Compared with cell sorting Tipifarnib FDA through surface biomarkers, sorting of SP cells is more convenient, less costly, and universally applicable. Moreover, the latter can be used to isolate SP cells with unknown surface biomarkers. Taken together, in the present study, we used FACS to isolate SP cells from the human cervical cancer cell line HeLa, and then examined the biological characteristics of the sorted SP cells. In 2004, Kondo et al. isolated SP cells from the HeLa cell line, which accounted for approximately 1. 2% of the total number of HeLa cells. In our study, we ob tained sorted SP cells that accounted for 1. 07% of the total number of HeLa cells. The result of SP cells in this study was mostly consistent with Kondos.

Microscopic observa tions showed that the sorted SP cells shared morphological characteristics with stem like cells, including smaller size, rounder shape and higher adherence than non SP cells, as well as colony like growth. This result suggests that SP cells may possess certain stem cell morphological characteristics. In addition, we tested the expression of surface markers of embryonic stem cells and hematopoietic stem cells in SP and non SP cells. The expression levels of Oct3/4 and CD133 in SP cells were significantly higher than those in non SP cells, which is consistent with previous findings. As suggested in many previous studies, high ex pression of ABCG2/BCRP on the cell membrane is a pre requisite for SP cells to extrude Hoechst 33342 dye and maintain their stem cell like characteristics.

In our study, immunocytochemical analysis showed that BCRP was highly expressed in the sorted SP cells, whereas non SP cells hardly expressed BCRP, which is consistent with the proposed molecular mechanism mentioned above. In addition to those surface markers, ALDH 1 has received increasing attention as a specific marker for CSCs. ALDH 1 of the ALDH family is a cytosolic en zyme that catalyzes the intracellular oxidation of acetal dehyde to acetate, and is involved in the differentiation and gene expression of a variety of tissues. It is also a specific marker of normal stem cells in tissues. In recent years, breast, lung, prostate, and pancreatic cancers have been found to be associated with high expression of ALDH 1 in a small number of cells with stem cell like characteristics.

In our study, no significant differ ences were observed in the expression of ALDH1 be tween sorted SP and non SP cells, which might due to the different origin and differentiation of tumor cells. Further investigation Brefeldin_A is needed to treat ALDH 1 as a cell surface stemness related marker of cervical cancer cells. According to the stem cell theory of cancer, CSCs have the potential for continuous differentiation and self renewal. SP cells can give rise to both SP and non SP cells through asymmetric division, whereas non SP cells can only differentiate into non SP cells, resulting in higher tumorigenicity of SP cells than non SP cells.

In brief, antibodies against histone isoforms were used to precipitate chromatin in sporo cysts, cercaria and adults. The resulting Cisplatin DNA was ana lyzed either by ChIP Seq or qPCR. ChIP Seq data are available at the NCBI SRA under accessions SRX088545, SRX088544, SRX088543 and SRX087825. For ChIP Seq analysis, a repeat pseudogenome was constructed in which each identified repeat sequence occurred only once. Then SOAP2 was used to align roughly 100,000 36 bp reads for miracidia of two strains, cercaria and adult couples to this pseudogenome. Hit counts for each repeat were normal ized by the total number of aligned reads and compared for the different stages. Introduction p21 was originally identified as a cell cycle regulator through inhibition of different cyclin/cyclin dependent kinase complexes.

p21 is a member of the Cip/Kip family of cell cycle inhibitors, which also includes p27Kip1 and p57Kip2. In addition to its role in cell cycle control, p21 is involved in the regulation of cellular senescence, gene transcription, apoptosis and actin cytos keleton. The role of p21 in breast cancer develop ment and progression has not been fully investigated. While p21 is involved in cell cycle control and is a down stream target of the tumor suppressor p53, it does not fulfill the classic definition of a tumor suppressor. Germline or somatic mutations in the p21 gene are not common in human cancers. Furthermore, in vivo stu dies using p21 knockout mice showed that, while loss of p21 expression efficiently blocked the ability of the cells to undergo G1 arrest following DNA damage, these animals developed normally.

Intriguingly, p21 is often overexpressed in aggressive tumors, including carcino mas of the pancreas, breast, prostate, ovary and cervix. Together these observations suggest that the role played by p21 in cancer is more complex than initially thought and that, in addition to its well known cell cycle regulatory effect, it may have uncharacterized roles in promoting carcinogenesis. Tumor cell migration and invasion are critical steps in the metastatic process and are regulated by numerous tumor secreted factors which modify the tumor microen vironment by acting on stromal recruitment and extracel lular matrix degradation, resulting in tumor cell migration and invasion. Among these tumor secreted factors, TGFb has been shown to play a pivotal role in promoting tumor metastasis.

The TGFb family regu lates asymmetric cell division and cell fate determination during embryogenesis and exerts profound effects on reproductive functions, immune responses, cell growth, bone formation, tissue remodeling and repair throughout adult life. The effects of TGFb in breast cancer are complex. TGFb Drug_discovery is thought to play a dual role in breast cancer progression, acting as a tumor suppressor in nor mal and early carcinoma, and as a pro metastatic factor in aggressive carcinoma.

At 120 minutes fol lowing fluorescein addition, basal media was placed in a Corning 96 well black assay plate and fluores cein was determined using a Typhoon Trio Plus. Western blot analysis Western blot analyses are processed inhibitor licensed using the following protocol. Briefly, to prepare total cellular protein MDCK cells are washed with cold PBS and lysed in buffer con taining 1% Triton X 100, 1% sodium deoxycholate, 0. 1% SDS, 2 mM EDTA, 0. 15 M NaCl, 0. 01 M NaPO4, mini Complete protease inhibitor with the following phosphatase inhibitors 2 mM Na3VO4 and 10 mM NaF. DNA is sheared using a small gauge nee dle, and insoluble material is precipitated by centrifuga tion. Supernatants were collected and stored at 20 C for analyses. The method to prepare Triton X 100 soluble and insolu ble fractions was adapted from Singh et.

al. with minor modifications. MDCK cells were scraped into lysis buffer and incubated for 20 min. at 4 C. Following centrifugation, supernatants were collected and considered the TX 100 soluble frac tions, the pellets were placed in lysis buffer containing 1% SDS and TX 100 insoluble proteins were released by three sonication pulses using a Branson Sonifier 450. Insoluble material was removed by centrifugation, supernatants were collected and stored at 20 C for analyses. Protein concentration was determined using the Pierce BCA microtiter plate protocol using albu min as a reference standard. Lysates are denatured at 95 C for 5 min in Lammeli sample buffer, electrophoresed on 10% SDS PAGE gels, and electroblotted to PVDF mem brane for immunodetection.

Tight junction specific antis era including anti occludin and claudin 1 and 3 were employed in this study. Immunoblots are processed by blocking non specific binding sites in 5% non fat milk in Tris buffered saline with 0. 1% Tween 20 for 30 minutes followed by incubation with diluted primary antibody for 2 hours at room temperature. Immunoblots are then washed three times in TBS T fol lowed by incubation with an HRP conjugated secondary antibody. Following extensive washing with TBS T, immunoblots are developed with a stable West Pico chemiluminescent substrate. The image was captured on the VersaDoc 3000 and ana lyzed with the integrated QuantityOne 1 D analysis soft ware. Immunofluorescent analysis MDCK cell monolayers were grown on culture treated cover slips and treated for 24 hours in one of the follow ing conditions media only, TNF IFN, or TNF IFN with U0126. Layers were rinsed once Carfilzomib with sterile PBS and placed on ice for ten minutes. Cells were permeabilized with an actin stabilizing permeabilization buffer containing 0. 2% Tri ton X100, 100 mM KCl, 3 mM MgCl2, 1. 3 mM CaCl2, 25 mM sucrose, and 2 mM HEPES, pH 7. 1 for 2 min on ice.

Therefore, we determined the effects of simultaneous inhibition of WEE1 and CHK1 on the cell selleck chemicals cycle profile of asynchron ously growing cell populations. We selected two cell lines where synergy was observed, NCI H2009 NSCLC and Su. 86. 86 pancreatic cancer cells, and used concentrations of the two inhibitors that had little effect on cell prolifera tion when dosed alone, but had a profound effect in combination. We also included normal human renal epi thelial cells and normal human mammary epithelial cells, which did not score high in the combin ation synergy screen and were unresponsive to the concentrations of inhibitors used. In all the cells tested, MK 1775 had no dis cernible effect on the cell cycle profile, whereas MK 8776 caused an increase in the number of G1 S phase cells in the cancer lines.

Notably, the combination of MK 1775 and MK 8776 led to a dramatic accumulation of cells with G1 S phase DNA content in both tumor lines. Again, this effect was not seen in either of the normal cell populations tested, consistent with published observations that WEE1 knockdown is more deleterious in transformed cells than in non transformed cells. Inhibition of WEE1 and CHK1 leads to synergistic accumulation of DNA damage Inactivating mutations of p53 can impair the G1 check point arrest that typically follows DNA damage. Due to a compromised G1 checkpoint, it is suggested that p53 deficient tumor cells are more dependent on the G2 checkpoint and therefore more likely to be sensitized to DNA damaging agents by G2 checkpoint modulators, i. e. WEE1 or CHK1 inhibitors.

Mutational status of p53 was found for 31 of the 39 cancer cell lines screened and these lines were analyzed for synergy of the MK 1775 and MK 8776 combination. Synergy scores ranged from 0 to 0. 4 among both p53 wild type and p53 mutant cell lines, but we failed to observed any difference in synergy among the two groups. The same cell panel did demonstrate a trend toward greater overall sensitivity to the MK 1775 plus MK 8776 combination among p53 mutant lines. Two p53 wild type or null isogenic cell line pairs demonstrated similar synergy and overall response to the MK 1775 and MK 8776 combination. Loss of either WEE1 or CHK1 function through siRNA depletion or small molecule inhibition results in an accumu lation of DNA damage. Therefore, we considered the likeli hood that combining MK 1775 with MK 8776 might result in increased DNA damage. To differentiate effects of the combination from effects of either single agent, we selected concentrations of MK 1775 and Carfilzomib MK 8776 that alone had limited effect in a cell proliferation assay, but when com bined led to 80% growth inhibition.

After hypoxia, apoptosis was analyzed using Annexin V FITC PI binding staining and caspase selleck chem 3 7 activity were mea sured by Cytomics FC500 flow cytometer. Total RNAs and protein were prepared for real time reverse transcription polymerase chain reaction and western blot analysis. RNA extraction and real time RT PCR Total RNA was extracted from cultured cells using Tri zol. The levels of mRNAs or miRNAs were measured by real time quantitative RT PCR using Bio Rad IQ5 system. For mRNA detection, reverse transcription was performed with Pri meScript RT reagent kit ac cording to the manufacturers instructions, and real time RT PCR was carried out using SsoFast EvaGreen Supermix kit with Bio Rad IQ5 real time PCR system.

The real time PCR reaction contained, 10 uL of SsoFast EvaGreen supermix, 1 uL of sense primer, 1 uL of anti sense primer, 2 uL of cDNA template, and 6 uL of H2O. The program of two step real time RT PCR was 95 C for 30 seconds, followed by 40 cycles of 95 C for 5 seconds, and 60 C for 10 seconds. The relative expres sion level of mRNAs was normalized to that of internal control B actin by using the 2 Ct cycle threshold method. Primer sequences were as follows, To detect the level of mature miR 494, the complementary DNA was synthesized using PrimeScript RT re agent kit and miRNA specific stem loop RT primers. The 10 uL of reaction contained, 2 uL of 5�� RT buffer, 0. 5 uL of Pri meScript RT Enzyme Mix, 1 uL of miR 494 RT primer, 1 uL of total RNA, and 5. 5 uL of H2O. The in cubation condition was 37 C for 15 minutes, followed by 85 C for 5 seconds.

Then qRT PCR was performed with SsoFast EvaGreen Supermix kit and Bio Rad IQ5 real time PCR system. The reaction contained, 10 uL of SsoFast EvaGreen supermix, 1. 5 uL of forward primer, 1. 5 uL of reverse primer, 2 uL of cDNA template, and 5 uL of H2O. The program was the same as that described above. Forward and reverse primers were designed from RiboBio. U6 small nuclear RNA was used as an internal control. Protein extraction and western blot analysis Cells were washed twice quickly with ice cold phosphate buffered saline after either hypoxic or normoxic incubation, solubilized in 1�� lysis buffer with protease inhibitors and phosphatase inhibitors on ice. Cell lysates were sonicated in an Ultrasonic Dismemberator on ice, followed by boiling for 5 minutes and centrifuging at 12000 g for 10 minutes at 4 C and the supernatants were retained.

Protein con centration was determined by a BCA Protein Assay kit. For western blot, equal amounts of total protein in spe cial condition were loaded for electrophpresis GSK-3 in sodium dodecyl sulfate polyacrylamide gels and then transferred to polyvinylidene fluoride microporous mem branes. After blocking for 1 hour at room temperature, the membranes were incubated with the primary antibodies overnight at 4 C.

It is noticeable that HL60 cells were selleckchem 17-DMAG more sensitive at 24 h than control cells. The Namalwa and Raji lines are mutant for p53 Namalwa cells are p53 double mutant cells that contain two non synonymous mutations in exon 7 of p53 gene, while Raji cells contain mutations in exon 6. Nevertheless, the mutated proteins are stabilized in these cells and can accu mulate to relatively high levels. As shown in Figure 6C, Namalwa cells were highly susceptible to DRB induced death within the 40 100 M range in a dose dependent manner and the death mechanism was apoptosis, as demonstrated by their pos itive staining for active caspase 3 and cell surface annexin V expression. It is noticeable that Namalwa cells were more sensitive at 24 h than control cells.

Raji cells were also susceptible to DRB induced cytotoxicity, albeit with some differences in the death pathway and kinetics DRB induced death occurred only after 48 hr and apoptosis was not the solely mechanism of death, as caspase 3 activation and cell surface annexin V expression were only detectable after 48 hr and in a low percentage of cells. Blast cells from a primary AML sample are highly sensitive to DRB treatment Lastly, the effect of DRB on cell viability and apoptosis induction was studied in a human primary AML sample. Leukemic blasts, selected through SSC CD45 parameters accounted for about 60% of total cells. To define the sensitivity of both tumour blasts and non malignant cells to this drug, we assessed the viability of treated cells in a dose response experiment using CD45 PI staining and flow cytometry.

Blast cells were susceptible to DRB induced death within the 10 100 M range in a dose dependent manner and the death mechanism Batimastat was apoptosis. Interestingly, blast cells were significantly more sensitive to DRB than non malignant cells at all doses analysed. Discussion Here we show that the cyclin dependent kinase inhibitor DRB efficiently leads normal and leukemic cells to apop tosis in a DNA replication and DNA damage independ ent manner. In T cells expressing wild type p53, DRB mia lymphomaDRB induced apoptosis in prototypic leukae induced a cytosolic p53 response that led the cells to apoptosis through Bax activation. Nevertheless, apoptosis was also induced in p53 deficient mutated leukemic cells through alternative pathways, possibly involving p73. Overall, the transcription stress response imposed by DRB is very powerful in inducing apoptosis independently of a cells p53 status, and could provide an attractive approach to the treatment of some forms of cancer.

It is known that receptor proteins that bind Gemcitabine molecular weight elicitors generate signals that are transmitted to the sites of gene expression via different components, such as Ca2 ion fluxes, medium alkalinization and cytoplasmic acidification, oxi dative burst, jasmonate and nitric oxide etc. Many CDPKs and MAPKs have been identified to play a role in defense responses and also secondary metabolite produc tion. The effect of UV B irradiation on expression of TIA biosyn thetic genes, Tdc and Str, and catharanthine production has been reported previously in C. roseus leaves. The transcription factor GT 1 binds to the promoter region of Tdc in vitro. The functional importance of GT 1 in the induction of Tdc expression by UV light has been demonstrated by point mutations in the GT 1 binding site.

However, the molecular basis of UV B signaling cas cades leading to the induction of expression of Tdc and Str genes and the production of TIAs is largely unknown. It has been observed that the polypeptide wound signal, sys temin specific cell surface receptors initiate a signal trans duction cascade upon UV B irradiation in L. peruvianum cell suspension cultures. In the present study, the sig naling pathways mediating UV B induced catharanthine accumulation in C. roseus suspension cultures were inves tigated. UV B induced alkalinization of the culture medium, generation of hydrogen peroxide, activation of CDPK and MBPK as well as accumulation of catharan thine and stimulation of transcription of Tdc and Str genes were studied.

Inhibitors of binding of ligand cell surface receptors, protein kinases and phosphatases, calcium fluxes and H2O2 were used to dissect the UV B signaling cascade. Results Alkalinization of C. roseus cell suspension medium in response to UV B irradiation and its inhibition by suramin Medium alkalinization an early event occurring in elici tor treated plant cell cultures, has been used as a marker of elicitor responses in studying elicitor binding sites in plant cells. Medium alkalinization is thought to result from elicitor stress induced depolarization of the plasma membrane and subsequent K H exchange with Ca2 influx Cl efflux. To determine whether medium alkalinization is involved in UV B signal transduction as an early event, six day old cells were exposed to UV B irra diation for various time periods and extracellular pH changes were measured in the cell sus pension medium for 120 min.

As shown in Figure 1a, the effect of UV B on medium alkalinization was not dose dependent. However, the kinetics and intensity of this response were Brefeldin_A dependent on their respective exposure times. C. roseus cells showed a rapid increase in the medium pH after UV B irradiation peaking at 10 min with an increase of about 0. 7 units in 5 min irradiated cells.

0 with bootstrap analysis on the phylogeny. fr server, and by using IQTree with fast bootstrap analysis. Bayesian MCMC sampling was carried out using MrBayes 3. 2 with model averaging. For tree reconstruction using quartet puzzling, kinase inhibitor 17-AAG Tree Puzzle 5. 2 was used with 100,000 puzzling steps. Tree Puzzle was in addition used for likelihood mapping. The resulting trees were visualized using iTOL. Consensus sequences of the untrimmed, gapped alignments were generated using WebLogo. Motif prediction To identify potential functional domains in the Dact proteins, protein sequences were searched using PSort and NetNes 1. 1. Embryos and in situ hybridization Fertilized chicken eggs were incubated in a humidified atmosphere at 38. 5 C. Embryos were staged according to.

Mice were obtained from the UoP animal resource centre and mated overnight. The appearance of a vaginal plug the next morning was taken as day 0. 5 of development. Zebrafish embryos were raised at 28 C in egg water to prevent pigmentation and staged according to. All animal experiments were conducted following the UK Animals Act and have been approved of by UoP AWERB. Embryos were harvested in 4% PFA and subjected to in situ hybridization as described in and. Probes for mouse Dact genes were kindly provided by R. Suriben, chicken Dact1 and Dact2 probes are detailed in. The dact1 probe recognizes dact1a and b, the dact3 probe recognized dact3 derived from both scaffold 110 and from scaffold 13803.

Probes for zebrafish dact1 and dact2 were synthesized using PCR products obtained from 36hpf embryo cDNAs, which were amplified using a gene specific forward primer and a reverse primer containing the T7 promoter sequence in addition to gene specific region. Background The most common short chain fatty acids are nat ural microbial fermentation products in the gastrointesti nal tract. SCFA, including propionic, butyric and valeric acids, each with three, four and five carbons, respectively, contribute to the energy balance of all mammalian species. The major sources of these carbohydrates are hemi celluloses and fiber, which consists of plant cell wall polysaccharides such as cellulose and pectins. In rumi nants, SCFA are a major energy source and contribute up to 70% of their energy requirements. Beyond their nutritional impact, SCFA, especially butyrate, have a mul titude of cellular regulatory effects that modulate cell dif ferentiation, proliferation, and motility.

All three major components of SCFA induce apoptosis and inhibit cell proliferation, however, AV-951 butyrate has the most potent effect. Roles for butyrate have been established in cell dif ferentiation, proliferation, motility and in particular induction of cell cycle arrest and apoptosis. Apopto sis is a genetically regulated cellular suicide mechanism that plays a crucial role in development and in the defense of homeostasis of animals.

30 minutes post injection, embryos were transferred to 0. 1X MMR and allowed to develop until stage 21 for in situ hybridization or stage 45 for organ placement score. www.selleckchem.com/products/Calcitriol-(Rocaltrol).html All experimental procedures involving the use of animals for experimental purposes were approved by the Institutional Animal Care and Use Committees and Tufts University Department of Lab Animal Medicine under the protocol number M2008 08. Scoring for Organ situs At stage 45, embryos were anesthetized with 5% tricaine and analyzed for position of 3 organs the heart, stomach, and gallbladder. Heterotaxia was defined as reversal in position of one or more organs. Only embryos with normal dorsoanterior development were scored to avoid scoring instances of secondary randomization due to errors in the dorso ventral or antero posterior axial patterning, and only clear left or right sided organs were scored.

Percent heterotaxia was calculated as the number with hetero taxia divided by the number of total scorable embryos, i. e. embryos normal in all other ways. A c2 test was used for further statistical analysis. Whole amount in situ hybridization Embryos were collected at different stages of development and fixed in MEMFA for 3 hours at room temperature and used for in situ hybridization as described in Harland. Plasmids containing Xenopus Mad3 and Xenopus HDAC cDNA were purchased from Open Biosystems and cloned in pCS2. The plasmids were linearized and anti sense probes for in situ hybridization were generated in vitro using DIG labeling mix from Invitrogen.

Xenopus embryo drug treatment Batches of embryos were separated into experimental and control groups and exposed to 0. 1X MMR or 0. 1X MMR containing 100 mM Sodium Butyrate during different stages of development. The drug was washed out and the embryos were allowed to develop until stage 21 for Nr1 in situ or until stage 45 for organ placement score. Western Blotting and Coimmunoprecipitation assay For Western blottings embryos were collected at stage 7 and homogenized in lysis buffer. The lysate was centrifuged for 15 minutes at 4 C and the supernatant was collected and frozen. The extracted proteins were subjected to SDS PAGE and blotted onto a PVDF membrane. After blocking with 5% skim milk and 0. 1% Tween 20 in PBS, membrane filters were incubated with an anti Mad3 or anti acetyl H4 overnight at 4 C.

Cilengitide The membranes were washed in PBT and incubated with secondary HRP conjugated antibody. Immunosignals were visualized with chemoluminiscence. For Co IP assays, embryos were injected at the 1 cell stage with Mad3WT flag or Mad3 5mut flag constructs and collected at stage 7 when 10 embryos were homogenized in lysis buffer. A total of 100 ul of embryo lysate was incubated with 2 ug anti 5HT or rabbit IgG for 3 hours at 4 C fol lowed by incubation with proteinA agarose for 1 hour at the same temperature. The beads were collected by centrifugation and washed in lysis buffer.

HDACi have demon strated potent activity against colon cancer cell lines in vitro and in xenograft models with little or no cytotoxicity reported against normal cells and clinical evaluations thus far have demonstrated favorable toxicity profiles. Several studies to date have demonstrated that HDACi induce alterations in the expression of multiple drug tar gets and/or metabolic pathways www.selleckchem.com/products/INCB18424.html that are critical molecular determinants for cancer therapeutics. Importantly combi nation treatment with additional agents targeting these modulated pathways has resulted in synergistic growth inhibitory effects on cancer cells in vitro and in vivo. It has been recently reported that HDACi synergize with 5 FU in vitro and in vivo in colon cancer cell line models through HDACi induced downregulation of the 5 FU target enzyme thymidylate synthase, providing a mechanis tic basis for the drug synergy.

The HDACi vorino stat is also reported to acetylate and markedly reduce the chaperone activity of HSP90 in T cell lymphoma models resulting in a synergistic interaction with the HSP90 inhibitor bortezomib. This combination was subse quently extended to colon cancer cell lines with similar synergistic anti proliferative effects. In addition, the HDACi vorinostat was demonstrated to induce tumor cell selective expression of the TRAIL death receptors 4 and 5 sensitizing breast cancer xenografts to the effects of a TRAIL agonistic antibody, an observation which is currently being clinically evaluated in lymphoma patients.

More recently, HDACi were also reported to enhance the apoptotic effects of EGFR inhibitors in lung cancer models and clinical evaluation of this is ongoing. Therefore, the identification of novel genes modulated by HDACi in colon cancer cells may provide pathway driven rationale for novel and urgently needed efficacious drug combinations. This study was designed to determine the effects of two clinically relevant HDACi, vorinostat and LBH589 on the growth characteristics of two cytogenetically distinct colon cancer cell line models HCT116 and HT29. In addi tion, HDACi induced alterations in global gene expres sion were analyzed using the Illumina Human 6 V2 BeachChip arrays and Ingenuity Pathway Analysis. Methods Compounds and Reagents LBH589 was provided by Novartis Pharmaceuticals. Vorinostat was provided by Merck and Co,Inc. and the National Cancer Institute.

CellTiter96 AQueous MTS rea gent was purchased from Promega. Cell Lines HCT116 AV-951 colon cancer cells were a generous gift of Prof. Bert Vogelstein and HT29 colon cancer cells were purchased from selleck compound ATCC. HCT116 and HT29 cell lines were maintained in McCoys 5A medium, supplemented with 10% fetal bovine serum, penicillin/streptomycin and sodium pyruvate. Cells were maintained in a humidi fied Hepa Class100 Incubator at 37 C and 5% CO2. Cell lines were routinely screened for mycoplasma using the MycoALERT Detection kit.