C. Peschel

Primary proliferating immature myeloid cells from CML patients are not resistant to induction of apoptosis by DNA damage and growth factor withdrawal

Induction of apoptosis by growth factor deprivation or gamma‐irradiation‐induced DNA damage was directly studied in proliferating primary haemopoietic cells derived from CD34‐positive cells of 13 CML patients and 12 normal controls. CD34‐positive cells were cultured in the presence of appropriate concentrations of SCF and G‐CSF for 5–7 d. After gamma irradiation with 500 rad or growth factor deprivation, the fraction of apoptotic cells was assessed by two independent methods applying either measurement of cells incorporating FITC‐labelled dUTP by terminal transferase or assessment of the fraction of cells with a less than 2N DNA content in flow cytometry.

Plasma levels of IL-1, TNF alpha, IL-6, IL-8, G-CSF, and IL1-RA during febrile neutropenia: Results of a prospective study in patients undergoing chemotherapy for acute myelogenous leukemia

Plasma levels of IL-1, IL-6, IL-8, IL1-RA, TNF alpha, and G-CSF were prospectively studied during 23 chemotherapy cycles of 20 patients suffering from acute myelogenous leukemia. Increased plasma levels of IL-6, IL-8, and G-CSF were observed in patients with febrile neutropenia and/or major infection. Plasma levels of IL-6, IL-1, TNF alpha and IL-1-RA measured 1 day before and 1 day after the onset of febrile episodes did not accurately predict the development of major infection. In contrast, IL-8 plasma levels were significantly higher in those patients who subsequently developed major infection. The question whether IL-8 plasma levels identify high risk or low risk patients with sufficient specificity and sensitivity has to be answered in large scale clinical trials.

Bcr-Abl kinase promotes cell cycle entry of primary myeloid CML cells in the absence of growth factors.

Cell cycle control of both immature and differentiated primary myeloid normal and chronic‐phase chronic myeloid leukaemia (CML) cells to growth factor deprivation was studied. CD34+ cells were cultured in liquid culture. After removal of growth factors for 48 h normal cells were very efficiently arrested with the fraction of cells in S phase reduced by 70.8 ± 6.5% in CD34+ and 50.5 ± 4.2% in CD34− cells. In contrast, a significantly higher proportion of leukaemic cells remained in S phase. The fraction of S‐phase cells was reduced by only 29.3 ± 5.7% in CD34+ CML cells and 21.2 ± 3.8% in CD34− cells. This abnormal negative cell cycle control in leukaemic cells was specific for growth factor deprivation. Reaction to IFN‐α and TNF‐α treatment was identical both in normal and CML cells. Equal quantities of the cytokines TNF‐α, IL‐1α, IL‐1RA and IL‐6 were produced by CML and normal cells. However, production of GM‐CSF, with a median of 11 ± 5 pg/ml, was found only in the supernatants of CML cells. But antibodies to GM‐CSF did not restore growth factor dependence of the leukaemic cells. The defect was completely corrected by the abl‐specific tyrosine kinase inhibitor CGP 57148 without effecting cell cycle control of normal cells. Our results demonstrate a directly Bcr‐Abl‐dependent defective response of both immature and differentiated primary myeloid CML cells to growth factor deprivation.

Influence of Interferon-α on Cytokine Expression by the Bone Marrow Microenvironment—Impact on Treatment of Myeloproliferative Disorders

Myeloproliferative disorders (MPD) are characterized by several common clinical and biological features, although at the molecular level, each disease entity exhibits distinct abnormalities. IFN-alpha exerts beneficial therapeutic effects in chronic myelogenous leukemia, polycythemia vera and essential thrombocythemia, resulting in control of hematopoietic hyperplasia and, in a minority of patients, in induction of cytogenetic remission. The mechanism of action of IFN-alpha in MPD is poorly defined. Recently published in vitro findings suggest that IFN-alpha interacts with the regulation of hematopoiesis by multiple ways. Its antiproliferative activity is well known for more than a decade, however, substantial growth inhibition is achieved only at relatively high concentrations. Defective adhesion of hematopoietic progenitor cells in CML to bone marrow stromal cells is corrected by IFN-alpha, which might expose CML progenitors to inhibitory cytokines produced by the bone marrow microenvironment. Recent work from our group demonstrated, that IFN-alpha potently interacts with the production of hematopoietic cytokines in bone marrow stromal cells. Expression of stimulatory cytokines, such as GM-CSF, G-CSF, IL-1 and IL-11 is inhibited by IFN-ct, whereas the production of negative regulators, such as IL-1RA and MIP-1 alpha, is stimulated. The combined action of IFN-alpha on paracrine expression of cytokines suggests an indirect antihematopoietic effect, which might contribute to its clinical activity in MPD.

Induction of interferon regulatory factors, 2′‐5′ oligoadenylate synthetase, P68 kinase and RNase L in chronic myelogenous leukaemia cells and its relationship to clinical responsiveness

The genes crucially determining the therapeutic response of chronic myelogenous leukaemia (CML) to interferon‐alpha (IFN‐alpha) are unknown. Recently, two independent IFN‐alpha signalling pathways were identified: the classic pathway mediates induction of 2′‐5′ oligoadenylate synthetase (2‐5 OAS), p68 kinase and IFN regulatory factor‐2 (IRF‐2), whereas the alternate pathway leads to activation of IFN regulatory factor‐1 (IRF‐1). We investigated whether deficient or imbalanced expression of components of these two pathways is associated with resistance of CML cells to antiproliferative action of IFN‐alpha/beta. Constitutive and IFN‐induced transcript levels of IFN‐dependent genes in mononuclear cells, granulocytes, monocytes, lymphocytes and CD34+ cells of chronic‐phase CML and blast crisis patients were assessed by Northern blot techniques and were correlated with subsequent clinical responses to IFN therapy. Our results demonstrated that IFN‐alpha or ‐beta treatment in vitro and in vivo leads to an enhanced expression of IRF‐1, IRF‐2, RNase L, p68 and 2‐5 OAS which was independent of the degree of cellular differentiation and clonal evolution of CML. Neither the magnitude of induction of these genes nor the IRF‐1/IRF‐2 mRNA balance differed between chronic‐phase CML patients responding or failing IFN‐alpha therapy. These results indicate that failure of IFN‐alpha treatment is not due to defects in mRNA induction of the above‐mentioned candidate genes for the direct antiproliferative response to IFN type I.

Effect of interleukin-3 pretreatment on granulocyte/macrophage colony-stimulating factor induced mobilization of circulating haemopoietic progenitor cells

Summary. Recombinant human colony stimulating factors (CSFs) as single agents are increasingly used for mobilizing peripheral blood progenitor cells (PBPCs) for stem cell transplantation. We have shown in rhesus monkeys that interleukin‐3 (IL‐3) pretreatment markedly potentiated the increase in PBPC numbers of subsequent administration of granulocyte/macrophage‐CSF (GM‐CSF). Here we studied the effect of IL‐3 pretreatment on GM‐CSF‐induced mobilization of PB progenitors in patients who were potential candidates for autologous stem cell transplantation (n=16). Patients were treated with GM‐CSF at a dose of 5 μg/kg/d for 5 d and after a treatment free interval received another cycle of GM‐CSF immediately following pretreatment with IL‐3 at different doses and duration: 2.5 μg/kg/d (n = 4), 5μg/kg/d (n = 3) and 10μg/kg/d (n = 3) for 3d, 5μg/kg/d for 7d (n = 4) and 5μg/kg/d for 14 d (n = 2), respectively. Only 7d pretreatment with IL‐3 showed consistent effects. Although IL‐3 did not mobilize by itself, pretreatment with 5μg/kg/d of IL‐3 for 7d significantly potentiated GM‐CSF‐induced mobilization of PB CFU‐GM numbers, leading to a mean increase in PB CFU‐GM numbers over baseline by 18.5 +5.2 (SEM) fold by IL‐3/ GM‐CSF as compared to a 4.7 + 1.7‐fold increase by GM‐CSF alone. A significant enhancement by the 7d IL‐3 pretreatment was also observed for erythroid (BFU‐E) and multi‐potential progenitor cells (CFU‐mix) which were 3.3 + 1.3‐and 3.4 + 0.9‐fold, respectively, mobilized by GM‐CSF alone, as compared to 8.5 + 2.3‐ and 19.2 + 3.4‐fold, respectively, by the IL‐3/GM‐CSF combination. Our results suggest that 7 d pretreatment with IL‐3 may be a useful mean to augment mobilization of circulating progenitors by more lineage‐restricted CSFs. These findings may be important for the design of mobilization strategies that use growth factors without preceding chemotherapy.

Regulation of immunomodulatory functions by granulocyte-macrophage colony-stimulating factor and granulocyte colony-stimulating factor in vivo

Abstract The present study was designed to investigate in vivo immunomodulatory properties of hematopoietic growth factors. The influence on the activation of cytokine synthesis and on the expression of surface antigens associated with cellular activation of G-CSF or GM-CSF was investigated in cancer patients receiving these factors. One single dose of growth factor was administered to patients with bladder cancer (G-CSF group) or small cell lung cancer (GM-CSF group) before chemotherapy. After cytoreductive chemotherapy patients received supportive therapy with G-CSF or GM-CSF. Peripheral blood mononuclear cells and plasma samples were obtained for flow cytometry, Northern blot analysis, and assessment of cytokine protein levels after single-dose as well as after continous cytokine administration. Our results demonstrate differences in the induction of biological activities by GM-CSF and G-CSF in vivo which correlate well with in vitro findings. Among mature hematopoietic cells the effect of G-CSF is restricted to the granulocyte lineage. With GM-CSF moderate but unequivocal modulation of monocyte function was observed. On peripheral blood monocytes expression of MHC class-II molecules and CD44 was markedly stimulated. After one single dose of GM-CSF, plasma levels of sCD25 and IL-1RA were significantly induced (p<0.0001, p=0.032, respectively) and a trend to increased IL-8 levels was observed. The changes in plasma proteins were not correlated with shifts of mRNA expression for IL-8 and IL-1RA. T-cell activation was not observed with either cytokine. These results suggest that immunomodulatory features are differentially regulated by G-CSF and GM-CSF. The clinical relevance of a selective use of both hematopoietic growth factors in various disease settings remains to be determined.

Biotherapy of chronic myelogenous leukemia

The aim of this review is to summarize the current knowledge on the clinical results of biotherapy of chronic myelogenous leukemia (CML) and potential mechanisms of the antitumor action of interferon alpha. IFN alpha treatment induces hematologic and cytogenetic remissions in patients with chronic phase CML. In addition, the duration of the chronic phase is prolonged by IFN alpha resulting in a significant survival benefit. In two randomized clinical trials this survival benefit was demonstrated in all chronic phase CML patients independent of their risk scores. Moreover, IFN treatment also delays the onset of clinical relapse after allogeneic bone marrow transplantation. The critical mechanisms of IFN action have not yet been identified. Both direct and indirect antiproliferative mechanisms have been described. In particular, differential regulation of growth promoting and growth inhibiting cytokines represents an attractive hypothetical mechanism of IFN action. Nevertheless, no leukemia specific IFN activities explaining cytogenetic remissions and/or delay of disease progression have been identified. Further research on that field are required to further improve biological CML therapies.

Induction of the pro-myelocytic leukaemia gene by type I and type II interferons

The physiological role of the pro-myelocytic leukaemia (PML) gene product is poorly defined. Among other functions, PML is involved in haematopoietic differentiation and in control of cell growth and tumorigenesis. We investigated the regulation of human PML expression by interferons (IFNs) and IL-1 in various human haematopoietic lines (U937, THP1, HL60, NB4), in human diploid fibroblasts and in human peripheral blood leukocytes. Cytokine-induced modulation of PML expression was assessed by Northern blot analyses, flow cytometry studies and in situ immunolabelling. Our data show that IFNs and IL-1 upregulate PML transcript and protein expression in a time and dose-dependent manner. In situ immunolabelling revealed that upregulation of protein expression by IFN-alpha is a consequence of a marked increase in both the number and the intensity of the staining of so-called PML nuclear bodies. Our data suggest that stimulation of PML expression by interferons and IL-1 may account for upregulation of PML proteins observed in inflammatory tissues and in proliferative states.

Cationic lipide mediated transfer of c-abl and bcr antisense oligonucleotides to immature normal myeloid cells: Uptake, biological effects and modulation of gene expression

Uptake and biochemical and biological effects of antisense oligodeoxynucleotides (ODN) specific for c-abl and bcr genes were studied in normal immature myeloid cells. CD34-positive cells were purified by positive and negative selection and cultured in liquid culture for 7 days. These cells were then incubated with ODNs, either alone or in combination with cationic lipids. The uptake of ODNs was enhanced by the use of cationic lipids. In addition, very low concentrations of ODNs in combination with cationic lipids were capable of specifically inhibiting the expression of the c-abl gene. In contrast, no effects were seen on the expression of bcr. However, despite the effective blocking of c-abl expression, no changes in cellular growth patterns were observed in liquid culture or in a colony-forming assay. We conclude that the use of cationic lipids might enhance the gene-regulatory effects of ODNs by increasing their uptake into normal hematopoietic cells.