Enhancement of replication of Epstein-Barr virus DNA in human lymphoblastoid cell lines by N-methyl-N''-nitro-N-nitrosoguanidine

N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG) is a potent carcinogenthat alkyiates nucleic acids. Interaction of MNNG with humanlymphoblastoid cell fines carrying Epstein-Barr virus (EBV)was studied. Treatment of virus-producing cells (P3HR-1) withMNNG resulted in an {small tilde}3-fold increase in EBV genomecopies per cell as determined by cRNA - DNA hybridization. Thiseffect was not observed in a non-virus-producer line (Raji).Dose-response studies indicated that the optimum concentrationwas between 0.5 µg and 2 µg/ml. This same dose rangewas most effective in inhibiting cell proliferation both ofP3HR-1 and Raji cells. Concomitant treatment of P3HR-1 cellswith MNNG and 12-O-tetradecanoyl-phorbol-13-acetate gave anadditive increase to 9-fold of the number of EBV genome copiesper cell. Pretreatment of Raji cells with MNNG followed by superinfectionwith P3HR-1 virus resulted in a 35% enhancement of EBV DNA replicationas analyzed by density centrifugation. In contrast, Raji cellssuperinfected with MNNG-treated EBV showed a marked reductionin EBV DNA replication which indicates that the lesions producedin the viral genome by the drug interfered with the infectiouspotential of the virus.     

氯化镧对人白血病细胞系K562生长的影响

 目的 了解稀土氯化镧（LaCl3）对白血病细胞系K562生长和凋亡的影响,为临床应用LaCl3治疗白血病提供理论依据。方法 用MTT法鉴定LaCl3对K562细胞生长增生的影响;显微镜下观察细胞形态的变化,流式细胞术及TUNEL法鉴定细胞凋亡情况;流式细胞术分析细胞周期的变化。半固体琼脂糖培养法观察LaCl3对正常骨髓细胞形成粒－巨噬细胞集落的影响。结果 在所研究的浓度范围内,随着LaCl3浓度升高,K562细胞生长抑制率增高、凋亡率增加,呈剂量依赖性关系。随着作用时间延长,1 mmol／L LaCl3对细胞的抑制率增高、凋亡率增加,呈时间依赖性关系;K562细胞G0／G1期细胞百分率升高而S期细胞百分率下降。0.1及0.5 mmol／L LaCl3显示出对正常骨髓祖细胞有轻度刺激增生效果,而较高浓度（1.5～2.0 mmol／L）,正常骨髓祖细胞形成集落数下降且含有较多巨噬细胞,集落细胞形态正常,没有显示凋亡形态。结论 在所研究浓度范围内,稀土LaCl3能抑制K562细胞生长并且诱导细胞凋亡,呈剂量及时间依赖性关系;但对正常骨髓造血细胞的影响还有待于进一步研究。     

Effects of human lymphoblastoid interferon on cultured breast cancer cells

The effect of human interferon on growth and fibronectin production in a range of cultured breast cancer cell lines has been investigated. The cell lines differ in their sensitivity to interferon's growth-inhibitory effect as determined from growth curves, but the degree of sensitivity varies according to the method of assay. When growth was measured in cultures seeded with fairly large numbers of cells, five out of seven lines examined showed a reduction of 50% or more in the number of attached cells after treatment for 8 days with 1,000 units/ml of interferon. Growth of the two cell lines least affected by interferon treatment in dense culture could, however, be markedly inhibited under conditions of clonal growth in agar or when sparsely seeded on plastic and this was due to inhibition of initiation of growth. In some breast epithelial lines, where the number of attached cells was regulated in part by cell shedding, it was found that interferon not only inhibited cell proliferation, but also increased cell shedding. To determine if interferon increased cell shedding by affecting fibronectin production, the total and cell associated fibronectin was estimated in control and interferon-treated cultures. Four of 10 cell lines examined produced detectable amounts of fibronectin, and in only one of these lines, Hs578T derived from a carcino-sarcoma, was the production and retention of fibronectin inhibited by interferon treatment. In those breast carcinoma lines which produced detectable amounts of fibronectin, the level of production and retention of the protein, expressed as a proportion of the total protein, did not change after interferon treatment, but showed an increase if expressed on a per cell basis. This is explained by the fact that the interferon-treated cells showed an increase in cell volume.     

Anti-proliferative effects of tumor necrosis factor, gamma interferon and 5-fluorouracil on human colorectal carcinoma cell lines

We have previously utilized a bank of diverse human colorectal carcinoma cell lines to assess the synergistic antiproliferative effect of tumor necrosis factor (TNF) and IFN-gamma (IFN-gamma) in combination. In this study, we used 3 of these cell lines (HCT 116, SKO1 and VACO 9P) to study the growth-inhibitory effects of TNF and IFN-gamma (TNF/IFN-gamma) on these cells when administered in conjunction with 5-fluorouracil (5-FU). All 3 cell lines were sensitive to suprapharmacologic concentrations of 5-FU. However, the 3 lines varied in their sensitivities to clinically achievable concentrations of 5-FU. Concentrations of 0.1 microgram/ml of 5-FU administered for 96 hr, and 50 micrograms/ml administered for 1 hr, inhibited the growth of HCT 116 cells by 20% and 77%, that of SKCO1 cells by 25% and 66%, and that of VACO 9P cells by 25% and 55%, respectively. All 3 cell lines were sensitive to the anti-proliferative effects of TNF/IFN-gamma in a dose-dependent and duration-dependent fashion. TNF/IFN-gamma was administered for 1 hr every other day on days 1, 3 and 5 to the 3 cell lines. Cells were also exposed to 5-FU, administered either concomitantly for 96 hr, or for 1 hr on day 1. The addition of TNF/IFN-gamma to clinically achievable concentrations of 5-FU in both schedules resulted in additive cytotoxicity. For example, the addition of 10 ng/ml of both TNF and IFN-gamma to 96 hr of 0.1 microgram/ml 5-FU resulted in 10%, 5%, and 20% of control growth for the HCT 116 cell line, SKCO1 cell line, and VACO 9P cell line, respectively. The addition of 10 ng/ml of both TNF and IFN-gamma to 1 hr of 50 micrograms/ml of 5-FU inhibited all cell growth in all 3 cell lines. We conclude that TNF/IFN-gamma and 5-FU can be combined to achieve higher anti-tumor activity than either 5-FU or TNF/IFN-gamma alone in this in vitro model, that the 3-drug combination has potent growth-inhibitory effects at pharmacologic concentrations which are not schedule-dependent, and that this combination warrants further study in clinical trials.     

in vitro effects of recombinant human interferon gamma on human mesothelioma cell lines

Malignant mesothelioma is a tumor arising from serous surfaces and often related to asbestos exposure. Malignant mesothelioma is resistant to various forms of therapy. Radiotherapy, surgery or chemotherapy only slightly improve prognosis. IFN-γ produces complete or partial responses in stage-l patients with malignant mesothelioma. The in vitro biological effect of IFN-γ on malignant mesothelioma cells remains poorly elucidated. In the present study, 32 well-characterized human mesothelioma cell lines (HMCL) were treated with r-hu IFN--γ at 4 doses and cell growth was determined by a colorimetric method (MTT assay). Among the 32 HMCLs tested, 11 exhibited significant cell-growth inhibition; 16 were insensitive to r-hu IFN-γ, and 5 were slightly inhibited. The sensitive cell lines were strongly inhibited by r-hu IFN-γ. Our results show that HMCL exhibit a large range of responses to r-hu IFN-γ, some of which can be compared with those obtained in vivo in humans.     

Role of 2'-5'-oligoadenylate synthetase in gamma-interferon-mediated growth inhibition of A431 cells

Interferons are regulatory secretory proteins that have a variety of biological activities, including antiproliferative actions against tumor cell lines and tumors in vivo. Among the several interferon-inducible genes, modulation of 2'-5'-oligoadenylate synthetase expression has been the focus of considerable attention because of its implication in growth regulation. In the studies presented here, we report growth inhibition of A431 cells by gamma-interferon and provide new evidence to support the possible involvement of 2'-5'-oligoadenylate synthetase. We observed that the antiproliferative effect of gamma-interferon on cultures of A431 cells was accompanied by prolongation of G0-G1 phase and profound morphological changes, which were progressively prominent from day 4 onward. The levels of 2'-5'-oligoadenylate synthetase enzyme activity and mRNA were significantly elevated following gamma-interferon treatment and correlated well with the kinetics of growth-inhibitory effects. There were differential induction and expression of different isoenzyme forms of 2'-5'-oligoadenylate synthetase: the Mr 100,000 and Mr 40,000 proteins were induced maximally to varying degree by day 4, with subsequent decline; in contrast, the Mr 67,000 protein was induced late and remained high up to day 9 after gamma-interferon treatment. In accord with these observations, fractionation of enzymatic activity also revealed differences in the enzyme activities of 2'-5'-oligoadenylate synthetase forms from control and treated cells. Therefore, these observations demonstrate the existence of possible differential regulation of 2'-5'-oligoadenylate synthetase isoenzymes and strengthen the concept that growth inhibition of cells may be mediated via induction of 2'-5'-oligoadenylate synthetase.     

白细胞介素-24 delE5对人类白血病细胞系K562的抑制作用

 目的　探索白细胞介素（IL）-24 delE5对人类白血病细胞系K562的抑制作用。方法　用TPA诱导白血病细胞系U937和HL-60向单核-巨噬细胞分化后,检测mda-7／IL-24及其选择性剪接体IL-24 delE5的表达。构建IL-24 delE5真核表达载体,稳定转染K562细胞。通过MTT法、集落形成实验、流式细胞术、Annexin-Ⅴ／PI检测及动物实验,观察IL-24 delE5对K562细胞增殖、集落形成、细胞周期、凋亡及体内致瘤性的影响;同时与mda-7／IL-24进行比较。结果　在TPA诱导分化的U937和HL-60细胞中发现IL-24 delE5的表达。稳定转染IL-24 delE5的K562增殖及集落形成明显下降,与空载体相比,G0／G1期细胞比例由（24.46±3.99）%增至（42.69±3.04）%,细胞周期阻滞于G0／G1期,体内实验证实K562细胞移植瘤生长明显被抑制。与mda-7／IL-24相比,上述各实验结果差异均无统计学意义。结论　IL-24 delE5与mda-7／IL-24一样对人类白血病细胞系K562具有明显的体内外抑制作用,该作用可能与IL-24 delE5所引起细胞周期G0／G1期阻滞有关。     

Expression of myeloid-specific antigens on two human erythroleukaemia cell lines,HEL and K562

The cell-surface phenotype of the human erythroleukaemia cell line, HEL, was investigated using a panel of lineage-specific monoclonal antibodies by indirect immunofluorescence. Antigens specific for the erythroid, granulocytic and monocytic lineages were detected, but no lymphoid- or platelet-specific antigens were found. The data obtained for HEL were compared to findings for the K562 human erythroleukaemia cell line and considerable similarities were demonstrated between the phenotypes of the two. In particular it was noted that the majority of granulocyte/monocyte markers present on HEL were also present on K562. The occurrence on HEL of markers of several haemopoietic lineages would suggest an origin from the non-lymphopoietic multipotential stem-cell or CFU-GEMM.     

Predominance of Mex= cells in newly-established human lymphoblastoid cell lines

Previous studies have demonstrated that approximately onethirdof human lymphoblastoid cell lines (LCLs) are deficient in removingO⁶-methylguanine residues because of the lack of O⁶-alkylguanine-DNAalkyltransferase (O⁶-AGT) activity. Such LCLs have been designatedMex^–, while the proficient LCLs are Mex⁺. Our determinationsof O⁶-AGT activity as a function of cellular protein concentrationon 37 previouslyestablished LCLs discolsed that the expressionof the enzyme was high in 14 (Mex⁺) and barely detectable in16 (Mex^–). The other seven LCLs showed intermediate activityof the enzyme. By contrast, all of the 28 LCLs that we newlyestablished contained high enzyme activity, implying that theyconsisted of mainly Mex⁺ cells. Since the conventional O⁶-AGTw y on Mex⁺ cell populations was not capable of detecting theco-existence of Mex^– cells as a minor component, we attemptedto determine the proportion of Mex^– phenotype in newly-immortalizedlymphoblastoid cell clones which had been established directlyon semisolid agar. All of the 15 independent clones derivedfrom a single blood sample also showed high O⁶-AGT activity,rendering it unlikely that Epstein - Barr virus transformationper se was responsible for the generation of Mex^– LCLs.These results collectively indicate that Mex⁺ in cells predominatein LCLs shortly after establishment and also suggest that thepossible growth advantage for Mex^– cells should play animportant role in the subsequent development of Mex^– LCIsduring the long-term culture in vifro.     

DIFFERENTIAL EXPRESSION OF GENES INVOLVED IN METABOLISM BETWEEN TUMORIGENITIC HUMAN LEUKEMIA CELL LINES K562 AND K562－n

Objective: To study the molecular mechanism of different tumorigenicity in nude mice of human leukemia cell lines K562-n and K562. Methods: To analyze the genes differently expressed between K562 and K562-n cells by using cDNA microarray technique. Results: Among the 12800 genes detected, some genes involved in material metabolism and material transport were differently expressed between K562-n and K562 cells. These genes include homo sapiens placenta-specific ATP-binding cassette transporter gene, dihydrodiol dehydrogenase gene,hepatic dihydrodiol dehydrogenase gene, NAD-dependent methylene tetrahydrofolate dehydrogenase cyclohydrolase,lysophosphatidic acid acyltransferase, alpha gene,argininosuccinate lyase gene, mitochondrial isocitrtated ehydrogenase, adhesion protein SQM1 gene,dimethylarginine dimethylamino-hydrolase gene, M1 subunit of ribonucleotide reductase and farnesyl pyrophosphate synthetase gene. Conclusion: The high tumorigenicity of K562-n cells is related to the different expression of some genes concerned with cell metabolism and material transpoert.     

Mechanism of interaction between cisplatin and human recombinant interferon gamma in human ovarian-cancer cell lines

Human ovarian carcinoma cells (2008 and its cisplatin-resistant sub-line 2008/C13*) were sensitized to cisplatin by treatment with human recombinant gamma interferon (IFNγ). IFNγ produced no significant change in the uptake of CDDP. Exposure of 2008 and 2008/C13* cells to IFNγ resulted in a time-dependent decrease of cellular glutathione and total glutathione-S-transferase activity, principally the π isoform. By contrast, the treatment of 2008 and 2008/C13* cell lines with IFNγ induced rather than suppressed metallothionein II_A mRNA levels. IFNγ changed neither the formation of total platinum-DNA adducts, nor DNA repair. A significant decrease in c-erbB-2 expression was observed both in sensitive and in resistant cell lines after treatment with IFNγ, and this decrease was dose-dependent. Our results indicate that the mechanism of IFNγ-induced sensitization in human ovarian-cancer cell lines is multifactorial. © 1995 Wiley-Liss, Inc.     

Effects of herbimycin A derivatives on growth and differentiation of K562 human leukemic cells.

Herbimycin A, a specific tyrosine kinase inhibitor, induced erythroid differentiation of human myelogenous leukemia K562 cells with a high level of bcr/abl tyrosine kinase. Several derivatives of herbimycin A were synthesized and their effects on cell proliferation and differentiation of K562 cells were examined. Of the compounds tested, 19-allylaminoherbimycin A was the most effective in inducing differentiation of K562 cells. However, the parent compound was the most potent growth inhibitor, suggesting that chemical modification of herbimycin A reduces the growth-inhibiting activity. The sensitivities of K562 cells to herbimycin derivatives were different from those of a rat kidney cell line infected with Rous sarcoma virus (v-src), suggesting that bcr/abl kinase may differ in sensitivity from other tyrosine kinases. These results indicate that a specific inhibitor of bcr/abl kinase could be an effective antitumor agent against chronic myelogenous leukemia.     

Effects of recombinant human interferon-alpha, beta and gamma on the antiproliferative activity of cytarabine in K562 human myeloid leukemia clonogenic cells.

The effects of recombinant human interferon-alpha, beta and gamma (IFN) on the antiproliferative activity of cytarabine (ara-C) in K562 human myeloid leukemia clonogenic cells were studied in an agar capillary microassay. The addition of IFN-alpha did not affect the antiproliferative activity of ara-C in K562 cultures treated with low concentrations of ara-C: 10-50 nM, whereas in those treated with high concentrations of ara-C: 100-500 nM, IFN-alpha significantly reduced the antiproliferative activity of ara-C. The addition of 3 x 10(4) U/ml IFN-alpha to ara-C-treated K562 cultures increased the IC50 of ara-C on day 5 from 102 to 214 nM, i.e. ara-C+IFN-alpha was about twofold less potent than ara-C alone. Low concentrations of IFN-beta and IFN-gamma did not affect the antiproliferative activity of ara-C on K562 colonies, but high concentrations of these two interferons reduced the antiproliferative activity of ara-C. The addition of 4 x 10(3) U/ml IFN-beta or 10(4) U/ml IFN-gamma increased the IC50 of ara-C on day 5 to 304 nM or to 316 nM, respectively, i.e. ara-C+IFN-beta or IFN-gamma was about threefold less potent than ara-C alone. The significant reduction of the desired antiproliferative activity of ara-C by the three interferons was reproduced in liquid suspension cultures of K562 cells on day 4 in the following order: IFN-gamma greater than IFN-beta greater than IFN-alpha. The present negative interactions of the three interferons with ara-C particularly at high concentrations, may caution against the clinical use of the combination of ara-C and interferon in the treatment of myeloid leukemia patients.     

Effects of inducers of erythroid differentiation of human leukemia K562 cells on vincristine-resistant K562/VCR cells

K562/VCR cells, which are resistant to the cytotoxicity of vincristine, were isolated from human erythroleukemia K562 cells. Various compounds that induce erythroid differentiation of K562 cells were tested on K562/VCR cells. Differentiation of K562/VCR cells was not induced by actinomycin D or adriamycin alone, but the resistance of these cells to the inducers was overcome by verapamil. In contrast, mitomycin C, butyric acid and hemin induced differentiation of K562/VCR cells as effectively as that of K562 cells. These results suggest that therapy by induction of differentiation of leukemic cells is effective for leukemic cells that have acquired resistance to therapeutic drugs.     

Effects of the antitumoural dequalinium on NB4 and K562 human leukemia cell lines. Mitochondrial implication in cell death

Dequalinium (DQA) is a delocalized lipophylic cation that selectively targets the mitochondria of carcinoma cells. However, the underlying mechanisms of DQA action are not yet well understood. We have studied the effects of DQA on two different leukemia cell lines: NB4, derived from acute promyelocytic leukemia, and K562, derived from chronic myeloid leukemia. We found that DQA displays differential cytotoxic activity in these cell lines. In NB4 cells, a low DQA concentration (2microM) induces a mixture of apoptosis and necrosis, whereas a high DQA concentration (20microM) induces mainly necrosis. However, K562 cell death was always by necrosis as the cells showed a resistance to apoptosis at all time-periods and DQA concentrations assayed. In both cell lines, the cell death seems to be mediated by alterations of mitochondrial function as evidenced by loss of mitochondrial transmembrane potential, O2*- accumulation and ATP depletion. The current study improves the knowledge on DQA as a novel anticancer agent with a potential application in human acute promyelocytic leukemia chemotherapy.     

The antileukemic alkaloid fagaronine and the human K 562 leukemic cells: effects on growth and induction of erythroid differentiation

In view of new antitumor compounds which could exert their therapeutic effect through a combination of cell growth inhibition and cell maturation, we describe here the effects of a novel antileukemic alkaloid, fagaronine, on the growth and the induction of hemoglobin synthesis in the K 562 cell line. We found that fagaronine, after 3 days, reduces in a concentration dependent relationship the cell growth rate without lethality and this effect on the cell growth is irreversible. Reducing the cell growth rate by 50% (IC50 = 3 X 10(-6)M) is sufficient to induce an optimal amount of hemoglobin synthesis (75% benzidine-positive cells, 13-15 pg hemoglobin/cell) after 4 days of culture. Considering the variation of the total intracellular protein content during the response, it appears that fagaronine stimulated mainly hemoglobin synthesis, and to a lesser extent non-hemoglobin proteins. These results suggest that the novel antileukemic alkaloid, fagaronine, can be considered as a potent inducer of differentiated-associated properties in the human K 562 leukemic cells.     

Grb2抑制剂对K562细胞生长增生的影响

目的 探讨针对Grb2-SH3的抑制剂peptidimer-c对K562细胞生长增生的影响.方法 应用N端基团保护的Fmoc化学,固相合成针对Grb2-SH3的二聚肽peptidimer-c,高压液相色谱技术(HPLC)分析肽的纯度,质谱法分析肽的结构,应用pull-down实验,观察peptidimer-c与K562细胞裂解物中Grb2分子的结合.应用锥虫蓝拒染法、WST-1法、克隆形成法观察peptidimer-c对K562细胞生长的抑制.通过克隆形成实验,探讨peptidimer-c与常用的CML治疗药物甲磺酸伊马替尼(商品名:格列卫)、羟基脲及阿糖胞苷联合应用的合并效应.结果 HPLC图谱上只见一样品峰而无其他杂峰.质谱分析显示,所合成的化合物与设计的肽是一致的.pull-down结果显示,peptidimer-c可与K562细胞中的Grb2分子特异性结合.锥虫蓝计数法结果提示,peptidimer-c可明显抑制K562细胞的生长,且在加药后短时间内(3～6h)即有明显作用,其对K562增生的抑制呈浓度依赖型,而非时间依赖型.WST-1检测结果显示,peptidimer-c杀伤K562细胞的半致死剂量为(17±2)μmol/L.几种化合物对K562克隆形成抑制的半致死剂量分别为:peptidimer-c(3.9±0.9)μmol/L,伊马替尼(0.03±0.02)μmol/L,羟基脲(15±7)μmol/L,阿糖胞苷(0.014±0.012)μmol/L.peptidimer-c分别与伊马替尼、阿糖胞苷、羟基脲联合应用时,对K562克隆形成抑制均表现为相加作用或协同作用,其中1.5 μmol/L peptidimer-c与0.05 μmol/L伊马替尼联合应用,表现协同作用,1.5 μmol/L peptidimer-c与0.006 μmol/L阿糖胞苷或0.01 μmol/L阿糖胞苷联用,也显示协同抑制效应.结论 peptidimer-c能有效抑制K562细胞的生长和增生.与其他类型的药物合用,表现为相加或协同效应,可提高抗肿瘤效应.