The nongenotropic synthetic ligand 4-estren-3alpha17beta-diol is a high-affinity genotropic androgen receptor agonist.

The nongenotropic ligand estren (Science 298:843-846, 2002) was evaluated for its transcriptional activity mediated by the human androgen receptor (AR). Our results show that estren can bind, translocate, transactivate, and regulate two known target genes of AR in androgen-responsive cell lines. Estren binds recombinant AR with 10-fold higher affinity than either estrogen receptor (ER)-alpha or ERbeta. Estren-bound AR can translocate AR to the nucleus and stimulate the androgen response element-luciferase reporter activity with an efficacy similar to that of androgen. Estren also increased the expression of prostate-specific antigen (PSA) in a dose-dependent manner in human LnCaP cells. Using chromatin immunoprecipitation analysis, we show that the estren-bound AR coimmunoprecipitates with a region of the PSA gene promoter. Therefore, cotreatment with an AR antagonist, bicalutamide, blocked the estren-induced increase in PSA expression. In contrast, phosphoinositol 3-kinase inhibitor wortmannin, or extracellular signal-regulated kinase inhibitor 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophynyltio)butadiene (U0126), and ER antagonist ICI-182780 failed to block the effects of estren. In vivo analysis of estren's action on male-orchidectomized ICR mice revealed estren's AR agonist actions on the levator ani and seminal vesicle target tissues. Taken together, our results reveal the hitherto unidentified genotropic action of estren mediated by AR in androgen-responsive cells and tissues.

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.