Ex vivo elimination of lymphoblastic leukemia cells from human marrow by mafosfamid

Studies were performed to evaluate the anti-tumor activity of mafosfamid, a new synthetic derivative of cyclophosphamide. We tested its ability to eliminate lymphoblastic leukemia cells from autologous bone marrow grafts following a 30 min preincubation in a highly sensitive clonogenic assay. Treatment with 50-100 micrograms mafosfamid/ml eliminated more than 4 logs of contaminating clonogenic tumor cells from a 200-fold excess of normal bone marrow. Flow cytometric studies showed differences in cell cycle kinetics between mafosfamid-resistant and mafosfamid-susceptible tumor cell clones. Compared to drug susceptible clonogenic tumor cells, clones that resisted treatment with 100 micrograms mafosfamid/ml exhibited a smaller percentage of cells in S-phase, indicating that mafosfamid is mostly cytotoxic to rapidly cycling tumor cells. The combination of mafosfamid and a target cell selective immunotoxin containing pokeweed anti-viral protein was superior to mafosfamid alone or immunotoxin alone for purging mafosfamid-resistant leukemic cells from human marrow.     

Elimination of clonogenic breast cancer cells from human bone marrow. A comparison of immunotoxin treatment with chemoimmunoseparation using 4-hydroperoxycyclophosphamide, monoclonal antibodies, and magnetic microspheres

Autologous bone marrow transplantation (ABMT) may aid in the management of breast cancer, but is currently limited to patients without bone marrow metastases. In earlier studies, 5 logs of malignant clonogenic breast cancer cells could be eliminated from human bone marrow using a combination of chemoseparation with 4-hydroperoxycyclophosphamide (4-HC) and immunoseparation with monoclonal antibodies and magnetic microspheres. In this report the authors compare chemoimmunoseparation to treatment with immunotoxins for elimination of tumor cells from human bone marrow and for the preservation of normal precursors. Breast cancer cells from each of five cell lines were mixed with a tenfold excess of irradiated human bone marrow cells. Treatment with a combination of five immunotoxins reduced clonogenic tumor cell growth by 1.8 to 5.5 logs depending upon the cell line. With two of the five cell lines, clonogenic tumor cells were eliminated quantitatively. Using the CAMA-1 breast cancer cell line, treatment with multiple immunotoxins was compared with chemoimmunoseparation with 4-HC, a panel of five unconjugated monoclonal antibodies and magnetic microspheres. Chemoimmunoseparation eliminated 3.5 to 5.4 logs of malignant cells, while preserving 21% of Colony-forming unit-granulocyte-macrophage (CFU-GM) and 37% of burst-forming unit-erythrocyte (BFU-E). No clonogenic breast cancer cells could be detected. Immunotoxin treatment eliminated 2.2 to 5.4 logs of clonogenic breast cancer cells, but had no effect on the bone marrow precursors. In seven of ten experiments, however, clonogenic breast cancer cells remained after immunotoxin treatment. Consequently, treatment with 4-HC, multiple murine monoclonal antibodies and magnetic microspheres provided more consistent elimination of tumor cells than separation with immunotoxins, but was significantly more toxic for marrow precursors.     

No preferential sensitivity of clonogenic AML cells to ASTA-Z-7557

ASTA-Z-7557, an in vitro active metabolite of cyclophosphamide, has recently been introduced to purge autologous bone marrow grafts of patients with AML. The rationale of this approach assumes a relatively higher sensitivity of leukemic cells to the drug as compared to that of normal marrow precursors. We have investigated in direct comparison the sensitivity to ASTA-Z-7557 of normal bone marrow progenitors (GM-CFC and BFU-E) and clonogenic leukemic cells (L-CFC). Normal bone marrow cells and purified leukemic blast cells were exposed to varying concentrations of the drug. Dose-response relationships did not indicate a selective cytotoxic susceptibility of L-CFC to ASTA-Z-7557. The recovery of bone marrow precursors following exposure to ASTA-Z-7557 depended on the cell concentration during exposure and was higher for 2 X 10(7) cells/ml than for 1 X 10(6)/ml. To mimic minimal residual leukemia cell mixtures of 95% irradiated normal bone marrow cells with 5% leukemic blast cells were exposed to ASTA-Z-7557. In this mixture killing of L-CFC was largely decreased. These data suggest that in vitro incubation of autologous bone marrow grafts of patients with minimal residual leukemia with ASTA-Z-7557 might not offer a therapeutic advantage.     

Increased efficacy in selective elimination of leukemic cell line clonogenic cells by a combination of monoclonal antibodies BA-1, BA-2, BA-3 + complement and mafosfamid (ASTA Z 7557)

Studies were undertaken to examine the efficacy of combining monoclonal antibodies BA-1, BA-2, BA-3 + complement with a stabilized derivative of cyclophosphamide (mafosfamid) for elimination of leukemic cell line clonogenic cells. In a series of experiments conducted with leukemic cell lines a combination of two cycles of treatment with BA-1, 2, 3 + complement combined with 25 micrograms/ml-1 mafosfamid was superior to either modality alone in killing clonogenic leukemic cells. Identical treatment of normal bone marrow had no effect on the monopotent stem cells CFU-E and CFU-GM, or the pluripotent stem cell CFU-MIX, but some inhibition of the monopotent stem cell CFU-MK did occur. These results suggest that consideration be given to incorporation of BA-1, 2, 3 + complement and mafosfamid combination treatment in autologous bone marrow transplantation for acute lymphoblastic leukemia.     

Elimination of clonogenic tumor cells from human bone marrow using a combination of monoclonal antibody:ricin A chain conjugates

Effective autologous bone marrow transplantation for leukemia and lymphoma is likely to depend upon the selective removal in vitro of malignant cells from normal human bone marrow precursors. Highly specific cytotoxic conjugates formed by coupling ricin A chain to monoclonal antibodies might prove useful for the selective elimination of malignant cells. Consequently, ricin A chain conjugates have been prepared with several different murine monoclonal antibodies and tested for their ability to eliminate clonogenic Burkitt's lymphoma cells from an excess of human bone marrow. The most active reagents included an antibody:A chain conjugate which bound to the nonpolymorphic chain of the la molecule and another which reacted with the mu heavy chain of cell surface immunoglobulin. Conjugates formed with anti-common acute lymphoblastic leukemia antigen, anti-Mr 26,000 glycoprotein, and anti-B1 were much less active on these Burkitt's cells, contrasting with results of complement-dependent tumor cell lysis. Tumor cell kill was partially inhibited by the addition of greater than 2 X 10(6) human bone marrow cells/ml but could be potentiated by increasing the concentration of conjugate or by the addition of 10 mM ammonium chloride. In the presence of ammonium chloride, at least 4 logs of clonogenic tumor cells could be eliminated within 24 h from a 20-fold excess of bone marrow using 10(-7) M ricin A chain linked to one or two different antibodies. Similar treatment of normal human bone marrow temporarily inhibited granulocyte-macrophage colony-forming units (cell) formation but did not compromise establishment of continuous bone marrow cultures. The degree of selective elimination of tumor cells with A chain antibody conjugates was comparable to that achieved with 4-hydroperoxycyclophosphamide or with multiple monoclonal antibodies and complement.     

Immunological and pharmacological removal of small cell lung cancer cells from bone marrow autografts

Autologous bone marrow transplantation is used in small cell lung cancer (SCLC) to reverse the hematological toxicity induced by high dose therapy even though the presence of cancerous cells in the graft is potentially dangerous by reinfusion of the disease along with the hematopoietic stem cells. The present studies were undertaken to examine the effectiveness of anti-SCLC rat monoclonal antibodies LCA1 and LC66 plus human complement combined with a derivative of cyclophosphamide (Asta-Z 7557) for the elimination of cancerous clonogenic cells from the graft. In a series of assays conducted with three SCLC cell lines, used alone or mixed with normal bone marrow cells, the addition of Asta-Z 7557 to two cycles of treatment with monoclonal antibodies plus complement results in a 4- to 5-logarithmic reduction of the clonogenic SCLC cells detectable by limiting dilution analysis. This was superior to either treatment used alone. When normal bone marrow was submitted to the same treatment, a median (range) of 44% (15-77%) of the colony-forming unit, granulocyte-macrophage was recovered. These results suggest that the association of immunological (LCA1 and LC66 plus human complement) and pharmacological (Asta-Z 7557) removal methods is effective for purging metastatic clonogenic cells from bone marrow of SCLC patients and could be considered before autologous bone marrow transplantation.     

Cyclophosphamide/X-ray: combined mode preparation for transplantation therapy.

Use of total body irradiation (TBI) and/or chemotherapy as a preparation for marrow transplantation in the treatment of leukemia has been only moderately successful in the clinic. Although cyclophosphamide (CY) has shown promise as a marrow ablative agent, leukemia relapses are often found, and optimal therapeutic protocols have not been established. Our transplantation therapy studies of murine leukemia with parental recipients and hybrid donors provide an excellent model for research aimed at improved survival of human transplant patients. Utilizing a murine leukemia induced by a virus, various doses of CY in combination with sub-lethal irradiation were compared to determine the optimal pretreatment for transplantation therapy. Both normal and leukemic mice were engrafted with virus resistant, histocompatible marrow following these preparations, then monitored for survival and long term effects. Leukemic mice were also evaluated for pluripotent as well as myeloid committed stem cells as a measure of the effectiveness of the treatment in elimination of leukemic cells. Leukemic groups were also compared for the percentage and time of leukemia relapse. All CY/X-ray combinations were more effective in elimination of stem cell populations than supralethal TBI alone. However, the best survival was obtained with lethal TBI alone or low dose CY in combination with 550 R.     

Elimination of malignant clonogenic cells from human bone marrow using multiple monoclonal antibodies and complement

A clonogenic assay has been developed that utilizes Burkitt's lymphoma tumor cell lines to detect elimination of up to 5 logs of tumor cell contamination within human bone marrow. Different Burkitt's lymphoma lines bear one or more of a group of markers, including common acute lymphoblastic leukemia antigen gp26 (glycoprotein with a molecular weight of 26,000), B1, surface membrane immunoglobulin, HLA, beta 2-microglobulin, and Ia. Burkitt's tumor cells of the Namalwa line have been mixed with a 20-fold excess of irradiated human bone marrow cells. After treatment with one or more monoclonal antibodies and rabbit complement (RC), mixtures have been grown on a monolayer of irradiated human bone marrow cells and tumor cells enumerated by limiting dilution. Multiple treatments with antibody and RC were more effective than a single treatment in destroying clonogenic tumor cells which bore relevant determinants. Human serum components inhibited the lytic activity of RC in the presence of murine monoclonal antibodies. The total concentration of bone marrow cells proved critical in determining the complete elimination of tumor. Incubation of the Namalwa tumor cell line with RC and the J2 anti-gp26 eliminated more than 3 logs of malignant cells from a 20-fold excess of human bone marrow. Combinations of two monoclonal antibodies were more effective than any single antibody in eliminating Namalwa cells. A combination of three monoclonal reagents was no more effective than a combination of J2 and B1 or J2 and J5 in eliminating Namalwa cells. Treatment of human bone marrow with three antibodies and RC did not, however, produce a selective loss of nonmalignant GM-CFU-C, CFU-E, or BFU-E.     

CD34 antigen expression in children with Philadelphia chromosome-positive acute lymphoblastic leukemia.

One characteristic of Philadelphia chromosome (Ph')-positive acute leukemia is the occasional presence of both lymphoid and myeloid features in the same leukemia. This phenomenon supports the theory that this subtype of acute leukemia arises from lymphoid-myeloid stem cell, pluripotent progenitors. Very few reports, however, describe the immunophenotype, especially CD34 antigen, of Ph'-positive acute lymphoblastic leukemia (ALL). It has been shown that CD34, the human progenitor cell antigen, is found on 1% or less of normal human bone marrow cells, approximately 30% of acute leukemias, and multipotent progenitor cells; CD34 is not found on normal peripheral blood cells. A high frequency of CD34 expression was found in children with Ph'-positive ALL: CD34 was positive for all six patients tested, and one had an acute mixed-lineage leukemia. These findings suggest the involvement of a pluripotent stem cell in Ph'-positive ALL.     

Activity of a monoclonal antibody-saporin-6 conjugate against B-lymphoma cells

A monoclonal antibody reactive with the immunoglobulin heavy chain (TEC IgM) has been conjugated to saporin-6 (SAP), which is the major ribosome-inactivating protein from the seeds of the plant Saponaria officinalis. Studies with Burkitt's lymphoma cell line Bjab 113 demonstrate that this immunotoxin is capable of killing 3 logs (99.9%) of clonogenic lymphoma cells after a 2-hour incubation. The presence of human bone marrow inhibits the activity of the conjugate. However, full potency of TEC IgM-SAP immunotoxin is restored by adding 1 mM amantadine to the incubation medium. The reaction is highly specific and is inhibited by the presence of excess anti-mu-antibody or human serum. Clonal growth of other Burkitt's lymphoma cell lines is inhibited to a lesser extent by the immunotoxin. The presence of surface IgM on the different cell lines is directly correlated to target cell killing by TEC IgM-SAP. Isolation of Bjab 113 clones surviving treatment demonstrates that only a minority are truly resistant and that the others randomly escape the treatment. The highly potent and specific activity of this conjugate in the presence of bone marrow buffy coat and its exceptionally rapid onset of action make this conjugate a good candidate for the ex vivo elimination of neoplastic cells from the bone marrow of non-Hodgkin's lymphoma patients.     

Use of a cocktail of monoclonal antibodies and human complement in selective killing of acute lymphocytic leukemia cells

Autologous remission bone marrow is a potential source of repopulative stem cells after ablative chemoradiotherapy of tumor patients. Even with remission bone marrow, one major obstacle to use of autologous bone-marrow support is the danger of reinfusing viable tumor cells. This report describes a purging protocol with human complement, which seems suitable for eliminating acute lymphatic leukemia (ALL) cells of the common ALL type. Lysis of ALL blasts is induced with a cocktail of 3 monoclonal antibodies of IgM type (termed VIB-pool). These are directed against the CALLA (CD 10) antigen (VIL-AI antibody) and against 2 different epitopes of the CD24 surface structure (VIB-C5 and VIB-E3 antibodies). The purging efficiency was evaluated with leukemic cell lines of the common ALL type (Reh-6 and Nalm-6) and with blast cells from common ALL patients. Optimal lysis was obtained with antibody and human serum concentrations as low as 1 microgram/ml and 7% respectively. As a standard purging protocol we propose one 20-min incubation at room temperature with antibody followed by two 30-min incubations at 37 degrees C with 25% human complement. In dye exclusion test 99% purging efficiency and in clonogenic assays detecting elimination of up to 5 logs of clonogenic tumor cells 99.99% (= 4 logs) purging efficiency were achieved. Treatment with VIB-pool and human complement had no negative effect on the growth of the normal hemopoietic progenitor cells CFU-GM, CFU-E and BFU-E.     

Elimination of malignant clonogenic breast cancer cells from human bone marrow

Autologous bone marrow transplantation is a promising approach to the treatment of breast cancer but is at present limited to patients without bone marrow metastases. To eliminate malignant clonogenic breast cancer cells from normal human bone marrow, immunomagnetic separation has been combined with chemoseparation using 4-hydroperoxycyclophosphamide. Breast cancer cell lines have been mixed with a 10-fold excess of irradiated human bone marrow from normal donors. Mixtures have been incubated with a combination of five different monoclonal antibodies which bind to epithelial cell surface antigens of Mr 42,000, 55,000, 72,000, 200,000, and greater than 200,000. Antiglobulin coated microspheres which contained magnetite were added, and tumor cells were trapped in a magnetic field. Elimination of tumor cells from the decanted marrow was measured in a limiting dilution assay. Two treatments with antibody and microspheres permitted elimination of 2-4 logs of clonogenic breast cancer cells, depending upon the cell line studied. Similar treatment of nonirradiated normal marrow failed to affect levels of colony forming units-granulocyte-macrophage significantly. Use of immunomagnetic purging in combination with 4-hydroperoxycyclophosphamide eliminated up to 5 logs of tumor cells but reduced the recovery of colony forming units-granulocyte-macrophage. If prompt engraftment is observed following reinfusion of similarly treated marrow in phase I trials, these techniques should permit extension of autologous bone marrow transplantation to a larger population of breast cancer patients.     

In vitro colony formation of transplantable rat leukemias in comparison with human acute myeloid leukemia.

In vitro colony formation in two different soft agar systems was studied with bone marrow from untreated patients suffering from acute myeloid leukemia (AML) and from rats in various stages of two different transplantable myeloid leukemias. In both the thin-agar-layer system, which uses a feeder layer of fetal fibroblasts, and the Robinson culture system, human AML marrow failed to produce colonies. A similar failure was observed with the BN rat leukemia. In contrast, the Shay rat leukemic marrow produced an abnormally large number of colonies in the later stages of the disease. Evidence was obtained that the colonies produced by the Shay leukemic marrow consisted of leukemic cells, and that the disappearance of colonies from human AML and from BN rat leukemic marrow is caused by the numerical disappearance of normal pluripotent hemopoietic stem cells from the marrow and by the inability of the clonogenic leukemic cells to produce colonies in vitro. The results indicate that the BN rat leukemia is a realistic animal model for human AML.     

Selective elimination of breast cancer cells from human bone marrow using an antibody-Pseudomonas exotoxin A conjugate

A pancarcinoma monoclonal antibody (NR-LU-10), homogeneously reactive with human breast cancer cells, was conjugated to Pseudomonas exotoxin A. The immunotoxin was evaluated for its potential for purging breast cancer cells from human bone marrow. The immunotoxin NR-LU-10 antibody did not react with normal bone marrow preparations yet readily detected 1% contamination of bone marrow by MCF-7 breast cancer cells added to normal bone marrow without significantly inhibiting the colony-forming ability of bone marrow progenitor cells. NR-LU-10-Pseudomonas exotoxin A has potential for purging bone marrow of breast cancer cells without impairing the growth of bone marrow progenitor cells.     

In vitro bone marrow purging of multidrug-resistant cells with a mouse monoclonal antibody directed against Mr 170,000 glycoprotein and a saporin-conjugated anti-mouse antibody.

Selective elimination of multidrug resistance-positive cells (LoVo/Dx) was obtained by using the monoclonal antibody MRK 16, which recognizes a surface epitope of the Mr 170,000 glycoprotein, and a sheep anti-mouse immunoglobulin antibody, conjugated to the ribosome-inactivating protein saporin 6. The killing was greatly decreased or even abolished by adding the monoclonal antibody at a 100-fold concentration. Both the MRK 16 and anti-mouse saporin 6 conjugate did not show any killing activity when they were used separately. In cell suspensions composed of 90% normal bone marrow cells and 10% multidrug resistance-positive cells, the monoclonal antibody MRK 16 followed by the anti-mouse immunotoxin caused the elimination of 99% multidrug resistance-positive cells, as revealed by immunofluorescence and immunocytochemistry as well as by a clonal assay. Human normal hematopoietic precursors (granulomonocytic colony-forming units, erythroid burst-forming units, and multipotent granulomonocytic, erythroid, and megakaryocytic-forming units) were not affected by the MRK 16 plus immunotoxin treatment. This technique might be suitable for ex vivo bone purging in an appropriate clinical setting, such as autologous bone marrow transplantation.     

Cytotoxic activity of an anti-transferrin receptor immunotoxin on normal and leukemic human hematopoietic progenitors

The process of cellular iron uptake involves a specific receptor for the plasma carrier transferrin and a pathway of receptor-mediated endocytosis. Transferrin receptor expression is closely related to the rate of cell proliferation, and conjugates between anti-transferrin receptor monoclonal antibodies and toxins have been shown to have potent cytotoxic activity. We have constructed an anti-transferrin receptor immunotoxin by conjugating the anti-transferrin receptor monoclonal antibody B3/25 to a ribosome-inactivating protein, the saporin-6 (SO6), which is derived from the seeds of the plant Saponaria officinalis. The immunotoxin B3/25-SO6 was tested for in vitro cytotoxic activity against the human cell lines K-562 and HL-60 and against normal human bone marrow hematopoietic progenitors and acute myeloid leukemia clonogenic cells. The immunotoxin proved to be an effective inhibitor of K-562 and HL-60 clonogenic cell growth, in vitro colony formation being completely inhibited at immunotoxin concentrations ranging from 10(-7) to 10(-10) M. B3/25-SO6 markedly reduced the recloning efficiency of HL-60 clonogenic cells at 10(-12) M. Exposure of HL-60 cells in suspension culture to 10(-9) M B3/25-SO6 for 48-72 h completely abolished their clonogenic potential. The immunotoxin was also found to be cytotoxic against normal human bone marrow progenitor cells (burst-forming unit-erythroid and colony-forming unit-granulocyte, macrophage) in a dose-dependent manner. However, exposure of normal colony-forming unit-granulocyte, macrophage in suspension culture to 10(-9) M B3/25-SO6 for 72 h resulted in only 50% suppression of their clonogenic potential. Finally, B3/25-SO6 was found to be a potent inhibitor of in vitro growth of acute myeloid leukemia clonogenic cells. The cytotoxic effects of B3/25-SO6 were shown to be specific, since both saporin alone and irrelevant immunotoxins did not have any effect in the cellular systems examined. We conclude that the immunotoxin B3/25-SO6 has dose-related cytotoxic effects on both normal and leukemic human hematopoietic progenitors. Since there are substantial differences between normal and leukemic progenitors with respect to the proportion of cycling cells and the expression of transferrin receptors, B3/25-SO6 or similar immunotoxins may have clinical application in bone marrow-purging procedures.     

Combined chemoseparation and immunoseparation of clonogenic T lymphoma cells from human bone marrow using 2'-deoxycoformycin, deoxyadenosine, 3A1 monoclonal antibody, and complement

Chemoseparation and immunoseparation techniques have been combined to eliminate malignant clonogenic T lymphoma cells from human bone marrow. Incubation with 5 microM 2'-deoxycoformycin and 500 microM deoxyadenosine has eliminated 2 logs of HSB-2 T lymphoma cells from a 20-fold excess of irradiated human bone marrow. Multiple incubations with 3A1 antibody and rabbit complement eliminated approximately 2 logs of HSB-2 cells from similar mixtures. Used in combination, the 2 techniques eliminated up to 4 logs of T lymphoma cells. Incubation of normal human bone marrow under similar conditions failed to affect growth of granulocyte-macrophage colony-forming cell units, burst-forming erythroid units, or multipotential erythroid-granulocyte-megakaryocyte-macrophage colony-forming hematopoietic progenitor cells units.     

Comparative in vitro effects of cyclophosphamide derivatives on murine bone marrow-derived stromal and hemopoietic progenitor cell classes

We investigated the in vitro effects of ASTA-Z-7595, ASTA-Z-7557, ASTA-Z-7654, and 4-hydroperoxycyclophosphamide (4HC) on murine stromal fibroblastoid colony-forming units, committed hemopoietic progenitors (erythroid burst-forming units and granulocyte/macrophage colony-forming units), and pluripotent hemopoietic stem cells assayed by the spleen colony-forming unit (CFU-s) assay. In general, the drugs showed a time-and dose-dependent effect on colony-forming unit survival, and the relative toxicities were in the order in which the drugs are listed above. We found a relative sparing of day 12 CFU-s compared with day 7 CFU-s and committed hemopoietic and stromal progenitors, although colony size of day 12 CFU-s was reduced. Our results support two possible mechanisms for delayed or inadequate hemopoietic reconstitution in clinical studies using bone marrow purged with 4-hydroperoxycyclophosphamide or ASTA-Z-7557, i.e., damage to (a) transplantable stromal cells or (b) the hemopoietic stem cells.     

Magnetic affinity colloid (MAC) cell separation of leukemia cells from autologous bone marrow aspirates

A colloidal suspension of Co₂B with avidin irreversibly adsorbed to the surface has been used with biotinylated antibodies and lectins to eliminate specific cell populations from mixtures with peripheral blood or bone marrows. Using the monoclonal antibodies CF-1 and PM-81 with this magnetic affinity colloid (MAC), we can eliminate five logs of K562 cells from mixtures with peripheral blood or marrow cells as determined by a linear limiting dilution clonogenic assay. We have also used this separation to eliminate clonogenic leukemia cells from fresh samples of peripheral blood and bone marrow from relapsed acute leukemia patients. Using CF-1 alone or in combination with PM-81, we eliminated two logs of colonies and clusters of leukemia cells from the fresh samples. The same antibodies used with MAC separation of hematologically normal marrows allow recovery of greater than 30% of the hematopoietic progenitors.     

Effects of tilorone on hemopoietic stem cells and on the development of Friend leukemia

Summary Hematological effects of tilorone, an interferon inducer, on the hematopoietic cell system of normal CBA/Ca mice and on the development of Friend virus (FV-P)-induced polycythemia in DBA/2 mice were studied. In normal mice 80 mg/kg IP had a marked depressive effect on pluripotent (CFU-S), granuloid committed (CFU-C), and erythroid committed (CFU-E) stem cells with regeneration between days 5 and 12. In bone marrow smears only lymphopenia was detected. Treatment of mice before FV-P infection caused a slight retardation in the development of the splenomegaly and the transformation of bone marrow cells to Ep independence. Repeated treatment after FV-P infection also reduced the increase in spleen weight and the development of reticulocytosis, but the Ep independence of bone marrow and spleen cells was not influenced. In vitro exposure of normal cells and cells from FV-P-infected animals to the drug showed the same sensitivity of colony growth in normal as well as in Ep-independent CFU-E. The action of the drug on Friend leukemia is at least in part considered a toxic effect on the hematopoietic stem cell system.