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PubMed2004

Formation and characterization of spontaneously formed heterokaryons between quail myoblasts and 3T3-L1 preadipocytes: correlation between differential plasticity and degree of differentiation.

Skeletal muscle cells and adipose cells have a close relationship in developmental lineage. Our previous study has shown that the heterokaryons between quail myoblasts and undifferentiated 3T3-L1 cells (preadipocytes) normally differentiated into myotubes, whereas the heterokaryons between myoblasts and differentiated 3T3-L1 cells (adipocytes) failed myogenic differentiation. These results suggest differences between preadipocytes and adipocytes. The purpose of this study was to clarify whether preadipocytes have flexibility in differentiation before terminal adipose differentiation. Presumptive quail myoblasts transformed with a temperature-sensitive mutant of Rous sarcoma virus (QM-RSV cells) and mouse 3T3-L1 cells (either preadipocytes or adipocytes) were co-cultured for 48 h under conditions allowing myogenic differentiation. On co-culture between myoblasts and undifferentiated 3T3-L1 cells, heterokaryotic myotubes formed spontaneously, but not on co-culture with differentiated 3T3-L1 cells. In addition, the heterokaryotic myotubes expressed mouse myogenin derived from the 3T3-L1 cell gene. Our previous study indicated that the fusion sensitivity of differentiating myoblasts change with decreasing cholesterol of the cell membrane during myoblast fusion. Thus we compared the level of membrane cholesterol between undifferentiated and differentiated 3T3-L1 cells. The result showed that the level of membrane cholesterol in 3T3-L1 cells increases during adipose differentiation. Corresponding to the increase in membrane cholesterol content, differentiated 3T3-L1 cells had lower sensitivity to HVJ (Sendai virus)-mediated cell fusion than undifferentiated 3T3-L1 cells. This study demonstrated that 3T3-L1 cells at an undifferentiated state have a capacity for spontaneous fusion with differentiating myoblasts following myogenic differentiation, and that the capacity is lost after terminal adipose differentiation.

3T3-L1 Cells↗
PubMed1984

Control of differentiation in heterokaryons and hybrids involving differentiation-defective myoblast variants.

Clones of differentiation-defective myoblasts were isolated by selecting clones of L6 rat myoblasts that did not form myotubes under differentiation-stimulating conditions. Rat skeletal myosin light chain synthesis was induced in heterokaryons formed by fusing these defective myoblasts to differentiated chick skeletal myocytes. This indicates that the structural gene for this muscle protein was still responsive to chick inducing factors and that the defective myoblasts were not producing large quantities of molecules that dominantly suppressed the expression of differentiated functions. The regulation of the decision to differentiate was then examined in hybrids between differentiation-defective myoblasts and differentiation-competent myoblasts. Staining with antimyosin antibodies showed that the defective myoblasts and homotypic hybrids formed by fusing defective myoblasts to themselves could in fact differentiate, but did so more than a thousand times less frequently than the 64% differentiation achieved by competent L6 myoblasts or homotypic competent X competent L6 hybrids. Heterotypic hybrids between differentiation-defective myoblasts and competent L6 cells exhibited an intermediate behavior of approximately 1% differentiation. A theoretical model for the regulation of the commitment to terminal differentiation is proposed that could explain these results by invoking the need to achieve threshold levels of secondary inducing molecules in response to differentiation-stimulating conditions. This model helps explain many of the stochastic aspects of cell differentiation.

Animals↗
PubMed1995

Differential regulation of Max and role of c-Myc during erythroid and myelomonocytic differentiation of K562 cells.

The protein Max binds to c-Myc and the heterodimer c-Myc/Max seems to be the active form in vivo. While the expression of c-myc is extensively regulated, no major changes in max expression have been reported so far with respect to differentiation. We have studied the expression of c-Myc and Max during in vitro differentiation of the bipotent human myeloid leukemia K562 cell line. This cell model system allowed us to compare c-Myc and Max expression during differentiation along erythroid (induced by 1-beta-D-arabinofuranosyl-cytosine) and myelomonocytic lineages (induced by 12-0-tetradecanoylphorbol-13-acetate). We found that c-myc expression decreased as a result of both differentiating treatments. The expression level of max remained unchanged during myelomonocytic differentiation. In contrast, max mRNA and protein were dramatically down-regulated during erythroid differentiation of K562 cells, thus demonstrating that max gene is subjected to regulation during differentiation. We also studied the expression of the other two described members of the c-Myc network: mxi1 and mad. mxi1 expression increased during erythroid differentiation but was strongly down-regulated during myelomonocytic differentiation of K562. mad was constitutively expressed during K562 erythroid differentiation and slightly increased during induction of the myelomonocytic pathway. We have obtained K562 sublines stably transfected with a zinc-inducible c-myc gene. In these clones the overexpression of c-Myc did not interfere with TPA-induced myelomonocytic differentiation. In contrast, erythroid differentiation was significantly inhibited upon c-myc induction despite the down-regulation of endogenous max expression. These results suggest a differential role for c-Myc in the human myeloid cell differentiation depending on the cell lineage.

Basic Helix-Loop-Helix Leucine Zipper Transcriptio↗
PubMed1995

Treatment of HL60 cells with various combinations of retinoids and 1 alpha,25 dihydroxyvitamin D3 results in differentiation towards neutrophils or monocytes or a failure to differentiate and apoptosis.

It is well documented that treatment of serum-grown HL60 cells with 10(-7) M all-trans retinoic acid (all-trans RA) induces neutrophil differentiation, whereas treatment with 10(-7) M 1 alpha,25 dihydroxyvitamin D3(D3) induces differentiation towards monocytes. In recent investigations, using serum-free grown HL60 cells, we observed that all-trans RA, at 10(-7) M, did not induce neutrophil differentiation and that all-trans RA, at 10(-8) M, reduced the D3 concentration required for monocyte differentiation to 5 x 10(-9) M. In this study, co-operative interactions between all-trans and 9-cis RA and D3 which promote neutrophil and monocyte differentiation of HL60 cells have been analysed in detail. Treatment of serum-free grown HL60 cells with 5 x 10(-7) M all-trans RA or 9-cis RA resulted in sub-optimal neutrophil differentiation (up to 25% mature cells). As shown for all-trans RA, 9-cis RA cooperated with D3 to promote monocyte differentiation. Culture of HL60 cells in 5 x 10(-7) M 9-cis RA together with a wide range of concentrations of D3 resulted in promotion of neutrophil differentiation at 10(-15)-10(-12) D3, a failure to differentiate and apoptosis at 10(-11)-10(-10) M D3, followed by co-operativity between 9-cis RA and 5 x 10(-9) M D3 in inducing monocyte differentiation in the absence of neutrophil differentiation. Similar results were obtained when HL60 cells were treated with 5 x 10(-7) all-trans RA together with a wide range of concentrations of D3. Cross titration analyses of the effects of 9-cis RA and D3 on HL60 cell differentiation were undertaken to determine the boundaries of the concentrations of each agent, alone and in combination, that give rise to optimal neutrophil and monocyte differentiation of HL60 cells. The observed cooperativities between either 9-cis RA or all-trans RA and D3 have important implications for the use of combinations of these agents in differentiation therapy.

Apoptosis↗
PubMed2005

Differential expression of CCN-family members in primary human bone marrow-derived mesenchymal stem cells during osteogenic, chondrogenic and adipogenic differentiation.

BACKGROUND: The human cysteine rich protein 61 (CYR61, CCN1) as well as the other members of the CCN family of genes play important roles in cellular processes such as proliferation, adhesion, migration and survival. These cellular events are of special importance within the complex cellular interactions ongoing in bone remodeling. Previously, we analyzed the role of CYR61/CCN1 as an extracellular signaling molecule in human osteoblasts. Since mesenchymal stem cells of bone marrow are important progenitors for various differentiation pathways in bone and possess increasing potential for regenerative medicine, here we aimed to analyze the expression of CCN family members in bone marrow-derived human mesenchymal stem cells and along the osteogenic, the adipogenic and the chondrogenic differentiation. RESULTS: Primary cultures of human mesenchymal stem cells were obtained from the femoral head of patients undergoing total hip arthroplasty. Differentiation into adipocytes and osteoblasts was done in monolayer culture, differentiation into chondrocytes was induced in high density cell pellet cultures. For either pathway, established differentiation markers and CCN-members were analyzed at the mRNA level by RT-PCR and the CYR61/CCN1 protein was analyzed by immunocytochemistry.RT-PCR and histochemical analysis revealed the appropriate phenotype of differentiated cells (Alizarin-red S, Oil Red O, Alcian blue, alkaline phosphatase; osteocalcin, collagen types I, II, IX, X, cbfa1, PPARgamma, aggrecan). Mesenchymal stem cells expressed CYR61/CCN1, CTGF/CCN2, CTGF-L/WISP2/CCN5 and WISP3/CCN6. The CYR61/CCN1 expression decreased markedly during osteogenic differentiation, adipogenic differentiation and chondrogenic differentiation. These results were confirmed by immuncytochemical analyses. WISP2/CCN5 RNA expression declined during adipogenic differentiation and WISP3/CCN6 RNA expression was markedly reduced in chondrogenic differentiation. CONCLUSION: The decrease in CYR61/CCN1 expression during the differentiation pathways of mesenchymal stem cells into osteoblasts, adipocytes and chondrocytes suggests a specific role of CYR61/CCN1 for maintenance of the stem cell phenotype. The differential expression of CTGF/CCN2, WISP2/CCN5, WISP3/CCN6 and mainly CYR61/CCN1 indicates, that these members of the CCN-family might be important regulators for bone marrow-derived mesenchymal stem cells in the regulation of proliferation and initiation of specific differentiation pathways.

Journal Article↗
PubMed2005

The CDK inhibitor p27 enhances neural differentiation in pluripotent NTERA2 human EC cells, but does not permit differentiation of 2102Ep nullipotent human EC cells.

Embryonal carcinoma (EC) cells, the stem cells of teratocarcinomas, are the malignant counterparts of pluripotent embryonic stem (ES) cells, but commonly exhibit a reduced ability to differentiate, presumably because of continual selection for genetic changes that alter the balance between self-renewal, differentiation and apoptosis in favour of self-renewal. To explore the nature of the genetic changes that promote nullipotency, we have compared two human EC cell lines, a 'nullipotent' line, 2102Ep, and a 'pluripotent' line, NTERA2. A hybrid derived by fusion of these cells differentiates in response to retinoic acid but, unlike the parental NTERA2 line, does not form terminally differentiated neurons. This implies that the nullipotent EC cell line, 2102Ep, differs in expression of at least two functions in comparison with the NTERA2 pluripotent line, one affecting commitment to differentiation, and one affecting terminal neural differentiation. We have now investigated the possible role of the CDK inhibitor, p27kip1 (p27) in commitment and terminal differentiation. In NTERA2, but not in 2102Ep cells, retinoic acid induces up-regulation of p27 expression, suggesting that 2102Ep cells lack this capacity. However, constitutive expression of a p27 transgene does not overcome the block to differentiation in the 2102Ep parental cells; commitment to differentiation must be blocked elsewhere. On the other hand, constitutive over-expression of p27 from a transgene enhances the neural differentiation of NTERA2 cells. Our results suggest that p27 plays a role in terminal neuronal differentiation of human EC cells, but not in their initial commitment to differentiation, and that other factors, possibly Cyclin D2, specifically limit its ability to promote neural differentiation.

Base Sequence↗
PubMed2003

Estrogen-regulated conditional oncoproteins: tools to address open questions in normal myeloid cell function, normal myeloid differentiation, and the genetic basis of differentiation arrest in myeloid leukemia.

Neutrophils, monocytes and dendritic cells are effectors of innate immunity and essential coactivators in the acquired immune response. Understanding the biochemical basis of their mature cell functions, their differentiation from hematopoietic progenitors, and the mechanisms by which myeloid leukemia oncogenes block their differentiation programs, continue to be areas of active research. Four major problems limit progress in these fields. First, the biochemical analysis of mature cells is limited by the time and cost of purifying neutrophils, monocytes, or dendritic cells from wild-type and genetically modified mouse strains. Second, while immortal myeloid cell lines are used to understand the transcriptional basis of normal terminal differentiation following their treatment with differentiationpromoting agents (e.g. G-CSF, IL-6, RA, TPA), these cells contain stable defects responsible for their immortalization, and the degree to which they model normal differentiation is often incomplete. Third, these same inducible cell lines are used as model systems to determine how myeloid oncoproteins prevent differentiation; however, oncoproteins that block differentiation of marrow progenitors cultured in GM-CSF or IL-3 but permit their differentiation in response to G-CSF or RA, do not score effectively in these assays (e.g. Hoxa9, Mll-Enl). Fourth, there is no reproducible method to derive myeloid progenitor lines that execute predictable terminal differentiation to neutrophils, monocytes, or dendritic cells. Developing this type of system is needed to evaluate how myeloid gene inactivation by knockout technologies alters lineage-specific differentiation and mature cell function. Conditional myeloid oncoproteins provide a tool to solve these research problems by providing a predictable and inexpensive means of expanding, in culture, GM-CSF- or IL-3-dependent myeloid progenitors from any genotype, and by permitting their synchronous differentiation to neutrophils, monocytes, or dendritic cells under defined culture conditions following inactivation of the conditional oncoprotein. This system of conditionally immortalizing normal bone marrow precursors provides the large numbers of normal cells required for analysis of cell biology and protein biochemistry, and further provides a model system in which to study the genetic mechanisms controlling terminal differentiation and how specific oncoproteins expressed in the cell lines prevent this differentiation program. The ability to derive conditionally-immortalized progenitor lines from knock-out mice provides cell lines for the reconstitution of knockout gene function and subsequent dissection of knockout protein function by mutational analysis. Finally, conditional myeloid cell lines can be established from both ES cells and from d10 fetal liver cells, allowing for the analysis of embryonic lethal mutants on both the maturation and terminal differentiation of mature myeloid cells. In this review,we summarize the importance and limitations of current approaches in myeloid cell research, and how estrogen-regulated conditional oncoproteins help to solve these problems.

Animals↗
PubMed2006

Neuroendocrine cell differentiation of poorly differentiated colorectal adenocarcinoma correlates with liver metastasis.

Poorly differentiated (PD) adenocarcinoma often retains the capacity for neuroendocrine (NE) cell differ-entiation; however, it is difficult to distinguish the NE cell differentiation by routine hematoxylin and eosin staining. It is important to detect the presence of NE cell differentiation in advanced colorectal carcinomas because these carcinomas have been shown to produce distant metastasis at the time of diagnosis and to have a particularly poor prognosis. In this study, the characteristics of PD adenocarcinoma with NE cell differentiation and its biological metastatic mechanisms were investigated. Forty-eight of 2204 colorectal cancer patients, diagnosed as having PD adenocarcinoma (2.2%) were enrolled in this study. Immunohistochemical analysis was performed with anti-chromogranin A anti-synaptophysin, anti-CD34, anti-D2-40, and anti-VEGF antibodies. The clinicopathological factors for PD adenocarcinoma with NE cell differentiation were compared with those for PD adenocarcinoma without NE cell differentiation. Microvessel density (MVD) was assessed using immunostained slides with anti-CD34 antibody and vascular endothelial growth factor (VEGF) expression in PD adenocarcinoma with NE cell differentiation was confirmed by in situ hybridization. By immunohistochemical staining for chromogranin A and synaptophysin, NE cell differentiation was detected in eight of 48 patients (16.7%) with PD adenocarcinoma. The frequency of liver metastasis at the time of diagnosis was significantly higher in patients having PD adenocarcinoma with NE cell differentiation (p=0.03). Moreover, MVD and VEGF expression level tended to be higher in patients having PD adenocarcinoma with NE cell differentiation (p=0.13 and 0.068, respectively). NE cell differentiation in PD adenocarcinoma may produce liver metastasis through microvessel formation in the tumor induced by VEGF. In PD colorectal adenocarcinoma, immunohistochemical analysis of NE markers is important for establishing the presence of NE cell differentiation and further study is necessary to evaluate the effectiveness of anti-angiogenic drugs to PD adenocarcinoma with NE cell differentiation.

Adenocarcinoma↗
PubMed2006

[Characteristics of morphology, differentiation related marker, and proliferation dynamics of differentiated brain tumor stem cells in vitro].

OBJECTIVE: To pursue the changes of cell morphology, expression of differentiation related markers, and proliferation cycles of brain tumor stem cells (BTSCs) after differentiation in vitro. METHODS: Tumor stem cells of the line CD133(+) were obtained from two specimens from one clinical case with anaplasia ependymocytoma during operation, one specimen being obtained during the first operation and then second specimen being obtained during the second operation 6 months later on the recurrent tumor. CD133(+) cells were acquired by using magnetic sorting and then cultured to differentiate in medium containing 10% fetal bovine serum. The morphology of the cells was observed under phase contrast microscope. Cells were collected respectively before differentiation and 3, 7, 10, and 21 days after the differentiation. The cell surface markers such as CD133, nestin, glial fibrillary acidic protein (GFAP), and beta-tubulin III were detected with flow cytometry. Proliferation cycles were examined before differentiation and in the 7th day after differentiation. Normal neural stem cells (NSCs) obtained from fetal brain tissues were used as controls. RESULTS: (1) The BTSCs were round shape at the beginning, then changed to short fusiform, polygon and long fusiform. Seven days later the cells reversed to short fusiform and round shape. The cells accumulated into cell spheres and floated in the culture medium again. While the NSCs differentiated along their routine rules. (2) Both the undifferentiated BTSCs and NSCs showed high level expression of CD133 and nestin. After differentiation the BTSCs expressed CD133 and nestin, the expression levels decreased first and then increased. The expression rates of CD133 and nestin were (3.65 +/- 0.17)% and (28.99 +/- 1.26)% in the 7th day, (14.63 +/- 1.16)% and (45.46 +/- 1.27)% in the 21st day. While the positive expression rate of GFAP was higher than that of beta-tubulin III. In the 10th day the NSCs under differentiation lost the expression of CD133 and nestin. The percentage of GFAP positive cells and beta-tubulin III positive cells were (88.94 +/- 1.23)% and (11.94 +/- 0.36)% respectively. (3) All undifferentiated BTSCs were hypodiploid. After differentiation majority of the BTSCs were hypodiploid or hyperdiploid, The percentages of S phase and G(2)-M phase cells in the BTSCs were higher than that in the NSCs. The cell composition of recrudescent BTSCs was more complex than that of the primary BTSCs. All NSCs were diploid whether differentiated or not. Most of the NSCs were G(0)-G(1) phase cells. CONCLUSION: The differentiation direction of BTSCs is quietly different from that of the NSCs. There is an obvious dysdifferentiation in BTSCs.

Animals↗
PubMed2000

Differential expression of multiple unexpected genes during U937 cell and macrophage differentiation detected by suppressive subtractive hybridization.

OBJECTIVE: The objective of this study was to identify new markers of myelomonocytic differentiation using a sensitive technique that permits detection of rare differential gene expression. MATERIALS AND METHODS: [corrected] Suppressive subtractive hybridization (SSH) was performed between the human myelomonocytic U937 cell line and 1 alpha, 25-dihydroxyvitamin D3 and transforming growth factor beta 1 differentiated U937 cells. cDNA clones with significant increased expression in differentiated U937 cells over nondifferentiated U937 cells were characterized by sequencing. [corrected] The pattern of differential gene expression obtained by SSH was confirmed by cDNA Southern and Northern blots on the undifferentiated vs. differentiated U937 cells, and by reverse transcriptase polymerase chain reaction on undifferentiated human CD34(+) stem cells isolated from bone marrow vs. peripheral blood CD14(+) mature monocytes. RESULTS: Seven cDNAs never associated with in vitro U937 cell myelomonocytic differentiation (prolactin, 11-beta hydroxysteroid dehydrogenase [11 beta-HSD)] haptoglobin alpha (2FS)-beta precursor, GLIPR, RTVP, the RNA helicase P68, and spermidine-spermine N1-acetyltransferase) were identified. The first five of these genes previously were associated with immune function and the last two are important for intermediary metabolism. Differential expression was confirmed in CD34(+)/CD14(+) monocyte differentiation for all genes but 11 beta-HSD. CONCLUSIONS: We identified six new markers of U937 cell differentiation, which also are differentially expressed during normal human myelomonocytic differentiation.

Acetyltransferases↗
PubMed1998

Inhibition of erythroid differentiation and induction of megakaryocytic differentiation by thrombopoietin are regulated by two different mechanisms in TPO-dependent UT-7/c-mpl and TF-1/c-mpl cell lines.

Thrombopoietin (TPO) regulates megakaryocytic (MK) maturation and platelet production. Molecular and cellular mechanisms of the TPO-induced MK differentiation are not totally understood. In order to develop cellular models to study these mechanisms, we introduced c-mpl into UT-7 and TF-1 cells by means of a retroviral vector and compared the effects of TPO on these two cell lines. UT-7 and TF-1 cell lines are two factor-dependent leukemic cell lines with an erythroid and MK phenotype. They proliferate in response to IL-3, GM-CSF and EPO, but not to TPO. The erythroid differentiation of both cell lines can be markedly increased by EPO. Several UT-7/c-mpl and TF-1/c-mpl cell clones which express different levels of the c-mpl protein (Mpl) were obtained and all became TPO-dependent for their proliferation. The UT-7/c-mpl clones, but not the TF-1/c-mpl clones, were capable of undergoing MK differentiation in response to TPO. This was demonstrated by the increase in MK markers (GPIIb, GPIIIa, GPIb alpha, GPIX and vWF), the appearance of cytoplasmic alpha-granules, intracellular membranes resembling demarcation membranes which were immunologically labeled with an GPIIb/IIIa anti-antibody, and a small percentage of polyploid cells (8N and 16N). In contrast, TPO inhibited the erythroid program of differentiation (glycophorin A, beta-globin and EPO receptor) as well as the differentiative activity of EPO in both UT-7/c-mpl and TF-1/c-mpl clones. It is noteworthy that the differentiative effect of EPO in TF-1/c-mpl cells was associated with an increase in GATA-1 transcripts which was totally suppressed by TPO. Overall the effects of TPO are the same as those of phorbol myristate acetate (PMA) which also induces MK differentiation and inhibits erythroid differentiation. These results suggest that: (1) Mpl expression is necessary but not sufficient for induction of MK differentiation; and (2) induction of Mk differentiation and inhibition of erythroid differentiation by TPO involve different signaling pathways; the pathway involved in the inhibition of erythroid differentiation might be related to a downregulation of GATA-1 expression in TF-1 cells.

Anti-Bacterial Agents↗
PubMed2002

Primary acute myeloid leukaemia blasts resistant to cytokine-induced differentiation to dendritic-like leukaemia cells can be forced to differentiate by the addition of bryostatin-1.

Primary acute myeloid leukaemia (AML) blasts can be induced to differentiate into dendritic-like leukaemia cells (DLLC) by culture with certain cytokine combinations. DLLC offer potential for use as autologous vaccines based on their ability to present putative leukaemia-specific antigens to T cells. It has been reported, however, that in around 30-50% of AML cases the leukaemia cells are not capable of undergoing DLLC differentiation. The purpose of this study was to identify the features that represent successful DLLC differentiation and, for those cases shown to be resistant to cytokine-induced differentiation, to use differentiating agents in an attempt to overcome the differentiation block. Leukaemia cells derived from 42 patients with AML were cultured in vitro with cytokines GM-CSF, IL-4 and TNFalpha/CD40L. In 22 cases the leukaemic cells underwent DLLC differentiation based on characteristic morphological changes and expression of costimulatory and dendritic cell-associated molecules. Four cases were not evaluable because of poor viability over the culture period. The remaining 16 cases failed to show evidence of DLLC differentiation. Many of these differentiation resistant cases were associated with poor risk karyotypic features. Nine of the resistant cases were selected for further study. Differentiating agents trichostatin (TSA), azacytidine (AZA) and bryostatin (BRYO) were used in combination with cytokines for the first 96 h of the culture period. Bryostatin (BRYO) alone was shown to be capable of overcoming differentiation resistance and allowing DLLC differentiation to proceed.

Acute Disease↗
PubMed1998

Protein kinase C isoforms play differential roles in the regulation of adipocyte differentiation.

In this study we first established, by immunoblotting with specific antibodies, the temporal changes in cellular levels of protein kinase C (PKC) isoforms during differentiation of 3T3-F442A pre-adipocytes. Both pre-adipocyte and adipocyte 3T3-F442A cells were found to express PKC-alpha, -gamma, -delta, -epsilon, -zeta and -mu. However we were unable to detect PKC-beta, -eta or -theta. The same PKC isoform expression profile was found in rat adipocytes. The alpha, delta and gamma isoforms displayed similar temporal patterns of expression during differentiation of 3T3-F442A cells; all increased rapidly, peaking at day 2 of differentiation. Subsequently, the expression of these isoforms decreased, resulting in lower levels in fully differentiated adipocytes than in pre-adipocytes. The expression of PKC-epsilon increased steadily during differentiation, resulting in markedly elevated levels in adipocytes. Although expression of PKC-mu increased during differentiation, this was attributable to prolonged confluence rather than to the differentiation process itself. No change was observed in PKC-zeta levels during adipocyte development. Anti-sense oligodeoxynucleotides (ODNs) were used to deplete selectively the individual PKC subtypes. Each of the ODNs used effectively depleted the specific isoforms to undetectable levels and did not affect expression of the other PKC subtypes. This approach indicated that pre-adipocyte differentiation is not dependent upon PKC-zeta but that PKC-alpha,-delta and -mu each exert an inhibitory influence upon differentiation. Use of anti-sense ODNs to deplete PKC-epsilon and -gamma revealed that pre-adipocyte differentiation is dependent upon each of these isoforms. However, PKC-gamma, but not PKC-epsilon, appeared to be necessary for the clonal expansion of differentiating cells, suggesting that PKC-epsilon is required at a later phase in the differentiation process, when its expression is elevated, for the attainment and maintenance of the adipocyte phenotype.

3T3 Cells↗
PubMed1996

Multilineage differentiation of cloned HRA-19 cells in serum-free medium: a model of human colorectal epithelial differentiation.

Colorectal epithelium is composed of polarised absorptive enterocytes, mucus-producing goblet cells and enteroendocrine cells. All these cell lineages are thought to arise from multipotential stem cells located near the base of the crypt, but the mechanisms which control differentiation and commitment of cells to a particular lineage are poorly understood. We have used the human rectal adenocarcinoma cell line, HRA-19, to investigate the regulation of expression of lineage-specific markers. HRA-19 cells have multipotential characteristics, forming absorptive, mucous and endocrine cells when grown as xenografts. However, HRA-19 cells grown in vitro in culture medium containing 10% foetal calf serum show negligible expression of the differentiated phenotypes observed in vivo. These findings initially suggested that the absence of positive stimuli from extracellular matrix, stromal cells and/or soluble factors present in vivo resulted in the lack of differentiation in vitro. The subsequent demonstration of a marked inhibitory effect of foetal calf serum on differentiation provided an alternative explanation for the differences between in vivo and in vitro differentiation. In addition, the inhibition of differentiation differed widely between batches of foetal calf serum and limited the usefulness of the system for studying the regulation of differentiation. This manuscript describes the development of chemically defined culture conditions (Dulbecco's Eagles medium supplemented with insulin, transferrin and ascorbic acid) which reproducibly induced the multilineage differentiation of HRA-19 cells into absorptive, mucous and endocrine cells. Morphological characteristics and the expression of lineage-specific markers, as determined by immunocytochemistry, identified absorptive, goblet and endocrine cells in HRA-19 monolayers grown in this serum-free medium. Differentiation of cloned HRA-19 cells in to the three cell lineages proceeds in the absence of stromal cells and without exogenous extracellular matrix, although these factors may subsequently be shown to modulate the rate of cell differentiation. These chemically defined culture conditions will facilitate the study of differentiation in the HRA-19 cell line in the absence of the complex mixture of growth factors, hormones and differentiation inhibitory factor(s) present in foetal calf serum.

Cell Differentiation↗
PubMed2004

Dual-regulated expression of C/EBP-alpha and BMP-2 enables differential differentiation of C2C12 cells into adipocytes and osteoblasts.

CCAAT/enhancer-binding proteins (C/EBPs) as well as bone morphogenic proteins (BMPs) play essential roles in mammalian cell differentiation in shaping adipogenic and osteoblastic lineages in particular. Recent evidence suggested that adipocytes and osteoblasts share a common mesenchymal precursor cell phenotype. Yet, the molecular details underlying the decision of adipocyte versus osteoblast differentiation as well as the involvement of C/EBPs and BMPs remains elusive. We have engineered C2C12 cells for dual-regulated expression of human C/EBP-alpha and BMP-2 to enable independent transcription control of both differentiation factors using clinically licensed antibiotics of the streptogramin (pristinamycin) and tetracycline (tetracycline) classes. Differential as well as coordinated expression of C/EBP-alpha and BMP-2 revealed that (i) C/EBP-alpha may differentiate C2C12 myoblasts into adipocytes as well as osteoblasts, (ii) BMP-2 prevents myotube differentiation, (iii) is incompetent in differentiating C2C12 into osteoblasts and (iv) even decreases C/EBP-alpha's osteoblast-specific differentiation potential but (v) cooperates with C/EBP-alpha on adipocyte differentiation, (vi) osteoblast formation occurs at low C/EBP-alpha levels while adipocyte-specific differentiation requires maximum C/EBP-alpha expression and that (vii) BMP-2 may bias the C/EBP-alpha-mediated adipocyte versus osteoblast differentiation switch towards fat cell formation. Dual-regulated expression technology enabled precise insight into combinatorial effects of two key differentiation factors involved in adipocyte/osteoblast lineage control which could be implemented in rational reprogramming of multipotent cells into desired cell phenotypes tailored for gene therapy and tissue engineering.

Adipocytes↗
PubMed2004

Differential roles of insulin receptor substrates in brown adipocyte differentiation.

Insulin promotes adipocyte differentiation via a complex signaling network involving multiple insulin receptor substrates (IRSs). In cultured brown preadipocytes, expression of IRS-1 and IRS-2 mRNAs and proteins was at relatively high levels before and after differentiation into mature fat cells, while IRS-3 transcript was not detectable in preadipocytes but increased during the course of differentiation, and IRS-4 mRNA was barely detected in both states. To determine more precisely the roles of various IRS proteins in adipogenesis, we established and characterized brown preadipocyte cell lines from wild-type and IRS knockout (KO) animals. While wild-type, IRS-2 KO, and IRS-4 KO cells fully differentiated into mature adipocytes, IRS-3 KO cells showed a moderate defect in differentiation and IRS-1 KO cells exhibited a severe defect in the process. Cells lacking both IRS-1 and IRS-3 completely failed to differentiate. Expression of the adipogenic markers peroxisome proliferator-activated receptor gamma (PPARgamma), CCAAT/enhancer-binding protein alpha, fatty acid synthase, glucose transporter 4, and the transcription factor signal transducer and activator of transcription 5, as well as the brown-fat-specific markers PPARgamma coactivator 1 alpha and uncoupling protein 1, mirrored the differentiation pattern. Reconstitution of the IRS-1 KO cells with IRS-1 and IRS-4, but not IRS-2 or IRS-3, compensated for the lack of differentiation in IRS-1 KO cells. A chimeric molecule containing the N terminus of IRS-1 and the C terminus of IRS-2, but not one with the N terminus of IRS-2 and the C terminus of IRS-1, also rescued differentiation. Expression of Wnt 10a, a molecule known to inhibit adipogenesis, was dramatically increased in the IRS-1 KO cells, and this could be reduced by overexpression of IRS-1 or IRS-4, which was correlated with restoration of differentiation. These data indicate that both IRS-1 and -3 play important roles in the differentiation of brown adipocytes and that the N terminus of IRS-1 is more important for this function of the molecule. Although IRS-4 is not essential for the process, overexpression of IRS-4 can compensate for the deficiency in differentiation in IRS-1 KO cells.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Gene expression in TGFbeta-induced epithelial cell differentiation in a three-dimensional intestinal epithelial cell differentiation model.

BACKGROUND: The TGFbeta1-induced signal transduction processes involved in growth and differentiation are only partly known. The three-dimensional epithelial differentiation model, in which T84 epithelial cells are induced to differentiate either with TGFbeta1 or IMR-90 mesenchymal cell-secreted soluble factors, is previously shown to model epithelial cell differentiation seen in intestine. That model has not been used for large scale gene expression studies, such as microarray method. Therefore the gene expression changes were studied in undifferentiated and differentiated three-dimensional T84 cultures with cDNA microarray method in order to study the molecular changes and find new players in epithelial cell differentiation. RESULTS: The expression of 372 genes out of 5188 arrayed sequences was significantly altered, and 47 of them were altered by both mediators. The data were validated and the altered genes are presented in ontology classes. For the genes tested the expressions in protein level were in accordance with the mRNA results. We also found 194 genes with no known function to be potentially important in epithelial cell differentiation. The mRNA expression changes induced by TGFbeta1 were bigger than changes induced by soluble factors secreted by IMR-90 mesenchymal cells. The gene expression data was depicted in already known signaling pathway routes. CONCLUSION: Our results reveal potential new signaling pathways and several new genes affected by TGFbeta in epithelial cell differentiation. The differentiation induced by TGFbeta1 appears to be more potent than the differentiation induced by mesenchymal cells. This study indicates that our cell culture model is a suitable tool in studying regulatory mechanisms during epithelial cell differentiation in intestine. Furthermore the present results indicate that our model is a good tool for finding new players acting in the differentiation of epithelial cells.

Cell Culture Techniques↗
PubMed2000

Differential expressions of BMP family genes during chondrogenic differentiation of mouse ATDC5 cells.

Clonal cell line ATDC5 enables the monitoring of the early- and late-phase chondrogenic differentiation in a single culture. Undifferentiated ATDC5 cells differentiate into type II collagen expressing chondrocytes through a cellular condensation stage (early-phase differentiation) and then to type X collagen-expressing hypertrophic chondrocytes (late-phase differentiation). Progression of cellular differentiation was accelerated by the activation of bone morphogenetic protein (BMP) signaling. ATDC5 cells expressed transcripts for at least four members of the BMP family. The BMP-4 transcripts were expressed in all stages of differentiation, as were transcripts for BMP type IA receptor (ALK-3) and BMP type II receptor. In contrast, transcripts for Growth/ Differentiation factor-S (GDF-5) were induced during a cellular condensation, and those for BMP-6 were induced during the formation of cartilage nodules, and declined as the differentiated ATDC5 cells became hypertrophic, and BMP-7 transcripts were only detected after cells became calcified. Exogenously added BMP-4 indeed promoted the early-phase differentiation. Late-phase differentiation of cells was also stimulated by BMP-4 and BMP-6. Thus, the cumulative increase in BMP signaling promoted the sequential transitions of differentiation steps of cells. These results indicate that the coordinated expressions of endogenous BMPs are involved in the progression of chondrogenic differentiation in ATDC5 cells.

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