Induced Cell Cycle Progression Research Articles

Antioxidants counteract lipopolysaccharide-triggered alterations of human colonic smooth muscle cells

Gut dysmotility develops in individuals during and after recovering from infective acute gastroenteritis and it is apparently due to a direct effect of circulating lipopolysaccharides (LPS). This is an endotoxin with a prooxidant activity derived from gram-negative bacteria. Due to the lack of human models available so far, the mechanisms underlying LPS-induced gut dysmotility are, however, poorly investigated. In the present work long-term effects of LPS and their reversibility have been assessed by means of different analytical cytology methods on pure primary cultures of human colonic smooth muscle cells. We found that LPS triggered the following alterations: (i) a redox imbalance with profound changes of contractile microfilament network, and (ii) the induction of cell cycle progression with dedifferentiation from a contractile to a synthetic phenotype. These alterations persisted also after LPS removal. Importantly, two unrelated antioxidants, alpha-tocopherol and N-acetylcysteine, were able to reverse the cytopathic effects of LPS and to restore normal muscle cell function. The present data indicate that LPS is capable of triggering a persistent and long-term response that could contribute to muscle dysfunction occurring after an infective and related inflammatory burst and suggest a reappraisal of antioxidants in the management of postinfective motor disorders of the gut.

Activated Ras Protein Accelerates Cell Cycle Progression to Perturb Madin-Darby Canine Kidney Cystogenesis

In a number of human cancer cells, K-RAS is frequently mutated and activated constitutively, culminating in the induction of continuous cell growth, a hallmark of cancer cells. It is still unclear, however, how the mutated K-RAS induces morphological abnormalities in cancerous tissues. To investigate the mechanism underlying the K-RAS-induced morphological changes, we utilized an auxin-dependent protein expression system, which enabled us to rapidly induce and evaluate constitutively active K-Ras in MDCK (Madin-Darby canine kidney) cysts, a model for polarized epithelial structure. Cells carrying the constitutively active KRasV12 gene were morphologically indistinguishable from normal cells in two-dimensional culture. However, in a gel of extracellular matrix, KRasV12-expressing cells failed to form a spherical cyst. When KRasV12 induction was delayed until after cyst formation, some cells in the cyst wall lost polarity and were extruded into and accumulated in the luminal space. With effector-specific mutants of KRasV12 and inhibitors for MEK and PI3-kinase, we found that both the Raf-MEK-ERK and PI3-kinase axes are necessary and sufficient for this phenotype. Live cell imaging with cell cycle indicators showed that KRasV12 expression promoted cell cycle progression, which was prevented by either MEK or PI3-kinase inhibitors. From these results, we provide a model wherein active-Ras induces cell cycle progression leading to apical cell extrusion through Raf and PI3-kinase in a cooperative manner. The system developed here can be applied to drug screening for various cancers originating from epithelial cells.

Multiple biomarker tissue arrays: A computational approach to identifying protein-protein interactions in the EGFR/ERK signalling pathway

BackgroundMany studies have demonstrated genetic and environmental factors that lead to renal cell carcinoma (RCC) and that occur during a protracted period of tumourigenesis. It appears suitable to identify and characterise potential molecular markers that appear during tumourigenesis and that might provide rapid and effective possibilities for the early detection of RCC. EGFR activation induces cell cycle progression, inhibition of apoptosis and angiogenesis, promotion of invasion/metastasis, and other tumour promoting activities. Over-expression of EGFR is thought to play an important role in tumour initiation and progression of RCC because up-regulation of EGFR has been associated with high grade cancers and a worse prognosis.MethodsCharacterisation of the protein profile interacting with EGFR was performed using the following: an immunohistochemical (IHC) study of EGFR, a comprehensive computational study of EGFR protein-protein interactions, an analysis correlating the expression levels of EGFR with other significant markers in the tumourigenicity of RCC, and finally, an analysis of the utility of EGFR for prognosis in a cohort of patients with renal cell carcinoma.ResultsThe cases that showed a higher level of this protein fell within the clear cell histological subtype (p = 0.001). The EGFR significance statistic was found with respect to a worse prognosis. In vivo significant correlations were found with PDGFR-β, Flk-1, Hif1-α, proteins related to differentiation (such as DLL3 and DLL4 ligands), and certain metabolic proteins such as Glut5. In silico significant associations gave us a panel of 32 EGFR-interacting proteins (EIP) using the APID and STRING databases.ConclusionsThis work summarises the multifaceted role of EGFR in the pathology of RCC, and it identifies EIPs that could help to provide mechanistic explanations for the different behaviours observed in tumours.

Dehydrocostuslactone Suppresses Angiogenesis In Vitro and In Vivo through Inhibition of Akt/GSK-3β and mTOR Signaling Pathways

The traditional Chinese medicine component dehydrocostuslactone (DHC) isolated from Saussurea costus (Falc.) Lipschitz, has been shown to have anti-cancer activity. Angiogenesis is an essential process in the growth and progression of cancer. In this study, we demonstrated, for the first time, the anti-angiogenic mechanism of action of DHC to be via the induction of cell cycle progression at the G0/G1 phase due to abrogation of the Akt/glycogen synthase kinase-3β (GSK-3β)/cyclin D1 and mTOR signaling pathway. First, we demonstrated that DHC has an anti-angiogenic effect in the matrigel-plug nude mice model and an inhibitory effect on human umbilical vein endothelial cell (HUVEC) proliferation and capillary-like tube formation in vitro. DHC caused G0/G1 cell cycle arrest, which was associated with the down-regulation of cyclin D1 expression, leading to the suppression of retinoblastoma protein phosphorylation and subsequent inhibition of cyclin A and cdk2 expression. With respect to the molecular mechanisms underlying the DHC-induced cyclin D1 down-regulation, this study demonstrated that DHC significantly inhibits Akt expression, resulting in the suppression of GSK-3β phosphorylation and mTOR expression. These effects are capable of regulating cyclin D1 degradation, but they were significantly reversed by constitutively active myristoylated (myr)-Akt. Furthermore, the abrogation of tube formation induced by DHC was also reversed by overexpression of Akt. And the co-treatment with LiCl and DHC significantly reversed the growth inhibition induced by DHC. Taken together, our study has identified Akt/GSK-3β and mTOR as important targets of DHC and has thus highlighted its potential application in angiogenesis-related diseases, such as cancer.

Cyclin D1 inhibits hepatic lipogenesis via repression of carbohydrate response element binding protein and hepatocyte nuclear factor 4α

Following acute hepatic injury, the metabolic capacity of the liver is altered during the process of compensatory hepatocyte proliferation by undefined mechanisms. In this study, we examined the regulation of de novo lipogenesis by cyclin D1, a key mediator of hepatocyte cell cycle progression. In primary hepatocytes, cyclin D1 significantly impaired lipogenesis in response to glucose stimulation. Cyclin D1 inhibited the glucose-mediated induction of key lipogenic genes, and similar effects were seen using a mutant (D1-KE) that does not activate cdk4 or induce cell cycle progression. Cyclin D1 (but not D1-KE) inhibited the activity of the carbohydrate response element-binding protein (ChREBP) by regulating the glucose-sensing motif of this transcription factor. Because changes in ChREBP activity could not fully explain the effect of cyclin D1, we examined hepatocyte nuclear factor 4α (HNF4α), which regulates numerous differentiated functions in the liver including lipid metabolism. We found that both cyclins D1 and D1-KE bound to HNF4α and significantly inhibited its recruitment to the promoter region of lipogenic genes in hepatocytes. Conversely, knockdown of cyclin D1 in the AML12 hepatocyte cell line promoted HNF4α activity and lipogenesis. In mouse liver, HNF4α bound to a central domain of cyclin D1 involved in transcriptional repression. Cyclin D1 inhibited lipogenic gene expression in the liver following carbohydrate feeding. Similar findings were observed in the setting of physiologic cyclin D1 expression in the regenerating liver. In conclusion, these studies demonstrate that cyclin D1 represses ChREBP and HNF4α function in hepatocytes via Cdk4-dependent and -independent mechanisms. These findings provide a direct link between the cell cycle machinery and the transcriptional control of metabolic function of the liver.

PD0332991, a CDK4/6 Inhibitor, Has An Anti-Leukemic Activity Against Lymphoid Crisis of CML and Ph+ALL Even with T315I Mutation in BCR-Abl; in Vitro Analysis

Abstract 1511Since BCR-ABL plays a central role in cell cycle progression of Philadelphia-chromosome positive (Ph+) leukemia cells and CDK4/6 critically involves in G1-progression of cell cycle, we analyzed sensitivity of Ph+ leukemia cell lines to compounds that act as specific CDK4/6 inhibitors. H3-thymidine uptake assay showed that both PD183812 and CBC219476 significantly inhibited cell growth of Ph+ lymphoid leukemia cell lines (n=9) in comparison with Ph+ myeloid leukemia cell lines (n=7) and Ph- ALL cell lines (n=26). Thus, we next tested the anti-leukemic activity of PD0332991, a potent CDK4/6 inhibitor that is under phase II clinical study for solid tumor patients, and found that 8 of 9 Ph+ lymphoid leukemia cell lines showed extremely higher sensitivity to PD0332991; median IC50 was <25 nM. IC50 of Ph+ lymphoid leukemia cell lines was significantly lower than that of Ph+ myeloid cell lines (200 nM, n=7) and Ph-ALL cell lines (100nM, n=25). PD0332991 effectively dephosphorylated Rb protein (pRb), and subsequently induced G1 arrest on all of Ph+ lymphoid leukemia cell lines. Moreover, PD0332991 gradually induced cell death in 4 Ph+ lymphoid leukemia cell lines. Since CDK4/6 inhibitor acts depending on intact pRb, we analyzed protein and gene expression status of Rb. Of note, all Ph+ lymphoid leukemia cell lines expressed intact pRb except for one cell line that showed relative resistance to PD0332991. In contrast, pRb was almost undetectable in Ph+ myeloid cell lines in spite of comparable level of Rb gene expression, which might be mechanism for resistance to PD0332991. However, most of Ph- ALL cell lines had intact pRb expression in spite of their relative resistance to PD0332991, indicating that Rb status alone did not explain higher PD0332991-sensitivity of Ph+ lymphoid leukemia cell lines. Thus, we assumed that Ph+ lymphoid leukemia cells showed higher PD0332991-sensitivity probably because BCR-ABL regulates CDK4/6 expression for cell cycle progression. To clarify this assumption, we treated Ph+ lymphoid leukemia cell lines with imatinib and performed immunoblot analysis of cell cycle machineries such as CDKs, cyclines, and CDK inhibitors. Of note, CDK4 expression level was frequently downregulated by imatinib in Ph+ lymphoid leukemia cell lines. Moreover, imatinib-induced downregulation of CDK4 in Ph+ lymphoid leukemia cell line was abrogated by the addition of IL-7 and FLT3 ligand, which stimulated cell cycle progression of imatinib-treated Ph+ ALL cell line. LY294002, a PI3K inhibitor, but not U0126, a MAPK inhibitor, and AG490, an inhibitor for JAK/STAT pathway, efficiently downregulated CDK4 expression in Ph+ lymphoid leukemia cell lines. Gene expression level of CDK4 in Ph+ lymphoid leukemia cell lines was downregulated by imatinib, and lactastatin, an inhibitor of protein degradation, partially inhibited imatinib-induced downregulation of CDK4 protein in Ph+ lymphoid leukemia cell lines, indicating that BCR-ABL regulates CDK4 expression both in gene expression level and in protein degradation level. These findings indicated that Ph+ lymphoid leukemia cell lines showed higher sensitivity to PD0332991 since BCR-ABL induces cell cycle progression of Ph+ lymphoid leukemia cells by regulating CDK4 as one of downstream pathways. Accordingly, we tested if PD0332991 shows anti-leukemic activity in Ph+ lymphoid leukemia cells that have a T315I mutation of BCR-ABL. SU/SR is an imatinib-resistant Ph+ ALL cell line with T315I mutation (IC50 for imatinib >10 mM), which was established from SU-Ph2, an imatinib-sensitive Ph+ ALL cell line (IC50 for imatinib <0.1 mM), after long-term culture in the presence of gradually increasing concentration of imatinib. Of note, PD0332991 effectively dephosphorylated pRb and inhibited cell growth of both SU/SR and SU-Ph2. Our findings provide a rationale for efficacy of PD0332991 in the context of anti-leukemic therapy for lymphoid crisis of CML and Ph+ ALL patients even with T315I mutation in BCR-ABL. Disclosures:No relevant conflicts of interest to declare.

Epidermal growth factor-stimulated human cervical cancer cell growth is associated with EGFR and cyclin D1 activation, independent of COX-2 expression levels

Cervical cancer constitutes the second most common cancer in women. It is evident from earlier studies that epidermal growth factor (EGF) is a mitogen, in that it mimics the function of estrogen by mediating cross-talk with other oncoproteins. Although epidermal growth factor receptor (EGFR) is highly expressed in breast and ovarian tumor tissues, its regulation by the exogenous source of its ligand EGF in human papillomavirus (HPV)-associated cervical cancer remains unclear. In this study, we addressed the question of whether EGF is required for the proliferation of HPV-positive cervical cancer cells and what mechanisms are involved. To determine this, we conducted a series of studies using HPV-positive human cervical cancer cells, CaSki and HeLa, and stimulated the cells with EGF. Our findings suggest that 6 h of stimulation with 10 ng/ml of EGF is sufficient to induce cell cycle progression associated with a significant increase in DNA synthesis, EGFR, COX-2 and cyclin D1 levels. Consistently, cellular localization and Western blot analysis for p-EGFR (Try-1045) protein showed an increase after EGF stimulation. Using siRNA gene knockdown assays we have shown that cyclin D1 siRNA has a significant negative effect on EGFR and inhibit cell growth independent of COX-2 levels. In summary, our findings reveal that an exogenous EGF stimulation may enhance HPV-related cervical cancer cell proliferation by activating EGFR and cyclin D1 that is independent of COX-2 levels, suggesting that the inhibitors of EGFR and cyclin D1 may be effective against cervical cancer cell proliferation.

Membrane attack by complement: the assembly and biology of terminal complement complexes

Complement system activation plays an important role in both innate and acquired immunity. Activation of the complement and the subsequent formation of C5b-9 channels (the membrane attack complex) on the cell membranes lead to cell death. However, when the number of channels assembled on the surface of nucleated cells is limited, sublytic C5b-9 can induce cell cycle progression by activating signal transduction pathways and transcription factors and inhibiting apoptosis. This induction by C5b-9 is dependent upon the activation of the phosphatidylinositol 3-kinase/Akt/FOXO1 and ERK1 pathways in a Gi protein-dependent manner. C5b-9 induces sequential activation of CDK4 and CDK2, enabling the G1/S-phase transition and cellular proliferation. In addition, it induces RGC-32, a novel gene that plays a role in cell cycle activation by interacting with Akt and the cyclin B1-CDC2 complex. C5b-9 also inhibits apoptosis by inducing the phosphorylation of Bad and blocking the activation of FLIP, caspase-8, and Bid cleavage. Thus, sublytic C5b-9 plays an important role in cell activation, proliferation, and differentiation, thereby contributing to the maintenance of cell and tissue homeostasis.

Comparisons between Murine Polyomavirus and Simian Virus 40 Show Significant Differences in Small T Antigen Function

Although members of a virus family produce similar gene products, those products may have quite different functions. Simian virus 40 (SV40) large T antigen (LT), for example, targets p53 directly, but murine polyomavirus LT does not. SV40 small T antigen (SVST) has received considerable attention because of its ability to contribute to transformation of human cells. Here, we show that there are major differences between SVST and polyomavirus small T antigen (POLST) in their effects on differentiation, transformation, and cell survival. Both SVST and POLST induce cell cycle progression. However, POLST also inhibits differentiation of 3T3-L1 preadipocytes and C2C12 myoblasts. Additionally, POLST induces apoptosis of mouse embryo fibroblasts. SVST reduces the proapoptotic transcriptional activity of FOXO1 through phosphorylation. On the other hand, SVST complements large T antigen and Ras for the transformation of human mammary epithelial cells (HMECs), but POLST does not. Mechanistically, the differences between SVST and POLST may lie in utilization of protein phosphatase 2A (PP2A). POLST binds both Aα and Aβ scaffolding subunits of PP2A while SVST binds only Aα. Knockdown of Aβ could mimic POLST-induced apoptosis. The two small T antigens can target different proteins for dephosphorylation. POLST binds and dephosphorylates substrates, such as lipins, that SVST does not.

Phosphodiesterase 3A (PDE3A) Deletion Suppresses Proliferation of Cultured Murine Vascular Smooth Muscle Cells (VSMCs) via Inhibition of Mitogen-activated Protein Kinase (MAPK) Signaling and Alterations in Critical Cell Cycle Regulatory Proteins

Cyclic nucleotide phosphodiesterase 3 (PDE3) is an important regulator of cyclic adenosine monophosphate (cAMP) signaling within the cardiovascular system. In this study, we examined the role of PDE3A and PDE3B isoforms in regulation of growth of cultured vascular smooth muscle cells (VSMCs) and the mechanisms by which they may affect signaling pathways that mediate mitogen-induced VSMC proliferation. Serum- and PDGF-induced DNA synthesis in VSMCs grown from aortas of PDE3A-deficient (3A-KO) mice was markedly less than that in VSMCs from PDE3A wild type (3A-WT) and PDE3B-deficient (3B-KO) mice. The reduced growth response was accompanied by significantly less phosphorylation of extracellular signal-regulated kinase (ERK) in 3A-KO VSMCs, most likely due to a combination of greater site-specific inhibitory phosphorylation of Raf-1(Ser-²⁵⁹) by protein kinase A (PKA) and enhanced dephosphorylation of ERKs due to elevated mitogen-activated protein kinase phosphatase 1 (MKP-1). Furthermore, 3A-KO VSMCs, compared with 3A-WT, exhibited higher basal PKA activity and cAMP response element-binding protein (CREB) phosphorylation, higher levels of p53 and p53 phosphorylation, and elevated p21 protein together with lower levels of Cyclin-D1 and retinoblastoma (Rb) protein and Rb phosphorylation. Adenoviral overexpression of inactive CREB partially restored growth effects of serum in 3A-KO VSMCs. In contrast, exposure of 3A-WT VSMCs to VP16 CREB (active CREB) was associated with inhibition of serum-induced DNA synthesis similar to that in untreated 3A-KO VSMCs. Transfection of 3A-KO VSMCs with p53 siRNA reduced p21 and MKP-1 levels and completely restored growth without affecting amounts of Cyclin-D1 and Rb phosphorylation. We conclude that PDE3A regulates VSMC growth via two complementary pathways, i.e. PKA-catalyzed inhibitory phosphorylation of Raf-1 with resulting inhibition of MAPK signaling and PKA/CREB-mediated induction of p21, leading to G₀/G₁ cell cycle arrest, as well as by increased accumulation of p53, which induces MKP-1, p21, and WIP1, leading to inhibition of G₁ to S cell cycle progression.

Viral-Mediated Noisy Gene Expression Reveals Biphasic E2f1 Response to MYC

Gene expression mediated by viral vectors is subject to cell-to-cell variability, which limits the accuracy of gene delivery. When coupled with single-cell measurements, however, such variability provides an efficient means to quantify signaling dynamics in mammalian cells. Here, we illustrate the utility ofthis approach by mapping the E2f1 response to MYC, serum stimulation, or both. Our results revealed an underappreciated mode of gene regulation: E2f1 expression first increased, then decreased as MYC input increased. This biphasic pattern was also reflected in other nodes of the network, including the miR-17-92 microRNA cluster and p19Arf. A mathematical model of the network successfully predicted modulation of the biphasic E2F response byserum and a CDK inhibitor. In addition to demonstrating how noise can be exploited to probe signaling dynamics, our results reveal how coordination of the MYC/RB/E2F pathway enables dynamic discrimination of aberrant and normal levels of growth stimulation.

Supraphysiological estrogen levels adversely impact proliferation and histone modification in human embryonic stem cells: possible implications for controlled ovarian hyperstimulation assisted pregnancy

Objective Controlled ovarian hyperstimulation (COH) results in supraphysiologic levels of maternal serum estradiol (E 2) during the luteal phase, thus promoting oocyte production at unknown risk to the subsequently developing embryo. Human embryonic stem cells (hESCs) have been identified as a model system to assess the impact of COH on early embryonic development, specifically 17β-estradiol mediated effects on proliferation, gene expression, and histone modification. Study design Cell proliferation and associated factors, such as HDAC1, as well as histone modification patterns were evaluated in ERα and β expressing hESCs after exposure to 17β-estradiol (1 × 10 −10 M to 1 × 10 −7 M), as well as in an untreated control. Results Resultant data revealed that while physiologically relevant E 2 levels (1 × 10 −9 M E 2) induced cell cycle progression from G1 to the proliferation phase, supraphysiologic levels akin to those observed after COH (1 × 10 −7 M E 2) adversely affected hESCs proliferation via down regulation of HDAC1. Modification of H3K9me2, PhH3S10, H4K5ac, and H2A.Z histone patterns were also dependent on 17β-estradiol concentration. Conclusion While physiologic levels of 17β-estradiol induced cell proliferation, possibly via HDAC1 involvement in histone modification, cell proliferation in hESCs was suppressed at supraphysiologic levels.

Cytogenetics and spectral Karyotyping analysis in CLL Patients: Correlation with FISH and ZAP70 Expression

AbstractAbstract 4844Cytogenetic abnormalities play an important role as prognostic factors in CLL. However, due the low mitotic index of CLL B cells in vitro, analysis of a set of subjects for the most commonly known aberrations is usually done by FISH on interphase cell. The objectives of this investigation were the use of the oligonucleotide DSP30 in combination with IL-2, as a B-cell mitogen for cytogenetic investigation in CLL and correlation among the karyotype analysis obtained (G-banding + SKY), FISH profile from unstimulated cells, ZAP70 expression and stratification status for each patient. For metaphase induction, peripheral blood mononuclear cells were cultured in RPMI 1640 medium with 20% fetal calf serum in the presence of the immunostimulatory CpG-oligonucleotide DSP30 and IL-2. Additionally, one set of cell culture was performed for each patient without any stimulant agent, for FISH analysis. The FISH panel included probes for the detection of +12, and deletions of 11q22.3 (ATM), 13q14 (D13S25 and D13S319), and 17p13 (TP53). The cut off levels for trissomy 12 (>2%), del(13q) (>2.4%), del(11q23.3) (>2.5%), del(17p13.1) (>3%) were established according to the iFISH patterns observed in a group of 4 age and sex-matched normal control peripheral blood samples studied with the same probes. Spectral karyotype analysis (SKY) was performed, according manufactures' instruction. The ZAP70 profile was obtained by flow cytometry analysis. In concordance with literature, the cut off value adopted for ZAP70 was 20%. In a group of 64 subjects studied, the cytogenetic analysis showed chromosomal aberrations in 52 patients (81.25%). The profile of abnormalities observed were del(6)(q24), +8(x2), del(11)(q13~q23), +12, +15(x2), del(12)(p13), -17, +21, +19, +18, del(13)(q31), del(14)(q24), del(17)(p13), +21, +4, +5, +11, t(1;12)(q31;p13), t(11;13)(q23;q12), t(15;18)(q11.1;q11), t(1;10)(p22;p14), t(14;22)(q32;q11), t(17;18)(q10;q10), t(9;13)(q21;q22), t(10;13)(q26;q14), t(9;12)(q12;p11), t(X;12)(p11.2;q24). Twelve patients exhibited normal karyotype (18.75%). All subjects presenting chromosomal abnormalities, by using G-banding analysis, were confirmed by SKY. In patients with normal cytogenetic, SKY analysis did not identified any criptic abnormality. Cells without any stimulant agent showed concordance with the cytogenetic profile obtained (FISH analysis). The ZAP70 expression did not show any relationship between the group of patients with chromosomal abnormalities and the group with normal karyotype. The use of the immunostimulatory oligonucleotide DSP30 in combination with IL-2 showed to be effective to induce cell cycle progression of CLL cells in vitro than others mitogens. Cytogenetic aberrations detected by G-banding in addition to FISH analysis were heterogeneous. The limited spectrum of chromosomal abnormalities seen by FISH analysis may contribute to underestimate the prognostic value, where others abnormalities may be present in patient's karyotype. These results indicate that classical cytogenetic analysis can contribute to the stratification of different subsets of CLL patients with complex karyotype associated with poor prognosis.Financial support: FAPESP (Proc. 07/52462-7). Disclosures:No relevant conflicts of interest to declare.

O9. Cross-talk between growth and differentiation pathways in cell fate determination in the developing brain

Proliferation of neural stem/progenitor cells (NSCs/NPCs) is closely linked to the inhibition of neuronal and glial differentiation. However, the molecular link between induction of cell cycle progression and inhibition of cell differentiation in NSCs/NPCs is poorly understood. Fibroblast growth factor 2 (FGF2) promotes proliferation of NSCs/NPCs while it inhibits their differentiation, which suggests that a molecular link between growth and differentiation pathways is functioning downstream of FGF2 signaling. We hypothesized that a component or components in the proliferative signaling pathway should also be involved in inhibition of NSCs/NPCs differentiation. We have found that the FGF2 signaling and the Wnt canonical pathway cooperate together, via inactivation of glycogen synthase kinase 3beta (GSK3beta) and subsequent induction of cyclin D1 expression, to promote proliferation of NSCs/NPCs. Interestingly, the GSK3beta inactivation-mediated beta-catenin accumulation in the nucleus is involved in the inhibition of neurogenesis from NSCs/NPCs by activation of hes1 and hes5 gene expression through the cooperation with a Notch pathway. As we have previously demonstrated, astrocyte differentiation from NSCs/NPCs is synergistically induced by a combination of LIF and BMP2. In this symposium, we also present our recent data showing that beta-catenin stabilization by a GSK3 beta inhibitor decreases the number of GFAP expressing astrocytes in the culture of NSCs/NPCs with LIF and BMP2. In addition, forced expression of cyclin D1, a common downstream target of FGF2 and Wnt signals, decreases the number of GFAP expressing astrocytes. We further show that knockdown of cyclin D1 relieves the inhibitory effect of the GSK3beta inhibitor. In conclusion, our findings indicate that cross-interactions among growth and differentiation pathways are important for the cell-fate determination in the developing brain.

Novel F141L Pre-S2 Mutation in Hepatitis B Virus Increases the Risk of Hepatocellular Carcinoma in Patients with Chronic Genotype C Infections

Several lines of evidence have suggested that some naturally occurring mutations of hepatitis B virus (HBV) play a critical role in hepatocellular carcinoma (HCC). Here, we describe a novel HCC-related pre-S2 mutation, F141L. To prove the relationship between the F141L mutation and HCC, molecular epidemiology studies using MboII PCR restriction analysis (PRA) were performed, and the molecular mechanism was investigated through construction of a stable hepatocyte cell line expressing the large surface HB protein (LHB) with the F141L mutation (F141L-LHB). Application of MboII PRA to samples from 241 Korean patients with chronic liver diseases of different clinical stages confirmed that F141L mutants were significantly related to HCC, even in comparison to liver cirrhosis (HCC, 26.3% of patients, or 26/99; liver cirrhosis, 3.8% of patients, or 2/52; P = 0.001). By studying stable cell lines, we found that F141L-LHBs could induce cell cycle progression by downregulating the p53 and p21 pathways and upregulating CDK4 and cyclin A. Furthermore, we found that in a colony-forming assay, the colony-forming rates in cell lines expressing F141L-LHBs were about twice as high as those of the wild type. In conclusion, our results suggest that F141L-LHBs may contribute importantly to the pathogenesis of HCC by inducing cell proliferation and transformation. So, the F141L mutation examined in this study could serve as a diagnostic marker for the prognosis of HCC.

Cdk11p58 regulation of cell cycle progression in cancer development

Abstract The Cdk11 protein kinases are members of the cyclin-dependent kinase superfamily. There exist two distinct but closely related human CDK11 genes (p110 and p58). Cdk11p58 is a Ser/Thr kinase involved in cell cycle progression, promoting the centrosome maturation; apoptotic signaling and tumorigenesis. Using immunoprecipitation and mass spectrometry approaches we demonstrate that GADD45, PDEF and cdk11p58 interact in a complex that might be involved in cell cycle regulation in cancer. GADD45 protein family members (α, β, and γ) are involved in the induction or prevention of apoptosis, cell cycle arrest and participate in the DNA repair machinery. Arrest at the G2/M phase mediated by GADD45 have been identified to rely on its capacity to inhibit the kinase activities such as the CDK1/cyclinB1 complex and the Aurora A, a serine/threonine kinase that play critical roles in the regulation of mitotic events. PDEF (prostate-derived ETS factor) is a transcriptional factor that plays a major role in cancer development and metastasis. In this study, the kinase activity of cdk11p58 over PDEF as well as the importance of the cdk11p58 binding to the GADD45 family members were evaluated and we demonstate that cdk11p58 phosphorilate PDEF and the phosphorilation process is inhibited by GADD45. Co-expression of PDEF and cdk11p58 in 293T cells leads to PDEF ubiquitination and degradation, which can be reverted by MG132, a proteasome inhibitor. Corroborating this data, confocal microscope analysis revealed that cdk11p58 co-localized with PDEF and ubiquitin into the nucleus. However, the process is partially blockage by the presence of GADD45 gene, which seems to prevent PDEF degradation mediated by cdk11p58. Deregulation of the ETS factors leads to altered expression of key genes required for cancer progression. PDEF acts as a negative regulator of tumor progression by modulating the expression of growth and migration promoting genes. The degradation of PDEF by cdk11p58 may be a critical step in cancer cell tumorigenesis and more experiments are under way to unravel the importance of this event in cancer cell migration and invasion. In order to gain further insights about the role of cdk11p58 and GADD45 in cell cycle regulation of cancer cells, we developed DU145 cell lines overexpressing the GADD45α (DU145-GADD45+) and the cdk11p58 (DU145-p58+) proteins. Additionally, DU145 with the cdk11p58 knocked-down (DU145-p58si) was obtained by transduction of lentivirus containing siRNA against the cdk11p58 gene sequence (Sigma). The biological effect of overexpression or know-down of the cdk11p58 gene in the cell cycle phase was assessed by flow cytometry (FACS) analysis of propidium iodide stained cells. FACS analysis revealed that DU145-p58+ cells progress faster when compared to a control DU145 cell line, while DU145-GADD45+ and DU145-p58si induced cell cycle arrest at the G2/M phase. Furthemore, overexpression of GADD45 in the DU145-p58+ cell line induced an arrest at the G2/M phase, comparable to the control group. Preliminary result of transient transfection of PDEF indicates that ovexpression of PDEF in 293T cells leads to arrest at the G1/S phase. Transient tranfections of cdk11p58 leads to induction of proliferation when compared with control cells while cells expressing the GADD45 α and PDEF genes demonstrate slower proliferation rates. In conclusion, PDEF was identified to be a novel target for the kinase activity of cdk11p58. Cdk11p58 induced cell cycle progression and cell proliferation of a prostate cancer cell line that can be inhibited by GADD45 contributing to cell cycle arrest at the G2/M phase.

VMY-1-103, a dansylated analog of purvalanol B, induces caspase-3-dependent apoptosis in LNCaP prostate cancer cells

The 2,6,9-trisubstituted purine group of cyclin dependent kinase inhibitors have the potential to be clinically relevant inhibitors of cancer cell proliferation. We have recently designed and synthesized a novel dansylated analog of purvalanol B, termed VMY-1-103, that inhibited cell cycle progression in breast cancer cell lines more effectively than did purvalanol B and allowed for uptake analyses by fluorescence microscopy.ErbB-2 plays an important role in the regulation of signal transduction cascades in a number of epithelial tumors, including prostate cancer (PCa). Our previous studies demonstrated that transgenic expression of activated ErbB-2 in the mouse prostate initiated PCa and either the overexpression of ErbB-2 or the addition of the ErbB-2/ErbB-3 ligand, heregulin (HRG), induced cell cycle progression in the androgen-responsive prostate cancer cell line, LNCaP. In the present study, we tested the efficacy of VMY-1-103 in inhibiting HRG-induced cell proliferation in LNCaP prostate cancer cells. At concentrations as low as 1 µM, VMY-1-103 increased both the proportion of cells in G1 and p21CIP1 protein levels. At higher concentrations (5 µM or 10 µM), VMY-1-103 induced apoptosis via decreased mitochondrial membrane polarity and induction of p53 phosphorylation, caspase-3 activity and PARP cleavage. Treatment with 10 µM Purvalanol B failed to either influence proliferation or induce apoptosis. Our results demonstrate that VMY-1-103 was more effective in inducing apoptosis in PCa cells than its parent compound, purvalanol B, and support the testing of VMY-1-103 as a potential small molecule inhibitor of prostate cancer in vivo.

Cover Picture: Mol. Nutr. Food Res. 6'2010

AbstractRegular issues provide a wide range of research and review articles covering all aspects of Molecular Nutrition &amp; Food Research.Selected topics of issue 06 are:Measurement of caffeic and ferulic acid equivalents in plasma after coffee consumption: small intestine and colon are key sites for coffee metabolismComparative resistance of food proteins to adult and infant in vitro digestion modelsGarlic components inhibit angiotensin II – induced cell cycle progression and migration: involvement of cell cycle inhibitor p27Kip1 and MAPKDiosgenin attenuates inflammatory changes in the interaction between adipocytes and macrophagesOleuropein and hydroxytyrosol inhibit MCF‐7 breast cancer cell proliferation interfering with ERK1/2 activation

Dominant roles of the Raf/MEK/ERK pathway in cell cycle progression, prevention of apoptosis and sensitivity to chemotherapeutic drugs

The effects of the Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR signaling pathways on cell cycle progression, gene expression, prevention of apoptosis and sensitivity to chemotherapeutic drugs were examined in FL/ΔAkt-1:ER*(Myr+) + ΔRaf-1:AR cells which are conditionally-transformed to grow in response to Raf-1 and Akt-1 activation by treatment with testosterone or tamoxifen respectively. In these cells we can compare the effects of normal cytokine vs. oncogene mediated signaling in the same cells by changing the culture conditions. Raf-1 was more effective than Akt-1 in inducing cell cycle progression and preventing apoptosis in the presence and absence of chemotherapeutic drugs. The normal cytokine for these cells, interleukin-3 induced/activated most downstream genes transiently, with the exception of p70S6K that was induced for prolonged periods of time. In contrast, most of the downstream genes induced by either the activate Raf-1 or Akt-1 oncogenes were induced for prolonged periods of time, documenting the differences between cytokine and oncogene mediated gene induction which has important therapeutic consequences. The FL/ΔAkt-1:ER*(Myr+) + ΔRaf-1:AR cells were sensitive to MEK and PI3K/mTOR inhibitors. Combining MEK and PI3K/mTOR inhibitors increased the induction of apoptosis. The effects of doxorubicin on the induction of apoptosis could be enhanced with MEK, PI3K and mTOR inhibitors. Targeting the Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR pathways may be an effective approach for therapeutic intervention in those cancers which have upstream mutations which result in activation of these pathways.

Abstract 4607: Novel role of PARP4 in hormone-dependent breast cancer progression and metastasis

Abstract Estrogen/estrogen receptor-mediated actions play an important role in regulating a variety of biological process including development, homeostasis and breast cancer progression. Even though systemic hormone therapies that either block local estrogen production by aromatase inhibitors (AI) or block actions of estrogen/estrogen receptor (ER) by antiestrogens (AE) is well tolerated, the development of resistance to these agents is still a major concern. Since multiple signaling pathways involved in ER activation, combination of endocrine and non endocrine agents that block these signaling pathways may delay the onset of resistance to hormonal therapy. Recent studies from our laboratory have identified Poly(ADP-ribose) polymerase 4(PARP4) as a novel ER co-regulator. We have investigated with hormone resistant breast cancer cells to demonstrate whether blocking of PARP4 results in resensitization to hormone therapies. PARP comprise of a family of enzymes which catalyses poly(ADPribosyl) action of DNA-binding proteins. PARP-1, the best characterized member, however PARP4 is an understudied protein with numerous capabilities. PARP4 has both N-terminal PXXP and PPXXP motifs which are known to interact with SH3 domain of c-Src as well as LXXLL motif which interacts with estrogen receptor (ER). We show that PARP4 is widely expressed in breast cancer cells (MCF-7, MDA-MB-231, ZR-75, and SKBR3) and its expression is significantly higher in these cells compared to non malignant MCF-10A cells. To understand the important role of PARP4-ER axis in hormonal resistance, metastasis and to test whether blocking of PARP4 mediated actions provide a therapeutic advantage to treat endocrine resistant breast tumors, we have used hormone resistant breast cancer cells that over-express PARP4 or and treated with PARP4 siRNA nanoparticles. The PLGA-siRNA nanoparticles were designed multifunctionally using chemical conjugation. The nanoparticles were characterized physiochemically. Our studies show that PARP4 functionally interacts with ER. As demonstrated by immunoprecipitation, PARP4 interacts with ER upon ligand (E2)-mediated stimulation. PARP4 induces cell cycle progression, local aromatase activity and promotes in vitro tumorigenic potential of breast cancer cells. siRNA nanoparticles of PARP4 inhibit the metastatic incidence in vivo. Our study also shows that PARP4-ER axis enhances multiple signaling cross-talk leading to hormonal independence/resistance and metastasis. Our results suggests that targeting PARP4 may provide a therapeutic advantage to treat endocrine resistant tumors and blocking the development of resistance to hormonal therapeutic agents and as well as decrease the metastatic potential in therapy resistant breast tumors. (Supported by NIH/NCI P30 CA 54174, RRT) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4607.