Differential hemoglobin synthesis in murine erythroleukemic cells: hemin effects

The addition of butyric acid to murine erythroleukemic cells (clone T3Cl2) induced the cells to differentiate, producing adult hemoglobin (A, alpha 2,beta 2) and an embryonic hemoglobin (E2, alpha 2Y2). The subsequent addition of hemin to the differentiating cells increased the synthesis of adult hemoglobin four-fold and the synthesis of embryonic hemoglobin two-fold; the relative synthesis of the alpha and beta globins increased more than the y globin. The embryonic hemoglobin was expressed prior to the adult hemoglobin in differentiating cells.     

Promoting effect of 5-azacytidine on the myogenic differentiation of bone marrow stromal cells

Bone marrow stromal cells (BMSC) can differentiate into various cell types including myocytes, which may be valuable in cellular therapy of myocardial infarction. In an attempt to increase the myogenic commitment of BMSC, we investigated the extent of conversion induced by the demethylation agent 5-azacytidine. BMSC isolated from the adult rat tibia were exposed in culture to 5microM 5-azacytidine for 24h, 1 day after seeding. The treatment was repeated at weekly intervals and the expression of muscle-specific proteins and genes was assessed. The results revealed that cultured cells lost the native expression of osteocalcin and alkaline phosphatase as a function of time and began to express connexin 43. Exposure to 5-azacytidine of BMSC induced, at 14 days, a myocyte-resembling phenotype that included the expression of muscle-specific proteins (sarcomeric alpha-actin, troponin T, desmin, alpha-actinin, and GATA-4) and genes (GATA-4, myoD, desmin, and alpha-actinin), numerous mitochondria and myofilaments; however, the latter did not form sarcomeres. Although some of these myogenic markers also appeared in untreated cells, exposure to 5-azacytidine induced an enhanced response of calcium channels, as well as a threefold increase in desmin and myoD gene expression and a twofold increase in alpha-actinin gene and protein expression above the control values. In conclusion, the results demonstrate a promoting effect of 5-azacytidine on the expression of muscle-specific proteins and genes in BMSC in culture. Notably, the myogenic differentiation takes place over a short period of time. Priming of mesenchymal cells to cardiomyogenic differentiation may have significant applications in cellular approaches to ameliorate muscle loss after myocardial ischemia.     

Effect of 5-Azacytidine on the Methylation Aspects of NMDA Receptor NR2B Gene in the Cultured Cortical Neurons of Mice

Our previous study revealed that the exposure of the drug 5-Azacytidine and ethanol to the cultured cortical neurons of mice causes demethylation of cytosine residues in the CpG island of the NMDA receptor NR2B gene (Marutha Ravindran and Ticku, Mol Brain Res 121:19-27, 2004). In the present study, we further analyzed methylation in the CpG island with various concentration frame and time frame of exposure of the cultured cortical neurons with 5-azacytidine to identify whether methylation in the NR2B gene is site specific or region specific. Methylation was studied by digesting the genomic DNA with methylation sensitive HpaII, MspI, AciI or HhaI enzyme following the exposure of cultured cortical neurons of mice with 5-azacytidine by performing PCR and Southern hybridization. We observed demethylation of DNA at 1, 3 and 5 μM concentrations of 5-azacytidine in the regions (5982–6155), (6743–7466) and at 3 and 5 μM concentrations of 5-azacytidine used in the region (6477–6763). Similarly in the time frame study with 5-azacytidine, demethylation of DNA was observed at 24 h and 36 h of incubation with 5-azacytidine in the regions (5982–6155), (6743–7466) and at 36 h of incubation with 5-azacytidine used in the region (6477–6763). Our experimental results demonstrate that the methylation in the CpG islands of the NR2B gene may not be site specific or region specific in the cultured cortical neurons of mice.     

Increase of histidine decarboxylase activity in murine myelomonocytic leukemia cells (WEHI-3B) in parallel to their differentiation into macrophages

When cells of mouse myelomonocytyc leukemia cell line, WEHI-3B, were cultured in the presence of actinomycin D plus the serum which was obtained from mice injected with bacterial endotoxin, i.e., lipopolysaccharide, their histidine decarboxylase (l-histidine carboxy-lyase, EC 4.1.1.22) (HDC) activity increased about 100-fold with a peak at 48 h. According to the increase in HDC activity, the expression of surface antigens associated with macrophages, such as Mac II, Mac III and Ia, increased markedly on WEHI-3B cells as well as their morphological changes to macrophages. Histamine levels in the culture medium increased concomitantly with the increase in the HDC activity in WEHI-3B cells, whereas the histamine contents inside the cells did not increase remarkably. Furthermore, the addition of lipopolysaccharide to the culture medium caused an additional 2-fold increase in the HDC activity of WEHI-3B cells. These results indicate that the increase in HDC activity in WEHI-3B cells may represent an event in the process of the differentiation to macrophages.     

Cell growth and hemoglobin synthesis in murine erythroleukemic cells propagated in high density microcapsule culture

Summary A new microencapsulation technology, developed for the encapsulation of living cells, has been demonstrated to be useful for the study of growth and differential gene expression using Friend erythroleukemic cells cultured at high cell densities. Using this technology, cultures of FL Clone 745 cells were encapsulated within semipermeable membranes composed of cross-linked alginic acid and poly-l-lysine. Cell growth studies measuring total cell number demonstrated an average generation time of 8.5 h in 5% (vol/vol) microcapsule cultures vs. 8.0 h in suspension cultures. Similar microcapsule cultures were serially propagated for more than 90 cell generations (13 sequential passages) with no significant change in this growth rate. In addition, final culture densities of greater than 1.0×10⁸ cells/ml of intracapsular volume were attained using a 3% (vol/vol) microcapsule culture in conjunction with a standard refeeding schedule. Comparison of the level of dimethyl sulfoxide-induced hemoglobin production in suspension and microcapsule cultures demonstrated that the total amount of hemoglobin produced on a per cell basis was comparable in both systems. Due to the retention characteristics of the semipermeable membrane, the concentration of detergent-released hemoglobin, relative to other released protein, was approximately twofold higher in microcapsule cultures than in control suspension cultures.     

Culturing and differentiation of murine embryonic stem cells in a three-dimensional fibrous matrix

Embryonic stem (ES) cells have indefinite self-renewal ability and pluripotency, and can provide a novel cell source for tissue engineering applications. In this study, a murine CCE ES cell line was used to derive hematopoietic cells in a 3-D fibrous matrix. The 3-D matrix was found to maintain the phenotypes of undifferentiated ES cells as indicated by alkaline phosphatase (ALP) activity and stage specific embryonic antigen-1 (SSEA-1) expression. In hematopoietic differentiation, cells from 3-D culture exhibited similar cell cycle distribution and SSEA-1 expression to those in the initial cell population. The Oct-4 expression was significantly down-regulated, which indicated the occurrence of differentiation, although the level was slightly higher than that in Petri dish culture. The expression of c-kit, cell surface marker for hematopoietic progenitor, was higher in the 3-D culture, suggesting a better-directed hematopoietic differentiation. Cells in the 3-D matrix tended to form large aggregates associated with fibers. For large-scale processes, a perfusion bioreactor can be used for both maintenance and differentiation cultures. As compared to the static culture, a higher growth rate and final cell density were resulted from the perfusion bioreactor due to better control of the reactor environment. At the same time, the differentiation capacity of ES cells was preserved in the perfusion culture. The ES cell culture in the fibrous matrix thus can be used as a 3-D model system to study effects of extracellular environment and associated physico-chemical parameters on ES cell maintenance and differentiation.     

DNA repair in murine embryonic stem cells and differentiated cells

Embryonic stem (ES) cells are rapidly proliferating, self-renewing cells that have the capacity to differentiate into all three germ layers to form the embryo proper. Since these cells are critical for embryo formation, they must have robust prophylactic mechanisms to ensure that their genomic integrity is preserved. Indeed, several studies have suggested that ES cells are hypersensitive to DNA damaging agents and readily undergo apoptosis to eliminate damaged cells from the population. Other evidence suggests that DNA damage can cause premature differentiation in these cells. Several laboratories have also begun to investigate the role of DNA repair in the maintenance of ES cell genomic integrity. It does appear that ES cells differ in their capacity to repair damaged DNA compared to differentiated cells. This minireview focuses on repair mechanisms ES cells may use to help preserve genomic integrity and compares available data regarding these mechanisms with those utilized by differentiated cells.     

Alterations in the transferrin receptor of human erythroleukemic cells after induction of hemoglobin synthesis

When K562 human erythroleukemic cells are induced to differentiate by addition of hemin to their medium, the number of binding sites for transferrin on the cell surface is substantially reduced. This reflects an internalization of receptors since no such reduction is observed when the total binding sites in soluble extracts of uninduced and differentiating cells are compared. The internalization of transferrin receptors has also been shown using lactoperoxidase-mediated radioiodination of cell surfaces and by immune precipitation of total and surface labeled receptors using an anti-receptor monoclonal antibody. Transferrin receptors from uninduced and differentiating cells were partially purified by affinity chromatography on transferrin-Sepharose and shown to be disulfide-bridged homodimers of a polypeptide with an apparent molecular weight of approximately 90,000. This protein is a phosphoprotein that can be resolved by isoelectric focusing into three major and two minor forms. By digestion with bacterial alkaline phosphatase, it was shown that at least four of these forms are probably phosphorylation variants of a single polypeptide. As differentiation proceeds, the proportions of the individual forms of the receptor change with a shift to the more phosphorylated polypeptides.     

Promotion of haematopoietic activity in embryonic stem cells by the aorta-gonad-mesonephros microenvironment

We investigated whether the in vitro differentiation of ES cells into haematopoietic progenitors could be enhanced by exposure to the aorta-gonadal-mesonephros (AGM) microenvironment that is involved in the generation of haematopoietic stem cells (HSC) during embryonic development. We established a co-culture system that combines the requirements for primary organ culture and differentiating ES cells and showed that exposure of differentiating ES cells to the primary AGM region results in a significant increase in the number of ES-derived haematopoietic progenitors. Co-culture of ES cells on the AM20-1B4 stromal cell line derived from the AGM region also increases haematopoietic activity. We conclude that factors promoting the haematopoietic activity of differentiating ES cells present in primary AGM explants are partially retained in the AM20.1B4 stromal cell line and that these factors are likely to be different to those required for adult HSC maintenance.     

Collagen synthesis is required for ascorbic acid-enhanced differentiation of mouse embryonic stem cells into cardiomyocytes

Ascorbic acid has been reported to promote the differentiation of embryonic stem (ES) cells into cardiomyocytes; however, the specific functions of ascorbic acid have not been defined. A stable form of ascorbic acid, namely, l-ascorbic acid 2-phosphate (A2-P), significantly enhanced cardiac differentiation; this was assessed by spontaneous beating of cardiomyocytes and expression of cardiac-specific markers obtained from mouse ES cells. This effect of ascorbic acid was observed only when A2-P was present during the early phase of differentiation. Treatment with two types of collagen synthesis inhibitors, l-2-azetidine carboxylic acid and cis-4-hydroxy-d-proline, significantly inhibited the A2-P-enhanced cardiac differentiation, whereas treatment with the antioxidant N-acetyl cysteine showed no effect. These findings demonstrated that ascorbic acid enhances differentiation of ES cells into cardiomyocytes through collagen synthesis and suggest its potential in the modification of cardiac differentiation of ES cells.     

Immunocytochemical localization of ornithine decarboxylase in cultured murine cells

Summary Antiserum elicited to ornithine decarboxylase (ODC) purified from murine RAW 264 macrophage-like cells has been employed to localize ODC in cultured murine cells. The antiserum immunoprecipitated 100% of the ODC activity from the cultured cells. The specificity of the antiserum was demonstrated by the immunoprecipitation from ³⁵S-methionine metabolically-labeled cell extracts of a single protein which migrated upon SDS-gel electrophoresis coincident with authentic ODC. Indirect immunofluorescence experiments were performed on paraformaldehyde-fixed RAW 264 cells and JB6 epidermal cells using the rabbit anti-ODC antiserum and FITC-conjugated goat anti-rabbit IgG. Little immunofluorescence was apparent in non-stimulated cells. Intense immunofluorescence was detectable in stimulated cells at times of peak cellular ODC activity. Antigenically-reactive ODC was localized diffusely in the cytoplasm and was absent in the nuclei of RAW 264 cells, whereas in the JB6 cells the immunodetectable enzyme protein was localized in a punctate pattern in both the cytoplasm and nucleoplasm and was absent in the nucleolus. The appearance and disappearance of immunoreactive ODC in both cell types after stimulation was consistent with the alterations in ODC activity.     

The genetics of the repair of 5-azacytidine-mediated DNA damage in the fission yeastSchizosaccharomyces pombe

We have recently demonstrated thatSchizosaccharomyces pombe cells treated with the nucleoside analogue 5-azacytidine (5-azaC) require previously characterised G2 checkpoint mechanisms for survival. Here we present a survey of known DNA repair mutations which defines those genes required for survival in the presence of 5-azaC. Using a combination of single-mutant and epistasis analyses we find that the excision, mismatch and recombinational repair pathways are all required in some degree for the repair of 5-azaC-mediated DNA damage. There are distinct differences in the epistatic interactions of several of the repair mutations with respect to 5-azaC-mediated DNA damage relative to UV-mediated DNA damage.     

DNA crosslinking induced by 1,2-diaminocyclohexanedichloroplatinum(II) in murine leukemia L1210 cells and comparison with other platinum analogues

1,2-Diaminocyclohexanedichloroplatinum(II) (DCDP) is an analogue of the clinically efficacious cancer chemotherapeutic drug cis-diamminedichloroplatinum(II) (cis-DDP). DCDP is presently undergoing clinical trials at least in part because a cis-DDP-resistant murine leukemia L1210 cell line is sensitive to its action. The alkaline elution technique was used to measure DNA-protein and DNA-interstrand crosslinks induced by DCDP in sensitive and resistant L1210 cells. This was compared to the action of cis-DDP and its clinically ineffective isomer trans-DDP. The action of DCDP was similar to that for cis-DDP with maximum crosslinking occurring between 6 and 12 h after a 1 h treatment. Both cis-DDP and DCDP exhibited proportionately higher levels of interstrand crosslinking than trans-DDP. Near complete removal of both classes of DCDP-induced crosslinks was seen by 72 h. While the extent of crosslinking was different for each compound, little difference between the two cell lines was noted with respect to crosslinking by either DCDP or trans-DDP. These cell lines exhibit a 2-fold resistance to both DCDP and trans-DDP and at equitoxic doses of both drugs the resistant cells demonstrated twice the interstrand crosslinks that were seen in the sensitive cells. The extent of crosslinking related directly to the concentration of drug. When treated with equitoxic doses of DCDP, cis-DDP or trans-DDP, the resistant cells consistently exhibited more interstrand crosslinks than sensitive cells, suggesting the existence of a more critical cytotoxic lesion which was not detectable by the alkaline elution technique. These lesions could be either intrastrand crosslinks or monofunctional platination. Resistance must be due to a differential sensitivity to the lesions that form, which may be due to an altered capacity to repair the lesions.     

A novel, efficient method to derive bovine and mouse embryonic stem cells with in vivo differentiation potential by treatment with 5-azacytidine

Pluripotent embryonic stem cells (ESCs) were first isolated nearly three decades ago from mice, yet efficient ESC isolation has been limited to rodents and primates to date. We report a novel and robust technique for isolating ESCs from mammalian pre-implantation embryos by altering the epigenotype of embryonic explants and using pressed zona pellucida-free blastocysts. We first examined this technique for murine ESC derivation. Compared with controls, murine ESCs were efficiently derived when explants were exposed to 1μM 5-azacytidine, an epigenetic modifier that causes DNA demethylation (56.1% vs 31.6%; P < 0.01). Mouse ESCs stained positively for alkaline phosphatase, expressed markers of pluripotency including Oct4, Rex1 and SSEA1 and formed teratomas when injected into Severe Combined Immuno-Deficient (SCID) mice. The approach was subsequently used for bovine ESC derivation. In bovine a higher concentration of 5-azacytidine (5 μM) was required to elicit a response. This technique resulted in up to 18 times more efficient isolation of pluripotent cells than traditional methods (71.4% vs 4.0%; P < 0.001). These putative bovine ESCs expressed OCT4, REX1 mRNA and SSEA-1 and SSEA-4 proteins; and were able to form embryoid bodies in vitro and teratomas when injected in Severe Combined Immuno Deficient (SCID) mice. This is the first report on derivation of ESCs with both in vitro and in vivo differentiation potential in a livestock species.     

Quantitative variations of the isoforms in haptoglobin 1-2 and 2-2 individual phenotypes

Haptoglobin is a hemoglobin-binding protein presenting in humans three distinct phenotypes (Hpt 1-1, Hpt 1-2, or Hpt 2-2). The Hpt 1-2 and Hpt 2-2 phenotypes are in turn represented by populations of isoforms. The relative amounts of the major isoforms of Hpt 1-2 and Hpt 2-2 were found to differ not only in different individuals, but also in the same individual before and after a physical effort. Exercise-dependent changes in the plasma concentrations of ascorbate, urate, alpha-tocopherol, retinol, and glutathione were also observed, but correlations between such changes and those of the amount for any isoform were not found. Samples of Hpt 1-2 or Hpt 2-2 were challenged with oxidants (H(2)O(2) with ferrous ions, spermine-NO, KO(2), and 3-morpholinosydnonimine), but the isoform levels were not altered. Hpt 2-2 isoforms were present in Hpt 1-2, as minor species. Furthermore, different isoforms exhibited different hemoglobin binding abilities. Thus, these parameters should also be taken into consideration in studies correlating Hpt phenotypes prevalence with pathologies or functional differences.