Colorectal Adenocarcinomas Elevated Expression of Bcl-X and Reduced Bak in Primary Updated Version

Expression of several members of the BCL-2 family of genes was investigated by immunohistochemical methods in 30 primary colorectal adenocarcinomas and 24 adenomatous polyps. When compared to the intensity observed in adjacent normal mucosa! epithelial cells, the inten sity of Bcl-X immunostaining was elevated in 18 of 30 (60%) carcinomas (P = 0.0001) and 12 of 24 (50%) adenomatous polyps (P = 0.0001). Immunoblot analysis of five pairs of tumors and adjacent normal colonie tissue indicated marked elevations in the relative levels of the anti-apoptotic Bcl-X, protein in all cases. In contrast to the increased Bcl-X expression, the intensity of Bcl-2 immunostaining was greater than that of normal colonie mucosa in only 3 of 30 (10%) carcinomas and, in fact, was lower than that of adjacent normal epithelial cells in 25 (83%) cases (P = 0.0001). Furthermore, the percentage of Bcl-2 immunopositive cells was generally lower in carcinomas than in adenomas (mean Â± SE, 44 Â±6% versus 73 Â±5%, respectively; P = 0.001) and in moderately or poorly differentiated tumors than in well-differentiated tumors (39 Â±6% versus 70 Â±11%, respectively; P = 0.045). In addition, the proportion of tumors in which the Bcl-2 immunointensity was more than or equal to that of normal colonie mucosa was significantly lower in carcinomas than adenomas (5 of 30 versus 15 of 24, respectively; /' < 0.001), suggesting that decreases in Bcl-2 expression represent a later event associated with the progression of colorectal cancers. When compared to that of normal adjacent colonie epithelium, the intensity of Mcl-1 immunostaining was reduced in 20 of 30 (67%) of carcinomas (P = 0.0001) compared to only 1 of 24 adenomas, suggesting that decreases in Mcl-1 expression represent a later event associated with progression from a benign to a malignant phenotype or with transition to a less-differentiated state, because most of the carcinomas evaluated here (25 of 30; 83%) were not well differenti ated. The intensity of immunostaining for the pro-apoptotic protein Bak was reduced compared to that of normal mucosa! epithelial cells in 27 of 30 (90%) carcinomas and 22 of 24 (92%) adenomas, suggesting that reductions in Bak expression occur early in colorectal tumor progression (/' = 0.0001). In contrast, the intensity of immunostaining for the proapoptotic protein Bax was not significantly altered in carcinomas; com pared to that of normal colonie mucosa, Bax immunointensity was re duced in only 7 of 30 (23%) carcinomas and 3 of 24 (13%) adenomas, and the percentage of Bax immunopositive cells was also not significantly different in any of the histolÃ3gica! subgroups. Taken together, these results suggest that expression of Ik-l-X, is increased in undifferentiated primary colorectal cancers, often with accompanying reciprocal decreases in the anti-apoptotic proteins Bcl-2 and Mcl-1 and the pro-apoptotic protein Bak, whereas Bax expression is relatively constant. Thus, a shift from expression of the anti-apoptotic proteins Bcl-2 and Mcl-1 to the Hcl-X, protein may occur during progression of colorectal tumors. Received 11/27/95; accepted 3/14/96. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1Supported in part by Grant CA-60181 from the National Cancer Institute, the U.S. National Dairy Council, and Grants DHP-32E and IRG-177C from the American Cancer Society. J. C. R. is a Scholar of the Leukemia Society of America. 2 To whom requests for reprints should be addressed, at The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037. Phone: (619) 455-6480, extension 3430; Fax: (619) 450-4693; E-mail: jreed@ljcrf.edu. INTRODUCTION Colorectal adenocarcinoma represents the fourth most common type of cancer and the second most frequent cause of cancer-associ ated death in the United States (1). Tumor kinetics studies have suggested that in vivo expansion of neoplastic colonie epithelial cells results not only from increased rates of cell proliferation but also from decreased rates of cell death due to apoptosis (2-4). Furthermore, decreased proportions of apoptotic cells have been documented during progression of these neoplasms from benign adenomatous polyps to invasive cancers (5). The Bcl-2 family of proteins regulates a distal step in an evolutionarily conserved pathway for programmed cell death and apoptosis (reviewed in Refs. 6-9). Some of the members of this gene family are blockers of cell death, including Bcl-2, Bcl-XL> and Mcl-1; whereas others are promoters of apoptosis, such as Bax, Bak, and Bcl-Xs. Furthermore, in many cases, these proteins can physically interact with each other in a network of homoand heterodimers in which the relative proportions of the anti-apoptotic and pro-apoptotic members of this family determine the ultimate sensitivity of cells to cell death stimuli (9-19). Of potential relevance to mechanisms of carcinogenesis and resistance to therapy, overexpression of Bcl-2 has been shown to provide protection against a wide variety of apoptotic insults, including growth factor deprivation, loss of cell attachment to extracellular matrix proteins, oncogenes (c-myc), tumor suppressor genes (p53), cytolytic T cells, radiation, and essentially all currently available chemotherapeutic drugs (reviewed in Refs. 6, 20, and 21). In this report, we characterized for the first time the expression of several Bcl-2 family proteins (including Bcl-2, Bcl-X, Mcl-1, Bax, and Bak) in a group of 30 colorectal adenocarcinomas and 24 adenomatous polyps. MATERIALS AND METHODS All patient materials represented randomly selected cases of colorectal carcinoma or adenoma derived from the St. Luke's-Roosevelt Center Hospital Department of Surgical Pathology or from snap-frozen colonie tissue obtained at surgical resection. Formalinor Bouin's-fixed, paraffin-embedded tumors were stained with H&E and classified historically by expert pathologists as well-differentiated adenocarcinoma (n = 5), moderately or poorly differenti ated cancer (n = 25), adenoma without severe dysplasia (n = 8), adenoma with severe dysplasia (n = 14), or hyperplastic adenomatous polyps (n = 2). Sections from these cases were immunostained for Bcl-2 family proteins exactly as described previously, using a microwave accentuation technique; antipeptide rabbit antisera specific for Bcl-2, Bcl-X, Mel-1, Bax, or Bak; and a diaminobenzidine colorimetrie detection method (22-26). All of these anti peptide antisera have been described previously in detail (22-26), with the exception of the anti-Bak antibody that was generated in rabbits by using a synthetic peptide corresponding to amino acids 14-36 of the human Bak protein (NH2-C-E-P-A-L-P-S-A-S-E-E-Q-V-A-Q-D-T-E-E-V-F-R-amide) conjugated to maleimide-activated keyhole limpet hemocyanin and ovalbumin carrier proteins (Pierce, Inc.). The immunostaining specificity was confirmed for all antibodies by preadsorption of antisera with specific peptides that completely abolished immunoreactivity with colorectal tissues and by the failure of irrelevant peptides to block immunostaining (data not shown). In all specimens, residual nonneoplastic epithelial cells, lymphocytes, or peripheral nerves were present that contained specific immunostaining and thus served as