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Biomedical subjects

F Grignani

Publications and source records attributed to F Grignani.

At least 55 records · Page 3Linked to original sources
PubMed1995

Characterization of a new monoclonal antibody (PG-M3) directed against the aminoterminal portion of the PML gene product: immunocytochemical evidence for high expression of PML proteins on activated macrophages, endothelial cells, and epithelia.

PG-M3 is a new monoclonal antibody (MoAb) specifically directed against a peptide sequence located in the aminoterminal region of the human PML protein. PML gene fuses with the retinoic acid receptor alpha (RAR alpha) gene during the t(15; 17) chromosomal translocation of acute promyelocytic leukemia (APL). The epitope recognized by PG-M3 is species-specific and fixative-resistant and is shared by most PML isoforms and PML/RAR alpha fusion proteins. PML is consistently located within the nucleus, although a minority of cells (about 20%), both in vitro and in vivo, show positivity for PML also in the cytoplasm. The nuclear staining pattern of PG-M3 varies from speckled (cells other than APL) to micropunctate (APL cells). Although two physiologically expressed PML isoforms are detectable by immunocytochemistry only or predominantly in the cytoplasm of transfected cells, the cytoplasmic localization of PML is a property also shared by the PML isoforms that predominantly localize to the nuclei. Immunohistologic analysis of normal human tissues with the PG-M3 MoAb showed variable PML expression, with the highest levels of the protein in postmitotic, differentiated cell types, such as endothelial cells, epithelia, and tissue macrophages, especially activated ones. In keeping with this in vivo finding, PML appears strongly upregulated in the U937 promonocyte cell line after exposure to agents that induce monocyte/macrophage activation (interferon gamma) or maturation (vitamin D3 and transforming growth factor beta 1).

3T3 Cells↗
PubMed1995

Promyelocytic leukemia-specific PML-retinoic acid alpha receptor fusion protein interferes with erythroid differentiation of human erythroleukemia K562 cells.

Acute promyelocytic leukemia (APL) is characterized by a t(15;17) chromosomal translocation with breakpoints within the retinoic acid alpha receptor (RAR alpha) gene on 17 and the PML gene, which encodes a putative transcription factor, on 15. A PML-RAR alpha fusion protein is formed as a consequence of the translocation. We show here that expression of the PML-RAR alpha protein in K562 erythroleukemia cells results in a reduced expression of erythroid differentiation markers and a reduced sensitivity to the erythroid differentiative action of heme. Overexpression of RAR alpha, but not of PML, elicited a similar inhibition of K562 erythroid differentiation. These findings indicate that overexpression of either RAR alpha or PML/RAR alpha interferes with erythroid differentiation and support the hypothesis that RAR alpha is involved in the regulation of normal hematopoiesis and alteration of the RAR alpha signaling by PML/RAR alpha is implicated in the promyelocytic leukemogenesis.

Cell Differentiation↗
PubMed1995

Acute promyelocytic leukaemia cells resistant to retinoic acid show further perturbation of the RAR alpha signal transduction system.

Acute promyelocytic leukaemia (APL) cell lines resistant to all-trans retinoic acid (ATRA) have been previously derived from the NB4 cell line, and characterized as having lost the expression of the intact pml/RAR alpha fusion protein. To confirm the association between ATRA-resistance and alteration in the fusion protein at the clonal level, 16 clones were generated from ATRA-resistant APL cell lines. All clones show immunological (HLA class I and II, CD11b and c, CD13 and 33), molecular and growth features similar to the parental cell lines. To investigate whether the irradiation protocol used to generate the previously reported retinoic acid-resistant NB4.306 cell line induced additional alterations that could render these cells able to escape the anti-proliferative effect of retinoic acid (ATRA), an additional ATRA-resistant APL cell line, [NB4.007/6], was generated, under the selective pressure of ATRA, from the NB4 cell line without previous radiation. This cell line shows resistance to the anti-proliferative and differentiating action of ATRA. The NB4.007/6 cell line contains the t(15;17) chromosome translocation, shows the usual pml/RAR alpha hybrid DNA but expresses no detectable amount of the usual pml/RAR alpha protein in Western blot analysis, similarly to the NB4.306 cell line. Finally, the relative resistance to ATRA of NB4.306 and NB4.007/6 was evaluated by comparing the phenotypic (CD11b) changes induced by ATRA in these two lines with those induced in the parental, ATRA-sensitive, NB4 cell line. It is estimated that NB4.306 and NB4.007/6 are about 300 and 70 times less sensitive to ATRA than the original NB4 cell line.

Cell Line↗
PubMed1995

The acute promyelocytic leukemia-specific PML/RAR alpha fusion protein reduces the frequency of commitment to apoptosis upon growth factor deprivation of GM-CSF-dependent myeloid cells.

PML/RAR alpha is the putative transforming sequence of acute promyelocytic leukemias. We investigated the effects of PML/RAR alpha on cell survival by expressing the fusion protein in the growth factor-dependent TF-1 cell line and analyzing the kinetics of cell death after GM-CSF deprivation. Results showed that PML/RAR alpha expression markedly delayed apoptotic cell death (3 weeks vs 1 week) without inducing growth factor independence. Growth factor deprivation caused rapid and massive apoptosis of control TF-1 cells (>95% apoptotic cells after 4-5 days). Factor-deprived control cells were synchronously and irreversibly committed to apoptosis as shown by their inability to re-enter the cell cycle after GM-CSF re-addition. The percentage of apoptotic cells in the PML/RAR alpha expressing cells was low (approximately 30-35%) and constant over the 4 weeks of factor deprivation. GM-CSF re-addition produced rapid increase in cell number at all time points during the 4 weeks of factor deprivation, suggesting that commitment to apoptosis was asynchronous and delayed in PML/RAR alpha-expressing TF-1 cells. We conclude that PML/RAR alpha interferes with the genetic pathways which regulate survival by reducing the frequency of commitment to apoptosis. This biological effect of PML/RAR alpha may contribute to its leukemogenetic potential.

Apoptosis↗
PubMed1995

IL-4 is able to reverse the CD2-mediated negative apoptotic signal to CD4-CD8- alpha beta and/or gamma delta T lymphocytes.

Activation of immature thymocytes or transformed T lymphocytes via T-cell receptor (TCR)/CD3 signalling can induce programmed cell death (apoptosis). Recent data indicate that anti-CD3/TCR monoclonal antibodies (mAb) also trigger apoptosis in activated (but not resting) mature peripheral blood T lymphocytes. Here we report that triggering of resting CD4-CD8-TCR alpha beta+ and/or TCR gamma delta+ via the alternative CD2-dependent activation pathway is able to induce programmed cell death. A pair of mitogenic anti-CD2 mAb provoked a dramatic rise in [Ca2+]i that was almost entirely sustained by extracellular fluxes, and the inhibition of membrane [Ca2+/Mg2+] ATPase. The resulting endonuclease activation was able to induce DNA fragmentation, as revealed by propidium iodide staining and gel electrophoresis. Induction of apoptosis was prevented by the presence of interleukin-4 (IL-4) as well as by endonuclease inactivation with 100 microM ZnCl2, but enhanced by the contemporary block of protein kinase C. Thus it seems that in resting T lymphocytes the strong calcium signal delivered by the alternative CD2 activation pathway may act as a negative apoptotic signal in both alpha beta and gamma delta T cells with low (non-major histocompatibility complex restricted) antigenic affinity, so limiting the extension of polyclonal T-cell growth.

Antibodies, Monoclonal↗
PubMed1995

Local expansion of allergen-specific CD30+Th2-type gamma delta T cells in bronchial asthma.

BACKGROUND: T lymphocytes infiltrating airways during the allergic immune response play a fundamental role in recruiting other specialized cells, such as eosinophils, by secreting interleukin 5 (IL-5), and promoting local and systemic IgE synthesis by producing IL-4. Whether these presumed allergen-specific T cells are of mucosal or systemic origin is still a matter of conjecture. MATERIALS AND METHODS: Immunophenotype, IL-4 production, and in vitro proliferative response to specific or unrelated allergens were analyzed in the bronchoalveolar lavage (BAL) fluid lymphocyte suspensions obtained from untreated patients with allergic asthma. Healthy subjects and patients affected by pulmonary sarcoidosis, a granulomatous lung disease characterized by infiltrating Th1 CD4+ lymphocytes, served as controls. RESULTS: The proportions of gamma delta T lymphocytes, mostly CD4+ or CD4- (-)CD8-, was higher in asthmatic subjects than in controls (p < 0.05). Most BAL gamma delta CD4+ lymphocytes of asthmatic patients displayed the T cell receptor (TCR)-gamma delta V delta 1 chain. While CD30 antigen coexpression on the surface of BAL alpha beta(+) T lymphocytes was low (ranging from 5 to 12%), about half of pulmonary gamma delta T cells coexpressed it. These cells produced IL-4 and negligible amounts of interferon-gamma (IFN gamma), and proliferated in vitro in response to purified specific but not unrelated allergens. In contrast, control or sarcoidosis gamma delta T cells never displayed the CD30 surface molecule or produced significant quantities of IL-4. CONCLUSIONS: These findings not only confirm our previous hypothesis that the allergen-specific Th2-type lymphocytes found in the lungs of asthmatic patients are gamma delta T cells belonging to airway mucosal immunocytes, but also strongly support the notion that asthma is a local rather than a systemic disease.

Adult↗
PubMed1994

PML/RAR alpha+ U937 mutant and NB4 cell lines: retinoic acid restores the monocytic differentiation response to vitamin D3.

We have analyzed the differentiation program of a U937 promonocytic leukemia clone transduced with the acute promyelocytic leukemia specific PML/RAR alpha fusion gene, the expression of which is under the control of the inducible metallothionine (MT) I promoter (MTPR9 clone). MTPR9 cells treated with Zn2+ hence exhibit levels of PML-RAR alpha protein as high as fresh acute promyelocytic leukemia blasts. In the absence of Zn2+, i.e., upon low level PML/RAR alpha expression, 1,25-dihydroxyvitamin D3 (D3) and particularly D3 plus transforming growth factor beta 1 (TGF-beta 1) induced terminal differentiation of MTPR9 cells (as observed in "wild-type" U937 cells), on the basis of morphology, membrane antigen pattern, and functional criteria. Conversely, in the presence of Zn2+, D3 and D3 plus TGF-beta 1 failed to induce terminal differentiation, as evaluated by the above parameters. Interestingly, retinoic acid (RA) treatment suppresses the differentiation blockade induced by high level PML-RAR alpha protein; indeed, Zn(2+)-treated MTPR9 cells incubated with RA plus D3 exhibited significant terminal monocytic maturation, comparable to that of cells treated with D3 alone or combined with RA in absence of Zn2+. Similar observations were made in NB4, a PML-RAR+ human acute leukemic line. As expected RA treatment of NB4 cells causes granulocytic differentiation. Interestingly, the cell line is only scarcely induced to mature monocytic cells by D3 or D3 plus TGF-beta 1 treatment, whereas it is effectively induced to monocytic maturation by combined treatment with D3 and RA. Accordingly, the rate of NB4 cell proliferation is only slightly affected by D3 or D3 plus TGF-beta 1 treatment, mildly inhibited by RA, and markedly decreased by D3 plus RA. These results indicate that in both U937 and NB4 cells high level PML/RAR alpha expression inhibits the monocytic terminal differentiation program triggered by D3 or D3 plus TGF-beta 1, whereas RA treatment effectively antagonizes this inhibitory PML-RAR alpha action and restores the D3 differentiative effect.

Antigens, CD↗
PubMed1994

Analysis of p53 gene mutations in acute myeloid leukemia.

We have previously reported the absence of mutations within exons 5-9 of the p53 gene in a panel of 30 cases of acute promyelocytic leukemia (APL), which represent the M3 FAB type of acute myeloid leukemia (AML). In the present report, we extend our analysis of p53 gene mutations to 70 cases of AML representative of the other FAB types of the disease, including M1 (16 cases), M2 (20 cases), M4 (17 cases), M5 (12 cases), and M6 (5 cases). DNAs were analyzed for p53 gene mutations in exons 5 to 9 by polymerase chain reaction (PCR), single-strand conformation polymorphism (SSCP), and direct sequencing of PCR-amplified products. Mutant p53 alleles were detected in 5 of 70 cases; 1 case in exon 5, 2 cases in exon 6, and 2 cases in exon 7. The alterations of the p53 gene were represented by point mutation leading to an amino acid substitution in four cases, and deletion in the remaining case. In four of the five cases, direct sequencing indicated the loss of the normal p53 allele; in the remaining case, two mutations were detected, presumably involving both p53 alleles. Three cases showed mutations at diagnosis; in the remaining two, the mutations were observed in clinical relapse but not at diagnosis. Our results confirm the relatively low incidence of p53 mutations in AML and further support the evidence that p53 plays a role in leukemogenesis through a recessive mechanism (two-hit model) of inactivation of tumor suppressor activity.

Acute Disease↗
PubMed1994

The natural tyrosine kinase inhibitor genistein produces cell cycle arrest and apoptosis in Jurkat T-leukemia cells.

Genistein, a natural isoflavonoid phytoestrogen, is a strong inhibitor of protein tyrosine kinases. We analyzed the effects of genistein on in vitro growth, cell-cycle progression and chromatin structure of Jurkat cells, a T-cell leukemia line with a constitutively increased tyrosine phosphorylation pattern. Exposure of in vitro cultured Jurkat cells to genistein resulted in a dose-dependent, growth inhibition. Cell-cycle analysis of genistein-treated cells revealed a G2/M arrest at low genistein concentrations (5-10 micrograms/ml), while at higher doses (20-30 micrograms/ml) there was also a perturbation in S-phase progression. The derangements in cell-cycle control were followed by apoptotic death of genistein-treated cells. Immunocytochemical analysis of cells stained with a FITC-conjugated anti-phosphotyrosine monoclonal antibody showed that 30 micrograms/ml genistein effectively inhibit tyrosine kinase activity in cultured Jurkat cells. Our results indicate that the natural isoflavone genistein antagonizes tumor cell growth through both cell-cycle arrest and induction of apoptosis and suggest that it could be a promising new agent in cancer therapy.

Antineoplastic Agents↗
PubMed1994

Growth-inhibitory effects of the natural phyto-oestrogen genistein in MCF-7 human breast cancer cells.

Genistein, a natural isoflavonoid phyto-oestrogen, inhibits the tyrosine kinase activity of growth factor receptors and oncogene products, as well as the in vitro growth of some tumour cell lines. The low incidence of breast cancer in countries with a flavonoid-rich soy-based diet and the protection afforded by soy-derived products against experimental mammary tumours in rats suggest that genistein and other isoflavonoid compounds may exert an anti-tumour activity. We analysed the effects of genistein on cell number and cell cycle progression (flow cytometric analysis of propidium iodide-stained nuclei) of human breast cancer cells (MCF-7) in vitro. Genistein produced a significant, dose-dependent inhibition of MCF-7 cell growth with an ID50 of approximately 40 microM after 72 h of incubation. Cell cycle analysis showed a reversible G2/M arrest in cell cycle progression at 10 microM genistein concentrations, whilst a marked fall in S-phase cell percentage associated with a persistent arrest in G2/M phase was observed in cultures treated with genistein doses equal to or greater than 50 microM. These effects were significant at 24 h of incubation; flow cytometric analysis at later times (48 and 72 h) revealed a population of cells with decreased DNA content and nuclear fragmentation characteristic of apoptosis. Thus, the growth inhibitory activity of genistein in MCF-7 cells results from the sum of cytostatic and apoptotic effects. Since the mitogenic action of insulin and insulin-like growth factor (IGF)-I in MCF-7 cells is a tyrosine kinase-dependent phenomenon, we analysed the genistein impact on S-phase entry produced by insulin in cultures partially synchronised in G0/G1 phase by serum deprivation. Insulin addition after a 36-h culture period in serum-free medium produced a strong increase in the percentage of S-phase cells (from 18.4 +/- 2.3 to 46.2 +/- 4.1 after 24 h) which was almost completely blocked by 100 microM genistein (20.1 +/- 3.1). Immunofluorescence analysis with a fluoresceine isothiocyanate (FITC)-conjugated anti-phosphotyrosine antibody revealed a strong increase in MCF-7 cell staining after insulin stimulation, but not when genistein was added with insulin. In conclusion, the dietary phyto-oestrogen genistein inhibits in vitro growth of MCF-7 human breast cancer cells through blocks in the "critical checkpoints" of cell cycle control and induction of apoptosis. These effects are likely to depend on impairment in the signal transduction pathway from tyrosine kinase receptor(s).

Breast Neoplasms↗
PubMed1994

Effect of the acute promyelocytic leukemia PML/RAR alpha protein on differentiation and survival of myeloid precursors.

Acute promyelocytic leukaemia is characterized by an expansion of haematopoietic precursors arrested at the promyelocytic stage (1). The differentiation block can be reversed by retinoic acid, which induces blast differentiation both in vitro (2) and in vivo (3-4). Acute promyelocytic leukaemia is also characterized by a 15;17 chromosome translocation (5) with breakpoints within the retinoic acid alpha receptor (RAR alpha) gene on 17 and within the PML gene, that encodes a putative transcription factor of unknown function (6-7), on 15 (8-10). As a consequence of the translocation a PML/RAR alpha gene is formed. It is transcriptionally active and encodes a PML/RAR alpha fusion protein detectable in all APL cases (11-14). We expressed the PML/RAR alpha protein in U937 myeloid precursor cell line and show that they: 1) lose the capacity to differentiate under the action of different stimuli (vitamin D3, transforming growth factor beta 1); ii) acquire enhanced sensitivity to retinoic acid; iii) exhibit a higher growth rate that is due to a reduction in apoptotic cell death. These results provide the first evidence of biological activity of PML/RAR alpha and recapitulate critical features of the promyelocytic leukemia phenotype.

Cell Differentiation↗
PubMed1993

N-(4-hydroxyphenyl)retinamide induces apoptosis of malignant hemopoietic cell lines including those unresponsive to retinoic acid.

N-(4-Hydroxyphenyl)retinamide (HPR) is a synthetic retinoid of particular clinical interest in cancer chemoprevention. We have examined the in vitro effects of HPR on lymphoid and myeloid malignant cell lines and found that at concentrations between 10(-5) and 3 x 10(-7) M it induces a dose-dependent growth inhibition (the peak plasma concentration in patients treated with HPR is 1 to 2 x 10(-6) M). The antiproliferative effect of HPR was, in all cell lines except K422, more potent than that induced by an equimolar dose of all-trans retinoic acid (RA). Also, this effect was irreversible on HL60 and DoHH2 cells that had been exposed to HPR (3 x 10(-6) M) for 24 h, but reversible on Raji and DHL4 exposed to the retinoid for 48 and 72 h, respectively. Time-course growth analysis showed that HPR at 3 x 10(-6) M or below induces a rapid fall of thymidine uptake and viability (> 90%), whereas between 10(-6) and 3 x 10(-7) M exhibits cytostatic effects. Interestingly, the RA-resistant HL-60R and NB306 cells, characterized by a point mutation in the retinoic acid receptor (RAR) and by the loss of the pml/RAR protein, respectively, were, like the parental RA-inducible HL-60 and NB4 cell lines, fully responsive to HPR, thereby suggesting that HPR and RA could act through different receptors or pathways. DNA flow-cytofluorimetric analysis revealed that HPR does not block cells in a specific phase of the cell cycle but triggers programmed cell death or apoptosis. This phenomenon was evidenced both by the visualization, on gel electrophoresis, of fragmented DNA, and by the "in cell" enzymatic labeling of DNA breaks with fluorescent dUTP. With the latter method, apoptotic cells become detectable by 6 h following exposure to 3 x 10(-6) M HPR. Ultrastructural examination of HPR-treated samples showed cells with chromatin compaction and cytoplasm condensation, characteristic of apoptotic cells. In conclusion, our study demonstrates that HPR suppresses malignant cell growth and induces apoptosis at pharmacologically relevant doses. The differential responsiveness by a number of cell lines, especially HL-60R and NB306, to HPR and RA indicates that these compounds may act through different receptors. The clinical use of HPR, particularly in retinoic acid-unresponsive acute promyelocytic leukemia patients, is suggested.

Apoptosis↗
PubMed1993

Occurrence of resistance to retinoic acid in the acute promyelocytic leukemia cell line NB4 is associated with altered expression of the pml/RAR alpha protein.

The mechanism(s) by which acute promyelocytic leukemia (APL) cells acquire resistance to all-trans retinoic acid (ATRA) is poorly understood. We describe here an APL cell line, named NB4.306, that shows resistance to the anti-proliferative action of ATRA. This cell line is also operationally resistant to most ATRA-induced phenotypic modifications (CD11b, CD11c, CD13, and CD33). No significant differences in ATRA intracellular accumulation, efflux, or metabolism were found between NB4.306 and the parent NB4 cell line that could explain the observed resistance of the NB4.306 line. The NB4.306 cell line was found to be positive for the t15;17 translocation and showed the usual pml/RAR alpha fusion bands in both Southern and Northern blot assays, but expressed no detectable amount of the usual pml/RAR alpha protein, as assayed by Western blot analysis using an anti-RAR alpha antibody. These results were confirmed in 14 of 14 clones obtained from the NB4.306 cell line, while 30 of 30 clones obtained from the parental NB4 line expressed the usual 110-Kd fusion polypeptide. It is concluded that the occurrence of resistance to ATRA in the NB4.306 cell line is closely associated to the loss of expression of the intact pml/RAR alpha protein.

Blotting, Southern↗
PubMed1993

The acute promyelocytic leukemia-specific PML-RAR alpha fusion protein inhibits differentiation and promotes survival of myeloid precursor cells.

Acute promyelocytic leukemia is a clonal expansion of hematopoietic precursors blocked at the promyelocytic stage. The differentiation block can be reversed by retinoic acid, which induces blast maturation both in vitro and in vivo. Acute promyelocytic leukemia is characterized by a 15;17 chromosome translocation with breakpoints within the retinoic acid alpha receptor (RAR alpha) gene on 17 and the PML gene, which encodes a putative transcription factor, on 15. A PML-RAR alpha fusion protein is formed as a consequence of the translocation. We expressed the PML-RAR alpha protein in U937 myeloid precursor cells and showed that they lost the capacity to differentiate under the action of different stimuli (vitamin D3 and transforming growth factor beta 1), acquired enhanced sensitivity to retinoic acid, and exhibited a higher growth rate consequent to diminished apoptotic cell death. These results provide evidence of biological activity of PML-RAR alpha and recapitulate critical features of the promyelocytic leukemia phenotype.

Animals↗
PubMed1993

Human CD4 lymphocytes specifically recognize a peptide representing the fusion region of the hybrid protein pml/RAR alpha present in acute promyelocytic leukemia cells.

Fusion proteins present in leukemic cells frequently contain a new amino acid at the fusion point. We tested whether a peptide (BCR1/25) encompassing the fusion region of the hybrid molecule pml/RAR alpha, which is selectively expressed by acute promyelocytic leukemia (APL) cells, can be recognized by human T lymphocytes in vitro. CD4+ lymphocytes, at both polyclonal and clonal level, recognized peptide BCR1/25 in an HLA-DR--restricted fashion on presentation by autologous antigen-presenting cell (APC) or by APC expressing the HLA-DR11 restricting molecule. Control peptides corresponding to the normal pml and RAR alpha proteins were not recognized. One clone (DEG5) also exerted a high and specific cytotoxicity against autologous cells pulsed with BCR1/25. The autologous DE LCL containing a transduced pml/RAR alpha fusion gene and expressing a bcr1 type of the pml/RAR alpha hybrid protein induced the proliferation of DE anti-BCR1/25 T cell clones. It is concluded that the bcr1 type-pml/RAR alpha fusion protein of APL contains an antigenic site, absent from the normal parent molecules and recognized by human CD4+ lymphocytes.

Amino Acid Sequence↗