Human Acute Myelocytic Leukemia Cell Research Articles

A polymer monolith composed of a perovskite and cucurbit[6]uril hybrid for highly selective enrichment of phosphopeptides prior to mass spectrometric analysis.

A hybrid monolith was prepared from perovskite and cucurbit[6]uril [poly(hydroxyethyl methacrylate-pentaerythritol triacrylate) monolith] for the enrichment of phosphopeptides. By coupling with mass spectrometry, three goals were simultaneously realized, viz. (a) selective enrichment of phosphopeptides from non-phosphopeptides, (b) identification of mono- and multi-phosphopeptides, and (c) recognition of tyrosine phosphopeptides. The perovskite introduced into the monolith warrants high selectivity for phosphopeptides even at a high (10,000:1) ratio of non-phosphopeptides to phosphopeptides, and and enables identification of eight mono- and multi-phosphopeptides from standard β-casein tryptic digests. Tyrosine phosphopeptides were specifically detected via the recognition capability of cucurbit[6]uril integrated into the monolith. The method has remarkably specific enrichment capacity for phosphopeptides from samples including human serum, nonfat milk, and human acute myelocytic leukemia cell lysate. Graphical abstractSchematic representation of a monolith integrated with perovskite and cucurbit[6]uril. The monolithic column was coupled with mass spectrometry and applied to the enrichment of phosphopeptides. The method has remarkably specific enrichment capacity for phosphopeptides from complex biological samples.

Establishment and characterization of a new human acute myelocytic leukemia cell line SH-2 with a loss of Y chromosome, a derivative chromosome 16 resulting from an unbalanced translocation between chromosomes 16 and 17, monosomy 17, trisomy 19, and p53 alteration

To report here a new acute myelocytic leukemia (AML) cell line SH-2 and describe its biological characteristics. Mononuclear cells isolated from a patient with AML-M2 subtype were passaged by liquid culture medium. Interleukin-3 and bone marrow stromal cells were used to support cell proliferation at the first 3 months. Various methods, including cytogenetic analysis, fluorescence in situ hybridization (FISH), multiplex FISH (M-FISH), reverse transcriptase polymerase chain reaction (RT-PCR), multiplex RT-PCR, short tandem repeat (STR)-PCR, direct sequencing of DNA, clonogenic assay, and tumorigenicity in nude and severe combined immunodeficient (SCID) mice were employed to identify and characterize SH-2 cell line. SH-2 cells were maintained without cytokine and stromal cells for 3 years. It had no Epstein-Barr virus or mycoplasma contamination. The SH-2 cell line showed typical myelocytic features in morphology and simultaneous strongly expressed myeloid antigens (CD13, 99.6% and CD33, 99.26%) and natural killer (NK)-related antigens (CD56, 99.5% and CD16/56, 99.62%) suggesting that SH-2 is an AML cell line with NK-antigen expression. SH-2 cell line initially showed a karyotype of 45, X, -Y, der(16)t(16;17)(q24;q12), -17, +19. During the passage period, the cells with a hypodiploid karyotype gradually decreased and were replaced by the near-tetraploid cells with a karyotype of 71-105(86), XX, -Y, -Y, der(16)t(16;17)x2, -17, -17, +19, +19. FISH and M-FISH delineated all abnormalities. SH-2 cells had the approximately same morphological, immunophenotypical, and cytogenetic features as the patient's leukemia cells had. STR-PCR provided powerful evidence for the derivation of SH-2 cell line from the patient's leukemia cells. SH-2 cells showed multiple drug resistance (MDR), which may be related to the p53 gene alteration, including the loss of one p53 allele due to the monosomy 17 and a point mutation of CAG to CAT at codon 576 of exon 5 in another p53 allele resulting in the loss of p53 gene function. In addition, SH-2 cell line did not express MDR-related genes, such as MDR1, multidrug resistance-related protein, and lung resistance protein, but expressed apoptosis-related genes, such as Bcl-2, Fas, glutathione S-transferase-pi, and p21, which were also related to the MDR. SH-2 cell line had tumorigenic capacities in nude and SCID mice. Because SH-2 cell line had a clear biology background, it will provide a useful tool for the study of the pathogenesis and treatment strategy of AML with MDR.

The Establishment and Characterization of a Human Acute Myelocytic Leukemia Cell Line, SH-2 Carrying t(16;17))(q24;q12) Translocation.

We report the establishment of a novel cell line from a patient with acute myelocytic leukemia. The initial growth of this cell line was dependent on the presence of the stromal cell and cytokine interleukine 3. Subsequently, a stroma and cytokine-independent cell line was established, named SH-2. The cell line has proliferated continuously in vitro for more than 24 months. The SH-2 cell line showed identical chromosomal alterations to that of the initial bone marrow aspiration specimen, demonstrated a t(16;17))(q24;q12) translocation accompanied by deletions of y and 17 and triple 19.Chromosome painting FISH and Multi-FISH conformed the karyotype.SH-2 cells expressed myeloid antigens CD13, CD33 and MPO, SH-2 co-expressed NK markers CD16 and CD56. The loss of one p53 allele were proven by chromosome painting, FISH. A point mutation of CAG to CAT at codon 576 of extron 5 in another p53 allele was found by direct sequencing of DNA. Neither Epstein-Barr virus nor mycoplasma was not detected in SH-2 cells. DNA fingerprinting confirmed the authenticity of the cell line. Cytokines of GM-CSF,IL-3,IL-6,SCF,IL-4 and IL-5 can improve the proliferation of SH-2. SH-2 has colony formality and tumorigenic capacity in nude mice. The breakpoints of chromosomes 16 and 17 were localized RP11-356C4(16q24.3)and between BAC clone RP1-161P9 and RP11-47L3 corresponding to 17q12. The establishment of an myelocytic leukemia cell line with t(16;17)(q24;q12) could be valuable for the study of leukemogenesis and for the research of cloning the new gene involved in the t(16;17)(q24;q12) translocation.

8-Hydroxydeoxyguanosine induces senescence-like changes in KG-1, human acute myelocytic leukemia cell line

8-Hydroxydeoxyguanosine (oh8dG) treatment induced senescence-like changes in KG-1 cells, a human acute myelocytic leukemia cell line. The oh8dG-treated cells stained positive for senescence associated β-galactosidase (SA-β-galactosidase) and had enlarged cell shape, both of which are senescence indexes. The oh8dG-treated cells were also cell growth inhibited and arrested at G1 in the cell cycle. The accumulation of cdk (cyclin dependent kinase) inhibitors, such as p16, p21, and p27, also implies that cellular senescence was induced in oh8dG-treated cells. However, these changes were not accompanied by cell differentiation or telomerase activity. Taken together, we conclude that oh8dG treatment of KG-1 cells induces cellular senescence.

Effects of anti-CXCR 4 monoclonal antibody 12G5 on proliferation and apoptosis of human acute myelocytic leukemia cell line HL-60

Objective To investigate the expression of CXCR 4 on HL-60 cell line and the proliferation. apoptosis of HL-60 cell line cocultured with bone marrow stromal cells, so as to assess the possibility of 12G5, an anti-CXCR 4 monoclonal antibody, in eradicating the minimal residual disease. Methods The activity of SDF-1 was inhibited by 10 μg/ml 12G5. After treatment with 12G5, the status of adhesion was observed, and the adhesion rates, apoptosis and cell cycles were detected after 24 h of treatment. Cell growth rates were measured by trypan blue exclusion. Cell growth curve was plotted, and the expression of PCNA and apoptosis related protein including PCNA, Bcl-2 and Fas were detected with immunohis-tochemical technique. Results (1) There was middling degree expression of CXCR 4 on HL-60 membrane. From 0 h to 6 h, as the time of 12G5 incubation along, the expression of CXCR 4 decreased gradually. (2) After treatment for 24 h, the adhesion rates in the experiment group and the control were (39.4 ± 7.9) and (51.4±5.9)%, respectively. (3) After treatment for 24 h, the percentage of HL-60 cells in G. G. phase were (55.21±4.9)%, and that in S phase and G 2/M phase were (30.40±4.1)%, and (14.39 = 5.2)%, respectively, with the corresponding proportions being (44.67±2.2)%, (45.30±3.7)%, and (10.03±2.6)% in the control. (4) The percentage of apoptotic HL-60 cells was (8.95±1.7)% in the experiment group, compared to (3.97±2.4)% in the control. (5) The survival rates of HL-60 cells decreased markedly at 48 h to 96 h, and the proliferation slowed down at this time duration. (6) The expression of PCNA and Bcl-2 down-regulated significantly, but the Fas protein expression was up-regulated. Conclusion 12G5 could inhibit the capability of adhesion and proliferation of HL-60 cells and it can induce more cells to enter G 0/G 1 phase and promote apoptosis. It may be helpful by inhibiting the bloactivity of SDF-1 with 12G5 in the therapy of marrow residual disease.

Histamine H2 receptors and histidine decarboxylase in normal and leukemic human monocytes and macrophages.

Spontaneous and all-trans-retinoic acid (RA)-induced differentiation of normal human monocytes and of leukemic THP-1 monocytes into macrophages resulted in a progressive loss of adenosine 3',5'-cyclic monophosphate production induced by histamine via typical H2 receptors (H2R). In THP-1 cells and in HL-60 human acute myelocytic leukemia cells, RA treatment increased the abundance of the 4.5-kb messenger RNA of the H2R gene fourfold, suggesting transcriptional control by a RA response element. Scatchard plots of [3H]tiotidine binding indicated the expression of H2R with similar affinity and binding capacity in THP-1 monocytes and macrophages, while the conversion of normal monocytes into macrophages decreased H2R density from 91.8 to 43.1 fmol/mg protein, with no change in affinity (Kd = 9.9 to 11.2 nM). In THP-1 macrophages, histamine inhibited 4 beta-phorbol 12-myristate 13-acetate (PMA)-induced H2O2 formation via the activation of H2 receptors. Expression of the H2R gene, histamine accumulation, and histidine decarboxylase activity were also demonstrated in normal human monocytes/macrophages and peripheral lymphocytes. Histamine and H2R may therefore affect, via intracrine, autocrine, and paracrine pathways, various immune and inflammatory responses of the lymphoid and myeloid progenitors and lineages in the bone marrow and peripheral tissues.

RDlx, a Novel Distal-less-like Homeoprotein Is Expressed in Developing Cartilages and Discrete Neuronal Tissues

From a rat chondrosarcoma we isolated a cDNA that encodes a novel homeoprotein rDlx. The homeodomain of rDlx shows a high degree of sequence identity with those of Drosophila Distal-less, mouse Dlx, and Xenopus Xdll proteins. Northern hybridization of rDlx revealed a 1.4- to 1.6-kb RNA species in a rat chondrosarcoma and a cell line derived from this tumor and in mouse C3H10T1/2 cells, but no rDlx RNA was detected in mouse NIH3T3 fibroblasts, rat skin fibroblasts, mouse C2 myoblasts, mouse myeloma S194 cells, human B-cell lymphoma Daudi cells, or human acute myelocytic leukemia cells. RNase protection assays showed that rDlx transcripts were present at high levels in 14-day-old rat embryos, 18-day-old rat embryo skeletal tissues, and adult rat brain. rDlx RNAs were present at lower levels in newborn rat rib cartilage, 18-day-old rat embryo soft tissues, newborn rat skin, and adult rat heart. rDlx transcripts were not detected in adult rat liver, spleen, lung, kidney, testis, or skeletal muscle. In situ hybridization of rat embryos at different stages revealed that rDlx transcripts were present in otic vesicle, branchial arches, apical ectodermal ridge of limb bud, developing cartilages, perichondria of mature cartilages, mesenchymal cells of developing membranous bones, developing teeth, ganglionic eminence of the telencephalon, diencephalon, olfactory epithelia, and epidermis of the skin. rDlx RNAs were also detected in the developing parasympathetic mesenteric ganglia of the gastrointestinal tract. Hence, rDlx RNAs are mainly expressed in several neuronal tissues and developing skeletal tissues.

Interleukin-3 and granulocyte-monocyte colony-stimulating factor receptors on human acute myelocytic leukemia cells and relationship to the proliferative response

Interleukin-3 (IL-3) and granulocyte-monocyte-colony-stimulating factor (GM-CSF) stimulate proliferation of human acute myeloid leukemia (AML) in vitro, although patterns of response among clinical cases are diverse. Whether regulatory abnormalities related to growth factor responses in human AML may establish the outgrowth of the neoplasm is unclear. We determined receptor numbers and affinity for IL-3 and GM- CSF on human AML cells using human recombinant IL-3 (rIL-3) and GM-CSF (rGM-CSF). In 13 of 15 cases of primary AML high-affinity (kd 26 to 414 pmol/L) receptors for IL-3 were demonstrable on the cells. The average numbers of IL-3 receptors ranged from 21 to 145 receptors per cell. Normal WBCs showed IL-3 receptors on their surface at similar densities. IL-3 receptor positivity often correlated with GM-CSF receptor positivity of AML; GM-CSF receptors were demonstrated on the cells of 11 of 15 cases, although average numbers of GM-CSF receptors were ten times greater. The in vitro response of the cells to exogenous IL-3 or GM-CSF was examined by measuring thymidine uptake. Because IL-3 and GM-CSF were potent inducers of DNA synthesis in vitro, apparently relatively few receptors are required to permit activation of growth. These experiments did not provide evidence for overexpression or increased receptor sensitivity as an explanation for AML growth. In a minority of cases, however, the cells were unable to respond to IL-3 (four of 15 cases) or GM-CSF (four of 15 cases) despite normal receptor availability on the cell surface.

Interleukin-3 and Granulocyte-Monocyte Colony-Stimulating Factor Receptors on Human Acute Myelocytic Leukemia Cells and Relationship to the Proliferative Response

Interleukin-3 (IL-3) and granulocyte-monocyte-colony-stimulating factor (GM-CSF) stimulate proliferation of human acute myeloid leukemia (AML) in vitro, although patterns of response among clinical cases are diverse. Whether regulatory abnormalities related to growth factor responses in human AML may establish the outgrowth of the neoplasm is unclear. We determined receptor numbers and affinity for IL-3 and GM-CSF on human AML cells using human recombinant IL-3 (rIL-3) and GM-CSF (rGM-CSF). In 13 of 15 cases of primary AML high-affinity (kd 26 to 414 pmol/L) receptors for IL-3 were demonstrable on the cells. The average numbers of IL-3 receptors ranged from 21 to 145 receptors per cell. Normal WBCs showed IL-3 receptors on their surface at similar densities. IL-3 receptor positivity often correlated with GM-CSF receptor positivity of AML; GM-CSF receptors were demonstrated on the cells of 11 of 15 cases, although average numbers of GM-CSF receptors were ten times greater. The in vitro response of the cells to exogenous IL-3 or GM-CSF was examined by measuring thymidine uptake. Because IL-3 and GM-CSF were potent inducers of DNA synthesis in vitro, apparently relatively few receptors are required to permit activation of growth. These experiments did not provide evidence for overexpression or increased receptor sensitivity as an explanation for AML growth. In a minority of cases, however, the cells were unable to respond to IL-3 (four of 15 cases) or GM-CSF (four of 15 cases) despite normal receptor availability on the cell surface.