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.
Previously, we reported that Chinese hamster ovary (CHO) cells transfected with murine mouse major histocompatibility complex class II genes, exhibit a unique antigen (Ag) processing defect whereby these cells are impaired in processing only Ag with disulfide bonds. Here, we examined various aspects of the intracellular reducing environment in the CHO cells to understand the underlying mechanism causing the defect. A cell hybrid generated by the fusion of CHO cells and L cell fibroblasts was used for comparison due to their competency in processing Ag. The transport pathway of cysteine within the CHO cells appeared normal. However, these cells had a significantly lower level of glutathione, a major physiological reducing thiol, compared to the cell hybrid. Treatment of the CHO cells with N-acetyl-L-cysteine did not augment their glutathione content nor their ability to process Ag. When the cell hybrid was treated with L-buthionine-(S,R)-sulfoximine (BSO), which significantly decreased their glutathione level, the hybrid poorly processed hen egg lysozyme (HEL) and ovalbumin, which have disulfide bonds. In contrast, BSO treatment did not affect the capacity of the hybrid to process pigeon cytochrome c and carboxymethylated HEL, which lack disulfide bonds. Therefore, low intracellular glutathione levels in antigen-presenting cells correlated with defective processing of Ag with disulfide bonds, indicating that this thiol may be a critical factor in regulating productive Ag processing.
Activation of CD4+ T cells requires processing of exogenous protein antigens by antigen-presenting cells (APC). A macrophage hybridoma and B cell lymphoma were comparable in their ability to process hen egg lysozyme (HEL), which involves reduction of its disulfide bonds. The intracellular levels of cysteine and glutathione, major physiological thiols, based on protein content were similar within these cell lines. In addition, the cysteine transport pathway in viable cells was assessed by 35S-cystine uptake. For macrophages, the majority of the radioactivity resided in high density subcellular fractions of Percoll gradients that comigrated with lysosomal beta-galactosidase (beta-gal). Besides the lysosomes, low density fractions cosedimenting with endosomes incorporated the radiolabel in the B cells. Both peaks of radioactivity disappeared when the B cells were incubated with unlabeled carboxymethyl-cysteine (CM-cysteine), a specific competitor of the plasma membrane CG transport system. The distinct gradient profiles of radiolabel uptake in the cells correlated with a difference in their capacity to process the transferrin-lysozyme conjugate (TF-HEL). TF-HEL was significantly more stimulatory than HEL in inducing a HEL-specific T cell response with the B cells as the APC. However, the potencies of TF-HEL and HEL were similar when the macrophages were the APC. Thus, the intracellular location of cysteine transport activity may be cell lineage-dependent, and its presence may, in part, determine whether an organelle is a productive site of processing antigens with disulfide bonds that is necessary for CD4+ cell activation.
Summary 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-a (TGF-a) 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 106/cell. Constitutive expression of TGF-a 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-a protein sythesis 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-a/EGF-R autocrine loop operating within cells that produce high local concentrations of TGF-a in the three-dimensional architecture of a spheroid.
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