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Tumor Biology

, Volume 34, Issue 2, pp 723–733 | Cite as

Gene expression levels of human shelterin complex and shelterin-associated factors regulated by the topoisomerase II inhibitors doxorubicin and etoposide in human cultured cells

  • Masahiro Kato
  • Masahiro Nakayama
  • Minako Agata
  • Kenichi Yoshida
Research Article

Abstract

Human telomerase reverse transcriptase (hTERT) is responsible for telomere elongation, and its activity is strongly related to the expression level of the hTERT gene; however, the transcriptional regulation of telomeric genes, which play a central role in telomere maintenance and protection by facilitating replication and regulating telomerase access, is poorly understood. In this study, we aimed to reveal the changes in the mRNA expression of six components of the shelterin complex and three shelterin complex-associated factors in topoisomerase II inhibitor-treated human cultured cells. Using a quantitative gene expression analysis, we found that a reduction in telomeric repeat-binding factor 1 (TRF1), protection of telomeres (POT1), and TRF1-interacting ankyrin-related ADP-ribose polymerase 1 (TNKS1) mRNAs was observed in etoposide- and doxorubicin-treated HeLa and U-2 OS cells, while an increased TRF2-interacting telomeric protein (RAP1) mRNA level was observed in U-2 OS cells. Furthermore, doxorubicin suppressed TRF1 and POT1 mRNAs in both Saos-2 and WI-38 cells and increased RAP1 mRNA in WI-38 cells. In agreement with the results obtained in the quantitative gene expression analysis in U-2 OS cells, the topoisomerase II inhibitors negatively and positively regulated the POT1 and RAP1 gene promoters, respectively. Taken together, these results suggest the successful identification of unique topoisomerase II inhibitor-inducible telomeric genes and provide mechanistic insight into the regulation of telomeric gene expression by chemotherapeutic agents.

Keywords

Telomere shelterin Gene expression Topoisomerase II inhibitor Doxorubicin Etoposide 

Abbreviations

ALT

Alternative lengthening of the telomere

DSBs

DNA double-strand breaks

GAPDH

Glyceraldehyde-3-phosphate dehydrogenase

hTERT

Human telomerase reverse transcriptase

NHEJ

Nonhomologous end joining

NSCLC

Nonsmall cell lung cancer

PCR

Polymerase chain reaction

PINX1

PIN2-interacting protein 1

POT1

Protection of telomeres

RAP1

TRF2-interacting telomeric protein

RT

Reverse transcription

TERC

Telomerase RNA component

TIN2

TRF1-interacting nuclear factor 2

TNKS1

TRF1-interacting ankyrin-related ADP-ribose polymerase 1

TPP1

POT1- and TIN2-organizing protein

TRF1

Telomeric repeat-binding factor 1

TSA

Trichostatin A

Supplementary material

13277_2012_600_Fig4_ESM.jpg (61 kb)
Supplementary Fig. 1

Gene silencing of TRF2 in U-2 OS-TetOn-short hairpin TRF2 (shTRF2) cells. U-2 OS-TetOn-shTRF2 cells were treated with 1 μg/mL of doxycycline for 7 days or 2 μg/mL of doxycycline for 10 days to induce TRF2 silencing. a Western blot analysis using TRF2 antibody. GAPDH was detected as a loading control. b Senescence-associated β-galactosidase (β-gal) assay was performed with a Senescence Detection kit (BioVision, Mountain View, CA). Representative microscopic appearance of doxycycline-untreated and doxycycline-treated U-2 OS-TetOn-shTRF2 cells (×100). β-gal-positive cells were marked with arrowheads. c Quantitative real-time RT-PCR analysis of TRF2 and CDKN1A mRNA levels in doxycycline-treated (2 μg/mL for 10 days) U-2 OS-TetOn-shTRF2 cells by comparison with a control (doxycycline-untreated U-2 OS-TetOn-shTRF2 cells). Values represent the mean ± SD. *P < 0.05 (n = 2, versus doxycycline-untreated U-2 OS-TetOn-shTRF2 cells) according to a Student’s t test. The TaqMan primer used for CDKN1A was Hs00355782_m1 (Life Technologies). (JPEG 60 kb)

13277_2012_600_MOESM1_ESM.tif (330 kb)
High-resolution image (TIFF 330 kb)
13277_2012_600_Fig5_ESM.jpg (38 kb)
Supplementary Fig. 2

Effect of doxorubicin on TRF1, POT1, and RAP1 proteins. U-2 OS cells were treated with 1 μM of doxorubicin or DMSO for 24 h, and a western blot using the a TRF1 mouse monoclonal antibody (TRF-78/NB100-1701; Novus Biologicals, Littleton, CO), b POT1 rabbit polyclonal antibody (NB500-176; Novus Biologicals), and c RAP1 antibody (A300-306A; Bethyl Laboratories, Montgomery, TX) was performed (40 μg of lysate/lane). TRF1, POT1, and RAP1 antibodies were used at 1/500, 1/500, and 1/1,000 dilution, respectively. The left side of the panel shows the position of the molecular weight marker (in kilodaltons). GAPDH was used as a loading control. (JPEG 37 kb)

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Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2012

Authors and Affiliations

  • Masahiro Kato
    • 1
  • Masahiro Nakayama
    • 1
  • Minako Agata
    • 1
  • Kenichi Yoshida
    • 1
  1. 1.Department of Life SciencesMeiji UniversityKawasakiJapan

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