Concurring with these results, tMSC showed the lowest levels of the active form of glutathione, the form of glutathione able to provide antioxidant power. Overall, these data indicate that transformation of MSC leads to a global transcriptional selleck chemical Z-VAD-FMK down regulation of the cellular antioxidant program. Nrf2 is repressed during cellular transformation via activation of RASRAFERK pathway Western blot experiments confirmed suppression of Nrf2 expression and its downstream target NQO1 that correlated with ST and H RasV12 induced activation of ERK and AKT pathways. To investigate the mechanism of Nrf2 repression during transformation, we focused in the last transformation step where the more pronounced down regulation of Nrf2 and ARE containing genes occurred.

We studied the roles of RAS and some RAS downstream effectors by expressing con stitutive active mutants of H RAS, RAF 1, and AKT in immortal MSC4. We found that activation of RAS and RAF, but not AKT, led to decreased expression of Nrf2 and NQO1. Recent reports showed that Nrf2 Inhibitors,Modulators,Libraries expression was de creased in certain human breast cancer cells and breast tumors when compared with normal mammary epithe lial cells or normal breast tissue. Interestingly, we found a reduction in Nrf2 and NQO1 expression when normal human mammary epithelial cells were transformed using the same oncogenic elements that we employed to transform MSC, suggesting that this mechanism for Nrf2 regulation is not restricted to adult MSC. Next we used chemical inhibitors to address whether Nrf2 expression is transcriptionally regulated via ERK or PI3KAKT pathways in the breast cancer cell lines MDA MB 231 and Inhibitors,Modulators,Libraries MCF 7.

While cell survival was not affected by the concentration of Inhibitors,Modulators,Libraries inhibitors used in this assay, treatment with the ERK inhibitor U0126 led to a significant increase in the transcription of Nrf2 and NQO1. How ever, inhibition of AKT with GSK690693, or PI3K with LY294002 and wortmannin did not induce expression Inhibitors,Modulators,Libraries of Nrf2 nor NQO1. The effect of these in hibitors on ERK and PI3KAKT Inhibitors,Modulators,Libraries pathways is shown in Figure 3E, where a modest but consistent activation of the Nrf2 pathway could be detected following only 16 hours treatment with U0126. Overall our data indicate that the RASRAFERK pathway mediates Nrf2 repres sion in these cancer cells. Nrf2 activity was found suppressed in tumor cells due to increased expression of the ubiquitin ligase Cul3 that, together with Keap1, targets Nrf2 for degradation by the proteasome.

However, expression of Keap1 and Cul3 did not increase in transformed MSC. Nrf2 protein stabilization by means of tert butylhydroquinone impairs MSC transformation To investigate whether Nrf2 down regulation contributes to increased ROS, we induced Nrf2 in tMSC by phosphatase inhibitor TBHQ, a chemical that stabilizes Nrf2 protein by impairing its pro teasomal degradation.

The AB42 was degraded by lysosome pathway To determine the fate of AB42 after its internalization, we examined the AB42 content in cellular fraction in NG2 cell line and cell http://www.selleckchem.com/products/BI6727-Volasertib.html culture supernatant. AB42 in the cellular fraction increased within 3 hours of AB42 incubation then decreased from 24 hours, indicating that AB42 was Inhibitors,Modulators,Libraries first in ternalized and then degraded. The AB42 in the culture supernatant decreased over time, supporting that AB42 was taken up by the cells. To determine whether lysosome pathway is involved in AB42 degradation, we investigated the intracellular distribution of AB42 after its internalization. NG2 cell line was exposed to HiLyte Fluor 488 labeled AB42 and lysotracker dye for visual izing lysosomes. As shown in Figure 5B, AB42 is local ized in lysosomes.

AB42 exposure also increased the mRNA expression of Lysosomal associated membrane Inhibitors,Modulators,Libraries proteins, LAMP1 and LAMP2. Leupeptin and pepstatin, the inhibitors for major cysteine and aspartyl Inhibitors,Modulators,Libraries proteases for lysosomal proteolysis, blocked the degradation of AB42 in the NG2 cells. These data suggested that internalized AB42 was transported into the lysosome and degraded by the lysosome dependent pathway. Autophagy involved in the degradation of AB42 in NG2 cells Since autophagy is involved in amyloid beta peptide me tabolism and clearance, we tested whether au tophagy regulated the degradation of AB42 in NG2 cells. AB42 treatment increased the expression of autophagy re lated proteins, including LC3 and beclin1. Furthermore, AB42 was co localized with LC3, suggesting that they were enclosed by autophagosomes.

AB42 treatment increased the number of mCherry LC3 puncta positive cells, suggesting an induction of au tophagy. Autophagy inhibitors, wortmannin and bafilomycin A1, decreased the AB42 degradation. The expression of LC3 Inhibitors,Modulators,Libraries II dec reased when treated with wortmannin and increased when treated with bafilomycin A1. Beclin1 did not show changes when treated with wort mannin or when treated with bafilomycin A1 in NG2 cells. Knockdown of beclin1 in NG2 cells using a beclin1 specific siRNA also increased the accumulation of AB42. Inhibitors,Modulators,Libraries Together, these results suggest that AB42 could induce autophagy which directed AB42 to lysosome dependent protein degrad ation in NG2 cells. Discussion Amyloid plaques, which consist of aggregates of AB in the brain, are the predominant pathological change in AD patients.

selleck chem Tipifarnib The overload of AB combined with hyperphos phorylated neurofibrillary tangles, neuronal loss, inflammation, and oxidative stress make synaptic dysfunc tion and behavioral changes. It is important to understand the mechanisms underlying the clearance of the amyloid protein in the brain. The levels of AB peptides within the brain are tightly regulated by mechanisms con trolling their generation and clearance. In human CNS, the rate for production and clearance of AB is 7. 6% per hour and 8. 3% per hour, respectively.

The drug tranilast, an anti allergic agent, has been applied for bronchial asthma, allergic rhinitis and atopic dermatitis, also suppresses collagen synthesis in keloid or hypertrophic scars. The inhibitory effect of tranilast in different cell types is probably by antagonizing selleck chemical Wortmannin and inhibiting synthesis and secretion of TGF B. Since tranilast responsibilities through TGF B pathway, it seems also tamoxifen influences this pathway, we hypothesize that combination of tamoxifen and tranilast may an appro priate therapeutic option Inhibitors,Modulators,Libraries for breast cancer management. In this Inhibitors,Modulators,Libraries paper, possible synergistic effect of tamoxifen with tranilast was examined in the hope of creating a more ef fective anti tumor treatment strategy.

Methods Cell lines drugs MCF 7 and MDA MB 231 ob tained from the National Cell bank of Iran, were grown in RPMI 1640 media supplemented with 10% fetal calf serum and penicillin/streptomycin antibi otics. Cultures were maintained at 37 C in a humidified atmosphere Inhibitors,Modulators,Libraries of 5% CO2 in air. TAM and tranilast were purchased from Enzo Life Sciences and dissolved in di methyl sulfoxide so that the final dimethyl sulf oxide concentration in experimental wells did not exceed 0. 5%. Aliquots of a 1000 uM stock solution of TAM and tranilast were stored in dark at ?70 C, defrosted and diluted with cell culture medium to the desired concentra tion before use. The concentrations used alone treatment were the fol lowing Inhibitors,Modulators,Libraries TAM 1, 2, 5, 10 and 20 uM. tranilast 10, 20, 50, 100 and 200 uM. The treatment combinations used were 2 uM of TAM with different concentrations of tranilast 10, 20, 50, 100, and 200 uM for 48 h.

Cell viability measurement Cytotoxic effect of TAM and tranilast was determined by MTT test. MCF 7 or MDA MB 231 cells were seeded in 96 well culture plates at 104 cells/well density. Cells were allowed Inhibitors,Modulators,Libraries to attach nevertheless for 24 h before drugs were added to the medium. All drug concentrations were tested in triplicate wells and the assays were performed in three separate experiments. Following 48 h exposure at 37 C and 5% CO2, 20 ul MTT solution was added to each well and in cubated for 4 h at 37 C. The medium with MTT were removed, and 100 ul DMSO was added to dissolve formazan crystals at room temperature for 30 min. The optical density of each well was measured using an ELISA reader at 570 nm. The percentage of cell viability was calculated according to the following equation Lactate dehydrogenase assay MCF 7 or MDA MB 231 cells were cultured in 96 well plates. The plates were incubated over night at 37 C and on the next day, 300 ul of culture media containing drug doses were added to each well, and the plates were incubated at 37 C in 5% CO2. 48 h later, 100 ul of medium from each well was carefully transferred to new plates.

This kinase assay approach may provide a novel strategy for the treatment of HRPC, particularly advanced prostate cancer in which the Vav3 signaling pathway is activated. Methods Cell culture and hypoxia induction LNCaP human prostate cancer cells were maintained in RPMI 1640 medium supplemented with 10% heat inactivated fetal bovine serum, 50 IU/ml penicillin, Inhibitors,Modulators,Libraries and 50 ug/ml streptomycin and cul tured at 37 C in a humidified atmosphere of 5% CO2. To establish chronic hypoxia conditioned LNCaP cells, LNCaP cells were cultured under hypoxia for 6 months. The experiments using LNCaPH cells were performed under hypoxic conditions. KPK13 human renal cell carcinoma cells were cultured in minimum essential medium supplemented with 10% FBS, with 50 IU/ml penicillin, and 50 ug/ml streptomycin in 5% CO2 at 37 C.

Immunocytochemistry Cultured cells were washed with PBS, fixed in methanol for 20 min, and incubated in 10% goat normal serum Inhibitors,Modulators,Libraries for 10 min at 37 C. Cells were in cubated in a primary antibody against Vav3 at room temperature in PBS with 1% BSA for 60 min. After incubation with the primary antibody, the secondary antibody fluorescein dye conjugated goat anti rabbit IgG was added in PBS with 1% BSA for 30 min. Cells were visualized using confocal laser microscopy Inhibitors,Modulators,Libraries followed by nuclear staining with 1 ug/ml 2,4 diamidino 2 phenylindole dihydrochloride n hydrate. Transient transfection of Vav3 siRNA Cells were transiently transfected with a Vav3 siRNA du plex or a control siRNA using Lipofectamine RNAiMAX according to the manufacturers instructions. The sequence of the siRNA against Vav3 synthesized.

Following transfection, cells were sub jected to growth inhibition, live/death, flow cytometric, and immunoblot analyses. Inhibitors,Modulators,Libraries Growth inhibition assay Cell viability was determined using a cell proliferation assay. In brief, exponentially growing cells were seeded in 6 well plates at 1 105 cells/well. After overnight cul ture, the culture medium was changed to fresh standard medium containing 5 nM docetaxel for 0 Inhibitors,Modulators,Libraries 72 h or various concentrations of docetaxel for 72 h in the presence or absence of si Vav3. After treatment, the cell number was counted with a hemocytometer. Live/death analysis Cells were treated with si Vav3, 5 nM docetaxel, or si Vav3 plus 5 nM docetaxel for 48 h. Live and dead cells though were detected using the Live/Death Viability/Cytotoxicity assay kit for which fluorescence was observed and pictures were taken at 4�� magnification. The data from three independent experiments were expressed as a mean percentage. Flow cytometric and DNA fragmentation analyses For cell cycle analysis, flow cytometric analysis of propidium iodide stained nuclei was performed.

The amount of open space covered by migrated cells increased from 34% up to 86%. Knockdown of RSK1 had little effect on spontaneous cell migration, but silencing RSK2 expression showed a moderate effect on spontaneous cell migration. In MSP induced cell migration, silencing RSK1 expression did inhibitor supplier not impair MSP induced cell migration, as more than 80% of the open space was still covered by migrated cells. In con trast, MSP induced cell migration Inhibitors,Modulators,Libraries was significantly impaired in RSK2 siRNA treated cells. In this case, only 27% of the open space was covered by migrated cells, which was similar to spontaneous migration. TGF b1 induced cell migration was not affected by knockdown of RSK1. The inhibitory effect was only observed in cells treated with specific RSK2 siRNA.

Moreover, we observed that silencing RSK2 expression also impairs cell migration Inhibitors,Modulators,Libraries synergized by combined MSP and TGF b1 stimulation. Thus, silencing RSK2 but not RSK1 by specific siRNA decreases MSP induced cell migration in L3. 6pl cancer cells. Discussion The purpose of this study is to identify the major signal ing molecule that controls MSP induced EMT in epithelial Inhibitors,Modulators,Libraries cells. Altered RON expression and activation contribute to malignant progression of various epithelial cancers. RON is overexpressed in various types of primary cancer samples including those from colon, breast, and pancreas. Aberrant RON activation also causes increased tumor cell proliferation, matrix inva sion, and drug resistance. Currently, the role of MSP and Inhibitors,Modulators,Libraries RON in regulating EMT under physiological conditions is largely unknown.

In contrast, MSP induced RON activation or RON overexpression have been shown to induce EMT in various cancer cells including colon, breast, and pancreas. The changes to mesenchymal phenotype in RON activated tumor cells have been considered as a molecular basis for increased tumor Inhibitors,Modulators,Libraries malignancy including cell migration, matrix invasion, and distance metastasis. Several upstream signaling proteins such as Erk1/2 have been implicated in MSP induced EMT . however, the major effector molecule that transduces RON signals leading to EMT is still unknown. Intracellular proteins such as b catenin and NF B have been identified as effector molecules in MSP induced EMT. Nevertheless, their significance is often limited to parti cular cell models.

Thus, identification of the major sig naling molecule is important not only for an understanding of the cellular mechanisms of EMT, but also for the development of potential therapies that tar get cancer cell migration and invasion. Results selleckchem Tubacin from this study indicate that RSK2 is a major determinant bridging RON signaling to EMT. This con clusion is supported by the following evidence. First, inhibition of RSK, as indicated in the cell shape based screen by using specific RSK inhibitor SL0101, comple tely prevented MSP induced spindle like morphology.

Thus, increased level of Cdk5 activator may facilitate Cdk5 mediated phosphorylation of overex pressed p53, which causes cell growth inhibition. The decreased level of p35 protein in HTet43GFP cells does not cause cell growth inhibition because of unavailability of its substrate. Though, Cdk5 plays an important role in activating overexpressed p53, as such it is not involved http://www.selleckchem.com/products/Bortezomib.html in Inhibitors,Modulators,Libraries the pro liferation of parental HeLa cells per se in spite of the fact that E6 expression leads to increase in Cdk5 protein expression. p53 executes its apoptotic function through intrinsic or extrinsic pathways. To further confirm the pathway involved, we investigated Bax, an important transcriptional target of p53 involved in promoting intrinsic mitochon drial apoptosis.

Bax translocates to mitochondrial outer membrane causing MOMP and releases cytochrome C into cytosol. Inhibitors,Modulators,Libraries Cells lacking Bax or those overexpressing Bcl 2 are profoundly resistant to a broad range of apoptotic stimuli, including chemotherapeutic drugs treat ment and serum starvation. In HPV positive cancers Bcl 2 overexpression and Bax degradation by E6 facilitates cancer progression. Here, we demonstrate that upre gulated Bax translocates to mitochondria upon PP2A inhi bition in p53 overexpressing cells which is dependent on Cdk5 activity. Thus, only phosphorylated p53 triggers Bax transcription to increase its levels and cause apoptosis. In addition, the cell cycle arrest caused by inhibition of PP2A in p53 overexpressing cells may be dependent on tran scriptional upregulation of p21 gene.

Collectively these Inhibitors,Modulators,Libraries data also provide Inhibitors,Modulators,Libraries evidence for reactivation of E6 disrupted p21 and Bax pathways in HPV positive cells. Finally, we propose that Cdk5 interacts with p53 and phosphorylates Ser20 and Ser46 residues. Phosphoryla tion restores the ability of overexpressed p53 to specifi cally bind on p21 and bax promoters. These findings provide novel insight into the regulation of p53 transactivation functions and propose PP2A to be a key player in modulating p53 functionality. The phosphory lated status of specific residues may be involved in pro moter selection and this proposition needs further investigations. Also, this is the first report which pro vides mechanism for functional activation of p53, and details the essential modifications necessary for non genotoxically overexpressed p53 to be able to execute its tumor suppressor functions in HPV positive cells. More over, activation of overexpressed p53 without targeting viral Inhibitors,Modulators,Libraries oncogenes may have implication next in the treatment of virus infected carcinomas. The efforts towards the newer approaches to target p53 pathway and usefulness of reactivation of p53 pathways in treatment of cancers are encouraging.

Cleaved bands greater than 5 fold over the control were shown at 3. 5M with all incubation periods. PARP can release apoptosis inducing factor, which induces chromatin condensation and large scale DNA fragmentation when released into the cytosol. Simi lar to PARP, AIF cleavage was shown at concentrations www.selleckchem.com/products/Nilotinib.html greater than 0. 35M. Cleaved bands Inhibitors,Modulators,Libraries of AIF greater than 2 fold and were shown at 3. 5M at 24 hrs. Determination of Anti lymphoma Effect of ApoG2 in SCID Mice Previous studies in this laboratory indicated that the MTD for ApoG2 could not be determined. We tested up to 800 mg/kg iv of ApoG2. testing beyond 800 mg/kg were not attempted, due to cost and other logistical issues. For this efficacy trial, 5 106 WSU FSCCL cells were injected into the intraperitoneal cavity of 7 mice per group.

Seven days post WSU FSCCL inoculation, 25 mg/kg of ApoG2 was injected into each animal either Inhibitors,Modulators,Libraries intravenously or intraperitoneally over 5 days. After 105 days, 42% of the i. v. treated Inhibitors,Modulators,Libraries animals, and 60% of the i. p. treated ani mals had died from FL. Statistical comparison of survival curves for i. v. treatment and untreated control show a chi square of 8. 005 and P 0. 0047. Statistical comparison of survival curves for i. p. treatment and untreated control show a chi square of Inhibitors,Modulators,Libraries 4. 397 and P 0. 0360. Pathological evaluation showed that ret roperitoneal lymph nodes were diffusely replaced by tumor cells in mice that died. The effec tiveness of ApoG2 was further demonstrated by bone marrow examination which was completely replaced by tumor cells by day 34 in control animals.

In Inhibitors,Modulators,Libraries con trast, ApoG2 treated mice showed normal bone marrow with no apparent tumor infiltration. Discussion FL has been increasing in incidence over the past three decades and is now the fifth most common malignancy in the United States. There are many approaches to the treatment of FL, but the goal of therapy has been to main tain the best quality of life and treat when a patient is at high risk or the disease progresses. Standard chemother apy regimens directed towards these low grade lympho mas still lack complete curative effects. This may be in part due to the overexpression of Bcl 2, a key molecule of resistance in indolent lymphoma. Overexpression of Bcl 2 has been implicated to play a significant role in the clin ical outcome of FL patients. A number of approaches have been sought to target overexpression of Bcl 2 in FL, e.

g. downregulation of Bcl 2 protein via antisense oligo nucleotides. Most recently, hydrophobic groove of the Bcl 2 family of anti apoptotic proteins has become a very attractive target for the design of SMIs. SMIs filling the hydrophobic groove mimic cognate proteins selleckchem such as Bax and Bid. SMIs directed against BH3 domains have been categorized into at least eight different chemi cal classifications. Laboratories, including this one, have been in the search to find novel small molecule inhibitors to Bcl 2.

Tumor http://www.selleckchem.com/products/MLN-2238.html necrosis selleck screening library factor alpha is a pleiotropic cytokine that plays an important role in immunity and in flammation as well as in the control of cell proliferation, differentiation, and apoptosis. TNF is produced mainly by macrophages Inhibitors,Modulators,Libraries and enhances tumor regression mediated by cytotoxic T Inhibitors,Modulators,Libraries cells. TNF has been Inhibitors,Modulators,Libraries implicated to play a role in advanced breast Vismodegib Hedgehog/Smoothened cancer and some other metastatic tumors. It induces tumor necrosis by initiating apoptotic cell or death affecting tumor vascularization. Paradoxically however, it can also promote tumor cell proliferation and progression.

In this Inhibitors,Modulators,Libraries study, we found that versican G3 expressing MC3T3 E1 cells showed Inhibitors,Modulators,Libraries enhanced cell survival in serum free AMEM Inhibitors,Modulators,Libraries medium, while lower cell viability was observed in serum free AMEM medium with TNF com pared to vector control cells.

Annexin V FITC apoptosis detection assays confirmed that versican G3 expressing MC3T3 E cells showed enhanced cell apoptosis in serum free AMEM Inhibitors,Modulators,Libraries medium with TNF when Inhibitors,Modulators,Libraries com pared to vector cells. Immunoblotting showed that G3 expressing MC3T3 E1 cells expressed enhanced pEGFR in serum free AMEM medium with or without TNF. When cultured in TNF, G3 expressing MC3T3 Inhibitors,Modulators,Libraries E1 cells also showed increased expression of pSAPK/JNK, although GSK 3B expres sion did not Inhibitors,Modulators,Libraries appear influenced. Selective SAPK/ JNK inhibitor SP600125 could also prevent versican G3 enhanced MC3T3 E1 cell apoptosis induced by TNF.

SP6000125 blocked G3 enhanced expression levels of pSAPK/JNK and had no effect on GSK 3B ex pression, when the cells were cultured in TNF medium.

These results indicated that versican Inhibitors,Modulators,Libraries Inhibitors,Modulators,Libraries G3 domain enhanced MC3T3 E1 Inhibitors,Modulators,Libraries cell apoptosis induced by TNF through enhanced expression of EGFR/JNK signaling. Select ive SAPK/JNK inhibitor SP6000125 blocked G3 enhanced expression of EGFR/JNK signaling observed in MC3T3 E1 cells and thereby prevented Inhibitors,Modulators,Libraries its enhanced effect on pre osteoblast cell apoptosis. Versican G3 domain Inhibitors,Modulators,Libraries modulated MC3T3 E1 cell differentiation, growth and apoptosis through epidermal growth factor like motifs There appears to be important functions of the EGF like motifs of versican G3 domain.

In transiently transfected breast cell lines 66c14 and 4T07 with G3 fragment lacking the EGF like motifs, the G3EGF expressing cells did not show enhanced cell growth and migration when compared to G3 transfected selleck compound cells.

We also stably transfected these constructs into 4T07 cells, and found that G3 expressing breast cancer cells showed enhanced cell migration and invasion to MC3T3 http://www.selleckchem.com/products/brefeldin-a.html E1 cells. But the G3EGF expressing cells did not show enhanced cell migration and invasion to MC3T3 E1 cells. In our experiments, we also stably transfected MC3T3 E1 CP127374 cells with a G3 construct, G3EGF, and vector. We found that G3EGF expressing MC3T3 E1 cells did not show enhanced cell growth inhibition induced by TGF B1 when compared to the G3 transfected cell group.

E2 increased sellectchem the expression of 897 genes by 1. 5 fold in MCF7 EGFR cells after 6 hr, while a similar number of Inhibitors,Modulators,Libraries genes was 1. 5 fold lower expressed compared to controls. The number of genes induced or decreased by EGF was slightly higher. As expected, TAM greatly reduced the number of genes 1. 5 fold up or down regulated by E2. TAM hardly affected the number of EGF regulated genes. TAM, however, had a significant effect on the number of genes regulated by combined E2 EGF exposure due to down regulation of E2 responsive genes. In order to further characterize the inhibitory effect of TAM on E2 regulated genes, we calculated the percentage of inhibition by TAM for each gene. The inhibition by TAM of E2 induced genes was large the expression of more than 65% of E2 up regulated genes was inhibited by TAM by 50%.

Interestingly, the effect of TAM on genes up regulated by Inhibitors,Modulators,Libraries E2 under Inhibitors,Modulators,Libraries the condition of combined E2 EGF exposure was almost as big as with exposure to E2 alone. This indicates that the inhibitory effect of TAM is only slightly affected by exposure of the cells to EGF. In general, similar observations were made for the inhibitory effect of TAM on E2 down regulated genes as for E2 up regulated genes. Further analysis of the E2 and EGF regulated genes showed that the identity of E2 and EGF induced genes are different most genes up regulated by E2 are not induced by EGF. Many known E2 regulated genes such as TFF1, PGR, GREB1 and MYC belong to this class. Similarly, the majority of EGF induced genes is not induced by E2. However, there is number of genes that is up regulated 1.

5 fold by both E2 and EGF, and for part of these, there is a synergistic effect of E2 and EGF. Analysis with Metacore software suggests that the most important transcription factors for these genes are AR, c JUN, c MYC, EGR1, ESR1, HIF1A, p53 and SP1, which is consistent with the cell proliferation pathways activated by E2 and EGF. Furthermore, there is a relatively Inhibitors,Modulators,Libraries large number of genes induced by combined E2 EGF exposure that is not induced by E2 or EGF Inhibitors,Modulators,Libraries alone. This most likely is also due to a synergistic effect of E2 and EGF because 60% of these genes are already induced by E2 or EGF alone but just below the threshold of 1. 5 fold. Conversely, there is also an antagonistic effect because best some of the E2 up regulated genes are down regulated by EGF, and visa versa. In conclusion, the majority of genes are uniquely induced by either E2 or EGF and only for a limited number of genes there is an agonistic or antagonistic effect. Similar conclusions can be drawn for E2 and EGF down regulated genes.