Abstract

Failure to maintain an appropriate balance between cell death and proliferation is partly due to derangements in the regulation of apoptosis. In this work, apoptosis and the expression of apoptosis regulating proteins were studied by 3′-end labeling of fragmented apoptotic DNA (TUNEL) and immunohistochemistry in a set of 147 tissue samples consisting of 44 biopsies of normal and dysplastic bronchial epithelium, and 103 non-small cell lung carcinomas.

The quantity of apoptotic cells and bodies, apoptotic index (AI%), is defined as a percentage of apoptotic cells in the entire tumor cell population. Changes in the apoptotic activity were already seen in the metaplasia-dysplasia-carcinoma sequence of the lung, where the AI% increased gradually until moderate epithelial dysplasia but started to decrease after that. Thus, the AI% for invasive NSCLC (1.20 for squamous cell carcinoma and 1.24 for adenocarcinoma) was slightly lower than in premalignant bronchial epithelium (mean 1.50), but clearly higher than in normal tissue (0.20 for normal bronchial epithelium and 0.24 for lung interstitial cells). 53% of SQCCs and 50% of ACs showed p53 positive nuclei indicative of mutated p53 protein. The immunostaining of bcl-2, bax and mcl-1 revealed diffuse, cytoplasmic staining and was present in most tissues studied. No statistically significant associations between the extent of apoptosis and the expression of p53, bcl-2, bax, or mcl-1 could be found, although. The immunostaining for caspases 3, 6 and 8 was restricted to the tumor areas, reflecting increased apoptotic activity in them. The AI% was significantly higher in NSCLCs in which the single-cell staining pattern for caspase-8 was dominant (P = 0.017), whereas the expression of caspases 3 and 6 had no association with apoptosis. The number of apoptotic cells was significantly higher in NSCLC tumors with a high number of CD3+ and CD8+ T-lymphocytes (P = 0.01) and B-cells (P = 0.05). By multivariate analysis, enhanced apoptosis in NSCLC showed a 1.9-fold risk (95% CI 1.04–3.60; P = 0.04) and p53 positivity a 2.3-fold risk (95% CI 1.30–4.10; P = 0.005) for a shortened survival. Both factors appeared as independent prognostic variables.

Apoptosis is clearly enhanced in premalignant and malignant lung tissue in comparison with normal tissue. Furthermore, the expression of the apoptosis-regulating genes is different in tumor tissue from that in normal tissue, and some of the changes in their expression can be seen even in the premalignant lesions of the bronchial epithelium. The expression of caspases seen only in tumor tissue implies the activation of the apoptotic mechanisms and, thus, the lowered treshold of tumor cells to undergo apoptosis. Even in the advanced stages of the disease, the immune defense is effective and the cytotoxic action of activated CD8+ T-cells clearly involves apoptosis. Based on these results it is concluded that alterations in the apoptotic activity and changes in the expression of apoptosis-regulating genes are associated with malignant transformation and growth in lung tissue.