Use of growth-attachment correlation plots to analyse human melanoma cell dependency on 2-oxocarboxylates and serum

Modulation of the population-dependent growth of the human melanoma line MM96 by serum and a series of 2-oxocarboxylates was analysed by a method offering several useful features. Initial cell attachment was measured by the use of cells prelabelled with [14C] thymidine (C) while the size of the replicating cell population on any particular day was monitored by [3H] thymidine (H) incorporation for that particular 24 hr. The 3H incorporation was normalized for the initial cell attachment as the 3H/14C ratio (H/C), which was found to be little affected by artifacts of precursor equilibration. The method allows large numbers of replicate samples so that quantitation of dose-response relationships is simple and statistically robust, and simultaneous monitoring of cell attachment and growth within the same sample is routine. Not only cell growth, but also attachment to the culture vessel surface was found to be increased by higher seeded population densities, by increasing serum concentration and by pyruvate supplementation. Further investigation of the pyruvate effect showed that all nine of the soluble 2-oxocarboxylates tested were effective for both parameters, with little difference between them which was attributable to molecular structure. Quantitative studies of the potentiation of growth and attachment by increasing cell density or a range of concentrations of 2-oxocarboxylates led to the finding that increments in the growth parameter (H/C) were directly proportional to increments in attachment (C). Such a relationship would be unexpected in ordinary logarithmically-growing cultures. The observation is consistent with the hypothesis that increases in initial cell attachment, whether induced by 2 oxocarboxylates or higher inoculation densities, lead to conditioning of the medium by cell factors which stimulate growth and are produced in amounts proportional to the attached cell numbers. Pyruvate, when present with higher serum concentrations, stimulated growth above the levels accounted for by concomitant increases in cell attachment. Serum also appears to have other potentiating factors in addition to those attributable to its content of 2-oxocarboxylates. Differences in the response to 2-oxocarboxylates of human melanoma cells and human diploid fibroblasts were noted, and suggest that the present observations may be due to unique properties of the melanoma cell.