Growth Arrest In Phase Research Articles

Quantitative and Temporal Proteome Analysis of Butyrate-treated Colorectal Cancer Cells

Colorectal cancer is one of the most common cancers in developed countries, and its incidence is negatively associated with high dietary fiber intake. Butyrate, a short-chain fatty acid fermentation by-product of fiber induces cell maturation with the promotion of growth arrest, differentiation, and/or apoptosis of cancer cells. The stimulation of cell maturation by butyrate in colonic cancer cells follows a temporal progression from the early phase of growth arrest to the activation of apoptotic cascades. Previously we performed two-dimensional DIGE to identify differentially expressed proteins induced by 24-h butyrate treatment of HCT-116 colorectal cancer cells. Herein we used quantitative proteomics approaches using iTRAQ (isobaric tags for relative and absolute quantitation), a stable isotope labeling methodology that enables multiplexing of four samples, for a temporal study of HCT-116 cells treated with butyrate. In addition, cleavable ICAT, which selectively tags cysteine-containing proteins, was also used, and the results complemented those obtained from the iTRAQ strategy. Selected protein targets were validated by real time PCR and Western blotting. A model is proposed to illustrate our findings from this temporal analysis of the butyrate-responsive proteome that uncovered several integrated cellular processes and pathways involved in growth arrest, apoptosis, and metastasis. These signature clusters of butyrate-regulated pathways are potential targets for novel chemopreventive and therapeutic drugs for treatment of colorectal cancer.

Mycelium differentiation and antibiotic production in submerged cultures of Streptomyces coelicolor.

Despite the fact that most industrial processes for secondary metabolite production are performed with submerged cultures, a reliable developmental model for Streptomyces under these culture conditions is lacking. With the exception of a few species which sporulate under these conditions, it is assumed that no morphological differentiation processes take place. In this work, we describe new developmental features of Streptomyces coelicolor A3(2) grown in liquid cultures and integrate them into a developmental model analogous to the one previously described for surface cultures. Spores germinate as a compartmentalized mycelium (first mycelium). These young compartmentalized hyphae start to form pellets which grow in a radial pattern. Death processes take place in the center of the pellets, followed by growth arrest. A new multinucleated mycelium with sporadic septa (second mycelium) develops inside the pellets and along the periphery, giving rise to a second growth phase. Undecylprodigiosin and actinorhodin antibiotics are produced by this second mycelium but not by the first one. Cell density dictates how the culture will behave in terms of differentiation processes and antibiotic production. When diluted inocula are used, the growth arrest phase, emergence of a second mycelium, and antibiotic production are delayed. Moreover, pellets are less abundant and have larger diameters than in dense cultures. This work is the first to report on the relationship between differentiation processes and secondary metabolite production in submerged Streptomyces cultures.

Kaempferol-7- O-β- d-glucoside (KG) isolated from Smilax china L. rhizome induces G 2/M phase arrest and apoptosis on HeLa cells in a p53-independent manner

Kaempferol-7- O-β- d-glucoside (KG), a flavonoid glycoside, isolated from Smilax china L. rhizome, displayed marked anticancer activity on a panel of established cancer cells, of which, HeLa human cervix carcinoma cells were the most sensitive. Meanwhile, the cytotoxic effects of KG on normal human cells (HEK293 embryonic kidney cells and L-02 embryonic liver cells) were much smaller than on cancer cells. This work studied the molecular mechanisms underlying KG induced growth inhibition in HeLa cells. The results showed that KG induced G 2/M phase growth arrest correlated with Cyclin B1 and Cdk1 decrease in a p53-independent manner, and also caused an increase in apoptosis, which was confirmed by characteristic morphological changes, evident DNA fragmentation, increased apoptotic sub-G1 population. Furthermore, inhibition of NF-κB nuclear translocation, up-regulation of Bax and down-regulation of Bcl-2, were observed in HeLa cells treated with KG, which indicated that the mitochondrial pathway was involved in the apoptosis signal pathway. In summary, KG displayed a significant anti-tumor effect through cell cycle arrest and apoptotic induction in HeLa cells, which suggested that KG might have therapeutic potential against cervix carcinoma.

Differential protein expression following low temperature culture of suspension CHO-K1 cells

BackgroundTo ensure maximal productivity of recombinant proteins (rP) during production culture it is typical to encourage an initial phase of rapid cell proliferation to achieve high biomass followed by a stationary phase where cellular energies are directed towards production of rP. During many such biphasic cultures, the initial phase of rapid cell growth at 37°C is followed by a growth arrest phase induced through reduction of the culture temperature. Low temperature induced growth arrest is associated with many positive phenotypes including increased productivity, sustained viability and an extended production phase, although the mechanisms regulating these phenotypes during mild hypothermia are poorly understood.ResultsIn this study differential protein expression in suspension CHO-K1 cells was investigated following a reduction of the culture temperature from 37°C to 31°C in comparison to standard batch culture maintained at 37°C using 2D-DIGE (Fluorescence 2-D Difference Gel Electrophoresis) and mass spectrometry (MS). There is only limited proteomic analysis of suspension-grown CHO cells describing a direct comparison of temperature shifted versus non-temperature shifted cultures using 2D-DIGE. This investigation has enabled the identification of temperature-dependent as well as temperature-independent proteomic changes. 201 proteins were observed as differentially expressed following temperature shift, of which 118 were up regulated. Of the 53 proteins identified by MALDI-ToF MS, 23 were specifically differentially expressed upon reduction of the culture temperature and were found related to a variety of cellular functions such as regulation of growth (HNRPC), cap-independent translation (EIF4A), apoptosis (importin-α), the cytoskeleton (vimentin) and glycoprotein quality control (alpha glucosidase 2).ConclusionThese results indicate the extent of the temperature response in CHO-K1 cells and suggest a number of key regulatory proteins and pathways that are involved in modulating the response of cells to mild hypothermia. Regulation of these identified proteins and pathways could be useful for future approaches to engineer CHO cells for improved recombinant protein production.

Initial identification of low temperature and culture stage induction of miRNA expression in suspension CHO-K1 cells

This paper describes the first miRNA analysis carried out on hamster cells specifically Chinese hamster ovary (CHO) cells which are the most important cell line for the manufacture of human recombinant biopharmaceutical products. During biphasic culture, an initial phase of rapid cell growth at 37 degrees C is followed by a growth arrest phase induced through reduction of the culture temperature. Growth arrest is associated with many positive phenotypes including increased productivity, sustained viability and an extended production phase. Using miRNA bioarrays generated with probes against human, mouse and rat miRNAs, we have identified 26 differentially expressed miRNAs in CHO-K1 when comparing cells undergoing exponential growth at 37 degrees C to stationary phase cells at 31 degrees C. Five miRNAs were selected for qRT-PCR analysis using specific primer sets to isolate and amplify mature miRNAs. During this analysis, two known growth inhibitory miRNAs, miR-21 and miR-24 were identified as being upregulated during stationary phase growth induced either by temperature shift or during normal batch culture by both bioarray and qRT-PCR. Sequence data confirmed the identity of cgr-miR-21, a novel Cricetulus griseus ortholog of the known miRNA miR-21. This study offers a novel insight into the potential of miRNA regulation of CHO-K1 growth and may provide novel approaches to rational engineering of both cell lines and culture processes to ensure optimal conditions for recombinant protein production.

Experimental study on the effect of trichostatin A on differentiation of human lung carcinoma cell

To investigate the influence of trichostatin A (TSA) on the proliferation and phase growth arrest of human lung carcinoma cells and on the expression of histone deacetylase 3 (HDAC3). Human lung carcinoma cells of the line A549 were cultured and treated with TSA of the concentrations of 5, 10, 20, and 40 microg/L. MTT assay was adopted to observe the proliferation of the cells. The cell cycle was determined by flow cytometry. RT-PCR was used to determine the mRNA expression of P21WAF1/CIP1 gene, a tumor suppressor gene, and histone deacetylase 3 (HDAC3) in the A549 cells. Trichostatin A treatment led to a time- and dose-dependent inhibition in carcinoma cells A549 proliferation (Twenty-four hours after the treatment of TSA The inhibition rates of the A549 cells in the control group and the cells treated with TSA of the concentrations of 5, 10, 20, and 40 microg/L were 6.2%+/-1.1%, 18.5%+/-2.3%, 28.9%+/-3.6%, 39.4%+/-3.7%, and 45.6%+/-2.7% respectively 24 hours later (P<0.05): 8.1%+/-2.3%, 26.9%+/-4.2%, 35.6%+/-3.8%, 56.5%+/-6.1%, and 69.8%+/-5.3% 48 h later (P<0.05); and 10.5%+/-1.3%, 28.4%+/-3.2%, 50.5%+/-5.8%, 70.5%+/-6.9%, and 78.6%+/-4.5% 72 h later (P<0.05). The percentages of the cells in the G0/G1 phase of the control group and the groups treated by TSA of different concentrations were 56.5%+/-8.1%, 70.5%+/-6.7%, 78.6%+/-4.6%, 82.4%+/-3.7%, and 85.6%+/-7.5% respectively (P<0.05). The mRNA expression of HDAC3 of the control group and the TSA-treated groups were 0.85, 052, 0.43, 0.32, and 0.25 respectively, P<0.05), and the mRNA expression of P21WAF1/CIP1 were 0.09, 0.17, 0.20, 0.27, and 0.35 respectively, P<0.05). TSA induces the expression of P21WAF1/CIP1 through inhibition of HADC3.

Effect Of ISS And TGF-β1 On MLg Lung Fibroblast Proliferation In Vitro

RATIONALE: Since CpG-rich immunostimulatory DNA sequences (ISS) reduced peribronchial fibrosis in the chronic ovalbumin murine asthma model of airway remodeling, we hypothesized that ISS might act in part through a TLR9-dependent effect on fibroblast proliferation.METHODS: RT-PCR was used to determine whether TLR-9 was expressed by MLg, a murine lung fibroblast cell line. MLg fibroblast proliferation was assayed with a BrdU ELISA at 48h. MLg fibroblasts were incubated after a growth arrest phase with ISS 10 μg/ml (a ligand for TLR9), or M-ODN 10 μg/ml (modified oligodeoxynucleotides, which are not TLR9 ligands) with or without TGF-β1 15 ng/ml.RESULTS: MLg fibroblasts expressed TLR-9 as assessed by RT-PCR. MLg fibroblasts incubated with ISS alone had increased proliferation relative to both M-ODN (p<0.01) and untreated media (p<0.02). In contrast, MLg fibroblasts incubated with the combination of ISS and TGF-β1 had reduced proliferation relative to media (p<0.03). The combination of M-ODN and TGF-β1 also inhibited MLg fibroblast proliferation (p<0.03). TGF-β1 alone did not significantly affect proliferation of MLg fibroblasts.CONCLUSIONS: MLg fibroblasts express TLR-9 as assessed by RT-PCR. In the absence of TGF-β1, ISS induced proliferation of MLg fibroblasts in a TLR-9 dependent manner. In the presence of TGF-β1, exogenous DNA (either ISS or M-ODN) inhibited proliferation of MLg fibroblasts in a DNA sequence-independent fashion. Thus in MLg fibroblasts, ISS may either induce proliferation, or inhibit proliferation dependent upon the presence of extracellular TGF-β1. RATIONALE: Since CpG-rich immunostimulatory DNA sequences (ISS) reduced peribronchial fibrosis in the chronic ovalbumin murine asthma model of airway remodeling, we hypothesized that ISS might act in part through a TLR9-dependent effect on fibroblast proliferation. METHODS: RT-PCR was used to determine whether TLR-9 was expressed by MLg, a murine lung fibroblast cell line. MLg fibroblast proliferation was assayed with a BrdU ELISA at 48h. MLg fibroblasts were incubated after a growth arrest phase with ISS 10 μg/ml (a ligand for TLR9), or M-ODN 10 μg/ml (modified oligodeoxynucleotides, which are not TLR9 ligands) with or without TGF-β1 15 ng/ml. RESULTS: MLg fibroblasts expressed TLR-9 as assessed by RT-PCR. MLg fibroblasts incubated with ISS alone had increased proliferation relative to both M-ODN (p<0.01) and untreated media (p<0.02). In contrast, MLg fibroblasts incubated with the combination of ISS and TGF-β1 had reduced proliferation relative to media (p<0.03). The combination of M-ODN and TGF-β1 also inhibited MLg fibroblast proliferation (p<0.03). TGF-β1 alone did not significantly affect proliferation of MLg fibroblasts. CONCLUSIONS: MLg fibroblasts express TLR-9 as assessed by RT-PCR. In the absence of TGF-β1, ISS induced proliferation of MLg fibroblasts in a TLR-9 dependent manner. In the presence of TGF-β1, exogenous DNA (either ISS or M-ODN) inhibited proliferation of MLg fibroblasts in a DNA sequence-independent fashion. Thus in MLg fibroblasts, ISS may either induce proliferation, or inhibit proliferation dependent upon the presence of extracellular TGF-β1.

Promotion of recombinant macrophage colony stimulating factor production by dimethyl sulfoxide addition in Chinese hamster ovary cells

A Chinese hamster ovary (CHO) cell line is the primary choice for the production of recombinant protein drugs with glycosylation modification. Recombinant protein productivity was enhanced by the addition of dimethyl sulfoxide (DMSO) in CHO cells. However, DMSO induced G0/G1 phase growth arrest and reduced cell growth rate. A two-stage process was developed to utilize the stimulatory effect of DMSO on recombinant protein production and mitigate the problem of growth inhibition in this study. In the first stage, cells are cultured without DMSO for a certain period in order to obtain a high cell density. Sequentially, DMSO is added to achieve a high specific productivity in the second stage. Using this approach, we found that by adding 1% DMSO after 24 h of growth, macrophage colony-stimulating factor (M-CSF) volumetric productivity is increased by 57% compared with the value obtained without the addition of DMSO.

2-(3-Fluorophenyl)-6-methoxyl-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (YJC-1) induces mitotic phase arrest in A549 cells

A 2-phenyl-4-quinolone (2-PQ) derivative, 2-(3-fluorophenyl)-6-methoxyl-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (YJC-1), was synthesized in our laboratory. In this study, we delineated the growth-inhibitory effect of YJC-1 in human lung carcinoma A549 cells. YJC-1 inhibited cell growth with an IC 50 value of about 4.8 μM via microtubule polymerization, causing growth arrest in the mitotic phase. Immunoblotting analysis revealed a dramatic induction of cyclin-dependent kinase (CDK) inhibitor p21 Cip1/Waf1 and down-regulation of Cdc25C phosphatase to inhibit the protein expression of cyclin B1 and CDK1. We also found that YJC-1 induced a profound time-dependent elevation in p21 Cip1/Waf1 gene expression in comparison with the negative control. In vivo, we also found that YJC-1 significantly suppressed tumor growth in mice inoculated with A549 cells. These findings suggest that YJC-1 can suppress A549 cell growth via mitotic phase arrest.

Ras Triggers Ataxia-telangiectasia-mutated and Rad-3-related Activation and Apoptosis through Sustained Mitogenic Signaling

Genetic evidence indicates that Ras plays a critical role in the initiation and progression of human thyroid tumors. Paradoxically, acute expression of activated Ras in normal rat thyroid cells induced deregulated cell cycle progression and apoptosis. We investigated whether cell cycle progression was required for Ras-stimulated apoptosis. Ras increased CDK-2 activity following its introduction into quiescent cells. Apoptotic cells exhibited a sustained increase in CDK-2 activity, accompanied by the loss of CDK-2-associated p27. Blockade of Ras-induced CDK-2 activity and S phase entry via overexpression of p27 inhibited apoptosis. Inactivation of the retinoblastoma protein in quiescent cells through expression of HPV-E7 stimulated cell cycle progression and apoptosis, indicating that deregulated cell cycle progression is sufficient to induce apoptosis. Ras failed to induce G1 phase growth arrest in normal rat thyroid cells. Rather, Ras-expressing thyroid cells progressed into S and G2 phases and evoked a checkpoint response characterized by the activation of ATR. Ras-stimulated ATR activity, as evidenced by Chk1 and p53 phosphorylation, was blocked by p27, suggesting that cell cycle progression triggers checkpoint activation, likely as a consequence of replication stress. These data reveal that Ras is capable of inducing a DNA damage response with characteristics similar to those reported in precancerous lesions. Our findings also suggest that the frequent mutational activation of Ras in thyroid tumors reflects the ability of Ras-expressing cells to bypass checkpoints and evade apoptosis rather than to simply increase proliferative potential.

Anandamide inhibits Cdk2 and activates Chk1 leading to cell cycle arrest in human breast cancer cells

This study was designed to determine the molecular mechanisms underlying the anti-proliferative effect of the endocannabinoid anandamide on highly invasive human breast cancer cells, MDA-MB-231. We show that a metabolically stable analogue of anandamide, Met-F-AEA, induces an S phase growth arrest correlated with Chk1 activation, Cdc25A degradation and suppression of Cdk2 activity. These findings demonstrate that Met-F-AEA induced cell cycle blockade relies on modulated expression and activity of key S phase regulatory proteins. The observed mechanism of action, already reported for well-known chemotherapeutic drugs, provides strong evidence for a direct role of anandamide related compounds in the activation of cell cycle checkpoints.

The intracellular genistein metabolite 5,7,3′,4′-tetrahydroxyisoflavone mediates G2-M cell cycle arrest in cancer cells via modulation of the p38 signaling pathway

The cellular actions of genistein are believed to mediate the decreased risk of breast cancer associated with high soy consumption. We have investigated the intracellular metabolism of genistein in T47D tumorigenic and MCF-10A nontumorigenic cells and assessed the cellular actions of resultant metabolites. Genistein selectively induced growth arrest and G2-M phase cell cycle block in T47D but not MCF10A breast epithelial cells. These antiproliferative effects were paralleled by significant differences in the association of genistein to cells and in particular its intracellular metabolism. Genistein was selectively taken up into T47D cells and was subject to metabolism by CYP450 enzymes leading to the formation of both 5,7,3′,4′-tetrahydroxyisoflavone (THIF) and two glutathionyl conjugates of THIF. THIF inhibited cdc2 activation via the phosphorylation of p38 MAP kinase, suggesting that this species may mediate genistein's cellular actions. THIF exposure activated p38 and caused subsequent inhibition of cyclin B1 (Ser 147) and cdc2 (Thr 161) phosphorylation, two events critical for the correct functioning of the cdc2–cyclin B1 complex. We suggest that the formation of THIF may mediate the cellular actions of genistein in tumorigenic breast epithelial cells via the activation of signaling through p38.

Contribution of Aldose Reductase to Diabetic Hyperproliferation of Vascular Smooth Muscle Cells

The objective of this study was to determine whether the polyol pathway enzyme aldose reductase mediates diabetes abnormalities in vascular smooth muscle cell (SMC) growth. Aldose reductase inhibitors (tolrestat or sorbinil) or antisense aldose reductase mRNA prevented hyperproliferation of cultured rat aortic SMCs induced by high glucose. Cell cycle progression in the presence of high glucose was blocked by tolrestat, which induced a G0-G1 phase growth arrest. In situ, diabetes increased SMC growth and intimal hyperplasia in balloon-injured carotid arteries of streptozotocin-treated rats, when examined 7 or 14 days after injury. Treatment with tolrestat (15 mg x kg(-1) x day(-1)) diminished intimal hyperplasia and decreased SMC content of the lesion by 25%. Although tolrestat treatment increased immunoreactivity of the lesion with antibodies raised against protein adducts of the lipid peroxidation product 4-hydroxy trans-2-nonenal, no compensatory increase in lesion fibrosis was observed. Collectively, these results suggest that inhibition of aldose reductase prevents glucose-induced stimulation of SMC growth in culture and in situ. Even though inhibition of aldose reductase increases vascular oxidative stress, this approach may be useful in preventing abnormal SMC growth in vessels of diabetic patients.

Cross-talk between DNA damage and cell survival checkpoints during G2 and mitosis: pharmacologic implications

In this study, we wanted to clarify the role of survivin-mediated survival signaling during G2 and M in tumor cells treated with DNA-damaging agents. As a cellular model, we selected MOLT-4 human T-cell lymphoblastic leukemia cells that overexpress survivin and nonfunctional p53. Treatment with melphalan, a classic DNA-damaging agent, led to the induction of the DNA damage checkpoint and growth arrest in the G2 phase of the cell cycle. Checkpoint abrogation by caffeine was accompanied by mitotic entry and rapid apoptotic cell death, whereas cells remaining in G2 remained viable during the same time interval. Unexpectedly, when the spindle checkpoint was activated following G2 abrogation, two different effects could be observed. If the microtubules of the melphalan-treated cells were destabilized by nocodazole, cells became arrested in prometaphase with low survivin levels and entered apoptosis. In contrast, if the microtubules of the melphalan-treated cells were stabilized by taxol, cells were still arrested in prometaphase, but apoptotic execution was inhibited. This effect is, most likely, directly mediated by survivin itself given its well-established antiapoptotic functions. In conclusion, depending on the way the spindle checkpoint was activated in cells with damaged DNA, cells could be either protected by survivin or die during mitosis. We suggest that the efficacy of DNA damage checkpoint abrogators used in combination with DNA-damaging agents may critically depend on whether DNA damage is able to invoke spindle checkpoint response and to activate survivin-associated survival signaling during mitosis.

New Concepts for the Functional Maturation of the Bovine Claw: Endothelial Biology, Angiogenesis and Ageing

Introduction: Endothelial dysfunction and impaired angiogenesis are connected to ageing. However, the mechanisms involved in age‐related alterations are not fully elucidated, whereby reduced VEGF‐expression and decreased mobilization of circulating endothelial progenitor cells are suggested as possible causes. When cultured in vitro, endothelial cells often enter a growth arrest phase called ‘cellular senescence’ that is consistent with many changes in age‐related vascular disease. Based on the decreasing lifespan observed in high yielding dairy cows, a hypothesis has been formulated that these cows, due to their enormous metabolic performance, show an accelerated process of ageing, resulting in typical production‐related diseases, particularly lameness. As a basis for further research into the role of endothelial cell biology in high producing cattle, the aim of this study was to set up a model of endothelial ageing. As a first step, the role of angiogenesis in the functional adaptation of the microvasculature of the bovine pododerma was examined.Material and methods: Scanning electron microscopy of vascular corrosion casts of healthy juvenile and adult claws as well as pathologically altered claws, and routine light microscopy of pododermal serial sections was compared to an in vitro model of angiogenesis based on cultured bovine microvascular endothelial cells. For detection of angiogenic molecular control mechanisms effective in both, the pododermal system and our cell culture model, lectin‐ and immunohistochemistry as well as RT‐PCR were carried out.Results and discussion: In the adolescent and adult claw, sprouting and intussusceptive angioadaptation was demonstrated. Evidence of angiogenic processes was also detected in the pathologically altered claws. Immunolocalization of angiogenic factors within the dermo‐epidermal system of the claw was established. The detected angioadaptation may be visible expression of the increased metabolic demands of the claw caused by the growing body‐weight‐load. Increase of the pododermal vascular density via angiogenesis was also the connectional reparative principle in pathologically altered claws.

Histone Deacetylase Inhibitors LBH589 and LAQ824 Deplete Ezh2 and Associated Polycomb Repressive Complex 2/3 Proteins Resulting in Downregulation of HOXA9 and MEIS1 Expression in Human Acute Leukemia Cells.

Human enhancer of Zeste homolog (Ezh2) protein belongs to Polycomb Repressive Complex (PRC) 2, which also includes Eed and Suz12. Ezh2 has been shown to promote cellular transformation, and increased Ezh2 expression has been strongly correlated with the invasiveness of prostate and breast cancers. The enzymatically-active Ezh2-containing, PRC2 complex possesses histone methyl transferase (HMTase) activity mediated by the SET domain of Ezh2, which is involved in the methylation of histone (H) 3, lysine (K)-27 and −9. Through this, the PRC2 complex regulates the expression of homeobox domain containing HOX family of transcription factors including HOXA9 and MEIS1, which have been shown to be involved in human leukemogenesis. Co-expression of HOXA9 and MEIS1 is common in acute myeloid leukemia and collaborates in the leukemogenesis. In the present studies, we determined the effect of hydroxamate histone deacetylase inhibitors LBH589 and LAQ824 on Ezh2 and PRC2 complex proteins and their activity in the cultured (K562, LAMA-84, U937 and HL-60) and primary human AML cells. Exposure to 10 to 100 nM of LBH589 or LAQ824 for 24 hours, in a dose dependent manner depleted the protein levels of Ezh2, as well as reduced Suz12 and Eed levels in the cultured and primary leukemia cells. This was associated with decreased levels of the tri- and dimethylated K27 and increased acetylation of K27, both on H3. Correspondingly, these H3 modifications were associated with a significant decline in the levels of HOXA9 and MEIS1. As has been previously reported, treatment with LBH589 and LAQ824 induced p21, as well as caused cell cycle growth arrest in the G1 phase and apoptosis of the cultured and primary AML cells. We next determined whether knockdown of Ezh2 by siRNA to Ezh2 also induces growth inhibitory and cytotoxic effects against the leukemia cells. In the cultured and primary acute leukemia cells, knockdown of Ezh2 expression (by ~80%) by Ezh2 siRNA, but not by the control siRNA, was associated with depletion of Suz12 levels and increased H3K27 acetylation. This was associated with increase in the % of cells in the G1 phase of the cell cycle and significant inhibition of their clonogenic survival in colony growth assays. Co-treatment with LBH589 and siRNA to Ezh2 caused further decline in the Ezh2 levels and increased loss of clonogenic survival of the leukemia cells. These findings suggest that down modulation of Ezh2 and PRC2 complex and its HMTase activity may inhibit clonogenic survival of human acute myeloid leukemia cells. Additionally, combined effect of the knockdown of Ezh2 and its activity along with treatment with LBH589 or LAQ824 may have an improved anti-leukemia efficacy, especially against human AML where HOXA9 and MEIS1 genes are deregulated.

Pan1p, an actin cytoskeleton-associated protein, is required for growth of yeast on oleate medium

Pan1p is a yeast actin cytoskeleton-associated protein localized in actin patches. It activates the Arp2/3 complex, which is necessary for actin polymerization and endocytosis. We isolated the pan1-11 yeast mutant unable to grow on oleate as a sole carbon source and, therefore, exhibiting the Oleate- phenotype. In addition, mutant cells are temperature-sensitive and grow more slowly on glycerol or succinate-containing medium but similarly to the wild type on ethanol, pyruvate or acetate-containing media; this indicates proper functioning of the mitochondrial respiratory chain. However, growth on ethanol medium is compromised when oleic acid is present. Cells show growth arrest in the apical growth phase, and accumulation of cells with abnormally elongated buds is observed. The growth defects of pan1-11 are suppressed by overexpression of the END3 gene encoding a protein that binds Pan1p. The morphology of peroxisomes and induction of peroxisomal enzymes are normal in pan1-11, indicating that the defect in growth on oleate medium does not result from impairment in peroxisome function. The pan1-11 allele has a deletion of a fragment encoding amino acids 1109-1126 that are part of (QPTQPV)7 repeats. Surprisingly, the independently isolated pan1-9 mutant, which expresses a truncated form of Pan1p comprising aa 1-859, is able to grow on all media tested. Our results indicate that Pan1p, and possibly other components of the actin cytoskeleton, are necessary to properly regulate growth of dividing cells in response to the presence of some alternative carbon sources in the medium.

Interferon beta increases c-Myc proteolysis in mouse monocyte/macrophage leukemia cells

Growth inhibitory activity of interferons (IFNs) has been attributed to several events. These include rapid induction of cyclin-dependent kinase inhibitors, such as those in the Cip/Kip and Ink4 families and down-regulation of c- myc mRNA and c-Myc transcriptional activity. Here, we report an additional mechanism, involving regulation of Myc protein levels, through which type 1 IFN may halt proliferation of cells. This was discovered using a cell line which constitutively expresses c- myc from a retrovirus vector and which was reported to have undergone deletion of genes encoding the Ink4 tumor suppressors p15 and p16. IFNβ caused a reduction in the steady state level of c-Myc protein by increasing degradation through the 26S proteasome. Our data, as well as that of others, indicate that multiple levels of c-Myc expression can be affected by IFN treatment and this contributes to rapid growth arrest in the G1 phase of the cell cycle.

Mutation of Cys105 Inhibits Dimerization of p12CDK2-AP1 and Its Growth Suppressor Effect

p12(CDK2-AP1) (p12) is a CDK2-associated protein that negatively regulates its kinase activity. Growth arrest of normal diploid cells by contact inhibition resulted in an induction of p27(kip1) and reduction of CDK2 levels. Interestingly, we observed concomitantly in growth-arrested cells, there was a reduction of nuclear p12 and the appearance of a nuclear 25-kDa molecule (p25) recognized by anti-p12 polyclonal antibody. Biochemical analysis showed that bacterial His-tagged p12 could be converted into a dimeric p25 in a reducing agent-dependent manner, and mutating the only cysteine residue of p12 (Cys(105) --> Ala(105)) abolished the dimerization. Transient transfection of wild type p12 into U2OS cells showed a reducing agent-sensitive dimerization that was also abolished by the C105A mutation. Furthermore, reduction of p12 expression by a short interfering RNA resulted in a parallel reduction of p25. These data supports the possibility that p25 is a homodimeric form of p12 through the cysteine residue. More interestingly, transient transfection of p12 (C105A) into the normal diploid lung fibroblast CCD18LU cells resulted in a reduction of the growth-inhibitory effect of p12 and abolished the inhibitory effect of p12 on CDK2 kinase activity. In addition, we found that the C105A mutation did not alter nuclear localization of p12, but it prevented association with CDK2. Taken together, our data suggest that p12 forms a nuclear homodimers in contact inhibited normal diploid cells and dimerization of p12 is a necessary process for the growth inhibition effect by p12.

Human and mouse mesotheliomas exhibit elevated AKT/PKB activity, which can be targeted pharmacologically to inhibit tumor cell growth

Malignant mesotheliomas (MMs) are very aggressive tumors that respond poorly to standard chemotherapeutic approaches. The phosphatidylinositol 3-kinase (PI3K)/AKT pathway has been implicated in tumor aggressiveness, in part by mediating cell survival and reducing sensitivity to chemotherapy. Using antibodies recognizing the phosphorylated/activated form of AKT kinases, we observed elevated phospho-AKT staining in 17 of 26 (65%) human MM specimens. In addition, AKT phosphorylation was consistently observed in MMs arising in asbestos-treated mice and in MM cell xenografts. Consistent with reports implicating hepatocyte growth factor (HGF)/Met receptor signaling in MM, all 14 human and murine MM cell lines had HGF-inducible AKT activity. One of nine human MM cell lines had elevated AKT activity under serum-starvation conditions, which was associated with a homozygous deletion of PTEN, the first reported in MM. Treatment of this cell line with the mTOR inhibitor rapamycin resulted in growth arrest in G1 phase. Treatment of MM cells with the PI3K inhibitor LY294002 in combination with cisplatin had greater efficacy in inhibiting cell proliferation and inducing apoptosis than either agent alone. Collectively, these data indicate that MMs frequently express elevated AKT activity, which may be targeted pharmacologically to enhance chemotherapeutic efficacy. These findings also suggest that mouse models of MM may be useful for future preclinical studies of pharmaceuticals targeting the PI3K/AKT pathway.