DNA demethylation during the differentiation of 3T3-L1 cells affects the expression of the mouse GLUT4 gene.
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GLUT4 is the major glucose transporter in adipose tissue and skeletal and cardiac muscles. We examined the mechanisms underlying GLUT4 gene expression in 3T3-L1 cells, which express the gene during their differentiation from preadipocytes to adipocytes. In transient transfections, the activity of a mouse GLUT4 promoter extending to -100 bp in the 5'-flanking region did not differ significantly between 3T3-L1 preadipocytes and adipocytes. Promoter activity up to -590 bp in preadipocytes and adipocytes showed a 70% lower and 228% higher activity, respectively, than promoter activity extending to -100 bp. We also examined methylation status of the GLUT4 promoter. Up to -100 bp, there were five CpG sites at -11, -30, -58, -63, and -75 bp. Two CpG sites at -11 and -30 bp were highly methylated in preadipocytes (60 and 92%, respectively) and highly demethylated in adipocytes (28.6 and 25%, respectively). Conversely, three CpG sites at -58, -63, and -75 bp were highly demethylated in both preadipocytes and adipocytes (<12%). In gel mobility-shift assays, a fragment extending from -40 to -1 bp generated a methylation-sensitive band with nuclear extracts from both preadipocytes and adipocytes when the CpG sites were methylated. Southwestern analysis identified a protein of approximately 55 kDa that bound strongly to the methylated probe. Furthermore, methylation of the CpG sites inhibited promoters extending to -50 or -70 bp. These results suggest that in addition to cell type-specific transcription factor, methylation of specific CpG sites and the methylation-sensitive transcription factor contribute to GLUT4 gene regulation during 3T3-L1 differentiation. (+info)
Activation of protein kinase B and induction of adipogenesis by insulin in 3T3-L1 preadipocytes: contribution of phosphoinositide-3,4,5-trisphosphate versus phosphoinositide-3,4-bisphosphate.
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Ectopic expression of activated protein kinase B (PKB) induces the differentiation of confluent 3T3-L1 preadipocytes into adipocytes. PKB is regulated by the lipid products of phosphoinositide 3-kinase (PI 3-kinase), phosphatidylinositol-3,4-bisphosphate [PI(3,4)P2], and phosphatidylinositol-3,4,5-trisphosphate [PI(3,4,5)P3]. However, the relative contribution of each 3-phosphorylated phosphoinositide species in activating PKB remains unclear. Treatment of intact 3T3-L1 preadipocytes with synthetic 3-phosphorylated phosphoinositides revealed that only PI(3,4)P2 stimulated PKB activity. PKB was also activated by insulin, in a dose- and time-dependent manner. This activation was associated with an isolated rise in PI(3,4,5)P3, without any detectable change in PI(3,4)P2, demonstrating that this lipid was sufficient to activate PKB. Wortmannin and LY294002, inhibitors of PI 3-kinase, reduced insulin-dependent activation of PKB, whereas rapamycin, an inhibitor of p70 S6 kinase, had no effect. Platelet-derived growth factor (PDGF), which is not adipogenic, stimulated the production of both 3-phosphorylated phosphoinositide species, and this was associated with a greater activation of PKB than that observed with insulin. A low dose of PDGF (1 ng/ml), which increased the production of only PI(3,4,5)P3 and mirrored the insulin effect, was unable to induce adipocyte differentiation. In summary, insulin and PDGF differ with respect to the accumulation of 3-phosphorylated phosphoinositides and to PKB activation in 3T3-L1 preadipocytes, but these responses do not themselves explain why insulin, but not PDGF, is adipogenic. (+info)
Multilineage potential of adult human mesenchymal stem cells.
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Human mesenchymal stem cells are thought to be multipotent cells, which are present in adult marrow, that can replicate as undifferentiated cells and that have the potential to differentiate to lineages of mesenchymal tissues, including bone, cartilage, fat, tendon, muscle, and marrow stroma. Cells that have the characteristics of human mesenchymal stem cells were isolated from marrow aspirates of volunteer donors. These cells displayed a stable phenotype and remained as a monolayer in vitro. These adult stem cells could be induced to differentiate exclusively into the adipocytic, chondrocytic, or osteocytic lineages. Individual stem cells were identified that, when expanded to colonies, retained their multilineage potential. (+info)
Z/AP, a double reporter for cre-mediated recombination.
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The Cre/loxP site-specific recombination system combined with embryonic stem cell-mediated technologies has greatly expanded our capability to address normal and disease development in mammals using genetic approaches. The success of this emerging technology hinges on the production of Cre-expressing transgenic lines that provide cell type-, tissue-, or developmental stage-specific recombination between loxP sites placed in the genome. Here we describe and characterize the production of a double-reporter mouse line that provides a convenient and reliable readout of Cre recombinase activity. Throughout all embryonic and adult stages, the transgenic animal expresses the lacZ reporter gene before Cre-mediated excision occurs. Cre excision, however, removes the lacZ gene, allowing expression of the second reporter, the human alkaline phosphatase gene. This double-reporter transgenic line is able to indicate the occurrence of Cre excision in an extremely widespread manner from early embryonic to adult lineages. It will be a valuable reagent for the increasing number of investigators taking advantage of the powerful tools provided by the Cre/loxP site-specific recombinase system. (+info)
Modulation of rat preadipocyte adipose conversion by androgenic status: involvement of C/EBPs transcription factors.
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Androgenic status affects rat preadipocyte adipose conversion from two deep intra-abdominal (epididymal and perirenal) fat depots differently. The aim of this study was to establish whether these site-specific alterations of adipogenesis are related to altered expressions of the transcriptional factors regulating proliferation and differentiation of preadipocytes, c-myc and CCAAT/enhancer binding proteins (C/EBPs: C/EBPalpha and beta). The increased proliferation of epididymal and perirenal preadipocytes from castrated rats was not linked to variations in c-myc mRNA and protein levels. The expression of the early marker of adipogenesis, lipoprotein lipase (LPL), was decreased by androgenic deprivation in epididymal cells but remained insensitive to the androgenic status in perirenal preadipocytes. In contrast, LPL expression increased in subcutaneous preadipocytes from castrated rats, an effect which was partly corrected by testosterone treatment. Expression of C/EBPbeta was unaffected by androgenic status whatever the anatomical origin of the preadipocytes. In contrast, the mRNA and protein levels of C/EBPalpha were greatly decreased by androgenic deprivation in epididymal cells, an alteration which could not be corrected by in vivo testosterone administration. Altogether these results demonstrated that in preadipocytes androgenic deprivation affects site-specifically the expression of LPL, an early marker of adipogenesis and of C/EBPalpha, a master regulator of adipogenesis. These observations contribute to an explanation of why castration induces defective adipose conversion in rat epididymal preadipocytes specifically. (+info)
Experimental cryptorchidism induces a change in the pattern of expression of LH receptor mRNA in rat testis after selective Leydig cell destruction by ethylene dimethane sulfonate.
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In the rat, the cytotoxic drug ethylene dimethane sulfonate (EDS) selectively eliminates mature Leydig cells (LCs) from testicular interstitium, activating a complex process of proliferation and differentiation of pre-existing LC precursors. We observed previously that after EDS treatment, the early LC precursors persistently express a truncated 1.8 kb form of LH receptor (LHR) mRNA. This prompted us to study whether experimental cryptorchidism, known to alter the process of LC repopulation, can influence the pattern of testicular LHR mRNA expression after EDS administration. EDS treatment completely eliminated mature LCs both in control and unilaterally cryptorchid (UC) rats. This response was followed by gradual reappearance of newly formed, functionally active LCs, as evidenced by the recovery in testicular LHR content and plasma testosterone levels in both experimental groups. Noteworthy, the rate of LC repopulation was higher in the abdominal testes of UC rats, in keeping with previous findings. Interestingly, the 1.8 kb LHR transcript was persistently expressed in scrotal testes at all time-points, but undetectable upon Northern hybridization in abdominal testes at early stages after EDS administration, when low levels of expression of truncated LHR transcripts could only be detected by semi-quantitative RT-PCR analysis. In addition, the faster LC repopulation in cryptorchid testes was associated with precocious recovery of the complete array of LHR mRNA transcripts, including the 1.8 kb species. These changes appeared acutely and irreversibly, as unilateral positioning of scrotal testes into the abdomen resulted in a rapid loss of expression of the 1.8 kb LHR transcript, whereas scrotal relocation of the UC testes failed to alter the pattern of LHR gene expression. In conclusion, experimental cryptorchidism changes the pattern of LHR mRNA expression in rat testis after selective LC destruction by EDS. This change, i.e. repression of the 1.8 kb LHR transcript after EDS administration, is acute and irreversible, and likely related to the impairment of testicular microenvironment following cryptorchidism. However, even though at low levels, the expression of truncated forms of LHR mRNA appears to be a universal feature of proliferating LC precursors. The UC testis may represent a good model for analysis of the regulatory signals involved in the control of LHR gene expression. (+info)
Transchromosomal mouse embryonic stem cell lines and chimeric mice that contain freely segregating segments of human chromosome 21.
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At least 8% of all human conceptions have major chromosome abnormalities and the frequency of chromosomal syndromes in newborns is >0.5%. Despite these disorders making a large contribution to human morbidity and mortality, we have little understanding of their aetiology and little molecular data on the importance of gene dosage to mammalian cells. Trisomy 21, which results in Down syndrome (DS), is the most frequent aneuploidy in humans (1 in 600 live births, up to 1 in 150 pregnancies world-wide) and is the most common known genetic cause of mental retardation. To investigate the molecular genetics of DS, we report here the creation of mice that carry different human chromosome 21 (Hsa21) fragments as a freely segregating extra chromosome. To produce these 'transchromosomal' animals, we placed a selectable marker into Hsa21 and transferred the chromosome from a human somatic cell line into mouse embryonic stem (ES) cells using irradiation microcell-mediated chromosome transfer (XMMCT). 'Transchromosomal' ES cells containing different Hsa21 regions ranging in size from approximately 50 to approximately 0.2 Mb have been used to create chimeric mice. These mice maintain Hsa21 sequences and express Hsa21 genes in multiple tissues. This novel use of the XMMCT protocol is applicable to investigations requiring the transfer of large chromosomal regions into ES or other cells and, in particular, the modelling of DS and other human aneuploidy syndromes. (+info)
Fludarabine-based non-myeloablative chemotherapy followed by infusion of HLA-identical stem cells for relapsed leukaemia and lymphoma.
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Many patients have not been offered potentially curative allogeneic marrow transplants because of the toxicity of myeloablative regimens in the setting of advanced age or organ dysfunction. We treated five patients, ineligible for myeloablative chemotherapy due to one of these criteria, with fludarabine-based non-myeloablative chemotherapy followed by reinfusion of G-CSF-mobilised allogeneic peripheral blood progenitor cells (PBPC). Two patients died early of multi-organ failure. Another patient with massive splenomegaly was infused with a suboptimal number of PBPC; no engraftment was documented. The remaining two patients demonstrated mixed chimerism early post-transplant, but by 3 and 6 months respectively, engraftment was almost entirely of donor origin. One of these patients, transplanted with relapsed AML, remains in remission with extensive chronic GVHD at 17 months. The other patient, transplanted with chemorefractory mantle cell lymphoma, progressed early post-transplant but entered remission coincident with the onset of severe GVHD following cessation of cyclosporin A, suggesting a powerful graft-versus-mantle cell lymphoma effect. These preliminary observations suggest this approach results in engraftment and GVHD/graft-versus-tumour effects similar to myeloablative regimens and may provide an alternative in patients ineligible for conventional conditioning regimens. (+info)