We have made NIH/3T3 cells tumorigenic by treatment with the ultimate carcinogen of benzo(a)pyrene, namely r-7, t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydro-benzo(a)-pyrene (anti-BPDE) and passaging in culture. Using these cells and mouse embryo fibroblasts (MEF) we propose to examine by somatic cell genetic approaches (1) suppression of tumorigenicity by fusion of cytoplasts of MEF or untreated NIH/3T3 cells to karyoplasts of BPDE-treated NIH/3T3 cells; (2) suppression of tumorigenicity of potentially transformed cells by fusion of cytoplasts of MEF or NIH/3T3 cells to karyoplasts of BPDE-NIH/3T3 cells which are not tumorigenic at passage 5; (3) induction of immortality of MEF by fusion of their karyoplasts to cytoplasts of BPDE-treated NIH/3T3 cells; and (4) induction of tumorigenicity by fusing cytoplasts derived BPDE-treated NIH/3T3 cells to karyoplasts of untreated NIH/3T3 cells. We have chosen to work with BPDE since it binds to mitochondrial DNA with a 50-100 times greater affinity than to nuclear DNA and produces tumors in rats and mice. Because many tumor cells have altered mitochondrial function, we will examine mitochondrial DNA for BPDE-induced mutations. Since it has been shown that BPDE introduces a specific mutation in the ras-Hal oncogene at codons 11 and 12 leading to a loss of an Mspl restriction site, we will initially analyze for loss of an Mspl restriction site in mitochondrial DNA obtained from BPDE-induced tumorigenic cells. To increase the probability of detection of such a mutation, mitochondrial DNA from tumorigenic BPDE-treated NIH/3T3 cells and nontumorigenic NIH/3T3 cells will be cloned. By examining which of the mitochondrial DNA eleven Mspl sites have been eliminated, we will be able to pinpoint the mutated gene(s).

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
2R01CA040065-04A1
Application #
3179523
Study Section
Chemical Pathology Study Section (CPA)
Project Start
1986-05-01
Project End
1993-07-31
Budget Start
1990-08-01
Budget End
1991-07-31
Support Year
4
Fiscal Year
1990
Total Cost
Indirect Cost
Name
University of Texas Sw Medical Center Dallas
Department
Type
Schools of Medicine
DUNS #
City
Dallas
State
TX
Country
United States
Zip Code
75390
Savre-Train, I; Piatyszek, M A; Shay, J W (1992) Transcription of deleted mitochondrial DNA in human colon adenocarcinoma cells. Hum Mol Genet 1:203-4
Shay, J W; Werbin, H (1992) New evidence for the insertion of mitochondrial DNA into the human genome: significance for cancer and aging. Mutat Res 275:227-35
Shay, J W; Baba, T; Zhan, Q M et al. (1991) HeLaTG cells have mitochondrial DNA inserted into the c-myc oncogene. Oncogene 6:1869-74
Shay, J W; Pierce, D J; Werbin, H (1990) Mitochondrial DNA copy number is proportional to total cell DNA under a variety of growth conditions. J Biol Chem 265:14802-7
Pierce, D J; Werbin, H; Shay, J W (1990) An improved procedure for quantitating mitochondrial DNA in cultured mammalian cells. Biotechniques 9:724-9
Shay, J W; Ishii, S (1990) Unexpected nonrandom mitochondrial DNA segregation in human cell hybrids. Anticancer Res 10:279-84
Kamimura, N; Ishii, S; Ma, L D et al. (1989) Three separate mitochondrial DNA sequences are contiguous in human genomic DNA. J Mol Biol 210:703-7
Iman, D S; Shay, J W (1989) Modification of myc gene amplification in human somatic cell hybrids. Cancer Res 49:4417-22
Shay, J W; Liu, Y N; Werbin, H (1988) Cytoplasmic suppression of tumor progression in reconstituted cells. Somat Cell Mol Genet 14:345-50
Shay, J W; Werbin, H (1988) Cytoplasmic suppression of tumorigenicity in reconstructed mouse cells. Cancer Res 48:830-3

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