Cell Line 32D Research Articles

Gab1 mediates hepatocyte growth factor-stimulated mitogenicity and morphogenesis in multipotent myeloid cells.

Hepatocyte growth factor (HGF)-stimulated mitogenesis, motogenesis and morphogenesis in various cell types begins with activation of the Met receptor tyrosine kinase and the recruitment of intracellular adaptors and kinase substrates. The adapter protein Gab1 is a critical effector and substrate of activated Met, mediating morphogenesis, among other activities, in epithelial cells. To define its role downstream of Met in hematopoietic cells, Gab1 was expressed in the HGF-responsive, Gab1-negative murine myeloid cell line 32D. Interestingly, the adhesion and motility of Gab1-expressing cells were significantly greater than parental cells, independent of growth factor treatment. Downstream of activated Met, Gab1 expression was specifically associated with rapid Shp-2 recruitment and activation, increased mitogenic potency, suppression of GATA-1 expression and concomitant upregulation of GATA-2 transcription. In addition to enhanced proliferation, continuous culture of Gab1-expressing 32D cells in HGF resulted in cell attachment, filopodia extension and phenotypic changes suggestive of monocytic differentiation. Our results suggest that in myeloid cells, Gab1 is likely to enhance HGF mitogenicity by coupling Met to Shp-2 and GATA-2 expression, thereby potentially contributing to normal myeloid differentiation as well as oncogenic transformation.

Over‐expression of EDAG in the myeloid cell line 32D: Induction of GATA‐1 expression and erythroid/megakaryocytic phenotype

Erythroid differentiation-associated gene (EDAG), a hematopoietic tissue-specific transcription regulator, plays a key role in maintaining the homeostasis of hematopoietic lineage commitment. However, the mechanism and genes regulated by EDAG remain unknown. In this study, we showed that overexpression of EDAG in a myeloid cell line 32D led to an erythroid phenotype with increased number of benzidine-positive cells and up-regulation of erythroid specific surface marker TER119. The megakaryocytic specific marker CD61 was also induced significantly. Using a genome-wide microarray analysis and a twofold change cutoff, we identified 332 genes with reduced expression and 288 genes with increased expression. Among up-regulation genes, transcription factor GATA-1 and its target genes including EKLF, NF-E2, Gfi-1b, hemogen, SCL, hemoglobin alpha, beta and megakaryocytic gene GPIX were increased. Silencing of EDAG by RNA interference in K562 cells resulted in down-regulation of these genes. Taken together, EDAG functions as a positive regulator of erythroid/megakaryocytic differentiation in 32D cells associated with the induction of GATA-1 and its target genes.

Re-programming of IL-10 signal transduction: an inflammation-induced mechanism to control T cell susceptibility to suppression (49.18)

Abstract Modulation of regulatory T cell (Treg) suppressive function may play an important role in the development of autoimmune diseases, tumor growth and transplant outcome. Currently, little is known about the molecular mechanisms that render T cells refractory to suppression. Since IL-10 is an important mediator of Treg activity, we investigated the influence of pro-inflammatory mediators on the IL-10 signaling pathway in T cells. The mouse CD4 T cell line D10 was incubated for various periods with the supernatant of bone marrow-derived dendritic cells stimulated overnight with the TLR4 ligand LPS (MATSup). We then measured the integrity of the IL-10 signaling pathway by assessing phosphorylation of the transcription factor STAT3 in response to titrated amounts of IL-10. Even after 24h incubation with MATSup, phosphorylated STAT3 was readily detected in T cells in response to IL-10. Surprisingly, measurement of cytoplasmic levels of total STAT3 and of pro-inflammatory STAT1 revealed short- term (3 to 6 hours) reduction of both transcription factors, followed by upregulation with an altered ratio between STAT3 and STAT1 in favor of the latter. This altered ratio appeared within 12 hours and was preserved for at least 24 hours. These results suggest that pro-inflammatory mediators re-program the IL-10 (and probably other) signaling pathway in T cells, reducing their susceptibility to suppression. Interference with this process may represent an effective immunomodulatory strategy.

Effects of aqueous garlic extract on oxidant/antioxidant status in 32D and 32Dp210 cell lines

To investigate the possible effects of aqueous garlic extract on the oxidant/antioxidant status and apoptosis in 32D (wild type mouse myeloid cell = normal) and 32Dp210 (BCR-ABL fusion gene (+) mouse myeloid cell = Chronic Myelocytic Leukemia cells) cell lines. Materials and methods: Aqueous garlic extract (10% w/v) was added into the cell line media with 2 different final concentrations (0.4% and 1%). At 0 h and at 24, 48, and 72 h later, the oxidant (malondialdehyde (MDA) level, and xanthine oxidase (XO) enzyme activity) and antioxidant (superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) enzyme activities) parameters were measured in the cell lines. Results: It was observed that the garlic extract caused no change in the XO and antioxidant enzyme activities, but it increased the MDA level in the 32D cell line. However, significant increases were found in the MDA level, XO, and antioxidant enzyme activities in the 32Dp210 cell line treated by the garlic extract. Additionally, it was shown that garlic extract had antiproliferative and apoptotic effects on both cell lines. The most effective apoptotic dose was found to be 0.4% (w/v), and at this concentration the death risk of the 32Dp210 cell line was calculated at 2.08 times higher than that of the 32D cell line. Conclusion: It has been suggested that garlic directly causes oxidant stress in the 32D cell line owing to its own oxidant ingredients, and that the oxidant stress created by garlic in the 32Dp210 cell line might occur through increased XO activity and/or its oxidant ingredients. Additionally, antioxidant enzyme activities were found to increase in the 32Dp210 cell line; it would seem that this compensatory change could not prevent the oxidant stress created. We think that the oxidant potential of garlic extract might play a part in its possible anticancer potential, previously supposed by several investigators.

Kinase Activity of RTKs Is Dispensable for Factor Independent Growth and Transformation of a Myeloid Cell Line 32D in the Presence of Cbl Mutants.

Abstract 3970Poster Board III-906 PurposeThe Cbl proto-oncogene products have emerged as important components of the signal transduction cascades downstream of both non-receptor and receptor tyrosine kinases (RTKs). By regulation of receptor trafficking and degradation, they have been shown to tightly regulate the intensity and amplitude of RTK activation. c-Kit belongs to the family of the class-III RTKs and plays an important role in the pathogenesis of acute myeloid leukemia (AML). So far, very little is known about the role of c-Cbl mutants in the role of c-Kit signaling. ResultsWe analyzed the interaction of c-Cbl with c-Kit and the functional relevance of this interaction in the IL-3-dependent murine myeloid progenitor cell line 32Dcl3. We recently identified the first c-Cbl mutation in human disease in an AML patient, named Cbl-R420Q. Co-expression of two different dominant negative mutants of c-Cbl (Cbl-R420Q or Cbl-70Z) with Kit induced cytokine-independent proliferation, survival and clonogenic growth. Importantly, transformation was observed also with kinase-dead forms of Kit and Flt3 in the presence of Cbl-70Z, but not in the absence of Kit or Flt3, suggesting a mechanism dependent on RTKs, but independent of their kinase activity. Instead, transformation appeared to depend on Src family kinases (SFKs), as c-Cbl co-immunoprecipitated with SFKs and SFK inhibition abolished transformation. ConclusionOur results indicate that c-Cbl has an important role in c-Kit signal mitigation. They demonstrate that disturbed mechanisms of c-Kit internalization have important implications for its transforming potential, possibly in the development of AML. Furthermore, these findings may explain primary resistance to tyrosine kinase inhibitors targeted at RTKs. Disclosures:No relevant conflicts of interest to declare.

5030 High resolution array Comparative Genomic Hybridization (aCGH) of breast carcinoma identifies Mouse double minutes 4 (Mdm4) as one of the early genetic changes in breast cancer development – Mdm4 is a new independent prognostic and predictive marker

To evaluate the effectiveness of mitigation of acute ionizing radiation damage by mitochondrion-targeted small molecules.We evaluated the ability of nitroxide-linked alkene peptide isostere JP4-039, the nitric oxide synthase inhibitor-linked alkene peptide esostere MCF201-89, and the p53/mdm2/mdm4 protein complex inhibitor BEB55 to mitigate radiation effects by clonogenic survival curves with the murine hematopoietic progenitor cell line 32D cl 3 and the human bone marrow stromal (KM101) and pulmonary epithelial (IB3) cell lines. The p53-dependent mechanism of action was tested with p53+/+ and p53−/− murine bone marrow stromal cell lines. C57BL/6 NHsd female mice were injected i.p. with JP4-039, MCF201-89, or BEB55 individually or in combination, after receiving 9.5 Gy total body irradiation (TBI).Each drug, JP4-039, MCF201-89, or BEB55, individually or as a mixture of all three compounds increased the survival of 32D cl 3 (p = 0.0021, p = 0.0011, p = 0.0038, and p = 0.0073, respectively) and IB3 cells (p = 0.0193, p = 0.0452, p = 0.0017, and p = 0.0019, respectively) significantly relative to that of control irradiated cells. KM101 cells were protected by individual drugs (p = 0.0007, p = 0.0235, p = 0.0044, respectively). JP4-039 and MCF201-89 increased irradiation survival of both p53+/+ (p = 0.0396 and p = 0.0071, respectively) and p53−/− cells (p = 0.0007 and p = 0.0188, respectively), while BEB55 was ineffective with p53−/− cells. Drugs administered individually or as a mixtures of all three after TBI significantly increased mouse survival (p = 0.0234, 0.0009, 0.0052, and 0.0167, respectively).Mitochondrial targeting of small molecule radiation mitigators decreases irradiation-induced cell death in vitro and prolongs survival of lethally irradiated mice.

Molecular variability of FLT3/ITD mutants and their impact on the differentiation program of 32D cells: Implications for the biological properties of AML blasts

FLT3 is the most frequently mutated gene in acute myeloid leukemia (AML), with internal tandem duplications (ITDs) accounting for up to 30% of its mutations. To analyze the impact of individual ITDs on the expression profile of immature myeloid cells, we have established 32D cell lines expressing nine different FLT3/ITDs isolated from AML patients and subjected them to whole genome expression profiling and 2DE/LC/MS proteomics. Our data indicate that in comparison to the controls, FLT3/ITD -positive 32D cells exhibit less mature expression profiles resembling early hematopoietic progenitors. Moreover, our results suggest that there exist biological differences among individual ITD variants.

Molecular Variability of FLT3/ITD Mutants and Their Impact on the Differentiation Program of 32D Cells: Implications for the Biological Properties of AML Blasts

FLT3 is the most frequently mutated gene in AML – up to 40% of AML harbor an activating mutation within FLT3 gene. Though AML is a relatively rare disease, such a high mutability rate, as observed with FLT3 gene, is striking. To elucidate the molecular background of this phenomenon, we have established nine unique FLT3/ITD - carrying 32D cell lines and a set of controls, and subjected them to whole genome expression analysis and 2DE LC/MS proteomics. Data obtained on this so far largest set of ITD mutants indicates that FLT3/ITD positive cells exhibit less mature expression profiles resembling ST-HSC and MkEP/CMP/LMPP progenitors. FLT3/ITD-positive 32D cells overexpress a combination of genes of pluripotency (Etv5, endoglin, EpoR, Mpl, Trfr2, GATA.2, Hemgn, Myb, Dok2, Gli1, Pim3, PML, Ikaros, Pum1, Dlx4, Hox-c8, HoxB9, Runx1, c-Myc, Shh, FL) and early erythroid precursors (EpoR, Trfr2, GATA.1, Klf1), implicating their less mature expression profile than 32D cell lines harboring wild type FLT3 or 32D controls. We hypothesize that FLT3/ITD might contribute not only to the proliferative advantage of FLT3/ITD positive cells, but also to their reprogramming towards less differentiated stages, thus strengthening their malignant properties. This finding might explain the pronounced mutation rate of the aberrantly expressed FLT3 gene in AML, and, also, the inferior prognosis of FLT3/ITD positive AML patients. Moreover, the microarray data has revealed biological differences among individual ITD variants – a finding supporting the recent clinical data on the prognostic impact of the size of individual ITDs.

Acetylated Resveratrol: A New Small Molecule Radioprotector

New small molecules with radioprotective capacity are required for treatment of radiation spills or as countermeasures against radiological terrorism. Small molecules which can be easily stored, easily transported, inexpensive to produce and easily administered are optimal. We have tested the dietary component resveratrol and an acetylated moiety for protection of cells against radiation damage. Irradiation survival curves were carried out with resveratrol or acetylated resveratrol. Cells from the murine hematopoietic progenitor cell line 32D cl 3 were incubated with resveratrol or acetylated resveratrol (10 μM) for 1 hr and then were irradiated to doses ranging from 0 to 8 Gy. The cells were suspended in methylcellulose, incubated for 7 days at 37oC, colonies of greater than 50 cells counted and data analyzed by linear quadratic and single-hit, multi-target models. In vivo studies were performed by injecting intraperitoneally female C57BL/6NHsd mice either 10 minutes before or 10 minutes after an LD50/30 dose of 9.5 Gy total body irradiation. We compared resveratrol or acetylated resveratrol (1, 10, or 25 mg/kg). The mice were followed for irradiation induced hematopoietic syndrome. Incubation of 32D cl 3 cells in resveratrol or acetylated resveratrol before irradiation resulted in an increase in radiation resistance by an increased shoulder on the survival curve of 33.2 ± 5.7 or 37.5 ± 9.9, respectively, compared to 6.9 + 1.8 for 32D cl 3 cells (p = 0.0122 or 0.0072, respectively). The mice injected with acetylated-resveratrol had increased survival at 10 and 25 mg/kg compared to control irradiated mice (p < 0.0001 or = 0.0004, respectively). In contrast, resveratrol did not increase mouse survival. Neither resveratrol nor acetylated resveratrol were effective mitigators when given after irradiation. Acetylated resveratrol administered before irradiation in a mouse model is radioprotective. Further studies are in progress to determine if acetylated resveratrol can be translated to clinical use as a radioprotective agent.

STI571 reduces NER activity in BCR/ABL-expressing cells

Nucleotide-excision repair (NER) is the most versatile mechanism of DNA repair, recognizing and dealing with a variety of helix-distorting lesions, such as the UV-induced photoproducts cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6–4) photoproducts. We investigated the influence of an anticancer drug, STI571, on the efficacy of NER in removing UV-induced DNA damage. STI571 is used mostly in the treatment of chronic myeloid leukemia and inhibits activity of the BCR/ABL oncogenic tyrosine kinase, which is a hallmark of this disease. NER activity was examined in the BCR/ABL-expressing cell lines K562 and BV173 of myeloid and lymphoid origin, respectively, as well as in CCRF-CEM cells, which do not express BCR/ABL. A murine myeloid parental 32D cell line and its counterpart transfected with the BCR/ ABL gene were also tested. NER activity was assessed in the cell extracts by use of an UV-irradiated plasmid as a substrate and by a modified single-cell gel electrophoresis (comet) assay on UV-treated nucleoids. Additionally, quantitative PCR was performed to evaluate the efficacy of the removal of UV-induced lesions from the p53 gene by intact cells. Results obtained from these experiments indicate that STI571 decreases the efficacy of NER in leukemic cells expressing BCR/ABL. Therefore, STI571 may overcome the drug resistance associated with increased DNA repair in BCR/ABL-positive leukemias.

A cell-based high-throughput screen for epidermal growth factor receptor pathway inhibitors

Epidermal growth factor receptor (EGFR) is a valid drug target for development of target-based therapeutics against non-small-cell lung cancer. In this study, we established a high-throughput cell-based assay to screen for compounds that may inhibit EGFR activation and/or EGFR-mediated downstream signaling pathway. This drug screening platform is based on the characterization of an EGFR-transfected 32D cell line (32D-EGFR). The expression of EGFR in 32D cells allowed cell proliferation in the presence of either epidermal growth factor (EGF) or interleukin 3 (IL-3) and provided a system for both screening and counterscreening of EGFR pathway-inhibitory compounds. After the completion of primary and secondary screenings in which 32D-EGFR cells were grown under the stimulation of either EGF or IL-3, 9 of 20,000 compounds were found to selectively inhibit the EGF-dependent proliferation, but not the IL-3-dependent proliferation, of 32D-EGFR cells. Subsequent analysis showed that 3 compounds of the 9 initial hits directly inhibited the kinase activity of recombinant EGFR in vitro and the phosphorylation of EGFR in H1299 cells transfected with EGFR. Thus, this 32D-EGFR assay system provides a promising approach for identifying novel EGFR and EGFR signaling pathway inhibitors with potential antitumor activity.

Dimerization by a Cytokine Receptor Is Necessary for Constitutive Activation of JAK2V617F

The majority of the BCR-ABL-negative myeloproliferative disorders express the mutant JAK2, JAK2V617F. Previously we showed that constitutive activation of this oncogenic JAK2 mutant in Ba/F3 or 32D cells requires coexpression of a cognate homodimeric cytokine receptor, such as the EpoR. However, overexpression of JAK2V617F in Ba/F3 cells renders them cytokine-independent for growth in the absence of an exogenous cytokine receptor. Here, we demonstrated that JAK2V617F domains required for receptor association are essential for cytokine-independent growth by overexpressed JAK2V617F, suggesting JAK2V617F is binding to an unknown endogenous cytokine receptor(s) for its activation. We further showed that disruption of EpoR dimerization by coexpressing a truncated EpoR disrupted JAK2V617F-mediated transformation, indicating that EpoR dimerization plays an essential role in the activation of JAK2V617F. Interestingly, coexpression of JAK2V617F with EpoR mutants that retain JAK2 binding but are defective in mediating Epo-dependent JAK2 activation due to mutations in a conserved juxtamembrane motif does lead to cytokine-independent activation of JAK2V617F. Overall, these findings confirm that JAK2V617F requires binding to a dimerized cytokine receptor for its activation, and that the key EpoR juxtamembrane regulatory motif essential for Epo-dependent JAK2 activation is not essential for the activation of JAK2V617F. The structure of the activated JAK2V617F is thus likely to be different from that of the activated wild-type JAK2, raising the possibility of developing a specifically targeted therapy for myeloproliferative disorders.

Isomerization of a Single Aspartyl Residue of Anti-Epidermal Growth Factor Receptor Immunoglobulin γ2 Antibody Highlights the Role Avidity Plays in Antibody Activity

A new isoform of the light chain of a fully human monoclonal immunoglobulin gamma2 (IgG2) antibody panitumumab against human epidermal growth factor receptor (EGFR) was generated by in vitro aging. The isoform was attributed to the isomerization of aspartate 92 located between phenylalanine 91 and histidine 93 residues in the antigen-binding region. The isomerization rate increased with increased temperature and decreased pH. A size-exclusion chromatography binding assay was used to show that one antibody molecule was able to bind two soluble extracellular EGFR molecules in solution, and isomerization of one or both Asp-92 residues deactivated one or both antigen-binding regions, respectively. In addition, isomerization of Asp-92 showed a decrease in in vitro potency as measured by a cell proliferation assay with a 32D cell line that expressed the full-length human EGFR. The data indicate that antibodies containing either one or two isomerized residues were not effective in inhibiting EGFR-mediated cell proliferation, and that two unmodified antigen binding regions were needed to achieve full efficacy. For comparison, the potency of an intact IgG1 antibody cetuximab against the same receptor was correlated with the bioactivity of its individual antigen-binding fragments. The intact IgG1 antibody with two antigen-binding fragments was also much more active in suppressing cell proliferation than the individual fragments, similar to the IgG2 results. These results indicated that avidity played a key role in the inhibition of cell proliferation by these antibodies against the human EGFR, suggesting that their mechanisms of action are similar.

The Cyclin Interactor p26INCA1 Regulates the Hematopoietic Stem Cell Pool Via CDK Inhibition.

Cell cycle progression is driven by the kinase activity of cyclin/CDK complexes. Dysregulation of the cell cycle leads to altered cell growth and contributes to tumorigenesis. Recently, we identified p26INCA1 as novel interaction partner of Cyclin A1/CDK2. Here, we characterize the phenotype of Inca1-null mice to uncover the cellular and molecular function of Inca1. Inca1-knockout mice were viable and fertile. FACS analyses revealed that aging mutant animals harbored an increased hematopoietic stem cell (HSC) pool. Bone marrow cells of young mice exhibited enhanced clonogenic replating efficiency in colony formation assays as compared to wildtype mice. Weekly administration of the myeloablative agent 5-fluorouracil (5-FU) led to a significantly shorter life span of Inca1−/ − mice compared to wildtype littermates. The increased 5-FU toxicity might thus be related to a higher number of cycling HSC in Inca1−/ − bone marrow. Analysis of the impact of Inca1 on cell cycle regulation demonstrated that the fraction of Inca1−/ − embryonic fibroblasts (MEFs) in S phase was significantly increased. Ectopic INCA1 expression reduced proliferation and colony formation of proliferating cells such as primary bone marrow cells, HeLa, HuTu80 and 32D cell lines. Serum starvation rapidly induced and mitogenic signals inhibited Inca1 expression providing a further link to cell cycle regulation. To identify the molecular mechanism of cell cycle regulation by Inca1, we investigated the influence of Inca1 on the direct inhibition of CDK2. In spleen lysates from Inca1-deficient mice, cellular CDK2 kinase activity towards Histone H1 was significantly induced compared to lysates of wildtype littermates. In in vitro kinase assays, recombinant INCA1 strongly inhibited CDK2 activity. In addition, we hypothesized that other cyclin kinase inhibitors (CKI) could partially compensate in vivo for the loss of Inca1 function. p21cip1/waf1 mRNA and protein expression were induced in Inca1−/ − MEFs compared to wildtype cells hinting at a partial compensation of the loss of Inca1 by induction of p21. Loss of Inca1 combined with p21 knockdown synergistically increased S-phase. These results indicate that Inca1 could be functionally related to p21 and that the rather mild phenotype observed in Inca1−/ − mice and the modest differences in Cdk activity observed in cell lysates lacking Inca1 could be due to compensatory induction of the CKI p21. In summary, loss of Inca1 increased cell proliferation, replating efficiency, S-phase progression, and Cdk2 activity whereas gain of Inca1 suppressed these cell functions. Inca1 expression was induced during cell cycle arrest. We conclude that Inca1 could be a novel cell cycle suppressor regulating the quiescence of HSCs through the inhibition of Cdk2.

Priming Effect with G-CSF Enhances In Vitro Apoptosis by Treatment with Ara-C and VP-16 in Leukemic Cell Line.

Introduction: We have recently reported that the effectiveness of low dose Ara-C, VP-16 and G-CSF (AVG therapy) for elderly AML patients who were ineligible for intensive chemotherapy (Hematol Oncol, in press). G-CSF has been reported to potentiate in vitro anti-leukemic effect of Ara-C. The mechanism of the potentiation is assumed to recruit quiescent G0 leukemic cells into cell cycle. We hypothesized that the enhanced cytotoxicity was due to the apoptosis by the effect of the priming of G-CSF, and the effect was depended on the cell cycle. In order to afford proof of this hypothesis, we assayed proliferation, apoptosis, and cell cycle in leukemic cell lines.Materials: Ara-C, VP-16, G-CSF was provided by Nippon Shinyaku, Nihonkayaku, Chugai pharmacy, respectively, Tokyo, Japan. 32D and HL-60 were obtained from RIKEN Bioresource Center Cell Bank (Ibaragi, Japan), Ba/F3 was generous gifts from Dr. Kume, Jichi medical school, Tochigi, Japan.Methods: 5 x 105/ml HL60, 32D and Ba/F3 were cultured with various concentrations of Ara-C and/or VP-16 in the presence or absence of G-CSF 50ng/ml for 3 days. At the end of the culture, cell proliferation and viability were determined by using the trypan blue. The Annexin V-binding capacity of treated cells was examined by flow cytometry using ANNEXIN V-FITC APOPTOSIS DETECTION KIT I purchased from BD Pharmingen™. Cell cycle analysis was done with BrdU Flow KIT purchased from BD Pharmingen™. The incorporated BrdU was stained with specific anti-BrdU fluorescent antibodies, and the levels of cell-associated BrdU are then measured by flow cytometory.Result:1.Ara-C and VP-16 inhibited proliferation and decreased viability of leukemic cell lines dose-dependently. Half killing concentration (IC50) was redused in combination of Ara-C and VP-16 than Ara-C or VP-16 alone. In G-CSF dependent cell line (32D), IC50 was redeced in the presence of G-CSF than absence of G-CSF at G-CSF, and there was no significant difference between with and without G-CSF in G-CSF independent cell lines (HL-60, Ba/F3) (p<0.05).2.In combined treatment of low dose Ara-C (10−7M) and VP-16 (10−7M), the percentage of apoptotic cells were increased to 20.67% from 13.04% by addition of G-CSF in 32D, and there was no significant differencebetween with and without G-CSF in HL-60 and Ba/F3 (p<0.05).3.At combined treatment of low dose Ara-C and VP-16, the percentage of G0/G1 phase cells were decreased to 43.94% from 35.63% and S phase cells were increased to 29.50% from 24.05% in 32D by addition of G-CSF, and there was no significant difference between with and without G-CSF in HL-60 and Ba/F3 (p<0.05).Discussion: We first showed a combination effect of Ara-C and VP-16. Next we demonstrated that the potentiation of the cytotoxicity was mediated through the mechanism of apoptosis, and apoptosis played an important role for eradicating leukemic cells by low dose Ara-C and VP-16. And G-CSF recruited cells G0/G1 phase into S phase in G-CSF dependent cells by addition of G-CSF. These results suggest that priming effect of G-CSF significantly potentiate the cytotoxicity mediated by AVG chemotherapy.Conclusion: The priming effect of G-CSF might be admitted at least of a part in AML cells.

Cbl Mutants Interact with c-Kit and Lead to Factor Independent Growth and Transformation of a Myeloid Cell Line 32D.

The Cbl proto-oncogene products have emerged as important components of the signal transduction cascades downstream of both non-receptor and receptor tyrosine kinases (RTKs). By regulation of receptor trafficking and degradation, they have been shown to tightly regulate the intensity and amplitude of RTK activation. c-Kit belongs to the family of the class-III RTKs and plays an important role in the pathogenesis of acute myeloid leukemia (AML). So far, very little is known about the role of c-Cbl mutants in the role of c-Kit signaling. We analyzed the interaction of c-Cbl with c-Kit and the functional relevance of this interaction in the IL-3-dependent murine myeloid progenitor cell line 32Dcl3. We analyzed the effect of c-Cbl and two different dominant negative mutants of c-Cbl (Cbl-70Z and Cbl-R420Q) on c-Kit-ligand-activated internalization. The transfection of c-Cbl mutants, but not of wildtype c-Cbl, significantly inhibited receptor internalization, as analyzed by FACS analysis. Expression of Cbl-70Z in 32Dcl3 cells severely inhibited apoptosis induced by growth factor deprivation, as has been described before. However, when coexpressed with wildtype c-Kit, 32Dcl3 cells also rapidly proliferated in the absence of any exogenously added growth factors. We furthermore analyzed SCF induced c-Kit ubiquitination in the presence and absence of c-Cbl mutants. SCF induced rapid ubiquitination of c-Kit that was strongly reduced in the presence of Cbl-70Z and abolished by Cbl-R420Q. Also, the Cbl mutants altered the amplitude and changed the quality of c-Kit dependent signaling events. In colony assays we were able to show ligand independent colony growths in methyl cellulose only in the presence of wildtype c-Kit together with a c-Cbl mutant. Our results indicate that c-Cbl has an important role in c-Kit signal mitigation. Furthermore, they demonstrate that disturbed mechanisms of c-Kit internalization have important implications for its transforming potential, possibly in the development of AML.

Alpha-, beta- and kappa-caseins inhibit the proliferation of the myeloid cell lines 32D cl3 and WEHI-3 and exhibit different differentiation properties

We have recently shown that sodium caseinate (CasNa) was able to inhibit the proliferation of the myeloid cell line 32D cl3 in a non-toxic way, and that it also induced the expression of macrophage colony-stimulating factor (M-CSF). Casein is the main protein present in milk and is composed of alpha ( α), beta ( β) and kappa ( κ) subunits. This work was undertaken to evaluate if any one casein is responsible for the proliferation and differentiation properties found for CasNa on myeloid cells. Taking into consideration that 32D cl3 cells are considered to be non-malignant and dependent on IL-3 for proliferation, we also included for this study a leukemic cell line, WEHI-3, that does not depend on any external growth factor for its proliferation in order to evaluate if the growth inhibitory effect of caseins is also present for malignant cells. Our results showed that all caseins were inhibitory for the proliferation of either 32D cl3 and WEHI-3 and that only the 32D cl3 cells were induced to differentiate into the monocyte-macrophage lineage. In order to evaluate if CasNa was able to inhibit the proliferation of other myeloid cells we used J774 and P388 and found that they were also inhibited. We also determined that the different caseins exhibit different differentiation properties, with α-casein being the only one able to induce the secretion of M-CSF. We consider this work to open a new field of research, where casein, or its components, can be studied for their possible role in hematopoiesis and on the inhibition of malignant cell proliferation for therapeutic use.

Dok-1 is a Positive Regulator of IL-4 Signalling and IgE Response

Interleukin-4 (IL-4) plays an essential role in the control of humoral immunity by regulating lymphocyte proliferation and differentiation, including the T helper type 2 lineage commitment of CD4(+) T cells as well as the isotype switching to IgE in B cells. The adaptor protein Dok-1 is known to have an essential role in the negative regulation of a variety of cytokine signalling events. However, here we have found that the loss of Dok-1 impaired the proliferative response of CD4(+) T cells and B cells to IL-4. Conversely, the forced expression of Dok-1 in the myeloid cell line 32D augmented the IL-4-induced proliferation, indicating a positive role for Dok-1. Tyrosine phosphorylation, and thereby the activation of Stat6 and IRS-2, is critical for IL-4 signalling; however, only the activation of Stat6, not the IRS-2-dependent phosphorylation of Akt, was perturbed in Dok-1-deficient cells stimulated with IL-4. Furthermore, mice lacking Dok-1 showed an impaired IgE response to thymus-dependent antigen. Thus, Dok-1 is a positive regulator of IL-4 signalling and IgE response.

Interleukin-3 and erythropoietin cooperate in the regulation of the expression of erythroid-specific transcription factors during erythroid differentiation

To characterize how interleukin-3 and erythropoietin regulate cell fate by modulating the expression of lineage-specific transcription factors. This study analyzed mRNA and protein levels, gene transcription rates, and mRNA and protein stabilities of erythroid-specific transcription factors in lineage-restricted cells derived from the 32D cell line cultured either in interleukin-3 or erythropoietin. Erythroid 32D subclones expressed levels of Idl, Gata-2, and Scl comparable and levels of Eklf and Gata-1 higher than those expressed by myeloid subclones. While maintained in interleukin-3, erythroid cells remained immature despite their high expression of Gata-1, Gata-2, Scl, Eklf, and Idl. Switching the erythroid cells to erythropoietin induced cell maturation (within 48 hours) and reduced expression of Gata-2 and Idl (in 24 hours) but did not alter the expression of Gata-1. The effects of interleukin-3 were mostly mediated by increases in transcription rates (Scl and Gata-2), and that of erythropoietin was apparently due to increased mRNA and protein (Gata-1, Scl, and Eklf) stability. In particular, erythropoietin increased the stability of the processed and transcriptionally more active form of GATA-1 protein. These results suggest that interleukin-3 and erythropoietin cooperate to establish the lineage-specific transcription factor milieu of erythroid cells: interleukin-3 regulates mainly gene transcription and erythropoietin consistently increases mRNA and protein stability.

Purification and identification of the STAT5 protease in myeloid cells.

STAT (signal transducer and activator of transcription) proteins are critical regulators of cytokine-induced cell proliferation, differentiation and survival. STAT functional activity can be variably regulated by post-translational modifications, including phosphorylation, acetylation, methylation and sumoylation. Additionally, limited proteolytic digestion of full-length STAT proteins (STATalpha) generates C-terminally truncated forms (STATgamma) in different cell lineages, which have significantly reduced transcriptional activity due to the lack of the transactivation domain. Previously, it has been shown that STAT5gamma, generated by an unidentified nuclear serine protease, plays an important role in myeloid cell differentiation and is aberrantly expressed in acute myeloid leukaemia. To better understand this regulatory mechanism for STAT5 function, we have purified the STAT5 protease from the immature myeloid cell line 32D and identified it by MS analysis as the granule-derived serine protease, CatG (cathepsin G). We show that purified CatG can specifically cleave full-length STAT5 to generate STAT5gamma, and this activity can be inhibited by AEBSF [4-(2-aminoethyl)benzenesulfonyl fluoride] in an in vitro protease assay. Importantly, preparation of nuclear and cytoplasmic extracts from immature myeloid cell lines, 32D and FDC-P1, in the presence of a specific inhibitor for CatG results in the identification of STAT5alpha only. These studies indicate that nuclear STAT5gamma does not naturally exist in immature myeloid cells and is artificially generated from STAT5alpha during the preparation of extracts due to the abundance of CatG in these cells. Therefore in contrast with earlier studies, our data suggest that STAT5alpha, rather than STAT5gamma is the active form in immature myeloid cells.