The p53 tumour suppressor protein is a critical mediator of the DNA damage response in mammalian cells. In response to DNA damage, wild type p53 protein transcriptionally modulates a battery of downstream genes involved in growth control, DNA repair and apoptosis. It is hypothesised that this 'p53 response' serves to preserve genomic integrity, and reflects the tumour suppressor properties of p53 relevant to human malignancy [1
]. There is marked tissue variability in the induction of particular p53-regulated responses, such that an equivalent dose of ionizing radiation, which induces DNA strand breaks, will induce p53-dependent apoptosis in murine thymocytes and small intestinal epithelial cells [4
], but not hepatocytes [6
]. Consequently, it is necessary to consider tissue context when determining roles of p53 in tumour suppression. Further, additional functions of p53 distinct from stress responses, including regulation of centrosome number and ploidy, have been reported and highlight additional, housekeeping functions of p53 in maintaining genomic integrity [7
]. There is controversy as to which of these functions of p53 (i.e. housekeeping or stress response) is dominant in suppressing tumour formation [9
], but given the tissue variability of the p53 response, a conservative expectation is that penetrance of the p53-null phenotype in either respect is cell-type dependent. This analysis reports on the effects of p53 deficiency in a subpopulation of murine lung epithelial cells, the Clara cells.
Clara cells are a non-ciliated population of airway epithelial cells which reside predominantly in the bronchioles and which are proficient in surfactant production and xenobiotic metabolism by virtue of expression of surfactant proteins A, B and D and a battery of cytochrome P450 isoenzymes, the nature of which vary between mammalian species [11
]. The observation that ciliated cells develop from Clara cells [12
] revived an interest in the Clara cell as a potential stem cell in the airways. The distal migration of Clara cells in the lungs of animals exposed to ozone, and the frequent 'bronchiolisation' of alveoli in lungs with chronic injury further suggest a role of the Clara cell in alveolar remodeling [16
]. However, it is noteworthy that Clara cells display marked interspecific heterogeneity in their distribution [17
]. Significantly, in rodents Clara cells occupy tracheal, bronchial, and bronchiolar epithelium, whereas primates accommodate Clara cells in only the epithelium lining terminal and respiratory bronchioles [19
]. It has been suggested that adenocarcinoma of the lung, a tumour of the small airways, represents malignancy of the Clara cell. Indeed, early-stage tumours of transgenic mice expressing SV40 T antigen from the small airway epithelial cell-specific promoter SP-C, a mouse model of human lung adenocarcinoma, frequently express the Clara cell markers in vivo [21
]. In human lung adenocarcinoma, where progression of the disease is histologically defined, mutation of p53 occurs early in tumour formation, possibly at a preneoplastic stage [22
]. Presumably, loss of p53 confers a selective advantage in preneoplastic lesions of the lung and is mutated accordingly. However, the mechanism by which p53 loss contributes to lung carcinogenesis is not understood.
In this analysis, the authors explored the hypothesis that Clara cells, as a major progenitor species of the small airways, are relevant to neoplasia of the lung, and that phenotypic effects of p53 deficiency would predominate in this cell type. A method for extracting, isolating, and culturing Clara cells from the mouse has been described previously [24
] and was incorporated into this analysis to facilitate a greater understanding of the role of p53 in small airway epithelial cells. 5-Bromo-2'-deoxyuridine (BrdU) incorporation assays were employed to assess baseline effects of p53 deficiency in proliferating airway epithelial cell cultures, phenomena difficult to measure in vivo due to the low turnover of small airway epithelium. In addition, BrdU incorporation assays were used to characterise the Clara cell response to ionizing radiation, with the intention of identifying DNA damage response functions of p53 in this cell type.