Open Collections

Isolation and characterization of retinoic acid-induced revertants of bovine papillomavirus 1 DNA-transformed mouse G127 cells

University of British Columbia

The action of all-trans-retinoic acid (PA), which is currently being tested as a chemopreventive and chemotherapeutic agent, was studied on bovine papillomavirus (BPV)-l DNA mediated transformation of mouse Cl27 cells. BPV-1 DNA-transformed cell lines B3 and BF3 were exposed to 5 pM of PA for 10 weeks. The copy number of BPV DNA in the transformed cells gradually decreased by PA treatment. After 10-week PA treatment, the cells were void of viral DNA, no longer exhibited a transformed morphology and lost the ability to form multilayered foci. These PA treated B3 and BF3 cells are PA-induced revertants and designated as B3RA1O and BF3RA1O. The revertant cells were resistant to retransforruation by BPV DNA. The transformation efficiency of the revertant cells is at least 7- to 13-fold less than that of C127 cells. The revertant cells were not resistant to transformation induced by a human Ha-ras gene. These two revertant cell lines have similar population doubling time and saturation density as C127 cells. Using cDNA cloning and differential hybridization technique, a eDNA sequence that is expressed differently in BF3 transformed cells and BF3PA1O revertants was cloned. DNA sequencing revealed that the cloned gene is the ND5 gene of mitochondria. In the transformed cells, the level of ND5 mNRA was dependent on the density of the cells. ND5 expression was 5- to 7- fold higher at 8% cell confluency than at 80% confluency. In the revertant cells, the amount of ND5 mRNA was independent of cell density. In Cl27 cells, the pattern of ND5 expression was similar to that in the revertant cells at subconfluency. However, after the cells reached confluency, ND5 gene was expressed the same as in transformed EF3 cells. PA increased the amount of ND5 mRNA in untransformed C127, transformed BF3, and revertant BF3RA1O cells by 8- to 10-fold at subconfluency. After the cells reached confluency, PA only induced a 2- to 3-fold increase in the level of ND5 mRNA. The results demonstrated that the reversion of the transformed phenotype of the transformed cells by PA treatment is associated with the elimination of BPV DNA and changes of cellular gene expression pattern. The different patterns of ND5 expression in transformed cells and PA-induced revertants suggest mitochondrial genes may play a role in cell transformation.

Thesis/Dissertation

application/pdf

2008-12-17

For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use.

http://hdl.handle.net/2429/3074

Zoology

University of British Columbia

UBCV

DSpace