G1 Phase Accumulation Research Articles

HOXA6: A Novel Candidate Gene in AML.

Molecular classification of acute myeloid leukemia (AML) has identified several candidate genes that could potentially define prognosis and response to therapy. One such candidate, identified from microarray studies, is the Class I homeobox gene HOXA9. The HOX gene network encodes master regulators of developmental processes including hemopoiesis. To quantify the contribution of this network of genes in AML, we carried out specific RQ-PCR analysis on twenty-four de novo patient samples using a subset of genes (12 HOX and MEIS1) selected on the basis of their recently reported expression in AML. HOXA6 was ranked, as the most highly expressed gene (range 1 x 103 – 2 x 107 copies per 50 ng RNA), substantially higher than HOXA9 (see Table). Further analysis identified high expression of HOXA6 in both human myeloid cell lines and CD34+ enriched primary progenitors. Parallel studies with murine progenitors (c-Kit+, Lin−) and cell lines also showed a preponderance of Hoxa6 expression over other family members including Hoxa9 and Hoxb4. Several hemopoietic cell lines, namely Ba/F3, EML, FDCP-Mix A4 and 32Dcl3 were subsequently used to investigate Hoxa6 regulation following differentiation or growth factor stimuli. Hoxa6 expression decreased with cell differentiation and growth factor depletion/replenishment studies indicated a cell-cycle component for Hoxa6 regulation. Direct evaluation of cell-cycle status, using Hoechst 33342 staining and cell sorting, identified peak expression of Hoxa6 during S-phase. Gene deletion studies involving Hox tend to result in either a moderate or no phenotype, presumably due to intrinsic compensatory mechanisms. We therefore overexpressed HOXA6 in the Ba/F3 cell line to gain functional insights. Ba/F3-A6 cells were compared to mock-transfected and vector controls on the basis of proliferation, maturation, cell-cycle status, growth factor-dependence and apoptosis. The Ba/F3-A6 cells displayed a growth advantage over normal cells in the presence of IL-3 and maturation was not impaired. Cell-cycle analysis showed a reduction in the number of cells in both G2M and S-phase, associated with accumulation in the pre G1-phase, indicative of increased apoptosis. IL-3 depletion studies of Ba/F3-A6 cells indicated substantial factor-independent growth compared to controls, implying oncogenic potential for HOXA6. In support of this, a recent report (Mamo et al, Blood. 2006 Jul 15;108(2):622–9) indicated Hoxa6 as a potential collaborator in a Meis1-induced model of AML. Taken together these findings identify Hoxa6 as a novel candidate gene in AML with the capacity to alter growth and survival of hemopoietic cells.Gene Expression Ranking of HOX and MEIS1 in AML.GENEEXPRESSION RANGEMEAN RANKS.D.OVERALL RANKHOXA61.2 x 103 – 1.7 x 1072.21.61HOXB39.3 x 101 – 8.4 x 1063.22.52HOXB2*7.9 x 102 – 5.4 x 1063.42.03HOXA94.0 x 101 – 5.3 x 1065.32.44MEIS10.6 x 101 – 8.4 x 1065.42.75HOXA10*2.4 x 102 – 1.7 x 1055.53.26HOXB41.5 x 102 – 7.8 x 1055.53.27HOXA7*5.3 x 103 – 1.8 x 1065.71.78HOXB62.3 x 101 – 8.8 x 1056.62.89HOXA44.1 x 101 – 1.1 x 1057.93.410HOXA5*3.4 x 101 – 4.3 x 1049.32.811HOXC61.0 x 101 – 3.2 x 1039.72.312HOXA11*4.0 x 101 – 6.1 x 10310.62.213Expression values (copies per 50 ng RNA) compiled from primary AML patient samples (n=24) or * (n=12). S.D = standard deviation.

Oncostatin M Induces Growth Arrest by Inhibition of Skp2, Cks1, and Cyclin A Expression and Induced p21 Expression

Oncostatin M has been characterized as a potent growth inhibitor for various tumor cells. Oncostatin M-treated glioblastoma cells cease proliferation and instigate astrocytal differentiation. The oncostatin M-induced cell cycle arrest in G(1) phase is characterized by increased level of the cyclin-dependent kinase (CDK) inhibitory proteins p21(Cip1/Waf1/Sdi1) and p27(Kip1). Induction of p21 protein corresponds to increased mRNA level, whereas p27 accumulates due to increased stability of the protein. Interestingly, stabilization of p27(Kip1) occurs even in S phase, showing that p27 stabilization is a direct consequence of oncostatin M signaling and not a result of the cell cycle arrest. Degradation of p27 in late G(1) and S phase is initiated by the ubiquitin ligase complex SCF-Skp2/Cks1. Oncostatin M inhibits expression of two components of this E3 ligase complex (Skp2 and Cks1). Although combined overexpression of Skp2 and Cks1 rescues p27 degradation in S phase, it can not override p27 accumulation in G(1) phase and cell cycle arrest by oncostatin M. In addition to increasing Cdk inhibitor level, oncostatin M also impairs cyclin A expression. Cyclin A mRNA and protein level decline shortly after oncostatin M addition. The accumulation of two CDK inhibitor proteins and the repression of cyclin A expression may explain the broad and potent antiproliferative effect of the cytokine.

Combined low dose ionizing radiation and green tea-derived epigallocatechin-3-gallate treatment induces human brain endothelial cells death

The microvasculature of brain tumors has been proposed as the primary target for ionizing radiation (IR)-induced apoptosis. However, the contribution of low dose IR-induced non-apoptotic cell death pathways has not been investigated. This study aimed to characterize the effect of IR on human brain microvascular endothelial cells (HBMEC) and to assess the combined effect of epigallocatechin-3-gallate (EGCg), a green tea-derived anti-angiogenic molecule. HBMEC were treated with EGCg, irradiated with a sublethal (< or =10 Gy) single dose. Cell survival was assessed 48 h later by nuclear cell counting and Trypan blue exclusion methods. Cell cycle distribution and DNA fragmentation were evaluated by flow cytometry (FC), cell death was assessed by fluorimetric caspase-3 activity, FC and immunoblotting for pro-apoptotic proteins. While low IR doses alone reduced cell survival by 30%, IR treatment was found more effective in EGCg pretreated-cells reaching 70% cell death. Analysis of cell cycle revealed that IR-induced cell accumulation in G2-phase. Expression of cyclin-dependent kinase inhibitors p21(CIP/Waf1) and p27(Kip) were increased by EGCg and IR. Although random DNA fragmentation increased by approximately 40% following combined EGCg/IR treatments, the synergistic reduction of cell survival was not related to increased pro-apoptotic caspase-3, caspase-9 and cytochrome C proteins. Cell necrosis increased 5-fold following combined EGCg/IR treatments while no changes in early or late apoptosis were observed. Our results suggest that the synergistic effects of combined EGCg/IR treatments may be related to necrosis, a non-apoptotic cell death pathway. Strategies sensitizing brain tumor-derived EC to IR may enhance the efficacy of radiotherapy and EGCg may represent such a potential agent.

Cell cycle and cell death regulation of neural progenitor cells in the 5-azacytidine (5AzC)-treated developing fetal brain

In the developing brain, neural progenitor cells are susceptible to many extrinsic stresses, including DNA damage. We treated pregnant rats with 5-azacytidine (5AzC), a DNA demethylating and damaging agent, to investigate the cellular responses of the fetal brain, focusing on the regulation of proliferation and cell death. 5AzC first induced the accumulation of cells in abnormal mitosis, G2-phase accumulation, and then apoptosis of the neural progenitor cells. Most of the apoptotic cells were in G1 phase. Cell cycle transition studies suggested that G2/M progression was blocked, after which the cells moved to G1 phase or underwent apoptosis. p53, a key factor for response to DNA damage, and some of its target genes showed increased expression in Western blot and DNA microarray analyses. In 5AzC-treated fetal brains of p53-deficient mice, apoptosis did not occur, although G2/M accumulation was induced. These results suggest that, in the developing brain, apoptosis is p53-dependent but that another mechanism governs the G2/M checkpoint. The G2/M regulator, Cdc2, was activated by dephosphorylation through G2/M accumulation, suggesting accelerated entry into mitosis leading to accumulation of cells showing abnormal mitosis. Furthermore, some cells may have died due to mitotic catastrophe. Throughout brain development, various cell cycle and cell death regulation mechanisms provide neural progenitor cells with options for defense from DNA damage.

Increased Levels of the Heat Shock Protein (hsp) 70 Contribute as a Pro-Survival and Pro-Growth Mechanism in the Transformation of Bone Marrow Progenitor Cells.

Hsp70 is an ATP-dependent molecular chaperone that assists in the folding of native proteins into active conformation and prevents aggregation of misfolded and mutated abnormal proteins. In normal non-transformed cells, the expression of hsp70 is low and largely stress-inducible due to misfolded and denatured proteins. Recent studies in our laboratory have demonstrated that human acute leukemia cells abundantly express hsp70, which exerts strong antiapoptotic effects upstream and downstream of the mitochondria. Additionally, as compared to the controls, the mouse myeloid 32D or BaF3 cells transformed by Bcr-Abl or FLT-3 also show increased expression of hsp70. To further elucidate the pro-survival and pro-growth effects of hsp70 and its role in the leukemia transformation, we created stable hsp70 transfectants of the IL-3-dependent 32D and BaF3 (normally maintained in culture in IL-3 containing 10% WEHI medium), i.e., 32D/hsp70 and BaF3/hsp70 cells. These cells displayed 3 to 5 fold higher levels of hsp70, as compared to the control 32D or BaF3 cells. Both 32D/hsp70 and BaF3/hsp70 cells showed significantly improved growth and survival supported by 10% WEHI medium. Following culture in 0%, and less so in 1%, WEHI medium for 24 hours, 32D and BaF3 cells undergo cell cycle G1 phase accumulation, with corresponding decline in the % of cells in the S phase. Following this exposure, they also show markedly increased apoptosis and loss of clonogenic survival, as determined by the colony growth assays in methylcellulose. In contrast, under similar conditions of exposure to reduced % of WEHI conditioned medium, 32D/hsp70 and BaF3/hsp70 cells displayed significantly less accumulation in G1 phase, as well as reduced loss of clonogenic survival and apoptosis (p<0.05). This was also associated with reduced loss of mitochondrial membrane potential and increased accumulation of reactive oxygen species. Notably, following IL-3 withdrawal, exposure to 10 ng/ml of G-CSF for 72 hours induced significantly less differentiation of 32D/hsp70 versus 32D cells, as determined by increase in the % of cells expressing CD11b and GR1 (determined by specific antibody staining and flow cytometry) or by evaluation of the morphologic features of differentiation (p<0.05). Western blot analyses demonstrated that both 32D/hsp70 and BaF3/hsp70 cells, compared to their controls, possessed significantly higher expression of IL-3β receptor (R) and pSTAT5. Importantly, the supernatants of hsp70 overexpressing cells, compared to their controls, also showed higher levels of IL-3, as detected by an ELISA. In addition, BaF3/hsp70, versus the control cells, showed increased DNA binding activity and transactivation by the AP1 transcription factor, utilizing a protein/DNA binding array assay (Panomics, Redwood City, CA) and AP-1-luciferase cis-reporting analysis (Stratagene, La Jolla, CA), respectively. These findings strongly suggest that increased hsp70 levels confer a growth and survival advantage through an IL3- IL-3βR-STAT5-dependent mechanism in the marrow progenitor cells, which may contribute to the transformation induced by leukemia associated oncoproteins.

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In Cryptococcus neoformans the DNA content of cells having tiny buds varied rather widely, depending on growth phases and strains used. Typically, buds of C. neoformans emerged soon after initiation of DNA synthesis in the early exponential phase. However, bud emergence was delayed to G2 during transition to the stationary phase, and in the early stationary phase budding scarcely occurred, although roughly half of the cells completed DNA synthesis. The timing of budding in C. neoformans was shifted to later cell cycle points with progression of the growth phase of the culture. Similarly, a deficit in oxygen was demonstrated to delay the timing of budding, prolong the G2 phase and cause accumulation of cells after DNA synthesis, but before commitment to budding. The C. neoformans homologue of the main cell cycle control gene CDC28/Cdc2 was isolated using degenerate RT-PCR. The full-length coding region was then amplified using primers to target the regions around the start and stop codons. The gene was called CnCdk1 and was found to have high homologies to S. cerevisiae CDC28 and S. pombe cdc2. To determine its function, its ability to rescue S. cerevisiae cdc28-temperature sensitive mutants was tested. S. cerevisiae cdc28-4 and cdc28-1N strains transformed with the pYES2-CnCdk1 construct exhibited growth at the restrictive temperature. Results of the sequence analysis and the ability of CnCdk1 to complement the S. cerevisiae cdc28-ts mutations support its assumed role as the CDC28/cdc2 homologue in C. neoformans.

Lectins as Bioactive Plant Proteins: A Potential in Cancer Treatment

Plant lectins, a unique group of proteins and glycoproteins with potent biological activity, occur in foods like wheat, corn, tomato, peanut, kidney bean, banana, pea, lentil, soybean, mushroom, rice, and potato. Thus, dietary intakes by humans can be significant. Many lectins resist digestion, survive gut passage, and bind to gastrointestinal cells and/or enter the circulation intact, maintaining full biological activity. Several lectins have been found to possess anticancer properties in vitro, in vivo, and in human case studies; they are used as therapeutic agents, preferentially binding to cancer cell membranes or their receptors, causing cytotoxicity, apoptosis, and inhibition of tumor growth. These compounds can become internalized into cells, causing cancer cell agglutination and/or aggregation. Ingestion of lectins also sequesters the available body pool of polyamines, thereby thwarting cancer cell growth. They also affect the immune system by altering the production of various interleukins, or by activating certain protein kinases. Lectins can bind to ribosomes and inhibit protein synthesis. They also modify the cell cycle by inducing non-apoptotic G1-phase accumulation mechanisms, G2/M phase cell cycle arrest and apoptosis, and can activate the caspase cascade. Lectins can also downregulate telomerase activity and inhibit angiogenesis. Although lectins seem to have great potential as anticancer agents, further research is still needed and should include a genomic and proteomic approach.

Fucoidan Extracted From Cladosiphon Okamuranus Tokida Induces Apoptosis of Human T-Cell Leukemia Virus Type 1-Infected T-Cell Lines and Primary Adult T-Cell Leukemia Cells

: Adult T-cell leukemia (ATL) is caused by human T-cell leukemia virus type 1 (HTLV-1) and remains incurable. The highest endemic area of HTLV-1 carriers in Japan is located in Okinawa, and novel treatments are urgently needed in this area. We extracted fucoidan, a sulfated polysaccharide, from the brown seaweed Cladosiphon okamuranus Tokida cultivated in Okinawa, Japan and examined its tumor-suppression activity against ATL. Fucoidan significantly inhibited the growth of peripheral blood mononuclear cells of ATL patients and HTLV-1-infected T-cell lines but not that of normal peripheral blood mononuclear cells. Fucoidan induced apoptosis of HTLV-1-infected T-cell lines mediated through downregulation of cellular inhibitor of apoptosis protein-2 and survivin and G1 phase accumulation through the downregulation of cyclin D2, c-myc, and hyperphosphorylated form of the retinoblastoma tumor suppressor protein. Further analysis showed that fucoidan inactivated NF-κB and activator protein-1 and inhibited NF-κB-inducible chemokine, C-C chemokine ligand 5 (regulated on activation, normal T expressed and secreted) production, and homotypic cell-cell adhesion of HTLV-1-infected T-cell lines. In vivo use of fucoidan resulted in partial inhibition of growth of tumors of an HTLV-1-infected T-cell line transplanted subcutaneously in severe combined immune deficient mice. Our results indicate that fucoidan is a potentially useful therapeutic agent for patients with ATL.

Anti-gene peptide nucleic acid specifically inhibits MYCN expression in human neuroblastoma cells leading to cell growth inhibition and apoptosis

We developed an anti-gene peptide nucleic acid (PNA) for selective inhibition of MYCN transcription in neuroblastoma cells, targeted against a unique sequence in the antisense DNA strand of exon 2 of MYCN and linked at its NH(2) terminus to a nuclear localization signal peptide. Fluorescence microscopy showed specific nuclear delivery of the PNA in six human neuroblastoma cell lines: GI-LI-N and IMR-32 (MYCN-amplified/overexpressed); SJ-N-KP and NB-100 (MYCN-unamplified/low-expressed); and GI-CA-N and GI-ME-N (MYCN-unamplified/unexpressed). Antiproliferative effects were observable at 24 hours (GI-LI-N, 60%; IMR-32, 70%) and peaked at 72 hours (GI-LI-N, 80%; IMR-32, 90%; SK-N-KP, 60%; NB-100, 50%); no reduction was recorded for GI-CA-N and GI-ME-N (controls). In MYCN-amplified/overexpressed IMR-32 cells and MYCN-unamplified/low-expressed SJ-N-KP cells, inhibition was recorded of MYCN mRNA (by real-time PCR) and N-Myc (Western blotting); these inhibitory effects increased over 3 days after single treatment in IMR-32. Anti-gene PNA induced G(1)-phase accumulation (39-53%) in IMR-32 and apoptosis (56% annexin V-positive cells at 24 hours in IMR-32 and 22% annexin V-positive cells at 48 hours in SJ-N-KP). Selective activity of the PNA was shown by altering three point mutations, and by the observation that an anti-gene PNA targeted against the noncoding DNA strand did not exert any effect. These findings could encourage research into development of an anti-gene PNA-based tumor-specific agent for neuroblastoma (and other neoplasms) with MYCN expression.

Gene Expression in Normal Human Bronchial Epithelial (NHBE) Cells Following In Vitro Exposure to Cigarette Smoke Condensate

Cigarettes that burn tobacco produce a complex mixture of chemicals, including mutagens and carcinogens. Cigarettes that primarily heat tobacco produce smoke with marked reductions in the amount of mutagens and carcinogens and demonstrate reduced mutagenicity and carcinogenicity in a battery of toxicological assays. Chemically induced oxidative stress, DNA damage, and inflammation may alter cell cycle regulation and are important biological events in the carcinogenic process. The objective of this study was to characterize and compare the effects of smoke condensates from cigarettes that burn tobacco and those that primarily heat tobacco on gene expression in NHBE cells. For this comparison, we used quantitative RT/PCR and further evaluated the effects on cell cycling using flow cytometry. Cigarette smoke condensates (CSCs) were prepared from Kentucky 1R4F cigarettes (a tobacco-burning product designed to represent the average full-flavor, low "tar" cigarette in the US market) and Eclipse (a cigarette that primarily heats tobacco) using FTC machine smoking conditions. The CSC from 1R4F cigarettes induced statistically significant increases in the mRNA levels of genes responsive to DNA damage (GADD45) and involved in cell cycle regulation (p21;WAF1/CIP1), compared to the CSC from Eclipse cigarettes. In addition, genes coding for cyclooxygenase-2 (COX-2) and interleukin 8 (IL-8), which are associated with oxidative stress and inflammation, respectively, were increased statistically significantly more by CSC from 1R4F than by that from Eclipse. Furthermore, a dose-dependent increase in IL-8 protein secretion into cell culture media was stimulated by 1R4F exposure, whereas minimal IL-8 protein was secreted after Eclipse treatment. The biological relevance of the differential effect on gene expression was reflected in differential cell cycle regulation, as cells exposed to 1R4F CSC exhibited more significant S phase and G2 phase accumulation than cells exposed to Eclipse CSC. These data indicate that the simplified smoke chemistry of the tobacco-heating Eclipse cigarette yields statistically significant reductions in the expression of key genes involved in DNA damage, oxidative stress, inflammatory response, and cell cycle regulation in normal human bronchial epithelial cells compared to a representative tobacco-burning cigarette.

Combination of the histone deacetylase inhibitor LBH589 and the hsp90 inhibitor 17-AAG is highly active against human CML-BC cells and AML cells with activating mutation of FLT-3

AbstractPresent studies show that LBH589, a novel cinnamic hydroxamic acid analog histone deacetylase inhibitor, induces acetylation of histone H3 and H4 and of heat shock protein 90 (hsp90), increases p21 levels, as well as induces cell-cycle G1 phase accumulation and apoptosis of the human chronic myeloid leukemia blast crisis (CML-BC) K562 cells and acute leukemia MV4-11 cells with the activating length mutation of FLT-3. In MV4-11 cells, this was associated with marked attenuation of the protein levels of p-FLT-3, FLT-3, p-AKT, and p-ERK1/2. In K562 cells, exposure to LBH589 attenuated Bcr-Abl, p-AKT, and p-ERK1/2. Treatment with LBH589 inhibited the DNA binding activity of signal transducers and activators of transcription 5 (STAT5) in both K562 and MV4-11 cells. The hsp90 inhibitor 17-allyl-amino-demethoxy geldanamycin (17-AAG) also induced polyubiquitylation and proteasomal degradation of FLT-3 and Bcr-Abl by reducing their chaperone association with hsp90. Cotreatment with LBH589 and 17-AAG exerted synergistic apoptosis of MV4-11 and K562 cells. In the imatinib mesylate (IM)-refractory leukemia cells expressing Bcr-Abl with the T315I mutation, treatment with the combination attenuated the levels of the mutant Bcr-Abl and induced apoptosis. Finally, cotreatment with LBH589 and 17-AAG also induced more apoptosis of IM-resistant primary CML-BC and acute myeloid leukemia (AML) cells (with activating mutation of FLT-3) than treatment with either agent alone. (Blood. 2005;105:1768-1776)

Anticancer effects of the novel 1α, 25-dihydroxyvitamin D3 hybrid analog QW1624F2-2 in human neuroblastoma

Vitamin D3 analogs are potential anti-cancer agents with theoretically wide therapeutic index, but there have been limited studies directed towards human neuroblastoma. The antiproliferative ability of the novel vitamin D3 hybrid analog QW-1624F2-2 (QW, 1-hydroxymethyl-16-ene-24, 24-F2-26, 27-bishomo-25-hydroxyvitamin D3) was examined in two human neuroblastoma-derived cell-lines. Analog QW inhibited cell-cycle progression of IMR5 cells with accumulation in G1 phase. QW induced the differentiation of CHP134 as evidenced by increased neurite length. These effects were accompanied by decreased expression of MYCN in both the cell-lines treated with QW. Furthermore, QW inhibited the migration of CHP134 cells in matrigel invasion assays, indicating its anti-invasive ability. In athymic nude mice, we found that QW was less calcemic than EB1089 (1alpha, 25-dihydroxy-22, 24-diene-24, 26,27-trishomovitamin D3). Systemic administration of QW in a mouse xenotransplantation model revealed that it is more effective than EB1089 in suppressing the growth of CHP134 flank tumors. In summary, the low-calcemic hybrid analog QW showed significant anti-tumor activity in vivo and thus exhibits potential as a novel cancer therapeutic.

Synergistic Cytotoxic Effects of a Combination of a Novel Tyrosine Kinase Inhibitor AMN107 and Histone Deacetylase Inhibitor LBH589 Against Bcr-Abl Expressing Human Leukemia Cells.

AMN107 (Novartis Pharmaceuticals, Basel, Switzerland) is a potent aminopyrimidine tyrosine kinase (TK) inhibitor, which is active at nanomolar concentrations against c-Kit, PDGFR as well as the wild type Bcr-Abl and common variety of mutant Bcr-Abl (e.g., M351T, F317L and E255V) TK. In a previous report, we demonstrated that treatment with the hydroxamic acid analogue histone deacetylase inhibitor LBH589 (Novartis) (20 to 100 nM) induces hsp90 acetylation and promotes polyubiquitylation and proteasomal degradation of Bcr-Abl, which is associated with apoptosis of the cultured Bcr-Abl expressing human chronic myeloid leukemia-blast crisis (CML-BC) K562 and LAMA-84 cells, as well as of primary CML-BC cells. In the present studies, we determined the cell cycle and apoptotic effects of AMN107 and/or LBH589 in K562, LAMA-84 and primary CML-BC cells. Treatment with AMN107 (20 to 100 nM) induced cell cycle G1 phase accumulation and apoptosis, exerting 10 to 20 fold more potent effect than imatinib mesylate (IM) in K562 and LAMA-84 cells. This was associated with marked induction of p27, inhibition of Bcr-Abl TK activity, as well as attenuation of the levels of p-STAT5, p-AKT, Bcl-xL, and c-Myc levels in a dose dependent manner. Co-treatment with LBH589 (50 nM) and AMN107 (100 nM) induced more G1 phase accumulation than either agent alone. The combination of AMN107 and LBH589 also exerted synergistic apoptotic effects in K562 cells, as determined by the median effect isobologram analysis. This was associated with more attenuation of the levels of pro-growth and pro-survival proteins, e.g., p-STAT5, p-ERK1/2, c-Myc and Bcl-xL, as well as greater induction of the levels of pro-death p27 and Bim proteins. Treatment of human AML HL-60 cells containing ectopic expression of the IM-refractory, mutant Bcr-AblT315I with LBH589 (50 nM for 24 hours) attenuated the mutant Bcr-Abl levels and induced apoptosis of HL-60/Bcr-AblT315I cells. Treatment with AMN107 (up to 2.0 μM) alone was ineffective in inducing loss of viability of HL-60/Bcr-AblT315I cells, and co-treatment with LBH589 (50 nM) and AMN107 did not induce more loss of cell viability of HL-60/Bcr-AblT315I cells than treatment with LBH589 alone. AMN107 levels ≥ 200 nM significantly inhibited survival of HL-60/Bcr-AblE255K and HL-60/Bcr-AblM351T cells. Consistent with this, the combined treatment with LBH589 and AMN107 also induced more loss of viability than treatment with either agent alone in 4 samples of IM-refractory primary CML-BC cells. These studies demonstrate that both AMN107 and LBH589 are active against IM-resistant CML-BC and the combination of the two exerts superior activity against IM-sensitive and IM-resistant, wild type and mutant Bcr-Abl expressing cultured and primary CML cells.

A combination of histone deacetylase inhibitor LBH589 and the hsp90 inhibitor 17-AAG is highly active against human CML-BC and AML cells with constitutively active mutant FLT-3 tyrosine kinase

6541 Background: We have reported that treatment with histone deacetylase inhibitors, LAQ824 and LBH589 (Novartis Pharma AG), and hsp90 inhibitor 17-AAG attenuates the levels of Bcr-Abl and FLT-3, as well induces apoptosis of human CML-BC and AML cells. In these studies we determined the antileukemia activity of the combination of LBH589 and 17-AAG. Methods: K562 (CML-BC), MV4–11 (AML cells with the activating length mutation of FLT-3) and primary AML (with FLT-3 mutation) were exposed to 0.1 to 2.0 uM 17-AAG and/or 5 to 100 nM LBH589 for 24 to 48 hours. Following this, the cell cycle status and apoptosis (by flow cytometry), as well as the expressions of p21, hsp70, p-FLT-3, FLT-3, p-AKT, p-ERK1/2 and p-STAT5 (by Western analyses) were determined. Results: LBH589 treatment induced p21 levels and mediated cell cycle G1 phase accumulation as well as apoptosis of MV4–11 and K562 cells. In MV4–11 cells, this was associated with marked attenuation of the protein levels of p-FLT-3, FLT-3, p-AKT, p-ERK1/2 and p-STAT5, as well as the DNA binding activity of STAT5. Furthermore, co-treatment with LBH589 and the FLT-3 kinase inhibitor PKC412 (Novartis Pharma AG) exerted synergistic cytotoxic effect against MV4–11 cells. In K562 cells, exposure to LBH589 attenuated Bcr-Abl, p-AKT, p-ERK1/2 and p-STAT5. Treatment with 17-AAG or LBH589 inhibited the chaperone association of hsp90 with FLT-3 and Bcr-Abl in MV4–11 and K562 cells, respectively, resulting in polyubiquitylation and proteasomal degradation of FLT-3 and Bcr-Abl. Significantly, co-treatment with LBH589 and 17-AAG induced more apoptosis of MV4–11 and K562 cells that either agent alone. The combination also attenuated the levels of mutant T315I Bcr-Abl and induced apoptosis of Gleevec-refractory primary CML-BC,as well as induced more apoptosis of primary AML cells with FLT-3 mutations. Conclusions: These studies demonstrate that the combination of LBH589 and 17-AAG may be highly effective in attenuating p-FLT-3 and Bcr-Abl, as well as inducing apoptosis of CML-BC and AML cells with FLT-3 mutations. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Novartis Corp. Novartis Corp.

A combination of histone deacetylase inhibitor LBH589 and the hsp90 inhibitor 17-AAG is highly active against human CML-BC and AML cells with constitutively active mutant FLT-3 tyrosine kinase

6541 Background: We have reported that treatment with histone deacetylase inhibitors, LAQ824 and LBH589 (Novartis Pharma AG), and hsp90 inhibitor 17-AAG attenuates the levels of Bcr-Abl and FLT-3, as well induces apoptosis of human CML-BC and AML cells. In these studies we determined the antileukemia activity of the combination of LBH589 and 17-AAG. Methods: K562 (CML-BC), MV4–11 (AML cells with the activating length mutation of FLT-3) and primary AML (with FLT-3 mutation) were exposed to 0.1 to 2.0 uM 17-AAG and/or 5 to 100 nM LBH589 for 24 to 48 hours. Following this, the cell cycle status and apoptosis (by flow cytometry), as well as the expressions of p21, hsp70, p-FLT-3, FLT-3, p-AKT, p-ERK1/2 and p-STAT5 (by Western analyses) were determined. Results: LBH589 treatment induced p21 levels and mediated cell cycle G1 phase accumulation as well as apoptosis of MV4–11 and K562 cells. In MV4–11 cells, this was associated with marked attenuation of the protein levels of p-FLT-3, FLT-3, p-AKT, p-ERK1/2 and p-STAT5, as well as the DNA binding activity of STAT5. Furthermore, co-treatment with LBH589 and the FLT-3 kinase inhibitor PKC412 (Novartis Pharma AG) exerted synergistic cytotoxic effect against MV4–11 cells. In K562 cells, exposure to LBH589 attenuated Bcr-Abl, p-AKT, p-ERK1/2 and p-STAT5. Treatment with 17-AAG or LBH589 inhibited the chaperone association of hsp90 with FLT-3 and Bcr-Abl in MV4–11 and K562 cells, respectively, resulting in polyubiquitylation and proteasomal degradation of FLT-3 and Bcr-Abl. Significantly, co-treatment with LBH589 and 17-AAG induced more apoptosis of MV4–11 and K562 cells that either agent alone. The combination also attenuated the levels of mutant T315I Bcr-Abl and induced apoptosis of Gleevec-refractory primary CML-BC,as well as induced more apoptosis of primary AML cells with FLT-3 mutations. Conclusions: These studies demonstrate that the combination of LBH589 and 17-AAG may be highly effective in attenuating p-FLT-3 and Bcr-Abl, as well as inducing apoptosis of CML-BC and AML cells with FLT-3 mutations. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Novartis Corp. Novartis Corp.

Cotreatment with 17-Allylamino-Demethoxygeldanamycin and FLT-3 Kinase Inhibitor PKC412 Is Highly Effective against Human Acute Myelogenous Leukemia Cells with Mutant FLT-3

Presence of the activating length mutation (LM) in the juxtamembrane domain or point mutation in the kinase domain of FMS-like tyrosine kinase-3 (FLT-3) mediates ligand-independent progrowth and prosurvival signaling in approximately one-third of acute myelogenous leukemia (AML). PKC412, an inhibitor of FLT-3 kinase activity, is being clinically evaluated in AML. Present studies demonstrate that treatment of human acute leukemia MV4-11 cells (containing a FLT-3 LM) with the heat shock protein 90 inhibitor 17-allylamino-demethoxy geldanamycin (17-AAG) attenuated the levels of FLT-3 by inhibiting its chaperone association with heat shock protein 90, which induced the poly-ubiquitylation and proteasomal degradation of FLT-3. Treatment with 17-AAG induced cell cycle G(1) phase accumulation and apoptosis of MV4-11 cells. 17-AAG-mediated attenuation of FLT-3 and p-FLT-3 in MV4-11 cells was associated with decrease in the levels of p-AKT, p-ERK1/2, and p-STAT5, as well as attenuation of the DNA binding activity of STAT-5. Treatment with 17-AAG, downstream of STAT5, reduced the levels of c-Myc and oncostatin M, which are transactivated by STAT5. Cotreatment with 17-AAG and PKC412 markedly down-regulated the levels of FLT-3, p-FLT-3, p-AKT, p-ERK1/2, and p-STAT5, as well as induced more apoptosis of MV4-11 cells than either agent alone. Furthermore, the combination of 17-AAG and PKC412 exerted synergistic cytotoxic effects against MV4-11 cells. Importantly, 17-AAG and PKC412 induced more loss of cell viability of primary AML blasts containing FLT-3 LM, as compared with those that contained wild-type FLT-3. Collectively, these in vitro findings indicate that the combination of 17-AAG and PKC412 has high level of activity against AML cells with FLT-3 mutations.

Gefitinib (‘Iressa’, ZD1839), an epidermal growth factor receptor tyrosine kinase inhibitor, up-regulates p27 KIP1 and induces G1 arrest in oral squamous cell carcinoma cell lines

High expression of epidermal growth factor receptor (EGFR) is frequently observed in many solid tumor types including oral squamous cell carcinomas (OSCC). Recently, the results of preclinical studies and early clinical trials targeting the EGFR have shown evidence of the activity. In this study, gefitinib (‘Iressa’, ZD1839), an EGFR-tyrosine kinase inhibitor, inhibited cell proliferation and upregulated p27 KIP1 in OSCC cells. Growth inhibition was observed in OSCC xenografts when mice were treated with gefitinib in vivo. A flow cytometric analysis demonstrated that treatment with gefitinib induced accumulation in G1 phase, accompanied by a decrease in the percentage of cells in S phase. Apoptosis was not seen in this study. Cell growth was inhibited by an increase of the cell cycle inhibitor p27 KIP1 and a decrease of its ubiquitin ligase subunit Skp2.

Anti-Epidermal Growth Factor Receptor Monoclonal Antibody 225 Upregulates p27KIP1 and p15INK4B and Induces G1 Arrest in Oral Squamous Carcinoma Cell Lines

Epidermal growth factor receptor (EGFR) regulates the growth and progression of human oral squamous cell carcinoma (SCC). Recently, the link between EGFR signaling and the cell cycle has been identified. Some reports have described that EGFR-blocking monoclonal antibody 225 (mAb225) induced G1 arrest and inhibited the growth of various cancer cells. The purpose of this study was to evaluate the effect of mAb225 on human oral SCC cell lines. Exposure to mAb225 in culture inhibited the growth of oral SCC cell lines in an EGFR number-independent manner, with the percent inhibition ranging from 13.8 to 76.6%. Flow-cytometric analysis demonstrated that treatment with mAb225 induced cell accumulation in G1 phase, accompanied by a decrease in the percentage of cells in the S phase. Apoptosis was not seen in this study. G1 arrest was accompanied by a decrease in CDK2-, CDK4-, and CDK6-associated histone H1 kinase activities, and an increase in the expression levels of cell cycle inhibitors p27^KIP1 and p15^INK4B. These results suggested that the antiproliferative effect of EGFR blockade by mAb225 in oral SCC may be mediated by p27^KIP1 and p15^INK4B.

Different effects of baicalein, baicalin and wogonin on mitochondrial function, glutathione content and cell cycle progression in human hepatoma cell lines.

The effects of the flavonoids from Scutellaria baicalensis Georgi (baicalein, baicalin and wogonin) in cultured human hepatoma cells (Hep G2, Hep 3B and SK-Hep1) were compared by MTT assay and flow cytometry. All three flavonoids dose-dependently decreased the cell viabilities accompanying the collapse of mitochondrial membrane potential and the depletion of glutathione content. However, the influence of baicalein, baicalin or wogonin on cell cycle progression was different. All three flavonoids resulted in prominent increase of G2/M population in Hep G2 cells, whereas an accumulation of sub G1 (hypoploid) peak in Hep 3B cells was observed. In SK-Hep1 cells, baicalein and baicalin resulted in dramatic boost in hypoploid peak, but wogonin made mainly in G1 phase accumulation. These data, together with the previous findings in other hepatoma cell lines, suggest that baicalein, baicalin and wogonin might be effective candidates for inducing apoptosis or inhibiting proliferation in various human hepatoma cell lines.

Arsenic Trioxide Induces Apoptosis in HTLV-I Infected T-cell Lines and Fresh Adult T-cell Leukemia Cells Through CD95 or Tumor Necrosis Factor α Receptor Independent Caspase Activation

Arsenic trioxide (As2O3) has been reported to induce apoptosis in human T-cell leukemia virus type-I (HTLV-I) infected T-cell lines and fresh adult T-cell leukemia (ATL) cells and to induce G1 phase accumulation in HTLV-I infected T-cell lines. The present study aimed to clarify the pathway of As2O3-induced apoptosis in HTLV-I infected T-cell lines, MT-1 and MT-2, and fresh ATL cells separated from peripheral blood of patients with acute or chronic type ATL. Cells were treated up to 72 h at clinically tolerable concentrations of As2O3 (1–2 µmol/l) shown to be safe in patients with acute promyelocytic leukemia (APL). Activation of caspases 3, 8, and 9, loss of mitochondrial transmembrane potential and cleavage of poly (adenosine diphosphate-ribose) polymerase (PARP) were observed during As2O3 treatment. Furthermore, prior exposure to a broad-spectrum caspase inhibitor blocked As2O3-induced apoptosis but not G1 phase accumulation. While pre-treatment with a CD95 receptor-blocking antibody (Ab) or a TNF-α neutralizing Ab did not show such inhibitions in these cells. In conclusion, As2O3 induces apoptosis in HTLV-I infected T-cell lines and fresh ATL cells through CD95 or TNF-α receptor independent caspase activation.