Subunit composition of plasma von Willebrand factor in patients with the myeloproliferative syndrome

In order to evaluate the role of proteolysis in acquired von Willebrand's disease (vWD) associated with the myeloproliferative syndrome, we have determined the relative quantity of von Willebrand factor (vWF) fragments as compared with the intact 225 kDa subunit in four patients. The plasma vWF of each individual lacked large multimers; each had a prolonged bleeding time; and both platelet and leukocyte counts were elevated. Plasma was obtained from blood drawn into 1 mmol/L leupeptin, 6 mmol/L N-ethylmaleimide, and 5 mmol/L EDTA to prevent in vitro proteolysis. vWF was isolated from plasma by immunoadsorbent chromatography, reduced, subjected to SDS-5% polyacrylamide gel electrophoresis, and immunoblotted with a mixture of 55 anti-vWF monoclonal antibodies. In three patients with essential thrombocytosis (ET) the 176 and 140 kDa fragments were increased in proportion to the intact 225 kDa subunit indicating increased proteolysis. Treatment of one ET patient with CCNU (Lomustine) decreased the platelet count and, to a lesser extent, the white blood cell count. This was associated with a correction of the bleeding time, a partial correction of the multimeric abnormality, and a lessening of vWF cleavage. In a patient with polycythemia rubra vera (PRV) the proportion of the 176 kDa fragment was increased to the upper limit of normal but there was no definite evidence of increased proteolysis. These studies provide evidence that proteolysis plays a role in the acquired von Willebrand's disease associated with the myeloproliferative syndrome. However, other mechanisms must also be considered.     

Elimination of drug-resistant myeloma tumor cell lines by monoclonal anti-P-glycoprotein antibody and rabbit complement

The effectiveness of ex vivo chemotherapy with drugs, such as vincristine, etoposide, and Adriamycin (doxorubicin, Adria Labs, Columbus, OH) for elimination of residual tumor cells from human bone marrow grafts could be undermined by the presence of multidrug- resistant tumor cells in the bone marrow. Therefore, to supplement chemoseparation, we investigated whether MRK-16, a monoclonal antibody (MoAb) to the surface moiety of multidrug resistance-associated P- glycoprotein antigen, can eliminate drug-resistant tumor cells in the presence of rabbit complement (RC). Two doxorubicin (DOX)-resistant human myeloma tumor cell line, 8226/DOX40 (resistant to 4 x 10(-7) mol/L DOX) and 8226/DOX6 (6 x 10(-8) mol/L DOX) with high and low amounts of cell surface P-glycoprotein, respectively, and the drug- sensitive parent cell line 8226/S were used as tumor models in this study. Using the limiting dilution assay, we have shown that three cycles of treatment with 25 micrograms/mL of MRK-16 MoAb and a 1:4 final dilution of RC eliminated 2.90 +/- 0.10 logs of 8226/DOX40 cells and 1.94 +/- 0.18 logs of 8226/DOX6 cells. One and two cycles of treatment were less effective, eliminating 0.47 +/- 0.40 and 1.94 +/- 0.36 logs of 8226/DOX40 and 0.12 +/- 0.20 and 1.63 +/- 0.58 logs of 8226/DOX6 cells, respectively. The 8226/S cell growth was unaffected by one to three cycles of treatment. The cell kill was not impaired when the antibody plus complement treatment was carried out on a mixture of 8226/DOX40 or 8226/DOX6 cells with a ninefold excess of irradiated bone marrow mononuclear cells (MNCs). The three cycles of treatment with antibody plus complement did not adversely affect granulocyte- macrophage colony-forming unit (GM-CFU) survival in hematologically normal marrows (92.5% to 104% survival) or in myeloma patient marrows (85% to 100%). These results show that it is possible to eliminate drug- resistant myeloma tumor cell lines from the admixed human bone marrow by treatment with MRK-16 MoAb plus RC. This method could prove to be effective for elimination of other drug-resistant tumor cell lines including those of leukemia and solid tumors, and will be further useful for supplementing chemopurging, and immunopurging of bone marrow with other antitumor cell antibodies.     

土贝母水提物对体外培养人肝癌细胞增殖及代谢的影响

目的研究中药土贝母(BPF)水提物对体外培养的人肝癌细胞的抑癌活性.方法 BPF经粉碎、浸提、超速离心、减压浓缩、冷冻干燥得BPF水提物.人肝癌SMMC-7721细胞经BPF提取物处理不同时间后以MTT法测其对人肝癌细胞线粒体代谢活性的影响,以细胞记数法观察了其对细胞增殖的影响.结果①经200 mg/L BPF提取物处理48 h,人肝癌SMMC-7721细胞线粒体活性为对照组的31.3%,处理72 h为21.4%;②以100mg/L BPF提取物处理7d中,人肝癌细胞数分别为0.57,0.316,0.29,0.285,0.57,1.287,1.2,对照FCS组分别为1.82,3.67,6.2,9.4,10.1,11,9,NON组分别为1.7,3.35,5.6,8.4,8.9,9.3,7.6,实验组人肝癌细胞数不仅低于对照组,而且低于处理前细胞数(P＜0.01);③经BPF提取物处理24 h后,即使更换为正常培养基,人肝癌细胞数在96 h的恢复培养中基本无增加.结论中药BPF提取物能显著抑制体外培养的肝癌细胞增殖和降低线粒体代谢活性.     

绞股蓝总苷对老龄大鼠的抗氧化作用观察

目的：观察口服绞股蓝总苷对老龄大鼠的抗氧化作用。方法：测定药后大鼠红细胞超氧化物歧化酶（ＳＯＤ）和全血谷胱甘肽过氧化物酶（ＧＳＨ－Ｐｘ）等抗氧化酶含量及肾上腺内维生素Ｃ（ＶｉｔＣ）含量。结果：绞股蓝总苷４０和２０ｍｇ／ｋｇ连续用药２０ｄ能升高大鼠红细胞ＳＯＤ和全血ＧＳＨ－Ｐｘ酶活力,且能降低大鼠肾上腺皮质中ＶｉｔＣ含量。结论：绞股蓝总苷能增强老龄大鼠机体抗氧化能力及提高肾上腺皮质的功能。     

The effect of DDAVP on plasma levels of von Willebrand antigen II in normal individuals and patients with von Willebrand's disease

The infusion of 1-deamino-(8-D-arginine)-vasopressin (DDAVP) causes not only an elevation in factor VIII-related antigen (FVIIIR:Ag), but also a marked elevation of plasma von Willebrand antigen II (vWAgII). vWAgII reaches a peak concentration at 60 min and is elevated 3-8-fold over basal levels in normal individuals and individuals with type I, IIA, and IIB von Willebrand's disease. As the mechanism of hemostatic alteration brought about by DDAVP might be due to release of endothelial cell proteins, endothelial cell cultures were performed. The cultures demonstrated synthesis and secretion of vWAgII, as evidenced by the incorporation of 35S-methionine into the vWAgII molecule. Thus, vWAgII, like FVIIIR:Ag, is an endothelial cell protein.     

国产阿克拉霉素A的体内外抗肿瘤作用

国产阿克拉霉素A(aclacinomycin;ACM-A)能明显抑制体外培养小鼠白血病P₃₈₈和人体胃癌SGC-7901细胞的生长。在0.01 μg/ml浓度时能抑制65.4%的克隆形成,ED₅₀为0.0085μg/ml。ACM-A对白血病小鼠有显著治疗作用,延长小鼠生命时间143%,并有半数小鼠获得治疗。对Ehrlich腹水癌和肉瘤-180也有较强的抑制作用,对肉瘤-180的作用与adriamycin相似。ACM-A主要抑制³H-TDR和。H-UR分别参人人体胃癌细胞的DNA和RNA;大剂量时对蛋白质合成也有影响。P₃₈₈细胞实验中证明ACM-A对DNA合成的抑制程度与adriamycin相似,低于daunomycin。     

维拉帕米对MDR1基因转染的Swiss－3T3细胞的化疗增敏作用

为进一步了解维拉帕米对抗药性逆转作用的待征，在人ＭＤＲ１基因转染的Ｓｗｉｓｓ－３Ｔ３多药抗药性细胞，观察了维拉帕米逆转幅度与阿霉素抗性水平的关系。各个转染细胞与母细胞相比，阿霉素毒性明显降低。非毒性浓度（３Ｕｍｏｌ·Ｌ－１）的维拉帕米对阿霉素毒性的增强作用，在转染细胞均高于母细胞，但逆转幅度与抗性水平成反比。Ｓｏｕｔｈｅｒｎ杂交显示，转染细胞基因组中有ＭＤＲ１ｃＤＮＡ整合。转染细胞的阿霉素蓄积障碍可被维拉帕米纠正。讨论了药物主动转运的饱和现象在维拉帕米增强效应中的作用，以及Ｐ－糖蛋白与药物相互作用的方式。     

维拉帕米对MDR1基因转染的Swiss-3T3细胞的化疗增敏作用

为进一步了解维拉帕米对抗药性逆转作用的特征,在人MDR1基因转染的Swiss-3T3多药抗药性细胞,观察了维拉帕米逆转幅度与阿霉素抗性水平的关系。各个转染细胞与母细胞相比,阿霉素毒性明显降低。非毒性浓度(3μmol·L^-1)的维拉帕米对阿霉素毒性的增强作用,在转染细胞均高于母细胞,但逆转幅度与抗性水平成反比。Southern杂交显示,转染细胞基因组中有MDR1 cDNA整合。转染细胞的阿霉素蓄积障碍可被维拉帕米纠正。讨论了药物主动转运的饱和现象在维拉帕米增强效应中的作用,以及P-糖蛋白与药物相互作用的方式。     

Stable overexpression of PML alters regulation of cell cycle progression in HeLa cells

Our previous studies demonstrated that PML is a growth suppressor that suppresses oncogenic transformation of NIH/3T3 cells and rat embryo fibroblasts. PML is a nuclear matrix-associated phosphoprotein whose expression is regulated during the cell cycle. Disruption of PML function by t(15;17) in acute promyelocytic leukemia (APL) plays a critical role in leukemogenesis. To further study the role of PML in the control of cell growth, we have stably overexpressed PML protein in the HeLa cell line. This overexpression of PML significantly reduced the growth rate of HeLa cells and suppressed anchorage-independent growth in soft agar. We consequently investigated several parameters correlated with cell growth and cell cycle progression. We found that, in comparison with the parental HeLa cells, HeLa/PML stable clones showed proportionally more cells in G1 phase, fewer cells in S phase and about the same number in G2/M phase. The HeLa/PML clones showed a significantly longer doubling time as a result of a lengthening of the G1 phase. No effect on apoptosis was found in HeLa cells overexpressing PML. This observation indicates that PML suppresses cell growth by increasing cell cycle duration as a result of G1 elongation. To further understand the mechanism of the effect of PML on HeLa cells, expression of cell cycle-related proteins in HeLa/PML and parental HeLa cells was analyzed. We found that Rb phosphorylation was significantly reduced in PML stable clones. Expression of cyclin E, Cdk2 and p27 proteins was also significantly reduced. These studies indicate that PML affects cell cycle progression by mediating expression of several key proteins that normally control cell cycle progression. These results further extend our current understanding of PML function in human cells and its important role in cell cycle regulation.