bluuebunny/crossref_metadata_2025_split huggingface dataset:
Study on DNA Methylation Status of WT1 Gene in Its Promoter Region in Hematologic Neoplasm Cell Lines.
Authors: Zhao Ye , Chen Zi-xing , Hu Shao-yan , Cen Jian-nong 
Published: October 2019

Abstract
The methylation at CpG island in the promoter region of a gene is one of the important epigenetic mechanism which regulates the gene activity. To study the DNA methylation pattern of WT1 gene promoter region within hematologic neoplastic cell lines and its correlation with WT1 gene expression by using the PCR-based methods. RT-PCR and Methylation-specific PCR were performed to study the WT1 gene expression in 8226, HL-60, Jurkat, K562, KG-1, NB4, Raji, SHI-1, U266 and U937 cell lines and the DNA methylation status in promoter region of WT1 gene. After treatment of U937 cell line by 5-aza-CdR, a demethylation inducing agent, the changes of WT1 gene expression level and the methylation status in its promter region in U937 cells was determined. Our Results showed that HL-60, K562, KG-1, NB4, SHI-1 cell lines demonstrated higher level of WT1 expression, while extremely low level was found in 8226, Jurkat, Raji, U266 and U937. The DNA hypermethylation in WT1 gene promoter region was identified in 8226, Jurkat, Raji, U266 and U937 cell lines. The WT1 gene expression in U937 was markedly enhanced after treatment with 5-aza-CdR in company with the decrease of methylated level and the increase of unmethylated level in its promoter region. These results indicate that modulation of the DNA methylation in WT1 promoter region is one of the epigenetic mechanisms to regulate its expression.
In-Depth Mutation Scanning of Signaling Pathway Genes Involved in the Development and Targeted Treatment of Hematopoietic Malignancies.
Authors: Lilleberg Stan L. , Durocher Jeffrey , Hempel Jill , Wasserburger Carrie , Koul Manika , Troy John , Nickerson Mike L. 
Published: October 2019

Abstract
The Raf/MEK/ERK, Wnt/beta-catenin, JAK/STAT and PI3K/Akt signal transduction pathways have key roles in the regulation of cell cycle progression and apoptosis, and are current focal points of therapeutic development and intervention strategies for hematopoietic neoplasias. These pathways have several regulatory components that keep proliferative and anti-apoptotic mechanisms in check, but which can also drive neoplastic processes when their functions are altered by genetic and epigenetic events. Links between critical pathways are also being established and, although only partially understood, may provide important clues for the development of more efficacious therapies that target multiple pathways. Although mutations in several pathway-associated genes known to contribute to the malignant phenotype have been discovered, the mutation status of the pathway as a whole in a particular blood cancer type remains to be determined. In this study, we applied novel mutation detection technologies (DHPLC, Surveyor Nuclease, and fluorescent DNA sequencing) in a combinatorial fashion to achieve high throughput, accuracy, and sensitivity for a comprehensive screening of signal transduction pathway genes. Key targets for the analysis included genes encoding receptor tyrosine kinases, cytoplasmic kinases, GTPases, transcription factors and tumor suppressors, with emphasis on Raf/MEK/ERK and PI3K/AKT pathways. We present both somatic alterations and inherited polymorphisms (SNPs) in these genes from the analyses of AML and CML sample sets. Functional analyses will confirm the causative nature of these genetic variants and their contributions to tumorigenesis. Other groups have focused on gene families for analysis of somatic mutations in tumors, such as kinases and phosphatases. This study takes a signaling pathway approach to group gene targets for analysis. We believe that pathway-based mutation analysis strategies offer significant potential to enhance understanding of cancer initiation, disease progression, response to therapy, and mechanisms of drug resistance.
Genetic Polymorphism of Thiopurine S-Methyltransferase in the Russian Population.
Authors: Samochatova Elena V. , Chupova Natalia V. , Roudneva Anastasia E. , Roumyantsev Alexander G. , Nasedkina Tatyana V. , Krynetskiy Evgeny Y. , Krynetskaya Natalia F. , Ribeiro Raul C. , Evans William E. 
Published: October 2019

Abstract
Thiopurine S-methyltransferase (TPMT) is a key enzyme in the catabolism of 6-mercaptopurine (6MP), a key component of therapy for childhood acute lymphoblastic and promyelocytic leukemias and lymphoblastic non-Hodgkin lymphoma. TPMT mutations reduce TPMT enzymatic activity; therefore, TPMT-deficient patients develop severe hematopoietic toxicity when treated with standard doses of 6MP (50–75 mg/m2/day). The frequency of TPMT alleles has not previously been evaluated in the Russian Federation. We determined the allele frequency and the pattern of mutant TPMT alleles in Russian children with hematologic malignancies (leukemia/lymphoma) and other diseases, and in healthy adults, in different areas of the Russian Federation. A microchip was designed to detect the main mutant alleles of human TPMT (TPMT*2, *3A, *3B, *3D, *7, and *8) and was used to assay 982 samples. Only 3 inactivating mutations of the TPMT gene were identified: 45 subjects (4.6%) had TPMT genotype *1/*3A, 8 (0.8%) had *1/*3C, and 2 (0.2%) had *1/*2. No homozygous genotypes and no mutant *3B, *3D, *7, or *8 alleles were found. The estimated frequency of the wild-type allele was 97.1% (95% CI, 93.8%–100%) and that of variant alleles was 2.9% (95% CI, 0%–6%). Most individuals with the *1/*3A genotype were of Slavic background, and genotype *1/*3C was found in a buryat (Mongolian race) and in residents of the North Caucasian and the Urals regions. These findings indicate ethnic differences in the frequency of nonfunctional TPMT alleles. The overall frequency of mutant TPMT genotypes in the Russian Federation is 5.6%, which is similar to that observed in the white populations of the United States and Europe.
Antiproliferative Effects of Tyrosine Kinase (STI 571) and Farnesyl Transferase Inhibitors (R115777 and SCH66336) on Acute Leukemia Human Tumor Cell Lines.
Authors: Russo Domenico , Michelutti Angela , Malagola Michele , Grafone Tiziana , Ottaviani Emanuela , Candoni Anna , Skert Cristina , Filì Carla , Castelli Maurizio , Martinelli Giovanni , Damiani Daniela , Baccarani Michele 
Published: October 2019

Abstract
The inhibition of the c-Kit signal transduction pathway by imatinib mesylate (STI571) and the inhibition of the post-translational modification of the N-K Ras proteins by farnesyl transferase inhibitors (FTIs) have shown potential efficacy in treating acute myeloid leukemias. We investigated the activity of (STI571) and of two distinct FTIs, R115777 and SCH66336, on two pairs of acute leukemia (AL) human tumor cell lines, each pair consisting of the parental cell line, HL60 and CCRF-CEM, and of its drug-selected multidrug resistant (MDR) Pgp-positive subline, HL60-DNR and CEM-VLB. The effectiveness of STI571, R115777 and SCH66336 in inhibiting cell proliferation of AL cell lines have been evaluated by colorimetric MTT assay. Cell growth was evaluated after a 7-day incubation at 37°C and 5% CO2 by using 50 microl per well of the MTT solution (5 mg/mL). The inhibition dose 50 (ID50) was defined as the drug dose that inhibited cell growth to 50% of the control. The results were correlated with the MDR phenotype and c-Kit expression. The toxic effect of STI 571 on all the acute leukemias derived cell lines was very low for both parental and MDR-Pgp+ sublines, ranging from 0.9 and more than 5 microM. STI571 activity was influenced by the c-Kit (CD117) expression and not by the MDR expression of cell lines. In fact, among these acute leukemia derived cell lines the higher toxic effect (ID50=0.9 microM) was obtained in HL60 DNR, the only cell line with a CD117 positivity. R115777 was more toxic than SCH66336 on all the tested AL cell lines. The Inhibition Dose 50 (ID50) of the two farnesyl transferase inhibitors ranged from 0.0065 to 0.21 microM for R115777 and from 0.3 to 6.5 microM for SCH66336. These results suggest that a potential synergistic effects of STI571 and R155777 combination could be explored in c-Kit positive acute myeloid leukemias.
Study of Cytotoxic Effects of K562/Adm Cells by Low Frequency Ultrasound Enchance Adriamycin.
Authors: Chen Bao-An , Meng Qing-Qi , Gao Chong , Ding Jia-Hua , Sun Yun-Yu , Wang Jun , Cheng Jian , Zhao Gang , Fei Fei , Chen Ning-Na 
Published: October 2019

Abstract
Objective The aim of this study is to investigate and analysis the effect of ultrasound potentiate the cytotoxicity of adriamycin and reverse drug resistance on leukemia drug resistance K562/Adm cell line in vitro, to find out the mechanism of the reverse effect of ultrasound exposure.
Methods Human leukemia adriamycin resistant strain K562/Adm as target cells, were treated by adriamycin singly in group Adm, by ultrasound exposure singly in group US and by adriamycin prior to ultrasound exposure in group Adm+US. The dosages that didn’t attribute to cell killing immediately were detected. Trypan blue dye exclusion test and MTT assay were used to determine the sensitivity of K562/Adm. Wright’s staining and transmission electron microscope were used to detect the apoptosis and structure change of K562/Adm cells. Flow cytometry was used to analysis intracellular drug concentration and electron microscopic scanning was used to observe the membrane change. Immunocytochemistrial method was used to evaluate the expressions of P-gp.
Results At 0.17W•cm−2 and lower acoustic intensity, ultrasound didn’t result in K562/Adm acute cells destruction; and 0.5 W•cm−2 ultrasonic intention could make cell killed rapidly after K562/Adm cells were irritated by ultrasound exposure singly. Significant differences were obtained between ultrasound treated and untreated cells in the presence of various concentrations of adriamycin. If the same concentration of cytotoxic agents were used, more cells were killed if sonication was applied. ultrasound for 30s at 20kHz, 0.17W•cm−2 intensity almost could not damage K562/Adm cells but dramatically decrease adriamycin concentration which induce cell achieve IC50;There were some morphological alterations in cells irradiated by ultrasound, Nearly all the treated cells by ultrasound exhibited small holes with diameter about 1~2 μm in the K562/Adm cell surface; the intracellular adriamycin accumulation in group Adm+US were prompted compared with Adm group and controlled group. Many apoptotic phenomena were observed in Adm+US group, show many vesicle and the form of apoptotic body, but there were no change in US group compared with controlled group. And the expression of P-gp protein had no significant difference between before and after ultrasound eradiated.
Conclusions
Higher ultrasound intensity could make K562/Adm cells killed rapidly, and lower ultrasonic level could potentiate the cytotoxicity of adriamycin to K562/Adm cells and reverse drug resistance on K562/Adm cells. Ultrasonic cavitation and sonoporation are the main mechanisms of the synergism between adriamycin and low-level sonication; the ultrasonically induced increase in intracellular drug accumulation. The expression of P-gp had no change in US group compare with controlled group.
Study on Effect of Low Frequency and Low Intensity Ultrasound on Leukemic Cell Lines K562 and Human Hematopoietic Cells.
Authors: Chen Bao-An , Ma Yan , Gao Chong , Ding Jia-Hua , Sun Yun-Yu , Wang Jun , Cheng Jian , Zhao Gang , Fei Fei , Chen Ning-Na 
Published: October 2019

Abstract
Objective: To investigate the effect of low frequency and low intensity ultrasound on leukemia cell lines K562 and to clarify the role of Caspase-3 in the effect and to study the effect of low frequency and low intensity ultrasound on human hematopoietic cells.
Methods: K562 cells in log phase were divided into 2 groups: control group and experimental group. The tensity of low frequency(20kHz) and low intensity ultrasound were 0.03W/m2 0.1W/m2 and 0.25W/m2 respectively. The exposure time were 30s and 60s respectively. K562 cells were incubated in 24-well culture plates for different time periods (6h,24h,48h) after sonication and the number of vital cells was tested by MTT assay. The morphology of apoptosis were analyzed by Wright’s stain and transmission electron microscope. The pencentage of apoptosis were studied by flow cytometry (FCM). The activation of Caspase-3 were tested by Caspase-3 kit with spectrophotometric method. All the hematopoietic cells were classified to two groups: control and experimental groups. The intensity of low frequency(20kHz) and low intensity ultrasound were 0.03W/m2 0.1W/m2 and 0.25W/m2 respectively. The exposure time were 30s and 60s respectively. The cells were incubated in 24-well culture plates for different time periods (6h, 24h, 48h) after sonication and the number of vital cells were tested by typhan blue exclusion. The ratio of apoptosis and morphology of apoptosis were analyzed by FCM, Wright’s stain and transmission electron microscope.
Results: The absorbance value of MTT decreased significantly after exposure. The highest apoptosis was found in 0.25W group after 30-s sonication and 6-h incubation. Morphological alterations observed in cells after exposure to ultrasound included: cell shrinkage, membrane blebbing, chromatin condensation, nuclear fragmentation, and apoptotic body formation. The result of FCM included: control 7.6%; 0.03W,30s,6h 11.57%; 0.1W,30s,6h 35.95%;0.25W,30s,6h 38.87%. There were no significant difference of activation of Caspase-3 between control and experimental group(0.25W group after 30-s sonication and 6-h incubation). The result of typhan blue exclusion was: the difference between each group was not significant. FCM, Wright’s stain and transmission electron microscope did not find apoptosis in each group.
Conclusions: Low frequency and low intensity ultrasound can induce apoptosis in K562 cell lines after exposure. The effect went significantly when the intensity of ultrasound increase, without relation to the exposure time. The low frequency and low intensity ultrasound has no significant effect on human hematopoietic cells.
Studies on the Effects of Berbamine on the Human Leukemia Cell Line NB4.
Authors: Zhao Xiao-ying , He Zhi-wen , Wu Dong , Xu Rong-zhen 
Published: October 2019

Abstract
Purpose: To study the effects of berbamine on the human leukemia cell line NB4
Methods: Berbamine compound was dissolved to the concentration of 1mg/ml with 0.9% sodium chloride and diluted to desired concentrations (as indicated in figure ledends 0.5,1,2,4,8,12,16ug/mL)with culture medium. Growth of NB4 cells was determined by MTT assay based upon the reduction of tetrazolium dye 3–4,5-dimethylthiazol-2,5-diphenyltetrazolium(MTT). Morphological observation was used to detect the characteristic changes of apoptosis in NB4 cells and DNA agarose electrophoresis were used to detect the characteristic changes of apoptosis in NB4 cells, while apoptosis rate was measured by flow cytometry assay. The _expression of PML/RARα gene was determined by nested-PCR.
Result: MTT results showed berbamine could inhibit the growth of NB4 cells significantly after treated with different concentration of berbamine for different time, IC50 was 3.86 ug•mL-1at 48 hours. Typical nuclear condensation and apotosis body were observed as early as 24 hours following a 8μgμg•mL-1 berbamine treatment;while the DNA agarose electrophoresis also show the typical DNA ladder,and the apoptosis rate of NB4 cell was increased from 2.83%to 58.84% after treated with 12ug•mL-1 berbamine for 48 hours. The _expression of PML/RARα gene wasn’t changed obviously.
Conclusion: Berbamin could inhibit the proliferation of NB4 cells and inducing apoptosis might be its one of mechanisms. The berbamine-induced apoptosis is not related to the _expression of PML/RARα gene.
Effect of Homoharringtonine on Differentiation and Apoptosis of Human Leukemic Cell.
Authors: Mai Wenyuan , Lin Maofang 
Published: October 2019

Abstract
Homoharringtonine (HHT) is a cephalotaxine ester derived from an evergreen tree found wildely throughout southern China, which has antileukemic activities against a variety of acute myeloid leukemic cells especially acute promyelocytic leukemia and acute monocytic leukemia. In order to illustrate the mechanisms of HHT in the treat of leukemia, we assessed the effect of HHT on several human leukemic cell lines, including promyelocytic leukemic cell HL60 and erythroblastic leukemic cell line K562. The differentiation of K562 cells and HL60 cell was determined by nitro tetrozolium blue reduction test (NBT) and differentiation antigens assay. The apoptosis of leukemic cells induced by HHT was analyzed by morphological observation, DNA gel electrophoresis, flow cytometry assay and terminal deoxyribonucleotidyl transferase-mediated dUTP-biotin nick and labeling method. Our results showed that the capacity of K562 and hL60 cells to reduce NBT was gradually increased after exposure to HHT at a lower concentration. The capacity of K562 cells to reduce NBT was higher after exposure to 0.01ug/ml of HHT than 0.005ug/ml of HHT. It indicated that the capacity of cells to reduce NBT was dependent on HHT concentration and time of exposure.
The fraction of HL60 cells displaying CD14 or CD11b increased significantly whereas that of displaying CD33 decreased after incubated with 0.001ug/ml of HHT for ten days. This data indicated that HHT induced hl60 cells to mature into monocyte-like cells.
Characteristic changes for apoptosis emerged in K562, and HL60 cells after exposured to HHT at a concentration 0.05–100ug/ml and 0.00505ug/ml respectively. The percentage of apoptotic cells in K562 and HL60 cells increased according to the concentration or exposure time of HHT. There was no DNA ladder in DNA gel electropheresis when K562 and HL60 cells incubated with HHT at a concentration lower than 0.05ug/ml and 0.005ug/ml respectively, even if the incubation time was long enough.
In this study, we confirmed that HHT has differentiative and apoptotic effects on human leukemic cells. The extent of HHT induced differentiation and apoptosis was dependent on drug concentration and time exposure.
Chronic Imatinib Mesylate Exposure Leads to Reduced Intracellular Drug Accumulation in K562 Cells Measured by High-Performance Liquid Chromatography.
Authors: Jia Yongqian , Yan Zhen , Zhu Huanling , Niu Ting , Zeng Tingting , Nan Feng , Liang Mengzhi 
Published: October 2019

Abstract
Imatinib mesylate is a selective tyrosine kinase inhibitor that is effective in the treatment of Philadelphia-positive chronic and acute leukemia. Unfortunately disease recurrence due to drug resistance is still the challenge to obtain cure. To investigate the possible mechanisms of imatinib resistance, we established a BCR/ABL+ cell line with resistance to imatinib (K562-R) by culturing a wild-type K562 cell line (K562-W) in gradually increased concentrations of imatinib over a period of 6 months, which can survive and was maintained in vitro at 3.0 umol/L of imatinib. K562-W and K562-R cells were cultured to logarithmic phase without imatinib, then treated with imatinib at 3.0 umol/L for 2 hours with or without cyclosporin A (2 ug/ml). The cells were washed thoroughly and incubated for further 0, 15, 30 and 60 minutes. Then cell suspensions containing 2×106 cells was measured for imatinib intracellular concentrations by High-Performance Liquid Chromatography (HPLC) after repeated freezing and thawing these cells. The imatinib concentration of K562-W cell is 0.31 umol/L, which is stable even after incubation for 1 hour. The imatinib concentration of K562-R is decreased significantly, being 0.21, 0.08, 0.06 and less than 0.01 umol/L at different incubation times mentioned above. The high level of cyclosporin A showed no effects on imatinib concentrations of both K562-W and K562-R cells. Flow cytometry showed there was no MDR expression of K562-R, whereas there was a small population (about 20%) of K562-W cells with dim expression of MDR. Compared to K562-W cells, the copies of BCR/ABL fusion genes determined by the real-time PCR showed that there were no amplifications of the fusion genes in K562-R cells and BCR/ABL cDNA sequencing showed no mutations of the imatinib ATP binding sites. The western blot assay showed that the expression of heat shock protein 90(Hsp90) was increased in K562-R cells. These results indicate that the intracellular eliminations of imatinib may be one of possible mechanisms for K562-R cells and Hsp90 as a critical molecular chaperone might be involved in the process.
Heme-Mediated Change in Sensitivity to Anti-Leukemia Reagents in BCR/ABL-Positive Cell Lines.
Authors: Ohmine Ken , Nagai Tadashi , Kikuchi Satoru , Miyoshi Takuji , Nakamura Makiko , Kondo Takahito , Komatsu Norio , Furuyama Kazumichi , Ozawa Keiya 
Published: October 2019

Abstract
Heme modulates various cellular functions as a component of hemoproteins. Recently, heme-mediated direct regulation of activity of transcription factors such as Bach1 has been shown. We reported in the last meeting that heme affects sensitivity to imatinib through, at least in part, regulation of Nrf2 transcription factor activity. In this study, we performed further analysis of the mechanisms of heme-mediated regulation of imatinib-sensitivity and the role of heme in the sensitivity to other small molecule compounds and conventional anti-leukemia drugs. IC50 values of imatinib against the BCR/ABL-positive cell line KCL22 were increased by addition of 1–100μM hemin, which eventually becomes heme in cells, in a dose-dependant manner. In the presence of hemin, imatinib-mediated increases in the levels of cleaved caspase 3, cleaved caspase 7, cleaved caspase 9 and cleaved PARP as well as the number of annexin V-positive cells were abrogated, suggesting that heme has an inhibitory effect on imatinib-induced apoptosis signaling. Luciferase reporter assay demonstrated that hemin increased MARE (Maf recognition element)-mediated γ-glutamylcystein synthetase (γ-GCS) light subunit gene promoter activity. Accordingly, the level of γ-GCS light subunit protein was increased after hemin treatment. We next examined the effect of suppression of Nrf2 expression on hemin-mediated change in imatinib sensitivity because Nrf2 might regulate MARE-mediated gene expressions. Transfection of Nrf2 siRNA resulted in partial abrogation of hemin-mediated upregulation of IC50 values of imatinib with suppression of hemin-mediated induction of γ-GCS light subunit expression, suggesting that suppression of Nrf2 partially restored the sensitivity to imatinib in hemin-treated cells. We also examined the effect of hemin on the sensitivity to conventional anti-leukemia drugs such as cytarabine, eoposide, hydroxyurea daunorubicin, idarubicin, doxorubicin and mitoxantrone in KCL22 cells. While IC50 values of cytarabine, etoposide and hydroxyurea were not changed by hemin treatment, addition of hemin resulted in significant increase in IC50 values of four anthracyclin anti-leukemia drugs. Furthermore, hemin also abrogated the sensitivity to U0126, which is a small molecule compound targeting MEK1/2. These results suggest that heme is an important molecule in the regulation of sensitivity to various anti-leukemia reagents.