The Study Of Proliferation Inhibition And Apotosis In Jurkat Cell By BRD4 Inhibitor JQ1

Objectives:Acute T lymphocytic leukemia(T- ALL) is derived from lymphocytic lineage of hematologic malignancy, which is characterized in cloning proliferation of malignant precursors. Poor therapeutic effects of chemotherapy,relapsing easily by regular treatment,and possibility of being prone to aggressing the central nervous system make the treatment to acute T lymphocytic leukrmia a clinical problem. Both the domestic and foreign researches show inactivation of c-myc gene induces sustained tumor regression,through the phenomenon termed “oncogene addiction”. Recent years, the study about domain structure of bromine protein demonstrates the inhibitor(JQ1) could inhibit and kill malignant cells coming from variety of tumors, including chronic myeloid leukemia,ovarian cancer, malignant glioma and lung denocarcinoma, et al. Our team in previous study also found that JQ1 can significantly reduce the c- myc m RNA expression level in the tumor cells, thus there is a positive therapeutic effect to acute B lymphocytic leukemia,which is characterized in BCR- ABL gene mutation.T-ALL is similar with B-ALL derived from thymus progenitor cells, then whether on earth Brd4 inhibitor JQ1 has the same effect is the focus of this research. This study intends to adopt T-ALL( Jurkat cell line) with JQ1,through observing cell morphology under linght microscopy, determined by MTT method to detect JQ1 role in Jurkat cell line with inhibitory concentration, flow cytometry instrument to detect cell apoptosis rate, by fluorescence quantitative PCR to detect Notch1 m RNA, c- myc m RNA, PTEN m RNA expression in NOTCH pathway, exploring possible mechanism of JQ1 on T-ALL cell line, putting forward a new thought and providing theoretical basis for clinical application.Methods:1. The Jurkat cell line proliferation inhibition rate interfered by JQ1 were detected by MTT assay;2. Jurkat cell line apoptosis rate interfered by JQ1 were detected by flow cytometry;3. Real-time fluorescence quantitative polymerase chain reaction(RT-PCR) detected Notch1 m RNA, c- myc m RNA, PTEN m RNA expression.Results:1. The results of MTT test showed that : Jurkat cell lines interfered by JQ1 were cultured lasted 48 h, 72 h, 96 h. Compared with control group, the drug in different concentrations could have obvious inhibitory effect on the proliferation of Jurkat cells.And the cell proliferation inhibition rate was rising with increased concentration and time expansion, characterized in a time- dose dependent manner. The 48 h half inhibitory concentration(IC50) of JQ1 was 2.67 μmol/L.2. The results of flow cytometry test showed that: Jurkat cells interfered by JQ1 in different concentrations were cultured lasted 48 h,72 h. The results tested by fluorescence staining flow cytometry instrument showed the average early apoptosis of control group after 48 h rate was(1.41 + 0.06) %, while the early apoptosis rate of observation groups termed 0.8, 1.6, 4 μmol/L of JQ1 were respectively(20.9 + 0.67) %,(25.27 + 0.32) %,(32.70 + 0.93) %. And early apoptosis rate of control group in control group after 72 h was(16.50 + 0.46) %, while early apoptosis rate in observation groups were respectively(26.43+ 0.72) %、(39.67 + 0.64) %、(61.23 + 1.15) %.Through the single factor analysis of variance of statistics, compared with control group, the drug in different concentrations could have obvious apoptosis rate of Jurkat cells. And the cell early apoptosis rate was rising with increased centration and time expansion, characterized in a time- dose dependent manner.(Time effect of the linear trend F = 373.845, P < 0.001, the doses of the linear trend F = 505.763, P < 0.001).3. The real-time fluorescent quantitative PCR detection displayed that: When Jurkatcell lines cultured respectively by different concentrations of JQ1 after 48 h, the dissolution curve of Notch1 m RNA, c- myc m RNA and PTEN m RNA were in a single amplification curve, which means good specificity. Study results showed that Notch1 m RNA、c-myc m RNA expression in observation groups quantity significantly decreased than the control group, while PTEN m RNA expression were gradually increasing compared with control group(F value were respectively 1624.83, 823.82, 4460.76, P < 0.001), there were statistically significant(P < 0.05).Conclusions:1.JQ1 could significantly inhibit Jurkat cell proliferation, characterized in a time-dose dependent manner.2.JQ1 could induce the apoptosis of Jurkat cells effectively, also characterized in a time-dose dependent manner.3.Bromine structure domain protein inhibitor JQ1 could significantly downregulate the expression of Notch signaling pathway related gene: Notch1, c- myc and upregulate PTEN m RNA level, which indicated that NOTCH signal pathway may be one of the pathways of JQ1 induced Jurkat cell line apoptosis.