Acute lung injury is implicated in many respiratory diseases, including lung
adenocarcinoma and emphysema. In this thesis, the hypothesis that the stress protein
p53 is important in acute lung injury was investigated. p53 is a short-lived, latent
transcription factor which is activated and stabilised in response to a wide range of
cellular stresses, including DNA damage. In certain cell types, wild type p53 protein
mediates a variety of DNA damage responses and transcriptionally modulates a
battery of downstream genes involved in DNA repair, growth control, and apoptosis.
The effects of p53 were investigated in a mouse model of acute lung injury and in
short-term primary cultures of isolated Clara cells. Gene targeted mice, germline
deficient in p53, were exposed to y-irradiation and compared to wild type controls.
The in vivo response to DNA damage was characterised in terms of growth arrest,
apoptosis, morphology, and gene expression. An acute stress response was observed
in vivo, and localised to a subpopulation of the lung epithelium, the bronchiolar cells.
p53 was stabilised in this population and was associated with transcriptional
induction of Bax, but not other bcl-2 family members. p53 deficient mice did not
display this rapid accumulation of Bax transcripts, as assessed by RNase Protection
Assay. Within wild type and p53 null mice, y-irradiation did not induce apoptosis in
lung epithelial cells at any timepoints studied, as assessed by morphology, but did
induce strand breaks that were detectable by TUNEL. Cell cycle activity, as assessed
by BrdU incorporation, was infrequent in the lung at all timepoints, regardless of p53
status, and hence an effect of p53 on cell cycle progression was not detected in vivo.
The effects of p53-deficiency were additionally investigated in short-term primary
cultures of murine bronchiolar Clara cells. Culturing of Clara cells allowed an
assessment of the functional consequences of p53 deficiency in proliferating cells.
Clara cells, isolated from gene-targeted p53-deficient mice, were compared to cells
derived from wild type littermates. Baseline proliferation rates, as determined by
BrdU incorporation, were similar irrespective of p53 status. p53 null cultures
displayed abnormal morphology; specifically, a high incidence of multinucleation,
which increased with time in culture. Multinucleated cells maintained expression of
the Clara cell marker CC10, and were proficient in S phase DNA synthesis, as
determined by BrdU incorporation. Nucleation defects in p53 -/- Clara cells
associated with abnormalities in mitosis and cytokinesis, as documented by timelapse videomicroscopy, and with increased centrosome number, determined by
confocal microscopy. Defects in centrosome homeostasis, mitotic fidelity, and
cytokinesis in p53-null Clara cells may reflect a novel role of p53 in preserving
genomic integrity in lung epithelium.
Effects of p53-deficiency were also studied following exposure to DNA damage. A
p53-dependent reduction in the BrdU index was observed in Clara cells following
ionizing radiation. The reduction in BrdU index in wild type cells displayed serumdependency, and occurred only in the absence of serum. Apoptosis was infrequent in
both genotypes, as determined by time-lapse videomicroscopy. Taken together,
these findings demonstrate that in murine primary Clara cell culture, growth arrest
but not apoptosis is a p53-mediated response to DNA damage, and that extracellular
factors, such as serum, influence this response.
In addition, a transgenic model employing lung-specific Cre/lox technology was
evaluated. The Cre/lox recombinase system evolved within bacteriophage PI as a
mechanism to maintain correct unit copy segregation of the prophage within host
cells. This thesis reports application of this system to regulate gene expression in
murine lung epithelial cells in vivo, with the eventual goal of generating improved
mouse models of acute lung injury. Transgenic mice expressing Cre from the lungspecific promoter human SP-C were crossed to a Floxed DNA Ligase I line, and
transgene stability and function assessed by PCR and Southern blot methodologies.
The SP-C Cre transgene was demonstrated as stable, but of low copy-number.
Excision of the floxed allele, as determined by PCR, was specific to the lung, and
was not observed in other tissues. However, the level of excision was poor as
assessed by Southern analysis of the excision event. Furthermore, Cre expression
was undetectable by RT-PCR. Low expression levels of Cre may reflect the low
copy-number of the SP-C/Cre transgene.
In summary, this thesis reports on the role of p53 in lung epithelial cells, and on the
feasibility of using Cre/lox technologies to regulate gene expression in the lung
epithelium of transgenic mice. Bax mRNA induction, but not apoptosis, is a DNA
damage response of small airway epithelial cells. Transactivation of Bax was p53-
dependent in irradiated lung, as determined by RNase protection assay, and did not
occur in p53-/- mice. To further investigate the role of p53 in the lung, a method for
extracting, isolating, and culturing Clara cells, a progenitor cell of the lung, was
established and incorporated into this analysis. Absence of p53 favours
multinucleation and loss of cell cycle arrest in primary Clara cell culture, and
highlight additional roles of p53 in cell division and growth control. In addition, this
thesis explored the feasibility of using Cre/lox technologies to regulate gene
expression in murine lung epithelium. SP-C/Cre mice were assessed in their ability
to excise a Floxed DNA Ligase I cassette in the tissues of double transgenic mice.
Cre-mediated excision was specific to lung epithelium, but infrequent.