Epigenetic and genetic alterations of p33ING1b in ovarian cancer

Dan-Hua Shen1,{dagger}, Kelvin Yuen-Kwong Chan2,3,{dagger}, Ui-Soon Khoo2, Hextan Yuen-Sheung Ngan3, Wei-Cheng Xue2, Pui-Man Chiu2, Philip Ip2 and Annie Nga-Yin Cheung2,*

1 Department of Pathology, People's Hospital, Peking University, Beijing, Republic of China, 2 Department of Pathology, S.H. Ho Foundation Research Laboratories in Pathology, and 3 Department of Obstetrics and Gynecology, Hong Kong Jockey Club Clinical Research Centre, The University of Hong Kong, Hong Kong, Republic of China

* To whom correspondence should be addressed at: Department of Pathology, The University of Hong Kong, Queen Mary Hospital, Pokfulam Road, Hong Kong, Republic of China. Tel: +852 28554876; Fax: +852 28725197; Email: anycheun{at}hkucc.hku.hk


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 
p33ING1b is a candidate tumor suppressor gene and a nuclear protein. We investigated whether genetic and epigenetic mechanisms affect p33ING1b expression in ovarian cancer thus contributing toward its pathogenesis. A total of 111 ovarian cancers collected from Beijing and Hong Kong were used for this study. Weak or negative p33ING1b protein expression was demonstrated by immunohistochemistry on tissue microarray in 28/111 cases. Real-time quantitative RT–PCR also showed overall significant reduction of p33ING1b mRNA expression (P = 0.0137), with 53.1% (17/32) cases showing 2- to 5-fold reduction and absence of expression. The reduction of mRNA expression in cancer correlated with decreased p33ING1b protein expression (P < 0.0001). While no p33ING1b mutation was found, allelic loss at the p33ING1b locus was demonstrated in 25% (8/32) cases. The allelic loss profiles also showed statistical significant correlation with reduction of p33ING1b protein and mRNA expression (P = 0.031 and 0.030). Promoter methylation as assessed by methylation specific PCR was found in 23.9% (21/88) cases analyzed. Bisulfite sequencing results confirmed the p33ING1b promoter methylation status of these methylation positive cases. Statistical significant correlation between methylation and mRNA expression (P = 0.006) was demonstrated. Treatment with demethylating drug, 5'-aza-2'-deoxycytidine, resulted in dosage-dependent elevated mRNA expression of p33ING1b in ovarian cancer cell lines. This is the first study reporting epigenetic mechanism regulating the p33ING1b expression. Our findings support that genetic and epigenetic alteration of p33ING1b are likely to contribute towards the pathogenesis of ovarian cancers.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 
p33ING1b, the inhibitor of growth 1b gene, has been identified as a novel growth inhibitor and tumor suppressor gene. It is located on chromosome 13q34, and encodes a 279-amino acid 32-kDa-nuclear protein (1). The p33ING1b is the most widely expressed isoform amongst the other isoforms in the ING1 family (24) and is the focus of the current study. Suppression of p33ING1b is associated with the loss of cellular growth control and immortalization, while its overexpression arrests cells in the G0/G1 phase of the cell cycle (5). Allelic loss of p33ING1b was reported in head and neck, and brain cancers (69); and reduced expression of it has been found in lymphoid malignancy, esophageal, gastric, brain and breast cancers (914), suggesting that the reduced expression of p33ING1b might contribute to tumorigenesis. However, mutation in the p33ING1b gene is rare (9,12,15,16), and hence, other mechanisms may have contributed to the reduced expression of this gene.

DNA methylation is an enzyme-induced chemical modification, which usually occurs in cytosine–guanine dinucleotide-rich regions, CpG islands and within the gene promoter regions (17). Altered methylation patterns have been observed in many cancers (1822). Decreased methylation may allow the expression of previously quiescent proto-oncogenes or imprinted genes to become active and induce cell proliferation. In contrast, increased methylation at the promoter regions of a tumor suppressor gene may result in silencing them through the inhibition of transcription and thereby reducing the suppression of cell proliferation (17). The p33ING1b gene promoter region (8) is rich in CpG dinucleotides. CpG islands have been identified by computational analysis by CpGPlot (http://www.bei.ac.uk/emboss/cpgplot/) and also illustrated in Ensembl Human Genome database (Ensembl gene: ENSG00000153487). It has been suggested that p33ING1b might serve as a ‘class 2 tumor suppressor’ by inactivating RNA expression rather than by mutation or genetic loss (11). The possible involvement of the epigenetic factor of CpG methylation affecting the transcript level of p33ING1b tumor suppressor gene has not been reported.

In the present study, we used methylation specific polymerase chain reaction (MSP), bisulfite sequencing (BSG) method as well as methylation-sensitive enzyme restriction PCR to analyze the p33ING1b methylation pattern in the ovarian cancers. As far as we know, we are the first group to study promoter CpG methylation status of p33ING1b in human cancers. Immunohistochemical study using monoclonal antibody (CAb1-4) recognizing p33ING1b performed on tissue microarray (TMA) were used to assess the protein expression pattern of these tumors. The mRNA expression was assessed by real-time quantitative reverse transcription polymerase chain reaction (RT–PCR) assay. Mutation analysis and allelic loss study were also performed.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 
Clinical samples and cell lines
The paraffin embedded tissues of ovarian cancer (111 cases—62 from Beijing and 49 from Hong Kong) were retrieved from the Departments of Pathology, People's Hospital, Peking University, Beijing, and Queen Mary Hospital, the University of Hong Kong, Hong Kong. Thirty-two of forty-nine ovarian cancers with available frozen samples of cancer and corresponding non-tumor blocks were also retrieved from the archival tissue bank of the latter department. Fallopian tube was chosen as the non-tumor counterpart to ovarian epithelium as both are of Müllerian duct origin (23). Normal ovarian tissue was collected from six patients, from individuals who had undergone total abdominal hysterectomy and bilateral salphingo-oophorectomy due to non-cancerous diseases. All the cases were verified by two surgical pathologists. The criteria used for histological type of tumor were based on the classification of the World Health Organization (Tables I and II). The clinical data of the patients from Hong Kong were retrieved and reviewed by the gynecological oncologist in the Department of Obstetrics and Gynecology, Queen Mary Hospital, the University of Hong Kong.


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Table I. The overall correlation between the result of p33ING1b immunohistochemistry and histological subtypes

 

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Table II. The overall correlation between the p33ING1b promoter methylation and histological subtypes

 
The human ovarian cancer cell lines, OVCA3 and SKOV3 were maintained in DMEM with 4 mM L-glutamine and McCoy's 5a with 1.5 mM L-glutamine (Life Technologies, Inc., Gaithersburg, MD, USA) respectively, supplemented with 10% fetal bovine serum in a humidified incubator at 37°C and an atmosphere of 5% carbon dioxide.

Tissue microarray
Before TMA construction, H&E-stained sections of each sample case were reviewed to define tumor representative regions. All tumor areas in the H&E sections were circled. Four to six cylindrical tissue cores with a diameter 0.6 mm were then punched randomly from previously defined tumor region of each tissue block and brought into a recipient paraffin block using DNA-TMA instrument (24,25). Forty to fifty six tissue samples were embedded in a single composite paraffin block.

Immunohistochemistry
TMA sections, 5 µm thick, were cut and mounted on 2% aminopropyltriethoxysilane-coated glass slides. One section from each TMA block was H&E-stained and another section was examined for p33ING1b expression. The monoclonal antibody (CAb1-4) recognizing p33ING1b (26,27) was supplied by Prof. Raibowol (Department of Biochemistry, University of Calgary, Canada). The sections were deparaffinized, and endogenous peroxidase activity was blocked using 1% hydrogen peroxide in methanol. The sections were then placed in a microwave oven for 15 min at 95°C in 10 mM Tris–HCl buffer (pH 7.4) for antigen retrieval. Non-specific antigen was blocked using 10% normal rabbit serum in 2% bovine serum albumin for 30 min. Sections were incubated using the CAb1-4 antibody overnight at 4°C. Biotin-labeled antimouse immunoglobulin was used as secondary antibody. The sections were then incubated with an avidin–biotin–peroxidase complex. 3,3-Diminobenzidine-hydrogen peroxide was used as chromogen. The sections were dehydrated in ethanol, cleared in xylene and mounted. A paraffin embedded tissue of normal stomach was used as a positive control (11), whereas a section incubated in rabbit serum instead of primary antibody was used for comparison as a negative control.

For assessment of immunoreactivity, both the intensity and percentage of positive staining were evaluated. Staining intensity was graded as – (negative),+ (weak positive) and ++ (strong positive). A case was considered to be negative if there was no staining or weak staining involving <10% cells were found.

DNA and RNA extraction
Twenty to thirty sections, 15 µm thick, were cut for each paraffin embedded tissue of cancer and deparaffinized for genomic DNA extraction using conventional phenol/chloroform preceding proteinase K digestion method. DNA extracted from the frozen tissues of 32 cancer cases was processed in a similar manner. Total RNA was also extracted from the frozen tumor and non-tumor counterpart tissues of the 32 cancer cases using TRIZOL (Invitrogen, Carlsbad, CA) according to the manufacturer's instruction. The dissected tissues were homogenized in 1 ml of TRIZOL and mixed with chloroform followed by isopropanol precipitation. The extracted RNA was suspended in RNase-free water. The quality and quantity of DNA and RNA were checked by agarose gel electrophoresis and spectrophotometry. All of the cancer tissue used were dissected and H&E stained, and they were confirmed by the clinical pathologist to have >75% tumor cells.

RNA expression study
One microgram of total RNA was synthesized to cDNA by reverse transcription. The synthesized cDNA was used for real–time quantitative RT–PCR by TaqMan assay. The TaqMan probe was 5' FAM-AGT GCT ACG AGC GCT TCA GTC GCG-TAMRA 3', and the primers were ING1bF 5'-GAG ATC GAC GCG AAA TAC CAA-3' and ING1bR 5'-CTC CTG GCT GCG GA TCA G-3', where the probe and primers were specific to the p33ING1b transcript. The quantitative expression levels of p33ING1b and the housekeeping gene, TBP, were measured against the calibrated curve constructed by the linearized plasmid containing the gene insert as described previously (23). The expression level of p33ING1b was normalized with that of TBP whose probe and primer sequences were described previously (23). The normalized value of p33ING1b in the tumor was compared with that in the non-tumor counterpart of each case for a total of 32 ovarian cancer cases. The real-time quantitation was performed on ABI 7700 Real-time PCR machine (Applied Biosystems, Foster City, CA), and every sample was triplicated for each run of measurement.

Allelic loss and mutation analyses
The 32 ovarian cases studied had genomic DNA available from tumor and non-tumor counterpart tissues. These paired cases (same cases used in RNA expression study) were used for p33ING1b allelic loss analysis. Three fluorescent labeled micorsatellite markers (5' flanking D13S278 and D13S796, and 3' flanking D13S285 and p33ING1b) were used. A PCR was performed in a 10 µl volume containing 1 x PCR Gold Buffer, 2 mM MgCl2, 250 µM dNTP, 400 µM primers, and 1 U AmpliTaq Gold DNA polymerase (Applied Biosystems). The PCR consisted of an initial denaturation step at 95°C for 10 min, 40 cycles of 15 s at 95°C, 1 min at 60°C and 30 s at 72°C, and an additional 10 min extension step at 72°C. The amplified microsatellite fragments were run on ABI PRISM 377 sequencer (Applied Biosystems), using GeneScan and Genotype software (Applied Biosystems) to analyze the allelic loss status of each case. In informative cases, allelic loss on the marker was defined as a decrease of 50% of the signal intensity of one of the alleles compared with that of another allele.

Primers were designed specific to flank and amplify the two fragments which encoded the exons 1 and 2 of p33ING1b. These two fragments encoded the complete coding region of p33ING1b: exon 1 ING1b-1F (forward) 5'-GGA GAG CGA GGG CTT TGC AT-3' and ING1b-1R (reverse) 5'-CTT CCT CCG GTC CAC GGC AG-3'; and exon 2 ING1b-2F (forward) 5'-GCG AGT GAC GCC TGT CCT TCT T-3' and ING1b-2R (reverse) 5'-CTT GCA CCT CAA CAA AGG CAG C-3'. The PCR consisted of an initial denaturation step at 95°C for 10 min, 40 cycles of 15 s at 95°C, 30 s at 58°C and 45 s at 72°C, and an additional 5 min extension step at 72°C. The PCR product was purified and then used for direct sequencing on ABI PRISM 377 sequencer (Applied Biosystems).

Methylation specific polymerase chain reaction and bisulfite sequencing
Two micrograms of DNA was modified by treatment with sodium bisulfite as described previously (23). Methylation and unmethylation-sensitive primers were specifically designed to detect methylation status of CpG dinucleotides in the CpG island identified by computational analysis to be located within 500 bp of the p33ING1b promoter upstream to the exon 1 of p33ING1b (Figure 1). The sequences of primers used to amplify and detect unmethylated p33ING1b promoter were (forward) 5'-TGG ATG GTG TAG GTG TGG GAG TT-3' and (reverse) 5'-CCA AAC ACA AAC AAA AAT AAC AAC ACA-3', whereas the sequences of methylation-sensitive primers were (forward) 5'-CGG ATG GCG TAG GCG CGG GAG TC-3' and (reverse) 5'-CCG AAC ACG AAC GAA AAT AAC GAC GC-3' (Figure 1). The PCR was performed under the following conditions: 94°C for 4 min, followed by 40 cycles of 94°C for 10 s, 58°C (unmethylation-sensitive primers) or 62.5°C (methylation-sensitive primers) for 30 s and 72°C for 30 s, finally 72°C for 3 min. To ensure completion of bisulfite chemical modification, universal methylated DNA and genomic DNA from normal placenta tissue were included in each sodium bisulfite treatment experiment. For each run of MSP test samples, they served as methylation-positive and unmethylation-positive controls. Genomic DNA not treated for bisulfite modification was used as reaction negative control. MSP products were separated on 2% agarose gel containing ethidium bromide. Representative cases of MSP products were subject to direct sequencing. All the methylation-positive cases detected by MSP were analyzed by BSG.



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Fig. 1. The region between –95 and –410 of the p33ING1b promoter was identified in silico to be a CpG island (shaded area). The solid arrows indicate the locations of the methylation and unmethylation specific primers used in the MSP assay, and the dotted arrows indicate the locations of the methylation unbiased primers for bisulfite sequencing. Circles denote the positions of CpG dinucleotides. Illustration was generated from CpG island prediction by MethPrimer (http://itsa.ucsf.edu/~urolab/methprimer).

 
BSG was carried out using methylation unbiased primers on sodium bisulfite modified tumor DNA to amplify the region harboring the MSP product fragment as shown in Figure 1. The primer sequences were as follows: (forward) 5'-GGG AGA YGA TAT AAA GGG AGG G-3' and (reverse) 5'-TAA ATA ATA CCC CCC RAA CTC TTA CTA CTA-3'. The reaction was performed at: 95°C for 4 min, followed by 40 cycles of 94°C for 10 s, 55°C for 30 s, and 72°C for 45 s, finally 72°C for 3 min. The amplified product was cloned into pGEM-T Easy vector and ten clones were randomly selected for sequencing.

Demethylation experiment and methylation-sensitive enzyme restriction PCR
The cell lines of OVCA3 and SKOV3 previously found to have methylation of p33ING1b by MSP and BSG were treated with a demethylating drug, 5'-aza-2'-deoxycytidine (5'-AC) (Sigma, St Louis, MO) (28). The cell lines that reached 80% confluence were treated with 5'-AC at concentration gradients of 0, 0.1, 1.0, 10, 50, 100 µM for 5 days and the experiment was repeated. The TRIZOL reagent was added directly to the cells for RNA extraction. The RNA expression of p33ING1b was quantitated using TaqMan real-time RT-PCR as described earlier. The genomic DNA from the cell lines was then isolated from the organic phase residue of the TRIZOL after RNA extraction. BSG was carried on these DNAs.

Methylation-sensitive enzymes (HpaII and HhaI) and methylation-insensitive enzyme (MspI) digested the DNA extracted from the ovarian cell lines OVCA3 and SKOV3. The digested DNAs were purified by ethanol precipitation. The purified DNA served as the template in PCR where the primers were designed to flank the enzyme restriction sites: RE-1F fragment (forward) 5'-CTCGGGCCTATCCACCTCTTCTG-3' and (reverse) 5'-ACCACCACTCCCAGCAGCCTAG-3' harbor HpaII, HhaI and MspI. The PCR reaction consisted of: 95°C for 10 min, 35 cycles of 15 s at 95°C, 1 min at 60°C and 30 s at 72°C, and an additional 5 min at 72°C. The amplified fragments were resolved by agarose gel electrophoresis, and then visualized under ultraviolet-transilluminator. A target region of an internal control gene (TBP) containing no such enzyme restriction sites was used as control (23).

Statistical analysis
Software of SPSS 10.1 for Windows (SPSS Inc., Chicago, IL) was used to perform the statistical analysis. Comparisons were made by Chi-square test, Fisher's Exact test and Wilcoxon test (two-sided) where applicable. P-value of <0.05 was taken to be statistically significant. Correlation between experimental data and clinical data was also performed. Kaplan–Meier analysis and log-rank test were used for disease free interval and survival analysis.


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 Materials and methods
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Immunohistochemical analysis
Positive p33ING1b staining was observed as a nuclear pattern. p33ING1b nuclear staining was seen in the normal gastric fundic glands epithelial cells, i.e. the positive control (Figure 2). Weak or negative p33ING1b staining was observed in 25.2% (28/111) of carcinoma cases (Tables I and II, Figure 2). The relationship between the tumor histological subtypes and the results of p33ING1b immunohistochemistry are shown in Tables I and II. While the mucinous subtype of ovarian carcinoma displayed the highest proportion of reduced p33ING1b protein expression compared with the other histological subtypes, there was no overall statistical significance of staining pattern with histological types and tumor stage (P-values = 0.70 and 0.056, chi-square and Fisher's exact tests, respectively) (Tables I and IV).



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Fig. 2. Tissue microarray immunohistochemistry. The differential protein expressions of p33ING1b in (OC) ovarian tumors. Upper panel: positive control. Lower panel: weak (left) and strong (right).

 

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Table IV. Correlation between clinical parameters (histological subtypes, tumor stages and chemosensitivity) and protein expression, mRNA expression, allelic loss and promoter methylation of the p33ING1b gene in ovarian epithelial tumors

 
mRNA expression
The mRNA expression of p33ING1b studied in 32 ovarian cancers showed significant overall reduced expression in tumor compared to that in non-tumor (P-value = 0.0137, Wilcoxon test) (Figure 3A). In 17 of 32 (53.1%) ovarian cancer cases a significant 2- to 5-fold reduction and absence of p33ING1b expression was demonstrated. By this 2-fold reduction criteria in tumor, the reduction of mRNA expression was found to correlate significantly with decreased p33ING1b protein expression in 12/32 cases of ovarian cancer (P-value < 0.0001, Fisher's exact test). However, the expression was not correlated with the clinical parameters of histological types and tumor stage (P-values = 0.46 and 0.50, Fisher's exact test) (Table IV).



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Fig. 3. (A) The real-time quantitative RT–PCR results. Overall reduced RNA expression of p33ING1b in the studied ovarian cancer cases with respect to their normal counterparts. The box extends from the 25th percentile to the 75th percentile, with a line at the median (the 50th percentile). The whiskers extend above and below the box to show the highest and lowest values. (B) (left) MSP results on representative ovarian (OC) tumors and their non-tumor (NT) counterparts and (right) ovarian cell lines of OVCA3 and SKOV3: (M) methylation, (U) unmethylation. (C) Methylation-sensitive enzyme PCR results on the ovarian cells, OVCA3 and SKOV3: upper panel represents the p33ING1b promoter and lower panel represents the internal control, a housekeeping gene TBP.

 
Allelic loss and mutation analyses
Allelic loss at the p33ING1b loci, 13q33-34, was observed in 8 out of 32 (25%) ovarian cancer cases in which at least one marker demonstrated allelic loss (Table III). Among these eight cases, six cases were found to have allelic loss in at least two informative loci in tumor. Overall, our allelic loss results showed statistical significant correlation with reduction of p33ING1b protein and mRNA expression but not promoter methylation (P-values = 0.031, 0.030 and 0.95, Fisher's exact test, respectively) in the studied ovarian tumors (Table III). In these 32 cases, no mutation was detected in either fragments corresponding to the coding sequence of p33ING1b (data not shown). No significant correlation was noted between allelic loss and the clinical data of the tumor histological types and stage (P-values = 0.10 and 0.43, Fisher's exact test, respectively) (Table IV).


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Table III. Summary of p33ING1b protein expression, mRNA expression, allelic loss and promoter methylation in 32 ovarian cancer cases

 
Methylation analysis
MSP analysis was successfully performed on 88 of 111 cases of paraffin embedded carcinoma tissues. Results showed p33ING1b promoter methylation in 21 out of 88 (23.9%) cases of tumors (Table II). Promoter methylation detected in paraffin embedded and frozen tissues were of similar proportion: 14/56 (25%) and 7/32 (22%), respectively. Promoter methylation could not be detected from non-tumor counterparts of cancer cases or from all six normal ovarian tissues by MSP and BSG. The BSG results of the tumor cases and the two cell lines found to be methylation positive by MSP were summarized in Table V. The regions of –213 to –235 and –339 to –361 were the primers region used in MSP assay. The CpGs in these regions were frequently methylated (Table V and Figure 4A and B), and their frequencies ranged from 0–75%, and predominantly 50–75% (Table V).


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Table V. Summary of BSG results of all of the ovarian tumor (OC) cases found to be methylation positive by MSP and of the five representative OC cases found to be methylation negative (i.e. unmethylation positive) by MSP assay

 


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Fig. 4. BSG results using methylation unbiased primers; (A) illustrates the forward and (B) the reverse MSP primer priming sites respectively. A(i) and B(i) are MSP methylation-positive cases and A(ii) and B(ii) MSP unmethylation-positive cases with the CpG dinucleotides (underlined) at the priming sites of the methylation and unmethylation specific primers respectively. (C) illustrates the direct sequencing result of MSP products of a representative case of ovarian tumor sample which was positive for methylation and unmethylation specific reactions. The sequence of the p33ING1b promoter is given at the top of the panel; CpG dinucleotides are in boxes. See online supplementary material for a colour version of this figure.

 
Overall statistical significant correlation between p33ING1b promoter methylation and mRNA expression was noted in the 32 studied frozen tissue samples (P-value = 0.006, Fisher's exact test). Seven cases with promoter methylation demonstrated a reduction in p33ING1b mRNA expression in the tumor. It was also observed that nearly all the cases with reduced p33ING1b mRNA or protein expression were associated with either allelic loss or promoter hypermethylation in tumor (Table III). Nevertheless, correlation between the p33ING1b protein expression and promoter methylation in the overall ovarian cancer cases failed to reach statistical significance (P-value = 0.77, Fisher's exact test) (Table II). There was no significant correlation between the promoter methylation and the clinical data of the tumor histological type and clinical stage (P-values = 0.77 and 0.31, Fisher's exact test, respectively) (Table IV).

We were able to demonstrate p33ING1b promoter methylation in the ovarian cancer cell lines OVCA3 and SKOV3. Heterogeneous promoter methylation pattern was detected in both cell lines, with both methylated and unmethylated alleles detected by MSP and BSG (Figure 4B and Table V). Methylated and unmethylated products from some representative cases were directly sequenced and confirmed (Figure 4C). Methylation-sensitive enzyme restriction PCR results showed that successful amplification was detected in the DNA digested by the enzymes HpaII and HhaI, but not in that digested by the enzyme MspI (Figure 4C). This implied the presence of the methylated and unmethylated CpGs in these methylation-sensitive enzyme restriction sites which concurred with the BSG results. Treatment by the demethylating drug 5'-AC on the cell lines was successfully performed. After 5'-AC treatment, the cell proliferative ability was apparently decreased. Using quantitative real-time PCR, the mRNA expression of p33ING1b was found to increase after the 5'-AC treatment. OVCA3 and SKOV3 were found to have highest elevated expression of p33ING1b after treatment by 5'-AC at the optimal concentrations of 50 and 0.1 µM, respectively (Figure 5). This suggested a dosage-dependent differential effectiveness of the demethylating drug on these cancer cell lines. BSG was performed on both cell lines after treatment with the optimal 5'-AC concentration. Most of the CpGs were unmethylated (Table V) and the 5'-AC could almost completely eliminate CpG DNA methylation.



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Fig. 5. The mRNA expression of p33ING1b in the ovarian cancer cells, OVCA3 and SKOV3, after the 5'-AC treatment. The expressions of p33ING1b normalized by the housekeeping gene, TBP, were measured in the cells treated with different concentration of 5'-AC. The demethylation experiment was repeated on two independent occasions and the measurement of the p33ING1b expression level was triplicated. The means and standard deviations were indicated by the bars and errors bars.

 
In the samples we studied, the patients sensitive to the chemotherapy treatment would have better survival outcome (P-value < 0.0001, log-rank test, data not shown). Clinical parameters of the studied cases were analyzed with the expression level, allelic loss and promoter methylation status of p33ING1b results. No significant correlation with tumor histological type and stage was shown. There was also no statistically significant correlation between the experimental results (the protein, mRNA expression level, allelic loss and promoter methylation status of p33ING1b) and other clinical parameters: the chemosensitivity (P-values = 0.82, 0.82, 0.95, 0.80, Fisher's exact test, respectively) (Table IV), patients' disease free interval (P-values = 0.35, 0.31, 0.24, and 0.31, log-rank test, respectively) (data not shown), and survival data (P-values = 0.51, 0.85, 0.88, and 0.85, log-rank test, respectively) (data not shown).


    Discussion
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 Abstract
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 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 
The p33ING1b gene has been proposed to be a candidate tumor suppressor. Its tumor suppressive functions, including G0/G1 cell cycle arrest, anchorage-dependent growth, senescence and apoptosis have been described (5,6). Thus, the functional loss of p33ING1b might contribute to tumorigenesis.

Recently, the reduced expression of p33ING1b has been determined in several tumors with variable frequencies (2,915). In breast cancer, for example, reduced p33ING1b expression was found in 58–80% of cases (2,13,14). Allelic loss in the p33ING1b gene has been found (79), but mutation is rare. However, there is no publication investigating the expression status of p33ING1b in ovarian epithelial tumors. In the present study, we have demonstrated the reduced protein and mRNA expression of p33ING1b by immunostaining on TMA and real-time quantitative RT–PCR, respectively. The protein expression pattern of p33ING1b was different in different histological subtypes with reduced expression most commonly observed in mucinous subtype. The reduced mRNA expression correlated well with the decreased levels to the absence of p33ING1b protein.

There was no mutation detected in p33ING1b of the 32 tumor samples we studied. Such findings concurred with the reports of Toyama et al. (13) regarding primary ovarian cancers. Only one germline missense alteration and three germline silent alterations of the p33ING1b gene were detected among 377 primary breast cancers. Such findings suggested that other inactivation mechanisms, such as allelic loss and promoter methylation, may be responsible for the reduced p33ING1b expression in cancers.

Allelic loss was found in 8 of the 32 ovarian cancer cases analyzed in our study, with significant correlation between reduction of p33ING1b protein and mRNA expression. The overall frequency of allelic loss of ovarian cancer in our study, however, is lower than that reported in esophageal squamous cell carcinoma (11), but is somewhat higher than in glioblastomas (9).

Promoter CpG methylation is a frequent event in ovarian cancers (2931). Strathdee et al. (30) demonstrated hypermethylation in seven of ten loci in primary ovarian tumors, including BRCA1, HIC1, MINT25, MINT31, MLH1, p73, and bTR. This suggested that promoter methylation is a major pathway leading to inactivation of tumor suppressor genes, which may facilitate the development of ovarian tumor. The promoter methylation of p33ING1b in human cancers has not been studied. We are hitherto the first group to explore this epigenetic mechanism in regulating the expression of p33ING1b.

The MSP and BSG assays, showed the methylation of the p33ING1b promoter in the ovarian carcinomas in our study. The incidence of methylation though different between the histological subtypes and the stages of ovarian epithelial tumors, showed no significant statistical difference. We were also able to demonstrate p33ING1b promoter methylation in ovarian cancer cell lines, OVCA3 and SKOV3, by MSP, BSG, and methylation sensitive enzyme restriction PCR assay. More importantly, our BSG results confirm that the CpGs at the primer sites for MSP are indeed frequently methylated. Our demethylation study on these cell lines lends support to this observation. The application of methylation inhibitor, 5'-AC, was able to reverse the methylation of p33ING1b and increase the mRNA expression of p33ING1b. Each cell line demonstrated a different dosage-dependent effectiveness of 5'-AC, beyond which lowering of the expression of p33ING1b was observed. This may be due to cytotoxicity of and/or the consequence ‘global’ demethylation by the demethylating drug to the cells (3234). The demethylation treatment would have globally reactivated genes, including p33ING1b, which might suppress the cell growth and such alteration might become lethal to the cells.

Clinico-pathological parameters including tumor histology, stage, chemosensitivity, patients' disease free interval and survival did not show statistical significant correlation with the expression, allelic loss and promoter methylation of p33ING1b. In our studied samples, the patients sensitive to the chemotherapy treatment would have better survival outcome but no association between p33ING1b expression or promoter methylation status and chemosensitivity of the cancer could be detected. Vieyra et al. (35) and Cheung and Li (36,37) showed that p33ING1b was involved in the hydrogen peroxide and UV-mediated apoptosis, but not in the campthothecin-induced cell death in vitro. Similarly, our findings did not indicate a significant role of p33ING1b in the chemotherapeutic drug-mediated apoptosis in the ovarian cancers, yet further investigation would be needed to elucidate this.

It is interesting to observe that all cases with reduced p33ING1b mRNA or protein expression were associated with either allelic loss or promoter hypermethylation, confirming that they are important mechanisms in regulating p33ING1b expression. Such findings confirmed the ‘class 2 tumor suppressor’ role of p33ING1b. However, promoter methylation and allelic loss are probably not the sole mechanisms regulating the expression of the p33ING1b gene. Other epigenetic mechanisms, such as histone deacetylation, may have contributed to p33ING1b gene silencing those cases which showed negative or reduced p33ING1b expression but no promoter hypermethylation or allelic loss.


    Supplementary material
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 Supplementary material
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Supplementary material can be found at: http://www.carcin.oupjournals.org/


    Notes
 
{dagger} The first two authors contributed equally to this work Back


    Acknowledgments
 
The monoclonal antibody (CAb1-4) was a generous and kind gift from Professor Karl Riabowol, Cancer Biology Research Group, Departments of Biochemistry, Molecular Biology and Oncology, University of Calgary, Calgary, Alberta, Canada. This study was supported by grants from the Research Grant Council Grant, Hong Kong SAR and the Committee on Research and Conference Grants from the University of Hong Kong.


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 References
 

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Received August 17, 2004; revised December 16, 2004; accepted December 18, 2004.





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