The dynamic process of apoptosis analyzed by flow cytometry using Annexin-V/propidium iodide and a modified in situ end labeling technique.

BACKGROUND: To study the apoptotic process in time, we used the following flow cytometric (FCM) techniques: phosphatidylserine (PS) translocation by Annexin-V (AnV), DNA fragmentation by in situ end labeling (ISEL), and propidium iodide (PI) staining. Because PS translocation is assumed to be an early feature of programmed cell death (PCD), we questioned if AnV positivity implies inevitable cell death. METHODS: Apoptosis was induced in Jurkat cells by gamma-irradiation, incubation with camptothecin (CPT), or cytosine beta-D-arabinofuranoside (Ara-C). At different time intervals, PCD was quantified by AnV/PI and ISEL. To analyze the influence of cell handling procedures on PCD, we applied these three FCM techniques on CD34+ bone marrow (BM) stem cells after selection and after a freeze-thaw procedure. Various AnV/PI- CD34+ fractions were cultured in a single-cell single-well (SCSW) assay. RESULTS: Jurkat cells under three different detrimental conditions showed essentially the same pattern of apoptosis in time. Initially developed AnV+/PI- cells subsequently (within 1 h) showed ISEL positivity, after which they turned into AnV+/PI++ cells with even higher levels of ISEL positivity (80-90%). Eventually, they lost some of their PI and ISEL positivity and formed the AnV+/PI+ fraction. Cell handling of CD34+ cells caused high and variable AnV+/PI- fractions (overall range 23-62%). Within total AnV+ and AnV+/PI- populations, only a minority of CD34+ cells showed ISEL positivity (range 4-8% and 0.8-6%, respectively). Different fractions of AnV+/PI- CD34+ cells did have clonogenic capacity. CONCLUSIONS: PCD of cell suspensions in vitro can be followed accurately in time by these three FCM techniques. PS translocation is followed rapidly (within 1 h) by oligo-nucleosomal DNA fragmentation, after which cell (and nuclear) membrane leakage occurs. Detection of PS asymmetry by AnV-fluorescein isothiocyanate (FITC) is not always associated with (inevitable) apoptosis, as can be concluded from the proliferative capacity of AnV+ /PI- CD34+ cells in the SCSW assay.

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 curve's 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 microM 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.

Triggering noncycling hematopoietic progenitors and leukemic blasts to proliferate increases anthracycline retention and toxicity by downregulating multidrug resistance.

Expression of the multidrug resistance (MDR) mechanisms P-glycoprotein (Pgp) and MDR-related protein (MRP) decrease cellular retention and consequently cytotoxicity of anthracyclines. MDR is expressed on normal human hematopoietic progenitors and leukemic blasts. Normal CD34(+) progenitors showed rhodamine efflux in 20% to 30% of the cells, which could be blocked by verapamil. These cells appeared noncycling, in contrast to the proliferating rhodamine bright (RhoB) cells. We postulated that MDR expression can be downregulated by proliferation induction. Triggering rhodamine dull (RhoD) CD34(+) cells to proliferate indeed resulted in a higher rhodamine retention and significantly decreased efflux modulation by verapamil (P =.04). Also in acute myeloid leukemia (AML), the proliferation rate (percentage S/G(2)+M and Iododeoxyuridine labelings index) was significantly less in the RhoD blasts (P

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.

Automatic analysis of growth onset, growth rate and colony size of individual bone marrow progenitors.

A method for automatic enumeration of proliferating bone marrow progenitors after single cell sorting is described. The system is based on regular inverse microscopy, recording with a video camera, and image analysis using dedicated software on an Apple computer. Single CD34+ progenitor cells were sorted in 96-well plates. Three times weekly phase-contrast video images of each well were stored and analyzed for the actual number of cells. From the subsequent counts growth curves were plotted for each individual progenitor. Enumeration by image analysis correlated very well with manual cell counting (r = 0.99, P < 0.0001). To show the capability of the method to analyze growth rate and growth delay, more differentiated (CD34+/CD13+/CD33+) progenitors were compared with more primitive (CD34+/CD13+/CD33g-) progenitors. Differences in the timing of colony outgrowth were shown to be based on delay in growth initiation. Initiation of growth was delayed 2.6-3.1 days in CD34+/CD13+/ CD33- fraction of 3 different donors (P < 0.0001). The growth rates of the progenitors in both fractions were not significantly different. The described method seems important to more accurately evaluate subpopulations of progenitors, the effect of growth promoting or inhibiting factors, and effects of cytotoxic drugs and irradiation.

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 microg/ml; adriamycin 0.03 microg/ml) compared to rhodamine bright cells (IC50 daunorubicin 0.0025 microg/ml; adriamycin 0.01 microg/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.

Anti-endothelial cell antibodies in sera of patients with autoimmune diseases: comparison between ELISA and FACS analysis.

In some patients suffering from rheumatoid arthritis (RA), vasculitis is a clear clinical manifestation, mentioned as rheumatoid vasculitis (RV). Autoantibodies directed against endothelial cells (AEA) have been implicated in the pathogenesis of this disorder, and it has been suggested in a number of studies that testing for AEA should be included in diagnosing RV. To test this hypothesis, we have evaluated the presence of AEA in sera of patients suffering from various autoimmune diseases, employing an ELISA with fixed cultured endothelial cells (EC). In all the groups of patients ELISA-positive sera were present. A significant difference in percentage of positivity was found between the RA and RV group (P < 0.05). In addition, our results indicated that not only antibodies directed against antigens on the EC membrane were detected, but also antibodies directed against intracellular components like DNA, histones and cytoskeletal components. Therefore, we also tested all these patient sera on unfixed intact EC using indirect immunofluorescence followed by FACS analysis. Whereas in the total patient population 34 out of 65 patients were AEA-positive as determined in the ELISA, only seven patients were weakly positive when examined by flow cytometry. We conclude that: (i) an ELISA on fixed EC does not specifically detect AEA. A positive test result is, however, to some extent correlated with the occurrence of vasculitis, and may therefore be helpful in diagnosing this disease; (ii) FACS analysis is a more suitable method than ELISA to measure the presence of membrane-specific AEA in patient sera; (iii) specific IgG-AEA are less common in patients suffering from autoimmune disorders than was assumed previously.

Diffuse CNS involvement in systemic lupus erythematosus: intrathecal synthesis of the 4th component of complement.

In extracerebral systemic lupus erythematosus (SLE), the complement system plays a prominent pathogenic role, and decreased serum concentration of the 4th component (C4) is a reliable indicator of systemic disease activity. In diffuse CNS-SLE, however, the pathogenic role of complement is less clear. In 12 patients with active diffuse CNS-SLE presenting with delirium (4), organic personality syndrome (3), or generalized seizures (5), we determined the CSF indexes of the complement components C3, C4, and factor B, and of IgG, IgA, and IgM. There was a significant increase of the C4 index in these patients compared with controls and a significantly higher CSF C4 index in patients with an increased IgM index. We conclude that intrathecal C4 is being produced in diffuse CNS-SLE.

Antiperinuclear factor: indicator of more severe disease in seronegative rheumatoid arthritis.

The specificity of antiperinuclear factor (APF) for patients with rheumatoid arthritis (RA) is well documented. It is unknown whether detection of these antibodies adds information to the detection of rheumatoid factor (RF). In a group of 132 patients with RA, 94 were RF positive. Of the 38 patients persistently negative for RF, 14 (37%) were positive for APF. These 14 proved to have a disease course similar to that of RF positive patients. This similarity was shown most impressively by radiological progression of the disease, and to a lesser extent, by the medication needed to control the disease and the number of extraarticular manifestations. No significant correlation was shown between APF and antinuclear antibodies. Among the RF positive patients with their generally poorer prognosis, APF identified the worst affected. Our study suggests that APF in serum of patients with RA is associated with a poor disease outcome, especially in RF negative patients.

Cross-reactivity of anti-DNA antibodies with proteoglycans.

Ten sera of patients with systemic lupus erythematosus (SLE) were tested in an enzyme linked immunosorbent assay for their ability to react with glycosaminoglycans, constituents of proteoglycans, in relation to their anti-DNA reactivity. The SLE sera reacted with hyaluronic acid and chondroitin sulphate and this reactivity correlated with the anti-DNA activity of these sera. By contrast, sera obtained from patients with other autoimmune diseases or normal sera lacked any of these reactivities. Anti-DNA antibodies purified by affinity chromatography with either oligo dT cellulose or Cibracon blue F3Ga Sepharose reacted with DNA as well as with hyaluronic acid. The cross-reactivity of anti-DNA antibodies could be confirmed by the reaction of a mouse monoclonal anti-DNA antibody with DNA, hyaluronic acid, and chondroitin sulphate. This pattern of cross-reactivities of anti-DNA antibodies suggests that several compounds can function as antigenic targets for these antibodies provided that their structures contain repeating negatively charged groups.

Anti-nuclear matrix antibodies in mixed connective tissue disease.

Purified serum antibodies of patients suffering from mixed connective tissue disease were tested for their immunological specificity against nuclear constituents of HeLa S3 cells. In the indirect immunofluorescent staining technique, using cells and nuclei as targets, a typical speckled intranuclear staining pattern was obtained, that persisted after degradation and extraction of all nucleic acids and their associated proteins. This treatment of nuclei with detergents, DNase, RNase and high salt concentrations leave intact only the so-called nuclear matrix which is an intranuclear proteinaceous network. Further proof that nuclear matrix proteins were targets of the autoimmune reaction was obtained after separation of these proteins by sodium dodecyl sulfate polyacrylamide gel electrophoresis and electrophoretic transfer to nitrocellulose (blotting). A specific number of blot-transferred matrix proteins reacted with purified serum antibodies of 10 patients with mixed connective tissue disease, whereas this reaction was negative with normal healthy individuals. IgG preparations of 7 patients with systemic lupus erythematosus showed a weak, if any, reaction with matrix constituents. Obviously, in some connective tissue diseases serum antibodies are expressed which are directed to specific nuclear matrix antigens.

Variations of the phosphorylation of 1-beta-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.

Cytosine arabinoside binding to human plasma proteins.

The interaction of cytosine arabinoside (Ara-C) with human plasma proteins was investigated by means of ultrafiltration and ultracentrifugation. The results obtained with both methods did not differ significantly. Ara-C binding was studied at plasma levels within the therapeutic range (0.005-1.0 mg/l). It appeared that 13.3% (SD: 2.2%) of Ara-C in the plasma was bound to proteins. The percentage of bound drug was independent of the drug concentration, at least in the therapeutic range.