Elsevier

Life Sciences

Volume 79, Issue 23, 2 November 2006, Pages 2202-2208
Life Sciences

Altered expression of connexins in urethane-induced mouse lung adenomas

https://doi.org/10.1016/j.lfs.2006.07.033Get rights and content

Abstract

Lung carcinogenesis is a multistep process whose molecular alterations can be studied in mouse models. Urethane, a specific lung tumor carcinogen, can induce adenomas in mice. Mouse lung alveolar cells reportedly generate lung neoplasms, and express connexins 26, 32, 43 and 46. The aim of the present study was to evaluate the expression of connexins in urethane-induced lung adenomas. Fifteen-day-old CD1 male mice received 2 i.p. injections of urethane (1.5 g/kg bw). The mice were euthanized 25 weeks after urethane injection, and lung adenomas were quantified. Lung tissue and lung adenomas were harvested and the RNA was extracted. The expression of connexins 26, 32, 43 and 46 was evaluated by Real-Time PCR, and these proteins were identified by Western blot. Immunohistochemistry revealed the distribution pattern of these connexins in lung tissue and adenomas. The treatment with urethane was associated with the downregulation of Cx26, 32 and 46 expressions, and with the upregulation of Cx43 expression in lung tissue. Surprisingly, in lung adenomas Cx32 and Cx43 expressions were not detected, although the expression of connexins 26 and 46 was present. Western blot and immunohistochemistry corroborated the RT-PCR data. These results may indicate a role of Cx32 and Cx43 in urethane-induced lung carcinogenesis, since their absence may contribute to the development of urethane induced lung tumors. The role of Cx26 and Cx46 is yet to be determined.

Introduction

Carcinogenesis is a multistep process involving alterations in genes related to cell growth and differentiation. In the molecular pathology era, the detection of genetic alterations in lung carcinogenesis is an important clue to the prevention or treatment of these tumors.

Only a few experimental models are available to study lung carcinogenesis in vivo. Urethane (ethylic ester of carbamic acid) is a chemical carcinogen which specifically promotes the development of lung tumors from alveolar type II pneumocytes in rodents (Manson et al., 2000). The development of lung adenomas can be observed 25 weeks after urethane injection in 15-day-old mice. Adducts of vinyl carbamate epoxies can be formed after the metabolization of urethane (Hoffler et al., 2003). Although the effects of urethane injection have long been described, the molecular alterations involved in urethane-induced lung carcinogenesis have not been explored.

Among the carcinogen-induced genetic alterations which may lead to benign or malignant neoplasms, decreased expression of proteins involved in the formation of communicating gap junctions has been reported (Cesen-Cummings et al., 1998, Loewenstein and Kanno, 1966, Weinstein et al., 1976). Connexins (Cx) belong to a large multigene family, and their role is to form the gap junctions that facilitate the exchange of small molecules (< 1 kDa) between the cytoplasms of adjacent eukaryotic cells. Such gene family members are named according to their molecular weights, and differ in both function and expression pattern (Willecke et al., 2002). The expression of connexins 26, 32, 43 and 46 has been described in mouse lung alveolar cells (Lee et al., 1997, Abraham et al., 1999, Abraham et al., 2001).

The purpose of the present study was to investigate the alterations in connexin expression in mouse lungs and lung adenomas after the treatment with urethane, and to verify which connexin(s) could play a role in lung carcinogenesis.

Section snippets

Mice and urethane injection

CD1 males from the Animal Facility of the Department of Pathology of the Faculty of Veterinary Medicine and Zootechny, University of São Paulo, were used. The animals were weaned at the age of 4 weeks and housed under controlled conditions (22 ±2 °C, 81 65 ± 15% relative humidity, air exchange rate 15 times/h, 12 h light–12 h dark cycle) in filter top cages, receiving a standard pellet diet (Lab Chow; Purina, Curitiba, Brazil) and tap water ad libitum throughout the study. At the age of 15 and

Number of lung adenomas in CD1 mice 25 weeks after the injection of urethane

The mean number of adenomas developed in CD1 male mice 25 weeks after the injection of urethane was 9.88 ± 5.43.Histologically, the adenomas were classified in papillary (23.2 ± 8.49%), mixed (35.7 ± 14.62%) or solid (41.1 ± 13.1%) (Fig. 1).

Connexins 26, 32, 43 and 46 mRNA levels reduced in mouse lungs after treatment with urethane

The results of mRNA quantification of connexin genes are presented in Table 1. Urethane treatment decreased Cx26 transcripts in lung tissue. However, urethane induced adenomas did not present any changes in Cx26 expression when compared with urethane-treated lung

Discussion

The present study was undertaken to investigate if urethane could alter Cx expression in lung tissue. Most studies on the role of Cxs have been performed on cultured cells, due to their usefulness for functional studies. However, in order to understand the potential role of Cxs in multistage carcinogenesis it is essential to study the effect on gap junction expression in in vivo models.

Based on our results, the development of lung adenomas is associated with a reduction in Cx32 and Cx43

Acknowledgments

The authors are grateful to Dr. Mike Koval, from the University of Pennsylvania, for kindly supplying the antibody for Cx46. This work is part of the PhD thesis of J.L. Avanzo in the Experimental and Comparative Pathology Program of the Faculty of Veterinary Medicine and Zootechny of the University of São Paulo. This research was supported by grant 01/06820-2 from the Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP). J.L. Avanzo was supported by a fellowship from FAPESP 01/06821-9.

References (18)

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