The tumor suppressor p53 protein is a transcription factor that plays a central role in the cellular response to DNA damage, and it can cause either G1 arrest or apoptosis. Recently, it was shown to induce the tumor suppressor p21Waf1/Cip1/Sdi1 (p21), which inhibits cyclin-CDK complex kinase activity. Although the etiology of idiopathic pulmonary fibrosis (IPF) is still uncertain, it is postulated that IPF begins with an initial inflammatory lesion localized to the alveolus and progresses on to chronic inflammation with alveolitis. We examined whether p53 and p21 are upregulated in association with chronic DNA damage in the bronchial and alveolar epithelial cells in patients with IPF in an attempt to repair the injury. We performed in situ detection of DNA strand breaks or apoptosis (TUNEL) in the tissues as well as immunohistochemistry (IHC) for p53 and p21. Positive signals by TUNEL were detected mainly in the bronchiolar and alveolar epithelial cells in 10 of 14 lung specimens from patients with IPF. On the other hand, no positive signal by TUNEL was detected in normal lung parenchyma or in specimens of pulmonary emphysema. The IHC demonstrated that p53 and p21 were expressed especially in hyperplastic bronchial and alveolar epithelial cells of lung tissues from all patients with IPF, except five specimens for p21. These results are consistent with those obtained by TUNEL. In normal lung parenchyma and specimens of pulmonary emphysema, p53 and p21 were not detected except in scattered alveolar macrophages and in the epithelial cells within localized fibrotic regions. These results suggest that p53 and p21 are upregulated in association with chronic DNA damage, resulting in either G1 arrest or apoptosis so that the DNA damage can be repaired in IPF. We speculate that chronic DNA damage and repair may lead to mutation of the p53 gene and tumorigenesis in IPF.

The incidence of apoptosis and the expression of Fas antigen (Fas)/Fas ligand (FasL) mRNA in bleomycin-induced pulmonary fibrosis in mice were examined. Male ICR mice were intratracheally instilled with bleomycin (5 U/kg of body weight). The controls were injected with sterile saline. The animals were anesthetized and killed at 1, 6, and 12 h, and 1, 3, 5, 7, 9, and 14 days after bleomycin instillation. We assessed the incidence of apoptosis in lung tissues by DNA fragmentation on agarose gel electrophoresis, terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end-labeling, and electron microscopy. The expression of Fas and FasL mRNA was detected by reverse transcription polymerase chain reaction (RT-PCR). The localization of Fas mRNA was analyzed by in situ hybridization and that of FasL mRNA was analyzed by RT in situ PCR. The results showed that (1) a single instillation of bleomycin leads to the rapid appearance of apoptosis in bronchial and alveolar epithelial cells, which resolves within 1 day, and (2) apoptosis reappears on day 7 and continues for over 14 days after bleomycin instillation. This was accompanied with a progression of fibrosis. Corticosteroid administration completely blocked both apoptosis and fibrosis. The expression of Fas mRNA was upregulated in the alveolar epithelial cells by the bleomycin instillation. FasL mRNA was also upregulated in infiltrating lymphocytes after bleomycin treatment, but not in the control mice. The administration of corticosteroids suppressed the expression of Fas and FasL mRNA as well as apoptosis and fibrosis. Although these results do not show that apoptosis mediated by the Fas/FasL system is directly linked to bleomycin-induced fibrosis, we speculate that excessive apoptosis and the Fas/FasL system play a role in the pathogenesis of bleomycin-induced lung injury.

The history of patients with idiopathic pulmonary fibrosis (IPF)shows that the disease may be preceded by a viral-like illness. Although viruses have not been demonstrated, it is possible that viruses were not detected in culture because they do not replicate during latency.We investigated the presence of adenovirus in IPF and interstitial pneumonia associated with collagen vascular disease (CVD-IP), using the nested polymerase chain reaction (PCR) and in situ hybridization (ISH) for the E1A region of the adenovirus genome. Studies were performed on lung tissues obtained by transbronchial lung biopsy from 19 patients with IPF, 10 patients with CVD-IP and, for comparison, from 20 patients with sarcoidosis.The E1A DNA was present in 3 out of 19 (16%) cases of IPF, in 5 of 10 (50%) cases of CVD-IP, and in 2 of 20 (10%) cases of sarcoidosis. The incidence of E1A DNA in CVD-IP was significantly higher than that in sarcoidosis (p<0.05). In patients with IPF and CVD-IP, E1A DNA was more prevalent in patients treated with corticosteroids (6 out of 9 cases; 67%) than in those without it (2 out of 20 cases; 10%) (p<0.01). ISH studies showed that 1 out of 8 cases of IPF and CVD-IP, in which E1A DNA was detected by PCR, was positive for E1A DNA.We conclude that adenovirus E1A is unlikely to be aetiologically involved in the pathogenesis of idiopathic pulmonary fibrosis or interstitial pneumonia associated with collagen vascular disease. However, a latent adenovirus infection may be reactivated or may newly infect the host following corticosteroid administration.

These results suggest that apoptosis may be associated with the resolution of inflammation and with tissue repair and also suggest the involvement of the Fas antigen/Fas ligand pathway in acute immune complex alveolitis in mice.

Group C adenovirus is latent in human tissues and can malignantly transform cells. The purpose of this study was to investigate the association between this virus and lung cancer. We investigated latent adenoviral infection using the nested polymerase chain reaction and in situ hybridization in transbronchial biopsy specimens from patients with small-cell lung cancer and non-small-cell lung cancer. The polymerase chain reaction was performed on DNA extracts with two sets of primers directed at a 261-base-pair target sequence of the E1A region of the adenoviral genome. In situ hybridization was performed on histological sections using DNA representing the entire adenovirus type 5 genome. E1A target DNA was present in 11 (31%) of 35 cases of small-cell lung cancer but in none of the 40 cases of non-small-cell lung cancer (P < 0.01). Of the 11 cases found positive by PCR, 8 were positive for adenovirus DNA by in situ hybridization. Adenovirus was prominent in tumor cells in 5 of the 8 cases, and in normal epithelial cells in the 3 remaining cases. Adenovirus DNA was not detected by in situ hybridization in specimens in which E1A DNA was not detected by the polymerase chain reaction. Small-cell lung cancer has mutations or deletions in the p53 and retinoblastoma genes more frequently than are found in non-small-cell lung cancer. Therefore, we speculate that adenovirus infection might participate in the pathogenesis of SCLC by producing mutation in these genes, rather than by inhibiting the function of these proteins.