Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Heterocyclic carbon compounds containing a hetero ring...
Reexamination Certificate
2001-06-06
2004-02-10
Ungar, Susan (Department: 1642)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Heterocyclic carbon compounds containing a hetero ring...
C514S183000, C424S009100, C424S009361, C435S007100
Reexamination Certificate
active
06689775
ABSTRACT:
1. BACKGROUND OF INVENTION
The present invention generally relates to the use of thioredoxin as, inter alia, a cell growth stimulator, as well as a screen for agents that are useful in reducing or eliminating thioredoxin-associated apoptosis inhibition and agents that are useful in inhibiting thioredoxin stimulated cell growth.
Thioredoxin is a low molecular weight (M
r
11,000-12,000) redox protein found in both prokaryotic and eukaryotic cells. (Holmgren A., J. Biol. Chem., 264:13963-13966, 1989), that undergoes reversible thiol reduction by the NADPH-dependent enzyme thioredoxin reductase. Human thioredoxin, which has 5 cysteine (Cys) residues, is a 11.5 kDa protein with 27% amino acid identity to
E. coli
thioredoxin. Human thioredoxin contains 3 additional Cys residues not found in bacterial thioredoxin that give it unique biological properties. (Gasdaska P Y, et al., Biochem. Biophys. Acta., 1218:292-296, 1994). Cys32 and Cys35 are the conserved catalytic site cysteine residues that undergo reversible oxidation to cystine. Cys92, Cys69 and Cys73 are found in mammalian but not in bacterial thioredoxins. Cys73 appears to be particularly important for maintaining the biological activity of thioredoxin in an oxidizing environment. Thioredoxin reduces a variety of intracellular proteins including enzymes such as ribonucleotide reductase which is important for DNA synthesis, and critical Cys residues in transcription factors such as NF-&kgr;B, AP-1 and the glucocorticoid receptor, thus, altering their binding to DNA. In addition to its intracellular actions, human thioredoxin has remaskable eztracellular celm growth stimulating properties. It has been reported (Gasdaska P Y, et al., Biochem. Biophys. Acta., 1218:292-296, 1994) that thioredoxion is identical to a growth factor reported to be secreted by human HTLV-1 transformed leukemia cell lines (Fox J A, et al., Proc.Natl. Accd. Sci. USA, 84:2663-2677, 1987). It has also been found that human recombinant thioredoxin will stimulate the growth of a wide variety of fibroblast and human solid tumor cell lines in culture (Gasdaska J R, et al., Cell Growth Differ., 6:1643-1650, 1995; Oblong J E, et al., J. Biol. Chem., 269:11714-11720, 1994).
E. coli
thioredoxin does not stimulate cell proliferation.
Thioredoxin was first studied for its ability to act as reducing co-factor for ribonucleotide reductase, the first unique step in DNA synthesis. (Laurent T C, et al., 15 J. Biol. Chem., 239:3436-3444, 1964). More recently thioredoxin has been shown to exert redox control over a number of transcription factors modulating their binding to DNA and thus, regulating gene transcription. Transcription factors regulated by thioredoxin include NF-&kgr;B (Matthews J R, et al., Nucl. Acids Res., 20:3821-3830, 1992), TFIIIC (Cromlish J A, et al., J. Biol. Chem., 264:18100-18109, 1989), BZLFI (Bannister 20 A J, et al., Oncogene, 6:1243-1250, 1991), the glucocorticoid receptor (Grippo J F, et al., J. Biol. Chem., 258:13658-13664, 1983) and, indirectly through a nuclear redox factor Ref-1/HAPE, thioredoxin can regulate AP-1 (Fos/Jun heterodimer) (Abate C, et al., Science 249:1157-1161, 1990). Thioredoxin is also a growth factor with a unique mechanism of action.
Human thioredoxin has been sequenced and cloned. (Gasdaska P Y, et al., Biochem. Biophys. Acta., 1218:292-296, 1994; Deiss L P, et al., Science 252:117-120, 1991). It has been shown that the deduced amino acid sequence of thioredoxin is identical to that of a previously known protein called eosinophil cytotoxicity stimulating factor (Silberstein D S. et al. J. Biol. Chem. 268:9138-9142, 1993) or adult T-cell leukemia-derived factor (ADF) (Gasdaska P Y, et al., Biochem. Biophys. Acta., 1218:292-296, 1994). ADF has been reported to be secreted by virally transformed leukemic cell lines and to stimulate their growth (Yodoi J, et al., Adv. Cancer Res., 57:381-411, 1991). These observations have been extended to show that human recombinant thioredoxin stimulates the proliferation of both normal fibroblasts and a wide variety of human solid and leukemic cancer cell lines. (Gasdaska J R, et al., Cell Growth Differ., 6:1643-1650, 1995); Powis G, et al., Oncol. Res., 6:539-544, 1994; Oblong J E, et al., J. Biol. Chem., 269:11714-11720, 1994). It has been shown that thioredoxin stimulates cell proliferation by increasing the sensitivity of the cells to growth factors secreted by the cells themselves. (Gasdaska J R, et al., Cell Growth Differ., 10 6:1643-1650, 1995).
Recombinant modified thioredoxins, otherwise called mutant thioredoxins, have been developed, but no indications of uses were known in the art for any particular mutant form. In a wild type thioredoxin, the cysteine (Cys) residues at the conserved -Cys32-Gly-Pro-Cys35-Lys active site of thioredoxin undergo reversible oxidation-reduction catalyzed by the NADPH-dependent flavoprotein thioredoxin reductase. (Luthman M, et al., Biochem., 21:6628-6633, 1982). It has been reported that mutation of the active site Cys32 and Cys35 residues to serine (Ser) residues, either singly or together (C32S/C35S thioredoxin), results in a compound that is redox inactive (i.e., it is not a substrate for reduction by thioredoxin reductase) and that does not stimulate cell proliferation (Oblong J E, et al., J. Biol. Chem., 269:11714-11720, 1994).
Thioredoxin mRNA has been found to be over expressed by some human tumor cells (Gasdaska P Y, et al., Biochem. Biophys. Acta., 1218:292-296, 1994; Grogan T, et al., Cancer Res., 1997, in press) and since it is secreted from cells by a leaderless secretary pathway (Rubartelli A. et al., J. Biol. Chem., 267:24161-24164, 1992) it could be a growth factor for some human cancers (Gasdaska J R, et al., Cell Growth Differ., 6:1643-1650, 1995). However, the mechanism for cell growth stimulation by thioredoxin mRNA has been examined and found not to promote cell growth. Recombinant human thioredoxin is not taken up by cells and does not bind to high affinity cell surface receptors but appears to enhance the sensitivity of cells to endogenously produced or other growth factors, a mechanism termed voitocrine (Greek, voithos =helper) (Gasdaska J R, et al., Cell Growth Differ., 6:1643-1650, 1995).
The in vitro cell growth stimulating activity of human thioredoxin has been previously reported for human lymphoid and solid tumor cancer cells (Gasdaska J R, et al., Cell Growth Differ., 6:1643-1650, 1995; Oblong J E, et al., J. Biol. Chem., 269:11714-11720, 1994) and for mouse fibroblast cells (Oblong J E, et al., J. Biol. Chem., 269:11714-11720, 1994). The production of a Cys
73
→Ser mutant thioredoxin has been previously reported. In one study it did not act like wild-type thioredoxin as a component of a complex cell growth stimulating factor called “early pregnancy factor” (Tonissen K, 10 et al., J. Biol. Chem., 268:22485-22489, 1993). In another study it was reported that Cys
73
→Ser mutant thioredoxin did not form a dimer, but cell growth stimulating activity by the mutant thioredoxin was not investigated in this study (Ren X, et al., Biochem., 32:9701-9705, 1993). However, the ability of the Cys73→Ser mutant and other mutant thioredoxins to stimulate cell proliferation has not been reported There have been no prior reports of administration of mutant thioredoxins in vivo.
It has been known that certain human tumor cells were found to over-express thioredoxin mRNA compared to normal lung tissue from the same subject. (Gasdaska P Y, et al., Biochem. Biophys. Acta., I 218:292-296, 1994). It has also been known that human primary colorectal tumors have exhibited elevated levels of thioredoxin mRNA compared to normal colonic mucosa (Berggren M, et al., Anticancer Res., 16:3459-3466, 1996). It has not been known that thioredoxin protein was present in certain human tumor cells, and it has not been known that thioredoxin protein played any role in preventing or enhancing tumor cell growth.
While thioredoxin itself is known, its use in identifying agents that inhibit cell growth stimulated by thioredoxin has not
Arizona Board of Regents, acting on behalf of the University of
Davis Minh Tam
Miller Raymond A.
Pepper Hamilton LLP
Ungar Susan
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