Very little is known about the apoptotic index of salivary gland neoplasms. We used two different anti-apoptotic indexes (AI-1 and AI-2) to calculate the apoptotic profile of these lesions. The higher these coefficients are, the more probable an anti-apoptotic profile is expected. In contrast, an index < 1 indicates an increase in BAX
or a decrease in BCL-2
mRNA transcription, favoring apoptosis. It has been shown that Bcl-2 protein forms heterodimers with the Bax protein such that Bcl-2-Bax inhibits apoptosis, whereas Bax-Bax homodimers favor it [19
]. Tumour growth depends on the balance between proliferation/apoptotic indexes. In the present paper we demonstrated that most of the salivary gland neoplasm samples showed a higher AI-1 and AI-2 when compared to normal salivary glands, suggesting a predominance of anti-apoptotic behavior in neoplastic cells, which in turn contributes to neoplasia growth (Figure ). This study is the first to demonstrate that salivary gland tumours present an anti-apoptotic transcriptional signature.
It was reported, using the 3'-end DNA labeling method (TUNEL), that in salivary gland neoplasms apoptosis is inversely associated with Bcl-2 expression, but not related to Bax expression [11
]. This result was strengthened by another publication using TUNEL method which described an inverse association between apoptosis and the expression of Bcl-2 in adenoid cystic carcinomas [15
]. However, in mucoepidermoid carcinomas such association did not exist [13
]. In the present paper we have shown an increased BCL-2
mRNA transcription in the salivary tumours compared to normal salivary glands in 78% of the samples. If we consider only the pleomorphic adenoma samples, BCL-2
overexpression was even higher, corresponding to 88% of these tumours. This result supports the above mentioned paper by Soini and colleagues (1998) [11
], which pointed to a very low apoptotic index (0.01%) in pleomorphic adenomas. Our results are in agreement with another study that described Bcl-2 immunopositivity in 33/35 samples of pleomorphic adenomas investigated [14
]. Also, all the pleomorphic adenomas exhibited AI-1 and/or AI-2 higher than normal salivary glands (Figure ). Altogether, the evidence points to Bcl-2 as an important factor in the salivary gland neoplasms pathogenesis and as a possible molecular target in salivary gland tumour treatment in the future.
We did not analyze other benign/malignant lesions in separate groups, because as they are rather unusual (eg. adenoid cystic carcinoma, cystadenocarcinoma, mucinous cystadenoma) we had only a few fresh samples included in the study. However, the expression profile of the two mucoepidermoid samples included in the study was unique, as one of them revealed an apoptotic tendency (#27) and in previous immunohistochemistry based publications, these lesions did not reveal a high percentage of Bcl-2 positive samples [11
We demonstrated an overall BCL-2 mRNA overexpression, as well as an increased AI-1 and AI-2 in the salivary tumours when compared to normal salivary glands, and in addition we found an association between increased tumour size and high cellular proliferation index. Although the malignant and benign group of samples did not show difference in the AI-1/AI-2, the malignant samples showed a statistical significant higher cellular proliferation index. Taking these findings together, it seems that while both, benign and malignant tumours, tend to evade apoptosis, the malignant samples in addition tend to have a higher cell proliferation activity, guaranteeing a growth advantage.
p53 positive samples showed higher BCL-2
transcription levels than the negative ones, indicating an association of p53 immunopositivity with an increased AI-1 and a predominantly anti-apoptotic profile. It has been shown that p53 induces apoptosis by repressing the transcription of the anti-apoptotic gene BCL-2
and activating the transcription of the apoptotic BAX
]. Such apoptotic function could be inactivated by p53 mutations. The mutated p53 is usually more stable than the wild-type leading to higher levels of p53 and to immunohistochemical detection of such protein. Therefore, p53 immunoexpression in the samples analyzed may reflect loss of apoptosis induction promoted by this protein, which may explain the increased anti-apoptotic activity found in these tumours [21
]. According to the last publication of the World Health Organization on Head and Neck tumours, the role of p53 in salivary gland neoplasms is an issue of controversy [1
]. In the present paper, we demonstrated an association between malignancy and high proliferation index and between proliferation and p53 positivity. This evidence suggests that p53 (clone DO7) positivity may be used as a marker in salivary tumours as it is associated not only with higher proliferation index but also with an anti-apoptotic profile, both contributing to tumour growth. This apparent importance of p53 staining as a potentially useful marker in salivary neoplasms empowers other findings of association between higher salivary p53 expression in salivary gland tumours and poor survival [17