Biochemical analysis of the stress protein response in human oesophageal epithelium

Abstract

Background—The oesophageal epithelium is exposed routinely to noxious agents in the environment, including gastric acid, thermal stress, and chemical toxins. These epithelial cells have presumably evolved effective protective mechanisms to withstand tissue damage and repair injured cells. Heat shock protein or stress protein responses play a central role in protecting distinct cell types from different types of injury. 
Aim—To determine (i) whether biochemical analysis of stress protein responses in pinch biopsy specimens from human oesophageal epithelium is feasible; (ii) whether undue stresses are imposed on cells by the act of sample collection, thus precluding analysis of stress responses; and (iii) if amenable to experimentation, the type of heat shock protein (Hsp) response that operates in the human oesophageal epithelium. 
Methods—Tissue from the human oesophagus comprised predominantly of squamous epithelium was acquired within two hours of biopsy and subjected to an in vitro heat shock. Soluble tissue cell lysates derived from untreated or heat shocked samples were examined using denaturing polyacrylamide gel electrophoresis for changes in: (i) the pattern of general protein synthesis by labelling epithelial cells with ³⁵S-methionine and (ii) the levels of soluble Hsp70 protein and related isoforms using immunochemical protein blots. 
Results—A single pinch biopsy specimen is sufficient to extract and analyse specific sets of polypeptides in the oesophageal epithelium. After ex vivo heat shock, a classic inhibition of general protein synthesis is observed and correlates with the increased synthesis of two major proteins of molecular weight of 60 and 70 kDa. Notably, cells from unheated controls exhibit a "stressed" biochemical state 22 hours after incubation at 37°C, as shown by inhibition of general protein synthesis and increased synthesis of the 70 kDa protein. These data indicate that only freshly acquired specimens are suitable for studying stress responses ex vivo. No evidence was found that the two heat induced polypeptides are previously identified Hsp70 isoforms. In fact, heat shock results in a reduction in the steady state concentrations of Hsp70 protein in the oesophageal epithelium. 
Conclusion—Systematic and highly controlled studies on protein biochemistry are possible on epithelial biopsy specimens from the human oesophagus. These technical innovations have permitted the discovery of a novel heat shock response operating in the oesophageal epithelium. Notably, two polypeptides were synthesised after heat shock that seem to differ from Hsp70 protein. In addition, the striking reduction in steady state concentrations of Hsp70 protein after heat shock suggests that oesophageal epithelium has evolved an atypical biochemical response to thermal stress. 



Keywords: oesophagus; heat shock; Hsp70; hyperthermia; stress responses; cancer

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Figure 1 .

: Ex vivo steady state protein concentrations derived from tissue biopsy samples. Soluble protein (10 µg) derived from tissue lysates was applied to a 10% SDS-polyacrylamide gel and total protein was detected by staining with Commassie blue. Before lysis, the tissue samples were treated as follows: lane 1, immediately frozen; lane 2, incubated in media at 0°C before freezing; lanes 3-5, transfered from 0°C to media at 37°C and then immediately incubated for 20 minutes at either 37°C (lane 3), 45°C (lane 4), or 55°C (lane 5). Then, samples were incubated at 37°C for four hours and were immediately frozen in liquid nitrogen. The arrow shows the migration of a predominant polypeptide whose concentrations were dramatically reduced ex vivo. 


Figure 2 .

: Protein synthesis in tissue biopsy samples after an in vitro heat shock at different temperatures. Samples from three representative patients (samples 1-3) were incubated for 20 minutes at 37°C (lanes 1, 4, and 7), 45°C (lanes 2, 5, and 8), or 55°C (lanes 3, 6, and 9), and then after four hours of equilibration at 37°C, the samples were incubated in fresh media containing ³⁵S-methionine for an additional 45 minutes at 37°C. Radiolabelled protein derived from cell lysates was visualised using SDS-polyacrylamide gel electrophoresis and fluorography. The arrows show the migration of the two major polypeptides synthesised after heat shock. 


Figure 3 .

: Prolonged incubation of tissue biopsy specimens induces a stress protein response in the absence of heat shock. Samples from three representative patients (samples 1-3) were incubated for 20 minutes at 37°C (lanes 1, 3, and 5) or 55°C (lanes 2, 4, and 6), and then after four hours (lanes 1-4) or 22 hours (lanes 5 and 6) of equilibration at 37°C, the samples were incubated in fresh media containing ³⁵S-methionine for an additional 45 minutes at 37°C. Radiolabelled protein derived from cell lysates was visualised using SDS-polyacrylamide gel electrophoresis and fluorography. The arrows show the migration of the two major polypeptides synthesised after heat shock. 


Figure 4 .

: Hsp70 protein concentrations decline after heat shock. Biopsy samples from three representative patients (samples 1-3) were incubated for 20 minutes at 37°C (lanes 4, 6, and 8) or 55°C (lanes 3, 5, and 7), and then after four hours (lanes 3-6) or 22 hours (lanes 7 and 8) of equilibration at 37°C, the samples were incubated in fresh media containing ³⁵S-methionine for an additional 45 minutes at 37°C. Hsp70 protein derived from soluble cell lysates (10 µg) was visualised using SDS-polyacrylamide gel electrophoresis and immunoblotting with a monoclonal antibody specific for Hsp70 protein. As a control, the concentrations of Hsp70 protein were measured in soluble lysates from normal (10 µg; lane 1) or heat shocked lung (lane 2; one hour after a mild hyperthermic induction in male Wistar rats). The arrows show the migration of Hsp70 protein. 


Figure 5 .

: Progressive decrease in Hsp70 protein concentrations with increasing thermal stress. Tissue biopsy samples were treated as follows: the cells were transfered from 0°C to media at 37°C and then incubated for 20 minutes at either 37°C (lane 3), 45°C (lane 2), or 55°C (lane 1). Then, samples were incubated at 37°C for four hours and were frozen in liquid nitrogen. Soluble protein (10 µg) derived from tissue lysates was applied to a 10% SDS-polyacrylamide gel and Hsp70 protein concentrations were quantified using SDS-polyacrylamide gel electrophoresis and immunoblotting with a monoclonal antibody specific for Hsp70 protein. The arrow shows the migration of Hsp70 protein. 


Figure 6 .

: Characterisation of a novel monoclonal antibody to Hsp70 protein isoforms. Protein immunoblots using a novel monoclonal antibody generated to Hsp70 protein (MB-H1) were performed with purified Hsp70 (lane 5), purified Hsc70 (lane 6) and the Hsp70/Hsc70 isoforms in four distinct tumour cell lines (lanes 1-4; BT549 (breast cancer); HS913T (fibrosarcoma); SK-UT-1 (leiomyosarcoma); and MCF7 (breast cancer), respectively). 


Figure 7 .

: Hsc70 protein concentrations remain constant after heat shock. Biopsy samples from three representative patients (samples 1-3) were incubated for 20 minutes at 37°C (lanes 4, 6, and 8) or 55°C (lanes 3, 5, and 7), then after four hours (lanes 3-6) or 22 hours (lanes 7 and 8) of equilibration at 37°C, the samples were incubated in fresh media containing ³⁵S-methionine for an additional 45 minutes at 37°C. Hsc70 protein and related isoforms derived from soluble cell lysates were visualised using SDS-polyacrylamide gel electrophoresis and immunoblotting with monoclonal antibody MB-H1. As a control, the concentrations of Hsp70 protein were determined in soluble lysates from normal (10 µg; lane 1) or heat shocked lung (lane 2; one hour after a mild hyperthermic induction in male Wistar rats). The arrows show the migration of Hsc70 protein and isoforms. 


Figure 8 .

: The effects of ethanol and acid exposure on the heat shock protein response. Biopsy samples were incubated for 20 minutes at 37°C in normal media (lane 3), low pH media (lane 2), or 4% ethanol (lane 1). After four hours of equilibration at 37°C in fresh media, the samples were incubated in media containing ³⁵S-methionine for an additional 45 minutes at 37°C. Left panel: radiolabelled protein derived from cell lysates was visualised using SDS-polyacrylamide gel electrophoresis and fluorography. Right panel: Hsp70 protein derived from cell lysates was visualised using SDS-polyacrylamide gel electrophoresis and immunoblotting with a monoclonal antibody specific for Hsp70. The arrow shows the migration of Hsp70 protein.