Characterization of 5-oxo-L-prolinase in normal and tumor tissues of humans and rats: a potential new target for biochemical modulation of glutathione.
5-Oxo-L-prolinase (5-OPase) is an enzyme of the gamma-glutamyl cycle involved in the synthesis and metabolism of glutathione (GSH), which is known to protect cells from the cytotoxic effects of chemotherapy and radiation. Previous studies on rats have shown that administration of the cysteine prodrug L-2-oxothiazolidine-4-carboxylate, a 5-oxo-L-proline analogue that is metabolized by 5-OPase, preferentially increases the GSH content of normal tissues while paradoxically decreasing it in the tumor and results in an enhanced in vivo tumor response to the anticancer drug melphalan. These observations initiated the present study of 5-OPase in experimental models and clinical specimens to investigate the potential role of this enzyme in the selective modulation of GSH in normal and tumor tissues. First, 5-OPase activity was measured in tissues of tumor-bearing rats, in the peripheral mononuclear cells of normal human subjects, and in surgically resected tumor and the adjacent normal tissues from patients. We found that the activity of 5-OPase in human kidney, liver, and lung is significantly lower than that found in rats. Second, we have raised a polyclonal IgG anti-5-OPase antibody by immunizing rabbits with purified 5-OPase from rat kidney. This antibody has very high affinity (shown by immunoprecipitation) and specificity (shown by Western blot) and cross-reacts with human 5-OPase (shown by Western blot and immunohistochemistry). It was then used to examine the distribution of 5-OPase in paired normal and neoplastic human specimens using Western blot and immunohistochemistry. Examination of paired normal and neoplastic tissues of stomach and lung revealed a significantly lower level of 5-OPase in tumor tissues than in the paired normal tissues. In colon tissues, there is no significant difference in 5-OPase level between the normal and tumor tissues. These findings could have implications for both carcinogenesis and therapy.
Chen
X
X
McGill Center for Translational Research in Cancer, Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital, McGill University, Montreal, Quebec, Canada.
Schecter
R L
RL
Griffith
O W
OW
Hayward
M A
MA
Alpert
L C
LC
Batist
G
G
D000818:Animals; D005260:Female; D005978:Glutathione / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D009369:Neoplasms / Q000201:enzymology*; D011752:Pyroglutamate Hydrolase / Q000276:immunology / Q000378:metabolism*; D011761:Pyrrolidonecarboxylic Acid; D051381:Rats; D011916:Rats, Inbred F344; D013844:Thiazoles / Q000494:pharmacology; D053778:Thiazolidines
D013844:Thiazoles; D053778:Thiazolidines; D011752:Pyroglutamate Hydrolase; D005978:Glutathione; D011761:Pyrrolidonecarboxylic Acid; C029536:2-oxothiazolidine-4-carboxylic acid