[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.

[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.

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.

Cisplatin or carboplatin? Neutrophil to lymphocyte ratio may serve as a useful factor in small cell lung cancer therapy selection.

The present study aimed to investigate the significance of the neutrophil to lymphocyte ratio (NLR) in peripheral blood of patients with small cell lung cancer (SCLC) when selecting a first-line treatment. A total of 73 patients with SCLC who had complete clinical data and sought treatment at Fujian Medical University Union Hospital between January 2014 and May 2016 were included. Data were retrospectively analyzed, utilizing a receiver operating characteristic curve to determine the NLR cut-off value. Out of the 73 patients, 39 were classified as high-NLR (NLR ≥3.80) and 34 as low-NLR (NLR <3.80). Compared with the high-NLR group, patients in the low-NLR group had a longer progression free survival (PFS); however, there was no statistically significant difference in overall survival (OS) time. Patients with a high NLR had a significantly longer PFS (P=0.021) and OS time (P=0.042) when treated with a etoposide/cisplatin (EP) therapy regimen, compared with those treated with etoposide/carboplatin (EC). PFS was the longest in the high-NLR patients with limited stage (LS; P=0.002). Among the patients receiving the EC regimen, the PFS of the low-NLR group was significantly longer compared with the high-NLR group (P=0.003). Patients in the low-NLR group who received thoracic radiotherapy had a longer PFS (P=0.011), when comparing patients in the low-NLR group who did not receive thoracic radiotherapy, and within this group the therapeutic effect of radiation was the greatest in LS patients. Compared with the high-NLR group, the low-NLR group patients who received cranial radiotherapy had a significantly longer PFS (P=0.039). For the initial evaluation of patients with SCLC, pre-treatment NLR may be of significance for selecting first-line chemotherapy agents. As the present study was retrospective and investigated a limited number of patients, further research and prospective studies are warranted.

Detection of circulating stage III-IV gastric cancer tumor cells based on isolation by size of epithelial tumor: using the circulating tumor cell biopsy technology.

BACKGROUND: To explore the correlations between circulating tumor cells (CTCs) and the patients' clinical characteristics and other tumor markers in stage III-IV gastric cancer patients, and assess the value of CTCs as a predictor of the efficacy of chemotherapy. METHODS: CTCs were measured in 42 patients with stage III-IV gastric cancer using the isolation by size of epithelial tumor (ISET) method. We analyzed the correlations between the number of CTCs, patients' pathological characteristics and peripheral blood tumor markers. Then divided the 42 patients into two groups, the progressive disease group (PD group) and the disease control rate group (DCR group), according to the efficacy of chemotherapy, and analyzed the differences in the CTC expression between the two groups. RESULTS: The threshold number of CTCs was closely related to the clinical stage (P=0.044), and was positively correlated with the value in U/mL of CA724 (P<0.05). The treatment response to cytotoxic chemotherapy in the high threshold number group was significantly poorer. CONCLUSIONS: CTCs technology based on ISET method has a high detection rate. CTCs are promising predictor for the evaluation and prediction of treatment responses in stage III-IV gastric cancer.

Role of miR-196 and its target gene HoxB8 in the development and proliferation of human colorectal cancer and the impact of neoadjuvant chemotherapy with FOLFOX4 on their expression.

The present study aimed to investigate the interaction between miR-196 and its target gene homeobox B8 (HoxB8) in colorectal cancer (CRC) cells, and the sensitivity of miR-196 and HoxB8 to fluorouracil, leucovorin and oxaliplatin (FOLFOX4) chemotherapy (1,200 mg/m2 fluorouracil, 200 mg/m2 leucovorin and 85 mg/m2 oxaliplatin). In total, 80 tissue samples were collected in the present study. In total, 50 patients undergoing preoperative chemotherapy completed at least 3 cycles (2 weeks per cycle) of 85 mg/m2 oxaliplatin (day 1) combined with a 2 h injection of 200 mg/m2 leucovorin (days 1 and 2), a bolus injection of 400 mg/m2 and 44 h continuous intravenous infusion of 1,200 mg/m2 fluorouracil. Complete response and partial response were included in the chemotherapy sensitive group (25 patients), and stable disease and progressive disease were included in the chemotherapy resistant group (25 patients). In addition, 30 patients without preoperative chemotherapy were examined for mRNA and protein expression of miR-196 and HoxB8. The expression of the mRNA and protein of miR-196 and HoxB8 was analyzed in 30 CRC and normal mucosa tissue samples. In addition, the expression of the mRNA and protein of miR-196 and HoxB8 was measured in 50 tissue samples obtained from patients that had received FOLFOX4 neoadjuvant chemotherapy. The expression levels of miR-196 and HoxB8 mRNA in CRC tissues were significantly increased compared with the corresponding normal mucosa tissue (P<0.05). The miR-196 mRNA was significantly correlated with lymph node metastasis, tumor stage and distant metastasis (P<0.05). miR-196 was indicated to be negatively correlated with HoxB8 mRNA expression (r=-0.458; P<0.05). The relative amount of miR-196 in the chemotherapy-sensitive group of patients was 0.949±0.691, which was increased compared with the chemotherapy-resistant group (0.345±0.536; P<0.01). The relative level of HoxB8 mRNA in the chemotherapy-sensitive group was 0.490±0.372, which was decreaesd compared with the chemotherapy-resistant group (0.725±0.438; P<0.05). HoxB8 protein expression level in the chemotherapy-sensitive group was decreased compared with the chemotherapy-resistant group (Z=-2.396; P=0.017). Overall, miR-196 was correlated with metastasis and prognosis, and HoxB8 was highly expressed in CRC tissues. The difference in the gene expression of miR-196 and HoxB8 may be associated with the sensitivity to FOLFOX4 for CRC patients. In addition, the highly expressed miR-196 increased the sensitivity of CRC cells to chemotherapy with FOLFOX4 by inhibiting HoxB8.

Knockdown of KLK11 inhibits cell proliferation and increases oxaliplatin sensitivity in human colorectal cancer.

It has been reported that kallikrein 11 (KLK11) is crucially involved in the development and progression of various types of cancer. However, the molecular mechanisms that underlie the involvement of KLK11 in aberrant colorectal cancer (CRC) cell growth remain largely unclear. The aim of the present study was to investigate the role of KLK11 and the effects of KLK11 on oxaliplatin (L-OHP) chemosensitivity by knocking down KLK11 in LOVO and HCT-8 cells. Loss-of-function assays revealed KLK11 inhibition significantly inhibited growth and induced apoptosis of CRC cells in vitro. Notably, further experiments found that knockdown of KLK11 expression increased the L-OHP chemosensitivity of CRC cells. KLK11 inhibition of increased L-OHP-induced apoptosis may be associated with activation of caspase-3 cleavage and the apoptosis signaling pathway. The present results indicated that KLK11 may be an potential target of interest for future research into therapies for CRC.

sMicroRNA-1290 inhibits cells proliferation and migration by targeting FOXA1 in gastric cancer cells.

BACKGROUND/AIMS: Gastric cancer is the third leading cause of cancer-related deaths in the world with high mortality rate due to the lack of markers in early detection and effective therapies. MicroRNAs (miRNAs), a critical part of epigenetic regulations in tumor, have been shown to be closely related to the initiation, development, invasion, metastasis and prognosis of gastric cancer. The present study aims to investigate the expression of miR-1290 in gastric tumor cells and to elucidate the target gene of miR-1290 in SGC-7901 gastric cancer cells. METHODOLOGY: The fluorescence in situ hybridization, real time PCR and Western blot were used to investigate the expression of miR-1290 in gastric tumor cells and clinical gastric tumor samples. The effect of miR-1290 expression on gastric tumor cells was studied using Synthetic miR-1290 inhibitor transfection, in vitro wound healing assay and flow cytometry analysis. Bioinformatics and Luciferase reporter assay were used to predict and validate the target gene of miR-1290. RESULTS: Our results revealed that miR-1290 was highly expressed in SGC-7901 gastric cancer cells as well as in clinical gastric cancer samples, which was correlated with clinical stages, depth of invasion and lymph node metastasis. Synthetic miR-1290 inhibitor transfection significantly inhibited the proliferation and migration of SGC-7901 cells. Bioinformatics analysis and luciferase reporter assay suggested that miR-1290 functioned in gastric cancer cells by targeting FOXA1 gene. CONCLUSION: miR-1290 promotes gastric tumor cells proliferation and metastasis through FOXA1, which could be used as a marker for diagnosis and a target for therapeutic intervention.

Paclitaxel combined with oxaliplatin as first-line chemotherapy for locally advanced or metastatic gastric cancer.

PURPOSE: This retrospective study was designed to evaluate the efficacy and safety of Paclitaxel (PTX) combined with Oxaliplatin (OXA) as first-line chemotherapy for locally advanced or metastatic gastric cancer (AGC). METHODS: Untreated patients with histologically confirmed AGC who received PTX at 135 mg/m(2) and OXA at 85 mg/m(2) every 2 weeks were studied. Antitumor activity was assessed by imaging and toxicities were evaluated. RESULTS: Thirty-nine (39) patients were enrolled. With 9.83 months median time of follow-up, 1 year OS rate was 42.0%. Complete response, partial response, stable disease and progressive disease was 2.6, 66.7, 17.9 and 12.0% respectively, the overall response rate was 69.2%. The mPFS was 8.5 months and the mOS 14.4 months. Grade 3/4 of toxicities included neutropenia (38.5%), febrile neutropenia (20.5%), vomiting (7.7%) and hypertransaminasemia (7.7%). Grade 2 peripheral neuropathy occurred in 33.3% patients. CONCLUSIONS: The combination of PTX combined with OXA is an active and safe regime for AGC and has a high overall response rate.

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.

Establishment of a predictive genetic model for estimating chemotherapy sensitivity of colorectal cancer with synchronous liver metastasis.

OBJECTIVE: We examined the whole genome expression profile in advanced colorectal cancer (ACC) patients who had received FOLFOX4 chemotherapy to establish a genetic biomarker model predicting chemotherapy sensitivity. METHODS: Eligible ACC patients were divided into two groups, based on postchemotherapy evaluation results: specifically, the sensitive group (experimental group) and the resistant group (control group). The genome expression profiles of colorectal cancer tissues were examined using DNA microarray analysis, and differential gene expression was identified using a significance analysis of the microarray. The probe signal log ratios were used to produce the area-under-the-curve, sensitivity, and specificity for candidate genes. Genes exhibiting differential expression and significant predictive power were used to simulate a genetic model for estimating chemotherapy sensitivity. RESULTS: Totally, 30 ACC patients were eligible for the study, 13 assigned to the experimental group and 17 to the control group. In total, 30 genes showing significant differential expression were identified. Seven candidate genes (NKX2-3, FXYD6, TGFB1I1, ACTG2, ANPEP, HOXB8, and KLK11), which exhibited positive or negative correlations, were incorporated into a genetic model, with an overall accurate predication rate of 93.3%. CONCLUSIONS: The predictive model involving the seven genes listed had high accuracy in estimating chemotherapy sensitivity to the FOLFOX4 regimen.

[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.

[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.

[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.

[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.

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.

[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.