小儿急性淋巴细胞白血病P16蛋白表达的研究

The expression of P16 protein in childhood acute lymphoblastic leukemia

目的 探讨P16蛋白表达异常在小儿急性淋巴细胞白血病 (ALL)发病中的意义。方法 应用免疫细胞化学SP法检测了 5 2例初治ALL、2 0例正常对照小儿的P16蛋白的表达。结果 初治ALL中P16蛋白表达阳性率为 42 % ,明显低于正常对照组 (95 % ) (P <0 .0 1)。正常对照P16蛋白弱阳性表达占 80 %。在ALL阳性表达者中 ,强阳性占 73% (16 / 2 2 ) ,弱阳性占 5 % (1/ 2 2 )。T系与B系ALL中P16蛋白缺失率分别为 80 % (8/ 10 )和 5 8% (18/ 31) (P >0 .0 5 )。在P16表达阳性的B系ALL中 ,强阳性占 77% (10 / 13)。结论 P16失活导致细胞周期G1/S检查点功能低下 ,在ALL的发生、发展过程中具有重要的作用。P16蛋白高表达亦为不利因素 ,可能通过抑制细胞凋亡参与部分B系ALL的发病 ;而P16蛋白失活导致细胞增殖加速 ,与T系ALL的发病更密切。

Objective The tumor suppressor gene P16 mapped at 9p21. P16 as a negative regulatory protein of cell cycle plays a role in inhibition of cell division and proliferation. Recently, it has been reported that the homozygous deletion of P16 gene was much higher in childhood acute lymphoblastic leukemia(ALL), especially in T ALL, than other types of leukemia. But it is difficult to find the abnormalities of the gene regulation sequence with the ordinary molecularbiological techniques. Study on protein activity could finally reflect gene function in tumor cells. Thereafter, we detected the expression of P16 protein, and explored the relationship with immunophenotypes in childhood ALL and the significance in the pathogenesis of ALL. Methods Fifty two recently diagnosed childhood ALL patients aged from 1 year 2 months to 14 years including 31 boys and 21 girls were selected as experiment group. Twenty healthy children aged from 2 months to 9 years, 13 boys and 7 girls, were in the control group. The patients included 10 T ALL, 9 early pre B ALL, 14 C ALL, 4 pre B ALL, and 4 B cell ALL. The biotin streptavidin method was used to detect the P16 protein. Cell samples were pre incubated with hydrogen peroxide (3%) for 20 min at room temperature, and with non immune goat serum (10%) for 20 min at 37℃. Primary antibodies (polyclonal antibody, Santa Crue, dilution 1∶50) were applied and incubated for 12 hr at 4℃. The secondary antibodies were biotinylated goat anti rabbit IgG (1∶50 dilution) and incubated for 30 min at 37℃, after that the streptavidin biotinylated peroxidase (SP) complex (dilution 1∶200, 30 min), 37℃ was added. Finally, peroxidase activity was visualized by adding DAB, which formed a yellowish brown product, counterstain was performed with hematoxylin. The staining was graded as negative (-), weakly positive (+), positive(++) and strongly positive (+++). Results The positive expression rate of P16 protein in childhood ALL patients was 42%, obviously lower than that in normal group (95%, P <0.001). The lower level expression (+) of P16 protein in normal group was 80%, while in ALL, the ratio of strongly positive (+++) over positive expressions was 73% (16/22), the ratio of weak positive (+) over positive expression was 5% (1/22). The deletion of P16 protein expression was found in 8/10 of T lineage ALL (80%) and in 18/31 of B lineage ALL (58%) patients, respectively ( P >0.05). In the subtype of B lineage ALL , the deletion of P16 protein expression occurred in 3/9 of early pre B ALL (33%), 7/14 of C ALL (50%), 4/4 of pre B ALL (100%) and 4/4 of B cell ALL (100%). The degree of P16 protein expression in pre B ALL and B cell was lower than that in pre B ALL and C ALL by Ridit analysis. The strong positive expression of P16 protein was revealed in 77% of B lineage ALL. Conclusion The P16 inactivation could suppress the function of G1/S checkpoint in cell cycle, which plays a very important role in the leukemogenesis and cancer progression. The enhanced expression of P16 protein may be related to the leukemogenesis in some groups of B lineage ALL by prolonging leukemic cell survival, in contrast, P16 inactivation which results in the higher proliferative activity may associate closely with the development of cancer in T lineage ALL.