AT-rich interactive domain1A determines sensitivity to oxaliplatin in gastric cancer cells.

BACKGROUND: Gastric cancer is a highly heterogeneous disease and its traditional histopathological classification is difficult to meet clinical needs. Oxaliplatin is an antitumor drug with high efficiency and low toxicity. Therefore, the insensitivity or secondary drug resistance of oxaliplatin to gastric cancer is vital for tumor progression. The aim of this study was to investigate the sensitivity of gastric cancer cells to oxaliplatin after ARID1A (AT-rich interactive domain1A gene) gene silencing. METHODS: MGC-803 and AGS cells were selected as gastric cancer cells for study. ARID1A protein and mRNA expression was detected by Western blot and quantitative reverse-transcription PCR (qRT-PCR). The short hairpin RNA (shRNA) fragment of ARID1A gene silencing was constructed and introduced into gastric cancer cells. The cell proliferation activity was calculated using CCK8 and the IC50 was calculated. The flow cytometry was used to detect the cell cycle and apoptosis rate. The ability of cell invasion was detected by transwell method. Cells were treated with different concentrations of oxaliplatin. RESULTS: The proliferation of gastric cancer cells was promoted by ARID1A gene silencing (P<0.01), the quantity of cells in S phase increased (P<0.05), and the invasive ability increased (P<0.05). After treatment with oxaliplatin at different concentrations, ARID1A gene silencing reduced the inhibition rate of oxaliplatin on gastric cancer cells and apoptosis rate (P<0.05), and increased IC 50 (P<0.01). CONCLUSIONS: ARID1A gene silencing, a factor promoting proliferation of gastric cancer cells, would reduce the sensitivity of gastric cancer cells to oxaliplatin, which can provide a basis for the exploration of targeted drugs for individualized treatment of gastric cancer.

Epigallocatechin-3-gallate and trichostatin A synergistically inhibit human lymphoma cell proliferation through epigenetic modification of p16INK4a.

DNA methylation and histone deacetylation play important roles in the occurrence and development of cancers by inactivating the expression of tumor suppressors, including p16(INK4a), a cyclin-dependent kinase inhibitor. The present study investigated the effect of epigallocatechin-3-gallate (EGCG) alone or in combination with trichostatin A (TSA) on p16(INK4a) gene expression and growth in human malignant lymphoma CA46 cells. CA46 cell viability and cell cycle were analyzed; methylation of the p16(INK4a) gene was assessed by nested methylation-specific PCR (n-MSP). p16(INK4a )mRNA and protein expression was determined by real-time quantitative PCR and western blot analyses, respectively. Both EGCG and TSA alone inhibited CA46 cell proliferation; the combined treatment (6 µg/ml EGCG and 15 ng/ml TSA) significantly reduced CA46 cell proliferation from 24 to 96 h (all P<0.001). Cells treated with 24 µg/ml EGCG or the combination treatment (6 µg/ml EGCG and 15 ng/ml TSA) had lower proliferative indices when compared to the other groups. Co-treatment with EGCG and TSA decreased p16(INK4a) gene methylation, which coincided with increased p16(INK4a) mRNA and protein expression. Thus, EGCG and TSA synergistically reactivate p16(INK4a) gene expression in part through reducing promoter methylation, which may decrease CA46 cell proliferation.

[Methylation status of IEX-1 gene promotor CpG island in malignant hematopoietic cell lines].

This study was to purposed to explore the methylation status changes of IEX-1 gene promoter CpG island and its relevance with occurrence of hematologic malignancies. The methylation status of IEX-1 gene promoter CpG island in 9 NB4, HL-60 U937, Raji, CA46, Jurkat, K562, CEM and Molt4 hematologic malignant cell lines was detected by using methylation-specific PCR, the methylation status of IEX-1 gene promoter CpG island in normal peripheral blood mononuclear cells treated by M. sssI enzyme and the methylation status of IEX-1 gene promoter CpG island in normal peripheral blood mononuclear cells untreated were used as positive and negative controls respectively. The results showed that the hypermethylation of IEX-1 gene promoter CpG islands was detected in NB4, Molt4 and Raji cell lines, as well as in normal peripheral blood mononuclear cells treated by M. sssl enzyme; the partial methylation status was found in CA46, CEM, U937, K562, HL-60 and Jurkat cell lines; the unmethylation status was observed in untreated normal peripheral blood mononuclear cells. It is concluded that the changes of methylation status of gene IEX-1 promoter CpG island correlates with hematologic malignancies to a certain extent.

[Mechanism of As(2)O(3) on hdpr1 gene demethylation in Jurkat cell line].

This study was purposed to investigate the effect of As(2)O(3) on the demethylation of anti-oncogene-hdpr1 gene of acute lymphoblastic leukemia cell line Jurkat in vitro and its mechanism. The inhibitory effect of As(2)O(3) on the proliferation of Jurkat cells was assayed by CCK-8; the change of Jurkat cell cycle was detected by flow cytometry before and after using As(2)O(3); the effect of As(2)O(3) on the methylation model of hdpr1 gene was analyzed by methylation-specific PCR, and the effect of As(2)O(3) on the expression of hdpr1 mRNA was analyzed by semiquantitative RT-PCR. The results showed that the proliferation rate of Jurkat cells was decreased significantly after being treated with As(2)O(3), and in dose-and time-dependent manner; As(2)O(3) blocked Jurkat cell cycle in G(0)/G(1) phase in dose-dependent manner. As(2)O(3) could reverse hypermethylation of hdpr1 gene and induce its mRNA reexpression, and down-regulate the dnmt1, dnmt3a, dnmt3b mRNA expression level also in dose-dependent manner. It is concluded that the As(2)O(3) suppresses the proliferation of Jurkat cells and blocks cell cycle is G(0)/G(1), its possible mechanism may be down-regulating mRNA expression level of dnmt1, dnmt3a and dnmt3b, induce demethylation of hdpr1 gene from abnormal hypermethylation status and activates its reexpression.

[The significance of methylation status of secreted frizzled-related protein gene promoter in acute leukemia].

OBJECTIVE: To investigate the relationship between promoter hypermethylation of secreted frizzled-related protein (SFRP) gene and acute leukemia (AL). METHODS: We examined the promoter methylation status of SFRP1, 2, 4 and 5 in primary or relapsed AL patients, cell lines (HL60, NB4, Molt-4 and Jurkat) and peripheral blood mononuclear cells from healthy people with methylation-specific PCR (MSP). RESULTS: None of the normal mononuclear cells showed methylation of any SFRP genes. The frequencies of aberrant methylation among the samples were 33.9% (20/59) for SFRP1, 23.7% (14/59) for SFRP2, 6.8% (4/59) for SFRP4 and 10.2% (6/59) for SFRP5 in acute myelocytic leukemia (AML), and 39.3% (11/28) for SFRP1, 28.6% (8/28) for SFRP2, 25.0% (7/28) for SFRP4 and 32.1% (9/28) for SFRP5 in acute lymphoblastic leukemia (ALL). Hypermethylation of SFRP1, 2, 5 genes were present in the 4 AL cell lines. SFRP4 was methylated in NB4, Molt-4 and Jurkat cell lines. However, methylation and unmethylation of SFRP4 were both detected in HL60. CONCLUSIONS: Hypermethylation of SFRP genes is a common event in the evolution of AL. Methylation of SFRP genes might serve as potential independent biomarkers for early detection of AL.

[Effect of triptolide on reversing hypermethylation of apc gene in Jurkat cells and its possible mechanisms].

This study was aimed to investigate the effect of traditional Chinese medicine, Triptolide (TPL) on reversing hypermethylation of antioncogene (apc gene) in acute lymphoblastic leukemia cell line Jurkat in vitro and to explore its mechanisms. The effects of TPL on cell growth, proliferation and cell cycle were detected by growth curve, MTT assay, colony formation test and flow cytometry, respectively. The effect of TPL on apc gene methylation of Jurkat cells was analyzed by nested methylation specific PCR; the expressions of apc gene, dnnt3a, dnmt3b mRNA were measured by RT-PCR; the protein expression of apc gene was detected by Western blot. The results showed that as compared with untreated control cells, the TPL of different concentrations could significantly inhibit growth and proliferation of Jurkat cells in dose-and time-dependent manners with IC50 19.7 ng/ml at 48 hours. All cytosines in CpG dinucleotides in untreated Jurkat cells had no changed, while all cytosines in Jurkat cells treated with TPL had been converted to thymidine suggesting the methylation of apc gene in Jurkat cells. The TPL could reverse hypermethylation of apc gene and induced the mRNA and protein expression of apc gene in dose-dependent manner. It is concluded that the small dose of TPL can obviously suppress the proliferation of Jurkat cells, activate and up-regulate the expression of apc gene through demethylation of apc gene resulting from DNMT and/or direct action, thereby inhibit the proliferation rate of Jurkat cells.

Arsenic trioxide inhibits DNA methyltransferase and restores expression of methylation-silenced CDKN2B/CDKN2A genes in human hematologic malignant cells.

Cyclin-dependent kinase inhibitors CDKN2B and CDKN2A are tumor suppressor genes that are frequently dysregulated in a variety of cancers. Aberrant regulation via DNA hypermethylation causes gene silencing. Arsenic trioxide has been successfully used to treat malignant, hematopoietic diseases and is known to act by induction of apoptosis and inhibition of cellular proliferation. However, arsenic trioxide has been recently reported to act via inhibition of DNA hypermethylation in some solid tumors. The goal of this study was to explore the mechanism of arsenic trioxide induced demethylation of the CDKN2B and CDKN2A promoters in the hematologic malignant cell lines Molt4, MUTZ-1, U937, U266 and CA46. We used bisulphate modification and nested-methylation specific PCR to determine the levels of methylated and unmethylated promoter sequences in untreated and As2O3-treated cells. We used semi-quantitative RT-PCR and immunoblotting to quantify CDKN2B and CDKN2A mRNA and protein levels, respectively. We measured DNMT activity in nuclear extracts of untreated and treated cells using radiolabeled SAM as a methyl donor. The CDKN2B promoter was hypermethylated in Molt4 and MUTZ-1 cells, while the CDKN2A promoter was hypermethylated in U937, U266 and CA46 cells. As2O3 treatment caused demethylation associated with an increase in mRNA levels of the CDKN2B and CDKN2A genes. We also demonstrated a concomitant inhibition in DNMT activity and DNMT mRNA levels in As2O3-treated cells. In summary, As2O3 restored expression levels of tumor suppressor genes in hematologic malignant cells by causing promoter demethylation along with an inhibition of DNMTs 1, 3a and 3b.

[Inhibitory effect of VPA on multiple myeloma U266 cell proliferation and regulation of histone acetylation].

This study was aimed to investigate the effects of sodium valproate (VPA) on the proliferation and regulation of histone acetylation of multiple myeloma cell line U266. U266 cells were treated with VPA. Cell proliferation was determined by CCK-8 assay, and cell cycle were analyzed by flow cytometry (FCM). The expression level of HDAC1 mRNA was detected by RT-PCR, and the protein levels of HDAC1 and histone H3, H4 acetylation was detected by Western blot. The results showed that the VPA inhibited the proliferation of U266 cells in concentration-and time-dependent manners.After exposure to different concentrations of VPA for 48 hours, the proportion of G(0)/G(1) cells increased, while the proportion of S phase cells decreased. The cell cycle was arrested obviously in G(0)/G(1) phase (p < 0.05). The expression of HDAC1 mRNA was inhibited, and the protein level of HDAC1 was down-regulated, while the histone H3/H4 acetylation was up-regulated in U266 cells. It is concluded that the VPA can inhibit cell proliferation of U266 and induce G(0)/G(1) phase arrest. The increase of histone H3/H4 acetylation resulting from inhibiting HDAC1 by VPA might be considered as a possible mechanism.

[Effect of epigallocatechin-3-galate on human acute monocytic leukemia cell line U937 and its relevant mechanism].

The purpose of this study was to explore the effect of epigallocatechin-3-galate (EGCG) on acute monocytic leukemia cell line U937 and its relevant mechanism. The viability of U937 cells were assayed by SRB method. The cell cycle of U937 cells was analyzed by flow cytometry. The mRNA and protein expression of p16 gene were detected by RT-PCR and Western blot, respectively. Methylation level of U937 cells was analyzed by n-MSP. The mRNA expression of DNA methyltransferase 1 (DNMT1), DNMT3A and DNMT3B genes were analyzed by RT-PCR. The results showed that EGCG could inhibit the growth of U937 cells significantly in dose-and time-dependent manners (r=0.71), and induce the G0/G1 arrest of U937 cells in dose-dependent manner. EGCG could up-regulate the mRNA and protein expression of P16 gene in U937 cells in dose-dependent manner. EGCG could down-regulate the methylation level of p16 gene in U937 cells in dose-dependent manner. EGCG could down-regulate the mRNA expression of DNMT3A, DNMT3B genes, while did not influence the mRNA expression of DNMT1 gene. It is concluded that EGCG can up-regulate the mRNA and protein expression of p16 gene by demethylation or/and by inhibiting DNMT3A and DNMT3B genes, leading, in turn, to G0/G1 arrest and growth inhibition of U937 cells.

[Arsenic trioxide reverses hypermethylation of p16 and activates its transcription in malignant lymphoma cell line CA46].

This study was aimed to investigate the reversing effect of arsenic trioxide (As2O3) on methylation status and the regulatory effect on transcription of malignant lymphoma cell line CA46 p16 gene as well as their possibe mechanisms. The hypermethylated malignant lymphoma cell line CA46 was used as a subject of experiment for studying relation of gene methylation with expression. The effect of As2O3 on the proliferation and viability of CA46 was detected by SRB method, the change of p16 methylation status after exposure to As2O3 was determined by nMSP, the expressions of p16, DNMT1, DNMT3A, DNMT3B mRNA were assayed by RT-PCR, the influence of As2O3 on CA46 cell cycle was analyzed by flow cytometry using analytical method for DNA ploidy. The results showed that the methylation level of p16 gene was obviously reduced after treatment with As2O3 for 72 hours and the hypermethylation of p16 gene was successfully reversed; the expression of p16 gene in untreated (control) group was low while it was enhanced in treated groups; the gray scale ratios of p16 gene to beta-actin in groups treated with As2O3 of concentration 0.5, 1.0 and 2.0 micromol/L were 0.33+/-0.10, 0.57+/-0.11 and 0.67+/-0.09 respectively, exhibiting a significant difference in comparison with 0.73+/-0.13 of positive control (p<0.01); as compared with the untreated group, the expression of DNMT3A and DNMT3B in treated groups was obviously down-regulated in a concentration-dependent manner, while expression of DNMT1 was nearly unchanged; as compared with control, all the 3 different concentrations of As2O3 could inhibit the proliferation of CA46 cells and increase the cell number in G0/G1 phase. It is concluded that the As2O3 may up-regulate the expression of p16 gene, recover the activity of p16 gene, thereby promote the regulatory function on cell cycle resul-ting in arrest of cells in G0/G1 phase and inhibit growth of tumor cells through depressing the expression of DNMT3A and DNMT3B and/or directly reversing the methylation status of p16 gene.

[The establishment and evaluation of diagnostic accuracy of AllGlo(TM) probe-based techniques for invasive aspergillosis.].

OBJECTIVE: To establish and evaluate a diagnostic technique based on the AllGlo(TM) probe for the invasive aspergillosis. METHODS: With the self-designed AllGlo(TM) probes and primers and the standards, two standard curves of the real-time PCR based on AllGlo(TM) probes were established respectively for A. flavus and A. fumigatus, then its specificity, sensitivity and reproducibility were evaluated respectively. RESULTS: The findings indicated that the standard curve of A. flavus was Y = -3.003X + 36.825, and A.fumigatus' was Y = -3.052X + 38.016, and their interassay coefficient of variation respectively were 15.60% and 12.94%, suggesting a good reproducibility. The lowest spore concentration they could be detected was 10 CFU/ml, which equated to 100 - 1000 copies of internal transcribed spacer (ITS)2 genes, suggesting a good sensibility. They didn't have cross-positive reaction with other fungus, human genome and bacteria, which indicated a good specificity. CONCLUSION: The diagnostic technique based on the AllGlo(TM) probe for the invasive aspergillosis possessed a good sensitivity, good specificity and deadly accuracy.

[Methylation status of CpG islands in secreted frizzled-related protein gene promoter region of malignant hematopoietic cell lines].

To investigate the methylation status of CpG islands of the secreted frizzled-related protein (SFRP) gene promoter region in malignant hematopoietic cell lines, and to explore the possible relationship of CpG abnormal methylation status with pathogenic mechanism of hematologic malignancies. Methylation-specific PCR was used to detect the status of SFRP gene promoter region in nine malignant hematopoietic cell lines and peripheral blood mononuclear cells from healthy people. The results indicated that hypermethylation of 2 genes coding for SFRP1 and 2 were present in nine malignant hematopoietic cell lines, however, methylation and unmethylation of SFRP4 were both detected in CA46, HL60 and U937 cell lines, and SFRP5 in U266 as well. None of the normal mononuclear cells showed methylation of SFRP 1-5 genes. It is concluded that the hypermethylation of SFRP genes is related to the evolution of malignant hematopoiesis. Methylation of SFRP genes may serve as potential independent biomarkers for early detection of hematologic malignancies.

[Detection of APC gene promoter methylation in hematological malignant cell lines by nested-methylation specific polymerase chain reaction].

This study was aimed to investigate the efficiency of nested methylation specific polymerase chain reaction (nMS-PCR) for detecting the APC gene promoter methylation and to clarify the roles of methylation in genesis and development of hematologic malignancies, as well as to screen the hematologic malignant cell lines with hypermethylation of APC gene promoter to use as an ideal cell model for exploring the relationship between gen methylation and gene expression. The genome DNA of 10 cell lines modified with bisulfide was amplified and the methylation status of APC gene promoter was detected by using nMS-PCR. The results showed that the methylation of APC gene promoter was detected in Jurkat cells, while could not be detected in CA46, U266, Molt4, K562, HL-60, CEM, AKR, U937 and Raji cell lines. In conclusion, APC gene methylation in hematological malignant cell lines can be accurately detected by nMS-PCP method, which is simple method for detecting methylation status of various hematological malignant cell lines.

[Detection of p16 gene methylation status in adult patients with acute leukemia by using n-MSP].

The study was aimed to explore the relationship between patterns of methylation or deletion and the development of acute leukemia, and further to clarify the possible mechanism in the development of adult acute leukemia. Nested methylation-specific polymerase chain reaction (n-MSP) was adopted to analyze p16 gene methylation or deletion patterns in 82 adult acute leukemia patients with different subtypes and stages. The results indicated that rate of p16 gene methylation was 39.0% in 82 adult acute leukemia patients, among them, 41.4% in acute myelogenous leukemia (AML) and 33.3% in acute lymphoblastic leukemia (ALL). It were found that 36.6% of de novo AL patients and 54.5% of relapsed AL patients developed the hypermethylation of p16 gene. Out of the 82 patients, 6 seemed to have deletion of p16 gene, including 1 AML (1.7%) and 5 ALL (20.8%). There were no hypermethylation or deletion of p16 gene in the 16 controls. It is concluded that methylation of p16 gene may play a more important role than homozygous deletion of p16 gene in the leukemogenesis and progression of adult acute leukemia.