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Potential Synergistic Effect with Altered P53 Protein on Proliferative Activity

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Potential Synergistic Effect with Altered P53 Protein on Proliferative Activity ICANCERRESEARCH56,5557—5562,December15, 19961 Advances in Brief Altered @16INK4andRetinoblastoma Protein Status in Non..Small Cell Lung Cancer: Potential Synergistic Effect with Altered p53 Protein on Proliferative Activity Ichiro Kinoshita, Hirotoshi Dosaka.Akita,' Takayuki Mishina, Kenji Aide, Motoi Nishi, Hiromitsu Hiroumi, Fumihiro Hommura, and Yoshikazu Kawakami First Department of Medicine, Hokkaido University School of Medicine Ii. K., H. D-A.. T. M., K. A., H. H., F. H., Y. K.J, and Department of Public Health, Sapporo Medical University School of Medicine [M. N.], Sapporo 060. Japan Abstract (4, 6), which is supported by observation of an inverse correlation between alteration of both proteins in several types of tumors (7—13). p16@―4protein (pl6) and retinoblastoma protein (pith), like p53 pro In NSCLCs, whereas the RB gene is shown to be inactivated in Win, are Important tumor suppressors that regulate the cell cycle. We 6—32%of the tumors (14—18),the frequency of alteration involving immunohistochemically examined fresh-frozen specimens of 114 resected non-small cell lung cancers (NSCLCs) for loss of p16 and pRB expression, the CDKN2 gene is controversial. Of NSCLC cell lines, homozygous together with aberrant accumulation of p53 proteIn and the proliferative deletion of the CDKN2 gene is observed in 20—50% and point activity determined by the Ki-67 Index. Three pith-positive tumors were mutation in 6—21% (2, 3, 10—12, 19—22). Recent reports showed uninterpretable for p16 status. Of the remaining 111 tumors, 30 (27%) DNA hypermethylation with CDKN2 gene silencing in about 30% of lacked p16 expression, and 10 (9%) lost pith expression. No tumors NSCLC cell lines (12). For primary NSCLCs, although there is a showed coincident loss of both proteins, supporting the hypothesis that study reporting 30% of intragenic mutations of the CDKN2 gene (23), they function In a single pathway. Of 25 tumors, including 4 p16-negative most studies showed that the abnormalities of the CDKN2 gene are tumors, examined by Southern blot analysis, only 2 p16-negative tumors rare ( 10, 21, 22, 24) and are largely restricted to metastatic sites (21, were considered to have reduced gene dosage consistent with possible 22). Nevertheless, the rare occurrence of CDKN2 alterations in pri homozygous deletion of the CDKN2 gene encoding pi6, suggesting that mary NSCLCs may be underestimated, not only because epigenetic immunohistochemistry is a sensitive and suitable method to screen for p16 alteration. Loss of p16 expression did not correlate with any clinical alterations, including DNA hypermethylation, has not been investi factors or p53 status, whereas loss of pRB expression correlated with gated thus far, but also because homozygous deletion may be ob heavy smoking (P 0.03 by Fisher's exact test and P = 0.01 by the scured by contamination due to normal stromal cells (13). Moreover, multivariate logistic regression analysis). Proliferative activity was con in NSCLCs, little is known about the clinical and cell biological siderably higher in p53-positive tumors than in p53-negative tumors implications of the inactivation of the pl6/pRB pathway and its (P < 0.001). Loss of p16 or pith expression was associated with a further association with the status of p53 protein (25), another tumor sup increase in proliferative activity In the p53-positive tumors (P 0.009) but pressor working at the G@checkpoint (26). not with proliferative activity in the p53-negativetumors. These results Therefore, in the present study, we immunohistochemically exam suggest that alteration of the pl6IpRB pathway is relatively frequentiy med primary NSCLCs for the loss of p16 and pRB expression, involved in the development and progression of NSCLCs and that its together with aberrant accumulation of p53 protein and the prolifer effect on the proliferative activity is potentially synergistic with altered p53 proteIn. ative activity determined by Ki-67 index. We show a relatively frequent loss of p16 in both early and late stages of NSCLCs. The Introduction reciprocal loss of p16 and pRB provides further support for the hypothesis that both proteins function in a single pathway. Further The CDKN2 and the RB2tumor suppressor genes play a pivotal role more, we demonstrate the potential synergistic effect of the altered in the regulation of the cell cycle at the G1 checkpoint. In normal cells, p16/pRB pathway with altered p53 protein on the proliferative active hypophosphorylated pRB prevents cells from transition across activity. the G1 checkpoint by sequestering transcription factors such as E2F, which is essential for DNA replication, but inactivation of pRB by Materials and Methods CDK-mediated phosphorylation allows cells to progress to the S phase through the G1 checkpoint (1). pl61@4 protein (p16), the Tissue Specimens. Primary tumor specimens from 114 NSCLCs were product of the CDKN2 gene, which has been recently found to be obtained by surgery from the Hokkaido University Medical Hospital, Sapporo deleted in a variety of tumor cells (2, 3), inhibits CDK4- and CDK6- Minami-Ichijo Hospital, and National Sapporo Minami Hospital during 1990 and 1995. Tumors were snap-frozen in liquid nitrogen and stored at —80CCin mediated phosphorylation of pRB (4, 5). Therefore, p16 and pRB are OCT compound (Miles, Elkhart, IN) for immunohistochemistry. For 25 spec suggested to function in a single regulatory pathway of the cell cycle imens, portions of the tumor were also snap-frozen and stored at —80°Cfor genomic DNA preparation. Adjacent non-frozen blocks were fixed in 10% Received 7/15/96; accepted 10/29/96. neutral buffered formalin and embedded in paraffin, and then several sections The costs of publication of this article were defrayed in part by the payment of page from each specimen were stained with H&E to observe histopathology and charges. This article must therefore be hereby marked advertisement in accordance with normal contamination. According to the 1981 WHO classification, they were 18 U.S.C. Section 1734 solely to indicate this fact. I To whom requests for reprints should be addressed, at First Department of Medicine, histopathologically diagnosed as adenocarcinoma (n 74), squamous cell Hokkaido University School ofMedicine, North 15,West 7, Kita-ku, Sapporo 060, Japan. carcinoma (n = 34), and large cell carcinoma (n 6). Phone:8l-(ll).7l6-ll6l,ext.5911;Fax:81-(ll)-727-6724. Immunohistochemistry for p16, pRB, and p53 Proteins. In a pilot study 2 The abbreviations used are: RB, retinoblastoma; pRB, retinoblastoma protein; to choose the mmmunostainingprotocol for p16 protein on frozen materials, CDK, cyclin-dependent kinase; p16. @16@'@K4proteinproduct of the CDKN2/MTSI gene; NSCLC, non-small cell lung cancer; GAPDH, glyceraldehyde-3-phosphate we used cytospinned p16-positive and -negative cell lines, NCI-H209 and dehydrogenase. HL6O,respectively (8), and tested three rabbit polyclonal anti-pl6 primary 5557 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1996 American Association for Cancer Research. p16 AND pRB IN NSCLCs resectedNSCLCsCharacteristicsp16expressionaPpRB Table 1plo and pRB expressionin 114 expression+—+—ppRB expression+ 00.04Age—71 1030 8.50.29SexMale60210.677670.54Female219283Smoking(mean ±SD)62.7 ±9.764.6 ±9.60.3662.9 ±9.766.3 ± years)―0—192390.853400.03205620689Histological(pack typeAdeno 53190.076680.52Squamous267322Large2460pT statusT120100.362830.72T,@6120767pN status'N048210.376840.09N,_3319346pM statusM075280.8696100.46M16280pStageI—LI48220.176850.26III—IV338365 p53 status Normal 46 19 0.53 6430.06407 Altered —@--____________ a Three pRB+ tumors could not be evaluated for p16 status (see text). b Smoking history data were not available for three cases. C Lymph nodes were not resected for two cases. antibodies, one against the entire human p16 protein (PharMingen, San Southern Blot Analysis. Genomic DNA was extracted from 25 NSCLC Diego, CA), and the other two against the NH2 terminus or COOH terminus tumor specimens, Southern blotted after digestion with EcoRI, and hybrid of human p16 proteins (Santa Cruz Biotechnology, Santa Cruz, CA), in ized to [a-32P]dCTP-labeled human CDKN2 cDNA (7, 10) and cardiac various concentrations, and three fixatives: methanol, acetone, and 10% actin cDNA. The final wash was performed twice in 0.2X SSC, 0.1% SDS buffered formalin. The protocol using anti-entire-human p16 antibody at 50°Cfor 20 mm. The blots were then exposed to Kodak XAR-5 film (PharMingen) as a primary antibody and methanol as a fixative yielded the (Kodak, Rochester, NY) and quantitatively analyzed by a densitometer. most nucleus-specific staining with the highest intensity in NCI-H209 cells, The autoradiographs were developed at various times to ensure that this whereas all of the combinations of antibodies and fixatives generated analysis was performed in the linear range of the film. The CDKN2 proper nuclear staining and weak cytoplasmic staining in the NCI-H209 signal intensity was normalized against the cardiac actin signal intensity. cells. For additional experiments, therefore, 5-p@m frozen sections of Because normal cells were present in tumor specimens, we considered a NSCLC specimens were fixed in methanol at 4°Cfor 5 mm and were tumor to have reduced gene dosage consistent with possible homozygous reacted with the anti-entire-human p16 primary antibody (PharMingen) at deletion of the CDKN2 gene in the tumor cells when the normalized a I:200 dilution or with control rabbit
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