Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Essential role of DNA methyltransferase 1-mediated transcription of insulin-like Growth Factor 2 in Resistance to Histone Deacetylase Inhibitors

doi: 10.1158/1078-0432.CCR-16-0534

Figure Lengend Snippet: (A) Viability of parental NSCLC cells vs. their sublines with acquired resistance to vorinostat in response to vorinostat treatment, determined by the MTT assay. The viability of cells treated with vorinostat (5 μM) was shown. (B) Anchorage-independent colony-forming ability of vorinostat-resistant NSCLC sublines which experienced siRNA-mediated knockdown of IGF-1R (Scr: scrambled siRNA control). Colony-forming ability of cells treated with vorinostat (1 μM) was shown. (C) Overexpression of IGF-1R led to a decreased sensitivity of H520 cells to vorinostat (assessed by the suppression of anchorage-independent colony formation following the treatment with vorinostat [1 μM for 2 weeks]) compared with the empty vector (EV) controls. (D, E) Viability (D) and anchorage-dependent colony-forming ability (E) of parental NSCLC cells vs. their sublines with acquired resistance in response to erlotinib, linsitinib, or their combination. Cells were treated with linsitinib (Linsi; 0.5 μM), alone or in combination with erlotinib (Erlo; 0.5 μM; D) or increasing concentrations of erlotinib (Erlo; 0.2, 0.5, and 1 μM; E). The data are presented as the mean ± SD (n = 3). Statistical significance was determined by Student’s t-test (*: P < 0.05; **: P < 0.01; ***: P < 0.001). Con: control; Vo: vorinostat.

Article Snippet: Human NSCLC lines (H460, H226B, H1299, H226Br, H520, H1944, A549, H358, and H322) and human lymphoma cells (HH) were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA) or kindly provided by Drs. Jack A. Roth and John V. Heymach (MD Anderson Cancer Center, Houston, TX, USA).

Techniques: MTT Assay, Knockdown, Control, Over Expression, Plasmid Preparation

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Essential role of DNA methyltransferase 1-mediated transcription of insulin-like Growth Factor 2 in Resistance to Histone Deacetylase Inhibitors

doi: 10.1158/1078-0432.CCR-16-0534

Figure Lengend Snippet: (A) Western blot and RT-PCR analyses of DNMT1 protein and mRNA levels in NSCLC sublines with acquired resistance to vorinostat. DNMT1, 3A and 3B and CTCF expression levels were compared between vorinostat-resistant (R) sublines H1944, H358 and H322 and their parental controls (P). (B, C) Western blot (B) and IHC (C) analyses of DNMT1 levels in xenograft tumors of H1944R and H1944 cells. (D) Immunoblot analyses of DNMT1, DNMT3A, and DNMT3B expression in the NSCLC cell lines. (E) Correlation between the level of DNMT1 transcription and NSCLC cell lines’ resistance to vorinostat was determined by calculating the Pearson correlation coefficient using Graphpad Prism 6. DNMT1 transcription was determined by real-time PCR, and the sensitivity of NSCLC cells to vorinostat treatment (IC50 values) was previously determined and shown in our previous report (17). (F) The DNMT1 promoter activity in sensitive (H1944), primary vorinostat-resistant (H226B, H226Br), and acquired vorinostat-resistant (H1944R, H358R, and H322R) cells (RLU: relative luminescence units). (G) Methylation in the CTCF6 site of the H19/IGF2 ICR determined by Methylation-specific PCR (top) and CTCF binding to this region determined by ChIP assay (middle) in H1944R cells with shRNA-mediated knockdown of DNMT1 (bottom). (H) shRNA-mediated knockdown of DNMT1 prevented the upregulation of IGF2 and the activation of the IGF-1R pathway in response to vorinostat (5 μM for 2 days) in vorinostat-resistant NSCLC cells (either acquired or intrinsic). The data of the luciferase reporter assay and the ChIP analysis are presented as the mean ± SD. Significance was determined by Student’s t-test (F, left and middle; G) and one-way ANOVA (F, right) (*: P < 0.05; ***: P < 0.001). Vo: vorinostat.

Article Snippet: Human NSCLC lines (H460, H226B, H1299, H226Br, H520, H1944, A549, H358, and H322) and human lymphoma cells (HH) were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA) or kindly provided by Drs. Jack A. Roth and John V. Heymach (MD Anderson Cancer Center, Houston, TX, USA).

Techniques: Western Blot, Reverse Transcription Polymerase Chain Reaction, Expressing, Real-time Polymerase Chain Reaction, Activity Assay, Methylation, Binding Assay, shRNA, Knockdown, Activation Assay, Luciferase, Reporter Assay

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Essential role of DNA methyltransferase 1-mediated transcription of insulin-like Growth Factor 2 in Resistance to Histone Deacetylase Inhibitors

doi: 10.1158/1078-0432.CCR-16-0534

Figure Lengend Snippet: (A) Decreases in the DNA methylation of the CTCF6 site in H1299 cells with shRNA-based knockdown of STAT3 expression. Left. The decreased level of STAT3 expression in H1299 cells stably transfected with STAT3 shRNAs. Right. The DNA methylation status was examined by methylation-specific PCR. (B) Downregulation of the transcription of STAT3, DNMT1, and IGF2 by H1299 cells with shRNA-based stable ablation of STAT3 expression, determined by real-time PCR. (C) DNMT1 transcription in the indicated NSCLC cells after treatment with vorinostat. Cells were treated with vorinostat (5 μM) for 2 days. The mRNA expression was analyzed by real-time PCR. (D) The siRNA-mediated knockdown of STAT3 attenuated the vorinostat-induced mRNA expression of DNMT1 in vorinostat-resistant NSCLC cells (either acquired or intrinsic). (E) Mutation of the lysine acetylation site of STAT3 (K/R) led to a decrease in vorinostat-induced DNMT1 expression in H1299 cells. The data are presented as the mean ± SD (n = 3). Significance was determined by Student’s t-test (*: P < 0.05; **: P < 0.01; ***: P < 0.001). Vo: vorinostat.

Article Snippet: Human NSCLC lines (H460, H226B, H1299, H226Br, H520, H1944, A549, H358, and H322) and human lymphoma cells (HH) were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA) or kindly provided by Drs. Jack A. Roth and John V. Heymach (MD Anderson Cancer Center, Houston, TX, USA).

Techniques: DNA Methylation Assay, shRNA, Knockdown, Expressing, Stable Transfection, Transfection, Methylation, Real-time Polymerase Chain Reaction, Mutagenesis

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Essential role of DNA methyltransferase 1-mediated transcription of insulin-like Growth Factor 2 in Resistance to Histone Deacetylase Inhibitors

doi: 10.1158/1078-0432.CCR-16-0534

Figure Lengend Snippet: (A) Viability of H226B cells with stable transfection with DNMT1 shRNA determined by the MTT assay. (B) Overexpression of DNMT1 led to a decreased sensitivity of H1944 cells to vorinostat (assessed by the suppression of colony formation following the treatment with vorinostat [1 μM for 2 weeks]) compared to the empty vector (EV) controls. (C) The results from the MTT assay indicated that co-treatment of decitabine (Deci) with vorinostat sensitized vorinostat-resistant NSCLC cells (either acquired or intrinsic) to vorinostat. (D) DNA methylation in the CTCF6 site of the H19/IGF2 ICR determined by methylation-specific PCR, and the CTCF binding to the CTCF6 site was determined by the ChIP assay. H1944R and H226B cells were daily treated with decitabine (5 μM) for 4 days. (E-G) Immunoblotting analyses indicating enhanced apoptotic cell death by the co-treatment of decitabine with HDIs (E: vorinostat; F: panobinostat [Pano]; G: romidepsin [Romi]) in vorinostat-resistant NSCLC cells (either acquired or intrinsic) compared with single treatments of vorinostat or decitabine. The data are presented as the mean ± SD (n = 3). Significance was determined by Student’s t-test (*: P < 0.05; ***: P < 0.001). Vo: vorinostat.

Article Snippet: Human NSCLC lines (H460, H226B, H1299, H226Br, H520, H1944, A549, H358, and H322) and human lymphoma cells (HH) were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA) or kindly provided by Drs. Jack A. Roth and John V. Heymach (MD Anderson Cancer Center, Houston, TX, USA).

Techniques: Stable Transfection, shRNA, MTT Assay, Over Expression, Plasmid Preparation, DNA Methylation Assay, Methylation, Binding Assay, Western Blot