Biomedical subjects

C J Guidos

Publications and source records attributed to C J Guidos.

At least 19 recordsLinked to original sources

PubMed2003

Notch signaling in development and disease.

Notch receptors and ligands were first identified in flies and worms, where they were shown to regulate cell proliferation, cell differentiation, and, in particular, binary cell fate decisions in a variety of developmental contexts. The first mammalian Notch homolog was discovered to be a partner in a chromosomal translocation in a subset of human T-cell leukemias. Subsequent studies in mice and humans have shown that Notch signaling plays essential roles at multiple stages of hematopoiesis, and also regulates the development or homeostasis of cells in many tissues and organs. Thus, it is not surprising that mutations which disrupt Notch signaling cause a wide range of cancers and developmental disorders. Perhaps because it is so widely used, Notch signaling is subject to many unusual forms of regulation. In this review, we will first outline key aspects of Notch signaling and its regulation by endocytosis, glycosylation, and ubiquitination. We will then overview recent literature elucidating how Notch regulates cell-lineage decisions in a variety of developmental contexts. Finally, we will describe the roles of dysregulated Notch signaling in causing several types of cancer and other pathologies.

Alagille Syndrome↗

PubMed2001

Subversion of the T/B lineage decision in the thymus by lunatic fringe-mediated inhibition of Notch-1.

Notch-1 signaling is essential for lymphoid progenitors to undergo T cell commitment, but the mechanism has not been defined. Here we show that thymocytes ectopically expressing Lunatic Fringe, a modifier of Notch-1 signaling, induce lymphoid progenitors to develop into B cells in the thymus. This cell fate switch resulted from Lunatic Fringe-mediated inhibition of Notch-1 function, as revealed by experiments utilizing lymphoid progenitors in which Notch-1 activity was genetically manipulated. These data identify Lunatic Fringe as a potent regulator of Notch-1 during the T/B lineage decision and show that an important function of Notch-1 in T cell commitment is to suppress B cell development in the thymus.

Animals↗

PubMed2001

Irradiation promotes V(D)J joining and RAG-dependent neoplastic transformation in SCID T-cell precursors.

Defects in the nonhomologous end-joining (NHEJ) pathway of double-stranded DNA break repair severely impair V(D)J joining and selectively predispose mice to the development of lymphoid neoplasia. This connection was first noted in mice with the severe combined immune deficient (SCID) mutation in the DNA-dependent protein kinase (DNA-PK). SCID mice spontaneously develop thymic lymphoma with low incidence and long latency. However, we and others showed that low-dose irradiation of SCID mice dramatically increases the frequency and decreases the latency of thymic lymphomagenesis, but irradiation does not promote the development of other tumors. We have used this model to explore the mechanistic basis by which defects in NHEJ confer selective and profound susceptibility to lymphoid oncogenesis. Here, we show that radiation quantitatively and qualitatively improves V(D)J joining in SCID cells, in the absence of T-cell receptor-mediated cellular selection. Furthermore, we show that the lymphocyte-specific endonuclease encoded by the recombinase-activating genes (RAG-1 and RAG-2) is required for radiation-induced thymic lymphomagenesis in SCID mice. Collectively, these data suggest that irradiation induces a DNA-PK-independent NHEJ pathway that facilitates V(D)J joining, but also promotes oncogenic misjoining of RAG-1/2-induced breaks in SCID T-cell precursors.

Animals↗

PubMed1997

The absolute number of trans-rearrangements between the TCRG and TCRB loci is predictive of lymphoma risk: a severe combined immune deficiency (SCID) murine model.

Pilot studies in human populations have demonstrated a correlation between the level of antigen receptor trans-rearrangements and risk (at the population level) of lymphoid malignancy. Irradiation of newborn severe combined immune deficiency mice results in an increased risk of subsequent development of thymic lymphoma (100% of mice so irradiated are dead of thymic lymphoma by 20 weeks of age). We, therefore, assayed the occurrence of trans-rearrangements in this well-controlled mouse mutant system and found a 50-100-fold increase in the absolute number of TCRGV-TCRBJ trans-rearrangements compared to unirradiated littermates (and a comparable fold increase over age-matched BALB/c mice) at 2 weeks following irradiation. We also found a marked disproportion in generating trans-rearrangements versus intralocus rearrangements in the severe combined immune deficiency system compared to BALB/c, independent of irradiation. The trans-rearrangements noted were polyclonal in nature. These data, again, suggest that the absolute level of antigen receptor trans-rearrangements may serve as a biomarker of lymphoma risk.

Animals↗

PubMed1997

TCR engagement of CD4+CD8+ thymocytes in vitro induces early aspects of positive selection, but not apoptosis.

Immature CD4/CD8 double-positive (DP) thymocytes expressing self MHC-restricted TCR are positively selected in response to TCR signals to survive and differentiate into functionally competent CD4 or CD8 single positive (SP) T cells. In contrast, DP precursors expressing autoreactive TCR are clonally deleted in response to TCR signals. We show here that in vitro TCR engagement of TCR(low) DP thymocytes rapidly triggers a variety of events considered to be hallmarks of positive selection in vivo. These include increased expression of CD5 and Bcl-2, termination of RAG-1 and pre-T(alpha) gene expression, and a switch in lck promoter usage. We also demonstrate that CD4- or CD28-mediated signals synergize with TCR signals to induce these outcomes. Finally, we show that the response of DP thymocytes to TCR engagement is selective in that clonal deletion, CD4/CD8 lineage commitment, and other events associated with maturation, such as changes in expression of Thy-1, HSA, MHC class I, and CD45-RB, were not induced. Thus, only subsets of maturational processes associated with positive selection in vivo were shown to be directly coupled to TCR signaling pathways at the DP stage. These observations support conclusions from in vivo systems suggesting that multiple, temporally separated TCR engagements are required to effect the entire spectrum of developmental changes associated with positive selection, and provide a conceptual and experimental framework for unraveling the complexity of positive selection.

Animals↗

PubMed1997

Essential and perilous: V(D)J recombination and DNA damage checkpoints in lymphocyte precursors.

V(D)J recombination generates a diverse array of antigen-binding specificities, but breakage and re-joining of DNA segments have grave implications for the maintenance of genomic stability and oncogenic risk. Exposure of eukaryotic cells to genotoxic agents activates a DNA damage checkpoint that induces cell-cycle arrest and DNA repair, or apoptosis. We discuss several lines of evidence implicating DNA-dependent protein kinase (DNA-PK), and the gene mutated in ataxia telangiectasia (ATM), two mammalian homologues of yeast DNA damage-checkpoint genes, in regulating the response to intrinsic DNA damage that occurs during V(D)J recombination.

Amino Acid Sequence↗

PubMed1996

V(D)J recombination activates a p53-dependent DNA damage checkpoint in scid lymphocyte precursors.

Double-stranded DNA breaks (DSBs) trigger p53-mediated cell cycle arrest or apoptosis pathways that limit the oncogenic consequences of exposure to genotoxic agents, but p53-mediated responses to DSB generated by normal physiologic events have not been documented. "Broken" V(D)J coding ends accumulate in scid lymphocyte precursors as a consequence of a mutation in DNA-dependent protein kinase (DNA-PK). The ensuing failure to rearrange efficiently antigen receptors arrests lymphoid development. Here we show that scid thymocytes express high levels of p53 protein, attributable to recombinase activating gene (RAG)-dependent generation of DSB adjacent to V, D, and J gene segments. To examine the functional importance of p53 expression in vivo, we bred p53-/- scid mice. The absence of p53 facilitated production of in-frame V(D)Jbeta coding joints and developmental progression of scid thymocytes, in addition to a dramatic accumulation of pro-B cells. All mice developed disseminated pro-B or immature T cell lymphoma/leukemia by 7-12 weeks of age. We present evidence that p53 deficiency prolongs the survival of scid lymphocyte precursors harboring broken V(D)J coding ends, allowing the accumulation of aneuploid cells. These results demonstrate that a p53-mediated DNA damage checkpoint contributes to the immune deficiency characteristic of the scid mutation and limits the oncogenic potential of DSBs generated during V(D)J recombination.

Animals↗

PubMed1996

Transient restoration of gene rearrangement at multiple T cell receptor loci in gamma-irradiated scid mice.

The developmental arrest of thymocytes from scid mice, deficient in variable, (diversity), and joining, or V(D)J recombination, can be overcome by sublethal gamma-irradiation. Since previous studies focused on restoration of rearrangement of the T cell receptor (TCR) beta locus, productive rearrangement of which is selected for, we sought to examine to what extent locus specificity and cellular selection contributed to the observed effects. We report here that irradiation of newborn scid mice induces normal V-D-J rearrangements of the TCR delta locus, which like TCR beta, is also actively rearranged in CD(4-)CD(8-) (double negative) thymocytes. In contrast, no complete V-J alpha rearrangements were detected. Instead, we detected substantial levels of hairpin-terminated coding ends at the 5' end of the J alpha locus, demonstrating that TCR alpha rearrangements manifest the effects of the scid mutation. Irradiation, therefore, transiently compensates for the effects of the scid mutation in a locus-nonspecific manner in thymocytes, resulting in a burst of normal TCR beta and delta rearrangements. Irradiation also allows the development of cells that can initiate but fail to complete V(D)J recombination events at the TCR alpha locus, which is normally inaccessible in scid thymocytes.

Animals↗

PubMed1996

Positive selection of CD4+ and CD8+ T cells.

Significant progress has been made in characterizing intermediates and defining individual steps of positive selection, providing important insights into mechanisms of CD4/CD8 lineage commitment. New evidence suggests that specific recognition of peptides may be important for positive selection of CD4+ T cells. Several studies have defined signal-transduction pathways important for positive selection and have provided evidence that distinct signaling pathways may regulate positive versus negative selection.

Animals↗

PubMed1996

Fyn can partially substitute for Lck in T lymphocyte development.

Lck, a Src family tyrosine kinase, transduces signals important for the development of alphabeta and gammadelta T cells. However, T cell development is only partially compromised in Lck-deficient mice, suggesting that other kinases may also transduce pre-TCR or TCR signals. One candidate is Fyn, a Src kinase coexpressed with Lck in immature and mature T cells. Here we show that T cell development is completely compromised in lck(-/-)fyn(-/-) mice. In addition, we demonstrate that expression of a gain-of-function mutant fyn(T) transgene completely restores production of immature CD4/CD8 double positive thymocytes and gammadelta T cells and improves the representation of CD4 or CD8 single positive thymocytes. These observations reveal that Fyn can subserve some Lck-like functions in T cell development.

Animals↗

PubMed1996

Inactivation of Fac in mice produces inducible chromosomal instability and reduced fertility reminiscent of Fanconi anaemia.

Fanconi anaemia (FA) is an autosomal recessive disease characterized by bone marrow failure, variable congenital malformations and predisposition to malignancies. Cells derived from FA patients show elevated levels of chromosomal breakage and an increased sensitivity to bifunctional alkylating agents such as mitomycin C (MMC) and diepoxybutane (DEB). Five complementation groups have been identified by somatic cell methods, and we have cloned the gene defective in group C (FAC)(7). To understand the in vivo role of this gene, we have disrupted murine Fac and generated mice homozygous for the targeted allele. The -/- mice did not exhibit developmental abnormalities nor haematologic defects up to 9 months of age. However, their spleen cells had dramatically increased numbers of chromosomal aberrations in response to MMC and DEB. Homozygous male and female mice also had compromised gametogenesis, leading to markedly impaired fertility, a characteristic of FA patients. Thus, inactivation of Fac replicates some of the features of the human disease.

Animals↗

PubMed1996

Lck dependence of signaling pathways activated by gamma-irradiation and CD3 epsilon engagement in RAG-1(-/-)-immature thymocytes.

The src family tyrosine kinase, Lck, transduces signals from the pre-TCR complex which regulate the development and expansion of CD4/CD8 double-positive (DP) thymocytes from CD25(+) CD4/CD8 double-negative progenitors. We and others have recently shown that sublethal gamma-irradiation bypasses the need for TCRbeta expression to promote the development and expansion of DP thymocytes in scid or recombinase-activating gene (RAG)-deficient mice. Here we demonstrate that gamma-irradiation activates an Lck-dependent signaling process in immature thymocytes similar to that initiated physiologically by the pre-TCR complex.

Animals↗

PubMed1996

Biochemical and genetic defects in the DNA-dependent protein kinase in murine scid lymphocytes.

The scid gene product has been identified as the 460-kDa catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs p460), a member of the phosphatidylinositol 3-kinase family. DNA-PK activity is undetectable in scid cells, but the molecular basis for this defect has not been identified. Here we report that expression of p460 in scid lymphocyte precursors is detectable but is reduced at least 10-fold relative to that in wild-type lymphocytes. In addition, we show that the scid mutation disturbs p460 nuclear association, presumably affecting its role in DNA repair pathways. To examine the molecular basis for our observations, we used a degenerate PCR strategy to clone the C-terminal p460 kinase domain from wild-type and scid thymocytes. Northern (RNA) analysis with these probes revealed normal steady-state p460 mRNA levels in scid cells, suggesting that the reduced abundance of p460 protein is due to a posttranscriptional defect. Sequence comparisons identified a single-base-pair alteration in the scid C-terminal p460 kinase domain, resulting in a premature stop codon. This mutation is predicted to truncate p460 by approximately 8 kDa, but it preserves the conserved motifs required for kinase activity in members of the phosphoinositidyl 3-kinase family. Despite a computed molecular weight alteration of less than 2%, we were able to visualize this difference by Western blot (immunoblot) analysis of wild-type and scid p460. These data demonstrate that the scid DNA-PKes mutation is not a null allele and suggest a molecular rationale for the well-described leakiness of the scid phenotype.

Amino Acid Sequence↗

PubMed1995

In vitro maturation of clonal CD4+CD8+ cell lines in response to TCR engagement.

Engagement of the TCR on immature CD4+CD8+ (DP) thymocytes by an appropriate peptide/MHC ligand evokes a complex program of maturation known as positive selection. As a result, DP thymocytes are rescued from programmed cell death, become committed to the CD4 or CD8 lineage, extinguish expression of V(D)J recombinase activity, and undergo further maturation. We describe here a panel of DP thymic lymphoma cell lines that, in response to in vitro TCR engagement, undergo many of the TCR-beta-induced maturation events that have been reported to accompany positive selection of DP thymocytes in vivo. These events include increased expression of CD5, CD69, CD45, TCR-alpha, and MHC class I, and decreased expression of Thy-1 and heat-stable Ag. In addition, we observed TCR-induced expression of the bcl-2 gene, a well described inhibitor of programmed cell death. Finally, TCR engagement decreased expression of recombinase-activating genes and terminal deoxynucleotidal transferase genes, as well as V(D)J recombinase activity. However, TCR engagement did not elicit demonstrable CD4/CD8 lineage commitment. These observations suggest that engagement of the TCR on these DP cell lines elicits multiple maturation events that are part of the positive selection developmental program, but not CD4/CD8 lineage commitment. Thus, these DP cell lines provide the opportunity to elucidate molecular mechanisms of maturation and CD4/CD8 lineage commitment in vitro.

Animals↗

PubMed1995

Development of CD4+CD8+ thymocytes in RAG-deficient mice through a T cell receptor beta chain-independent pathway.

Antigen-binding diversity is generated by site-specific V(D)J recombination of the T cell receptor (TCR) and immunoglobulin loci in lymphocyte precursors. Coordinate expression of two structurally distinct recombinase activating genes, RAG-1 and RAG-2, is necessary for activation of site-specific V(D)J recombination. In mice bearing targeted disruptions of either the RAG-1 or RAG-2 genes, T and B lymphocyte development is arrested at the CD4-8- double negative (DN) thymocyte or B220+/CD43+ pro-B cell stage. Development of CD4+CD8+ double positive (DP) thymocytes is restored by expression of a functionally rearranged TCR beta transgene, suggesting that TCR beta expression is critical for this developmental transition. We have found that treatment of adult or newborn RAG-deficient mice with a single sublethal dose of gamma-irradiation rescues the DN to DP transition in early thymocytes, and this is accompanied by a dramatic increase in thymus cellularity. In contrast to the observed induction of thymocyte maturation, there was no phenotypic or functional evidence of coincident B lymphocyte development in irradiated RAG-deficient mice. Interestingly, maturation of DP thymocytes occurred without expression of TCR beta protein in the cytoplasm or on the cell surface. These results suggest an in vivo pathway for DP thymocyte development which is TCR beta chain independent.

Animals↗

PubMed1994

Rescue of T cell-specific V(D)J recombination in SCID mice by DNA-damaging agents.

Assembly of antigen receptor V (variable), D (diversity), and J (joining) gene segments requires lymphocyte-specific genes and ubiquitous DNA repair activities. Severe combined immunodeficient (SCID) mice are defective in general double-strand (ds) DNA break repair and V(D)J coding joint formation, resulting in arrested lymphocyte development. A single treatment of newborn SCID mice with DNA-damaging agents restored functional, diverse, T cell receptor beta chain coding joints, as well as development and expansion of thymocytes expressing both CD4 and CD8 coreceptors, but did not promote B cell development. Thymic lymphoma developed in all mice treated with DNA-damaging agents, suggesting an interrelation between V(D)J recombination, dsDNA break repair, and lymphomagenesis.

Animals↗

PubMed1990

T cell receptor-mediated negative selection of autoreactive T lymphocyte precursors occurs after commitment to the CD4 or CD8 lineages.

To identify the maturational stage(s) during which T cell receptor (TCR)-mediated positive and negative selection occurs, we followed the development of CD4+8- and CD4-8+ T cells from TCRlo CD4+8+ thymic blasts in the presence of different positive and negative selecting (major histocompatibility complex or Mls) elements. We describe novel lineage-committed transitional intermediates that are TCRmed CD4+8lo or TCRmed CD4lo8+, and that show evidence of having been positively selected. Furthermore, negative selection is not evident until after cells have attained one of the TCRmed transitional phenotypes. Accordingly, we propose that negative selection in normal mice occurs only after TCRlo CD4+8+ precursors have been positively selected into either the CD4 or CD8 lineage.

Animals↗

PubMed1990

A cell cycle analysis of growth-related genes expressed during T lymphocyte maturation.

Fetal liver or bone marrow-derived T lymphocyte precursors undergo extensive, developmentally regulated proliferation in response to inductive signals from the thymic microenvironment. We have used neonatal mouse thymocytes size-separated by centrifugal elutriation to study the cell cycle stage-specific expression of several genes associated with cell proliferation. These include genes involved in the biosynthesis of deoxyribonucleotide precursors, such as dihydrofolate reductase (DHFR), thymidylate synthase (TS), and the M1 and M2 subunits of ribonucleotide reductase, as well as c-myc, a cellular oncogene of unknown function. Using nuclear run-on assays, we observed that the transcription rates for these genes, with the exception of TS, are essentially invariant not only throughout the cell cycle in proliferating cells, but also in noncycling (G0) cells. The TS gene showed a transient increase in transcription rate in cells which bordered between a proliferating and nonproliferating status. Studies of an elutriated T cell line, S49.1, yielded similar results, indicating that the process of immortalization has not affected the transcriptional regulation of these genes. Analysis of steady-state mRNA levels using an RNase protection assay demonstrated that the levels of DHFR and TS mRNA accumulate as thymocytes progress through the cell cycle. In contrast, only the M2 subunit of ribonucleotide reductase showed cyclic regulation. Finally, in contrast to cultured cell models, we observed an abrupt fivefold increase in the steady-state level of c-myc mRNA in the transition from G1 to S-phase. We conclude from these studies that the transcriptional regulation of specific genes necessary for cellular proliferation is a minor component of the developmental modulation of the thymocyte cell cycle.

Animals↗