Altered expression of Ape1/ref-1 in germ cell tumors and overexpression in NT2 cells confers resistance to bleomycin and radiation.
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 therapy and may serve as a diagnostic marker for refractory disease. To our knowledge, this is the first example of overexpressing Ape1/ref-1 in a mammalian system resulting in enhanced protection to DNA-damaging agents.
Robertson
K A
KA
Herman B. Wells Center for Pediatric Research, Department of Pediatrics, James Whircomb Riley Hospital for Children, Indiana University School of Medicine, Indianapolis 46202, USA.
Bullock
H A
HA
Xu
Y
Y
Tritt
R
R
Zimmerman
E
E
Ulbright
T M
TM
Foster
R S
RS
Einhorn
L H
LH
Kelley
M R
MR
D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D001761:Bleomycin / Q000494:pharmacology*; D019757:Carbon-Oxygen Lyases / Q000096:biosynthesis* / Q000235:genetics; D018236:Carcinoma, Embryonal / Q000188:drug therapy / Q000378:metabolism* / Q000532:radiotherapy; D004260:DNA Repair; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D043223:Deoxyribonuclease IV (Phage T4-Induced); D019008:Drug Resistance, Neoplasm; D018014:Gene Transfer Techniques; D018237:Germinoma / Q000188:drug therapy / Q000378:metabolism* / Q000532:radiotherapy; D006801:Humans; D011836:Radiation Tolerance / Q000502:physiology*; D012190:Retroviridae / Q000235:genetics; D014407:Tumor Cells, Cultured
D000964:Antimetabolites, Antineoplastic; D001761:Bleomycin; D043223:Deoxyribonuclease IV (Phage T4-Induced); D019757:Carbon-Oxygen Lyases; C085108:APEX1 protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase