Skip Navigation

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (36)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Tan, L.-w.
Right arrow Articles by Dobrovic, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Tan, L.-w.
Right arrow Articles by Dobrovic, A.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Carcinogenesis, Vol. 23, No. 2, 231-236, February 2002
© 2002 Oxford University Press

CANCER BIOLOGY

Variable promoter region CpG island methylation of the putative tumor suppressor gene Connexin 26 in breast cancer

Lor-wai Tan,2,3, Tina Bianco and Alexander Dobrovic,1,3

Department of Haematology–Oncology and University of Adelaide Department of Medicine, The Queen Elizabeth Hospital, Woodville South, SA 5011, Australia


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Intercellular communication via gap junctions is a mechanism for tumor suppression. Connexin 26 (Cx26) is a structural component of gap junctions expressed by breast epithelial cells. Expression levels of Cx26 are reduced in many breast tumors. Methylation-sensitive single-stranded conformation analysis showed variable methylation in the promoter region CpG island in 11 out of 20 (55%) breast cancer patients. Heterogeneity in methylation patterns was observed both between and within tumors. The degree of methylation ranged from a few CpG dinucleotides to almost all the CpG dinucleotides in the analyzed region. The most frequently methylated CpG was in an Sp1 site known to be important for Cx26 gene expression. One of eight breast cancer cell lines (MD-MBA-453) was methylated in the promoter region and did not express Cx26. Treatment of MDA-MB-453 with 5-aza-2'-deoxycytidine resulted in the re-expression of Cx26 mRNA. Methylation of the promoter region is likely to be an important mechanism in modulating the expression of Cx26 in breast cancer.

Abbreviations: Cx26, Connexin 26; MS-SSCA, methylation-specific single-stranded conformational analysis; RT–PCR, reverse transcription polymerase chain reaction.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Gap junctions are specialized cell membrane channels connecting adjacent cells and facilitate the transfer of metabolites, nucleotides and small regulatory molecules between cells (1,2). The basic unit of gap junctions is the connexon, which consists of a hexameric arrangement of transmembrane proteins called connexins. Connexins are members of a multigene family and multiple connexins are expressed in tissue specific patterns. Connexin 26 (Cx26), originally identified by subtractive hybridization, is one such connexin that is expressed in normal human mammary ductal epithelial cells but was reported to be absent in some breast cancer cell lines (3).

Loss of communication via gap junctions appears to play a role in oncogenesis with a number of connexins implicated in tumor suppression (1–5). In vivo and in vitro studies have shown that restoration of connexin expression and functional communication in tumor cells retards tumor cell growth and restores normal phenotype (2). Transfection of the Cx26 gene into gap junction-deficient human cancer cell lines reduced their growth rate and tumorigenicity and thus Cx26 can be considered a putative tumor suppressor gene (6,7).

DNA methylation occurs almost exclusively at cytosines located within CpG dinucleotides. However, clusters of CpG dinucleotides known as CpG islands, which are associated with the promoter region of many genes, are generally unmethylated. The methylation of a CpG island in the promoter region of a gene is associated with the transcriptional silencing of that gene (8,9). Methylation contributes to the repression of the inactivated genes as the genes can be reactivated by inhibitors of DNA methylation (10).

Methylation of promoter associated CpG islands is a mechanism for transcriptional silencing of a number of tumor suppressor genes during breast cancer development including the p16, ECAD and BRCA1 genes (11–14). Many of these methylated genes are known to be important in the biology of breast cancer and the re-introduction of a functional copy into a cell line reverses the tumorigenicity. In this report, we assess the methylation status of the Cx26 CpG promoter region in breast cancer biopsies and breast cancer cell lines using methylation-specific single-stranded conformational analysis (MS-SSCA) (15) and genomic sequencing (16).


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Tissue samples and cell lines
Breast cancers from 20 patients with no family history of breast cancer (`sporadic' cancers) and eight breast cancer cell lines (Hs578t, MDA-MB-231, MDA-MB-453, MDA-MB-468, MCF7, T47D, PMC-42 and ZR-75-1) were used for methylation analyses. The cell lines were maintained in RPMI or DMEM (Life Technologies Inc., Rockville, MD) supplemented with 10% fetal calf serum (Commonwealth Serum Laboratories, Melbourne, Australia) and glutamine. Cultures were kept at 37°C in a humidified 5% CO2 atmosphere.

DNA/RNA extraction
Genomic DNA was prepared by resuspending 5–20x106 cells or 10 µm sections of frozen biopsies in TES (10 mM Tris–HCl pH8.0, 1mM EDTA, 100mM NaCl) with 300µg Proteinase K and 20% SDS followed by an overnight incubation at 37°C. Then 3 M NaCl was added to precipitate proteins and the supernatant was collected by centrifugation. The DNA was pelleted using absolute ethanol, washed in 70% ethanol, air dried and resuspended in UltraPure water (Fisher Biotec, Perth, Australia). RNA was extracted using Trizol (Life Technologies) according to the manufacturer's directions.

PCR amplification of bisulfite modified DNA
Bisulfite treatment was modified from the method of Bianco et al. (15). Briefly, ~1 µg of genomic DNA was denatured with 0.3 M NaOH for 15 min at 37°C in a final volume of 20 µl. Then 278 µl of 6.24 M urea (Ajax Chemicals, Sydney, Australia) and 4 M sodium bisulfite (Sigma, St Louis, MO) solution and 2 µl of freshly prepared 100 mM hydroquinone (Sigma) were added. The 6.24 M urea/4 M sodium bisulfite solution was made by dissolving 7.5 g urea in 10 ml of sterile water followed by 7.6 g of sodium bisulfite, adjusting the pH to 5 with 10 M sodium hydroxide and adding sterile water to a final volume of 20 ml. The final concentrations of the urea, sodium bisulfite and hydroquinone were 5.8 M, 3.7 M and 6.67 mM, respectively.

The DNA was subjected to 20 cycles at 55°C for 15 min followed by denaturation at 95°C for 30 s in a thermocycler essentially as described by Paulin et al. (17). The modified DNA was then desalted using the CONCERT Rapid PCR Purification System (Life Technologies) according to the manufacturer's instructions. A desulfonation step followed where 5.5 µl of 3 M sodium hydroxide was added and the DNA incubated at 37°C for 15 min. The sample was then co-precipitated with 5 µg/µl molecular biology grade mussel glycogen (Roche, Mannheim, Germany) in sodium acetate/ethanol at –20°C for 16 h. The pellet was then resuspended in 50 µl of Ultrapure water and stored at 4°C until used.

Primers were designed to amplify both bisulfite modified methylated and unmethylated DNA but not unmodified DNA. If CpG sites could not be avoided, the CpG sites were placed as far as possible to the 5' end of the primer. The target fragment of 245 bp (–128 to + 119) spans 30 CpG dinucleotides, 15 of which are upstream of exon 1 (+1 to +160) (GenBank accession no. U43932). The target fragment was amplified using the primer pair: 5'-ATTCGGGAAGTTTTGAGGATTTAGAGGT-3' (sense) and 5'-GTTAAAATCTCTA CGCTAAAACTCCTAC-3' (antisense). PCR was performed in a final volume of 50 µl using 5–10 µl of sodium bisulfite modified DNA, 100 ng of each primer, 2 mM MgCl2 and 0.5 U of HotStarTaq DNA polymerase in buffer supplied by the manufacturer (Qiagen, Valencia, CA). The first cycle was preceded by a polymerase activation step at 95°C for 15 min. Amplification was performed for 45 cycles with an initial 10 cycles of 94°C for 1 min, touch down annealing temperature step from 72 to 62°C for 45 s and 72°C for 45 s followed by 35 cycles of 94°C for 1 min, 62°C for 1 min and 72°C for 1 min. Ten microliters of the PCR product were electrophoresed at 100 V for 30 min on a 1.6% agarose gel to verify amplification.

Single-stranded conformational analysis and sequencing
SSCA and sequencing of the target fragments was according to Bianco et al. (15). Gels were stained with SYBR Gold (Molecular Probes, Eugene, OR) and visualized on an UV transilluminator. Bands to be sequenced were stabbed with a pipette tip and placed directly into a pre-mixed PCR solution using primers as above. Amplification was performed for 35 cycles at 94°C for 1 min, 60°C for 1 min and 72°C for 1 min. PCR products were cycle sequenced using Big Dye Terminator Cycle Sequencing kit (Applied Biosystems, Foster City, CA) and an ABI 373 automated DNA sequencer.

5-aza-2'-Deoxycytidine treatment
MDA-MB-453 cells were plated in 10 cm Petri dishes at a density of 5x105 cells/ml. After 6 h, 5-aza-2'-deoxycytidine (Sigma) at a final concentration of 1 µM in MilliQ water was added to the medium for 24 h. The cells were then washed with phosphate-buffered saline and fresh medium was added. The treated cells were harvested after 48, 72 and 96 h for reverse transcription polymerase chain reaction (RT–PCR).

RT–PCR
Between 100 ng and 1 µg of total RNA was reversed transcribed using 500 ng of random hexamers (Amersham Pharmacia, Uppsala, Sweden) and 200 U MMLV reverse transcriptase (Life Technologies) for first strand cDNA synthesis in a total volume of 50 µl. A 2 µl aliquot of the cDNA was used for PCR amplification of Cx26 with a 242 bp target sequence spanning intron 1 using the primers: 5'-GCAGAGACCCCAACGCCGAGAC-3' and 5'-GCAGACAAAGTCGGCCTGCTCATC-3'. The ubiquitously expressed nucleoporin 98 gene (NUP98) (18) was used for the positive control. The primers N1428F (5'-GGCATCTTTGTTTGGGAACAACC-3') and N1681R (5'-CCTTCTTCTTA-GGGTCTGACATC-3') gave a product of 250 bp. All RT–PCRs included negative controls where the reverse transcriptase enzyme was omitted.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Cx26 methylation in sporadic breast tumors and breast cancer cell lines
The Cx26 promoter is found in a large CpG island. We selected a 245 bp section of this island spanning the promoter region for further analysis by MS-SSCA (Figure 1Go). We assessed the methylation status of 20 sporadic breast tumors and eight breast cancer cell lines (Figure 2Go). From the 20 tumor samples, we were able to obtain matched tumor and normal samples for seven specimens. MS-SSCA of the 245 bp PCR product showed 11 of 20 (55%) patients and one of eight (12.5%) breast cancer cell lines with variant bands indicative of methylation.



View larger version (6K):
[in this window]
[in a new window]
  		Fig. 1. Schematic diagram of the promoter region of Cx26. Vertical lines correspond to each CpG dinucleotide pair. The location of exon 1 (solid bar), the CpG island (hatched bar), the region undergoing MS-SSCA (unfilled bar) and Sp1 sites (S) are indicated.

 



View larger version (154K):
[in this window]
[in a new window]
  		Fig. 2. (A) MS-SSCA of breast cancer patients. Breast tumor DNA from 20 patients was modified by sodium bisulfite treatment. Primers were designed to amplify both methylated and unmethylated Cx26 promoter regions. Variant bands are indicated by arrow heads. In the case of tumor samples six and 20, the decreased fidelity of the reproduced image obscured the variant bands. The multiple variant bands in patient 15 marked by the arrowheads correspond reading downwards to bands I–VI as discussed in the text. (B) MS-SSCA of breast cancer cell lines. Lane 1, Hs578t; lane 2, MDA-MB-231; lane 3, MDA-MB-453; lane 4, MDA-MB-468; lane 5, MCF7; lane 6, T47D; lane 7, PMC-42; lane 8, ZR-75-1.

 
Aberrant bands were individually sequenced to ascertain the degree of methylation of the target region (Figure 3Go). We were able to confidently assess the methylation status of 26 CpG dinucleotides between positions – 93 and +91. All eight breast cancer cell lines were sequenced. MDA-MB-453 was fully methylated across the entire target region whereas no methylation was observed with the other cell lines.



View larger version (43K):
[in this window]
[in a new window]
  		Fig. 3. Methylation patterns of bands from aberrantly methylated breast tumors and the breast cancer cell line MDA-MB-453 after MS-SSCA. All patient tumors were histologically assessed as ductal carcinomas except for tumor 19 (lobular carcinoma). Sequencing of these bands revealed a heterogenous methylation pattern for the patients examined. Unmethylated CpG dinucleotides are indicated by an open circle; methylated CpG dinucleotides are indicated by a closed circle. (The first and last two CpG dinucleotides were omitted as we were unable to determine fidelity of the sequencing data at these regions.)

 
Variable patterns of methylation were observed between patients. Patients 9 and 15 exhibited methylation on virtually every CpG dinucleotide across the target region whereas some other patients showed fairly light methylation. There was little consensus in the patterns seen in different patients, although the CpG dinucleotides at –81 and –93 were methylated in most tumors.

A few patients showed multiple bands. We examined patient 15 in greater detail by sequencing each of the six individual variant bands. Bands I–V were heavily methylated while band VI was lightly methylated. The methylation patterns of bands III and IV were identical and probably arose from the opposite strands of the same sequence (Figure 3Go).

RT–PCR
RT–PCR showed that all breast cancer cell lines expressed Cx26 except MDA-MB-453 (Figure 4Go). The degree of expression by the non-methylated cell lines was variable after 30 cycles. MDA-MB-453 was the only cell line not to express Cx26 and this remained the case when amplification was extended to 45 cycles.



View larger version (68K):
[in this window]
[in a new window]
  		Fig. 4. RT–PCR of seven breast cancer cell lines examined for Cx26 expression. Lane 1, Hs578t; lane 2, MDA-MB-231; lane 3, MDA-MB-453; lane 4, MDA-MB-468; lane 5, MCF7; lane 6, T47D; lane 7, PMC-42; lane 8, ZR-75–1. Only MDA-MB-453 (lane 3) did not express Cx26. All cell lines expressed the housekeeping gene NUP98 which was used to verify RNA integrity and successful RT–PCR.

 
We examined the expression levels of Cx26 on the seven fresh tumor/normal pairs (Figure 5Go). Four of the seven tumor samples showed variant methylation patterns but lack of Cx26 expression in the tumor sample was observed in only patients 15 and 19. The tumor samples were grossly dissected and the expression of Cx26 may have reflected the presence of normal breast epithelium. All samples were positive for the housekeeping gene NUP98 used to verify RNA integrity and successful reverse transcription.



View larger version (50K):
[in this window]
[in a new window]
  		Fig. 5. RT–PCR of patients 14–20 from whom fresh frozen tumor (T) and normal (N) material was available. Only the tumor from patient 15 with variant methylated bands as shown by MS-SSCA did not express Cx26. All tumor samples expressed NUP98 (data not shown).

 
Demethylation of MDA-MB-453 by 5-aza-2'-deoxycytidine
Re-expression of Cx26 in the hypermethylated breast cancer cell line MDA-MB-453 was detected after treatment with the demethylating agent, 5-aza-2'-deoxycytidine (Figure 6Go). The relative degree of expression increased with recovery time after the initial exposure to 5-aza-2'-deoxycytidine. Cells harvested at 48 and at 72 h after treatment showed increasing levels of Cx26 re-expression with cells sampled at 96 h showing the strongest amplification signal (Figure 6Go lane 5).



View larger version (75K):
[in this window]
[in a new window]
  		Fig. 6. Expression of Cx26 in the breast cancer cell line MDA-MB-453 after exposure to 5-aza-2' deoxycytidine was determined by RT–PCR using primers which amplified a 242 bp fragment. The integrity of RNA and success of the reverse transcription was verified by RT–PCR using a NUP98 primer pair that yielded a 250 bp fragment. Lanes 1 and 2, untreated MDA-MB-453 cells; lane 3, 48 h; lane 4, 72 h; lane 5, 72 h. Cx26 re-expression after the appropriate recovery times; RT–PCR controls of specimens from corresponding samples using the NUP98 primer pair.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
Cx26 has been implicated as a tumor suppressor gene for its part in gap junction intercellular communication, as reviewed by Locke (2). Lee et al. (3) showed the expression of Cx26 in normal human mammary epithelial but not breast cancer cells. Significantly, they established that lack of expression was not due to any physical loss of the gene. Transfection of the Cx26 gene into the gap junction-deficient human cancer cell lines HeLa (6) and MDA-MB-435 (7) reduced their growth rate and tumorigenicity. We therefore examined whether Cx26 was silenced by methylation in breast tumors.

We found Cx26 promoter methylation in 11 out of 20 (55%) of the breast cancer patients examined. Considerable inter-patient variation in the methylation patterns in the Cx26 promoter region was observed. Some tumors showed hypermethylation of the region with almost every site in the promoter methylated whereas other tumors showed only partial methylation over the region examined. However, methylation tended to occur in discrete clusters of CpG dinucleotides along the target fragment in each tumor. These areas of clustering were either the 5', mid or 3' end of the fragment. Tu and Kiang (19) identified the region around the –93 and particularly the –81 Sp1 sites as being critical for transcription (Figure 1Go). Our sequencing data showed that position –81 was methylated in seven of these samples (Patients 4, 6, 7, 9, 13, 15 and 19), representing the single most common methylated site whereas position –93 was methylated in six of the samples (Patients 4, 6, 9, 13, 15 and 20). Intra-tumor variation in methylation patterns was most apparent in patient 15. Of the six bands sequenced in patient 15, three were methylated at position –93 and four at position –81.

Heterogeneous methylation patterns have also been observed in other genes, e.g. the RB promoter in retinoblastomas (20) and the p15 promoter in acute myeloid leukaemia (21). Stirzaker et al. (20) concluded that hypermethylation of the entire promoter region was unnecessary for silencing of the RB gene. They proposed that heterogeneity in the patterns of RB promoter methylation was due to a lack of fidelity in the maintenance of the original pattern (20). Cells sampled at various stages of tumor progression or cells undergoing clonal evolution could also give rise to heterogeneous methylation patterns.

Lack of Cx26 expression by RT–PCR was detected in only two of the four methylated samples (patients 15 and 19; Figure 5Go). As none of the tumor samples had been microdissected, it was possible that contaminating RNA from normal breast epithelium had also been extracted. We were able to demonstrate for one paired sample (patient 19) that Cx26 was expressed in the normal but not the methylated tumor. However, no expression of Cx26 was observed in the `normal' adjacent breast tissue of patient 15. This suggests that inactivation of Cx26 may precede methylation in some patients.

Methylation as detected by aberrant MS-SSCA banding patterns was present in one of eight breast cancer cell lines. Genomic sequencing confirmed that MDA-MB-453 was hypermethylated at the Cx26 promoter while the other cell lines were unmethylated. Treatment of MDA-MB-453 with the demethylating agent 5-aza-2'-deoxycytidine showed that re-expression of Cx26 was inducible after a brief exposure to 5-aza-2'-deoxycytidine. This directly implicates hypermethylation of the promoter region with the lack of Cx26 expression in MDA-MB-453.

The comparative rarity of Cx26 promoter methylation in breast cancer cell lines is notable in the light of reports showing widespread CpG island methylation in cultured cell lines (22–24). The cell lines reported by Lee et al. (3) to lack Cx26 expression by northern analysis were MCF7, MDA-MB-231, MDA-MB-468, T47D and ZR-75-1. In this study we found these cell lines to be unmethylated. Moreover, our RT–PCR analysis showed expression of Cx26 in the above cell lines as well as Hs578t and PMC-42 at 30 and 45 cycles. Lee et al. reported that treatment of two of the cell lines (MDA-MB-231 and ZR-75-1) with the phorbol ester PMA, which activates protein kinase C, resulted in re-expression of Cx26 (3). However, our results indicate that what was seen was probably an up-regulation.

Our results are also at variance with a recent report examining Cx26 methylation in breast cancer cell lines (25). These showed undetectable expression in the MCF7 and T47D cell lines by northern analysis. Hypermethylation was observed in MCF7 but 5-aza-2'-deoxycytidine treatment did not induce Cx26 expression. In contrast, our RT–PCR analysis easily detected Cx26 expression in MCF7 and showed low levels of expression in T47D. The very sensitive MSP (methylation-specific PCR) methodology used by Singal et al. (25) may have detected small methylated subpopulations of MCF7 and this would explain why 5-aza-2'-deooxycytidine treatment did not lead to re-expression.

Some of these findings are reminiscent of those for the tumor suppressor gene, BRCA1. Expression of BRCA1 is low or absent in most ductal carcinomas and cell lines (26). Methylation occurs in ~20% of patients but much less frequently in cell lines (13,27–30). This suggests that while methylation is an important mechanism leading to inactivation of tumor suppressor genes, other mechanisms may also play a pivotal role in preventing gene transcription. For the gelsolin gene, histone deacetylation seems to be the primary mechanism in repressing gene expression (31), whereas methylation is the more important inactivation mechanism for the MLH1, TIMP3, p15 and p16 genes (32) and this also seems to be the case for the Cx26 gene.

Methylation of the promoter CpG island region is a common epigenetic mechanism for transcriptional silencing of tumor suppressor genes (8,9). The alterations in methylation seen during the development of breast cancer are the genesis for variation and the basis for selection of tumorigenic cells that are able to proliferate in an environment conducive to their survival. In breast cancer, a number of tumor suppressor genes have been shown to be methylated in a significant proportion of tumors. This report now adds the candidate tumor suppressor gene Cx26 to this list of genes.


   Notes
 
1 Present addresses: Department of Pathology, Peter MacCallum Cancer Institute, Locked Bag 1, A'Beckett Street, East Melbourne, VIC 8006, Australia and Back

2 Department of Otolaryngology, The Queen Elizabeth Hospital, Woodville, SA 5011, Australia Back

3 To whom correspondence should be addressed Email: alexander.dobrovic{at}adelaide.edu.au or lorwai.tan{at}adelaide.edu.au Back


   Acknowledgments
 
We thank Dr Damian Hussey for providing NUP98 primers. We thank Professor R.E.Sage and Mr David Walsh for their support. We thank Dr Sally-Anne Stephenson for critically reading this manuscript and Ms Margaret Colbeck for assistance with obtaining patient records. L.W.T. is supported by the Esther Caldwell Cook Postdoctoral Research Fellowship. This work was funded by grants from National Health and Medical Research Council (to A.D.) and The Queen Elizabeth Hospital Research Foundation (to A.D. and L.W.T.).


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Laird,D.W., Fistouris,P., Batist,G., Alpert,L., Huynh,H.T., Carystinos,G.D. and Alaoui-Jamali,M.A. (1999) Deficiency of Connexin 43 gap junctions is an independent marker for breast tumours. Cancer Res., 59, 4101–4110.
  2. Locke,D. (1998) Gap junctions in normal and neoplastic mammary glands. J. Pathol., 186, 343–349.[Web of Science][Medline]
  3. Lee,S.W., Tomasetto,C., Paul,D., Keyomarsi,K. and Sager,R. (1992) Transcriptional downregulation of gap-junction proteins blocks junctional communication in human mammary tumour cell lines. J. Cell Biol., 118, 1213–1221.[Abstract/Free Full Text]
  4. Jamieson,S., Going,J.J., D'arcy,R. and George,W.D. (1998) Expression of gap junction proteins Connexin 26 and Connexin 43 in normal human breast and in breast tumours. J. Pathol., 184, 37–43.[Web of Science][Medline]
  5. Li,G.-Y., Lin,H.H., Zheng,J.T. and Kiang,D.T. (1998) Gap junction Cx26 gene modulation by phorbol esters in benign and malignant human mammary cells. Gene, 209, 139–147.[Web of Science][Medline]
  6. Mensil,M., Krutovskikh,V., Piccoli,C., Elfgang,C., Traub,O., Willecke,K. and Yamasaki,H. (1995) Negative growth control of HeLa cells by connexin genes: connexin species specificity. Cancer Res., 55, 629–639.
  7. Hirschi,K.K., Xu,C.E., Tsukamoto,T. and Sager,R. (1996) Gap junction genes Cx26 and Cx43 individually suppress the cancer phenotype of human mammary carcinoma cells and restore differentiation potential. Cell Growth Diff., 7, 861–870.[Abstract]
  8. Jones,P.A. and Laird,P.W. (1999) Cancer epigenetics comes of age. Nature Genet., 21, 163–167.[Web of Science][Medline]
  9. Baylin,S.B., Herman,J.G., Graff,J.R., Vertino,P.M. and Issa,J.-P. (1998) Alterations in DNA methylation: a fundamental aspect of neoplasia. Adv. Cancer Res., 72, 141–196.[Web of Science][Medline]
  10. Mohandas,T., Sparkes,R.S. and Shapiro,L.J. (1981) Reactivation of an inactive human X chromosome: evidence for X inactivation by DNA methylation. Science, 211, 393–396.[Abstract/Free Full Text]
  11. Herman,J.G., Merlo,A., Mao,L., Lapidus,R.G., Issa,J.-P.J., Davidson,N.E., Sidransky,D. and Baylin,S.B. (1995) Inactivation of the CDKN2/p16/MTS1 gene is frequently associated with aberrant DNA methylation in all common human cancers. Cancer Res., 55, 4525–4530.[Abstract/Free Full Text]
  12. Yoshiura,K., Kanai,Y., Ochiai,A., Shimoyama,Y., Sugimura,T. and Hirohashi,S. (1995) Silencing of the E-cadherin invasion-suppressor gene by CpG methylation in human carcinomas. Proc. Natl Acad. Sci., 92, 7416–7419.[Abstract/Free Full Text]
  13. Dobrovic,A. and Simpfendorfer,D. (1997) Methylation of the BRCA 1 gene in sporadic breast cancer. Cancer Res., 57, 3347–3350.[Abstract/Free Full Text]
  14. Yang,X., Yan,L. and Davidson,N.E. (2001) DNA methylation in breast cancer. Endocrine-Related Cancer, 8, 115–127.[Abstract]
  15. Bianco,T., Hussey,D. and Dobrovic,A. (1999) Methylation-sensitive, single-strand conformation analysis (MS-SSCA): a rapid method to screen for and analyse methylation. Hum. Mutat., 14, 289–293.[Web of Science][Medline]
  16. Clark,S.J. and Frommer,M. (1997) Bisulphite genomic sequencing of methylated cytosines. In Taylor,G.R. (ed.) Laboratory Methods for the Detection of Mutations and Polymorphisms. CRC Press, Boca Raton, FL, pp. 151–162.
  17. Paulin,R., Grigg,G.W., Davey,M.W. and Piper,A.A. (1998) Urea improves efficiency of bisulfite-mediated sequencing of 5'-methylcytosine in genomic DNA. Nucleic Acids Res., 26, 5009–5010.[Abstract/Free Full Text]
  18. Hussey,D.J., Nicola,M., Moore,S., Peters,G.B. and Dobrovic,A. (1999) The (4;11) (q21;p15) translocation fuses the NUP98 and RAP1GDS1 genes and is recurrent in T-cell acute lymphocytic leukemia. Blood, 94, 2072–2079.[Abstract/Free Full Text]
  19. Tu,Z.J. and Kiang,D.T. (1998) Mapping and characterization of the basal promoter of the human Connexin 26 gene. Biochem. Biophys. Acta, 1443, 169–181.[Medline]
  20. Stirzaker,C., Millar,D.S., Paul,C.L., Warnecke,P.M., Harrison,J., Vincent, P.C., Frommer,M. and Clark,S.J. (1997) Extensive DNA methylation spanning the Rb promoter in retinoblastoma tumours. Cancer Res., 57, 2229–2237.[Abstract/Free Full Text]
  21. Dodge,J.E., List,A.F. and Futscher,B.W. (1998) Selective variegated methylation of the p15 CpG island in acute myeloid leukaemia. Int. J. Cancer, 78, 561–567.[Web of Science][Medline]
  22. Antequera,F., Boyes,J. and Bird,A. (1990) High levels of de novo methylation and altered chromatin structure at CpG islands in cell lines. Cell, 62, 503–514.[Web of Science][Medline]
  23. Huang,T.H.-M., Perry,M.R. and Laux,D.E. (1999) Methylation profiling of CpG islands in human breast cancer cells. Hum. Mol. Genet., 8, 459–470.[Abstract/Free Full Text]
  24. Smiraglia,D.J., Rush,L.J., Fruhwald,M.C. et al. (2001) Excessive CpG island hypermethylation in cancer cell lines versus primary human malignancies. Hum. Mol. Genet., 10, 1413–1419.[Abstract/Free Full Text]
  25. Singal,R., Tu,Z.J., Vanwert,J.M., Ginder,G.D. and Kiang,D.T. (2000) Modulation of the Connexin 26 tumour suppressor gene expression through methylation in human mammary epithelial cell lines. Anticancer Res., 20, 59–64.[Web of Science][Medline]
  26. Thompson,M.E., Jensen,R.A., Obermiller,P.S., Page,D.L. and Holt,J.T. (1995) Decreased expression of BRCA1 accelerates growth and is often present during sporadic breast cancer progression. Nature Genet., 9, 444–450.[Web of Science][Medline]
  27. Bianco,T., Chenevix-Trench,G., Walsh,D.C.A., Cooper,J.E. and Dobrovic,A. (2000) Tumour-specific distribution of BRCA1 promoter region methylation supports a pathogenetic role in breast and ovarian cancer. Carcinogenesis, 21, 147–151.[Abstract/Free Full Text]
  28. Catteau,A., Harris,W.H., Xu,C.-F. and Solomon,E. (1999) Methylation of the BRCA1 promoter region in sporadic breast and ovarian cancer: correlation with disease characteristics. Oncogene, 18, 1957–1965.[Web of Science][Medline]
  29. Esteller,M., Silva,J.M., Dominguez,G. et al. (2000) Promoter Hypermethylation and BRCA1 inactivation in sporadic breast and ovarian tumours. J. Natl Cancer Inst., 92, 564–569.[Abstract/Free Full Text]
  30. Rice,J.C., Massey-Brown,K.S. and Futscher,B.W. (1998) Aberrant methylation of the BRCA 1 CpG island promoter is associated with decreased BRCA 1 mRNA in sporadic breast cancer cells. Oncogene, 17, 1807–1812.[Web of Science][Medline]
  31. Mielnicki,L.M., Ying,A.M., Head,K.L., Asch,H.L. and Asch,B.B. (1999) Epigenetic regulation of gelsolin expression in human breast cancer cells. Exp. Cell Res., 249, 161–176.[Web of Science][Medline]
  32. Cameron,E.E., Bachman,K.E., Myohanen,S., Herman,J.G. and Baylin,S.B. (1999) Synergy of demethylation and histone deacetylase inhibition in the re-expression of genes silenced in cancer. Nature Genet., 21, 103–107.[Web of Science][Medline]
Received October 31, 2001; accepted November 2, 2001.


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Nucleic Acids ResHome page
L. S. Kristensen, T. Mikeska, M. Krypuy, and A. Dobrovic
Sensitive Melting Analysis after Real Time- Methylation Specific PCR (SMART-MSP): high-throughput and probe-free quantitative DNA methylation detection
Nucleic Acids Res., April 1, 2008; 36(7): e42 - e42.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
R. A. Hinshelwood, L. I. Huschtscha, J. Melki, C. Stirzaker, A. Abdipranoto, B. Vissel, T. Ravasi, C. A. Wells, D. A. Hume, R. R. Reddel, et al.
Concordant Epigenetic Silencing of Transforming Growth Factor- Signaling Pathway Genes Occurs Early in Breast Carcinogenesis
Cancer Res., December 15, 2007; 67(24): 11517 - 11527.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Gastrointest. Liver Physiol.Home page
K. K. Nagothu, A. K. Rishi, R. Jaszewski, O. Kucuk, and A. P. N. Majumdar
Folic acid-mediated inhibition of serum-induced activation of EGFR promoter in colon cancer cells
Am J Physiol Gastrointest Liver Physiol, September 1, 2004; 287(3): G541 - G546.
[Abstract] [Full Text] [PDF]

Home page
 Toxicol SciHome page
J. K. Chipman, A. Mally, and G. O. Edwards
Disruption of Gap Junctions in Toxicity and Carcinogenicity
Toxicol. Sci., February 1, 2003; 71(2): 146 - 153.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (36)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Tan, L.-w.
Right arrow Articles by Dobrovic, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Tan, L.-w.
Right arrow Articles by Dobrovic, A.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?