IMMORTAL, NONTUMORIGENIC MOUSE MAMMARY OUTGROWTHS EXPRESS HIGH-LEVELSOF CYCLIN B1 AND ACTIVATION OF CYCLIN B1 CDC2 KINASE

Neoplastic transformation of mouse mammary epithelial cells is the res ult of several identifiable phenotypic changes which presumably requir e sequential genetic alterations. In our model system, mammary cells p rogress from a mortal state (virgin duct) to several morphologically d istinct intermediate states. The intermediate states are distinct cell populations that are phenotypically identified as immortal, non-tumou rigenic (i.e. EL11), weakly tumourigenic ductal/alveolar hyperplasia ( i.e. EL12) and moderately tumourigenic alveolar hyperplasiaa (i.e. TM1 2) to invasive tumours (i.e. EL12T/TM12T). We have studied the changes in total cyclin A and B1 levels, cyclin A and B1 complexed to cdc2, c yclin B1cdc2 kinase activity and cyclin D proteins in EL11 and EL12 im mortalized outgrowth lines. Results revealed increased levels in total cyclin B1(>5-fold), cyclin B1/cdc2 (3-4-fold) and cyclin B1/cdc2 kina se activity (2-3.5-fold) in EL11 and EL12 phenotypes when compared to control mammary gland (virgin). No changes in the levels of total cycl in A or cycln A associated to cdc2 were observed. Cyclin D1, D2 and D3 protein levels were low in the EL11 immortal-ductal outgrowth. Exposu re to hormones via a pituitary isograft stimulated the synthesis of cy clin D1 and D2 but not D3 associated to cdk4 as well as total cdk4 pro teins. Bromodeoxyuridine (BrdUrd) labelling indices showed marked incr eases in immortal ductal outgrowths (EL11 and EL12) when compared to v irgin, suggesting that epithelial cells are cycling in these cell popu lations. Even in the-presence of hormone stimulation, EL11 outgrowths were not tumourigenic, suggesting-that other events are necessary to d rive the cells to a tumourigenic phenotype. The results suggest that i ncreased levels of cyclin B1 and cyclin B1-cdc2 kinase activities are early events and may be an important marker for the immortalized pheno type.