CDK10 Is Not a Target for Aberrant DNA Methylation in Breast Cancer

Patients and Methods

Patients and tumor specimens. Ninety six breast cancer patients surgically treated at the Department of Surgery, Medical University of Vienna, from May 2006 to June 2008, were included in the study. A representative formalin-fixed, paraffin-embedded (FFPE) tumor block from each patient was collected after obtaining appropriate Institutional Review Board permission and written informed consent from the patients. All the tumor specimens had been obtained at the time of surgery before adjuvant therapy and the paraffin blocks had been stored at room temperature at the Department of Pathology, Medical University of Vienna. From each tumor block sections were cut at 4 to 10 μm. One 4 μm section was stained by hematoxylin and eosin to confirm the presence of invasive carcinoma histologically and further sections were used as described.

Nucleic acid isolation and bisulfite treatment of genomic DNA. The genomic DNA was isolated from the FFPE breast cancer samples using a QIAamp DNA FFPE Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Four 5 μm sections from each tumor block were used for the DNA preparation. In addition, genomic DNA from MCF-7 breast cancer cells and from human B lymphocytes was isolated by digestion with proteinase K followed by standard phenol-chloroform extraction and ethanol precipitation as reported previously (16). Afterwards, 2 μg of genomic DNA were modified by treatment with sodium bisulfite using an EpiTect 96 Bisulfite Kit (Qiagen).

The total RNA was extracted with a Siemens, silica bead-based and fully automated isolation method for RNA from one 10 μm whole FFPE tissue section on a Hamilton MICROLAB STARlet liquid handling robot (17). The robot, buffers and chemicals (unless indicated differently) were part of a Siemens VERSANT® kPCR Molecular System (Siemens Healthcare Diagnostics, Tarrytown, NY; not commercially available in the USA). Briefly, 150 μl FFPE buffer (Buffer FFPE, research reagent, Siemens Healthcare Diagnostics, not commercially available) were added to each section and incubated for 30 minutes at 80°C with shaking to melt the paraffin. After cooling down, proteinase K was added and incubated for 30 minutes at 65°C. After lysis, residual tissue debris was removed from the lysis fluid by a 15 minutes incubation step at 65°C with 40 μl silica-coated iron oxide beads. The beads with surface-bound tissue debris were separated with a magnet and the lysates were transferred to a standard 2 ml deep well-plate (96 wells). There, the total RNA and DNA was bound to 40 μl unused beads and incubated at room temperature. Chaotropic conditions were produced by the addition of 600 μl lysis buffer. Then, the beads were magnetically separated and the supernatants were discarded. Afterwards, the surface-bound nucleic acids were washed three times followed by magnetization, aspiration and disposal of supernatants. Afterwards, the nucleic acids were eluted by incubation of the beads with 100 μl elution buffer for 10 minutes at 70°C with shaking. Finally, the beads were separated and the supernatant incubated with 12 μl DNase I Mix (2 μL DNase I (RNase free); 10 μl 10x DNase I buffer; Ambion/Applied Biosystems, Darmstadt, Germany) to remove contaminating DNA. After incubation for 30 minutes at 37°C, the DNA-free total RNA solution was aliquoted and stored at −80°C.

mRNA expression analysis by reverse transcription kinetic PCR (RT-kPCR). All the samples were analyzed with one-step RT-kPCR for the gene expression of one reference gene, RPL37A (ribosomal protein L37a) and three target genes CDK10, CCND1 (cyclin D1) and ESR1 (estrogen receptor 1) in an ABI PRISM® 7900HT (Applied Biosystems, Darmstadt, Germany). The SuperScript® III Platinum® One-Step Quantitative RT-PCR System with ROX (6-carboxy-X-rhodamine) (Invitrogen, Karlsruhe, Germany) was used according to the manufacturer's instructions. The PCR conditions were as follows: 30 minutes at 50°C, 2 minutes at 95°C followed by 40 cycles of 15 seconds at 95°C and 30 seconds at 60°C. All the PCR assays were performed in triplicate. As surrogate marker for RNA yield, the housekeeper gene, RPL37A cycle threshold (Ct) value was used as described elsewhere (17). The relative gene expression levels of the target genes were calculated by the ΔCt method using the formula: 20 - (Ct_target - Ct_RPL37A). The sequences of the primers and probes were as follows: RPL37A: forward: 5′-TGTGGTTCCTGCATGAAGACA-3′, reverse: 5′-GTGACAGCG GAAGTGGTATTGTAC-3′, probe: 5′-TGGCTGGCGGTGCCT GGA-3′; CDK10: forward: 5′-GCACGCC CAGTGA GAACAT-3′, reverse: 5′-CAGGTTGTTGTAGGGCTG CTT-3′, probe: 5′-CCGGGCTTTTCCAAGCTGCCA-3′; CCND1: forward: 5′-CACGCGCAGACCTTCGTT-3′, reverse: 5′-CCGCTGCC ACCATGGA-3′, probe: 5′-TGTGCCACAGATGTGAAGTTCATT TCC-3′ and ESR1: forward: 5′-GCCAAATTGTGTTTGATGG ATTAA-3′, reverse: 5′-GACAAAACCGAGTCACATCAGTAATAG-3′, probe: 5′-ATGCCCTTTTGCCGATGCA-3′. All the RT-kPCR assays were performed using 1 μl of eluate in a 10 μl reaction volume and run in triplicate. The mean of the triplicates is reported.

Methylation-specific PCR (MSP). The methylation status of CDK10, RASSF1A (Ras association domain family 1A) and DAL-1 (differentially expressed in adenocarcinoma of the lung) was determined by MSP (18). Bisulfite-treated DNA was subjected to PCR amplification using primers designed to anneal specifically to the methylated or unmethylated bisulfite-modified DNA sequence within the gene. CDK10 MSP was performed with two different primer sets. Primer set A amplified a 328 bp fragment and was used as reported previously (14). In addition, a search for CpG islands in the 5' region of CDK10 (ENSG00000185324, www.ensembl.org, release 52) was performed using the CpGplot tool (http://www.ebi.ac.uk/Tools/emboss/cpgplot/index.html) which found a CpG island located at -225 bp to +632 bp relative to the transcription start site of CDK10. The sequences of CDK10 MSP primer set B yielding a 126 bp fragment were designed using MethPrimer (19). The primers were designed to amplify a region within a predicted promoter region at -138 bp to -12 bp relative to the transcription start site of CDK10 (Figure 1). The forward and reverse primer sequences of CDK10 MSP primer set B were: 5′-AGGTTCGAATTG TAGTAGTCGGAG-3′ and 5′-GACGCAAACGCGAAAACTCC TTCC-3′. The PCR conditions were as follows: initial denaturation for 12 minutes at 95°C followed by 40 cycles of denaturation for 30 seconds at 95°C, annealing for 40 seconds at 60°C and extension for 30 seconds at 72°C with a final extension for 7 minutes at 72°C. The MSP primer sequences and PCR conditions for RASSF1A and DAL-1 were used as reported previously (20-22). The MSP products were separated in 2% agarose gels stained with GelRed™ (Biotium, Hayward, CA, USA) and visualized under UV spectrophotometry. The DNA extracted from the normal B-lymphocytes and the breast cancer cell line MCF-7 was treated with Sss1 methylase (New England Biolabs, Beverly, MA, USA) and used as a positive control for the methylated alleles. Furthermore, DNA from FFPE breast tissue was treated with Sss1 methylase (New England Biolabs) and used as a positive control for the methylated alleles to ensure the efficiency of MSP on fragmented DNA. Water blanks were used as the negative controls.

Statistical analyses. Comparison of CDK10, CCND1 and ESR1 mRNA expression with the clinico-pathological parameters was performed using the Mann-Whitney U-test or the Kruskal-Wallis test. Correlations among CDK10, CCND1 and ESR1 mRNA levels were assessed using the Spearman's Correlation Coefficient Method. All the p-values are the result of two-sided tests. A p-value equal to or less than 5% was considered statistically significant. The SPSS 15.0 software (SPSS Inc., Chicago, IL, USA) was used for the calculations.

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Figure 1.

CpG island search and location of MSP primer sets A and B in the CDK10 5′ region. A 1000 bp fragment which included the transcription start site of CDK10 was analyzed for the presence of a CpG island using the EMBOSS CpGplot program. CpG sites are shown as vertical bars. MSP primer binding sites of primer set A and B are shown as arrows.