Breast tissue samples (n = 170) used for methylation and mRNA expression analyses were obtained from patients treated by primary surgery for breast cancer at the Departments of Gynaecology at the University Hospitals of Aachen, Jena, Regensburg and Düsseldorf, Germany, with institutional review board approval. All patients gave informed consent to the study for retention and analysis of their tissue for research purposes. Part of the tumour material and macroscopically normal breast was snap-frozen in liquid nitrogen after surgery. Hematoxylin and eosin (H&E)-stained sections were prepared for assessment of the percentage of tumour cells, only samples with >70% tumour cells were selected. The normal breast tissue used for standardisation contained approximately 40% of epithelial cells. For patient characteristics see Table 1.

The human breast cell lines BT20, MDA-MB231, MCF7 and T47D used for this study were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA) and cultured under recommended conditions.

Frozen tissue samples were dissolved in lysis buffer for subsequent DNA isolation using the blood and cell culture DNA kit (Qiagen, Hilden, Germany) or for RNA isolation by using TRIzol^® (Gibco-BRL, Glasgow, UK) according to the protocol supplied by the manufacturer.

Of the total RNA, 1 μg was reverse transcribed using the Reverse Transcription System (Promega, Madison, WI, USA). To improve transcription rate we mixed oligo-dT and pdN₍₆₎-Primers 1:2. For PCR, 1 μl cDNA was amplified using ID4, (For 5'-CGC TCA CTG CGC TCA ACA C-3' Rev 5'-CTA ACT TCT GCT CTT CCC CC-3', product size 148 bp) and Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) (For 5'-GAA GGT GAA GGT CGG AGT CA-3' Rev 5'-TGG ACT CCA CGA CGT ACT CA-3', product size 289 bp) primers. Reactions were initiated as "Hot Start" PCR at 95°C for 5 min and held at 80°C before addition of 1 unit of Taq DNA polymerase (Roche, Mannheim, Germany). Cycle conditions applied for both genes were: 94°C for 5 min, 38 cycles of 95°C for 1 min, 58°C for 1 min, 72°C for 1 min and a final extension at 72°C for 10 min. PCR analyses were carried out in a PTC-200 cycler (Bio-Rad, formerly MJ Research, Hercules, CA, USA). The amplification products were analysed on a 2% agarose gel containing ethidium bromide under UV-light.

Semi-quantitative PCR was performed using the LightCycler system together with the LightCycler DNA Master SYBR Green I Kit (Roche, Mannheim, Germany) as previously described 22. Reaction volumes of 20 μl consisted of the following components: 3 mM MgCl₂, 10 μM forward primer, 10 μM reverse primer, 2 μl LightCycler DNA Master SYBR Green I and 1 μl of cDNA as PCR template. For primer sequences of GAPDH and ID4 amplification, see Reverse Transcription PCR section. To assure maximum specificity of ID4 mRNA detection a touchdown PCR program was designed (see additional file 1). Gene expression was quantified by the comparative CT method, normalising CT-values to the housekeeping gene GAPDH and calculating relative expression values 23. Post amplification melting curve analyses were performed to assure product specificity. Relative ID4 expression levels were standardised in comparison to the expression level of pooled normal breast tissue samples. To ensure experiment accuracy, all reactions were performed in triplicates.

Bisulphite-modification and methylation-specific PCR (MSP) were performed as previously described 222425. Of the genomic DNA, 1 μg was bisulphite-treated using the EZ DNA Methylation Kit™ (Zymo Research, Orange, CA, USA) according to the manufacturer's specifications. For MSP, 1 μl of modified DNA was amplified using MSP primers that specifically recognise the unmethylated (U For 5'-GGT AGT TG GAT TTT TTG TTT TTT AGT ATT-3' Rev 5'-AAC TAT ATT TAT AAA ACC ATA CAC CCC A-3, product size 157 bp) or methylated (M For 5'-TAG TCG GAT TTT TCG TTT TTT AGT ATC-3' Rev 5'-CTA TAT TTA TAA AAC CGT ACG CCC CG-3', product size 161 bp) ID4 promoter sequence after bisulphite conversion. DNA derived from human carcinoma cell line MDA-MB231 was bisulphite-treated to serve as a control for the unmethylated ID4 promoter sequence. DNA derived from human mammary carcinoma cell line BT20 was used as a positive control for methylated ID4 sequences as described elsewhere 20. Amplification products were visualised by UV-illumination on 3% low range ultra agarose gel (Bio-Rad Laboratories, Hercules, CA, USA) containing ethidium bromide.

Cells were plated at a density of 3 × 10⁴ cells/cm² in a 6-well plate on day 0. The demethylating agent DAC 26 (Sigma-Aldrich, Deisenheim, Germany) was added to a final concentration of 1 μM in fresh medium on days 1, 2 and 3. Additionally, 300 nM TSA (Sigma-Aldrich) was added on day 3. Cells were harvested on day 4 for RNA and DNA extraction. Control cells were incubated without the addition of DAC or TSA and fresh medium was supplied on days 1, 2 and 3.

Sections of three micrometers were dried for 30 min at 72°C, deparaffinised in xylene, rehydrated in a decreasing ethanol series and subsequently boiled for 35 min in Tris-EDTA buffer (pH 9.0) for antigen retrieval. Polyclonal ID4 rabbit anti-human antibody (Sc291, Santa Cruz, CA, USA) was utilised in 1:150 dilution and sections were incubated for 90 min. IHC was performed by using the ChemMate Envision Kit (DAKO, Hamburg, Germany). Sections were counterstained with Mayer's hematoxylin and embedded in Entellan^® mounting medium (EMS Diatome, Fort Washington, PA, USA). Sections of normal and tumorous colon tissues were used for positive controls as described elsewhere 13 (for details, see additional file 2). The application of primary antibody to tissue sections was omitted in negative controls.

Statistical analyses were carried out by using SPSS version 14.0 (SPSS, Chicago, IL). Differences were considered statistically significant when P-values were located below 0.05. The two-sided, non-parametric Dunn's-Multiple-Comparison-Test was used in order to compare the delta CT values of the realtime RT-PCR results of the breast cancer group with the normal breast group as well as the different methylation groups. Two-sided Log-rank tests were performed in order to correlate RFS/OS with ID4 methylation and other clinicopathological parameters. A multivariate Cox-regression analysis was performed in order to test the independent prognostic relevance of ID4 methylation. The limit for reverse selection procedures was P = 0.2. The proportionality assumption for all variables was assessed with log-negative-log survival distribution functions.

First, we established a methylation-specific PCR (MSP) for the ID4 gene, using MSP primers which are complementary to the central CpG island of the ID4 promoter region (Figure 1A). The designed MSP primers amplify the ID4 promoter sequence starting approximately 30 bp upstream of the transcription start site (TSS). In order to demonstrate that ID4 promoter methylation may be associated with ID4 gene silencing, we performed demethylation analyses with four human breast cancer cell lines (MDA-MB231, BT20, MCF7 and T47D). For this purpose, these cell lines were treated with the demethylating agent DAC and the histone deacetylase inhibitor TSA. ID4 expression was measured 72 h later by performing realtime PCR (Figure 1B). We found that in all methylated cell lines (BT20, MCF7, and T47D) ID4 mRNA expression was restored after the treatment. The increase of ID4 expression after promoter demethylation was 119-fold in T47D cells, 38-fold in MCF7 cells and 19-fold in BT20 breast cancer cells. The unmethylated cell line MBA-MD231 showed just a marginal alteration of its ID4 mRNA levels.

Recently we have demonstrated that ID4 mRNA expression is downregulated in 78% (39/50) of human primary breast carcinomas 27. Umetani et al. had shown before that promoter hypermethylation is implicated to be an effective mechanism of ID4 inactivation in human breast cancer, albeit this group only analysed small sized (T1) breast tumours 20. In order to determine the exact methylation frequency of the ID4 promoter in a clinical relevant spectrum of human breast cancer we analysed genomic DNA from 170 primary breast cancer patients by MSP technology.

Representative results are shown in Figure 1C. In total ID4 promoter methylation was found in 68.9% (117/170) of breast cancer specimens. Accordingly, 31.1% of the breast cancer specimens (53/170) exhibited no ID4 promoter methylation. Normal breast tissues (n = 13) were analysed by MSP as well and did not exhibit any ID4 promoter methylation (data not shown), indicating that this is a tumour-specific process.

Next, we wanted to analyse whether ID4 promoter methylation consequently led to silencing of the ID4 promoter as measured by realtime PCR analysis of the gene transcript. For this purpose, a part of the same breast cancer cohort (n = 46) used previously for methylation analysis was re-assessed (Figure 2A). Compared to a normal breast tissue standard (12 pooled normal breast tissues) loss of ID4 mRNA expression in unmethylated breast cancer specimens was marginal (median fold change = 1.3). In contrast, methylated breast cancer specimens exhibited a highly significant (P < 0.001) loss of ID4 expression (median fold change = 12.3). Thus, these data clearly indicate that ID4 promoter methylation is associated with ID4 gene silencing. The comparison of ID4 expression in breast tumours versus normal breast tissues resulted in 82.6% downregulation in tumour samples by the fold change two (FC 2) approach. In order to confirm that promoter methylation also affects loss of ID4 protein, we performed a parallel analysis of ID4 promoter methylation, mRNA and protein expression in three matched samples with normal breast tissue and corresponding tumour tissue (Figure 3A, B, C). Breast cancer specimens (T) with unmethylated ID4 promoter exhibited only a marginal decline (FC = 1.1) in ID4 mRNA expression. In accordance with the mRNA data, the abundance of ID4 protein in the tumour was very similar to that found in the corresponding normal tissue (N). Breast cancer specimens exhibited strong ID4 mRNA downregulation (16-fold and 71-fold, respectively) in comparison to their corresponding normal tissues depending on clear ID4 promoter methylation. Note, that in these tumour tissues nearly complete loss of ID4 protein expression was evident (Figure 3B, C).

Finally, descriptive Fisher's exact tests were performed in order to correlate ID4 methylation with clinicopathological patient characteristics (Table 2). A hypermethylated ID4 promoter was significantly associated with positive lymph node status (P = 0.03) and loss of ID4 mRNA expression (P < 0.001). No associations were found with age at diagnosis, tumour size, histological grade/type and oestrogen/progesterone receptor status. A comparison between recurrence-free survival (RFS)/overall survival (OS) and ID4 methylation status is shown in Table 3. We found an increased risk for tumour recurrence in breast cancer patients with ID4 promoter methylation (P = 0.036) compared to patients with lack of ID4 methylation (Figure 2B). Estimation was accomplished by the method of Kaplan-Meier. ID4 promoter methylation is considerably associated with a low 10-years RFS rate (47%) while patients without ID4 promoter methylation have a 10-years RFS rate of 71%. Cox regression models including factors possibly influencing RFS in relation to ID4 promoter methylation, failed significance in confirming the prognostic value of ID4 promoter methylation as an independent marker, probably due to its close relation to positive lymph node status (data not shown).