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Results: 5

1.

Fig 4. RASSF1A siRNA inhibited IFN-induced apoptosis in DNMT1 depleted ACHN cells and in IFN sensitive WM9 melanoma cells. From: Expression of RASSF1A, an epigenetically silenced tumor suppressor, overcomes resistance to apoptosis induction by interferons.

A): After 8 daily transfections with DNMT1 antisense (DNMT1 AS) or mismatch control oligonucleotide (DNMT1 MM) at 40 nM, ACHN cells were plated for transfection with RASSF1A siRNA or control siRNA (40 nM). 4h after siRNA transfection, cells were replated and treated with IFN-beta (50 U/ml) 16h later. Apoptosis was measured by TUNEL assay after 4 days of IFNs. Frequency of apoptotic cells was reduced from 63.9 +/− 9.19 to 35 +/− 4.1 % (mean +/− SD) in RASSF1A siRNA pretreated cells. Oligonucleotides and siRNAs alone did not cause apoptosis (<5% TUNEL +). Immunoblotting after 24h of IFN-β treatment (50 U/ml) confirmed suppression of RASSF1A protein by siRNA. B): IFN sensitive WM9 cells were transfected with RASSF1A siRNA or control siRNA (40 nM). 4h after siRNA transfection, cells were replated and treated with IFN-beta (500 U/ml) 16h later. TUNEL analysis after 4 days of IFN identified reduction of IFN-induced apoptosis. siRNAs alone did not cause apoptosis (<5% TUNEL +). RASSF1A immunoblotting after 24h of IFN-β (500 U/ml) confirmed RASSF1A protein suppression by siRNA. On TUNEL graphs the means and standard deviations (error bars) of independent experiments are displayed.

Frederic J. Reu, et al. Cancer Res. ;66(5):2785-2793.
2.

Fig 1. 5-AZA-dC effects on resistance of ACHN (A), SK-RC-45 (B), A375 (C), and normal melanocytes and kidney epithelial cells (D) to IFN-induced apoptosis. From: Expression of RASSF1A, an epigenetically silenced tumor suppressor, overcomes resistance to apoptosis induction by interferons.

TUNEL assay: (FITC) positive DNA was used to assess apoptosis of cells that had been treated with IFNs over 4–5 days 16h after plating. All three malignant cell lines were resistant to apoptosis induction by up to 500 U/ml of IFN-α2 or IFN-β (A–C). In renal carcinoma (A, B) and melanoma (C) cells pretreatment with 200 nM 5-AZA-dC (AZA), daily over 2–6 days before IFN treatment overcame resistance to apoptosis induction by 50 to 100 U/ml IFN-α2 or IFN-β, while causing little to moderate apoptosis alone (5–20% TUNEL + cells). Normal neonatal human melanocytes (NHEM) and normal kidney epithelial cells (NKE) did not become sensitive to apoptosis induction by IFN-β (100 U/ml) after pretreatment with 200 nM 5-AZA-dC (AZA) daily over 4 days, which alone caused little apoptosis (up to 10% TUNEL + cells) (D). Reduction in DNMT1 protein was confirmed in whole cell lysates isolated after 4 days of 5-AZA-dC at 200 nM and subjected to SDS-PAGE and western blot analysis (A-D). 5-AZA-dC markedly decreased free DNMT1 protein in ACHN, SK-RC-45, A375, NHEM, and NKE cells (A-D). TUNEL graphs show the means and standard deviations (error bars) of independent experiments.

Frederic J. Reu, et al. Cancer Res. ;66(5):2785-2793.
3.

Fig 2. Depletion of DNMT1 by oligonucleotide antisense overcomes resistance to IFN-induced apoptosis. From: Expression of RASSF1A, an epigenetically silenced tumor suppressor, overcomes resistance to apoptosis induction by interferons.

ACHN (A) and SK-RC-45 (B) cells were transfected daily with 40 nM DNMT1 AS or mismatch oligonucleotide (MM) or lipofectin transfection reagent only (L or Lipo). Every other day, 4h after the preceding transfection, cells were replated at 15 000 cells/cm2 (ACHN) or 5000 cells/cm2 (SK-RC-45) to keep confluencies optimal for transfection efficiency. A): Near-complete depletion of DNMT1 was achieved by day 4 of AS treatment and maintained to day 8 allowing for cell divisions in the absence of the maintenance DNA methyltransferase. Expression of stat1, stat2, and stat3 was not affected. After 6–8 days cells were plated at 5000 cells/cm 2 for IFN treatment 16 h later. Apoptosis was assessed by immunoblotting for cleaved caspase 3 after 48 hr of IFN (following 8 days of DNMT1 AS) and by TUNEL assay to detect cells containing fragmented DNA after 5 days of IFN (following 6 and 8 days of DNMT1 AS). Duration of DNMT1 depletion correlated with apoptosis induction by 50 U/ml IFN-α 2 or IFN-β as determined by TUNEL assay, while MM did not sensitize. B) DNMT1 AS (AS) at 40nM was sufficient for near-complete depletion of DNMT1 protein by day 4 in SK-RC-45 cells. Resistance to apoptosis induction by 500 U/ml of IFN-α 2 or IFN-β over 4 days was overcome by pretreatment with DNMT1 AS over 6 days. On TUNEL graphs the means and standard deviations (error bars) of independent experiments are displayed.

Frederic J. Reu, et al. Cancer Res. ;66(5):2785-2793.
4.

Fig. 5. Effect of lentivirus expression of RASSF1A on IFN and Apo2L/TRAIL -induced apoptosis in ACHN cells. From: Expression of RASSF1A, an epigenetically silenced tumor suppressor, overcomes resistance to apoptosis induction by interferons.

A): Map of lentiviral RASSF1A construct. B): ACHN cells were transduced with RASSF1A carrying lentivirus or empty virus. 72h later selective antibiotic was added and the population of clones was grown in selection media for 14 days before assessing apoptotic response to IFN-β (50 U/ml over 5 days) by TUNEL assay. After confirmation of stable expression of RASSF1A by immunoblotting, clonal selection was performed to identify clones that expressed similar amounts of RASSF1A protein as achieved by DNMT1 AS (40 nM) over 8 days. Treatment with DNMT1 mismatch control oligonucleotide (MM) and empty lentivirus transduced cells served as negative controls. In clones expressing RASSF1A at levels comparable to DNA demethylating treatment, IFN-β (50 U/ml over 5 days) resulted in 20–40% apoptotic cells while stable transduction with empty lentivirus (kept in selective antibiotic for equal amounts of time) did not. C): Concurrent treatment of ACHN RASSF1A clone 1.3 with 2 μg/ml TRAIL neutralizing antibody (TRAIL-N AB) and IFN-β (50 U/ml) over 5 days inhibited IFN-induced apoptosis compared to cotreatment with 2 μg/ml control rabbit immunoglobulin (CTRL AB). D): RASSF1A expression markedly increased sensitivity to apoptosis induction by Apo2L/TRAIL in ACHN cells. Normal kidney epithelial (NKE) cells, even after pretreatment with 5-AZA-dC (200 nM) over 4 days, which alone resulted in moderate apoptosis induction, remained resistant to the apoptosis inducing effects of Apo2L/TRAIL. TUNEL graphs display the means and standard deviations (error bars) of independent experiments.

Frederic J. Reu, et al. Cancer Res. ;66(5):2785-2793.
5.

Fig 3. Effects of DNMT1 inhibition on expression of RASSF1A and its promoter region and influence of IFNs on RASSF1A protein expression. From: Expression of RASSF1A, an epigenetically silenced tumor suppressor, overcomes resistance to apoptosis induction by interferons.

A) To reactivate RASSF1A mRNA expression indicated cell lines were treated daily with 40 nM DNMT1 AS (AS) over 4–8 d (ACHN) or 6d (SK-RC-45). Mismatch control oligonucleotide (MM) or lipofectin transfection reagent alone (L) were used as control treatments. Alternatively RASSF1A mRNA expression was reactivated by daily 5-AZA-dC treatments at 200 nM over 2–6 (ACHN) or 4 d (SK-RC-45, A375). For RASSF1A RT-PCR 500 ng RNA transcribed into cDNA was amplified over 35 cycles with RASSF1A primers; for GAPDH detection 250 ng RNA, transcribed into cDNA were amplified over 20 cycles. HeLa cells served as positive controls (19, 21, 23, 24) and WM9 cells as well as normal melanocytes (NHEM) and kidney epithelial (NKE) cells were found to express RASSF1A mRNA at baseline. B): Approximately 100 ng of bisulfite modified DNA from ACHN cells was used for methylation specific PCR. Bisulfite modified DNA from HeLa cells was unmethylated control. Forty nM DNMT1 AS over 8 days led to partial demethylation of RASSF1A promoter CpG island in ACHN cells. C): ACHN cells were transfected daily with 40 nM DNMT1 antisense (DNMT1 AS) or mismatch control oligonucleotide (MM) over 8 days before treatment with IFNs over 48 hr. Floating and adherent cells were harvested for RASSF1A (mAB) and MST1 (pAB) immunoblotting. DNMT1 AS reactivated RASSF1A protein expression, which was further augmented by IFNs. Cleavage (activation) of MST1 by IFNs occurred only in RASSF1A expressing DNMT1 pretreated ACHN cells. Augmentation of RASSF1A protein expression by IFN was also observed in A375 cells pretreated with 5-AZA-dC (AZA) at 200 nM daily over 4 days, and without pretreatment in WM9 cells, known to be sensitive to apoptosis induction by IFNs (27). Similar results were obtained in independent experiments.

Frederic J. Reu, et al. Cancer Res. ;66(5):2785-2793.

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