EDR is a stress-related survival factor from stroma and other tissues acting on early haematopoietic progenitors (E-Mix)

The erythroid differentiation regulator (EDR) is a highly conserved autocrine factor produced in many tissues. Its haemoglobin synthesis-inducing activity for human and murine erythroleukaemia cell lines had been detected in WEHI-3 conditioned medium. EDR functions were analysed in detail. It is released from cells immediately in response to various stressful conditions and enhances cell survival particularly at a lower concentration range and low cell density. At high cell density and high EDR concentration the opposite effect of an increase in cell death was observed. Its essential function within a tissue is considered to be the maintenance of growth homeostasis. Cells kept in culture for weeks show a decreasing responsiveness to EDR supply. This was also noted in freshly cloned EDR-responsive mouse erythroleukaemia cells pointing to a molecular adaptation process. Human haematopoietic progenitors were amplified 7-fold by EDR when kept at low cytokine levels. At saturating levels progenitors giving rise to at least two lineages in semisolid medium (E-mix) respond to EDR with an average 1.87-fold increase in colony numbers and a bell-shaped dose-response curve. Of the more mature BFU-E compartment a response was observed particularly in cases with low colony numbers. Given the release from irradiated stromal cells and the ability to partly substitute for stromal cells in the Burkitt's lymphoma cell line BL-70, EDR functions as a stromal survival factor for stroma-responsive cells.     

CUX1 transcription factors: from biochemical activities and cell-based assays to mouse models and human diseases

Oxidored nitro domain containing protein 1 (NOR1) is usually restrictively expressed in the brain and testis. Detection of altered NOR1 expression could help us to identify its functions in cell growth, differentiation, metabolism, or even carcinogenesis. In this study, NOR1 homologues were identified in multiple species through GenBank search. NOR1 is a novel protein conserved in multiple species. Mouse NOR1 shared high homology with human NOR1. Furthermore, NOR1 expression was analyzed in mouse tissues by using RT-PCR, Western blot, and immunohistochemistry. The data showed that NOR1 is broadly expressed in neurons of mouse brain and the expression profile changes during postnatal development of the mouse brain. Moreover, in non-nervous tissues, strong immunostaining for NOR1 protein was observed in the testis, epididymis and trachea. In addition, expression of human NOR1 protein in different normal and cancerous human tissues was analyzed via search of the human RNA and protein databases; the data showed that although most malignant cells weakly stained or were negative for NOR1 expression, the liver cancer cells displayed moderate to strong expression of NOR1. These data suggested that NOR1 might serve as a cancer/testis/brain antigen in cells, and that altered NOR1 expression in liver cancer may help us to elucidate the functions of NOR1 protein in liver carcinogenesis.     

K-Cl cotransporter gene expression during human and murine erythroid differentiation

The K-Cl cotransporter (KCC) regulates red blood cell (RBC) volume, especially in reticulocytes. Western blot analysis of RBC membranes revealed KCC1, KCC3, and KCC4 proteins in mouse and human cells, with higher levels in reticulocytes. KCC content was higher in sickle versus normal RBC, but the correlation with reticulocyte count was poor, with inter-individual variability in KCC isoform ratios. Messenger RNA for each isoform was measured by real time RT-quantitative PCR. In human reticulocytes, KCC3a mRNA levels were consistently the highest, 1-7-fold higher than KCC4, the second most abundant species. Message levels for KCC1 and KCC3b were low. The ratios of KCC RNA levels varied among individuals but were similar in sickle and normal RBC. During in vivo maturation of human erythroblasts, KCC3a RNA was expressed consistently, whereas KCC1 and KCC3b levels declined, and KCC4 message first increased and then decreased. In mouse erythroblasts, a similar pattern for KCC3 and KCC1 expression during in vivo differentiation was observed, with low KCC4 RNA throughout despite the presence of KCC4 protein in mature RBC. During differentiation of mouse erythroleukemia cells, protein levels of KCCs paralleled increasing mRNA levels. Functional properties of KCCs expressed in HEK293 cells were similar to each other and to those in human RBC. However, the anion dependence of KCC in RBC resembled most closely that of KCC3. The results suggest that KCC3 is the dominant isoform in erythrocytes, with variable expression of KCC1 and KCC4 among individuals that could result in modulation of KCC activity.     

Uridine kinase: altered subunit size or enzyme expression as a function of cell type, growth stimulation, or mutagenesis

Using antibody prepared against pure uridine kinase from Ehrlich ascites cells, we have measured the expression of enzyme protein by the Western blot technique. Variations were observed in the Mr of the enzyme subunit for uridine kinase from different species: 32,000 (mouse Ehrlich ascites cells), 30,000 (normal human lymphocytes), 28,000 (mouse tissues), 27,500 (rat tissues). For different normal tissues from the same species, there was no significant variation in the subunit size. Transformed human and mouse cell lines, selected for a deficiency of uridine kinase activity in the presence of inhibitors activated by this enzyme, expressed two cross-reacting proteins, one with a normal (30,000) and one with a smaller (21,000) subunit molecular weight than was found in the parental cell line (human lymphoma), or only a smaller protein of Mr 25,000 (mouse lymphoma). Our results show that selection protocols using metabolite inhibitors do not always repress the expression of the enzyme but instead may lead to selection of those cells that have a mutation in the uridine kinase gene, resulting in the expression of an inactive enzyme. The expression of uridine kinase protein changes when cells are stimulated to divide. For both mouse fibroblasts and human lymphocytes, expression of uridine kinase protein as well as activity clearly increased after cells were stimulated to grow. In fibroblasts, increases are seen by 3 hr after stimulation, and plateau after 9 hr at a sevenfold increase. In lymphocytes, no change is seen until 12 hr after stimulation, and a plateau is not reached until 72 hr, with a total increase of approximately 50-fold. There has been considerable interest in the possibility of uridine kinase isozymes. Except for cells that have been mutagenized, the present results show that, as judged by subunit molecular weight, there appears to be only one enzyme form in normal and neoplastic cells or in cells in which uridine kinase activity is induced.     

Protein Synthesis in Hybrid Cells Derived from Fetal Rat × Mouse Chimeric Organs

Abstract. Malignant hybrid cells (As3) derived from fusion of rat hepatoma cells (Fu5AH) with mouse teratocarcinoma cells (OTT6050) were injected into genetically marked mouse blastocysts which were subsequently transferred into pseudopregnant surrogate mothers. From a total of 61 fetuses developed, four normally differentiated fetuses at day 18 of gestation showed hybrid cell contributions in their livers and a few other organs of endo-mesodermal origin. The chimeric tissues were briefly cultured in vitro and then further investigated for their protein synthesis using two-dimensional gel electrophoresis. After comparison of the protein patterns obtained from the corresponding normal rat and mouse organs, several rat-specific polypeptides were detected in the cultured chimeric tissues illustrating functional xenogeneic gene expression during in situ differentiation. In addition, some other rat proteins characteristic of the parental hybrid cell line disappeared. The tumorigenicity of the chimeric tissues was tested by subcutaneous transplantation into immunodeficient nude mice. Tumors originating from two of the four chimeric organs differed histologically from those formed by cells of the hybrid As3 line since they also contained muscle-like structures resembling rhabdomyosarcomas. The tumors were analyzed for their protein synthesis and compared with the three malignant cell lines of parental origin. The morphologic differences between the tumors derived from the chimeric organs and those developed from the As3 cell line were also reflected in characteristic differences of their protein synthesis patterns. Our results demonstrate that interspecific rat × mouse hybrid cells, when implanted into early mouse embryos, participate in fetal tissue differentiation and selectively repress certain rat gene products typical of the malignant parental cells as well as functionally reactivate other rat genes presumably required for normal development.     

CD40 ligand rescues inhibitor of differentiation 3-mediated G1 arrest induced by anti-IgM in WEHI-231 B lymphoma cells

The engagement of membrane-bound Igs (mIgs) results in growth arrest, accompanied by apoptosis, in the WEHI-231 murine B lymphoma cells, a cell line model representative of primary immature B cells. Inhibitor of differentiation (Id) proteins, members of the helix-loop-helix protein family, functions in proliferation, differentiation, and apoptosis in a variety of cell types. In this study, we analyzed the involvement of Id protein in mIg-induced growth arrest and apoptosis in WEHI-231 cells. Following stimulation with anti-IgM, expression of Id3 was up-regulated at both the mRNA and protein levels; this up-regulation could be reversed by CD40L treatment. Retrovirus-mediated transduction of the Id3 gene into WEHI-231 cells resulted in an accumulation of the cells in G(1) phase, but did not induce apoptosis. E box-binding activity decreased in response to anti-IgM administration, but increased after stimulation with either CD40L alone or anti-IgM plus CD40L, suggesting that E box-binding activity correlates with cell cycle progression. WEHI-231 cells overexpressing Id3 accumulated in G(1) phase, which was accompanied by reduced levels of cyclin D2, cyclin E, and cyclin A, and a reciprocal up-regulation of p27(Kip1). Both the helix-loop-helix and the C-terminal regions of Id3 were required for growth-suppressive activity. These data suggest that Id3 mimics mIg-mediated G(1) arrest in WEHI-231 cells.     

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.     

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

When cells of mouse myelomonocytic 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 Iad, 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.     

Erythroid differentiation in a friend erythroleukemic cell x lymphoma hybrid cell line is limited, possibly due to reduced hem levels

Induction of erythroid differentiation has been investigated in a cell hybrid formed between an inducible Friend cell and a lymphoma line (L5178Y) derived from the same strain of mouse (DBA/2). Although globin messenger RNA (mRNA) is induced by DMSO to a level similar to that in the inducible Friend cell parent (about 9 000 molecules/cell) haemoglobin does not accumulate in detectable amounts, nor do morphological changes characteristic of terminal differentiation occur. This failure to accumulate haemoglobin in response to DMSO is due to a reduced rate of globin chain synthesis (6% of total protein synthesis, compared to 25% for the parental Friend cell), and partly to inability of the globin chains synthesized to form tetrameric haemoglobin molecules. Globin chain instability is not the reason why haemoglobin does not accumulate. In comparison, treatment of the hybrid cells with haemin induces about 14% globin synthesis and about 13 000 globin mRNA molecules. These values are somewhat higher than with DMSO. Treatment of hybrid cells with haemin plus DMSO is even more effective; it induces 25% globin synthesis and about 30 000 globin mRNA molecules and terminal differentiation also occurs normally. Whether treated with DMSO or haemin or both, virtually all the globin mRNA molecules seem to be present in polysomes and are therefore presumably in the process of being translated. These results suggest that failure of differentiation in these hybrid cells is due to haem limitation which also prevents the expression of other co-ordinated erythroid functions.     

Genes newly identified as regulated by glucocorticoids in murine thymocytes

Glucocorticoids induce dramatic biochemical and morphological changes in lymphocytes through an unknown process that requires RNA and protein synthesis. In order to identify genes involved in this response, we previously isolated 11 cDNA clones from the murine WEHI-7TG thymoma cell line that correspond to mRNAs induced by glucocorticoids. We now report the isolation of two new cDNA clones whose gene expression is regulated by glucocorticoids in WEHI-7TG cells. We further characterize the two new cDNA clones, as well as those described previously, by examining the response of each of the corresponding mRNAs to glucocorticoids in murine thymocytes. With the exception of two, all cDNAs correspond to genes that are induced by glucocorticoids in murine thymocytes within 4 h of treatment. We previously identified two of the cDNAs as the mouse VL30 retrovirus-like element and the mouse homolog of chondroitin sulfate proteoglycan core protein. We have now identified four additional cDNA clones that correspond to the genes for calmodulin, mitochondrial phosphate carrier protein, immunoglobulin (Ig)-related glycoprotein (GP-70), and the 70 kilodalton autoantigen for Lupus and Graves diseases. Two other cDNA clones represent previously undescribed genes: one shares a high similarity to known sequences for the family of G-protein-coupled receptors and the other to a human placental-specific protein, PP11. Another cDNA appears to contain sequences for an unknown gene and the remnants of a mouse transposon. ETn. The remaining clones represent new, unidentified genes induced by glucocorticoids in murine thymocytes and in the WEHI-7TG cell line.     

Role of human ribosomal RNA (rRNA) promoter methylation and of methyl-CpG-binding protein MBD2 in the suppression of rRNA gene expression

The methylation status of the CpG island located within the ribosomal RNA (rRNA) promoter in human hepatocellular carcinomas and pair-matched liver tissues was analyzed by bisulfite genomic sequencing. Significant hypomethylation of methyl-CpGs in the rRNA promoter was observed in the tumor samples compared with matching normal tissues, which was consistent with the relatively high level of rRNA synthesis in rapidly proliferating tumors. To study the effect of CpG methylation on RNA polymerase I (pol I)-transcribed rRNA genes, we constructed pHrD-IRES-Luc (human rRNA promoter-luciferase reporter). In this plasmid, Kozak sequence of the pGL3-basic vector was replaced by the internal ribosome entry site (IRES) of encephalomyocarditis viral genome to optimize pol I-driven reporter gene expression. Transfection of this plasmid into HepG2 (human) cells revealed reduced pol I-driven luciferase activity with an increase in methylation density at the promoter. Markedly reduced luciferase activity in Hepa (mouse) cells compared with HepG2 (human) cells showed that pHrD-IRES-Luc is transcribed by pol I. Site-specific methylation of human rRNA promoter demonstrated that methylation of CpG at the complementary strands located in the promoter (-9, -102, -347 with respect to the +1 site) inhibited luciferase activity, whereas symmetrical methylation of a CpG in the transcribed region (+152) did not affect the promoter activity. Immunofluorescence studies showed that the methyl-CpG-binding proteins, MBD1, MBD2, MBD3, and MeCP2, are localized both in the nuclei and nucleoli of HepG2 cells. Transient overexpression of MBD2 suppressed luciferase activity specifically from the methylated rRNA promoter, whereas MBD1 and MBD3 inhibited rRNA promoter activity irrespective of the methylation status. Chromatin immunoprecipitation analysis confirmed predominant association of MBD2 with the endogenous methylated rRNA promoter, which suggests a selective role for MBD2 in the methylation-mediated inhibition of ribosomal RNA gene expression.     

IL-6 is a differentiation factor for M1 and WEHI-3B myeloid leukemic cells

IL-6 has multiple biologic activities in different cell systems including both the ability to support cell proliferation and to induce differentiation. We reported previously the isolation and functional expression of a mouse IL-6 (mIL-6) cDNA clone derived from bone marrow stromal cells. In this paper, we show that mIL-6 is a potent inducer of terminal macrophage differentiation for a mouse myeloid leukemic cell line, M1. Addition of mIL-6 to cultures of M1 cells rapidly inhibits their proliferation and induces phagocytic activity and morphologic changes characteristic of mature macrophages. These phenotypic changes are accompanied at the molecular level by a decrease in proto-oncogene c-myc mRNA accumulation and increases in Fc gamma R, proto-oncogenes c-fos and c-fms (CSF-1R) mRNA expression. Furthermore, IL-6 enhances the expression of Fc gamma R and c-fms in differentiation-responsive D+, but not unresponsive D- sublines of mouse myelomonocytic leukemic WEHI-3B cells. Together with our previous observation that IL-6 stimulates colony formation by normal myeloid progenitors, these results strongly suggest an important regulatory role for IL-6 in myeloid cell growth and differentiation.     

Characterization of matriptase expression in normal human tissues.

Matriptase is a type II transmembrane serine protease that has been implicated in the progression of epithelium-derived tumors. The role of this protease in the biology of normal epithelial cells remains to be elucidated. Matriptase mRNA has been detected by Northern analysis in tissues rich in epithelial cells, and the protein is expressed in vivo in normal and cancerous breast, ovarian, and colon tissues. However, a systematic analysis of the distribution of matriptase protein and mRNA in normal human tissues rich in epithelium has not been reported. In this study we characterized the expression of the protease in a wide variety of normal human tissues using a tissue microarray and whole tissue specimens. Significant immunoreactivity and mRNA expression were detected in the epithelial components of most epithelium-containing tissues. Matriptase expression was found in all types of epithelium, including columnar, pseudostratified columnar, cuboidal, and squamous. Distinct spatial distributions of reactivity were observed in the microanatomy of certain tissues, however. This suggests that although matriptase is broadly expressed among many types of epithelial cells, its activity within a tissue may be regulated in part at the protein and mRNA levels during the differentiation of selected epithelia.     

TACC3 expression is tightly regulated during early differentiation

Transforming acidic coiled-coil (TACC) proteins are hypothesized to play a role in normal cellular growth and differentiation and to be involved in centrosomal microtubule stabilization. Our current studies aim to delineate the expression pattern of TACC3 protein during cellular differentiation and in a variety of normal human tissues. TACC3 is known to be upregulated in differentiating erythroid progenitor cells following treatment with erythropoietin and is required for replication of hematopoietic stem cells. However, we demonstrate that a dramatic upregulation of TACC3 also occurs during the early differentiation of NIH 3T3-L1 cells into adipocytes and PC12 cells into neurons, indicating that TACC3 mediates cellular differentiation in several cell types. Using real-time PCR, we quantitated the mRNA levels of TACC3 compared to TACC1 and TACC2 in various human adult tissues. We observed the highest expression of TACC3 mRNA in testis, spleen, thymus and peripheral blood leukocytes, all tissues undergoing high rates of differentiation, and a lower level of expression in ovary, prostate, pancreas, colon, small intestine, liver and kidney. In contrast, TACC1 and TACC2 mRNA levels are more widespread. By immunohistochemistry, we confirm that the TACC3 protein localizes to differentiating cell types, including spermatocytes, oocytes, epithelial cells, bone marrow cells and lymphocytes. Thus, these observations are concordant with a basic role for TACC3 during early stages of differentiation in normal tissues.     

Differentially expressed genes in endothelial differentiation

By screening differentially expressed genes in mouse embryonic stem (ES) cells by subtractive hybridization, we identified three conserved but uncharacterized genes encoding bromodomain containing 3 (BRD3), protein lysine methyltransferase (PLM), and kelch domain containing 2 (KLHDC2), which were downregulated during endothelial differentiation. An RNA blot study showed that these genes were markedly expressed in undifferentiated ES cells, whereas the expression was reduced upon endothelial differentiation; a study of mouse endothelium showed a significant reduction in the expression of BRD3. A study of human BRD3, located on chromosome 9 at q34, a region susceptible to genomic rearrangement, showed an altered expression in 4 of 12 patients with bladder cancer, compared with adjacent noncancerous tissues. Taken together with the result of siRNA inhibition showing the positive regulation of cell proliferation by BRD3, it is suggested that this molecule plays a role in allowing cells to enter the proliferative phase of the angiogenic process.     

Developmental regulation of a constitutively expressed mouse mRNA encoding a 72-kDa heat shock-like protein

Multiple heat shock cognate (hsc70) cDNA clones were isolated from the mouse embryonal carcinoma cell line F9. They all encode a single 72-kDa protein, which is constitutively expressed in all mouse cell lines and tissues tested, and which is only slightly induced by hyperthermia. hsc70 RNA is very abundant in F9 stem cells and brain, but very little is found in 14-day-old embryos. Upon differentiation of F9 stem cells induced by retinoic acid and cyclic AMP, expression of the hsc70 gene decreases only slightly, suggesting that hsc70 is highly expressed in early mouse development and is then down-regulated towards the end of embryogenesis. In adult tissues only the brain retains the high level of hsc70 gene expression found in F9 stem cells. We also show that expression of hsc70 protein and clathrin is uncoupled in F9 cells, indicating that the uncoating activity of coated vesicles may not be the only function of hsc70 protein.