Flow cytometric monitoring of anthracycline accumulation after anti-neoplastic ether phospholipid treatment.

Ether phospholipids are new anti-neoplastic drugs that have been found active against a variety of tumor cell lines, including drug-resistant sublines. We have characterized the antiproliferative activity of three ether phospholipids, i.e. ET-18-OCH3 (Edelfosine), BM 41.440 (limofosine) and a new aza-derivative (BN 52205), on three leukemic cell lines, i.e. K562 (chronic myeloid leukemia, blast crisis), HL60 (promyelocytic acute leukemia) and CEM (T cell leukemia), and their respective drug-resistant sublines, i.e. K562-ADR (adryamicin resistant), HL60-DNR [daunorubicin (DNR) resistant] and CEM-VLB (vinblastin resistant). These resistant sublines have been found to express the multidrug-resistant phenotype, revealed by the presence of the P-glycoprotein (PgP) using different monoclonal antibodies. Increased cellular accumulation of the fluorescent anthracycline has been found in both sensitive and resistant cell lines after different ether phospholipid treatment times. In resistant cells, the ether phospholipid effect on DNR accumulation has also been found after blocking the PgP function by verapamil and cyclosporin A. These results confirm that the ether phospholipid action is closely linked with the membrane biochemical composition and that these new anti-tumor drugs are able to change the dynamic structural organization of the tumor cell membrane.

In vitro effect of rGM-CSF on proliferation and maturation of leukemic cells from patients with acute myelogenous leukemia.

We investigated the effect of rGM-CSF on the proliferation/differentiation balance of the leukemic cells maintained in liquid cultures during 7 days, from 16 patients with acute myelogenous leukemia (AML). Cell proliferation was measured by tritiated thymidine (3HT) incorporation, and by the plating efficiency (PE) observed after 7 days of culture. Differentiation was measured by the ability of cells to reduce nitroblue tetrazolium (NBT) and by the percentage of immature myeloid cells. After 7 days of culture without rGM-CSF, an increase of 3HT incorporation (p = 0.01 compared to control) was observed in 8 cases. In these patients, an absence of PE was noted in only one case. Among the 8 patients with decreased 3HT incorporation (p = 0.01 compared to control), 6 exhibited functional maturation (increase of % of NBT + cells, p = 0.01), and 4 showed no PE. Seven days exposure to 50 ng/ml rGM-CSF increased the leukemic cell proliferation in 9 cases, induced complete functional differentiation in 4, and enhanced the CFU-L recovery in 6 cases. These effects were mainly observed in the "proliferative" group, where 7 of the cases had an increase of 3H-T. However, two of the "non proliferative" cases were also stimulated by GM-CSF. An absence of proliferation was generally accompanied by functional maturation.

Effect of differentiating agents on modulation of MDR1 gene expression in multidrug-resistant hematopoietic HL60/DNR cell line.

The phenomenon of multidrug resistance (MDR) is frequently encountered in clinical situations, and could contribute to the failure of chemotherapy in acute leukemia. Preliminary studies have suggested that MDR1 gene expression in normal hematopoietic stem cells might be downregulated during differentiation. In the present study, we induced a multidrug-resistant promyelocytic leukemia cell line, HL60/DNR, to myeloid differentiation by exposure to all-trans retinoid acid and dimethyl sulfoxide (DMSO). We found that HL60/DNR cells retained the ability to respond to the differentiation stimulus. However, although MTT assays revealed a slight decrease of IC50 in differentiated cells, neither efflux of daunorubicin (DNR), nor expression of P-glycoprotein (P-gp), nor quantity of MDR1 mRNA has been downregulated in differentiated cells. We can conclude, therefore, that MDR1 gene expression in this multidrug-resistant myeloid cell line is not modified by induction of its differentiation.

Cyclosporin A as a modifier agent in the salvage treatment of acute leukemia (AL).

Sixteen adult patients with relapsed (7 patients) or refractory (9 patients) acute leukemia received mitoxantrone (10 mg/m2 per day for 3 days) and etoposide (200 mg/m2 per day for 3 days) with escalating dose of cyclosporin A (CsA) from a loading dose of 2 mg to 6.5 mg/kg per 2 h followed by 3 days continuous infusion of 5-15 mg/kg per day. The major toxicities were stomatitis and prolonged aplasia, occurring for 15 mg/kg per day of CsA. Transient conjugated hyperbilirubinemia occurred in all patients, and was CsA dose-dependent (r = 0.7). Adequate serum levels of CsA (> 1 microgram/ml) were obtained in 3/6 patients treated with 10 mg/kg per day and 4/4 patients with 15 mg/kg per day. The pharmacokinetic of mitoxantrone showed an unusual increase of carboxylic metabolites, parallel to CsA levels. We observed six responses (two complete and four partial remissions), and eight resistances. Two patients died at days 3 and 8 from sepsis. Before treatment, 7/16 patients tested for P-gp with C219 were positive (> 10% positive cells). 3/6 responders were P-gp-positive. At time of leukemic regrowth, cells expressing P-gp before therapy reverted to P-gp-negative cells.

Daunorubicin uptake by leukemic cells: correlations with treatment outcome and mdr1 expression.

The in vitro daunorubicin (DNR) cell uptake was investigated by flow cytometry in K562/DOX resistant cell line and in 42 patients with acute myeloid leukemia (AML). The proportion of cells able to take up DNR was higher in untreated patients (50% +/- 30) than in previously treated patients (31% +/- 31) (p = 0.04). We noted a good correlation (p < 0.001) between the drug uptake after exposure to 0.1 microM DNR and achievement of complete remission. Cyclosporin A (CsA, 1 microgram/ml) and verapamil (5 micrograms/ml), but not cefoperazone (10 mM), completely reversed (CsA) or partially reversed (verapamil) the DNR efflux from K562/DOX mdr1(+) cell line. CsA significantly increased (p < 0.01) the DNR uptake of fresh leukemic cells, but not consistently, with no relationship to mdr1 mRNA cellular level. This absence of correlation was explained by the fact that several patients with no mdr1 gene expression exhibited a low in vitro DNR uptake, showing that the MDR phenotype is not the only mechanism responsible for the alteration of DNR pharmacokinetics in AML.

Treatment of relapsing and refractory adult acute myeloid leukemia according to in vitro clonogenic leukemic cell drug sensitivity.

In an attempt to improve the results of treatment in patients with relapsing or refractory acute myeloid leukemia (AML), we conducted a pilot study using a two-drug salvage regimen according to the in vitro clonogenic leukemic cell (CFU-L) drug sensitivity. Fourteen patients were included in the study, 10 with relapsing and 4 with refractory AML. Drug exposure was assessed for daunorubicin, cytosine arabinoside, etoposide, mitoxantrone and amsacrine. The two drugs with the best inhibitory effect on CFU-L were selected for the treatment. A complete remission was achieved in only 4 patients and a partial remission in 3 patients. Although the number of patients is too small for appropriate statistical analysis and for a definite conclusion to be drawn, the observed response rate with directed therapy did not appear better than that expected from empiric salvage regimen.

Cyclin A expression in human hematological malignancies: a new marker of cell proliferation.

Several in vitro studies have shown that the cyclin A gene is expressed and plays an important role in both the S and G2-M phases of the cell cycle. We analyzed cells from the blood and bone marrow of patients with various, mostly neoplastic, hematological disorders to determine whether (a) the cyclin A protein level correlated with that of cyclin A RNA and (b) cell distribution among the different phases of the cell cycle correlated with cyclin A RNA expression. Thirty-eight patients were studied by means of dot blot and Western blot techniques for cyclin A RNA and protein accumulation, and 21 were also studied for cell cycle distribution by using flow cytometric analysis. Semiquantitative studies were based on densitometric computerized evaluation of dot and Western blots. There was a very strong positive correlation between cyclin A RNA and protein expression (r = 0.99; P < 0.00005), indicating that cyclin A accumulation is regulated in these cells at a transcriptional level. There was also a highly significant positive correlation between cyclin A RNA expression and the cumulative percentage of cells in S plus G2-M phase (r = 0.98; P < 0.00005). Therefore, this in vivo study shows that the expression of cyclin A RNA and protein in human hematological malignancies correlates with the percentage of cells in S plus G2-M phase and identifies cyclin A as a new potential cell proliferation index in oncology.

Multidrug resistance (MDR) gene expression in acute non lymphoblastic leukemia: sequential analysis.

Sequential evaluation of P-glycoprotein expression was performed in 29 patients with acute nonlymphoblastic leukemia using immunocytochemistry with the C219 antibody. At diagnosis, 32% of the patients exhibited more than 5% of the P-gp(+) leukemic cells. Under chemotherapy, 62% of the patients eventually expressed a subset of P-gp positive leukemic cells. After conventional doses of cytosine-arabinoside (Ara-C) and daunorubicin or mitoxantrone, positive P-gp cells were noted in 65% of the cases. This percentage was significantly higher (p = 0.002) than the proportion of positive cases (15%) observed after regimens containing either intermediate doses of Ara-C or cyclosporine A, a P-gp modulator.

Relevance of mdr1 gene expression in acute myeloid leukemia and comparison of different diagnostic methods.

In an attempt to determine the incidence and clinical relevance of mdr1 gene expression in acute myeloid leukemia (AML), we examined 126 specimens obtained from adult patients with de novo AML by slot blot and immunocytochemistry. We found a high incidence of mdr1 gene expression in newly diagnosed patients (27% by immunocytochemistry and 43% by slot blot). No difference was observed between newly diagnosed patients and relapsed patients. However, patients with resistant disease showed statistically higher incidence of mdr1 gene expression compared to the untreated and relapsing patients (60% versus 27% by immunocytochemistry, p 0.005; and 73% versus 45% by slot blot, p less than 0.05). The expression of mdr1 gene correlated significantly with clinical drug resistance: 62% of patients positive for mdr1-mRNA and 68% of patients positive for P-glycoprotein (P-gp) eventually developed resistance to chemotherapy, while this was the case for a lower percentage of patients who did not express mdr1 gene (only 23% by slot blot analysis, p = 0.0052, or 24% by immunocytochemistry, p = 0.0009). A combined parameter, mdr1-mRNA/P-gp, had a very high prognostic value in terms of specificity and sensitivity. All nine patients (100%) who were mdr1-mRNA+/P-gp+ progressed to clinical drug resistance afterward, whereas 11 of 13 (85%) patients who were mdr1-mRNA-1 P-gp- entered complete remission and only two patients later developed drug resistance (p = 0.0005). It could thus be used as a reliable parameter in clinical settings.

Molecular analysis of 12 patients with the t(8;21) translocation and M2 acute myelogenous leukemia.

The t(8;21)(q22;q22) is a nonrandom cytogenetic abnormality associated with acute myelogenous leukemia of the M2 subtype (FAB classification). The 8q- and 21q+ derivative chromosomes have previously been isolated in somatic cell hybrids and used to map the anonymous sequences D21S65 and D21S17, which were proximal and distal, respectively, to the breakpoint on chromosome 21. DNA from a series of 12 t(8;21) patients and 7 controls was analyzed by pulsed field gel electrophoresis. Physical linkage of probes D21S65 and D21S17 on a 2100 kb NruI fragment was established by partial digestion experiments. In all the patients, the translocation generated a rearranged D21S65 NruI fragment of 650 to 750 kb, suggesting heterogeneity in the breakpoints. This heterogeneity was confirmed by using BssHII, SacII, and EagI enzymes. Our results are consistent with the presence of a 100 Kb breakpoint cluster region on chromosome 21 encompassing the AML1 gene. Interestingly, in half of the patients, demethylation of an NruI site located 7 kb proximal to the last exon of the AML1 gene occurred on the nontranslocated chromosome 21.

Prognostic value of in vitro uptake and retention of cytosine arabinoside in acute myelogenous leukemia.

In vitro uptake and retention of 3H-cytosine arabinoside (ara-C) was studied in 68 acute myelogenous leukemia (AML) patients (ten were studied twice) treated with a regimen containing conventional (54 patients) or high doses (24) of ara-C. Drug uptake and retention after four hours were measured following 30 minutes exposure to 1 and 50 micrograms/mL of ara-C. A good correlation was observed between high uptake in the acid soluble (AS) fraction (P less than .01), or high retention in the acid insoluble (Al) fraction (P less than .03) of 1 microgram, but not 50 micrograms, of the drug and obtainment of complete remission (CR) in patients treated with conventional doses of ara-C. Patients with a high percentage of ara-C retained in the AS fraction (greater than 26%) have a significantly longer CR duration than patients with a lower level of retention (P less than .02). In 17 patients who achieved a CR in spite of a low tritiated ara-C uptake, the clonogenic leukemic (CFU-L) cells were especially sensitive to drugs (15 of 17 v three of 11 among resisting patients).

Prognostic value of clonogenic assay for induction and duration of complete remission in acute myelogenous leukemia.

The validity of an in vitro clonogenic drug sensitivity assay to predict the induction and the duration of complete remission was evaluated in a group of 81 patients with acute myelogenous leukemia treated with chemotherapy including an anthracycline drug (daunorubicin or adriamycin) and cytosine arabinoside (Ara-C). The inhibition of bone marrow clonogenic leukemic cells by in vitro exposure to anthracyclines 10(-5) and 10(-6) M, Ara-C 10(-5) M, and daunorubicin 10(-6) M + Ara-C 10(-7) M was significantly correlated with the achievement of a complete remission, but not with the duration of remission. A high second plating efficiency was correlated with short duration of complete remission, reflecting the poor prognosis of a high self-renewal capacity.

DNA ligases in human leukemia.

Following partial purification on sucrose gradient and/or phosphocellulose chromatography, DNA ligase was tested in peripheral white blood and bone marrow cells of nearly 100 patients with various kinds of leukemias, mainly acute leukemias. Terminal deoxynucleotidyl transferase (TdT) was tested in parallel. DNA ligase of acute myeloblastic leukemia (AML) was extracted with the same sedimentation coefficient (5.5S) on sucrose gradient, and eluted with the same KCl molarity (0.3 M) than the one extracted from normal lymphocytes. Acute lymphoblastic leukemias (ALL) were characterized by no detectable DNA ligase activity--in most T or non T-non B-ALL, or a low activity in pre-B and B (Burkitt type) ALL, with levels similar to the one observed in chronic lymphocytic leukemia (CLL). An inverse relationship was observed between DNA-ligase and TdT in ALL, ligase being undetectable in cells positive for TdT and being present in some T or non T-non B, and in all pre-B and B-ALL negative for TdT. AML and chronic myelocytic leukemia (CML) were characterized by a markedly higher DNA-ligase activity. This activity was higher in the most differentiated subtypes--M2, M3 and M4 subtypes of FAB classification--and in CML. Moreover a high degree of correlation was observed in AML between the DNA ligase activity and the S phase fraction measured by 3 H-thymidine autoradiography or flow cytophotometry on the total cell sampling. Besides their clinical interest, these results are discussed in relation with the role of DNA-ligase in DNA replication and repair.