Uev1A promotes breast cancer cell migration by up-regulating CT45A expression via the AKT pathway.

BACKGROUND: UEV1A encodes a ubiquitin-E2 variant closely associated with tumorigenesis and metastasis, but its underlying mechanism in promoting metastasis remains to be investigated. METHODS: In this study, we experimentally manipulated UEV1A and CT45A gene expression and monitored their effects on cancer-related gene expression, cell migration and the signal transduction cascade. RESULTS: It was found that UEV1A overexpression induces CT45A family gene expression in breast cancer cells. Indeed, ectopic expression of UEV1A was sufficient to induce CT45A and its downstream genes involved in tumorigenesis, epithelial-mesenchymal transition (EMT), stemness and metastasis, and to promote cell migration and EMT signaling. Consistently, depletion of CT45A abolished the above effects, indicating that CT45A is a critical downstream effector of Uev1A. The Uev1A-induced cell migration and EMT signaling was dependent on AKT but independent of NF-κB, indicating that CT45A acts downstream of the AKT pathway. CONCLUSIONS: Based on previous reports and observations in this study, we propose that the Ubc13-Uev1A complex activates AKT through K63-linked polyubiquitination, which leads to enhanced CT45A expression, stimulated cell migration and EMT signaling in breast cells. Since similar effects were also observed in a colorectal cancer cell line, the Ubc13/Uev1A-AKT-CT45A axis may also promote tumorigenesis and metastasis in other tissues.

Experimental estimation of stored stress within spherical microtissues : What can and cannot be inferred from cutting experiments.

Biological tissues accumulate mechanical stress during their growth. The mere measurement of the stored stress is not an easy task. We address here the spherical case and our experiments consist in performing an incision of a spherical microtissue (tumor spheroid) grown in vitro. On the theoretical part we derive a compatibility condition on the stored stress in spherical symmetry, which imposes a relation between the circumferential and radial stored stress. The numerical implementation uses the hyperelastic model of Ciarlet and Geymonat. A parametric study is performed to assess the influence of each parameter on the shape of the domain after the incision. As a conclusion, the total radial stored stress can be confidently estimated from the measurement of the opening after incision. We validate the approach with experimental data.

The ENmix DNA methylation analysis pipeline for Illumina BeadChip and comparisons with seven other preprocessing pipelines.

BACKGROUND: Illumina DNA methylation arrays are high-throughput platforms for cost-effective genome-wide profiling of individual CpGs. Experimental and technical factors introduce appreciable measurement variation, some of which can be mitigated by careful "preprocessing" of raw data. METHODS: Here we describe the ENmix preprocessing pipeline and compare it to a set of seven published alternative pipelines (ChAMP, Illumina, SWAN, Funnorm, Noob, wateRmelon, and RnBeads). We use two large sets of duplicate sample measurements with 450 K and EPIC arrays, along with mixtures of isogenic methylated and unmethylated cell line DNA to compare raw data and that preprocessed via different pipelines. RESULTS: Our evaluations show that the ENmix pipeline performs the best with significantly higher correlation and lower absolute difference between duplicate pairs, higher intraclass correlation coefficients (ICC) and smaller deviations from expected methylation level in mixture experiments. In addition to the pipeline function, ENmix software provides an integrated set of functions for reading in raw data files from mouse and human arrays, quality control, data preprocessing, visualization, detection of differentially methylated regions (DMRs), estimation of cell type proportions, and calculation of methylation age clocks. ENmix is computationally efficient, flexible and allows parallel computing. To facilitate further evaluations, we make all datasets and evaluation code publicly available. CONCLUSION: Careful selection of robust data preprocessing methods is critical for DNA methylation array studies. ENmix outperformed other pipelines in our evaluations to minimize experimental variation and to improve data quality and study power.

Focused PCR screen reveals p53 dependence of nitric oxide-induced apoptosis and up-regulation of maspin and plasminogen activator inhibitor-1 in tumor cells.

We investigated p53-dependent gene expression in nitric oxide (NO)-induced apoptosis of two tumor cell types. Seventy-seven putative p53-regulated genes were screened for NO-mediated expression changes. Twenty-four genes were up-regulated and three genes were down-regulated significantly by NO in human neuroblastoma cells. Genes known to be involved in apoptosis, which were up-regulated by > or = 2-fold, included FAS, CASP-1, BIK, PUMA, DR4 and the serpins maspin (SERPINB5), and plasminogen activator inhibitor-1 (PAI-1). Real-time PCR confirmed maspin and PAI-1 mRNAs exhibited the greatest NO-induced induction, which occurred in a p53-dependent manner. The substantial NO-mediated up-regulation of these serpins mRNAs correlated with large increases in their protein levels, which occurred before or coinciding with apoptosis. p53-deficient neuroblastoma cells were largely resistant to NO killing and showed much reduced maspin and PAI-1 mRNA and protein levels after NO treatment. p53 was activated by NO mainly in the nuclei of neuroblastoma cells. p53(-/-) HCT116 colon carcinoma cells were strongly resistant to NO-induced apoptosis and failed to up-regulate maspin and PAI-1 (in contrast to p53(+/+) HCT116 cells). Our results suggest that both apoptosis and induction of the two serpins by NO require the transcriptional activity of p53. Because maspin is a tumor suppressor and PAI-1 can promote senescence and regulate cell death, it will now be worth investigating whether their p53-mediated expression contributes to the NO-induced p53-dependent death of tumor cells.

[Fascination with natural glycosides].

Recent progress in glycobiology reveals that sugar-chains play a crucial role in cell-cell recognition among immunity, inflammation, and malignant tumor in the living body. To study the mechanism of action requires a sample of sufficient quantity. However, isolating a sugar chain and a glycoside in pure form, difficulty follows sugar chain composition again, and studies have been limited to a few sugar chains. Glycosides are a group of compounds known to be the active principle of a natural drug. We have isolated triterpene glycosides from a Leguminosae plant, which improved liver disorder, and steroid glycosides from a plant of the Solanaceous with cytotoxic activity against human cancer cell lines. A biological activity test suggested an important role of the aglycone part and the sugar chain part of those glycosides. Although many studies of sugar chains of glycoprotein and glycolipid are known, there are few examples of studies of the sugar chain function of a glycoside of a natural drug, and the role of a sugar chain of a glycoside for its pharmacologic action expression is unknown. Therefore, as a biological tool investigating the function of a sugar chain of a natural glycoside, we have begun to synthesize a useful "glycoside" which can be utilized as a lead compound having activity.

Maintenance of HCT116 colon cancer cell line conforms to a stochastic model but not a cancer stem cell model.

The cancer stem cell (CSC) model, in which a small population of cells within a tumor possesses the ability to self-renew and reconstitute the phenotype of primary tumor, has gained wide acceptance based on evidence over the past decade. It has also been reported that cancer cell lines contain a CSC subpopulation. However, phenotypic differences between CSCs and non-CSCs in cancer cell lines are not better defined than in primary tumors. Furthermore, some cell lines do not have a CSC population, revealed as a side population and expression of CD133. Thus, the identification of CSCs in cancer cell lines remains elusive. Here, we investigated the CSC hierarchy within HCT116 colon cancer cells, which do not have a CD133-positive subpopulation. We examined the expression of alternative CSC markers epithelial specific antigen (ESA) and CD44 in floating-sphere-derived cells, which are known to be the cells of enriching CSCs. Sphere-derived HCT116 cells exhibited heterogeneous expression of ESA and CD44. The two major subpopulations of HCT116 sphere cells (ESA(low)CD44(-/low) and ESA(high)CD44(high)) exhibited a biological/proliferative hierarchy of sphere-forming and soft agar colony-forming activity. However, there was no difference between the two subpopulations in the incidence of xenograft tumors. When ESA(low)CD44(-/low) cells were allowed to aggregate and re-form floating-spheres, the biological/proliferative hierarchy of parental HCT116 spheres was reconstituted, in terms of ESA and CD44 expression. Thus, HCT116 cells have plasticity when they are set in floating-spheres, suggesting that maintenance of the HCT116 cell line conforms to a stochastic model, not a CSC model.

A mastic gum extract induces suppression of growth of human colorectal tumor xenografts in immunodeficient mice.

BACKGROUND: We recently reported that ethanol and hexane extracts of the plant product, mastic gum (MG), contain constituents which can induce p53- and p21-independent G1-phase arrest followed by apoptosis of human HCT116 colon cancer cells in vitro. Herein, we extended these studies to investigate the in vivo anticancer activity of the hexane extract of MG (He-MG) against human colon tumor. The in vivo anticancer activity of He-MG was assessed in a human colon cancer/immunodeficient mouse model. MATERIALS AND METHODS: Control and HCT116 tumor bearing SCID mice were injected intraperitoneally with He-MG at different administration schedules and doses ranging from 100 to 220 mg/kg body weight and tumor growth (size) was monitored. RESULTS: He-MG administered at a dose of 200 mg/kg administered daily for 4 consecutive days (followed by 3 days without treatment) inhibited tumor growth by approximately 35% in the absence of toxicity (side-effects) after 35 days. CONCLUSION: He-MG was found to possess antitumor activity against human colorectal cancer under the experimental conditions of this study. The extent of suppression and toxicity by a specific He-MG dose depends on the schedule of administration.

Evaluation of high-throughput genotoxicity assays used in profiling the US EPA ToxCast chemicals.

Three high-throughput screening (HTS) genotoxicity assays-GreenScreen HC GADD45a-GFP (Gentronix Ltd.), CellCiphr p53 (Cellumen Inc.) and CellSensor p53RE-bla (Invitrogen Corp.)-were used to analyze the collection of 320 predominantly pesticide active compounds being tested in Phase I of US. Environmental Protection Agency's ToxCast research project. Between 9% and 12% of compounds were positive for genotoxicity in the assays. However, results of the varied tests only partially overlapped, suggesting a strategy of combining data from a battery of assays. The HTS results were compared to mutagenicity (Ames) and animal tumorigenicity data. Overall, the HTS assays demonstrated low sensitivity for rodent tumorigens, likely due to: screening at a low concentration, coverage of selected genotoxic mechanisms, lack of metabolic activation and difficulty detecting non-genotoxic carcinogens. Conversely, HTS results demonstrated high specificity, >88%. Overall concordance of the HTS assays with tumorigenicity data was low, around 50% for all tumorigens, but increased to 74-78% (vs. 60% for Ames) for those compounds producing tumors in rodents at multiple sites and, thus, more likely genotoxic carcinogens. The aim of the present study was to evaluate the utility of HTS assays to identify potential genotoxicity hazard in the larger context of the ToxCast project, to aid prioritization of environmentally relevant chemicals for further testing and assessment of carcinogenicity risk to humans.

Screening for compounds that induce apoptosis of cancer cells grown as multicellular spheroids.

Screening and initial characterization of anticancer drugs are typically performed using monolayer cultures of tumor cells. It is well established that such monolayer cultures do not represent the characteristics of 3-dimensional solid tumors. The multicellular tumor spheroid model is of intermediate complexity between in vivo tumors and in vitro monolayer cultures and would be more suitable for drug screening. The authors describe a procedure in which multicellular spheroids are used to screen for compounds that induce tumor cell apoptosis. Multicellular spheroids were generated in 96-well plates, and apoptosis was determined using the M30-Apoptosense enzyme-linked immunosorbent assay method. A Z' factor of approximately 0.5 was observed for HCT116 colon carcinoma spheroids using staurosporine to induce apoptosis. This procedure is attractive for secondary screening of hits from larger cell-based screens.

The apoptotic effect of nanosilver is mediated by a ROS- and JNK-dependent mechanism involving the mitochondrial pathway in NIH3T3 cells.

Nanomaterials and nanoparticles have received considerable attention recently because of their unique properties and diverse biotechnology and life sciences applications. Nanosilver products, which have well-known antimicrobial properties, have been used extensively in a range of medical settings. Despite the widespread use of nanosilver products, relatively few studies have been undertaken to determine the biological effects of nanosilver exposure. The purpose of this study was to evaluate the toxicity of nanosilver and to elucidate possible molecular mechanisms underlying the biological effects of nanosilver. Here, we show that nanosilver is cytotoxic, inducing apoptosis in NIH3T3 fibroblast cells. Treatment with nanosilver induced the release of cytochrome c into the cytosol and translocation of Bax to mitochondria, indicating that nanosilver-mediated apoptosis is mitochondria-dependent. Nanosilver-induced apoptosis was associated with the generation of reactive oxygen species (ROS) and JNK activation, and inhibition of either ROS or JNK attenuated nanosilver-induced apoptosis. In nanosilver-resistant HCT116 cells, up-regulation of the anti-apoptotic proteins, Bcl-2 appeared to be associated with a diminished apoptotic response. Taken together, our results provide the first evidence for a molecular mechanism of nanosilver cytotoxicity, showing that nanosilver acts through ROS and JNK to induce apoptosis via the mitochondrial pathway.

Heterozygous inactivation of human Ku70/Ku86 heterodimer does not affect cell growth, double-strand break repair, or genome integrity.

Ku, the heterodimer of Ku70 and Ku86, plays crucial roles in non-homologous end-joining (NHEJ), a major pathway for repairing DNA double-strand breaks (DSBs) in mammalian cells. It has recently been reported that heterozygous disruption of the human KU86 locus results in haploinsufficient phenotypes, including retarded growth, increased radiosensitivity, elevated p53 levels and shortened telomeres. In this paper, however, we show that heterozygous inactivation of either the KU70 or KU86 gene does not cause any defects in cell proliferation or DSB repair in human somatic cells. Moreover, although these heterozygous cell lines express reduced levels of both Ku70 and Ku86, they appear to maintain overall genome integrity with no elevated p53 levels or telomere shortening. These results clearly indicate that Ku haploinsufficiency is not a commonly observed phenomenon in human cells. Our data also suggest that the impact of KU70/KU86 mutations on telomere metabolism varies between cell types in humans.

Phenethyl isothiocyanate suppresses cancer stem cell properties in vitro and in a xenograft model.

BACKGROUND: Cancer stem cells (CSCs) are a subset of cells within the bulk of a tumor that have the ability to self-renew and differentiate, and are thus associated with cancer invasion, metastasis, and recurrence. Phenethyl isothiocyanate (PEITC) is a natural compound found in cruciferous vegetables such as broccoli and is used as a cancer chemopreventive agent; however, its effects on CSCs are little known. PURPOSE: To evaluate the effect of PEITC on CSCs in this study by examining CSC properties. METHODS: NCCIT human embryonic carcinoma cells were treated with PEITC, and the expression of pluripotency factors Oct4, Sox-2, and Nanog were evaluated by luciferase assay and western blot. Effect of PEITC on self-renewal capacity and clonogenicity were assessed with the sphere formation, soft agar assay, and clonogenic assay in an epithelial cell adhesion molecule (EpCAM)-expressing CSC model derived from HCT116 colon cancer cells using a cell sorting system. The effect of PEITC was also investigated in a mouse xenograft model obtained by injecting nude mice with EpCAM-expressing cells. RESULTS: We found that PEITC treatment suppressed expression of the all three pluripotency factors in the NCCIT cells, in which pluripotency factors are highly expressed. Moreover, PEITC suppressed the self-renewal capacity and clonogenicity in the EpCAM-expressing CSC model. EpCAM was used as a specific CSC marker in this study. Importantly, PEITC markedly suppressed both tumor growth and expression of three pluripotency factors in a mouse xenograft model. CONCLUSION: These results demonstrate that PEITC might be able to slow down or prevent cancer recurrence by suppressing CSC stemness.

Resveratrol analogue, HS-1793, induces apoptotic cell death and cell cycle arrest through downregulation of AKT in human colon cancer cells.

Resveratrol, a polyphenolic compound, is a naturally occurring phytochemical and is found in a variety of plants, including grapes, berries and peanuts. It has gained much attention for its potential anticancer activity against various types of human cancer. However, the usefulness of resveratrol as a chemotherapeutic agent is limited by its photosensitivity and metabolic instability. In this study the effects of a synthetic analogue of resveratrol, HS-1793, on the proliferation and apoptotic cell death were investigated using HCT116 human colon cancer cells. Although this compound has been reported to have anticancer activities in several human cancer cell lines, the therapeutic effects of HS-1793 on human colon cancer and its mechanisms of action have not been extensively studied. HS-1793 inhibited cell growth and induced apoptotic cell death in a concentration-dependent fashion. Induction of apoptosis was determined by morphological changes, cleavage of poly(ADP-ribose) polymerase, alteration of Bax/Bcl-2 expression ratio, and caspase activations. Flow cytometric analysis revealed that HS-1793 induced G2/M arrest in the cell cycle progression in HCT116 cells. Furthermore, HS-1793 showed more potent anticancer effects in several aspects than resveratrol in HCT116 cells. In addition, HS-1793 suppressed Akt and the phosphatidylinositol-3 kinase/Akt inhibitor LY294002 was found to enhance its induction of apoptosis. Thus, these findings suggest that HS-1793 have potential as a candidate chemotherapeutic agent against human colon cancer.

Combination of chrysin and cisplatin promotes the apoptosis of Hep G2 cells by up-regulating p53.

Cisplatin is a chemotherapy drug commonly used for the treatment of human cancers, however, drug resistance poses a major challenge to clinical application of cisplatin in cancer therapy. Recent studies have shown that chrysin, a natural flavonoid widely found in various plants and foods, demonstrated effective anti-cancer activity. In the present study, we found that the combination chrysin and cisplatin significantly enhanced the apoptosis of Hep G2 cancer cells. Combination of chrysin and cisplatin increased the phosphorylation and accumulation of p53 through activating ERK1/2 in Hep G2 cells, which led to the overexpression of the pro-apoptotic proteins Bax and DR5 and the inhibition of the anti-apoptotic protein Bcl-2. In addition, combination of chrysin and cisplatin promoted both extrinsic apoptosis by activating caspase-8 and intrinsic apoptosis by increasing the release of cytochrome c and activating caspase-9 in Hep G2 cells. Our results suggest that combination of chrysin and cisplatin is a promising strategy for chemotherapy of human cancers that are resistant to cisplatin.

LIN28B promotes colon cancer migration and recurrence.

LIN28B is involved in "stemness" and tumourigenesis by negatively regulating the maturation of let-7 microRNA family members. In this study, we showed that LIN28B expression promotes migration and recurrence of colon cancer. Immunohistochemistry and reverse-transcription polymerase chain reactions were performed to detect LIN28B expression in colon cancer tissue microarrays, paraffin-embedded surgical resected tissues and cancer cells. Loss-of-function, migration and proliferation analyses were performed to delineate the potential roles of LIN28B in colon cancer. LIN28B was upregulated in colon cancer tissue compared to normal mucosa, and its overexpression correlated with reduced patient survival and increased tumour recurrence. LIN28B suppression inhibited the migration of SW480 colon cancer cells and facilitated the cytotoxicity induced by oxaliplatin in SW480 and HCT116 colon cancer cells. In conclusion, LIN28B overexpression contributes to colon tumourigenesis, and LIN28B may serve as a diagnostic tool and therapeutic target for colon cancer.

Deficiency in the 15 kDa selenoprotein inhibits human colon cancer cell growth.

Selenium is an essential micronutrient for humans and animals, and is thought to provide protection against some forms of cancer. These protective effects appear to be mediated, at least in part, through selenium-containing proteins (selenoproteins). Recent studies in a mouse colon cancer cell line have shown that the 15 kDa selenoprotein (Sep15) may also play a role in promoting colon cancer. The current study investigated whether the effects of reversing the cancer phenotype observed when Sep15 was removed in mouse colon cancer cells, were recapitulated in HCT116 and HT29 human colorectal carcinoma cells. Targeted down-regulation of Sep15 using RNAi technology in these human colon cancer cell lines resulted in similarly decreased growth under anchorage-dependent and anchorage-independent conditions. However, the magnitude of reduction in cell growth was much less than in the mouse colon cancer cell line investigated previously. Furthermore, changes in cell cycle distribution were observed, indicating a delayed release of Sep15 deficient cells from the G(0)/G(1) phase after synchronization. The potential mechanism by which human colon cancer cells lacking Sep15 revert their cancer phenotype will need to be explored further.

CPUYJ039, a newly synthesized benzimidazole-based compound, is proved to be a novel inducer of apoptosis in HCT116 cells with potent KSP inhibitory activity.

OBJECTIVES: This study investigated the antiproliferative and apoptotic activities of CPUYJ039, a newly synthesized benzimidazole-based kinesin spindle protein (KSP) inhibitor, on HCT116 cell lines. METHODS: KSP-inhibitory activity was tested using the malachite-green method. The in-vitro cell proliferation inhibitory activity of the sample was measured using WST reagent. Flow-cytometric evaluation of cellular DNA content was performed. Apoptotic cells were quantified by annexin V-FITC-PI double staining. To confirm that the cytotoxic activity was a consequence of KSP inhibition, microtubule morphology and DNA segregation were observed by double staining of microtubules and DNA. The expression of Bcl-2 and Bax in CPUYJ039-treated HCT116 cells was detected by Western blotting. KEY FINDINGS: CPUYJ039 was evaluated and proved to have potent inhibitory activities in the KSP ATPase and HCT116 cell proliferation assays. CPUYJ039 inhibited the proliferation of HCT116 cells in a dose- and time-dependent manner and markedly induced G2/M phase cell-cycle arrest with characteristic monoastral spindles and subsequent cell death in HCT116 cells, which was associated with an increase of the Bax/Bcl-2 ratio. CONCLUSIONS: These results suggest that CPUYJ039 may be a novel inducer of apoptosis in HCT116 cells with potent KSP inhibitory activity.

Contextual synthetic lethality of cancer cell kill based on the tumor microenvironment.

Acute and chronic hypoxia exists within the three-dimensional microenvironment of solid tumors and drives therapy resistance, genetic instability, and metastasis. Replicating cells exposed to either severe acute hypoxia (16 hours with 0.02% O(2)) followed by reoxygenation or moderate chronic hypoxia (72 hours with 0.2% O(2)) treatments have decreased homologous recombination (HR) protein expression and function. As HR defects are synthetically lethal with poly(ADP-ribose) polymerase 1 (PARP1) inhibition, we evaluated the sensitivity of repair-defective hypoxic cells to PARP inhibition. Although PARP inhibition itself did not affect HR expression or function, we observed increased clonogenic killing in HR-deficient hypoxic cells following chemical inhibition of PARP1. This effect was partially reversible by RAD51 overexpression. PARP1(-/-) murine embryonic fibroblasts (MEF) showed a proliferative disadvantage under hypoxic gassing when compared with PARP1(+/+) MEFs. PARP-inhibited hypoxic cells accumulated γH2AX and 53BP1 foci as a consequence of altered DNA replication firing during S phase-specific cell killing. In support of this proposed mode of action, PARP inhibitor-treated xenografts displayed increased γH2AX and cleaved caspase-3 expression in RAD51-deficient hypoxic subregions in vivo, which was associated with decreased ex vivo clonogenic survival following experimental radiotherapy. This is the first report of selective cell killing of HR-defective hypoxic cells in vivo as a consequence of microenvironment-mediated "contextual synthetic lethality." As all solid tumors contain aggressive hypoxic cells, this may broaden the clinical utility of PARP and DNA repair inhibition, either alone or in combination with radiotherapy and chemotherapy, even in tumor cells lacking synthetically lethal, genetic mutations.

p53 suppresses structural chromosome instability after mitotic arrest in human cells.

The p53 tumor suppressor inhibits the proliferation of cells that undergo prolonged activation of the mitotic checkpoint. However, the function of this antiproliferative response is not well defined. Here, we report that p53 suppresses structural chromosome instability after mitotic arrest in human cells. In both HCT116 colon cancer cells and normal human fibroblasts, DNA breaks occurred during mitotic arrest in a p53-independent manner, but p53 was required to suppress the proliferation and structural chromosome instability of the resulting polyploid cells. In contrast, cells made polyploid without mitotic arrest exhibited neither significant structural chromosome instability nor p53-dependent cell cycle arrest. We also observed that p53 suppressed both the frequency and structural chromosome instability of spontaneous polyploids in HCT116 cells. Furthermore, time-lapse videomicroscopy revealed that polyploidization of p53(-/-) HCT116 cells is frequently accompanied by mitotic arrest. These data suggest that a function of the p53-dependent postmitotic response is the prevention of structural chromosome instability after prolonged activation of the mitotic checkpoint. Accordingly, our study suggests a novel mechanism of tumor suppression for p53, as well as a potential function for p53 in the outcome of antimitotic chemotherapy.

p53-Dependent p21-mediated growth arrest pre-empts and protects HCT116 cells from PUMA-mediated apoptosis induced by EGCG.

The tumor suppressor protein p53 plays a key role in regulation of negative cellular growth in response to EGCG. To further explore the role of p53 signaling and elucidate the molecular mechanism, we employed colon cancer HCT116 cell line and its derivatives in which a specific transcriptional target of p53 is knocked down by homologous recombination. Cells expressing p53 and p21 accumulate in G1 upon treatment with EGCG. In contrast, same cells lacking p21 traverse through the cell cycle and eventually undergo apoptosis as revealed by TUNEL staining. Treatment with EGCG leads to induction of p53, p21 and PUMA in p21 wild-type, and p53 and PUMA in p21(-/-) cells. Ablation of p53 by RNAi protects p21(-/-) cells, thus indicating a p53-dependent apoptosis by EGCG. Furthermore, analysis of cells lacking PUMA or Bax with or without p21 but with p53 reveals that all the cells expressing p53 and p21 survived after EGCG treatment. More interestingly, cells lacking both PUMA and p21 survived ECGC treatment whereas those lacking p21 and Bax did not. Taken together, our results present a novel concept wherein p21-dependent growth arrest pre-empts and protects cells from otherwise, in its absence, apoptosis which is mediated by activation of pro-apoptotic protein PUMA. Furthermore, we find that p53-dependent activation of PUMA in response to EGCG directly leads to apoptosis with out requiring Bax as is the case in response to agents that induce DNA damage. p21, thus can be used as a molecular switch for therapeutic intervention of colon cancer.