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.

Breast tumor contamination of PBSC harvests: tumor depletion by positive selection of CD34(+) cells.

BACKGROUND: Positive selection of CD34(+) cells may reduce or eliminate tumor cells contaminating PBSC harvests of breast cancer (BrCa) patients. However, to assess tumor purging accurately methods may be needed that are of higher sensitivity than standard immunocytochemistry (ICC) assays. METHODS: BrCa-cell depletion, resulting from CD34(+) cell selection, was evaluated using a novel, highly sensitive assay based upon immunomagnetic enrichment with ICC detection in 36 BrCa patients undergoing highdose chemotherapy with autologous PBSC support. RESULTS: The prevalence of BrCa-cell contamination was significantly lower (P = 0.0078) in selected CD34(+) cell fractions (17/35, 49%) from apheresis collections compared with CD34(-) cell fractions (25/35, 71%). In 8/34 (24%) patients, BrCa cells were detected in CD34(-) cell fractions, but not in paired CD34(+) cell fractions. Significantly lower total numbers (P < 0.0005) of BrCa cells were enumerable in CD34(+) cell fractions compared with corresponding apheresis harvests. The median total BrCa-cell content of selected CD34(+) cell fractions with measurable contamination was 22 BrCa cells (range, 6-73 BrCa cells), compared with 3297 BrCa cells (range, 10-98 400 BrCa cells) in apheresis harvests. The median log depletion of BrCa cells achieved by positive CD34(+) cell selection in specimens with detectable contamination both before and after selection was 2.2 (range, 1.7-4.0). Total pre-selection BrCa cell number was significantly predictive (P = 0.004) of residual detectable post-selection contamination. DISCUSSION: Positive CD34(+) cell selection is an effective tumor purging strategy. The prevalence of PBSC contamination in BrCa patients is substantially higher than formerly appreciated.

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.

Enhanced analytical sensitivity of a quantitative PCR for CMV using a modified nucleic-acid extraction procedure.

Accurate and rapid diagnosis of CMV disease in immunocompromised individuals remains a challenge. Quantitative polymerase chain reaction (QPCR) methods for detection of CMV in peripheral blood mononuclear cells (PBMC) have improved the positive and negative predictive value of PCR for diagnosis of CMV disease. However, detection of CMV in plasma has demonstrated a lower negative predictive value for plasma as compared with PBMC. To enhance the sensitivity of the QPCR assay for plasma specimens, plasma samples were centrifuged before nucleic-acid extraction and the extracted DNA resolubilized in reduced volume. Optimization of the nucleic-acid extraction focused on decreasing or eliminating the presence of inhibitors in the pelleted plasma. Quantitation was achieved by co-amplifying an internal quantitative standard (IS) with the same primer sequences as CMV. PCR products were detected by hybridization in a 96-well microtiter plate coated with a CMV or IS specific probe. The precision of the QPCR assay for samples prepared from untreated and from pelleted plasma was then assessed. The coefficient of variation for both types of samples was almost identical and the magnitude of the coefficient of variations was reduced by a factor of ten if the data were log transformed. Linearity of the QPCR assay extended over a 3.3-log range for both types of samples but the range of linearity for pelleted plasma was 20 to 40,000 viral copies/ml (vc/ml) in contrast to 300 to 400,000 vc/ml for plasma. Thus, centrifugation of plasma before nucleic-acid extraction and resuspension of extracted CMV DNA in reduced volume enhanced the analytical sensitivity approximately tenfold over the dynamic range of the assay.

Clinical analysis of the novel breast cancer serum assay BT1.

In an effort to define possible clinical relationships to the BT1 serum assay, a retrospective study population including both Caucasian and African American breast cancer patients was tested. Test results were compared to clinical information provided by the Virginia Cancer Registry. The 64 patients, ages 29 through 69, had a wide range of BT1 values which were not attributable to race or age. The majority of these patients had infiltrating duct carcinoma, with eight additional morphology of neoplasm diagnoses represented in the entire group. No morphology code was associated with either a reactive or non-reactive BT1 result. Staging information was available for 26 patients, diagnosed with stages I, II, and III breast cancer. Positive BT1 values were found throughout these diagnosis stages. In addition, multiple serum samples were available from some patients. Analysis of these longitudinal samples showed different patterns, with test values remaining unreactive in 4 patients, and reactive in 9 others. Interestingly, 5 patients showed values increasing from negative to positive while 3 patients went from positive to negative over time.

A randomized phase 3 study of peripheral blood progenitor cell mobilization with stem cell factor and filgrastim in high-risk breast cancer patients.

This randomized study compared the number of leukaphereses required to collect an optimal target yield of 5 x 10(6) CD34(+) peripheral blood progenitor cells/kg, using either stem cell factor (SCF) at 20 micrograms/kg/d in combination with Filgrastim at 10 micrograms/kg/d or Filgrastim alone at 10 micrograms/kg/d, from 203 patients with high-risk stage II, III, or IV breast cancer. Leukapheresis began on day 5 of cytokine administration and continued daily until the target yield of CD34(+) cells had been reached or a maximum of 5 leukaphereses performed. By day 5 of leukapheresis, 63% of the patients treated with SCF plus Filgrastim (n = 100) compared with 47% of those receiving Filgrastim alone (n = 103) reached the CD34(+) cell target yield. There was a clinically and statistically significant reduction (P <.05) in the number of leukaphereses required to reach the target yield for the patients receiving SCF plus Filgrastim (median, 4 leukaphereses) compared with patients receiving Filgrastim alone (median, 6 or more leukapherses; ie, <50% of patients reached the target in 5 leukaphereses). All patients receiving SCF were premedicated with antihistamines, albuterol, and pseudoephedrine. Treatment was safe, generally well tolerated, and not associated with life-threatening or fatal toxicity. In conclusion, SCF plus Filgrastim is a more effective peripheral blood progenitor cell (PBPC)-mobilization regimen than Filgrastim alone. In addition to the potential for reduced leukapheresis-related morbidity and costs, SCF offers additional options for obtaining cells for further graft manipulation.

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.

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.

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.

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.

Induction of apoptotic DNA fragmentation and c-jun downregulation in human myeloid leukemia cells by the permeant Ca2+ chelator BAPTA/AM.

The permeant Ca2+ chelator acetoxymethyl-1,2-bis(2-aminopheoxy)ethane- N,N,N',N'-tetraacetic acid (BAPTA/AM), an agent previously used to characterize drug-induced apoptosis in neoplastic cells, has been examined with respect to induction of DNA fragmentation and cytotoxicity in the human leukemia cell lines HL-60 and U937. Exposure of cells to various concentrations of BAPTA/AM for 6 h resulted in a biphasic induction of internucleosomal DNA cleavage, with maximal damage occurring at 10-microM concentrations. Higher BAPTA/AM concentrations were associated with the loss of internucleosomal cleavage products, but with the appearance of larger (i.e., 50-kilobase) fragments on pulsed-field gel electrophoresis. Cells exposed to 10 microM BAPTA/AM exhibited classic apoptotic morphology, whereas cells exposed to 50-microM concentrations displayed atypical features (e.g., cell swelling, chromatin clumping); in each case, substantial cytotoxicity was noted. The actions of BAPTA/AM did not depend upon the presence of extracellular Ca2+, nor were they affected by impermeant Ca2+ chelators. Measurement of cytosolic Ca2+ by Fura-2/AM or Indo-1 revealed late but not early increases in intracellular Ca2+ in BAPTA/AM-treated cells. Finally, BAPTA/AM-induced apoptosis was accompanied by the concentration-dependent downregulation of the immediate early response gene c-jun. These findings suggest a complex role for Ca2+ chelators such as BAPTA/AM in the regulation of human myeloid leukemic cell apoptosis, and indicate that this agent may selectively antagonize internucleosomal DNA fragmentation without interfering with other aspects of the apoptotic response and/or cell lethality.

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.

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.

Magnetic resonance imaging of cardiac rhabdomyosarcoma. Quantifying the response to chemotherapy.

This report illustrates the use of cardiac magnetic resonance imaging (MRI) to quantify the initial extent of a cardiac rhabdomyosarcoma and, more importantly, its response to chemotherapy. Image slices spanning the heart and adjacent structures were analyzed using Simpson's rule applied to the image slices to estimate the tumor volume initially, then after 5 weeks, and again after 4 months of chemotherapy. A substantial, progressive reduction in tumor volume during chemotherapy was shown. After chemotherapy was discontinued, an increase in tumor volume was shown. It is suggested that, in addition to being useful in patient care, the technique may be useful in clinical investigations by providing an objective, quantitative measure of tumor response to therapy.

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.

Expression of protein-associated DNA damage in the alkaline elution assay in the absence of enzymatic deproteinization.

In the alkaline elution assay, expression of protein-associated DNA damage induced by topoisomerase II antagonists is facilitated by proteinase K digestion of the drug-stabilized topoisomerase-II-DNA complex. In the absence of this enzymatic deproteinization step, drug-induced DNA strand breaks are masked by the binding of the topoisomerase-II-DNA complex to the synthetic filter from which DNA is eluted subsequent to alkaline denaturation. In this manuscript, we report that as the number of cells lysed on the filter is increased, binding of the topoisomerase-II-DNA complex to the filter is compromised, permitting expression of DNA damage in the absence of enzymatic deproteinization.

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.