The T Cell Receptor γ Chain Alternate Reading Frame Protein (TARP), a Prostate-specific Protein Localized in Mitochondria

Abstract

We previously showed that mRNA encoding TARP (T cell receptor ␥ chain alternate reading frame protein) is exclusively expressed in the prostate in males and is up-regulated by androgen in LNCaP cells, an androgen-sensitive prostate cancer cell line. We have now developed an anti-TARP monoclonal antibody named TP1, and show that TARP protein is up-regulated by androgen in both LNCaP and MDA-PCa-2b cells. We used TP1 to determine the subcellular localization of TARP by Western blotting following subcellular fractionation and immunocytochemistry. Both methods showed that TARP is localized in the mitochondria of LNCaP cells, MDA-PCa-2b cells, and PC-3 cells transfected with a TARP-expressing plasmid. We also transfected a plasmid encoding TARP fused to green fluorescent protein into LNCaP, MDA-Pca-2b, and PC-3 cells and confirmed its specific mitochondrial localization in living cells. Fractionation of mitochondria shows that TARP is located in the outer mitochondrial membrane. Immunohistochemistry using a human prostate cancer sample showed that TP1 reacted in a dot-like cytoplasmic pattern consistent with the presence of TARP in mitochondria. These data demonstrate that TARP is the first prostate-specific protein localizing in mitochondria and indicate that TARP, an androgen-regulated protein, may act on mitochondria to carry out its biological functions.Prostate cancer is the most frequently occuring solid cancer in men and the second cause of death from cancer in the United States. Current statistics estimate that 220,900 new cases were diagnosed in 2003 and that about 28,900 males die of this disease (1). Most forms of prostate cancer are initially androgendependent, but the response to androgen ablation therapy is transient. After a few years, most cases of metastatic prostate cancer relapse to the status of androgen independence, resulting in death. No effective treatment options exist against androgen-independent prostate cancers. Improvement in this clinical situation requires novel therapies based on a better understanding of the biochemical basis of prostate cancer initiation and progression.One approach for understanding the progression mechanism of prostate cancer is to identify new prostate-specific genes whose expression is altered in prostate cancer. These prostatespecific genes should enable us to understand more about normal prostate function and contribute to our understanding of prostate cancer progression. Prostate-specific genes also can be useful targets for molecular based therapies using antibodies, vaccines, and small inhibitory molecules. Because the prostate is not a vital organ, its destruction by antibodies or vaccines that target prostate-specific antigens should not adversely affect the prognosis of the patient. Also, prostate-specific genes can be useful diagnostic markers to predict the presence, the spread, and the prognosis of prostate disease using samples such as blood or urine. These three aspects: cancer progression, pharmacology, and diagnostics, have encouraged many research...

Recognition of Prostate and Breast Tumor Cells by Helper T Lymphocytes Specific for a Prostate and Breast Tumor-Associated Antigen, TARP

“…HTL line (7D) from an HLA-DR4/DR9, HLA-DR53 normal male individual. Moreover, the peptide-induced response of this HTL line was inhibited by anti -HLA-DR mAb (L243) but not by anti -HLA class I mAb (W6/32), indicating that this HTL line recognized the peptide TARP [1][2][3][4][5][6][7][8][9][10][11][12][13][14] presented in the context of MHC II (Fig. 1A).…”

“…We first selected and synthesized three peptides from the TARP sequence, TARP [1][2][3][4][5][6][7][8][9][10][11][12][13][14] (MQMFPPSPLFFFLQ), TARP [14][15][16][17][18][19][20][21][22][23][24][25][26][27] (QLLKQSSRRLEHTF), and TARP 27-41 (FVFLRNFSLMLLRYI), based on results (data not shown) derived a prediction algorithm that identifies potential promiscuous MHC II binding peptides for three common human allele products, HLA-DR1, HLA-DR4, and HLA-DR7 (25). In the past, this approach has allowed us to identify several HTL epitopes for renowned TAA, including HER-2/neu, gp100, MAGE-A3, PSA, EBNA2, CEA, and the HTLV-1 envelope protein (18 -24).…”

“…An HTL line was selected from an HLA-DR4/DR9, HLA-DR53 normal male individual by weekly stimulation of CD4 + Tcells with peptide and APC as described in Materials and Methods. A , using autologous PBMC as APC, theT-cell response to peptideTARP [1][2][3][4][5][6][7][8][9][10][11][12][13][14] was inhibited by anti^HLA-DR mAb L243 but not by anti^HLA class I mAb W6/32 (both used at 10 Ag/mL). B, when mouse fibroblast (L cells) transfected with HLA-DR genes were used as APC, it became evident that the HTL line recognized peptideTARP [1][2][3][4][5][6][7][8][9][10][11][12][13][14] (used at 3 Ag/mL) in the context of HLA-DR53.…”

“…Complete culture medium for all procedures consisted of AIM-V medium (Invitrogen Life Technologies, Carlsbad, CA) supplemented with 3% human male AB serum. [1][2][3][4][5][6][7][8][9][10][11][12][13][14] . An HTL line was selected from an HLA-DR4/DR9, HLA-DR53 normal male individual by weekly stimulation of CD4 + Tcells with peptide and APC as described in Materials and Methods.…”

“…The T-cell receptor g alternate reading frame protein (TARP) is a 7-kDa mitochondrial product that is specifically expressed by normal and transformed prostatic epithelial cells, which has also been found present in some breast cancer cell lines (11,12). Recently, two peptide epitopes from TARP were described (TARP [4][5][6][7][8][9][10][11][12][13] and TARP [27][28][29][30][31][32][33][34][35] ), which were shown to induce antitumor CTL responses in vitro (13,14). Furthermore, circulating CD8 + T lymphocytes from peripheral blood of prostate cancer patients were able to recognize these CTL epitopes, suggesting that immune responses to TARP are present in vivo (13).…”