bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of a Bone Marrow Derived Mesenchymal Cell Line from Nude Rat Assembling Tumor Stem/Progenitor Cell Properties In Vitro and In Vivo.
Authors: Wang Mengyu , Ju Jingfang , Saeboe-Larssen Stein , Mikalsen Svein-Ole , Olsen Elena , Nesland Jahn M. , Fodstad Oystein , Maelandsmo Gunhild , Kvalheim Gunnar
Published: April 2020

Here we report the in vitro and in vivo properties of a spontaneously transformed nude rat mesenchymal stem cells (rTMSCs) following culture passages 23. When this cell line was compared with early passages of mesenchymal cells (rMSCs) from the same animal, no apparent differences could be seen with regard to the phenotype and Gap junctions’ connexin 43. However, the telomerase activity in rTMSCs is lower than in the rMSCs. Karyotyping of rTMSC gave a trisome 6 phenotype. Microarry analysis of rTMSC shows over expression of the transforming growth factor family including TGF-beta and BMp6. Both rMSC and rTMSC had equal capability to differentiate into adipocytic and “neural like” linage. In contrast rTMSC was unable to differentiate into bone and only this cell line form spheroids in cultures.

Subcutaneously injection of rTMSC in doses down to 2x 104 into nude/nude mice formed tumor in all animals tested. Examination of the tumor assembles morphology like an immature sarcoma. When 100–200 SP + cells were administered to the animals, tumor could also be formed. After intravenous injections and direct injections into left heart ventricle all animals developed metastasis in the lung; abdominal cavity; bone and skin. The rTMSC has been stabled marked with reporter gene GFP and Leucipherase. Currently the proliferation and migration capacity of the linage-differentiated rTMSC is studied in vivo by imaging and these results will be presented. We conclude that our normal and transformed rMSCs could be potential useful for further biological studies on mesenchymal cells both in vitro and in vivo.
Active Oral Regimen for Elderly Adults with Newly Diagnosed Acute Myelogenous Leukemia (AML): Phase I Trial of Oral Tipifarnib (T) Combined with Oral Etoposide (E) for Adults ≥Age 70 Who Are Not Candidates for Traditional Cytotoxic Chemotherapy (TCC).
Authors: Karp Judith E. , Feldman Eric J. , Morris Lawrence , Greer Jacqueline , Ironside Valerie , Smith B. Douglas , Ritchie Ellen , Gore Steven D. , Levis Mark J. , Kaufmann Scott H. , Malek Sami , Wright John J.
Published: April 2020

T is a Farnesyltransferase inhibitor with clinical activity in elderly adults with newly diagnosed AML who are not candidates for TCC. For adults ≥ age 65 with poor-risk AML, T 600 mg BID for 21 out of every 28–63 days induces complete remissions (CR) in 15%, median duration 7.3 mos, median survival 18 mos. In vitro, T+E exhibits synergistic anti-AML effects. In an attempt to increase CR rates in elderly AML pts, we are conducting a Phase I trial of oral T+E, with escalating doses of both drugs and 14 vs. 21 days of T every 28–63 days (median time to beginning cycle 2 is Day 31, range 29–43). To date, 60 adults ≥ age 70 (median 77, range 71–91) have been treated at 10 dose levels of T (300, 400 or 600 mg BID, 14 or 21 days) + E (100, 150 or 200 mg daily days 1–3 and 8–10) and have received 145 cycles (median 3, range 1–7). A total of 35 (58%) have had secondary AML and 31 (52%) have had adverse cytogenetics. Marrow blast percent was median 55% (range 20–96%), with 75% >30,000 blasts. Nonhematologic toxicities include oropharyngeal mucositis (15% ≥ grade 2), CNS (overall 30%, with 80% of those ≤ grade 2), renal (6%), hyperbilirubinemia (6%), rash (20%) and fatigue (20%). Toxicities occurred across all T+E dose levels tested to date. Documented infections occurred in 16 (27%) pts during cycle 1 and 22/145 (14%) cycles. Twenty six pts (43%) required hospitalization during cycle 1 for median 7 days (range 2–28). Overall hospitalization rate for all cycles was 28% (41/145). Ten pts (17%) died on study: 6 during cycle 1 (4 infection, 1 emphysema, 1 cerebrovascular) and 4 during cycles 2–4 (3 acute myocardial infarctions, 1 ventricular arrhythmia). For 56 evaluable pts, 12 (21%) have achieved CR and 11(20%) have achieved partial remission/hematologic improvement (PR/HI). All pts achieving CR have done so within 2 cycles (9/12 after cycle 1). Eight of 30 pts (27%) receiving T ≥ 400 mg BID have achieved CR. Median age of CR pts is 79 (range 71–86), 8 (67%) have secondary AML, and 5 (42%) have complex cytogenetics. CR durations to date range from 2+ to 13.5+ mos, with median 7+ months. Three of 12 have relapsed at 3, 10, and 11 mos, 2 have been retreated with T+E, and both have achieved 2nd CRs. In sum, the oral regimen of T+E is tolerable and feasible on an outpatient basis for elderly adults with newly diagnosed AML who are not candidates for TCC, with the suggestion of improvement of CR rates over T alone.
Src Kinase Inhibitors Enhance ATRA-Mediated Differentiation of Myeloid Cells.
Authors: Miranda Michelle B. , Johnson Daniel E.
Published: April 2020

Myeloid leukemias are characterized by a blockade in differentiation resulting in accumulation of proliferating progenitor cells and a lack of mature granulocytes and monocytes. In acute promyelocytic leukemia (APL), a subtype of AML, treatment with all-trans retinoic acid (ATRA) has been shown to overcome this blockade in differentiation and is the current standard of care for APL patients. However, other forms of myeloid leukemia show no response to ATRA. Src family kinases (SFK) have been shown to be activated during normal myeloid development by both proliferation-inducing cytokines, such as IL-3, and differentiation-promoting factors, such as G-CSF. To elucidate the role of src family kinases during myeloid differentiation, we examined the impact of SFK inhibitors, PP1 and PP2 on ATRA-mediated differentiation of myeloid cell lines. Interestingly, PP1 potentiated ATRA-mediated myeloid differentiation of HL-60 cells, inducing a two-fold increase in differentiated cells at 72 hours as determined by CD11b staining, NBT reduction and morphological analysis. To determine whether enhancement of ATRA-mediated differentiation was specific for PP1, or could be promoted by a different src family kinase inhibitor, we evaluated the impact of PP2. PP2 was found to exhibit similar effects on ATRA-induced myeloid differentiation of HL-60 cells. Additionally, both PP1 and PP2 enhanced ATRA-induced monocytic differentiation of U937 cells and granulocytic differentiation of NB-4 cells. Interestingly, PP1 was found to enhance ATRA-induced differentiation even when added 24 hours after addition of ATRA. The impact of PP1 on ATRA-mediated myeloid differentiation was dependent on signaling via the MEK/ERK pathway since pre-treatment with U0126, a MEK-1/-2 inhibitor, attenuated myeloid differentiation induced by the combination of RA and PP1. Reporter assays utilizing an RARE-luciferase construct indicate that PP1 and PP2 do not enhance ATRA-mediated differentiation by promoting the transcriptional activity of the retinoic acid receptor (RAR). Together, our results demonstrate that PP1 and PP2 potently enhance ATRA-induced myeloid differentiation of AML cell lines and suggest that src inhibition in combination with ATRA may be a useful treatment for AML. Studies evaluating the impact of src inhibition on ATRA-mediated differentiation of primary AML cells are currently underway.
Tumor Necrosis Factor-Mediated Inflammation Exacerbates Genomic Instability and Promotes Leukemia Development.
Authors: Li June , Sejas Daniel P. , Zhang Xiaoling , Rani Reena , Pang Qishen
Published: April 2020

A correlation has been shown between elevated circulating pro-inflammatory cytokines and anemia in patients with leukemia-related diseases but direct evidence for the mechanistic link between inflammation and leukemia is lacking. We have investigated the role of the pro-inflammatory cytokine tumor necrosis factor a (TNF-a) in leukemic development using the disease model of Fanconi anemia, a genetic disorder clinically characterized by congenital anomalies, progressive bone marrow failure, and a high risk of developing leukemia and other cancers. We demonstrate that long-term TNF-a exposure of Fanconi bone marrow progenitors enhances inflammatory response, promotes clonal proliferation, and ultimately leads to leukemia development. NF-kB activation is required for TNF-a-promoted progenitor growth and early stage of leukemia development but is dispensable for the maintenance of leukemic transformation. Pharmacological elimination of TNF-a-induced reactive oxygen species reduces inflammation and delays leukemia development, suggesting that oxidative damage may serve as a link between inflammation and leukemia. In addition, TNF-a-promoted leukemic cells show persistent DNA damage response and increased genomic instability. Furthermore, correction of Fanconi genetic deficiency prevents clonal progenitor proliferation and leukemic transformation by eliminating oxidative DNA damage. Thus, inflammation can exacerbate genomic instability and contribute to leukemia development. This may explain, at least in part, why cells with genomic instability have high predisposition to cancer. Our study underscores therapeutic value of anti-oxidants and anti-inflammation towards tumorigenesis.
Expression of CD271 in Normal and Malignant Hematopoietic Progenitor and Stem Cells.
Authors: Jiang Ying , Powers Jennifer , Muschler George F. , Maciejewski Jaroslaw P.
Published: April 2020

The relationship between mesenchymal (MSC) and hematopoietic stem cells (HSC) involves close interaction in the marrow microenvironment. They also may share certain phenotypic features and even possibly a multipotent common precursor. CD271 (also known as LNGFR, p75NTR) has been reported to be a suitable marker of resting primitive MSCs. CD271 and its ligands, neurotrophins (NTs, e.g. nerve growth factor) also play important roles in hematopoiesis, such as enhancement of colony formation by human bone marrow progenitor cells. Dysregulated expression or mutations of NT receptors were associated with the initiation and progression of various malignancies. Based on expression of CD271 in the mesenchymal stem cell compartment, we have hypothesized that this antigen may also be present on a specific subset of normal or malignant hematopoietic progenitor and stem cells. First, we investigated the expression and function of CD271 in normal bone marrow. In controls (n=11), expression of surface CD271 was present on 1.67 ± 1.26% and 2.78 ± 4.50% of CD45+ and CD45− cell populations, respectively. Within the immature hematopoietic compartment, CD271 was expressed on 2.16 ± 1.16% cells defined by CD45 and CD34 expression. When the properties of CD271-expressing CD34 cells were studied in methylcellulose colony assays, those sorted for CD271 antigen showed a much lower clonogenic potential as compared to the CD271-CD34+ cell fractions (10.5 ± 2.12 vs. 183.5 ± 17.68). Subsequently, we investigated whether leukemic cell lines express CD271. We have analyzed HL60, U937, UT7, KG1, K562, HEL, MKN95, NB4 and Kasumi cells using flow cytometry. CD271 was found to be strongly expressed (72.8%) on KG1 AML. Smaller but clearly distinct subpopulations of HEL cells (7.8%) and NB4 cells (5.2%, APL cells) expressed CD271 suggesting either aberrant differentiation in some subpopulations of these cells or the presence of an early precursor population. We then assessed whether CD271 is expressed by CD34+ blasts in patients with myelodysplastic syndrome (MDS n=9) and AML (n=9). CD271 antigen was found on blast cells in 5/9 MDS (1 CMML, 1 RARS, 2 MDS-derived sAML, 1 RA) patients (7.16 ± 1.97% of CD271+ cells within blasts) and 3/9 AML patients (9.70 ± 3.65% of CD271+ cells within blast gate) suggesting that this fraction may differ functionally from the remaining majority of blasts. This is the first report that relates CD271 expression to MDS. The properties of a distinct CD34+ cell population expressing CD271 in myeloid malignancies are currently under investigation. Results of these studies may point towards the presence of a specific subset of precursor/stem cells with clinical and prognostic implications.
Comparison of Differential microRNA Expression Profiles between CD34+CD38− and CD34+CD38+ Cells in Both Umbilical Cord Blood and Acute Myeloid Leukemia.
Authors: Li Xin , Zhang Jiahua , Liu Jun , Huang Shiang
Published: April 2020

Recent studies demonstrated that some miRNAs played an important role in human normal and malignant hematopoiesis. MicroRNA expression is tissue and developmental stage restricted. Therefore, in order to address the roles of miRNAs in the regulation and maintenance of normal hematopoietiesis and leukemogenesis, in this study, we performed an analysis of differential genome-wide miRNA expressions of CD34+CD38− and CD34+CD38+ cells from human umbilical cord blood(CB) and acute myeloid leukemia(AML) bone marrow, respectively.

Methods Mononuclear cells from cord blood (CB) and AML bone marrow were stained with monoclonal antibodies. CD34+CD38− and CD34+CD38+ cells were sorted using FACSVantageSE. The mRNA were extracted and hybridized to miRCUURY array chip (Exiqon). The data were analyzed with GeneSpring and informatics technique. Real-time PCR for several miRNAs were performed to confirm the results obtained by miRNA chip analysis.

Results Based on the criteria of two or more fold changes of miRNA expression level, 73 miRNA overexpressions and 14 miRNA downexpressions were observed within CD34+CD38+ cells in comparison with CD34+CD38− cells in CB. Interestingly, only one overexpression and10 downexpressions of miRNAs were found within CD34+CD38+ cells as compared with CD34+CD38− cells in AML. This might suggest the less differential and maturational activities during the transition from CD34+CD38− cells to CD34+CD38+ cells in AML as compared with their normal counterpart. In comparison between CB and AML, 94 over-expressed and 28 down-expressed miRNAs were displayed in AML CD34+CD38− cells. More specifically, miR-155, -190 were down-expressed in both CB and leukemic CD34+CD38+ subsets, while miR-220, -499 were over-expressed in CB CD34+CD38+ cells and down-expressed in leukemic CD34+CD38+ cells. Putative hematopoiesis-specific target genes were analyzed with informatics technique. We found that each miRNA had several hundred genes as putative targets and each target gene had more than one miRNA predicted to be its regulator. For example, hsa-miR-509 has 561 putative target genes, of which IL1A, IL3RA1, FGFR2, SMAD2 are hematopoiesis-specific, and SMAD2 has 34 miRNAs as its regulators.

Conclusion: The miRNAs play important roles in the developments of both hematopoiesis and leukemogenesis. The analysis of differential microRNA expression profiles may be a powerful tool to allow us insight on the mechanisms of regulations of hematopoietic stem cells and leukemic stem cells.
Establishment of a NOD/SCID/γc−/−-Dependent Lymphoid Leukemia Cell Line, D593, with Novel RUNX1-LAF4 Fusion Gene.
Authors: Abe Akihiro , Ninomiya Manabu , Imagama Shizuka , Suzuki Momoko , Hayakawa Fumihiko , Katsumi Akira , Kiyoi Hitoshi , Kojima Seiji , Naoe Tomoki
Published: April 2020

We established a NOD/SCID/γc−/−(NOG mouse)-dependent human lymphoid leukemia cell line, D593, by repeated xenotransplantation of pediatric T-cell acute lymphoblastic leukemia cells with the translocation t(2;21). The cell line, D-593, could be serially transplanted from mouse to mouse over a 2-year period. D593 had the same immuno-phenotype as the original leukemia cells: positive for CD2, 5, 7, 14, and 34, and negative for CD3, 4, 8, 19, and 41a. Cytoplasmic CD3 was positive and the rearrangement of T-cell receptor was detected by Southern blot analysis. A previously unreported translocation of t(2;21)(q11;q22) was observed in both the original patient sample and D593. The split signal of RUNX1 was detected by fluorescence in site hybridization in D593 indicating the involvement of RUNX1. Using 3′-RACE and RT-PCR analysis, we identified novel chimeric transcripts of RUNX1-LAF4 joining exon 7 of RUNX1 to exon 4 of LAF4. In the transplanted NOG mice, D593 homed into the trabecular endosteal region of bone marrow (BM), and proliferated from the endosteum to medulla. At the late stage of engraftment, the BM was filled with human lymphoblasts and metastases into the trabecular of the spleen and Glisson’s sheath of the liver were also observed. These findings suggest that D593 is a useful cell line to study not only the leukemia-related biology of RUNX1-LAF4 but also the novel therapeutic model against core-binding factor (CBF) leukemia.
Inv(11)(q21q23) Fuses MLL to the NOTCH Co-Activator Mastermind-Like 2 in Secondary T Cell Acute Lymphoblastic Leukemia.
Authors: Metzler Markus , Zuna Jan , Staege Martin S. , Harder Lana , Meyer Claus , Flohr Thomas , Meerpohl Joerg , Fronkova Eva , Langer Thorsten , Harbott Jochen , Trka Jan , Siebert Reiner , Marschalek Rolf , Niemeyer Charlotte Marie , Rascher Wolfgang
Published: April 2020

Development of a secondary leukemia after chemotherapeutic treatment for childhood acute leukemia is associated with rearrangements of the MLL gene on chromosome 11q23 and characteristically results in acute myeloid leukemia (AML). We identified the NOTCH co-activator Mastermind-like 2 as MLL fusion partner in two cases of pediatric secondary leukemia. The MLL-MAML2 fusion results from a cryptic inv(11)(q21q23) only detectable by interphase fluorescence in situ hybridization with an MLL split signal probe. With a latency of twenty months and five years after initial diagnosis for MLL negative AML and precursor B acute lymphoblastic leukemia (ALL), respectively, both patients developed a therapy-related T-cell acute lymphoblastic leukemia.

MAML2 is the first MLL fusion partner involved in human Notch-signaling and was only recently identified as recurrent translocation fusion partner in a subset of salivary gland tumors. The genomic MLL breakpoint shows similar localization and sequence features described for etoposide induced treatment-related AML. MLL-MAML2 positive cells were detectable up to two years prior to clinical apparent secondary leukemia in one case. The discrepant dynamics of clone expansion quantified by either the genomic fusion sequence or Ig/TCR gene rearrangements as clone specific markers suggests a selection process within the inversion positive population and the need for additional mutation events to promote overt leukemic disease. Whole genome expression profiles demonstrated differential expression of both typical MLL and NOTCH downstream genes, which suggests a modulatory role of the MAML2 transcriptional activation domain in MLL leukemogenesis and lineage assignment induced by the MLL-MAML2 fusion protein.

MSM21620813

WSF3401600.
Screening and Identifying of the Protein Tyrosine Phosphatase Superfamily Members Related to Chronic Myeloid Leukemia and Exploration of Its Potential Function.
Authors: Du Qingfeng , Zhang Song , Li Rong , Gong Junmei , Zhu Hongqian , Wang Yu , Yao Feng , Fan Wei , Liu Xiaoli , Zhou Shuyu
Published: April 2020

Chronic myeloid leukemia (CML) is characterized by formation of a BCR-ABL fusion gene, which encodes a chimeric protein, P210BCR-ABL. The protein tyrosine kinase(PTK) activity of P210BCR-ABL is aberrantly regulated and lead to catalyzes the phosphorylation of tyrosine residues in specific sites of P210BCR-ABL itself and a host of substrates, which is considered as the sufficient and necessary factor in the pathogenesis of CML. Current studies suggest that the tyrosine phosphorylation is a reversible dynamic procedure which is governed by the coordinated and competing actions of PTK and protein tyrosine phosphatases(PTP). As the balance between PTK and PTP are broken by P210BCR-ABL in CML, we assume that some responsive regulations must been done by the cells in order to antagonize the impact of PTK activity of P210BCR-ABL. Among those regulations, the changes at the transcriptional level of many important genes including PTP will be most direct and efficient. In order to screen and identify PTP related to CML and explore its potential function, we firstly induced apoptosis of K562 cells with STI571 and analyze the differential expression of PTP with BioStarH40s expression profile cDNA array. Among the 21 PTP included in the BioStarH40s cDNA microarray, 2 PTP, FAP1(NM_006264, ration=2.417) and SHP1(M77273, ration=5.012) showed differential expression. The results of cDNA microarray confirmed by semi-quantitative RT-PCR suggested that FAP1and SHP1 may involve the apoptotic signal transduction pathway triggered by STI571. Secondly, the mRNA level of SHP1 in CML was analyzed with semi-quantitative RT-PCR. The results detected that the mRNA expression level of SHP1 in CML-CP (7 cases) show no statistic difference with normal control. But in contrast, the mRNA expression had down-regulated in CML-BC(4 cases) and show significant difference with normal control. Furthermore, we cloned the full length cDNA sequence of SHP1 with RT-PCR and sub-cloned it into the mammalian expression vector pcDNA3.0. The orientation and the sequence of pcDNA3-SHP1 were further validated by restrictive enzyme digestion analysis and DNA sequence analysis. Thirdly, the potential functions of the SHP1, such as triggering apoptosis and inducing differentiation, were explored by over-expressing the candidate gene in K562 cells with lipofectin transfection technique. The results showed that over-expression of SHP1 in K562 cells is sufficient for inducing apoptosis and erythroid differentiation that may be strengthened by the additional of STI571 treatment.
Characterization of the NAD-Dependent Human Histone Deacetylases Gene Sirtuin 1 and Its Implications on Aging and the Development of Malignant Disease.
Authors: Mahlknecht Ulrich R. , Voelter-Mahlknecht Susanne F.
Published: April 2020

A dysregulation of the tightly controlled equilibrium of acetylation and deacetylation plays a causative role in the generation as well as in the suppression of cancer. Histone acetylation modifiers are therefore gaining increasing attention as potential targets in the treatment of cancer. Sirtuin 1 (SIRT1) is a nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase, which belongs to the silent information regulator 2 (Sir2) family of sirtuin histone deacetylases (HDACs). The yeast Sir2 protein and its mammalian derivatives play a central role in epigenetic gene silencing, DNA repair and recombination, cell-cycle, microtubule organization, and in the regulation of aging. We have isolated and characterized the human Sirt1 genomic sequence and identified a number of SIRT1 protein interaction partners, which will be important for the further understanding its physiological role. The Sirt1 gene spans a region of 33,660 bp and is located on chromosome 10q21.3. The protein is localized in the nucleus, and interacts with and deacetylates a growing number of proteins, such as p53, the Forkhead transcription factor FOXO3A, PML, BCL6, TAFI68, HES1, and CTIP2. SIRT1 has been shown to be essential for embryonic development, muscle differentiation and is an important mediator of organismal longevity through a number of different mechanisms such as the induction of cell cycle arrest, resistance to oxidative stress and the inhibition of apoptosis.

Some histone deacetylase genes have been shown to be rearranged in the context of chromosomal translocations in human acute leukemias and solid tumors, where fusions of regulatory and coding regions of a variety of transcription factor genes result in completely new gene products, which may interfere with regulatory cascades that control cell growth and differentiation. On the other hand, some histone acetylation modifying enzymes have been located within chromosomal regions that are particularly prone to chromosomal breaks. In these cases gains and losses of chromosomal material may affect the availability of functionally active HATs and HDACs, which in turn disturbs the tightly controlled equilibrium of histone acetylation. A deletion of Sirt1 would therefore most probably shift the steady state toward acetylation at the level of specific genes targeted by SIRT1 and either upregulate or downregulate transcriptional events. Such dysregulation might represent a critical event in the multistep pathway leading to full cellular transformation and the development of malignancy.
Protein Interaction Partners of PDZ-Binding Kinase (PBK) Implicate a Possible Role in Leukemogenesis.
Authors: Nandi Asit K. , Ford Tamara , Fleksher Daniel , Neuman Brian , Rapoport Aaron P.
Published: April 2020

Considering the fact that multiple genetic hits are required for the development of human cancers, we focused to examine the possible complementation by a novel mitotic kinase called PDZ- binding kinase (PBK) in the backdrop of deregulated c-Myc expression which either as a primary or a secondary genetic event is responsible for a wide variety of cancers including hematological malignancies. PBK, a mitotic kinase and a MAP kinase kinase homolog was originally identified in the laboratory as a differentially expressed gene in Burkitt’s lymphoma cell line GA10 compared to hyperplastic tonsillar B cells. We earlier found that PBK was upregulated in 9 out of 12 primary samples of acute myelogenous leukemia (AML) as well as several other leukemia and lymphomas while CD34+ immunoselected bone marrow stem/ progenitor cells expressed very little PBK. PBK was found to be coregulated with c-Myc during macrophage differentiation of promyelocytic leukemia cells HL60 and during growth arrest in the presence of low doses of an antineoplastic compound doxorubicin. We further demonstrated that binding by the cell cycle- specific transcription factors E2F and CREB/ ATF respectively at −146 bp and −312 bp positions upstream of the transcription start site of PBK is required for optimal PBK expression. In order to understand the functional implications of PBK expression in oncogenically stimulated cells, we have analyzed the protein- protein interactions involving PBK in the human bone marrow cells utilizing yeast two- hybrid approach. To this end, a constitutively active PBK mutant was expressed in yeast as a fusion protein with GAL4 DNA- binding domain. Utilizing GAL4- PBK as bait, we have isolated several cDNA clones including those encoding the high- mobility group nucleosomal binding protein 2 (HMGN2) and ret finger protein 2 (RFP2) as potential interaction partners for PBK. It was reported that mitotic phosphorylation of the nucleosomal binding domain (NBD) present in the HMGN1, another member of the related protein family, regulates interaction with 14.3.3 proteins and entry into the nuclei. In a separate study, it was reported that a 31 amino acid synthetic peptide from HMGN2 overlapping to NBD when injected intravenously was able to direct homing to the nuclei of tumor cells. RFP2 is a candidate tumor suppressor gene residing in the critical deleted region (CDR) 13q14 as it occurs in several different hematological malignancies such as B-cell chronic lymphocytic leukemia (CLL), mantle cell lymphoma, diffuse large cell lymphoma, myeloma including solid tumors such as head and neck cancer, oral tumors and prostate carcinoma. Validation of these interactions in the mammalian cell environment is currently in progress.
Tetraspanins Affect Myeloma Cell Fate Via Akt Signaling and FoxO Activation.
Authors: Drucker Liat , Zismanov Victoria , Tohami Tali , Tartakover-Matalon Shelly , Lishner Michael
Published: April 2020

The significance of cellular interaction with microenvironmental components to tumor progression is increasingly acknowledged. In myeloma (MM), the involvement of bone marrow is critically important to cell growth, survival and drug resistance. It is currently perceived that effective disease treatment must address these interactions. We have previously shown that re-expression of the eGFP fusion proteins CD81-N1 and CD82-N1 tetraspanins in MM cell lines (CAG, RPMI 8226) caused cell stress and massive death. Here we demonstrate that the anti-myeloma effect of CD81-N1 and CD82-N1 compared to mock involves: a down-regulation of Akt(Ser473) [20%–40%]; activation of FoxO transcription factors [evidenced in decreased phosphorylation (20%–40%) and elevated transcription of Gadd45A (70%–80%)]; and is accompanied by a decrease in phosphorylated mTOR(Ser2448) [30%] and mTOR/rictor activity [demonstrated in phosphorylation of Akt at Ser473 position] (p<0.05). Next, we investigated the involvement of PI3K, the prime activator of Akt. Our results show that addition of the PI3K inhibitor wortmannin, affected both the mock and tetraspanin transfected myeloma cells in the same manner. We deduce from these results that the CD81-N1/CD82-N1 induced death is independent of the PI3K signaling. This finding further supports the importance of mTOR/rictor rather than PI3K in the regulation of Akt activity under these circumstances. Based on publications that attribute JNK a regulatory role in FoxO4 activity, especially in stress related scenarios, we decided to examine the consequences of JNK inhibition on tetraspanin induced cell death. Indeed, a most significant rescue of 26% of the cells was demonstrated in the CD81-N1 transfected CAG. Moreover, we conjectured that the cell death was due to myeloma dependence on Akt activation. Hence, in a breast cancer cell line model characterized with variegated levels of Akt activation we demonstrated that the tetraspanin effect was also mediated via Akt and dependent on the ‘cell’s addiction’ to its activity. Significantly higher rates of cell death were observed in the Akt dependent MDA-MB-231 breast cancer cell line compared to Akt independent MCF-7 with both CD81-N1 and CD82-N1 overexpression (70% versus 50% and 50% versus 10%, respectively). Moreover, in CD82-N1 transfected MDA-MB-231 we observed a significant reduction in Akt(Ser473). We suggest that tetraspanins have modulator potential in MM and constitute a promising frontier to be explored in Akt dependent pathological conditions. Moreover, targeting the pathways (via the ‘tetraspanin switch’) rather than blocking a single factor may present a promising therapeutic strategy in a complex drug resistant disease such as myeloma.
Delayed Achievement of Molecular Responses Is Associated with Increased Risk of Progression among Patients (pts) with Chronic Myelogenous Leukemia (CML) In Chronic Phase (CP) Treated with Imatinib (IM).
Authors: Quintas-Cardama Alfonso , Kantarjian Hagop M. , Jones Dan , Garcia-Manero Guillermo , O’Brien Susan , Shan Jenny , Ravandi Farhad , Wierda William G. , Cortes Jorge
Published: April 2020

Background: Achieving a cytogenetic (CG) response or a molecular response after imatinib therapy has been associated with improved event-free (EFS) and transformation-free survival (TFS). It is unclear whether achieving these responses earlier confers an advantage. A recent update of the IRIS trial suggests that achieving a major CG response (MCyR) at 12, 18 or 24 months (mo) confers a similar TFS advantage. Another report (J Clin Oncol2006;24:454) suggests that achieving a complete CG response (CCyR) at 12 or 24 mo confers equal prognosis to patients. Although some patients may indeed improve their response with continued therapy, a pt not in CCyR faces the competing possibilities of eventually achieving a CCyR vs progressing.

Methods: We analyzed the risks of improving the CG response vs progressing for pts not in CCyR at different times to determine whether early responses confer an advantage. 258 pts with CML CP treated with IM were analyzed. Progression was defined as transformation to AP or BP, loss of CHR or major CG response, or a doubling of the white cell count to more than 20x109/L.

Results: After 3 mo of IM therapy, 77 (74%) of 104 assessable pts for CG response had a CCyR whereas 17 (16%) progressed. These differences were consistently significant at 6 and 12 mo (p=0.04) (Table 1).

We then analyzed the long term risk of progression vs the probability of achieving CCyR according to the molecular response at different time points (Table 2).

Patients with Bcr-Abl/Abl transcript levels >1–10 after 3 mo of therapy had a 92% probability of achieving CCyR with continued therapy, similar to the 98% for those with ≤1. However, they have a significantly higher risk of eventual progression that is more similar to that of pts with values of >10. The risk of progression increases to 23% if transcript levels >10 at 6 mo.

Conclusion: These results suggest that while the risk of progression may be similar for pts who achieve a CCyR regardless of the time at which that is attained, those who fail to obtain a CCyR within the first 12 mo of IM therapy have higher rates of disease progression. This risk is discernible as early as 3 mo into IM therapy and may provide a rationale for therapies that induce higher rates of early molecular response (e.g. high-dose imatinib, new tyrosine kinase inhibitors).

Table 1 Time on imatinib No. not in CCyR No. with eventual outcome with continued therapy (%) CCyR Progression p value 3 months 104 77 (74) 17 (16) 6 months 47 28 (60) 12 (26) 0.04 12 months 26 16 (62) 8 (31)

Table 2 % Probability Months %Bcr-Abl/Abl No. CCyR Progression p value ≤ 1 87 98 2 3 > 1–10 76 92 11 0.04 > 10 30 67 13 ≤ 1 140 99 4 6 > 1–10 34 91 9 0.005 > 10 13 62 23
The Study of Effect and Mechanism by Vitamin K2 on Human MDS Cell Line MUTZ-1.
Authors: Chen Bao-An , Xu Xin , Shao Ze-Ye , Ding Jia-Hua , Xia Guo-Hua , Gao Chong , Sun Yun-Yu , Chen Ning-Na , Fei Fei
Published: April 2020

The myelodysplastic syndromes (MDS) are characterized by hemopoietic insufficiency associated with cytopenias leading to serious morbidity and the additional risk of leukemic transformation. Vitamin K2(VK2) is reported to induce apoptosis or differentiation of leukemic cell lines in vitro. For investigating the effects and mechanism of VK2 on human MDS cell line MUTZ-1 in vitro,we observed the changes of morphologic features of MUTZ-1 cells by exposing to VK2.The transmission electron microscope was used to observe the apoptosis of MUTZ-1 cells. Cellular proliferation was determined by the MTT assay. The flow cytometry was used to analysis apoptosis rate and the change of cell cycle. The expression of apoposis-related genes bcl-2, survivin and bax were detected by reverse transcriptase polymerase chain reaction(RT-PCR).The activity of caspase-3 was detected by chemiluminescence assay. After exposing to 10μmol L−1 and higher concentration of VK2, it could inhibit MUTZ-1 cells proliferation in a dose-and time-dependent manner(p<0.05). At concentration of 5μmol/l VK2 treatment, it might accelerate cellular proliferation, but there’s no significant difference compared with control group. Apoptosis peak on FCM and positive Annexin-V FITC/PI on cell membrane showed that VK2 induced apoptosis of MUTZ-1 cells in a dose-and-time-dependent manner, G0/G1 cell cycle arrest, significantly dow-regulated the expression of bcl-2 and survivin, but had no effect on the expression of bax.The activities of caspase-3 were significantly increased. Low concentration of VK2 could facilitate cell proliferation. The higher concentration of VK2 could induce apoptosis of MUTZ-1 cells. These results indicate that VK2 induces MUTZ-1 cells apoptosis by activating caspase-3 pathway, the apoptosis related genes bcl-2, survivin down-regulated might play an important role in this process.
Prognostic Significance of Multidrug Resistance Proteins Expression in Acute Myeloid Leukemia.
Authors: Zakharov Oleg D. , Rybalkina Ekaterina U. , Stavrovskaya Alla A. , Volkova Maya
Published: April 2020

Background: Conventional induction chemotherapy induces complete remission (CR) in 65–75% of adults with de novo acute myeloid leukemia (AML), and 15–20% of the patients have refractory disease. We investigated the prognostic significance of multidrug resistance proteins (P-glycoprotein (Pgp), BCRP, MRP1 and LRP) expression on AML blast cells before treatment.

Methods: We included in analysis 30 patients (pts) with de novo AML. Expression of multidrug resistance proteins (MDR) was detected by indirect immunofluorescence technique and flow cytometry on bone marrow blast cells before chemotherapy. Expression of MDR proteins was considered as positive if at least 25% of the blast cells were stained by anti-MDR protein antibody. All pts received standard induction therapy (cytarabine, etoposide and idarubicin or daunorubicine).

Results: Blast cells was defined as Pgp-positive in 64.3% of cases, BCRP+ in 42.9%, MRP1+ in 46.4%, and LRP+ in 64.3% of cases. After induction therapy 20 (66,7%) pts achieved CR and 10 pts (33.3%) were resistant. MDR proteins expression was observed more frequently in resistant group, then in a sensitive one (70% vs 61% for Pgp, 70% vs 33% for MRP1, 100% vs 44% for LRP, 80% vs 22% for BCRP, respectively), difference for LRP and BCRP was statistically significant (p=0.004). Blast cells of all resistant pts expressed 2–4 MDR proteins (all studied proteins − 40%, 3 of them − 40% and 2 proteins − 20%). In a group of pts archived CR the blast cells expressed 3 proteins only in 2 cases (10%), and the expression of all 4 proteins we observed only in 1 patient (5%) with very short CR duration (3 months). Other pts from this group express only one studied protein.

According to chromosome analysis 18.2% of pts had favorable, 50% - intermediate and 31.8% unfavorable cytogenetic. Blast cells of all pts in cytogenetically unfavorable group expressed more then 1 protein, 3 or 4 MDR proteins expressed in 71,4% of cases. In favorable and intermediate cytogenetic groups blast cells expressed 1or 2 MDR proteins in 73,2% of cases, 3 or 4 proteins in 20%.

Conclusions: The expression of MDR proteins in AML has a prognostic value with respect to CR achievement in pts receiving standard antracycline-Ara-C regimens. The detection of any single protein didn’t have prognostic significance, only co-expression of 2 and more proteins predict unfavorable treatment outcome. We observed a correlation between the cytogenetic and the MDR phenotype.
Tetra-O-Methyl Nordihydroguaiaretic Acid Inhibits Growth and Induces Death of Leukemia Cells Independent of Cdc2 and Survivin.
Authors: Carter Bing Z. , Mak Duncan H. , Schober Wendy D. , Heller Jonathan , Andreeff Michael
Published: April 2020

Tetra-O-methyl nordihydroguaiaretic acid (M4N) was shown to induce G2 arrest in mammalian cells and suppress the growth of human xenograft tumors by inhibiting Cdc2 and survivin expression. We have previously shown that inhibition of survivin expression by antisense oligonucleotide induced G2 block and subsequent cell death in HL-60 cells. In this study, we examined the effect of M4N on leukemic cells and found that M4N inhibited growth and induced apoptosis in various leukemic cell lines and blasts from AML patients at concentrations from 5μM to 40μM. However, Cdc2 and survivin levels were not significantly affected. In agreement with these results, no G2 arrest was observed. Cell death and cell growth inhibition induced by M4N in leukemia cells were dependent neither on XIAP, Bcl-2, or Bcl-XL levels nor on caspase-8. Although M4N induced the loss of mitochondrial membrane potential in HL-60 cells, overexpression of Bcl-2 or Bcl-XL did not attenuate cell growth inhibition and cell death induced by M4N. M4N did not promote cell differentiation in HL-60 cells. Interestingly, significant inhibition of AKT and to a lesser degree of ERK phosphorylation was observed at 24 hours in OCI-AML3 cells treated with M4N. Collectively, our data showed that M4N inhibited cell growth and induced cell death in both leukemic cell lines and AML patient sample through a mechanism not mediated by Cdc2 and survivin inhibition and independent of death receptor and mitochondrial apoptotic pathways.
Effect of Valproic Acid on the Expression of P27Kip1 and Multidrug Resistance in HL60 Cells.
Authors: Yin Songmei , Nie Danian , Xie Shuangfeng , Li Yiqing , Wang Xiuju , Ma Liping
Published: April 2020

Multidrug resistance (MDR) was the main cause of treatment failure in leukemic chemotherapy. P170, the protein expressed from MDR-1 gene, play an important role in MDR. P27Kip1 was a member of cyclin dependent kinase inhibitor family. It was also a negative regulator in cell generation cycle. It also promoted cells apoptosis. Valproic acid (VPA) was a kind of histone deacetylace inhibitor which could inhibit cell cycle and induce apoptosis. In the current study, we investigated the effects of VPA on the expression of P27Kip1 and P170 of HL60 cells in vitro. HL60/HT cells were induced from HL60 cells by harringtonine (HT) in gradient concentrations. The P27Kip1 expression and P170 expression were tested in flow cytometry. We tested the expression of P27Kip1 and P170 in normal mononuclear cells, HL60 cells and HL60/HT cells, also the effects of VPA on P27Kip1 expression and P170 expression in HL60 cells and HL60/HT cells. The 50% inhibiting concentration (IC50) of vincristine for HL60/HT cells was 5.2 times higher than that for HL60 cells (0.078ug/ml vs. 0.015ug/ml). The IC50 of HT for HL-60/HT cells was 9.3 times higher than that for HL-60 cells (0.14μg/ml vs. 0.015μg/ml). The IC50 of Cytarabine for HL60/HT cells was 3.65 times higher than that for HL60 cells (0.73ug/ml vs. 0.20ug/ml). The expression of P27Kip1 in normal mononuclear cells was the highest, while that in HL60 cells was higher than HL60/HT cells (96.18±3.13% vs. 85.83±4.65% vs. 35.91±3.13%, one way ANOVA, P<0.05). The expression of P170 in normal mononuclear cells and HL60 cells had no significant difference (0.54±0.32% vs. 0.62±0.31%, P>0.05). But the P170 expression of HL60/HT cells was 6.71±1.61%, which was much higher than that of normal mononuclear cells or HL60 cells (P<0.05). In HL60 cells, the expression of P27Kip1 increased after cultured with 1mmol/L VPA (92.21±3.43% vs. 85.83±4.65%, P<0.05), but the expression of P170 didn’t change significantly (0.65±0.18% vs. 0.62±0.31%, P>0.05). In HL60/HT cells, the expression of P27Kip1 increased after cultured with VPA (66.20±5.07% vs. 35.91±3.13%, P<0.05), and the expression of P170 didn’t change significantly either (6.40±1.59% vs. 6.71±1.61%, P>0.05). These results indicate that the P27Kip1 expression in normal mononuclear cells were higher than that in leukemia cells (HL60 cells), and the P27Kip1 expression in common HL60 cells were higher than that in MDR HL60/HT cells. VPA could increase the expression of P27Kip1 in HL60 cells and HL60/HT cells. VPA had no effect on the expression of P170 in HL60 cells or HL60/HT cells. That means, The MDR-reverse effect of VPA may not come from the P170 descent mechanism.
siRNA Targeting bcl-2 Gene Improve H and RA Drug-Sensitivity in HL-60 Cells.
Authors: Hu Haiyan , Zhang Yuan , Kunyuan Guo
Published: April 2020

The anti-apoptosis gene, bcl-2, is also related to drug-resistance, which expresses markedly higher on all kinds of leukemia. small interference RNA can silence the targeted mRNA expression.

Objective: To study the effect of G3139 and siRNA targeting bcl-2 gene which increase the sensitivity of HL-60 cell to HT, Ara-C, As2O3 and RA.

Method:Incubate leukima cell HL-60 with H, Ara-C, As2O3 and RA, meanwhile transfected siRNAs or G3139 in it by lipofectamine2000 with OPTI-MEM cultural medium for 6h, then add equal volume RPMI-1640 culture medium containing 20% inactive newborn calf serum. After transfection for 24,48 and 72h, cell proliferations were determined by MTT method. After transfection for 48h, we test the level of Bcl-2 protein, ROS and membrane potential of mitochondrion in HL-60 cell by FCM.

Result:

Compare with control group, the G3139 and siRNA targeting bcl-2 combine with H or Ara-C or As2O3 have significant effects on inhibiting the proliferation of HL-60(P <0.05); After transfecting 48h, the level of Bcl-2 protein and ROS decrease significantly, meanwhile membrane potential of mitochondrion in HL-60 cells increase markedly (P<0.01). Compare with control group, the G3139 and siRNA targeting bcl-2 combine with RA have significant effects on inhibiting the proliferation of HL-60 and the level of Bcl-2 protein decrease, but there were not difference between test groups and control group on the level of ROS and membrane potential of mitochondrion (P<0.01).

Conclusion: The effective siRNA2, targeting bcl-2, can increased the drug-sensitivity though inhibiting the expression of Bcl-2 protein. Different drug has different mechanism to induce the leukimic cell apoptosis.
The Requirement of Reactive Oxygen Species Generation in the Apoptosis of Leukemia Cells Induced by 2-Methoxyestradiol.
Authors: Kunyuan Guo , She Miaorong , Hu Haiyan , Niu Xinqing , Tu Sanfang , Lu Xiaoxun
Published: April 2020

2-Methoxyestradiol (2-ME) is a new anticancer agent currently under investigation for treatment of leukemia. We evaluated the effects of 2-ME-induced apoptosis in two myeloid leukemia cell lines (U937 and HL-60) in association with reactive oxygen species (ROS) generation. We found that 2-ME resulted in viability decrease in a dose-dependent manner, generated ROS: nitric oxide and superoxide anions, and mitochondria damage. 2-ME-induced apoptosis correlated with increase in ROS. Quenching of ROS with N-acetyl-L-cysteine protected leukemia cells from the cytotoxicity of 2-ME and prevented apoptosis induction by 2-ME. Furthermore, addition of manumycin, a farnesyltransferase inhibitor, demonstrated by our previous studies that induced apoptosis of leukemic cells and induced ROS, significantly enhanced the apoptosis-induced by 2-ME. In conclusion, cellular ROS generation play an important role in the cytotoxic effect of 2-ME. It is possible to use ROS-generation agents such as manumycin to enhance the antileukemic effect. Such a combination strategy need the further in vivo justify and may have potential clinical application.
Functional CXCR4 Expressing Microparticles and SDF-1 Correlate with Circulating AML Cell Counts.
Authors: Kalinkovich Alexander , Tavor Sigal , Avigdor Abraham , Kahn Joy , Brill Alexander , Petit Isabelle , Goichberg Polina , Tesio Melania , Netzer Neta , Naparstek Elizabeth , Hardan Izhar , Nagler Arnon , Resnick Igor , Tsimanis Alexander , Lapidot Tsvee
Published: April 2020

Stromal cell-derived factor-1 (SDF-1/CXCL12) and its receptor CXCR4 are implicated in the pathogenesis and prognosis of AML. Cellular microparticles (MPs), the submicron vesicles shed from the plasma membrane of various circulating cells, are associated with numerous human disorders. In the present study, we studied the putative relationships between CXCR4/SDF-1 axis and MPs in AML. We detected CXCR4 expressing MPs (CXCR4+MPs) in the peripheral blood and bone marrow plasma samples of normal donors (n=24) and newly diagnosed adult AML patients (n=26). The majority of CXCR4+MPs in AML patients were CD45+ whereas in normal individuals they were mostly CD41+. In samples from AML patients, the levels of CXCR4+MPs and total SDF-1 were significantly elevated as compared to normal individuals. Importantly, we found a strong correlation between the levels of CXCR4+MP and white blood cell (WBC) counts in the peripheral blood and bone marrow plasma obtained from the AML patients. Of interest, functional, non-cleaved SDF-1 levels were reduced in these patients compared to normal individuals, and also strongly correlated with the WBC counts. Furthermore, our data indicate N-terminal truncation of the CXCR4 molecule in the MPs of AML patients. This was found also in MPs obtained from the conditioned media of normal human CD34+ progenitors lentiviarlly transduced with CXCR4 vector in vitro. Appearance of MPs possessing N-terminally truncated CXCR4 in AML patients is likely to be dependent on proteolytic enzymes, such as elastase, which was elevated. However, MPs isolated from AML patients were capable of transferring functional CXCR4 molecule to the AML-derived HL-60 cells, enhancing their migration to SDF-1 in vitro and increasing their homing to the bone marrow of irradiated NOD/SCID mice. The CXCR4 antagonist AMD3100 reduced the increased migration and homing of MP treated HL-60 cells. Taken together, these findings suggest that functional CXCR4+MPs and SDF-1 are involved in the progression of AML. We propose that their levels are potentially valuable as an additional diagnostic AML parameter. Moreover, our findings suggest also the need for CXCR4- and SDF-1-target therapeutic approaches, clinically relevant in AML in the near future.