Commitment to ploidy conversion of 3Y1 cells during metaphase arrest by colcemid

Diploid rat 3Y1 fibroblasts proliferate to a saturation density, where they are arrested with a 2N DNA content. After treatment to induce ploidy conversion, the conversion rate can be estimated by determining the fraction of cells with a 4N DNA content in the confluent culture using flow cytometry. Using this method it was found that during mitotic inhibition with colcemid, 3Y1 cells were converted to tetraploids with a high efficiency (above 80%); the optimum colcemid concentration and exposure period were 40 ng/ml and 8 hr, respectively. When metaphase cells were reseeded with 40 ng/ml of colcemid, they delayed anchorage to a dish; 6 hr was required for complete adhesion (in the absence of colcemid only 1 hr was required). When reseeded metaphase cells were exposed to 40 ng/ml of colcemid for 5 hr followed by its removal, a greater fraction of the cells anchored to the substratum were converted to tetraploids, whereas most of the floating cells were not. A greater fraction of the anchored cells had formed nuclei, whereas most of the floating cells preserved condensed metaphase chromosomes. These results indicate that the cells which have formed nuclear structure without chromosome separation during mitotic inhibition are irreversibly committed to ploidy conversion, with restoration of anchorage.