Mcm2 deficiency results in short deletions allowing high resolution identification of genes contributing to lymphoblastic lymphoma.

Mini-chromosome maintenance (Mcm) proteins are part of the replication-licensing complex that is loaded onto chromatin during the G1-phase of the cell cycle and required for initiation of DNA replication in the subsequent S-phase. Mcm proteins are typically loaded in excess of the number of locations that are used during S-phase. Nonetheless, partial depletion of Mcm proteins leads to cancers and stem cell deficiencies. Mcm2 deficient mice, on a 129Sv genetic background, display a high rate of thymic lymphoblastic lymphoma. Here array comparative genomic hybridization is used to characterize the genetic damage accruing in these tumors. The predominant events are deletions averaging less than 0.5 Mbp, considerably shorter than observed in prior studies using alternative mouse lymphoma models or human tumors. Such deletions facilitate identification of specific genes and pathways responsible for the tumors. Mutations in many genes that have been implicated in human lymphomas are recapitulated in this mouse model. These features, and the fact that the mutation underlying the accelerated genetic damage does not target a specific gene or pathway a priori, are valuable features of this mouse model for identification of tumor suppressor genes. Genes affected in all tumors include Pten, Tcfe2a, Mbd3 and Setd1b. Notch1 and additional genes are affected in subsets of tumors. The high frequency of relatively short deletions is consistent with elevated recombination between nearby stalled replication forks in Mcm2-deficient mice.

DNA damage response and tumorigenesis in Mcm2-deficient mice.

Minichromosome maintenance proteins (Mcm's) are components of the DNA replication licensing complex. In vivo, reduced expression or activity of Mcm's has been shown to result in highly penetrant early onset cancers (Shima et al., 2007; Pruitt et al., 2007) and stem cell deficiencies (Pruitt et al., 2007). Here we use mouse embryonic fibroblasts from an Mcm2-deficient strain of mice to show by DNA fiber analysis that origin usage is decreased in Mcm2-deficient cells under conditions of hydroxyurea (HU)-mediated replication stress. DNA damage responses (DDRs) resulting from HU and additional replication-dependent and replication-independent genotoxic agents were also examined and shown to function at wild-type (wt) levels. Further, basal levels of many components of the DDR were expressed at wt levels, showing that there is no acute replicative stress under normal growth conditions. Only very modest, 1.5- to 2-fold increases in the basal levels of gamma-H2AX, p21(cip1) and 53bp foci were found, consistent with a slight chronic elevation in DDR pathways. The one condition in which a larger difference between wt- and Mcm2-deficient cells was found occurred after ultraviolet irradiation and may reflect the role of Chk1-mediated suppression of dormant origins. In vivo, abrogating p53-mediated DDR in Mcm2-deficient mice results in increased embryonic lethality and accelerated cancer formation in surviving mice. Further, p53 mutation rescues the negative effect of Mcm2 deficiency on the survival of neural stem cells in vitro; however, the enhanced survival correlates with increased genetic damage relative to Mcm2 wt cells carrying the p53 mutation. Together these results show that even relatively minor perturbations to primary or dormant replication origin usage contribute to accelerated genetic damage in vivo. In addition, these studies show that tumor types resulting from Mcm2 deficiency are strongly affected by interaction with both genetic background and p53.

Current issues in the management of acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL) is caused by one of four genetic lesions that disrupt the alpha receptor for retinoic acid, RAR alpha. The fusion protein responsible for greater than 99% of APL cases, PML-RAR alpha, inhibits PML-dependent apoptotic pathways in a dominant negative fashion and blocks myeloid differentiation by direct transcriptional inhibition of retinoic acid target genes. This transcriptional inhibition is mediated by recruitment of co-repressor proteins and resultant deacetylation of histones in the promoter regions of genes (yet to be identified) that control promyelocyte development. In the presence of high levels of all-trans retinoic acid (ATRA), both PML-dependent apoptotic mechanisms and myeloid-specific gene expression programs are reactivated. In the clinic, the combination of anthracycline-based chemotherapy plus ATRA cures approximately 80% of APL patients, and a high percentage of relapsed patients can achieve second remissions with arsenic trioxide. With the publication of results from the European APL 93 trial, the 'standard-of-care' for induction treatment of APL now includes ATRA plus concurrent anthracycline-based chemotherapy. The amount and type of consolidation therapy necessary for an individual APL patient remains somewhat of an open question, but at present should include at least two cycles of chemotherapy. Based on recent trials that demonstrate a benefit of maintenance ATRA (+/- low-dose chemotherapy), all APL patients should probably receive some type of maintenance therapy. While the above approach currently cures the majority of APL patients, future improvements in the treatment of this disease will require risk-adapted protocols that incorporate real-time molecular monitoring and appropriate introduction of novel therapeutic agents.

Identification of B94 (TNFAIP2) as a potential retinoic acid target gene in acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL) is characterized by a block to myeloid differentiation caused by expression of the fusion oncoprotein promyelocytic leukemia-retinoic acid receptor alpha (PML-RARalpha). The purpose of this study was to identify genes that are regulated in a PML-RARalpha-dependent fashion by retinoic acid (RA), because such genes may be integrally involved in APL pathogenesis and/or myeloid differentiation. A cDNA microarray approach was used to identify genes induced in response to RA in TF1 myeloid leukemia cells expressing PML-RARalpha (TF1-PR cells). The B94 gene (TNFAIP2; Unigene Hs.101382), originally identified as a tumor necrosis factor alpha-inducible gene in endothelial cells, was one of several genes found to be induced by RA specifically in TF1-PR cells, but not in TF1-neo (control) cells. The induction of B94 was most pronounced in cells expressing the PML-RARalpha short isoform and was negligible in cells that expressed a mutant PML-RARalpha protein containing a deletion of the PML coiled-coil domain. B94 induction by RA occurred within 1 h, did not require new protein synthesis, and was inhibited by actinomycin D, suggesting rapid transcriptional activation. B94 was also induced by RA in NB4, UF1, and HL-60 cells, but not in other hematopoietic cell lines tested, suggesting that its up-regulation by RA may be specific to cells that express PML-RARalpha or are at the late myeloblast or promyelocyte stage of myeloid development. A screen of bone marrow cells from normal donors or patients with acute myelogenous leukemia showed that B94 was highly expressed in normal marrow and in marrow from patients with acute myelogenous leukemia French-American-British subtypes M0-M2, but was repressed in marrow cells from APL patients. Treatment of APL blasts in vitro with all-trans-RA resulted in up-regulation of B94 mRNA. These results suggest that B94 plays a role in myeloid development and support the hypothesis that B94 is a target gene of PML-RARalpha in APL.

The beta3A subunit gene (Ap3b1) of the AP-3 adaptor complex is altered in the mouse hypopigmentation mutant pearl, a model for Hermansky-Pudlak syndrome and night blindness.

Lysosomes, melanosomes and platelet-dense granules are abnormal in the mouse hypopigmentation mutant pearl. The beta3A subunit of the AP-3 adaptor complex, which likely regulates protein trafficking in the trans - Golgi network/endosomal compartments, was identified as a candidate for the pearl gene by a positional/candidate cloning approach. Mutations, including a large internal tandem duplication and a deletion, were identified in two respective pearl alleles and are predicted to abrogate function of the beta3A protein. Significantly lowered expression of altered beta3A transcripts occurred in kidney of both mutant alleles. The several distinct pearl phenotypes suggest novel functions for the AP-3 complex in mammals. These experiments also suggest mutations in AP-3 subunits as a basis for unique forms of human Hermansky-Pudlak syndrome and congenital night blindness, for which the pearl mouse is an appropriate animal model.

Mutations in orthologous genes in human spondyloepimetaphyseal dysplasia and the brachymorphic mouse.

The osteochondrodysplasias are a genetically heterogeneous group of disorders affecting skeletal development, linear growth and the maintenance of cartilage and bone. We have studied a large inbred Pakistani family with a distinct form of recessively inherited spondyloepimetaphyseal dysplasia (SEMD) and mapped a gene associated with this dwarfing condition to chromosome 10q23-24, a region syntenic with the locus for the brachymorphic mutation on mouse chromosome 19. We identified two orthologous genes, ATPSK2 and Atpsk2, encoding novel ATP sulfurylase/APS kinase orthologues in the respective regions of the human and mouse genomes. We characterized a nonsense mutation in ATPSK2 in the SEMD family and a missense mutation in the region of Atpsk2 encoding the APS kinase activity in the brachymorphic mouse. ATP sulfurylase/APS kinase catalyses the metabolic activation of inorganic sulfate to PAPS, the universal donor for post-translational protein sulfation in all cell types. The cartilage-specificity of the human and mouse phenotypes provides further evidence of the critical role of sulfate activation in the maturation of cartilage extracellular matrix molecules and the effect of defects in this process on the architecture of cartilage and skeletogenesis.

Mouse models of Hermansky Pudlak syndrome: a review.

Hermansky Pudlak Syndrome (HPS) is a recessively inherited disease affecting the contents and/or the secretion of several related subcellular organelles including melanosomes, lysosomes, and platelet dense granules. It presents with disorders of pigmentation, prolonged bleeding, and ceroid deposition, often accompanied by severe fibrotic lung disease and colitis. In the mouse, the disorder is clearly multigenic, caused by at least 14 distinct mutations. Studies on the mouse mutants have defined the granule abnormalities of HPS and have shown that the disease is associated with a surprising variety of phenotypes affecting many tissues. This is an exciting time in HPS research because of the recent molecular identification of the gene causing a major form of human HPS and the expected identifications of several mouse HPS genes. Identifications of mouse HPS genes are expected to increase our understanding of intracellular vesicle trafficking, lead to discovery of new human HPS genes, and suggest diagnostic and therapeutic approaches toward the more severe clinical consequences of the disease.

The mouse pale ear (ep) mutation is the homologue of human Hermansky-Pudlak syndrome.

The recessive mutation at the pale ear (ep) locus on mouse chromosome 19 was found to be the homologue of human Hermansky-Pudlak syndrome (HPS). A positional cloning strategy using yeast artificial chromosomes spanning the HPS locus was used to identify the HPS gene and its murine counterpart. These genes and their predicted proteins are highly conserved at the nucleotide and amino acid levels. Sequence analysis of the mutant ep gene revealed the insertion of an intracisternal A particle element in a protein-coding 3' exon. Here we demonstrate that mice with the ep mutation exhibit abnormalities similar to human HPS patients in melanosomes and platelet-dense granules. These results establish an animal model of HPS and will facilitate biochemical and molecular analyses of the functions of this protein in the membranes of specialized intracellular organelles.

An integrated genetic map of the pearl locus of mouse chromosome 13.

We have used a Mus domesticus/spretus congenic animal and two interspecific backcross panels to map genetically 30 sequence-tagged sites (STSs) and 13 genes to the vicinity of the pearl locus on mouse chromosome 13. The STSs defining the mapped region are from D13Mit9 to D13Mit37, spanning 10.6 cM. Genes mapped to this region include Versican (Cspg2), GTPase activating protein (Rasa), dihydrofolate reductase (Dhfr), arylsulfatase (As-1), thrombin receptor (Cf2r), hexosaminidase b(Hexb), 3-hydroxy-3-methylglutaryl coenzyme A reductase (Hmgcr), microtubule associated protein 5/1b (Mtap5), phosphodiesterase (Pde), phosphatidylinositol 3' kinase (Pik3rl), rat integrin a1-subunit (Itga1), collagen receptor a2-subunit (Itga2), and 5-hydroxytryptamine 1a receptor (Htr1a). This high resolution genetic map of the pearl region of chromosome 13 establishes the order of multiple markers, including genes whose human homologs are located within a limited region of human chromosome 5, with respect to the phenotypic anchor marker pearl.

Molecular markers near the mouse brachymorphic (bm) gene, which affects connective tissues and bleeding time.

Several inherited skeletal/connective tissue defects are associated with hemorrhagic disorders in humans. Accordingly, three mouse mutants (brachymorphic [bm], hemimelic extra toes [Hx], and ulnaless [Ul]), with inherited skeletal abnormalities, were analyzed for hemorrhagic tendencies. All three had prolonged bleeding times. Platelet numbers, size, and function, as well as common soluble plasma clotting factors, were not measurably affected. To further define the bm mutation, its chromosomal location relative to 19 other molecular markers was determined to a high resolution in a large interspecific backcross. Several microsatellite markers were found to be very closely linked to bm and should provide useful entry points for the eventual identification of this gene by positional/candidate cloning techniques. These results suggest that inherited skeletal abnormalities and bleeding tendencies are associated more frequently in both humans and animal models than is commonly recognized. Identification of these genes may reveal novel relationships between osteogenesis and hemostasis.

The beta-glucuronidase propeptide contains a serpin-related octamer necessary for complex formation with egasyn esterase and for retention within the endoplasmic reticulum.

beta-Glucuronidase is retained within the endoplasmic reticulum (ER) via complex formation with esterase-22 (egasyn), which in turn has a COOH-terminal HTEL ER retention sequence. To identify the regions of glucuronidase that interact with egasyn, complex formation was assayed in COS cells cotransfected with egasyn cDNA and with either deletion constructs of glucuronidase or with constructs containing specific glucuronidase propeptide sequences appended to the carboxyl terminus of a rat secretory protein alpha 1-acid glycoprotein. The region of glucuronidase essential for complex formation is a linear octamer sequence at the COOH terminus of the propeptide. A portion of this octamer is similar to a sequence near the reactive site of serpins. This and associated data indicate that an interaction related to that between serine proteinases and their serpin inhibitors retains beta-glucuronidase within the ER. Further, attachment of this octamer sequence provides an alternative method of targeting proteins to the ER lumen of any cell that contains egasyn. These and related results demonstrate that complex formation with esterases/proteinases within the ER is important in the subcellular targeting and/or processing of certain proteins.

The carbohydrate structure of the asparagine-linked oligosaccharides of rat plasma thiostatin.

The complete carbohydrate structure of the asparagine-linked oligosaccharides of rat plasma thiostatin was elucidated through chemical and enzymatic methods including gas chromatography-mass spectrometry (GC-MS) and lectin affinity chromatography. Pronase digestion of thiostatin yielded a major glycopeptide fraction with asparagine the most abundant amino acid present. Based on one mole of aspartic acid, the following molar ratios obtained for the four major amino acids: aspartic acid (1.0), threonine (0.53), glycine (0.48) and serine (0.30). Neutral sugar analysis yielded a 3:2 molar ratio for mannose to galactose based on an assigned value to mannose of 3. On this basis, the fraction also contained 3 residues of sialic acid and, on average, 0 to 1 residue of fucose. GC-MS of partially methylated alditol acetates from the glycopeptide fraction identified the presence of biantennary and triantennary structure. Analyses of the neutral sugar and amino-acid composition, together with methylation data, support a biantennary N-linked structure for this major glycopeptide fraction and a triantennary N-linked structure as a lesser component. Sequencing of the desialyated 14C-labelled glycopeptide fraction by sequential exoglycosidase digestion and lectin affinity chromatography uncovered the following saccharide order: terminal galactose, N-acetylglucosamine and pentasaccharide inner core. This sequence is consistent with the N-linked glycan structures demonstrated by methylation and compositional analyses.

Purification and characterization of acute phase rat plasma thiostatin.

Thiostatin was purified from acute phase plasma of turpentine-treated rats by a novel, single-step carboxymethyl-papain Sepharose 4B column chromatographic procedure. Purified thiostatin appeared as a single band in SDS-PAGE with an estimated molecular weight of 68,000. Western blot with polyclonal rabbit anti-thiostatin IgG confirmed a homogeneous immuno-reactive 68 kDa species. Specific activity, as determined by enzyme-linked immunosorbent assay (ELISA), was 0.972 mg kininogen equivalent per mg protein. The yield of thiostatin exceeded 60% and the protein was purified 10.7-fold.

Role of carbohydrate in rat plasma thiostatin: deglycosylation destroys cysteine proteinase inhibition activity.

Rat plasma thiostatin, a 68kDa glycoprotein cysteine proteinase inhibitor, was chemically deglycosylated with trifluoromethanesulfonic acid. Both native and deglycosylated species were characterized with regard to amino acid and carbohydrate composition, homogeneity by SDS-PAGE, immunoreactivity by Western blot, and cysteine proteinase inhibitor activity. Deglycosylation of thiostatin did not alter the integrity of the protein backbone nor change the immunologic recognition of the molecule by polyclonal rabbit anti-rat thiostatin. However, deglycosylation did destroy cysteine proteinase inhibitor activity activity as measured against papain.