Promoter characterization and structure of the gene encoding mouse lysosomal alpha-d-mannosidase.

Mouse lysosomal alpha-d-mannosidase (EC 3.2.1.24) is an enzyme involved in the catabolism of N-linked glycoproteins. The gene is differentially expressed in mouse tissues, and the highest level of mRNA is found in the epididymis. The expression of mannosidase in the epididymis may be hormonally regulated, since its activity increases with age. To understand the factors affecting the expression of mouse mannosidase, we isolated and characterized the promoter and determined the exon-intron structure. The gene is about 15 kb, consists of 24 exons, and the 5' flanking region contains GC-rich regions, TATA boxes, CAAT boxes, and putative binding sites for the transcription factors Sp1, AP2, and PEA3. PEA3 factor may participate in the transcriptional control of mannosidase expression in the mouse epididymis. In fact, it has been demonstrated that the PEA3 motif is spatially and temporally expressed within the mouse epididymis, and its accumulation is controlled by androgens and testicular factors. A 1279-bp fragment from the initiation codon had the strongest promoter activity, and three different transcription start sites were identified at positions -131, -149, and -174.

Glutamine synthetase gene expression in a glioblastoma cell-line of clonal origin: regulation by dexamethasone and dibutyryl cyclic AMP.

We investigated the expression of glutamine synthetase (GS), an enzyme involved in astroglial metabolism and marker of astroglial functional maturity, in a glioblastoma cell-line (GL-15) of clonal origin. In spite of their phenotypic immaturity, evidenced in a mosaic fashion by a poor glial fibrillary acidic protein (GFAP) expression, the level of GS-mRNA is high in GL15 cells and the considerable amount of GS biological activity can be further induced and stabilized by glucocorticoids. A correlation between the induction by dexamethasone of the GS-mRNA level and the GS biological activity suggests a transcriptional regulation of GS expression by the aforesaid hormone. Under this hormonal action, changes in cell morphology occur and they are correlated with an overexpression of the GFAP, a marker of astroglial differentiation. On the contrary, dibutyryl cyclic AMP (dbc AMP) down-regulates the GS-mRNA expression and decreases GS activity. These results suggest that GL-15 cells have a common glucocorticoid dependent mechanism able to induce GS and GFAP as well as morphological changes. However in these cells AMPc responsive elements are involved in the negative modulation of the GS expression, contrary to what occurs in normal astroglial cells.

Putative role of antioxidant enzymes and glyoxalases in carcinogenesis.

The effects various drugs exert on antioxidant enzyme and glyoxalase activity in rat livers were studied. All drugs tested provoked a marked reduction in glutathione peroxidase and a small drop in both glyoxalase I and II activity. It is hypothesized that the substances tested support tumour development by neutralizing organic peroxides, thereby favouring the oxidation of carcinogens and, as a consequence, the formation of metabolites that trigger neoplastic transformation. The reduction in glyoxalase activity is probably attributable to the enhanced cell proliferation induced by the treatment.

Effects of dimethylsulfoxide on Friend erythroleukemic cell proliferation and on the activity of enzymes involved in this process.

Variations in catalase, glutathione peroxidase (GP) and adenylate cyclase (AC) activity in murine erythroleukemic (MEL) cells were studied during multiplication and dimethylsulfoxide (DMSO)-induced differentiation. The results demonstrated that, although DMSO favors the incorporation of 3H-thymidine into DNA of treated cells, it slows down cell multiplication. Increased incorporation was also observed in superoxide dismutase (SOD)-treated cells. DMSO also determined an early and significant drop in AC activity and a late fall in catalase activity, whereas there was no significant variation in GP activity in parallel with the decreased cell multiplication that accompanied cell differentiation. We hypothesize that DMSO and SOD favor 3H-thymidine incorporation by neutralizing the reactive forms of oxygen and that the reduction in catalase and AC activity is closely related to the mitotic activity of MEL cells.

Antioxidant enzymes and proliferative activity of cell lines of different origin.

The effects of catalase treatment were studied in two in vitro passaged ascites tumour lines (ATP C+ and EAT) and in three in vitro established human myeloid leukemia cell lines (HL-60; KG-1; KG-1a) characterized by the arrest of cells at different stages of maturation. The results demonstrate that catalase treatment favoured proliferation in the in vitro passaged ascites tumour cells, but not in the in vitro established leukemia lines. Enzyme assays on five in vitro cell lines revealed that catalase was only present in HL-60. Although glutathione peroxidase activity was initially found in all five cell lines, it disappeared from two ascites tumour cells when they were transferred in culture. It is hypothesized that catalase treatment favours ascites tumour cell proliferation because it replaces glutathione peroxidase in eliminating H2O2.

The role of antioxidant enzymes in benzo(a)pyrene-induced carcinogenesis.

Chick embryo hepatocytes were cultured in the presence of benzo(a)pyrene in order to study the effects of this carcinogen on catalase, glutathione peroxidase and superoxide dismutase activity. The results demonstrate that benzo(a)pyrene is incapable of modifying the activity of these enzymes, even though it is taken up by cultured cells to form benzo(a)pyrene-DNA adducts. The effect of culturing, however, caused a marked reduction in the activity of these enzymes. The significance of these activity variations in benzo(a)pyrene in vitro carcinogenesis is discussed.

Possible role of certain antioxidant enzymes in dimethylnitrosamine-induced liver carcinogenesis.

An investigation was carried out to establish whether the reduction in catalase, glutathione peroxidase and superoxide dismutase activity, normally observed in liver tumours, is an early event and therefore of pathogenetic importance, or whether it is a late occurrence. Experiments performed on dimethylnitrosamine-treated hepatectomized and non-hepatectomized rats show that the decrease in activity of these enzymes is entirely due to hepatectomy, since the tumour-inducing doses of dimethylnitrosamine failed to provoke variations in the activity of these enzymes, in either normal or regenerating liver.

Replicative behaviour of ascites tumour cells transplanted in BALB C mice of different sex.

The present research demonstrates that cells from an ascites tumour (ATP C+) multiply more actively in the peritoneum of male mice, provided they are maintained alive in this environment for long periods of time by weekly transplants in animals of the same sex. Solid tumours obtained by inoculating ATP C+ cells, removed from the peritoneum of male mice, into the subcutaneous dorsal region of castrated male mice, grew more rapidly than those obtained by inoculating the same cells removed from the peritoneum of female mice, always provided that the cells had been passaged at length in animals of the same sex. Cytogenetical studies of these two cell subpopulations revealed that cells reproduced for 2 years in males had a less stabile karyotype and a greater incidence of acrocentric associations.

Different behaviour of normal and neoplastic cells cultured in vitro in the presence of catalase and superoxide dismutase.

Chicken embryo fibroblasts and hepatocytes were studied in the presence of catalase and superoxide dismutase in order to establish whether these enzymes had the capacity to favour cell multiplication as previously shown for in vitro tumour ascites cells (ATP C+). The results indicate that, unlike ATP C+ cells, both fibroblasts and hepatocytes are inhibited in their multiplication by superoxide dismutase. Similar effects are exerted on hepatocytes by catalase, whereas the multiplication of fibroblasts is favoured by high doses of this enzyme. Enzyme determinations revealed high levels of catalase and superoxide dismutase in hepatocytes, whereas both enzymes were poor in fibroblasts and ATP C+.

Antagonism between catalase and ascorbic acid in control of normal and neoplastic cell multiplication.

The authors studied the effects of a treatment with ascorbic acid on in vitro multiplication of ascites tumour cells (ATP C+), of fibroblast-like cells and of hepatocytes from chick embryos, by measuring [3H]thymidine incorporation into DNA. The results obtained show that the ATP C+ cells are the most sensitive to the toxic effects of the experimental treatment, while the hepatocytes are the most resistant cell population. A treatment with catalase was able to greatly reduce the damage caused by ascorbic acid on the ATP C+ cells. It is hypothesized that ascorbic acid inhibits cell multiplication by the H2O2 formed by its oxidation and that the cells having the highest level of catalase are more resistant to its toxic effects.

Cell multiplication of an ascites tumour in the presence of superoxide dismutase and catalase.

Superoxide dismutase and catalase were demonstrated to favour the multiplication of ascites tumour cells in vitro. It is proposed that these enzymes neutralize the O2-. and H2O2 that may accumulate in the neoplastic cell and that cell damage occurs because the cellular levels of both enzymes are low.

Inhibition of the binding of 7,12-dimethylbenz[a]anthracene to DNA by ascorbic acid, reduced glutathione and cysteine in chick embryo cells cultured in vitro.

A study on the capacity of ascorbic acid, reduced glutathione and cysteine to interfere with 3H-7,12-dimethylbenz[a]anthracene (3H-DMBA) binding to DNA in cultured fibroblast-like cells from 11-day-old chick embryos showed that, although the total amount of 3H-DMBA in the treated cells was greater than in the untreated cells, the DNA-bound 3H-DMBA was less. Comparisons between the various experimental groups demonstrated that the greater 3H-DMBA in the ascorbic acid-, reduced glutathione-, and cysteine-treated groups could not be attributed to an initially higher number of cells, nor to a treatment-induced increase in DNA synthesis. It is proposed that the three substances examined inhibit the oxidative degradation of 3H-DMBA, thereby favoring its accumulation within the cell and reducing the formation of DNA-binding metabolites.

Influence of sexual hormones on solid tumor growth and ATP C+ cell replication.

Solid tumors obtained by implanting ATP C+ cells subcutaneously into the back of BALB/cf/Had/Se substrain mice developed more rapidly in males than females. Experiments conducted on gonadectomized animals demonstrated that female gonads inhibit the development of this tumor. Studies on ATP C+ cells cultured in vitro with various doses of male and female hormones showed that mainly progesterone, but to a lesser extent also estradiol, significantly inhibit cell proliferation with respect to testosterone. The treatment of normal and gonadectomized animals with testosterone and progesterone confirms the results obtained in vitro. Analyses carried out on ATP C+ cells do not reveal presence of estradiol and progesterone receptors.

Effects of ascorbic and dehydroascorbic acid on the multiplication of tumor ascites cells in vitro.

The effects of AA and DHA on ATP C+ cell multiplication in vitro were studied by measuring incorporation of 3H thymidine into DNA. The results obtained demonstrate that both AA and DHA have the same effects: they favor cell multiplication at low doses and inhibit it at high doses. Experiments carried out with serial doses of both these substances revealed that AA is more efficient in determining both stimulating and inhibiting effects. The lesser efficiency of DHA may be attributed to its limited stability in culture medium. Studies on the effect of high doses of AA and DHA added to the culture medium in single or fractionated doses revealed that fractionated administration is more efficient in inhibiting cell multiplication than single administration.

Absence of accumulation phenomena in normal and tumoral tissues of mice treated with ascorbic acid.

The authors studied the effects of an energetic and prolonged ascorbic acid (AA) treatment on AA concentration in a solid tumor obtained by transplanting ascites tumor cells in the dorsal subcutaneous region of Balb/C1/Had/Se substrain mice. The results demonstrate that this tumor, whose growth is favored by AA, not only is incapable of accumulating AA, but surprisingly presents a lowering of its concentration as the result of treatment with very high doses of AA. The levels of AA in various organs and tissues of mice bearing the aforesaid tumor show no significant difference to those observed in organs and tissues of healthy mice. An energetic and prolonged treatment with AA not only is incapable of favoring accumulation, but also causes a lowering of its concentration in the liver and kidney, similar to that observed in tumors. Therefore, the hypothesis may be advanced that this decrease is caused by the adaptation of some enzymatic system, which results in a greater utilization of AA. This greater consumption could be the cause of the decreased AA levels once esogenous administration is arrested.

[Derivation of a velocity equation for a reaction model witwh two binding sites for acetylcholinesterase]. / Derivazione dell'equazione di velocita' di un modello di reazione a due siti di legame per.l'acetilcolinesterasi.

The authors suggest a kinetic model with two binding sites for acetylcholinesterase; it is suitable to study particular enzyme forms that show a kinetic pattern which cannot be explained by the model with a single binding site of Krupka and Laidler. The velocity equation was obtained using the algorithm of King and Altman and carrying out elementary calculations.