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 viability, 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.

Summary The buoyancy of suspension‐grown Mastocytoma P815 X‐2 cells in albumin‐rich Cohn fraction V protein (CFVP) density gradients was found to be affected by prior incubation of the cells in pancreatin‐EDTA salt solution. Whereas in pH 5·2 CFVP, pancreatin‐EDTA treated cells behaved as if of reduced density when compared with the control ‘undigested’ group, in pH 7·3 CFVP they behaved as if of increased density. By contrast, pancreatin‐EDTA treatment had no effect on the buoyancy of mastocytoma cells in polyvinylpyrrolidone‐coated colloidal silica (PVP‐CS, Percoll T.M.) density gradients of either pH 5·2 or pH 7·3. As cell size determinations failed to reveal alterations in cell size either as a direct result of pancreatin‐EDTA treatment or as a combined consequence of such treatment and exposure to CFVP either with or without centrifugation, a mechanism involving a change in cell density other than during the centrifugation process itself seems unlikely. Binding studies employing 125I‐CFVP, although indicating that CFVP bound to cells at 4°, failed to reveal a pancreatin‐EDTA treatment‐related difference in the avidity of this binding. Although the mechanism of the pancreatin‐EDTA‐induced buoyancy shift in CFVP remains obscure, the absence of such an effect in PVP‐CS suggests that the latter cell separation solution may more accurately be used to determine cell density.

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An investigation was made of the sequential biophysical and morphological changes that occur as cultured human melanoma cells (MM96) outgrow their supply of nutrient. Simultaneous buoyant-density and velocity-sedimentation fractionation experiments were used to characterize cells from 3 kinetically differing types of culture. Cells from exponential cultures were large, moderately dense and rapidly sedimenting; cells from post-exponential cultures were of intermediate size, less dense and much more slowly sedimenting; and dye-excluding cells from reproductively non-viable late post-exponential cultures were small, of widely variable though generally high density and sedimented moderately rapidly. Although reproductive viability was high in cells from both exponential and post-exponential cultures, depletion of clonogenic cells was seen at the extremes of the distribution profiles of cells fractionated by either method. This was particularly evident at the low-density extreme of the buoyant-density profiles where cells retained viability despite their loss of proliferative potential. As cells became post-exponential, nuclear size diminished in parallel with cell size, the number of microvilli declined, mitochondria condensed, cytoplasm vacuolated, the frequency of osmiophilic vacuolar inclusions rose, chromatin clumped and nucleoli became prominent. Progression to a reproductively non-viable late post-exponential state resulted in a continued parallel fall in nuclear size, increased cytoplasmic blebbing, further mitochondrial condensation, an increased proportion of cytoplasmic vacuoles containing osmiophilic material, the major part of which was melanin, and further clumping and margination of chromatin. Cells progressed rapidly from this newly described pre-apoptotic state to death by apoptosis, a process characterized by the budding and division of cells into a number of ultrastructurally well-preserved membrane-bound fragments.