Single nucleotide polymorphism in PTEN-Long gene: A risk factor in chronic myeloid leukemia.

The BCR-ABL1 oncogene is associated with chronic myeloid leukemia (CML) pathogenesis, but the molecular mechanisms that initiate leukemogenesis are still unclear. Cancer pathogenesis has been associated with genetic alterations that may lead to inactivation of tumor suppressor genes. Phosphatase and tensin homolog (PTEN) is frequently deleted or inactivated in various tumors. A recently discovered variant of PTEN, PTEN-Long (PTEN-L), results from an alternative translation initiation site located upstream of the canonic AUG and generates a protein of 576 amino acids instead the expected protein of 403 amino acids. A 16 bp perfect palindromic motif centered on the PTEN-L CUG513 start codon is required for translation initiation. A single nucleotide polymorphism (SNP) of PTEN-L gene rs12573787 is located on the first exon respect to the CUG initiation site. In this case-control study we evaluated the association of genetic variants in PTEN-L with CML risk and therapy response in the Argentine population. The allele A of SNP rs12573787 was found to be associated with CML risk OR (95% CI) 1.71 (1.11-2.63) p = 0.016, which resulted consistent by multivariate analysis adjusted by gender and age. According to previous evidence that CML is more frequent in males, we found that the genetic risk of CML was confined to this gender. Unexpectedly, we also found this association confined to CML patients older than 45 years old. To our knowledge, this is the first time that PTEN-L rs1257378 was studied in CML suggesting that the variant A allele is a risk factor for CML development but, no association with the failure to TKIs treatment was found.

Polymorphic variants of GSTM1, GSTT1, and GSTP1 genes in childhood acute leukemias: A preliminary study in Argentina.

BACKGROUND AND AIM: Despite recent major advances in leukemia research, the etiopathogenesis of childhood leukemias remains far elusive. Individual predisposing factors, including polymorphisms in detoxification enzymes, have been implicated in the molecular pathogenesis and heterogeneity of the disease. Genetic polymorphisms of glutathione S-transferases (GSTs) that alter enzyme activity could be an additional factor that increases the risk of acute leukemia, but data are lacking in Argentina. We assessed the association of GST polymorphisms and the susceptibility to childhood leukemia in Argentina by conducting an exploratory case-control study and correlated patients' genotype to clinical and biological features. METHODS: Deletion polymorphisms in GSTM1 and GSTT1 genes and the single nucleotide polymorphism in GSTP1 c.313A>G (rs1695; p.105Ile>Val) were genotyped by PCR-RFLP in 36 patients and 133 healthy individuals. RESULTS: GSTM1-null genotype was associated with a lower risk of developing acute leukemia (P = 0.013; OR: 0.31; CI: 0.12-0.80), while GSTP1-GG variants displayed an increased risk (P = 0.01; OR: 3.9; CI: 1.85-8.2). However, no differences were found for GSTT1 gene. Conclusion These preliminary results, to be validated in a larger population from Argentina, suggest that the development of pediatric leukemia may be differentially influenced by polymorphic variants in GST genes.

The proteasome inhibitor MLN-273 blocks exoerythrocytic and erythrocytic development of Plasmodium parasites.

Protein degradation is regulated during the cell cycle of all eukaryotic cells and is mediated by the ubiquitin-proteasome pathway. Potent and specific peptide-derived inhibitors of the 20S proteasome have been developed recently as anti-cancer agents, based on their ability to induce apoptosis in rapidly dividing cells. Here, we tested a novel small molecule dipeptidyl boronic acid proteasome inhibitor, named MLN-273 on blood and liver stages of Plasmodium species, both of which undergo active replication, probably requiring extensive proteasome activity. The inhibitor blocked Plasmodium falciparum erythrocytic development at an early ring stage as well as P. berghei exoerythrocytic progression to schizonts. Importantly, neither uninfected erythrocytes nor hepatocytes were affected by the drug. MLN-273 caused an overall reduction in protein degradation in P. falciparum, as demonstrated by immunoblots using anti-ubiquitin antibodies to label ubiquitin-tagged protein conjugates. This led us to conclude that the target of the drug was the parasite proteasome. The fact that proteasome inhibitors are presently used as anti-cancer drugs in humans forms a solid basis for further development and makes them potentially attractive drugs also for malaria chemotherapy.

RGS18 is a myeloerythroid lineage-specific regulator of G-protein-signalling molecule highly expressed in megakaryocytes.

Myelopoiesis and lymphopoiesis are controlled by haematopoietic growth factors, including cytokines, and chemokines that bind to G-protein-coupled receptors (GPCRs). Regulators of G-protein signalling (RGSs) are a protein family that can act as GTPase-activating proteins for G(alphai)- and G(alphaq)-class proteins. We have identified a new member of the R4 subfamily of RGS proteins, RGS18. RGS18 contains clusters of hydrophobic and basic residues, which are characteristic of an amphipathic helix within its first 33 amino acids. RGS18 mRNA was most highly abundant in megakaryocytes, and was also detected specifically in haematopoietic progenitor and myeloerythroid lineage cells. RGS18 mRNA was not detected in cells of the lymphoid lineage. RGS18 was also highly expressed in mouse embryonic 15-day livers, livers being the principal organ for haematopoiesis at this stage of fetal development. RGS1, RGS2 and RGS16, other members of the R4 subfamily, were expressed in distinct progenitor and mature myeloerythroid and lymphoid lineage blood cells. RGS18 was shown to interact specifically with the G(alphai-3) subunit in membranes from K562 cells. Furthermore, overexpression of RGS18 inhibited mitogen-activated-protein kinase activation in HEK-293/chemokine receptor 2 cells treated with monocyte chemotactic protein-1. In yeast cells, RGS18 overexpression complemented a pheromone-sensitive phenotype caused by mutations in the endogeneous yeast RGS gene, SST2. These data demonstrated that RGS18 was expressed most highly in megakaryocytes, and can modulate GPCR pathways in both mammalian and yeast cells in vitro. Hence RGS18 might have an important role in the regulation of megakaryocyte differentiation and chemotaxis.

Suppressive effects of TNF-alpha, TGF-beta1, and chemokines on megakaryocytic colony formation in CD34+ cells derived from umbilical cord blood compared with mobilized peripheral blood and bone marrow.

CD34+ cells from human umbilical cord blood (CB) were isolated and investigated for megakaryocytic (MK) colony formation in response to recombinant human (rh) stimulatory and suppressive cytokines and compared with their counterparts in normal BM and G-CSF-mobilized peripheral blood (mPBL). First, we observed that IL-11 by itself at any dosage had no stimulator activity on MK colony formation derived from CD34+ cells in CB, mPBL, and BM. IL-3, steel factor (SLF), or thrombopoietin (Tpo) alone stimulated numbers of colony-forming unit-megakaryocyte (CFU-MK) in a dose-dependent fashion. Maximum growth of MK progenitor cells was noted in the presence of a combination of cytokines: IL-11, IL-3, SLF, and Tpo. The frequency of CFU-MK in CB and mPBL was significantly greater than that in BM, and the size of colonies in CB and mPBL was significantly greater than that in BM, and the size of colonies was larger as well. In addition, an increased number of big mixed colonies containing MK were observed in CB and mPBL. In the presence of IL-11, IL-3, SLF, and Tpo, CFU-MK derived from CB, mPBL, and BM was suppressed by tumor necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta1 (TGF-beta1). CFU-MK derived from normal BM was inhibited by some chemokines evaluated, whereas CFU-MK derived from CB was suppressed only by platelet factor-4 (PF-4), IFN-inducible protein-10 (IP-10), Exodus-1, Exodus-2, and Exodus-3, but to a lesser degree. In CB, unlike granulocyte-macrophage (CFU-GM), erythroid (BFU-E), high-proliferative potential (HPP-CFC), or multipotential (CFU-GEMM) progenitors, at least a subpopulation of MK progenitors are in S-phase. Therefore, CB MK progenitors respond to the suppressive effects of some members of the chemokine family. Similar results were noted for burst-forming unit-MK (BFU-MK). Our results indicate that CB and mPBL are rich sources of MK progenitors and that MK progenitors in CB are responsive to the suppressive effects of TNF-alpha and TGF-beta1 and some members of the chemokine family.

Recombinant human interleukin-11 synergizes with steel factor and interleukin-3 to promote directly the early stages of murine megakaryocyte development in vitro.

The authors studied the role that interleukin (IL)-11 plays during the early stages of megakaryocyte (MK) development by investigating its in vitro effects on cell subpopulations enriched for bone marrow primitive progenitor cells and early and late committed progenitor cells. Progenitor subpopulations were isolated from bone marrow of normal or 5-fluorouracil (5FU)-treated mice and separated by sorting based on the surface antigens Sca-1, c-kit, and CD34. Functional analysis of the cell subpopulations, 5FU Lin(-)Sca-1(+)c-kit(+) or normal bone marrow (NBM) Lin(-)Sca-1(+)c-kit(+)CD34(-)cells, indicated that exposure of these cells to recombinant human (rh)IL-11 in combination with steel factor (SF) stimulates the formation of colonies in methylcellulose and their proliferation in single cell-containing liquid cultures. Kinetic studies of MK progenitor generation, in response to SF and rhIL-11, demonstrated that a significant number of the progenitors produced are committed to the MK lineage. RhIL-11 also synergized with both SF and IL-3 to stimulate MK colony growth from NBM Lin(-)Sca-1(+)c-kit(+) cells (early progenitors) and NBM Lin(-)Sca-1(-)c-kit(+) cells (committed late progenitors). In the presence of IL-3, NBM, Lin(-)Sca-1(-)c-kit(+) cells responded more strongly to rhIL-11 than SF. Consistent with these results is the observation that IL-11 receptor alpha chain mRNA is present in all the progenitor cells from which the MKs are derived. This cell culture and RNA analysis suggest that murine bone marrow primitive progenitor cells and early and late progenitor cells are direct targets of rhIL-11 and that rhIL-11 has the potential to promote megakaryocyte development at several very early stages. (Blood, 2000;95:503-509) (Blood. 2000;95:503-509)

Recombinant human interleukin-11 directly promotes megakaryocytopoiesis in vitro.

We have investigated the mechanism of action of the thrombopoietic cytokine, recombinant human interleukin-11 (rhIL-11), on megakaryocytopoiesis in vitro. We have shown that rhIL-11-induced murine and human megakaryocytopoiesis are not mediated by thrombopoietin (Tpo). Murine megakaryocytes (MKs) were produced from bone marrow (BM) mononuclear cells cultured with rhIL-11, IL-3, and a combination of the two cytokines. Conditioned media (CM) were collected and assayed for the presence of biologically active Tpo. Tpo activity was not detected in any of the CMs tested. Next, human BM CD34+ cells were cultured in serum-free fibrin clot medium with rhIL-11, IL-3, or rhIL-11 plus IL-3 and an antibody that neutralizes human Tpo activity. No inhibition of either burst-forming unit-MK- or colony-forming unit-MK-derived colony formation was observed. The antibody did partially inhibit steel factor-induced MK-colony formation, suggesting that the actions of this cytokine are mediated, in part, by Tpo. We determined that MKs can be direct targets of rhIL-11 by showing the expression of functional IL-11 receptor on these cells. Total RNA was prepared from cultured human BM CD41+CD14- cells (MKs) and IL-11 receptor alpha chain mRNA was detected in the MKs by reverse transcription-polymerase chain reaction. Analysis of single-sorted CD41+CD14- cells confirmed that the observed IL-11 receptor expression was not due to contaminating CD41- cells in the pool. The presence of rhIL-11 receptor alpha chain protein in the cells was established by Western blot analysis. After a short exposure of purified BM MKs to rhIL-11, enhanced phosphorylation of both its signal transduction subunit, gp130, and the transcription factor, STAT3 was detected, showing a direct activation of receptor signaling by the cytokine. Consistent with the lack of effect of rhIL-11 on platelets in vivo, IL-11 receptor alpha chain mRNA and protein were not detected in isolated human platelets. These data indicate that rhIL-11 acts directly on MKs and MK progenitors but not on platelets.

Molecular cloning and characterization of murine interleukin-11.

Human interleukin-11 (IL-11) has been shown to have pleiotropic action on hematopoietic, hepatic, stromal, epithelial, neural, and osteoclast cells. In the present work, the murine IL-11 cDNA has been isolated from a fetal thymic cell line, and its structure and function compared with human IL-11. The murine protein was demonstrated to have identical actions on the proliferation of a murine plasmacytoma cell line, murine primitive bone marrow progenitor cells, and megakaryocyte precursors. The murine IL-11 protein was synthesized as a soluble thioredoxin-IL-11 fusion in Escherichia coli and the expression of murine IL-11 was examined by pulse-chase radiolabeling in COS cells. The chromosomal location of the murine IL-11 gene was assigned to the proximal arm of chromosome 7.

The role of recombinant interleukin 11 in megakaryocytopoiesis.

Recombinant human interleukin 11 (rHuIL-11) is a multifunctional cytokine with activities on a broad range of hematopoietic cells including primitive stem cells and mature progenitor cells. Analysis of rHuIL-11 in vitro has revealed that its hematopoietic activities are predominantly a result of synergistic interactions with other early-acting factors such as IL-3 and Steel factor. Studies indicate that rHuIL-11 acts directly on purified stem and progenitor cell populations and can support the growth of colony forming units-megakaryocyte in these cultures. In normal animals, rHuIL-11 has a potent effect on cells of the megakaryocyte (MK) lineage. Administration of rHuIL-11 results in a two- to threefold increase in circulating platelets, stimulation of bone marrow (BM) and spleen progenitor numbers, and enhanced MK maturation as measured by a shift to higher ploidy values. rHuIL-11 administration in preclinical models of myelosuppression induced by chemotherapy and/or irradiation has shown a reproducible acceleration of platelet recovery and, in some models, enhanced neutrophil and red blood cell recovery. rHuIL-11 has been tested in a non-human primate myelosuppression model using carboplatin. Administration of rHuIL-11 following carboplatin treatment was found to eliminate the period of severe thrombocytopenia (<20,000 platelets/ml) and enhance the recovery of platelets to normal levels (>100,000/ml). Recently, human clinical trials conducted with rHuIL-11 in patients treated with chemotherapy have demonstrated its potent thrombopoietic activity, including improved platelet nadirs, enhanced platelet recovery and a significant decrease in the number of patients who require platelet transfusions. Combined with the preclinical results, these studies confirm that this cytokine will be an effective agent in the treatment of myelosuppression and thrombocytopenia associated with cancer chemotherapy and BM transplantation.

Influence in vitro of IL-3/Epo fusion proteins compared with the combination of IL-3 plus Epo in enhancing the proliferation of single isolated erythroid and multipotential progenitor cells from human umbilical cord blood and adult bone marrow.

Human interleukin-3/erythropoietin (IL-3/Epo) fusion protein have been constructed, expressed, and tested for biological activity. These fusion proteins were previously shown to be active on erythroid progenitors (BFU-E) from unseparated human bone marrow. We evaluated if these fusion proteins could stimulate erythroid and multipotential progenitor cells directly at the single-cell level. Two IL-3/Epo fusion proteins containing short (SL-3E, two amino acids) and long (LL-3E, 23 amino acids) linker sequences as well as a short linker Epo/IL-3 sequence (SL-E3, three amino acids) were tested. Highly enriched CD34 or BFU-E enriched CD34 CD33- cells from human umbilical cord blood or CD34 HLA-DR+CD33- cells from normal adult bone marrow were sorted as single cells into single wells. The combination of Epo plus IL-3 synergized to enhance the proliferation of BFU-E and multipotential progenitors (CFU-GEMM) in comparison to the individual effects of these cytokines. The three fusion proteins also enhanced proliferation of BFU-E and CFU-GEMM at the single-cell level and were at least as active as the combination of Epo and IL-3, demonstrating that IL-3/Epo fusion proteins directly stimulate proliferation of BFU-E and CFU-GEMM and that biological activity of IL-3 and Epo in vitro can be maintained when these proteins are fused. The activity of the combination of Epo and IL-3 or the fusion proteins was partially neutralized by preincubation with monoclonal antibodies to either Epo or IL-3 and was neutralized by greater than 90% by the combination of both antibodies, suggesting that the Epo and IL-3 components of the fusion proteins were both involved in the enhancing activity of these proteins. Additionally, use of monoclonal antibody to the human Epo receptor completely blocked the stimulating/enhancing activity of Epo alone, Epo plus IL-3, or the fusion proteins for stimulation of colony formation by BFU-E and CFU-GEMM but not for granulocyte-macrophage progenitors (CFU-GM), suggesting that the enhancing effects of the fusion proteins are most likely mediated, at least in part, by the Epo receptor.

Interleukin-6 is required in vivo for the regulation of stem cells and committed progenitors of the hematopoietic system.

The development of blood cells from hematopoietic stem cells is controlled by multiple cytokines. These growth factors influence survival, cell cycle status, differentiation into lineage-committed progenitors, final maturation into blood cells, and perhaps self-renewal of stem cells. The specific contribution of IL-6 to these processes in vivo was evaluated in mice with a targeted disruption of the IL-6 gene. Decreases in the absolute numbers of CFU-Sd12 and preCFU-S, as well as in the functionality of LTRSC in these mutant mice, suggests a role for IL-6 in the survival, self-renewal, or both of hematopoietic stem cells and early progenitors. In addition, as a result of the IL-6 deficiency, the control between proliferation and differentiation of the progenitor cells of the granulocytic-monocytic, megakaryocytic, and erythroid lineages into mature blood cells is altered, leading to abnormal levels of committed progenitors of these lineages and to a slow recovery from hematopoietic ablation.

Interleukin-3/erythropoietin fusion proteins: in vitro effects on hematopoietic cells.

Erythropoietin (Epo) acts synergistically with interleukin-3 (IL-3) to induce proliferation and differentiation of erythroid progenitors. This synergy occurs at IL-3 concentrations that have little or no effect alone. To determine whether optimal expansion of erythroid cells results when they are targeted by a molecule with both IL-3 and Epo activities, fusion proteins were generated and analyzed. Expression vectors were constructed in which the coding regions of human IL-3 and Epo cDNAs were joined by either a short (2 to 3 amino acids) or long (23 amino acids) linker sequence and expressed in Chinese hamster ovary (CHO) cells. Analysis of equilibrium binding properties of the IL-3 and Epo moieties revealed that in all fusion proteins each retained the ability to bind receptor. When IL-3 was connected to Epo by a short linker, the binding affinity of the IL-3 moiety was lower. In vitro proliferative activity of each moiety was observed on cell lines responsive to IL-3, Epo or a combination of the two cytokines. Fusion of IL-3 to Epo through its amino terminus was found to result in partial loss of its function. All the fusion proteins were biologically active on human bone marrow. When IL-3 was located at the amino domain of the protein, induction of erythroid colonies was similar to that of a mixture of IL-3 and Epo. These results indicate that biological integrity of both IL-3 and Epo can be maintained when these cytokines are fused, but that enhancement of erythropoiesis over that observed with a mixture of the two cytokines cannot be achieved by their fusion alone. Other requirements such as the coexpression of the IL-3 and Epo receptors and the sharing of a receptor subunit are likely to be needed for an optimal cell response to the fusion growth factors.

Erythropoietin induces Raf-1 activation and Raf-1 is required for erythropoietin-mediated proliferation.

Erythropoietin mediates the rapid phosphorylation of Raf-1 in the murine cell lines HCD-57 and FDC-P1/ER, which proliferate in response to this cytokine. Phosphorylation occurs at both serine and tyrosine residues and as such is similar to the Raf-1 phosphorylation seen after interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor, and interleukin-2 stimulation in other murine cell lines. Such data suggest that these growth factors may share a common mechanism(s) of Raf-1 phosphorylation. Furthermore, in association with Raf-1 phosphorylation, erythropoietin induces a 2-3-fold increase in Raf-1 kinase activity as measured in immune complex kinase assays in vitro. Finally, a c-raf antisense oligodeoxyribonucleotide, which specifically decreases intracellular Raf-1 levels, also substantially inhibits both erythropoietin and IL-3-directed DNA synthesis. Together, these results provide evidence that activated Raf-1 is a necessary component of erythropoietin and IL-3 growth signaling pathways.

Characteristics of erythroleukemia cells selected for vincristine resistance that have accelerated inducer-mediated differentiation.

The induction of murine erythroleukemia cells (MELC; DS19/Sc9) to terminal differentiation by hexamethylenebisacetamide (HMBA) is characterized by a latent period of 10-12 hr before onset of commitment to terminal-cell division and increased transcription of globin genes. MELC variants, derived from this parental cell line, selected for resistance to vincristine (VC), can be induced to differentiate with little or no latent period. This study shows that accelerated HMBA-induced commitment is characteristic of MELC with a low level (2- to 5-fold) of VC resistance in four independently derived cell lines. Both resistance to VC and accelerated differentiation are stable phenotypes for at least 50 passages (approximately 5 months) in the absence of VC. Low-level VC-resistant MELC do not display increased levels of P-glycoprotein or mdr1, mdr2, and mdr3 mRNAs, nor do they exhibit cross-resistance to colchicine or doxorubicin. These cells do show (i) increased level of protein kinase C activity, (ii) reduced accumulation of [3H]VC, and (iii) restoration of VC sensitivity in the presence of verapamil. MELC selected for higher levels of VC resistance (approximately 500-fold) do express high levels of P-glycoprotein and the mdr3 gene. During HMBA-induced differentiation, DS19/Sc9 decrease [3H]VC accumulation, but P-glycoprotein content does not change. A VC-transport-associated protein, also critical for the process of induced differentiation, may be constitutively present in VC-resistant MELC, accounting for their enhanced sensitivity to inducer. This protein accumulates by exposure of VC-sensitive cells to HMBA, contributing to their differentiation and decreased level of VC accumulation.

Relationship between globin mRNA accumulation and commitment to terminal cell differentiation in inducer sensitive and resistant erythroleukemia variants.

The relationship between the kinetics of commitment to terminal cell differentiation and the rates of accumulation of globin mRNA has been examined during the induction of erythroid differentiation by polar/apolar chemical inducers in murine erythroleukemia cells (MELC), under conditions of more and less rapid commitment. Two differentiation inducers and three MELC variants have been studied. Hexamethylene bisacetamide (HMBA) initiates more rapid commitment than does dimethylsulfoxide (Me2SO). MELC variant DR10 is resistant to induction by Me2SO and responds sluggishly to HMBA, in comparison with the DS19-Sc9 variant. V3.17, an MELC variant resistant to low concentrations of vincristine, shows increased sensitivity to the inducers and an accelerated rate of commitment to terminal differentiation compared with DS19-Sc9. It is demonstrated that commitment and the actual expression of differentiation, as measured by the accumulation of alpha-, beta maj-, and beta min-globin mRNA, are temporally coordinated functions during induced differentiation of a transformed cell line by exposure to polar/apolar agents.

Vincristine-resistant erythroleukemia cell line has marked increased sensitivity to hexamethylenebisacetamide-induced differentiation.

Hexamethylenebisacetamide (HMBA)-induced murine erythroleukemia (MEL) differentiation is a multistep process. Commitment is the capacity to express terminal cell division and characteristics of the differentiated phenotype even after the cells are removed from culture with inducer. Culture of MEL cell line 745A.DS19 (DS19) with HMBA causes commitment to terminal differentiation after a latent period of about 10-12 hr. Previous studies have shown that during this latent period, HMBA causes a number of metabolic changes, including modulation in expression of certain protooncogenes. We now report the development of a MEL cell line (designated V3.17) derived from DS19 that is resistant to vincristine and is (i) markedly more sensitive to HMBA, (ii) induced to commitment without a detectable latent period, and (iii) resistant to the effects of phorbol ester and dexamethasone, which are potent inhibitors of HMBA-mediated DS19 differentiation. We suggest that this V3.17 MEL cell line may express a factor that circumvents HMBA-mediated early events, which prepare the cells for commitment to terminal differentiation.

Regulation of murine alpha-, beta major-, and beta minor-globin gene expression.

Transcriptional properties of the mouse globin genes and their promoters were examined in COS cells using transient expression assays. Transfected intact mouse alpha 1-globin genes generate transcripts, whereas beta maj and beta min globin transcripts are detected only if the genes are associated with a strong exogenous (SV40) enhancer sequence. Under these conditions the ratio of accumulated beta maj and beta min mRNA sequences approximates the 4:1 ratio observed in reticulocytes and murine erythroleukemia cells (MELC) induced to differentiate by hexamethylene bisacetamide (HMBA). As determined using hybrid genes consisting of globin gene 5' regions fused to the chloramphenicol acetyltransferase (CAT) structural gene, this 4:1 ratio appears dependent upon the relative activities of the two 5' promoter regions.

Hypomethylation of DNA during differentiation of Friend erythroleukemia cells.

DNA from mammalian cells has been shown to contain significant amounts of 5-methyl cytosine resulting from enzymatic transfer of methyl groups from s-adenosylmethionine to cytosine residues in the DNA polymer. The function of this modification is not known. We have found that DNA synthesized during chemically induced differentiation of friend erythroleukemia cells is hypomethylated, as measured by its ability to accept methyl groups transferred by homologous DNA methyltransferases in vitro. The extent of hypomethylation detected by this sensitive method is small, a decrease of less than 1.6 percent in 5-methylcytosine content. Hypomethylated DNA can be isolated from friend erythroleukemia cells grown in the presence of dimethyl sulfoxide, butyrate, hexamethylene-bis- acetamide, pentamethylene-bis acetamide, and ethionine. However, hypomethylated DNA is found only under conditions where differentiation is actually induced. DNA isolated from cells of a dimethyl sulfoxide- resistant subclone grown in the presence of that agent is not hypomethylated, although DNA of these cells becomes hypomethylated after growth in the presence of inducers that can trigger their differentiation. We also find that the DNA of friend erythroleukemia cells does not become hypomethylated when the cells are exposed to inducing agents in the presence of substances that inhibit differentiation. These results suggest a close link between genome modification by methylation and differentiation of friend erythroleukemia cells.