Paul Baines

The MEK inhibitor, PD98059, reduces survival but does not block acute myeloid leukemia blast maturation in vitro

Abstract: The appearance of blasts in acute myeloid leukemia (AML) reflects a shift from cellular processes inducing maturation and cell death to those favouring survival and accumulation. We have monitored changes in the growth factor signalling molecule MAPKinase, in the cytoprotective protein Bcl‐2 and in the cell death protein Bax, during maturation of proliferating and non‐proliferating AML blasts in vitro. Eighteen AML samples were cultured for 7 d in serum‐free medium with or without a supplement of recombinant cytokines comprising c‐kit ligand, IL3 and GMCSF. Maturation of AML blasts, as assessed by morphology on Romanowsky‐stained slides of 7/18 samples and by changes in surface CD markers on all 18 leukemias, occurred in both the absence and presence of cytokines. Cell numbers decreased to a mean of 71% after 7 d of cytokine‐free culture, but increased to 210% in cytokine‐supplemented cultures. The proportion of CD15‐positive cells, assessed by flow cytometry, increased over 7 d in 17/18 samples, from a mean of 22% to 68% in cytokine‐free cultures and to 72% in cytokine‐supplemented cultures (p=<0.0001 for both). By immunofluorescence/flow cytometry, there was no significant change in Bcl‐2 over 7 d of culture, while Bax increased, particularly in cytokine‐free cultures (2.2‐fold), which led to a significant decrease in the Bcl‐2/Bax ratio. Immunoblotting demonstrated that ERK was briefly phosphorylated after seeding AML blasts into culture. PD98059, an inhibitor of MAPKinase kinase (MEK) which activates MAPKinase, inhibited this transient ERK phosphorylation but was unable to block maturation as measured by acquisition of CD15 in samples from 12 patients with low starting numbers of CD15‐positive cells. PD98059, however, reduced cell numbers in 7‐d liquid culture and, in cytokine‐supplemented cultures, this was associated with a 1.3‐fold increase in Bcl‐2 (p=0.012) and a 1.4‐fold increase in Bax (p=0.02). Overall, these data demonstrate that most leukemic populations can partially differentiate in vitro without the need for cytokines or inducers. The MAPKinase pathway is not required for this maturation, but it does maintain cell viability in the absence or presence of cytokines. A rise in Bcl‐2 may not protect AML blasts in the face of elevated Bax.

Characterization of malignant cell populations in MNU-induced leukaemia of mice.

Abstract A surface marker analysis was found to be of value in classifying leukaemias of poorly differentiated blasts induced by MNU. Thy-1+ve(T cell) lymphoma commonly arose in intact animals with the thymus frequently, though not invariably, enlarged. Thy-1−ve, Ig—ve leukaemias arose in a low percentage of intact mice and in 8 16 leukaemias of thymectomized mice that were tested. These “markerless” leukaemias remain uncharacterized. Of the remaining leukaemias that developed in thymectomized mice, 4 16 were Ig+ve (or B cell) lymphomas and 4 16 showed low-intensity staining for both Thy-1 and Ig. The mechanism of thymic involvement in MNU-leukaemogenesis is discussed. Leukaemias of intact and thymectomized mice do not appear to arise from a common target cell since Thy-1+ve leukaemias rarely arose in thymectomized mice given implants of neonatal thymus tissue shortly after MNU-treatment. In fact, MNU-induced thymic lymphoma arises from an intra-thymic target population.

Multidrug resistance in leukaemia.

Summary Multidrug resistance hampers successful chemotherapy in many haematological neoplasms and is mediated by several cellular proteins. In some cases, the genes encoding these proteins have been shown to confer resistance on transfer to drug-sensitive cell lines. This is true for the efflux pump product of the MDR 1 gene, P-170. Upregulation of enzymes such as GST has been observed, although the contribution of this enzyme in drug resistance expressed by malignant haematopoietic cells is still uncertain. Cells also appear to be able to downregulate enzymes which are drug targets. Examples include the decrease in Topo II which accompanies the resistance shown by cells to VP-16 and VM-26. Although many reports include both presentation and relapsed patients, there are few data on samples drawn from the same patients before and after chemotherapy. While P-170 and GST appear to be raised more often in cells from resistant and relapsed disease, it is quite clear that such mechanisms can be active in de novo malignancy and do not necessarily emerge as a consequence of prior chemotherapy. Methods of detecting drug resistance are reviewed here; these include in vitro cellular assays for drug toxicity, and molecular, immunological and functional detection of P-170 or Topo II. The clinical evaluation of such assays is only just beginning and some of the data are contradictory. To some extent, this may reflect the complex way in which the various resistance mechanisms may interact. Nevertheless, there are some encouraging early signs that the application of these assays to clinical material will yield valuable data on the relative contributions of these mechanisms and on ways in which they may be overcome. At present, much attention has focused on the potential of agents which prevent the P-170 efflux pump from exporting cytotoxics from the cell. This is likely to be only the first of new therapies arising from an improved understanding of multidrug resistance. More immediately, assays for multidrug resistance and its parameters may find their place as routine diagnostic and prognostic tools in the laboratory.

Haemopoietic colony-forming cells from peripheral blood stem cell harvests: cytokine requirements and lineage potential

Summary. We have measured the in vitro growth requirements of progenitor cells released into the blood of cancer patients following administration of chemotherapy and cytokines. In order to distinguish the direct effects of cytokines on progenitors from those activating acessory cells, we have comparied clonogenic grwoth before and after CD34‐positive selection of progenitors, in serum‐free conditions. CD34 selection had little effect on the cytokine requirements of erythroid colony‐forming cells and single cytokines, particularly interleukin‐3, could support considerable colony growth in both mononulear and CD34+ cell suspensions. Optimal erythroid colony grwoth, however, usually required the addition of a combination of stem cells factor and interleukin‐3, in addition to erythoropoietin, which was was always required. Maximal numbers of granulocyte monocyte progenitors in mononuclear cell cultures, could be achieved with a mixture of stem cell factros, ionterleukin‐3 and granulocyte‐monocyte colony stimulating factor. How ever, after CD34 selection, full myeloid colony growth was only achieved when granulocyte colony stimulating factor was added to the above mixture. This presumably reflects loss of accessory cells, during CD34 selection, which produced this cytokine. When transplanted after 8 d of culutre. 16/22 myeloid colonies from erythorpoietin‐free cultures of peripheral blood stem cell harvests, could generate secondary multipotential. However, surface marker analysis of individual erythroid colonies revealed only the occasional presence of granulocytes and monocytes. These date demonstrate that cytokine mixtures are required for optimal colony growth, particuarly after CD34 selection, a nd that most mobilizied, blood clonogenic cells are multipotential.

Complete replacement of serum in cultures of murine primitive erythroid and multipotential progenitor cells absolute requirement for spleen conditioned medium

We describe a serum-free medium for the formation of erythropoietic bursts by murine bone marrow cells. Iscoves modified Dulbeccos medium supplemented with bovine serum albumin, iron-saturated transferrin, soybean phospholipids and cholesterol supported burst formation. The further addition of hemin increased burst numbers to above those obtained in serum-containing cultures. With or without hemin, a source of burst-promoting activity (BPA) (crude or partially purified spleen conditioned medium) and erythropoietin were essential. This system provides a sensitive assay for BPA. Of all colonies developing in these cultures, 16% were pure erythroid, 17% mixed erythroid/myeloid, 36% macrophage, 19% macrophage/basophil and macrophage/neutrophil, 9% basophil and 2% neutrophil.

Clonal growth of haemopoietic progenitor cells from myelodysplastic marrow in response to recombinant haemopoietins

The growth factor requirements of granulocyte-macrophage (GM) and erythroid marrow progenitor cells from 12 myelodysplastic (MDS) patients have been analysed. GM progenitors from two of six patients who grew normal numbers of colonies in response to conditioned medium + erythropoietin (5637CM + Epo) showed defective responses to either GMCSF and/or IL-3. Of all the recombinant factors tested (IL-3, IL-1, GCSF, GMCSF, MCSF), GMCSF was the strongest stimulator of myeloid clonal growth, inducing normal numbers of GM colonies from marrow of six patients (two of whom were neutropenic). Erythroid colonies were low in 5637CM + Epo-supplemented cultures of marrow from all but one patient and remained poor in the presence of any of the haemopoietins. tested. Supraoptimal doses (for normal marrow) of these haemopoietins improved colony growth in only one patient (GM colonies in response to IL-3). Combinations of factors were also largely ineffective at raising myeloid or erythroid colony numbers. These data indicate that the defective response of MDS progenitor cells to growth factors is not amenable to experimental manipulation of recombinant factor levels or combinations. Clonal assays might suggest a role for GMCSF therapy in a subpopulation of neutropenic MDS patients but their potential now needs to be evaluated in association with clinical trials.

Enrichment of haemopoietic progenitor cells from the marrow of patients with myelodysplasia.

Myeloid and erythroid progenitors from myelodysplastic marrows have been separated from accessory cell populations likely to influence their in‐vitro growth. Myeloid colony‐forming cells were enriched 23‐fold and erythroid progenitors 5‐7‐fold but, in both cases, retained their abnormal growth characteristics. After enrichment and removal of lymphocytes and monocytes, erythroid burst formation became markedly more dependent on the addition of 5637 bladder carcinoma conditioned medium as an exogenous source of haemopoietic growth factors. This suggests that lymphocytes and monocytes usually support erythropoiesis in cultures of myelodysplastic marrow and demonstrates that erythroid burst‐forming progenitor cells from myelodysplastic patients can respond to these populations in vitro. Haemopoietic failure in myelodysplasia appears to result from a defect within the preleukaemic clonogenic cell, rather than from an aberration in exogenous factors. The procedure outlined here can be used as a first step towards the isolation of these abnormal progenitors.

Increased circulating normal and BCR-ABL+Ve progenitor numbers in Philadelphia chromosome-positive acute myeloid leukaemia

We recorded elevated numbers of circulating myeloid and erythroid colony-forming cells in 15 adult patients with acute myeloid leukaemia (AML) who presented with high blood white cell counts. Since leukaemic blasts from three of these patients were Philadelphia chromosome-positive (Ph+), we were able to determine if blood progenitors from these particular patients arose from the leukaemic clone or from residual normal progenitors. Blasts and colonies were intensively investigated using a combination of cell surface marker analysis by flow cytometry, RT-PCR and interphase fluorescence in situ hybridization (FISH). FISH detected rearrangements within the major breakpoint BCR (M-BCR) region in blasts and in some myeloid and erythroid colonies from patients 1 and 2. The minor breakpoint (m-BCR) region was detected in blasts and in some myeloid and erythroid colonies from patient 3. RT-PCR detected long b2a2 BCR-ABL transcripts in blasts from patients 1 and 2, although misspliced short e1a2 transcripts were also seen in patient 1. Only e1a2 transcripts were found in blasts from patient 3. Flow sorting demonstrated the B-cell marker CD19 on blasts and on a proportion of myeloid and erythroid progenitors from patients 1 and 3. RT-PCR also detected IgH rearrangements, further evidence of B-cell differentiation, in blasts from these two patients. We conclude that both normal and clonal circulating progenitor numbers can be raised in both M-BCR and m-BCR Ph+ AML. The underlying cause, perhaps efflux from a congested marrow, may be common to AML patients with a high blood white cell count.

GCSF augments post-progenitor proliferation in serum-free cultures of myelodysplastic marrow while ATRA enhances maturation

All-trans retinoic acid (ATRA) and granulocyte colony stimulating factor (GCSF) are potential inducers of myeloid progenitor cell growth and neutrophil differentiation in myelodysplasia (MDS). We have compared the effects of ATRA and GCSF on the colony growth of 10 MDS marrows, in semi-solid and liquid serum-free mononuclear cell (MNC) cultures, supplemented with a mixture of stem cell factor (SCF), interleukin 3 (IL-3) and granulocyte-monocyte colony stimulating factor (GmCSF) (SIGm mix), which is fully-supportive for myeloid and erythroid (with erythropoietin (EPO)) colony formation in normal marrow. Only 1/10 MDS patients produced normal granulocyte-macrophage colony-forming cell (GmCFC) numbers, under SIGm conditions and erythroid colonies (ECFC) were subnormal in all patients. ATRA (10(-7) M) increased GmCFC numbers (P=0.05) in semi-solid cultures of normal, but not MDS marrow MNC and decreased erythroid colonies in cultures of marrow from either source (P=0.008 and P=0.0001 for normal and MDS, respectively). ATRA enhanced neutrophilic maturation in liquid cultures of both normal and myelodysplastic CD34 + ve cells, as detected by conventional morphology and acquisition of CD15. In contrast to ATRA, GCSF increased Gm colony size but not numbers in semi-solid cultures of normal marrow MNC, which suggests the cytokine augments post-progenitor amplification. This would explain why GCSF increased cell yields in liquid cultures of normal and MDS MNC while GmCFC accumulation remained unchanged. GCSF, though, increased Gm colony numbers in semi-solid cultures of MDS marrow MNC (P=0.014) so that 4/10 patients now grew colonies within the normal range. This was again probably due to increasing clone size, so that some clusters, the numbers of which may be elevated in MDS, were now scored as colonies. Overall, these data indicate that ATRA can enhance the maturation of the progeny of MDS GmCFC whilst GCSF can augment their amplification.

Overgrowth of a leukemic culture by a minor CD34+ population

We have investigated the differentiation potential of blast cells in a case of acute myeloid leukemia which comprised a majority CD34- population and a minor (2%) CD34+ fraction. Blasts were cultured for 2 weeks in a combination of cytokines--c-Kit ligand, interleukin 3 and granulocyte macrophage colony-stimulating factor (SIGm mix)--together with all-trans retinoic acid or 1alpha ,25-dihydroxy vitamin D3. Maturation of blasts was assessed by morphology on Romanowsky-stained slides, changes in surface CD markers and clonogenic culture. After 7 days of culture of unseparated blasts in SIGm, most maturation was monocytic, but with retinoic acid 63% of blasts had matured into granulocytes. Vitamin D3 enhanced monocytic differentiation, with 60% of cells becoming monocytic. The percentage of CD14 and CD15 positive cells decreased over 7 days in SIGm (from 62% to 17% and from 76% to 39% for CD14 and CD15, respectively). CD14+ cell numbers were maintained, or recovered, in cultures supplemented with vitamin D3 (59% at day 7), and CD15+ cell numbers, too, remained unchanged in the presence of retinoic acid (67%) or vitamin D3 (66%). Aberrant markers CD7 and CD56 declined under any conditions. When separated, both the CD34- and CD34+ fractions showed similar changes in morphology and surface maturation markers, suggesting that these two populations may be closely related. However, only a few CD34+ cells expressed the aberrant markers present on the majority blast population. The CD34- population declined in culture while the CD34+ fraction rapidly expanded. This probably reflects the difference in progenitor content; high numbers of colony-forming cells were concentrated in the CD34+ subpopulation. We conclude that both CD34- and CD34+ populations can differentiate but only the CD34+ fraction proliferates. Primitive clonogenic CD34+ cells from this patient may generate occasional aberrant CD34+ blasts which could then differentiate into the accumulating aberrant CD34- blast population.