ß-Sitosterol modulates TLR4 receptor expression and intracellular MyD88-dependent pathway activation in J774A.1 murine macrophages.

Recent evidence has shown that dietary phytosterols (PS) possess anti-inflammatory properties both in vivo and in vitro. Our previous work shows that PS ß-Sitosterol (SIT), may function by down-regulating pro-inflammatory transcription factors NF-kB and STAT1 in response to LPS stimulation, possibly through modulation of the TLR4 pathway. The objective in this study was to determine the effects of SIT on TLR4 surface expression and localization into lipid rafts, as well as to investigate its effects on intracellular MyD88 dependent pathway activation. J774A.1 macrophages were pre-treated with cyclodextrin vehicle loaded with cholesterol or SIT, then stimulated with LPS (100 ng/ml) for 30 min. ImageStream cytometry demonstrated that SIT down-regulates TLR4 expression without affecting lipid raft distribution. Western blot demonstrated that SIT down-regulated the adaptor protein MyD88 and the activity of IRAK1 but increased SOCS3 expression. Together, these results provide evidence that SIT may indeed elicit anti-inflammatory properties by down-regulating some components of the TLR4 pathway.

Breast cancer resistance protein (BCRP/MXR/ABCG2) in acute myeloid leukemia: discordance between expression and function.

Data on breast cancer resistance protein (BCRP, MXR, ABCG2) expression in acute myeloid leukemia (AML) have been inconsistent, possibly due to use of different assays in different studies. BCRP mRNA was studied by the reverse-transcription polymerase chain reaction and BCRP protein expression (BXP-21, BXP-34 or anti-ABCG2 antibody, with anti-CD34 and anti-CD33) and function (fumitremorgin C modulation of mitoxantrone retention) by flow cytometry in eight cell lines and in pretreatment blasts from 31 AML patients. BCRP mRNA levels, antibody staining and function correlated strongly in cell lines (Pearson r values, 0.73-0.97), but not in AML samples. AML sample BCRP mRNA levels were between those in parental 8226 and 35-fold mitoxantrone-resistant 8226/MR20 cells in all but one case, and BCRP mRNA had the wild-type sequence at codon 482 in all. In AML, unlike in cell lines, BCRP protein expression or function, when present, was only detected in small subpopulations. BCRP mRNA and protein expression did not correlate, nor did staining with different BCRP antibodies, and function did not correlate with mRNA nor protein expression. Presence of BCRP only in subpopulations and discordance among BCRP measurements suggest complex biology of BCRP in AML and incomplete modeling by cell lines.

Erythropoietin-dependent transformation of myelodysplastic syndrome to acute monoblastic leukemia.

Acute monoblastic leukemia (acute myeloid leukemia [AML], French-American-British type M5a) with leukemia cutis developed in a patient 6 weeks after the initiation of erythropoietin (EPO) therapy for refractory anemia with ringed sideroblasts. AML disappeared from both marrow and skin after the discontinuation of EPO. Multiparameter flow cytometric analysis of bone marrow cells demonstrated coexpression of the EPO receptor with CD45 and CD13 on the surface of blasts. The incubation of marrow cells with EPO, compared to without, resulted in 1.3- and 1.6-fold increases, respectively, in tritiated thymidine incorporation and bromodeoxyuridine incorporation into CD13(+) cells. Clinical and laboratory findings were consistent with the EPO-dependent transformation of myelodysplastic syndrome (MDS) to AML. It is concluded that leukemic transformation in patients with MDS treated with EPO may be EPO-dependent and that management should consist of the discontinuation of EPO followed by observation, if clinically feasible.

Time-lapse video reveals immediate heterogeneity and heritable damage among human ileocecal carcinoma HCT-8 cells treated with raltitrexed (ZD1694).

BACKGROUND: Cellular heterogeneity in drug response has important clinical implications, and is believed to develop over many generations during clonal evolution in human tumors. The purpose of this study was to determine the level of heterogeneity exhibited by sister cells soon after their birth. METHODS: Human ileocecal carcinoma cells (HCT-8) were followed up to 11 days in vitro after a 2-h exposure to 1 microM raltitrexed (IC(95)) in a time-lapse video system. RESULTS: Over five experiments, 414 cells were followed after exposure to raltitrexed. Immediate sterility occurred in 74% of treated cells. Only 6% of cells could produce more than two generations of offspring, and heterogeneity in drug response was seen. Comparing sister cells < 24 h old, the more proliferative sibling produced up to 73 times more offspring, with a median ratio of 9.0 (control median = 1.19). Offspring of prolific drug-treated cells had a decreased probability of division (68% compared with 92%) and an increased average interdivision time (19.0 h compared with 15.1 h). CONCLUSIONS: Short-term exposure to raltitrexed resulted in increased interdivision times and production of sterile offspring extending seven generations. Cellular heterogeneity (difference in proliferation potential comparing drug-treated sister cells) was evident without a period of clonal evolution.

Characterization of protein kinase chk1 essential for the cell cycle checkpoint after exposure of human head and neck carcinoma A253 cells to a novel topoisomerase I inhibitor BNP1350.

Cellular topoisomerase I is an important target in cancer chemotherapy. A novel karenitecin, BNP1350, is a topoisomerase I-targeting anticancer agent with significant antitumor activity against human head and neck carcinoma A253 cells in vitro. As a basis for future clinical trials of BNP1350 in human head and neck carcinoma, in vitro studies were carried out to investigate its effect on DNA damage and cell cycle checkpoint response. The treatment of A253 cells with BNP1350 caused biphasic profiles of DNA fragmentation displayed from 0 to 48 h after 2-h exposure. Pulsed-field gel electrophoresis demonstrated that the first wave of DNA damage was mainly megabase DNA fragmentation, but the second wave of DNA damage was 50- to 300-kb DNA fragmentation in addition to megabase DNA damage. The cell cycle checkpoint response was characterized after exposure to 0.07 and 0.7 microM concentrations of BNP1350, the IC(50) and IC(90) values, respectively. After exposure to a low concentration of BNP1350 (IC(50)), A253 cells accumulated primarily in G(2) phase. In contrast, treatment with a high concentration of BNP1350 (IC(90)) resulted in S phase accumulation. The concentration-associated cell cycle perturbation by BNP1350 was correlated with different profiles of cell cycle-regulatory protein expression. When treated with the low concentration of BNP1350, cyclin B/cdc2 protein expression was up-regulated, whereas with the high concentration, no significant change was observed at 24 and 48 h. In addition, increased phosphorylation of a G(2) checkpoint kinase chk1 was observed when cells were treated with a low concentration of BNP1350, whereas only slight inhibition of chk1 activity was found in the cells treated with the higher concentration. Altered chk1 phosphorylation after DNA damage appears to be associated with specific phases of cell cycle arrest induced by BNP1350. Because A253 cells do not express the p53 protein, the drug-induced alterations of the G(2) checkpoint kinase chk1 are not p53-dependent.

Mechanism of action of the dual topoisomerase-I and -II inhibitor TAS-103 and activity against (multi)drug resistant cells.

UNLABELLED: TAS-103 is a recently developed dual inhibitor of topoisomerase-I (topo-I) and topoisomerase-II (topoII). TAS-103 has documented cytotoxicity in vitro and antitumor activity against a variety of mouse, rat, and human xenografts in vivo. PURPOSE: To determine TAS-103 activity against (multi)drug resistant cells in vitro and to delineate its mechanism of action. METHODS: TAS-103 was evaluated for activity against three human multidrug-resistant cell lines representing resistance mediated by P-glycoprotein (Pgp)-, multidrug resistance protein (MRP), and lung resistance protein (LRP) as well as one camptothecin-resistant cell line associated with a mutated topo-I enzyme. Drug sensitivity following short (2 h), intermediate (6-8 h) and long term (24 h) exposures were compared. The mechanism of action was studied by evaluating inhibition of topoisomerase-I and -II specific DNA relaxation assays, drug-induced DNA/protein cross-link formation, and competitive DNA intercalation with ethidium bromide. RESULTS: Increasing the exposure time only modestly potentiated TAS-103 cytotoxicity (3-5 fold) demonstrating a lack of strong exposure duration dependency. TAS-103 cytotoxicity was not affected by the presence of any of the drug resistance mechanisms studied. TAS-103 inhibits topo-I and -II activity in DNA relaxation assays, but in our assay system TAS-103 was found to have only a weak ability to induce DNA-protein crosslinks. DNA migration patterns in agarose gel electrophoresis indicate that TAS-103 can interact directly with DNA. Also its ability to displace ethidium bromide which has intercalated into the DNA provides an indication on the nature of drug-DNA interaction. CONCLUSIONS: TAS-103 cytotoxicity is not affected by the presence of Pgp, MRP, LRP or mutations in the CAM binding region of the topo-I enzyme and its growth-inhibitory effect appears to be weakly dependent on exposure duration. The presented evidence suggest that the inhibitory effects of TAS-103 on topo-I and -II may in part be related to its DNA binding rather than primarily through stabilization of topo-I or -II intermediates with DNA through specific binding to the enzymes.

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.

Topoisomerase-I inhibitor SN-38 can induce DNA damage and chromosomal aberrations independent from DNA synthesis.

BACKGROUND: SN-38 is the active metabolite of the topoisomerase-I (topo-I) inhibitor Irinotecan (CPT-11). Generally, topo-I inhibitors stabilize the complex between topo-I and DNA which collide with moving DNA replication forks, eventually leading to double stranded DNA damage. Therefore, topo-I inhibitors are regarded as S-phase specific. The present study investigated S-phase dependent and independent effects of SN-38. MATERIALS AND METHODS: Effects of exposure of A2780 cells to SN-38 (2 hours) were studied by assessing DNA/protein crosslinks, DNA damage and cytogenetic aberrations. RESULTS: A close correlation (r2 = 0.97) was established between drug-induced DNA/protein crosslinks and double stranded DNA breaks. Cytogenetic analysis revealed near maximum clastogenic effects already evident immediately following 2 hours drug exposure. However, qualitatively, chromatid breaks at 24 hours were different from those at 0 hours, in that at 24 hours they were associated with radial chromosome configurations and sister chromatid exchanges. CONCLUSION: The data corroborate that the S-phase dependent mechanism of action of topo-I inhibitors is also applicable to SN-38. The cytogenetic data indicate two distinct interactions of SN-38 with DNA: immediate induction of chromatid breaks independent from DNA synthesis, and induction of chromatid breaks associated with radial chromosome configurations dependent on DNA synthesis.

Rational design of irinotecan administration based on preclinical models.

Most clinical drug regimens for irinotecan (CPT-11 [Camptosar]) have been empirically based on classic in vivo pharmacokinetic and pharmacodynamic considerations. We propose an alternative approach that attempts to provide a rationally designed schedule of irinotecan administration based on preclinical data. HL60 cells grown in suspension or as subcutaneously implanted solid xenografts in nude mice served as in vitro and in vivo models to rest the activity of irinotecan or its active metabolite, SN-38. For SN-38, within an effective drug concentration range, scheduling drug administration based on duration of DNA synthesis inhibition significantly potentiated cell kill in vitro, and increasing drug concentrations at suboptimal scheduling did not result in additive cell kill. These data suggested that even though high drug doses may be attainable in vivo, they may not be required to achieve maximum antitumor activity. To test this hypothesis, a sensitive in vivo model to test the toxicity and antitumor activity of CPT-11 is required, which is provided in the human myeloid HL60 xenograft model grown in nude mice. In this model, CPT-11 at a dose 50 mg/kg, daily x5 (MTD) achieved 100% complete tumor regression. This model should be useful to test the hypothesis that for irinotecan, administration of a minimum effective dose (MED) at an optimal schedule can achieve maximum antitumor activity and should therefore prevail over the classic approach of administering the MTD.

Evaluation of topoisomerase I catalytic activity as determinant of drug response in human cancer cell lines.

The prognostic value of topoisomerase I (Topo I) catalytic activity and expression of the multidrug resistance (MDR) marker P-glycoprotein (Pgp) and multidrug resistance protein (MRP) for in vitro sensitivity to Topo I interactive agents were evaluated. The efficacy of short term (2 h) and long term (24 h) exposures of camptothecin (CPT), two CPT derivatives (SN-22, SN-38) and the indolocarbazole compound NB-506, was determined against human ovarian carcinoma (A2780 and A2780 DX5), human fibrosarcoma (HT1080 and IIT1080/DR4) and human ileocecal carcinoma (HCT-8). For each cell line the Topo I protein levels and catalytic activity were determined and correlated with drug-induced cytotoxicity. In general, the Topo I protein levels correlated with Topo I catalytic activity. Drug-induced cytotoxicity increased significantly with prolongation of the exposure time. With the 2 h exposure, the multidrug resistant A2780 DX5 cell line (Pgp+, MRP-) was moderately resistant to all four drugs compared to its parental cell line. In case of CPT and SN-22 but not for SN-38 and NB-506, this resistance was no longer detectable following 24 h drug exposure. No resistance was detectable for the multidrug resistant HT1080/DR4 (Pgp-, MRP+) cell line when compared to its parental cell line. With short term exposures a strong trend was observed toward increased cytotoxicity with increased Topo I catalytic activity, especially if this correlation was studied between derivative cell lines (A2780 vs. A2780 DX5 and HT1080 vs. HT1080/DR4). This correlation weakened when all 5 cell lines and both exposure conditions were considered. Thus, although overall the correlation between Topo I catalytic activity and sensitivity to Topo I interactive drugs between different cell lines is weak, this correlation may be stronger when comparing derivative cell lines. For CPT and SN-22 but not for SN-38 and NB-506, the moderate resistance levels observed in the Pgp-expressing cell line could be negated by prolongation of exposure duration. MRP expression did not effect drug efficacy. The data demonstrate that the importance of Topo I catalytic activity as single prognostic factor for drug response to Topo I interactive agents is weak and that additional mechanisms affecting drug response have to be taken into consideration.

A unique human ovarian carcinoma cell line expressing CD34 in association with selection for multidrug resistance.

BACKGROUND: The role of P-glycoprotein (Pgp) in multidrug resistance (MDR) is uncontested. Expression of Pgp on hematopoietic cells has been correlated with CD34 expression. For acute myeloid leukemia, the prognostic value of Pgp for clinical response is at best equivalent to that of CD34. The current study investigated whether expression of CD34 can be associated with selection for drug resistance. METHODS: Several established MDR cell lines were screened by flow cytometry for expression of CD34. Human ovarian carcinoma cells (A2780), which simultaneously expressed CD34 and Pgp, were identified. Subsequent cloning resulted in a new cell line (A2780-Dx5c) that expressed CD34 in the absence of Pgp. Involvement of non-Pgp-mediated MDR mechanisms was assessed by immunohistochemistry (MRP and LRP), enzyme activity studies (glutathione pathway), cross-resistance patterns, and Northern blot (type II alpha topoisomerase). RESULTS: A2780-Dx5c was cross-resistant to doxorubicin, daunorubicin, idarubicin, and VP-16. However, unlike the Pgp-expressing cells, it was not cross-resistant to vincristine or amsacrine. The drug resistance was correlated with a decreased level of type II alpha topoisomerase in the A2780-Dx5c cell line compared with the parental cell line. No evidence was found of involvement of MRP, LRP, or the glutathione pathway with drug resistance in this cell line. CONCLUSIONS: A new cell line of nonhematopoietic and nonvascular endothelial origin that expresses CD34 in association with selection for MDR was cloned. A study of MDR mechanisms in this cell line revealed that reduced type II alpha topoisomerase levels were likely responsible for the MDR observed. A study of the causal relation between the selection of drug resistance and the expression of CD34 may provide insight into why CD34 correlates with poor clinical response in patients with acute myeloid leukemia.

d,l-buthionine-(S,R)-sulfoximine potentiates in vivo the therapeutic efficacy of doxorubicin against multidrug resistance protein-expressing tumors.

Intracellular glutathione (GSH) has been implicated as a regulatory determinant of multidrug resistance protein (MRP) function. The objective of the present study was to evaluate in vivo the ability of d,l-buthionine-(S,R)-sulfoximine (d,l-BSO), a potent inhibitor of GSH biosynthesis, to reverse MRP-mediated drug resistance to doxorubicin. Athymic nude mice (nu/nu) bearing advanced parental human fibrosarcoma HT1080 and MRP-expressing HT1080/DR4 tumors were treated with the maximum tolerated dose of doxorubicin (10 mg/kg, i. v. push). This therapy produced an overall response rate of 50% (20% complete response and 30% partial response) in mice bearing parental HT1080 xenografts, whereas no significant antitumor activity against HT1080/DR4 tumors was observed. Treatment of mice bearing HT1080 and HT1080/DR4 xenografts with a continuous i.v. infusion of nontoxic doses of d,l-BSO (300 and 600 mg/kg/day) produced a 60% reduction of GSH plasma levels and greater than 95% reduction in GSH tumor levels in both parental and multidrug-resistant tumors; however, this treatment possessed no in vivo antitumor activity by itself. Under these treatment conditions, a combination of d,l-BSO with the maximum tolerated dose of doxorubicin administered at 24 h during a 48-h i.v. infusion of d,l-BSO completely restored the response of MRP-expressing HT1080/DR4 tumors to doxorubicin (overall response rate, 63%; complete response rate, 38%) with no potentiation of host toxicity. The d,l-BSO-induced in vivo reversal of MRP-mediated drug resistance correlated in vitro with the restoration of intracellular doxorubicin retention in cultured HT1080/DR4 cells. Depletion of GSH by d,l-BSO in drug-sensitive HT1080 tumors that do not express MRP did not alter the in vivo response to doxorubicin. Using the same treatment schedule, dose, and administration of doxorubicin with and without d,l-BSO in nude mice bearing P-170 glycoprotein-expressing A2780/Dx5 tumors, no potentiation of the therapeutic index of doxorubicin was found, demonstrating the in vivo selectivity of d, l-BSO-induced GSH depletion on MRP-function. The data reported herein indicate that in vivo function of MRP as a mediator of doxorubicin resistance requires the presence of sufficient GSH pools. d,l-BSO may provide an example of an effective in vivo modulator of MRP-mediated drug resistance.

DiOC2(3) is not a substrate for multidrug resistance protein (MRP)-mediated drug efflux.

Multidrug resistance (MDR) is often related to expression of P-glycoprotein (Pgp) or Multidrug Resistance Protein (MRP). Pgp-mediated MDR can be evaluated by determining cellular retention of fluorescent substrates by flow cytometry. This study determined if agents used to evaluate Pgp function also can be used to evaluate MRP function. Cellular retention of doxorubicin (Dox), Rhodamine-123 (Rh-123), and 3,3'-diethyloxacarbocyanine iodide (DiOC2(3)) were studied in MRP-expressing cell lines (HL60/Adr and HT1080/DR4), whereas a Pgp expressing cell line (A2780/Dx5) served as a positive control. Overexpression of Pgp correlated inversely with retention of Dox, Rh-123, and DiOC2(3); however, under identical experimental conditions (1 h reincubation in drug-free medium), no retention difference of the three agents was detected between parental and MRP-expressing resistant cells. Upon extending the reincubation time to 4 h, an efflux of Rh-123 and Dox in the resistant lines became apparent and even more pronounced after 24h; however, still no efflux was detectable for DiOC2(3). Incubation of the cells with a modulator of MDR, PAK-104P, negated the observed drug efflux in Pgp and MRP expressing cells, which correlated with increased sensitivity of the MDR lines to doxorubicin. Thus both Dox and Rh-123 can be used to evaluate MRP-function, but DiOC2(3) can not.

Biological effects of recombinant human granulocyte colony-stimulating factor in patients with untreated acute myeloid leukemia.

Hematopoietic growth factors are being administered to patients with acute myeloid leukemia (AML) both to shorten the duration of chemotherapy-induced neutropenia and in an attempt to increase cytotoxicity of cell cycle-specific agents. However, limited information is available concerning the effects of growth factors in AML patients. To examine the in vivo effects of recombinant human granulocyte colony-stimulating factor (G-CSF) on AML cells, laboratory studies were performed before and after a 72-hour intravenous infusion of G-CSF (10 micrograms/kg/d) administered to 28 untreated AML patients. Twenty-seven patients (96%) showed increases in at least one of the following parameters after G-CSF: blood blasts, bone marrow (BM) blasts, leukemia cells in S phase or interphase cells with leukemia-specific markers shown by fluorescence in situ hybridization. The median paired change in absolute blast count was +2.7 x 10(9)/L (P = .0001) after G-CSF, as compared with 0.0 during the 72 hours before initiation of G-CSF. The median percentage of BM leukemia cells in S phase increased from 6.0% to 10.7% after G-CSF (median change, %5.9%; P = .009). Interphase BM cells with trisomy 8 or monosomy 7 increased in 6 of 6 patients with these abnormalities (P = .02) with a median percent increase of 47%. Blood neutrophil counts also increased during G-CSF (median paired change, +2.8 x 10(9)/L; P < .0001). Trisomy 8 or monosomy 7 was shown by fluorescence in situ hybridization in post-G-CSF blood neutrophils from 4 of 6 patients but was also present in neutrophils before G-CSF. We conclude that the percentage of leukemia cells in S phase increases and that leukemia cell populations undergo expansion during short-term administration of G-CSF in almost all AML patients.

PAK-104P, a pyridine analogue, reverses paclitaxel and doxorubicin resistance in cell lines and nude mice bearing xenografts that overexpress the multidrug resistance protein.

Multidrug resistance (MDR) is considered multifactorial and has been associated with overexpression of the multidrug resistance protein (MRP). However, effective compounds for reversal of MRP-related MDR are limited. In the present study, the modulatory activity of the novel pyridine analogue PAK-104P on MRP-mediated resistance to doxorubicin and paclitaxel was investigated in two doxorubicin-selected human tumor cell lines [HT1080/DR4 (sarcoma) and HL60/ADR (leukemia)] and compared with the nonimmunosuppressive cyclosporine analogue PSC-833. In cell lines HT1080/DR4 (MRP/lung resistance-related protein phenotype) and HL60/ADR (MRP phenotype), doxorubicin resistance was significantly higher (250-fold and 180-fold, respectively) than that to paclitaxel (6-fold and 9-fold, respectively). With noncytotoxic concentrations of PAK-104P (1 and 5 microM), the reversal of doxorubicin resistance was significant but partial in HT1080/DR4 and HL60/ADR cells (dose-modifying factor for 5.0 microM PAK-104P, 25.0 and 31.2, respectively), whereas complete reversal of paclitaxel resistance was achieved in HL60/ADR cells. In contrast, PSC-833 modulation of doxorubicin and paclitaxel resistance was modest. Cellular drug uptake and retention studies by flow cytometry analysis demonstrated that PAK-104P was effective in restoring cellular doxorubicin concentrations in resistant cells to levels comparable to those obtained in parental cells. In athymic nude mice, PAK-104P significantly potentiated the therapeutic efficacy of doxorubicin and paclitaxel against resistant HT1080/DR4 xenografts. Of significance is that the maximum tolerated doses of doxorubicin and paclitaxel were administered in combination with PAK-104P, documenting improvement in the therapeutic index of these agents. In addition to reversing P-glycoprotein-mediated MDR, the pyridine analogue PAK-104P provides an example of an effective in vivo modulator of MRP-mediated MDR.

Detection of incorporated iododeoxyuridine in colonies by immunoperoxidase staining: a novel method to measure the proportion of cycling colony-forming cells.

In vitro suicide by tritiated thymidine (3H-TdR), hydroxyurea (HU), or cytosine arabinoside (Ara-C) is assumed to reflect the proportion of colony-forming cells in S-phase at the time of exposure. However, these techniques are not always accurate. Nonradioactive iododeoxyuridine (IdUrd) is incorporated into DNA during S-phase and can be detected by monoclonal antibodies. In the present study, a new IdUrd application was developed to investigate the kinetics of hematopoietic progenitor cells. After incubation with IdUrd, colony-forming cells were cultured in semisolid assay. An immunoperoxidase staining protocol was developed to detect IdUrd in cells of colonies in agar. Colony-forming cells in S-phase during the IdUrd exposure were postulated to give rise to IdUrd+ colonies, whereas non-S-phase cells would generate IdUrd- colonies. Toxicity, sensitivity, and IdUrd inactivation studies indicated that progenitor cells could safely be pulse-labeled for 2 hours with 40 microM IdUrd, whereas prolonged labeling with 1 microM IdUrd was at least feasible for 5 days. Molt-4 cells and normal bone marrow cells were used to compare IdUrd pulse-labeling with 3H-TdR suicide. Part of the Molt-4 cells were enriched for G1- and S-phase cells by counterflow centrifugation. The bone marrow cells were either unstimulated or stimulated with growth factors. As a result, the accuracy of both techniques could be tested in populations with different quantities of S-phase cells. Wide confidence intervals of the suicide technique contrasted with the small confidence intervals obtained with IdUrd pulse-labeling. For instance, the fraction of Molt-4 cells with 27.8% S-phase cells contained 17.7% (confidence interval -8.2 to 43.6%) clonogenic cells in S-phase when determined with 3H-TdR suicide. Of this fraction, the percentage of clonogenic cells in S-phase was 30.6% with a confidence interval of 25.5 to 36.2% when determined with IdUrd pulse-labeling. In our hands, the IdUrd pulse-labeling was more accurate than the 3H-TdR suicide technique. Thus far, kinetic studies of progenitors have been limited to the determination of the fraction of S-phase cells by suicide techniques. By prolonged IdUrd labeling, it is now possible to determine the proliferating fraction of progenitor cells.

Schedule-dependent efficacy of idarubicin (Ida) and doxorubicin (Dox).

Schedule dependency of idarubicin (Ida) and doxorubicin (Dox) toxicity was investigated in vitro using the K562 human leukemia cell line. For Dox, repeated exposure to the IC30 (d x 3) resulted in comparable survival as single exposure to the total accumulative dose (20%). For Ida, repeated exposure to the IC30 (d x 3) decreased survival to 5%, while single exposure to the total accumulative dose reduced survival only to 20%. Total cellular accumulation of Dox was independent of schedule of exposure, while for Ida, repeated exposure resulted in a significantly higher drug accumulation compared to the single exposure to the accumulative dose. The data indicate that the schedule-dependent differences in cytotoxicity for the two compounds can be accounted for almost exclusively by an increased cellular uptake and retention of Ida with repeated drug exposure.

Toxicity of idarubicin and doxorubicin towards normal and leukemic human bone marrow progenitors in relation to their proliferative state.

The effect of growth stimulation on the sensitivity of normal and leukemic human bone marrow progenitors to idarubicin and doxorubicin was studied. Clonogenic assays from colony-forming units of the granulocyte-macrophage lineage (CFU-GM) and leukemic clonogenic cells (CFU-L) were applied using human placenta conditioned medium (HPCM) as source of growth factors. Before seeding cells in clonogenic assay they were exposed to the anthracyclines for 2 h without preincubation, or following a 48 h preincubation period in the presence of HPCM. Drug concentrations used ranged from 0.001-0.1 microgram/ml for idarubicin and from 0.1-1.5 microgram/ml for doxorubicin. In addition, a limited number of bone marrow samples were exposed to the cytostatically active metabolite of idarubicin; idarubicinol (Idol; range 0.001-0.1 microgram/ml). Proliferation of CFU-GM and CFU-L during 48 h was measured by iododeoxyuridine (IdUrd) incorporation. Spontaneous proliferation of CFU-GM increased from 38 to 88% after 48 h stimulation by HPCM. The mean number of proliferating CFU-L increased from 40 to 77% when stimulated with HPCM. Doxorubicin inhibited colony formation of CFU-GM and CFU-L to 50% (IC50CFU-GM, IC50CFU-I) at mean concentrations of 0.355 microgram/ml and 0.103 microgram/ml when applied before preincubation with HPCM, and 0.108 microgram/ml and 0.055 microgram/ml when applied after preincubation. Idarubicin appeared the most potent drug in all experiments regardless of the preincubation procedure or sample origin, with average IC50CFU-GM and IC50CFU-L of 0.008 microgram/ml and 0.006 microgram/ml, respectively, when applied before preincubation with HPCM, and 0.006 microgram/ml and 0.005 microgram/ml when applied after preincubation with HPCM. Idarubicinol showed intermediate potency with average IC50CFU-GM of 0.022 microgram/ml and 0.023 microgram/ml when applied before and after preincubation with HPCM, respectively. In order to assess the effect of growth stimulation on drug sensitivity, samples were evaluated pair-wise (sensitivity of the same sample before vs. after preincubation with HPCM) and were submitted to the Wilcoxon test for matched pairs. Statistically significant enhancement of cytotoxicity was demonstrated for Doxorubicin vs. CFU-GM (p < or = 0.021) and a strong trend versus CFU-L (p = 0.06). The data further demonstrate that proliferation-dependency of doxorubicin toxicity is more pronounced for CFU-GM than for CFU-L. These data also show that idarubicin and idarubicinol toxicity is proliferation-independent. Idarubicin is relatively more potent than doxorubicin in suppressing the growth potential of low or nonproliferating progenitors.(ABSTRACT TRUNCATED AT 400 WORDS)

Idarubicin-related side effects in recipients of T-cell-depleted allogeneic bone marrow transplants are schedule dependent.

The influence of three different dosage schedules of anthracycline (idarubicin or daunorubicin)-intensified preparative therapy prior to T-cell-depleted allogeneic bone marrow transplantation (BMT) on (1) the severity and duration of oral toxicity (mucositis), (2) the duration of bone marrow aplasia, and (3) overall survival, relapse, and disease-free survival was studied in 99 BMT patients with standard- or high-risk hematologic malignancies. A further 146 patients who did not receive the anthracycline-intensified conditioning served as (historic) controls. All patients received cyclophosphamide (total dose, 120 mg/kg) on days -6 and -5 and total body irradiation on days -2 and -1 prior to BMT. The 99 patients who received the anthracycline-intensified preparative regimen were given either idarubicin (total dose, 42 mg/m2; n = 88) or daunorubicin (total dose, 156 mg/m2; n = 11) by continuous intravenous infusion between days -7 and -1 prior to BMT in 59 cases (cohort 1), on days -8 and -7 in 17 cases (cohort 2), and on days -12 and -11 in 23 cases (cohort 3). The occurrence of severe oral mucositis and delayed bone marrow recovery was schedule dependent, being substantially lower with earlier administration of the anthracycline-intensified regimen on days -12 and -11 before BMT (cohort 3), in comparison with later administration (cohorts 1 and 2). Plasma drug and metabolite concentrations were measured in 11 patients who received idarubicin. At the time of allogeneic bone marrow infusion (day 0), patients in cohorts 1 and 2 had plasma concentrations of idarubicin and idarubicinol (its active metabolite) in the range of in vitro cytotoxicity. However, in cohort 3, plasma concentrations on day 0 were much lower, which correlated with the lower maximum intensity and shorter duration of mucositis in these patients. In terms of overall survival, relapse rate, and disease-free survival in standard-risk patients, the anthracycline-intensified regimen proved to be very effective. Transplant-related mortality was 25% in the anthracycline group compared with 32% in the controls. The probability of relapse also was significantly less in the anthracycline group in comparison with controls (17% v 46%; P < .001), and the probabilities of long-term overall and disease-free survival were significantly greater (71% v 37% [P < .01] and 63% v 32% [P < .01], respectively). Only three patients in the idarubicin group experienced cardiotoxicity (one in each cohort); the causative relationship with anthracyclines was considered likely in one, possible in one, and doubtful in the third.

Cell cycle related uptake, retention and toxicity of idarubicin, daunorubicin and doxorubicin.

Exponentially growing Molt-4 cells were separated by means of counterflow centrifugation into fractions enriched for cells in early G1, late G1, S, or G2+M phase of the cell cycle. Subsequently, cells were exposed for 2 h to Idarubicin (Ida, 0.02-0.15 microgram/ml). Daunorubicin (Dnr, 0.1-0.75 microgram/ml) or Doxorubicin (Dox, 0.1-0.75 microgram/ml). Drug uptake, measured by flow cytometry, increased progressively with cell cycle traverse from early G1-to M-phase. The relative fraction of drug lost following an extensive wash procedure was 72% in case of Ida and 23% for Dnr and Dox and was independent of the cell cycle phase. Inhibition of DNA synthesis was determined by qualitative flow cytometric analysis of 5-iodo-2'-deoxyuridine (IdUrd) incorporation into DNA. The three drugs showed a similar gradual increase of inhibition of DNA synthesis from G1 to G2+M phase, demonstrating that cell cycle phase dependency of drug toxicity applies to all three anthracyclines studied.