冈田酸对全反式维甲酸诱导NB4、MR2细胞分化的影响

Effect of okadaic acid on differentiation of NB4 and MR2 cells induced by all-trans retinoic acid

目的探讨丝（苏）氨酸蛋白磷酸化酶2A（PP2A）抑制剂冈田酸（Okadaic acid，OKA）对全反式维甲酸（ATRA）诱导白血病细胞分化的影响，探讨PP2A活性与白血病细胞ATRA耐药的关系。方法用ATRA、OKA、ATRA＋OKA分别作用于急性早幼粒细胞白血病细胞系NB4细胞及其耐药细胞系MR2细胞，采用瑞特染色法和NBT还原实验观察细胞形态变化，流式细胞术分析细胞表面CD11b表达，丝（苏）氨酸蛋白磷酸化酶试剂盒检测细胞内PP2A活性，Westernblot法检测细胞内PP2A各亚基表达。结果①细胞形态学、NBT还原实验及流式细胞术检测结果均显示，加入OKA抑制蛋白磷酸化酶活性能促进ATRA诱导的NB4细胞分化，并协同ATRA能诱导其耐药细胞MR2细胞发生部分分化。②酶活性分析显示，未处理的NB4细胞PPA活性为（959±83）pmol磷酸盐·min^-1·μg蛋白^-1；ATRA诱导NB4细胞分化过程中，PP2A活性明显下降[（534±43）pmol磷酸盐·min^-1·μg蛋白^-1]，ATRA＋OKA组PP2A活性下降更加明显[（229±23）pmol磷酸盐·min^-1·μg蛋白^-1]；单用ATRA作用于MR2细胞，则PP2A活性无明显影响，而ATRA＋OKA诱导MR2细胞分化中，PP2A活性下降。③Westemblot结果显示，ATRA作用5d的NB4细胞中PP2A-C亚基表达较对照组明显减少，而A和B亚基表达与对照组比较变化不明显；在MR2细胞中，A、B和C亚基表达在处理组与对照组之间无明显差别。结论ATRA诱导的NB4细胞分化过程中细胞内PP2A-C表达减少，PP2A活性降低。ATRA不能有效抑制MR2细胞内PP2A的活性，可能是MR2细胞对ATRA耐药的机制之一。

Objective To study the changes in expression and activity of protein phosphatases type 2A（ PP2A ） during differentiation of NB4 and NB4-MR2 cells induced by all-trans retinoic acid （ATRA）, and evaluate the role of PP2A in MR2 resistance to ATRA. Methods ATRA, okadaic acid （ OKA ） and ATRA ＋ OKA at the same dosage were incubated with NB4 and MR2 cells respectively. Wright＇ s staining and NBT reduction test were employed to evaluate the change in the cells. The CD11 b expression was measured by flow cytometry. The activity of PP2A was evaluated by serine/threonine phosphatase assay system, and the level of PP2A subunits was detected by Western blot. Results① Wright＇s staining, NBT reduction test and flow cytometry results showed OKA could augment the differentiation of NB4 induced by ATRA, and OKA ＋ ATRA induced slight differentiation of MR2 cells . ②Phosphatase assay showed a decrease in PP2A phosphatase activity [ （534±43）pmol·min^-1·μg protein^-1] in NB4 after ATRA treatment, companed with that activity [ （959±83 ） pmol·min^-1·μg protein^-1] in untreated NB4 cells. OKA enhanced the inhibitory effect of ATRA on the activity in NB4. When OKA ＋ ATRA was incubated with MR2, PP2A in the cells was significantly decreased[ （229±23）pmol·min^-1·μg protein^-1].③Western blot analysis showed that the level of PP2A catalytic subunit （ PP2A/C ） was decreased during the course of ATRA-induced NB4 cell differentiation, whereas expressions of every subunits of PP2A in MR2 cells were somewhat unaltered. Conclusion Expression of PP2A/C and activity of PP2A is decreased during differentiation of NB4 induced by ATRA, and no repression of the PP2 activity maybe related to MR2 resistance to ATRA.