All quantifications Tipifarnib leukemia were performed in duplicate for three independent experiments and normalized with re spect to the endogenous PDGF mRNA levels. Target cDNA expression was quantified using the Ct method. Vali dated primers were obtained from Qiagen. Western Blot Western Blots were performed with the following anti bodies anti Akt total, anti Erk total, anti phospho Akt, anti phospho Erk. anti HIF 1alpha, anti actin. Flow cytometry For each condition, 106 cells were washed in PBS 1X at 4 C. Fluorescence was analyzed on a FACScan cytometer, and the data were analyzed with CellQuest. The measurements were performed twice in two independent experiments. Immunohistochemistry Immunohistochemistry used standard procedures. Briefly, tumors were fixed in 4% paraformaldehyde and embedded in paraffin.

Sections were deparaffinized and heated for 10 minutes in 10 mmol L citrate buffer, pH 6. 2, for antigen Inhibitors,Modulators,Libraries retrieval. They were stained with Harris solution and Eosin for histological examination and immunostained using the primary antibodies raised against CXCR4 and CXCR7. Slides were then incubated for 30 min with secondary biotinylated anti mouse antibody. Immunostaining was developed with a liquid DAB substrate kit according to the manufacturers instructions. Background The RAS RAF MEK ERK and PI3K AKT sig naling pathways regulate gene expression programs that promote cell growth, proliferation, motility, and survival. Mutations that cause constitutive RAS ERK or PI3K AKT signaling are among the most common alter ations in human cancer and both pathways are often acti vated in the same tumor.

PI3K AKT activation is common in prostate cancer, often due to loss of a suppres sor of the pathway, PTEN. However, unlike other car cinomas, prostate cancers rarely have activating mutations in RAS or RAF, and thus, the mechanisms that allow transcriptional activation of RAS ERK target genes in this malignancy are not fully understood. RAS ERK signaling can be initiated Inhibitors,Modulators,Libraries by tyrosine kinase receptors that activate RAS, followed by the RAF MEK ERK kinase cascade, resulting in phosphorylated ERK. pERK, in turn, phosphorylates transcription fac tors, including some members of the ETS family, leading to increased transcriptional activation of target genes. PI3K phosphorylates phosphoinositides leading to activation of downstream proteins such as the kinase AKT.

Inhibitors,Modulators,Libraries PTEN, a phosphatase, Inhibitors,Modulators,Libraries can reverse this process and acts as a tumor suppressor. Activated AKT has mul tiple functions, one being the activation of the mTOR containing signaling complex mTORC1, Inhibitors,Modulators,Libraries which alters translational control of gene expression. AKT also acti vates the mTORC2 complex, which provides positive feedback by phosphorylating and activating AKT. The RAS ERK and PI3K AKT pathways are highly intercon than nected. For example, RAS can activate PI3K, and AKT can phosphorylate and inhibit RAF.