Existence of mutations in the homeodomain-encoding region of NKX2.5 gene in Iranian patients with tetralogy of Fallot.

BACKGROUND: Tetralogy of Fallot (TOF), the most common cyanotic heart defect and one of the most common congenital heart diseases, occurs mostly sporadically and nonsyndromically. The underlying molecular genetic mechanism is not known. Therefore, the existence of mutations in the homeodomain-encoding region of NKX2.5 gene in Iranian patients with tetralogy of Fallot is evaluated. MATERIALS AND METHODS: In the present study, we analyzed the peripheral blood samples of27 patients in order to find any mutation in the 180 bp homeodomain-encoding region of NKX2.5 gene, which is known to be involved in heart development and diseases. DNA was extracted and all the samples were amplified by polymerase chain reaction (PCR) and sequenced. RESULTS: Twenty-seven patients were included in the study. Twenty-five of them were infants and children (6 days to 11 years of age), one was a teenager (14-years of age), and another was a 33-year-old man [mean ± standard deviation (SD): 5.80 ± 3.90 years]. Thirteen patents were males (mean ± SD: 6.587077 ± 5.02 years) and 14 were females (mean ± SD: 5.0726 ± 2.81 years). One synonymous variant, i.e., c.543G>A was identified in one patient. CONCLUSION: Mutations in the homeodomain-encoding region of NKX2.5 gene may not have an outstanding role in etiology of tetralogy of Fallot patients in Iran.

Dysregulation of the mevalonate pathway promotes transformation.

The importance of cancer metabolism has been appreciated for many years, but the intricacies of how metabolic pathways interconnect with oncogenic signaling are not fully understood. With a clear understanding of how metabolism contributes to tumorigenesis, we will be better able to integrate the targeting of these fundamental biochemical pathways into patient care. The mevalonate (MVA) pathway, paced by its rate-limiting enzyme, hydroxymethylglutaryl coenzyme A reductase (HMGCR), is required for the generation of several fundamental end-products including cholesterol and isoprenoids. Despite years of extensive research from the perspective of cardiovascular disease, the contribution of a dysregulated MVA pathway to human cancer remains largely unexplored. We address this issue directly by showing that dysregulation of the MVA pathway, achieved by ectopic expression of either full-length HMGCR or its novel splice variant, promotes transformation. Ectopic HMGCR accentuates growth of transformed and nontransformed cells under anchorage-independent conditions or as xenografts in immunocompromised mice and, importantly, cooperates with RAS to drive the transformation of primary mouse embryonic fibroblasts cells. We further explore whether the MVA pathway may play a role in the etiology of human cancers and show that high mRNA levels of HMGCR and additional MVA pathway genes correlate with poor prognosis in a meta-analysis of six microarray datasets of primary breast cancer. Taken together, our results suggest that HMGCR is a candidate metabolic oncogene and provide a molecular rationale for further exploring the statin family of HMGCR inhibitors as anticancer agents.

Optimization of experimental design parameters for high-throughput chromatin immunoprecipitation studies.

High-throughput, microarray-based chromatin immunoprecipitation (ChIP-chip) technology allows in vivo elucidation of transcriptional networks. However this complex is not yet readily accessible, in part because its many parameters have not been systematically evaluated and optimized. We address this gap by systematically assessing experimental-design parameters including antibody purity, dye-bias, array-batch, inter-day hybridization bias, amplification method and choice of hybridization control. The combined performance of these optimized parameters shows a 90% validation rate in ChIP-chip analysis of Myc genomic binding in HL60 cells using two different microarray platforms. Increased sensitivity and decreased noise in ChIP-chip assays will enable wider use of this methodology to accurately and affordably elucidate transcriptional networks.

The c-Myc oncogene directly induces the H19 noncoding RNA by allele-specific binding to potentiate tumorigenesis.

The product of the MYC oncogene is widely deregulated in cancer and functions as a regulator of gene transcription. Despite an extensive profile of regulated genes, the transcriptional targets of c-Myc essential for transformation remain unclear. In this study, we show that c-Myc significantly induces the expression of the H19 noncoding RNA in diverse cell types, including breast epithelial, glioblastoma, and fibroblast cells. c-Myc binds to evolutionarily conserved E-boxes near the imprinting control region to facilitate histone acetylation and transcriptional initiation of the H19 promoter. In addition, c-Myc down-regulates the expression of insulin-like growth factor 2 (IGF2), the reciprocally imprinted gene at the H19/IGF2 locus. We show that c-Myc regulates these two genes independently and does not affect H19 imprinting. Indeed, allele-specific chromatin immunoprecipitation and expression analyses indicate that c-Myc binds and drives the expression of only the maternal H19 allele. The role of H19 in transformation is addressed using a knockdown approach and shows that down-regulation of H19 significantly decreases breast and lung cancer cell clonogenicity and anchorage-independent growth. In addition, c-Myc and H19 expression shows strong association in primary breast and lung carcinomas. This work indicates that c-Myc induction of the H19 gene product holds an important role in transformation.

Determinants of sensitivity to lovastatin-induced apoptosis in multiple myeloma.

Statins, commonly used to treat hypercholesterolemia, have been shown to trigger tumor-specific apoptosis in certain cancers, including multiple myeloma (MM), a plasma cell malignancy with poor prognosis. In this article, we show that of a panel of 17 genetically distinct MM cell lines, half were sensitive to statin-induced apoptosis and, despite pharmacodynamic evidence of drug uptake and activity, the remainder were insensitive. Sensitive cells were rescued from lovastatin-induced apoptosis by mevalonate, geranylgeranyl PPi, and partially by farnesyl PPi, highlighting the importance of isoprenylation. Expression profiling revealed that Rho GTPase mRNAs were differentially expressed upon lovastatin exposure in sensitive cells, yet ectopic expression of constitutively active Rho or Ras proteins was insufficient to alter sensitivity to lovastatin-induced apoptosis. This suggests that sensitivity involves more than one isoprenylated protein and that statins trigger apoptosis by blocking many signaling cascades, directly or indirectly deregulated by the oncogenic lesions of the tumor cell. Indeed, clustering on the basis of genetic abnormalities was shown to be significantly associated with sensitivity (P = 0.003). These results suggest that statins may be a useful molecular targeted therapy in the treatment of a subset of MM.

Analysis of Myc bound loci identified by CpG island arrays shows that Max is essential for Myc-dependent repression.

The c-myc proto-oncogene encodes a transcription factor, c-Myc, which is deregulated and/or overexpressed in many human cancers. Despite c-Myc's importance, the identity of Myc-regulated genes and the mechanism by which Myc regulates these genes remain unclear. By combining chromatin immunoprecipitation with CpG island arrays, we identified 177 human genomic loci that are bound by Myc in vivo. Analyzing a cohort of known and novel Myc target genes showed that Myc-associated protein X, Max, also bound to these regulatory regions. Indeed, Max is bound to these loci in the presence or absence of Myc. The Myc:Max interaction is essential for Myc-dependent transcriptional activation; however, we show that Max bound targets also include Myc-repressed genes. Moreover, we show that the interaction between Myc and Max is essential for gene repression to occur. Taken together, the identification and analysis of Myc bound target genes supports a model whereby Max plays an essential and universal role in the mechanism of Myc-dependent transcriptional regulation.

Blocking the Raf/MEK/ERK pathway sensitizes acute myelogenous leukemia cells to lovastatin-induced apoptosis.

The statin family of drugs are well-established inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase and are used clinically in the control of hypercholesterolemia. Recent evidence, from ourselves and others, shows that statins can also trigger tumor-specific apoptosis by blocking protein geranylgeranylation. We and others have proposed that statins disrupt localization and function of geranylgeranylated proteins responsible for activating signal transduction pathways essential for the growth and/or survival of transformed cells. To explore this further, we have investigated whether the mitogen-activated protein kinase (MAPK) signaling cascades play a role in regulating statin-induced apoptosis. Cells derived from acute myelogenous leukemia (AML) are used as our model system. We show that p38 and c-Jun NH2-terminal kinase/stress-activated kinase MAPK pathways are not altered during lovastatin-induced apoptosis. By contrast, exposure of primary and established AML cells to statins results in significant disruption of basal extracellular signal-regulated kinase (ERK) 1/2 phosphorylation. Addition of geranylgeranyl PPi reverses statin-induced loss of ERK1/2 phosphorylation and apoptosis. By establishing and evaluating the inducible Raf-1:ER system in AML cells, we show that constitutive activation of the Raf/MAPK kinase (MEK)/ERK pathway significantly represses but does not completely block lovastatin-induced apoptosis. Our results strongly suggest statins trigger apoptosis by regulating several signaling pathways, including the Raf/MEK/ERK pathway. Indeed, down-regulation of the Raf/MEK/ERK pathway potentiates statin-induced apoptosis because exposure to the MEK1 inhibitor PD98059 sensitizes AML cells to low, physiologically achievable concentrations of lovastatin. Our study suggests that lovastatin, alone or in combination with a MEK1 inhibitor, may represent a new and immediately available therapeutic approach to combat tumors with activated ERK1/2, such as AML.

MYC interaction with the tumor suppressive SWI/SNF complex member INI1 regulates transcription and cellular transformation.

MYC is a key driver of cellular transformation and is deregulated in most human cancers. Studies of MYC and its interactors have provided mechanistic insight into its role as a regulator of gene transcription. MYC has been previously linked to chromatin regulation through its interaction with INI1 (SMARCB1/hSNF5/BAF47), a core member of the SWI/SNF chromatin remodeling complex. INI1 is a potent tumor suppressor that is inactivated in several types of cancers, most prominently as the hallmark alteration in pediatric malignant rhabdoid tumors. However, the molecular and functional interaction of MYC and INI1 remains unclear. Here, we characterize the MYC-INI1 interaction in mammalian cells, mapping their minimal binding domains to functionally significant regions of MYC (leucine zipper) and INI1 (repeat motifs), and demonstrating that the interaction does not interfere with MYC-MAX interaction. Protein-protein interaction network analysis expands the MYC-INI1 interaction to the SWI/SNF complex and a larger network of chromatin regulatory complexes. Genome-wide analysis reveals that the DNA-binding regions and target genes of INI1 significantly overlap with those of MYC. In an INI1-deficient rhabdoid tumor system, we observe that with re-expression of INI1, MYC and INI1 bind to common target genes and have opposing effects on gene expression. Functionally, INI1 re-expression suppresses cell proliferation and MYC-potentiated transformation. Our findings thus establish the antagonistic roles of the INI1 and MYC transcriptional regulators in mediating cellular and oncogenic functions.

Identifying genes regulated in a Myc-dependent manner.

The c-myc proto-oncogene can direct a diverse array of biological activities, including cell cycle progression, apoptosis, and differentiation. It is believed that Myc can affect this wide variety of activities by functioning as a regulator of gene transcription, although few targets have been identified to date. To delineate the molecular program regulated downstream of Myc, we used a cDNA microarray approach and identified 52 putative targets out of >6000 cDNAs analyzed. To further distinguish the subset of genes whose regulation was dependent upon Myc per se from those regulated in response to activation of general mitogenic or apoptotic programs, the putative cDNA targets were then screened by a series of assays. By this approach 37 putative targets were ruled out and 15 Myc target genes were uncovered. Interestingly, comparing our results with other high throughput screens reveals that certain putative Myc targets previously reported are shown not to be regulated downstream of Myc (e.g. ribosomal proteins, HSP90beta), whereas others are further supported by our analyses (e.g. pdgfbetar, nucleolin). The identity of genes specifically regulated downstream of Myc provides the critical tools required to understand the role Myc holds in the transformation process and to delineate how Myc functions as a regulator of gene transcription.