Inhibition of BCR-ABL expression with antisense oligodeoxynucleotides restores beta1 integrin-mediated adhesion and proliferation inhibition in chronic myelogenous leukemia hematopoietic progenitors

Chronic myelogenous leukemia (CML) is characterized by the continuous proliferation and abnormal circulation of malignant hematopoietic progenitors. This may be related to the unresponsiveness of CML progenitors to beta1 integrin adhesion receptor-mediated inhibition of progenitor proliferation by the marrow microenvironment. In hematopoietic cell lines, the BCR-ABL oncogene product, p210(BCR-ABL), interacts with a variety of cytoskeletal elements important for normal integrin signaling. We studied the role of p210(BCR-ABL) in abnormal integrin function in CML by evaluating the effect of inhibition of BCR-ABL expression with antisense oligodeoxynucleotides (AS-ODNs) on integrin-mediated adhesion and proliferation inhibition of malignant primary progenitors from CML marrow. Preincubation of CML CD34(+)HLA-DR+ (DR+) cells with breakpoint-specific AS-ODNs significantly increased adhesion of CML progenitors to stroma and fibronectin (FN). Pretreatment with breakpoint-specific ODNs also resulted in significant inhibition of CML progenitor proliferation after ligand or antibody-mediated beta1 integrin engagement. Breakpoint-specific ODNs were significantly more effective in restoring CML progenitor adhesion and proliferation inhibition than control ODNs. BCR-ABL mRNA and p210(BCR-ABL) levels in CML CD34(+) cells were significantly reduced after incubation with breakpoint-specific AS-ODN. These studies indicate a role for BCR-ABL in abnormal circulation and defective integrin-dependent microenvironmental regulation of proliferation of CML hematopoietic progenitors.     

Gene therapy for chronic myelogenous leukemia (CML): a retroviral vector that renders hematopoietic progenitors methotrexate-resistant and CML progenitors functionally normal and nontumorigenic in vivo

Chronic myelogenous leukemia (CML) is a malignant disease of the human hematopoietic stem cell caused by the BCR/ABL gene rearrangement. The only curative therapy is allogeneic transplantation. Although autologous transplants may prolong survival, most patients relapse because of disease persisting in the host and in the graft. Continued administration of chemotherapy after transplant could reduce the incidence of relapse provided that the autograft can be protected by transfer of a drug-resistance gene. However, CML autografts will almost certainly contain malignant stem cells that will also be rendered drug-resistant. The presence of the BCR/ABL oncoprotein is necessary and sufficient for malignant transformation seen in CML. We thus hypothesized that transfer of a vector that combines a drug-resistance gene with anti-BCR/ABL antisense (AS) sequences may allow for posttransplant chemotherapy to decrease persistent disease while rendering inadvertently transduced CML stem and progenitor cells functionally normal. We constructed a retroviral vector, LasBD, that combines the methotrexate (MTX)-resistant tyrosine-22 dihydrofolate-reductase (tyr22-DHFR) gene and AS sequences directed at the b3a2 BCR/ABL breakpoint. b3a2 BCR/ABL containing 32D and MO7e cells were transduced with LasBD and selected in MTX for 14 days. Expression of the AS sequences reduced BCR/ABL mRNA and p210(BCR/ABL) protein levels by 6- to 10-fold in most cells. This subsequently led to the restoration of normal function of BCR/ABL cDNA+ cells: they grew significantly slower in the presence of interleukin-3 (IL-3); they underwent apoptotic cell death when cultured without IL-3; and they had restored expression and function of adhesion receptors. These effects were specific, because LasBD-containing AS sequences directed at the b3a2 BCR/ABL breakpoint did not affect p190(BCR/ABL)-containing cells. LasBD also rendered 20% to 30% of primary Ph- and Ph+ CD34(+) cells MTX-resistant and decreased BCR/ABL mRNA levels in MTX resistant Ph+ CD34(+) cells by 10-fold. Expression of the MTX-resistant DHFR gene and the AS sequences has been stable for at least 1 year in vitro and for more than 70 days in vivo. Finally, LasBD decreased tumorigenicity of 32DBCR/ABL cells in vivo by 3 to 4 logs. In conclusion, the tyr22-DHFR gene in the LasBD vector can protect normal hematopoietic cells from MTX-mediated toxicity, whereas the AS sequences in LasBD can suppress expression of the BCR/ABL gene and restore normal function of BCR/ABL cDNA-containing cells. The LasBD vector may therefore prove to be an extremely useful adjunct in autologous transplantation for CML.     

T cell receptor- and beta 1 integrin-mediated signals synergize to induce tyrosine phosphorylation of focal adhesion kinase (pp125FAK) in human T cells

The beta 1 subfamily of integrins is thought to play an important role in both the adhesion/migration and proliferation/differentiation of T cells. beta 1 integrins can provide T cell costimulation through interaction of very late antigen (VLA) 4 (VLA-4) (alpha 4 beta 1) and VLA-5 (alpha 5 beta 1) with the extracellular matrix protein fibronectin (FN), or by VLA-4 binding to its cell surface ligand, vascular cell adhesion molecule (VCAM) 1. The mechanism by which beta 1 integrin members transduce T cell-costimulatory signals is poorly understood. Studies in non-T cells have demonstrated regulation of the tyrosine focal adhesion kinase pp125FAK by beta 1 integrin engagement and, most recently, indicate a role for pp125FAK in linking integrin-mediated signal transduction to the Ras pathway (Schaller, M. D., and J. T. Parsons, 1994, Curr. Opin. Cell. Biol. 6: 705-710; Schlaepfer, D. D., S. K. Hanks, T. Hunter, and P. Van der Geer. 1994. Nature (Lond.), 372:786-790). Although pp125FAK kinase occurs in T cells, there are no reports on its regulation in this cell type. The studies described in this article characterize novel regulation of pp125FAK by the T cell receptor (TCR)-CD3 antigen complex and beta 1 integrins, and provide the first account, in any cell type, of integrin alpha 4 beta 1-mediated pp125FAK tyrosine phosphorylation. We demonstrate a rapid and sustained synergistic increase in tyrosine phosphorylation of human pp125FAK in Jurkat T cells after simultaneous (a) triggering of the TCR-CD3 complex, and (b) alpha 4 beta 1 and alpha 5 beta 1 integrin-mediated binding of these cells to immobilized FN or alpha 4 beta 1 integrin-mediated binding to immobilized VCAM-1. Studies with normal peripheral blood-derived CD4+ human T blasts confirm the synergistic action of a TCR-CD3 complex-mediated costimulus with a FN- or VCAM-1-dependent signal in the induction of T cell pp125FAK tyrosine phosphorylation. In vitro kinase assays performed on pp125FAK immunoprecipitates isolated from Jurkat cells and normal CD4+ T cells identified a coprecipitating 57-kD tyrosine-phosphorylated protein (pp57), distinct from pp59fyn or pp56lck. These results indicate, for the first time, the involvement of a specific kinase, pp125FAK, in alpha 4 beta 1- and alpha 5 beta 1-mediated T cell-costimulatory signaling pathways. In addition, the data demonstrate novel regulation of pp125FAK tyrosine phosphorylation by the TCR-CD3 complex.     

Big BU/CY is associated with a favorable long-term outcome in patients allotransplanted for chronic myelogenous leukemia in chronic phase

Twenty-six adult patients, median age 36 years (range 21-53) with chronic myeloid leukemia in first chronic phase were allotransplanted between October 1989 and May 1995. The preparative regimen consisted of busulphan 16 mg/kg and cyclophosphamide 200 mg/kg (big BU/CY). Cyclosporin A and methotrexate were used for GVHD prophylaxis. Twenty-two donors were HLA-identical siblings and four donors were mismatched for one antigen of class I. The global incidence of acute GVHD was 50%, that of severe aGVHD (grades 3-4) was 11%; the global incidence of chronic GVHD was 30%. No patients developed veno-occlusive disease of the liver or interstitial pneumonia. Five patients died, one of relapse, four of transplant-related causes, mostly related to aGVHD; thus, the transplant-related mortality was 16%. Twenty-one patients are alive, in remission, with a median follow-up of 55 months (range 24-90); actuarial probability of survival is 78% (CI 64-96). Our study shows that this conditioning regimen is relatively easy to administer and seems to be as effective as, if not superior to, regimens containing TBI, in patients with chronic myeloid leukemia in chronic phase and the transplant-related mortality is not excessive even in older patients.     

Increased expression of intercellular adhesion molecule 1, CD11/CD18 cell surface adhesion glycoproteins and alpha 4 beta 1 integrin in a rat model of chronic interstitial lung fibrosis

The expression of the intercellular adhesion molecule 1 (ICAM-1), and the integrins CD49, CD11b/c, and CD11a (LFA-1 alpha chain) was analyzed in an experimental model of pulmonary fibrosis. Adult rats were exposed to 75% oxygen during 10 weeks, and to 2.0 mg/kg of paraquat twice weekly. Rats were sacrificed at 2 days, and at 2 and 10 weeks after the first injection of paraquat. Lungs were fixed in 4% paraformaldehyde and used for histology and immunohistochemistry. At 2 days the lungs showed a diffuse inflammation composed of a mixed polymorphonuclear and mononuclear cell infiltrate. Afterwards, the inflammatory process was predominantly mononuclear, and an increasing fibroblast proliferation was observed. Early inflammatory events (48 h) correlated with a moderate increased expression of ICAM-1, LFA, and CD11b/c in epithelial cells as well as a pronounced expression of ICAM-1 and CD11b/c in macrophages. At 2 and 10 weeks, there was a progressive increased expression of CD11b/c and ICAM-1 by macrophages, as well as of LFA in epithelial cells, and of ICAM-1 and CD49 by epithelial and interstitial cells. Lymphocytes showed a slight increased expression of LFA at 2 weeks, and of CD49 at 2 and 10 weeks. These results suggest that macrophages expressing ICAM-1, CD11b/c, and CD49 are involved in the earlier and late phases of the disease whereas fibroblast and epithelial cells expressing ICAM-1 and CD49 might play a role in the cell interactions involved in the fibrotic phase.     

Macrophage-inflammatory protein-3 beta/EBI1-ligand chemokine/CK beta-11, a CC chemokine, is a chemoattractant with a specificity for macrophage progenitors among myeloid progenitor cells

Chemoattractants are potential factors influencing cell migration. Stromal cell-derived factor-1, a CXC chemokine, is the only chemokine reported to have chemotactic activity for hemopoietic progenitor cells (HPC). We report in this work another chemokine of the CC subfamily, which is chemotactic for HPC. Macrophage-inflammatory protein (MIP)-3 beta/EBI1-ligand chemokine/CK beta-11 attracted bone marrow and cord blood CD34+ cells. In contrast to stromal cell-derived factor-1, which attracts multiple types of HPC, MIP-3beta attracted mainly CFU granulocyte macrophage, but not other HPC such as burst-forming unit erythrocyte or CFU granulocyte, erythrocyte, macrophage, and megakaryocyte. Chemoattracted CD34+ cells formed CFU granulocyte macrophage-like colonies, which were morphologically determined as large macrophages. These progenitors were selectively responsive to stimulation by macrophage CSF, demonstrating that MIP-3 beta attracts macrophage progenitors. Expression of CCR7, the receptor for MIP-3 beta, was detected at a mRNA level in the attracted CD34+ cells as well as input CD34+HPC. Expression of MIP-3 beta mRNA was not constitutive, but was inducible in bone marrow stromal cells by inflammatory agents such as bacterial LPS, IFN-gamma, and TNF-alpha. Taken together, our findings suggest that MIP-3 beta is expressed in the bone marrow environment after induction with certain inflammatory cytokines and LPS, and may play a role in trafficking of macrophage progenitors in and out of the bone marrow in inflammatory conditions.     

Upmodulation of alpha v beta 1 integrin expression on human tumor cells by human interleukin for DA cells/leukemia inhibitory factor and oncostatin M: correlation with increased cell adhesion on fibronectin

Integrins belong to a large family of heterodimeric membrane glycoproteins which mediate cell-cell or cell-extracellular matrix interactions. These interactions could play a major role during the migration of tumor cells across the extracellular matrix and vascular endothelium and would thus appear to be requisite for the metastatic process. Pretreatment of the Foss human melanoma cell line with HILDA/LIF or OSM, two cytokines involved in acute-phase response, increased the expression of membrane alpha v beta 1 1.5-2-fold. The same phenomenon was observed on the SK-N-SH human neuroblastoma cell line. alpha v beta 1 upmodulation was concomitant with improved tumor cells attachment to the fibronectin matrix. This greater adhesion of tumor cells to fibronectin was inhibited by specific monoclonal antibodies against alpha v or beta 1 integrin subunits. Similar results were obtained after TNF-alpha treatment. Our findings demonstrate the ability of HILDA/LIF and OSM to modulate tumor cell capacity to adhere to the matrix component, suggesting a potential role for these cytokines in modulation of tumoral progression.     

Petunia p34cdc2 protein kinase activity in G2/M cells obtained with a reversible cell cycle inhibitor, mimosine

Protoplasts isolated from petunia leaf mesophyll are non-cycling cells mostly with 2C content. Cells regenerating from protoplast culture enter mitosis after 48 h. This experimental model is used to relate p34cdc2 kinase activity to cell cycle phase. Our results show that the histone H1 phosphorylation, and hence p34cdc2 kinase activity, peaks with G2+early M cell cycle phase. However, a trace kinase activity was already present when most cells were entering S phase. To obtain a maximum of cells in G1+S phases, the protoplast culture was treated with the rare amino acid, mimosine. Mimosine blocked plant cells derived from protoplast culture both at G1 and in early and mid S phase. Despite the increased G1+S level, p34cdc2 kinase activity did not increase. This suggests that the trace activity appearing when the majority of cells are entering S does not correspond to any putative p34cdc2 activation at G1/S transition but to the activation of the minor 4C population initially present in the leaf: the hypothesis remains that p34cdc2 kinase activity is solely related to G2+M phase in petunia.     

Reduction in levels of the cyclin-dependent kinase inhibitor p27(kip-1) coupled with transforming growth factor beta neutralization induces cell-cycle entry and increases retroviral transduction of primitive human hematopoietic cells

Successful gene therapy depends on stable transduction of hematopoietic stem cells. Target cells must cycle to allow integration of Moloney-based retroviral vectors, yet hematopoietic stem cells are quiescent. Cells can be held in quiescence by intracellular cyclin-dependent kinase inhibitors. The cyclin-dependent kinase inhibitor p15(INK4B) blocks association of cyclin-dependent kinase (CDK)4/cyclin D and p27(kip-1) blocks activity of CDK2/cyclin A and CDK2/cyclin E, complexes that are mandatory for cell-cycle progression. Antibody neutralization of beta transforming growth factor (TGFbeta) in serum-free medium decreased levels of p15(INK4B) and increased colony formation and retroviral-mediated transduction of primary human CD34(+) cells. Although TGFbeta neutralization increased colony formation from more primitive, noncycling hematopoietic progenitors, no increase in M-phase-dependent, retroviral-mediated transduction was observed. Transduction of the primitive cells was augmented by culture in the presence of antisense oligonucleotides to p27(kip-1) coupled with TGFbeta-neutralizing antibodies. The transduced cells engrafted immune-deficient mice with no alteration in human hematopoietic lineage development. We conclude that neutralization of TGFbeta, plus reduction in levels of the cyclin-dependent kinase inhibitor p27, allows transduction of primitive and quiescent hematopoietic progenitor populations.     

ER-34122, a novel dual 5-lipoxygenase/cyclooxygenase inhibitor with potent anti-inflammatory activity in an arachidonic acid-induced ear inflammation model

OBJECTIVE AND DESIGN: To investigate the effect of ER-34122, a novel pyrazole derivative, on 5-lipoxygenase (LOX) and cyclooxygenase (COX) metabolite production in vitro, ex vivo and in vivo. MATERIAL: In vitro, lysate of rat basophilic leukemia cells, the microsome fraction of sheep seminal vesicles, human polymorphonuclear leukocytes, human synovial cells, and human monocytes. Ex vivo and in vivo, male Balb/c mice or SD rats. TREATMENT: In ex vivo study, ER-34122 (0.03-1 mg/kg) was orally administered 1 h before withdrawal of blood samples. In carrageenin-induced paw edema, ER-34122 (3-100 mg/kg) and indomethacin (1-10mg/kg) were orally administered 1 h before carrageenin injection. In arachidonic acid-induced ear inflammation, ER-34122 (0.3-10mg/kg), zileuton (10-100mg/kg) and indomethacin (0.3-3mg/kg) were orally administered 1 h before arachidonic acid application. METHODS: 5-Hydroxyeicosatetraenoic acid and other eicosanoids were determined by using an HPLC method and enzyme immunoassay, respectively. Rat hind paw edema and mouse ear edema were assessed by measuring paw volume and ear thickness, respectively. Myeloperoxidase (MPO) activity and eicosanoid content of the ear tissue were also determined. RESULTS: ER-34122 inhibited both LOX and COX product generation in vitro, and ex vivo. ER-34122 and indomethacin inhibited carrageenin-induced paw edema formation. In the arachidonic acid-induced ear inflammation, ER-34122 inhibited inflammatory responses (edema formation and MPO accumulation) as well as eicosanoids (LTB4, LTC4 and PGE2) generation. A representative LOX inhibitor, zileuton, also inhibited these inflammatory responses, while a COX inhibitor, indomethacin, did not suppress them though it completely inhibited PGE2 generation. CONCLUSIONS: The anti-inflammatory characteristics of ER-34122 are considered to be superior to those of COX inhibitors such as indomethacin, because in addition to its inhibitory activity on the COX pathway, ER-34122 inhibits LOX products generation, as revealed by the inhibition of edema formation or polymorphonuclear leukocyte infiltration in the arachidonic acid-induced ear inflammation model.     

A novel mitogenic signaling pathway of bradykinin in the human colon carcinoma cell line SW-480 involves sequential activation of a Gq/11 protein, phosphatidylinositol 3-kinase beta, and protein kinase Cepsilon

The signaling routes connecting G protein-coupled receptors to the mitogen-activated protein kinase (MAPK) pathway reveal a high degree of complexity and cell specificity. In the human colon carcinoma cell line SW-480, we detected a mitogenic effect of bradykinin (BK) that is mediated via a pertussis toxin-insensitive G protein of the Gq/11 family and that involves activation of MAPK. Both BK-induced stimulation of DNA synthesis and activation of MAPK in response to BK were abolished by two different inhibitors of phosphatidylinositol 3-kinase (PI3K), wortmannin and LY 294002, as well as by two different inhibitors of protein kinase C (PKC), bisindolylmaleimide and Ro 31-8220. Stimulation of SW-480 cells by BK led to increased formation of PI3K lipid products (phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3, 4-bisphosphate) and to enhanced translocation of the PKCepsilon isoform from the cytosol to the membrane. Both effects of BK were inhibited by wortmannin, too. Using subtype-specific antibodies, only the PI3K subunits p110beta and p85, but not p110alpha and p110gamma, were detected in SW-480 cells. Finally, p110beta was found to be co-immunoprecipitated with PKCepsilon. Our data suggest that in SW-480 cells, (i) dimeric PI3Kbeta is activated via a Gq/11 protein; (ii) PKCepsilon is a downstream target of PI3Kbeta mediating the mitogenic signal to the MAPK pathway; and (iii) PKCepsilon associates with the p110 subunit of PI3Kbeta. Thus, these results add a novel possibility to the emerging picture of multiple pathways linking G protein-coupled receptors to MAPK.     

Anticarcinogenic effect of a flavonoid antioxidant, silymarin, in human breast cancer cells MDA-MB 468: induction of G1 arrest through an increase in Cip1/p21 concomitant with a decrease in kinase activity of cyclin-dependent kinases and associated cyclins

There is an increasing interest in identifying potent cancer preventive and therapeutic agents against breast cancer. Silymarin, a flavonoid antioxidant isolated from milk thistle, exerts exceptionally high to complete anticarcinogenic effects in tumorigenesis models of epithelial origin. In this study, we investigated the anticarcinogenic effect of silymarin and associated molecular mechanisms, using human breast carcinoma cells MDA-MB 468. Silymarin treatment resulted in a significantly high to complete inhibition of both anchorage-dependent and anchorage-independent cell growth in a dose- and time-dependent manner. The inhibitory effects of silymarin on cell growth and proliferation were associated with a G1 arrest in cell cycle progression concomitant with an induction of up to 19-fold in the protein expression of cyclin-dependent kinase (CDK) inhibitor Cip1/p21. Following silymarin treatment of cells, an incremental binding of Cip1/p21 with CDK2 and CDK6 paralleled a significant decrease in CDK2-, CDK6-, cyclin D1-, and cyclin E-associated kinase activity with no change in CDK2 and CDK6 expression but a decrease in G1 cyclins D1 and E. Taken together, these results suggest that silymarin may exert a strong anticarcinogenic effect against breast cancer and that this effect possibly involves an induction of Cip1/p21 by silymarin, which inhibits the threshold kinase activities of CDKs and associated cyclins, leading to a G1 arrest in cell cycle progression.     

An evaluation of the prognostic significance of antigen CD95(Fas/APO-1) expression on the cells of patients with a myelodysplastic syndrome, acute myeloid leukemia and chronic myeloleukemia

AIM: The expression of CD95(Fas/APO-1) antigen was studied on bone marrow cells of 19 MDS patients, peripheral blood blast cells of 15 acute myeloid leukemia (AML) patients, blast cells and granulocytes of 68 patients with chronic myeloid leukemia (CML)--24 in chronic, 9 in accelerated phase and 35 in blastic crisis (BC)--by indirect surface immunofluorescence assay using flow cytometry (FACScan, Becton Dickinson, USA). RESULTS: CD95(Fas/APO-1) antigen was revealed on bone marrow cells of 8 out of 19 (36.8%) MDS patients; the percentage of antigen-positive cells was 38.1 +/- 19.2%; on 45.5 +/- 22.8% of cells in 6(45%) of 15 AML patients. Fas/APO-1 antigen was totally absent in CML chronic stage; its expression was found in 34% (12 of 35) of our patients with CML BC on peripheral blood blasts and in 56% (5 of 9) on peripheral blast cells of CML patients in acceleration phase. CONCLUSION: The data on overall survival of CD95-positive MDS patients suggest that the presence of Fas antigen is a favorable prognostic sign for patients with MDS. The patients from CD95-negative group represent a risk group both for survival and AML transformation. In CML BC group the survival does not depend upon Fas-antigen expression.     

Glucose repression/derepression in budding yeast: SNF1 protein kinase is activated by phosphorylation under derepressing conditions, and this correlates with a high AMP:ATP ratio

BACKGROUND: Genetic studies of Saccharomyces cerevisiae have shown that Snf1p and Snf4p, which together form the SNF1 complex, are essential for gene derepression on removal of glucose from the medium. However the metabolic signal(s) involved, and the exact role of SNF1, have remained enigmatic. Recently, the AMP-activated protein kinase (AMPK) was shown to be the mammalian homologue of SNF1. AMPK is activated by the elevation of the cellular AMP:ATP ratio, which occurs during cellular stress in mammalian cells. The mechanism of activation involves phosphorylation of AMPK by an upstream protein kinase (AMPKK). We have investigated whether a similar mechanism might explain the role of SNF1 in yeast in the response to the stress of glucose starvation. RESULTS: The protein kinase activity of SNF1 was dramatically and rapidly activated by phosphorylation on removal of glucose from the medium. SNF1 was not activated directly by AMP, but could be inactivated by protein phosphatases and reactivated by mammalian AMPKK. We also demonstrated that an endogenous SNF1-reactivating factor, most likely an upstream protein kinase, is present in yeast extracts. Under a variety of different growth conditions, there was a correlation between cellular adenine nucleotide levels and the activation state of SNF1. CONCLUSIONS: Apart from the lack of direct allosteric activation of SNF1 by AMP, the regulation of the mammalian AMPK and yeast SNF1 protein kinase cascades is highly conserved. Adenine nucleotides are now good candidates for metabolic signals which indicate the lack of glucose in the medium, triggering activation of SNF1 and derepression of glucose-repressed genes.