Tyrosine Kinases Inhibitor A77 1726 Inhibits The Expression Of C-fos And C-myc In Dami Cells Induced By RhIL-13

AIM: It is to investigate A77 1726 inhibits the expression of proto-oncogene c-fos and c-myc in Dami cells induced by rhIL-13, partly elucidated the signal transduction mechanism of IL-13 and A77 1726.METHODS: 1. Cell culture: Dami cells were cultured in RPMI 1640 supplemented with 10% calf serum at 37℃with 5% CO2 . Before the treatment of rhIL-13, Dami cells were grown to a density of 5×108~1×109 /L and cultured without serum for 20 h. 2. Experiment of dosage-efficiency: The diagram of curves were made according to the growth of Dami cells and the absorbency value of Dami cells was mensurated. The least effective concentration of A77 1726 on the growth of Dami cells was measured. 3. Isolation of total RNA: Total RNA was extracted from those cells incubated with or without rhIL-13 100μg/L and A77 1726 50μM respectively for various time points. The RNA quality was measured by means of RNA agar gel electrophoresis. 4. Reverse transcription polymerase chain reaction (RT-PCR) was used to examine the expression of some genes including that:①the expression of c-fos mRNA in Dami cells incubated with rhIL-13 for 15, 30, 60 and 120 min;②the expression of c-myc mRNA in Dami cells incubated with rhIL-13 for 0, 2, 4 and 6 h;③the expression of c-fos mRNA in Dami cells incubated both with rhIL-13 and A 77 1726 for 15, 30, 60 and 120 min;④the expression of c-myc mRNA in Dami cells incubated both with rhIL-13 and A77 1726 for 2, 4 and 6 h. 5. Western Blotting: After Dami cells were cultured in the medium of serum-free for 20 h, these cells were incubated with rhIL-13 for 15, 30, 60, 120, 240 and 360 min, and RIPA Buffer was used for the lysis of Dami cells. The expression of c-Fos and c-Myc protein were observed by Western Blotting after the isolation of protein.RESULTS: 1. The least effective concentration of A77 1726 on the growth of Dami cells was 50μM. 2. rhIL-13 induced the expression of c-fos mRNA in Dami cells (P<0.05), reaching a peak of the expression in 30 min (P<0.01). 3. rhIL-13 down -regulated the expression of c-myc mRNA in Dami cells (P<0.05). 4. rhIL-13 induced the expression of c-Fos protein in Dami cells (P<0.05), reaching a peak of the expression in 60 min (P<0.01). 5. rhIL-13 down-regulated the expression of c-Myc protein of Dami cells (P<0.05). 6. A77 1726 inhibited the expression of c-fos mRNA in Dami cells induced by rhIL-13. 7. A77 1726 inhibited that rhIL-13 down–regulated the expression of c-myc mRNA in Dami cells. 8. A77 1726 inhibited the expression of c-Fos protein of Dami cells induced by rhIL-13. 9. A77 1726 inhibited that rhIL-13 down–regulated the expression of c-Myc protein in Dami cells. CONCLUSION: 1. IL-13 induced the expression of c-fos mRNA and protein in Dami cells. 2. IL-13 down-regulated the expression of c-myc mRNA and protein in Dami cells. 3. A77 1726 inhibited the expression of c-fos mRNA and protein in Dami cells induced by IL-13. 4. A77 1726 inhibited that IL-13 down-regulated the expression of c-myc mRNA and protein in Dami cells.