Loss of 21WAF1/CiP1 Protein Expression Accompanies Progression of Sporadic Colorectal Neoplasms but not Hereditary Nonpolyposis Colorectal Cancers’

p2! (p21WAF1/CiP1), a cyclin-dependent kinase inhibitor, induces G1 arrest and can inhibit the activity of the proliferating cell nuclear antigen (PCNA). We analyzed p21 expression during colorectal tumorigenesis, its association with its transcriptional regulator p53, and its relationship to rates of cell proliferation and apoptosis. p2! and p53 protein expression were examined in sporadic tumors and hereditary nonpolyposis colorectal cancers (HNPCCs) by immunohistochemistry (LHC) and immunoblotting. Apoptosis was examined using a DNA nick end-labeling assay, and cell proliferation was examined by PCNA staining. In normal colorectal epithelia, nuclear p2! staining was uniformly detected in crypt cells of the superficial compartment (upper one-third) that stained negatively for PCNA. p21 and PCNA expression were, therefore, mutually exclusive. In sporadic cases, a decrease in the frequency of p2! expression accompanied adenoma development and progression to carcinoma. Specifically, p21 was detected in 12 of 16 (75%) adenomas and 10 of 32 (31 %) carcinomas. In contrast to sporadic cases, HNPCCs with known mutations in DNA mismatch repair genes expressed p21 in 12 of 15 (80%) carcinomas. An inverse relationship between p2! and p53 was observed wherein mutant p53 proteins were detected in 4 of 15 (27%) HNPCCs versus 22 of 32 (69%) sporadic carcinomas. Although p2! + carcinoma cells were generally negative for p53, IHC revealed that some carcinoma cells expressed both p2! and p53 proteins. Furthermore, p53Received 10/15/97; revised 1/6/98: accepted 2/19/98. 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. I This work was supported by a grant from the Glaxo Institute of Digestive Health (to F. A. S.). F. A. S. is a recipient of a Career Development Award from the American Cancer Society. 2 To whom requests for reprints should be addressed, at Box 78, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: (713) 792-2828; Fax: (713)745-1163. mutated SW480 colon carcinoma cells were found to coexpress p21 and p53, suggesting that p2! can also be activated by a p53-independent mechanism. No association was found between p2! or PCNA and apoptotic labeling indices in adenomas or carcinomas. In conclusion, a decrease in p2! expression accompanies neoplastic progression in sporadic cases but not in HNPCCs. This finding appears related to p53 status in that the frequency of p53 expression was significantly reduced in HNPCCs compared to sporadic cases, suggesting a difference in their molecular pathways of tumorigenesis. INTRODUCTION The p21 (p21WAFI/CIPI) gene is located on chromosome 6p and encodes the p21 protein, which functions as a potent inhibitor of cdk activity ( 1 , 2). Members of a family of cdks function as key positive regulators of the cell cycle that promote cell proliferation. p21 induces G, arrest and blocks entry into S phase by inhibiting cdks3 (1, 2) or by binding to the PCNA, resulting in a blockade in DNA replication (3, 4). p21 is a downstream target effector of the wt p53 protein, which tramscriptionaiiy activates p21 (5, 6). wt p53 proteins accumulate as a consequence of DNA damage and can bind to specific sites on the p21 promoter, resulting in induction of p21 expression (5). Mutational inactivation of p53, a frequent event in coborectal cancers (7), with resultant failure to tramsactivate p21, may thereby produce uncontrolled cell proliferation. Transfection of the p21 cDNA and overexpressiom of the p21 protein have been shown to inhibit the growth of cultured colon, brain, and lung cancer cells, as well as leukemia cells (5, 8, 9). Furthermore, p21 overexpression reduced the tumorigenicity of colon carcinoma cells when injected into nude mice (9). Studies in colon cancer cells (6) and in p21-deficient murine fibroblasts ( 10) have shown that p21 is necessary for p53mediated G1 arrest induced by DNA damage. The requirement for p21 in p53-mediated growth arrest suggests that p21 plays a critical role in p53-mediated tumor suppression (6, 1 1 ). Suppression of tumor growth may occur secondary to cell cycle arrest or, alternatively, by induction of apoptosis (12). Interestingly, loss of p53 function has been associated with attenuated apoptotic rates in vivo (12). Although p53 appears to be a key regulator of p2 1 , abundant evidence indicates that p2 1 can also be induced independently of p53 (13-16). In most normal tis3 The abbreviations used are: cdk, cyclin-dependent kinase; HNPCC, hereditary nonpolyposis coborectal cancer; PCNA, proliferating cell nuclear antigen: wt. wild-type; TUNEL. terminal deoxynucleotidyl transferase-mediated nick end labeling; MMR. mismatch repair; IHC. immunohistochemistry; RT, room temperature: MAb. monocbonal antibody; DAB, diaminobenzidine: Al, apoptotic index; LI, labeling index. Research. on December 27, 2017. © 1998 American Association for Cancer clincancerres.aacrjournals.org Downloaded from Table 1 Analysis of HNPCC cases for mutations in hMLHI and hMSH2 genes determined by DNA sequencing (23) or hMSH2 protein expression. p21 and p53 expression were also examined by IHC