Biochemical analysis of the stress protein response in human oesophageal epithelium

D Hopwood,^a S Moitra,^a B Vojtesek,^b D A Johnston,^a J F Dillon,^a T R Hupp^a

^a Department of Molecular and Cellular Pathology, Ninewells Hospital and Medical School, Dundee University, Dundee DD1 9SY, ^b Department of Cellular and Molecular Oncology, Masaryk Memorial Cancer Institute, Zluty kopec 7, 656 53 Brno, Czech Republic

Correspondence to: Dr T R Hupp.

Accepted for publication 25 March 1997

BackgroundThe 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.
AimTo 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.
MethodsTissue 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.
ResultsA 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.
ConclusionSystematic 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.
(GUT 1997;41:156-163)

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