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Biomedical subjects

A Albini

Publications and source records attributed to A Albini.

At least 109 records · Page 6Linked to original sources
PubMed1996

Altered production of laminin and nidogen by high and low metastatic variants of murine melanoma cells.

An altered ability to interact with and degrade extracellular matrix molecules is a common feature of the malignant phenotype. Although changes in the expression of matrix proteins in metastases in vivo are relatively well documented, little is known about the changes in matrix production by malignant cells in culture. Here we have examined the synthesis of the basement membrane components laminin and nidogen (entactin) by low and high metastatic variants of the K-1735 murine melanoma cells. Protein deposition was examined by western blotting as well as immunofluorescence; protein synthesis was examined by immunoprecipitation with specific antibodies. Gene expression was also evaluated by measuring steady-state mRNA levels using cDNA probes on northern and dot-blots. Laminin gamma 1 levels appeared to be similar in both high and low metastatic lines; however, the high metastatic lines had reduced levels of the laminin beta 1 chain. On the contrary, nidogen expression was observed only in the high metastatic lines. Traces of a laminin alpha chain were present only in immunoprecipitates of the low metastatic cells and could not be detected in the high metastatic cells. Both high and low metastatic cells deposited an extracellular matrix of basement membrane components, with laminin deposition decreased in high metastatic cells. Modified expression, production, and deposition of basement membrane components in high metastatic melanoma cells could be involved in their altered interactions with the extracellular matrix.

Animals↗
PubMed1996

Vascular endothelial growth factor regulates angiogenesis and vascular permeability in Kaposi's sarcoma.

Abundant vasculature with increased permeability is a prominent histological feature of Kaposi's sarcoma (KS), a multifocal, cytokine-regulated tumor. Here we report on the role of vascular endothelial growth factor (VEGF) in AIDS-KS angiogenesis and vascular permeability. We demonstrate that different cytokines, which were previously shown to be active in KS development, modulate VEGF expression in KS spindle cells and cooperate with VEGF on the functional level. Northern blot analysis as well as studies on single cells using in situ hybridization revealed that VEGF expression in cultivated AIDS-KS spindle cells is up-regulated by platelet-derived growth factor-B and interleukin-1 beta. Western blot and enzyme-linked immunosorbent assay analysis of cell culture supernatants demonstrated that the VEGF protein is secreted by stimulated AIDS-KS spindle cells in sufficiently high amounts to activate proliferation of human dermal microvascular endothelial cells. Basic fibroblast growth factor did not increase VEGF expression but acted synergistically with VEGF in the induction of angiogenic KS-like lesions in a mouse model in vivo. Angiogenesis and cellularity of KS-like lesions were clearly increased when both factors were injected simultaneously into the flanks of mice, compared with separate injection of each factor. A comparable angiogenic reaction as obtained by simultaneous injection of basic fibroblast growth factor and VEGF was observed when cell culture supernatants of AIDS-KS spindle cells were used for these experiments. Finally, analysis of primary human AIDS-KS lesions revealed that high amounts of VEGF mRNA and protein were present in KS spindle cells in vivo. These data provide evidence that VEGF, in concert with platelet-derived growth factor-B, interleukin-1 beta, and basic fibroblast growth factor, is a key mediator of angiogenesis and vascular permeability in KS lesions in vivo.

Animals↗
PubMed1995

Effect of osteoblast supernatants on cancer cell migration and invasion.

Malignant tumor cells of different origin seem to have preferential sites for metastasis. Breast cancer, prostate cancer and certain melanomas have bone as one of their preferential targets for metastasis. Bone is continuously being remodelled, a process largely controlled by local growth factors. A possible explanation for malignant cell recruitment to bone is that osteoblast products, directly secreted or released from the matrix by osteoclast resorbing activity, are able to stimulate cancer cell migration. To test this hypothesis we have utilized an in vitro system of differentiating osteoblasts which in culture progress all the way to the formation of mineralized nodules. Conditioned media obtained from these osteoblast cultures at different stages were able to induce chemotactic migration and invasion of both melanoma and breast cancer cells. The migratory and invasive phenotype was accompanied by enhanced gelatinolytic activity of osteoblast stimulated cancer cells. Our data suggest that osteoblasts secrete potent factors able to direct tumor cell migration towards remodelling bone.

Animals↗
PubMed1995

Angiogenic properties of human immunodeficiency virus type 1 Tat protein.

Extracellular human immunodeficiency virus type 1 (HIV-1) Tat protein promotes growth of spindle cells derived from AIDS-associated Kaposi sarcoma (AIDS-KS), an angioproliferative disease very frequent in HIV-1-infected individuals. Normal vascular cells, progenitors of AIDS-KS cells, proliferate in response to Tat after exposure to inflammatory cytokines, whose levels are augmented in HIV-1-infected individuals and in KS lesions. Here we show that Tat also promotes AIDS-KS and normal vascular cells to migrate and to degrade the basement membrane and stimulates endothelial cell morphogenesis on a matrix substrate. These effects are obtained at picomolar concentrations of exogenous Tat and are promoted by the treatment of the cells with the same inflammatory cytokines stimulating expression of the receptors for Tat, the integrins alpha 5 beta 1 and alpha v beta 3. Thus, under specific circumstances, Tat has angiogenic properties. As Tat and its receptors are present in AIDS-KS lesions, these data may explain some of the mechanisms by which Tat can induce angiogenesis and cooperate in the development of AIDS-KS.

Acquired Immunodeficiency Syndrome↗
PubMed1995

Inhibition of invasion, gelatinase activity, tumor take and metastasis of malignant cells by N-acetylcysteine.

The thiol N-acetylcysteine (NAC) is currently considered one of the most promising cancer chemopreventive agents by virtue of its multiple and coordinated mechanisms affecting the process of chemical carcinogenesis. Recent studies have shown that an unpaired cysteine residue in the propeptide plays a key role in inactivation of latent metastasis-associated metalloproteinases: the present study was designed to assess whether NAC could also affect tumor take, invasion and metastasis of malignant cells. As assessed by zymographic analysis, NAC completely inhibited the gelatinolytic activity of type-IV collagenases in the cells tested (gelatinases A and B). Moreover, NAC was efficient in inhibiting the chemotactic and invasive activities of tumor cells of human (A2058 melanoma) and murine origin (K1735 and B16-F10 melanoma cells as well as C87 Lewis lung carcinoma cells) in Boyden-chamber assays, which are predictive of the invasive and metastatic properties. Reduced glutathione (GSH) had a similar, although less effective activity. The number of lung metastases decreased sharply when B16-F10 murine melanoma cells, injected i.v. into nude mice, were pre-treated with NAC and resuspended in medium supplemented with 10 mM NAC. In other experiments NAC was given in drinking water, starting 48-72 hr before subcutaneous inoculation of either B16-F10 cells or of their highly metastatic variant B16-BL6, or intramuscular injection of LLC cells. In all experiments NAC treatment decreased the weight of the locally formed primary tumor and produced a dose-related delay in tumor formation. Spontaneous metastasis formation by B16-F10 and B16-BL6 tumors was slightly yet significantly reduced by oral administration of NAC. However, this was not observed for Lewis lung tumors. These data indicate that NAC affects the process of tumor-cell invasion and metastasis, probably due to inhibition of gelatinases by its sulfhydryl group, with the possible contribution of other mechanisms, including the potent antioxidant activity of this thiol.

3T3 Cells↗
PubMed1995

A single cis-acting element in a short promoter segment of the gene encoding the interphotoreceptor retinoid-binding protein confers tissue-specific expression.

Interphotoreceptor retinoid-binding protein (IRBP) is the major protein component of the interphotoreceptor matrix. IRBP has a highly restricted tissue-specific expression in retinal photoreceptor cells and in a subgroup of pinealocytes. With the purpose of understanding how transcriptional regulation contributes to the expression of human IRBP, we have studied a short promoter fragment (from -123 to +18, relative to the transcription start site). We demonstrate, by analysis of the expression of the lacZ reporter gene fused to this short promoter fragment in transgenic mice, that it is sufficient to confer tissue-specific expression in retinal photoreceptors and in pinealocytes. DNA/protein binding assays, performed to identify binding sites for tissue-specific trans-acting factors, have shown that an element between -45 and -58 binds a factor present only in nuclear extracts of retinoblastoma-derived cell lines, which express IRBP. An element further upstream, between -86 and -106, binds apparently ubiquitous factors. Site-directed mutagenesis was performed to disrupt a GATTAA motif included in the -45 to -58 binding site and a second inverted GATTAA motif present shortly upstream. In transgenic mice bearing the mutated version of the promoter fragment, the expression of the reporter gene was completely abolished, thus suggesting that this element is essential for tissue-specific expression. A GATTAA motif appears in the 5'-flanking regions of several photoreceptor-specific genes, suggesting that this could be the recognition site for a photoreceptor-specific factor.

Animals↗
PubMed1995

Chemopreventive properties and mechanisms of N-Acetylcysteine. The experimental background.

The thiol N-acetylcysteine (NAC), now under clinical trial for cancer chemoprevention both in Europe (project Euroscan) and in the US (National Cancer Institute), has been shown during the past decade to exert protective effects in a variety of experimental test systems. NAC inhibited spontaneous mutagenicity and that induced by a number of chemical compounds and complex mixtures. Moreover, NAC significantly decreased the incidence of neoplastic and preneoplastic lesions induced by several chemical carcinogens in rodents (mice, rats, hamsters), e.g., in lung, trachea, colon, liver, mammary gland, Zymbal gland, bladder and skin. Our studies provided evidence that multiple mechanisms contribute to NAC antimutagenicity and anticarcinogenicity. They include extracellular mechanisms, such as detoxification of reactive compounds due to the nucleophilic and antioxidant properties of NAC, inhibition of nitrosation products, and enhancement of thiol concentration in intestinal bacteria; trapping and enhanced detoxification of carcinogens in long-lived non-target cells, such as erythrocytes and bronchoalveolar lavage cells; mechanisms working in the cytoplasm of target cells, such as replenishment of GSH stores, modulation of metabolism of mutagens/carcinogens, blocking of electrophiles, and scavenging of reactive oxygen species; and nuclear effects, such as inhibition of DNA adduction by metabolites of carcinogens, inhibition of "spontaneous" mutations, attenuation of carcinogen-induced DNA damage, and protection of nuclear enzymes, such as poly(ADP-ribose) polymerase.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcysteine↗
PubMed1995

The alpha 3 beta 1 integrin is involved in melanoma cell migration and invasion.

The VLA3 (alpha 3 beta 1) integrin receptor recognizes several ligands; however, the function of this integrin is still debated. Expression of VLA3 appears to be increased in malignant melanoma and correlates with the degree of dermal invasiveness. Here we have studied the role the alpha 3 integrin subunit in malignant melanoma cell migration and invasion into extracellular matrices. The 2/14 clone of the Me665/2 human melanoma cell line, which expresses high levels of VLA integrins, was highly migratory and invasive, while the low integrin expressing 2/56 clone showed limited migration and was not invasive. Antibodies to the beta 1 subunit inhibited adhesion, migration, and invasion of two different malignant melanoma cell lines, the 2/14 clone and A2058 cells, indicating a crucial role for VLA integrins in these phenomena. While anti-alpha 6 antibodies inhibited adhesion to laminin and anti-alpha 5 antibodies inhibited adhesion to fibronectin, antibodies to the alpha 3 subunit did not inhibit adhesion of these cells to laminin, fibronectin, or collagen i.v. In contrast, the P1B5 anti-alpha 3 antibodies were good inhibitors of the migration of these cells toward laminin, fibronectin, and collagen IV and also blocked invasion of these cells through a reconstituted basement membrane matrix (Matrigel). Another anti-alpha 3 antibody, F4, did not effect migration, while both the P1B5 and F4 antibodies induced cellular aggregation on Matrigel. Our data suggest a specific role for alpha 3 beta 1 in the migration and invasion of melanoma cells.

Antibodies, Monoclonal↗
PubMed1995

Osteoblastic cells from rat long bone. I. Characterization of their differentiation in culture.

We here report the characterization for osteogenic markers alkaline phosphate (AP), osteocalcin, and mineral deposition of osteoblast cultures derived from cells migrating out from seven-day-old rat tibia fragments. The cells outgrown from bone fragments responded to stimulation with PTH with cAMP increase. We show in these cultures a high level of biosynthesis of type III collagen and osteonectin, and report the stimulatory effect that conditioned serum-free media (CM) from these cultures exert on the migration of endothelial cells (EA hy 926). The cultures were kept for the initial seven days in a Coon's modified F12 medium, then were switched to a medium containing ascorbic acid and beta-glycerophosphate (BGP), and cultured for another 41 days. They showed constitutive and timely restricted osteoblast markers and mineralization. Maximal AP activity occurred in concomitance with cell doubling, then fell to low levels by the time cultures were stationary. 45Ca incorporation in the monolayer increased after four weeks of culture, in concomitance with the appearance of unmineralized nodules, and remained high throughout the phase of mineral deposition. Biosynthesis of collagens type I, type III, and type V was detected at all times; secreted newly synthesized collagens decreased overall, relative to total secreted newly synthesized proteins, and on a per cell basis, with progression of the culture, while the ratio of collagen type III/collagen type I increased. Osteonectin was detected by immunohistochemistry and high amounts of osteonectin were synthesized constitutively. Osteocalcin was detected on virtually all cells tested at 21 and 28 days. A preliminary step in neoangiogenesis is the migration of endothelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaline Phosphatase↗
PubMed1995

Production of angiogenesis inhibitors and stimulators is modulated by cultured growth plate chondrocytes during in vitro differentiation: dependence on extracellular matrix assembly.

Secretion of angiogenesis inhibitors and stimulators is modulated during in vitro differentiation of embryonic chick growth plate chondrocytes. Supernatants from dedifferentiated cells undergoing maturation to hypertrophic chondrocytes in suspension progressively inhibited vascular cell random migration and invasion of basement membrane matrix by endothelial cells. Maximal inhibition was exhibited by conditioned medium from hypertrophic chondrocytes. The same medium also repressed vascular cell migration induced by highly angiogenic Kaposi's sarcoma cell supernatants and prevented formation of an anastomosed network of tube-like structures by endothelial cells plated on matrigel. On the contrary, when the suspension culture of hypertrophic chondrocytes was supplemented with ascorbic acid, a condition leading to the formation of a mineralized tissue similar to calcified cartilage, a dramatic switch to production of angiogenic activity was observed. Medium conditioned by osteoblast-like cells derived from hypertrophic chondrocytes also induced vascular cell migration and invasion of basement membrane matrix. The presence of angiogenic activity in the conditioned medium was assessed also by an in vivo assay in mice using reconstituted basement membrane associated with heparin. Therefore, interactions of chondrocytes with their extracellular matrix are an absolute requirement for the expression of angiogenic activities by hypertrophic chondrocytes at late developmental stages.

Animals↗
PubMed1994

Retinoic acid negatively regulates beta 4 integrin expression and suppresses the malignant phenotype in a Lewis lung carcinoma cell line.

Retinoic acid (RA) is a potent inhibitor of the malignant phenotype and of tumour cell growth. We observed that in vitro RA treatment of a highly metastatic lung carcinoma cell line (C87) induced a marked reduction in the amount of the beta 4 integrin subunit. The downregulation of this adhesion molecule was assessed by immunofluorescence, immunoprecipitation, and northern analysis. In order to investigate the effects of RA on the malignant phenotype in C87 cells we performed morphological and functional analysis after RA treatment. We found that RA was able to produce marked changes in C87 cell shape, increasing the number of flat cells (90% of the total cell population), and significantly inhibiting the malignant and invasive phenotype of C87 cells. RA treatment suppressed their clonogenic potential in soft agar (control, 20 +/- 5; RA, 0), and strongly reduced their chemotactic and chemoinvasive capacity (chemotaxis: control, 231 +/- 5; RA, 28 +/- 0; chemoinvasion: control, 132 +/- 11; RA = 2 +/- 1). FACS analysis and cell count, however, indicated that RA reduced the growth of C87 cells only partially. After 72 h of treatment we observed only a 10% reduction in the S phase fraction of the cell population. Finally, the reduced lung colony-forming ability, observed after i.v. injection of RA-treated cells (lung foci/animal: RA-treated cells, 1 +/- 0.1; untreated, 8.5 +/- 0.8), further supports the conclusion that in this murine lung carcinoma cell line a marked reduction in the expression of the beta 4 integrin subunit is associated with a marked inhibition of the malignant phenotype.

Animals↗
PubMed1994

Expression of beta 1B integrin isoform in CHO cells results in a dominant negative effect on cell adhesion and motility.

The integrin subunit beta 1B, a beta 1 isoform with a unique sequence at the cytoplasmic domain, forms heterodimers with integrin alpha chains and binds fibronectin, but it does not localize to focal adhesion sites (Balzac, F., A. Belkin, V. Koteliansky, Y. Balabanow, F. Altruda, L. Silengo, and G. Tarone. 1993. J. Cell Biol. 121:171-178). Here we analyze the functional properties of human beta 1B by expressing it in hamster CHO cells. When stimulated by specific antibodies, beta 1B does not trigger tyrosine phosphorylation of a 125-kD cytosolic protein, an intracellular signalling pathway that is activated both by the endogenous hamster or the transfected human beta 1A. Moreover, expression of beta 1B results in reduced spreading on fibronectin and laminin, but not on vitronectin. Expression of beta 1B also results in severe reduction of cell motility in the Boyden chamber assay. Reduced cell spreading and motility could not be accounted for by preferential association of beta 1B with a given integrin alpha subunit. These data, together with our previous results, indicate that beta 1B interferes with beta 1A function when expressed in CHO cells resulting in a dominant negative effect on cell adhesion and migration.

Animals↗
PubMed1994

Angiogenic potential in vivo by Kaposi's sarcoma cell-free supernatants and HIV-1 tat product: inhibition of KS-like lesions by tissue inhibitor of metalloproteinase-2.

OBJECTIVE: To determine the neoplastic nature of Kaposi's sarcoma (KS). A highly vascularized lesion, KS is frequently associated with AIDS, indicating HIV products may be involved. DESIGN AND METHODS: We determined the angiogenic properties of KS cell-secreted products and the HIV-1-tat gene product in vivo. Cell-free secreted products (KS-CM) from cultured epidemic and sporadic KS spindle cells or recombinant (r) HIV-1 tat protein were injected into mice with a matrix support (Matrigel). RESULTS: KS-CM produced lesions carrying all the phenotypic hallmarks of KS, as observed by light and electron microscopy: spindle-shaped cells, haemorrhages and an inflammatory infiltrate, as well as Factor VIII-positive endothelial cells lining new blood vessels. Electron microscopy indicated an initial granulocyte invasion, with spindle-cell migration and neocapillary formation in the centre of the matrix. These lesions required the cofactor heparin; KS-CM or heparin alone were poorly angiogenic. A less intense angiogenesis, with lower cellularity and few granulocytes, was observed in basic fibroblast growth factor (bFGF)/heparin lesions, indicating that factors other than bFGF are present in the KS spindle-cell products. When the collagenase inhibitor tissue inhibitor of metalloproteinases (TIMP)-2 was added to the sponges, KS-CM-induced angiogenesis was reduced by approximately 65% and bFGF-induced angiogenesis inhibited completely. Recombinant HIV-1 tat protein, a growth factor for KS cells, induced vascularization that was also enhanced by heparin, implying that HIV-1 tat could contribute to the aetiology of HIV-associated KS. CONCLUSIONS: KS-like lesions were obtained by injecting cell-free secreted products, suggesting that KS is a 'self-propagating' proliferative lesion caused by a cytokine imbalance and not a true neoplasm. Heparin-binding factors appear to be involved, and HIV-1 tat angiogenic properties implicate this molecule in AIDS-associated KS. Inhibition of KS-CM-induced KS-like lesions by TIMP-2 suggests that metalloproteinase inhibitors could be potential therapeutic agents for KS.

Animals↗
PubMed1994

Inhibition of AIDS-Kaposi's sarcoma cell induced endothelial cell invasion by TIMP-2 and a synthetic peptide from the metalloproteinase propeptide: implications for an anti-angiogenic therapy.

In the initial phases of angiogenesis, endothelial cells must degrade and cross the vessel basement membrane, as do tumor cells during invasion and metastasis formation. Various metalloproteinases have been implicated in tumor cell invasion, in particular MMP-2 (72 kDa collagenase IV, gelatinase A), which has been demonstrated to be associated with tumor metastasis formation. Supernatants from AIDS-Kaposi sarcoma (KS) cells induce normal endothelial cells to invade through a reconstituted basement membrane (Matrigel) in vitro, which correlates with the angiogenic potential of KS cells in vivo. Here we demonstrate that two specific inhibitors of MMP-2, TIMP-2 and a peptide from the MMP-2 propeptide region (peptide 74), inhibit endothelial cell invasion induced by AIDS-KS cell supernatants. Smooth muscle cells were much less sensitive to these inhibitors. These data suggest that MMP-2 activation is a key event in endothelial cell invasion, the initial phase of tumor-associated neoangiogenesis. Inhibition of this enzyme could be an effective treatment for KS and tumor-associated angiogenesis.

Acquired Immunodeficiency Syndrome↗
PubMed1994

Oncostatin M activates phosphatidylinositol-3-kinase in Kaposi's sarcoma cells.

Oncostatin M (OM) is a polypeptide cytokine that induces autocrine and paracrine effects on AIDS-Kaposi's sarcoma (KS) cells (Nair et al., Science, 255, 1430-1432, 1992; Miles et al., Science, 255, 1432-1434, 1992). The signalling pathways underlying this activation are largely unknown. We have found that OM binding to KS cell lines in vitro identifies a higher affinity binding site (Kd 10-20 pM) with a lower affinity (Kd 1.5 nM), high capacity binding site. The binding of OM to its receptor at the KS cell surface stimulates a rapid tyrosine phosphorylation of multiple proteins, including the p85 regulatory subunit of phosphatidylinositol-3-kinase (PI3K). In addition OM can stimulate the in vivo activation of PI3K and increases the PI3K activity in anti-phosphotyrosine and anti-src kinase family antibody directed immunoprecipitates. Genistein, an inhibitor of tyrosine kinases, inhibits the synthesis of phosphatidylinositol 3,4-biphosphate and the growth of KS cells. Finally, OM enhances tyrosine kinase activity in immune complex kinase assay performed with antibody anti-src kinase family. These data suggest that in KS cells OM can stimulate formation of tyrosine kinase co-ordinate signalling complexes, containing at least src kinase family and PI3K, which can drive the accumulation of the putative second-messengers D3-phosphorylated phosphoinositides.

Antineoplastic Agents↗