Replication-deficient adenovirus expression vectors were used to introduce a recombinant DNA construct containing enhanced green fluorescent protein (EGFP) under control of a truncated, human heat shock promoter into human prostate cancer cells growing either exponentially or in plateau phase. This was done to measure controlled, heat shock-induced EGFP expression under conditions relevant to treating human cancers with heat-activated gene therapy. Both the temporal duration and magnitude of EGFP expression increased proportionately with stronger heat shocks (time at temperature) up to maximum values that were induced by 4 h at 41.0 degrees C or 2 h at 42.0 degrees C. Longer heat shocks at either temperature yielded no additional EGFP expression and ultimately reduced it. Maximal EGFP expression was induced in exponential cultures by heat shocks delivered 12-24 h after virus infection. Induction at progressively later postinfection times induced increasingly lower, peak EGFP expression. Maximal EGFP expression could not be induced until 48 h after infection of plateau phase cultures but could still be induced 180 h after virus infection. However, peak EGFP levels in plateau cultures were approximately 25-50% of those observed in identically induced exponential cultures. Ostensibly, the differences in expression from the heat shock promoter observed in exponential and plateau cultures were attributable to cell division diluting the vector within exponential cultures and the lower metabolic activity in serum-starved plateau cultures. For all experimental conditions, EGFP expression induced from the heat shock promoter was comparable with or higher than that from the constitutively active cytomegalovirus promoter over any 24-h period. The experimental results demonstrated that EGFP expression from the heat shock promoter was controllable in both exponential and plateau phase cultures and support the plausibility of using controlled heat shock activation of this promoter as a means of regulating both the spatial and temporal expression of therapeutic DNA constructs within human tissues. The ability to localize and regulate expression from the heat shock promoter may prove particularly advantageous for many cancer applications, especially if the therapeutic products are highly toxic, e.g., proteotoxins or cytokines. However, the results of this study suggest that differential growth conditions within tumors could markedly affect the expression of recombinant DNA under control of both inducible and constitutive promoters. Consequently, inducing schemes may need to be spatially adjusted to obtain the desired therapeutic results in all tumor domains using heat-activated gene therapy.