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D D Ross

Publications and source records attributed to D D Ross.

At least 55 records · Page 3Linked to original sources
PubMed1994

Synergistic reversal of multidrug-resistance phenotype in acute myeloid leukemia cells by cyclosporin A and cremophor EL.

Cremophor (Crem) EL, the vehicle for intravenous delivery of cyclosporin A (CsA), has been reported to counteract multidrug resistance (MDR) in P-glycoprotein (Pgp)-over-expressing cell lines. Because of this, we sought to determine whether Crem functions independently as a modulator of MDR in blast cells obtained from acute myelogenous leukemia (AML) patients, and the nature of its interaction in combination with CsA in reversing an MDR phenotype. In the phenotypically classical MDR AML cell lines HL-60/Vinc (overexpresses Pgp) or HL-60/AR (does not overexpress Pgp), the dose causing half-maximum enhancement (D50) of daunorubicin (DNR, 1 micrograms/mL, 3 hours) accumulation was achieved by the combination of CsA and Crem (CsA/Crem) at 1.2 mumol/L CsA. In contrast, the D50 for Crem alone was approached at an amount that would be needed to suspend 6.2 mumol/L CsA for HL-60/Vinc, and 81 mumol/L CsA for HL-60/AR. The D50 concentrations for CsA alone (dissolved in ethanol, which does not alter DNR accumulation) were also higher than those for CsA/Crem, being 6.5 mumol/L for HL-60/Vinc, and 3.1 mumol/L for HL-60/AR. The maximum absolute level of enhancement of DNR accumulation (Emax) in each cell line was approximately equivalent for CsA/Crem or CsA alone, and was equal to the 3 hr intracellular DNR accumulation observed in parental, drug sensitive HL-60/W cells. For Crem alone, HL-60/AR and HL-60/Vinc cells showed markedly different responses: HL-60/Vinc cells attained intracellular DNR content comparable to HL-60/W, whereas HL-60/AR cells achieved only approximately 35% of this level. Multiple-drug effects were analyzed by calculation of the Combination Index (Chou and Talalay, Adv Enzyme Regul 22:27, 1984), which indicated that CsA and Crem are synergistic in causing enhancement of DNR accumulation in these MDR HL-60 cell lines. In blasts from AML patients, 5 mumol/L CsA/Crem or an equivalent amount of Crem alone each caused significant (P < .001) enhancement of DNR accumulation (60 AML-patient marrow samples) or DNR retention (51 AML-patient marrows). Similarly, CsA/Crem or Crem alone caused significant (P < .01) enhancement of the cytotoxicity of DNR in 36 AML blast cell specimens. The degree of enhancement of accumulation/retention or cytotoxicity by CsA/Crem was approximately equivalent to that obtained with Crem alone. These studies indicate that Crem can reverse an MDR phenotype in patient AML blast cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Cell Survival↗
PubMed1993

Enhancement of daunorubicin accumulation, retention, and cytotoxicity by verapamil or cyclosporin A in blast cells from patients with previously untreated acute myeloid leukemia.

This work was designed to discern the frequency of expression of classical multidrug resistance (MDR) in acute myeloid leukemia (AML) at the time of diagnosis, using Western blotting for P-glycoprotein (Pgp) and functional assays for an MDR phenotype (enhancement of daunorubicin [DNR] accumulation/retention and cytotoxicity by the known MDR modulators verapamil, cyclosporin A, and progesterone). Blast cells were studied from 49 newly diagnosed AML patients who were subsequently treated with the "3 and 7" combination of cytosine arabinoside (ara-C) and DNR. DNR accumulation (1 microgram/mL, 3 hours) and retention (16 hours) were determined by flow cytometry. Cyclosporin A (CsA, 5 mumol/L) or verapamil (6.6 mumol/L) each caused significant enhancement of DNR accumulation and retention in these blast cell samples (P < .001, Wilcoxon's test). Verapamil or CsA caused greater than 20% enhancement of DNR accumulation or retention in over 25% or 50% of these patients, respectively; however, there was no correlation with the presence or degree of enhancement and response to treatment. Progesterone (10 mumol/L) caused no significant enhancement of DNR accumulation or retention. The effects of the MDR modulators on the cytotoxicity of DNR was also determined in blast cells from 40 of the patients, using a flow cytometric assay. CsA alone was cytotoxic (caused an approximate 20% decrease in cell survival compared with control, P < .001); CsA or verapamil caused enhancement of 1 mumol/L DNR cytotoxicity (P < .001). Greater than 40% enhancement of cell kill by CsA or verapamil was observed in over 75% of patients studied. There was no difference in the degree of enhancement of cytotoxicity between patients clinically sensitive or resistant to treatment. Progesterone caused no enhancement in DNR cytotoxicity. In contrast to the functional assays, highly sensitive immunoblots using the C219 antibody to Pgp showed evidence of low level expression of Pgp in blast cells from only 3 of these patients: 1 was chemotherapy resistant, 2 were sensitive. Thus, although the functional assays suggest a high frequency of expression of a classic MDR phenotype in AML patients at the time of diagnosis, with enhancement by CsA obtained at a clinically relevant concentration (5 mumol/L), the frequency of Pgp expression detectable by C219 Western blots was low in these patients. This could be interpreted either that the method used was not sufficiently sensitive to detect Pgp in all of the blast cell specimens that actually overexpressed mdr1, or that the accumulation/efflux-based MDR phenotype observed is not always mediated by Pgp in these previously untreated patients.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1992

Mechanistic implications of alterations in HL-60 cell nascent DNA after exposure to 1-beta-D-arabinofuranosylcytosine.

To improve our understanding of the mechanism of 1-beta-D-arabinofuranosylcytosine (ara-C) incorporation into DNA, we investigated the physical properties (size, position of nucleoside incorporation) of small fragments of nascent DNA (nDNA) obtained by pH-step alkaline elution of intact HL-60 cells following their exposure to ara-C. In the pH-step alkaline elution procedure, the smallest fragments of nDNA elute at pH 11. Anion-exchange high-performance liquid chromatography (HPLC) of nDNA obtained by 1 h elution at pH 11.0 of lysed HL-60 cells revealed a preponderance of nDNA fragments ranging from 0.5 to 40 kb in control ([3H]-dThd-labeled) cells. Exposure of cells to ara-C (0.8-1 microM) resulted in a loss of the preponderance of radiolabel in fragments of 0.5-40 kb along with redistribution of the radiolabel (from [3H]-dThd or [3H]-ara-C) into smaller nDNA fragments (predominantly < 100 bases in length) as determined by HPLC. We used the ability of pH-step alkaline elution to provide these small nDNA fragments produced by ara-C to investigate the paradoxical behavior of ara-C as a chain terminator in cell-free DNA synthetic systems while being incorporated into an internucleotide position in intact cells. Following the digestion of purified nDNA with micrococcal nuclease and spleen phosphodiesterase II, the proportion of radiolabel in 3'-dNMP (indicating an internucleotide position) or free nucleoside (indicating a chain terminus position) was determined by reverse-phase HPLC. In digests of prelabeled genomic DNA, as expected, > 90% of the radiolabel from [14C]-dThd or [3H]-ara-C was found to exist in an internucleotide position (as determined by co-chromatography with authentic 3'-dTMP or 3'-ara-CMP). In contrast, digests of nDNA that eluted at pH 11.0 revealed a significantly higher proportion of radiolabel in the chain terminus position (29%-35%) when the nDNA was obtained from cells exposed to 1 microM [3H]-ara-C as compared with cells exposed to [3H]-dThd or [3H]-dCyd alone (< 10%). These data obtained from pH-step alkaline elution of intact cells suggest that by causing the inhibition of chain elongation while failing to inhibit the formation of new nDNA replication intermediates, ara-C exposure leads to the production of very small nDNA fragments. This relative chain-terminating effect of ara-C is most apparent in the small nDNA replication fragments that elute at pH 11.0.(ABSTRACT TRUNCATED AT 400 WORDS)

Carbon Radioisotopes↗
PubMed1991

Interaction of N,N',N''-triethylenethiophosphoramide and N,N',N''-triethylenephosphoramide with cellular DNA.

The antineoplastic agents N,N',N''-triethylenethiophosphoramide (thioTEPA) and N,N',N''-triethylenephosphoramide (TEPA) were studied for their interaction with the DNA of L1210 cells in the presence and absence of rat hepatic microsomes and NADPH. Alkaline elution was used to study 3 types of DNA lesions. When L1210 cells were incubated with thioTEPA alone, or with thioTEPA in the presence of microsomes and NADPH, no single-strand breaks were detected. However, incubation of L1210 cells for 2 h with thioTEPA, at concentrations greater than or equal to 100 microM, caused a dose-dependent increase in interstrand cross-linking that reached a maximum by 2 h after drug exposure. In the presence of rat hepatic microsomes and NADPH, this cross-linking was eliminated, but a different DNA lesion, alkali-labile sites, was produced. These alkali-labile sites were partially reparable with maximum repair achieved by 2 h after removal of drug. ThioTEPA was greater than 85% consumed by the microsomal incubation conditions employed, and TEPA was the only product of the microsomal metabolism of thioTEPA. Alkaline elution studies of L1210 cells that had been incubated with TEPA, alone or in the presence of microsomes and NADPH, demonstrated an elution pattern identical to that produced by thioTEPA in the presence of microsomes and NADPH. Lymphoblastoid cell lines derived from patients with Fanconi's anemia were far more sensitive to thioTEPA and mechlorethamine hydrochloride than were lymphoblasts derived from normal humans, but this hypersensitivity was not noted with TEPA or bleomycin. This is consistent with the known hypersensitivity of cells from patients with Fanconi's anemia to agents that produce interstrand cross-links and with the alkaline elution studies described above. In contrast, lymphoblastoid cell lines derived from patients with ataxia telangiectasia were no more sensitive to thioTEPA than were lymphoblasts derived from normal humans but were far more sensitive to bleomycin. One of these cell lines proved hypersensitive to TEPA, whereas the other was no more sensitive to TEPA than were lymphoblasts from normal humans. Our data imply that thioTEPA produces interstrand cross-links but that TEPA, the primary metabolite of thioTEPA, produces DNA lesions that are alkali labile.

Animals↗
PubMed1991

Use of plasma cytotoxic activity to model cytotoxic pharmacodynamics of anticancer drugs.

We have developed a pharmacokinetic/pharmacodynamic approach that integrates the disposition, cytotoxic activity and interaction of anticancer drugs. Fundamental to this approach is the measurement of the cytotoxicity, against a "target" cell line, of patient plasma collected at different times after administration of the anticancer agent(s). To illustrate this approach, we have studied the plasma cytotoxic activity (PCA), against HL-60 cells, of plasma from 11 acute myeloblastic leukemic patients treated with daunorubicin (DNR). Plasma, obtained before and serially for 24 h after DNR treatment, was assayed by HPLC for DNR and daunorubicinol (DNRol), its active metabolite. The corresponding observed PCA values (PCAobs) against HL-60 cells were also measured with a flow-cytometric cell-survival assay that we had developed previously. The pharmacodynamics, i.e. PCA, were co-modeled (dual Hill equation with an interaction term to allow synergism or antagonism) with the pharmacokinetics. The integration of the PCA profile provided the area under the observed PCA versus time curve (AUCobs). For each patient, we also generated an "interaction panel", by adding known amounts of DNR and DNRol to his or her pretreatment plasma. The corresponding cytotoxicities were measured, and then applied to the pharmacodynamic model. This provided a standard surface from which the PCA of each sample obtained after therapy was predicted (PCAprd), on the basis of assayed concentrations of DNR and DNRol in that sample. For plasma samples obtained after treatment, the model simultaneously fit all three outputs, i.e. PCA and DNR/DNRol concentration, very well. We observed substantial interpatient variability in HL-60 growth rate in medium containing patient pretreatment plasma, in DNR activity in pretreatment plasma, and in the in vitro activity (PCA) of plasma obtained after DNR treatment. We also compared the AUCprd to the AUCobs for each patient, and we identified a subset of 4/11 acute myeloblastic leukemic patients who had developed much more PCA after DNR administration that could be explained by the measured concentrations of DNR and DNRol. This may be due to unidentified active metabolites or to factors produced in the plasma in response to the treatment. This pharmacokinetic/pharmacodynamic model is promising to describe pharmacodynamics and interactions of anticancer drugs in cancer patients.

Adult↗
PubMed1991

Membrane glycoprotein changes associated with anthracycline resistance in HL-60 cells.

The glycoproteins on the surface of HL-60/S wild-type, drug-sensitive human leukemia cells and HL-60/AR anthracycline-resistant cells which do not overexpress the P-glycoprotein, were characterized by labeling with [35S]-methionine, NaB[3H4], phosphorus 32, or sodium iodide I 125. HL-60/S and HL-60/AR cell lysates and membrane fractions tagged with [35S]-methionine or phosphorus 32 showed no significant differences in their protein patterns as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and by autoradiography. HL-60/S cells labeled with NaB[3H4] yielded glycoproteins that were smeared predominantly in the molecular-weight range of 210,000 and 160,000 Da, with pI values ranging between pH 4 and pH 4.4. In contrast, NaB[3H4]-labeled HL-60/AR cells showed 7-8 discrete glycoproteins within a molecular-weight range of 170,000 and 140,000 Da, with pI values also ranging between pH 4 and pH 4.4. In addition, [3H]-glucosamine incorporation into HL-60/S and HL-60/AR cells revealed that the latter showed lower uptake of [3H]-glucosamine than did the former. Following treatment with tunicamycin, [3H]-glucosamine uptake in HL-60/S cells decreased, whereas that in HL-60/AR cells remained unchanged. Surface-membrane radioiodination of HL-60/S and HL-60/AR cells showed two distinct protein electrophoretic patterns, with differences being observed in both the high-(220-95 kDa) and low-molecular-weight ranges (21 kDa). Flow cytometric analysis of HL-60/S and HL-60/AR cells using myeloid and lymphoid antigen-specific antibodies demonstrated no antigenic differences between HL-60/S and HL-60/AR cells. HL-60/S cells incubated in the presence of tunicamycin, an inhibitor of N-linked glycosylation, or the protein kinase C agonist phorbol 12-myristate 13-acetate (PMA) developed a glycoprotein pattern similar to that observed in HL-60/AR cells. In addition, tunicamycin treatment of HL-60/S cells decreased daunorubicin (DNR) retention and altered its intracellular distribution as compared with that in HL-60/AR cells. These data indicate that HL-60/AR cells do not possess either de novo or amplified high-molecular-weight surface-membrane proteins; instead, existing proteins are hypoglycosylated. These results also show that HL-60/AR cells exhibit the multidrug-resistant phenotype in association with altered membrane glycoproteins of both high (220-95 kDa) and low molecular weight (21 kDa), but without overexpression of the P-glycoprotein. Furthermore, in HL-60/S cells, the multidrug-resistant phenotype is partially inducible by inhibition of N-linked glycosylation of cell-surface proteins.

Antibiotics, Antineoplastic↗
PubMed1991

Cellular and pharmacologic aspects of drug resistance in acute myeloid leukemia.

Modern treatments and supportive care regimens for acute myeloid leukemia have produced some cures of what was once a uniformly fatal disease. To enhance the cure rate, intense efforts are now being put forth to understand the mechanisms of resistance to chemotherapeutic regimens that actually arise in clinical cases of acute myeloid leukemia, and to develop ways and means to circumvent such resistance. This review focuses on such efforts involving daunorubicin and cytarabine, the two most effective agents currently available for treatment of the disease. Most current published studies of daunorubicin have focused on detecting the classic form of multidrug resistance. For cytarabine, pharmacologically directed treatment approaches and enhancement of cytarabine leukemic cell kill by increasing the distribution of cells in S-phase and manipulating cellular cytarabine transport are currently under investigation.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1990

Effects of 1-beta-D-arabinofuranosylcytosine on DNA replication intermediates monitored by pH-step alkaline elution.

The pH-step alkaline elution method enables the isolation and quantification of nascent DNA (nDNA) replication intermediates, including Okazaki fragments, short length nDNA from replicon origins, longer lengths of nascent but subgenomic length nDNA (molecular weight, 20-30 x 10(6)), and full (or genomic) length nDNA (L. C. Erickson et al., Chromosoma, 74: 125-139, 1979). We utilized this technique to study, in HL-60 cells, the effects of 1-beta-D-arabinofuranosylcytosine (ara-C) on the formation of these replication intermediates and the kinetics of transit of radiolabel from [3H]thymidine ([3H]dThd) or [3H]-ara-C through these nDNA fragments and into full length nDNA. In the continuous presence of [3H]-ara-C (4 microM), the majority of radiolabel (greater than 85%) remained in the nascent subgenomic fractions, with 30-50% remaining in Okazaki fragments. These proportions did not change substantially with increasing time of exposure to [3H]-ara-C (from 1 to 24 h), although the total amount of [3H]-ara-C incorporated into DNA continued to increase with increasing time of exposure. In contrast, when cells were exposed to [3H]-ara-C for 1 h, placed in drug-free medium, and studied by the pH-step method at various times thereafter, the transit of radiolabel through progressively larger nDNA intermediates and into full length nDNA was rapid and equal to that of [3H]dThd in cells not exposed to drug. The observed elution of [3H]-ara-C in the subgenomic-length DNA fragments was not due to ara-C-induced breaks in template (parental) DNA and subsequent incorporation of [3H]-ara-C into the template strand, since ara-C treatment of cells prelabeled with [14C]dThd failed to cause substantial elution of the 14C label at the various pH steps used. In studies of the effects of ara-C on [3H]dThd incorporation into nDNA, concentrations of 1 to 10 microM ara-C inhibited total incorporation of radiolabel into DNA by greater than 90% and incorporation into full length nDNA by greater than 97%. In contrast, these concentrations of ara-C failed to decrease the amount of [3H]dThd incorporated into Okazaki fragments or other non-mitochondrial low molecular weight nDNA, compared to control. These studies demonstrate that, in HL-60 cells, ara-C causes profound inhibition of nascent chain elongation, does not cause chain termination, and does not inhibit initiation. In fact, ara-C may stimulate initiation, leading credence to recent theories proposing endoreduplication or reinitiation as consequences of ara-C incorporation into DNA.

Arabinonucleotides↗
PubMed1989

Estimation of cell survival by flow cytometric quantification of fluorescein diacetate/propidium iodide viable cell number.

We report a flow cytometric method to quantify the number of viable cells remaining in suspension culture following exposure to cytotoxic drugs. Cell viability is assessed by flow cytometric measurement of cellular fluorescence after staining with fluorescein diacetate and propidium iodide in isotonic solution. The number of viable cells per ml of culture is determined by a timed count of viable cells and from knowledge of the flow cytometer sample flow rate. P388 murine or HL-60 human leukemia cells in culture were used as model systems. This method can quantify accurately viable cell concentrations in suspension culture from 100 cells/ml to 1 million cells/ml. The sensitivity of the method as a cytotoxicity assay increases if, following brief (1-4-h) exposure to drug, greater time is allowed for cell death and lysis to occur prior to flow cytometric counting of viable cells. If the viability assessment is deferred for at least 72 h following drug (daunorubicin, actinomycin D, vincristine) exposure, results were obtained approximating those obtained from the soft agar clonogenic assay or the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. In studying the cytotoxic effects of vincristine, actinomycin D, 1-beta-D-arabinofuranosylcytosine, and daunorubicin on P388 or HL-60 cells sensitive and resistant to these agents, reasonable results were obtained by flow cytometric counting of viable cell number. We have been able to perform this flow cytometric viability assay with ease using bone marrow blast cells obtained from patients with acute myelogenous leukemia. The method is facile, relatively rapid, and since it is ideal for studying cells in suspension culture, its potential as a predictor of chemotherapeutic response in leukemia warrants further evaluation.

Animals↗
PubMed1989

Improvement of flow-cytometric detection of multidrug-resistant cells by cell-volume normalization of intracellular daunorubicin content.

To improve the ability of flow cytometry to detect multidrug-resistant cells, we studied the extent to which cell volume heterogeneity accounts for the variance of intracellular daunorubicin (DNR) content. For P388 murine or HL-60 human leukemia cells exposed to DNR (1 micrograms/ml, 60 min), log intracellular DNR content varied in direct proportion to log cell volume measured by flow cytometry, with a correlation coefficient of .9. This relationship was confirmed by cell sorting based on intracellular DNR content with subsequent volume determination of the sorted cells. Normalization of intracellular DNR content for cell volume (thus obtaining intracellular DNR concentration) was accomplished by subtracting log cell volume from log intracellular DNR content for each cell. This resulted in a 34% decrease (range 23-58%) in standard deviation compared to DNR content measurements without volume normalization for all cell types tested. Following exposure to DNR (as above), intracellular DNR content of drug-sensitive P388 or HL-60 cells measured by flow cytometry was 12- and 8-fold greater than that of the multidrug-resistant sublines P388/ADR and HL-60/AR, respectively. However, because of the variance of intracellular DNR content, the predictive value of flow-cytometric determination of intracellular DNR content as a discriminant assay for detecting the frequency of drug-resistant cells in a mixed population was acceptable only when the frequency of resistant cells in the population exceeded 10%. In contrast, volume normalization of intracellular DNR content enhanced the ability of the flow-cytometric assay to discriminate resistant cells by 10-fold for P388 cells and 100-fold for HL-60 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Killing lung cancer cells at cell-cycle phase by a new indium-111-bleomycin complex.

The efficacy of killing small cell lung cancer (SCLC) cells at the G1, S, and G2-M phase of the cell-cycle by a new 111In-bleomycin complex (111In-BLMC) was investigated. SCLC cells (N417, H526, H209) were synchronized by double thymidine block and assessed by DNA content with flow cytometry, and the period for the maximal accumulation of cells in S, G1, or G2-M phase was determined. Cells in different cell cycle phases were exposed to 0.9% NaCl, BLM, or 111In-BLMC for 1 hour and observed for colony formation. The survival of H526 cells treated with 111In-BLMC was 71% (for enriched S phase), 46% (G1), and 31% (G2-M). For N417 cells, it was 25% (S), 20% (G1), and 8% (G2-M) for 111In-BLMC and 18% (S), 33% (G1), and 10% (G2-M) for BLM. These results indicated that SCLC cells in G2-M were most sensitive and those in S phase were least sensitive to 111In-BLMC; cells in G1 phase were the least sensitive to BLM.

Bleomycin↗
PubMed1989

Mycobacterial disease associated with aplastic anaemia.

Haematological changes associated with infectious disease are relatively common but true aplastic anaemia secondary to infection is rare. We describe a patient wit disseminated Mycobacterium avium-intracellulare infection and who had a histologically proven remission of his aplastic anemia accompanying antimycobacterial therapy. We also review the literature on the haematological changes associated with mycobacterial infections and other infectious causes of aplastic anaemia.

Aged↗
PubMed1988

Bromodeoxyuridine enhancement of 1-beta-D-arabinofuranosylcytosine metabolic activation and toxicity in HL-60 leukemic cells.

We tested whether bromodeoxyuridine (BrdUrd), an analogue of thymidine (dThd), enhances 1-beta-D-arabinofuranosylcytosine (ara-C) metabolic activation, as does dThd. HL-60 cells were exposed to 10, 100, or 1000 nM ara-C for 3 h. Simultaneous exposure of log phase HL-60 cells to BrdUrd (1-1000 microM) and ara-C for 3 h resulted in enhancement of ara-C incorporation into DNA, with a doubling of incorporation in response to 10 nM ara-C occurring at concentrations of BrdUrd greater than 100 microM. Preexposure of cells to BrdUrd for 16 h followed by addition of ara-C for 3 h resulted in even greater ara-C incorporation into DNA. This increase was most marked at the lower concentrations of ara-C (10 and 100 nM), where approximately 3-fold enhancement of ara-C incorporation was observed in response to BrdUrd concentrations greater than 100 microM. Intracellular pools of 1-beta-D-arabinofuranosyl-CTP increased significantly (up to 3-fold) following 16-h exposure to BrdUrd (30, 100, or 300 microM) at all concentrations of ara-C tested. The ara-C phosphorylating activity of cell-free extracts obtained following 16-h exposure of cells to BrdUrd increased 1.5- to 2.3-fold over control. Intracellular dCTP pools fell to approximately 50% of control after exposure to 750 microM BrdUrd or dThd. Exposure to BrdUrd for 16 h caused a concentration-dependent increase in cells with S-phase DNA content, as assessed by flow cytometry, with a doubling of cells in S phase (to 60%) observed in response to 500 microM BrdUrd. HL-60 cells exposed to identical conditions of BrdUrd for 3 h showed no significant alteration in cell cycle phase distribution. Thus, although BrdUrd does increase cells in S phase, the increased ara-C incorporation caused by BrdUrd cannot be explained solely on a cytokinetic basis since enhancement of incorporation was observed after a 3-h exposure of cells to BrdUrd and ara-C. The combination of ara-C (100 nM) and BrdUrd (100-1000 microM) exhibited cytotoxic synergism, as measured by the fluorescein diacetate/propidium iodide method. These data demonstrate a clear potential for BrdUrd modulation of ara-C metabolism in human leukemia. Additionally, the interaction of BrdUrd and ara-C should be considered in the interpretation of studies of the effects of ara-C on DNA synthesis as measured by flow cytometric quantification of incorporated BrdUrd.

Arabinofuranosylcytosine Triphosphate↗
PubMed1988

Isolation of highly multidrug-resistant P388 cells from drug-sensitive P388/S cells by flow cytometric cell sorting.

To investigate the spontaneous frequency of occurrence of stable multidrug-resistant cells in a population of drug-sensitive cells, we exposed drug sensitive P388/S cells to daunorubicin (dnr) for 1 h, then used fluorescence-activated cell sorting based on intracellular dnr fluorescence to isolate cells within P388/S having different intracellular content of drug. One of the sort windows chosen (low dnr content sort window) isolated only P388/S cells with intracellular drug content equal to or less than that of the known multidrug-resistant subline P388/adr. This sort window constituted approximately 3% of P388/S cells with lowest dnr content. By such a procedure we were able, on one of seven attempts, to isolate and cultivate stable, highly multidrug-resistant cells (comparable to that of P388/adr) from the P388/S cells obtained from the low dnr-content sort window. Net growth of cells in culture was observed 15-20 days after sorting, indicating that of the P388/S cells collected from the low dnr-content sort window, very few were actually highly drug-resistant. On no occasion could resistant cells be cultivated from cells sorted from P388/S with higher dnr content, as would be expected if mutation to a multidrug-resistant phenotype had occurred as a result of exposure to drug. The resistant cells isolated from P388/S by sorting (called P388/LoSort) displayed low intracellular accumulation of dnr that was enhanced by verapamil, were cross-resistant to vincristine and actinomycin-D, and distinct from P388/S, possessed a 150- to 160-kD membrane species identified by Vinca alkaloid photoaffinity labeling.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Metabolism of ara-C by blast cells from patients with ANLL.

The dose-response relationship between extracellular concentration of cytosine arabinoside (ara-C) and intracellular formation of the putative active metabolites of ara-C [ara-C incorporation into DNA and intracellular pools of ara-C in triphosphate form (ara-CTP)] was investigated in blast cells obtained from patients with acute nonlymphocytic leukemia (ANLL) by exposing these cells in vitro to 10, 100, or 1,000 nmol/L of ara-C. We studied 23 untreated patients who subsequently achieved complete remission (CR) with a regimen using daunorubicin and conventional doses of ara-C (ara-C-sensitive group), and 30 patients judged to be ara-C-resistant either by failing initial induction therapy (16 patients) or by having relapsed on an ara-C-containing maintenance regimen (14 patients). In both patient groups, ara-C incorporation into DNA and intracellular ara-CTP both displayed statistically significant increases in response to increasing extracellular concentrations of ara-C (P = .0001 in both cases), with the rate of increase of ara-CTP greater than that of ara-C incorporation. Moreover, blast cells from all patients, even those who were most clinically resistant to ara-C, were able to form ara-CTP and to incorporate ara-C into DNA. Each tenfold increment in extracellular ara-C concentration caused an 8.5-fold increase in ara-CTP, but only a 3.6-fold increase in ara-C incorporation into DNA. Thus, the efficiency of incorporation of ara-C into DNA (defined as the ratio of ara-C incorporation to ara-CTP pools) decreased by 58% with each tenfold increment in the extracellular concentration of ara-C (P less than .0001), presumably as a result of the inhibitory effect of ara-CTP on DNA polymerase. Using an analysis of covariance, modest differences were found in the levels of the ara-C metabolite variables in the ara-C-sensitive group as compared with the resistant group. However, because there was considerable overlap in ara-C metabolite formation among the patient groups, it was not possible to predict clinical outcome by these in vitro assessments of ara-C metabolism.

Acute Disease↗
PubMed1986

Effects of verapamil on in vitro intracellular accumulation and retention of daunorubicin in blast cells from patients with acute nonlymphocytic leukemia.

The effects of verapamil on the intracellular pharmacokinetics of daunorubicin (DNR) in blast cells from the bone marrows of patients with acute nonlymphocytic leukemia (ANLL) were studied to determine whether verapamil was capable of enhancing intracellular accumulation and retention of DNR in ANLL, as has been observed in murine P388 cells resistant to DNR. Seventeen marrows from ANLL patients were studied, 13 of which were from patients who were considered to be clinically refractory to DNR. We took care to include such patients in this study since, in the P388 model, verapamil enhancement of DNR uptake is observed only in cells resistant to DNR. Intracellular accumulation of DNR was studied by exposing blast cells to DNR (1 microgram/mL) +/- verapamil (6.6 mumol/L) for up to 4 hours. Following a 2-hour exposure of cells to DNR +/- verapamil, intracellular retention of DNR was studied by incubating the cells in DNR-free medium for 3 hours. Intracellular DNR/10(6) cells was quantified by fluorometry. In 12 of 15 patient marrows, verapamil failed to enhance intracellular accumulation of DNR. Three patient marrows had a very small increment in DNR uptake in response to verapamil (approximately 14% greater than DNR alone) that was significant (P less than .05) by paired t test. Intracellular retention of DNR (t 1/2) and the area under the intracellular DNR v time curve (AUC) were studied in 17 patients marrow specimens. No significant alterations in these parameters were observed in response to verapamil. These data indicate either that verapamil did not substantially enhance DNR uptake or retention in blast cells obtained from ANLL patients who are clinically resistant to DNR, or that the frequency of DNR-resistant cells (ie, verapamil-responsive cells) among the blast cells obtained from these patients was too low to influence the population mean of intracellular DNR as measured in these studies.

Acute Disease↗
PubMed1984

Synergistic inhibition of human leukemia cell growth by deoxyguanosine and 1-beta-D-arabinofuranosylcytosine.

We studied the ability of 2'-deoxyguanosine (dGuo) to influence 1-beta-D-arabinofuranosylcytosine (ara-C) inhibition of soft agar cloning of the cultured human leukemia cell line K562. Ara-C alone inhibited cloning in concentrations of greater than 10 nM, with a steep drop in colony formation observed between 10 and 100 nM. dGuo and ara-C synergistically inhibited cloning; the combination of ineffective concentrations of dGuo (10-50 microM) and ara-C (less than or equal to nM) inhibited cloning by 40-70%. In K562 cells, dGuo is metabolized by both nucleoside kinase and purine nucleoside phosphorylase (PNP), resulting in augmentation of both the GTP pool (to more than 200% of control after a 3 hr incubation with 500 microM dGuo) and the dGTP pool (to more than 2700% of control after 3 hr with 500 microM dGuo). dGuo (50-500 microM) caused a decrease in the dCTP and dTTP pools and an increase in the dATP pool. Synergistic concentrations of dGuo plus 10 nM ara-C augmented the ara-CTP pool up to 800% of control after 3 hr to levels equivalent to those observed after incubation with 500 nM ara-C alone. Incorporation of 10 nM ara-CTP into DNA also increased in the presence of dGuo (up to a maximum of 300% of control), but only to a level that approximated the value observed with nM ara-C alone. The disparity between enlargement of the ara-CTP pool and augmentation of ara-C incorporation into DNA is consistent with the observation of Steinberg et al. [Cancer Res. 39, 4330 (1979)] that high concentrations of dGTP may inhibit DNA polymerase activity. Thus, synergy between dGuo and ara-C is multifactorial, possibly involving inhibition of DNA polymerase by elevated dGTP and ara-CTP pools and augmented incorporation of ara-C into DNA.

Cell Line↗