Down-Regulation of Molecular Chaperone 78-kd Glucose-Regulated Protein/Immunoglobulin-Binding Protein Expression Involved in Enhancement of Human RS Cell Mutability

Objectives: Enhancement of cell mutability via extracellular materials of cancer cells is a crucial event leading to the development of cancers; however, the activation process of mutability is still not well understood. In this study, to identify the regulatory mechanism of cell mutability, we investigated mutability modulated in response to human pancreatic cancer cell-conditioned medium and identified the candidates for cellular molecules involved in the mutability modulation. Methods: To test the mutation-modulating effects of the conditioned medium, human RS cells were cultured with medium derived by culturing human pancreatic cancer KP-4 cells, followed by irradiation with UV (mainly 254 nm in wavelength). Mutations were detected by phenotypic ouabain resistance and genetic base substitution of K-ras codon 12. Messenger RNA differential display was used to identify genes that were differentially expressed between conditioned medium-treated and mock-treated RSa cells. The influence of 78-kd glucose-regulated protein/immunoglobulin-binding protein (GRP78/BiP) expression on mutability was assessed by the down-regulation of GRP78/BiP using antisense oligonucleotides or antisense complementary DNA. Results: The UV-induced mutagenicity in RS cells was strengthened by preculture with KP-4 cell-conditioned medium. Messenger RNA differential display revealed that GRP78/BiP expression was suppressed in RS cells after treatment of the conditioned medium. Furthermore, the level of UV-induced mutations was elevated significantly in GRP78/BiP down-regulated cells. Conclusions: Culture of human RS cells with pancreatic cancer KP-4 cell-conditioned medium resulted in increased UV mutagenicity, possibly via the down-regulation of GRP78/BiP. Abbreviations: EMEM - Eagle minimal essential medium, DMEM/F12 - Dulbecco modified Eagle medium/nutrient mixture F12, OuaR - ouabain resistant, UV - far-ultraviolet ray, GAPDH - glyceraldehyde 3-phosphate dehydrogenase, PNA - peptide nucleic acid, GRP78 - 78-kd glucose-regulated protein, BiP - immunoglobulin-binding protein, ER - endoplasmic reticulum