Summary 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 hypoxiainduced stress protein is discussed.Hypoxia is an important environmental stress encountered in some solid tumours that can influence the effectiveness of radiation, hyperthermia and chemo-therapy (Coleman, 1988;Heacock & Sutherland, 1990). Poor vascularisation of tumours is common, and this results in impaired delivery of oxygen, glucose and other nutrients to cells distant from blood vessels, as well as inefficient removal of metabolic wastes (Sutherland, 1988). Among the many changes in tumour cellular physiology that occur under hypoxic stress, it has been observed that a group of proteins, called oxygen regulated proteins or ORPs, can be induced to undergo enhanced rates of synthesis depending on the severity and duration of the stress (Heacock & Sutherland, 1990;Anderson et al., 1979;Heacock & Sutherland, 1986). Moreover, the kinetics of the development of resistance to the drug adriamycin in vitro have been reported to correlate with the enhanced expression of some of these ORPs Subjeck & Shyy, 1986). Another phenotype associated with chronic hypoxia is enhanced radiation sensitivity following reoxygenation. Although low oxygen is known to give rise to radiation resistance, through a diminishment of the phenomenon known as the oxygen effect (von Sonntag, 1987), upon reoxygenation chronically hypoxic cells exhibit enhanced sensitivity to gamma radiation (Kwok & Sutherland, 1989a;Kwok & Sutherland, 1989b). The mechanistic basis of this effect is not understood, although it may be a consequence of the stress caused by reoxygenation rather than by hypoxia per se.The molecular weights assigned to five major ORPs are 260, 150, 100, 80 and 33 kilodaltons (Heacock & Sutherland, 1986 In experiments designed to determine a time course for the induction of heme oxygenase under hypoxia, the chambers were opened, the plates were placed on ice, the media was removed by aspiration and the cells were immediately lysed as above by the addition of cold lysis buffer.Polyacrylamide gel electrophoresis and western blotting The labelled Triton soluble proteins were analyzed by twodimensional SDS-polyacrylamide gel electrophoresis accordCorrespondence: R.M. Sutherland.

Summary 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.As a tumour grows, heterogeneities of cellular microenvironments occur, such as the development of oxygen gradients in the tumour as a consequence of deficient vascularisation, and cause hypoxic cells that may be resistant to radiotherapy (Sutherland, 1988). Several studies using monolayer cultures (Smith et al., 1980;Teicher et al., 1981Teicher et al., , 1985 and the multicell spheroid system (Sutherland et al., 1979) Conditions of hypoxic exposure Before being gassed with N2, the cells were supplied with 5 ml of fresh complete medium and allowed to equilibrate in a humidified 37°C incubator. Cells undergoing hypoxic stress were isolated in specially designed hypoxic chambers at room temperature (Sutherland et al., 1982). The chambers were repeatedly evacuated and filled every 15 min for 2.25 h with the appropriate gas mixtures certified to contain less than 10 ppm 02. The sealed chambers were then removed to a warm room (37'C) at a time point, t, referred to hereafter as '0' hours of hypoxia.Preparation of drugs and conditions of drug exposure All drugs used were obtained from Sigma Chemical Company. Stock solutions of adriamycin (ADR), vincristine, and actinomycin D (ACTD) were prepared with phosphatebuffered saline (PBS). Solutions of 5-fluorouracil (5-FU), colchicine, and cisplatin were made before each experiment. The solvents used were PBS for ADR, ACTD and cisplatin and distilled water for 5-FU. Absolute ethanol was used as a solvent for colchicine in order to obtain a sufficiently high concentration for the experiments. The final concentration of alcohol in the medium was 1%. As a control for the alcohol solvent, we established that 1% of absolute ethanol in cultures for 2 h had no effect on plating efficiency. Drug treatment was started under aerobic conditions at 37'C in the incubator after culture dishes were removed from the chambers at the zero time. Exposure times for ADR, ACTD and cisplatin were 1 h. A 2 h exposure time was used for 5-FU, colchicine and vincristine to cause significant cell killing. Exposure times were limited to 1-2 h to avoid the effect of release from a ORPs-induced state,...

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