Cyclin A1 promoter hypermethylation in human papillomavirus-associated cervical cancer
© Kitkumthorn et al; licensee BioMed Central Ltd. 2006
Received: 23 November 2005
Accepted: 08 March 2006
Published: 08 March 2006
The aim of this study was to evaluate epigenetic status of cyclin A1 in human papillomavirus-associated cervical cancer. Y. Tokumaru et al., Cancer Res 64, 5982-7 (Sep 1, 2004)demonstrated in head and neck squamous-cell cancer an inverse correlation between cyclin A1 promoter hypermethylation and TP53 mutation. Human papillomavirus-associated cervical cancer, however, is deprived of TP53 function by a different mechanism. Therefore, it was of interest to investigate the epigenetic alterations during multistep cervical cancer development.
In this study, we performed duplex methylation-specific PCR and reverse transcriptase PCR on several cervical cancer cell lines and microdissected cervical cancers. Furthermore, the incidence of cyclin A1 methylation was studied in 43 samples of white blood cells, 25 normal cervices, and 24, 5 and 30 human papillomavirus-associated premalignant, microinvasive and invasive cervical lesions, respectively.
We demonstrated cyclin A1 methylation to be commonly found in cervical cancer, both in vitro and in vivo, with its physiological role being to decrease gene expression. More important, this study demonstrated that not only is cyclin A1 promoter hypermethylation strikingly common in cervical cancer, but is also specific to the invasive phenotype in comparison with other histopathological stages during multistep carcinogenesis. None of the normal cells and low-grade squamous intraepithelial lesions exhibited methylation. In contrast, 36.6%, 60% and 93.3% of high-grade squamous intraepithelial lesions, microinvasive and invasive cancers, respectively, showed methylation.
This methylation study indicated that cyclin A1 is a potential tumor marker for early diagnosis of invasive cervical cancer.
Cervical cancer (CC) is an important health problem and is a leading cause of cancer mortality worldwide in women.  When exposed to and infected by one of the high-risk human papillomaviruses (HPV), vulnerable cervical epithelium may enter a complex multistep process and develop an invasive carcinoma. [2–4] The spectrum of histologic alterations during the intricate processes of multistep carcinogenesis can be classified as premalignant lesions, including low-grade and high-grade squamous intraepithelial lesions (SILs), and malignant invasive cervical cancers.  Despite its strong association with CC, HPV infection alone is not sufficient for the cervical epithelium to fully develop an invasive cervical cancer. Persistent HPV infection contributes to the development of SILs, with viral oncoproteins facilitating the dysregulation of cellular proliferation and the apoptotic process. However, additional accumulation of mutations, as well as epigenetic alterations in the crucial oncogenes and tumor suppressor genes, is required before these premalignant lesions fully transform into invasive cancers. 
The aim of this study was to evaluate DNA methylation status of cyclin A1 (CCNA1) in HPV-associated CC. CCNA1, a second A-type cyclin, has been shown to be essential for entry into metaphase of male meiosis I[7, 8] Consistent with this function, CCNA1 is highly expressed in testis and hematopoietic progenitor cells, but is present at low levels in most other tissues.  No phenotype other than male infertility has been reported in mice lacking CCNA1.  Surprisingly, several lines of evidence suggest that CCNA1 may be a potential epithelial tumor suppressor gene. First, the expression of CCNA1 has been demonstrated to be downregulated in several cancers, such as nasopharyngeal carcinoma and head and neck squamous-cell cancer (HNSCC). [11–13] Second, CCNA1 plays an important role in DNA double-strand break repair following radiation damage by activation of the non-homologous end-joining process that confers DNA stability.  Finally, the promoter, similar to several key tumor suppressor genes, is frequently hypermethylated in colon cancer and HNSCC. [13, 15]
Expression of CCNA1 has been shown to be correlated with the activation of TP53. In a HNSCC model, there is an inverse relationship between CCNA1 promoter methylation and TP53 mutation status in HNSCC tissues.  Similar to HNSCC, the majority of CC is of squamous cell origin and its molecular carcinogenesis strongly correlates with impaired TP53 function. [16–18] However, unlike HNSCC, the functional loss of TP53 in CC is not ascribed to gene mutation, but is processed by viral and host protein-protein interaction. CC is strongly associated with infection by high-risk HPV types and its oncoprotein E6 has the ability to associate with and neutralize the function of TP53. [17, 18] E6 binds to TP53 and catalyzes multi-ubiquitination and degradation of TP53. Consequently, the majority of CC cells have a wild-type TP53, but the protein levels are decreased. Therefore, in comparison with HNSCC, it was of interest to determine if CCNA1 is methylated in HPV-associated squamous cell CC.
Cell lines and tissue samples
SiHa and two HeLa CC cell lines from different sources were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. All three cells were purchased from ATCC. SiHa, HeLa (S), and HeLa (K) were grown and maintained in laboratories of Dr. Ponglikitmongkol M, Mahidol University, Dr. Gutkind JS, NIH, USA and Dr. Ruxrungthum K, Chulalongkorn University, respectively.
With approval of ethical committee, faculty of medicine, Chulalongkorn university, normal cervical tissues, cancer tissues and blood samples were obtained and prepared as previously described. [19, 20] Cervical tissues were obtained by punch biopsy of lesions under direct visualization or under colposcopic examination. Specimens were divided in two. The first sample was submitted to routine histological examination, and the second was reserved for DNA isolation. Blood samples were obtained by venipuncture from CC patients and healthy blood donors. All HPV-positive premalignant lesions were exfoliated cells, selected from routine cytological screening. In brief, cervical cells were collected with a cervical sampler (Digene Corporation, Gaithersburg, MD, USA) using the cervical cytobrush technique, and were divided into three parts. The first was reserved for routine cytological diagnosis. The second was tested for the presence of high-risk HPV (types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68) DNA by Hybrid Capture 2 (Digene Corporation, Gaithersburg, MD, USA).  In cases of positive high-risk HPV and complete histological tissue evaluation, the third part was subjected to CCNA1 methylation analysis. DNA extraction was performed using Tris/SDS and proteinase K at 50°C overnight, followed by phenol/chloroform extraction and ethanol precipitation.
Cervical biopsy specimens and Papanicolaou smears were examined and reviewed by at least two gynecologic pathologists to ensure good quality control of the final pathology results. All CCs contained 20–95% malignant cells. The histological diagnoses distinguished among normal epithelium, low-grade SILs, high-grade SILs, microinvasive and invasive cancer. In case of invasive cancer, only those samples classified as squamous-cell lesions were used for further analysis.
Additional six OTC-embedded frozen CCs and five normal cervices, obtained from hysterectomy specimens, were microdissected as previously described.22 Histologically normal epithelium, connective tissue and malignant cells were subjected to CCNA1 methylation and expression studies.
HPV detection and typing
HPV L1, E6 gene amplification and dot blot hybridization were performed as previously described[19, 22, 23] Briefly, each L1 amplification reaction contained the L1 degenerate primers MY11 and MY09. The E6 reactions contained WD72, WD66, WD154, WD67 and WD76. Both reactions were used to amplify genomic DNA during 40 PCR cycles. To analyze the amplicons for the presence of high-risk HPV, we applied dot blot hybridization using the HPV type-specific oligo probes, WD170, WD132, RR1, RR2, WD103, WD165, WD, consensus L1, MY12/13, WD126, WD128, MY16, WD133/134, MY14 and WD174. The membranes were subjected to analysis by a phosphoimager. Results for L1 and E6 dot blots were scored independently. Duplicate filters were prepared for all specimens.
Sodium bisulfite modification and duplex methylation-specific PCR (MSP)
Oligonucleotide sequences and conditions for PCR analyses
Amplicon size (bp)
Annealing temperature (°C)
RNA preparation and analysis
Expression of CCNA1 in the CC cell lines was examined by RT-PCR. Total RNA was extracted using the TRIZOL reagent (Invitrogen, Singapore) according to the manufacturer's specifications and 5 μg of each sample was subjected to cDNA synthesis using MMLV reverse transcriptase (Fermentas, Hanover, MD, USA). PCR mixtures contained 10× PCR buffer, 0.2 mM dNTPs, 0.4 μM each of primers CCNA1cDNAF and CNA1cDNAR, 1 U of HotStartaq and 80 ng cDNA. GAPDH served as the internal control (Table 1). Aliquots of 10 μl of the PCR products were subjected to electrophoresis on a 2% agarose gel stained with ethidium bromide on preparation, and were visualized by a UV trans-illuminator.
Bisulfite genome sequence analysis
Some CCNA1 methylation-positive CCs were selected for sequence analysis. The bisulfited DNAs were amplified using CCNA1cloningF and CCNA1cloningR (Table 1). The amplified fragments were cloned using the PGemT easy vector and sequenced.
The aim of this study was to determine if the CCNA1 promoter is methylated in CC and to elucidate how the epigenetic alteration occurs during multistep CC development. The experiments conducted comprised of: first, establishment of CCNA1 MSP; second, identification of the methylation status and correlation with expression in CC cell lines, normal cervix and CC; and finally, investigation of the frequency of methylation in normal tissues, high-risk HPV-associated low SILs, high SILs, microinvasive and invasive squamous cell CC.
CCNA1 methylation in CC cell lines
Previously, Carsten Müller-Tidow et al. [26, 27] extensively studied the role of CCNA1 methylation and found that CCNA1 was methylated in several non-expressing tumor cell lines, including HeLa. To confirm this particular finding in CC cell lines, we investigated methylation and expression in HeLa and SiHa cells. Our preliminary study in HeLa, HeLa(S), revealed complete non-methylation, which contradicts the previous report (Fig. 1B). To settle this controversy, we attempted to further evaluate additional CC cell lines, including HeLa(K) grown in a different laboratory, and SiHa. The result confirmed the Carsten Müller-Tidow et al. [26, 27] finding, in that the majority of Hela(K) cells, as well as all SiHa cells, were hypermethylated. CCNA1 RT-PCR confirmed the inverse relation between DNA methylation and gene expression. CCNA1 RNA levels were high, intermediate and low in HeLa(S), HeLa(K) and SiHa cells, respectively (Fig. 1). These data indicate that CCNA1 methylation is common in CC cell lines and its physiological role is to decrease gene expression. The absence of methylation in HeLa(S) might indicate a demethylation process that occurs under different cell culture and maintenance conditions.
CCNA1 methylation and expression in cervical tissues
CCNA1 methylation incidence during multistep cervical carcinogenesis
CCNA1 methylation and clinico-pathological correlation
Total number of cases
CCNA1 promoter hypermethylation
Squamous cell CC
FIGO stage I-IIA
FIGO stage IIB-IV
Grade 1, keratinized type
Grade 2, non-keratinized type
This study demonstrated that: (i) CCNA1 promoter hypermethylation in HPV-associated squamous cell CC is unusually common; (ii) it is specific to CC; and (iii) the methylation is more common in invasive phenotypes compared to other histopathological stages during multistep carcinogenesis. This finding identifies both the interesting biology of CC and a potential clinical application of CCNA1 methylation as an additional molecular marker for the early diagnosis of invasive CC.
Annual cytology screening has dramatically increased the effectiveness of early CC detection. Nonetheless, additional tests will help to improve the sensitivity and specificity of a single Papanicolaou smear for histological analysis. Recently, testing for oncogenic HPVs has been introduced to aid in the triage of women with atypical squamous cells of undetermined significance (ASCUS).  However, because the majority of patients with HPV-associated lesions do not progress to invasive cancer, several studies have attempted to add a panel of tumor suppressor gene methylations to improve the effectiveness of molecular cytological diagnosis. [29, 30] Since the frequency of CCNA1 methylation is high and specific to invasive CC, this gene should be a good candidate to increase the coverage rate for early cancer detection.
In HNSCC, CCNA1 promoter hypermethylation is inversely related to TP53 mutation.  Nonetheless, the frequency of CCNA1 promoter hypermethylation in CC is high, whereas the function of TP53 in CC is usually impaired as a consequence of protein degradation induced by binding of the viral E6 protein.  This observation may be due to either differences in tissue types or pathophysiological outcomes of TP53 between mutations and diminution of the protein function subsequent to E6 binding. We prefer the latter hypothesis, since TP53 and CCNA1 have been shown to augment each other's expression. [13, 14] Consequently, the CCNA1 protein could help to increase physiologic TP53 to counter the function of E6, except for cases of TP53 mutation. In other words, alterations of both CCNA1 and TP53 in HNSCC will be redundant. In contrast, in CC, a decrease in CCNA1 protein should prevent the increment of TP53 that would have compensated for the protein destruction by E6.
Multistep process analysis revealed that CCNA1 methylation is remarkably specific for cervical carcinogenesis. The biological function of CCNA1 is to activate DNA breakage repair by mechanisms depending on CDK2 activity and Ku proteins.  It is interesting to hypothesize why the genomic instability, triggered by impairment of the CCNA1 function, is crucial as an early event in CC development. Perhaps the rate of spontaneous mutations in cervical epithelial cells is too low to accumulate sufficient malignancy-transformation-dependent oncogene and tumor suppressor gene mutations if the cells possess fully functional CCNA1. Therefore, the frequency of invasive CC devoid of CCNA1 methylation is limited.
This study demonstrates the strong association between CCNA1 promoter hypermethylation and invasive HPV-associated CC indicates that this gene could serve as an effective molecular marker. Moreover, our finding, in comparison with previous reports, [13, 14] also suggests that there is a possible molecular link between oncogenic HPVs, TP53 and CCNA1 promoter hypermethylation.
cervical cancer, CCNA1: cyclin A1, SILs: squamous intraepithelial lesions, HPV: Human papillomavirus, WBC: white blood cell
We would like to thank the entire staff of the Department of Obstetrics and Gynecology for their assistance in collecting and providing the tissue samples, Drs. Virote Sriuranpong and Gutkind JS, Kiat Ruxrungthum, and Mathurose Ponglikitmongkol for the CC cell lines, HeLa(S), HeLa(K) and SiHa, respectively, peripheral nerve research unit for assisting in microdissection and Dr. Virote Sriuranpong, Ms Petra Hirsch, Mrs. Nita Suyarnsestakorn and Asia Science Editing for language editing the manuscript. This work has been supported by the National Center for Biotechnology and Genetic Engineering (Thailand), The Royal Golden Jubilee Ph.D. program, the Thailand Research Funds and the Molecular Biology and Genetics of Cancer Development research unit, Chulalongkorn University.
- Pisani P, Parkin DM, Bray F, Ferlay J: Estimates of the worldwide mortality from 25 cancers in 1990. Int J Cancer. 1999, 83 (1): 18-29. 10.1002/(SICI)1097-0215(19990924)83:1<18::AID-IJC5>3.0.CO;2-M.View ArticlePubMedGoogle Scholar
- Durst M, Glitz D, Schneider A, zur Hausen H: Human papillomavirus type 16 (HPV 16) gene expression and DNA replication in cervical neoplasia: analysis by in situ hybridization. Virology. 1992, 189 (1): 132-140. 10.1016/0042-6822(92)90688-L.View ArticlePubMedGoogle Scholar
- Bosch FX, Manos MM, Munoz N, Sherman M, Jansen AM, Peto J, Schiffman MH, Moreno V, Kurman R, Shah KV: Prevalence of human papillomavirus in cervical cancer: a worldwide perspective. International biological study on cervical cancer (IBSCC) Study Group. J Natl Cancer Inst. 1995, 87 (11): 796-802.View ArticlePubMedGoogle Scholar
- zur Hausen H: Papillomaviruses and cancer: from basic studies to clinical application. Nat Rev Cancer. 2002, 2 (5): 342-350. 10.1038/nrc798.View ArticlePubMedGoogle Scholar
- The 1988 Bethesda System for reporting cervical/vaginal cytological diagnoses. National Cancer Institute Workshop. Jama. 1989, 262 (7): 931-934. 10.1001/jama.262.7.931.
- Ferenczy A, Franco E: Persistent human papillomavirus infection and cervical neoplasia. Lancet Oncol. 2002, 3 (1): 11-16. 10.1016/S1470-2045(01)00617-9.View ArticlePubMedGoogle Scholar
- Sweeney C, Murphy M, Kubelka M, Ravnik SE, Hawkins CF, Wolgemuth DJ, Carrington M: A distinct cyclin A is expressed in germ cells in the mouse. Development. 1996, 122 (1): 53-64.PubMedGoogle Scholar
- Liu D, Matzuk MM, Sung WK, Guo Q, Wang P, Wolgemuth DJ: Cyclin A1 is required for meiosis in the male mouse. Nat Genet. 1998, 20 (4): 377-380. 10.1038/3855.View ArticlePubMedGoogle Scholar
- Yang R, Morosetti R, Koeffler HP: Characterization of a second human cyclin A that is highly expressed in testis and in several leukemic cell lines. Cancer Res. 1997, 57 (5): 913-920.PubMedGoogle Scholar
- van der Meer T, Chan WY, Palazon LS, Nieduszynski C, Murphy M, Sobczak-Thepot J, Carrington M, Colledge WH: Cyclin A1 protein shows haplo-insufficiency for normal fertility in male mice. Reproduction. 2004, 127 (4): 503-511. 10.1530/rep.1.00131.View ArticlePubMedGoogle Scholar
- Maxwell SA, Davis GE: Differential gene expression in p53-mediated apoptosis-resistant vs. apoptosis-sensitive tumor cell lines. Proc Natl Acad Sci U S A. 2000, 97 (24): 13009-13014. 10.1073/pnas.230445997.View ArticlePubMedPubMed CentralGoogle Scholar
- Sriuranpong V, Mutirangura A, Gillespie JW, Patel V, Amornphimoltham P, Molinolo AA, Kerekhanjanarong V, Supanakorn S, Supiyaphun P, Rangdaeng S, Voravud N, Gutkind JS: Global gene expression profile of nasopharyngeal carcinoma by laser capture microdissection and complementary DNA microarrays. Clin Cancer Res. 2004, 10 (15): 4944-4958. 10.1158/1078-0432.CCR-03-0757.View ArticlePubMedGoogle Scholar
- Tokumaru Y, Yamashita K, Osada M, Nomoto S, Sun DI, Xiao Y, Hoque MO, Westra WH, Califano JA, Sidransky D: Inverse correlation between cyclin A1 hypermethylation and p53 mutation in head and neck cancer identified by reversal of epigenetic silencing. Cancer Res. 2004, 64 (17): 5982-5987. 10.1158/0008-5472.CAN-04-0993.View ArticlePubMedGoogle Scholar
- Muller-Tidow C, Ji P, Diederichs S, Potratz J, Baumer N, Kohler G, Cauvet T, Choudary C, van der Meer T, Chan WY, Nieduszynski C, Colledge WH, Carrington M, Koeffler HP, Restle A, Wiesmuller L, Sobczak-Thepot J, Berdel WE, Serve H: The cyclin A1-CDK2 complex regulates DNA double-strand break repair. Mol Cell Biol. 2004, 24 (20): 8917-8928. 10.1128/MCB.24.20.8917-8928.2004.View ArticlePubMedPubMed CentralGoogle Scholar
- Xu XL, Yu J, Zhang HY, Sun MH, Gu J, Du X, Shi DR, Wang P, Yang ZH, Zhu JD: Methylation profile of the promoter CpG islands of 31 genes that may contribute to colorectal carcinogenesis. World J Gastroenterol. 2004, 10 (23): 3441-3454.View ArticlePubMedPubMed CentralGoogle Scholar
- Somers KD, Merrick MA, Lopez ME, Incognito LS, Schechter GL, Casey G: Frequent p53 mutations in head and neck cancer. Cancer Res. 1992, 52 (21): 5997-6000.PubMedGoogle Scholar
- Tommasino M, Accardi R, Caldeira S, Dong W, Malanchi I, Smet A, Zehbe I: The role of TP53 in Cervical carcinogenesis. Hum Mutat. 2003, 21 (3): 307-312. 10.1002/humu.10178.View ArticlePubMedGoogle Scholar
- Thomas M, Pim D, Banks L: The role of the E6-p53 interaction in the molecular pathogenesis of HPV. Oncogene. 1999, 18 (53): 7690-7700. 10.1038/sj.onc.1202953.View ArticlePubMedGoogle Scholar
- Mutirangura A, Sriuranpong V, Termrunggraunglert W, Tresukosol D, Lertsaguansinchai P, Voravud N, Niruthisard S: Telomerase activity and human papillomavirus in malignant, premalignant and benign cervical lesions. Br J Cancer. 1998, 78 (7): 933-939.View ArticlePubMedPubMed CentralGoogle Scholar
- Pornthanakasem W, Shotelersuk K, Termrungruanglert W, Voravud N, Niruthisard S, Mutirangura A: Human papillomavirus DNA in plasma of patients with cervical cancer. BMC Cancer. 2001, 1 (1): 2-10.1186/1471-2407-1-2.View ArticlePubMedPubMed CentralGoogle Scholar
- Hubbard RA: Human papillomavirus testing methods. Arch Pathol Lab Med. 2003, 127 (8): 940-945.PubMedGoogle Scholar
- Resnick RM, Cornelissen MT, Wright DK, Eichinger GH, Fox HS, ter Schegget J, Manos MM: Detection and typing of human papillomavirus in archival cervical cancer specimens by DNA amplification with consensus primers. J Natl Cancer Inst. 1990, 82 (18): 1477-1484.View ArticlePubMedGoogle Scholar
- Bauer HM, Ting Y, Greer CE, Chambers JC, Tashiro CJ, Chimera J, Reingold A, Manos MM: Genital human papillomavirus infection in female university students as determined by a PCR-based method. Jama. 1991, 265 (4): 472-477. 10.1001/jama.265.4.472.View ArticlePubMedGoogle Scholar
- Herman JG, Graff JR, Myohanen S, Nelkin BD, Baylin SB: Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci U S A. 1996, 93 (18): 9821-9826. 10.1073/pnas.93.18.9821.View ArticlePubMedPubMed CentralGoogle Scholar
- Chalitchagorn K, Shuangshoti S, Hourpai N, Kongruttanachok N, Tangkijvanich P, Thong-ngam D, Voravud N, Sriuranpong V, Mutirangura A: Distinctive pattern of LINE-1 methylation level in normal tissues and the association with carcinogenesis. Oncogene. 2004, 23 (54): 8841-8846. 10.1038/sj.onc.1208137.View ArticlePubMedGoogle Scholar
- Muller-Tidow C, Bornemann C, Diederichs S, Westermann A, Klumpen S, Zuo P, Wang W, Berdel WE, Serve H: Analyses of the genomic methylation status of the human cyclin A1 promoter by a novel real-time PCR-based methodology. FEBS Lett. 2001, 490 (1-2): 75-78. 10.1016/S0014-5793(01)02128-7.View ArticlePubMedGoogle Scholar
- Muller C, Readhead C, Diederichs S, Idos G, Yang R, Tidow N, Serve H, Berdel WE, Koeffler HP: Methylation of the cyclin A1 promoter correlates with gene silencing in somatic cell lines, while tissue-specific expression of cyclin A1 is methylation independent. Mol Cell Biol. 2000, 20 (9): 3316-3329. 10.1128/MCB.20.9.3316-3329.2000.View ArticlePubMedPubMed CentralGoogle Scholar
- Levi AW, Kelly DP, Rosenthal DL, Ronnett BM: Atypical squamous cells of undetermined significance in liquid-based cytologic specimens: results of reflex human papillomavirus testing and histologic follow-up in routine practice with comparison of interpretive and probabilistic reporting methods. Cancer. 2003, 99 (4): 191-197. 10.1002/cncr.11507.View ArticlePubMedGoogle Scholar
- Widschwendter A, Gattringer C, Ivarsson L, Fiegl H, Schneitter A, Ramoni A, Muller HM, Wiedemair A, Jerabek S, Muller-Holzner E, Goebel G, Marth C, Widschwendter M: Analysis of aberrant DNA methylation and human papillomavirus DNA in cervicovaginal specimens to detect invasive cervical cancer and its precursors. Clin Cancer Res. 2004, 10 (10): 3396-3400. 10.1158/1078-0432.CCR-03-0143.View ArticlePubMedGoogle Scholar
- Feng Q, Balasubramanian A, Hawes SE, Toure P, Sow PS, Dem A, Dembele B, Critchlow CW, Xi L, Lu H, McIntosh MW, Young AM, Kiviat NB: Detection of hypermethylated genes in women with and without cervical neoplasia. J Natl Cancer Inst. 2005, 97 (4): 273-282.View ArticlePubMedGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/6/55/prepub
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.