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Recent evidence has resurrected the concept of specialized populations of T lymphocytes that are able to suppress an antigenspecific immune response. T-regulatory cells (T-reg) have been characterized as CD4 þ CD25 þ T cells. Previous reports describing differential gene expression analysis have shown that the glucocorticoid-induced tumor necrosis family receptor family-related gene (GITR) is upregulated in these cells. Furthermore, antibodies specific for GITR have been shown to inhibit the T-suppressor function of CD4 þ CD25 þ T-reg. The ligands for both mouse and human GITR have been cloned recently. We have inserted the sequences for natural, membrane-bound GITR-ligand (GITR-L) and a truncated secreted form of GITR-L (GITR-Lsol) into the adenovirus-5 genome. Coculture experiments show that cells infected with Ad-GITR-L and supernatants from cells infected with Ad-GITR-Lsol can increase the proliferation of both CD4 þ CD25-and CD8 þ T cells in response to anti-CD3 stimulation, in the presence, as well as in the absence, of CD4 þ CD25 þ T cells. The virus constructs were injected into growing B16 melanoma tumors. Ad-GITR-L was shown to attract infiltration with both CD4 þ and CD8 þ T cells. Both constructs were shown to inhibit tumor growth.

While studying the effect of steroids on the growth of the yeast Saccharomyces cerevisiae, we found that pregnenolone was converted into the acetate ester. This reaction was identified as a transfer of the acetyl group from acetyl-CoA to the 3b-hydroxyl group of pregnenolone. The corresponding enzyme, acetyl-CoA:pregnenolone acetyltransferase (APAT) is specific for D 5 -or D 4 -3b-hydroxysteroids and short-chain acyl-CoAs. The apparent K m for pregnenolone is <0.5 mm. The protein associated with APAT activity was partially purified and finally isolated from an SDS/polyacrylamide gel. Tryptic peptides were generated and N-terminally sequenced. Two peptide sequences allowed the identification of an open reading frame (YGR177c, in the S. cerevisiae genome database) translating into a 62-kDa protein of hitherto unknown function. This protein encoded by a gene known as ATF2 displays 37% identity with an alcohol acetyltransferase encoded by the yeast gene ATF1. Disruption of ATF2 led to the complete elimination of APAT activity and consequently abolished the esterification of pregnenolone. In addition, a toxic effect of pregnenolone linked to the disruption of ATF2 was observed. Pregnenolone toxicity is more pronounced when the atf 2-D mutation is introduced in a yeast strain devoid of the ATP-binding cassette transporters, PDR5 and SNQ2. Our results suggest that Atf 2p (APAT) plays an active role in the detoxification of 3b-hydroxysteroids in association with the efflux pumps Pdr5p and Snq2p.

In mammals, the final 1 IF-hydroxylation step of the hydrocortisone biosynthesis pathway is performed by a mitochondrial enzyme, namely cytochrome P-450, Ip, together with the electron carriers adrenodoxin and NADPH adrenodoxin oxidoreductase. Successful production of a functional steroid 1 1p-hydroxylase activity was obtained in recombinant yeast in vivo. This conversion was achieved by coexpression of a mitochondrially targeted adrenodoxin and a modified bovine P-450, ,/, whose natural presequence was replaced by a yeast presequence, together with an unexpected yeast endogenous NADPHadrenodoxin-reductase-like activity. Adrenodoxin and P-450, behave as a mitochondrial matrix and membrane protein, respectively. Saccharomyces cerevisiae apparently produces a mitochondrial protein which is capable of transferring electrons to bovine adrenodoxin, which in turn transfers the electrons to P-450, , , + The endogenous adrenodoxin oxidoreductase gains electrons specifically from NADPH. The notion that a yeast microsomal NADPH P-450 oxidoreductase can transfer electrons to mammalian microsomal P-450s can be extended to mitochondria, where an NADPH adrenodoxin oxidoreductase protein transfers electrons to adrenodoxin and renders a mitochondrial mammalian P-450 functional in vivo. The physiological function of this yeast NADPH adrenodoxin oxidoreductase activity is not known.Keywords: cytochrome P-450; mitochondria; yeast; bioconversion ; electron transport.In the adrenal cortex of vertebrates, hydrocortisone is synthesized from cholesterol in a transformation that requires five enzymic steps. Four out of the five reactions are carried out by enzymes belonging to the superfamily of cytochrome P-450 proteins (i.e. encoded by the CYP gene family; for a review, see [I] and [2]). Almost all the P-450 proteins from eukaryotes are anchored to the endoplasmic reticulum membrane facing the cytosol [3]. Only a few members of this family are localized to the inner mitochondrial membrane; among them are the sidechain-cleaving P-450 (P-450,,,) and the 1 1p-hydroxylase (P-450,,,); these enzymes catalyze the first and the last steps, respectively, in hydrocortisone biosynthesis. In a complex reaction sequence, P-450," removes the side chain of cholesterol, generating pregnenolone as the first step of the pathway, whereas P-450,,, hydroxylates I I-deoxycortisol at position C1 I as the ultimate step in hydrocortisone biosynthesis. The latter enzyme can hydroxylate various corticosteroids in positions C18 or C19, it also has an inherent aldosterone synthase activity with 1 %hydro-Correspondence to T. Achstetter, TRANSGENE, 11 rue de MolsFax: +33 88 22 58 07. Abbreviations. P-450, , , $, P-450,,, and P-450,,,,,, cytochromes P-450 110-18 hydroxylase, P-450 cholesterol side chain cleavage and P-450 vitamin D, 25-hydroxylase; ADR, NADPH-adrenodoxin oxidoreductase; matADX, mature form of ADX; CoxVI,,,, cytochrome oxidase subunit VI prepeptide; TEFl, transcription elongation factor 1 ; CYCl isocytochrome C,.(ECl.14.15.4); NADPH-adrenodoxin oxido...

Based on the observation that the growth of solid tumors is dependent on the formation of new blood vessels, therapeutic strategies aimed at inhibiting angiogenesis have been proposed. A number of proteins with angiostatic activity have been described, but their development as therapeutic agents has been hampered by difficulties in their production and their poor pharmacokinetics. These limitations may be resolved using a gene therapy approach whereby the genes are delivered and expressed in vivo. Here we compared adenoviral delivery of endostatin, proliferin-related protein (PRP), and interferon-inducible protein 10 (IP10) genes. Recombinant adenoviruses carrying the three angiostatic genes express biologically active gene products as determined in vitro in endothelial cell proliferation and migration assays, and in vivo by inhibition of neoangiogenesis in rat chambers. Eradication of established tumors in vivo, in the murine B16F10 melanoma model in immunocompetent mice, was not achieved by intratumoral injection of the different vectors. However, the combination of intravenous plus intratumoral injections allowed rejection of tumors. Ad-PRP or Ad-IP10 were significantly more efficient than Ad-endostatin, leading to complete tumor rejection and prolonged survival in a high proportion of treated animals. These data support the use of in vivo gene delivery approaches to produce high-circulating and local levels of antiangiogenic agents for the therapy of local and metastatic human tumors.