Down-regulation of molecular chaperone 78-kd glucose-regulated protein/immunoglobulin-binding protein expression involved in enhancement of human RS cell mutability.
OBJECTIVE
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.
Hirano
Junko
J
Department of General Surgery, Graduate School of Medicine, Chiba University, Chiba, Japan.
Kita
Kazuko
K
Sugaya
Shigeru
S
Ichimura
Yoshinobu
Y
Yamamori
Hideo
H
Nakajima
Nobuyuki
N
Suzuki
Nobuo
N
D015152:Blotting, Northern; D002460:Cell Line; D045744:Cell Line, Tumor; D017077:Culture Media, Conditioned / Q000737:chemistry*; D016373:DNA, Antisense / Q000235:genetics; D015536:Down-Regulation; D000091342:Endoplasmic Reticulum Chaperone BiP; D005333:Fetus; D005347:Fibroblasts; D015870:Gene Expression; D020869:Gene Expression Profiling; D006360:Heat-Shock Proteins / Q000235:genetics*; D006801:Humans; D015151:Immunoblotting; D018832:Molecular Chaperones / Q000235:genetics*; D009153:Mutagens / Q000494:pharmacology; D009154:Mutation; D010042:Ouabain / Q000494:pharmacology; D010190:Pancreatic Neoplasms / Q000378:metabolism*; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D014466:Ultraviolet Rays
D017077:Culture Media, Conditioned; D016373:DNA, Antisense; D000091342:Endoplasmic Reticulum Chaperone BiP; C000717247:HSPA5 protein, human; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D009153:Mutagens; D012333:RNA, Messenger; D010042:Ouabain