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ACTION DE RECHERCHE CONCERTEE

2009-2014

Effect of Genotoxic Agents and Viral Infection on Alternative Splicing

Acronym: Alternate

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1. Table of contents

1. Table of contents 22. Proposal title 33. Network composition 34. Summary of the proposal 45. Project description 6 A. State of the art and description of the proposal 6 Alternative splicing and diseases 6 DNA damage and damage-induced signalling 7 Alternative splicing induced by DNA damages 8 a. Physical agents 11 b. Chemical agents 11 b1: environmental compounds 11 b2: Therapeutic compounds 13 b3: Bile and acid salts 13 c. Biological agents 14 c1: Viruses and DNA damage 14 c2: Viruses and alternative splicing 15 c3: Selection of viruses to be investigated 15 Goals of the study 16 Motivation of the present proposal 20 Expected advances 21 B. Milestones of the project 22 C. Schedule and procedures 34 D. Participation of the partners in the different work packages 35 Network management 38 a. Coordination 38 b. Work package leader 38 c. Meetings 38 d. Ethic 396. Budget 40 A. Global budget 40 B. Pluri-annual budget 42 C. Composition of the partners laboratories 437. List of equipment 458. Annexe 1: Partners 469. Annexe 2: Referees 67

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2. Proposal title Effect of genotoxic agents and viral infection on alternative splicing Keywords : DNA damage, gene splicing, genome-wide, pollutant, chemotherapy, virus, Barrett’s oesophagus

3. Network composition Coordinator – Partner 1 Dr. Alain Colige Laboratory of Connective Tissues Biology GIGA-Research Tour de Pathologie B23/3 University of Liège B-4000 Liège Tel.: +32 4 3662738 Fax: +32 4 3662457 Email: acolige@ulg.ac.be Website: http://www.lctb.ulg.ac.be

Co-Investigator Partner 1 Dr. Charles Lambert Laboratory of Connective Tissues Biology GIGA-Research Tour de Pathologie B23/3 University of Liège B-4000 Liège Tel.: +32 4 3662458 Fax: +32 4 3662457 Email: c.lambert@ulg.ac.be Website: http://www.lctb.ulg.ac.be

Partner 2 Dr. Yvette Habraken Laboratory of Fundamental Virology GIGA-Research Tour GIGA, B34/2 University of Liège B-4000 Liège Tel.: +32 4 3662447 Fax : +32 4 3664534 Email : yvette.habraken@ulg.ac.be Website : http://www.virofond.ulg.ac.be/ Partner 3 Dr. Philippe Delvenne Laboratory of Pathology GIGA-research Tour de Pathologie B23/4 University of Liège B-4000 Liège Tel.: +32 4 3662564 Fax: +32 4 3662919 Email : P.Delvenne@ulg.ac.be

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4. Summary of the proposal Human genome contains roughly 25.000 genes. However, it encodes for at least 100.000

proteins with unique aminoacid sequences, a number probably largely underestimated. The determination of the complete repertoire of proteins is one of the challenges of the post-genomic era. The increased complexity of the proteome versus genome is mainly due to a post-transcriptional process of alternative splicing (AS) which concerns 90% of the genes. Molecular mechanisms controlling AS contribute to acquired and genetic diseases and could represent relevant therapeutic targets.

Living organisms are continuously challenged with DNA damaging agents either physical

(UV-B and ionizing radiations) or chemical (chemotherapeutic agents and pollutants). We and others have previously shown that DNA damaging agents induce an aberrant splicing of a number of genes, including VEGF-A and genes involved in the cell cycle and survival. The expression of aberrant variants likely helps cells to escape apoptosis induced by DNA damage, what would allow the hazardous transmission of altered genes to daughter cells.

In addition to these recognized DNA damaging agents, viruses can also activate DNA damage

signalling pathways. This activation is observed with retro-virus like HIV-1 that integrates its genome in cellular host genetic material, but also by viruses that do not require genome integration. A limited number of reports also indicate that virus infection affects the splicing of host genes or the expression or activity of proteins involved in pre-mRNA splicing. Our recent data suggest that, infection by alpha herpes virus Varicella Zoster Virus (VZV) induces the phosphorylation and AS of ATM, a central player in the recognition of DNA double-strand breaks (DSB). Together these data suggest that viral infection modulates AS by a mechanism that involves DNA damage recognition. These processes might enable viruses to escape cell vigilance and defence. In addition, aberrant gene splicing might contribute to the severity of the diseases induced by infection.

In-depth investigations of aberrant splicing induced by DNA damage and viral infection are

still lacking. Our project aims at:

1) Evaluating the effect of viral infection and physical or chemical agents on the activation of DNA damage signalling pathways. In particular, we will evaluate ATM/ATR activation and the regulation of selected downstream signalling pathways. Alterations induced by UV, γ-rays, a series of widely-distributed pollutants and DNA damaging agents used in therapy, and four types of viruses (HIV, HPVs, HTLV-1 and VZV) will be analyzed in this study. They represent a considerable health problem and are of particular interest for the processes described here. 2) Analyzing the effect of viral infection and DNA damage on the aberrant splicing of a number of genes and on the expression and the activity of factors involved in AS. Genes aberrantly spliced in response to DNA damaging agents or involved in the pathogenesis of genetic diseases will be investigated in priority. Due to their central role in DNA damage recognition, a particular attention will be paid to AS of ATM and ATR, and to the activity and binding partners of their various splice variant. A genome-wide analysis (spliceomic) will be realised using ‘all-exons’ DNA chips and deep sequencing. Tissue samples of mice treated with genotoxic agents or infected with viruses will be analysed in parallel to in vitro investigations. 3) investigating the effect of a set of pharmacological agents -known to alter AS- on and on the aberrant splicing induced by viruses and DNA-damaging agents and on viral infectivity. A small, though increasing, number of chemicals able to interfere with aberrant splicing is available. We already showed that epigallocathechin gallate and resveratrol inhibit the expression of a specific VEGF variant induced by DNA damaging agents. The effect of analogues and of new compounds is under investigations and will be further analyzed.

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Alternate: The acronym of the proposal directly refers to its main subject aiming to analyze the regulation of alternative splicing mechanisms. However, it also stresses the fact that our proposal provides a novel, alternate view on the pre-mRNA splicing that could represent, alongside with transcriptome and proteome, a “moving target” highly regulated during development and during physiological and pathological processes.

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5. Project description A. State of the art and description of the proposal

Alternative splicing and diseases

In eukaryotes, the sequence of most genes encoding for proteins is composed of exons

interspersed by introns. Exons and introns are transcribed into a single pre-mRNA but only exon sequences are retained during maturation into mRNA, explaining why genes and their respective mRNA are not colinear. This process is referred to as constitutive splicing (Fig. 1). 15% of human genetic diseases are associated with mutations at the splice sites (Krawczak et al., 2007), underscoring the importance of the process in health. Aberrant splicing is observed and likely involved in acquired pathologies such as, among others, a large number of cancers (Fig. 2).

Fig. 1: Different patterns of alternative splicing. Exons are represented by boxes and introns by solid black lines. Dashed lines represent different possibilities for splice site joining. Constitutive regions are shown in gray while alternative ones in a punctuated pattern. (from Blaustein et al., 2007)

Human genome contains roughly 25.000 genes while the proteome contains more than 100.000 different proteins (meaning proteins with unique amino-acid sequences), a number probably largely underestimated. This protein repertoire larger than that predicted from the genome stems from a process called alternative splicing (Fig. 1). This process, concerning more than 90 % of the genes, is developmentally, physiologically and pathologically regulated. It enables the exclusion or the inclusion of one, several, or part of exons in the fully maturated mRNA. In some cases the mRNA is degraded, during a process called nonsense mediated mRNA decay, resulting in the reduced expression of the natural protein encoded by the gene. Alternatively the different proteins (splice variants) produced from one gene have distinct and sometimes opposite functions. Constitutive and alternative splicing are

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performed through a large array of cis-acting splicing factors (> 300), including the SR proteins (arginine and serine rich proteins). The activity of the SR proteins may be regulated by phosphorylation/dephosphorylation of the serine residues within the SR domain by a number of kinases and phosphatases. These reversible phosphorylations link pre-mRNA splicing to signal transduction pathways (Stamm, 2008).

A better understanding of the regulation of alternative splicing mechanisms, together with the identification and characterization of all the splice variants is one of the challenges of the post-genomic era.

Fig. 2: Aberrant splicing of mRNA can give rise to production of protein isoforms with oncogenic properties. (from Pajares et al., 2007) DNA damage and damage-induced signalling

DNA is a continuously damaged by physical or chemical agents, endogenous as well as exogenous, resulting in many types of alterations as base alkylation, oxidation or fragmentation, cross-links, single or double-strand breaks,…. These damage cause a real threat to the cell homeostasis by affecting genome integrity and stability. Damage accumulation leads to numerous diseases such as neurodegenerescence and cancer. Knowledge about cellular responses to DNA damage is growing rapidly but appears to be much more complex than initially expected. In the past, DNA repair systems were studied independently of any other cellular processes. Gradually, it was established that DNA damage responses are not limited to DNA repair but also include signalling cascades leading to cell

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cycle arrest, activation of transcription factor p53 and NF-kB, apoptosis and chromatin remodelling. The last newcomer, in this ever expending field of cellular responses induced by genotoxic lesions, is the alternative splicing of pre-mRNA of a growing number of genes.

Each type of DNA lesion initiates a specific set of responses (identification of the damage, repair and signalling cascades). Cellular responses to DNA double-strand breaks (DSB) are under intense scrutiny because DSB are the most dangerous lesions a cell can withstand and because any mishap in repair process of any kinds of damage will ultimately lead to a double-strand break. If not repaired correctly, these breaks induced genome wide consequences that can lead to chromosomal instability, tumour formation or cell death.

ATM (ataxia telangiectasia mutated) orchestrates most of the cellular responses elicited by double-strand breaks (Shiloh, 2006). It is a high molecular weight serine /threonine protein kinase belonging to the family of phosphoinositide 3-kinase (PI3K) related protein kinase. In cells with an intact genome, ATM is kept inactive as a dimer or multimer. Once double-strand breaks are detected, ATM is very rapidly monomerized and activated by autophosphorylation and acetylation (reviewed in Lavin, 2008). The NBS/Mre11/Rad50 complex recognizes DSB and is necessary for ATM activation. Activated P-ATM can then integrate structures named “nuclear foci” that form at the break site. It contains : NBS/Mre11/Rad50, MDC1, H2AX-P, 53BP1. MDC1 (mediator of DNA damage protein checkpoint 1) is a scaffold protein necessary to the stability of the nuclear foci and recruitment of P-ATM to the breaks (Bekker-Jensen et al., 2006, Lou et al., 2006). Only a fraction of P-ATM is recruited to the nuclear foci, the majority remaining free in the nucleoplasm. Nuclear foci are indispensable for downstream signalling of P-ATM leading to cell cycle arrest.

ATR (Ataxia telangiectasia and Rad3 related protein) and ATM are members of the same kinase family and share some common substrates. ATR is mainly activated upon UV irradiation, alkylation, single-strand breaks and replication arrest, but not by DSB. Its activation depends on the presence of RPA-coated single-strand stretches of DNA. RPA (Replication Protein A) is recruited to DNA single-strand stretches that are generated during replication process as well as repair processes. It is hyperphosphorylated upon DNA damage. ATR dependent signalling has been less characterized that ATM related signalling. Its investigation has long been impeded by the absence of cellular model as ATR deficient cells do not divide. ATR controls cell cycle arrest through the phosphorylation of Chk1. Cross talks are observed between ATM and ATR (Hurley and Bunz, 2007; Myers and Cortez, 2006).

Several publications have indicated that the AS observed after DNA damage is either

ATM or ATR dependent (Chandler et al., 2006, Katzenberger et al., 2006, Mineur et al., 2007). A global proteomic analysis of the phosphoproteins induced by ionizing irradiation has revealed a surprisingly large number of ATM or ATR substrates. 700 different proteins phosphorylated by either ATM or ATR were identified. Among these proteins several are related to pre-mRNA splicing (Matsuoka et al. 2007). Alternative splicing induced by DNA damage

We and others have shown that DNA damage impact on the splicing of a number of genes. They include VEGF-A (Mineur et al., 2007), NBS1 (a sensor of DSB, Takai et al., 2008), CD44 (Filippov et al., 2007) as well as a series of genes participating in the p53

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signalling as PIG-3 (Nicholls et al., 2004, and personal observation), SIRT1 (Xiao et al., 2007, and personal observation), PA26 (Velasco-Miguel et al., 1999), MDM-2 (Chandler et al., 2006, and personal observation) and MDM-X (Chandler et al., 2006), or in apoptosis as Bax (Schmitt et al., 2000), Bcl-X (personal observation), and caspase-2 (Solier et al., 2004). As an example, we have demonstrated that a new VEGF isoform (VEGF111) is produced by a large array of cells treated with physical or chemical genotoxic agents such as UV-B, camptothecin, mitomycin C, Cisplatin and doxorubicin (Mineur et al., 2007, and unpublished observation). The VEGF111 variant is biologically active and, as opposed to other isoforms, highly resistant to proteolysis. A robust angiogenic response was observed around the tumours formed by transplantation of human cells expressing this isoform into nude mice. Preliminary investigations performed in our laboratories on signalling pathways involved in the AS of VEGF induced by DNA damage demonstrated a role for ATM and protein phosphatase-1 (PP1) (Mineur et al., 2007).

Reports indicating that DNA damage regulate splicing are found in the literature;

however, no systematic in depth study of alternative splicing or its impact on human health has been undertaken so far. This impact might be significant, and a better knowledge of mechanisms regulating splicing might help to rescue the effects of DNA damaging agents. A number of environmental and occupational factors, or agents used in chemotherapy and

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radiotherapy are able to induce gene damage directly or indirectly, and therefore are likely to alter gene splicing. These factors are of chemical, physical and biological nature. Table I: List of agents used in this study. IARC (International Agency for Research on Cancer) group 1: carcinogenetic to human; 2A: probably carcinogenetic to human; 2B: possibly carcinogenetic to human. Agents IARC

group DNA damage Biological effect

Physical: UV-B 1 Pyrimidine dimer,

6-4 photoproduct Melanoma

γ-irradiation 1 Base fragmentation, base oxidation,single and double-strand breaks

Various cancers

Environmental and occupational chemicals:

Aflatoxin B1 1 Alkylation of guanine Liver cancer

Carbon nanomaterials Not listed

Oxidative damage, AP site, DNA breaks

Under reviewed by IARC

Dioxins

1 Oxidative damage, DNA breaks, DNA adduct

Various cancer

Formaldehyde 1 DNA protein/cross link nasopharyngeal cancer, leukemia and sinonasal cancer

Heavy metals (2) 1-2 Oxidative stress. Inhibition of DNA repair.

various

Microcystines 2B Oxidative stress, DNA breaks Liver cancer Pesticides (glyphosate) Not

listed DNA damage Lymphoma

Polycyclic aromatic hydrocarbons (benzo(a)pyrene)

1 DNA strand breaks Various cancer

Chemotherapeutical: Camptothecin

Not listed

Single-strand breaks converted to DSB in S phase of cell cycle

Cisplatin 2A Nucleotide alkylation and DNA crosslinks

Doxorubicin 2A DSB, oxidative damage Etoposide 2A Double-strand breaks Lomustine Not

listed DNA adduct, cross link

Mitomycin C 2B DNA Cross link Temozolomide Not

listed Base methylation, including O6- methylguanine

Other chemical agents: Bile salts and acid pH Not

listed Oxidative stress Barrett’s associated

oesophagal carcinoma Biological : HPV (16-18) 1 ROS Cervix HIV 1 Transient DNA breaks Kaposi HTLV-1 1 Genomic instability Adult T-cell leukaemia VZV Not

listed Varicella, Zoster

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a. Physical agents

UV-B are the main factor responsible for the occurrence of melanoma. The increasing prevalence of this very malignant skin cancer is likely due to changes in habits and possibly to the destruction of the ozone layer. They induce DNA damages such as pyrimidine dimers and the 6-4 photoproduct. The impact of UV-B on splicing is demonstrated in a number of studies in vitro. They alter the splicing of ribonucleotide reductase R2 in cells from Aedes albopictus (Jayachandran et al., 2004), PIG3 (Nicholls et al., 2004), MDM-2 (Chandler et al., 2006), and VEGF in a number of human cells (Mineur et al., 2007).

Ionizing radiation (IR) can alter DNA directly or indirectly via H2O radiolysis that generates free radicals. Multiple DNA damage are generated: base fragmentation or oxidation and DNA breaks (single- and double-strand). Exposure to IR is limited, for most people, to medical applications. Excessive expure to IR has multiple detrimental biological effects such as burns, sterility, blood disorder and cancers. Radiotherapy is commonly used to treat cancer cells as DNA damage induce apoptosis in rapidly dividing cells. Little is known however on the impact of IR on alternative gene splicing and on its potential adverse effects. Katzenberger et al. (2006) established that Drosophila TAF1 pre-mRNA alternative splicing was dependent of ATM/Chk2 after IR induced DNA lesion. Other publications have reported that IR and other types of genotoxic agents do not induce the same alternative splicing signature. For example, MDM2 alternate spliced form, which lacks p53 binding domain and the ARF domain, is induced following UV irradiation or Cisplatin treatment but not following IR in MCF7 breast cancer cells (Chandler et al., 2006). Similarly, the alternate splice variant VEGF111 is not detected after IR in these cells (unpublished). b. Chemical agents

Many chemicals are known to damage DNA, either directly or indirectly, and to induce cancer. These agents are environmental or of therapeutical use. Some of them will be evaluated in this study. Bile salts and acidic pH were also included as they are involved in the Barrett’s oesophagus.

b1. Environmental compounds

A high number of agents from the natural or occupational environment are classed group 1 or 2 by IARC. Some of the most pertinent agents are listed in Table I in regard of their present or foreseen wide distribution in the environment.

Aflatoxin B1, produced by Aspergillus parasiticus, is not toxic by itself, but its

metabolization in the liver generates toxic compounds. It can be found in food inappropriately stocked, especially in Africa and East Asia, and more than 98 % of people tested in Gambia, Guinea, Senegal and Nigeria were contaminated.

Carbon nanomaterials are increasingly used in electronics and cosmetics, and are

recognized as DNA damaging agents (Lindberg et al., 2008). Their carcinogenicity is being reviewed by IARC.

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Dioxins are a class of compounds, the most studied being the 2,3,7,8-tetrachlorodibenzo-para-dioxin (2,3,7,8-TCDD, or Seveso dioxin) which constitutes the reference. It also needs to be metabolized to become toxic. Dioxins are genotoxic but so far there is no clear link between DNA damage and cancer and other diseases.

Formaldehyde is widely used in industry and in pathology labs, and is released in air by burning wood, natural gas, kerosene and cigarette. It was shown to induce DNA damage and alternative splicing of NMDA receptor 2A in rat (Gaunitz et al., 2002).

Humans are exposed to heavy metals through air, drinking water, food, contaminated

soil, etc. Heavy metals belong to IARC group 1 or 2B. They provoke DNA damage via the induction of oxidative stress. Arsenic and lead widely contaminate our environment. Epidemiological studies have shown that arsenic can cause different types of cancer via exposure to contaminated drinking water and/or ambient air (reviewed by Yoshida et al. 2004). In humans, inorganic arsenic prevalent in drinking water as arsenite or arsenate is metabolised in the liver. Occupational exposure to arsenic, primarily by inhalation, is causally associated with lung cancer. Shi et al. provided evidence in 2004, that arsenic generates free radicals that lead to cell damage and death. Arsenic alters the splicing of GADD45alpha, generating a new isoform that antagonizes the effect of classical GADD45alpha isoform on cell cycle (Zhang et al., 2008). The toxicity of lead has been documented for centuries but its carcinogenetic potential was recognized more recently. At concentrations comparable to those encountered with human exposure, two mechanisms may underlie lead-induced genotoxicity. The major one results from (i) a disruption of the pro-oxidant/anti-oxidant balance by decreasing the anti-oxidant defence and (ii) an interference with different DNA repair systems. Lead inhibits nucleotide excision repair and base excision repair by affecting the AP-endonuclease, therefore leading to AP (apyrimidinic/apurinic) sites accumulation. Moreover it also inhibits NHEJ double-strand repair and affects ATM signalling (reviewed by Beyersmann and Hartwig 2008, Gastaldo 2007).

Microcystines are produced by cyanobacteria and contaminate water. Chronic low-

level exposure is suspected to cause human hepatocellular carcinoma in China (Harada et al., 1996) and have been held responsible for the death of patients after renal dialysis with water contaminated with the toxin in Brazil (Jochimsen et al., 1998).

Anti-herbal compounds are widely used pesticides. Among them, glyphosate

(Roundup®) is actually the most popular. The genotoxicity of its metabolite AMPA has been demonstrated (Manas et al., 2008).

Polycyclic aromatic hydrocarbons (PAH) are a class of compounds produced as

byproducts of fossil fuel or biomass burning and present in cooked foods such as grilled meats. Benzo(a)pyrene is the reference. It is highly carcinogen and requires metabolic activation before damaging DNA. Its metabolite reacts with DNA to create bulky adducts, but this compound is also known to generate oxidative stress and double-strand breaks (Delgado et al., 2008).

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b2. Therapeutic compounds

Numerous genotoxic chemicals are used in chemotherapy to kill the rapidly dividing cancer cells. All the compounds described below initiate ATM and/or ATR signalling cascades.

Camptothecin and its soluble derivatives, Topotecan and Irinotecan (CPT-11), are potent antineoplastic compounds with efficacy against a large range of tumour cells (Pommier et al. 1998, Pommier 2006). These DNA topoisomerase I inhibitors act by stabilizing the cleavage complex “DNA / topoisomerase I”, thus inducing single-strand break. Subsequently, the progression of the replication fork during the S phase of the cell cycle converts that single-strand break into a double-strand break, a more severe lesion. Several examples of alternative splicing initiated by camptothecin are reported (Mineur et al 2007; Solier et al. 2004; Katzenberger et al. 2006).

Etoposide and doxorubicin are DNA topoisomerase II inhibitors that stabilize the covalent enzyme-cleaved DNA complex mostly throughout S, G2 and M phases (Burden and Osheroff 1998, Mc Clendon and Osheroff 2007). Both compounds generate DSB independently of cell cycle. However, these two drugs do not generate identical DNA lesions as doxorubicin generates oxygen free radicals (ROS) and intercalates into DNA, whereas etoposide does not (Campbell et al 2006). Both compounds have been reported to initiate alternate splicing of caspase 2 pre-mRNA (Solier et al 2004). Etoposide is currently used to treat leukemias, small cell lung cancers, lymphomas and germ-line malignancies. Doxorubicin treats a variety of soft-tissue sarcomas, non-Hodgkin lymphoma and osteosarcomas.

Mitomycin C, is a potent DNA cross-linker used in the treatment of solid tumours. It was reported to induce the alternate splice variant VEGF111 in vitro (Mineur at al 2007).

Cis-diaminedichloroplatinum(II) (Cisplatin) has been for the last 30 years the drug of choice to treat advanced ovarian cancers and other gynaecologic solid tumours. It is a DNA cross-linking agent that forms both intra- and inter-strand cross links. Chandler et al. (2006) demonstrated that Cisplatin induces the alternate splicing of hDM2 pre-mRNA into hDM2ALT1.

Temozolomide is a DNA methylating agent, producing methylpurine (70%), N-3 methyladenine (10%) and O6 methylguanine (5%). It is used alone or in combination with IR to treat high grade glioblastoma. In addition, it induces single and double-strand breaks in cells with an efficient mismatch repair system (Taverna et al, 2001). Lomustine is also an alkylating agent, belonging to the family of the haloethylnitrosurea. It creates many DNA lesions, including base alkyl adducts and inter-chain cross links. It is has been used to treat glioblastoma since many years. So far, they are no report in the literature of pre-mRNA alternative splicing induced by these two alkylating compounds. b3: Bile and acid salts

DNA damage can also be induced by bile and acid salts of the gastro-oesophageal reflux (Jolly et al., 2004). This process causes mucosal injury and initiates chronic inflammation. It represents a risk factor for Barrett’s oesophagus, which is characterized by

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intestinal metaplasia replacing the normal squamous oesophageal epithelia. Barrett’s oesophagus is a pre-malignant lesion predisposing to dysplasia and finally to adenocarcinoma. DNA damage resulting from the effect of bile and acid salts occurs via oxidative stress mechanisms (Dvorak et al., 2007). Indeed, deoxycholic acid (DCA), a secondary bile acid, is implicated in the generation of ROS leading to chromosomal damage (Jenkins et al., 2008). AS may be induced in Barrett’s oesophagus as suggested by the differential expression of growth hormone-releasing hormone receptor and its splice variant (SV-1) in human normal and malignant mucosa of oesophagus (Hohla et al., 2008). Novel splice variants of MAGE-A10 were also identified in oesophageal adenocarcinoma (Lin et al., 2004).

c. Biological agents

At a first glance, it could seem inconsistent to include biological agents in the present study. However, a growing number of evidences indicate that viruses interact with, or use to their advantage, the DSB signalling pathways and repair machineries of their host cells. They can either activate DNA damage recognition pathways or alter them (reviewed in Weitzman et al. 2004, Lilley et al., 2007). A few reports indicate that viruses modify gene splicing in infected cells. Hence, their study has as an added value in the present proposal.

c1: Viruses and DNA damage DNA damage responses elicited by viruses start being characterized, but deserve a

more thorough interest. Human immunodeficiency virus type 1 (HIV-1) was suggested to hijack host cell DSB

repair pathways to complete its integration. Several studies have linked HIV-1 virus or its protein Vpr (known to induce DSB and to block cell cycle at the G2/M border) to components of the DSB repair pathways. ATM and ATR inhibitors were shown to suppress HIV-1 infection (Nunnari et al 2005, Lau et al. 2005). Smith et al. (2008) have established that NBS is required for an efficient integration of HIV-1 DNA.

Herpes simplex virus type 1 (HSV-1) triggers the phosphorylation of ATM and NBS

upon replication (Gregory and Bachenheimer, 2008) and the loss of Mre11 upon infection. ICP0, an IE gene product with multiple functions, is required for ATM-dependent Chk2 and CDC25C phosphorylation (Li et al 2008). It promotes cell cycle arrest, known to be part of the classical DSB responses induced by chemical compounds.

Varicella Zoster Virus (VZV) establishes life-long latency in dorsal root ganglia. We

have observed that a productive infection leads to (i) ATM phosphorylation both in Mewo and Vero cell lines, and (ii) a drastic reduction of NBS expression (unpublished observations).

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are known to

induce DNA damage. In particular 8-oxo-7,8-dihydro-2’-deoxyguanosine and 8-nitroguanine are formed (Wiseman and Halliwell, 1996; Yermilov et al., 1995). These two types of DNA damage are also observed in patients with CIN II or III induced by high risk HPV (Hiraku et al., 2007). Indeed, some high-risk HPVs (notably HPV16, HPV33 and HPV52) are associated with DNA lesions in epithelial cells, leading to cell proliferation, dysplastic changes and carcinogenesis. Moreover, the level of 8-oxo-7,8-dihydro-2’-deoxyguanosine has been shown

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to be correlated with the grade of cervical lesions (Romano et al., 2000). The protein E6 of HPV-1, -8 and 16 binds to and inhibits XRCC1, a scaffold protein important for both single-strand break repair and base excision repair (Iftner et al. 2002).

HTLV-1 infection induces genomic instability. It was demonstrated that Tax protein

attenuates the ATM-mediated DNA damage response, therefore allowing the replication of the damaged host cells genome before the complete repair of the lesions. HTLV-1 infection induces the premature dephosphorylation of ATM following DNA damage and thus inactivates its kinase activity (Chandhasin et al 2008). Tax acts by disrupting the binding of MDC1 to γH2AX, interrupting the chromatin-dependent ATM amplification loop

c2: Viruses and alternative splicing

Viruses use the splicing machinery of the cells in order to orchestrate the splicing of

their own RNA and the shift from early to late genes expression. In addition they are able to subvert the splicing machinery of the cells. Different types of HPV seem to induce alternative spliced WAPL variants (Oikawa et al., 2008). Several specific additional spliced WAPL variants have indeed been isolated depending on the grade of cervical lesions. Epstein-Barr virus (EBV) alter the splicing of CD44 and DNA polymerase-β (Kryworuckho et al., 1995, Li et al., 2003), and HIV that of CD44 and Cdk9 (Giordanengo et al., 1996; Shore et al., 2003). Cell transformation with the T-antigen from SV40 induces alternative splicing of TEF1 (Zuzarte et al., 2000). Kaposi’s sarcoma associated herpesvirus/human herpesvirus-8 (KSHV/HHV-8) also regulates the splicing of DNA polymerase B, Bcl-X and CD45 (Li et al., 2003). The molecular mechanisms used by viruses to affect the splicing machinery remain largely unexplored, but reports indicate that cellular splicing factors are positively or negatively controlled by viral infection (Huang et al., 2002). For example HPV16 regulates the expression of SF2/ASF and hnRNPA1 (Cheunim et al., 2008), and HIV that of SC35 and proteins of the hnRNP of A/B and H groups (Dowling et al., 2008). The viral early-to-late switch of gene expression is closely tied to another cellular splicing factor, SRp20 and more specifically to its function and its differential expression in HPV16-infected keratinocytes (Jia et al., 2009). Some viruses also encode for splicing factors (Törmänen et al., 2006).

In some instances the altered splicing is likely to limit cell defence or immune

surveillance. HTLV-1 induces the expression of an IL-2Rα chain lacking exons 5 to 7 and having an altered reading frame (Horiuchi et al., 1997). Mouse hepatitis virus type 3 also induces alternative splicing of STAT1 and STAT3, which are essential players in the expression of IFN-γ and IFN-α (Ning et al., 2003). Finally, the Sendai virus induces the expression of a dominant negative isoform of TBK1, resulting in reduced expression of IFN-β (Deng et al., 2008). Hence these data suggest that targetting the splicing induced by viruses might reduce their infectivity. c3: Selection of viruses to be investigated

HPV are small, double-stranded DNA viruses which infect epithelial tissues. They can be subdivided into high risk and low risk depending on the frequency with which they induce tumours (Munoz et al., 2003; Munoz et al., 2006). Indeed, there is a strong association between high risk subtypes of HPV and genital cancers. Cervical cancer evolves from pre-existing noninvasive malignant lesions (CINs for cervical intraepithelial neoplasias) ranging

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from CIN I (mild dysplasia; also called L-SIL for low grade squamous intraepithelial lesion) to CIN II (moderate dysplasia), CIN III (severe dysplasia/carcinoma in situ) and H-SIL (for high grade squamous intraepithelial lesion and carcinoma). HPV16 is responsible of a large number of cancers, in particular in developing and emerging countries. Expression of the HPV16 E6 oncogene interferes with the p53-dependent response to DNA damage (Filippov et al., 2007). HIV-1 is a retrovirus belonging to the lentiviridae family. Its genetic material is composed of single-stranded RNA. During the productive cycle, this single-stranded RNA is retro-transcribed into double-stranded viral DNA which integrates into the cellular genome. The evolution of the disease is divided into three steps: the primo-infection, the asymptomatic phase and the AIDS (acquired immunodeficiency syndrome). Indeed, a significant number of HIV-infected patients develop AIDS and die from opportunistic infections and diseases. An estimated 10-20 million people are infected with HTLV-1. A significant fraction (about 2-3 %) will develop ATL (adult T cell leukemia) or HAM/TSP (HTLV-associated myelopathy/tropical spastic paraparesis) (Yoshida, 2005). ATL is the third most frequent malignancy amongst T cell neoplasms. This disease has a very poor prognosis due to frequent relapse of tumour cells. HAM/TSP is characterized by an infiltration of the central nervous system (CNS) by T lymphocytes leading to an inflammatory response and axonal degeneration of the spinal cord. VZV is an alpha herpes virus having a DNA double-stranded genome. It is the etiological agent of two clinically distinct diseases; varicella (chicken pox) as primary infection and zoster (shingles) after reactivation of latent virus from the dorsal root ganglia (Flisser and Tapia-Cornier, 1999). Varicella VZV enters the host via the respiratory mucosal epithelium. In our country, 90% of the 8 year old children have been exposed to VZV (Khoshnood et al., 2006). Shingles is usually observed in elderly or immuno-compromised patients. Forty percent of patients after bone marrow transplant will reactivate the virus within one year of the transplantation. Goals of our study: Although a number of genotoxic stresses or viruses are known to affect the splicing of several genes, a systematic and comparative study is still lacking. We will evaluate the effect of various agents on splicing and on DNA damage in parallel experiments. The agents of interest are those listed in the table I. Their choice has been established based on the most recent scientific data from the literature and our on-going complementary research programmes. The physical and chemical agents listed are a representative fraction of the DNA damaging agents encountered in the environment or used in cancer therapy. The four viruses used as models are also of outstanding importance. Together these agents are responsible for a significant number of diseases worldwide.

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Our studies aim at: 1) Evaluating the effect of viral infection and physical or chemical agents on the activation of DNA damage signalling pathways.

The DNA damage response pathways coordinate lesions repair to cell cycle arrest,

activation of transcription factors, and apotposis. This signalling is almost always transduced by ATM or ATR kinases. ATM and ATR are primarily activated by DSB and a structure containing single-stranded DNA, respectively. The nature of the DNA lesion does not only determine the identity of the kinase activated at first, but also affects the kinetic of activation and the selection of downstream substrates. Downstream substrates of ATM and ATR can be either specific or common, and, when common, be phosphorylated at the same or at specifically targeted residue(s), in function of the nature of the initial DNA alteration, and thus differently modulating their biochemical properties.

The originality of this study resides in the comparison of the response to viral, physical and chemical agents. Little is known on the DNA damage response pathways initiated by the selected viruses. The chemical and physical agents selected induce a broad panel of damage that are repaired by different mechanisms. We plan to monitor the phosphorylation of key proteins involved in the DNA damage signalling pathways after viral infections or physical and chemical treatments. This extensive evaluation will allow us to establish a minimal response signature for each tested compounds. These minimal response signatures will then be correlated with the alternate splicing profile of the selected genes. 2) Analyzing the effect of viral infection and DNA damage on the aberrant splicing of a number of genes and on the expression and the activity of factors involved in AS.

Genes of interest are those already known to be alternatively spliced in cells treated with DNA damaging agents or viruses (Table II), as well as genes being aberrantly spliced in acquired and genetic diseases (Table III). A particular attention will be paid to the splicing of ATM and ATR pre-mRNA themselves. Indeed, these proteins are central players in the recognition of DNA damage and the signalling cascade that they induce (see above). We already demonstrated that ATM is involved in aberrant splicing of VEGF-A induced by genotoxic agents. Moreover our data indicate that a short isoform of ATM is produced in MeWo cells infected with VZV or treated with camptothecin. A low number of representative samples will be selected for genome-wide (spliceomic) analysis of alternative splicing. The steps of the signal cascade between ATM/ATR activation and alternative splicing will be identified.

We already demonstrated that several SR proteins are hypophosphorylated in cells treated with DNA damaging agents (Ch. Lambert, unpublished). The expression and phosphorylation of splicing factors will be investigated in parallel with aberrant pre-mRNA splicing and DNA damage signaling cascades (see above).

It may seem paradoxical to include in our choice gene products aberrantly spliced in

acquired or inherited diseases. However the rationale of this approach is supported by several observations:

(1) The expression of the aberrant isoform of lamin A, responsible of premature ageing in progeria, is also induced during normal ageing (McClintock et al., 2007);

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(2) Genotoxic stress induces the expression of a truncated variant of MDM-2 that is also observed in a number of rhabdomyosarcoma (Chandler et al., 2006);

(3) Altered splicing of CD44 is observed in cancer cells and is as well induced by chemotherapeutical agents and viruses;

(4) epigallocathechin gallate (EGCG) corrects the aberrant splicing of IKAP in familial dysautosomia (Anderson et al., 2003) as well as the genotoxic stress-induced splicing of VEGF (personal observation). Table II: Genes alternatively spliced upon treatment with DNA damaging agents or viruses. Gene Agents References ATM Chemotherapeutical agents, VZV Personal

observation Bcl-X * UV-B, chemotherapeutical agents,

KSHV/HHV-8 Li et al., 2003 ; personal observation,

Caspase-2 Chemotherapeutical agents Solier et al., 2004 CD44 Mitomycin-C, EBV, HIV, HPV Filipov et al., 2007;

Kryworukho et al., 1995 ; Giordanengo et al., 1996 ; Dall et al., 1996

CD45 KSHV/HHV-8 Li et al., 2003

Cdk9 HIV Shore et al., 2003 DNA polymerase β EBV, KSHV/HHV-8 Li et al., 2003 GHRH Bile and acid salts Hohla et al., 2008 IL-2Rα HTLV-1 Horiuchi et al., 1997MDM-2 * UV-B, chemotherapeutical agents MDM-X UV-B, chemotherapeutical agents

Chandler et al., 2006

NBS1 γ-irradiation , methyl methane sulfonate Takai et al., 2008 PA26 γ-irradiation, chemotherapeutical agents Velasco-Miguel et

al., 1999 PIG3 * UV-B ; chemotherapeutical agents Nicholls et al.,

2004 ; personal observation

SIRT1 * Chemotherapeutical agents Personal observation

STAT1, STAT3 Mouse hepatitis virus Ning et al., 2003 TBK1 Sendai virus Deng et al., 2008 TEF1 SV40 Zuzarte et al., 2000 VEGF * UV-B, chemotherapeutical agents Mineur et al., 2007 *: Primer pairs already designed.

The effect of the most significant agents, either physical, chemical or biological, will

be tested in animal models or in human samples in vivo and ex vivo. Samples of interest are: - tissues from animals with acute exposure to genotoxic agents - cervix biospies from patients with HPV - samples from patients prior to and during chemotherapy (hair follicles, sputum,

lymphocytes) - samples (sputum, lymphocytes, hair follicles) from workers exposed to occupational

agents (formaldehyde, glyphosate) - transgenic mice carrying the genome of HPV16

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Table III: Genes with aberrant splicing in acquired and genetic diseases Gene Function / altered splicing in References ATR Seckel syndrome O’Driscoll et al.,

2003 BRCA1/BRCA2 Tumour suppressor. Splicing altered in various cancers. Srebrow and

Kornblihtt, 2006 CD44 Cell proliferation, differentiation and migration.

Angiogenesis. Splicing altered in cancers Caballero et al., 2001

CD45 Autoimmune and infectious diseases Tchilian and Beverley, 2006

CDH17 Cell adhesion. Splicing altered in hepatocellular carcinoma Srebrow and Kornblihtt, 2006

Cyclin D1 Proto-oncogene. Splicing altered in cancer. Knudsen et al., 2006FGF-receptors Splicing altered in various cancers Brinkman, 2004 fibronectin Adhesion glycoproteinSplicing altered in cancer Ebbinghaus et al.,

2004 HAS1 Splicing altered in multiple myeloma Adamia et al., 2005 Kit Oncogene. Splicing altered in gastrointestinal stromal tumour Srebrow and

Kornblihtt, 2006 KLF6 Tumour suppressor. Splicing altered in prostate cancer. Srebrow and

Kornblihtt, 2006 LKB1 Tumour suppressor. Splicing altered in Peutz-Jeghers

syndrome Srebrow and Kornblihtt, 2006

MDM2 Regulation of p53. Splicing altered in rhabdomyosarcoma. Chandler et al., 2006

Prolactin receptor Splicing altered in breast cancer. Meng et al., 2004 PSA and other kallikreins

Splicing altered in various tumours Brinkman, 2004

Rac1 GTPase. Involved in cell migration and MMP overexpression. Splicing altered in cancer

Srebrow and Kornblihtt, 2006

Ron Tyrosine kinase receptor. Splicing altered in human gastric carcinoma cells

Srebrow and Kornblihtt, 2006

TSG101 Acute myeloid leukaemia Lin et al., 1998 WT1 Transcriptional regulator. Splicing altered in Wilm’s tumours Brinkman, 2004

3) investigating the effect of a set of pharmacological agents known to alter AS on the aberrant splicing induced by viruses and DNA-damaging agents and viral infectivity

A number of pharmacological agents are able to interfere with the pre-mRNA splicing (http://www.eurasnet.info/scientists/alternative-splicing-and-disease/substances).They include (1) Na butyrate, known to correct the aberrant splicing of CTFR pre-mRNA (Chang et al., 2001; Nissim-Rafinia and Kerem, 2006); (2) valproic acid and aclarubicin, able to induce the splicing of SMN2 exon 7 (Andreassi et al., 2001; Brichta et al., 2003); (3) EGCG and kinectin, able to correct the aberrant splicing of IKAP1 in familial dysautosomia (Anderson et al., 2003; Slaugenhaupt et al., 2004); (4) LiCl, able to correct the splicing of Tau; (5) IDC16 and SRPIN340, that impairs correct splicing of HIV (Bakkour et al., 2007) and propagation of Sindbis virus (Fukuhara et al., 2006), respectively. Some of these compounds are known to regulate the expression of proteins involved in alternative splicing. Most of them are commercially available.

We demonstrated that the expression of VEGF111 in cells treated with

chemotherapeutical agents can be inhibited by EGCG and resveratrol in vitro (Lambert et al.,

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article in preparation), showing that genotoxic stress-induced splicing may be pharmacologically corrected. Agents known to correct splicing will be tested on the most significant altered splicing events described in this study. The finding of some effective agents might allow the reversion of the splicing defect in diseases. In particular the effect of pharmacological agents on the splicing of genes involved in cancer progression, in infectivity of viruses and host resistance will be tested, as a first screening for identifying potential new therapeutic strategies and molecules. Motivation for the present proposal

Living organisms are continuously threatened with a number of DNA damaging agents and viruses that have the potency to modulate pre-mRNA splicing, and therefore their proteins repertoire. These modifications potentially participate to the evolution toward the disease state. For example, the expression of aberrant proteins might help cells to escape apoptosis induced by DNA damage, allowing the transfer of altered genes to daughter cells and the acquisition of a transformed phenotype. Chemotherapeutical agents can induce the expression of protein variants that can participate to the acquisition of drug resistance by cancer cells. As an example, the DNA damage-induced expression of the protease resistant VEGF111 might help cells bearing a VEGF receptor to survive, as well as to promote tumour angiogenesis. Some viruses are also believed to affect the splicing of several genes in order to decrease cell resistance to infection.

Induction of alternative splicing by genotoxic agents and viruses is a novel

concept that may be significant in the acquisition and progression of many diseases. Investigation of the mechanisms involved in this process and the regulation of aberrant splicing by pharmacological agents are new and promising fields of research that provide significant opportunities of development. Besides expected publications in high level international journal, data and concepts generated during this project could be of valuable importance for future medical applications and for biopharmaceutical companies, as they could lead to the design of new therapeutics and the development of new diagnostic tools.

Our consortium takes advantages of the complementary expertise of the three

laboratories, covering all the involved specific domains as illustrated at the end of the proposal in the section describing the partners’ expertise. For example, partner 3 has a long standing experience in epithelial metaplasia, Barrett’s oesophagus and HPV, and is a reference laboratory for the anatomopathological analysis of various pathologies. Partner 2’s laboratory is expert in DNA damage signalling and in VZV study, while partner 1 has a strong experience in transcriptomic analysis and in the study of intracellular pathways of regulation (Lambert et al., 1999, 2001a, b, Mineur et al., 2007; Deroanne et al., 2005). The broad interest of the project and the capacity of partner 1 to address these issues is further illustrated by the selection by the European Space Agency of a program (rated A) aiming at investigating the alternative splicing events induced by high LET particles as found in cosmic radiations or used in hadron cancer therapy.

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Expected advances:

Fundamental questions will be addressed to gain a better understanding of acquired and inherited diseases related to altered regulation of the splicing machinery. Gene products that are alternatively spliced in response to genotoxic agents will be extensively identified using high throughput approaches such as “all exons” µ-arrays and deep sequencing. Deep sequencing and bioinformatics tools will be performed in collaboration with the GIGA-Genomic and GIGA-Bioinformatics platforms. This will allow to establish various correlations between the type of the initial DNA damage, the induced signalling cascade, the activition status of splicing factors, the alternative splicing signature, the function of the protein variants and the biological consequences at the cellular level.

These analyses might trigger a re-appraisal of the biological effects of the numerous genotoxic agents present in the environment.

These comparative analyses will also enable to predict the overall expected effect induced by a specific agent only on the basis of the induced DNA damage or from the study of the splicing signature of a small cohort of gene products. It might also be indicative of adverse cooperative effects of various agents as, for example, a possible reduction of resistance to viral infection in specific environmental conditions (pollutants, exposure to chemicals, ...).

The finding of the VEGF111 variant in cells treated with DNA damaging agents was the basis of a patent deposited by the University of Liège. The systematic investigation proposed in this project should enable the description of new protein variants displaying novel and specific functions. This will increase our knowledge of the human proteome and possibly lead to the identification of variants with medical and/or commercial interest. The comparison of DNA damage and aberrant splicing induced by chemical agents ex vivo using non-invasive procedure such as hair plucking could be used for evaluating individual sensitivities and detecting subjects with particular sensitivity to a specific, or a class of, genotoxic agents. This detection might be of particular importance for the selection of chemotherapeutical treatments as well as for the follow-up of people concerned by occupational hazard.

A small number of compounds able to regulate aberrant splicing in specific genetic diseases are already used in clinics for other indications, as LiCl and valproic acid. EGCG and resveratrol are natural compounds extracted from green tea and grapes. They are commonly used as food additives, and toxicological data are already available. These agents might therefore be rapidly tested for their effect in genotoxic stress-induced diseases in human. As alternative splicing mechanisms help cells to survive by some aspects, inhibition of aberrant splicing might increase the efficiency of chemotherapy or reduce its adverse effects. This hypothesis deserves a careful investigation. Other potential applications of this program address (i) the treatment of accidental acute exposure to environmental or occupational hazardous agents, (ii) patients with Barrett’s oesophagus or (iii) patients infected with the viruses listed above. Finally the finding of new agents interfering with gene splicing might also open the way to new treatments for acquired or genetic diseases caused by aberrant splicing.

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Our project may look ambitious and overload by the numerous tasks listed above. In this context, we want to put forward the fact that this project will be integrated in ongoing related programs as that recently funded by the “Fondation contre le Cancer” or selected by ESA (see above). It is clear however that some specific aspects will be developed more thoroughly. Priorities will be established and regularly reviewed on the basis of the most recent literature and according to the more promising data generated by our ongoing related projects. B. Milestones of the project. The work has been divided in different interconnected work packages (WP): WP1: Induction of DNA damage in vitro WP2: DNA damage signalling: selection of pertinent agents and cells WP3: Alternative splicing induced by genotoxic agents and viruses: selection of pertinent

agents and cells WP4: Signalling pathways induced from DNA damage to the splicing machinery in selected

agents and cells WP5: Functionality of ATM splice variants induced by DNA damage WP6: Correction of aberrant splicing induced by pharmacological agents WP7: Alternative splicing induced by genotoxic agents and viruses ex vivo and in vivo WP8: Correction of aberrant splicing induced by genotoxic agents and viruses ex vivo and in

vivo Workpackage 1 : Induction of DNA damage in vitro

The goal of this WP is an extensive characterization of the different conditions inducing DNA damages and/or alternative splicing that will be tested respectively in WP2 and WP3. Cells will be treated with agents listed in Table 1.

Data from partner 1 indicate that UV-B, camptothecin, doxorubicin, mitomycin C and

Cisplatin affect the splicing of some genes listed in Table II (PIG3, MDM-2, and VEGF-A; unpublished data and Mineur et al., 2007). Therefore, they will be used as reference agents and genes for comparative experiments using other agents listed in Table IV. When possible the conditions will be chosen in order to work at equitoxic levels as determined by biological functional assays.

Table IV reports also cells that will be analyzed after exposure to the various agents, and the partner in charge of this specific task. Selected cells are either cell types naturally exposed to the respective agents or cell lines previously tested and characterized in order to include in the study both positively and negatively responding cells. The requirement of several cell lines is evidenced by the variable level of the UVB-induced expression of VEGF111 in 20 different cell lines (Fig. 4). MCF-7 cells, known to respond to a number of agents by inducing aberrant splicing, will be challenged by the various physical and chemical agents for a comparative purpose.

All cell lines listed in Table IV are available and already used in the laboratories of the consortium or at the GIGA, with the exception of Seg-1, OE33 and Flo-1. MCF-7 cells derive

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from epithelial cells, HaCat from skin keratinocytes, HEK293 from kidney, HepG2 from liver, BEAS2B from lung, Jurkat, MT2, Champ and EVA from T-lymphocytes, LN18 and U87 from glioma, SiHa, CaSKi, HeLa, CK2, C33a and W12 from cervix keratinocytes, and MeWo from human melanoma. When required, a L3 facility is available at the GIGA.

Table IV: Agents, cells and partners

Samples will be harvested at various times and distributed between the partners for

investigating DNA damage recognition signalling (WP2) and alternative splicing of genes (WP3), in order to limit experimental variations. This will enable us to select appropriate cells and agents for determining DNA damage induced signature by genome wide analysis (WP3) and investigating the signalling pathways between DNA lesions and the splicing machinery (WP4).

Agents Cells Partner Physical: UV-B MCF-7, HaCat, primary keratinocytes 1 γ-irradiation MCF-7, HEK293, HepG2 2 Environmental and occupational chemicals:

Aflatoxin B1 MCF-7, HepG2, Jurkat 1 Carbon nanomaterials MCF-7, HepG2, Jurkat 1 Dioxins MCF-7, HepG2, Jurkat 1 Formaldehyde MCF-7, BEAS2B 1 Heavy metals MCF-7, HepG2, Jurkat 1 Microcystines MCF-7, HepG2, Jurkat 1 Pesticides (glyphosate) MCF-7, HepG2, Jurkat 1

Polycyclic aromatic hydrocarbons (benzo-a-pyrene)

MCF-7, HepG2, Jurkat 1

Chemotherapeutical: Camptothecin MCF-7, HEK293 2 Cisplatin MCF-7, HEK293 2 Doxorubicin MCF-7, HEK293 2 Etoposide MCF-7, HEK293 2 Lomustine MCF-7, LN18, U87 2 Mitomycin C MCF-7, HEK293 2 Temozolomide MCF-7, LN18, U87 2 Other chemical agents: Bile salts and acid pH MCF-7, Seg-1, OE33 and Flo-1 3 Biological : HPV (16, 18, 33) SiHa, CaSki (positive for HPV16), HeLa (HPV18),

CK2 ( HPV33), C33a (negative control, no HPV), W12, HaCat, primary keratinocytes from cervix.

3

HIV Jurkat, PBMC 3 HTLV-1 MCF-7, MT2, Champ, EVA 3 VZV MeWo, HEK293 2

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Partner 1 will be in charge of cell treatment with UV-B and environmental and

occupational chemicals.

Partner 2 will be in charge of the cells treatment by γ-rays using a Gammacell 40 (Nordion) and chemotherapeutical agents, and of in vitro infection by VZV in collaboration with Dr. Catherine Sadzot from the Laboratory of Fundamental Virology. Dr. Sadzot has been working with this pathogen for many years. VZV is instable in the extracellular medium and remains associated to cells. Therefore, infected cells are added to a layer of non contaminated cells. The effect of VZV will be tested on two different cell lines: HEK293 (El Mjiyad et al, 2007) and Mewo (melanoma) as preliminary experiments suggest an increased phosphorylation of ATM after VZV infection in this cell lines.

Partner 3 will be in charge of cell treatment with bile and acid salts, and of the culture

of different cell lines, the set up of models and the preparation of the different extracts from HPV positive and control cells. More precisely, extracts from different keratinocyte cell lines positive for different high risk types of HPV (for example SiHa and CaSki positive for HPV16; CK2 positive for HPV33, HeLa infected by HPV18,…) and keratinocytes not infected by HPV (C33a, HaCat and primary keratinocytes isolated from foreskin or cervix) will be provided in order to study the alternative splicing and DNA damage which are important for the infectivity of the high risk HPV. Moreover, the effect of HPV integration on these two processes will be analyzed in the W12 cell line. Indeed, in this cell line, HPV16 is under an episomal form at early stages whereas it is integrated in the cellular genome at late stages. This will allow to follow genes splicing and DNA damage mechanisms during the different stages of infectivity. Furthermore, stably transfected keratinocytes with plasmids encoding the different HPV proteins will be performed to identify which HPV proteins are important for the investigated mechanisms. Finally, organotypic cultures of HPV-transformed keratinocytes will also be used in order to study the aberrant splicing and DNA damage induced by HPV in a more relevant context, notably during the stratification of keratinocytes.

T-lymphocytes will be infected by HIV-1 in collaboration with Dr. Souad Rahmouni (Laboratory of Immunopathology, University of Liège).

Cell line # Fig. 4 : The expression of VEGF111 mRNA is expressed as the % versus total VEGF mRNA in 20 cells lines treated with UV-B. MCF-7 cells are #1 (Mineur et al., 2007).

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Extracts will be performed in three HTLV-1 infected cell lines : MT2 (HTLV-1 positive T-cell line), Champ (ATL derived T-cell line) and Eva (HAM/TSP-derived T cell line), by partner 3 in collaboration with Luc Willems (Molecular and Cellular Biology lab, GIGA). Furthermore, these cells will also be used to infect MCF-7 cells with this virus.

A total of ~22 agents (2 physical agents, ~16 chemicals and 4 viruses) will be tested for induction of DNA damage and alteration of gene splicing in WP2 and WP3. As different cell types and two durations of treatment will be tested, an estimate number of 100 samples will be analyzed in WP2 and WP3. This number is manageable as the analytical techniques are routinely used in the laboratories of the consortium.

Workpackage 2: Study of DNA damage signalling: selection of pertinent agents and cells Partner 2 will monitor, by western blotting, the activation of selected key proteins for DNA damage signalling pathways using cell extracts derived from WP1. These proteins and the related processes are: - phosphorylation of ATM, H2AX (the phosphorylation of H2AX on Ser 139 is an indication of the presence of DSB) and downstream substrates of ATM and ATR involved in the control of cell cycle (p53, Chk1 and Chk2). The activation of ATR is monitored through its downstream substrate Chk1. - phosphorylation of AKT (known to phosphorylate SR proteins involved in mRNA splicing). - detection of markers of apoptosis. - expression and phosphorylation of splicing factors. The study of the activity of these key proteins should allow identifying the nature of the DNA damage signalling pathways activated and of the splicing factors involved in aberrant splicing. Some of the agents listed in Table 1 are already known to activate these signalling pathways, and will serve as positive controls in the study. Partner 2 has a long experience in the study of proteins involved in DNA damage responses. The expertise and most of the tools and reagents required for this study are therefore already available. Workpackage 3: Investigation of alternative splicing induced by genotoxic agents and viruses in vitro: selection of pertinent agents and cells

Partner 1 will be in charge of this WP. Alternative splicing of a limited number of gene products (Table II) will be evaluated from cells prepared in WP1 by the three partners. This study will be performed by analysis of RT-PCR after gel electrophoresis. Pairs of primers for amplification of the mRNA labelled with a * in Table II are already available in the laboratory. The others are being designed. These data will be analyzed and compared with respects to the data from DNA damage analysis (WP2).

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The expression of mRNA splice variants from the genes described in Table III will be investigated in selected samples. A survey of the literature will allow us to find new genes potentially interesting for the present study. Replicates of the experiments will be performed for experimental setting of culture conditions (WP1). Dose-response analysis will be done.

Cells, genotoxic agents and conditions of treatment (duration and concentration) that demonstrated both DNA damage and significant modification of alternative splicing of several gene products will be selected for genome-wide analysis using ‘All-exons’ DNA chips (Affymetrix). Alternatively or additionaly, deep sequencing will be performed. The first technique allows the study of splicing of already identified exons. The second is more time consuming but enables the discovery of new exons. New splicing events evidenced by genome-wide analysis will be back-tested by RT-PCR and gel electrophoresis. New detected RNA sequences will be confirmed by sequencing of their cDNA.

In WP2, we will establish a partial “response signature” for each compound or agent tested. These results will be confronted with the results obtained in WP3 and entered in a comparison matrix. A correlation between the nature of the damage, of the splicing factors involved, and of the spliced gene detected may emerge.

Workpackage 4: Study of the signalling pathways between DNA lesions and the pre-mRNA splicing machinery. Partner 2, interacting with Partner 1, will be in charge of this WP. WP4 will be divided into two parts. The first part will deal with events upstream or quasi simultaneous to ATM or ATR activation (loop), and the second part with events clearly downstream of these kinases. The study will be carried on a small number of selected agents to allow the comparison and/or generalisation of the observed processes. The results obtained in WP2 and 3 will dictate the final choice of DNA damaging agents and of the targeted alternatively spliced gene products used as reporters.

First part.

Two proteins will be investigated in priority: NBS (sensor) and MDC1 (adaptor). The role of NBS, required for ATM activation after IR, will be assessed by comparing

the alternative splicing of the selected genes in NBS deficient and proficient cell lines (Kraakman-Van Der Zwet et al., 1999; Habraken et al., 2003). NBS is also important for ATR-dependent hyperphosphorylation of RPA (Manthey et al., 2007). NBS deficient cells are more sensitive to many DNA damaging compounds than their isogenic corrected conterparts.

The importance of the nuclear foci, adjacent to the DSB and essential for checkpoint

activation, will be assessed by removing the scaffold protein MDC1 by siRNAs (technique routinely used in the three laboratories). Reducing MDC1 protein expression alters to formation of the nuclear foci and thereof the recruitment of P-ATM into these structural entities. The phosphorylation of Chk2 is thus prevented. It is important to notice that the

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destruction of the foci does not affect ATM phosphorylation in itself (Lou et al., 2006) neither the phosphorylation of ATM substrates that do not need to be recruited to the foci. Though the role of MDC1 for the recruitment of ATR to chromatin is less understood, it was shown that MDC1 is an upstream regulator of Chk1 phosphorylation (Lou et al., 2006; Sivasubramaniam et al., 2008).

Second part.

The downstream molecular cascade linking ATM or ATR to the activity of splicing

factors and the alternative splicing machinery is largely unknown. It will be investigated here by means of siRNA, pharmacological inhibitors and the use of deficient cells. In particular, the importance of Chk1 and Chk2 in this signalling cascade, suggested by the work of Katzenberger 2006, will be analysed. Similarly, the importance of AKT - a substrate of ATM known to phosphorylate the SR proteins involved in splicing - will be also investigated. The working hypothesis linking ATM directly to proteins involved in the alternative splicing will be also tested. Indeed, a broad survey reported by the group of Elledge has identified several proteins belonging to or regulatinging the spliceosome (Table V). In vitro kinase assay will be performed to determine if SR proteins are direct ATM substrates and the target serine or threonine will be identified. Once the identity of the targeted residue confirmed, it will be mutated and the effect of the mutation on alternative splicing investigated in cells.

The techniques described in this WP are currently used in the laboratory of Partner 2. The results obtained in this WP should shed some light on the signalling cascade transducing the signal from DNA lesion to the alternative splicing machinery. Table V: Splicing factors phosphorylated upon ATM activation. ________________________________________________________________________________ SF3A1 (SPLICING FACTOR 3A, SUBUNIT 1, 120KDA) SF3B2 (SPLICING FACTOR 3B, SUBUNIT 2, 145KDA) SF3B5 (SPLICING FACTOR 3B, SUBUNIT 5, 10KDA) SFRS2IP (SRRP129 PROTEIN SPLICING FACTOR, ARGININE/SERINE-RICH 2, INTERACTING PROTEIN) SFRS8 (SPLICING FACTOR, ARGININE/SERINE-RICH 8) SFRS14 (SPLICING FACTOR, ARGININE/SERINE-RICH 14) ____________________________________________________________________ Workpackage 5: Functionality of ATM splice variant induced by DNA damage This WP will be performed by partner 2. If an ATM splice variant specifically induced by genotoxic lesions is identified in WP3, its functionality will be assessed. Its kinase potential will be assessed by in vitro kinase assay on synthetic GST-p53 (WT or mutated) in presence of absence of a specific ATM inhibitor. Its ability to complement an ATM deficient cell line for cell cycle checkpoint, cell survival, NF-κB and p53 activation will be verified by stable tranfection. This WP is potentially central to the study due to the crucial role of ATM for DNA repair. However, by its nature, it will be performed only if interesting splice variant of ATM are identified in WP3. If ATM is not relevant, ATR could be investigated as an alternative plan.

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Workpackage 6: Correction of aberrant splicing induced by pharmacological agents.

A number of agents known to regulate aberrant splicing will be tested for inhibition of the splicing events induced by genotoxic agents or viruses. We already showed that epigallocathechin gallate (EGCG, the main polyphenol from green tea) and resveratrol (extracted from grapes) inhibit the doxorubicin-induced expression of VEGF111, while valproic acid, kinetin, curcumin and SAHA (a HDAC inhibitor) had no effect. Other polyphenols from green tea, and synthetic analogues of EGCG and resveratrol will be included in the study. Collaborative works with chemists from various Universities (Dr. G. Hanquet, University of Strasbourg; Dr. V. Kren, University of Prague; Dr. S. Haroutounian, University of Athens) have already started to analyse the effect of analogues of EGCG and resveratrol on VEGF111 expression, in the frame of the European Community COST Action C0602. The inhibitory potency of these agents will be tested in a couple of cells and gene splicing events. In addition the involvement of splicing factors suggested in WP2 and 4 will be tested by means of RNA interference. The most effective agents will be selected for ex vivo and in vivo assay (WP8). Partner 1 will be in charge of this study, Partners 2 and 3 being responsible for providing the samples. Workpackage 7 : Alternative splicing induced by genotoxic agents and viruses ex vivo and in vivo

Lymphocytes and hair follicles from a small cohort of healthy volunteers will be challenged in culture with several genotoxic agents. DNA damage and alternative splicing will be compared. These data should allow determining potential individual sensitivities to these agents. Partners 1 and 2 will be in charge of this study.

A few genotoxic agents selected and fully characterized in vitro and ex vivo will be

further tested in vivo in mice but only after verifying that the aberrant splicing observed in human samples affects also the mice orthologs. It could include skin treated with UV-B or chemicals by topical application, and organs and hair follicles (see below) of animals treated with IR and chemicals used in systemic application (including chemotherapeutical agents). Partner 1 will be in charge of this study; however Partners 2 and 3 will provide some samples of treated animals.

Alternative splicing and DNA damage will be analyzed in biological samples from

HPV-infected patients. HPV biological samples are available in the Human Tissue Bank of the University of Liège (liquid cytology and biopsy samples from patients without lesion or with L-SIL, H-SIL or cancer). Partner 3 will be in charge of the extractions necessary for subsequent analysis of DNA damage signalling and splicing in these samples by Partners 2 and 1, respectively. Furthermore, clinical and biological parameters (progression/regression of lesions, recurrence, HVP type, viral load,…) will be correlated to the results of gene splicing analysis (Partner 3).

Aberrant splicing will be investigated in patients prior to and during chemotherapy, as well as in workers exposed to occupational agents as formaldehyde. Samples of interest are lymphocytes, sputum and hair follicles as they are sampled by low or non invasive techniques. Hair follicles cells are sensitive to chemotherapy as demonstrated by frequent hair loss. We already demonstrated that hair follicles treated with chemotherapeutical agents ex vivo have aberrant splicing of VEGF-A (personal observation). We also already succeeded in

29

preparation of RNA from sputum and from low number (~10) of hair follicles. Partner 1 will be in charge of this study.

Workpackage 8 : Correction of aberrant splicing induced by genotoxic agents and viruses ex vivo and in vivo.

Data from WP6 might define potential drugs and siRNA acting against aberrant splicing in vitro. Their effect will be tested in animal models and ex vivo models of interest as defined by WP7. Partner 1 will be in charge of this study; however Partners 2 and 3 will provide some samples of treated animals. For HPV, the effects of the inhibitors of gene splicing will be analyzed by injecting specific inhibitors in transgenic mice encoding the whole genome HPV16 (K14-HPV16, Arbeit et al., 1994) and in control mice (partner 3) in order to confirm the results obtained in primary keratinocytes. As a summary of the WP structure, studies performed in the WP1-3 will enable to characterize alternative splicing signatures that will be useful for the subsequent studies of DNA damage-induced signalling cascade (WP4,5) and splicing correction in vitro (WP6) and in vivo (WP7,8). References - Adamia S, Reiman T, Crainie M, Mant MJ, Belch AR, Pilarski LM. (2005). Intronic splicing of hyaluronan synthase 1 (HAS1): a biologically relevant indicator of poor outcome in multiple myeloma. Blood 105:4836-4844. - Anderson SL, Qiu J, Rubin BY. (2003). EGCG corrects aberrant splicing of IKAP mRNA in cells from patients with familial dysautonomia. Biochem Biophys Res Commun 310:627-633. - Andreassi C, Jarecki J, Zhou J, Coovert DD, Monani UR, Chen X, Whitney M, Pollok B, Zhang M, Androphy E, Burghes AH. (2001). Aclarubicin treatment restores SMN levels to cells derived from type I spinal muscular atrophy patients. Hum Mol Genet 10:2841-2849. - Arbeit JM, Munger K, Howley PM, Hanahan D. (1994). Progressive squamous epithelial neoplasia in K14-human papillomavirus type 16 transgenic mice. J Virol 68:4358-4368. - Bakkour N, Lin YL, Maire S, Ayadi L, Mahuteau-Betzer F, Nguyen CH, Mettling C, Portales P, Grierson D, Chabot B, Jeanteur P, Branlant C, Corbeau P, Tazi J. (2007). Small-molecule inhibition of HIV pre-mRNA splicing as a novel antiretroviral therapy to overcome drug resistance. PLoS Pathog 3:1530-1539. - Bekker-Jensen S, Lukas C, Kitagawa R, Melander F, Kastan MB, Bartek J, Lukas J. (2006). Spatial organization of the mammalian genome surveillance machinery in response to DNA strand breaks. J Cell Biol 173:195-206. - Beyersmann D, Hartwig A. (2008). Carcinogenic metal compounds: recent insight into molecular and cellular mechanisms. Arch Toxicol 82:493-512. - Blaustein M, Pelisch F, Srebrow A. (2007). Signals, pathways and splicing regulation. Int J Biochem Cell Biol 39:2031-2048. - Blaustein M, Pelisch F, Tanos T, Munoz MJ, Wengier D, Quadrana L, Sanford JR, Muschietti JP, Kornblihtt AR, Caceres JF, Coso OA, Srebrow A. (2005). Concerted regulation of nuclear and cytoplasmic activities of SR proteins by AKT. Nat Struct Mol Biol 12:1037-1044. - Brichta L, Hofmann Y, Hahnen E, Siebzehnrubl FA, Raschke H, Blumcke I, Eyupoglu IY, Wirth B. (2003). Valproic acid increases the SMN2 protein level: a well-known drug as a potential therapy for spinal muscular atrophy. Hum Mol Genet 12:2481-2489. - Brinkman BM. (2004). Splice variants as cancer biomarkers. Clin Biochem 37:584-594. - Burden DA, Osheroff N. (1998). Mechanism of action of eukaryotic topoisomerase II and drugs targeted to the enzyme. Biochim Biophys Acta 1400:139-154.

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- Caballero OL, de Souza SJ, Brentani RR, Simpson AJ. (2001). Alternative spliced transcripts as cancer markers. Dis Markers 17:67-75. - Campbell KJ, O'Shea JM, Perkins ND. (2006). Differential regulation of NF-kappaB activation and function by topoisomerase II inhibitors. BMC Cancer 6:101. - Chandhasin C, Ducu RI, Berkovich E, Kastan MB, Marriott SJ. (2008). Human T-cell leukemia virus type 1 tax attenuates the ATM-mediated cellular DNA damage response. J Virol 82:6952-6961. - Chandler DS, Singh RK, Caldwell LC, Bitler JL, Lozano G. (2006). Genotoxic stress induces coordinately regulated alternative splicing of the p53 modulators MDM2 and MDM4. Cancer Res 66:9502-9508. - Chang JG, Hsieh-Li HM, Jong YJ, Wang NM, Tsai CH, Li H. (2001). Treatment of spinal muscular atrophy by sodium butyrate. Proc Natl Acad Sci U S A 98:9808-9813. - Cheunim T, Zhang J, Milligan SG, McPhillips MG, Graham SV. (2008). 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- Lin J, Lin L, Thomas DG, Greenson JK, Giordano TJ, Robinson GS, Barve RA, Weishaar FA, Taylor JM, Orringer MB, Beer DG. (2004). Melanoma-associated antigens in esophageal adenocarcinoma: identification of novel MAGE-A10 splice variants. Clin Cancer Res 10:5708-5716. - Lin PM, Liu TC, Chang JG, Chen TP, Lin SF. (1998). Aberrant TSG101 transcripts in acute myeloid leukaemia. Br J Haematol 102:753-758. - Lindberg HK, Falck GC, Suhonen S, Vippola M, Vanhala E, Catalan J, Savolainen K, Norppa H. (2008). Genotoxicity of nanomaterials: DNA damage and micronuclei induced by carbon nanotubes and graphite nanofibres in human bronchial epithelial cells in vitro. Toxicol Lett. - Lou Z, Minter-Dykhouse K, Franco S, Gostissa M, Rivera MA, Celeste A, Manis JP, van Deursen J, Nussenzweig A, Paull TT, Alt FW, Chen J. (2006). MDC1 maintains genomic stability by participating in the amplification of ATM-dependent DNA damage signals. Mol Cell 21:187-200. - Manas F, Peralta L, Raviolo J, Garcia Ovando H, Weyers A, Ugnia L, Gonzalez Cid M, Larripa I, Gorla N. (2008). Genotoxicity of AMPA, the environmental metabolite of glyphosate, assessed by the Comet assay and cytogenetic tests. Ecotoxicol Environ Saf. - Manthey KC, Opiyo S, Glanzer JG, Dimitrova D, Elliott J, Oakley GG. (2007). NBS1 mediates ATR-dependent RPA hyperphosphorylation following replication-fork stall and collapse. J Cell Sci 120:4221-4229. - Matsuoka S, Ballif BA, Smogorzewska A, McDonald ER, 3rd, Hurov KE, Luo J, Bakalarski CE, Zhao Z, Solimini N, Lerenthal Y, Shiloh Y, Gygi SP, Elledge SJ. (2007). ATM and ATR substrate analysis reveals extensive protein networks responsive to DNA damage. Science 316:1160-1166. - McClendon AK, Osheroff N. (2007). DNA topoisomerase II, genotoxicity, and cancer. Mutat Res 623:83-97. - McClintock D, Ratner D, Lokuge M, Owens DM, Gordon LB, Collins FS, Djabali K. (2007). The mutant form of lamin A that causes Hutchinson-Gilford progeria is a biomarker of cellular aging in human skin. PLoS ONE 2:e1269. - Meng J, Tsai-Morris CH, Dufau ML. (2004). Human prolactin receptor variants in breast cancer: low ratio of short forms to the long-form human prolactin receptor associated with mammary carcinoma. Cancer Res 64:5677-5682. - Mineur P, Colige AC, Deroanne CF, Dubail J, Kesteloot F, Habraken Y, Noel A, Voo S, Waltenberger J, Lapiere CM, Nusgens BV, Lambert CA. (2007). Newly identified biologically active and proteolysis-resistant VEGF-A isoform VEGF111 is induced by genotoxic agents. J Cell Biol 179:1261-1273. - Munoz N, Bosch FX, de Sanjose S, Herrero R, Castellsague X, Shah KV, Snijders PJ, Meijer CJ. (2003). Epidemiologic classification of human papillomavirus types associated with cervical cancer. N Engl J Med 348:518-527. - Munoz N, Castellsague X, de Gonzalez AB, Gissmann L. (2006). Chapter 1: HPV in the etiology of human cancer. Vaccine 24 Suppl 3:S3/1-10. - Myers JS, Cortez D. (2006). Rapid activation of ATR by ionizing radiation requires ATM and Mre11. J Biol Chem 281:9346-9350. - Nicholls CD, Shields MA, Lee PW, Robbins SM, Beattie TL. (2004). UV-dependent alternative splicing uncouples p53 activity and PIG3 gene function through rapid proteolytic degradation. J Biol Chem 279:24171-24178. - Ning Q, Berger L, Luo X, Yan W, Gong F, Dennis J, Levy G. (2003). STAT1 and STAT3 alpha/beta splice form activation predicts host responses in mouse hepatitis virus type 3 infection. J Med Virol 69:306-312. - Nissim-Rafinia M, Kerem B. (2006). Splicing modulation as a modifier of the CFTR function. Prog Mol Subcell Biol 44:233-254. - Nunnari G, Argyris E, Fang J, Mehlman KE, Pomerantz RJ, Daniel R. (2005). Inhibition of HIV-1 replication by caffeine and caffeine-related methylxanthines. Virology 335:177-184. - O'Driscoll M, Ruiz-Perez VL, Woods CG, Jeggo PA, Goodship JA. (2003). A splicing mutation affecting expression of ataxia-telangiectasia and Rad3-related protein (ATR) results in Seckel syndrome. Nat Genet 33:497-501. - Oikawa K, Akiyoshi A, Tanaka M, Takanashi M, Nishi H, Isaka K, Kiseki H, Idei T, Tsukahara Y, Hashimura N, Mukai K, Kuroda M. (2008). Expression of various types of alternatively spliced WAPL transcripts in human cervical epithelia. Gene 423:57-62. - Pajares MJ, Ezponda T, Catena R, Calvo A, Pio R, Montuenga LM. (2007). Alternative splicing: an emerging topic in molecular and clinical oncology. Lancet Oncol 8:349-357. - Pommier Y. (2006). Topoisomerase I inhibitors: camptothecins and beyond. Nat Rev Cancer 6:789-802. - Pommier Y, Pourquier P, Fan Y, Strumberg D. (1998). Mechanism of action of eukaryotic DNA topoisomerase I and drugs targeted to the enzyme. Biochim Biophys Acta 1400:83-105. - Romano G, Sgambato A, Mancini R, Capelli G, Giovagnoli MR, Flamini G, Boninsegna A, Vecchione A, Cittadini A. (2000). 8-hydroxy-2'-deoxyguanosine in cervical cells: correlation with grade of dysplasia and human papillomavirus infection. Carcinogenesis 21:1143-1147.

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C : Schedule and Procedures Efforts will be done to keep with the schedule shown in the Gantt chart (Table VI). However if delays are detected, meeting will be organized in order to identify the problems and find appropriate solutions. Table VI: Gantt chart.

Year 2009 2010 2011 2012 2013 2014 Trimester 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3

WP1 WP2 WP3

WP4 WP5 WP6

WP7 WP8

Color code : Green = partners 1, 2 and 3 ; yellow = partner 2 ; red = partner 1 Techniques and methodologies listed below are routinely used in the research units of the three partners. WP1 : Cell culture, including organotypic culture of keratinocytes. UV-B and γ -irradiation

Virus infection: VZV (in collaboration with Dr. C. Sadzot), HPV, HIV (in collaboration with Dr. S. Rahmouni, GIGA-R) and HTLV-1 (in collaboration with Dr. L. Willems, GIGA-R)

Cell transfection WP2: Western blotting siRNA tranfection

WP3: RNA purification. RT-PCR

Micro-arrays on All-exon chips Affymetrix (in collaboration with R. Benotmane, Radiobiology Unit, Belgian Nuclear Research Center, SCK-CEN).

cDNA sequencing (GIGA platform) Deep sequencing (Animal Genomics, GIGA-R) WP4: Cell culture

Western blotting Immunohistochemistry, immunofluorescence Pharmacological inhibition siRNA transfection In vitro kinase assay Site-directed mutagenesis Immunoprecipitation

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WP5: Cell culture

Western blotting FACS analysis Electrophoretic mobility shift assay Immunofluorescence (epifluorescence, confocal) In vitro kinase assay

Cell transfection qRT-PCR

WP6: Pharmacological inhibition Cell culture

RNA purification RT-PCR WP7 : RNA purification Mice use RT-PCR Western blotting WP8 : Pharmacological inhibition mice use

Cell culture RNA purification

RT-PCR Western blotting D. Participation of the partners in the different work packages Partner 1 will be in charge of:

- cell treatments with environmental DNA damaging agents (WP1, 3, 6-8). - detection of the AS of the different selected genes (WP3). - the genome wide analysis of splicing (spliceomic) (WP3). - testing pharmacological agents potentially able to correct aberrant splicing (WP6-8).

Partner 1 will hire a technician for 2 years to perform the screening for alternative forms of pre mRNAs of the selected genes described in Table 2, WP3 and subsequent WP. One PhD student will be hired by partner 1 (proposed PhD thesis title: Alternative splicing induced by DNA damaging agents and viral infection). This student will be supervised and guided by Drs A. Colige and Ch. Lambert and other scientific members of the laboratory. Partner 2 will be in charge of:

- Cell treatment with the anticancer drugs and cell infection by VZV (WP1, 6-8). - Identification of the activation of key elements of DNA damage signalling cascade

(WP2).

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- In depth investigation of the signalling cascade from DNA lesions to alternative splicing (WP4).

- Characterization of the function of splice variants of ATM or ATR (WP5). -

Partner 2 will hire one PhD student (proposed PhD thesis title: Identification of the DNA damage signalling cascade leading to alternative splicing). The student will be under the supervision of Dr Y. Habraken and will have many interactions with other research fellows or post-doctoral fellows present in her laboratory. Partner 3 will be in charge of:

- providing cell samples infected by HPVs (WP1, 6-8) - coordinating the viral infection by HTLV-1 and HIV (WP1, 6-8). - providing cell samples treated with bile and acid salts, and samples of patients with

HPV infection or Barrett’s oesophagus (WP1, 7). - Establishment of a correlation between AS signature and clinical data (WP7). - Monitoring the effect of aberrant splicing inhibitors in HPV-infected mice models

(WP8). Parner 3 will hire one post-doctoral fellow. The participation of the three partners in the different WP is summarized in Table VII. The interactions between the differents WP is illustrated in the figure 5. Table VII: Partner’s participation in the different work packages Partner WP1 WP2 WP3 WP4 WP5 WP6 WP7 WP8 1 2 3

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WP1WP1

WP2WP2 WP3WP3

WP4WP4

WP5WP6WP6 WP7WP7

WP8WP8

Partner 2Partner 2Partner 1Partner 1 All partnersAll partners

Fig. 5: Interactions chart. Arrows indicate dependencies between the various WPs. Bi-directional arrows (between WP1 and WP2, and between WP1 and WP3) illustrate the partly iterative relationship between these WPs, data from WP2 and WP3 being used to refine conditions developed in WP1. Blue arrows related to WP5 indicate that this WP will be performed only in case of positive results regarding ATM and/or ATR in WP3. The number of arrows originating from or going to WP3 are indicative of the crucial importance of characterizations of the alternative splicing signature. The central and final position of WP8 reflects the ultimate goal of the entire project. WP1: Induction of DNA damages in vitro WP2: DNA damage signaling (selection of pertinent agents and cells) WP3: Alternative splicing induced by genotoxic agents and viruses WP4: Signaling pathways induced from DNA damages to the splicing machinery in selected agents and cells WP5: Functionality of ATM splice variant(s) induced by DNA damages WP6: Correction of aberrant splicing induced by genotoxic agents and viruses in vitro WP7: Investigation of alternative splicing induced by genotoxic agents and viruses ex vivo and in vivo WP8: Correction of aberrant splicing induced by genotoxic agents and viruses ex vivo and in vivo

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Network management

The structure and the success of the proposed study are based on strong interactions and interdependencies between the various work packages. Since the three partner laboratories are geographically very close and possess complementary expertises, it was decided to take advantage of this situation by establishing a flexible, although well-designed, management. Besides the formal management required for an ambitious project, efforts will be made to favor informal discussion between the young researchers directly or indirectly involved in ALTERNATE. We strongly believe that this particular type of project will strongly benefit from such a bottom-up approach.

a. Coordination

The coordinator (Alain Colige, Partner 1) is the promoter and scientific coordinator of two multi-laboratories projects funded by the Walloon Region. He will be responsible for administrative and financial matters with the support of Christine Clabeq at the LCTB office. He will be in charge of organizing the kick off and the biannual meetings (see below). Together with Charles Lambert (Partner 1), Yvette Habraken (Partner 2) and Philippe Delvenne (Partner 3), he will manage:

Problems related to communication within the consortium and ULg, and possibly towards external laboratories or public bodies.

Potential problems or advantages regarding collaborations with other laboratories

Intellectual property matter, in collaboration with “Interface” , ULg, for (i) evaluating opportunities of protection and (2) writing agreement between the partners

The writing (and distribution within the consortium) of the annual reports from data provided by Work Package Leaders (WPL) (see below).

b. Work Package Leaders (WPL)

For each WP a WPL will be designated based on his experience and specific implication in the involved tasks. He will be responsible for coordinating, monitoring and reporting the activities of his WP. He will also interact with other WPL. All the WPL, together with the coordinator, will form a Steering Committee that will analyze the overall progress of the project, put forward priorities and scientific plans for the next 6 months periods and, eventually, suggest remedial actions.

c. Meetings

Three types of meetings are foreseen

A general assembly will be held every 6 months. All researchers directly and indirectly involved in the project will be invited to present their more recent data (even negative results). This will allow reaching a critical mass and stimulating interactions at all level of the consortium and integration within the other ongoing related researches. Outstanding speakers

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in the field of the project could be invited on an “opportunity” basis. Attention will be paid however to Intelectual Property Protection, if required.

WP-focused meetings will be held on a 3-months basis. These meetings will be, by essence, informal and will aim to prevent management problems inside or between the WPs.

The steering committee will meet during the general assembly day or on a demand basis.

d. Ethic

The coordinator, with the help of the involved WPL, will be responsible for obtaining approval from the “Ethic Committee for use of animals” (ULg) and from the “Clinical Ethical Committee” (ULg) concerning the use of human cells or tissues (mainly blood and hair follicles). If required, an Informed Consent document will be established for approval by patients. The three laboratories have numerous other projects, sometimes related to this proposal, that have been approved by the ethical committes. Moreover, the coordinator and other researchers of the groups have experience and the required diploma for using laboratory animals. All the experiments will be performed in one of the two animal facilities of the ULg where laboratory partners are already housing and using animals. Furthermore, L3 rooms for viruses manipulation and housing infected animals are also available. The different partners are well aware of the ethical issues related to human beings (Declaration of Helsinki) and to research on animal, such as the 3R rule (Reduction, Refinement, Replacement). As examples,

the various genotoxic agents will be first extensively characterized in relevant models in vitro before to be tested in animals;

cohort containing minimal, but statistically sufficient, number of animals will be established (with the help of the Statistic department);

animal will be housed in optimal conditions and any effort will be made to prevent stress and reduce pain.

For all the abovementioned reasons, we do not foresee problems for obtaining formal approval for any of the WP.

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6. Budget A. Global Budget See explanation on next page

3

months 12

months 12

months 12

months 12

months 9

months year 1 year 2 year 3 year 4 year 5 year 6 Total 2009 2010 2011 2012 2013 2014 Personnel Partner 1 research fellow 7.800 30.800 31.500 32.100 38.000 37.500 177.700Partner 1 Technician * 9.900 39.500 35.550 84.950Partner 2 research fellow 7.800 30.800 31.500 32.100 38.000 37.500 177.700Partner 3 post doc fellow 17.400 71.300 72.000 58.000 218.700Partner 3 technician 0 0 0 sub-total personnel 42.900 172.400 170.550 122.200 76.000 75.000 659.050 Operating cost Small Equipment Partner 1 7.500 7.500Partner 2 7.500 7.500 Sub total small equipment 15000 0 0 0 0 0 15000 Consommables Partner 1 6.000 35.000 55.000 35.000 35.000 25.000 191.000Partner 2 6.000 35.000 35.000 35.000 35.000 25.000 171.000Partner 3 4.000 22.000 22.000 20.000 68.000 Sub total consommables 16.000 92.000 112.000 90.000 70.000 50.000 430.000 Travels/meetings Partner 1 2.000 2.000 2.000 2.000 8.000Partner 2 2.000 2.000 2.000 2.000 8.000Partner 3 2.000 2.000 4.000 Sub total travels 0 6.000 6.000 4.000 4.000 0 20.000 Sub total Operating cost 31.000 98.000 118.000 94.000 74.000 50.000 465.000 Overhead ( 5%) Partner 1 1.560 5.365 6.203 3.455 3.750 3.125 23.458Partner 2 1.065 3.390 3.425 3.455 3.750 3.125 18.210Partner 3 1.070 4.765 4.800 3.900 0 0 14.535 Sub total overhead 3.695 13.520 14.428 10.810 7.500 6.250 56.203

TOTAL 77.595 283.920 302.978 227.010 157.500 131.250 1.180.253

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*A technician will be hired for two years, from Ocober 2009 to September 2011. Research fellows will realise a PhD thesis. They will be hired for four years with a tax-exempt fellowship and a fifth year to complete the work and allow the redaction of their theses with a regular salary. The technician will be hired the first two years to help with the progress of WP2 and WP3. Partner’s 1 operating cost for 2011 include part of the exons chips purchase, the remaining expenses being in charge of other related projects (ESA, Fondation contre le Cancer). Small equipments consist in electrophoresis devices and power supplies. Patrick Roncarati, permanent member of partner’s 3 lab, will be trained by the post-doctoral fellow in order to ensure the completion of the project. The PhD fellow in the laboratory of partner 3 will be mainly responsible for the set up of new culture conditions and assays related to viruses. By the end of his three years contract, he will train P. Roncarati, a permanent technician of the laboratory who will be in charge of the less demanding follow up of the project.

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B. Pluri-annual Budget

Dr A. ColigeDr Y.

Habraken Dr P.

Delvenne

YEAR POST PARTNER

1 PARTNER

2 PARTNER

3 P1+P2+P3

2009 Personnel 17.700 7.800 17.400 42.900

Operating cost 13.500 13.500 4.000 31.000

Overhead 1.560 1.065 1.070 3.695 Sub Total 32.760 22.365 22.470 77.595

2010 Personnel 70.300 30.800 71.300 172.400

Operating cost 37.000 37.000 24.000 98.000

Overhead 5.365 3.390 4.765 13.520 Sub Total 112.665 71.190 100.065 283.920

2011 Personnel 67.050 31.500 72.000 170.550

Operating cost 57.000 37.000 24.000 118.000

Overhead 6.203 3.425 4.800 14.428 Sub Total 130.253 71.925 100.800 302.978

2012 Personnel 32.100 32.100 58.000 122.200

Operating cost 37.000 37.000 20.000 94.000

Overhead 3.455 3.455 3.900 10.810 Sub Total 72.555 72.555 81.900 227.010

2013 Personnel 38.000 38.000 0 76.000

Operating cost 37.000 37.000 0 74.000

Overhead 3.750 3.750 0 7.500 Sub Total 78.750 78.750 0 157.500

2014 Personnel 37.500 37.500 0 75.000

Operating cost 25.000 25.000 0 50.000

Overhead 3.125 3.125 0 6.250 Sub Total 65.625 65.625 0 131.250 TOTAL 492.608 382.410 305.235 1.180.253

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C. Composition of the partner’s laboratories The individuals listed below are members of partner’s laboratories that will support the progress of this project. Their scientific expertise and/or technical help will be necessary for the accomplishment of the project. Partner 1 Head: Dr. Colige Alain (Skills: Cell biology, molecular biology, RNA

interference) Post doctoral researchers :

Dr. Lambert Charles (Skills: gene regulation, alternative splicing, inhibition of aberrant splicing)

Dr. Deroanne Christophe (Skills: gene regulation, cell signalling, microarrays, siRNA)

MD, PhD. B. Ahmed (Skills: use of laboratorys animals) PhD Students :

Mineur Pierre (Skills: gene regulation, alternative splicing, inhibition of aberrant splicing)

Neutelings Thibaud (Skills: gene regulation, alternative splicing) Technicians: Nix Marie-Jeanne (Skills: mRNA extraction, RT-PCR, gel electrophoresis)

Hoffmann Audrey (Skills: mRNA extraction, RT-PCR, gel electrophoresis, cell culture)

Heyeres Antoine (Skills: mRNA extraction, RT-PCR, gel electrophoresis) Partner 2 Head : Prof. Piette Jacques (skills : signal transduction, VZV) Permanent post-doctoral researchers:

Dr. Sadzot-Delvaux Catherine (skills : VZV, immunology and signal transduction) Dr. Dejardin Emmanuel (skills : inflammation and signal transduction) Dr. Habraken Yvette (skills: DNA damage, DNA damage signalling, ATM

interactome) Dr. Legrand-Poels Sylvie (skills : immunology and signal transduction)

Temporary post-doctoral researchers:

Dr. Gloire Geoffrey (skills : oxidative stress, signal transduction and apotposis),

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Dr. Bontems Sebastien (skills : VZV, photodynamic therapy) Dr. Divalentin Emmanuel (skills : VZV, asthma, transcriptome analysis, bio-

informatic analyses) PhD students :

Sabatel Hélène (skills: DNA damage, DNA damage signalling) Ulens Emilie (skills : DNA damage, ATM interactome) Vandevenne Patricia (skills : VZV, signal transduction) Lebrun Marielle (skills : VZV, viral genome BAC and mutation) Ote Isabelle (skills : VZV, signalling, mRNA splicing)

Technicians:

Renotte Nathalie (skills: Cell culture, protein analysis, IF) Lassence Cédric (skills: Cell culture, site directed mutagenesis, PCR)

Partner 3 Head: Prof. Delvenne Philippe (Skills : HPV biology, anatomopathology) Post doctoral researchers :

Dr. Horion Julie (Skills : metaplasia, molecular and cellular biology), Dr. Kustermans Gaëlle (Skills : primary cells culture, HPV, molecular and cellular

biology) Dr. Guenin Samuel (Skills : metaplasia, HPV)

PhD Students :

- Herfs Michael (Skills : metaplasia, HPV, immunology) - Somja Johan (Skills : metaplasia)

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7. List of equipment

The 3 partners are member of the GIGA Research Center and have access to the same equipment. This center has several facilities ((http://www.giga.ulg.ac.be/extranet/services.htm)) including: - Classical culture laboratories, L2 and L3 facilities.

- Genomic plateform with ABI 3730 capillary sequencers, ABI 7900 Taqman, Affimetrix

Genechip System, Illumina Beadstation 500 GX.

- Proteomic plateform with Ion Trap and Maldi Mass spectrometers

- Imaging plateform: FACS Canto and FACS Vantage, Leica Confocal Microscope

- Bio-informatics plateform with a Firewall-protected computer grid and a database system

- Mouse transgenic plateform.

The Laboratory of partner 3 (http://www.crce.ulg.ac.be/biotheque/biotheque.html) also hosts the Human Tissue Bank of the University of Liège. The GIGA offers also the possibility to work with radioactive isotopes in a controlled local and has a Gammacell 40 to irradiate the cell. The three laboratories are well equipped with smaller material such as PCR and qRT-PCR, refrigerated centrifuges, ultracentrifuges, DNA and protein electrophoresis apparatus, epifluorescence microscopes …..