Clearance of senescent cells reverts the cigarette smoke-induced lung senescence and airspace enlargement in p16-3MR mice

Cigarette smoke (CS) leads to increased oxidative stress, inflammation, and exaggerated senescence, which are involved in the pathogenesis of chronic obstructive pulmonary disease (COPD). While the role of cellular senescence in COPD is known, it is not clear if the removal of senescent cells could alleviate the disease symptoms. To test this, we used the novel mouse model-p16-3MR, and studied the effect of ganciclovir (GCV)-mediated removal of senescent cells after chronic CS (3 months) and environmental tobacco smoke (ETS) (6 months) exposure to CS. Our results showed the reversal of CS-induced cellular senescence on the clearance of p16⁺ senesced cells by GCV treatment. Interestingly, the clearance of p16⁺ senescent cells via GCV led to a decrease in the neutrophil counts in the BALF of GCV-treated CS-exposed p16-3MR mice, as well as reversal of CS-mediated airspace enlargement in p16-3MR mice. Mice exposed to low dose ETS caused insignificant changes in the SA-β-Gal⁺ senescent cells and airspace enlargement. Overall, our data provide evidence for the role of lung cellular senescence on smoke exposure and clearance of senescent cells in p16-3MR mice in the reversal of COPD/emphysema pathology with a possibility of senolytics as therapeutic interventions in COPD.     

Cellular senescence increases expression of bacterial ligands in the lungs and is positively correlated with increased susceptibility to pneumococcal pneumonia

Cellular senescence is an age-associated phenomenon that promotes tumor invasiveness owing to the secretion of proinflammatory cytokines, proteases, and growth factors. Herein we demonstrate that cellular senescence also potentially increases susceptibility to bacterial pneumonia caused by Streptococcus pneumoniae (the pneumococcus), the leading cause of infectious death in the elderly. Aged mice had increased lung inflammation as determined by cytokine analysis and histopathology of lung sections. Immunoblotting for p16, pRb, and mH2A showed that elderly humans and aged mice had increased levels of these senescence markers in their lungs vs. young controls. Keratin 10 (K10), laminin receptor (LR), and platelet-activating factor receptor (PAFr), host proteins known to be co-opted for bacterial adhesion, were also increased. Aged mice were found to be highly susceptible to pneumococcal challenge in a PsrP, the pneumococcal adhesin that binds K10, dependent manner. In vitro senescent A549 lung epithelial cells had elevated K10 and LR protein levels and were up to 5-fold more permissive for bacterial adhesion. Additionally, exposure of normal cells to conditioned media from senescent cells doubled PAFr levels and pneumococcal adherence. Genotoxic stress induced by bleomycin and oxidative stress enhanced susceptibility of young mice to pneumonia and was positively correlated with enhanced p16, inflammation, and LR levels. These findings suggest that cellular senescence facilitates bacterial adhesion to cells in the lungs and provides an additional molecular mechanism for the increased incidence of community-acquired pneumonia in the elderly. This study is the first to suggest a second negative consequence for the senescence-associated secretory phenotype.     

Effects of sustained exercise on pulmonary clearance of aerosolized 99mTc-DTPA

The effects of intensive prolonged exercise on the pulmonary clearance rate of aerosolized 99mTc-labeled diethylenetriaminepentaacetate (99mTc-DTPA) and pulmonary mechanics were studied in seven healthy nonsmoking volunteers. 99mTc-DTPA clearance and pulmonary mechanics (lung volumes and compliance) were assessed before and after 75 min of constant-load exercise performed on a treadmill, corresponding to 75% of maximal O2 uptake. Because both clearance measurements were made in similar conditions of pulmonary blood flow, respiratory rate, and tidal volume, changes in clearance rate can be assumed to represent changes of alveolar epithelial permeability. After exercise, total, apical, and basal clearance were significantly increased (P less than 0.01, 0.05, and 0.05, respectively) and the increases in total clearance and tidal volume observed during exercise were significantly correlated (P less than 0.05). In contrast, no significant change was found in pulmonary mechanics. These results show that prolonged intensive exercise induces an increase in epithelial permeability, which appears to be related to the mechanical effects of sustained increased ventilation. Because no change was evidenced in pulmonary volumes or in lung elasticity, our results suggest that this increase may result from alteration of the intercellular tight junctions rather than from a surfactant deficiency.     

Altered pulmonary epithelial permeability in canine radiation lung injury

A radioaerosol scanning technique measuring regional clearance of sodium pertechnetate (99mTcO-4) and 99mTc-labeled diethylenetriaminepentaacetate (99mTc-DTPA) was used to assess changes in canine pulmonary epithelial permeability following lung irradiation. Doses of 2,000 cGy (11 dogs), 1,000 cGy (2 dogs), and 500 cGy (2 dogs) were given in one fraction to either the entire right hemithorax (500 cGy) or the right lower lung (1,000 and 2,000 cGy). Radioaerosol scans, chest roentgenograms, and computerized tomograms (CT) were obtained before and serially after irradiation. A dose of 2,000 cGy resulted in a decrease in regional pulmonary epithelial permeability to both 99mTcO4- and 99mTc-DTPA; both showed significant decreases from the 2nd wk postirradiation onward. In comparison, CT and chest roentgenogram did not become abnormal until 7.1 +/- 2.8 (SD) and 8.2 +/- 2.6 wk, respectively. Doses of 1,000 and 500 cGy produced reversible decreases in 99mTcO4- clearance. Lung morphology showed definite changes of radiation pneumonitis after 2,000 and 1,000 cGy but not after 500 cGy at approximately 9, 17, and 12 wk postirradiation, respectively. These results suggest that dose-dependent changes in pulmonary physiology may precede obvious structural alterations in radiation lung injury.     

Sequential changes in lung metabolism, permeability, and edema after ANTU

Lung injury and pulmonary edema were induced in rats after intraperitoneal injection of 10 mg/kg alpha-naphthylthiourea (ANTU). The time course of development of lung injury was assessed by the clearance of 99mTc-diethylenetriamine pentaacetate (99mTcDTPA) from the lung into the blood, the pharmacokinetics of tritiated prostaglandin E2 [( 3H]PGE2) in the isolated perfused lung, and by increase in the weight ratio (wet-to-dry) of lung. Two hours after ANTU administration, the clearance of 99mTcDTPA was significantly faster than in untreated animals and implied an increase in permeability of the alveolar-capillary barrier. This change preceded the increase in wet-to-dry weight ratio of lung, which was not significant until 5 h after ANTU administration. The pharmacokinetics of [3H]PGE2 were significantly altered after ANTU and these changes persisted beyond the time when both lung weight ratio and 99mTcDTPA clearance had recovered to normal values. We conclude that both 99mTcDTPA clearance and PGE2 pharmacokinetics change in ANTU-induced lung injury but with different time courses. In the progressive phase of lung injury due to ANTU, the early change in clearance of 99mTcDTPA suggests that an increased permeation of the alveolar capillary barrier by this small molecule precedes pulmonary edema due to an increased colloid permeability of the barrier. Abnormal metabolism in the pulmonary microvasculature persists when the permeability defect and edema have recovered.     

Elimination of p19ARF‐expressing cells protects against pulmonary emphysema in mice

Senescent cells accumulate in tissues during aging and are considered to underlie several aging‐associated phenotypes and diseases. We recently reported that the elimination of p19^ARF‐expressing senescent cells from lung tissue restored tissue function and gene expression in middle‐aged (12‐month‐old) mice. The aging of lung tissue increases the risk of pulmonary diseases such as emphysema, and cellular senescence is accelerated in emphysema patients. However, there is currently no direct evidence to show that cellular senescence promotes the pathology of emphysema, and the involvement of senescence in the development of this disease has yet to be clarified. We herein demonstrated that p19^ARF facilitated the development of pulmonary emphysema in mice. The elimination of p19^ARF‐expressing cells prevented lung tissue from elastase‐induced lung dysfunction. These effects appeared to depend on reduced pulmonary inflammation, which is enhanced after elastase stimulation. Furthermore, the administration of a senolytic drug that selectively kills senescent cells attenuated emphysema‐associated pathologies. These results strongly suggest the potential of senescent cells as therapeutic/preventive targets for pulmonary emphysema.     

Time course of changes in lung permeability and edema in the rat exposed to 100% oxygen

Rats were exposed to 100% oxygen for up to 60 h to determine early changes in lung permeability leading to the development of pulmonary edema. The time course of development of increased solute flux was assessed by the clearance of 99mTc-labeled diethylenetriamine pentaacetate (99mTc-DTPA) from the lung and the accumulation of 125I-labeled albumin (125I-albumin) in the lung. These end points were related to the development of pulmonary edema by the measurement of the wet-to-dry weight ratio of the lung and the weight of fluid in the pleural cavity. No significant changes occurred until 48 h of hyperoxia, when sharp increases in both indexes of lung permeability and wet-to-dry weight ratio occurred. By 60 h of exposure, pleural effusions had developed. The volume of this effusion was significantly correlated to both 99mTc-DTPA clearance and 125I-albumin flux.     

Changes in lung permeability after chronic pulmonary artery obstruction.

We have shown that left pulmonary artery ligation (LPAL) in mice causes a prompt angiogenic response, with new systemic vessels from intercostal arteries penetrating the pleura within 6 days. Because angiogenic vessels in other organs have been shown to exhibit increased permeability, we studied vascular permeability (Evans blue dye extravasation, lung wet weight-to-dry weight ratio, and lavaged protein) in naive C57BL/6 mice and 4 h, and 14 and 21 days after LPAL (4-6 mice/time point). We also measured radiolabel clearance as an index of functional perfusion after LPAL. Tracer clearance from the left lung was maximal by 6 days after LPAL and not different from right lungs. Thus a functional vasculature is established before 6 days of LPAL that results in normal tracer clearance. By 21 days after LPAL, Evans blue-albumin was significantly increased in the left lung relative to both 4 h (no vasculature) and 14 days after LPAL. Only after 21 days of LPAL was left lung wet weight-to-dry weight ratio significantly different from naive lungs. Additionally, lavaged protein was significantly increased both 4 h and 21 days after LPAL relative to control mice. Thus, using three different methods, results consistently demonstrated increased permeability to protein and water 21 days after LPAL. Although changes in surface area of perfusion might affect the interpretation of these results, blood flow measured with labeled microspheres indicated no change in left lung perfusion between 14 and 21 days of LPAL. Thus the lung vasculature, remodeled as a consequence of chronic pulmonary artery obstruction, demonstrates increased water and protein permeability.     

New Developments in the Pathogenesis of Smoke Inhalation-Induced Pulmonary Edema

Smoke inhalation causes most of the deaths in fire-related injuries, with pulmonary edema as a major determinant in the outcome of smoke-inhalation injury. The pathophysiology of pulmonary edema is thought to be related to the products of incomplete combustion. Damage to the integrity of the alveolar epithelium is one of the determinants of the development of smoke-induced pulmonary edema. In recent studies using lung clearance of aerosolized pentetic acid (DTPA [diethylenetriaminepentaacetic acid]) labeled with technetium Tc 99m to assess the permeability of the alveolar epithelium, several factors were identified that may increase a person''s susceptibility to smoke-induced acute lung injury. These are increased initial alveolar permeability and alterations in the number and activity of alveolar macrophages. Clinical measurement of ^99mTcDTPA clearance may provide a sensitive and convenient method for the early detection and serial assessment of smoke-induced alveolar epithelial permeability changes.     

BMP4 signaling induces senescence and modulates the oncogenic phenotype of A549 lung adenocarcinoma cells

Lung cancer is the most common visceral malignancy in males, with rapidly increasing incidence in females, and a devastatingly poor prognosis. Transforming growth factor (TGF)-beta has been shown to induce senescence in A549 lung cancer cells, and both TGF-beta and bone morphogenetic protein (BMP) 2 can suppress the transformed phenotype of A549 cells in vitro. We examined the effects of BMP4, another member of the TGF-beta superfamily, on specific oncogenic properties of A549 cancer cells. When A549 cancer cells were treated continuously with 100 ng/ml of BMP4, a senescent phenotype was observed after 2 wk of treatment. The BMP-treated cells appeared larger than untreated cells, grew more slowly, had more senescence-associated beta-galactosidase activity, and had less telomerase activity, as measured by the telomeric repeat amplification protocol assay. Invasion through Engelbreth Holm-Swarm matrix was inhibited in the senescent cell population. Senescent BMP4-treated cells had lower ERK activation, VEGF expression, and Bcl2 expression than wild-type cells, consistent with a less proliferative, less angiogenic phenotype with increased susceptibility to death by apoptosis. BMP4 treatment also resulted in sustained elevation of Smad1. In vivo xenograft studies in the flanks of nude mice confirmed that the BMP-treated cells were significantly less tumorigenic than untreated cells. Direct overexpression of Smad1 using adenoviral constructs resulted in cell death within 5 days. These studies suggest that BMP4 pathway signaling can induce senescence and thus negatively regulate the growth of A549 lung cancer cells.     

Variation in senescent-dependent lung changes in inbred mouse strains.

Previous studies from our laboratories showed lung development differences between inbred strains of mice. In the present study, the C57BL/6J (B6) and DBA/2J (D2) strains were examined for senescent-dependent differences with respect to the lung structure and function. Specifically, we hypothesize that senescent changes in lung vary between strains due to identifiable gene expression differences. Quasi-static pressure-volume curves and respiratory impedance measurements were performed on 2- and 20-mo-old B6 and D2 mice. Lung volume at 30 cm H(2)O (V(30)) pressure was significantly (P < 0.01) increased with age in both strains, but the increase was proportionally greater in D2 (68%) than in B6 (40%) mice. In addition, decreased elastic recoil pressure at 50% of V(30) and a reduction in airway resistance as a function of positive end-expiratory pressure were observed in 20-mo-old D2 mice but not in B6 mice. Morphometric analysis of lung parenchyma showed significant decreases in elastic fiber content with age in both strains, but the collagen content was significantly (P < 0.01) increased with age in D2 but not B6 mice at 20 mo. Furthermore, using quantitative RT-PCR methods, gene expression differences between strains suggested that D2 mice significantly (P < 0.05) downregulated the expressions of elastin (Eln) and procollagen I, III, and VI (Col1a1, Col3a1, and Col6a3) in lung tissue at 20 mo of age. These age-dependent changes were accompanied by an increased gene expression in matrix metalloproteinase 9 (Mmp9) in D2 and an increase in tissue inhibitor of matrix metalloproteinase (Timp1 and Timp4) in B6 mice. In conclusion, the results from the present study demonstrate that lung mechanics of both strains show significant age-dependent changes. However, changes in D2 mice are accelerated relative to B6 mice. Moreover, gene expression differences appear to be involved in the strain-specific changes of lung mechanic properties.     

Basal lung mechanics and airway and pulmonary vascular responsiveness in different inbred mouse strains.

Little is known about interstrain variations in baseline lung functions or smooth muscle contractility in murine lungs. We therefore examined basal lung mechanics and airway, as well as vascular reactivity to methacholine, thromboxane (using U-46619), and endothelin-1 (ET-1), A/J, AKR, BALB/c, C3H/HeN, C57BL/6, and SCID mice. All experiments were performed with isolated perfused mouse lungs. Except AKR mice (which were excluded from further analysis), all other strains showed stable pulmonary compliance, pulmonary resistance, and pulmonary arterial pressure within a control period of 45 min. Among these strains, C3H/HeN mice exhibited higher dynamic pulmonary compliance and lower pulmonary resistance, whereas SCID mice had higher baseline pulmonary resistance than the other strains. Concentration-response experiments with methacholine showed a lower airway reactivity for C57BL/6 mice compared with the other strains. Perfusion with 1 microM U-46619 or 100 nM ET-1 revealed a similar pattern: the agonist-inducible broncho- and vasoconstriction was lower in C57BL/6 mice than in all other strains, whereas it tended to be higher in SCID mice. The present study demonstrates a correlation between airway and vascular responsiveness in all tested strains. SCID mice are hyperreactive, whereas C57BL/6 mice are hyporeactive, to smooth muscle constrictors. Lung mechanics, as well as airway and vascular responsiveness, appear to be genetically controlled.     

Age-dependent changes of airway and lung parenchyma in C57BL/6J mice.

In the current study, we hypothesize that senescent-dependent changes between airway and lung parenchymal tissues of C57BL/6J (B6) mice are not synchronized with respect to altered lung mechanics. Furthermore, aging modifications in elastin fiber and collagen content of the airways and lung parenchyma are remodeling events that differ with time. To test these hypotheses, we performed quasi-static pressure-volume (PV) curves and impedance measurements of the respiratory system in 2-, 20-, and 26-mo-old B6 mice. From the PV curves, the lung volume at 30 cmH(2)O pressure (V(30)) and respiratory system compliance (Crs) were significantly (P < 0.01) increased between 2 and 20 mo of age, representing about 80-84% of the total increase that occurred between 2 and 26 mo of age. Senescent-dependent changes in tissue damping and tissue elastance were analogous to changes in V(30) and Crs; that is, a majority of the parenchymal alterations in the lung mechanics occurred between 2 and 20 mo of age. In contrast, significant decreases in airway resistance (R) occurred between 20 and 26 mo of age; that is, the decrease in R between 2 and 20 mo of age represented only 29% (P > 0.05) of total decrease occurring through 26 mo. Morphometric analysis of the elastic fiber content in lung parenchyma was significantly (P < 0.01) decreased between 2 and 20 mo of age. To the contrary, increased collagen content was significantly delayed until 26 mo of age (P < 0.01, 2 vs. 26 mo). In conclusion, our data demonstrate that senescent-dependent changes in airway and lung tissue mechanics are not synchronized in B6 mice. Moreover, the reduction in elastic fiber content with age is an early lung remodeling event, and the increased collagen content in the lung parenchyma occurs later in senescence.     

Influence of murine leukemia proviral integrations on development of N-methyl-N-nitrosourea-induced thymic lymphomas in AKR mice.

The AKR mouse strain is characterized by a high incidence of spontaneous thymic lymphoma that appears in older animals (greater than 6 months of age) and is associated with novel provirus integrations of ecotropic and recombinant murine leukemia viruses (MuLVs). Treatment of 4- to 6-week-old AKR/J mice with the carcinogen N-methyl-N-nitrosourea (MNU) results in thymic lymphomas that arise as early as 3 to 4 months of age and contain novel somatically acquired MuLV provirus integrations. The AKR/J strain develops MNU-induced lymphoma with a higher incidence and shorter latency than has been observed for other inbred mouse strains. To determine whether provirus integrations of endogenous MuLV account for the enhanced susceptibility of the AKR strain, the incidence and latency of MNU-induced lymphoma development was compared in AKR/J and AKR.Fv-1b mice. The restrictive b allele of the Fv-1 locus restricts integration and replication of endogenous N-tropic MuLV; therefore, AKR-Fv-1b mice have a very low incidence of spontaneous lymphoma. In contrast, AKR.Fv-1b mice develop MNU-induced lymphomas with an incidence and latency similar to those of the AKR/J strain. Furthermore, thymic lymphomas from both strains express an immature CD4-8+ phenotype, indicating neoplastic transformation of the same thymocyte subset. Southern blot analysis confirmed that lymphoma DNA from AKR.Fv-1b mice did not contain somatically acquired provirus integrations. These results demonstrate that provirus integration does not contribute to the predisposition of AKR mice to develop a high incidence of early MNU-induced lymphomas. Nevertheless, MNU treatment stimulated high-level expression of infectious ecotropic MuLV in AKR.Fv-1b as well as in AKR/J mice, suggesting that viral gene products might enhance lymphoma progression.     

Pharmacological clearance of senescent cells improves survival and recovery in aged mice following acute myocardial infarction

Cardiovascular disease is the leading cause of death in individuals over 60 years old. Aging is associated with an increased prevalence of coronary artery disease and a poorer prognosis following acute myocardial infarction (MI). With age, senescent cells accumulate in tissues, including the heart, and contribute to age‐related pathologies. However, the role of senescence in recovery following MI has not been investigated. In this study, we demonstrate that treatment of aged mice with the senolytic drug, navitoclax, eliminates senescent cardiomyocytes and attenuates profibrotic protein expression in aged mice. Importantly, clearance of senescent cells improved myocardial remodelling and diastolic function as well as overall survival following MI. These data provide proof‐of‐concept evidence that senescent cells are major contributors to impaired function and increased mortality following MI and that senolytics are a potential new therapeutic avenue for MI.     

Lung edema clearance: 20 years of progress: invited review: alveolar edema fluid clearance in the injured lung.

Resolution of pulmonary edema involved active transepithelial sodium transport. Although several of the cellular and molecular mechanisms involved are relatively well understood, it is only recently that the regulation of these mechanisms in injured lung are being evaluated. Interestingly, in mild-to-moderate lung injury, alveolar edema fluid clearance is often preserved. This preserved or enhanced alveolar fluid clearance is mediated by catecholamine-dependent or -independent mechanisms. This stimulation of alveolar liquid clearance is related to activation or increased expression of sodium transport molecules such as the epithelial sodium channel or the Na(+)-K(+)-ATPase pump and may also involve the cystic fibrosis transmembrane conductance regulator. When severe lung injury occurs, the decrease in alveolar liquid clearance may be related to changes in alveolar permeability or to changes in activity or expression of sodium or chloride transport molecules. Multiple pharmacological tools such as beta-adrenergic agonists, vasoactive drugs, or gene therapy may prove effective in stimulating the resolution of alveolar edema in the injured lung.     

Adiposity-Related Biochemical Phenotype in Senescence-Accelerated Mouse Prone 6 (SAMP6)

Senescence-accelerated mouse prone 6 (SAMP6) is a model of senile osteoporosis. From 10 to 22 wk of age, SAMP6 mice were heavier than age-matched AKR/J and SAMR1 mice. Body mass indices of 10- and 25-wk-old SAMP6 mice were higher than those of age-matched AKR/J and SAMR1 mice, indicating obesity in the SAMP6 animals. We compared the blood biochemical values among SAMP6, SAMR1, and AKR/J mice to assess whether the SAMP6 strain has abnormal obesity-related parameters. Plasma glucose, triglyceride, insulin, and leptin levels were higher in 10-wk-old SAMP6 mice than in age-matched SAMR1 and AKR/J mice, whereas plasma glucagon and adiponectin levels in 25-wk-old SAMP6 were lower compared with those in age-matched SAMR1 and AKR/J. Total cholesterol levels in SAMR1 and SAMP6 mice at 10 and 25 wk of age were higher than those in AKR/J mice. Hepatic lipid levels were higher in 10- and 25-wk-old SAMP6 mice compared with age-matched AKR/J and SAMR1 animals. These results indicate that SAMP6 mice exhibit obesity and hyperlipidemia, suggesting that the SAMP6 strain is a potential tool for the study of hyperlipidemia.Abbreviations: BMI, body mass indexThe senescence-accelerated mouse strains were developed through selective breeding of AKR/J mice based on graded scores for senescence and pathologic phenotypes.⁴⁴ The 9 senescence-prone (SAMP) strains all have a shortened lifespan and display an early onset of senescence after normal development and maturation, whereas the 3 senescence-resistant (SAMR) strains are resistant to early senescence and serve as controls. Among the SAMP strains, SAMP8 and SAMP10 exhibit deficits in learning and memory at a relatively early stage in their lifespan.^6,30 In contrast, SAMP6 mice are considered to be a model of senile osteoporosis, with their low bone mass and slow bone loss;²⁴ the bone mineral density of SAMP6 mice decreases after 4 mo of age.^14,17Our regular measurement of body weight revealed that SAMP6 mice were significantly higher between 10 and 22 wk of age than were age-matched SAMR1 and AKR/J. Based on this observation, we decided to compare body mass indices (BMIs), blood biochemical values, and liver sections among mice of these strains at 10 and 25 wk of age, which respectively correspond to the beginning and end of a period of significant body weight gain in SAMP6 mice compared with age-matched SAMR1 and AKR/J. Increased BMIs of SAMP6 mice at 10- and 25 wk compared with those of age-matched AKR/J and SAMR1 animals would indicate obesity in the SAMP6. In addition, because osteoblasts and adipocytes are thought to share a common precursor cell, osteoporosis and enhanced adipogenesis may be related. For example, adipogenesis in the bone marrow increases with aging and during osteoporosis,^15,33,34 and increased bone turnover occurs in hypercholesterolemic or dyslipidemic patients.²² Therefore obesity in SAMP6 mice might be due at least in part to enhanced adipogenesis. We measured and compared blood biochemical values among SAMP6, SAMR1, and AKR/J (the founder for the SAM strains) mice to assess whether the SAMP6 strain has abnormalities in blood biochemical markers, such as triglycerides or cholesterol.     

The study of correlation between the K-ras genotype and murine susceptibility to chemically induced lung neoplasms

The susceptibility of the ICR, DD/He and CC57/BR mice to urethane-induced lung tumors was analyzed in comparison with the A/He (highly susceptible) and AKR (resistant) lines of mice. Allelic variants of the K-Ras gene intron 2 in these lines have been determined. Susceptibility of the ICR mice was similar to that of the A mice, and intron 2 of the K-Ras ICR gene carried the 37-bp deletion analogous to that described in the A/He line. The DD mice intron 2 also contained the deletion, but despite the presence of the "susceptible" K-Ras allele, the DD/He mice were resistant to urethane induction of lung tumors. The CC57BR line carried the deletion and demonstrated relatively high susceptibility. Our findings indicate that the K-Ras gene may be important in the chemical induction of lung tumors.