John Pinto

Cloning, expression, genomic localization, and enzymatic activities of the mouse homolog of prostate-specific membrane antigen/NAALADase/folate hydrolase

Abstract. Human Prostate Specific Membrane Antigen (PSMA), also known as folate hydrolase I (FOLH1), is a 750-amino acid type II membrane glycoprotein, which is primarily expressed in normal human prostate epithelium and is upregulated in prostate cancer, including metastatic disease. We have cloned and sequenced the mouse homolog of PSMA, which we have termed Folh1, and have found that it is not expressed in the mouse prostate, but primarily in the brain and kidney. We have demonstrated that Folh1, like its human counterpart, is a glutamate-preferring carboxypeptidase, which has at least two enzymatic activities: (1) N-acetylated α-linked l-amino dipeptidase (NAALADase), an enzyme involved in regulation of excitatory signaling in the brain, and (2) a γ-glutamyl carboxypeptidase (folate hydrolase). The 2,256-nt open reading frame of Folh1 encodes for a 752-amino acid protein, with 86% identity and 91% similarity to the human PSMA amino acid sequence. Cells transfected with Folh1 gained both NAALADase and folate hydrolase activities. Examination of tissues for NAALADase activity correlated with the mRNA expression pattern for Folh1. Fluorescent in situ hybridization (FISH) revealed Folh1 maps to only one locus in the mouse genome, Chromosome 7D1-2.

Effect of epidermal growth factor on the growth and radiation sensitivity of human breast cancer cells in vitro

PURPOSEnTo study the effect of epidermal growth factor (EGF) on the radiation sensitivity of MCF-7 breast cancer cells.nnnMETHODS AND MATERIALSnRadiation dose survival curves were generated for MCF-7 cells under conditions of hormonal deprivation. Epidermal growth factor and/or a monoclonal antibody to its receptor (mAb-225) were added prior to irradiation. Cell cycle distribution was determined by flow cytometry and cellular glutathione (GSH) levels were measured by a glutathione reductase assay.nnnRESULTSnUnder hormonal deprivation (control), more than 90% of the MCF-7 cells were arrested in G0/G1 phase and the D(o) of their survival curve was 0.66 +/- .01 Gy. The addition of EGF resulted in (a) growth stimulation; (b) increased percentage of cells in the S-phase of the cell cycle; (c) increased radioresistance (D(o) = 0.81 +/- .04 Gy; p < .05, compared with controls); (d) increased cellular GSH level. The EGF effect on radiation response was observed in a time- and dose-dependent manner. The addition of mAb-225 blocked the ability of EGF to enhance growth and radioresistance (D(o) = 0.68 +/- .03 Gy).nnnCONCLUSIONnEpidermal growth factor stimulates the growth and when administered prior to irradiation increases the radioresistance of hormone-deprived MCF-7 cells. These effects are inhibited by a specific antibody to the EGF receptor. Epidermal growth factor concomitantly increased the fraction of S-phase cells and intracellular GSH levels. This system of growth factor-altered radiosensitivity in human breast cancer cells provides a useful model for the study of the radiation response mechanisms in human malignancy.

Helicobacter pylori decreases gastric mucosal glutathione

Activation of oxidative stress pathways may contribute to gastric epithelial damage and mutagenesis caused by Helicobacter pylori. We measured the effect of H. pylori on the concentrations of reduced glutathione (GSH), an important endogenous defense against oxidant damage, in gastric epithelial cells in vivo and in vitro. GSH concentrations were significantly lower in gastric biopsies from 19 H. pylori-infected patients than 38 normal controls, and correlated inversely with inflammatory cell numbers. In vitro, H. pylori initially increased GSH levels in AGS cells, but subsequently depleted intracellular GSH stores completely after 24 h. No GSH was detected in H. pylori. Our data suggest that diminished GSH levels with H. pylori colonization of the gastric mucosa may be due to a direct effect of the bacterium as well as through the associated inflammatory response.

Alterations of prostate biomarker expression and testosterone utilization in human LNCaP prostatic carcinoma cells by garlic-derived S-allylmercaptocysteine

This study determined the effects of S‐allylmercaptocysteine (SAMC), a phytoconstituent from garlic, on the expression of androgen‐responsive biomarkers, prostate specific antigen (PSA), and prostate specific membrane antigen (PSMA), in human prostatic carcinoma cells (LNCaP).

Relationship of glutathione and glutathione-S-transferase to cisplatin sensitivity in human head and neck squamous carcinoma cell lines

Factors controlling glutathione metabolism may govern sensitivity to chemotherapeutic agents such as cisplatin. Using a battery of cell lines derived from previously untreated head and neck squamous cell carcinomas, we examined cisplatin resistance relative to (a) glutathione-S-transferase (GST)-pi gene amplification and expression, (b) basal and inducible GST-total and GST-pi enzymatic activity, and (c) cellular levels of reduced glutathione (GSH). Using Southern blot analysis and northern blot hybridization, no relationship between GST-pi gene amplification, mRNA expression and drug resistance could be identified. Despite the capacity of cisplatin to induce GST enzyme activity, the response was variable and unrelated to cisplatin responsiveness. However, an inverse relationship between GSH levels and cisplatin sensitivity was identified. To further clarify these effects, cells were treated with S-allyl cysteine (SAC), a thioallyl derivative isolated from garlic (Allium sativum), which altered cellular GSH in a biphasic manner. Pretreatment with SAC to lower cellular GSH levels followed by exposure to cisplatin significantly enhanced the cytotoxic effects of cisplatin, while SAC alone had no effect on cell growth.

ANTIMALARIAL EFFECTS OF RIBOFLAVIN DEFICIENCY

La carence en riboflavine modifie lactivite de plusieurs enzymes du metabolisme intermediaire, ce qui a pour effet une protection vis-a-vis du paludisme. Laddition de riboflavine aux milieux de culture inhibe lactivite in vitro de plusieurs medicaments sur P. falciparum. Plusieurs produits qui induisent chez lanimal des carences en riboflavine ont une activite antipaludique

Tissue autofluorescence as an intermediate endpoint in NMBA-induced esophageal carcinogenesis

The capacity to identify subclinical neoplastic disease of the upper aerodigestive tract (UADT) using tissue auto-fluorescent spectroscopy would significantly contribute to cancer screening. Rats received N-nitrosomethyl benzylamine (NMBA), a carcinogen shown to cause esophageal malignancies. Following sacrifice at early weekly intervals, gross assessment of esophageal mucosa of NMBA-exposed rats was indistinguishable from saline-treated controls. Histopathologic evaluation, however, revealed NMBA-induced preneoplastic changes in the epithelium. Concurrent with these changes, the NMBA-exposed rats demonstrated specific alterations in autofluorescence. These results demonstrate that NMBA-induced esophageal premalignancy can be distinguished by autofluorescent properties. The capacity to detect alterations in autofluorescence may allow more sensitive screening of UADT mucosa at risk for cancer development.

Acute ethanol exposure alters hepatic glutathione metabolism in riboflavin deficiency

Since acute ethanol consumption and riboflavin deficiency each induces oxidative stress within tissues, we examined whether their combined effects compromise the major antioxidative system in liver, namely, reduced glutathione (GSH) metabolism. Four hours before sacrifice, half the riboflavin-deficient (RD) and riboflavin-sufficient (RS) rats were treated with ethanol (3 g/kg). Livers were excised and analyzed for GSH and enzymes that control its metabolism. In RD rats, GSH increased while glucose-6-phosphate dehydrogenase (G6PD) activity decreased. Ethanol had no effect on these measurements in RS rats. In RD rats, ethanol administration decreased GSH along with the activities of GSH peroxidase, glutathione reductase, and G6PD. These data suggest that riboflavin deficiency alone does not compromise hepatic GSH metabolism. By contrast, ethanol consumption together with riboflavin deficiency depletes hepatic GSH, blunts enzyme activities controlling GSH metabolism and may enhance alcohol-induced liver injury.

New approaches to the possible prevention of side effects of chemotherapy by nutrition

In an effort to develop new methods for preventing side effects of chemotherapy, the authors initiated studies to determine whether Adriamycin (doxorubicin) inhibits the metabolism of riboflavin (vitamin B2). Adriamycin has been shown to form a 1:1 stoichiometric complex with riboflavin, as well as to compete for binding to tissue proteins. Adult rats treated with Adriamycin in clinically relevant doses were compared to control animals in ability to convert riboflavin into flavin adenine dinucleotide (FAD), the active flavin coenzyme derivative, in heart, skeletal muscle, liver, and kidney. Rats treated with Adriamycin exhibited diminished formation of carbon 14 (14C)FAD in skeletal muscle to nearly 50% that of controls, and in heart to about 70% to 80% of controls. Under these conditions, (14C)FAD formation in liver and kidney was largely unaffected by Adriamycin. In preliminary studies, riboflavin‐deficient animals treated with Adriamycin had accelerated mortality rates compared to those of food restricted controls treated with similar doses of Adriamycin. The data as a whole suggest a potential mechanism for Adriamycin‐induced cardiac and skeletal myopathy, i.e., inhibition of synthesis of FAD, a flavin coenzyme which is involved in electron transport, lipid metabolism, and energy generation. These findings in an animal model raise the possibility that defects of riboflavin nutriture, either dietary or drug‐induced, may be a determinant of Adriamycin toxicity. Further studies are required to explore the potential for preventing side effects due to Adriamycin by administration of this vitamin.

ETHACRYNIC ACID: A NOVEL RADIATION ENHANCER IN HUMAN CARCINOMA CELLS

PURPOSEnBecause agents that interfere with thiol metabolism and glutathione S-transferase (GST) functions have been shown to enhance antitumor effects of alkylating agents in vitro and in vivo, the present study was conceived on the basis that an inhibitor of GST would enhance the radiation response of some selected human carcinoma cells. Ethacrynic acid (EA) was chosen for the study because it is an effective inhibitor of GST and is a well known diuretic in humans.nnnMETHODS AND MATERIALSnExperiments were carried out with well-established human tumor cells in culture growing in Eagles minimum essential medium (MEM) supplemented with 10% fetal calf serum (FCS). Cell lines used were MCF-7, MCF-7 adriamycin resistant (AR) cells (breast carcinoma), HT-29 cells (colon carcinoma), DU-145 cells (prostate carcinoma), and U-373 cells (malignant glioma). Cell survival following the exposure of cells to drug alone, radiation alone, and a combined treatment was assayed by determining the colony-forming ability of single plated cells in culture to obtain dose-survival curves. The drug enhancement ratio was correlated with levels of GST.nnnRESULTSnThe cytotoxicity of EA was most pronounced in MCF-7, U-373, and DU-145 cells compared to MCF-7 AR and HT-29 cells. The levels of GST activity were found to be lower in those EA-sensitive cells. A significant radiation enhancement was obtained with EA-sensitive cells exposed to nontoxic concentrations of the drug immediately before or after irradiation. The sensitizer enhancement ratio (SER) of MCF-7 cells was 1.55 with EA (20 micrograms/ml), while the SER of MCF-7 AR was less than 1.1. Based on five different human tumor cells, a clear inverse relationship was demonstrated between the magnitude of SER and GST levels of tumor cells prior to the combined treatment.nnnCONCLUSIONnThe present results suggest that EA, which acts as both a reversible and irreversible inhibitor of GST activity, could significantly enhance the radiation response of human cancer cells and the level of GST in tumor cells may predict the magnitude of radiation enhancement with EA. Ethacrynic acid would be an excellent drug as a radiosensitizer for further in vivo tumor study.