Depletion of DNA methyltransferase 1 and/or DNA methyltransferase 3b mediates growth arrest and apoptosis in lung and esophageal cancer and malignant.

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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 Edmund S. Kassis, MD, Ming Zhao, MD, Julie A. Hong, MS, G. Aaron Chen, MS, Dao M. Nguyen, MD, David S. Schrump, MD The Journal of Thoracic and Cardiovascular Surgery Volume 131, Issue 2, Pages 298-306.e2 (February 2006) DOI: 10.1016/j.jtcvs.2005.05.022 Copyright © 2006 The American Association for Thoracic Surgery Terms and Conditions

Figure 1 Trypan blue exclusion analysis of proliferation of CALU-6, A549, BIC, SKGT5, H2373, and H2052 (A) and normal human bronchial epithelial cells (NHBE; B) after antisense oligo (ASO) transfection. Day 0 counts represent the number of viable cells immediately before initial exposure to DNA methyltransferase (DNMT) ASOs, mismatch (MM) oligos, or lipofectin. Day 3 counts represent cell numbers at the 72-hour time point for normal media (NM), lipofectin (lipofectin alone), MM oligos, DNMT1 ASOs (25, 50, and 75 nmol/L), DNMT3b ASOs (25, 50, and 75 nmol/L), and combination DNMT1/DNMT3b ASOs (25, 50, 75 nmol/L). NHBE cells were exposed only to 75 nmol/L DNMT 1 or DNMT 3b. The Journal of Thoracic and Cardiovascular Surgery 2006 131, 298-306.e2DOI: (10.1016/j.jtcvs.2005.05.022) Copyright © 2006 The American Association for Thoracic Surgery Terms and Conditions

Figure 2 ApoBrdU analysis of CALU-6 (A), A549 (B), BIC (C), and H2373 (D) cell lines exposed to normal media (NM), lipofectin alone, mismatch oligonucleotides, or DNA methyltransferase (DNMT) 1, DNMT3b, or both antisense oligos (ASOs; 25 or 75 nmol/L). The percentage of apoptotic cells for each exposure is indicated in the corresponding box. SKGT5 cells exhibited a pattern of apoptosis similar to that observed for CALU-6 and A549 cells, whereas results pertaining to H2052 cells were similar to those observed for BIC cells (data not shown). The Journal of Thoracic and Cardiovascular Surgery 2006 131, 298-306.e2DOI: (10.1016/j.jtcvs.2005.05.022) Copyright © 2006 The American Association for Thoracic Surgery Terms and Conditions

Figure 3 ApoBrdU analysis of apoptosis in A549 cells exposed to DNA methyltransferase (DNMT) 1 antisense oligo (ASO) alone, DNMT1 ASO plus Z-VAD, or DNMT1 ASO plus DEVD (A) or A549 cells exposed to DNMT3b ASO alone, DNMT3b ASO plus Z-VAD, or DNMT3b ASO plus DEVD (B). Cells were also treated with control exposure to normal media (NM), lipofectin alone, and mismatch (MM) oligos. The x axis indicates the percentage of apoptotic cells. Both caspase inhibitors partially abrogated the apoptotic effects of DNMT1 and DNMT3b ASOs. The Journal of Thoracic and Cardiovascular Surgery 2006 131, 298-306.e2DOI: (10.1016/j.jtcvs.2005.05.022) Copyright © 2006 The American Association for Thoracic Surgery Terms and Conditions

Figure E1 Western blot analysis of DNA methyltransferase (DNMT) 1 expression in A549 lung cancer cells after DNMT antisense oligo (ASO) transfection by using protocols and reagents described by Beaulieu and colleagues.19 Shown are treatment groups NM (normal media), lipofectin, ASO to DNMT1 (25 and 75 nmol/L), and ASO to DNMT 3b (75 nmol/L). A dose-dependent and specific inhibitor of DNMT1 was observed after transfection with the DNMT1 ASO. The Journal of Thoracic and Cardiovascular Surgery 2006 131, 298-306.e2DOI: (10.1016/j.jtcvs.2005.05.022) Copyright © 2006 The American Association for Thoracic Surgery Terms and Conditions