Alternatively, inhibition of specific SASP factors could also be achieved, as is the case for IL-6, IL-8 and TNF

Alternatively, inhibition of specific SASP factors could also be achieved, as is the case for IL-6, IL-8 and TNF. of NRC-AN-019 therapy-induced senescence in cancer and highlight the great promise of two-step strategies in anticancer therapies. showed stem-like features, in which the expression of and was markedly increased. is a master transcription factor of cellular self-renewal.55 Moreover, senescent human and mouse cancer cells could also exhibit proliferation potential and senescence characteristics at the same time.11,19 In epithelial ovarian cancer (EOC), Nacarelli et al NRC-AN-019 found that cisplatin-induced senescence upregulated ALDH1 protein levels and the expression of along with mRNA, which is also the cancer stem-like cell (CSC) signature. When cisplatin-treated EOC cells rather than EOC cells were inhibited, the EOC CSC signature was suppressed and the survival of EOC-bearing mice was improved.56 By lineage tracing the p21+ senescent cells in the p21-CreER mouse model, some of the previously labeled senescent cells were found to re-entered the cell cycle and proliferate rather than being cleared at the late embryonic stage.57 In agreement with previous observations, the researchers from Charit University Medicine found that E-Myc-Bcl2-overexpressing lymphoma cells induced senescence by chemotherapy exhibited both senescence and stem cell-like characteristics. Based on single-cell tracking experiments, SA–gal-positive cells were found to incorporate 5-ethynyl-2-deoxyuridine (EdU), which suggested that senescent cells restarted DNA synthesis. More importantly, the senescent tumor cells of mouse and human origin were allowed to escape cellular senescence and resume sustainable proliferation when senescence-essential mediators such as Suv39 h1 or p53 were acutely inactivated. Strikingly, compared to cells that are never senescent, tumor cells that escape from the senescent state can re-enter the cell cycle with higher aggressive growth and tumor-initiation potential by activating the WNT pathway.19 In addition, Yu et al showed that loss of the H3K9 me3 mark, such as elevate activity of the H3K9 demethylase JMJD2C, could make OIS cell re-entry into Rabbit Polyclonal to OR cell cycle in melanocyte models.58 These findings indicate that senescent cells have the potential to repossess their self-renewal capability. TIS may result in senescence-associated reprogramming, which promotes cells to escape from the arrested condition and cancer recurrence (Figure 1B). However, more direct evidence about the stemness recovery of senescent tumor cells remains to be investigated in the future. Senescence-Associated Signaling Crosstalk Between Tumor Cells and Their Niche Cells Senescent cells can secrete a complex mixture of proinflammatory cytokines, chemokines, growth factors NRC-AN-019 and other cytokines, which is also called as SASP.10 These secretomes might be an important mediator of the cross-talk between senescent cells and nonsenescent cells (Figure 1C). The Effects of Senescent Cancer Cells on Neighboring Cells At the early stage of tumors, SASPs contribute to increased tumor growth control by reinforcing and/or spreading senescence-associated cell cycle exit via autocrine and paracrine signaling. SASPs might be required for stable cell cycle arrest. For example, IL-6 and IL-8, the major components of the SASP, have been NRC-AN-019 shown to restrain the proliferation of malignant cells by reinforcing the senescence growth arrest in an autocrine fashion.59 PAI-1 was also found to be essential for the induction of senescence even in the absence of p53.60 In other studies, senescent MCF-7 breast cancer cells triggered by adriamycin were observed to have an antiproliferative effect in which they induced a senescent state of cell cycle arrest in naive MCF-7 cells by secreting factors in vitro.61 Paracrine-induced senescence by SASPs might be important for the inhibition of tumor progression. Moreover, secreted SASP factors can directly modulate the immune response by signaling to the immune system. As described NRC-AN-019 above, senescent cancer cells recruit immune cells to remove the senescent tumor cells, consequently limiting tumor progression via engagement of the innate and adaptive immune systems. Other than frequently acting as an anticancer mechanism, senescent cells have recently been considered beneficial to cancer progression and relapse in some settings.10,18 Their contribution includes creating an immunosuppressive and protumor tissue microenvironment that protects some cancer cells from being cleared and permits tumor cell residues, thus positively favoring cancer relapse and progression. One of the functions of SASPs is that they can suppress the tumor immune response, which contributes to cancer promotion. For example, a recent study found that chemotherapy-induced senescent cells were able to circumvent immune clearance through concerted MMP-mediated shedding of NKG2D ligands and paracrine.

Moreover, BRCA2 expression in mice subcutaneous xenograft showed consistency with the expression with p53BER2 (Fig

Moreover, BRCA2 expression in mice subcutaneous xenograft showed consistency with the expression with p53BER2 (Fig. reverse nutlin-3-induced G1-arrest and senescence in TP53-WT cell lines. What is more, the knockdown of p53BER2 showed resistance to nutlin-3 treatment in vivo. Additionally, we found BRCA2 could be regulated by p53BER2 in vitro and vivo; further experiment showed p53BER2 could induce cell-cycle arrest and DNA repair by mediating BRCA2. In summary, the Rabbit Polyclonal to CHST10 p53-associated enhancer RNA-p53BER2 mediates the cell cycle and senescence of p53 in TP53-WT renal cancer cells. (%)0.7640.382?55452025? 55392217Gender, (%)0.2230.637?Male592930?Female251312T stage, (%)4.4020.111?T1a421824?T1b?+?T2291415?T313103Fuhrman grade, (%)9.9730.007?G130822?G2432617?G3?+?G41183 Open in a separate window renal cell carcinoma, cycle threshold, Fuhrman nuclear grade. P53BER2 could be specifically expressed in TP53-WT renal cancer cell lines Since p53BER2 is an enhancer RNA mediated by wild-type p53, we wonder whether p53BER2 is mediated in wild-type p53 in renal cancer cells. First, as shown in Fig. ?Fig.1A,1A, we could find that p53BER2 expression in TP53 mutant cells (786-O) is the lowest, consistent with our conjecture. To further understand the relationship between p53BER2 and wild-type p53 protein, we used the p53 protein activator nutlin3a to treat p53 wild-type and TP53 mutant kidney cell lines. Western Blot showed that nutlin3 was effective in inducing p53 expression and its target- p21 in Caki-1, but not in the 786-O cell line (Fig. ?(Fig.2A).2A). qPCR results indicated that nutlin3a could induce p53BER2 in p53 wild-type cells (OSRC-2, ACHN, CAKI-1), but did not induce TP53 mutant cell expression (Fig. ?(Fig.2B).2B). Also, we included p21 and PUMA as the positive control, and the results showed the expression of p21 and PUMA were upregulated with nutlin-3 treatment (Fig. ?(Fig.2A2A and Supplementary Fig. 1C, D). But the PAPPA expression, the previous target of p53BER222, did not change with nutlin-3 treatment, which might be due to different biological mechanisms in different tissue and cells (Supplementary Fig. 1E). To further explore the relationship between p53 and p53BER2, we got the pLX313-TP53-WT and pLX313-TP53-P278A, then we used the corresponding lentivirus and empty vector (EV) lentivirus to infect H1299, which were found as a p53-null cell26. Then we used WB and qPCR to test the expression of p53 and p53BER2; the results showed that overexpression of p53 induced an obviously increased expression of AZD9898 p53BER2 in WT-TP53 H1299 cells, but not in the AZD9898 TP53-P278A H1299 (Fig. 2C, D). Then we used si-P53 to transfect H1299-wt-TP53; qPCR results revealed that downregulation of p53 could decrease the expression level of p53BER2 (Supplementary Fig. 2A, B). p53BER2 reporter could detect the expression of p53BER2 in TP53-wt RCC cell lines It has been reported in the literature that wild-type p53 can bind to p53BER2 to enhance promoter expression. Using this principle, we designed the p53BER2 reporter to further investigate whether P53 initiates the promoter by direct binding to p53BER2. First, we combined the p53BER2-specific sequence with the minimal promoter to form a p53 promoter that specifically recognizes the wild-type p53 protein. Further, we used the GAL4-UAS system to enhance promoter efficiency and use dual to report the AZD9898 gene, and finally form the p53BER2 reporter (Fig. ?(Fig.2E).2E). Since 293T and HK2 have a basic expression of wt-p5327,28 and p53, 786-O express a relatively low level of mut-p53 protein. Here we transfected the p53 reporter and control reporters into the HK2, 293T, and 786-O cell lines, respectively,.

Programmed cell death 1 (PD-1), CD127 and CD27/CD28 expression among CD8+ T cell subsets

Programmed cell death 1 (PD-1), CD127 and CD27/CD28 expression among CD8+ T cell subsets. were defined as naive [CD45RA+human leucocyte antigen D-related (HLA-DRC)], early effector (CD45RACHLA-DRC) and mature Treg (CD45RACHLA-DR+). Overall Treg frequency was increased significantly among CD3+CD4+ T cells in lenalidomide- (median = 167%) untreated (median = 81%) patients (= 741E-06). Among Treg mature cells were increased significantly in lenalidomide-treated patients (median = 282%) compared to untreated patients (median = 148%, = 0001) at the expense of naive Treg (lenalidomide median = 34%, untreated median = 118%, = 0003). (b) Absolute numbers of CD4+ T cells and Tregs including subsets in the indicated patient groups. Significant decrease of CD4+ T cells in lenalidomide-treated (median = 215/l) compared to untreated patients (median = 447/l, = 0006). Naive Treg Rabbit Polyclonal to MMP-11 were decreased (lenalidomide median = 110/l, untreated median = 492/l, = 0021) and mature Treg were increased (lenalidomide median = 122/l, untreated median = 45/l, = 0001). (c) Mature Treg frequencies during (-)-p-Bromotetramisole Oxalate lenalidomide therapy: lenalidomide induction or therapy stop in single patients. Boxes depict the interquartile range (IQR), the line the median and the whiskers the 95% confidence interval (CI). cei0177-0439-SD2.jpg (603K) GUID:?301BE568-67C4-4B1B-8D52-C8E1A7315894 Fig. S3. Influence of lenalidomide on natural killer (NK) cell frequency and activation status in multiple myeloma (MM) patients. (a) Box plots for NK cell frequency among all lymphocytes (left) and NKp46, NKp30 and NKG2D (right) represent untreated MM patients (= 36) and lenalidomide-treated patients (= 17). NKp46 [median mean fluorescence intensity (MFI) ut = 338, Lena = 486] and NKp30 (median MFI ut = 348, Lena = 553) were elevated significantly (NKp46: = 0028, NKp30: = 0029) on NK cells, whereas there was no difference in NKG2D expression (median MFI ut = 181, Lena = 180). Boxes depict the interquartile range (IQR), the line the median and the whiskers the 95% confidence interval (CI). (b) Correlation between NKp30 and NKp46 expression of NK cells was evaluated using the non-parametric Spearman’s rank test; Spearman’s correlation coefficient (are poorly defined. In an observational study we assessed the impact of lenalidomide on different populations of immune cells in multiple myeloma patients. Lenalidomide therapy was associated with increased amounts of a CD8+ T cell subset, phenotypically staged between classical central memory T cells (TCM) and effector memory T cells (TEM), consequently termed TCM/TEM. The moderate expression of perforin/granzyme and phenotypical profile of these cells identifies them as not yet terminally differentiated, which makes them promising candidates for the anti-tumour response. In addition, lenalidomide-treated patients showed higher abundance of CD14+ myeloid cells co-expressing CD15. This population was able to inhibit both CD4+ and CD8+ T cell proliferation and could thus be defined as a so far undescribed novel myeloid-derived suppressor cell (MDSC) subtype. We observed a striking correlation between levels of TCM/TEM, mature regulatory T cells (Tregs) and CD14+CD15+ MDSCs. (-)-p-Bromotetramisole Oxalate In summary, lenalidomide induces both activating and inhibitory components of the immune system, indicating the existence of potential counter-regulatory mechanisms. These findings provide new insights into the immunomodulatory action of lenalidomide. and [11]. (-)-p-Bromotetramisole Oxalate Reports on MDSC in human MM are scarce. To date, two publications report this cell type in (-)-p-Bromotetramisole Oxalate MM patients, one describing an increased frequency of human leucocyte antigen D-related (HLA-DR)lo monocytes in patients with MM at various stages of their disease [14] and the other observing granulocytic MDSCs in the peripheral blood and bone marrow of MM patients [15]. A promising approach to counteract immunoinhibitory effects in MM is the implementation of immunotherapeutic agents such as lenalidomide (CC-5013, IMiD3, Revlimid), which is an effective drug in the treatment of newly diagnosed and relapsed MM. Furthermore, it has been employed successfully for maintenance therapy after high-dose chemotherapy of MM patients [16]..

These data indicate that BI 2536 attenuates the autophagy process by inactivation the AMPK signaling pathway

These data indicate that BI 2536 attenuates the autophagy process by inactivation the AMPK signaling pathway. Discussion There is now a growing body of evidence showing that PLK1 has attractive therapeutic potential in treatment of various types of cancers, including neuroblastoma. that PLK1 is definitely highly indicated in almost all of the 8 neuroblastoma cell lines except NGP cells. In addition, the status of MYCN amplified or not does not seem Cyantraniliprole D3 to impact PLK1 manifestation. Next, to evaluate whether PLK1 could be regarded as a potential restorative target in NB, we analyzed PLK1 mRNA transcripts in neuroblastoma tumor samples by using the R2: Genomics Analysis and Visualization Platform ( R2 is definitely a web-based microarray and RNA-seq database which contains a large amount of data units publicly available. In SEQC-498 cohorts comprising 498 neuroblastoma individuals’ samples, high PLK1 manifestation ( median) was impressive associated with both poor relapse free and overall survival of individuals (Number ?(Number1C).1C). Related results were found in Versteeg-88 dataset including 88 neuroblastoma samples (Number ?(Number1D),1D), demonstrating that PLK1 could be Mouse monoclonal to MAP2K4 served like a potential predictor in NB individuals’ outcome. Open in a separate window Number 1 PLK1 was over-expressed and inhibition of PLK1 by BI 2536 reduced viability in neuroblastoma cell lines. (A) Quantification of PLK1 mRNA manifestation of neuroblastoma cell lines. (B) Western blot analysis of PLK1 manifestation in neuroblastoma cell lines. (C) Overall survival and event free survival storyline generated from SEQC-498 cohorts in R2: Genomics Analysis and Visualization Platform ( (D) Overall survival and event free survival storyline generated from Versteeg-88 cohorts in R2: Genomics Analysis and Visualization Platform ( (E) Molecular structure of BI 2536 and IC50 value of BI 2536 in neuroblastoma cell lines. The IC50 ideals were derived after plotting proliferation ideals on a logarithmic curve. Experiments were performed in quadruplicate and repeated twice. (F) Proliferation rate of neuroblastoma cell lines treated with BI 2536. NB cells (2 104) were seeded in 96-well plates over night and incubated with DMSO or increasing concentrations of BI 2536 (1, 2.5, 5, 10, 25, 50 or 100nM) for 24 h. Cell proliferation rate was determined as a percentage of the DMSO treated control wells. BI 2536 inhibits cell proliferation of neuroblastoma cells In order to evaluate the effect of PLK1 inhibition, BI 2536, a specifically pharmacological inhibitor of PLK1, was applied (Number ?(Figure1E).1E). We treated a panel of NB cell lines with BI 2536 and evaluated cellular viability by CCK8 assay. As demonstrated in Figure ?Number1F,1F, BI 2536 significantly reduced cell viability with escalating doses of BI 2536 treatment in all NB cell lines tested, with the half-maximal inhibitory concentration (IC50) in the nanomolar range (Number ?(Figure1E).1E). Furthermore, to observe the long-term effect of BI 2536 on cell proliferation, we select two MYCN- amplied NB cell lines (SK-N-BE(2) and NGP cells) and two MYCN non-amplied NB cell lines (SH-SY5Y and SK-N-SH cells) for clone formation assay. The results showed that cell colonies decreased significantly after BI 2536 administration (Number ?(Number2A2A & B). Taken together, these results demonstrate that BI 2536 potently inhibits proliferation and viability of neuroblastoma cells. Open in a separate window Number 2 BI 2536 inhibited clone formation ability in neuroblastoma cell lines. (A) Clone formation assay of SH-SY5Y, SK-N-SH, NGP and SK-N-BE(2) cells incubated with DMSO or different concentrations of BI 2536(10 or 25 nM) for 2 weeks. (B) Clones quantity of SH-SY5Y, SK-N-SH, NGP and SK-N-BE(2) cells incubated with indicated concentration of BI 2536 or DMSO. * 0.01 and *** 0.001. ideals were determined by two-tailed t checks. All data are representative of three self-employed experiments with n = 3-6 per group and are means s.e.m. BI 2536 disturbs cell cycle progress in neuroblastoma cells In particular, since BI 2536 showed probably the most pronounced anti-proliferation effects in SH-SY5Y and SK-N-BE(2) cells, we selected them for further studies. BI 2536 treatment resulted in significant cell morphology switch, appearing as cell floating and shrinkage (Number ?(Figure3A).3A). As PLK1 is definitely part of the regulatory network controlling CDK1/cyclin B complex activity which settings access into mitosis in the G2/M transition 31, we next examined the effect of BI 2536 treatment on cell cycle. Not surprisingly, cell cycle analysis displayed build up of cell populations in the G2 phase Cyantraniliprole D3 from 12.761.33% to 63.643.28% in SH-SY5Y cells in response to 5nM BI 2536 treatment for 24 hr. At the same time, a decrease in the population of G1 and S phase cells was observed. Higher concentration of BI 2536 administration induced more serious mitosis disorder. In related, the G2 human population Cyantraniliprole D3 was improved from 6.063.66% to 18.947.14%, with G1 fraction decreased from 56.304.63% to 46.014.54 % in SK-N-BE(2) cells exposed upon 10nM BI 2536 (Figure ?(Number3B3B & C). In addition, GFP- Histone was used to track the mitotic arrest. As demonstrated in Number (3D, in control group (SH-SY5Y cells treated with DMSO), GFP-Histone was dispersed in the nucleus, which means most cells are.

Altogether, these data showed that our culture model maintained a diversity of the mTEC subpopulations comparable with that in global thymuses

Altogether, these data showed that our culture model maintained a diversity of the mTEC subpopulations comparable with that in global thymuses. Open in a separate window Figure 2 Primary cultured human thymic cells display medulla thymic epithelial cell features. and and Values were obtained using the ANOVA test. Asterisks indicate significant differences (**and and thymic expression and 87% of thymic expression were due to the medullary microdissected areas of human thymuses CCK2R Ligand-Linker Conjugates 1 while K8 was mainly cortical (Figure S3 in Supplementary Material). The compared analysis of the gene expressions and their ratios in TEC cultures versus thymic biopsies, confirmed that our culture method sustained the growth of cells expressing predominantly medullary markers such as and agglutinin-1 (UEA) lectin (27, 48, 49), a marker of highly proliferative mTECs expressing autoimmune regulator (AIRE) protein (45). Figure ?Figure22 showed that cultured cells exhibited positive labeling for K5/14, for CLAUDIN 4 (Figures ?(Figures2ACC)2ACC) as compared with thymic biopsies (Figures ?(Figures2DCF).2DCF). These labeling mirrored the medulla compartment of the thymus tissue (Figures ?(Figures2DCF).2DCF). The UEA antibody labeled few cultured mTECs (Figures ?(Figures2GCI).2GCI). Similarly, few mTECs in human thymic sections were stained with this antibody (Figures ?(Figures2JCL).2JCL). The percentage of positive cells in cultured mTECs and in CCK2R Ligand-Linker Conjugates 1 the thymic medullary areas is shown for the different markers in Figure ?Figure2M,2M, and no statistical differences were observed. Altogether, these data showed that our culture model maintained a diversity of the mTEC subpopulations comparable with that in global thymuses. Open in a separate window Figure 2 Primary cultured human thymic cells display medulla thymic epithelial cell features. Representative pictures of a primary cultured human thymic epithelial cells (TECs) (day 7) (ACC) and human thymus (DCF) co-labeled with an anti-Claudin 4 antibody (red), anti-keratin 5, and 14 antibodies (green). Representative pictures of primary cultured human TECs (GCI) and human thymus (JCL) co-labeled with an agglutinin I lectin (UEA) (red), anti-keratin 5 and 14 antibodies (green). The percentage of positive cells in primary cultured human TECs represented the number of KERATIN 5/14, CLAUDIN 4, or UEA positive cells versus the total cell number (M). For thymic sections, the surface of KERATIN 5/14 or CLAUDIN 4 positive areas was measured and compared with the thymic medulla. Images were acquired with a Zeiss Axio Observer Z1 Inverted Microscope using 20 magnification. The counting was done as previously described in Dragin et al. (50). ImageJ software was used to display the digital pictures and to count manually the labeled cells. Graph bar represents the results obtained with four different human biopsies and primary cultured human TECs. The non-parametric MannCWhitney test was used for statistical analyses. Human Primary Cultured mTECs Express Factors Involved in T Cell Negative Selection Process Medulla thymic epithelial cells play a major role in immune tolerance by expressing and presenting TSAs to developing T cells. TSAs expression in mTECs is controlled by various transcription factors among them AIRE, FEZf2, and PRDM1. We CCK2R Ligand-Linker Conjugates 1 evaluated the ability of cultured primary TECs to express such tolerance markers. At day 7, we observed that primary cultured TECs expressed (Figure ?(Figure3A)3A) and various TSAs, such as the -acetylcholine receptor (Values were obtained using the non-parametric MannCWhitney test. Asterisks indicate significant differences (*(Figure ?(Figure4A),4A), tumor growth factor- ((Figure ?(Figure4C),4C), and (Figure ?(Figure4D)4D) compared with the other cell types. Of course, in human thymuses, different cell types may express Values were obtained Rabbit polyclonal to AKAP5 using the MannCWhitney test. Asterisks indicate significant differences (*mRNA expression is CCK2R Ligand-Linker Conjugates 1 regulated by RANK/CD40 and lymphotoxin beta receptor signaling pathways (56C58). We observed a significant increase of AIRE mRNA expression (Figure ?(Figure5A)5A) suggesting that the CCK2R Ligand-Linker Conjugates 1 cultured cells conserved their ability to overexpress.


GZN1201804). Authors contributions X.X., Q.L., and C.S. when ectopically expressed, can attenuate p53 activation-induced EGFR reduction and cellular senescence. We further showed that the increased degradation of DYRK1A caused by p53 activation was mediated by MDM2. MDM2 was found to physically interact with and ubiquitinate DYRK1A, ultimately leading to its proteosomal degradation. Importantly, administration of Nutlin-3a, which disrupts the binding of MDM2 to p53, but not that of MDM2 to DYRK1A, reduced the levels of DYRK1A and EGFR, induced senescence, and inhibited growth of tumor xenografts formed by U87 glioblastoma cells. Ectopic expression of EGFR in tumor xenografts attenuated senescence and tumor reduction caused by Nultin-3a. Our findings thus established a novel link between p53 and EGFR and may have implications in p53 activation-based therapies. Introduction Upregulation of epidermal growth factor receptor (EGFR), in the forms of amplification and activating point mutation, was commonly detected in lung cancer1C3, gliblastomas4, esophageal squamous cell cancers5, and many other types of cancer6. The gain of function in EGFR plays a critical role in driving the Meclofenoxate HCl proliferation and survival of many types of cancer cells, via upregulating the AKT and MAPK pathways. Correspondingly, treatment of lung cancers bearing EGFR mutations with EGFR tyrosine kinase inhibitors Gefitinib and Erlotinib has been shown to be much more effective than chemotherapy7C9. In addition, upregulation of EGFR in tumor stroma also mediates angiogenesis and resistance to vascular endothelial growth factor (VEGF) inhibitor10. Cancer cells can even transfer activated EGFR to macrophages and thereby suppress innate immunity11. Therefore, inhibition of EGFR signaling by RTK inhibitor or Rabbit polyclonal to ABHD12B antibodies has far-reaching clinical implications. is the most commonly mutated tumor suppressor gene in human cancer12. p53, the protein encoded by has been shown to be either up- or downregulated by p53 at the transcription level, depending on cell lines or cell types under study22C25. Many factors were also identified to regulate EGFR turnover at protein level26C28. Dual-specificity tyrosine-phosphorylated and tyrosine-regulated kinase 1A, or DYRK1A, was shown to promote the stabilization of EGFR by phosphorylating SPRY2, which interferes with the Cbl-mediated ubiquitination of Meclofenoxate HCl EGFR29. Interestingly, DYRK1A can be negatively regulated by p53 via miR-124630. Therefore, diverse mechanisms may govern the regulation of EGFR by p53. Downregulation of EGFR-MEK-ERK signaling pathway is sufficient to induce cellular senescence in glioblastoma cells31. In an effort to elucidate the mechanisms underlying the cellular senescence induced by p53 activation, we found that downregulation of EGFR can also mediate p53-induced senescence in a subset of cancer cell lines. The downregulation of EGFR by p53 is achieved at both the transcriptional level and protein level. Even in cells in which transcription is enhanced by p53 activation, EGFR protein level can still be reduced. DYRK1A, which is required for the maintenance of EGFR stability, is downregulated by p53. We further showed that the downregulation of DYRK1A is mediated by p53 target gene was increased. A luciferase reporter containing EGFR promoter showed a reduction in luciferase activity when treated by Nutlin-3a (Fig.?S3A), indicating that p53 could negatively regulate transcription. However, in contrast to the reduction of EGFR at the protein level, transcription showed a positive response to p53 activation in U2OS and A2780 cells (Fig.?S3B and S3C). mRNA levels were reduced by Nutlin-3a in A172 and HT1080 cells (Fig.?S3D and S3E). These results suggest that while repression of transcription may contribute to the downregulation of EGFR when p53 is activated, reduction in EGFR can occur in the presence of increased transcription. On the other Meclofenoxate HCl hand, while the protein amount of EGFR was elevated in A549 cells in response to Nutlin-3a treatment, mRNA level was reduced (Fig.?S4). These results suggest that post-transcriptional regulation likely plays an important role in determining the eventual amount of EGFR. Downregulation of EGFR mediates cellular senescence induced by p53 activation The activation of p53 can either lead to apoptosis or cellular senescence depending on cell types. We next examined the fates of the cells in which EGFR was downregulated by p53 activation. Nutlin-3a treatment strikingly induced cellular senescence in U87 and U2OS cells, as shown by positive senescence-associated -galactosidase (SA–gal) staining, reduction of lamin B1, and reduced 5-ethynyl-2-deoxyuridine (EdU) incorporation, p16 (Fig.?2aCc, Figs.?S5AC5D). Consistently, depletion of p53 by RNA interference (RNAi) greatly attenuated the Nutlin-3a-induced senescence (Fig.?2d, e). No increase in the level of apoptosis was detected (data not shown). The senescent cells appeared to be arrested at the G0 or G2 phase (data not shown). We previously showed that berberine-induced senescence of U87 glioblastoma cells is mediated by the downregulation of EGFR and RNAi of EGFR alone can induce.