Acute Myelogenous Leukemia Blasts Research Articles

Coculture of native human acute myelogenous leukemia blasts with fibroblasts and osteoblasts results in an increase of vascular endothelial growth factor levels

Angiogenesis seems important in the development of acute myelogenous leukemia (AML). Proangiogenic vascular endothelial growth factor (VEGF) is constitutively secreted by the AML blasts for a subset of patients, but it can also be released by non-leukemic bone marrow cells. VEGF levels were determined after coculture of native human AML blasts with fibroblast lines, osteoblastic sarcoma cell lines, normal bone marrow stromal cells and normal osteoblasts. Cultures were prepared with leukemic and non-leukemic cells separated by a semipermeable membrane or in direct contact. The non-leukemic cells usually showed higher spontaneous VEGF release than AML cell populations. Coculture of AML blasts with HFL1 fibroblasts caused a supra-additive increase of VEGF levels when the cell populations were cultured separately, and the increase was also observed when cells were cultured in direct contact. An increase was also observed when AML blasts were cultured with osteoblastic sarcoma cells, normal bone marrow stromal cells and normal osteoblasts. Coculture had divergent effects on VEGF mRNA levels both for leukemic and non-leukemic cells, but increased mRNA levels were commonly observed especially for the non-leukemic cells. Cytokine inhibition experiments suggest that IL1 is important for the VEGF-increasing crosstalk, whereas the mechanisms are probably heterogeneous for coculture with osteoblasts. The bi-directional crosstalk via local cytokine networks between AML blasts and non-leukemic cells results in increased local VEGF levels, an observation suggesting that VEGF-targeting antiangiogenic therapy should be considered as a general therapeutic strategy in AML.

The CXCR4 Score: A New Prognostic Marker in Acute Myelogenous Leukemia.

Despite major improvements in the understanding and treatment of certain leukemias during the past years, the overall survival of patients with acute myelogenous leukemia (AML) remains poor and new prognostic markers and therapeutic strategies are urgently needed. The chemokine stromal cell-derived factor-1 (SDF-1) and its receptor CXCR4 play key roles in retention of hematopoietic cells in the marrow microenvironment and release into the circulation. Rambouts and colleagues recently reported that CXCR4 expression on AML cells correlates with Flt3 mutations and prognosis (Blood 104: 550-57, July 2004). The aim of our study is to evaluate the prognostic significance of CXCR4 surface expression by AML blasts. Ninty newly diagnosed patients with AML were were divided into three groups based on expression of CXCR4 by AML blasts (CXCR4 score A–C). Group A contains patients with a mean fluorescence intensity ratio (MFIR) for CXCR4 <5 (n=32), group B: MFIR 5–10 (n=26), and group C: MFIR >10 (n=32). MFIR was calculated by divding the mean flourescence intensity for CXCR4 by that for the non-specific isotype control of the respective sample and indicates the level of CXCR4 expression. The characteristics for each group are summarized in Table 1.Previously, we demonstrated that CXCR4 receptors on AML cells mediate adesion and induce migration beneath marrow stromal cells (Burger JA,et al., Br J Haematol. 122: 579–89, August 2003). Because adhesion to stromal elements can affect survival and induce adhesion-mediated drug resistance, antagonists of the CXCR4/SDF-1 axis may increase the efficacy of current treatments. We conclude that CXCR4 expression at time of diagnosis correlates with leukocytosis, LDH, blast persistence after first therapy, relapse rate, and survival. Therfore, CXCR4 expression in AML may be an important progostic factor for complete remission and survival. As such, CXCR4/SDF-1 interaction may provide the basis for a novel therapeutic approach in AML.CharacteristicScore A (N=32)Score B (N=26)Score C (N=32)Median CXCR4 MFIR (range)2.8 (1.0–4.9)6.9 (5.1–9.4)17.2 (10.3–73.4)Median Age (range)62.0 (21–89)59.5 (18–80)65.0 (32–93)Median WBC (range)2.3 (1.0–14.0)10.2 (10.2–17)30.0 (2.7–240)Median PLT (range)56.0 (8–211)86.0 (13.6–265)82.5 (4–218)Median LDH (range)251.5 (140–2637)368.0 (187–1489)357.0 (144–2760)Unfavorable Cytogenetics4/2810/247/27blast persistence after first therapyN=6, 22.2%N=13, 65%N=14, 73.7%Relapse Rate13.8%52.4%40%Total DeathsN=7, 21.9%N=13, 50%N=17, 53.1%Mean overall survival (months)12.4±11.910.3±9.88.3±7.4Mean follow-up (months)12.0±19.7Median Overall Survival (months)9.0 (2–52)6.5 (0–42)7.0 (0–25)Median follow-up (months)7.0 (0–52)Table

Pediatric AML primary samples with FLT3/ITD mutations are preferentially killed by FLT3 inhibition

AbstractPediatric acute myelogenous leukemia (AML) has a poor prognosis, and novel therapies are needed. The FLT3 tyrosine kinase represents a promising target in pediatric AML. FLT3 is constitutively activated either by an internal tandem duplication (ITD) or by a point mutation (PM) in 17% to 24% of pediatric AML cases. Autocrine stimulation of wild-type (WT) FLT3 by coexpressed FLT3 ligand (FL) occurs in many other cases. FLT3/ITD mutations confer a particularly poor prognosis in pediatric AML patients. Inhibitors of FLT3 are being tested in adult AML patients, with promising preliminary results. In this study, cytotoxicity and apoptosis assays were performed on 44 diagnostic pediatric AML blast samples (14 FLT3/WT, 15 FLT3/ITD, 15 FLT3/PM) using CEP-701, a potent and selective FLT3 inhibitor. Pronounced cytotoxicity and induction of apoptosis were observed in a higher percentage of FLT3/ITD samples (93%) than FLT3/PM (27%) or FLT3/WT (29%). The cytotoxicity was greatest in samples with a high FLT3/ITD mutant-to-wild-type allelic ratio. The addition of FL enhanced the survival and augmented the sensitivity to FLT3 inhibition for the CEP-701–responsive subset of FLT3/WT and FLT3/PM samples. Clinical testing of FLT3 inhibitors as molecularly targeted agents for the improvement of outcome of pediatric AML patients is warranted.

In vitro crosstalk between fibroblasts and native human acute myelogenous leukemia (AML) blasts via local cytokine networks results in increased proliferation and decreased apoptosis of AML cells as well as increased levels of proangiogenic Interleukin 8

Interactions between native human acute myelogenous leukemia (AML) blasts and nonleukemic cells in the bone marrow microenvironment seem important both for disease development chemosensitivity. Native human AML blasts from consecutive patients were cultured with normal human bone marrow stromal cells and two fibroblast lines (HFL1 and Hs27) separated by a semipermeable membrane. This bidirectional crosstalk via the cytokine network between AML blasts and fibroblasts caused (i) increased proliferation, (ii) mediated antiapoptotic signalling and (iii) increased local levels of proangiogenic IL8.

Treatment of myelodysplastic syndromes with valproic acid alone or in combination with all-transretinoic acid

AbstractValproic acid (VPA) has been shown to inhibit histone deacetylase activity and to synergize with all-transretinoic acid (ATRA) in the differentiation induction of acute myelogenous leukemia (AML) blasts in vitro. We treated 18 patients with myelodysplastic syndromes (MDS) and AML secondary to MDS (sAML/MDS) with VPA monotherapy (serum concentrations 346-693 μM [50-100 μg/mL]). Five patients received VPA and ATRA (80 mg/m2/d, days 1-7, every other week). Response according to international working group (IWG) criteria was observed in 8 patients (44%) on VPA monotherapy, including 1 partial remission. Median response duration was 4 months (range, 3-9 months). Four of 5 patients relapsing were treated with VPA + ATRA, 2 of them responding again. Among 5 patients receiving VPA + ATRA from the start, none responded according to IWG criteria, but 1 patient with sAML/MDS achieved a marked reduction in peripheral and marrow blasts. Thus, VPA is of therapeutic benefit for patients with MDS, and ATRA may be effective when added later.

In vitro effects of native human acute myelogenous leukemia blasts on fibroblasts and osteoblasts

Bone marrow stromal cells constitute a heterogeneous population, and in the present study we investigated intercellular crosstalk via release of soluble mediators between native human AML blasts and fibroblasts/osteoblasts. Coculture of nonleukemic stromal cells and AML blasts separated by a semipermeable membrane decreased proliferation of the fibroblast line HFL1, and the inhibition was maintained when HFL1 and AML cells were cultured in direct contact. A similar inhibitory effect was observed for osteoblastic sarcoma cell lines (Cal72, SJSA-1) and normal osteoblasts. GM-CSF was released by both nonleukemic cells and a subset of AML blast populations, and increased levels of GM-CSF were detected in AML cocultures with fibroblasts and osteoblastic sarcoma cells when testing AML cell populations with constitutive GM-CSF release. Furthermore, constitutive IL-1beta secretion by AML blasts was detected only for a subset of patients, whereas relatively high levels of IL-1RA were observed for all patients; coculture of AML blasts with HFL1 fibroblasts and osteoblastic sarcoma cells increased IL-1beta levels for patients with constitutive IL-1beta secretion, whereas IL-1RA levels were slightly decreased but still generally higher than IL-1beta levels (tested only for HFL1 fibroblasts). The bidirectional crosstalk between AML blasts and stromal cells with increased release of AML growth factors may be important in leukemogenesis, whereas the decreased stromal cell proliferation combined with the persistent release of IL-1RA may in addition inhibit remaining normal hematopoiesis and thereby contribute to bone marrow failure in AML.

Cotreatment with 17-Allylamino-Demethoxygeldanamycin and FLT-3 Kinase Inhibitor PKC412 Is Highly Effective against Human Acute Myelogenous Leukemia Cells with Mutant FLT-3

Presence of the activating length mutation (LM) in the juxtamembrane domain or point mutation in the kinase domain of FMS-like tyrosine kinase-3 (FLT-3) mediates ligand-independent progrowth and prosurvival signaling in approximately one-third of acute myelogenous leukemia (AML). PKC412, an inhibitor of FLT-3 kinase activity, is being clinically evaluated in AML. Present studies demonstrate that treatment of human acute leukemia MV4-11 cells (containing a FLT-3 LM) with the heat shock protein 90 inhibitor 17-allylamino-demethoxy geldanamycin (17-AAG) attenuated the levels of FLT-3 by inhibiting its chaperone association with heat shock protein 90, which induced the poly-ubiquitylation and proteasomal degradation of FLT-3. Treatment with 17-AAG induced cell cycle G(1) phase accumulation and apoptosis of MV4-11 cells. 17-AAG-mediated attenuation of FLT-3 and p-FLT-3 in MV4-11 cells was associated with decrease in the levels of p-AKT, p-ERK1/2, and p-STAT5, as well as attenuation of the DNA binding activity of STAT-5. Treatment with 17-AAG, downstream of STAT5, reduced the levels of c-Myc and oncostatin M, which are transactivated by STAT5. Cotreatment with 17-AAG and PKC412 markedly down-regulated the levels of FLT-3, p-FLT-3, p-AKT, p-ERK1/2, and p-STAT5, as well as induced more apoptosis of MV4-11 cells than either agent alone. Furthermore, the combination of 17-AAG and PKC412 exerted synergistic cytotoxic effects against MV4-11 cells. Importantly, 17-AAG and PKC412 induced more loss of cell viability of primary AML blasts containing FLT-3 LM, as compared with those that contained wild-type FLT-3. Collectively, these in vitro findings indicate that the combination of 17-AAG and PKC412 has high level of activity against AML cells with FLT-3 mutations.

Cotreatment with histone deacetylase inhibitor LAQ824 enhances Apo-2L/tumor necrosis factor-related apoptosis inducing ligand-induced death inducing signaling complex activity and apoptosis of human acute leukemia cells.

Present studies demonstrate that treatment with the histone deacetylases inhibitor LAQ824, a cinnamic acid hydroxamate, increased the acetylation of histones H3 and H4, as well as induced p21(WAF1) in the human T-cell acute leukemia Jurkat, B lymphoblast SKW 6.4, and acute myelogenous leukemia HL-60 cells. This was associated with increased accumulation of the cells in the G(1) phase of the cell cycle, as well as accompanied by the processing and activity of caspase-9 and -3, and apoptosis. Exposure to LAQ824 increased the mRNA and protein expressions of the death receptors DR5 and/or DR4, but reduced the mRNA and protein levels of cellular FLICE-inhibitory protein (c-FLIP). As compared with treatment with Apo-2L/tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or LAQ824 alone, pretreatment with LAQ824 increased the assembly of Fas-associated death domain and caspase-8, but not of c-FLIP, into the Apo-2L/TRAIL-induced death-inducing signaling complex. This increased the processing of caspase-8 and Bcl-2 interacting domain (BID), augmented cytosolic accumulation of the prodeath molecules cytochrome-c, Smac and Omi, as well as led to increased activity of caspase-3 and apoptosis. Treatment with LAQ824 also down-regulated the levels of Bcl-2, Bcl-x(L), XIAP, and survivin. Partial inhibition of apoptosis due to LAQ824 or Apo-2L/TRAIL exerted by Bcl-2 overexpression was reversed by cotreatment with LAQ824 and Apo-2L/TRAIL. Significantly, cotreatment with LAQ824 increased Apo-2L/TRAIL-induced apoptosis of primary acute myelogenous leukemia blast samples isolated from 10 patients with acute myelogenous leukemia. Taken together, these findings indicate that LAQ824 may have promising activity in augmenting Apo-2L/TRAIL-induced death-inducing signaling complex and apoptosis of human acute leukemia cells.

Involvement of reactive oxygen species in adaphostin-induced cytotoxicity in human leukemia cells

AbstractAdaphostin (NSC 680410), an analog of the tyrphostin AG957, was previously shown to induce Bcr/abl down-regulation followed by loss of clonogenic survival in chronic myelogenous leukemia (CML) cell lines and clinical samples. Adaphostin demonstrated selectivity for CML myeloid progenitors in vitro and remained active in K562 cells selected for imatinib mesylate resistance. In the present study, the mechanism of action of adaphostin was investigated in greater detail in vitro. Initial studies demonstrated that adaphostin induced apoptosis in a variety of Bcr/abl-cells, including acute myelogenous leukemia (AML) blasts and cell lines as well as chronic lymphocytic leukemia (CLL) samples. Further study demonstrated that adaphostin caused intracellular peroxide production followed by DNA strand breaks and, in cells containing wild-type p53, a typical DNA damage response consisting of p53 phosphorylation and up-regulation. Importantly, the antioxidantN-acetylcysteine (NAC) blunted these events, whereas glutathione depletion with buthionine sulfoximine (BSO) augmented them. Collectively, these results not only outline a mechanism by which adaphostin can damage both myeloid and lymphoid leukemia cells, but also indicate that this novel agent might have a broader spectrum of activity than originally envisioned. (Blood. 2003;102:4512-4519)

Synthetic triterpenoids activate a pathway for apoptosis in AML cells involving downregulation of FLIP and sensitization to TRAIL.

Acute myelogenous leukemia (AML) remains a deadly disease for most adult patients, due primarily to the emergence of chemoresistant cells. Defects in apoptosis pathways make important contributions to chemoresistance, suggesting a need to restore apoptosis sensitivity or to identify alternative pathways for apoptosis induction. Triterpenoids represent a class of naturally occurring and synthetic compounds with demonstrated antitumor activity, including 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid (CDDO) and its methyl ester (CDDO-m). We explored the effects of CDDO and CDDO-m in vitro on established AML cell lines (HL-60, U937, AML-2) and on freshly isolated AML blasts. CDDO and CDDO-m reduced the viability of all AML cell lines tested in a dose-dependent manner, with effective doses for killing 50% of cells (ED(50)) within 48 h of approximately 1 and 0.5 muM, respectively. CDDO or CDDO-m also induced substantial increases in cell death in five out of 10 samples of primary AML blasts. Cell death induced by CDDO and CDDO-m was attributed to apoptosis, based on characteristic cell morphology and evidence of caspase activation. Immunoblot analysis demonstrated proteolytic processing of caspase-3, -7, and -8, but not caspase-9, suggesting the involvement of the 'extrinsic' pathway, linked to apoptosis induction by TNF-family death receptors. Accordingly, CDDO and CDDO-m induced concentration-dependent reductions in the levels of FLIP protein, an endogenous antagonist of caspase-8, without altering the levels of several other apoptosis-relevant proteins. Reductions in FLIP were rapid, detectable within 3 h after exposure of AML cell lines to CDDO or CDDO-m. CDDO and CDDO-m also sensitized two of four leukemia lines to TRAIL, a TNF-family death ligand. The findings suggest that synthetic triterpenoids warrant further investigation in the treatment of AML, alone or in combination with TRAIL or other immune-based therapies.

The angioregulatory phenotype of native human acute myelogenous leukemia cells: influence of karyotype, Flt3 abnormalities and differentiation status.

The cytogenetic abnormalities and the response to induction therapy have been regarded as the most important prognostic parameters in acute myelogenous leukemia (AML) patients. Recent studies have demonstrated that internal tandem duplications and specific D-835 point mutations of the Flt3 gene, as well as the angioregulatory phenotype represent additional adverse prognostic factors. The aim of the study was to investigate possible associations between genetic abnormalities, differentiation status and angioregulatory phenotype in native human AML blasts. Native AML blasts derived from consecutive patients were cultured in vitro and concentrations of angioregulatory molecules determined in the supernatants. Most patients released at least two different angioregulatory mediators. Pro-angiogenic interleukin 8 (IL8) was released at relatively high levels for most patients, many of these patients showed additional release of pro-angiogenic vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF). High release of anti-angiogenic IL12 was associated with high release of pro-angiogenic IL8 and VEGF. Furthermore, patients with D-835 mutations showed increased IL12 release, whereas patients with normal karyotype had decreased HGF release. Myelomonocytic differentiation was associated with IL18 release and CD34 expression with low IL12 release. Our results suggest that native human AML blasts have a pro-angiogenic phenotype. Although the investigated genetic abnormalities are associated with variation in the in vitro release of angioregulators, these differences are relatively small and do not quantitatively involve the most important IL8 release. It therefore seems unlikely that this phenotypic variation can explain the prognostic impact of the genetic abnormalities.

Effects of angiogenic regulators on in vitro proliferation and cytokine secretion by native human acute myelogenous leukemia blasts.

Angiogenesis seems to be important in the pathogenesis of acute myelogenous leukemia (AML). The endothelial cell proliferation and microvessel formation are regulated by a wide range of soluble mediators, including angiogenin, angiopoietin-2, basic fibroblast growth factors, vascular endothelial growth factor (VEGF), VEGF-D, angiostatin and endostatin. In the present study, it has been investigated whether these mediators have an additional direct effect on the proliferation and cytokine release by native human AML blasts. AML cells derived from a large group of consecutive patients were investigated. All these mediators could alter the proliferation and cytokine release [interleukin (IL) 1beta, IL6, IL8, tumor necrosis factor alpha] for a minority of patients. Alteration of spontaneous proliferation by at least one mediator was detected in five of 38 patients; whereas, altered cytokine (Flt3-ligand, granulocyte-macrophage colony-stimulating factor, stem cell factor)-dependant proliferation was observed for 10 patients. Growth enhancement was most frequently observed, whereas growth inhibition was uncommon. The effects on AML blast proliferation were often dependant on or were modulated by the presence of the three hematopoietic growth factors. Based on the present results, it is concluded that angioregulatory mediators have additional growth-enhancing effects directly on the AML blasts for certain patients. However, based on the results from this investigation and previous studies it is suggested that their major contribution to the pathogenesis of AML is through their effects on regulation of bone marrow angiogenesis, and future studies of these mediators in AML should probably focus on these effects.

Bone marrow purging studies in acute myelogenous leukemia using the recombinant anti-CD33 immunotoxin HuM195/rGel

This study was designed to determine the effect of immunotoxin HuM195/rGel on normal human bone marrow before clinical purging. HuM195/rGel is composed of the recombinant plant toxin gelonin (rGel) chemically coupled to the anti-CD33 human chimeric antibody HuM195. The CD33 antigen is of significant interest as a target for therapy of acute myelogenous leukemia because it is present in leukemic blasts of most patients but absent in the earliest progenitor bone marrow cells. HuM195/rGel was optimally cytotoxic to acute myelogenous leukemia HL60 cells with 24 hours of exposure. We developed an in vivo purging model by mixing mobilized peripheral blood progenitor cells with HL60 cells to simulate a remission in bone marrow. Cells were treated with 10 nmol/L of HuM195/rGel either with or without exposure to freeze/thaw procedure, which has been reported to act synergistically with HuM195/rGel to produce cytotoxic effect. When clonogenic cell recovery rates were determined, HuM195/rGel alone did not affect normal peripheral blood progenitor cells, whereas HuM195/rGel plus freeze/thaw provided 2 logs of tumor cell elimination in our purging model. We also observed similar results under conditions used in the transplantation setting. We concluded that for acute myelogenous leukemia blasts expressing CD33, HuM195/rGel could be useful as a purging reagent for autologous transplantation.

Effects of TNFα on the growth and sensitivity to cytosine arabinoside of blast progenitors in acute myelogenous leukemia with special reference to the role of NF-κB

Leukemic blasts in acute myelogenous leukemia (AML) are derived from a minor population of cells called blast progenitors. Hematopoietic growth factors (HGFs) stimulate their growth and simultaneously sensitize them to cytosine arabinoside (Ara-C), a cell-cycle-specific cytotoxic drug. Since tumor necrosis factor α (TNFα) modifies HGF activities, we examined the effects of TNFα in combination with HGFs on in vitro growth and Ara-C sensitivity of AML blast progenitors in patient samples. TNFα variably affected HGF-supported colony formation and the self-renewal of blast progenitors. However, the combination of TNFα with IL-3 uniformly rendered blast progenitors more resistant to Ara-C irrespective of whether TNFα suppressed or augmented IL-3-supported growth, indicating that TNFα regulates the Ara-C sensitivity of leukemic progenitors independently of their cell cycle status. Since nuclear factor-κB (NF-κB) is activated by TNFα and induces expression of prosurvival genes, effects of the antisense oligodeoxynucleotides to NF-κB subunits, p65 and p50, were examined. Antisense oligodeoxynucleotides sensitized HL60 cells to Ara-C but rendered leukemic progenitors in patient samples even more resistant to Ara-C in the presence of TNFα and IL-3 in combination, indicating that NF-κB is involved in the Ara-C sensitivity of leukemic blast progenitors but may exert opposite dual functions, namely protection from and induction of apoptosis, under different conditions.

Quantification and cytokine production of circulating lymphoid and myeloid cells in acute myelogenous leukaemia.

A simple assay was developed to assess the potential of patients with acute myelogenous leukaemia (AML) to respond to immunotherapy. Lymphocytes, monocytes and leukaemic blasts with their corresponding intracellular cytokine profiles were evaluated by four-colour flow cytometry. In 50 microl samples of whole blood, surface labelling for CD45, CD8 and CD3 was used for cell identification prior to intracellular staining for interleukin (IL)-4, IL-10, IL-12 and interferon (IFN)-gamma. Absolute numbers of CD8(+) and CD8(-) (putative CD4(+)) T-cells, NK cells (CD8(+)/CD3(-)) and monocytes were determined by reference to a fixed number of added fluorescent beads. The absolute numbers of CD8(-) and CD8(+) T-cells in the blood of patients with AML were similar to those of normal controls. More of the lymphocytes in the blood of leukaemic patients spontaneously produced cytokines compared with those of controls. Furthermore, primary AML blasts secreted predominantly IFN-gamma. After recovery from chemotherapy, lymphocyte counts tended to be lower than in normals and reduction of NK cells reached significance after the second chemotherapy (P=0.01). A prominent CD8(lo)/CD3(lo-int) lymphocyte subset appeared after recovery in some patients. This laboratory application of the study of cell subsets and intracellular cytokines in patients undergoing treatment may be helpful in monitoring immunological responses in AML.

Flavonoid effects on normal and leukemic cells

Quercetin and flavopiridol, both flavonoids which influence oxidative milieu, proliferation, and apoptosis of various cell types, were examined for their effects on acute myelogenous leukemic cells and normal progenitors. Both quercetin and flavopiridol inhibited the growth and viability of various acute myelogenous leukemia (AML) cell lines and AML blasts isolated afresh from patients with AML of various subtypes. The effects on inhibition of proliferation and decreased viability were also significant in normal CD34+ cells isolated from normal marrow donors. In certain AML cases, the effects of flavopiridol appeared to be mediated through activation of caspase 3, offering one possible mechanism for the apoptosis evident after exposure to flavopiridol as measured by annexin V expression. These flavonoid compounds might find use in various therapeutic settings in AML.

Chemokine/chemokine receptor nomenclature.

Chemokine/chemokine receptor nomenclature.

Sensitivity of myeloid leukemia cells to calcium influx blockade: application to bone marrow purging.

The aim of this study was to assess the potential of store-operated Ca(2+) channel (SOC) antagonists as purging agents for leukemia cells. Clonogenic, limiting dilution, and nuclear condensation assays were used to evaluate SOC antagonist efficacy. SOC activity and endoplasmic reticulum Ca(2+) content were measured by flow cytometry. Murine bone marrow transplantation was used to determine purging efficacy and effects on hemopoietic reconstitution. Econazole (Ec) and ketotifen (Ke) were variably effective against human and murine leukemia cell lines after 24 hours of incubation. However, a 2-hour serum and bovine serum albumin-free treatment protocol with Ec was found to maximize differential sensitivity between leukemic cells and normal hemopoietic progenitors. Primary acute myelogenous leukemia blast cell viability was reduced 4.2 to 5.1 logs by 2-hour Ec treatment as measured by limiting dilution. An inverse relationship between endoplasmic reticulum Ca(2+) content and Ke sensitivity in leukemia and untransformed cells was observed. Nuclear condensation, an index of apoptosis, which occurred after 24-hour treatments with either Ec or Ke, was not observed after 2-hour serum- and bovine serum albumin-free Ec exposures; however, condensed nuclei were observed after an additional 10-hour incubation in growth medium without drug. Using bone marrow deliberately contaminated with 1% P815 cells, we showed that highly effective in vitro purging can be accomplished using Ec with no adverse effects on bone marrow reconstitution in mice. These studies suggest that SOC antagonists have potential as purging agents for residual leukemia cells present in bone marrow in the context of high-dose chemotherapy and autologous transplantation for leukemia.

Prolonged administration of all-trans retinoic acid in combination with intensive chemotherapy and G-CSF for adult acute myelogenous leukemia: single-centre pilot study in different risk groups

An exploratory trial was conducted to evaluate toxicity and potential therapeutic role of all trans-retinoic acid (ATRA) given long-term together with chemotherapy and G-CSF to adult patients with acute myelogenous leukemia (AML). ATRA was administered orally at 45 mg/m(2)/day on days 1-14 and 25 mg/m(2)/day on days 15-28 of two standard cycles (idarubicin, etoposide, cytarabine, G-CSF) and of up to three high-dose courses (cytarabine, G-CSF). The results obtained in 19 patients enrolled in the ATRA trial were compared with those from 29 comparable cases treated with the same schedule without ATRA, according to patient risk class and an in vitro study. ATRA was administered for a median of 52 days to the patients selected for study who achieved a remission. ATRA-related toxicity was mostly non-severe apart from high incidence of headache in conjunction with high-dose cytarabine. Complete remission (CR) rate after cycle 1 (54%), kinetics of hematological recovery, postremission treatment realization, disease-free survival (DFS 37.5% at three years) and overall survival (30% at three years) were not different between ATRA-treated and untreated patients. The only significant prognostic factor was the patient risk class, as defined by cytogenetics and other clinical criteria: DFS rate was 57% at three years in standard-risk cases compared to only 19% in the high-risk group, with no influx by ATRA in either category. The in vitro study, in patients with a definite clinical response, failed to document any inhibitory or pro-apoptotic effect of ATRA on AML blast cells. As a consequence to these results, the pilot ATRA phase was closed. This study does not suggest a significant role for the present ATRA schedule as an adjunct to standard antileukemic therapy in adult AML.

Serum levels of angiogenin, basic fibroblast growth factor and endostatin in patients receiving intensive chemotherapy for acute myelogenous leukemia

Angiogenesis seems to be important both in the pathogenesis of acute myelogenous leukemia (AML) and for the susceptibility of AML blasts to chemotherapy. Recent clinical studies even suggest that antiangiogenic therapy can induce disease control in patients with AML relapse. In this context we have investigated the profile of the systemic component of angiogenic regulation in AML by characterizing the serum levels of (i) the angiogenic regulators angiogenin, basic fibroblast growth factor (bFGF) and endostatin; (ii) the endothelial cell marker soluble (s) E-selectin. Patients with untreated AML had increased levels of angiogenin, endostatin and sE-selectin, whereas the levels of bFGF were not significantly altered. The systemic levels of the proangiogenic bFGF, the antiangiogenic endostatin and the endothelial cell marker sE-selectin showed significant correlations, whereas angiogenin and sE-selectin levels were not correlated. Furthermore, intensive chemotherapy resulted in decreased systemic levels of the 2 proangiogenic mediators angiogenin and bFGF, whereas endostatin levels remained high after treatment. Although angiogenin normally is a part of the acute phase reaction, its systemic levels were not altered when patients with chemotherapy-induced cytopenia developed complicating bacterial infections. Our results suggest that intensive chemotherapy can modulate the systemic component of angiogenic regulation in AML patients.