Gene expression profile by inhibiting Raf-1 protein kinase in breast cancer cells.
Raf-1 protein serine-threonine kinase plays an important role in cell growth, proliferation, and cell survival. Previously, we and others have demonstrated that antisense raf oligonucleotide-mediated inhibition of Raf-1 expression leads to tumor growth arrest, radiosensitization and chemosensitization in vivo. Raf-1 inhibition is also associated with apoptotic cell death. In this study, we inhibited Raf-1 using an antisense raf oligonucleotide (AS-raf-ODN) to identify downstream targets of Raf-1 using microarray gene expression analysis. Treatment of MDA-MB-231 breast cancer cells with 250 nM AS-raf-ODN led to significant inhibition of Raf-1 protein (75.2 +/- 9.6%) and c-raf-1 mRNA levels (86.2 +/- 3.3%) as compared to untreated control cells. The lipofectin control or mismatch oligonucleotide had no effect on Raf-1 expression. To determine the changes in gene expression profiles that were due to inhibition of Raf-1, we simultaneously compared the gene expression patterns in AS-raf-ODN treated cells with untreated control cells and cells treated with lipofectin alone or MM-ODN. A total of 17 genes (4 upregulated and 13 down-regulated) including c-raf-1 were identified that were altered after AS-raf-ODN treatment. Functional clustering analysis revealed genes involved in apoptosis (Bcl-XL), cell adhesion (paxillin, plectin, Rho GDIalpha, CCL5), metabolism (GM2A, SLC16A3, PYGB), signal transduction (protein kinase C nu), and transcriptional regulation (HMGA1), and membrane-associated genes (GNAS, SLC16A3). Real-time PCR, Northern analysis and Western analysis confirmed the microarray findings. Our study provides insight into Raf-1 related signaling pathways and a model system to identify potential target genes.
Mewani
Rajshree R
RR
Department of Radiation Medicine, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC 20057, USA.
Tian
Song
S
Li
Bihua
B
Danner
Malika T
MT
Carr
Theresa D
TD
Lee
Sung
S
Rahman
Aquilur
A
Kasid
Usha N
UN
Jung
Mira
M
Dritschilo
Anatoly
A
Gokhale
Prafulla C
PC
D015152:Blotting, Northern; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000201:enzymology / Q000235:genetics* / Q000473:pathology; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D005260:Female; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D019908:Proto-Oncogene Proteins c-raf / Q000037:antagonists & inhibitors* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D019908:Proto-Oncogene Proteins c-raf