bluuebunny/crossref_metadata_2025 huggingface dataset:
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

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

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

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

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

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

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

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

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.
Comparison of Uptake and Intracellular Induced Structural Changes of Arsenic Trioxide, an Inorganic Compound, and Organic Arsenic Derivative S-Dimethylarsino-Glutathione (SGLU; ZIO-101) in NB4 Acute Promyelocytic Leukemia (APL) Cells.
Authors: Manshouri Taghi , Kala Subbarao V. , Ashoori Faramarz , Zingaro Ralph , Freireich Emil J. , Andreeff Michael , Kantarjian Hagop M. , Verstovsek Srdan
Published: October 2019

Arsenic trioxide (As2O3; ATO) inhibits proliferation and induces apoptosis of APL cells. However, little is known about the intracellular structural changes associated with arsenic exposure. We studied effects of 2 arsenic compounds, ATO and S-dimethylarsino-glutathione (SGLU; ZIO-101) on NB4 APL cells. ZIO-101 is novel water-soluble organic arsenic derivative currently in Phase I clinical studies. Exposure of cancer cells to ZIO-101 results in G2/M cell cycle arrest and apoptosis. NB4 cells were exposed to 10, 50, 100 and 250 μM ZIO-101 or ATO for 1 h and intracellular arsenic content was determined by inductively coupled plasma mass spectrometry (ICP/MS). ZIO-101-treated cells contained 5–8 fold more arsenic then ATO-treated cells. Electron microscopy of NB4 cells exposed to 1 μM ZIO-101 or ATO for 24–72 h revealed different structural changes. ATO treated cells showed time-dependent mitochondrial hypotrophy and apoptosis, including cytoplasmic vacuolization, nuclear condensation and cell blebs. In contrast, ZIO-101 treated cells showed time-dependent mitochondrial atrophy, mitochondrial matrix condensation, and apoptosis. These data suggest that ZIO-101 is more specific mitochondrial toxin than ATO. Studies are in progress to understand the exact mechanism through which ZIO-101 affects mitochondria. Because these arsenic derivatives have different mechanism of action they may have different spectrums of activity against cancers; this should be tested in clinical trials.
A Phase I Open-Label Trial To Determine the Pharmacokinetics of Glucarpidase in Subjects with Normal and Impaired Renal Function.
Authors: Phillips Marc A. , Auton Tim R. , Ward Suzanne B. , Smith William B. , Balan Guhan
Published: October 2019

Methotrexate (MTX) is used in high doses in the treatment of cancers such as NHLs, leukemias and osteosarcoma. In some patients it produces renal damage which delays its elimination from the body. Prolonged exposure to high concentrations of MTX results in serious toxic effects. Glucarpidase (Voraxaze™, formerly carboxypeptidase G2) contains a recombinant enzyme which rapidly and predictably breaks down MTX in the blood within 15 minutes of administration. Multiple studies to date have evaluated glucarpidase as an intervention for patients at risk or experiencing toxicities related to MTX overexposure. Continuing studies seek to evaluate the use of glucarpidase in a planned way to circumvent toxicity and prevent prolonged hospitalization. The current study examined the pharmacokinetics of glucarpidase in subjects who had both normal and severely impaired renal function, similar to that of the intended patient population.

Methods: Twelve male and female subjects participated in this study; 8 with normal renal function (calculated creatinine clearance >80 mL/min) and 4 with severely impaired renal function (calculated creatinine clearance <30 mL/min). Each subject received a single intravenous dose of glucarpidase 50 units/kg (equivalent to 114.5 μg/kg), infused over 5 min. Serum glucarpidase profiles were evaluated for all subjects using 7 mL blood samples collected at the following time points: pre-dose (prior to start of glucarpidase IV dose), end of 5-minute infusion, and 0.25, 0.5, 1, 2, 4, 6, 8, 12, 18, 24, 48, 72, and 96 hours following the start of the infusion. The study protocol was approved by the Crescent City Institutional Review Board IRB (New Orleans, LA) and all subjects gave their written informed consent prior to inclusion in the study.

Results: The mean (SD) Cmax for glucarpidase in renally impaired subjects was 2.9 μg/mL (0.83), the mean half-life was 10.0 (2.1) h and mean AUC0-∞ was 24.5(9.4) μg.h/mL. Similar values were found in subjects with normal renal function (mean Cmax 3.1 μg/mL, mean t1/2 9.0 h and mean AUC0-∞ 23.4 μg.h/mL). No adverse events were recorded in the study.

Conclusions: The results indicated little effect of renal impairment on the serum pharmacokinetics of glucarpidase. Evaluations of adverse events, clinical laboratory assessments, vital signs, ECGs, and physical examinations indicated that a single 50 unit/kg dose of glucarpidase was safe and well-tolerated when administered to subjects with normal renal function and subjects with severe renal impairment. PK parameters found in this study may be relevant to cancer patients treated with 50 units/kg glucarpidase, including those with MTX-induced renal toxicity.
Copper-Complexed Melphalan (MOC*M), a Member of a New Class of Anti-Leukemic and Anti-Tumouric Substances for Treatment of Primary and Relapsed Acute Lymphoblastic Leukemia in Childhood.
Authors: Prokop Aram , Frias Corazon , Henze Guenter , Sadolinnaya Swetlana , Tatarskiy Valeriy
Published: October 2019

Acute lymphoblastic leukemia (ALL) is the most frequent malignant disease of childhood. Despite a relatively good prognosis (survival 80 %), approximately ¼ of the patients suffer from relapses with a much poorer prognosis (survival 40 %). If a complete remission through chemotherapy is not achieved, the patients will not survive. Thus, the search for new cytostatic substances which can break the resistance against conventional cytostatic drugs is of great interest.

We developed a new class of copper-containing cytostatic agents with apoptosis-inducing properties. The present study deals with 20 children, who suffer from de novo ALL or relapsed ALL. In vitro measurement of DNA-fragmentation in primary lymphoblasts of the children showed, that the copper-complexed cytostatic drugs are considerably more effective, compared to conventional analogues and other cytostatic drugs (cytarabine p<0.002, vincristine p< 0.006) used against childhood ALL. Furthermore, the new copper-containing analogues overcome drug resistance against doxorubicin (p<0.001) in vitro. In addition, the prototype of copper-complexed drug analogues, MOC*M, a melphalan-copper-acetoacetonate-complex, has synergistic effects in apoptosis induction combined with melphalan or conventional drugs in therapy of ALL in childhood like vincristin, doxorubicin and cytarabine.

Experiments revealed that MOC*M specifically induces apoptosis, as evidenced by DNA fragmentation and dissipation of the mitochondrial membrane potential. MOC*M induces cell death, which was functionally characterized by the use of different cellular model systems being devoid of defined molecular parts of the apoptosis machinery. MOC*M triggers apoptosis in a Bcl-2-independent manner in the multi-resistant melanoma cell line MelHO with a 30-fold over-expression of Bcl-2. In vitro and in vivo experiments on mice with tumors sarcom S-180, melanoma B-16 and adenocarcenom in the large intestine proved a high anti-tumor activity of MOC*M with anti-metastasis and immunizing properties without any side effects in kidney or liver. Thus, MOC*M is able to prolong the life of animals with leucosis L-1210 and P-388.

We could show that the accumulation of the tritium-labelled MOC*M compounds took place mainly in the tumor cells in vivo. Moreover, MOC*M is also inhibiting glycolysis in the tumor cells.

The result of pre-clinical tests with MOC*M preparations, tested on a limited quota of oncological patients with different tumors, was a very large spectrum of anti-tumor and anti-leukemic activities. Further MOC*M has an immense tolerability in vivo. All in all, copper-containing cytostatic drugs comprise an innovative, highly promising class of cytostatic agents for cancer and leukemia therapy, especially for the therapy of relapsed ALL in childhood.
Synergistic Effects of Bortezomib with Low Dose Melphalan Against Human AML Cells.
Authors: Gautier Marc , Bubis Jeffrey A. , Wu Jia-Yan , Meehan Kenneth R.
Published: October 2019

The treatment of AML in the elderly population is difficult given the inherent resistant disease, the toxic effects of therapy and the presence of co-morbid conditions. We proposed preclinical studies to investigate the potential synergy of bortezomib with melphalan against AML cells, with the anticipation of developing a clinical trial. To test the killing potential of bortezomib or melphalan, limiting dilution experiments were performed with subsequent analysis using the MTT assay. Two human AML cell lines, GDM-1 and Kasumi-1, were used as targets. Tumor cells (7.5 x 103 cells/well) were plated on 96-well tissue culture plates and incubated overnight at 37 °C with 5% CO2. At 24 hrs, varying concentrations of melphalan (doses: 100 uM to 1 uM) or bortezomib (doses: 100 nM to 1 nM) were added to each well. After 48 hrs of culture, the MTT assay was performed. Each test was run in triplicate. When using the GDM-1 cell line, the addition of melphalan alone (1 uM) resulted in 80% viability (+/− 1.7%), while bortezomib alone (1 nM) yielded 86% viability (+/− 3.6%). An increased dose of each medication decreased the viability of the GDM-1 cells. An increased dose of Melphalan (3 uM) reduced the viability to 23% (+/− 4.2 %). The viability dropped to 62% (+/− 4.9 %) with an increased dose of Bortezomib (3 nM). After combining the medications, the inhibitory activity against the GDM-1 cells required lower doses then either drug alone. For example, melphalan (1uM) with bortezomib (1 nM) reduced the viability of GDM-1 cells to 20% (+/− 2.8%); While melphalan (3 uM) and bortezomib (3 nM) further suppressed the viability to 3.7 % (+/−2.5 %). Similar results were found using the Kasumi-1 AML cell line. These results demonstrate the potential synergy of the combination of bortezomib with melphalan against AML cells. These preclinical results are currently being tested in a clinical trial using low dose melphalan with bortuzimab in elderly AML and MDS patients.
Guggulsterones Induce Apoptosis and Differentiation in AML: Identification of Isomer-Specific Antileukemic Activities of the Pregnanedienedione Structure.
Authors: Samudio Ismael , Konopleva Marina , Safe Stephen , Andreeff Michael
Published: October 2019

The gum resin from the guggul tree, commiphora mukul, has been used in Ayurvedic medicine for centuries to treat inflammatory and lipid disorders, and an ethylacetate extract of the resin, termed guggulipid, has been reported to have an anti-obesity and antilipidemic effect in clinical trials with no significant toxicity. In this study, we investigate the antileukemic effects of the active components in guggulipid, namely cis- and trans-guggulsterone (cGS and tGS, respectively), and compare their effects to those of the unrelated structural isomer 16-dehyroprogesterone (P). Our results demonstrate that all three compounds inhibited the proliferation of HL60 and U937 cells with IC50 values ranging from 3.6 - 10.9 μM after treatment for 6 days. These growth inhibitory effects correlated with externalization of phosphatidyl serine (PS) and loss of mitochondrial membrane potential (ΔψM) suggesting that these isomeric steroids induce apoptosis in leukemia cells. Z-VAD-fmk prevented PS externalization, but not ΔψM loss indicating that mitochondrial dysfunction occurred in the absence of caspase activation. Interestingly, while all three compounds increased the generation of reactive oxygen species and decreased phosphorylation of ERK, only cGS induced a rapid depletion of reduced glutathione levels and oxidation of the mitochondrial phospholipid cardiolipin. Curiously, the potent antioxidant n-acetylcysteine (NAC) only reversed the cytotoxicity of P suggesting that this structural isomer induces a different cytotoxic response from that of cGS and tGS. P and tGS induced differentiation of HL60 and NB4 cells as evidenced by increased surface expression of CD11b and/or CD14, and all three steroids rapidly induced mitochondrial dysfunction and PS externalization of CD34-positive blasts from primary leukemic samples. This study is the first to demonstrate that guggulsterones and 16-dehydroprogesterone exert antileukemic effects via the induction of apoptosis and differentiation, and more importantly, identifies the pregnadienedione structure as a potential chemotherapeutic scaffold for the development of novel antileukemic agents.
Cytotoxic Activity of the Proteasome Inhibitor MLN-273 but Not of Bortezomib Is Inhibited by MDR1.
Authors: Rumpold Holger , Wolf Anna M. , Untergasser Gerold , Gastl Guenther , Gunsilius Eberhard , Wolf Dominik
Published: October 2019

Proteasome inhibitors as bortezomib have been included in standard treatment regimens in several hematological malignancies, especially multiple myeloma. As resistance mechanisms depending on MDR1 expression have not been evaluated in detail for proteasome-inhibitors it was the aim of our study to investigate resistance of cells to Bortezomib and MLN-273 conferred by MDR1.

To address that question K562Dox cells have been long-term cultured in low concentrated doxorubicin to obtain MDR1 overexpression. These cells were stably transfected with a Sleeping-Beauty vector system carrying an siRNA targeting the mRNA of PgP. With that approach, PgP was reduced to 10% compared to the wild-type cell line. PgP-positive and PgP-negative cells were exposed to bortezomib and MLN-273 and proliferation measured was in a 3H-Thymidine incorporation assay. This showed a dose-dependent reduction of DNA-synthesis in cells exposed to Bortezomib, regardless of PgP-expression. In contrast, MLN-273 showed a PgP-dependency by inhibiting cells expressing PgP (K562Dox: EC 50 of 5,6 ng/ml and vector control cells K562Dox-Co: EC 50 of 29 ng/ml) less than MDR1-knock-down cells (K562Dox-siMDR1: EC50 of 0.3 ng/ml). In MDR1 positive cells this growth inhibition was reveresed by addition of verapamil, a MDR1 blocking substance (K562Dox: EC50 of 2.4 ng/ml and K562Dox-Co: EC50 of 2.8 ng/ml). This growth inhibitory effect was due to apoptosis induction, which has been shown by staining for Annexin V and propidium iodide. The direct effect of MDR1 on proteasome activity was measured by western blotting IkBα, a substrate of the proteasome, which was still present in MLN-273 treated MDR1-negative cells but not in MDR1-positive cells incubated with MLN-273.

These results show that MLN-273 highly likely is a substrate for MDR1, which may gain clinical significance in treating hematological malignancies in the future.
A Novel Multiparameter Flow Cytometric Cytotoxicity Assay Detects p53 Deletion as the Major Cause of In Vitro Resistance to Fludarabine in B-Cell Chronic Lymphocytic Leukemia.
Authors: Huang James Z. , Bakke Antony C. , Fan Guang , Braziel Rita , Gatter Ken M. , Maziarz Richard T. , Leis Jose F. , Zhong Yanping
Published: October 2019

Individual patients with B-CLL demonstrate variable responses to standard induction and salvage therapeutic regimens. It would be highly desirable to develop a predictable and reproducible laboratory diagnostic strategy that guides the selection of appropriate drugs and/or regimens based on the drug sensitivity and resistance profiles of leukemic cells for individual patients. As a first step towards this goal, a study was designed to investigate the differences of in vitro drug sensitivity profiles of leukemic cells with different cytogenetic abnormalities from CLL patients. CLL cells from 43 patients were incubated in vitro with four commonly used chemotherapeutic agents (fludarabine, chlorambucil, cladribine or prednisolone) individually or in combination. Multiparameter flow cytometry was utilized to determine the decrease in leukemic cell viability after drug exposure. Both fresh and cryopreserved samples were assessed and were found to be equivalent for assay, regardless of the percentage of B-CLL cells or the degree of spontaneous apoptosis. The highest in vitro resistance to fludarabine, was seen in all seven cases of B-CLL cells with deletions of p53, a cytogenetic abnormality associated with poor clinical outcome. Interestingly, in vitro response to chlorambucil and prednisolone was seen some CLL cases with p53 deletion and correlated with clinical response to these drugs. In CLL cases without p53 deletion, a marked variability in vitro drug sensitivity CLL cells was observed but no significant difference was detected among cases with normal cytogenetics (n=13), ATM deletion (n=4), trisomy 12 (n=3), or 13q deletion (n=7). Our findings provide direct evidence of cellular resistance to fludarabine in CLL associated with p53 deletion, confirming prior clinical observations. In vitro drug sensitivity assay may prove useful in guiding choices for therapy for CLL patients based on the drug sensitivity profile of leukemic cells in individuals.
High Incidence of Bcl-2/IgH Rearrangement in Mexican Patients With Follicular Lymphoma and Its Relevance as a Marker for Minimal Residual Disease.
Authors: Arana Rosa M. , Sobrevilla-Calvo Pedro , Rivas-Vera Silvia , Baez Enrique , Baltazar Severiano , Duque Jorge , Gregorio Ignacio , Tripp Francisco , Garces Oscar , Aguilar Luis , Flores Luis
Published: October 2019

Follicular lymphoma is frequently associated with the chromosomal rearrangement t(14;18)(q32;q21), which joints one of the JH segments of the heavy chains of immunoglobulins (IgH) gene in 14q32 with the Bcl-2 gene in 18q2, originating a chimeric protein. The frequency of this marker is unknown in the Mexican population.

OBJECTIVE: To determine the incidence of the Bcl2-IgH rearrangement in Mexican patients with follicular lymphoma and its frequency as a marker of minimal residual disease after therapy.

PATIENTS AND METHODS: 200 patients (102 male and 98 female) were evaluated; the analysis was made in peripheral blood samples (PB) in 64 cases (32%) or bone marrow (BM) in 136 (68%). Genomic DNA was obtained and the Bcl-2/IgH rearrangement was amplified by both PCR and nested PCR using primers for JH and exon-intron 3 region of Bcl-2 (MBR and MCR regions). The Bcl-2/IgH rearrangement was used as a marker to determine minimal residual disease (MDR) in 90 out of 200 patients in clinical remission, with a follow up ranging from 12 to 60 months; The incidence and tissue type analysis were compared using chi-square statistics.

RESULTS AND DISCUSSION: We found a positive Bcl-2/IgH rearrangement in 80% of the Follicular NHL cases, with a breakage in the MBR region in 160 cases and in MCR in 10 cases, in the remaining 30 patients the Bcl-2/IgH was negative. We detected the Bcl-2/IgH rearrangement more frequently in the BM samples (86%) than in the PB (42%) (p<0.0001). Fifty of 90 initially Bcl-2/IgH positive patients converted to negative PCR, after treatment. It is also of interest that in this population the molecular response rates in BM were lower (18/50=36%) than in the PB (32/50=64%) (p<0.0001). In conclusion we found a high frequency of Bcl-2/IgH rearrangement in cases of follicular lymphoma in Mexican patients, highlighting the relevance of the molecular analysis at diagnosis and its use as a marker for molecular minimal residual disease.
Expression of Leukemia-Associated Antigens (LAA) in Patients with Acute Myelogenous Leukemia (AML).
Authors: Schoettker Bjoern , Reimer Peter , Kunzmann Volker , Einsele Hermann , Weissinger Florian
Published: October 2019

Introduction: Immunotherapeutic approaches may be effective additional options to conventional chemotherapy and stem cell transplantation regimens in the treatment of patients with acute myelogenous leukaemia (AML). Without eliciting significant side effects they may help to prolong disease-free survival or at least may delay the time to progression. Immuniotherapeutic strategies comprise vaccination with professional antigen-presenting dendritic cells or transfusion of T-cells with specific anti-leukemic activity. Leukemia-associated antigens are useful targets as they are expressed predominantly in the malignant cells and constitutively not detectable in normal tissue, or as they are at least significantly overexpressed in the leukemic blasts. To determine potential targets for specific immunotherapy in our patients with AML, we started to analyze the antigen profiles of these patients’ leukemia cells using the previously characterized leukemia-associated antigens survivin, WT1, and proteinase-3 (with the HLA-A2-specific nonameric peptide PR-1), and c-Ski, an antigen recently found to be predominantly but not exclusively overexpressed in AML with deletion or monosomy of chromosome 7, indicating a poor prognosis.

Methods: The mRNA expression of the above mentioned antigens was examined by conventional RT-PCR. Blasts from patients with AML were collected at diagnosis or at first relapse from peripheral blood and analyzed.

Results: The antigen expression pattern in comparison to the AML cell lines HL-60 and K-562 is shown in Table 1.

Conclusions: Although treatment of AML patients became more effective during the last decades, high relapse rates contribute to the poor prognosis of the disease. Additional therapeutic strategies are needed to help and prevent disease relapse. Immunotherapy directed to LAA might elicit specific CTL responses effectively eliminating minimal residual disease. Using the LAA profile shown above, we should be able to determine at least one antigen in each patient with AML to serve as a target for individual specific immunotherapy with antigen-loaded autologous dendritic cells or /and specific autologous T-cells.

Table 1: Expression pattern of the LAA survivin, WT1, proteinase-3 und c-Ski in AML cell lines HL-60 and K-562, and in seven patients with AML. +/++/+++ positive, - negative, n.d. not determined yet -actinβ survivin WT-1 PRO3 c-ski HL-60 +++ +++ ++ + +++ K-562 +++ ++ ++ − − B.R. +++ +++ ++ +++ +++ B.G. +++ − ++ + − B.C. +++ − n.d. n.d. n.d. E.B. +++ − − n.d. n.d. E.E. +++ +++ +++ ++ + S.T. +++ +++ +++ ++ + V.B. +++ +++ n.d. n.d. n.d.
Identification of Novel Type of ETV6-TTL Fusion Gene in Pediatric Myeloid/NK Cell Precursor Acute Leukemia Having t(12;13)(p13;q14).
Authors: Taki Tomohiko , Hayashi Yasuhide , Taketani Takeshi , Sako Masahiro , Taniwaki Masafumi
Published: October 2019

The t(12;13)(p11-13:q11-14) is a recurring chromosomal abnormality found in different types of hematological malignancies. Three genes on 13q12-14, CDX2, FLT3, and TTL have been identified as fusion partners of ETV6(TEL) on 12p13. These genes create different forms of fusion transcripts with ETV6. In the present study, we identified novel types of ETV6-TTL fusion transcripts in a case of a 9 year-old female who developed myeloid/NK cell precursor acute leukemia with t(12;13)(p13;q14). Cytogenetic analyses of the leukemic cells of the patient using a regular G-banding revealed 47, XX, del(2)(q?), -9, der(12)t(12;13)(p13;q14), add(13)(q1?2), +mar1, +mar2 [13/20]. Fluorescence in situ hybridization using YAC clone revealed that ETV6 gene was split in metaphase chromosomes of patient’s leukemic cells. We performed reverse-transcriptase-polymerase chain reaction (RT-PCR) analysis to detect the fusion transcripts of ETV6-CDX2, ETV6-FLT3 or TTL-ETV6, however, no fusion transcripts of previously described types were detected. We next performed RT-PCR analysis using various sets of primers to detect unknown types of fusion transcripts involving these genes, and detected novel types of fusion transcripts of ETV6-TTL. These fusion transcripts consisted of exons 1 to 5 of ETV6 and exons 5 to 8 of TTL, and exons 1 to 6 of ETV6 and exon 9 or exons 8 to 9 of TTL. No reciprocal fusion transcripts were detected. Predicted fusion proteins consisted of N-terminal ETV6 lacking whole or part of ETS-binding domain and C-terminal TTL. Previous report showed major type of TTL-ETV6 fusion transcript consisted of exons 1 to 5 of TTL and exons 2 to 8 of ETV6 which contained both helix-loop-helix and ETS-binding domains. These results suggested that novel types of ETV6-TTL act as different fusion proteins from previously reported TTL-ETV6 in leukemogenesis. At the cytogenetic level, it may be difficult to distinguish ETV6-CDX2, ETV6-FLT3, and ETV6-TTL. Further accumulation of the patients with t(12;13) and further analysis of these novel types of fusion transcripts may clarify the pathogenesis of t(12;13)-leukemia.
Acute Myeloid Leukemia (AML-M2) with a Novel Translocation t(5;11)(q35;q13) and Normal Expression of Cyclin D1.
Authors: de Oliveira Fabio M. , dos Santos Guilherme A.S. , Marinato Andre F. , Araujo Amelia G. , Lima Ana S.G. , Jacomo Rafael H. , Falcao Roberto P. , Tone Luis G. , Simoes Belinda P. , Rego Eduardo M.
Published: October 2019

Rearrangements of the long arm of chromosome 11 are commonly associated with acute leukemias, with the breakpoints clustered mainly to the 11q23 region, frequently leading to the rearrangement of the MLL gene. CCND1 (previously PRAD1, BCL1), the gene encoding cyclin D1, is located at 11q13. Overexpression of cyclin D1 represents one of the common genetic alterations in human neoplasia, leading to a change in G1-S transition and uncontrolled cell growth. Here we describe the case of a patient with acute myelogenous leukemia (AML) harboring a chromosomal translocation involving 11q13 and a new partner, chromosome 5. A 30-year-old male presented with dyspnea for one month and peripheral blood counts of: hemoglobin 4.9 g/dL, platelets 62 x 109/L and leukocytes 29.5 x 109/L (with 45% of blasts). Bone marrow examination showed a hypercellular marrow with 82% blasts positive for myeloperoxidase and negative for non-specific esterase. The case was classified as AML M2 according to FAB. Classical cytogenetics and spectral karyotyping (SKY) studies performed by unsynchronized culture of the marrow cells revealed an abnormal clone of 46,XY, t(5;11)(q35;q13)[20]. RT-PCR for the rearrangements MLL/AF9, MLL/AF6, MLL/AF4, MLL/ENL, and MLL/ELL were performed and were negative for all of them. Cyclin D1 overexpression was not detected in bone marrow cells by Real Time PCR. The patient was submitted to induction chemotherapy with Daunorrubicin and Cytarabine but obtained only partial remission after 2 cycles of chemotherapy (10% of blasts in bone marrow after second induction). He was than submitted to an allogeneic stem cell transplantation (SCT) from his HLA identical sister. On day +30 after SCT he was in complete hematological remission. The classical cytogenetics study after chemotherapy and bone marrow transplantation revealed karyotypes 46,XY[20] and 46,XX[15]/46,XY[15]chi, respectively. The t(5;11)(q35;q13) represents a recurrent abnormality in renal oncocytoma (benign tumors that occur predominantly in the kidney) and leads frequently to cyclin D1 overexpression. Nevertheless, this cromossomal translocation has never been described in AML.
Quantitative Analysis of Wilms’ Tumor Gene 1(WT1) Expression in Acute Lymphoblastic Leukemia (ALL) of Adults Reveals Lower WT1 Levels Than in Acute Myeloid Leukemias (AML) and Varying Expression Levels between Individual Patients among Different ALL Subgroups.
Authors: Siehl Jan M. , Thiel Eckhard , Hoelzer Dieter , Schwartz Stefan , Keilholz Ulrich
Published: October 2019

Background: Overexpression of the embryonic transcription factor WT1 is common in human acute leukemias. Therefore WT1 became an interesting target mainly for immunotherapeutic approaches in AML. In previously published data from qualitative PCR analysis the frequency of WT1 overexpression in ALL is lower than in AML. But so far only limited data from quantitative analysis of WT1 expression in ALL are available.

Methods: In the present study we analysed bone marrow or blood samples of 238 adult ALL patients at diagnosis for the expression levels of WT1 by real-time RT - PCR (LightCycler). All patients were treated in the German Multicenter Study for the Treatment of ALL (GMALL 07/2003). Only samples containing at least 60 % blasts were used for analysis. Expression levels were compared to previously published data of WT1 expression levels in AML samples.

Results: WT1 expression was found in 219 out of 238 ALL samples (92 %). Compared to AML WT1 expression levels in ALL were significantly lower. There were no differences between the various ALL immuno phenotypes of the B or T lineage, but lymphoblasts with a coexpression of myeloid markers had significantly higher WT1 expression levels than those without a coexpression of myeloid markers. Surprisingly, patients who were bcr-abl negative had significantly higher WT1 expression levels than bcr-abl positive patients.

Conclusions: Using quantitative RT-PCR technology WT1 expression can be found in most ALL cases. There are usually less intense expression patterns compared to AML and expression varies among different ALL subgroups. Thus WT1 has promise to be an interesting target also in ALL therapy but in a more individually determined fashion. The relation of WT1 expression level and treatment outcome is currently being analysed.