Growth Of CCRF-CEM Cells Research Articles

Effect of Regulating A20 Expression on NF-κB Expression and Biological Characteristics of Jurkat Cells

To explore the effect of regulating A20 expression on NF-κB and biological characteristics of Jurkat cells with glucocorticoid (GC) resistance. CCRF CEM and Jurkat cells were treated with dexamethasone (DEX) at concentrations of 100、10、1、0.1、0.01 and 0.001 μmol/L, and cultured for 24、48 and 72 h. The proliferation inhibition rate of Jurkat cell was detected by CCK-8. A20 plasmid was constructed, A20-siRNA was designed and synthesized, and transfected into Jurkat cells by liposome. CCK-8 was used to detect the proliferation rates of Jurkat cells in different concentrations of DEX group, DEX combined with A20 plasmid group and A20-siRNA group. The mRNA expression level of NF-κB was detected by RT-qPCR, the protein expression level of NF-κB was detected by Western blot, and the apoptosis of Jurkat cells was examined by flow cytometry. The inhibitory effects of DEX at different concentrations on the growth of CCRF CEM cells were time-dependent (r=0.984, P＜0.05) and concentration-dependent (r=0.966, P＜0.05). At the point of 24 hour, the IC50 approached 1 μmol/L in CCRF CEM cells. Great large differences began to appear between 1 and 10 μmol/L, the proliferation rate of Jurkat cells treated with 1 μmol/L DEX did not show a significant change. Therefore, 1 μmol/L was selected as control group. The cell proliferation rate of A20 plasmid transfection combined with different concentrations of DEX group was lower than that of DEX group and A20-siRNA combined with DEX group. After transfection of A20 plasmid, the expression level of NF-κB was significantly lower than that of control group (P＜0.05), and the apoptotic rate was significantly higher than that of control group (P＜0.05). After transfection of Jurkat cells with A20-siRNA, the expression level of NF-κB was significantly higher than that of control group (P＜0.05). The apoptotic rate of cells in A20-siRNA group was not significantly changed (P＞0.05). Jurkat cells are resistant to DEX. A20 overexpression combined with DEX can increase sensitivity of Jurkat cells with GC resistance and decrease the proliferation rate of Jurkat cells, down-regulate the expression level of NF-κB and promote the apoptosis of Jurkat cells.

Potent Inhibition of Human Folylpolyglutamate Synthetase by Suramin

Suramin, a bis-hexasulfonated napthylurea, was studied as an inhibitor of human folylpolyglutamate synthetase (FPGS), a crucial enzyme in folate metabolism. Suramin is a more potent (IC50, 0.9 μM) inhibitor of FPGS partially purified from CCRF-CEM human leukemia cells than is bromosulfophthalein (IC50, 17 μM), the first reported nonsubstrate-analog inhibitor of FPGS (J. J. McGuireet al., Adv. Exptl. Med. Biol.163, 199, 1983). FPGS inhibition by suramin is reversed by bovine serum albumin (which binds suramin). Suramin is a noncompetitive inhibitor with aminopterin (Kii= 0.9 μM;Kis= 1.1 μM) and glutamic acid (Kii= 1.0 μM;Kis= 5.2 μM) as the variable substrates; suramin inhibition tends toward being competitive with respect to the third FPGS substrate, ATP (Kii= 3.4 μM;Kis= 0.35 μM), since the major effect is on itsKm. Suramin is a much less potent inhibitor of two other folate-dependent enzymes, dihydrofolate reductase (IC50, 38 μM; methotrexate (MTX), 0.6 nM) and thymidylate synthase (IC50, 87 μM; MTX, 48 μM). The effects of suramin on growth of CCRF-CEM cells and a MTX-resistant subline (R30dm) expressing low levels of FPGS activity were determined. R30dm is slightly collaterally sensitive to suramin consistent with FPGS inhibition contributing to the cytotoxic mechanism. These data, and those of Rideoutet al.(Int. J. Cancer61, 840, 1995), demonstrating that the reduced folate carrier system of CCRF-CEM is inhibited, suggest that inhibition of folate metabolism could be involved in the mechanism of action of suramin.

"Reverse" and "symmetrical" analogues of actinomycin D: metabolic activation and in vitro and in vivo tumor growth inhibitory activities.

Two new classes of actinomycin D analogues, tetracyclic "reverse" analogues and a tricyclic "symmetrical" analogue of actinomycin D, are reported. These analogues bind to DNA and the binding does not occur by an intercalation mechanism. The analogues inhibit the synthesis of DNA and RNA in P388 tumor cells and the growth of CCRF-CEM cells in vitro at nanomolar concentrations. The tetracyclic "reverse" analogues, which are structurally related to the previously reported actinomycin D oxazolyl analogues, are metabolized in the presence of rat hepatic microsomes and tumor cell homogenates. The metabolism takes place with the loss of the oxazole ring; thus the "reverse" analogues produce a major metabolite which is the "symmetrical" analogue; the actinomycin oxazolyl analogues generate 7-hydroxyactinomycin D. Further, the microsomes activate the analogues to free-radical states which catalyze the production of superoxide as shown by stimulation of epinephrine oxidation and also indicated by electron paramagnetic resonance studies. The "symmetrical" and "reverse" analogues also demonstrate very high activities in these systems. In in vivo studies using P388/S, P388/ADR leukemia, and B16 melanoma in mice, the analogues showed increased activity and superior therapeutic index values, in comparison to actinomycin D.