Mechanism of cell proliferation in acute T cell leukemia with high expression of Notch1 gene

Abstract

Objective To investigate the mechanisms of cell proliferation of acute T cell leukemia (T-ALL) with high expression of Notch1 gene and explore the potential of targeted drug intervention. Methods A3, MOLT-4, Jurkat cell lines and peripheral blood mononuclear cell (PBMC) derived from 20 healthy volunteers were selected as subjects. All the cells were treated with palbociclib at different concentrations(0, 50, 100 nnol/L). The cell proliferation status was detected by CCK-8 method. Inhibitory concentration 50 (IC50) values of different concentrations of palbociclib were calculated. The cell morphology was observed, and the cell cycle changes of T-ALL cell line and PBMC were analyzed by flow cytometry. Real-time quantitative (RT)-PCR was used to detect the mRNA relative expression level of related genes. The t-test was used to compare the mRNA relative expression of related genes in T-ALL cells and PBMC after treatment with different concentrations of palbociclib. Results ① The gray scale percentages of Notch1 gene expression in A3 and MOLT-4 cell lines were (62.3±3.40)% and (77.6±4.71)%, which were significantly higher than that of PBMC with (20.8±1.53)%. And the differences were significantly (t=6.236, 10.034; P=0.025, 0.008). There were no significant difference between (28.5±1.36)% of Jurkat cell line and PBMC (t=1.257, P=0. 430). ② Compared with PBMC, the proliferation activity of MOLT-4 cell line was significantly inhibited by palbociclib, followed by A3 cell line.And that inhibition of cell proliferation activities of all cells was time- and concentration-dependent.After 48 h, the IC50 values of A3 and MOLT-4 cell lines were (0.43±0.12) μmol/L and (0.10±0.16) μmol/L respectively, which were lower than that of PBMC with (0.83±0.15)μmol/L, and the differences were significantly (t=12.354, 7.486; P=0.006, 0.015). ③ Microscopically, compared with PBMC, the proliferation of A3 and MOLT-4 cell lines were inhibited significantly at 100 nmol/L palbociclib. ④ In the analysis of cell cycle, compared with palbocicib 0 nmol/L, the S+ G2 phase of A3 and MOLT-4 cell lines decreased significantly at palbocicib 50 nmol/L (t=5.358, 7.096; P=0.039, 0.017) , the S+ G2 phase of PBMC didn′t change significantly (t=2.324, P=0.160) at palbocicib 50 nmol/L. The cell count in S+ G2 phase of A3 and MOLT-4 cell lines and PBMC were significantly lower than that in the treatment with palbocicib 0 nmol/L, and the differences were statistically significant (t=10.024, 12.022, 6.102; P= 0.008, 0.006, 0.032). ⑤ When treated with palbocicib 0 nmol/L, the mRNA relative expression levels of CDK4, CDK6, Notch1 and Myc in A3 and MOLT-4 cell lines were significantly higher than those of PBMC (A3 cell lines vs. PBMC: t=6.953, 6.058, 7.210, 4.692 , P=0.021, 0.034, 0.026, 0.045; MOLT-4 cell lines vs. PBMC: t=8.541, 5.2671, 0.536, 8.735, P=0.019, 0.038, 0.007, 0.015). The mRNA relative expression levels of CDK4, CDK6 and Myc in MOLT-4 cell line treated with palbocicib 50 and 100 nmol/L were significantly lower than those treated with palbocicib 0 nmol/L, and the differences were statistically significant (palbocicib 50 nmol/L vs. palbocicib 0 nmol/L: t=7.210, 6.053, 4.725, P=0.026, 0.035, 0.039; palbocicib 100 nmol/L vs. palbocicib 0 nmol/L: t=13.579, 12.025, 12.019, P= 0.005, 0.006, 0.006). The mRNA relative expression levels of CDK4, CDK6 and Myc in A3 cell line were significantly lower than those treated with palbocicib 0 nmol/L. The differences were statistically significant (t=7.164, 7.280, 4.823; P=0.029, 0.023, 0.041). Compared with palbocicib 0 nmol/L, there were no significant differences in the mRNA relative expression levels of Notch1 in A3 and MOLT-4 cell lines treated with palbocicib 50 nmol/L (t=1.524, 2.065; P=0.375, 0.149). When treated with palbocicib 100 nmol/L, the mRNA relative expression levels of Notch1 in A3 and MOLT-4 cell lines were significantly higher than those treated with palbocicib 0 nmol/L, and the differences were statistically significant (t=5.162, 6.206; P=0.037, 0.028). Conclusions T-ALL cell lines with high Notch1 expression overexpressed CDK4、CDK6、Myc genes.Palbociclib could inhibit the expression of CDK4/6 gene and arrest the proliferation of T-ALL cells in G1 phase, so palbociclib could indirectly resist the promotion of tumor cells caused by Notch1 activation. Key words: Leukemia, T-Cell; Acute disease; Cell proliferation; Receptor, Notch1; Palbociclib