PSF易位细胞膜负调控血液肿瘤多药耐药的作用与分子机制

Accumulating evidence suggests that a variety of changes on genes and proteins and multiple complex mechanisms maybe involved, in the emergence and development of multidrug resistance (MDR) in various cancers. During a study on monoclonal antibodies reacted with MDR associated membrane proteins, we first confirmed relocation of nucleoprotein PSF on cell surface in neoplastic hematologic disorder, and PSF overexpression on HL60 cell surface compared to its MDR HL60/ADR cells. We also observed that the sensitivity and proliferation were improved significantly by antibody treatment. Our results suggest that relocated membrane PSF may be a new negative regulator of MDR protein in hematologic neoplasa. In this study, we will reconstruct PSF expression on the non-expression of haematological tumor cell lines, and further investigate MDR mediated by relocated membrane PSF including the MDR mechanisms and drug spectrum, MDR in animal models and patients. We will also study the molecular mechanisms of the PSF relocation to the cell membrane. The investigations cloud further reveal the MDR molecular mechanism mediated by relocated membrane PSF, which will provide the experimental evidence for befitting targets of reversal agents and MDR clinical prediction.

肿瘤多药耐药是涉及细胞内多种基因和蛋白改变、多种机制参与的复杂生物学过程。申请人在白血病耐药相关膜蛋白抗体的研究中通过一株单抗在国内外首次证实细胞核蛋白PSF可易位表达于血液肿瘤细胞表面，且在敏感HL60细胞膜上表达高于耐药HL60/ADR细胞，经该抗体处理后，HL60细胞对阿霉素敏感性和增殖能力显著改变，推测膜易位PSF可能为一新的负调控血液肿瘤多药耐药蛋白。本研究将构建细胞膜蛋白易位表达载体，使PSF在血液肿瘤细胞膜高表达，进一步研究PSF易位细胞膜情况下介导的血液肿瘤耐药及其耐药谱；在耐药动物模型和临床样本中验证PSF膜易位介导的耐药；研究PSF易位细胞膜的分子机制及通过调控血液肿瘤细胞增殖相关生物学特性负调控肿瘤耐药的分子机制。该研究将深入揭示由易位膜蛋白介导的血液肿瘤多药耐药的分子机制，为设计合理的耐药逆转剂和多药耐药预测靶点提供实验基础。

项目首先构建能在细胞膜稳定表达PSF的慢病毒载体，感染细胞并筛选稳定克隆。通过改造的基因工程细胞株体外研究，发现细胞膜过表达PSF能提高敏感细胞IC50，介导DOX, CAR, VP-16, VCR耐药，但不改变耐药株IC50。持续转染PSF检测膜PSF表达水平，敏感株膜表面PSF表达持续增高，耐药株膜PSF不表达PSF或略有降低。提示PSF在敏感株倾向于膜定位，在耐药株倾向于胞内积累。随后我们设计了含SBP系列载体，通过co-IP和LC-MS/MS发现：敏感株中K18和PSF发生直接互作，介导PSF的膜转运，敲除K18后PSF不发生膜定位；耐药株中PSF和K18不发生结合和互作。结合位点的初步研究发现K18和PSF-C端结合。.PSF在细胞膜过表达促进敏感株细胞的增殖并轻微抑制凋亡，但不改变耐药株的增殖和凋亡特性。而对于K18敲除的敏感株过表达PSF后，不发生PSF的膜易位，其促增殖和抗凋亡的特性也显著增强。这说明PSF发生胞内积累是其介导耐药的原因，其易位细胞膜的水平越高，细胞对化疗药越敏感。.PSF易位细胞膜的临床转化研究发现：健康供者骨髓和外周血白细胞膜上几乎不表达PSF；PSF膜易位现象发生在AML-M0-M5各型患者的骨髓和外周血白细胞膜上，其中低危组患者初诊时PSF发生高度膜易位，CCR后PSF膜表达降低至正常人水平；高危组和部分中高危患者、移植后复发患者PSF膜易位水平极低，部分复杂核型伴FLT3/ITD等不良预后融合基因的患者甚至初发时就未检测到PSF膜易位。.对PSF易位细胞膜维持敏感性/介导耐药的机制和临床转化的研究显示，PSF的易位表型能很好的区分了预后好（化疗敏感）和预后差（化疗耐药）的患者，有望成为白血病残留监测和危险度分层的分子学标记，对临床确定化疗方案、是否移植和移植时机具有重药的意义，也为未来开发药物提供了新靶点。