Myeloperoxidase is involved in H2O2-induced apoptosis of HL-60 human leukemia cells.

We examined the mechanism of H(2)O(2)-induced cytotoxicity and its relationship to oxidation in human leukemia cells. The HL-60 promyelocytic leukemia cell line was sensitive to H(2)O(2), and at concentrations up to about 20-25 micrometer, the killing was mediated by apoptosis. There was limited evidence of lipid peroxidation, suggesting that the effects of H(2)O(2) do not involve hydroxyl radical. When HL-60 cells were exposed to H(2)O(2) in the presence of the spin trap alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN), we detected a 12-line electron paramagnetic resonance spectrum assigned to the POBN/POBN(.) N-centered spin adduct previously described in peroxidase-containing cell-free systems. Generation of this radical by HL-60 cells had the same H(2)O(2) concentration dependence as initiation of apoptosis. In contrast, studies with the K562 human erythroleukemia cell line, which is often used for comparison with the HL-60, and with high passaged HL-60 cells (spent HL-60) studied under the same conditions failed to generate POBN(.). Cellular levels of antioxidant enzymes superoxide dismutase, glutathione peroxidase, and catalase did not explain the differences between these cell lines. Interestingly, the K562 and spent HL-60 cells, which did not generate the radical, also failed to undergo H(2)O(2)-induced apoptosis. Based on this we reasoned that the difference in H(2)O(2)-induced apoptosis might be due to the enzyme myeloperoxidase. Only the apoptosis-manifesting HL-60 cells contained appreciable immunoreactive protein or enzymatic activity of this cellular enzyme. When HL-60 cells were incubated with methimazole or 4-aminobenzoic acid hydrazide, which are inhibitors of myeloperoxidase, they no longer underwent H(2)O(2)-induced apoptosis. Hypochlorous acid stimulated apoptosis in both HL-60 and spent HL-60 cells, indicating that another oxidant generated by myeloperoxidase induces apoptosis and that it may be the direct mediator of H(2)O(2)-induced apoptosis. Taken together these observations indicate that H(2)O(2)-induced apoptosis in the HL-60 human leukemia cell is mediated by myeloperoxidase and is linked to a non-Fenton oxidative event marked by POBN(.).

Nitric oxide inhibits iron-induced lipid peroxidation in HL-60 cells.

Nitric oxide ((*)NO) can protect cells against the detrimental effects of reactive oxygen species. Using low-density lipoprotein as well as model systems, it has been demonstrated that (*)NO can serve as a chain-breaking antioxidant to blunt lipid peroxidation. To test the hypothesis that (*)NO can serve as a chain-breaking antioxidant in cell membranes, we examined the effect of (*)NO on iron-induced lipid peroxidation in human leukemia cells. We exposed HL-60 cells to an oxidative stress (20 microM Fe(2+)) and monitored the consumption of oxygen as a measure of lipid peroxidation. Oxygen consumption was arrested by the addition of (*)NO as a saturated aqueous solution. The duration of inhibition of oxygen consumption by (*)NO was concentration-dependent in the 0.4-1.8 microM range. The inhibition ended upon depletion of (*)NO. The addition of (*)NO prior to initiation of peroxidation delayed the onset of peroxidation; the nearer in time it was before Fe(2+) addition, the longer the inhibition. Depletion of cellular glutathione levels by d, l-buthionine-S,R-sulfoximine prior to Fe(2+) addition resulted in a more rapid initial rate of oxygen depletion and a shorter time for the (*)NO-induced inhibition of oxygen consumption. Complementary studies of this iron-induced lipid peroxidation, using thiobarbituric acid reactive substances as a marker, also demonstrated the protective effects of (*)NO. This protection of cells against lipid peroxidation also manifested itself as a reduction in trypan blue uptake, an observation demonstrating the protective effects of (*)NO on membrane integrity. We conclude that (*)NO protects HL-60 human leukemia cells from lipid peroxidation and that this protection ameliorates the toxicity of the oxidation processes initiated by Fe(2+) and dioxygen.

Phase I clinical study of fish oil fatty acid capsules for patients with cancer cachexia: cancer and leukemia group B study 9473.

The purpose of this study was to determine the maximum tolerated dose and dose-limiting toxicities of fish oil fatty acid capsules containing omega-3 fatty acid ethyl esters. Twenty-two patients with neoplastic disease not amenable to curative therapy who had lost 2% of body weight over a previous 1 month time period were given an escalating dose of fish oil fatty acids. The maximum tolerated dose was found to be 0.3 g/kg per day of this preparation. This means that a 70-kg patient can generally tolerate up to 21 1-g capsules/day containing 13.1 g of eicosapentaenoic acid + docosahexaenoic acid, the two major omega-3 fatty acids. Dose-limiting toxicity was gastrointestinal, mainly diarrhea, and a poorly described toxicity designated as "unable to tolerate in esophagus or stomach." A patient with chronic lymphocytic leukemia taking the fish oil provided an unusual opportunity to perform a detailed biochemical study of the effect of fish oil capsules on the lipids of malignant cells at several sequential time points in treatment. Studies of the malignant lymphocytes, serum, and whole blood of this one patient revealed an increase in eicosapentaenoic acid, the major component of the fish oil capsules, during fish oil capsule treatment. This study provides a scientific basis for the selection of omega-3 fatty acid doses for future studies in cancer. The maximum tolerated dose found is considerably higher than anticipated from published studies of many human diseases. The observation of a modification of the lipids of leukemic cells, serum, and blood in a patient with chronic leukemia provides a biochemical basis for a possible effect of fish oil supplements on cancer cachexia and tumor growth.

Sensitivity of K562 and HL-60 cells to edelfosine, an ether lipid drug, correlates with production of reactive oxygen species.

Edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine; ET-18-OCH3), a membrane-targeting anticancer ether lipid drug has been shown previously in vitro to be capable of initiating oxidative processes in cells. Here we study two human leukemia cell lines (HL-60 and K562) that have different sensitivities to edelfosine; HL-60 cells are more sensitive than K562 cells. To determine whether edelfosine alters the sensitivity of these lines to an oxidative stress, cells were subjected to the oxidative stress of iron(II) plus ascorbate and then monitored for free radical formation, membrane integrity, and cytotoxicity. The HL-60 cell was sensitive to the ether lipid drug in clonogenic and dye exclusion assays; a lipid-derived free radical was generated by this sensitive cell in the presence of small amounts of Fe2+ and ascorbate as detected by electron paramagnetic resonance and the spin trap alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone. There was also simultaneous generation of an ascorbate-free radical, which has been shown to estimate cellular oxidative flux. In contrast, the K562 cell was resistant to edelfosine cytotoxicity in all assays and did not generate either lipid-derived or ascorbate-free radicals. Subcellular homogenates of the HL-60 cell generated both radicals when exposed to the drug, but homogenates of K562 did not generate either, suggesting that differential drug uptake or intracellular drug localization is not the cause of the difference in oxidation. Trypan blue uptake by the HL-60, but not the K562 cells, measured under the same conditions as the oxidation experiments, demonstrated a loss of membrane impermeability with similar time and concentration dependence, suggesting a causal relationship of membrane damage and radical generation. Complementary studies of HL-60 cell membrane integrity with propidium iodide impermeability and light scatter using the flow cytometer showed a concentration dependence that was similar to radical generation. Biochemical studies of the fatty acids of the HL-60 cell revealed more highly polyunsaturated lipids in the cells. Cellular antioxidant enzymes and vitamin E contents of the two cell lines were similar. We conclude that there is a time- and concentration-dependent generation of important oxidations by the sensitive HL-60 cells exposed to the membrane-targeted ether lipid, but the resistant K562 cells are oxidatively silent. This may be due in part to the differences in fatty acid polyunsaturation of the cellular membranes. The difference in oxidative susceptibility could be the basis for drug resistance to this membrane-specific anticancer agent.

Randomized study of prophylactic platelet transfusion threshold during induction therapy for adult acute leukemia: 10,000/microL versus 20,000/microL.

PURPOSE: We designed and conducted a randomized single-institution trial comparing two common prophylactic platelet transfusion thresholds in patients undergoing induction therapy for acute leukemia. PATIENTS AND METHODS: Seventy-eight patients undergoing induction therapy for acute leukemia were randomized to receive prophylactic apheresis platelet concentrates when the platelet count was either < or = 10,000/microL or < or = 20,000/microL. RESULTS: There was no significant difference in the total number of bleeding episodes per patient with a median of four in the < or = 10,000/microL arm and two in the < or = 20,000/microL arm (25th to 75th percentiles of 2, 7 and 1, 5, respectively; P = .12). Patients randomized to the < or = 10,000/microL arm received more platelet transfusions for bleeding [one (0, 2) v zero (0, 0); P = .0003]. In contrast, patients on the < or = 20,000/microL arm received more platelet transfusions for prophylactic indications [10 (5, 14) v six (3, 8); P = 0.001], as would be expected, but less for bleeding. Nevertheless, the total number of platelet transfusions given to patients on the < or = 20,000/microL arm was higher and nearly significant [11 (6, 15) v seven (5, 11); P = .07]. There were no statistically significant differences between the groups with regard to RBC transfusion requirements, febrile days, days hospitalized, days thrombocytopenic, need for HLA-matched platelets, remission rate, or death during induction chemotherapy. No patient in either group died from hemorrhage or underwent major surgery for bleeding complications. CONCLUSION: Giving prophylactic platelets at a threshold of < or = 10,000/microL compared with < or = 20,000/microL can decrease the total utilization of platelets with only a small adverse effect on bleeding, and no statistically significant effect on morbidity.

Production of lipid-derived free radicals in L1210 murine leukemia cells is an early oxidative event in the photodynamic action of Photofrin.

Photofrin photosensitization initiates a sequence of oxidative events that begins with singlet oxygen formation and ultimately leads to cell death. We hypothesize that membrane lipid-derived free radical formation is an early event in this process. In the presence of iron and ascorbate, lipid free radicals are generated during cellular photosensitization of L1210 cells as detected by electron paramagnetic resonance spin-trapping techniques. Tocopherol levels decline in an inverse manner to lipid radical formation. Trypan blue dye exclusion by membranes also decreases inversely to lipid radical formation but at an initially slower rate than alpha-tocopherol depletion. Propidium iodide nuclear staining as an alternative measure of cell integrity was a later event, occurring when alpha-tocopherol levels had fallen by 90%, trypan blue survival had decreased to below 10%, and lipid radical formation was nearing plateau levels. Likewise, the formation of cellular debris did not occur substantially until alpha-tocopherol was virtually exhausted and radical intensity had nearly reached a maximum. These temporal observations suggest the following sequence of events that leads to Photofrin photosensitization-induced cytotoxicity in the presence of iron and ascorbate: (1) singlet oxygen-derived lipid hydroperoxide formation and subsequent radical production; (2) cellular alpha-tocopherol depletion; (3) trypan blue-detectable membrane leakage; (4) nuclear exposure to propidium; (5) cell disintegration. These observations are consistent with membrane lipid-derived free radical formation being an early and perhaps seminal event in photosensitization by Photofrin, which leads to a concatenated series of events terminating in cell destruction.

Increased efficacy of in vitro Photofrin photosensitization of human oral squamous cell carcinoma by iron and ascorbate.

Photofrin, a photosensitizer used in the photodynamic therapy of cancer, selectively localizes in cellular membranes. Upon exposure to visible light, Photofrin produces singlet oxygen (1O2), which reacts with membrane polyunsaturated fatty acids forming lipid hydroperoxides. Transition metals, such as Fe2+, catalyze the production of cytotoxic free radicals from lipid hydroperoxides. Ascorbate reduces ferric to ferrous iron, further augmenting lipid peroxidation. Therefore, to increase the efficacy of Photofrin photosensitization, we added 20 microM ferrous sulfate and 100 microM ascorbic acid, in an aqueous layer over SCC-25 oral squamous cell carcinoma cells during in vitro illumination. In electron paramagnetic resonance spin trapping experiments, using POBN (alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone), we observed that the presence of this pro-oxidant combination greatly increases the production of membrane-derived lipid free radicals. The effect was time dependent but only partially concentration dependent. Trypan blue dye exclusion demonstrated that this increase in lipid radical formation correlated with cytotoxicity. These observations support the hypothesis that Photofrin photosensitization leads to lipid hydroperoxide formation, which increases the cell's susceptibility to iron-induced Fenton chemistry. The resulting free radical-mediated lipid peroxidation results in cell death. From these data we hypothesize that the efficacy of photodynamic therapy of superficial cancer might be increased by the topical application of the pro-oxidant combination of iron and ascorbate. Furthermore, their use will probably allow lower doses of Photofrin without compromising antitumor effect.

Vitamin E slows the rate of free radical-mediated lipid peroxidation in cells.

Much of what is known about the antioxidant mechanism of vitamin E has been learned from studies of lipid dispersions, solutions, or subcellular organelles. We have investigated the effect of vitamin E supplementation on intact live eucaryotic cells. L1210 murine leukemia cells were exposed to an oxidative stress induced by 20 microM Fe2+ and 100 microM ascorbic acid introduced immediately before oxidative measurements were begun, and the kinetics of the generation of lipid-derived free radicals, as measured by EPR spin trapping (a product) and O2 consumption (a reactant) were measured. Cells grown for 24 h with supplemental (5-100 microM) vitamin E in their media had a slower rate of lipid radical generation compared to cells grown without vitamin E supplementation; this inhibition in the rate of oxidation was generally dependent upon the amount of vitamin E supplementation. In complementary studies measuring O2 consumption, 5-100 microM vitamin E slowed the rate of oxidation (10-fold with 100 microM supplemental vitamin E) consistent with the EPR studies. The membrane active drug edelfosine accentuated the vitamin E effects; vitamin E introduced a discernible lag phase (time delay) in both lipid radical generation and O2 consumption that was not seen in the absence of edelfosine. Vitamin E supplementation of cells also altered the kinetics of ascorbate free radical formation. We conclude that vitamin E inhibits lipid peroxidation in cells by slowing the rate of lipid peroxidation; but with iron/ascorbate as the initiating system, vitamin E does not delay the onset of peroxidation. Of special interest is that these free radical peroxidation events parallel cell membrane damage as detected using trypan blue exclusion. These observations are consistent with the free radical events preceding and causing the observed membrane damage.

Relative alpha-tocopherol deficiency in cultured cells: free radical-mediated lipid peroxidation, lipid oxidizability, and cellular polyunsaturated fatty acid content.

We propose that most cultured cells are deficient in vitamin E. Using our optimized assay for tocopherol, we find that L1210 lymphoblastic leukemia cells, cultured in standard growth media, contain only 2.3 +/- 0.03 micrograms of tocopherol/10(8) cells, whereas when they are transplanted and grown for the same time in the ascites fluid of mice fed standard diets, this increases to 5.8 +/- 0.6 micrograms of tocopherol/10(8) cells. This apparent tocopherol deficiency in cultured cells is likely due to the low concentrations of tocopherol contained in most tissue culture media, even with the addition of serum. To further study this apparent deficiency and the relationship of cellular tocopherol to membrane lipid bis-allylic hydrogen positions, we supplemented the growth media of L1210 lymphoblastic leukemia cells with alpha-tocopherol and compared the resultant cellular tocopherol content to the degree of unsaturation of cellular lipids, alpha-Tocopherol was incorporated by cells in a time- and concentration-dependent manner with plateaus at 24 h and 100 microM, respectively. A maximum 400% increase in cellular tocopherol was easily achieved. By experimentally modifying the fatty acid content of cellular lipids, we were able to determine that cellular tocopherol uptake and content is not a function of cellular lipid composition; cells enriched with polyunsaturated lipids incorporated tocopherol to the same extent as those enriched with more saturated lipids. Thus, as the cellular polyunsaturated fatty acid content increases, the tocopherol:bis-allylic position ratio in the cells decreases, resulting in less antioxidant protection for each lipid double bond. Consequently, when polyunsaturated fatty acid-enriched cells are exposed to an oxidative stress, such as Fe2+, their tocopherol levels decline much faster than cells enriched with saturated fatty acids. This decline is consistent with their respective tocopherol:bis-allylic position ratio. These results provide a basis, at the cellular level, for investigators to consider vitamin E when studying cell response to oxidative stress.

A five-drug remission induction regimen with intensive consolidation for adults with acute lymphoblastic leukemia: cancer and leukemia group B study 8811.

The goal of this phase II multicenter clinical trial was to evaluate a new intensive chemotherapy program for adults with untreated acute lymphoblastic leukemia (ALL) and to examine prospectively the impact of clinical and biologic characteristics on the outcome. One hundred ninety-seven eligible and evaluable patients (16 to 80 years of age; median, 32 years of age) received cyclophosphamide, daunorubicin, vincristine, prednisone, and L-asparaginase; 167 patients (85%) achieved a complete remission (CR), 13 (7%) had refractory disease, and 17 (9%) died during induction. A higher CR rate was observed in younger patients (94% for those < 30 years old, 85% for those 30 to 59 years old, and 39% for those > or = 60 years old, P < .001) and in those who had a mediastinal mass (100%) or blasts with a T-cell immunophenotype. Eighty percent of B-lineage and 97% of T-cell ALL patients achieved a CR (P = .01). The coexpression of myeloid antigens did not affect the response rate or duration. Seventy percent of those with cytogenetic or molecular evidence of the Philadelphia (Ph) chromosome and 84% of those without such evidence achieved a CR (P = .11). Patients in remission received multiagent consolidation treatment, central nervous system prophylaxis, late intensification, and maintenance chemotherapy for a total of 24 months. After a median follow-up time of 43 months, the median survival for all 197 patients is 36 months; the median remission duration for the 167 CR patients is 29 months. Favorable pretreatment characteristics relative to remission duration or survival are younger age, the presence of a mediastinal mass or lymphadenopathy, a white blood cell count (WBC) less than 30,000/microL, L1 morphology, T or TMy immunophenotype, and the absence of the Ph chromosome. The estimates of the proportion surviving at 3 years are 69% for patients less than 30 years old, 39% for those 30 to 59 years old, 89% for those who had a mediastinal mass, 59% with WBC less than 30,000/microL, 63% with L1 morphology, 69% for T or TMy antigen expression, and 62% for those who lack the Ph chromosome. Fifteen patients (8%) had no unfavorable prognostic factors and have an estimated probability of survival at 5 years of 100% (95% confidence interval, 77% to 100%). This intensive chemotherapy regimen produces a high remission rate and a high proportion of durable remissions in adults with ALL.

Bone marrow morphology during induction phase of therapy for acute myeloid leukemia (AML).

Sequential bone marrow aspirates and sections from patients with acute myeloid leukemia (AML) were examined to determine if a correlation exists between bone marrow morphology during induction phase of therapy and outcome. Of 95 patients of AML diagnosed between July 1987 and December 1991, 53 uniformly treated patients (induction therapy with cytosine arabinoside and daunorubicin) had sequential bone marrow examinations performed in the 2- to 5-week period following initiation of induction therapy. Four morphologic patterns were recognized in these 53 patients: Group I (22 patients)--hypocellularity or normal regeneration (> or = 15% cellularity and < 5% blasts) on the initial 2-week marrow followed by marrows showing normal regeneration; Group II (10 patients)--hypocellularity followed by "reactive myeloblastosis" (> or = 15% cellularity, 5% to 34% blasts, with promyelocytes = or > blasts); Group III (12 patients)--residual blasts (> or = 5% blasts with blasts >> promyelocytes) in the initial posttherapy marrow; Group IV (9 patients)--atypical patterns not fitting any of the other categories. Complete remission was achieved in all 32 patients in Groups I and II without additional induction therapy, but was achieved eventually in only 10 of 21 patients in Groups III and IV combined (p < 0.005), 15 of whom received additional induction therapy. Remission duration and actuarial survival for each group were as follows: Group I: 344/596 days; Group II: 443 days/> 660 days; Group III and IV combined: 351/311 days (p = 0.017 for actuarial survival). Seven of 21 patients in Groups III and IV had unfavorable initial morphology (MO, hypoplastic AML and AML preceded by myelodysplasia) compared to only 3 of 32 patients in Groups I and II (p = 0.039). It was thus observed that "reactive myeloblastosis" with up to 34% blasts on the third or fourth week bone marrow following an initial hypocellular marrow does not require additional induction therapy to achieve durable remissions or favorable survival. Also, residual blasts that outnumber promyelocytes, and atypical patterns of regeneration correlate with lower remission induction rates, shortened survival, and unfavorable morphology on the initial diagnostic bone marrow.

Free radical-mediated lipid peroxidation in cells: oxidizability is a function of cell lipid bis-allylic hydrogen content.

Oxidizability of lipids in homogeneous solution varies linearly with the extent of their unsaturation. In vitro cellular, as well as in vivo, studies of oxidizability have generally relied upon chemical indicators of peroxidation such as thiobarbituric acid-reactive substances. To examine the oxidizability of lipids in cells, we have measured oxygen uptake and, using electron paramagnetic resonance spin trapping with alpha-(1-oxo-4-pyridyl)-N-tert-butylnitrone (POBN), the real time generation of lipid-derived free radicals. We have used our experimental in vitro cellular lipid modification model to examine the rate and extent of lipid peroxidation versus the degree of lipid unsaturation in L1210 murine leukemia cells. Lipid peroxidation was stimulated using the prooxidants iron, ascorbate, and the ether lipid compound 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine. We did a total cellular lipid analysis to determine the number of lipid carbon-carbon double bonds contained in L1210 cells enriched with eight fatty acids of different degrees of unsaturation. We found in cellular lipids that (i) lipid chain length had no apparent effect on the rate or extent of radical formation; (ii) the maximum amount of lipid radical generated increases with the total number of bis-allylic positions in the cellular lipids; and, most importantly, (iii) the rate of cellular lipid peroxidation increases exponentially with the number of bis-allylic positions. Our quantitative results clearly demonstrate, for the first time, that the number of bis-allylic positions contained in the cellular lipids of intact cells determines their susceptibility, i.e., oxidizability, to free radical-mediated peroxidative events.

Acute neutrophilic dermatosis with myeloblastic infiltrate in a leukemia patient receiving all-trans-retinoic acid therapy.

We report a case of leukemia-associated acute febrile neutrophilic dermatosis (Sweet's syndrome) that is unique because its initial histologic findings mimicked leukemia cutis. Otherwise, the clinical manifestations and response to corticosteroid therapy were typical of Sweet's syndrome. The onset of the dermatosis coincided with the onset of neutrophilic differentiation induced by single-agent leukemia therapy with all-trans-retinoic acid (ATRA). Subsequent exacerbation of the manifestations of Sweet's syndrome and the ultimate conversion of the histologic picture to the expected mature neutrophilic dermal infiltrate coincided with the completion of neutrophilic differentiation in the peripheral blood and bone marrow. The ability of immature neutrophil precursors to induce cutaneous lesions of Sweet's syndrome may indicate an ATRA-induced functional maturation that slightly precedes its effect on morphologic maturation. We conclude that a cutaneous infiltrate of early neutrophil precursors does not preclude a diagnosis of Sweet's syndrome in patients with acute leukemia who respond to ATRA therapy.

Membrane lipid free radicals produced from L1210 murine leukemia cells by photofrin photosensitization: an electron paramagnetic resonance spin trapping study.

We have detected membrane lipid-derived free radicals from neoplastic cells subjected to Photofrin photosensitization. The presence of the prooxidants iron or iron plus ascorbate in the L1210 cell system increased the intensity of the spin-trapped lipid radical electron paramagnetic resonance spectra and correspondingly decreased cell survival. In addition, raising the proportion of unsaturated lipids in the cell membranes by supplementation of the growth medium with docosahexaenoic acid increased lipid radical formation and decreased cell survival when the L1210 cells were subjected to Photofrin and light. These data educe the hypothesis that the extent of radical generation as well as the efficacy of photodynamic therapy can be increased when prooxidant conditions, which enhance free radical processes, are present in conjunction with photosensitizers that target membrane lipids.

Unidirectional membrane uptake of the ether lipid antineoplastic agent edelfosine by L1210 cells.

We have studied the cellular uptake of edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine; ET-18-OCH3), a membrane active anticancer drug of the ether lipid family, by L1210 murine leukemia cells. Initial unidirectional linear uptake velocity was 1.1 nmol/min per 2 x 10(6) cells; at about 30 min it reached a steady-state phase of accumulation of approximately 5 nmol/2 x 10(6) cells. Concentration studies indicated no saturation kinetics from 0 to 40 microM. Studies with metabolic inhibitors displayed no energy dependence. There was no effect of chloroquine, monensin or cytochalasin B, which are known inhibitors of endocytosis. The inhibitory effect of lower temperature on uptake was moderate in extent and compatible with passive diffusion. There was no efflux of drug from preloaded cells which indicates intense binding of incorporated drug to cells. In human serum, edelfosine bound to several protein components, primarily high density lipoprotein and albumin, and this may explain why cellular uptake was slowed considerably by the presence of serum or albumin in the incubation medium. We conclude that the lipophilic ether lipid derivative edelfosine is taken up by passive diffusion by the L1210 cell. It is tightly bound to cellular structures, probably by insertion into the membrane lipid bilayer.

Minimally differentiated acute leukemia.

We have studied 35 adult patients with morphologically undifferentiated peroxidase-negative acute leukemia that failed to meet the criteria for acute lymphoblastic leukemia and compared them to patients with FAB M1-M7 seen by the same physicians. The diagnosis of minimally differentiated acute leukemia (MD-AL) was associated with a higher incidence of prior hematologic disease, lower WBC, fewer blood blasts, lower marrow cellularity and a tendency towards older age. Of all patients treated with AML since January 1983, those with MD-AL were less likely to get a complete remission than those with other subtypes (35 vs 64%, p = 0.03). Treatment failure was usually due to resistant disease. Analysis of outcome as a function of drugs used during induction therapy showed an advantage for regimens containing vincristine and prednisone. The leukemic blast cells of nine patients were immunophenotyped for myeloid, lymphoid and megakaryoblast/platelet antigens. Although there were too few for a full statistical analysis as was applied to the larger group of 35 patients with MD-AL, these patients had a lower bone marrow cellularity as compared to FAB M1-M7 and a low remission rate. Eight of these were found to have positive myeloid markers and met the criteria for FAB M0. We conclude that patients with MD-AL form a distinct group with characteristic presenting features and a low response rate. Outcome data suggest that vincristine and prednisone should be included in experimental induction programs.

Increased generation of lipid-derived and ascorbate free radicals by L1210 cells exposed to the ether lipid edelfosine.

Using the spin trap alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone, we have detected a lipid-derived carbon-centered free radical generated from intact L1210 lymphoblastic leukemia cells that were exposed to 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (edelfosine or ET-18-OCH3) and oxidative stress. The spectral characteristics, including hyperfine splitting constants of aN = 15.61G and aH = 2.65G, were consistent with the spin trapping of an alkyl radical. Radical detection required iron and prior enrichment of cellular components with the polyunsaturated fatty acid docosahexaenoic acid; unmodified cells failed to generate detectable free radical. Ascorbate further enhanced radical generation. The detection of lipid-derived free radicals when intact cells are exposed to edelfosine provides further evidence that oxidative stress may play an important role in the cytotoxic mechanism of this class of anticancer drug.

Effects of exogenous lipids on cancer and cancer chemotherapy. Implications for treatment.

The addition of fatty acids to the diets of tumour-bearing animals results in specific and defined structural modification of the tumour membrane lipids without disrupting the cell. Furthermore, fatty acids at higher concentrations may act directly as anticancer agents, and there is evidence of selective sensitivity of neoplastic cells. Similarly, experimental enrichment of the diets with specific lipids modulates carcinogen-induced tumorigenesis at the initiation or promotion steps, and in some animal models, the growth rate of established tumours. Therefore, anticancer therapies which utilise lipid-based strategies may be useful clinically. Although dietary strategies used alone may have some favourable effect, it seems likely that the combination of diet with anticancer drugs has the best possibility of providing the extent of cytotoxicity required for tumour eradication. Such combinations could take advantage of an additive effect of each, or could act synergistically such as by the influence of dietary fatty acid modification on drug transport. However, dietary lipids may also increase the toxicity of anticancer drugs to normal tissues and decrease the therapeutic index. Further research is needed to define the role of lipids in future chemotherapy.

Congenital dyserythropoietic anemia type II diagnosed in a 69-year-old patient with iron overload.

Congenital dyserythropoietic anemia type II is a rare disorder that is often diagnosed in patients before age 20 years. Patients with this disorder, which is also called hereditary erythroblastic multinuclearity associated with a positive acidified serum lysis test, may have symptoms of iron overload. The purpose of this case report is to alert physicians to consider the diagnosis of congenital dyserythropoietic anemia type II in elderly patients who have anemia and iron overload.

Membrane peroxidative damage enhancement by the ether lipid class of antineoplastic agents.

The ether lipid antineoplastic agents have no known interaction with DNA, but rather they appear to target membranes. The primary mechanism of action is unknown but effects on membrane biology are documented. We have studied the effect of two ether lipids on membrane lipids and examined the hypothesis that membrane peroxidative damage may be involved in their mechanism of action. With the use of cells having membranes enriched in polyunsaturated fatty acids of the omega-3 family of fatty acids, we have demonstrated that the prototypical ether lipid 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine and a thioether lipid analogue, 1-O-hexadecylmercapto-2-methoxymethyl-rac-glycero-3-phosphocholine , increase membrane lipid peroxidation and cytotoxicity in a time- and drug concentration-dependent manner. The oxidative cofactors Fe2+ and ascorbic acid were required. The pattern of cell death did not fully correspond to the peroxidation, since cofactors were required for peroxidation but not cytotoxicity. However, the rate of decrease in cell viability after exposure to the drug and cofactors corresponded to the peroxidation rate. In addition, when L1210 cells modified with the monounsaturated fatty acid oleic acid or unmodified cells were used, there was no ether lipid-enhanced peroxidation, and the cells were significantly less sensitive to the drug, with or without cofactors. The lipid-soluble antioxidant vitamin E inhibited 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine peroxidation and cytotoxicity in a concentration-dependent manner in the presence of cofactors but not consistently without them. Depletion of cellular glutathione content of L1210 cells using L-buthionine-(SR)-sulfoximine resulted in 40% augmentation of cofactor-facilitated cytotoxicity of 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine and a borderline effect on peroxidation. Another ether lipid, the thio compound 1-O-hexadecylmercapto-2-methoxymethyl-rac-glycero-3-phosphocholine , enhanced peroxidation in the presence of cofactors with kinetics corresponding to those of cytotoxicity. In the presence of ether lipid and cofactors the intensity of ascorbate free radical increased, consistent with oxidative stress. We conclude that the ether lipids stimulate membrane lipid peroxidation in a time- and drug concentration-dependent manner in the presence of oxidative cofactors. Even though peroxidation may not fully explain the cytotoxic effect of the ether lipid class of anticancer drugs, this observation provides further information on the nature of the membrane damage induced by the drugs. Since the ether lipids generate no known free radical intermediates directly, this suggests that membrane damage indirectly results in a process involving a peroxidative reaction.