鲍氏威酸诱导急性早幼粒白血病细胞分化与PML/RARα关系的研究

目的：研究鲍氏威酸（BC4）诱导不同融合基因PML／RARα基因型的急性早幼粒白血病（APL）细胞分化的能力及对APL细胞分化相关基因c-myc的表达影响，探讨鲍氏威酸诱导分化作用的机制。方法：采用RT-PCR检测APL细胞PML／RARα的基因型；运用骨髓干、祖细胞集落培养技术分析各基因型APL细胞分别在生理盐水（NS）对照组，鲍氏威酸（BC4）组，全反式维甲酸（ATRA）组培养体系中增殖与分化能力，测定各组集落与丛的形成率，形态分化率，NBT还原率以及粒细胞吞噬率，培养前后各组APL细胞c-myc表达阳性率。结果：与NS对照比较，BC4和ATRA均使PML-RARα^＋组APL细胞丛形成率明显增高（P〈0.01），而集落形成率无明显变化（P〉0．05），3项细胞分化指标增高（0．01〈P〈0．05），c-myc表达阳性率降低（P〈0．05）；BC4对PML／RARα^＋（L型）诱导分化作用强于PML-RARα^＋（S型）APL，BC4对部分ATRA治疗后复发耐药病例有诱导分化能力。BC4及ATRA对PML／RARα^＋APL细胞的各项诱导分化指标无明显改变。结论：BC4对APL具有诱导分化作用，下调c-myc基因表达，其诱导分化能力与PML／RARα^-基因型有关，并能诱导部分ATRA耐药细胞分化     

早幼粒细胞白血病核体研究进展

早幼粒细胞白血病（PML）基因位于15号染色体上，其表达产物PML蛋白定位于一种被称为核体或POD的核内亚微结构中，具有抑制肿瘤、抑制生长及调节转录等作用。急必早幼粒细胞白血病（APL）患者形成PML－RARα融合基因，表达融合蛋白，使正常核体结构发生改变。本文重要介绍PML蛋白与其它POD相关蛋白的功能，相互作用及APL发病的分子机制研究进展。     

急性早幼粒细胞白血病FLT3基因内部串联重复突变研究

目的 分析急性早幼粒细胞白血病（APL）患者骨髓细胞FLT3基因内部串联重复（ITD）突变情况。方法 采用PCR方法分析103例初发APL患者骨髓单个核细胞FLT3基因的ITD突变，分析ITD阳性患者的临床特征。结果 103例初发APL患者FLT3-ITD突变20例（19．4％）。FLT3-ITD阳性与PML-RARα融合基因短型和变异型异构体密切相关，FLT3-ITD阳性患者中PML—RARα融合基因短型16例，变异型2例，长型2例（P〈0．0001）。FLT3-ITD阳性患者外周血WBC高于FLT3-ITD阴性患者（P〈0．01），其中PML—RAR仅短型和（或）变异型伴FLT3-ITD阳性患者WBC明显高于FLT3-ITD阴性患者（P=0．015）。而PML-RARα长型伴FLT3-ITD阴性患者与阳性患者WBC比较，差异无统计学意义。FLT3-ITD阳性APL患者的诱导缓解率为90％，随访16例（2例失访），中位随访时间26（11～47）个月，均处于第1次完全缓解。结论 FLT3-ITD是APL患者常见的基因突变，其发生与PML—RARα短型和变异型相关。伴FLT3-ITD阳性PML—RARα短型和变异型与发病时WBC增高具有相关性，而对近期疗效无明显影响。     

急性早幼粒细胞白血病患儿PML／RARα融合基因罕见型2例

目前认为急性早幼粒细胞白血病（APL ）属于急性髓细胞白血病（AML ）的特殊类型［1］,由于 APL 细胞存在 t （15;17）（q22;q21）,该易位使第15号染色体长臂2区2带的早幼粒细胞白血病基因（PML）和第17号染色体长臂2区1带的维甲酸受体‐α（RARα）基因形成 PML ／RARα融合基因,该基因在产生过程中因断裂点不同及 mRNA 的剪切、拼接不同会产生不同的异构体并在 APL 发病中起到重要作用［2］。本院收治2例APL 患儿出现新的 PML ／RARα融合基因变异体,现报道如下。     

一种新的早幼粒细胞白血病/维甲酸受体α融合基因S亚型变异易位

目的：逆转录聚合酶链反应（RT－PCR）检测早幼粒细胞白血病／维甲酸受体α（PML／RARα）融合基因，筛查变异易位并对变异易位产物测序，以进一步了解变异易位的特点及临床意义。方法：取急性早幼粒细胞白血病（APL）患者骨髓，RT－PCR检测L亚型和S亚型，发现的变异易位经全自动测序仪测定其碱基序列。结果：11例PML／RARα融合基因表达的患者中，S亚型1例、L亚型8例、变异S亚型合并L亚型2例；变异S亚型产物测序得到206bp的碱基序列，检索证实是一种新的变异易位。结论：发现了一种新的S亚型变异易位，证实同一个体可有S和L二种不同亚型并存；提示PML／RARα融合基因的RT－PCR检测中，识别变异易位有重要意义。     

含PML/RARα（L型）与ABL双基因质粒标准品的制备及应用

目的建立一个含PML/RARα（L型）与ABL双基因质粒标准品,用于PML/RARαL型）的定量检测。方法从NB4细胞中提取总RNA并逆转录为cDNA,以此为模板,设计PML/RARα（L型）与ABL基因特异性引物进行PCR,PCR产物经琼脂糖电泳、胶回收、PCMV4载体克隆后转化DH5α工程菌,提取质粒进行酶切、测序鉴定,提取质粒用于制作荧光定量PCR标准曲线,同时对质粒的稳定性能进行评价。结果结果NB4细胞提取的总RNA完整性良好,构建的质粒经酶切鉴定后与目的条带一致,测序结果与目的片段几乎完全一致,且质粒的原始浓度为9×1012copies/ml,倍比稀释至9×104copies/ml,均能得到良好的标准曲线（R2≈1）,且质粒的稳定性好,提示含PML/RARa（L型）与ABL双基因荧光定量PCR质粒标准品构建成功。结论本研究建立了制备含PML/RARα（L型）与ABL双基因质粒标准品方法,并成功制备出稳定的含PML/RARα（L型）与ABL双基因质粒标准品。     

应用实时定量逆转录聚合酶链反应方法检测急性早幼粒细胞白血病患者PML-RARα基因转录本的临床意义

急性早幼粒细胞白血病（APL）特征性染色体改变形成了t（15；17），涉及PML—RARα融合基因。由于全反式维甲酸、砷剂的使用，目前APL患者的生存期大大延长，但是缓解期的患者仍存在复发的危险性。我们利用实时定量RT—PCR方法检测了56例APL患者PML—RARα融合基因转录本的表达，以便动态观察这些患者的治疗效果，提高治愈率。现将结果报告如下。     

荧光原位杂交技术在急性髓系白血病诊断中的应用

目的探讨荧光原位杂交技术（FISH）在急性髓系白血病（AML）的诊断中的意义。方法对初发的经骨髓常规形态学、细胞化学染色和免疫分型初步诊断的10例AML-M2、19例AML-M3，11例AML不能确定为M2或者M3的患者，进行FISH技术检测AML1／ETO和／或PML／RARα融合基因，进而协助诊断和指导治疗。结果10例AML-M2中AML1／ETO融合基因阳性率40％（4／10）；19例AML-M3中PML／RARα融合基因阳性率89％（17／19），1例AML1／ETO融合基因阳性，确诊为AML-M2；11例AML中AML1／ETO融合基因阳性率18％（2／11），PML／RARα融合基因阳性率45％（5／11），其余4例未检测到以上两种融合基因。结论FISH是一种敏感的分子遗传学的新技术，具有高效、快速、灵敏等优点。对AML的诊断分型具有重要帮助，进一步指导临床治疗。     

实时定量PCR检测46例初诊急性早幼粒细胞白血病患者PML/RARαmRNA的分子表达

本研究探讨实时定量PCR（Q-PCR）检测PML／RARα mRNA的方法学，并对46例初诊急性早幼粒细胞白血病（APL）患者骨髓标本进行检测。构建PML／RARα的bcr1型和ber3型转录本以及内参照abl基因转录本的阳性标准品质粒；利用ABI Prism 7500型Q-PCR仪对46例初诊APL患者和40例非APL患者骨髓标本进行检测，PML／RARα mRNA定量结果以校正比值（NQ）表示，NQ=PML／RARα mRNA拷贝数／ABLmRNA拷贝数；应用四色流式细胞术检测免疫表型。结果显示，Q-PCR结果的日间差和日内差平均变异系数（CV）分别为1．58％和0．88％。可重复敏感度为可以检测5 copies／100ng RNA。40例非APL患者PML／RARα mRNA均为阴性。46例初诊APL患者PML／RARα mRNA表达量NQ中位值为0．450（0．084—1．082）。比较32例bcr1型和14例bcr3型两组患者的特征表明，PML／RARα mRNANQ中位值分别为0.454（0.084—1、082）和0．386（0.151—0．848）（P〉0．05）。形态学诊断M3v的患者比例分别为9.40％和48.96％（P〈0.05）；初诊时WBC中位数分别为2．15（0．2—59．6）和9．35（0．91—122．8）（P〈0．05），而在性别、年龄、初诊时血红蛋白和血小板计数、骨髓中APL细胞比例、DIC指标等方面无差异。流式细胞仪术检测时，CD45／SSC射门情况下，APL细胞群分布可以分为两类：高侧向角（L-SSC，粗颗粒）和非高侧向角（NL-SSC，细颗粒）两类。bcr1型患者中85．70％表现为L—SSC，而bcr3型患者中64．29％表现为NL-SSC。结论：建立的Q—PCR方法稳定可靠，敏感度高；berl型和bcr3型APL患者的PML／RARetmRNA表达量无差异，bcr3型APL患者中形态学M3v比例和WBC数比bcr1型患者高；PML／RARα不同转录本类型和免疫表型以及细胞形态学之间有一定的相关性。     

PML-RARα及RARα融合蛋白在三硫化二砷诱导的NB4细胞凋亡中的作用研究

为研究PML-RARα及RARα融合蛋白在三硫化二砷（As2S3)诱导的人早幼粒细胞白血病细胞系NB4细胞凋亡中的作用，本研究应用细胞形态观察、流式细胞术测定及DNA电泳等方法观察三硫化二砷对NB4细胞的诱导凋亡作用，用染色体G显带技术、反转录PCR及Western印迹测定这一过程中PML－RARα融合基因及其表达产物、野生型RARα蛋白的变化。结果表明，三硫化二砷在0．5－2μmol/L之间能诱导NB4细胞凋亡，在此过程中PML－RARα融合基因无明显变化，但PML－RARα融合蛋白和野生型RARα蛋白明显减少。结论提示PML－RARα融合蛋白和野生型RARα蛋白的降解在三硫化二砷诱导的NB4细胞凋亡过程中可能起了重要作用。     

胞质中早幼粒细胞白血病基因功能的研究进展

早幼粒细胞白血病（PML）基因是在急性早幼粒细胞白血病（APL）发病机制的研究中发现命名,其编码的PML蛋白位于核内小体（nuclearbody）,并可在细胞核-细胞质之间穿梭（shuttlebetweenthenucleusandthecyto—plasm）,完成许多调节功能。有关胞核PML的功能研究较多,但近年来国外资料表明胞质PML基因在血液系统肿瘤及实体肿瘤中具有重要的意义。现就胞质中PML基因生物学功能进行综述。     

Role of promyelocytic leukemia (PML) sumolation in nuclear body formation, 11S proteasome recruitment, and As2O3-induced PML or PML/retinoic acid receptor alpha degradation.

Promyelocytic leukemia (PML) is the organizer of nuclear matrix domains, PML nuclear bodies (NBs), with a proposed role in apoptosis control. In acute promyelocytic leukemia, PML/retinoic acid receptor (RAR) alpha expression disrupts NBs, but therapies such as retinoic acid or arsenic trioxide (As2O3) restore them. PML is conjugated by the ubiquitin-related peptide SUMO-1, a process enhanced by As2O3 and proposed to target PML to the nuclear matrix. We demonstrate that As2O3 triggers the proteasome-dependent degradation of PML and PML/RARalpha and that this process requires a specific sumolation site in PML, K160. PML sumolation is dispensable for its As2O3-induced matrix targeting and formation of primary nuclear aggregates, but is required for the formation of secondary shell-like NBs. Interestingly, only these mature NBs harbor 11S proteasome components, which are further recruited upon As2O3 exposure. Proteasome recruitment by sumolated PML only likely accounts for the failure of PML-K160R to be degraded. Therefore, studying the basis of As2O3-induced PML/RARalpha degradation we show that PML sumolation directly or indirectly promotes its catabolism, suggesting that mature NBs could be sites of intranuclear proteolysis and opening new insights into NB alterations found in viral infections or transformation.     

UBE1L represses PML/RAR{alpha} by targeting the PML domain for ISG15ylation

Acute promyelocytic leukemia (APL) is characterized by expression of promyelocytic leukemia (PML)/retinoic acid (RA) receptor alpha (RARalpha) protein and all-trans-RA-mediated clinical remissions. RA treatment can confer PML/RARalpha degradation, overcoming dominant-negative effects of this oncogenic protein. The present study uncovered independent retinoid degradation mechanisms, targeting different domains of PML/RARalpha. RA treatment is known to repress PML/RARalpha and augment ubiquitin-activating enzyme-E1-like (UBE1L) protein expression in NB4-S1 APL cells. We previously reported RA-induced UBE1L and the IFN-stimulated gene, 15-kDa protein ISG15ylation in APL cells. Whether the ubiquitin-like protein ISG15 directly conjugates with PML/RARalpha was not explored previously and is examined in this study. Transient transfection experiments with different PML/RARalpha domains revealed that RA treatment preferentially down-regulated the RARalpha domain, whereas UBE1L targeted the PML domain for repression. As expected, ubiquitin-specific protease 18 (UBP43/USP18), the ISG15 deconjugase, opposed UBE1L but not RA-dependent PML/RARalpha degradation. In contrast, the proteasomal inhibitor, N-acetyl-leucinyl-leucinyl-norleucinal, inhibited both UBE1L- and RA-mediated PML/RARalpha degradation. Notably, UBE1L induced ISG15ylation of the PML domain of PML/RARalpha, causing its repression. These findings confirmed that RA triggers PML/RARalpha degradation through different domains and distinct mechanisms. Taken together, these findings advance prior work by establishing two pathways converge on the same oncogenic protein to cause its degradation and thereby promote antineoplastic effects. The molecular pharmacologic implications of these findings are discussed.     

早幼粒细胞白血病蛋白与结直肠腺瘤癌变的相关性及机制初探

目的 分析结直肠腺瘤、结直肠癌组织及肠癌细胞中早幼粒细胞白血病蛋白(promyelocytic leukemia protein,PML)的表达情况,探讨PML在结直肠癌发生发展中的作用.方法 采用免疫组化、Western印迹法检测PML在结直肠癌、结直肠腺瘤及正常结直肠组织中的表达情况.检测10种不同肠癌细胞株的PM...     

Promyelocytic leukemia protein (PML) and Daxx participate in a novel nuclear pathway for apoptosis

The promyelocytic leukemia protein (PML) gene of acute promyelocytic leukemia (APL) encodes a cell growth and tumor suppressor essential for multiple apoptotic signals. Daxx was identified as a molecule important for the cytoplasmic transduction of the Fas proapoptotic stimulus. Here, we show that upon mitogenic activation of mature splenic lymphocytes, Daxx is dramatically upregulated and accumulates in the PML nuclear body (NB) where PML and Daxx physically interact. In the absence of PML, Daxx acquires a dispersed nuclear pattern, and activation-induced cell death of splenocytes is profoundly impaired. PML inactivation results in the complete abrogation of the Daxx proapoptotic ability. In APL cells, Daxx is delocalized from the NB. Upon retinoic acid treatment, which induces disease remission in APL, Daxx relocalizes to the PML NBs. These results indicate that PML and Daxx cooperate in a novel NB-dependent pathway for apoptosis and shed new light in the role of PML in tumor suppression.     

Role of promyelocytic leukemia (PML) protein in tumor suppression

The promyelocytic leukemia (PML) gene encodes a putative tumor suppressor gene involved in the control of apoptosis, which is fused to the retinoic acid receptor alpha (RARalpha) gene in the vast majority of acute promyelocytic leukemia (APL) patients as a consequence of chromosomal translocations. The PMLRARalpha oncoprotein is thought to antagonize the function of PML through its ability to heterodimerize with and delocalize PML from the nuclear body. In APL, this may be facilitated by the reduction to heterozygosity of the normal PML allele. To determine whether PML acts as a tumor suppressor in vivo and what the consequences of deregulated programmed cell death in leukemia and epithelial cancer pathogenesis are, we crossed PML(-/-) mice with human cathepsin G (hCG)-PMLRARalpha or mammary tumor virus (MMTV)/neu transgenic mice (TM), models of leukemia and breast cancer, respectively. The progressive reduction of the dose of PML resulted in a dramatic increase in the incidence of leukemia, and in an acceleration of leukemia onset in PMLRARalpha TM. By contrast, PML inactivation did not affect neu-induced tumorigenesis. In hemopoietic cells from PMLRARalpha TM, PML inactivation resulted in impaired response to differentiating agents such as RA and vitamin D3 as well as in a marked survival advantage upon proapoptotic stimuli. These results demonstrate that: (a) PML acts in vivo as a tumor suppressor by rendering the cells resistant to proapoptotic and differentiating stimuli; (b) PML haploinsufficiency and the functional impairment of PML by PMLRARalpha are critical events in APL pathogenesis; and (c) aberrant control of programmed cell death plays a differential role in solid tumor and leukemia pathogenesis.     

Review of progressive multifocal leukoencephalopathy

Progressive multifocal leukoencephalopathy (PML) is a fatal demyelinating disease of the central nervous system caused by a reactivation of the JC virus (JCV). PML occurs almost exclusively in the setting of cellular immune deficiency and its prevalence has recently increased greatly, in line with the AIDS (acquired immunodeficiency syndrome) epidemic in Western countries. A Japanese epidemiological survey showed an increasing trend in the number of AIDS-PML cases. To date, there are no effective and specific therapies for PML. As patients with AIDS-PML may benefit from combined antiretroviral therapy, efforts to improve the immune status of such patients is therefore considered to be a key factor in achieving a successful outcome for PML cases. However, PML deficits are nevertheless expected to be permanent and most cases of PML result in death within a few months. It is therefore hoped that improvements in diagnostic modalities can be made which can eventually allow for the earlier detection of PML while, at the same time, improved curative therapies for PML can also be developed in the near future.     

PML and rheumatology: the contribution of disease and drugs

Progressive multifocal leukoencephalopathy (PML), a rare, typically fatal, opportunistic infection caused by the JC virus, is becoming relevant to physicians in multiple specialties, including those who prescribe biologic agents for the treatment of autoimmune disorders. Reports of PML have led to US Food and Drug Administration alerts and warning letters regarding four immunosuppressive agents in recent years (natalizumab, rituximab, efalizumab, and mycophenolate mofetil). Consequently, informed clinical decision-making requires understanding the risk of PML associated with these therapies. An estimate of the relative frequency of PML associated with specific rheumatic conditions has been generated. Systemic lupus erythematosus appears to be associated with susceptibility to PML that cannot be fully explained by the intensity of immunosuppressive therapy. Further, the use of rituximab in patients with rheumatic disease has raised concerns. However, definitive attribution of cause is precluded by the limitations of the currently available data. All patients with rheumatic disease, regardless of the intensity of their current immunosuppressive therapy, should be considered potentially at risk of PML. With an evolving understanding of a greater clinical heterogeneity of PML, advances in diagnostic methods, and significant implications for therapy, PML should be considered in the differential diagnosis of neurologic manifestations of rheumatic diseases.     

Drug‐induced progressive multifocal leukoencephalopathy: a case/noncase study in the French pharmacovigilance database

Progressive multifocal leukoencephalopathy (PML) is an often fatal demyelinating disease of the central nervous system. As effective treatment is unavailable, identification of all drugs that could be associated with PML is essential. The objective of this study was to investigate the putative association of reports of PML and drugs. We used the case/noncase method in the French PharmacoVigilance database (FPVD). Cases were reports of PML in the FPVD between January 2008 and December 2015. Noncases were all other reports during the same period. To assess the association between PML and drug intake, we calculated an adverse drug report odds ratio (ROR) with its 95% confidence interval. We have studied the delay of onset of PML for each drug concerned. Among the 101 cases of PML, 39 drugs were mentioned as suspect. The main therapeutic classes suspected with significant ROR were antineoplastic agents (n = 85), immunosuppressants (n = 67), and corticosteroids. A latent interval from the time of drug initiation to the development of PML is established: the median time to onset was 365 days (123–1095 days). The onset of PML is highly variable and differs among drug classes [from 1 to 96 months (IQR: 39.0–126)]. An association between PML and some immunosuppressant drugs was found as expected, but also with antineoplastic agents and glucocorticoids. An important delay of PML onset after stopping treatment is suspected and should alert prescribers. Prescribers but also patients should be informed about the potential associations with all these drugs. Monitoring could be necessary for many drugs to early detect PML.