Cell kinetics after high dose cytosine arabinoside in patients with acute myelocytic leukemia

In this study 10 patients with acute myelocytic leukemia (AML) each received a rapid intravenous injection of high dose cytosine arabinoside (HD Ara-C; 1 g/m2). Bone marrow aspirates were obtained before and after Ara-C administration to determine the percentage of cells in S-phase measured by flow cytometry. In 5 out of 10 cases synchronization of the leukemic cells in S-phase of the cell cycle was observed. However, the time of maximum synchronization turned out to be difficult to predict. Therefore, the strong correlation between percentage of cells in S-phase at diagnosis and the time of maximal accumulation of S-phase cells after Ara-C administration, as observed by others in childhood AML, could not be confirmed for adult AML patients. Although synchronization of AML cells after in vivo Ara-C administration could be demonstrated in at least half of the patients, the practical consequences are such that clinical application was hampered.     

Distribution of the cytoskeletal protein,Nestin, in acute leukemia

Abstract

Nestin is a neuroepithelial stem cell marker that is expressed in some types of tumor cells. Recent reports suggest that Nestin may be closely related to malignant cell proliferation and migration. Acute leukemia (AL) is characterized by a lack of differentiation, which results in uncontrolled proliferation in the bone marrow and accumulation of immature cells. The expression and function of Nestin in AL is unclear. We investigated Nestin immunohistochemical patterns of 87 patients that included 47 cases of acute myeloid leukemia (AML) and 40 cases of acute lymphoblastic leukemia (ALL), and 20 patients in complete remission (CR) from AML or ALL. We also investigated the clinico-pathological features of 87 cases of AL and their CR and overall survival (OS). Nestin was expressed in leukemic blasts and mature granulocytic cells in most cases (39/47) of AML. Conversely, Nestin was expressed in mature granulocytic cells in fewer cases (6/40) of ALL, but not in blasts. Nestin expression appeared in leukemic blasts of AML, but not ALL. Nestin expression in AML blast cells was not associated with CR or OS. We provide evidence that Nestin is expressed in AL and might be a useful immunohistochemical marker for identifying AML and ALL.     

Auer-rod positive myelocytic leukemia cells in diffusion chambers: differentiation along the eosinophilic pathway

Auer-rod positive acute myelocytic leukemia (AML) blasts from a 33-year-old patient were cultured in diffusion chambers (DC) in order to test their differentiation potential. The cells were labeled with anti-human granulocyte anti-serum known to be negative for eosinophils, and evaluated using the unlabeled peroxidase-antiperoxidase (PAP) method. Parallel to a decline in the number of leukemic blasts there was an increase of up to 86% in the number of granulocytic cells belonging to the eosinophilic series. Auer-rod bodies were found in the eosinophilic cells even after 20 days in culture. Staining with anti-granulocyte antiserum failed to demonstrate positive cells at any time during DC culture. Based on the negative reaction with the anti-granulocytic antibodies already at an early stage of development evidence is provided for the existence of a progenitor cell exclusively committed to the eosinophilic pathway.     

Recombinant human colony stimulating factor-granulocyte/macrophage and -granulocyte, but not macrophage induce the development of a respiratory burst in primary human myeloid leukemic cells in vitro

Respiratory burst develops in myeloid blast cells if they differentiate functionally along the monocytic or granulocytic lineage. Using the nitroblue tetrazolium (NBT) assay we studied the effects of recombinant human granulocyte/macrophage colony stimulating factor (rhuGM-CSF), rhuG-CSF and rhuM-CSF on development of respiratory burst activity in primary blast cells from patients with myeloid leukemia. Assessing suspension cultures containing cells from patients with acute myeloid leukemia (AML, n = 13) or myeloid-blast crisis (myBC) of chronic myeloid leukemia (CML, n = 5) it was found that the percentage of NBT positive cells was increased by at least 20% as compared to control cultures by rhuGM-CSF in 6/17 cases, by rhuG-CSF in 7/17 cases and by rhuM-CSF in 0/16 cases, representing in 'responders' a mean increase of 267% and 270% in the absolute number of NBT positive cells by rhuGM-CSF and rhuG-CSF, respectively. Morphological examination of cultured cells from 'responders', as compared to controls, showed decreased blast cell content but generally no evidence of terminal differentiation. The demonstration of Auer rods in NBT positive cells indicates that respiratory burst developed in a leukemic clone. These findings may be of physiological, pathophysiological and clinical relevance.     

Differentiation of myeloid cells in liquid culture. 1. Progenitor cells found in normal peripheral blood

In order to establish a method for studying myeloid differentiation, light density, non-adherent, T-cell depleted mononuclear cells prepared from 26 normal peripheral blood buffy-coats were cultured in McCoy'5A medium supplemented with 15 per cent fetal calf serum (FCS) for three weeks at 37 degrees C in a humidified 5 per cent CO2 atmosphere. The total number of viable cells in the cultures on weeks 1 and 2 represented 73 +/- 10 per cent and 98 +/- 41 per cent of the initial number of viable cells seeded. After one week, blasts represented 26 +/- 10 per cent of the initial number of viable cells while all the initially contaminating mature granulocytes had disappeared. After two weeks, granulocytic differentiation was noted in most cultures and viable myelocytes and more mature cells represented 45 +/- 26 per cent of the initial number of viable cells. The differentiation was independent on the lot of FCS used. The addition of PHA stimulated leukocyte conditioned medium to the cultures did not enhance granulocytic differentiation. The granulocytic differentiation observed in the absence of exogenous CSF persisted after removing the cells adhering to the bottom of the flasks on day 2 of the culture. An endogenous colony stimulating activity was detected in the cultures on week 3 but its intensity did not clearly correlate with the degree of granulocytic differentiation.     

Reactivity patterns of monoclonal antibodies positive on myelomonocytic leukemia cells as defined by esterase isoenzyme analysis

Summary The reactivity with monoclonal antibodies (MoAbs) specific for myelomonocytic cells and the expression of a particular esterase isoenzyme were analyzed in 159 cases of acute myeloid leukemias. The incidence of positivity of 16 MoAbs (MCS-2, MCS-1, OKM1, My-1, Leu-M1, Leu-M3, CA-2-38, MY4, MY7, MY8, MY9, VIM-D2, VIM-D5, Mo1, Mo2, 63D3) was studied using the indirect immunofluorescence technique. A carboxylic esterase isoenzyme which can be inhibited completely and selectively by sodium fluoride (NaF) was demonstrated by isoelectric focusing on horizontal polyacrylamide gels. This NaF-sensitive isoenzyme indicated the monocytic origin of the blast cells as it is specific for this cell lineage. Prior to the immunological-isoenzymatic analysis all cases were categorized into two subtypes according to morphological criteria of the FAB classification system: 147 cases of AML (FAB M1-3) and 12 cases of AMMoL/AMoL (FAB M4/5). However, 15 out of 147 cases of AML expressed the NaF-sensitive isoenzyme and were therefore assigned to the group AMMoL/AMoL. Likewise, 1 case, diagnosed morphologically as AMMoL, was negative for this marker isoenzyme and was assigned to the other leukemia subtype. The incidence of reactivity varied widely for the MoAbs tested regarding the overall results on all cases and the positivity on cases of either AML or AMMoL/AMoL. The MoAbs were grouped into four classes depending on the pattern of reactivity with myeloblastic or monoblastic or both subtypes of acute myeloid leukemia. The MoAbs MCS-2, MY7, Leu-M1, and MY9 detected the vast majority of cases with either myelocytic or monocytic involvement (group-I: pan-myelomonocytic reactivity). The MoAbs MCS-1, OKM1, VIM-D5, and Mo1 showed a predominance in their staining pattern for monocytic variants, but were also positive on a substantial percentage of nonmonocytic cases (group-II: predominantly reactive with monocytic, but also myelocytic cases). The MoAbs Leu-M3, MY4, VIM-D2, Mo2, and MY8 reacted with the large majority of AMMoL/AMoL cases and with a small number of AML cases (group-III: monocyte-specific reactivity). The MoAbs of group-I are useful in differentiating acute lymphoid from acute myeloid leukemias. The MoAbs of group-III, and to a lower extent those of group-II, will be of considerable value in the subtyping of acute myeloid leukemias. The results show that (1) accuracy of leukemia classification might not always be achieved by morphology alone, but that immunological and biochemical aspects should be included as well, and (2) several MoAbs are very useful tools for classification and subtyping of acute myeloid leukemias.     

Expression of immunological markers on leukemic cells before and after cryopreservation and thawing

We have studied the effects of cryopreservation on the viability and on the expression of surface antigens of acute leukemia cells. Marrow samples were obtained at initial diagnosis from 89 patients with acute myeloid leukemia (AML), acute undifferentiated leukemia (AUL), and acute lymphoid leukemia (ALL). In AML, the mean viability was greater than 90% in the types M1, M4, and M5 of the French-American-British classification, 79% in M2, and 3% in M3 types. The viability was 74% in AUL. In ALL, the viability was 95% for pre-B leukemias, but only 2% in T-cell leukemias. The expression of myeloid antigens was studied before and after freezing and thawing using three monoclonal antibodies (NHL30.5, against poorly differentiated granulocytic leukemias, VIMC6 against differentiated granulocytic leukemias and granulocytes; and UCHM1 or CRIS-6, against monocytic leukemias and monocytes). The percentage of cells stained by NHL30.5 and UCHM1 or CRIS-6 was very similar before and after cryopreservation. For VIMC6, the mean staining after cryopreservation was 60% of the initial one. In pre-B ALL, the stainings by anti common ALL antigen before and after cryopreservation were also very similar. We conclude that leukemic cryopreserved cells are suitable for immunologic studies. The recovery is, however, very low in promyelocytic AML and T-cell ALL.     

Short-term culture of myeloid leukemic cells allows efficient transduction by adenoviral vectors

BACKGROUND: Ex vivo gene therapy of acute myeloid leukemia (AML) requires efficient transduction of leukemic cells. Recombinant adenovirus has been reported to be a poorly efficient vector in leukemic cells. We investigated leukemic cell culture as a possible method of improving the efficacy of this vector. METHODS: Leukemic cell lines and primary cultured AML cells were incubated with adenoviral vectors carrying GFP, LacZ, or IL-12 cDNA. Transduction efficiency was evaluated by measuring adenoviral genome copy number and transgene expression in leukemic cells. The expression of the coxsackie/adenovirus receptor (CAR), CD29, CD49e, and CD51/61 was measured, as was the effect of blocking integrin on adenoviral transduction. RESULTS: Increasing the multiplicity of infection (MOI) to 300 plaque-forming units per cell enhanced transduction of leukemic cell lines and to a lesser degree of AML cells. Analysis of adenoviral genome copy per cell showed only a partial correlation between gene transfer efficiency and transgene expression. Culture of AML cells for 3 days prior to adenoviral transduction increased both adenoviral copy number per cell and the percentage of transgene-expressing cells. CD29, CD49e, and CD51/61 but not CAR expression increased in cultured AML cells between days 0 and 3 and integrin-blocking experiments showed inhibition of transduction in two of four AML samples tested. CONCLUSIONS: Efficient ex vivo gene transfer in primary cultured AML cells can be achieved by short-term culture of leukemic cells prior to gene transfer with adenoviral vectors at a high MOI. This effect appears to be at least partially mediated by enhanced integrin expression.     

Opposite regulation of vitamin D receptor by ATRA in AML cells susceptible and resistant to vitamin D-induced differentiation

Some leukemic cell lines can be driven to differentiate to monocyte-like cells by 1,25-dihydroxyvitamin D(3) (1,25D) and to granulocyte-like cells by all-trans retinoic acid (ATRA). Acute myloid leukemias (AMLs) are heterogeneous blood malignancies characterized by a block at various stages of hematopoietic differentiation and there are more than 200 known chromosome translocations and mutations in leukemic cells of patients diagnosed with AML. Because of the multiplicity in the genetic lesions causing the disease, AMLs are particularly difficult to treat successfully. In particular, various AML cells to a variable degree respond to 1,25D-based differentiation and only one type of AML undergoes successfully ATRA-based differentiation therapy. In this paper we describe that AML cell line KG-1 is resistant to 1,25D-induced monocytic differentiation, while sensitive to ATRA-induced granulocytic differentiation. We show that KG-1 cells have very low level of VDR protein and that expression of VDR mRNA is upregulated by ATRA. We show for the first time that this regulation is cell context-specific, because in another AML cell line, HL60, VDR mRNA is downregulated by ATRA. ATRA-induced VDR protein in cytosol of KG-1 cells can be further activated by 1,25D to induce monocytic differentiation of these cells.     

Programmed cell death-4 tumor suppressor protein contributes to retinoic acid-induced terminal granulocytic differentiation of human myeloid leukemia cells

Programmed cell death-4 (PDCD4) is a recently discovered tumor suppressor protein that inhibits protein synthesis by suppression of translation initiation. We investigated the role and the regulation of PDCD4 in the terminal differentiation of acute myeloid leukemia (AML) cells. Expression of PDCD4 was markedly up-regulated during all-trans retinoic acid (ATRA)-induced granulocytic differentiation in NB4 and HL60 AML cell lines and in primary human promyelocytic leukemia (AML-M3) and CD34(+) hematopoietic progenitor cells but not in differentiation-resistant NB4.R1 and HL60R cells. Induction of PDCD4 expression was associated with nuclear translocation of PDCD4 in NB4 cells undergoing granulocytic differentiation but not in NB4.R1 cells. Other granulocytic differentiation inducers such as DMSO and arsenic trioxide also induced PDCD4 expression in NB4 cells. In contrast, PDCD4 was not up-regulated during monocytic/macrophagic differentiation induced by 1,25-dihydroxyvitamin D3 or 12-O-tetradecanoyl-phorbol-13-acetate in NB4 cells or by ATRA in THP1 myelomonoblastic cells. Knockdown of PDCD4 by RNA interference (siRNA) inhibited ATRA-induced granulocytic differentiation and reduced expression of key proteins known to be regulated by ATRA, including p27(Kip1) and DAP5/p97, and induced c-myc and Wilms' tumor 1, but did not alter expression of c-jun, p21(Waf1/Cip1), and tissue transglutaminase (TG2). Phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway was found to regulate PDCD4 expression because inhibition of PI3K by LY294002 and wortmannin or of mTOR by rapamycin induced PDCD4 protein and mRNA expression. In conclusion, our data suggest that PDCD4 expression contributes to ATRA-induced granulocytic but not monocytic/macrophagic differentiation. The PI3K/Akt/mTOR pathway constitutively represses PDCD4 expression in AML, and ATRA induces PDCD4 through inhibition of this pathway.     

Leukomogenic factors downregulate heparanase expression in acute myeloid leukemia cells

Heparanase is a heparan sulfate-degrading endoglycosidase expressed by mature monocytes and myeloid cells, but not by immature hematopoietic progenitors. Heparanase gene expression is upregulated during differentiation of immature myeloid cells. PML-RARalpha and PLZF-RARalpha fusion gene products associated with acute promyelocytic leukemia abrogate myeloid differentiation and heparanase expression. AML-Eto, a translocation product associated with AML FAB M2, also downregulates heparanase gene expression. The common mechanism that underlines the activity of these three fusion gene products involves the recruitment of histone deacetylase complexes to specific locations within the DNA. We found that retinoic acid that dissociates PML-RARalpha from the DNA, and which is used to treat acute promyelocytic leukemia patients, restores heparanase expression to normal levels in an acute promyelocytic leukemia cell line. The retinoic acid effects were also observed in primary acute promyelocytic leukemia cells and in a retinoic acid-treated acute promyelocytic leukemia patient. Histone deacetylase inhibitor reverses the downregulation of heparanase expression induced by the AML-Eto fusion gene product in M2 type AML. In summary, we have characterized a link between leukomogenic factors and the downregulation of heparanase in myeloid leukemic cells.     

Acute leukemia in adults: assessment of remission induction with combination chemotherapy by clinical and cell-culture criteria.

Remission induction was assessed by clinical and cell-culture criteria for 65 patients with acute myelogenous leukemia (AML), 11 patients with chronic myelogenous leukemia (CML) in blast crisis and 19 patients with acute lymphoblastic leukemia (ALL). Cyclophosphamide, cytosine arabinoside and vincristine (CAV) therapy resulted in complete remission in 23 of 50 previously untreated patients with AML and in 3 of the 11 patients with CML. Fourteen patients with ALL responded to vincristine-prednisone induction therapy and two to induction therapy with CAV. The median duration of survival of the responding patients was 2.2 years, compared with 4 months for the patients who did not respond to treatment. Granulopoietic colony formation, assessed by assay of colony-forming units dependent on colony-stimulating activity in culture (CFU-C), was abnormal in 37 of 42 bone marrow aspirates from patients with AML before treatement. CFU-C concentration increased when leukocyte-conditioned medium (LCM) was added to the cultures; 13 cultures had normal or elevated CFU-C concentration with LCM. Marrow cells of patients with ALL or CML in blast crisis demonstrated a similar pattern. Serial studies of marrow CFU-C concentration of 31 patients with AML demonstrated a change to a normal pattern with successful remission induction. Results of this study suggest that administration of purified LCM to leukemic patients might increase granulocyte production from potential but unstimulated granulopoietic precursors. This therapy would lessen the probability of death from infection during remission induction.     

Interaction between leukemic-cell VLA-4 and stromal fibronectin is a decisive factor for minimal residual disease of acute myelogenous leukemia

Bone-marrow minimal residual disease (MRD) causes relapse after chemotherapy in patients with acute myelogenous leukemia (AML). We postulate that the drug resistance is induced by the attachment of very late antigen (VLA)-4 on leukemic cells to fibronectin on bone-marrow stromal cells. We found that VLA-4-positive cells acquired resistance to anoikis (loss of anchorage) or drug-induced apoptosis through the phosphatidylinositol-3-kinase (PI-3K)/AKT/Bcl-2 signaling pathway, which is activated by the interaction of VLA-4 and fibronectin. This resistance was negated by VLA-4-specific antibodies. In a mouse model of MRD, we achieved a 100% survival rate by combining VLA-4-specific antibodies and cytosine arabinoside (AraC), whereas AraC alone prolonged survival only slightly. In addition, overall survival at 5 years was 100% for 10 VLA-4-negative patients and 44.4% for 15 VLA-4-positive patients. Thus, the interaction between VLA-4 on leukemic cells and fibronectin on stromal cells may be crucial in bone marrow MRD and AML prognosis.     

Growth of normal versus leukemic bone marrow cells in long term culture from acute lymphoblastic and myeloblastic leukemias

The ability of the in vitro long-term bone marrow culture (LTBMC) system to impair the survival of leukemic cells and to enhance the growth of normal progenitors has been studied. Bone marrow cells from 19 acute lymphoblastic leukemia (ALL) and 30 acute myeloid leukemia (AML) patients at diagnosis were grown in LTBMC for 4-10 weeks. In half of the cases the leukemic population declined down to undetectable levels and was replaced by putative normal hemopoietic precursors, both in ALL and in AML. In the remaining cases, leukemic cells persisted throughout the culture time and few if any normal hemopoietic cells were detected. These data led us to extend to the lymphoid compartment the previous observation of decreasing leukemic myeloid blasts in LTBMC. The potential of such cultures as an in vitro purging system for autologous bone marrow transplantation in selected poor-prognosis lymphoid malignancies should be explored, as has been done for acute and chronic myeloid leukemias.     

LRRC25 plays a key role in all-trans retinoic acid-induced granulocytic differentiation as a novel potential leukocyte differentiation antigen

Leukocyte differentiation antigens (LDAs) play important roles in the immune system, by serving as surface markers and participating in multiple biological activities, such as recognizing pathogens, mediating membrane signals, interacting with other cells or systems, and regulating cell differentiation and activation. Data mining is a powerful tool used to identify novel LDAs from whole genome. LRRC25 (leucine rich repeat-containing 25) was predicted to have a role in the function of myeloid cells by a large-scale “omics” data analysis. Further experimental validation showed that LRRC25 is highly expressed in primary myeloid cells, such as granulocytes and monocytes, and lowly/intermediately expressed in B cells, but not in T cells and almost all NK cells. It was down-regulated in multiple acute myeloid leukemia (AML) cell lines and bone marrow cells of AML patients and up-regulated after all-trans retinoic acid (ATRA)-mediated granulocytic differentiation in AML cell lines and acute promyelocytic leukemia (APL; AML-M3, FAB classification) cells. Localization analysis showed that LRRC25 is a type I transmembrane molecule. Although ectopic LRRC25 did not promote spontaneous differentiation of NB4 cells, knockdown of LRRC25 by siRNA or shRNA and knockout of LRRC25 by the CRISPR-Cas9 system attenuated ATRA-induced terminal granulocytic differentiation, and restoration of LRRC25 in knockout cells could rescue ATRA-induced granulocytic differentiation. Therefore, LRRC25, a potential leukocyte differentiation antigen, is a key regulator of ATRA-induced granulocytic differentiation.     

Surface markers in acute non-lymphoid leukemia: analysis with a panel of 36 monoclonal antibodies

The reactivity of a panel of monoclonal antibodies was studied in fifty-four cases of acute myeloid (AML) or undifferentiated (AUL) leukemias. Thirty-six antibodies from the Myeloid section of the Second Workshop on Human Leukocyte Differentiation Antigens were used in an indirect immunofluorescence assay. The antibodies could be classified into three groups recognizing respectively granulocytic, monocytic or granulomonocytic leukemias. Most antibodies stained erythroblastic and megakaryoblastic leukemias. In each group, it was possible to define antibodies staining either the less differentiated forms (FAB M 1 and M 5 a) or the more differentiated forms (M 2, M 3, M 4 and M 5 b). Six out of eight AUL were stained by some of the antibodies (mainly from the monocytic group). However, a heterogeneity of stainings in a same blast population was observed.     

Rac1 signaling protects monocytic AML cells expressing the MLL-AF9 oncogene from caspase-mediated apoptotic death

We investigated the relevance of signaling mechanisms regulated by the Ras-homologous GTPase Rac1 for survival of acute myeloid leukemia (AML) cells harbouring the MLL-AF9 oncogene due to t(9;11)(p21;q23) translocation. Monocytic MLL-AF9 expressing cells (MM6, THP-1) were hypersensitive to both small-molecule inhibitors targeting Rac1 (EHT 1864, NSC 23766) (IC_50EHT ~12.5 μM) and lipid lowering drugs (lovastatin, atorvastatin) (IC_50Lova ~7.5 μM) as compared to acute myelocytic leukemia (NOMO-1, HL60) and T cell leukemia (Jurkat) cells (IC_50EHT >30 μM; IC_50Lova >25 μM). Hypersensitivity of monocytic cells following Rac1 inhibition resulted from caspase-driven apoptosis as shown by profound activation of caspase-8,-9,-7,-3 and substantial (~90 %) decrease in protein expression of pro-survival factors (survivin, XIAP, p-Akt). Apoptotic death was preceded by S139-posphorylation of histone H2AX (γH2AX), a prototypical surrogate marker of DNA double-strand breaks (DSBs). Taken together, abrogation of Rac1 signaling causes DSBs in acute monocytic leukemia cells harbouring the MLL-AF9 oncogene, which, together with downregulation of survivin, XIAP and p-Akt, results in massive induction of caspase-driven apoptotic death. Apparently, Rac1 signaling is required for maintaining genetic stability and maintaining survival in specific subtypes of AML. Hence, targeting of Rac1 is considered a promising novel strategy to induce lethality in MLL-AF9 expressing AML.     

Detection of minimal disease in acute leukemia using flow cytometry: studies in a rat model for human acute leukemia

The study was made using a rat model for human acute myelocytic leukemia (BNML), which shows striking similarities with human acute myelocytic leukemia (AML). A monoclonal antibody (MCA-Rm124), raised against BNML cells, allowed the recognition of the leukemic cell fraction. The discriminative capacity of the monoclonal antibody is based on differences in labeling intensities between normal and leukemic cells. After i.v. cellular transfer of leukemia, the growth of the leukemic cell population in the bone marrow, the liver, and the spleen was monitored using MCA-Rm124 and flow cytometry. For the bone marrow and the liver, a clonogenic assay for leukemic cells was used to quantify the cell content in these organs. A good correlation was found between the bioassay-derived data and the flow-cytometry-derived data. The doubling times of the leukemic cell population were not equal for the two organs studied, indicating that a number of different processes contribute to the net cell production per organ. Apart from their application in the detection of residual leukemia, monoclonal antibodies might be employed in the analysis of the growth kinetics of the "invisible" leukemic cell population.