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Cyclin D1 Amplification Is Independent of P16 Inactivation in Head And

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Cyclin D1 Amplification Is Independent of P16 Inactivation in Head And Oncogene (1999) 18, 3541 ± 3545 ã 1999 Stockton Press All rights reserved 0950 ± 9232/99 $12.00 http://www.stockton-press.co.uk/onc Cyclin D1 ampli®cation is independent of p16 inactivation in head and neck squamous cell carcinoma K Okami1,3, AL Reed1, P Cairns1, WM Koch1, WH Westra2, S Wehage1, J Jen1 and D Sidransky*,1 1Department of OtolaryngologyÐHead & Neck Surgery, Division of Head and Neck Cancer Research, Johns Hopkins University School of Medicine, 818 Ross Research Building, 720 Rutland Avenue, Baltimore, Maryland, MD 21205-2196, USA; 2Department of Pathology, 7181 Meyer Building, Johns Hopkins Hospital, 600 North Wolfe Street, Baltimore, Maryland, MD 21287, USA; 3Department of Otolaryngology, Yamaguchi University, Ube 755, Japan Progression through the G1 phase of the cell cycle is kinase (cdk) 4 complexes. This cdk4 phosphorylation mediated by phosphorylation of the retinoblastoma activity is negatively regulated by the tumor suppressor protein (pRb) resulting in the release of essential gene p16 (a CDK inhibitor), and positively by cyclin transcription factors such as E2F-1. The phosphorylation D1. Each individual component of this p16-cyclin D1- of pRb is regulated positively by cyclin D1/CDK4 and Rb pathway is commonly targeted in various malig- negatively by CDK inhibitors, such as p16 (CDKN2/ nancies (Weinberg, 1995; Sherr, 1996). We previously MTS-1/INK4A). The p16 /cyclin D1/Rb pathway plays reported a high frequency (83%) of p16 inactivation in a critical role in tumorigenesis and many tumor types human HNSCC (Reed et al., 1996) and less frequent display a high frequency of inactivation of at least one inactivation (13%) of pRb (Yoo et al., 1994). Cyclin D1 component of this pathway. In order to determine the ampli®cation has been reported in 22 ± 64% of HNSCC overall contribution of these three components to based on several dierent genetic or IHC methods progression of head and neck squamous cell carcinoma (Jares et al., 1994; Kyomoto et al., 1997; Olshan et al., (HNSCC), we examined p16 inactivation, cyclin D1 1997; Michalides et al., 1995). However, few reports ampli®cation, and pRb expression in 23 primary have simultaneously examined all components of this HNSCC tumors and ®ve cell lines. p16 inactivation critical pathway in HNSCC (Olshan et al., 1997; Lukas was detected in 19/23 (83%) primary tumors by detailed et al., 1995). genetic analysis and was con®rmed by immunohisto- p16 and pRb inactivation have been reported to be chemistry (IHC). Absence of Rb protein expression inversely correlated in some tumor types (Shapiro et indicative of pRb inactivation was identi®ed in 2/23 (9%) al., 1995; Parry et al., 1995; Sakaguchi et al., 1996; tumors. In this set of tumors, there was a perfect inverse Andl et al., 1998). It has thus been assumed that each correlation between p16 and pRb inactivation. Using component of this pathway is an alternative target for ¯uorescence in situ hybridization (FISH) cyclin D1 abrogation of a common pathway and that alterations ampli®cation was identi®ed in 4/5 (80%) cell lines and of multiple components in the same pathway is not 4/11 (36%) primary tumors. However, 2/4 cell lines and necessary for tumor progression (Serrano et al., 1996). all four primary tumors with cyclin D1 ampli®cation We thus undertook a comprehensive study of all contained a concomitant alteration of p16. Therefore 21/ potential alterations within the p16-cyclin D1-Rb 23 (91%) of primary HNSCC contained at least one pathway in cell lines and primary HNSCC. We found alteration in the p16/cyclin D1/Rb pathway. Although a high frequency of p16 inactivation and cyclin D1 p16 and Rb alteration are apparently exclusive, cyclin D1 ampli®cation in cell lines and primary HNSCC. ampli®cation occurs concomitantly with the loss of p16 Moreover, a signi®cant proportion of primary tumors suggesting an additional role for this ampli®cation in contained concomitant alterations of p16 and cyclin D1 HNSCC. suggesting that unlike p16 and pRb, alterations of these two components are not mutually exclusive. Keywords: cyclin D1; p16; head and neck cancer Results and dscussion Introduction p16 status The G1 phase of the cell cycle is controlled by The p16 gene status in these tumors has been reported phosphorylation of the retinoblastoma protein (pRb) previously (Reed et al., 1996). p16 inactivation was and mediated in part by cyclin D-cyclin dependent detected by IHC in 19 out of 23 (83%) cases (Table 1). The IHC results were con®rmed by genetic analysis as previously reported (12 had HD at the p16 locus, ®ve contained methylation of the promoter region of p16, and one had a frameshift mutation in exon 1; Reed et al., 1996). IHC analysis of p16 appears to be a reliable and practical method to determine p16 *Correspondence: D Sidransky Received 20 October 1998; revised 8 January 1999; accepted 5 March inactivation in fresh tissue as reported (Reed et al., 1999 1996). p16, Cyclin D1 and Rb in head and neck cancer KOkamiet al 3542 the BAC probe on chromosome 11q13 (data not pRb IHC shown). In order to determine the incidence of cyclin pRb IHC showed strong nuclear staining in both D1 gene ampli®cation, FISH analysis was carried out tumor and normal cells (Figure 1a). pRb inactivation on both HNSCC cell lines and primary tumors. We was observed in two cases out of 23 (9%) exhibiting a identi®ed gene ampli®cation in four out of ®ve (80%) good contrast between the negative staining tumor area cell lines (Table 1). The FaDu cell line contained four and the positive staining normal areas (Figure 1b). The control signals and more than 20 cyclin D1 signals incidence of pRb inactivation was consistent with our (Figure 1a). We found two typical appearances of previous study on primary HNSCC (Yoo et al., 1994; gene ampli®cation in cell lines 011, 012 and 022. Cell Andl et al., 1998). line 011 had 4 ± 6 control signals and 10 ± 12 cyclin D1 signals, and in the metaphase spreads of chromosome 11, it exhibited a homogeneously staining region Cyclin D1 ampli®cation by FISH (HSR, Brodeur and Hogarty, 1998) of the cyclin D1 FISH was ®rst performed on normal lymphocyte gene (Figure 2b). Cell line 012 and 022 metaphase spreads which con®rmed the chromosomal location of spreads displayed 4 ± 6 cyclin D1 signals due to a duplication of the long arm of chromosome 11 seen in each nucleus (Figure 2c). In the primary tumors, we found four cases of gene ampli®cation out of 11 (36%) cases available (Table 1). Figure 2d shows a primary tumor with four control signals and 12 cyclin D1 signals. Previously, ampli®cation of cyclin D1 was reported in 22 ± 64% of HNSCC (Jares et al., 1994; Kyomoto et al., 1997; Olshan et al., 1997; Michalides et al., 1995). These studies used various methods to detect the ampli®cation including Southern blot analysis, Northern blot hybridization, dierential PCR, or IHC. Although the frequency of amplifica- tion detected by these various approaches is somewhat Table 1 Aberrations of p16, cyclin D1 and pRb in HNSCC primary tumors and cell lines Primary Cyclin D1 Fold tumors/ p16 p16 pRB ampli®- ampli®- Cell line statusa IHC IHC cationb cationc 1 Methylated 7 + N/A 2 HD 7 + N/A 3 Methylated 7 + N/A 4 Methylated 7 + 7 5 Methylated 7 + + 3 6 HD 7 + N/A 7 HD 7 + N/A 8 HD 7 + N/A 9 HD 7 + N/A 10 HD 7 + N/A 11 HD 7 + 7 12 HD 7 + 7 13 HD 7 + N/A 14 Retained + + 7 15 LOH + 7 7 16 Frame shift in 7 + + 3 exon 1 17 HD 7 + + 2 18 HD 7 + N/A 19 HD 7 + N/A 20 LOH 7 + N/A 21 Retained + + 7 22 LOH + 7 7 23 Methylated 7 + + 5 FaDu Point mutation 7 + + 6 Cell line 011 wt + + + 3 Cell line 012 Methylated 7 + + 2 Cell line 022 wt + + + 2 Cell line 029 HD 7 7 7 ap16 status was assessed by microsatellite analysis, sequencing, and promoter methylation assay (see text; Cairns et al. (1994)). Methylated, promoter region was methylated; HD, homozygous Figure 1 pRb IHC in primary HNSCC. (a) Rb-positive tumor b showing retention of nuclear pRb staining in neoplastic cells deletion; LOH, loss of heterozygosity; wt, wildtype. Cyclin D1 ampli®cation was assessed by FISH. N/A, fresh specimen not (arrow) and the normal interstitial tissue (arrow head). (b) Rb- c negative tumor demonstrating absence of nuclear staining in available for FISH. Fold ampli®cation compared to control cancer cells (arrows) with retention of staining in normal region centromeric probe+weak staining, protein expression con®rmed by (arrow heads) Western blot (Liggett et al., 1996) p16, Cyclin D1 and Rb in head and neck cancer KOkamiet al 3543 consistent, there is the limitation of quantitation by all Relationship between p16, cyclin D and pRb of these methods. FISH is the most reliable method to 1 analyse gene ampli®cation (Brodeur and Hogarty, Table 2 shows the correlation between the p16, pRb 1998) because it can demonstrate the minimal region and cyclin D1 status. As reported previously (Shapiro et of ampli®cation in situ and can detect the relative gain al., 1995; Parry et al., 1995; Sakaguchi et al., 1996; of gene copy number compared to a control. One cell Andl et al., 1998), inactivation of p16 and pRB had an line (029) in our study had four signals for both inverse correlation which was signi®cant by Fisher's chromosome 11 and cyclin D1 indicative of a simple exact test (P=0.0397).
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