A Unique Microglia Type Associated with Restricting Development of Alzheimer’s Disease

PLoS BIOLOGY Senescence-Associated Secretory Phenotypes Reveal Cell-Nonautonomous Functions of Oncogenic RAS and the p53 Tumor Suppressor Jean-Philippe Coppe 1 , Christopher K. Patil 1[ , Francis Rodier 1,2[ , Yu Sun 3 , Denise P. Mun oz 1,2 , Joshua Goldstein 1¤ , Peter S. Nelson 3 , Pierre-Yves Desprez 1,4 , Judith Campisi 1,2* 1 Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, United States of America, 2 Buck Institute for Age Research, Novato, California, United States of America, 3 Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America, 4 California Pacific Medical Center Research Institute, San Francisco, California, United States of America Cellular senescence suppresses cancer by arresting cell proliferation, essentially permanently, in response to oncogenic stimuli, including genotoxic stress. We modified the use of antibody arrays to provide a quantitative assessment of factors secreted by senescent cells. We show that human cells induced to senesce by genotoxic stress secrete myriad factors associated with inflammation and malignancy. This senescence-associated secretory phenotype (SASP) developed slowly over several days and only after DNA damage of sufficient magnitude to induce senescence. Remarkably similar SASPs developed in normal fibroblasts, normal epithelial cells, and epithelial tumor cells after genotoxic stress in culture, and in epithelial tumor cells in vivo after treatment of prostate cancer patients with DNA- damaging chemotherapy. In cultured premalignant epithelial cells, SASPs induced an epithelial–mesenchyme transition and invasiveness, hallmarks of malignancy, by a paracrine mechanism that depended largely on the SASP factors interleukin (IL)-6 and IL-8. Strikingly, two manipulations markedly amplified, and accelerated development of, the SASPs: oncogenic RAS expression, which causes genotoxic stress and senescence in normal cells, and functional loss of the p53 tumor suppressor protein. Both loss of p53 and gain of oncogenic RAS also exacerbated the promalignant paracrine activities of the SASPs. Our findings define a central feature of genotoxic stress-induced senescence. Moreover, they suggest a cell-nonautonomous mechanism by which p53 can restrain, and oncogenic RAS can promote, the development of age-related cancer by altering the tissue microenvironment. Citation: Coppe JP, Patil CK, Rodier F, Sun Y, Mun oz DP, et al. (2008) Senescence-associated secretory phenotypes reveal cell-nonautonomous functions of oncogenic RAS and the p53 tumor suppressor. PLoS Biol 6(12): e301. doi:10.1371/journal.pbio.0060301 Introduction Cancer is a multistep disease in which cells acquire increasingly malignant phenotypes. These phenotypes are acquired in part by somatic mutations, which derange normal controls over cell proliferation (growth), survival, invasion, and other processes important for malignant tumorigenesis [1]. In addition, there is increasing evidence that the tissue microenvironment is an important determinant of whether and how malignancies develop [2,3]. Normal tissue environ- ments tend to suppress malignant phenotypes, whereas abnormal tissue environments such at those caused by inﬂammation can promote cancer progression. Cancer development is restrained by a variety of tumor suppressor genes. Some of these genes permanently arrest the growth of cells at risk for neoplastic transformation, a process termed cellular senescence [4–6]. Two tumor suppressor pathways, controlled by the p53 and p16INK4a/pRB proteins, regulate senescence responses. Both pathways integrate multiple aspects of cellular physiology and direct cell fate towards survival, death, proliferation, or growth arrest, depending on the context [7,8]. Several lines of evidence indicate that cellular senescence is a potent tumor-suppressive mechanism [4,9,10]. Many poten- tially oncogenic stimuli (e.g., dysfunctional telomeres, DNA PLoS Biology | www.plosbiology.org damage, and certain oncogenes) induce senescence [6,11]. Moreover, mutations that dampen the p53 or p16INK4a/pRB pathways confer resistance to senescence and greatly increase cancer risk [12,13]. Most cancers harbor mutations in one or both of these pathways [14,15]. Lastly, in mice and humans, a senescence response to strong mitogenic signals, such as those delivered by certain oncogenes, prevents premalignant lesions from progressing to malignant cancers [16–19]. Academic Editor: Julian Downward, Cancer Research UK, United Kingdom Received June 27, 2008; Accepted October 22, 2008; Published December 2, 2008 Copyright: O 2008 Coppe et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Abbreviations: CM, conditioned medium; DDR, DNA damage response; ELISA, enzyme-linked immunosorbent assay; EMT, epithelial–mesenchymal transition; GSE, genetic suppressor element; IL, interleukin; MIT, mitoxantrone; PRE, presenescent; PrEC, normal human prostate epithelial cell; REP, replicative exhaustion; SASP, senescence-associated secretory phenotype; SEN, senescent; shRNA, short hairpin RNA; XRA, X-irradiation * To whom correspondence should be addressed. E-mail: jcampisi@lbl.gov [ These authors contributed equally to this work. ¤ Current address: Genomics Institute of the Novartis Research Foundation, San Diego, California, United States of America December 2008 | Volume 6 | Issue 12 | e301

Senescence-Associated Secretory Phenotypes Reveal Cell-Nonautonomous Functions of Oncogenic RAS and the p53 Tumor Suppressor

Mini Mental State

There are a number of ways in which a clinical diagnosis of dementia of the Alzheimer type can be made – the application of clinical criteria is the commonest but ancillary techniques such as neuroima

Clinical Diagnosis of Alzheimer's Disease

Despite mortality due to communicable diseases, poverty, and human conflicts, dementia incidence is destined to increase in the developing world in tandem with the ageing population Current data from developing countries suggest that age-adjusted dementia prevalence estimates in 65 year olds are high (>or=5%) in certain Asian and Latin American countries, but consistently low (1-3%) in India and sub-Saharan Africa; Alzheimer's disease accounts for 60% whereas vascular dementia accounts for approximately 30% of the prevalence Early-onset familial forms of dementia with single-gene defects occur in Latin America, Asia, and Africa Illiteracy remains a risk factor for dementia The APOE epsilon4 allele does not influence dementia progression in sub-Saharan Africans Vascular factors, such as hypertension and type 2 diabetes, are likely to increase the burden of dementia Use of traditional diets and medicinal plant extracts might aid prevention and treatment Dementia costs in developing countries are estimated to be US$73 billion yearly, but care demands social protection, which seems scarce in these regions

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Alzheimer's disease and vascular dementia in developing countries: prevalence, management, and risk factors

190 Punjabis (Hindus and Sikhs) of Chandigarh and 152 Hindus of Jodhpur (Rajasthan) were examined for six genetic markers, four of which (APO C-II, C6, C7 and FXIIIA) were not studied before in Asiatic Indians. For APO C-II and C7 only the common phenotype was found in a total of 229 and 99 subjects, respectively. For the remaining four markers the two samples were pooled since the gene frequency estimates were not significantly different: FXIIIA*2 = 0.205 +/- 0.016; C6*B = 0.366 +/- 0.037; PGM1*2 = 0.247 +/- 0.017; PGD*C = 0.041 +/- 0.008. These data may contribute to evaluate the extent of the Mongoloid genetic admixture into the Caucasoid gene pool of the Punjab and Rajasthan Hindu population.

A survey of six genetic markers on the populations of Punjab and Rajasthan (India).

Human placental alkaline phosphatase (ALP) activity has been determined in specimens obtained from 562 Italian subjects. The mean activities of the three common homozygotes (Pl 2 = 4.70 ± 0.24, Pl 1 = 4.09 ± 0.08, and Pl 3 = 2.15 ± 0.71 μmol of p-nitrophenol produced) were significantly different.

The differences among the various allelic forms account for 10% of the total quantitative variation of the human placental alkaline phosphatase.

Human placental alkaline phosphatase electrophoretic alleles: Quantitative studies

In a survey on 2405 placentae of different ethnic origin evidence for at least 11 different mutated alleles at Pi locus was obtained by identifying 60 rare variants of human placental alkaline phosphatase. An attempt has been made to compare our results with those previously reported in the literature. Placental alkaline phosphatase is a polymorphic tissue glycoprotein normally produced during foetal life. A fraction of alkaline phosphatase which has physical, chemical and immunological properties very similar to those of placental phosphatase has been detected in a number of adults with malignant tumors (Fishman, 1974). The enzyme synthesis is controlled by an autosomal locus (Pi) with three common codominant alleles Pi1, Pi2, Pi3 (or old nomenclature Plsl, Plfl, PI11). Since the first report on electrophoretic variations of placental alkaline phosphatase (Robson and Harris, 1965; 1967) many populations have been examined for this system and all have been found polymorphic. The world distribution of its gene frequencies shows that it is a good anthropological marker (Lucarelli, et al. 1979). In most cases, besides the six common phenotypes 1, 2, 3, 1-2, 1-3 and 2-3, unusual patterns have been observed. In this paper we describe the rare variants found by us and confirm the exceptional degree of genetic variability of this system. Materials and Methods Placental samples of Negro (291), English (97), Italian (171), Mixed White of European extraction (667), Jewish (164) and Puerto Rican (69) subjects were collected consecutively during the period from September 1968 to May 1971 at the Yale New Haven Hospital and sent to Rome for analysis. Placental samples of Italian subjects (946) were collected on several occasions in different Italian districts. Italian, English, Jewish and 1National Research Council, Center for Evolutionary Genetics, Department of Genetics, University of Rome, School of Science, Rome, Italy. Human Biology, September 1979, Vol. 51, No. 3, pp. 341-352. ® Wayne State University Press, 1979 This content downloaded from 157.55.39.210 on Sun, 31 Jul 2016 05:11:48 UTC All use subject to http://about.jstor.org/terms 342 R. Palmarini), R. M. Corbo, R. Scacchi and P. Lucarelli Mixed White of European origin subjects were pooled and combined with the Italians of Italy to constitute the Caucasian population. The results obtained from this material, not concerning rare variants, have already been published (Bottini et al. 1970; Lucarelli et al. 1970; Bottini et al. 1972a; Bottini et al. 1972b; Palmarino et al. 1975; Lucarelli et al. 1978). Tissue extracts were prepared as described by Boyer (1963); starch-gel electrophoresis at pH 6.0 and 8.6 were performed according to Robson and Harris (1965) and enzymatic activity was developed by Boyer's method (1961). Nomenclature: we have denoted the three common Pi alleles as Pi1, Pi2 and Pi3 according to Donald and Robson (1974) and the rare ones have been numbered in order of their discovery from Pi4' to Pi29' . The index has been used to differentiate our classification numbers from those previously used in the literature (Donald and Robson, 1974).

Human placental alkaline phosphatase: analysis of genetically determined rare variants.

The effect of three thiol reagents on erythrocyte adenosine deaminase (ADA) activity has been studied. Oxidized glutathione and iodoacetate do not alter ADA activity, while the treatment with p-chloromercuribenzoate at similar concentrations results in a reduction of enzymatic activity which is statistically significant only for ADA 1, but not ADA 2-1 phenotype haemolysates.

Effect of some thiol reagents on erythrocyte adenosine deaminase (ADA) activity.

The genetic structure of Rajasthan Hindus and Punjab-Haryana Hindus and Sikhs has been studied for ABO, RH, APOC2, C6, C7, F13A, F13B, HP, ORM1, ACP1, ADA, AK1, ESD, GLO1, PGD, PGM1 subtyping, and PGP This is the first genetic survey on Hindus of Rajasthan Furthermore, many of these markers have never been studied on Hindus before (APOC2, C6, C7, F13A, F13B, ORM1, PGP) These data, together with those previously available for Hindus, have been utilized to analyze the within-Hindus genetic heterogeneity by RST statistic and correspondence analysis The genetic relationships of Hindus to other Causcasoid populations were also investigated In the first analysis, two eastern states (Orissa and Andhra Pradesh) were found to be quite separate from each other and clearly distinct from the northwestern and western states Out of the markers which could not be utilized in this analysis, PGM1 subtyping turned out to discriminate between the Dravidian-speaking and the Indo-Aryan-speaking Hindus The second analysis shows a clear-cut separation of Hindus from Europeans, with Near Eastern and Middle Eastern populations genetically in an intermediate position

Genetic heterogeneity among the Hindus and their relationships with the other "Caucasoid" populations: new data on Punjab-Haryana and Rajasthan Indian states.

Rosa Maria Corbo

Papers

A survey of six genetic markers on the populations of Punjab and Rajasthan (India).

Human placental alkaline phosphatase electrophoretic alleles: Quantitative studies

Human placental alkaline phosphatase: analysis of genetically determined rare variants.

Effect of some thiol reagents on erythrocyte adenosine deaminase (ADA) activity.

Genetic heterogeneity among the Hindus and their relationships with the other "Caucasoid" populations: new data on Punjab-Haryana and Rajasthan Indian states.