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Results for “clonal modeling”

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PubMed1999

Neuroprotection and neuronal differentiation studies using substantia nigra dopaminergic cells derived from transgenic mouse embryos.

The major pathological lesion of Parkinson's disease (PD) is the selective cell death of dopaminergic (DA) neurons in substantia nigra (SN). Although the initial cause and subsequent molecular signaling mechanisms leading to DA cell death underlying the PD process remain elusive, brain-derived neurotrophic factor (BDNF) is thought to exert neuroprotective as well as neurotrophic roles for the survival and differentiation of DA neurons in SN. Addressing molecular mechanisms of BDNF action in both primary embryonic mesencephalic cultures and in vivo animal models has been technically difficult because DA neurons in SN are relatively rare and present with many heterogeneous cell populations in midbrain. We have developed and characterized a DA neuronal cell line of embryonic SN origin that is more accessible to molecular analysis and can be used as an in vitro model system for studying SN DA neurons. A clonal SN DA neuronal progenitor cell line SN4741, arrested at an early DA developmental stage, was established from transgenic mouse embryos containing the targeted expression of the thermolabile SV40Tag in SN DA neurons. The phenotypic and morphological differentiation of the SN4741 cells could be manipulated by environmental cues in vitro. Exogenous BDNF treatment produced significant neuroprotection against 1-methyl-4-phenylpyridinium, glutamate, and nitric oxide-induced neurotoxicity in the SN4741 cells. Simultaneous phosphorylation of receptor tyrosine kinase B accompanied the neuroprotection. This SN DA neuronal cell line provides a unique model system to circumvent the limitations associated with primary mesencephalic cultures for the elucidation of molecular mechanisms of BDNF action on DA neurons of the SN.

1-Methyl-4-phenylpyridinium↗

PubMed2003

The population genetics of clonal and partially clonal diploids.

The consequences of variable rates of clonal reproduction on the population genetics of neutral markers are explored in diploid organisms within a subdivided population (island model). We use both analytical and stochastic simulation approaches. High rates of clonal reproduction will positively affect heterozygosity. As a consequence, nearly twice as many alleles per locus can be maintained and population differentiation estimated as F(ST) value is strongly decreased in purely clonal populations as compared to purely sexual ones. With increasing clonal reproduction, effective population size first slowly increases and then points toward extreme values when the reproductive system tends toward strict clonality. This reflects the fact that polymorphism is protected within individuals due to fixed heterozygosity. Contrarily, genotypic diversity smoothly decreases with increasing rates of clonal reproduction. Asexual populations thus maintain higher genetic diversity at each single locus but a lower number of different genotypes. Mixed clonal/sexual reproduction is nearly indistinguishable from strict sexual reproduction as long as the proportion of clonal reproduction is not strongly predominant for all quantities investigated, except for genotypic diversities (both at individual loci and over multiple loci).

Alleles↗

PubMed1991

Retinoic acid modulation of mRNA levels in malignant, nontransformed, and immortalized osteoblasts.

Clonal cell lines presumably "arrested" at a particular stage of differentiation are useful models to study the processes of differentiation in osteoblasts. UMR-201 is a presumptive preosteoblastic nontransformed rat clonal cell line with a limited life span in culture. Two immortalized cell lines, UMR-201-10A (10A) and UMR-201-10B (10B), were derived from UMR-201 by stable transfection with simian virus (SV) 40 large T antigen. This study compares the growth and profile of gene expression of the immortalized cell lines with those of UMR-201 and UMR-106-06, a rat clonal cell line with well-defined osteoblast-like phenotypic characteristics. All four cell lines constitutively expressed the mRNA for the gamma, alpha, and beta receptors for retinoic acid (RA), the growth hormone receptor, pro-alpha 1(I) collagen, osteonectin, bone proteoglycan I, and bone morphogenetic proteins (BMP) 1 and 2A. Alkaline phosphatase mRNA was absent in the preosteoblast cell lines but was induced by treatment with 10(-6) M RA, which also increased the steady-state levels of mRNA for osteopontin and BMP1. mRNA for matrix gla protein was constitutively present and further induced by RA in UMR-201 and 10B only. Messenger RNA for bone sialoprotein and bone morphogenetic protein 3 were constitutively expressed in UMR-106-06 and UMR-201 but absent in the immortalized cell lines. None of the cell lines expressed measurable mRNA for bone gla protein or bone proteoglycan II. 10B grew more rapidly than UMR-201, but unlike UMR-201, it was also able to proliferate in serum-free medium and exhibit anchorage-independent growth. In summary, this study identifies novel retinoic acid effects on gene expression in these cells. Differences noted in the expression of mRNAs between UMR-106-06 and the other cell lines may provide some insight into the sequence of expression of these phenotypic characteristics as osteoblasts differentiate.

Alkaline Phosphatase↗

PubMedDate not supplied

Stem cell kinetics and intestinal cancer.

The conventional view that intestinal stem cells divide every few days so that each stem cell undergoes several thousand divisions in a lifetime is challenged. It is proposed that fully differentiated cells, even in old age, are no more than sixty divisions from the zygote. This concept leads to a model in which the intestinal crypts are maintained by a linear hierarchy of stem cells. The model is consistent with experimental data on the clonal origin of intestinal crypts and the observed crypt mutant frequency following exposure to chemical carcinogens. It also provides an explanation for an important paradox in cancer which is that cancer incidence rises rapidly with age but less rapidly with carcinogen dose even though both are thought to act through the same mechanism.

Aged↗

PubMed1996

Receptors on T cells escaping superantigen-mediated deletion lack special beta-chain junctional region structural characteristics.

The TCR V beta element is pivotal for superantigen recognition; however, not all T cells bearing a particular V beta element respond to an individual superantigen. Recent evidence has indicated that the TCR V alpha element also contributes to recognition of superantigen/MHC class II complexes. To determine whether the TCR beta-chain junctional regions influence recognition of a superantigen encoded by mouse mammary tumor virus (MMTV) proviral integrant Mtv-1, we have analyzed these regions in T cells that have survived superantigen-mediated negative selection in B10.BR-Mtv-1 mice. Our data indicate: 1) no TCR J beta skewing, 2) no difference in the length of the third complementarity-determining region (CDR3), and 3) no outstanding structural features that are shared among the junctional regions of the V beta 3+ T cells that escape thymic clonal elimination in superantigen-expressing mice. Several possible models for TCR engagement of viral superantigen/MHC class II complexes are discussed.

Amino Acid Sequence↗

PubMed1981

Blast cells in acute myeloblastic leukemia: a model.

A model of the evolution of acute myeloblastic leukemia (AML) as a clonal hemopathy is presented and reviewed in light of the available data on the behavior of leukemic blast-stem cell colonies. The model postulates that determination of a pluripotent stem cell of a leukemic clone occurs abruptly with intermediate stem cell classes being rare. Transformations occurring in committed myelopoietic progenitors are not self-maintaining. Leukemic clones in AML originate by genetic transformation to a lineage independent of myelopoietic differentiation. The biological behavior of AML clones changes with time as a result, in the short term, of environmental changes, and in the long term, of genetic changes. Sensitivity of leukemic blast colonies to adriamycin and Ara-C, although variable from patient to patient, is relatively stable for one clone during a major portion of the course of the disease.

Animals↗

PubMed1993

Comparison of cytogenetic and restriction fragment length polymorphism analyses for the detection of loss of chromosome material in clonal hemopoietic disorders.

Loss of chromosome material, as manifested by monosomy or partial deletion, is commonly found in neoplastic cells. We have undertaken a systematic comparison of standard cytogenetic analysis and molecular analysis for the detection of such loss, using as a model loss of chromosome 7 in 72 patients with a clonal myeloid malignancy. A large number of probes was used to screen three regions of chromosome 7 for loss by restriction fragment length polymorphism (RFLP) analysis. There were nine cases in which loss of chromosome 7 was detected by both techniques, but seven in which loss was detected by only one of the methods, demonstrating the complementary nature of these two techniques.

Aged↗

PubMed1994

A minority of carcinoma cells producing acidic fibroblast growth factor induces a community effect for tumor progression.

It is generally accepted that primary tumors become heterogeneous as a consequence of tumor-cell genetic instability. Clonal dominance has been shown to occur in some experimental models allowing a subpopulation of cells to overgrow the primary heterogeneous tumor and to metastasize. Alternatively, interactions among coexisting tumor subpopulations may contribute to the emergence of a malignant invasive primary solid tumor. We asked the question whether emergence of carcinoma cells producing a growth/dissociating factor within a tumor cell population may be a determinant for tumor progression and for clonal dominance. To mimic such a situation, we have investigated the impact of tumor subpopulation heterogeneity in an in vivo model in which mixtures of carcinoma cells that differ in their ability to produce acidic fibroblast growth factor are injected into nude mice. Our data indicate that a growth-factor-producing cell subpopulation can confer increased tumorigenicity to an entire cell population and subsequently elicit a shorter delay for appearance of metastasis. A community effect via cell interactions may account for a heterogeneous tumor cell population rather than clonal dominance during progression of certain tumor types.

Animals↗

PubMed1995

The clonal composition of myelin basic protein-reactive encephalitogenic T cell populations is influenced both by the structure of relevant antigens and the nature of antigen-presenting cells.

Studies of experimental autoimmune encephalomyelitis (EAE) in rodents have revealed that encephalitogenic T cell lines reactive with myelin basic protein (BP) are frequently dominated by clones expressing a restricted T cell receptor repertoire. Using the rat EAE model, we have begun to examine the basis for clonal dominance within BP-reactive T cell lines. We find that variations introduced into the standard protocol of periodic antigen stimulation produce marked shifts in the representation of different clones within encephalitogenic T cell populations. For example, altering the source of antigen-presenting cells (APC), while holding antigen (BP) constant, and substituting BP from guinea pig (GPBP) for that of the rat antigen (RBP) with constant APC, both cause shifts in the composition of the dominant clones within BP-reactive T cell lines. Our results suggest that: (i) adherence to an invariant protocol of antigen challenge may lead to an underestimation of the diversity of BP-reactive encephalitogenic T cell populations; and (ii) the minor structural differences between GPBP and RBP not only cause the weak immunogenicity of RBP but also result in the alteration of different T cell subsets. These observations indicate that apparent restrictions upon the repertoire of autoimmune T cells should be interpreted with caution when such cells are elicited by immunization with foreign antigens.

Amino Acid Sequence↗

PubMed2001

Clonal evolution of a radon-induced rat lung tumor.

Radon gas may represent a source of pulmonary radio-contamination either in mine or in domestic conditions. Since epidemiological studies are controversial, as long as biological markers of the exposure to such agents will not be identified, the question will remain open. We have previously shown a direct dose-dependent relationship between lung cancer occurrence and radon inhalation of rats. In this study, we report a cytogenetic study of a radon-induced rat lung tumor. Chromosome banding and chromosome specific paintings were performed on cultures of both fresh and xenografted tumors. We found by analyzing 17 sub-clones that all karyotypes presented a translocation involving rat chromosomes (RNO) 8 and 20, and a terminal deletion of RNO 15p suggesting a monoclonal origin of this tumor. RNO 15 is homologous to numerous human chromosomes (HSA), in particular to HSA 3p14.2, 3p22-p24.1 and 3p24.2-p24.3, this human chromosome being frequently lost in human lung carcinomas. Besides sharing chromosome alteration involving common features with those found in human lung cancer, this rat lung carcinoma represents a useful model to study tumor progression with respect to clonal evolution.

Animals↗

PubMed2001

Streptococcus iniae virulence is associated with a distinct genetic profile.

Streptococcus iniae causes meningoencephalitis and death in commercial fish species and has recently been identified as an emerging human pathogen producing fulminant soft tissue infection. As identified by pulsed-field gel electrophoresis (PFGE), strains causing disease in either fish or humans belong to a single clone, whereas isolates from nondiseased fish are genetically diverse. In this study, we used in vivo and in vitro models to examine the pathogenicity of disease-associated isolates. Strains with the clonal (disease-associated) PFGE profile were found to cause significant weight loss and bacteremia in a mouse model of subcutaneous infection. As little as 10(2) CFU of a disease-associated strain was sufficient to establish bacteremia, with higher inocula (10(7)) resulting in increased mortality. In contrast, non-disease-associated (commensal) strains failed to cause bacteremia and weight loss, even at inocula of 10(8) CFU. In addition, disease-associated strains were more resistant to phagocytic clearance in a human whole blood killing assay compared to commensal strains, which were almost entirely eradicated. Disease-associated strains were also cytotoxic to human endothelial cells as measured by lactate dehydrogenase release from host cells. However, both disease-associated and commensal strains adhered to and invaded cultured human epithelial and endothelial cells equally well. While cellular invasion may still contribute to the pathogenesis of invasive S. iniae disease, resistance to phagocytic clearance and direct cytotoxicity appear to be discriminating virulence attributes of the disease-associated clone.

Animals↗

PubMed2002

Clonality analysis of synchronous lesions of cervical carcinoma based on X chromosome inactivation polymorphism, human papillomavirus type 16 genome mutations, and loss of heterozygosity.

One of the most common forms of carcinoma in women, cervical invasive squamous cell carcinoma (CIC), often coexists with multiple lesions of cervical intraepithelial neoplasia (CIN). CIC and CIN show heterogeneity with respect to both histopathology and biology. To understand the causes, origin, and model of progression of cervical carcinoma, we assessed the clonality of a case with multiple synchronous lesions by analyzing X chromosome inactivation polymorphism, human papillomavirus type 16 (HPV16) sequence variation/mutations, and loss of heterozygosity (LOH). Microdissection was performed on 24 samples from this case, representing the entire lesional situation. The combination of different X chromosome inactivation patterns, two HPV16 point mutations, and LOH at three genomic microsatellite loci, led to the identification of five different "monoclonal" lesions (CIN II, CIN III, and invasive carcinoma nests) and five different "polyclonal" areas (CIN II and normal squamous epithelium). This finding indicated that CIC can originate from multiple precursor cells, from which some clones might progress via multiple steps, namely via CIN II and CIN III, whereas others might develop independently and possibly directly from the carcinoma precursor cells. Our results also supported the view that HPV16 as a "field factor" causes cervical carcinoma, which is probably promoted by the loss of chromosomal material as indicated by the LOH.

Adult↗

PubMed1995

A neuron-specific enhancer targets expression of the gonadotropin-releasing hormone gene to hypothalamic neurosecretory neurons.

The molecular mechanisms specifying gene expression in individual neurons of the mammalian central nervous system have been difficult to study due to the cellular complexity of the brain and the absence of cultured model systems representing differentiated central nervous system neurons. We have developed clonal, differentiated, neuronal tumor cell lines of the hypothalamic GnRH-producing neurons by targeting tumorigenesis in transgenic mice. These cells (GT1 cells) provide a model system for molecular studies of GnRH gene regulation. Here we present the identification and characterization of a neuron-specific enhancer responsible for directing expression of the rat GnRH gene in GT1 hypothalamic neurons. This approximately 300 base pair (bp) upstream region (-1571 to -1863) confers enhancer activity to a short -173-bp GnRH promoter or to a heterologous promoter only in GT1 cells. The enhancer is bound by multiple GT1 nuclear proteins over its entire length. Deletion of more than 30 bp from either end dramatically reduces activity, and even large internal fragments carrying seven of the eight DNAse I-protected elements show decreased activity. Scanning replacement mutations demonstrate that several of the internal elements are required for activity of the enhancer. Thus, the GnRH gene is targeted to hypothalamic neurons by a complex multicomponent enhancer that relies on the interaction of multiple nuclear-protein binding enhancer elements.

Animals↗

PubMed2002

Immune tolerance to combined organ and bone marrow transplants after fractionated lymphoid irradiation involves regulatory NK T cells and clonal deletion.

Immune tolerance to organ transplants has been reported in laboratory animals and in humans after nonmyeloablative conditioning of the host and infusion of donor bone marrow cells. We examined the mechanisms of immune tolerance to mouse cardiac allografts in MHC-mismatched hosts that developed mixed chimerism after posttransplant conditioning with a 2-wk course of multiple doses of lymphoid tissue irradiation, depletive anti-T cell Abs, and an infusion of donor bone marrow cells. When CD1(-/-) or J(alpha)281(-/-) hosts with markedly reduced NK T cells were used instead of wild-type hosts, then the conditioning regimen failed to induce tolerance to the heart allografts despite the development of mixed chimerism. Tolerance could be restored to the CD1(-/-) hosts by infusing enriched T cells from the bone marrow of wild-type mice containing CD1-reactive T cells but not from CD1(-/-) host-type mice. Tolerance could not be induced in either IL-4(-/-) or IL-10(-/-) hosts given the regimen despite the development of chimerism and clonal deletion of host T cells to donor MHC-Ags in the IL-10(-/-) hosts. We conclude that immune tolerance to bone marrow transplants involves clonal deletion, and tolerance to heart allografts in this model also involves regulatory CD1-reactive NK T cells.

Animals↗

PubMed1992

Microanatomic clonality of gamma delta T cells in human leishmaniasis lesions.

T cells bearing gamma delta Ag receptors accumulate in the lesions of patients with localized American cutaneous leishmaniasis (LCL), and are thought to be involved in immunity to the parasite. To obtain clues as to the nature of the Ag recognized by these cells, we analyzed the diversity of the TCR delta-chain in LCL lesions. Using mAb against variable (V) encoded determinants with immunoperoxidase, both V delta 1 and V delta 2 subpopulations were identified in the dermal granulomas. However, within the epidermis of LCL lesions, the majority of the gamma delta T cells were V delta 1 positive. PCR analysis of lesion-derived DNA using oligonucleotide primers for V and junctional (J) gene segments revealed preferential usage of J delta 1 in lesions compared with the peripheral blood of these patients. Nucleotide sequence analysis of the V-J junction indicated limited diversity of gamma delta T cells within specific microanatomic regions. In addition, use of a single diversity (D) gene segment, D delta 3, in V delta 2 cells in lesions was observed, as opposed to multiple D delta gene segment usage in the blood of the same individuals. The distribution, gene segment usage and clonality of gamma delta T cells in lesions of leishmaniasis was remarkably similar to that observed in leprosy. Therefore, gamma delta T cells responding to infection may recognize a limited set of nominal Ag, perhaps common to distinct pathogens and/or those expressed by the host. Our findings are most consistent with a model in which specific gamma delta T cells are clonally selected by these Ag in lesions and undergo oligoclonal expansion within a microanatomic region.

Amino Acid Sequence↗

PubMed2000

Regulation of T-cell apoptosis: a mixed lymphocyte reaction model.

Despite the capacity for antigen-specific activation and rapid clonal expansion, homeostatic mechanisms ensure that the mature immune system contains a relatively stable number of T cells. In recent years, it has become apparent that this stability is a consequence of apoptotic death of most of the specific T cells generated during an immune response. Clearly this process must be tightly regulated in order to retain sufficient T-cell progeny to mediate an effective response, whilst allowing the rapid deletion of these cells at the end of the response to prevent lymphadenopathy and cross-reactive autoimmunity. In this study, the factors that regulate the sensitivity of T cells to apoptosis were investigated in vitro after the induction of primary T-cell activation within a mixed lymphocyte reaction (MLR). It was found that activated T cells rapidly acquire the expression of both Fas and Fas ligand (FasL) on their surface and contain high levels of the precursor form of the pro-apoptotic enzyme, caspase 8 (FLICE). However, these T cells were resistant for up to 5 days to apoptosis following the stimulation of Fas; a maximal apoptotic response was observed after 7 days. This time point coincided with a marked reduction in expression of the FLICE inhibitory protein (FLIP) and maximal activity of caspase 8. At time points beyond day 7, the number of viable cells in the MLR decreased further despite a reduction in the expression of FasL. However, the expression of interleukin-2 (IL-2) at these late time points was low, resulting in a decrease in expression of the anti-apoptotic protein Bcl-2. This can produce apoptosis by allowing leakage of cytochrome-c from mitochondria resulting in direct activation of the caspase cascade. In this study, it is shown that T cells are resistant to apoptosis for the first 5 days after activation as a consequence of insensitivity of the Fas pathway and the presence of intracellular Bcl-2. After between 5 and 7 days, the cells become sensitive to Fas-mediated apoptosis while retaining Bcl-2 expression. At later time points, Fas ligation is reduced but the cells respond to a decreased availability of IL-2 by reducing Bcl-2 expression; this encourages further apoptosis by allowing the direct activation of caspase enzymes.

Adult↗

PubMed1993

Murine models of autoimmune disease and Sjögren's syndrome.

Murine models of autoimmune disease exhibit heterogenous features that are now being understood in terms of their basic molecular defects. Defects in apoptosis genes or genes that contribute to B-cell activation, T-cell tolerance loss, and development of autoimmune disease, including glomerulonephritis, have been identified. The interaction of primary and secondary predisposing genes leads to a diverse spectrum of autoimmune features that is different in different strains of mice. The challenge in the future will be to correlate these primary and secondary autoimmune genes with specific environmental triggers to explain the diverse spectrum of autoimmune disease. The ability to correct the apoptosis defect and autoimmune disease in CD2-Fas transgenic MRL-lpr/lpr mice brings hope that genetic therapy will eventually be possible in humans.

Animals↗

PubMed1991

Hematopathologic features of Epstein-Barr virus-induced human B-lymphoproliferation in mice with severe combined immunodeficiency. A model of lymphoproliferative diseases in immunocompromised patients.

The anatomical distribution, morphology, and clonality, of 'non-Hodgkin's lymphomas' in immunocompromised patients are usually distinctly different from NHL occurring in the general population. Mosier DE, Gulizia RJ, Baird SM, Wilson DB: Nature (London) 335:256, 1988 have described lymphoproliferative disease (LPD) of human B cell origin in mice with severe combined immunodeficiency (scid mice) after transfer of human peripheral blood mononuclear cells from Epstein-Barr virus-seropositive individuals. Reported herein is detailed information regarding the morphology, phenotypes, and clonality of LPD lesions in 10 of 18 scid mice that had developed LPD after transfer of peripheral blood mononuclear cells. These lesions were diffuse and monomorphic proliferations of immunoblastoid cells. They were invasive in their growth and often necrotic. Human B cell-related and activation-associated antigens were found on the LPD lesions, although the numbers of cells with the latter antigens were relatively small. Immunofixation electrophoresis for human immunoglobulins in sera of the majority of mice revealed oligoclonal populations, however, phenotypic and cytogenetic analyses showed no definite monoclonality. This scid mouse model is beneficial for understanding the early phases in the pathogenesis of LPD in immunocompromised patients.

Animals↗