Fausto Grignani

A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry

Corticosteroids, calcium ionophores and anti-CD3 monoclonal antibodies kill mouse thymocytes incubated in vitro. Cell death is preceded by extensive DNA fragmentation into oligonucleosomal subunits. This type of cell death (apoptosis), which physiologically occurs in the intrathymic process of immune cell selection, is usually evaluated by either electrophoretic or colorimetric methods which measure DNA fragmentation in the nuclear extracts. These techniques are unable to determine the percentage of apoptotic nuclei or recognize the apoptotic cells in a heterogeneous cell population. We have developed a flow cytometric method for measuring the percentage of apoptotic nuclei after propidium iodide staining in hypotonic buffer and have compared it with the classical colorimetric and electrophoretic techniques using dexamethasone (DEX)-treated mouse thymocytes. Apoptotic nuclei appeared as a broad hypodiploid DNA peak which was easily discriminable from the narrow peak of thymocytes with normal (diploid) DNA content in the red fluorescence channels. When the DEX-induced apoptosis was inhibited by either low-temperature (4 degrees C) incubation or cycloheximide treatment, no hypodiploid DNA peak appeared. Similarly, thymocyte death induced by sodium azide, a substance with cell-killing activity through non-apoptotic mechanisms, did not result in any variation in the normal DNA peak. The flow cytometric data showed an excellent correlation with the results obtained with both electrophoretic and colorimetric methods. This new rapid, simple and reproducible method should prove useful for assessing apoptosis of specific cell populations in heterogeneous tissues such as bone marrow, thymus and lymph nodes.

A novel transforming protein (SHC) with an SH2 domain is implicated in mitogenic signal transduction.

A new SH2-containing sequence, SHC, was isolated by screening cDNA libraries with SH2 representative DNA probes. The SHC cDNA is predicted to encode overlapping proteins of 46.8 and 51.7 kd that contain a single C-terminal SH2 domain, and an adjacent glycine/proline-rich motif with regions of homology with the alpha 1 chain of collagen, but no identifiable catalytic domain. Anti-SHC antibodies recognized three proteins of 46, 52, and 66 kd in a wide range of mammalian cell lines. These SHC proteins complexed with and were phosphorylated by activated epidermal growth factor receptor. The physical association of SHC proteins with activated receptors was recreated in vitro by using a bacterially expressed SHC SH2 domain. NIH 3T3 mouse fibroblasts that constitutively overexpressed SHC acquired a transformed phenotype in culture and formed tumors in nude mice. These results suggest that the SHC gene products couple activated growth factor receptors to a signaling pathway that regulates the proliferation of mammalian cells.

The acute promyelocytic leukemia-specific PML-RARα 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.

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.

Cooperation between the RING+B1-B2 and coiled-coil domains of PML is necessary for its effects on cell survival

PML/RARα is the abnormal protein product of the Acute Promyelocytic Leukemia-specific 15;17 translocation. Both the PML and RARα components are required for the PML/RARα biological activities, namely its capacity to block differentiation and to increase survival of haematopoietic precursors. The physiological role of PML and its contribution to the function of the fusion protein are unknown. PML localizes to the cytoplasm and within specific nuclear bodies (NBs). In vitro, overexpression of PML correlates with suppression of cell transformation. The PML aminoterminal portion retained within the PML/RARα protein contains the RING finger, two newly defined cystein/histidine-rich motifs called B-boxes (B1 and B2) and a coiled-coil region. We report here that PML has a growth suppressive activity in all the cell lines tested, regardless of their transformed phenotype, and that the cellular basis for the PML growth suppression is induction of apoptotic cell death. Analysis of various nuclear and cytoplasmic PML isoforms showed that the PML growth suppressive activity correlates with its nuclear localization. Analysis of the localization and growth suppressive activity demonstrated that: (i) the Ring+B1-B2 and coiled-coil regions are both indispensable and sufficient to target PML to the NBs; (ii) individual deletions of the various PML domains have no effect on its growth suppressor activity; (iii) the Ring+B1-B2 region exerts a partial growth suppressor activity but its fusion with the coiled-coil region is sufficient to recapitulate the suppressive function of wild type PML. These results indicate that PML is involved in cell survival regulation and that the PML component of the fusion protein (Ring+B1-B2 and coiled-coil regions) retains intact biological activity, thereby suggesting that the effects of PML/RARα on survival derive from the activation of the incorporated PML sequence.

Increased Allergen-Specific, Steroid-Sensitive γδ T Cells in Bronchoalveolar Lavage Fluid from Patients with Asthma

Macrophages are the main cellular component in bronchoalveolar lavage fluid specimens obtained from normal patients. Other cell types, including T lymphocytes (CD3+) of both helper (CD4+) and suppressor-cytotoxic (CD8+) T-cell subsets, are also present, but the percentage of these cell types usually does not exceed 10%. Eosinophils, basophils and mast cells, and T lymphocytes have been found to be increased in bronchoalveolar lavage fluid samples in studies that have attempted to quantify the magnitude of the airway inflammatory response in patients with asthma [1, 2]. Recent findings suggest that T lymphocytes play a fundamental role in the induction of allergic inflammation. In fact, these cells not only recruit other specialized cells, such as eosinophils, by secreting interleukin-5 [1], they also promote local and systemic synthesis of IgE through the production of interleukin-4 [3, 4]. Lymphocytes with such functional activities are currently termed T-helper 2 (Th2), whereas the non-overlapping cell counterpart that secretes interferon- or interleukin-2 or both is termed T-helper 1 (Th1) and is involved in the delayed hypersensitivity immune reactions [3]. Although Th2-like activated T lymphocytes have been detected in the lungs of atopic asthmatic patients [5], it is still unclear whether these cells bear the or the less common T-cell receptor for antigen on their surface. This doubt is legitimated by the fact that many CD3+ intraepithelial lymphocytes found in the nasal mucosa of patients with allergic rhinitis are T cells that display the T-cell receptor heterodimer [6]. These findings prompted us to determine whether allergen-specific T lymphocytes are present in the bronchoalveolar lavage fluid of asthmatic patients and that, in these patients, they will be the major T-cell subset to disappear after systemic steroid treatment. Interestingly, our in vivo and in vitro studies provide evidence that apoptosis is the basic mechanism through which corticosteroid treatment acts on this T-lymphocyte type, thereby confirming the results of our previous investigations [7]. Methods Patients We studied 12 mildly symptomatic patients (6 children and 6 adults) with chronic asthma (FEV1 70% of that predicted for persons of their age and height) who were not receiving therapy with inhaled or oral corticosteroids, sodium cromoglycate, theophylline, or 2-agonists. Results of skin prick tests with purified Dermatophagoides pteronyssinus, D. farinae, and Parietaria judaica allergen extracts (Neo Abello, Madrid, Spain) were positive; results of an enzyme-linked immunosorbent assay (DPC Corporation, Los Angeles, California) for circulating allergen-specific IgE were also positive. None of the patients was a smoker or had had antecedent upper respiratory tract infection. Bronchoalveolar lavage fluid collection and fiberoptic bronchoscopy were done as previously described [8]. Ten healthy non-atopic, nonsmoking volunteers and patients diagnosed as having pulmonary sarcoidosis (n = 5) or extrinsic allergic alveolitis (n = 4), two pathologic conditions known to be associated with over-expanded lung CD4+ or CD8+ T-cell populations, were used as adult control groups. Age-matched uninfected children with cystic fibrosis (n = 5) or anatomic malformation (n = 4) of the airways served as controls for the cohort of asthmatic children. Bronchoalveolar lavage was repeated in 3 asthmatic patients after 1 week of therapy with deflazacort (Flantadin, Lepetit, Milano, Italy), 60 mg twice daily (a dose equivalent to 50 mg of prednisone), and then 3 weeks after therapy. We administered corticosteroids to these patients for exacerbation of their disease. We obtained informed consent from all study participants, and the clinical research was conducted in accordance with the local ethical committee guidelines. Lymphocyte Typing Pulmonary T cells were phenotyped with the following monoclonal antibodies: phycoerythrin-conjugated anti-CD3 (Ortho Pharmaceutical Corporation, Raritan, New Jersey), which recognizes up to 90% of T cells bearing the or T-cell receptor heterodimer; anti-CD4 and anti-CD8 (Ortho), which stain helper/inducer and cytotoxic T-cell subsets; and fluorescein-conjugated anti-TCR 1, anti-V1(a), and anti-V2(a) (T Cell Sciences, Cambridge, Massachusetts), which identify all T lymphocytes and the reciprocal subtypes expressing V1+ and V2+ gene products. In the analysis of two-color cytofluorimetric data (FACScan, Becton-Dickinson, Mountain View, California), we used the Lysis II program (Becton-Dickinson) to optimize gating of lymphocytes and to provide an objective means of excluding both debris and other cell types. T-Cell Enrichment In accordance with the manufacturers instructions (Dynal, A. S., Oslo, Norway), pulmonary mononuclear cells from 3 D. pteronyssinus-sensitive patients were enriched in TCR1-reactive T-cell subsets by negative magnetic immunoselection, using a mixture of anti- T-cell receptor monoclonal antibodies. This procedure yielded a + T-cell population of greater than 85%, which was used for culture and apoptotic cell death experiments. Proliferation Assay To assess the allergen-specificity of bronchoalveolar lavage T cells, 1.2 105 mononuclear cells and 4 104 -enriched lung T cells (the latter co-cultured with 8 104 autologous macrophages) from three D. pteronyssinus-sensitive patients and, because of the very small percentage of T cells in normal bronchoalveolar lavage fluid, 1.2 105 mononuclear cells from three controls were seeded in microplates and cultured in medium alone (RPMI-1640 [Gibco, Grand Island, New York] supplemented with fetal calf serum, L-glutamine, Hepes buffer, and antibiotics) or in the presence of 1 mug/mL purified D. pteronyssinus (Neo Abello, Madrid, Spain) or unrelated allergen Lolium perenne (purified L. perenne, Neo Abello) from the Graminaceae family. After 60 hours of culture, the cells were pulsed for 16 hours with 0.5 muCi [3H]-thymidine and were harvested, and the radioactivity was measured by liquid scintillation. Results are expressed as net cpm (counts per minute) [3H]-thymidine incorporation and reflect absolute cpm [3H]-thymidine uptake minus background cpm (cpm incorporated in the absence of allergen). Evaluation of Apoptotic Cell Death Apoptotic cells were qualitatively evaluated by agarose gel DNA electrophoresis [7] and quantitatively assessed as described previously [9]. Briefly, 1 106 TCR1+ pulmonary T cells were incubated for 24 hours with medium alone or dexamethasone (107 M), resuspended in 1.5 mL hypotonic propidium iodide solution and propidium iodide fluorescence of individual nuclei measured in a FACScan flow cytometer (Becton-Dickinson). We carried out control experiments using macrophage-depleted pulmonary T cells from healthy persons. Statistical Analysis Study populations were stratified according to age class. We used the Kruskall-Wallis one-way analysis of variance (with a significance level of 0.05) for statistical evaluation of the differences in bronchoalveolar lavage T-lymphocyte subset distributions. Pairwise comparisons for measuring the significance of the differences among mean values ( SE) calculated in the various groups (asthmatic patients compared with controls) were done with the Mann-Whitney U-test. In asthmatic patients (n = 12), the overall relation between serum IgE levels (IU/mL) and the percentages of pulmonary T cells was analyzed using the Spearman correlation test. The Statistical Package for the Social Sciences (SPS, Chicago, Illinois), version 4.0, was used for all statistical computations. Results As shown in Table 1, a significant proportion of CD3+ T lymphocytes in patient samples double-stained for the TCR1 monoclonal antibody, which defines + T lymphocytes. More than half of these T cells were CD4+, primarily of the V1+ subset, whereas the remainder of T cells found in the lung of our asthmatic patients were CD4 CD8, as shown by the fact that they never reacted with anti-CD8 monoclonal antibody. In addition, the percentage of T cells expressing the V2 isoform of the T-cell receptor was negligible in all samples tested (data not shown). Statistically significant differences were also obtained when the absolute numbers of pulmonary T cells from patients with asthma (mean count in adults, 29.6 2.5 104; in children, 23.2 3.1 104) were compared with those of the corresponding controls (mean count in adults, 0.2 0.1 104; P < 0.001; in children, 0.3 0.1 104; P < 0.001). Table 1. Bronchoalveolar Lavage T-Lymphocyte Subset Distribution in Patients with Allergic Asthma* The bronchoalveolar lavage T-cell concentration was not correlated with either specific serum IgE levels or any other clinical or functional (spirometric) variables (data not shown). In contrast, a relation was found between total serum IgE concentrations and pulmonary T-lymphocyte numbers, but this did not reach statistical significance (r = 0.21; P = 0.06). In the three in vitro experiments that we carried out, pulmonary T cells from D. pteronyssinus-sensitive patients proliferated in response to the specific allergen, with a net [3H]-thymidine incorporation ranging from 2400 to 4850 cpm, but did not proliferate in the presence of unrelated (L. perenne) allergen extract, with a [3H]-thymidine incorporation ranging from 120 to 450 cpm. Furthermore, lung T cells abruptly decreased and + CD4+ and CD8+ T lymphocytes increased in these patients after 1 week of treatment with glucocorticoids. Interestingly, the absolute numbers of T cells did not increase again when therapy was discontinued (Figure 1). The T-cell subpopulation was sensitive to steroid-induced programmed cell death in vitro. In fact, 24-hour dexamethasone incubation followed by flow-cytometric analysis with propidium iodide staining showed that the propidium iodide fluorescence profiles of apoptotic nuclei increased from 7% to 35% in dexamethasone-treated bronchoalveolar -enriched T cells, but not in control

Intensified and high-dose chemotherapy with granulocyte colony-stimulating factor and autologous stem-cell transplantation support as first-line therapy in high-risk diffuse large-cell lymphoma.

PURPOSE In our previous study with MACOPB, we identified a high-risk group of patients with a poor 3-year survival rate of 29%. These patients were defined as having at diagnosis advanced-stage disease with high tumor burden (TB) and elevated lactate dehydrogenase (LDH) level or bone marrow (BM) involvement. A novel therapeutic scheme was investigated to improve the outcome of these patients. PATIENTS AND METHODS Fifty patients with high-risk diffuse large-cell lymphoma (DLCL) were enrolled. The therapeutic scheme includes three phases: induction with 8 weeks of MACOPB; intensification with a 3-day course of mitoxantrone 8 mg/m2 plus high-dose cytarabine (HDARA-C) 2 g/m2 every 12 hours plus dexamethasone 4 mg/m2 every 12 hours (MAD protocol) and granulocyte colony-stimulating factor (G-CSF) 5 microg/kg on days 4 to 17 to harvest peripheral-blood progenitor cells (PBPC); consolidation with carmustine (BCNU), etoposide, ARA-C, and melphalan (BEAM) regimen; plus autologous stem-cell transplantation (ASCT) with PBPC, marrow, or both. RESULTS Thirty-six patients (72%) achieved a complete response (CR), 11 (22%) showed no response (NR), and three (6%) died of toxicity. Among the 22 PRs or NRs after the induction phase, 56% of patients achieved a CR with subsequent intensified therapy. With a median follow-up duration of 32 months, the overall survival and failure-free survival rates were 56% and 50%, respectively. The disease-free survival rate is 69% at 32 months. Leukapheresis after MAD and G-CSF yielded a median of 32 x 10(6)/kg CD34+ cells and 80 x 10(4)/kg granulocyte-macrophage colony-forming units (CFU-GM). Thirty-nine patients were autografted and 11 did not undergo ASCT: six because of disease progression, four due to toxicity, and one because of patient refusal. The median times to achieve engrafment were 11 days (range, 7 to 19) to a neutrophil count greater than 0.5 x 10(9)/L and 12 days (range, 8 to 60) to a platelet count greater than 50 x 10(9)/L. CONCLUSION This sequential scheme with intensified and high-dose chemotherapy with ASCT is feasible with moderate toxicity and may improve the outcome in high-risk DLCL.

Formation of PML/RARα high molecular weight nuclear complexes through the PML coiled-coil region is essential for the PML/RARα-mediated retinoic acid response

Retinoic Acid (RA) treatment induces disease remission of Acute Promyelocytic Leukaemia (APL) patients by triggering terminal differentiation of neoplastic cells. RA-sensitivity in APL is mediated by its oncogenic protein, which results from the recombination of the PML and the RA receptor α (RARα) genes (PML/RARα fusion protein). Ectopic expression of PML/RARα into haemopoietic cell lines results in increased response to RA-induced differentiation. By structure-function analysis of PML/RARα-mediated RA-differentiation, we demonstrated that fusion of PML and RARα sequences and integrity of the PML dimerization domain and of the RARα DNA binding region are required for the effect of PML/RARα on RA-differentiation. Indeed, direct fusion of the PML dimerization domain to the N- or C-terminal extremities of RARα retained full biological activity. All the biologically active PML/RARα mutants formed high molecular weight complexes in vivo. Functional analysis of mutations within the PML dimerization domain revealed that the capacity to form PML/RARα homodimers, but not PML/RARα-PML heterodimers, correlated with the RA-response. These results suggest that targeting of RARα sequences by the PML dimerization domain and formation of nuclear PML/RARα homodimeric complexes are crucial for the ability of PML/RARα to mediate RA-response.

Description of a sequential staining procedure for double immunoenzymatic staining of pairs of antigens using monoclonal antibodies

This paper describes a sequential staining procedure for double immunoenzymatic staining of pairs of antigens in frozen tissue sections and cell smears using monoclonal antibodies. This technique involves performance of an indirect immunoperoxidase sandwich (including development of the enzyme reaction) followed by an unlabelled immuno-alkaline phosphatase sandwich (the APAAP method). The two enzyme labels are revealed using DAB/H2O2 for peroxidase and naphthol AS-MX plus fast blue or fast red for alkaline phosphatase. When compared with a hapten-sandwich/biotin-avidin system, the sequential staining procedure proved to be simpler and more sensitive and was also more suitable for double immunoenzymatic staining when monoclonal antibodies were only available in small amounts. The sequential staining procedure is particularly useful for the identification of antigens distributed in different cell populations or in different sites (e.g., nucleus and cytoplasm or cell surface) of the same cell. In contrast, this method does not appear to be very suitable for demonstrating two antigens located in the same site (e.g., surface membrane) of the same cell for which purpose double immunofluorescence remains the first choice.

A prospective analysis of chemotherapy following surgical resection of clinical stage I-II small-cell lung cancer.

Thirty-seven (37) consecutive patients with clinical Stage I (T1–2NO, Mo), and Stage II (T1–2N1 Mo) central small-cell lung cancer (SCLC) underwent complete surgical resection of the primary tumor. Ten patients were subsequently pathologically Stage I, 14 patients were Stage II, and 8 were Stage III (T3;N2). The pathologically Stage I, II, and III patients were then treated with chemotherapy consisting of cyclophosphamide (1 g/m2), doxorubicin (50 mg/m2), and vincristine 2 mg (CAV) every 3 weeks for six courses followed by prophylactic cranial irradiation (2000 cGy in 10 fractions). Median survival in Stage I patients is 162 weeks and calculated 5-year survival is 50%; for Stage II patients, median survival is 86 weeks and calculated 5-year survival is 35%. T3;N2 patients have a median survival of 63 weeks; calculated 5-year survival is 21%. Our data suggest surgery plus adjuvant chemotherapy and cranial irradiation results in long-term survival in early central SCLC. These data support the need for randomized surgical trials in Stage I, II, and III central SCLC.