JaLCDOI 10.18926/AMO/30807
FullText URL fulltext.pdf
Author Iwamoto, Ryota| Fushimi, Kazuo| Hiraki, Yoshio| Namba, Masayoshi|
Abstract <p>&#8195;This study was conducted to evaluate the frequency of DNA transfection into human cells following X-ray irradiation. We transfected plasmid DNA (pSV2neo) into human cells, HeLa and PA-1, by either calcium phosphate precipitation or the lipofection method immediately after irradiating the cells with various doses of X-rays. The transfection frequency was evaluated by counting the number of G418-resistant colonies. When circular plasmid DNA was used, irradiation up to a dose of 2 Gy dose-dependently increased the transfection frequency, which reached a maximum of 5 to 10-fold that of the control unirradiated cells. When linear plasmid DNA was used, the transfection frequency was 2 times higher than that of circular DNA. All five of the clones that were randomly chosen expressed the transfected neo gene. In addition, the pSV2neo gene was randomly integrated into the genomic DNA of each clone. These findings indicate that X-ray treatment can facilitate foreign DNA transfer into human cells and that radiation-induced DNA breaks may promote the insertion of foreign DNA into host DNA. The enhancement of DNA transfection with X-rays may be instrumental in practicing gene therapy.</p>
Keywords DNA transfection neo gene X-ray irradiation
Amo Type Article
Published Date 1997-02
Publication Title Acta Medica Okayama
Volume volume51
Issue issue1
Publisher Okayama University Medical School
Start Page 19
End Page 23
ISSN 0386-300X
NCID AA00508441
Content Type Journal Article
language 英語
File Version publisher
Refereed True
PubMed ID 9057931
Web of Science KeyUT A1997WL24600004
JaLCDOI 10.18926/AMO/30789
FullText URL fulltext.pdf
Author Mihara, Koichiro| Miyazaki, Masahiro| Kondo, Tadashi| Fushimi, Kazuo| Tsuji, Toshiya| Inoue, Yusuke| Fukaya, Kenichi| Ishioka, Chikashi| Namba, Masayoshi|
Abstract <p>We used a yeast functional assay (functional analysis of separated alleles in yeast: FASAY) to determine the p53 gene status of human cell lines maintained in our laboratory. This assay enables the researcher to score wild-type p53 expression on the basis of the ability of expressed p53 to transactivate the reporter gene HIS 3 via the p53-responsive GAL 1 promoter in Saccharomyces cerevisiae. The cell lines examined were ten hepatoma, two hepatoblastoma, three in vitro immortalized fibroblast, two osteosarcoma, a chondrosarcoma, an ovarian teratocarcinoma and a colon cancer cell line. Out of 20 cell lines, 11 cell lines had mutations in both alleles of the p53 gene, and another 8 cell lines had no mutation in the p53 gene. Thus, 55% of the cell lines examined had mutations in the p53. Interestingly, PA-1 cells had both the normal and the mutant p53 alleles, showing that FASAY is a useful method for detecting the wild-type and mutated p53 genes simultaneously. As for the three liver cell lines harboring HBsAg, there was no relationship between their p53 gene status and the presence of HBsAg. Two cell lines were normal for p53 status, while the other had a mutation of the p53 gene.</p>
Keywords p53 mutation FASAY cultured human cells
Amo Type Article
Published Date 1997-10
Publication Title Acta Medica Okayama
Volume volume51
Issue issue5
Publisher Okayama University Medical School
Start Page 261
End Page 265
ISSN 0386-300X
NCID AA00508441
Content Type Journal Article
language 英語
File Version publisher
Refereed True
PubMed ID 9359923
Web of Science KeyUT A1997YD65300004
JaLCDOI 10.18926/AMO/30772
FullText URL fulltext.pdf
Author Pu, Hong| Tsuji, Toshiya| Kondo, Asami| Fushimi, Kazuo| Ohashi, Ryuichiro| Inoue, Yusuke| Mimura, Tetsushige| Hamazaki, Keisuke| Miyazaki, Masahiro| Namba, Masayoshi|
Abstract <p>Characteristics of human hepatoma cell lines with the wild-type p53 were compared with those of human hepatoma cell lines with the mutant-type p53. The p21 protein located downstream of p53 was expressed in cell lines with the wild-type p53 but was not expressed in cell lines with the mutant-type p53. As to other tumor suppressor genes such as p16 and p27, there was no difference in their expression between both types of cell lines. In addition, no marked difference was observed in the activities of CDK2 and CDK4 between cell lines with the wild-type and the mutant-type p53. Phosphorylated Rb protein was detected in all cell lines except the HLE line, indicating that this cell line may have a deletion of and/or a mutation of the Rb gene. These results indicate that abnormalities of tumor suppressor genes other than p53, p16, p27, and Rb may be involved in hepatocarcinogenesis. The population doubling time of the wild-type p53 cells was significantly longer than that of the mutant p53 cells. Neither type of cell line showed a specific chromosome distribution which would indicate karyotype instability. The cell lines expressing the wild-type p53 produced tumors at lower frequency than those with the mutant p53 gene. Although there was no significant difference in effects of TGF-&#946;1, EGF, cholera toxin, and db-cAMP on cell growth between the two types of cells, all three cell lines with the wild-type p53 were resistant to cytotoxicity of TNF-&#945;, while two of the three with the mutant p53 were very sensitive to its cytotoxic effects.</p>
Keywords hepatoma p53 p21?p16?p27?Rb TNF-?
Amo Type Article
Published Date 1997-12
Publication Title Acta Medica Okayama
Volume volume51
Issue issue6
Publisher Okayama University Medical School
Start Page 313
End Page 319
ISSN 0386-300X
NCID AA00508441
Content Type Journal Article
language 英語
File Version publisher
Refereed True
PubMed ID 9439773
Web of Science KeyUT 000071183400004