Radiation-induced upregulation of gamma-glutamyltransferase in colon carcinoma cells is mediated through the Ras signal transduction pathway.

The activity of gamma-glutamyltransferase (GGT) is frequently upregulated in tumor cells after oxidative stress and may thus increase the availability of amino acids needed for biosynthesis of the antioxidant glutathione. As gamma-radiation of tumor cells can result in oxidative stress, we investigated whether such treatments modulate the enzyme level in colon carcinoma CC531 cells. Radiation of these cells blocked cell proliferation, increased cellular size, initiated apoptosis and upregulated GGT activity and protein levels in a dose- and time-related manner. A slight but significant increase in the cellular level of reactive oxygen species (ROS) was found directly after radiation but appeared not to cause the GGT elevation. Thus, other mechanisms than cellular oxidative stress appear to be responsible for the radiation-induced upregulation of GGT. Stable transfection of activated Ras in a human colon carcinoma cell line expressing wild-type Ras resulted in an increased GGT level, while a reduced enzyme level was demonstrated in another cell line with constitutively activated Ras after stably transfection with a dominant-negative Ras mutant. Moreover, addition of specific protein kinase inhibitors that blocked downstream targets PI-3K and MEK1/2 of Ras, prior to and after radiation, attenuated the radiation-induced activation of GGT. These results support a role for Ras, being frequently activated after radiation, in regulating the level of GGT and also indicate that GGT participates in radioresistance.

Radiomodulatory effect of liposome encapsulated AK-2123 on tumor in mice exposed to hepatocarcinogen.

An attempt was made to evaluate the whole body gamma-radiation effect on tumor in the presence of free and liposome encapsulated AK-2123, a hypoxic cell radiosensitizer that has widely been used in combination with a number of cancer therapies such as thermotherapy, chemotherapy and radiotherapy. Entrapment efficiency of AK-2123 into liposome was determined by LASER Raman spectroscopy. Cancer induction in mice was carried out by repeated exposure of N-nitrosodiethylamine (DEN) in combination with partial hepatectomy. Parameters such as marker enzymes activities (GGT and AChE), rates of nucleic acid synthesis, viability modification factor and the histology of liver tissues monitored, supported the induction of cancer in liver. In addition, the effect of free as well as liposome encapsulated AK-2123 on haemopoietic parameters were also studied. It was observed that AK-2123 after incorporation into liposome afforded more efficient radiomodulatory effects than that of free AK-2123 as determined by the above-mentioned parameters. Neither free AK-2123 nor liposome encapsulated AK-2123 showed any detectable toxic effects on the mice. Thus, it is seen that treatment of cancer with a combination of radiation, a radiomodifier and a drug delivery system, opens a wide scope for exploitation for the improvement of existing cancer therapies.

[Gamma-glutamyltransferase (EC 2.3.2.2) activity in the course of brachytherapy of the uterine cervix and corpus carcinoma]. / Aktywnosc gamma-glutamylotransferazy (EC 2.3.2.2) w monitorowaniu brachyterapii raka szyjki i trzonu macicy.

The gamma-glutamyltransferase (EC 2.3.2.2) activity was measured in the blood serum obtained from 25 female patients with the uterine cervix carcinoma 10 and from 21 women with the uterine corpus carcinoma 10, treated by brachytherapy. The enzyme activity was determined in the five time points: before the brachytherapy, in the middle of the treating course, immediately after the application of the full dose of radium and in the seventh and the fourteenth day after the finishing the treatment. It was assumed the changes in the enzyme activity during the treatment course, and the highest values were observed immediately after the brachytherapy. It was noticed, that there is the direct connection between the enzyme activity changes and used brachytherapy method.

[The effect of chronic x-ray radiation at low doses on the gamma-glutamyl transpeptidase activity in rat organs and tissues]. / Vliianie khronicheskogo rentgenovskogo izlucheniia v malykh dozakh na aktivnost' gamma-glutamiltranspeptidazy v organakh i tkaniakh krys.

Effect of chronic low dose X-ray irradiation (total doses of 2.58, 5.16, 6.46, 7.75, 10.32 and 12.92 mC/kg) on gamma-glytamyltranspeptidase activity was investigated in rat organs and tissues which were different by radiosensitivity. Enzymatic activity was measured one hour after exposure. The increase in enzymatic activity was found in brain, liver, lungs, spleen, blood serum and partially in kidneys under all exposure doses. Exposure to 2.58, 5.16 and 12.92 mC/kg inhibited gamma-glutamyltranspeptidase activity in small intestines.

Increase of brain ammonia after microwave irradiation and its mechanism.

Microwave irradiation at 4kW for 0.4 sec applied to the heads of mice produced an increase in brain ammonia. This increase resulted from stimulated breakdown of glutamine initiated by microwave irradiation and proceeded until freezing of the brain. Two glutamine-related enzymes, in the brain, phosphate-dependent glutaminase (assayed in the presence of 200mM phosphate) and glutamine synthetase, were inactivated by microwave irradiation in a similar fashion. On the other hand, glutaminase activity in the presence of 10mM phosphate increased. This is considered to be a probable cause of the increase in brain ammonia. The increased 10mM-phosphate glutaminase activity remained stable in the precipitate fraction even after 5% Triton X-100 treatment.

Radiation inactivation probe of membrane-bound enzymes: gamma-glutamyltranspeptidase, aminopeptidase N, and sucrase.

gamma-Glutamyltranspeptidase (GGT), aminopeptidase N (AP-N), and sucrase in purified rabbit intestinal brush border membrane vesicles were irradiated in situ at -135 degrees C using high energy electrons. Surviving activities of the enzymes were measured as a function of radiation dose, and the functional unit target sizes (corresponding to carbohydrate-free polypeptides) were determined using target analysis. The in situ functional unit sizes were GGT 59 kDa, AP-N 59 kDa, and sucrase 63 kDa. Together with biochemical data determined previously, it is concluded that the noncovalently attached large (approximately 40 kDa) and small (approximately 25 kDa) subunits of GGT are both required for catalytic activity. Furthermore, these data suggest that (i) the membrane-bound form of AP-N consists of one or more noncovalently attached subunits of 59 kDa, each of which is enzymatically active; and (ii) in situ sucrase activity is associated with a subunit of 63 kDa which is noncovalently attached within the sucrase-isomaltase complex.

Radiation inactivation of membrane proteins: molecular weight estimates in situ and after Triton X-100 solubilization.

Target size analysis by radiation inactivation is widely used for molecular weight determination of membrane enzymes and receptors in situ without the need for prior solubilization or purification. However, since most molecular weight data available in the literature on membrane proteins involve the use of detergents for solubilization, the target sizes of membrane proteins in situ and after solubilization by detergent treatment have been compared. Using data from the literature and personal results, three different types of behavior of membrane proteins in presence of detergents were found: (i) uncoupling of subunits (electric eel acetylcholinesterase, placental steroid sulfatase, and human nonspecific beta-glucosidase); (ii) coupling of protein molecules (mouse liver neuraminidase, and rat liver insulin receptor regulatory component); and (iii) no major change in quaternary structure (rat liver insulin receptor, kidney gamma-glutamyltransferase, asialoglycoprotein receptor, insulin degrading enzyme, and human leucocyte neuraminidase). For all these proteins, there is a statistically significant increase in target size of about 24% over the value obtained in situ without detergent. A relatively large body of literature data involving a variety of membrane proteins, membrane types, and irradiation conditions (electron accelerators or 60Co sources, and proteins irradiated in lyophilized form or frozen solution) was examined, and it was concluded that target sizes of membrane proteins, irradiated in the presence of Triton X-100, should be diminished by a factor of about 24% to obtain the molecular weight value.

In situ determination of the molecular weight of hepatic gamma-glutamyl transferase and gamma-glutamyl hydrolase activities.

The molecular weight of gamma-glutamyl transferase from normal rat liver and hepatoma tissue was determined by radiation-inactivation and found to be approx 100000 in each case. The molecular weight previously reported for the subunit containing the gamma-glutamyl binding site (22000) is considerably less than that of the holoenzyme, suggesting that in situ the large subunit is implicated in both transferase and hydrolase activities.