Effects of simian virus 40 T-antigens on normal human mammary epithelial cells reveal evidence for spontaneous alterations in addition to loss of p16(INK4a) expression.
Under standard culture conditions, normal human mammary epithelial cells (HMECs) divide a limited number of times before proliferation ceases in a growth-arrested state referred to as selection. Cells that have undergone spontaneous loss of p16(INK4a) expression due to hypermethylation of the p16(INK4a) CpG island emerge from selection and proliferate for an extended, but limited, period before senescence. Here we show, as expected, that selection was bypassed by expression of SV40 large T-antigen proteins containing an intact pRb-binding domain in preselection cells. These cells were immortalized with high efficiency (seven of nine separate cultures). Also as expected, postselection cells were immortalized by expression of the human papillomavirus-16 E6 oncoprotein (four of four cultures), which inactivates p53 protein. In contrast, we found that expression of SV40 large T-antigen protein, which also inactivates p53, was poorly maintained in postselection cultures due to its growth-suppressive effects; consequently, these cells became immortalized at low efficiency (one of 11 cultures). Reexpression of p16(INK4a) in postselection HMECs by the demethylating agent, 5-azacytidine, or transfection of a p16(INK4a) expression plasmid did not restore the ability of these cells to undergo SV40-induced transformation. Postselection HMECs are a widely used in vitro model system, but these observations indicate they have undergone changes in gene expression in addition to loss of p16(INK4a) expression.
Huschtscha
L I
LI
Children's Medical Research Institute, 214 Hawkesbury Road, Westmead, NSW 2145, Australia.
Neumann
A A
AA
Noble
J R
JR
Reddel
R R
RR
D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics*; D001374:Azacitidine / Q000494:pharmacology; D001940:Breast / Q000166:cytology; D002471:Cell Transformation, Neoplastic; D002478:Cells, Cultured; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000235:genetics*; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D005260:Female; D005786:Gene Expression Regulation; D006801:Humans; D009856:Oncogene Proteins, Viral / Q000235:genetics; D011506:Proteins / Q000235:genetics; D012097:Repressor Proteins; D016160:Retinoblastoma Protein / Q000378:metabolism; D013539:Simian virus 40 / Q000235:genetics; D014162:Transfection; D025803:Tumor Suppressor Protein p14ARF
D000952:Antigens, Polyomavirus Transforming; D019941:Cyclin-Dependent Kinase Inhibitor p16; C068651:E6 protein, Human papillomavirus type 16; D009856:Oncogene Proteins, Viral; D011506:Proteins; D012097:Repressor Proteins; D016160:Retinoblastoma Protein; D025803:Tumor Suppressor Protein p14ARF; D001374:Azacitidine