Activation of Raf-1 in human pancreatic adenocarcinoma.
Point mutations in the Ras oncogene cause Ras to remain in its active GTP-bound state sending signals downstream continuously. Since 75 to 90% of all human pancreatic ductal adenocarcinomas harbor activating mutations at codon 12 of the K-ras oncogene it was our belief that Raf-1-MEK-MAPK will be activated in the majority of human pancreatic cancers. The aim of this study was to confirm activation of Raf-1 in K-ras mutant human pancreatic cancer. Additionally, we sought to determine if Raf-1 activation differed in K-ras mutant and nonmutant pancreatic cancer. Furthermore, we were interested in determining if Raf-1 activation in pancreatic cancer led to subsequent activation of downstream effectors such as MAP kinase. The presence of mutations in codon 12 of the K-ras oncogene in 14 human pancreatic adenocarcinoma cell lines was determined by use of mutant allele-specific PCR restriction fragment length polymorphism analysis. Raf-1 expression of quiescent cells was determined by immunoblotting using a rabbit anti-human polyclonal antibody and enhanced chemiluminescence. MAP kinase activity was determined by measuring the incorporation of phosphate into Myelin Basic Protein. Seven cell lines were noted to have mutations in codon 12 of K-ras while seven cell lines did not. There was no difference in expression of the 74 kDa-activated form of Raf-1 in K-ras mutant vs K-ras nonmutant cell lines. However, there was a significant increase in MAP kinase activity in the nonmutant cell lines compared to the cell lines with Ras mutations (P = 0.026). We conclude that Raf-1 is expressed in its active form in human pancreatic cancer regardless of K-ras status. However, signalling downstream of Raf-1 differs in cell lines with K-ras mutations compared to those cell lines without K-ras mutations.
Berger
D H
DH
Department of Surgery, Allegheny University of the Health Sciences, MCP/Hahnemann School of Medicine, Philadelphia, Pennsylvania 19129, USA. Berger@Allegheny.edu
Jardines
L A
LA
Chang
H
H
Ruggeri
B
B
D000230:Adenocarcinoma / Q000201:enzymology / Q000235:genetics*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D004789:Enzyme Activation; D005786:Gene Expression Regulation; D011905:Genes, ras; D006801:Humans; D009154:Mutation; D010190:Pancreatic Neoplasms / Q000201:enzymology / Q000235:genetics*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000235:genetics* / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf; D014407:Tumor Cells, Cultured
D011518:Proto-Oncogene Proteins; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D017871:Calcium-Calmodulin-Dependent Protein Kinases