Showing papers by "Arnold J. Levine published in 2015"

The Sequence of the Human Genome

Abstract: A 2.91-billion base pair (bp) consensus sequence of the euchromatic portion of the human genome was generated by the whole-genome shotgun sequencing method. The 14.8-billion bp DNA sequence was generated over 9 months from 27,271,853 high-quality sequence reads (5.11-fold coverage of the genome) from both ends of plasmid clones made from the DNA of five individuals. Two assembly strategies—a whole-genome assembly and a regional chromosome assembly—were used, each combining sequence data from Celera and the publicly funded genome effort. The public data were shredded into 550-bp segments to create a 2.9-fold coverage of those genome regions that had been sequenced, without including biases inherent in the cloning and assembly procedure used by the publicly funded group. This brought the effective coverage in the assemblies to eightfold, reducing the number and size of gaps in the final assembly over what would be obtained with 5.11-fold coverage. The two assembly strategies yielded very similar results that largely agree with independent mapping data. The assemblies effectively cover the euchromatic regions of the human chromosomes. More than 90% of the genome is in scaffold assemblies of 100,000 bp or more, and 25% of the genome is in scaffolds of 10 million bp or larger. Analysis of the genome sequence revealed 26,588 protein-encoding transcripts for which there was strong corroborating evidence and an additional ∼12,000 computationally derived genes with mouse matches or other weak supporting evidence. Although gene-dense clusters are obvious, almost half the genes are dispersed in low G+C sequence separated by large tracts of apparently noncoding sequence. Only 1.1% of the genome is spanned by exons, whereas 24% is in introns, with 75% of the genome being intergenic DNA. Duplications of segmental blocks, ranging in size up to chromosomal lengths, are abundant throughout the genome and reveal a complex evolutionary history. Comparative genomic analysis indicates vertebrate expansions of genes associated with neuronal function, with tissue-specific developmental regulation, and with the hemostasis and immune systems. DNA sequence comparisons between the consensus sequence and publicly funded genome data provided locations of 2.1 million single-nucleotide polymorphisms (SNPs). A random pair of human haploid genomes differed at a rate of 1 bp per 1250 on average, but there was marked heterogeneity in the level of polymorphism across the genome. Less than 1% of all SNPs resulted in variation in proteins, but the task of determining which SNPs have functional consequences remains an open challenge.

TAp73 opposes tumor angiogenesis by promoting hypoxia-inducible factor 1α degradation

Abstract: Tumor hypoxia and hypoxia-inducible factor 1 (HIF-1) activation are associated with cancer progression. Here, we demonstrate that the transcription factor TAp73 opposes HIF-1 activity through a nontranscriptional mechanism, thus affecting tumor angiogenesis. TAp73-deficient mice have an increased incidence of spontaneous and chemically induced tumors that also display enhanced vascularization. Mechanistically, TAp73 interacts with the regulatory subunit (α) of HIF-1 and recruits mouse double minute 2 homolog into the protein complex, thus promoting HIF-1α polyubiquitination and consequent proteasomal degradation in an oxygen-independent manner. In human lung cancer datasets, TAp73 strongly predicts good patient prognosis, and its expression is associated with low HIF-1 activation and angiogenesis. Our findings, supported by in vivo and clinical evidence, demonstrate a mechanism for oxygen-independent HIF-1 regulation, which has important implications for individualizing therapies in patients with cancer.

Distinguishing the immunostimulatory properties of noncoding RNAs expressed in cancer cells.

Abstract: Recent studies have demonstrated abundant transcription of a set of noncoding RNAs (ncRNAs) preferentially within tumors as opposed to normal tissue. Using an approach from statistical physics, we quantify global transcriptome-wide motif use for the first time, to our knowledge, in human and murine ncRNAs, determining that most have motif use consistent with the coding genome. However, an outlier subset of tumor-associated ncRNAs, typically of recent evolutionary origin, has motif use that is often indicative of pathogen-associated RNA. For instance, we show that the tumor-associated human repeat human satellite repeat II (HSATII) is enriched in motifs containing CpG dinucleotides in AU-rich contexts that most of the human genome and human adapted viruses have evolved to avoid. We demonstrate that a key subset of these ncRNAs functions as immunostimulatory "self-agonists" and directly activates cells of the mononuclear phagocytic system to produce proinflammatory cytokines. These ncRNAs arise from endogenous repetitive elements that are normally silenced, yet are often very highly expressed in cancers. We propose that the innate response in tumors may partially originate from direct interaction of immunogenic ncRNAs expressed in cancer cells with innate pattern recognition receptors, and thereby assign a previously unidentified danger-associated function to a set of dark matter repetitive elements. These findings potentially reconcile several observations concerning the role of ncRNA expression in cancers and their relationship to the tumor microenvironment.

Involvement of tumor suppressors PTEN and p53 in the formation of multiple subtypes of liposarcoma

Abstract: Liposarcoma (LPS) is a type of soft tissue sarcoma that mostly occurs in adults, and in humans is characterized by amplifications of MDM2 and CDK4. The molecular pathogenesis of this malignancy is still poorly understood and, therefore, we developed a mouse model with conditional inactivation of PTEN and p53 to investigate these pathways in the progression of the disease. We show that deletion of these two tumor suppressors cooperate in the formation of multiple subtypes of LPS (from well-differentiated LPS to pleomorphic LPS). In addition, progression of the tumors is further characterized by the expression of D cyclins and CDK4/6, which allow for continued cell division. Microarray analysis also revealed novel genes that are differentially expressed between different subtypes of LPS, which could aid in understanding the disease and to unravel potential new therapeutic targets.

The Evolution of Tumors in Mice and Humans with Germline p53 Mutations

Abstract: Mice with a homozygous p53 gene deletion develop thymic lymphomas by 9 wk of age. Using the sequence of the rearranged T-cell receptor gene from each clone of cells in the thymus, one can determine the number of independent transformation events. These tumors are oligoclonal, occurring at a frequency of 0.13-0.8 new cancer clones per day. By 20 wk only a few clones are detected, indicating competition among transformed cell clones. DNA sequencing of these tumors demonstrates a point mutation frequency of one per megabase and many genes that are consistently amplified or deleted in independent tumors. The tumors begin with an inherited p53 gene deletion. Next is a PTEN mutation in a stem cell or progenitor cell, before the rearrangement of the T-cell receptor. After that, the T-cell clone selects gene amplifications in cyclin D and cdk-6, and in Ikaros in the Notch pathway. Humans heterozygous for the p53 mutant gene in the germline (Li-Fraumeni syndrome) develop cancers at an early age. The penetrance of heterozygous p53 mutations is ∼93% of individuals developing tumors over their lives. At older ages the remaining 7% of this Li-Fraumeni population actually have a lower risk of developing tumors than the population at large with wild-type p53 genes.

Stem cells, aging and cancers

Abstract: Recently tissue specific stem cells from mammals have been isolated, characterized and grown in three-dimensional cultures producing faithful organoids composed of all the cell and tissue types of the organ [1] In all cases of diverse tissue specific stem cells the WNT signal transduction pathway functions to promote replication of the stem cells and is regulated by R-spondins acting upon G-linked receptors, LGR 4, 5 and 6, which prove to be excellent biomarkers for these stem cells [1) John Cairns first pointed out [2] that the stem cells in rapidly renewing tissues of animals with long life spans compete for space and nutrients in their nitches and they will undergo mutations and natural selection for variants that propagate well and have reduced potentials for differentiation leading to the precursors of cancers Based upon this he proposed that such stem cells required excellent DNA repair processes that reduce the mutation rate to prevent or lower the incidence of cancers Indeed it has been possible to introduce mutations (APC, RAS, TGF-beta and p53) into normal colon stem cells and reproduce adenocarcinomas of the colon [3] In support of the stem cell hypothesis as the initiator of cancers p53 mutations have been isolated from clones (derived from stem cells) of epithelial cells of the esophagus in patients with Barrett's esophagus and in hematopoetic stem cells and progenitor cells from patients treated for AML [4] A second line of support for this idea comes from the observations measuring the number of hematopoetic stem cells in mice, cats or humans [5] and surprisingly the number of these stem cells increases with age (selection of more fit cells for replication) but the ability to transplant these cells into a recipient declines with age (selection of less fit for differentiation) All these results are consistent with the hypothesis that mutations arise in tissue specific stem cells that favor cell division and reduce the probability of commitment to differentiation What is the nature of these mutations? In stem cells a splice variant of the p53 protein (delta-40-p53) is produced which, deletes the first 40 amino acids of p53 and the first transactivation domain so that the delta-40- p53 protein fails to transcribe efficiently the genes that full length p53 normally regulates (like p21) [6] Restoration of the full- length p53 protein and its activation in stem cells stops cell division (via p21) and triggers differentiation of the stem cell [7] The activation of full- length p53 in stem cells followed by differentiation has been observed in tissue regeneration of planarians and salamanders, as well as in mouse IPS cells and cancerous teratocarcinoma stem cells [7] In mice with inherited p53 mutations the T-cell lineage stem cell acquires and selects PTEN mutations prior to entry of the stem cell into the thymus and the V-D-J recombination steps and the production of differentiated T-cells [8] While p53 and possibly PTEN are examples of mutations in stem cells that promote cell division and slow differentiation, we can expect that an entire class of mutations in genes that mediate epigenetic changes should also produce these results While mutations residing and accumulating in tissue specific stem cells over a lifetime can give rise to cancers, they can also produce abnormalities in tissue functions that contribute to the diseases of the elderly One might predict a decline in the balance and efficiency of the immune system, abnormal cytokine production, poor metabolic functions and neurodegeneration all could result from mutations that may accumulate in the tissue specific stem cells over a lifetime It is ironic that the very body plan of vertebrates employing stem cells to regenerate our tissues, give us longevity and eliminate large numbers of poorly functioning cells through terminal differentiation and cell death relies upon the presence of stem cell populations that compete over a life time to produce the fittest stem cell with a mutational profile that maximizes replication and reduces the efficiency of differentiation These ideas bring together stem cells, cancers and longevity employing a common evolutionary process acting in both an individual and in populations