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Tumor Biology |
Departments of Oncology, [E. E., D. K., D. M. L., S. S.], Surgery [C. B. U.], and Radiology [V. R.], Johns Hopkins University School of Medicine, Baltimore, Maryland 21231; Division of Medicine, Imperial College School of Medicine, London W12ONN, United Kingdom [B. N., L. B.]; Northwestern University Medical School, Chicago, Illinois 60611 [S. A. W.]; and Duke University Medical Center, Durham, North Carolina 27710 [J. M.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Given the critical role of the D-type cyclins in cell cycle regulation, their abnormal or untimely expression could disrupt the cell cycle and therefore render them growth-promoting genes or oncogenes. It has been indicated that cyclin D1 is a putative proto-oncogene and is overexpressed in a number of tumor types including lymphomas, breast cancer, and thyroid carcinoma (1) . Aberrant expression of cyclin D2 has been noted in human ovarian granulosa cell tumors and testicular germ cell tumor cell lines (4) . Recently, evidence was presented suggesting that cyclin D2 is a direct target of Myc, and that accumulation of cyclin D2 contributes to sequestration of the cell cycle inhibitor p27 and to cell cycle entry (5 , 6) . A recent analysis of myc-induced genes using oligonucleotide microarrays also revealed cyclin D2 as a target of c-myc in primary human fibroblasts (7) . Furthermore, in B-lymphocytic tumors such as Burkitt’s lymphoma, transcriptional activation by chromosomal translocation renders c-myc a potent inducer of cell proliferation by promoting the expression of cell cycle activators such as cyclin D2, cyclin E, and cdk4 (8) .
Although well known for their proliferation-promoting activity, the D-type cyclins were also shown to have growth-inhibitory effects, based on their ability to induce a senescence-like phenotype (reviewed in Ref. 9 ) and to inhibit cell proliferation (10) . In fact, Cyclin D2 was unique among the three D-type cyclins, being up-regulated manyfold under conditions of growth arrest in phenotypically normal human and murine fibroblasts. Furthermore, ectopic overexpression of cyclin D2 effectively blocked cell cycle progression, suggesting an alternate role for cyclin D2 in promoting exit from the cell cycle and maintaining a nonproliferative state. The fact that high levels of cyclin D2 are expressed in normal human tissues composed primarily of nonproliferating contact-inhibited cells such as breast and brain (10) is consistent with those in vitro observations.
At least four previous studies have demonstrated that cyclin D2 mRNA and protein were absent in almost all breast cancer cell lines examined, whereas cultured normal breast epithelial cells had abundant expression (11, 12, 13, 14) . Furthermore, our serial analysis of gene expression and array analysis revealed low or absent cyclin D2 mRNA expression in primary breast cancers as compared with finite life span HMECs (15) , again suggesting that cyclin D2 expression is incompatible with proliferation in mammary epithelial cells. The significance of the differential expression pattern in HMECs compared with breast cancer cell lines was questioned by a report stating that cyclin D2 is expressed in the myoepithelial cells that form the outer lining of the mammary ducts but not in the luminal epithelial cells (13) . This distribution could explain the apparent tumor-specific loss of cyclin D2, because most carcinomas arise from the luminal cells (13) . We have now reinvestigated the expression of cyclin D2 in these two cell types from the normal mammary gland and found that cyclin D2 mRNA is expressed in both luminal and myoepithelial cells. We also demonstrated lack of cyclin D2 mRNA and protein in the majority of breast carcinomas and showed that silencing of gene expression correlated with hypermethylation of the cyclin D2 promoter.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Primary breast tumor tissues were obtained after surgical resection at the Johns Hopkins University and Duke University, and stored frozen at -80°C. Samples containing >50% tumor cells were selected after microscopic examination of representative tissue sections from each tumor. Microdissection of carcinoma and DCIS lesions was performed on 8-µm cryosections using a laser capture microscope (20) or by manually scraping the cells with a 25-gauge needle under x40 magnification (21) . Genomic DNA was extracted by incubating the microdissected cells at 55°C for 12 h in 50 µl of buffer containing 10 mM Tris Cl (pH 8.0), 1 mM EDTA, 0.1% Tween 20, and 0.5 µg/µl proteinase K. The extract was heat inactivated at 85°C for 5 min and used directly for sodium bisulfite treatment (22) .
MSP.
One µg of genomic DNA or the 50-µl extract of microdissected cells (as described above) was treated with sodium bisulfite as described by Herman et al. (22)
and was analyzed by MSP using primer sets located within the CpG-rich island in the cyclin D2 promoter. Primers specific for unmethylated DNA were 5'-GTTATGTTATGTTTGTTGTATG-3' (sense, -1616 to -1594) and 5'-TAAAATCCACCAACACAATCA-3' (antisense, -1394 to -1414) and yielded a 222-bp PCR product. Primers specific for methylated DNA were 5'-TACGTGTTAGGGTCGATCG-3' (sense, -1427 to -1409) and 5'-CGAAATATCTACGCTAAACG-3' (antisense, -1152 to -1171) and yielded a 276-bp PCR product. The PCR conditions were as follows: 1 cycle of 95°C for 5 min "hot start," then addition of 1 µl of Taq polymerase (RedTaq; Sigma); 35 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 45 s; and 1 cycle of 72°C for 5 min. The PCR products were resolved by electrophoresis in a 2% agarose gel.
Treatment of Cells with 5-aza-dC and TSA.
Cells were seeded at a density of 1 x 106 cells/100-mm plate. Twenty-four h later, cells were treated with 0.75 µM 5-aza-dC (Sigma) or with 100 ng/ml of TSA (Sigma; Refs. 23
and 24
). Total cellular DNA and RNA were isolated at 0, 3, and 5 days after addition of 5-aza-dC and at 0, 24, and 48 h after addition of TSA, as described above.
RT-PCR.
Total RNA was treated with RNase-free DNase (Boehringer-Mannheim; 0.5–1 units/µl) for 30 min at 37°C, followed by heat inactivation at 65°C for 10 min. Reverse transcription reactions contained 2 µg of DNase-treated RNA, 0.25 µg/µl pdN6 random primers (Pharmacia), 1x first-strand buffer (Life Technologies, Inc.), 1 mM of each deoxynucleotide triphosphate (Pharmacia), and 200 units of Moloney murine leukemia virus-RT (Life Technologies, Inc.), and were incubated for 1 h at 37°C, followed by heat inactivation at 75°C for 5 min. PCR was performed using the primers 5'-CATGGAGCTGCTGTGCCACG-3' (sense) and 5'-CCGACCTACCTCCAGCATCC-3' (antisense) for cyclin D2 and primers 5'-AGCCATGGAACACCAGCTC-3' (sense) and 5'-GCACCTCCAGCATCCAGGT-3' (antisense) for cyclin D1. Coamplified products of 36B4, a "housekeeping" ribosomal protein gene, were used as an internal control, using primers 5'GATTGGCTACCCAACTGTTGCA-3' (sense) and 5'-CAGGGGCAGCAGCCACAAAGGC-3' (antisense). The 25-µl reactions contained 1x buffer (2x Reaction Mix; BRL) and 100 nM of each primer. The PCR conditions were: 1 cycle of 94°C for 1 min "hot start," then addition of 1 unit of Taq polymerase (RedTaq; Sigma); 1 cycle of 94°C for 2 min; 35 cycles of 94°C for 15 s, 55°C for 30 s, 72°C for 45 s, and finally, 72°C for 5 min. The PCR samples were resolved by electrophoresis on a 2% agarose gel.
Western Blot Analysis.
Proteins were extracted from cell pellets and from 8-µm cryosections of primary breast tumors in buffer containing 20 mM Tris (pH 7.5), 150 mM NaCl, and phenylmethylsulfonyl fluoride (100 µg/ml), and sonicated. Ten µg of protein was fractionated on 12.5% SDS-PAGE and transferred by electrophoresis to a nylon membrane. The blot was incubated with anti-cyclin D2 antibody (Ab-4, "cocktail" mouse monoclonal antibodies; Neomarkers, San Diego, CA) diluted 1:200 in 5% skim milk for 2 h at room temperature. Horseradish peroxidase-conjugated antibody antimouse IgG (Amersham) was used at 1:1000, and binding was revealed using enhanced chemiluminescence (Amersham).
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Table 1)
. The two immortalized HMEC lines lacking cyclin D2 expression were HBL100 and MCF10A (data not shown; Table 1
). In contrast, 10 of 11 breast cancer cell lines showed no detectable expression of cyclin D2. Only one breast cancer cell line, HS578T, expressed a low but detectable level of cyclin D2 (Fig. 1A
; Table 1
). Likewise, the results with primary tumors reflected the findings in cultured cells. Eighteen of 24 primary breast carcinomas expressed significantly lower levels of cyclin D2 mRNA (Fig. 1B
and data not shown) as compared with finite life span HMEC 184 and five other HMECs (Fig. 1A
; Table 1
). As an additional control for cyclin D2 expression, we analyzed the expression of cyclin D1 in the same panels of cell lines and tumors. Consistent with the observations of others (11, 12, 13, 14
, 25
, 26)
, cyclin D1 mRNA was detectable in all of the cell lines and primary breast tumors tested (Fig. 1)
. Thus, in both breast cancer cell lines and primary tumors, specific loss of cyclin D2 mRNA expression but not of cyclin D1 expression was observed. These results extend and confirm our data (15)
and others (11
, 13
, 14
, 25
, 27)
.
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; Table 1
). Of note, primary breast carcinomas that expressed very low or undetectable levels of cyclin D2 mRNA (nos. 03, 11, 30, 38, 40, 63, and 70 in Fig. 1B
) showed no detectable cyclin D2 protein (Fig. 2)
. HMECs that were derived from normal breast tissue and expressed high levels of cyclin D2 mRNA (Fig. 1A)
showed clearly detectable levels of cyclin D2 protein as well (Fig. 2)
.
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. However, one luminal epithelial cell sample expressed a noticeably lower level of cyclin D2 (Fig. 3
, Lane 3) compared with the other three. Additionally, another finite life span HMEC, 184, also stained for luminal cell markers, cytokeratins 8 and 18 and mucin, but not for the myoepithelial cell marker cytokeratin 14.4 As seen in Fig. 1A
, HMEC 184 and its three derivatives also expressed cyclin D2 mRNA (Fig. 1A)
. Thus, cyclin D2 mRNA was expressed in all five of five luminal and four of four myoepithelial cell preparations from the normal breast. Immunohistochemical analysis of normal breast tissue using anti-cyclin D2 antibodies (Neomarkers) was attempted but was unsuccessful, because nonspecific staining was observed.
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80% of the CpGs are methylated. Exceptions to this are the CpG islands in the promoter region of many genes. CpG islands are CG-rich regions of DNA, 1 kb in length, present in the promoters of >60% of human genes. Normally, CpG islands are unmethylated, and the chromatin in those sites is enriched in hyperacetylated histone and deficient in histone H1, characteristics of active chromatin. Both unmethylated and methylated DNA are assembled into nucleosomes (reviewed in Refs. 28
and 29
).
The cyclin D2 promoter contains a CpG-rich region at 1000–1600 bp 5' to the translation start site (Fig. 4A)
. To test whether loss of cyclin D2 expression in breast cancer is associated with aberrant methylation, we designed primers for MSP to rapidly screen for cyclin D2 promoter methylation. Hypermethylation at the CpG rich region was detected in 11 of 11 breast cancer cell lines (data not shown; Table 1
) that lacked expression of cyclin D2 mRNA (Fig. 1A)
and protein (11
, 13
, 14
, 25
, 27) . Hypermethylation was also noted in 49 of 106 (46%) primary breast carcinomas (Table 1
; Fig. 4B
; data not shown).
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; Table 1
).
To further confirm that cyclin D2 hypermethylation is confined to tumors and does not occur in the normal breast, we examined normal mammary epithelial cells prepared by a variety of techniques. By MSP analysis, cyclin D2 promoter was found to be unmethylated in seven mammary organoid preparations from reduction mammoplasties and in four HMECs cultured from nonmalignant breasts (Fig. 4D)
. The only exception to this finding was in immortalized HMECs HBL100 and MCF10A, which contained hypermethylated cyclin D2. As expected, these HMECs were the only two that did not express cyclin D2 mRNA (Table 1
; data not shown).
To rule out the contribution of inflammatory blood cells present in breast cancer specimens as the source of methylated cyclin D2, we tested 10 samples of peripheral blood lymphocytes from noncancer patients. All 10 peripheral blood lymphocytes contained unmethylated cyclin D2 alleles (Fig. 4E)
.
As shown in Fig. 2
, expression of cyclin D2 protein was undetectable in 10 of the 13 primary breast cancers tested (tumor panel 1; Table 1
). By MSP analysis, hypermethylation of the cyclin D2 promoter was observed in 8 of 13 primary breast carcinomas (data not shown). In seven of eight tumors (nos. 30, 38, 40, 63, 70, 17, and 72), methylation of cyclin D2 correlated with lack of cyclin D2 protein. In the one exception (tumor 36), both mRNA and protein expression was seen, despite the presence of a hypermethylated cyclin D2 gene. Five of 13 tumors contained unmethylated cyclin D2 sequences. Among these, two (tumor nos. 13 and 35) showed expression of mRNA and protein, whereas three (tumor nos. 03, 11, and 21) did not. Thus, 87% of the tumors showed a correlation between hypermethylation of the cyclin D2 promoter and silencing of gene expression. The observation that some tumors lacked protein expression despite the unmethylated status of the gene suggests that although methylation may constitute a major pathway for silencing of cyclin D2 expression in breast cancers, alternative pathways account for the loss of the protein in a proportion of these tumors.
Cyclin D2 Promoter Hypermethylation in Preneoplasia.
DCIS is a preneoplastic lesion with a potential for progression to invasive cancer. To determine whether hypermethylation of the cyclin D2 promoter occurs early in the evolution of breast cancer, we performed MSP analysis on samples from 13 primary breast tumors that contained both invasive and noninvasive components. One or more areas of carcinoma (n = 17) and adjacent DCIS (n = 18) were carefully microdissected from the same section. Hypermethylated cyclin D2 was present in 8 of 17 carcinomas and in 8 of 18 DCIS samples ( Table 2
; Fig. 4F
; data not shown). In all of the 13 cases, the methylation status of both carcinoma and DCIS lesions from the same tumor was concordant. This finding suggests that alteration of cyclin D2 expression is an early event and may precede transformation to the fully malignant stage of invasive carcinoma.
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or protein (11, 12, 13)
. If silencing of expression was mediated by promoter methylation and/or altered chromatin conformation, then demethylation of the gene by exposure to 5aza-dC or treatment with the histone deacetylase inhibitor TSA should result in removal of the repressive mechanism and reexpression of the gene. Indeed, when MDA-MB231 and MCF7 cells were exposed to 5-aza-dC in culture, the cyclin D2 promoter was partially demethylated (as analyzed by MSP), and cyclin D2 mRNA expression was restored (as analyzed by RT-PCR; Fig. 5A
). Although the unmethylated cyclin D2 band is visible by day 3 of treatment with 5-aza-dC, the mRNA was seen only at the day 5 time point (Fig. 5A)
. There was a lag period between demethylation of the promoter sequences and mRNA expression of cyclin D2 (Fig. 5A)
, indicating that demethylation of the gene precedes gene expression. Furthermore, exposure to TSA also led to reexpression of the cyclin D2 mRNA (Fig. 5B)
, but no change in the methylation status of the gene was observed (data not shown). These results suggest that methylation and histone deacetylation at the promoter region may play a functional role in suppressing the expression of cyclin D2 in breast cancer.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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It has been suggested previously that cyclin D2 is expressed in myoepithelial but not in luminal epithelial cells of the breast (13)
. If this were the case, lack of expression of cyclin D2 in breast cancers would be anticipated, because the vast majority of these tumors originate from luminal rather than myoepithelial cells. However, by RT-PCR, we detected cyclin D2 expression in four mammary luminal epithelial cultures of the 184 series (Fig. 1A)
and in all four of four purified luminal epithelial cell extracts (Fig. 3)
, as well as in four of four myoepithelial cell extracts (Fig. 3)
. Therefore, we believe that the gene is expressed in both of these cell subtypes. One could attribute the presence of cyclin D2 RT-PCR product to the high sensitivity of the assay in amplifying even a few myoepithelial cells contaminating the luminal cell preparations. Our Western analysis of multiple HMEC samples (including the 184 series) counters this argument, because this less-sensitive technique detected cyclin D2 protein in all seven samples tested (Fig. 2)
. Of note, of four fractionated luminal cell preparations, one expressed a lower level of cyclin D2 mRNA (Fig. 3
, sample 3), in concordance with the previous report. It should be pointed out that the published report was based on the findings in one luminal cell preparation (13)
. Possibly, cyclin D2 is down-regulated under certain circumstances, such as stress, hormonal fluctuations, or culture conditions. Much larger panels of fractionated cells need to be examined, and further research is warranted to explore the impact of physiological influences on cyclin D2 levels in normal mammary cells.
Searching for a mechanism underlying the consistent loss of cyclin D2 expression in breast cancers, we tested the cyclin D2 promoter for CpG hypermethylation as a possible cause of gene silencing. CpG hypermethylation is an epigenetic, heritable change that appears to be tightly associated with the formation of repressive chromatin (28 , 29) . By MSP analysis, cyclin D2 promoter hypermethylation was detected in 49 of 106 (46%) of the tumors.
The strength of the correlation between hypermethylation of the promoter and silencing of gene expression would provide an assessment of the importance of DNA methylation in cyclin D2 gene regulation. Methylation of cyclin D2 promoter, mRNA, and protein expression were studied in a panel of 13 primary breast carcinomas. In 7 tumors, hypermethylation of the gene correlated with low or no cyclin D2 mRNA and lack of protein expression (Figs. 1B
and 2
). The one exception to this finding was tumor 36, which showed expression of mRNA and protein but contained hypermethylated cyclin D2. This observation of lack of correlation between hyper-methylation and expression, albeit in one tumor, has precedence in the published literature (23
, 24 , 33
, 34)
. In this tumor, competing influences of transcription complexes with histone acetylase and histone deacetylase activities appear to have favored the former, allowing gene transcription to occur despite promoter methylation. Interestingly, treatment of breast cancer cells, MDA-MB231, with TSA, a histone deacetylase inhibitor, resulted in reexpression of the cyclin D2 mRNA within 24 h with no change in the methylation status (Fig. 5B)
. Altogether, these findings indicate that an appropriate conformation of the chromatin in which the cyclin D2 promoter is embedded is sufficient for transcription and can override the effects of DNA hypermethylation.
The results of this study show that in breast cancers, cyclin D2 protein expression is undetectable in 80% of the tumors, and that silencing of cyclin D2 gene expression may be attributed to tumor-specific methylation in 50% of the tumors tested. In fact, methylation was proposed previously as the mechanism suppressing cyclin D2 expression in resting primary B lymphocytes. In that instance, infection of these cultured cells with EBV led to the demethylation and reexpression of the cyclin D2 gene (35)
. Other repressive mechanisms likely contribute to silencing of the gene in the remaining 30% of cyclin D2-nonexpressing tumors. The high rate of aberrant methylation in DCIS (44%) also suggests that loss of cyclin D2 may be an early event in the malignant transformation of breast cancers.
The frequency of cyclin D2 promoter hypermethylation in breast cancer and its absence in normal breast tissue and blood cells, as shown in this report, make it an excellent candidate marker for breast malignancy. On the basis of the high sensitivity of PCR-based assays for detection of methylated genes, cyclin D2 may prove to be useful for detection of cancer in minute samples such as ductal fluid, fine needle biopsies, pathologically negative sentinel lymph nodes, and blood from metastatic breast cancer patients. We are currently exploring the applications of methylated alleles of cyclin D2 as a tumor marker.
Finally, the fact that cyclin D2 expression is lost in breast tumors strongly suggests that the function of this protein is not limited to its well-known role in G1-S transition during the cell cycle. Breast cancer-specific loss of cyclin D2 expression, confirmed by the present study, sets the stage for investigation of a possible role for cyclin D2 in the terminal differentiation and senescence of human breast epithelial cells and exploration of the part its loss may play in the evolution of breast cancer.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by Grant BCTR 2000 577 (to S. S.) from the Susan G. Komen Foundation and Grant P50 CA88843-01 from the NIH Specialized Programs of Research Excellence in Breast Cancer. 
2 To whom requests for reprints should be addressed, at Johns Hopkins Oncology Center, BBCRB Room 410, 1650 Orleans Street, Baltimore, MD 21231. Phone: (410) 614-2479; Fax: (410) 614-4073; E-mail: saras{at}jhmi.edu
3 The abbreviations used are: cdk, cyclin-dependent kinase; MSP, methylation-specific PCR; RT-PCR, reverse transcription-PCR; HMEC, human mammary epithelial cell; 5-aza-dC, 5'-aza-2'-deoxycytidine; TSA, trichostatin A; DCIS, ductal carcinoma in situ.
4 Internet address: http://www.lbl.gov/LBL-Programs/mrgs/review.html.
Received 11/22/00. Accepted 1/17/01.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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C. Qiu, L. Shan, M. Yu, and E. G. Snyderwine Deregulation of the Cyclin D1/Cdk4 Retinoblastoma Pathway in Rat Mammary Gland Carcinomas Induced by the Food-derived Carcinogen 2-Amino-1- methyl-6-phenylimidazo[4,5-b]pyridine Cancer Res., September 15, 2003; 63(18): 5674 - 5678. [Abstract] [Full Text] [PDF]
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H. Matsubayashi, N. Sato, N. Fukushima, C. J. Yeo, K. M. Walter, K. Brune, F. Sahin, R. H. Hruban, and M. Goggins Methylation of Cyclin D2 Is Observed Frequently in Pancreatic Cancer but Is Also an Age-related Phenomenon in Gastrointestinal Tissues Clin. Cancer Res., April 1, 2003; 9(4): 1446 - 1452. [Abstract] [Full Text] [PDF]
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 B. Terris, E. Blaveri, T. Crnogorac-Jurcevic, M. Jones, E. Missiaglia, P. Ruszniewski, A. Sauvanet, and N. R. Lemoine Characterization of Gene Expression Profiles in Intraductal Papillary-Mucinous Tumors of the Pancreas Am. J. Pathol., May 1, 2002; 160(5): 1745 - 1754. [Abstract] [Full Text] [PDF]
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U. Lehmann, F. Langer, H. Feist, S. Glockner, B. Hasemeier, and H. Kreipe Quantitative Assessment of Promoter Hypermethylation during Breast Cancer Development Am. J. Pathol., February 1, 2002; 160(2): 605 - 612. [Abstract] [Full Text] [PDF]
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V. Shridhar, A. Sen, J. Chien, J. Staub, R. Avula, S. Kovats, J. Lee, J. Lillie, and D. I. Smith Identification of Underexpressed Genes in Early- and Late-Stage Primary Ovarian Tumors by Suppression Subtraction Hybridization Cancer Res., January 1, 2002; 62(1): 262 - 270. [Abstract] [Full Text] [PDF]
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B. Li, J. Goyal, S. Dhar, G. Dimri, E. Evron, S. Sukumar, D. E. Wazer, and V. Band CpG Methylation as a Basis for Breast Tumor-specific Loss of NES1/Kallikrein 10 Expression Cancer Res., November 1, 2001; 61(21): 8014 - 8021. [Abstract] [Full Text] [PDF]
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S. Dhar, R. Bhargava, M. Yunes, B. Li, J. Goyal, S. P. Naber, D. E. Wazer, and V. Band Analysis of Normal Epithelial Cell Specific-1 (NES1)/Kallikrein 10 mRNA Expression by in Situ Hybridization, a Novel Marker for Breast Cancer Clin. Cancer Res., November 1, 2001; 7(11): 3393 - 3398. [Abstract] [Full Text] [PDF]
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