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

Dysregulated cell proliferation is one phenotypic change associated withneoplasia. Key protein complexes involved in regulating cell division arecomposed of cyclins, cyclin-dependent kinases (CDK) and CDK inhibitors(CDI). Many virally transformed cells in culture exhibit disruptedcyclin-CDK-CDI complexes, suggesting that such changes may play amechanistic role in viral transformation. To determine whether similaralterations may be involved in chemical carcinogenesis we characterizedcyclin D1-CDK-CDI protein complexes in a non-tumorigenic mouse liver cellline and investigated whether complexes were altered after transformationwith the genotoxic carcinogens N-methyl-N'-nitro-N- nitrosoguanidine (MNNG)or 3-methylcholanthrene (MC). In non- tumorigenic mouse liver cells cyclinD1 associated with CDK6, CDK4 or CDK2 to form binary (cyclin D1-CDK),tertiary (cyclin D1-CDK-p27KIP1) or quaternary (cyclin D1-CDK-p21WAF1-PCNA)complexes. After chemical transformation of mouse liver cells with eitherMC or MNNG, select cyclin D1-CDK-CDI protein complexes were altered. InMC-transformed cells formation of various binary, tertiary and quaternarycyclin D1- CDK-(CDI) protein complexes was reduced, resulting in decreasedCDK4 kinase activity. Interestingly, CDK6 kinase activity was dramaticallyelevated due to high levels of cyclin D3 in association with CDK6. InMNNG-transformed cells select cyclin D1-CDK6-CDI and cyclin D1-CDK2-CDIprotein complexes were altered but CDK6 and CDK4 kinase activity remainedunaffected. Distinct changes in cyclin D1-CDK-CDI complexes found betweenthe two chemically transformed mouse liver cell lines suggest that eachcell line harbored unique mutations or alterations that differentiallycontributed to stabilization of cyclin D1-CDK-CDI holoenzymes. p53 genemutations were not detected in the MC- or MNNG- transformed mouse livercell lines and thus were not involved in disrupting cyclin D1-CDK-CDIprotein complexes. In summary, this study presents evidence that D-type CDKprotein complexes can be altered physically and functionally after chemicaltransformation with genotoxic carcinogens, suggesting that components ofthe cell cycle machinery can be targeted during chemical carcinogenesis.