Studies on a human melanoma cell line: Effect of cell crowding and nutrient depletion on the biophysical and kinetic characteristics of the cells

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Abstract
In an investigation of the changes that occur in cultured neoplastic cells as they outgrow their supply of nutrient, MM96 human melanoma cells were found to diminish in size and to proliferate more slowly. These changes were accompanied by a moderate increase in the proportion of cells with a G1-like DNA content. When replated under favorable conditions, many of these cells gradually resumed active proliferation. Continuing adverse culture conditions led to a continued fall in cell size, loss of reproductive viabllity, and finally to rapid cell death. Simultaneous buoyant-density and velocity-sedimentation-fractionation experiments showed that cells from exponential cultures were moderately dense and rapidly sedimenting, cells from postexponential cultures were less dense and much more slowly sedimenting, and dye-excluding cells from reproductively nonviable, late postexponential cultures were of widely variable though generally high density, and were moderately rapidly sedimenting. Although neither fractionation method resulted in significant enrichment of clonogenic cells, depletion was seen at both extremes of both types of profile. Cells fractionated by velocity were sorted according to DNA content and hence location in the cell cycle. The relationship between sedimentation rate and cell-cycle location was reflected in the continuous thymidine labeling patterns of the separated cells. Study of these patterns suggested that cycle durations lengthened as crowding increased and nutrient became depleted, and shortened upon reseeding at low density into fresh medium.