This score was used as the cut off for identifying gene networks significantly affected by tipifarnib. Real Time RT PCR The genes and primers used for RT PCR are listed in Table 2. Due to the limited amount total RNA from the patient samples, RNA that had been through one round of linear amplification was used. The Roche Molecular LightCycler with Syber Green I system detection was used for real time PCR. PCR thermocycling consisted of denaturation at 95 C for 45 seconds, followed by 30 cycles at 62 C for 10 seconds, and 72 C for 12 seconds. Samples were run in triplicate with both test primer sets and the control gene eukaryotic elongation factor 1 alpha. This gene was used to control for differences in the amount of target material since initial microarray experiments found that expression of the EEF1A1 gene did not vary significantly between drug treated and control cells.

A standard curve was also run in each PCR reaction. Fold changes were calculated by normalizing the test crossing thresholds with the EEF1A1 amplified control Ct. Results and Discussion Response of AML like cell lines to tipifarnib Tipifarnib inhibited the growth of 4 human AML cell lines in a dose dependent manner. The IC50 of these cell lines when treated with tipifarnib ranged from 19 to 134 nM. The mutation status of the ras oncogenes in the AML cell lines are also shown. These data indicate that the four AML like cell lines are sensitive to tipifarnib treat ment at concentrations well below the micromolar con centrations that is achievable in the bone marrow of leukemia patients.

However, there was no correlation between the type of ras mutation and sensitivity to the drug. These data are consistent with the activity of tipi farnib in vivo and allowed for further characterization of gene expression changes in these cells after treatment with pharmacologically relevant drug concentrations. Identification of genes differentially Batimastat expressed in tipifarnib treated AML cells We next asked what genes are modulated following treat ment of AML cells with tipifarnib and if there are differ ences between the affected gene networks in cell lines compared to primary cells from patients. To this end we first selected the three most sensitive cell lines and treated them with tipifarnib or vehicle alone over a 6 day time course.

A standard concentration of 100 nM tipifarnib was chosen to ensure exposure within the pharmacologically active range of the compound. Samples for RNA analysis were harvested daily from duplicate cell cultures. Message RNA was isolated, amplified and hybridized to the cDNA microarrays containing approximately 7000 genes. Based on scatter plot analysis the microarray data was found to be highly reproducible between duplicate samples. A one way ANOVA was employed to identify genes that were significantly changed over the 6 day time course compared to time matched controls.