Caspase 8 and maspin are downregulated in breast cancer cells due to CpG site promoter methylation.
BACKGROUND
Epigenetic changes associated with promoter DNA methylation results in silencing of several tumor suppressor genes that lead to increased risk for tumor formation and for progression of the cancer.


METHODS
Methylation specific PCR (MSP) and bisulfite sequencing were used for determination of proapoptotic gene Caspase 8 (CASP8) and the tumor suppressor gene maspin promoter methylation in four breast cancer and two non-tumorigenic breast cell lines. Involvement of histone H3 methylation in those cell lines were examined by CHIP assay.


RESULTS
The CpG sites in the promoter region of CASP8 and maspin were methylated in all four breast cancer cell lines but not in two non-tumorigenic breast cell lines. Demethylation agent 5-aza-2'-deoxycytidine (5-aza-dc) selectively inhibits DNA methyltransferases, DNMT3a and DNMT3b, and restored CASP8 and maspin gene expression in breast cancer cells. 5-aza-dc also reduced histone H3k9me2 occupancy on CASP8 promoter in SKBR3cells, but not in MCF-7 cells. Combination of histone deacetylase inhibitor Trichostatin A (TSA) and 5-aza-dc significant decrease in nuclear expression of Di-methyl histone H3-Lys27 and slight increase in acetyl histone H3-Lys9 in MCF-7 cells. CASP8 mRNA and protein level in MCF-7 cells were increased by the 5-aza-dc in combination with TSA. Data from our study also demonstrated that treatment with 5-FU caused a significant increase in unmethylated CASP8 and in CASP8 mRNA in all 3 cancer lines.


CONCLUSIONS
CASP8 and maspin expression were reduced in breast cancer cells due to promoter methylation. Selective application of demethylating agents could offer novel therapeutic opportunities in breast cancer.
Division of Cancer Research and Training, Department of Internal Medicine, Charles R, Drew University of Medicine and Science, Los Angeles, CA 90059, USA.
Yanyuan
Y
Wu
Monica
M
Alvarez
Dennis J
DJ
Slamon
Phillip
P
Koeffler
Jaydutt V
JV
Vadgama
D001943:Breast Neoplasms / Q000235:genetics* / Q000378:metabolism*; D053181:Caspase 8 / Q000096:biosynthesis*; D045744:Cell Line, Tumor; D047369:Chromatin Immunoprecipitation; D018899:CpG Islands; D019175:DNA Methylation; D044127:Epigenesis, Genetic; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology; D008954:Models, Biological; D011401:Promoter Regions, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015843:Serpins / Q000096:biosynthesis*
D006877:Hydroxamic Acids; C085344:SERPIN-B5; D015843:Serpins; C012589:trichostatin A; D053181:Caspase 8