Altered expression of Ape1/ref-1 in germ cell tumors and overexpression inNT2 cells confers resistance to bleomycin and radiation

The human AP endonuclease (Ape1 or ref-1) DNA base excision repair (BER) en zyme is a multifunctional protein that has an impart on a wide variety of i mportant cellular functions including oxidative signaling, transcription fa ctor 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 (F os-Jun, nuclear factor-kappaB, Myb, ATF/cyclic AMP-responsive element bindi ng protein family, HIF-1 alpha, HLF, PAX, and p53) through a redox mechanis m and thus represents a novel component of signal transduction processes th at regulate eukaryotic gene expression. Ape1/ref-1 has also been shown to b e closely linked to apoptosis associated with thioredoxin, and altered leve ls 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 hist ologies including seminomas, yolk sec 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 re sponsible 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/D 1 using retroviral gene transduction with the vector LAPESN. Using an oligo nucleotide cleavage assay and immunohistochemistry to assess Ape1/ref-1 rep air activity and expression, respectively, we found that the repair activit y 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 endonu clease activity in the oligonucleotide cleavage assay, and this was reflect ed in a 2-3-fold increase in protection against bleomycin, Lesser protectio n was observed with gamma -irradiation, We conclude that: (a) Ape1/ref-l 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 cert ain therapeutic agents; and (c) Ape1/ref-1 expression in GCT cell lines det ermined by immunohistochemistry and repair activity assays parallels the le vel of protection from bleomycin, We further hypothesize that elevated Ape1 /ref-1 levels observed in human testicular cancer may be related to their r elative resistance to therapy and may serve as a diagnostic marker for refr actory disease. To our knowledge, this is the first example of overexpressi ng Ape1/ref-1 in a mammalian system resulting in enhanced protection to DNA -damaging agents.