PubMed Health⌕ Search

Biomedical subjects

A Passaniti

Publications and source records attributed to A Passaniti.

15 recordsLinked to original sources
PubMed1992

The neurotoxin 1-methyl-4-phenylpyridinium: a selective cytostatic agent in small-cell lung cancer cell lines with neuroendocrine properties.

BACKGROUND: Small-cell lung cancer (SCLC) is a common malignancy that is usually fatal, since it metastasizes and recurs even after aggressive chemotherapy. While the cellular origin of this cancer is not well established, the cells of certain tumors exhibit neuroendocrine markers, including L-dopa decarboxylase. PURPOSE: We designed in vitro and in vivo studies to investigate whether the neuroendocrine features in classic SCLC cell lines were sufficient to make them sensitive to 1-methyl-4-phenylpyridinium (MPP+), a known neurotoxin that destroys nigrostriatal dopaminergic neurons. METHODS: Both classic SCLC cell lines (NCI-H345, NCI-H510, NCI-H187, and NCI-H146) and variant SCLC cell lines (NCI-H417, NCI-H82, NCI-H446, and NCI-H524) were exposed to MPP+ (0-512 microM) for 3 days. Inhibition of DNA synthesis was determined by [3H]thymidine incorporation assays. In a related experiment, MPP+ was removed from the classic cell line culture, and the incorporation of [3H]thymidine was determined. In the in vivo study, male athymic nude mice received subcutaneous injections of 0.5 mL tumor cells with matrigel for 10 days to enhance tumor growth, followed by MPP+ at doses of 100-400 micrograms/d given intraperitoneally for 2 days. RESULTS: All four classic SCLC cell lines showed great sensitivity to MPP+, with detachment from laminin substrates and inhibition of DNA synthesis. MPP+ interfered with [3H]thymidine incorporation and, thus, with DNA synthesis in classic SCLC cell lines at low doses (median +/- SD, 12 +/- 4 microM), whereas much higher doses (median, > 512 microM) were required to inhibit [3H]thymidine incorporation in the variant lines. Treated cells excluded trypan blue dye, showing that inhibition of DNA synthesis was not due to cytotoxicity, and the cells incorporated [3H]thymidine when MPP+ was removed from the culture medium, demonstrating that the inhibition was reversible. MPP+ inhibited the growth of the classic NCI-H187 and variant NCI-H417 cell lines implanted in nude mice. CONCLUSIONS: These results suggest that MPP+ differentially interferes with DNA synthesis in SCLC cell lines in vitro; the selective inhibitory effect on classic cell lines suggests that the neuroendocrine properties expressed by classic SCLC cells may be responsible for the differential effect. IMPLICATIONS: MPP+ exerts a cytostatic effect on these cell lines, and the differential sensitivity observed in vitro is maintained in vivo, suggesting that MPP+ or other pyridinium compounds may be of therapeutic value in SCLC.

1-Methyl-4-phenylpyridinium↗
PubMed1992

Stimulation of human prostatic carcinoma tumor growth in athymic mice and control of migration in culture by extracellular matrix.

The tumorigenicity, migration, growth and invasiveness of certain tumor cells is stimulated by basement membranes. Here we have examined the effect of Matrigel, an extract of basement membrane proteins, on the behavior of several prostate cancer cell lines, testing their growth and invasiveness in vitro and in vivo. Cells of the Tsu-prI line were more invasive than PC-3, Du-145, or LNCaP cells. Peptide inhibitors implicated laminin in the migration and invasion of these cells. When these cells were suspended in Matrigel and injected into nude mice, their growth was greatly enhanced, since large tumors formed in athymic nude mice whereas virtually no tumors were observed in the absence of Matrigel. The growth of a slowly growing line, LNCaP, was increased by exogenous basic fibroblast growth factor when injected with Matrigel. A laminin cell adhesion peptide, YIGSR, was a potent inhibitor of Matrigel-stimulated tumor growth implicating cell-laminin interactions in this process. These results suggest that tumor growth of prostate adenocarcinoma cells may be dependent both on cellular growth factors and on cell-matrix interactions mediated by laminin which facilitate the development of transplanted tumors in nude mice.

Animals↗
PubMed1992

Adrenal carcinoma tumor progression and penultimate cell surface oligosaccharides.

Many previous studies have implicated cell surface saccharides, and sialylglycoconjugates in particular, as important mediators of tumor cell metastasis. In this report, we have used three different specific sialidases and a highly sensitive high-performance liquid chromatographic sialic acid assay to probe the cell surfaces of several murine adrenal carcinoma variants. In contrast to several earlier studies on other metastatic variants, we find no significant differences in the overall levels of cell surface or total cellular sialic acid among three Y1 murine adrenal carcinoma variants with widely different metastatic phenotypes. However, using highly purified, linkage-specific sialyltransferases, in conjunction with V. cholerae sialidase, to probe the cell surface saccharide topography of specific penultimate oligosaccharides, we do find striking differences in oligosaccharide structures underlying the sialic acid moieties. Two tumorigenic and metastatic variants (F2 and F4) contain about 6-fold more penultimate Gal beta 1----4GlcNAc sialylation sites than a related tumorigenic but nonmetastatic variant (HSR) when CMP-[3H]-N-acetylneuraminic acid and the Gal beta 1----4GlcNAc alpha 2,6 sialyltransferase are used to probe the adrenal carcinoma cell surfaces. The metastatic variants also are found to contain 4- to 4.5-fold more Gal beta 1----3GalNAc sialylation sites than the nonmetastatic variant when the Gal beta 1----3GalNAc alpha 2,3 sialyltransferase is used as a cell surface probe. Earlier work, which used the same sialyltransferase probes on sialidase-treated murine melanoma variants (A. Passaniti and G. W. Hart, J. Biol. Chem., 263: 7591-7603, 1988), also showed similar quantitative differences in penultimate structures between metastatic variants. However, in contrast to the adrenal carcinoma cells, the highly metastatic melanoma cells have severalfold lower levels of sialylatable penultimate Gal beta 1----4GlcNAc and Gal beta 1----3GalNAc saccharides compared to their nonmetastatic counterparts. Thus, while the precise structural alterations or surface accessibilities of penultimate saccharides appear to be cell type dependent, these results suggest that pronounced changes in penultimate cell surface sialo-oligosaccharide moieties occur during progression to a malignant phenotype in two widely different tumor systems. These types of alterations in the underlying penultimate oligosaccharide structures of cell surface sialoglycoconjugates may be a common feature of highly metastatic cells arising from very different tumor cell types.

Acetylgalactosamine↗
PubMed1992

A simple, quantitative method for assessing angiogenesis and antiangiogenic agents using reconstituted basement membrane, heparin, and fibroblast growth factor.

BACKGROUND: Blood vessel growth is necessary for normal tissue homeostatis and contributes to solid tumor growth. Methods to quantitate neovascularization should be useful in testing biological factors and drugs that regulate angiogenesis or to induce a vascular supply to promote wound healing. EXPERIMENTAL DESIGN: An extract of basement membrane proteins (Matrigel) was found to reconstitute into a gel when injected subcutaneously into C57/BL mice and to support an intense vascular response when supplemented with angiogenic factors. RESULTS: New vessels and von Willebrand factor antigen staining were apparent in the gel 2-3 days after injection, reaching a maximum after 3-5 days. Hemoglobin content of the gels was found to parallel the increase in vessels in the gel allowing ready quantitation. Angiogenesis was obtained with both acidic and basic fibroblast growth factors and was enhanced by heparin. Several substances were tested for angiostatic activity in this assay by coinjection in Matrigel with fibroblast growth factor and heparin. Platelet-derived growth factor BB, interleukin 1-beta, interleukin-6, and transforming growth factor-beta were potent inhibitors of neovascularization induced by fibroblast growth factor. Tumor necrosis factor-alpha did not alter the response but was alone a potent inducer of neovascularization when coinjected with Matrigel and heparin. Consistent with the previously demonstrated importance of collagenase in mediating endothelial cell invasion, a tissue inhibitor of metalloproteinases that also inhibits collagenases was found to be a potent inhibitor of fibroblast growth factor-induced angiogenesis. CONCLUSIONS: Our assay allows the ready quantitative assessment of angiogenic and anti-angiogenic factors and should be useful in the isolation of endothelial cells from the capillaries that penetrate into the gel.

Angiogenesis Inducing Agents↗
PubMed1992

Experimental models that mimic the differentiation and dedifferentiation of vascular cells.

Endothelial and smooth muscle cells normally exist in a quiescent differentiated state. After injury to the vessel, these cells dedifferentiate, migrate, and proliferate as needed for repair. In culture on plastic, both endothelial and smooth muscle cells exhibit the dedifferentiated phenotype. We have found that laminin and reconstituted basement membrane proteins (Matrigel) induce a very rapid cessation of endothelial cell proliferation followed by alignment and subsequent reorganization into tubelike structures. We have also found that smooth muscle cells in culture exhibit a differentiated phenotype when exposed to Matrigel. The molecular mechanisms involved in smooth muscle differentiation resemble those of skeletal muscle, in which proliferation and differentiation appear to be mutually exclusive states controlled by both positive and negative transcriptional regulators. The dedifferentiated smooth muscle cells produce proteases and exhibit a migratory and invasive phenotype capable of destroying normal tissue architecture. These studies suggest that the modulation of endothelial and smooth muscle cells between a differentiated and dedifferentiated phenotype is regulated by extracellular matrix components as well as by cytokines. Model systems such as those described here should allow the identification of molecular events controlling the differentiation of vascular cells and facilitate the development of therapeutic agents that maintain healthy vessels.

Basement Membrane↗
PubMed1991

[Bolus nicardipine in the hemodynamic assessment for heart transplantation of patients with severe failure of ischemic origin and high pulmonary resistance].

Nicardipine i.v. bolus (5 mg/5 min) was administered in the pulmonary artery trunk in 13 patients (2 f, 11 m), mean age 48 +/- 8 yrs, affected by ischemia congestive heart failure, with pulmonary hypertension (pulmonary vascular resistances greater than 6 U.W. and/or systolic pulmonary artery blood pressure greater than or equal to 60 mmHg). The vasodilatation induced by nicardipine caused a rapid improvement of all hemodynamic parameters, with a significant reduction of systemic and pulmonary pressures and resistances; in addition, cardiac output increased significantly. Even if heart rate decreased and mean right atrial pressure fell, their variation did not reach statistical significance. These beneficial effects are attributable to the vasodilator action of nicardipine on the systemic and pulmonary vascular districts. Therefore, in the hemodynamic evaluation of patients with ischemic cardiomyopathy proposed for heart transplantation, we propose the employment of nicardipine in testing the vascular reactivity in cases with secondary pulmonary hypertension.

Adult↗
PubMed1990

Metastasis-associated murine melanoma cell surface galactosyltransferase: characterization of enzyme activity and identification of the major surface substrates.

Surface galactosyltransferase (GT) has been described on a variety of cells where it is believed to be involved in cell-cell and cell-substratum adhesion. Here we show that B16 metastatic murine melanoma cells exhibit a 5-fold higher cell surface GT activity than their nonmetastatic counterparts, although total GT activity in NP-40 solubilized cells is similar for both melanoma variants. Interestingly, on living cells, this cell surface GT almost exclusively galactosylates an endogenous glycoprotein (Mr = 110,000). This metastasis-associated GT is specific for terminal D-N-acetylglucosamine, catalyzes the formation of a beta 1-4 linkage, does not recognize polylactosaminoglycans, and has its specificity altered from D-N-acetylglucosamine to D-glucose by alpha-lactalbumin; yet the Mr = 110,000 protein is not a major substrate when exogenous bovine GT is used on the outside of living cells. In addition to this protein-specific endogenous GT activity, another cell surface GT activity that selectively galactosylates glucosylceramide is also prominent. Endogenous galactosylation of both protein and glycolipid substrates is reduced when the membrane is solubilized by the detergent NP-40 but remains unaltered in the presence of digitonin, which permeabilizes but does not dissolve the membrane. These data suggest that the GTs and their substrates are associated on the cell surface. Chloroquine treatment of intact cells leads to a 4-fold and a 3-fold increase in galactosylation of the Mr = 110,000 protein and glucosylceramide, respectively, suggesting that these two substrates normally reside mostly in the lysosomal or Golgi compartments. The increased expression of lysosomal membrane proteins on the surfaces of highly metastatic cells may, in part, also explain the galactosylation differences observed. These studies further suggest that increased surface localization of certain glycosyltransferases with highly restricted in situ substrate specificities may be a common feature of highly metastatic tumor cells.

Animals↗
PubMed1989

Purification of chicken liver ferritin by two novel methods and structural comparison with horse spleen ferritin.

Ferritin was purified from chicken liver by two different methods: gel filtration on controlled-pore glass beads, and immunoaffinity chromatography employing a chicken ferritin-specific monoclonal antibody that did not cross-react with horse spleen ferritin. This antibody recognizes intact ferritin and an oligomeric 240 kDa form of the molecule after protein transfer to nitrocellulose, but not the 22 kDa chicken ferritin subunit. Chicken liver ferritin purified by these methods exhibited reduced migration on non-denaturing polyacrylamide gels compared with horse spleen ferritin. These results were consistent with the difference in calculated isoelectric points of chicken and horse ferritin subunits. By two-dimensional gel electrophoresis, chicken ferritin 22 kDa subunits exhibited isoelectric points from 6.1 to 6.6 whereas horse spleen ferritin subunits exhibited isoelectric points of 5.8-6.3. The 240 kDa form of the chicken ferritin molecule had an isoelectric point of 6.6 whereas the 210 kDa form of the horse ferritin molecule had isoelectric points of 5.1 and 4.9. Intact chicken liver ferritin particles were 13.4 +/- 0.8 nm (controlled-pore glass-purified) and 12.5 +/- 0.9 nm (affinity-purified) in diameter when viewed by electron microscopy. Horse spleen ferritin consisted of slightly smaller particles with an average diameter of 11.0 +/- 0.7 nm. However, ferritin from chicken liver and horse spleen co-migrated with an apparent molecular mass of 470 kDa when analysed by Sepharose 4B gel filtration chromatography. These results indicate that, consistent with results from other published purification methods, the chicken ferritin purified by the methods reported here exhibits both structural similarities to, and differences from, horse spleen ferritin.

Animals↗
PubMed1989

In vitro galactosylation of a 110-kDa glycoprotein by an endogenous cell surface galactosyltransferase correlates with the invasiveness of adrenal carcinoma cells.

We have examined the role of a cell surface galactosyltransferase, laminin, and laminin-binding protein (receptor) in the invasion of clonal derivatives of a murine adrenal carcinoma cell line. Although a 10-fold variation was found in the ability to invade a reconstituted basement membrane matrix, levels of intracellular laminin and the laminin-binding protein were shown to be present and secreted equally in all lines. Of the eight lines tested, seven showed a correlation between invasion and the incorporation of [3H]galactose from UDP-[3H]galactose into a 90- to 110-kDa protein. One noninvasive line (clone HSR), however, retained high galactosyltransferase activity yet could not galactosylate the endogenous 90- to 110-kDa substrate. Interestingly, this clone was unable to attach to laminin. Although high galactosyltransferase activity can be consistent with cells of high invasiveness, our results suggest that the galactosylation status of a 90- to 110-kDa Y1 cell surface glycoprotein is most indicative of invasion potential.

Adrenal Gland Neoplasms↗
PubMed1989

Coated vesicles from chicken liver bind ferritin.

The iron-storage protein ferritin was found to be associated with highly purified coated vesicles (CV) from chicken liver. Chicken liver ferritin was morphologically similar to ferritin from horse spleen and could be isolated using a specific anti-ferritin monoclonal antibody. This antibody recognized a 240 X 10(3) Mr form of chicken ferritin but not the 22 X 10(3) Mr ferritin subunit after protein transfer to nitrocellulose. CV purified by controlled-pore glass-bead chromatography also contained ferritin when assayed by monoclonal anti-ferritin antibody using a sensitive enzyme-linked assay. Ferritin remained associated with CV even after re-chromatography. Ferritin particles were observed to be associated with CV by electron microscopy. CV-associated ferritin could be quantitatively removed from CV by treatment of the CV with 0.5 M-Tris-HC1 + 2M-urea at pH 8.5, conditions that also lead to dissociation of the clathrin lattice. Triton X-100 detergent treatment did not affect the association of ferritin with CV. These results indicate that purified CV from chicken liver contain ferritin in association with the clathrin lattice. The possible functional significance of this association is discussed.

Animals↗
PubMed1988

Cell surface sialylation and tumor metastasis. Metastatic potential of B16 melanoma variants correlates with their relative numbers of specific penultimate oligosaccharide structures.

Numerous investigations suggest that cell surface glycoconjugates, and in particular sialic acids, are directly involved in determining the metastatic phenotype. To further evaluate this hypothesis, we have used a variety of techniques to probe the cell surfaces of several metastatic variants of the murine B16 melanoma that were selected for experimental lung-colonizing ability (Fidler, I. (1973) Nature 242, 148-149) or for their ability to spontaneously metastasize from the site of a subcutaneous injection (Stackpole, C. W., Alterman, A. L., and Fornabaio, D. M. (1985) Invasion & Metastasis 5, 125-142). Using a highly sensitive high performance liquid chromatography sialic acid assay in conjunction with Vibrio cholerae sialidase, we find that none of these metastatic variants differ significantly in their overall levels of cell surface sialic acid. Using highly purified, linkage-specific sialyltransferases, in conjunction with specific glycosidases, to probe the cell surface saccharide topography of specific penultimate oligosaccharides, we also find no significant differences between the efficient lung-colonizing variant, B16-F10 and the poorly-colonizing B16-F1 or B16-Flr variants. In contrast, the spontaneously metastatic variants examined contain substantially different levels of specific penultimate sialylation sites. The tumorigenic but nonmetastatic B16-LM3/G3.26 variant contains 4-fold more penultimate Gal beta 1-3GalNAc sialylation sites than the tumorigenic and highly metastatic B16-LM3/G3.12 variant when CMP[3H]NeuAc and the alpha 2-3Gal beta 1-3GalNAc sialyltransferase are used to probe the melanoma cell surfaces. Several prominent glycoconjugates of apparent Mr 43,000, 40,000, and 30,000 are especially evident upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the nonmetastatic cells. The nonmetastatic variant also contains 2-fold more Gal beta 1-4GlcNAc sialylation sites than the metastatic variant when the alpha 2-6Gal beta 1-4GlcNAc sialyltransferase is used as a cell surface probe. In this case, glycoconjugates of apparent Mr 74,000, 45,000, and 43,000 are more prominently observed on the cell surfaces of the nonmetastatic variant. These data indicate that the differences in lung-colonizing abilities of B16 melanoma metastatic variants do not correlate with the numbers or sialylation states of specific penultimate oligosaccharide structures on their surfaces. However, the relative levels of specific penultimate saccharide structures do correlate with the ability of the cells to undergo spontaneous metastasis from a subcutaneous tumor.

Animals↗
PubMedDate not supplied

New models to define factors determining the growth and spread of human prostate cancer.

The incidence of many cancers shows a sharp increase with age. This is particularly true of prostate cancer, which arises in many older males. Little or no morbidity is observed as the tumor develops in situ in the prostate. However, once malignant cells escape from the primary lesion and metastasize, the disease assumes a much more serious course. Here we report on the activity of human prostate cancer cells in culture as well as their behavior when transplanted into nude mice. In vitro, several lines of prostate carcinoma cells obtained from metastatic lesions were embedded in reconstituted basement membrane proteins (Matrigel) and found to exhibit highly invasive activity as observed with malignant cells from other types of tumors. Also, we report an improved method for obtaining an increased growth of human prostate cancer cells in nude mice by injecting these cells in Matrigel. Since there are no adequate animal models of prostate cancer, the systems described here may prove useful in defining events underlying the development and progression of the tumor cells to malignant status, as well as facilitate the analyses of novel therapeutic agents to prevent the growth and the spread of this cancer.

Animals↗