Phosphorothioate BCR-ABL antisense oligonucleotides induce cell death, but fail to reduce cellular bcr-abl protein levels.

The bcr-abl oncogene is a fusion gene resulting from a reciprocal translocation which forms the hallmark of chronic myeloid leukemia (CML). Antisense oligonucleotides complementary to the two possible mRNA breakpoints were found to inhibit cell growth of CML patient cells and cell lines, but doubt exists about their specificity. In order to test the specificity, phosphorothioate and 3' phosphorothioate capped antisense BCR-ABL oligonucleotides of different length were used. Stability, cellular uptake of oligonucleotides and effect on cell growth were studied in two CML cell lines, BV173 and LAMA-84. Phosphorothioate antisense BCR-ABL oligonucleotides were most stable, showed the highest uptake and induced cell death in BV173 but not in LAMA-84 cells. We selected the most effective antisense oligonucleotide for further analysis. The BV173 and LAMA-84 cell lines do not express the normal c-abl protein, we therefore used a c-abl specific monoclonal antibody for the detection of p210bcr-abl expression by flow cytometry. Dead cells found after treatment were gated out of analysis. Although BCR-ABL antisense oligonucleotides can induce apoptosis, no reduction of p210bcr-abl levels could be detected in living cells after treatment with antisense oligonucleotides. We conclude that antisense mediated inhibition of translation of mRNA into p210bcr-abl is not the mechanism responsible for the induction of apoptosis in cell line BV173.

Effect of isotype on internalization and cytotoxicity of CD19-ricin A immunotoxins.

We analyzed the effect of isotype variation on effectiveness of B-cell CD19 immunotoxins (IT) by using class switch variants (CLB-B4-IgG1 and CLB-B4-IgG2a) conjugated to ricin A. Notably, IgG1-IT appeared to be approximately 100-fold more potent than IgG2a-IT toward B-cell lines Daudi and KM3. Binding and internalization studies with 125I-labeled monoclonal antibodies (mAbs) revealed a higher cellular uptake of IgG1 compared to IgG2a, despite similar binding affinities. Following removal of the Fc part, both mAbs internalized at the same rate as IgG2a, indicating that the Fc part of IgG1 is involved in enhanced cellular uptake. The involvement of Fc gamma RII (CD32) in this process was demonstrated by a decreased cytotoxicity of IgG1-IT (and not IgG2a-IT) in the presence of Fc gamma RII-blocking mAbs AT10 or IV.3. To identify the isoform responsible for this phenomenon, internalization of IgG1 and IgG2a in 11 B-cell lines and malignant B-cells of 8 patients was compared with expression of Fc gamma RII subclasses. In addition to four cell lines (Daudi, KM3, Nalm6, and Raji), the malignant B-cells of two patients showed enhanced uptake of IgG1 relative to IgG2a. Only the Fc gamma RIIa transcript was found in all B-cells. Furthermore, enhanced uptake of IgG1 correlated with rosetting of erythrocytes sensitized with anti-glycophorin A mAb of IgG1 isotype rather than with Fc gamma RIIa membrane expression levels. These data support the idea that functional Fc gamma RIIa is involved in the enhanced IgG1 uptake observed in a subset of B-cells. Our study, therefore, points to an important role for the Fc region of IT in the delivery of cytotoxic effects.

Antisense BCR-ABL oligonucleotides induce apoptosis in the Philadelphia chromosome-positive cell line BV173.

BCR-ABL antisense oligonucleotides can specifically reduce colony formation of early hematopoietic progenitor cells from chronic myeloid leukemia (CML) patients. Little is known about the mechanism of this inhibition. We studied the inhibition of the bcr-abl oncogene using fluorescein-labeled phosphorothioate oligonucleotides in the Philadelphia chromosome-positive cell line BV173. Oligonucleotide stability, uptake, bcr-abl mRNA degradation, inhibition of cell proliferation, and cell death were studied. The oligonucleotide uptake was directly dependent on the extracellular concentration and was constant over the first 18 h of incubation. After that the uptake rate decreased. We detected a decrease in bcr-abl mRNA after 3 days of treatment with antisense oligonucleotides, but much less in controls. The controls used in the experiments were the sense oligonucleotide, equimolar amounts of sense and antisense, and an untreated control. Antisense oligonucleotides completely inhibited cell growth of BV173 cells and did not inhibit growth of HL-60 cells, whereas control oligonucleotides had no such effect on either cell line. An oligonucleotide specific for the other CML breakpoint was also effective in reducing cell growth of BV173. By the use of a DNA double staining technique to discriminate between necrotic and apoptotic cells, we detected a large number of apoptotic cells in antisense treated BV173 cultures after 5 days of treatment as compared to controls. We conclude that antisense BCR-ABL oligonucleotides reduce bcr-abl mRNA expression in BV173 cells mainly in a sequence-specific manner and induce apoptosis.

Detection of point mutations in DNA using capillary electrophoresis in a polymer network.

The use of capillary electrophoresis (CE) in a polymer network for single-strand conformation polymorphism (SSCP) is investigated. SSCP is a method to detect DNA point mutations, essential in the diagnosis of several diseases. The PCR (polymerase chain reaction) amplified p53 gene, a tumour suppressor gene known to be frequently mutated in malignant cells, was subjected to CE analysis. Two single-strand DNA fragments of 372 bp in length differing in only one nucleotide could be separated. We conclude that SSCP using CE in a polymer network is a powerful method for the detection of point mutations in DNA sequences.

Proliferation patterns in acute myeloid leukemia: leukemic clonogenic growth and in vivo cell cycle kinetics.

In a prospective study of 33 newly diagnosed patients with acute myeloid leukemia (AML), we analyzed the relationship of proliferation parameters with clinical parameters, response to induction therapy, and survival. The median follow-up was 26 months. The proliferative capacity of the leukemic progenitor cells was studied using colony-forming assays (number of colony-forming units, growth pattern, and spontaneous clonogenic growth capacity). The cell kinetic parameters of the bone marrow blasts were determined by in vivo labeling with iododeoxyuridine and subsequent flow cytometry: labeling index (LI), DNA synthesis time (Ts), potential doubling time. No or only weak relationships were observed between the experimental and clinical parameters such as age, sex, % blasts, white blood cell count, FAB subtype, cytogenetics, and % CD 34+ cells. This suggests that clonogenic growth and cell cycle kinetics of bone marrow blasts are independent cell biologic properties of AML. No association between the proliferation parameters and induction response rate was noticed. Analysis of the overall survival and event-free survival revealed trends to longer survival rates in patients with a below-median LI (< or = 7.6%) and below-median Ts value (< or = 14.3 h). These trends were more pronounced in the group of de novo AML (n = 23), where the prolonged event-free survival in patients with below-median Ts reached statistical significance (p = 0.02). None of the other parameters appeared significantly correlated with survival, although there was a trend to longer survival rates in patients who had no spontaneous clonogenic growth capacity (p = 0.13). In conclusion, proliferation parameters in leukemic cells provide additional information on the cell biologic characteristics of AML, and these parameters may have prognostic value for response and duration of survival in AML.

Cell cycle kinetics in malignant lymphoma studied with in vivo iododeoxyuridine administration, nuclear Ki-67 staining, and flow cytometry.

Cell cycle kinetics of malignant lymphoma were investigated using in vivo labeling with iododeoxyuridine (IdUrd) and subsequent flow cytometry (FCM) of IdUrd/DNA and Ki-67/DNA. This approach provides an extensive cell kinetic profile from only one single tumor biopsy, including data upon the percentage of S-phase cells, the IdUrd labeling index (LI), Ki-67-derived growth fraction, duration of the S-phase, duration of the G1-phase, potential doubling time, cell production rate, and total cell cycle time. Tissue samples from 33 patients were studied: non-Hodgkin's lymphoma (NHL; n = 22), Hodgkin's disease (HD; n = 7), and reactive hyperplasia (n = 4). In NHL, the percentage of S-phase cells, LI, growth fraction, duration of the S-phase, and cell production rate were significantly correlated with the histologic malignancy grade according to the Working Formulation (P < or = .02). Data found in HD were not essentially different from those in low-grade NHL and reactive hyperplasia. Remarkably, the duration of the S-phase, the duration of the G1-phase, and the total cell cycle time appeared to be rather independent of histologic malignancy grade within the NHL category. A significant correlation was observed between the IdUrd LI and the percentage of S-phase cells, the growth fraction, the potential doubling time, and the cell production rate (P < .001), but not with the duration of the separate cell cycle phases (P > .05). Our data show (1) that it is feasible to obtain detailed information on the in vivo growth characteristics of malignant lymphoma; and (2) that the transition time through the different cell cycle phases widely varies, even within distinct histologic subgroups.

Cytotoxicity of CD3-ricin A chain immunotoxins in relation to cellular uptake and degradation kinetics.

The cytotoxicity of WT32 (CD3)-ricin A immunotoxin (IT) to the acute lymphoblastic leukemia T-cell line Jurkat was compared with the rate of internalization and intracellular degradation of WT32 and WT32-ricin A during continuous exposure. Moreover, the influence of NH4Cl and monensin on these processes was studied. Based on protein synthesis inhibition ([3H]leucine incorporation), it appeared that cytotoxicity was not fully expressed directly after exposure to IT due to a delay in either the internalization of membrane-bound IT or the action of intracellular ricin A. Varying the duration of incubation and postponing [3H]leucine addition for up to 24 h after initiation showed that cytotoxicity occurred in two phases, rapid internalization of initially bound IT followed by a continuous but slower uptake, possibly due to reexpression of the CD3 antigen. No differences were found in the rate of internalization and degradation of 125I-labeled WT32 and WT32-ricin A. Internalization started rapidly after binding at 37 degrees C, was fastest during the first 12 h (+/- 360,000 molecules/cell), and continued for at least 24 h (+/- 420,000 molecules/cell). Exocytosis of intracellularly degraded molecules became measurable after 1 to 2 h of incubation at 37 degrees C and increased to approximately 400,000 molecules/cell in 24 h. After 4 h of incubation at 37 degrees C the number of internalized molecules exceeded the amount of WT32 that could maximally bind to the cell membrane (+/- 150,000 molecules/cell), confirming reexpression of antigen. The addition of NH4Cl and monensin enhanced the cytotoxicity of WT32-ricin A, probably due to an increased intracellular amount of IT. These agents appeared to reduce strongly the degradation of internalized WT32, resulting in an accumulation of intracellular molecules. NH4Cl was most effective during the first 12 h of incubation, whereas monensin increased the amount of intracellular WT32 molecules after 2 to 24 h. Our observations suggest that incubation conditions for the optimal cytotoxicity of IT treatment can be predicted by studying the internalization and degradation of the IT or respective monoclonal antibody.

Simultaneous measurement of DNA content and cell-surface immunofluorescence of human bone marrow cells using a single laser flow cytometer.

This paper describes the measurement of S phase DNA content in human bone marrow subpopulations using a single laser method for bivariate analysis of DNA content and cell-surface immunofluorescence (s-IF). Low density (less than 1.077 g/ml) bone marrow cells were labeled with a panel of unconjugated monoclonal antibodies (MoAb) for the lymphoid (CD2 + CD19), T-lymphoid (CD2), B-lymphoid (CD19), erythroid (anti-glycophorin-A), myelomonocytic (CD13, CD33; single and as cocktail) and monocytic (CD14) lineages. A fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse label was used as second step. Unfixed, MoAb-labeled cells were incubated for 24 h with a hypotonic propidium iodide solution for DNA staining. Cells were analysed on a single-laser flow cytometer, operating at 488 nm. The effect of the combined staining protocol upon both s-IF and DNA stainability was evaluated. Only a slight decrease (mean: 29.0%) in s-IF intensity was observed after DNA staining. The percentages of immunofluorescent cells in the bone marrow samples of 10 normal individuals before and after DNA staining were essentially unchanged for all the MoAbs used. The DNA histograms of the immunophenotypically defined subpopulations were of excellent quality with a mean coefficient or variation of 1.8%. This procedure allows the assessment of very low levels of S-phase DNA content, as measured in normal low density blood cells of 8 healthy volunteers (mean 0.07%).(ABSTRACT TRUNCATED AT 250 WORDS)

In vivo and in vitro pharmacokinetic differences between four structurally closely related anthracyclines in hematopoietic cell subtypes in humans.

Minor differences in chemical structure of adriamycin (ADM), 4'-epiadriamycin (E-ADM), daunomycin (DNM), and 4-demethoxydaunomycin (D-DNM) lead to large differences between cellular and plasma pharmacokinetic parameters in vivo, as well as in cellular drug handling. Anthracyclines accumulated in cells to several hundred-fold the plasma concentration. Half-lives, as well as the ratio of parent drug/metabolite, differed markedly. The slopes of the in vivo cellular concentration-time curves after the end of the bolus injection resembled the efflux curves observed after a 5-min exposure in vitro. In vivo, the area under the cellular concentration-time curve (AUCc) for equimolar dosages was largest for ADM and smallest for D-DNM. In vitro however, cellular drug levels and AUCc were highest for D-DNM, followed by DNM, E-ADM, and ADM. Final cellular drug concentrations were 300-2500 times the medium concentration, with clearly higher values observed after the 360-min exposure. Minor structural differences were related to considerable variations in cellular drug handling, with different patterns in vivo and in vitro. These studies point to difficulties occurring in the in vitro experimental studies of in vivo pharmacokinetic properties of anthracyclines and stress the need for direct determination of target cell drug concentrations in vivo, in the search for the understanding of cell drug handling-related mechanisms of action of the anthracyclines.

In vivo cellular adriamycin concentrations related to growth inhibition of normal and leukemic human bone marrow cells.

Inhibition of clonogenicity of normal and leukemic human hematopoietic progenitor cells was studied after in vivo and in vitro exposure of bone marrow to adriamycin (ADM). Flow cytometric determination of cellular ADM concentrations in blast cells, expressed in fluorescence units/cell (FU/cell), correlated well with the extent of cytotoxicity. After 2 h in vitro exposure to 500 ng ADM/ml, the ADM concentration of leukemic (n = 7) and normal (n = 4) bone marrow blast cells amounted to 231 +/- 180 and 249 +/- 53 FU/cell respectively, producing moderate decreases in clonogenicity by 44 +/- 30 and 54 +/- 27%. Exposure to 2000 ng/ml produced ADM concentrations of 1184 +/- 472 FU/cell for leukemic blast cells and 1024 +/- 281 FU/cell for normal blast cells. Inhibition of clonogenicity was 96 +/- 7% in leukemic blasts and 99 +/- 1% in normal blasts. In vivo ADM concentrations in leukemic blast cells at 1-2 h after administration were 216 +/- 98 FU/cell (n = 8 patients). This implies that inhibition of clonogenicity after administration of conventional dosages of ADM will be approx. 60-70% for both leukemic and normal bone marrow progenitor cells. Such values were noted in four patients of whom bone marrow was cultured, which was obtained shortly after ADM monotherapy.

Leukemic cell and plasma daunomycin concentrations after bolus injection and 72 h infusion.

The effect of the duration of daunomycin (DNM) infusion on leukemic cell drug concentrations was evaluated. Cellular and plasma DNM concentrations were measured in 20 patients with acute non-lymphocytic leukemia. DNM 45 mg/m2 was administered either as a bolus injection or as a 4-, 8- or 72-h constant-rate infusion during 3 consecutive days. Peak plasma DNM levels amounted to 227 +/- 116 ng/ml after bolus injection and were only 16 +/- 6 ng/ml after 72-h DNM infusions. Terminal plasma DNM half-lives were 14 +/- 4 h. Peak leukemic cell DNM concentrations at the 3rd day of administration were 16810 +/- 2580 ng/10(9) cells (bolus injections) and 10310 +/- 5510 ng/10(9) cells (72-h infusions). The areas under the cellular curve were similar and independent of the duration of the DNM infusion. Peak leukemic cell daunomycinol (DNMol) concentrations were respectively 3500 +/- 1600 ng/10(9) cells and 2850 +/- 1720 ng/10(9) cells. Cellular DNM terminal half-life was 13 +/- 4 h. DNM concentrations in nucleated blood and bone marrow cells correlated well (r = 0.93, n = 26). Long-term infusion produced less severe side effects. Therapeutic efficacy was maintained during long-term infusion.

Cellular and plasma adriamycin concentrations in long-term infusion therapy of leukemia patients.

To determine whether long-term adriamycin (ADM) infusions resulted in cellular ADM concentrations at least comparable to those observed after bolus injections, ADM cellular and plasma concentrations were measured in 18 patients with leukemia. ADM was administered at 30 mg/m2 per day for 3 days, either as bolus injections or as 4-, 8-, or 72-h infusions. Negligible accumulation of plasma ADM was observed. Peak plasma ADM concentrations after bolus injections were 1640 +/- 470 ng/ml (n = 7). Maximum levels were 176 +/- 34 ng/ml during 4-h infusion (n = 5); 85 +/- 50 ng/ml during 8-h infusion (n = 4); and 47 +/- 5 ng/ml (n = 2) after 72-h infusion. ADM concentrations in nucleated blood and bone marrow cells correlated well (r = 0.82, n = 47). ADM accumulated in leukemic cells up to 30-100 times the plasma concentrations. The shorter the administration time-span, the higher the peak leukemic cell concentration and the greater the loss of drug immediately after the end of the administration. The final cellular ADM half-life was approximately 85-110 h. After long-term infusion and bolus injection of the same dose, similar areas under the curve for plasma or leukemic blast cell ADM concentrations were attained. Since comparable therapeutic efficacy was observed in all regimens, the antileukemic effect appeared not to be related to the peak plasma concentrations, while acute toxicity phenomena decreased with increasing duration of the infusion. Long-term ADM infusion deserves more attention in the treatment of patients with anthracyclines.

Plasma and human leukemic cell pharmacokinetics of oral and intravenous 4-demethoxydaunomycin.

On 3 consecutive days, 4-demethoxydaunomycin (D-DNM) was administered orally (30 mg/m2) as bolus injection and 4- or 24-hour infusion to seven patients with acute leukemia. Cellular (nucleated blood and bone marrow cells) and plasma drug concentrations were studied. After bolus injection, peak plasma D-DNM concentrations were about 50 mg/ml. D-DNM plasma t1/2s were 0.4 +/- 0.3 hours (T1/2 alpha) and 16.4 +/- 4.7 hours (T1/2 beta). D-DNM concentrations in nucleated blood and bone marrow cells were on the same order of magnitude and amounted to more than 400 times the plasma concentration, whereas 4-demethoxydaunomycinol (D-DNMol) concentrations were about 200 times higher. Cellular D-DNM concentrations were maximal at the end of intravenous dosing and at 2 to 4 hours after D-DNM ingestion. D-DNMol concentrations increased more slowly and accumulated on subsequent treatment days in cells and plasma; D-DNM and D-DNMol cellular t1/2 times were 42 and 72 hours, respectively. Antileukemic activity was observed.

Quantitation of anthracyclines in human hematopoietic cell subpopulations by flow cytometry correlated with high pressure liquid chromatography.

The major white cell subpopulations present in bone marrow and peripheral blood can be discriminated by forward and perpendicular light scatter two-parameter flow cytometry (FCM). Fluorescent properties of anthracycline antibiotics allow measurement of the concentration of these cytotoxic drugs in hematopoietic cells by FCM as a third parameter. Analysis of scatter-gated fluorescence histograms provides quantitative information about the cellular concentration of at least four cell categories in human blood and bone marrow cells. A good correlation was found between the mean cellular fluorescence measured by FCM and the overall cellular concentration of adriamycin, daunomycin, and their main metabolites determined with high-pressure liquid chromatography (HPLC). In incubation experiments with human hematopoietic tissues, the final concentration of various anthracyclines in subpopulations of white cells appeared to be dependent on cell density, incubation time, temperature, and type of compound and its concentration. FCM analysis is a rapid, sensitive, and quantitative method for measurement of cellular anthracycline concentrations in subpopulations and therefore provides an useful new tool in monitoring chemotherapy.

Incorporation of nucleic acid and protein precursors in enriched populations of proliferating normal and leukemic cells.

Nucleic acid and protein synthesis was determined in immature myeloid cells isolated from seven normal bone marrow samples and compared to blasts from nine patients with fullblown acute non-lymphocytic leukemia. Samples obtained from normal bone marrow contained a mean of 82% immature myeloid cells with 29.9% S-phase cells. These cells incorporated [3H]thymidine with a mean of 17,500 counts/min [3H]uridine with a mean of 24,000 counts/min and [3H]leucine with a mean of 2100 counts/min per 0.2 x 10(6) cells. Leukemic bone marrow cells could be separated in fractions with different proliferative activities. Leukemic samples with a mean of 3.6% S-phase cells incorporated [3H]thymidine with a mean of 1100 counts/min, [3H]uridine with a mean of 15,700 counts/min and ]3H]leucine with a mean of 2600 counts/min per 0.2 x 10(6) cells. For leukemic samples with a mean of 30.6% S-phase cells these values were: [3H]thymidine 22,200 counts/min, [3H]uridine 49,700 counts/min and [3H]leucine 6700 counts min per 0.2 x 10(6) cells. The incorporation studies were carried out for the first time in normal and leukemic cells with a comparable proliferative activity. Non-lymphocytic leukemic blastic cells showed a two-fold increase in RNA synthesis and a three-fold increase in protein synthesis compared to enriched samples of normal early myeloid cells with the same proliferative activity.

The influence of splenectomy on the invitro lymphocyte response to phytohemagglutinin and pokeweed mitogen in Hodgkin's disease.

Using tissue culture techniques, the 14C-thymidine incorporation of peripheral lymphocytes in 17 Hodgkin's patients was tested before and after splenectomy under stimulation with phytohemagglutinin and pokeweed. Incorporation under phytohemagglutinin stimulation about 10 days after splenectomy was not affected in Hodgkin's patients with pathologic Stages I and II, but was significantly (p less than 0.005) increased in those with Stages III and IV. The total PHA stimulation potency, i.e. the product of the lymphocyte count and PHA stimulation, increased slightly in both groups. Incorporation under pokeweed stimulation after splenectomy did not significantly differ from that before the operation in the two groups. Although the number of cases studied is rather small, it is concluded that splenectomy causes no demonstrable untoward effect on the cellular immunologic potency. The immunologic state is more likely to be favorably influenced than unfavorably.