Cytotoxicity of doxorubicin for normal hematopoietic and acute myeloid leukemia cells of the rat

Summary The dose response curves of doxorubicin for hematopoietic rat bone marrow cells were investigated and compared with the dose-response curves of doxorubicin for leukemia cells from bone marrow and spleen of rats inoculated with an acute myelocytic leukemia (BNML). Various assays were used to determine the cytotoxicity of doxorubicin. It was found that the inhibition of DNA synthesis by doxorubicin compared well with results obtained with in vivo assays for the determination of clonogenic hematopoietic (CFU-S) and leukemic (LCFU-S) cells. It was found that doxorubicin at concentrations ranging from 0.1–1.0 g · 10^-7 cells inhibits DNA synthesis of leukemic cells to 60% and that of hematopoietic cells to 90%. Higher doxorubicin concentrations further inhibit DNA synthesis of only hematopoietic cells. These results were confirmed with clonogenic assays. Pre-treatment with the S phase-specific drug arabinoside cytosine (ara-C) increased the efficacy of doxorubicin in vitro significantly. In view of the doxorubicin concentrations in bone marrow obtained in vivo (1 g · 10^-7 cells), it is concluded that dosage reduction may reduce toxicity with no concomitant decrease of antileukemic activity of doxorubicin.     

Modulation of induced resistance to adriamycin in two human breast cancer cell lines with tamoxifen or perhexiline maleate

Summary The clinical utility of adriamycin in the treatment of patients with metastatic breast cancer is oftenlimited by the development of drug resistance. It has been recognized that in addition to the development of primary resistance against adriamycin, malignant cells can simultaneously develop cross-resistance to other agents. An adriamycin-resistant human breast cancer cell line (MCF 7^Ad) was developed by exposing the parent line (MCF 7) to gradually increasing concentrations of adriamycin while the cells were being grown in monolayer. Using these lines in a clonogenic assay, the relative drug sensitivities to adriamycin, vinblastine, melphalan, 5-fluorouracil and methotrexate were studied. MCF 7^Ad was 12.5-fold more resistant to adriamycin than MCF 7 and 500-fold cross-resistant to vinblastine. There was no cross-resistance to melphalan, 5-fluorouracil or methotrexate. The resistance of MCF 7^Ad was decreased by simultaneous exposure to tamoxifen (by a factor of 3.33) or perhexiline maleate (by a factor of 7.50). This decreased resistance was evidenced by a shift to the left of the sensitivity curves. However, there was no consistent change in the sensitivity curves of MCF 7. At the selected concentration of tamoxifen and perhexiline maleate, the cloning efficiency of MCF 7 and MCF 7^Ad was 80%–90% of control values in medium without tamoxifen, perhexiline maleate or cytotoxic drugs. The resistance of MCF 7^Ad to adriamycin was associated with a lower accumulation of [¹⁴C]adriamycin than exhibited by the sensitive MCF 7 line. There was no consistent change in [¹⁴C]adriamycin accumulation in MCF 7 or MCF 7^Ad when tamoxifen was added, but when perhexiline maleate was added the [¹⁴C] accumulation increased. These results suggest that the tamoxifen-induced change in MCF 7^Ad adriamycin resistance was not due to an increase in the amount of cell-associated adriamycin, but rather to some other mechanism that increased the cytotoxicity of the adriamycin.Abbreviations RPMI 1640 Roswell Park Memorial Institute medium 1640 - FBS fetal bovine serum - PBS phosphate buffered saline - trypsin-EDTA trypsin (w/0.05%) plus ethylenediaminetetraacetic acid (w/0.02%) - IC₅₀ concentration of each drug, extrapolated from concentration versus colony formation curve, that inhibits colony formation by 50% as compared to controls without exposure to the drug     

Intracellular uptake and cytotoxic effect in vitro of doxorubicin and epirubicin in human leukemic and normal hematopoietic cells

Summary Leukemic cells from patients presenting with acute nonlymphoblastic leukemia and normal hematopoietic bone marrow cells from healthy donors for allogeneic bone marrow transplantation were incubated for 3 h with doxorubicin and epirubicin at different concentrations. The intracellular uptake at the end of the incubation was determined by photofluorometry in leukemic cells from 15 patients and in normal cells from 9 donors for bone marrow transplantation. Cytotoxicity in vitro against granulocyte/macrophage colony-forming units (CFU-GM) was determined in normal cells from 7 donors, and in vitro toxicity against leukemic cells was determined by a clonogenic technique in cells from 6 patients and by vital dye staining (DiSC) following 4 days' culture in cells from 15 patients. Epirubicin was significantly less toxic than doxorubicin to normal hematopoetic cells (72%±20% survival of cells for epirubicin vs 45%±13% for doxorubicin at a concentration of 0.2 m;P0.005). As analyzed by the DiSC assay, 0.2 m epirubicin was slightly more toxic to leukemic cells than was the same concentration of doxorubicin (47% vs 61% survival,P0.01), but the clonogenic assay revealed no difference in toxicity to leukemic cells. At a concentration of 0.2 m, the mean intracellular uptake of epirubicin in leukemic cells was 0.43±0.26 nmol/mg protein as compared with 0.33±0.14 nmol/mg protein for doxorubicin (not significant). In normal cells, the uptake of epirubicin at a concentration of 0.2 m was 0.47±0.25 nmol/mg protein as compared with 0.31±0.21 nmol/mg protein for doxorubicin (not significant). The reduced myelotoxicity observed in vitro together with the retained toxicity to leukemic cells indicates that the therapeutic index of epirubicin is better than that of doxorubicin.     

Purge of malignant cells from bone marrow by hematoporphyrin derivatives and light exposurein vitro

During autologous bone marrow graft in treatment of malignant diseases, it is critical to purge malignant cells from the marrow. In the present study, the sensitivity to photodynamic inactivation of 3 leukemic cell lines was compared with their counterpart normal hematopoietic cells. After mouse leukemic L₁₂₁₀ cells were treated with a preparation of hematoporphyrin derivatives, YHpD, 10 μg/ml for 1 hr. and irradiated with blacklight (peak wavelength 395 nm, light intensity 0.6 mW/cm²) for 5 minutes, the survival rate of clonogenic cells decreased to <10%, while that of bone marrow granulocyte-macrophage progenitor cells (CFU-GM) in DBA/2 mice remained at nearly normal level (>80%). Similar results were obtained when human leukemic HL-60 cells were compared with human CFU-GM and mouse leukemic L₆₁₅ cells with CFU-GM in 615 strain mice. It is suggested that hematoporphyrin photoradiation may be useful for selectively killing leukemic cells in bone marrow.     

Harvesting Blood Stem Cells from Cranial Bone at Craniotomy – A Preliminary Study

Primary brain tumors seldom infiltrate into the cranium, even if they are invasive in the central nervous system. In this study, we examined whether blood stem cells can be harvested from cranial bone at craniotomy. Bone marrow cells in cranial bone were counted in 181 craniotomy specimens after staining with hematoxylin and eosin. Marrow volume was measured in 37 specimens using three-dimensional computed tomography (CT). In 10 cases, viable cells collected from very small bone pieces at craniotomy were cultured to examine granulocyte/macrophage colony-forming units (CFU-GM). In 2 cases, bone marrow cells were practically harvested from removed bone at surgery. The weight of bone flap at craniotomy was 35.0 ± 18.0 g. Bone flap marrow contained 1.5 × 10⁹cells/ml. CT examination showed that bone flap volume was 35.0 ± 9.0ml and marrow ratio was 65.1 ± 13.5%. Thus, at craniotomy, a typical bone flap contained about 3.4 × 10¹⁰ cells. Bone marrow cell count gradually decreased as subject age increased. The bone pieces obtained at craniotomy contained 3.1 ± 3.4 × 10⁶cells/g, and CFU-GM count was 0.4 × 10⁵cells/g. In one case, we collected 3.4 × 10⁸cells, including 1.8 × 10⁶ CFU-GM colonies. In another case, we collected 9.7 × 10⁸ cells, including 4.8 × 10⁶ CFU-GM colonies. These findings indicate that, at craniotomy, the number of blood stem cells in a typical bone flap is sufficient for autologous blood stem cell rescue.     

Heat sensitivity,thermotolerance and protein synthesis of granulocyte and macrophage progenitors from mice and from long-term bone marrow cultures

Ex-vivo purging of leukaemic cells by single or fractionated heating during bone marrow transplantation for acute myeloid or lymphoblastic leukaemias may be possible since many leukaemic cells may be more sensitive to killing by heat than normal bone marrow colony-forming unit-granulocyte and macrophages (CFU-GM). In these studies we have compared heat response, thermotolerance response and heat shock protein (HSP) synthesis of bone marrow progenitors from intact mice or bone marrow obtained from 37°C or 33°C long-term bone marrow cultures (LTBMCs). Such studies were done to examine whether CFU-GM responses to heat are influenced by in vitro growth conditions. In terms of heat response CFU-GM progenitors from LTBMCs showed increased heat sensitivity when compared to CFU-GM from mice, with CFU-GM from 33^CC cultures being more heat-sensitive than those from 37°C cultures. The kinetics of thermotolerance development and decay in CFU-GM were similar from all three systems. Thermotolerance was maximum at 3–9 h, began to decay by 24 h and was absent by 48 h from cells obtained from mice and LTBMCs grown at 37°C. However, CFU-GM from 33°C LTBMCs showed a delay in decay of thermotolerance. The kinetics of die development and decay of thermotolerance remained as above and was independent of incubation conditions whether bone marrow was left in situ or incubated in vitro in medium containing various colony-stimulating factors (CSF) or medium containing no CSF. The synthesis of HSPs was measured by one-and two-dimensional gel electrophoresis. Synthesis of HSP-70 kd was detected following a triggering dose of heat at 0–3 h for bone marrow heated in vivo and 0–6 h for bone marrow from LTBMCs. These studies indicate that CFU-GM heat and thermotolerance response was influenced by the temperature at which the cultures were maintained but not by other differences present in their microenvironment.     

Opposite effect of tumor necrosis factor alpha on granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor-dependent growth of normal and leukemic hemopoietic progenitors.

The effect of recombinant human tumor necrosis factor alpha (TNF-alpha) on normal and chronic myeloid leukemia granulocyte-macrophage progenitors (CFU-GM) growing in semisolid agar cultures in the presence of recombinant granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor was studied. Granulocyte-macrophage colony-stimulating factor-dependent growth of normal and chronic myeloid leukemia bone marrow CFU-GM was greatly enhanced by TNF-alpha at doses of 0.1 to 100 units/ml. Growth enhancement included neutrophil, eosinophil, and monocyte-macrophage colonies and clusters at 7 and 14 days of culture. Since similar results were achieved with highly enriched progenitor cell populations, devoid of accessory cells, an indirect effect on CFU-GM growth through the release by accessory cells of other cytokines upon TNF-alpha stimulation was thus ruled out. By contrast, the same doses of TNF-alpha inhibited the growth of normal CFU-GM in granulocyte colony-stimulating factor-dependent cultures. Taken together, our findings indicate that the final effect of TNF-alpha on normal bone marrow granulocyte-macrophage progenitor growth is dependent on the specific growth factor interacting with it, and that both normal and chronic myeloid leukemia CFU-GM are equally responsive to the combined effects of TNF-alpha and a given colony-stimulating factor.     

Effects of recombinant human tumor necrosis factor on highly enriched hematopoietic progenitor cell populations from normal human bone marrow and peripheral blood and bone marrow from patients with chronic myeloid leukemia

Previous studies using unseparated normal human bone marrow cells have indicated that recombinant tumor necrosis factor alpha (rTNF-alpha) can inhibit the in vitro colony growth by normal granulocyte/macrophage (CFU-GM) and erythroid (BFU-E) progenitor cells in a dose-dependent manner. In the present studies, by using very low numbers of highly enriched normal bone marrow progenitor cell populations as target cells, we have extended these previous findings to provide convincing evidence that erythroid and myeloid colony growth suppression by rTNF-alpha is manifested by a direct interaction between rTNF-alpha and CFU-GM and BFU-E progenitor cells. In addition, the sensitivity of normal peripheral blood and chronic myeloid leukemia bone marrow CFU-GM and BFU-E colony growth to inhibition by rTNF-alpha was examined and found to be comparable with that of normal bone marrow CFU-GM and BFU-E. Although the continuous presence of high doses of rTNF-alpha (5000 units/ml) was required in methylcellulose cultures for maximal CFU-GM (90%) and BFU-E (70%) colony suppression, short-term exposure (24 to 72 hr) of normal bone marrow-enriched progenitor cells to rTNF-alpha, in the absence of hematopoietic growth factors, was sufficient to irreversibly suppress up to 50 to 65% of CFU-GM colony growth. In contrast, the number of BFU-E colonies was increased under these conditions. If, however, hematopoietic growth factors (Mo-T-cell-conditioned medium and erythropoietin) were present during preincubation of the cells with rTNF-alpha, BFU-E were then slightly suppressed while the extent of CFU-GM inhibition remained essentially the same. The suppressive effect of rTNF-alpha on erythroid and myeloid progenitor cell growth appears to be most pronounced on the more primative stages of committed progenitor cell development, since inhibition of CFU-GM- and BFU-E-derived colony growth progressively decreased with the delayed addition of rTNF-alpha to methylcellulose cultures. [3H]Thymidine incorporation was also inhibited by rTNF-alpha in normal bone marrow-enriched progenitor cell populations stimulated to proliferate in liquid culture by colony-stimulating factors. This effect was transient, however, since the activity of rTNF-alpha declined after the first 24 h of culture at 37 degrees C, particularly at low doses of rTNF-alpha where the activity was completely lost after 48 h of culture. This loss of activity appeared to be due to a decreased sensitivity of progenitor cells to the antiproliferative effects of tumor necrosis factor (TNF) after an initial exposure rather than a lack of available TNF.(ABSTRACT TRUNCATED AT 400 WORDS)     

Additive apoptotic effect of STI571 with the cytoprotective agent amifostine in K-562 cell line

Purpose To study the apoptotic effect of the 2-phenylaminopyrimidine derivative STI571 in combination with antioxidant agents on K-562 cell line derived from a Philadelphia chromosome-positive chronic myeloid leukemia patient.Materials and methods K-562 (BCR/ABL+), U-937, and HL60 (BCR/ABL–) leukemic cell lines were incubated with STI571 and the antioxidant agents catalase, glutathione, superoxide dismutase, and amifostine (AMI). Apoptotic effect was analyzed by morphological and flow cytometric criteria.Results STI571 at concentrations higher than 0.25 mol L^–1 produced apoptosis (P<0.05) in K-562 cells only after treatment for 72 h. At the mentioned concentrations, STI571 also induced an increase in the loss of mitochondrial transmembrane potential from 24.6 to 40%. Combination of STI571 (0.5 mol L^–1) with antioxidant agents showed that the cytoprotective agent AMI (0.75 mg mL^–1) produced an additive effect in the proapoptotic activity of STI571 in K-562 cells at nuclear (58.8%±2.0 vs. 28.9%±3.3) and mitochondrial (53.3%±3.6 vs. 29.5%±1.2) levels.Conclusions Our results show that only AMI in combination with STI571, at submicromolar concentration, has an additive effect in K-562 cell line, and it does not have severe toxic effects on Philadelphia chromosome negative cells.     

Selection and immunomagnetic purging of peripheral blood CD34+ cells for autologous transplantation in B-cell non-Hodgkin's lymphomas

Background: Clonogenic tumor cells in the hematopoietic progenitor cell harvest may contribute to relapse after high dose therapy for B-cell malignancies. Purging of the HPC harvest requires large amounts of anti-B-cell antibodies, whereas CD34-selection enriches self renewing HPC's but malignant cells are still detectable in many CD34+ fractions.Patients and methods: We examined the feasability and safety of a CD34-selection followed by purging with anti-B-cell antibodies in 11 patients with B-cell non-Hodgkin's lymphomas undergoing high-dose therapy with cyclophosphamide, BCNU and etoposide with retransfusion of autologous HPC's.Results: A mean number of 340 × 10⁸ mononuclear cells was used for CD34-selection and immunomagnetic purging. CD34+ cells were enriched from a mean of 1.7% (range 0.2%–4.5%) to a mean of 68% (range 49%–87%) with a mean recovery of 27% (range 15%–43%). The mean number of retransfused CD34+ cells was 1.2 × 10⁶/kg (range 0.6–2.2 × 10⁶/kg) body weight with a median of 11 days (range 10–13 days) to neutrophil recovery of 0.5 × 10⁹/l and 17 days (range 13–25 days) to platelet recovery of 50 × 10⁹/l. Mean number of intravenous antibiotics and inpatient days were 8 (range 0–14) and 22 (range 19–26) respectively. Major toxicity consisted in four septicemias.Conclusions: CD34-selected and purged HPC's are safe and mediate rapid hematological recovery after high dose therapy for B-cell non-Hodgkin's lymphomas.     

Effects of recombinant growth factors on radiation survival of human bone marrow progenitor cells

The purpose of this study was to evaluate the individual radioprotective effects of 4 distinct purified recombinant human hematopoietic growth factors, namely recombinant human granulocyte-macrophage colony stimulating factor (rGM-CSF), recombinant human granulocyte colony stimulating factor (rG-CSF), recombinant human interleukin 1 (rIL-1), and recombinant human interleukin 2 (rIL-2) on human myeloid (CFU-GM) and erythroid (BFU-E) bone marrow progenitor cells. We demonstrate that (a) preconditioning with rGM-CSF, rG-CSF, or rIL-1 enables CFU-GM to repair sublethal radiation damage and renders CFU-GM less radiosensitive, (b) preconditioning with rGM-CSF or rIL-1 enables BFU-E to repair sublethal radiation damage, and (c) preconditioning with rIL-2 does not increase the radiation survival of CFU-GM or BFU-E. The effects of recombinant growth factors, in particular rGM-CSF, on the radiation damage repair, radiosensitivity, and proliferative activity of bone marrow progenitor cells resulted in a substantial increase in the mean numbers of progenitor cell-derived hematopoietic colonies in irradiated marrow samples. The effects of rGM-CSF on the radiation response of CFU-GM and BFU-E, and the effects of rG-CSF as well as rIL-1 on the radiation response of CFU-GM did not appear to require the presence of T-cells/T-cell precursors, NK-cells, B-cells/B-cell precursors, monocytes, macrophages, MY8 antigen positive non-CFU-GM myeloblasts, promyelocytes, myelocytes, metamyelocytes, granulocytes, or glycophorin A positive erythroid cells since virtually identical results were obtained with unsorted marrow samples or highly purified fluorescence activated cell sorter (FACS) isolated progenitor cell suspensions. To our knowledge, this report represents the first study on recombinant human growth factor-induced modulation of the radiation responses of normal human bone marrow progenitor cells.     

Increased hematopoietic progenitor cell maintenance in long-term bone marrow cultures containing minimal numbers of contaminating breast cancer cells

The maintenance of hematopoietic progenitor cells as assayedin the mixed colony (CFU-GEMM) assay in humanlong-term bone marrow cultures was compared between normalallogeneic marrow transplantation donor collections and those fromcandidates for high-dose therapy and autologous bone marrowtransplantation (ABMT). To be eligible for ABMT, patientswere required to have a histologically normal appearingbone marrow and therefore any tumor contamination wasat minimal levels and detectable only after evaluationof the cultured harvests. Marrow from 15 normaldonors, 36 patients with breast cancer, and 30patients with Hodgkin's disease was evaluated. The numberof mononuclear cells placed in culture was standardized.In all groups, significantly more progenitor cells wererecovered at 4–6 weeks of culture than at12–14 weeks. At 4–6 and 12–14 weeks, therewere no significant differences in the number ofprogenitor cells recovered from the cultures of normaldonors and tumor negative cultures of breast canceror Hodgkin's disease patients. However, following 4–6 and12–14 weeks of culture, progenitor cell numbers ofcultures which contained breast cancer cells were significantlyhigher than the pooled values for cultures fromthe concurrent normal controls, and those from breastcancer and Hodgkin's disease patients with tumor negativecultures. These results suggest that minimal breast cancercell contamination of the bone marrow can influencethe production of marrow progenitor cells. Exposure toprior chemotherapy or radiation therapy does not appearto be the cause of this effect. Themost likely mechanism is the local production ofcytokines by the tumor cells, although a processinvolving direct adhesive contact of the tumor cellswith hematopoietic cells, which is sometimes observed insemisolid cultures, cannot be excluded.     

Antigenic characteristics of circulating CFU-GM in chronic granulocytic leukaemia resemble those of CFU-GM in normal marrow and differ from those in normal blood

We studied the surface antigenic determinants of myeloid progenitor cells (Day 7 CFU-GM, Day 14 CFU-GM and BFU-E) in the peripheral blood and bone marrow of patients with chronic granulocytic leukaemia (CGL) and normal subjects by complement-mediated cytotoxicity with a panel of 8 selected murine monoclonal antibodies (McAbs) followed by culture in methyl cellulose. All classes of progenitor cell studied expressed HLA-DR antigens and also expressed other antigens recognized by two of the McAbs (S3-13 and S17-25) with myeloid specificity. Two other McAbs (R1.B19 and WGHS.29.1). Recognized antigens on Day 14 CFU-GM derived from normal marrow but not on those from normal blood. The pattern of reactivity of Day 14 CFU-GM from the blood of patients with CGL resembled to a considerable extent that of CFU-GM from normal marrow and differed from that of CFU-GM from normal blood. BFU-E from the blood of patients with CGL reacted with these McAbs in a manner very similar to that of BFU-E from normal blood; however the same two McAbs (R1.B19 and WGHS.19.1) reacted with a much higher proportion of the BFU-E from the marrows of CGL patients than of normal subjects. Our data are consistent with the hypothesis that normal blood-derived CFU-GM are more primitive than marrow-derived CFU-GM; however the CFU-GM in the circulation in CGL differ from those in normal blood, perhaps because they reflect overflow from or exchange with a hyperplastic marrow population.     

Hematopoietic progenitor cell collection and neoplastic cell contamination in breast cancer patients receiving chemotherapy plus granulocyte-colony stimulating factor (G-CSF) or G-CSF alone for mobilization

Background: We compared hematopoietic progenitor cell (HPC) collection and neoplastic cell contamination in breast cancer patients given cyclophosphamide (CTX) plus granulocyte-colony stimulating factor (G-CSF) or G-CSF alone for mobilization.Patients and methods: In 57 stage II–III breast cancer patients, CD34+ cells, colony-forming units-granulocyte macrophage (CFU-GM), early HPC and breast cancer cells were counted in HPC collections obtained after CTX plus G-CSF (n = 27) or G-CSF-alone mobilization (n = 30).Results: The CD34+ cell collection was about two-fold greater after CTX plus G-CSF mobilization (11.0 ± 7.9 vs. 5.8 ± 3.5 × 10⁶/kg, P < 0.001). Similarly, the total number of CFU-GM, CD34+CD38– cells and of week-5 cobblestone area forming cells (CAFC) collected was significantly higher in patients mobilized with CTX plus G-CSF. Breast cancer cells were found in the apheresis products of 22% of patients mobilized with CTX plus G-CSF and in 10% of patients mobilized with G-CSF alone (P = 0.36). Of seven patients who failed G-CSF-alone mobilization and eventually underwent chemotherapy plus G-CSF mobilization, none had cytokeratin-positive cells after G-CSF mobilization, whereas four out of seven had cytokeratin-positive cells after chemotherapy plus G-CSF (P = 0.07 by ² test).Conclusion: The CTX plus G-CSF mobilization protocol was associated with a significantly higher HPC collection. However, this benefit was not accompanied by a reduction in the incidence of tumor-contaminated HPC graft.     

The relative sensitivity of peripheral blood T-lymphocyte colony forming cells and bone marrow CFU-GM to deoxyadenosine and 2'deoxycoformycin

The effects of the nucleoside deoxyadenosine (AdR) towards 2'deoxycoformycin (dCF) treated peripheral blood T-lymphocyte and bone marrow myeloid progenitor cells has been studied. Mononuclear cell preparations were exposed to dCF and AdR in suspension culture prior to washing and culture in a medium free of exogenous nucleosides. The combination of dCF and AdR was found to be highly toxic to peripheral blood T lymphocyte colony forming cells (CFU-TL) following prolonged (18 h) incubation. CFU-GM were markedly less sensitive to the combination of dCF and AdR, concentrations of dCF (10(-5) M) and AdR (10(-5) M) which produced an almost total inhibition of CFU-TL only inhibited CFU-GM by a mean of 10%.     

A multi-center prospective phase II study of high-dose chemotherapy in germ-cell cancer patients relapsing from complete remission

Purpose: To prospectively determine the efficacy of repeated high-dose alkylating chemotherapy to salvage patients with germ-cell tumors who relapsed after adequate first-line chemotherapy.Patients and methods: Patients with germ-cell cancers relapsing from a first, second or third complete remission induced by chemotherapy were offered to participate in a Dutch national prospective trial with broad entry criteria. The salvage treatment began with a conventional dose of ifosfamide (4 g/m² on day 1) and etoposide (100 mg/m² on days 1, 2 and 3) followed by daily s.c. administration of G-CSF (10 µg/kg) until peripheral blood progenitor cells had been harvested. Immediately after bone marrow recovery, an intermediate dose chemotherapy course of carboplatin (target AUC: 10 mg · ml^–1 · min on day 1) and etoposide (500 mg/m² on days 1, 3 and 5) was given with G-CSF daily s.c. After bone marrow recovery, two subsequent courses of high-dose CTC chemotherapy were given, each containing cyclophosphamide (6 g/m²), thiotepa (480 mg/m²) and carboplatin (target AUC: 20 mg · ml^–1 · min). The high-dose chemotherapy was administered as 30–60-minute infusions, divided over 4 days and the stem-cell transplants were given 48–72 hours after the last chemotherapy infusion. Whenever possible, residual masses were resected at the end of treatment.Results: Thirty-five patients were treated between January 1994 and October 1997. The toxicity of the treatment was manageable. Second CTC courses were administered in 25 patients and were associated with hemorrhagic cystitis and veno-occlusive disease in 3 and 4 patients, respectively. One patient who had recently undergone a partial hepatectomy, died of veno-occlusive disease. At the time of analysis, the median follow-up of the surviving patients was 37 months (range 12–56 months). The median progression-free survival for all patients was 44 months, and the median overall survival has not been reached. According to the internationally accepted criteria for predicting the outcome of salvage chemotherapy in germ-cell cancer (Beyer et al. J Clin Oncol 1996; 14: 2638–45), 30 patients had good risk criteria. Of these, 29 received high-dose chemotherapy. Of this group, the salvage rate at two years was 65% (95% confidence interval: 49.5%–85.1%).Conclusions: Over half of the germ-cell cancer patients relapsing from a chemotherapy-induced complete remission can be salvaged by a treatment strategy that incorporates high-dose chemotherapy, even when treatment is given in a multi-center setting. These data confirm the international prognostic model proposed by Beyer et al. in a prospectively studied, independent patient group and provide further evidence that high-dose therapy has a role in the salvage setting of patients with germ-cell cancer.     

Differential cytotoxicity of deoxyguanosine and 8-aminoguanosine for human leukemic cell lines and normal bone marrow progenitor cells

The inhibitory effect of deoxyguanosine (GdR) alone or in combination with the purine nucleoside phosphorylase (PNP) inhibitor, 8-aminoguanosine (AG) was tested on human T, B, cALL and myeloid leukaemia cell lines and on normal human bone marrow haemopoietic progenitor cells. GdR was found to be toxic to T-leukaemia cells. AG (100μm ) alone did not have any inhibitory effect, but when used with GdR (2·5 × 10^?5m ) a synergistic effect was seen towards T cells. Incubation with GdR and AG resulted in a marked decrease in cell viability (> 90 per cent in three and > 75 per cent in four of 5 T leukaemic cell lines tested at 72 h). This drug combination did not inhibit the growth of non-T leukaemic cells and was also non-toxic to normal bone marrow multipotent progenitor cells (CFU-GEMM) in vitro. Adenosine deaminase (ADA) acts consecutively with PNP in purine degradation. The addition of an ADA inhibitor, deoxycoformycin and deoxyadenosine, however, did not enhance the toxicity of GdR and AG for T cell leukaemia. The possibility of using GdR and AG for in vitro removal of residual T leukaemic blasts with the sparing of normal bone marrow cells, prior to autologous bone marrow transplantation should be further explored.     

The anti-proliferative effect of suramin towards tamoxifen-sensitive and resistant human breast cancer cell lines in relation to expression of receptors for epidermal growth factor and insulin-like growth factor-I: Growth stimulation in the presence of tamoxifen

Background: A significant proportion of breast cancer patients receiving tamoxifen therapy relapse during treatment following acquisition of tamoxifen-resistant or oestrogen-independent phenotypes. The mechanism behind this rapid progression to oestrogen autonomy is at present unclear and further treatment modalities are limited. Suramin represents a novel potential second line therapy. The mechanism of the antineoplastic activity of suramin is not completely understood, although the drug binds to many growth factors including epidermal growth factor and insulin-like growth factors and can also dissociate growth factors from their receptors. In this study we have related suramin sensitivity to the expression of receptors for epidermal growth factor and insulin-like growth factor-I in a number of breast cancer cell lines including lines resistant to tamoxifen.Materials and methods: The anti-proliferative effects of suramin were investigated in two oestrogen dependent breast cancer lines (ZR-75-1 and MCF-7), oestrogen independent (ZR-PR-LT) and tamoxifen resistant (ZR-75-9a1) variants of ZR-75-1 and a tamoxifen resistant (LY2) variant of MCF-7. Full dose response curves were constructed and IC⁵⁰values determined for each cell line. Sensitivity to suramin was correlated with the level of expressio n of receptors for epidermal growth factor (EGFR) and insulin-like growth factor-I (IGFR). On observing stimulation of cell proliferation by suramin in the tamoxifen resistant cell lines in the presence of tamoxifen we also investigated the possible role of suramin sequestration of transforming growth factor- in mediating this effect.Results: All cell lines exhibited a dose- and time-dependent response to suramin treatment. Tamoxifen resistant ZR-75-9a1 cells (day 6 IC⁵⁰85 µg ml^–1) were more resistant to suramin than oestrogen independent ZR-PR-LT cells (day 6 IC⁵⁰45 µg ml^–1), and the parent ZR-75-1 line (day 6 IC⁵⁰56 µg ml^–1). Increased sensitivity to suramin was associated with increased expression of IGFR and decreased expression of EGFR. Tamoxifen resistant LY2 cells were significantly more sensitive to suramin (day 6 IC⁵⁰70 µg ml^–1) than MCF-7 cells (day 6 IC⁵⁰350 µg ml^–1). Both IGFR and EGFR expression by LY2 cells was lower than in the parent line. The antioestrogen-resistant ZR-75-9a1 and LY2 lines grown in the presence of 8 µM tamoxifen were growth stimulated by concentrations of the drug below 100 µg/ml. As growth stimulation observed in the presence of tamoxifen may have been due to suramin sequestration of tamoxifen induced TGF-1 secretion we also investigated the response of the cells to this peptide in the presence and absence of suramin. All cell lines were growth inhibited by TGF-1 except ZR-75-9a1 which was unresponsive. Responses to TGF-1 were modified in the presence of 100 µg suramin ml^–1 although TGF-1 was unable to mimic the ability of tamoxifen to stimulate proliferation in the presence of suramin.Conclusions: These results suggest that for ZR-75-1 cells and variants, increased sensitivity to suramin is associated with an increase in expression of IGFR and a decrease in EGFR numbers. However, tamoxifen resistant LY2 cells, in which both IGFR and EGFR expression is reduced were considerably more sensitive than parental MCF-7 cells suggesting that there is no clear relationship between EGFR and IGFR expression and suramin sensitivity. The unexpected stimulation of cell proliferation of the tamoxifen resistant variants by suramin in the presence of tamoxifen could not be explained by suramin sequestration of transforming growth factor- and the mechanism of this interaction remains unclear.     

Cellular and plasma pharmacokinetics of weekly 20-mg 4′-epi-adriamycin bolus injection in patients with advanced breast carcinoma

Summary Weekly low-dose injections of 20 mg 4-epiadriamycin (E-ADM) were given to 12 patients with advanced postmenopausal breast cancer for at least 8 weeks. In advance, all patients were given hormonal therapy and polychemotherapy not containing anthracyclines. E-ADM concentrations in plasma and urine and in blood and bone marrow cells were determined during 8 consecutive weeks. Plasma concentrations in the range of a few nanograms per milliliter were seen up to 72–96 h. Cellular concentrations, and were 190±66 ng/10⁹ cells on day 8, before the next injection was given. Nevertheless, no serious bone marrow toxicity was observed. In two patients with an increased plasma bilirubin concentration, cellular E-ADM concentrations were 20%–40% higher than those observed in the other patients. Plasma concentrations of E-ADM and 4-epi-adriamycinol showed terminal half-lives 2–3 times longer and could be followed throughout the week. In three patients biopsies of skin metastases were examined. In two patients E-ADM could be demonstrated in the tumor tissue up to 7 days after the last injection. Although the number of patients investigated is too small to relate the drug kinetics to clinical response, it is of interest that the two patients with the highest cellular E-ADM concentrations responded better than the others.This work was supported by the Queen Wilhelmina Foundation, The Netherlands Cancer Foundation (KWF) and, in part, by Farmitalia Carlo Erba, Milan, Italy     

Intracellular concentrations of anti cancer drugs in leukemic cells in vitro vs in vivo

Summary A comparison of intracellular concentrations of daunorubicin, doxorubicin and ara-C in myeloid blast cells was carried out in vivo and in vitro. In vivo, blood samples were obtained from 27 patients with acute nonlymphoblastic leukemia during and up to 4 days after drug infusion. Leukemic cells were isolated and drug concentrations were determined by HPLC. Before treatment, leukemic cells from 21 patients were isolated from blood and bone marrow, and in vitro incubations were done with anthracyclines for 1–3 h at concentrations of 0.1–1.0 M and with ara-C for 1 h to 5 days at concentrations of 0.5–5.0 M. The cells were cultured for 5 days, during which cell samples were taken for drug determination. The results showed that incubation with 0.2 M daunorubicin for 1 h and 0.2 M doxorubicin for 3 h and continuous exposure to 0.5 M ara-C gave intracellular concentration curves similar to those obtained in vivo. After 5 days' culture, the cytotoxic effect was determined by vital dye staining with fast green, the addition of an internal standard of fixed goose erythrocytes, cytospin centrifugation and counter-staining of living cells with haematoxylin/eosin (DiSC). Incubations at the above-mentioned concentrations exerted a cytotoxic effect of approximately 50%. We conclude that in mimicking the in vivo situation, it is important to consider differences in intracellular pharmacokinetics.