Heide Hellebrand

Christian Sutter

Ellen Honisch

Eva Gross

Barbara Wappenschmidt

Christian Schem

Helmut Deissler

Nina Ditsch

Verena Gress

Marion Kiechle

Helmut Deißler

Claus R Bartram

Rita K Schmutzler

Dieter Niederacher

Norbert Arnold

Alfons Meindl
email: alfons.meindl@lrz.tum.de

Germline mutations in the PALB2 gene are

Keywords: breast cancer, DNA repair, PALB2, population genetics Breast cancer, DNA repair, PALB2, Population Genetics

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Screening of more than 5000 German breast and ovarian cancer families by PCR based techniques revealed that in high risk groups, mutation frequencies in the two known BRCA genes reach between 35-50% [START_REF] Meindl | Comprehensive analysis of 989 patients with breast or ovarian cancer provides BRCA1 and BRCA2 mutation profiles and frequencies for the German population[END_REF]. In contrast, mutations in other predisposing genes like CHEK2 [START_REF] Dufault | Limited relevance of the CHEK2 gene in hereditary breast cancer[END_REF], ATM [START_REF] Dork | Spectrum of ATM gene mutations in a hospital-based series of unselected breast cancer patients[END_REF] and RAD51C [START_REF] Meindl | Germline mutations in breast and ovarian cancer pedigrees establish RAD51C as a human cancer susceptibility gene[END_REF] were detected with low prevalence. As a significant proportion of our families lacking mutations in the BRCA1, BRCA2, RAD51C, CHEK2 or ATM genes, respectively, suggests a strong genetic background, we decided to screen for mutations in PALB2, an additional breast cancer predisposing gene described recently.

PALB2 encodes for a protein functioning as Partner and Localizer of BRCA2 (PALB2 [MIM:610355]). It has been recently associated with recessive mutations in Fanconi anemia [MIM:227650] [START_REF] Xia | Fanconi anemia is associated with a defect in the BRCA2 partner PALB2[END_REF][START_REF] Reid | Biallelic mutations in PALB2 cause Fanconi anemia subtype FA-N and predispose to childhood cancer[END_REF] and heterozygous mutations in familial breast cancer [START_REF] Rahman | PALB2, which encodes a BRCA2interacting protein, is a breast cancer susceptibility gene[END_REF]. Moreover, a recurrent founder mutation has been reported for the Finnish population [START_REF] Erkko | A recurrent mutation in PALB2 in Finnish cancer families[END_REF]. Interestingly, truncating PALB2 mutations have also been found in familial pancreatic cancer cases [START_REF] Jones | Exomic sequencing identifies PALB2 as a pancreatic cancer susceptibility gene[END_REF].

The relative contribution of PALB2 mutations to familial breast cancer cases for most populations, including the German one, is still largely unknown. Besides the Finnish founder mutation only one rare founder mutation has been described in a small study with French-Canadian breast cancer families [START_REF] Foulkes | Identification of a novel truncating PALB2 mutation and analysis of its contribution to early-onset breast cancer in French-Canadian women[END_REF]. Complete analysis of the PALB2 gene in 95 Spanish breast cancer families revealed only one novel mutation, which was not present in another 725 high risk breast cancer cases [START_REF] Garcia | Analysis of FANCB and FANCN/PALB2 fanconi anemia genes in BRCA1/2-negative Spanish breast cancer families[END_REF]. Moreover, sequencing of the entire PALB2 gene in 360 Chinese women with early-onset breast cancer revealed only two novel truncating mutations present in three out of 360 families, which were further validated in a larger set of BRCA1/2 negative Chinese samples [START_REF] Cao | The prevalence of PALB2 germline mutations in BRCA1/BRCA2 negative Chinese women with early onset breast cancer or affected relatives[END_REF]. Finally, two novel aberrations were identified very recently in Italian families [START_REF] Papi | A PALB2 germline mutation associated with hereditary breast cancer in Italy[END_REF][START_REF] Balia | PALB2: a novel inactivating mutation in a Italian breast cancer family[END_REF]. Here we present the complete screening for mutations in the PALB2 gene in 818 familial cases of breast cancer from Germany.

MATERIALS AND METHODS

Patients and Families

Index patients from 818 unrelated breast cancer families were recruited through a clinicogenetic counselling setting at the Familial Breast and Ovarian Cancer Centers in Cologne, Düsseldorf, Heidelberg, Kiel, Munich and Ulm. They had been categorized in different risk groups: Group A: three or more affected females with breast cancer, at least two of them diagnosed before the age of 50 ys (n = 330); Group B: three or more affected females independent of age (including families with one breast cancer case <50ys, n = 229); Group C: two cases of breast cancer diagnosed before the age of 50 ys (n = 68); Group E: at least one case of breast and one ovarian cancer, respectively (n = 143). All affected index patients had been screened for small nucleotide alterations in BRCA1 and BRCA2 by the PCR-based mutation detection techniques dHPLC and/or direct DNA-sequencing [START_REF] Meindl | Comprehensive analysis of 989 patients with breast or ovarian cancer provides BRCA1 and BRCA2 mutation profiles and frequencies for the German population[END_REF] and tested negative for pathogenic mutations. DNAs from 450 control samples from healthy women aged older than 50 years were collected in Kiel (100), Düsseldorf (150) and Munich (200).

Direct sequencing

PCR and sequencing pimers are listed in Supp. Table S1. DNAs were amplified using AmpliTaq Gold or Platinum Taq DNA polymerase (Invitrogen) or the Expand High Fidelity PCR system (Roche). Sequencing was carried out after PCR amplification of all 13 exons of the PALB2 gene by using the same primers in the chain terminating reaction. Sequence reactions were electrophoresed on an ABI 3100 automated DNA sequencer (Applied Biosystems, Darmstadt, Germany). Sequences were analysed by using Sequence Pilot software (JSI-Medisys, Kippenheim, Germany) and by visual inspections of chromatograms. Reference sequence was obtained from NM_024675.3 using the first nucleotide of the ATG translation start as +1.

Denaturing high pressure liquid chromatography (dHPLC) and high resolution melting analysis (HRM)

The detection of sequence variants in exons 1, 2, 6, and 8-13 was carried out by dHPLC analysis as described elsewhere [START_REF] Xiao | Denaturing high-performance liquid chromatography: A review[END_REF]. In brief, the mutation analysis was done on a Wave DNA fragment analysis system (Transgenomic, Omaha, NE) under partially denaturing conditions. PCR products were heated up to 95°C and slowly cooled down to generate heteroduplex molecules in the case of heterozygous DNA samples. The optimal system performance was checked by standard samples (Transgenomic) and positive controls generated by in vitro mutagenesis (see below).

Exons 3, 4, 5 and 7 were screened by HRM analysis on a Lightcycler 480 instrument (Roche, Penzberg, Germany) using the Gene scanning software module of the system and a sensitivity setting of 0.30. Melting differences were visualized by Difference plots.

In vitro mutagenesis

To supply positive controls for a dHPLC/HRM screening, mutations were generated by in vitro mutagenesis (see asterisks in Fig. 1). We used the Quick change R II Site-Directed Mutagenese kit (Stratagene, Heidelberg, Germany).

Prediction of pathogenicity of missense variants

Potential consequences of missense mutations were obtained by using the prediction softwares SIFT (http://sift.jcvi.org/), PolyPhen (http://genetics.bwh.harvard.edu/pph), and Mutation Taster (http://www.mutationtaster.org/).

RESULTS

Mutation analysis of the PALB2 gene in familial breast cancer cases

In order to determine PALB2 mutation frequencies already identified in other populations (Fig. 1a) and/or to detect novel PALB2 sequence variations in the German population, we decided to sequence in a first step all 13 coding exons of the entire PALB2 gene in 108 familial breast cancer cases, which were enriched for high risk families from group A and E. Two novel truncating mutations in exon 4 (c.1240C>T, p.R414X) and exon 5 (c.2145_2146delTA, p.D715EfsX2) were found (Fig. 1b), whereas none of the published clearly pathogenic PALB2 mutations were detected (Table 1). In addition, we identified a novel, probably disease causing missense variant in exon 13 (c.3428T>C; p.L1143P, see below). Additional 710 families were screened for PALB2 mutations using the dHPLC and HRM high throughput techniques (see Fig. 1c). To establish the assay conditions, at least one positive mutational control was used. As for some of the exons mutated genomic DNAs were not available, we generated additional mutated templates by in vitro mutagenesis for an effective and reliable screening with respect to all melting domains of the amplicons (see Fig. 1, mutations with asterisks).

None of the known published truncating mutations [START_REF] Erkko | A recurrent mutation in PALB2 in Finnish cancer families[END_REF][START_REF] Cao | The prevalence of PALB2 germline mutations in BRCA1/BRCA2 negative Chinese women with early onset breast cancer or affected relatives[END_REF][START_REF] Garcia | Analysis of FANCB and FANCN/PALB2 fanconi anemia genes in BRCA1/2-negative Spanish breast cancer families[END_REF][START_REF] Rahman | PALB2, which encodes a BRCA2interacting protein, is a breast cancer susceptibility gene[END_REF][START_REF] Foulkes | Identification of a novel truncating PALB2 mutation and analysis of its contribution to early-onset breast cancer in French-Canadian women[END_REF][START_REF] Sluiter | PALB2 sequence variants in young South African breast cancer patients[END_REF][START_REF] Adank | PALB2 analysis in BRCA2-like families[END_REF][START_REF] Dansonka-Mieszkowska | A novel germline PALB2 deletion in Polish breast and ovarian cancer patients[END_REF][START_REF] Balia | PALB2: a novel inactivating mutation in a Italian breast cancer family[END_REF], except one single nonsense mutation in exon 5, c.2257C>T (p.R753X) [START_REF] Papi | A PALB2 germline mutation associated with hereditary breast cancer in Italy[END_REF], as well as none of our two novel mutations and the one putative pathogenic missense variation identified by direct sequencing, could be found in the 710 additional familial cases. However, we could identify one novel splice mutation in exon 1 (c.48 +1G>C), one novel frameshift mutation in exon 3 (c.172_175delTTGT, p.Q60RfsX7), and two further novel nonsense mutations in exon 4 (c.503C>A), and exon 9 (c.2962C>T) causing direct introduction of premature stop codons at the positions p.S168X and p.Q988X, respectively. The family history and histopathology of the tumor tissues of our index patients is indicated in Table 1.

Moreover, we identified by the dHPLC and HRM method additional 11 novel missense variants (Supp. Table S2). The application of three different prediction programs and conservation profiles indicated that only one of them might be disease associated (c.3128G>C; p.G1043A).

Several observations support the classification of the missense mutations L1143P and G1043A as pathogenic: a) not present in 450 healthy controls, b) highly evolutionary conserved, and c) all the three prediction algorithms PolyPhen, SIFT and Mutation Taster predicted a damaging effect on the protein. Nevertheless, they still have to be considered as unclassifed variants and functional analyses are warranted. This also applies for the missense mutations G998E and L939W, which are both present in patients and controls, however, overrepresentation of both changes in patients cannot be ruled out currently. Finally, one complex change in the 5`UTR, c.-214_236delins10, was found both once in patients and controls, but RNA analysis is required for a final characterization. This might also be indicated for two intronic aberrations (c.49-15t>c; c.109-12t>a), which were found each once in patients, but not in controls.

Patients' and family characteristics of identified mutation carriers

In total, we identified seven truncating mutations and two putative pathogenic missense mutations in the 818 familial cases (Table 1). As an extensive segregation analysis was available only in two families (e. g. Fig. 2), calculation of penetrance for our families revealed to be difficult. However, based on the obtained and partially documented family data, it can be considered as high, supporting the observation of higher ORs of clearly pathogenic PALB2 mutations within a familial context [START_REF] Byrnes | Are the so-called low penetrance breast cancer genes, ATM, BRIP1, PALB2 and CHEK2, high risk for women with strong family histories?[END_REF]; Southey et al., 60 th Annual meeting of the ASHG, Washington DC, 2010). The appearance of a PALB2 mutation might not be associated with breast cancer only, but also with colon/ovarian cancer. Concerning histology, PALB2 mutations can cause either hormone receptor positive, but equally hormone receptor negative breast cancer. As previously observed [START_REF] Tischkowitz | PALB2/FANCN: recombining cancer and Fanconi anemia[END_REF], estrogen/progesterone receptor negative carcinomas appear therefore to be slightly overrepresented in PALB2-related tumors compared to all breast cancers.

▼ ▼ ▼ ▼ ▼ ▼ ▼ ▼ ▼ ▼ ▼ ▼ ▼▼ ▼ ▼ ▼ ▼ ▼ p.

DISCUSSION

The PALB2 gene product is one of approximately ten proteins which directly complex with or bind to BRCA1 or BRCA2. The PALB2 protein interacts at the site of four WD40 domains with the very N-terminus of BRCA2 and at the very N-terminal site with a heptad coiled coil stretch in BRCA1, thus stabilizing its nuclear functions in DNA repair [START_REF] Xia | Control of BRCA2 cellular and clinical functions by a nuclear partner, PALB2[END_REF]Sy et al., 2009b;[START_REF] Zhang | PALB2 functionally connects the breast cancer susceptibility proteins BRCA1 and BRCA2[END_REF][START_REF] Zhang | PALB2 functionally connects the breast cancer susceptibility proteins BRCA1 and BRCA2[END_REF]. Patients lacking a functional PALB2 protein on the basis of inactivating mutations either develop Fanconi anemia (FANCN) or familial breast cancer. Moreover, truncating mutations have also been reported for familial pancreatic cancer [START_REF] Jones | Exomic sequencing identifies PALB2 as a pancreatic cancer susceptibility gene[END_REF]. More than 20 distinct clearly pathogenic mutations have been reported in the literature (most of them summarized in ref. [START_REF] Jones | Exomic sequencing identifies PALB2 as a pancreatic cancer susceptibility gene[END_REF]; quite novel ones in ref. [START_REF] Dansonka-Mieszkowska | A novel germline PALB2 deletion in Polish breast and ovarian cancer patients[END_REF][START_REF] Papi | A PALB2 germline mutation associated with hereditary breast cancer in Italy[END_REF][START_REF] Balia | PALB2: a novel inactivating mutation in a Italian breast cancer family[END_REF] or in international databases, e.g. HGMD, so far. However, only 16 of them have been identified in breast cancer families (see Fig. 1a). Based on these findings we were prompted to study 818 unrelated German familial breast cancer patients tested negative for BRCA1/2, together with 450 unaffected controls. In addition to the recently and quite actually published data [START_REF] Papi | A PALB2 germline mutation associated with hereditary breast cancer in Italy[END_REF][START_REF] Dansonka-Mieszkowska | A novel germline PALB2 deletion in Polish breast and ovarian cancer patients[END_REF][START_REF] Balia | PALB2: a novel inactivating mutation in a Italian breast cancer family[END_REF], we could add six novel truncating mutations and two novel putative disease contributing missense variants.

Population and ethnicity specific mutation profiles for the PALB2 gene

Our data convincingly support the evidence of population-specific mutational profiles for the PALB2 gene. Only one out of the truncating mutations described here has been found before in other breast cancer families from different geographic or ethnic origin, nor were we able to find any of the published mutations in our population. Likewise, in addition, we could exclude the occurrence of the recurrent Finnish founder mutation [START_REF] Erkko | A recurrent mutation in PALB2 in Finnish cancer families[END_REF], the four distinct truncating PALB2 mutations found in nine British breast cancer families of mixed ethnical background [START_REF] Rahman | PALB2, which encodes a BRCA2interacting protein, is a breast cancer susceptibility gene[END_REF], and the recurrent mutation (c.2323C>T) in the French-Canadian population [START_REF] Foulkes | Identification of a novel truncating PALB2 mutation and analysis of its contribution to early-onset breast cancer in French-Canadian women[END_REF], in our 818 familial cases. The nonsense mutation c.2257C>T, found in a breast cancer family from Italy, had been published after finding this mutation in our cohort.

So far, the identified mutations in the PALB2 gene are scattered among the entire gene and seem to be of very low frequency and distinct for different populations. The lack of recurrent founder mutations for most of the Caucasian populations or other ethnicities seems therefore to require initial comprehensive mutation screenings for the different populations or ethnicities. However, large genomic rearrangements in the PALB2 gene do not appear to contribute to the hereditary breast cancer susceptibility as reported by two independent publications [START_REF] Ameziane | Lack of large genomic deletions in BRIP1, PALB2, and FANCD2 genes in BRCA1/2 negative familial breast cancer[END_REF][START_REF] Pylkas | Analysis of large deletions in BRCA1, BRCA2 and PALB2 genes in Finnish breast and ovarian cancer families[END_REF].

Unclassified variants in the PALB2 protein

The prevalence of truncating mutations in the PALB2 gene is in the range of about 1.0% (Fig. 1 andref. [START_REF] Turnbull | Genetic predisposition to breast cancer: past, present, and future[END_REF]). The importance of PALB2 as a predisposing gene for familial breast cancer might on the other hand be increased by the detection and characterization of additional protein affecting missense mutations. For example, it has been reported that the PALB2 protein directly interacts with the very 5´end of the BRCA2 protein [START_REF] Xia | Control of BRCA2 cellular and clinical functions by a nuclear partner, PALB2[END_REF]Sy et al., 2009b;[START_REF] Zhang | PALB2 functionally connects the breast cancer susceptibility proteins BRCA1 and BRCA2[END_REF], and recently it has been shown that the aminoterminal part of the PALB2 protein is required for binding a coiled-coiled domain of BRCA1 (Sy et al., 2009a;[START_REF] Zhang | PALB2 functionally connects the breast cancer susceptibility proteins BRCA1 and BRCA2[END_REF]. Obviously, amino acid substitutions in these binding domains might be pathogenic. In agreement with these observations, we could find two missense mutations in this study, which are most likely pathogenic (p.T1030I and p.L1143P) and located in a domain of the PALB2 protein, which is interacting with BRCA2 (Sy et al., 2009a). Functional studies are underway to systematically investigate these putative pathogenic missense mutations by complementation assays [START_REF] Takata | Chromosome instability and defective recombinational repair in knockout mutants of the five Rad51 paralogs[END_REF]. On the other hand, we identified four novel aberrations in the 5´UTR and intronic part of the gene, which might cause either aberrant expression or splicing of the gene. RNA analysis experiments are underway. Two missense mutations (L939W, G998E) were found in patients and controls. Both seemed to be slightly overrepresented in patients, both are highly conserved and both were predicted to be deleterious by three algorithms. Their putative character as low risk variants should be clarified by the application of an extensive case-control study.

Impact on therapy and genetic counseling

As BRCA1-and BRCA2-deficient cells have been found to be highly sensitive to poly(ADP-ribose) polymerrase (PARP) inhibition, it has been assumed that this would be true as well for tumors lacking PALB2. Indeed, it could be shown recently that PARP inhibitor AZD2281 was cytotoxic to PALB2-mutant lymphoblasts [START_REF] Buisson | Cooperation of breast cancer proteins PALB2 and piccolo BRCA2 in stimulating homologous recombination[END_REF]. Hence, regarding the favorable treatment options by PARP inhibitors observed in familial BRCA1/2associated breast cancer cases [START_REF] Fong | Inhibition of poly(ADP-ribose) polymerase in tumors from BRCA mutation carriers[END_REF], the presence of PALB2 mutations should be of clinical relevance for providing tailored therapy regimens to the respective patients.

Figure 1 :

 1 Figure 1: Truncating mutations in the PALB2 gene. a) Localization of the previously reported deleterious mutations in the PALB2 gene identified in breast cancer families; published mutations indicated by an asterisk were additionally prepared by in vitro mutagenesis for the purpose of using them as positive controls for the dHPLC screening (see 1c). b) Depiction of novel mutations found by direct sequencing in 108 familial cases tested negative for BRCA1/2 mutations; c) Mutation sites in the PALB2 gene found in 710 familial cases analysed by dHPLC and HRM analysis.

  Figure. 2: Segregation of the nonsense mutation p.Q988X in a breast cancer family. a) Pedigree of the family. b) Corresponding sequence alteration c.2962C>T (p.Q988X) detected in exon 9 of the PALB2 gene. Additional pedigrees of the PALB2 mutation carriers are available in Supp.Figure S1.

Table S1 . Primer sequences covering the coding region of the PALB2 gene

 S1

	c.48+1G>C		Q60RfsX7		S168X
	Exon	Forward Primer	+93y	Reverse Primer	+72y	91y
	1	5' -ACTGGGACTCATCGACAGC -3' BC 90y	5' -TGGTCAGATGATACTGCTGC -3'	73y	73y
	2	5' -TTATGGACCAGTGCTACTCC -3'	5' -AGCTCTTTGGGCACGCTAG -3'	BC 61y
		74y	70y	71y		+82y
	BC 75	BC 63y c.48+1 49y BC 43y F o r P e e r R e v i e w 46y BC 60y p.Q60RfsX7 55y 51y 39y 52y 47y BC 46y CC 68y p.Q60RfsX7 BC 38y D715EfsX2 35y OC 70y BC bil. 42y BC 64y R414X OC 50y BC 44y p.R414X BC 64y BC 58y OC 46y BC 33y 60y p.R753X 58y	WT 52y 19y 42y [grandmother BC 43y] 45y BC 39y p.S168X 8y 38y 10y BC 63y 66y p.R753X R753X 11y 19y 23y 24y	4y	17y 41y 6y
				p.D715EfsX2

Supp

. Figure. S1: Pedigrees of the breast cancer families harbouring PALB2 mutations Page 14 of 13 John Wiley & Sons, Inc. Human Mutation

John Wiley & Sons, Inc.Human Mutation

Acknowledgements

We are indebted to Dr. D. Schäfer, Institute of Human Genetics, University of Frankfurt for providing one family. We thank Stefanie Engert, Jochen Meyer, Farnoosh Fathali-Zadeh, Maike Kreikemeier, Doris Karow, Birgit Teegen and Regina Grunewald for expert technical assistance.

Web Resources

The Human Gene Mutation Database at the Institute of Medical Genetics in Cardiff: http://www.hgmd.org/ Online Mendelian Inheritance in Man (OMIM): http://www.ncbi.nlm.nih.gov/Omim/ Mutation Taster: http://www.mutationtaster.org/ SIFT: http://sift.jcvi.org/ PolyPhen: http://genetics.bwh.harvard.edu/pph

Contract grant sponsor: German Cancer Aid, grant number: Me 107352

F o r P e e r R e v i e w


Murugan Kalimutho

Antonella Minutolo

Sandro Grelli

Amanda Formosa

Giulia Sancesario

Alessandra Valentini

Giorgio Federici

Sergio Bernardini
email: bernardini@med.uniroma2.it

Dr Murugan

cell cycle arrest and potentiates apoptosis via multiple death pathways in colorectal

Keywords: Satraplatin. CRC. Apoptosis. Platinum. p53. Oxaliplatin. miR-34a CRC: Colorectal cancer, DISC: death-inducing signalling complex, miR: MicroRNA, Satraplatin: JM-216, IC: Inhibitory concentration, CRPC: Castration-Resistant Prostate Cancer, UTR: Untranslated Region, platinum: p

Introduction

The median progression-free survival of advanced colorectal cancer (CRC) patients has increased since the introduction of oxaliplatin in combination with 5-Fluorouracilleucovorin in systemic chemotherapy treatment. However, as a single agent, oxaliplatin has been less effective against transforming cancerous cells. Despite its proven activity in combination treatments, the acquisition of drug resistance remains a major obstacle in CRC management, ultimately leading to patients death [START_REF] Goldberg | A randomized controlled trial of fluorouracil plus leucovorin, irinotecan, and oxaliplatin combinations in patients with previously untreated metastatic colorectal cancer[END_REF].

Thus, many attempts have been made to scrutinize the mechanisms of chemo-resistance.

The mechanisms include increased DNA repair, impaired DNA adducts formation, overexpression of copper transporters, enhanced drug detoxification and increased tolerance for DNA damage [START_REF] Hector | In vitro studies on the mechanisms of oxaliplatin resistance[END_REF][START_REF] Desoize | Particular aspects of platinum compounds used at present in cancer treatment[END_REF][START_REF] Gourdier | Drug specific resistance to oxaliplatin is associated with apoptosis defect in a cellular model of colon carcinoma[END_REF][START_REF] Chen | Combined modalities of resistance in an oxaliplatin-resistant human gastric cancer cell line with enhanced sensitivity to 5-fluorouracil[END_REF].

The p53 gene takes part in cellular responses to DNA damage by regulating cell cycle progression, cell death and DNA repair [START_REF] Lane | p53, guardian of the genome[END_REF]. It is mutated in more than 50% of colorectal tumours [START_REF] Arango | Molecular mechanism of action and prediction of response to oxaliplatin in colorectal cancer[END_REF]. Furthermore, the functional prominence of this genome guardian was correlated to cellular sensitivity and/or resistance to platinum agents. Although a clear interpretation is lacking, it has been suggested that the decreased sensitivity to platinum agents is a consequence of reduced or unfound expression of the p53 protein in these tumour models [START_REF] Manic | Mechanisms controlling sensitivity to platinum complexes: role of p53 and DNA mismatch repair[END_REF].

One major process leading to chemotherapy resistance is the ability of cancerous cells to evade apoptosis [START_REF] Mitsiades | Matrix metalloproteinase-7-mediated cleavage of Fas ligand protects tumor cells from chemotherapeutic drug cytotoxicity[END_REF]. The development of drugs resistant cells resembles the stages of tumour progression where molecular changes cause the evolution of cancerous cells that proliferate due to their ability to escape programmed cell death [START_REF] Micheau | Sensitization of cancer cells treated with cytotoxic drugs to fas-mediated cytotoxicity[END_REF][START_REF] Peter | Does CD95 have tumor promoting activities?[END_REF].

Thus attempts have been made to synthesize a platinum agent with more efficient binding sites in combination with other molecules to overcome the problems that arise with current chemotherapy regimens, including oxaliplatin cross resistance. For instance, satraplatin has been studied in a phase III clinical trial for Castration-Resistant Prostate Cancer (CRPC) [START_REF] Mark | New-generation platinum drugs in the treatment of cisplatin resistant cancers Expert[END_REF]. Satraplatin (also known as JM-216) or bis (acetato) ammine dichloro (cyclohexylamine) platinum (IV) is the first platinum analogue available for oral administration. Satraplatin exhibits a higher cytotoxic activity in vitro when compared to other platinum compounds and also exhibits activity against some cisplatin-resistant human tumour models [START_REF] Kelland | Preclinical antitumor evaluation of bis-acetato-ammine-dichloro-cyclohexylamine platinum (IV): an orally active platinum drug[END_REF][START_REF] Sharp | Transport of cisplatin and bis-acetatoammine-dichlorocyclohexylamine platinum (IV) (JM-216) in human ovarian carcinoma cell lines: identification of a plasma membrane protein associated with cisplatin resistance[END_REF].

As earlier developed platinum agents typically demonstrate significant de novo resistance and/or acquired resistance in tumour cells, satraplatin"s uniqueness dwells on its putative lack of cross-resistance [START_REF] Moore | Phase II evaluation of weekly cisplatin in metastatic hormone-resistant prostate cancer: a southeastern cancer study group trial[END_REF][START_REF] Mckeage | Phase I and pharmacokinetic study of an oral platinum complex given daily for 5 days in patients with cancer[END_REF][START_REF] Fokkema | Phase II study of oral platinum drug JM216 as first-line treatment in patients with small-cell lung cancer[END_REF]. Based on preliminary cell culture investigations on various tumour types, satraplatin exhibited the ability to create DNA adducts that are more resistant to recognition by DNA nucleotide excision repair enzymes and DNA mismatch repair proteins [START_REF] Fink | The role of DNA mismatch repair in platinum drug resistance[END_REF][START_REF] Fokkema | JM216, JM118, and cisplatin induced cytotoxicity in relation to platinum-DNA adduct formation, glutathione levels and p53 status in human cell lines with different sensitivities to cisplatin[END_REF][START_REF] Sternberg | Phase III trial of satraplatin, an oral platinum plus prednisone vs. prednisone alone in patients with hormone-resistant prostate cancer[END_REF]. Moreover, satraplatin-induced adducts are more stable and do not bind to high mobility group 1 proteins, which recognize DNA damage.

Satraplatin also inhibits trans-lesion replication by certain DNA polymerases [START_REF] Vaisman | Effect of DNA polymerases and high mobility group protein 1on the carrier ligand specificity for translesion synthesis past platinum-DNA adducts[END_REF]. These differences may provide a mechanism to overcome resistance to treatment observed in other platinum compounds. In an effort to investigate the underlying mechanism associated with the cytotoxicity of satraplatin, we examined the efficacy of satraplatin compared to oxaliplatin using colorectal cancer cell lines with regards to their p53 phenotype. Moreover to further demonstrate the mechanisms of satraplatin and its effect to overcome the chemo resistance of oxaliplatin in the absence of p53 gene, we further our analysis on three representative CRC cells exclusively one with p53 wildtype (HCT116), one for mutant p53 (HT29) and the final one for p53 knockout (HCT116). We further analyzed the cycle perturbation and basic molecular mechanisms of apoptosis induced by satraplatin in these cells.

Materials and Methods

Drugs, chemicals and reagents

Oxaliplatin was purchased from Sigma (09512-5MG) and satraplatin was kindly gifted by Agennix AG (formerly GPC Biotech AG), Fraunhoferstr, Germany. Stock solutions of satraplatin and oxaliplatin were prepared in 0.09% saline solution stored at -20°C.

Antibodies were purchased from appropriate companies as indicated: anti-p53 and anti-Bid (BD Pharmingen), anti-β-actin (Sigma), anti-Bax (Millipore), anti-BCL2 (Sigma), anti-p21 Cip1/WAF1 , anti-c-ABL, anti-Fas and anti-Cyclin-B1 (Santa Cruz), and anti-Caspase 8 (Alexis Biochemical). Secondary antibody of anti rabbit and mouse were purchased from DakoCytomation, Denmark A/S. ECL detection reagents from Perkin-Elmer. miRNA analysis primer sets from Applied Biosystems.

Cell culture

HT29 were kindly provided by Dr. Isabella Faroni, University of Rome "Tor Vergata" and HCT116, HCT116 p53 -/-and HCT15 cells were obtained from Dr. Soddu, Regina Elena Cancer Institute, Italy. WiDr and LoVo cells were given by Dr. Rossana Supino from Instituto Nazionale Tumori, Milan, Italy and HCT116 p21 -/-were obtained from Prof. Bert Vogelstein, from Ludwig Centre at John Hopkins, USA. All the cell lines were tested for mycoplasma infection and were grown in monolayer cultures in DMEM (HCT116, HCT116 p53 -/-, HT29, HCT116 p21 -/-), DMEM: F12 (WiDr), RPMI 1640 (HCT15) and Ham"s Nutrients mix F12 (LoVo) supplemented with 10-20% FBS and 1% penicillin streptomycin according to UKCCCR guidelines [START_REF] Masters | UKCCCR Guidelines for the Use of Cell Lines in Cancer Research[END_REF]. No antibiotics were added to the medium prior to the treatment. The cells were trypsinized and passed once a week and all the experiments were conducted at early passages.

Cytotoxicity evaluation assay

The concentration of oxaliplatin and satraplatin resulting in 50% inhibition of control growth (IC 50 ) was calculated with an MTS assay (Promega). Three thousand cells per well were seeded in 96-well plates and 24h later the cells were exposed to 200 µl of 0-250 µM oxaliplatin and satraplatin. After 72 h of incubation, 20 µl of MTS solution was added and the IC 50 concentrations were calculated using CalcuSyn Version 2.0. Since this analysis is concerned with a comparison between oxaliplatin and satraplatin, the analysis was done with drug concentrations controlled to be below IC50 value (10 µM) for practical considerations. At this concentration for both drugs, the percentage cell death lies between 20%-40% for all cell lines tested.

Intracellular platinum quantification

Intracellular platinum (Pt) levels were measured following 2h incubation with 6.25 µM, 12.5 µM, 25 µM, 50 µM and 100 µM of satraplatin or oxaliplatin. Approximately 1x10 6 cells were washed twice with 5 ml of cold phosphate-buffered saline (PBS) and the monolayer was scraped into 2 ml PBS. Cellular Pt content was determined by flameless atomic absorption spectroscopy (FAAS) using a Perkin-Elmer AAnalyst 800 (Beaconsfield, Bucks, UK). Cell aliquots were dissolved in 0.2% nitric acid and automatically injected into the graphite furnace. The concentration of Pt in ng/mL was measured by comparing Pt standards ranging from 5 to 500 ng/ml. Under these conditions the limit of detection was between 5 and 10 ng Pt/ml. The cellular platinum contents were then expressed as nmol Pt/ml normalized to 1 x 10 6 cells.

Cell cycle analysis

For cell cycle analysis 5 x 10 4 cells of HCT116, HCT116 p53 null and HT29 were seeded in six well plates in triplicate and allowed to attach for 24h. For time response studies, cells were treated with 10 µM satraplatin and oxaliplatin for 48h and 72h. Both attached and floating cells were harvested, washed twice with 1ml of PBS and re-suspended in PBS containing 10 mg/ml of RNAse and incubated for 1 hour. Later, 10-mg/ml propidium iodide (Sigma p4170-100 mg) was added prior to the analysis. Cells were analysed for DNA content using a FACScan flow cytometry (BD Biosciences, Mountain View, CA). The proportion of cells in G0/G1, S phase and G2/M were quantified using CellQuest TM software (BD Biosciences, San Jose, CA). The software directly calculated the percentage of cells in each phase.

Evaluation of apoptosis

For apoptosis, assays cells were plated in 6-well plates and treated with 10 µM of satraplatin or oxaliplatin. Isolated nuclei were then analysed after 24, 48 and 72h of treatment, using a FACScan flow cytometry (BD Biosciences, Mountain View, CA) as previously described [START_REF] Matteucci | Identification of nuclei from apoptotic, necrotic, and viable lymphoid cells by using multiparameter flow cytometry[END_REF]. Briefly, detectors and amplifier gains for forward and orthogonal scatter were adequately selected in order to simultaneously detect nuclei from viable, apoptotic and necrotic cells. Events were gated on forward versus orthogonal scatter in such a way that degraded DNA from cell debris or from doublets was excluded and nuclei from viable, apoptotic and necrotic cells were assayed. Data acquisition and analysis was performed using CellQuest TM software (BD Bioscences, San Jose, CA) on a minimum of 5,000 events for each samples. The second method for evaluation of apoptosis consisted in double staining of the cells with fluorescent annexin V and with PI solution for the FACS data confirmation. To this purpose, the "Annexin V-FITC Apoptosis Detection Kit" (BD-Bioscience Pharmingen, San Diego, CA, USA) was used according to manufacturer instructions. Briefly, 5 x10 5 cells were incubated for 15 minutes with annexin-V-fluorescein isothiocyanate (FITC) and then washed in annexin buffer. Cells were then stained with PI solution and analysed immediately after staining by flow cytometry using a FACScan flow cytometry and Cell Quest TM software. This method is widely accepted to distinguish between early apoptosis and necrosis.

Quantitative PCR

RNA isolation, cDNA synthesis and RT-PCR

Total RNA was isolated with TRIzol reagent (Invitrogen, CA). The integrity of total RNA was determined by 1% agarose gel electrophoresis. cDNA synthesis was carried out with the Superscript III cDNA synthesis kit (Invitrogen, CA) using 1 μg of total RNA as the template and specific reverse primers (Table 1) according to manufacturer instructions. The PCR amplification was carried out in a final volume of 25μl containing 12.5 μl of SYBR green (2X), 0.5 μl both primers (10 μM), 1 μl of cDNA. Cycling conditions included preheating at 95°C for 10 minutes and then 40 cycles of: 95°C for 30 seconds, 60°C for 30 seconds and 72°C for 45 minute, followed by standard dissociation curve. Analysis below 35 cycles was considered for the 2^-ΔΔCq calculation according to the MIQE guidelines [START_REF] Bustin | The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments[END_REF].

miRNA reverse transcription and qRT-PCR analysis

Enriched microRNAs were extracted with mirVana™ Isolation Kit (Ambion Inc. TX).

Based on TARGET scan and PIC target, miRNAs that target our molecules were selected. Based on the top predicted target, we identified 14 miRNAs involved with our selected gene group focusing on cell cycle analysis, cell death and DNA repair system.

Applied Biosystems qRT-PCR primer sets for miRNA specific reverse transcription including hsa-miR-150 (p53/ERCC1), hsa-miR-152 (MLH1/GADD45), hsa-miR-128a (MLH1/BAX), hsa-miR-34a (BCL2), hsa-214 (BAX), hsa-miR-218 (BRCA1), hsa-miR-21 (MSH2), hsa-miR-106a (p21), hsa-miR-20, hsa-miR-148a (GADD45), hsa-miR-370 (CLU), hsa-miR-326 (Tp73), hsa-miR-128b, hsa-miR-17-3p and endogenous control RNU19 and RNU6B were utilized according to the manufacturer"s protocol. Briefly, the reaction master mix containing 5 × RT Buffer, 5 × RT Primer, Array-Script™ Enzyme Mix and nuclease-free water was mixed with 50 ng of input microRNA. The mixture was incubated for 30 min at 37°C, and then 10 min at 95°C. qPCR was carried out using the Stratagene Real-Time PCR System and Applied Biosystems qRTPCR miRNA Detection Kit (Ambion Inc. TX). The PCR master mix containing Applied Biosystems 2× PCR Buffer (with FAM labeled Taqman probe), 1µL FAM labeled specific primers and cDNA from process miRNA. RT products was processed as follows: 95°C for 10 min, and then 95°C for 15 sec and 60°C for 30 sec for up to 40 cycles (n = 2). Gene expression was calculated via a 2^-ΔΔCq method.

Western blotting

All cells were seeded in 75 cm 2 flasks and treated with 10 µM oxaliplatin or satraplatin for 24, 48 or 72 h, then rinsed with PBS twice, harvested and re-suspended in 150 ml of Chaps 0.1% lysis buffer (1 M Tris-Cl pH 7.5, 5 M NaCl, 10% CHAPS, ddH 2 O). The cells suspension was vortexed and kept on ice for 30 min before cell debris was pelleted and the supernatant transferred to a new tube. SDS-polyacrylamide gel electrophoresis sample loading buffer was added to 20 µg aliquots, heated at 95 o C for 5 minute and then loaded on 10% Tris-HCl gels (BioRad, Hercules, CA, USA). Proteins were transferred to a nitrocellulase membrane (Amersham, Piscataway, NJ, USA) and blocked with 1% nonfat milk and 1% BSA for at least 2 h. Membranes were then probed at 4 o C with the appropriate primary antibody in 0.1% T-TBS overnight with the following antibodies: anti-p53 (1/500), anti-p21 Cip1/WAF1 (1/200) and anti-β-actin (1/1000) anti-Bax (1/500), anti-BCL2 (1/200), anti-c-ABL (1/200), anti-Fas (1/500), anti-Cyclin-B1 (1/200), anti-Caspase 8 (1/500) and anti-Bid (1/500). Membranes were washed three times with washing buffer (PBS with 0.1% Tween-20 for 10 minute interval). Then the filter was probed with the appropriate peroxidase-conjugate secondary antibody for 1 h (from DakoCytomation, Denmark A/S). The secondary antibody was washed three times with washing buffer and the signal was developed using ECL Plus Western Blotting Detection Method (Perkin-Elmer). Detection was carried out using a Molecular Imager ChemiDoc XRS System, Bio-Rad laboratories (UK) Ltd. Protein levels were standardized using the signal from the β-actin probe.

Statistical analysis

The gene expression ΔCq values of mRNA and miRNAs from each sample were calculated by normalizing with internal control of ACTB and B 2 M (for mRNA) and RNU19 and RNU6B (for microRNAs) and relative quantitation values were calculated according to the 2^ΔΔCq method. The statistical studies were performed using a statistical software system (SPSS, version 12.0 for windows, Chicago, IL, USA). The statistically significant differences in expression level between control and treated samples for each target were calculated using a paired Wilcoxon test. Comparison of mean values of proliferation and apoptosis levels in response to satraplatin and oxaliplatin treatment was carried out using the Bonferroni post hoc multiple comparisons ANOVA test. *p≤0.05 value was considered significant while ** p≤0.001 value was considered highly significant.

Results

Cytotoxic effect of satraplatin in the panel of colorectal cancer cells

In order to evaluate the effective drug concentrations, a panel of six CRC cell lines with different p53 genotypes were treated with various concentrations (ranging from 0 -250 µM) of oxaliplatin or satraplatin for 72 h, before MTS assay. The IC 50 value of each cell line is shown in Table 2. The IC 50 value ranged from 13 to 27 µM for satraplatin, and from 19 to 96 µM for oxaliplatin. All p values between these two treatment groups showed a significant difference in IC50 value except in HT29 cells where there was no significant difference as shown in table 2. The general trend of MTS assays for HCT116, HCT116 p53 -/-and HT29 cells are shown in Fig 1 .1 (A-C). In all three cell lines, satraplatin showed a higher percentage of cell inhibition based on MTS assay, compared to oxaliplatin. For example, there was a statistically significant difference between both drugs at doses from 10-50 µM in HCT116, p=0.001. To investigate the consequences of satraplatin treatment on cell cycle and apoptosis, we used three different CRC cell lines with different p53 status; HCT 116 (wild type), HCT 116 (p53 -/-) and HT29 (mutant).

Satraplatin treatment results in higher accumulation of platinum in CRC cells, compared to oxaliplatin

To investigate the cellular contents of platinum (Pt) in our model, we assessed different concentrations of drug (0-100 µM) based on FAAS. Following the exposure of satraplatin and oxaliplatin, the accumulation of satraplatin was higher in the cells tested compared to oxaliplatin (Fig 2). For instance, when the cells were treated with 100 µM of both drugs accumulation of satraplatin was 387.4 ng/ml vs. 29.13 ng/ml for oxaliplatin in HCT116 cells, 316 ng/ml for satraplatin vs. 18.96 ng/ml for oxaliplatin in HCT16 p53 -/-cells.

Based in this graph, we found that the platinum incorporation into cells at 10 µM, which was the evaluation dosage for the subsequent experiments, was approximately 4 fold higher for satraplatin compared to oxaliplatin.

Treatment of CRC cells with satraplatin and oxaliplatin alters cell cycle profile

To analyse the effect of satraplatin and oxaliplatin treatment on cell cycle, we treated CRC cells with 10 uM of each drug for 48 or 72 h. The cell cycle analysis of 3 CRC cell lines following treatment with oxaliplatin and satraplatin are shown in table 3.

Satraplatin noticeably increased the relative number of cells in G2-M phase (57.08% vs.

14.42% for untreated cells at 72h, **p<0.001) in HCT116 cells while a minor alteration was observed with oxaliplatin treatment. We also observed a similar alteration of cell cycle in p53 -/-HCT116 cells following satraplatin treatment (50.85 % vs. 28.52 % for untreated control at 72h, **p<0.001), while a similar pattern of cell perturbation was observed with oxaliplatin treatment at 72 h where the cells accumulated in S phase. On the other hand, in HT29 cells both drug treatments induced S phase cell cycle arrest, Table 3.

Satraplatin induces apoptosis in CRC cells

To assess the effect of satraplatin treatment on apoptosis the three CRC cell lines were treated with 10 µM satraplatin and oxaliplatin for 24, 48 and 72 h and apoptosis was assessed by flow cytometry. The results shown are the mean values ± standard error (SE) obtained from three independent experiments. In HCT116 cells no significant increase in apoptosis was detected following treatment with satraplatin for 24h, compared to untreated and oxaliplatin treated cells (data not shown). However following treatment for 48h and 72h, a substantial increase in sub-G1 population was detected as compared to untreated cells (48h: 2.8 fold changed vs.

14.48%, untreated control; 72 h: 10.9 fold changed with satraplatin vs. 7.7 fold changed with oxaliplatin to untreated control, p< 0.001 (Fig 3). A similar apoptotic profile was also observed in HT29 and HCT116 p53 -/-cells, although the difference between satraplatin and oxaliplatin was also profound in HT29 cells following 48h and 72h treatment. There were significant differences of apoptosis profile between these 3 cells lines tested as shown in fig 3 .      We confirmed these results by counting Annexin-V-PI (+) cells following satraplatin treatment. This method also allowed us to detect early apoptotic (annexin-V+/PI-) and late apoptotic/necrotic (annexin-V +/PI+) cells [START_REF] Matteucci | Identification of nuclei from apoptotic, necrotic, and viable lymphoid cells by using multiparameter flow cytometry[END_REF].

In HTC116 cells, 24 h of treatment with 10 µM satraplatin, resulted in 0.20% annexin-V+PI+ cells and 22.67 % annexin-V+/PI-cells vs. 0.14% annexin-V+PI+ cells and 7.55% annexin-V+/PI-cells with oxaliplatin treatment, while in untreated controls 0.23% annexin-V+PI+ and 9.32 % annexin-V+/PI-cells were observed. Similar results were obtained in HT29 mutant cells and in p53 -/-cells as showed in Fig 4, which demonstrates the efficacy of satraplatin on cell death induction as compared to oxaliplatin. These data also indicate that satraplatin induces cell death in cells mainly, if not exclusively, by apoptosis, with no or very little contribution by necrosis.

To study the biological effect of satraplatin on CRC cells cytotoxicity, cell cycle distribution and apoptosis, first we analysed the p53 protein expression since our models differ in p53 status. We observed a significant increase in p53 protein expression after 48 h of treatment of both drugs in HCT116 cells whereas no p53 protein accumulation was observed in HT29 cell line as shown in Fig 5A . We observed a down regulation of p53 protein expression with both treatments in HT29 cell line. No p53 expression has been shown in HCT116 p53 -/-cell line as expected. To clarify p53 expression and the action of its downstream genes dependent and independently based on these drugs treatment, first we analysed gene expression patterns at mRNA levels.

Satraplatin initiates p53 signalling pathway independently

To further investigate the molecular cascade of events following the exposure to both drugs, we selected appropriate genes involved in cell cycle regulation, DNA damage response and transcription factor control based on the p53 signalling pathway. Using this small array with a 15-gene simultaneous analysis after 48 h of treatment, we detected significant changes in mRNA modulation. We detected a minor alteration in p53 mRNA levels in HCT1116 (satraplatin: 1.6, p= 0.016 fold vs. oxaliplatin: 1.53 fold, p=0.007) but not in HT 29 (satraplatin: -1.05 fold vs. Oxaliplatin: -1.07 fold). We also detected the changes in expression levels of ERCC1, MSH2, BRCA1, GADD45, CLU and Bax genes (Table 4.1). BCL2 expression was much more down regulated in HCT116 cells with 2 fold lower than oxaliplatin treatment whereas 12 fold lower in p53 -/-cells. Table 4.1 shows the fold-changed expression level in the three cells tested. Furthermore, we noticed an up-regulation in Bax gene expression, further suggesting the apoptosis induction in these cells by p53-dependent and independent manner.

Effect on the levels of cell cycle related protein expression

To elucidate the role of cell cycle regulating proteins, we investigated the expression of cyclin B1 involved in G2/M cell cycle arrest. We found a marked increase in cyclin B1 in HCT116 and p53 null cells exposed to 48 h satraplatin. A very low expression was detected by oxaliplatin in HCT116 wild type and null p53 lines, whereas we could detect the same expression for HT29 by both drugs (Fig 5B). We also detected a marked increase in p21 expression at 48 h in HCT116 and the expression is much higher with satraplatin treatment than oxaliplatithe (Fig 5C). To confirm the G0/G1 arrest by oxaliplatin, we looked at the expression of c-ABL gene. The expression is much higher after oxaliplatin than after satraplatin treatment in HCT116 and null p53. This might suggest that the G0/G1 block was activated upon the treatment with oxaliplatin, whereas very low expression was detected after satraplatin treatment (Fig 5C ).

Effect on the levels of cell death pathway related protein expression

Bax relocation to the mitochondrial, accumulation of cytochrome c in the cytoplasm, caspase 8 activation and Bcl2 down-regulation are all events consisted with intrinsic pathway of apoptosis activation. To elucidate the mitochondrial associated apoptosis molecules, Bcl2 and Bax protein expression was noticed in our cell models. Bcl2 expression in both HCT116 and p53 null cells was much more reduced after 48 h with satraplatin than oxaliplatin treatment. No Bcl2 protein expression was detected in HT29 with the same result as gene expression. On the other hand, the expression of Bcl2 on oxaliplatin treatment was significantly reduced but a greater reduction was observed with satraplatin treatment (Fig 6A ,D). This further suggests that satraplatin induced apoptosis might be by p53-dependent as well as an -independent manner. We also detected Bax protein expression as well as an activation of truncated Bid in all the cells tested. Fas

Differential expression of miR-34a exhibits apoptosis signals in CRC cells

We conducted a differential miRNA expression analysis after satraplatin and oxaliplatin based treatments. The expression of miR-34a which target molecule is Bcl2 is higher after satraplatin than oxaliplatin treatment in HCT116 cells (2.65-vs. 1.89-fold). While a down-regulation of miR-34a levels was observed in HT29 cells for both drug treatments, in p53 null cells, we observed a 1.69-fold up-regulation with satraplatin, while a downregulation was observed with oxaliplatin treatment (Fig 6C). For both drugs, we also detected a down-regulation of hsa-miR-214 that targets the UTR of Bax gene. HCT116 and HT29 cells also exhibited a down-regulation of hsa-miR-214 but very low expression was observed in HCT116 p53 null cells. The differential expression of other miRNAs analysed was pronounced in table 4.2

Discussion

Colorectal cancer demonstrates elevated resistance to chemotherapy agents compared with other human malignancies [START_REF] Blijham | Chemotherapy of colorectal cancer[END_REF]. However, in a combination of 5-fluorouracil leucovorin-oxaliplatin patients have gradually achieved improvement in clinical outcomes along with increased median progression-free survival [START_REF] De Gramont | Leucovorin and fluorouracil with or without oxaliplatin as first-line treatment in advanced colorectal cancer[END_REF]. Despite this improvement most platinum compounds eventually suffer from de nova resistance or acquired resistance to the treatment over the time. In this study, we showed the effects of oxaliplatin versus an orally administered platinum drug, satraplatin in CRC cell lines which differ in p53 status on fundamental cellular pharmacological levels.

As documented before for prostate and other cancers [START_REF] Kelland | Ammine/amine platinum (IV) dicarboxylates: a novel class of platinum complexes exhibiting selective cytotoxicity to intrinsic cisplatin resistant human ovarian carcinoma cell lines[END_REF][START_REF] Wosikowski | Preclinical antitumor activity of the oral platinum analog satraplatin[END_REF], satraplatin exhibited comparable anti-proliferation effects on cell growth. Demonstrations of anti-proliferation effect on our clonogenic model further support the action of satraplatin in a p53dependent and -independent manner.

There was a significant discrepancy in the uptake of platinum into the cells across the three cell lines investigated. When we examined the contents of 10 μM of both drugs, the intracellular Pt levels were four fold higher with satraplatin. The cells displayed a greater ability to tolerate higher levels of Pt with satraplatin compared to oxaliplatin. This may be due to the greater lipophilicity of satraplatin enhancing its intracellular accumulation.

Flow cytometry analysis revealed that a slowdown in S phase transit was a prominent cell cycle effect of satraplatin in these cells. This was accompanied by a marked accumulation of cells in G2 phase in HCT116 and p53 null cells after 72 h. However, in HT29 cells, the majority of the cells were still in S phase with no evidence of build up of cells in G2 phase at this point. Similar cell cycle perturbations were observed with cisplatin [START_REF] Ormerod | Cisdiamminedichloroplatinum(II)-induced cell death through apoptosis in sensitive and resistant human ovarian carcinoma cell lines[END_REF] and other platinum agents [START_REF] Vaisman | Cell cycle changes associated with the formation of Pt-DNA adducts in human ovarian carcinoma cells with different cisplatin sensitivity[END_REF]31]. Taken together, these data indicate that the accumulation of cells in S phase is a general cell cycle effect of platinum drugs and depending on the nature of the cell type as well as based on dose dependent which showed to be associated with G2 cell cycle arrest [START_REF] Vaisman | Cell cycle changes associated with the formation of Pt-DNA adducts in human ovarian carcinoma cells with different cisplatin sensitivity[END_REF][31][START_REF] Neill | Apoptotic and non apoptotic cell death induced by cis and trans analogues of a novel ammine (cyclohexylamine) dihydroxodichloroplatinum (IV) complex[END_REF]. In the p53 wild type and p53 null cells, accumulation of cells in G2 coincided with the point at which significant apoptosis was occurring at 48 and 72 hours. It has been suggested that G2 arrest facilitates repair of DNA damage prior to mitosis and depending on the success of repair or extent of DNA damage, cells emerging from G2 either begin to cell cycle normally or engage in apoptosis [START_REF] Sorenson | Influence of cis-diamminedichloroplatinum (II) on DNA synthesis and cell cycle progression in excision repair proficient and deficient Chinese hamster ovary cells[END_REF][START_REF] Paola | To arrest or not to G2-M cell cycle arrest[END_REF]. Our data suggests that the latter may be occurring in the HT29 cells, despite the repair of Pt lesions induced by satraplatin.

Relatively more apoptotic cells were measured after satraplatin than oxaliplatin treatment suggesting that satraplatin is a stronger inducer of apoptosis. To confirm our apoptosis data measured by hypodiploid nuclei assessed by PI staining, we evaluated the percentage of annexin and PI which the values were quite similar. When we correlated our apoptosis data with the p53 gene status, we observed that even if HT29 cells that have reduced p53 protein expression, satraplatin still induced apoptosis. This suggests that the differences of p53 status in the cells play a role in the cell cycle arrest and apoptosis inductions and adds further weight to the evidence that this process may be p53-dependent as well as p53-independent.

The cell cycle determination and their validation at the protein level suggested that two different mechanisms are responsible for oxaliplatin-induced G1 and satraplatin-induced G2 transition control. Satraplatin induced an elevated protein expression of cyclin B1 after 48 h compared to oxaliplatin. It is now widely accepted that the G2-M transition is partly governed by the CDK1-cyclin B complex [START_REF] Yamori | Repression of cyclin B1 expression after treatment with Adriamycin, but not cisplatin in human lung cancer A549 cells[END_REF][START_REF] Voland | Repression of cell cycle-related proteins by oxaliplatin but not cisplatin in human colon cancer cells[END_REF]. Another fact is that p21 waf1/cip1 exerts a key role in controlling the G2 arrest by inhibiting the CDK1-cyclin B complex.

Protein analysis showed that p21waf1 /cip1 expression was up regulated under both drug treatments. Based on this data we assumed that the p21 waf1/cip1 pathway may be involved in early events of the satraplatin-induced G2 block and vice versa. [START_REF] Hata | Role of p21 waf1/cip1 in effects of oxaliplatin in colorectal cancer cells[END_REF] has also suggested that p21 waf1/cip1 might play a role in exerting the activity of oxaliplatin in a p53-dependent and -independent manner [START_REF] Hata | Role of p21 waf1/cip1 in effects of oxaliplatin in colorectal cancer cells[END_REF].

In the present study, we also found that HCT116 p53 null cells did not show an induction of p21waf1 /cip1 expression as measured in p53 wild type cells, suggesting that the lack of a functional p53 protein could lead to reduced p21 protein levels in response to oxaliplatin and satraplatin exposure. To clarify the role of p21 waf1/cip1 activation by mean of both drug treatments on elevated cytotoxicity, HCT116 p21 waf1/cip1 null cells were also tested. Satraplatin again exhibited a higher anti-proliferation effect on oxaliplatin compared to oxaliplatin. Indeed the IC 50 value of satraplatin was 6 μM vs. 37.5 μM for oxaliplatin (data not shown). This further suggests that the cells after satraplatin treatment endure apoptosis via a p21 waf1/cip1 -independent pathway.

To better understand the apoptosis cascade, we evaluated cell death associated protein expression. It well known that p53 is mutated in more than 50% in colorectal tumors, and has been shown to have a role in modulating tumour sensitivity to a wide number of chemotherapeutic agents [START_REF] Arango | Molecular mechanism of action and prediction of response to oxaliplatin in colorectal cancer[END_REF]. Here we show that satraplatin/oxaliplatin-induced apoptosis was associated with an up-regulation of p53 protein levels, detected at 48 h of treatment.

Using HCT116 p53 null cells we have shown that the targeted inactivation of p53 resulted in a 3-fold increase in the IC 50 in oxaliplatin-but not in satraplatin-treated cells.

Based on the p53 null model, we demonstrated that loss of p53 may acquire resistance towards oxaliplatin but not towards satraplatin treatment. It was already published that cell types having a mutant or inactive p53 and/or Bax gene(s) show increased resistance to oxaliplatin treatment [START_REF] Arango | Molecular mechanism of action and prediction of response to oxaliplatin in colorectal cancer[END_REF][START_REF] Manic | Mechanisms controlling sensitivity to platinum complexes: role of p53 and DNA mismatch repair[END_REF].

Furthermore, modulations of genes involved in the apoptosis response, such as Bax and Fas, are part of each platinum drug mode of action signature, indicating a common cell death mechanism [START_REF] Voland | Repression of cell cycle-related proteins by oxaliplatin but not cisplatin in human colon cancer cells[END_REF]. We have demonstrated a down regulation of Bcl2 and an up regulation of Bax and Bid after exposure of both drugs in a subset of cell lines. These results demonstrate an important functional intrinsic pathway in the apoptotic cascade of events initiated by both drugs and are in agreement with previous publications [START_REF] Gourdier | Drug specific resistance to oxaliplatin is associated with apoptosis defect in a cellular model of colon carcinoma[END_REF][START_REF] Gourdier | Drug specific resistance to oxaliplatin is associated with apoptosis defect in a cellular model of colon carcinoma[END_REF].

We also observed that satraplatin induced an apoptotic response through activation of the extrinsic pathway by promoting Fas expression which in turn leads to the formation of the death-inducing signalling complex (DISC) and the auto-catalytic activation of procaspase 8. This phenomenon was further enforced by the involvement of the extrinsic pathway by oxaliplatin-induced apoptosis in colon cancer cells [START_REF] Hayward | Enhanced oxaliplatin-induced apoptosis following antisense Bclxl down-regulation is p53 and Bax dependent: genetic evidence for specificity of the antisense effect[END_REF].

In addition to the basic molecular and biochemical analysis, we also examined the expression of certain miRNA on the response of cells to the platinum agent. MicroRNAs are small non-coding RNA molecules, which are able to control protein expression. It has been suggested that modification of miRNA gene expression could be another important factor in the responses to drug treatment [START_REF] Marchetti | Apoptosis induced by oxaliplatin in human colon cancer HCT15 cell line[END_REF]. As documented before, hsa-miR-34a was shown to induce apoptosis, cell cycle arrest, and senescence by p53 mediated activation [START_REF] Rossi | Modification of miR gene expression pattern in human colon cancer cells following exposure to 5-fluorouracil in vitro[END_REF]. Thus, we have detected high expression of miR-34a with satraplatin treatment than oxaliplatin in which miR-34a targeting Bcl2 protein expression. In fact we observed a lower expression of Bcl2 with satraplatin than oxaliplatin. In addition, we also detected a down-regulation of miR-214, which in turn targets Bax protein expression. This finding further suggests the role of miRNAs in chemotherapy responses based on p53-dependent and -independent routes. In summary, this study highlights the molecular mechanisms underlying the cytotoxic effects of satraplatin for the first time in colorectal cancer cells in an attempt to identify different means of predicting response to this oral platinum agent. Our data suggests that the effect of satraplatin on proliferating colorectal cancer cells induces a G2/M cell cycle arrest and a molecular cascade of events consistent with an intrinsic-extrinsic mechanism of apoptosis. Moreover, the cytotoxic effects of satraplatin were shown to be both p53dependent and -independent using an isogenic in vitro system. Further analysis is necessary to determine if this orally available platinum agent has an effect in combination with other chemotherapy agents compared with oxaliplatin-based combinations. This could be a better solution for malignant cells that acquired resistance or de nova resistance against the currently available chemotherapy regimens. Satraplatin in comparison to oxaliplatin resulted in more accumulation of Pt in each cell line tested in reference to p53 gene status. This demonstrates that the uptake of platinum is more efficient with satraplatin than oxaliplatin. Based on 10 µM treatment with both drugs, the accumulation of satraplatin was 35-40 ng/ml whereas with oxaliplatin only expected 3-10 ng/ml platinum for 10 6 cells. The accumulation of satraplatin in the cells was 4 fold higher than after oxaliplatin treatment. Error bars= S.E of two independent experiment. . A. Over expressed p53 protein by satraplatin and oxaliplatin treatment in HCT116 while reduced expression levels were observed in HT29 cells with both treatments. B.

Expression of p53 downstream genes, p21 waf/cip1, and Cyclin B1, c-ABL were examined following 48 h of treatment with both drugs at 10 µM concentration. The expression of p21 waf/cip1 is higher with satraplatin treatment while c-ABL expression was higher with oxaliplatin treatment. C. The expression of cyclin B1 is evidence at 48 which the expression is stronger with satraplatin treatment in HCT116 and p53 null cells while same levels of expression observed with oxaliplatin treatment in HT29 cells.

  Fig 1.2 (a-f) shows the cell morphological changes at 10 µM of both drugs in two of the cell lines tested as described in the figure legend.

  protein expression was detected by both drug treatments mainly in HCT116 cells suggesting that these cells might also undergo Fas-induced apoptosis (Fig 6A). To further illustrate the activation of caspase family, we choose pro-caspase 8 in our analysis. Procaspase 8 cleavage was observed with satraplatin treatment whereas only a very low signal with oxaliplatin treatment (Fig 6 B).

Figure 1 2 :

 12 Figure 1 Effect of satraplatin and oxaliplatin on cytotoxicity of representative colorectal

Figure 2 :

 2 Figure 2: Quantification of cellular platinum accumulation by Atomic Absorbance

Figure 3 :

 3 Figure3: Apoptosis analysis using PI as described early by us[START_REF] Matteucci | Identification of nuclei from apoptotic, necrotic, and viable lymphoid cells by using multiparameter flow cytometry[END_REF]

Figure 4

 4 Figure 4 Evaluation of apoptosis analysis in representative colorectal cell lines after the

Figure 5

 5 Figure 5 Effect of oxaliplatin and satraplatin on p53 protein and cell cycle related protein

Figure 6 :

 6 Figure 6: Effect of both drugs on cell death related protein and microRNA expression of

Table 1

 1 Primers list for mRNA RT-PCR

	Genes	Primer Set	Length (bp)

Table 2

 2 IC 50 value of Satraplatin and oxaliplatin in a panel of colorecal cancer cell lines

	IC50µM	IC 50 p values

Table 3

 3 Effect of satraplatin and oxaliplatin on cell cycle perturbation in representative colorectal cancer cells at 10 µM

			HCT116			HCT116 Tp53 -/-			HT29
		Untreated Oxaliplatin Satraplatin Untreated Oxaliplatin Satraplatin Untreated Oxaliplatin Satraplatin
						48 hours
	G0/G1	69.95	56.04	23,13**	67.47	65.32	26,56**	62.2	37.52*	17.64**

Table 4

 4 mRNA and miRNA of satraplatin vs. oxaliplatin at 48 hour post treatment

	4.1

Acknowledgment

This work was supported by grants from University Hospital Tor Vergata and Department of Internal Medicine, University of Rome, "Tor Vergata". We thank to Dr, Isabella Faraoni, University of Rome Tor Vergata, Dr. Soddu and Dr. Maurizio Franculli from Regina Cancer Institute, Rome, Italy, Dr. Rossana Supino from Instituto Nazionale Tumori, Milan, Italy and Prof. Bert Vogelstein from Ludwig Center at John Hopkins, USA for providing us the cell lines for our analysis. We also thank Agennix for providing us with satraplatin drug. Thank to Letizia Franzo" for her invaluable technical assistance in atomic absorbance spectrometry assay.

Conflict of interest statement

The authors stated that there are no conflicts of interest regarding the publication of this article.


Laura Robotham

Disa A Sauter

Anne-Catherine Bachoud-Lévi

Iris Trinkler

The impairment of emotion recognition in Huntington's disease extends to positive emotions Running page heading: recognition of positive emotions in HD

Keywords: Huntington's disease, Emotion, Vocalizations, Affect

. Although HD patients performed above chance for each emotion, they were found to be impaired in both positive and negative emotions, including pleasure, fear and anger. These findings complement previous work by demonstrating that impairments in emotion recognition in HD extend to positive and negative emotions, which may imply a general deficit.

Introduction

Huntington's disease (HD) is a rare, inherited, neurodegenerative disease characterized by motor impairments. It is associated with numerous cognitive and attentional deficits, as well as difficulty in processing and interpreting emotions. But although it is well established that patients with HD are impaired in the recognition of emotional signals, there is little agreement about the nature and extent of this impairment. Earlier studies found an impairment that disproportionately affected the recognition of disgust expressions in clinical (e.g. [START_REF] Sprengelmeyer | Loss of disgust: perception of faces and emotions in Huntington's disease[END_REF] and a circumscribed deficit for disgust in pre-clinical individuals with HD (e.g., [START_REF] Gray | Impaired recognition of disgust in Huntington's disease gene carriers[END_REF]. However, several studies have documented a more general impairment encompassing several negative emotions in clinical patients [START_REF] Calder | The relation between anger and different forms of disgust: Implications for emotion recognition impairments in Huntington's disease[END_REF] or virtually all negative emotions across both pre-clinical and clinical individuals (e.g., [START_REF] De Gelder | Huntington's disease impairs recognition of angry and instrumental body language[END_REF][START_REF] Johnson | Predict-HD Investigators of the Huntington Study Group. Beyond disgust: impaired recognition of negative emotions prior to diagnosis in Huntington's disease[END_REF][START_REF] Snowden | Emotion recognition in Huntington's disease and frontotemporal dementia[END_REF]. Some authors have argued that the selective impairment for disgust stimuli found in some studies may have been a consequence of item difficulty [START_REF] Milders | Differential deficits in expression recognition in gene-carriers and patients with Huntington's disease[END_REF]. Recently, [START_REF] Snowden | Emotion recognition in Huntington's disease and frontotemporal dementia[END_REF] employed ten different tasks assessing recognition of emotion from facial and vocal cues in patients with manifest HD. They found that patients were impaired in the recognition of several negative emotions across their extensive battery of tasks and concluded that HD may cause a general impairment in the identification of negative emotions.

A consistent finding across previous studies investigating emotion recognition in HD patients is that the recognition of positive emotions has been spared in the recognition of facial as well as vocal stimuli (e.g., [START_REF] Calder | The relation between anger and different forms of disgust: Implications for emotion recognition impairments in Huntington's disease[END_REF][START_REF] Snowden | Emotion recognition in Huntington's disease and frontotemporal dementia[END_REF] see [START_REF] Sprengelmeyer | Loss of disgust: perception of faces and emotions in Huntington's disease[END_REF]. However, one limitation is that they have all included only a single positive emotion category (joy/happiness). Recent work, however, has shown that a range of positive states can be reliably communicated using non-verbal signals such as posture, voice, and touch (see [START_REF] Sauter | More Than Happy: The Need for Disentangling Positive Emotions[END_REF]. In particular, non-verbal vocalizations, such as laughs, cheers, and sighs, reliably signal joyful emotions including amusement, achievement, and pleasure [START_REF] Sauter | Perceptual cues in non-verbal vocal expressions of emotion[END_REF][START_REF] Sauter | More than one kind of happiness: Can we recognize vocal expressions of different positive states?[END_REF], as well as negative emotions such as anger, disgust, fear and sadness. Nonverbal vocalizations thus provide a class of emotional signals in which the recognition of a range of both positive and negative emotions can be tested.

The aim of the current study was to examine emotion recognition in HD using several negative and positive emotions. In order to test the specificity of the impairment, we employed an emotion identification task using previously validated non-verbal affective vocalization stimuli of positive and negative emotions [START_REF] Sauter | Perceptual cues in non-verbal vocal expressions of emotion[END_REF][START_REF] Sauter | More than one kind of happiness: Can we recognize vocal expressions of different positive states?[END_REF]. This allowed us to address two hypotheses. One possibility is that the recognition of positive emotions is in fact spared in HD, as previous authors have found. Alternatively, previous studies may have failed to detect the impairment in the recognition of positive emotional stimuli because of the use of only a single class of positive emotional stimuli. This study examined the recognition of more than one type of positive emotional signal in HD.

Methods and Materials

Participants

The participants consisted of 14 genetically confirmed patients with HD (6 female, mean age = 51.29 (± 7.69) years; mean years of formal education = 12.29 (± 3.36)), and 15 control participants (7 female, mean age = 46.80 (± 11.18) years; mean years of formal education = 12.53 (± 2.64)). The patients and controls were matched for age (t 27 = -1.25, p > 0.2) and years of education (t 27 = .22, p > 0.8). Participants were native French speakers recruited through the biomarker program approved by the ethical committee of Henri Mondor Hospital, Paris and gave informed consent to taking part in the study. The HD patients had no previous neurological or psychiatric history other than HD. None of the participants reported hearing problems in everyday life and their basic auditory perception was assessed using a computer beep; all participants comfortably perceived the test sound at a volume of 3.5 of 10.

Background neurological and neuropsychological assessment of HD patients

The patients were at early stages of the disease, according to the Total Functional Capacity Scale [START_REF] Shoulson | Huntington disease: Functional capacities in patients treated with neuroleptic and antidepressant drugs[END_REF], presenting with apparent but mild movement and cognitive impairments. None of the patients showed signs of depression. Data from the neurological and neuropsychological assessments of the HD patients are summarized in Table 1.

Emotional vocalization stimuli

The emotional vocalization stimuli were obtained from a previously validated set of affective non-verbal vocalizations, such as laughs, sighs, and screams, lasting approximately one second per stimulus [START_REF] Sauter | Perceptual cues in non-verbal vocal expressions of emotion[END_REF][START_REF] Sauter | More than one kind of happiness: Can we recognize vocal expressions of different positive states?[END_REF].

The categories included in the current study were from three positive emotions (achievement, amusement, and pleasure), and four negative emotions (anger, disgust, fear, and sadness). Ten tokens were used per emotion category, resulting in a set of 70 stimuli, with equal numbers of male and female vocalizations.

Procedure

Participants were played the stimuli in a random order via headphones from a computer running a purpose-written E-prime script. After each stimulus, a list of the response alternatives was displayed on the screen (1: "accomplissement" (achievement), 2: "amusement" (amusement), 3: "colère" (anger), 4: "dégoût" (disgust), 5: "peur" (fear), 6: "plaisir" (pleasure), and 7: "tristesse" (sadness)), and participants were asked to judge the emotion expressed in the vocalization they heard.

Participants responded by typing in a number on the keyboard, and no time limit was enforced.

Statistical Analysis

In forced-choice tasks, performance for a particular category can be artificially inflated by the disproportionate use of that response. 'Hu' scores are unbiased hit rates that take into account each participant's use of the different response alternatives [START_REF] Wagner | On measuring performance in category judgment studies of nonverbal behavior[END_REF]. A score of zero denotes chance performance and a score of one perfect performance. Hu scores were calculated for patients' and controls' recognition rates for each emotion category, and tested against (Hu-score calculated-) chance level. Due to group sizes and heterogeneous population variances in the data, nonparametric analyses were employed for all analyses. Like [START_REF] Snowden | Emotion recognition in Huntington's disease and frontotemporal dementia[END_REF] and several previous authors, we employed Mann-Whitney U tests for pairwisecomparisons, and Friedman tests for within-group comparisons. Further, like [START_REF] Snowden | Emotion recognition in Huntington's disease and frontotemporal dementia[END_REF], correction procedures were not employed in order to avoid loss of power in these small samples.

Results

Both the HD patients and the controls were able to identify the emotions from the non-verbal vocalizations: They performed better than chance for the task overall (controls: z = -3.41, p < 0.001; HD patients: z = -3.30, p < 0.001). However, the controls performed better than the HD patients (average performance for controls: 0.52 (± 0.12), and for patients: 0.28 (± 0.16), U = 21, z = -3.67, p < 0.001). Confusion matrices are shown in Table 2. Errors were largely consistent across patients and controls, and included some cross-valence confusions. Common errors included sad sounds mistaken for pleasure, anger sounds classified as disgust, and achievement sounds perceived as signaling amusement.

INSERT TABLE 1 ABOUT HERE Discussion

The results of this study show that patients with HD have reduced sensitivity in the identification of emotional signals overall, for positive as well as negative emotions.

Importantly however, HD patients (as well as controls) were able to perform the task at better-than-chance levels, overall and for each category of non-verbal emotional vocalizations. Additionally, the relative difficulty and confusion errors made by the patients largely mirrored those of the controls (see Table 2). This pattern suggests that the patients' impairment was not due to a failure to understand or perform the task, but rather a consequence of a reduced sensitivity to emotional cues in both positive and negative affective vocal signals. Although the current study did not include a control task testing higher order auditory processing for non-emotional stimuli, the general ability of the patients to perform the task and the similarity of their performance to that of the control participants, suggests that the emotion recognition impairment in HD was not due to primary auditory deficits. A previous study has also found intact performance in HD patients on auditory tasks including phoneme processing [START_REF] Teichmann | The role of the striatum in phonological processing: Evidence from early stages of Huntington's disease[END_REF].

The results from the current study are consistent with previous work that has found impaired recognition of several negative emotions in HD [START_REF] De Gelder | Huntington's disease impairs recognition of angry and instrumental body language[END_REF][START_REF] Johnson | Predict-HD Investigators of the Huntington Study Group. Beyond disgust: impaired recognition of negative emotions prior to diagnosis in Huntington's disease[END_REF][START_REF] Milders | Differential deficits in expression recognition in gene-carriers and patients with Huntington's disease[END_REF][START_REF] Snowden | Emotion recognition in Huntington's disease and frontotemporal dementia[END_REF], but our findings go beyond earlier research in demonstrating an impairment that extends to positive affective signals, thus highlighting the importance of examining the identification of positive emotion in more depth.

While further work will be needed to confirm whether HD patients are also impaired in the recognition of positive emotional signals in other modalities, our results suggest that the emotion recognition deficit in HD is not specific to a single emotion or to negative emotions. This finding lends some support to the hypothesis that emotion processing may be impaired at a general level in HD. Given that HD patients are impaired in the recognition of emotional cues from facial, bodily, and vocal signals [START_REF] De Gelder | Huntington's disease impairs recognition of angry and instrumental body language[END_REF][START_REF] Snowden | Emotion recognition in Huntington's disease and frontotemporal dementia[END_REF], this impairment likely occurs at postperceptual processing levels.

Although little is known about HD patients' understanding of their own emotions, recent work has suggested that patients' general semantic representations are not impaired [START_REF] Hayes | Disgust and Huntington's disease[END_REF][START_REF] Snowden | Emotion recognition in Huntington's disease and frontotemporal dementia[END_REF], highlighting the need to examine other mechanisms that may play a causal role in the emotion recognition impairment in HD. It is worth noting that, although our results suggest an overall impairment in the recognition of both positive and negative emotions, the impairment for certain emotions of both valence types appear to be particularly severe. The recognition of anger and fear was especially impaired in the HD patients compared to control participants in our study, like in [START_REF] Calder | The relation between anger and different forms of disgust: Implications for emotion recognition impairments in Huntington's disease[END_REF], who attributed this pattern to insensitivity to social disapproval in HD. However, insensitivity to social disapproval is unlikely to explain the strong impairment for pleasure signals also found in the current study.

By contrast, given the motor symptomatology in HD, one candidate mechanism may be affective motor representations, which have been suggested to underlie emotion recognition across modalities [START_REF] Niedenthal | Embodying emotion[END_REF].

Another with some finding an impairment (e.g., [START_REF] Kan | Recognition of emotion from facial, prosodic and written verbal stimuli in Parkinson's disease[END_REF], and others not (e.g., [START_REF] Adolphs | Intact recognition of facial emotion in Parkinson's disease[END_REF].For auditory stimuli impaired recognition has been observed for a number of negative emotions (e.g., [START_REF] Lloyd | Comprehension of prosody in Parkinson's disease[END_REF][START_REF] Pell | On the receptive prosodic loss in Parkinson's disease[END_REF], as well as for positive prosody [START_REF] Pell | Processing emotional tone from speech in Parkinson's disease: a role for the basal ganglia[END_REF]. A recent meta-analysis of emotion recognition in PD concluded that the identification of negative emotions is disproportionately impaired in this group of patients [START_REF] Gray | A meta-analysis of performance on emotion recognition tasks in Parkinson's Disease[END_REF]. However, no study to date has used more than one category of positive emotion with PD patients. [START_REF] Shoulson | Huntington disease: Functional capacities in patients treated with neuroleptic and antidepressant drugs[END_REF], MADRS = Montgomery and Asberg Depression Rating Scale [START_REF] Montgomery | A new depression scale designed to be sensitive to change[END_REF]; FHVLT = French Hopkins Verbal Learning Test [START_REF] Rieu | French adaptation of the Hopkins Verbal Learning Test[END_REF]; MDRS = Mattis Dementia Rating Scale [START_REF] Mattis | Mental status examination for organic mental syndrome in elderly patients[END_REF]; normal range norms from [START_REF] Guelfi | L'évaluation clinique standardisée en psychiatrie[END_REF];

TMT B = Trail Making Test part B [START_REF] Tombaugh | Trail Making Test A and B: Normative data stratified by age and education[END_REF].

Table 2. Confusion matrices for controls and Huntington's patients in percentages.

Achieve = Achievement, Amuse = Amusement, Plea = Pleasure. Stimuli categories in rows and response categories in columns. All rows add to 100. Correct responses are marked in bold.

  Performance varied across the emotion types in the controls, (χ 2 r = 29.5, p < 0.001), and there was a trend for variability across emotion categories in the patient group (χ 2 r = 11.6, p = 0.07). Considering each emotion separately, both controls and HD patients performed above chance for all emotions (controls : z = -3.35 for achievement, z = -3.41 for amusement, z = -3.41 for pleasure, z = -3.41 for anger, z = -3.42 for disgust, z = -3.41 for fear, and z = -3.42 for sadness, all p < 0.001) and HD: (z = -2.67 for achievement; z = -3.30 for amusement; z = -3.23 for pleasure; z = -3.23 for anger; z = -3.11 for disgust; z = -3.23 for fear; and z = -3.23 for sadness; all p < 0.001, for achievement: p = 0.004). Performance per emotion and group is shown in Figure1.INSERT FIGURE 1 ABOUT HERERecognition performance in HD patients was lower than in controls for achievement (U = 56.0, z = -2.1, p = 0.032), anger (U = 34.0, z = -3.1, p = 0.002), disgust (U = 48.5, z = -2.5, p = 0.012), fear (U = 14.5, z = -4.0, p < 0.001), and pleasure (U = 43.5, z = -2.7, p = 0.006), with a trend also for sadness (U = 60.5, z = -2.0, p = 0.051), and amusement vocalizations (U = 66.5, z = -1.7, p = 0.093). Importantly, recognition performance in HD patients was lower for the average of positive (U = 45.5, z = -2.6, p=0.009) as well as the average of negative emotions (U = 21.5, z = -3.6, p<0.001).

  important factor in understanding the underlying mechanisms in causing this impairment is the neuropathology associated with HD. Comparisons of the emotion recognition problems in HD patients with those of other patient groups with brain pathology affecting the striatum, such as Parkinson's Disease (PD), could inform our understanding of the role of striatal regions in emotion processing. Studies on PD patients' emotion recognition in the visual modality have yielded inconclusive results,

Future

  comparisons of emotion recognition of a wide range of emotions in HD and PD patients may elucidate the contribution of the basal ganglia to emotion processing. The results from the current study are the first to show an impairment of recognition of positive emotional signals from non-verbal vocal signals in HD. The results are consistent with recent findings that have failed to find a specific impairment in the recognition of disgust in HD, and instead point to a global impairment of emotion recognition in HD, affecting both positive and negative emotional signals.

Figure 1 .

 1 Figure 1. Performance (Hu scores) in the recognition of non-verbal vocalizations of

Table 1 .

 1 Background neuropsychological data for the HD patients, with published normal ranges where available. Notes: UHDRS = Unified Huntington's Disease

	Rating Scale (UHDRS; Huntington Study Group, 1996); Motor and Behavioral sub-
	scales and TFC (Total Functional Capacity) scale

Table 1 .

 1 Cut-off was calculated as mean minus two standard deviations from the published control data (11.3 ± 0.8).

	Test	Sub-scale/domain	HD patients	Published normal
				cut-off
	UHDRS	TFC (max 13)	11.4 (1.2)	-
		Motor (max124)	26.3 (10.9)	-
		Behavioural (max 16)	1.9 (2.8)	-
	MADRS	Depression (max 60)	11.0 (6.7)	< 15
	FHVLT (C)	Delayed recognition (max 12)	8.1 (3.8)	>9.7 a
	MDRS	Total (max144)	128.9 (11.7)	>136
		Attention	35.6 (1.5)	>31
		Initiation	29.6 (7.4)	>28
	TMT B	Executive function	161.9 (58.7)	> 135

a

Acknowledgements

This work was supported by the GIS (Groupement d'Intérêt Scientifique) Institut Maladies Rares (A04159JS), the Dorothy M. and Maurice C. Shapiro Traveling Fellowship, and a Swiss National Funds post-doc grant to Iris Trinkler. The authors would like to thank Maryline Couette and Marie-Françoise Boissé for the general neuropsychological assessment, Katia Youssov for neurological assessment of the HD patients, and Severine Devignevielle for assistance with participant recruitment.