LATS1 Promotes B-ALL Tumorigenesis by Regulating YAP1 Phosphorylation and Subcellular Localization.

OBJECTIVE: YAP1 plays a dual role as an oncogene and tumor suppressor gene in several tumors; differentiating between these roles may depend on the YAP1 phosphorylation pattern. The specific function of YAP1 in B cell acute lymphoblastic leukemia (B-ALL), however, is currently unclear. Thus, in the present study, the role of YAP1 in B-ALL was investigated using relevant cell lines and patient datasets. METHODS: The effects of shRNA-mediated knockdown on YAP1 and LATS1 levels in the NALM6 and MOLT-4 cell lines were examined using Western blotting, quantitative real-time polymerase chain reaction, flow cytometry, immunostaining, and nude mouse subcutaneous tumorigenesis experiments. Gene expression levels of Hippo pathway-related molecules before and after verteporfin (VP) treatment were compared using RNA-Seq to identify significant Hippo pathway-related genes in NALM6 cells. RESULTS: Patients with ALL showing high YAP1 expression and low YAP1-Ser127 phosphorylation levels had worse prognoses than those with low YAP1 protein expression and high YAP1-Ser127 phosphorylation levels. YAP1-Ser127 phosphorylation levels were lower in NALM6 cells than in MOLT-4 and control cells; YAP1 was distributed in the nuclei in NALM6 cells. Knockdown of YAP1 inhibited MOLT-4 and NALM6 cell proliferation and arrested the NALM6 cell cycle in the G0/G1 phase. Before and after VP treatment, the expression of the upstream gene LATS1 was upregulated; its overexpression promoted YAP1-Ser127 phosphorylation. Further, YAP1 was distributed in the plasma. CONCLUSION: LATS1 may downregulate YAP1-Ser127 phosphorylation and maintain B-ALL cell function; thus, VP, which targets this axis, may serve as a new therapeutic method for improving the outcomes for B-ALL patients.

Genistein-induced Anticancer Effects on Acute Leukemia Cells Involve the Regulation of Wnt Signaling Pathway Through H4K20me1 Rather Than DNA Demethylation.

OBJECTIVE: To investigate the effects and mechanisms of genistein on the gene expression in the Wnt pathway in acute leukemia (AL) cells. METHODS: The expression of Wnt pathway genes and cell cycle-related genes were analyzed in two AL cell lines. Pyrophosphate sequencing was performed to determine the methylation degree. Then, the enrichment of H4K20me1 and H3K9ac was determined using ChIP-qPCR. Flow cytometry was used to analyze the cell cycle. RESULTS: The IC50 of genistein in the two AL cell lines was lower than that for the bone marrow mesenchymal stem cell line. Genistein upregulated H4K20me1, KMT5A and Wnt suppressor genes, including Wnt5a, and downregulated the downstream target genes of Wnt, such as c-myc and ß-catenin. The methylation degree and H3K9ac enrichment in the Wnt5a promoter region remained unchanged. However, the enrichment of H4K20me1 in the Wnt5a promoter and coding regions increased. In addition, genistein upregulated Phospho-cdc2, Myt1, Cyclin A, Cyclin E2, p21 and Phospho-histone H3, but downregulated Phospho-wee1. Cell cycle arrest was induced in the G2/M phase. CONCLUSION: Genistein inhibits the activation of the Wnt pathway by promoting the expression of Wnt5a through the activation of KMT5A and enrichment of H4K20me1 in the Wnt5a gene promoter and coding regions, rather than demethylation. Genistein also blocks the cell cycle in the G2/M phase. Therefore, genistein is a potential anti-leukemia drug.

Adjunctive Nonconvulsive Electrotherapy for Patients with Depression: a Systematic Review.

The efficacy and safety of adjunctive nonconvulsive electrotherapy (NET) for patients with depression are undetermined. This systematic review was conducted to examine the efficacy and safety of adjunctive NET for patients with depression. Chinese (WanFang and Chinese Journal Net) and English (PubMed, EMBASE, PsycINFO and the Cochrane Library) databases were systematically searched from their inception until Jan 27, 2021 by three independent investigators. One randomized controlled trial (RCT) with 3 treatment arms (n = 108) and two observational studies (single-group, before-after design, n = 31) were included. In the RCT, the antidepressant efficacy of NET on depression was similar to that of electroconvulsive therapy (ECT) (P > 0.05) but with significantly fewer neurocognitive impairments as measured by the Repeatable Battery for the Assessment of Neuropsychological Status (RBANS) (P < 0.05). In two observational studies, the 17-item Hamilton Depression Rating Scale (HAMD-17) scores decreased significantly from baseline to post-NET (all Ps < 0.05), without adverse neurocognitive effects. In the RCT, adverse drug reactions (ADRs) were not separately reported among the 3 treatment arms but a similar rate of discontinuation was reported. The currently available limited evidence from 3 studies suggests that NET as an adjunctive treatment may be a safe, well-tolerated, effective therapy for depression without serious neurocognitive impairments.

[A Case-Control Study on Receptor Gene Polymorphism and Risk Suffering from Adult Acute Leukemia in Fujian Area].

OBJECTIVE: To investigate the correlation of receptor gene (P2X7, VDR and SLC19A1) polymorphisms with risk suffering from acute leukemia (AL) in Fujian area. METHODS: Ninety-three cases of newly diagnosed AL as AL group and 90 persons not suffered from hematologic and other tumors as control group were selected and used for comparative analysis of receptor gene polymorphisms and risk suffering from AL between case and control groups. The bone marrow and peripheral blood were collected, from which the DNA was extracted. The PCR-RFLP was used to detect 8 SNP sites (P2X7: rs208294, rs2230911, rs3751143; VDR: rs2228570, rs7975232; SLC194A1: rs1051266, rs1131596, rs3788200) of receptor genes related with the environment response, and the genotypes analysis was used to the correlation of receptor gene polymorphisms with risk suffering from adult AL. RESULTS: The unvariate logistic analysis showed that as compared with control group, P2X7 rs208294 T>C mutation and rs3751143 A>C mutation in codominant model, dominant model and over-dominant model were higher in case group, moreover the differences were statistically significant (P<0.01, P<0.05 and P<0.05, respectively), which suggested that they could reduce the risk suffering from AL. The recessive inheritance model showed that SLC1941 rs1131596 G>A mutation could increase the risk suffering from AL (P<0.05). The stepwise multivariate logistic regression analysis showed that there was still statistically significant difference in P2X7 rs208294 mutation between case group and control group (P<0.05), moreover, the heterozygous mutation (CT) could decrease the risk suffering from AL, showing the better protective effect, compared with homozygous mutation(CC). CONCLUSION: The P2X7 rs208294 T>C mutation is one of protective factors against adult acute leukemia.

Erratum to: Genistein-induced Anticancer Effects on Acute Leukemia Cells Involve the Regulation of Wnt Signaling Pathway Through H4K20me1 Rather Than DNA Demethylation.

The article "Genistein-induced Anticancer Effects on Acute Leukemia Cells Involve the Regulation of Wnt Signaling Pathway Through H4K20me1 Rather Than DNA Demethylation", written by Hua-rong ZHOU, Jian-zhen SHEN, Hai-ying FU, Feng ZHANG was originally published electronically on the publisher's internet portal on October 2021 without open access. With the author(s)' decision to opt for Open Choice, the copyright of the article is changed to © The Author(s) 2021 and the article is forthwith distributed under a Creative Commons Attribution 4.0 International License ( https://creativecommons.org/licenses/by/4.0/ ), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.The original article has been corrected.

Upregulation of long non-coding RNA MALAT-1 confers poor prognosis and influences cell proliferation and apoptosis in acute monocytic leukemia.

Metastasis-associated lung adenocarcinoma transcript 1 (MALAT-1), a long non-coding RNA, has been documented to be a new prognostic marker and gene regulator in several types of cancer, but its potential involvement in acute myeloid leukemia (AML) remains unclear. This study investigated the expression and functional role of MALAT-1 in AML. MALAT-1 expression was assessed by real-time quantitative PCR. After lentiviral-mediated MALAT-1 knockdown, the proliferation of AML cells was determined by CCK-8 and colony formation assays. Cell cycle progression and apoptosis were evaluated by flow cytometry and the expression of caspase-3, -8 and -9 was assessed by western blot analysis. We found that MALAT-1 expression in patients with acute monocytic leukemia (M5) was significantly increased when compared with that of healthy controls, and the overall survival of M5 patients with high MALAT-1 expression was markedly reduced when compared with the overall survival of patients with low MALAT-1 expression. The analysis of cellular experiments showed that MALAT-1 silencing decreased the proliferation of M5 cells (U-937 and THP-1), inhibited cell cycle progression and increased apoptosis. Taken together, these findings suggest that high MALAT-1 expression is closely associated with poor prognosis in M5 patients and may play a role in leukemia cell proliferation and apoptosis, and may serve as a promising theranostic marker.

Relationship between epigenetic changes in Wnt antagonists and acute leukemia.

The present study was designed to investigate the relationship among epigenetic changes in Wnt antagonists, histone H4K20me1 and the expression of tumor-suppressor genes in acute leukemia (AL) to better understand the pathogenesis of leukemia. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed to detect messenger RNA (mRNA) expression levels of Wnt antagonists (Wnt5a, HDPR1, DKK1 and DKK3) in patients with AL and in normal controls; pyrophosphate sequencing was performed to detect the methylation status of the Wnt5a promoter; and western blotting was performed to detect the overall expression levels of Wnt5a protein and histone H4K20me1 in patients with acute myeloid leukemia (AML) and in normal controls. The relationship between Wnt5a protein expression and histone H4K20me1 was analyzed. Chromatin immunoprecipitation-qPCR (ChIP-qPCR) was performed to investigate the recruitment of H4K20me1 and SET8 to the Wnt5a promoter and coding regions. Our results demonstrated that the expression levels of Wnt antagonists were generally low in AML, but showed differential expression in acute lymphocytic leukemia (ALL). In most cases of AML, methylation of the Wnt5a promoter was observed and Wnt5a protein expression was low. In some cases of AML, the overall level of H4K20me1 protein was higher than that in normal controls. In addition, Wnt5a expression was positively correlated with H4K20me1 expression and was unrelated to the methylation status of its promoter. Moreover, H4K20me1 and SET8 were enriched in the Wnt5a promoter region and coding region. By contrast, Wnt5a expression was unrelated to H4K20me1 expression in normal controls. Moreover, we observed that the methylation of Wnt antagonists was often found in patients with AL, particularly those with AML, whereas the extent of methylation was variable in ALL patients. Wnt5a expression was positively correlated with the enrichment of H4K20me1 and SET8 at the Wnt5a promoter and coding regions. H4K20me1 increased Wnt5a expression by promoting transcription initiation and elongation.

[Application of Bisulfite Sequencing PCR in Detecting the Abnormal Methylation of Suppressor Gene of Wnt Signaling Pathway in Acute Promyelocytic Leukemia].

OBJECTIVE: To detect the abnormal methylation of the CPG island in the suppressor gene promoter region of the Wnt signaling pathway in cell strain NB4 of the acute promyelocytic leukemia by using the bisulfite sequcucing PCR(BSP), to screan the hyper-methylated suppressor gene of the Wnt signaling pathway and to evaluatc the potency of BSP in the methylation study. METHODS: The strain NB4 cells of the acute promyelocytic leukemia patients were used as the object, the mononuclear cells from 20 normal persons were used as the controls. The DNA was extracted and processed by bisulfite, the target sequences were amplified with PCR, then the abnormal methylation of the suppressor genes of the Wnt signaling pathway in the NB4 cells was analyzed by BSP, and the advantage and disadvantage of BSP were evaluated by comparison with the Methylation specific PCR and Pyrosequencing. RESULTS: The methylated rate of suppressor genes of the Wnt signaling pathways in the NB4 cells detected by BSP was as follows: the gene WIF1 95.26%, the gene DKK3 86%, the gene SFRP1 81.67%, the gene SFRP2 95.71%, the gene SFRP4 85%, and the gene SFRP5 95%; while the methylations in the control group were respectively as follows: the gene WIF-1 1.5%, the gene DKK3 4.2%, the gene SFRP1 0%, the gene SFRP2 0.9%, the gene SFRP4 2.5%, and the gene SFRP5 1.75%. A more significant methylation happened in the suppressor genes promoter of the Wnt signaling pathway in the NB4 cells as compared with the control group. CONCLUSION: Many hypermethylated suppressor genes are found in the Wnt signaling pathway of the acute promyelocytic leukemia NB4 cells, which may be served as one of the early diagnosis index and therapeutic target of the acute promyelocytic leukemia.

Role of HOTAIR in the diagnosis and prognosis of acute leukemia.

HOX antisense intergenic RNA (HOTAIR), a long non-coding RNA, plays an important role in the development of many types of cancers. Its function in acute leukemia (AL), however, has not been examined. The present study investigated the role of HOTAIR and its downstream genes in AL, and determined whether it could act as a molecular marker for prediction of leukemia development and prognosis. Real-time quantitative PCR was used to examine the expression of each gene in the HOTAIR signaling pathway in AL patients. The relationship between expression of HOTAIR and downstream genes and AL prognosis was analyzed. Expression of HOTAIR in patients with acute monocytic leukemia (M5) was increased as compared to controls (P<0.05). Compared to patients with low HOTAIR expression, overall survival and event-free survival of patients with high HOTAIR expression was significantly reduced. In addition, the expression of downstream genes in the HOTAIR signaling pathway including EZH2, LSD1, DNMT3A and DNMT3B was significantly increased in AL patients, and showed a significant positive correlation with high expression of HOTAIR (P<0.05). In conclusion, HOTAIR was closely related with a poor prognosis in AL patients. It may be involved in the development of leukemia by mediating methylation of DNA and histones.

[Analysis of varieties and standards of Primulaceae medicinal plants used in Tibetan medicine].

In this paper, the varieties and origin of Primulaceae plants that used in Tibetan medicine were analyzed. The results showed that there were 3 genera and 44 species (including the varieties) of Primulaceae plants were recorded in the relevant literatures. Among them, 17 varieties were recorded in Tibetan names, 24 varieties were recorded in Chinese names and 1 variety was used in both of them. In current quality criteria of standards at all levels in China country, 6 varieties were recorded in Tibetan names and 6 original plants were involved, which were 35% and 14% of them respectively. Seventeen varieties were recorded in Chinese name and 7 original plants were involved, which were 30% and 16% of them respectively. In Tibetan medicine standards and literatures, there were big differences between Tibetan names and Chinese names which were translated from Tibetan names and its original plants. There were only regulations of morphological identification and microscopic authentication, so the standards were very inadequate. Therefore, through literatures research, resources and current situation investigations, combining the research and specification of the name and original of Tibetan medicine, the level of normalization and standardization could be enhanced, the stable and controllable safety and utility in clinical medication could be ensured to promote advancement of industry technology Tibetan medicine.

[Analysis of varieties and standards of labiatae medicinal plants used in Tibetan medicine].

In this paper, an analysis was made on the varieties and standards of labiatae medicinal plants used in Tibetan medicine. The results showed 71 species of labiatae plants in 21 genera (including varieties) recorded in relevant literatures, involving 44 varieties of medicinal materials. Specifically, seven species (9.9%) were intersected with traditional Chinese medicines (TCM), 19 varieties (43%) were recorded in Chinese medicinal material standards at all levels, and 27 species (38%) were source plants. In Tibetan medicine standards and literatures, there are great differences between Tibetan names and translated Chinese names and among varieties of source plants. Apart from a few of varieties intersected with traditional Chinese medicines had complete standards and regulations in Chinese Pharmacopoeia, most of species only had characters, microscopic, physical and chemical identifications in Standards Issued by Ministry of Health-Tibetan Medicine, Tibetan Medicine Standard and local standards. Therefore, the Tibetan medicinal material variety-source specification and quality standard system shall be promoted on the basis of literatures research, investigations for resources and current applications and modern pharmaceutical studies.

[Traightened on Chinese endemic seed plant species of medicine plants used in Tibetan medicine].

This paper is in order to discussion with the composition and characteristics of Tibetan medicine plant resources, and promote the reasonable protection and utilization of the resources of Tibetan materia medica. Statistical analysis of species, distributions, and others of Chinese endemic seed plant from Tibetan medicine plants and usually used in the clinic of Tibetan medicine. The results showed that there are 523 species (25%) of Chinese endemic seed plant, belonging to 65 families and 162 genera, in about 2 000 varieties of Tibetan medicine plants recorded in relevant literatures. There are 180 Chinese endemic seed plant species (28%) belonging to 42 families and 72 genera from 625 medicine plants usually used in the clinic of Tibetan medicine. Specifically, the most of these Chinese endemic seed plant species are characteristic crude drug used in Tibetan medicine, and mainly or only distributed in Qinghai-Tibet Plateau. And a few species of them were intersected with traditional Chinese medicines (TCM) and other ethnic medicines. In addition, about 10% are listed in China Species Red List. The Qinghai-Tibet Plateau is the most abundant areas of Areal-types of the Chinese endemic seed plant. This is the biological and ecological reason formation the characteristics of Tibetan medicine plant resources. Therefore, strengthen the research of Chinese endemic seed plants used in Tibetan medicine is great significance for the reasonable protection and utilization of Tibetan medicine plant resources.

Overexpression of microRNA-143 inhibits growth and induces apoptosis in human leukemia cells.

Microrna-143 (miR-143) has been suggested to be a tumor suppressor, yet its role in hematological tumors has not been determined. Thus, we aimed to explore the expression and function of miR-143 in leukemia cells. miR-143 expression was assessed in bone marrow samples from 63 leukemia patients and 15 healthy controls using q-PCR, and its correlation with DNMT3A expression was determined. In addition, after lentiviral-mediated miR-143 overexpression, K562 cell proliferation was evaluated using CCK-8 analysis; cell cycle progression and apoptosis were determined using flow cytometry. The expression of Bcl-2 and pro-caspase-3 and -9 was assessed by q-PCR and western blot analysis, respectively. Leukemia patients had significantly lower relative miR-143 expression than healthy controls (P=0.004), and the expression levels of miR143 and DNMTA3A were negatively correlated (r=-0.663, P=0.001). Overexpression of miR-143 decreased DNMT3A mRNA and protein expression, and significantly reduced K562 cell proliferation at 72 and 96 h (both P ≤ 0.018). In addition, reduced colony formation and cell cycle progression were observed upon miR-143 overexpression. Flow cytometric analysis revealed that the early apoptosis rate was higher in the miR-143 group than the rate in the NC group. Bcl-2 mRNA expression and pro-caspase-3 and -9 protein expression were reduced in the miR-143-expressing cells. These findings suggest that miR-143 plays an important role in leukemia cell proliferation and apoptosis, possibly through silencing of DNMT3A. Further studies are necessary to determine the prognostic value and therapeutic potential of targeting miR-143.

CpG island methylator phenotype and its relationship with prognosis in adult acute leukemia patients.

OBJECTIVE: To investigated the relationship between CpG island methylator phenotype (CIMP) and prognosis in adults with acute leukemia. METHODS: Bone marrow samples from 53 acute myeloid leukemia and 50 acute lymphoblastic leukemia patients were collected. The methylation status of 18 tumor suppressor genes was determined using methylation-specific polymerase chain reaction. RESULTS: Greater than 30% of acute leukemia patients had methylated p15, p16, CDH1, CDH13, RUNX3, sFRP1, ID4, and DLC-1 genes; methylation of ≥4 were defined as CIMP positive. Age, type of leukemia, white blood cell count, and CIMP status were significantly associated with recurrence-free survival (RFS) and overall survival (OS) (P < 0.05). CIMP status was an independent prognostic factor for OS (hazard ratio: 2.07, 95% confidence interval: 1.03-4.15, P = 0.040). CIMP-negative patients had significantly improved RFS and OS (P < 0.05). p16 and DLC1 methylation was significantly associated with RFS and OS (P < 0.05). CONCLUSIONS: CIMP may serve as an independent risk factor for evaluating the prognosis of patients with acute leukemia.

[Community characters of Lophatherum gracile in Yongchuan district of Chongqing].

To understand Lophatherum gracile plant community's structural characteristics, a survey of community structure and species diversity was conducted through quadrat sampling in Yongchuan district of Chongqing. The results showed that there were 386 species vascular plants, belonging to 117 families and 229 genera. Based on habitat, community structure and species composition, L. gracile were found in three community types: Pinus massoniana community, banboo community, shurb community. Vertical structure was composed of three layers, including tree layer, shrub layer and herb layer. Species in shrub layer was the richness. P. massoniana is the only dominant species of the community, it can not regenerate naturally, the shrub layer has a greater effect on the community of L. gracile in the future. In addition, the banboo community and shurb community is not stable because of human's activity. Therefore, the community characters of L. gracile should be taken care of conservation when the resources are utilized.

[MicroRNA-193b expression in newly diagnosed leukemia patients and its significance].

This study was aimed to quantitatively detect the levels of microRNA-193b (miR-193b) in leukemia patients and explore its significance. Real time fluorescent quantitative PCR was used to detect the relative expression level of miR-193b. The expression changes of miR-193b in various types of leukemia were analyzed. Then the relationship among miR-193b expression, parts of laboratory index and the response to chemotherapy was analyzed as well. The results showed that miR-193b expression level in acute promyelocytic leukemia (APL) and chronic myeloid leukemia (CML) patients was not lower than that in normal group (P > 0.05). Except for APL, miR-193b expression level in acute myeloid leukemia (AML) patients was lower than that in normal group (P < 0.05). In AML (except for APL) patients, there was no correlation between white blood cell count (P > 0.05), the expression of CD34 (P > 0.05) and miR-193b expression level, but there was negative correlation between chemotherapy response and miR-193b expression level (P < 0.05). It is concluded that miR-193b expression level may be correlated with susceptibility of cells to chemotherapy in AML (except for APL) patients. miR-193b maybe become a new target in AML (except for APL) therapy.

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.

Methylation of secreted frizzled related protein gene in acute leukemia patients in China.

BACKGROUND: DNA methylation of CpG islands within the promoters of specific genes may play roles in tumor initiation and progression. It has been suggested such events may serve as critical check points. METHODS: The present study analyzed the methylation status of CpG islands within the promoters of secreted frizzled-related proteins (SFRPs) in 87 acute leukemia (AL) patients, 20 normal controls, and four AL cell lines. 5-aza-2'- deoxycytidine (5-Aza-CdR), an inhibitor of DNA methylation, was employed to determine its effect on SFRP expression. RESULT: Methylation of at least one SFRP promoter was observed in 69% of the AL patients analyzed. In addition, methylation of all four SFRP promoters was observed in Molt-4, Jurkat, HL60 and NB4 cells. In Jurkat cells, methylation levels of four SFRP promoters decreased in a dose-dependent manner upon treatment with 5-Aza-CdR, which coincided with increased mRNA expression. With increasing 5-Aza-CdR concentrations, the expression of DNA methyltransferases, DNMT3A and DNMT3B, significantly decreased in a dose-dependent manner. CONCLUSION: The present study demonstrated that SFRP gene methylation may be involved in AL progression, with a possible epigenetic mechanism influencing Wnt signaling.

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.