Hypermethylation of calcitonin gene regulatory sequences in human breast cancer as revealed by genomic sequencing

Hakkarainen, M; Wahlfors, Jarmo; Myöhänen, Sanna; Hiltunen, Mikko; Eskelinen, Matti; Johansson, Risto; Jänne, Juhani

doi:10.1002/(sici)1097-0215(19961220)69:6<471::aid-ijc9>3.0.co;2-1

Cited by 16 publications

(8 citation statements)

References 21 publications

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“…By using this method, we showed that the calcitonin gene was distinctly hypermethylated in breast cancer (Hakkarainen et al, 1996). Here, we show that the promoter of the calcitonin gene is likewise hypermethylated in colorectal cancer.…”

Section: Discussionmentioning

confidence: 83%

“…For the amplification, 100 ng of recently modified DNA was used in a 50-jl PCR mixture containing: 20 pmol of the upper primer (5'-GATAGTTTTA-GAAGTAAGAGTTAGATTTAAG-3'), 20 pmol of the lower primer (5'-CCTAACTACCCTCAACTTCCC-3'), 1.0 mM magnesium chloride, 0.2 mm dNTPs and 1 x buffer. The conditions for the first PCR cycle were +96°C for 4 min for denaturation and British Journal of Cancer (1997) 76(9) The amplification was carried out according to our recently developed method (Hakkarainen et al, 1996). In brief, primers for first PCR were selected to cover a 517-nucleotide region (GenBank accession number X15943).…”

Section: Dna Extraction and Bisulphite Modificationmentioning

confidence: 99%

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Hypermethylation of the WT1 and calcitonin gene promoter regions at chromosome 11p in human colorectal cancer

Hiltunen

Koistinaho²,

Alhonen³

et al. 1997

Br J Cancer

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Summary The short arm of the chromosome 11, known to harbour a number of putative and established tumour-suppressor genes, is frequently hypermethylated in various human neoplasms. We subjected the promoter regions of two genes residing at lip, namely the tumour-suppressor gene WT1 (Wilms' tumour gene) (11p13) and the calcitonin gene (11p15.5), to methylation analysis in human sporadic colorectal cancer using genomic sequencing. Both genes showed significant hypermethylation of CpG sites within their promoter regions in adenomas and carcinomas compared with normal colonic mucosa. Although the WT1 promoter region was significantly hypermethylated, two CpG sites located in Spl motifs were unmethylated in the majority of cases (68-74% of carcinomas). The expression of WT1 gene, as revealed by in situ hybridization, showed no differences between normal colonic mucosa and malignant carcinoma. Together with earlier observations, our present results support the view that the short arm of human chromosome 11 is subjected to widespread regional hypermethylation in various human malignancies.

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Section: Discussionmentioning

confidence: 83%

Section: Dna Extraction and Bisulphite Modificationmentioning

confidence: 99%

Hypermethylation of the WT1 and calcitonin gene promoter regions at chromosome 11p in human colorectal cancer

Hiltunen

Koistinaho²,

Alhonen³

et al. 1997

Br J Cancer

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“…A number of studies have found both tumor and metastasis suppressor genes to be abnormally methylated in breast cancer (11,(23)(24)(25)(26)(27)(28). Among these, E-cadherin and MDGI (mammary derived growth factor inhibitor) are the most frequently hypermethylated genes, with Ϸ50% of breast tumors exhibiting these epigenetic alterations (23,24).…”

Section: Discussionmentioning

confidence: 99%

High frequency of hypermethylation at the 14-3-3 σ locus leads to gene silencing in breast cancer

Ferguson

Evron

Umbricht

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

415

418

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Expression of 14-3-3 () is induced in response to DNA damage, and causes cells to arrest in G2. By SAGE (serial analysis of gene expression) analysis, we identified as a gene whose expression is 7-fold lower in breast carcinoma cells than in normal breast epithelium. We verified this finding by Northern blot analysis. Remarkably, mRNA was undetectable in 45 of 48 primary breast carcinomas. Genetic alterations at such as loss of heterozygosity were rare (1͞20 informative cases), and no mutations were detected (0͞34). On the other hand, hypermethylation of CpG islands in the gene was detected in 91% (75͞82) of breast tumors and was associated with lack of gene expression. Hypermethylation of is functionally important, because treatment of -non-expressing breast cancer cell lines with the drug 5-aza-2-deoxycytidine resulted in demethylation of the gene and synthesis of mRNA. Breast cancer cells lacking expression showed increased number of chromosomal breaks and gaps when exposed to ␥-irradiation. Therefore, it is possible that loss of expression contributes to malignant transformation by impairing the G 2 cell cycle checkpoint function, thus allowing an accumulation of genetic defects. Hypermethylation and loss of expression are the most consistent molecular alterations in breast cancer identified so far.A lthough many studies have identified critical genetic and epigenetic changes that mark the transformation of cells in tissues such as colon, pancreas, and lung, similar studies in breast cancer have met with limited success. In this paper we report the identification of a gene, 14-3-3 (), whose expression is undetectable in 94% (45͞48) of breast tumors.was originally identified as an epithelial-specific marker, HME1, which was down-regulated in a few breast cancer cell lines but not in cancer cell lines derived from other tissue types (1). Later studies showed that protein (also called stratifin) was abundant in differentiated squamous epithelial cells, but decreased by 95% in simian virus 40-transformed epithelial cells and in primary bladder tumors (2-4).We investigated the molecular mechanism underlying the low expression of in breast cancers. We find that genetic alterations such as loss of heterozygosity (LOH) and intragenic mutations are not major contributing mechanisms for lack of expression. Instead, we show that hypermethylation of the CpG-rich region in the gene is associated with its transcriptional silencing in the majority of breast tumors. Treatment of breast cancer cell lines that do not express with the DNA methyltransferase inhibitor, 5-aza-2Ј-deoxycytidine (5-aza-dC), leads to partial demethylation of this CpG island and synthesis of mRNA. Thus, hypermethylation appears to be responsible for silencing of gene expression.Recent studies have shed light on the function of . It was originally identified as a p53-inducible gene that is responsive to DNA damaging agents (5). apparently sequesters the mitotic initiation complex, cdc2-cyclin B1, in the cytoplasm after DNA damage (6). This prevents cdc...

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“…It is clear that a number of speci®c loci are hypermethylated in breast tumours or cell lines. These include the 5' CpG islands of genes encoding calcitonin (Hakkarainen et al, 1996), p16 , estrogen and progesterone receptors (Ottaviano et al, 1994;Ferguson et al, 1995;Lapidus et al, 1996) and E-cadherin (Gra et al, 1995). Methylation-sensitive restriction ®ngerprinting has identi®ed two further hypermethylated loci, HBC-1 and HBC-2 associated with breast cancer (Huang et al, 1997).…”

Section: Discussionmentioning

confidence: 99%

Methylation of the BRCA1 promoter region in sporadic breast and ovarian cancer: correlation with disease characteristics

Catteau¹,

Harris

Xu³

et al. 1999

Oncogene

307

181

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Reduced expression of BRCA1 has been reported in sporadic breast cancer, although the mechanisms underlying this phenomenon remain unclear. Abnormal methylation leading to silencing of tumour suppressor genes has been implicated in tumorigenesis in a wide range of sporadic cancers. Therefore, we sought to determine the frequency of methylation within the BRCA1 promoter region in a large group of sporadic invasive breast (n=96) and ovarian (n=43) carcinomas using Southern analyses. Overall, methylation was detected in 11% of breast cancer cases and in 5% of ovarian tumours. Methylation of the BRCA1 promoter region was strongly correlated with lack of estrogen and progesterone receptor expression. It is clear from the frequency of abnormal methylation of the BRCA1 promoter region, that this cannot be the sole mechanism mediating the reduced expression of BRCA1 that has previously been reported to occur in the majority of invasive sporadic breast tumours. Nevertheless this study suggests that abnormal methylation of the BRCA1 promoter may be important in tumorigenesis in a subset of sporadic breast and ovarian cancers.

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Hypermethylation of calcitonin gene regulatory sequences in human breast cancer as revealed by genomic sequencing

Cited by 16 publications

References 21 publications

Hypermethylation of the WT1 and calcitonin gene promoter regions at chromosome 11p in human colorectal cancer

Hypermethylation of the WT1 and calcitonin gene promoter regions at chromosome 11p in human colorectal cancer

High frequency of hypermethylation at the 14-3-3 σ locus leads to gene silencing in breast cancer

Methylation of the BRCA1 promoter region in sporadic breast and ovarian cancer: correlation with disease characteristics

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