Béatrice Eymin

Camille Leduc

Jean-Luc Coll

Elisabeth Brambilla

Sylvie Gazzeri
email: sylvie.gazzeri@ujf-grenoble.fr

p14 ARF induces G 2 arrest and apoptosis independently of p53 leading to regression of tumours established into nude mice

Keywords: apoptosis, G 2 arrest, tumour regression, ARF, p53

Until recently, the ability of ARF (human p14 ARF , murine p19 ARF ) tumour suppressor protein, encoded by the INK4A/ARF locus, to inhibit cell growth in response to various stimuli was related to its ability to stabilize p53 through the so-called ARF/MDM2/p53 pathway. However, recent data have demonstrated that ARF is not implicated in this unique p53-dependent pathway. By use of transient and stable expression, we show here that human p14 ARF inhibits the growth of human tumoral cells lacking functional p53 by inducing a transient G 2 arrest and subsequently apoptosis. This p14 ARF -induced G 2 arrest was correlated with inhibition of CDC2 activity, inactivation of CDC25C phosphatase and induction of the CDK inhibitor p21 WAF1 . Apoptosis was demonstrated using Hoechst 33352 staining, proteolytic activation of caspase-3 and PARP cleavage. Similar results were obtained in experiments with cells synchronized by hydroxyurea block. Importantly, we were able to reproduce these effects "in vivo" by showing that p14 ARF inhibits the growth of p53 nullizygous human tumours in nude mice and induces the regression of p53 -/-established tumours. In these experiments, tumoral regression was associated with inhibition of cell proliferation as well as induction of apoptosis confirming the data obtained in cell lines.

Introduction

The INK4A/ARF locus encodes two structurally distinct proteins, p16 INK4a and ARF, translated from alternatively spliced mRNAs (Quelle et al., 1995;Sherr, 1998). Both products are expressed in response to inappropriate proliferative signals and activate pivotal pathways implicated in cell-cycle control and tumour suppression (Chin et al., 1998;Sherr, 1998;Sherr, 2001). The cyclin-dependent kinase inhibitor (CDK-I) p16 INK4a prevents phosphorylation and functional inactivation of the retinoblastoma (RB) protein by cyclin-dependent kinases (Serrano et al., 1993). On the other hand, ARF protects against cellular transformation and immortalization by activating the p53 tumour suppressive protein (Sherr, 1998). Expression of ARF is induced in response to activated oncogenes such as Ras (Palmero et al., 1998;Serrano et al., 1997), c-myc (Zindy et al., 1998), E1A (de Stanchina et al., 1998), Abl (Cong et al., 1999;Radfar et al., 1998), and E2F-1 (Bates et al., 1998;Dimri et al., 2000) as well as during replicative senescence (Sherr, 1998). Because ARF-null mice are highly tumour-prone (Kamijo et al., 1997) and die early in life (Kamijo et al., 1999), ARF has been proposed to be most important in tumour surveillance.

Consistent with its role as a tumour suppressor, ARF can induce cell cycle arrest in both G 1 and G 2 phases and/or cell death (Kamijo et al., 1997;Quelle et al., 1995;Stott et al., 1998). Until recently, the growth suppressive function of ARF was related to its ability to stabilize p53 leading to the transcriptional activation of well-known p53-target genes such as p21 WAF1 or 14.3.3σ (Weber et al., 2002). However, recent data have pointed to additional p53independent ARF functions on cell growth but the mechanisms involved remain unclear. In murine systems, p19 ARF can suppress colony formation of p53 -/-cells by affecting the RB pathway (Carnero et al., 2000). Furthermore, mice lacking both p53 and p19 ARF develop a wilder spectrum of tumours than animals lacking either gene alone, and reintroduction of p19 ARF in mouse embryo fibroblasts (MEF) lacking p53, MDM2 and p19 ARF induces cell growth inhibition (Weber et al., 2000). In human, p53-independent ARF activity has been recently ascribed to its ability to impede S-phase progression (Yarbrough et al., 2002) and to induce p53 and Bax-independent apoptosis (Hemmati et al., 2002).

Because we previously showed that human p14 ARF can induce growth inhibition of p53-null cells (Eymin et al., 2001), we were interested in better characterizing the pathway(s) involved in the p53-independent functions of p14 ARF . To this aim, we transiently or stably expressed p14 ARF in human carcinoma cell lines lacking functional p53. Our data show that p14 ARF induces a transient G 2 arrest followed by apoptosis in these p53-deprived cells. This G 2 arrest correlates with inhibition of CDC2 activity, inactivation of CDC25C phosphatase, a major upstream regulator of CDC2, and induction of the CDK-I p21 WAF1 . On the other hand, apoptosis is associated with activation of procaspase-3 and PARP cleavage. Importantly, we also demonstrate that p14 ARF inhibits the growth and induces the regression of p53 nullizygous human tumours established in nude mice through inhibition of cell proliferation and induction of apoptosis, demonstrating the "in vivo" relevance of our data.

Results p14 ARF induces cell cycle arrest in transiently transfected p53-deficient cells

In order to investigate the p53-independent functions of p14 ARF , we transiently transfected two p53-deficient human cell lines derived from bronchioloalveolar lung adenocarcinoma using a full lenght p14 ARF expression vector. In these cell lines, p53 was inactivated either by homozygous deletion (H358 cells) or single point mutation (H322 cells).

In both cell lines, p14 ARF strongly inhibited cell growth after 7 days of G418 selection, as a 50% decrease in cell number was observed upon p14 ARF transfection as compared to cells transfected with the empty vector (Figure 1A). In these cells, p14 ARF was expressed in the nucleus where it resided predominantly in the nucleoli (Figure 1B). In addition, dual-color flow cytometry revealed that both H358 and H322 cells accumulated with a G 2 /M DNA content when transfected with p14 ARF . Since on visual observation there was no evidence of increased mitotic cells content in this population (data not shown), this pattern is likely to represent a G 2 arrest. Altogether, these results demonstrate the ability of human p14 ARF to inhibit cell growth by inducing a G 2 arrest in cells deprived of functional p53.

Inducible p14 ARF expression leads to G 2 arrest in stable transfectants clones

In order to confirm the results of transient transfections, we then established stable, p14 ARF -inducible clones in the H358 cell line using the doxycyclin-inducible expression system (Tet-On). Of note, p14 ARF is expressed at a low level in H358 cells (Figure 1A).

Several stable transfectant clones were obtained and further analyses were performed on four of them. Since all clones were found to give similar results, representative data of only clones 19 and 38 are presented here. As shown in Figure 2A, a strong expression of p14 ARF was detected after 24 hours of doxycyclin induction, which persisted even after 9 days of continuous induction. In contrast, little or no expression of the exogenous p14 ARF was observed in the uninduced state (Figure 2A). In these stable inducible clones, expression of p14 ARF correlated with its nucleolar accumulation (Figure 2B). As shown in Figure 2C (upper panels), a strong cell growth inhibition was observed upon 9 days of p14 ARF induction in both clones. This growth inhibition was significant as early as 72 hours after doxycyclin induction in clone 19 while 6 days were necessary for the clone 38 (Figure 2C, lower panels). Growth inhibition was not detected in an empty tet-on control clone, even in the presence of doxycyclin. Since on the basis of western blot and immunofluorescence analyses, expression of p14 ARF was lower in clone 38 than in clone 19 (data not shown), the differences of inhibition kinetics between clones 19 and 38 was likely related to the different p14 ARF expression levels between the two clones. Consistent with these time course studies of cell growth inhibition, a G 2 /M arrest was observed following 72 hours of doxycyclin induction in clone 19, and 6 days in clone 38 (Figure 2D). Again, no increase in mitotic cells content was detected in these populations based on visual observation, in agreement with G 2 -arrested cells.

Interestingly, we noticed the presence of cells with a DNA content greater than 4n upon p14 ARF induction (data not shown). Taken together, these results confirm, in stable transfectants clones, the ability of p14 ARF to induce a G 2 arrest in a p53-independent manner.

Modulation of cell-cycle protein expression associated with p14 ARF -induced G 2 arrest

As entry in mitosis is timely controlled by the cyclin B 1 /CDC2 complex (Figure 3A; (O'Connell et al., 2000;Takizawa & Morgan, 2000;Weinert, 1997), we analyzed the ability of p14 ARF to modulate its activity. Cyclin B 1 /CDC2 complexes were recovered by immunoprecipitation using anti-CDC2 antibody and their ability to phosphorylate histone H1 "in vitro" was tested in p14 ARF -induced cells. A strong inhibition of cyclin B 1 /CDC2 activity was observed in p14 ARF -expressing cells as compared to uninduced cells (Figure 3B).

Consistent with our cell cycle data (Figure 2D), this inhibition was observed after 72h or 6 days of p14 ARF induction, and persisted during 6 or 9 days in clones 19 and 38 respectively (Figure 3B). Under the same conditions, we did not detect any significant change in cyclin B 1 and CDC2 expression levels in p14 ARF -induced cells immunoblotting when compared to uninduced cells (Figure 3C) and to H358/Tet-On control cells (data not shown).

Before mitosis, cyclin B 1 /CDC2 complexes are maintained in an inactive state by phosphorylation of CDC2 on its Thr14 and Tyr15 residues [Figure 3A; (Blasina et al., 1997;Coleman & Dunphy, 1994;Norbury et al., 1991;Ye et al., 1996)]. Dephosphorylation of both residues causes activation of CDC2 and pushes the cells toward mitosis [ (Takizawa & Morgan, 2000), for review]. In addition, full activation of CDC2 also requires the phosphorylation of a conserved threonine residue (Thr161) (Draetta, 1997;Russo et al., 1996). CDC2 phosphorylation status was then examined using specific antibodies recognizing either Tyr15 or Thr161 phosphorylated residues. As a specific phospho-Tyr14 antibody was not available, CDC2-P Thr14 status could not be studied. As compared to uninduced cells, a marked decrease of CDC2-P Thr161 level was observed in p14 ARF -expressing cells suggesting that CDC2 was hypophosphorylated on this residue (Figure 3C). In contrast, the same apparent level of CDC2-P Tyr15 was detected in both cases (with or without doxycyclin induction ; Figure 3C). As dephosphorylation of CDC2 at tyrosine 15 occurs only in mitotic cells, the level of CDC2-P Tyr15 in a population of asynchronous cells, which contains few mitotic cells, is expected to be high. Therefore, we cannot exclude that this could mask an increased level of phosphorylated CDC2 at Tyr15 residue in the population of p14 ARFexpressing cells principally arrested in G 2 . Taken together, these results suggest that p14 ARF maintains CDC2 into an inactive state, dephosphorylated at Thr161 residue.

Then, we investigated the status of some upper regulators of the cyclin B 1 /CDC2 complexes. At the onset of mitosis, dephosphorylation of both Thr14 and Tyr15 residues on CDC2 is regulated by the CDC25C phosphatase [Figure 3A; (Kumagai & Dunphy, 1991;Millar et al., 1991;Strausfeld et al., 1991)]. CDC25C activity is itself controlled by a complex set of activatory/inhibitory phosphorylations [ (Takizawa & Morgan, 2000), for review].

Throughout cell cycle, except in mitosis, CDC25C is maintained inactivated by phosphorylation on its Ser216 residue that creates a binding site for small phosphoserinebinding proteins called 14-3-3 which inactivate CDC25C by sequestration into the cytoplasm (Peng et al., 1997;Takizawa & Morgan, 2000). In p14 ARF -expressing cells, we detected a slight decrease in CDC25C protein level which was not observed in the H358/Tet-On control cells in the same conditions (data not shown), as well as an increase of its inactive phosphorylated form at Ser216 (Figure 3C andD). This effect was specific for CDC25C as the level of CDC25B and 14.3.3σ proteins did not appear to vary (data not shown). These data strongly suggest that p14 ARF inactivates CDC25C by reducing its total expression level and increasing its inactive phosphorylated form. Furthermore and consistent with a biological inactivation, a cytoplasmic accumulation of CDC2, CDC2-P Tyr15 and CDC25C-P Ser216 was observed in p14 ARF -expressing cells (Figure 3E). In contrast, such accumulation never occured in uninduced cells or in H358/Tet-On control cells in agreement with the property of these proteins to shuttle continuously from the cytoplasm to the nucleus of proliferating cells.

Finally, as CDC2 activity and phosphorylation status can also be controlled by the p21 WAF1 CDK-inhibitors family (Dulic et al., 1998;Harper et al., 1995;Niculescu et al., 1998;Smits et al., 2000), expression of some of them was analyzed. An increase of p21 WAF1 expression level was detected in p14 ARF -expressing cells (Figure 3C) whereas p27 KIP1 level remained unchanged (data not shown).

Overall, these results suggest that p14 ARF induces a G 2 arrest independently of p53 by preventing the activation of cyclin B 1 /CDC2 complexes, at least in part through inactivation of CDC25C phosphatase and induction of p21 WAF1 .

Inhibition of G 2 /M phase progression in synchronized p14 ARF -expressing cells

To confirm the molecular events implicated in the p14 ARF -induced G 2 arrest, synchronized cells were used. Hydroxyurea (HU) treatment was carried out to block the cells in late G 1 and S phases of the cell cycle, allowing a more precise analysis of cellular DNA content in late stages of the cell cycle after block release, particularly between S and mitosis.

In these experiments, p14 ARF was induced 24 hours before HU block. As shown in figures 4A and 4B, uninduced control cells accumulated with a G 2 /M DNA content until 9 hours after block release and started to enter mitosis about 13 hours after replating in fresh medium.

Twenty four hours after the release, they were predominantly in G 0 /G 1 (Figures 4A &4B). In contrast, and consistent with a G 2 /M arrest, a continuous accumulation of p14 ARF -induced cells in G 2 /M phases of the cell cycle was observed between 9 and 13 hours, and persisted even 24 hours (Figures 4A &4B) and 48 hours (data not shown) after the release. As expected, accumulation of control uninduced cells in G 2 /M 13 hours after the block release correlated with a slight upregulation of cyclin B 1 and CDC2 whereas CDC25C protein level did not change (Figure 4C). At this time, and consistent with passage through mitosis, CDC25C was dephosphorylated at Ser216 and CDC2 was activated by dephosphorylation at Tyr15 and phosphorylation at Thr161 residues (Figure 4C). In these control cells, p21 WAF1 was never induced. In contrast, although p14 ARF -expressing cells also accumulated with a G 2 /M DNA content 13 hours after the block release, CDC2 remained phosphorylated on Tyr15 and hypophosphorylated on Thr161 residues (Figure 4C). These data suggest that CDC2 was in an inactive state in these cells. In addition, concomittant downregulation of CDC25C (Figure 4C and D) and upregulation of inactive CDC25C-P Ser216 and of p21 WAF1 protein expression levels (Figure 4C) were also observed. As this specific pattern of protein expression persisted 24 hours (Figure 4C) and even 48 hours (data not shown) after the block release, these data are in agreement with the absence of passage through mitosis of most of the p14 ARF -expressing cells.

We noticed that a small proportion of cells was still able to overpass this G 2 arrest. This might be linked to the shorter time (24 hours) of p14 ARF induction in synchronization experiments compared to those performed on asynchronous cells.

Taken together, these data with synchronized cells confirmed the results obtained in asynchronous p14 ARF -expressing cells and demonstrate the ability of p14 ARF to induce a G 2 arrest independently of p53 by preventing cyclin B 1 /CDC2 activation, at least in part through inactivation of CDC25C phosphatase and induction of p21 WAF1 .

G 2 arrest precedes apoptosis in p14 ARF -expressing cells

Since DNA damage checkpoints may delay cell cycle progression and lead to apoptosis if the cells are unable to undertake repair (Elledge, 1996;Hartwell & Weinert, 1989;Walworth, 2000), we asked whether the p14 ARF -induced G 2 arrest might ultimately lead to apoptosis in our cells. Using Hoechst 33352 staining, we observed an apoptotic pattern in 20% of the p14 ARF -expressing cells after 6 or 9 days of doxycyclin induction in clones 19 and 38 respectively. In contrast, this was never detected in uninduced control cells (Figures 5A &5B), as well as in an inducible control clone, even after 15 days of continuous doxycyclin treatment (data not shown). Under the same conditions, the proteolytic activation of procaspase-3 with appearance of the active p12 and p17 fragments as well as PARP (poly-ADP ribose polymerase) cleavage were specifically observed in p14 ARF -induced cells (Figure 5C). Apoptosis was confirmed in HU-synchronization experiments, since sub-G 1 DNA content, caspase-3 activation and PARP cleavage were specifically detected in p14 ARFexpressing cells 48 hours after the block release (Figure 5D). Overall, these results demonstrate that p14 ARF is also able to induce apoptosis independently of p53. Moreover, as G 2 arrest was clearly observed before apoptosis could take place (Figures 5A &5D), our data suggest that G 2 arrest precedes apoptosis in p14 ARF -expressing cells.

Antitumoral activity of p14 ARF in nude mice is associated with cell cycle arrest and apoptosis

In order to determine whether the activity associated with p14 ARF expression in cells grown in culture could be reproduced in tumours, H358/Tet-On/p14 ARF clones 19 and 38 or H358/Tet-On cells were injected subcutaneously into immunodeficient nude mice. Of note, the growth kinetics of all cells was very similar (data not shown). As identical results were obtained using both clones, representative data of clone 19 are presented here.

We first tested whether the ability of p14 ARF to induce G 2 arrest and apoptosis in cell lines might influence the tumoral progression. Whereas the uninduced H358/Tet-On/p14 ARF cells (mice drinking water) gave rise to rapid tumour growth, a marked inhibition was observed in mice that received doxycyclin (p=0,0043) (Figure 6A). This effect was specific of p14 ARF induction since growth inhibition was not detected in mice injected with H358/Tet-On cells and that received doxycyclin (Figure 6A).

We then asked whether p14 ARF was able to prevent the growth of pre-existing tumours lacking p53. H358/Tet-On/p14 ARF cells were injected into nude mice and tumours were allowed to grow until they reach 50 to 70 mm 3 volume. At this time, doxycyclin was added or not in their drinking water. A doxycyclin treatment of mice with established H358/Tet-On/p14 ARF tumours caused a highly significant tumoral regression after 80 days of treatment (p=0.0043) when compared to mice with same tumours but drinking water only or to mice with established H358/Tet-On tumours and drinking doxycyclin (Figure 6B). This antitumoral activity became statistically significant after 35 days of doxycyclin treatment (p=0.0043).

Immunostaining revealed a clear accumulation of p14 ARF in H358/Tet-On/p14 ARF tumours arising from doxycyclin-treated mice as compared to untreated mice, as well as an inhibition of tumor cell proliferation as determined by KI-67 staining and increased apoptosis as detected by active caspase-3 (Figure 7A) or TUNEL staining (data not shown). When the proliferative index to the apoptotic index was calculated, a lower figure was observed (1.66 versus 4.66) in tumours overexpressing p14 ARF than in those with a physiological level of p14 ARF , in agreement with cell growth inhibition and apoptosis induction. Furthermore, by using western blot analyses on whole tumour extracts, we demonstrated a marked downregulation of total CDC25C which was not detected in the control H358/Tet-On tumours (data not shown) and an apparent stable level of CDC2-P Tyr15 (Figure 7B). However, we could not demonstrate any change in CDC25C-P Ser216 or p21 WAF1 protein levels and were unable to detect CDC2-P Thr161 in treated or untreated tumours (data not shown). On the other hand, clear activation of procaspase-3 and PARP cleavage were specifically observed in treated tumours confirming the existence of apoptosis (Figure 7B). Overall, these results demonstrate the ability of p14 ARF to inhibit cell growth and to induce apoptosis "in vivo".

Discussion

Until recently, the ARF tumour suppressive protein was only ascribed to a welldefined ARF/MDM2/p53 linear pathway. In this model, ARF induces the stabilization and activation of p53 leading to a dual block in G 1 and G 2 phases of the cell cycle and/or cell death (Kamijo et al., 1997;Quelle et al., 1995;Tao & Levine, 1999;Weber et al., 2002;Weber et al., 1999;Zhang & Xiong, 1999). In contrast and despite the existence of many contradictory data, it is now well admitted that ARF also acts onto p53-independent pathways to inhibit cell growth. However, the exact nature of the molecular mechanisms involved in these processes is still unclear and the targets remain unknown. In this study, we demonstrate that p14 ARFexpressing cells undergo a G 2 arrest in the absence of functional p53 suggesting that p53 might be necessary for G 1 checkpoint but dispensable for G 2 arrest in response to p14 ARF induction. Consistently, it was previously reported that p53 -/-cells can accumulate in G 2 following DNA damage (Fan et al., 1995;Li et al., 1995;Lowe et al., 1993;Powell et al., 1995). However, as a prolonged G 2 -M arrest requires p53 (Bunz et al., 1998), this could explain why p14 ARF induces only a transient G 2 arrest that precedes apoptosis in the absence of active p53. According to our data, the ability of p14 ARF to induce p53-independent apoptosis was recently described in p53-deleted SAOS-2 cells and HCT116 p53-/-colon cancer cells (Hemmati et al., 2002). However, cell cycle analysis was not performed in these cells. On the other hand, Weber et al. recently reported a strict dependency of an intact p53 pathway for p14 ARF -mediated cell cycle arrest following adenovirus infection (Weber et al., 2002).

However, in this last paper, all experiments were performed in a 24-48 hours time frame and no long-term effects were analyzed in contrast to our study. Therefore, as mentionned by the authors, p14 ARF might be able to induce arrest or apoptosis in a p53-negative background once activated over a longer time period.

A key step in regulating the entry of eukaryotic cells into mitosis is the activation of the cyclin B 1 -CDC2 complex [ (Takizawa & Morgan, 2000), for review]. Consistently, a strong inhibition of CDC2 activity was associated with G 2 arrest in p14 ARF -expressing cells.

When we investigated the mechanisms involved in this inhibition, we observed that CDC2 was markedly hypophosphorylated on Thr161 and apparently hyperphosphorylated on Tyr15.

The inhibitory phosphorylations on Tyr15 and Thr14 of CDC2 are counteracted by the CDC25C phosphatase (Kumagai & Dunphy, 1991;Millar et al., 1991;Strausfeld et al., 1991).

In our p14 ARF -expressing cells, the level of CDC25C protein was reduced. Total CDC25C protein level is not significantly affected during cell cycle progression (Goldstone et al., 2001) in contrast to its phosphorylation status. Therefore, it is unlikely that the specific decrease of CDC25C level following p14 ARF induction could represent the sole consequence of G 2 arrest. Interestingly, as for CDC25A which is downregulated during G 1 checkpoint in response to DNA damage (Mailand et al., 2000), downregulation of CDC25C has been already reported in response to UVB radiations (Athar et al., 2000), γ radiations (Wang et al., 2000) or infection by adenovirus (Raj et al., 2001) and related to a decrease of transcription (Wang et al., 2000) or a degradation by the proteasome complex (Raj et al., 2001). Whether these mechanisms could be implicated in the downregulation of CDC25C protein following p14 ARF induction remains to be determined. On the other hand, overexpression and cytoplasmic accumulation of the CDC25C-P Ser216 inactive form was observed upon p14 ARF induction.

Maintenance of the inhibitory phosphorylation of CDC2 on Thr14/Tyr15 mediated by a CHK-CDC25C pathway has been previously implicated in a G 2 arrest following DNA damage (Peng et al., 1997;Poon et al., 1997;Rhind et al., 1997;Sanchez et al., 1997). In such conditions, inactive phosphorylation of CDC25C on Ser216 by CHK1 and CHK2 kinases was observed, allowing binding to the proteins of the 14.3.3 family and preventing CDC2 activation (Furnari et al., 1997;Graves et al., 2000;Peng et al., 1997;Sanchez et al., 1997). In this way, it would be interesting to study the implication of CHK1 and CHK2 kinases in the p14 ARF -mediated phosphorylation of CDC25C on Ser216. Overall, although further studies are required to assess the role of CDC25C in p14 ARF -mediated G 2 arrest, we propose that p14 ARF might inactivate CDC25C by both down-regulating its global expression and increasing its inhibitory phosphorylation on Ser216, both mechanisms cooperating to reinforce the G 2 cell cycle checkpoint. Therefore, although we cannot exclude that Tyr15 (and Thr14) phosphorylations of CDC2 could simply be a result rather than a cause of cyclin B 1 -CDC2 inactivation linked to multiple feedback loops controlling the activity of p34 CDC2 complexes (Poon et al., 1997), it is reasonnable to assume that inactivation of CDC25C is implicated in the apparent accumulation of inactive CDC2-P Tyr15 upon p14 ARF expression.

Concomitantly with CDC25C inactivation, a strong induction of the cyclin-CDK inhibitor p21 WAF1 was observed in p14 ARF -expressing cells. Under normal growth conditions, p21 WAF1 protein accumulates in G 2 (Dulic et al., 1998;Li et al., 1994) and promotes a pause in late G 2 (Dulic et al., 1998). However, several studies have also demonstrated the efficient role of p21 WAF1 in G 2 checkpoint (Agarwal et al., 1995;Niculescu et al., 1998). Interestingly, Bunz et al. have reported that p21 WAF1 is essential to sustain rather than to initiate the G 2 checkpoint after DNA damage in human cells (Bunz et al., 1998). Despite its low affinity for the cyclin B 1 -CDC2 complexes (Dulic et al., 1998;Harper et al., 1995;Winters et al., 1998), p21 WAF1 can inhibit CDC2 activity (Yu et al., 1998) and has been recently involved in the inhibition of its activating phosphorylation on Thr161 (Smits et al., 2000). Whether p21 WAF1 affects directly or not the phosphorylation status of CDC2 and mediates some of the p53-independent effects of p14 ARF remains to be determined. Overall, although we are aware that further studies are required to extend the mechanisms by which p14 ARF inhibits cell growth and to clearly identify direct targets, our data suggest that p14 ARF induces a G 2 arrest independently of p53 by preventing CDC2 activity through both induction of p21 WAF1 and inactivation of CDC25C.

Finally and perhaps the most significant aspect of this work was the finding that p14 ARF conditional expression inhibits tumour growth and induces the regression of p53-/human tumours established in nude mice. Importantly, these biological events were associated with an inhibition of cell proliferation as well as an increased apoptosis. Furthermore, although we were unable to reproducibly detect variations in the level of p21 WAF1 and CDC25C-P Ser216 , CDC2, CDC2-P Tyr15 , CDC25C, caspase-3 and PARP protein levels varied similarly "in vitro" and "in vivo" strengthening our data on cell lines. Taken together, our results reinforce the notion that p14 ARF can affect pathways and targets other than p53 and demonstrate for the first time that these p53-independent effects can contribute to the tumour suppressive functions of p14 ARF "in vivo". They also predicts that loss of p14 ARF in tumours that have already inactivated p53, might confer an additive deleterious effect on cell growth. Consistently, Weber et al recently reported that tumours arising in mice lacking ARF and p53 are more aggressive than those in mice lacking either gene alone (Weber et al., 2000). Furthermore, we previously reported the double inactivation of p14 ARF and p53 in a large proportion of high grade NE lung tumours (Gazzeri et al., 1998), the most aggressive lung tumours, a concept that received now full agreement in numerous other cancer types (Esteller et al., 2000;Lindstrom et al., 2000;Sanchez-Cespedes et al., 1999). Therefore, reintroduction of p14 ARF in these tumours could restore some of the normal checkpoint pathways and thus constitute a major goal of future therapy.

Materials and methods

Cell lines, transfection, proliferation, apoptosis and synchronization assays

H358 and H322 human bronchioloalveolar lung carcinoma cell lines were cultured in RPMI-1640 medium (GIBCO, Cergy Pontoise, France) supplemented with 10% (v/v) heatinactivated FCS, 2 mM L-glutamine and 100 units/ml penicillin/streptomycin in 5% CO 2 at 37°C. To ensure exponential growth, cells were resuspended in fresh medium 24 hours before transfection. Transient transfections were carried out using FUGENE 6 (Roche Diagnostic) according to the manufacturer's protocol. The total amount of DNA used for transfection was held constant by adding empty control vector. In the growth suppression assay, geneticin (G418; 800 µg/ml) was added to the media 48h post transfection and trypan blue excluding cells were counted one week later. Cell cycle analysis of transient transfection experiments was performed 4 days after same geneticin treatment. For clonogenic assays using p14 ARF inducible clones, cells were fixed in 100% ethanol at the indicated times, stained with 1% methylene blue in 0.01 M Borate buffer, pH 8.5 and optical density was measured at 630 nm after elution in HCl 0.1N. Evaluation of apoptosis in stable transfectants clones was carried out after 48h, 72h, 6 days and 9 days of doxycyclin treatment (1 µg/ml) using Hoechst 33352 staining (5 µg/ml, 30 min at 37°C). The percentage of apoptotic cells, identified by their fragmented chromatin, was scored on at least 300 cells using fluorescence microscopy analysis. In the synchronized assays, exponentially H358/Tet-On/p14 ARF clones were pretreated during 24 hours with or without 1 µg/ml doxycyclin and further cultured in the presence of 1 mM hydroxyurea (HU) (Sigma). 24 hours after HU treatment, cells were washed twice with PBS and cultured in fresh medium in the presence or absence of doxycyclin for additional indicated times. Cells were then collected and further analyzed for DNA content and protein expression. Cell-cycle and western blot analyses were processed as described below.

Generation of stable p14 ARF inducible clones

Obtention of stable p14 ARF inducible clones was performed using a modified tetracyclin-regulated inducible expression system (Tet-On System, Clontech, Ozyme, Saint Quentin en Yvelines, France) according to the manufacturer's protocol. Briefly, H358 cells were transfected in a first round with the pTet-On plasmid which contains the reverse doxycyclin repressor (rtTA) expression cassette, using TFX-50 (Promega, Charbonnières, France) and transferrin (Tfe) (Becton Dickinson, le Pont de Claix, France), and selection of transfectants was performed in the presence of G418 (800 µg/ml). Stable H358/Tet-On clones were screened using the Promega Luciferase Assay System after transient transfections with pTRE-Luc plasmid. A second round of tranfection with pTRE-p14 ARF and pTK-Hyg plasmids was then carried out using FUGENE 6. The pTRE-p14 ARF plasmid was constructed by subcloning wild-type p14 ARF cDNA downstream of the Tet-regulated promoter into pTRE plasmid. Double transfectants were then selected in the presence of hygromycin (200 µg/ml) and geneticin (100 µg/ml), and screened for p14 ARF expression by western blotting following 48 hours of doxycyclin induction (1 µg/ml). Four clones were arbitrarily selected for further experiments.

Antibodies

Monoclonal antibodies (mAbs) used included anti-CDC2/CDK1 (Ab-3, Oncogene Research), anti-CDC2 p34 (sc-54, Santa-Cruz, TEBU, Le Perray-en-Yvelines), anti-CDC25C (67211A, Pharmingen), anti-phospho-CDC25C (Ser216) (9D1, Cell Signaling), anti-cyclin B 1 (14541A, Pharmingen), anti-human KI67 antigen (clone Ki-67, DAKO) and anti-p21 WAF1 (Ab-1, clone EA10, Oncogene Research). Polyclonal antibodies used included anti-actin (20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)Sigma,Saint Quentin Fallavier,France), anti-caspase-3 (65906E, Pharmingen), antihuman caspase-3 active (R&D System), anti-phospho-CDC2 (Tyr15) (Cell Signaling), antiphospho-CDC2 (Thr161) (Cell Signaling), anti p14 ARF (Ab-1, Oncogene Research) and anti-PARP (Roche Diagnostics).

Cell-cycle analysis

Cells were washed twice in PBS and fixed in ice-cold acetone for 5 min at -20°C.

After two PBS washes, cells were saturated with 2% BSA, 2% goat serum in PBS for 30 min at room temperature and incubated overnight at +4°C with gentle rotation in presence of anti-p14 ARF (Ab-1, 1/200). After extended washes in PBS, Alexa TM 488 goat anti-rabbit-IgG (H+L) conjugate (2 mg/ml; 1/500, Interchim, Montluçon) was added for 30 min at room temperature.

Cells were then washed twice in PBS, incubated at 37°C for 30 min with 200 U/ml of RNAse A (Sigma) and then with propidium iodide (10 µg/ml in PBS). Cell-cycle distribution of p14 ARF -expressing cells was determined by dual-color flow cytometry using the Cellfit software (Becton Dickinson, Grenoble, France).

Indirect immunofluorescence 50x10

3 cells/well were seeded onto 4-wells LAB-TEK R . At indicated times, cells were fixed in 4% paraformaldehyde for 5 min at room temperature (RT), washed three times, saturated in presence of 2% BSA, 2% goat serum in PBS for 30 min at RT and further incubated with anti-p14 ARF (Ab-1, Oncogene Research; 1/1000) for 2 hours. After three PBS washes, Alexa TM 488 goat anti-rabbit IgG (H+L) conjugate (2 mg/ml; 1/500, Interchim, Montluçon) was added for 30 min at RT and the cells were then whashed twice in PBS.

Normal rabbit IgG at the same concentration as the primary antibody served as a negative control. Cells were counterstained with Hoechst and observed using an Olympus microscope (20x magnification). Images were captured with a Coolview CCD camera (Photonic Science) and digitally saved using Visilog software.

Immunoblotting

Cells or frozen tumour tissues were lysed in RIPA buffer for 30 min on ice, pelleted and the protein concentration was determined using the Biorad Dc protein assay. 20-40 µg protein were then separated by SDS-PAGE in 7-12% gels and electroblotted onto PVDF membranes (Amersham Pharmacia, France). Membranes were incubated overnight at +4°C with primary antibody and proteins were detected using horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA) and enhanced chemiluminescence detection system (Amersham, Les Ulis, france). To confirm the slight variation of CDC25C expression level, immunoblots were analyzed by densitometry using the NIH 1.6 software. Further quantification was performed by normalizing the signals with respect to that of the actin loading control. Results were expressed as a CDC25C/actin ratio which was arbitrarily normalized to a 100% expression level in uninduced cells at each time studied.

For cytoplasmic extracts, cells were incubated for 30 min into an ice-cold buffer containing 250 mM Sucrose, 20 mM HEPES-KOH, pH 7.5, 1.5 mM MgCl 2 , 1 mM EDTA, 1 mM EGTA, 1 mM DTT and protease/phosphatase inhibitors. Permeabilization was carried out by serial passages of the cell suspension through a 25G seringue until the obtention of 70-80% trypan blue positive cells. Centrifugation at 1700 rpm was then performed for 5 min and supernatants were ultracentrifugated for 30 min at 55.000 rpm in order to obtain the cytosolic fraction.

"In vitro" kinase reaction

Cells were washed three times with PBS, resuspended in lysis buffer (150 mM NaCl, 1mM EDTA, 1 mM EGTA, 50 mM Tris-HCl pH 7.4, 1% Triton 100X, 0.2 mM Na 3 VO 4 , 0.5% Nonidet P40, 0.1 mM PMSF, 2.5 µg/ml pepstatin, 10 µg/ml aprotinin, 5 µg/ml leupeptin), incubated on ice for 30 min and centrifugated for 20 min at 10.000 rpm. Protein concentration was adjusted to 200 µg in 200 µl NET buffer (150 mM NaCl, 50 mM Tris-HCl pH 7.5, 0.1% NP40, 1 mM EDTA pH 8, 0.25% gelatin, 0.02% sodium azide) supplemented with protease inhibitors before use and incubated overnight at +4°C with anti-cyclin B 1 or anti-CDC2 antibodies. 1 mg of protein A/protein G agarose beads was added to each reaction and samples were incubated for 1 hour at + 4°C with gentle rotation. After two washes in NET buffer, beads were subjected to a final wash in kinase buffer (50 mM Tris-HCl, pH 7.6, 10 mM MgCl 2 , 1 mM DTT). 10 µl aliquots were added to 40 µl of kinase buffer containing 50 µM ATP, 10 µCi (γ-32 P) ATP (3000 Ci/mmol) and 50 µg of histone H1 and incubated 30 min at 30°C. Samples were boiled in the presence of 50 µl SDS-PAGE loading buffer and subjected to electrophoresis on a 12% polyacrylamide gel. CDC2 activity was visualized by autoradiography.

Tumour treatment

"In vivo" experiments were conducted onto six-weeks-old female Swiss nude mice (Iffa Credo, Marcy l'Etoile, France). In all experiments, 20x10 6 H358/Tet-On/p14 ARF cells were injected subcutaneously into the nude mice and randomized into two groups, one receiving doxycyclin (2 mg/ml) in their drinking water and the second one receiving water

only. An additional group of nude mice injected with 20x10 6 H358/Tet-On cells and treated with doxycyclin was used as control. The tumour volumes were measured once a week in two perpendicular diameters and calculated as follows: a x b 2 x 0.4, where "a" and "b" are the largest and smallest diameters, respectively. All experiments were repeated twice for both 19 and 38 clones. In the tumour uptake assay, doxycyclin was provided immediately after cells injection. Each group was composed of 8 animals and difference in tumour volume was assessed using a non parametric Mann-Whitney t test. Study of tumour regression was performed on 10 animals per group that received doxycyclin or water when tumour volumes ranged from 50 to 70 mm 3 (about two or three weeks later). Mice were sacrified at differents times and tumours were divided into two parts before frozing in order to perform western blotting or immunohistochemistry. Statistical analysis between the tumour volumes of the different groups was performed using a non parametric Mann-Whitney t test on 6 animals per group.

Immunohistochemical staining

Immunostaining of p14 ARF , active caspase-3 and KI67 was performed on frozen sections, as previously described for p14 ARF and KI67 (Gazzeri et al., 1998;Labat-Moleur et al., 1998) and according to the manufacturer's protocol for caspase-3. Briefly, three step immunohistochemical method was applied on 5 µM thick frozen sections. After fixation, non specific binding sites were blocked by incubating the sections with 0.1% (w/v) BSA in phosphate-buffered saline (PBS) for 30 minutes at room temperature. The sections were subsequently incubated with primary antibodies overnight at 4°C, washed in PBS several times and exposed to biotinylated secondary antibodies consisting of either anti-rabbit biotinylated donkey F(ab')2 (1/1000; The Jackson Laboratory; West Grove; PA) or antimouse biotinylated donkey F(ab')2 (1/500; The Jackson Laboratory) for 1 hour at room temperature. Slides were then washed in PBS and incubated with the streptavidin-biotinperoxydase complex (1/200; Strept-AB complex; DAKO) for 1 hour at room temperature.

The slides were counterstained with Harris' hematoxylin. Normal rabbit or mouse IgG at the same concentration as the primary antibodies served as negative controls. H358/Tet-On/p14 ARF clone 19 was pretreated during 24 hours with (Dox +) or without (Dox -)

Legends to figures

1 µg/ml doxycyclin and further cultured during 24 hours in the presence of 1 mM hydroxyurea (HU). HU block was then released and cells were cultured in fresh medium with (Dox +) or without (Dox -) doxycyclin. Six hours (T6), 9 hours (T9), 13 hours (T13) and 24 hours (T24) after the block release, the cells were collected and processed for dual-color flow cytometry using the Cellfit software (Becton Dickinson) (A, B) and western-blot analysis (C).

Representative results of three independent experiments are presented. (D) Quantification of CDC25C expression level by densitometry in the presence (DOX+) or absence (DOX-) of doxycycline. Analysis was performed as explained in Figure 3D.

Figure

  Figure 1: p14 ARF inhibits cell growth and induces a G 2 arrest in transiently transfected p53-null cell lines.

Figure 2 :

 2 Figure 2: Inducible p14 ARF expression leads to G 2 arrest in stable transfectants clones. In all experiments, expression of p14 ARF was either uninduced (Dox -) or induced (Dox +) in H358/Tet-On/p14 ARF clones 19 and 38 in presence of 1 µg/ml of doxycyclin and experiments were performed at the indicated times. (A) Western blotting of p14 ARF protein expression in H358/Tet-On/p14 ARF clones 19 and 38. (B) Nucleolar accumulation of p14 ARF in both clones

Figure 4 :

 4 Figure 4: p14 ARF inhibits the G 2 /M phase transition in HU-synchronized cells.

Figure 5 :

 5 Figure 5: p14 ARF induces apoptosis independently of p53.In all experiments, H358/Tet-On/p14 ARF clones 19 and 38 were cultured with (Dox +) or

Figure 6 :

 6 Figure 6: p14 ARF inhibits tumour progression and induces tumour regression in nude

Figure 7 :

 7 Figure 7: p14 ARF inhibits cell proliferation and induces apoptosis in nude mice.H358/Tet-On/p14 ARF cells (clone 19) were sub-cutaneously injected into nude mice. After 3

G0

  + Dox -Dox + Dox -Dox + Dox -Dox + Dox -Dox +

Acknowledgements

This work was supported by a grant to E.B. from La Ligue Nationale Contre le Cancer as an « équipe labellisée » and by the Association pour la Recherche sur le Cancer. B.E. was supported by INSERM (Poste Accueil). We thank Christiane Oddou, Pascal Perron, Christine Claraz and Sylvie Veyrenc for technical assistance.

SD: standard deviation. (D) Cell-cycle analysis of the p14 ARF positive cells was performed using dual-color flow cytometry. Representative data of at least three independent experiments are shown. Similar results were obtained on the whole cell population after propidium iodide staining (data not shown).


Béatrice Eymin

Sylvie Gazzeri

Christian Brambilla

Elisabeth Brambilla
email: elisabeth.brambilla@ujf-grenoble.fr

Pr Elisabeth Brambilla

Distinct pattern of E2F1 expression in human lung tumours: E2F1 is upregulated in small cell lung carcinoma

Keywords: carcinogenesis, cell cycle, E2F1, expression, lung tumour

The transcription factor E2F1 is a key component of cell cycle that acts to transactivate genes required for S phase entry. Thus, it plays an important role in cellular proliferation, oncogenesis and differentiation. In order to investigate its potential implication in human lung carcinogenesis, we studied E2F1 protein expression by western blotting and immunohistochemistry in a series of 58 human lung tumours of all histological types. We showed that E2F1 product was overexpressed in 92% (24/26) of small cell lung carcinoma (SCLC) and in 50% (5/10) of large cell neuroendocrine carcinoma (LCNEC) whereas it was undetectable in 90% (10/11) of adenocarcinoma and 82% (9/11) of squamous carcinoma when compared to corresponding normal lung. No amplification was found but an increase in E2F1 mRNA expression was detected in 75% (18/24) of SCLC overexpressing E2F1 product. In these tumours and in contrast with NSCLC, upregulation of E2F1 product was associated with its nuclear accumulation and with overexpression of several of its target-genes. Moreover, E2F1 overexpression in NE lung tumours was significantly associated with a high KI67 index (p<0.0001) as well as a Bcl-2:Bax ratio > 1 (p<0.001). Overall, these results demonstrate a distinct pattern of E2F1 expression in human lung tumours and suggest that its deregulation could be involved in the carcinogenesis of SCLC.

Introduction

E2F1, a critical regulator of cell cycle progression, was the first identified member of a family of transcription factors generically referred to as E2F (Nevins, 1992). This protein plays a pivotal role in G 1 to S phase transition by transactivating specific targetgenes (Dyson, 1998;Helin, 1998 for review). The majority of E2F1-regulated genes encode proteins involved in DNA replication and/or in S phase progression such as DNA polymerase α, thymidine kinase (TK), dihydrofolate reductase (DHFR), Mini Chromosome Maintenance 2 to 7 (MCM), cdc6, cyclin A and cyclin E, cdc2, c-myc and E2F1 itself (DeGregori et al., 1995;Helin, 1998). E2F1 function is tightly regulated during cell cycle by the product of the retinoblastoma tumour suppressor gene (pRb). In G 1 , unphosphorylated pRb represses the transactivating activity of E2F1/DP1 heterodimeric complexes [START_REF] Flemington | Proc.Natl.Acad.Sci.USA[END_REF]Weintraub et al., 1995;Zhang et al., 1999). In late G 1 , phosphorylation of pRb by cyclin-dependent kinases (cdk) induces its dissociation from E2F1/DP1 heterodimers and progression into S phase (Mittnacht, 1998). In late S, cyclin A/cdk2 complex binds to E2F1 and phosphorylates DP1, thereby inactivating E2F1/DP1 activity.

Ectopic expression of E2F1 induces DNA synthesis in quiescent immortal rodent cells (Johnson et al., 1993;[START_REF] Qin | Proc.Natl.Acad.Sci.USA[END_REF][START_REF] Shan | [END_REF][START_REF] Shan | Proc.Natl.Acad.Sci.USA[END_REF], confers neoplastic properties to immortalized cells [START_REF] Degregori | Proc.Natl.Acad.Sci.USA[END_REF], and increases tumorigenesis in transgenic mice that lack p53 function [START_REF] Pierce | Proc.Natl.Acad.Sci.USA[END_REF]. Moreover, old transgenic mice overexpressing E2F1 developp spontaneous tumours in a variety of tissues [START_REF] Pierce | [END_REF]. Altogether, these results confer oncogenic properties to E2F1.

Paradoxically, mice lacking E2F1 also developp lymphoma or pulmonary adenocarcinoma suggesting that E2F1 can function as a tumour suppressor (Field et al., 1996;Yamasaki et al., 1996), probably via its ability to induce p53-dependent or independent apoptosis [START_REF] Wu | Proc.Natl.Acad.Sci.USA[END_REF][START_REF] Degregori | Proc.Natl.Acad.Sci.USA[END_REF]Phillips et al., 1999). Thus, the ability of E2F1 to promote cell cycle progression and tumour growth or apoptosis and tumour suppression might be tissue-specific.

Human lung cancers are divided into two different sub-classes based on the 1999 WHO classification of lung cancer [START_REF] Travis | WHO International Histological Classification of Tumours: Histological Typing of Lung and Pleural Tumours[END_REF]: (1) non small cell lung carcinoma (NSCLC) that include squamous carcinoma, adenocarcinoma and large cell carcinoma;

(2) neuroendocrine (NE) lung tumours that include carcinoid (typical and atypical), large cell neuroendocrine carcinoma (LCNEC) and small cell lung carcinoma (SCLC), the last one being the most aggressive lung tumours. Pathways involving p53 and pRb tumour suppressor genes are frequently deregulated during lung tumorigenesis. p53 is inactivated in 70% of human lung tumours either by missense [START_REF] Chiba | [END_REF]Brambilla et al., 1993) or null mutations [START_REF] Gazzeri | [END_REF]. pRb pathway is differentially disrupted in NSCLC and high grade NE tumours. Whereas loss of pRb protein expression (through mutations and/or loss of transcription) occurs in 85% of SCLC and LCNEC (Gouyer et al., 1998), pRb function is abolished in 85% of NSCLC either through loss of p16 INK4a protein expression [START_REF] Xu | [END_REF]Gazzeri et al., 1998), and/or cyclin D1 overexpression (Brambilla et al., 1997). As E2F1 is one key component of these two pathways (O'Connor et al., 1995[START_REF] Bates | p14 ARF links the tumour suppressors RB and p53[END_REF]Kowalik et al., 1998), any alterations affecting them, especially pRb inactivation, are thought to deregulate its activity. However, since E2F1 activation could contribute to either proliferation or apoptosis depending on the tissular context, the consequences of pRb inactivation on E2F1 expression and activity might differ during tumorigenesis according to each category of tumour.

E2F1 was poorly analyzed in lung tumours [START_REF] Demuth | [END_REF]Volm et al., 1998). To get further into its putative role in lung carcinogenesis, we studied E2F1 protein expression and mRNA in a series of 58 lung tumours of all histological types, in comparison with normal lung tissue. To assess E2F1 transactivating activity in each tumour category, we also studied the expression level of several of its known targetgenes. Our results identify a distinct pattern of E2F1 expression between NSCLC and SCLC and suggest that E2F1 upregulation and hyperactivity could contribute to the carcinogenesis of SCLC.

Results

Distinct pattern of E2F1 expression in human lung tumours

E2F1 protein expression was studied on a panel of 58 lung tumour tissue samples by western-blotting using two monoclonal antibodies (Table 1, Figure 1). E2F1 product was undetectable in all normal lung tissues studied (Figure 1A) and poorly detectable in 2/11 (18%) squamous carcinoma and 1/11 (9%) adenocarcinoma (Figure 1B & 1C). In contrast, when compared to normal lung, a significative overexpression of E2F1 was observed in 24/26 (92%) small cell lung carcinoma and 5/10 (50%) large cell NE carcinoma (Table 1, Figure 1D). Same results were obtained with the two anti-E2F1 antibodies used. Overall, E2F1 product was overexpressed in 29 of 36 (81%) NE lung carcinoma whereas it was undetectable in our conditions in 19 of 22 (87%) NSCLC. Immunohistochemical (IHC) analysis was performed using KH95 monoclonal antibody on 47 cases of this series where a frozen block was available. When compared to normal lung cells, overexpression of E2F1 was observed in 26/30 (86%) high grade NE tumors and in only 1/17 (6%) NSCLC, demonstrating that our unability to detect E2F1 protein by western blotting in NSCLC was not linked to a high proportion of contaminating stromal cells. IHC results were consistent with those of western blotting in 91% (43/47) of cases (Table 1, Figure 2). Four cases displayed discordance: in 3 of them where necrosis was intense, E2F1 staining was positive using IHC analysis whereas Western analysis was negative. In the last case, a faint band was observed by Western blot but tumour cells were negative in IHC analysis. Taken together, our results demonstrated a distinct pattern of E2F1 protein expression in human lung tumours.

Subcellular distribution of E2F1 in human lung tumours

As subcellular localization of E2F members could influence their activity, their interaction with target-genes as well as their degradation by the ubiquitin-proteasome pathway (Hateboer et al., 1996;Hofmann et al., 1996;[START_REF] Lindeman | Proc. Natl. Acad. Sci. USA[END_REF]Vigo et al., 1999), we next investigated the subcellular distribution of E2F1 in lung tumours on whole imprint preparations using indirect immunofluorescence in addition to classical immunohistochemistry revealed with peroxydase on tissue sections. Twenty of the 58 samples included in this study comprising 1 normal lung tissue, 5 SCLC and 5 LCNEC that overexpressed E2F1 product, 5 squamous carcinoma and 5 adenocarcinoma that expressed undetectable level of E2F1 by western blotting were analyzed. In all NSCLC tested, E2F1 product was never detected in the nucleus of tumour cells (Figure 3B).

Absence of nuclear staining was confirmed by counterstaining with Hoechst. However, we observed a faint E2F1 cytoplasmic staining in many NSCLC cells (Figure 3B) when compared to negative control. In contrast, all SCLC cells exhibited a strong nuclear staining thereby confirming overexpression of E2F1 in these set of tumours (Figure 3C).

Neither normal lung cells (Figure 3A) nor stromal cells exhibited nuclear staining, although the last cells might display some cytoplasmic staining. Same results were obtained using IHC analysis (see Figure 2).

Overexpression of E2F1 is associated with increased E2F1 mRNA transcripts

We next investigated if E2F1 protein upregulation in SCLC could be the consequence of E2F1 gene amplification and/or increase in E2F1 gene transcription.

Using Southern blotting, we did not detect any amplification of E2F1 gene in all SCLC tested when compared to normal lung tissues (data not shown). Analysis of E2F1 mRNA was performed by Northern blotting in the 24 SCLC overexpressing E2F1 protein. High levels of E2F1 transcripts were detected in 18 of those tumours (75%) when compared to normal lung tissues where E2F1 transcripts were undetectable using this technique (Figure 4). In the 6/24 remaining cases, E2F1 transcriptional level was undetectable as in normal lung despite high level of E2F1 protein expression.

Overexpression of E2F1 correlates with upregulation of some of its target-genes

To further analyze E2F1 activity in lung tumours, we studied by western blot analysis the expression of several well-known E2F1-target genes. When compared to normal lung tissues (Figure 5A), high levels of cyclins A and E, cdc2 and DHFR products were detected in 22/24 (90%) SCLC and 5/5 (100%) LCNEC that overexpressed E2F1 (Figure 5B). In one case (4%) SCLC, cyclin A was undetectable despite overexpression of other target-genes. In contrast, expression of these four proteins was weak in 17/19 (95%) NSCLC that exhibited undetectable E2F1 product (Figure 5C & 5D). Again, in 3/11 (27%) squamous lung carcinoma, cyclin A was slightly detectable despite low level of E2F1 expression (Figure 5D; lanes 9-11) suggesting that additional factors might regulate its expression in this subset of tumour. Taken together, these results indicated that upregulation of E2F1 protein expression in SCLC was associated with overexpression of some E2F1-targets genes.

Relationships between E2F1, p53, pRb and hp14 ARF status in lung carcinoma

Status of p53, pRb and hp14 ARF had been previously analyzed in this series of tumours (Brambilla et al., 1993;[START_REF] Gazzeri | [END_REF]Gazzeri et al., 1998a;Gazzeri et al., 1998b;Gouyer et al., 1998). In these tumours, pRb function was inactivated, either by loss-of-function mutations or p16 INK4a loss and/or cyclin D1 overexpression in 29/36 (80%) NE tumours and 18/22 (82%) NSCLC respectively. However, we did not find any correlation between the status of E2F1 and pRb inactivation in all tumours studied (data not shown). There was no inverse nor direct correlation linking E2F1 and hp14 ARF or p53 expression in any histological tumour class. However, we noticed that overexpression of E2F1 was frequently associated with inactivation of hp14 ARF [as detected by negative immunostaining (Gazzeri et al., 1998b)] (20/36, 69%) and/or of p53 [as indicated by p53positive immunoreactivity [START_REF] Gazzeri | [END_REF]] (18/36, 50%) in NE lung tumours (Table 2).

Correlation between E2F1 , KI67 and Bcl-2:Bax levels in human lung carcinoma

In human lung tumours, E2F1 might mediate either apoptosis or cellular proliferation depending on the context. Thus, we finally investigated in each tumoral subtype, the relationships between E2F1 protein expression, the well-known proliferative index KI67 (Gerdes et al., 1984) and the equilibrium of two main factors of cell susceptibility to apoptosis, Bcl-2 and Bax (Brambilla et al., 1996). KI67 proliferative index was significantly higher in NE lung tumours when compared to NSCLC (44 ± 19 versus 21 ± 13; p<0.0001; Table 3). Furthermore, a high KI67 index correlated with overexpression of E2F1 product in all histological subtypes (p<0.0001).

Susceptibility to apoptosis was investigated through IHC analysis of both Bcl-2 and Bax products. Indeed, we had previously demonstrated that a Bcl-2:Bax ratio > 1 correlated with a lower apoptotic index in NE lung tumours (Brambilla et al., 1996). We showed that Bcl-2 mean score was significantly higher in NE lung tumours when compared to NSCLC (202 ± 109 versus 41 ± 54; p<0.0001; Table 3). Furthermore, a Bcl-2:Bax ratio > 1 was significantly associated with NE phenotype (p<0.001) and E2F1 overexpression (p<0.001) whereas NSCLC exhibited a Bcl-2:Bax ratio < 1 and low level of E2F1 product (Table 3). Overall, these results strongly suggest that E2F1 overexpression in high grade neuroendocrine lung tumours might mediate cellular proliferation rather than apoptosis.

Discussion

We have investigated E2F1 protein expression in a series of 58 human lung tumours and demonstrated that NSCLC and NE carcinoma displayed distinct pattern of E2F1 expression. Whereas E2F1 product was undetectable by western blot analysis and IHC in nearly all NSCLC analyzed, it was upregulated in high grade NE lung tumours. As we previously showed that pRb function was invalidated (either by mutations or loss of p16 INK4a and/or cyclin D1 overexpression) in 80% of all lung tumours tested in this study (Gazzeri et al., 1998a;Gouyer et al., 1998), our present data suggest that inactivation of pRb pathway does not predict deregulated levels of E2F1 expression, at least during lung tumorigenesis.

To get clues onto the mechanism of E2F1 upregulation in SCLC, we analyzed DNA amplification and mRNA expression. Although amplification was not found, we detected an increase in E2F1 mRNA expression in the majority of those tumours overexpressing E2F1 protein. An increase in E2F1 transcription normally occurs in late G 1 in response to growth stimuli [START_REF] Sherr | [END_REF] but can also be detected in response to oncogenic stimuli such as myc (Leone et al., 1997). Moreover, E2F1 has been reported to stimulate its own transcription (Johnson et al., 1994;Neuman et al., 1994). These data and the present study suggest that, in SCLC, deregulated oncogenic signals could induce a primary increase of E2F1 gene transcription further amplified by protein accumulation.

Interestingly, some tumours exhibited a high E2F1 protein level despite absence of mRNA overexpression. E2F1 protein level is tightly controlled by ubiquitin-proteasome mediated proteolysis [START_REF] Campanero | Proc. Natl. Acad. Sci. USA[END_REF][START_REF] Vandel | [END_REF] and association with pRb has been shown to protect E2F1 from degradation (Hateboer et al., 1996;Hofmann et al., 1996). Since pRb was lost in 80% of the NE tumours studied, E2F1 accumulation in these tumours is probably not the result of protection by pRb. Additionally, the protein p45 SKP2 was recently shown to directly interact with E2F1 and to negatively regulate its stability [START_REF] Marti | [END_REF]Wade-Harper and Elledge, 1999). We have studied p45 SKP2 protein expression by IHC in our series of tumours but we could not find any correlation between p45 SKP2 and E2F1 expression (data not shown). Thus, it remains to elucidate if accumulation of E2F1 in SCLC could be associated with a defect in its proteolysis.

We observed E2F1 accumulation in the nuclei of SCLC tumoral cells that was consistent with the expression of an active E2F1 product (de la Luna et al., 1996;Allen et al., 1997) and found a strong correlation between the expression of E2F1 and several of its target-genes including cyclins A and E. Interestingly, in SCLC overexpressing E2F1, we also detected heterodimeric complexes between E2F1 and DP1 or E2F1 and mdm2 (data not shown). Since E2F1 transactivating activity is highly enhanced upon dimerization with DP1 (Wu et al., 1995) and binding of mdm2 (Martin et al., 1995), our results highly suggest that E2F1 is not only upregulated but also transcriptionally active in SCLC. In contrast and interestingly, absence of E2F1 nuclear staining correlated with weak level of E2F1 target-genes expression in NSCLC. Thus, despite loss of pRb repression, free E2F1 product might be transcriptionally inactive in this subset of tumors.

Therefore, what might be the biological consequences of E2F1 expression in lung carcinogenesis? During tumorigenesis, E2F1 has been implicated both as an oncogene (Johnson et al., 1993;[START_REF] Qin | Proc.Natl.Acad.Sci.USA[END_REF][START_REF] Pierce | Proc.Natl.Acad.Sci.USA[END_REF][START_REF] Pierce | [END_REF] or a tumour suppressor gene (Field et al., 1996;Yamasaki et al., 1996;[START_REF] Pierce | [END_REF] depending on the context and on its ability to induce cell proliferation and/or p53-dependent orindependent apoptosis. In lung tissues, we noticed that E2F1 protein expression displayed opposite patterns between NSCLC and SCLC making attractive the possibility that this protein could play a distinct role during tumorigenesis of these two tumour subsets. Since E2F1 knock-out mice developp pulmonary adenocarcinoma (Field et al., 1996), its inactivation in NSCLC could contribute to a defective apoptosis of tumoral cells.

Conversely, unbalance between apoptosis and cell proliferation could be implicated in the tumorigenesis of SCLC. In this subset of tumour, we observed a correlation between E2F1 expression and KI67 proliferative index suggesting that E2F1 might be involved in their high proliferative pattern. In contrast, the ability of E2F1 to induce apoptosis might be counteracted. Consistent with this, we previously showed that p53 is inactivated and/or hp14 ARF lost in most SCLC (Gazzeri et al., 1998b) and although statistically not significant, a correlation between E2F1 overexpression and p53 inactivation and/or hp14 ARF loss was detected in SCLC. As E2F1 can induce p53-dependent apoptosis via its ability to transactivate hp14 ARF [START_REF] Bates | p14 ARF links the tumour suppressors RB and p53[END_REF], inactivation of p53 or hp14 ARF could give an advantage to the proliferative effects of E2F1 versus its pro-apoptotic functions.

Moreover, as we observed that high grade neuroendocrine lung tumours including SCLC expressed a Bcl-2:Bax ratio > 1 (Brambilla et al., 1996 and this study), Bcl-2 could further inhibit the apoptosis of SCLC tumor cells overexpressing E2F1 and contribute to their proliferation. According to this, sensitivity of oesophageal cancer cells to E2F1mediated apoptosis was recently related to differential expression of Bcl-2 family member proteins (Yang et al., 2000).

Overall, our data identify a distinct pattern of E2F1 protein expression in human lung tumours and demonstrate its upregulation in SCLC, the most proliferative and aggressive lung tumours. In these tumours, E2F1 could therefore act as an oncogene being an essential actor of uncontrolled cellular proliferation, for example by counteracting the negative effects of cyclin-cdk inhibitors. This could explain why these tumours are so actively proliferative despite their high content in p16 INK4a (Kelley et al., 1997; data not shown) and p27 Kip1 products (Yatabe et al., 1998;data not shown). These findings have obvious conceptual implications in the promising strategic therapy targeting E2F1 function. They should aim at neutralizing E2F1 production in SCLC in order to inhibit their proliferation and inducing its expression in NSCLC in order to restitute apoptosis.

Material and methods

Tissue samples

Tissue samples were taken at surgical resection of lung tumours or node metastases at mediastinoscopy. They were immediately frozen and kept at -80°C until use. They consisted of 11 squamous carcinoma; 11 adenocarcinoma; 10 LCNEC and 26 SCLC according to the 1999 WHO international histological classification of lung tumours [START_REF] Travis | WHO International Histological Classification of Tumours: Histological Typing of Lung and Pleural Tumours[END_REF]. 300 cells count. Bcl-2 and Bax immunostaining were performed as previously described (Brambilla et al., 1996) using a Bcl-2 monoclonal antibody from Dako (1/100) and a Bax polyclonal antibody N-19 from Santa-Cruz (1/200). Scores of immunostaining were calculated by multiplying the percentage of labeled cells with the intensity (1+, 2+, 3+) of staining for a total score gain from 0 to 300. The Bcl-2/Bax ratio was established by dividing the 2 total scores.

Indirect immunofluorescence

Whole imprint preparations were performed by gentle pressing tumoural frozen tissue to silane-coated glass slides, air dried and stored at -80°C until use. Slides were treated for 10 min at room temperature in ice-cold nuclei isolation buffer [5 mM HEPES (pH 8), 50 mM KCl, 10 mM MgSO 4 , and 3 mM DTT], incubated at -20°C during 10 min in nuclei isolation buffer supplemented with 0.25% Triton 100X, washed twice with PBS and fixed for 5 min in acetone at -20°C. After fixation, slides were washed, saturated and incubated overnight with primary antibody (anti-human E2F1, Ab-4, 1/200) at 4°C. After three PBS washes, slides were exposed to Alexa TM 488 goat anti-mouse-IgG (H+L) conjugate (2 mg/ml; 1/500, Interchim, Montluçon) for 30 min at room temperature and washed twice in PBS. Normal mouse IgG at the same concentration as the primary antibody served as a negative control. The slides were counterstained with Hoechst and preparations were observed using an Olympus microscope (20x magnification), captured by a Coolview CCD camera (Photonic Science) and digitally saved using Visilog. The images were further processed by using Photoshop (Adobe).

Western-blot analysis

Tissues samples were lysed in ice-cold lysis buffer [5 mM EDTA, 150 mM NaCl, 100mM

Tris (pH 8), 0.5% Na deoxycholate, 0.5% NP40, 0.5% SDS, 0.1% aprotinin, 2 µg/ml leupeptin, 2 µg/ml pepstatin, and 1 mM PMSF] for 30 min and centrifugated (30 min, 15 000g). In order to ensure appropriate detection, a molecular weight marker was loaded onto each gel. Proteins (40 µg) were denatured in Laemmli buffer [60 mM Tris HCl (pH 6.8), 20% glycerol, 10% ß-mercaptoethanol, 4.6% SDS, and 0.003% bromophenol blue], separated by 10-12% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) and electroblotted on PVDF membrane (Hybond P, Amersham, Les Ulis, France). The membrane was then incubated for 2-3 h at room temperature with primary Ab in 2% nonfat milk TPBS, washed three times, incubated with horseradish peroxydase-conjugated goat anti-mouse or anti-rabbit Abs (Jackson ImmunResearch Laboratories , West Grove, PE) for 30 min and revealed using enhanced chemoluminescence detection kit (ECL; Amersham).

Northern Blot Analysis

Total RNA was extracted according to the method of Chomczynski and Sacchi (Chomczynski and Sacchi, 1987). 10 µg of total RNA were fractionated by electrophoresis in 1% agarose-formaldehyde gels and transferred onto a nylon membrane (Hybond N, Amersham). Equivalent RNA loading was verified by ethidium-bromide staining of gels before transfer onto membranes. A Bam H1-1.35-kb full lenght human E2F1 cDNA fragment (kindly provided by Dr. K. Helin, Milan) was used as a probe and was radiolabeled using the random primed DNA labeling kit (Roche Diagnostics). The membranes were prehybridized and hybridized in 50% formamide, 5X Denhardt's solution, 50 mM phosphate buffer (pH 6.5), 5X SSC, 0.1% SDS, and 2.5 µg/ml salmon sperm DNA for 42 h at 42°C. The most stringent final wash was for 20 min at 56°C in 0.2X SSC, and 0.1% SDS. Membranes were exposed for autoradiography at -80°C for at least 3 days.

Aknowledgements

Legends to figures

  This work was supported by grants from the Association pour la Recherche sur le Cancer, from the Comité Départemental de la Ligue contre le Cancer de l'Isère and from the Groupement des Entreprises Francaises dans la Lutte contre le Cancer. B.E. was supported by INSERM (Poste Accueil).

Figure 1 .

 1 Figure 1. E2F1 expression in normal lung tissues, small cell lung carcinoma (SCLC) and

Figure 2 .

 2 Figure 2. E2F1 immunostaining of lung cancer tissue on frozen sections using anti-E2F1

Figure 3 .

 3 Figure 3. Indirect immunofluorescence study of E2F1 subcellular localization. E2F1

Figure 4 .

 4 Figure 4. Expression of E2F1 transcripts by northern blot analysis. Some representative

Figure 5 .

 5 Figure 5. E2F1, cyclin A, cyclin E, DHRF and cdc2 protein expressions in normal lung

Antibodies

Monoclonal antibodies (mAbs) included anti-cdc2p34 (sc-54, Santa-Cruz Biotechnology, Santa Cruz, CA), anti-cyclin E (clone HE12, Euromedex, Souffelweyersheim), anti-DHFR (Dihydrofolate Reductase; clone 49, Transduction Laboratories, Lexington, KY) and two anti-human E2F1 (clone Ab-4, Neomarkers and clone KH95, Pharmingen).

Rabbit polyclonal antibodies included anti-actin (20-33, Sigma-Aldrich, L'Isle d'Abeau) and anti-cyclin A (C-22, sc-160, Santa-Cruz).

Immunohistochemistry (IHC)

E2F1, KI67, Bcl-2 and Bax immunostaining were performed on frozen sections. For E2F1 immunostaining, two monoclonal E2F1 antibodies KH95 (Santa-Cruz; 1/100) and Ab-4 (Neomarkers; 1/50; data not shown) were used. Three step immunohistochemical method was applied on 5 µm thick frozen sections taken in the immediate vicinity of samples used for western-blot and immunoprecipitation studies. After E2F1 primary antibodies, the secondary antibody was anti-mouse (1/1250) and third step was ABC complex (Streptavidin-Biotin peroxidase; Dako). Cases were recovered as positive when at least 20% of tumour cells showed obvious nuclear staining. Only nuclear staining was considered to assess immunoreactivity. Cytoplasmic staining was never considered in the assessment of E2F1 expression. It was observed in addition to nuclear staining in a subset of squamous carcinoma and adenocarcinoma. Normal lung cells, lymphocytes and vessels were all negative. KI67 immunostaining was performed using a mouse monoclonal antibody from Immunotech (1/100). KI67 percentage of positive cells was established on