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.     

Determination of intracellular reduced glutathione and glutathione related enzyme activities in cisplatin-sensitive and resistant experimental ovarian carcinoma cell lines

The level of GSH in ovarian carcinoma cells which were sensitive and resistant to cisplatin was serially determined following tumor removal from the animals, in addition, activities of GSH-reductase and GSH-S-transferase were assessed. The GSH level in the resistant line (O-342/DDP) was almost twice as high as that in its sensitive counterpart (O-342), when determined immediately following removal of the tumor (1.55 +/- 0.47 vs. 0.81 +/- 0.32 nmol/10(6) cells). Culturing the cells resulted in a decrease of GSH levels in both cell lines during the first 4 h. Thereafter, GSH levels in both cell lines increased up to 24 h. At this time the GSH level was higher in O-342 than in O-342/DDP. GSH-reductase activity in O-342/DDP cells was significantly higher than in O-342 cells when the enzyme was determined immediately after tumor removal; at the same time there was no difference in activity of GSH-S-transferase between two cell lines. After 24 h in culture, no significant difference between O-342 and O-342/DDP cells could be observed in the activity of the two enzymes.     

Tumor antigen on benign adenomas and on murine lung carcinomas quantitated by a two-site monoclonal antibody assay

TSP-180 is a Mr 180,000 cell surface protein found on several murine lung carcinomas but not on fibroblast or sarcoma cell lines. Monoclonal antibody 135-13C binds to TSP-180 with high affinity but could not be used to quantitate the protein in tumors and normal tissue (Cancer Res., 41: 3465-3470, 1981). TSP-180 was purified from a transplantable BALB/c carcinoma (line 1 cells) by immunoaffinity chromatography and used as an immunogen to produce another monoclonal antibody (346-11A) to a different epitope on the molecule. A two-site solid-phase radioimmunoassay for TSP-180 was developed to quantitate TSP-180 in tissue extracts. The assay can detect as little as 3 ng of TSP-180 in samples up to 10 mg of protein. Analyses of several lung carcinoma cell lines confirmed that TSP-180 is present in all (five of five) cell lines ranging from 40 to 800 ng per mg of cell protein extract. Mouse tissues from normal and tumor-bearing mice were analyzed for TSP-180. Values for line 1 tumors growing i.m. were about 70 ng of TSP-180 per mg of protein. Normal tissue from normal and tumor-bearing mice contained low levels of TSP-180 from less than 0.3 ng/mg of protein for liver to a high of about 11 ng/mg of protein for leg muscle. Finally, small benign urethane-induced adenomas (1 to 2 mm) from BALB/c mice had moderate amounts of TSP-180 (11 ng/mg of protein), while two primary lung adenocarcinomas in the same animals had 20 and 47 ng/mg of protein, respectively, suggesting that TSP-180 expression may increase with increasing cancer of these tumors. Analyses of individual urethane-induced lung tumors from A/J mice showed that 11 of 13 carcinomas had high levels of TSP-180, while only 1 of 6 adenomas had a detectable amount of TSP-180, and that was at a moderate level. Specific quantitation of tumor markers in normal, benign, and malignant lung tissue may be helpful in identifying different levels of gene expression as tumors progress to more malignant states.     

Profiles of prostaglandin biosynthesis in sixteen established cell lines derived from human lung, colon, prostate, and ovarian tumors

The profiles of prostanoid biosynthesis from endogenous arachidonic acid in 16 established cell lines derived from 4 histological classes of human carcinomas were determined by capillary gas chromatography-mass spectrometry. Detectable quantities of prostanoids were isolated from the culture medium of cell lines representative of the different histological classes of human tumors: colorectal adenocarcinomas (one of three cell lines); ovarian adenocarcinomas (one of three cell lines); prostate adenocarcinomas (zero of two cell lines); non-small cell carcinomas of the lung (four of five cell lines); and small cell carcinomas of the lung (zero of three cell lines). Prostaglandins E2 and F2 alpha were the only prostanoids synthesized in detectable quantities. Prostaglandin E2 biosynthesis (mean +/- SD), pmol/10(6) cells, n = 4) in cell lines exhibiting positive prostaglandin H synthase activity was: LoVo (colorectal adenocarcinoma, 0.4 +/- 0.1); A2780 (ovarian adenocarcinoma, 1.3 +/- 0.3); NCI-H322 (bronchioloalveolar cell carcinoma, 8.4 +/- 3.1); NCI-H358 (bronchioloalveolar cell carcinoma, 7.8 +/- 2.4); EKVX (adenocarcinoma of the lung, 21.3 +/- 5.5); and A427 (large cell undifferentiated carcinoma of the lung, 12.6 +/- 2.8). Prostaglandin F2 alpha production (pmol/10(6) cells +/- SD) was: LoVo (0.3 +/- 0.1); NCI-H322 (0.6 +/- 0.2); NCI-H358 (0.4 +/- 0.1); EKVX (1.8 +/- 0.4); and A427 (11.1 +/- 3.1). These findings suggest that within certain limitations cultured tumor cells provide simplified experimental systems for determination of prostaglandin biosynthetic characteristics of human tumors and that prostanoid biosynthesis may be particularly characteristic of certain non-small cell carcinomas of the lung.     

Glutathione depletion causes cell growth inhibition and enhanced apoptosis in pancreatic cancer cells

BACKGROUND: Recent studies have demonstrated that various tumors express enhanced levels of the radical scavenger glutathione (GSH). Moreover, there are grounds for claiming that GSH plays a crucial role in cell proliferation and tumor resistance. In the current study, we investigated the relation between cell growth and GSH levels in the pancreatic adenocarcinoma cell line, AsPC-1, and the significance of GSH in tumor resistance to chemotherapy. METHODS: Cell growth in AsPC-1 was initiated through transforming growth factor-alpha (TGF-alpha) or fetal calf serum (FCS). Then, cell cycle, cell proliferation, and cellular GSH content were analyzed at different times in the presence or absence of buthionine sulfoximine (BSO). The impact of GSH on chemotherapy-induced apoptosis was studied using 5-fluorouracil or melphalan in the presence or absence of BSO. Finally, we compared the GSH content of 15 pancreatic tumor specimens with 10 normal pancreatic tissue specimens. RESULTS: Analysis of GSH in pancreatic tissues demonstrated increased GSH levels in cancerous compared with normal tissue (17.5 +/- 2.3 vs. 8. 8 +/- 1.4 nmol/mg protein; P < 0.004). Incubation of AsPC-1 with TGF-alpha or FCS resulted in cell proliferation and cell cycle activity, whereas GSH content was not altered. Incubation of GSH-depleted cells with TGF-alpha did not stimulate cell growth. In addition, GSH-depletion resulted in an increased rate of apoptosis after melphalan (6.3 +/- 0.3 % vs. 11.2 +/- 0.3 %; P < 0.001), but not after 5-fluorouracil treatment. CONCLUSIONS: Taken together, our results show enhanced GSH levels in pancreatic carcinoma and an essential role of GSH in cell proliferation and in resistance of AsPC-1 cells. Therefore, GSH-depletion may improve the efficacy of adjuvant therapy in pancreatic carcinoma.     

Heterogeneity in the radiation survival curves and biochemical properties of human lung cancer cell lines

Human lung cancers of distinct histology exhibit different responses to radiation therapy in vivo. For examination of the basis of this phenomenon, the radiation survival curves and levels of relevant enzymes were determined in 16 lung cancer cell lines derived from tumors of different histology. These included lines from 5 adenocarcinomas, 7 small cell tumors, 3 variant small cell tumors, and 1 large cell tumor. These findings were compared to those obtained with the use of a normal skin fibroblast cell line. Whether cloned in liquid culture or soft agarose, cell lines had similar radiation survival curves. These curves were consistent with the apparent in vivo radiation responsiveness of the tumors. Although considerable heterogeneity in radiation survival curves was observed among the cell lines, cells from large cell lines and small variant lines had pronounced shoulders and extrapolation numbers (n) from 5.6 to 14. In contrast, cells from small cell lines and adenocarcinoma cell lines were more "sensitive" (-n values of 1-3.3). In these cell lines, levels of DNA polymerase beta, glutathione (GSH), GSH transferase, GSH reductase (NAD(P)H), gamma-glutamyltransferase did not correlate with radiation parameters of sensitivity. DNA polymerase beta and GSH levels were, however, higher than those in a line of normal skin fibroblasts. These cell lines may be useful in identifying the basis of the variable responsiveness of human lung cancer cells to ionizing radiation.     

Determinants of drug response in a cisplatin-resistant human lung cancer cell line

To elucidate the mechanism(s) of cisplatin resistance, we have characterized a human non-small cell lung cancer cell line (PC-9/CDDP) selected from the wild type (PC-9) for acquired resistance to cisplatin. PC-9/CDDP demonstrated 28-fold resistance to cisplatin, with cross resistance to other chemotherapeutic drugs including chlorambucil (X 6.3), melphalan (X 3.7) and 3-[(4-amino-2-methyl-5-pyrimidinyl)]methyl-1-(2-chloroethyl)-1-nitros our ea (ACNU) (x 3.9). There was no expression of mdr-1 mRNA in either wild-type or resistant cells. The mRNA and protein levels of glutathione S-transferase (GST) pi were similar in the two lines. A GST-mu isozyme was present in equal amounts and the activities of selenium-dependent and independent glutathione peroxidase and glutathione reductase were unchanged. The mRNA level of human metallothionein IIA and the total intracellular metallothionein levels were reduced in the resistant cells. Significantly increased intracellular glutathione (GSH) levels were found in the resistant cells (20.0 vs 63.5 nmol/mg protein) and manipulation of these levels with buthionine sulfoximine produced a partial sensitization to either cisplatin or chlorambucil. Increased GSH probably also played a role in determining cadmium chloride resistance of the PC-9/CDDP, even though this cell line had a reduced metallothionein level. Also contributing to the cisplatin resistance phenotype was a reduced intracellular level of platinum in the PC-9/CDDP. Thus, at least two distinct mechanisms have been selected in the resistant cells which confer the phenotype and allow degrees of cross resistance to other electrophilic drugs.     

Variation in sensitizing efficiency for SR 2508 in human cells dependent on glutathione content

The observation by several authors and ourselves that manipulation of glutathione (GSH) levels in cultured cells would change the relative sensitization of hypoxic cells by nitroimidazole sensitizers led to the hypothesis that natural variation in GSH level could influence the sensitizer enhancement ratio (SER). Since many human tumor cell lines have shown high GSH levels, the A549 lung line, the HeLa line and a skin fibroblast line, the AG1522, were studied in monolayer culture in glass flasks. The GSH levels (nmol/mg protein) measured were 324 for A549, 103 for HeLa, and 32 for 1522 cells. For V-79 cells the level was 25 nmol/mg. In control experiments there was no effect of up to 10 mM SR 2508 in air but BSO (D,L-buthionine-S-R-sulfoximine) treatment modestly sensitized the A549 in air and nitrogen. Calculation of SER values at 1% survival revealed very little sensitization at 0.1, 0.5, and 1.0 mM SR 2508 for A549 cells (SER not significantly different than 1). At 5 mM the SER was 1.5 and at 10 mM 1.65. In contrast, the SER for fibroblasts was 1.65 at 0.5 mM and 2.2 at 1.0 mM SR 2508. The results for HeLa were intermediate, 1.25 and 1.45 at the same concentrations. Thus, the SER relates to the GSH levels in the cells. Treatment with BSO increased the sensitization of A549 to a level similar to that seen for AG1522 at 0.5 and 1.0 mM. These findings could explain the negative results of some sensitizer clinical trials.     

The effect of hOGG1 and glutathione peroxidase I genotypes and 3p chromosomal loss on 8-hydroxydeoxyguanosine levels in lung cancer

Polymorphic genes for the peroxide scavenger glutathione peroxidase I (GPX1) and 8-hydroxydeoxyguanosine (8-OHdG) DNA glycosylase/apurinic (AP) lyase (hOGG1) map to loci on chromosome 3p which are subject to frequent loss of heterozygosity (LOH) in lung tumours. Levels of the pro-mutagenic, oxidative DNA lesion 8-OHdG, were measured in 37 paired normal and tumorous lung specimens using HPLC with electrochemical detection. Lung tumours were also analysed for 3p LOH by fluorescent PCR with Genescan analysis. No significant difference was observed between 8-OHdG levels in tumour [7.7 +/- 6.7 (mean +/- SE) 8-OHdG/10(6) 2'-deoxyguanosine (dG)] and normal (8.1 +/- 8.8 8-OHdG/10(6) dG) lung tissue. Adduct levels in normal lung tissue DNA were not associated with constitutive hOGG1 genotype although there was a trend towards lower 8-OHdG levels in individuals possessing the ALA6 GPX1 polymorphism. Lung tumours exhibiting 3p LOH (40%) contained higher levels of 8-OHdG adducts (10.9 +/- 2.6 8-OHdG/10(6) dG) (P = 0.05) and lower GPX1 enzyme activity [45.5 nmol glutathione (GSH)/min/mg] (P = 0.09) when compared with tumours without LOH at these sites (5.55 +/- 0.87 8-OHdG/10(6) dG and 63.6 nmol GSH/min/mg, respectively). In conclusion, tumours with 3p LOH at loci associated with hOGG1 and GPX1 appear to have compromised oxidative defence mechanisms as measured by reduced GPX1 enzyme activity and elevated 8-OHdG levels and this may affect the prognosis of lung cancer patients.     

c-erbB-2 protein in serum of primary lung cancer patients

We determined c-erbB-2 protein level in serum of 86 primary lung cancer patients (78 non-small cell lung carcinomas (NSCLC), 3 small cell carcinomas, 5 not histologically defined) and in 61 controls. Aim of this study was to evaluate the clinical usefulness of c-erbB-2 as marker for lung cancer diagnosis. The protein was measured with a commercially available sandwich enzyme immunoassay. Mean levels of c-erbB-2 were 72.8 +/- 122.3 fmol/ml in lung cancers and 64.6 +/- 17.5 fmol/ml in controls (P = 0.2). No association was found between c-erbB-2 levels and histotype, tumor stage, sex and smoking habits. Among NSCLC, only four patients showed a c-erbB-2 concentration higher than the selected cut-off value of 99.6 fmol/ml. Subjects with levels higher than the 75th percentile in tumors (73 fmol/ml) had a shorter median survival than those with lower levels (6.3 months versus 10.0 months, P = 0.003). Our results indicated that serum c-erbB-2 protein is not a reliable diagnostic marker. There is, however, a suggestion of a possible clinical usefulness in terms of survival prediction.     

Parathyroid hormone-related protein varies with sex and androgen status in nonsmall cell lung cancer

BACKGROUND: In nonsmall cell lung cancer, tumor parathyroid hormone-related protein (PTHrP) expression predicts longer survival in women but not in men. To explain the sex-dependent survival effect, the authors proposed that hormonal influences decrease PTHrP in men versus women, that PTHrP inhibits tumor growth, and that the effect is greater in women than in men. The objectives of this study were to compare lung carcinoma PTHrP expression and carcinoma growth in male and female mice and to determine whether gonadal steroids regulate PTHrP in lung cancer cells. METHODS: Tumor PTHrP content was measured by immunoassay, and tumor burden was assessed with multiple measures in BEN squamous cell orthotopic lung carcinomas in athymic mice. In addition, lung adenocarcinoma PTHrP messenger RNA (mRNA) values determined by microarray analyses were compared between men and women. Cultured lung cancer cells were assayed for PTHrP after treatment with estradiol or R1881, a synthetic androgen. RESULTS: Lung carcinomas contained approximately 3 times more PTHrP in female mice than in male mice. Similarly, levels of PTHrP mRNA were significantly greater in adenocarcinomas from patients who were women than from patients who were men. Male mice had greater tumor burden than female mice. Androgen treatment reduced PTHrP in 3 lung cancer lines. Estradiol had no effect. Testosterone treatment also reduced lung carcinoma PTHrP in female mice. CONCLUSIONS: Lung carcinomas in females expressed more PTHrP than in males possibly because of negative regulation by androgens in males. Female mice with higher tumor PTHrP content had significantly less tumor burden than male mice, supporting the hypothesis that PTHrP inhibits tumor growth.     

Cellular heterogeneity of DNA/total-protein content in human lung tumors, as determined by flow cytometry.

With the aim of distinguishing neoplastic cell sub-populations of different prognostic and diagnostic significance, dual-parameter measurements (DNA/protein) have been simultaneously determined in a (256, 256) channel matrix in lung samples derived from 110 patients affected by neoplastic and non-neoplastic lung diseases. Biparametric analysis demonstrated that cells with abnormally high red fluorescence (i.e., protein content), which is indicative of unbalanced growth, were often observed in malignant tumors as compared with normal lung samples. Furthermore, the dual-parameter analysis allowed recognition of additional aneuploid tumor-cell lines, indicating that the frequency of cytometrically determined diploid tumor is lower than that previously described by DNA monoparametric analysis. The recognition of aneuploid subpopulations by dual-parameter analysis in clinically and histologically negative one-parameter flow-cytometric "diploid" samples assumes important diagnostic value. The results have also shown the presence of multiple protein sub-populations in clones with the same ploidy value, indicating a higher level of cellular heterogeneity than demonstrated by DNA monoparametric measurements.     

Detoxification ability and toxicity of quinones in mouse and human tumor cell lines used for anticancer drug screening

The in vitro testing of antitumor drugs involves the use of mouse and human tumor cells. In particular, there is interest in developing agents active against human solid tumors. We examined several biochemical parameters that may contribute to the differential sensitivity of the cell lines used in our laboratory to the toxic effects of antitumor compounds. The tumor cell lines examined were of mouse (colon 38, L1210 leukemia, and C1498 leukemia) and human origin (CEM leukemia, CX1 colon, H116 colon, HCT8 colon and H125 lung). Quinone reductase activity was markedly different between leukemia and solid-tumor cell lines of either mouse or human origin, with increased activity being observed in the solid-tumor cell lines relative to the leukemia lines. GSH transferase activity also was generally increased in solid-tumor relative to leukemia cell lines. Superoxide dismutase activity and thiol levels were similar in leukemia and solid-tumor cell lines, except that thiol levels were very low in colon 38. Mouse cell lines from in vitro passage had somewhat higher activity of superoxide dismutase and thiol levels than did cells maintained in vivo, indicating relatively increased antioxidant defenses. The toxicity of 2,3-dimethoxy-1,4-naphthoquinone, a model quinone that exerts its toxic effects via production of reactive oxygen species, was significantly lower in mouse lines maintained in vitro than in those tested in vivo, whereas the toxicity of another quinone, menadione, was just slightly lower. Quinone reductase activity, GSH transferase activity, and thiol levels were significantly higher in the human lines than in the mouse lines. Accordingly, the toxicity of both quinones tended to be lower in the human lines than in the mouse lines.Abbreviations GSH Glutathione - S-15 15,000-g supernatant This work was supported by grants 1PO1 CA46560 and CA22453 from the NIH. We thank Chiab Panchapor, Susan Pugh, Kathryn White, Julia Dieckman, and Michael Fortuna for maintenance the tumor cells, transplantation of the tumors used, and toxicity testing of the quinones.     

Suppression of lung cancer with siRNA targeting PTTG

Pituitary transforming gene (PTTG) is frequently expressed at high levels in malignant tumors. We report high levels of expression of PTTG in various lung tumors and tumor-derived cell lines. For a better understanding of its role in maintaining the cancer phenotype, we used RNA interference (RNAi) directed against PTTG. Transfection of H1299 cells with PTTG siRNA duplex (5'-UGG GAG AUC UCA AGU UUC A-3') to a final concentration of 100 nmol/l resulted in almost complete depletion of PTTG mRNA within 24 and 48 h when compared to expression in untransfected cells or cells transfected with control siRNA. Western blot analysis showed nearly a 60% reduction in PTTG protein within 48 h of transfection. In phenotype analysis, we investigated the effect of PTTG siRNA on colony formation on soft agar, and tumor development in nude mice. Transfection of H1299 cells with PTTG siRNA duplex significantly reduced colony formation compared to untransfected cells or cells transfected with control siRNA. Mice injected with H1299 cells transfected with PTTG siRNA followed by injection of siRNA developed no tumors within two weeks of injection, but developed small tumors (67.85+/-45.87 mg) within 4 weeks of injection, whereas untransfected cells, or cells transfected with control siRNA developed large tumors (232.12+/-102.78 and 231.57+/-83.76 mg respectively). Suppression of PTTG as well as Ki67, bFGF and CD34 was observed in H1299 tumors treated with PTTG siRNA compared to untreated and control-treated siRNA in nude mice. In conclusion, decreasing PTTG expression through PTTG siRNA inhibits tumor growth both in vitro and in vivo. Future studies are needed to test whether PTTG expression can be efficiently depleted by siRNA expressed from a DNA-based expression vector combined with a specific-promoter, such that RNAi can specifically target PTTG in cancer cells without affecting normal cells.     

Expression of the neu gene-encoded protein (P185neu) in human non-small cell carcinomas of the lung

The neu protooncogene is a recently described transforming gene originally isolated from ethylnitrosourea-induced rat neuroblastomas. We have examined the expression of the neu gene in human non-small cell lung carcinomas using immunoprecipitation and immunohistochemistry. The neu protein product (p185neu) was present in eight of 22 non-small cell carcinoma cell lines derived from human lung tumors. Expression of p185neu was found in all histological subtypes of non-small cell carcinomas including large cell carcinomas, squamous cell carcinomas, and adenocarcinomas. Extension of these data to biopsy specimens of human lung tumors demonstrated that normal ciliated bronchial epithelium of the peripheral airways expressed p185neu at low levels. Neoplastic cells in four of 12 adenocarcinomas and three of five squamous cell carcinomas also expressed p185neu at levels higher than the normal ciliated bronchial epithelium. Together these studies indicate that p185neu expression is a common feature of human lung tumors.     

p185neu expression in human lung adenocarcinomas predicts shortened survival

p185neu is the protein product of the HER2/neu protooncogene. This protein has characteristics of a tyrosine kinase growth factor receptor and is postulated to be important in human carcinogenesis. To define the significance of the expression of this protein in human non-small cell lung cancer, 55 tumors from patients with squamous cell carcinoma (16), adenocarcinoma (29), or large cell carcinoma (10) of the lung were examined for p185neu using immunohistological methods. Five of 16 squamous cell carcinomas and 10 of 29 adenocarcinomas were found to overexpress p185neu relative to levels of expression seen in uninvolved bronchiolar epithelium. For the adenocarcinomas, p185neu expression was associated with older age (66.6 +/- 10.1 versus 57.5 +/- 10.8 years) (P = 0.04) and shortened survival (83.7 +/- 94.1 versus 188.5 +/- 120 weeks) (P = 0.01). In this group, using Cox's multivariate survival analysis, p185neu expression was found to be a significant determinant of survival (P = 0.04) even after accounting for the effect of tumor stage. For the squamous cell carcinomas, p185neu expression was not correlated with any of our clinicopathological parameters. Our findings indicate that non-small cell lung cancers which express p185neu do so at levels higher than that found in normal bronchiolar epithelium, and expression in adenocarcinomas of the lung is independently associated with diminished survival intervals.     

Cholinesterase activity of human lung tumours varies according to their histological classification

The probable involvement of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) in cancer and the relevance of cholinergic responses for lung cancer growth prompted us to study whether cholinesterase activity of human lung is altered by malignancy. Surgical pieces of non-small lung carcinomas (NSLC) and their adjacent non-cancerous tissues (ANCT) were analysed for AChE and BChE activities. AChE activity in adenocarcinoma (AC) was 7.80 +/- 5.59 nmol of substrate hydrolysed per min and per mg of protein (mU/mg), the same as in their ANCT (8.83 +/- 4.72 mU/mg; P = 0.823); in large cell carcinoma (LCC), 7.52 +/- 3.32 mU/mg, approximately 50% less than in their ANCT (15.39 +/- 5.66 mU/mg; P = 0.043); and in squamous cell carcinoma (SCC), 1.39 +/- 0.58 mU/mg, 80% less than in ANCT (6.08 +/- 2.88 mU/mg; P = 0.003). BChE activity was 5.85 +/- 3.20 mU/mg in AC and 9.56 +/- 3.38 mU/mg in ANCT (P = 0.022); 2.94 +/- 2.01 mU/mg in LCC and 6.50 +/- 6.63 mU/mg in ANCT (P = 0.068); and 4.49 +/- 2.30 mU/mg in SCC and ANCT 6.56 +/- 4.09 mU/mg (P = 0.026). Abundant AChE dimers and fewer monomers were identified in lung and, although their distribution was unaffected by cancer, the binding with concanavalin A revealed changes in AChE glycosylation between SCC and their ANCT. The fall in BChE activity affected all molecules, with a strong decrease of the amphiphilic tetramers. Western blotting revealed protein bands with the expected mass of the principal AChE subunits, and the deeper intensity of the protein signal in SCC than in healthy lung, in lanes loaded with the same units of AChE activity, supported an augment in the amount of AChE protein/unit of AChE activity in SCC. The increased availability of acetylcholine in neoplastic lung, resulting from the fall of cholinesterase activity, may enhance cholinergic signalling and contribute to tumour progression.     

多西紫杉醇对非小细胞肺癌细胞株生长及对COX-2蛋白表达的影响

目的：在体外研究不同浓度的多西紫杉醇（泰索帝）对非小细胞肺癌（NSCLC）细胞株生长及对胞内环氧化酶-2（COX-2）蛋白表达的影响。方法：采用人NSCLC细胞株A549和NCI-H460作为研究对象,体外药物敏感试验（MTT）检测不同浓度和作用时间的泰索帝对细胞增殖的抑制效应;应用免疫细胞化学的方法观察分别经泰索帝1、2、4nmol／L作用48小时后,细胞内COX-2蛋白的表达情况。结果：泰索帝住体外对NSCLC细胞株A549的生长抑制作用在药物浓度加大到10nmol／L后处于平台期,对NCI-H460存浓度加大到4nmol／L后处于平台期;两株细胞分别经泰索帝1、2、4nmol／L作用48小时后,细胞内COX-2蛋闩的表达同对照组（不加药物组）无统计学差异（P〉0．05）。结论：泰索帝住体外对NSCLC细胞株A549和NCI-H460的生长有明显的抑制作用,但不影响细胞内环氧化酶-2蛋白的表达。     

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.