Chemical transformation of mouse liver cells results in altered cyclin D CDK protein complexes

Dysregulated cell proliferation is one phenotypic change associated with neoplasia. Key protein complexes involved in regulating cell division are composed of cyclins, cyclin-dependent kinases (CDK) and CDK inhibitors (CDI), Many virally transformed cells in culture exhibit disrupted cyclin-CDK-CDI complexes, suggesting that such changes may play a mechanistic role in viral transformation. To determine whether similar alterations may be involved in chemical carcinogenesis we characterized cyclin D-1-CDK-CDI protein complexes in a non-tumorigenic mouse liver cell line and investigated whether complexes were altered after transformation with the genotoxic carcinogens N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or 3-methylcholanthrene (MC), In non-tumorigenic mouse liver cells cyclin D-1 associated,vith CDK6, CDK4 or CDK2 to form binary (cyclin D-1-CDK), tertiary (cyclin D-1-CDK-p27(KIP1)) or quaternary (cyclin D-1-CDK-p21(WAF1)-PCNA) complexes. After chemical. transformation of mouse liver cells with either MC or MNNG, select cyclin D-1-CDK-CDI protein complexes were altered, In MC-transformed cells formation of various binary, tertiary and quaternary cyclin D1-CDK-(CDI) protein complexes was reduced, resulting in decreased CDK4 kinase activity. Interestingly, CDK6 kinase activity was dramatically elevated due to high levels of cyclin D-3 in association with CDK6, In MNNG-transformed cells select cyclin D-1-CDK6-CDI and cyclin D-1-CDK2-CDI protein complexes were altered but CDK6 and CDK4 kinase activity remained unaffected. Distinct changes in cyclin D-1-CDK-CDI complexes found between the two chemically transformed mouse liver cell lines suggest that each cell line harbored unique mutations or alterations that differentially contributed to stabilization of cyclin D-1-CDK-CDI holoenzymes, p53 gene mutations were not detected in the MC- or MNNG-transformed mouse liver cell lines and thus were not involved in disrupting cyclin D1-CDK-CDI protein complexes. In summary, this study presents evidence that D-type CDK protein complexes can be altered physically and functionally after chemical transformation with genotoxic carcinogens, suggesting that components of the cell cycle machinery can be targeted during chemical carcinogenesis.