G1 Growth Arrest Research Articles

Inhibitors of Heat Shock Protein 72 Induction Enhance Apoptosis Induced by Hsp90 Inhibitors

Exposure of myeloma cells to Hsp90 inhibitors (eg 17-AAG) or proteasome inhibitors (eg Bortezomib) results in a time-dependent induction of heat shock protein 72 (Hsp72), protecting cells from drug-induced apoptosis. Hsp72 is a member of the heat shock protein 70 (Hsp70) family and enables cells to cope with harmful aggregations of proteins during and after stress, protecting cells from apoptosis in addition to acting as a chaperone for proteins such as immunoglobulin. We show that inhibition of Hsp72 upregulation induced by these anti-myeloma drugs, by either inhibitors of Hsp70 induction or siRNA, increases susceptibility of myeloma cells to drug-induced apoptosis. This provides a rationale for using such inhibitors in combination with anti-myeloma drugs. A number of commercially available inhibitors of Hsp70 induction, including KNK437 and triptolide, were used to inhibit stress-induced upregulation of Hsp72. Combining Hsp90 inhibitors (Hsp90i) with these compounds increased cell death to 73% from 33% when Hsp90i were used alone. Cell cycle analysis indicated that combining these compounds with Hsp90 inhibitors overcame the G2 growth arrest induced by Hsp90 inhibitors and increased the proportion of cells in sub G1, indicative of apoptosis. Analysis of their anti-proliferative effects demonstrated that, in H929 cells, Hsp90 inhibitor/triptolide combinations had additive effects whilst in U266 cells their effects were synergistic. To determine the underlying mechanisms responsible for the effects of the combination, key proteins involved in endoplasmic reticulum (ER) stress, Unfolded Protein Response and caspase-dependent and independent signalling pathways were analysed. We show that ER stress signalling through IRE1 and PERK pathways, measured by cleavage of XBP1 and induction of CHOP, were initiated earlier and sustained longer, when cells were treated with the combination. Similarly, analysis of caspase-dependent and independent signalling demonstrated that the combination induced early cleavage of caspases 9, 8 and 3 which was sustained. Nuclear translocation of AIF, the caspase-independent cell death signal, was strongly induced suggestive of strong activation of this pathway. We demonstrate that death induced by triptolide or its combination with 17-AAG is p53-independent using of a panel of myeloma cell lines. To confirm our findings, we used lentiviral siRNA constructs targeting the inducible forms of Hsp70 and then analysed response to Hsp90 inhibitors. These experiments confirm the results generated using the chemical inhibitors of Hsp70 expression. In conclusion, inhibiting drug-induced Hsp72 expression appears to overcome cellular resistance to Hsp90 inhibitors, supporting the clinical development of Hsp70 inhibitors and their combination with Hsp90 and proteasome inhibitors.

Jab1 is overexpressed in human breast cancer and is a downstream target for HER-2/neu

Jab1 is a coactivator of AP-1 transcription factor and the fifth subunit of the COP9 signalosome. This protein is a potential oncogene and is involved in the mediation of nuclear exportation and degradation of the tumor suppressor p27Kip1. However, control of Jab1 gene expression and its de-regulation in cancer cells are largely unknown. In this study, we demonstrated that Jab1 is overexpressed in 53 (80.3%) of a series of 66 human breast tumor tissues. In addition, its expression is significantly correlated with HER-2/neu overexpression (P=0.0318). HER-2/neu-overexpressing MDA-MB-453 human breast cancer cells exhibited higher expression of Jab1 than that of MCF-7 breast cancer cells. Promoter activity assay suggested that HER-2/neu oncogene upregulated Jab1 via transcriptional activation. Inhibition of HER-2/neu activity by Herceptin or AG825 significantly attenuated Jab1 expression in HER-2/neu-overexpressing MDA-MB-453 cells. On the contrary, ectopic expression of HER-2/neu stimulated Jab1 expression in MCF-7 cells. Knockdown of Jab1 expression by siRNA resulted in p27Kip1 upregulation and G1 growth arrest in Jab1-overexpressing MDA-MB-453 cells. Taken together, our results suggest that Jab1 is a downstream target for HER-2/neu and its overexpression is linked with HER-2/neu expression in breast cancer.

PTEN Nuclear Localization Is Regulated by Oxidative Stress and Mediates p53-Dependent Tumor Suppression

The tumor suppressor gene PTEN (phosphatase and tensin homologue deleted on chromosome 10) is frequently mutated or deleted in various human cancers. PTEN localizes predominantly to the cytoplasm and functions as a lipid phosphatase, thereby negatively regulating the phosphatidylinositol 3-kinase-AKT signaling pathway. PTEN can also localize to the nucleus, where it binds and regulates p53 protein level and transcription activity. However, the precise function of nuclear PTEN and the factors that control PTEN nuclear localization are still largely unknown. In this study, we identified oxidative stress as one of the physiological stimuli that regulate the accumulation of nuclear PTEN. Specifically, oxidative stress inhibits PTEN nuclear export, a process depending on phosphorylation of its amino acid residue Ser-380. Nuclear PTEN, independent of its phosphatase activity, leads to p53-mediated G(1) growth arrest, cell death, and reduction of reactive oxygen species production. Using xenografts propagated from human prostate cancer cell lines, we reveal that nuclear PTEN is sufficient to reduce tumor progression in vivo in a p53-dependent manner. The data outlined in this study suggest a unique role of nuclear PTEN to arrest and protect cells upon oxidative damage and to regulate tumorigenesis. Since tumor cells are constantly exposed to oxidative stress, our study elucidates the cooperative roles of nuclear PTEN with p53 in tumor suppression.

Comparative analysis of wild‐type and cAMP‐deathless S49 lymphoma cells identifies genes and pathways of cAMP/PKA‐promoted apoptosis

The second messenger cAMP acts via protein kinase A (PKA) to induce apoptosis by mechanisms that are poorly understood. Here, we assessed a role for mitochondria and analyzed gene expression in cAMP/PKA‐promoted apoptosis by comparing wild‐type (WT) S49 lymphoma cells and the S49 variant, D‐ (cAMP‐deathless), which lacks cAMP‐promoted apoptosis but that has WT levels of PKA activity as well as cAMP‐promoted G1 growth arrest. Treatment of WT, but not D‐, S49 cells with 8‐CPT‐cAMP for 24 h induced loss of mitochondrial membrane potential, mitochondrial release of cytochrome c and Smac and increase in caspase‐3 activity. Gene expression analysis (using Affymetrix 430 2.0 Arrays) revealed that WT and D‐ cells incubated with 8‐CPT‐cAMP have similar, but non‐identical, extents of cAMP‐regulated gene expression at 2h (~800 transcripts) and 6h (~1000 transcripts) (|Fold|>2, P<0.06); by contrast, at 24h ~2500 and ~1100 transcripts were changed in WT and D‐ cells, respectively. WT and D‐cells differed in CREB phosphorylation, expression of the transcriptional inhibitor Icer and cAMP‐regulated expression of genes in the Inhibitor of apoptosis (IAP) and Bcl families. We conclude that cAMP/PKA‐promoted apoptosis of lymphoid cells occurs via mitochondrial‐mediated events and that such apoptosis likely involves gene networks in multiple biochemical pathways. (Supported by the Leukemia and Lymphoma Society and the NIH).

Prematurely senescent ARPE-19 cells display features of age-related macular degeneration

The etiology of age-related macular degeneration (AMD), the leading cause of blindness in the developed world, remains poorly understood, but may be related to cumulative oxidative stress. The prime target of the disease is the retinal pigmented epithelium (RPE). To study the molecular mechanisms underlying RPE degeneration, we investigated whether repetitive oxidative stress induced premature senescence in RPE cells from the human ARPE-19 cell line. After exposure to 8 mM tert-butylhydroperoxide (tert-BHP) for 1 h daily for 5 days, the cells showed four well-known senescence biomarkers: hypertrophy, senescence-associated β-galactosidase activity, growth arrest, and cell cycle arrest in G1. A specific low-density array followed by qRT-PCR validation allowed us to identify 36 senescence-associated genes differentially expressed in the prematurely senescent cells. Functional analysis demonstrated that premature senescence induced amyloid β secretion, resistance to acute stress by tert-BHP and amyloid β, and defects in adhesion and transepithelial permeability. Coculture assays with choroidal endothelial cells showed the proangiogenic properties of the senescent RPE cells. These results demonstrate that chronic oxidative stress induces premature senescence in RPE cells that modifies the transcriptome and substantially alters cell processes involved in the pathophysiology of AMD. Oxidative stress-induced premature senescence may represent an in vitro model for screening therapeutics against AMD and other retinal degeneration disorders.

Identification and characterization of CCAAT/Enhancer Binding proteindelta (C/EBPdelta) target genes in G0 growth arrested mammary epithelial cells.

BackgroundCCAAT/Enhancer Binding Proteinδ (C/EBPδ) is a member of the highly conserved C/EBP family of leucine zipper (bZIP) proteins. C/EBPδ is highly expressed in G0 growth arrested mammary epithelial cells (MECs) and "loss of function" alterations in C/EBPδ have been associated with impaired contact inhibition, increased genomic instability and increased cell migration. Reduced C/EBPδ expression has also been reported in breast cancer and acute myeloid leukemia (AML). C/EBPδ functions as a transcriptional activator, however, only a limited number of C/EBPδ target genes have been reported. As a result, the role of C/EBPδ in growth control and the potential mechanisms by which "loss of function" alterations in C/EBPδ contribute to tumorigenesis are poorly understood. The goals of the present study were to identify C/EBPδ target genes using Chromatin Immunoprecipitation coupled with a CpG Island (HCG12K) Array gene chip ("ChIP-chip") assay and to assess the expression and potential functional roles of C/EBPδ target genes in growth control.ResultsChIP-chip assays identified ~100 C/EBPδ target gene loci which were classified by gene ontology (GO) into cell adhesion, cell cycle regulation, apoptosis, signal transduction, intermediary metabolism, gene transcription, DNA repair and solute transport categories. Conventional ChIP assays validated the ChIP-chip results and demonstrated that 14/14 C/EBPδ target loci were bound by C/EBPδ in G0 growth arrested MCF-12A MECs. Gene-specific RT-PCR analysis also demonstrated C/EBPδ-inducible expression of 14/14 C/EBPδ target genes in G0 growth arrested MCF-12A MECs. Finally, expression of endogenous C/EBPδ and selected C/EBPδ target genes was also demonstrated in contact-inhibited G0 growth arrested nontransformed human MCF-10A MECs and in mouse HC11 MECs. The results demonstrate consistent activation and downstream function of C/EBPδ in growth arrested human and murine MECs.ConclusionC/EBPδ target genes were identified by a global gene array approach and classified into functional categories that are consistent with biological contexts in which C/EBPδ is induced, such as contact-mediated G0 growth arrest, apoptosis, metabolism and inflammation. The identification and validation of C/EBPδ target genes provides new insights into the mechanistic role of C/EBPδ in mammary epithelial cell biology and sheds new light on the potential impact of "loss of function" alterations in C/EBPδ in tumorigenesis.

Deficiency of G1 regulators P53, P21Cip1and/or pRb decreases hepatocyte sensitivity to TGFβ cell cycle arrest

BackgroundTGFβ is critical to control hepatocyte proliferation by inducing G1-growth arrest through multiple pathways leading to inhibition of E2F transcription activity. The retinoblastoma protein pRb is a key controller of E2F activity and G1/S transition which can be inhibited in viral hepatitis. It is not known whether the impairment of pRb would alter the growth inhibitory potential of TGFβ in disease. We asked how Rb-deficiency would affect responses to TGFβ-induced cell cycle arrest.ResultsPrimary hepatocytes isolated from Rb-floxed mice were infected with an adenovirus expressing CRE-recombinase to delete the Rb gene. In control cells treatment with TGFβ prevented cells to enter S phase via decreased cMYC activity, activation of P16INK4A and P21Cip and reduction of E2F activity. In Rb-null hepatocytes, cMYC activity decreased slightly but P16INK4A was not activated and the great majority of cells continued cycling. Rb is therefore central to TGFβ-induced cell cycle arrest in hepatocytes. However some Rb-null hepatocytes remained sensitive to TGFβ-induced cell cycle arrest. As these hepatocytes expressed very high levels of P21Cip1 and P53 we investigated whether these proteins regulate pRb-independent signaling to cell cycle arrest by evaluating the consequences of disruption of p53 and p21Cip1. Hepatocytes deficient in p53 or p21Cip1 showed diminished growth inhibition by TGFβ. Double deficiency had a similar impact showing that in cells containing functional pRb; P21Cip and P53 work through the same pathway to regulate G1/S in response to TGFβ. In Rb-deficient cells however, p53 but not p21Cip deficiency had an additive effect highlighting a pRb-independent-P53-dependent effector pathway of inhibition of E2F activity.ConclusionThe present results show that otherwise genetically normal hepatocytes with disabled p53, p21Cip1 or Rb genes respond less well to the antiproliferative effects of TGFβ. As the function of these critical cellular proteins can be impaired by common causes of chronic liver disease and HCC, including viral hepatitis B and C proteins, we suggest that disruption of pRb function, and to a lesser extend P21Cip1 and P53 in hepatocytes may represent an additional new mechanism of escape from TGFβ-growth-inhibition in the inflammatory milieu of chronic liver disease and contribute to cancer development.

Aminopeptidase Inhibition as a Targeted Treatment Strategy in Myeloma.

Myeloma cells are highly dependent on the unfolded protein response to assemble folded immunoglobulins correctly. Therefore targeting protein handling within a myeloma cell by inhibiting the aminopeptidase enzyme system that catalyses the hydrolysis of amino acids from the N terminus of proteins may be a novel therapeutic approach. The effect of the aminopeptidase inhibitor CHR-2797 on myeloma cell proliferation and survival, gene expression, protein turnover, cell migration and myeloma-bone marrow stromal cell interactions was determined on a panel of myeloma cell lines and patient cells. CHR-2797 is able to inhibit the proliferation of myeloma cell lines and primary patient cells, whereas its derivative CHR-79888, an acid metabolite with low cell membrane permeability fails to induce myeloma cell death. This occurs though apoptosis as demonstrated by trypan blue exclusion and annexin V/PI staining, and is proceeded by G1 growth arrest. Western blot analysis demonstrates apoptosis occurs via a non-caspase dependant mechanism. Importantly CHR-2797 is able to induce apoptosis in cells known to be resistant to conventional chemotherapeutic agents. Analysis of the pathways involved using Affymetrix gene expression arrays demonstrates CHR-2797 causes an upregulation of many genes involved in the proteasome/ubiquitin pathway, as well as amino acid deprivation response genes and some aminopeptidases. A further mechanism contributing to cell death is activation of the unfolded protein response with activation of all three UPR pathways demonstrated by splicing of XBP1 to its active from XBP1s, an increase in CHOP with activation of the PERK pathway and cleavage of ATF6. Cytoplasmic inclusions are also present on light microscopy suggestive of the build up of misfolded proteins within the cytoplasm. CHR-2797 causes minimal inhibition of the proliferation of bone marrow stroma, but is able to overcome the protective effects of the micro-environment on myeloma cells, as the drug is still able to inhibit the proliferation of myeloma cells when they are bound to bone marrow stromal cells. Aminopeptidase inhibition is also able to inhibit the increase VEGF that occurs when myeloma cells and bone marrow stroma are bound together. Combination experiments of CHR-2797 with dexamethasone demonstrate synergy, in keeping with the different mechanisms of action the two drugs. CHR-2797 in combination with the proteasome inhibitor bortezomib demonstrates an additive effect. Although both drugs target intracellular protein turnover, gene expression studies of cells treated with CHR-2797 or bortezomib show deregulation of a number of genes specific to aminopeptidase inhibition, as well as a series of genes characteristic of protein turnover. These differences in the mechanism of action of the two drugs are also reflected in the Western blot analysis that demonstrates a predominately non-caspase mediated cell death in CHR2797 compared to a caspase mediated cell death with bortezomib. In summary inhibiting intracellular protein turnover using the aminopeptidase inhibitor CHR-2797 results in myeloma cell death and represents a novel therapeutic approach for the treatment of myeloma. A phase 1 clinical trial has been initiated in haematological malignancies and the results will also be presented at this meeting.

SIRT3 is Pro-Apoptotic and Participates in Distinct Basal Apoptotic Pathways

SIRT3, one of seven mammalian sirtuins, is a NAD-dependent deacetylase. SIRT3 localises to mitochondria where it deacetylates and thus activates acetyl-CoA synthetase 2 (AceCS2), indicating a role for SIRT3 in metabolism. Here we provide evidence that SIRT3 also impacts upon apoptosis and cell growth control. Using RNAi under basal (non-stress) conditions we show that SIRT3 is required for apoptosis induced by selective silencing of Bcl-2 in HCT116 human epithelial cancer cells. Identical treatment of ARPE19 epithelial non-cancer cells induces G1 growth arrest which also proved to be SIRT3-dependent. Previously we have identified SIRT1 and JNK2 as constitutive suppressors of apoptosis in HCT116 cells. We now demonstrate that SIRT3 functions in JNK2-regulated apoptosis but is dispensable for SIRT1-regulated apoptosis. SIRT3 is also dispensable for stress-induced apoptosis. Thus the pro-apoptotic functioning of SIRT3 is selectively coupled with defined pathways regulating cell survival under basal conditions.

BRAF mutation predicts MEK-dependence in non-small cell lung cancer (NSCLC)

10523 Background: Constitutive ERK signaling is common in human cancer and is often the result of activating mutations in BRAF, RAS and upstream receptors. Missense BRAF kinase domain mutations are frequently observed in melanoma, colon and thyroid cancers and less frequently in lung and other cancer types. The vast majority (>90%) involve a glutamic acid for valine substitution at codon 600 (V600E), which results in elevated BRAF kinase activity. BRAF kinase domain mutations with intermediate and impaired kinase activity have also been identified, most frequently in NSCLC. We have previously reported that tumors with V600E BRAF mutation are selectively sensitive to MEK inhibition. Methods: Using the potent and selective MEK1/2 inhibitor PD0325901 (Pfizer), we examined a panel of NSCLC cell lines with mutant EGFR, KRAS, and/or low, intermediate and high-activity BRAF kinase domain mutations, with regard to MEK dependence. In all but one case, EGFR, KRAS and BRAF mutations were mutually exclusive. Results: Consistent with our prior results, NSCLC cells with V600E BRAF mutation were exquisitely sensitive to MEK inhibition (PD0325901 IC50 of 2nM). The proliferation of cells with non-V600E mutations, including those with high (G469A), intermediate (L597V) and impaired (G466V) kinase activities, was also MEK dependent with IC50's ranging between 2.7 and 80 nM. Inhibition of MEK in these cells resulted in downregulation of cyclin D1 and G1 growth arrest, with variable induction of apoptosis. Despite high basal ERK activity, NSCLC tumor cells with EGFR mutation were uniformly resistant to MEK inhibition (at doses of up to 500nM), despite effective inhibition of ERK phosphorylation. Tumor cells with RAS mutation had a more variable response, with some cell lines demonstrating sensitivity, while others were completely resistant. There was no correlation between basal ERK activity and sensitivity to MEK inhibition. A strong inverse correlation between AKT phosphorylation/ activity and PD0325901 sensitivity was observed. Conclusions: These results suggest that MEK inhibition may be useful therapeutically in tumors with V600E and non-V600E BRAF kinase domain mutations. The results also suggest that inhibition of both MEK and AKT signaling may be required in NSCLC tumors with high basal AKT activity. No significant financial relationships to disclose.

A bioactive withanolide Tubocapsanolide A inhibits proliferation of human lung cancer cells via repressing Skp2 expression

Withanolides are generally defined as C(28) steroidal lactones built on an intact or rearranged ergostane skeleton and have been shown to exhibit antiproliferative activity on various types of cancer cells. In this study, we investigated the effect of a new withanolide Tubocapsanolide A isolated from Tubocapsicum anomalum and addressed its molecular action. Tubocapsanolide A inhibited proliferation of A549, H358, and H226 human lung cancer cells via induction of G(1) growth arrest. We found that Tubocapsanolide A treatment led to up-regulation of cyclin E, p21, and p27, whereas other cyclins and cyclin-dependent kinases were not affected in A549 cells. Conversely, Skp2, the F-box protein that is implicated in the mediation of degradation of p21 and p27, was significantly down-regulated. Chromatin immunoprecipitation assay suggested that Tubocapsanolide A suppressed Skp2 expression by inhibiting the binding of Rel A to the nuclear factor-kappaB site of Skp2 gene promoter. In addition, we showed that inhibition of Skp2 is a critical step for the suppression of cell proliferation by Tubocapsanolide A because ectoexpression of Skp2 effectively reversed Tubocapsanolide A-induced p27 up-regulation and growth inhibition in human lung cancer cells. Collectively, we have identified Skp2 as a molecular target for Tubocapsanolide A and suggest that this withanolide may be useful for the prevention or treatment of cancer cells with Skp2 overexpression.

Vanadate-Induced Inhibition of BCR-Triggered Apoptosis is Coupled with Tyrosine Phosphorylation and Induction of G2M Growth Arrest in Ramos-BL B Cells

The regulation of the tyrosine phosphorylation of key signaling molecules by tyrosine kinases and phosphatases is essential for BCR-triggered signaling cascades during B cell selection process. We used the non-selective tyrosine phosphatase inhibitor vanadate to study the importance of the late regulation of the tyrosine phosphorylation for BCR-triggered G1 growth arrest and apoptosis in Ramos-BL B cells. Vanadate induces G2M growth arrest in a dose-dependent manner and prevents BCR-triggered apoptosis. Vanadate-induced upregulation of the tyrosine phosphorylation is concomitant with increased expression of cyclin B and inhibition of caspase-3 activation and PARP cleavage. The anti-apoptotic effect of vanadate was observed even when added up to 6 hours after the treatment of Ramos-BL B cells with anti-IgM. Vanadate increases BCR-triggered tyrosine phosphorylation of the cytosolic tyrosine phosphatases, SHP-1 and SHP-2 after 24 hours. Co-stimulation with anti-CD40 prevents anti-IgM-triggered tyrosine phosphorylation of these phosphatases and up-regulates the expression of SHP-1. We conclude that the regulation of the tyrosine phosphatase activity is indispensable for BCR-triggered execution of the apoptosis in Ramos-BL B cells.

Structure and Implied Functions of Truncated B-Cell Receptor mRNAs in Early Embryo and Adult Mesenchymal Stem Cells: Cδ Replaces Cμ in μ Heavy Chain-Deficient Mice

Stem cells exhibit a promiscuous gene expression pattern. We show herein that the early embryo and adult MSCs express B-cell receptor component mRNAs. To examine possible bearings of these genes on the expressing cells, we studied immunoglobulin mu chain-deficient mice. Pregnant mu chain-deficient females were found to produce a higher percentage of defective morulae compared with control females. Structure analysis indicated that the mu mRNA species found in embryos and in mesenchyme consist of the constant region of the mu heavy chain that encodes a recombinant 50-kDa protein. In situ hybridization localized the constant mu gene expression to loose mesenchymal tissues within the day-12.5 embryo proper and the yolk sac. In early embryo and in adult mesenchyme from mu-deficient mice, delta replaced mu chain, implying a possible requirement of these alternative molecules for embryo development and mesenchymal functions. Indeed, overexpression of the mesenchymal-truncated mu heavy chain in 293T cells resulted in specific subcellular localization and in G(1) growth arrest. The lack of such occurrence following overexpression of a complete, rearranged form of mu chain suggests that the mesenchymal version of this mRNA may possess unique functions.

A Humanized Anti-IGF-1R Monoclonal Antibody (AVE1642) Synergizes with Bortezomib To Induce Apoptosis in CD45neg Myeloma Cells.

IGF-1 is a well-known growth factor for human myeloma cells. Thus, therapeutic strategies targeting IGF-1R have been proposed for the treatment of multiple myeloma. In this study, we investigated the effect of a humanized antagonistic monoclonal antibody to the IGF-1R (AVE1642). We showed that AVE1642 selectively inhibits IGF-1R but not insulin signaling in human myeloma cell lines (HMCL). Since we had previously shown the functional relevance of CD45 expression in the growth of myeloma cells, and the association of CD45 negative (CD45neg) status with a less favorable clinical outcome, both CD45 positive (CD45pos) and CD45neg HMCL were studied. We found that AVE1642 strongly inhibited the growth of CD45neg HMCL, leading to a G1 growth arrest, whereas it had almost no effect on the growth of CD45pos HMCL. Indeed, by cell counting over four days in standard culture conditions, AVE1642 induced a significant growth inhibition of the CD45neg LP-1 HMCL of 90% whereas it had no effect on the growth of the CD45pos XG-1 HMCL. Furthermore, AVE1642 binding induced a significant reduction of IGF-1R expression. In western blot, a decrease of 60% the IGF-1R expression of the LP-1 CD45neg HMCL was observed. This decrease was also confirmed by flow cytometry analysis. Finally, a stable CD45 silencing XG-1 cell line was generated and became sensitive to AVE1642. By cell counting over four days in standard culture conditions, AVE1642 inhibited the growth of the shCD45neg XG-1 HMCL of 56% whereas it had almost no effect on the growth of the shLuci-XG-1 HMCL. Thus, for the first time, we provided direct evidence that the expression of CD45 renders myeloma cells resistant to IGF1R inhibition. Furthermore, AVE1642 in combination with Bortezomib strongly increased the apoptosis induced by Bortezomib alone on CD45negHMCL. The CD45neg LP-1 HMCL was considered as a modestly sensitive cell line since 10nM of Bortezomib, which is in the range of therapeutic concentrations, induced 40±16% of apoptosis. Bortezomib in combination with AVE1642 was found to induce 80±1% of apoptosis in the CD45neg LP-1 HMCL. Apoptosis induction by AVE1642/Bortezomib combination was associated with an important induction of Noxa, a BH3-only pro-apoptotic protein that was increased by the addition of AVE1642. We also found a strong increase of caspase-3, -8 activation under this combination compared to Bortezomib alone. Taken together, these results support that therapy directed against IGF-1R associated with Bortezomib, could be beneficial in treating CD45neg patients, a population known for poor clinical outcome.

Downregulation of PCNA potentiates p21-mediated growth inhibition in response to hyperoxia

Prolonged exposure to hyperoxia inhibits cell proliferation in G1 via increased expression of p21. While p21 inhibits proliferating cell nuclear antigen (PCNA)-dependent DNA synthesis, it can also directly lower PCNA abundance; however, it is unclear whether loss of PCNA contributes to growth arrest. Here, we investigate how PCNA loss affects ability of p21 to exert G1 growth arrest of lung epithelial cells exposed to hyperoxia. In A549 cells that express p21 and growth arrest in G1 during hyperoxia, small interfering RNA (siRNA) knockdown of p21 led to G1 checkpoint bypass, increased cell death, and restoration of PCNA expression. Conditional overexpression of the PCNA binding domain of p21 in H1299 cells that do not normally express p21, or exposure to hyperoxia, caused a time-dependent loss of PCNA. Titrating PCNA levels using siRNA to approximate the low amount observed in cells expressing p21 resulted in S phase arrest. While lowering PCNA by itself caused S phase arrest, the combination of hyperoxia and siRNA against PCNA dramatically reduced PCNA abundance resulting in G1 arrest. G1 growth arrest was markedly enhanced upon the addition of p21 to these cells. Our findings suggest a model in which reducing expression of the abundant protein PCNA allows the less abundant protein p21 to be more effective at suppressing the processivity functions of remaining PCNA, thereby fully exerting the G1 checkpoint. Given that high p21 expression is often associated with lower PCNA abundance, our findings are suggestive of a global growth inhibitory mechanism involving p21-mediated PCNA suppression.

Rac1 GTPase Regulates Cell Genomic Stability and Senescence

The Rho family small GTPase Rac1 has been shown to play multiple roles in cell regulation, including actin cytoskeleton organization, transcriptional activation, microtubule dynamics, and endocytosis. Here, we report a novel role of Rac1 in regulating genomic stability and cell senescence. We observed in primary mouse embryonic fibroblasts that deletion of rac1 by gene targeting, as well as expression of the constitutively active Rac1 mutant L61Rac1, led to decreased cell growth that was associated with altered cell cycle progression at both G(1)/S and G(2)/M phases, increased apoptosis, and premature senescence. The senescence induction by either loss or gain of Rac1 activity was due at least in part to an increase in cellular reactive oxygen species (ROS). rac1 gene deletion caused a compensatory up-regulation of a closely related family member, Rac3, in mouse embryonic fibroblasts, the activity of which induced ROS production independently of Rac1. Furthermore, the Rac1-regulated ROS production and senescence correlated with the extent of DNA damage in the Rac1(-/-) and L61Rac1 cells. Treatment of these cells with a ROS inhibitor inhibited phospho-H2AX-positive nuclear focus formation. Finally, phospho-Ser(15) p53 was significantly increased in L61Rac1 and Rac1(-/-) cells, and genetic deletion of p53 from these cells readily reversed the senescence phenotype, indicating that Rac1 is functionally dependent on p53 in regulating cell senescence. Taken together, our results show that Rac1 activity serves as a regulator of cell senescence through modulation of cellular ROS, genomic stability, and p53 activity.

Indole-3-carbinol mediated cell cycle arrest of LNCaP human prostate cancer cells requires the induced production of activated p53 tumor suppressor protein

Indole-3-carbinol (I3C), a dietary compound found naturally in cruciferous vegetables of the Brassica genus such as broccoli and brussels sprouts, induces a G1 growth arrest of human reproductive cancer cells. We previously reported that in LNCaP prostate cancer cells, I3C down-regulated cyclin-dependent kinase (CDK) 2 activity. In our current study, Western blotting and quantitative RT-PCR demonstrated that I3C treatment increased both the transcripts and protein levels of the CDK2 inhibitor p21 waf1/cip1 (p21). Transfection of luciferase reporter plasmids containing wild-type and mutated p21 promoter fragments revealed that I3C induced p21 gene transcription through a p53 DNA binding element. Oligonucleotide precipitation showed that I3C increased the level of activated p53 nuclear protein that is competent to bind its DNA target site on the p21 promoter. Ablation of p53 production using short interfering RNA (siRNA) prevented that the I3C induced G1 arrest and up-regulation of p21 expression. Western blots using p53 phospho-specific antibodies revealed that I3C treatment increased the levels of three phosphorylated forms of p53 (Ser15, Ser37, Ser392) that are known to contribute to p53 protein stability and greater transactivation potential. Taken together, our results establish that the I3C induced G1 arrest of human prostate cancer cells requires the induced production of the activated phosphorylated forms of p53, which stimulate transcription of the CDK2 inhibitor p21.

Stable expression of temperature-sensitive p53: A suitable model to study wild-type p53 function in pancreatic carcinoma cells

Pancreatic adenocarcinoma is an extremely aggressive malignancy with a dismal prognosis. Inactivation of the p53 tumor-suppressor gene occurs in approximately 50% of primary tumors and is thought to account for a failure of the tumor cells to undergo growth arrest and apoptosis in response to chemotherapy. Hence, it is of interest to study the consequences of the restoration of wild-type (wt) p53 function in pancreatic carcinoma cells. Therefore, we retrovirally transduced temperature-sensitive (ts) human p53 into the p53-null pancreatic carcinoma cell line AsPC-1. ts p53 has a mutant phenotype at 37.5 degrees C, and a wt conformation at 32.5 degrees C. Stable expression of p53 in wt conformation caused upregulation of the p53 responsive gene p21Waf1/Cip1, and G1 growth arrest, but failed to induce Bax expression or apoptosis. In addition, we examined the effect of wt p53 expression on DNA damaging treatment. Interestingly, the doxorubicin- and radiation-induced S-/G2-phase arrests were suppressed by p53 in wt conformation. These results demonstrate that the ts p53/AsPC-1 model is suitable to investigate the effect of wt p53 restoration in pancreatic carcinoma cells.

CD45neg but Not CD45pos Human Myeloma Cells Are Sensitive to the Inhibition of IGF-1 Signaling by a Murine Anti-IGF-1R Monoclonal Antibody, mAVE1642

Insulin-like growth factor 1 (IGF-1) is a well-known growth factor for myeloma cells. Thus, therapeutic strategies targeting IGF-1R have been proposed for multiple myeloma treatment. In this study, we investigated the effect of the antagonistic anti-IGF-1R murineAVE1642 Ab (mAVE1642). We show that mAVE1642 selectively inhibits IGF-1R but not insulin signaling in human myeloma cell lines. Since we have previously shown the functional relevance of CD45 expression in the growth of myeloma cells and the association of CD45-negative (CD45neg) status with a less favorable clinical outcome, both CD45-positive (CD45pos) and CD45neg myeloma cell lines were selected for our study. We found that mAVE1642 strongly inhibits the growth of CD45neg myeloma cell lines, leading to a G1 growth arrest, whereas it has almost no effect on the growth of CD45pos myeloma cell lines. Furthermore, mAVE1642 binding induced a significant reduction of IGF-1R expression. We next demonstrated that the overexpression of IGF-1R in the CD45pos myeloma cell line increased Akt phosphorylation but was not sufficient to sensitize these cells to mAVE1642. In contrast, we generated a stable CD45-silencing XG-1 cell line and showed that it became sensitive to mAVE1642. Thus, for the first time, we provided direct evidence that the expression of CD45 renders cells resistant to mAVE1642. Taken together, these results support that therapy directed against IGF-1R can be beneficial in treating CD45neg patients.

Gene amplification and overexpression of protein phosphatase 1α in oral squamous cell carcinoma cell lines

Gene amplification of chromosomal band 11q13 is observed frequently in oral squamous cell carcinomas (OSCC). Several genes have been identified in the 11q13 amplicon, including FGF3, FGF4, CCND1, EMS1 and TAOS1. Some of these genes show good correlation between gene copy number and gene expression, and are thought to play a role in driving 11q13 amplification. The PPP1CA gene, which encodes the catalytic subunit of serine/threonine protein phosphatase protein phosphatase 1alpha (PP1alpha), is also located in 11q13. Protein phosphatase 1alpha, one of the isoforms of PP1, regulates critical cellular events, such as cell cycle progression, and apoptosis. We sought to explore the possibility that PPP1CA was amplified and overexpressed in OSCC cells. Indeed, some OSCC cell lines had PPP1CA gene amplification, as analysed by fluorescence in situ hybridization. We have also demonstrated that PPP1CA gene copy number is increased in 21% of the OSCC cell lines determined by quantitative microsatellite analysis. PP1alpha RNA expression determined by quantitative reverse transcription-polymerase chain reaction was significantly higher in OSCC cell lines with 11q13 amplification compared to those without 11q13 amplification (P=0.011). The difference was even more significant between cell lines with at least three copies of the PPP1CA gene and those with less than three copies of the gene (P=0.00045). Relative PP1alpha protein levels were also significantly associated with PPP1CA gene copy number (P=0.014). Furthermore, knockdown of PP1alpha and/or cyclin D1 by small interfering RNA suppressed OSCC cell growth, at least in part by modulating pRB phosphorylation, resulting in G0 growth arrest. These data suggest that like the cyclin D1 gene, CCND1, amplification and overexpression of the PP1alpha gene, PPP1CA, may be involved in OSCC tumorigenesis and/or progression.