Biomedical subjects

R Di Primio

Publications and source records attributed to R Di Primio.

At least 19 recordsLinked to original sources

PubMed1996

Dimethyl sulfoxide induces programmed cell death and reversible G1 arrest in the cell cycle of human lymphoid pre-T cell line.

In human B- and T-differentiated lymphoid cell lines DMSO was found to arrest the proliferation at the G1 stage of the cell cycle, without any detectable differentiation and DMSO itself was found to prevent apoptosis. Programmed cell death, or apoptosis, is now thought to be an important regulatory process in normal hemopoiesis and in the lymphoid system this program is started in the immune process such as autoreactive T-cell elimination in the thymus, and antigen-driven B-cell selection in the terminal centre. For this purpose, we have analysed the effect of DMSO using undifferentiated pre-B (KM-3) and pre-T (RPMI-8402) human lymphoid cells. Results obtained by multiparametric analyses show that DMSO affect only the pre-T cell line inducing a reversible G1 arrest of the cell cycle with a significant presence of apoptotic cells and modification of terminal transferase (TdT) expression. Pre-B cell line is resistant to DMSO treatment. These data provide evidence of a new model for the study of the selective cell type depending effect of DMSO in the immune system.

Apoptosis↗

PubMed1996

Intracellular distribution of polyamines in human lymphoblastoid cell line during phorbol ester-induced differentiation.

Polyamines such as putrescine, spermidine and spermine play an important role in nucleic acid metabolism. These aliphatic amines display a key role in cell-induced transformation by carcinogen substances. In particular, one of these, the phorbol myristate acetate, provokes cell differentiation and gives an increase of ornithindecarboxylase activity; enzyme regulating the pathways of polyamines. In this study we analyse the trend of the polyamines at cytoplasmic and nuclear level during phorbol treatment. Our results show a correlation between nuclear and cytoplasmic spermine, 3H-Thymidine, 3H-Leucine incorporation and cell cycle phases. These data remark that the polyamines are differently distributed into the cell during the phorbol myristate acetate-mediated differentiation process and that the spermine is down-regulated for to supply the increased protein biosynthesis.

B-Lymphocytes↗

PubMed1995

Terminal deoxynucleotidil transferase is a nuclear PKC substrate.

Protein phosphorylation is the regulatory mechanism of many cellular events in response to changes in metabolic activity and environmental conditions. Seeing that PKC and TdT levels in cells are both regulated by PMA, we sought particularly intriguing to investigate TdT phosphorylation in vivo, utilizing KM-3 cells, a TdT-positive human pre-B cell line treated with PMA and in vitro, employing purified PKC and human recombinant TdT. Our data show that TdT is a substrate for PKC activity, suggesting that TdT phosphorylation could play a key role in the pathway affecting the control of gene transcription and protein synthesis during lymphoid cells differentiation.

Autoradiography↗

PubMed1995

Polyamines and terminal deoxynucleotidyl transferase expression in KM 3 pre-B cell line during phorbol ester induced differentiation.

The aliphatic polyamines, putrescine, spermine and spermidine belong to a category of molecules implicated in DNA replication. Their synthesis is strongly activated during the G1 period and they have been implicated in the regulation of cell proliferation and differentiation. Terminal transferase is a DNA polymerase present in pre-T and pre-B cells and its expression can be modulated by phorbol ester treatment. In this study we have monitored the relationship of intracellular polyamine levels with terminal deoxynucleotidyl transferase down-regulation induced by 12-O-tetradecanoyl phorbol myristate 13-acetate treatment in the human pre-B KM-3 cell line. Phorbol myristate acetate can cause an increase, at 4 and 8 hours of differentiation, of intracellular levels of putrescine as well as a decrease in terminal deoxynucleotidyl transferase synthesis showing the probable involvement that polyamines have in the differentiation process.

B-Lymphocytes↗

PubMed1995

Nuclear translocation of beta II PKC isoenzyme in phorbol ester-stimulated KM-3 pre-B human leukemic cells.

Members of the protein kinase C (PKC) family play a key role in regulating cell growth and differentiation in response to several stimuli, including hormones, neurotransmitters, and growth factors. The different properties and substrate specificity of the PKC isoforms are not fully understood, and they are assumed to have specific functions in intracellular signaling. In lymphoid cells, the effects of PMA and Ca2+ ionophore, singly or in combination, on activation and expression of Ca(2+)-dependent PKC at the level of protein and messenger RNA have been examined. Starting from these observations and the possibility that differential isoenzyme expression might contribute to the differences in phorbol ester sensitivity of lymphoid cells, it seemed worthwhile to investigate the expression and the modulation of PKC isoforms in KM-3 cells, a human pre-B cell line, upon treatment with phorbol 12-myristate 13-acetate (PMA). Using multiparametric analysis we detected three PKC isoforms in the KM-3 cell line: alpha, beta II, and zeta. PMA treatment causes an intranuclear translocation of the beta II isoform, via the nuclear pore complex, associated with the interchromatinic regions. These data suggest that the beta II isoenzyme may play a strategic role in signal transduction and regulation of specific gene expression in B lymphocytes.

Biological Transport↗

PubMed1995

Terminal transferase positive rat thymocytes are resistant to steroid-induced apoptosis.

Apoptosis is a prominent mechanism of programmed cell death in the immune system. In the thymus apoptosis is responsible for the deletion of autoreactive T-cells during thymic differentiation. The typical features of apoptosis are characterized by nuclear and cytoplasmic morphologic changes, along with cleavage of chromatin at regularly spaced sites. Terminal deoxynucleotidyl transferase (TdT) is a DNA polymerizing enzyme found at an early stage of T and B lymphocyte differentiation, which generates diversity in the DNA sequence of immunoglobulin (Ig) or T cell receptor (TCR). The combined evaluations of thymocyte morphological features, immune phenotype and thymic topography associated to TdT expression allow the recognition of three different thymocyte subpopulations, characterized by small-size, intermediate-size and large-size. The results of this study show that dexamethasone (Dx)-treatment induces cell death via apoptosis involving distinct transformations related to differentiation stages of thymic subpopulations. Intermediate and small-size thymocytes that are TdT-negative or weakly positive at nuclear level are Dx sensitive. In contrast the large-size thymocytes, highly TdT positive, corresponding to the undifferentiated cells, do not show significant morphological modifications and TdT positivity to Dx-treatment. Immunocytochemical analysis shows that Dx-treatment does not affect TdT synthesis but morphological changes, occurring during apoptotic process, are responsive to intracellular movement and intranuclear arrangement of the TdT.

Animals↗

PubMed1995

Cytokines and programmed cell death in burkitt lymphoma cells.

Tumour necrosis Factor (TNF), Interleukin 1alpha and beta (IL-alpha and IL-beta), Interleukin 7 (IL-7) and Stem Cell Factor (SCF) are cytokines synthesized by immune system cells under stimulation of various agents. Apoptosis, or programmed cell death, is a process that appears in response to specific stimuli, apparently following an intrinsic program. In this work we examined, in RA-1 human lymphoblastoid B cell line, the effect induced by different cytokines in cell proliferation and in programmed cell death. After 48 hours of treatment is present an antiproliferative affects, detected by 3H-thymidine incorporation and morphological changes related to apoptotic process.

Apoptosis↗

PubMed1994

Interferon-gamma (IFN-gamma) induces programmed cell death in differentiated human leukemic B cell lines.

Interferons (IFNs) are cytokines that exert an antiviral effect on target cells and possess immunomodulatory and antitumor properties. In this study we have investigated the effects of human recombinant IFN-gamma on human leukemic B cell lines at different stages of maturation. Our data show that in Burkitt's lymphoma RAMOS-1 B cells IFN-gamma induces a reduction of cell growth and a clonal selection via programmed cell death; in contrast, IFN-gamma treatment of KM-3 pre-B cells does not induce biochemical and morphological changes as shown by electron microscope analysis and DNA gel electrophoresis.

Apoptosis↗

PubMed1994

Protein kinase C modulation in apoptotic rat thymocytes: an ultrastructural analysis.

Numerous events in the cell, such as gene expression, cell growth and metabolism are regulated by signal transduction pathways involving protein kinase C (PKC). Recent data indicate that a PKC-dependent mechanism also underlies the apoptotic death of cells induced by glucocorticoid hormones. In this report we have analysed the changes of PKC during dexamethasone-induced apoptosis in thymocytes by means of immunocytochemical and immunochemical analysis. The data obtained show an increase and intracellular movement of protein kinase C, which is translocated to the nucleus and linked to the nuclear matrix during the apoptotic process.

Animals↗

PubMed1994

Human leukemic pre-B line (KM-3) treated with phorbol-ester: trend of polyamines during cell differentiation.

Aliphatic polyamines, putrescine, spermine and spermidine present in bacteria and eukaryotic cells are essential for cell growth. Generally, polyamine levels are elevated in rapidly growing normal and pathological systems. Since polyamines belong to the category of molecules whose synthesis is strongly activated during the G1 period they have been implicated in the cell's preparation for DNA replication. In our experiments, we have differentiated by phorbol-ester, the human pre-B leukemic cell line KM-3. Biochemical and flow cytometric analysis show an increase, in treated cells, of polyamines pathway related to G1 and early S-phase of cell cycle during B cell differentiation.

B-Lymphocytes↗

PubMed1993

Phorbol ester-induced effects on cell cycle progression and terminal deoxynucleotidyltransferase (TdT) activity in KM-3 pre-B cell line.

Phorbol myristic acetate (PMA) is a tumor-promoting agent that has been shown to induce differentiation of human leukemia cells and of normal lymphoid cells. We have investigated the ability of PMA to induce inhibition of cell growth of the human KM-3 pre-B leukemic cell line by multiparametric analysis. Our results show that PMA treatment induces cell differentiation with the disappearance of terminal deoxynucleotidyltransferase and a decrease of cell growth, as evaluated by [3H]thymidine uptake. Flow cytometric analysis of BrdU incorporation shows that PMA is able to induce a modification of the cell cycle with a sharp decrease of the percentage of S-phase cells, which is more evident after 24 h of treatment. Comparison between the cell growth kinetics and TdT synthesis and activity shows that differentiated cells are still able to proliferate to a certain extent and that the TdT disappearance and the initial decrease of cell proliferation are two independent effects of PMA.

B-Lymphocytes↗

PubMed1993

Inositol lipid-mediated intranuclear signalling: a comparative analysis of in vivo labelling in interferon alpha-sensitive and -resistant Daudi lymphoma cells.

Changes in inositol lipid and diacylglycerol metabolism have been analysed in Daudi lymphoma cells treated up to 24 h with human DNA recombinant interferon alpha. Results showing a different response of nuclear phosphoinositides and diacylglycerol, compared to whole cells, suggest that the intranuclear signalling system activated by interferon in Daudi cells involves nuclear inositol lipid metabolism. A well-characterized clone of Daudi cells selected for resistance to the antiproliferative action of interferon provided controls for the specificity of results.

Adolescent↗

PubMed1993

Decrease in nuclear phospholipids associated with DNA replication.

Lipid metabolism in nuclei is very active and appears involved in the transduction of signals to the genome in response to agonists acting at the plasma membrane level. However, the precise topology of nuclear lipid metabolism and the relationship between nuclear lipids and crucial events of the cell function, such as DNA replication, have not been fully elucidated. By using a recently developed cytochemical method for detecting phospholipids inside the nucleus of intact cells at the electron microscope level, we have analyzed the changes in intranuclear phospholipids in DNA-replicating versus resting cells, which are both present in the same sample of regenerating liver after partial hepatectomy. The pattern of DNA synthesis in replicating cells has been monitored by electron microscope immunocytochemistry after bromodeoxyuridine (BrdU) labeling. The data obtained, which allow a fine localization and a quantitative analysis of both DNA synthesis and phospholipid distribution, indicate a significant reduction in the phospholipids detectable inside the nucleus in all steps of the S phase. This could depend on an increased nuclear phospholipid hydrolysis, whose products should in turn activate some of the enzymes involved in the control of DNA replication.

Animals↗

PubMed1993

Phorbol ester induces changes in the synthesis of nuclear polyphosphoinositides and expression of terminal deoxynucleotidil transferase (TdT) in nuclei of KM-3 cells.

Terminal deoxynucleotidyl Transferase (TdT) play an essential role in the immune system differentiation. KM-3 cells are lymphoblastoid cells expressing the TdT and when induced to differentiate by phorbol ester (PMA) they loose this enzyme. Therefore, because of the suggested involvement of polyphosphoinositide in controlling the nuclear events it has been analyzed the phosphorylation of nuclear polyphosphoinositides during KM-3 differentiation. When the differentiated state is reached the phosphorylation level of PIP2 increases in isolated nuclei and this is accompanied by a concomitant decrease of PIP and PA, hinting at a correlation between polyphosphoinositide metabolism and TdT expression.

Cell Differentiation↗

PubMed1992

Mouse and human hemopoietic cell lines of erythroid lineage express lamins A,B and C.

Using monoclonal antibodies, we have studied the expression of lamins A,B,C and vimentin in mouse and human erythroleukemia cells. We have found that in contrast with previous reports these cells have all three lamins. Mouse cells lack vimentin, whereas human cells express it. Lamins B and C are the most abundant lamins, whereas considerably less lamin A is detectable. Our results argue that some mouse and human hemopoietic cells can express all three lamins and that production of vimentin does not necessarily precede that of lamins A/C, as other reports have suggested in the past. The data also show that the absence of a salt resistant inner nuclear matrix is not always related with the lack of lamins A/C and vimentin, as recently proposed.

Animals↗

PubMed1992

Intracellular localization of terminal transferase during the cell cycle.

Changes in the localization of terminal transferase during the cell cycle in random cultures of human pre-T leukemia line RPMI-8402 were examined by light and electron microscopy on immunoperoxidase-stained preparations. Paraformaldehyde-fixed and saponin-permeabilized human cells were used with a monoclonal anti-human terminal deoxynucleotidyl transferase (TdT) primary reagent to demonstrate changes in enzyme distribution occurring between interphase and mitosis. Nuclear localization is found uniformly during interphase. At metaphase, however, the majority of TdT staining appears randomly distributed in the cytoplasm and traces of TdT staining remain associated with mitotic chromatin. At later phases, when the daughter cells are forming, the enzyme again appears to be restricted to the new nuclear structure.

Cell Cycle↗

PubMed1992

Ultrastructural localization of Terminal deoxynucleotidyl Transferase (TdT) in rat thymocytes.

TdT positive cells in rat thymus belong to distinct subsets as shown by light and electron microscopic immunocytochemistry. Using polyclonal antibodies to calf TdT and peroxidase labeled goat anti-rabbit IgG it has been possible to identify several subpopulations of TdT positive thymocytes. Large blasts corresponding to thymocytes at early maturational stages, are strongly positive for TdT which is diffusely distributed in both the nucleus and the cytoplasm. Smaller cells which correspond presumably to more advanced stages of maturation display nuclear TdT only, or are negative. Ultrastructural analyses of TdT indicate that the localization of the enzyme is related to the morphological features of the cells and TdT expression corresponds to maturational stages of T-cells.

Animals↗

PubMed1991

Association between nuclear matrix and terminal transferase: an electron microscope immunocytochemical analysis.

Nuclear matrix extracted from KM-3, a human pre-B leukemia cell line, appears to have a site of linkage for terminal deoxynucleotidyl transferase (TdT). The immunocytochemical analysis of the distribution of TdT using a rabbit polyclonal antibody which recognizes human terminal transferase, shows that the nuclear framework of these cells contains sites of immunoreactivity that appear uniformly distributed on the matrix fibres, while the nucleolar region is unreactive. This evidence points out the possibility that TdT could reside in the proteinaceous scaffold of the nucleus defined as nuclear matrix, thus strengthening the evidence for the metabolic and regulatory roles ascribed to this nuclear framework.

Cell Line↗