Masahide Ishibashi

Cloning and characterization of human MCM7 promoter

MCM7 is a member of the MCM protein family which has been implicated in the regulatory machinery allowing DNA to replicate only once during S phase. In quiescent cells, human MCM7 (hMCM7) mRNA is almost undetectable. Stimulation of cells to enter the cell cycle results in induction of hMCM7 expression. Here, we report cloning and characterization of the hMCM7 promoter. We isolated and sequenced a 0.5 kb genomic fragment that contains putative transcription factor binding sites including three E2F sites, three GC boxes and an E box. Several transcription start sites, which were used upon growth stimulation, were identified. The minimal promoter region required for transcription of a luciferase reporter gene was delineated, and it contained an E box and one E2F site, which were important for promoter activity. Interestingly, the cloned sequence appears to act as a promoter for mu-adaptin-related protein 2 (mu-ARP2) gene in the opposite orientation.

Immunolocalization of hCDC47 protein in normal and neoplastic human tissues and its relation to growth

hCDC47 is a human member of the MCM family, which has been implicated in the regulatory machinery causing DNA to replicate once per cell cycle. We examined its protein expression and localization in normal human tissues, using immunostaining with polyclonal antibodies. Positive nuclei were found in the proliferative components of lymph nodes, bone marrow, epidermis and mucosa. Immunohistochemical analysis was also performed for 3 types of cutaneous keratinocytic tumor originating from same cell type but showing different grades of malignancy. In seborrheic keratosis, a benign condition, cells with hCDC47‐positive nuclei were located in the outermost layers of the tumor lobules, while in Bowens disease, carcinomas in situ and squamous‐cell carcinomas, they were present throughout the lesions. The percentages of hCDC47‐positive cells were 65.4% in squamous‐cell carcinomas, 60.9% in Bowens disease, 12.6% in seborrheic keratosis and 3.9% in normal epidermis (n = 5 in all cases). Further expansion of the analysis to include malignant tumors from several other organs revealed that all malignant lesions tested contained more nuclear hCDC47‐positive cells than their normal counterparts. Our findings indicate that hCDC47 plays a role in normal and neoplastic cell growth in vivo and that hCDC47 immunolocalization could be used as an index of cell proliferation in tissue sections.Int. J. Cancer 74:180–184, 1997.

Adenoviruses of Animals

The opening quotation is a part of the description of the first epizootic of adenovirus, in which the etiological agent then named fox encephalitis virus, and now known as canine adenovirus type 1, was isolated. It was more than 20 years later that a group of human viruses were isolated and reported as adenoid-degenerating agents (Rowe et al., 1953) or respiratory-illness agents (Hilleman and Werner, 1954), and the name adenovirus was devised for the group of viruses (Enders et al., 1956). In retrospect, therefore, the canine adenovirus is the first member of the adenovirus family.

Differences in transforming activity and coded amino acid sequence among E6 genes of several papillomaviruses associated with epidermodysplasia verruciformis

Using the polymerase chain reaction technique, we cloned and sequenced DNA fragments containing the E6 genes of the epidermodysplasia verruciformis (EV)-associated HPVs 5, 8, 14, 20, 21, 25, and 47, of which only the sequences of HPVs 5, 8, and 47 have previously been reported. Based on the deduced amino acid sequence homology (57.3 to 83.0%), these HPVs could be divided into two clusters: HPVs 5, 8, and 47, and HPVs 14, 20, 21, and 25. The E6 genes of three HPVs from each cluster were examined for transforming activity toward a cultured rat fibroblast cell line, 3Y1, using the retrovirus-mediated gene transfer technique, and all were found to induce morphological transformation. However, the E6 genes of the first cluster were more potent than those of the second. Since HPVs 5 and 8 are the most frequently found HPVs in malignant lesions of EV patients, the observed in vitro transforming activities of the E6 genes may reflect their oncogenic potential in humans.

Genome organization and taxonomic position of human papillomavirus type 47 inferred from its DNA sequence.

The complete nucleotide sequence of human papillomavirus type 47 (HPV-47) DNA isolated from the lesion of epidermodysplasia verruciformis (EV) was determined. The computer-aided comparison of HPV-47 with other EV-associated viruses using the available sequence data on them revealed that HPV-47 resembles both HPV-5 and HPV-8 as much as HPV-5 and HPV-8 resemble each other, and it led us to regard these three viruses as one cluster and HPV-19 and HPV-25 as another. The conclusion implies that HPV-47 as well as HPV-5 and HPV-8 is associated with the cancer occurrence in EV. Two sets of splicing donor and acceptor sequences in HPV-47, which were previously shown to work in vivo, are also conserved in HPV-5 and HPV-8. One of them allows formation of an ORF predicted to encode an E1/E4 fused protein.

The primary structure of major viral RNA in a rat cell line transfected with type 47 human papillomavirus DNA and the transforming activity of its cDNA and E6 gene

A transformed cell line (RC335) showing higher saturation cell density was obtained from 3Y1 cells (a fibroblastic cell line of rat) transfected with DNA of human papillomavirus type 47 (HPV-47), an epidermodysplasia verruciformis-associated virus, which our laboratory reported previously. The cell line was found to produce a major and several minor species of viral RNAs. The primary structure of the major viral RNA in RC335 was extensively studied and found to consist of two exons and contain open reading frames (ORFs) E6, E7, and a fused ORF, E1/E4. The major RNA was indicated to play an important role in the transformation of RC335 by an experiment with the recombinant retrovirus designed to produce the RNA containing these ORFs, i.e., the recombinant virus induced transformation similar to that in RC335 upon infection of 3Y1 cells. Furthermore the experiments with recombinant viruses carrying a nonsense mutation or large deletion in the above ORF(s) indicated that E6 was necessary and sufficient for the transformation.

The oncogenicity of avian adenoviruses III. In situ DNA hybridization of tumor line cells localized a large number of a virocellular sequence in few chromosomes

Abstract The arrangement of the viral DNA in a CELO virus (an avian adenovirus)-induced rat tumor cell line was analyzed at the molecular and the cellular level. Blot hybridization of restriction endonuclease-digested tumor cell DNA and quantitative hybridization of nondigested tumor cell DNA with 32 P-labeled restriction endonuclease-generated fragments of the viral DNA indicate the following: (1) A contiguous portion of the viral DNA comprising 33% of the viral DNA molecules [i.e., 14.1 kilobases (kb)] is flanked at each terminus by a cellular DNA sequence. This viral DNA sequence, together with >6.1 kb of one of the flanking cellular DNA sequences and >6.8 kb of the other, forms repetition unit (total length >27kb). (2) During the evolution of the original tumor, this unit has attained as many as 160 copies per diploid amount of cellular DNA. In situ DNA hybridization of the cultured tumor cells with 3 H-labeled viral DNA indicates that most of the repetition units are constituents of chromosomal DNA and their distribution is limited to only a few chromosomes per hypotetraploid tumor cell. The majority of chromosomes carrying a large number of repetition units are abnormal chromosomes longer than rat chromosome No. 1.

Temperature-sensitive conditional-lethal mutants of an avian adenovirus (CELO): I. Isolation and characterization

Abstract Forty-nine temperature-sensitive ( ts ) mutants which scarcely grew at 40°, but grew well at 31° were isolated from a wild-type clone, A26, of chicken embryo lethal orphan virus (an avian adenovirus). The stocks of all the ts mutants, except one with a high content of ts + revertants, were tentatively classified into five groups on the basis of the morphological changes they induced in infected chicken kidney cells at 40°. Group I (22 mutants) caused preferential accumulation of viral antigen in the nucleus like the wild-type, A26. Group II (5 mutants) caused accumulation of antigen in both the nucleus and cytoplasm. Group III (11 mutants) caused retention of viral antigen in the cytoplasm with little in the nucleus. Group IV (9 mutants) produced little or no viral antigen. The mutants in groups I to IV, like A26, formed characteristic intranuclear inclusions, and enhanced nuclear Feulgen reaction in infected cells. Group V (1 mutant) produced no viral antigen or intranuclear inclusions. The Feulgen reaction was not enhanced in the nucleus of cells infected with this mutant. At 31°, all the ts mutants caused morphological changes, as did A26.

Chick embryo lethal orphan (CELO) virus-induced early and late polypeptides

Abstract Polypeptides of chick embryo lethal orphan virus (an avian adenovirus) were analyzed with SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Virions ( p = 1.338 g/cm 3 ) and virion-like particles having lower density (light particles, p = 1.289 g/cm 3 ) were composed of at least 11 and 14 types of polypeptides, respectively. At least 23 virus-induced polypeptides in infected cells (primary monolayer culture of chicken kidney cells) were detected; eight polypeptides (early polypeptides) were synthesized even in the presence of 1-β- d -arabinofuranosyl cytosine, but the others (late polypeptides) were not. On the basis of migration in SDS-PAGE, 5 polypeptides were found to be common to virions and infected cells; 5, to light particles and infected cells; 10, to virions and light particles; and 4, to all three. Scarcely any of the early polypeptides were found in the soluble fraction when extracted with the low salt buffer. Some of the early polypeptides were solubilized with the high salt buffer. Other early polypeptides were solubilized by sodium deoxycholate; three of these were found in the so-called “M-band,” as are early polypeptides of human adenoviruses. An experiment with radioactive glucosamine indicated that one of early polypeptides found in the M-band was a glycoprotein.

E7 Proteins of four groups of human papillomaviruses, irrespective of their tissue tropism or cancer association, possess the ability to transactivate transcriptional promoters E2F site dependently

In an experimental system in which an expression vector including the E7 gene of a given human papillomavirus (HPV), together with a luciferase reporter plasmid including the adenovirus E2 (Ad E2) promoter, was transiently transfected into cultured mouse NIH3T3 fibroblastic cells, we obtained the signal indicating that E7 proteins of HPV type 5, 12, 14, 20, 21, 25, and 47, which are associated with epidermodysplasia verruciformis (EV), can transactivate the Ad E2 promoter, as previously reported for E7 proteins of other HPVs. Because the underlying mechanism of the transactivation had not been analyzed, except for transactivation by E7 gene of cervical cancer-associated HPV-16, we compared the E7 genes of representatives of three other groups of HPVs (HPV -1, -11, and -47) with that of HPV-16 with regard to their transactivating activity toward artificially constructed promoters. The experiment with a shortened AdE2 promoter carrying only the E2F sites and TATA box provided evidence that all four E7 proteins can transactivate the shortened promoter and that this phenomenon is E2F site dependent. Further experiments with the reporter gene constructs carrying basal promoters or more complex forms with or without linked E2F sites, (a) confirmed previous finding by others that in cells producing no transactivator, the transcriptional level from promoters linked to E2F sites is rather repressed in comparison with the level of the corresponding promoters that are not linked to the E2F sites, and (b) demonstrated, for the first time, that in cells expected to produce the E7 protein of any one of the four HPVs, transcription from the promoter linked to the E2F sites was released from repression. In other words, the present results reveal that E7 proteins of any of the four HPVs can remove the E2F site-dependent repression, probably by modulating E2F complexes from repressing forms to activating ones.