Apoptotic cell death triggered by camptothecin or teniposide. The cell cycle specificity and effects of ionizing radiation

Abstract. We have previously observed that the DNA topoisomerase I inhibitor camptothecin (CAM), or DNA topoisomerase II inhibitors teniposide (TEN) and amsacrine (m-AMSA) trigger endonucleolytic activity in myelogenous (HL-60 or KGl), but not lymphocytic (MOLT-4) leukaemic cell lines. DNA degradation and other signs of apoptotic death were seen as early as 2–4 h after cell exposure to these inhibitors. Cells replicating DNA (S phase) were selectively sensitive whereas cells in G₁ were resistant; the sensitivity of G₂ or M cells could not be assessed in these studies. The present studies were aimed at revealing whether DNA repair replication induced by ionizing radiation can sensitize the cells, and to probe the sensitivity of cells arrested in G₂ or M, to these inhibitors. The data show that γ-irradiation (0.5–15 Gy) of HL-60 cells does not alter their pattern of sensitivity, i.e. G₁ cells, although engaged in DNA repair replication, still remain resistant to CAM compared with the S phase cells. Likewise, irradiation of MOLT-4 cells also does not render them sensitive to either CAM or TEN, regardless of their position in the cell cycle. Irradiation, however, by slowing the rate of cell progression through S, increased the proportion of S phase cells, and thus made the whole cell population more sensitive to CAM. HL-60 cells arrested in G₂ either by irradiation or treatments with Hoechst 33342 or doxorubicin appear to be more resistant to CAM relative to S phase cells. Also resistant are cells arrested in M by vinblastine. The data suggest that some factor(s) exist exclusively in S phase cells, which precondition them to respond to the inhibitors of DNA topoisomerases by rapid activation of endogenous nuclease(s) and subsequent death by apoptosis. HL-60 cells in G₁, G₂ or M, or MOLT-4 cells, regardless of the phase of the cycle, appear to be protected from such a mechanism, and even induction of DNA repair replication cannot initiate DNA degradation in response to DNA topoisomerase inhibitors. These data, together with the evidence in the literature that topoisomerase I may be involved in DNA repair, suggest that a combination of these inhibitors with treatments that synchronize cells in the S phase and/or recruit quiescent cells to proliferation, including radiation, may be of value in the clinic.     

Design and synthesis of novel nitrogen mustard-evodiamine hybrids with selective antiproliferative activity

A series of novel nitrogen mustard-evodiamine hybrids were synthesized and evaluated for their antitproliferative properties. The antiproliferative activities of 10a–d, 11a–d, and 12a–d against four different kinds of human cancer cell lines (PC-3, HepG2, THP-1 and HL-60) and human normal peripheral blood mononuclear cells (PBMC) were determined. The results showed that all the target hybrid compounds exhibited antiproliferative activities against tested human tumor cell lines to some extent and no antiproliferative activities (>200?μM) against human normal PBMC cells. The antiproliferative selectivity between tumorous and normal cells was very useful for further antitumor drug development. Among the target compounds, 12c showed the strongest cytotoxicity against two tumor cell lines (THP-1 and HL-60) with IC₅₀ values of 4.05?μM and 0.50?μM, respectively, and selected for further mechanism study in HL-60 cells. The results showed that 12c could induce HL-60 cells apoptosis and arrest at G₂ phase at low sub-micromolar concentrations via mitochondria-related pathways.     

Effect of staurosporine on MOLT-4 cell progression through G2 and on cytokinesis

Staurosporine (SSP) is an inhibitor of a variety of protein kinases with an especially high affinity towards protein kinase C. Whereas SSP has been shown to halt the cell cycle progression of various normal, nontransformed cell types in G₁, most virus transformed or tumor cells are unaffected in G₁ but arrest in G₂ phase. SSP has also been observed to increase the appearance of cells with higher DNA content, suggestive of endoreduplication, in cultures of tumor cells. Using multivariate flow cytometry (DNA content vs. expression of cyclin B, nucleolar p120 protein, or protein reactive with Ki-67 antibody) which makes it possible to discriminate cells with identical DNA content but at different phases of the cycle, we have studied the cell cycle progression of human lymphocytic leukemic MOLT-4 cells in the presence of 0.1 μM SSP.MOLT-4 cells did not arrest in G₁ or G₂ phase in the presence of the inhibitor. Rather, they failed to undergo cytokinesis, entering G₁ phase at higher DNA ploidy (tetraploidy; G_1T), and then progressed through S_T (rereplication) into G_2T and M_T. The rates of entrance to G₂ and G_2T were essentially identical, indicating that the rates of cell progression through S and S_T as well as through G₂ and G_2T, respectively, were similar. Cells entrance to mitosis and mitotic chromatin condensation were also similar at the diploid and tetraploid DNA content level and were unaffected by 0.1 μM SSP. No evidence of growth imbalance (altered protein or RNA to DNA ratio) was observed in the case of tetraploid cells. The data show that, in the case of MOLT-4 cells, all events associated with the chromosome or DNA cycle were unaffected by SSP; the only target of the inhibitor appears to be kinase(s) controlling cytokinesis. © 1994 Wiley-Liss, Inc.     

In-vitro cytotoxic and genotoxic effects of arsenic trioxide on human leukemia (HL-60) cells using the MTT and alkaline single cell gel electrophoresis (Comet) assays

Although arsenic trioxide (ATO) has been the subject of toxicological research, in vitro cytotoxicity and genotoxicity studies using relevant cell models and uniform methodology are not well elucidated. Hence, the aim of the present study was to evaluate the cytotoxicity and genotoxicity induced by ATO in a human leukemia (HL-60) cell line using the MTT [3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] and alkaline single cell gel electrophoresis (Comet) assays, respectively. HL-60 cells were treated with different doses of ATO for 24 h prior to cytogenetic assessment. Data obtained from the MTT assay indicated that ATO significantly (P < 0.05) reduced the viability of HL-60 cells in a dose-dependent manner, showing a LD₅₀ value of 6.4 ± 0.6 μg/mL. Data generated from the comet assay also indicated a significant dose-dependent increase in DNA damage in HL-60 cells associated with ATO exposure. We observed a significant increase (P < 0.05) in comet tail-length, tail arm and tail moment, as well as in percentages of DNA cleavage at all doses tested, showing an evidence of ATO-induced genotoxic damage in HL-60 cells. This study confirms that the comet assay is a sensitive and effective method to detect DNA damage caused by heavy metals like arsenic. Taken together, our findings suggest that ATO exposure significantly (P < 0.05) reduces cellular viability and induces DNA damage in HL-60 cells as assessed by MTT and alkaline single cell gel electrophoresis assays, respectively.     

Single-strand breaks, cell cycle arrest and apoptosis in HL-60 and LLCPK1 cells exposed to 1,2-dibromo-3-chloropropane

We investigated 1,2-dibromo-3-chloropropane (DBCP)-induced DNA damage, cell cycle alterations and cell death in two cell lines, the human leukemia HL-60 and the pig kidney LLCPK₁, both of which are derived from potential target sites for DBCP-induced toxicity. DBCP (30–300 µmol/L) caused a concentration-dependent increase in the levels of DNA single-strand breaks in both cell lines as well as in cultured human renal proximal tubular cells. After extended DBCP exposure in LLCPK₁ cells (100 µmol/L, 30 h), the level of DNA breaks returned almost to control values. Incubation for 48 h showed a clear reduction of growth with DBCP concentrations as low as 10 µmol/L. Flow cytometric analysis showed that DBCP (1–10 µmol/L) exposure for 24 h caused an accumulation of LLCPK₁ cells in the G₂/M-phase. In HL-60 cells the accumulation in G₂/M-phase was less marked, and at higher concentrations the cells accumulated in S-phase. Flow cytometric studies of HL-60 and LLCPK₁ cells exposed to 100–500 µmol/L DBCP showed increased number of apoptotic cells/bodies with a lower DNA content than that of the G₁ cells. Microscopic studies revealed that there were increased numbers of cells with nuclear condensation and fragmentation, indicating that apoptosis was the dominant mode of death in these cell lines, following exposure to DBCP. The characteristic ladder pattern of apoptotic cells was observed when DNA from DBCP-treated HL-60 cells and LLCPK₁ cells was electrophoresed in agarose. The finding that DBCP can cause an accumulation of cells in G₂/M-phase and induce apoptosis in vitro may be of importance for the development of DBCP-induced toxicity in vivo.     

Regulation of platelet-derived growth factor gene expression by transforming growth factor beta and phorbol ester in human leukemia cell lines.

We studied the expression of the genes encoding the A and B chains of platelet-derived growth factor (PDGF) in a number of human leukemia cell lines. Steady-state expression of the A-chain RNA was seen only in the promonocytic leukemia cell line U937 and in the T-cell leukemia cell line MOLT-4. It has previously been reported that both PDGF A and PDGF B genes are induced during megakaryoblastic differentiation of the K562 erythroleukemia cells and transiently during monocytic differentiation of the promyelocytic leukemia cell line HL-60 and U937 cells. In this study we show that PDGF A RNA expression was induced in HL-60 and Jurkat T-cell leukemia cells and increased in U937 and MOLT-4 cells after a 1- to 2-h stimulation with an 8 pM concentration of transforming growth factor beta (TGF-beta). PDGF A RNA remained at a constant, elevated level for at least 24 h in U937 cells, but returned to undetectable levels within 12 h in HL-60 cells. No PDGF A expression was induced by TGF-beta in K562 cells or in lung carcinoma cells (A549). Interestingly, essentially no PDGF B-chain (c-sis proto-oncogene) RNA was expressed simultaneously with PDGF A. In the presence of TGF-beta and protein synthesis inhibitors, PDGF A RNA was superinduced at least 20-fold in the U937 and HL-60 cells. PDGF A expression was accompanied by secretion of immunoprecipitable PDGF to the culture medium of HL-60 and U937 cells. The phorbol ester tumor promoter tetradecanoyl phorbol acetate also increased PDGF A expression with similar kinetics, but with a mechanism distinct from that of TGF-beta. These results suggest a role for TGF-beta in the differential regulation of expression of the PDGF genes.     

Cytostatic and Cytotoxic Effects of Pannon (P-30 Protein), A Novel Anticancer Agent

P-30 Protein is a novel protein, of molecular weight approximately 15 kD, obtained from the extract of a vertebrate tissue showing in vivo antitumour activity. Cytostatic and cytotoxic effects of this product in its purified form (P-30 Protein) or in partially purified extracts (Pannon) were studied in vitro on human leukaemic HL-60, human submaxillary carcinoma A-253, human colon adenocarcinoma Colo 320 CM and murine erythroleukaemia (Friend leukaemia) cell lines. of these cells, HL-60, A-253 and Colo 320 CM were sensitive and Friend leukaemia resistant to this agent. the effects were time- and concentration-dependent. During the initial 24–48 h of treatment, a slowdown in cell proliferation was apparent but cell death was not extensive. After 24–48 h, there was a reduction in the proportion of cells in S phase of the cell cycle and the cells became preferentially arrested in G₁ phase. the G₁ cells showed high heterogeneity with respect to RNA content and some cells were characterized by very low RNA content. Progressive cell death occurred in cultures maintained with Pannon for up to 7 d in proportion to its concentration. Reductions of 50 and 90% in clonogenicity of A-253 cells were observed during their growth in the presence of 0.13 and 1.5 μg/ml of this protein, respectively. Exponentially growing cells were more sensitive to Pannon compared with cells from confluent cultures. Colonies of A-253 cells growing in the presence of Pannon were much smaller in size compared with control colonies, indicating that the rate of proliferation of clonogens is reduced by this agent. It appears that P-30 Protein induces cytostatic effects via modulation of cell transition to quiescence or differentiation. the mechanism of its cytotoxic activity is unclear.     

N-acetyl tryptophan glucopyranoside (NATG) provides radioprotection to murine macrophage J774A.1 cells

ABSTRACT

Ionizing radiation induced perturbations in cellular redox homeostasis are manifested as cell cycle arrest, inflammatory response, and apoptosis. Present study was focused on determination of radioprotective efficacy of a secondary metabolite N-acetyl-l-tryptophan glucoside (NATG) isolated from radioresistant bacterium Bacillus sp. INM-1. Macrophage J774A.1 cells were treated with NATG (0.025–200?μg/ml) before γ-irradiation (10-50?Gy) and radioprotective efficacy in terms of cellular metabolic activity was assessed using MTT assay. Radiation-induced intracellular ROS generation and its inhibition by NATG (0.25?μg/ml) pretreatment was evaluated using 2',7'-dichlorodihydrofluorescein diacetate (H₂DCFDA) probe. Effects of NATG pretreatment with or without γ-irradiation (20?Gy) on cell cycle perturbations and apoptosis regulation was evaluated using flowcytometry. Results of the study displayed 0.25 and 50?μg/ml as effective dose (ED) and lethal dose (LD₅₀) dose of NATG. A significant (p?<?0.05) decrease in DCFDA fluorescence corresponding to decreased ROS levels was observed in NATG pretreated irradiated cells as compared to irradiated alone cells. Cell cycle progression analysis demonstrated decrease (～10%) in G_1, G₂, and S phase cells at 24?h post-treatment time interval in NATG pretreated cells as compared to control group. However, radiation-induced or NATG-induced treatment did not cause any alterations in G₂/M arrest. APO-BrDU analysis demonstrated significant (p?<?0.05) reduction in apoptosis level at 24?h time interval in NATG pretreated irradiated cells as compared to alone irradiated cells. Conclusively, present study suggests that NATG offers radioprotection by apoptosis inhibition mechanism without altering cell-cycle progression in J774A.1 cells. Further studies to evaluate detailed molecular mechanisms of radioprotection offered by NATG are ongoing.     

Target ROS to induce apoptosis and cell cycle arrest by 5,7-dimethoxy-1,4-naphthoquinone derivative

The 1,4-naphthoquinone derivatives bearing 5,7-dimethoxyl moiety were designed, synthesized, and tested as the antitumor agents against five human cancer cell lines (A549, Hela, HepG2, NCI-H460 and HL-60). All the compounds are described herein for the first time. The structure-activity relationships indicated that the presence of chlorine atom at the 2-position was crucial for the antiproliferative activity. Further, the electrochemical properties of the representative compounds (7e, 8e and 9e) were evaluated and a definite correlation between the redox potential and the antiproliferative activity. The most potent compound 9e displayed significant anti-leukemic activity with IC₅₀ value of 3.8?μM in HL-60 cells and weak cytotoxicity with IC₅₀ of 40.7?μM in normal cells WI-38. In mechanistic study for 9e, the increased numbers of apoptotic cells and increased cell population at G2/M phase correlated with ROS generation. Together, our results suggested that the derivatives of 2-chlorine-1,4-naphthoquinone might be the promising candidates for the treatment of promyelocytic leukemia.     

Retinoic Acid-Induced blr1 Expression Promotes ERK2 Activation and Cell Differentiation in HL-60 Cells

Retinoids are known to induce the differentiation and cell cycle arrest of human myeloid leukemia cells in vitro. Differential display was used to identify putative early regulatory genes that are differentially expressed in HL-60 human promyelocytic leukemia cells treated with retinoic acid. One of the cDNAs cloned encodes sequences identifying Burkitt's lymphoma receptor 1 (BLR1), a recently described chemokine receptor. Northern blot analysis demonstrates that blr1 mRNA expression increases within 9 h of retinoic acid treatment, well before functional differentiation or G₁/G₀ growth arrest at 48 h or onset of morphological changes, suggesting a possible regulatory function. The expression of blr1 mRNA is transient, peaking at 72 h when cells are differentiated. blr1 mRNA also is induced by other differentiation-inducing agents, 1α,25-dihydroxyvitamin D₃ and DMSO. Induction of blr1 mRNA by retinoic acid is not blocked by the protein synthesis inhibitor cycloheximide. In HL-60 cells stably transfected with blr1 cDNA, ectopic expression of blr1 causes an increase in ERK2 MAPK activation and promotes retinoic acid-induced G₁/G₀ growth arrest and cell differentiation. The early expression of blr1 mRNA during differentiation, its ability to increase ERK2 activation, and its enhancement of retinoic acid-induced differentiation suggest that blr1 expression may be involved in retinoic acid-induced HL-60 differentiation.     

氟苯达唑对人急性髓系白血病HL-60细胞的增殖抑制作用研究

目的:研究氟苯达唑对人急性髓系白血病HL-60细胞增殖的抑制作用,明确氟苯达唑对HL-60细胞周期,凋亡发生的作用机制。方法:噻唑蓝法(MTT)检测氟苯达唑对人急性髓系白血病HL-60细胞的生长抑制作用,流式细胞术检测氟苯达唑对HL-60细胞周期,DNA片段化的影响,免疫印迹法检测Caspase, Raf, Bcl-2家族蛋白表达。结果:氟苯达唑抑制人急性髓系白血病HL-60细胞生长,HL-60细胞G2/M期增加,与阴性对照组相比,在一定的剂量和时间内,差别具有显著统计学意义;DNA片段化上升,0.25,0.5,1μM组与对照组相比差别具有显著统计学意义,促使Cleaved PARP,Cleaved-caspase 3,Cleaved-caspase 9蛋白表达量趋势增加;Bag-1和Bcl-2蛋白表达量降低;b-raf,c-raf磷酸化蛋白表达水平逐渐降低。结论:氟苯达唑通过诱导HL-60细胞阻滞于G2/M期,增加DNA片段化水平,激活Caspase, Raf, Bcl-2家族介导的凋亡相关通路抑制人急性髓系白血病HL-60细胞增殖,诱导人急性髓系白血病HL-60细胞发生凋亡而发挥抗肿瘤作用。     

Two new coumarins and a new xanthone from the leaves of Rhizophora mucronata

Two new coumarins (1, 2) and a new xanthone (3), together with 14 known compounds—eight coumarins (4, 5, 9, 10, 12–15), three xanthones (11, 16, 17), a benzoic acid (6) and two flavonones (7, 8)—were isolated from the leaves of Rhizophora mucronata. The structures of the compounds were elucidated by spectroscopic (IR, MS, and NMR) analyses. The isolated compounds were tested for cytotoxicity against human cancer cell lines HL-60 and HeLa. Among these compounds, only compound 16 inhibited the growth of both HeLa (IC₅₀?=?4.8?μM) and HL-60 (IC₅₀?=?1.0?μM) cells. Compounds 4, 7, 10, and 12 exhibited moderate activity against HeLa cells (IC₅₀?=?3.8–8.3?μM). Compounds 5, 9, 11, and 17 showed moderate activity against HL-60 cells (IC₅₀?=?2.2–6.3?μM). Higher selectivity against HL-60 cell lines was observed for compounds 5, 9, 11, and 16 with SI values (NIH 3T3/HL-60) of 8.6, 19.2, 9.4, and 10.2, respectively.     

Mycoplasmal lipoproteins induce toll-like receptor 2- and caspases-mediated cell death in lymphocytes and monocytes

Lipoproteins of Mycoplasma salivarium and Mycoplasma fermentans preferentially induced necrotic cell death in lymphocytic cell lines, MOLT-4 and Raji, and in one monocytic cell line, THP-1, whereas they preferentially induced apoptotic cell death in another monocytic cell line, HL-60. These findings were also supported by ultrastructural observations by the use of scanning and transmission electron microscopes and by agarose gel electrophoresis of the genomic DNA. The lipoproteins activated caspase-3 in both MOLT-4 and HL-60 cells, which was assessed by the cleavage of the synthetic substrate DEVD-pNA and the endogenous substrate poly(ADP-ribose) polymerase. The cytotoxicity to MOLT-4 and HL-60 cells was inhibited by various caspase inhibitors, Ac-DMQD-CHO, Ac-IETD-CHO, and Z-VAD-FMK. The cytotoxicity was also partially suppressed by the monoclonal antibody to Toll-like receptor 2. Thus this study demonstrated that mycoplasmal lipoproteins induce caspases-dependent necrotic and apoptotic cell death in lymphocytes and monocytes/macrophages, which is partially induced by TLR2-mediated signaling.     

Nuclear phosphoinositide 3-kinase C2β activation during G2/M phase of the cell cycle in HL-60 cells

The activity of nuclear phosphoinositide 3-kinase C2β (PI3K-C2β) was investigated in HL-60 cells blocked by aphidicolin at G₁/S boundary and allowed to progress synchronously through the cell cycle. The activity of immunoprecipitated PI3K-C2β in the nuclei and nuclear envelopes showed peak activity at 8 h after release from the G₁/S block, which correlates with G₂/M phase of the cell cycle. In the nuclei and nuclear envelopes isolated from HL-60 cells at 8 h after release from G₁/S block, a significant increase in the level of incorporation of radiolabeled phosphate into phosphatidylinositol 3-phosphate (PtdIns(3)P) was observed with no change in the level of radiolabeled PtdIns(4)P, PtdIns(4,5)P₂ and PtdIns(3,4,5)P₃. On Western blots, PI3K-C2β revealed a single immunoreactive band of 180 kDa, whereas in the nuclei and nuclear envelopes isolated at 8 h after release, the gel shift of 18 kDa was observed. When nuclear envelopes were treated for 20 min with μ-calpain in vitro, the similar gel shift and increase in PI3K-C2β activity was observed which was completely inhibited by pretreatment with calpain inhibitor calpeptin. The presence of PI3K inhibitor LY 294002 completely abolished the calpain-mediated increase in the activity of PI3K-C2β but did not prevent the gel shift. When HL-60 cells were released from G₁/S block in the presence of either calpeptin or LY 294002, the activation of nuclear PI3K-C2β was completely inhibited. These results demonstrate the calpain-mediated activation of the nuclear PI3K-C2β during G₂/M phase of the cell cycle in HL-60 cells.     

Expression of proliferation-associated nuclear autoantigens, p330d/CENP-F and PCNA, in differentiation and in drug-induced growth inhibition using two-parameter flow cytometry

p330^d/CENP-F is a recently described nuclear autoantigen that was detected in PHA-stimulated but not in resting peripheral lymphocytes. This protein accumulates in the nucleus during S-phase and reaches maximum levels during the G₂ and M phases of the cell cycles. We compared the expression of p330^d/CENP-F and proliferating cell nuclear antigen (PCNA) during the induction of terminal myeloid differentiation of HL-60 tumour cells. HL-60 cells were induced to differentiate with retinoic acid (RA), dimethyl sulfoxide (DMSO), and 3-nitrobenzothiazolo [3,2-]quinolinium (NBQ), and collected at different intervals. Control and treated cells were analyzed by two-parameter flow cytometry using propidium iodide and antibodies to p330^d/CENP-F and PCNA. The percentage of p330^d/CENP-F and PCNA positive cells was found to be proportional to the percentage of proliferating cells. After two cell cycles (65 h), the percentage of p330^d/CENP-F and PCNA positive cells was reduced proportionately to the number of cells that had differentiated. Reduction in the expression of both antigens was completed after 120 h when 80% to 85% of the cells were arrested in G₁ and displayed the mature phenotype. The expression of p330^d/CENP-F and PCNA was also assessed in the growth inhibition of HT-29 cells induced by various concentrations of camptothecin (CPT), etoposide (VP-16), and aphidicolin (APH). There was a dose-dependent displacement of cells to late S-phase by CPT while VP-16 induced cells to accumulate in G₂+ M, and as expected these effects caused a strong increase in the cellular levels of both antigens. The arrest of cells in G₁ by APH led to a significant decrease in their expression. The dramatic reduction in p330^d/CENP-F levels during differentiation, and the correlation of its expression with the cell cycle effects of the cytotoxic drugs are consistent with the behaviour expected for a proliferation marker.     

Design,synthesis, in vitro antiproliferative activity and apoptosis-inducing studies of 1-(3′,4′,5′-trimethoxyphenyl)-3-(2′-alkoxycarbonylindolyl)-2-propen-1-one derivatives obtained by a molecular hybridisation approach

Inhibition of microtubule function using tubulin targeting agents has received growing attention in the last several decades. The indole scaffold has been recognized as an important scaffold in the design of novel compounds acting as antimitotic agents. Indole-based chalcones, in which one of the aryl rings was replaced by an indole, have been explored in the last few years for their anticancer potential in different cancer cell lines. Eighteen novel (3′,4′,5′-trimethoxyphenyl)-indolyl-propenone derivatives with general structure 9 were synthesized and evaluated for their antiproliferative activity against a panel of four different human cancer cell lines. The highest IC₅₀ values were obtained against the human promyelocytic leukemia HL-60 cell line. This series of chalcone derivatives was characterized by the presence of a 2-alkoxycarbonyl indole ring as the second aryl system attached at the carbonyl of the 3-position of the 1-(3′,4′,5′-trimethoxyphenyl)-2-propen-1-one framework. The structure–activity relationship (SAR) of the indole-based chalcone derivatives was investigated by varying the position of the methoxy group, by the introduction of different substituents (hydrogen, methyl, ethyl or benzyl) at the N-1 position and by the activity differences between methoxycarbonyl and ethoxycarbonyl moieties at the 2-position of the indole nucleus. The antiproliferative activity data of the novel synthesized compounds revealed that generally N-substituted indole analogues exhibited considerably reduced potency as compared with their parent N-unsubstituted counterparts, demonstrating that the presence of a hydrogen on the indole nitrogen plays a decisive role in increasing antiproliferative activity. The results also revealed that the position of the methoxy group on the indole ring is a critical determinant of biological activity. Among the synthesized derivatives, compound 9e, containing the 2-methoxycarbonyl-6-methoxy-N-1H-indole moiety exhibited the highest antiproliferative activity, with IC₅₀ values of 0.37, 0.16 and 0.17?μM against HeLa, HT29 and MCF-7 cancer cell lines, respectively, and with considerably lower activity against HL-60 cells (IC₅₀: 18?μM). This derivative also displayed cytotoxic properties (IC₅₀ values ～1?μM) in the human myeloid leukemia U-937 cell line overexpressing human Bcl-2 (U-937/Bcl-2) via cell cycle progression arrest at the G₂-M phase and induction of apoptosis. The results obtained also demonstrated that the antiproliferative activity of this molecule is related to inhibition of tubulin polymerisation. The presence of a methoxy group at the C5- or C6-position of the indole nucleus, as well as the absence of substituents at the N-1-indole position, contributed to the optimal activity of the indole-propenone-3′,4′,5′-trimethoxyphenyl scaffold.     

IFI 16 gene encodes a nuclear protein whose expression is induced by interferons in human myeloid leukaemia cell lines

We have characterized the induction of mRNA and protein products of the human IFI 16 gene in response to IFN-γ, IFN-α, and IFN-β₂ (IL-6). We demonstrate that the IFI 16 gene product is a novel nucleoprotein expressed in association with the differentiation of myeloid precursor cell lines. In Northern blots, IFI 16 mRNA was increased ～25-fold above barely detectable levels in unstimulated promyelocytic HL-60 cells, in response to IFN-γ. Other myeloid cell lines, U937 and K562, also demonstrated a marked IFN-γ-inducibility of IFI 16 mRNA. However, all three cell lines were far less responsive to IFN-α, and there was no response to IL-6. By comparison, a panel of T and B cell lines demonstrated high constitutive expression of IFI 16 mRNA that was not regulated by these cytokines. Culture of HL-60 cells in medium containing dimethylsulfoxide, retinoic acid, and 1,25 dihydroxyvitamin D₃, agents that stimulate the differentiation of HL-60 along myeloid pathways, also caused the induction of IFI 16 mRNA. To characterize the protein product of IFI 16, a monoclonal antibody was raised against a recombinant bacterial protein comprising the amino terminal 159 amino acids of IFI 16 fused to glutathione S-transferase. The antibody, designated 1G7, was used in Western blotting to demonstrate the strong induction of a cluster of proteins of 85–95 kDa in the nuclear extracts of IFN-γ-treated HL-60. The nuclear localization of IFI 16 antigen was confirmed by immunohistochemical staining of HL-60 cells treated with IFN-γ, dimethylsulfoxide, and retinoic acid. IFI 16 was also detected in the nuclei of monocytes, neutrophils, and lymphocytes in normal peripheral blood. Database comparisons of the IFI 16 amino acid sequence revealed 51% identity with the recently cloned myeloid cell nuclear differentiation antigen (MNDA), and extensive similarity to protein products of the Gene 200 cluster of IFN-inducible genes, Ifi 202 and Ifi 204. The amino terminal domain of IFI 16 encodes a putative nuclear localization signal, ¹²⁴PGAQKRKK, which is strongly conserved in MNDA and 204. Nuclear IFI 16 was able to bind double-stranded DNA in vitro and exhibited a similar elution profile from DNA-cellulose as previously observed for MNDA and 204. Therefore, IFI 16 and MNDA are members of a novel family of human DNA-binding proteins whose expression is associated with myeloid cell differentiation induced by cytokines and chemical agents.     

Cell Cycle Arrest during Measles Virus Infection: a G0-Like Block Leads to Suppression of Retinoblastoma Protein Expression

One of the major mechanisms by which measles virus (MV) infection causes disease and death is suppression of the immune response. The nonresponsiveness of MV-infected human lymphocytes to mitogens and a partial block in the G₀/G₁ phase of the cell cycle observed in vitro is thought to reflect in vivo immunosuppression. In order to molecularly dissect MV-induced immunosuppression, we analyzed expression of surface activation markers and cell cycle-regulatory proteins in MV-infected human T lymphocytes. MV Edmonston (MV-Ed) could induce and maintain a high level of the early activation marker CD69 in the absence of proliferation. Expression of cyclins D3 and E, which positively control entry into S phase, was also significantly decreased. Analysis of inhibitors of progression into S phase showed that a high level of p27 was maintained in the G₀/G₁-blocked subpopulation of MV-Ed-infected cells compared to the proliferating MV-infected cells. Furthermore, cell cycle-related upregulation of retinoblastoma (Rb) protein synthesis did not occur in the MV-Ed-infected lymphocytes. Acridine orange staining, which distinguishes cells in G₀ from cells in G₁, showed that RNA levels were not upregulated following activation, which is consistent with cells remaining in a G₀ state. Although expression of surface activation markers indicated entry into the cycle, intracellular Rb and RNA levels suggested a quiescent state. These results indicate that MV can uncouple activation of T lymphocytes from transition of G₀ to G₁.