Effect of in vitro interferon treatment on the rapid clearance of murine leukemia cells in vivo.

Previous work showed that interferon (IFN) can protect target cells from NK mediated lysis in vitro. In the present study we investigate the effect of IFN alpha/beta or IFN gamma treatment of three different murine leukemia cell lines. For this purpose FLC-745 (susceptible to the antiproliferative activity of IFN alpha/beta and gamma), FLC-3C18 (IFN alpha/beta -resistant and IFN gamma - susceptible) of DBA/2 origin and EL-4 (IFN alpha/beta - susceptible and IFN gamma - resistant) leukemia of C57B1/6 origin were treated with IFN alpha/beta or gamma in vitro and assayed for their susceptibility to natural resistance measured in vivo as organ rapid clearance 4 hr after iv injection into syngeneic mice. Using young or Poly I:C stimulated hosts, but not mice with low levels of natural resistance (i.e. older animals or mice treated with cyclophosphamide), slower elimination of treated cells was observed with: (a) FLC-745 cells treated with IFN alpha/beta and IFN gamma and (b) FLC 3C18 treated with IFN gamma. Such a delayed clearance was not observed with: (a) FLC-3C18 cells treated with IFN alpha/beta and (b) EL-4 leukemia cells preincubated with IFN alpha/beta or IFN gamma. These results suggest that under selected conditions IFNs can protect leukemic cells from in vivo natural reactivity.     

Natural resistance in mice against Friend leukemia cells. I. Studies with in vitro passaged interferon-sensitive and interferon-resistant cell clones

Anti-lymphoma natural resistance (NR) has been detected in DBA/2 mice inoculated intravenously (iv) with syngeneic Friend leukemia cells (FLC). Interferon-sensitive 745 or interferon-resistant 3Cl-8 clones, passaged in vitro and exhibiting "low" tumorigenicity in syngeneic DBA/2 mice, were used. NR, measured as rapid clearance of radiolabeled cells from lung and liver of recipient mice, was age-dependent, was boosted by host pretreatment with polyinosinic-polycytidylic (poly I:C) acid or Friend leukemia virus, and was decreased by mice pretreatment with cyclophosphamide or i-carrageenan. Treatment of "target" FLC with interferon suppressed the susceptibility of 745 FLC, but not that of 3Cl-8 FLC to host's NR. These data suggest that the "low" in vivo tumorigenicity of in vitro passaged FLC is, at least in part, due to host's NR directed against target structures associated with leukemia cells.     

Susceptibility of Friend virus antigen-modulated erythroleukemic cells to lysis by T lymphocytes from mice with dormant Friend virus infections.

Spleen cells from DBA/2 mice with dormant Friend leukemia virus (FLV) infections or from mice immunized with x-irradiated FLC-745 erythroleukemic cells are not cytolytic for FLC-745 cells when tested directly, but acquire cytolytic activity in vitro by cultivation with x-irradiated FLC-745 cells. Spleen cells cultured from normal mice do not acquire such cytolytic activity. Cytolytic activity resides in the T lymphocyte population. Alloantiserum, but not antisera against FLV-virion polypeptides, inhibited the lysis of FLC-745 cells by cytolytic T lymphocytes. To understand further the role of cellular and humoral immune anti-FLV responses in mice with dormant FLV-infections, in vitro experiments were conducted that mimicked the in vivo FLV-specific immune environment of these mice. We found that FLV-immune serum from mice with dormant FLV-infections modulated FLV-antigen expression on the surfaces of FLC-745 cells without affecting their susceptibility to lysis by cytolytic T lymphocytes. These results suggest that cytolytic T lymphocyte restrain the outgrowth of FLV-antigen-modulated erythroleukemic cells in mice with dormant FLV infections and that the targets for cell-mediated lysis may be H-2d-associated antigens.     

In vivo natural antitumor resistance against murine EL-4 lymphoma cells in lethally irradiated syngeneic C57Bl/6 mice

Natural resistance has been detected in lethally irradiated C57Bl/6 (B6) mice inoculated intravenously with the ascites form of a syngeneic B6 leukemia. EL-4 cells were injected into lethally irradiated (800 R) B6 mice and tumor cell proliferation was evaluated by 125IUdR uptake in different organs 4 days after the challenge. Differential growth of lymphoma cells was observed when young mice were injected as compared with older mice and when mice were treated with agents known to interfere with natural resistance (e.g., poly(I:C), FLV-P, carrageenan, cyclophosphamide, high doses of irradiated cells). Similar results were obtained by measuring rapid clearance of 125IUdR-labeled EL-4 cells from lungs of intact B6 mice. In vivo cold competition studies, employing EL-4 and several other tumor lines of the same or different haplotype, showed that only EL-4 and RBL-5 cells were capable of inhibiting syngeneic resistance against EL-4 tumor. On the contrary, YAC-1 lymphoma cells, the most susceptible target to natural killer-mediated cytotoxicity in vitro, did not compete. These results suggest that EL-4 cells express membrane determinants not detectable on normal H-2b parental bone marrow cells and are susceptible to natural resistance against hemopoietic tumor cells in lethally irradiated syngeneic B6 mice.     

Natural resistance against hematopoietic cells in lethally-irradiated mice infected with Friend leukemia virus

Summary The influence of in vivo infection with the polycythemic substrain of Friend leukemia virus on noninducible (natural) resistance against allogeneic normal or malignant grafts was studied in lethally irradiated mice. Parallel studies were performed on the NK system in the same experimental conditions. The results indicate that FLV-P infection of mice with full (DBA/2) vs partial (BALB/c and CD2F1) susceptibility did not suppress their in vivo natural resistance against bone marrow or El-4 leukemia cells. On the other hand, a decline in NK activity paralleled the progression of leukemic disease in the more susceptible DBA/2 hosts. Abbreviations used: FLV-P, N-tropic polycythemic substrain of Friend Leukemia Virus Complex; NR, natural resistance; NR in vivo, natural resistance against normal or malignant hemopoietic grafts occurring in vivo in lethally irradiated mice; NK, natural killer; (¹²⁵I)IUdR, ¹²⁵I-labeled 5-iodio-2-deoxyuridine; IV, intravenous     

Targeting myeloid leukemia with a DT(390)-mIL-3 fusion immunotoxin: ex vivo and in vivo studies in mice.

The IL-3 receptor was expressed on a high frequency of myeloid leukemia cells and also on hematopoietic and vascular cells. We previously showed that a recombinant IL-3 fusion immunotoxin (DT(390)IL-3) expressed by splicing the murine IL-3 gene to a truncated diphtheria toxin (DT(390)) gene selectively killed IL-3R(+) expressing cells and was not uniformly toxic to uncommitted BM progenitor cells (Chan,C.-H., Blazar,B.R., Greenfield,L., Kreitman,R.J. and Vallera,D.A., 1996, Blood, 88, 1445-1456). Thus, we explored the feasibility of using DT(390)IL-3 as an anti-leukemia agent. DT(390)IL-3 was toxic when administered to mice at doses as low as 0.1 microg/day. The dose limiting toxicity appeared to be related to platelet and bleeding effects of the fusion toxin. Because of these effects, DT(390)IL-3 was studied ex vivo as a means of purging contaminating leukemia cells from BM grafts in a murine autologous BM transplantation. In this setting, as few as 1000 IL-3R-expressing, bcr/abl transformed myeloid 32Dp210 leukemia cells were lethal. An optimal purging interval of 10 nM/l for 8 h eliminated leukemia cells from 32Dp210/BM mixtures given to lethally irradiated (8 Gy) C3H/HeJ syngeneic mice. Mice given treated grafts containing BM and a lethal dose of 32Dp210 cells survived over 100 days while mice given untreated grafts did not survive (P < 0.00001). DT(390)IL-3 may prove highly useful for ex vivo purging of lethal malignant leukemia cells from autologous BM grafts.     

Immunity to virus-free syngeneic tumor cell transplantation in the BALB/c mouse after immunization with homologous tumor cells infected with type C virus.

Syngeneic tumor cell lines free of endogenous type C virus or viral antigen antigen expression were derived from spontaneously occurring tumors of the BALB/cCr mouse. Two cell lines free of endogenous type C virus were examined and found to be highly tumorigenic in tumor growth kinetic studies. In vitro inoculation of these cell lines with Rauscher-murine leukemia virus (R-MuLV) resulted in their chronic infection in which 95 to 100% of the cells were scored as virus positive. These infected lines showed a highly significant increase in their immunogenicity as compared to their uninfected controls. Animals in which these virus-positive tumors regressed were then shown to be highly resistant to challenge with the uninfected tumor cell lines as well as to live R-MuLV. This observed resistance to uninfected tumor cell lines could not be induced by immunization of the mouse with uninfected tumor cells and R-MuLV simultaneously at the same injection site, nor could it be induced with lethally irradiated virus-infected tumor cells, subtumorigenic doses of uninfected cells, or inactivated R-MuLV or Gross leukemia virus (G-MuLV). Cell-mediated cytotoxicity studies revealed that spleen cells obtained from animals whose virus-infected tumors regressed were cytotoxic to homologous infected and uninfected tumor cells as well as to other uninfected tumor cell lines syngeneic to the BALB/c mouse. Correlation of in vitro cytotoxicity with in vivo immunity was provided by the Winn assay, by inoculation into susceptible mice of immune and nonimmune spleen cells premixed with uninfected tumor cells. The immune cells were highly effective in preventing this tumor cell transplantation. It was concluded that type-C virus infection of these syngeneic tumor cells resulted in their acquiring strong transplantation antigens that were in part due to the virion, but were at least in part due to alterations of antigens or haptens that are present in a less immunogenic form on the uninfected tumor cell.     

Dietary dehydroepiandrosterone inhibits bone marrow and leukemia cell transplants: role of food restriction

Dietary dehydroepiandrosterone (DHEA) inhibits the proliferation of syngeneic bone marrow cells (BMC) infused into lethally irradiated mice. Potential mechanisms for suppression of hematopoiesis were evaluated and the findings were as follows: (i) depletion of NK, T, B or macrophage cells failed to reverse suppression by DHEA; (ii) stem cell stimulation by erythropoietin, growth hormone, interleukin-2, Friend leukemia virus, or cyclophosphamide failed to reverse suppression; (iii) supplementation of fatty acids, mevalonate, or deoxyribonucleotides, which are dependent upon glucose-6-phosphate dehydrogenase function, did not enhance BMC growth in mice fed DHEA; (iv) DHEA downstream metabolites 4-androstenedione and 17beta-estradiol, as well as the synthetic steroid, 16alpha-chloroepiandrosterone (but not testosterone or 5-androstene-3beta,17beta-diol), also inhibited BMC growth. Tamoxifen antagonized the effects of 17beta-estradiol but not DHEA; (v) dietary DHEA causes hypothermia, but housing of DHEA-fed mice at 34 degrees C to maintain normal body temperature did not reverse suppression; (vi) DHEA leads to a decrease in food intake in rodents. Pair-feeding control diet to mice fed DHEA mimicked the effects of dietary DHEA; (vii) adrenalectomy and orchiectomy decrease the levels of stress and sex hormones, respectively. Neither procedure affected the ability of food restriction or DHEA feeding to inhibit hematopoiesis; (viii) growth of GR-3 NM pre-B leukemia cells in unirradiated mice was also suppressed by DHEA or food restriction. We conclude that DHEA, by reducing food intake in mice, inhibits bone marrow and leukemia cell growth. The precise mechanism(s) by which reduced food intake per se inhibits hematopoiesis is not known, but may involve an increased rate of cellular apoptosis.     

Induction of immune resistance against L1210 lymphatic leukemia in mice after lethal irradiation and reconstruction with fetal liver cells.

The immunohematopoietic potential of syngeneic fetal liver cells (SFLC) was examined and compared with syngeneic bone marrow cells (SBMC). SFLC generated about 3 times less 12th-day spleen colonies (CFU-S) than adult SBMC did. To test the SFLC ability for reconstitution of the immune system, mice were lethally total body irradiated (TBI) and transplanted i.v. with 3 x 10(7) SFLC or 1 x 10(7) SBMC. Thus, injected hematopoietic cells contained the same number of CFU-S. On days 28, 35, 42, and 49 after transplantation the mice were injected i.p. with 10(6) immunogenic L1210-Maf cells (L1210 leukemia cells treated in vitro with mafosfamide for inhibition of their growth in vivo) to test the ability for generation of immune response against L1210 leukemia. On day 56 the animals were challenged with 10(3) L1210 leukemia cells. Strong resistance against the leukemia was induced in TBI + SFLC and TBI + SBMC mice, suggesting that the SFLC similarly as SBMC are able to reconstitute immune system of the TBI host.     

Transferrin receptors and iron utilization in DMSO-inducible and -uninducible friend erythroleukemia cells

Dimethylsulfoxide (DMSO) induces hemoglobin synthesis and erythroid differentiation of Friend erythroleukemia cells in vitro. Induction is accompanied by increased transferrin-binding activity which is necessary for the cellular acquisition of iron from transferrin for hemoglobin synthesis. There are Friend cell variants in which hemoglobin synthesis is not induced by DMSO unless exogenous hemin is also present. In this study we have compared the inducibility of transferrin receptors and iron incorporation in DMSO-inducible (745) and -uninducible (M-18 and TG-13) Friend cell lines. Cellular transferrin-binding sites were estimated by Scatchard analysis of data obtained from specific binding of [125I]transferrin by the cells. Our results show that unlike 745, DMSO treatment of the variant cell lines M-18 and TG-13 does not result in increased transferrin-binding activity. The number of transferrin-binding sites and the rate of iron uptake is similar in uninduced 745 and DMSO-treated M-18 and TG-13 cells. Although exposure of M-18 cells to DMSO and hemin induces hemoglobinization, this treatment does not cause induction of transferrin receptors. These results indicate that the primary defect in M-18 cells may be the uninducibility of transferrin receptors. We have also shown that exposure of 745 cells to hemin during DMSO treatment prevents the induction of transferrin receptors, suggesting that hemin may control the expression of transferrin receptors in erythroid cells.     

Cell-mediated immunity to friend virus-induced leukemia. IV. In vitro generation of primary and secondary cell-mediated cytotoxic responses.

Primary and secondary cell-mediated cytotoxic responses to FBL-3 cells, a syngeneic Friend virus-induced leukemia in C57BL/6 mice, could be generated by in vitro techniques as tested by the 125IUdR release assay. The specificity of the cytotoxic reactions appeared to be directed against the Friend type-specific antigen and the FMR (Friend, Moloney, Rauscher) antigen which were also the major antigens for transplantation immunity to FBL-3. In comparison to the primary cytotoxic response, the secondary cytotoxic response was accelerated (detected at an earlier time after sensitization), enhanced (gave much higher levels of cytotoxicity), was also longer lasting, and could be induced by a wide dose range of tumor cells. The secondary response could only be induced with lymphocytes obtained from regressors that were resistant to FBL-3 challenge; lymphocytes from mice with progressive tumor growth had no detectable secondary response. It was found that both induction phase and the effector phase of cytotoxic responses were T cell dependent. The characteristics of these reactions were thus very similar to those obtained with in vivo immunization or challenge, providing a good correlation with in vivo tumor immunity.     

Effect of mouse interferon alpha/beta on the expression of H-2 (class I) antigens and on the levels of 2'-5' oligoadenylate synthetase activity in interferon-sensitive and interferon-resistant Friend leukemia cell tumors in mice

DBA/2 mice were injected intraperitoneally (i.p.) with interferon-sensitive 745 or interferon-resistant 3C1-8 Friend erythroleukemia cells (FLC) and then injected i.p. with mouse interferon alpha/beta. Interferon enhanced the expression of histocompatibility (H-2) antigens on individual 745 FLC within the peritoneum, but did not alter the expression of H-2 antigens on individual 3C1-8 FLC. Likewise, interferon treatment resulted in an increase in the level of 2'-5' oligo-adenylate (2-5A) synthetase activity in 745 FLC, but did not affect the level of activity in 3C1-8 FLC. These results provide evidence that the phenotype of interferon sensitivity or resistance of FLC does not change within the peritoneum. An incidental finding was that the basal level of 2-5A synthetase activity of in vivo passaged 745 cells was greater than that of 3C1-8 FLC. The finding that injection of mice bearing 745 FLC with antibody to mouse interferon alpha/beta reduced the level of 2-5A synthetase activity in these cells, but did not alter the level of 2-5A activity in 3C1-8 FLC, suggests that endogenous interferon in the peritoneum may have been the responsible factor.     

Multistage Friend erythroleukemia: independent origin of tumor clones with normal or rearranged p53 cellular oncogenes.

The erythroleukemia induced by Friend virus complex in adult mice is a multistage malignancy characterized by the emergence, late in the disease, of tumorigenic cell clones. We have previously shown that a significant proportion of these clones have unique rearrangements in their cellular p53 oncogene. The clonal relationships among Friend tumor cells isolated in the late stages of Friend erythroleukemia were analyzed by examining the unique integration site of Friend murine leukemia virus and the unique rearrangement in their cellular p53 oncogene. The majority of clones isolated from individual mice infected with Friend virus were clonally related as judged by the site of Friend murine leukemia virus integration. However, Southern gel analysis of DNA from individual Friend cell clones indicated that all of the clones with a normal p53 gene from the same mice were clonally related, but were unrelated to the Friend cell lines with a rearranged p53 gene. These results suggest that Friend tumor cells with rearrangements in their p53 gene arise as the result of a unique transformation event, rather than by progression from already existing tumor cells with a normal p53 gene. They also suggest that such rearrangements in the p53 gene confer a strong selective advantage to these cells in vivo.     

Myogenic potential of muscle side and main population cells after intravenous injection into sub-lethally irradiated mdx mice.

Muscle side population (SP) cells have demonstrated hematopoietic and myogenic activities in vivo upon intravenous (IV) injection into lethally irradiated mdx mice. In contrast, muscle main population (MP) cells were unable to rescue the bone marrow of lethally irradiated mice and, consequently, their in vivo myogenic potential could not be assessed using this method. In the current study, muscle SP or MP cells derived from syngeneic wild-type male mice were delivered to sub-lethally irradiated mdx female mice by single or serial IV injections. Recipient mice were euthanized 12 weeks after transplantation at which time the quadriceps and diaphragm muscles were analyzed for the presence of donor-derived cells. Mice injected with 10(4) muscle SP cells or with 10(6) MP cells appeared to have similar numbers of dystrophin-positive myofibers containing fused donor nuclei. Analysis of the remaining tissue via real-time quantitative PCR indicated that mice injected with muscle SP cells had a higher percentage of donor-derived Y-DNA in the quadriceps than mice injected with MP cells, suggesting that muscle SP cells may be enriched for progenitors able to engraft dystrophic skeletal muscles from the circulation. Although the overall engraftment did not reach therapeutically significant levels, these results indicate that further optimization of cell delivery techniques may lead to improved efficacy of cell-mediated therapy using muscle SP cells.     

Induction of erythroid differentiation in Friend leukemia cells by bromodeoxyuridine

Treatment of Friend leukemia cells with BrdU, the thymidine analog which interferes with DMSO induced differentiation in these cells as well as the expression of differentiated character in many other cell systems, is capable of inducing erythroid differentiation. Globin mRNA, as assayed by hybridization to globin cDNA, increases 2.5- to 30-fold after appropriate treatment with BrdU. This effect was observed with several different subclones of three independent Friend tumor cell lines. After BrdU treatment, globin mRNA content may reach up to 10-20% of the levels in DMSO induced cultures. The induction of erythroid differentiation is also apparent when accumulated heme content or the appearance of benzidine positive cells is monitored. One Friend cell line (745) we examined was not induced by BrdU although it incorporated an amount of BrdU into its DNA comparable to that incorporated by the other cell lines. In addition, BrdU did interfere with DMSO induction in this cell line. These results suggest that two different mechanisms may be operative in regulating erythroid differentiation in Friend leukemia cells. While BrdU interferes with the mechanism activated by DMSO treatment, this analog could independently activate an alternative mechanism.     

Adoptively transferred antigen-specific T cells can be grown and maintained in large numbers in vivo for extended periods of time by intermittent restimulation with specific antigen plus IL-2

The aim of the current study was to determine whether cultured tumor Ag-specific T cells could be induced to grow and maintained functional in large numbers in vivo by intermittent restimulation in vivo with specific Ag plus IL-2. T cells derived from spleens of B6 mice (Thy-1.2) immune to FBL-3, a Friend virus-induced leukemia, were activated by in vitro stimulation with irradiated FBL-3 and expanded by culture for 14 days with low concentrations of IL-2. The resultant FBL-3-specific T cell lines were adoptively transferred into cyclophosphamide pretreated congenic hosts (B6/Thy-1.1), and restimulated every 14 days by an injection of irradiated FBL-3 plus a 7-day course of IL-2. Donor T cells residing in the host were identified and quantified by use of antibody to the Thy-1.2 allele. The results confirmed that stimulation with FBL-3 on the day of transfer (day 0) plus IL-2 on days 0 to 6 induced rapid growth of donor T cells to approximately an 11-fold increase in total donor T cell number recoverable from host ascites and spleen by day 7. However, prolonging the course of IL-2 administration to 35 days did not maintain the number or the specific cytolytic function of donor T cells. By contrast, intermittent restimulation with specific Ag plus IL-2 induced intermittent regrowth of donor T cells in vivo, maintained the number of donor T cells in vivo at greater than the number input for longer than 1 mo, and allowed detection of substantially augmented donor T cell-mediated specific antitumor function over that period of time.     

Terminal differentiation in cultured Friend erythroleukemia cells.

Two populations of differentiated, hemoglobin-containing cells have been identified in cultures of Friend murine erythroleukemia cells (Friend cells): terminally differentiated benzidine-positive (B+) cells that are no longer capable of proliferation and are arrested in the G1 phase of the cell cycle, and their precursors, traversing B+ cells which undergo two or three cell divisions before reaching their terminally differentiated state. Thus Friend cells in suspension culture retain a limited capacity to synthesize DNA and divide after commitment to erythroid differentiation. We identified terminally differentiated cells using autoradiography after benzidine staining. We also developed a quantitative flow microfluorometric assay to distinguish cells that are terminally differentiated from those cells committed to differentiation but still capable of proliferation.We developed a purification procedure to isolate terminally differentiated Friend cells. Their DNA content was the same as that of the undifferentiated cells in G1 by both the diphenylamine reaction and a fluorescence assay. No loss of DNA was detected during the differentiation of Friend cells. As many as 72% of the total cells in a culture induced with DMSO (88% B+) were differentiated cells arrested in G1. As a control, a DMSO-resistant line derived from 745A neither differentiated nor arrested in G1 after growth in the presence of DMSO. The results of these studies were obtained using several compounds that induce differentiation and three independently isolated clones of 745A. We also observed arrest of differentiated cells in G1 with the two other well characterized, independently derived erythroleukemia cell lines, F4-1 and T3-C1-2.     

Specific esterase activity induced in mouse Friend erythroleukemia cells by dimethylsulfoxide.

Three cell lines of mouse erythroleukemia transformed by Friend virus (FLC), namely 745, F4-1, and 3BM-78, were grown for six days in the absence or in the presence of 1.5% (v/v) dimethylsulfoxide (DMSO) and compared cytochemically for naphtol-AS D-chloroacetate esterase (E), alkalinephosphatase (AP), myeloperoxidase (MP) and periodic acid Schiff (PAS) reaction activity. In the absence of inducer only 1–2% of slightly E positive cells could be found. E positivity greatly increased in 3BM-78 and F4-1 but poorly in 745 cells, after treatment with DMSO. Unlike E reaction, AP and MP reactions were positive in about 5% 3BM-78 and F4-1 cells without DMSO, but there were no positive cells after DMSO treatment. All three lines were always PAS negative. Hemoglobin synthesis (benzidine staining) was intensively induced by DMSO in all three lines. Morphologically after DMSO treatment, FLC matured displaying characteristics of basophilic megaloblastoid cells. The emergence of specific esterase activity, a marker of granulocytes, in FLC differentiating along the erythroid pathway, suggests that in these leukemia cells the genetic determinants for leukopoietic differentiation are retained and capable of being expressed phenotypically.     

Synthesis of erythrocyte-specific proteins in cultured friend leukemia cells

We have studied synthesis of specific proteins in two permanent ilness of Friend virus-induced erythroleukemia cells (Friend line 745 and Ostertag line FSD-1, both derived from DBA/2 mice). By 96 hr following treatment with 1–2% dimethyl sulfoxide (Me₂SO), up to 25% of the protein being synthesized by both these cultures is hemoglobin. At that time, hemoglobin constitutes up to 10% of the cellular soluble protein. Both lines synthesize heme and globin coordinately, and α and β globin chains in a nearly balanced 1:1 ratio. However, the ratio of β^Major:β^Minor chains synthesized by these induced Friend leukemia (FL) cells is approximately 9 in the FSD-1 line and 1.3 in the Friend Clone 745 line, whereas it is 4 in normal adult DBA/2 mouse erythrocytes. Evidence for the latter conclusion was obtained by electrophoresis of FL hemoglobins on cellulose acetate membranes, and also by chromatographic separation of α, β^Major, and β^Minor globins on carboxymethylcellulose in 8 M urea at 20°C. Carbonic anhydrase activity per mg protein is 3 times higher in induced than in control cultures. 2,3-diphosphoglyceric acid is not found in induced FL cells. Induced and control FL cells agglutinate strongly and equally with Phaseolus vulgaris phytohemagglutinin. The developmental process in these cultured leukemia cells appears to be an aberrant erythropolesis.