Gene Expression in the Lung of p53 Mutant Mice Exposed to Cigarette Smoke

Point mutations and deletion in the p53 gene are the most common genetic lesions in human cancer (1, 2, 3, 4), and germ-line inactivation of one p53 allele is a hallmark of Li-Fraumeni syndrome. This syndrome is characterized by occurrence of cancers in multiple organs, among which there is an extraordinarily high incidence of lung cancer in smokers (3).

A p53 transgenic mouse model was obtained recently by crossing UL53–3 mice to A/J mice, carrying the pulmonary adenoma susceptibility 1 (Pas 1) locus (5) and having a deletion in the EcoR1-generated 0.55-kb K-ras fragment (6). These genetic alterations render the A/J strain highly susceptible to the development of lung tumors, of which the yield increases “spontaneously” with age (7). The (UL53–3 × A/J)F₁ p53 mutant mice retain both copies of the normal p53 alleles, but introduction of a p53 transgene expressing the mutant p53 oncoprotein (Val₁₅₃) inactivates endogenous wild-type (wt) p53 (3, 8). A significant enhancement of lung tumor yield was observed in these mutant mice treated with the tobacco carcinogens benzo(a)pyrene or 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (3).

Despite the major role played by cigarette smoke in the epidemiology of lung cancer (9, 10, 11), it is difficult to reproduce the lung tumorigenicity of this complex mixture in animal models. A weak induction of tumors was observed in various strains of mice exposed whole-body to environmental cigarette smoke (12, 13). Removal of damaged cells via apoptosis represents one of the mechanisms hampering the formation of lung tumors in cigarette smoke-exposed rodents. In fact, exposure to cigarette smoke results in a strong induction of apoptosis in the respiratory tract of both mice (14) and rats (15).

On the basis of these premises, we recently performed a study in wt and p53 mutant (UL53–3 × A/J)F₁ mice, either sham-exposed or exposed to environmental cigarette smoke (16). Although the response of both wt and p53 mutant mice was not striking, this study provided evidence that p53 mutations play a role in smoke-related carcinogenesis not only in humans but also in A/J mice. In fact, mutant mice exhibited an enhanced susceptibility to environmental cigarette smoke in the medium term (28 days of exposure), as shown by a decreased apoptosis in bronchial epithelial cells, an increased formation of bulky DNA adducts in both heart and lungs, and an increased frequency of micronuclei in both pulmonary alveolar macrophages and peripheral blood erythrocytes. In the long term (5 months of exposure to environmental cigarette smoke followed by 4.5 months of recovery in filtered air), mutant mice were more sensitive than wt mice in developing lung tumors (16). Furthermore, even in sham-exposed mice the presence of the p53 mutation was associated with an early stimulus of bronchial cell proliferation and an age-related increase of bulky DNA adducts in both heart and lungs (16). In addition, irrespective of exposure to environmental cigarette smoke, mutant mice exhibited significantly higher incidences of forestomach keratosis and papillomas and glandular stomach hyperplasia.⁴ These findings suggest the following: (1) p53 plays a physiologic protective role in A/J mice, (2) the effect of p53, having been detected in lungs, heart, forestomach, glandular stomach, and peripheral blood (where it reflects alterations in bone marrow), has a multiorgan distribution, and (3) the p53 mutation affects the response to environmental cigarette smoke.

We report herewith the results of an additional study in which we evaluated multigene expression in the lung of both wt and p53 mutant A/J mice, either sham-exposed or exposed for 28 days to environmental cigarette smoke. The same lung samples used in the previous study (16) were analyzed for the expression of 1,185 genes by cDNA array and, for a limited number of genes, by reverse transcription-PCR (RT-PCR) and real-time quantitative-PCR. The aim of the present study was to explain at a transcriptional level the patterns of molecular alterations, cytogenetical changes, and lung tumors observed in the previous study (16). Due to the huge amount of work involved and to the fact that our major goal was to assess the influences of the p53 status and of exposure of mice to environmental cigarette smoke rather than the interindividual variability, we decided to analyze in triplicate the lung pools of the animals belonging to each experimental group. The results obtained highlight the role of both p53 status and environmental cigarette smoke exposure in affecting the expression of genes belonging to a variety of functional categories.

Twenty female p53 transgenic (UL53–3 × A/J)F₁ mutant mice (p53+/−) and 20 female wt littermate controls carrying 99.9% A/J background (p53+/+), which contained 5% of FVB genome, were bred at Ohio State University (Columbus, OH). Mutant mice were rederived into the A/J background after five generations by backcrossing from original p53 transgenic mice carrying a 135 val p53 mutation in exon 5 on a FVB/J mouse background, as described previously (4, 8, 16).

At the age of 4 to 5 weeks, the mice were shipped to the University of Genoa, where the experiment was conducted. The mice were housed in Makrolon cages on sawdust bedding and maintained on standard rodent chow (MIL, Morini, S. Polo d’Enza, Italy) and tap water ad libitum. The temperature of the animal room was 23 ± 2°C, with a relative humidity of 55% and a 12-hour day–night cycle. The housing and treatments of animals were in accordance with national and institutional guidelines.

After 20 days of acclimatization, the mice were divided into four experimental groups, each one composed of 10 mice, including sham-exposed or environmental cigarette smoke-exposed p53 mutant mice and sham-exposed or environmental cigarette smoke-exposed wt mice. Sham-exposed mice were kept for 28 days in filtered air. Environmental cigarette smoke-exposed mice were exposed whole-body for 28 consecutive days to the smoke generated by 1R3 reference cigarettes (Tobacco Research Institute, University of Kentucky, Lexington, KY), having a declared content of 27.1 mg total particulate matter, 22.8 mg tar, and 1.46 mg nicotine each. A mixture of sidestream cigarette smoke (89%) and mainstream cigarette smoke (11%), mimicking an exposure to environmental cigarette smoke, was produced by using a smoking machine (model TE-10, Teague Enterprises, Davis, CA), burning 5 cigarettes at one time, 6 hours a day divided in two 3-hour rounds with a 3-hour interval. The smoldering cigarette was puffed for 2 seconds, once every minute, for a total of 8 puffs per cigarette, at a flow rate of 1.05 L/min to provide a standard puff of 35 cm³. Under these conditions, the average total particulate matter in the exposure chambers was 113 mg/m³, and the average CO concentration was 580 ppm.

After 28 days, all of the mice were deeply anesthetized with diethyl ether and killed by cervical dislocation. The lungs were removed and immediately stored at −80°C.

Lung samples (100 mg) were pooled within each one of the four experimental groups and homogenized at 4°C in a guanidinium-thiocyanate containing buffer (Perkin-Elmer, Boston, MA). After digestion with proteinase K (Boehringer Mannheim, Mannheim, Germany), the material was sequentially treated with phenol and chloroform, digested with RNase-free DNase I (Sigma Chemical Co., St. Louis, MO), and treated again with phenol and chloroform. Total RNA was pelleted on nitrocellulose filters (Precipitette Cartridge, Perkin-Elmer) by adding isopropanol and washed three times with 80% EtOH. The whole extraction procedure was performed in a helium atmosphere by using an automated nucleic acid extractor (Genepure 341, Applied Biosystems, Foster City, CA). RNA concentration and purity were evaluated by spectrophotometric analysis. RNA structural integrity was assessed by gel electrophoresis. The presence of the rRNA 28S and 18S bands, corresponding to lengths of 4.5 and 1.9 kb, respectively, was assumed to be an indicator of absence of RNA degradation.

Gene expression was evaluated, as described previously (17), by synthesizing mRNA-complementary radiolabeled nucleotide sequences, hybridized with cDNA probes immobilized on a nylon membrane. ³²P-labeled mRNA-complementary nucleotide sequences were synthesized by incubating 15 μg total RNA with 200 units of reverse transcriptase (RT, superscript II RT, Life Technologies, Inc., Gaithersburg, MD) at 48°C for 140 minutes in the presence of gene-specific primers (Clontech Lab., Palo Alto, CA) and AT-α-³²P (Amersham, Buckinghamshire, United Kingdom). Radioactive nucleotide sequences were purified on ChromaSpin-200 diethyl pyrocarbonate-H₂O columns (Clontech), and their radioactivity was measured by a ³²P imager (InstantImager, Packard, Meriden, CT). The samples tested in each experiment were accurately equalized by total amounts of counts (7 × 10⁶ cpm/sample) and mixed with Cot-1 DNA (Clontech). Commercially available cDNA arrays spotted with the products of 1,185 genes (Clontech, ATLAS Mouse 1.2 Cancer Array) were prehybridized with fragmented salmon sperm DNA (Sigma) and incubated overnight at 68°C with ³²P-containing nucleotide sequences in a hybridization oven (Bibby Stuart, Staffordshire, United Kingdom). Hybridized arrays were washed six times with SDS-SSC solutions and analyzed by ³²P imager. Data analysis was performed by using ATLAS Image and ATLAS Navigator 1.5 software (Clontech). The array used included both positive controls (9 housekeeping genes) and negative controls [M13 mp18(+) strand DNA, λDNA, and pUC18 DNA]. Information on each spotted gene is available at the Clontech⁵ and Swiss-Prot⁶ websites. Three independent experiments were performed. For each gene, the ratio of signal intensity to background levels was quantified, and the mean (± SE) result obtained for each gene in the three experiments was calculated. The results were normalized by referring to the average values of the expression of all genes in all of the experiments.

p53 gene expression was also evaluated by semiquantitative RT-PCR. Gene-specific primers for p53 mRNA, designed by a commercially available software (Primer Premier 4, Premier Biosoft International, Palo Alto, CA), were used to synthesize cDNAs. The RT-PCR reaction was as follows: 1 μL (2 pmol) specific antisense primer, 1 μL 10 mmol/L deoxynucleotide triphosphate mix, 9 μL H₂O, and 1 μL RNA (1 μg/μl). The mixture was heated at 65°C for 5 minutes and quickly chilled in ice. Four μL of 5× first-strand buffer, 2 μL 0.1 mol/L DTT, and 1 μL (40 units/μl) RNase inhibitor (RNase OUT, Invitrogen) were added. The reaction mixture was incubated at 42°C for 2 minutes, after which 1 μL (200 U) of Superscript II RT (Invitrogen) was added. The reaction was carried out for 50 minutes at 42°C and stopped by heating at 70°C for 15 minutes. cDNA was amplified by PlatinumTaq polymerase (Invitrogen) reaction resulting in a 392-bp cDNA fragment. The number of PCR cycles was optimized to ensure that the product intensity fell within the linear phase of amplification. Specific reaction conditions, as related to primer sequences, were set up according to a commercially available software application (Primer Premier 4). The reaction product was separated by 1% agarose gel electrophoresis and identified by comparison with a DNA marker (pUC18DpnI Digest, Sigma). cDNA amounts were quantified by densitometric analysis using digital acquisition equipment (DC 120 Zoom Digital Camera, Eastman Kodak, Rochester, NY) and specifically designed software (1D Image Analysis Software, Eastman Kodak). The results were expressed in terms of arbitrary densitometric units indicating the intensity of fluorescence emitted by DNA-bound ethidium bromide after background subtraction. Three experiments were run for each tested sample. The expression of ubiquitin (GenBank code: X51703), a housekeeping gene, was evaluated in parallel in the same samples to normalize the data.

To additionally validate the results of cDNA array analyses, we performed a two-step real-time quantitative-PCR analyses of the expression of the transforming growth factor β 1 gene (GenBank code: M13177), the macrophage inflammatory protein 1 α type gene (GenBank code: M23447), and the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GenBank code: M32599), used to normalize the data. Two sequential reactions were performed. The first reaction was a reverse transcription procedure performed as described for p53 but using gene-specific antisense primers. This procedure results in the formation of first-strand cDNAs complementary to specific mRNAs. The second reaction was a real-time quantitative-PCR performed as follows: 12.5 μL ABsolute QPCR SYBR (Abgene, Epson, Surrey, United Kingdom), 0.5 μL sense primer (10 μmol/L), 0.5 μL antisense primer (10 μmol/L), 10.5 μL H₂O, and 1 μL cDNA. The mixture was incubated in a MyiQ Single Color real-time quantitative-PCR Detection System (Bio-Rad, Hercules, CA) at 95°C for 15 minutes to activate Taq polymerase (hot start reaction), followed by 40 amplification cycles. An internal reference standard was set up according to Rajeevan et al. (18). RNA-, cDNA-, and reverse transcription-free reactions were performed as negative controls. Three experiments were run for each tested sample.

Lung nuclei were separated from whole homogenates by differential centrifugation, fragmented by freezing and sonication, and nuclear proteins were recovered by centrifugation and supernatant collection. Proteins were quantified by the bicinchoninic acid method, and a standardized amount (100 μg) for each sample was transferred to an acrylamide gel. After the electrophoretic run, the gel was blotted to an Immun-Blot polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA) and then labeled with primary and secondary antibodies. Primary polyclonal rabbit antibodies (Novocastra Laboratories Ltd., Newcastle upon Tyne, United Kingdom) and secondary goat polyclonal antirabbit IgG-horseradish peroxidase antibodies (Santa Cruz Biotechnology Inc., Santa Cruz, CA) were used. Signals were detected by reaction with ECL Light Plus (Bio-Rad) and chemoluminescence detection (ChemiDoc, Bio-Rad). Densitometric data were processed by Quantity One 4.1.1 software (Bio-Rad). An authentic p53 positive reference standard (Becton and Dickinson, San Diego, CA) was used to confirm the identity of the p53 protein band. Amido black staining was executed to confirm that an equalized protein amount was present in each lane. Three separate experiments were performed.

All data were expressed as means ± SE of triplicate analyses. It should be noted that, having used pooled samples, the variability does not refer to variability between experiments but to variability of the arrays. The reproducibility of the data generated by cDNA arrays in three separate experiments was satisfactory, the average SE of the three data per gene among all 1,185 of the tested genes being the 17% of the corresponding mean values. For each one of the 42 genes reported in Table 1, mean and median values were compared, and distribution skewness was calculated within the 12 available data (4 experimental groups x 3 replicates). Both approaches indicated that the data are normally distributed. Accordingly, the statistical significance of differences between wt mice and mutant mice was evaluated by Student’s t test for unpaired data. To evaluate environmental cigarette smoke-related variations in gene expression, as detected by cDNA array, we considered increases in the environmental cigarette smoke to sham ratio >2 to be relevant. We preferred to assume this cut-off value as a biological criterion of significance. This approach, which is broadly used in the literature, is in our experience more restrictive than comparison of data by statistical analysis. In any case, we checked the significance of all of the environmental cigarette smoke-related variations >2 by statistical analysis (Student’s t test for unpaired data).

Table 2 summarizes the results relative to the analysis of p53 gene expression and p53 nuclear protein in the lung, as related to the p53 status of mice and their exposure to environmental cigarette smoke for 28 days. Both cDNA array and RT-PCR analyses showed that p53 gene expression is neither significantly affected by the germ-line p53 mutation nor by exposure to environmental cigarette smoke. However, the amounts of p53 protein were significantly higher in both sham-exposed mice (4.4-fold) and environmental cigarette smoke-exposed mutant mice (2.2-fold) compared with wt mice. Environmental cigarette smoke induced a significant increase of p53 protein in wt mice (2.4-fold) but not in mutant mice (1.2-fold). In addition to p53, the used cDNA array included the related genes mdm2 and p53-binding protein 2. Irrespective of the p53 status and exposure of mice to environmental cigarette smoke, no variation in the expression of these genes was detected in either wt mice or mutant mice (data not shown).

Table 1 reports the list of the 42 genes of which the expression, evaluated by cDNA array, was either induced >2-fold after exposure to environmental cigarette smoke and/or was significantly different in wt and p53 mutant mice. The genes are identified according to their name, GenBank code, and Atlas code. They are classified into functional categories, keeping in mind that several genes possess various functions, and accordingly they could be classified into different categories. The effect of the exposure to environmental cigarette smoke was considered to be biologically significant when the average ratio of environmental cigarette smoke to sham signal was >2 (bold characters in Table 1). All of the environmental cigarette smoke-related variations >2 were also statistically significant (P < 0.05). The differences in gene expression between wt and p53 mutant mice were assessed by statistical analysis. Upward arrows in Table 2 denote a significant increase of gene expression, whereas downward arrows denote a significant decrease of gene expression in mutant mice, compared with wt mice. The identity of the remaining 1,143 genes can be inferred, by exclusion, from the list relative to the ATLAS Mouse 1.2 Cancer Array available on the internet.⁵

In sham-exposed mice, the expression of 5 genes (0.4%) was significantly higher in mutant mice compared with wt mice, whereas the expression of 10 genes (0.8%) was significantly lower. Irrespective of the p53 status, none of the 1,185 tested genes was decreased >2-fold in environmental cigarette smoke-exposed mice, compared with sham-exposed mice. In contrast, there was a >2-fold increase of gene expression, attributable to environmental cigarette smoke, for 15 genes (1.3%) in wt mice and 31 genes (2.6%) in mutant mice. Induction by environmental cigarette smoke of 22 genes (1.9%) was significantly higher in mutant mice, whereas 6 genes (0.5%) were more environmental cigarette smoke-inducible in wt mice (Table 1).

In particular, as shown in Table 1, within the category “Apoptosis” environmental cigarette smoke up-regulated 2 proapoptotic genes in wt mice only. Within the category “Cell cycle”, 7 positive regulators (cyclin D1, 3 CDC25s, 2 MAPKs, and protein tyrosine phosphatase 4a1) were induced by environmental cigarette smoke in mutant mice only. Three other positive regulators (a MAPK, protein phosphatase 5 catalytic subunit, and src-related kinase) were more expressed in sham-exposed mutant mice than in wt mice and, irrespective of the p53 status, did not respond to environmental cigarette smoke. On the other hand, a negative regulator of the cell cycle (p21) was induced by environmental cigarette smoke in wt mice only.

A selective effect of environmental cigarette smoke in mutant mice was detected for 3 PKCs involved in “signal transduction”. The genes encoding for 8 “growth factors” were induced by environmental cigarette smoke. In particular, environmental cigarette smoke induced angiogenin-related protein in both wt and mutant mice, angiopoietin in wt mice only, and 6 factors (VEGF A and B, a PDGF, GRB2, and 2 TGF-related genes) in mutant mice only. The basal expression of all of these 6 factors, with the exception of GRB2, was significantly lower in mutant mice than in wt mice. The environmental cigarette smoke-related overexpression of TGF β 1, as assessed by cDNA array, was confirmed by real-time quantitative-PCR. When using this technique, the environmental cigarette smoke to sham ratio was 1.81 in wt mice and 5.10 in mutant mice.

All 3 of the genes involved in “xenobiotic metabolism”, encoding for a transmembrane activity (MDR1), a microsomal Phase I activity (CYP1A2), and a microsomal Phase II activity (UDPGT1) were induced to the same extent in wt mice and mutant mice. Similarly, with few exceptions, wt mice and mutant mice responded to environmental cigarette smoke in the same manner by overexpressing 7 genes involved in “protein repair, removal, and folding.” They included 4 HSPs, 1 HST, and 2 T-complex protein 1 subunits. Under basal conditions, 1 of them (T-complex protein 1 epsilon subunit) was significantly more expressed in mutant mice, which, however, did not respond to the environmental cigarette smoke stimulus.

Seven genes belonging to the category “Inflammation and immune response” were induced by environmental cigarette smoke. In particular, IL6 was selectively overexpressed in wt mice. The basal expression of 4 genes (MIP 1 α and 1β, mast cell factor, and prothymosin β 4) was significantly lower in mutant mice. However, their expression was selectively increased in mutant mice after exposure to environmental cigarette smoke. A similar selective response of mutant mice to environmental cigarette smoke was observed with 2 additional genes (cytokine inducible SH2-containing protein 2 and PPAR gamma), of which the basal expression was comparable in wt mice and mutant mice. In contrast, 1 gene (lymphocyte antigen 68) displayed a higher basal expression in mutant mice but, irrespective of the p53 status, did not respond to environmental cigarette smoke. The environmental cigarette smoke-related overexpression of MIP 1 α in mutant mice, as assessed by cDNAarray, was confirmed by real-time quantitative-PCR. When using this technique, the environmental cigarette smoke to sham ratio was 0.86 in wt mice and 2.44 in mutant mice.

The present study had two major goals. The first one was to evaluate, by cDNA array technology, the effect produced by cigarette smoke in mice having an A/J background, which are characteristically susceptible to pulmonary carcinogenesis (5, 6, 7). Despite the overwhelming impact of cigarette smoke on human health, the comprehension of the mechanisms involved is hampered by the complexity of this mixture, containing many agents that have convincingly been shown to cause lung tumors in laboratory animals and/or humans (19). Indeed, very few studies have investigated in vivo the effect of cigarette smoke on multigene expression. For instance, the expression of 2,031 genes was evaluated in the nasal epithelium and lungs of Sprague-Dawley rats exposed to mainstream cigarette smoke (20). In our laboratory, the expression of 746 toxicologically relevant genes was evaluated in the liver of fetuses of Swiss albino mice exposed to environmental cigarette smoke throughout pregnancy (17) and in the skin and lungs of SKH-1 hairless mice exposed to environmental cigarette smoke, either individually or in combination with other agents (21). An additional problem when working in vivo with inhalable agents is that the lung contains a number of cellular types. Thus, the results obtained when testing whole lung preparations reflect the effect of cigarette smoke on a mixed-cell population. Certainly, the comparative assay of environmental cigarette smoke-related effect in individual cells of the respiratory tract warrants additional studies.

The second goal of the present study was to evaluate the influence of the p53 status on multigene expression in the lung, both under basal conditions and after exposure to environmental cigarette smoke. The comparison of wt and p53 mutant mice allowed us to detect the differential expression of many genes involved in a variety of cell functions. It is noteworthy that the expression of p53 and the related genes mdm2 and p53-binding protein 2 was neither affected by the p53 status of mice nor by their exposure to environmental cigarette smoke. These data suggest that differences between wt and mutant mice in p53 functions do not occur at the level of gene expression but at a post-transcriptional level. In fact, in mutant mice the basal levels of p53 nuclear protein were strikingly higher but not inducible by environmental cigarette smoke, which conversely increased p53 levels in the lung of wt mice. The higher levels of p53 protein in mutant mice can be ascribed to the fact that p53 mutation results in the accumulation of mutated oncoprotein in the nucleus (22). The lack of p53 increase in environmental cigarette smoke-exposed mutant mice may be related to the fact that mutant p53 protein acts as a dominant-negative inhibitor of wt p53 production (23). In contrast, the p53 increase in environmental cigarette smoke-exposed wt mice is in line with the notion that wt p53 is phosphorylated and becomes stabilized in response to DNA damage (22).

It is well known that p53 protects the cell from DNA damage by inducing apoptosis and/or by slowing down cell replication via G₁ arrest (1, 2). In fact, different responses between mutant and wt mice were observed in the expression of genes involved in both apoptosis and cell cycle regulation. Interestingly, even in sham-exposed mutant mice some genes involved in the positive regulation of the cell cycle were selectively overexpressed. One of them was src-related kinase, which is known to be increased in proliferating epithelia (24). This conclusion is in agreement with our previous finding that, in the bronchial epithelium of the same sham-exposed mice, there was a significant increase of proliferating cell nuclear antigen, detected by immunohistochemistry (16). Proliferating cell nuclear antigen is required for DNA replication as a component of the DNA repair machinery (25). The differences between wt and mutant mice were more evident after exposure to environmental cigarette smoke. First of all, the negative regulator of the cell cycle p21 was induced by environmental cigarette smoke in wt mice only. p21 is the main effector of p53 for cell cycle arrest, as shown by the findings that p53 fails to induce G₁ arrest in the absence of p21 expression (26) and that p21 mRNA is undetectable in immortalized Li-Fraumeni cells homozygous for mutant p53 (27). Interestingly, p21 expression was found to be increased in alveolar cells exposed to cigarette smoke (28) and in alveolar macrophages and bronchial epithelial cells from smokers (29). The observed inability of p53 mutant mice to activate p21 indicates that these mice are not able to activate the defense mechanism of cell growth arrest as a response to environmental cigarette smoke-induced DNA damage. At the same time, environmental cigarette smoke up-regulated, but only in mutant mice, 7 genes involved in the positive regulation of the cell cycle. Among them, G₁-S–specific cyclin D1 is known to be down-regulated by p21 (30), 3 CDC25 isoforms are M-phase inducers of which the activity is antagonized by p53 (31), and 2 MAPKs have been related to the degradation of p53 protein via the proteasome pathway (32). Up-regulation of MAPKs by cigarette smoke, which has already been shown to occur in vitro in hamster fetal pulmonary cells (33), can be interpreted as an attempt to remove the abnormal accumulation of mutated p53 protein in mutant mice.

In parallel, environmental cigarette smoke selectively induced apoptosis in wt but not in p53 mutant mice, as shown by up-regulation of two caspase-related genes. The same patterns were pointed out by measuring the frequency of apoptotic cells in the bronchial epithelium (16). Therefore, it appears that apoptosis, which represents a crucial defense mechanism against cigarette smoke-related lung tumors (14, 15), is defective in p53 mutant mice. At variance with apoptosis, DNA repair mechanisms are poorly involved in protecting lung cells from cigarette smoke genotoxicity. In fact, irrespective of the p53 status, no DNA repair-related gene was induced in environmental cigarette smoke-exposed mice. The poor inducibility by environmental cigarette smoke of DNA repair-related genes had been observed previously in our laboratory in the liver of fetuses of Swiss albino mice exposed throughout pregnancy (17) and in the skin and lungs of SKH-1 hairless mice (21). Similarly, none of the typical oncogenes or tumor suppressor genes included in the tested cDNA array was modulated in its expression either by the p53 status or by exposure of mice to environmental cigarette smoke.

A selective environmental cigarette smoke-related overexpression of 3 PKC genes involved in signal transduction was observed only in p53 mutant mice. In vitro studies have demonstrated that PKCs possess an antagonistic role toward the p53 pathway (34, 35, 36). Accordingly, PKC activation results in an increased cell proliferation and decreased apoptosis. PKC had been found previously to be transiently activated in the lung of rats exposed to sidestream cigarette smoke, which may contribute to the promotion of cigarette smoke-related lung cancer (37). TGF β 1-related genes exhibited significantly lower basal levels in the lung of p53 mutant mice, but their expression was selectively induced after exposure to environmental cigarette smoke. TGF β 1, of which the expression was also increased in bronchial biopsies from smokers as compared with nonsmokers (38), is an upstream activator of p53 in response to DNA damage (39) and induces cell cycle inhibition and growth arrest in a p53-independent manner in lung epithelial cells (40). It is conceivable that the environmental cigarette smoke-related increase of TGF β 1 expression observed in mutant mice may represent an attempt to delay the cell cycle as an alternative to the p53 pathway.

Several genes involved in angiogenesis, including 2 angiogenic proteins, 2 VEGFs, PDGF, and GRB, were up-regulated by environmental cigarette smoke. This finding suggests that blood vessel proliferation could represent a response to environmental cigarette smoke-induced hypoxia in lung. It is established that nicotine stimulates angiogenesis, and VEGF plays a role in this effect (41). The environmental cigarette smoke-related induction of angiogenin-related protein was similar in wt mice and mutant mice, and induction of angiopoietin was attenuated in mutant mice. In contrast, the basal expression of VEFG A, VEGF B, and PDGF was higher in wt mice but was induced by environmental cigarette smoke only in mutant mice. A relationship between p53 status and VEGF expression has been demonstrated previously, mutant p53 being a potent inducer of VEGF expression (42). PDGF and its functionally related receptor GRB exert a potent promitotic activity by stimulating the cells to grow and to heal wounds. It is likely that the selective activation of PDGF by environmental cigarette smoke in mutant mice may represent an attempt to activate the p53 pathway, because this growth factor is involved in p53 phosphorylation, a critical event in the up-regulation of p53 during cellular stress (43). On the whole, our findings provide evidence that, after exposure to environmental cigarette smoke, p53 mutant mice are more prone than wt mice to angiogenesis, a mechanism that could contribute to the development of environmental cigarette smoke-induced lung tumors.

The expression of Phase I (CYP1A2) and Phase II (UDPGT 1) microsomal activities involved in the metabolism of xenobiotics, after exposure to environmental cigarette smoke, was up-regulated to a similar extent in wt and p53 mutant mice. The CYP1A1 gene, which has been found to be induced by mainstream cigarette smoke in the nasal epithelium and lung of Sprague-Dawley rats (20) and by environmental cigarette smoke in skin and lungs of SKH-1 hairless mice (21), was not included in the cDNA array used. The poor effect of p53 mutation on the metabolic activation or detoxification of environmental cigarette smoke components is in agreement with the finding that the metabolic activation of nitrosamines was not altered in p53+/− knockout mice (44). Moreover, the expression of CYP1A2 and GST-α genes was similar in the liver of wt and p53+/− mice (45).

As for membrane activities, environmental cigarette smoke up-regulated the expression of MDR1 in both wt and mutant mice. MDR protein is responsible for the extrusion outside cells not only of cytostatic drugs, thus impairing their efficacy, but also for the removal of toxic agents, thus protecting the cell (46). These results confirm our previous data generated in the liver of Swiss albino mouse fetuses exposed transplacentally to environmental cigarette smoke (17) and in both skin and lung of SKH-1 hairless mice exposed whole body to environmental cigarette smoke (21). These findings support the role of MDR1 as a p53-independent defense mechanism against cigarette smoke. In contrast, MDR1 up-regulation by cytostatic drugs has been reported to be expressed more efficiently in p53 mutant mice (47).

Wt and p53 mutant mice behaved in a similar way with regard to the environmental cigarette smoke-related up-regulation of 7 genes involved in the repair and removal of damaged proteins. The up-regulated activities also included 4 HSPs, of which the expression has been shown to be induced in vitro by cigarette smoke (48, 49). On the other hand, mutant mice were more susceptible than wt mice regarding the induction by environmental cigarette smoke of 6 genes involved in inflammation and immune response, which generally exhibited lower levels in mutant mice under basal conditions. Only one IL6-related gene was more intensely expressed in the lung of wt mice. These patterns indicate that the immune system reacts to environmental cigarette smoke exposure by inducing an inflammatory response in the lung, which is affected by the p53 status. The selective overexpression of PPARγ in mutant mice is likely to be related to the occurrence of lipid peroxidation in the lung of environmental cigarette smoke-exposed mice, because PPARγ is involved in fatty acid catabolism. In addition, PPARγ has been shown to possess a p53-dependent proapoptotic role that was only active in wt but not in p53 mutant cells in vitro (50).

On the whole, as we reported previously for molecular alterations, cytogenetical damage, and lung tumors (16), the effect of environmental cigarette smoke on multigene expression of mice carrying an A/J background was not dramatic but well evident. The p53 mutation appears to affect per se some physiologic functions and greatly enhances the response to environmental cigarette smoke, at both genomic and postgenomic levels, thus explaining the increased susceptibility of mutant mice to smoke-related alterations of intermediate biomarkers and development of lung tumors.