Development and characterization of an adriamycin-resistant sw480 human colon adenocarcinoma cell-line

An adriamycin resistant Sw480 human colon adenocarcinoma cell line (Sw480 Adr) was developed. Characteristics of the Sw480 Adr cells were compared with that of the parent Sw480 (Sw480 WT) cell line. Sw480 Adr cells were cross-resistant to mitoxantrone but not to cisplatin. Glutathione S-transferase enzyme activity as well as glutathione S-transferase pi content were increased 1.5 fold in the resistant cells, whereas P-170 glycoprotein content was increased several hundredfold. Sw480 Adr cells could be partly resensitised towards adriamycin by prior incubation with verapamil (1.65-3.3 mug/ml) and completely resensitised by cyclosporin A (2.5-5.0 mug/ml). Incubation of Sw480 Adr cells with various concentrations of adriamycin (200-1600 nM) during one week, did not change the P-170 glycoprotein levels. In addition, Sw480 Adr cells cultured in adriamycin-free medium for ten weeks (and passaged each week), showed no significant change in P-170 glycoprotein content. These results indicate that the most important mechanism for adriamycin resistance in Sw480 Adr human colon adenocarcinoma cells is overexpression of the P-170 glycoprotein, an ATP-driven drug efflux pump located in the plasma membrane. This overexpression of the P-170 glycoprotein in Sw480 Adr cells does not seem to be reversible.     

Glutathione and glutathione S-transferases in Barrett's epithelium.

Glutathione content, enzyme activity and isoenzyme composition of glutathione S-transferases were assayed in normal and Barrett''s esophageal epithelium of ten patients with Barrett''s esophagus. In addition, gastric and duodenal specimens from the same patients were also investigated. Glutathione content, glutathione S-transferase enzyme activity as well as glutathione S-transferase pi content were all significantly lower in Barrett''s epithelium as compared to normal esophageal mucosa. In contrast, glutathione S-transferase class alpha enzymes are markedly expressed in Barrett''s epithelium, whereas only low amounts are present in normal esophageal epithelium. Glutathione and glutathione S-transferase composition in Barrett''s epithelium show striking similarities with gastric epithelium, whereas duodenal epithelium is provided with considerable higher amounts of glutathione and glutathione S-transferases, except for levels of glutathione S-transferase class pi, which are lower. A significant negative correlation exists between glutathione S-transferase enzyme activity in the mucosa along the gastrointestinal tract, and the tumour incidence. Since glutathione and glutathione S-transferase are correlated with protection against cellular or cytogenetic damage, the low content of glutathione and glutathione S-transferases in the Barrett''s esophagus may be a factor of relevance for the increased tumour risk in this tissue.     

Biochemical characterization of resistance to mitoxantrone and adriamycin in Caco-2 human colon adenocarcinoma cells: a possible role for glutathione S-transferases.

Cytotoxicity of Adriamycin on human colon adenocarcinoma cell lines was investigated. Concentrations of Adriamycin producing 50% inhibition were very similar in HT29, Sw480, Sw620, and Sw1116 cells, whereas Caco-2 cells were relatively insensitive. As compared to the Sw1116 cell line, Caco-2 cells were also insensitive to mitoxantrone. Sensitivity to cisplatin, 5-fluorouracil, or ethacrynic acid was comparable in both cell lines. To find the mechanism for this mitoxantrone and Adriamycin resistance, several potential Adriamycin-detoxifying systems were characterized and quantified in both Sw1116 and Caco-2 cells. No dramatic differences in glutathione content and expression of both selenium dependent- and independent glutathione peroxidase, UDP-glucuronyltransferase, and cytochrome P-450 were found. However, highly significant differences in glutathione S-transferase activity were present, the expression of both class pi and class alpha glutathione S-transferases being much higher in the Caco-2 cell line. In addition, a slightly higher content of P-170 glycoprotein was present in the Caco-2 cells. These findings suggest that glutathione S-transferases, and to a lesser extent the P-170 glycoprotein, may be involved in mitoxantrone and Adriamycin resistance of Caco-2 colon carcinoma cells.     

Eradication of Helicobacter pylori restores glutathione S-transferase activity and glutathione levels in antral mucosa.

Glutathione S-transferases (GST) and glutathione peroxidases (GPO) are important in detoxification. GST activity in the mucosa of the gastrointestinal tract is inversely correlated with the development of gastrointestinal cancer. Helicobacter pylori (H. pylori) infection has been associated with gastric cancer. We studied GST activity and the substrate glutathione (GSH) in patients with H. pylori-associated gastritis. GST activity and isoenzyme levels, GPO activity and GSH levels were studied in antral biopsies of 38 H. pylori-positive patients, before and after eradication treatment. In 31 patients in whom H. pylori was successfully eradicated, antral GST enzyme activity before therapy was 532 (465 - 598) nmol / mg protein. min (mean and 95% confidence interval) and that after therapy was 759 (682 - 836) nmol / mg protein. min (P < 0.0001). Correspondingly, levels of GST alpha and GST-P1 were higher after eradication (P < 0.001). GSH concentration significantly increased: 21.2 (16.2 - 26.2) nmol / mg protein before and 27.1 (23.6 - 30.6) nmol / mg protein after therapy (P < 0.05). In 7 patients in whom H. pylori was not eradicated, GST activity was 671 (520 - 823) nmol / mg protein. min and 599 (348 - 850) nmol / mg protein before and after treatment respectively (P = 0.32). GSH levels were 17.4 (9.0 - 25.7) nmol / mg protein and 18.2 (9.1 - 27.3) nmol / mg protein, respectively (P = 0.84). No differences in antral GPO enzyme activity, both of selenium (Se)-dependent and total GPO, before and after successful treatment were found. Eradication of H. pylori infection increases GST activity and GSH levels in antral mucosa. Low GST activity and GSH concentration due to H. pylori infection might play a role in gastric carcinogenesis.     

Eradication of Helicobacter pylori Restores Glutathione S-Transferase Activity and Glutathione Levels in Antral Mucosa

Glutathione S-transferases (GST) and glutathione peroxidases (GPO) are important in detoxification. GST activity in the mucosa of the gastrointestinal tract is inversely correlated with the development of gastrointestinal cancer. Helicobacter pylori (H. pylori) infection has been associated with gastric cancer. We studied GST activity and the substrate glutathione (GSH) in patients with H. pylori-associated gastritis. GST activity and isoenzyme levels, GPO activity and GSH levels were studied in antral biopsies of 38 H. /pyfori-positive patients, before and after eradication treatment. In 31 patients in whom H. pylori was successfully eradicated, antral GST enzyme activity before therapy was 532 (465–598) nmol/mg protein-min (mean and 95% confidence interval) and that after therapy was 759 (682–836) nmol/mg protein-min ( P <0.0001). Correspondingly, levels of GST α and GST-P1 were higher after eradication ( P <0.001). GSH concentration significantly increased: 21.2 (16.2–26.2) nmol/mg protein before and 27.1 (23.6–30.6) nmol/mg protein after therapy ( P <0.05). In 7 patients in whom H. pylori was not eradicated, GST activity was 671 (520–823) nmol/mg protein min and 599 (348–850) nmol/mg protein before and after treatment respectively ( P =0.32). GSH levels were 17.4 (9.0–25.7) nmol/mg protein and 18.2 (9.1–27.3) nmol/mg protein, respectively ( P =0.84). No differences in antral GPO enzyme activity, both of selenium (Se)-dependent and total GPO, before and after successful treatment were found. Eradication of H. pylori infection increases GST activity and GSH levels in antral mucosa. Low GST activity and GSH concentration due to H. pylori infection might play a role in gastric carcinogenesis.     

Glutathione S-transferase and glutathione peroxidase expression in normal and tumour human tissues

Glutathione S-transferases play a central role in drug detoxification and have been implicated in the sensitivity of tumour cells to anticancer drugs. In this study, glutathione S-transferase (GST) isozyme expression in normal and tumour tissue from human lung, colon, stomach, breast, kidney and liver tissue has been quantified using sensitive and subunit specific radioimmunoassays (RIA), together with Western blot analysis and measurement of substrate metabolism. Glutathione S-transferase pi was the predominant GST in the majority of the tumours examined. The concentration of this enzyme was increased significantly in tumour tissue relative to normal lung, colon, and stomach tissue. A strong correlation was observed (r = 0.77, P less than 0.01) between GST activity and GST pi levels in those tumour samples. The concentrations of the alpha class GST, the predominant isoenzymes in normal stomach, kidney and liver, decreased dramatically in tumour tissue from these organs. Western blot analysis revealed the presence of novel polypeptides that cross-reacted with antisera raised against alpha and mu class GST. Our data demonstrates that although GST pi is the predominant GST isoenzyme in many tumours, significant levels of the other GST subunits are also present and collectively can represent a significant proportion of the GST content. Therefore the properties of all the GST isoenzymes need consideration when assessing the role of these proteins in drug resistance. Selenium-dependent glutathione peroxidase, an enzyme activity also implicated in the mode of action of certain antitumour agents, was also studied and shown to be the predominant glutathione-dependent peroxidase in all tumours except the hepatoma.     

Metallothionein content correlates with the sensitivity of human small cell lung cancer cell lines to cisplatin.

We have established cis-diamminedichloroplatinum(II) (cisplatin) resistant human small cell lung cancer cell lines, H69/CDDP0.2 and H69/CDDP, to investigate the mechanism of acquired resistance to cisplatin. H69/CDDP0.2 and H69/CDDP were 6- and 11-fold resistant to cisplatin compared with the H69 parental cell line. H69/CDDP was also resistant to cadmium chloride (2-fold), cis-diammine(glycolato)platinum (4-fold), 4-hydroperoxycyclophosphamide (3-fold) and 3-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-1-(2-chloroethyl)-1-nitrosour ea (4-fold) if the drug concentrations that inhibit cell growth by 50% from growth inhibition assay were compared. There was no significant difference in the cisplatin accumulation among these cell lines. Although DNA interstrand cross-link formations, determined by filter elution assay in H69/CDDP0.2 and H69/CDDP, was decreased to 20 to 30% of that in H69 parental cells, the repair capacity of DNA interstrand cross-links was equivalent in all three cell lines. Intracellular glutathione content was equal in all cell lines. H69/CDDP had the highest glutathione S-transferase activity (H69, 11 nmol/min/mg protein, H69/CDDP0.2, 12 nmol/min/mg protein; H69/CDDP, 74 nmol/min/mg protein, respectively) and an overexpression of glutathione S-transferase pi mRNA. The drug concentrations that inhibit cell growth by 50% for cisplatin in all cell lines were decreased by treatment with ethacrynic acid, an inhibitor of glutathione S-transferase pi, but this did not alter the relative degree of resistance. Intracellular metallothionein content (H69, 14 pmol/mg protein, H69/CDDP0.2, 22 pmol/mg protein; H69/CDDP, 33 pmol/mg protein, respectively) and expression of metallothionein mRNA were correlated with the drug concentrations that inhibit cell growth by 50% of the three cell lines for cisplatin and cadmium chloride. The present study suggested the importance of metallothionein in the mechanisms of cisplatin resistance.     

Differential effects on the metabolism of dimethylnitrosamine and aflatoxin B1 by hepatic microsomes from senescent rats

The ability of hepatic microsomes from senescent rats to metabolize the two potent hepatocarcinogens dimethylnitrosamine (DMN) and aflatoxin B1 (AFB1) was investigated. Seven and 24-month-old male Sprague-Dawley rats were used. Liver weights, and microsomal protein per gram tissue weight were higher, whereas cytochrome P-450 and cytochrome b5 were significantly lower in older rats. Glutathione S-transferases and NADPH cytochrome c reductase activities were dramatically reduced in senescent rats. There was no difference in the formation of formaldehyde from DMN in vitro (31 vs. 34 pmol/nmol P-450) between the young and old rats. In contrast, increased microsome mediated binding of AFB1 to DNA was observed in older rats (116 vs. 228 pmol/nmol P-450) suggesting the possibility of either quantitative or qualitative changes in P-450 species. Additionally the cytoplasmic GSH S-transferases from older rats affected lower inhibition of binding of AFB1 to DNA. These results indicated differential abilities in the hepatic microsomal metabolism of these two carcinogens which may cause differential effects of these carcinogens in senescent rats.     

Complementary DNA cloning, messenger RNA expression, and induction of alpha-class glutathione S-transferases in mouse tissues.

Glutathione S-transferases (EC 2.5.1.18) are a multigene family of related proteins divided into four classes. Each class has multiple isoforms that exhibit tissue-specific expression, which may be an important determinant of susceptibility of that tissue to toxic injury or cancer. Recent studies have suggested that alpha-class glutathione S-transferase isoforms may play an important role in the development of cancers. Several alpha-class glutathione S-transferase isozymes have been characterized, purified, and cloned from a number of species, including rats, mice, and humans. Here we report on the cloning, sequencing, and mRNA expression of two alpha-class glutathione S-transferases from mouse liver, termed mYa and mYc. While mYa was shown to be identical to the known alpha-class glutathione S-transferase complementary DNA clone pGT41 (W. R. Pearson et al., J. Biol. Chem., 263: 13324-13332, 1988), the other clone, mYc, was demonstrated to be a novel complementary DNA clone encoding a glutathione S-transferase homologous to rat Yc (subunit 2). The mRNA for this novel complementary DNA is expressed constitutively in mouse liver. It also is the major alpha-class glutathione S-transferase isoform expressed in lung. The levels of expression of the butylated hydroxyanisole-inducible form (mYa) are highest in kidney and intestine. Treatment of mice with butylated hydroxyanisole had little effect on the expression levels of mYc but strongly induced mYa expression in liver. Butylated hydroxyanisole treatment increased expression levels for both mYa and mYc to varying degrees in kidney, lung, and intestine. The importance of the novel mouse liver alpha-class glutathione S-transferase isoform (mYc) in the metabolism of aflatoxin B1 and other carcinogens is discussed.     

CARCINOGENESIS: Expression of cytochrome P450s and glutathione S-transferases in human esophagus with squamous-cell carcinomas

In order to clarify the expression of cytochrome P450 and glutathioneS-transferase in human esophagus, 41 samples of human esophaguswith squamous-cell carcinoma were investigated by immunoblotanalysis and enzyme assays. Cytochrome P450 1A2/1 was clearlyexpressed in micro-somes, and the amount in samples with tumoroustissue was significantly greater than that in samples withouttumorous tissues or in liver; cytochrome P450 2B6 and 3A4/3were expressed polymorphically. Aryl hydrocarbon hydroxylaseactivity was detected in microsomes and was greater in samplesfrom smokers than non-smokers. Patients who both smoked anddrank alcohol, however, had activity similar to that of patientswithout these habits. Glutathione S-transferase Ml and A1/2protein existed polymorphically in cytosol, and glutathioneS-transferase Pl-1 was detected in all samples.The frequencyof expression of the glutathione S-transferase A1/2 proteinwas greater in patients with Ml protein than in those without;no difference in the expression was seen for glutathione S-transferasePl-1. Neither smoking nor drinking influenced the expressionor activity of glutathione S-transferase. Our data support theidea that some carcinogens can be directly activated or inactivatedin human esophageal epithelium.     

Human glutathione S-transferase P1 polymorphisms: relationship to lung tissue enzyme activity and population frequency distribution

The association between glutathione S-transferase (GST) activity as measured by 1-chloro-2,4-dinitrobenzene (CDNB) conjugation and genotype at exon 5 and exon 6 of the human GSTP1 gene was investigated in normal lung tissue obtained from 34 surgical patients. These samples were genotyped for previously identified polymorphisms in exon 5 (Ile105Val) and exon 6 (Ala114Val) by PCR-RFLP and direct sequencing. GST enzyme activity was significantly lower among individuals with the 105 Val allele. Homozygous Ile/Ile samples (n = 18) had a mean cytosolic CDNB conjugating activity of 74.9 +/- 3.8 nmol/mg per min; heterozygotes (n = 13) had a mean specific activity of 62.1 +/- 4.2 nmol/mg per min and homozygous Val/Val (n = 3) had a mean specific activity of 52.5 +/- 4.5 nmol/mg per min. The CDNB conjugating activity measured for the Ile/Ile genotype group was significantly different from that observed in the Ile/Val group (P = 0.03), and from Ile/Val and Val/Val genotypes combined (P = 0.009). Mean GST activity values were consistently lower in individuals with genotypes containing the 105 valine allele, regardless of smoking exposure. Genotypes at codon 114 were also assessed but the mean GST activity was not significantly lower in individuals with the 114 valine allele. A new haplotype, present in two samples who were homozygous 105Ile and had a 114Val, was identified and proposed as GSTP1*D. Frequencies of the exon 5 and exon 6 polymorphisms were determined in samples obtained from European-Americans, African- Americans and Taiwanese. The differences observed were highly significant suggesting the possibility of GSTP1 genotype-associated, ethnic differences in cancer susceptibility and chemotherapeutic response.      

Antioxidant and xenobiotic-metabolizing enzyme gene expression in doxorubicin-resistant MCF-7 breast cancer cells

We investigated the expression of the genes for several antioxidant and xenobiotic-detoxifying enzymes in the multidrug-resistant variant of the human breast cancer cell line MCF-7, MCF-7/Dox. MCF-7/Dox is greater than 500-fold resistant to doxorubicin by clonogenic assay. Enzyme activity determinations in the cytoplasmic compartment of MCF-7/Dox revealed a 25-fold increase in glutathione peroxidase level compared to the parent line (mean +/- SD, 10 +/- 2.8 versus 0.4 +/- 0.24 nmol/min/mg; P less than 0.005). The activity of the other major hydrogen peroxide-detoxifying enzyme, catalase, was diminished in MCF-7/Dox (2.0 +/- 0.4 versus 4.8 +/- 1.4 mumol/min/mg; P less than 0.025 compared to MCF-7). Superoxide dismutase activity did not differ between the two cell lines. The specific activity of the xenobiotic-detoxifying enzyme DT-diaphorase was 4-fold lower in MCF-7/Dox compared to MCF-7 (DT-diaphorase, 117 +/- 45 versus 509 +/- 123 nmol/min/mg; P less than 0.005). Daunorubicinol-producing carbonyl reductase activity was equal in the two lines. Northern blot analysis demonstrated a 0.9-kilobase band of glutathione peroxidase mRNA in MCF-7/Dox; no glutathione peroxidase mRNA was detected in MCF-7. A 2.4-kilobase catalase and 0.7- and 1.4-kilobase superoxide dismutase mRNAs were detectable in MCF-7/Dox and MCF-7. When normalized to 28S RNA, no difference in the mRNA levels of catalase and superoxide dismutase in MCF-7/Dox and MCF-7 could be determined. DT-diaphorase mRNAs of 1.4 and 2.7 kilobases were found in both MCF-7/Dox and MCF-7 cells. A 1.2-kilobase mRNA homologous to the putative carbonyl reductase cDNA was also easily detectable in both MCF-7 and MCF-7/Dox. The amount of mRNA for both xenobiotic-detoxifying enzymes was decreased 2- to 4-fold in the doxorubicin-resistant cells. Southern blot analysis of PstI- and MspI-restricted genomic DNA revealed no evidence for amplification or rearrangement of the glutathione peroxidase gene. These results indicate that, in addition to the previously described overexpression of anionic glutathione S-transferase in MCF-7/Dox cells, an augmented glutathione peroxidase mRNA level is the major alteration in antioxidant and xenobiotic-detoxifying enzyme expression that could contribute to doxorubicin insensitivity in these multidrug-resistant breast cancer cells.     

Expression of glutathione S-transferases in normal gastric mucosa and in gastric tumors

Glutathione S-transferases from both normal gastric mucosa and its matched gastric tumors from 10 different patients were investigated. The transferases were purified and subsequently the isoenzyme composition was studied. Glutathione S-transferase (GST)-pi was present in all specimens in large amounts. Class alpha GSTs were present in 9 out of 10 normal specimens and in six tumors. In malignant tissue, expression of GST-pi was increased at the expense of class alpha GST. In six patients, the ratio GST-pi/GST-alpha was higher in tumorous versus normal tissue. On a Western blot, using a monoclonal antibody, GST-mu was shown to be present in both normal and malignant tissue from four patients, the other six patients completely missed the enzyme in their gastric tissue. When present, GST-mu amounts to only a few per cent of total GST protein. GST-pi was quantified by densitometric analysis of Western blots, treated with a monoclonal antibody against GST-pi. Both total GST enzyme activity as well as the absolute amounts of GST-pi protein were significantly higher in the tumors, as compared to its matched normal mucosa. The importance of this overexpression of GST-pi was previously unknown. However, the frequent occurrence of this phenomenon in many refractory tumors, and as shown now also in gastric cancers, suggests a role for GST-pi in the mechanism of anti-cancer drug resistance.     

Cellular glutathione (GSH) and glutathione S-transferase (GST) activity in human ovarian tumor biopsies following exposure to alkylating agents.

In vitro studies have suggested that elevated levels of the thiol glutathione (GSH) may be associated with acquired alkylating agent resistance, but there is currently little data on the relationship between elevated GSH and glutathione S-transferase levels and clinical alkylating agent resistance. In this study, GSH and glutathione S-transferase levels have been determined in 23 human ovarian tumor samples obtained prior to the onset of combination chemotherapy, and in 23 samples obtained after the development of acquired chemoresistance. GSH levels were 10-fold greater in human ovarian tumor cells obtained after alkylating agent resistance developed, than in biopsy samples obtained prior to treatment. No significant changes in the expression of total glutathione S-transferases were seen in relation to prior drug exposure.     

Resistance mechanisms in three human small cell lung cancer cell lines established from one patient during clinical follow-up

Mechanisms for resistance were studied in three classic type, human small cell lung cancer cell lines, GLC14, GLC16, and GLC19, that were established from one patient during clinical follow-up. Clinically the tumor changed from sensitive (GLC14) to completely resistant to (chemo)therapy (GLC19) during this period. The stain with JSB-1 antibody, detecting the Mr 170,000 multidrug resistance associated glycoprotein, was most pronounced in GLC16 and absent in GLC19. Intracellular Adriamycin (Adr) concentrations were decreased in GLC16 and GLC19 versus GLC14. Glutathione levels were 12.9, 15.5, and 16.6 micrograms/mg protein; total sulfhydryl groups were 36.5, 45.7, and 48.8 micrograms/mg protein; and glutathione S-transferase activity was 13, 29, and 43 nmol I-chloro-2,4-dinitrobenzene/min/mg protein for GLC14, GLC16, and GLC19, respectively. Incubation with DL-buthionine-S,R-sulfoximine increased Adr and cisplatin induced cytotoxicity, whereas X-ray induced cytotoxicity remained the same. Catalase activity increased from 0.88 to 1.73 to 3.83 mumol H2O2/min/mg protein in, respectively, GLC14, GLC16, and GLC19. Compared to GLC14 and GLC16, Adr induced a higher amount of DNA strand breaks in GLC19. In none of the three cell lines could Adr induced DNA strand breaks be repaired. X-ray induced a comparable amount of DNA strand breaks in all three cell lines but all cell lines were capable of repairing the X-ray induced DNA strand breaks within 90 min. It is concluded that a number of different mechanisms are operative and that some but not all of the observed changes in mechanisms for drug resistance in these lines correlate with the clinical data.     

Human leukemic myeloblasts and myeloblastoid cells contain the enzyme cytidine 5'-monophosphate-N-acetylneuraminic acid:Gal beta 1-3GalNAc alpha (2-3)-sialyltransferase

We have examined the role of CMP-NeuAc:Gal beta 1-3GalNAc-R alpha(2-3)-sialyltransferase in fresh leukemia cells and leukemia-derived cell lines. Enzyme activity in normal granulocytes using Gal beta 1-3GalNAc alpha-o-nitrophenyl as substrate was 1.5 +/- 0.7 nmol/mg/h whereas activity in morphologically mature granulocytes from 6 patients with chronic myelogenous leukemia (CML) was 4.2 +/- 1.6 nmol/mg/h (P less than 0.05). Myeloblasts from 5 patients with CML in blast crisis showed enzyme activity levels of 6.5 +/- 2.5 nmol/mg/h. From 2 patients with CML, both blasts and granulocytes were obtained, with higher enzyme activity in the patients' blasts (7.1 nmol/mg/h) than in their granulocytes (4.9 nmol/mg/h) in both cases, suggesting that the increase in enzyme activity is related to the differentiation or proliferation status of the CML cells. However, similarly high enzyme levels were also seen in myeloblasts from acute myeloblastic leukemia patients (5.6 +/- 1.4 nmol/mg/h) and in some acute myeloblastic leukemia-derived cell lines (KG1a and HL60), suggesting that increased levels of this enzyme are not directly correlated with the presence of the Ph1 chromosome. This alpha(2-3)-sialyltransferase activity can also be detected in normal peripheral blood lymphocytes and exhibits increased activity in chronic lymphocytic leukemia cells and acute lymphoblastic leukemia. These data suggest that the level of enzyme activity may vary with growth rate and maturation status in myeloid and lymphoid hemopoietic cells. Finally, we have identified a glycoprotein in acute myeloblastic leukemia cells that serves as a substrate for the alpha(2-3)-sialyltransferase. The desialylated form of the glycoprotein was resialylated in vitro by the purified placental form of this alpha(2-3)-sialyltransferase and exhibits a molecular weight of about 150,000.     

Habitual consumption of fruits and vegetables: associations with human rectal glutathione S-transferase

The glutathione (GSH)/glutathione S-transferase (GST) system is an important detoxification system in the gastrointestinal tract. A high activity of this system may benefit cancer prevention. The aim of the study was to assess whether habitual consumption of fruits and vegetables, especially citrus fruits and brassica and allium vegetables, is positively associated with parameters reflecting the activity of the GSH/GST enzyme system in human rectal mucosa. GST enzyme activity, GST isoenzyme levels of GST-alpha (A1-1, A1-2 and A2-2), -mu (M1-1) and -pi (P1-1), and GSH levels were measured in rectal biopsies from 94 subjects. Diet, lifestyle, GSTM1 and GSTT1 null polymorphisms were assessed. Mean GST enzyme activity was 237 nmol/min/mg protein (SD = 79). Consumption of citrus fruits was positively associated with GST enzyme activity [difference between high and low consumption: 28.9 (95% confidence interval (CI) = 9.3-48.6) nmol/min/mg protein], but was not associated with the other parameters. A positive association with brassica vegetables was found among carriers of the GSTM1-plus genotype [difference between high and low consumption: 22.6 (95% CI = 0.2-45.0) nmol/min/mg protein], but not among GSTM1-null individuals (-25.8 nmol/min/mg protein, 95% CI = -63.3-11.8). This is in line with a positive association between consumption of brassica vegetables and GSTM isoenzyme level [difference between high and low consumption: 67.5%, 95% CI = (6.8-162.7)]. Consumption of allium vegetables was not associated with GST enzyme activity, but negatively with GSTP1-1 levels [difference between high and low consumption: -23.3%, 95% CI = (-35.5; -8.6)]. Associations were similar among those with the GSTT1-plus and GSTT1-null genotype. In conclusion, variations in habitual consumption of fruits, particularly citrus fruits, and of vegetables, in particular brassica vegetables, among those with the GSTM1-plus genotype, may contribute to variations in human rectal GST enzyme activity.     

Glutathione transferase isoenzymes in normal and neoplastic human kidney tissue.

Glutathione transferase (GST) activity in the cytosolic fractions of renal cortex tumour was found to be significantly lower (215 +/- 156 mU/mg) than that present in the corresponding non-tumour (466 +/- 278 mU/mg) tissues. Using the immunoblotting technique, glutathione transferase isoenzymes expression in both tumour and non-tumour kidney was investigated. Alpha and pi class glutathione transferases were the most abundant enzymes in non-tumour kidney and were expressed by all samples investigated. Immunofluorescence analysis indicated that the pi class enzymes are localized mainly in the distal convoluted tubules, whereas alpha class enzymes are localized in the proximal tubules. In the tumour moiety the alpha class GST appears to be absent or expressed at low level as compared with non-tumour samples. On the contrary, no significant differences in the expression of pi class GST were found in tumour as compared with non-tumour tissues. Mu class GST protein was detected in 12 of 26 samples tested. When present, mu class GST constitutes a few per cent of total GST protein. Immunofluorescence studies indicate that mu class GSTs are localized within the distal convoluted tubules. According to the electrophoretic mobility at least two different mu GST subunits (26.5 and 27.5 kd) were found. In one sample only the faster mu class GST subunit was present, two samples expressed both types of GST subunits, whereas nine samples expressed only the slower GST subunit. With the exception of one sample, a reduction of mu class GST expression was seen in tumour as compared with non-tumour tissues. The decrease of activity seen in the cytosolic fraction of tumour kidney must be ascribed mainly to a reduction or to a lack of expression of alpha class GST and to a lesser extent of mu class GST.     

Glutathione S-transferases in small intestinal mucosa of patients with coeliac disease.

Patients with villous atrophy due to coeliac disease have an increased risk of developing small intestinal malignancies. Intestinal glutathione (GSH) and glutathione S-transferases (GST) are involved in the protection against carcinogenesis. The aim of this study was to evaluate GSH content and GST enzyme activity in small intestinal mucosa of untreated coeliacs compared to controls. We evaluated GSH content and GST enzyme activity, including the levels of GST classes alpha, mu, pi and theta, in small intestinal biopsies of untreated coeliacs (flat mucosa, Marsh IIIC, n = 12) compared to normal subjects (n = 23). Next, we evaluated GSH and GST's in coeliacs in remission (Marsh 0 - I, n = 11), coeliacs with persisting villous atrophy while on a gluten-free diet (partial villous atrophy, Marsh IIIA (n = 5); subtotal villous atrophy, Marsh IIIB (n = 6)) and patients with infiltrative / crypt-hyperplastic Marsh II lesions (n = 4). Total GST enzyme activity and content of GSTalpha are markedly suppressed in Marsh IIIC lesions compared to controls (resp. 220 +/- 79 vs. 464 +/- 189 nmol / mg protein*min (P < 0.001) and 2.79 +/- 2.46 vs. 6.47 +/- 2.29 mg / mg protein (P < 0.001)). In coeliacs in remission these levels normalized. Total GST enzyme activity and GSTalpha levels are proportionately lowered according to the degree of mucosal pathology in Marsh II, IIIA and IIIB. (Spearman's sigma correlation coefficient for total GST, -0.596, P < 0.001; GSTalpha, -0.620, P < 0.001). GSTmu, pi and theta and GSH levels are not significantly different in the selected study groups of mucosal pathology compared to controls. Total GST enzyme activity and content of GSTalpha in small intestinal mucosa are significantly lower in untreated coeliac disease compared to controls. In Marsh II, IIIA and IIIB, GST enzyme activity and GSTalpha content are proportionally lower according to the degree of mucosal pathology. Normal values are seen in coeliacs in remission. This correlation between coeliac disease and a suppressed GSH / GST detoxification system may explain in part the carcinogenic risk in untreated coeliac disease.     

鼻咽癌患者外周血血浆可的松水平和全血还原型谷胱甘肽含量的昼夜节律

背景与目的:谷胱甘肽与细胞对抗癌药物的解毒作用以及对放射性损伤的保护机制密切相关。本文通过对鼻咽癌患者和健康志愿者外周血血浆可的松水平和全血还原型谷胱甘肽含量的昼夜节律进行研究,从而为鼻咽癌患者进行肿瘤的时间治疗提供参考资料。方法:13名鼻咽癌患者(实验组)和14名健康志愿者(正常对照组)参加本项研究。每位受试者均从中午12点开始抽取外周静脉血4.5mL,每隔4h抽血一次,在24h内共抽血6次。血浆可的松水平的检测采用放射免疫法,全血还原型谷胱甘肽含量则采用高效液相色谱法进行检测。结果:鼻咽癌患者组和正常对照组的血浆可的松水平均呈现出明显的昼夜节律的特征,且节律特性相似。两组的血浆可的松水平的峰值均出现在早晨,而谷值均出现在午夜。不同时间点时,两组的全血还原型谷胱甘肽含量不同,且均具有显著性差异(重复测量的方差分析,F=5.18,P=0.02)。余弦分析表明,鼻咽癌患者组呈现出昼夜节律变化趋势(P=0.06),峰值出现在早晨(05∶02),正常对照组也呈现出明显的昼夜节律的特征,峰值出现在早晨(07∶44±01∶56)(P<0.01)。鼻咽癌患者组外周全血中还原型谷胱甘肽的节律调整均值为(19.60±1.11)nmol/mgprotein,正常对照组为(8.95±0.46)nmol/mgprotein。结论:包括晚期患者在内的鼻咽癌患者仍然具有正常的生物节律。鼻咽癌患者全血中还原型谷胱甘肽水平呈现出昼夜节律变化的趋势,与正常对照组节律特性相似。这为临床上选择恰当的时间对患者进行化疗和放疗提供了有价值的参考资料。