Since the gene encoding Id1 was cloned in 1990, Id proteins have been implicated in regulating a variety of cellular processes, including cellular growth, senescence, differentiation, apoptosis, angiogenesis, and neoplastic transformation. The development of knockout and transgenic animal models for many members of the Id gene family has been particularly useful in sorting out the biologic relevance of these genes and their expression during normal development, malignant transformation, and tumor progression. Here we review the current understanding of Id gene function, the biologic consequences of Id gene expression, and the implications for Id gene regulation of cell growth and tumorigenesis.
Id proteins are helix-loop-helix transcription factors that regulate tumor angiogenesis. In order to identify downstream effectors of Id1 involved in the regulation of angiogenesis, we performed PCR-select subtractive hybridization on wild-type and Id1 knockout mouse embryo fibroblasts (MEFs). Here we demonstrate that thrombospondin-1 (TSP-1), a potent inhibitor of angiogenesis, is a target of transcriptional repression by Id1. We also show that Id1-null MEFs secrete an inhibitor of endothelial cell migration, which is completely inactivated by depletion of TSP-1. Furthermore, in vivo studies revealed decreased neovascularization in matrigel assays in Id1-null mice compared to their wild-type littermates. This decrease was completely reversed by a TSP-1 neutralizing antibody. We conclude that TSP-1 is a major target for Id1 effects on angiogenesis.
The OGG1 gene encodes a highly conserved DNA glycosylase that repairs oxidized guanines in DNA. We have investigated the in vivo function of the Ogg1 protein in yeast mitochondria. We demonstrate that inactivation of ogg1 leads to at least a 2-fold increase in production of spontaneous mitochondrial mutants compared with wild-type. Using green fluorescent protein (GFP) we show that a GFP-Ogg1 fusion protein is transported to mitochondria. However, deletion of the first 11 amino acids from the N-terminus abolishes the transport of the GFP-Ogg1 fusion protein into the mitochondria. This analysis indicates that the N-terminus of Ogg1 contains the mitochondrial localization signal. We provide evidence that both yeast and human Ogg1 proteins protect the mitochondrial genome from spontaneous, as well as induced, oxidative damage. Genetic analyses revealed that the combined inactivation of OGG1 and OGG2 [encoding an isoform of the Ogg1 protein, also known as endonuclease three-like glycosylase I (Ntg1)] leads to suppression of spontaneously arising mutations in the mitochondrial genome when compared with the ogg1 single mutant or the wild-type. Together, these studies provide in vivo evidence for the repair of oxidative lesions in the mitochondrial genome by human and yeast Ogg1 proteins. Our study also identifies Ogg2 as a suppressor of oxidative mutagenesis in mitochondria.
Id genes regulate tumor angiogenesis and loss of Id1 inhibits tumor xenograft growth in mice. Here we evaluate the role of Id1 in a more clinically relevant tumor model system using a two-step chemical carcinogenesis protocol. Remarkably, we find that Id1-/- mice are more susceptible to skin tumorigenesis compared to their wild-type counterparts. Cutaneous neoplasms in Id1-/- mice show increased proliferation without alterations in tumor angiogenesis; however, Id1-/- mice possess 50% fewer cutaneous gammadelta T cells than their wild-type counterparts due to an intrinsic migration defect associated with loss of expression of the chemokine receptor, CXCR4. We suggest that there are important differences between the mechanisms of angiogenesis in transplanted and autochthonous tumors and that these findings will have significant implications for the potential utility of antiangiogenic therapies in cancer.
Mitochondrial dysfunction is a profound feature of cancer cells and is also known to cause several mitochondrial diseases. Mutations in mitochondrial DNA (mtDNA) have been reported frequently in these diseases. Although many environmental agents are known to cause damage to mitochondria, rapid methods need to be developed for testing agents that cause mitochondrial dysfunction and are involved in the development of mitochondrial and other diseases. Using Saccharomyces cerevisiae, we describe the development of a colorimetric method that identifies both physical and chemical agents that cause mitochondrial dysfunction and mutation of the mitochondrial genome. This method utilizes the previously reported ade2 mutant of S.cerevisiae that produces red colonies. However, when they lose mitochondrial function the colonies turn white. This colorimetric method has helped quantify the vulnerability of mtDNA to oxidative agents. Our study reveals that the oxidative agent adriamycin causes both mutation and extensive damage to mtDNA, which leads to loss of mtDNA. Our study also reveals that the lost mtDNA fragments migrate to the nucleus and integrate into the nuclear genome. Furthermore, our analysis reveals that loss of mtDNA leads to resistance to oxidative agents. The method described in this paper should aid in the rapid identification of environmental and other agents that cause mitochondrial dysfunction and mutagenesis, agents that may be involved in the development of mitochondrial and other diseases.
CD1d is a nonclassical Ag-presenting molecule that presents glycolipid Ags to NKT cells that are involved in immune defense and tumor rejection. It also plays a role in immunoregulatory functions in the epidermis. The mechanisms controlling the expression of CD1d are not well understood. Therefore, we cloned the CD1d gene promoter and characterized its activities in primary human keratinocytes and other cell lines of epithelial origin. We found that a CCAAT box in the CD1d promoter is required for its expression in keratinocytes. We show here that transcription factor C/EBP-β binds to the CCAAT box in the CD1d promoter in vitro and in vivo. Consistent with these observations, deletion of the gene encoding for C/EBP-β caused a loss of CD1d expression. The in vivo regulation of CD1d has significant implications for the pathologic mechanisms of certain immunologic skin diseases in which NKT cells play a role, such as allergic contact dermatitis and psoriasis. Together, these data show a central role for C/EBP-β in regulating CD1d transcription.
Background: Cyclins play a regulatory role in cell cycle progression, associated with cyclin-dependent kinases. We have investigated the structurefunction relationships of cyclin A, mainly using Xenopus egg extracts in vitro. To further analyse the function and structure of cyclin A in vivo, we expressed Xenopus cyclin A1 in the budding yeast Saccharomyces cerevisiae.
ABSTRACT. A strain of Saccharomyces cerevisiae that contains an integrated copy of a Xenopus cyclin Al gene under the control of the GAL1promoter has been constructed. On inducing expression of cyclin Al, the nuclear migration that occurs prior to division becomes aberrant. Instead of migrating to the neck between the mother cell and daughter bud, the nucleus, the short mitotic spindle and its associated two spindle pole bodies entered the daughter bud. This phenotype was induced by expression of an indestructible cyclin mutant, but not by a mutated cyclin Al unable to activate Cdc28 kinase. The nuclear abnormality induced by cyclin Al was overcome by cdc28 mutations that abolish its ability to bind cyclin Al. Both yeast cyclin Clb3 and Xenopus mitotic cyclin B produced the same phenotype, whereas Gl cyclin Cln2 did not. The results suggest that the proper movementof the nucleus through the spindle function during mitosis requires the appropriate activity of Cdc28 kinase mediated by specific cyclins.One essential function of the cell cycle is the equal division of chromosomes into the mother and the daughter cell. After DNAreplication in the budding yeast Saccharomycescerevisiae, the nucleus migrates to the mother-daughter junction prior to nuclear division during mitosis. In concert with bud emergence and initiation of DNAreplication, nuclear movementand division occurs as sequential events; the nucleus movestoward the neck site of the mother cell, translocates to the position in which half is in the mother and half in the daughter, and then the nucleus is pulled and divided to two nuclear lobes in the mother and the daughter (for reviews see 8, 18, 53). Unlike higher eukaryotes, the nuclear envelope of budding yeast does not break down during mitosis and the cell contains the nucleus throughout the cell cycle (7). Prior to nuclear migration, the spindle pole body embeddedin the nuclear envelope undergoes duplication and the duplicated SPBs move to positions 180°apart, the mitotic spindle runs through the nucleus between the SPBs; initially linear it spans most of the mother and the daughter cells (19, 51). The opposing forces of mitotic spindles are generated by the dynamic instability of microtubules and by molecular motors such as kinesin and dynein (1, 16 (12, 34, 35) and dynein (25, 28), nuclear and kinetochore components (ll, 44). Although, in most of these mutants, the failure of migration or separation results in the majority of nuclear DNAremaining in the mother cell, there are some exceptions; e.g. the espl mutation, which appears to cause a SPB defect, results in the chromosomal DNAeither to the mother cell or to the daughter bud (2, 27). In higher eukaryotes it is clear that the cyclin-p34cdc2 kinases are directly and indirectly responsible for the events that occur during mitosis; nuclear breakdown, chromosomal condensation and spindle formation (20).The kinase activity depends upon both A-and B-type cyclins. Cyclin B is known to be a regulatory subunit of M-phase promoting factor and the cyclin ...
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