Prolonged c-jun expression in irradiated ataxia telangiectasia fibroblasts

Dennis E. Hallahan, Edward Dunphy, Jaya Kuchibhotla, Andrew Kraft, Tito Unlap, Ralph R. Weichselbaum

Research output: Contribution to journalArticle

16 Citations (Scopus)

Abstract

Purpose: Ataxia telangiectasia (AT) is an autosomal recessive disorder associated with radiation sensitivity and an increased incidence of leukemia, lymphoma, and some solid tumors. After exposure to ionizing radiation, cells from patients with AT demonstrate an attenuated G1-phase checkpoint. Because c-jun is known to regulate, in part, the exit from G1 and the onset of DNA replication, we analyzed c-jun transcription in irradiated AT fibroblasts. Methods and Materials: AT5BI fibroblasts were irradiated and RNA was extracted and assayed for c-jun expression by Northern blot analysis. Transcriptional regulation of c-jun was evaluated by use of the 5' untranslated region of the jun promoter linked to the chloramphenicol acetyl transferase (CAT) reporter gene. Deletion mutants of the RSRF, SP-1, AP-1, and CCAAT domains within the jun promoter linked to the CAT reporter were transfected into AT5BI cells. Transfectants were irradiated, and CAT expression was quantified. After x-irradiation, nuclear protein binding to CCAAT was evaluated by an electrophoretic mobility shift assay. Results: X- ray-mediated c-jun expression was sustained in AT5BI cells as compared to only transient expression in irradiated normal diploid fibroblasts. Mutation of either the AP-1 or CCAAT domains within the c-jun promoter reduced transcription by 50% and combined deletion of both AP-1 and CCAAT cis-acting elements entirely eliminated radiation-mediated transcriptional activation. Electrophoretic mobility gel shift assay of the nuclear proteins isolated from irradiated AT fibroblasts demonstrated their increased binding to the CCAAT sequence at 30 min after irradiation. Competition for nuclear protein binding to the CCAAT sequence with excess cold CCAAT demonstrated that protein binding to this sequence was specific. These findings were distinct from induction by phorbol esthers in that the RSRF cis-acting element and DNA segments upstream of -132 base pairs do participate in c-jun induction by phorbol esthers but not by radiation. Conclusions: Radiation-mediated transcriptional regulation of c-jun is prolonged in AT fibroblasts and is regulated in combinatorial control by the AP-1 and CCAAT domains, and transcriptional regulation is distinct from that induced by phorbol esthers.

Original languageEnglish (US)
Pages (from-to)355-360
Number of pages6
JournalInternational Journal of Radiation Oncology Biology Physics
Volume36
Issue number2
DOIs
StatePublished - Sep 1 1996
Externally publishedYes

Fingerprint

ataxia
Ataxia Telangiectasia
fibroblasts
Transcription Factor AP-1
Fibroblasts
Chloramphenicol
Transferases
Nuclear Proteins
Protein Binding
proteins
deletion
Electrophoretic Mobility Shift Assay
radiation
Radiation
induction
deoxyribonucleic acid
cells
G1 Phase Cell Cycle Checkpoints
irradiation
leukemias

Keywords

  • Ataxia telangiectasia
  • c-jun
  • CCAAT box
  • Radiation

ASJC Scopus subject areas

  • Oncology
  • Radiology Nuclear Medicine and imaging
  • Radiation

Cite this

Prolonged c-jun expression in irradiated ataxia telangiectasia fibroblasts. / Hallahan, Dennis E.; Dunphy, Edward; Kuchibhotla, Jaya; Kraft, Andrew; Unlap, Tito; Weichselbaum, Ralph R.

In: International Journal of Radiation Oncology Biology Physics, Vol. 36, No. 2, 01.09.1996, p. 355-360.

Research output: Contribution to journalArticle

Hallahan, Dennis E. ; Dunphy, Edward ; Kuchibhotla, Jaya ; Kraft, Andrew ; Unlap, Tito ; Weichselbaum, Ralph R. / Prolonged c-jun expression in irradiated ataxia telangiectasia fibroblasts. In: International Journal of Radiation Oncology Biology Physics. 1996 ; Vol. 36, No. 2. pp. 355-360.
@article{37cc4f015bb645ab94d7fac939b01b93,
title = "Prolonged c-jun expression in irradiated ataxia telangiectasia fibroblasts",
abstract = "Purpose: Ataxia telangiectasia (AT) is an autosomal recessive disorder associated with radiation sensitivity and an increased incidence of leukemia, lymphoma, and some solid tumors. After exposure to ionizing radiation, cells from patients with AT demonstrate an attenuated G1-phase checkpoint. Because c-jun is known to regulate, in part, the exit from G1 and the onset of DNA replication, we analyzed c-jun transcription in irradiated AT fibroblasts. Methods and Materials: AT5BI fibroblasts were irradiated and RNA was extracted and assayed for c-jun expression by Northern blot analysis. Transcriptional regulation of c-jun was evaluated by use of the 5' untranslated region of the jun promoter linked to the chloramphenicol acetyl transferase (CAT) reporter gene. Deletion mutants of the RSRF, SP-1, AP-1, and CCAAT domains within the jun promoter linked to the CAT reporter were transfected into AT5BI cells. Transfectants were irradiated, and CAT expression was quantified. After x-irradiation, nuclear protein binding to CCAAT was evaluated by an electrophoretic mobility shift assay. Results: X- ray-mediated c-jun expression was sustained in AT5BI cells as compared to only transient expression in irradiated normal diploid fibroblasts. Mutation of either the AP-1 or CCAAT domains within the c-jun promoter reduced transcription by 50{\%} and combined deletion of both AP-1 and CCAAT cis-acting elements entirely eliminated radiation-mediated transcriptional activation. Electrophoretic mobility gel shift assay of the nuclear proteins isolated from irradiated AT fibroblasts demonstrated their increased binding to the CCAAT sequence at 30 min after irradiation. Competition for nuclear protein binding to the CCAAT sequence with excess cold CCAAT demonstrated that protein binding to this sequence was specific. These findings were distinct from induction by phorbol esthers in that the RSRF cis-acting element and DNA segments upstream of -132 base pairs do participate in c-jun induction by phorbol esthers but not by radiation. Conclusions: Radiation-mediated transcriptional regulation of c-jun is prolonged in AT fibroblasts and is regulated in combinatorial control by the AP-1 and CCAAT domains, and transcriptional regulation is distinct from that induced by phorbol esthers.",
keywords = "Ataxia telangiectasia, c-jun, CCAAT box, Radiation",
author = "Hallahan, {Dennis E.} and Edward Dunphy and Jaya Kuchibhotla and Andrew Kraft and Tito Unlap and Weichselbaum, {Ralph R.}",
year = "1996",
month = "9",
day = "1",
doi = "10.1016/S0360-3016(96)00327-6",
language = "English (US)",
volume = "36",
pages = "355--360",
journal = "International Journal of Radiation Oncology Biology Physics",
issn = "0360-3016",
publisher = "Elsevier Inc.",
number = "2",

}

TY - JOUR

T1 - Prolonged c-jun expression in irradiated ataxia telangiectasia fibroblasts

AU - Hallahan, Dennis E.

AU - Dunphy, Edward

AU - Kuchibhotla, Jaya

AU - Kraft, Andrew

AU - Unlap, Tito

AU - Weichselbaum, Ralph R.

PY - 1996/9/1

Y1 - 1996/9/1

N2 - Purpose: Ataxia telangiectasia (AT) is an autosomal recessive disorder associated with radiation sensitivity and an increased incidence of leukemia, lymphoma, and some solid tumors. After exposure to ionizing radiation, cells from patients with AT demonstrate an attenuated G1-phase checkpoint. Because c-jun is known to regulate, in part, the exit from G1 and the onset of DNA replication, we analyzed c-jun transcription in irradiated AT fibroblasts. Methods and Materials: AT5BI fibroblasts were irradiated and RNA was extracted and assayed for c-jun expression by Northern blot analysis. Transcriptional regulation of c-jun was evaluated by use of the 5' untranslated region of the jun promoter linked to the chloramphenicol acetyl transferase (CAT) reporter gene. Deletion mutants of the RSRF, SP-1, AP-1, and CCAAT domains within the jun promoter linked to the CAT reporter were transfected into AT5BI cells. Transfectants were irradiated, and CAT expression was quantified. After x-irradiation, nuclear protein binding to CCAAT was evaluated by an electrophoretic mobility shift assay. Results: X- ray-mediated c-jun expression was sustained in AT5BI cells as compared to only transient expression in irradiated normal diploid fibroblasts. Mutation of either the AP-1 or CCAAT domains within the c-jun promoter reduced transcription by 50% and combined deletion of both AP-1 and CCAAT cis-acting elements entirely eliminated radiation-mediated transcriptional activation. Electrophoretic mobility gel shift assay of the nuclear proteins isolated from irradiated AT fibroblasts demonstrated their increased binding to the CCAAT sequence at 30 min after irradiation. Competition for nuclear protein binding to the CCAAT sequence with excess cold CCAAT demonstrated that protein binding to this sequence was specific. These findings were distinct from induction by phorbol esthers in that the RSRF cis-acting element and DNA segments upstream of -132 base pairs do participate in c-jun induction by phorbol esthers but not by radiation. Conclusions: Radiation-mediated transcriptional regulation of c-jun is prolonged in AT fibroblasts and is regulated in combinatorial control by the AP-1 and CCAAT domains, and transcriptional regulation is distinct from that induced by phorbol esthers.

AB - Purpose: Ataxia telangiectasia (AT) is an autosomal recessive disorder associated with radiation sensitivity and an increased incidence of leukemia, lymphoma, and some solid tumors. After exposure to ionizing radiation, cells from patients with AT demonstrate an attenuated G1-phase checkpoint. Because c-jun is known to regulate, in part, the exit from G1 and the onset of DNA replication, we analyzed c-jun transcription in irradiated AT fibroblasts. Methods and Materials: AT5BI fibroblasts were irradiated and RNA was extracted and assayed for c-jun expression by Northern blot analysis. Transcriptional regulation of c-jun was evaluated by use of the 5' untranslated region of the jun promoter linked to the chloramphenicol acetyl transferase (CAT) reporter gene. Deletion mutants of the RSRF, SP-1, AP-1, and CCAAT domains within the jun promoter linked to the CAT reporter were transfected into AT5BI cells. Transfectants were irradiated, and CAT expression was quantified. After x-irradiation, nuclear protein binding to CCAAT was evaluated by an electrophoretic mobility shift assay. Results: X- ray-mediated c-jun expression was sustained in AT5BI cells as compared to only transient expression in irradiated normal diploid fibroblasts. Mutation of either the AP-1 or CCAAT domains within the c-jun promoter reduced transcription by 50% and combined deletion of both AP-1 and CCAAT cis-acting elements entirely eliminated radiation-mediated transcriptional activation. Electrophoretic mobility gel shift assay of the nuclear proteins isolated from irradiated AT fibroblasts demonstrated their increased binding to the CCAAT sequence at 30 min after irradiation. Competition for nuclear protein binding to the CCAAT sequence with excess cold CCAAT demonstrated that protein binding to this sequence was specific. These findings were distinct from induction by phorbol esthers in that the RSRF cis-acting element and DNA segments upstream of -132 base pairs do participate in c-jun induction by phorbol esthers but not by radiation. Conclusions: Radiation-mediated transcriptional regulation of c-jun is prolonged in AT fibroblasts and is regulated in combinatorial control by the AP-1 and CCAAT domains, and transcriptional regulation is distinct from that induced by phorbol esthers.

KW - Ataxia telangiectasia

KW - c-jun

KW - CCAAT box

KW - Radiation

UR - http://www.scopus.com/inward/record.url?scp=0030250629&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0030250629&partnerID=8YFLogxK

U2 - 10.1016/S0360-3016(96)00327-6

DO - 10.1016/S0360-3016(96)00327-6

M3 - Article

C2 - 8892460

AN - SCOPUS:0030250629

VL - 36

SP - 355

EP - 360

JO - International Journal of Radiation Oncology Biology Physics

JF - International Journal of Radiation Oncology Biology Physics

SN - 0360-3016

IS - 2

ER -