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Multicellular tumour spheroids are cellular aggregates that can be prepared from many types of tumour cells. These three-dimensional structures provide a model for analysing the effects of cell-cell contact and intercellular microenvironments on phenomena such as autocrine regulation of growth factor synthesis. Autoregulation of the synthesis of transforming growth factor-alpha (TGF-alpha) was investigated at the message and protein levels in spheroid and monolayer cultures prepared from the A431 human squamous carcinoma cell line. The epidermal growth factor receptor (EGF-R) of these monolayer A431 cells had an average surface density of 2.2 x 10(6)/cell. Constitutive expression of TGF-alpha mRNA was an average of 3-fold greater in A431 spheroids than in monolayers, even for densely packed, confluent monolayers. This effect did not depend on hypoxic stress within the spheroids. TGF-alpha protein synthesis was enhanced in comparison with that in monolayer culture, reaching a value of up to 2-fold greater on a per cell basis. These results are discussed in the context of a TGF-alpha/EGF-R autocrine loop operating within cells that produce high local concentrations of TGF-alpha in the three-dimensional architecture of a spheroid.

Chronic hypoxia increases the expression of a set of stress proteins (oxygen regulated proteins or ORPs) which is implicated in the development of drug resistance and radiation sensitivity in tumour cells. Five major ORPs have been documented, and two, ORP 80 and ORP 100, are considered to be identical to the glucose regulated stress proteins GRP78 and GRP94, respectively. We report here that ORP 33 is a form of the heme catabolic enzyme, heme oxygenase, using evidence obtained from northern blotting, two-dimensional polyacrylamide gel electrophoresis and western analysis. Heme oxygenase is believed to be an important component of the cellular response to oxidative stress. The significance of heme oxygenase as a hypoxia-induced stress protein is discussed.

In this report, we investigate several examples of hypoxia-induced drug resistance and compare them with P-glycoprotein associated multidrug resistance (MDR). EMT6/Ro cells exposed to drugs in air immediately after hypoxic treatment developed resistance to adriamycin, 5-fluorouracil, and actinomycin D. However, these cells did not develop resistance to colchicine, vincristine or cisplatin. When the cells were returned to a normal oxygen environment, they lost resistance. There was no correlation between the content of adriamycin and the development of adriamycin resistance induced by hypoxia. There was no difference between the efflux of adriamycin from aerobic cells and that from hypoxia-treated cells. The mRNA for P-glycoprotein was not detected in the hypoxia-treated cells. These results suggest that hypoxia-induced drug resistance is different from P-glycoprotein associated multidrug resistance.