Retinoid-induced histone deacetylation inhibits telomerase activity in estrogen receptor-negative breast cancer cells

Abstract

Background: Multiple mechanisms regulate cancer-associated telomerase activity at the level of human telomerase reverse transcriptase (hTERT) transcription which may serve as novel targets for anticancer approaches.

Materials and methods: The effects of prolonged all-trans retinoic acid (ATRA) exposure on hTERT regulation in estrogen receptor-negative SK-BR-3 breast cancer cells were examined.

Results: ATRA had a profound effect on the morphology and proliferation rate of the SK-BR-3 cells. ATRA also hindered the ability of these cancer cells to grow independently, rendering them more like normal somatic cells. The effect of ATRA on the decrease of telomerase activity was found to be associated with a rapid decrease in histone H3-lysine 9 acetylation (H3-K9-Ac) of the hTERT promoter. Extended-exposure to ATRA in these cells also caused the initiation of a putative compensatory mechanism, counteracting the induced surge in apoptosis.

Conclusion: A rapid decrease of H3-K9 acetylation at the hTERT promoter could be an important mechanism by which ATRA shuts down telomerase activity and mediates its antitumor effects in estrogen receptor-negative breast cancer cells.

Figure 1

Effect of ATRA exposure on morphology and proliferation in cultured breast cancer cells. Representative photographs, at 200X magnification of SK-BR-3 cells not exposed to ATRA (A) or exposed to ATRA for 6 (B) or 12 days (C). Black arrows in (B) indicate cells undergoing apoptosis. (D) Graphical representation of the means of cell counts taken during 12-day ATRA treatment. Means are +/− s.d. of triplicate experiments. (E) Representative SK-BR-3 cells colony formation.

Figure 2

Effect of ATRA on histone acetylation and DNA methylation at the hTERT promoter and telomerase activity. (A) Chromatin immunoprecipitation. Chromatin complexes were precipitated with anti-H3-K9-Ac and resultant purified DNA was amplified by PCR with primers specific for the hTERT promoter. “Input” represents 1% of samples removed prior to the immunoprecipitation and amplified by hTERT primers. (B) Schematic of the 24 CpG sites in the hTERT promoter region represented by boxes. Underdetermined: methylation status could not be determined (which generally indicates partially methylated CpGs). (C) TRAP assay. The height and intensity of the ladder correlates with telomerase activity. IC: internal control.

Figure 3

Effect of ATRA on anchorage-independent growth. (A) Representative colonies in soft agar assay. (B) Quantification of colony counts. Treatment number: separate ATRA treatments at the indicated concentrations. Means +/− s.d. of triplicate experiments.

Figure 4

Effect of ATRA on cellular apoptosis measured by FACS. (A) The lower left quadrant represents live cells, the upper and lower right quadrants represent late and early apoptotic cells respectively and the upper left quadrant indicates dead (necrotic) cells. (B) Graphical representation of cell numbers in the early and late apoptotic quadrants.