imatinib and up regulated by NGF treatment for 2 h. To derive a biological meaning from the individual lists of V560G c Kit and NGF TrkA selleck chem 17-AAG modulated genes, we uploaded these lists to Ingenuity Pathways Analysis application for biological function and path way analysis. Out of a total of 524 genes modulated by imatinib treatment 510 genes mapped to the IPA knowl edge database with 428 genes for functions pathways analysis. Out of 117 NGF induced genes accepted by the IPA knowledge database, 106 mapped for functions pathways analysis. A comparison of the two gene lists revealed a common set of 58 genes out of 117 NGF upregulated genes that were also down modulated by imatinib. This suggests that NGF TrkA signaling regulates many genes the expres sion of which are also regulated by V560G c Kit in HMC 1 cells.

NGF TrkA activation does not enhance expression of genes involved in immune related functions that were downregulated by imatinib treatment Since the expression of 452 genes out of 510 genes which were downregulated by imatinib treatment for 4 h was not restored by the stimulation with NGF for 2 h, we next analyzed the two datasets using PANTHER protein class analysis which measures the significance of certain functional categories among these targets by their enrichment relative to the total numbers in their respective categories. As shown in Table 2, immune response component genes such as cytokines and cytokine receptors genes were significantly downre gulated by imatinib treatment. In contrast, NGF TrkA does not acti vate receptor genes significantly.

In addi tion, NGF TrkA activated cytokine genes but drastically fewer genes than c Kit mediated gene modulation. These data suggest that NGF can take over the proliferation signal but not immune related function induced by c Kit. More than 67% of NGF TrkA upregulated genes are involved in cell survival and proliferation We next analyzed genes which are upregulated by NGF TrkA signaling by IPA analysis. Significantly, over 67% of upregulated genes are involved in survival and prolifera tion. Although the immediate early response genes, such as EGR4, c FOS, or FOSB are also down stream of c Kit signaling, these genes are not constitu tively expressing. Therefore, several c Kit inducible genes were not downregulated by imatinib treatment.

To con firm the micro array data, we performed quantitative reverse transcriptase polymerase chain reaction to examine the relative expression level of c FOS, JUNB, EGR1, and c MYC. HMC 1 cells were incubated without serum for 17 h and were then treated with imatinib for 4 h. Cells were stimu lated with NGF for 30 and 120 min. All samples Batimastat were standardized by expression level of glucuronidase beta mRNA. In agreement with the micro array data, qRT PCR analysis revealed that immediate selleck Pacritinib early response genes, such as c FOS JUNB, and EGR1, were upregulated upon stimulation with NGF compared to expression level in untreated HMC 1 cells. Standardization of RNA level using