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. 2003 Feb 19;129(2):71–75. doi: 10.1007/s00432-002-0400-z

Increased susceptibility to the tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in transgenic mice overexpressing c-myc and epidermal growth factor in alveolar type II cells

A Ehrhardt 1,4,✉, T Bartels 2, R Klocke 1,3, D Paul 1, R Halter 1
PMCID: PMC12161925  PMID: 12669230

Abstract

Purpose

As previously described, SPC/myc transgenic mice developed bronchioloalveolar adenocarcinomas derived from alveolar type II (AT II) cells within 10–14 months, whereas SPC/IgEGF transgenic mice developed hyperplasias. Our purpose was to determine the potential interplay of environmental and genetic factors in lung tumorigenesis.

Materials and methods

Six-week-old SPC/myc and SPC/IgEGF transgenic mice, overexpressing c-myc and a secretable form of the epidermal growth factor (IgEGF) under the control of the surfactant protein C (SPC) promoter, were treated with a single dose of the tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). As control groups, SPC/myc and SPC/IgEGF transgenic mice were treated with NaCl and non-transgenic littermates were treated with NNK or NaCl, respectively.

Results

After 6 months, none of the NaCl-treated transgenic littermates showed bronchioloalveolar hyperplasia and adenocarcinoma formation, whereas 100% of the NNK-treated SPC/myc transgenic mice did. The effect of NNK on SPC/IgEGF transgenic mice was less pronounced, inducing hyperplasia in the lung in only 16.7% of them. In 90% of the NNK-treated non-transgenic littermates no neoplastic changes were detected in the lung.

Conclusions

These results demonstrate that the progression of pulmonary bronchioloalveolar adenocarcinomas, induced by expression of c-myc as a transgene, was accelerated by NNK, suggesting that c-myc cooperates with NNK-induced mutations.

Keywords: c-myc, EGF, NNK, Lung cancer, Transgenic mice

Introduction

Lung cancer is one of the most common cancer diseases worldwide and is the leading cause of death by cancer in the US (U.S. Surgeon General 1982). It is estimated, that more than 85% of lung cancers are related to cigarette smoking (International Agency for Research on Cancer 1986). NNK is a potent tobacco carcinogen, and its carcinogenicity at doses comparable to those received by smokers during their lifetime has been shown in rats, Syrian golden hamsters, and mice (Rivenson et al. 1988; Yang et al. 1997; Hecht et al. 1983). Tumors induced by NNK in the mouse lung are classified as carcinomas with papillary or solid growth pattern (Belinsky et al. 1992), which represent a subtype of non-small cell lung carcinomas (NSCLC). The usual animal model to study carcinogen-induced pulmonary tumors as well as cancer chemoprevention are A/J mice. NNK is known to be activated in the lung, producing methylating and pyridyloxobutylating agents, that attack DNA leading to mutations (Nettesheim 1991). NNK-induced lung carcinomas in A/J mice frequently show activation of the K-ras proto-oncogene due to GC-to-AT transition at the second base of codon 12 (Kawano et al. 1996).

Recently, we have established new transgenic mouse models to study the tumorigenesis of pulmonary carcinomas derived from alveolar type II (AT II) cells (Ehrhardt et al. 2001). The two transgenic mouse lines overexpressed c-myc and IgEGF under the control of AT II cell-specific surfactant protein C (SPC)-promoter. SPC/myc transgenic mice developed multifocal hyperplasias, and bronchioloalveolar adenomas and carcinomas, whereas SPC/IgEGF transgenic mice developed hyperplasias. Hemizygous SPC/myc transgenic mice developed pulmonary bronchioloalveolar carcinomas derived from AT II cells within 10–14 months, whereas SPC/IgEGF transgenic mice developed hyperplasias at the average age of 19 months. We observed early stages in tumor development in SPC/myc transgenic mice at the average age of 7 months, whereas in SPC/IgEGF transgenic mice at the same age no morphologic changes were observed (Ehrhardt et al. 2001). Histopathology revealed that this early stage of tumor development in SPC/myc transgenic was characterized by multifocal hyperplasias originated in the alveolar epithelium.

Cancer is a multistep process, including activation of proto-oncogene and silencing of tumor suppressor genes (Vogelstein and Kinzler 1993; Weinberg 1991). To address the question whether NNK-induced mutations support the two oncogenes c-myc and IgEGF in lung tumorigenesis, we treated SPC/myc and SPC/IgEGF transgenic mice with a single dose of NNK.

Materials and methods

NNK treatment

The establishment and characterization of SPC/myc (transgenic line 8.2) and SPC/IgEGF transgenic mice (transgenic line 6.2) was previously described (Ehrhardt et al. 2001).

Six-week-old SPC/myc and SPC/IgEGF transgenic mice, non-transgenic littermates, and A/J mice (Jackson Laboratory, Bar Harbor, Me., USA) were treated either with a single dose of NNK (103 mg/kg, i.p.; Chemsyn Science Laboratories, Lenexa, Kan., USA) or with a 0.9% NaCl solution. NNK and NaCl-treated mice of all groups were killed and analyzed 6 months after treatment.

Histopathology

For histopathological analyses lungs were fixed in 10% formalin, embedded in paraffin, and stained with hematoxylin and eosin using standard protocols. The pulmonary hyperplasias or tumors, respectively, were classified in accordance to the International Classification of Rodent Tumors - The Mouse (Dungworth et al. 2001).

Results

Transgenic mouse lines and NNK treatment

To address the question whether NNK treatment of our transgenic mouse lines would result in an accelerated lung tumor formation, six-week-old SPC/myc and SPC/IgEGF transgenic mice, A/J mice, and non-transgenic littermates were injected with a single dose of NNK. A/J mice—which are known to be susceptible to NNK, thereby developing lung tumors—were used as positive controls. The transgenic mouse lines were propagated in a hybrid outbred mouse strain (CD2F1) which is not susceptible to NNK, enabling us to use the non-transgenic littermates as negative controls.

NNK-induced tumor development

Mice of all groups were killed after 6 months of NNK and NaCl treatment, respectively, and tumor development was determined by histopathology. Six months after NNK treatment, 100% of SPC/myc transgenic mice (n=4) and 16.7% of SPC/IgEGF (n=6) transgenic mice developed bronchioloalveolar hyperplasias, adenomas or carcinomas in the lung. Of the NNK-treated SPC/myc transgenic mice, 75% developed bronchioloalveolar adenocarcinomas. In sharp contrast, and in concordance with our previous findings (Ehrhardt et al. 2001), no later stages of tumor development were observed in SPC/myc transgenic mice which were treated with 0.9% NaCl as a negative control. Only the combination of NNK and the overexpression of the proto-oncogene myc was sufficient to cause later stages of tumor development in SPC/myc transgenic mice at the age of 7 months. In 90% of NNK-treated non-transgenic littermates (n=8), no lung tumor formation nor hyperplasias could be observed, and 100% of the NNK-injected A/J mice (n=6) showed either bronchioloalveolar carcinomas or bronchioloalveolar hyperplasias in the lung. No lung tumor formation was observed in NaCl-treated transgenic littermates and A/J mice. The average number of macroscopically counted lung tumor nodules in A/J mice was 6.3±1.5 and 3.75±2.1 in SPC/myc transgenic mice. A summary of the histological results of all treated mice is shown in Table 1. Our findings demonstrated a distinct acceleration of lung tumor formation by NNK in SPC/myc transgenic mice over a period of 6 months. Taken together, these findings may suggest that the NNK-induced mutations in the lung potentially cooperate with the overexpressed proto-oncogene in our transgenic mice during the process of lung tumor development.

Table 1.

Summary of histopathological findings six months after treatment. Six-week-old mice were treated with either a single dose of NNK or with a 0.9% NaCl solution. Mice were killed and analysed 6 months post-treatment. The lesions per mouse were analyzed by histopathology and expressed in mean±SD. (NNK 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, NaCl 0.9% sodium chloride solution)

Group Treatment
NNK NaCl
Histopathology Histopathology
% of mice with bronchiolo-alveolar hyperplasia (%) % of mice with bronchiolo-alveolar adenoma/carcinoma (%) Total number of bronchiolo-alveolar carcinomas per mouse (mean±SD) % of mice with bronchiolo-alveolar hyperplasia (%) % of mice with bronchiolo-alveolar adenoma/carcinoma (%)
SP-C/IgEGF transgenica (n=10) (n=6) (n=4)
16.7 0 0 0 0
SP-C/myc transgenics (n=8) (n=4) (n=4)
25 75 3.75+/-2.1 0 0
A/J mice (n=9) (n=6) (n=3)
16.7 83.3 6.3+/-1.5 0 0
Non-transgenic littermates (n=15) (n=8) (n=7)
10 0 0 0 0
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Histopathology

NNK-treated SPC/myc and SPC/IgEGF transgenic mice developed bronchioloalveolar hyperplasias, adenomas, and carcinomas derived from AT II cells. Figure 1A shows bronchioloalveolar adenoma in a NNK-treated SPC/myc transgenic mouse with papillary protrusions. Figure 1B shows bronchioloalveolar carcinomas of NNK-treated SPC/myc transgenic mice. The histopathology of NNK-induced hyperplasias and lung tumors in A/J mice were similar to that observed in SPC/myc transgenic mice (not shown). In Fig. 1C, the lung of an SPC/myc transgenic mouse is shown after NaCl treatment without any signs of abnormalities. This phenotype was found in all NaCl-treated as well as in 90% of NNK-treated non-transgenic littermates (not shown).

Fig. 1. A.

Fig. 1. A

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Bronchioloalveolar adenoma in SP-C/myc NNK-treated mice: regular papillary growth pattern of two small-sized formations in the lung periphery. The nodules are clearly demarcated from the adjacent parenchyma. B Bronchioloalveolar carcinoma in SP-C/myc NNK-treated mice: the irregularly growing neoplasia is poorly demarcated from the adjacent parenchyma. Cuboidal to columnar cells forming papillary projections indicate the well-differentiated growth pattern. C Normal lung parenchyma of SP-C/myc transgenic mice after NaCl treatment (bar = 100 µm)

Discussion

The results shown in this study demonstrate a distinct acceleration of lung hyperplasia and tumor formation in SPC/myc transgenic mice as a consequence of NNK treatment, which was less pronounced in SPC/IgEGF transgenic mice. Six months after application of NNK, 100% of SPC/myc and 16.7% of SPC/IgEGF transgenic mice developed neoplastic changes in the lung, whereas all NaCl-treated and 90% of the NNK-treated non-transgenic control mice showed neither hyperplasias nor lung carcinoma formation (Table 1). All mice in this study were evaluated at the age of 7 months. At this age spontaneous lung tumors are a rare event, whereas the incidence of lung tumors in common increases considerably in mice older than 18 months. However, specified data, in terms of the age of mice used here and for the hybrid strain CD2F1 serving as the genetic background of our transgenic mice, are not available (Rittinghausen et al. 1996).

C-myc, EGF, and TGFa are among the most frequently deregulated oncogenes in human lung carcinomas (Broers et al. 1993; Moody 1996). There is strong evidence that the K-ras proto-oncogene is a candidate for the major mouse pulmonary susceptibility locus Pas-1 (Lin et al. 1998). Several studies in NNK-treated A/J mice demonstrated that the K-ras oncogene is activated in 100% of the induced lung tumors by GC-to-AT transition at the second base of codon 12 (Kawano et al. 1996). The cooperation of K-ras and c-myc is known to occur in cell culture with various cell lines (Leone et al. 1997). Based on the assumption that NNK activates K-ras in our transgenic and non-transgenic littermates we speculate that the synergistic effect of c-myc and NNK in tumor formation might be due to activation of K-ras by NNK, leading to cooperation of K-ras and c-myc and thereby inducing the acceleration of lung tumor formation. Further studies need to be done regarding why NNK alone was not sufficient to induce lung tumor formation in non-transgenic littermates over a time period of 6 months (Table 1).

As previously described, SPC/IgEGF transgenic mice developed hyperplasias at the average age of 19 months. However, the individuals in the present study were killed and analyzed at the age of 7 months and only in 16.7% of the NNK-treated and none of the NaCl-treated SPC/IgEGF transgenic mice were morphological changes detected. The findings in NaCl-treated SPC/IgEGF individuals are in concordance with our previous data. We speculate that the effects of NNK in SPC/IgEGF transgenic mice may require a longer period of time to develop NNK-induced tumors. The EGF-receptor is known to activate Ras in response to ligand binding (Lo et al. 2001; Carpenter 2000). Consequently, expression of the transgene IgEGF and NNK treatment would result in either activation of K-ras by ligand binding or mutation of K-ras by NNK.

We demonstrated a synergistic effect in vivo of the tobacco carcinogen NNK and the oncogene c-myc in lung tumor formation. Similar results for a synergistic effect of carcinogen application and proto-oncogene overexpression were shown in transgenic mouse models developing tumors in esophageal squamous epithelium (Jenkins et al. 1999) and in the mammary gland (Yao et al. 1999). Because the source lines (DBA/2, Balb/C) of the hybrid mouse strain CD2/F1 used for generation and propagation of the transgenic lines are not susceptible to lung tumor formation (Dragani et al. 1991; Wardlaw et al. 2000), we concluded that expression of the transgene c-myc was necessary to facilitate susceptibility for NNK. Thus, the SPC/myc transgenic mice will be useful to screen for new susceptibility genes for lung cancer and may provide an animal model for short-term carcinogenicity studies in the lung.

Acknowledgements

We thank Claudia Beyer for technical support. This work was supported by grants from the Deutsche Krebshilfe (10-0936-Ha I), the Deutsche Forschungsgemeinschaft (DFG) (III GK-GRK 139/3-99), and from BMH4-CT96-0254 (EUROCYP).

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