P53 content in relation to cell growth and proliferation in murine L1210 leukemia and normal lymphocytes

The protein p53 has been reported to be associated with cell transformation and/or proliferation. Using p53 monoclonal antibodies we estimated by flow cytometry the relative content of this protein in individual L1210 leukemic cells from exponentially growing and plateau-phase cultures and compared it with that in normal thymocytes of parental DBA/2 mice and in mitogen-stimulation and nonstimulated human lymphocytes. Simultaneous differential staining of p53 vs DNA and p53 vs RNA, followed by bivariate analysis, made it possible to estimate p53 with respect to cell position in the cell cycle and correlate it with RNA (predominantly rRNA;) content. The data show that in exponentially growing L1210 cells p53 is being progressively accumulated during the G₁ S and G₂ phases and that the content of p53 and RNA are highly correlated. In plateau L1210 cultures most cells are arrested in G₁ some cells, however, still continue to progress through S and G₂. In these cultures the p53 content of all cells, regardless of the phase of the cell cycle, is diminished and the decrease in p53 is more pronounced than that of RNA or total protein content. The normal thymocytes as well as the stimulated lymphocytes show bimodal distribution with respect to p53 expression, compatible with the assumption that the cycling cells have increased expression of this protein related to the G₀ cells. Some cycling cells, however, have minimal p53. The quantitative p53 immunofluorescence data were confirmed by the immunoprecipitation and gel electrophoresis. The results suggest that expression of p53 in leukemic and normal cells is more correlated with cell growth than with entrance to the cell cycle or progression through particular phases of the cycle.     

Mode of Estrogen Action on Cell Proliferative Kinetics in CAMA-1 Cells. I. Effect of Serum and Estrogen

Both serum and estrogen affect cell proliferation in CAMA-1 cells. Their effects on cell cycle kinetics are being investigated with partially synchronized cells following 48 hours of serum deprivation. By comparing the growth kinetics of synchronized (serum-deprived) cells with asynchronized (normal-fed) cells, we observed that there was a delay of cell proliferation for approximately 25 hr for synchronized cells and that estrogen only induced cell proliferation in serum-supplemented culture. A serum protein (RPF), precipitated by ammonium sulfate, dialyzed in 3,500 daltons cut off membrane and reconstituted in culture medium, was shown to stimulate cell proliferation in a dose-related manner. In the absence of RPF, estrogen had no effect on cell growth and S-phase formation in serum-free medium, but significantly induced cell growth in the presence of RPF. Experiments conducted with a synchronized cell population showed that estrogen increased the proportion of G₁ phase cells to enter S phase, shortened the G, phase duration, and ultimately increased the proportion of mitotic cells per cycle. Collectively, these results show that (a) estrogen effects of cell proliferation in vitro requires a serum component, a “G₁/S-promoting factor” and (b) estrogen-induced tumor growth is a result of an accelerated rate of G₁/5 transition and an increased number of dividing cells per cycle. The knowledge of the interaction of estrogen with the G₁/S promoting factor on cell growth is of both fundamental and therapeutic importance.     

Effects of aclacinomycin on cell survival and cell cycle progression of cultured mammalian cells

The effects of aclacinomycin (ACM; NSC 208734) on cell viability, growth, and colony formation were investigated in suspension (Friend leukemia and L1210) and adherent (Chinese hamster ovary) cell systems. Cell cycle progression and the effect of the drug on various transition points in the cell cycle (i.e. G1 to S phase, through a window in early S phase and G2 phase to mitosis) were monitored by flow cytometry. Formation of Chinese hamster ovary cell colonies was inhibited by 50% following 24 hr of exposure to 0.05 micrograms ACM per ml whereas 1 hr of exposure to 1.0 micrograms ACM per ml reduced colony formation by only 30%. Stationary cultures required a drug concentration more than 5 times higher to reduce colony formation by an equivalent amount when present for 24 hr. Short-term (1-hr) exposure to drug concentrations up to 1.0 micrograms/ml had no effect on colony formation of stationary-phase Chinese hamster ovary cells. Cell growth was inhibited by 50% in suspension cultures of Friend leukemia and L1210 cells when exposed for 24 hr to 0.024 and 0.053 micrograms ACM per ml, respectively. Continuous drug exposure of Friend leukemia and L1210 cells to ACM concentrations of 0.05 to 0.1 micrograms/ml led to a slow down in cell progression manifested as an accumulation of cells in G2 + M phase by 24-hr and then in G1 phase by 48-hr culture. However, brief (1-hr) exposure of L1210 cells to 0.5 micrograms/ml resulted in an irreversible accumulation of cells in G2 + M phase. A more detailed examination of drug effects on the cell cycle determined that 0.1 micrograms ACM per ml resulted in a slow down in L1210 cells leaving G1 phase and entering mitosis and an accumulation of cells in G2 phase, although early S-phase cells appeared unaffected. At a 5 times higher drug concentration, exit of cells from G1 was almost completely halted, passage of cells through early S was slowed, and the entrance of cells into mitosis plateaued 3.5 hr after addition of the drug; G2-phase cells were only mildly affected. The RNA content of all cells examined was reduced by 35 to 50% depending upon dose and time of exposure. These findings are discussed in terms of the known biochemical effects of ACM on RNA and protein synthesis.     

Effects of ellipticine on cell survival and cell cycle progression in cultured mammalian cells

The effects of ellipticine [5,11-dimethyl-6H-pyrido(4,3-b)carbazole; NSC 71795] on cell viability, growth, and colony formation were investigated in suspension (Friend leukemia and L1210) and adherent [Chinese hamster ovary (CHO)]tumor cell systems as well as in mitogen-stimulated human peripheral blood lymphocyte cultures. Cell cycle progression and the terminal point of action of the drug were monitored by flow cytometry. Ellipticine was cytostatic for all cell lines tested, blocking cells in G2 phase following 24 hr constant exposure at concentrations in the range of 1.0 microgram/ml. A 10 times higher drug concentration was required to block cells in G2 if the cells were exposed for only 30 min to the drug followed by 23.5 hr culture in drug-free medium. Formation of CHO cell colonies was inhibited by 50% following exposure to ellipticine for 2 hr at 6.0 microgram/ml or for 24 hr at 0.3 microgram/ml. Fifty % cell kill in asynchronously growing Friend leukemia and L1210 cells was obtained following exposure to ellipticine for 24 hr at 2.0 microgram/ml and 1.15 microgram/ml, respectively, whereas human peripheral blood lymphocytes required 66 hr exposure to 1.0 microgram/ml to kill 50% of the cells. Phytohemagglutinin-stimulated lymphocytes were remarkably resistant to the cytotoxic effect of ellipticine but did display a dose-dependent inhibition of stimulation and accumulation in G2 whether the drug was added prior to our during active cell proliferation. Ellipticine, at cytostatic concentrations, had a marked effect on cellular RNA content. Friend leukemia cells, blocked in G2 by the drug, doubled their RNA content compared to control cells. L1210 and CHO cells, but not lymphocytes, also increased in RNA content following ellipticine treatment. Drug concentrations which blocked cells in G2 also led in the case of Friend leukemia and L1210 but not CHO cells to an increase in the proportion of cells with greater than 4C amounts of DNA.     

Cytokinetics of a Human Neuroblastoma Cell Line Following Treatment with Dianhydrogalactitol

Studies were carried out on the effect of dianhydrogalactitol on a human neuroblastoma cell line in culture. The drug was cytotoxic at concentrations of 5 μg/ml and 10 μ/ml for 1 hour, but not at 1 μ/ml. Flow cytometry of DNA showed that cell kill was preceded by a transient accumulation of cells in early S at 10 hours, and in late S at 36 hours. Smaller increases in the percent of cells in S phase were seen after treatment with 1 μg/ml, without cell kill. It may be concluded that dianhydrogalactitol causes an S-phase block, followed by death of cells in late S at concentrations above 5 μg/ml.

*Assistant Attending Physician, Solid Tumor Service, Department of Medicine; Research Associate, Laboratory of Biophysics, Recipient, American Cancer Society Junior Faculty Clinical Award.     

Killing of Human Lung Cancer Cells Using a New [111In]Bleomycin Complex [111In]BLMC

The ability of a [¹¹¹In]bleomycin complex ([¹¹¹In]BLMC) to kill five cell lines of human lung cancer (small cell lung cancer) was investigated. Cells were exposed to either 0.9% NaC1, [¹¹¹In]C1₃, BLM, [¹¹¹In]BLMC, nonradioactive InC1₃, or In-BLMC for 60 minutes, plated in soft agarose, and assessed for colony formation. [¹¹¹In]BLMC (40-200 μ;C) carried by 15-25 μ;g BLM/ml) was more cytotoxic than BLM (15-25 μ;g BLM/ml) by a factor of 1.6-5.3 for five cell lines. The percent survival of N417 cells was 28.4 for [¹¹¹In]BLMC (40 μ;Ci/15 μ;g BLM/ml) and 54.3 for BLM (15 μ;g/ml); 1.9 for [¹¹¹In]BLMC (200 μ;Ci/25 μ;g BLM/ml), and 10.0 for BLM (25 μ;g/ml). ¹¹¹InC1₃ (200 μ;Ci/ml) and nonradioactive InC1₃ failed to inhibit colony formation. The new [¹¹¹In]BLMC may be useful for therapy of some lung cancer patients.     

Antiproliferative Activity of Mammalian Lignan Derivatives Against the Human Breast Carcinoma Cell Line, ZR-75-1

The effect of each of twelve mammalian lignun derivatives on the growth of human mammary tumor ZR-75-1 cells was examined. At a concentration less than 10 μ g/ml, tumor cell growth was inhibited from 18-68%. The effect of 2,3-dibenzylbutane-1,4-diol (hattalin) was found to be strongest, inhibiting growth by 50% at a concentration (EC₅₀ of 2.1 μ g/ml. Hattalin inhibited membrane Na^%, K^%-ATPase of canine kidney cortex. It also inhibited the ATPase of the plasma membrane fraction from both cultured cells and a section of human breast cancer tissue at a concentration ranging from 0.5 to 2.0 mM. However, only a few percent of membrane ATPase from either ZR-75-1 cells or breast carcinoma tissue was inhibited by 2.0 mM of ouabain, suggesting that the target ATPase of hattalin was other than ouabainsensitive ATPase. The relative incorporation of [³H]thymidine per 1 10⁵ cells into the acid-precipitable fraction of ZR-75-1 cells was not affected by 1-50 μg/ml of hattalin, while a marked decrease resulted from 1-10 μg/ml of 5-fluorouracil (5-FU). These results suggest that the suppressive effect of hattalin on tumor cell growth my not occur through inhibition of DNA synthesis but rather partly by inhibition of the plasma membrane ATPase other than Na^% and K^% -dependent ones.     

In Vivo Accumulation of Etoposide in Peripheral Leukemic Cells in Patients Treated for Acute Myeloblastic Leukemia; Relation to Plasma Concentrations and Protein Binding

Since etoposide interacts with the nuclear enzyme topoisomerase II, the drug concentrations in the malignant cells during chemotherapy may have clinical correlates. Plasma protein binding of etoposide is extensive (94%) and alterations of the non-proteinbound fraction affect pharmacokinetic behavior of the drug. The pharmacokinetics of etoposide was therefore studied in plasma, total and non-proteinbound concentrations, and in leukemic cells isolated from peripheral blood samples from 22 patients after the first dose of the induction treatment for acute myelocytic leukemia. Fourteen patients received 100 mg/m² and eight patients 200 mg/ m² as a 1 h infusion. The mean area under the concentration versus time curve AUC_(0-x) in plasma was at the lower dose level 78.4 ± 29.1 (mean ± S.D.) μg/ml x h and 201.0 ± 56.5 μg/ml x h at the higher dose level. The fraction of non-proteinbound etoposide in plasma was 5.2 ± 3.4 and 5.4 ± 2.1% in the two treatment groups. AUC_(0-16h) in leukemic cells was 8.4 ± 8.7 and 22.4 ± 12.1 μ/ml x h at the two dose levels, respectively. The cellular etoposide concentration was 12.1 ± 7.9 and 14.7 ± 5.1% of the plasma concentration at the end of the infusion. The interpatient variability in cellular drug levels was considerable and exceeded the variability in plasma concentrations. Cellular accumulation of etoposide could be important for treatment outcome.     

Neuraminidase induced loss in the transplantability of murine leukaemia L 1210, induction of immunoprotection and the transfer of induced immunity to normal DBA-2 mice by serum and peritoneal cells

Leukaemia L 1210 cells preincubated in vitro with neuraminidase preparations derived either from Vibrio cholerae (VCN) or Clostridium perfringens (CPN) lost their i.p. transplantability for normal DBA/2 mice. This loss of transplantability could not be observed when the enzyme treated cells were implanted in mice whose immune status was suppressed by prior cyclophosphamide treatment. Mice receiving i.p. implants of enzyme treated leukaemia cells developed immunity to subsequent i.p. challenge with untreated L 1210 cells but not to a challenge with Ehrlich ascites tumour cells. The magnitude of immune response evoked by L 1210 cells preincubated with 250 u/ml of VCN or 35 μg/ml of CPN for 60 minutes was of relatively low level when compared with the immunity induced by leukaemia cells preincubated with 50 u/ml of VCN or 15 μg/ml of CPN for 30 or 60 minutes. Evidence is presented to show that the induced immunity can be transferred passively with the serum and with the peritoneal cells from the mice implanted with VCN treated L 1210 cells to normal DBA/2 mice. The significance of the neuraminidase induced increase in the immunogenicity of treated tumour cells is discussed.     

雪灵芝水提物对人胃癌MGC-803细胞增殖及细胞周期的影响

目的探讨雪灵芝水溶性提取物(arenaria kansuensis aqueous extract,AKAE)对人胃癌细胞株MGC-803细胞增殖及细胞周期的影响。方法体外培养的MGC-803细胞经不同浓度AKAE处理,采用MTT法检测受试细胞增殖水平;应用流式细胞术检测AKAE处理12 h后各组细胞的细胞周期;Western blot检测不同浓度、不同时间AKAE处理组MGC-803细胞中cyclin D1蛋白表达水平;实时定量 PCR检测各组MGC-803细胞中cyclin D1、p16和p21基因的mRNA相对表达量。结果AKAE对体外培养的MGC-803细胞增殖具有浓度依赖性抑制作用（P<0.05）,IC₅₀为(0.134±0.005)mg/ml;经AKAE处理12 h,0.8 mg/ml组MGC-803细胞的 G₁期细胞比例高于对照组、S期细胞比例低于对照组（P<0.05）;(0.2～0.8) mg/ml浓度的AKAE处理,对MGC-803细胞中cyclin D1的蛋白及mRNA表达具有明显抑制作用（P<0.05）;AKAE促进MGC-803细胞中p16、p21基因mRNA 表达,0.2 mg/ml处理组p16、p21 mRNA相对表达量分别为对照组的3.3倍和18.5倍。结论雪灵芝水提物(AKAE)对MGC-803细胞增殖具有抑制作用,其机制与G₁期阻滞和对G₁期相关因子表达的影响有关。     

Effects of chartreusin on cell survival and cell cycle progression

Chartreusin was lethal to both L1210 and P388 cells in culture with 90% of the cells being killed after a 24-hr exposure to 1.1 and 2.6 microgram/ml, respectively. The lethality of the drug increased in direct proportion to dose and exposure time. Both L1210 and Chinese hamster ovary cells in S phase were more sensitive to the lethality of the drug than were their corresponding non-S-phase cells. L1210 cells were partially synchronized by exposing an asynchronous culture to [methyl-3H]thymidine (20 Ci/mmol) and Colcemid for 3 hr. Synchronous culture of Chinese hamster ovary cells was established by planting mitotic cells. The progression of cells through the cell cycle was studied with flow microfluorometry both in the presence of the drug and after the drug had been washed off. In the presence of chartreusin the progression of mitotic cells into G1 was not affected. The movement of G1 cells into S was slower, and the movement of G2 cells into mitosis was blocked. When the drug was removed, the G2 to M block persisted for at least 4 hr but the progression of G1 cells to S was no longer inhibited.     

Tocopherols and saponins derived from Argania spinosa exert, an antiproliferative effect on human prostate cancer

The aim of our study is to evaluate the antiproliferative effect of tocopherols obtained from alimentary virgin argan oil extracted from the endemic argan tree of Morocco and of saponins extracted from argan press cake on three human prostatic cell lines (DU145, LNCaP, and PC3). The results were compared to 2-methoxyestradiol as antiproliferative drug candidates. Cytotoxicity and antiproliferative effects were investigated after cells' treatment with tocopherols and saponins compared to 2-Methoxyoestradiol as the positive control. Tocopherols and saponins extracted from argan tree and 2-methoxyestradiol exhibit a dose-response cytotoxic effect and an antiproliferative action on the tested cell lines. The best antiproliferative effect of tocopherols is obtained with DU145 and LNCaP cell lines (28 μg/ml and 32 μg/ml, respectively, as GI₅₀). The saponins fraction displayed the best antiproliferative effect on the PC3 cell line with 18 μg/ml as GI₅₀. Our results confirm the antiproliferative effect of 2-methoxyestradiol and show for the first time the antiproliferative effect of tocopherols and saponins extracted from the argan tree on hormone-dependent and hormone-independent prostate cancer cell lines. These data suggest that argan oil is of potential interest in developing new strategies for prostate cancer prevention.     

西妥昔单抗对肺腺癌A549细胞株辐射增敏作用的研究

目的探讨西妥昔单抗(Cetuximab, C225)对人肺腺癌A549细胞株的辐射增敏作用及其机制。方法 体外培养人肺腺癌A549细胞株, 经不同浓度的C225作用后, 使用CCK-8法测定细胞增殖抑制率, 计算出C225的半数抑制浓度(IC₅₀), 并将IC₅₀的1/5作为后续实验的浓度。采用克隆形成方法观察C225对细胞辐射敏感性的影响, 按多靶单击模型拟合细胞存活曲线, 计算辐射增敏比(SER)。流式细胞仪检测细胞凋亡以及细胞周期情况。结果 C225均抑制了细胞的增殖, 并呈现明显的量-效关系, 其IC₅₀为18.24μg/mL。与单独照射组相比, 加药照射组的SF₂、D₀、D_q均下降(P＜0.05), SER_D0为1.40。C225联合X线照射明显增强了辐射诱导的细胞凋亡(P＜0.05)。C225使细胞阻滞于G₀/G₁期(P＜0.05), 单独照射组G₂/M期细胞比例增加(P＜0.05), C225+照射组同时出现G₀/G₁、G₂/M期细胞阻滞(P＜0.05);与对照组相比, C225组、单独照射组以及C225+照射组S期细胞比例均下降(P＜0.05)。结论 C225对A549细胞株具有辐射增敏作用, 其机制可能与C225抑制细胞的生长增殖和受照射后亚致死性损伤的修复, 增加细胞凋亡以及诱导细胞G₀/G₁期阻滞有关。     

Heterogeneity of peripheral blood T-cell colony-forming cells in patients with T-cell malignancies

Some peripheral blood clonogenic T-cells from patients with T-cell malignancies can generate colonies in methylcellulose in the absence of added growth factors or mitogen stimulation (T-CFC_S). T-CFC from these patients were also able to form colonies in semi-solid media in the presence of added growth factors (T-CFC_i. T-CFC_S, in contrast to T-CFC_i, were highly clonogenic cells possessing self-renewal capacity in the absence of added growth factors. T-CFC₅ were in cell cycle and more sensitive to Ara-C or ADM (D₁₀ = 0.009 and 0.025 μg/ml respectively) than T-CFCi (D₁₀ = 1 and 2 μg/ml respectively). Furthermore, T-CFC₅ were more radiosensitive (D_o < 1.1 Gy) than T-CFC. (D_o <5 Gy). T-cell precursors from patients with immature blast cells (E⁻, OKT3⁻, OKT6⁺, OKT10⁺) were independent of added growth factors for their in vitro proliferation whereas in cases with mature blast cells (E⁺, OKT3⁺) T-CFC were significantly more dependent. These observations strongly suggest that T-CFC₅ and T-CFC_i represent different cell subsets.

The phenotype of pooled induced and spontaneous T-cell colonies was highly individualized. However, colonies contained a significant proportion of relatively immature T-cells as assessed by the proportion of OKT6⁺, OKT10⁺, OKT3⁺ and E⁺ cells. The phenotype of colony cells was quite similar to that observed on fresh leukemic cells suggesting a defect of the in vitro differentiation of both T-CFC₅ and T-CFC_i.     

维生素C联合三氧化二砷对膀胱癌T-24细胞株的作用

 目的 探讨维生素C联合三氧化二砷（As2O3）对膀胱癌T-24细胞的影响及其机制。方法 体外培养T-24细胞,分为6组,对照组不做处理,实验组分别加入不同浓度的As₂O₃ （0.4、4、40 μg/ml）组,维生素C（400 μg/ml）组、维生素C（400 μg/ml）+ As₂O₃ （0.4 μg/ml）组（联合组）,采用细胞增殖曲线检测T-24细胞生长差异情况,用流式细胞术检测细胞周期及凋亡率变化,RT-PCR、Western blot技术检测分析caspase-3、survivin mRNA及蛋白的表达情况。结果 联合组、维生素C（400 μg/ml）组、As₂O₃ （4 μg/ml）组及As₂O₃ （40 μg/ml）组均对膀胱癌T-24细胞增殖有显著抑制作用;联合组将细胞阻止在G₀/G₁期,且凋亡率显著高于其他五组（P<0.05）;RT-PCR及Western blot结果显示,维生素C（400 μg/ml）组、联合组的caspase-3 mRNA和蛋白表达升高,survivin mRNA和蛋白表达降低。结论 维生素C联合As₂O₃ 可抑制膀胱癌T-24细胞增殖,促进细胞凋亡,其机制可能与上调caspase-3 mRNA、蛋白,下调survivin mRNA、蛋白表达有关。     

人参皂苷Rd抑制人非小细胞肺癌A549细胞的生长及作用机制

目的 探讨人参皂苷Rd(GS-Rd)对人非小细胞肺癌(NSCLC)细胞株A549细胞增殖、凋亡、侵袭和周期的影响及可能机制.方法 采用MTT法,观察不同浓度(12.5、25、50、100μmol/L)GS-Rd体外作用NSCLC细胞系A549不同时间后对其细胞活力的影响;利用高内涵分析软件,观察不同浓度、不同时间作用于...     

17β-Estradiol metabolites affect some regulators of the MCF-7 cell cycle

The activity of p34^cdc2 plays a key role in the regulation of the eukaryotic cell cycle. Another cell cycle associated molecule is PCNA. We investigated the effects of 2-hydroxy-17β-estradiol, a cell proliferator, and 2-methoxy-17β-estradiol, a potent inhibitor of cell growth, on the levels and activity of p34^cdc2 and on the levels of PCNA, as well as on protein phosphorylation in MCF-7 cells. 2-Hydroxyestradiol increased p34^cdc2 activity at G₁/S and elevated PCNA levels during S-phase. 2-Methoxyestradiol caused unscheduled activation of p34^cdc2 in S-phase and decreased levels of p34^cdc2 and PCNA during G₂/M. We conclude that 2-hydroxy- and 2-methoxyestradiol have definite, though different regulatory functions during the cell cycle.     

Phase I study on 5-aza-2′-deoxycytidine in children with acute leukemia

A Phase I study with 5-aza-2′-deoxycytidine (5-AZA-CdR) was performed on children with acute leukemia who were resistant to conventional chemotherapy. The objective of this study was to find the dose-schedule of 5-AZA-CdR that produced a potent antileukemic effect and to evaluate its toxicity. At doses of 0.75–30 mg/kg administered as 12–30-h i.v. infusion and 10 mg/kg administered as i.v. bolus the antileukemic effect produced by 5-AZA-CdR was of short duration. A more potent antileukemic effect was observed when the dose of 5-AZA-CdR was increased from 36 to 80 mg/kg administered as 36–44-h i.v. infusion. At this dose range two of nine patients obtained an M₁ marrow and one of nine patients obtained an M₂ marrow. Clearing of meningeal leukemia occurred in two patients. Using an in vitro test which measures the inhibition of DNA synthesis in blood and marrow leukemic cells by cytosine arabinoside, five of nine patients showed signs of drug resistance after 5-AZA-CdR treatment at the higher doses. A bioassay which quantitates the growth inhibitory effects of 5-AZA-CdR on L1210 leukemic cells was used to estimate the concentration of this agent in the body fluids. At an infusion rate of 1.0 mg/kg/h the steady state plasma concentration was estimated to be 0.5 μg/ml. The plasma half-life of 5-AZA-CdR was 12 min. In two patients inhibitory concentrations of 5-AZA-CdR were detected in the cerebral spinal fluid.     

Effects of 5-aza-2'-deoxycytidine on survival and cell cycle progression of L1210 leukemia cells

The in-vitro effects of the antileukemic agent 5-aza-2'-deoxycytidine (5-aza-dCyd), on DNA synthesis, growth, cloning in agar, and cell cycle traverse of L1210 leukemia cells were studied. 5-Aza-dCyd at 0.1 microgram/ml for 10 hr (cytotoxic concentration) did not inhibit DNA synthesis but produced a very potent growth inhibition, and changed markedly the DNA flow cytometric histograms. A 5-h continuous exposure to the drug at concentrations ranging from 0.1 to 10 micrograms/ml caused an accumulation of cells in the S portion of the DNA histograms indicating a slowing of the progression of cells in the S phase. A longer exposure time (10 h) at the same concentrations led to a bimodal DNA distribution (peaks at G1 and G2-M) and a depletion of the S phase. When the exposure time to 5-aza-dCyd (0.1 microgram/ml) was extended to 15 and 20 h, there was a decrease in the G2-M peak and an augmentation of the G1 peak. To determine if 5-aza-dCyd produced a block in cell cycle progression, L1210 cells were treated for 10 h with colcemid and 5-aza-dCyd simultaneously for 10 h. Colcemid alone, or colcemid in combination with 5-aza-dCyd produced an accumulation of cells under a single G2-M peak. This indicates that 5-aza-dCyd did not block the progression of L1210 cells through S phase, but only produced a slowing down of this event. These results, indicating that 5-aza-dCyd does not block cell cycle progression and that its cytotoxic action is not self-limiting, are of importance for designing future clinical trials.     

Bcl-2 overexpression attenuates SP600125-induced apoptosis in human leukemia U937 cells

SP600125 is a specific inhibitor of c-Jun N-terminal kinase (JNK) that is known to strongly induce apoptosis and block cell cycle progression in G₂/M phase. In this study, we demonstrated that treatment of U937 cells with SP600125 resulted in significant G₂/M cell cycle arrest that was due to decreased cyclin B1 and cdc25c protein levels. Moreover, SP600125 promoted LDH release and DNA fragmentation that was associated with caspase-3 activation and degradation of its substrates. In contrast, overexpression of the antiapoptotic protein Bcl-2 rendered leukemia cells resistant to SP600125-induced apoptosis, but more sensitive to G₂/M phase arrest and endoreduplication (>4N DNA). Overexpression of Bcl-2 significantly inhibited SP600125-induced caspase-3 activation and degradation of its substrates, and sustained expression levels of the IAP-2 proteins following SP600125 treatment. The inhibitory effect of Bcl-2 on apoptosis was attenuated by treatment with the small molecule Bcl-2 inhibitor, HA14-1. These data provide important mechanistic insights related to Bcl-2-mediated resistance to SP600125-induced apoptosis, and induction of G₂/M phase arrest and endoreduplication.