Nickel selectively damages heterochromatin and transforms male Chinese hamster embryo cells at a higher frequency that female cells. About 40% of the heterochromatin in this species is localized on the long arm of the X- chromosome. A high percentage of male transformed cell lines examined thus far exhibit a deletion of this heterochromatic DNA as their primary chromosomal aberration. We will continue to study the incidence of transformation to anchorage-independent growth with nickel compounds and 3- methyl-cholanthrene in male and female cultures. Transformed clones will be karyotyped and characterized for their ability to grow in soft agar and form tumors in nude mice. The incidence of tumor formation in male and female Chinese hamsters will also be examined following treatment with crystalline nickel sulfide and 3-methylcholanthrene. The data thus far suggests that nickel may be inducing transformation by causing the loss of a """"""""transforming"""""""" suppressor gene associated with the long arm of the X- chromosome. This hypothesis is supported by recent findings demonstrating that 42 separate clones of male nickel-transformed cells, having a deletion of the long arm of the X-chromosome, all senesced upon introduction of a normal X-chromosome by microcell fusion. We will test the effect of introduction of the normal X-chromosome into nickel-transformed cells without a deletion in the heterochromatic long arm, as well as the effect of introduction of Chinese hamster X sequences from mouse A-9 cells harboring fragments of the Chinese hamster X-chromosome to attempt to localize the tumor suppressor gene. Clones that did not senesce when the X-chromosome was introduced did exhibit a substantially reduced capacity to grow in soft agar. All hybrids prior to and following testing of their growth in agar, transformation, etc., will be carefully karyotyped to assure that the effects observed can be related to the desired chromosome change. In addition to studies of the Chinese hamster X-chromosome, we are also interested in determining whether the human X-chromosome has similar suppressing activity as has been demonstrated for the Chinese hamsters. Mouse A-9 lines harboring fragments in the human X-chromosome will allow preliminary positional estimation of the presence of a possible tumor suppressing gene on the human X. These studies address the mechanism of nickel carcinogenesis involving deletions of tumor suppressor genes, focussing specifically on the X-chromosome, and will form the basis for future, more detailed studies at the level of single genes.
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