ABSTRACT
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.
