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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 GDI·, 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. Introduction Raf-1, a cytosolic serine/threonine protein kinase, plays an important role in mitogen and stress-induced signaling responses, cell survival and proliferation (1,2). Activated Raf-1 triggers a kinase cascade that includes phosphorylation and activation of MEK, which in turn activates ERK. These effects have generally been associated with cell proliferation and survival by inducing transcription factors such as AP-1, Elk-1, Ets and stimulation of the activity of promoters containing NF-κB sites (3-5). Recent studies have shown that Raf-1 also regulates the transcription of a number of diverse molecules (6-8). Although activating ras mutations have been reported in a variety of solid tumors and leukemias with resulting Raf-1/MEK/ERK activation, the complexity of Raf-1 regulation extends beyond its interaction with Ras (9,10). Recent reports using c-raf-1 gene-knockouts have observed MEK/ERK independent functions of Raf-1 in cell survival (11,12). Studies have linked the anti-apoptotic function of Raf-1 to a re-localization of Raf-1 to the mitochondria (13). Raf-1 is reported to exert prosurvival effects upstream of cytochrome c release by phosphorylating the pro-apoptotic Bcl-2 family member, BAD (13). Raf-1 also regulates the antiapoptotic transcription factor, NF-κB (14), which has been shown to participate in the transcriptional regulation of IAPs as well as c-FLIP, an inhibitor of caspase-8 activation (15). Raf-1 apparently induces NF-κB activation by degradation of IκB, an inhibitor of NF-κB, via MEKK-1, independent of MEK/ERK (16). Raf-1 activity has been demonstrated both in solid tumors and hematological malignancies. Previously, it was shown that overexpression or activation of Raf-1 protein kinase is associated with morphological transformation of immortalized cells and resistance to radiation and chemotherapeutic drugs (1,17-19). Interestingly, activation of Raf has been shown to induce transcription from the human mdr-1 promoter (19). Consequently, overexpression of Raf-1 was found to induce the multi-drug resistance phenotype, P-gp, making the cells resistant to chemotherapeutic drugs. Previously, we and others have demonstrated significant antitumor activity, radiosensitization and chemosensitization by antisense raf oligonucleotide-mediated inhibition of Raf-1 expression in various tumor cell types (20-23). Inhibition of Raf-1 also has been associated with apoptotic cell death (24,25). INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 17: 457-463, 2006 457 Gene expression profile by inhibiting Raf-1 protein kinase in breast cancer cells RAJSHREE R. MEWANI, SONG TIAN, BIHUA LI, MALIKA T. DANNER, THERESA D. CARR, SUNG LEE, AQUILUR RAHMAN, USHA N. KASID, MIRA JUNG, ANATOLY DRITSCHILO and PRAFULLA C. GOKHALE Department of Radiation Medicine, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, 3970 Reservoir Road, NW, Washington DC 20057, USA Received August 16, 2005; Accepted September 19, 2005 _________________________________________ Correspondence to: Dr Prafulla C. Gokhale, Department of Radiation Medicine, E207, The Research Building, Georgetown University Medical Center, 3970 Reservoir Road, NW, Washington