Decreased Cyclin B1 Research Articles

The Aurora A Kinase Inhibitor, Alisertib, Has Broad Activity In Nonclinical Models Of T-Cell Lymphoma and Is Highly Synergistic With Romidepsin, But Not With Pralatrexate Or The Proteasome Inhibitor, Ixazomib

Aurora A kinase (AAK), a serine-threonine protein kinase, regulates mitotic entry, spindle formation, and cytokinesis. Alisertib is a selective AAK investigational inhibitor with demonstrated clinical activity in acute myeloid leukemia, peripheral t-cell lymphoma (PTCL), DLBCL and other heme-lymphatic cancers. Here we report the cytotoxicity and apoptotic effect of Alisertib in a panel of T-cell-derived lymphoma cell-lines (TCL) (CTCL, HTLV+, T-ALL) and B-cell lymphoma cell-lines (DLBCL-ABC, DLBCL-GCB, MCL) alone and in combination with romidepsin, pralatrexate (PDX) and ixazomib, a proteasome inhibitor.Single agent concentration and time effect relationships were generated for 8 TCL, 4 DLBCL (2 ABC, 2 GCB) and 4 MCL cell-lines. The mean IC50 of alisertib in TCL was 350 nM (range 100-1000nM) and in B-cell lymphoma lines (DLBCL, MCL) was 200 nM (range 20-300 nM) at 48 hours, measured by growth inhibition. In all cell lines evaluated, there was a consistent 2-log fold decrease in IC50 values at 72 hours. Combination studies evaluating synergy were performed testing schedule, concentration, and time effect relationships. Interestingly, simultaneous exposure of combined alisertib and romidepsin at their IC10, IC20,and IC30 demonstrated marked synergy in TCL. Deepest synergy was observed at 72 hours with synergy coefficients ranging from 0.2 to 0.7. This synergistic interaction was restricted to the TCL cell-lines, with no benefit demonstrated in DLBCL or MCL cell lines. (Table 1) Similarly, alisertib did not demonstrate synergy in TCL, DLBCL or MCL cell lines at any concentration, combination, or time schedule with PDX or ixazomib (simultaneous incubation of alisertib + PDX, 24 hour pre-exposure alisertib followed by PDX, 24 hour pre-exposure PDX followed by alisertib; simultaneous incubation of alisertib + ixazomib).Evidence for apoptosis was confirmed for alisertib in combination with romidepsin in the TCL cell-line, H9 after 48 hours of exposure by increased caspase 3 and PARP cleavage, acetylated H3 expression, and decreased Cyclin B1, P27, and BCL2 expression; suggesting that cell death occurred through apoptosis. AnnexinV/propridium iodide via FACS analysis confirmed induction of apoptosis.These data support the observation that alisertib produces broad single-agent activity in models of TCL and demonstrates marked synergy with romidepsin. Interestingly this effect is restricted to TCL and there is no synergistic effect with ixazomib or PDX in TCL. Further evaluation of the mechanism of action with alisertib in combination with romidepsin as well as in vivo modeling of these combinations is ongoing. Disclosures:Off Label Use: Aurora kinase inhibitors are not approved for T-Cell lymphoma. Amengual:Acetylon Pharmaceuticals, INC: Membership on an entity's Board of Directors or advisory committees, Research Funding. O'Connor:Millennium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Spectrum Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Allos Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene Pharmaceuticals: Consultancy.

MiR-379 Regulates Cyclin B1 Expression and Is Decreased in Breast Cancer

MicroRNAs are small non-coding RNA molecules that control gene expression post-transcriptionally, and are known to be altered in many diseases including breast cancer. The aim of this study was to determine the relevance of miR-379 in breast cancer. miR-379 expression was quantified in clinical samples including tissues from breast cancer patients (n=103), healthy controls (n=30) and patients with benign breast disease (n=35). The level of miR-379 and its putative target Cyclin B1 were investigated on all breast tissue specimens by RQ-PCR. Potential relationships with gene expression and patient clinicopathological details were also determined. The effect of miR-379 on Cyclin B1 protein expression and function was investigated using western blot, immunohistochemistry and proliferation assays respectively. Finally, the levels of circulating miR-379 were determined in whole blood from patients with breast cancer (n=40) and healthy controls (n=34). The level of miR-379 expression was significantly decreased in breast cancer (Mean(SEM) 1.9 (0.09) Log10 Relative Quantity (RQ)) compared to normal breast tissues (2.6 (0.16) Log10 RQ, p<0.01). miR-379 was also found to decrease significantly with increasing tumour stage. A significant negative correlation was determined between miR-379 and Cyclin B1 (r=-0.31, p<0.001). Functional assays revealed reduced proliferation (p<0.05) and decreased Cyclin B1 protein levels following transfection of breast cancer cells with miR-379. Circulating miR-379 was not significantly dysregulated in patients with breast cancer compared to healthy controls (p=0.42). This data presents miR-379 as a novel regulator of Cyclin B1 expression, with significant loss of the miRNA observed in breast tumours.

Abstract 5474: Epidermal growth factor inhibition of HPV positive head and neck cancer cells and primary tumorgrafts results in significant growth inhibition mediated by apoptosis.

Abstract Purpose: Cetuximab, a monoclonal antibody targeting the epidermal growth factor receptor (EGFR), is approved for use in the treatment of head and neck cancer (HNC). One recent study (SPECTRUM) suggested that patient with recurrent or metastatic HPV+ HNC do not benefit from treatment with EGFR targeted therapy. However, most studies of EGFR inhibition have been performed with HPV-negative cells and clinical trials have been performed without regard to HPV status. We performed this study to determine whether HPV+ HNC cells and patient-derived tumorgrafts respond to cetuximab and to examine the mechanisms through which cetuximab affects HPV+ HNC. Methods: Four HPV+ cell lines (UD-SCC2, UM-SCC47, UPCI-SCC90, 93-VU-147T) were assessed for EGFR expression by western blot. Sensitivity to cetuximab was tested by assessing cell density 2, 4, 6, and 8 days after cetuximab treatment and by assessing colony formation 2 weeks after plating. Apoptosis was measured by caspase activation, flow cytometry for Annexin V and propidium iodide staining, and immunoblot. Cell cycle was assessed by immunoblot for cyclin D1, cyclin B1, and p27Kip-1 and confirmed by flow cytometry for propidium iodide stained cells. Subcutaneous flank xenografts and tumorgrafts were performed in Hsd:athymic Nude-Foxn1nu female mice treated with intraperitoneal cetuximab (0.2mg/mouse) delivered twice weekly for 2 weeks. Time to tumor quadrupling from baseline was assessed by Kaplan-Meier method. Results: Significant variation in EGFR expression was seen in HPV+ cells. Cetuximab treatment resulted in significant delay in cell proliferation (p&amp;lt;0.005 for all lines) and decrease in colony formation (p&amp;lt;0.04 for all lines). In these cell lines, cetuximab caused an increase in apoptosis as measured by caspase activity, Bax activation, and Annexin V labeling. In addition, as previously seen in HPV- cell lines, cetuximab increased p27Kip-1 and decreased cyclin B1 and cyclin D1 coinciding with in a G1 cell cycle arrest. Using both a cell line xenograft model and a direct-from-patient tumorgraft model of HPV+ HNCs, cetuximab resulted in significant tumor growth delay (median time to tumor quadrupling: 15 vs. 24 days, p=0.02; and 42 vs. 89 days, p=0.0001, respectively). Conclusions: Epidermal growth factor receptor inhibition by cetuximab appears to be effective in slowing proliferation and inducing apoptosis in HPV+ HNC. The proposed mechanism of action appears to be similar to that shown over a decade ago in HPV- HNC. These results suggest that cetuximab may play a role in the management of patients with HPV+ HNC. Citation Format: Grace C. Blitzer, Molly A. Smith, Alexandra D. Torres, Eric A. Armstrong, Paul M. Harari, Paul F. Lambert, Randall J. Kimple. Epidermal growth factor inhibition of HPV positive head and neck cancer cells and primary tumorgrafts results in significant growth inhibition mediated by apoptosis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5474. doi:10.1158/1538-7445.AM2013-5474

Abstract 1731: Decreased cyclin B1 expression contributes to mitochondrial apoptosis in medulloblastoma.

Abstract Medulloblastoma, the most common malignant pediatric brain tumor, remains difficult to cure. Previously, we have shown that downregulation of MMP-9 and uPAR induces Chk1-mediated G2/M cell-cycle arrest in medulloblastoma cells. In this study, we observed that downregulation of MMP-9 in nude mice D283 xenografts downregulated the expression of cyclin B1. Increasing evidence indicates that the deregulation of cyclin B1 is involved in neoplastic transformation, suggesting that inhibition of cyclin B1 could be an attractive strategy for anti-tumor therapy. To determine the effect of downregulation of cyclin B1, we treated medulloblastoma cells with nocodazole, which is an anti-neoplastic agent. Nocodazole-treated cells have undergone G2/M cell-cycle arrest because of the mitotic spindle arrest. Prolonged arrest of cells in mitosis due to nocodazole treatment typically results in cell death by apoptosis. Hence, we confirmed the induction of apoptosis in medulloblastoma cells by TUNEL analysis. Immunoblot analysis of cell cycle checkpoint proteins Chk1, Chk2 and cyclin B1 revealed that they were downregulated in combination treatments of shMMP-9 with radiation. Further, apoptotic proteins BAD, BAX and BAK were highly upregulated, which shows the involvement of mitochondria-mediated apoptosis in our treatments. In vivo analysis of BAD molecules in D283 nude mice xenografts confirmed that downregulation of cyclin B1 induces BAD-mediated apoptotic death in medulloblastoma tumors treated with either shMMP-9 alone or in combination with radiation. With this study, we demonstrate that downregulation of cyclin B1 is an effective and specific approach to induce apoptosis for effective anti-tumor therapy against medulloblastoma. Citation Format: Venkata Ramesh Dasari, Swapna Asuthkar, Arun Kumar Nalla, Jasti S. Rao. Decreased cyclin B1 expression contributes to mitochondrial apoptosis in medulloblastoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1731. doi:10.1158/1538-7445.AM2013-1731

Pomegranate Juice Metabolites, Ellagic Acid and Urolithin A, Synergistically Inhibit Androgen-Independent Prostate Cancer Cell Growth via Distinct Effects on Cell Cycle Control and Apoptosis

Ellagitannins (ETs) from pomegranate juice (PJ) are bioactive polyphenols with chemopreventive potential against prostate cancer (PCa). ETs are not absorbed intact but are partially hydrolyzed in the gut to ellagic acid (EA). Colonic microflora can convert EA to urolithin A (UA), and EA and UA enter the circulation after PJ consumption. Here, we studied the effects of EA and UA on cell proliferation, cell cycle, and apoptosis in DU-145 and PC-3 androgen-independent PCa cells and whether combinations of EA and UA affected cell proliferation. EA demonstrated greater dose-dependent antiproliferative effects in both cell lines compared to UA. EA induced cell cycle arrest in S phase associated with decreased cyclin B1 and cyclin D1 levels. UA induced a G2/M arrest and increased cyclin B1 and cdc2 phosphorylation at tyrosine-15, suggesting inactivation of the cyclin B1/cdc2 kinase complex. EA induced apoptosis in both cell lines, while UA had a less pronounced proapoptotic effect only in DU-145. Cotreatment with low concentrations of EA and UA dramatically decreased cell proliferation, exhibiting synergism in PC-3 cells evaluated by isobolographic analysis and combination index. These data provide information on pomegranate metabolites for the prevention of PCa recurrence, supporting the role of gut flora-derived metabolites for cancer prevention.

BTG2 Overexpression Increases the Radiosensitivity of Breast Cancer Cells In Vitro and In Vivo

Antiproliferative gene B-cell translocation gene, member 2 (BTG2) is a member of the BTG/TOB antiproliferative gene family. In this study, we investigated the effect of BTG2 gene overexpression on the radiosensitivity of breast cancer cells in vitro and in vivo. Results show that in human breast cancer cell line MCF-7 stably overexpressing BTG2 gene, cell sensitivity to ionizing radiation increased. The MCF-7-BTG2 cells were more susceptible to radiation-caused apoptosis with decreased cyclin B1, cyclin D1, Ku70, FEN-1, and XRCC1 protein expression as well as increased BAX protein expression. The findings indicate for the first time that BTG2 can improve the radiosensitivity of breast cancer cells by affecting cell cycle distribution, enhancing radiation-induced apoptosis, and inhibiting DNA repair-related protein expression.

Euphol from Euphorbia tirucalli selectively inhibits human gastric cancer cell growth through the induction of ERK1/2-mediated apoptosis

Gastric cancer is one of the most common malignancies worldwide, and the main cause of cancer-related death in Asia. The present study assessed the anticancer effects of euphol, a triterpene alcohol with anti-inflammatory and antiviral activities on human gastric cancer cells. Euphol showed higher cytotoxicity activity against human gastric CS12 cancer cells than against noncancer CSN cells. In addition, it up-regulated the pro-apoptotic protein BAX and down-regulated the prosurvival protein Bcl-2, causing mitochondrial dysfunction, possibly by caspase-3 activation. The anti-proliferative effects of euphol were associated with the increased p27kip1 levels and decreased cyclin B1 levels. Inhibition of ERK1/2 activation by PD98059 reversed euphol-induced pro-apoptotic protein expression and cell death. Taken together, these findings suggest that euphol selectively induced gastric cancer cells apoptosis by modulation of ERK signaling, and could thus be of value for cancer therapy.

Sigma-2 ligands induce tumour cell death by multiple signalling pathways

Background:The sigma-2 receptor has been identified as a biomarker of proliferating cells in solid tumours. In the present study, we studied the mechanisms of sigma-2 ligand-induced cell death in the mouse breast cancer cell line EMT-6 and the human melanoma cell line MDA-MB-435.Methods:EMT-6 and MDA-MB-435 cells were treated with sigma-2 ligands. The modulation of multiple signaling pathways of cell death was evaluated.Results:Three sigma-2 ligands (WC-26, SV119 and RHM-138) induced DNA fragmentation, caspase-3 activation and PARP-1 cleavage. The caspase inhibitor Z-VAD-FMK partially blocked DNA fragmentation and cytotoxicity caused by these compounds. These data suggest that sigma-2 ligand-induced apoptosis and caspase activation are partially responsible for the cell death. WC-26 and siramesine induced formation of vacuoles in the cells. WC-26, SV119, RHM-138 and siramesine increased the synthesis and processing of microtubule-associated protein light chain 3, an autophagosome marker, and decreased the expression levels of the downstream effectors of mammalian target of rapamycin (mTOR), p70S6K and 4EBP1, suggesting that sigma-2 ligands induce autophagy, probably by inhibition of the mTOR pathway. All four sigma-2 ligands decreased the expression of cyclin D1 in a time-dependent manner. In addition, WC-26 and SV119 mainly decreased cyclin B1, E2 and phosphorylation of retinoblastoma protein (pRb); RHM-138 mainly decreased cyclin E2; and 10 μM siramesine mainly decreased cyclin B1 and pRb. These data suggest that sigma-2 ligands also impair cell-cycle progression in multiple phases of the cell cycle.Conclusion:Sigma-2 ligands induce cell death by multiple signalling pathways.

The nucleosome binding protein NSBP1 is highly expressed in human bladder cancer and promotes the proliferation and invasion of bladder cancer cells

NSBP1 is a recently identified member of the HMGN protein family which binds to nucleosomes and regulates gene transcription through chromatin remodeling. In this study, we aimed to investigate the potential role of NSBP1 in human bladder cancer. We examined NSBP1 expression in 114 surgically removed bladder cancer specimens as well as 11 human bladder cell lines by immunohistochemistry and Western blot analysis, and found that NSBP1 level was correlated with the increased tumor grade and pathologic stage, and lymph node metastasis. RNAi-mediated knockdown of NSBP1 in EJ cells, a bladder cancer cell line that overexpressed NSBP1, resulted in moderate decrease of cell viability, moderate blockage of cell cycle at G2/M phase, and decreased cyclin B1 expression, but had no effects on apoptosis. Moreover, NSBP1 knockdown led to reduced activity of MMP-9 but not MMP-2. Taken together, these results suggest that NSBP1 promotes the viability of bladder cancer cells through increased cell proliferation but not decreased apoptosis, and increases the invasion ability of metastatic bladder cancer cells through the upregulation of MMP-9 activity. Our findings not only provide a molecular understanding of the role of NSBP1 in bladder cancer, but also suggest NSBP1 RNAi as a novel therapeutic approach for bladder cancer.

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 G 2/M phase. In this study, we demonstrated that treatment of U937 cells with SP600125 resulted in significant G 2/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 G 2/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 G 2/M phase arrest and endoreduplication.

Manganese superoxide dismutase activity regulates transitions between quiescent and proliferative growth

In recent years, the intracellular reactive oxygen species (ROS) levels have gained increasing attention as a critical regulator of cellular proliferation. We investigated the hypothesis that manganese superoxide dismutase (MnSOD) activity regulates proliferative and quiescent growth by modulating cellular ROS levels. Decreasing MnSOD activity favored proliferation in mouse embryonic fibroblasts (MEF), while increasing MnSOD activity facilitated proliferating cells' transitions into quiescence. MnSOD +/- and -/- MEFs demonstrated increased superoxide steady-state levels; these fibroblasts failed to exit from the proliferative cycle, and showed increasing cyclin D1 and cyclin B1 protein levels. MnSOD +/- MEFs exhibited an increase in the percentage of G(2) cells compared to MnSOD +/+ MEFs. Overexpression of MnSOD in MnSOD +/- MEFs suppressed superoxide levels and G(2) accumulation, decreased cyclin B1 protein levels, and facilitated cells' transit into quiescence. While ROS are known to regulate differentiation and cell death pathways, both of which are irreversible processes, our results show MnSOD activity and, therefore, mitochondria-derived ROS levels regulate cellular proliferation and quiescence, which are reversible processes essential to prevent aberrant proliferation and subsequent exhaustion of normal cell proliferative capacity. These results support the hypothesis that MnSOD activity regulates a mitochondrial 'ROS-switch' favoring a superoxide-signaling regulating proliferation and a hydrogen peroxide-signaling supporting quiescence.

Histone deacetylase inhibitor, suberoylanilide hydroxamic acid (Vorinostat, SAHA) profoundly inhibits the growth of human pancreatic cancer cells

Tumor suppressor genes are often silenced in human cancer; this can occur by transcriptional repression by deacetylation in the promoter regions, mediated by histone deacetylase (HDAC). HDAC inhibitors can block cancer cell growth by restoring expression of tumor suppressor genes. In this study, we investigated the effects of a HDAC inhibitor, suberoylanilide hydroxamic acid (SAHA) on pancreatic cancer cells. SAHA inhibited the growth of 6 pancreatic cancer cell lines in a dose-dependent manner as measured by MTT and clonogenic assays (ED(50) approximately 10(-6) M) associated with induction of apoptosis, G2 cell cycle arrest and also induced differentiation as indicated by morphology and increased expression of cytokeratin 7. It increased expression of p21(WAF1) (independent of the mutational status of p53), C/EBPalpha, RARalpha and E-cadherin; these genes have been associated with decreased proliferation in other cancers. SAHA decreased cyclin B1 expression; this cyclin normally promotes progression through G2 of the cell cycle. SAHA mediated acetylation of histone H3 globally, as well as, associated with the p21(WAF1) promoter, as measured by chromatin immunoprecipitation. SAHA also decreased levels of c-myc and cyclin D1, independent of an active beta-catenin pathway. In further studies, the combination of SAHA and an inhibitor of DNA methylation, 5-Aza-2'-deoxycytidine, had an enhanced antiproliferative effect on pancreatic cancer cells. In summary, SAHA inhibited the growth of human pancreatic cancer cells by inducing apoptosis, differentiation and cell cycle arrest, as well as increase in the expression of several tumor suppressor genes. SAHA is a novel, promising therapeutic agent for human pancreatic cancers.

The human synMuv-like protein LIN-9 is required for transcription of G2/M genes and for entry into mitosis

Regulated gene expression is critical for the proper timing of cell cycle transitions. Here we report that human LIN-9 has an important function in transcriptional regulation of G2/M genes. Depletion of LIN-9 by RNAi in human fibroblasts strongly impairs proliferation and delays progression from G2 to M. We identify a cluster of G2/M genes as direct targets of LIN-9. Activation of these genes is linked to an association between LIN-9 and B-MYB. Chromatin immunoprecipitation assays revealed binding of both LIN-9 and B-MYB to the promoters of G2/M regulated genes. Depletion of B-MYB recapitulated the biological outcome of LIN-9 knockdown, including impaired proliferation and reduced expression of G2/M genes. These data suggest a critical role for human LIN-9, together with B-MYB, in the activation of genes that are essential for progression into mitosis.

Induction of G2/M arrest and apoptosis in human gastric epithelial AGS cells by aqueous extract of Agaricus blazei

Agaricus blazei is well known as a traditional medicinal mushroom and it has been shown to exhibit immunostimulatory and anti-cancer activity. However, the cellular and molecular mechanism of cell cycle arrest and apoptosis of cancer cells is poorly understood. In this study, we have investigated whether A. blazei extract (ABE) exerts anti-proliferative and apoptotic effects on human gastric epithelial AGS cells. It was found that ABE could inhibit cell growth in a dose-dependent manner, which was associated with the arrest of G2/M phase and the induction of apoptotic cell death. Flow cytometric analysis showed that ABE could cause an arrest at the G2/M phase of the cell cycle, which is closely correlated to decreased cyclin B1 and cdc2 levels. Furthermore, this compound induced apoptosis through up-regulation of Bax and the activation of caspases with down-regulation of XIAP and cIAP-1, but not cIAP-2, and a capase-3 inhibitor could block cell death and apoptotic body formation. These data clearly indicate that ABE-induced apoptosis is associated with caspase-3 activation. In summary, the growth inhibition of ABE is highly related to cell cycle arrest at the G2/M phase and the induction of caspase-3-dependent apoptosis in human gastric epithelial AGS cells.

Myb Family Transcription Factors Contribute to G2/M Cell Cycle Transition in Normal and Malignant Hematopoietic Cells by Direct Regulation of Cyclin B1 Expression.

Myb family transcription factors are ubiquitously expressed, and are known to play a critical role in regulating G1/S cell cycle transition. Recently, Myb-like proteins have been found to regulate G2/M transit in plants, yeast, and Drosophila. A recent study in human T98G ganglioblastoma cells revealed that E2F, together with B-Myb, but not c-Myb, regulated cyclin B1 expression directly. However, in hematopoietic cells Myb's role in regulating cell cycle check points other than G1/S is less well defined. Herein we report that c-Myb, as well as B-Myb, up-regulates cyclin B1 expression in normal and malignant human hematopoietic cells, thereby contributing to G2/M cell cycle progression. Our initial experiments revealed a direct relationship between Myb and cyclin B1 expression. We then attempted to show causality using a variety of experimental approaches. First, ChIP assays demonstrated that c-Myb protein directly bound the cyclin B1 promoter in K562 and Mo7e cells. Second, a cycle 3 GFP reporter construct, driven by the cyclin B1 promoter, was upregulated in cells co-tranfected with a c-myb expression vector. Third, a conditionally active c-Myb restored cyclin B1 mRNA expression in K562 human leukemia cells in presence of cycloheximide in 6 hours. All these assays strongly suggest that c-myb directly regulates cyclin B1. Finally, cyclin B1 expression decreased by 85–90 % in Mo7e human leukemia cells in which c-myb had been silenced with siRNA. siRNA targeted to B-myb also decreased cyclin B1 expression, while neither siRNA species decreased cdc2 or cyclin A. The biologic significance of this relationship was revealed by two independent lines of experimentation. First, silencing B-myb resulted in a delay of cell cycle progression from S to G2/M, and an accumulation of cells in M phase, in HCT116 cells and K562 cells respectively. These abnormalities could be rescued, at least partially, by expression of exogenous c-myb. This observation conflicts with the report that c-Myb does not regulate cyclin B1 or G2/M progression in T98G cells suggesting that Myb functions could well be cell type specific. Additional analysis using PCR array showed that the absence of B-myb decreased the expression of 19 of 84 cell cycle related genes. Exogenous c-myb expression partially rescued 11 genes including cyclin B1, cyclin B2, cdc2, cdc20, CKS1B, p15INK4b and Ki-67, but not cyclin D1. In another experiment an inducible dominant negative c-Myb protein decreased cyclin B1 expression in K562 human leukemia cells, and the expected consequence of this, accelerated exit from the M phase, was observed. In activated primary human T-lymphocytes with IL-2 and CD34+ bone marrow cells, expression of c-Myb and cyclin B1 increased concordantly and silencing c-myb expression resulted in decreased cyclin B1 expression. We conclude from these studies that c-Myb in addition to B-myb plays a heretofore unappreciated role in G2/M cell cycle transition in normal and malignant human hematopoietic cells by directly regulating cyclin B1 expression.

Genistein inhibits radiation-induced activation of NF-κB in prostate cancer cells promoting apoptosis and G2/M cell cycle arrest

BackgroundNew cancer therapeutic strategies must be investigated that enhance prostate cancer treatment while minimizing associated toxicities. We have previously shown that genistein, the major isoflavone found in soy, enhanced prostate cancer radiotherapy in vitro and in vivo. In this study, we investigated the cellular and molecular interaction between genistein and radiation using PC-3 human prostate cancer cells.MethodsTumor cell survival and progression was determined by clonogenic analysis, flow cytometry, EMSA analysis of NF-κB, and western blot analysis of cyclin B1, p21WAF1/Cip1, and cleaved PARP protein.ResultsGenistein combined with radiation caused greater inhibition in PC-3 colony formation compared to genistein or radiation alone. Treatment sequence of genistein followed by radiation and continuous exposure to genistein showed optimal effect. Cell cycle analysis demonstrated a significant dose- and time-dependent G2/M arrest induced by genistein and radiation that correlated with increased p21WAF1/Cip1 and decreased cyclin B1 expression. NF-κB activity was significantly decreased by genistein, yet increased by radiation. Radiation-induced activation of NF-κB activity was strongly inhibited by genistein pre-treatment. A significant and striking increase in cleaved PARP protein was measured following combined genistein and radiation treatment, indicating increased apoptosis.ConclusionA mechanism of increased cell death by genistein and radiation is proposed to occur via inhibition of NF-κB, leading to altered expression of regulatory cell cycle proteins such as cyclin B and/or p21WAF1/Cip1, thus promoting G2/M arrest and increased radiosensitivity. These findings support the important and novel strategy of combining genistein with radiation for the treatment of prostate cancer.

C-Myb Plays a Role InG2/M Cell Cycle Transition by Direct Regulation of Cyclin B1 Expression in Hematopoietic Cells.

Myb family transcription factors are found throughout the phyla, and recent studies have demonstrated that Drosophila myb, as well as plant and yeast c-myb-like transcription factors, play an important role in regulating transition though the G1/S and G2/M phases of the cell cycle. Myb's ability to regulate passage through G2/M is due at least in part to its ability to induce Cyclin B1 expression. A recent study in human T98G ganglioblastoma cells revealed that E2F, together with B-Myb, regulated cyclin B1 expression. Though c-myb was expressed in these cells, it was not found in immunoprecipitated E2F-B-Myb protein complexes and for this reason was felt not to participate in cyclin B1 expression in these cells. Since c-myb plays such a critical role in regulating hematopoietic cell proliferation, and its role in regulating G2/M in blood cells has not previously been explored, we investigated whether c-myb was important is regulating this phase of the cell cycle using K562 and Mo7e cells, as well as PHA stimulated human T lymphocytes. In distinct contrast to findings reported for T98G cells, we now report that in normal and malignant human hematopoietic cells, c-Myb directly upregulates cyclin B1 expression. Several lines of evidence support this claim. First, cyclin B1 expression decreased in Mo7e human leukemia cells in which c-myb had been silenced with siRNA. siRNA targeted to B-myb also decreased cyclin B1 expression, while neither siRNA species decreased cdc2 or cyclin A in these cells. As expected, siRNA targeted against c-myb or B-myb impaired Mo7e cell proliferation. Simultaneous exposure to both siRNA blocked proliferation completely. Second, using an alternative strategy, an inducible dominant negative c-Myb protein also decreased cyclin B1 expression in K562 human leukemia cells. The expected consequence of this, accelerated exit from the M phase, was also observed. Third, we examined c-Myb expression in human T cells by western and Real Time PCR, pre and post PHA stimulation. c-Myb expression began to gradually increase in the G1 phase of cell cycle, continued to increase after S phase, with the maximal protein level being found in G2/M phase, and concordant with cyclin B1 expression. These results indicated a correlation between c-Myb and cyclin B1 expression but did not indicate if c-Myb regulated cyclin B1 expression directly. To address this question, several additional experiments were carried out. A CAT assay showed that overexpressing c-Myb protein could increase activity when driven by a cyclin B1 promoter construct ~5X compared to K562 control cells. Next, examination of the cyclin B1 promoter showed eight potential c-Myb binding sites. Two were canonical [5′-pyrimidine AACG/TG-3′] and located upstream of 6 others which were [5′-AACNG-3′] in type. An in vitro c-Myb binding assay revealed that c-Myb bound the canonical sites. We then performed a Chromatin Immunoprecipitation (ChIP) Assay with anti-c-Myb antibody and specifically enriched cyclin B1 promoter DNA sequences which strongly suggested that c-Myb bound the cyclin B1 promoter in vivo. A control antibody was inactive. Finally, a conditionally active c-Myb restored cyclin B1 mRNA expression in K562 human leukemia cells in presence of cycloheximide within 6 hours. Therefore, in addition to its role in regulating G1/S cell cycle transition, c-Myb also regulates cyclin B1 expression and therefore transition through the G2/M phase in human hematopoietic cells.

Small molecules that delay S phase suppress a zebrafish bmyb mutant

Bmyb is a ubiquitously expressed transcription factor involved in cellular proliferation and cancer. Loss of bmyb function in the zebrafish mutant crash&burn (crb) results in decreased cyclin B1 expression, mitotic arrest and genome instability. These phenotypic observations in crb mutants could be attributed to the decreased expression of cyclin B1, a cell-cycle regulatory protein that is responsible for driving cell progression from G2 through mitosis. To identify small molecules that interact with the bmyb pathway, we developed an embryo-based suppressor screening strategy. In 16 weeks we screened a diverse approximately 16,000 compound library, and discovered one previously unknown compound, persynthamide (psy, 1), that suppressed bmyb-dependent mitotic defects. Psy-treated embryos showed an S-phase delay, and knockdown of the cell-cycle checkpoint regulator ataxia telangiectasia--and Rad-related kinase (ATR) abrogated the suppression of crb. The DNA synthesis inhibitors aphidicolin (2) and hydroxyurea (3) also suppressed crb. S-phase inhibition upregulated cyclin B1 mRNA, promoting the progression of cells through mitosis. Our study demonstrates that chemical suppressor screening in zebrafish can identify compounds with cell-cycle activity and can be used to identify pathways that interact with specific cell-cycle phenotypes.

HYAL1 Hyaluronidase in Prostate Cancer: A Tumor Promoter and Suppressor

Hyaluronidases degrade hyaluronic acid, which promotes metastasis. HYAL1 type hyaluronidase is an independent prognostic indicator of prostate cancer progression and a biomarker for bladder cancer. However, it is controversial whether hyaluronidase (e.g., HYAL1) functions as a tumor promoter or as a suppressor. We stably transfected prostate cancer cells, DU145 and PC-3 ML, with HYAL1-sense (HYAL1-S), HYAL1-antisense (HYAL1-AS), or vector DNA. HYAL1-AS transfectants were not generated for PC-3 ML because it expresses little HYAL1. HYAL1-S transfectants produced < or = 42 milliunits (moderate overproducers) or > or = 80 milliunits hyaluronidase activity (high producers). HYAL1-AS transfectants produced <10% hyaluronidase activity when compared with vector transfectants (18-24 milliunits). Both blocking HYAL1 expression and high HYAL1 production resulted in a 4- to 5-fold decrease in prostate cancer cell proliferation. HYAL1-AS transfectants had a G2-M block due to decreased cyclin B1, cdc25c, and cdc2/p34 expression and cdc2/p34 kinase activity. High HYAL1 producers had a 3-fold increase in apoptotic activity and mitochondrial depolarization when compared with vector transfectants and expressed activated proapoptotic protein WOX1. Blocking HYAL1 expression inhibited tumor growth by 4- to 7-fold, whereas high HYAL1 producing transfectants either did not form tumors (DU145) or grew 3.5-fold slower (PC-3 ML). Whereas vector and moderate HYAL1 producers generated muscle and blood vessel infiltrating tumors, HYAL1-AS tumors were benign and contained smaller capillaries. Specimens of high HYAL1 producers were 99% free of tumor cells. This study shows that, depending on the concentration, HYAL1 functions as a tumor promoter and as a suppressor and provides a basis for anti-hyaluronidase and high-hyaluronidase treatments for cancer.

Cadmium-induced changes in growth and cell cycle gene expression in suspension-culture cells of soybean

The toxic effects of cadmium on growth and development of living organisms are well documented. However, the molecular mechanisms responsible for the inhibition of plant growth by cadmium are still not completely understood. We determined the effects of cadmium concentrations in the range of 1–11 μM on the growth of Glycine max L. cv. Navico suspension-culture cells, as well as on the expression of two cell cycle genes: cyclin B1 and cyclin-dependent type A kinase (CDK-A). There was no detectable decrease in cell viability at any tested Cd 2+ concentrations. The lower concentrations of Cd 2+ (1–4 μM) stimulated cell culture growth; however, this did not correspond with increased expression of cell cycle genes. The inhibition of cell growth was observed at concentration of Cd 2+ higher than 6 μM. Interestingly, it correlated well with the decreased cyclin B1 mRNA levels, but had no significant effect on the levels of CDK-A mRNA.