We have investigated the effects of the growth of A431 human squamous carcinoma cells as three-dimensional aggregates (multicellular tumor spheroids) on the expression and enzyme activity of heme oxygenase (HO). We demonstrate that A431 squamous carcinoma cells grown as day 4 spheroids selectively increase the expression of heme oxygenase 1 (HO-1), caused, directly or indirectly, by three-dimensional cell-cell contact effects. Steady-state levels of both mRNA and protein are significantly enhanced in spheroids compared with day 4 monolayers (approximately 13-fold). Because of the similarity of apparent half-lives between monolayers (2.7 h) and spheroids (2.1 h), it appears that the increases are caused at least partly by altered transcriptional rates. Total HO enzyme activity, measured by carbon monoxide production, is also up-regulated (2.6-fold) in spheroids, compared to that in monolayers. This increase indicates that the up-regulation in HO-1 protein expression corresponds to an increase in functional enzyme levels. We propose that HO may play a more complex role in cellular metabolism than would be evident from studies using two-dimensional monolayer cultures.
Supported by grants from the NIH (CA57692 to B. J. M., CA20329 to R. M. S., and HD14426 to D. K. S.).