Induction of cells with cancer stem cell properties from nontumorigenic human mammary epithelial cells by defined reprogramming factors.

Cancer stem cells (CSCs), a small and elusive population of undifferentiated cancer cells within tumors that drive tumor growth and recurrence, are believed to resemble normal stem cells. Although surrogate markers have been identified and compelling CSC theoretical models abound, actual proof for the existence of CSCs can only be had retrospectively. Hence, great store has come to be placed in isolating CSCs from cancers for in-depth analysis. On the other hand, although induced pluripotent stem cells (iPSCs) hold great promise for regenerative medicine, concern exists over the inadvertent co-transplantation of partially or undifferentiated stem cells with tumorigenic capacity. Here we demonstrate that the introduction of defined reprogramming factors (OCT4, SOX2, Klf4 and c-Myc) into MCF-10A nontumorigenic mammary epithelial cells, followed by partial differentiation, transforms the bulk of cells into tumorigenic CD44(+)/CD24(low) cells with CSC properties, termed here as induced CSC-like-10A or iCSCL-10A cells. These reprogrammed cells display a malignant phenotype in culture and form tumors of multiple lineages when injected into immunocompromised mice. Compared with other transformed cell lines, cultured iCSCL-10A cells exhibit increased resistance to the chemotherapeutic compounds, Taxol and Actinomycin D, but higher susceptibility to the CSC-selective agent Salinomycin and the Pin1 inhibitor Juglone. Restored expression of the cyclin-dependent kinase inhibitor p16INK4a abrogated the CSC properties of iCSCL-10A cells, by inducing cellular senescence. This study provides some insight into the potential oncogenicity that may arise via cellular reprogramming, and could represent a valuable in vitro model for studying the phenotypic traits of CSCs per se.

Allogeneic amniotic membrane-derived mesenchymal stromal cell transplantation in a porcine model of chronic myocardial ischemia.

Introduction. Amniotic membrane contains a multipotential stem cell population and is expected to possess the machinery to regulate immunological reactions. We investigated the safety and efficacy of allogeneic amniotic membrane-derived mesenchymal stromal cell (AMSC) transplantation in a porcine model of chronic myocardial ischemia as a preclinical trial. Methods. Porcine AMSCs were isolated from amniotic membranes obtained by cesarean section just before delivery and were cultured to increase their numbers before transplantation. Chronic myocardial ischemia was induced by implantation of an ameroid constrictor around the left circumflex coronary artery. Four weeks after ischemia induction, nine swine were assigned to undergo either allogeneic AMSC transplantation or normal saline injection. Functional analysis was performed by echocardiography, and histological examinations were carried out by immunohistochemistry 4 weeks after AMSC transplantation. Results. Echocardiography demonstrated that left ventricular ejection fraction was significantly improved and left ventricular dilatation was well attenuated 4 weeks after AMSC transplantation. Histological assessment showed a significant reduction in percentage of fibrosis in the AMSC transplantation group. Injected allogeneic green fluorescent protein (GFP)-expressing AMSCs were identified in the immunocompetent host heart without the use of any immunosuppressants 4 weeks after transplantation. Immunohistochemistry revealed that GFP colocalized with cardiac troponin T and cardiac troponin I. Conclusions. We have demonstrated that allogeneic AMSC transplantation produced histological and functional improvement in the impaired myocardium in a porcine model of chronic myocardial ischemia. The transplanted allogeneic AMSCs survived without the use of any immunosuppressants and gained cardiac phenotype through either their transdifferentiation or cell fusion.

Defining cell identity by comprehensive gene expression profiling.

The human body is composed of 60 trillion cells, which have their origin in a fertilized egg. During development, the potential of a cell or tissue can be achieved by environmental manipulation. Then, what molecular determinants underlie or accompany the potential of the cells? To obtain a broader understanding of these problems, it is important to analyze all transcripts / genes in a wide selection of cell types. The development of microarray technologies, which allow us to undertake parallel analyses of many genes, has led to a new era in medical science. In this review, we show that the global expression data have clearly elucidated discernible major trends of the phenomenon in preimplantation development and epithelial-mesenchymal transition, and of the character of marrow stromal cells, which are attracting a great deal of attention as they represent a valuable source of cells for regenerative medicine. One of the interesting results is obtained from microarray data of marrow stromal cells: OP9 cells that have been recognized as a type of niche-constituting preadipocyte derived from marrow stroma, are found to be chondroblasts. We also describe what effect each type of expression data would bring to reproductive and regenerative medicine, as well as offering an excellent model of cell differentiation in biology.

Synovial fluid-derived mesenchymal stem cells increase after intra-articular ligament injury in humans.

OBJECTIVE: The existence of mesenchymal stem cells (MSCs) in SF was previously reported. However, the behaviour and properties of MSCs derived from SF have not been fully elucidated. METHODS: Human SFs were obtained from 19 knee joints with anterior cruciate ligament injury around the time of reconstruction surgery, and from three healthy volunteers. SF was plated, cultured and examined for colony-forming number, in vitro differentiation, surface epitopes and gene profiles. Also, rabbit synovium-MSCs were injected into the knee joint in a rabbit partial anterior cruciate ligament defect model, and the injected cells were traced. RESULTS: SF-MSCs from IA ligament injury patients were 100 times more in number than those from healthy volunteers. Total colony number was positively correlated with post-injury period. No significant differences were observed among the cells derived from SF around the time of the surgery in relation to surface epitopes and differentiation potentials. Cluster analysis of gene profiles demonstrated that SF-MSCs were more similar to synovium MSCs than bone marrow MSCs. In rabbit experiments, the MSCs injected into the knee in which IA ligament was partially defective were observed more on the defected area than on the intact area of the ligament at 24 h. CONCLUSION: We demonstrated that SF-MSCs, similar to synovium MSCs, increased in number after IA ligament injury and surgery without marked alteration of the properties.

Myogenic transdifferentiation of menstrual blood-derived cells.

Cells with myogenic potential are present in many tissues, and these cells readily form skeletal muscle in culture. We here focus on menstrual blood as another cell source for regenerative medicine. Menstrual blood-derived cells have high replicative ability, similar to progenitors or stem cells, and transdifferentiate or meta-differentiate into myocytes in vitro at unexpectedly high frequencies. This unique phenotype can be explained by histological and embryological aspects of the endometrium. The remarkable myogenic capability of these cells enables us to "rescue" dystrophied myocytes of the mdx model of Duchenne muscular dystrophy through cell fusion and transdifferentiation. Endometrial cells supplied as a form of menstrual blood-tissue mixture can be used for cell-based therapy in addition to a place for embryo implantation.

Brain transplantation of genetically modified bone marrow stromal cells corrects CNS pathology and cognitive function in MPS VII mice.

Current therapies for lysosomal storage diseases (LSDs), enzyme replacement therapy and bone marrow transplantation are effective for visceral organ pathology of LSD, but their effectiveness for brain involvement in LSDs is still a subject of controversy. As an alternative approach, we transplanted genetically modified bone marrow stromal (BMS) cells to lateral ventricle of newborn mucopolysaccharidosis VII (MPS VII) mice. MPS VII is one of LSDs and caused by deficiency of beta-glucuronidase (GUSB), resulting in accumulation of glycosaminoglycans (GAGs) in brain. At 2 weeks after transplantation, the GUSB enzyme-positive cells were identified in olfactory bulb, striatum and cerebral cortex, and the enzymatic activities in various brain areas increased. The GAGs contents in brain were reduced to near normal level at 4 weeks after transplantation. Although GUSB activity declined to homozygous level after 8 weeks, the reduction of GAGs persisted for 16 weeks. Microscopic examination indicated that the lysosomal distention was not found in treated animal brain. Cognitive function in MPS VII animals as evaluated by Morris Water Maze test in treated mice showed a marked improvement over nontreated animals. Brain transplantation of genetically modified BMS cells appears to be a promising approach to treat diffuse CNS involvement of LSDs.

Newly developed 3D endoscopic system: preliminary experience.

Although there have been earlier reports on the use of three-dimensional endoscopic systems in the gastrointestinal tract, real-time three-dimensional imaging has not previously been described. This paper presents experience in three cases with a novel endoscopic system for three-dimensional observation in clinical situations. Case 1 was a hyperplastic gastric polyp, the shape of which was considerably enhanced in comparison with two-dimensional endoscopic viewing. Case 2 was an advanced esophageal cancer, in which the sense of depth was improved in the esophagus. Case 3 was a depressed early gastric cancer, the shape of which was also enhanced. Between July 2001 and March 2002, three experienced endoscopists also used this new system in a further 34 patients. A three-dimensional endoscopic environment was visible with the system in all patients, and all of the endoscopists were able to obtain visual information regarding spatial depth. This experience suggests that the three-dimensional video-endoscopic system used here can be of value in gastrointestinal endoscopy.

Expression of interleukin-6 in cerebral neurons and ovarian cancer tissue in Trousseau syndrome.

Interleukin-6 (IL-6) is reportedly increased in serum and CSF from acute stroke patients. However, the cellular origin and possible role of IL-6 in CNS after stroke are unclear. We describe a woman with recurrent stroke, disseminated intravascular coagulation (DIC) and non-bacterial thrombotic endocarditis (NBTE) caused by ovarian cancer (Trousseau syndrome). The patient died 50 days after the final episode of cerebral embolism. The immunohistochemical study revealed IL-6 protein to have been expressed both in cerebral neurons spared from ischemic insult and in epithelial cells of the ovarian tumor. We speculate that IL-6 produced in ovarian cancer may be associated with the hypercoagulable state and the development of NBTE in this patient. In contrast, IL-6 induction in cerebral neurons may contribute to the survival of these neurons after a stroke.

Enhancement of osteogenesis on hydroxyapatite surface coated with synthetic peptide (EEEEEEEPRGDT) in vitro.

Some dental implants are coated with hydroxyapatite (HA), which preferentially binds to bone. Several matrix proteins have an arginine-glycine-aspartic acid (RGD) sequence where cells attach via an integrin receptor. We hypothesized that coating an HA surface with an RGD-containing peptide might enhance the attachment and differentiation of osteoblasts. The HA disks (diameter 34 mm, thickness 1 mm) were treated with a solution (50 mM Tris/HCl and 150 mM NaCl, pH 7.4) containing the peptide EEEEEEEPRGDT, in which the E repetition exerts a high affinity to HA. After washing with phosphate-buffered saline, KUSA/A1 mouse osteoblastic cells were inoculated onto the HA surface and cultured. After 30 min, the number of cells attached to the surface was counted. The DNA content and alkaline phosphatase (ALP) activity were measured after 10 days in culture. Expression of bone matrix proteins was also examined by means of reverse transcriptase-polymerase chain reaction at 7 days; the mineralized area of the culture was also evaluated by staining with Alizarin Red S after 10 days. Treatment with the peptide stimulated cell attachment and increased DNA content and ALP activity. Furthermore, matrix protein expression and mineralized nodule formation were enhanced to a greater extent on the peptide-treated surface than on the nontreated surface. Our results indicate that coating an HA surface with RGD-containing peptide enhances osteoblast attachment and differentiation. This peptide treatment of HA-coated implants may stimulate the osseointegration of the implants.

Light induced apoptosis is accelerated in transgenic retina overexpressing human EAT/mcl-1, an anti-apoptotic bcl-2 related gene.

BACKGROUND/AIM: EAT/mcl-1 (EAT), an immediate early gene, functions in a similar way to bcl-2 in neutralising Bax mediated cytotoxicity, suggesting that EAT is a blocker of cell death. The aim of this study was to determine the effect of overexpression of the human EAT gene on light induced retinal cell apoptosis. METHODS: EAT transgenic mice incorporating the EF-1alpha promoter were utilised, and expression of human EAT was detected by RT-PCR. Light damage was induced by raising mice under constant illumination. Two groups of animals, EAT transgenic mice (n=14) and littermates (n=13), were examined by ERG testing and histopathology at regular time points up to 20 weeks of constant light stimulation. Electrophysiological and histopathological findings were evaluated by established systems of arbitrary scoring as scores 0-2 and scores 0-3, respectively. RESULTS: The mean score (SD) of ERG response was significantly lower in EAT transgenic mice (0.79 (0.89)) than in littermates (1.69 (0.48)) (p<0.01). Although the differences between the two survival curves did not reach statistical significance (p=0.1156), the estimated incidence of electrophysiological retinal damage was higher in EAT mice (0.0495/mouse/week; 95% confidence interval (CI) 0.0347-0.0500) than in littermates (0. 0199/mouse/week; 95% CI 0.0035-0.0364). The mean scores (SD) for histopathological retinal degeneration were 2.31 (0.63) in littermates and 1.43 (1.22) in EAT transgenic mice (p=0.065). However, Kaplan-Meier curves for histopathological failure in two groups of mice showed that retinal photoreceptor cells were preserved significantly against constant light in the littermate compared with transgenic mice (p=0.0241). The estimated incidence of histopathological retinal damage was 0.0042/mouse/week in the littermates (95% CI 0-0.0120) and 0.0419/mouse/week in the EAT mice (95% CI 0.0286-0.0500). CONCLUSION: Retinal photoreceptor cell apoptosis under constant light stimulation is likely to be accelerated in transgenic retina overexpressing EAT.

A novel differentiation-inducing therapy for acute promyelocytic leukemia with a combination of arsenic trioxide and GM-CSF.

Arsenic trioxide (As2O3) effectively induces clinical remission via apoptosis in relapsed acute promyelocytic leukemia (APL). However, because this new anti-leukemic drug is also considered to be a poison, its possible adverse effects are a highly important issue related to its clinical use. We here investigated, both in vitro and in vivo, the effects of a combination of As2O3 and GM-CSF as a novel therapeutic approach for the treatment of APL. Treatment of both retinoic acid (RA)-sensitive and -resistant APL cell lines (NB4 and UF-1 cells, respectively), as well as primary APL cells with a combination of As2O3 and GM-CSF for 4 days resulted in inducing differentiation, but not apoptosis, to mature granulocytes. In addition, a combination of both agents induced degradation of the PML/RARalpha protein. GM-CSF was found to be associated with increased tyrosine phosphorylation of Jak2 kinase in both NB4 and UF-1 cells, and a specific inhibitor of Jak2, AG490, completely blocked the ability of GM-CSF to prevent apoptosis and induce differentiation of As2O3-treated UF-1 cells. In in vivo analysis, As2O3 induced differentiation of APL cells in a RA-resistant APL model of human GM-CSF-producing transgenic SCID mice that had a high level of human GM-CSF in their sera. In contrast, As2O3 alone diminished tumors in UF-1 cells transplanted into NOD/SCID mice via induction of apoptosis. In conclusion, a combination of As2O3 and GM-CSF appears to be a novel differentiation-inducing therapy in patients with APL, including relapsed or RA-resistant cases.

Nesidioblastosis and mixed hamartoma of the liver in Beckwith-Wiedemann syndrome: case study including analysis of H19 methylation and insulin-like growth factor 2 genotyping and imprinting.

An infant with persistent hyperinsulinemic hypoglycemia, diffuse nesidioblastosis, and mixed hamartoma of the liver (MHL), in addition to demonstrating clinical, pathologic, and molecular manifestations of Beckwith-Wiedemann syndrome (BWS), is the subject of this report. H19 methylation assay and allelic expression analysis for insulin-like growth factor 2 (IGF2) indicated that the patient was mosaic for paternal isodisomic cells and normal cells in lung tissue, nontumoral liver tissue, tissue from the MHL, and pancreatic tissue. We propose that abundant IGF2 expression during development due to paternal isodisomy resulted in hepatomegaly and islet cell hyperplasia, which led to nesidioblastosis. MHL, by contrast, may have resulted from a decrease in disomic cells, compared with nontumoral liver tissue, which showed an increase in disomic cells. Thus, somatic mosaicism may result in unbalanced tissue growth, which may contribute to the formation of MHL in BWS.

EAT/mcl-1 expression in the human embryonal carcinoma cells undergoing differentiation or apoptosis.

Differentiation and apoptosis are precisely regulated events in early embryogenesis. Retinoic acid-induced differentiation in the embryonal carcinoma (EC) cell line NCR-G3 triggers concurrent induction of apoptosis. Using this system, which serves as a model of early embryogenesis, the expression of various bcl-2-related genes was analyzed as these genes display either positive or negative regulatory effects on apoptosis. EAT/mcl-1, an antiapoptotic bcl-2-related gene and immediate early gene, was dramatically expressed at an early stage of NCR-G3 differentiation. Bcl-xL, another antiapoptotic gene, was induced at a middle stage of differentiation and then gradually decreased to basal level. Expression of Bax, a proapoptotic molecule, was detected at a high level and remained relatively constant. Meanwhile, Bcl-2 and Bcl-xS were below detectable levels throughout the various stages of differentiation. As the balance of bcl-2 genes is a crucial regulatory step in apoptosis, the results suggest that EAT and Bax likely regulate apoptosis in the early stages of differentiation. In later stages of differentiation, down-regulation of EAT was found to coincide with a gradual increase in apoptosis of NCR-G3 cells. Furthermore, use of the monoclonal antibody (3A2) specific to EAT revealed that EAT is localized to the outer mitochondrial membrane in human EC cells. In addition, EAT immunoreactivity was not detected in apoptotic NCR-G3 cells while it was observed in nearly all viable cells. The findings suggest that rapid induction of EAT may prevent NCR-G3 cells from undergoing apoptosis, thereby supporting viability at the early stage of differentiation.

Mcl-1, an early-induction molecule, modulates activin A-induced apoptosis and differentiation of CML cells.

Activin A, one member of the transforming growth factor (TGF)-beta superfamily, is known to be a commitment factor for cell death and differentiation. In the present study, we demonstrate that human chronic myeloid leukemia (CML) cell lines, KU812 and K562 cells, either induced apoptosis or differentiation, respectively, by treatment with activin A. During these cell fate decisive events caused by activin A, rapid and transient up-regulation of Mcl-1 was observed in both cell lines. In activin A-induced apoptosis of KU812 cells, continuous up-regulation of Bax was observed. After the decrease in Mcl-1 expression had occurred, activation of caspase-9 and caspase-3 and cleavage of DFF45 were shown to take place in KU812 cells, resulting in the fragmentation of the genomic DNA of the cells. In contrast, the down-regulation of Mcl-1 without up-regulation of Bax caused accumulation of hemoglobin (Hb) contents in activin A-treated K562 cells. Interestingly, erythropoietin (EPO) prevented activin A-induced apoptosis with continuous expression of Mcl-1 and caused KU812 cells to undergo erythroid differentiation. To address the role of Mcl-1 in activin A-treated CML cells, KU812 and K562 cells were stably transfected with cDNA encoding Mcl-1 (designated as KU812/mcl and K562/mcl cells). As in combined effect of activin A and EPO on the parental KU812 cells, activin A induced differentiation, but not apoptosis, of KU812/mcl cells without modulating Bax levels. Activin A-treated K562/mcl cells, as well as parental cells, were only differentiated to erythroid cells. These results suggest that Mcl-1 is an early inducible gene activated by the activin A signaling pathway for both cellular differentiation and apoptosis, and continuous expression of Mcl-1 may be contributed to differentiation signals to the erythroid lineage in CML cells.

Epigenetic mark sequence of the H19 gene in human sperm.

We have investigated the epigenetic mark in the human H19 gene. The H19 promoter is methylation-free in human sperm, but it is methylated in the paternally derived allele of most adult tissues. Consequently, the H19 gene is exclusively transcribed from the maternal allele. It was demonstrated that the differentially methylated region (DMR) located 2 kb upstream from mouse H19 is essential for the imprinting of H19. A 39 bp sequence in DMR has a high degree of similarity between humans, mice and rats. The highly conserved 15 bp core region of the consensus sequence contains four methylatable sites, and thus has been proposed as a potential imprinting mark region. In this study, fine epigenetic sequencing analysis was performed on the sperm DNA in comparison with other adult organs. Interestingly, the conserved sequence of the potential mark region was methylated in almost all the sperm genomes analyzed. Furthermore, the single dinucleotide CpG, whose methylation affects the accessibility of the element to CTCF, was methylated in the conserved core in the human sperm. These results suggest that the human core sequences may act as an imprinting center in the reciprocal monoallelic expression of H19.

Brain from bone: efficient "meta-differentiation" of marrow stroma-derived mature osteoblasts to neurons with Noggin or a demethylating agent.

Bone marrow stromal cells are able to differentiate into adipogenic, chondrogenic, myogenic, osteogenic, and cardiomyogenic lineages, all of which are limited to a mesoderm-derived origin. In this study, we showed that neurons, which are of an ectoderm-origin, could be generated from marrow-derived stromal cells by specific inducers, fibronectin/ornithine coating, and neurosphere formation. The neurons generated from marrow stroma formed neurites, expressed neuron-specific markers and genes, and started to respond to depolarizing stimuli as functional mature neurons. Among stromal cells, isolated mature osteoblasts which had strong in vivo osteogenic activity could be efficiently converted into functional neurons. This transdifferentiation or meta-differentiation was enhanced by Noggin, an inhibitor of bone morphogenetic proteins, in comparison with 5-azacytidine, a demethylating agent capable of altering the gene expression pattern. Marrow stroma is therefore a potential source of cells for neural cell transplantation.

Involvement of EAT/mcl-1, an anti-apoptotic bcl-2-related gene, in murine embryogenesis and human development.

Apoptosis plays an important regulatory role in mammalian embryogenesis and development. EAT/mcl-1 (EAT), an anti-apoptotic bcl-2-related gene, was isolated during the early differentiation of a human embryonal carcinoma cell line, an event which serves as a model of early embryogenesis. EAT is involved in apoptotic regulation and is believed to also function as an immediate-early gene. Thus it was hypothesized that EAT would be expressed during early embryogenesis and would be involved in the regulation of apoptosis during this critical period. To clarify this early expression, two antibodies to EAT were generated by use of immunizing oligopeptide (aa 37-55) and recombinant protein (aa 31-229) for use in immunohistochemistry and immunoblotting, respectively. With these antibodies, we then determined EAT expression during murine embryogenesis and in human development, using human fetal tissue of 6 to 23 gestational weeks. During murine embryogenesis, the EAT protein was found to be rapidly induced after fertilization, to peak at the 2-cell stage, to remain constant until the 8-cell stage, and then to decrease to below unfertilized egg levels in blastocysts. EAT expression patterns in early human development were found to essentially overlap those observed in adult tissues which suggest that EAT expression continues until adulthood in terminally differentiated tissues. Among tissues distinct to fetal development, EAT was detected in the mesonephric (Wolffian) duct and paramesonephric (Müllerian) duct. It is also noteworthy that prominent EAT immunoreactivity was also observed in large primary oocytes in 21-week fetal ovary, but was not detected in primordial germ cells in 23-week fetal testis. In summary, EAT expression was detected in hematopoietic, epithelial, neural, endocrine, and urogenital cells; this provides evidence that EAT, as an anti-apoptotic molecule, possibly functions to regulate apoptosis during development in these systems.

Involvement of EAT/mcl-1, a bcl-2 related gene, in the apoptotic mechanisms underlying human placental development and maintenance.

The EAT/mcl-1 gene was isolated during the early differentiation of a retinoic acid-induced human embryonal carcinoma cell line to the trophectoderm lineage. EAT/mcl-1, a bcl-2 related gene, is involved in the genetic pathway of apoptosis; this suggests a role for apoptosis and the involvement of this gene in early placental development. In the current investigation, we analysed expression of EAT/mcl-1 at the mRNA level by Northern blot analysis and in situ hybridization, as well as at the protein level, by immunoblot analysis and immunohistochemistry. Our results identified constant expression of this gene in the placenta throughout pregnancy as well as a shift in its localization from the cytotrophoblast in the first trimester to the syncytiotrophoblast in the third trimester. In addition, there was an inverse correlation between EAT/mcl-1 expression and TaT-mediated deoxyuridine triphosphate nick-end labelling (TUNEL) reactivity in trophoblasts in the first trimester. These results suggest a role for EAT/mcl-1 in both early placental development in regulating trophoblast differentiation as well as a role for this gene in placental maintenance in regulating the process of trophoblast turnover.

Arsenic trioxide (As2O3)-induced apoptosis and differentiation in retinoic acid-resistant acute promyelocytic leukemia model in hGM-CSF-producing transgenic SCID mice.

Recent clinical studies in China and USA showed that arsenic trioxide (As2O3) is an effective treatment of acute promyelocytic leukemia (APL) patients refractory to all-trans retinoic acid (RA). We here investigate the effects of As2O3 on RA-resistant APL in vivo and in vitro using our RA-resistant APL model system. As2O3 can induce inhibition of cellular growth of both RA-sensitive NB4 and RA-resistant UF-1 APL cells via induction of apoptosis in vitro. The expression of BCL-2 protein decreased in a dose- and time-dependent manner in NB4 cells. Interestingly, the levels of BCL-2 protein were not modulated by As2O3, but it did upregulate BAX protein in UF-1 cells. UF-1 cells (1x10(7)) were transplanted into hGM-CSF-producing transgenic SCID mice and successfully formed subcutaneous tumors. After 40 days of implantation, mice were treated with As2O3, all-trans RA and PBS for 21 days. In all-trans RA- and PBS-treated mice, tumors grew rapidly, with a 4.5-fold increase in volume at day 21 compared to the initial size. In marked contrast, tumor size was decreased to half of the initial size by the treatment of As2O3, which resulted in cells with the typical appearance of apoptosis. Interestingly, one of the As2O3-treated mice showed mature granulocytes in the diminished tumor, suggesting that As2O3 had dual effects on RA-resistant APL cells in vivo: both inducing apoptosis and differentiation of the leukemic cells. We conclude that our RA-resistant APL model will be useful for evaluating novel therapeutic approaches to patients with RA-resistant APL, and for further investigation of the metabolism of As2O3 in vivo.