Elimination of leukemic cells by the combined use of ether lipids in vitro.

Two ether lipids, CP-46,665-1 (4-aminomethyl-1-[2,3-(di-n-decyloxy)-n- propyl]-4-phenylpiperidine) and ET-18-OCH3 (racemic 1-O-octadecyl-2-O-methylglycero-3-phosphocholine) have been shown to possess antileukemic activity in vitro. To explore the possible use of these compounds for purging remission bone marrow cells of leukemic cells, we examined the cytotoxic effect of these compounds on normal hematopoietic progenitor cells and leukemic cell line cells (HL-60, K-562, KG-1a, KG-1, and Daudi) by using the clonogenic assay. When cells were treated with CP-46,665-1 or ET-18-OCH3 (50 micrograms/ml for 1 h), these compounds did not inhibit the growth of normal progenitors, whereas the growth of the clonogenic leukemic cells was inhibited with differences in their sensitivities to the cytotoxic effect of CP-46,665-1 and ET-18-OCH3. Incubation of leukemic cells (HL-60 and Daudi cells) with both CP-46,665-1 (50 micrograms/ml) and ET-18-OCH3 (50 micrograms/ml) for 1 h resulted in a greater reduction of clonogenic leukemic cells than treated with each compound alone. Approximately a 3 log killing of clonogenic HL-60 cells and a 5 log killing of Daudi cells was achieved; however, the combined treatment of normal bone marrow cells with CP-46,665-1 and ET-18-OCH3 did not alter the growth of normal progenitors. This combined treatment also selectively eliminated the leukemic cells (HL-60 and Daudi cells) from a mixture (1000:1) of normal bone marrow cells and leukemic cells. It is conceivable that the pronounced difference in sensitivity to this combined treatment can be exploited for the elimination of residual leukemic cells in autologous remission marrow grafts.

Effects of phorbol ester on translocation and down-regulation of protein kinase C and phosphorylation of endogenous proteins in human acute myeloid leukemia cell line KG-1 and its phorbol ester-resistant subline KG-1a.

12-O-Tetradecanoylphorbol-13-acetate (TPA) induced decreases in the catalytic activity and immunoreactivity of protein kinase C (PK-C) in the soluble fraction, accompanied by increases in their activities in the particulate fraction, of a human myeloid leukemia cell line KG-1. TPA also caused a similar down-regulation and translocation of PK-C in KG-1a, a cloned subline shown to be resistant to the differentiating effect of TPA. The activity levels of enzyme in the soluble and particulate fractions from KG-1 cells, however, were about three times higher than those from KG-1a cells. Immunocytochemical studies showed that, when KG-1 cells were treated with 10 nM TPA for 30 min, PK-C was translocated to the plasma membrane in the adherent subpopulation of cells, whereas the enzyme remained largely in the cytoplasm and perinuclear area of the nonadherent cells. TPA, in contrast, caused a PK-C translocation primarily to the perinuclear region in KG-1a cells. Phosphorylation patterns of PK-C substrate proteins in the two cell lines were similar, except that phosphorylation of the Mr 33,000 and 97,000 proteins were predominant in KG-1 and KG-1a cells, respectively. The present findings showed existence of certain differential effects of TPA on the PK-C system in the two leukemia cell lines, suggesting a molecular basis for the selective resistance of KG-1a cells to the differentiating action of TPA.

Effects of thioether phospholipid BM 41.440 on protein kinase C and phorbol ester-induced differentiation of human leukemia HL60 and KG-1 cells.

The synthetic thioether phospholipid BM 41.440 (1-S-hexadecyl-2-methoxymethyl-rac-glycero-3-phosphocholine) was found to inhibit protein kinase C (PKC) activity competitively with respect to phosphatidylserine, with an apparent Ki value of about 6.4 microM. The agent also inhibited the enzyme activated by diolein or 12-O-tetradecanoylphorbol-13-acetate (TPA), without affecting binding of [3H]phorbol-12,13-dibutyrate to the enzyme. Myosin light chain kinase and cAMP-dependent protein kinase were not inhibited by BM 41.440, indicating a specificity of the action of the agent. BM 41.440 partly blocked the TPA-induced depletion of soluble PKC in HL60 and KG-1 cells (responsive to the differentiating effect of TPA) but not in K562 cells (resistant to the TPA effect). The thioether inhibited the phosphatidylserine/Ca2+-dependent phosphorylation of several common proteins in the solubilized homogenates of HL60 and KG-1 cells, and that of apparently distinct proteins in the preparation of K562 cells. The TPA-induced differentiation of HL60 and KG-1 cells was inhibited by BM 41.440 at a concentration inhibitory to PKC, but differentiation of HL60 cells promoted by dimethyl sulfoxide, retinoic acid, and 1,25-dihydroxyvitamin D3, on the other hand, was not affected. The present data suggested that PKC inhibition might partly account for the antineoplastic effect of BM 41.440, and that the agent could be useful in studying involvements of the PKC system in cellular processes.

Effects of alkyl-lysophospholipids on human leukemic cell lines measured by nuclear magnetic resonance.

Part of the cytotoxic action of alkyl-lysophospholipids (ALP) on leukemic cells is known to result from the lack of an O-alkyl cleavage enzyme and its antimetabolic effect which results in a toxic lysophospholipid buildup. Further, ALP (5 micrograms/ml) suppresses clonogenicity and tritiated thymidine uptake in HL60 cultures after 24 hr of exposure. The effect of ALP on two leukemic cell lines, HL60 and K562, measured by two nuclear magnetic resonance (NMR) techniques and examined by electron microscopy is reported. 31P-NMR spectroscopy indicates that the adenosine 5'-triphosphate:adenosine 5'-diphosphate ratios are unaffected after 24 hr, as is mitochondrial morphology, judging by electron micrographs. However, cell membrane integrity in HL60 is altered at that time. The earliest ALP effects occur in NMR internal water relaxation at 1 hr after ALP exposure, followed by a small reduction in tritiated thymidine uptake at 4 hr. No effect is observed in K562 cell cultures in morphology or NMR measurements. No new 31P-labeled metabolites were detected in either cell line as a result of drug treatment.

Phospholipid-sensitive Ca2+-dependent protein phosphorylation system in various types of leukemic cells from human patients and in human leukemic cell lines HL60 and K562, and its inhibition by alkyl-lysophospholipid.

Phospholipid-sensitive Ca2+-dependent protein kinase (PL-Ca-PK), its endogenous substrate proteins, and regulation of the enzyme system by an antitumor agent alkyl-lysophospholipid were investigated in various types of leukemic cells (chronic myelocytic, acute myelocytic, and acute monocytic) from patients and in two cultured human leukemic cell lines (HL60 and K562). Exceedingly high levels of PL-Ca-PK, largely localized in the particulate fraction, were found in all types and lines of leukemic cells; much lower levels of cyclic adenosine 3':5'-monophosphate-dependent protein kinase and cyclic guanosine 3':5'-monophosphate-dependent protein kinase were also found. Although numerous and similar endogenous substrates for PL-Ca-PK were found in all cell types and lines examined, substrates specific for certain leukemic cells appeared to be present. Substrate proteins for calmodulin-sensitive Ca2+-dependent protein kinase, cyclic adenosine 3':5'-monophosphate-dependent protein kinase, and cyclic guanosine 3':5'-monophosphate-dependent protein kinase, in comparison, were much fewer or undetected. The PL-Ca-PK activity and the phosphatidylserine-Ca2+-stimulated phosphorylation of endogenous proteins from leukemic cells were inhibited by alkyl-lysophospholipid, which acted as a competitive inhibitor of the phospholipid cofactor of the enzyme. The findings suggested that the PL-Ca-PK system is a predominant protein phosphorylation system in leukemic cells and that this enzyme system may represent a site of cytotoxic action of alkyl-lysophospholipid.

Isolation of a novel glycoprotein (EDC1) from the urine of a patient with acute myelocytic leukemia.

Gel filtration of urine from Patient ED with acute myelocytic leukemia showed a prominent protein peak with elution position corresponding to molecular weights of 20,000 to 35,000. The protein (EDC1) was isolated in pure form by sequential gel filtration and ion-exchange chromatography. Molecular weight of purified EDC1 was 27,000; it contained 27% carbohydrate and was rich in half-cystine (5% of residues). EDC1 was antigenically and chemically distinct from the recognized glycoproteins of normal plasma. With a specific rabbit antiserum and 125l-labeled EDC1, a radioimmunoassay for the glycoprotein was developed. Both noncancer and cancer plasmas contained immunoreactive material. In noncancer plasma, all the immunoreactivity was eluted from Sephadex G-75 and G-200 in position corresponding to molecular weights of 60,000 to 100,000 (Peak 1). In cancer plasma, an additional peak of immunoreactivity was eluted in the position corresponding to EDC1 (M.W., 20,000 to 30,000; Peak 2). Eighty-six % of urines from patients without clinical cancer were nonreactive in radioimmunoassay (less than 0.1 microgram immunoreactive EDC1 per ml); 11 and 3%, respectively, contained immunoreactivity equivalent to 0.1 to 0.9 and 1 to 9 microgram EDC1 per mi, entirely of Peak 1 type. Ninety-one % of urines from patients with disseminated cancer contained immunoreactivity equivalent to 10 to 9,999 microgram EDC1 per ml, primarily of Peak 2 type.

Abnormal profile of serum proteinase inhibitors in cancer patients.

We reported previously that an antitryptic glycoprotein, EDC1 (Mr 27,500), which is immunologically related to the normal serum proteinase inhibitor, inter-alpha-trypsin inhibitor (IATI), is excreted in large quantities in the urine of metastatic cancer patients. We have now measured immunoreactive titers of urinary EDC1 and five serum proteinase inhibitors (including IATI) in 16 patients with hematological cancers, 9 patients with various solid tumors, and 32 healthy subjects. The mean urinary EDC1 levels were 22-fold greater in all cancer patients as compared to normals [187.0 +/- 136.6 (S.D.) versus 8.4 +/- 8.2 mg/g creatinine; p less than 0.001]. In the cancer group, serum levels of immunoreactive alpha 1-proteinase inhibitor (also called alpha 1-antitrypsin), alpha 1-antichymotrypsin, and C1 inactivator averaged 152, 237, and 165% of the normal values, respectively (p less than 0.01). Immunoreactive alpha 2-macroglobulin levels were unchanged, and immunoreactive IATI levels were depressed (75% of the normals; p less than 0.01). The lower levels of IATI and elevated levels of EDC1 are consistent with the latter being derived from the former. In spite of the increased immunoreactive alpha 1-proteinase inhibitor level, the serum antitryptic capacity of the cancer group averaged only 50% of the normal group (p less than 0.01; range, 5 to 110% of normal average). This suggests that about 70% of the serum alpha 1-proteinase inhibitor in the cancer group is functionally inert.

Effects of selenium compounds on phospholipid/Ca2+-dependent protein kinase (protein kinase C) system from human leukemic cells.

Selenium compounds (selenium dioxide, selenious acid, and selenic acid) were found to inhibit phospholipid/Ca2+-dependent protein kinase (protein kinase C) and the phorbol ester-stimulated phosphorylation of endogenous substrate proteins from HL60 cells. Kinetic analysis indicated that selenium dioxide (SeO2) inhibited the enzyme noncompetitively with respect to phosphatidylserine (apparent Ki, 60 microM) and Ca2+ (apparent Ki, 68 microM). The inhibitory effect of SeO2 on protein kinase C was additive to that of another inhibitor of the enzyme (alkyl-lysophospholipid) when present together. SeO2 was also equally inhibitory to myosin light chain kinase, a calmodulin/Ca2+-dependent class of protein kinase. It, however, affected only very slightly cyclic adenosine 3':5'-monophosphate-dependent protein kinase. It is suggested that inhibition of Ca2+-dependent reactions might be related to the anticarcinogenic property of selenium.

Influence of 1-beta-D-arabinofuranosylcytosine conjugates of lipids on the growth and metastasis of Lewis lung carcinoma.

Five different lipid conjugates of 1-beta-D-arabinofuranosylcytosine (ara-C) were tested for their therapeutic activity on the growth and metastasis of Lewis lung carcinoma (3-LL). Among 1 ester-linked lipid conjugate (Ara-CDP-L-dipalmitin), 3 1-O-alkyl-lipid conjugates (ara-CDP-D,L-PBA, ara-CDP-D,L-PCA, ara-CDP-D,L-MBA) and a thioether-lipid conjugate (ara-CDP-D,L-PTBA) ara-CDP-D,L-PTBA, ara-CDP-D,L-PBA, and ara-CDP-L-dipalmitin produced the strongest tumor growth inhibition and increase of surviving animals in C57Bl6-mice bearing i.p.-implanted 3-LL. Furthermore these conjugates were superior to the parent compounds alone, or equimolar mixtures of the alkyllysophospholipid derivatives ET-18-OCH3 and ara-C. Successful therapeutic regimen consisted of 80-100 mg conjugate/kg, given i.p. daily for five consecutive days. Similar regimen injected shortly after the surgical removal of the primary tumor as adjuvant chemotherapy inhibited the metastasis of 3-LL to the lungs of the animals as demonstrated by an increase of survival time and the number of surviving animals.

Differential effects of various protein kinase C activators on protein phosphorylation in human acute myeloblastic leukemia cell line KG-1 and its phorbol ester-resistant subline KG-1a.

Human myeloid leukemia KG-1 cells are induced to differentiate to macrophage-like cells by tumor-promoting phorbol esters, such as 12-O-tetradecanoylphorbol-13-acetate (TPA). Cells from the cloned subline, KG-1a, unlike the parental line, are resistant to the differentiating effect of TPA. In the present studies, we investigated in these cells protein phosphorylation stimulated by various protein kinase C activators, including 1-oleoyl-2-acetylglycerol in the presence of the diacylglycerol kinase inhibitor R59022, TPA, mezerein, and bryostatin. All the agents stimulated, to a greater extent and with a higher potency, phosphorylation of several proteins in KG-1 cells than in KG-1a cells. On the other hand, these agents markedly stimulated phosphorylation of other proteins in KG-1a cells compared to that in KG-1 cells. The findings indicated that the actions of the diacylglycerol, 1-oleoyl-2-acetylglycerol, and the non-metabolizable activators (TPA, mezerein, and bryostatin) were very similar but not fully equivalent; and that KG-1a cells exhibited altered (increased or decreased) phosphorylation patterns, perhaps related to the TPA resistance characteristic of this subline of cells.

The disposition and pharmacokinetics in humans of 5-azacytidine administered intravenously as a bolus or by continuous infusion.

The disposition of 5-[4-14C]azacytidine, administered i.v. as a bolus or continuous infusion, was studied in cancer patients. After bolus, plasma 14C levels exhibited as multiphasic disappearance pattern; half-life (t1/2, beta phase) = 3.4 to 6.2 hr. Of 14C in plasma, less than 2% was associated with 5-[4-14C]azacytidine 30 min after dose. The ratios of 14C levels were: red cells/plasma, approximately 0.8; leukocytes/plasma, 1.1 to 2.3; nucleic acids/leukocytes, 0.2 to 0.43; sputum/plasma, 0.05 to 0.17. Urinary excretion (3 days) accounted for 73 to 98% of 14C, LEss than 1% in feces. The relative concentration of 5-azacytidine in plasma with continuous infusion stayed higher than with bolus; urinary excretion was similar. Fewer side effects were observed with continuous infusion than with bolus. The stability of 5-azacytidine was determined in various media at several temperatures by thin layer chromatography and nuclear magnetic resonance. At 20 degrees in Ringer's lactate (pH 6.2), the t1/2 was 94 to 100 hr. Stability increased with lowering of temperature and pH. From our data we conclude that 5-azacytidine should be given by continuous infusion rather than as a bolus.

Inhibition of protein kinase C, (sodium plus potassium)-activated adenosine triphosphatase, and sodium pump by synthetic phospholipid analogues.

The effects and modes of action of certain antineoplastic phospholipid analogues (racemic 1-O-octadecyl-2-O-methyl glycero-3-phosphocholine, BM 41.440, JH-1, CV-3988, and HePC) on (sodium plus potassium)-activated adenosine triphosphatase (Na,K-ATPase) and sodium pump activities were investigated. Inhibition of Na,K-ATPase in purified rat brain synaptosomal membranes by these lipids, in contrast to ouabain, was subject to membrane surface dilution and unaffected by whether the reaction was started with KCl, NaCl, or ATP. Kinetic analysis indicated that the analogues, again dissimilar to ouabain, were likely to interact directly or indirectly with sodium-binding sites of Na,K-ATPase located at the intracellular surface of the plasma membrane, a conclusion also supported by studies using the inside-out vesicles of human erythrocyte membranes. The studies also showed that ouabain (but not the lipids) increased the affinity constant of Na,K-ATPase for K+, whereas the lipids (but not ouabain) increased that for Na+. The lipids also inhibited 86Rb uptake by intact human leukemia HL60 cells at potencies quite comparable to those seen for inhibition of purified protein kinase C or Na,K-ATPase. It is suggested that Na,K-ATPase (sodium pump) might represent a hitherto unrecognized site of action for the lipid analogues, and that the antineoplastic effects of the agents might be due to, in part, inhibition of both protein kinase C and Na,K-ATPase and perhaps other membrane-associated enzymes.

A phase III trial comparing idarubicin and daunorubicin in combination with cytarabine in acute myelogenous leukemia: a Southeastern Cancer Study Group Study.

PURPOSE: A randomized clinical trial was undertaken to compare the therapeutic effectiveness of idarubicin (IDR) to daunorubicin (DNR), and both were given in combination with cytarabine (CA) in acute myelogenous leukemic (AML) patients. PATIENTS AND METHODS: Newly diagnosed patients were given a daily infusion of CA (100 mg/m2) for 7 days and were assigned randomly to receive DNR (45 mg/m2) or IDR (12 mg/m2) daily for the first 3 days. Those patients who achieved a complete remission (CR) were given three consolidation courses that consisted of CA (100 mg/m2 intravenously [IV]) and thioguanine (TG; 100 mg/m2 orally) every 12 hours for 5 days and either DNR (50 mg/m2) or IDR (15 mg/m2) on the first day of each cycle. After consolidation, patients received late intensification, which consisted of the same drugs used for induction except that the CA was given for 5 days and the anthracycline for 2 days. Four courses were planned at 13-week intervals. RESULTS: The CR rates were 75 of 105 (71%) on the IDR arm and 65 of 113 (58%) on the DNR arm (P = .03). The median survival and median remission durations were 297 and 433 days, respectively, on the IDR arm. The median survival and median remission durations were 277 and 328 days, respectively, on the DNR arm. Six deaths occurred during late intensification, five on IDR and one on DNR; this approach was abandoned after 47 patients were entered. The median survival was significantly longer for patients who received late intensification. CONCLUSION: This trial demonstrated that IDR was more effective than DNR in remission induction in AML.

Phase II clinical trial of carboplatin in relapsed and refractory leukemia.

Carboplatin is a second-generation platinum complex drug which has demonstrated activity against a variety of neoplasms including acute leukemia, particularly when given by continuous intravenous (i.v.) infusion. Adults with acute myelogenous leukemia (AML) or acute lymphoblastic leukemia (ALL), either refractory or in first or second relapse, were given a continuous i.v. infusion of carboplatin at a dose of 315 mg/m2 daily for 5 days. A second course was given if the bone marrow at day 14 showed persistent leukemia. If the marrow was hypoplastic, treatment was delayed until marrow recovery was documented. Those with residual leukemia were given a second course. Those achieving complete remission (CR) were given an additional course as consolidation. Of the 46 eligible patients entered (36 AML and 10 ALL) eight achieved CR (17%) of which 6 were AML and 2 ALL. Of nine primary refractory patients, two achieved CR, one AML and one ALL. Excluding the inevaluable patients (protocol violations, patient refused further therapy, early deaths prior to day 14, the CR rate was eight of 28 (29%). All except two CRs required two courses of induction. The non-hematologic toxicity was minimal except for renal and auditory toxicity. Renal toxicity greater than grade 2 was seen in 17 patients and was associated with concomitant use of nephrotoxic antibiotics. In two patients, renal failure was a major factor in the cause of death. Ototoxicity was observed in 11 patients, but was grade 3 in only three. There were 18 deaths during the study. Fourteen died of infection, two died of infection and hemorrhage, one died of hemorrhage while aplastic, and one died of other causes. This trial indicates that carboplatin is an active agent in acute leukemia and warrants further investigation.

A phase III trial of high-dose cytosine arabinoside with or without etoposide in relapsed and refractory acute myelogenous leukemia. A Southeastern Cancer Study Group trial.

A phase III clinical trial was developed to test whether the addition of etoposide to a high-dose cytosine arabinoside regimen would improve the remission rate, duration of remission, and survival in relapsed and refractory patients with acute myelogenous leukemia. One hundred and thirty-one patients stratified by age, performance status, percentage of marrow blasts, platelet count, bilirubin and presence or absence of clinical infection, refractory or relapsed (+/- 9 months) were randomized to receive high-dose cytosine arabinoside, 3 g/m2 every 12 h for 6 days with or without three doses of etoposide, 100 mg/m2 days 7-9. Of 67 patients randomized to cytosine arabinoside alone, 31% obtained a complete remission with a median remission duration of 11.9 months. Of 66 patients randomized to the combination regimen, 38% obtained a complete remission with a median duration of 25 months. None of these differences were statistically significant. Significantly (p = 0.036) longer survival was seen in patients on the combination regimen under the age of 50. There was no difference in overall survival. Six and 8%, respectively, of patients were free of disease at 5 years. The addition of etoposide to a high-dose cytosine arabinoside regimen had at best a marginal effect at the expense of some increase in toxicity.

Acute tumor lysis in T-cell leukemia induced by amsacrine.

The acridine orange derivative amsacrine (National Services Center No. 249992) was used to treat a patient with T-cell acute lymphoblastic leukemia (ALL). A 17-year-old boy had a tumor lysis syndrome after a single 200 mg/sq m dose of amsacrine. This was followed by a prolonged period of marrow hypoplasia leading to death from infection. Review of the literature and our own experience would suggest that T-cell ALL may be exquisitely sensitive to amsacrine.

Acquired hypomegakaryocytic thrombocytopenic purpura. Occurrence in a patient with absent thrombopoietic stimulating factor.

A 49-year-old woman had purpura and thrombocytopenia not associated with drugs or identifiable underlying disease. The platelet survival was normal and the marrow showed a sharp reduction in megakaryocytes with preservation of other cell lines. There was no response to steroids or infusion of fresh frozen plasma. Lithium carbonate therapy similarly had no effect. Thrombopoietic activity was absent in serum and urine samples. Erythropoietin activity was normal. In vitro formation of granulocyte-macrophage colonies in soft agar was normal. The case represents a unique incidence of selective megakaryocytic hypoplasia, though to result from a failure in stem cell differentiation.

Clostridium difficile. Colonization and toxin production in a cohort of patients with malignant hematologic disorders.

We examined 45 (80%) of 56 consecutive adult patients with malignant hematologic disorders who were hospitalized during a 15-week period at Emory University Hospital, Atlanta. Stool samples for Clostridium difficile culture and cytotoxin assay were obtained on admission and then weekly during each patient's hospitalization. On admission, four patients had detectable C difficile in their stool samples, which was associated with prior antimicrobial use but not with prior cancer chemotherapy. One of the four patients with positive stool samples also had toxin present in the stool sample and was the only one with diarrhea. Eight (36%) of 22 patients hospitalized for one or more weeks had C difficile isolated from at least one stool specimen. The positive cultures showed no clustering in time, and no risk factors were identified for colonization. Only seven of 15 culture-positive stool samples and three of seven toxin-positive samples were associated with diarrhea.

Enrichment of colony forming cells by methotrexate and isokinetic density gradient centrifugation.

Purification of target cells responsive to granulopoietic regulators would be of importance in understanding their mechanism of action. Previous studies have shown that mouse marrow could be enriched 3- to 7-fold with respect to agar colony forming cells (CFU-C) by appropriately scheduled doses of Methotrexate. To further increase this enrichment, such marrow has been subjected to centrifugation through an isokinetic gradient of Ficoll in culture medium. Under optimal conditions (17.5 min at 77.5 X g) the combination of chemotherapy and velocity sedimentation yielded a 17-fold enrichment of CFU-C's.

Effect of cryopreservation on purging of leukemic marrow with alkyl-lysophospholipids.

Alkyl-lysophospholipids (ALP) are reported to be selectively cytotoxic to neoplastic cells and have been demonstrated to be an effective purging agent for autologous marrow transplantation in a murine model, WEHI-3B. We studied the effect of in vitro treatment of normal bone marrow cells with ALP and of cryopreservation on spleen colony-forming units (CFU-s) in irradiated, syngeneic Balb/c mice. We found that exposure of cells to doses of ALP of 5 or 20 micrograms/ml followed by cryopreservation had no effect on CFU-s, but that a dose of 100 micrograms/ml was toxic. A simulated remission marrow was prepared by mixing normal murine bone marrow cells with WEHI-3B myelomonocytic leukemic cells to give a 1%-2% concentration of leukemic cells. These were exposed to 0- to 100-micrograms/ml concentrations of ALP for 24 h, cryopreserved, thawed, and injected into the tail veins of lethally irradiated syngeneic mice, and survival measured. It was found that concentrations of ALP from 10 to 50 micrograms/ml resulted in long-term disease-free survivors. These results established the fact that cryopreservation did not alter the effectiveness of ALP in purging marrows of residual leukemic cells.