Jocelyn Evans

Gilles Ivaldi

Building a local powerbase? The Rassemblement National in the 2020 municipal elections in France

Keywords:

The 2020 municipal elections represent a further stage in the institutionalisation of the Rassemblement National at the level of local governance, and at the same time underline the limits to the party's organisational capacity. Within a context of significant political and socio-economic upheaval, the party's capacity to consolidate its 2014 victories, and build upon these through local mobilisation of political networks and exploitation of traditional opposition weaknesses, was limited to its existing strongholds in the North-East and South of the country. In particular, these successes were helped through the distancing of the local notables from national party labelling, and indicated the strength of pragmatic municipal governance in convincing voters to endorse candidates distancing themselves from the RN's extremist profile. Yet, only where a complex interplay of local socioeconomic factors, candidate profile and opposition weakness obtained could the party secure a limited number of victories.

Introduction

The 2020 municipals represented an important step forward in the institutionalization of the Rassemblement National (RN, formerly FN) in France. In 2017, Marine Le Pen had won 21.3 per cent of the presidential vote and progressed into the second-round run-off where she had received 33.9 per cent against the centrist and pro-EU candidate Emmanuel Macron. In May 2019, the RN's list led by Jordan Bardella topped the European elections with 23.3% of the vote and 23 seats, while the La République en Marche! (LREM) list led by Nathalie Loiseau came second with 22.4%, effectively making the RN the main opposition to Macron and the Philippe government.

Following these national successes, the 2020 municipal elections were seen as a real-life test of the local strength of the RN. Examining the party's performance in the 2020 municipal elections, we start from the premise that local elections in France are traditionally fought over both local and national issues. In 2020, the national context produced by the Covid-19 pandemic following months of political unrest during the Giles jaunes (Yellow Vests) movement and pension reform strikes was propitious to protest voting and higher abstention, potentially resulting in stronger support for the RN. During the campaign, Marine Le Pen sought to represent national issues over local ones, stirring up discontent with the Macron presidency and Philippe's government. Simultaneously, a crucial aspect of the RN campaign was to promote and defend their incumbent mayors' track record since 2014, which focused on local governance and issues.

The context of the 2020 elections

The 2020 municipal elections took place in the context of the outbreak of Covid-19 pandemic. The first round took place on 15 March, a few days before Edouard Philippe's government declared a state of health emergency and total lockdown of the country, while deciding to postpone the second round originally scheduled for 22 March. Amidst fears of infection, abstention rose to an historic 55.3%, an increase of about 20 points on the previous elections of 2014. The rapid unfolding of the COVID-19 epidemic was the main factor behind the dramatic decline in participation: according to IFOP, no less than 55% of French voters said they had abstained because they worried about the coronavirus, which compared with only a fifth in the previous survey conducted just one week before the election 1 .

The Covid-19 health crisis occurred in the wake of one of the longest running political crises in France after the emergence of the 'yellow vests' (gilets jaunes) nation-wide working and lower middle-class protest movement against fuel tax rises and high living costs in October 2018. Social unrest continued into most of 2019, eventually leading to the organization by President Macron of a 'great national debate' in mid-January 2019 -a three-month long exercise of direct democracy at local level -and the passing of anti-crisis measures and tax cuts to prop up purchasing power and appease protesters.

Political unrest and pressure from the street continued in opposition to the government's pension reform in May 2019, resulting in mass protests and one of the country's biggest strikes in decades. One of Macron's key campaign promises, the pension reform, was widely rejected by the public, with polls showing a majority in favour of the strikes and opposed to the government's plan to profoundly change the country's post-war pension system. This eventually led PM Philippe to scrap the most controversial part of the reform, making concessions in particular on the so-called 'pivot age' which could be implemented after 2027.

A 'mixed' party strategy: local versus national

Amidst political unrest, the RN's municipal campaign was officially launched at the La Rochelle party national council in June 2019, followed by the party convention in mid-January 2020 which set up the main objectives and targets of the party in the forthcoming municipals. The campaign reflected the attempt by the RN to achieve a balance between local and national issues, all the while highlighting the organizational weaknesses of the party and its difficulty in expanding its powerbase locally beyond its established strongholds. Meanwhile, the 2020 municipal campaign implemented the so-called 'de-demonization' strategy at the local level, whereby RN candidates and mayors would move further away from the party's emblems and far right reputation, as well as its national leadership.

National issues and organizational weaknesses

During her only campaign meeting on 6 March 2020 in Marseilles, Marine Le Pen strongly emphasized national issues such as immigration, linking them with crime and insecurity, and denouncing the "abject compromise with Islamism and communitarianism", while warning against the "hundreds of thousands, if not millions" of migrants who were preparing to land in Europe. "The link between insecurity and massive immigration is no longer in doubt for people of good faith -I am telling the truth to the French", she said, while demanding zero tolerance, and pledging that "families of delinquents should be evicted from social housing" 2 . The party's intention to set a national agenda for the municipal campaign was further revealed in Le Pen's call to "vote a real motion of censure against Macron" and her announcement in January 2020 that she would run for the 2022 presidential election, thus seeking already to establish herself as the main opponent to President Macron. "Emmanuel Macron and myself," Le Pen explained, "are the symbols of the globalist versus nationalist cleavage that has emerged in the 2017 presidential election, and which is replacing the divide between the right and the left" 3 .

The focus on security issues in the RN's local platform, together with immigration and the threat of so-called Muslim 'communitarianism' (communautarisme), was very similar to that in the party's municipal charter of 2014, while reflecting the set of issues that traditionally top the party's national agenda. The RN's municipal campaign convention held in January 2020 emphasized such issues, pledging that municipal management by RN mayors should reflect the main political orientations of the party. Local policies would for instance include "zero tolerance for anti-social behaviour and acts of delinquency, the fight against communitarianism, defence of France's secularism, the absolute refusal of migrant settlements, the effective reduction of taxes, and a prioritisation of local employment" 4 . As explained by Stéphane Ravier, RN Senator and candidate in Marseille: "I am going to talk about streets and roadworks, but trust me, I won't forget to talk about immigration!" 5 Amidst the Covid-19 health crisis, Le Pen sought to mobilize fears and to stir up anger with the government's crisis management, decisively turning her back on Macron's calls for national unity. Le Pen pursued an aggressive strategy of attacking the government for its "grave mistakes", while 2 https://www.lemonde.fr/politique/article/2020/03/07/a-marseille-marine-le-pen-met-l-immigration-au-c-ur-de-sa-campagne_6032144_823448.html accusing Macron and Philippe of "lying about absolutely everything" amidst controversies about the lack of tests and masks, endorsing conspiracy theories and adopting a strong anti-elite and antisystem tone 6 . The RN leader advocated closing all borders, linking the epidemic with globalization, while criticising the EU for being unresponsive and calling for France to regain its national sovereignty.

However, the focus by the RN on national issues did little to mask the organizational weaknesses of the party and lack of support for Le Pen nationally. Popularity data suggested that Marine Le Pen was slowly, albeit only partially, recovering from her failure in the 2017 presidential election. Her poor second-round debate against Macron had revealed the difficulty for Le Pen to bring credibility to her candidacy, resulting in the collapse of positive opinion in the polls (Evans and Ivaldi 2018, chap 8). According to TNS-SOFRES, Le Pen's popularity rose from a low of 14% of positive opinions in February 2018 -down from 29% just before the presidential runoff -to 20% on the eve of the March municipal elections. But this remained below her 27% of positive opinions just before the 2014 municipal ballot, suggesting that many of the 2020 RN local performances were independent of a 'Marine' effect at the national level 7 .

Whilst the yellow vests and pension reform movements had produced a propitious political context for the RN, there was little evidence that the party had benefited electorally from such widespread discontent with the Macron presidency despite the many attempts by Le Pen to try and take the lead over the protest movement by claiming to speak for the "France of the forgotten". The RN leader had also opposed the government's pension reform calling for a referendum and pledging that she would keep retirement age at 60, creating internal tensions between free-market advocates such as Nicolas Bay and Louis Aliot and economically left-leaning elites such as Sébastien Chenu and Philippe Olivier within the RN.

Additionally, the RN was hit by major party finance scandals. The party was put on trial and suspected of deliberately inflating campaign expenses in the 2017 elections to increase public reimbursement. The trial involved the Riwal communication agency, led by a friend close to Marine Le Pen, Frédéric Chatillon, as well as the Jeanne micro party led by Marine Le Pen, Axel Loustau, and other party apparatchiks such as former party treasurer Wallerand de Saint-Just and Jean-François Jalkh 8 . The party's financial difficulties persisted into the 2020 elections to the point of forcing the RN to call for private donations while under threat from a Russian court over the non-repayment of a 9.14 million euro loan contracted in Russia in 2014 9 . In February, Marine Le Pen announced the imminent launch of a new campaign to borrow money from the French to finance her party's election campaign 10 .

While the Riwal judgement of June 2020 brought unexpected relief for the RN's finances, with the party being fined a mere 18,750 euros, when the public prosecutor's office had asked for 500,000 euros and the state lawyers 11.6 million euros in damages, the RN was significantly weakened financially and organisationally in the 2020 elections, its accounts showing no less than 24.4 million euros of debt, to which one should add the 6.8 million that the European Parliament is still demanding for the jobs of MEPs' assistants that the FN had been accused of employing as party staff between 2009 and 2017 11 .

Between 2017 and 2020, RN membership declined from an estimated 50,000 down to about 27,000. This reflected growing political disgruntlement among RN's supporters after Marine Le Pen's defeat in the 2017 second-round runoff and the internal crisis which had followed, ultimately leading to Florian Philippot's departure from the party in September 2017. As will be discussed below, the decrease in number of RN lists across the country reflected the difficulty for the party of mobilizing at the local level and recruiting middle-level party elites to run the lists -even in departments where the RN list had polled over 30% of the vote in the 2019 European elections12 . This difficulty could still primarily be accounted for by the party's far right reputation13 .

Finally, an important aspect of party strategy in the RN municipal campaign of 2020 concerned the party's ability to build coalitions with the mainstream right locally. The yearly RN Barometer published by Kantar in early March 2020 showed some improvement in the party's reputation in public opinion, with signals of less negative views of the RN. A majority of the French -56%, up 9 points in one year -believed the RN could come to power in the future, while only 51% said that Le Pen's party represented a threat to democracy -down 7 points from 2017, reflecting an increasing institutionalisation and normalization of the RN in French politics14 .

Nevertheless, the RN largely failed to escape local political isolation. Overall, 'black-blue' coalitions emerged in fewer than 20 municipalities across the country, principally in the Mediterranean south where the ideological and organizational borders between the mainstream right and far right have historically been more 'porous' [START_REF] Blöss | Le vote Front National dans les Bouches-du-Rhône : "laboratoire" de l'alliance entre la droite et l'extrême-droite ?[END_REF]. This was exemplified in municipalities such as Frontignan (Hérault), where RN candidate Gérard Prato led a right-wing coalition endorsed by the RN, Nicolas Dupont-Aignan's Debout la France (DLF) and Thierry Mariani's Droite Populaire, a minor splinter group from LR formed in 2010. In the city of Lunel (Hérault), the RN list led by Julia Plane made room for a number of former members of Les Républicains (LR)15 , while in Perpignan, Louis Aliot received support from a former member of LREM as well as LR deputy mayor of Nîmes, Patricia Fourquet. Other examples of electoral alliances between the RN and the mainstream right in the Southern region included Agde, Sète (Hérault), Uchaud, Milhaud (Gard), Menton (Alpes-Maritimes) and Vidauban, Six-Fours-les-plages (Var) 16 .

Prominent figures of the right, such as Robert Ménard, mayor of Béziers, and Thierry Mariani, who had joined the RN during the 2019 European election campaign, played an important role in building links with the RN. As explained by Mariani, the objective was to "help build a large rightwing alliance around the RN, to provide a political alternative to disgruntled voters of the right (…) and to break the cordon sanitaire as quickly as possible" 17 . In the lead-up to the elections, the Droite Populaire endorsed 16 RN candidates in cities such as Annecy, Avignon, Douai, Marseille 11 https://www.lemonde.fr/politique/article/2020/06/17/jugement-riwal-un-repit-inespere-pour-les-finances-durassemblement-national_6043145_823448.html and Rennes. This followed the Convention of the Right which was held in September 2019, bringing together leaders of the Droite Populaire and former députés Jean-Paul Garraud and Nicolas Dhuicq, Marion Maréchal and right-wing intellectual Eric Zemmour.

Finally, in Lyon (Rhône), the RN endorsed former regional radical left LFI councillor Andrea Kotarac who ran on a ticket with Agnès Marion. In 2014, Kotarac had run as a La France Insoumise (LFI) municipal candidate in the 8 th arrondissement. He had left LFI in May 2019 over disagreement with the leadership's strategy. Overall, the number of cases of candidates defecting from the populist left to join the RN remained negligible, however.

'De-demonizing' the RN locally

The 2020 RN campaign pushed the so-called strategy of 'de-demonization' locally. A first crucial aspect was to promote and defend the FN mayors' track record since 2014, thereby emphasizing local governance and issues 18 . In early 2020, the RN under the direction of Gilles Pennelle, national director for the municipal campaign, published a booklet reviewing the performances of all RN mayors since 2014, which painted an extremely flattering picture of the "action of determined mayors who care about the well-being of their city and community" and their "common sense management" 19 . In particular, the booklet emphasized local tax cuts and security -i.e. video surveillance and reinforcing local municipal police -as well as economic support for local entrepreneurs, while providing a detailed record of all actions conducted since 2014. The booklet also claimed growing popular support for the incumbents across all RN municipalities. In March 2015, a survey conducted by IPSOS across a sample of voters living in FN municipalities had revealed that the vast majority (74%) were satisfied with the municipal management of the (then) FN mayors. The latter were rated highly positively on valence traits such as authority (82%) and dynamism (81%), which contrasted with the previous deficiency in local government credibility in FN cities in the late 1990s 20 .

The emphasis on pragmatic governance by RN mayors was part of a broader attempt to dedemonize the party locally. During the late 1990s, the experience of FN municipal management strongly suggested that the ability of the party to consolidate its local positions was dependent upon a number of adaptative strategies and policy alterations by its mayors. The model of local governance that had dominated in particular in Marignane and Orange had been notable for its shedding political flags and party affiliation in favour of a more pragmatic and apolitical style of management, which contrasted with the more ideological approach in Vitrolles and Toulon (Viard 1996[START_REF] Martin | Toulon la noire: le Front national au pouvoir[END_REF][START_REF] Evans | The Front national vis-à-vis power in France: factors of political isolation and performance assessment of the Extreme Right in municipal office[END_REF].

In 2008/2009, the FN conducted such non-partisan and locally based campaigns in the municipal elections in the northern city of Hénin-Beaumont. Priority was given to the party's well entrenched local representative, Steeve Briois, a well-known enfant du pays (born-and-bred) figure of the regional political scene and municipal councillor since the mid-1990s, who ran a low-profile campaign, distancing himself from the national party. By 2014, such strategic attempts by FN representatives at locally playing down the partisan card were closely associated with personality factors and candidate profile in the field. In cities won by the FN such as Fréjus, Hénin-Beaumont and Béziers, no reference was made to either the FN or the Rassemblement Bleu Marine (RBM).

Elsewhere, the FN had promoted the rising stars of 'de-demonization' such as Florian Philippot in Forbach, Gilbert Collard in Saint-Gilles, and Valérie Laupies in Tarascon.

In 2020, many RN candidates ran similar low-profile campaigns, essentially playing the local notable card. Across many municipalities, RN representatives would keep their distance from the most controversial elements of the party's ideology and seek to detach themselves from its extremist reputation, by removing the RN's name and symbols from their election material, thus emulating Robert Ménard's winning strategy in Béziers in 2014. The most notable example was that of Louis Aliot, MP and member of the RN's executive bureau, who successfully ran in Perpignan by dropping the RN's name and logo21 . Another interesting example of such a strategy is that of the city of Nice where RN activists distributed a free tabloid, Le Journal des Municipales, with no apparent political affiliation or mention of the RN, but making strong political claims clearly biased towards the local RN candidate, Philippe Vardon22 .

An analysis of the names of the RN's lists in the 2014 and 2020 municipal elections shows significant differences (see Table 1). In 2014, the vast majority of FN candidates (87%) had run under the 'Bleu Marine' label that had been formed two years earlier as an umbrella for local coalitions in the 2012 legislatives. References to the party's name were largely absent in 2020, with less than 5% mentioning the Rassemblement national. A majority (54%) of RN lists had names with no reference to the party while another third (36%) would only mention the term 'Rassemblement' (rally) which traditionally refers to a broader coalition cross-cutting the traditional left-right cleavage in French politics -e.g. the Gaullist Rassemblement du Peuple français after WWII.

RN lists in the 2020 municipals

The strategy that was laid out at the mid-January 2020 party convention clearly prioritized "quality over quantity", declaring the RN's intention to focus on a limited number of municipalities with the best chance of RN wins, while emphasizing the need to secure the few cities that the party had won in 2014. Based on the 2019 electoral returns, a total of 100 municipalities across a dozen departments were seen as the main targets for the RN, of which 20 could be potential wins for the party in March 2020 24 . These notably included the larger city of Perpignan (120,000 inhabitants) in the South, as well as a number of smaller urban and periurban municipalities which, for the vast majority, were situated in the two regional strongholds in the Midi and industrial North-East, such as Carpentras (Vaucluse), Tarascon (Bouches-du-Rhône), Brignolles (Var), Denain (Nord) and the former socialist city of Lens (Pas-de-Calais).

In 2014, the FN had targeted cities with significantly higher electoral support, which included some of its traditional strongholds in the Midi and North-East, along with a few towns in major urban peripheries, and a handful along the Eastern border. Leaving aside Paris, Lyon and Marseille, the average 2012 first round legislative score for the FN across all contested municipalities was 17.5 per cent, well above its 2012 national figure of 13.6 per cent, which confirmed that the party was targeting those areas with apparent electoral potential for 2014. Similarly in 2020, RN lists were chosen by the party strategically, as locations where evidence suggested it could make an impact: on average, in these chosen communes the FN had outperformed its overall national score by 6 points in the 2017 legislatives and by 5.9 points in the 2019 European elections. As will be discussed, these were municipalities located around well-established RN electoral strongholds as for instance exemplified in Pas-de-Calais where the party invested candidates across a number of municipalities surrounding its bastion of Hénin-Beaumont, such as Lens, Bruay-la-Buissière, Wingles, Carvin, Grenay, Mazingarbe, Beuvry and Noyelles-Godault, where the party had achieved some of its best performances in the 2019 European elections 25 .

In 2020, the party officially endorsed in total 478 lists26 . These included five lists overseas in La Réunion as well as 30 lists in small communes with less than 3,500 inhabitants. A total of 443 RN lists were found in metropolitan communes with more than 3,500 inhabitants, which compared with 582 in 2014. Overall, the RN managed to run 15,825 candidates compared with 20,096 six years earlier 27 .

24

These were identified from a larger pool of 193 communes with more than 3,500 inhabitants where the RN had won over 40% in the 2019 European elections. See https://www.lemonde.fr/politique/article/2019/09/13/municipales-lescommunes-cibles-du-rassemblement-national_5509890_823448.html

25

In 2019, RN voting strengths in the former mining area were well in evidence in communes such as Mazingarbe (57.6%), Hénin-Beaumont (55.9%), Grenay (54.4%), Calonne-Ricouart (53.9%), Marles-les-Mines (53.7%), Sains-en-Gohelle (52.2%), Courcelles-lès-Lens (52%), Divion (51.6%), Hersin-Coupigny (51.3%), Noyelles-Godault (50.7%), Guînes (50.3%), Wingles (50.2%).

Of those 15,825 RN candidates, 640 had been registered as 'other right' (DVD), 497 as 'extreme-right' (EXD) while another 513 simply had no party label.

Regional clustering

Typically, support for the RN clusters in the Northern and Eastern regions of Hauts-de-France, and Grand Est, as well as in the Mediterranean South-East in Provence-Alpes-Côte-d'Azur (PACA) and Occitanie. In the 2019 European elections, the RN list received 33.5% of the vote in HDF, 30.5% in PACA, 28.2% in Grand-Est and 25.7% in Occitanie, which compared for instance with 17.3% in Bretagne and 18.8% in Pays-de-la-Loire 28 .

In March 2020, PACA had the largest number of RN lists (N=90), followed by Hauts-de-France (N=80). Together the northern (HDF and Grand Est) and southern (PACA and Occitanie) regions totalled 253 of the 443 lists, representing 57%, a percentage higher than six years earlier when those four regions alone accounted for 51% of all FN lists, further confirming that the party was concentrating on its established areas of electoral strength (see Table 2). Compared with 2014, RN presence was stable or decreased in some of the party's mission priorities such as Bretagne, Paysde-la-Loire and Centre-Val de Loire. As was the case six years earlier, the RN campaign largely left aside small villages with fewer than 1,000 inhabitants where the party had no incentive to invest time or financial resources. nationally. This was replicated in the 2019 European elections with the RN list receiving just over 7% of the vote in the capital city, 10.2% in Lyon and 9.4% in Bordeaux for instance.

Reflecting the drift in RN support away from larger urban centres, and despite the party running across all sectors of the 'big PLM three' -i.e. Paris (17 sectors), Lyon (9 sectors) and Marseille (8 sectors) where it needed to be seen -in 2020, RN lists and candidates were found in 301 municipalities with more than 10,000 inhabitants, which compared with 417 in 2014, that is a drop of about 28% (see Table 3). In some cases, the RN's lack of implantation in large urban centres was reflected in the party strategy to run campaigns seeking to unite 'small' and mid-sized communes in opposition to the 'big' city centre. The main objective would be to win seats in smaller communes that form part of intercommunalities (intercommunalités) to influence local policy making and the provision of public services 29 . Under the provisions of the RCT and NOTRe laws, intercommunalities have fiscal power and they increasingly represent an important locus of local policy making and public administration, in particular in metropoles and urban communities built around larger urban municipalities. There were a few other cases of such a strategy by the RN. In the department of Var, the party targeted smaller cities around its stronghold of Fréjus such as Puget-sur-Argens and Roquebrunesur-Argens for the purpose of increasing its presence within the community of agglomeration. A similar strategy was discernible in the Gard where the RN targeted cities around Beaucaire with the objective of winning the communauté de communes de Petite Camargue, and near the city of Perpignan (Pyrénées Orientales) where the party ran candidates across the neighbouring municipalities of Le Soler, Canet-en-Roussillon, Argelès-sur-Mer, Claira, Le Barcarès and St-Laurent-de-la-Salanque. In the Nice Côte d'Azur metropolis where the RN fielded candidates in 15 neighbouring communes and villages, such as Cagnes-sur-Mer, Saint-Laurent-du-Var and La Trinité, running on a common platform against the local metropolitan tax created by the Mayor of Nice, Christian Estrosi, and the political hegemony of the big city, calling for a "fair distribution of financial aid" between municipalities and a "rotating presidency of the metropolitan authority" 30 . Finally, consistent with the geography of RN lists, the largest pools of candidates were found in the southern areas -PACA and Occitanie totalling over 5,000 candidates, compared with about 4,000 in the northern regions of HDF and Grand Est. In 2020, the RN largely failed however to draw from its existing pool of local elites. In 2014, the FN had run 20,096 candidates. Of those, only 2,229 ran again in the 2020 municipal elections, which accounted for just under 15% of all RN candidates. Of 29 Intercommunalité refers to an intermediate institutional layer in France, in the form of a group of communes cooperating for the provision of a number of public services such as water supply, public transport, economic planning and urban development.

https://france3-regions.francetvinfo.fr/provence-alpes-cote-d-azur/alpes-maritimes/nice/municipales-rassemblementnational-presente-ses-tetes-liste-metropole-nice-cote-azur-1751689.html the 1,497 FN representatives who had been elected six years earlier, only 364 ran again on RN lists in the 2020 elections. Since the 2014 municipal success, a significant proportion, estimated at about 30% of elected FN municipal councillors, have resigned31 , underlining the instability and turnover in the party's local powerbases. Of the 2020 municipal candidates, a total 419 had also run in the regional elections of December 2015 (out of a total 1,912 FN candidates) and 495 had been on a RN binôme in the 2015 departmental elections a few months earlier (out of 3,818 FN candidates).

Results

In the first round of the 2020 municipal elections, the RN lists received 385,042 votes across the 443 metropolitan communes where they were running. Six years earlier, the FN had totalled 1,047,900 votes across 582 communes, which represented a substantial improvement on its 2008 electoral nadir with just 1% of the national vote in the 122 municipalities in which the FN had fielded candidates. At the national level, such a dramatic drop in electoral support for the RN in 2020 reflected both the smaller number of communes with a RN list and the exceptionally high level of abstention in the municipal election. On average, in the communes where it was present in 2020, the RN won 14.4% of the valid vote -ranging from less than 1% up to nearly three quarters (74.2%) of the vote in its 'promised land' of Hénin-Beaumont.

In 2014, the FN had improved its performance in regions where support for the party was lower, while losing support in some of its established strongholds, most notably in the South. On average, the difference in score between the first round of the 2012 legislatives and the first round of the municipals was just -0.1, but, as Table 4 shows, territorially, the party had lost about -3.5 points on average in the Mediterranean region. Comparable data for 2020 suggest that the RN lost about 5 points on average since the 2014 and 2017 elections. Interestingly, in 2020 the RN performed relatively worse in its 2017 legislative strongholds of the north and south, with losses of -7.7 and -6.3 points respectively, as opposed to -2.5 points in the other regions. The party showed relatively greater resilience however in its established strongholds of 2014 with losses of -4.7 and -3.7 points in the North-East and Mediterranean respectively, against -5.3 in the rest of the country. As will be discussed further below, this primarily illustrates the process of electoral consolidation by the RN in its municipal 'bastions' of 2014.

Territorial divide and unemployment

The FN vote has been linked at aggregate and individual level to unemployment levels, particularly in regard to blue-collar workers and those in vulnerable socioeconomic strata voting for the party through reasons of economic threat from migration, and rejection of parties of governments' respective failure in economic policy. In a period of heightened saliency of economic issues, following Yellow Jackets protests over fuel tax, pension reforms and other welfare cuts from Macron's government, there were strong incentives for the party to target communes where such issues at both national and local level, thereby including social housing and other municipal social services, are widespread, and where typical RN working and lower-middle class voters tend to cluster.

Plotting unemployment and median income, using 2017 census data (Figure 1) 32 , we can see a clear pattern of the majority of RN lists in areas with lower median incomes and higher rates of unemployment, nearly all above the country's average of about 10%. Distinguishing between lists which stood in the first round only, and those which reached the ballotage (second-round runoff), those lists in higher income areas which were required for purposes of visibility, rather than winnability, in other words the prosperous sectors of the main urban conurbations, including the Paris, Lyon and Marseille (PLM) metropolises, are notable in not making it past the first round. These differences are illustrated further in Table 5 which reports the average unemployment and median income levels in four exclusive types of commune in the 2020 election: a) with no RN list; b) with a losing RN list in the first round; c) with a losing RN list in the second round; d) which the RN won. The division between communes having RN lists and not is clear on the unemployment variablesome 7 percentage points on average. There is some evidence that higher unemployment is characteristic of more successful RN lists, although the differences are not great. On median income, however the differences are negligible. The median income is skewed right for the RN first-round lists because of the inclusion of Paris and its rich suburbs -Versailles, Saint-German-en-Laye and Levallois-Perret, for example. However, even without these, the income differentials are negligible, in terms of progression to the second round, and victory. Overall, then, whilst we can use unemployment to identify likely RN targets, we cannot explain the extent of the lists' success, and certainly not their victory, on socio-economic context alone.

Abstention and the RN vote

Previous studies of the FN vote have noted a significant obstacle to party support from high abstention rates. Despite paradoxically strong partisan loyalty across electoral periods, evident in the 1990s [START_REF] Shields | La politique du pire: The Front National and the 1997 legislative elections[END_REF] early 2000s [START_REF] Mayer | Ces Français qui votent Le Pen[END_REF], and, after the 2007 switch to Sarkozy [START_REF] Mayer | Comment Nicolas Sarkozy a rétréci l'électorat Le Pen[END_REF] still evident at the commune level between the 2017 presidential first round and the 2019 European elections -Figure 2 panel A -second round performances have suffered from fall-off in turnout, and legislative elections, increasingly seen by voters as mere 'confirmatory' elections of the presidentials [START_REF] Dupoirier | Four rounds in a row: the impact of presidential election outcomes on legislative elections in France[END_REF], have particularly seen this phenomenon in the wake of the RN presidential candidates' elimination. In the 2014 municipals, FN sympathisers were the second-most likely group of party supporters to abstain [START_REF] Muxel | La vague de l'abstention[END_REF] Table 2).

However, in the 2020 municipals, there is only very partial evidence of an abstention penalty for the RN. As Figure 2, panels B and C show, neither round registered any substantial decline on average in RN vote share by the abstention. The first-round correlation is weak, but significant (r = -0.17, p < .001), indicating that higher levels of abstention may have penalised the RN to some extent. The correlation found at aggregate level is partially confirmed by polls conducted on election day, which suggest that RN supporters may have been less prone to turn out in the first round33 . However, neither second-round abstention (panel C) nor the change in abstention across rounds (panel D) reaches significance. Only at the extremes of abstention and change in turnout can we see a marked effect -the majority of cases show little pattern.

Second-round and Front Républicain

The RN found itself in 119 of the postponed second-round run-offs in June 2020, but with little hope of victory in the vast majority of these. Having secured eight town halls in the first round -all of them 2014 incumbents -the RN's principal chances of success lay in towns where a first-round victory had been missed by the narrowest of margins, for example Bédarrides (Vaucluse, 47.9%), or where no withdrawal or merger of lists by third-and lower-placed candidates would allow the formation of the so-called front républicain -a tactical bloc to defeat the RN. In the most recent sub-national elections, the 2015 regionals, the front républicain had denied the FN control of regional councils in its two heartlands of Provence-Alpes-Côte d'Azur and Hauts-de-France.

Similarly, in the 2017 presidentials, opposition to Marine Le Pen from nearly all losing candidates resulted in a de facto 'barrage' securing Emmanuel Macron's victory.

However, since the early 2010s, the mainstream right Union pour un Mouvement Populaire/Les Républicains had shown increasing ambivalence towards this strategy, often promoting a 'neither nor' ('ni…ni…') position, or denouncing the FN without expressly calling for their voters to move tactically to a leftist candidate. Such weakening of the front républicain was discernible in 2020. In towns such as Moissac (Tarn-et-Garonne), the withdrawal of the Divers Droite list was not accompanied by its endorsement of the remaining left list. In the small commune of Mazan (Vaucluse), the lack of withdrawal by either moderate left or right list would lead to the victory of the RN list led by Louis Bonnet with well under 40% of the second-round vote. On the left, the disastrous experience of a four-way run-off in Mantes-la-Ville (Yvelines) in 2014 did not lead to a clear endorsement for the Divers Centre candidate, supported by LREM and conservative right lists, against RN incumbent Cyril Nauth by the third-and fourth-placed left lists.

Yet, as discussed above, the spectre of the breaching of the cordon sanitaire -conservative Right allying with the RN -remained a highly localised phenomenon with limited effect. In Marles-les-Mines (Pas-de-Calais), one of the RN's 2017 legislative victories, the merger of the RN and centrist lists to face the incumbent left list could not secure a victory despite the presence of a second Communist list. Only in two other communes, both in Vaucluse, did Divers Droite lists merge with the RN -on a total of 644 merged lists in towns over 3500 inhabitants. In 2020, the RN's highest-profile victory came in the Southern city of Perpignan (Pyrénées-Orientales), where Louis Aliot, vice-president of the party and one of the cities two députés, defeated the front républicain formed by the LREM and Green lists stepping down in favour of the 34 https://www.lemonde.fr/les-decodeurs/article/2020/06/10/municipales-2020-seulement-13-6-des-listes-lrm-qui-ontfusionne-l-ont-fait-avec-des-listes-de-gauche_6042387_4355770.html 35 The absence of agreement did not always result in a far right victory. For example, Ligue du Sud incumbent Marie-France Bompard in Bollène (Vaucluse) failed to defeat the leftist Anthony Zilio despite the withdrawal sans accord of the PCF candidate. And conversely, in Bruay-La-Buissière (Pas de Calais), the withdrawal of the PS incumbent Olivier Switaj to faire barrage against the RN candidate, Ludovic Pajot, was insufficient.

LR incumbent, Jean-Marc Pujol. In many ways, this victory epitomised the conditions for not just RN, but any party's success -a high-profile, locally implanted challenger; a long-term, ailing incumbent; a fragmented and politically divided opposition; and a challenging socio-economic environment (Perpignan has 25% unemployment, and one-third of the population living below the poverty line36 ). Symbolically, Aliot's personal separation from Marine Le Pen and discarding of any explicit link to the RN in his campaign, together with support from Robert Ménard, similarly sans etiquette in nearby Béziers, helped consolidate the personal vote against Pujol's party apparatchik positioning in an ostensible sinecure as maire adjoint since 1989.

FN wins in 2020

In 2014, the FN had won 12 cities with more than 1,000 inhabitants, including Béziers and the small city of Camaret-sur-Aigues where local candidates had been endorsed by the FN from outside the party. Additionally, the party had won a small town with less than 200 inhabitants in the department of Oise (Le Hamel), as well as around 1,550 councillors across the country. The 2014 performance had eclipsed the 2008 municipal setback where the party had won a mere 59 seats, while improving on the previous apogee of 1995, where a highly concentrated set of lists provided over 1,300 seats. Additionally, 459 FN representatives had won seats in the municipal corporations (intercommunalités) of larger conurbations, extending their influence further. The biggest takes were the 7th sector of Marseille with more than 150,000 inhabitants, as well as the cities of Béziers and Fréjus37 .

In 2020, the RN managed to retain eight of its previous 2014 cities, losing Cogolin, Le Luc, Mantes-la-Ville and the 7 th sector of Marseille, while winning another six -Bédarrides, Bruay-la-Buissière, Mazan, Moissac, Morières-lès-Avignon and, most importantly, the larger city of Perpignan (Table 6). Overall, the party secured a total of just over 1,000 municipal seats, down by a third from its 2014 pool of local councillors. Similarly, with only 330 seats, the RN largely failed to extend its influence across intercommunalities. A similar axis obtains between Béziers, the self-styled laboratoire frontiste of Robert Ménard, and Perpignan. In the South-West of the country, Perpignan has always been an island of support for the radical right, as Pierre Sergent's fiefdom in the 1980s. The high-profile candidacy of Louis Aliot and distancing from the national party, in a similar socio-economic environment, echoed Ménard's campaign in 2014, despite differences between the two mayors on the relative importance of national profile and local specificity.40 However, the map underlines the relative failure of the RN's tache d'huile strategy in the Pyrénées-Orientales, with no additional RN win in the area of the Narbonnaise along the Mediterranean coast. Finally, in the Var, RN hegemonic position over the Fréjus-Le Luc-Cogolin triangle did not produce any additional city for the party in 2020, which suggests that the magnitude of the tache d'huile phenomenon should not be exaggerated and may be context dependent.

Map 1. RN vote change (2014-2020, first round) around key FN town halls

Conclusion

Following a series of electoral successes in the 2017 presidential and legislative elections, and the 2019 European elections, the 2020 municipals were seen as a confirmation of the RN's ability to consolidate its party system position, while responding to the imperative to establish a more solid power-base throughout the country in order to prepare for future electoral expansion. For the party, electoral consolidation at the municipal level was seen as crucial for further development of its network of local cadres and grassroots, and ability to prove itself as a party of local government in the few cities it had won in 2014, which would eventually allow Le Pen's party to achieve greater credibility.

Looking at the balance of national and local issues, our analysis suggests that the campaign and issues behind the RN vote were primarily local. Looking at the 443 metropolitan municipalities where the RN ran in 2020, we find little evidence of any relationship between RN performances and abstention levels across the country. Our findings point to the importance of local factors relating to the party's consolidation and entrenchment in some -but not all -of its well-established strongholds. First, we see in the heartlands of RN support in the Nord-Pas-de-Calais and Provence-Alpes-Côte d'Azur a process of diffusion -the so-called 'tache d'huile' (oil stain) -from the party's bastions to neighbouring communes. Second, we see an ostensible personal vote in cities with incumbent RN mayors, where support for the RN was substantially higher in 2020. Candidate effects are important factors of local party politics and elections in France [START_REF] Brouard | L'effet candidat lors des élections parlementaires: l'exemple des élections législatives 2012 en France[END_REF] and they are associated with candidate notability [START_REF] Brack | Conclusion to special issue: "To the district… and beyond![END_REF]). In the case of the RN, such an effect may be partly accounted for by those mayors' strategy of moving away from the party's emblems, names and far right reputation, a strategy pursued by many in 2014, and suggesting a move to 'de-demonize' the RN in local politics.

We would conclude that, while demonstrating the RN's learning curve of local governance, the 2020 municipals represented another demonstration of the limits of RN success. Our analysis illustrates a possible paradox of RN consolidation at the local level. On the one hand, unlike the short-lived experience of FN cities during the 1990s, current RN mayors have built strong local constituencies and secured substantial pools of loyal voters in their municipalities, demonstrating governing credibility and the ability to consolidate locally. On the other hand, despite these local success stories, the RN still remains extremely weak organizationally and lacks the local resources and grassroots to expand its local base of power beyond its few bastions. Most notably, beside Perpignan, Fréjus and, to a lesser extent, Béziers, the RN still has difficulties establishing itself in larger urban municipalities, which would be essential to providing the political resources and legitimacy needed by Marine Le Pen to break the 'glass ceiling' of national power in the 2022 presidential election.

Figure 1 .

 1 Figure 1. Scatterplot of median income and unemployment in French communes (identifiers for first-and second-round RN lists)

Figure 2 .

 2 Figure 2. RN vote share and abstention (by commune)

Table 1 . Names of RN's lists in the 2014 and 2020 municipal elections

 1 Additionally, in 2020, the RN sought to prioritize younger candidates. As explained by Philippe Olivier, RN MEP, "we will promote young shoots, you will see, real little Bardella kids" 23 . Jordan Bardella, leader of the RN's youth movement Génération Nation (former FNJ) since June 2018, was indeed seen as the incarnation of the attempt to attract younger voters to the party and revitalize its membership base in preparation for the upcoming local, regional and, come 2022, presidential elections. Younger candidates were found across a number of RN's targeted municipalities, such as Arnaud de Rigné, 23 years old (Carvin), Thomas Morelle, 22 (Wingles), Marc-Alexandre de Fleurian, 30 (Calais) in the Northern region, Dorian Muñoz, 28, La Seyne-sur-Mer (Var), Kévin Pfeffer, 29, in Stiring-Wendel (Alsace), and the youngest député in the Assemblée Nationale, Ludovic Pajot, 26, in Bruay-La-Buissière (Pas de Calais).

	2014			2020
	Name	N	%	Name	N	%
	Bleu Marine	505 86.8 Rassemblement	159 35.9
	Front national 29	5.0 Rassemblement national 19	4.3
	Fait front	24	4.1 Bleu Marine	13	2.9
	D'abord	14	2.4 D'abord	8	1.8
	Ville française 6	1.0 Ville française	3	0.7
	Other	4	0.7 Other	241 54.4
		582 100.0		443 100.0

Table 2 . RN lists in 2014 and 2020 by Region

 2

	Region	FN lists 2014	%	RN lists 2020	%
	Auvergne-Rhône-Alpes	63	10.8	38	8.6
	Bourgogne-Franche-Comté	17	2.9	15	3.4
	Bretagne	9	1.5	13	2.9
	Centre-Val de Loire	20	3.4	13	2.9
	Corse	1	0.2	0	0.0
	Grand Est	47	8.1	33	7.4
	Hauts-de-France	77	13.2	80	18.1
	Île-de-France	88	15.1	39	8.8
	Normandie	34	5.8	24	5.4
	Nouvelle-Aquitaine	39	6.7	37	8.4
	Occitanie	67	11.5	50	11.3
	Pays de la Loire	14	2.4	11	2.5
	Provence-Alpes-Côte d'Azur	106	18.2	90	20.3
	Total	582	100.0	443	100.0
	3.2. The metropolitan divide
	Since the early 2000s, electoral support for the FN has decreased in large metropolitan centres. In
	2017, the FN vote peaked in periurban areas characterised by 'multicentricity' i.e. adjacent to larger
	urban centres and multiple nodes of economic activity and employment, showing a significant drop

in support in France's largest metropolitan areas such as Lyon (8.9%), Toulouse (9.4%), Bordeaux (7.4%) and Nantes (7.2%). In Paris, Marine Le Pen won just 5% of the vote, compared with 21.3%

28

In the first round of the 2017 presidential election, Marine Le Pen won her highest scores in PACA and HDF with 28.2 and 31% of the vote respectively, and topped the polls in Grand-Est (27.8%) and Occitanie (23%).

Table 3 . RN lists in 2014 and 2020 by size of municipality Size FN lists 2014 % RN lists 2020 % Diff

 3

	Less than 3500	42	7.2	26	5.9 -16
	3500-10000	123	21.1	116	26.2 -7
	10000-30000	224	38.5	151	34.1 -73
	30000-50000	94	16.2	62	14.0 -32
	50000+	99	17.0	88	19.9 -11
	TOTAL	582		443

Table 4 . RN legislative and municipal performances in 2012-2014 and 2017-2020; change in RN municipal performance, 2014-2020

 4

		2012-2014			2017-2020			2014-2020
	Region of	Mean Standard	N	Mean Standard	N	Mean Standard	N
	France	deviation			deviation			deviation
	North-East -0.03 6.92	123	-7.68	8.57	113	-4.74	8.78	69
	South	-3.52 7.33	160	-6.34 11.11	132	-3.68 10.30	94
	Other	1.70. 4.30	299	-2.45	6.00	164	-5.28	3.99	133
	All	-0.10 6.25	582	-5.15	8.91	409	-4.65	7.67	296
	Note: Metropolitan France. 2017-2020 figures exclude electoral sectors in Paris, Lyon and Marseille which do not map onto 2017
	legislative constituencies.

Table 5 . Communal unemployment and income levels by RN list success

 5

	List type	Unemployment (%) Median income	n
			(euros p.a.)
	No RN list	10.8	19013	35,144
	Losing RN list in 1 st round	16.9	20530	317
	Losing RN list in 2 nd round	18.0	19250	112
	Victorious RN list	18.6	18686	14

  34 In Carpentras (Vaucluse), pressure from the RN candidate to form a union of right-wing lists mobilised the Parti Chrétien-Démocrate and Centre National des Indépendants et Paysans, but could not bring over the Republican list, allowing the re-election of the Socialist incumbent. Similarly, the attempt by the head of the Union des Démocrates et Indépendants list, Patrice Novelli, in Menton (Nice) to merge with the RN list resulted in his suspension from the party and from his role as délégué départemental, allowing the re-election of the LR incumbent. In probably one of the highest profile victories of 2014 -Marseille's 7 th sector -the withdrawal of the leftist Printemps Marseillais list controversially allowed victory for the LR challenger over RN candidate Stéphane Ravier.35

Table 6 . FN / RN wins in 2014 and 2020 38

 6 The party strategy of consolidating its 2014 wins, and building locally on these victories to build local networks has been described as a tache d'huile (an oil stain) strategy, spreading outwards into peripheral areas from the 2014 stronghold. 39 However, the extent to which this accounts for RN successes in the election is variable. Map 1 shows four areas of RN resilience -2014 mayoral victories in bold, 2020 victories in normal type -with choropleth shading by change in vote between 2014 and 2020. Any commune with a decrease in its vote is hatched-shaded. Grey communes indicate increased vote share, darkening with increase.Moving clockwise in Map 1, the Vaucluse-Gard cluster presents the clearest evidence of the 'tache d'huile'. Three 2014 victories in Beaucaire, Le Pontet and Camaret show consolidation in increased first-round vote share in 2020, accompanied by increases in neighbouring communes, and three additional town halls in Bédarrides, Mazan and Morières. Nonetheless, as many communes in the interstices saw a diminishing of RN support. Similarly, around Hénin-Beaumont, a line of communes saw increased first-round RN voting, with a single victory in Bruay-la-Buissière. Again, however, as many adjacent communes saw drops in vote share for the RN. In Bruay-la-Buissière, Ludovic Pajot's prominence, as the benjamin of the Assemblée Nationale, and close ties to Steeve Briois in Hénin-Beaumont, allowed the mobilisation -including a rise in turnout between the two rounds -of sufficient support to displace the Socialist incumbent.

	FN wins in 2014 (N=12)
	Municipality	Population % FN round 1 % FN	Configuration Lost in
					round 2	2020
	Beaucaire	15,894	32.8	39.8	4-Way
	Béziers	71,432	44.9	47.0	3-Way FN-
						Left-Right
	Camaret-sur-		4,556	31.2	36.6	3-Way FN-
	Aigues					Left-Right
	Cogolin	11,119	39.0	53.1	Duel FN-Right Left the
							party
	Fréjus	52,344	40.3	45.6	3-Way FN-
						Right-Right
	Hayange	15,730	30.4	34.7	4-Way
	Hénin-Beaumont	26,868	50.3	--	First round
	Le Luc		9,532	36.9	42.0	4-Way	Lost
	Le Pontet	16,899	34.7	42.6	3-Way FN-
						Right-Right
	Mantes-la-Ville	19,839	21.7	30.3	4-Way	Lost
	Marseille Sect. 7	151,327	32.9	35.3	3-Way FN-	Lost
						Left-Right
	Villers-Cotterêts	10,411	32.0	41.5	3-Way FN-
						Right-Other
	RN wins in 2020 (N=14)
	Municipality		Population	% FN round 1	% FN round	Configuration
						2
	Beaucaire		15,882		59.51	--
	Bédarrides		5,086		47.8	49.14	3-Way RN-Other-
							Centre
	Béziers		76,493		68.74	--
	Camaret-sur-Aigues		4,556		70.20	--
	Bruay-la-Buissière		22,230		38.57	51.99	Duel RN-Other
	Fréjus		53,168		50.61	--
	Hayange		15,776		63.14	--
	Hénin-Beaumont		25,901		74.21	--
	Le Pontet		17,556		57.21	--
	Mazan		5,944		28.06	37.99	3-Way RN-Right-
							Left
	Moissac		12,652		47.03	62.45	Duel RN-Left
	Morières-lès-Avignon	8,295		45.94	55.42	Duel RN-Left
	Perpignan		121,875		35.66	53.09	Duel RN-Right
	Villers-Cotterêts		10,694		53.47	--

https://www.franceinter.fr/politique/municipales-dans-la-majorite-des-departements-marine-le-pen-a-du-mal-atrouver-des-candidats

13https://www.lemonde.fr/politique/article/2020/02/12/au-rassemblement-national-le-logo-complique-les-municipales_6029355_823448.

html 14 https://fr.kantar.com/opinion-publique/politique/2020/barometre-d-image-du-rassemblement-

national-2020/ 15 https://www.lemonde.fr/politique/article/2020/01/15/municipales-a-lunel-six-elus-de-droite-rejoignent-la-liste-durassemblement-

national_6025938_823448.html 16Let us note that in a few other municipalities, the RN endorsed non-affiliated incumbents. This was the case for instance in cities such as Dourges (Pas-de-Calais), Aimargues (Gard) and Thor (Vaucluse).

https://www.valeursactuelles.com/politique/municipales-thierry-mariani-veut-rassembler-autour-du-rn-113591

https://www.lemonde.fr/politique/article/2019/10/26/a-perpignan-louis-aliot-joue-la-carte-robert-menard_6017009_823448.html

22

https://rassemblementnational.fr/retrouvez-la-liste-des-candidats-du-rassemblement-national-ou-soutenus-par-lerassemblement-national/

https://oeilsurlefront.liberation.fr/les-intox/2017/10/30/elus-municipaux-au-fn-les-premiers-partis-de-france_1606877

https://www.ipsos.com/sites/default/files/ct/news/documents/2020-03/comprendre_le_vote_premier_tour_municipales_2020.pdf

https://www.franceinter.fr/politique/municipales-voici-pourquoi-perpignan-risque-de-basculer-au-rn

We also include here Camaret-sur-Aigues where the Ligue's candidate, Philippe de Beauregard, had received official support from Marion Maréchal-Le Pen in the runoff -a consolidation of a far right's Provençal axis since 2012.

Additionally, in Vaucluse, Jacques Bompard's Ligue du Sud gained an additional two municipalities in Orange and Bollène.

https://www.lemonde.fr/politique/article/2020/01/27/municipales-de-la-dediabolisation-a-lanotabilisation-la-strategie-aliot-pour-conquerir-perpignan_6027389_823448.html; https://www.republicain-lorrain.fr/edition-thionville-hayange/2020/05/02/val-de-fensch-lhypothese-d-un-vice-president-rn-fait-

debat 40 https://www.lefigaro.fr/politique/bisbilles-entre-les-allies-du-sud-louis-aliot-et-robert-menard-20200727

du-vote-15032020/ https://www.lemonde.fr/politique/article/-pen-se-declarecandidate-a-la-presidentielle-de-2022_6026133_823448.html 4 https://www.lemonde.fr/politique/article/2020/01/12/municipales-marine-le-pen-lance-la-campagne-du-rn-et-l-opasur-l-electorat-de-droite_6025623_823448.html 5 https://www.lemonde.fr/politique/article/2019/09


A Alahmadi
email: analahmadi@kau.edu.sa

A Alkathiry
email: aakathiry@uqu.edu.sa

A Altassan
email: aaltassan@kau.edu.sa

A Bonnecaze
email: alexis.bonnecaze@univ-amu.fr

H Shoaib
email: hashoaib@kau.edu.sa

P Sol
email: sole@enst.fr

Keywords: 2010 Mathematics Subject Classification. Primary: 49B05; Secondary: 16A10 unital rings, self-orthogonal codes, Type IV codes

1. Introduction. The build-up method is a powerful technique to construct selfdual codes over fields and rings [START_REF] Han | Construction of self-dual codes over F 2 + uF 2[END_REF][START_REF] Kim | Euclidean and hermitian self-dual MDS codes over large finite fields[END_REF]. Starting from a self-dual code of length n, it builds a self-dual code of length n + 2 by a simple recursion. This method was used successfully over a non-unital non commutative ring in [START_REF] Alahmadi | The buildup construction of quasi self-dual codes over a non-unital ring[END_REF]. In this article, we adapt this method to generate quasi self-dual (QSD) codes over the ring I, a non-unital, commutative ring of order 4 [START_REF] Alahmadi | Quasi Type IV codes over a non-unital ring[END_REF]. QSD codes are defined in that reference as self-orthogonal codes of length n, and size 2 n . This special concept plays an analogous role in I to that played by self-dual codes over fields and unital rings. Of special interest is the subclass of Type IV codes, namely QSD codes with all weights even, which exists for several rings of order four [START_REF] Dougherty | Type IV self-dual codes over rings[END_REF][START_REF] Alahmadi | Type IV codes over a non-unital ring[END_REF]. A relaxation of that concept that is special to the ring I is that of quasi Type IV codes (QTIV), that is to say QSD codes with an even torsion code [START_REF] Alahmadi | Quasi Type IV codes over a non-unital ring[END_REF]. While the build up constructions do not seem to preserve the class of Type IV codes, they are shown here to preserve the wider class of QTIV codes. As a result, we classify QSD codes of length at most 6. We also classify Type IV codes and QTIV codes of length n ≤ 6. The material is organized as follows. The next section collects some necessary facts and notations about codes, rings, modules, and duality. Section 3 derives the main construction. Section 4 contains numerical data. Section 5 concludes the article, and points out some open problems.

2. Background material.

2.1. Binary codes. Denote by wt(x) the Hamming weight of x ∈ F n 2 . The dual of a binary linear code C is denoted by C ⊥ and defined as

C ⊥ = {y ∈ F n 2 | ∀x ∈ C, (x, y) = 0}
, where (x, y) = n i=1 x i y i , denotes the standard inner product. A code C is selforthogonal if it is included in its dual: C ⊆ C ⊥ . A code is even if all its codewords have even weight. All self-orthogonal binary codes are even, but not all even codes are self-orthogonal. Two binary codes are equivalent if there is a permutation of coordinates that maps one to the other. 2.2. Rings. Following [START_REF] Fine | Classification of finite rings of order p 2[END_REF] we define a ring on two generators a, b by its relations From this table, we infer that this ring is commutative, and without an identity element for the multiplication. It is local with maximal ideal J = {0, b}, and residue field F 2 = {0, 1}, the finite field of order 2. Thus we have a b-adic decomposition as follows. Every element i ∈ I can be written

I = a, b | 2a = 2b = 0, a 2 = b, ab = 0, .
i = as + bt,
where s, t ∈ F 2 and where we have defined a natural action of F 2 on I by the rule r0 = 0r = 0 and r1 = 1r = r for all r ∈ I. Thus a = 1a, c = 1c and c = a1+b1. Note that for all r ∈ I, this action is "distributive" in the sense that r(s ⊕ t) = rs + rt, where ⊕ denote the addition in F 2 . On occasion we will use the inner product notation (x, r)

for x ∈ F n 2 , r ∈ I n to denote (x, r) = n i=1 x i r i = xi=1 r i .
Denote by α : I → I/J F 2 the map of reduction modulo J. Thus α(0) = α(b) = 0, and α(a) = α(c) = 1. This map is extended in the natural way in a map from I n to F n 2 .

Modules.

A linear I-code C of length n is an I-submodule of I n . It can be described as the I-span of the rows of a generator matrix. With that code we associate two binary codes of length n :

1. the residue code defined by res(C) = {α(y) | y ∈ C}, 2. the torsion code defined by tor(C) = {x ∈ F n 2 | bx ∈ C}. It is easy to check that res(C) ⊆ tor(C) [START_REF] Alahmadi | Quasi Type IV codes over a non-unital ring[END_REF]. It is traditional to denote by k 1 the dimension of the residue code and k 1 + k 2 that of the torsion code.

A simple application of the first isomorphism theorem [START_REF] Alahmadi | Quasi Type IV codes over a non-unital ring[END_REF], shows that

|C| = |res(C)||tor(C)| = 2 2k1+k2 .
An additive code of length n over F 4 is an additive subgroup of F n 4 . It is an F 2 vector space with 4 k elements for some k ≤ n (here 2k is an integer, but k may be half-integral). Using a generator matrix G, such a code can be cast as the F 2 -span of its rows. To every linear I-code C is attached an additive F 4 -code φ(C) by the alphabet substitution

0 → 0, a → ω, b → 1, c → ω 2 ,
where

F 4 = F 2 [ω], extended naturally to F n 4 .
It can be checked that for all x ∈ I n , we have T r(φ(x)) = α(x), and thus res(C) = T r(φ(C)), where T r() denotes the usual trace from F 4 down to F 2 . Similarly, we see that tor(C) is the so-called subfield subcode of φ(C) that is

F n 2 ∩ φ(C).
We use the Magma notation ( Cf. the Handbook section of [16])

[< 0, 1 >, • • • , < i, A i >, • • • , < n, A n >]
for the weight distribution of a quaternary code, where A i is the number of codewords of weight i. Two I-codes are permutation equivalent if there is a permutation of coordinates that maps one to the other.

C ⊥ = {y ∈ I n | ∀x ∈ C, (x, y) = 0}.
Thus the dual of a module is a module. A code is self-dual if it is equal to its dual.

Remark 1. The repetition code of length 2 is defined by R 2 := {00, aa, bb, cc}. Its dual is R ⊥ 2 = {00, aa, bb, cc, 0b, b0, ac, ca}, a supercode of R 2 of size 8. In length one, we have J ⊥ = I.

A code C is self-orthogonal if ∀x, y ∈ C, (x, y) = 0. Clearly, C is self-orthogonal iff C ⊆ C ⊥ . A code of length n is quasi self-dual if it is self-orthogonal and of size 2 n .
Following a terminology from [START_REF] Dougherty | Type IV self-dual codes over rings[END_REF], a quasi self-dual code over I with all weights even is called a Type IV code.

Remark 2. The repetition code of length 2 is quasi self-dual over I and is of Type IV. This shows, by taking direct sums of codes, that Type IV codes over I exits for all even lengths. We see that J is a quasi self-dual code over I. This shows, again by taking direct sums, that QSD codes exist for all integer lengths. Following a terminology introduced in [START_REF] Alahmadi | Quasi Type IV codes over a non-unital ring[END_REF], we shall call a QSD code with an even torsion code quasi Type IV (QTIV). Every Type IV code is quasi Type IV but not conversely as the next example shows.

Construction.

In this section we discuss two kinds of construction methods for quasi self-dual codes over I. The following theorem is the first one, constructing quasi self-dual codes of length increased by two, with one more generator.

Theorem 1. Let C 0 denote a quasi self-dual code of length n and over I, with generating set r 1 , . . . , r k . Let x be a fixed vector in F n 2 satisfying (x, x) = 1. (Thus any x of odd Hamming weight works). Write

y i = (x, r i ) for 1 ≤ i ≤ k. The I-span of the following k + 1 vectors is a quasi self-dual code C of length n + 2.
(a, 0, ax), (y 1 , y 1 , r 1 ), . . . , (y k , y k , r k ).

Proof. First, we check that C is self-orthogonal.

• the first vector is orthogonal to itself by definition of x, since a 2 +a 2 (x, x) = 0.

THE BUILD-UP CONSTRUCTION OVER

A COMMUTATIVE NON-UNITAL RING 5

• the last k vectors are orthogonal to each other and to themselves by selforthogonality of C 0 , since y i y j + y i y j + (r i , r j ) = 0. • the first vector is orthogonal to the last k vectors by definition of the y i 's since ay i + (ax, r i ) = ay i + a(x, r i ) = ay i + ay i = 0.

Hence C is self-orthogonal. We claim that |C| = 4|C 0 | = 2 n+2 . Indeed define C 0 as the generator span of the last k generators. Write S y = (y, 0, yx), for all y ∈ I.

Then it can be seen that the construction in the theorem is equivalent to

C = ∪y∈I (S y + C 0 ),
where ∪ denotes disjoint union. Thus C is quasi self-dual of length n + 2.

The next result shows that this construction preserves the quasi Type IV property.

Corollary 1. If C 0 is QTIV then C obtained from the previous theorem is also QTIV.

Proof. Since C 0 is quasi Type IV, then tor(C 0 ) is an even code. If tor(C 0 ) is even so is tor( C 0 ), since we are only adding two equal coordinates. Because S b = b(1, 0, x) we see that tor(C) = tor( C 0 ) ∪((1, 0, x) + tor( C 0 )). Since (x, x) = 1, the vector (1, 0, x) has even weight. Thus tor(C) is even, and C is quasi Type IV.

The second kind of construction also constructs QSD codes of length two more, but with two more generators. Theorem 2. Let C 0 be a quasi self-dual code of length n over I and G 0 = (r i ) be a k × n generator matrix for C 0 , where r i is the i-th generator of G 0 , 1 ≤ i ≤ k. Let x be a fixed vector in F n 2 . For 1 ≤ i ≤ k, let y i = ((x, r i ), (x, r i )) be a vector of length 2. Then the following generators (b, 0, bx), (0, b, bx), (y 1 , r 1 ), . . . , (y k , r k ). generate a quasi self-dual code C over I of length n + 2

Proof. Firstly, we show that C is self-orthogonal.

• The first generator is orthogonal to itself since b 2 + b 2 (x, x) = 0. Similarly the second generator is orthogonal to itself. • The first generator is orthogonal to the second generator as b 2 (x, x) = 0.

• The first (or, the second) generator is orthogonal to any one of the last k generators since b(x, r i ) + (bx, r i ) = 0. • The last k generators are orthogonal to each other and to themselves by selforthogonality of C 0 since (y i , y j ) + (r i , r j ) = (x, r i )(x, r j ) + (x, r i )(x, r j ) = 0 Hence, the set of k generators generates a self-orthogonal code C. We claim that |C| = 4|C 0 | = 2 n+2 . Indeed define C 0 as the row span of the last k generators. Write S y = (y, 0, yx), T y = (0, y, yx) for all y ∈ I. Then it can be seen that the construction in the theorem is equivalent to C = ∪y,z∈J (S y + T z + C 0 ). Therefore, C is quasi self-dual code of length n + 2 Like for the first construction, we have a result on quasi Type IV codes.

  Thus, I has characteristic two, and consists of four elements I = {0, a, b, c}, with c = a + b. The addition table is immediate from these definitions + 0 a b c 0 0 a b c a a 0 c b b b c 0 a c c b a 0 The multiplication table is as follows. × 0 a b c 0 0 0 0 0 a 0 b 0 b b 0 0 0 0 c 0 b 0 b THE BUILD-UP CONSTRUCTION OVER A COMMUTATIVE NON-UNITAL RING 3

4 A 2 . 4 .

 424 . ALAHMADI, A. ALKATHIRY, A. ALTASSAN, A. BONNECAZE, H. SHOAIB, P. SOL É Duality. Define an inner product on I n as (x, y) = n i=1 x i y i . The dual C ⊥ of C is the module defined by

Example 1 .

 1 The code with the three generators  a b a b 0 b b 0 b 0 0 b   is QSD but notType IV as the sum of first and second row has odd weight. But its torsion code is an even code. Thus, it is quasi Type IV.

A. ALAHMADI, A. ALKATHIRY, A. ALTASSAN, A. BONNECAZE, H. SHOAIB, P. SOL É

A. ALAHMADI, A. ALKATHIRY, A. ALTASSAN, A. BONNECAZE, H. SHOAIB, P. SOL É

Corollary 2. If C 0 is QTIV then C obtained from the previous theorem is also QTIV if (x, x) = 1.

Proof. Since C 0 is quasi Type IV, tor(C 0 ) is even. If tor(C 0 ) is even so is tor( C 0 ). Because both S b = b(1, 0, x) and T b = b(0, 1, x) are multiples of a binary vector by b, we see that

Since, by hypothesis, (x, x) = 1, the vectors (1, 0, x) and (0, 1, x) have even weight. Thus, tor(C) is even, and C is QTIV.

Remark 3. The number of codes generated from a given C 0 depends on the number of choices for x. Thus Theorem 1 generates 2 n-1 codes, and Theorem 2 generates 2 n codes.

4. Numerical results. In this section, we continue the classification of inequivalent codes given in [START_REF] Alahmadi | Quasi Type IV codes over a non-unital ring[END_REF] for n < 4, by means of the methods described in this paper up to n = 6. It is an open problem to know if the build-up methode can produce enough inequivalent codes in higher lengths.

We take for granted that QSD codes containing odd weight vectors cannot be Type IV.

Recall from [START_REF] Alahmadi | Quasi Type IV codes over a non-unital ring[END_REF] the following results.

• For n = 1 there is just one code generated by the matrix (b).

• For n = 2 there are two Type IV codes with weight distribution [< 0, 1 >, < 2, 3 >]. • For n = 3 there are four QSD codes with weight distributions In view of the super-exponential number of codes generated by this method, more computing power might be needed to extend the numerical results to higher lengths .


Ioana Ferecatu

Rahamata Ali-Boina

Keywords: Retinoblastoma protein, Rb cleavage, caspase, apoptosis, transfection, GFP

Several caspase-cleaved forms of the retinoblastoma protein have been described. Here, we compared the effect of full-length Rb versus the truncated p76 Rb and p100 Rb proteins on cell death regulation in five human cell lines. Interestingly, we observed that p76 Rb triggers cell death in all tested cell lines and that p100 Rb protects two cell lines against etoposide or TNF--induced cell death, whereas full-length Rb has no apoptotic effect. These results show that truncated forms of Rb can have specific activities in the regulation of cell death. They also suggest that caspase-cleavage of Rb should not be simply assimilated to a degradation process. Finally, we show that cell death induced by p76 Rb is Bax-dependent and is diminished by Bcl-2 overexpression or by caspase inhibition and that p100 Rb could inhibit cell death by decreasing both p53 stability and caspase activity.

The p76 Rb and p100 Rb truncated forms of the Rb protein exert antagonistic roles on cell death regulation in human cell lines Nathalie Le Floch a,1 , Vincent Rincheval a,1,* , Ioana Ferecatu a , Rahamata Ali-Boina a , Flore Renaud a , Bernard Mignotte a and Jean-Luc Vayssière a a Université de Versailles / St Quentin-en-Yvelines, Laboratoire de génétique et biologie cellulaire / Ecole Pratique des Hautes Etudes, Laboratoire de génétique moléculaire et physiologique, 45 avenue des Etats-Unis, 78035 Versailles cedex, France.

Introduction

The retinoblastoma protein (Rb) was the first identified tumour suppressor protein. Its loss of function is linked to the development of many human cancers. It has been shown that overexpression of Rb can induce growth arrest in a major G1 checkpoint blocking S-phase entry. The Rb protein governs cell cycle progression by controlling the expression of E2Fdependent genes in two ways: by directly binding and blocking the activation domain of E2F proteins or by active repression through chromatin remodelling.

Evidences for the importance of Rb in cellular differentiation and cell death come from studies of Rb knock-out mice, where the disruption of the Rb gene causes death by day 14 of gestation associated with defects in the development of the haematopoietic system, as well as in the central and peripheral nervous systems [START_REF] Lee | Mice deficient for Rb are nonviable and show defects in neurogenesis and haematopoiesis[END_REF][START_REF] Jacks | Effects of an Rb mutation in the mouse[END_REF][START_REF] Clarke | Requirement for a functional Rb-1 gene in murine development[END_REF]. Interestingly, abolition of Rb function results in apoptosis involving both p53-dependent and -independent pathways [START_REF] Morgenbesser | p53-dependent apoptosis produced by Rb-deficiency in the developing mouse lens[END_REF][START_REF] Macleod | Loss of Rb activates both p53-dependent and independent cell death pathways in the developing mouse nervous system[END_REF].

The transcription factor E2F-1 was proposed to be one of the possible links between Rb and p53. Indeed, free E2F-1 can transcriptionally activate p19 ARF , which stabilizes p53 by inhibiting the function of its regulator MDM2 [START_REF] Pomerantz | The Ink4a tumor suppressor gene product, p19Arf, interacts with MDM2 and neutralizes MDM2's inhibition of p53[END_REF]. Overexpression of Rb, which leads to a sequestration of E2F-1, can therefore inhibit apoptosis induced by p53.

A pro-apoptotic function of Rb has also been observed but is less well documented.

Most of these studies show a direct role of Rb in the induction of apoptosis either by transcriptional activation or repression [START_REF] Bowen | Radiation-induced apoptosis mediated by retinoblastoma protein[END_REF][START_REF] Zhao | RB activation and repression of C-MYC transcription precede apoptosis of human prostate epithelial cells[END_REF][START_REF] Bowen | Retinoblastoma protein-mediated apoptosis after gamma-irradiation[END_REF]. One model describes that Rb interacts with p53 and MDM2 in a trimeric complex that stabilizes both p53 and MDM2, enhancing p53mediated transrepression which can trigger apoptosis [START_REF] Hsieh | RB regulates the stability and the apoptotic function of p53 via MDM2[END_REF]. More recently, two distinct Rb-E2F-1 complexes have been described, one of which activates transcription from proapoptotic genes to directly promote cell death [START_REF] Ianari | Proapoptotic function of the retinoblastoma tumor suppressor protein[END_REF]. These data suggest that the pro-apoptotic activities of Rb might be related to the complexes that it could form with its partners. Indeed, our studies in drosophila have shown that the capacity of the drosophila homolog of Rb to be pro-apoptotic depends on the proliferation status of the cells in which it is expressed [START_REF] Milet | The Drosophila retinoblastoma protein induces apoptosis in proliferating but not in post-mitotic cells[END_REF].

During apoptosis, Rb was shown to be cleaved by caspases, either at a C-terminal caspase-3-like consensus site generating a p100 Rb form, lacking the last 42 amino-acids [START_REF] Janicke | Specific cleavage of the retinoblastoma protein by an ICE-like protease in apoptosis[END_REF][START_REF] Chen | Apoptosis is associated with cleavage of a 5 kDa fragment from RB which mimics dephosphorylation and modulates E2F binding[END_REF][START_REF] Boutillier | Caspase-dependent cleavage of the retinoblastoma protein is an early step in neuronal apoptosis[END_REF], or at an internal cleavage site producing two fragments of 68 and 48 kDa [START_REF] An | Cleavage of retinoblastoma protein during apoptosis: an interleukin 1 beta-converting enzyme-like protease as candidate[END_REF].

Transgenic mice containing a germline mutation (RB-MI) at the caspase cleavage site generating p100 Rb were created. These mice exhibit resistance to apoptosis in selective tissues in response to specific death stimuli [START_REF] Chau | Signal-dependent protection from apoptosis in mice expressing caspase-resistant Rb[END_REF] and it was shown that this resistance involves a defect in Bid cleavage [START_REF] Huang | Blockade of tumor necrosis factor-induced Bid cleavage by caspase-resistant Rb[END_REF]. Recently, we identified the previously undescribed cleavage of Rb, at a LExD site, generating the p76 Rb form, which antagonizes p53-induced apoptosis in rat embryo fibroblasts [START_REF] Lemaire | Caspase-9 can antagonize p53-induced apoptosis by generating a p76(Rb) truncated form of Rb[END_REF].

In this article, we compared the effect of full-length Rb versus p76 Rb and p100 Rb , in several human cell lines. Interestingly, we report that although full length Rb does not have any apoptotic effect, p76 Rb triggers cell death in the absence of any other apoptosis inducers in all the tested cell lines and p100 Rb protects two cell lines against etoposide or TNF- induced cell death. These results show that truncated forms of Rb can have specific activities in the regulation of cell death if compared to the full-length protein. Finally, we show on the one hand that p76 Rb -induced cell death is Bax-dependent and can be diminished by Bcl-2 overexpression or caspase inhibition and on the other hand that p100 Rb could inhibit cell death by decreasing p53 protein level and caspase activity.

Materials and methods

Reagents. Emetine (E), mouse tumour necrosis factor- (TNF-) and etoposide (eto) were from Sigma. Chloromethyl-X-rosamine (CMX-ros) was from Molecular Probes. z-Val-Ala-DL-Asp-Fluoromethylketone (zVAD) was from Bachem. Stock solutions were prepared as follows: emetine and TNF- in serum free medium, etoposide and CMX-ros in dimethyl sulfoxide (DMSO), zVAD in methanol. All stock solutions were stored at -20°C.

Cell lines, cell culture and induction of cell death. We used the following human cell lines: HT1080, HEK293T, SAOS-2, TC7 and HT29. All cell lines were cultured at 37°C in a humidified atmosphere containing 5% CO 2 and in DMEM/F12 medium supplemented with 10% fetal bovine serum together with penicillin (100 g/ml), streptomycin (100 U/ml) and glutamax (1% v/v) from Invitrogen. Cell death was induced by the addition to exponentially growing adherent cells of etoposide or TNF- plus emetine during 24h. The concentrations of apoptosis inducers were determined to induce between 30% and 60% cell death after a 24 h incubation. The following doses were used: 125 g/ml etoposide for HEK293T, 30 g/ml etoposide or 0.4 g/ml emetine plus 2.5 ng/ml TNF- for HT1080, 1.5 g/ml emetine plus 12.5 ng/ml TNF- for SAOS-2, HT29 and TC7. zVAD was used at 100 M.

Plasmids. The GFP-Rb (pEGFPC1-Rb) construct was previously described [START_REF] Markiewicz | Lamin A/C binding protein LAP2alpha is required for nuclear anchorage of retinoblastoma protein[END_REF] with Rb cDNA of human origin. cDNAs corresponding to truncated p100 Rb and p76 Rb were subcloned into pGEM-T vectors (Promega) after PCR amplification. PCR was performed on human wild type Rb coding sequence (NM_000321) contained in pEGFPC1-Rb plasmid.

Primers used for PCR amplification are 5′-TCTCGAGCTATGCCGCCCAAAACCCCCCGAAAA-3′ and 5′-GAAGCTTTCAATCTGCTTCATCTGATCCTTCA-3′ for p100Rb; 5′-TCTCGAGCTATGACAAGAATTATTGAAGTTC-3′ and 5′-GAAGCTTTCATTTCTCTTCCTTGTTTGAGGT-3′ for p76 Rb (italic: XhoI or HindIII restriction sites, bold: start or stop codon introduced when necessary, underlined: specific Rb sequence). PCR was performed using standard methods with DyNAzymeTM EXT DNA polymerase (Finnzymes), performing 5 PCR cycles at 37°C (denaturation at 94°C for 30s, annealing at 37°C for 30 s and extension at 72°C for 3 min) followed by 25 PCR cycles at 55°C (denaturation at 94°C for 30 s, annealing at 55°C for 30 s and extension at 72°C for 3 min), except for p100 Rb amplification which required 30 cycles at 50°C instead of 25 cycles at 55°C. PCR products were extracted from agarose gel with JETsorb kit and ligated into pGEM-T vector using T4-DNA-ligase according to manufacturer's instructions (pGEM-T kit, Promega). pGEM-T-p100 Rb and -p76 Rb vectors were next digested using XhoI and HindIII enzymes and purified p100 Rb and p76 Rb fragments were ligated into XhoI/HindIII linearized pEGFPC1 vector (Invitrogen). Fusion cDNA sequences were checked by sequencing both strands using appropriate primers (MWG biotech). The use of pEGFPC1 vector conferred a GFP tag at the N-terminus of Rb, p76 Rb and p100 Rb .

Transfection. The nucleofection technique was performed according to the manufacturers' instructions (Amaxa) and cells were transfected with the following combinations of nucleofection solutions and programs: HEK293T (L / A-020), SAOS-2 (V / D-024), TC7 (T / T-030), HT29 (R / W-017). The HT1080 cells were transfected with the lipofectamin 2000 technique according to the manufacturer's instructions (Invitrogen). Induction of cell death was performed 24 h after the beginning of transfection.

SiRNA treatments. Cells were seeded 24 h before transfection with control non silencing (QIAGEN) or Bax siRNA (Hs BAX 10 HP validated siRNA, QIAGEN).

Lipofection was made using HiPerfect reagent (QIAGEN) and 25 nM siRNA according to the manufacturer's instructions. The cells were incubated with siRNA for 24h at 37°C before transfection with GFP-Rb or GFP-p76 Rb encoding vectors, as described above. Experiments (protein extraction for western-blot analysis and flow cytometry analysis) were carried out 24h to 48h later.

Flow cytometry. Flow cytometric measurements were performed using a XL3C flow cytometer (Beckman-Coulter). Cells expressing GFP-tagged proteins were identified by the emission of green fluorescence (525 nm) and mitochondrial membrane potential (m) was assessed by the retention of CMX-ros (620 nm). This fluorochrome, at low doses, specifically accumulates into the mitochondria depending on m. As a drop in m characterises all types of cell death [START_REF] Lemasters | The mitochondrial permeability transition in cell death: a common mechanism in necrosis, apoptosis and autophagy[END_REF], cell viability was determined by measuring the percentage of cells with low CMX-ros fluorescence. Cell death was also checked by assessing the decrease in cell size / forward scatter that reflects cell shrinkage during cell death. These measurements were performed in GFP positive cells in order to characterize the effect of exogenous proteins on cell viability. The expression level of the fusion proteins was systematically assessed by measuring GFP fluorescence intensity .Briefly, the cells were trypsinized and pooled with their corresponding medium and centrifuged (200g, 5 min). Cells were then resuspended in complete medium with 150 M CMX-ros and analyzed under FACS after 30 min incubation at 37°C. Western blot analysis. Western blotting was performed as previously described [START_REF] Bouleau | Fibroblast Growth Factor 1 inhibits p53dependent apoptosis in PC12 cells[END_REF].

The primary antibodies used were mouse monoclonal anti-p53 (Pab 122, gift from Dr. E.

May, IRSC, Villejuif, France), anti-MDM2 (SMP40, Santa Cruz), anti-enolase (gift from N.

Lamande, College de France, Paris), anti-cleaved PARP (Asp214, BD Pharmingen), anti-Rb (G3-245, BD Pharmingen), rabbit polyclonal anti-p53-P (Ser-15, Santa Cruz), anti-caspase 9 (Cell Signaling) and anti-cleaved caspase-3 (Asp175, Cell Signaling) as well as goat polyclonal anti-PUMA- (N-19, 1:200, Santa Cruz). Secondary antibodies (peroxidaseconjugated) were anti-mouse, anti-rabbit or anti-goat immunoglobulin (Biosystem).

Immunoreactive spots were detected by ECL or ECLplus kits depending on signal intensity (Amersham).

Results

Our aim was to characterise potential roles of p100 Rb and p76 Rb in several human cell lines. To perform this study, we have used GFP-tagged proteins in order to assess cell viability only in cultured cells that were successfully transfected. We have also used cell lines with distinct p53 status (summarized in Fig. 1A) in order to test if p100 Rb and p76 Rb activities depended on p53. Each cell line was transfected with vectors encoding the three forms of Rb (full length Rb, p100 Rb and p76 Rb ) and 24 hours later, the cells were incubated or not with apoptosis inducers (emetine + TNF- (E/TNF) or etoposide) during another 24 hours. The cells were then subjected to flow cytometric analysis to evaluate their viability (see Materials and Methods). Etoposide is a topoisomerase II inhibitor which can trigger the intrinsic pathway of apoptosis [START_REF] Karpinich | The course of etoposide-induced apoptosis from damage to DNA and p53 activation to mitochondrial release of cytochrome c[END_REF] whereas TNF- is a cytokine which can trigger the extrinsic pathway of cell death [START_REF] Thorburn | Death receptor-induced cell killing[END_REF]. TNF- was used in combination with emetine, an inhibitor of protein synthesis which (at non toxic concentrations) potentiates the pro-apoptotic effect of this cytokine [START_REF] Sidoti-De Fraisse | TNFalpha activates at least two apoptotic signaling cascades[END_REF][START_REF] Dumay | The superoxide dismutase inhibitor diethyldithiocarbamate has antagonistic effects on apoptosis by triggering both cytochrome c release and caspase inhibition[END_REF]. HT29, TC7 and SAOS-2 were not sensitive to etoposide (probably because of their deficient p53 status) whereas HEK293T cells were insensitive to E/TNF (data not shown). The HT1080 cell line was the only one to be sensitive to inducers of both intrinsic and extrinsic apoptotic pathways. After drugs incubation, cellular viability was measured on GFP positive cells by characterizing the mitochondrial membrane potential (m) which has been shown to decrease during apoptosis, autophagy or necrosis [START_REF] Lemasters | The mitochondrial permeability transition in cell death: a common mechanism in necrosis, apoptosis and autophagy[END_REF]. Cell shrinkage was also assessed by flow cytometry to confirm the results (data not shown). Fig. 1A shows that the full-length Rb protein has no significant effect (if compared to GFP alone) on cellular viability, in the presence or in the absence of apoptosis inducers, in all tested cell lines. This is also the case for p100 Rb in the absence of apoptosis inducers.

Interestingly, we show here that p76 Rb significantly induces cell death on its own in the five tested cell lines (compare GFP or GFP-Rb to GFP-p76 Rb ). This suggests that the effect of p76 Rb is independent of the status of p53. p76 Rb also increases the level of cell death in the presence of apoptosis inducers, except in HT29 cells. We have also observed that p100 Rb inhibits apoptosis in HT1080 + etoposide and SAOS-2 + E/TNF (compare GFP or GFP-Rb to GFP-p100 Rb ). The results obtained with the three forms of Rb are very interesting since they show that the truncated forms of Rb could have new and specific activities. Fig. 2B shows that the effects of these proteins are not related to a difference in their expression levels, indicated by the measurements of GFP fluorescence intensities. The fact that p76 Rb appears to be a little less expressed may be due to its specific toxicity.

In order to better understand how p76 Rb and p100 Rb regulate cell death, we tried to characterize several aspects of their mechanism of action. Fig. 2A shows that the broad spectrum caspase inhibitor zVAD significantly inhibits p76 Rb induced cell death in TC7 and HT29 cell lines, demonstrating that this cell death can be caspase-dependent. We have also used a variant of the SAOS-2 cell line conditionally overexpressing bcl-2 (Tet off system from Clontech) and showed that Bcl-2 significantly inhibits p76 Rb induced cell death (Fig. 2B).

Finally, we have tested the effect of Bax silencing on p76 Rb induced cell death in HT1080 cells. Fig. 2C (left panel) shows the efficiency of the siRNA treatment and points out that p76 Rb is able to trigger the accumulation of the pro-apoptotic Bax protein. Interestingly, it can also be observed from the corresponding analysis of cellular viability (right panel) that Bax siRNA decreases p76 Rb induced cell death, showing that it is Bax-dependent. Taken together, these results strongly suggest that GFP-p76 Rb triggers cell death by inducing the mitochondrial (intrinsic) pathway of apoptosis.

Next, we have investigated the mechanism underlying the anti-apoptotic role of p100 Rb in the HT1080 cells incubated with etoposide. We show in Fig. 3 that p100 Rb inhibits the accumulation of both total and the active p53 (phosphorylated on serine 15), triggered by etoposide. Interestingly, this phenomenon occurs very early (between 5 and 8h of incubation) and is no more visible after 16h (coinciding with a decrease in p100 Rb amount). Nevertheless, it seems to have consequences on the p53 transcriptional target and pro-apoptotic protein PUMA since its accumulation is specifically inhibited afterward. Interestingly, p100 Rb also seems to inhibit processing of caspase-9 and caspase-3 as well as caspase-3 activity, visualised by PARP cleavage, after 16 h of incubation. It is thus tempting to hypothesize that the upstream events seen with p53 stability are responsible for the caspase inhibition and the anti-apoptotic activity of p100 Rb .

Discussion

Here, we have shown that p76 Rb and p100 Rb have specific roles on cell death regulation when compared to full-length Rb. In particular, we observed that p76 Rb induces cell death in the five tested human cell lines, probably via the activation of a mitochondrialdependent apoptotic pathway. We have also observed an anti-apoptotic activity of p100 Rb in two cell lines. We have shown in the HT1080 cell line that p100 Rb interferes with the stability and activity of the p53 protein whereas this mechanism appears impossible in SAOS-2 cells, since they are p53-deficient. This matter of fact strongly suggests that p100 Rb could act at multiple levels to inhibit cell death. Indeed, if we consider that p100 Rb inhibits both p53 and caspase activity, we can hypothesize that the inhibition of caspase activity could be partly independent from the effect on p53. Concerning the interplay between p100 Rb and p53, two partners of Rb were already described to be involved in the regulation of p53 stability: MDM2 and E2F-1 [START_REF] Pomerantz | The Ink4a tumor suppressor gene product, p19Arf, interacts with MDM2 and neutralizes MDM2's inhibition of p53[END_REF][START_REF] Godefroy | p53 and Retinoblastoma protein (pRb): a complex network of interactions[END_REF]. First, it has been reported that p100 Rb exhibit an enhanced E2F binding [START_REF] Chen | Apoptosis is associated with cleavage of a 5 kDa fragment from RB which mimics dephosphorylation and modulates E2F binding[END_REF], suggesting that p100 Rb could inhibit E2F-1-dependent expression of p19 ARF and consequently p53 stability [START_REF] Pomerantz | The Ink4a tumor suppressor gene product, p19Arf, interacts with MDM2 and neutralizes MDM2's inhibition of p53[END_REF]. Second, it has been shown that, in contrast to full-length Rb, p100 Rb cannot interact with MDM2 [START_REF] Janicke | Specific cleavage of the retinoblastoma protein by an ICE-like protease in apoptosis[END_REF]. Thus, the C-terminal cleavage of Rb could prevent the formation of the Rb-MDM2-p53 trimeric complex which is known to stabilize p53 [START_REF] Hsieh | RB regulates the stability and the apoptotic function of p53 via MDM2[END_REF].

In conclusion, implications of both E2F-1 and MDM2 proteins could be in agreement with the anti-apoptotic role of p100 Rb .

Finding that p76 Rb significantly induces cell death in all tested human cell lines is interesting since it suggests that this effect is not cell type dependent. The effect of p76 Rb is probably independent from p53 since it occurs in both wild type (HT1080) and p53-deficient (TC7, HT29, SAOS-2) cell lines (see Fig. 1A). However, we cannot exclude that several pathways could be triggered by p76 Rb , some pathways being p53-dependent or independent. Finally, by using GFP-tagged proteins, we have observed that full-length Rb, p100 Rb and p76 Rb are located into the nucleus (data not shown). The fact that these three forms of Rb exhibit distinct activities suggests that they interact with different partners into this compartment to regulate the mitochondrial-dependent pathway of apoptosis. Thus a differential regulation of gene expression is probably central to the activity of the p76 Rb and p100 Rb truncated forms of the retinoblastoma protein. However the specific partners and the precise mechanism of this regulation remain to be determined.

The role of full length Rb in cell death regulation is complex since Rb was shown to exert opposite effects, depending on the cellular context. Our study suggests that such a complexity could exist for truncated forms of Rb since we have shown a few years ago that p76 Rb can inhibit a p53-dependent cell death in immortalized or primary rodent fibroblasts [START_REF] Lemaire | Caspase-9 can antagonize p53-induced apoptosis by generating a p76(Rb) truncated form of Rb[END_REF]. The pro-apoptotic effect of p76 Rb was first found in the human HEK293T cell line in which p53 may be inactivated by the presence of two viral oncoproteins: E1B and LT. Thus we have hypothesized that a difference in p53 activity could be responsible for the discrepancies between our two studies. However, we have tested the effect of p76 Rb on human cell lines with distinct p53 status, and found that p76 Rb was intrinsically and systematically pro-apoptotic. Since this new activity of p76 Rb was a general observation in human cell lines, we now postulate that cell death regulations by the Rb pathway might be different between rodent and human cells, explaining the opposite roles of p76 Rb in our two independent studies. Such an hypothesis would be in agreement with several of our previous studies, which showed that caspase inhibition can paradoxically accelerate cell death in rodent cells [START_REF] Lemaire | Caspase-9 can antagonize p53-induced apoptosis by generating a p76(Rb) truncated form of Rb[END_REF][START_REF] Rincheval | Inhibition of Bcl-2-dependent cell survival by a caspase inhibitor: a possible new pathway for Bcl-2 to regulate cell death[END_REF][START_REF] Godefroy | p53 can promote mitochondria-and caspaseindependent apoptosis[END_REF][START_REF] Godefroy | Transcriptional repression by p53 promotes a Bcl-2insensitive and mitochondria-independent pathway of apoptosis[END_REF] whereas we have never retrieved this effect in human cells. Finally, we are currently using Drosophila melanogaster in our laboratory as an in vivo model to study the role of truncated forms of the Rb homolog. Our results suggest that p76 Rb accelerates cell death in specific tissues if compared to full length Rb (unpublished data), reinforcing the significance of the present study. *Statistically significant difference in the viability of cells transfected with vectors were indicated (-zVAD versus +zVAD, -Bcl-2 versus +Bcl-2). Statistical analysis was determined using Student's t test. P  0.05 was accepted as significant. Fig. 3. Effect of p100 Rb on p53 stability and caspase activation. The HT1080 cells were transfected with the GFP and GFP-p100 Rb encoding vectors and were incubated 24 hours later for different times with etoposide. Western blots were performed to assess the role of p100 Rb on p53 accumulation and activity, as well as caspase activation and activity.

Legends to figures

Fig. 1 .

 1 Fig. 1. Effect of GFP-p76 Rb and GFP-p100 Rb versus GFP-Rb and GFP on cellular viability in five human cell lines. (A) The four vectors encoding GFP, GFP-Rb, GFP-p76 Rb and GFP-p100 Rb were transfected in human cell lines with different p53 status (in HEK293T cells, two

Fig. 2 .

 2 Fig. 2. Regulation of p76 Rb -induced cell death. All cell lines used here were transfected with

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Acknowledgments

This work was supported in part by grants from the Association pour la Recherche contre le Cancer (#3819) and from the Ligue Nationale Contre le Cancer. We thank Simon Heath for his critical reading of the manuscript. We are grateful to Christopher J. Hutchison who has provided the pEGFPC1-Rb plasmid.