Wnt5a修饰的bMSCs对白血病小鼠造血及白血病细胞生长的影响

探讨Wnt5a基因修饰的骨髓间充质干细胞（bMSCs）对急性髓系白血病小鼠体内造血和白血病细胞生长的影响。方法:外周血瑞氏染色、骨髓流式细胞仪鉴定移植性HL60人白血病重度联合免疫缺陷(SCID)小鼠模型。造模成功的SCID小鼠,随机分为实验组（A组:Ad5-Wnt5a-bMSCs+模型小鼠）;对照组（B组:Ad5-GFP-bMSC+模型小鼠;C组:bMSC+模型小鼠;D组:模型小鼠）。RT-PCR检测外源性Wnt5a基因经骨髓腔移植入白血病小鼠模型后的定植情况。骨髓MSC及CFU-Mix培养、流式细胞仪、组织细胞化学及组织病理检测白血病小鼠移植前后各组外周血、骨髓、肝、脾、肾中的白血病细胞以及骨髓造血的变化。结果:成功建立急性髓性白血病模型;外源性Wnt5a基因转染人bMSCs,转染率达37.38%。外源性Wnt5a基因成功定植于受体骨髓中。Wnt5a基因修饰bMSCs移植后,与对照组比较,实验组SCID小鼠生存率明显提高,差异有统计学意义（P<0.05）;外周血有核细胞及瘤细胞计数明显降低,差异有统计学意义（P<0.05）;CFU-Mix和MSC集落生长恢复较快,集落数明显增多,差异有统计学意义（P<0.05）;小鼠骨髓、肺、肝、脾、肾CD33阳性率明显降低,差异有统计学意义（P<0.05）。结论:Wnt5a基因修饰的bMSCs在体内能有效地抑制白血病细胞生长,支持骨髓造血。      

Transport Mechanisms of Idarubicin, an Anthracycline Derivative, in Human Leukemia HL60 Cells and Mononuclear Cells, and Comparison with Those of Its Analogs

Transport mechanisms of idaruhicin (IDA) in HL60 cells, as leukemia cells, and human mononuclear cells (MNCs), as normal cells, were investigated, and compared with those of its analogs. The uptake of IDA by both cell types was temperature- and concentration-dependent, was inhibited competitively by daunorubicin (DNR) and noncompetitively by adriamycin (ADR), and was stimulated by preloading of the cells with DNR and ADR, indicating the partial involvement of a carrier-mediated mechanism. On pretreatment of the cells with 2,4-dinitrophenol, IDA uptake by HL60 cells increased, but that by MNCs decreased, suggesting that IDA was partially taken up into HL60 cells via an energy-independent carrier system, and into MNCs via an energy-dependent one. We speculated that in HL60 cells the carrier concerned with IDA uptake was common to DNR and ADR, and that the binding site of IDA on the carrier was the same as that for DNR, but not that for ADR, while in MNCs the carrier system consisted of, at least in part, a carrier for DNR uptake and one for ADR uptake, and the binding site of IDA was identical to that for DNR in the former, but different from that for ADR in the latter. It appeared that the uptake of IDA was greater than those of pirarubicin, DNR and ADR in both HL60 cells and MNCs, and that IDA was incorporated into MNCs more efficiently than into HL60 cells because of the higher uptake efficacy of the carrier(s).     

Contribution of the Nucleoside Transport System to Doxorubicin Transport in HL60 Cells but Not in Mononuclear Cells

Previously, we reported that pirarubicin (THP), an anthracycline, was transported, at least in part, via a nucleoside transport system in human leukemic HL60 cells, but not in mononuclear cells (MNCs). In this study, the contribution of the nucleoside transport system to the transport of other anthracyclines, doxorubicin (DOX), daunorubicin (DNR) and idarubicin (IDA), in HL60 cells and MNCs was investigated. The experiments were performed after both types of cells had been pretreated with a metabolic inhibitor, 2,4-dinitrophenol, to deplete cellular ATP. The DOX uptake by HL60 cells was partially inhibited by inhibitors of equilibrative nucleoside transporters. In HL60 cells, moreover, the uptake of DOX depended on an inwardly directed Na⁺-gradient, and was inhibited by concentrative nucleoside transporters, but there was no change in the DNR or IDA uptake under any of these conditions. On the other hand, the uptake of the three drugs by MNCs was not affected by any inhibitors of the nucleoside transporters, and there was no dependence of the uptake on an Na⁺-gradient. These results suggested that DOX, but not DNR or IDA, was partially transported in HL60 cells via the nucleoside transport system, whereas in MNCs the system did not contribute to the uptake of any of these three drugs. Thus, nucleoside transport systems contributing to the transport of anthracyclines may be different among different derivatives and cell types.     

人干扰素对HL60,K562细胞生长及细胞周期的影响

用人干扰素(α和γ)与HL60、K562细胞共同培养后,对细胞生长有不同程度抑制作用。IFN_r对细胞生长抑制作用强于IFN_r,IFN_r和IFN_r联合应用有协同作用,在K562细胞,细胞在细胞周期中的分布也发生改变,G_0/G_1期细胞比例减少,S期细胞比例增高,在HL60细胞则无明显的细胞周期再分布情况。提示细胞在S期的堆积是细胞生长受抑的原因之一。     

c—myc反义RNA转录体的构建及其对HL60细胞恶性表型的影响

Fujiwara T等的利用逆转录病毒载体介导的野生型p53基因疗法对肺癌的治疗作用的工作(见38页)引起了同道们的关注,Carbone DP及Minna JD对此专门作了精采评论,现摘译其重点内容以飨读者.     

c—myc反义RNA转录体的构建及其对HL60细胞恶性表型的影响

本研究构建了c—myc反义RNA真核细胞可诱导表达质粒PGXC．用PGXC及PSV2neo质粒共转染入早幼粒细胞白HL60，经G418抗性筛选得到转染细胞HL60^R，与亲本细胞相比其生长速率明显减低：细胞周期中G1期细胞增多，而S期细胞相应减少；细胞形态及功能呈现分化诱导；在软琼脂上不能形成集落，该细胞在裸鼠体内的致瘤性亦显著减弱以至消失。     

三硫化二砷诱导 HL-60细胞凋亡与坏死的实验研究

目的：研究三硫化二砷（As2S3)能否诱导急性粒细胞白血病细胞株（HL－60）凋亡与坏死。方法：应用细胞生长测定、形态学观察及流式细胞仪检测等多种方法,在体外研究As2S3对HL－60细胞凋亡和增殖抑制的作用。结果：2．5μmol/L As2S3作用18h后诱导HL－60小部分细胞凋亡和大部分细胞坏死,7．5μmol/L As2S3作用8h后可引起HL－60细胞凋亡。结论：As2S3可诱导HL－60细胞凋亡与坏死,有必要进一步探索其对急性粒细胞白血病治疗的价值。     

人食管鳞癌EC9706细胞线粒体DNA中ND1基因突变的检测及意义

目的通过对人食管鳞癌EC9706细胞线粒体DNA（mtDNA）中ND1基因进行检测,分析其基因突变的意义。方法培养人食管鳞癌EC9706细胞,提取其mtDNA中的ND1基因,并测序。结果测序发现,人食管鳞癌EC9706细胞mtDNA中ND1基因的3971处出现点突变,由C突变为T。结论人食管鳞癌EC9706细胞中mtDNA的ND1基因的突变与食管癌发生、发展关系密切。     

Synergistic Effects of Highly Unsaturated Fatty Acid-containing Phosphatidyl-ethanolamine on Differentiation of Human Leukemia HL-60 Cells by Dibutyryl Cyclic Adenosine Monophosphate

Highly unsaturated fatty acid-containing phospholipid (HUFA-PL) has many nutritional and medical applications. We investigated the effect of HUFA-PL on differentiation of human leukemia HL-60 cells induced by dibutyryl cyclic adenosine monophosphate (dbcAMP). HUFA-containing phosphatidylethanolamine (HUFA-PE), such as salmon testis PE, significantly enhanced dbcAMP -induced cell differentiation. A combined treatment of 200 μM dbcAMP with 50 μ M HUFA-PE increased the nitroblue tetrazolium (NBT)-reducing activity, which is an indicator of differentiation, to a level comparable to that in the case of 500 μ M dbcAMP treatment. In contrast, HUFA-lyso PE (a monoacyl form) did not exert an enhancing effect on dbcAMP-induced differentiation. The enhancing effect of HUFA-PE was suppressed by a protein kinase C inhibitor, staurosporine, while a protein kinase A inhibitor, H-8, did not suppress the enhancing effect. These findings suggest that HUFA-PE might enhance dbcAMP-induced differentiation through modulation of the protein kinase C signaling pathway in HL-60 cells.     

IDA对白血病骨髓基质培养体系中HL-60细胞杀伤效应的观察

目的 :研究细胞毒药物对体外残留白血病模型中HL 6 0细胞的杀伤效应。方法 :采用Dexter型骨髓培养体系形成白血病骨髓基质细胞贴壁层 ,接种HL 6 0细胞共培养 ,用去甲氧基柔红霉素 (IDA)处理后 ,动态观察HL 6 0细胞的活性变化。结果 :随着IDA剂量的增加及培养时间的延长 ,HL 6 0细胞活力逐渐减弱 ,去甲氧基柔红霉素 (IDA)与骨髓基质细胞层或单纯的培养基体外孵育对HL 6 0细胞杀伤能力减弱。结论 :急性白血病骨髓基质有助于HL 6 0细胞逃避化疗药物杀伤 ,对残留白血病的形成具有重要作用。     

Possibility of Contribution of Nucleoside Transport Systems to Pirarubicin Uptake by HL60 Cells but Not Mononuclear Cells

Previously, we reported that pirarubicin (THP), an anthracycline, was taken up, at least in part, by both human leukemic HL60 cells and mononuclear cells (MNCs) via a carrier-mediated system. In this study, the possibility of a contribution of nucleoside transport systems to the uptake of THP by HL60 cells and MNCs was investigated. The experiments were performed after both types of cells had been pretreated with a metabolic inhibitor, 2, 4-dinitrophenol, to deplete cellular ATP. In HL60 cells, THP uptake was increased and decreased significantly by treatment with equilibrative nucleoside transport inhibitors, nitrobenzylthioinosine (NBMPR), nitrobenzylthioguanosine and dilazep, in the presence and absence, respectively, of an inwardly directed Na⁺-gradient. THP uptake by HL60 cells showed an overshoot in the presence of the gradient, and was decreased by treatment of the cells with monensin, indicating that the uptake partially depended on the Na⁺-gradient. In HL60 cells in which equilibrative nucleoside transport was inhibited by NBMPR, THP uptake in the presence of the gradient was inhibited by Na⁺-dependent concentrative nucleoside transport inhibitors, but no inhibition was observed in the absence of the gradient. In MNCs, conversely, there was no effect of any equilibrative nucleoside transport inhibitor or the Na⁺-gradient on THP uptake. These results suggested that THP was taken up, at least in part, via both equilibrative and concentrative nucleoside transport systems in HL60 cells, but not in MNCs.     

二氟甲基鸟氨酸及反义bcl-2协同诱导HL60细胞凋亡

目的　观察二氟甲基鸟氨酸 (DFMO)及反义bcl 2调控bcl 2基因表达对HL6 0细胞凋亡的诱导作用。方法　构建反义bcl 2逆转录病毒重组体 ,建立产病毒细胞系 ,转染HL6 0细胞 ,用形态观察、生长曲线、FCM分析、集落形成、DNA电泳图谱、分子杂交及免疫组化等方法研究细胞生长特性及细胞凋亡诱导。结果　成功地构建了反义bcl 2逆转录病毒重组体及产病毒细胞系。以此转染HL6 0细胞 ,引起了bcl 2mRNA及蛋白表达下降 ,但生长速率、细胞周期分布及鸟氨酸脱羧酶 (ODC)基因表达水平与亲本细胞比较差别不大 ;无明显的细胞凋亡 ,仅集落形成能力有所减弱。用小剂量DFMO处理反义bcl 2转染的细胞 ,不仅生长受到明显抑制 ,而且可诱导细胞凋亡。结论　DFMO与反义bcl 2对HL6 0细胞增殖抑制及凋亡诱导有协同作用。抑制bcl 2表达能提高肿瘤细胞对DFMO作用的敏感性。     

Expression of the Annexin VIII Gene Acute Promyelocytic Leukemia

Annexin VIII is preferentially expressed in APL, but its level of expression in other subtypes of AML is much lower. Annexin VIII was originally found to be a vascular anticoagulant, but evidence obtained from our recent studies suggests that it does not play a role in hemorrhage diathesis in APL. The specific expression of annexin VIII in APL may relate to its possible role in hematopoietic cell differentiation. The expression of annexin VIII is developmentally regulated in APL-derived NB4 cells. It can be downregulated as a response to induction by ATRA, an agent which is also capable of inducing maturation of NB₄ cells. Our current understanding is that annexin VIII is most likely involved in signal transduction and may have a role as a modulator of PKC. A change in cellular PKC activity is expected to have a significant impact on cell differentiation and proliferation. The biological function of annexin VIII is currently unknown, but its expression in APL and its possible role in differentiation and proliferation of the leukemia cells would provide an excellent model system to study and elucidate this intriguing question.     

Bcl-2反义肽核酸诱导HL60细胞凋亡

目的　探讨不同结构的反义药物对HL6 0细胞系生物学活性的影响。方法　应用细胞计数、细胞形态观察和流式细胞术观察并比较反义肽核酸和反义寡核苷酸对白血病细胞HL6 0生物学活性的影响。结果　 10 μmol/L靶向bcl mRNA蛋白编码区的反义肽核酸能有效地抑制HL6 0细胞的生长、下调bcl 2蛋白的水平及诱导细胞凋亡。 10μmol/L同样靶点的反义肽核酸和反义寡核苷酸作用HL6 0细胞 72小时 ,细胞凋亡的百分率分别为 17.8± 1.5 3 ,13.17± 1.12 ,统计学上有显著性差异。结论　反义Bcl 肽核酸能诱导HL6 0细胞的调亡 ,比反义寡核苷酸有更好的反义作用。     

Skewed Rearrangement of the VH4-21 Gene During Pre-B Acute Lymphoblastic Leukemia

Thirty-six pre-B acute lymphoblastic leukemias (ALL) were studied for VH family expression. Among the 35 detected rearrangements, VH1 family genes were expressed in 7, VH2 in 1, VH3 in 18, VH4 in 6 and VH6 in 3. This expression is close to that expected according to the complexity of the system. The complete sequence of the 6 VH4 genes was examined in order to determine whether there is a skewed rearrangement of individual genes in this family. Our results indicate rearrangement of VH4-21 in 3 cases, 71 -4 in one, 58P2 in one case and probably of a new germinal VH4 gene for the sixth case. All the genes were displaying an almost complete homology with their germinal VH counterparts. The 6 sequenced genes associated with 6 different D gene segments displaying a close homology with their germinal counterpart. JH4 segment was expressed in 3 cases and JH6 in the remaining 3. These results associated with previous results obtained by others indicate that there is skewed rearrangement of the VH4-21 gene in pre-B ALL. It is presently unknown whether this phenomenon is the consequence of a selective process or whether it reflects what normally occurs in the normal human functional repertoire, which could be more limited than the germline repertoire.     

1,25-dihydroxyvitamin D3 inhibits proliferation of human promyelocytic leukaemia (HL60) cells and induces monocyte-macrophage differentiation in HL60 and normal human bone marrow cells

1,25-dihydroxyvitamin D₃ induces monocyte-macrophage differentiation and inhibits proliferation of cells from the human promyelocytic leukaemia cell line HL60. Similarly human bone marrow progenitor cells differentiate preferentially along the monocyte-macrophage pathway when incubated in the presence of 1,25-dihydroxyvitamin D₃. We suggest that the inhibition of growth which occurs after addition of the vitamin to HL60 might be paralleled in vivo by inhibition of proliferation of leukaemic cells; also we speculate that the vitamin may be involved in the control of both monocyte-macrophage and osteoclast production in vivo.     

Transport mechanism of anthracycline derivatives in human leukemia cell lines: uptake and efflux of pirarubicin in HL60 and pirarubicin-resistant HL60 cells

 We studied the transport mechanism of pirarubicin (THP) in HL60 and its THP-resistant (HL60/THP) cells, which showed no expression of mdr1 mRNA on Northern blot analysis. Under physiological conditions, the uptake of THP by both types of cell was time- and temperature-dependent. The amount of drug transport in the resistant cells was significantly less than that in the parent cells within 3 min of incubation. THP uptake was significantly higher in the presence than in the absence of 4 mM 2,4-dinitrophenol (DNP) in glucose-free Hanks’ balanced salt solution in both HL60 and HL60/THP cells and the increases were approximately equal. In the presence of DNP, the uptake of THP by both types of cell was concentration-dependent, and there were no significant differences in the apparent kinetic constants (Michaelis constant (K _m), maximum velocity (V _max) and V _max/K _m) for THP uptake between HL60 and HL60/THP cells. Additionally, THP transport was competitively inhibited by its analogue doxorubicin. The efflux of THP from HL60/THP cells was significantly greater than that from HL60 cells, and the release from both types of cell was completely inhibited by decreasing the incubation temperature to 0°C and by treatment with DNP in glucose-free medium. In contrast, the P-glycoprotein inhibitors verapamil and cyclosporin A did not inhibit THP efflux. However, genistein, which is a specific inhibitor of multidrug resistance-associated protein (MRP), increased the THP remaining in the resistant cells, and the value was approximately equal to that of the control group in the sensitive cells. These results suggest that THP is taken up into HL60 and HL60/THP cells via a common carrier by facilitated diffusion, and then pumped out in an energy-dependent manner. Furthermore, the accelerated efflux of THP by a specific mechanism, probably involving MRP, other than the expression of P-glycoprotein, resulted in decreased drug accumulation in the resistant cells, and was responsible, at least in part, for the development of resistance in HL60/THP cells. Received: 28 July 1994/Accepted: 10 February 1995