trichostatin-a and Chromosome-Deletion

Since it is extremely difficult to establish an animal model for human chromosomal abnormalities, induced pluripotent stem cells (iPSCs) provide a powerful alternative to study underlying mechanisms of these disorders and identify potential therapeutic interventions. In this study we established iPSCs from a young girl with a hemizygous deletion of Xq27.3-q28 who exhibited global developmental delay and intellectual disability from early in infancy. The deletion site on the X chromosome includes Fragile X Mental Retardation 1 (FMR1), the gene responsible for fragile X syndrome, which likely contributes to the patient's neurodevelopmental abnormalities. The FMR1 gene was expressed in approximately half of the iPSC clones we generated while it was absent in the other half due to the random inactivation of normal and abnormal X chromosomes. The normal or absent expression pattern of the FMR1 gene was not altered when the iPSCs were differentiated into neural progenitor cells (NPCs). Moreover, chromosome reactivating reagents such as 5-aza-2-deoxycytidine, trichostatin A, and UNC0638, were tested in an attempt to reactivate the suppressed FMR1 gene in affected iPSC-NPCs. The affected and control isogenic iPSCs developed in this study are ideal models with which to identify downstream consequences caused by the Xq27.3-q28 deletion and also to provide tools for high-throughput screening to identify compounds potentially improving the well-being of this patient population.

Topics: Cell Differentiation; Cells, Cultured; Child, Preschool; Chromosome Deletion; Chromosomes, Human, X; Decitabine; Developmental Disabilities; Female; Fragile X Mental Retardation Protein; Fragile X Syndrome; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Induced Pluripotent Stem Cells; Intellectual Disability; Quinazolines

Deletion of 11q23-q24 is frequent in a diverse variety of malignancies, including breast and colorectal carcinoma, implicating the presence of a tumor suppressor gene at that chromosomal region. We examined a 6-Mb region on 11q23 by high-resolution deletion mapping, using both loss of heterozygosity analysis and customized microarray comparative genomic hybridization. LARG (leukemia-associated Rho guanine-nucleotide exchange factor) (also called ARHGEF12), identified from the analysed region, is frequently underexpressed in breast and colorectal carcinomas with a reduced expression observed in all breast cancer cell lines (n=11), in 12 of 38 (32%) primary breast cancers, 5 of 10 (50%) colorectal cell lines and in 20 of 37 (54%) primary colorectal cancers. Underexpression of the LARG transcript was significantly associated with genomic loss (P=0.00334). Hypermethylation of the LARG promoter was not detected in either breast or colorectal cancer, and treatment of four breast and four colorectal cancer cell lines with 5-aza-2'-deoxycytidine and/or trichostatin A did not result in a reactivation of LARG. Enforced expression of LARG in breast and colorectal cancer cells by stable transfection resulted in reduced cell proliferation and colony formation, as well as in a markedly slower cell migration rate in colorectal cancer cells, providing functional evidence for LARG as a candidate tumor suppressor gene.

Topics: Antimetabolites, Antineoplastic; Azacitidine; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Chromosome Deletion; Chromosome Mapping; Chromosomes, Human, Pair 11; Colorectal Neoplasms; Decitabine; DNA Methylation; Female; Guanine Nucleotide Exchange Factors; Humans; Hydroxamic Acids; Male; Nucleic Acid Hybridization; Promoter Regions, Genetic; Protein Synthesis Inhibitors; Rho Guanine Nucleotide Exchange Factors; Transfection; Tumor Suppressor Proteins

Identification of a homozygous deletion in cancer cells provides strong evidence for the location of a tumor suppressor gene (TSG). We analyzed the 2p24 homozygous deletion of a non-small-cell lung cancer (NSCLC) cell line, NCI-H2882, and found that the deletion size was 3.7 Mbp. Since RhoB, which has been suggested to be a candidate TSG, was located in this region, we analyzed RhoB for alterations in NSCLC. Although we found no mutations in 48 cell lines including 20 NSCLCs, a loss of heterozygosity (LOH) analysis in 128 primary NSCLCs showed that 25 of 62 informative samples had LOH at the RhoB locus. Northern blot analysis of 28 cell lines (including 15 NSCLCs) indicated that RhoB expression was downregulated in 27. We analyzed RhoB expression in 112 primary NSCLCs with immunohistochemistry and found no or a weak RhoB expression in 33 (42%) of 78 adenocarcinomas, whereas we found it in 29 (94%) of 31 squamous cell carcinomas. No or a weak expression of RhoB was more frequently observed in poorly- or moderately-differentiated adenocarcinomas than in well-differentiated ones (p = 0.0014). Furthermore, no or a weak expression of RhoB indicated a tendency to poor patient prognosis. Although hypermethylation was not found at the promoter region, the RhoB expression in NSCLC cell lines was induced by histone deacetylase inhibition, suggesting that RhoB downregulation may be due to histone modification. The present study demonstrates that RhoB expression is frequently downregulated in NSCLCs by multiple mechanisms, suggesting that RhoB is a candidate TSG for NSCLC.

Topics: Aged; Blotting, Northern; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Chromosome Deletion; Chromosome Mapping; Chromosomes, Human, Pair 2; DNA Methylation; Down-Regulation; Female; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Immunohistochemistry; Kaplan-Meier Estimate; Loss of Heterozygosity; Lung Neoplasms; Male; Microsatellite Repeats; Middle Aged; Prognosis; Promoter Regions, Genetic; Reverse Transcriptase Polymerase Chain Reaction; rhoB GTP-Binding Protein

Interstitial loss of all or part of the long arm of chromosome 5, or del(5q), is a frequent clonal chromosomal abnormality in human myelodysplastic syndrome (MDS, a preleukemic disorder) and acute myeloid leukemia (AML), and is thought to contribute to the pathogenesis of these diseases by deleting one or more tumor-suppressor genes. Although a major commonly deleted region (CDR) has been delineated on chromosome band 5q31.1 (refs. 3-7), attempts to identify tumor suppressors within this band have been unsuccessful. We focused our analysis of gene expression on RNA from primitive leukemia-initiating cells, which harbor 5q deletions, and analyzed 12 genes within the CDR that are expressed by normal hematopoietic stem cells. Here we show that the gene encoding alpha-catenin (CTNNA1) is expressed at a much lower level in leukemia-initiating stem cells from individuals with AML or MDS with a 5q deletion than in individuals with MDS or AML lacking a 5q deletion or in normal hematopoietic stem cells. Analysis of HL-60 cells, a myeloid leukemia line with deletion of the 5q31 region, showed that the CTNNA1 promoter of the retained allele is suppressed by both methylation and histone deacetylation. Restoration of CTNNA1 expression in HL-60 cells resulted in reduced proliferation and apoptotic cell death. Thus, loss of expression of the alpha-catenin tumor suppressor in hematopoietic stem cells may provide a growth advantage that contributes to human MDS or AML with del(5q).

Topics: Acute Disease; alpha Catenin; Blotting, Western; Cell Line; Cell Line, Tumor; Cell Transformation, Neoplastic; Chromosome Deletion; Chromosomes, Human, Pair 5; DNA Methylation; Flow Cytometry; Gene Expression Regulation, Neoplastic; Green Fluorescent Proteins; HL-60 Cells; Humans; Hydroxamic Acids; In Situ Hybridization, Fluorescence; K562 Cells; Leukemia, Myeloid; Mutation; Myelodysplastic Syndromes; Myeloid Progenitor Cells; Reverse Transcriptase Polymerase Chain Reaction; Transfection; U937 Cells

Chromosome 1p is frequently deleted in neuroblastoma (NB) tumours. The commonly deleted region has been narrowed down by loss of heterozygosity studies undertaken by different groups. Based on earlier mapping data, we have focused on a region on 1p36 (chr1: 7 765 595-11 019 814) and performed an analysis of 30 genes by exploring features such as epigenetic regulation, that is DNA methylation and histone deacetylation, mutations at the DNA level and mRNA expression. Treatment of NB cell lines with the histone deacetylase inhibitor trichostatin A led to increased gene transcription of four of the 30 genes, ERRFI1 (MIG-6), PIK3CD, RBP7 (CRBPIV) and CASZ1, indicating that these genes could be affected by epigenetic downregulation in NBs. Two patients with nonsynonymous mutations in the PIK3CD gene were detected. One patient harboured three variations in the same exon, and p.R188W. The other patient had the variation p.M655I. In addition, synonymous variations and one variation in an intronic sequence were also found. The mRNA expression of this gene is downregulated in unfavourable, compared to favourable, NBs. One nonsynonymous mutation was also identified in the ERRFI1 gene, p.N343S, and one synonymous. None of the variations above were found in healthy control individuals. In conclusion, of the 30 genes analysed, the PIK3CD gene stands out as one of the most interesting for further studies of NB development and progression.

Topics: Adaptor Proteins, Signal Transducing; Azacitidine; Cell Line, Tumor; Chromosome Deletion; Chromosomes, Human, Pair 1; Class I Phosphatidylinositol 3-Kinases; Decitabine; DNA Methylation; DNA Mutational Analysis; DNA-Binding Proteins; DNA, Neoplasm; Exons; Genetic Variation; Histones; Humans; Hydroxamic Acids; Mutation; Neuroblastoma; Phosphatidylinositol 3-Kinases; Polymorphism, Genetic; Retinol-Binding Proteins, Cellular; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transcription Factors; Transcription, Genetic; Tumor Suppressor Proteins; Up-Regulation