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. 1920 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). 21 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 L1, E6 gene amplification and dot blot hybridization were performed as previously described192223 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.

The DNA samples were subjected to bisulfite treatment. 2425 Briefly, 2 μg of genomic DNA was denatured with NaOH (final concentration 0.2 M). Subsequently, 10 mM hydroquinone and 3 M sodium bisulfite were added and incubated at 50°C for 16 h. The modified DNA was then purified using Wizard DNA purification resin (Promega, Madison, WI, USA) followed by ethanol precipitation. Duplex MSPs were performed to identify the CCNA1 methylation status of all samples. The duplex PCR mixtures contained 10× PCR buffer (Qiagen, Chuo-ku, Tokyo), deoxynucleotide triphosphates (0.2 mM), primers CCNA1metF, CCNA1metR, CCNA1unmetF and CCNA1unmetR (final concentration 0.4 μM each per reaction) (Table 1), 1 U of HotStarTaq (Qiagen, Chuo-ku, Tokyo) and bisulfited DNA (80 ng). The amplification reaction was carried out for 30 cycles in a 2400 Perkin Elmer thermal cycler. Then 10-μl aliquots of the PCR products were stained with cyber green, run on an 8% non-denaturing polyacrylamide gel. The band intensity was visualized and measured by using a phosphoimager.

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.

Duplex MSP for CCNA1 was designed according to the sequence in Figure 1A. The methylated sequence comprised of 46 bp and the non-methylated sequence, 67 bp, shown as the lower and the upper amplicons, respectively.

Previously, Carsten Müller-Tidow et al. 2627 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. 2627 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.

We validated the reliability of this duplex MSP by performing calibration experiments using SiHa mixed with HeLa(S), CCNA1 completely hypermethylated and non-methylated cells, respectively (Fig. 2A). With at least three replicates for each experiment, the result demonstrates the consistency of the current approach, with minimal intra- and inter-assay variations (Fig. 2B). It is noteworthy that the correlation between measured and actual CCNA1 methylation percentages was not linear, but exponential.

The discovery of an inverse correlation between CCNA1 methylation and expression in CC lines suggested possibility of the same situation in vivo. To test this hypothesis, we evaluated the epigenetic control in vivo. Six frozen OTC-embedded CCs and five normal cervices were microdissected and subjected to duplex MSP and CCNA1 RT-PCR. Figure 3 shows examples of typical in vivo results. First, whereas no methylation could be observed, CCNA1 mRNA was discoverable by RT-PCR in normal cervix from both epithelium and connective tissue cells (Fig. 3A). In contrast, epigenetic control was detectable in cervical epithelia of CC patients from both malignant cells and adjacent histologically normal cervical epithelia. Nonetheless, in matched cases, a higher degree of methylation could be demonstrated in cancer than in normal cells. From all CCs, no CCNA1 mRNA was detectable. Interestingly, even if methylation was detected, CCNA1 was expressed in malignancy-adjacent histologically normal cervical tissues. Moreover, an inverse correlation between the methylation level and mRNA quantity was observed. CCNA1 expression in methylated malignancy-adjacent histologically normal cervical epithelium may be due to normal cell contamination or partial methylation at the promoter according to CC multistep progression. Whereas complete methylation could be observed in most cancer cells, partial and non-methylated CCNA1 was discovered in the adjacent epithelia (Fig. 3B). In conclusion, this experiment evaluating cervical tissue in vivo led to three conclusions. First, CCNA1 methylation was exclusively associated with cervical carcinogenesis. Second, the epigenetic alteration occurred earlier than morphological transformation of the cellular phenotype. Finally, methylation may play a role in this gene inactivation.

Cervical intraepithelial neoplasia provides a crucial model to study the multistep process of carcinogenesis. Therefore, we evaluated the frequency of CCNA1 methylation in several cervical epithelial tissues with a distinctive degree of malignant transformation, normal cervix, CIN, microinvasive and CC, respectively. We selected 43, 25 and 30 cases of white blood cells (WBC), normal cervical biopsies and invasive CCs, respectively (Table 2). Among these samples, 13 WBC samples and 6 normal cervical samples, located at least 3 cm from the tumor margin and showing the absence of HPV DNA, originated from CC patients. For all cases, when a methylated amplicon was visible and the methylation percentage measured exceeded 5%, the test was deemed positive. All selected CCs were squamous and positive for HPV. Of the cases, 24 harbored HPV type 16, 4 had HPV type 18 and 2 cases displayed unclassifiable HPV types. Interestingly, a high frequency of methylation was exclusively present in CCs, i.e., 28 cases or 93.3% (Fig. 4A,B and Table 2). To reveal multistep carcinogenesis, we included 24 cases of SILs and 5 microinvasive cancers from exfoliated cervical cells. All cases were positive for oncogenic HPV, analyzed by Hybrid Capture 2. Whereas 60% and 36.6% of the microinvasive cancers and high SILs, respectively, demonstrated CCNA1 methylation, none of the HPV-associated low SILs exhibited these epigenetic changes (Fig. 4B and Table 2).