B cell differentiation: B cell subsets and factors, organs or structures involved in B lymphocyte genesis.
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Human CD34+ fetal liver stem cells differentiate to T cells in a mouse thymic microenvironment.
Hematopoietic stem cells differentiate in the thymus to T cells along precisely defined intermediates. This process is thymic epithelium dependent and involves cytokines and cell-cell interactions between thymic stroma and T-cell precursors. Here we report that highly purified human CD34++ fetal liver stem cells differentiate to mature T cells, when seeded into isolated fetal thymic lobes of severe combined immunodeficient mice, and subsequently cultured in vitro. The human stem cells differentiate sequentially into CD4+CD8-CD3-, CD4+CD8+CD3-, CD4+CD8+CD3+, and finally, CD4+CD8-CD3+4 and CD4-CD8+CD3++ cells. Phenotypic analysis for additional maturation markers showed that these CD4 and CD8 single-positive thymocytes are fully maturate cells. By immunochemistry, human HLA-DR+ cells with a dendritic morphology could be detected. This novel chimeric human-mouse fetal thymus organ culture offers a tool to study human T-cell ontogeny in vitro and is a rapid and reliable test method for T-cell precursor activity of cultured or transfected human stem cells.
Animals↗
Proliferating cells versus differentiated cells in tissue engineering.
The efficiency of cell or tissue cultures is usually judged by how quickly confluence is reached within a Petri dish or on a scaffold. Growth factors and fetal bovine serum are employed to drive cultured cells from one mitosis to the next as quickly as possible. The tissue specific interphase is extremely short under these conditions, so that the degree of differentiation desired in tissue engineering cannot be achieved. To reach the goal of functional differentiation in vitro mitosis and interphase must be separated experimentally and tailored to the specific requirements of the cell-type used. This could be achieved by a three step concept for tissue-engineering in vitro as we present here. The expansion phase is followed by a phase in which tissue differentiation is initiated. The final phase serves to express and maintain histotypical differentiation of the generated tissue.
Animals↗
"The critical role of the nucleolus in cell differentiation and stem cell development - the concept as it applies to the malignant cell.".
Whereas growth prior to organ formation in the embryo is by replication of primitive cells, growth subsequent to organ formation is assumed to be due to replication of differentiated cells. The replication of both primitive cells and differentiated cells is said to conform to the classical mitotic cycle. But the cycle does not show how differentiation takes place, and to remedy this an alternative cycle incorporating clonal division is postulated. In order to explain malignant growth on the basis of the mitotic cycle, traditionalists have had to introduce the concept of dedifferentiation but the concept does not quite stand up to a critical analysis. Malignant change appears to be due to a nucleolar fault rather than a chromosomal one, and the behaviour of the malignant cell in adenocarcinoma of the lung confirms this. Smears made from the tumour mass indicate that the malignant stem cells are transformed into "specialised cells" by the same process of clonal division as already postulated for normal tissue growth. The presence of differentiating cells in adenocarcinoma greatly lessens the likelihood of dedifferentiation playing a part in malignancy. On the other hand, the presence of abnormal stem cells with bizarre nucleoli strengthens the case for malignancy being due primarily to a nucleolar fault.
Adenocarcinoma↗
Adhesive properties of human basal epidermal cells: an analysis of keratinocyte stem cells, transit amplifying cells, and postmitotic differentiating cells.
The basal layer of human epidermis is a heterogeneous population of proliferative and differentiating cells that can be divided into at least three functionally discrete compartments: keratinocyte stem cells, transit amplifying cells, and postmitotic differentiating cells. Basal cells adhere to the underlying basement membrane via integrins, and although decreased adhesion is a key event in epidermal differentiation, the specific role of particular integrins is poorly understood. We report here on the comparative expression and function of the beta1 versus alpha6beta4 integrins in keratinocyte stem cells, transit amplifying cells, and postmitotic differentiating cells of neonatal human foreskin epidermis. Adhesion assays demonstrate that both keratinocyte stem cells and transit amplifying cells comprise rapidly adhering cells that exhibit high levels of functional beta1 and alpha6beta4 integrins. Interestingly, a proportion of basal cells that have begun to differentiate in vivo within the basal layer as determined by their expression of the differentiation-specific markers K10 and involucrin also retain high levels of activated beta1 integrin, but downregulate alpha6beta4 expression selectively (termed alpha6dimbeta1bri). These cells also retain their adhesive capacity, indicating that induction of differentiation in vivo does not correlate with decreased beta1 integrin expression or function. We have previously reported on the use of alpha6 integrin in conjunction with a proliferation associated marker (10G7 ag) to separate keratinocyte stem cells (phenotype alpha6bri10G7dim) from other basal cells (Li et al. Proc Natl Acad Sci 95:3902-3907 1998). A comparison of the long-term proliferative potential of beta1bri10G7dim cells with alpha6bri10G7dim showed that selection of alpha6bri10G7dim allows the isolation of a purer fraction of keratinocyte stem cells.
Antigens, CD↗
Cell differentiation: reciprocal regulation of Apaf-1 and the inhibitor of apoptosis proteins.
The molecular mechanisms by which differentiated cells combat cell death and injury have remained unclear. In the current issue, it has been shown in neurons that cell differentiation is accompanied by a decrease in Apaf-1 and the activity of the apoptosome with an increased ability of the inhibitor of apoptosis proteins (IAPs) to sustain survival (Wright et al., 2004). These results, together with earlier ones, deepen our understanding of how cell death and the apoptosome are regulated during differentiation and in tumor cells.
Animals↗
DNA-protein interaction sites in differentiating cells. I. Two-dimensional mapping of modulated sites.
We have previously shown that DNA-protein attachment sites form during the induction of hematopoietic cell differentiation. Affinity phase-partitioning studies of DNA/protein complexes demonstrated that the DNA involved is not randomly distributed throughout the genome. The object of this study was to use filter binding followed by two-dimensional (2D) polyacrylamide gel electrophoresis (using a neutral 6% gel in the first dimension and a denaturing gradient gel in the second dimension) to gain insight into changes in DNA-protein interactions during induced granulocytic and monocytic differentiation of HL60 cells. Nitrocellulose filter-binding enriched samples for protein-associated DNA sufficiently to change the pattern of DNA spots on 2D gels. The patterns of spots obtained was reasonably reproducible between experiments and highly reproducible within experiments. Gels obtained from cells induced to differentiate by either phorbol ester or all-trans retinoic acid (RA) showed identical patterns for the majority of spots but changes in a small proportion of spots with respect to uninduced controls. Both intensification and reduction/disappearance of spots was observed, demonstrating the existence of both invariant and variant DNA/protein attachment sites during the early stages of hematopoietic cell differentiation. Previous studies have implicated DNA topoisomerase II in chromatin structural changes that are necessary for induction of granulocytic differentiation. We therefore examined the filter-binding DNA preparation by 5'-exonuclease digestion (since topoisomerase II is known to bind covalently to the 5'termini on either side of its cleavage sites). The filter-associated DNA exhibited increased 5' exonuclease protection (with respect to filter flow-through DNA), and the degree of protection increased significantly with exposure to phorbol ester and less markedly with retinoic acid. However, since not all filter DNA was 5' protected, it remains unresolved whether the specific differentiation-associated DNA-protein interactions revealed here involve DNA topoisomerase II or some other protein.
Binding Sites↗
Nonterminally differentiated cells express decreased growth factor responsiveness.
In 3T3 T mesenchymal stem cells, at least four types of biological states exist that can mediate the control of cell differentiation and/or proliferation. These include the predifferentiation growth arrest state, the nonterminal differentiation state, the terminal differentiation state, and a growth arrest state induced by growth factor/serum deficiency. The current studies were performed to investigate the relative mitogenic responsiveness of cells at these four states and specifically to determine if nonterminally differentiated cells show decreased responsiveness to specific mitogens. Twenty-five different serum, plasma, and growth factor combinations were evaluated. The results show that undifferentiated, growth-arrested cells are highly responsive to numerous mitogens and that by definition terminally differentiated cells are not responsive to any mitogens. In contrast, nonterminally differentiated cells demonstrate a unique pattern of mitogenic responsiveness. Whereas nonterminally differentiated cells can be stimulated to proliferate by high concentrations of serum or plasma supplemented with growth factors, they cannot be stimulated to proliferate by combinations of multiple purified growth factors. These results suggest that the process of nonterminal differentiation is associated with a significant change in factors/cofactors required to stimulate cell proliferation and that these factors/cofactors are present in plasma.
Animals↗
Expression patterns of TEL genes in Poaceae suggest a conserved association with cell differentiation.
Poaceae species present a conserved distichous phyllotaxy (leaf position along the stem) and share common properties with respect to leaf initiation. The goal of this work was to determine if these common traits imply common genes. Therefore, homologues of the maize TERMINAL EAR1 gene in Poaceae were studied. This gene encodes an RNA-binding motif (RRM) protein, that is suggested to regulate leaf initiation. Using degenerate primers, one unique tel (terminal ear1-like) gene from seven Poaceae members, covering almost all the phylogenetic tree of the family, was identified by PCR. These genes present a very high degree of similarity, a much conserved exon-intron structure, and the three RRMs and TEL characteristic motifs. The evolution of tel sequences in Poaceae strongly correlates with the known phylogenetic tree of this family. RT-PCR gene expression analyses show conserved tel expression in the shoot apex in all species, suggesting functional orthology between these genes. In addition, in situ hybridization experiments with specific antisense probes show tel transcript accumulation in all differentiating cells of the leaf, from the recruitment of leaf founder cells to leaf margins cells. Tel expression is not restricted to initiating leaves as it is also found in pro-vascular tissues, root meristems, and immature inflorescences. Therefore, these results suggest that TEL is not only associated with leaf initiation but more generally with cell differentiation in Poaceae.
Amino Acid Sequence↗
Ketogenic HMGCS2 Is a c-Myc target gene expressed in differentiated cells of human colonic epithelium and down-regulated in colon cancer.
HMGCS2, the gene that regulates ketone body production, is expressed in liver and several extrahepatic tissues, such as the colon. In CaCo-2 colonic epithelial cells, the expression of this gene increases with cell differentiation. Accordingly, immunohistochemistry with specific antibodies shows that HMGCS2 is expressed mainly in differentiated cells of human colonic epithelium. Here, we used a chromatin immunoprecipitation assay to study the molecular mechanism responsible for this expression pattern. The assay revealed that HMGCS2 is a direct target of c-Myc, which represses HMGCS2 transcriptional activity. c-Myc transrepression is mediated by blockade of the transactivating activity of Miz-1, which occurs mainly through a Sp1-binding site in the proximal promoter of the gene. Accordingly, the expression of human HMGCS2 is down-regulated in 90% of Myc-dependent colon and rectum tumors. HMGCS2 protein expression is down-regulated preferentially in moderately and poorly differentiated carcinomas. In addition, it is also down-regulated in 80% of small intestine Myc-independent tumors. Based on these findings, we propose that ketogenesis is an undesirable metabolic characteristic of the proliferating cell, which is down-regulated through c-Myc-mediated repression of the key metabolic gene HMGCS2.
Acetylation↗
Specific alterations in the pattern of histone-3 synthesis during conversion of human leukemic cells to terminally differentiated cells in culture.
The presence of nano- to micromolar concentrations of 12-0-tetradecanoyl-phorbol-13-acetate (TPA) in suspension cultures of human promyelocytic leukemia cells, HL-60, or human monocytic leukemia cells, THP-1, resulted in the appearance of macrophage-like cells attached to the substratum. The terminally TPA-differentiated cells continued to synthesize histones at a low rate even though DNA replication had ceased. The pattern of synthesis of histone variants in differentiated cells differed from that in undifferentiated cells and resembled that of quiescent or density-arrested cells. In undifferentiated cells, all three histone-H3 variants are synthesized, while in quiescent cells, only the H3.3 variant is synthesized. When TPA-differentiated macrophages were placed in normal medium, the pattern of histone synthesis was not altered, thus substantiating previous findings that the differentiation is irreversible. Further, TPA-differentiated macrophages and macrophages isolated from a normal human donor exhibited identical pattern of histone synthesis. Altogether, the results indicate that changes in the synthetic rates of histones during the TPA-induced maturation of human leukemic cells is not directly due to TPA or terminal cell differentiation per se but is due to the cessation of cell proliferation and DNA replication.
Acetamides↗
Augmentation of interferon production after cell-differentiation of U937 cells by TPA.
Persistent infections with mumps virus were established in several human lymphoid cells of T-cell origin (Molt-4, TALL-1, and CCRF-CEM) and human monocyte cells (U937 and THP-1). 2',5'-Oligoadenylate synthetase (2-5AS) activity was demonstrated to be only slightly induced by interferon (IFN) or TPA (12-O-tetradecanoyl-phorbol-13-acetate) treatment in these cells. Treatment of the persistently infected cells with IFN or TPA did not stimulate an increase in the amount of synthetase mRNA. Induction of cell differentiation and augmentation of IFN production by TPA were demonstrated in U937 cells persistently infected with mumps virus (U937-MP). Similar results for IFN production were obtained from differentiated U937 cells. It is suggested that cell differentiation of U937 cells might be associated with the development of IFN inducibility.
2',5'-Oligoadenylate Synthetase↗
Higher levels of organization in the interphase nucleus of cycling and differentiated cells.
The review examines the structured organization of interphase nuclei using a range of examples from the plants, animals, and fungi. Nuclear organization is shown to be an important phenomenon in cell differentiation and development. The review commences by examining nuclei in dividing cells and shows that the organization patterns can be dynamic within the time frame of the cell cycle. When cells stop dividing, derived differentiated cells often show quite different nuclear organizations. The developmental fate of nuclei is divided into three categories. (i) The first includes nuclei that undergo one of several forms of polyploidy and can themselves change in structure during the course of development. Possible function roles of polyploidy is given. (ii) The second is nuclear reorganization without polyploidy, where nuclei reorganize their structure to form novel arrangements of proteins and chromosomes. (iii) The third is nuclear disintegration linked to programmed cell death. The role of the nucleus in this process is described. The review demonstrates that recent methods to probe nuclei for nucleic acids and proteins, as well as to examine their intranuclear distribution in vivo, has revealed much about nuclear structure. It is clear that nuclear organization can influence or be influenced by cell activity and development. However, the full functional role of many of the observed phenomena has still to be fully realized.
Animals↗
The intranuclear amount of phospholipase C beta 1 decreases following cell differentiation in Friend cells, whereas gamma 1 isoform is not affected.
The existence of a signal transduction system in the nucleus, based on polyphosphoinositide breakdown mediated by specific phosphoinositidases (PLC), has been widely documented. In different cell systems, nuclear PLCs can be modulated, in response to agonists, either by enhancing or by down-regulating their activity, thus leading to DNA replication or to cell differentiation. Friend cells, induced to erythroid differentiation by dimethyl sulfoxide (DMSO), show a down-regulation of PLC beta 1 isoform, as indicated by the reduction of the transcription of its mRNA and of the in vitro synthesis of its translation product. The intracellular localization and the amount of different PLC isoforms have been evaluated by electron microscope immunocytochemistry. In untreated Friend cells, PLC beta 1 and gamma 1 isoforms are both present within the nucleus, whereas mainly the gamma 1 isoform is detected in the cytoplasm. The small amount of cytoplasmic PLC beta 1 is probably representative only of the newly synthesized enzyme. Quantitative immunolabeling analyses demonstrate that erythroid differentiation is associated with a significant decrease of the PLC beta 1 amount in the nucleus and with an almost complete disappearance of that isoform in the cytoplasm, whereas the PLC gamma 1 isoform is unaffected. The two PLC isoforms, moreover, appear to be differently associated with the nuclear components, PLC beta 1 being steadily bound to the inner nuclear matrix, whereas PLC gamma 1 is almost completely soluble.
Animals↗
Lymphokine-induction of memory B-cell differentiation: differential stimulation of large virgin and memory B-cell differentiation.
In order to compare and contrast the requirements of virgin and memory B cells for B-cell differentiation factors, a model system was developed in which low-density rat B cells isolated from 4-week primed antigen-draining lymph nodes were cultured in vitro. This large low-density cell population contained B cells which were 90% surface IgM positive and 60% IgD positive and showed moderately elevated Ia staining. When the cell population was stimulated with antigen plus lymphokines or lymphokines alone, antigen-specific IgG antibody was secreted; this was used as a measure of memory cell differentiation. When the cell population was stimulated with mitogen (lipopolysaccharide plus dextran sulfate) plus lymphokines, polyclonal IgG and IgM secretion was seen and was used as a measure of virgin B-cell differentiation. Using this system, we found that lymphokines contained in a Con A-induced rat spleen cell supernatant (CSN) were sufficient to drive both memory and virgin B-cell differentiation. In contrast, lymphokines contained in the supernatant from the murine T-cell hybridoma B151K12 (B151CFS) were able to induce large amounts of polyclonal IgM and IgG secretion but did not support memory B-cell differentiation. When recombinant human IL-2 was added to these cultures, it acted synergistically to augment virgin B-cell differentiation, but this combination of lymphokines was still not able to support memory B-cell differentiation. Furthermore, recombinant rat interferon-gamma and a commercial source of human BCGF, with or without IL-2, were unable to promote significant virgin or memory B-cell differentiation. These data support the hypothesis that memory B cells and virgin B cells differ in their lymphokine requirements for differentiation into antibody-secreting cells.
Animals↗
Cell differentiation and cell-cycle alterations by tyrosine kinase inhibitors in human melanoma cells.
Differentiation therapy is an attractive option for malignant melanoma, as traditional forms of chemotherapy seem to have little effect on this type of tumour. Among the several pathways for the experimental induction of differentiation of melanoma, we have focused on signal transduction mediated by protein kinases. We have examined the effects of calphostin C (a protein kinase C inhibitor), genistein and methyl 2,5-dihydroxycinnamate (tyrosine kinase inhibitors), and exogenous phosphotyrosine (an activator of protein tyrosine phosphatases) on the growth, morphology and differentiation of malignant melanomas in vitro. All four compounds tested were able to inhibit cell proliferation, but only genistein and methyl 2,5-dihydroxycinnamate were able to induce morphological changes, yielding a more dendritic or a rounder phenotype, respectively. The latter two drugs were also able to induce specific cell-cycle alterations, in contrast to calphostin C and phosphotyrosine. Melanin content was increased greatly in phophotyrosine treated cells and, to a smaller extent, in cells treated with genistein. RNA expression of specific genes encoding cytolytic T-cell antigens was not altered by the two tyrosine kinase inhibitors, in spite of the phenotypic changes observed. Together, these results suggest that tyrosine kinases are involved in cell cycle, growth, and differentiation pathways in malignant melanomas; however, these pathways may not be co-dependent. The results also suggest that these pathways may be sensitive to specific tyrosine kinase inhibitors or activators of protein tyrosine phosphatases.
Antigens, Neoplasm↗
Chicken calmodulin promoter activity in proliferating and differentiated cells.
A 1218-base pair (bp) portion of the chicken calmodulin promoter was sequenced and assayed for promoter activity. This portion of the promoter was found sufficient to produce accurate transcriptional initiation. The promoter sequence was GC rich, particularly in the 700 bp region 5' to the cap site. Eight plasmids were prepared containing the first calmodulin exon and 30-1218 bp of the promoter, ligated to the reporter gene chloramphenicol acetyl transferase. In chicken embryonic fibroblasts and proliferating BC3H-1 cells promoter activity increased progressively with increasing promoter length up to 617 bp. Extension of the promoter beyond 617 bp inhibited expression, as did sequences within the first calmodulin exon. In BC3H-1 cells differentiation was found to reduce calmodulin mRNA levels approximately 3-fold. Activity of the calmodulin promoter constructs also decreased by a similar extent with differentiation. Sequences up to 234 bp 5' to the calmodulin cap site were markedly less effective in elevating chloramphenicol acetyl transferase activity in differentiated BC3H-1 cells than in proliferating cells and may account for the lower overall activity of the calmodulin promoter in these cells. Within this region several sequences were identified, including an extensive homology to the rat calmodulin I gene promoter that could be significant in regulation of calmodulin expression.
Animals↗
Alternative modes of c-myc regulation in growth factor-stimulated and differentiating cells.
We have analysed the regulation of c-myc expression in murine fibroblasts and F9 teratocarcinoma cells. The initiation of c-myc transcription is induced to similar levels after serum stimulation of confluent and subconfluent Balb/c A31 fibroblasts while intragenic pausing within the gene's first exon remains unaffected. Sense c-myc transcription continues unabated for at least 18 hours in subconfluent cells, whereas in confluent cells it rapidly falls to pre-induced levels. Cytoplasmic c-myc mRNAs accumulate within 1-2 hours of serum addition to subconfluent cells and reach a higher level than expected from the degree of induction of sense transcription. However, c-myc mRNA levels fall close to pre-induced levels by 18 hours demonstrating that c-myc expression is initially subject to strong positive and then eventually strong negative post-transcriptional control. Anti-sense transcription within the c-myc locus was found to be constitutive under all these physiological states, thereby demonstrating that c-myc transcriptional control is strand specific. Epidermal growth factor stimulates c-myc transcription in a way different from that of serum: (1) initiation of transcription is not significantly enhanced, but intragenic pausing is significantly abrogated; and (2) post-transcriptional mechanisms do not enhance the degree of c-myc mRNA accumulation. In contrast to our results in fibroblastic cells, differentiating F9 teratocarcinoma cells down-regulate c-myc expression entirely at the post-transcriptional level. Our findings indicate that different cell types preferentially employ different modes of myc control depending on their physiological status.
Animals↗