Methylation of the p16Ink4a tumor suppressor gene 5′-CpG island in breast cancer

Cancer Letters 163 (2001) 59±69

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Methylation of the p16 Ink4a tumor suppressor gene 5 0 -CpG island in breast cancer N.H. Nielsen a,b,*, G. Roos a, S.O. Emdin c, G. Landberg a b

a Department of Medical Biosciences, UmeaÊ University, S-901 87 UmeaÊ, Sweden Department of Pathology, Diagnostic Radiology, Oncology and Radiation Physics, UmeaÊ University, S-901 87 UmeaÊ, Sweden c Department of Perioperative Sciences and Surgery, UmeaÊ University, S-901 87 UmeaÊ, Sweden

Received 18 May 2000; received in revised form 12 October 2000; accepted 16 October 2000

Abstract Methylation of the p16 Ink4a tumor suppressor gene 5 0 CpG island was analyzed in 104 primary breast cancer specimens using Southern blotting and methylation speci®c polymerase chain reaction (PCR) (MSP). Eight and four tumors, respectively, showed methylation, and all MSP positive tumors were detected by Southern blotting. To investigate possible methylation not detectable by these methods, bisulphite genomic sequencing was performed in 220 clones from 14 selected tumors. Absent methylation or methylation of single CpG dinucleotides prevailed in all tumors, but of the MSP positive tumors, three contained alleles with methylation of 31 or 32 of the 32 analyzed CpG dinucleotides in the island. Partially methylated alleles were also observed. In a group with low p16 Ink4a expression determined by Western blotting, four randomly selected tumors contained several identical clones with methylation of 15 CpG dinucleotides by bisulphite genomic sequencing but with a methylation pattern that did not support detection by either Southern blotting or MSP, increasing the potential signi®cance of p16 Ink4a methylation in breast cancer. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: p16 Ink4a; 5 0 -CpG methylation; Breast cancer; Cell cycle

1. Introduction Tumor associated alterations of cytosine methylation in CpG dinucleotides include increased global hypomethylation and regional hypermethylation localized to high density CpG regions, or CpG islands, in promoters of critical growth regulatory genes. As autosomal genes, apart from certain imprinted genes, in normal cells in general are hypomethylated independently of expression the signi®cance of hypomethylation in cancer is unclear. Abnormal methylation of genes involved in cellular transformation has been linked to transcriptional inactivation by both * Corresponding author. Tel.: 146-90-7852408; fax: 146-907852829.

correlative data and various experimental studies, reviewed in [1,2]. The mechanisms that account for gene inactivation associated with hypermethylation are allele speci®c depending on altered structure of the methylated allele and not on a general incapacity of transcription. Thus it has been demonstrated that one methylated and transcriptional inactive normal allele can coexist with a hypomethylated active other allele containing a frame shift mutation rendering the gene product non-functional [3]. Furthermore, inactivating coding region mutations are usually not accompanied by hypermethylation of the same allele. Recently, a protein that binds methylated DNA, MeCP2, has been found in complexes with histone deacetylase. This interaction may interfere with the activity of histone deacetylase and favor formation

0304-3835/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(00)00674-1

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of acetylated histones and condensed DNA that is rendered transcriptionally inactive [4,5]. Despite the clear correlation between hypermethylation and gene inactivation the underlying cellular alterations responsible for this state of abnormal methylation in cancer are unknown. Increased activity of the enzyme required to maintain methylation during DNA replication is a common ®nding in tumors and may contribute to the methylation imbalance found in cancer cells [2]. In addition, reduced availability of certain transacting factors, such as Sp1, which could protect against methylation, may be involved in the initial stages of the process [6,7]. Hypermethylation of the p16 Ink4a tumor suppressor gene 5 0 -CpG island has been implicated as an important mechanism of gene inactivation alone with nonepigenetic alterations in diverse types of cancers [8± 10]. For breast cancer, mutations of the p16 Ink4a gene are uncommon [11±15] while con¯icting data concerning deletions has been reported [12,13,16± 18], and hypermethylation could offer an alternate explanation to the often observed lack of p16 Ink4a protein expression in breast tumors [19±21]. In a study of 16 breast cancer specimens, ®ve were found to harbour hypermethylation as determined by Southern blotting considering one methylation sensitive restriction site [8]. While a recent study detected a comparable frequency of p16 Ink4a methylation in breast cancer, an even higher frequency of methylated alleles that was accompanied by repressed p16 Ink4a was observed in normal breast tissue, whereas premalignant lesions and carcinomas displayed successive loss of methylation and increased p16 Ink4a expression raising serious doubts about the role of p16 Ink4a methylation in breast cancer pathogenesis [22]. Further studies are therefore clearly needed to elucidate the extent of p16 Ink4a methylation in breast cancer, and in the present report we have thoroughly investigated the methylation status in 104 primary breast cancer specimens using various techniques.

2. Materials and methods 2.1. Patient material One hundred and four cryo-preserved tumor specimens from patients with primary breast cancer were

included in the study. These were obtained from a collection of 114 consecutively collected tumors used previously for cell cycle analysis [23] and were considered representatively for primary breast cancer concerning disease stage and other clinical parameters. No patient received any anti-tumoral treatment prior to tumor sampling. 2.2. Analysis of p16 Ink4a gene methylation by Southern blotting Tumors were digested in SDS (sodium dodecyl sulphate)-containing buffer by proteinase-K, DNA extracted by phenol/chloroform, ethanol precipitated and then resuspended in 10 mM Tris buffer (pH 8.0). Five micrograms DNA was digested overnight with 50 units of Sac II (New England Biolabs Inc., USA) according to the manufacturer's instructions, DNA puri®ed by a desalting column (DNA clean-up System, Promega Inc., USA) and further digested with 25 units of EcoR I (New England Biolabs Inc.). DNA fragments were separated on 1% agarose gels, blotted onto nylon membranes (Hybond-N, Amersham Ltd., UK) by capillary transfer in 20 £ SSC solution and immobilized by UV-cross linking. A 336 bp cloned fragment of the 5 0 -untranslated region and part of exon one of the p16 Ink4a gene was labelled by random priming (Megaprime System, Amersham Ltd., UK) and hybridization performed in QuickHyb solution (Stratagene Inc., USA) as recommended by the manufacturer with a ®nal high stringency wash in 0.1 £ SSC and 0.1% SDS buffer at 638C for 30 min. Films were exposed between 3 days and 1 week. 2.3. Analysis of p16 Ink4a gene methylation by MSP and bisulphite genomic sequencing Bisulphite processing of DNA was performed in principle as described by Frommer et al. [24] and the modi®cations introduced by Clark et al. [25]. Shortly, 1 mg of genomic DNA was digested by EcoR I and denatured in 0.35 M NaOH at 378C for 20 min. The bisulphite reaction was carried out in 3.2 M sodium bisulphite and 0.5 mM hydroquinone (Sigma Chemical Co., USA) at 558C for 20±22 h. DNA was recovered by a desalting column (DNA Clean-Up System, Promega Inc., USA) and desulphonated in 0.2 M NaOH at 378C for 15 min, neutralized

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by ammonium acetate, alcohol precipitated, dried and then resolved in 20 ml 10 mM Tris buffer (pH 8.0). Primers speci®c for bisulphite modi®ed and completely methylated (p16-M) or unmethylated (p16-U) DNA at the centre of the CpG island were as described by Herman et al.[26]. Ampli®cation was performed in a standard polymerase chain reaction (PCR) buffer (Boehringer±Mannheim, GmbH, Germany) containing 5% DMSO, 1.5 mM MgCl2 and 1 ml of bisulphite modi®ed DNA for 35 cycles with denaturation at 958C for 1 min, annealing at 658C (p16-M primers) or 608C (p16-U primers) for 45 s and extension at 728C for 30 s. Ampli®cation products were resolved on 3% agarose gels that were stained with ethidium bromide. For bisulphite genomic sequencing the sense strand of a 336 bp fragment of the p16 Ink4a gene CpG island corresponding to nucleotides 2128 to 1208 relatively to the translational initiating site [27] was ampli®ed with primers which did not contain cytosine in a CpG context and consequently annealed to the DNA template independently of the methylation status of the island. Forward primer, 5 0 -AAAGAGGAGGGGTTGGTTGGTTATTA and backward primer, 5 0 -TACCTAATTCCAATTCCCCTACAAACT. (Forward primer position 5±30 and reverse primer position 310±336 in Genbank accession number U12818). The PCR-reaction was performed in buffer containing 10 mM Tris±HCl (pH 9.0), 50 mM KCl, 0.1% Triton X100, 5% DMSO, 1.75 mM MgCl2, 0.2 mM of each dNTP and 1 ml bisulphite treated DNA. The ampli®cation was carried out for 30 cycles with denaturation at 958C for 1 min, annealing at 628C for 1 min and extension at 728C for 30 s. Gel-puri®ed ampli®ed DNA fragments were ligated into pT7blueT (Novagen Inc., USA), transformed into competent Escherichia coli, and for every tumor analyzed 15±25 white colonies were randomly picked for plasmid puri®cation and dideoxy nucleotide cycle sequencing using Taq DNA polymerase (PRISM ready reaction dye deoxy terminator cycle sequencing kit, Perkin±Elmer Corp., USA) and an automated sequence analyzer (ABI377A, Applied Biosystem Inc., USA). In addition to methylation of CpG and CpNpG sites, occasional methylation of cytosine in CpNpNpG and CpNpNpNpG nucleotides was seen as previously reported in tumor specimens [28,29], but comprising less than 1% of all methylated cytosine. Due to unknown biological importance and whether these

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methylation sites represent true cytosine methylation and not are caused by shortcomings of the bisulphite method, clones with cytosine methylation in a CpG or CpNpG context only were accepted in the study. 3. Results Methylation of a 336 bp segment of the 5 0 -untranslated region and ®rst exon of the p16 Ink4a gene which include the most densely packed CpG fragment of the island containing 32 CpG dinucleotides was subjected to analysis using Southern blotting, MSP (methylation speci®c PCR) and bisulphite genomic sequencing (Fig. 1A). Patient characteristics from whom tumor samples were obtained have been reported previously [23] and consisted of primary breast cancers cases with a representative distribution of disease stages and other clinical parameters. 3.1. Methylation of the 5 0 CpG island of the p16 Ink4a gene as determined by Southern blotting Genomic DNA was double digested with the methylation sensitive enzyme Sac II and the methylation insensitive enzyme EcoR I according to Section 2. Of the 104 tumors analyzed, eight (8%) were found to be methylated on a Sac II restriction site in the core of the CpG island and of these were six (6%) methylated on an additional Sac II site that resides 220 bp upstream in the promoter region (Fig. 1B). All tumors with methylation exhibited strong hybridization signal corresponding to DNA with absent methylation of the two Sac II restriction sites and may represent non-cancerous tissue in the sample and/or the presence of various tumor cells clones with different methylation status. The hybridization signal speci®c for methylated alleles varied from very weak but still discernible to an intensity similar to that of hypomethylated DNA (Fig. 1B). All tumors with methylation were redigested to con®rm the results. 3.2. Methylation of the 5 0 CpG island of the p16 Ink4a gene as determined by MSP To independently con®rm the Southern blot result and detect rare species of methylated alleles, the MSP method was employed [30]. This relies on bisulphite modi®cation of DNA with conversion of cytosine, but

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Fig. 1. (A) Map of the 5 0 -end of the p16 Ink4a gene with details of restriction sites, the probe used in Southern blotting, the region analyzed by bisulphite genomic sequencing and location of the primers used in MSP. (B) Southern blot analysis of the methylation status of the Sac II restriction site at the core of the p16 Ink4a gene 5 0 -CpG island. DNA from breast tumors was double digested with EcoR I and Sac II and detected with a p16 Ink4a-gene speci®c probe. Methylation at the Sac II restriction site in the CpG island and absent methylation at an additional Sac II site 220 bp upstream in the promoter region is seen as a 3.6 kb fragment (tumor U249) while methylations at both restriction sites are seen as 4.3 kb fragments (tumor U158, U165, U236 and U261). All tumors showed strong hybridization corresponding to DNA with absent methylation at the two Sac II restriction sites (3.4 kb fragment). Normal, non-tumor DNA.

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not 5-methylcytosine, to uracil and in the subsequent PCR to thymine. The primers used detected the sense strand and speci®cally ampli®ed either completely methylated or unmethylated DNA within the primer sequences of which the downstream primer covered the Sac II site used in the Southern blot analysis. Four tumors (4%) were found to be methylated by MSP (Fig. 2A) all showing distinct ampli®cation and were all also detected by Southern blotting by which three tumors were methylated on both Sac II sites and one on only the 3 0 -site. In agreement with the Southern blot results all tumors showed distinct ampli®cation with primers speci®c for unmethylated DNA con®rming that a signi®cant fraction of DNA, even in samples with hypermethylated alleles, were in the hypomethylated form. In view of the often heterogeneous nature of methylation reported in tumors,

Fig. 2. (A) MSP analysis of the p16 Ink4a gene 5 0 -CpG island. The tumors U158, U165, U177 and U261 contained alleles with CpG hypermethylation as demonstrated by positive ampli®cation using primers speci®c for methylated DNA (M). All tumors showed in addition distinct ampli®cation using primers speci®c for unmethylated DNA (U). Control, PCR without DNA. (B) Detection level of methylated p16 Ink4a alleles by MSP. Various amount of DNA from the cell line T47D1 containing one methylated allele was mixed with DNA from the unmethylated tumor U142. Under the employed assay conditions it was possible to detect approximately one methylated out of 1000 unmethylated alleles.

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experiments were performed to estimate the detection level of hypermethylated alleles under the given assay conditions by the admixture of varying amounts of DNA from the cell line T47D1 containing completely methylated alleles [8] with DNA from a tumor samples that were hypomethylated (U142). As shown in Fig. 2B it was possible to detect one methylated out of approximately 1000 hypomethylated alleles verifying the scarcity of p16 Ink4a hypermethylation in breast cancer. 3.3. Bisulphite genomic sequencing of the 5 0 CpG island of the p16 ink4a gene. Southern blotting enables the detection of restriction site containing methylations while MSP detects the complete methylation of DNA fragments encompassing the primer sequences, in the present study six CpG dinucleotides. The low frequency of p16 Ink4a CpG methylation found could therefore be explained by the occurrence of methylation positioned outside regions detectable by these methods. To pursue this possibility the methylation status of all CpG dinucleotides within the 336 bp region was investigated in selected tumors by bisulphite genomic sequencing. After DNA bisulphite modi®cation and PCR ampli®cation between 15 and 25 clones from every tumor were sequenced. To unravel the extent of CpG methylation in tumors showing methylation by Southern blotting the analysis was ®rst performed in these eight tumors, and a summary of the ®ndings are presented in Table 1. For all tumors the prevailing pattern was either absent methylation or methylation of single CpG dinucleotides, but several tumors contained additional alleles with more extensive methylations (Fig. 3). Four tumors contained clones with methylation encompassing the Sac II restriction site and the positive reaction by Southern blotting in the other tumors were probably caused by rare methylational events and comply with low intensity on the Southern blot of methylated DNA (Fig. 1B and Table 1). In the four MSP positive tumors it was possible to detect clones with 31 or 32 methylated alleles in three tumors (Table 1) while no completely methylated alleles were found in one tumor despite the sequencing of 16 clones, potentially explainable by the high sensitivity of the MSP method. In the four tumors with methylation of the Sac II site on Southern blot, but

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lated tumors in contrast to hypomethylated tumors that showed more variable protein levels. The ®nding above of partially but relatively profoundly methylated alleles provoked selection of four tumors with low expression by Western blotting and negative for p16 Ink4a methylation by Southern blotting and MSP for bisulphite genomic sequencing. Two tumors with defect pRB and high p16 Ink4a protein expression were in addition included in the analysis (Table 1). The tumors with high p16 Ink4a expression were completely hypomethylated or contained occasionally methylated CpG dinucleotides in contrast to one of the four randomly selected tumors that contained several identical clones with methylation of 14 of the 32 CpG dinucleotides (Fig. 5).

negative by MSP, no completely methylated clones were detected, but one tumor showed partially methylated alleles containing 15 CpG dinucleotides (Table 1), however, with a pattern that did not support ampli®cation under the used MSP assay conditions. Cytosine methylation in a non-CpG context has been described in eucaryotic cells [31], and in the present study prevalent cytosine methylation was noticed at CpNpG sites consisting of either mCpApG or m CpTpG. Several tumors contained clones with identical patterns of methylation within a tumor. For example in U158, four out 16 analyzed clones contained mCpTpG trinucleotides with an identical position. Totally, 29 mCpTpG and 4 mCpApG trinucleotides were detected in the 220 analyzed clones with an average rate of 29 per 10 (2.9 £ ) and 6 per 4 (1.5 £ ) positive clone, respectively. 3.3. Association between methylation of the p16 gene 5 0 CpG island and protein expression

4. Discussion

Ink4a

This is the ®rst comprehensive analysis of the methylation status of the p16 Ink4a tumor suppressor gene 5 0 -CpG island in a larger collection of breast cancer specimens. In contrast to previous studies in which 17±48% of analyzed tumors showed partial or complete methylation [8,17,22,33] the frequency in this study was somewhat lower by both Southern blot-

Expression of p16 Ink4a protein by Western blotting has been described elsewhere [32] and the protein levels in methylated and non-methylated tumors as determined by Southern blotting are shown in Fig. 4. p16 Ink4a protein expression was low in all methy-

Table 1 Number of clones in the p16 Ink4a gene 5 0 CpG island that contained methylated CpG-dinucleotides a Tumor number

U158 U165 U177 U215 U236 U249 U258 U261 U156 U250 U145 U167 U179 U185

Number of methylated CpG-dinucleotides 0

1

9, 4* 11, 4* 12, 1* 9, 2* 14 10 10, 4* 10 11, 5* 12, 2* 2.3* 8 7.4* 11.3*

1, 1, 1 1, 1, 1 1, 1 1, 1 2, 1, 1 1, 1, 1 1, 1* 2 1, 1 2, 1, 1* 9 2 1, 1

2

5

1, 1

1

1 4 1

14

15

1

1

1

1

3

29

31

1

Southern blotting

MSP

WB

1 1 1 1 1 1 1 1 2 2 2 2 2 2

1 1 1 2 2 2 2 1 2 2 2 2 2 2

0, 03 0, 06 0, 01 0, 09 0, 31 0, 38 0, 05 0, 21 1, 14 0, 78 0, 03 0, 01 0, 01 0, 00

32 3

1

a Asterisk denotes clones with additional CpNpG-methylation. In cases where a tumor contained clones with identical patterns of methylation the number of each of these is indicated. WB: p16 Ink4a expression as determined by Western blotting and expressed relative to a HeLa cell line extract given an arbitrary value of 1.

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Fig. 3. Bisulphite genomic sequencing of the p16 Ink4a gene 5 0 -CpG island. Three different methylation patterns are shown. In the tumor U165, both unmethylated (A) and completely methylated alleles (B) were detected, whereas the tumor U177 contained partially methylated alleles (C). The sequences shown correspond to the reverse strand of the p16 Ink4a gene and methylated CpG sites are indicated.

Fig. 4. Protein levels of p16 Ink4a as determined by Western blotting [23] in tumors with CpG methylation or unmethylation at the Sac II restriction site in the p16 Ink4a 5 0 -CpG island.

Fig. 5. Methylation of the p16 Ink4a 5 0 -CpG island in tumor U145. Of the 13 analyzed clones, three contained an identical pattern of methylation in 14 out of the 32 CpG dinucleotides in the island. Enzyme restriction sites and primers in the MSP analysis are indicated. F, forward primer; R, reverse primer. X, mCpG, W, CpG.

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ting and MSP (8 and 4%, respectively). In addition to some methodological differences including the utilization of different restriction enzymes in the various studies and certain arbitrariness in the detection of partially methylated CpGs by Southern blotting, our material represents consecutively collected tumor specimens obtained before possible adjuvant therapy and therefore represents primary untreated breast cancer patients, whereas in the above studies no information on tumor stages or other clinical parameters were reported. In case p16 Ink4a is inactivated during tumor progression major impact on the frequency of methylation can be expected depending on tumor selection. Besides the relatively small number of tumors analyzed in the previous reports, another cause of difference in the stated frequency of p16 Ink4a methylation could be demographic diversity of patients; all of our patients were Caucasian from Northern Europe. Southern blotting faithfully detected MSP-positive tumors documenting agreements of results with the two methods as previously reported in a comparative study on melanomas [34]. MSP detects complete methylation within the primer sequences but can be designed to detect whatever methylation pattern and is preferable over Southern blotting because of much higher sensitivity and less consumption of DNA. A comparison of Figs. 1A and 2B, however, also reveals the non-quantitative nature of the MSP assay. Furthermore, as bisulphite genomic sequencing demonstrated, several tumors contained partially methylated alleles which could be widespread and thus of possible functional importance but not detectable by either Southern blotting or MSP identifying shortcomings of these methods (Fig. 5). An extension of the present work would be to use microdissected tumor samples in the molecular analysis in order to avoid admixture of non-cancerous cells. The fraction of normal tissue in the tumor samples varied, but based on histological assessment, at least 50% of the tissue consisted of tumor (data not shown) and our Southern blotting and bisulphite genomic sequencing data thus apply to entire tumor samples. Bisulphite genomic sequencing revealed variable extent of methylation from absent to complete methylation of an allele and the occurrence of tumors with alleles that showed different patterns of methylation. The strong hybridization signal from hypomethylated

DNA by Southern blotting (Fig. 1B) argue that a fraction of this possibly was derived from tumor DNA even in samples with hypermethylated alleles. This heterogeneity in methylation pattern should be appreciated in the perspective of classical genetic alterations and the multistep nature of neoplastic progression. During tumor evolution epigenetic changes participate with genetic alterations such as mutations and deletions in the generation and expansion of various tumor cell clones. Methylation is believed to be a discrete process that commences at occasional CpG-dinucleotides at the boundaries of the island and later spreads to cover the entire island. Relatively intense methylation of the p16 Ink4a promoter is permissive for transcription [35] and the partially methylated alleles that were detected in several tumors could thus during tumor progression attain importance if on course toward complete methylation, increasing the abundance and potential importance of p16 Ink4a methylation in breast cancer. The only study hitherto of p16 Ink4a methylation alterations during tumor progression found surprisingly diminishing amount of methylation from normal breast tissue to metastatic lesions [22], but this study has to be reproduced with inclusion of additional methylation sites in the 5 0 end of the gene in the analysis before de®nitive conclusions can be drawn. Furthermore, studies on the role of p16 Ink4a methylation in lifespan extension and escape from moralization, two key events that are believed to occur during development of all cancers, uncovered overall hypomethylation of p16 Ink4a in human mammary epithelial cells accompanied by detectable p16 Ink4a expression in early passage cultures [36,37]. During escape from senescence, increasing amount of p16 Ink4a methylation was detected that paralleled decreasing amount of protein, and may thus re¯ect the inherent process that is thought to occur in the course of tumor development [36,37]. Our ®nding of methylated cytosine at non-CpG sites, especially in CpTpG trinucleotides, is in accordance with another study on breast cancer [28] and in a study on lymphomas of the brain [29]. Caution, however, should be exercised in the interpretation of non-CpG methylation because of the relative resistance of cytosine in some situation in a non-CpG context to conversion by the bisulphite method [38]. Both maintenance and de novo methylation of cytosine at CpNpG sites are supported by the methylation

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machinery of mammalian cells [29]. Methylation of the outer 5 0 cytosine in the consensus binding motif for the transcription factor Sp1 has been shown to interfere with Sp1 binding and transcription [39] suggesting a potential biological role of non-CpG methylation in gene regulation. Whether this type of methylation is altered in cancer and has importance for the down regulation of p16 Ink4a in breast cancer are topics of further interest. All eight tumors with methylation by Southern blotting and especially those with more widespread methylation as judged by MSP and bisulphite genomic sequencing showed low p16 Ink4a expression by Western blotting attesting to the relationship between promoter methylation and inhibition of transcription (Table 1). Two tumors (U236 and U249) that were methylated by Southern blotting but not by MSP express detectable levels of p16Ink4a, explainable by possible tumor heterogeneity (Table 1). A much larger fraction of tumors, however, showed categorically low protein expression by Western blotting yet unmethylated by the methods used to analyze methylation. The distribution of p16 Ink4a protein by Western blotting shows groups with low, intermediate and high protein expressions of which the former was proposed to represent aberrant expression [30]. The expression of p16 Ink4a is probably low in non-transformed and non-proliferative tissues [27,40] making discrimination of the group with abnormal low p16 Ink4a expression dif®cult. To con®rm the prevalent down regulation of p16Ink4a in breast cancer, immunohistochemistry was performed with good agreement between the methods, and clearly a distinct sub-population of tumors with undetectable nuclear and cytoplasmatic staining was identi®ed (data not shown). The ®nding in this study of 50% of tumors with low p16 Ink4a expression is comparable with other studies [19,20] and raises question about the underlying mechanisms responsible. Mutations of p16 Ink4a seem to be rare in breast cancer [11±14] while reports on homozygous deletions are con¯icting. Occasional examples have been found by Southern blotting [12,13,18] whereas reports using FISH [16,17] and closely spaced microsatellite markers spanning the 9p21 region [16] have detected between 15 and 30% homozygous deletions. FISH and LOH allows the detection of microdeletions in contrast to Southern blotting, and applying these methods on larger series

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of primary cancers with representative distribution of disease stages should allow the determination of the real abundance of homozygous deletions in breast cancer. In conclusion, downregulation of p16 Ink4a expression is a reproducible cell cycle aberration in a substantial fraction of primary breast cancers while the occurrence of heavily methylated alleles of the p16 Ink4a 5 0 -CpG promoter seems less frequent. Partially methylated alleles, however, could be more widespread but dif®cult to detect by Southern blotting and MSP, are nonetheless of importance in tumor induction and progression.

Acknowledgements Anna Ljunqvist is appreciated for skilful performance of the DNA sequence analysis. This work was supported by grants from the Swedish Cancer Society, 3813-B98-03XAC, and Lion's Cancer Research Foundation, UmeaÊ University.

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