Biomedical subjects

N Zini

Publications and source records attributed to N Zini.

At least 19 recordsLinked to original sources

PubMed1997

Increase of nuclear phosphatidylinositol 4,5-bisphosphate and phospholipase C beta 1 is not associated to variations of protein kinase C in multidrug-resistant Saos-2 cells.

The multidrug resistance (MDR) phenotype that is mediated by an overexpression of P-glycoprotein, has been suggested to be related also to an increased activity of protein kinase C (PKC) and to changes in phospholipid pattern. By electron microscope quantitative immunocytochemistry, we investigated whether PKC and other elements of the polyphosphoinositide signal transduction system are affected in an MDR variant of the human osteosarcoma cell line Saos-2. These cells, which are characterized by an increased expression of P-glycoprotein not only at the plasma membrane but also at the nuclear level, showed increased intranuclear amounts of phosphatidylinositol 4,5-bisphosphate and of phospholipase C beta 1, while both the amount and activity of both nuclear and cellular PKC were not modified with respect to sensitive cells. These results suggest that, in this model, the changes observed in the elements of nuclear signal transduction could be related to previously reported modifications of the MDR phenotype, but that P-glycoprotein phosphorylation is not dependent from increased PKC activity.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1997

Establishment and characterization of two new cell lines derived from human metastatic breast carcinomas.

Two human cancer cell lines (MA 2 and MA 3) were established from pleural effusions of infiltrating ductal carcinomas of the breast. The lines were maintained in continuous monolayer culture with doubling times of 70 (MA 2) and 78 (MA 3) hr for more than two years and possessed extensively rearranged abnormal karyo-types with modal chromosome number of 83 (MA 2) and 81 (MA 3) and DNA index values of 1.65 and 1.77, respectively. No amplifications or rearrangements were evident in the c-myc, int-2, c-erb B2, c-Ha-ras, or hst 1 genes in MA 2 and MA 3 cell lines. The clinical histories of the patients from whom the cell lines were derived are reported and compared with the results observed in the cell lines in vitro. The presence of CEA, CA 15-3, and MCA tumor markers observed in the primary tumor tissues was retained by the established cell lines. While the primary tumor tissues were ER+/PgR borderline+ (MA 2) and ER-/PgR+ (MA 3), the MA 2 line was ER+/PgR- and the MA 3 line remained ER-/PgR+. The MDR P-glycoprotein was not expressed either in primary tumor tissues or in the respective cell lines. High expression of cytokeratins 7, 18, and 19 was evident by immunohistochemical analysis in each cell line. whereas cytokeratins 8 and 17 were poorly or not at all expressed. The treatment history of the patients from whom the cell lines were derived involved CMF followed six months later by novantrone and cisplatin plus VP 16 (MA 2) and FEC followed four years later by CMF (MA 3). The chemosensitivity pattern assay of the cell lines indicated that the MA 2 line was sensitive to doxorubicin, cisplatin, and vinblastine, whereas the MA 3 line was sensitive to doxorubicin and cisplatin. The characteristics of these cell lines indicate them to be a good experimental model to investigate breast cancer biology and anticancer drug response.

Adult↗

PubMed1997

Protein kinase C isoforms undergo quantitative variations during rat spermatogenesis and are selectively retained at specific spermatozoon sites.

Signal transduction elements, including protein kinase C, have been identified in mammalian spermatozoa. In order to evaluate the pattern of expression and the subcellular localization of nine different protein kinase C isoforms in the course of spermatogenesis, we utilized quantitative electron microscopy immunocytochemistry on thin sections of rat seminiferous tubules. The results indicate a progressive reduction of the protein kinase C isoforms present in the early stages of spermatogenesis, so that in late spermatids none of them is present in the nucleus, while the isoforms alpha, gamma and beta II are specifically retained in the acrosome, the isoforms beta I and zeta in the neck, and the isoform epsilon in the tail. These isoforms, except for beta II, are maintained at the same sites in spermatozoa. Western blotting analysis indicates the presence of alpha and gamma isoforms in the head subfraction, and of beta I, zeta and epsilon isoforms in the tail subfraction of spermatozoa. These findings suggest that specific protein kinase C isoforms may be functionally involved in some events of spermatozoa differentiation and, eventually, in the fertilization process.

Animals↗

PubMed1996

A new cell line from human infiltrating ductal carcinoma of the breast: establishment and characterization.

We established a novel cancer cell line (MAST) from the ascitic fluid of a metastatic infiltrating ductal carcinoma of the breast. The epithelial and neoplastic nature of the MAST cells was confirmed by ultrastructural analysis. The cell line was maintained as a monolayer with a doubling time of about 68 h, and it possessed an abnormal karyotype with a modal chromosome number of 60, a trisomy of chromosome 18 and other unidentified rearranged chromosomes. Among the markers consistently found in MAST metaphases, we noted a t(14; 14) and a very large subtelocentric, a large satellited acrocentric and a very large submetacentric chromosome with striking fluorescent bands. Immunoenzymatic assay demonstrated that the MAST cell line was positive for estrogen and progesterone receptors. The in vitro drug-sensitivity assay showed a marked resistance of the cell line to 5-fluorouracil and 4-hydroperoxycyclophosphamide and a moderate resistance to etoposide and 4'-epidoxorubicin. The molecular analysis showed a four-to sixfold amplification of the c-myc gene and no amplification or rearrangement of the int-2, c-erbB-2, c-Ha-ras, c-mos and hst-1 genes.

Adult↗

PubMed1996

Influence of specimen preparation on the identification of phospholipids by the phospholipase A2-gold method in mineralizing cartilage and bone.

The role of phospholipids in biological mineralization has been hypothesized but not fully elucidated. In order to identify phospholipids at the ultrastructural level in the mineralizing extracellular matrix, rat epiphyseal cartilage and metaphyseal bone have been labeled with the phospholipase A(2) (PLA(2))-gold method. The specificity and the efficiency of phospholipid detection have been evaluated by postembedding labeling of sections from epoxy- or hydrophilic resin-embedded samples, and by preembedding labeling of cryosectioned samples. The efficiency of the labeling was higher in cryosections than in hydrophilic resin-embedded specimens, while lower efficiency was found in epoxy resin-embedded samples. A 2- to 6-fold increase of the labeling density in calcified with respect to uncalcified areas of cartilage and bone has been found, depending on the specimen preparation used. The labeling intensity was significantly higher, at the periphery of the calcifying nodules in the epiphyseal cartilage matrix and in the calcifying osteoid, while the fully calcified bone matrix presented a weak labeling. Matrix vesicles, which are considered a possible source of extracellular phospholipids, appeared labeled in cryosections and in epoxy resin-embedded samples after a preincubation with PLA(2), which also increased the labeling of the intracellular membranes. The localization of phospholipids in the areas of initial mineralization suggests some hypotheses on the possible involvement of these molecules in the mineral phase deposition process.

Animals↗

PubMed1996

Interleukin-1 alpha induces variations of the intranuclear amount of phosphatidylinositol 4,5-bisphosphate and phospholipase C beta 1 in human osteosarcoma Saos-2 cells.

Some key elements of signal transduction have been identified within the nucleus and demonstrated to be responsive to specific agonists in numerous cell types. In particular, mitogenic stimuli have been reported to induce a transient increase of the nuclear phospholipase C beta 1 activity, causing the release of inositide-derived second messengers, whereas differentiating stimuli induced a decrease of the enzyme activity and an increase of nuclear phosphatidylinositol 4,5-bisphosphate (PIP2). Recently, we reported evidence, in human osteosarcoma Saos-2 cell lines, on the presence of specific nuclear phospholipase C isoforms and on the activation of phospholipase C beta 1 in the nucleus following the exposure to interleukin-1 alpha. In this study we report immunocytochemical ultrastructural evidence on quantitative variations of PIP2 and phospholipase C beta 1 amounts in the nucleus of Saos-2 cells at different times of exposure to interleukin-1 alpha. After short periods of culture in the presence of the agonist, the intranuclear amount of PIP2 is decreased, while a translocation of phospholipase C beta 1 occurs from the cytoplasm to the nucleus, in correspondence with the increased hydrolyzing activity of the enzyme. After longer periods of incubation with interleukin-1 alpha, on the other hand, the intranuclear amount of PIP2 is restored to initial level, while the amount of phospholipase C beta 1 is increased both at the nuclear and cytoplasmic level, when its activation is no longer effective. The results, compared with those obtained in other cell types responsive to given agonists, account for a cell-specific modulation of signal transduction based on polyphosphoinositide breakdown at the nuclear level.

Bone Neoplasms↗

PubMed1996

Cytoplasmic and nuclear localization sites of phosphatidylinositol 3-kinase in human osteosarcoma sensitive and multidrug-resistant Saos-2 cells.

The intracellular localization of phosphatidyl-inositol 3-kinase (PI 3-kinase) has been analyzed by western blotting, confocal, and electron microscopy immunocytochemistry in human osteosarcoma Saos-2 cells. By western blotting, the enzyme appears to be present in both the cytoplasmic and nuclear subfractions. By confocal microscope immunocytochemistry, the cytoplasmic fluorescence is localized in the perinuclear region and on a network of filaments, while a diffused signal is present in the nucleus, except for the nucleolar areas. Ultrastructural analyses on whole cells and on in situ matrix preparations reveal that nuclear PI 3-kinase is localized in interchromatin domains, in stable association with inner nuclear matrix components, while the enzyme diffused in the cytosol is partly associated with the cytoskeletal filaments. Quantitative evaluations indicate that, in a multidrug-resistant variant obtained by continuous exposure of Saos-2 cells to doxorubicin, the amount of nuclear and cytoplasmic PI 3-kinase is significantly lower than in the sensitive parental cell line. The nuclear localization of PI 3-kinase and its variation in multidrug-resistant cells, characterized by a reduced mitotic index, are consistent with the data on the existence of a nuclear inositol lipid cycle, which could also utilize 3-phosphorylated inositides to modulate signal transduction for the control of some key functional activities.

Blotting, Western↗

PubMed1996

Transfected Saos-2 cells overexpressing phosphoinositidase C beta 1 isoform accumulate it within the nucleus.

The subcellular partitioning of the phosphoinositidase C (PIC) isoforms involved in signal transduction, with the selective localization of the PIC beta 1 isoform in the nucleus, represents a crucial aspect of the complex mechanism of cell response to agonists. In order to further elucidate this phenomenon, we utilized human osteosarcoma Saos-2 cells, transfected with the cDNA for rat PIC beta 1. In the cells overexpressing this isoform, immunocytochemical analyses at the electron microscope level reveal an increased synthesis at the cytoplasm and a significant accumulation within the nucleus of the protein. Interestingly, the sites of intranuclear localization are, as in wild type cells, the interchromatin domains. These results indicate that the transfected cells maintain the capability of accumulating the enzyme within the nucleus and can be considered a model for functional studies on the nuclear signal transduction also in response to specific agonists.

Cell Nucleus↗

PubMed1996

Combined use of malachite green fixation and PLA2-gold complex technique to localize phospholipids in areas of early calcification of rat epiphyseal cartilage and bone.

Two cytochemical methods (malachite green fixation and PLA2-gold complex technique) were combined in order to detect the presence and ultrastructural distribution of phospholipids in epiphyseal cartilage and metaphyseal bone of 25-day-old rats. Chondrocytes and osteoblasts showed a more intense PLA2-gold complex labeling of rough endoplasmic reticulum than mitochondria and plasma membranes. Roundish osmiophilic, electron-dense, lightly labeled structures were visible at the periphery of the cells. In areas of early mineralization of cartilage, not all the matrix vesicles were labeled by PLA2-gold complex. Calcification nodules showed intense labeling in comparison with the surrounding, lightly labeled uncalcified matrix; gold particles were chiefly found at their periphery. Calcification nodules of metaphyseal bone were deeply electron-dense after glutaraldehyde-malachite green fixation; PLA2-gold labeling was mainly found in connection with crystals, whereas the uncalcified matrix was weakly labeled. Calcified bone matrix showed a heavy labeling with randomly scattered gold particles. When pre-embedding decalcification was carried out in the presence of malachite green, a good preservation of organic component was obtained. The PLA2-gold positivity of decalcified areas of cartilage and bone confirmed the presence of phospholipids in mineralized matrix.

Animals↗

PubMed1996

Nuclear phosphoinositide-specific phospholipase C, phosphatidylinositol 4,5-bisphosphate and protein kinase C during rat spermatogenesis.

Presence and intracellular distribution of phosphoinositide-specific phospholipase C, phosphatidylinositol 4,5-bisphosphate and protein kinase C have been investigated in rat maturing germ cells and spermatozoa. The isoforms beta 1 and gamma 1 of phosphoinositide-specific phospholipase C were immunologically identified and found to be predominantly nuclear or cytoplasmic and nuclear, respectively. The two enzymes were present in the maturing cell lineage of the seminiferous tubule, except for the nucleus of late spermatids, and absent in spermatozoa, in which, however, a phosphoinositide-specific phospholipase C activity persisted, due to yet uncharacterized enzyme(s). Protein kinase C paralleled these developmental changes, and was completely down-regulated in both total cell homogenates and isolated nuclei obtained from spermatozoa. On the contrary, phosphatidylinositol 4,5-bisphosphate, present at the nuclear level in all cell types, accumulated in the nuclei of late spermatids and spermatozoa. These data support the contention that the spermatozoon nucleus stores a lipid-dependent signaling apparatus which could be reactivated either during sperm maturation or at fertilization.

Animals↗

PubMed1996

Ultrastructural features and P-glycoprotein immunolocalization in Saos-2/DX580 multidrug-resistant human osteosarcoma cells.

The multiple drug type of resistance to anticancer agents (MDR) is mediated by an over-expression of the MDR1 gene product, the P-glycoprotein. This is largely present at the cell surface of MDR cells, mediating the active efflux of cytotoxic molecules, but may be found also intracellularly. In this paper, using Saos-2 human osteosarcoma cells as a model, we provide further evidence of increased presence of P-glycoprotein at the plasma membrane and in the nucleus of MDR cells, where it is closely bound to the nuclear matrix. The structural changes observed in Saos-2 MDR cells, including an increase of the cell surface by the formation of blebs, and a peculiar clustering of chromatin, which are similar to those observed in other MDR cell lines, are likely to be associated with the observed overexpression of the P-glycoprotein at the cell membrane and nuclear level. These findings suggest the existence of more complex, still undetermined, mechanisms underlying the MDR phenomenon.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1995

Immunocytochemical evaluation of protein kinase C translocation to the inner nuclear matrix in 3T3 mouse fibroblasts after IGF-I treatment.

The complex pathway which links the agonist-cell membrane receptor binding to the response at the genome level involves, among other elements, protein kinase C (PKC). Agonists acting at the cell membrane can affect an autonomous nuclear polyphosphoinositide signaling system inducing an activation of nuclear phosphoinositidase activity and a subsequent translocation of PKC to the nuclear region. The fine localization of PKC has been investigated by means of electron microscopy quantitative immunogold labeling in 3T3 mouse fibroblasts, mitogenically stimulated by IGF-I. The enzyme, which in untreated cells is present in the cytoplasm, except for the organelles, and in the nucleoplasm, after IGF-I treatment is reduced in the cytoplasm and almost doubled in the nucleus. The PKC isoform translocated to the nucleus is the alpha isozyme, which is found not only associated with the nuclear envelope but mainly with the interchromatin domains. By using in situ matrix preparations, PKC appears to be retained at the nuclear matrix level, both at the nuclear lamina and at the inner nuclear matrix, suggesting a direct involvement in the phosphorylation of nuclear proteins which are responsible for the regulation of DNA replication.

3T3 Cells↗

PubMed1995

Immunocytochemical detection of the intranuclear variations of phosphatidylinositol 4,5-bisphosphate amount associated with changes of activity and amount of phospholipase C beta 1 in cells exposed to mitogenic or differentiating agonists.

The intracellular localizations of phosphatidylinositol 4,5-bisphosphate (PIP2) and of its hydrolyzing enzyme phospholipase C (PLC; in this case the beta 1 isoform) have been evaluated by electron microscope immunocytochemistry in cells exposed to mitogenic or differentiating agents. These cells have been previously demonstrated to present a signal transduction system based on the polyphosphoinositide hydrolysis localized at the nuclear level, which can be specifically modulated by agonists. The results demonstrate that in Swiss 3T3 mouse fibroblasts mitogenically stimulated by insulin-like growth factor I (IGF-I), a rapid and transient decrease of the PIP2 detectable by immunogold labeling occurs at the nuclear interior. This effect appears due to the activation of the PLC beta 1 isozyme already present in the nucleus, since no significant variations of the enzyme amount and distribution can be detected by immunolabeling. However, after 30 min of exposure to IGF-I, when the PLC beta 1 activity is returned to basal level, a slight but significant increase of the enzyme amount is detected both in the nucleus and in the cytoplasm. On the other hand, an increased accumulation of PIP2 in the nucleus, accompanied by a decrease of the intranuclear amount of PLC beta 1 isozyme, have been observed in mouse erythroleukemia Friend cells, induced to erythroid differentiation by dimethylsulfoxide (DMSO). These results indicate that quantitative immunocytochemistry represents an increment in the available methodologies to investigate the complex regulation of nuclear PI-signalling.

3T3 Cells↗

PubMed1995

Multidrug-resistance (MDR) phenotype of human osteosarcoma cells evaluated by quantitative morphological and electron microscopy analyses.

Multidrug-resistant (MDR) variants of a human osteosarcoma cell line (U-2 OS) have been recently obtained by continuous exposure to doxorubicin (DX). The growth and phenotypic characteristics of these cell lines have been demonstrated to be related to the level of expression of P-glycoprotein. In this work, the morphological changes associated with MDR have been evaluated by quantitative image analysis and transmission electron microscopy. Resistant cells present morphological changes with respect to sensitive cells at both cytoplasmic and nuclear level. Some of these changes appear to be related to the degree of resistance but not to the direct presence of DX, since deprived cells maintain some modified characters, while others are partly lost. These findings suggest that DX exposure affects cell metabolism causing progressive changes of the cell morphotype.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1995

Immunocytochemical detection of phosphatidylinositol 4,5-bisphosphate localization sites within the nucleus.

Phosphatidylinositol 4,5-bisphosphate (PIP2) is a key element of signal transduction, being the preferential substrate of specific phospholipases that produce second messengers such as inositol trisphosphate (IP3) and diacylglycerol (DG). Because PIP2 has been cytochemically identified by monoclonal antibodies not only in the cytoplasmic membranes but also in the nuclear envelope and within the nucleus, we performed a study by immunoblotting and by confocal and electron microscopic immunocytochemistry to identify the nuclear sites of PIP2 localization and to exclude any cross-reactivity of the antibody with other nuclear molecules. The results confirm the specificity of the immunolabeling and indicate that PIP2 is localized at precise intranuclear sites both in in situ and in isolated nuclei. They also show that a significant amount of the phospholipid is retained by the cytoskeleton and by the inner nuclear matrix in in situ matrix preparations. Moreover the sensitivity of the immunocytochemical reaction is capable of detecting quantitative variations of PIP2 nuclear content induced by agonists that modulate the signal transduction system at the nuclear level.

3T3 Cells↗

PubMed1995

Immunocytochemical detection of phosphatidylinositol 4,5-bisphosphate in Epon embedded specimens.

A comparison between the time-consuming freeze-substitution Lowicryl HM 20 embedding procedure with the Epon embedding method for the immunocytochemical detection of phosphatidylinositol 4,5-bisphosphate (PIP2) by means of a monoclonal antibody is described. The results indicate that the intracellular localization of the phospholipid and the efficiency of its identification are similar, opening the possibility of performing the immunoreaction also on samples that are routinely processed for pathological investigations.

Animals↗

PubMed1995

The intranuclear amount of phospholipase C beta 1 decreases following cell differentiation in Friend cells, whereas gamma 1 isoform is not affected.

The existence of a signal transduction system in the nucleus, based on polyphosphoinositide breakdown mediated by specific phosphoinositidases (PLC), has been widely documented. In different cell systems, nuclear PLCs can be modulated, in response to agonists, either by enhancing or by down-regulating their activity, thus leading to DNA replication or to cell differentiation. Friend cells, induced to erythroid differentiation by dimethyl sulfoxide (DMSO), show a down-regulation of PLC beta 1 isoform, as indicated by the reduction of the transcription of its mRNA and of the in vitro synthesis of its translation product. The intracellular localization and the amount of different PLC isoforms have been evaluated by electron microscope immunocytochemistry. In untreated Friend cells, PLC beta 1 and gamma 1 isoforms are both present within the nucleus, whereas mainly the gamma 1 isoform is detected in the cytoplasm. The small amount of cytoplasmic PLC beta 1 is probably representative only of the newly synthesized enzyme. Quantitative immunolabeling analyses demonstrate that erythroid differentiation is associated with a significant decrease of the PLC beta 1 amount in the nucleus and with an almost complete disappearance of that isoform in the cytoplasm, whereas the PLC gamma 1 isoform is unaffected. The two PLC isoforms, moreover, appear to be differently associated with the nuclear components, PLC beta 1 being steadily bound to the inner nuclear matrix, whereas PLC gamma 1 is almost completely soluble.

Animals↗

PubMed1995

Nuclear immunolocalization of P-glycoprotein in multidrug-resistant cell lines showing similar mechanisms of doxorubicin distribution.

The MDR1 gene product P-glycoprotein is a plasma membrane efflux pump which is responsible for multiple drug resistance of cancer cells. Although the ability of multidrug-resistant cells to exclude drugs from the nucleus is a distinctive and possibly the main mechanism for resistance against a number of drugs, including doxorubicin, this phenomenon is not entirely understood. In this paper, the relationship between doxorubicin subcellular distribution and P-glycoprotein activity at different cell sites has been investigated by different techniques. Cytofluorometry and confocal microscopy were used to study doxorubicin subcellular distribution in U-2 OS human osteosarcoma cells and in the multidrug-resistant variant U-2 OS/DX580. Stable levels of doxorubicin accumulation were found in the nuclei of sensitive cells, whereas the absence of detectable levels of drug in the nuclei of resistant cells could be attributed to an energy-dependent mechanism. Moreover, in resistant cells, inhibition of P-glycoprotein activity was able to induce drug accumulation in the nuclei of resistant cells and to achieve cytotoxic effects comparable to those observed in sensitive cells. Similar results were also found in isolated nuclei from U-2 OS/DX580 cells. The expression of P-glycoprotein in U-2 OS/DX580 and in two other multidrug-resistant cell lines (SW948-R-300 and LoVo-R-100) was investigated by confocal microscopy and immunoelectron microscopy, by using a panel of monoclonal antibodies directed against this protein. Higher levels of P-glycoprotein expression, not only in the plasma membrane and inside the cytoplasm, but also in the nucleus, were found in U-2 OS/DX580 and in LoVo-R-100 multidrug-resistant cells compared to their corresponding sensitive cells. SW948-R-300 cells, featuring increased amounts of MDR1 mRNA but lacking P-glycoprotein expression at the cell surface, showed a higher P-glycoprotein immunolabeling only in the nucleus and in the cytoplasm. The localization of P-glycoprotein in the nucleus of multidrug-resistant cells was confirmed also by studies on isolated nuclei and nuclear matrices, and by Western blot analysis on total cell and isolated nuclear extracts. These findings, suggesting the possible involvement of nuclear P-glycoprotein in the regulation of subcellular doxorubicin distribution in multidrug-resistant cells, open new insights on the mechanisms of P-glycoprotein-mediated resistance to anticancer drugs.

ATP Binding Cassette Transporter, Subfamily B, Mem↗