Resistance to chemotherapeutic agents remains one of the major impediments to a successful treatment of chronic myeloid leukemia (CML). Misregulation of the activity of a specific group of ...ATP-binding cassette transporters (ABC) is responsible for reducing the intracellular concentration of drugs in leukemic cells. Moreover, a consistent body of evidence also suggests that ABC transporters play a role in cancer progression beyond the efflux of cytotoxic drugs. Despite a large number of studies that investigated the function of the ABC transporters, little is known about the transcriptional regulation of the ABC genes. Here, we present data showing that the oncoprotein c-MYC is a direct transcriptional regulator of a large set of ABC transporters in CML. Furthermore, molecular analysis carried out in CD34+ hematopoietic cell precursors of 21 CML patients reveals that the overexpression of ABC transporters driven by c-MYC is a peculiar characteristic of the CD34+ population in CML and was not found either in the population of mononuclear cells from which they had been purified nor in CD34+ cells isolated from healthy donors. Finally, we describe how the methylation state of CpG islands may regulate the access of c-MYC to ABCG2 gene promoter, a well-studied gene associated with multidrug resistance in CML, hence, affecting its expression. Taken together, our findings support a model in which c-MYC-driven transcriptional events, combined with epigenetic mechanisms, direct and regulate the expression of ABC genes with possible implications in tumor malignancy and drug efflux in CML.
Histone deacetylase (HDAC) inhibitors reactivate tumor suppressor gene transcription; induce cancer cell differentiation, growth arrest, and programmed cell death; and are among the most promising ...new classes of anticancer drugs. Myc oncoproteins can block cell differentiation and promote cell proliferation and malignant transformation, in some cases by modulating target gene transcription. Here, we show that tissue transglutaminase (TG2) was commonly reactivated by HDAC inhibitors in neuroblastoma and breast cancer cells but not normal cells and contributed to HDAC inhibitor-induced growth arrest. TG2 was the gene most significantly repressed by N-Myc in neuroblastoma cells in a cDNA microarray analysis and was commonly repressed by N-Myc in neuroblastoma cells and c-Myc in breast cancer cells. Repression of TG2 expression by N-Myc in neuroblastoma cells was necessary for the inhibitory effect of N-Myc on neuroblastoma cell differentiation. Dual step cross-linking chromatin immunoprecipitation and protein coimmunoprecipitation assays showed that N-Myc acted as a transrepressor by recruiting the HDAC1 protein to an Sp1-binding site in the TG2 core promoter in a manner distinct from it's action as a transactivator at E-Box binding sites. HDAC inhibitor treatment blocked the N-Myc-mediated HDAC1 recruitment and TG2 repression in vitro. In neuroblastoma-bearing N-Myc transgenic mice, HDAC inhibitor treatment induced TG2 expression and demonstrated marked antitumor activity in vivo. Taken together, our data indicate the critical roles of HDAC1 and TG2 in Myc-induced oncogenesis and have significant implications for the use of HDAC inhibitor therapy in Myc-driven oncogenesis.
CENP-C is a fundamental component of functional centromeres. The elucidation of its structure-function relationship with centromeric DNA and other kinetochore proteins is critical to the ...understanding of centromere assembly. CENP-C carries two regions, the central and the C-terminal domains, both of which are important for the ability of CENP-C to associate with the centromeric DNA. However, while the central region is largely divergent in CENP-C homologues, the C-terminal moiety contains two regions that are highly conserved from yeast to humans, named Mif2p homology domains (blocks II and III). The activity of these two domains in human CENP-C is not well defined. In this study we performed a functional dissection of C-terminal CENP-C region analyzing the role of single Mif2p homology domains through in vivo and in vitro assays. By immunofluorescence and Chromatin immunoprecipitation assay (ChIP) we were able to elucidate the ability of the Mif2p homology domain II to target centromere and contact alpha satellite DNA. We also investigate the interactions with other conserved inner kinetochore proteins by means of coimmunoprecipitation and bimolecular fluorescence complementation on cell nuclei. We found that the C-terminal region of CENP-C (Mif2p homology domain III) displays multiple activities ranging from the ability to form higher order structures like homo-dimers and homo-oligomers, to mediate interaction with CENP-A and histone H3. Overall, our findings support a model in which the Mif2p homology domains of CENP-C, by virtue of their ability to establish multiple contacts with DNA and centromere proteins, play a critical role in the structuring of kinethocore chromatin.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract
The MYC family of transcription factors is a major driver of human cancer and potential therapeutic target. However, no clinically viable drugs have been yet developed that are able to ...directly tackle MYC oncoproteins. In our laboratory, we are exploring alternative approaches aiming to disturb signalling downstream of MYC. MYCN is frequently activated in neuroblastoma, a paediatric solid malignancy that, in its metastatic form, has a very poor prognosis. An important pathway regulated by MYC is the CKS1/SKP2/p27
kip1
axis. In this study, we have repurposed the anti-psychotic drug Prozac to disrupt CKS1/SKP2/p27
Kip1
signalling and assess its potential as an anti-neuroblastoma agent in vitro and in vivo. Using DNA editing technology, we show that stabilisation of p27
Kip1
operated by Prozac in MYC-activated cells is essential for the anti-neuroblastoma activity of the drug. Furthermore, dosing mice with a concentration of Prozac equivalent to that used in long-term clinical trials in children with psychiatric disorders caused a significant reduction of metastatic disease in two models of high-risk neuroblastoma. The favourable toxicity profile of Prozac suggests that long-term treatments might be implemented in children with MYC/CKS1
high
neuroblastomas.
Research over the past decade has confirmed that epigenetic alterations act in concert with genetic lesions to deregulate gene expression in acute myeloid leukemia and myelodysplastic syndromes. ...Epigenetic alterations may serve as markers of disease, and may potentially be used for classification, prognostication and to monitor minimal residual disease. In addition, we now have the capability to pharmaceutically target epigenetic modifications, and there is an urgent need for early validation of the efficacy of the drugs. Also, an improved understanding of the functionality of epigenetic modifications may further pave the road towards individualized therapy. The recent advances in biotechnology and bioinformatics provide a plethora of novel tools for characterizing the epigenome in clinical samples, but at this point the practical, clinical utility of these methodologies needs further exploration. Here, we provide the pros and cons of the currently most feasible methods used for characterizing the methylome in clinical samples, and give a brief introduction to novel approaches to sequencing that may revolutionize our abilities to characterize the genomes and epigenomes in acute myeloid leukemia and myelodysplastic syndrome patients.
CLU (clusterin) is a tumor suppressor gene that we have previously shown to be negatively modulated by the MYCN proto-oncogene, but the mechanism of repression was unclear. Here, we show that MYCN ...inhibits the expression of CLU by direct interaction with the non-canonical E box sequence CACGCG in the 5′-flanking region. Binding of MYCN to the CLU gene induces bivalent epigenetic marks and recruitment of repressive proteins such as histone deacetylases and Polycomb members. MYCN physically binds in vitro and in vivo to EZH2, a component of the Polycomb repressive complex 2, required to repress CLU. Notably, EZH2 interacts with the Myc box domain 3, a segment of MYC known to be essential for its transforming effects. The expression of CLU can be restored in MYCN-amplified cells by epigenetic drugs with therapeutic results. Importantly, the anticancer effects of the drugs are ablated if CLU expression is blunted by RNA interference. Our study implies that MYC tumorigenesis can be effectively antagonized by epigenetic drugs that interfere with the recruitment of chromatin modifiers at repressive E boxes of tumor suppressor genes such as CLU.
Background: The neuroblastoma oncogene MYCN and the PRC2 members EZH2 and SUZ12 are regulators of gene transcription.
Results: MYCN and PRC2 form a repressive complex on the promoter of the tumor suppressor gene CLU.
Conclusion: PRC2 members are recruited by MYCN to repress gene expression and induce tumorigenesis.
Significance: Reactivation of MYCN-PRC2-repressed genes by epigenetic drugs could be of clinical value in neuroblastoma.
Nitric oxide (NO) exerts its function in several cell and organ compartments. Recently, several lines of evidence have been accrued showing that NO can play a critical role in oncogenesis. Here we ...summarize some of these findings and highlight the role of NO as a possible target for antineoplastic drugs. Specifically, NO appears to affect some aspects of neuronal tumour progression, particularly the chemoresistance phenotype, through inhibition of MYC activity and expression of a large set of ATP binding cassette transporters. Here we provide lines of evidence supporting the view that MYCN can alter expression of several members of the ABC transporter family thus influencing the chemoresistance phenotype of neuroblastoma cells. Furthermore, we show that increased intracellular NO concentration either through addition of NO donors to culture medium or through forced expression of nNOS in neuroblastoma cells leads to decreased expression of MYCN and ABC drug transporter genes. Overall, data reviewed here and novel results presented, unveil a NO-MYCN-ABC transporters axis with important implication on development and control of the chemoresistance phenotype in neuronal tumours.
The transcription factor cAMP-response element-binding protein (CREB) mediates survival in many cells, including neurons. Recently, death of cerebellar granule neurons due to nitric oxide (NO) ...deprivation was shown to be accompanied by down-regulation of CREB activity (1). We now provide evidence that overproduction of endogenous NO or supplementation with exogenous NO renders SK-N-BE human neuroblastoma cells more resistant to apoptosis induced by serum deprivation. Parental cells underwent apoptosis after 24 h of serum deprivation, an outcome largely absent in clones overexpressing human neuronal nitric oxide synthase (nNOS). This protective effect was reversed by the inhibition of NOS itself or soluble guanylyl cyclase, pointing at cGMP as an intermediate effector of NO-mediated rescue. A slow-releasing NO donor protected parental cells to a significant extent, thus confirming the survival effect of NO. The impaired viability of serum-deprived parental cells was accompanied by a strong decrease of CREB phosphorylation and transcriptional activity, effects significantly attenuated in nNOS-overexpressing clones. To confirm the role of CREB in survival, the ectopic expression of CREB and/or protein kinase A largely counteracted serum deprivation-induced cell death of SK-N-BE cells, whereas transfection with a CREB negative mutant was ineffective. These experiments indicate that CREB activity is an important step for NO-mediated survival in neuronal cells.
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