Daria Terescenco
email: daria.terescenco@univ-lehavre.fr

Geraldine Savary
email: geraldine.savary@univ-lehavre.frflorence.clemenceau@airliquide.com

Celine Picard
email: celine.picard@univ-lehavre.fr

Florence Clemenceau

Emmanuelle Merat

Michel Grisel
email: michel.grisel@univ-lehavre.fr

Influence of the emollient on emulsions containing lamellar liquid crystals: from molecular organization towards applicative properties

Keywords: Emulsion / Formulation / stability / liquid crystals/ α-gel / emollients / molecular organization / applicative properties alkyl polyglucoside CCT, caprylic capric triglycerides Cosmetic, Toiletry and Fragrance Association DTG, thermogravimetric derivative IHD, isohexadecane PMMA, poly(methyl methacrylate) SD, standard deviation SLS, static light scattering TGA, thermogravimetric analysis WAXD, wide-angle X-ray diffraction

The sensory perception of cosmetic emulsions is complex as it is governed by an important number of parameters like the choice of raw materials, their interactions, the structural organisation of the system, etc.

The aim of the present work was to go further in the interpretation of the emollient-surfactant interactions, towards the emulsions applicative properties. For this purpose, two systems containing liquid crystals of the lamellar type were formulated, differing only in the selected emollient.

METHODS:

First, the liquid crystals types were checked using different tools like the optical microscopy under the bright and polarized light, the wide-angle X rays diffraction and, finally, thermogravimetric analysis.

Next, two sensory attributes, namely compression force and difficulty of spreading, were evaluated by a sensory panel. In addition to that, complementary instrumental characterizations (flow tests, textural analysis and contact angle measurements) were performed in order to understand how the panel could discriminate the products.

RESULTS:

The results showed that isohexadecane emollient induces the α-gel structures, while caprylic capric triglycerides favour the formation of the lamellar liquid crystals near to α-gel.

For the compression force, the results point out that there is no direct interaction between the oil phase and the skin. For this attribute, depending on its chemical structure, emollient impacts the human perception only by changing the lamellar phase type. Concerning the difficulty of spreading, both the emulsion structure and the emollient properties should be considered. Immediate perception is impacted by the emulsions destruction, making the droplet roll one on each other. Then, once the droplets monolayer is disrupted, the emollient comes into direct contact with the skin. In this case, the perception is governed by the direct affinity of the emollient with the skin, nonpolar emollients being easier to spread if compared to polar ones.

CONCLUSION:

The sensory perception is guided not only by the choice of the raw materials but also by their interactions. It was shown that the chemical structure of the emollients affected the molecular organization of liquid crystals present in the emulsion and, consequently, directly or indirectly its sensory perception.

Introduction

Most of the skin care formulations are of emulsion type. An emulsion is a system in which droplets of a liquid are dispersed in a second liquid, the two liquids being partially or totally immiscible (water/oil) [START_REF] Knowlton | Handbook of Cosmetic Science and Technology[END_REF]. One of the most efficient ways to stabilize an emulsion is the use of the surfactant, by means of the interfacial tension decrease. Moreover, depending on its structure, the surfactant may organize itself in liquid crystals of either hexagonal, lamellar or cubic type [START_REF] Tadros | Phase behaviour of surfactant systems[END_REF]. These structures can provide a better stability to the system and are able to change its microscopic and macroscopic properties. But at the same time, the liquid crystals organization can be affected by other ingredients present in the formulation.

Our previous study [START_REF] Terescenco | Influence of the emollient structure on the properties of cosmetic emulsion containing lamellar liquid crystals[END_REF], based on the surfactant of the alkyl polyglucoside (APG) type, demonstrated its ability to form lamellar liquid crystals. Several types of lamellar phases, L α , L β , L γ , L β′ , L δ , L s , and P β′ were observed in lyotropic phase diagrams [START_REF] Tardieu | Structure and polymorphism of the hydrocarbon chains of lipids: A study of lecithin-water phases[END_REF] but two main lamellar organizations are prevailing: lamellar liquid crystals (L α ) and α-gel (L β ). The alkyl polyglucoside surfactant, Cetearyl alcohol (C 16 -C 18 OH) / Cetearyl glucoside (C 16 -C 18 APG), forms exclusively α-gel in binary systems (composed only of water and surfactant). In this case, the hydrocarbon chains remain stiff, perpendicular to the plane of the lamellae, with axes organized as a two-dimensional hexagonal lattice [START_REF] Neto | The physics of lyotropic liquid crystals : phase transitions and structural properties[END_REF]. Considering together the theory and our previous results, the next step of our work consisted in the investigation of the behaviour of the lamellar organization through the addition of the emollient to the binary system.

Emollients are defined by CTFA dictionary as "Cosmetic ingredients which help to maintain the soft, smooth, and pliable appearance of the skin. Emollients function by their ability to remain on the skin surface or in the stratum corneum to act as a lubricant, to reduce flaking, and to improve the appearance of the skin" [START_REF] Wenninger | International Cosmetic Ingredient Dictionary and Handbook[END_REF]. Due to their properties, emollients are part of the emulsions oil phase. Different studies [START_REF] Chorilli | Structural characterization and in vivo evaluation of retinyl palmitate in non-ionic lamellar liquid crystalline system[END_REF][START_REF] Lodén | Role of Topical Emollients and Moisturizers in the Treatment of Dry Skin Barrier Disorders[END_REF][START_REF] Stojiljković | Investigation of the effects of different emollients on the structure and skin moisturizing potential of the cosmetic creams[END_REF][START_REF] Savary | Impact of emollients on the spreading properties of cosmetic products: A combined sensory and instrumental characterization[END_REF] demonstrated that the chemical structure and the polarity of emollients affect emulsions organisation, sensory properties as well as interactions with the skin. As a function of their chemical structure, they can be either "nonpolar" (e.g. paraffin and isoparaffin) or "polar" substances (e.g. esters and triglycerides), although this polarity parameter is seldom determined or given [START_REF] Zocchi | Skin Fel Agents. Handbook of Cosmetic Science and Technology Second Edition[END_REF].

In previous work, seven studied emollients were divided into two groups depending on the presence of heteroatom in their chemical structure. Heteroatom-free oils were more likely to keep the α-gel organization in the system. But once a heteroatomcontaining emollient is introduced to the matrix, a complementary, lamellar liquid phase (L α ) is formed. In the L α phase, the hydrocarbon chains of the surfactant are in a liquid-like state [START_REF] Neto | The physics of lyotropic liquid crystals : phase transitions and structural properties[END_REF]. As a consequence, we could establish that both the microscopic and macroscopic properties of the emulsions are strongly related by the emollient structure.

The objective of the present study is to go further in the exploration of the lamellar phases properties formed in cosmetic emulsions through the applicative characterization. For this purpose, two systems were considered. The first one was prepared using isohexadecane as emollient, to assure the α-gel formation. The second one was formulated with caprylic capric triglycerides, a polar emollient with a heteroatomic structure. In addition to the α-gel, this second emulsion also contained lamellar liquid crystals. Two sensory attributes were selected to describe the applicative properties of these emulsions, namely: compression force and difficulty of spreading. These attributes were evaluated using both sensory and instrumental approach. Then, interdependency between the emulsions organization and its applicative properties were investigated and discussed.

Materials and methods

Materials

Cetearyl Glucoside and Cetearyl alcohol mixture, used as emulsifier, as well as the Phenoxyethanol and Methylparaben and Ethylparaben and Propylparaben and Butylparaben, used as preservative, were provided by SEPPIC, France.

Isohexadecane and Caprylic capric glycerides were used as emollients. Their properties were already investigated [START_REF] Terescenco | Influence of the emollient structure on the properties of cosmetic emulsion containing lamellar liquid crystals[END_REF] and the main results, describing their density, surface tension and interfacial tension values are resumed in Table I.

Methods

Emollients contact angle measurement

The test was performed using a portative goniometer PGX+ (ScanGaule, Gravigny, France) connected to the PGPlus software. It is equipped with a high-resolution camera to acquire images, with a specific lighting system associated with a mirror to visualize the liquid droplet deposited onto the surface. A syringe (Ø=0.77mm) was used to depose the first droplet on the skin surface and its volume was increased by the addition of five successive drops to approximately 7µL. A photograph was taken after each liquid addition and the advanced contact angle was determined on the identified triple point (intersection of the liquid, solid and vapour phases using the software). Advancing contact angle was measured at least in triplicate for each emollient.

Emulsions formulation

Emulsions were prepared as follows: first, the oil phase (20% of emollient) and 5% of emulsifier were heated up to 75°C under mechanical stirring (400rpm); then, the oily phase was added to the water phase previously heated at 75°C. The mixture was then homogenized at 10,000 rpm for one minute using a T25 digital ultra-turrax (IKA, Freiburg, Germany) equipped with the dispersing head S25N-25F. Finally, the formulations were left cooling down to 30°C under mechanical stirring (400rpm) and then the preservative was added. Once prepared, the mixtures were split into three samples, stocked at 4°C, 40°C and ambient temperature, respectively, for stability monitoring.

Optical microscopy

The emulsions microstructure was observed without sample dilution by means of a photomicroscope (Leica DMLP microscope) equipped with a digital camera at a magnification of x200: first, under the bright field and secondly, under the polarized light (using cross-polarizers) in order to evidence the presence and characterize the liquid-crystal phases. Leica IM 1000 software was used to analyse the micrographs.

Droplet size distribution

The emulsion droplets size were measured by static light scattering (SLS) using a laser diffraction particle size analyser SALD-7500 nano (Shimadzu Co., Ltd, Japan) equipped with a violet semiconductor laser (405 nm) and a reverse Fourier optical system (the beam converge before encountering the sample). The emulsions were diluted with deionized water to achieve the absorption parameter equal to 0.2. Once introduced into the batch cell (7 cm 3 ), the emulsions were stirred during the measurement to assure homogenous dispersion. Data were analysed using software Wing SALD II and the droplets mean values for each emulsion were acquired.

Rheological measurements

A continuous flow ramp was performed using a controlled stress rheometer (HR1, TA instruments).

Emulsions analysis.

Flow curves were obtained by recording shear stress and viscosity values at increasing shear rates ranging from 0.001 to 8000s -1 (continuous ramp, logarithmic mode) for 300s. A cone-plate aluminium device was used, with a diameter of 40 mm, a cone angle of 1°59'38'' and a gap of 47µm.

Emollients analysis. Flow properties were obtained by recording shear stress and viscosity values at increasing shear rates ranging from 0.1 to 50000 s -1 (continuous ramp, logarithmic mode) for 300 s. For this test, an aluminium plane geometry (60 mm) was used.

All the measurements were carried out in duplicate at 25°C. The solvent trap was used to prevent sample drying. Once loaded, the samples were left at rest for two minutes prior to any measurement. Samples were analysed once and then changed prior to further analysis.

Wide-angle X-ray diffraction (WAXD)

Samples were analysed in a reflection mode by X-ray diffraction (XRD) using a PANalytical Xpert Powder diffractometer (PANalytical B.V., Almelo, The Netherlands) with a CoK a (λ = 1.7902 Å) monochromatic radiation source, and operating voltage and current maintained at 40 kV and 40 mA, respectively. A 15 mm mask was used. The detector was a linear PIXcel1D detector. Data was acquired by steps of 0.02° for 2θ values ranging from 2 to 50°. The formulations were pressed under Capton foil, avoiding air bubbles.

From diffraction angle theta (θ) the interlayer spacing was calculated according to Bragg's law.

Thermogravimetric analysis

Thermogravimetric analysis (TGA) was performed on a Setsys apparatus (Setaram, Caluire-et-Cuire, France) with the Setsoft software. Around 35 mg of emulsion were weighed and heated under the air atmosphere by the following procedure:

-Isotherm: 300 sec at 25°C; -Ramp: 25°C to 130°C at 2°C/min; -Isotherm: 120 sec at 130°C.

The weight loss of the sample is registered as a function of temperature (% mass loss/ total mass). The first derivative of the TGA curve (the DTG curve) was also plotted to visualise the water evaporation dynamics (%/min).

Texture analysis

Texture analysis was performed using a TA.XT Plus (Stable Micro Systems, Cardiff, UK). The apparatus was equipped with a friction module A/FR (ASTM-D 1894-90) to perform the spreading test. The protocol was adapted from Gilbert [START_REF] Gilbert | Caractérisation physico-chimique et sensorielle d'ingrédients cosmétiques: une approche méthodologique[END_REF] and it represents the spreading of 200µL of the cream between two surfaces, polypropylene plastic sheet and Helioplate ™ HD 2 (Helioscreen, Marseille, France), over 120mm distance and at a constant speed (3mm/sec). The force required to spread the sample is registered as a function of time.

The area under the curve A+ (g.sec), describing the difficulty to spread the product was then calculated. Also, the slope of the force curve was estimated, to interpret the evolution of the spreading of the product function of time. Finally, the affinity of the product with the polypropylene surface was evaluated through the shape of the force curve, namely through the calculation of the noise amplitude. Three repetitions were performed for each sample.

Sensory analysis

Two attributes corresponding to different phases of a cosmetic emulsions application were evaluated (Table II). A positive displacement pipette equipped with capillary pistons, Microman, was used to deliver an exact amount of the emulsion on the skin.

The recruited panel was composed of 10 volunteers aged from 22 to 36 years with Caucasian skin type. Two training sessions were organized for each attribute to acquire the definition, the evaluation procedure, the rating scale and the commercial references. Then, each product was evaluated twice.

Table II. Definitions and scales used to describe the selected attributes (compression force and difficulty of spreading) for the sensory analysis.

Phases Attributes Definitions Scales

Pick-up Compression force

Force required to fully compress product between thumb and forefinger 0 (soft) → 7 (hard)

Rub-out

Difficulty of spreading Force required to move the product over the skin 0 (soft) → 7 (hard)

Data analysis

Results were presented as the mean value ± standard deviation (SD).

XLSTAT software (Addinsoft, Paris, France) was used to perform the statistical analyses of the collected data. The ANOVA test (two-way analysis of variance) was applied to the results to spot the significant differences between the emulsions (P<0.05). Mean intensities were compared using the Tukey multiple comparison tests to distinguish different groups of products.

Results and discussion

Evidence of the presence of liquid crystals in the emulsions

The first part of the present work focuses on the detailed characterization of the emulsions. It should be mentioned that in the previous study the quantity of the APG surfactant was higher than usual in order to induce an abundant presence of liquid crystals. Here, the main goal was to obtain systems at limited emulsifier level for a topical application but still keeping α-gel organization. Therefore, Cetearyl alcohol/Cetearyl glucoside emulsifier was fixed at 5% near to 20% of emollient in emulsions containing: isohexadecane (EM_IHD) or caprylic/capric triglycerides (EM_CCT). The occurrence of liquid crystals structures was checked with several analyses as presented afterwards.

Microscopy and static light scattering

Firstly, the two distinct emollient containing emulsions show classical oil droplets dispersed in the continuous aqueous phase. As visible in Fig. 1, the emulsion prepared with non-polar oil IHD owns larger droplets when compared to the one prepared with polar emollient CCT. This result is consistent with previous observations obtained with higher surfactant content, thus confirming that oil polarity governs the emulsion's microstructure [START_REF] Terescenco | Influence of the emollient structure on the properties of cosmetic emulsion containing lamellar liquid crystals[END_REF]. Liquid crystals formation was confirmed through the microscopy under the polarized light observations. The "onion rings" observed for both emulsions undoubtedly pointed at the organization of the surfactant in lamellas [START_REF] Fairhurst | Lyotropic surfactant liquid crystals[END_REF][START_REF] Müller-Goymann | Liquid crystals in drug delivery[END_REF]. As expected, these structures were formed due to the presence of alkyl polyglucoside and fatty alcohol mixed emulsifier, which is known for favouring the lamellar phase formation [START_REF] Savic | The impact of urea on the colloidal structure of alkylpolyglucoside-based emulsions: Physicochemical and in vitro/in vivo characterization. Colloids in Cosmetics and Personal Care[END_REF][START_REF] Savić | An alkylpolyglucoside surfactant as a prospective pharmaceutical excipient for topical formulations: The influence of oil polarity on the colloidal structure and hydrocortisone in vitro/in vivo permeation[END_REF]. Next, a wide angle X-rays analysis was performed to access the molecular organization of the matrix and to interpret the extent of the surfactant -emollient interactions.

Wide angle X-rays diffraction

As explained earlier, the two main lamellar organizations are lamellar liquid crystals (L α ) and α-gel (L β ), respectively. In the L α phase, the hydrocarbon chains are in a liquid-like state while in the L β phase, the hydrocarbon chains remain stiff, perpendicular to the plane of the lamellae, with the axes organized as a two-dimensional hexagonal lattice [START_REF] Neto | The physics of lyotropic liquid crystals : phase transitions and structural properties[END_REF]. Both organizations may be efficiently observed by WAXD [START_REF] Eccleston | Synchrotron X-ray investigations into the lamellar gel phase formed in pharmaceutical creams prepared with cetrimide and fatty alcohols[END_REF].

Fig. 2 depicts the WAXD patterns for both EM_IHD and EM_CCT emulsions. The most representative sharp peak at 0.412 nm corresponds to α-gel as reported in the literature [START_REF] Fairhurst | Lyotropic surfactant liquid crystals[END_REF][START_REF] Kalnin | Monitoring fat crystallization in aerated food emulsions by combined DSC and time-resolved synchrotron X-ray diffraction[END_REF]. Meanwhile, the presence of the lamellar liquid crystals (L α ), with "fluid-like" characteristics, can also be evidenced at 0.46 nm [START_REF] Rand | X-Ray diffraction study in water of lipids extracted from human erythrocytes[END_REF][START_REF] Larsson | Crystal and liquid crystal structures of lipids[END_REF], but only for EM_CCT. Thus, these results confirm that there are significant differences at the molecular level between the samples depending on the oil polarity. Although both emulsions contain α-gel structures, the lamellar liquid phase is only present in case of "polar" emollients (containing heteroatoms). Such results are of primary importance to whom interested in understanding the liquid crystals occurrence in emulsions; thus, the following explanations, together with schematic representation for the corresponding liquid crystals structures, are proposed. IHD emollient being completely hydrophobic, mainly inserts between the alkyl part of the surfactant layer (Fig. 3a), with no or fair disruption for the alkyl chains organization as illustrated in Fig. 3b. On the contrary, for CCT sample, interactions with the surfactants polar head occur, in addition to the nonpolar interactions with the surfactant alkyl part, causing reorganization for the surfactant-emollient system. Thus the formation of lamellar liquid phase is induced as schematized on Fig. 3c. To sum up, the above results confirmed the occurrence of surfactant lamellar liquid crystals for the emulsions containing polar emollient. The effect of the emollients chemical structure on the α-gel thus remains unchanged when compared to previous results obtained with higher surfactant content. As expected, such structural differences do imply different microscopic and macroscopic properties for the corresponding emulsions as evidenced through rheological tests. A higher elastic modulus G' for the α-gel containing samples (G'EM_IHD = 820±11 Pa) versus those possessing complementary lamellar liquid crystals (G'EM_CCT = 556±8 Pa) was reached.

Nevertheless, it is important reminding that water remains the main component for the studied emulsions. Although water plays a key role in the continuous phase properties, it may also be entrapped inside the surfactant-oil lamellar organization.

Therefore, it is crucial to fully understand the water distribution within the emulsion matrix. The different types of water were investigated through thermogravimetric analysis experiments.

TGA analysis

Several examples in the literature evoke thermogravimetric technique as a good methodology to describe the types of water present in emulsified systems [START_REF] Prestes | Rheological measurements and thermal characterization of lamellar gel phase emulsions developed with cetearyl alcohol/nonionic ethoxylated surfactants[END_REF][START_REF] Nesseem | Formulation and evaluation of itraconazole via liquid crystal for topical delivery system[END_REF][START_REF] Savic | Colloidal microstructure of binary systems and model creams stabilized with an alkylpolyglucoside non-ionic emulsifier[END_REF], all being based on the work by Junginger et al [START_REF] Junginger | The ratio of interlamellarly fixed water to bulk water in O in W creams[END_REF]. The authors described the thermal behaviour of an emulsion containing crystalline gel structures. Three distinct steps of water evaporation were specified in this work depending on the corresponding temperature range:

-Up to 56°C: bulk water evaporation; water mechanically entrapped in the cetostearyl alcohol gel-network, -Between 56°C (melting of the cetostearyl alcohol monohydrate) and 72°C: evaporation of the semihydrate water from cetostearyl alcohol and of hydrophilic gel phase, associated with the "secondary" water, -Above 72°C: evaporation of interlamellar fixed water after melting of hydrophilic gel phase.

In our case, unlike the Junginger example, the water evaporation occurred in a single step for both emulsions. Inspired by the literature examples based on the same surfactant [START_REF] Savic | Colloidal microstructure of binary systems and model creams stabilized with an alkylpolyglucoside non-ionic emulsifier[END_REF][START_REF] Savić | Vehicles based on a sugar surfactant: Colloidal structure and its impact on in vitro/in vivo hydrocortisone permeation[END_REF], we have also estimated the water loss as a function of three temperature ranges of evaporation. The water evaporation percentages of two emulsions depending on the studied zone are presented in Table III. Water samples were analysed in the same conditions and lead to the similar water loss profiles as emulsions (Table III). It should be mentioned that the third stage of EM_IHD is also influenced by the beginning of the IHD evaporation. It is interesting to notice that the pure water evaporation was less pronounced during the two first ranges compared to emulsions evaporation. As profile was similar for each sample, the slopes of the TG curves were also calculated between 30°C and 100°C to estimate the evaporation speed over the analysed temperature ramp.

The IHD sample shows higher evaporation speed, with the slope value equal to -1.231 ± 0.066. The result is closer to the pure water evaporation -1.237 ± 0.055, when compared to the CCT sample that possesses an evaporation rate equal to -1.141 ± 0.015. If transposing these results to the characteristics of liquid crystals in the emulsions (see wide angle X-Rays diffraction data), the lower water release rate from CCT containing samples may be explained by the presence of lamellar liquid crystals present in this matrix. This result and interpretations are consistent with the specific organization of such liquid crystals that, therefore, appear efficient for keeping the water entrapped when compared to the other structures.

This observation can be confirmed by the rheological temperature sweep test (Fig. 4). The slope value of the water evaporation also depends on the destruction of the system due to thermal effect. The "melting" of the pure α-gel intervenes in "one step" at 51°C (tan δ=1), while for the lamellar liquid crystals the temperature of melting is higher, at 64°C. This can be explained by both the interactions between L β and water molecules and its higher resistance due to its flexibility. It is obvious that the presence of heteroatoms in the surfactant-CCT structure present in the L β phase induces polar intermolecular interaction, these last being favourable for water retention. On the contrary, such interactions do not exist in the case of IHD-surfactant, so consequently, this last system does not retain water molecules that much. Otherwise, as evidenced earlier in this paper, the relative disorganization of the surfactant bilayers when associated with CCT makes it more flexible when compared to the IHD surfactant mixture, and therefore more resistant to temperature thus slowing down the water release. Based on the literature [START_REF] Tadros | Phase behaviour of surfactant systems[END_REF][START_REF] Junginger | The ratio of interlamellarly fixed water to bulk water in O in W creams[END_REF][START_REF] Junginger | Ointments and creams as colloidal drug delivery systems[END_REF][START_REF] Junginger | Pharmaceutical emulsions and creams. Emulsions -A Fundamental and Practical Approach[END_REF] and on the results previously described herein, a schematic representation of the two formulated emulsions can be proposed. On the one hand, the emulsion containing the nonpolar emollient is composed only of α-gel (Fig. 5a). On the other hand, if considering polar emollient, the lamellar liquid crystalline phase (L α ) is formed around the droplet near to the α-gel, this last being still present in the emulsion (Fig. 5b). Such a schematic view of the effect of emollient properties onto the emulsion's structure is consistent with the whole observations exposed above: the droplets size and shape as observed by microscopic techniques, liquid crystals formation as observed by polarized optical microscopy and XRD experiments, mechanical characteristics determined by oscillatory rheology and, finally, results issued from TGA measurements. Once the emulsions substantially described, the second part of the project consisted in the sensory and instrumental analysis of their applicative properties. Compression force and difficulty of spreading were the most discriminative textural properties and correspond to two different application steps: pick up and rub-out. Some specific tests, developed to mimic the sensory analysis, were described in the works by Laura Gilbert [START_REF] Gilbert | Caractérisation physico-chimique et sensorielle d'ingrédients cosmétiques: une approche méthodologique[END_REF][START_REF] Gilbert | Rheological and textural characterization of cosmetic emulsions containing natural and synthetic polymers: relationships between both data[END_REF], where different correlations between physical and sensory tests were established. In the present work, our interest was focused on the spreading test. Both Savary et al. [START_REF] Savary | Impact of emollients on the spreading properties of cosmetic products: A combined sensory and instrumental characterization[END_REF] and Gilbert et al. [START_REF] Gilbert | Caractérisation physico-chimique et sensorielle d'ingrédients cosmétiques: une approche méthodologique[END_REF] proposed optimal conditions for measuring the spreading properties of emulsions using either a PMMA plate and a polypropylene sheet as surfaces for spreading tests. The same conditions were used in the present study to elucidate whether the spreading properties of emulsions are only governed by the oil phase or by the oil/surfactant interaction. Measurements were performed on both the pure emollients and the emulsions.

In order to transpose the emollients behaviour from the instrumental towards the sensory perception, emollients were characterized according to their contact angle formed on the human skin (Fig. 8). The IHD, as nonpolar oil, showed higher spreading that CCT as illustrated by spontaneous larger contact surface on the skin and, as a consequence, a smaller contact angle when compared to CCT. Results for the contact angle can be linked to the conditions of the instrumental spreading test. Because the surface energy of the polypropylene and PMMA is equal to 30.1mN/m and 41.1mN/m respectively [START_REF] Gooch | Surface energy. Encyclopedic Dictionary of Polymers[END_REF], these two materials can be considered as fairly comparable to the skin surface in terms of surface energy, the skin owning a surface energy around 38.7mN/m [START_REF] Agache | Presentation of the skin surface ecosystem[END_REF].

Then the friction test was performed in two steps. First, the spreading profile was drafted for the pure emollients and then, for the corresponding emulsions. Once the experiments performed, several data were calculated on the basis of the force evolution monitored over time (Fig. 9). The following parameters were determined:

-A+ (g.sec), the positive area under the force curve; -Slope value on the force curve; -

The amplitude of the force variation during the test (g).

Fig. 9 illustrates that the emollients force versus time curves present a typical, standard profile. The areas under the curves were higher when compared to the baseline, meaning that the presence of emollients induces friction forces more important than between both surfaces without product. The positive area under the force curve (2326±49 g.sec for IHD and 2751±62 g.sec for CCT) showed the easier spreading of the nonpolar emollient versus the polar one. Interestingly, the affinity of emollient with the surface material was estimated through the noise values as illustrated by Fig. 9c: the larger the noise amplitude, the more the sample withstands the surface material. Here, the surface used for the textural characterization was non-polar (polypropylene) and, as expected, the pure raw materials clearly developed specific interactions with this support. This could be estimated by the "noise amplitude parameter": IHD one was 9g while CCT one was 30g. These observations were consistent with the contact angle measurements, the nonpolar oil (IHD) owning higher affinity with the polypropylene sheet when compared to the polar CCT one. The force curve was analysed over the studied 120 mm of the spreading distance. All the emollients gave a negative force slope value, with -0.69 for IHD and -0.61 for the CCT, respectively. This suggests that the force decreases during the test, thus the spread becomes easier once the quantity of the product between the two surfaces decreases and the values tend to reach a constant curve value. One can conclude that these results are not only influenced by the emollient viscosity, but also by the emollient/solid support affinity.

Considering the emulsions, the evolution is far different from the one observed for the pure oils. As an illustration for IHD and CCT based emulsions, Fig. 10 shows that the emulsion first favours the spreading of the sled, the force values being lower than the baseline. Next, once the thickness of the cream becomes thin enough but still visible, the spreading becomes more difficult (after 20 seconds of spreading). The slope values obtained for the studied emulsions are presented in Table V. The differences between the slopes of the products appear statistically significant. One can notice that slope I and II are also quite different depending on the studied emulsion. The slope I corresponds to the first step of spreading when the droplets are rolling one on another without being disrupted. The slope I value of the EM_CCT is higher than the EM_IHD, arriving at the same baseline. Consequently, lamellar liquid crystals are easier to spread at the very beginning of the test, contrary to the α-gel.

The second step of the spreading test is shown by the slope II. It represents the interaction between IHD or CCT emollient with the support once the droplets are disrupted. Logically, the spreading of the CCT polar emollient is more difficult on the nonpolar surface, making the EM_CCT slope II superior to the EM_IHD.

The information obtained from this test are consistent with the sensory analysis scores and follow the conclusions based on the oils contact angle results. When the contact angle increases (which means affinity with nonpolar surface decreases) the spreading becomes more difficult and requires the application of a higher mechanical force. So, as illustrated for the attribute "difficulty of spreading", one may consider that this attribute evaluated during application actually depends on both oil phase/matrix interactions (first stage) and oil phase individual properties once the matrix is disrupted (second stage).

Conclusion

This study was based on cosmetic emulsions containing liquid crystals of lamellar type. Liquid crystals structures were obtained using a mixed emulsifier Cetearyl alcohol/ Cetearyl glucoside. Two emollients were selected as oil phases. Isohexadecane (IHD) was chosen as representative nonpolar oil, while Caprylic capric triglycerides (CCT), a widely used cosmetic emollient, was selected as the polar one. The difference of polarity was mainly due to the absence (IHD) or the presence (CCT) of heteroatom. Emulsions were formulated with 20% (w/w) of oil and 5% (w/w) of the emulsifier, thus making them suitable for topical application.

Due to the chemical differences between the emollients, two different molecular organisations were obtained for the emulsions. The formation of α-gel was assured in both the IHD and CCT containing emulsions (EM_IHD and CCT_EM, respectively). In addition to that, only EM_CCT emulsion showed the presence of the lamellar liquid crystals near to α-gel. Such distinct molecular organizations markedly impact both the microscopic and macroscopic properties of the systems. EM_CCT formed oil droplets of a smaller size when compared to EM_IHD. Also, the polar oil containing emulsion showed a lesser dynamic viscosity, but a better thermal resistance than the nonpolar one. Finally, the whole results allowed demonstrating that the matrix type impacted the water organization and, consequently, the rate of the water release. This result was explained by higher water retention within the disorganized and more hydrophilic lamellar structures evidenced for emulsions containing polar emollient.

This work brings new approaches to the interpretation of surfactant/oil interactions, by elucidating the role of the molecular organization in the macroscopic and sensory properties of cosmetic emulsions. The proposed interpretations are always supported by a combination of analytical techniques and, therefore, are largely reliable.

For the compression force attribute, the sensory perception was coupled to the dynamic viscosity analysis. It was shown that emollients physical properties do not directly affect the pick-up perception of the emulsions. Meanwhile, the oil phase affects this attribute indirectly, through building specific structures together with the surfactant, like lamellar liquid crystal phase and αgel.

To describe the difficulty of spreading attribute, sensory and texture analysis, as well as emollients contact angle measurements were used. As for the previous parameter, the first step of the spreading test mainly depends on the emulsions molecular organization. Furthermore, the most noticeable perception occurs during the second step, once reached the disruption of the emulsion's structure. Then, as the emollient enters in the direct contact with the skin, the spreading perception and evaluation become fully dependent on the physicochemical affinity of the pure emollient with the skin.

The importance of this work lies in the full control of the emulsions composition and structure. Due to a specific multiscale approach and to a thoughtful choice of raw materials we managed to form the different emulsions with a full understanding and control of their molecular organization. On this basis, it was possible not only to describe, but also to interpret and then to explain both the macroscopic and the applicative properties of the emulsions.

Figure 1 .

 1 Figure 1. Bright field and polarized light micrographs of the emulsions containing isohexadecane as nonpolar oil and caprylic/capric triglycerides as polar emollient at 20% with the median droplet size values for each sample.

Figure 2 .

 2 Figure 2. WAXD patterns of IHD and CCT emulsions. The intensity (a,u,) is represented as a function of the 2θ°. The sharp peak at 0.412nm corresponds to the α-gel (Lβ) formation in both samples, while the diffuse halo at 0.45 nm present in EM_CCT sample is a sign of the lamellar liquid phase formation.

Figure 3 .

 3 Figure 3. Schematic representation of the (a) initial α-gel formed by the APG mixed emulsifier; (b) once isohexadecane is added to the system without disturbing stiff hydrocarbon chains; (c) when heteroatom-containing molecules of caprylic capric triglycerides are inserted between alkyl chains, making them more flexible and thus forming lamellar liquid phase.

Figure 4 .

 4 Figure 4. Temperature sweep test results for EM_IHD (black) and EM_CCT (grey). EM_IHD shows one-step destruction, with tanδ maximal value at 51°C. The destruction of the EM_CCT is more progressive with maximal tanδ value at 64°C.

Figure 5 .

 5 Figure 5. Schematic representation of the emulsion containing (a) nonpolar emollient. The oil droplets and the bulk water are entrapped by the α-gel. For the (b) polar emollient, the droplets are complementarily surrounded by the lamellar liquid crystals (Lα). In both cases, the semihydrate water is retained by the fatty alcohol molecules, while the interlamellar water is placed between the α-gel layers.

Figure 7 .

 7 Figure 7. Sensory scores of EM_IHD and EM_CCT as a function of the difficulty of spreading.

Figure 8 .

 8 Figure 8. Droplet shape of IHD and CCT emollients on the human skin and mean values of the emollients contact angle versus skin (°).

Figure 9 .

 9 Figure 9. Spreading profile of a) pure IHD emollient and b) CCT emollient with its c) noise amplitude example, with the baseline plotted in light grey.

Figure 10 .

 10 Figure 10. Spreading profile of a) EM_IHD and b) EM_CCT emollient with the baseline plotted in light grey. The slope value of the force curve was calculated over the whole distance. Also, complementary slope I and slope II were calculated and correspond to the spreading between 0 and 20 seconds (lower to the baseline) and between 20 and 40 seconds (superior to the baseline).

Table I .

 I INCI, trade name, the supplier and the codes of two selected emollients, near to of the density measurement results, surface tension, as well as interfacial tension values.

	INCI	Trade name	Supplier Code Density	Surface tension (mN/m)	Interfacial tension versus water (mN/m)
	Isohexadecane	Isohexadecane	IMCD	IHD	0.787	24.40±0.03	43.61±2.72
	Caprylic/Capric Triglyceride	Myritol 318	BASF	CCT	0.948	28.95±0.04	13.49±0.22

Table III .

 III Percentage of the water loss / total water amount over the specified temperature ranges. The data should be compared by column. Two different letters in each column indicate the statistically significant differences between the products.

	Sample	% (water loss/water amount) 30°C-50°C 50°C-70°C 70°C-110°C
	EM_IHD	8.8	A	26.3	A	75.6	A
	EM_CCT	6.7	B	25.8	A	61.8	B
	Water	5.1	B	16.6	B	76.1	A

Table V .

 V Slope values of the force curve obtained during the total test time, slope I corresponds to the spreading between 0 and 20 seconds (lower to the baseline) and between 20 and 40 seconds (superior to the baseline).

		EM_IHD	EM_CCT
	Slope	0.506±0.06 1.095±0.036
	Slope I	0.549±0.107 0.816±0.041
	Slope II	0.507±0.053 1.027±0.139

Acknowledgements

This work has been carried out in the context of the ALGRAAL, selected as Project of Excellence by the General Directorate for Competitiveness Industry and Services (DGCIS) in the 16th Appeal FUI (Fonds Unique Interministériel). The authors gratefully acknowledge the financial support of the FUI and the Région Normandie.

Applicative properties: compression

The results of the compression force sensory test are presented in Fig. 6. The compression force is the evaluation of the products firmness during the pick-up phase.

The sensory panel felt the difference between the two products. At this stage, one can question whether the significant difference in perception is due to the emollient or to the specific structures created by the surfactant -emollient interaction. Thus, a complementary instrumental analysis was envisaged in order to answer this question. Several papers describe correlations between the sensory perception and the instrumental analysis for cosmetic products on the basis of rheology and texture analysis [START_REF] Gilbert | Predicting sensory texture properties of cosmetic emulsions by physical measurements[END_REF][START_REF] Dubuisson | Influence de la phase grasse et des polymères naturels sur les paramètres physicochimiques en lien avec la perception tactile de l'émulsion[END_REF][START_REF] Estanqueiro | Comparison between sensory and instrumental characterization of topical formulations: impact of thickening agents[END_REF][START_REF] Moravkova | Relation between sensory analysis and rheology of body lotions[END_REF]. For the "compression force" sensory attribute, Gilbert et al. [START_REF] Gilbert | Predicting sensory texture properties of cosmetic emulsions by physical measurements[END_REF] described a good correlation (R² = 0.961) with the test of compression with a texture analysis. Or, according to Barnes [START_REF] Barnes | Chapter 18 The rheology of emulsions[END_REF], the various physical operations with emulsions can be also expressed by some typical shear rate ranges. As an example, the shear rate corresponding to the compression force attribute can be placed around 10² s -1

. Thus, the apparent viscosity of both emulsions and the pure IHD and CCT emollients at this share rate was measured during a flow test experiment. As visible in Table IV, the dynamic viscosities of the emulsions are much higher than those of pure emollients and do not follow the same order. This illustrates the major role of the emulsion's structure and organization when compared to the components taken alone. The compression parameter appears mainly governed by the molecular organization of the emulsion. The viscosity of the emollient alone does not affect the pick-up parameter. This is due to the fact that the droplets are not disrupted during this test.

Instead, the indirect impact on the organization of the liquid crystals and, as consequence, on the compression values is observed. Compression force appears strongly related to the rigidity of the surfactant bi-and multi-layers organization, which depends on the emollient molecular peculiarities.

Applicative properties: spreading profile

The sensory perception of the "difficulty of spreading" attribute is presented in Fig. 7. This attribute does not follow the same ranking as compression attribute exposed above. EM_CCT appears more difficult to spread on the skin than EM_IHD. In this case, in order to understand which parameters are involved in this texture perception, the spreading property was evaluated through an instrumental test inspired by the literature.

Conflicts of interest

There are no conflicts to declare.


Béatrice Eymin
email: beatrice.eymin@ujf-grenoble.fr

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

Christian Brambilla

Elisabeth Brambilla

Mdm2 overexpression and p14 ARF inactivation are two mutually exclusive events in primary human lung tumors

Keywords: carcinogenesis, cell cycle, Mdm2, lung tumor, p14 ARF Alternative Reading Frame, IHC, immunohistochemistry, LCNEC, Large Cell NE Carcinoma, Mdm2, Murine Double Minute 2, NE, Neuroendocrine, NLS, Nuclear Localization Signal, NrLS, Nucleolar Localization Signal, NSCLC, Non-Small Cell Lung Carcinoma, PBS, Phosphate Buffer Saline, PMSF, Phenylmethylsulfonate, pRb, Retinoblastoma protein, SCLC, Small Cell Lung carcinoma, SDS, Sodium Dodecyl Sulfate, TPBS, Tween Phosphate Buffer Saline

Pathways involving p53 and pRb tumor suppressor genes are frequently deregulated during lung carcinogenesis. Through its location at the interface of these pathways, Mdm2 can modulate the function of both p53 and pRb genes. We have examined here the pattern of expression of Mdm2 in a series of 192 human lung carcinomas of all histological types using both immunohistochemical and western blot analyses and four distinct antibodies mapping different epitopes onto the Mdm2 protein. Using Immunohistochemistry (IHC), Mdm2 was overexpressed as compared to normal lung in 31% (60/192) of all tumors analyzed, whatever their histological types. Western blotting was performed on 28 of the 192 tumoral samples. Overexpression of p85/90, p74/76 and p57 Mdm2 isoforms was detected in 18% (5/28), 25% (7/28) and 39% (11/28) of the cases respectively. Overall, overexpression of at least one isoform was observed in 14/28 (50%) lung tumors and concomittant overexpression of at least two isoforms in 7/28 (25%) cases. A good concordance (82%) was observed between immunohistochemical and western blot data. Interestingly, a highly significant inverse relationship was detected between p14 ARF loss and Mdm2 overexpression either in NSCLC (p=0.0089) or in NE lung tumors (p<0.0001). Furthermore, a Mdm2/p14 ARF >1 ratio was correlated with a high grade phenotype among NE tumors overexpressing Mdm2 (p=0.0021). Taken together, these data strongly suggest that p14 ARF and Mdm2 act on common pathway(s) to regulate p53 and/or pRb-dependent or independent functions and that the Mdm2:p14 ARF ratio might act as a rheostat in modulating the activity of both proteins.

Introduction

Bronchogenic carcinomas represent the most frequent fatal malignancy in males in Europe and in both sex in United States. They can be divided into different sub-classes based on the 1999 WHO classification of lung cancer [START_REF] Travis | Ed Springer[END_REF]. These various histological types emerge from pluripotent stem cells that can follow distinct, often overlapping, carcinogenic pathways involving sequential alterations in oncogenes and tumor suppressor genes. p53 and pRb tumor suppressor genes are frequently inactivated during lung tumorigenesis (Brambilla et al., 1993;Brambilla et al., 1999;Chiba et al., 1990;Gazzeri et al., 1994;Gazzeri et al., 1998b;Gouyer et al., 1998;Xu et al., 1996) emphasizing that molecular regulators of these pathways could conceivably be targeted as well.

The Mdm2 protein acts as a bridge over p53 and pRb (Yap et al., 1999). The Mdm2 gene was initially identified as an amplified gene on a murine double minute chromosome in the spontaneously transformed BALB/c 3T3 cells (Cahilly-Snyder et al., 1987). This oncogenic property was further demonstrated in « in vitro » experiments where overexpression of Mdm2 was able to increase the tumorigenic potential (Finlay, 1993) and proliferative rate (Martin et al., 1995) of cultured cells. As well as being a transcriptional target for p53, Mdm2 can also antagonize p53dependent transcriptional activation and growth arrest by direct binding via its Nterminal region (Chen et al., 1993;Momand et al., 1992;Oliner et al., 1993).

Furthermore, Mdm2 can promote the degradation of p53, acting as an ubiquitinprotein ligase to ubiquitinate p53 (Haupt et al., 1997;Kubbutat et al., 1997) and triggering its nuclear export (Geyer et al., 2000;Roth et al., 1998;Tao & Levine, 1999a) and degradation in cytoplasmic proteasomes (Freedman & Levine, 1998;Roth et al., 1998). The inhibitory effect of Mdm2 towards p53 is counteracted by human p14 ARF , a tumor suppressor gene that acts as a sensor of hyperproliferative signals emanating from oncoproteins (Bates et al., 1998;Zindy et al., 1998). Direct binding of p14 ARF to Mdm2 inhibits p53 degradation by blocking p53-Mdm2 nuclear export (Tao & Levine, 1999b;Zhang & Xiong, 1999), sequesters Mdm2 into the nucleolus (Kamijo et al., 1997;Quelle et al., 1995;Weber et al., 1999) and inhibits its ubiquitin ligase activity (Honda & Yasuda, 1999;Midgley et al., 2000;Pomerantz et al., 1998).

By so doing, p14 ARF prevents the negative-feedback regulation of p53 by Mdm2 and leads to the activation of p53 in the nucleoplasm. Conversely, high levels of Mdm2 relocalize endogenous p14 ARF from nucleoli to nucleoplasm (Zhang & Xiong, 1999) suggesting that balance between both protein levels and their respective subcellular location might be important to regulate their effects.

In addition to its clear role in the regulation of p53, Mdm2 is also able to promote tumorigenesis by interacting with pRb and to inhibit its growth regulatory function through a mechanism(s) actually unknown (Sun et al., 1998;Xiao et al., 1995). In addition, more recent studies have shown that pRb can form a trimeric complex with Mdm2 and p53 and thereby blocks the anti-apoptotic activity of Mdm2 by preventing the degradation of p53 (Hsieh et al., 1999). Thus, depending on the context, interaction between pRb and Mdm2 proteins might mediate distinct effects.

The Mdm2 gene generates various Mdm2 products, of which only a subset complexes with p53 protein. In addition to the full length protein (p85/90), p54/57 and p74/76 isoforms have been also described as Mdm2 gene products (Gudas et al., 1995;Haines et al., 1994;Olson et al., 1993). Eventhough the function of these Mdm2 isoforms remains to be elucidated, it is noteworthy that the p74/76 isoforms lacking the N-terminal domain of the full lenght protein do not bind to p53 (Haines et al., 1994;Olson et al., 1993) and can antagonize the ability of p90 to target p53 destruction (Perry et al., 2000). In contrast, the p54/57 isoforms bind to p53 but lack C-terminal epitopes (Olson et al., 1993).

Abnormalities of Mdm2 expression have been reported in human tumors, specially in sarcomas where Mdm2 gene amplification is commonly observed (Cordon-Cardo et al., 1994;Oliner et al., 1992). In other tumor types, gene amplification is much less common and a variable frequency of Mdm2 overexpression has been described using either immunohistochemical or immunoblot studies (Foulkes et al., 1995;Horie et al., 2001;Lianes et al., 1994;O'Neill et al., 1998). In human lung tumors, amplification of Mdm2 gene has been reported in only a few cases of non small cell lung cancer (NSCLC) (Higashiyama et al., 1997;Marchetti et al., 1995) and mutations were never found (Mariatos et al., 2000). In contrast, aberrant expression of Mdm2 product has been variably appreciated since 24 to 70% of NSCLC and 40 to 70% of small cell lung carcinoma (SCLC) were reported to overexpress Mdm2 proteins (Aikawa et al., 2000;Gorgoulis et al., 1996a;Gorgoulis et al., 1998;Gorgoulis et al., 2000;Gorgoulis et al., 1996b;Higashiyama et al., 1997;Stefanaki et al., 1998). Furthermore, a clear overexpression of Mdm2 product has been observed in preneoplastic lung lesions suggesting that alteration of Mdm2 could be an early event during lung carcinogenesis (Rasidakis et al., 1998).

In this study, we investigated the pattern of Mdm2 expression in a large series of human lung cancers of all histological types with known p53, pRb and p14 ARF status.

By using several antibodies recognizing all Mdm2 isoforms and both immunohistochemical and western blot techniques, we show that Mdm2 is overexpressed in 31% of human lung tumors as compared to normal lung, whatever their histological subtypes. More interestingly, our data demonstrate that Mdm2 overexpression and p14 ARF loss are two mutually exclusive events (p<0.0001) suggesting that both proteins are located onto common pathway(s) to regulate p53 and/or pRb functions.

Results

Immunohistochemical analysis of Mdm2 protein in human lung tumors.

Mdm2 expression was studied by immunohistochemistry using 4 antibodies mapping distinct epitopes onto the Mdm2 protein (Figure 1A) on a panel of 192 tumor tissue samples of all histological types.

The results were recorded independently by two investigators (EB, CB) who assessed the percentage of positive cells and the intensity of staining. Normal lung parenchyma adjacent to tumor on sections was considered as internal control, whereas 3 normal lung tissues taken for diagnosis in patients without history of cancer were taken as external controls. These tissues contained both positive epithelial and endothelial cells and negative lymphocytes that were the gold standard for comparison in our study. Nuclear staining only was considered to assess Mdm2 immunoreactivity.

In normal lung that was present in the vicinity of the tumors as well as in control normal lung tissues, a similar intensity of staining was observed on the entire target population of epithelial and endothelial cells using all antibodies.

Because Mdm2 immunostaining was heterogeneous among lung tumors, differential scores were ascribed in each case and for each antibody according to the intensity of staining and the percentage of stained cells. A mean score was calculated and the resulting data were grouped as described in the methods section into the 4 following classes of staining: 0, undetectable; 1, faint; 2, moderate; and 3, high.

According to this, Mdm2 immunostaining in normal alveolar and bronchiolar epithelium and stromal cells (endothelial cells and fibroblasts) was uniformaly distributed between classes 1 and 2 (30 to 80 in extreme score values). Tumors displaying a mean score of class 3 were considered as Mdm2 overexpressing cases since their level of staining was definitely higher than the maximum level reached by normal lung cells. According to this, Mdm2 was overexpressed in 31 of 90 (34%) NSCLC and in 29 out of 102 (28%) NE lung tumors (Table 1 andFigure 2). No significant difference in the pattern of Mdm2 staining was observed between the distinct histological sub-classes of lung tumors. In contrast, Mdm2 overexpression was associated with an extended stage in adenocarcinomas (p=0.0248, data not shown).

Possible discordance between antibodies reactivity was investigated according to the localization of their epitope onto the Mdm2 protein. SMP14 and 2A10 antibodies, both recognizing mid-region epitopes in the acidic zinc region of the molecule, always gave a similar score of Mdm2 immunostaining that was lower than those obtained with the two other antibodies in 8% (16/192) of the cases. Absence or abnormal low level of Mdm2 staining was observed in 3% (5/192) and 7% (13/192) of the cases with N20 and C-Ter antibodies respectively, as compared with the high level of reactivity using the 3 others antibodies. Overall, 100% of the tumors analyzed displayed the same pattern of Mdm2 immunostaining (intensity and percentage of positive tumor cells) with at least two antibodies and 88% (168/192) with at least 3 distinct antibodies indicating that a good concordance existed between the antibodies reactivity.

Western blot analysis of Mdm2 in human lung tumors.

In order to validate the IHC data, a western blot analysis was performed onto 28 of the 192 lung tumors using the same four Mdm2 antibodies. The tumors consisted in 6 squamous cell carcinomas, 7 adenocarcinomas, 3 basaloids carcinomas, 4 LCNEC and 8 SCLC and included 7 Mdm2 overexpressing cases based on the IHC analysis.

Using our panel of antibodies and as previously described (Gudas et al., 1995;Haines et al., 1994;Olson et al., 1993), three major Mdm2 isoforms were detected based on their molecular weight of approximately 90kDa (p85/90), 74/76 kDa (p74/76) and 57 kDa (p57/58) (Table 2; Figures 1B &3). All antibodies reacted with the full-length p85/90 Mdm2 product. Additionally, both SMP14 and C-Ter antibodies recognized the 74/76 isoform whereas N-20 antibody detected the p57 isoform.

The pattern of Mdm2 expression was firstly analyzed in the normal lung. In all samples studied, p85/90 and p57 isoforms were expressed at a relatively low level, whatever the antibody used for p85/90 isoform (Figure 3 & Table 2). A similar low level of p74/76 expression was observed using C-Ter antibody but a higher basal level was found with SMP14 in all these samples suggesting a higher reactivity of SMP14 than that of C-Ter antibody with p74/76 Mdm2 isoform (Table 2). Overall, the pattern of Mdm2 expression in the normal lung using western blotting was consistent with that obtained by IHC.

Also consistent with the IHC data was the wide distribution of Mdm2 expression levels in lung tumors as tumor samples displayed undetectable or low (+), moderate (++), or high (+++) levels of Mdm2 isoforms when compared to normal lung. Of the 28 cases analyzed, 5 (18%; T6, T9, T14, T19, T24) displayed a high level of p85/90 isoform with at least two antibodies (Table 2 &Figure 3). Overexpression of p85/90 was not associated with a specific histological subtype. Discordances of reactivity with one antibody as compared to the others was observed in 11% (3/28) of the cases for C-Ter, 18% (5/28) for N20 and 14% (4/28) for SMP14/2A10 antibodies (Table 2).

A high level of p74/76 isoforms was detected in 7 out of 28 lung tumors (25%) using both SMP14 (1/28, T23) and C-Ter (6/28, T1, T6, T8, T10, T19, T22) antibodies. The lower incidence of p74/76 overexpression using SMP14 is probably linked to its ability to detect a higher level in the normal lung, making difficult the detection of a clear overexpression in tumor samples. Although not statistically significant, overexpression of p74/76 was more frequent in SCLC (3/8, 38%) than in other histological subtype (4/20, 20%).

Finally, p57 isoform was quantitatively more expressed than the two other isoforms as high levels of p57 were observed in 11/28 (39%) lung tumors using N-20 antibody. Interestingly, we noticed that p57 was more frequently overexpressed in SCLC (6/8, 75%; p=0.014) than in other histological sub-types.

Overall, overexpression of all isoforms was observed in 2 cases (T6, T19). p85/90 and p57 were co-overexpressed in 2 cases (T9, T14) and p74/76 and p57 in 3 cases (T1, T8, T10). One case displayed high level of p85/90 only (T24), two others p74/76 only (T22, T23) and four others p57 only (T5, T12, T16, T27). Thus, overexpression of at least one isoform was observed in 14/28 (50%) lung tumors and overexpression of at least two Mdm2 isoforms was detected in 7/28 (25%) cases.

Moreover, we noticed that nearly all the tumors (5/7) with high level of p74/76 cooverexpressed the p57 isoform. Taken together, these data suggest that aberrant expression of several Mdm2 isoforms might occur simultaneously during lung tumorigenesis.

Comparison of IHC and western blot data

Results of western blotting were considered concordant with those of IHC when the same status of Mdm2 was detected with at least two distinct antibodies, whatever the isoform. In these conditions, of the 21 samples considered as Mdm2 nonoverexpressing cases using IHC (Table 2), 17 (81%) displayed concordant results by western blotting. In the four other cases (T1, T8, T10, T23), overexpression of Mdm2 was detected using western blot analysis. In two of these samples (T8, T23) however, discordances were observed between the antibodies used in the immunohistochemical analysis. In the last two cases (T1, T10), all antibodies gave concordant IHC data and discordance with western blotting was general.

On the other hand, of the 7 samples considered as Mdm2 overexpressing cases using IHC, 6 (86%, T6, T9, T14, T19, T22, T24) displayed concordant results by western blotting. Interestingly, nearly all these tumors (T6, T9, T14, T19, T22) overexpressed at least two isoforms as detected by western blot analysis. In the last case (T16), overexpression was detected with only one antibody. However, this sample contained more than 50% of stromal cells suggesting that overexpression of Mdm2 in tumor cells was masked by the contamination with low expressing stromal cells.

Taken together, these data showed a good concordance (23/28, 82%) between both techniques.

Relationship between Mdm2 and p53 expression in human lung tumors.

The results of Mdm2 immunostaining were compared to those of p53 previously carried out on the same series of tumor samples (Brambilla et al., 1993;Gazzeri et al., 1994). There was no inverse or direct correlation linking Mdm2 and p53 expression in NSCLC and NE lung tumors (Table 3). However, although not statistically significant, we noticed that more than half of the tumors exhibiting Mdm2 overexpression (32/60, 53%), specially adenocarcinomas (11/13, 85%), also displayed a p53 mutant immunophenotype. These results suggest that Mdm2 overexpression can be induced by p53-independent pathway(s).

Relationship between Mdm2 expression and pRb/p16

INK4a /cyclin D1 status in human lung tumors.

Alterations of pRb pathway either through pRb loss or p16 INK4a loss and/or cyclin D1 overexpression had been previously studied in this series of tumor samples (Brambilla et al., 1999;Gazzeri et al., 1998b;Gouyer et al., 1998) (Table 4). No correlation was found between Mdm2 status and alterations of either pRb pathway or both p53 and pRb pathways in NSCLC or in NE lung tumors (Table 4). However, we noticed that 61% (8/13) of adenocarcinomas exhibited both Mdm2 overexpression and deregulation of p53 and pRb pathways (data not shown).

Comparison of Mdm2 and p14 ARF expression in human lung tumors.

Results of p14 ARF immunostaining performed previously on the same series of tumor samples (Gazzeri et al., 1998a) have been presented again to allow comparison between the expression of both proteins. Mdm2 overexpression and p14 ARF loss were inversely correlated in NSCLC (p=0.0089) and even more significantly in NE tumors (p<0.0001) (Table 5). Alteration of either Mdm2 or p14 ARF expression was never correlated with p53 status. In contrast, it was highly associated with inactivation of pRb pathway in NE lung tumors (p=0.0017; Table 5).

As equilibrium between Mdm2 and p14 ARF expression is thought to modulate their activity, Mdm2 and p14 ARF scores were compared in all samples analyzed using a paired t test. Although statistically not significant, the mean score of p14 ARF was globally higher than that of Mdm2 in all NSCLC and carcinoïds tested (Figure 4A).

Conversely, the mean score of Mdm2 was statistically higher than that of p14 ARF in LCNEC (p=0.001) and in SCLC (p<0.0001, Figure 4A). Moreover, even when only Mdm2 overexpressing tumors were considered, the mean score of Mdm2 was significantly higher than that of p14 ARF in SCLC (p=0.0021) whereas the level of both proteins was not statistically different in all other histological sub-types (Figure 4B).

Discussion

High levels of Mdm2 product are frequently observed in both human cancer cell lines (Gudas et al., 1995) and tumor samples (Foulkes et al., 1995;Gorgoulis et al., 1996a;Gorgoulis et al., 1996b;Horie et al., 2001;Lianes et al., 1994). In most studies, Mdm2 overexpression was assessed by using immunohistochemical studies with one or two antibodies mapping different epitopes onto Mdm2 protein. However, analysis of Mdm2 status is complicated by the existence of various isoforms which detection depends on the antibody used, rendering hazardous the functional interpretation of immunostaining data without any concomittant biochemical analysis. In this study, we analyzed Mdm2 protein status by using both Immunohistochemical and Western blot techniques and four Mdm2 antibodies allowing detection of all Mdm2 isoforms.

Because we found a good concordance between the antibodies reactivity in the immunohistochemical analysis as well as between both IHC and western blot techniques, we are confident that our evaluation of Mdm2 status in our series of human lung tumors is reliable.

We showed a high level of Mdm2 product in 31% of all lung tumors analyzed whatever their histological sub-type. In bronchogenic carcinomas, Mdm2 overexpression was previously reported as a consequence of increased transcription rather than gene amplification (Gorgoulis et al., 1996a;Higashiyama et al., 1997).

Beside its constitutively expressed promoter, the Mdm2 gene contains an additional p53-responsive promoter (Barak et al., 1994). Some previous studies have reported overexpression of Mdm2 transcript in human tumor cell lines regardless of the endogenous p53 status (Gudas et al., 1995;Ries et al., 2000b). Furthermore, we and others [this study and (Gorgoulis et al., 1996a;Gorgoulis et al., 1998;Gorgoulis et al., 1996b)] showed concomittant Mdm2 overexpression and p53 mutant immunophenotype in lung tumors. Taken together, these data suggest that Mdm2 overexpression can be mediated by p53-independent pathways. A recent study has shown that the Mdm2 promoter is also a target of the Ras/Raf pathway (Ries et al., 2000a). Accordingly, we and others [this study; (Higashiyama et al., 1997)] observed a slightly more frequent Mdm2 overexpression in lung adenocarcinoma that are known to frequently express a constitutively active Ras mutant (Rodenhuis & Slebos, 1992).

Other studies have reported that Mdm2 is also regulated at a post-transcriptional level (Gudas et al., 1995;Khosravi et al., 1999;Maya et al., 2001) as well as by the proteasome (Buschmann et al., 2000;Honda & Yasuda, 1999;Honda & Yasuda, 2000). Overall, these data indicate that several mechanisms might contribute to Mdm2 accumulation during lung tumorigenesis.

According to previous data, we detected three Mdm2 isoforms in lung tissues using western blot analysis. We did not find any correlation between overexpression of either isoform and clinical pathological features, consistent with previous reports (Gorgoulis et al., 1996b;Ralhan et al., 2000). However, overexpression of p57 isoform was more frequent in SCLC (75%; p=0.0144) as compared to other histological subtypes suggesting its potential implication in the carcinogenesis of high grade NE lung tumors. Interestingly, same predominant overexpression of p57 has been previously described in breast carcinomas (Bueso-Ramos et al., 1996) and chronic lymphocytic leukemias (Haidar et al., 1997). Co-overexpression of at least two isoforms was observed in half of the tumors analyzed indicating that several Mdm2 isoforms can be concomitantly deregulated. The role of the Mdm2 isoforms in tumorigenesis remains unclear. Although p90 and p57 bind to p53 and inhibit its nuclear accumulation (Haines et al., 1994;Olson et al., 1993), p74/76 can stabilize p53 by inhibiting the ability of p90 to stimulate its degradation without affecting the p90/p53 interaction (Perry et al., 2000). Thus, it would be possible that the ratio between the three Mdm2 isoforms determines their effects onto p53 or other cell-cycle regulatory targets.

We did not find any inverse or direct correlation linking Mdm2 and p53 status.

However, alteration of p53, as reflected by abnormally stabilized p53 protein, was frequently observed among adenocarcinoma overexpressing Mdm2 protein (11/13, 85%). As overexpression of Mdm2 was statistically associated with extended stages in this histological subtype (this study), our data suggest that alteration of both p53 and Mdm2 expression might contribute to more aggressive features in this subset of NSCLC. Beside its function towards p53, Mdm2 is also able to interact with pRb thereby inactivating its negative growth regulatory function (Sun et al., 1998;Xiao et al., 1995). In high grade NE lung tumors (LCNEC and SCLC), pRb pathway is often inactivated whereas carcinoids exhibit intact pRb pathway. Therefore, Mdm2 overexpression might be a mechanism of pRb impairment in carcinoids whereas both alterations could confer an additive growth advantage for high grade NE lung tumors. p14 ARF and Mdm2 products are functionally closely related. p14 ARF acts as an inhibitor of Mdm2 function towards p53 (Honda & Yasuda, 1999;Midgley et al., 2000;Pomerantz et al., 1998;Tao & Levine, 1999b;Zhang & Xiong, 1999). On the other hand, Mdm2 effect(s) onto p14 ARF functions remains controversial. Indeed, it was recently shown that loss of Mdm2 could either suppress (Carnero et al., 2000) or restore [START_REF] Weber | [END_REF] the ability of murine p19 ARF to induce p53-independent cell cycle arrest. Thus, depending on the context (cellular types, upstream signals, expression level), Mdm2 could behave either as a mediator or an inhibitor of p14 ARF function onto cell proliferation. We found here a striking inverse relationship between Mdm2 and p14 ARF status, strongly suggesting that Mdm2 and p14 ARF act onto a same pathway, according to the pRb/p16 INK4a model. Furthermore, alteration of either Mdm2 or p14 ARF expression was significantly associated with inactivation of pRb pathway in high grade NE lung tumors. Altogether, these data suggest that the Mdm2/p14 ARF pathway might regulate target genes other than p53 and pRb, such as E2F1 (Eymin et al., 2001). Alternatively, the multiple impairments of the Mdm2/p14 ARF /p53/pRb network in these tumors could reflect a "gain of function" phenotype to which the Mdm2/p14 ARF balance is the limiting step.

Nucleolar sequestration of Mdm2 by p14 ARF leads to p53 accumulation (Kamijo et al., 1997;Quelle et al., 1995;Weber et al., 1999). In contrast, high levels of Mdm2 relocate endogenous p14 ARF from nucleoli to nucleoplasm (Zhang & Xiong, 1999). Thus, the balance between both proteins levels might control their subcellular localization and activity. Accordingly, p14 ARF promoter methylation was recently associated with Mdm2 cytoplasmic localization in human cell lines and tumors (Esteller et al., 2001). In our series of human lung tumors, we did not find any correlation between p14 ARF status and Mdm2 subcellular localization (cytoplasmic or nuclear, data not shown). However, we showed that Mdm2 expression levels were significantly higher than those of p14 ARF in high grade NE lung tumors (LCNEC, SCLC) whereas no significant difference was observed in all other histological subtypes. As Mdm2 overexpression can overcome p19 ARF -mediated growth-arrest (Carnero et al., 2000), our data suggest that imbalance between p14 ARF and Mdm2 proteins might contribute to the carcinogenesis of high grade NE tumors. Furthermore, when only Mdm2-overexpressing tumors were considered, Mdm2 level was statistically higher than that of p14 ARF only in SCLC, the most aggressive tumors among lung cancers. Thus, it is conceivable that in NSCLC and low grade NE lung tumors (carcinoids) the high level of p14 ARF counteracts Mdm2 overexpression, whereas in SCLC the absence or low level of p14 ARF predicts its unability to antagonize the oncogenic properties of Mdm2.

In conclusion, we show for the first time a strong inverse relationship between Mdm2 and p14 ARF expression in human tumors. These results reinforce the functional link between both proteins and suggest that they act onto same pathway(s) to regulate p53 and/or pRb-dependent or independent functions. A recent study reported that p53 null/Mdm2 heterozygous mice display longer tumor latency, fewer incidence of lymphoma but higher incidence of sarcoma than p53/Mdm2 double null mice (McDonnell et al., 1999). Therefore, depending on the levels of Mdm2 being expressed in the cell and the tissue type, Mdm2 appears to play dual roles "in vivo" either as a tumor suppressor or an oncogene. These data endorse the notion of a Mdm2 gene dosage effect (McDonnell et al., 1999;[START_REF] Weber | [END_REF]. According to this, our results suggest that the Mdm2:p14 ARF ratio might regulate, as a rheostat, the activity of both proteins thereby modulating the functional consequences of Mdm2 expression. This could explain why overexpression of Mdm2 transcript or protein "per se" is not always associated with an adverse prognostic in lung tumors (Higashiyama et al., 1997;Ko et al., 2000).

Material and methods

Tissue samples

One hundred and ninety two lung tumors were included in this study. Tissue samples were taken at surgical resection of lung tumors or at mediastinoscopy of node metastases in order to establish the diagnosis and extension of the disease in non operable patients. Tumor tissues were immediately frozen and kept at -80°C until use. 38 males, 9 females, 4 stages I/II, 43 stages III/IV) according to the 1999 WHO international histological classification of lung tumors [START_REF] Travis | Ed Springer[END_REF].

Antibodies

Mdm2 protein isoforms were studied using four distinct antibodies mapping different epitopes onto Mdm2. The monoclonal antibodies SMP-14 (Santa Cruz, TEBU, Le Perray en Yvelines) and 2A10 (kindly provided by A. Levine) are directed against residues 154-167 of the acidic domain and 294-339 of the zinc finger domain, respectively. The rabbit polyclonal antibodies N-20 (Santa Cruz, TEBU) and carboxy terminal C-Ter (NCL-Mdm2P, Novocastra, TEBU) recognize residues 1-26 of the amino terminus and an epitope localized near the carboxy terminus of Mdm2 protein, respectively. Antibody to p14 ARF (a gift from C. Larsen) has been previously described (Della Valle et al., 1997).

Immunohistochemistry (IHC)

Mdm2 immunostaining was performed on 6 µm thick frozen sections that were previously fixed in 0.4% paraformaldehyde for 10 minutes. Three step immunohistochemical method was applied. Non specific binding sites were blocked by incubating the sections with 0.1% (w/v) bovine serum albumin in phosphatebuffered saline (PBS) for 30 minutes at room temperature. The sections were subsequently incubated with the primary antibody overnight at 4°C using either N-20, C-Ter, SMP14 or 2A10 at 1:4000, 1:2000, 1:500 and 1:2000 dilutions, respectively.

Slides were then washed in PBS several times and the primary antibody was detected using biotinylated secondary antibodies consisting of either anti-rabbit biotinylated donkey F(ab')2 (1:1000; The Jackson Laboratory; West Grove; PA) or anti-mouse 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 streptavidinbiotin-peroxydase complex (1:200; Strept-AB complex; DAKO) for 1 hour at room temperature. The chromogenic substrate of peroxydase was a solution of 0.05% 3.3diaminobenzidine tetrahydrochloride, 0.03% H 2 O 2 , and 10 mM imidazole in 0.05M Tris buffer (pH 7.6). The slides were counterstained with Harris' hematoxylin. Normal rabbit or mouse IgG at the same concentration as the primary antibodies served as negative controls. Only nuclear staining was considered to assess immunoreactivity.

Cytoplasmic staining was never considered in the assessment of Mdm2 expression.

The intensity of immunohistochemical staining was evaluated by two independent observers as well as in distinct areas of the slide sections for correlation and confirmation of tissues analysis. Scores of immunostaining were calculated by multiplying the percentage of labeled cells with the intensity of staining (1+, 2+, 3+) and tumors were graded into four classes based on their score (class 0: no staining; class 1: < 50; class 2: 50-100; class 3 ≥ 100). Tumors with more than 50% of tumor cells displaying moderate or strong nuclear staining were considered as tumors overexpressing Mdm2 (score ≥ 100; class 3) when compared to normal tissues. p53, pRb and p14 ARF immunostaining have been previously described (Brambilla et al., 1993;Brambilla et al., 1999;Gazzeri et al., 1998a ;Gouyer et al., 1998).

Western-blot analysis

Fifteen normal lung tissues and 28 representative tumor samples of the IHC series were analyzed using western blotting. These samples were taken at the immediate vicinity of those studied by immunohistochemistry. Tissues were lysed in ice-cold lysis buffer (5 mM EDTA, 150 mM NaCl, 100mM Tris (pH 8), 0.5% sodium deoxycholate, 0.5% nonidet 40, 0.5% sodium dodecyl sulfate (SDS), 0.1% aprotinin, 2 µg/ml leupeptin, 2 µg/ml pepstatin, and 1 mM PMSF) for 30 min and centrifugated for 30 min at 15000g. Supernatants were then collected and frozen at -80°C until use.

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 7-10% 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 hours at room temperature with primary antibody in 2% non-fat milk TPBS, washed three times in TPBS, incubated with secondary horseradish peroxydase-conjugated goat anti-mouse or anti-rabbit antibodies (The Jackson Laboratory, West Grove, PA) for 30 minutes and revealed using enhanced chemoluminescence detection kit (ECL; Amersham). Dilutions of the primary antibodies were 1:10.000, 1:20.000, 1:20.000 and 1:5000 for 2A10, N-20, SMP14 and C-Ter respectively. To ensure equal loading and transfer of proteins, the membranes were subsequently probed with a polyclonal actin antibody (1:500; Sigma-Aldrich; L'Isle d'Abeau). Mdm2 and p14 ARF scores were evaluated in each tumor sample using immunohistochemistry. Mean scores were calculated for each histological sub-type of lung tumors and compared using a paired t-test. Mdm2 and p14 ARF mean scores are presented ± SD (standard deviation) and compared in all samples (A) and in samples overexpressing Mdm2 protein (B). SCC, Squamous Cell Carcinoma; ADK, Adenocarcinoma; LCNEC, Large Cell Neuroendocrine Carcinoma; SCLC, Small Cell Lung Carcinoma; NS, non significant.

Legends to figures

For

  histological classification, tumor samples were fixed in formalin and/or alcoholic Bouin's fixative. They consisted of 44 squamous carcinomas (42 males, 2 females; 21 stages I/II, 23 stages III/IV), 28 adenocarcinomas (21 males, 7 females; 17 stages I/II, 11 stages III/IV), 18 basaloids carcinomas (18 males, no female; 9 stages I/II, 9 stages III/IV), 26 typical and atypical carcinoids (13 males, 13 females; 24 stages I/II, 2 stages III/IV), 29 Large Cell Neuroendocrine Carcinomas (LCNEC; 27 males, 2 females; 16 stages I/II, 13 stages III/IV) and 47 Small Cell Lung Carcinomas (SCLC;

Figure 1 .

 1 Figure 1. Reactivity of Mdm2 antibodies onto Mdm2 protein. A, Major functional domains of Mdm2 protein. Binding sites of some Mdm2 targets p53, pRb and p14 ARF are shown at the top. The hatched boxes indicate nuclear localization (NLS), export (NES) and nucleolar localization signals (NrLS) as mentionned. The black boxes indicate zinc fingers. The acidic domain and the RING finger are also represented. At the bottom, reactivity of N20, SMP14, 2A10 and C-Ter antibodies with the full length Mdm2 protein epitopes is indicated. B, Representation of the three major Mdm2 isoforms, p85/90, p74/76 and p57/58. p85/90 contain all Mdm2 epitopes, p74/76 is devoided of N-terminal epitope that maps between residues 19 to 50 (region required for binding to p53) and p57 has deletion of the C-terminal epitope.

Figure 2 .

 2 Figure 2. Immunohistochemical analysis of Mdm2 in normal lung and lung tumors. A, Mdm2 nuclear immunostaining with SMP14 antibody in normal bronchial basal and suprabasal cells (score 50) (x200); B, Normal lung alveolar parenchyma stained with N20 antibody. Type II cells show moderate nuclear staining (arrow) (score 80) (x200); C, Squamous cell carcinoma immunostained with C-Ter antibody showing a faint Mdm2 nuclear expression (score 20) (x200). Note the moderate Mdm2 expression in entrapped type II cells (arrow); D, Squamous cell carcinoma exhibiting a strong nuclear staining with 2A10 antibody (score 150) (x200); E, Small cell lung carcinoma immunostained with N20 antibody (score 50) (x200); F, Large cell neuroendocrine carcinoma immunostained with SMP14 antibody (score 180) (x200); G-H, Small cell lung carcinoma showing discordances between a high nuclear staining

Figure 3 .

 3 Figure 3. Representative western blots showing the expression of Mdm2 isoforms in lung tumors. Expression of Mdm2 isoforms in tumor samples (T) was compared to that of normal lung (NL). A, Immunoblotting with 2A10 antibody. T6 and T19 are representative of lung tumors that overexpress p85/90 isoform. B, Immunoblotting with N20 antibody.T6 overexpress both p85/90 and p57 isoforms and T16 p57 isoform only. C, Immunoblotting with SMP14 antibody. Overexpression of p85/90 in T6 sample according to 2A10 and N20 staining. T23 is the only tumor that overexpress p74/76 isoform using SMP14. D, Immunoblotting with C-ter antibody. T8 and T22 are representative of tumors that overexpress p74/76 isoform. T24 exhibits high levels of p85/90 only.

Figure 4 .

 4 Figure 4. Comparison of Mdm2 and p14 ARF mean scores according to the histological type of human lung tumors.

Statistical Analyses

All statistical correlations were based on the chi-square (X 2 ) test. Additional Student paired-T test was performed as mentionned in the text.

Aknowledgments