Intracellular detection of Bcl-2 and p53 proteins by flow cytometry: comparison of monoclonal antibodies and sample preparation protocols.

Several techniques have been proposed for flow cytometric evaluation of intracellular antigens. This approach is particularly important for detection at the single cell level of proteins which correlate to tumour progression. Bcl-2 and p53 are two of the most relevant proteins. In the present study we have compared five different cell fixation-permeabilisation protocols and nine fluorochrome-conjugated (FITC or PE) monoclonal antibodies (mAb): four mAb directed against Bcl-2 and five against p53. For detection of Bcl-2 we have analysed three Bcl-2 positive cell lines (K562, Daudi and MCF-7), and peripheral blood samples obtained from nine healthy subjects. To distinguish internal positive (lymphocytes) and negative control cells (granulocytes), it was necessary to perform simultaneous detection of surface and intracellular antigens. For detection of p53 three cell lines, two p53 positive (Raji and CEM) and one p53 negative (HL-60), were analysed. Using these cells we have performed a combined analysis of the efficiency of monoclonal antibodies and sample preparation techniques. In conclusion, clones 124-FITC and Bcl-2/100-PE (Bcl-2), and clones BP53,12-FITC and G59-12-PE (p53) provided the highest specific fluorescence intensity of the respective markers independent of cell preparation protocols. Importantly, our results show that mAb background may depend on the specific fixation/permeabilisation kit and that mAb titration using negative and positive control cells is essential to determine the specificity and the sensitivity of the mAb used.

Interleukin 2 and interleukin 15 differentially predispose natural killer cells to apoptosis mediated by endothelial and tumour cells.

Human natural killer (NK) cells constitutively express the beta- and gamma-chains of the interleukin 2 (IL-2)/IL-15 receptor, and both IL-2 and IL-15 are able to activate NK cell proliferation and cytotoxicity. When IL-2-primed human NK cells are exposed to sensitive targets (i.e. K562) they undergo apoptosis mediated by the beta(2)-integrin CD18. Here, we demonstrate that: (i) endothelial cells, similar to K562 tumour target cells, induce apoptosis of IL-2-primed NK cells; (ii) endothelial- and K562 cell-induced apoptosis is significantly lower in IL-15 than in IL-2-stimulated NK cells; (iii) a critical role in the apoptosis of IL-2-primed NK cells is played by the alpha-chain of the IL-2 receptor. Our data show for the first time that IL-2-activated NK cells can die by apoptosis upon contact with the vascular endothelium, which is a necessary step for their extravasation, with a direct pathophysiological relevance on the strategy of adoptive immunotherapy of cancer. On the other hand, IL-15, although generating a similar level of activation of NK cells, largely prevents their apoptotic fate. Therefore, IL-15 produced early in the immune response, when T cells are not yet activated, generates lymphokine-activated killer cells that are efficient killers relatively protected from apoptosis. Once activated, T cells produce IL-2 that overcomes the effect of IL-15 on NK cells, paving the way for their death by apoptosis.

Ionizing radiation sensitizes erythroleukemic cells but not normal erythroblasts to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)--mediated cytotoxicity by selective up-regulation of TRAIL-R1.

Cytotoxic activity of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo-2 ligand), used alone or in different combinations with either a low (1.5 Gy) or a high (15 Gy) single dose of ionizing radiation (IR), was investigated on erythroleukemic cells (K562, HEL, Friend, primary leukemic erythroblasts) and on primary CD34(+)-derived normal erythroblasts. Human recombinant TRAIL alone variably affected the survival/growth of erythroleukemic cells; K562 cells were the most sensitive. Moreover, all erythroleukemic cells were radio-resistant, as demonstrated by the fact that cytotoxicity was evident only after treatment with high-dose (15 Gy) IR. Remarkably, when IR and TRAIL were used in combination, an additive effect was noticed in all erythroleukemic cells. Augmentation of TRAIL-induced cell death by IR was observed with both low and high IR doses and required the sequential treatment of IR 3 to 6 hours before the addition of TRAIL. Conversely, both TRAIL and IR showed a moderate cytotoxicity on primary CD34(+)-derived normal erythroblasts when used alone, but their combination did not show any additive effect. Moreover, the cytotoxicity of IR plus TRAIL observed in erythroleukemic cells was accompanied by the selective up-regulation of the surface expression of TRAIL-R1 (DR4), and it was completely blocked by the z-Val-Ala-Asp (OMe)-CH(2) (z-VAD-fmk) caspase inhibitor. On the other hand, the surface expression of TRAIL-R1 in CD34(+)-derived normal erythroblasts was unaffected by IR, which induced the up-regulation of the decoy TRAIL-R3. These data demonstrate that treatment with IR provides an approach to selectively sensitize erythroleukemic cells, but not normal erythroblasts, to TRAIL-induced apoptosis through the functional up-regulation of TRAIL-R1.

Assays of natural killer (NK) cell ligation to target cells.

Assays for natural killer cells have long been established in the flow repertoire, but successful application of a flow-based protocol requires attention to many details. This unit provides these details in a comprehensive manner. In particular, the assay is designed for simple bench-top cytometers, rather than for more complex research instruments. Following this protocol, even a novice user should be able to achieve successful completion of an NK assay.

Infection of CD34(+) hematopoietic progenitor cells by human herpesvirus 7 (HHV-7).

To investigate the tropism of the T-lymphotropic human herpesvirus 7 (HHV-7) for hematopoietic progenitors, cord blood CD34(+) cells were inoculated in vitro with HHV-7 and then induced to differentiate along the granulocytic and erythroid lineages by the addition of appropriate cytokine cocktails. In semisolid assays, HHV-7 modestly affected the growth of committed (granulocytic/macrophagic and erythroid) progenitors, whereas it significantly decreased the number of pluripotent (granulocytic/erythroid/ monocytic/megakaryocytic) progenitors. Such inhibitory effect was completely abrogated by incubating HHV-7 inoculum with anti-HHV-7 neutralizing serum. In liquid cultures, HHV-7 hastened maturation along the myeloid but not the erythroid lineage, as demonstrated by the up-regulation of CD33 early myeloid antigen at day 7 of culture, and of CD15 and CD14 antigens at day 15. Moreover, HHV-7 messenger RNA was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) in cells maturating along both the myeloid and the erythroid lineages. To evaluate the relevance of these in vitro findings, the presence of HHV-7 was investigated in bone marrow (BM) unfractionated mononuclear cells (MCs) as well as in purified CD34(+) and CD34(-) cell subsets, obtained from 14 normal adult donors. HHV-7 DNA was detected by DNA-PCR in 4 of 7 BMMC samples, and it was found to be associated with both the CD34(-) (2 of 7) and the CD34(+ )(1 of 7) fractions. These data indicate that HHV-7 infects BM cells in vivo and shows the ability to affect the survival/differentiation of CD34(+) hematopoietic progenitors in vitro by inhibiting more ancestral progenitors and perturbing the maturation of myeloid cells.

TNF-related apoptosis-inducing ligand (TRAIL) as a negative regulator of normal human erythropoiesis.

The impact of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) on normal hematopoietic development was investigated using adult peripheral blood CD34(+) hematopoietic progenitor cells, induced to differentiate along the erythroid, megakaryocytic, granulocytic, and monocytic lineages by the addition of specific cytokine cocktails. TRAIL selectively reduced the number of erythroblasts, showing intermediate levels of glycophorin A (glycophorin A(interm)) surface expression, which appeared in liquid cultures supplemented with stem cell factor + interleukin 3 + erythropoietin at days 7-10. However, neither immature (day 4) glycophorin A(dim) erythroid cells nor mature (day 14) glycophorin A(bright) erythroblasts were sensitive to TRAIL-mediated apoptosis. Moreover, pre-exposure to TRAIL significantly decreased the number and size of erythroid colonies in semisolid assays. These adverse effects of TRAIL were selective for erythropoiesis, as TRAIL did not significantly influence the survival of cells differentiating along the megakaryocytic, granulocytic, or monocytic lineages. Furthermore, TRAIL was detected by Western blot analysis in lysates obtained from normal bone marrow mononuclear cells. These findings indicate that TRAIL acts in a lineage- and stage of differentiation-specific manner, as a negative regulator of normal erythropoiesis. (Blood. 2000;95:3716-3724)

Ultrastructural characterization of maturation, platelet release, and senescence of human cultured megakaryocytes.

The aim of this study was to evaluate the ultrastructural features of human megakaryocytes cultured in vitro. For this purpose, pluripotent CD34(+) (cluster of differentiation 34) hematopoietic progenitor cells, obtained from the peripheral blood of healthy adult donors, were differentiated along the megakaryocytic lineage in liquid cultures by the addition of the megakaryocyte-specific growth factor thrombopoietin (TPO, 100 ng/ml). After only 6-8 days, virtually all of the CD34-derived cells expressed the early megakaryocytic CD61 antigen, while, after 15-16 days, most cells also expressed the late megakaryocytic CD42a antigen. Ultrastructural analysis of cells obtained after 7 days of culture showed aspects typical of developing megakaryocytes (MK), such as formation of platelet territories and cytoplasmic fragmentation. At later (15-16 day) culture times, two distinct cell populations were observed: fully developed megakaryocytes releasing platelets into the culture medium and senescent megakaryocytes, characterized by morphological features of apoptosis. Analysis of DNA fragmentation in these cells revealed that apoptosis in megakaryocytes occurred in the absence of the internucleosomic cleavage, which is characteristic of most, but not all, types of apoptosis in cells of hematopoietic origin. On the other hand, flow cytometry of the DNA content of senescent megakaryocytes showed a subdiploid peak that was likely due to a loss of micronuclei during processing.

Lineage-related susceptibility of human hemopoietic cell lines to apoptosis.

Apoptosis plays a fundamental role in shaping normal hematopoiesis. We have investigated the relationship existing between susceptibility to apoptosis and lineage commitment in hemopoietic cells. The presence and degree of apoptosis were investigated in myeloid (HL-60 and K562), T (Jurkat and MOLT-4), and B (CESS and Raji) lymphoid cell lines by using a variety of techniques-transmission electron and light microscopy, flow cytometry and DNA gel electrophoresis. The major achievement of this study is that hematopoietic cells respond to different chemical (staurosporin, tiazofurin, camptothecin) and physical (hyperthermia or hypothermia) stimuli by apoptosis in a lineage-related way. Moreover, with respect to the methods used to detect apoptosis, a strong correlation was observed between the presence of the hypodiploid peak determined by flow cytometry and the DNA laddering evaluated by gel electrophoresis, but both techniques failed to demonstrate the presence of apoptosis in some cases. We conclude that cells of different hematopoietic lineages mostly show a lineage-related behaviour in their apoptotic response to different stimuli, suggesting that the lineage commitment and the stage of differentiation can confer different sensitivities to specific apoptotic stimuli. Moreover, morphological techniques still represent the most reliable approach to detect apoptosis in hemopoietic cells.

Natural killer (NK) cell-mediated cytotoxicity: differential use of TRAIL and Fas ligand by immature and mature primary human NK cells.

Mature natural killer (NK) cells use Ca2+-dependent granule exocytosis and release of cytotoxic proteins, Fas ligand (FasL), and membrane-bound or secreted cytokines (tumor necrosis factor [TNF]-alpha) to induce target cell death. Fas belongs to the TNF receptor family of molecules, containing a conserved intracytoplasmic "death domain" that indirectly activates the caspase enzymatic cascade and ultimately apoptotic mechanisms in numerous cell types. Two additional members of this family, DR4 and DR5, transduce apoptotic signals upon binding soluble TNF-related apoptosis-inducing ligand (TRAIL) that, like FasL, belongs to the growing TNF family of molecules. Here, we report that TRAIL produced or expressed by different populations of primary human NK cells is functional, and represents a marker of differentiation or activation of these, and possibly other, cytotoxic leukocytes. During differentiation NK cells, sequentially and differentially, use distinct members of the TNF family or granule exocytosis to mediate target cell death. Phenotypically immature CD161(+)/CD56(-) NK cells mediate TRAIL-dependent but not FasL- or granule release-dependent cytotoxicity, whereas mature CD56(+) NK cells mediate the latter two.

A novel surface marker (B203.13) of human haemopoietic progenitors, preferentially expressed along the B and myeloid lineages.

We used a monoclonal antibody (mAb) (B203.13, IgM) generated from a mouse immunized with the human B/myeloid bi-phenotypic B1b cell line, to analyse haemopoietic cells. The antigen recognized by this mAb is expressed on most adult and umbilical cord blood CD21+ B cells, at minimal density on mature monocytes, and is undetectable on granulocytes, T, natural killer (NK) cells, and erythrocytes. Within umbilical cord blood and adult bone marrow haemopoietic progenitor cells, the B203.13 mAb recognized a surface marker, present on progenitor cells of several haemopoietic lineages, that was transiently expressed on early erythroid and T/NK progenitors, and was preferentially maintained on cells of the B and myeloid lineages. Within the CD34+ cells, B203.13 was expressed on early committed myeloid (CD33+) and erythroid (CD71dim) progenitor cells, as confirmed in colony formation assays. The mAb also reacted with cells of B and myeloid chronic leukaemias and cell lines. These data define B203.13 mAb as a novel reagent useful for the characterization of haemopoietic progenitors and leukaemias.

Kinetics of in vitro natural killer activity against K562 cells as detected by flow cytometry.

Natural killer (NK) cells bind to K562 tumor target cells in vitro and kill them. The binding and cytotoxic activities of NK cells are tightly related to each other: degranulation of the cytotoxic effector is the basis for target cell damage and a consequence of effector-target recognition and binding. However, the two phases of NK activity, binding and killing, have always been measured separately by various methodologies and under different experimental conditions, because of the lack of a comprehensive methodology able to measure both of them at one time. Here we describe the simultaneous measurement of the binding and killing activities against K562 of resting and cytokine (IL-2 or IL-12)-stimulated NK cells by flow cytometry. NK, K562 and conjugates can be identified and measured by flow cytometry on the basis of NK mAb staining and target cells autofluorescence (Binding Plot). Within each population of the binding plot, killed targets can be identified and measured by their scatter characteristics (Cytotoxicity Plot). We show that i) the conjugate formation is enhanced in cytokine-stimulated cells, even at relatively short co-incubation times; ii) the conjugate release is also accelerated by cytokines; iii) the conjugate release is always quicker than the induction of the morphological changes in the target cell that generate its modified scattering properties.

IMP dehydrogenase inhibitor, tiazofurin, induces apoptosis in K562 human erythroleukemia cells.

Tiazofurin, an anticancer drug which inhibits IMP dehydrogenase, decreases cellular GTP concentration, induces differentiation and down-regulates ras and myc oncogene expression, caused apoptosis of K562 cells in a time- and dose-dependent fashion. Apoptotic cells were detected by (1) flow cytometry, (2) electron microscopy, and (3) fluorescence in situ nick translation and confocal microscopy, while the DNA ladder was not detectable. The induced apoptosis was abrogated by guanosine which replenishes GTP pools through the guanosine salvage pathways, while it was enhanced by hypoxanthine, a competitive inhibitor of GPRT. The tiazofurin-mediated apoptosis may therefore be linked with the decrease of GTP and the consequent impairment of specific signal transduction pathways. Tiazofurin induced apoptosis also in lymphoblastic MOLT-4 cells, suggesting that this action is not confined to cells of the myeloid lineage, where the differentiating effects of the drug are more pronounced.

Anti-BrdUrd labeling of newly synthesized DNA in HL-60 cells triggered to apoptosis.

Apoptosis is an active process that takes place during pre- and postnatal life. It can be viewed as the essential counterpart to cell proliferation, both phenomena being aimed at the maintenance of tissue and organ homeostasis. Because apoptosis often takes place during the S phase of the cell cycle, we describe the spatial and temporal correlation between DNA synthesis and DNA cleavage taking place in the same nucleus at the same time as a result of the action of camptothecin on proliferating HL-60 cells in vitro. The relationship between DNA synthesis and DNA fragmentation was studied at the single-cell level by bromodeoxyuridine (BrdUrd) incorporation revealed by flow cytometry, electron microscopy, and confocal microscopy. Most HL-60 cells are triggered to apoptosis during the first hour of treatment with camptothecin, and only cells in early-middle S phase are sensitive to the drug effect, whereas late S phase cells appear insensitive to camptothecin-induced apoptosis. Our data, therefore, reinforce the hypothesis of a DNA strand break threshold that may exist in the cell, beyond which the apoptotic program is activated. Moreover, DNA synthesis activity in the nucleus committed to apoptosis is gradually downregulated; after 6 h of camptothecin treatment, virtually no residual DNA replication activity can be detected in micronuclei. DNA repair does not appear to be involved in bromode-oxyuridine incorporation during the apoptotic process.

Definition of a natural killer NKR-P1A+/CD56-/CD16- functionally immature human NK cell subset that differentiates in vitro in the presence of interleukin 12.

Human natural killer (NK) cell differentiation from immature lineage negative (Lin-) umbilical cord blood cells was examined in vitro. Cells expressing differentiation antigens of mature NK cells (CD56, CD16, CD2, CD8, NKR-P1A) were generated from Lin- cells cultured with interleukin (IL)-2 and a murine bone marrow stromal cell line expressing the human membrane-bound form of stem cell factor. Two subsets of NK cells were identified in these cultures: one expressed both NKR-P1A and CD56 and, in variable proportions, all other NK cell differentiation antigens; the second subset expressed only NKR-P1A and, unlike the former, was not cytotoxic. Neither subset expressed interferon (IFN)-gamma mRNA even after stimulation with phorbol di-ester and Ca2+ ionophore, but both expressed tumor necrosis factor alpha mRNA and the cytotoxic granule-associated proteins TIA-1, perforin, and serine esterase-1. After 10-d culture with IL-2, IL-12, and irradiated B lymphoblastoid cells, approximately 45% of the NKR-P1A+/ CD56- cells became CD56+, and the same cultures contained cells capable of cytotoxicity and of IFN-gamma production. These results indicate that NKR-P1A expression in the absence of other NK cell markers defines an intermediate, functionally immature stage of NK cell differentiation, and that effector functions develop in these cells, concomitantly with CD56 expression, in the presence of IL-12. These cells likely represent the counterpart of a CD3-/NKR-P1A+/ CD56-/CD16- cell subset that, as shown here, is present both in adult and neonatal circulating lymphocytes.

Supravital exposure to propidium iodide identifies apoptotic cells in the absence of nucleosomal DNA fragmentation.

By flow cytometry, we have quantitatively evaluated HL-60, MOLT-4, and P815 apoptotic cells induced with camptothecin, staurosporine, and hyperthermia, respectively. Apoptosis was measured by two different flow cytometry techniques, using propidium iodide (PI) uptake in permeabilized and nonpermeabilized cells. Apoptosis also has been analyzed by electron microscopy and DNA cleavage by in situ nick translation and DNA gel electrophoresis. We demonstrate that supravital exposure to propidium iodide without prior permeabilization identifies apoptotic cells, and clearly distinguishes them from necrotic cells in all the cases examined. This capability is independent of the nucleosomal (180-200 bp) fragmentation of DNA (which does not take place in MOLT-4 cells), which is the basis for detection of apoptosis both as a "ladder" by DNA gel electrophoresis and as a hypodiploid peak by flow cytometry. Therefore, alterations in membrane permeability, on which PI uptake in living cells is based, allow distinction of apoptotic cells from necrotic and living cells independently of the heterogeneous biochemical patterns involved in programmed cell death, which may or may not lead to DNA oligonucleosomal fragmentation.

Early events of liver regeneration in rats: a multiparametric analysis.

5-Bromodeoxyuridine (BrdU), a synthetic analogue of thymidine, has been utilized in vivo to detect the proliferation which occurs in the liver after two-thirds surgical hepatectomy. Immunocytochemical detection of BrdU incorporation has been carried out at both the morphological and flow cytometrical level, while structural changes of regenerating liver have been investigated, using Mallory-Azan-stained paraffin sections, by means of an image analyser. The results obtained show that in vivo DNA synthesis progression throughout S phase follows a pattern similar to that previously described in vitro in both 3T3 fibroblasts and Friend erythroleukemia cells and also demonstrate a precise correlation between morphological patterns of BrdU incorporating cells and their lobular distribution. Moreover, the activation of at least two proliferation waves can be detected from 18 to 34 h after hepatectomy: the former, starting from adjacent regions of contiguous lobules, apparently induces an irregular increase of lobular dimension; the latter, involving both inner and peripheral lobular domains, seems to be correlated with the appearance of nodule-like structures at the lobule periphery. In view of these results the role of the hepatic acinus and the hypothesis of a streaming of parenchymal cells during liver regeneration have been discussed.

Ionizing radiation-induced apoptosis and DNA repair in murine erythroleukemia cells.

A morphological study of DNA repair and apoptotic patterns in relationship with cell cycle events was performed on murine erythroleukemia cells. The presence and distribution of DNA replicon sites were evaluated through the BrdU-anti BrdU immunofluorescence and immunogold techniques in light and electron microscopy. Different patterns of labelling and percentages of BrdU positive cells were observed depending on irradiation dose (up to 60 Gy) and time in post-irradiation culture (up to 24 hours). An enlargement of the S phase of the cell cycle was evidenced 18 hours post-irradiation as determined by flow cytometry analysis. The high resolution approach showed that, in spite of several morphological alterations, BrdU labelling was present even in cells displaying early and late apoptotic features.

Tiazofurin induces a down-modulation of ICAM-1 expression on K562 target cells impairing NK adhesion and killing.

Tiazofurin treatment of K562 leukemia cells in vitro depletes the metabolites of the guanylate biosynthetic pathway, inducing erythroid differentiation, that, in turn, alters the phenotypic profile. As a consequence, K562 cells possibly modify their interaction with immune cells. Here we describe the binding and killing activity of peripheral blood NK cells against differentiating K562 cells and the correlation between their altered binding capacity and ICAM-1 expression levels in differentiating K562 cells. We found that decreased percentages of NK (and T) cells were bound to differentiating K562 cells generating a decreased cytotoxic activity. This corresponded to decreased expression of ICAM-1, as detected by FACS analysis and Western blot. Erythroid differentiation, binding and killing reduction, and ICAM-1 down-modulation were completely abrogated by guanosine treatment. Tiazofurin causes a decrease in lymphocyte recognition and binding to K562 target cells. This can be ascribed to the down-modulation of ICAM-1 expression on target cells, which, therefore, can escape killing, acquiring a selective survival advantage.

Nuclear pores in the apoptotic cell.

During apoptosis, nuclear pores undergo strong modifications, which are described here in five different apoptotic models. Conventional electron microscopy, supported by freeze-fracture analysis, showed a constant migration of nuclear pores towards the diffuse chromatin areas. In contrast, dense chromatin areas appear pore-free and are frequently surrounded by strongly dilated cisternae. A possible functional significance of this pore behaviour during apoptosis is discussed.