Swine manure injection with low-disturbance applicator and cover crops reduce phosphorus losses.

Injection of liquid swine manure disturbs surface soil so that runoff from treated lands can transport sediment and nutrients to surface waters. We determined the effect of two manure application methods on P fate in a corn (Zea mays L.)-soybean [Glycine max (L.) Merr.] production system, with and without a winter rye (Secale cereale L.)-oat (Avena sativa L.) cover crop. Treatments included: (i) no manure; (ii) knife injection; and (iii) low-disturbance injection, each with and without the cover crop. Simulated rainfall runoff was analyzed for dissolved reactive P (DRP) and total P (TP). Rainfall was applied 8 d after manure application (early November) and again in May after emergence of the corn crop. Manure application increased soil bioavailable P in the 20- to 30-cm layer following knife injection and in the 5- to 20-cm layer following low-disturbance injection. The low-disturbance system caused less damage to the cover crop, so that P uptake was more than threefold greater. Losses of DRP were greater in both fall and spring following low-disturbance injection; however, application method had no effect on TP loads in runoff in either season. The cover crop reduced fall TP losses from plots with manure applied by either method. In spring, DRP losses were significantly higher from plots with the recently killed cover crop, but TP losses were not affected. Low-disturbance injection of swine manure into a standing cover crop can minimize plant damage and P losses in surface runoff while providing optimum P availability to a subsequent agronomic crop.

Human hematopoietic precursors in long-term culture: single CD34+ cells that lack detectable T cell, B cell, and myeloid cell antigens produce multiple colony-forming cells when cultured with marrow stromal cells.

CD34+ human marrow cells not expressing T cell-, B cell-, and myeloid cell-associated antigens (TBM-) were cloned by two-color cell sorting into culture wells containing irradiated marrow stromal cells. After 4 wk of culture, 3.7 +/- 2.1% of these cells generated colony-forming cells (CFC), with each of these cells generating 6.3 +/- 5.3 CFC. This was not due to the 0.5 +/- 0.5% CFC present in the purified CD34+ TBM- cells, as less than 1% of CFC persist in these cultures. This is the first demonstration that single immature precursor cells in human long-term cultures generate multiple CFC progeny. The immature nature of these clonable CD34+ TBM- precursors suggests their candidate status as human hematopoietic stem cells.

Precursors of colony-forming cells in humans can be distinguished from colony-forming cells by expression of the CD33 and CD34 antigens and light scatter properties.

We determined whether human marrow cells that directly form colonies in vitro could be distinguished from cells that generate or become CFC only after LTMC in the presence of irradiated marrow stromal cells. In previous studies, an anti-CD33 antibody, L4F3, and complement (C') were used to lyse nearly all CFC in marrow, and the remaining cells generated CFC in LTMC. In the present studies, marrow cells were treated with L4F3 + C' and the remaining CD33- cells were separated into CD34+ and CD34- populations and placed in LTMC. Only the CD34+ cells were found to generate significant numbers of CFC. To compare the CD33-CD34+ and CD33+CD34+ cells, we isolated each cell population using two-color FACS. Only LTMCs of the CD33-CD34+ cells generated CFC for greater than 5 wk. In contrast, cells that expressed both the CD33 and CD34 antigens, which contained most of the CFC, generated few CFC in LTMC. Fractionation of marrow cells based on right angle and forward light scattering suggested that precursors for CFC have low right angle and low forward light scattering properties. The CD33-CD34+ marrow cells were therefore further fractionated based on light scatter characteristics. Cells with low right angle and low forward light scatter formed few or no colonies on direct culture, yet generated greater numbers of CFC after 4 wk of LTMC than did cells with low right angle and high forward light scatter. Most (87-98%) CFC generated in the LTMCs that were initiated with CD33-CD34+ cells were found to express the CD33 antigen. Thus, hematopoietic progenitors with differing proliferative and differentiative potentials can be directly separated on the basis of their expression of CD33 and CD34 cell surface antigens and their light scatter properties.

Phosphatidic acid signaling mediates lung cytokine expression and lung inflammatory injury after hemorrhage in mice.

Because phosphatidic acid (PA) pathway signaling may mediate many basic reactions involving cytokine-dependent responses, we investigated the effects of CT1501R, a functional inhibitor of the enzyme lysophosphatidic acid acyltransferase (LPAAT) which converts lysophosphatidic acid (Lyso-PA) to PA. We found that CT1501R treatment not only prevented hypoxia-induced PA increases and lyso-PA consumption in human neutrophils, but also prevented neutrophil chemotaxis and adherence in vitro, and lung injury and lung neutrophil accumulation in mice subjected to hemorrhage and resuscitation. In addition, CT1501R treatment prevented increases in mRNA levels and protein production of a variety of proinflammatory cytokines in multiple lung cell populations after blood loss and resuscitation. Our results indicate the fundamental role of PA metabolism in the development of acute inflammatory lung injury after blood loss.

Phase III trial comparing paclitaxel poliglumex vs docetaxel in the second-line treatment of non-small-cell lung cancer.

Paclitaxel poliglumex (PPX), a macromolecule drug conjugate linking paclitaxel to polyglutamic acid, reduces systemic exposure to peak concentrations of free paclitaxel. Patients with non-small-cell lung cancer (NSCLC) who had received one prior platinum-based chemotherapy received 175 or 210 mg m(-2) PPX or 75 mg m(-2) docetaxel. The study enrolled 849 previously treated NSCLC patients with advanced disease. Median survival (6.9 months in both arms, hazard ratio=1.09, P=0.257), 1-year survival (PPX=25%, docetaxel=29%, P=0.134), and time to progression (PPX=2 months, docetaxel=2.6 months, P=0.075) were similar between treatment arms. Paclitaxel poliglumex was associated with significantly less grade 3 or 4 neutropenia (P<0.001) and febrile neutropenia (P=0.006). Grade 3 or 4 neuropathy (P<0.001) was more common in the PPX arm. Patients receiving PPX had less alopecia and did not receive routine premedications. More patients discontinued due to adverse events in the PPX arm compared to the docetaxel arm (34 vs 16%, P<0.001). Paclitaxel poliglumex and docetaxel produced similar survival results but had different toxicity profiles. Compared with docetaxel, PPX had less febrile neutropenia and less alopecia, shorter infusion times, and elimination of routine use of medications to prevent hypersensitivity reactions. Paclitaxel poliglumex at a dose of 210 mg m(-2) resulted in increased neurotoxicity compared with docetaxel.

Response of simian virus 40 (SV40)-transformed, cultured human marrow stromal cells to hematopoietic growth factors.

The response of marrow stromal cells transformed with wild-type simian virus 40 to recombinant growth factors was examined. When transformed stromal cells were plated in semisolid medium without the addition of growth factors, only 0.4% of cells formed colonies while with the addition of recombinant factors such as interleukin 1 (IL-1) or tumor necrosis factor (TNF), up to 10% of the cells formed colonies. Colonies were individually plucked and cell lines were developed that could be analyzed for expression of growth factors. The data show that unstimulated marrow stromal cells lines produced no detectable colony-stimulating activity. However, cell lines derived from "autonomously growing colonies" and from colonies grown with T cell-conditioned medium, with IL-1 alpha or beta, or with TNF alpha produced colony-stimulating activity and transcripts for granulocyte/macrophage-colony-stimulating factor (CSF), granulocyte-CSF, and IL-1 beta. A novel feature of the cell lines derived from colonies was that the production of growth factors was constitutive and persisted in excess of 4 m.

Chronic myelogenous leukemia: in vitro studies of hematopoietic regulation in a patient undergoing intensive chemotherapy.

A patient heterozygous for the X-linked enzyme glucose-6-phosphate dehydrogenase and with Philadelphia chromosome-positive chronic myelogenous leukemia (CML) was treated with combination chemotherapy and had a partial loss of Philadelphia chromosome accompanied by partial restoration of nonclonal hematopoiesis as determined by glucose-6-phosphate dehydrogenase. Studies of in vitro hematopoiesis were performed after chemotherapy to evaluate the influences of neoplastic stem cells on normal cells and to determine whether there were physical and cell kinetic differences between leukemic stem cells and their normal counterparts. The data revealed the following: (a) The frequencies of normal committed granulocytic stem cells (CFU-C) and erythroid stem cells (BFU-E) in blood did not differ from the frequencies in marrow. (b) Normal late erythroid progenitors (CFU-E) were found at a significantly lower frequency that the more primitive BFU-E. Calculations indicated that not only was there a decrease in CFU-E production by normal BFU-E, but there was also abnormal clonal expansion of CML BFU-E (CFU-E:BFU-E ratio for normal progenitors was 1.1, whereas for the CML clone it was 11.5). (c) No increase in frequency of normal CFU-C was found after marrow cells were exposed to high specific activity tritiated thymidine. (d) Normal CFU-C and those from the CML clone were not separable on the basis of density. (e) The frequency of normal BFU-E was consistently greater than that of CFU-C, suggesting that regulatory differences influence the commitment of normal progenitors to the two pathways.

Polycythemia vera. Further in vitro studies of hematopoietic regulation.

Further in vitro studies of hematopoietic regulation were carried out in two patients with polycythemia vera who were also heterozygotes (Gd(B)/Gd(A)) for glucose-6-phosphate-dehydrogenase (G-6-PD). While only G-6-PD type A was detectable in circulating erythrocytes, granulocytes and platelets, cultures of peripheral blood and marrow from one patient revealed a substantial number of G-6-PD type B erythroid burst-forming units (BFU-E) and granulocyte/macrophage colony-forming units. Detailed analysis demonstrated: (a) where detectable, normal BFU-E and granulocyte/macrophage colony-forming units were found with similar frequencies; (b) the same frequencies for normal progenitors characterized cultures of peripheral blood and marrow; (c) inhibition of normal erythroid differentiation between BFU-E and the more mature erythroid colony-forming unit; (d) a decline in the prevalence of normal colony-forming units with time, suggesting that disease progression is associated with further suppression of normal hematopoiesis by products of the abnormal clone.

Polycythemia vera. Increased expression of normal committed granulocytic stem cells in vitro after exposure of marrow to tritiated thymidine.

In previous studies of two patients with polycythemia vera (PV) and heterozygous at the X-linked locus for glucose-6-phosphate dehydrogenase (G-6-PD), only type A isoenzyme was found in non-lymphoid hematopoietic cells. However, some granulocytic and erythrocytic colonies grown in vitro had type B G-6-PD and therefore arose from presumably normal progenitors. In this study we exposed marrow cells from these same two patients to high-specific activity tritiated thymidine (3HTdR) before culture to kill cells actively synthesizing DNA. Individual granulocytic colonies were plucked and tested for G-6-PD after 14 d of culture. The frequency of type B colonies rose after exposure to 3HTdR from 8/101 to 11/36 in patient 1 and from 0/32 to 6/31 in patient 2 (P less than 0.003). No increase in the frequency of normal erythroid bursts after 3HTdR exposure was seen, implying that in PV, early granulopoiesis, and erythropoiesis are regulated differently. The results demonstrated that only type A granulocytic colonies, arising from the abnormal clone, were removed by the 3HTdR. In addition, for patient 2, statistical analysis indicated there was an absolute increase in normal granulocytic colonies detected in culture. Thus, PV clonal colony-forming units in culture (CFU-C) cycle more rapidly than do normal CFU-C and may suppress proliferation of normal CFU-C in vitro.

Polycythemia vera. Physical separation of normal and neoplastic committed granulocyte-macrophage progenitors.

In previous studies of two patients with polycythemia vera (PV) who were heterozygous at the X-linked locus for glucose-6-phosphate dehydrogenase (G6PD), only A type enzyme was found in nonlymphoid blood cells. However, some erythroid and granulocytic colonies grown in vitro were type B and therefore arose from presumably normal progenitors. One patient had enough type B colonies (8%) that studies of the physical characteristics of normal and PV clonal colony-forming cells could be undertaken. When marrow cells were separated by velocity sedimentation at unit gravity, most PV clonal granulocyte-macrophage progenitors (CFU-C) (type A G6PD) sedimented between 6.4 and 7.2 mm/h, whereas most residual normal, type B CFU-C sedimented less than or equal to 5.9 mm/h (P = 0.04)., When blood cells were separated over a discontinuous buoyant density gradient, PV clonal CFU-C equilibrated at densities < 1.065 g/ml, whereas residual normal CFU-C were found greater than or equal to 1.065 g/ml (P < 0.01). PV clonal and residual normal erythroid burst-forming progenitors were not separable by either method. Thus PV clonal CFU-C are larger and less dense cells than are residual normal CFU-C.

Treatment of acute myeloid leukemia cells in vitro with a monoclonal antibody recognizing a myeloid differentiation antigen allows normal progenitor cells to be expressed.

Monoclonal antibody L4F3 reacts with most acute myeloid leukemia (AML) cells and virtually all normal granulocyte/monocyte colony-forming cells (CFU-GM). Our objective was to determine whether lysis of AML cells with L4F3 and complement allowed expression of normal myeloid progenitors. The five glucose-6-phosphate dehydrogenase (G6PD) heterozygous patients with AML studied manifested only a single G6PD type in blast cells and in most or all granulocyte colony-forming cells, indicating that the leukemias developed clonally. The cells remaining after L4F3 treatment from two of the patients gave rise to granulocytic colonies that expressed the G6PD type not seen in the leukemic clone, indicating that they were derived from normal progenitors (CFU-GM). L4F3-treated cells from these two patients cultured over an irradiated adherent cell layer from normal long-term marrow cultures also gave rise to CFU-GM, which were shown by G6PD analysis to be predominantly nonleukemic. In the other three patients, the progenitor cells remaining after L4F3 treatment were derived mainly from the leukemic clone. The data suggest that in vitro cytolytic treatment with L4F3 of cells from certain patients with AML can enable normal, presumably highly immature progenitors to be expressed.

Effect of 5-azacytidine on gene expression in marrow stromal cells.

When exposed to 5-azacytidine, marrow stromal cells from active long-term marrow cultures and cell lines derived from simian virus 40-transformed stromal cells rapidly upregulated c-abl and interleukin-6 transcripts while downregulating the expression of collagen I, a major matrix protein. Similar effects occurred with interleukin-1 alpha and tumor necrosis factor alpha, although the time course was considerably prolonged.

Effect of recombinant alpha-interferon on the expression of the bcr-abl fusion gene in human chronic myelogenous human leukemia cell lines.

Recent investigations have shown a therapeutic and cytogenetic response in chronic myelogenous leukemia (CML) patients treated with recombinant alpha 2-interferon (IFN alpha 2). Philadelphia chromosome-positive (and many Ph1-negative) chronic myelogenous leukemia cells transcribe a novel bcr-abl fusion mRNA which may confer a growth advantage upon these cells. We investigated the effect of IFN alpha 2 on the levels of bcr-abl transcript expression in three Ph1-positive CML cell lines, EM2, KCL22, and K562. Although IFN alpha 2 inhibited cell proliferation in all three CML cell lines, IFN alpha 2 had no effect on the level of bcr-abl mRNA expression in any of the CML cell lines. In contrast, IFN alpha 2 increased the expression of class I HLA gene products. We conclude that while the bcr-abl fusion gene and its transcript undoubtedly play key roles in the pathogenesis of CML, the antiproliferative effect of IFN alpha 2 in CML cell lines relies upon genetic mechanisms other than modulation of bcr-abl expression.

Location and distribution of difucoganglioside (VI3NeuAcV3III3Fuc2nLc6) in normal and tumor tissues defined by its monoclonal antibody FH6.

The distribution of a novel difucoganglioside (6B ganglioside, NeuAc alpha 2----3Gal beta 1----4[Fuc alpha 1----3]GlcNAc beta 1----3Gal beta 1----4[Fuc alpha 1----3]GlcNAc beta 1----3Gal beta 1----4Glc beta 1----1Cer) in various normal adult and fetal tissues, as well as in cancer tissues, has been studied by immunoperoxidase staining with a specific monoclonal antibody, FH6, directed to this antigen. A large variety of embryonic and fetal tissues (stomach, colon, small intestine, pancreas, esophagus, lung, and heart) showed a diffuse, weakly positive staining, particularly in the epithelial layer, up to the 70th to 80th day of gestation. However, no staining was observed in various normal adult tissues, including gastrointestinal and glandular epithelial tissues which were stained positively by antibody N-19-9 (directed to sialyl-Lea) or CSLEXI (directed to sialyl-Lex). FH6-positive loci were limited to the proximal convoluted tubuli in kidney and granulocytes. In contrast, 44 of 76 cases of cancer tissue tested, including gastric, colonic, lung, breast, and renal cancers, showed clearly positive staining. The intensity of staining in gastric and colonic cancer tissues by FH6 antibody was weaker and less frequent, although the incidence of positive staining for lung (50%) and breast cancer (86%) was significantly higher than that of the antigen stained by monoclonal antibody FH4 (Y. Fukushi, S. Hakomori, and T. Shepard, J. Exp. Med., 159: 506-520, 1984), which is directed to the asialo core of the FH6 antigen. The antigen levels in the serum of patients with various cancers, inflammatory diseases, and normal subjects were determined by radioimmunoassay. The antigen level was found to be significantly higher in the serum of some patients with cancer, particularly lung, liver, and pancreatic cancers, as compared with the serum levels in other types of cancer, noncancerous diseases, and normal subjects.

Lisofylline inhibits transforming growth factor beta release and enhances trilineage hematopoietic recovery after 5-fluorouracil treatment in mice.

The effectiveness of endogenous or exogenously administered colony-stimulating factors may be modulated by the presence of hematopoietic inhibitory molecules. Cytotoxic therapy may result in the induction of hematopoietic inhibitors contributing to prolonged myelosuppression, whereas preventing the induction of such inhibitors may accelerate multilineage recovery. Lisofylline [LSF; (R)-1-(5-hydroxyhexyl)-3,7, dimethyl-xanthine], inhibits the signaling and/or release of certain hematopoietic inhibitory molecules such as tumor necrosis factor alpha, macrophage inflammatory protein 1 alpha, transforming growth factor beta, and IFN-gamma. Treatment of murine bone marrow cells with the cytotoxic agent 5-fluorouracil (5-FU) results in the release of a nondialyzable inhibitor of progenitor (colony-forming unit-granulocyte macrophage; CFU-GM) proliferation. When murine bone marrow cells were treated with 5-FU plus LSF, release of this inhibitor of CFU-GM proliferation was blocked. Neutralizing antibody and Western blot analysis indicated that the inhibitor was TGF-beta. We tested the effect of LSF (100 mg/kg i.p., b.i.d.) on multilineage regeneration after high-dose 5-FU or thiotepa treatment in BALB/c mice. In 4 of 5 experiments, LSF significantly accelerated neutrophil recovery (P < or = 0.05, Wilcoxon paired-signed test). In addition, platelet, reticulocyte, and CFU-GM regeneration were significantly accelerated in mice treated with LSF compared to control mice (P < or = 0.05). LSF had no significant effects on the ability of 5-FU to kill hematopoietic progenitor cells, nor did LSF stimulate or inhibit proliferation of CFU-GM. LSF had no effect on chemotherapy-induced killing of tumor cells in vitro, nor on the antitumor activity of 5-FU or thiotepa in BALB/c mice implanted with P388 leukemia cells. Inhibition of hematopoietic inhibitor release may accelerate multilineage recovery after cytotoxic therapy and, as such, may represent an alternative or additional therapy to the use of positively acting lineage specific colony-stimulating factors.

Pharmacological inhibition of phosphatidylcholine biosynthesis is associated with induction of phosphatidylinositol accumulation and cytolysis of neoplastic cell lines.

De novo production of phosphatidic acid (PA) in tumor cells is required for phospholipid biosynthesis and growth of tumor cells. In addition, PA production by phospholipase D has been cited among the effects of certain oncogenes and growth factors. In this report, it has been demonstrated that enhanced phospholipid metabolism through PA in tumor cells can be exploited pharmacologically for development of anticancer agents, such as CT-2584, a cancer chemotherapeutic drug candidate currently in Phase II clinical trials. By inhibiting CTP:choline-phosphate cytidylyltransferase (CT), CT-2584 caused de novo phospholipid biosynthesis via PA to be shunted away from phosphatidylcholine (PC) and into phosphatidylinositol (PI), the latter of which was doubled in a variety of CT-2584-treated tumor cell lines. In contrast, cytotoxic concentrations of cisplatin did not induce accumulation of PI, indicating that PI elevation by CT-2584 was not a general consequence of chemotherapy-induced cell death. Consistent with this mechanism of action, propranolol, an inhibitor of PA phosphohydrolase and phosphatidylcholine biosynthesis, was also cytotoxic to tumor cell lines, induced PI accumulation, and potentiated the activity of CT-2584 in cytotoxicity assays. As expected from biophysical properties of anionic phospholipids on cellular membranes, CT-2584 cytotoxicity was associated with disruption and swelling of endoplasmic reticulum and mitochondria. We conclude that CT-2584 effects a novel mechanism of cytotoxicity to cancer cells, involving a specific modulation of phospholipid metabolism.

Cloning and sequencing of the human c-abl 3' untranslated region.

The human c-abl oncogene gives rise to different mRNA transcripts which vary primarily in that they possess alternative first exons. In the present study, we present the sequence for the human c-abl 3' untranslated region (3'utr) and show that while human and murine c-abl cDNA sequences are generally homologous, human c-abl transcripts unlike the murine c-abl transcripts do not differ significantly in their 3' utrs.

Phase I study of busulfan and cyclophosphamide in preparation for allogeneic marrow transplant for patients with multiple myeloma.

PURPOSE: To study the toxicity and potential efficacy of busulfan (BU) and cyclophosphamide (CY) as a conditioning regimen before allogeneic bone marrow transplantation (ABMT) in patients with multiple myeloma (MM). PATIENTS AND METHODS: Twenty patients with MM underwent conditioning, which was followed by ABMT from 16 HLA-identical donors, three one-antigen-mismatched donors, and one HLA A, B, D-identical unrelated donor. Four levels of BU plus CY were evaluated. RESULTS: Severe regimen-related toxicity occurred in two of five patients who received BU 16 mg/kg and CY 120 mg/kg, in none of the four patients who received BU 14 mg/kg and CY 120 mg/kg, in one of eight patients who received BU 14 mg/kg and CY 147 mg/kg, and in two of three patients who received BU 14 mg/kg and CY 174 mg/kg. Twelve of 15 (80%) assessable patients achieved a complete remission with the disappearance of M-protein and the return of normal marrow morphology. Ten patients died of complications related to the ABMT, and two patients died of progressive or relapsed MM. Overall, eight of 20 patients were alive; seven (35%) were in complete remission 190 to 1,271 days after ABMT. CONCLUSIONS: The maximum-tolerable dose given in this setting was BU 14 mg/kg and CY 147 kg/mg. These results suggest that this regimen may have significant antimyeloma activity. Further phase II studies are warranted.

Differential expression of collagenase by human fibroblasts and bone marrow stromal cells.

The bone marrow stroma, represented in long-term marrow culture by cells of the adherent layer, is composed of a heterogenous mixture of macrophages and mesenchymal cells, including fibroblasts, endothelial cells and adipocytes, in association with a proteoglycan matrix. This matrix, which is synthesized by the stroma, is capable of binding hematopoietic growth factors, and likely plays a major role in hematopoietic regulation. Clonally-derived non-transformed bone marrow stromal cells, propagated in the presence of basic fibroblast growth factor, were studied for expression of collagenase, an enzyme whose substrate, collagen, is a major component of the extracellular matrix. Expression of steady-state collagenase mRNA was undetectable in both unstimulated dermal fibroblasts and non-transformed marrow stromal cells. However, stimulation with interleukin 1 alpha (10 U/ml) for 24 h resulted in marked accumulation of collagenase mRNA in dermal fibroblast cells, yet failed to elicit a similar response in bone marrow stromal cells. Both marrow stromal cells and dermal fibroblasts constitutively expressed transcripts of collagen I, and rhIL-1 alpha upregulated transcripts of interleukin 6 in both these cells as well. Although similar in morphology, these data indicate that bone marrow stromal cells differ from fibroblasts in their response to IL-1. In the marrow microenvironment, where IL-1 may be secreted by a variety of cell types, such suppression of collagenase expression may serve to prevent unwanted mobilization of collagen from the glycoprotein matrix by marrow stromal cells.

The effect of long-term marrow culture on the origin of colony-forming cells in acute myeloblastic leukemia: studies of two patients heterozygous for glucose-6-phosphate dehydrogenase.

Long-term marrow cultures (LTMCs) provide a selective growth advantage for cytogenetically normal cells in patients with acute and chronic myeloid leukemias. In the present study, LTMCs were established from two patients with newly diagnosed acute myeloid leukemia (AML) who were heterozygous for the X-linked enzyme glucose-6-phosphate dehydrogenase (G6PD). Initially only leukemic clusters grew from cells plated in semisolid medium, but after 1 or more weeks in LTMC, morphologically normal granulocyte-macrophage colonies were detected. Nonetheless, in one of the patients, more than 80% of these colonies expressed the G6PD type observed in the leukemic blast cells, indicating a probable neoplastic derivation for many of them. In the second patient, colonies cultured during the first 3 weeks of the LTMC were predominantly derived from clonal progenitors, whereas after week 4 the colonies were derived from normal stem cells. Colonies derived from clonal or normal stem cells were not morphologically distinguishable. These data support the conclusion that LTMC has a selective anti-leukemic effect on marrow cells from some patients. However, normalization of colony growth is by itself not a sufficient criterion for determination of whether committed progenitor cells from patients with AML are derived from normal or leukemic stem cells.