title: Depletion of DNA methyltransferase 1 and/or DNA methyltransferase 3b mediates growth arrest and apoptosis in lung and esophageal cancer and malignant pleural mesothelioma cells.
abstract: OBJECTIVE
  DNA methyltransferase (DNMT)1, DNMT3b, or both, facilitate malignant transformation through chromatin remodeling mechanisms. The present study was undertaken to examine the effects of antisense-mediated inhibition of DNMT expression in cultured thoracic malignancies.
  
  
  METHODS
  CALU-6 and A549 lung cancer, SKGT5 and BIC esophageal adenocarcinoma, and H2373 and H2052 malignant pleural mesothelioma (MPM) cells, as well as normal human bronchial epithelial (NHBE) cells, were transfected with phosphorothioate-modified antisense oligos targeting DNMT1, DNMT3b, or both, or mismatch oligos. Quantitative reverse transcription-polymerase chain reaction, Western blotting, trypan blue exclusion, and ApoBrdU techniques were used to evaluate DNMT expression, proliferation, and apoptosis after antisense oligo transfections. Gene expression profiles were assessed by using long-oligo array techniques.
  
  
  RESULTS
  Antisense oligos mediated specific and dose-dependent depletion of DNMT1 and DNMT3b, resulting in pronounced inhibition of proliferation of all thoracic cancer lines, but not NHBE cells. Depletion of DNMT1 or DNMT3b coincided with dramatic, caspase-dependent, p53-independent apoptosis in 4 of the 6 thoracic cancer lines. The antiproliferative effects of the antisense oligos were not attributable to induction of RASSF1A, p16, or p21 tumor suppressor genes, and did not coincide with demethylation of genes encoding cancer-testis antigens. DNA methyltransferase knockdown mediated induction of numerous genes regulating response to genotoxic stress. Gene expression profiles after DNMT1, DNMT3b, or combined DNMT1/3b depletion were remarkably similar, yet distinctly different from expression profiles mediated by 5 aza 2' deoxycytidine.
  
  
  CONCLUSIONS
  Antisense oligos targeting DNMT1 and DNMT3b induce genomic stress, and mediate potent growth inhibition in lung and esophageal cancer and MPM cells. These findings support further evaluation of DNMT knockdown strategies for cancer therapy.
authors.affiliation: Thoracic Oncology Section, Surgery Branch, Center for Cancer Research, National Cancer Institute, Bethesda, Md 20892, USA.
authors.forename: Edmund S
authors.initials: ES
authors.lastname: Kassis
authors.forename: Ming
authors.initials: M
authors.lastname: Zhao
authors.forename: Julie A
authors.initials: JA
authors.lastname: Hong
authors.forename: G Aaron
authors.initials: GA
authors.lastname: Chen
authors.forename: Dao M
authors.initials: DM
authors.lastname: Nguyen
authors.forename: David S
authors.initials: DS
authors.lastname: Schrump
mesh_terms: D000230:Adenocarcinoma / Q000473:pathology / Q000503:physiopathology*; D017209:Apoptosis / Q000502:physiology*; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000502:physiology*; D004938:Esophageal Neoplasms / Q000473:pathology / Q000503:physiopathology*; D015870:Gene Expression; D006801:Humans; D008175:Lung Neoplasms / Q000473:pathology / Q000503:physiopathology*; D008654:Mesothelioma / Q000473:pathology / Q000503:physiopathology*; D020319:Oligodeoxyribonucleotides, Antisense / Q000494:pharmacology; D010997:Pleural Neoplasms / Q000473:pathology / Q000503:physiopathology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D014407:Tumor Cells, Cultured; D000097584:DNA Methyltransferase 3B
chemical_list: D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; D020319:Oligodeoxyribonucleotides, Antisense; D000097584:DNA Methyltransferase 3B; C000619380:DNMT1 protein, human