Altered expression of Ape1/ref-1 in germ cell tumors and overexpression in NT2 cells confers resistance to bleomycin and radiation

• Published in: Cancer research (Chicago, Ill.) Ill.), 2001-03, Vol.61 (5), p.2220-2225
• Main Authors: ROBERTSON, Kent A, BULLOCK, Heather A, YI XU, TRITT, Renee, ZIMMERMAN, Erika, ULBRIGHT, Thomas M, FOSTER, Richard S, EINHORN, Lawrence H, KELLEY, Mark R
• Format: Article
• Language: English
• Subjects: Antimetabolites, Antineoplastic - pharmacology; Antineoplastic agents; Apel protein; Biological and medical sciences; bleomycin; Bleomycin - pharmacology; Carbon-Oxygen Lyases - biosynthesis; Carbon-Oxygen Lyases - genetics; Carcinoma, Embryonal - drug therapy; Carcinoma, Embryonal - metabolism; Carcinoma, Embryonal - radiotherapy; Chemotherapy; Deoxyribonuclease IV (Phage T4-Induced); DNA Repair; DNA-(Apurinic or Apyrimidinic Site) Lyase; Drug Resistance, Neoplasm; endonuclease; Gene Transfer Techniques; Germinoma - drug therapy; Germinoma - metabolism; Germinoma - radiotherapy; Humans; Medical sciences; Pharmacology. Drug treatments; Radiation Tolerance - physiology; ref-1 gene; Retroviridae - genetics; Tumor Cells, Cultured
• ISSN: 0008-5472; 1538-7445

The human AP endonuclease (Ape1 or ref-1) DNA base excision repair (BER) enzyme is a multifunctional protein that has an impact on a wide variety of important cellular functions including oxidative signaling, transcription factor regulation, and cell cycle control. It acts on mutagenic AP (baseless) sites in DNA as a critical member of the DNA BER repair pathway. Moreover, Ape1/ref-1 stimulates the DNA-binding activity of transcription factors (Fos-Jun, nuclear factor-kappaB, Myb, ATF/cyclic AMP-responsive element binding protein family, HIF-1alpha, HLF, PAX, and p53) through a redox mechanism and thus represents a novel component of signal transduction processes that regulate eukaryotic gene expression. Ape1/ref-1 has also been shown to be closely linked to apoptosis associated with thioredoxin, and altered levels of Ape1/ref-1 have been found in some cancers. In a pilot study, we have examined Ape1/ref-1 expression by immunohistochemistry in sections of germ cell tumors (GCTs) from 10 patients with testicular cancer of various histologies including seminomas, yolk sac tumors, and malignant teratomas. Ape1/ref-1 was expressed at relatively high levels in the tumor cells of nearly all sections. We hypothesized that elevated expression of Ape1/ref-1 is responsible in part for the resistance to therapeutic agents. To answer this hypothesis, we overexpressed the Ape1/ref-1 cDNA in the GCT cell line NT2/D1 using retroviral gene transduction with the vector LAPESN. Using an oligonucleotide cleavage assay and immunohistochemistry to assess Ape1/ref-1 repair activity and expression, respectively, we found that the repair activity and relative Ape1/ref-1 expression in GCT cell lines are directly related. NT2/D1 cells transduced with Ape1/ref-1 exhibited 2-fold higher AP endonuclease activity in the oligonucleotide cleavage assay, and this was reflected in a 2-3-fold increase in protection against bleomycin. Lesser protection was observed with gamma-irradiation. We conclude that: (a) Ape1/ref-1 is expressed at relatively high levels in some GCTs; (b) elevated expression of Ape1/ref-1 in testicular cancer cell lines results in resistance to certain therapeutic agents; and (c) Ape1/ref-1 expression in GCT cell lines determined by immunohistochemistry and repair activity assays parallels the level of protection from bleomycin. We further hypothesize that elevated Ape1/ref-1 levels observed in human testicular cancer may be related to their relative resistance to thera