Cell differentiation and therapeutic effect of low doses of cytosine arabinoside in human myeloid leukemia

Bone marrow cells from 2 patients over 60 years of age with acute myeloblastic (AML) of monoblastic (AMoL) leukemia were cultured in the presence of a low dose of cytosine arabinoside. In the cells from the AML patient this treatment induced differentiation to metamyelocytes and a decrease in the number of blasts, so that there was an 11 -fold increase in the ratio of differentiated myeloid cells to blasts. In the patient with AMoL there was differentiation to monocytes and macrophages and only a 3-fold increase in the ratio of differentiated myeloid cells to blasts. In the latter patient actinomycin D was a more potent inducer of differentiation than cytosine arabinoside, daunomycin was similar to cytosine arabinoside and adriamycin showed the lowest response. Four courses of low dose treatment with cytosine arabinoside produced remission in the patient with AML and in another patient with AMoL whose cells were not tested in culture. No remission was induced by this low dose treatment in the patient with AMoL whose cells showed only a small decrease in blast cells in culture with cytosine arabinoside. It is suggested that prescreening for effective compounds in patients with myeloid leukemias and the use of low dose therapy can be of help in obtaining remission without serious side effects. This could be especially useful in patients where there may be severe toxic effects after high dose chemotherapy.     

Triple combination of retinoic acid + low concentration of cytosine arabinoside + hexamethylene bisacetamide induces differentiation of human AML blasts in primary culture

Differentiation induction therapy provides an alternative therapeutic approach for patients with acute myeloid leukemia (AML) who are either unsuitable for or unresponsive to conventional cytotoxic chemotherapy. The effect of a triple combination of retinoic acid (RA) + low concentration of cytosine arabinoside (Ara-C) + hexamethylene bisacetamide (HMBA) on differentiation of blasts from 24 AML patients was studied. Non-adherent mononuclear cells were seeded at a concentration of 5 x 10(5) cells per ml in 24-well tissue culture plates containing RPMI 1640 culture medium with 20 per cent fetal calf serum and 10 per cent 5637-conditioned medium and incubated with 10(-6) M retinoic acid, 10(-6) M cytosine arabinoside and/or 2 mM hexamethylene bisacetamide for six days at 37 degrees C in a humidified incubator under 5 per cent CO2. Morphological, cytochemical and functional differentiation into mature cells were induced in blasts from 22 out of the 24 AML patients following exposure to the triple combination of 10(-6) M RA + 10(-6)M Ara-C + 2 mM HMBA in primary culture. These effective results justify a clinical trial of such triple combination for AML patients who are either unsuitable for or unresponsive to conventional cytotoxic chemotherapy.     

Monocytic differentiation induced by side-chain modified analogs of vitamin D in ex vivo cells from patients with acute myeloid leukemia

The differentiation-inducing potential of side-chain modified analogs of vitamins D, compared to the reference compound, 1,25-dihydroxyvitamin D₃, was studied in blast cells from patients with acute myeloid leukemia and in cell lines. Analogs PRI-1906 and PRI-1907 showed increased cell-differentiation activities, PRI-1907 even at a very low concentration. Our study revealed a high variability of individual patients’ blasts in their susceptibility to vitamin D analogs. The blasts of the patients with normal karyotype and with mutated NPM1 reacted to analogs with stronger differentiation than the blasts of the remaining patients, while the blasts with mutated FLT3 receptor reacted with weaker differentiation than the remaining blasts.     

Control of in vivo differentiation of myeloid leukemic cells. iv. inhibition of leukemia development by myeloid differentiation-inducing protein

It is shown that a 5-day schedule of two injections per day of the myeloid differentiation-inducing protein MGI-2 inhibited the in vivo development of leukemia in SL and SJL/J mice with different syngeneic MGI⁺D⁺ clones of myeloid leukemic cells. With this schedule of treatment high levels of MGI-2 were maintained in the serum for long periods. In contrast to these results with MGI-2, the same schedule of injections of the myeloid growth-inducing protein MGI-1 did not affect the in vivo development of leukemia in mice with MGI⁺D⁺ myeloid leukemic cells, but stimulated normal myelopoiesis in the bone marrow. Different forms of MGI-1 including MGI-1M and MGI-1G had different serum half-lives, and the form of MGI-1 with the shortest serum half-life showed the smallest in vivo effect on normal myelopoiesis. MGI-2 injections did not inhibit the in vivo development of differentiation-defective WEHI-3B myelomonocytic leukemic cells or YAC lymphoma cells. The results indicate that the in vivo inhibitory effect of MGI-2 on the development of myeloid leukemia correlated with its differentiation-inducing potential on the leukemic cells. It is concluded that this approach of inhibiting leukemia development by inducing differentiation should also be applied to human leukemic patients, whose cells have been shown to be inducible for differentiation in culture by human MGI-2 or by other differentiation-inducing compounds.     

Retinoic acid alone and in combination with cytosine arabinoside induces differentiation of human myelomonocytic and monoblastic leukaemic cells

The effect of retinoic acid (RA) alone and in combination with cytosine arabinoside (Ara-C) on differentiation of fresh human myeloid leukaemic cells from patients with AML was studied. Cells from six patients: three with acute myelomonocytic leukaemia AMMoL and three with acute monoblastic leukaemia AMoL with a percentage of blasts greater than 70, were treated in an in vitro primary suspension culture with retinoic acid (10(-7) M), cytosine arabinoside (100 ng/ml) or both in combination. Non-adherent mononuclear cells were seeded at a concentration of 5 x 10(5) cells/ml in RPMI 1640 culture medium supplemented with 20 per cent fetal bovine serum and 10 per cent (PHA-LCM) phytohaemagglutinin leucocyte conditioned medium and incubated for 6 days at 37 degrees C in a humidified incubator containing 5 per cent CO2 in air. Morphological and functional differentiation into terminal mature elements was induced in all leukaemia cells of the six patients following exposure to the combination of both agents. These results suggest the potential usefulness of the combination of a differentiating agent (retinoic acid) and an antileukaemic drug (cytosine arabinoside) in the treatment of acute myeloid leukaemias: AMMoL and AMoL. This combination warrants a clinical trial.     

Triple combination of retinoic acid plus actinomycin D plus dimethylformamide induces differentiation of human acute myeloid leukaemic blasts in primary culture

Differentiation induction therapy provides an alternative for treatment of acute myeloid leukaemia (AML) patients who are either unsuitable for or unresponsive to conventional cytotoxic chemotherapy. The effect of a triple combination of retinoic acid (RA) + actinomycin D (Act-D) + dimethylformamide (DMF) on differentiation of blasts from 24 AML patients was studied. Non-adherent mononuclear cells were seeded at a concentration of 5 x 10(5) cells/ml in 24-well tissue-culture plates containing RPMI 1640 culture medium with 20% fetal calf serum, 10% autologous serum and 10% 5637-conditioned medium and incubated with 10(-6) M retinoic acid, 5 nM actinomycin D and/or 100 mM dimethylformamide alone and in combination with each other for 6 days at 37 degrees C in a humidified incubator and an atmosphere containing 5% CO2. The triple combination of 10(-6) M retinoic acid + 5 nM actinomycin D + 100 mM dimethylformamide induced 90% of the blasts from 22 of the 24 AML patients to differentiate. The combination of N-methylformamide (a compound similar to dimethylformamide) with cyclophosphamide significantly increased the in vivo activity with no concomitant increase in its reversible hepatotoxicity. Since several polar compounds related to dimethyl-formamide, e.g. hexamethylene bisacetamide and N-methylformamide, are currently undergoing phase II clinical trials, it may be feasible to combine one of these with retinoic acid and/or actinomycin D in the treatment of AML patients.     

Isolation and characterization of HL-60 cell variants with different potentials for spontaneous differentiation

Although HL-60 cells, an in vitro established cell line derived from a patient with acute promyelocytic leukemia, are blocked at the promyelocytic stage of myeloid differentiation, certain chemicals can induce the cells to undergo terminal differentiation into either granulocytes or macrophages. Moreover, a small fraction of the cell population undergoes differentiation spontaneously without the addition of any inducing agent. In this paper it is demonstrated that this cell line is heterogeneous with respect to the ability of the cells to differentiate spontaneously: some clones (SD+) have a higher tendency to do so than others (SD-). In semi-solid medium, SD+ cells developed diffused colonies containing mature monocytes and macrophages, whereas SD- cells developed compact colonies of promyelocytes. Based on these morphological differences the various clones were isolated and analysed. Although only a small fraction of the population actually became differentiated at any particular time, practically all the cells in the SD+ clones had the potential to differentiate spontaneously. The clones also differ in their response to differentiation inducers; whereas some agents induced complete differentiation in both types of clones, others (e.g. actinomycin C and cytosine arabinoside) induced only SD+ clones, suggesting that differentiation induced by the latter agents is related to the ability of the cells to differentiate spontaneously. Thus the potential of leukemic cells to undergo spontaneous differentiation may be an important factor when considering differentiation-inducing therapy for leukemic patients.     

Study of differentiation of fresh myelogenous leukemic cells by compounds that induce a human promyelocytic leukemic line (HL-60) to differentiate

The human promyelocytic leukemia cell line known as HL-60 can be triggered to mature to functional granulocytes and/or macrophages after exposure to a variety of compounds. The findings have generated enthusiasm for possible therapy of leukemia using compounds that induce leukemic cell differentiation. We investigated whether five compounds known to trigger HL-60 differentiation to granulocytes could trigger the maturation of blast cells from 12 patients with myelogenous leukemia. Maturation was judged by morphology, superoxide production, phagocytosis, expression of Fc receptors, and development of alpha-napthyl acetate esterase activity. The blast cells from most patients showed little morphological, histological or functional maturation after exposure to the various compounds as compared to the blast cells cultured without the compounds. Actinomycin was able to induce significant maturation of leukemic cells of some patients when maturation was analyzed by several statistical methods. Our study suggests that many compounds which trigger differentiation of promyelocytic leukemia cells may not trigger differentiation of less mature myeloid leukemic cells.     

Indirect induction of differentiation of normal and leukemic myeloid cells by recombinant interleukin 1

Different clones of myeloid leukemic cells can be induced to differentiate to mature macrophages or granulocytes by different normal hematopoietic regulatory proteins. The present experiments with recombinant IL-1 alpha and recombinant IL-1 beta show that, (a) that there are clones of myeloid leukemic cells which can be induced to differentiate to mature cells by the myeloid cell differentiation-inducing protein MGI-2 and can also be induced to differentiate to mature macrophages and granulocytes by both types of IL-1; (b) this IL-1-induced differentiation is mediated by endogenous production of differentiation-inducing protein MGI-2; (c) IL-1 and MGI-2 induce production of GM-CSF in these leukemic cells; and (d) IL-1 also induces cell differentiation and production of MGI-2 and GM-CSF in normal myeloid precursor cells. The results indicate that IL-1 induces differentiation indirectly.     

Early clearance of peripheral blood blasts predicts response to induction chemotherapy in acute myeloid leukemia

BACKGROUND:

Early marrow blast clearance 14 days after induction chemotherapy is an independent prognostic indicator of outcomes in acute myeloid leukemia (AML).

METHODS:

We evaluated the relationship between time to peripheral blood blast clearance after induction and disease status as assessed by day 14 and day 30 marrow biopsies in 162 patients with AML. Day 6 after induction was the optimal cutoff point determined by a receiver operating characteristic analysis and was selected to divide patients into early blast clearance (EBC; ≤6 days; n = 119) and delayed blast clearance (DBC; >6 days; n = 43) groups.

RESULTS:

DBC patients were older, but otherwise the 2 groups were comparable. Marrow blast clearance on day 14 after induction chemotherapy was observed in 84% of patients in the EBC group and 60% in the DBC group. With a median follow‐up of 1538 days, both relapse‐free survival (RFS) (442 vs 202 days, P = .0017) and overall survival (OS) (930 vs 429 days, P < .0001) were longer in the EBC group, and a multivariable analysis showed that EBC independently predicted clearance of marrow blasts at day 14 (P = .0018), remission (P = .0179), RFS (P = .0171), and OS (P = .0122).

CONCLUSIONS:

Early clearance of peripheral blood blasts after induction chemotherapy predicts for early marrow blast clearance, complete remission, RFS, and OS. Cancer 2012. © 2012 American Cancer Society.     

TP53-altered acute myeloid leukemia and myelodysplastic syndrome with excess blasts should be approached as a single entity

TP53-altered myelodysplastic syndrome with excess blasts and TP53-altered acute myeloid leukemia should be considered under one unifying classification term for their study in clinical trials. Ultimately, such a unification would simplify the screening processes for clinical trials and allow a focus on treating the patient for a genetically defined disorder rather than one based on an arbitrary blast threshold.     

Regulation of cell-surface receptors for hematopoietic differentiation-inducing protein MGI-2 on normal and leukemic myeloid cells

The normal myeloid hematopoietic regulatory proteins include 4 different growth-inducing proteins (IL-3, MGI-1GM = GM-CSF, MGI-1G = G-CSF, and MGI-1M = M-CSF = CSF-1). There is also another type of normal myeloid regulatory protein (MGI-2) with no MGI-1 (CSF or IL-3) activity, which can induce differentiation of normal myeloid precursors and certain clones of myeloid leukemic cells. Studies on the binding of MGI-2 to differentiation-competent (D+) and differentiation-defective (D-) clones of mouse myeloid leukemic cells and to normal cells indicate that: (1) D+ clones of myeloid leukemic cells had about 2,500 high-affinity surface receptors per cell, like mature normal myeloid cells, and the bound MGI-2 was rapidly internalized with its cell-surface receptors at 37 degrees C causing down-regulation of MGI-2 receptors in both the normal and leukemic cells; (2) in some D- clones, the number and internalization of MGI-2 receptors were similar to those of D+ clones whereas other D- clones had only 0-100 MGI-2 receptors per cell; (3) normal thymus and lymph-node lymphocytes and T lymphoma cells did not show detectable MGI-2 receptors; (4) there was an independent expression of receptors for MGI-2 and for the 4 myeloid growth-inducing proteins on different clones of myeloid leukemic cells; and (5) none of the 4 myeloid growth-inducing proteins IL-3, MGI-1GM, MGI-1G, or MGI-1M, inhibited binding of MGI-2 to its receptors. The cytotoxic proteins lymphotoxin and tumor necrosis factor did not induce differentiation of the mouse myeloid leukemic cells and also did not inhibit binding of MGI-2 to its receptors. These results show that the myeloid differentiation-inducing protein MGI-2 binds to cell-surface receptors that are different from the receptors for the 4 myeloid growth-inducing proteins and these cytotoxic proteins.     

Coupling of growth and differentiation in normal myeloid precursors and the breakdown of this coupling in leukemia

Normal myeloid precursors are dependent on the macrophage and granulocyte growth-inducing protein MGI-I for cell viability and multiplication. MGI-I also induces production of the differentiation-inducing protein MGI-2, and this induction of a differentiation-inducing protein by a growth-inducing protein provides a mechanism for the normal coupling of growth and differentiation. It is shown that this induction of MGI-2 by MGI-I occurs in the myeloid precursors and not in some other cells in the normal bone marrow, that the induced MGI-2 can be detected 6 h after the addition of MGI-I, and that MGI-2 can be induced in these cells by purified MGI-I. There are clones of myeloid leukemic cells that no longer require MGI-I for cell viability and multiplication, but in which this requirement for MGI-I can be restored after induction of differentiation by MGI-2. A similar concentration of MGI-I was required for the optimum induction of growth in these differentiating leukemic cells and in normal myeloid precursors. In the presence of MGI-I these differentiating leukemic cells multiplied and then lost their differentiation-associated properties. In contrast to normal myeloid cells, MGI-I did not induce MGI-2 in the MGI-I requiring differentiating myeloid leukemic cells. This lack of induction of MGI-2 by MGI-I occurred in cells cultured in serum-containing or serum-free-medium, and can explain the loss of differentiation-associated properties. The results indicate that there has been a genetic breakdown of the normal coupling mechanism between growth and differentiation in these leukemic cells so that MGI-I can no longer induce MGI-2.     

Regulation of the genes for interleukin-6 and granulocyte-macrophage colony stimulating factor by different inducers of differentiation in myeloid leukemic cells

Different clones of myeloid leukemic cells can be induced to differentiate to mature macrophages and/or granulocytes by hematopoietic regulatory proteins and by other compounds. We now show that induction of differentiation in different clones of myeloid leukemic cells with the normal hematopoietic proteins granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), or interleukin 3 and by compounds such as dexamethasone or cytosine arabinoside (ara C) induces the expression of genes for the myeloid differentiation inducing protein MGI-2 that we have shown is interleukin 6 (IL-6) and for GM-CSF. We have previously shown that induction of differentiation with interleukin-1, IL-6, or bacterial lipopolysaccharide (LPS) also induces IL-6 and GM-CSF gene expression. Treatment of these leukemic clones with hematopoietic proteins that do not induce differentiation did not induce IL-6 or GM-CSF gene expression. The results indicate that induction of IL-6 and GM-CSF gene expression is part of the normal differentiation program in myeloid cells and support our previous evidence that there is transregulation of gene expression between different hematopoietic regulatory proteins.     

Discrepancy between in-vitro and in-vivo inductions of differentiation by retinoids of human acute promyelocytic leukemia cells in relapse

In-vitro studies of leukemic cells with retinoic acid and a therapeutic clinical trial with its derivative, etretinate, in a 58 yr-old male patient with 15;17 translocation-positive acute promyelocytic leukemia (APL) in relapse are reported. Actinomycin D was used in combination. Bone marrow promyelocytes from the patient prior to etretinate and actinomycin D matured morphologically in the liquid culture with retinoic acid; 98% were matured myeloid cells after 6 days at a concentration of 10⁻⁶ M of retinoic acid as compared with 2% of the control culture. Positive NBT reactions were seen in none of the cells in the latter but in 95% of the cells in the former. Actinomycin D, when added alone, only induced NBT positivity, but, when used in combination with retinoic acid, increased both NBT positivity and morphologically matured cells. The patient was treated daily with 2 μg/kg of actinomycin D (or 20 mg/m² or 33 mg/m² cytosine arabinoside after the 6th day) in 24-h infusions and per oral 90 mg/body of etretinate. No effectiveness was observed both morphologically and clinically. The patient expired 15 days after the initiation of etretinate. Thus, a discrepancy existed in the response of leukemic cells from this relapsed patient with APL to the in-vitro and in-vivo attempts to include differentiation by retinoids and actinomycin D.     

Regulation of in-vivo differentiation of myeloid leukemic cells by antigen-specific helper T lymphocytes

There are clones of myeloid leukemic cells that can be induced to differentiate in vitro and in vivo by normal macrophage and granulocyte differentiation-inducing protein MGI-2 (= DF). The differentiation of these myeloid leukemic cells in vivo is regulated by a cell mediated immune response which requires T lymphocytes. We now show that differentiation of myeloid cells in vivo can be induced by antigen-specific helper T lymphocytes and that this is associated with the ability of the helper T cells to produce myeloid cell differentiation-inducing protein MGI-2. Antigen specific helper T cells can accumulate at a site that contains the antigen. It is suggested that migration in response to antigen of helper T cells producing differentiation factors may play an important role in inducing in vivo differentiation of leukemic cells.     

The effect of anti-CD44 monoclonal antibodies on differentiation and proliferation of human acute myeloid leukemia cells

Acute myeloid leukemia (AML) is a clonal malignant disease characterized by an increasing number of immature myeloid cells arrested at various stages of granulocytic and monocytic differentiation. The stage of the blockage defines distinct AML subtypes (AML1 to AML5 are the most frequent ones). There is increasing evidence that the malignant clone is maintained by rare AML stem cells endowed with self-renewal capacity, which through extensive proliferation coupled to partial differentiation, generate leukemic progenitors and blasts, of which the vast majority have limited proliferative capacity. Contrarily to chemotherapy alone, which is still unable to cure most AML patients, the differentiation therapy, which consists in releasing the differentiation blockage of leukemic blasts, has succeeded, when it is combined with chemotherapy, to greatly improve the survival of AML3 patients, using retinoic acid as differentiating agent. However, this molecule is ineffective in other AML subtypes, which are the most frequent. We have shown that specific monoclonal antibodies (mAbs, H90 and A3D8) directed to the CD44 cell surface antigen, that is strongly expressed on human AML blasts, are capable of triggering terminal differentiation of leukemic blasts in AML1 to AML5 subtypes. These results have raised the perspective of developing a CD44-targeted differentiation therapy in most AML cases. Interestingly, these anti-CD44 mAbs can also induce the differentiation of AML cell lines, inhibit their proliferation and, in some cases, induce their apoptotic death. These results suggest that H90 and/or A3D8 mAbs may be capable to inhibit the proliferation of leukemic progenitors, to promote the differentiation of the leukemic stem cells at the expense of their self-renewal, and, perhaps, to induce their apoptotic death, thereby contributing to decrease the size of the leukemic clone. The challenges of an anti-CD44 based differentiation therapy in AML, and its importance in relation to the new other therapies developed in this malignancy, are discussed in this review.     

Modulation of cytotoxic effect of anticancer drugs by dipyridamole in HeLa cells in vitro

Exponentially growing HeLa cells were treated with various antitumor drugs and dipyridamole (DP), and the cell growth inhibition ratio was determined. Enhanced growth inhibition was found in combined treatment with DP and 5-fluorouracil, 1-hexylcarbamoyl-5-fluorouracil, methotrexate, adriamycin, daunomycin, 4'-0-tetrahydropyranyl-adriamycin, actinomycin D and vincristine. In contrast, reduction of the cytotoxic effect of cytosine arabinoside and enocitabine was found when these were combined with DP.     

A Comparative Study of DCMP versus DCcMP Combination Chemotherapy for Adult Acute Leukemia

An experimental study using L-1210 mouse leukemia indicatedthat daunomycin, cyclocytidine, and 6-mercaptopurine (DCcM)combination chemotherapy was more effective than daunomycin,cytosine arabinoside, and 6-mercaptopurine (DCM). Based on theseresults, a clinical study comparing daunomycin, cytosine arabinoside,6-mercaptopurine, and predni-solone (DCMP) combination chemotherapywith daunomycin, cyclocytidine, 6-mercaptopurine, and prednisolone(DCcMP) in the treatment of adult acute nonlymphocytic leukemiawas conducted. Patients were randomly allocated to receive eitherregimen. Ten of 15 patients treated with DCMP had complete remissionfor a median duration of 43 weeks and one had partial remission.Seven of 15 patients treated with DCcMP had complete remissionfor a median duration of 18 weeks and two had partial remission.The patient characteristics before chemotherapy, the toxicityof the chemotherapeutic regimens and the causes for differencesin antitumor activity between the experimental study and theclinical study are discussed.     

A study of multidrug resistance and cell kinetics in a child with near-haploid acute lymphoblastic leukemia

Marked hypodiploidy is found in a small group of patients with acute lymphoblastic leukemia (ALL) and is associated with very poor prognosis. Cells from a patient with near-haploid ALL (karyotype: 27 XY, DNA index = 0.5) were investigated by multiparameter flow cytometry at relapse and at multiple time-points during reinduction chemotherapy. The cell cycle of these near-haploid leukemic blasts and their chromatin structure was studied by acridine orange (AO) DNA/RNA flow cytometric assays. Most leukemic cells were in "G0", and no recruitment of the bone marrow cells into the G1 phase of the cell cycle was found during reinduction therapy with high dose cytosine arabinoside. After two cycles of chemotherapy, the patient achieved clinical remission, but persistent haploid cells were identified by flow cytometry and he relapsed after 8 weeks and died after 16.7 weeks. The leukemic blasts expressed very high levels of a 180 kd p-glycoprotein associated with multidrug resistance and daunomycin efflux could be blocked by verapamil. Expression of gp 180 and the verapamil effect on intracellular daunomycin concentration indicate multidrug resistance. We conclude that cell kinetic quiescence and multidrug resistance may both be factors responsible for the poor prognosis of this patient with near-haploid ALL. Further studies of this patient group should determine if these mechanisms are indeed responsible for the poor prognosis associated with near-haploid leukemia.