Differential specificity of monochlorobimane for isozymes of human and rodent glutathione S-transferases

Monochlorobimane (MCB) has been used as a glutathione (GSH) specific fluorescent probe capable of delineating GSH heterogeneity in cellular systems. Generally, low concentrations of MCB (less than 50 microM) have been used to quantitatively label GSH in rodent cell lines. Incubation of the hamster cell lines, CHO AB1 and V79, with 10 microM MCB labeled 75 and 39% of the reduced GSH pool, respectively. In contrast, incubation of 7 different human cell lines with 10 microM MCB labeled less than 4% of the total reduced GSH pool. The human cell lines required 1000 microM MCB to label an average of 73% of the GSH pool (range, 60-88%). When using 1000 microM MCB to label GSH, flow cytometry results from 7 different cell lines (human and rodent) were in good agreement with high performance liquid chromatography and standard spectrophotometric analysis with regards to a rank ordering of the GSH content determined for each cell line. The human glutathione S-transferases B2B2, B1B2, psi, pi, and the rat transferases 1-2, 3-3, and 3-4 were isolated and purified for steady state kinetic analysis with MCB and GSH as the primary substrates. The human basic transferases, B1B2 and B2B2, had Km values for MCB of 354 and 283 microM and Vmax values of 33.3 and 34.6 mumol bimane-GSH/min/mg protein, respectively. The rat basic transferase 1-2 showed similar kinetic results with a Km of 199 microM and a Vmax of 35.5 mumol bimane-GSH/min/mg protein. The human neutral transferase (psi) had a Km for MCB of 204 microM with a Vmax of 6.5 mumol bimane-GSH/min/mg protein. In contrast, MCB has a high affinity for the rat neutral transferase with a Km of 2.6 microM and a Vmax of 35.1 mumol bimane-GSH/min/mg protein. The human acidic transferase (pi), the predominate transferase found in most human tumor cell lines, has a Km of 264 microM for MCB and a Vmax of 1.99 mumol bimane-GSH/min/mg protein. The kcat/Km values indicated that MCB is an excellent substrate for the rat neutral transferases while the human pi glutathione S-transferase showed the least reactivity. Collectively the data indicate that MCB fails to label GSH at lower concentrations (less than 50 microM) in human cell lines because of the reduced affinity of MCB for the human transferases and possibly also due to differences in glutathione S-transferase isozyme expression between rodent and human cell lines.     

Quantification of cellular viability by automated microscopy and flow cytometry

Cellular viability is usually determined by measuring the capacity of cells to exclude vital dyes such as 4′,6-diamidino-2-phenylindole (DAPI), or by assessing nuclear morphology with chromatinophilic plasma membrane-permeant dyes, such as Hoechst 33342. However, a fraction of cells that exclude DAPI or exhibit normal nuclear morphology have already lost mitochondrial functions and/or manifest massive activation of apoptotic caspases, and hence are irremediably committed to death. Here, we developed a protocol for the simultaneous detection of plasma membrane integrity (based on DAPI) or nuclear morphology (based on Hoechst 33342), mitochondrial functions (based on the mitochondrial transmembrane potential probe DiOC₆(3)) and caspase activation (based on YO-PRO^®-3, which can enter cells exclusively upon the caspase-mediated activation of pannexin 1 channels). This method, which allows for the precise quantification of dead, dying and healthy cells, can be implemented on epifluorescence microscopy or flow cytometry platforms and is compatible with a robotized, high-throughput workflow.     

石蜡包埋组织DNA含量的流式细胞分析对恶性淋巴瘤的诊断价值

 目的 探讨流式细胞术（FCM）对恶性淋巴瘤的DNA含量分析在肿瘤诊断、临床分期、分类中的意义。方法 采用流式细胞仪检测48例恶性淋巴瘤（ML）和30例淋巴结反应性增生（RLN）,石蜡包埋组织,并分析它们的DNA倍体及细胞周期变化。结果 48例恶性淋巴瘤石蜡包埋组织样本中17例为异二倍体,31例为二倍体,30例RLN为二倍体。恶性淋巴瘤的S期细胞比率（SPF）、增生指数（PI）均高于淋巴结反应性增生（P＜0.05）。结论 异倍体的出现对恶性淋巴瘤的诊断具有较高特异性。SPF,PI对恶性淋巴瘤和淋巴结反应性增生的鉴别具有重要意义。     

What role do glutathione S-transferases play in the cellular response to ionizing radiation?

The glutathione S-transferases (GST's) are cytosolic dimeric proteins that are composed of three family members, alpha, pi, and mu, and a fourth microsomal member. These four family members are primarily involved in cellular detoxification of xenobiotics and hydroperoxides. Recently, a strong correlation has been found between the overexpression of GST's and resistance to chemotherapeutic drugs. In comparison to chemotherapy, little is known about the role of GST's in the cellular response to ionizing radiation. To determine which GST's may be involved in this response, we have identified Chinese hamster ovary cell lines that possess different levels of alpha and pi GST isozyme activity. The survival of these cell lines to ionizing radiation was similar to that of wild-type Chinese hamster ovary-KI cells from which they were derived. Although differences in GST levels did not affect ionizing radiation sensitivity per se, we found that ionizing radiation decreased the amount of cytosolic pi GST without affecting alpha GST levels. Taken together, these data suggest that GST's are involved in the cellular response against oxidative stress generated by ionizing radiation.     

New sensitivity test using flow cytometry

Summary Flow cytometry (FCM) has been used to evaluate not only the malignancy of tumor cells but also the effects of chemotherapy. Here a new application of FCM for selecting the best antineoplastic agent in the chemotherapy for brain tumors is reported.Through our preliminary study using established brain tumor cell lines, the system for this sensitivity test was developed. Antineoplastic agents were placed in contact with monolayer-cultured cells; then cell viability and changes in the DNA histogram were analyzed by FCM. Cell viabilities were measured with the fluorescein diacetate (FDA) staining method, and the DNA histogram was analyzed by the propidium iodide (PI) staining method. The best antineoplastic agent was determined based on changes in cell viability and cell cycle. In other words, when markedly decreased viability as compared with that of the control, is measured by FCM, then the agents can be considered to have had a cytocydal effect on the tumor cells, and thus the sensitivity of the agents is able to be evaluated. If the viability of the tumor cell is observed to be similar to that of the control, the cytostatic effects of the agents are able to be evaluated only if a marked change is observed in the DNA histogram.After the preliminary study, this system was applied clinically to malignant brain tumor cases, resulting in success in selecting the best antineoplastic agent for each individual case. Our sensitivity test using this FCM established in vitro system has much potential value for clinical use.     

Modulation of human glutathione S-transferases by botanically defined vegetable diets.

Glutathione S-transferases (GSTs) conjugate activated xenobiotics with glutathione; thus, GST induction may improve detoxification and excretion of potentially harmful compounds. Using a randomized cross-over design, we tested the hypothesis that, in humans, serum GST-alpha concentration (GST-alpha) and GST activity increase with vegetable consumption and that this effect is GSTM1 genotype dependent. Twenty-one men (10 GSTM1-null and 11 GSTM1+) and 22 women (15 GSTM1-null and 7 GSTM1+), nonsmokers, 20-40 years of age and not on medications, ate four 6-day controlled diets: basal (vegetable-free), and basal supplemented with three botanically defined groups of vegetables (i.e., brassica, allium, and apiaceous). Fasting blood samples, collected on the last 2 days of each feeding period, were analyzed for GST-alpha, serum GST activity [against 1-chloro-2,4-dinitrobenzene (CDNB) and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl)] and peripheral-lymphocyte GST-mu activity (against trans-stilbene oxide). The brassica, but not allium or apiaceous, vegetable diets (relative to the basal diet) increased GST-alpha by 26% (P = 0.005) and GST (NBD-Cl) activity by 7% (P = 0.02) in the GSTM1-null individuals, particularly the women. Apiaceous vegetable supplementation decreased GST-alpha in the GSTM1+ men (P = 0.03). Among the GSTM1+ women, both brassica and the allium diets increased GST-mu activity by 18% (P = 0.02) and 26% (P = 0.001), respectively. The vegetable diets had no effect on GST (CDNB) activity, irrespective of GSTM1 genotype or sex. These results demonstrate that GSTM1 genotype has a significant effect on GST responses to diet and that brassica vegetables are most effective at inducing GST-alpha, whereas both brassica and allium vegetables induce GST-mu. GST responses were more pronounced in women than men, but it is not clear from this study whether this is a dose-per-body-weight or a sex-specific effect.     

Use of monochlorobimane for glutathione measurements in hamster and human tumor cell lines

The use of monochlorobimane (MCIB) as a fluorescence label for glutathione (GSH) quantitation was investigated in human tumor cell lines. When MCIB was used with a hamster fibroblast cell line under conditions where GSH was either depleted or elevated, an excellent correlation between bimane-GSH fluorescence and the standard cyclic GSH reductase assay (Tietze's) was accomplished. When the MCIB technique was applied to a human lung adenocarcinoma cell line, little or no GSH labeling was noted even at MCIB levels 10X higher than that used for the hamster line. HPLC analysis suggested that the source of the problem may be the affinity for MCIB to glutathione S-transferase. By using higher dye concentrations and longer staining times, adequate staining was possible. While the MCIB technique may have problems quantitating GSH levels between cell types, the possibility of examining GSH heterogeneity in solid tumor biopsies remains feasible.     

Hematopoietic cellular therapy: implications for the flow cytometry laboratory

Advances in hematopoietic stem cell transplantation, gene therapy, and immunotherapy have necessitated a host of novel monitoring procedures. Cell sorting is also coming of age as a clinical procedure designed to organize hematopoietic grafts for specificity of cellular components designed for individual patients or diseases. This article has focused on these novel developments in their historical context. The next generation of transplantation flow cytometry promises to be an exciting one.     

Chlorambucil-monoglutathionyl conjugate is sequestered by human alpha class glutathione S-transferases.

The spontaneous reaction of 110 microM chlorambucil (4-[p-[bis(2-chloroethyl)amino]phenyl]-butanoic acid; CHB) with 5 mM GSH at 37 degrees C in physiological phosphate-buffered saline for 35 min gave primarily the monoglutathionyl derivative, 4-[p-[N-2-chloroethyl,N-2-S-glutathionylethyl]amino]phenyl]-butano ic acid; CHBSG) and the diglutathionyl derivative, 4-[p-[bis(2-S-glutathionylethyl]amino]phenyl]-butanoic acid (CHBSG2) with small amounts of the hydroxy-derivatives: 4-[p-[N-2-chloroethyl,N-2-hydroxy-ethyl]amino] phenyl-butanoic acid (CHBOH) and 4-[p-[N-2-S-glutathionylethyl-2-hydroxyethyl]amino]phenyl]-butanoi c acid (CHBSGOH). The inclusion of approximately physiological amounts of human glutathione S-transferases (GSTs) A1-1, A2-2, P1-1, M1a-1a M3-3 or P1-1 (for nomenclature see Mannervik et al., 1992, Biochem. J., 282, 305) had little or no catalytic effect on these reactions as determined by loss of CHB. However, GTSs A1-1 and A2-2 were associated with a significant increase of CHBSG at the expense of CHBSG2 + CHBSGOH suggesting that these GTs sequestered CHBSG at the active site. This interpretation was supported by inhibition studies which showed that CHBSG was a pure competitive inhibitor of the activity of GSTs A1-1 and A2-2 towards 1-chloro-2,4-dinitrobenzene with Ki's of 1.3 and 1.2 microM respectively. GSH transferases P1-1 and M1a-1a were inhibited by CHBSG above 10 microM. Incubation of 2 microM CHB, a concentration which may be of more significance for chemotherapy, in the presence or absence of GST A1-2 (20-50 microM) showed catalysis of GSH monoconjugation equivalent to 18% of the spontaneous rate. However, the dominant effect again was the sequestration of CHBSG which reached 74.3 +/- 1.5 (SEM)% of the total reactants at 60 min compared to 28.9 +/- 0.3(SEM)% in controls. CHBSG, although possessing a potential electrophilic centre, showed no detectable alkylation of plasmid DNA but indirect evidence was obtained that it alkylated other cellular macromolecules. It is concluded that the contribution of GSTs to catalysis of CHB detoxication will depend on factors not previously considered, namely the relative molarities of CHB, CHBSG and GSTs, and the cellular capacity to excrete CHBSG to relieve product inhibition.     

Quantification of induction of rat oesophageal, gastric and pancreatic glutathione and glutathione S-transferases by dietary anticarcinogens

Four dietary, naturally occurring anticarcinogens (flavone,coumarin,     

Enhancement of rat hepatic and gastrointestinal glutathione and glutathione S-transferases by {alpha}-angelicalactone and flavone

The naturally occurring anticarcinogens flavone and     

Modulation of glutathione S-transferases and glutathione peroxidase by the anticarcinogen butylated hydroxyanisole in murine extrahepatic organs.

Induction of glutathione S-transferases (GST) by the anticarcinogen butylated hydroxyanisole (BHA) has been examined in lung, kidney and small intestine of male and female BALB/c mice. BHA produced maximal induction of GST in the gut and although it increased GST levels in the kidney, it had little effect on pulmonary GST. Dietary BHA induced Alpha (Ya and Yk), Mu (Yb) and Pi (Yf) class GST subunits at least 10-fold in the small intestine but, by contrast, selenium-dependent glutathione peroxidase activity was reduced by approximately 4-fold in this organ following BHA treatment. In the kidney, all of the GST subunits, apart from Yk in males, showed modest levels of induction by BHA. However, a pronounced sex difference in the expression of renal alpha class subunits in both control and BHA-treated mice was observed, with female mice expressing approximately 4-fold greater levels of Ya and Yk than male mice. All renal GST were localized primarily in the proximal tubules. Dietary BHA was found to have the least inductive effect in the lung, where the GST were localized solely in the bronchi. The pulmonary Mu class GST subunits were induced approximately 2-fold by BHA; the expression of other GST was marginally increased by this inducer. Alpha class GST was also subject to sexual differentiation in the lung with female mice possessing higher levels of Yc and Yk than males. The Ya-type subunit was not detected in the lung nor was it induced by BHA.     

The glutathione S-transferases: influence of polymorphism on cancer susceptibility.

The glutathione S-transferase supergene family is an important part of cellular enzymic defence against endogenous and exogenous chemicals, many of which have a carcinogenic potential. However, while a wide variety of chemicals can act as substrates for different members of the supergene family, the precise function of these enzymes remains unclear. The supergene family comprises several gene families that include polymorphic loci, prompting the hypothesis that allelic variants associated with less effective detoxification of potential carcinogens can confer an increased susceptibility to cancer. For example, the null genotypes at the mu class GSTM1 and theta class GSTT1 loci have attracted particular interest, and recently identified allelic variants at the mu class GSTM3 and pi class GSTP1 loci are also putative susceptibility candidates. However, while associations between GSTM1 and GSTT1 genotypes and risk have been observed in some case-control studies in lung, bladder and colon cancers, other studies have reported contrary findings, and the importance of these polymorphisms in mediating the risk of smoking-related cancers remains generally unproven. We describe the influence of glutathione S-transferase polymorphisms on the risk of several cancers, including basal cell carcinoma of skin. In the latter cancer, associations between tumour numbers, site and accrual have been observed, suggesting a role for GST enzymes in the detoxification of the products of ultraviolet radiation-induced oxidative stress. We review below current knowledge of polymorphism in GST loci, possible in vivo GST substrates, and the difficulties of determining the role of this complex gene family on the basis of available epidemiological data.     

A predictive assay for human tumor cellular response to hyperthermia using dansyl lysine staining and flow cytometry

The heat response of five human tumor biopsies has been examined using the fluorescent probe dansyl lysine and multiparameter flow cytometry. Dansyl lysine has previously been shown to possess specificity for heat killed mammalian cells. The human tumors tested included a cervical squamous cell carcinoma, malignant melanoma, colon adenocarcinoma, ovarian carcinoma, and a mesothelioma. The samples were excised, mechanically disrupted into single cell suspensions and heated in vitro for various lengths of time at 45 degrees C. The cells were returned to 37 degrees C incubation for 12 to 15 hours prior to staining with dansyl lysine. The fraction of cells staining dansyl lysine was quantitated by flow cytometry after gating on high forward angle light scatter and 90 degrees C light scatter. This gate excluded much of the normal cell contamination within the tumor sample. The data show that the heat response of human tumor biopsies varied significantly, with cervical carcinoma and malignant melanoma being the most resistant and the mesothelioma and ovarian carcinoma the most heat sensitive. Finally, evidence is presented for the expression of thermotolerance in ovarian carcinoma and mesothelioma biopsies pre-heated in vitro. Dansyl lysine appears to be useful in measuring the intrinsic cellular heat sensitivity of human tumors and in determining the kinetics of decay of thermotolerance following an initial heat exposure.     

Simultaneous detection of cellular ras p21 oncogene product and DNA content by two-parameter flow cytometry

The rapid identification of the expression of oncogene products in specific cell types could be useful to investigate normal and malignant cell proliferation. We have developed a sensitive fixation - permeabilization technique (with 70% ethanol and 0.01% Triton x 100) for the detection of p21 ras oncoprotein and DNA content. Cell suspensions with negligible cell clumping, bright specific immunofluorescent staining were obtained with this method. Bivariate flow cytometry was then used to quantitate simultaneously the distribution of anti p21 ras oncoprotein (with a specific FITC-labeled antibody) and of total DNA (with propidium iodide). The study was carried out in human leukemic cell lines HL-60 and K562, human breast carcinoma cell line MCF-7 and fresh neoplastic cells from human acute leukemia. The p21 ras oncoprotein was found in all phases of cell cycle. The degree of its expression, however, varied widely in diploid (C0/G1) cells from different samples, which could be related to differences in the relative proportion of G0 and G1 cells. Compared to the conventional gel electrophoretic technique, the use of bivariate FCM is feasible, fast, requires fewer cells per sample (2 x 10(6] and allows both the ras oncogene expression in intact cell populations as well as its relationship with cell cycle phases to be studied.     

Measurements of cellular proliferation by H-3-thymidine-labeling index and flow cytometry in breast cancer patients

In 50 node negative breast cancer patients, tumor S-phase fraction (SPF) was determined by H-3-thymidine labeling index ((HTLI)-H-3) or flow cytometry (FC). Forty-five patients had tumor cell kinetics measured by both techniques. Twenty-three patients were classified as having high proliferative activity and 22 low by (HTLI)-H-3, while 32 as highly proliferating and 13 low proliferating by FC. In 30 patients only, both indices agreed on identifying high or low proliferative activity. These results suggest the need of more careful attention to standardization and quality control of cell kinetic data before carrying out clinical trials based on these parameters.     

胃镜活检胃癌组织DNA与RNA含量流式细胞分析的诊断意义

目的:探讨胃癌基因学的诊断标准及准确率。方法:用FCM对32例术前经胃镜取材的胃癌组织细胞DNA、RNA含量进行定量分析,经用胃镜活检取材的肿瘤组织,采用流式细胞光度分析技术。结果DNA异倍率为71 .43% (23/32), RNA增高率为85 .71% (27/32); 用“标准DI”和“标准RI”为指标判断胃癌的准确率分别为78. 57% (25/32)和83 .33%(27/32);且双指标同时应用准确率为92 .86% (30/32),高于任一单指标。结论:“标准DI”和“标准RI”是判断胃肿瘤良、恶性生物学性质的理想指标。     

Autofluorescence of gastric cancer--study by flow cytometry

Autofluorescence from cancer cells and non-cancerous cells of the stomach was measured by flow cytometry. The number of cells with fluorescence and its intensity was higher in cancer than in the surrounding mucosa. Preliminary results suggest that, if fluorescence emission from non-cellular substances is eliminated, measurement of fluorescence excited by laser illumination would permit earlier detection of gastric cancer.