Pituitary 6: 127–133, 2003 C  2004 Kluwer Academic Publishers. Manufactured in The Netherlands. Correlation of Bcl-2 and Bax with Apoptosis in Human Pituitary Adenomas Demetrios Sambaziotis 1 , Nikiforos Kapranos 2 , and George Kontogeorgos 1 1 Department of Pathology G. Gennimatas General Hospital and 2 Department of Molecular Pathology, MITERA Maternity and Surgical Center of Athens, Athens, Greece Abstract. Bcl-2 oncogene and Bax gene play an important role in regulating apoptosis. In the present study, the ex- pression of bcl-2 and bax was investigated and correlated with apoptosis in a series of 81 pituitary adenomas. Bcl-2 and bax proteins were localized by immunohistochemistry and the histoscore (HSC) was assessed by multiplying the immunohistostaining grade (1 to 4) by the staining inten- sity grade (1 to 3). According to bcl-2/bax HSC the tumors were separated in group A when ≥1 and group B when < 1. The apoptotic labeling index (ALI) was accessed by the in situ end-labeling (ISEL) technique. Bcl-2 protein was equally detected in functioning and nonfunctioning adeno- mas with statistically significant higher HSC in nonfunc- tioning tumors ( P < 0.03). Bax protein was immunopositive in the substantial majority of adenomas with significantly higher HSC in functioning as compared to nonfunctioning adenomas ( P < 0.0009). The ALI was significantly higher in functioning adenomas as compared to nonfunctioning ade- nomas ( P < 0.04). In addition, ALI was significantly higher in group B than in group A ( P < 0.004) and it was corre- lated with bax HSC ( P < 0.004). Finally, the group B of bcl-2/bax significantly predominated in nonfunctioning tu- mors ( P < 0.0009) and in microadenomas ( P = 0.05), as compared with functioning adenomas and macroadenomas respectively. In conclusion, our findings suggest that bcl-2 and bax molecules play a role in the regulation of apoptotic mechanisms in pituitary adenomas. Key Words. apoptosis, bax, bcl-2, in situ, pituitary Introduction Apoptosis or programmed cell death represents an en- ergy requiring mechanism in normal and neoplastic cells with morphologic and biochemical characteristics dis- tinctive from classic necrosis [1]. The protein products of a large number of genes regu- late apoptosis acting as molecular death switches. One of the main genes is the bcl-2 gene family. Bcl-2 is a 24–26 KDa protein located mainly on the outer mito- chondrial membrane, originally discovered as a chro- mosomal translocation in B cell follicular lymphoma [2]. Bcl-2 was found to contribute to the neoplastic pro- gression by inhibiting apoptosis induced by hormones and cytokines and thus, extending cell survival [3]. Re- cent studies have shown that bcl-2 blocks the release of mitochondrial enzyme cytochrome c [4,5], which is necessary for activation of caspaces and thus mediates apoptosis [6]. Bax (bcl-2-associated X protein) is the first protein to be isolated showing homology with bcl-2, through- out two highly conserved regions. Bax can dimerize with itself or with bcl-2 and when overproduced, bax homodimers promote apoptosis [7]. In contrast, when bcl-2 is in excess, bcl-2 homodimers predominate and cells are protected from cell death. Bcl-2 inhibits apop- tosis without increasing cell proliferation and negatively regulates the apoptotic activity of bax by formation of bcl-2/bax heterodimers. Bcl-2/bax ratio represents a cell death switch, which predetermines cell life or death re- sponse to an apoptotic stimulus [7]. Low bax expres- sion in breast cancer cells contributes to progression of the disease [8]. In contrast, bax deficient mice demon- strate lymphoid hyperplasia and male germ cell death as well [9]. These data suggest that bax may promote or inhibit cell death depending upon the cell type in which it is expressed. Loss of bax also promotes tumor forma- tion in mice [5] and probably in humans [10]. Studies on bcl-2 and bax mRNA and protein indicate that bax is more widely expressed than bcl-2, suggesting that bax has additional functions besides inhibiting bcl-2 [11,12]. For example, the tumor suppressor gene p53 directly in- duces Bax expression in radiation sensitive cells; thus, p53 may play a role in the apoptotic elimination of cells after their exposure to DNA-damaging agents. In addi- tion, it was shown that p53 can activate apoptosis in part through transcriptional transactivation of bax [13]. Although the ultrastructural spectrum of apoptotic changes is conclusive, it is not always feasible to detect single cell death by light microscopy [14,15]. Demonstra- tion of intanucleosomal DNA fragmentation by the in situ end-labeling (ISEL) technique represents alterna- tive approach to investigate apoptosis [14,16]. Immuno- cytochemical localization of bcl-2 and bax gene prod- ucts can provide additional information on the status and regulation of apoptosis. Given the limited number of mainly experimental studies on apoptosis in pituitary Address correspondence to: George Kontogeorgos, M.D., Ph.D., Department of Pathology, G. Gennimatas Athens General Hospital, KOFKA Bulding, 1st Floor, 154 Messogion Avenue, 115 27 Athens, Greece. Tel.: (+201) 778-4302; Fax: (+201) 778-4302 or 770-4765; E-mail: gkonto@med.uoa.gr