For kinase inhibitor therapy, inhibitors were added into culture media twenty minutes just before radiation. cells remained in 37 C incubator through the 20 minutes deal with ment. Culture media have been then eliminated and cells were exposed to UVR. Fresh media was added into irradiated cells without having even further washing to depart residue kinase inhibitors in the media. All mutations were con firmed by DNA sequencing. The QCXIP GFP vector was generated by ligating GFP coding sequence from pEGFP N1 to the BamH I webpage on QCXIP vector. The p21WAF1 CIP1 pro moter construct was a variety gift from Dr. Wafik El Deiry, The Mish1 and Mish2 shRNA plasmids have been obtained from Open Bio methods. These plasmids had been co transfected with pMD2G and pSPAX2 plasmids into 293T cells for virus manufacturing, Transduction was carried out while in the presence of 10 ug ml of protamine, utilizing the filtered 293T media as virus source.
Movement cytometry and cell cycle analysis Cells were trypsinized and washed when with one? PBS, fixed in cold 70% ethanol overnight or right up until use. Cells had been incubated in Propidium Iodide staining solu tion in dark Dovitinib solubility for thirty minutes. 50 ug ml PI, 0. 1% sodium citrate, 50 ug ml RNase A, 0. 03% NP 40 in 1? PBS. ten,000 total events have been counted for each sample. Cell populations from each and every phase have been calculated in accordance to CellQuest directions, Cell lysate and western blot evaluation Cell pellet was lysed inside a lysis 250 buffer and quan tified by the Bradford protein assay system, Western blot was performed using antibodies towards MiTF C5 plus D5, p21, p27, p53 DO 1, p84 plus a tubulin, ubi quitin, All western blots were repeated at the very least twice, one particular representative blot is shown in figures.
Quite a few growth things together with vascular endothelial growth issue and basic fibroblast development component, in association with their receptor tyrosine kinase receptors, play supplier ARN-509 a vital position in angiogenesis in normal and pathological settings, Very important to most RTK mediated signaling is the activation on the extracel lular signal regulated kinase mitogen activated protein kinase signaling cascade. This cascade is precisely controlled by the exercise of various regulatory proteins, such as members in the Sprouty protein family. SPRY was initially described as an antagonist of Breathless FGF receptor signaling while in tracheal branching in Drosophila, 4 mammalian homologs are actually described and therefore are extensively expressed in embryonic and grownup tissues, except for SPRY3 whose expression is believed for being restricted for the brain and testes in grownups, All SPRY proteins share a really conserved, cysteine wealthy C terminal domain in addition to a extra variable N terminal domain. They’re topic to tight manage at multiple amounts. differential localization, submit translational modification, and regulation of protein levels.

cDNA was ready utilizing the initial strand cDNA synthesis kit accord ing towards the manufacturers instructions. Actual time PCR was performed together with the Light Cycler Rapidly Start out DNA SYBR Green kit. All primers have been obtained from Search LC, The calculated variety of exact transcripts was normalized to 10,000 copies from the housekeeping gene PPIB, and expressed as variety of copies per ul of input cDNA. Statistical comparisons have been manufactured by Mann Whitney U test. A P worth much less than 5% was thought to be important. Immunohistochemistry and immunocytochemistry analyses IHC was carried out as described in advance of, Antibodies used as well as dilution ratios are proven in Table 1. Immu nocytochemistry evaluation was performed employing a previ ously described protocol, Immunoblot analysis and densitometry To keep passage dependent variations at minimum, third passages of PSC and HSC have been employed for analysis.
Stellate cells had been grown in Petri dishes until finally 80% confluency below normal ailments. Protein extraction was automobile ried out as published in advance of, Immunoblot analyses along with the densitometric evaluation in the blots have been accomplished as described previously, Antibodies employed along with the ideal dilution are proven in Table one. Graphic outcomes selleck are expressed after normalization for the housekeeping gene GAPDH as % change on the appropriate con trol. Cells had been grown in 10 cm plates till 80% confluence. Serum no cost medium was stored about the cells Tubastatin for 48 h. Super natants and total cell lysates have been extracted and frozen at 80 C right up until use. For the quantification of CCL2, a com mercially offered ELISA kit was implemented according to the producers recommendations.
Benefits Evaluation of gene expression profiles Morphology and purity of stellate cells was routinely checked by immunocytochemistry, In order to identify organ and illness certain transcriptome signatures of stellate cells, a microarray analysis was manufactured, The obtained gene expression profiles were visualized as heatmaps, Red signifies large gene expression, whereas black signifies intermediate gene expression and green signifies reduced gene expression from the corresponding gene in contrast for the reference, respectively. Every single row represents one particular gene, every single column represents one particular sample. The dendrograms about the left side in the heat maps define how the genes cluster soon after applying hierarchical cluster analysis, Expressions of genes are expressed making use of log2 ratios, exactly where the indicator signifies which channel had the increased intensity. Therefore, an expression ratio of 2 represents a 4 fold up regulation of a gene underneath condi tion A in contrast to this gene beneath condition B, an expression ratio of two represents a four fold down regula tion. Organ exact profile A total of 160 annotated genes had been recognized as differen tially expressed amongst pancreas and liver derived stel late cells.

Quite a few epithelial cancers have already been located to overexpress EGFR, like head and neck, breast, colon, lung, prostate, kidney and bladder, Research present that antibodies that block the EGF binding site of EGFR inhibit tumor cell proliferation, There fore, blocking EGFR together with standard cancer ther apies can be an attractive anti tumor strategy. Erbitux, a chimeric human murine mono clonal antibody, competitively binds for the available extracellular domain of EGFR and inhibits dimerisation and subsequently inhibits cell proliferation, tumor development and metastasis, In most scientific studies, the use of Erbitux, as an anti EGFR therapy in blend with chemotherapy and radiotherapy has demonstrated signif icant clinical efficacy, as a result of its excellent tolerability and non overlapping toxicities, Also, in vivo therapies with Erbitux and chemotherapy medicines resulted inside a higher regression of bladder tumor development in contrast with either agent alone, While in the present study we’ve got evaluated the anti tumor result of Erbitux in combination with PDT on bladder carcinoma xenograft model.
Our findings indicate that combining PDT and Erbitux substantially enhances the anti tumor exercise, by inhibiting order Gemcitabine EGFR expression, rising apoptosis and by dephosphorylat ing critical EGFR tyrosine websites. These effects might professional vide a rationale for evaluating the mixture of PDT and Erbitux being a cancer therapy modality in the clinical setting. Benefits Tumor regression To investigate the long run effectiveness of PDT and Erbitux, we employed MGH bladder tumor xenograft model in athymic nude mice. Tumors have been permitted to grow to sizes of 6 seven mm in diameter in advance of PDT treatment method was carried out and were measured three times per week and charted for 90 days, The complete tumor volume for every group equals the sum of person tumor volumes, which in our situation were 8 10 person tumors.
Tumor inhibition was calculated on day 29 once the handle tumors reached highest volume of two cm3. The imply relative tumor inhibition of 93% was observed in tumors handled with all the combi nation treatment of PDT plus Erbitux when compared with manage tumors. Every week soon after remedy, accelerated tumor growth was observed selleckchem inside the combination therapy group, but there was a lower thereafter in tumor size, leading to comprehensive tumor regression. The tumors handled with PDT only and Erbitux only, exhibited 57. 8% and 74. 8% indicate tumor inhibition respectively. In contrast to regulate, the general tumor response was higher inside the monotherapy groups of PDT only and Erbitux only, however the differ ence in between the monotherapy groups weren’t signifi cant.

For instance, stereotactic radiosurgery and novel chemotherapy agents are more and more utilized to treat the recurrent tumors. Since of those remedy advances, outcomes have changed drastically for appropriately picked sufferers. Past scientific studies have exposed that pretreat ment prognostic variables perform a position in clinical outcomes, Quite a few tiny studies have recognized clinical, tumor and treatment method connected prognostic things that influence outcomes, The clinical factors of age, Karnofsky performance standing, extent of surgical resection, publish operative radiation remedy, degree of necrosis inside of the resection pathology, plus the degree of en hancement on preoperative and postoperative MR im aging scientific studies happen to be shown to influence survival, Identifying diverse prognostic subgroups of patients could aid tailor distinct remedy regimens to enhance outcomes.
The goal of this study is always to much better analyze vital pretreatment and remedy variables in the Usa population connected with make improvements to ments in total survival for individuals with glioblastoma. Procedures All data were acquired through the 1997 to 2008 constrained use databases from the Surveillance, Epidemiology, and Finish Results selleck chemical pf-562271 System from the US Nationwide Cancer Insti tute, which contains information from geographically specified U.s. spots that span a population of approximately thirty million people. Registry information are submitted without personal identifiers, therefore, patient informed consent and ethics committee approval were not essential to complete this analysis. We examined 34,664 patients aged 20 many years or older by using a diagnosis of glioblastoma multiforme be tween 1973 and 2008. The patients were grouped by race, diagnosis 12 months, radi ation treatment method, extent of surgery and age at diagnosis, Individuals younger than 50 years have been even more sub grouped into ten 12 months age bands.
Chemotherapy being a prognostic variable was not integrated due to the fact facts regarding this weren’t readily available during the SEER database. Numbers of sufferers during the racial groups, diagnosis 12 months groups and age groups are shown in Table one and numbers of patients within the ra diation treatment method groups and extent of surgical treatment groups are shown in Table two. Similarly, zafirlukast the distributions of sufferers younger than 50 years into 10 12 months age bands for these similar groupings are proven in Tables three and 4. Survival curves were estimated employing the Kaplan Meier method and employed to compare racial groups, diagnosis yr groups, radiation treatment method groups, extent of sur gery groups and age groups. Andersen 95% confidence intervals for that median survival time with the groups have been constructed. Petos log rank tests were employed to de termine if there is certainly statistical evidence of variations be tween the survival curves in the groups.

On top of that, these mechanisms are most likely differentially lively concerning cell lines as they is going to be dependent on which receptors and kinases are expressed or preferentially activated inside a cell. Quite a few members with the relatives of Src kinases have been also uncovered for being correlated with radiosensitivity. SFKs have already been proven to be concerned in pathways that manage cell division and survival and Src is implicated in AKT activation immediately after radiotherapy, Having said that, dasatinib was only capable to reduce survival immediately after ra diotherapy in UT SCC24A cells in an additive way. This really is in contrast with a latest examine by Raju et al, which showed that dasatinib enhances radiosensitivity in HNSCC cells via inhibition of radiation induced DNA fix.
A doable reason for this discrepancy is that as a consequence of differential sensitivity our panel of three cell lines was as well tiny to detect the radiosensitizing result of dasatinib. selelck kinase inhibitor Namely, within the examine of Raju et al. only two out of six cancer lines showed radiosensitization by dasatinib, None theless, these information with each other suggest that dasatinib can radiosensitize tumors, but that dasatinib is likely not helpful from the majority of HNSCC patients. In contrast to dasatinib, inhibition of MEK1 2 did lead to decreased survival right after radiotherapy in all cell lines, using a supra additive impact in UT SCC24A. MEK1 two and its downstream kinases ERK1 two are actually implicated in radioresistance in HNSCC prior to, though the impact of pathway inhibition on radiosensitivity is in steady, On this study, MEK1 two inhibition was made use of to inhibit downstream phosphorylation of MSK1 2, which was correlated with radiosensitivity.
Even though clear inhibition of pERK1 two was detected in all cell lines, pMSK1 selleck screening compounds was only decreased in UT SCC40, which only showed an additive impact of MEK inhibition. Consequently, these data suggest that the radiosensitizing effect of MEK inhibition is not really regulated by means of MSK. Unique inhib ition of MSK is going to be needed to even more investigate the role of MSK in radioresistance in HNSCC. Interestingly, the cell line that showed synergism between MEK inhi bition and radiotherapy, also showed a synergistic effect of p38 inhibition. Also with this inhibitor no reduce of pMSK1 amounts was observed.
MEK and p38 each belong towards the relatives of mitogen activated protein ki nases, Therefore, MEK and p38 could activate a further common pathway which is critical for survival just after radiotherapy in UT SCC24A cells, for instance the two MEK and p38 can activate MNK1 and therefore regulate mRNA translation, Surprisingly, enhanced pMEK1 two levels have been observed in all cell lines after MEK inhibition, and in addition p p38 was greater by p38 inhibition inside the cell line that showed decreased survival just after radiotherapy, Upregulation of pMEK1 2 after MEK inhibition has also been observed by Turke et al.

To find out no matter if the JNK3 signaling pathway is right involved in the mechanism of action of PTX, usual epithelial cells have been handled with distinct concentrations expression. In three independent tumor cell cultures we observed that PTX had no effect on ATP1AL1 gene expression. Nonetheless 0. six ng ml PTX led to down regulation in the gene, Interestingly, down regulation of ATP1AL1 gene expression didn’t progress when greater PTX concentrations have been utilized. Rather the of your cell permeable pyrazolourea compound that acts being a potent and JNK3 particular inhibitor. Subsequently the cells have been exposed to PTX. Lastly, cell viability was assessed in comparison to normal epithelial cells treated with PTX but within the absence from the inhibitor.
As shown in Figure 5B major loss of cell viability selleckchem Palbociclib was currently observed at a dose of 20 nM pyrazolourea and PTX mediated toxicity was continuously greater with in creasing concentration of pyrazolourea in contrast to non pyrazolourea taken care of cells, Cells remained unaffected when handled with pyrazolourea alone indicat ing that loss of cell viability is solely attributed to PTX. The data presented here give sturdy evidence that the repression of JNK3 gene expression is vital for expanding PTX toxicity, suggesting the MAPK JNK signalling cascades pathway has a critical purpose in the resist ance of HNSCC cells to PTX. Discussion The data presented right here demonstrate that regular epithelial proven on tumor cells suggests that their morphology can be made use of as an index of PTX toxicity.
Morphological alter in tumor cells also correlated with LDH release indicating a reduction of cellular function, mostly the mem brane integrity as would be anticipated in response to PTX that is known to have an impact on the plasma membrane, It’s apparent that many of the pharmacological LY500307 results of PTX are attributable to your effect of this substance on trans membrane ion transfer. PTX features a exceptional action around the Na,K ATPase, converting the pump into an ion channel and leading to K efflux, Na influx and membrane depolarization, PTX can in vitro trigger lysis of mouse spleen cells which is attributed to a PTX induced increase in cellular calcium levels, The toxicity of PTX in mammals is strongly dependent upon the route of administration, PTX is most toxic by intra venous injection, the LD50 in mice amounted to 0. 15 0. 53 ug kg, The PTX toxicity by ip adminis tration is lower than that by iv injection, with values of 0. 31 one. 5 ug kg becoming reported for mice, PTX is considerably less toxic orally than soon after iv or ip administration. Benefits from your number of existing studies reviews an oral LD50 from 510 ug kg to 767 ug kg in mice, PTX is described being a tumor promoter, This may misleadingly propose that it really is capable of triggering tumors.

Inside a modest cohort of 51 CRC, Oikonomou E et al. have reported a considerably decrease incidence of KRASG12 13 mutations and also have concluded that there’s clear correlation involving these mutations and upregulation of TRAIL R1 and TRAIL R2. Despite lack of statistical significance they’ve con cluded that CRC with mutations in KRAS or BRAF gene had substantially upregulated both TRAIL death recep tors. In our earlier review KRAS gene mutations have been noticed in 80 285 CRC and have been an indepen dent prognostic marker for bad survival. Interestingly we have observed a considerably larger expression of TRAIL R2 in CRC subgroup lacking KRAS mutations as compared on the CRC subgroup with KRAS mutations. In view of the current locate ings of KRAS mutations and PIK3CA mutations contri buting to resistance to EGFR inhibitors like Cetuximab, a much better comprehending of your TRAIL process with context to KRAS mutations could be helpful.
The KRAS gene has two choice fourth exon variants that consequence from differential splicing and activating mutations have an impact on both isoforms, Studies in animals indicate that KRAS4A promotes apoptosis even though KRAS4B inhi bits it, and KRAS4B promotes differentiation, In our study, KRAS 4A a pro apoptotic isoform, in particular was discovered to become an independent prognostic marker for superior survival in all great post to read CRC patients. Even while in the CRC subgroup lacking KRAS mutations KRAS4A was related with far better survival. On top of that, we have now observed a very considerable association of KRAS4A and both the TRAIL receptors. TRAIL R1 and TRAIL R2. Thinking about the tight linkage amongst TRAIL R1 and KRAS4A potential research should be conducted to understand the associa tion in between these markers. In summary, our research demonstrates substantial TRAIL R1 expres sion to be an independent prognostic marker for greater survival in colorectal cancer.
Substantial TRAIL R1 or TRAIL R2 expression was linked having a less aggressive phenotype characterized by early AJCC stage, nicely differentiated tumors, microsatellite stable cancers, absence of KRAS mutations and expression of professional apop totic molecules. KRAS4A, p27kip1 and cleaved caspase three. Even more perform is needed to elucidate the biological signif icance of high TRAIL R1 expression and greater outcome, and to set up the RO4929097 association among TRAIL R1 expression and response to therapy that tar gets this receptor. The biological results of TRAIL in CRC versions, its enhancement of chemosensitivity with normal chemotherapeutic agents along with the result of endogenous TRAIL receptor levels on survival make TRAIL an really appealing therapeutic target. Patient variety and tissue microarray construction 4 hundred forty eight patients with CRC diagnosed in between 1990 and 2006 were chosen from King Faisal Specialist Hospital and Investigation Centre.

Immu nocytochemistry analysis was performed utilizing a previ ously described protocol, Immunoblot evaluation and densitometry To keep passage dependent variations at minimum, third passages of PSC and HSC were employed for analysis. Stellate cells have been grown in Petri dishes until 80% confluency beneath conventional circumstances. Protein extraction was vehicle ried out as published in advance of, Immunoblot analyses along with the densitometric examination with the blots have been finished as described previously, Antibodies utilised as well as the proper dilution are shown in Table 1. Graphic final results are expressed immediately after normalization on the housekeeping gene GAPDH as % adjust of the proper con ELISA Cells were grown in ten cm plates right up until 80% confluence. Serum absolutely free medium was kept about the cells for 48 h. Super 80 C right up until use. For that quantification of CCL2, a com mercially accessible ELISA kit was utilised in accordance for the producers suggestions.
Outcomes Evaluation of gene expression profiles Morphology and purity of stellate cells was routinely checked by immunocytochemistry, In order PCI-32765 molecular weight to recognize organ and condition precise transcriptome signatures of stellate cells, a microarray evaluation was created, The obtained gene expression profiles were visualized as heatmaps, Red signifies large gene expression, when black signifies intermediate gene expression and green signifies lower gene expression of the corresponding gene when compared to the reference, respectively. Each and every row represents one particular gene, every column represents one sample. The dendrograms on the left side from the heat maps define how the genes cluster soon after applying hierarchical cluster examination, Expressions of genes are expressed making use of log2 ratios, where the sign signifies which channel had the larger intensity.
Hence, an expression ratio of 2 represents a four fold up regulation of a gene below condi tion A in comparison with this gene below ailment B, an expression ratio of 2 represents a 4 fold down regula tion. Organ specific profile A total of 160 annotated genes had been recognized as differen tially expressed concerning pancreas and liver derived stel late cells. To get a clear and properly defined matrix, these genes have been compared as. downregulated in pancreatic additional hints stellate cells in comparison to hepatic stellate cells or upregulated in pancreatic stellate cells in comparison to hepatic stellate cells, A group of selected genes are presented in Table 2. Signifi cantly distinctive genes in each and every group with large differential expression ratios had been additional analyzed by quantitative serious time PCR, immunoblotting, immunocytochemistry and immunohistochemistry in all sufferers. Pancreatic stellate cell certain genes On this group, collagen type XI alpha 1 was probably the most unique gene with a 13.

They alternate among GTP type and GDP kinds, Hsp90 is needed for Rab recycling in the early exocytic pathways. Many of the interactors of Hsp90 involved in trafficking such as, Rab1 GTPases, Rab5b and Rab6 show up regulation in all of the clusters. Only the important thing regulator GDI is extremely expressed in cluster 2 along with Hsp90. This really is sugges tive of enhanced Hsp90 dependent trafficking in cluster two. Hsp90 regulates the function of ribosome by most important taining the stability of 40S ribosomal subunit compo nents and 60S ribosomal subunit components, Hsp90 machinery is regarded to activate eIF2a kinases, HRI is definitely the significant eIF2 a kinase responsible for your greater eIF2 a phosphorylation on heat shock in erythroid cells, PfPK4 is often a protein kinase relevant to eIF 2alpha kinases. It is actually up regulated in all clusters and peaks in cluster two.
Glycogen Synthase Kinase GSK3 is marginally up regulated in clusters 1 and three and present at basal degree in selleck chemical TW-37 cluster 2, AKT is often a ser thr kinase which inhibits professional apoptotic signal. Hsp90 interacts with AKT and prevents its dephosphorylation by PP2A, Homologs of Hsp90, PI3K and PP2A are existing in Plas modium. AKT is located to be up regulated in every one of the clusters but exhibits greatest expression in cluster1, PI3K is up regulated in the many clusters, large in clusters 2 and three. Interestingly PP2A, the inhibitor of PKB kinase is extremely up regulated in cluster one followed by clusters 2 and three. Hsp90 also regulates MAP kinase pathways, concerned in cell proliferation, cell differentiation, cell motion and cell death. Raf homologs in Plasmodium are PFL0080c and PFB0520w.
Homologues of MAP1 2 are PF14 0294 and PF11 0147 and MEK are Pfnek, The many components of MAP kinase path way are up regulated in each of the clusters however the pathway could be most lively in cluster two due to the up regu lation of the master regulator Hsp90 during the similar, The up regulation of AKT and MAP kinase pathway in cluster two implies an increased Hsp90 dependent anti apoptotic pathway and cell prolif eration pathway on this cluster, PfHsp70 Odanacatib loved ones P. falciparum encodes for 6 genes that belong towards the Hsp70 loved ones PF08 0054, PFI0875w, MAL7P1. 228, PF11 0351, PF07 0033, and MAL13P1. 540. Out of these 6 genes, transcript information for only 4 are identified inside the clinical isolates. Hsp70 I, the cytosolic isoform of para internet site Hsp70 is extensively explored as an antigen plus a vaccine candidate for malaria, Hsp70 C transcript is discovered for being up regulated only in some cluster two parasites. Nevertheless, a lot of of its interact ing partners present up regulation in different clusters. As with other chaperones Hsp70 C interactors that exhib ited greatest transcript levels in cluster 2 patients are also uncovered for being really expressed in cluster 3 sufferers.

Usual untransformed cell lines had been significantly less sensitive to your growth inhibiting effects of U0126, with all the quantity of cells dropping by 12% in C2C12 and 18% in NIH3T3. These success indicated that in ordinary untransformed cell lines U0126 inhibited development somewhat, whereas failed to induce extended lasting phospho ERK inhibi tion. In addition, the colony forming assay in soft agar showed the colony formation from the IGR39, SW403 and PC3 tumor cell lines was abolished by U0126, whereas numer ous, significant colonies had been existing during the untreated cells, These information demonstrate that cell transformation of different tumor derived cell lines is halted by inhibition of MEK ERK pathway followed by c Myc down regulation.
Discussion The pharmacological inhibitors of Ras MEK ERK signal ling are arousing significant interest on account of their potential therapeutic employs, On this paper, we addressed kinase inhibitor the problem of if MEK ERK inhibition, by focusing on c Myc, prevents the transformed phenotype expression in RD cells as well as inside a amount of tumor cell lines that express a mutated model of ras and above express c Myc. The effective development inhibition induced from the MEK inhibitor U0126 in RD, colon carcinoma, pros tate and melanoma cell lines obviously demonstrates the MEK ERK pathway can be a pre requisite for that aberrant development of those cells. Indeed, U0126 completely inhib its phospho ERKs in all tumor cell lines implemented. It is actually note cells, U0126 is also able to abolish anchorage independ ent development. The failure of TPA to abolish anchorage inde pendent development could be explained by its inability to induce p21WAF1 and its constructive effects on c Myc and cyclin D1 expression in non adherent RD cultures.
Conversely, the U0126 mediated arrest of development in non adherent cul tures is usually due to the drastic c Myc down regulation and cyclin D1, identified to become concerned in cell transformation, On top of that, the experiment in suspension cul tures propose that ATP-competitive ALK inhibitor MEK ERK inhibitor, U0126, may have cytostatic results, These success demonstrate the mere inhibition of development prospective is not sufficient to prevent the transformed phenotype expression. Recent studies during the literature report, over the one particular hand, that MAPKs and c Myc cooperate in marketing invasive development and, over the other, that targeted disruption of c Myc suppresses cell transformation and tumor forma tion, The Ras MAPK pathways are, however, presently receiving attention owing for the treatment likely they worthy that each c Myc phosphorylation and c Myc expression itself decreased in RD cells as well as in all the non muscle tumor cell lines examined following MEK ERK inhibition.