Changes In Cell Cycle Proteins Research Articles

Benznidazole modulates cell proliferation in acute leukemia cells

Context: We have previously reported that benznidazole (BZL), known for its trypanocidal action, has anti-proliferative activity against different cell lines like HeLa and Raw 264.7 among others. At the moment, it has not been reported if the anti-proliferative effect of BZL is similar for non-adherent hematopoietic cells like was reported for adherent cancer cell lines.Objective: We aimed to investigate the efficacy of BZL on the growth of the leukemic cell lines THP-1 and OCI/AML3.Materials and methods: We evaluated cell proliferation by [3H]-thymidine incorporation and MTT reduction as well as cell death by lactate dehydrogenase (LDH) activity. We assessed apoptosis by flow cytometry for detection of annexin V-positive and propidium iodide-negative cells, along with nuclear morphology by diamidino-2-phenolindole (DAPI) staining. Western blot studies were performed to evaluate changes in cell cycle proteins in BZL-treated cells.Results: BZL significantly reduced proliferation of both cell lines without inducing cell death. Likewise it produced no significant differences in apoptosis between treated cells and controls. In addition, flow cytometry analysis indicated that BZL caused a larger number of THP-1 cells in G0/G1 phase and a smaller number of cells in S phase than controls. This was accompanied with an increase in the expression of the CDK inhibitor p27 and of cyclin D1, with no significant differences in the protein levels of CDK1, CDK2, CDK4, cyclins E, A and B as compared to controls.Conclusion: BZL inhibits the proliferation of leukemic non-adherent cells by controlling cell cycle at G0/G1 cell phase through up-regulation of p27.

Mirk/Dyrk1B mediates G0/G1 to S phase cell cycle progression and cell survival involving MAPK/ERK signaling in human cancer cells

BackgroundMirk/Dyrk1B contributes to G0 arrest by destabilization of cyclin D1 and stabilization of p27kip1 to maintain the viability of quiescent human cancer cells, and it could be negatively regulated by mitogenic-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signaling. This study was performed to investigate the effect of Mirk/Dyrk1B on cell cycle and survival of human cancer cells involving MAPK/ERK signaling.MethodsThe correlations between Mirk/Dyrk1B expression and active ERK1/2 detected by western blot in both ovarian cancer and non-small cell lung cancer (NSCLC) cells were analyzed by simple regression. Mirk/Dyrk1B unique phosphopeptides with sites associated with Mirk/Dyrk1B protein were isolated and quantitated by liquid chromatography coupled to tandem mass/mass spectrometry (LC-MS/MS) proteomics analysis. The human cancer cells were treated with small interfering RNAs (siRNAs) and/or U0126, an inhibitor of MEK for indicated duration, followed by investigating the alterations of cell cycle and apoptosis as well as related proteins examined by flow cytometry and Western blot, respectively.ResultsOur study demonstrated the widely expressed Mirk/Dyrk1B proteins in the human cancer cells were positively correlated with the levels of activated ERK1/2. Moreover, Mirk/Dyrk1B protein expressions consistent with the tyrosine autophosphorylated levels in the human cancer cells were increased by U0126 or growth factor-depleted culture. Conversely, knockdown of Mirk/Dyrk1B by siRNA led to up-regulated activation of c-Raf-MEK-ERK1/2 pathway and subsequent changes in cell cycle proteins (cyclin D1, p27kip1), accompanied by increased growth rate and cells from G0/G1 into S of cell cycle which could be blocked by U0126 in a dose-dependent manner, indicating Mirk/Dyrk1B may sequester MAPK/ERK pathway, and vice versa. Whereas, combined Mirk siRNA and U0126 induced cell apoptosis in the human cancer cells.ConclusionsThese data together show that Mirk/Dyrk1B mediates cell cycle and survival via interacting with MAPK/ERK signals and simultaneous inhibition of both pathways may be a novel therapeutic target for human cancer.

Abstract 905: Mirk/Dyrk1B mediates cell survival and G0/G1 phase of cell cycle via interacting with MAPK/ERK pathway in human cancer cells

Abstract Minibrain-related kinase (Mirk) known as the dual specificity tyrosine-phosphorylation-regulated kinase (Dyrk) 1B is a serine/threonine kinase. Our recent studies have also demonstrated that knockdown of Mirk by small interfering RNA (siRNA) resulted in reduction of viable cells and cell apoptosis in both ovarian cancer and non-small cell lung cancer (NSCLC) cell lines. Furthermore, it has been reported that Mirk/Dyrk1B can be negatively regulated by mitogenic-activated protein (MAP) / extracellular signal-regulated kinase (ERK) kinase signaling. In this study, we demonstrated the widely overexpressed Mirk protein in variant cell lines of both human ovarian cancer and NSCLC was correlated with the levels of activated ERK examined by western blot analysis. Interestingly, knockdown of Mirk by siRNA in those ovarian cancer cells (OVCAR3, OVCAR10, OVCAR1063, MDAH2774), and NSCLC cells (H292, H441, H1299, PC9) led to increased cell number and cells from G0/G1 into S phase of cell cycle accompanied by up-regulated activation of ERK and its upstream signals, increased cyclin D1, reduced p27, and no evidence of cleaved PARP which are not consistent with our previous findings in the other cell lines. We hypothesized that the increased activation of ERK after Mirk siRNA treatment may block the anti-proliferative effects of Mirk siRNA in some lines or may counterbalance cell loss through apoptosis in human cancer. To examine this further in both OVCAR3 and H292 cells, the increase in cell proliferation induced by Mirk siRNA could be blocked by the MEK inhibitor U0126 in a dose-dependent manner. MEK inhibition abrogated the activation of ERK and subsequent changes in cell cycle proteins (cyclin D1, p27) induced by Mirk knockdown. Combined Mirk siRNA and U0126 induced cell apoptosis in both OVCAR3 and H292 cells. In summary, Mirk may mediate cell survival and G0/G1 phase of cell cycle via interacting with MAPK/ERK pathway in human cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 905. doi:1538-7445.AM2012-905

Parathyroid hormone‐related protein is a hypertrophy factor for human mesangial cells: Implications for diabetic nephropathy

Hypertrophy of human mesangial cells (HMC) is among the earliest characteristics in patients with diabetic nephropathy (DN). Recently, we observed the upregulation of parathyroid hormone (PTH)-related protein (PTHrP) in experimental DN, associated with renal hypertrophy. Herein, we first examined whether PTHrP was overexpressed in human DN, and next assessed the putative role of this protein on high glucose (HG)-induced HMC hypertrophy. As previously found in mice, kidneys from diabetic patients showed an increased tubular and glomerular immunostaining for PTHrP. In HMC, HG medium increased PTHrP protein expression associated with the development of hypertrophy as assessed by cell protein content. This effect was also induced by PTHrP(1-36). HG and PTHrP(1-36)-induced hypertrophy were associated with an increase in cyclin D1 and p27Kip1 protein expression, a decreased cyclin E expression, and the prevention of cyclin E/cdk2 complex activation. Both PTHrP neutralizing antiserum (α-PTHrP) and the PTH/PTHrP receptor antagonist (JB4250) were able to abolish HG induction of hypertrophy, the aforementioned changes in cell cycle proteins, and also TGF-β1 up-regulation. Moreover, the capability of both HG and PTHrP(1-36) to induce HMC hypertrophy was abolished by α-TGFβ1. These data show for the first time that PTHrP is upregulated in the kidney of patients with DN. Our findings also demonstrate that PTHrP acts as an important mediator of HG-induced HMC hypertrophy by modulating cell cycle regulatory proteins and TGF-β1.

Prodigiosins as Anti Cancer Agents: Living Upto Their Name

Prodigiosins are a family of bright red colored bacterial pigment and derive their name from the miraculous (prodigious) events associated with their occurrence. They indeed seem to be living upto their name as a host of activities such as anti-microbial, anti-malarial, anti-cancer and immunosuppressive have been associated with them. Out of these, immunosuppressive and anti-cancer activity has received more importance as it has a clinical promise. Prodigiosins, isolated mostly from Gram negative bacteria are characterized by a common pyrryldipyrrylmethene structure with varying side chains. The review discusses the mechanisms involved in the anti-cancer activity of this class of compounds. In vitro, prodigiosins have been shown to primarily target the cancer cells independently of the p53 status while little or no effect has been observed on normal cells. In addition, prodigiosins are effective in cancer cells with multidrug resistance phenotype and defects in the apoptotic pathways. These make prodigiosins attractive candidates for further development. Though the molecular targets of prodigiosins have not been clearly defined, they have been found to target different signaling pathways possibly through induction of DNA double strand breaks and/ or neutralization of pH gradients leading to changes in cell cycle proteins and apoptosis. The review will discuss the recent findings related to the mechanism involved in the anti-cancer activity of this class of molecules.

Oocyte cryopreservation an analysis of cyclin B and map kinase protein levels as an indicator for oocyte activation following cryopreservation

This study evaluated cell cycle protein dynamics in oocytes following cryopreservation by either vitrification in calcium containing and calcium free mediums or controlled rate freezing. Using a murine model, the effect on oocyte survival and activation was assessed using western blot analysis to evaluate changes in cell cycle proteins. Prospective comparative study. Oocyte-cumulus complexes isolated from superovulated FVB mice were denuded with 80 U/ml Cumulase™. Metaphase II oocytes (n = 830) were alternately allocated into four groups: 1) Control – not cryopreserved, 2) Controlled-rate, 3) Vitrification with calcium (Vit with Ca+), and 4) Vitrification without calcium (Vit without Ca+). Western Blot analysis was performed on groups of five oocytes per treatment for quantitative assessment of cyclin B and MAPK protein levels. Comparisons between cryopreservation protocols are presented in the table; individual variables represent means ± SEM after normalization to non-cryopreserved oocytes. Vitrified oocytes had a significantly higher survival rate compared to oocytes frozen by controlled rate (P<0.01a,b). Vitrification without calcium resulted in a decrease in cyclin B at 0 hours post thaw compared to vitrification with calcium and controls (P=0.06c,d). MAPK levels did not differ statistically between treatments (P=0.24). Tabled 1Table 1TreatmentOocytes ThawedOocytes Survived (%)0 hr post-thaw Cyclin B4 hr post-thaw Cyclin B0 hr post-thaw MAPK4 hr post-thaw MAPKControls (Fresh)—238 (100)100100100100Vit with Ca+182174 (96)a106 ± 38c65 ± 40132 ± 26172 ± 63Vit without Ca+178168 (94)a29 ± 7d231 ± 138163 ± 40132 ± 74Controlled Rate23274 (32)bn/an/a69 ± 6103 ± 22 Open table in a new tab Results show that vitrification without calcium caused a reduction in cell cycle proteins 0 hours after cryopreservation, indicating cell cycle activation. A trend for higher levels of cyclin B is seen in the same treatment four hours post-warming, suggesting that the oocytes were able to restore prior protein levels. This indicates that oocyte activation was minimized, as a high level of cyclin B would prevent premature activation. MAPK levels were unaffected by these cryopreservation protocols. The novel approach of measuring changes in cell cycle proteins following cryopreservation can be used to determine cryodamage and degree of oocyte activation.

Lipoxins Inhibit Akt/PKB Activation and Cell Cycle Progression in Human Mesangial Cells

Lipoxins (LX) are endogenously produced eicosanoids with a spectrum of bioactions that suggest anti-inflammatory, pro-resolution roles for these agents. Mesangial cell (MC) proliferation plays a pivotal role in the pathophysiology of glomerular inflammation and is coupled to sclerosis and tubulointerstitial fibrosis. We have previously reported that LXA4 acts through a specific G-protein-coupled-receptor (GPCR) to modulate MC proliferation in response to the proinflammatory mediators LTD4 and platelet-derived growth factor (PDGF). Further investigations revealed that these effects were mediated by modulation of receptor tyrosine kinase activity. Here we have explored the underlying mechanisms and report inhibition of growth factor (PDGF; epithelial growth factor) activation of Akt/PKB by LXA4. LXA4 (10 nmol/L) modulates PDGF-induced (10 ng/ml, 24 hours) decrements in the levels of cyclin kinase inhibitors p21Cip1 and p27Kip1. PDGF-induced increases in CDK2-cyclin E complex formation are also inhibited by LXA4. The potential of LXA4 as an anti-inflammatory therapeutic is compromised by its degradation; this has been circumvented by synthesis of stable analogs. We report that 15-(R/S)-methyl-LXA4 and 16-phenoxy-LXA4 mimic the native compound with respect to modulation of cell proliferation and PDGF-induced changes in cell cycle proteins. In vivo, MC proliferation in response to PDGF is associated with TGFbeta1 production and the subsequent development of renal fibrosis. Here we demonstrate that prolonged (24 to 48 hours) exposure to PDGF is associated with autocrine TGFbeta1 production, which is significantly reduced by LXA4. In aggregate these data demonstrate that LX inhibit PDGF stimulated proliferation via modulation of the PI-3-kinase pathway preventing mitogen-elicited G1-S phase progression and suggest the therapeutic potential of LX as anti-fibrotic agents.

Cell cycle aberrations by α-synuclein over-expression and cyclin B immunoreactivity in Lewy bodies

α-Synuclein is a presynaptic protein that accumulates abnormally in Lewy bodies of Parkinson’s disease (PD) and dementia with Lewy bodies (DLB). Its physiological function and role in neuronal death remain poorly understood. Recent immunohistochemical studies suggest that cell cycle-related phenomena may play a role in the pathogenesis of Alzheimer’s disease and perhaps other neurodegenerative disorders. In this investigation, we examined the effects of α-synuclein expression levels on cell cycle indices in PC12 cells engineered to conditionally induce α-synuclein expression upon withdrawal of doxycycline. Over-expression of α-synuclein resulted in enhanced proliferation rate and enrichment of cells in the S phase of the cell cycle. This was associated with increased accumulation of the mitotic factor cyclin B and down-regulation of the tumor suppressor retinoblastoma 2. Additionally, ERK1/2, key molecules in proliferation signaling, were highly phosphorylated. Immunohistochemical studies on postmortem brains revealed intense cyclin B immunoreactivity in Lewy bodies in cases with DLB and to a lesser extent in PD. We propose that elevated expression of α-synuclein causes changes in cell cycle regulators through ERK activation leading to apoptosis of postmitotic neurons. These changes in cell cycle proteins are also associated with ectopic expression of cyclin B in Lewy bodies.

Atrial natriuretic peptide type C induces a cell-cycle switch from proliferation to differentiation in brain-derived neurotrophic factor- or nerve growth factor-primed olfactory receptor neurons.

With the discovery of postnatal stem cells within the brain, it has become important to understand how extracellular factors might affect the maturation of neuronal precursors in the postnatal brain. Neurotrophic factors are known to play a role in neuronal development but display pleiotrophic effects, in part because of their physiological interactions with other factors. One factor positioned to interact with neurotrophins in the brains of postnatal animals is atrial C-type natriuretic peptide (CNP). In this study, we used olfactory receptor neurons (ORNs) as a model, because their precursors demonstrate the most robust and functional postnatal neurogenesis of those systems thus far described. We examined the effects of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) and the interactions of these neurotrophins and CNP in postnatal olfactory neuronal precursors. Results obtained using mice with targeted deletion of the gene for BDNF indicated that BDNF is a neuroproliferation-inducing and survival factor for ORN precursors. These roles were confirmed in vitro using primary cultures of ORNs. NGF was found to be a proliferation-inducing factor but not a survival factor. The addition of CNP to either BDNF- or NGF-treated neuronal precursors resulted in an inhibition of proliferation and the promotion of maturation. These effects were accompanied by changes in cell-cycle proteins that suggest possible mechanisms for these effects. Thus, CNP may function in the postnatal brain to regulate the exit from the cell cycle in neuronal precursor cells.

Atrial Natriuretic Peptide Type C Induces a Cell-Cycle Switch from Proliferation to Differentiation in Brain-Derived Neurotrophic Factor- or Nerve Growth Factor-Primed Olfactory Receptor Neurons

With the discovery of postnatal stem cells within the brain, it has become important to understand how extracellular factors might affect the maturation of neuronal precursors in the postnatal brain. Neurotrophic factors are known to play a role in neuronal development but display pleiotrophic effects, in part because of their physiological interactions with other factors. One factor positioned to interact with neurotrophins in the brains of postnatal animals is atrial C-type natriuretic peptide (CNP). In this study, we used olfactory receptor neurons (ORNs) as a model, because their precursors demonstrate the most robust and functional postnatal neurogenesis of those systems thus far described. We examined the effects of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) and the interactions of these neurotrophins and CNP in postnatal olfactory neuronal precursors. Results obtained using mice with targeted deletion of the gene for BDNF indicated that BDNF is a neuroproliferation-inducing and survival factor for ORN precursors. These roles were confirmed in vitro using primary cultures of ORNs. NGF was found to be a proliferation-inducing factor but not a survival factor. The addition of CNP to either BDNF- or NGF-treated neuronal precursors resulted in an inhibition of proliferation and the promotion of maturation. These effects were accompanied by changes in cell-cycle proteins that suggest possible mechanisms for these effects. Thus, CNP may function in the postnatal brain to regulate the exit from the cell cycle in neuronal precursor cells.

Cell Cycle Proteins Exhibit Altered Expression Patterns in Lentiviral-Associated Encephalitis

Cell cycle proteins regulate processes as diverse as cell division and cell death. Recently their role in neuronal death has been reported in several models of neurodegeneration. We have reported previously that two key regulators of the cell cycle, the retinoblastoma susceptibility gene product (pRb) and transcription factor E2F1, exhibit altered immunostaining patterns in simian immunodeficiency virus encephalitis (SIVE). Here we show that E2F1 and the inactivated, hyperphosphorylated form of pRb (ppRb) also exhibit altered immunostaining patterns in human immunodeficiency virus encephalitis (HIVE). Quantification of E2F1 and ppRb staining by immunofluorescent confocal microscopy confirms a significant increase in E2F1 and ppRb in both HIVE and the simian model. This increase in E2F1 and ppRb staining correlates with an increase in the presence of activated macrophages, suggesting a link between changes in cell cycle proteins and the presence of activated macrophages. Changes in ppRb and E2F1 staining in SIVE also correlate with alterations in E2F/DNA binding complexes present in the nuclear and cytoplasmic fractions from both midfrontal cortex and basal ganglia. These findings suggest that changes in cell cycle proteins occur in both HIVE and the simian model and that these changes have functional implications for gene expression in neural cells under encephalitic conditions mediated by macrophage activation or infiltration.