to 10-fold amplification of the cyclin Dl gene in 16 of the 50 .... W$I qp -. 4.0 o w -2.2. 2.0. 97. 97. 69. 46. 41f 4g,. 4oww. W .. -*. IgG. 36 kDa. (cyclin Dl). - Ha-ras. Li.
Proc. Natl. Acad. Sci. USA Vol. 90, pp. 9026-9030, October 1993 Medical Sciences
Altered expression of the cyclin Dl and retinoblastoma genes in human esophageal cancer (gene amplIfIcatIon/cell cycle)
WEI JIANG*, YU-JING ZHANG*, SCOTT M. KAHN*, MONICA C. HOLLSTEINt, REGINA M. SANTELLA*, SHIH-HsIN Lut, CURTIS C. HARRIS§, RUGGERO MONTESANOt, AND I. BERNARD WEINSTEIN*¶ *Comprehensive Cancer Center and Institute of Cancer Research, Columbia University, College of Physicians and Surgeons, New York, NY 10032; tInternational Agency for Research on Cancer, 150 Courts Albert Thomas, 69372 Lyon Cedex 08, France; tCancer Institute, Chinese Academy of Medical Sciences, Bebijng, People's Republic of China; and §Laboratory of Human Carcinogenesis, National Cancer Institute, National Institutes of Health, Bethesda,
MD 20892
Communicated by Richard Axel, April 22, 1993
the cyclin Dl gene had been mapped to the chromosome 11q13 locus close to the INT2 and HSTI genes (17). Cyclins form a family of proteins that complex with cyclindependent protein kinases (CDKs) to govern key transitions in the cell cycle (for review, see refs. 18 and 19) Cyclin Dl was isolated as a gene that is rearranged in parathyroid adenomas (PRADI) and certain B-cell leukemias (BCL-J) (17, 20). It also complements a Saccharomyces cerevisiae strain that is mutant in three known Gl cyclins (21, 22) and is induced in the late G1 phase following the treatment of a growth-arrested macrophage cell line with colony-stimulating factor 1 (23). The product of the cyclin Dl gene is expressed at high levels and forms a complex with a CDK during the G1 phase of the cell cycle in sychronized cells (23). In a recent study we detected amplification of the cyclin Dl gene in about 25% of primary esophageal tumors from China (24). The RB gene, which is located on chromosome 13q14, was the first tumor suppressor gene to be isolated. Loss of heterozygosity and loss of expression ofthe RB gene are seen during the development of retinoblastoma (RB) and several other types of human cancers (25). The product of the RB gene is a 110-kDa nuclear phosphoprotein. This protein is hypophosphorylated during the G1 phase and hyperphosphorylated in the S, G2, and M phases of the cell cycle. It is thought that this phosphorylation blocks an inhibitory function of the RB protein on progression through the later phases of the cell cycle (26). Alternatively, oncoproteins encoded by certain DNA tumor viruses can bind to the RB protein and block its inhibitory function (26). In vitro studies demonstrate that several serine and threonine sites in the RB protein can be phosphorylated by specific CDK/cyclin complexes (27, 28). Of particular interest to the present studies are recent results indicating that cotransfection of cyclin Dl with RB can partially override the cell growth inhibition obtained with RB alone in a RB-negative cell line (29). Although cyclin Dl can enhance the phosphorylation of RB protein in vitro (30), there is conflicting evidence on the actual mechanism of action of cyclin Dl in vivo (31, 32). In view of the above findings, in the present study we have examined, in parallel, cyclin Dl amplification and cyclin Dl and RB expression in human esophageal carcinoma cell lines and also in a series of primary human esophageal carcinomas from high-incidence regions in China, Italy, and France.
We have examined DNA from four human ABSTRACT esophageal carcinoma cell lines and 50 primary esophageal carcinomas obtained from China, Italy, and France for amplification of the cyclin Dl gene. We also examined 36 of these 50 carcinomas for expression of the cydin Dl and retinoblastoma (RB) proteins by immunohistochemistry. We found a 3to 10-fold amplification of the cyclin Dl gene in 16 of the 50 (32%) tumors and in two of the four cell lines. Cyclin Dl protein was overexpressed in 12 of 13 tumors and the two cell lines that showed gene amplification when compared to normal controls. Studies on RB protein expression indicated that 6 of the 36 (17%) tumor samples examined and one cell line did not show detectable expression of this protein. The tumors and cell lines that had cyclin Dl gene amplification and overexpression exhibited normal levels of expression of RB protein. By contrast, the tumors and cell line that did not appear to express the RB protein did not show ampliffcation of the cycin Dl gene and expressed only low levels of the cyclin Dl protein (P = 0.03). These results suggest that the inhibitory effect of RB on cell cycle progression can be abrogated during tumor development either by loss of expression of the RB gene or by increased expression of the cyclin Dl gene. Human esophageal cancer is one of the most common worldwide cancers and occurs at very high frequencies in certain areas of China, South Africa, France, and Italy (1). Environmental and nutritional factors as well as cultural habits are thought to be important contributing factors to esophageal carcinogenesis (2, 3). Despite these epidemeologic findings, the precise etiologic factors and their mechanisms of action still remain unknown. Cytogenetic and molecular studies have revealed chromosomal abnormalities including a homogeneously staining band in the region of 11q12 (4), amplification of the cellular protooncogenes MYC, EGFR (5), INT2, and HSTI (6-8), and loss of heterozygosity of the tumor suppressor genes RB (9), p53 (10-12), MCC (13) and APC (13). Point mutations in the p53 gene occur in about 40% of esophageal cancers (10-12). However, activation of RAS oncogenes by point mutation appears to be a very rare event in this type of cancer (12, 14-16). Despite the fact that amplification of the HSTI and INT2 genes on chromosome 11q13 has been found in about 20-50% of esophageal cancers, little or no expression of these two genes has been detected in the corresponding cells (6-8). These findings suggest that an additional gene(s) at the chromosome 11q13 locus is involved in the development of esophageal cancer. These considerations attracted our interest in a recent report that
Cell Lines and Tissues. Four human esophageal carcinoma cell lines, EC17 (33), EC109 (33), HCE4 (34), and HCE7 (34),
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Abbreviations: RB, retinoblastoma; CDK, cyclin-dependent protein kinase. $To whom reprint requests should be addressed.
MATERIALS AND METHODS
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were grown in cell culture in RPMI 1640 medium plus 10% fetal calf serum. A total of 50 primary human esophageal carcinomas and some adjacent nontumorous specimens were obtained from patients who underwent surgery in the People's Republic of China, Italy, or France. Clinical and histological data on these tumors are available from the authors. A normal esophageal sample was also obtained (24). DNA and RNA Analysis. DNA and RNA were isolated from the cell lines and the frozen tissue specimens as described (24). Southern and Northern blot filters were hybridized with the following [32P]dCTP-labeled probes: human cyclin Dl, c-Ha-ras, -t-actin, and RB, as described (24). The intensities of the radioactive bands on the blots were quantitated using a Betascope instrument (Betagen, Waltham, MA.). Immunopricepitation and Western Blots. Immunopricepitation and Western blots were performed with an anti-human cyclin D polyclonal antibody (Upstate Biotechnology, Lake Placid, NY) or an anti-human RB monoclonal antibody, PMG3-245 (PharMingen) (21), using the ECL Western blotting detection system (Amersham). Immunohistochemistry. Subconfluent cell cultures grown on glass slides were fixed in acetone at -20°C. Cell cultures and deparaffinized microtome sections of the tumors were quenched for endogenous peroxidase activity in 0.3% hydrogen peroxide in methanol. The slides were incubated with either the anti-human cycin D polyclonal antibody or the anti-human RB monoclonal antibody at 4°C overnight. After washing, immunostaining was performed using biotinylated anti-rabbit or anti-mouse antibody kits (Vector Laboratories) and diaminobenzidene (1 mg/ml) and 0.02% hydrogen peroxide (35). As a control, the slides were stained without the primary antibody to monitor background staining.
trophoresed, and hybridized to RB probes, no gross abnormalities were seen in any of the four cell lines (data not shown). RNA samples from the same cell lines were examined by Northern blot analysis using the cyclin Dl and RB probes. The cell lines HCE4 and HCE7 contained very high levels of 4.7- and 1.8-kb cyclin Dl transcripts when compared to the EC17 and EC109 cell lines. The cell lines EC109, HCE4, and HCE7 expressed about equal levels of an expected RB transcript-i.e., 4.7 kb. The EC17 cell line, however, expressed a slightly shorter 4.5- to 4.6-kb RB transcript (data not shown). In view of the above results it was of interest to examine the levels of expression of the corresponding proteins in these cell lines. The cyclin Dl and RB proteins were immunoprecipitated from cell lysates obtained from proliferating cell cultures, using the corresponding antibodies, and then analyzed by Western immunoblotting. We detected high levels of the 36-kDa cyclin Dl protein (Fig. 1B) in the two cell lines (HCE4 and HCE7) that also displayed DNA amplification and increased expression at the RNA level of the cyclin Dl gene. A low level of expression of this protein was seen in the EC109 cells (Fig. 1B), which also expressed cyclin Dl mRNA but did not show gene amplification (Fig. 1A and ref. 24). Cyclin Dl protein was not detected in the EC17 cell (Fig. 1B), which is consistent with the fact that this gene was not amplified in these cells and these cells expressed very low levels of cyclin Dl mRNA (Fig. 1A and ref. 24). Similar results were obtained when Western blot analysis was done on a cell lysate without prior immunoprecipitation (data not shown). We also detected abundant levels of the normal 110-kDa RB protein in the EC109, HCE4, and HCE7 cell lines (Fig. 1C). This is consistent with our finding that these three cell lines also expressed abundant levels of the normal 4.7-kb RB mRNA transcript. On the other hand, EC17 cells, which expressed a shorter and presumably aberrant transcript, did not express detectable levels of the RB protein (Fig. 1C). Immunohistochemistry with the cyclin D antibody demonstrated a high nuclear content of cyclin D protein in the two cell lines (HCE4 and HCE7) in which this gene was amplified and overexpressed but weak or no staining in the EC109 and EC17 cell lines in which this gene was not amplified. Representative photos of the EC17 and HCE4 cell lines are shown in Fig. 2 A and B, respectively. Thus, the immunostaining results correlate in a general way with the immunoprecipitation and DNA amplification results. To our knowledge, there has been no previous report that cyclin Dl is present in the nucleus in mammalian cells, although this is not surprising in view of its putative function(s). It is also of
RESULTS Studies on Cyclin Dl and RB in Human Esophageal Carcinoma Cell Lines. Genomic DNAs from the four esophageal carcinoma cell lines were digested with EcoRI and examined by Southern blot analysis using a 32P-labeled probe for cyclin Dl (Fig. 1A). The cell lines EC17 and EC109 displayed no abnormalities, but the HCE4 and HCE7 cell lines displayed 4-fold and 3-fold amplification, respectively, of cyclin Dl DNA. As a control for DNA loading and copies of chromosome 11 in these cells, the same blots were also hybridized to a human c-Ha-ras probe (Fig. 1A). The results indicated that the increased intensities of the cyclin DI DNA bands are due to gene amplification rather than an increased number of copies of chromosome 11 in these cells. When the DNAs from these four cell lines were digested with HindIII, elecrl-
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FIG. 1. (A) Southern blot analysis of cyclin Dl in human esophageal carcinoma cell lines. The molecular sizes of the cyclin Dl DNA fragments in an EcoRI digest are indicated. (B and C) Immunoprecipitation and Western blot analysis of protein extracts from these cell lines using anti-cyclin D antibody (B) or anti-RB antibody (C). Molecular mass markers are indicated on the left.
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Immunohistochemical analysis of the RB protein in the same cell lines indicated that the EC109, HCE4, and HCE7 cells, but not the EC17 cells, displayed prominent nuclear staining of this protein. These results are in agreement with the Northern blot analysis and immunoprecipitation results described above. Representative photos are shown in Fig. 2 C and D. In contrast to the marked variation in staining ofthe cell population seen with cyclin Dl (Fig. 2B), staining of the RB protein was seen throughout the cell population in the EC109, HCE4, and HCE7 cell lines (Fig. 2D). This is consistent with previous evidence that the RB protein is present throughout the cell cycle (37). Studies in Primary Human Esophageal Carcinomas. We recently reported that 5 of 20 primary esophageal tumors obtained from a high esophageal carcinoma incidence region in China demonstrated amplification of the cyclin Dl gene (24). It was of interest to see if amplification of this gene also occurs in esophageal tumors obtained from high-incidence regions in other parts of the world. Therefore, 26 tumor samples were obtained from cases in Italy and 4 were from cases in France. A representative Southern blot obtained after digestion of the DNA with EcoRI is shown in Fig. 3 and the total set of data is summarized in Table 1. We found that 16 of the 50 (32%) tumors examined displayed a 3- to 10-fold amplification the cyclin Dl gene. In this limited sample set, there was no statistically significant difference in the frequencies of gene amplification between the cases from China, Italy, and France. To verify the biological significance of the above findings, we examined whether there is increased expression of the cyclin Dl protein in primary tumors that display amplification of the gene. Suitable histologic sections were available from 36 of the 50 tumors listed in Table 1 and from several normal esophageal samples. Thirteen of the 36 tumor samples were cases that displayed amplification of the cyclin Dl gene by Southern blot analysis. Twelve of these 13 cases displayed much more intense cyclin Dl staining than the esophageal epithelial cells in the normal control samples. Representative photos are shown in Fig. 4 A and B, and the results on these tumor samples are summarized in Table 2. In view of recent evidence suggesting an interaction between cyclin Dl and RB in cell cycle control and the results we obtained with the tumor cell lines (Fig. 1 B and C), we also used immunohistochemistry to examine the expression of the RB protein in the same set of 36 tumor samples. Representative photos are shown in Fig. 5. We found that all of the 13 tumors that had amplification of the cyclin Dl gene (12 of which also demonstrated increased expression of the cyclin FIG. 2. Immunohistochemical staining ofhuman cyclin Dl (A and B) and RB (C and D) in the EC17 (A and C) and HCE4 (B and C) esophageal carcinoma cell lines. (x 140.)
interest that even in the cells that carry amplified copies of this gene, only a subpopulation of the cells showed nuclear staining. The polyclonal anti-human cyclin D antibody used in these studies was generated with a specific sequence contained within the C-terminal domain of human cyclin Dl (19) and reacts with cyclin Dl and D2 but not D3 (36). In Northern blot analyses, we have found that these four esophageal carcinoma cell lines express relatively high levels of cyclin Dl and D3 but only trace levels of D2 (data not shown). These findings, taken together with our results indicating that the level of expression of cyclin D protein detected with this antibody correlated, in general, with amplification of the cyclin Dl gene, make it likely that the Western blot and immunostaining results obtained with this antibody do reflect the relative abundance of cyclin Dl protein.
NM 2
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FIG. 3. Representative Southern blot analysis demonstrating amplification of the cyclin Dl gene in human primary esophageal carcinomas. DNA was extracted from the indicated tumors obtained from cases in Italy and from a normal sample of esophageal mucosa (see Materials and Methods and Table 1), digested with EcoRI, and subjected to Southern blot analysis. Tumor number is indicated above each lane; NM, normal mucosa. The molecular sizes of the bands that hybridized to the cyclin Dl probe are indicated.
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Table 1. Frequency of amplification of the cyclin Dl gene in human esophageal carcinomas No. with amplified Source of tumors No. of samples cyclin Dl (%)* 5 (25)t 20 China 26 10 (38) Italy 1 (25) 4 France 50 16 (32) Total *Number of cases in which the tumor DNA displayed a 3- to 10-fold amplification of cyclin Dl. For details, see the text. tTaken from ref. 24.
Dl protein) were positive for the RB protein. Of the remaining 23 tumors, none of which had cyclin Dl amplification, 6 had undetectable RB protein staining, while the remaining 17 were RB positive. Our studies do not, however, exclude the possibility that in some of the RB-positive cases a mutant protein is expressed. Thus, amplification of the cyclin Dl gene was almost always associated with persistent expression ofthe RB protein, whereas about one-third of the tumors that lacked cyclin Dl amplification had apparently lost the expression of the RB protein. The failure to find tumor or tumor-derived cell lines that displayed cyclin Dl amplification and loss of RB expression (Fig. 1 and Table 2) was statistically significant when assessed by the Fisher exact test (P = 0.03).
DISCUSSION The present studies extend our previous evidence (24) that the cyclin Dl gene is frequently amplified in primary esophageal cancers from China to esophageal tumors obtained from high-incidence regions in Italy and France. Taken together, the overall incidence of cyclin Dl amplification in the 50 tumors examined thus far is about 30%o. Amplification of this gene is also frequently seen in esophageal cancers in Japan (M. Terada, personal communication). Other genetic lesions found with a significant frequency in esophageal tumors from these disparate regions of the world are discussed in the Introduction. Since it appears that the risk factors for devel-
FIG. 4. Immunohistochemical staining of cyclin Dl in a normal esophageal mucosa sample (A) and a primary esophageal carcinoma in which the cyclin Dl gene was amplified 10-fold (B). (x 140.)
Proc. Natl. Acad. Sci. USA 90 (1993)
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Table 2. Correlation of cyclin Dl and RB expression in human esophageal carcinomas No. of tumors No. of tumors with cycin Dl positive for RB No. of tumors amplificationt expressiont Group per group* 1 13 13 13 2 6 0 0 17 17 0 3 *Complete data on cyclin Dl and RB were obtained on 36 of the 50 primary human esophageal cancers examined. tAmplification of cyclin Dl was demonstrated by Southern blot analysis (see text and Table 1). All but 1 of the 13 samples that showed cyclin Dl amplification also showed high cyclin Dl expression by immunohistochemistry. Representative examples of high expression are shown in Fig. 4. None of the samples in group 2 displayed amplification or increased expression of the cyclin Dl. None of the samples in group 3 displayed amplification of cyclin D1; they varied considerably in the extent of expression of cyclin Dl. tRB expression was determined by immunohistochemistry. A representative sample is illustrated in Fig. 5.
oping esophageal cancer differ considerably in these four regions of the world, it seems likely that amplification of the cyclin Dl gene, along with the other genetic lesions, are inherent in the stepwise evolution of esophageal cancer, rather then hallmarks or "fingerprints" of the causative agent(s). Since the amplified copies of the cyclin Dl gene seen in a subset of esophageal cancers are contained within a much larger amplicon that can encompass the entire chromosome 11q13 region (38), it could be argued that one or more other genes in this amplicon play a critical role in esophageal cancer, rather than the cyclin Dl gene. This seems unlikely for several reasons. In the present study we demonstrated that amplification of the cyclin Dl gene is associated with increased expression of the related protein, both in esophageal cell lines and in primary tumors. This is in contrast to the HSTI and INT2 genes that are coamplified with cyclin Dl but are not expressed in esophageal tumors (6, 24). In addition, it seems likely that aberrant expression of cyclin Dl perturbs
FIG. 5. Immunohistochemical staining of RB in an esophageal carcinoma in which the cyclin Dl gene was not amplified and highly expressed (A) and a tumor in which cyclin Dl was amplified and overexpressed (B). (x 140.)
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growth control in human tumors since characteristic rearrangements of this gene are seen in parathyroid tumors and certain B-cell leukemias and lymphomas. These rearrangements are also associated with increased expression of cyclin Dl (17, 38, 39). Amplification of the human chromosome 11q13 region, including the cyclin Dl gene, has also been seen in human tumors of the breast, bladder, liver, head and neck, and poorly differentiated squamous lung carcinomas (40, 41). Overexpression of the cyclin Dl gene in tumors might perturb G1-related events, at least in part, through interactions with the RB tumor suppressor protein, which has been implicated in negative control of cell cycle progression (26). Previous studies have demonstrated that loss of heterozygosity of the RB gene locus occurs in a subset of esophageal cancers (9), and in the present study we found that about 17% of the esophageal tumors examined fail to express detectable levels of the RB protein when examined by immunohistochemistry (Fig. 5B and Table 2). A striking association found in the present study was that all of the tumors that overexpressed cycin Dl retained the expression of the RB protein (Table 2). Although our findings are based on a limited number of cases, they suggest the following model. During the multistep evolution of esophageal tumors, the normal inhibitory role of the RB protein in cell cycle progression can be abrogated by various mechanisms, including (i) increased levels of cyclin Dl (group 1 tumors, Table 2); (ii) loss of expression of the RB protein, presumably due to mutations or deletions in the RB gene (group 2 tumors, Table 2); or (iii) other mechanisms not yet revealed that override the function of RB (group 3 tumors, Table 2 and refs. 42-45). According to this model there would be no selective advantage for mutations in cyclin Dl and RB genes. Alternatively, overexpression of cyclin Dl in combination with loss of RB function might be toxic to cells. Therefore, during tumor development there would be selection against cells that sustain both events. The first mechanism is consistent with a recent study indicating that cotransfection of cyclin Dl with RB into a RB-negative cell line can reverse the growth inhibition obtained with RB alone (29). This antagonism could be mediated through phosphorylation of the RB protein (30) or other mechanisms that remain to be determined (31). The above model would explain why amplification and overexpression of cyclin Dl could be a critical event in the multistage development of a subset (about 30%o) of esophageal cancers. The remaining tumors might evolve through other mechanisms that abrogate growth control, including loss of expression of the RB gene. Obviously, mutations in numerous additional genes play a role in the carcinogenic process (46), but this example provides a paradigm for explaining why tumors of the same histologic type show heterogeneity at the genetic level even though a common regulatory pathway is deregulated. We thank Drs. C. Sherr for the cyclin D2 and D3 probes; Wen-Hwa Lee for the human RB probe; A. Perracchia, L. Chieco-Bianchi, and L. Partensky for the tumor specimens; and Wei-Yann Tsai for valuable assistance in the statistical analysis. This research was supported by National Institutes of Health Grant CA02111 and awards from the American Cancer Society (Special Institute Grant 13), the Lucille Markey Charitable Trust, and the National Foundation for Cancer Research to I.B.W. 1. 2. 3. 4.
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