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Effect of all-trans-retinoic acid on bovine papillomavirus (BPV)-1 DNA-induced mouse C127 cell transformation and number of BPV DNA copies Li, Gang


Bovine papillomavirus (BPV)-l DNA-induced transformation of mouse C127 cells was used as a model for examining the action of all-trans -retinoic acid (RA), which is currently being tested as a chemopreventive and chemotherapeutic agent. Prior to studying the action of RA, the transformation frequency and the nature of transformed clones were studied. Transformed colonies formed two weeks after viral DNA transfection. The transformation frequency was proportional to the amount of viral DNA used for transfection. The 8 transformed colonies examined contained 20 to 100 copies of viral genomes per cell on average. The BPV DNA copy number was stably inherited over 19 passages. The transformed cells had a heterogeneous number of BPV DNA copies. BPV DNA was present extrachromosomally in more than 95% of the subclones containing the viral genomes. Transformed cells became elongated after the cells reached confluency. The effect of RA was examined on (1) transformation induced in C127 cells by transfection with plasmid pdBPV-1 (142-6), which contains DNA of bovine papillomavirus (BPV), (2) the capacity of transformed BPV DNA-containing clones to form colonies with transformed properties (i.e., piling up into multilayered colonies), and (3) the number of BPV DNA copies in transformed cells. At non-toxic doses ranging from 10⁻⁷ to 10⁻⁵M, RA reduced the frequency of transformed foci in a dose-dependent manner. The extent of inhibition depended on the length of RA treatment and on the time that elapsed between DNA transfection and RA treatment. RA exerted only a slight inhibitory effect during the first days after transfection. Complete inhibition was observed when the cells were continuously exposed after transfection to RA at doses ranging from 0.5 to 1 x 10⁻⁵M. The inhibitory effect of RA on transformation was reversible. Transformed foci started to form after withdrawal of RA treatment. RA reduced the saturation density of the transformed cells without reducing their growth rate. RA specifically suppressed the piling-up phenotype of the transformed cells. The doses for complete suppression of transformed phenotype is similar to those for complete inhibition of viral DNA-induced transformation. The number of BPV DNA copies gradually decreased when the cells were grown over several generations in the presence of RA (5 x 10⁻⁶M). After five weeks of treatment (approximately 30 cell generations), the cell cultures appeared to have less than one copy of BPV DNA. The number of BPV-1 DNA copies was examined after withdrawal of RA. The low number of BPV DNA copies in the RA-treated cell population did not increase for at least 10 passages when the cells were subcultured before reaching confluence. After 5 weeks treatment with RA, the transformed cell populations that contained less than one BPV DNA copy lost the transformed phenotype. However, a tiny fraction of cells (1 in 13,000) still had more than 10 BPV DNA copies and retained the capacity to develop into transformed colonies. The tiny fraction of cells were not resistant to RA. After the RA treatment was extended to 10 weeks, transformed cells completely lost BPV DNA. The BPV DNA free cells do not express a transformed phenotype. The elongated morphology of the transformed cells were also reversed to normal (star-shaped) after 10 week treatment with RA. The above results recommend the use of retinoids as chemopreventive and chemotherapeutic agents for papillomavirus-induced tumours.

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