High Levels Of c-Myc Research Articles

C-Myc Regulates RNA Splicing of the A-Raf Kinase and Its Activation of the ERK Pathway

A-Raf kinase can inhibit apoptosis by binding to the proapoptotic mammalian sterile 20-like kinase (MST2). This function relies on expression of hnRNP H, which ensures the correct splicing of a-raf mRNA needed to produce full-length A-Raf protein. Here, we showed that expression of hnRNP H and production of full-length A-Raf is positively controlled by c-Myc. Low c-Myc reduces hnRNP H expression and switches a-raf splicing to produce A-Raf(short), a truncated protein. Importantly, A-Raf(short) fails to regulate MST2 but retains the Ras-binding domain such that it functions as a dominant negative mutant suppressing Ras activation and transformation. Human colon and head and neck cancers exhibit high hnRNP H and high c-Myc levels resulting in enhanced A-Raf expression and reduced expression of A-Raf(short). Conversely, in normal cells and tissues in which c-Myc and hnRNP H are low, A-Raf(short) suppresses extracellular signal regulated kinase activation such that it may act as a safeguard against oncogenic transformation. Our findings offered a new paradigm to understand how c-Myc coordinates diverse cell functions by directly affecting alternate splicing of key signaling components.

Identification of c-myc-dependent proteins in the medulloblastoma cell line D425Med

High c-myc levels are linked to poor prognosis in medulloblastoma (MB), and it was the aim of the current study to search for c-myc-dependent proteins in the MB cell line D425Med. For this purpose D425Med cells and cells with knocked-down c-myc (by siRNA) were analysed by a gel-based differential proteomics study using mass spectrometry. Heterogeneous nuclear ribonucleoproteins C1/C2, heterogeneous nuclear ribonucleoprotein A/B, stathmin, endoplasmic reticulum protein ERp29 precursor and guanidinoacetate N-methyltransferase were c-myc dependently expressed. Signalling, the protein machinery, metabolism and endoplasmic reticulum function may be affected and these results enable studying tumour tissue for these proteins as potential dignity markers or pharmacological targets.

Abstract 2906: Investigating the role of high c-Myc levels in the resistance to TGFβ-mediated tumor suppression in melanoma

Abstract Resistance to transforming Growth factor (TGF) β-mediated tumor suppression represents a major step in melanoma aggressiveness. At the cell surface, TGF-β assembles a complex of transmembrane receptor serine/threonine kinases (types I and II) and induces transphosphorylation and activation of the type I receptor (TβR-I, ALK5) by the type II receptor kinase (TβR-II). The activated type I receptor phosphorylates the downstream effectors Smad2 and Smad3, which then associate with a common Smad4, and these activated complexes translocate into the nucleus, where they regulate transcription of target genes. Smad2 and Smad3 activities mediate TGFβ growth inhibitory effects by 1) downregulation of c-myc, CDC25A and Id family members; and 2) upregulation of p15 and p21 cyclin-dependent kinase inhibitors. In order to investigate whether high levels of c-myc could play a role in TGFβ resistance, we analyzed c-myc expression in 13 human melanoma cell lines. 6 melanoma cell lines demonstrated high levels of the protein c-myc, a repressor of p21 and p15 expression. Interestingly, these cell lines also had low levels of p21, raising the possibility that high c-myc levels were maintaining low levels of this cyclin-dependent kinase inhibitor in these lines. We were then interested to know whether low levels of Smad2 or/and Smad3 could potentially be responsible for high c-myc levels in the cells. Our preliminary data shows that while all 13 melanoma cell lines had comparable levels of Smad2 expressed, 7 out of the 13 had lower expression of Smad3. Interestingly, 6 of the 7 with low Smad3 expression were the lines with the highest levels of c-myc and the lowest levels of p21. We therefore suggest that these cell lines may have selected for a lower Smad3 expression as a way to render themselves insensitive to TGFβ-mediated growth suppression. In order to directly address the role of high c-myc levels in the resistance to TGFβ-mediated growth inhibition, we are knocking down the expression of c-myc in the resistant melanoma cell lines with high levels of the protein. We are analyzing these cells to determine if they are now less resistant to TGFβ. On the other hand, we are over-expressing c-myc in TGFβ sensitive melanoma cell lines (Radial Growth Phase melanomas) with low levels of c-myc, with the expectation that these melanoma lines will become more resistant to TGFβ. We will correlate these results to the expression levels of p15 and p21. In order to determine whether reduction of Smad3 expression could represent a mechanism leading to high c-myc expression, we will over-express Smad3 in the melanoma cell lines with low Smad3 expression and concomitant high c-myc and low p21 levels and determine whether we can restore sensitivity to TGFβ-mediated growth inhibition. Taken together our results allude to a novel mechanism used by melanoma cells which may involve utilizing c-myc to evade TGFβ mediated suppression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2906. doi:10.1158/1538-7445.AM2011-2906

Suppression of multidrug resistance by treatment with TRAIL in human ovarian and breast cancer cells with high level of c-Myc

In this study, we investigated the role of c-Myc in overcoming multidrug resistance (MDR) in human ovarian and breast cancer cells by TRAIL. We showed that P-gp expressing MDR variants (Hey A8-MDR and MCF7-MDR cells) with high level of c-Myc were highly susceptible to TRAIL treatment when compared to their drug-sensitive parental human ovarian cancer Hey A8 and breast MCF-7 cells, respectively. Up-regulation of DR5 TRAIL receptor and down-regulation of c-FLIP and the promotion of caspase-dependent cell death, which contribute to TRAIL sensitization of MDR cells, were regulated by the over-expressed c-Myc in the MDR cells. After targeted inhibition of c-Myc with specific siRNA, these responses to TRAIL disappeared and TRAIL-induced apoptosis was also suppressed in MCF7-MDR cells. Treatment with TRAIL significantly reduced P-glycoprotein (P-gp)-mediated efflux of rhodamine123 in both Hey A8-MDR and MCF7-MDR cells. Furthermore, TRAIL significantly potentiated the cytotoxicity of vinblastine, vincristine, doxorubicin and VP-16 that are P-gp substrate anticancer drugs in both MDR cells, which resulted in the reversal effect of TRAIL on the MDR phenotype. The present study shows for the first time that elevated c-Myc expression in the MDR cells plays a critical role in overcoming MDR by TRAIL that can act as a specific sensitizer for P-gp substrate anticancer drug.

Abstract 2069: Regulation of nucleostemin by reactive oxygen species

Abstract Nucleostemin (NS) is a nucleolar-nucleoplasmic shuttle protein that is a key regulator of cell proliferation, binds p53 and Mdm2, and is highly expressed in tumor cells and embryonic stem cells. We have identified NS as a previously unreported target of redox regulation in transformed hematopoietic cells. NS oligomerization is present in HL-60 leukemic cells and Raji B lymphoblasts that express high levels of c-Myc and have high intrinsic levels of reactive oxygen species (ROS); exposure of these cells to reducing agents including N-acetylcysteine results in the dissociation of NS into monomers and dimers. On the other hand, exposure of other cell types that have relatively low levels of intrinsic ROS to low levels of H2O2 induces disulfide bond-mediated oligomerization of NS and, with prolonged exposure, immobilizes NS within the nucleolus, preventing its translocation to the nucleoplasm in response to nucleolar stress. Increasing levels of ROS also result in significantly increased NS protein stability. The redox-and oncogene-mediated regulation of NS was validated in a murine lymphoma tumor model in which c-Myc is overexpressed and in CD34+ cells from four patients with chronic myelogenous leukemia in blast crisis in which the BCR-ABL oncogene is expressed. In both instances, increased intrinsic ROS levels were associated with markedly increased expression of NS protein and oligomerization that was reversed by exposure of cells to N-acetylcysteine. These results demonstrate that NS is a highly redox-sensitive protein. Increased intracellular ROS levels, such as those that result from oncogenic transformation in hematopoietic malignancies, increase the oligomerization of NS, enhance its stability and most likely impair its ability to regulate Mdm2-p53 interactions in the nucleus. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2069. doi:10.1158/1538-7445.AM2011-2069

Reactive Oxygen Species Regulate Nucleostemin Oligomerization and Protein Degradation

Nucleostemin (NS) is a nucleolar-nucleoplasmic shuttle protein that regulates cell proliferation, binds p53 and Mdm2, and is highly expressed in tumor cells. We have identified NS as a target of oxidative regulation in transformed hematopoietic cells. NS oligomerization occurs in HL-60 leukemic cells and Raji B lymphoblasts that express high levels of c-Myc and have high intrinsic levels of reactive oxygen species (ROS); reducing agents dissociate NS into monomers and dimers. Exposure of U2OS osteosarcoma cells with low levels of intrinsic ROS to hydrogen peroxide (H(2)O(2)) induces thiol-reversible disulfide bond-mediated oligomerization of NS. Increased exposure to H(2)O(2) impairs NS degradation, immobilizes the protein within the nucleolus, and results in detergent-insoluble NS. The regulation of NS by ROS was validated in a murine lymphoma tumor model in which c-Myc is overexpressed and in CD34+ cells from patients with chronic myelogenous leukemia in blast crisis. In both instances, increased ROS levels were associated with markedly increased expression of NS protein and thiol-reversible oligomerization. Site-directed mutagenesis of critical cysteine-containing regions of nucleostemin altered both its intracellular localization and its stability. MG132, a potent proteasome inhibitor and activator of ROS, markedly decreased degradation and increased nucleolar retention of NS mutants, whereas N-acetyl-L-cysteine largely prevented the effects of MG132. These results indicate that NS is a highly redox-sensitive protein. Increased intracellular ROS levels, such as those that result from oncogenic transformation in hematopoietic malignancies, regulate the ability of NS to oligomerize, prevent its degradation, and may alter its ability to regulate cell proliferation.

C-MYC expression sensitizes medulloblastoma cells to radio- and chemotherapy and has no impact on response in medulloblastoma patients

BackgroundTo study whether and how c-MYC expression determines response to radio- and chemotherapy in childhood medulloblastoma (MB).MethodsWe used DAOY and UW228 human MB cells engineered to stably express different levels of c-MYC, and tested whether c-MYC expression has an effect on radio- and chemosensitivity using the colorimetric 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS) assay, clonogenic survival, apoptosis assays, cell cycle analysis, and western blot assessment. In an effort to validate our results, we analyzed c-MYC mRNA expression in formalin-fixed paraffin-embedded tumor samples from well-documented patients with postoperative residual tumor and compared c-MYC mRNA expression with response to radio- and chemotherapy as examined by neuroradiological imaging.ResultsIn DAOY - and to a lesser extent in UW228 - cells expressing high levels of c-MYC, the cytotoxicity of cisplatin, and etoposide was significantly higher when compared with DAOY/UW228 cells expressing low levels of c-MYC. Irradiation- and chemotherapy-induced apoptotic cell death was enhanced in DAOY cells expressing high levels of c-MYC. The response of 62 of 66 residual tumors was evaluable and response to postoperative radio- (14 responders (CR, PR) vs. 5 non-responders (SD, PD)) or chemotherapy (23 CR/PR vs. 20 SD/PD) was assessed. c-MYC mRNA expression was similar in primary MB samples of responders and non-responders (Mann-Whitney U test, p = 0.50, ratio 0.49, 95% CI 0.008-30.0 and p = 0.67, ratio 1.8, 95% CI 0.14-23.5, respectively).Conclusionsc-MYC sensitizes MB cells to some anti-cancer treatments in vitro. As we failed to show evidence for such an effect on postoperative residual tumors when analyzed by imaging, additional investigations in xenografts and larger MB cohorts may help to define the exact function of c-MYC in modulating response to treatment.

Viral-Mediated Noisy Gene Expression Reveals Biphasic E2f1 Response to MYC

Gene expression mediated by viral vectors is subject to cell-to-cell variability, which limits the accuracy of gene delivery. When coupled with single-cell measurements, however, such variability provides an efficient means to quantify signaling dynamics in mammalian cells. Here, we illustrate the utility ofthis approach by mapping the E2f1 response to MYC, serum stimulation, or both. Our results revealed an underappreciated mode of gene regulation: E2f1 expression first increased, then decreased as MYC input increased. This biphasic pattern was also reflected in other nodes of the network, including the miR-17-92 microRNA cluster and p19Arf. A mathematical model of the network successfully predicted modulation of the biphasic E2F response byserum and a CDK inhibitor. In addition to demonstrating how noise can be exploited to probe signaling dynamics, our results reveal how coordination of the MYC/RB/E2F pathway enables dynamic discrimination of aberrant and normal levels of growth stimulation.

High susceptibility of metastatic cells derived from human prostate and colon cancer cells to TRAIL and sensitization of TRAIL-insensitive primary cells to TRAIL by 4,5-dimethoxy-2-nitrobenzaldehyde

BackgroundTumor recurrence and metastasis develop as a result of tumors' acquisition of anti-apoptotic mechanisms and therefore, it is necessary to develop novel effective therapeutics against metastatic cancers. In this study, we showed the differential TRAIL responsiveness of human prostate adenocarcinoma PC3 and human colon carcinoma KM12 cells and their respective highly metastatic PC3-MM2 and KM12L4A sublines and investigated the mechanism underlying high susceptibility of human metastatic cancer cells to TRAIL.ResultsPC3-MM2 and KM12L4A cells with high level of c-Myc and DNA-PKcs were more susceptible to TRAIL than their poorly metastatic primary PC3 and KM12 cells, which was associated with down-regulation of c-FLIPL/S and Mcl-1 and up-regulation of the TRAIL receptor DR5 but not DR4 in both metastatic cells. Moreover, high susceptibility of these metastatic cells to TRAIL was resulted from TRAIL-induced potent activation of caspase-8, -9, and -3 in comparison with their primary cells, which led to cleavage and down-regulation of DNA-PKcs. Knockdown of c-Myc gene in TRAIL-treated PC3-MM2 cells prevented the increase of DR5 cell surface expression, caspase activation and DNA-PKcs cleavage and attenuated the apoptotic effects of TRAIL. Moreover, the suppression of DNA-PKcs level with siRNA in the cells induced the up-regulation of DR5 and active caspase-8, -9, and -3. We also found that 4,5-dimethoxy-2-nitrobenzaldehyde (DMNB), a specific inhibitor of DNA-PK, potentiated TRAIL-induced cytotoxicity and apoptosis in relatively TRAIL-insensitive PC3 and KM12 cells and therefore functioned as a TRAIL sensitizer.ConclusionThis study showed the positive relationship between c-Myc expression in highly metastatic human prostate and colon cancer cells and susceptibility to TRAIL-induced apoptosis and therefore indicated that TRAIL might be used as an effective therapeutic modality for advanced metastatic cancers overexpressing c-Myc and combination of TRAIL therapy with agent that inhibits the DNA-PKcs/Akt signaling pathway might be clinically useful for the treatment of relatively TRAIL-insensitive human cancers.

Abstract 3314: Side population in renal cell carcinoma cell line is enriched with drug resistant, but not tumor initiating cells

Abstract Several lines of evidences suggested the presence of tumor initiating cells in renal cell carcinoma (RCC) and side population (SP) may be enriched with these “cancer stem cells (CSC)”. Herein we demonstrated the presence of Side population in 10 RCC cell lines ranging from 0.19 - 36.1%. Lineage tracing experiments showed that the SP can be enriched through sequential sorting and can differentiate into non-SP (NSP) which constitutes the bulk of the cells. The NSP can also formed back the original population of the SP in CRL1611 cells. Interestingly, the presence of NSP seem to prevent the SP from differentiating into NSP. The SP cells have about 15% higher proliferation rate and 50% greater colony forming efficiency which are reported characteristics of CSC. SP expresses higher level of cMyc, but lower level of CD105 as compared to the NSP. On the contrary, there is no difference in tumorigenic potential between SP and NSP using both correlation and in vivo studies. However, we verified that SP are 10% more resistance to mTOR inhibitor, RAD001 which coincide with higher expression of drug resistance membrane transporters ABCB1 and ABCG2 in SP cells. Treatment with RAD001 increases the amount of SP concomitantly up regulate ABCB1 and ABCC1 significantly. Taken together, Hoechst dye exclusion did not define tumor initiating cells, but is a valuable technique for identifying drug resistance cells in RCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3314.

Dihydroartemisinin accelerates c-MYC oncoprotein degradation and induces apoptosis in c-MYC-overexpressing tumor cells

Artemisinin and its derivatives (ARTs) are effective antimalarial drugs and also possess profound anticancer activity. However, the mechanism accounted for its distinctive activity in tumor cells remains unelucidated. We computed Pair wise Pearson correlation coefficients to identify genes that show significant correlation with ARTs activity in NCI-55 cell lines using data obtained from studies with HG-U133A Affymetrix chip. We found c-myc is one of the genes that showed the highest positive correlation coefficients among the probe sets analyzed ( r = 0.585, P < 0.001). Dihydroartemisinin (DHA), the main active metabolite of ARTs, induced significant apoptosis in HL-60 and HCT116 cells that express high levels of c-MYC. Stable knockdown of c-myc abrogated DHA-induced apoptosis in HCT116 cells. Conversely, forced expression of c-myc in NIH3T3 cells sensitized these cells to DHA-induced apoptosis. Interestingly, DHA irreversibly down-regulated the protein level of c-MYC in DHA-sensitive HCT116 cells, which is consistent to persistent G1 phase arrest induced by DHA. Further studies demonstrated that DHA accelerated the degradation of c-MYC protein and this process was blocked by pretreatment with the proteasome inhibitor MG-132 or GSK 3β inhibitor LiCl in HCT116 cells. Taken together, ARTs might be useful in the treatment of c-MYC-overexpressing tumors. We also suggest that c-MYC may potentially be a biomarker candidate for prediction of the antitumor efficacies of ARTs.

B Cell Activating Factor and c-Myc Regulate the Progression of Chronic Lymphocytic Leukemia.

Abstract 359▪▪This icon denotes an abstract that is clinically relevant.Chromosomal translocations involving c-Myc are frequently found in high grade lymphoma and multiple myeloma. In contrast, c-Myc translocations rarely occur in low-grade lymphomas/leukemias like chronic lymphocytic leukemia (CLL), but when present they are associated with rapid disease progression and bad prognosis. Overexpression of c-myc may also be the result of increased transcription by several proto-oncogene transcription factors, including NF-kB. Mice with c-Myc de-regulation at different stages of B cell development develop either aggressive B cells lymphomas or plasma cell neoplasm. So far, no c-Myc mouse model developed low-grade lymphoma/leukemia. iMycCa mice develop an expansion of CD5+ peritoneal B1 cells, as compared with WT littermates mice. These mice have a normal life-span and very rarely develop B cell lymphoma at older age. Interestingly, in iMycCa mice mature B cells, but not plasma cells,could be rescued from apoptosis by administration of B cell-activating factor belonging to the TNF family (BAFF). To our surprise, double transgenic iMycCa/Baff-Tg (Myc/Baff) mice developed a disease resembling human CLL, with dramatically shorter mean survival than parental strains, due to early onset and rapid clonal expansion of a mature CD5+B220low B cell population. Those cells transferred the disease into Baff-Tg (Baff) mice with marked infiltration in lymphoid organs and bone marrow. Gene-expression analyses revealed that among the genes altered in Myc/Baff CD5+B220lowleukemia cells were those with known relevance to human CLL disease, including elevated anti-apoptotic Bcl2 family members. Apart from studies on individual genes, sub-network analysis was performed which showed enrichment of apoptosis-related and stress-induced gene sets in Myc/Baff CD5+CD3- leukemia cells. The NF-kB gene set, a major target downstream of BAFF signaling, was also enriched in Myc/Baff CD5+CD3- leukemia cells. We observed a continuum in levels of c-MYC mRNA in 166 samples using Affymetrix array analyses. Changes in c-Myc protein expression were confirmed by immunoblot analyses and correlated with disease progression. In accordance with the functions of c-Myc as a promoter of cell cycle progression, as well as apoptosis, we found enhanced spontaneous cell death in vitro in CLL cells expressing high levels of c-Myc, which could be abrogated by co culture with BAFF expressing nurse-like cells (NLC) or recombinant BAFF. In addition to its anti-apoptotic role, BAFF treatment of primary human CLL cells led to dramatically enhanced expression of c-Myc through the IKK/NF-kB pathway. Inhibition of the NF-kB pathway significantly reduced viability of both Myc/Baff CD5+CD3- leukemia cells and human CLL cells co-cultured with NLC. Also it significantly lowered CD5+B220low leukemia cell population in blood and spleen, and prevented the infiltration of leukemia cells into lymph nodes and bone marrow of transplanted mice. This study demonstrates a potential pathologic role for c-Myc, in the pathogenesis and progression of CLL. Disclosures:No relevant conflicts of interest to declare.

Abstract 5631: Isolation of a Resident and Dynamic Oct4+ Stem Cell Population From Adult Skin

The transcription factor Oct3/4 is a critical regulator of pluripotency in embryonic stem cells and induced pluripotent stem (iPS) cells and iPS generation requires re-activation of endogenous Oct4 expression. Since Oct4 might be a critical regulator of somatic stem cell pluripotency we have employed a genetic, bona-fide reporter strategy to determine the temporo-spatial expression of Oct4 in adulte mice. RESULTS: Postnatal Oct4 expression was determined in transgenic Oct4::eGFP reporter mice by flow cytometry, for hearts and skin the response to injury was also evaluated (n=9 –28). The fidelity of the Oct4-GFP reporter strain was confirmed by immunofluorescence confocal microscopy and by RT-PCR. No GFP+ cells were detected in bone marrow or spleen, nor in the atria or ventricles of the myocardium under baseline and post-ischemic conditions. Importantly, Oct4+ cells resided in skin, where levels increased postnatally over time until puberty and returned to low levels in the adult (Oct4+/500.000 cells: puberty 400±268; adult 53±23). Furthermore, the Oct4+ cell pool increased robustly after wound injury (289±156, n=9, p&lt;0.05) even in remote skin areas. In situ analysis revealed the existence of GFP+ niches associated with distinct regions of hair follicle. Oct4+ cells after FACS-sorting revealed a phenotype of c-kit lo, sca-1 +, CXCR4 lo, alpha6 integrin lo, beta1 integrin -, and expressed by qRT low levels of Oct4, Sox2, Klf4, and high levels of c-myc, factors involved in iPS induction. The differentiation potential of Oct4+ skin cells was evaluated by transplanting dual reporter cells (GFP+; lacZ+) into nude mice following hindlimb ischemia and analysis of contribution to tissue lineages. 4 weeks after transplantation, LacZ+ cells contributed to muscle cells, adipose tissue and vascular structures. CONCLUSION: Genetic reporter studies reveal no evidence for Oct4+ cells in bone marrow or heart but, unexpectedly, show persistence of resident Oct4+ stem cells in adult skin, which contribute to mesodermal cell lineage after transplantation. These findings suggest adult skin as an important source for regenerative cell therapy.

RNA interference-mediated c-MYC inhibition prevents cell growth and decreases sensitivity to radio- and chemotherapy in childhood medulloblastoma cells

BackgroundWith current treatment strategies, nearly half of all medulloblastoma (MB) patients die from progressive tumors. Accordingly, the identification of novel therapeutic strategies remains a major goal. Deregulation of c-MYC is evident in numerous human cancers. In MB, over-expression of c-MYC has been shown to cause anaplasia and correlate with unfavorable prognosis.MethodsTo study the role of c-MYC in MB biology, we down-regulated c-MYC expression by using small interfering RNA (siRNA) and investigated changes in cellular proliferation, cell cycle analysis, apoptosis, telomere maintenance, and response to ionizing radiation (IR) and chemotherapeutics in a representative panel of human MB cell lines expressing different levels of c-MYC (DAOY wild-type, DAOY transfected with the empty vector, DAOY transfected with c-MYC, D341, and D425).ResultssiRNA-mediated c-MYC down-regulation resulted in an inhibition of cellular proliferation and clonogenic growth, inhibition of G1-S phase cell cycle progression, and a decrease in human telomerase reverse transcriptase (hTERT) expression and telomerase activity. On the other hand, down-regulation of c-MYC reduced apoptosis and decreased the sensitivity of human MB cells to IR, cisplatin, and etoposide. This effect was more pronounced in DAOY cells expressing high levels of c-MYC when compared with DAOY wild-type or DAOY cells transfected with the empty vector.ConclusionIn human MB cells, in addition to its roles in growth and proliferation, c-MYC is also a potent inducer of apoptosis. Therefore, targeting c-MYC might be of therapeutic benefit when used sequentially with chemo- and radiotherapy rather than concomitantly.

C-myc suppressor FBP-interacting repressor for cancer diagnosis and therapy

Based on the genetic background of cancer, we have been trying to develop novel diagnostic and therapeutic strategies against human cancers. c-myc gene activation has been detected in many human cancers, indicating a key role of c-myc in tumor development. Thus targeting c-myc gene suppression is a promising strategy for cancer treatment. Recently, an interaction between FIR (FUSE-Binding Protein-Interacting Repressor) and TFIIH/p89/XPB helicase was found to repress c-myc transcription and so might be important for suppressing tumor formation. Previously, we have shown that the expression of splicing variant of FIR is elevated in colorectal cancer tissues and promotes tumor development by disabling FIR-repression to sustain high levels of c-Myc, opposing apoptosis in cancer cells. In this study, FIR recombinant adenovirus vector induces tumor growth suppression against tumor xenografts in animal model experiment. Together, one clue to the development of cancer diagnosis and therapies directed against c-Myc may go through FIR and its splicing variant.

Epigenetic Down-Regulation of ARF Expression Is a Selection Step in Immortalization of Human Fibroblasts by c-Myc

The transcription factor c-Myc is implicated in the pathogenesis of many cancers. Among the multiple functions of c-Myc, activation of hTert and other genes involved in cellular life span contributes to its role as an oncogene. However, the ability of c-Myc to directly immortalize human cells remains controversial. We show here that overexpression of c-Myc reproducibly immortalizes freshly isolated human foreskin fibroblasts. c-Myc-immortalized cells displayed no gross karyotypic abnormalities but consisted of an oligoclonal population, suggesting that additional events cooperated to achieve immortalization. Levels of p53 and p16 were increased, but both p53-dependent DNA damage response and growth arrest in response to p16 overexpression remained intact. A marked decrease in expression of the tumor suppressor ARF occurred in several independently established c-Myc-immortalized cell lines. Methylation-specific PCR showed that the ARF gene was methylated in immortalized but not early-passage c-Myc cells, whereas p16 was unmethylated in both cell populations. Restoration of ARF expression by treatment with a demethylating agent or overexpression by a retroviral vector coincided with inhibition of proliferation and senescence of c-Myc-immortalized cells. Our findings predict that epigenetic events play a significant role in human tumors that express high levels of c-Myc.

C-MYC Correlates with Expression of Multidrug Transporters ABCC1 and ABCC4.

C-MYC is an oncogene with protean effects and there is some speculation that aberrant overexpression in cancer may be associated with drug resistance by increased expression of the Multi-Drug Resistance Transporters, for example ABCC4 (MRP4) and ABCC1 (MRP1). A relationship between N-MYC and these transporters has been established in the limited context of childhood neuroblastoma and it would be tantalising to extrapolate this to the wider scope of adult tumours. The corollory would be that modulating the levels of C-MYC might have a beneficial secondary effect of making a tumour more sensitive to chemotherapeutic treatments. We have determined that the ABCC4 promoter directly responds in vitro to C-MYC and N-MYC contransfection and that this effect can be abolished by the progressive disruption of the C-MYC binding sites i.e. mutation of the E-boxes. Transient or stable overexpression of C-MYC or N-MYC did not increase multidrug transporter protein expression in 5 tumour cell lines, possibly due to the methylation status of the E-boxes or that the transporters present are already maximally expressed. However, knocking-down C-MYC by shRNA results in a concomitant reduction in ABCC1 and ABCC4. Together, these results suggest both direct and indirect regulation may be important in the functional control of these transporters. A survey was performed of Chronic Myeloid Leukaemias (CML, n=26) and Myelomas (n=23), assessing the expression of C-MYC, ABCC4 and ABCC1 by quantitative RT-PCR. The genes of interest were normalised to housekeeping genes and linear regression applied to examine the relationship of C-MYC levels to transporter expression. In CML, a direct relationship was seen between C-MYC and both ABCC4 and ABCC1. In Myeloma, a correlation was noted between C-MYC and ABCC1 but was not seen with ABCC4, as the levels detected were very low. The inference is that high levels of C-MYC may be associated with elevated multidrug transporter levels in other tumours and may be of use in predicting transporter-mediated drug resistance.

Plakoglobin‐dependent disruption of the desmosomal plaque in pemphigus vulgaris

We recently reported that the pathogenesis of pemphigus vulgaris (PV), an autoimmune blistering skin disorder, is driven by the accumulation of c-Myc secondary to abrogation of plakoglobin (PG)-mediated transcriptional c-Myc suppression. PG knock-out mouse keratinocytes express high levels of c-Myc and resemble PVIgG-treated wild-type keratinocytes in most respects. However, they fail to accumulate nuclear c-Myc and loose intercellular adhesion in response to PVIgG-treatment like wild-type keratinocytes. This suggested that PG is also required for propagation of the PVIgG-induced events between augmented c-Myc expression and acantholysis. Here, we addressed this possibility by comparing PVIgG-induced changes in the desmosomal organization between wild-type and PG knock-out keratinocytes. We found that either bivalent PVIgG or monovalent PV-Fab (known to trigger blister formation in vivo) disrupt the linear organization of all major desmosomal components along cell borders in wild-type keratinocytes, simultaneously with a reduction in intercellular adhesive strength. In contrast, PV-Fab failed to affect PG knock-out keratinocytes while PVIgG cross-linked their desmosomal cadherins without significantly affecting desmoplakin. These results identify PG as a principle effector of the PVIgG-induced signals downstream of c-Myc that disrupt the desmosomal plaque at the plasma membrane.

Role for Brm in Cell Growth Control

Recently, we have shown implication of Brm, the catalytic subunit of the SWI/SNF chromatin remodeling complex, in repression of cyclin A expression in quiescent cells. Here, we have examined the fate of cells lacking Brm throughout the cycle. We find that despite elevated levels of cyclins A and E, these cells can respond to serum starvation, however, without reaching a canonical G(0) phase as they continue to express high levels of c-Myc and have an abnormally large average size. The response to serum starvation can be correlated with increased levels of Rb proteins p130 and p107 as well as increased association of p27 with the cyclin-dependent kinases, possibly compensating for the higher levels of G(1) cyclins by reducing their associated kinase activity. After serum stimulation, reentry into the cycle occurs normally, but the S phase is delayed and shorter. In addition, the M phase has an increased duration, and we observed frequent faulty chromosome segregation events in anaphase. Altogether, our data suggest that cells can partially overcome the absence of Brm by activating several compensatory mechanisms to control the cell cycle. However, they remain profoundly affected, unable to enter a canonical quiescent state, presenting a shorter S phase, and finally unable to perform correct chromosome segregation.

An Essential Role of Alternative Splicing of c-myc Suppressor FUSE-Binding Protein–Interacting Repressor in Carcinogenesis

Elevated expression of c-myc has been detected in a broad range of human cancers, indicating a key role for this oncogene in tumor development. Recently, an interaction between FUSE-binding protein-interacting repressor (FIR) and TFIIH/p89/XPB helicase was found to repress c-myc transcription and might be important for suppressing tumor formation. In this study, we showed that enforced expression of FIR induced apoptosis. Deletion of the NH(2)-terminal repression domain of FIR rescued the cells from apoptosis as did coexpression of c-Myc with FIR; thus, repression of Myc mediates FIR-driven apoptosis. Surprisingly, a splicing variant of FIR unable to repress c-myc or to drive apoptosis was frequently discovered in human primary colorectal cancers but not in the adjacent normal tissues. Coexpression of this splicing variant with repressor-competent FIR, either in HeLa cells or in the colon cancer cell line SW480, not only abrogated c-Myc suppression but also inhibited apoptosis. These results strongly suggest the expression of this splicing variant promotes tumor development by disabling FIR repression and sustaining high levels of c-Myc and opposing apoptosis in colorectal cancer.