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Tobacco Carcinogen-Induced Cellular Transformation Increases Activation of the Phosphatidylinositol 3'-Kinase/Akt Pathway in Vitro and in Vivo

¹ Cancer Therapeutics Branch,
² Cell and Cancer Biology Branch, and
³ Laboratory of Human Carcinogenesis, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland, and
⁴ Lovelace Respiratory Research Institute, Albuquerque, New Mexico

	ABSTRACT

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The role of the phosphatidylinositol 3'-kinase (PI3K)/Akt pathway during tobacco carcinogen-induced transformation is unknown. To address this question, we evaluated this pathway in isogenic immortalized or tumorigenic human bronchial epithelial cells in vitro, as well as in progressive murine lung lesions induced by a tobacco-specific carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. Compared with immortalized cells, tumorigenic cells had greater activation of the PI3K/Akt pathway, enhanced survival, and increased apoptosis in response to inhibition of the pathway. In vivo, increased activation of Akt and mammalian target of rapamycin was observed with increased phenotypic progression. Collectively, these results support the hypothesis that maintenance of Akt activity is necessary for survival of preneoplastic as well as transformed lung epithelial cells and suggest that inhibition of the PI3K/Akt pathway might be a useful approach to arrest lung tumorigenesis.

	Introduction

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Lung cancer remains the leading cause of cancer death throughout the world, causing approximately 1.2 million deaths annually (1) . Although approximately 90,000,000 current or former smokers in the United States have a permanently increased relative risk for developing lung cancer (and are at increased risks for developing other tobacco-related diseases), only 10–18% of this group will ultimately develop lung cancer (2) . Thus, the identification of early molecular events inherent to lung tumorigenesis would provide a basis for interventions aimed at preventing the phenotypic progression of lung cancer. Alterations in tumor suppressor genes or oncogenes were the first molecular events to be identified in lung tumorigenesis, but changes in activation of signal transduction pathways may also be important (3) .

Akt is likely intrinsic to the biology of lung cancer because our laboratory has shown that Akt is active in non-small cell lung cancer cells and promotes cellular survival and resistance to chemotherapy or radiation (4) . Moreover, we recently demonstrated that administration of nicotine or a tobacco-specific carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), induced activation of Akt in a dose-and time-dependent manner in primary human lung epithelial cells of both large and small airway origin (5) . Activation of Akt in these primary cells conferred protection against different cellular stresses such as DNA damage, serum withdrawal, and cell detachment. Moreover, daily or bolus dosing with nicotine decreased contact inhibition, dependence on extracellular growth factors, and adherence to the extracellular matrix, all properties of a transformed phenotype. Cumulatively, these results suggested that rapid Akt activation by either nicotine or NNK may be an early event caused by smoking and that Akt activation may play a role in tobacco-induced lung cancer.

Because Akt increases the survival of cells at each end of the phenotypic spectrum of lung tumorigenesis (in established lung cancer cells and primary lung epithelial cells), we sought to extend these studies and examine Akt activation at additional intermediate steps in carcinogenesis. Therefore, we analyzed the phosphatidylinositol 3'-kinase (PI3K)/Akt pathway in isogenic, immortalized (BEAS2B) or tumorigenic (NNK-BEAS2B) human lung bronchial epithelial cells in vitro and in a spectrum of NNK-induced murine lung lesions in vivo. Progressive activation of the PI3K/Akt pathway correlated with phenotypic progression of lung epithelial cells in both mammalian systems, thereby strengthening the hypothesis that Akt activity plays a role in lung tumorigenesis.

	Materials and Methods

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Cell Culture.
Immortalized BEAS2B and isogenic, fully transformed NNK-BEAS2B cells were generated and cultured as described previously (6 , 7) . Briefly, these SV40-immortalized, telomerase-positive cells were maintained at 37°C, 3.5% CO₂ in serum-free LHC-8 media plus the following growth supplements: bovine pituitary extract; epidermal growth factor; epinephrine; hydrocortisone; insulin; and transferrin (BioSource, Camarillo, CA). For growth factor-free (GFF) conditions, cells were cultured in serum-free DMEM/HAM’s F-12 (1:1; Life Technologies, Inc., Gaithersburg, MD) plus 1% penicillin/streptomycin (Life Technologies, Inc.).

Reagents.
Phospho-specific antibodies directed against serine 473 of Akt, serine 241 of PDK-1, serine 2448 of mammalian target of rapamycin (mTOR), serine 166 of Mdm2, serine 256 of FKHR/AFX, serine 389 of p70/S6K1, serine 21 of glycogen synthase kinase (GSK)-3, serine 9 of GSK-3ß, threonine 1462 of TSC2, native antibodies against Akt, and antibodies against the cleaved form of poly(ADP-ribose) polymerase were obtained from Cell Signaling Technology (Beverly, MA). Antibodies against -tubulin, PTEN, and green fluorescence protein were obtained from Sigma (Milwaukee, WI), Oncogene Research (EMD Biosciences, Inc., San Diego, CA), and Zymed Laboratories, Inc. (South San Francisco, CA), respectively. Antibodies against cyclin D1, p27^kip, and p21^cip were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). LY294002 and U0126 were obtained from CalBiochem (EMD Biosciences, Inc.). PIA24 was synthesized as described previously (8) .

Proliferation Assay.
BEAS2B or NNK-BEAS2B cells were plated under normal growth conditions in sextuplet in 96-well plates at 5 x 10³ cells/well and allowed to recover overnight. Cells were then deprived of growth factors, and, at the indicated times, the 96-well plates were processed and analyzed using a plate reader as described previously (5) .

Apoptosis.
For measurement of apoptosis, BEAS2B and NNK-BEAS2B cells were plated in complete media or media devoid of growth factors for 48 h. Pharmacological inhibitors [LY294002 (10 µM) or PIA24 (2.5 µM)] were added at the time of growth factor deprivation. After treatment, apoptosis was assessed by flow cytometry as described previously (4) .

Adenoviral Infection of BEAS2B and NNK-BEAS2B Cells.
For measurement of apoptosis caused by adenoviruses containing dominant negative Akt, BEAS2B and NNK-BEAS2B cells were plated and infected with adenoviral particles encoding either the ß-galactosidase gene or dominant negative Akt (graciously supplied by Dr. Kenneth Walsh). The preparation and characterization of these adenoviral vectors have been described previously (9) . After infection, cells were placed in complete media or GFF media for 48 h. Apoptosis was assessed by flow cytometry as described above. Biochemical analysis of apoptosis and Akt pathway signaling after infection with adenovirus encoding ß-galactosidase dominant negative or constitutively active Akt was performed as described below.

Immunoblotting.
After different treatments, cell extracts were prepared, and SDS-PAGE and immunoblot analysis were performed as described previously (4) .

Immunohistochemical Analysis of NNK-Induced Tumors from A/J Mice.
For immunohistochemical analysis of Akt, mTOR, and GSK3ß phosphorylation or total levels of GSK3ß, A/J mice were given PBS or 9.1 mg of the tobacco-specific N-nitrosamine NNK in drinking water for 8 weeks and serially sacrificed (10) . Lung tissues were fixed in 10% neutral formalin and embedded in paraffin, and 5-µm sections were stained for phospho-Akt, phospho-mTOR, phospho-GSK3ß, or total GSK3ß. Specificity of these antibodies in immunohistochemistry has been demonstrated previously (5) .

Statistical Analysis.
Statistical comparison of mean values was performed using Student’s t test. All Ps are two tailed. Statistical evaluation of scoring of immunohistochemical specimens was performed using the Mann-Whitney sum test.

	Results

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Tobacco-Carcinogen Transformed Human Bronchial Epithelial Cells Have Increased Activation of the PI3K/Akt Pathway, Increased Proliferation, and Increased Survival.
The ability of cells to survive in the absence of serum or exogenous growth factors is one hallmark of cell transformation (11) , and this can be promoted by Akt activation (12) . To investigate the status of the PI3K/Akt pathway in immortalized BEAS2B and tumorigenic NNK-BEAS2B cells, we directly compared the responses of BEAS2B and NNK-BEAS2B cells with growth factor withdrawal. Immunoblot analysis of cell extracts from BEAS2B and NNK-BEAS2B cells using a phospho-specific Akt substrate antibody demonstrated that under normal growth conditions, the phosphorylation of substrates downstream of Akt is decreased in BEAS2B cells in comparison with NNK-BEAS2B cells (Fig. 1A) . When growth factors were removed from the media, phosphorylation of Akt substrates generally decreased in both cell types, but the phosphorylation of high molecular weight substrates (M_r >132,000) was maintained to a greater extent in NNK-BEAS2B cells. These results using the phospho-specific Akt substrate antibody suggest that after growth factor withdrawal, Akt activity is maintained to a greater extent in NNK-BEAS2B cells as compared with BEAS2B cells.

View larger version (48K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. Transformed lung epithelial cells [NNK-BEAS2B (NNKB)] demonstrate increased Akt signaling, increased proliferation, and decreased apoptosis compared with immortalized cells [BEAS2B (B)]. A, BEAS2B and NNK-BEAS2B cells were deprived of growth factors for 48 h, and extracts were prepared for immunoblot analysis using a phospho-specific antibody directed against the consensus Akt phosphorylation motif. B, cells were treated as described in A, and immunoblotting was performed using phospho-specific antibodies to serine 241 of PDK1, serine 473 or threonine 308 of Akt, serine 166 of Mdm2, serine 265 of FKHR/AFX, threonine 1462 of TSC2, serine 2448 of mTOR, serine 349 of p70/85S6K, serine 21/9 of glycogen synthase kinase 3/ß, or serine 65 of 4EBP-1 or native antibodies to Akt, PTEN, p27kip, p21cip, and -tubulin. C, for proliferation assays, cells were plated in 96-well plates, and proliferation was measured at the indicated times by absorbance at 540 nm using a 96-well plate reader (left panel). For apoptosis assays, cells were treated as described in A, and parallel samples were prepared for immunoblot analysis using antibodies specific for the cleaved form of poly(ADP-ribose) polymerase and native -tubulin (top right panel) or flow cytometric analysis of formation of sub-2N DNA (bottom right panel). Ps comparing apoptosis for each cell type under either growth condition were <0.05 (*). To assess the effect of signaling inhibitors, immunoblot and flow cytometric analysis (D and E, respectively) were performed on cells treated as described in A, except that LY294002 (10 µM) or PIA24 (2.5 µM) was added to the cells at the time of growth factor deprivation. Ps are shown. F, the relative increase in apoptosis for the BEAS2B and NNK-BEAS2B cells in response to the small molecule inhibitors was obtained by setting the value of either cell line in growth factor-free (GFF) media to 1 and comparing the rest of the data against this value. Ps for LY294002 versus GFF and PIA24 versus GFF are <0.05 (*). The Ps for comparison of apoptosis induced by LY294002 or PIA24 in BEAS2B or NNK-BEAS2B cells are shown. Immunoblotting experiments were performed three times, and the graphs in C, E, and F are representative of experiments performed three times in triplicate.

The ability of NNK-BEAS2B cells to maintain Akt pathway signal transduction and cyclin D1 levels with growth factor deprivation suggests that these cells may be more likely to proliferate and survive this cellular stress. To test this, we compared cellular proliferation under GFF conditions (Fig. 1C , left panel). NNK-BEAS2B cells, but not BEAS2B cells, were able to proliferate for 8 days. To compare apoptosis, we cultured BEAS2B or NNK-BEAS2B cells under GFF conditions for 48 h. Increased apoptosis was observed in BEAS2B cells, as assessed by poly(ADP-ribose) polymerase cleavage or flow cytometric analysis. NNK-BEAS2B cells demonstrated very little cell death under GFF conditions, compared with normal growth conditions. These data suggest that the ability of NNK-BEAS2B cells to maintain Akt pathway activity is linked to their ability to proliferate and resist growth factor withdrawal-induced apoptosis, which may contribute to the conversion to a tumorigenic phenotype.

Using Small Molecule Inhibitors to Show Dependence of Immortalized and Tumorigenic Lung Epithelial Cells on the PI3K/Akt Pathway for Cellular Survival.
To determine whether the maintenance of Akt signaling in NNK-BEAS2B cells during growth factor withdrawal was responsible for the increase in cellular survival observed under these conditions, we treated BEAS2B or NNK-BEAS2B cells with inhibitors of PI3K (LY294002) or Akt (PIA24) and assessed pathway inhibition and apoptosis (Fig. 1, D and E) . The addition of LY294002 or PIA24 completely inhibited phosphorylation of Akt and decreased phosphorylation of downstream substrates in both the BEAS2B and NNK-BEAS2B cells under GFF conditions.

We next investigated the effects of these molecules on cellular survival. Treatment of BEAS2B or NNK-BEAS2B cells with the small molecule inhibitors LY294002 or PIA24 potentiated apoptosis in response to growth factor withdrawal (Fig. 1E) , showing that immortalized or tumorigenic bronchial epithelial cells depend on the PI3K/Akt pathway for survival. A specific role for the PI3K/Akt pathway in survival of these cells was also supported by the fact that inhibition of the mitogen-activated protein/ERK kinase (MEK)/mitogen-activated protein kinase pathway by a MEK-specific small molecule inhibitor, U0126, had no effect on BEAS2B or NNK-BEAS2B apoptosis (data not shown). To determine whether the immortalized or tumorigenic lung epithelial cells were differentially sensitive to LY294002 or PIA24, we calculated the relative increase in apoptosis in response to these inhibitors using the data from Fig. 1E . As shown in Fig. 1F , the administration of LY294002 or PIA24 to BEAS2B cells caused an approximate 1.6–2.0-fold increase in apoptosis, whereas the addition of these inhibitors resulted in a 2.2–4.4-fold increase in apoptosis in the NNK-BEAS2B cells (Fig. 1F) . This suggested that the tumorigenic NNK-BEAS2B cells are more sensitive to the inhibition of the PI3K/Akt pathway.

Genetic Inhibition of Akt by a Dominant Negative Mutant Increases Apoptosis of BEAS2B or NNK-BEAS2B Cells.
The results presented above using pharmacological inhibitors of the PI3K/Akt pathway strongly implicate Akt signaling in the resistance of BEAS2B or NNK-BEAS2B cells to growth factor deprivation-induced apoptosis. Because small molecule inhibitors may have nonspecific effects, we wanted to confirm these results by genetically modifying the Akt pathway. BEAS2B or NNK-BEAS2B cells were infected with adenoviral dominant-negative Akt (Ad-dn Akt) or adenoviral encoded ß-galactosidase (Ad-ßGal) at a multiplicity of infection of 100, and apoptosis was assessed under normal or GFF conditions (Fig. 2A) . Apoptosis was greater in BEAS2B or NNK-BEAS2B cells expressing dominant negative Akt versus ß-galactosidase under normal or GFF conditions. In BEAS2B cells, apoptosis was 17% for Ad-dn Akt and 7% for Ad-ßGal under normal growth conditions and 30% versus 17% under GFF conditions, respectively. In NNK-BEAS2B cells, apoptosis was 14% for Ad-dn Akt and 5% for Ad-ßGal under normal growth conditions and 19% versus 8% under GFF conditions, respectively. The expression of dominant negative Akt in each cell type was demonstrated by immunoblot analysis of the epitope tags hemagglutinin and green fluorescence protein and Akt itself (Fig. 2A , bottom left panels). To assess relative sensitivity of these cells, we calculated the induction of apoptosis in BEAS2B and NNK-BEAS2B cells by Ad-dn Akt (Fig. 2A , bottom right panel). In normal media, Ad-dn Akt caused equal amounts of apoptosis in BEAS2B and NNK-BEAS2B cells (2.5-fold over the ß-galactosidase control). However, under GFF conditions, Ad-dn Akt induced more apoptosis in NNK-BEAS2B cells as compared with BEAS2B cells (2.0-fold versus 1.6-fold; Fig. 2A , bottom right panel). These data confirm the results of previous experiments using small molecule inhibitors; NNK-BEAS2B cells are more prone to apoptosis caused by inhibition of the PI3K/Akt pathway.

View larger version (39K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. Inhibition of Akt by adenoviruses containing dominant-negative Akt alters survival of immortalized (BEAS2B) and transformed (NNK-BEAS2B) lung epithelial cells. A, BEAS2B or NNK-BEAS2B cells were infected at a multiplicity of infection of 100 with either adenoviral dominant-negative Akt (Ad-dn Akt) or adenoviral ß-galactosidase (Ad-ßGal). After infection, cells were placed in either normal media or growth factor-deprived media for 48 h. Parallel samples were collected and analyzed by flow cytometry to evaluate apoptosis (top panel) or immunoblotted with antibodies that specifically recognize the hemagglutinin or green fluorescence protein epitope tags, native Akt, and -tubulin (bottom left panels). Ps for comparing apoptosis induced by Ad-dn Akt or Ad-ßGal were <0.05 (*). The induction of apoptosis was calculated using the data shown in A by setting apoptosis observed with Ad-ßGal under each experimental condition equal to 1 (bottom right panel). Ps are shown. B, BEAS2B and NNK-BEAS2B cells were infected at the indicated multiplicities of infection with either Ad-dn Akt or Ad-ßGal. The cells were left in infection media for 24 h, and apoptosis was measured 48 h later using flow cytometry (left panels). To verify infection, immunoblot analysis was performed on BEAS2B and NNK-BEAS2B cells infected with Ad-dn Akt using antibodies to native Akt, green fluorescence protein, and -tubulin (right panels). Induction of apoptosis by Ad-dn Akt at each multiplicity of infection is shown in bottom panel, where apoptosis caused by Ad-ßGal was set to 1. Comparisons of apoptosis caused by Ad-dn Akt in BEAS versus NNK-BEAS2B cells yielded Ps < 0.05 (*). Experiments were performed three times, and the graphs in A and B are representative of experiments performed three times in triplicate.

Increased Activation of the Akt Pathway in Preneoplastic and Neoplastic Murine Lung Lesions in Vivo.
The results from the BEAS2B/NNK-BEAS2B in vitro model presented above suggest that Akt activation is progressive during lung tumorigenesis. To further define the role of activation of the Akt pathway in lung tumor formation, we examined the well-characterized A/J mouse model of lung tumorigenesis, which uses systemic administration of NNK to induce lung tumors (14) . We performed an immunohistochemical analysis of paraffin-embedded lung tissue samples using phospho-specific antibodies against serine 473 of Akt, serine 2448 of mTOR, and serine 9 of GSK3ß. Staining for active Akt, mTOR, and GSK3ß was predominantly cytoplasmic and was observed in bronchial epithelium and in progressive histological alterations including type II cell hyperplasias (T2Hs), atypical adenomatous hyperplasias (AAH), adenomas, and adenocarcinomas (Fig. 3) .

View larger version (90K): [in this window] [in a new window] [Download PPT slide]   Fig. 3. Representative staining of bronchial epithelium, type II alveolar hyperplasias, adenomas, and adenocarcinomas using activation state-specific antibodies against Akt, mTOR, and glycogen synthase kinase 3ß. A–C illustrate intense expression of phospho-Akt in premalignant changes. Reactive type II pneumocytes in A and atypical alveolar hyperplasia (AAH) in B reveal strong immunoreactivity (arrows), whereas the epithelium surrounding the airway lumen (Lu) remains less reactive. C, papillary intraluminal growth also reveals more intense staining than untransformed epithelium (arrowhead in the bottom right corner). Immunoreactivity of mTOR is variable and weak in the histologically normal airway epithelium and type II cells (D) but intense in adenocarcinomas (E), whereas immunoreactivity remains low for phospho-glycogen synthase kinase ß in adenocarcinomas (F). Bar, 25 µm.

	Discussion

Top ABSTRACT Introduction Materials and Methods Results Discussion REFERENCES

One of the hallmarks of cell transformation is the acquired ability of cells to survive in the absence of exogenous growth factors. NNK-BEAS2B cells have been shown previously to form colonies in the presence of high serum concentrations, but their ability to survive in the absence of growth factors has not been thoroughly investigated. Our results indicate that on the withdrawal of growth factors, NNK-BEAS2B cells retain more active Akt compared with BEAS2B cells, as evidenced by sustained phosphorylation of Akt at serine 473 and threonine 308, as well as substrates downstream of Akt. Interestingly, these substrates consisted of components of the protein translational apparatus (TSC2, mTOR, 4EBP-1, and p70/85^S6K), cell metabolism (GSK3/ß), and regulators of apoptosis (Mdm2) and transcription of proapoptotic genes (forkhead family members FKHR/AFX).

Akt has been shown to phosphorylate both Mdm2 and forkhead family members, resulting in regulation of their activity and subsequent protection from apoptosis (16, 17, 18, 19) . Mdm2 controls the levels of p53 through regulation of proteasome-dependent p53 degradation, whereas the forkhead family of transcription factors controls the levels of the proapoptotic proteins Fas ligand, Bim, insulin-like growth factor-binding protein 1, and p27^kip (19, 20, 21) . Therefore, it is likely that the ability of NNK-BEAS2B cells to maintain Akt activity during the insult of growth factor deprivation potentially results in the inhibition of p53 and FKHR/AFX-dependent cell death, thereby increasing cell survival. These results are further supported by earlier work from our laboratory showing that in primary airway epithelial cells, Akt activation in response to nicotine and NNK increased phosphorylation of these same substrates and was necessary for survival during the imposition of different forms of cellular stress (5) . Although it is possible that inhibition of other modulators of cell death, in addition to p53 and the forkhead family, are also involved in NNK-BEAS2B survival during growth factor withdrawal, analysis of apoptosis-regulated signaling kinase 1, which controls p38/stress-activated protein kinase activation and has been shown to be negatively regulated by Akt-dependent phosphorylation (22) , demonstrated no change in NNK-BEAS2B cells compared with BEAS2B (data not shown).

Our present work with the BEAS2B/NNK-BEAS2B in vitro model system suggests that Akt activity is integral to the process of transformation and may be an early event. Indeed, increased phosphorylation of Akt in vitro has been observed in different tumorigenic cell lines derived from BEAS2B cells (23) . We provide direct support for this hypothesis by showing increased staining in a spectrum of lung lesions with phospho-specific antibodies directed against Akt and mTOR, but not GSK3ß. The mechanism for loss of GSK3 phosphorylation with progression from adenoma to adenocarcinoma is unclear. Loss of a phosphorylated epitope in an immunohistochemical analysis has also been observed by Tsao et al. (24) , who showed that although Akt phosphorylation at serine 473 was increased in dysplastic lesions from smokers compared with normal bronchial epithelium, it was decreased in tumors compared with dysplastic lesions. It is notable that we saw increased staining in reactive type II cells and AAH, which are likely the progenitors for tumors in the A/J mouse model. Even if phosphorylation of one or more components is lost in the final step of transformation, the ability to detect active components in the PI3K/Akt/mTOR pathway in premalignant lesions in vivo has identified new targets that could be considered for chemoprevention.

The possibility that inhibiting Akt at multiple steps in the transformation process might effectively kill premalignant cells makes Akt an interesting target for lung cancer chemoprevention, but the ability to exploit Akt activation in this way will depend on the toxicity of inhibiting the PI3K/Akt pathway in surrounding normal tissues. In that regard, inhibition of Akt by LY294002 alone in primary human bronchial or small airway epithelial cells did not increase apoptosis (5) . The compounds that target components of the PI3K/Akt pathway that are furthest along in clinical development are inhibitors of mTOR such as rapamycin, CCI-779, and RAD-001. Interestingly, administration of rapamycin to BEAS2B or NNK-BEAS2B cells inhibited mTOR and p70S6K activity but did not increase apoptosis (data not shown). Therefore, testing various inhibitors of the PI3K/Akt pathway in animal models of lung carcinogenesis will best establish efficacy and determine the feasibility of using these compounds for chemoprevention.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: Phillip A. Dennis, National Cancer Institute/Naval Medical Oncology, Building 8, Room 5101, 8901 Wisconsin Avenue, Bethesda, Maryland 20889-55105.

Received 10/15/03. Accepted 11/21/03.

	REFERENCES

Top ABSTRACT Introduction Materials and Methods Results Discussion REFERENCES

 

1. Mackay J., Eriksen M. . The Tobacco Atlas, WHO Geneva 2002.
2. Jemal A., Murray T., Samuels A., Ghafoor A., Ward E., Thun M. J. Cancer statistics, 2003. CA Cancer J. Clin., 53: 5-26, 2003.[Abstract/Free Full Text]
3. Hahn W. C. Immortalization and transformation of human cells. Mol. Cells, 13: 351-361, 2002.[Medline]
4. Brognard J., Clark A. S., Ni Y., Dennis P. A. Akt/protein kinase B is constitutively active in non-small cell lung cancer cells and promotes cellular survival and resistance to chemotherapy and radiation. Cancer Res., 61: 3986-3997, 2001.[Abstract/Free Full Text]
5. West K. A., Brognard J., Clark A. S., Linnoila I. R., Yang X., Swain S. M., Harris C., Belinsky S., Dennis P. A. Rapid Akt activation by nicotine and a tobacco carcinogen modulates the phenotype of normal human airway epithelial cells. J. Clin. Investig., 111: 81-90, 2003.[CrossRef][Medline]
6. Reddel R. R., Ke Y., Gerwin B. I., McMenamin M. G., Lechner J. F., Su R. T., Brash D. E., Park J. B., Rhim J. S., Harris C. C. Transformation of human bronchial epithelial cells by infection with SV40 or adenovirus-12 SV40 hybrid virus, or transfection via strontium phosphate coprecipitation with a plasmid containing SV40 early region genes. Cancer Res., 48: 1904-1909, 1988.[Abstract/Free Full Text]
7. Klein-Szanto A. J., Iizasa T., Momiki S., Garcia-Palazzo I., Caamano J., Metcalf R., Welsh J., Harris C. C. A tobacco-specific N-nitrosamine or cigarette smoke condensate causes neoplastic transformation of xenotransplanted human bronchial epithelial cells. Proc. Natl. Acad. Sci. USA, 89: 6693-6697, 1992.[Abstract/Free Full Text]
8. Kozikowski A. P., Sun H., Brognard J., Dennis P. A. Novel PI analogues selectively block activation of the pro-survival serine/threonine kinase Akt. J. Am. Chem. Soc., 125: 1144-1145, 2003.[CrossRef][Medline]
9. Suhara T., Kim H. S., Kirshenbaum L. A., Walsh K. Suppression of Akt signaling induces Fas ligand expression: involvement of caspase and Jun kinase activation in Akt-mediated Fas ligand regulation. Mol. Cell. Biol., 22: 680-691, 2002.[Abstract/Free Full Text]
10. Castonguay A., Pepin P., Stoner G. D. Lung tumorigenicity of NNK given orally to A/J mice: its application to chemopreventive efficacy studies. Exp. Lung Res., 17: 485-499, 1991.[Medline]
11. Hanahan D., Weinberg R. A. The hallmarks of cancer. Cell, 100: 57-70, 2000.[CrossRef][Medline]
12. Nicholson K. M., Anderson N. G. The protein kinase B/Akt signalling pathway in human malignancy. Cell Signalling, 14: 381-395, 2002.[CrossRef][Medline]
13. Datta S. R., Brunet A., Greenberg M. E. Cellular survival: a play in three Akts. Genes Dev., 13: 2905-2927, 1999.[Free Full Text]
14. Malkinson A. M. Molecular comparison of human and mouse pulmonary adenocarcinomas. Exp. Lung Res., 24: 541-555, 1998.[Medline]
15. Aoki M., Blazek E., Vogt P. K. A role of the kinase mTOR in cellular transformation induced by the oncoproteins P3k and Akt. Proc. Natl. Acad. Sci. USA, 98: 136-141, 2001.[Abstract/Free Full Text]
16. Ashcroft M., Ludwig R. L., Woods D. B., Copeland T. D., Weber H. O., MacRae E. J., Vousden K. H. Phosphorylation of HDM2 by Akt. Oncogene, 21: 1955-1962, 2002.[CrossRef][Medline]
17. Mayo L. D., Donner D. B. A phosphatidylinositol 3-kinase/Akt pathway promotes translocation of Mdm2 from the cytoplasm to the nucleus. Proc. Natl. Acad. Sci. USA, 98: 11598-11603, 2001.[Abstract/Free Full Text]
18. Biggs W. H., III, Meisenhelder J., Hunter T., Cavenee W. K., Arden K. C. Protein kinase B/Akt-mediated phosphorylation promotes nuclear exclusion of the winged helix transcription factor FKHR1. Proc. Natl. Acad. Sci. USA, 96: 7421-7426, 1999.[Abstract/Free Full Text]
19. Brunet A., Bonni A., Zigmond M. J., Lin M. Z., Juo P., Hu L. S., Anderson M. J., Arden K. C., Blenis J., Greenberg M. E. Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor. Cell, 96: 857-868, 1999.[CrossRef][Medline]
20. Oren M., Damalas A., Gottlieb T., Michael D., Taplick J., Leal J., Maya R., Moas M., Seger R., Taya Y., Ben-Ze’ev A. Regulation of p53: intricate loops and delicate balances. Biochem. Pharmacol., 64: 865-871, 2002.[CrossRef][Medline]
21. Dijkers P. F., Medema R. H., Pals C., Banerji L., Thomas N. S., Lam E. W., Burgering B. M., Raaijmakers J. A., Lammers J. W., Koenderman L., Coffer P. J. Forkhead transcription factor FKHR-L1 modulates cytokine-dependent transcriptional regulation of p27(KIP1). Mol. Cell. Biol., 20: 9138-9148, 2000.[Abstract/Free Full Text]
22. Kim A. H., Khursigara G., Sun X., Franke T. F., Chao M. V. Akt phosphorylates and negatively regulates apoptosis signal-regulating kinase 1. Mol. Cell. Biol., 21: 893-901, 2001.[Abstract/Free Full Text]
23. Chun K. H., Kosmeder J. W., II, Sun S., Pezzuto J. M., Lotan R., Hong W. K., Lee H. Y. Effects of deguelin on the phosphatidylinositol 3-kinase/Akt pathway and apoptosis in premalignant human bronchial epithelial cells. J. Natl. Cancer Inst. (Bethesda), 95: 291-302, 2003.[Abstract/Free Full Text]
24. Tsao A. S., McDonnell T., Lam S., Putnam J. B., Bekele N., Hong W. K., Kurie J. M. Increased phospho-AKT (Ser⁴⁷³) expression in bronchial dysplasia: implications for lung cancer prevention studies. Cancer Epidemiol. Biomark. Prev., 12: 660-664, 2003.[Abstract/Free Full Text]

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				J. A. Crowell, V. E. Steele, and J. R. Fay Targeting the AKT protein kinase for cancer chemoprevention Mol. Cancer Ther., August 1, 2007; 6(8): 2139 - 2148. [Abstract] [Full Text] [PDF]

				L. Kopelovich, J. R. Fay, C. C. Sigman, and J. A. Crowell The Mammalian Target of Rapamycin Pathway as a Potential Target for Cancer Chemoprevention Cancer Epidemiol. Biomarkers Prev., July 1, 2007; 16(7): 1330 - 1340. [Abstract] [Full Text] [PDF]

				P. Song, H. S. Sekhon, A. Lu, J. Arredondo, D. Sauer, C. Gravett, G. P. Mark, S. A. Grando, and E. R. Spindel M3 Muscarinic Receptor Antagonists Inhibit Small Cell Lung Carcinoma Growth and Mitogen-Activated Protein Kinase Phosphorylation Induced by Acetylcholine Secretion Cancer Res., April 15, 2007; 67(8): 3936 - 3944. [Abstract] [Full Text] [PDF]

				C. A. Granville, N. Warfel, J. Tsurutani, M. C. Hollander, M. Robertson, S. D. Fox, T. D. Veenstra, H. J. Issaq, R. I. Linnoila, and P. A. Dennis Identification of a Highly Effective Rapamycin Schedule that Markedly Reduces the Size, Multiplicity, and Phenotypic Progression of Tobacco Carcinogen-Induced Murine Lung Tumors Clin. Cancer Res., April 1, 2007; 13(7): 2281 - 2289. [Abstract] [Full Text] [PDF]

				J. Shen, C. Behrens, I. I. Wistuba, L. Feng, J. J. Lee, W. K. Hong, and R. Lotan Identification and Validation of Differences in Protein Levels in Normal, Premalignant, and Malignant Lung Cells and Tissues Using High-Throughput Western Array and Immunohistochemistry Cancer Res., December 1, 2006; 66(23): 11194 - 11206. [Abstract] [Full Text] [PDF]

				P. Adiseshaiah, D. V. Kalvakolanu, and S. P. Reddy A JNK-Independent Signaling Pathway Regulates TNF{alpha}-Stimulated, c-Jun-Driven FRA-1 Protooncogene Transcription in Pulmonary Epithelial Cells J. Immunol., November 15, 2006; 177(10): 7193 - 7202. [Abstract] [Full Text] [PDF]

				V. Dasari, M. Gallup, H. Lemjabbar, I. Maltseva, and N. McNamara Epithelial-Mesenchymal Transition in Lung Cancer: Is Tobacco the "Smoking Gun"? Am. J. Respir. Cell Mol. Biol., July 1, 2006; 35(1): 3 - 9. [Full Text] [PDF]

				K. El-Bayoumy, A. Das, B. Narayanan, N. Narayanan, E. S. Fiala, D. Desai, C. V. Rao, S. Amin, and R. Sinha Molecular targets of the chemopreventive agent 1,4-phenylenebis (methylene)-selenocyanate in human non-small cell lung cancer Carcinogenesis, July 1, 2006; 27(7): 1369 - 1376. [Abstract] [Full Text] [PDF]

				S. Y. Lee, E. J. Kang, G. Y. Hur, K. H. Jung, H. C. Jung, S. Y. Lee, J. H. Kim, C. Shin, K. H. In, K. H. Kang, et al. Peroxisome proliferator-activated receptor-{gamma} inhibits cigarette smoke solution-induced mucin production in human airway epithelial (NCI-H292) cells Am J Physiol Lung Cell Mol Physiol, July 1, 2006; 291(1): L84 - L90. [Abstract] [Full Text] [PDF]

				P. Dasgupta, R. Kinkade, B. Joshi, C. DeCook, E. Haura, and S. Chellappan Nicotine inhibits apoptosis induced by chemotherapeutic drugs by up-regulating XIAP and survivin PNAS, April 18, 2006; 103(16): 6332 - 6337. [Abstract] [Full Text] [PDF]

				Q. Zhang, P. Adiseshaiah, D. V. Kalvakolanu, and S. P. Reddy A Phosphatidylinositol 3-Kinase-regulated Akt-Independent Signaling Promotes Cigarette Smoke-induced FRA-1 Expression J. Biol. Chem., April 14, 2006; 281(15): 10174 - 10181. [Abstract] [Full Text] [PDF]

				J.-T. Chen, T.-S. Lin, K.-C. Chow, H.-H. Huang, S.-H. Chiou, S.-F. Chiang, H.-C. Chen, T.-L. Chuang, T.-Y. Lin, and C.-Y. Chen Cigarette Smoking Induces Overexpression of Hepatocyte Growth Factor in Type II Pneumocytes and Lung Cancer Cells Am. J. Respir. Cell Mol. Biol., March 1, 2006; 34(3): 264 - 273. [Abstract] [Full Text] [PDF]

				P. Martinez-Moreno, S. Nieto-Ceron, J. Torres-Lanzas, F. Ruiz-Espejo, I. Tovar-Zapata, P. Martinez-Hernandez, J. N. Rodriguez-Lopez, C. J. Vidal, and J. Cabezas-Herrera Cholinesterase activity of human lung tumours varies according to their histological classification Carcinogenesis, March 1, 2006; 27(3): 429 - 436. [Abstract] [Full Text] [PDF]

				H.-Y. Lee, S.-H. Oh, J. K. Woo, W.-Y. Kim, C. S. Van Pelt, R. E. Price, D. Cody, H. Tran, J. M. Pezzuto, R. M. Moriarty, et al. Chemopreventive Effects of Deguelin, a Novel Akt Inhibitor, on Tobacco-Induced Lung Tumorigenesis J Natl Cancer Inst, November 16, 2005; 97(22): 1695 - 1699. [Abstract] [Full Text] [PDF]

				J. Tsurutani, S.S. Castillo, J. Brognard, C. A. Granville, C. Zhang, J. J. Gills, J. Sayyah, and P. A. Dennis Tobacco components stimulate Akt-dependent proliferation and NF{kappa}B-dependent survival in lung cancer cells Carcinogenesis, July 1, 2005; 26(7): 1182 - 1195. [Abstract] [Full Text] [PDF]

				F. R. Hirsch and S. M. Lippman Advances in the Biology of Lung Cancer Chemoprevention J. Clin. Oncol., May 10, 2005; 23(14): 3186 - 3197. [Abstract] [Full Text] [PDF]

				E. S. Fiala, O. S. Sohn, C.-X. Wang, E. Seibert, J. Tsurutani, P. A. Dennis, K. El-Bayoumy, R. S. Sodum, D. Desai, J. Reinhardt, et al. Induction of preneoplastic lung lesions in guinea pigs by cigarette smoke inhalation and their exacerbation by high dietary levels of vitamins C and E Carcinogenesis, March 1, 2005; 26(3): 605 - 612. [Abstract] [Full Text] [PDF]

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