Down-regulation of ATM Protein Sensitizes Human Prostate Cancer Cells to Radiation-induced Apoptosis

Jean-Philip Truman,Nuri Gueven,Martin Lavin,Steven Leibel,Richard Kolesnick,Zvi Fuks,Adriana Haimovitz-Friedman

doi:10.1074/jbc.m503701200

Jean-Philip Truman, Nuri Gueven + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m503701200

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Abstract

Treatment with the protein kinase C activator 12-O-tetradecanoylphorbol 12-acetate (TPA) enables radiation-resistant LNCaP human prostate cancer cells to undergo radiation-induced apoptosis, mediated via activation of the enzyme ceramide synthase (CS) and de novo synthesis of the sphingolipid ceramide (Garzotto, M., Haimovitz-Friedman, A., Liao, W. C., White-Jones, M., Huryk, R., Heston, D. W. W., Cardon-Cardo, C., Kolesnick, R., and Fuks, Z. (1999) Cancer Res. 59, 5194-5201). Here, we show that TPA functions to decrease the cellular level of the ATM (ataxia telangiectasia mutated) protein, known to repress CS activation (Liao, W.-C., Haimovitz-Friedman, A., Persaud, R., McLoughlin, M., Ehleiter, D., Zhang, N., Gatei, M., Lavin, M., Kolesnick, R., and Fuks, Z. (1999) J. Biol. Chem. 274, 17908-17917). Gel shift analysis in LNCaP and CWR22-Rv1 cells demonstrated a significant reduction in DNA binding of the Sp1 transcription factor to the ATM promoter, and quantitative reverse transcription-PCR showed a 50% reduction of ATM mRNA between 8 and 16 h of TPA treatment, indicating that TPA inhibits ATM transcription. Furthermore, treatment of LNCaP, CWR22-Rv1, PC-3, and DU-145 human prostate cells with antisense-ATM oligonucleotides, which markedly reduced cellular ATM levels, significantly enhanced radiation-induced CS activation and apoptosis, leading to apoptosis at doses as a low as 1 gray. These data suggest that the CS pathway initiates a generic mode of radiation-induced apoptosis in human prostate cancer cells, regulated by a suppressive function of ATM, and that ATM might represent a potential target for pharmacologic inactivation with potential clinical applications in human prostate cancer.

Highlights

Prostate cancer is regarded as relatively resistant to the lethal effects of radiation [3, 4]
To exclude the possibility that the effect of tetradecanoylphorbol 12-acetate (TPA) represents a LNCaP cell-specific phenomenon, we carried out experiments with another androgen-responsive prostate cancer cell line, CWR22-Rv1
Consistent with this pattern, a full-time analysis of ceramide levels in CWR22-Rv1 cells stimulated by 10 ng/ml TPA (Fig. 1D) showed that ceramide levels increased by 32 h from a base line of 103 Ϯ 17 pmol/106 cells to 225 Ϯ 45 pmol/106 cells (p Ͻ 0.01, Wilcoxon rank sum) and remained elevated until 66 h post-treatment (Fig. 1D)

Summary

EXPERIMENTAL PROCEDURES

Materials—The human prostate cancer cell lines LNCaP, PC-3, and DU-145 were obtained from ATCC. A total of 75 ␮g of microsomal membrane fraction was incubated with 1 ml of reaction buffer containing 2 mM MgCl2, 20 mM HEPES (pH 7.4), 20 ␮M defatted bovine serum albumin, 70 ␮M unlabeled palmitoyl coenzyme A, 3.6 ␮M (0.1 ␮Ci) [1-14C]palmitoyl-coenzyme A, and escalating amounts of dihydrosphingosine (0, 1, 2.5, 5, and 10 ␮M). 35 fmol of 32P-labeled (using T4 polynucleotide kinase) double-stranded Sp1 consensus sequence (5Ј-ATTCGATCGGGGCGGGGCGAGC-3Ј) was added to 10 ␮g of nuclear extract in the presence of 1 ␮g of herring sperm DNA, 10 ␮g of bovine serum albumin, and binding buffer consisting of 10 mM HEPES pH 7.9, 50 mM KCl, 0.5 mM EDTA, 10% glycerol, 0.1% Nonidet P-40, 5 mM dithiothreitol, and 0.2 mM phenylmethylsulfonyl fluoride. LNCaP cells were incubated with anti-bromodeoxyuridine fluorescein isothiocyanate antibody, and total DNA was stained using 7-amino-actinomycin D, as per the manufacturer’s instructions. Colonies with at least 50 cells were scored, and the results were analyzed by the CellFit version 2.5 program [46]

RESULTS

DISCUSSION

Ceramide level