CLONAL NATURE OF SPONTANEOUSLY IMMORTALIZED 3T3 CELLS

Mouse embryo fibroblasts (MEFs), when plated at appropriate densities, proliferate vigorously for several passages, and then the growth rate of the culture slows considerably. If the cells are plated at a high enough density and continuously passed, the cultures will eventually o vercome this ''crisis'' period and resume rapid growth. Here, we have addressed the question of what the changes are that cells undergo in o vercoming the growth restraints of crisis. Primary MEF cells were infe cted with a retrovirus which confers G418 resistance and selected in G 418. The resultant precrisis population comprised cells which each con tained a retrovirus integrated at a unique genomic location. These cel ls were then passed according to the 3T3 protocol until immortal, rapi dly growing cells emerged. The integration pattern of the retrovirus i n the immortal population was examined. In two independent experiments , the immortal population of cells grown in the presence of G418 compr ised two independent clones of cells, with additional clones undetecta ble at the level of detection of the assays used. The integration patt ern was also examined in parallel infected cultures grown in the absen ce of selection. In one experiment the unselected immortal population contained the same labeled clone that appeared in the sister infected culture, indicating that an immortal precursor was present in the prec risis population. These results are consistent with the idea that a mu tation is responsible for the immortal phenotype. (C) 1996 Academic Pr ess, Inc.