White blood cells were washed twice with PBS, resuspended in FBS with 10% DMSO and immediately frozen at 80 C. Some characteristics of the two patient samples used in the present study are shown in Table 1. Ras mutational status Analysis of activating mutations in N ras, K ras, and H ras codons was determined by PCR and RFLP analysis as pre viously described. Microarray analysis Total RNA was isolated using the Qiagen RNeasy kit and treated with DNase1 to remove any residual genomic DNA. Probe preparation was performed as previously described. Linear amplification was performed on total RNA to obtain at least 15 ?g of amplified RNA. Cell line mRNA and patient sample mRNA underwent one and two rounds of linear amplification respectively. Microar rays were generated and probes hybridized as described.

Samples were hybridized to arrays that contained 7452 cDNAs from the IMAGE consortium and Incyte libraries. The intensity level of each microarray was scaled so that the 75th percentile of the expression levels was equal across micro arrays. To control for chip errors, replicate clones on each chip that displayed a coefficient of variance greater than 50% of the mean were excluded from the analysis. Since back ground intensity was a maximum of 30 relative fluores cent units for all experiments, a threshold of 30 RFU was assigned to all clones exhibiting an expression level lower than this. The microarray data were then nor malized by quantile normalization and logarithmically transformed before further analysis.

Statistical analysis Analysis of variance and t tests were used to investigate the effect of drug treatment and time and their interactions for Anacetrapib each gene. Multiple hypotheses testing was controlled by applying the false discovery rate algorithm. All statistical analyses were performed in S Plus 6. 1. Ratio matrices were generated based on pair wise analysis of treated versus control samples. Hierarchical clustering was performed using a correlation metric and complete linkage. Pathway analysis A total of 1198 genes that had a false discovery rate 0. 1 in at least one cell line were used for the pathway analysis. Gene refseq accession numbers were imported into the Ingenuity Pathway Analysis software. 898 of these genes were mapped to the Ingenuity database. Seventy two of these genes were also affected in patient samples and were, therefore considered to be significantly regulated by tipifarnib.

The identified genes were mapped to genetic networks available in the Ingenuity database and were then ranked by score. The score is the probability that a collection of genes equal to or greater than the number in a network could be achieved by chance alone. A score of 3 indicates that there is a 1/1000 chance that the focus genes are in a network due to random chance. Therefore, scores of 3 or higher have a 99.