Phenotype-rescue of cyclin-dependent kinase inhibitor p16/INK4A defects in a spontaneous canine cell model of breast cancer.
Mammary cancer is among the most frequently observed canine tumors in unspayed female dogs resulting in death due to metastatic disease. These tumors are excellent models of human breast cancer but until recently there was only anecdotal evidence regarding underlying genetic defects. We recently reported expression defects in the cyclin-dependent kinase p21/Cip1 and p53 among three independent canine mammary tumor (CMT) cell lines derived from spontaneous canine mammary cancers. We investigated further defects in the same three cell lines focusing on additional tumor suppressor gene defects in cyclin-dependent kinase inhibitors. p27/KIP1 appeared normally expressed and did not appear to encode inactivating mutations. In contrast, expression of p16/INK4A was defective/absent in two cell lines and normal/slightly induced in the third cell line. To determine if defects were causative in maintaining the transformed phenotype, a p16/INK4A transgene was permanently transfected followed by selection and single cell cloning. CMT/p16 clones were characterized for transgene expression, p16 protein content and phenotype including proliferation rate, cell cycle phase distribution, contact inhibition, substrate dependent cell growth and cell morphology. All cell lines appeared unique yet clear indications of phenotype rescue due to p16/INK4A transgene complementation were observed suggesting that defects in p16 expression were present in all three. In some cases cellular senescence also appeared to be induced. These data provide evidence supporting p16/INK4A mutations as causative defects promoting transformation in canine mammary cancer and further characterizes tumor suppressor gene defects with functional consequences in these cells supporting their application as spontaneous animal models of human disease.
DeInnocentes
Patricia
P
Department of Pathobiology, College of Veterinary Medicine, Auburn University, Alabama 36849, USA.
Agarwal
Payal
P
Bird
R Curtis
RC
D000818:Animals; D001483:Base Sequence; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000235:genetics / Q000378:metabolism*; D004195:Disease Models, Animal; D004285:Dogs; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D010641:Phenotype; D010802:Phylogeny; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D013379:Substrate Specificity; D019076:Transgenes / Q000235:genetics
D019941:Cyclin-Dependent Kinase Inhibitor p16