Published in Cancer Detection and Prevention 2000; 24(Supplement 1).

Switch Controlling of PTHrP gene expression: methylation state and transcription factor Ets1 transactivation in breast cancer cell lines

M Rousiere MD 1, C Cataisson 1, J Foley PhD 2, J Taboulet PhD 1, Z Bouizar PhD PharmD 1

1 INSERM 349, Paris, France, 2 Medical Sciences, Indiana University School of Medicine, Bloomington, IN,

AIM: Parathyroid hormone related peptide (PTHrP) is the agent responsible for humoral hypercalcemia of malignancy. PTHrP is commonly overexpressed in breast cancers, however, mechanisms underlying increased PTHrP expression are largely unknown. We investigated the influence of DNA methylation state and trans-acting factors (specifically Ets1) upon regulation of PTHrP gene expression in human breast cancer cell lines. METHODS: The methylation status of PTHrP promoter region in a series of immortalized breast epithelial and breast cancer cell lines was investigated by methylation-sensitive restriction enzyme digestion, and correlated with PTHrP mRNA expression determined by semiquantitative RT-PCR. Next, cell lines were treated with a methylation inhibitor (5-azacytidine) and subsequently PTHrP gene expression was evaluated. Using a series of human 5' PTHrP reporter constructs, critical transacting sequences within the PTHrP gene were identified. RESULTS: In cell lines that failed to express or expressed low levels of PTHrP (S1T6, S1T3, T47D and MCF7), the PTHrP promoter region was hypermethylated, whereas high PTHrP-expressing lines (S2T2 and NS2T2A1) were hypomethylated. Treatment with 5-azaC increased PTHrP gene mRNA levels in the low PTHrP-expressing lines. Transient transfection experiments indicated that an Ets1 site in close proximity to the P3 promoter was critical for high levels of reporter gene activity in the lines that expressed abundant PTHrP mRNA. CONCLUSIONS: Our data provide evidence that methylation may silence the PTHrP gene in human breast cell lines, possibly by preventing access of positive-acting transcription factors to specific regulatory sequences.

KEY WORDS: PTHrP metastasis.

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Paper presented at the International Symposium on Impact of Biotechnology on Cancer Diagnostic & Prognostic Indicators; Geneva, Switzerland; October 28 - 31, 2000; in the section on metastasis & prevention.