G. Vierwinden

A low but functionally significant MDR1 expression protects primitive haemopoietic progenitor cells from anthracycline toxicity

Pgp is expressed on normal haemopoietic progenitor cells. The significance of the efflux pump in protecting normal progenitors for anthracycline toxicity is not defined and is the subject of this study. Pgp was measured in CD34+ progenitors with a rhodamine efflux assay. A high efflux, modulated by verapamil, was only found in a distinct subpopulation (20–30%). Pgp measured by the monoclonal antibody antibody (MoAb) MRK‐16 was low in the rhodamine dull, but significantly (P < 0.04) higher than in the rhodamine bright cells. Reverse transcriptase polymerase chain reaction (RT‐PCR) of MDR1 mRNA showed a very weak signal in both populations. In a single‐cell clonogenic assay, rhodamine dull cells appeared less sensitive to anthracyclines (IC50 daunorubicin 0.005 μg/ml; adriamycin 0.03 μg/ml) compared to rhodamine bright cells (IC50 daunorubicin 0.0025 μg/ml; adriamycin 0.01 μg/ml). Furthermore, verapamil significantly (P < 0.05) potentiated anthracycline toxicity only in the rhodamine dull cells, proving its Pgp‐specific modulating effect. Rhodamine dull cells gave larger and more mixed colonies compatible with a more primitive origin. Although detection with MoAbs and RT‐PCR revealed a low Pgp level, functionally this Pgp appeared to be very important in protecting primitive progenitors against anthracycline toxicity. This protection can be jeopardized by administration of Pgp modulators.

Cyclosporin increases cellular idarubicin and idarubicinol concentrations in relapsed or refractory AML mainly due to reduced systemic clearance

The feasibility of adding both the multidrug resistance modulator cyclosporin (CsA) and granulocyte colony-stimulating factor (G-CSF) to a standard salvage regimen of idarubicin (IDA) and cytarabine was evaluated in patients with resistant or relapsed acute myeloid leukemia and myelodysplastic syndrome. Three patients received IDA 12 mg/m2/day, the next four patients 9 mg/m2/day. The dose of CsA was 16 mg/kg/day. Six patients showed Pgp expression and none MRP1 expression. Grade III or IV toxicity (CTC-NCIC criteria) was registered in six patients for gastrointestinal, two patients for cardiovascular and one patient for neurological complications. Three patients died in hypoplasia and three patients showed leukemic regrowth. Three control patients were treated with IDA 12 mg/m2/day and cytarabine, but no CsA and G-CSF. The plasma IDA and idarubicinol (ida-ol) area under the curves of patients treated with IDA 12 mg/m2 plus CsA were higher (P < 0.05) than in controls. cellular ida concentrations were almost similar, but cellular ida-ol concentrations were significantly higher (P < 0.05) in the presence of csa than in controls. we conclude that the toxicity either with ida 12 or 9 mg/m2/day was too high. The modulating effect of CsA was mainly based on changes in plasma kinetics of IDA and ida-ol, although ida-ol cellular clearance was delayed in the presence of CsA.

Idarubicin DNA intercalation is reduced by MRP1 and not Pgp.

Currently available data regarding the substrate specificity of the multi-drug resistance (MDR) mechanisms P-glycoprotein (Pgp) and MDR-associated protein (MRP1) for idarubicin are inconclusive. A multiparameter flow cytometry method was developed which allows simultaneous quantitative measurement of total cellular fluorescence and the amount of anthracyclines intercalated into the DNA. Anthracycline DNA intercalation was measured by fluorescence resonance energy transfer (FRET) between Hoechst 33342 and anthracyclines. Daunorubicin and idarubicin accumulation were studied and compared in established cell lines expressing Pgp and MRP1. The data demonstrate that daunorubicin DNA intercalation is affected by both Pgp and MRP1 whereas idarubicin DNA intercalation is affected only by MRP1. MRP1 and Pgp function could be blocked completely by 5 μM PAK 104P, while higher concentrations of verapamil, PSC 833 and cyclosporin A were necessary to attain complete blocking of MRP1 compared to Pgp. Daunorubicin DNA intercalation correlates better with cell survival and is more sensitive at physiological MDR expression as observed in hematopoietic progenitors than daunorubicin levels measured by total cellular fluorescence. In conclusion, idarubicin DNA intercalation is reduced by MRP1 but not by Pgp. PAK-104P is an effective modulator for both Pgp and MRP1 and may further improve idarubicin efficacy.

Variations of the phosphorylation of 1-β-d-arabinofuranosylcytosine (ara-C) in human myeloid leukemic cells related to the cell cycle

Bone marrow cells of five patients with acute myeloid leukemia were fractionated by means of counterflow centrifugation (elutriation). The different fractions were enriched with cells belonging to subsequent stages of the cell cycle. Cytokinetic evaluation of these cell fractions was performed by [3H]thymidine autoradiography, [3H]thymidine incorporation and DNA/RNA-flow cytometry. Phosphorylation of cytosine arabinoside (ara-C, 1-beta-D-arabinofuranosylcytosine) in the different fractions was measured by incubation of the cells for 30 min with 1.07 microM [3H]ara-C. Phosphorylation of ara-C in the whole bone marrow samples ranged from 5.9 to 33.2 pmol/10(6) cells. In the fractions containing only G1-phase cells, phosphorylation ranged from 1.2 to 19.5 pmol/10(6) cells. The phosphorylation seems to increase before DNA synthesis starts. Maximal activities were found in the fractions enriched with cells in late G1- or S-phase of the cell cycle. In these fractions the ara-C phosphorylating activity was 1.5-8 times higher compared to the fractions with the lowest activity. One may therefore assume that not only S-phase cells are killed by ara-C, but that G1-phase cells which can phosphorylate ara-C, may also be doomed when they enter S-phase, since the elimination of the intracellular cytosine arabinoside tri-phosphate (ara-CTP) is a relatively slow process. The fraction of G1-phase cells phosphorylating ara-C, may be an important determinant in the extent of the cell-killing effect of ara-C treatment in the different leukemias.

A sensitive bio-assay for pharmacokinetic studies of cytosine arabinoside in man

Abstract A sensitive bio-assay is presented for the determination of plasma levels of cytosine arabinoside (Ara-C). The test is based on the inhibition of the incorporation of tritiated thymidine into DNA of rat bone marrow cells in the presence of Ara-C. The accuracy and the sensitivity of the test is demonstrated. Unknown plasma concentrations of Ara-C as low as 0·003 mg/l can be measured. The plasma disappearance of Ara-C after an intravenous bolus injection in a dose of 100 mg/m 2 , is biphasic. The half life times of these two phases vary for individuals which is illustrated in two patients with acute leukemia in which the half life time of Ara-C in the first phase was 1·8 and 1·4 min, and in the second phase 16·0 and 9·2 min respectively.

The role of the different CD34 epitopes in detection and positive selection of CD34+ bone marrow and peripheral blood stem cells.

Positive selection of CD34+ cells is an attractive approach to reduce tumour cell contamination in bone marrow (BM) and peripheral blood progenitor cell (PBPC) autografts in malignancies not expressing CD34. All current selection methods use monoclonal antibodies (MoAbs) specific for the class I or class II CD34 epitopes, while for detection most investigators use class III MoAbs. Since the distribution of the different CD34 epitopes on haematopoietic progenitors differs, we studied their significance in CD34+ selection procedures. Testing MoAbs against class I, II and III CD34 epitopes on normal BM we observed that ± 23% of class III positive cells was class I negative. A higher expression of the class III epitope compared with classes I or II was observed on the KG1 cell line, whereas no differences in binding capability were found. The class III epitope anti‐CD34, 561, was compared with the class I epitope anti‐CD34, BI‐3C5, both coupled to M450 Dynabeads. The yield of CD34+ cells obtained with the 561 beads was 1.7% of the mononuclear cells versus 0.95% using the class I epitope, a 1.95‐fold increase (1.3–2.7), whereas the purity was similar (96% in both cases). The absolute number of CD34+ cells was therefore twofold higher after 561 selection, including cells with a more mature phenotype. In single cell assay comparable numbers of highly proliferative progenitors but higher numbers of differentiated colonies per phenotypic subfraction were measured. In conclusion, M450 beads coated with the 561 anti‐class III CD34 epitope are more efficient in isolating CD34+ cells from bone marrow, probably due to a broader distribution of the class III epitope.

The transcription factor nuclear factor Y regulates the proliferation of myeloid progenitor cells

The transcription factor nuclear factor Y (NF-Y) consists of three subunits; NF-YA, -B and -C, and this complex binds DNA and the basal transcription machinery through binding of TBP.[1][1] NF-Y targets include cell cycle related genes and hematopoietic (stem) cell genes such as cd34 and hoxb4 .[2][