Cell Cycle Retardation Research Articles

Activation of GPER Induces Differentiation and Inhibition of Coronary Artery Smooth Muscle Cell Proliferation

BackgroundVascular pathology and dysfunction are direct life-threatening outcomes resulting from atherosclerosis or vascular injury, which are primarily attributed to contractile smooth muscle cells (SMCs) dedifferentiation and proliferation by re-entering cell cycle. Increasing evidence suggests potent protective effects of G-protein coupled estrogen receptor 1 (GPER) activation against cardiovascular diseases. However, the mechanism underlying GPER function remains poorly understood, especially if it plays a potential role in modulating coronary artery smooth muscle cells (CASMCs).Methodology/Principal FindingsThe objective of our study was to understand the functional role of GPER in CASMC proliferation and differentiation in coronary arteries using from humans and swine models. We found that the GPER agonist, G-1, inhibited both human and porcine CASMC proliferation in a concentration- (10−8 to 10−5 M) and time-dependent manner. Flow cytometry revealed that treatment with G-1 significantly decreased the proportion of S-phase and G2/M cells in the growing cell population, suggesting that G-1 inhibits cell proliferation by slowing progression of the cell cycle. Further, G-1-induced cell cycle retardation was associated with decreased expression of cyclin B, up-regulation of cyclin D1, and concomitant induction of p21, and partially mediated by suppressed ERK1/2 and Akt pathways. In addition, G-1 induces SMC differentiation evidenced by increased α-smooth muscle actin (α-actin) and smooth muscle protein 22α (SM22α) protein expressions and inhibits CASMC migration induced by growth medium.ConclusionGPER activation inhibits CASMC proliferation by suppressing cell cycle progression via inhibition of ERK1/2 and Akt phosphorylation. GPER may constitute a novel mechanism to suppress intimal migration and/or synthetic phenotype of VSMC.

TCH-1030 targeting on topoisomerase I induces S-phase arrest, DNA fragmentation, and cell death of breast cancer cells

Camptothecin (CPT) and its derivatives are powerful anticancer agents, but these compounds are chemically unstable due to their α-hydroxy lactone six-membered E-ring structure, which is essential for trapping topoisomerase I (topo I)-DNA cleavage complexes. Moreover, the reversibility of trapping the topo I-DNA cleavage complex and the tight binding of CPTs to human serum albumin limit the levels of available active drug. CPT analogs are the only clinically available drugs that target topo I. Owing to the clinical importance of CPT analogs, the development of new anticancer agents which inhibit topo I is urgently needed. In the present study, we report the synthesis, biologic evaluation, and molecular mechanism of a series of substituted indeno[1,2-c]quinoline derivatives against the growth of several human cancer cell lines. We found that 9-methoxy-6-(piperazin-1-yl)-11H-indeno[1,2-c]quinoline-11-one O-3-(dimethylamino)propyl oxime (TCH-1030) intercalated into DNA and preferentially inhibited DNA topo I relaxation. Flow cytometric analysis and BrdU incorporation assays indicate that TCH-1030 alters cell cycle progression, induces S-phase arrest, and causes DNA polyploidy (>4N) that is distinct from the typical G2-M arrest reported with known topoisomerase toxins. Our data indicate that TCH-1030 induces caspase 3 activation, PARP cleavage, γ-H2AX phosphorylation, and, consequently, DNA fragmentation and apoptosis. We also demonstrated that treatment with TCH-1030 significantly inhibits tumor growth in a BT483-xenograft nude mouse model. Taken together, we conclude that the primary mechanism of action of TCH-1030-induced cell cycle retardation and apoptosis-mediated DNA damage involves DNA binding and intercalation as well as topo I inhibition.

Suppression of human HCC cell growth in vitro by siRNA silencing of forkhead box M1 expression.

173 Background: Development of targeted therapy for hepatocellular carcinoma (HCC) remains a major challenge. The transcription factor forkhead box M1 (FOXM1) is a member of the forkhead family, and it induces the expression of genes involved in the execution of mitosis. Liver regeneration studies that used the albumin promoter/enhancer cre recombinase (Alb-Cre) transgene to mediate hepatocyte-specific deletion of the mouse Foxm1 LoxP/LoxP targeted allele (Foxm1fl/fl) demonstrated that Foxm1 is required for hepatocyte DNA replication and mitosis. Methods: We explored the possibility of FoxM1 being a potential therapeutic target for HCC.To inactivate target gene expression, Huh7 and HepG2 cells were treated with the various doses of three different siRNAs (FoxM1-1, FoxM1-2 and FoxM1-3). The change of FoxM1 expression in mRNA level was then determined by quantitative real-time PCR. Cell growth was analyzed by MTT assay, and apoptosis was estimated by ELISA for the detection of denatured ssDNA only formed during apoptotic progression. Results: Among the three tested siRNA molecules, the FoxM1-1siRNA was the most effective in inhibiting HCC cell growth. Both Huh7 and HepG2 cells transfected with 15nM of FoxM1-1siRNA showed more than 60% growth inhibition by 4 days. Inhibition of cell growth was due to increased rate of apoptotic cell death which was about 2-fold higher than in untreated control cells. Upregulation of apoptosis directly correlated with down-regulation of FoxM1 mRNA level. Furthermore, siRNA-mediated silencing of FoxM1 expression in HCC cells retarded cell cycle progression. Conclusions: These results indicate that FoxM1 plays a pivotal role in HCC pathogenesis and maybe an attractive molecular target for systemic HCC therapy.

Chemopreventive Effects of Pterostilbene on Urethane-Induced Lung Carcinogenesis in Mice via the Inhibition of EGFR-Mediated Pathways and the Induction of Apoptosis and Autophagy

Lung cancer is the most commonly diagnosed cancer and the leading cause of cancer deaths globally. Due to the lack of successful chemopreventive agents for lung cancer, there is an emerging need to evaluate new and effective agents for lung cancer prevention. Pterostilbene, a naturally occurring analogue of resveratrol, has been reported to be an effective chemopreventive agent against many cancers. The aim of this study is to investigate the chemopreventive effects of pterostilbene in urethane-induced murine lung tumors. Pretreatment with pterostilbene at 50 or 250 mg/kg significantly reduced tumor multiplicity by 26 and 49%, respectively. Pterostilbene also significantly inhibited tumor volume by 25 and 34% and decreased the tumor burden per mouse by 45 and 63%, respectively. The mechanisms by which pterostilbene suppresses lung tumorigenesis have been investigated in lung tissues and homogenates. The results indicate that the pterostilbene-mediated chemopreventive effects in vivo were a result of the inhibition of epidermal growth factor receptor (EGFR) and its downstream pathways, leading to retarded cell cycle progression, and of the induction of apoptosis and autophagy during urethane-induced lung tumorigenesis.

Decreased expression of LATS1 is correlated with the progression and prognosis of glioma

BackgroundLATS1 is a tumor suppressor genes implicated in the pathogenesis of certain types of tumors, but its role is not known in human glioma.MethodsUsing real-time PCR and immunohistochemistry, we detected the mRNA and protein expression of LATS1 in glioma. The effect of LATS1 on cell growth and invasion were investigated.ResultsWe found that mRNA and protein of LATS1 expression is significantly downregulated in glioma compared with normal control brain tissues. Furthermore, reduced LATS1 expression was markedly negatively correlated with WHO grade and KPS (p<0.001 and p<0.001) in glioma patients. Patients with lower LATS1 expression had a significantly shorter overall survival time than did patients with higher LATS1 expression. Multivariate analysis suggested that the level of LATS1 expression was an independent prognostic indicator (p<0.001) for the survival of patients with glioma. Forced expression of LATS1 in glioma U251 cells not only significantly suppressed cell growth, migration and invasion, but retarded cell cycle progression from G2/M to G1 in vitro. Finally, we found that overexpressed LATS1 markedly inhibited the expression level of cell cycle factor CCNA1.ConclusionThese results indicate that LATS1 is an important candidate tumor suppressor and its downregulated expression may contribute to glioma progression.

Differential responses of endogenous adult mouse neural precursors to excess neuronal excitation

Adult neurogenesis in the subgranular zone of the hippocampus (SGZ) is enhanced by excess as well as mild neuronal excitation, such as chemoconvulsant-induced brief seizures. Because most studies of neurogenesis after seizures have focused on the SGZ, the threshold of neuronal excitation required to enhance neurogenesis in the subventricular zone (SVZ) is not clear. Therefore, we examined the responses of SVZ precursors to brief generalized clonic seizures induced by a single administration of the chemoconvulsant pentylenetetrazole (PTZ). Cell cycle progression of precursors was analysed by systemic administration of thymidine analogues. We found that brief seizures immediately resulted in cell cycle retardation in the SVZ. However, the same effect was not seen in the SGZ. This initial cell cycle retardation in the SVZ was followed by enhanced cell cycle re-entry after the first round of mitosis, leading to precursor pool expansion, but the cell cycle retardation and expansion of the precursor pool were transient. Cell cycle progression in the PTZ-treated group returned to normal after one cell cycle. The numbers of precursors in the SVZ and new neurons in the olfactory bulb, which are descendants of SVZ precursors, were not significantly different from those in control mice more than 2 days after seizures. Because similar effects were observed following electroconvulsive seizures, these responses are likely to be general effects of brief seizures. These results suggest that neurogenesis in the SVZ is more tightly regulated and requires stronger stimuli to be modified than that in the SGZ.

Alberto Abbruzzese (1952–2011) Scientist who studied from amino acids to nanoparticles in anti-cancer therapy

Alberto Abbruzzese (1952–2011) Scientist who studied from amino acids to nanoparticles in anti-cancer therapy

Activation of G‐protein‐coupled estrogen receptor (GPER) inhibits coronary artery smooth muscle cell proliferation

One aspect of the beneficial influences of 17β‐estradiol on vascular smooth muscle cells (VSMC) is inhibition of VSMC proliferation following vascular injury. GPER is a potential mediator of estrogen action on coronary arteries, but whether GPER plays a role in coronary artery smooth muscle proliferation has never been reported. The objective of our study was to test the functional role of GPER in human and porcine coronary (CASMC) proliferation by applying flow cytometric analysis and Western blotting. The results are as follows: G‐1 (GPER agonist) inhibited both human and porcine CASMC proliferation in a concentration and time‐dependent manner (10−8 to 10−5 M). Flow cytometry revealed that G‐1 significantly decreased the proportion of S‐phase and G2/M cells in the growing cell population (stimulated by 10% charcoal/dextron striped FBS or 10 nM PDGF‐BB), suggesting that G‐1‐induces cell proliferation by slowing down the progression of the cell cycle. Western blot revealed that G‐1‐induced cell cycle retardation was associated with decreased expression of cyclinB, up‐regulation of cyclinD1, and concomitant induction of p21. G‐1 treatment also inhibited the phosphorylation of ERK1/2 and Akt. We conclude that GPER activation inhibits CASMC proliferation by retardation of cell cycle progression via inhibiting ERK1/2 and Akt activities.

The RhoA-ROCK-PTEN pathway as a molecular switch for anchorage dependent cell behavior

The proliferation of anchorage-dependent cells of mesenchymal origin requires the attachment of the cells to substrates. Thus, cells that are poorly attached to substrates exhibit retarded cell cycle progression or apoptotic death. A major disadvantage of most polymers used in tissue engineering is their hydrophobicity; hydrophobic surfaces do not allow cells to attach firmly and, therefore, do not allow normal proliferation rates. In this study, we investigated the molecular mechanism underlying the reduced proliferation rate of cells that are poorly attached to substrates. There was an inverse relationship between the activity of the small GTPase RhoA (RhoA) and the cell proliferation rate. RhoA activity correlated inversely with the strength of cell adhesion to the substrates. The high RhoA activity in the cells poorly attached to substrates caused an increase in the activity of Rho-associated kinase (ROCK), a well-known effector of RhoA that upregulated the activity of phosphatase and tensin homolog (PTEN). The resulting activated PTEN downregulated Akt activity, which is essential for cell proliferation. Thus, the cells that were poorly attached to substrates showed low levels of cell proliferation because the RhoA-ROCK-PTEN pathway was hyperactive. In addition, RhoA activity seemed to be related to focal adhesion kinase (FAK) activity. Weak FAK activity in these poorly attached cells failed to downregulate the high RhoA activity that restrained cell proliferation. Interestingly, reducing the expression of any component of the RhoA-ROCK-PTEN pathway rescued the proliferation rate without physico-chemical surface modifications. Based on these results, we suggest that the RhoA-ROCK-PTEN pathway acts as a molecular switch to control cell proliferation and determine anchorage dependence. In cells that are poorly attached to substrates, its inhibition is sufficient to restore cell proliferation without the need for physico-chemical modification of the material surface.

Lowered Nudix type 5 (NUDT5) expression leads to cell cycle retardation in HeLa cells

The molecule 8-oxo-7,8-dihydroguanine (8-oxoGua), an oxidized form of guanine, can pair with adenine or cytosine during nucleic acid synthesis. Moreover, RNA containing 8-oxoGua causes translational errors, thus leading to the production of abnormal proteins. Human NUDT5, a MutT-related protein, catalyzes the hydrolysis of 8-oxoGDP to 8-oxoGMP, thereby preventing misincorporation of 8-oxoGua into RNA. To investigate the biological roles of NUDT5 in mammalian cells, we established cell lines with decreased level of NUDT5 expression. In NUDT5 inhibited cells, the RNA oxidation was not significantly higher than that of normal cells. However, the cell cycle G1 phase was significantly delayed, and cell numbers in both S and G2/M phases were reduced, indicating that cell proliferation was hampered by NUDT5 suppression. Key proteins for preventing the G1-S transition, including p53, p16, and Rb were increased, while the Rb phosphorylation was decreased. These results suggested that the NUDT5 protein may play significant roles in regulating the G1-S transition in mammalian cells.

Abstract 1634: Targeting L1 cell adhesion molecule using lentivirus-mediated short hairpin RNA interference reverses aggressiveness of oral squamous cell carcinoma

Abstract Oral cancer consistently ranks as one of the ten most frequently diagnosed cancers in the world. Despite multidisciplinary treatment with surgery, chemotherapy, and radiation, more than 50% of patients with oral squamous cell carcinoma (OSCC) receiving therapy will eventually develop recurrent and metastatic disease, which carries a particularly poor prognosis. Characterization of the specific molecular alternations associated with OSCC cell growth, invasion, and metastasis can advance our understanding of the molecular mechanisms underlying cancer induction and progression as well provide a basis for the development of new and effective therapeutic treatments for cancer patients. The L1 cell adhesion molecule (L1CAM) has been implicated in tumor progression of many types of cancers, but its role in OSCC has not been investigated. In the present study, we demonstrated overexpression of L1CAM in OSCC cells, but not in normal keratinocytes, using both clinical specimens and cell lines. This overexpression demonstrated a strong correlation with less differentiation and a higher invasion potential of cancer cells, supporting the significance of L1CAM in human OSCC tumor progression. Targeting L1CAM gene expression in SCC4 cells overexpressing L1CAM using a lentivirusmediated small hairpin RNA (shRNA) led to a significant reduction in cell proliferation in vitro via retardation of cell cycle at the G1 phase. In addition, shRNA knockdown of L1CAM strongly attenuated the migration and invasion of SCC4 cells, and this was also observed to parallel increased E-cadherin levels and decreased levels vimentin, fibronectin, and Snail-family transcription factors, indicating that L1CAM expression was related to the epithelial-mesenchymal transition. Furthermore, while mice receiving orthotopically placed control SCC4 cells died within 40 days due to invasive tumor growth and regional lymph node metastasis, prolonged animal survival and complete suppression of tumor progression was observed in mice implanted with L1CAM-deficent SCC4 cells, further substantiating the fundamental importance of L1CAM in OSCC pathophysiology. Our findings suggested that L1CAM is a critical mediator of tumor progression in OSCC, and targeting L1CAM using lentivirus-mediated shRNA may be a useful molecular pharmaceutical approach for the treatment of advanced OSCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1634. doi:10.1158/1538-7445.AM2011-1634

A cell-death-defying factor, anamorsin mediates cell growth through inactivation of PKC and p38MAPK

Anamorsin (AM) plays crucial roles in hematopoiesis and embryogenesis. AM deficient (AM KO) mice die during late gestation; AM KO embryos are anemic and very small compared to wild type (WT) embryos. To determine which signaling pathways AM utilizes for these functions, we used murine embryonic fibroblast (MEF) cells generated from E-14.5 AM KO or WT embryos. Proliferation of AM KO MEF cells was markedly retarded, and PKCθ, PKCδ, and p38MAPK were more highly phosphorylated in AM KO MEF cells. Expression of cyclinD1, the target molecule of p38MAPK, was down-regulated in AM KO MEF cells. p38MAPK inhibitor as well as PKC inhibitor restored expression of cyclinD1 and cell growth in AM KO MEF cells. These data suggest that PKCθ, PKCδ, and p38MAPK activation lead to cell cycle retardation in AM KO MEF cells, and that AM may negatively regulate novel PKCs and p38MAPK in MEF cells.

In Vitro Study on Apoptosis Induced by Strontium‐89 in HumanBreast Carcinoma Cell Line

Many radiopharmaceuticals used for medical diagnosis and therapy are beta emitters; however, the mechanism of the cell death caused by beta-irradiation is not well understood. The objective of this study was to investigate the apoptosis of human breast carcinoma MCF-7 cell lines induced by Strontium-89 (89Sr) and its regulation and control mechanism. High-metastatic Breast Carcinoma MCF-7 cells were cultured in vitro using 89Sr with different radioactive concentration. The inhibition rate of cell proliferation was measured by MTT color matching method. The cell cycle retardation, apoptosis conditions, mitochondrion transmembrane potential difference and Fas expression were tested and analyzed. The genes P53 and bcl-2 expressions was also analyzed using immunity histochemical analysis. After being induced by 89Sr with various of radioactive concentration, it was found that the inhibition of cell proliferation of MCF-7 cells was obviously, the retardation of cell cycle occurred mainly in G2-M. It was also found that the obvious apoptosis occurred after being induced by 89Sr, the highest apoptosis rate reached 46.28%. The expressions of Fas acceptor and P53 gene increased, while bcl-2 gene expression decreasesd. These findings demonstrate that in the ranges of a certain radioactive concentration, the inhibition rate of MCF-7 cell proliferation and retardation of cell cycle had positive correlation with the concentration of 89Sr. And the mitochondrion transmembrane potential decrease would induce the apoptosis of MCF-7 cell notably, which were controlled by P53 and bcl-2 genes, involved with the Fas acceptor.

A Cell-Death-Defying Factor, Anamorsin, Contributes Cell Growth through Binding with PICOT and Inactivation of PKCs and p38MAPK.

AbstractAbstract 1552 [Background]Anamorsin (also called CIAPIN-1) is a cell-death-defying factor, which was originally isolated as a molecule that conferred resistance to apoptosis induced by growth factor starvation. Anamorsin is ubiquitously expressed in various organs, including hematopoietic tissues like bone marrow, spleen, and thymus. Anamorsin-deficient (AM KO) mice die in late gestation. AM KO embryos are anemic and the size of the embryos is very small. It is thought that anamorsin plays a crucial role in hematopoiesis during late and/or terminal stages of differentiation and embryogenesis. Anamorsin does not show any homology to known apoptosis and cell growth regulatory molecules such as Bcl-2 family, caspase family, or signal transduction molecules. (J Exp Med 199: 581–592, 2004) Anamorsin is composed of a methyltransferase domain in the N-terminal region and a hypothetical Zn-ribbon-like motif in the C-terminal region, however, the precise biological effects of anamorsin remained to be elucidated. [Methods & Results]In an attempt to clarify the mechanisms of the anamorsin functions, we have performed the yeast-two-hybrid assay to identify anamorsin-interacting molecules and found that PICOT (PKCθ interacting cousin of thioredoxin) preferentially bound to anamorsin. Next, we tried to determine the binding sites of anamorsin and PICOT with the yeast-two-hybrid assays by using their several deletion mutants and found that the N-terminal region (11-180aa) of anamorsin and the N-terminal region (18-117aa) of PICOT were essential for binding each other. Furthermore, we tried to examine the signaling pathways by using murine embryonic fibroblast (MEF) cells produced from E-14.5 AM KO or wild type (WT) embryos. The proliferation of AM KO MEF cells was quite retarded compared with that of WT MEF cells. It is found that the phosphorylation states of ERK1/2, NFkB, and AKT were similar both in AM KO MEF cells and WT MEF cells, while PKCθ, PKCδ and p38 MAPK were more phosphorylated in AM KO MEF cells than in WT MEF cells. The expression of cyclin D1, the target molecule of p38 MAPK, was down-regulated in AM KO MEF cells. The PKC inhibitor, Rottlerin, blocked p38 MAPK phosphorylation and the p38 MAPK inhibitor, SB203580, restored the expression of cyclin D1 and the cell growth of AM KO MEF cells. [Conclusion]P38 MAPK, the stress activated MAPK, and PKCs have been known to link to cell growth, differentiation, and apoptosis, and also to be essential for cell survival in response to various stimuli. From our results, it was thought that PKCθ, PKCδ, and p38 MAPK activation lead to cell cycle retardation of AM KO MEF cells and anamorsin might negatively regulate PKCθ, PKCδ, and p38 MAPK cooperatively with PICOT in MEF cells. This study clarified a novel mechanism of the anamorsin functions. Disclosures:No relevant conflicts of interest to declare.

Targeting L1 Cell Adhesion Molecule Using Lentivirus-Mediated Short Hairpin RNA Interference Reverses Aggressiveness of Oral Squamous Cell Carcinoma

The L1 cell adhesion molecule (L1CAM) has been implicated in tumor progression of many types of cancers, but its role in oral squamous cell carcinoma (OSCC) has not been investigated. In the present study, we demonstrated overexpression of L1CAM in OSCC cells, but not in normal keratinocytes, using both clinical specimens and cell lines. This overexpression demonstrated a strong correlation with less differentiation and a higher invasion potential of cancer cells, supporting the significance of L1CAM in human OSCC tumor progression. Targeting L1CAM gene expression in SCC4 cells overexpressing L1CAM using a lentivirus-mediated small hairpin RNA (shRNA) led to a significant reduction in cell proliferation in vitro via retardation of cell cycle at the G1 phase. In addition, shRNA knockdown of L1CAM strongly attenuated the migration and invasion of SCC4 cells, and this was also observed to parallel increased E-cadherin levels and decreased levels of vimentin, fibronectin, and Snail-family transcription factors, indicating that L1CAM expression was related to the epithelial-mesenchymal transition. Furthermore, while mice receiving orthotopically placed control SCC4 cells died within 40 days due to invasive tumor growth and regional lymph node metastasis, prolonged animal survival and complete suppression of tumor progression was observed in mice implanted with L1CAM-deficent SCC4 cells, further substantiating the fundamental importance of L1CAM in OSCC pathophysiology. Our findings suggested that L1CAM is a critical mediator of tumor progression in OSCC, and targeting L1CAM using lentivirus-mediated shRNA may be a useful molecular pharmaceutical approach for the treatment of advanced OSCC.

Transfer of 4-hydroxynonenal, a inhibitory hemozoin (HZ) product, from HZ or HZ-laden phagocytes to developing human erythroid cells. A model for erythropoiesis inhibition in malaria anemia

Blood stage P. falciparum (Pf) accumulates hemozoin (HZ), the crystal of dimerized undigested heme. HZ wrapped up in the food vacuole membrane binds saturated and polyunsaturated fatty acids (PUFA) and proteins, generates bioactive ligands and is expelled in the blood during schizogony as residual body. HZ contains redox active iron that peroxidizes PUFA to produce bioactive hydroxy-PUFA (HETEs; HODEs) and terminal aldehydes (4-hydroxynonenal, HNE). HETEs is responsible for some of the HZ effects: (1) down-modulation of oxidative burst; (2) inhibition of dendritic cells (DC) differentiation-maturation; and (3) enhancement of expression and enzyme activity of metalloproteinase MMP-9. Low-micromolar HETE recapitulated effects (2) and (3). Recapitulation was abrogated by PPAR-gamma-receptor inhibitors. Other effects, such as inhibition of erythropoiesis, were due to the transfer of HNE to differentiating human erythroid precursors. Erythropoiesis was inhibited by transferred HNE via blockage of cell cycle and the down-regulation of protein expression of crucial receptors (erythropoietinR, transferrinR and stem-cell-factorR). Malarial dyserythropoiesis, which plays an important role in malaria anaemia (MA) is characterized by (1) decreased growth and differentiation of erythroid precursors; (2) low reticulocyte response; and (3) normal production of erythropoietin (EPO). In human bone marrow (BM) the erythroblastic island (EI) is the elementary unit of erythropoiesis in which a central macrophage (MAC) is surrounded by differentiating erythroid cells. In malaria anaemia, HZ-laden central MACs are closely adjacent to developing erythroid cells. HZ and HZ-laden human monocytes inhibited growth of co-cultivated human erythroid cells and produced HNE that diffused to adjacent cells generating HNE-protein adducts. Co-cultivation with HZ or treatment with low-micromolar HNE inhibited growth of erythroid cells interfering with cell cycle without apoptosis. Following HZ/HNE treatment, two critical proteins in cell cycle regulation, p53 and p21, were increased and the retinoblastoma protein, central regulator of G1-to-S-phase transition, was consequently hypophosphorylated, while GATA-1, master transcription factor in erythropoiesis was reduced. The resul was decreased expression of cyclin A and D2 retarded cell cycle progression in erythroid cells and the K562 cell line. As a second major effect, HZ and HNE inhibited protein expression of crucial receptors (R): transferrin R1, stem cell factorR, interleukin-3R and erythropoietinR. The reduced receptor expression and the impaired cell cycle activity decreased the production of cells expressing glycophorin-A and hemoglobin. In conclusion, present data confirm the inhibitory role of HZ, identify HNE as one HZ-generated inhibitory molecule and describe molecular targets of HNE in erythroid progenitors possibly involved in erythropoiesis inhibition in malaria anemia. Supported by Regione Piemonte, IARC and Compagnia di San Paolo grants to ES, WP and PA.

Regulation of cell division and growth in roots of Lactuca sativa L. seedlings by the Ent-Kaurene diterpenoid rabdosin B.

Rabdosin B, an ent-kaurene diterpenoid purified from the air-dried aerial parts of Isodon japonica (Burm.f) Hara var. galaucocalyx (maxin) Hara, showed a biphasic, dose-dependent effect on root growth and a strong inhibitory effect on root hair development in lettuce seedlings (Lactuca sativa L.). Lower concentrations of rabdosin B (20-80 microM) significantly promoted root growth, but its higher levels at 120-200 microM, by contrast, had inhibitory effects. Additionally, all tested concentrations (10-40 microM) inhibited root hair development of seedlings in a dose-dependent manner. Further investigations on the underlying mechanism revealed that the promotion effect of rabdosin B at the lower concentrations resulted from increasing the cell length in the mature region and enhancing the mitotic activity of meristematic cells in seedlings' root tips. In contrast, rabdosin B at higher concentrations inhibited root growth by affecting both cell length in the mature region and division of meristematic cells. Comet assay and cell cycle analysis demonstrated that the decrease of mitotic activity of root meristematic cells was due to DNA damage induced cell cycle retardation of the G(2) phase and S phase at different times.

Induction of G1 arrest in glioma cells by T11TS is associated with upregulation of Cip1/Kip1 and concurrent downregulation of cyclin D (1 and 3)

In our laboratory, a novel therapeutic probe, T11TS, a membrane glycoprotein, was isolated which had antineoplastic activity against experimental glioma. Development of a novel therapeutic strategy with T11TS has unearthed a newer dimension of its mechanism of action: modulation of the cell cycle. In this study, we have presented evidence to support the finding that T11TS induces G1 cell cycle arrest of rat glioma cells. Results of flow cytometric studies showed that the treatment produced a marked increase in the proportion of cells in the G1 phase. Flow cytometry, immunoblotting, immunoprecipitation, and kinase assays were performed for investigating the involvement of G1 cell cycle regulators. T11TS induces downregulation of the cyclin-D (1 and 3) expression with the concurrent upregulation of p21 and p27 and their concomitant association with cyclin-dependent kinase 4, proliferating cell nuclear antigen and cyclin E respectively leading to a decrease in cyclin-dependent kinase 4 kinase activity. A transient rise in retinoblastoma protein level and coordinated binding of retinoblastoma protein with E2F coincided with the accumulation of cells in G1 phase. Thus, our observations have uncovered an antiproliferative pathway for T11TS, causing retardation of glioma cell cycle.

Short 42 C heat shock induces phosphorylation and degradation of Cdc25A which depends on p38MAPK, Chk2 and 14.3.3

The effects of heat shock (HS; 42 degrees C) on the cell cycle and underlying molecular mechanisms are astonishingly unexplored. Here, we show that HS caused rapid Cdc25A degradation and a reduction of cell cycle progression. Cdc25A degradation depended on Ser75-Cdc25A phosphorylation caused by p38MAPK and Chk2, which phosphorylated Ser177-Cdc25A that is specific for 14.3.3 binding. Upon HS, Cdc25A rapidly co-localized with 14.3.3 in the perinuclear space that was accompanied with a decrease of nuclear Cdc25A protein levels. Consistently, a 14.3.3 binding-deficient Cdc25A double mutant (Ser177/Ala-Tyr507/Ala) was not degraded in response to HS and there was no evidence for an increased co-localization of Cdc25A with 14.3.3 in the cytosol. Therefore, upon HS, p38, Chk2 and 14.3.3 were antagonists of Cdc25A stability. On the other hand, Cdc25A was protected by Hsp90 in HEK293 cells because the specific inhibition of Hsp90 with Geldanamycin caused Cdc25A degradation in HEK293 implicating that Cdc25A is an Hsp90 client. Specific inhibition of Hsp90 together with HS caused and accelerated degradation of Cdc25A and was highly cytotoxic. The results presented here show for the first time that Cdc25A is degraded by moderate heat shock and protected by Hsp90. We describe the mechanisms explaining HS-induced cell cycle retardation and provide a rationale for a targeted hyperthermia cancer therapy.

Nelfinavir induces the unfolded protein response in ovarian cancer cells, resulting in ER vacuolization, cell cycle retardation and apoptosis

Proteasome inhibitors and protease inhibitors are currently being discussed to be useful to sensitize drug-resistant cancer cells to chemotherapeutic agents or to act independently as single agents on drug resistant cancer cells. We tested the effect of the clinically applied HIV protease inhibitor nelfinavir on ovarian cancer cells. Nelfinavir efficiently induced cell death in carboplatin-sensitive (SKOV3, OV-GH-5) and carboplatin-resistant (OVCAR3, OV-GH-1) ovarian cancer cell lines as well as in cancer biopsies and ascites samples from patients with recurrent ovarian cancer. Nelfinavir significantly changed the morphology of ovarian cancer cells, resulting in formation of large ER-derived vacuoles and induced upregulation of the hsp70 heat shock family member BiP (GRP78) which accumulated within swollen ER membranes. Upregulation of BiP and phosphorylation of eIF2alpha indicated induction of the unfolded protein response, which can cause cell cycle arrest and apoptosis. Correspondingly, we observed downregulation of cell cycle regulatory proteins after nelfinavir treatment, especially that of cyclin D3, and induction of apoptosis as confirmed by annexin binding. Because nelfinavir represents an already approved drug for use in humans with HIV infection, it could rapidly be tested in clinical studies as a potential treatment strategy against drug-resistant ovarian cancer.