Therapy for disseminated germ cell tumors (GCT) has been successful with 70-80% of patients being cured with front line chemotherapy. However, for those 20-30% of patients with extra-gonadal primaries or refractory disease, the response to therapy is poor with only 3-30% surviving disease-free after second line agents. One approach to treating resistant disease is the development of strategies to augment the chemotherapeutic agents that have been so successful in the majority of GCT patients. Little is known about the role of DNA repair systems in GCT's except that efficient repair appears to make tumor cells resistant to therapy. We have observed that GCT's express high levels of Ape1/ref-1 compared to normal tissues. Ape1/ref-1 is a multifunctional protein with DNA base excision repair (BER) activity and redox activity required for activation of specific transcription factors including Fos, Jun, NFkappaB, HIF-1alpha (hypoxia inducible factor), p53, and PAX5. This novel combination of functions links Ape1/ref-1 with resistance to many of the therapeutic agents (bleomycin, cisplatin, radiation, and VP-16) used to treat GCT's by acting as direct substrates for BER or indirectly by altering signaling through transcription factors regulated by Ape1/ref-l. Based on this information, we hypothesize: High level expression of Ape1/ref-1 in GCT's is a functional marker of disease which 1) is predictive of high risk disease and 2) can be manipulated to gain a therapeutic advantage. The major thrust of this proposal is to characterize the molecular biology of Ape1/ref-1 in GCT's as it relates to the clinical course of patients and the response of GCT cells to therapeutic agents. To approach this goal, we have developed four Specific Aims:
Specific Aim1 : Determine the relative expression of, Ape1/ref-1in good prognosis and high-risk GCT's. This takes advantage of the wealth of clinical GCT material available at Indiana University.
Specific Aim 2 : What is the role of Ape1/ref-1 in GCT progression and development, including cell growth, apoptosis, cell cycle, and differentiation? That is how does the high level expression of Ape1/ref-1, including over-expression of repair, redox, and nuclear localization domain mutants independently affect the ability of GCT cells to grow as cancer cells.
Specific Aim 3 : How do changes in the redox status of Ape1/ref-1 affect repair function? Using site-specific mutants, determine which cysteine residues specifically control repair function of Ape1/ref-l? Specific Aim 4: How do alterations in the repair and redox functioning of Ape1/ref-1 affect the response of GCT cells to therapeutic agents? Using what we learn in Aims 2 & 3, how can we alter the resistance of GCT cells to therapeutic agents. Through these analyses, we hope to determine the underlying mechanisms by which Ape1/ref-1 function is linked to the progression of testicular cancer. If any mutants are shown to sensitize the GCT cell lines to chemo-/IR agents, as expected, this will set the stage for future gene therapy approaches to sensitize GCT's to therapy.
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