Proliferation kinetics of subpopulations of human marrow cells determined by quantifying in vivo incorporation of [2H2]-glucose into DNA of S-phase cells

This report investigated in vivo turnover kinetics of marrow hematopoietic progenitors and precursors using a recently developed stable isotope-mass spectrometric technique (SIMST). Human subjects were administered a 2-day infusion of 6,6-[2H2]-glucose, a nontoxic stable isotope-labeled form of glucose, which becomes incorporated into DNA of all S-phase cells. The percent [2H2]-glucose incorporated into DNA in the form of [2H2]-deoxyadenosine (%[2H2]-dA enrichment) was determined by gas chromatography-mass spectrometry. The rate constant of replacement of unlabeled by labeled DNA strands (labeling kinetics) was used to calculate population turnover kinetics of CD34+ cells, CD133+ cells, and CD133-CD34+ cells. The observed mean replacement half-life (t1/2) was 2.6 days for CD34+ cells, 2.5 days for CD133-CD34+ cells, and 6.2 days for CD133+ cells. Results from the estimated rate constant of replacement of labeled by unlabeled DNA (delabeling kinetics) also demonstrated slower turnover rates for CD133+ cells than for CD133-CD34+ cells. Although there was a relatively rapid initial decrease in the %[2H2]-dA enrichment, low levels of labeled DNA persisted in CD34+ cells for at least 4 weeks. The results indicate the presence of subpopulations of CD34+ cells with relatively rapid turnover rates and subpopulations with a slower t1/2 of 28 days. Results also demonstrate that in vivo [2H2]-glucose-SIMST is sensitive enough to detect differences in turnover kinetics between erythroid and megakaryocyte lineage cells. These studies are the first to demonstrate the use of in vivo [2H2]-glucose-SIMST to measure in vivo turnover kinetics of subpopulations of CD34+ cells and precursors in healthy human subjects.     

Demonstration of clonal heterogeneity in adenocarcinomas on Barrett's esophagus by flow cytometric study of cellular DNA content]

Flow cytometry was used to examine the spatial distribution of nuclear DNA content in Barrett's mucosa, in one patient with high grade dysplasia and in 6 patients with Barrett's adenocarcinoma. All tumors were aneuploid. Each adenocarcinoma but the most advanced seemed to arise from a single clone of aneuploid or near-tetraploid cells which was found in all biopsy specimens taken from the tumor. Multiple aneuploid populations of cells were seen in the larger tumors. Eight clones were individualized in the most advanced case of cancer. In all patients with carcinoma, the mucosa surrounding the tumor was aneuploid. Some areas were characterized by the same DNA index as in the tumor, others contained distinct aneuploid cell populations. The spatial distributions of aneuploid clones and dysplastic areas were not perfectly superimposed. These data suggest that neoplastic progression in Barrett's esophagus is associated with genomic instability preceding the development of malignancy. Clonal heterogeneity in Barrett's adenocarcinoma is more marked when compared to other tumors and suggests a majoration of genomic instability during tumor progression.     

Inhibition of DNA biosynthesis by vincristine and pentoxifylline in murine P388 leukemia cells resistant to doxorubicin

Pentoxifylline (PTX) is a methylxanthine used clinically in the treatment of intermittent claudication. It is an active hemorheological agent used for the treatment of defective microcirculation. The use of the anticancer agent vincristine is limited by its toxicity to normal body tissues. The data presented in the present paper show that it is possible to achieve greater cell-kill by using vincristine in combination with pentoxifylline. The effect of pentoxifylline alone and in combination with vincristine was studied using membrane filtration technique in P388 leukemia (P388) and its subline P388/DOX resistant to doxorubicin and cross-resistant to vincristine. Pentoxifylline (100 mumol/l) had minimal inhibitory effect on DNA biosynthesis in P388 leukemia cells. Vincristine, at the concentration employed in this study did not show significant inhibition of DNA biosynthesis confirming multidrug resistant nature of P388/DOX cells. Pentoxifylline had a dose-sparing effect, wherein it enhanced the antiproliferative activity of vincristine at a clinically achievable concentration. The studies on reversibility of inhibition of DNA biosynthesis in P388/DOX cells pretreated with vincristine and pentoxifylline showed the irreversible nature of the effect of combination of vincristine and pentoxifylline. This observation warrants the possible use of pentoxifylline as an adjuvant in cancer chemotherapy.     

Inhibition of phytohemagglutinin-induced DNA synthesis and of adenosine deamination by 5-azacytidine and 5-aza-2'-deoxycytidine in intact human cells

5-Azacytosine nucleosides block phytohemagglutinin-induced thymidine incorporation into DNA in intact human blood lymphocytes in a dose-dependent manner at 1-100 micrograms/ml. Maximal inhibition was observed when mitogen and 5-azacytidine or 5-aza-2'-deoxycytidine were added to the culture in admixture. Both analogues also inhibit deamination of exogenous adenosine in stimulated cells without affecting the synthesis of nucleotides from adenosine, adenine or hypoxanthine. Similar inhibitory effect of 5-aza-2'-deoxycytidine on the metabolism of adenosine was observed in normal (WI-38) and SV40 virus-transformed (VA-13) human fibroblasts.     

Effect of SiO_2 nanoparticles exposure on microRNA expression level in human bronchial epithelial cells

<正>Objective To investigate the effect of short and long term exposure to Si O2nanoparticles on microRNA expression level in human bronchial epithelial cells(16HBE cells).Methods The 16HBE cells were exposed to 5,10,15,20,25,30 and 40μg/ml Si O2nanoparticles for24 h to detect the cell viability by using CCK-8 assay.     

Cytotoxicity of floxuridine and 5-fluorouracil in human T-lymphoblast leukemia cells: enhancement by leucovorin

The inhibitory effects of leucovorin (LV) combined with 5-fluorouracil (FUra) or floxuridine (FdUrd) on growth of human T-lymphoblast leukemia cells (CCRF-CEM) were determined as a function of time, dose, and sequence of exposure. Exposure of CCRF-CEM cells in exponential growth to LV (1-100 microM) for 4 hours and to FUra (100 microM) or FdUrd (0.5 microM) during the last 2 hours resulted in synergistic inhibitory effects on cell growth. Synergism was dependent on LV dose (100 greater than 10 greater than 1 microM) and did not occur at 0.1 microM. No clear dependence of synergy on sequence was observed with FUra and LV combinations. With LV and FdUrd combinations, synergism was dependent on sequence of exposure (LV + FdUrd and LV----FdUrd were synergistic, but FdUrd----LV was not). Thymidine (0.1 microM), added after drug treatment, substantially rescued CCRF-CEM cells from LV----FUra cytotoxicity. Concomitant hypoxanthine (100 microM) only partially protected CCRF-CEM cells from the toxicity of this combination. These results are consistent with the hypothesis that the mechanism by which LV potentiates fluoropyrimidine cytotoxicity is the enhancement of complex formation between thymidylate synthase and 5-fluorodeoxyuridylate, presumably as a consequence of an increase of intracellular levels of 5,10-methylenetetrahydrofolate generated from LV. Also, enhanced stability of this complex in the presence of high levels of the folate coenzyme may contribute to the synergy observed. These data also provide a rationale for use of FUra and especially FdUrd and LV in the treatment of lymphoid malignancies in man.     

多克隆抗胸腺细胞球蛋白对白血病细胞增殖抑制及凋亡诱导作用

目的:探讨多克隆兔抗人胸腺细胞球蛋白(ATG)对白血病细胞的生长抑制以及诱导凋亡作用。方法:体外培养Jurkat、Raji、HL-60、NB4、K562、U937细胞株以及15例患者原代白血病细胞。用50、100、150、200、250μg/mL终浓度的ATG在不同时间分别作用于以上细胞,采用细胞计数试剂盒(CCK-8)比色法检测细胞增殖抑制率,应用流式细胞术测定细胞凋亡率。结果:ATG对Jurkat、Raji、HL-60细胞有明显的增殖抑制以及诱导凋亡作用,且高浓度ATG抑制作用更强。ATG对NB4、K562、U937细胞增殖抑制与诱导凋亡作用均不明显。15例初发白血病患者中7例的原代细胞经ATG作用后明显凋亡,凋亡率均>30%。结论:ATG具有广谱抗白血病作用,尤其是对淋巴细胞白血病作用较强,为其在异基因造血干细胞移植中的应用提供了实验依据。     

Dup-697, a specific COX-2 inhibitor,suppresses growth and induces apoptosis on K562 leukemia cells by cell-cycle arrest and caspase-8 activation

This investigation was designed to assess the effect of DuP-697 on growth and apoptosis in a human chronic myeloid leukemia (CML) cell line (K562 cells) and primary CML cells from CML patient bone marrow. DuP-697 significantly suppressed K562 cells and primary CML cells growth and induced apoptosis in a concentration-dependent manner and the growth-inhibiting effect was independent on Philadelphia chromosome. The IC50 of DuP-697 at 36 h was 31.7 μM. It arrested G1-S phase transmit on cell cycle and its apoptosis activity was partially abrogated by pretreating K562 cells with Z-IETD-fmk, a specific inhibitor of caspase-8. This study suggested that Dup-697 suppresses growth and induces apoptosis on K562 leukemia cells by cell-cycle arrest and caspase-8 activation.     

Cell cycle compartments of adult mouse hepatocytes identified by flow cytometric analysis of total cellular and nuclear RNA content: Effect of aging on G1 substates

A model of the hepatocyte cell cycle was computer-constructed using two-parameter cytochemical data obtained by flow cytometric measurements of the RNA and DNA content of individual liver cells (or isolated liver cell nuclei). In young mice (under 10 weeks post-partum), the diploid (2C DNA) G₁ phase was resolved into 3 distinct subpopulations of liver cells distinguishable on the basis of RNA content. These substates were conventionally designated G_1Q (quiescent non-cycling cells; lowest RNA content), G_1A (cells of the indeterminate phase; do not directly enter the DNA synthetic compartment; intermediate RNA content) and G_1B (immediate pre-DNA synthetic cells; highest G₁ RNA content). An early G₁-associated event during postnatal aging of the mouse liver (pre-10 weeks post-partum) involved a shift from a predominant G_1A/B substate to a predominant G_1Q substate. Later stages in aging were characterized by a gradual decline in the percentage of G_1A/B cells in the 2C G₁ compartment. Greater than 80% of the total 2C G₁ liver cell population of animals approaching 2 years of age consisted of GIQ hepatocytes. This increase in the quiescent cell population (G_1Q) and decrease in the cycling G₁ substates (G_1A/B) within the mouse liver as a function of age may be involved in the aging-associated diminution of the hepatic compensatory regenerative response in mammals.     

Antileukemic drugs increase death receptor 5 levels and enhance Apo-2L-induced apoptosis of human acute leukemia cells

In present studies, treatment with tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL, also known as Apo-2 ligand [Apo-2L]) is shown to induce apoptosis of the human acute leukemia HL-60, U937, and Jurkat cells in a dose-dependent manner, with the maximum effect seen following treatment of Jurkat cells with 0.25 microg/mL of Apo-2L (95.0% +/- 3.5% of apoptotic cells). Susceptibility of these acute leukemia cell types, which are known to lack p53(wt) function, did not appear to correlate with the levels of the apoptosis-signaling death receptors (DRs) of Apo-2L, ie, DR4 and DR5; decoy receptors (DcR1 and 2); FLAME-1 (cFLIP); or proteins in the inhibitors of apoptosis proteins (IAP) family. Apo-2L-induced apoptosis was associated with the processing of caspase-8, Bid, and the cytosolic accumulation of cytochrome c as well as the processing of caspase-9 and caspase-3. Apo-2L-induced apoptosis was significantly inhibited in HL-60 cells that overexpressed Bcl-2 or Bcl-x(L). Cotreatment with either a caspase-8 or a caspase-9 inhibitor suppressed Apo-2L-induced apoptosis. Treatment of human leukemic cells with etoposide, Ara-C, or doxorubicin increased DR5 but not DR4, Fas, DcR1, DcR2, Fas ligand, or Apo-2L levels. Importantly, sequential treatment of HL-60 cells with etoposide, Ara-C, or doxorubicin followed by Apo-2L induced significantly more apoptosis than treatment with Apo-2L, etoposide, doxorubicin, or Ara-C alone, or cotreatment with Apo-2L and the antileukemic drugs, or treatment with the reverse sequence of Apo-2L followed by one of the antileukemic drugs. These findings indicate that treatment with etoposide, Ara-C, or doxorubicin up-regulates DR5 levels in a p53-independent manner and sensitizes human acute leukemia cells to Apo-2L-induced apoptosis. (Blood. 2000;96:3900-3906)     

重组改构人肿瘤坏死因子与多柔比星对Raji细胞增殖和凋亡作用

目的:观察重组改构人肿瘤坏死因子(rmhTNF-NC)与多柔比星(DOX)对Raji细胞增殖、凋亡的影响。方法:体外培养Raji细胞,分为对照组、rmhTNF-NC组、DOX组和rmhTNF-NC联合DOX组。应用四甲基偶氮唑盐(MTT)法检测细胞增殖抑制率,用流式细胞仪分析细胞凋亡率。结果:体外实验中不同浓度(0、100、500、1 000、5 000、10 000 U/mL)rmhTNF-NC处理的Raji细胞24、48、72 h细胞增殖抑制率、凋亡率均无明显增加(P>0.05);1μg/mL DOX与10 000 U/mL的rmhTNF-NC联合作用24、48 h后细胞增殖抑制率及凋亡率较单用DOX组及单用rmhTNF-NC都明显增高(P0.05),但细胞完全死亡率增加(P<0.05),且各浓度组的细胞增殖抑制率和凋亡率随培养时间延长而增高(P<0.05)。结论:rmhTNF-NC与DOX联合应用可使细胞增殖抑制率、凋亡率增加,并促进细胞死亡,具有协同作用。     

Effect of recombinant human tumor necrosis factor on the colony growth of human leukemia progenitor cells and normal hematopoietic progenitor cells

The effects of recombinant human tumor necrosis factor (rH-TNF) on the colony growth of human leukemia progenitor cells (L-CFU), granulocyte- macrophage progenitor cells (CFU-GM), and erythroid progenitor cells (BFU-E) were studied. L-CFU was assayed with leukemia cells obtained from patients with acute myelogenous leukemia. CFU-GM and BFU-E were assayed with bone marrow cells obtained from hematologically normal donors and patients with acute leukemia or non-Hodgkin's lymphoma in complete remission. A dose-dependent growth inhibition of L-CFU as well as CFU-GM and BFU-E was observed by rH-TNF at concentrations of 1 to 100 U/mL. The inhibitory effect on L-CFU was significantly greater than that on CFU-GM. No correlation was observed between the inhibitory effect on L-CFU and the number of colonies formed in the cultures without rH-TNF. Preincubation of the progenitor cells in culture medium containing 20% fetal calf serum with up to 1,000 U/mL of rH-TNF for 24 hours did not result in the inhibition of colony growth of L-CFU or CFU- GM. The inhibitory effect of rH-TNF was neutralized by an anti-rH-TNF murine monoclonal antibody.     

Effect of matrine on autophagy and apoptosis of human leukemia K562/IM cells

正Objective To study effects of matrine on autophagy and apoptosis of human leukemia K562/IM cells.Methods(1)Detection of cell proliferation and autophagy by matrine and imatinib:the K562 and K562/IM cells were divided into the blank control group,the different concentrations matrine(0.25,0.50,1.00,2.00,5.00mg/m L)group and imatinib(0.05,0.25,0.50,     

Effects of dose and duration of exposure on 5-aza-2'-deoxycytidine cytotoxicity for L1210 leukemia in vitro

We have investigated the effects of 5-aza-2'-deoxycytidine (DAC) on the growth and clonogenic potential of L1210 leukemia in vitro. Cells were exposed to DAC (0.001-100 micrograms/ml) for periods of 1-120 hrs. Following drug removal, cell growth in suspension culture was measured for up to 7 days, and cell survival was estimated by a colony-formation assay. When cell clonogenicity was plotted against DAC concentration in log-log axis, curves for each exposure time were linear between 0.01 and 0.5 micrograms/ml of DAC, but survival leveled off to a constant percentage for drug concentrations greater than 0.5 micrograms/ml. Percent survival decreased as exposure time increased up to 24 hrs; however, increases in exposure time greater than 24 hrs did not consistently decrease survival any further. At the lower concentrations this leveling of cytotoxicity is due to the spontaneous decomposition of DAC and to the lack of cytotoxicity of the breakdown products. At the higher concentrations the cause of the leveling remains uncertain. Incubation of L1210 with DAC at concentrations greater than 0.5 micrograms/ml for greater than or equal to 24 hrs resulted in total inhibition of measurable cell growth for 72-96 hrs following drug removal. Sequential colony-formation assays at various intervals following drug removal demonstrated a time-dependent increase in cell clonogenicity at a rate approximating the growth rate of untreated L1210 cells. This suggests that despite total cytostasis of the major population of cells, a small fraction of cells is capable of dividing at a near normal rate if removed from the drug environment. Implications of these results for in vivo applications of DAC are discussed.     

Expression of Bcl-2 by human bone marrow mast cells and its overexpression in mast cell leukemia

Bcl-2 protein plays a major role in the prevention of programmed cell death of differentiating cells. In the present study, the expression of cytoplasmic bcl-2 by human Bone Marrow Mast Cells (BMMC) from both normal and pathological bone marrow samples was examined. A total of 35 subjects corresponding to 9 healthy volunteers, 8 cases of adult indolent systemic mast cell disease (SMCD), 4 cases of pediatric mastocytosis (PM), 11 cases of hematological malignancies (HM), 2 cases of reactive bone marrow, and 1 case of mast cell leukemia (MCL) were analyzed. The expression of bcl-2 was studied using quantitative three-color flow cytometry. We also studied the molecular configuration of the bcl-2 gene and other relatives by Southern blot and polymerase chain reaction (PCR) in the MCL case. Bcl-2 expression was detected in BMMC from all samples analyzed. No significant differences on the expression of bcl-2 were detected between BMMC from healthy subjects and patients with SMCD, PM, HM, and reactive bone marrow. By contrast, bcl-2 protein was overexpressed in BMMC from MCL patient without gene rearrangement. Our results show that bcl-2 protein was constitutively expressed by BMMC. BMMC from MCL display overexpression of bcl-2, which could not be related to molecular rearrangements involving the bcl-2 gene. The expression of this protein by mature MC may play a role in the prevention of MC apoptosis and thus help to explain the long survival of these cells. The overexpression of bcl-2 by BMMC in MCL may help to explain their resistance to chemotherapy-induced apoptosis.     

Effect of manganese ions on the incorporation of dideoxynucleotides by bacteriophage T7 DNA polymerase and Escherichia coli DNA polymerase I.

Incorporation of dideoxynucleotides by T7 DNA polymerase and Escherichia coli DNA polymerase I is more efficient when Mn2+ rather than Mg2+ is used for catalysis. Substituting Mn2+ for Mg2+ reduces the discrimination against dideoxynucleotides approximately 100-fold for DNA polymerase I and 4-fold for T7 DNA polymerase. With T7 DNA polymerase and Mn2+, dideoxynucleotides and deoxynucleotides are incorporated at virtually the same rate. Mn2+ also reduces the discrimination against other analogs with modifications in the furanose moiety, the base, and the phosphate linkage. A metal buffer, isocitrate, expands the MnCl2 concentration range effective in catalyzing DNA synthesis. The lack of discrimination against dideoxynucleoside triphosphates using T7 DNA polymerase and Mn2+ results in uniform terminations of DNA sequencing reactions, with the intensity of adjacent bands on polyacrylamide gels varying in most instances by less than 10%.