Promoter methylation represents one of the major epigenetic mechanisms responsible for the regulation of gene expression. Hypomethylating drugs are currently approved for the treatment of myelodysplastic syndromes and acute myeloid leukemia, and some studies have recently been carried out on diffuse large B cell lymphoma (DLBCL). DLBCL is a type of Non-Hodgkin lymphoma. The aim of the present study was to assess the role of DNA methyltransferase (DNMT)1 in mediating the epigenetic regulation of some key targets previously emerged as hypermethylated in Non-Hodgkin lymphoma. Reverse transcription-quantitative PCR, genome-wide arrays and methylation-specific PCR were used to determine the level of methylation of specific targets. Gene silencing, gene expression and immunoblotting were used to investigate the role of DNMT1 and DNMT3a in lymphoma cells. The present study showed that lymphoma cell lines displayed a completely different methylation profile on selected targets compared with primary B lymphocytes and peripheral blood mononuclear cells. 5′-aza-cytidine (5AZA) and 5′-aza-2-deoxycitidine (decitabine) exerted their activity through, at least in part, mechanisms independent of DNMT1 downregulation. Despite a global hypomethylating effect of 5AZA and decitabine, DNMT1 was not found to be necessary to maintain the hypermethylation of Krüppel-like factor 4 (KLF4), death associated protein 1 (DAPK1) and spastic paraplegia 20 (
Lymphomas represent the vast majority of tumors of the lymphoid system and 4.3% of all new cancer cases in the United States (
We previously described a novel panel of genes that are differentially methylated in human B lymphocytes purified from follicular lymphoma (FL) and diffuse large B cell lymphoma (DLBCL) (
The present study aimed to elucidate the profile of promoter methylation of these selected target genes upon treatment with the two widely used hypomethylating agents 5′-aza-cytidine (5AZA) and 5′-aza-2-deoxycitidine (decitabine) (
Thus, the current study investigated DNMT1 and DNMT3a in response to 5AZA or decitabine and by assessing the effects of their specific silencing. The results were aimed at evaluating the effects on promoter methylation of
The Toledo cell line (ATCC-CRL-2631) was purchased from American Type Culture Collection. NU-DUL-1 cells (ACC 579) were purchased from GSMZ-German Collection of Microorganisms and Cell Cultures GmbH. The GNE-587170 cell line was supplied by Dr Mark Minden's laboratory (Ontario Cancer Institute, Toronto, Canada). Non-Hodgkin lymphoma-derived cell lines were cultured in RPMI-1640 medium (Euroclone SpA) with penicillin-streptomycin at 10 U/ml (Euroclone SpA) and 10% FBS (Gibco; Thermo Fisher Scientific, Inc.). Cells were routinely tested for mycoplasma contamination using the MycoAlert™ Mycoplasma Detection Kit (Lonza Group, Ltd.). Cell lines were authenticated by PCR-single-locus-technology (Eurofins Scientific).
Hypomethylating drugs were used at a concentration ranging between 0.05–10 µM, which is specified in each figure. The drugs were dissolved in water and the control is represented by untreated cells. Cells were incubated in a humidified incubator at 37°C with 5% CO2 for the indicated time each experiment.
Viability assays were performed by using a Cell Counting Kit-8 (CCK-8; Boster Biological Technology). Briefly, 20,000 cells were seeded in a 96-well plate and, after 48 h of incubation, 10 µl/well tetrazolium salt was added. Spectrophotometric reading at 450 nm with a 620 nm filter was performed after 1 h of incubation.
Time-course apoptosis assays following either 5AZA or decitabine treatment were performed using an IncuCyte® S3 (Essen Bioscience). Cells were seeded at a density of 20,000 cells/well. The reagents used to label the cells were IncuCyte® Nuclight Rapid Red and Cell Health Reagent Caspase-3/7 (both used at a 1:2,000 final dilution in the wells; Essen Bioscience). Cells were incubated in a humidified incubator at 37°C with 5% CO2. Each condition was performed in triplicate.
The cytologic specimen was prepared using Cytospin™ 4 (Thermo Fisher Scientific, Inc.) and the glass was fixed with 95% ethanol for 20 min at room temperature. Immunocytochemistry was performed with Monoclonal Mouse Anti-Human Ki-67 Antigen Clone MIB-1 (1:100; cat. no. M724001-2; Dako; Agilent Technologies, Inc.) for 40 min at room temperature. The slides were pre-treated with heat antigen retrieval in EDTA buffer for 64 min. The immunocytochemical staining was developed on Ventana BenchMark ULTRA platform using the pre-diluted UltraView DAB Detection Kit (cat. no. 05269806001; Ventana Medical Systems, Inc.; Roche Diagnostics) for 8 min at 36°C. Slides were counterstained with hematoxylin for 3 min at room temperature. Slides were observed on a Nikon Eclipse 80i light microscope (magnification, ×40; Nikon Corporation).
Quantitative gene-specific methylation was performed via EpiTect Methyl II qPCR (Qiagen S.r.l.) as previously described (
Global chromatin methylation was performed using the MethylFlash Methylated DNA Quantification kit (EpiGentek Group, Inc.) with 100 ng DNA/sample, according to the manufacturer's instructions. Methylation-specific PCR (MS-PCR) was performed with 50 ng bisulfite-converted genomic DNA. Primers and PCR conditions are presented in
Total RNA was extracted using TRIzol (cat. no. 15596026; Invitrogen; Thermo Fisher Scientific, Inc.) with the RNA isolation protocol (W.M. Keck Foundation Biotechnology microarray Research Laboratory at Yale University) and RT was performed with the iScript™ cDNA Synthesis kit (Bio-Rad Laboratories, Inc.) according to the manufacturer's protocol. Gene expression was performed via qPCR with the SsoFast™ EvaGreen® Supermix (Bio-Rad Laboratories, Inc.) following the manufacturer's protocols. The Cq value of 37 cycles was chosen as the lower threshold (cutoff) for gene expression using the 2−∆∆Cq method of quantification (
Immunoblotting was performed as previously described (
Gene silencing was performed through 4D-Nucleofector™ Core Unit (Lonza Group, Ltd.). The buffer used was the Amaxa™ SF cell line 4D Nucleofector™ X kit (cat. no. V4XC-2024; Lonza Group, Ltd.). Small interfering (si)RNAs were purchased from Ambion (Thermo Fisher Scientific, Inc.), diluted in molecular biology grade water and used at a final concentration of 100 nM. The cells were either transfected with siRNAs against DNMT1 (cat. nos. 4390824, s4216 and s4217; Ambion; Thermo Fisher Scientific, Inc.), DNMT3a (cat. nos. 4392420 and s200426; Ambion; Thermo Fisher Scientific, Inc.) or both. Negative control #2 siRNA (cat. no. 4390846) was used as the control (scramble). Sequences are listed in
PBMCs were purified from the peripheral blood using Histopaque®−1077 (Sigma-Aldrich; Merck KGaA) according to the manufacturer's instructions. Blood samples from healthy donors were collected at the Azienda Unità Sanitaria Locale - IRCCS di Reggio Emilia (Reggio Emilia, Italy), between October 2017 and June 2018. The age range was 19–60 years (five female patients and 22 male patients). The use of PBMCs upon written informed consent was previously approved by the Local Ethics Committee of Reggio Emilia (Comitato Etico Provinciale; approval no. 2016/0025053).
IncuCyte® S3 Software 2020B (Essen Bioscience) was used to analyze and graph the time-course fluorescence data. The probes specific to
Data analysis was performed with GraphPad Prism 5.0 (GraphPad Software, Inc.). One-way ANOVA followed by Bonferroni's post hoc test (multiple comparisons) were performed. Data are represented as the mean ± standard error of the mean. Figures were prepared with Adobe Illustrator CS4 (Adobe Systems, Inc.). P<0.05 was considered to indicate a statistically significant difference.
It was observed that lymphoma cell lines displayed a characteristic profile of promoter methylation on specific targets, including tumor suppressors. A panel of nine target promoters were previously selected and assessed on a series of primary B lymphocytes sorted from healthy and tumor lymph nodes (
The epigenetic regulation of these targets was initially evaluated through the use of hypomethylating agents. Apoptosis was triggered by 5AZA and decitabine, as demonstrated by caspase-3/7 activation after 24 h (
DNMT1 was downregulated and PARP was cleaved upon treatment with either 5AZA or decitabine (
To assess the specific activity of hypomethylating agents in this study, global chromatin methylation was measured via a fluorescence immunoenzymatic assay with anti-5mC antibody. As expected, chromatin was demethylated upon treatment with 5AZA in both cell lines after 48 h and up to 7 days later (
The effect of decitabine treatment was also evaluated. Global chromatin methylation decreased after 48 h in the Toledo and NU-DUL-1 cell lines compared with the NT groups, but the levels of treated samples returned to the same levels observed in the NT groups after 7 days (
The gene expression of the three genes of interest was assessed by RT-qPCR. Only
These data collectively suggested that 5AZA and decitabine selectively affected only some regions of the genome, which was consistent with previous findings on colon cancer cells (
To investigate the role of specific DNMTs in the maintenance of methylation at the level of the selected targets, DNMT1 and DNMT3a were individually silenced and promoter methylation was assessed.
DNMT1 and DNMT3a were consistently and specifically downregulated at the RNA and protein levels 48 h after transfection with siDNMT1 and siDNMT3a compared with the scramble siRNA group (
Gene expression analysis showed that none of the KLF4, DAPK1 and SPG20 targets were affected by any of the silencing conditions (
To assess whether proliferation was affected by the knockdown of DNMT, Ki67 expression was determined by immunocytochemistry. The majority of the nuclei were Ki67-positive in all the conditions tested after 48 h of incubation (
It was demonstrated that genomic DNA was strongly demethylated by 5AZA and decitabine (
The data collected thus far demonstrated that the
To investigate the whole SPG20 locus, the data from an Infinium Human Methylation 450K BeadChip array was analyzed. The β-values for 28 DLBCL cell lines were filtered for the
To confirm the methylation status of this specific open sea region, primers were designed for MS-PCR on the probe cg15754752, located upstream of the TSS (
It is noteworthy to underline that the data obtained with an older type of array (Illumina 27K) in a colon cancer cell line and available through the repository ‘ArrayExpress’ confirm our findings (
Once it had been demonstrated that the
Hypomethylating drugs exhibit antitumoral activity in hematological cancers (
These previous studies indicate how gene and protein expression can be influenced by the methylation of CpG islands in a complex and context-dependent manner. The data presented in the present study are no exception to this since it demonstrated that the mechanism of action of DNMTs upon silencing are not entirely predictable and some specific targets associated with lymphoma proliferation are differentially and stably methylated compared with circulating, healthy PBMCs.
First of all, the observed dose-dependent downregulation of DNMT1 upon treatment with hypomethylating agents did not affect the methylation status of the key
In the model of the current study, silencing of either DNMT1, DNMT3a or both did not affect the levels of methylation nor the expression levels of
The present study focused on
Here, it was demonstrated, for the first time, that lymphoma cell lines displayed stable
Most importantly, the present study demonstrated that human PBMCs expressed Spartin protein (the product of
A meta-analysis recently published on patients with lymphoma evidenced how the hypermethylation of
The present data showed that
To conclude, the current data indicated that three loci,
The authors would like to thank Ms. Marina Grassi, Ms. Tonia de Simone (Laboratory of Translational Research, Azienda Unità Sanitaria Locale IRCCS, Reggio Emilia, Italy) and Dr Ione Tamagnini (Pathology Unit, Azienda Unità Sanitaria Locale IRCCS, Reggio Emilia, Italy) for their technical support. They are also thankful to Dr Minden's laboratory (Ontario Cancer Institute, Canada) for the gift of GNE-587170 cells.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. The datasets generated and/or analyzed during the current study are available in the Gene Expression Omnibus repository,
RF conceived the present study, performed most of the experiments and wrote the manuscript. VYC and MP performed the experiments. LaC provided the blood samples and performed some of the experiments. LuC obtained the genome-wide methylation data. FB and FM supervised the manuscript and contributed to data interpretation. All authors have read and approved the final manuscript. RF and VYC confirm the authenticity of all the raw data.
The use of PBMCs from healthy donors upon written informed consent was approved by the Local Ethics Committee of Reggio Emilia (Comitato Etico Provinciale; approval no. 2016/0025053).
Not applicable.
The authors declare that they have no competing interests.
Quantitative promoter methylation in two diffuse large B cell lymphoma cell lines and effects of hypomethylating agents. (A) Toledo and NU-DUL-1 cell lines were analyzed via EpiTect Methyl II qPCR. Data are reported as % promoter methylation and error bars represent SD (n=3). (B) Immunoblotting showing DNMT1, PARP and β-actin after 5AZA treatment for 48 h. (C) Immunoblotting showing DNMT1 and PARP and β-actin after decitabine treatment for 48 h. (D) Immunoblotting showing DNMT1, PARP and β-actin after 5AZA treatments for 7 days. (E) Immunoblotting showing DNMT1 and PARP and β-actin after decitabine treatments for 7 days. n=2. DNMT1, DNA methyltransferase 1; PARP, poly (ADP-ribose) polymerase; NT, not treated; KLF4, Krüppel-like factor 4; DAPK1, death associated protein 1; SPG20, spastic paraplegia 20; 5AZA, 5′-aza-cytidine; decitabine, 5′-aza-2-deoxycitidine.
Global chromatin methylation, gene-specific quantitative methylation and gene expression after 5AZA and decitabine treatment. (A) Chromatin methylation after 48 h or 7 days from a single administration of the indicated drug. ***P<0.0001 (n=3; one-way ANOVA). (B) Quantitative promoter methylation of
Effects of DNMT1- and DNMT3a-selective silencing on gene-specific quantitative methylation and expression. (A) Silencing efficiency after 48 h from transfection in Toledo and NU-DUL-1 cell lines as determined by RT-qPCR. *P<0.05, **P<0.01, ***P<0.0001 (n=3; one-way ANOVA). (B) Silencing efficiency 48 h after transfection in Toledo and NU-DUL-1 cell lines as determined by immunoblotting (n=2). (C) Gene expression levels of
Genome-wide methylation analysis by Infinium Human Methylation 450K BeadChip array and MS-PCR. (A) Heatmap of the β-values of