Effects of verapamil on daunomycin cellular retention and cytotoxicity in P388 leukemic cells.

We have utilized digitized video microscopy to investigate the influence of verapamil, a calcium channel blocker, on the in vitro effects of daunomycin in P388 sensitive and resistant sublines. In this study, verapamil enhanced the uptake of daunomycin and its cytotoxicity in both sublines, but the effect was more pronounced in the resistant cells. Flow cytometry showed a pronounced accumulation of resistant cells at G2-M when treated with daunomycin in the presence of verapamil, with no effects observed in the absence of verapamil. Analysis of individual resistant cells using the digitized video fluorescence microscopy technique demonstrated that the influence of verapamil on daunomycin uptake affected all cells and was not restricted to certain subpopulations of cells.

Differences in daunomycin retention in sensitive and resistant P388 leukemic cells as determined by digitized video fluorescence microscopy.

Cellular uptake and binding of daunomycin were studied using the digitized video fluorescence microscopy technique, in a sensitive and a resistant subline of P388 leukemic ascites tumor cells. When a 60-min time course of uptake was monitored, the sensitive cells had a 4-fold greater uptake than did the resistant cells. When the cells were perfused with drug-free medium, identical exchangeable levels of the drug were lost from both sublines. The difference in drug uptake could be accounted for entirely on the basis of differences in a slowly exchanging drug fraction which probably represents bound intracellular drug. In glucose-free medium, uptake of daunomycin was accelerated by metabolic inhibition to a greater extent in resistant than in sensitive cells. Furthermore, there was minimal decrease in the fluorescence when both sublines were perfused with drug-free medium. The addition of glucose to this medium induced a significant decrease in fluorescence in resistant but not in sensitive cells. These data raise the possibility that decreased drug uptake in resistant cells associated with decreased slowly exchanging drug fraction may be associated with an inherent defect in drug binding which is reversed by inhibition of energy metabolism. Parallel in vitro and in vivo studies revealed the presence of uptake heterogeneity; both sensitive and resistant cells contained subpopulations (20 to 30%) that have less or more fluorescence than the predominant pattern. This observation demonstrates the possible use of the digitized video fluorescence microscopy for recognizing subsets of cells with different drug susceptibility and to monitor the emergence of anthracycline-resistant cell populations.

Digitized video fluorescence microscopy studies of adriamycin interaction with single P388 leukemic cells.

We have evaluated a new fluorescent method, the digitized video fluorescence microscopy technique, for the analysis of Adriamycin drug levels in single-cell suspension. This method uses a Leitz microscope equipped with an HBO 50 watt mercury source; the vertical body of the microscope is attached to an intensified silicone intensifier video camera with its output coupled to a video cassette recorder and to an Apple II microcomputer equipped with a video image digitizer. Using this technique, we were able to corroborate previous findings of decreased uptake and increased efflux in resistant as compared to sensitive P388 leukemic cells. This instrument may have wide applications in the study of anthracycline cell interaction or of any other drug with fluorescent properties.

Characterization of a K562 multidrug-resistant cell line.

A daunorubicin-resistant variant of the K562 human leukemia cell line (K562-R), which demonstrates cross-resistance to other anthracycline antibiotics and Vinca alkaloids, has been developed in vitro by continuous exposure to daunorubicin. Cross-resistance to anthracyclines and Vinca alkaloids is reversed when cells are exposed to drugs in the presence of verapamil, a calcium channel blocker. The K562-R cell line overexpresses a 4.5-kilobase mRNA, which is thought to code for the Mr 170,000 membrane glycoprotein associated with multidrug resistance. Transport studies indicate reduced intracellular accumulation and retention of daunorubicin in the K562-R cells as compared to the parent cell line. These studies further suggest the presence of distinct cellular pools composed of both rapidly and slowly exchanging drug, with the rapidly exchanging pool being more pronounced in the resistant line. The development of multidrug resistance in the K562-R cell line is also associated with the overexpression of five different cell surface membrane proteins ranging in molecular weight between 50,000 and 210,000, whose function remains to be defined.

Characteristics of uptake and cytotoxicity of a low-density lipoprotein-daunomycin complex in P388 leukemic cells.

We have investigated the in vitro drug-cell interaction and therapeutic effects of a low-density lipoprotein (LDL)-daunomycin complex as compared to free daunomycin. Uptake and retention of daunomycin were significantly enhanced in sensitive and resistant P388 leukemia cells when they were perfused with LDL-daunomycin complex relative to free drug. The interaction of the LDL-daunomycin complex with P388 cells appeared to be through a LDL-specific pathway since competition with free LDL but not high-density lipoprotein significantly reduced daunomycin uptake. The LDL-daunomycin complex was cytotoxic to both daunomycin-sensitive and daunomycin-resistant P388 sublines. Colony growth studies showed the LDL-daunomycin complex to be more cytotoxic at shorter exposure times relative to free drug. Resistant cells showed 55 and 12% colony growth with 10-min and 2-hr exposures, respectively, to 0.4 microgram daunomycin/ml of the LDL-daunomycin complex. Free drug at 0.4 microgram/ml drug concentration and similar exposure times resulted in no loss in colony growth. The resistant cells were subjected to cell cycle analysis based on DNA content and showed an accumulation of cells at the G2-M phase of the cell cycle when treated with the LDL-daunomycin complex, with no effects being observed with free daunomycin.

Resistance to natural killer cell-mediated cytolysis by a pleiotropic drug-resistant human erythroleukemia (K562-R) cell line.

K562-R, a pleiotropic drug-resistant cell line established in vitro, and K562-S, the chemotherapy-sensitive parent line, were used as targets in the natural killer cell (NK) system. At each of the effector:target ratios studied, the K562-R demonstrated a decrease in their susceptibility to both unstimulated and interferon-activated NK cells, as compared to the K562-S line. This difference did not appear to be related to a variable expression of NK target structures, as the number of effector:target conjugates was similar, and both lines competed equally well in cold target inhibition experiments. The K562-R cells were not resistant to complement or monocyte-mediated killing, suggesting a relatively specific resistance to cell-mediated killing. These results are compatible with an NK postbinding defect in the K562-R cells and suggest that greater tumorigenicity for pleiotropic drug-resistant cells may in part be due to altered susceptibility to host defense.

Evidence for inhibition of growth related to compromised DNA synthesis in the interaction of daunorubicin with H-35 rat hepatoma.

The H-35 rat hepatoma is relatively insensitive to the anthracycline antibiotic, daunorubicin (DNR), requiring 0.25 microM daunorubicin for inhibition of cell proliferation by 50%. Studies were undertaken to investigate the basis for the apparent intrinsic resistance in this cell line. The relative insensitivity of the H-35 cells to DNR is not a function of metabolic inactivation of DNR to deoxyaglycone derivatives; after a 2-h incubation, less than 10% of drug is metabolized, exclusively by conversion to daunorubicinol. The limited toxicity of DNR to the rat hepatoma may be explained, in part, by the absence of DNA strand breaks at daunorubicin concentrations up to 1 microM while higher (supraclinical) DNR concentrations (5 and 10 microM) produce direct, "non-protein-associated" DNA strand breaks. Limited daunorubicin toxicity in this tumor cell line may also be related to the apparent absence of free radical-mediated cellular damage as the free radical scavengers dimethyl sulfoxide, catalase, methanol, and mannitol fail to reverse the inhibitory effect of 1 microM DNR on cell proliferation. Daunorubicin does not induce leakage of the cytoplasmic enzyme, glutamic oxaloacetate transaminase, or diminish mitochondrial enzyme function. Conversely, while drug effects on RNA synthesis are small, and protein synthesis is minimally diminished, inhibition of cell proliferation corresponds closely with inhibition of DNA synthesis.

Dynamic parameters of membrane lipids in normal and leukemic human lymphocytes isolated from peripheral blood and bone marrow.

The degree of microviscosity (eta), and lipid fluidity (LFU) of cellular membranes of normal and leukemic lymphocytes obtained from peripheral blood and bone marrow of normal donors and acute lymphatic leukemic (ALL) patients was quantitatively monitored by fluorescence polarization analysis with the aid of the fluorescent lipophilic probe 1,6-diphenyl-1,3,5-hexatriene when embedded in cellular membranes of intact cells. The results have shown a marked decrease in eta and a significant increase in LFU in lymphocytes obtained from both peripheral blood and bone marrow of ALL patients at admission when compared to both T- and B-lymphocytes obtained from peripheral blood of normal donors. Moreover, both dynamic parameters, eta and LFU, show normal characteristic values in lymphocytes obtained from bone marrow of ALL patients in complete hematological remission. Since in few cases a decrease in eta and an increase in LFU were observed in bone marrow lymphocytes isolated from ALL patients in remission, the possibility that these dynamic parameters may serve as a diagnostic tool for an early detection of a new relapse is discussed.

Preparation and interaction of a low-density lipoprotein:daunomycin complex with P388 leukemic cells.

A low-density lipoprotein (LDL):daunomycin complex was prepared which contains, on the average, 8 nmol of daunomycin/mg of apolipoprotein. LDL was not perturbed to any significant degree by association with daunomycin, and the complex remained stable at 4 degrees. Fluorescence quenching with DNA and potassium iodide, antibody precipitation, and density flotation studies suggested at least two domains for localization of the daunomycin, the surface region and the hydrophobic core of the LDL particle. Interaction of the LDL:daunomycin complex with P388 leukemic cells which were sensitive or resistant to daunomycin occurred rapidly in both sublines, with quantitatively larger amounts of the drug being cell associated than when free drug was used alone. The cells appeared morphologically intact, with no nuclear damage after short-term incubation with LDL:daunomycin. Subfractionation of cell membranes and organelles from the sensitive and resistant cell lines incubated with LDL:daunomycin showed accumulation of drug in the plasma membrane and microsomal-lysosomal-mitochondrial membranes and an insoluble nuclear chromatin material. Both 125l-labeled apoprotein and daunomycin from a 125l-LDL:daunomycin complex showed enrichment in similar membrane:organelle fractions of the P388 cells.

Interaction of anthracyclines with nucleotides and related compounds studied by spectroscopy.

Interaction of the antitumour anthracyclines with mononucleotides and related compounds can be assessed through the perturbation of the spectral properties of the drugs. Purine-derived compounds induce spectral changes more efficiently than pyrimidine derivatives. No marked differences are observed when mono-, di- or triphosphate derivatives, deoxy forms, nucleosides or free nitrogen bases are used for the experiments. Visible absorbance data indicate the existence of a drug/purine nucleotide complex in solution. Assuming a simple equilibrium, this complex would be of low affinity (Keq 100 M-1). Circular dichroism spectra of daunomycin in the presence of ATP suggest that the resulting daunomycin/ATP complexes are not comparable to those formed by intercalation of the anthracycline into DNA. 31P-NMR of ATP in the presence of daunomycin does not support the notion that anthracycline/nucleotide complex formation involves interaction through the phosphate group(s) of the nucleotide. Analysis of the quenching of the drug's intrinsic fluorescence in the presence of nucleotides indicates a predominantly collisional, dynamic quenching mechanism. Values in the 2-6 mM and 85-100 mM range, respectively, are estimated for the reciprocal of the Stern-Volmer quenching constant for a variety of purine and pyrimidine derivatives. This indicates that purine derivatives are highly efficient quenchers of the fluorescence of anthracyclines, while pyrimidine derivatives are not. The fluorescence lifetime of daunomycin in the absence of quencher and the Stern-Volmer quenching constants obtained for different nucleotides are used to calculate the apparent bimolecular rate constants for collisions between fluorophore and quencher to occur. Values of (2-3) X 10(11) and 1 X 10(10) M-1 X s-1 are obtained, respectively, for purine and pyrimidine derivatives. This suggests a combination of static and dynamic quenching processes for purine compounds, which is consistent with the drug/purine nucleotide complex formation detected by visible absorbance. Because of the high intracellular concentration of certain nucleotides, particularly ATP, the above processes are predicted to be highly significant 'in vivo'.

Randomized placebo-controlled multicenter evaluation of diethyldithiocarbamate for chemoprotection against cisplatin-induced toxicities.

PURPOSE: Diethyldithiocarbamate (DDTC) blocks cisplatin-induced toxicities in animal models without inhibiting antitumor effects. DDTC chemoprotection was tested in a randomized, multicenter, double-blind comparison versus placebo (PB) in patients with lung or ovarian cancer. Primary end points were nephrotoxicity, ototoxicity, neuropathy, and completion of therapy. PATIENTS AND METHODS: Between April 1990 and February 1992, 221 patients were registered with small-cell lung cancer (SCLC), non-small-cell lung cancer (NSCLC), or ovarian cancer. Cisplatin (100 mg/m2) and cyclophosphamide (in ovarian cancer) or etoposide (in lung cancer) were administered with either DDTC (1.6 g/m2 over 4 hours) or PB intravenously, every 4 weeks for a planned six cycles. RESULTS: At an interim safety analysis, data were available for 195 patients from the combined lung and ovarian cancer populations (PB, 99 patients; DDTC, 96 patients). Withdrawal for chemotherapy-induced toxicities occurred in 9% of PB-treated patients and 23% of DDTC-treated patients (P = .008). The mean cisplatin delivered dose-intensity (DDI) was 23 mg/m2/wk on both arms. However, the mean cisplatin cumulative dose delivered (CDD) was 379 mg/m2 on the PB arm, compared with 247 mg/m2 on the DDTC arm (P = .0001). At the time of interim analysis, 28% of PB-treated patients had completed all six cycles of therapy, compared with only 6% of DDTC-treated patients (P < .001). Although, clinical hearing loss, neuropathy, emesis, and myelosuppression were equivalent in the two treatment arms, DDTC-treated patients had more nephrotoxicity as determined by changes in serum creatinine concentration. Toxicities related to DDTC infusion included transient hypertension, flushing, and hyperglycemia. DDTC did not compromise response rates in either tumor type. CONCLUSION: This study did not demonstrate a significant chemoprotective effect against cisplatin-induced toxicities with the DDTC dose schedule tested. Patients who received DDTC received lower cumulative doses of cisplatin, but were more likely to be withdrawn from treatment early due to chemotherapy-related toxicities.

Randomized trial of vinorelbine compared with fluorouracil plus leucovorin in patients with stage IV non-small-cell lung cancer.

PURPOSE: This prospective randomized trial was performed to compare the effectiveness of intravenous vinorelbine tartrate with intravenous fluorouracil and leucovorin (5-FU/LV) on the primary end points of survival, quality of life (QOL), and relief of cancer-related symptoms in patients with advanced non-small-cell lung cancer (NSCLC). Secondary end points included tumor response rates and time to treatment failure. In addition, the safety of both treatment regimens was evaluated in this multicenter study. PATIENTS AND METHODS: Two hundred sixteen patients with stage IV NSCLC were enrolled onto this study from 18 centers. Vinorelbine was administered at a dose of 30 mg/m2/wk. 5-FU/LV was administered at a dose of 425 mg/m2 and 20 mg/m2, respectively, for 5 consecutive days every 4 weeks. Patients with progressive disease or toxicity were removed from study while responding and stable patients were continued on therapy. RESULTS: The median survival time of patients who received vinorelbine was 30 weeks, with 25% of patients alive at 1 year, compared with a median survival time of 22 weeks and 16% of patients alive at 1 year for those treated with 5-FU/LV (P = .03, log-rank test). This improvement in survival was associated with a higher objective response rate (12% v 3%) and time to treatment failure (10 weeks v 8 weeks) for vinorelbine versus 5-FU/LV. The dose-limiting toxicity of vinorelbine was granulocytopenia, with 54% of patients experiencing grade 3/4 granulocytopenia. Nonhematologic toxicity of vinorelbine was generally grade 1 or 2. The most common grade 3 toxicities were related to injection-site reactions. CONCLUSION: This trial confirms the efficacy of vinorelbine in patients with advanced NSCLC. The clinical activity and relatively favorable toxicity profile of this agent make it a reasonable and useful treatment option in the management of patients with this disease.

Economic analysis of a randomized clinical trial to compare filgrastim-mobilized peripheral-blood progenitor-cell transplantation and autologous bone marrow transplantation in patients with Hodgkin's and non-Hodgkin's lymphoma.

PURPOSE: High-dose chemotherapy (HDC) with peripheral-blood progenitor cell (PBPC) and autologous bone marrow (ABM) transplant (T) has documented survival benefits for relapsed Hodgkin's disease (HD) and non-Hodgkin's lymphoma (NHL). Treatment costs associated with HDC and its supportive care have restricted its use both on and off clinical trial. In a prospective randomized clinical trial, filgrastim-mobilized PBPCT resulted in faster recovery of bone marrow function, with less hospitalization and supportive care than ABMT. This study was undertaken to analyze the costs of the two strategies using prospectively collected data from a randomized clinical trial that compared filgrastim-mobilized PBPCT versus ABMT. PATIENTS AND METHODS: Clinical results and resource utilization from a randomized clinical trial that compared filgrastim-mobilized PBPCT versus ABMT following carmustine, etoposide, cytarabine, and melphalan (BEAM) HDC for HD and NHL are presented. The trial was performed in six centers in Germany, the United Kingdom, and Belgium. Resource utilization data were used to project costs and Massay Cancer Center (MCC) in the United States incurred the cost of treating the cohort. Costs were projected to the United States, because the economic implications to United States centers are significant, costs of care vary markedly among countries but resource utilization on this trial did not, and a randomized trial is unlikely to be performed in the United States. RESULTS: Fifty-eight patients with relapsed HD or NHL underwent HDC with BEAM. The PBPCT and ABMT groups had similar short-term survival after BEAM. PBPCT patients had a shorter hospitalization (median, 17 v 23 days; P = .002), neutrophil recovery (11 v 14 days; P = .005), platelet recovery to > or = 20 x 10(9)/L (16 v 23 days; P = .02), and days of platelet transfusions (6 v 10; P < .001). Estimated costs were $8,531 for ABM harvest and $5,760 for PBPC collection, including filgrastim mobilization. The total estimated average cost was $59,314 for each ABMT patient versus $45,792 for each PBPCT patient. Cost savings of $13,521 (23%) were due to shorter hospitalizations with less supportive care. CONCLUSION: PBPCT is as safe and more effective than ABMT for HD and NHL in the short term. PBPCT represents a significant cost savings due to lower autograft collection costs, shorter hospital stays, and less supportive care. The savings exceed the costs for filgrastim mobilization and PBPC collection. Actual savings will vary depending on local practice patterns, charges, and costs.

Economic analysis of unrelated allogeneic bone marrow transplantation: results from the randomized clinical trial of T-cell depletion vs unmanipulated grafts for the prevention of graft-versus-host disease.

Unrelated-donor marrow transplantation is a potential option for transplant candidates lacking a compatible related donor. The T-cell Depletion Study compared the 3-year disease-free survival for patients receiving T-cell-depleted (TCD) donor marrow (n = 203) vs unmanipulated donor marrow with methotrexate and cyclosporine (M/C) (n = 207). Hospital costs during index admission were documented with billing data, while hospital costs during subsequent 6-month follow-up were estimated from case report forms. Patients with index admission billing were included in the analysis (TCD = 119, M/C = 127). Total hospital length of stay (LOS) was similar across groups, with medians 47.0 days for TCD and 52.0 days for M/C (P = 0.72). Total hospital costs were comparable, 145,115 dollars vs 141,981 dollars (P = 0.63) for TCD and M/C, respectively. However, controlling for site and patient characteristics, TCD was associated with a 12.1% reduction in LOS for the index admission (95% CI -19.4%, -4.3%). Independent of treatment, HLA matching (6/6) was associated with an 8.6% (95% CI -17.4%, +1.2%) reduction in the index admission LOS, while cost was lower by 15.8% (95% CI -26.7%, -3.3%). Treatment costs were similar for TCD and M/C study groups. Savings on reduced cost for treating acute graft-versus-host disease were likely offset by increase in serious infections in the TCD arm.

Characterization of a multidrug resistant human erythroleukemia cell line (K562) exhibiting spontaneous resistance to 1-beta-D-arabinofuranosylcytosine.

We have assessed the response of a previously characterized multidrug resistant (MDR) human erythroleukemia cell line (K562R) to the nucleoside analog antimetabolite 1-beta-D-arabinofuranosylcytosine (ara-C). This cell line has been subjected to selection pressure by intermittent exposure to daunorubicin, but not ara-C, since its initial isolation. In comparison to the parental line (K562S), K562R were approximately 15-fold more resistant to ara-C as determined by 3H-dThd incorporation, MTT dye reduction and clonogenicity. Following a 4-h exposure to 10 microM ara-C, K562S accumulated approximately seven times more ara-CTP, and incorporated approximately 250% more ara-C into DNA than their resistant counterparts. The intracellular generation of ara-CTP was not significantly influenced by the cytidine deaminase inhibitor THU or the deoxycytidylate deaminase inhibitor dTHU (1 mM each) in either cell line. Rates of dephosphorylation of ara-CTP were equivalent in sensitive and resistant cells, as were intracellular levels of both ribonucleotide and deoxyribonucleotide triphosphates. However, K562R displayed a significant (ie 70%) reduction in the level of activity of the pyrimidine salvage pathway enzyme, deoxycytidine kinase (dCK), compared to K562S cells. In contrast to U937 leukemic cells, DNA extracted from K562S and K562R cells following exposure to 10 microM ara-C for 6 h did not exhibit the characteristic internucleosomal DNA cleavage on agarose gel electrophoresis typical of drug-induced apoptosis. Lastly, Northern analysis revealed equivalent levels of dCK message in the two cell lines. K562R represents an unusual example of a classical multidrug resistant human leukemic cell line exhibiting spontaneous cross-resistance to the antimetabolite ara-C, and may prove of value in attempts to understand the mechanism(s) by which human leukemic myeloblasts survive in vivo exposure to combination chemotherapeutic regimens containing drugs that are not classically associated with the multidrug resistance phenomenon.

Metabolism of the anthracycline antibiotic daunorubicin to daunorubicinol and deoxydaunorubicinol aglycone in hepatocytes isolated from the rat and the rabbit.

In the rat and rabbit hepatocyte in suspension, daunorubicin was metabolized primarily to deoxydaunorubicinol aglycone and daunorubicinol. Little deoxydaunorubicin aglycone was observed in either species. High levels of daunorubicinol in the rabbit hepatocyte reflected the greater affinity of rabbit hepatic aldo-keto reductase for the substrate, daunorubicin. Conjugates of the anthracyclines were not observed with hepatocytes from either species. The relative formation of deoxydaunorubicinol aglycone and deoxydaunorubicin aglycone suggests that daunorubicinol is a preferred substrate for reductive deglycosidation. Consequently, the presence of daunorubicinol in the circulation of patients undergoing chemotherapy with daunorubicin may be a factor in the therapeutic efficacy of this antineoplastic agent.

Metabolism of adriamycin in hepatocytes isolated from the rat and the rabbit.

Metabolism of adriamycin, an anthracycline antibiotic, was characterized in both rat and rabbit hepatocytes under aerobic conditions. Adriamycin was the predominant fluorescent species within the cell in hepatocytes from both the rat and the rabbit. In the rat hepatocyte, the primary intracellular metabolite was deoxyadriamycin aglycone, while significant levels of deoxyadriamycinol aglycone were also synthesized; little adriamycinol was observed in the cells or the incubation medium. In contrast, in the rabbit hepatocyte, significant levels of adriamycinol as well as deoxyadriamycinol aglycone were formed; deoxyadriamycinol aglycone was the primary intracellular metabolite while low levels of deoxyadriamycin aglycone were observed. The relative formation of deoxyadriamycinol aglycone and deoxyadriamycin aglycone suggests that adriamycinol may be metabolized more effectively to the deoxyaglycone derivative than the parent drug. Conjugates of adriamycin were not observed in hepatocytes from either the rat or the rabbit or in the incubation medium.

Components of intrinsic drug resistance in the rat hepatoma.

A carcinogen-transformed rat hepatoma cell line (Reuber H-35) was utilized as a model system for investigation of the biochemical factors which may limit the effectiveness of chemotherapy in intrinsically resistant tumors such as hepatocellular carcinoma. Northern blotting demonstrated expression of mRNA coding for the P-170 membrane-glycoprotein associated with the multi-drug resistance phenotype, while Western blotting identified the P-170 glycoprotein in the hepatoma cell membrane. Consistent with these observations, tumor cell sensitivity to the vinca alkaloids, vincristine and vinblastine, to the anthracycline antibiotics, Adriamycin and daunorubicin, and to the demethylepipodophyllotoxin derivative, VM-26, was enhanced by continuous incubation in the presence of the calcium channel antagonist, verapamil. Verapamil produced a minimal change in cell sensitivity to the demethylepipodophyllotoxin derivative, VP-16, and to the aminoacridine, m-AMSA. Relatively high detoxification potential via the glutathione metabolic pathway was also observed in the hepatoma cell. The capacity of topoisomerase II in nuclear extracts from the hepatoma cell to mediate cleavable complex formation stimulated by VM-26, VP-16 and m-AMSA appeared to be at least comparable to, if not greater than that from drug-sensitive HL-60 cells, suggesting that drug resistance may not occur at the level of this enzyme. Consistent with findings in a number of tumor cell lines resistant to antineoplastic drugs, the antiproliferative activity of the topoisomerase II inhibitors VM-26, VP-16 and m-AMSA appeared to be dissociable from the induction of DNA strand breaks, suggesting that such lesions in DNA may fail to fully account for the antiproliferative activity of these agents in the hepatoma cell.

Transplantation of CD34+ peripheral blood cells selected using a fully automated immunomagnetic system in patients with high-risk breast cancer: results of a prospective randomized multicenter clinical trial.

Tumor contamination of autologous peripheral blood stem/progenitor cell grafts occurs in a substantial proportion of high-risk breast cancer patients, and the possibility that such contamination may contribute to relapse has focused attention on methods for removal of the contaminating cells prior to transplantation. One such approach is positive selection of CD34+ cells. A fully automated immunomagnetic cell selection system has recently been introduced to facilitate the positive selection process. A multicenter randomized clinical trial was designed to evaluate the capacity of CD34+ cells isolated using the fully automated system to support prompt hematopoietic reconstitution following high-dose chemotherapy in high-risk breast cancer patients, as well as to assess the safety and tolerability of the CD34+ cell transplants. In recipients of isolated CD34+ cells, the median time to an absolute neutrophil count > or =500/microl was 10 days, a value identical to that observed in patients receiving unfractionated apheresis collections. In the isolated CD34+ cell recipients median time to a platelet count > or =20 000/microl was 12 days, compared with 10 days in the unfractionated cell group. There were no statistically significant differences between the groups in median time to neutrophil or platelet engraftment. Infusion of autologous cells was well tolerated by the study groups. There were no inter-group differences in the incidence of infections, need for platelet transfusions, or duration of hospitalization. Isolated CD34+ cells were high in purity and sufficient in number for use in autologous transplantation. The fully automated immunomagnetic cell selection system affords an efficient and time-saving option for isolation of CD34+cells to be used as autologous grafts in high-risk breast cancer patients, and the isolated CD34+ cells support undelayed hematopoietic reconstitution.

Variable effects of tamoxifen on human hematopoietic progenitor cell growth and sensitivity to doxorubicin.

To determine the influence of tamoxifen on the drug sensitivity of normal human hematopoietic progenitor cells, T-cell- and adherent-cell depleted human bone marrow mononuclear cells (T-, Ad-) were exposed in vitro to 5 microM tamoxifen for 24 h. The effects of tamoxifen were highly variable, as exposure to tamoxifen produced an increase (97% +/- 12.3%) in the growth of day-12 committed myeloid progenitors (CFU-GM) in only four of ten experiments utilizing bone marrow from different donors. When T-, Ad- myeloid progenitor cells treated with tamoxifen were subsequently exposed to doxorubicin, 7 of 14 experimental samples studied demonstrated a net increase in the number of surviving clonogenic cells as compared with cells exposed to doxorubicin alone. Tamoxifen also stimulated the growth of a more purified (CD34(+)-selected) progenitor cell population in four of four experiments (by 62.5% +/- 4.9%) but did not increase the survival of these cells upon exposure to doxorubicin; in fact, in five of ten experimental samples, tamoxifen enhanced cell sensitivity to doxorubicin. Taken together, these observations indicate that tamoxifen produces variable stimulation of committed myeloid progenitor cell growth in vitro. Furthermore, while under some circumstances, tamoxifen appears to have the capacity to enhance CFU-GM survival in the presence of doxorubicin, this drug combination may also result in enhanced toxicity to normal bone marrow progenitors.