3 and Bik. A mutant lacking the nuclear export signal of E4orf6 failed to inhibit apoptosis and to translocate BNIP3 protein from the mitochondria. Moreover, it was also established that E4orf6 was able to interact with BNIP3 and Bik. In BNIP3 protein, the region required for the interaction included the transmembrane domain, which is required for the localization of BNIP3 to the mitochondria. These results suggest that E4orf6 is exported from the nucleus to the cytoplasm, enabling it to interact with BH3-only proteins, eventually leading to the inhibition of apoptotic activity.

Nuclear factor I stimulates transcription of the adenovirus 12 E1A gene in a cell-free system

Binding to the cis-acting region of NF-I-like protein and/or NF-III-like protein was previously suggested to be responsible for the preferential stimulation of transcription from distal start-site of the adenovirus 12 E1A gene in a cell-free system. In this study, nuclear extracts of Ehrlich ascites tumor cells depleted of NF-I-like protein were found to lose activity to stimulate the E1A gene transcription. This activity was recovered when NF-I purified from HeLa cells with no contamination of NF-III was supplemented. It is thus evident that NF-I is involved in stimulating distal transcription of the adenovirus 12 E1A gene. Moreover, activities for both stimulating the E1A gene transcription and binding to a region recognized by NF-I did not apparently exist in nuclear extracts of a cell line expressing the adenovirus 12 E1A gene. These results suggest that transcription of the adenovirus 12 E1A gene may possibly be autoregulated at least in part through modulation of the activity of NF-I.