P21 Cip1 Protein Research Articles

Persistent P21Cip1 Induction Mediates G1 Cell Cycle Arrest by Methylseleninic Acid in DU145 Prostate Cancer Cells

The induction of G(1) cell cycle arrest and apoptosis by second-generation selenium compounds (e.g., methylselenol precursors such as methylseleninic acid, MSeA) may contribute to their anti-cancer activities. We have documented previously induction of G(1) arrest and apoptosis by MSeA in association with upregulation of cyclin-dependent kinase inhibitor (CDKI) proteins p21Cip1 and/or p27Kip1 in DU145 prostate cancer cells. However, whether these CDKIs play a critical mediator role in G(1) arrest and apoptosis by MSeA has not been addressed. In the present work, we show exposure of p53-mutant DU145 cells to sub-apoptotic concentrations of MSeA induced p21cip1 mRNA (3 h) and protein (6 h) much faster than p27kip1 mRNA (12 h) and protein (12 h). Knocking down of p21 by siRNA completely abolished G(1) arrest induction by MSeA in DU145 cells, yet si-p27 RNA had no attenuation effect on the G(1) arrest. Depletion of p21Cip1 alone or both p21Cip1 and p27Kip1 increased MSeA-induced caspase-mediated apoptosis. Immunoprecipitation detected increased binding of p21Cip1 to CDK2 and CDK6 in MSeA-exposed DU145 cells. In DU145 xenografts from mice acutely treated with MSeA p.o., the induction of p21Cip1 was observed at 72 h of daily exposure. In p53-wild type LNCaP PCa cells and p53-null PC-3 PCa cells, MSeA modestly and transiently upregulated p21Cip1 protein level, subsiding to basal level by 24 h, without affecting P27Kip1 abundance in the same duration. Si-p21 RNA knockdown in these cells have only a partial effect to reverse G(1) arrest induction by MSeA. Together, our data support persistent, p53-independent, p21Cip1 induction as a critical mediator of MSeA-induced G(1) arrest in DU145 PCa cells, however, p21Cip1 induction and G(1) arrest were not necessary for, and may antagonize, caspase-mediated apoptosis.

Abstract 2882: Penta-O-galloyl-beta-D-glucose induces DNA replicative S arrest and G1 arrest independent of p21Cip1, p27Kip1 and p53 in human breast cancer cells and is orally active against breast cancer xenograft

Abstract Natural herbal compounds with novel actions different from existing breast cancer targeting modalities are attractive for improving treatment and prevention efficacy and safety. We have recently shown that penta-1,2,3,4,6-O-galloyl-beta-D-glucose (PGG), a naturally occurring gallotannin polyphenol compound, induced S-phase arrest in prostate cancer cells through inhibiting DNA replicative synthesis, in addition to the induction of G1 arrest and cell deaths at higher levels of exposure. We and others have shown that PGG through i.p. injection exerts a strong in vivo growth suppression of human prostate cancer xenograft models in athymic nude mice. We hypothesize that the novel DNA replication targeting action and the cellular arrest and death effects of PGG are applicable to breast cancer cells and PGG could be a novel agent for treatment or prevention of breast cancer, especially those without proven drugable targets such as triple-negative breast cancer. In cell culture studies, exposure to serum achievable level of PGG induced rapid S arrest in p53-wild type ER-dependent MCF-7 breast cancer cells and in p53-mutant ER-/PR- and Her2-negative (triple-negative) MDA-MB-231 breast cancer cells as indicated by the lack of BrdU incorporation into S-phase cells. Higher levels of PGG induced more caspase-mediated apoptosis in MCF-7 than in MDA-MB-231 cells. In addition to S-arrest, PGG induced G1 arrest in both cell lines. Contrary to a study by others with questionable design, we have found no induction of CDK inhibitory proteins p21Cip1 and p27Kip1 with G1 arrest. PGG treatment led to decreased cyclin D1 in both cell lines and over-expressing cyclin D1 abolished G1 arrest and hastened S arrest. In serum-starvation synchronized MCF-7 cells, we observed a close association of down-regulation of cyclin D1 and de-phosphorylation of Rb by PGG shortly before G1-S transition stimulated by serum. Though PGG inhibited estrogen-stimulated cell growth in MCF-7 cells, it had little effect on estrogen receptor-alpha level. To test the in vivo efficacy of PGG, we treated female athymic mice inoculated with MDA-MB-231 xenograft with 10 mg/kg body weight by daily gavage and compared with weekly paclitaxel treatment at the same dosage. Oral administration of PGG led to a better growth inhibitory efficacy than the taxane drug. Together, our in vitro and in vivo data support PGG as a potential chemopreventve or treatment agent for breast cancer with novel targeting actions. 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 2882.

A novel signaling pathway associated with Lyn, PI 3-kinase and Akt supports the proliferation of myeloma cells

Interleukin-6 (IL-6) is a growth factor for human myeloma cells. We have recently found that in myeloma cells the activation of both signal transducer and activator of transcription (STAT) 3 and extracellular signal-regulated kinase (ERK) 1/2 is not sufficient for the IL-6-induced proliferation, which further requires the activation of the src family kinases, such as Lyn. Here we showed that the Lyn-overexpressed myeloma cell lines had the higher proliferative rate with IL-6 and the enhanced activation of the phosphatidylinositol (PI) 3-kinase and Akt. The IL-6-induced phosphorylation of STAT3 and ERK1/2 was not up-regulated in the Lyn-overexpressed cells, indicating that the Lyn-PI 3-kinase-Akt pathway is independent of these pathways. The PI 3-kinase was co-precipitated with Lyn in the Lyn-overexpressed cells of which proliferation with IL-6 was abrogated by the specific inhibitors for PI 3-kinase or Akt, suggesting that the activation of the PI 3-kinase-Akt pathway associated with Lyn is indeed related to the concomitant augmentation of myeloma cell growth. Furthermore, the decreased expression of p53 and p21 Cip1 proteins was observed in the Lyn-overexpressed cells, implicating a possible downstream target of Akt. This study identifies a novel IL-6-mediated signaling pathway that certainly plays a role in the proliferation of myeloma cells and this novel mechanism of MM tumor cell growth associated with Lyn would eventually contribute to the development of MM treatment.

Acetylbritannilactone induces G 1 arrest and apoptosis in vascular smooth muscle cells

Background The present study was designed to determine the effects of Acetylbritannilactone (ABL), a naturally occurring Inula britannica L., on vascular smooth muscle cell (VSMC) proliferation and apoptosis. Methods In vitro experiments were performed to evaluate the effects of ABL on the VSMC cycle and apoptosis stimulated by chemoattractant. In addition, to examine the effects of ABL in vivo, balloon injury to rat carotid arteries was performed. Results ABL treatment inhibited platelet-derived growth factor (PDGF) induced DNA synthesis and proliferation in cultured VSMC. Such growth-inhibitory effects of ABL were associated with G 1 phase arrest, which were correlated with reduction of cyclins D1, A, and E expression and cyclin-dependent kinase (CDK) 2, CDK4, and CDK6 proteins, increased the CDK inhibitory protein p21 cip1 expression, and enhanced the binding of p21 cip1 to CDKs. In addition, ABL also induced apoptosis in proliferative VSMCs, as evidenced by the induction of a higher ratio of Bax/Bcl-2, activation of caspase-9, caspase-3, and the cleavage of endogenous substrate Poly (ADP-ribose) polymerase. However, pretreatment with pan-caspases inhibitor (z-VAD-fmk) only partially reversed ABL-induced apoptosis, suggesting the involvement of both caspase-dependent and caspase-independent pathways in these processes. Furthermore, the effects of ABL on VSMCs were associated with the downregulation of extracellular signal-regulated kinase (ERK) 1/2 signaling pathways. In vivo, ABL (26 mg/kg/day) significantly suppressed injury-induced ERK1/2 phosphorylation, and increased VSMC apoptosis 14 days after balloon injury. Conclusions Our findings demonstrated that ABL was capable of suppressing the abnormal VSMC proliferation, accompanied by the induction of apoptosis in vivo and in vitro. It suggested that ABL could be considered a pharmacological candidate for the prevention of restenosis after balloon angioplasty.

KG-135, enriched with selected ginsenosides, inhibits the proliferation of human prostate cancer cells in culture and inhibits xenograft growth in athymic mice

Sun ginseng (SG) was recently developed as a heat-processed form of ginseng. The Rg3, Rk1, and Rg5 ginsenosides are its main ginsenoside components. SG has been reported to have more potent pharmacological activities than red ginseng (RG), where these pharmacological activities include vasodilatory, anti-oxidant and anti-tumorigenic effects. In the present study, we investigated KG-135, the ginsenoside-rich fraction of SG and demonstrated that this fraction inhibits proliferation of human prostate cancer cells both in vitro and in vivo. KG-135 caused a significant growth inhibition of DU145 and PC-3 human prostate cancer cells. KG-135 induced cell cycle arrest in the G1 phase and caused an associated increase in the p21 Cip1 protein levels. When KG-135 was fed to mice that had been xenografted with DU145 tumors, a time-dependent inhibition of tumor growth was noted without any observed toxicity. Immunohistochemical analysis of the tumor tissues showed that KG-135 led to a decrease in the expression of proliferating cell nuclear antigen (PCNA). Microarray analysis of the tumors revealed that KG-135 inhibited tumor growth and also caused changes in the expression levels of multiple cancer-related genes. These data suggest that KG-135 effectively inhibits prostate cancer cell proliferation. Its mechanism of action likely involves cyclin inhibition and regulation of the expression of the TNFRSF25 and ADRA2A genes.

Dipeptidyl Peptidase 2 is an essential survival factor in the regulation of cell quiescence

Most cells in the body are in a resting state and undergo cell cycle progression only upon growth factor stimulation or activation. While much research on proliferation and activation has been performed, very little about signals that maintain quiescent cells in G0 is known, preventing cell cycle entry or apoptosis. In this study, the pathways of apoptosis induction in quiescent peripheral blood cells and fibroblasts mediated by inhibition or down-regulation of Dipeptidyl Peptidase 2 (DPP2) have been explored. A decrease in DPP2 activity was found to cause resting cells to exit from G0, accompanied by a decrease in p130, p27Kip1 and p21Cip1 protein levels. In addition, DPP2-inhibited or down-regulated cells exhibit an increase in early G1/S progressors, with increases in the levels of retinoblastoma (pRb), p107 and cyclin D proteins. Furthermore, decrease of DPP2 activity leads to an increase in c-Myc and a decrease in Bcl-2, two events that have been associated with apoptosis induction. This apoptosis by DPP2 down-regulation is prevented in p53-/- cells or by ectopic expression of proteins that suppress p53 or c-Myc activity. Thus, DPP2 is essential for maintaining lymphocytes and fibroblasts in G0, and its inhibition results in apoptosis mediated by induction of c-Myc and p53.

Extracellular Signal-Regulated Kinase 2-Dependent Phosphorylation Induces Cytoplasmic Localization and Degradation of p21Cip1

p21(Cip1) is an inhibitor of cell cycle progression that promotes G(1)-phase arrest by direct binding to cyclin-dependent kinase and proliferating cell nuclear antigen. Here we demonstrate that mitogenic stimuli, such as epidermal growth factor treatment and oncogenic Ras transformation, induce p21(Cip1) downregulation at the posttranslational level. This downregulation requires the sustained activation of extracellular signal-regulated kinase 2 (ERK2), which directly interacts with and phosphorylates p21(Cip1), promoting p21(Cip1) nucleocytoplasmic translocation and ubiquitin-dependent degradation, thereby resulting in cell cycle progression. ERK1 is not likely involved in this process. Phosphopeptide analysis of in vitro ERK2-phosphorylated p21(Cip1) revealed two phosphorylation sites, Thr57 and Ser130. Double mutation of these sites abolished ERK2-mediated p21(Cip1) translocation and degradation, thereby impairing ERK2-dependent cell cycle progression at the G(1)/S transition. These results indicate that ERK2 activation transduces mitogenic signals, at least in part, by downregulating the cell cycle inhibitory protein p21(Cip1).

Inhibition of Notch pathway prevents osteosarcoma growth by cell cycle regulation

The study shows constitutive activation of the Notch pathway in various types of malignancies. However, it remains unclear how the Notch pathway is involved in the pathogenesis of osteosarcoma. We investigated the expression of the Notch pathway molecules in osteosarcoma biopsy specimens and examined the effect of Notch pathway inhibition. Real-time PCR revealed overexpression of Notch2, Jagged1, HEY1, and HEY2. On the other hand, Notch1 and DLL1 were downregulated in biopsy specimens. Notch pathway inhibition using γ-secretase inhibitor and CBF1 siRNA slowed the growth of osteosarcomas in vitro. In addition, γ-secretase inhibitor-treated xenograft models exhibited significantly slower osteosarcoma growth. Cell cycle analysis revealed that γ-secretase inhibitor promoted G1 arrest. Real-time PCR and western blot revealed that γ-secretase inhibitor reduced the expression of accelerators of the cell cycle, including cyclin D1, cyclin E1, E2, and SKP2. On the other hand, p21cip1 protein, a cell cycle suppressor, was upregulated by γ-secretase inhibitor treatment. These findings suggest that inhibition of Notch pathway suppresses osteosarcoma growth by regulation of cell cycle regulator expression and that the inactivation of the Notch pathway may be a useful approach to the treatment of patients with osteosarcoma.

PKG induces the expression of tumor suppressor genes in colon cancer cells

The role of cyclic guanosine 3',5′‐monophosphate (cGMP)‐dependent protein kinase (PKG) as an anticancer messenger emerged recently, but there is little known about its action. In this study, we examined the role of PKG‐Iβ on the beta‐catenin pathway and the expression of tumor suppressor proteins in human colon cancer cells. Our results revealed that cells overexpressing PKG‐Iβ showed β‐catenin translocation to the nucleus compared to control cells. This translocation of β‐catenin was accompanied with an increase in T‐cell factor (TCF)‐luciferase activity and β‐catenin binding to TCF oligonucleotide as tested by elecromobility shift assays in both LoVo and SW480 cells. We analyzed the expression of the tumor suppressor retinoblastoma (Rb), p27KIP1, p53, and p21CIP1 at both the protein levels and promoter activities. Cells overexpressing PKG‐Iβ showed an increase in expression of the p21CIP1, p27KIP1, and Rb proteins. Both p21CIP1 and p27KIP1 promoters were activated in LoVo or SW480 cells overexpressing PKG as demonstrated by luciferase reporter assays. Specific PKG inhibitors (DT2/DT3) as well as phosphatase inhibitors (okadaic acid, Calyculin A) reduced β‐catenin accumulation in the nucleus as well as the promoter activities of p21 and Rb luciferase reporter constructs. These results suggest that PKG is stabilizing β‐catenin, probably through activation of a phosphatase. These effects of PKG activation on the expression of tumor suppressor genes may explain, at least in part, the anticancer effects of activation of PKG.

Aspirin inhibits camptothecin-induced p21CIP1 levels and potentiates apoptosis in human breast cancer cells

The ability of aspirin to trigger apoptosis in cancer cells is well known and is consistent with the clinical and epidemiological evidence on its chemopreventive effects in curtailing epithelial cancers, including breast cancer. We hypothesized that the anticancer effects of aspirin may involve acetylation of the tumor suppressor protein p53, a known regulator of apoptosis. In the present study, we determined if aspirin at the physiologically achievable concentration of 100 microM acetylates p53 and modulates the expression of p21CIP1, a protein involved in cell cycle arrest, and Bax, a pro-apoptotic protein. Using MDA-MB-231 human breast cancer cells, we demonstrate that aspirin at 100 microM concentration markedly acetylated the p53 protein, which was primarily localized to the nucleus. Aspirin induced p21CIP1 protein levels in a transient fashion in contrast to the sustained induction of Bax. The induction of p21CIP1 protein levels began at 3 h and was maximal at 6-8 h; however, it decreased to control levels by 30 h. In contrast, the anticancer drug, camptothecin (CPT) induced a steady accumulation of p21CIP1 protein. Remarkably, when cells were co-treated with aspirin and CPT, p21CIP1 levels were drastically downregulated, and this phenomenon was observed in many cancer cell lines. Incubation of recombinant p21 with cytoplasmic extracts from aspirin-treated cells caused its degradation suggesting the involvement of proteases in the disappearance of p21CIP1. Consistent with this data, aspirin decreased the survival of CPT-treated cells and greatly increased the extent of apoptosis. Our observation that aspirin has the ability to inhibit p21CIP1 after its initial induction has important implications in chemotherapy, and suggests its potential use to increase the efficacy of anticancer agents.

Continuous elimination of oxidized nucleotides is necessary to prevent rapid onset of cellular senescence

Reactive oxygen species (ROS) appear to play a role in limiting both cellular and organismic lifespan. However, because of their pleiotropic effects, it has been difficult to ascribe a specific role to ROS in initiating the process of cellular senescence. We have studied the effects of oxidative DNA damage on cell proliferation, believing that such damage is of central importance to triggering senescence. To do so, we devised a strategy to decouple levels of 8-oxoguanine, a major oxidative DNA lesion, from ROS levels. Suppression of MTH1 expression, which hydrolyzes 8-oxo-dGTP, was accompanied by increased total cellular 8-oxoguanine levels and caused early-passage primary and telomerase-immortalized human skin fibroblasts to rapidly undergo senescence, doing so without altering cellular ROS levels. This senescent phenotype recapitulated several salient features of replicative senescence, notably the presence of senescence-associated beta-galactosidase (SA beta-gal) activity, apparently irreparable genomic DNA breaks, and elevation of p21(Cip1), p53, and p16(INK4A) tumor suppressor protein levels. Culturing cells under low oxygen tension (3%) largely prevented the shMTH1-dependent senescent phenotype. These results indicate that the nucleotide pool is a critical target of intracellular ROS and that oxidized nucleotides, unless continuously eliminated, can rapidly induce cell senescence through signaling pathways very similar to those activated during replicative senescence.

Stabilization of N-Myc Is a Critical Function of Aurora A in Human Neuroblastoma

In human neuroblastoma, amplification of the MYCN gene predicts poor prognosis and resistance to therapy. In a shRNA screen of genes that are highly expressed in MYCN-amplified tumors, we have identified AURKA as a gene that is required for the growth of MYCN-amplified neuroblastoma cells but largely dispensable for cells lacking amplified MYCN. Aurora A has a critical function in regulating turnover of the N-Myc protein. Degradation of N-Myc requires sequential phosphorylation by cyclin B/Cdk1 and Gsk3. N-Myc is therefore degraded during mitosis in response to low levels of PI3-kinase activity. Aurora A interacts with both N-Myc and the SCF(Fbxw7) ubiquitin ligase that ubiquitinates N-Myc and counteracts degradation of N-Myc, thereby uncoupling N-Myc stability from growth factor-dependent signals.

Role of polyamines in p53-dependent apoptosis of intestinal epithelial cells

Although p53 is known to play a critical role in the proliferation of gastrointestinal epithelia, the role of the Mdm2/p53 pathway in response to inducers of apoptosis in intestinal epithelial cells is unknown. Our data show that camptothecin (CPT)-induced apoptosis correlated with increased p53, p21Cip1, and Mdm2 protein levels, with a simultaneous increase in ATR Ser428, p53 Ser15 and Mdm2 Ser166 phosphorylation in IEC-6 cells. Increased p53 levels and its phosphorylation increased Bax protein, caspase-9, -3 activation and apoptosis. However, TNF-α/CHX-mediated apoptosis was independent of p53 protein levels and phosphorylation. The translation inhibitor, cycloheximide (CHX), prevented CPT-induced apoptosis. CHX completely prevented CPT-induced p53 phosphorylation and synthesis of p21Cip1, Bax and Bcl-xL proteins without altering p53 levels. The p53 activator, RITA, augmented CPT-induced apoptosis. The Mdm2 antagonist, Nutlin-3, significantly increased apoptosis, which was accompanied by increased p53, Mdm2 and p21Cip1 protein levels. The ATM/ATR kinase inhibitor, CGK733, blocked CPT-induced p53 Ser15 phosphorylation and protected cells from CPT-induced apoptosis. Inhibition of ornithine decarboxylase (ODC) with α-difluromethylornithine (DFMO) and subsequent depletion of intracellular polyamines increased p53 protein, Mdm2 Ser166 phosphorylation and conferred resistance to CPT-induced apoptosis. However, polyamine depletion had no effect on p53 phosphorylation. Nutlin-3 reversed the protective effect of DFMO and sensitized cells to CPT-induced apoptosis. These results suggest that p53 stabilization and accumulation in response to polyamine depletion predominantly modulate cell cycle checkpoints via p21Cip1 expression and inhibit transcription of target genes responsible for apoptosis. In contrast, phosphorylation and stabilization of p53 in response to DNA-damage lead to apoptosis, which indicates different roles of p53 during DNA damage and polyamine depletion.

Adult neurogenesis, cell cycle and drug discovery in psychiatry

For many years the production of new neurons in mammalian brain was thought to be restricted to development. It is now clear that neurogenesis does occur in adult mammals, including humans (Eriksson et al, 1998). Antidepressant drugs and procedures that reduce depression, such as electroconvulsive shock and exercise, increase neurogenesis. The relationships among adult neurogenesis, antidepressant drugs, and depression have generated considerable interest and controversy (Duman, 2004; Scharfman and Hen, 2007). p21Cip1, a cyclin-dependent kinase inhibitor, restrains cell-cycle progression and proliferation. It is found in neuroblasts and newly developing neurons in the subgranular zone of the hippocampus (Pechnick et al, 2008). Chronic treatment with the tricyclic antidepressant imipramine decreases p21Cip1 transcript and protein levels and stimulates neurogenesis in this region. Moreover, mice lacking p21Cip1 have increased rates of hippocampal neurogenesis. Thus, p21Cip1 restrains neurogenesis in the hippocampus, and antidepressant-induced stimulation of neurogenesis might be due to decreased p21Cip1 expression. Cell-cycle regulation occurs downstream from the primary site of action of antidepressants, suggesting that new therapeutic strategies might directly target cell-cycle proteins. Currently, neurogenesis is a phenomenon in search of a function. There are four key questions that must be answered prior to the implementation of effective treatment strategies directed at altering neurogenesis. First, what is the role of adult neurogenesis in normal brain function? In humans, neurogenesis occurs in the hippocampus and olfactory bulb (Gould, 2007). Advances in imaging technology would help establish the conditions and pathological states under which neurogenesis is altered and whether neurogenesis is a latent process in other brain regions. This information is important because drug-induced stimulation of neurogenesis could disrupt fundamental neurobiological processes. Second, are changes in behavior and/or functional deficits in any disease state due to decreased (or increased) neurogenesis? Excessive neurogenesis could result in inappropriate integration into existing neural networks and could underlie pathological conditions such as epilepsy (Scharfman and Hen, 2007). Drug-induced stimulation of neurogenesis might have unforeseen adverse consequences. Third, are basal and drug-induced neurogenesis age-dependent in humans? In rodents, the rate of neurogenesis decreases from adolescence to adulthood, and the decline is very steep (Abrous et al, 2008). If the rate of neurogenesis is profoundly decreased in older humans, then drugs targeted at stimulating neurogenesis might have limited efficacy in that population. Fourth, are there adverse consequences associated with long-term stimulation of neurogenesis? Long-term and unrelenting stimulation of mitosis without appropriate differentiation and migration could lead to unexpected problems. In addition, it is possible that adult neural stem cells have finite proliferation potentials. Long-term stimulation of neurogenesis might eventually produce premature exhaustion of neuronal precursors, the subsequent loss of therapeutic efficacy and premature ‘aging’ in the system. There is a growing list of drugs and behavioral procedures that can stimulate or decrease neurogenesis. Modulating neurogenesis could be a new therapeutic target for the treatment of psychiatric disorders; however, more fundamental information on neurogenesis in humans needs to be obtained to design rational therapeutic strategies and avoid unforeseen adverse consequences.

Phase I Trial of Vorinostat (SAHA) in Combination with Alvocidib (Flavopiridol) in Patients with Relapsed, Refractory or (Selected) Poor Prognosis Acute Leukemia or Refractory Anemia with Excess Blasts-2 (RAEB-2)

Based on preclinical findings demonstrating marked antileukemic synergism between the HDAC inhibitor vorinostat and the CDK inhibitor flavopiridol (Almenara et al., Leukemia 16:1331–43, 2002), a Phase I trial of these agents has been initiated in patients with refractory AML/MDS. Eligible patients (pts) have acute leukemia (AL) refractory to standard therapy or de novo AL with poor risk factors (age >60 or antecedent myelodysplastic syndrome), imatinib-refractory chronic myeloid leukemia in blast crisis, or RAEB-2. Pts are excluded for prior auto- or allogeneic marrow or stem cell transplantation, significant comorbidities or organ dysfunction, and QTc interval >0.470 seconds. All pts receive oral vorinostat (V) 200 mg 3 times daily. In the initial stages of the trial, pts also received flavopiridol (F) by 1-hour bolus infusion on days 1–5 out of a 21-day cycle. Initial F dose levels were (mg/m2): 10, 20, 30, 40. Subsequently, a “hybrid” F infusion schedule (30 minute load followed by 4 hour infusion) was adopted based on recent evidence of activity of this schedule in CLL. With the hybrid schedule, F is given on a combination of days (d) 1, 3, 8 and 11. Targeted F dose levels are, in mg/m2 (Fload/Finfusion): 20/20 on d1 and 8; 30/20 on d1 and 8; 30/30 on d1 and 8; 30/50 on d1 and 8; 30/50 on d1, 3 and 8; 30/50 on d 1,3, 8 and 11. Dose limiting toxicity (DLT) is defined as NCI CTCAE grade 4 non-hematologic (heme) toxicity (except that related to cytopenias, infection or hyperbilirubinemia), grade 3 non-heme toxicity which lasts >7 days, or grade 4 ANC/platelet toxicity that persists for > 6 weeks in absence of leukemia. 28 pts have been enrolled. With the bolus schedule 14 pts were treated at 4 dose levels without identification of an MTD, after which development of the hybrid schedule was begun. Since initiation of the hybrid schedule, 8 pts have been treated at 3 dose levels. One DLT (infectious colitis with sepsis) was seen on level 4 of the bolus schedule and one DLT (gr 3 atrial fibrillation) was seen at the 3rd hybrid dose level. The MTD of the hybrid schedule has not been reached. Non-DLT toxicities include diarrhea, anorexia, nausea, weight loss, fatigue, electrolyte abnormalities, hyperbilirubinemia, dyspnea, confusion, febrile neutropenia and infectious complications (including atypical infections). Of 22 pts treated, 20 are evaluable for response. Thus far, there have been no complete remissions in this heavily pretreated population. However, 10 pts experienced some clinical benefit despite meeting criteria for “remission induction failure” – of those, 3 were treated with ≥ 5 cycles. 10 pts had a best response of progressive disease. One heavily pre-treated pt with refractory AML, who progressed after FLAG x2 followed by gemtuzumab ozogamycin, remains in treatment following 7 cycles with improved PS and QOL, a marked improvement in peripheral WBC, and a normocellular marrow with 6% blasts. To date, hyperacute tumor lysis has not been seen with the hybrid schedule, but aggressive prophylaxis and monitoring remain integral to the treatment plan. QTc evaluation and monitoring has been implemented because of V interactions with concurrent medications commonly used in this population (azole antifungals, among others). Correlative laboratory studies reveal a regular increase in acetylated tubulin but heterogeneous post-treatment effects on p21CIP1, Mcl-1, and other proteins in leukemic blasts. Collectively, these findings indicate that a regimen combining vorinostat and flavopiridol is well tolerated in patients with refractory or high-risk AML, and may have some activity in patients highly resistant to standard therapy. Pending identification of the MTD and RPTD (recommended phase II dose), Phase II evaluation of this therapeutic strategy should define its activity more definitively.

Multiple Cyclin Kinase Inhibitors Promote Bile Acid-induced Apoptosis and Autophagy in Primary Hepatocytes via p53-CD95-dependent Signaling

Previously, using primary hepatocytes residing in early G1 phase, we demonstrated that expression of the cyclin-dependent kinase (CDK) inhibitor protein p21Cip-1/WAF1/mda6 (p21) enhanced the toxicity of deoxycholic acid (DCA) + MEK1/2 inhibitor. This study examined the mechanisms regulating this apoptotic process. Overexpression of p21 or p27(Kip-1) (p27) enhanced DCA + MEK1/2 inhibitor toxicity in primary hepatocytes that was dependent on expression of acidic sphingomyelinase and CD95. Overexpression of p21 suppressed MDM2, elevated p53 levels, and enhanced CD95, BAX, NOXA, and PUMA expression; knockdown of BAX/NOXA/PUMA reduced CDK inhibitor-stimulated cell killing. Parallel to cell death processes, overexpression of p21 or p27 profoundly enhanced DCA + MEK1/2 inhibitor-induced expression of ATG5 and GRP78/BiP and phosphorylation of PKR-like endoplasmic reticulum kinase (PERK) and eIF2alpha, and it increased the numbers of vesicles containing a transfected LC3-GFP construct. Incubation of cells with 3-methyladenine or knockdown of ATG5 suppressed DCA + MEK1/2 inhibitor-induced LC3-GFP vesicularization and enhanced DCA + MEK1/2 inhibitor-induced toxicity. Expression of dominant negative PERK blocked DCA + MEK1/2 inhibitor-induced expression of ATG5, GRP78/BiP, and eIF2alpha phosphorylation and prevented LC3-GFP vesicularization. Knock-out or knockdown of p53 or CD95 abolished DCA + MEK1/2 inhibitor-induced PERK phosphorylation and prevented LC3-GFP vesicularization. Thus, CDK inhibitors suppress MDM2 levels and enhance p53 expression that facilitates bile acid-induced, ceramide-dependent CD95 activation to induce both apoptosis and autophagy in primary hepatocytes.

Activation of Protein Kinase G Increases the Expression of p21CIP1, p27KIP1, and Histidine Triad Protein 1 through Sp1

The anticancer role of cyclic guanosine 3',5'-monophosphate (cGMP)-dependent protein kinase G (PKG) has become of considerable interest, but the underlying mechanisms are not fully established. In this study, we examined the effects of activation of PKG on the expression of three tumor suppressor proteins in human SW480 colon cancer cells. Our results revealed that treatment with cell permeable cGMP derivatives, or the cGMP phosphodiesterase inhibitor sulindac sulfone (exisulind, aptosyn, hereafter called exisulind) led to increased expression of the tumor suppressor proteins p21(CIP1), p27(KIP1), and Histidine triad protein 1 (HINT1), and their corresponding mRNAs. Overexpression of PKG Ibeta also caused increased expression of the p21(CIP1), p27(KIP1), and HINT1 proteins. Both the p21(CIP1) and p27(KIP1) promoters contain Sp1 binding sites and they were activated by PKG in luciferase reporter assays. Specific Sp1 sites in the p21 and p27 promoters were sufficient to mediate PKG-induced luciferase reporter activity, suggesting an interaction between Sp1 and PKG. Indeed, we found that PKG can phosphorylate Sp1 on serine residue(s) and this resulted in transcriptional activation of Sp1. Knockdown of Sp1 expression with siRNA inhibited the increased expression of p21(CIP1), p27(KIP1), and HINT1 induced by the cGMP derivative 8-pCPT-cGMP in SW480 cells. These novel effects of PKG activation on the expression of three tumor suppressor genes may explain, at least in part, the anticancer effects of activation of PKG. They also provide a rationale for further developing activators of PKG for the prevention and treatment of cancer.

Nox2 modulation of cell cycle inhibitory protein p21 cip1 expression in endothelial cells

The abilities of endothelial cells (EC) to proliferate, to be quiescent and to undergo apoptosis during remodelling are important determinants relating to angiogenesis, wound healing and many cardiovascular diseases. EC express constitutively an NADPH oxidase, which is a major source of superoxide production. The catalytic subunit of NADPH oxidase has several isoforms (Nox1–5). However, their individual roles in endothelial function remain unknown. In this study, we investigated the role of Nox2 in nutrient deprivation-induced cell cycle arrest and apoptosis.

Inositol trisphosphate 3-kinase B (InsP3KB) as a physiological modulator of myelopoiesis.

Inositol trisphosphate 3-kinase B (InsP3KB) belongs to a family of kinases that convert inositol 1,4,5-trisphosphate (Ins(1,4,5)P3 or IP3) to inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4). Previous studies have shown that disruption of InsP3KB leads to impaired T cell and B cell development as well as hyperactivation of neutrophils. Here, we demonstrate that InsP3KB is also a physiological modulator of myelopoiesis. The InsP3KB gene is expressed in all hematopoietic stem/progenitor cell populations. In InsP3KB null mice, the bone marrow granulocyte monocyte progenitor (GMP) population was expanded, and GMP cells proliferated significantly faster. Consequently, neutrophil production in the bone marrow was enhanced, and the peripheral blood neutrophil count was also substantially elevated in these mice. These effects might be due to enhancement of PtdIns(3,4,5)P3/Akt signaling in the InsP3KB null cells. Phosphorylation of cell cycle-inhibitory protein p21(cip1), one of the downstream targets of Akt, was augmented, which can lead to the suppression of the cell cycle-inhibitory effect of p21.

S-Phase-specific Activation of PKCα Induces Senescence in Non-small Cell Lung Cancer Cells

Protein kinase C (PKC) has been widely implicated in positive and negative control of cell proliferation. We have recently shown that treatment of non-small cell lung cancer (NSCLC) cells with phorbol 12-myristate 13-acetate (PMA) during G1 phase inhibits the progression into S phase, an effect mediated by PKC delta-induced up-regulation of the cell cycle inhibitor p21 Cip1. However, PMA treatment in asynchronously growing NSCLC cells leads to accumulation of cells in G2/M. Studies in post-G1 phases revealed that PMA induced an irreversible G2/M cell cycle arrest in NSCLC cells and conferred morphological and biochemical features of senescence, including elevated SA-beta-Gal activity and reduced telomerase activity. Remarkably, this effect was phase-specific, as it occurred only when PKC was activated in S, but not in G1, phase. Mechanistic analysis revealed a crucial role for the classical PKC alpha isozyme as mediator of the G2/M arrest and senescence, as well as for inducing p21(Cip1) an obligatory event for conferring the senescence phenotype. In addition to the unappreciated role of PKC isozymes, and specifically PKC alpha, in senescence, our data introduce the paradigm that discrete PKCs trigger distinctive responses when activated in different phases of the cell cycle via a common mechanism that involves p21 Cip1 up-regulation.