Induction and apoptotic regression of lung adenocarcinomas by regulation of a K-Ras transgene in the presence and absence of tumor suppressor genes

Abstract

To investigate the role of an activated K-Ras gene in the initiation and maintenance of lung adenocarcinomas, we developed transgenic mice that express murine K-Ras4b(G12D) under the control of doxycycline in type II pneumocytes. Focal proliferative lesions of alveolar type II pneumocytes were observed as early as seven days after induction with doxycycline; after two months of induction, the lungs contained adenomas and adenocarcinomas, with focal invasion of the pleura at later stages. Removal of doxycycline caused a rapid fall in levels of mutant K-Ras RNA and concomitant apoptotic regression of both the early proliferative lesions and the tumors. Tumor burden was dramatically decreased by three days after withdrawal, and tumors were undetectable after one month. When similar experiments were performed with animals deficient in either the p53 gene or the Ink4A/Arf locus, tumors arose more quickly (within one month of exposure to doxycycline) and displayed more obvious histological features of malignancy; nevertheless, these tumors also regressed rapidly when the inducer was removed, implying that continued production of mutant K-Ras is necessary to maintain the viability of tumor cells in the absence as well as the presence of tumor suppressor genes. We also show that the appearance and regression of these pulmonary tumors can be readily monitored in anesthetized transgenic animals by magnetic resonance imaging.

Figure 1

Organization of transgenes and expression patterns. (A) Transgenic constructs used to generate the tetracycline operator-regulated K-Ras4b^G12D (Tet-op-K-Ras4b^G12D) responder mice (upper) and the Clara cell secretory protein-tet activator (CCSP-rtTA) mice (lower). The origins of the component are described in the text and their sizes are indicated in kilobases (kb). (B) RT–PCR tests for K-Ras4b^G12D RNA. Total RNA was isolated from the right accessory lobe of 18 different mice and 0.25 μg was used for RT–PCR analysis of K-Ras4b^G12D and actin RNAs as described in Materials and Methods. Each sample was run in parallel with a sample lacking RT. Samples were prepared from animals that did not receive doxycycline (nontransgenics, lanes 1,2; bitransgenics, lanes 3,4), from animals that received doxycycline for 9 d (bitransgenic, lane 5; responder transgene only, lane 6), and from bitransgenic mice that received doxycycline for 2 mo (lanes 7–9) or for 2 mo followed by withdrawal of the inducer for 3 d (lanes 10–14) or for 8 d (lanes 15–18).

Figure 2

Appearance of proliferative foci and tumors after induction of mutant K-Ras with doxycycline. (A) Kinetics of induction of histological changes. Samples of lung tissue were stained with hematoxylin and eosin after exposure to inducer for 7 d, 14 d, and 1, 2, and 4 mo, as indicated. (B) Trichrome stain of tumor from a bitransgenic mouse treated for 4 mo shows invasion of stroma by tumor cells. (C, D) Cell-type-specific staining of a tumor from a mouse treated for 2 mo with doxycycline indicates properties of type II pneumocytes. Positive staining for Prosurfactant protein C (C) and negative staining of the tumor but positive staining of the airways for Clara cell secretory protein (CCSP; D). (E) Photographs of intact lungs from three bitransgenic mice induced with doxycycline for 3 mo (top row), compared to lungs from non-induced bitransgenic mice (bottom row).

Figure 3

Induction of mutant K-Ras with doxycycline promotes lung cell proliferation. The relative proportions of proliferating lung cells were determined by injection of Brdu and immunohistochemical staining after 3 h incorporation in bitransgenic mice that did not receive inducer (A), that received inducer for 14 d (B) or 2 mo (C), and that had inducer withdrawn for 3 d following 2 mo of treatment (D).

Figure 4

Effects of withdrawal of doxycycline and deinduction of mutant K-Ras on lungs of bitransgenic mice. (A) Tumor regression. Sections of lungs from bitransgenic mice treated with doxycycline for 2 mo and after removal of the inducer for 3 d, 7 d, or 1 mo were stained with H&E. (B) Appearance of apoptotic cells. Lung tumor samples were assayed for apoptotic cells using the TUNEL procedure after 2 mo on doxycycline (left) and 3 d after removal of the inducer (right). Examples of TUNEL-positive cells are indicated by red arrowheads. (C) “Pitting” on the surface of an intact lung from a bitransgenic mouse that received doxycycline for 5 mo and then had the inducer withdrawn for 3 d. The sample was fixed and dehydrated before photography.

Figure 5

Increase and decrease in the weight of lungs from K-Ras bitransgenic mice treated with doxycycline and subjected to withdrawal of the inducer. Weights were determined after excision, fixation in 4% paraformaldehyde, and dehydration in 70% ethanol. The bitransgenic mice were from a mixed genetic background (A), a mixed background with a mutation affecting Ink4A/Arf (B), FVB/N (C), and an FVB/N background with a mutation affecting p53 (D).

Figure 6

Characterization of tumors arising in K-Ras bitransgenic mice in backgrounds of or Ink4A/Arf deficiencies. (A) Malignant features of histopathology of tumors in the presence and absence of tumor suppressor genes. Samples of H&E-stained tumor-bearing lungs from indicated mouse strains after induction with doxycycline are shown at 40× magnification; for description, see text. (B) Tumors in tumor-suppressor-gene-deficient backgrounds appear more quickly during induction but regress similarly after doxycycline withdrawal. Lung slices were stained with H&E after exposure of bitransgenic mice to doxycycline for 1 or 2 mo and after withdrawal for 7 d. Bitransgenics in a wild-type background are shown on the left; in a p53-deficient background in the middle; and in an Ink4A/Arf-deficient background on the right, all as labeled.

Figure 7

Magnetic resonance imaging of tumor regression. The pictures show sagittal (upper) and transverse (lower) images of lung fields from a bitransgenic mouse without known tumor suppressor gene mutations (left) and another bitransgenic mouse with a mutation affecting Ink4A/Arf (right). Each animal was anesthetized and subjected to MRI after induction with doxycycline for the indicated times and again after 8 d without the inducer. Arrows indicate positions of the posterior thoracic wall (upper, sagittal sections) and the outer myocardial wall (lower, transverse sections).