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Results for “clonal modeling”

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PubMed2003

Restricted clonal expression of IL-2 by naive T cells reflects differential dynamic interactions with dendritic cells.

Limited frequencies of T cells express IL-2 in primary antigenic responses, despite activation marker expression and proliferation by most clonal members. To define the basis for restricted IL-2 expression, a videomicroscopic system and IL-2 reporter transgenic model were used to characterize dendritic cell (DC)-T cell interactions. T cells destined to produce IL-2 required prolonged interactions with DCs, whereas most T cells established only transient interactions with DCs and were activated, but did not express IL-2. Extended conjugation of T cells with DCs was not always sufficient to initiate IL-2 expression. Thus, there is intrinsic variability in clonal T cell populations that restricts IL-2 commitment, and prolonged engagement with mature DCs is necessary, but not sufficient, for IL-2 gene transcription.

Animals↗

PubMed2003

High simian T-cell leukemia virus type 1 proviral loads combined with genetic stability as a result of cell-associated provirus replication in naturally infected, asymptomatic monkeys.

Simian T-cell leukemia virus type 1 (STLV-1) is a primate T cell leukemia virus of the group of oncogenic delta retroviruses. Sharing a high level of genetic homology with human T cell leukemia virus type 1 (HTLV-1), it is etiologically linked to the development of simian T cell malignancies that closely resemble HTLV-1 associated leukemias and lymphomas and might thus constitute an interesting model of study. The precise nature of STLV-1 replication in vivo remains unknown. The STLV-1 circulating proviral load of 14 naturally infected Celebes macaques (Macaca tonkeana) was measured by real-time quantitative PCR. The mean proportion of infected peripheral mononuclear cells was 7.9%, ranging from <0.4% to 38.9%. Values and distributions were closely reminiscent of those observed in symptomatic and asymptomatic HTLV-1 infected humans. Sequencing more than 32 kb of LTRs deriving from 2 animals with high proviral load showed an extremely low STLV-1 genetic variability (0.113%). This paradoxical combination of elevated proviral load and remarkable genetic stability was finally explained by the demonstration of a cell-associated dissemination of the virus in vivo. Inverse PCR (IPCR) amplification of STLV-1 integration sites evidenced clones of infected cells in all infected animals. The pattern of STLV-1 replication in these asymptomatic monkeys was indistinguishable from that of HTLV-1 in asymptomatic carriers or in patients with inflammatory diseases. We conclude that, as HTLV-1, STLV-1 mainly replicates by the clonal expansion of infected cells; accordingly, STLV-1 natural monkey infection constitutes an appropriate and promising model for the study of HTLV-1 associated leukemogenesis in vivo.

Animals↗

PubMed1991

Mutation of the p53 gene in human acute myelogenous leukemia.

Heterogeneity of p53 protein expression is seen in blast cells of patients with acute myelogenous leukemia (AML). p53 protein is detected in the blasts of certain AML patients but not in others. We have identified p53 protein variants with abnormal mobility on gel electrophoresis and/or prolonged half-life (t 1/2). We have sequenced the p53 coding sequence from primary blast cells of five AML patients and from the AML cell line (OCIM2). In OCIM2, a point mutation in codon 274 was identified that changes a valine residue to aspartic acid. A wild type p53 allele was not detected in these cells. Two point mutations (codon 135, cysteine to serine; codon 246, methionine to valine) were identified in cDNA from blasts of one AML patient. Both mutations were present in blast colonies grown from single blast progenitor cells, indicating that individual leukemia cells had sustained mutation of both p53 alleles. The cDNAs sequenced from blast samples of four other patients, including one with prolonged p53 protein t 1/2 and one with no detectable p53 protein, were fully wild type. Thus, the heterogeneity of p53 expression cannot be explained in all cases by genetic change in the p53 coding sequence. The prolonged t 1/2 of p53 protein seen in some AML blasts may therefore reflect changes not inherent to p53. A model is proposed in which mutational inactivation of p53, although not required for the evolution of neoplasia, would confer a selective advantage, favoring clonal outgrowth during disease progression.

Base Sequence↗

PubMed1983

Characterization of a cultured human T-cell line with genetically altered ribonucleotide reductase activity. Model for immunodeficiency.

From human CCRF-CEM T-cells growing in continuous culture, we have selected, isolated, and characterized a clonal cell line, APHID-D2, with altered ribonucleotide reductase activity. In comparative growth rate experiments, the APHID-D2 cell line is less sensitive than the parental cell line to growth inhibition by deoxyadenosine in the presence of 10 microM erythro-9-(2-hydroxy-3-nonyl)adenine, an inhibitor of adenosine deaminase. The APHID-D2 cell line has elevated levels of all four dNTPs. The resistance of the APHID-D2 cell line to growth inhibition by deoxyadenosine and the abnormal dNTP levels can be explained by the fact that the APHID-D2 ribonucleotide reductase, unlike the parental ribonucleotide reductase, is not normally sensitive to inhibition by dATP. These results suggest that the allosteric site of ribonucleotide reductase which binds both dATP and ATP is altered in the APHID-D2 line. The isolation of a mutant clone of human T-cells which contains a ribonucleotide reductase that has lost its normal sensitivity to dATP and which is resistant to deoxyadenosine-mediated growth inhibition suggests that a primary pathogenic target of accumulated dATP in lymphocytes from patients with adenosine deaminase deficiency may be the cellular ribonucleotide reductase.

Cell Division↗

PubMed1998

Development of a model for evaluating the interaction between human pre-B acute lymphoblastic leukemic cells and the bone marrow stromal cell microenvironment.

Clonal expansion of B-cell precursor acute lymphoblastic leukemia (ALL) is potentially regulated by survival, growth, and death signals transduced by the bone marrow (BM) microenvironment. Using a human BM stromal cell culture that supports the growth of normal human B-cell precursors, we established a pre-B ALL cell line designated BLIN-2. BLIN-2 has a clonal rearrangement of the Ig heavy chain locus, a dic(9;20) chromosomal abnormality, and a bi-allelic deletion of the p16(INK4a) and p19(ARF) genes. The most interesting feature of BLIN-2 is an absolute dependence on adherent human BM stromal cells for sustained survival and growth. BLIN-2 cultured in the absence of BM stromal cells undergo apoptosis, and direct contact with viable BM stromal cells is essential for optimal growth. BLIN-2 cells also grow on vascular cell adhesion molecule-1 (VCAM-1)-negative human skin fibroblasts, making it unlikely that a very late antigen-4 (VLA-4)/VCAM-1 interaction is required for BLIN-2 growth. Western blot analysis of BLIN-2 cells cultured in the presence or absence of BM stromal cells demonstrates that contact of BLIN-2 with BM stromal cells induces hyperphosphorylation of Rb. In contrast, the pre-B ALL cell line BLIN-1, which has a bi-allelic deletion of p16(INK4a) p19(ARF) but does not require BM stromal cells for growth, does not undergo Rb phosphorylation after BM stromal cell contact. The BLIN-2 cell line will facilitate identification of ligand/receptor interactions at the B-cell precursor/BM stromal cell interface and may provide new insight into microenvironmental regulation of leukemic cell survival and growth.

Apoptosis↗

PubMed2001

Identification of a new iron-regulated virulence gene, ireA, in an extraintestinal pathogenic isolate of Escherichia coli.

Our laboratory is studying an extraintestinal pathogenic isolate of Escherichia coli (CP9) as a model pathogen. We have been using human urine, ascites, and blood ex vivo to identify genes with increased expression in these media relative to expression in Luria-Bertani (LB) broth. Such genes may represent new or unrecognized virulence traits. In this study, we report the identification of a new gene, ireA (iron-responsive element). This gene has an open reading frame of 2,049 nucleotides, and its peptide has a molecular mass of 75.3 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its expression is increased a mean of 3.6-fold in human urine, 16.2-fold in human ascites, and 6.6-fold in human blood relative to expression in LB medium, and it is Fe repressible. IreA also exhibits peptide similarities (48 to 56%) to previously identified proteins that function as siderophore receptors, suggesting that IreA is involved in iron acquisition. PCR-based analysis of ireA's phylogenetic distribution detected ireA in none (0%) of 14 fecal isolates that represented probable commensal strains, but in 13 (26%) of 50 random urine and blood clinical isolates (P = 0.05) and in 5 (100%) of 5 representatives of the J96-like, clonal group of which CP9 is a member (P < 0.001). In a mouse urinary tract infection model, the presence of ireA contributed significantly to CP9's ability to colonize the bladder (P < 0.02), evidence that IreA is a urovirulence factor. Taken together, these findings demonstrate that ireA encodes a new virulence factor, which is likely involved in Fe acquisition.

Animals↗

PubMed2001

Development of allergic inflammation in a murine model of asthma is dependent on the costimulatory receptor OX40.

Asthma is thought to result from an abnormal expansion of CD4 T cells reactive with airborne allergens, and pathology is controlled by several cytokines of the T helper type 2 (Th2) family. The exact molecules which are involved in generating allergen-reactive T cells are not clear. Studies with blocking reagents or knockout animals have shown that the CD28/B7 interaction partially controls development of allergic asthma in mouse models, but may not be the sole molecule involved. In this report, we have investigated the role of the tumor necrosis factor receptor family member OX40 in allergic inflammation using OX40-deficient mice. OX40 has been shown to participate in regulating clonal expansion and memory development of CD4 T cells and may synergize with CD28. Our studies demonstrate that OX40(-/)- mice, primed with the model allergen ovalbumin and challenged through the airways with aerosolized antigen, are severely impaired in their ability to generate a Th2 response characterized by high levels of interleukin (IL)-5, IL-4, and immunoglobulin E. Moreover, OX40(-/)- mice exhibit diminished lung inflammation, including an 80-90% reduction in eosinophilia and mucus production, less goblet cell hyperplasia, and significantly attenuated airway hyperreactivity. These studies highlight the potential importance of OX40 in development of allergic asthma and suggest that targeting OX40 may prove useful therapeutically.

Allergens↗

PubMed1996

Tal-1 induces T cell acute lymphoblastic leukemia accelerated by casein kinase IIalpha.

Ectopic activation of the TAL-1 gene in T lymphocytes occurs in the majority of cases of human T cell acute lymphoblastic leukemia (T-ALL), yet experiments to date have failed to demonstrate a direct transforming capability for tal-1. The tal-1 gene product is a serine phosphoprotein and basic helix-loop-helix (bHLH) transcription factor known to regulate embryonic hematopoiesis. We have established a transgenic mouse model in which tal-1 mis-expression in the thymus results in the development of clonal T cell lymphoblastic leukemia/lymphoma. Thus, overexpression of tal-1 alone can be transforming, verifying its pathogenic role in human T-ALL. In addition, leukemogenesis is accelerated dramatically by transgenic co-expression of tal-1 and the catalytic subunit of casein kinase IIalpha (CKIIalpha), a serine/threonine protein kinase known to modulate the activity of other bHLH transcription factors. Although tal-1 is a substrate for CKII, the synergy of the tal-1 and CKIIalpha transgenes appears to be indirect, perhaps mediated through the E protein heterodimeric partners of tal-1. These studies prove that dysregulated tal-1 is oncogenic, providing a direct molecular explanation for the malignancies associated with TAL-1 activation in human T-ALL.

Animals↗

PubMed2005

The Caco-2 cell line as a model of the intestinal barrier: influence of cell and culture-related factors on Caco-2 cell functional characteristics.

The human intestinal Caco-2 cell line has been extensively used over the last twenty years as a model of the intestinal barrier. The parental cell line, originally obtained from a human colon adenocarcinoma, undergoes in culture a process of spontaneous differentiation that leads to the formation of a monolayer of cells, expressing several morphological and functional characteristics of the mature enterocyte. Culture-related conditions were shown to influence the expression of these characteristics, in part due to the intrinsic heterogeneity of the parental cell line, leading to selection of sub-populations of cells becoming prominent in the culture. In addition, several clonal cell lines have been isolated from the parental line, exhibiting in general a more homogeneous expression of differentiation traits, while not always expressing all characteristics of the parental line. Culture-related conditions, as well as the different Caco-2 cell lines utilized in different laboratories, often make it extremely difficult to compare results in the literature. This review is aimed at summarizing recent, or previously unreviewed, data from the literature on the effects of culture-related factors and the influence of line sub-types (parental vs. different clonal lines) on the expression of differentiation traits important for the use of Caco-2 cells as a model of the absorptive and defensive properties of the intestinal mucosa. Since the use of Caco-2 cells has grown exponentially in recent years, it is particularly important to highlight these methodological aspects in order to promote the standardization and optimisation of this intestinal model.

Autocrine Communication↗

PubMed2004

Glomerular T cells are of restricted clonality and express multiple CDR3 motifs across different Vbeta T-cell receptor families in experimental autoimmune glomerulonephritis.

Experimental autoimmune glomerulonephritis (EAG) is an animal model of Goodpasture's disease which can be induced in Wistar-Kyoto (WKY) rats by a single intramuscular injection of collagenase-digested rat glomerular basement membrane (GBM) in adjuvant. This model is characterised by anti-GBM antibody production, accompanied by focal necrotising glomerulonephritis with crescent formation and glomerular infiltration by T cells and macrophages. Previous work has shown that EAG is a T-cell-dependent disease. We proposed that intraglomerular T cells might be directly involved in pathogenesis and would be oligoclonal. In this study, EAG was induced by standard methods, the kidneys perfused with saline at week 2 and week 4, and the glomeruli separated by a sieving method. Glomerular RNA was extracted and reverse transcribed. RT-PCR showed overexpression of an average of two Vbeta families in each kidney analysed. However, no predominant single Vbeta family was overexpressed in any of the experimental animals. CDR3 spectratyping of Fam-labelled PCR products showed a marked restriction involving different Vbeta families. Sequencing demonstrated multiple CDR3 motifs, each expressed in association with different Vbeta gene segments. Our results show that glomerular T cells are of restricted clonality and suggest a role for antigen-specific effector T cells in the pathogenesis of EAG.

Animals↗

PubMed2006

A pseudolikelihood approach for simultaneous analysis of array comparative genomic hybridizations.

DNA sequence copy number has been shown to be associated with cancer development and progression. Array-based comparative genomic hybridization (aCGH) is a recent development that seeks to identify the copy number ratio at large numbers of markers across the genome. Due to experimental and biological variations across chromosomes and hybridizations, current methods are limited to analyses of single chromosomes. We propose a more powerful approach that borrows strength across chromosomes and hybridizations. We assume a Gaussian mixture model, with a hidden Markov dependence structure and with random effects to allow for intertumoral variation, as well as intratumoral clonal variation. For ease of computation, we base estimation on a pseudolikelihood function. The method produces quantitative assessments of the likelihood of genetic alterations at each clone, along with a graphical display for simple visual interpretation. We assess the characteristics of the method through simulation studies and analysis of a brain tumor aCGH data set. We show that the pseudolikelihood approach is superior to existing methods both in detecting small regions of copy number alteration and in accurately classifying regions of change when intratumoral clonal variation is present. Software for this approach is available at http://www.biostat.harvard.edu/ approximately betensky/papers.html.

Algorithms↗

PubMed2003

Gene expression profiling of ciliary neurotrophic factor-overexpressing rat striatal progenitor cells (ST14A) indicates improved stress response during the early stage of differentiation.

Neuronal progenitor cells delivering neurotrophic factors are a promising therapeutic tool for treatment of neurodegenerative diseases. Although several promising results have come from studies in different animal models, detailed knowledge of the action of neurotrophic factors in the CNS is still lacking. A clonally derived, immortalized rat striatal cell line (ST14A) expressing ciliary neurotrophic factor (CNTF) offers a stable and controlled background with which to analyze CNTF actions on the transcriptional level in CNS progenitor cells. To identify early transcriptional changes induced by CNTF expression, we transfected the CNTF gene into ST14A cells, which differentiate at the nonpermissive temperature of 39 degrees C via suppression of the immortalizing SV40 large T antigen. This shows a CNTF-dependent hypoxic/ischemic stress response during the earliest stage of differentiation, with expression of specific transcripts and evidence of translational repression leading to decreased protein synthesis in the transfected cells. This process is mediated by the Ras/MAP kinase pathway and is accompanied by impaired proliferation and metabolism as well as signs of neuronal differentiation. The stress-like response in the early stage of differentiation improves the ability of the transfected cells to respond to and cope with a stressful environment in vivo. The present data indicate higher viability, longer life, and greater differentiation capacity of CNTF-ST14A cells if they are used for transplantation. We conclude that the stress-like response during the early stage of differentiation improves the ability of the CNTF-ST14A cells to respond and adapt to a stressful environment, which renders them useful candidate cells for in vivo trials of treatment for neurodegenerative diseases in animal models, e.g., of Huntington's disease.

Animals↗

PubMed2002

Transduction of donor hematopoietic stem-progenitor cells with Fas ligand enhanced short-term engraftment in a murine model of allogeneic bone marrow transplantation.

Fas-mediated apoptosis is a major physiologic mechanism by which activated T cells are eliminated after antigen-stimulated clonal expansion generates a specific cellular immune response. Because activated T cells are the major effectors of allograft rejection, we hypothesized that genetically modifying allogeneic bone marrow (BM) cells prior to transplantation could provide some protection from host T-cell attack, thus enhancing donor cell engraftment in bone marrow transplantation (BMT). We undertook studies to determine the outcome of lentiviral vector-mediated transduction of Fas ligand (FasL) into lineage antigen-negative (lin(-)) mouse BM cells (lin(-) BMs), in an allogeneic BMT model. FasL-modified lin(-) BMs killed Fas-expressing T cells in vitro. Mice that received transplants of allogeneic FasL(+) lin(-) BMs had enhanced short-term engraftment, after nonmyeloablative conditioning, as compared to controls. We observed no major hepatic toxicity or hematopoietic or immune impairment in recipient mice at these time points. These results suggest potential therapeutic approaches by manipulating lymphohematopoietic stem-progenitor cells to express FasL or other immune-modulating genes in the context of BMT.

Animals↗

PubMed1998

Cerebral ischemia and CNS transplantation: differential effects of grafted fetal rat striatal cells and human neurons derived from a clonal cell line.

Stroke mortality has declined over recent decades, prompting a demand for the development of effective rehabilitative therapies for stroke survivors. This effort has been facilitated by significant progress in replicating the behavioral and neuropathological changes of authentic human cerebral ischemia using relevant animal models. Since the rodent model of middle cerebral artery occlusion mimics several motor abnormalities seen in clinical cerebral ischemia, we have utilized this model to investigate treatment strategies for stroke. The present study explored the potential benefits of neural transplantation of fetal rat striatal cells or human neurons derived from a clonal embryonal carcinoma cell line to correct the abnormalities associated with cerebral ischemia. We report here that ischemia-induced behavioral dysfunctions were ameliorated by the neural grafts as early as 1 month post-transplantation. Of note, transplantation of human neurons induced a significantly more robust recovery than fetal rat striatal grafts. Thus, the logistical and ethical concerns about the use of fetal striatal cells for transplantation therapy can be eliminated by exploiting cell line-derived human neurons as alternative graft sources. Transplantation of human neurons has a therapeutic potential for treatment of behavioral deficits associated with cerebral ischemia.

Animals↗

PubMed2001

Karyotypic similarity identified by multiplex-FISH relates four prostate adenocarcinoma cell lines: PC-3, PPC-1, ALVA-31, and ALVA-41.

Recently developed molecular cytogenetic techniques for karyotyping are providing new and important insights regarding the chromosomal changes that occur in solid tumors. We used multiplex-FISH to analyze four adenocarcinoma cell lines, PC-3, PPC-1, ALVA-31, and ALVA-41, in which the characterization of a large number of rearranged chromosomes was partially or substantially inconclusive by G-banding. Although the original descriptions of these lines depict them as distinct entities established from different patients, this study demonstrates that these four lines share numerous, highly rearranged chromosomes, strongly supporting the conclusion that they are derived from the same patient material. Our analysis indicates that PPC-1, ALVA-31, and ALVA-41 were derived from PC-3 through mechanisms involving clonal progression represented by sequential changes and clonal diversion represented by differing patterns of changes. Extensive cellular heterogeneity was detected in all four lines, and most rearrangements included segments derived from multiple chromosomes. Each line also showed a set of unique derivative chromosomes. However, a limited number of metaphase cells (approximately 10) was analyzed for each line, and numerous single-cell abnormalities were detected in all of them. Therefore, it is plausible that the number of clonal, shared, and/or unique rearrangements has been underestimated. These cell lines have been utilized as models for understanding the biology of prostate cancer and reportedly differ in their cell physiology. Rather than detracting from their value, a complete understanding of the interrelationships of these lines to one another may provide the opportunity to define the molecular changes that have led to their individual malignant phenotypes.

Adenocarcinoma↗

PubMed1998

Use of a reverse transcriptase-polymerase chain reaction assay to analyze allele-specific expression in individual hippocampal neurons.

We report here a single-cell RT-PCR assay for allele-specific gene expression that can be used to probe for somatic variability within the CNS. Such variability, arising from epigenetic (nonmutational) events or somatic mutation early in development, may give clues as to clonal origin and may also affect the inheritance pattern of some CNS disorders. As a model system, we used reciprocal F1 hybrids of the cross Mus musculus C57BL/6J x Mus musculus castaneus. RNA was isolated from individual dissociated pyramidal neurons from hippocampi of F1 pups. For each gene of interest, single base polymorphisms were identified between the two parental strains by automated sequencing of RT-PCR products. Allele-specific expression was then analyzed by means of the previously described quantitative RT-PCR single nucleotide primer extension (SNuPE) assay (Singer-Sam et al., PCR Methods Appl. 1:160-163, 1992). Individual neurons showed monoallelic expression of the two control genes, X-linked Rps4, and the imprinted gene Snrpn; in contrast expression of Ncam and F3cam, coding for neural cell adhesion molecules, was found to be biallelic.

Alleles↗

PubMed1992

X-linked glucose-6-phosphate dehydrogenase (G6PD) and autosomal 6-phosphogluconate dehydrogenase (6PGD) polymorphisms in baboons.

Electrophoretic polymorphisms of glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD) were examined in captive colonies of five subspecies of baboons (Papio hamadryas). Phenotype frequencies and family data verified the X-linked inheritance of the G6PD polymorphism. Insufficient family data were available to confirm autosomal inheritance of the 6PGD polymorphism, but the electrophoretic patterns of variant types (putative heterozygotes) suggested the codominant expression of alleles at an autosomal locus. Implications of the G6PD polymorphism are discussed with regard to its utility as a marker system for research on X-chromosome inactivation during baboon development and for studies of clonal cell proliferation and/or cell selection during the development of atherosclerotic lesions in the baboon model.

Alleles↗

PubMed2002

The mutator phenotype theory can explain the complex morphology and behaviour of cancers.

Almost all solid malignancies exhibit complex cytological and architectural abnormalities, which vary from cell to cell and area to area within the same tumour, and between tumours of the same type. The degrees of these abnormalities do not correlate perfectly with the biological behaviour (especially growth rate and metastatic potential) among the various tumour types. These features of tumours have long been considered to invalidate simple mutational or 'abnormal gene expression' (epigenetic) theories of carcinogenesis. The 'mutator phenotype/clonal selection' hypothesis is based on the now well-established phenomenon of genetic instability of cancer cells, and proposes that this instability is an essential requirement for the development of tumours, and not an irrelevant side-effect of some other process. This paper argues that this hypothesis can provide a satisfactory explanation for the diverse histological and biological features of solid malignancies. Further, because virtually all solid tumours are histologically abnormal, genetic instability is likely to be essential for the malignant process. The concepts of mutator phenotype and clonal selection are therefore supported.

Animals↗