Cytokinetic and cytotoxic effects of urea on HeLa cells in suspension cultures.

Total and viable cell counts, differential mitotic cell counts, and incorporation of tritiated thymidine were used to study the kinetics of suspension cultures of HeLa cells exposed to urea concentrations of 0.5-1.5%. Aside from any nonspecific osmotic effects, urea concentrations of 1.0-1.4% exhibited significant cytokinetic and cytotoxic effects. Most characteristically, mitotic cells arrested in metaphase began to accumulate 4-6 hours after addition of urea and reached a peak at 15-18 hours. Thus when the cells were in the S-phase or at the S/G2 boundary at the time of addition of urea, they exhibited metaphase arrest. Subsequently, cultures continuously exposed to urea showed a decline in the mitotic index, indicating that the entry rate of cells into mitosis is lower than the rate at which cells escape from the mitotic block. Such cultures exhibited numerous abnormal and abortive mitoses and a decrease in growth and viability of the cell populations. In contrast to the initial single wave of arrested mitosis seen with continuous exposure to urea, intermittent exposure on alternate days resulted in successive waves of arrested metaphases and had considerably more pronounced effects on the growth and viability of the cultures.

Chromosomal aberrations in early stages of development of methotrexate resistance in HeLa cells.

High incidence of dicentrics, rings, double rings, acentric rod-like fragments and double minutes in polyploid cells during the early stages of development of methotrexate resistance in HeLa cells suggests that hypertetraploid cells are precursors of cells with higher resistance levels.

Cytogenetical changes during early stages of development of methotrexate resistance in HeLa cells.

HeLa cells cultured in ever-increasing doses of methotrexate (MTX) acquired resistance to this drug. Chromosomal changes occurring at early stages during the development of resistance to various doses (0.1, 0.3, 0.5, 1.0, 2.1, 3.0, 10.0 and 100.0 micrograms/ml) of MTX included: (a) an increase in the percentage of hypertetraploid cells (greater than 92 chromosomes) at all doses, and most profoundly at 1.0, 2.1 and 3.0 micrograms/ml; (b) an increase in the percentage of cells with structural abnormalities; (c) a remarkable increase in the percentage of hypertetraploid cells containing dicentrics, particularly at MTX dose levels 0.5-3.0 micrograms/ml and (d) emergence of increasing numbers of double minutes per cell with increasing MTX doses. At dose levels 2.1 and 3.0 micrograms/ml the modal chromosome number increased to 82, while at 0.1-1.0 microgram/ml it was similar to the mode of sensitive HeLa (64, 66) and at 10.0 and 100.0 micrograms/ml dropped to 62, 63. The results obtained suggest that polyploidization and formation of dicentrics are associated with the earlier stages of development of resistance to methotrexate.