Chemistry: analytical and immunological testing – Cancer
Reexamination Certificate
1998-09-03
2001-10-09
Warden, Jill (Department: 1743)
Chemistry: analytical and immunological testing
Cancer
C436S813000, C436S119000, C436S120000, C435S004000
Reexamination Certificate
active
06300136
ABSTRACT:
FIELD OF THE INVENTION
The field of the invention is cysteine levels in mammalian tissues.
BACKGROUND OF THE INVENTION
Low molecular weight thiol-containing compounds play a key role in protecting cells from the toxic effects of ionizing radiation, other free-radical-generating reactions, reactive oxygen species and chemical toxins. The only thiol-containing amino acid, cysteine, is considered to be a relatively minor cellular component in its free form, often reported to be present at concentrations of only a few micromolar in normal tissues (Loh et al., 1990, Rad. Res. 121:98-106). The presence of cysteine in the tripeptide glutathione (GSH) provides a much greater reserve of intracellular thiols than does free cysteine. Total intracellular concentrations of glutathione are typically more than 1 mM and have been reported to be as high as 25 mM in some cells (Post et al., 1983, Biochem. Biophys. Res. Comm. 114:737-742). Intracellular protein thiols comprise an additional 25 mM cysteine, while extracellular proteins, or the extracellular portion of transmembrane proteins, contain predominantly cysteine-cysteine disulfides (cystine) (Jocelyn, 1972,
Biochemistry of the sulfhydryl group
, Academic Press, New York). Enzymes such as glutathione-S-transferase and glutathione peroxidase have evolved to use glutathione, not cysteine, as the nucleophilic acceptor or reductant in cellular protection against toxic xenobiotics (particularly electrophiles) (Reed, 1990, Ann. Rev. Pharm. Toxicol. 30:603-663; Fahey and Sundquist, 1991, Adv. Enzym. 64:1-53).
Studies on the contrasting roles of radiation sensitization by oxygen versus protection by aminothiols have, in similar fashion, emphasized the role of glutathione. Despite this emphasis on the role of glutathione, it remains important to consider the auxiliary role of free cysteine, particularly in protecting cells from damage produced by ionizing radiation. This is because cysteine, due to its neutral charge and smaller size, is expected to have greater access to the negatively charged DNA and, therefore, is expected to provide greater protection from the damaging effects of ionizing radiation. (Zheng et al., 1988, Rad. Res. 114:11-27; Bump et al., 1992, Rad. Res. 132:94-104). However, the prior art does not disclose that tumor cells might have increased concentrations of free cysteine or that this might be a possible mechanism of radiation resistance in tumor cells.
Studies concerning the role of free cysteine in protecting cells from ionizing radiation have been hampered by the fact that cysteine is inherently difficult to measure except as part of the total non-protein sulfhydryl (NPSH) pool (Ellman, 1959, Arch. Biochem. Biophys. 82:70-77). For instance, there are no sensitive biochemical assays for the measurement of free cysteine analogous to the recycling assay for measuring glutathione levels developed by Tietze (1969, Anal. Biochem. 27:502-522). Further, development of more specific assays, based on HPLC, has been hindered by the fact that cysteine and its derivatives are extremely hydrophillic. Thus, cysteine and its derivatives tend to elute with the solvent and salt fronts used in HPLC rendering them difficult to resolve using this method (Allison and Shoup, 1983, Anal. Chem. 55:8-12; Fahey et al., 1983, In:
Radioprotectors and anticarcinogens
, pp. 103-120, Academic Press, New York). Another difficulty hampering the measurement of free cysteine during sample preparation is the presence of enzymes which degrade glutathione, such as y-glutamyltranspeptidase, to cysteine. Indeed, previous studies have suggested that the high cysteine levels observed in tissues such as kidney and liver are artifacts caused by y-glutamyltranspeptidase degradation of glutathione (Standeven and Wetterhahn, 1991, Toxicol. Appl. Pharmacol. 107:269-284).
There has been a long-felt but unfilled need for methods to diagnose tumors in mammals, particularly in the case of tumors which avoid diagnosis using conventional methods. There is also a need to elucidate and measure resistance of tumors to therapies, such as radiation and chemotherapy, and to adjust the therapy for treatment of such tumors. The present invention meets these needs.
SUMMARY OF THE INVENTION
The invention relates to a method of detecting a tumor in a mammalian tissue. The method comprises measuring the concentration of free cysteine in the tissue, wherein a concentration of free cysteine in the tissue higher than about 80 &mgr;M to about 250 &mgr;M is an indication that the tissue bears a tumor.
In one aspect, the mammalian tissue is selected from a group consisting of lung tissue, esophageal tissue, gastro-esophageal tissue, and cervical tissue.
In one embodiment, the mammalian tissue is lung tissue and the concentration of free cysteine in the tissue higher than 200 &mgr;M is an indication that the tissue bears a tumor.
In another embodiment, the mammalian tissue is esophageal tissue and the concentration of free cysteine in the tissue higher than 100 &mgr;M is an indication that the tissue bears a tumor.
In yet another embodiment, the mammalian tissue is gastro-esophageal tissue and the concentration of free cysteine in the tissue higher than 100 &mgr;M is an indication that the tissue bears a tumor.
In yet another embodiment, the mammalian tissue is cervical tissue and the concentration of free cysteine in the tissue higher than 100 &mgr;M is an indication that the tissue bears a tumor.
The invention also includes a method of detecting the growth, remission, or stasis of a tumor in mammalian tissue before, during or after administration of antitumor therapy. The method comprises measuring the concentration of free cysteine in the tissue before, during or after administration of the antitumor therapy. A higher concentration of free cysteine in the tissue compared with the concentration of free cysteine in the tissue before administration of the antitumor therapy is an indication of the growth and/or metastasis of a tumor in a mammal before, during or after administration of antitumor therapy.
In one aspect, the antitumor therapy is selected from a group consisting of chemotherapy and radiation therapy.
The invention also includes a method of detecting the growth, remission, or stasis of a tumor in a mammalian tissue before, during or after administration of antitumor therapy. The method comprises of measuring the concentration of free cysteine in the tissue before, during or after administration of antitumor therapy. The same or a lower concentration of free cysteine in the tissue compared with the concentration of free cysteine in the tissue before the administration of antitumor therapy is an indication of the remission, decrease in size, slowing of growth, reduction or cessation of metastasis, or stasis of a tumor in a mammal before, during or after administration of antitumor therapy.
In one aspect, the antitumor therapy is selected from a group consisting of chemotherapy and radiation therapy.
The invention includes a method of assessing the resistance of tumor cells to antitumor therapy. The method comprises measuring the concentration of free cysteine in the tumor cells and measuring the concentration of free cysteine in non-tumor cells where there is a direct correlation between the concentration of free cysteine in the tumor cells and the resistance of the tumor cells to antitumor therapy such that a higher concentration of free cysteine in a tumor cell compared with the concentration of free cysteine in an otherwise identical tumor cell is an indication that the tumor cell is more resistant to the antitumor therapy.
In one aspect, the tumor cells are in a tumor tissue.
In another aspect, the antitumor therapy is selected from a group consisting of chemotherapy and radiation therapy.
In one embodiment, the antitumor therapy is radiation therapy.
The invention also includes a method of identifying a compound which affects the concentration of free cysteine in a mammalian tissue. The method comprises contacting the tissue with a test compound and comparing the concentratio
Evans Sydney M.
Koch Cameron J.
Cole Monique T.
Morgan, Lewis & Bockius, L.L.P.
The Trustees of the University of Pennsylvania
Warden Jill
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