Murine Erythroleukemia Cells Research Articles

1-Methylnicotinamide stimulates cell growth and inhibits hemoglobin synthesis in differentiating murine erythroleukemia cells

Exposure of murine erythroleukemia cells (MELCs) to nicotinamide (NA) or its synthetic analog N′-methylnicotinamide (N′-MN) reduces cell growth and induces terminal differentiation, marked by increased heme and globin accumulation. On the contrary, 1-methylnicotinamide (1-MN), the primary metabolite of excess NA, was found to stimulate cell growth and reduce spontaneous differentiation of cultured MELCs. Log phase MELCs exhibited up to 50% higher cell density above untreated cells when cultured for up to 96 h with 2.5 mM 1-MN. When combined with NA or several chemically-unrelated inducers of hemoglobin synthesis in cultured MELCs, 1-MN reduced the globin mRNA levels and heme accumulation by 40–80%. 1-MN was able to inhibit heme production if present during only the first 24–48 h after NA exposure. Pre-treatment with 1-MN could not confer resistance of cells to effects of NA, suggesting the inhibition is reversible. Commitment to differentiate in semisolid medium by the most potent inducer, 5 mM N′-MN, was inhibited up to 95% by 2.5 mM concentrations of 1-MN. It appears that 1-MN has opposing effects on growth and induction of differentiation than those seen in MELC cultures exposed to NA or N′-MN.

PU.1 is dispensable to block erythroid differentiation in Friend erythroleukemia cells

Friend murine erythroleukemia cell lines derive from erythroblasts transformed with the Friend complex where the spleen-focus forming virus integrated in the vicinity of the Sfpi-1 locus. Erythroleukemia cells do not differentiate and grow indefinitely in the absence of erythropoietin. Activation of the transcription factor PU.1, encoded by the Sfpi-1 gene, is thought to be responsible for the transformed phenotype. These cells can overcome the blockage and reinitiate their differentiation program when exposed to some chemical inducers such as hexamethylene bisacetamide. In this study, we established cell cultures that were capable to proliferate unconstrained in the presence of the inducer. Resistant cell lines restart erythroid differentiation, though, if forced to exit the cell cycle or by overexpressing the transcription factor GATA-1. Unexpectedly, expression of PU.1 was suppressed in the resistant clones albeit the spleen-focus forming virus was still integrated in the proximity of the Sfpi-1 locus. Exposure to 5-Aza-2′-deoxycytidine activates PU.1 expression suggesting that the PU.1 coding gene is highly methylated in the resistant cells. Altogether these results suggest that PU.1 is dispensable to block erythroid differentiation.

The role of peroxiredoxin III on late stage of proerythrocyte differentiation

Peroxiredoxin III (Prdx III), the mitochondrial peroxidase, was preferentially expressed in murine erythroleukemia (MEL) cells. However, the mechanisms by which Prdx III regulates erythroid differentiation are unknown. In this study, K562 cells were differentiated by Ara-C treatment, and Prdx III was dramatically increased until day 5. We also investigated Prdx III expression pattern on in vitro erythropoiesis of human CD34 + cells. When human CD34 + cells became proerythrocyte on day 7, Prdx III was diminished, and then augmented on day 12. We established the stable sublines of Prdx III overexpression (O/E), and dominant-negative (D/N). The intracellular ROS level of Prdx III O/E cell line was lower than D/N stable cell lines. Moreover, Prdx III O/E cell line was placed in G1-arrest, but not D/N cell lines. Finally, the expression level of β-globin and GATA-1 was dramatically increased in Prdx III O/E cell line.

Benzo[b]thiophene-based histone deacetylase inhibitors

Benzo[b]thienyl hydroxamic acids, a novel class of histone deacetylase (HDAC) inhibitors, were identified via a targeted screen of small molecule hydroxamic acids. Various substitutions were explored in the C5- and C6-positions of the benzo[b]thiophene core to characterize SAR and develop optimal inhibitors. It was determined that substitution at the C6-position of the benzo[b]thiophene core with a three-atom spacer yielded optimal HDAC1 inhibition and anti-proliferative activity in murine erythroleukemia (SC-9) cells.

In VitroandIn VivoExpression of Human Erythrocyte Pyruvate Kinase in Erythroid Cells: A Gene Therapy Approach

Human pyruvate kinase deficiency (PKD), an autosomal recessive disorder produced by mutations in the PKLR gene, is the most common cause of chronic nonspherocytic hemolytic anemia. Transduction of wild-type erythroid (R-type) pyruvate kinase (RPK) cDNA into deficient hematopoietic stem cells could be of potential use as rescue therapy in severe clinical cases. In this study, gammaretroviral vectors expressing human RPK were designed as possible gene therapy candidates for this disease. Through real-time quantitative reverse transcriptase-polymerase chain reaction, Western blotting, and flow cytometric analysis, we demonstrate stable RPK expression in both undifferentiated and differentiated murine erythroleukemia cells. In this in vitro assay, the proportion of transduced cells and the intensity of expression of the transgene remained unaltered after 6 months of culture. Moreover, transplanting human RPK-transduced Lin(-)Sca-1(+) mouse cells in myeloablated primary and secondary recipients rendered high proportions of erythroid precursors and mature erythrocytes expressing RPK, without inducing hematopoietic effects. These findings suggest that retroviral vectors could be useful for the delivery and expression of RPK in erythroid cells, and provide evidence of the potential use of gene therapy strategies to phenotypically correct erythroid PKD.

Exploring cellular memory molecules marking competent and active transcriptions

BackgroundDevelopment in higher eukaryotes involves programmed gene expression. Cell type-specific gene expression is established during this process and is inherited in succeeding cell cycles. Higher eukaryotes have evolved elegant mechanisms by which committed gene-expression states are transmitted through numerous cell divisions. Previous studies have shown that both DNase I-sensitive sites and the basal transcription factor TFIID remain on silenced mitotic chromosomes, suggesting that certain trans-factors might act as bookmarks, maintaining the information and transmitting it to the next generation.ResultsWe used the mouse globin gene clusters as a model system to examine the retention of active information on M-phase chromosomes and its contribution to the persistence of transcriptional competence of these gene clusters in murine erythroleukemia cells. In cells arrested in mitosis, the erythroid-specific activator NF-E2p45 remained associated with its binding sites on the globin gene loci, while the other major erythroid factor, GATA-1, was removed from chromosome. Moreover, despite mitotic chromatin condensation, the distant regulatory regions and promoters of transcriptionally competent globin gene loci are marked by a preserved histone code consisting in active histone modifications such as H3 acetylation, H3-K4 dimethylation and K79 dimethylation. Further analysis showed that other active genes are also locally marked by the preserved active histone code throughout mitotic inactivation of transcription.ConclusionOur results imply that certain kinds of specific protein factors and active histone modifications function as cellular memory markers for both competent and active genes during mitosis, and serve as a reactivated core for the resumption of transcription when the cells exit mitosis.

The notch pathway positively regulates programmed cell death during erythroid differentiation

Programmed cell death plays an important role in erythropoiesis under physiological and pathological conditions. In this study, we show that the Notch/RBPjkappa signaling pathway induces erythroid apoptosis in different hematopoietic tissues, including yolk sac and bone marrow as well as in murine erythroleukemia cells. In RBPjkappa(-/-) yolk sacs, erythroid cells have a decreased rate of cell death that results in increased number of Ter119(+) cells. A similar effect is observed when Notch activity is abrogated by incubation with the gamma-secretase inhibitors, DAPT or L685,458. We demonstrate that incubation with Jagged1-expressing cells has a proapoptotic effect in erythroid cells from adult bone marrow that is prevented by blocking Notch activity. Finally, we show that the sole expression of the activated Notch1 protein is sufficient to induce apoptosis in hexametilene-bisacetamide-differentiating murine erythroleukemia cells. Together these results demonstrate that Notch regulates erythroid homeostasis by inducing apoptosis.

An approach to cell membrane diagnosis: laser-induced `reformation' of the murine erythroleukaemia cell from a spherical to planar membrane

Detection of the cytoplasmic domain of transmembrane oncoproteins attracts primary attention in cancer diagnosis, since the diagnostically marked extracellar domain is shed off along with increased malignancy. The shedding problem severely inhibits the conventional cell diagnosis, like immunomicroscopy from outside the cells. If detection on the cytoplasmic surface of each cell membrane becomes as extensive as the detection on the whole cell membrane and as efficient as that done within seconds, it will be in high demand. We propose here a new application of femtosecond laser, where a single cell is ruptured as a laserinduced cavity expands in cytosol and the whole cell membrane is thus projected over a flat surface in a short period of time (< 10 s). Using this configuration, we achieved projection of the cytoplasmic surface of the cell membrane for 57% of murine erythroleukemia cells irradiated, including 34% of them at full extension of the membrane. The inside-out projection was confirmed by immunostaining both -actinin in murine erythroleukemia cells and an oncoprotein c-erbB2 in human adenocarinoma cells. This projection requires only a small area (< 1000 μm2) and a short irradiation interval on a microscope slide, thus leaves room for a high throughput serial process.

Discovery and development of SAHA as an anticancer agent

The path to the discovery of suberoylanilide hydroxamic acid (SAHA, vorinostat) began over three decades ago with our studies designed to understand why dimethylsulfoxide causes terminal differentiation of the virus-transformed cells, murine erythroleukemia cells. SAHA can cause growth arrest and death of a broad variety of transformed cells both in vitro and in vivo at concentrations that have little or no toxic effects on normal cells. It was discovered that SAHA inhibits the activity of histone deacetylases (HDACs), including all 11 known human class I and class II HDACs. HDACs have many protein targets whose structure and function are altered by acetylation including histones and non-histone proteins component of transcription factors controlling gene expression and proteins that regulate cell proliferation, migration and death. SAHA is in clinical trials and has significant anticancer activity against both hematologic and solid tumors at doses well tolerated by patients. A new drug application has been approved for SAHA (vorinostat) treatment of cutaneous T-cell lymphoma.

Exogenous Wild‐Type p53 Gene Improved Survival of Nude Mice Injected with Murine Erythroleukemia Cell Line Through Amelioration of Hemorheological Changes

Previous investigations have shown that human wild-type p53 gene (WTp53) inhibits the growth of leukemia and tumor cells in vitro. In the present study, the authors used nude mice and examined the therapeutic role of p53 gene for erythroleukemia in vivo in the absence of MHC effects. The nude mice were injected with murine erythroleukemia cells (MEL), MEL cells transfected with wild-type p53 gene (MEL-W), and MEL cells transfected with mutated p53 gene (MEL-M). Abnormalities were found in the hemorheological and biophysical properties of red blood cells in all 3 groups of animals, but the abnormalities were lesser in degree and later in appearance in MEL-W group than in MEL and MEL-M groups. Furthermore, the nude mice in MEL-W group lived longer than those in MEL and MEL-M groups. The results showed that WTp53 restrained the growth of erythroleukemia cells in vivo and reduced the erythroleukemia tumorigenesis in the microcirculation by improving the hemorheological and biophysical properties of MEL cells, which helped to prolong the life span of nude mice suffering from erythroleukemia. These results contribute to our knowledge on the use of wild-type p53 gene for the treatment of erythroleukemia disease.

Severe Hypoxia Defines Heterogeneity and Selects Highly Immature Progenitors Within Clonal Erythroleukemia Cells

We showed that resistance to severe hypoxia defines hierarchical levels within normal hematopoietic populations and that hypoxia modulates the balance between generation of progenitors and maintenance of hematopoietic stem cells (HSC) in favor of the latter. This study deals with the effects of hypoxia (0.1% oxygen) in vitro on Friend's murine erythroleukemia (MEL) cells, addressing the question of whether a clonal leukemia cell population comprise functionally different cell subsets characterized by different hypoxia resistance. To identify leukemia stem cells (LSC), we used the Culture Repopulating Ability (CRA) assay we developed to quantify in vitro stem cells capable of short-term reconstitution (STR). Hypoxia strongly inhibited the overall growth of MEL cell population, which, despite its clonality, comprised progenitors characterized by markedly different hypoxia-resistance. These included hypoxia-sensitive colony-forming cells and hypoxia-resistant STR-type LSC, capable of repopulating secondary liquid cultures of CRA assays, confirming what was previously shown for normal hematopoiesis. STR-type LSC were found capable not only of surviving in hypoxia but also of being mostly in cycle, in contrast with the fact that almost all hypoxia-surviving cells were growth-arrested and with what we previously found for HSC. However, quiescent LSC were also detected, capable of delayed culture repopulation with the same efficiency as STR-like LSC. The fact that even quiescent LSC, believed to sustain minimal residual disease in vivo, were found within the MEL cells indicates that all main components of leukemia cell populations may be present within clonal cell lines, which are therefore suitable to study the sensitivity of individual components to treatments. Disclosure of potential conflicts of interest is found at the end of this article.

Proteomic analysis of erythroid differentiation induced by hexamethylene bisacetamide in murine erythroleukemia cells

Murine erythroleukemia (MEL) cells are transformed erythroid precursors that are arrested in an immature and proliferating state. These leukemic cells can be grown in cell cultures and induced to terminal erythroid differentiation by a treatment with a specific chemical inducer such as N,N'-hexamethylene bisacetamide. MEL cells then re-enter their original erythroid program and differentiate along the erythroid pathway into non-dividing hemoglobin-rich cells resembling orthochromatophilic normoblasts. To deepen our understanding of the molecular mechanisms underlying and erythroid differentiation and leukemia we monitored changes in protein expression in differentiating MEL cells. In our effort to find new candidate proteins involved in the differentiation of MEL cells, we embraced a proteomic approach. Employing two-dimensional (2D) electrophoresis combined with mass spectrometry, we compared protein expression in non-induced MEL cells with MEL cells exposed to N,N'-hexamethylene bisacetamide for 48 h. From 700 proteins spots observed, 31 proteins were differentially expressed. We successfully identified 27 of the differentially expressed molecules by mass spectrometry (MALDI-MS). In addition to proteins involved in heme biosynthesis, protein metabolism, stress defense and cytoskeletal organization, we identified 3 proteins engaged in regulation of cellular trafficking and 7 proteins important for regulation of gene expression and cell cycle progression including 3 components of chromatin remodeling complexes. Many of the identified molecules are associated with erythroid differentiation or leukemia for the first time. To our knowledge, this is the first study applying a modern proteomic approach to the direct analysis of erythroid differentiation of leukemic cells.

Purification and Identification of Proteins That Bind to the Hereditary Persistence of Fetal Hemoglobin –198 Mutation in the γ-Globin Gene Promoter

Expression of the gamma-globin gene is silenced in adult humans. However, certain point mutations in the gamma-globin gene promoter are capable of maintaining expression of this gene during adult erythropoiesis, a condition called non-deletion hereditary persistence of fetal hemoglobin (HPFH). Among these, the British form of HPFH carrying a T-->C point mutation at position -198 of the Agamma-globin gene promoter results in 4-10% fetal hemoglobin in heterozygotes. In this study, we used nuclear extracts from murine erythroleukemia cells to purify a protein complex that binds the HPFH -198 gamma-globin gene promoter. Members of this protein complex were identified by mass spectrometry and include DNMT1, the transcriptional coactivator p52, the protein SNEV, and RAP74 (the largest subunit of the general transcription factor IIF). Sp1, which was previously considered responsible for HPFH -198 gamma-globin gene activation, was not identified. The potential role of these proteins in the reactivation and/or maintenance of gamma-globin gene expression in the adult transcriptional environment is discussed.

Altered regulation of β-like globin genes by a redesigned erythroid transcription factor

Targeted regulation of beta-like globin genes was studied using designer zinc finger transcription factors containing the DNA binding domain of the red cell specific transcription factor erythroid Kruppel-like factor (EKLF) fused to repression domains. Globin gene expression was analyzed after introduction of the modified transcription factors into cell lines, embryonic stem cells and transgenic mice. As would be predicted, when introduced transiently into cells these transcription factors were effective in repressing the adult beta-globin promoter CACCC element, which is the natural target for EKLF. In murine erythroleukemia cells repression of the adult beta-globin gene was accompanied by a reactivation of the endogenous embryonic betaH1-globin gene. Studies in differentiated embryonic stem cells and transgenic mice confirmed the reactivation of embryonic gene expression during development. Our studies support a competition model for beta-globin gene expression and underscore the importance of EKLF in the embryonic/fetal-to-adult globin switch. They also demonstrate the feasibility of designer zinc finger transcription factors in the study of transcriptional control mechanisms at the beta-globin locus and as potential gene therapy agents for sickle cell disease and related hemoglobinopathies.

Activation of Prn‐p gene and stable transfection of Prn‐p cDNA in leukemia MEL and neuroblastoma N2a cells increased production of PrPC but not prevented DNA fragmentation initiated by serum deprivation

Prion protein (PrP(C)) via its isoform PrP(SC) is involved in the pathogenesis of transmissible spongiform encephalopathies (TSEs). We observed that murine erythroleukemia (MEL) cells arrested in phase G(1) undergo transcriptional activation of Prn-p gene. Here, we explored the potential role of activation of Prn-p gene and cytosolic accumulation of PrP(C) in growth arrest, differentiation, and apoptotic DNA fragmentation by stably transfecting MEL and N2a cells with Prn-p cDNA. Stably transfected MEL cells (clones # 6, 12, 20, 38, and 42) were assessed for growth and differentiation, while clones N2a13 and N2a8 of N2a cells for growth and apoptosis by flow cytometry using Annexin V and propidium iodide (PI). Our results indicate that (a) Induction of terminal differentiation of stably transfected MEL cells led to growth arrest, activation of Prn-p gene, concomitant expression of transfected Prn-p cDNA, suppression of bax gene, cytosolic accumulation of PrP(C), and DNA fragmentation. The latter was also induced in non-differentiated MEL cells growing under serum-free conditions; (b) similarly, serum deprivation promoted growth arrest, apoptosis/necrosis associated with DNA fragmentation in parental N2a and N2a13 cells that produced relative high level of PrP(C) and not PrP(SC). These data indicate that activation of Prn-p gene and expression of transfected Prn-p cDNA in cells of both hematopoietic and neuronal origin occurred concomitantly, and led to cytosolic accumulation of PrP(C) and DNA damage induced by serum deprivation. PrP(C) production failed to protect DNA fragmentation induced by serum deprivation. The question how does PrP(C) contribute to growth arrest and DNA fragmentation is discussed.

Global modulation of chromatin dynamics mediated by dephosphorylation of linker histone H1 is necessary for erythroid differentiation

Differentiation of metazoan cells involves dramatic changes in gene expression patterns and proliferative capacity driven primarily by epigenetic mechanisms. Here we used in vivo photobleaching techniques and biochemical assays to investigate the contribution of alterations in chromatin dynamics to the differentiation of murine erythroleukemia (MEL) cells, a model system for erythroid development. As MEL cells differentiate the binding affinity of all linker histone variants increases, indicative of an overall decrease in chromatin flexibility. Changes in H1(0) binding properties depend on phosphorylation at one or more of three cyclin-dependent kinase sites. The presence of constructs mimicking constitutively phosphorylated H1 results in a significant inhibition in the acquisition of commitment to terminal cell division and the expression of erythroid-specific properties. These data indicate that the progressive loss of cdk activity associated with MEL cell differentiation leads to the accumulation of dephosphorylated linker histones and restricted chromatin flexibility and that these are necessary events in the progression of erythroid differentiation. We present additional data indicating that the presence of phosphorylated H1 has a dominant effect on the binding behavior of other linker histones and propose a model for the role of linker histone phosphorylation in which these modifications act within the context of assembled chromatin.

The study of sequence configuration and functional impact of the (AC)n(AT)xTy motif in human β‐globin gene promoter

In this report we examine the (AC)n(AT)xTy motif residing -530 bp 5' upstream of the beta-globin gene in Chinese thalassaemic patients. This motif is a putative binding site for a repressor protein, termed beta protein 1 (BP1) (Berg et al., Nucleic Acids Res 1989;17:8833-8852). Variations in the (AC)n(AT)xTy repeats affect the binding affinity of BP1, thereby altering the expression of the beta-globin gene. Eight different configurations of this repeat motif are identified in our population of Chinese beta-thalassaemia patients. A (AC)3(AT)7T5 motif was identified among these thalassaemia patients and its influence in beta-globin gene expression was studied using stable transfection assay in murine erythroleukemia (MEL) cells. Our data demonstrated that the (AC)3(AT)7T5 motif has a moderately strong repressor effect on the expression of the cis-linked beta-globin gene. The high affinity of BP1 for this motif may result in the suppression of the transcription of the beta-globin gene (Berg et al., Am J Hematol 1991;36:42-47). We postulate that silencer elements in the beta-globin promoter play an important role in modifying the clinical presentation of the disease.

The transcription factor KLF11 can induce γ‐globin gene expression in the setting of in vivo adult erythropoiesis

Previous studies in a fetal erythroid cell line demonstrated that the transcription factor, Krüppel-like factor 11 (KLF11), could specifically induce transcription from a gamma-globin gene promoter, and that this induction was mediated through a specific canonical CACCC cis-DNA binding motif. We report here that ectopic expression of KLF11 can also induce fetal gamma-globin gene expression in the setting of adult erythropoiesis both in vitro and in vivo. Studies in an adult-stage murine erythroleukemia (MEL) cell line demonstrated that retrovirus vector-mediated transduction of KLF11 could increase both the amount of expression from a basally active, but not from a overtly silenced, recombinant gamma-globin transgene, as well as the frequency of cells expressing this transgene. A similar pattern of gamma-globin gene induction was also observed both in vitro and in vivo following KLF11 transduction of bone marrow from mice containing a basally active gamma-globin transgene. These studies provide the first evidence that ectopic expression of a transcription factor can induce gamma-globin gene expression in vivo during adult erythropoiesis.

Hypoxia Induces Hemoglobin Production in Human Erythroleukemia Cells by Increasing the Secretion of Transforming Growth Factor -β1 and the Expression of Mitoferrin.

Hypoxia induces erythropoiesis by stimulating erythropoietin secretion from kidney in vivo. Hypoxia also stimulates hemoglobin production in cultured murine erythroleukemia cells, but its mechanism is still unknown. We have found that hypoxia induces hemoglobin production in human erythroleukemia cell line, YN-1, and have analyzed the mRNA expression profile of YN-1 cells under hypoxic conditions (1% O2, 5% CO2 and 94% N2) using DNA microarrays, and compared it with that of normoxic conditions (5% CO2 and 95% room air). For DNA microarray analysis, total RNA was prepared from YN-1 cells, which were incubated under hypoxic or normoxic conditions for 6 hours. We thus found the increased mRNA expression for several genes involved in erythroid differentiation, including transforming growth factor β1 (TGF-β1) and mitoferrin (mitochondrial iron importer, previously known as SLC25A37). Among these candidates, we have focused on TGF-β1, since TGF-β1 is able to induce erythroid differentiation of several erythroleukemia cell lines, including YN-1 cells and K562 cells. We initially confirmed the increased mRNA expression of TGF-β1 under hypoxic conditions by Northern blotting analysis in YN-1 cells and YN-1-0A cells, which is a daughter cell line of YN-1 cells and is maintained in serum-free medium. Likewise, the hypoxic induction of mitoferrin mRNA in YN-1 cells was confirmed by Real Time PCR analysis. Then, using ELISA system, we measured the TGF-β1 concentration in the culture medium of YN-1-0A cells, and have found that hypoxia stimulates TGF-β1 secretion from the cells. Notably, the hypoxia-mediated increases in hemoglobin production and γ-globin mRNA expression were significantly suppressed in YN-1-0A cells, when TGF-β1 activity in the culture medium was neutralized with anti-TGF-β1 antibody. These results suggest that hypoxia stimulates TGF-β1 secretion from YN-1 cells and YN-1-0A cells, which in turn induces the terminal differentiation of erythroid cells in an autocrine manner. Interestingly, in contrast to the hemoglobin production, addition of TGF-β1 into the culture medium did not influence mitoferrin expression, suggesting that hypoxia induces mitoferrin expression by a mechanism independent of TGF-β1. Since several putative hypoxia responsive elements are present in the promoter region of the human mitoferrin gene, hypoxia inducible factor 1 (HIF-1) may be involved in inducible expression of mitoferrin under hypoxic conditions. The hypoxia-mediated induction of mitoferrin expression is important for effective supply of iron for heme biosynthesis in erythroid cells, as several missense mutations of the zebrafish mitoferrin gene are associated with hypochromic anemia and erythroid maturation arrest (Shaw G.C. et al., Nature, vol.440, p96–100, 2006). Taken together, we suggest that hypoxia stimulates hemoglobin production in erythroid cells by coordinately increasing the iron supply into mitochondria and the γ-globin expression, which may be achieved in part by the induction of mitoferrin and the enhanced secretion of TGF-β1, respectively.

Microarrays for undergraduate classes

A microarray experiment is presented that, in six laboratory sessions, takes undergraduate students from the tissue sample right through to data analysis. The model chosen, the murine erythroleukemia cell line, can be easily cultured in sufficient quantities for class use. Large changes in gene expression can be induced in these cells by erythropoietic agents such as DMSO over a 72-h time period. Students isolate total RNA from control (0 h) and 72-h DMSO-treated murine erythroleukemia cells. From this, they synthesize a cDNA copy incorporating amino-allyl dUTP, which is then coupled to either a Cy5 or a Cy3 dye. Equal amounts of the two labeled cDNA samples are then applied to a standard cDNA microarray, which is then hybridized, washed, and scanned. Up- and down-regulated genes are selected using an "in-house" user-friendly data base program. Quality control checks are included at various stages throughout the procedure and, as the process of erythropoiesis is well characterized, a number of erythroid sequences serve as internal controls on the validity of the array data. Through this experiment, students gain experience in a wide range of molecular biology techniques, the use of controls to check a multistep process, validation of results, and strategies to manage the large amount of data generated. Most importantly, it provides undergraduate students with an opportunity to carry out experiments using cutting edge techniques normally found only in research laboratories.