Protective effect of vaginal application of neutralizing and nonneutralizing inhibitory antibodies against vaginal SHIV challenge in macaques.

Definition of antibody (Ab) functions capable of preventing mucosal HIV transmission may be critical to both effective vaccine development and the prophylactic use of monoclonal Abs. Although direct antibody-mediated neutralization is highly effective against cell-free virus, increasing evidence suggests an important role for immunoglobulin G (IgG) Fcγ receptor (FcγR)-mediated inhibition of HIV replication. Thus, a panel of well-known neutralizing (NAbs) and nonneutralizing Abs (NoNAbs) were screened for their ability to block HIV acquisition and replication in vitro in either an independent or FcγR-dependent manner. Abs displaying the highest Fc-mediated inhibitory activity in various in vitro assays were selected, formulated for topical vaginal application in a microbicide gel, and tested for their antiviral activity against SHIVSF162P3 vaginal challenge in non-human primates (NHPs). A combination of three NAbs, 2G12, 2F5, and 4E10, fully prevented simian/human immunodeficiency virus (SHIV) vaginal transmission in 10 out of 15 treated NHPs, whereas a combination of two NoNAbs, 246-D and 4B3, although having no impact on SHIV acquisition, reduced plasma viral load. These results indicate that anti-HIV Abs with distinct neutralization and inhibitory functions differentially affect in vivo HIV acquisition and replication, by interfering with early viral replication and dissemination. Therefore, combining diverse Ab properties may potentiate the protective effects of anti-HIV-Ab-based strategies.

HIV-1 replication in Langerhans and interstitial dendritic cells is inhibited by neutralizing and Fc-mediated inhibitory antibodies.

Langerhans cells (LCs) and interstitial dendritic cells (IDCs) may be among the first human immunodeficiency virus type 1 (HIV-1) targets after sexual transmission. We generated cells of these types by differentiation of purified CD34(+) cord blood cells. After in vitro infection with R5-tropic strains, we obtained similar percentages of infected cells for both dendritic cell (DC) subsets. Moreover, LC infection was not increased by blockage of langerin by antilangerin. These results indicate that, under our experimental conditions, there was no evidence of any preference of HIV replication in LCs versus IDCs. The inhibitory activity of HIV-1-specific IgAs and IgGs against HIV-1 replication in LCs and IDCs was analyzed. We found that neutralizing antibodies inhibit HIV-1 infection of both DC subsets. Interestingly, HIV-1 was inhibited more efficiently by the IgGs than the corresponding IgA, due to an Fcγ receptor-dependent mechanism. Moreover, nonneutralizing inhibitory IgGs were able to inhibit infection of both LCs and IDCs. These results underline the importance of HIV-1 inhibition by the binding of the Fc part of IgGs to Fcγ receptors and suggest that the induction of neutralizing and nonneutralizing inhibitory IgGs in addition to neutralizing IgAs at mucosal sites may contribute to protection against sexual transmission of HIV-1.

The major population of PHA-stimulated PBMC infected by R5 or X4 HIV variants after a single cycle of infection is predominantly composed of CD45RO+CD4+ T lymphocytes.

PHA-stimulated peripheral blood mononuclear cells (PBMCs) are widely used for investigating replication and neutralization of HIV primary isolates in vitro. The objective of this study was to identify the T lymphocyte subset(s) that are found infected after one replication cycle by either R5- or X4-HIV-1 variants in PHA-stimulated PBMCs from healthy donors. Infected T lymphocytes were detected by intracellular p24 staining and characterized by cell surface immunophenotyping using flow cytometry. The predominant lymphocyte subset expressing p24 after 24 h of infection with either R5 or X4 HIV-1 strains was found to exhibit mainly the memory CD45RO phenotype, a greater percentage of CD62L(+)CD45RO(+) central memory T lymphocytes was infected with X4 HIV strains. Although some CD45RA(+) lymphocytes were also infected, these cells co-expressed CD45RO(+). The proportion of lymphocytes expressing CD4 and CD4/CD45RO decreased by 20% after 24 h of infection. A 2-fold decrease of CD4(+)CD8(+) T lymphocytes could also be recorded, even though this subset accounted for less than 5% of total lymphocytes in control cultures. Moreover, CD4(+)CD8(+) T cells further decreased by 90% after 4 days of infection, a time at which they scored p24(+). Therefore, our results indicate that the in vitro infection system of PHA-stimulated PBMC utilized in neutralization assays provides an appropriate model for the study of infected CD45RO(+) lymphocytes but not CD45RA(+) lymphocytes.

Polyamine metabolism in primary human colon adenocarcinoma cells (SW480) and their lymph node metastatic derivatives (SW620).

The natural polyamines are multifunctional constituents of all eucaryotic cells. The objective of this work was to compare aspects of polyamine metabolism in two related cell lines with the idea to investigate whether metabolic differences can be attributed to functional differences of the cells. The human colon carcinoma-derived cell lines SW480 and SW620 were chosen as models. SW480 cells were isolated from the primary tumour, SW620 cells from a lymph node of the same patient. SW620 cells grow faster, and the key regulatory enzymes of polyamine biosynthesis (ODC and AdoMetDC) are more active in the metastatic cells. Moreover, their ability to accumulate polyamines from the environment is more important than of SW480 cells. Likewise polyamine concentrations were markedly higher in SW620 cells, although they are much smaller than SW480 cells, and have a particularly small cytoplasmic space. Both cell lines show a striking diminution of ODC and AdoMetDC activities and changes in the polyamine patterns at the transition from exponential to non-exponential growth--most probably as a consequence of high cell density. Depletion of putrescine and spermidine due to inactivation of ODC by DFMO causes accumulation of cells in G1, and a proportional decrease of S-phase cells in both cell lines. Based on morphologic and other criteria SW480 and SW620 cells were typified as poorly differentiated. In agreement with their low grade of differentiation they exhibit a low alkaline phosphatase activity. However, the time-dependent decrease of alkaline phosphatase is not typical of differentiation patterns of other adenocarcinoma-derived cell lines or of normal enterocytes. The high capacity of de novo polyamine biosynthesis and of polyamine uptake is presumably a prerequisite for the rapid growth and invasiveness. The fact that these properties were more accentuated in the case of SW620 cells and paralleled enhanced metastatic properties indicate relationships between basic parameters of polyamine metabolism and malignancy.

Cytotoxic effects of the polyamine oxidase inactivator MDL 72527 to two human colon carcinoma cell lines SW480 and SW620.

N1,N4-bis(2,3-butadienyl)-1,4-butanediamine (MDL 72527) was considered to be a selective inactivator of FAD-dependent tissue polyamine oxidase. Recently MDL 72527 was reported to induce apoptosis in transformed hematopoietic cells through lysosomotropic effects. Since it is the only useful inhibitor of polyamine oxidase available at present, the re-evaluation of its properties seemed important. Human colon carcinoma-derived SW480 cells and their lymph node metastatic derivatives (SW620) were chosen for our study because they differ in various aspects of polyamine metabolism but have similar polyamine oxidase activities. MDL 72527 inhibited cell growth in a concentration-dependent manner, depleted intracellular polyamine pools, and caused the accumulation of N1-acetyl derivatives of spermidine and spermine. SW620 cells were more sensitive to the drug than were SW480 cells. At 150 micromol/L MDL 72527, SW620 cells accumulated in S-phase of the cell cycle, showed decreased polyamine transport rate, and showed no increase of polyamine N1-acetyltransferase activity. In contrast, SW480 cells were not arrested in a particular phase of the cell cycle, showed enhanced polyamine uptake, and showed a mild induction of acetyltransferase. The results suggest that MDL 72527 retains its value as a selective tool in short-term experiments only at concentrations not exceeding those necessary for the inactivation of polyamine oxidase. At concentrations above 50 micromol/L and at exposure times longer than 24 h, it may derange cell functions nonspecifically, and thus blur the results of studies intended to elucidate polyamine oxidase functions.

Induction of apoptosis by high linear energy transfer radiation: role of p531.

The involvement of the tumor suppressor p53 gene in the sensitivity of many cell types towards low linear energy transfer (LET) radiation is now well established. However, little information is available on the relationship between p53 status of tumor cells and their ability to undergo apoptosis following exposure to high-LET radiation. Here we present the results of experiments carried out with the human lymphoblastoid cell line TK6 and its p53 knock-out counterpart NH32. Cells were irradiated at doses ranging from 0.25 to 8 Gy with fast neutrons (65 MeV), carbon ions (95 MeV/nucleon), and X rays (15 MV). For both cell lines, the occurrence of apoptosis, determined by the quantification of hypodiploid particles as well as the activation of several caspases, was compared with their sensitivity towards high-LET radiation. Results indicate that p53 is involved in the response of TK6 cells to fast neutrons and carbon ions, as measured by cell proliferation and occurrence of apoptosis. However, p53-deficient cells are still able to undergo apoptosis following irradiation. This suggests that heavy ions and fast neutrons induce cellular damage that is not under the control of p53. The involvement of executioner caspases in high-LET radiation induced apoptosis was also evaluated by use of specific inhibitors.

Involvement of TP53 in apoptosis induced in human lymphoblastoid cells by fast neutrons.

We investigated the involvement of TP53 in apoptosis induced by fast neutrons in cells of three human B-lymphoblast cell lines derived from the same donor and differing in TP53 status: TK6 (wild-type TP53), WTK1 (mutant TP53) and NH32 (knockout TP53). Cells were exposed to X rays or to fast neutrons at doses ranging from 0.5 to 8 Gy. Apoptosis was determined by measurements of the sub-G0 /G1-phase DNA content and by the externalization of phosphatidylserine. Fast neutrons induced extensive apoptosis in TK6 cells, as shown by the formation of hypodiploid particles, the externalization of phosphatidylserine, and the activation of caspases. In contrast, cell death was triggered at a significantly lower rate in cells lacking functional TP53. However, TP53-independent cell death also expressed the morphological and biochemical hallmarks of apoptosis. Proliferation tests and clonogenic assays showed that fast neutrons can nevertheless kill WTK1 and NH32 cells efficiently. The absence of functional TP53 only delays radiation-induced cell death, which is also mediated by caspases. These results indicate that fast-neutron irradiation activates two pathways to apoptosis and that the greater relative biological effectiveness of fast neutrons reflects mainly an increase in clonogenic cell death.

[Induction of apoptosis of splenic lymphocytes in mice by accelerated carbon ions]. / Induction de l'apoptose dans les lymphocytes spléniques de souris par un faisceau d'ions carbone.

To assess the capacity of heavy ions to induce apoptosis in lymphocytes, mice have been irradiated with accelerated carbon ions (95 MeV/nucleon) at doses ranging from 0.1 to 4 Gy. Their spleens were removed 24 h later and gently dissociated to prepare a single cell suspension. Mononuclear cells were then maintained in culture at 37 degrees C, and the occurrence of apoptosis in these cells was analysed 24 h later. Lymphocytes were also irradiated in vitro, in the presence of Ac-DEVD-CHO, a potent caspase-3 and -7 inhibitor. Results from three experiments performed at the Grand Accelerateur National d'Ions Lourds (GANIL, Caen, France) are reported here. They indicate that carbon ions induce a marked, dose-dependent, reduction of the spleen weight and cellularity. However, in sharp contrast with spleen cells prepared from X-ray irradiated mice, only a slight increase of apoptosis is evidenced in cultured lymphocytes from mice irradiated with heavy ions. The significance of such results is discussed. So far, few data exist concerning the biological effects of heavy ions, in particular their capacity to induce apoptosis in lymphocytes; the present study provides useful clues for further investigations.

Prevention of nitrogen mustard-induced apoptosis in normal and transformed lymphocytes by ebselen.

The alkylating agent, nitrogen mustard (HN2), is thought to cause apoptosis through production of free oxygen radicals. To explore the mechanism of HN2-induced apoptosis, we utilized ebselen, a selenoorganic compound with potent antioxidant activity. We examined whether ebselen would inhibit apoptosis in BALB/c mouse spleen lymphocytes and human MOLT-4 leukemia cells treated with HN2 (2.5 microM) in vitro. Non-toxic concentrations (<50 microM) of ebselen were found to prevent HN2-induced apoptosis of murine lymphocytes in a dose-dependent manner, as measured by cell viability, hypodiploid DNA formation, and phosphatidylserine externalization. However, ebselen was ineffective at preventing spontaneous apoptosis in these cells, pointing to the selectivity of its action. Furthermore, pretreatment with ebselen at 1-10 microM for 72 hr protected MOLT-4 cells from HN2-induced apoptosis and maintained cell viability and proliferation as monitored by the above-mentioned parameters. This was accompanied by the preservation of mitochondrial transmembrane potential and elevated glutathione levels and by a blockage of caspase-3 and -9 activation. In vivo, ebselen also had a marked protective effect against spleen weight loss associated with lymphocyte apoptosis in mice treated by HN2. Therefore, ebselen provides an efficient protection against HN2-induced cell death in normal and tumoral lymphocytes and might prove useful as an antidote against alkylating agents.

Modulation of the antiproliferative activity of anticancer drugs in hematopoietic tumor cell lines by the poly(ADP-ribose) polymerase inhibitor 6(5H)-phenanthridinone.

Poly (ADP-ribose) polymerase (PARP) is involved in the cellular responses to genotoxic damage and its inhibition has been proposed as potentiating anticancer drug activity. Here, we evaluated the ability of the PARP inhibitor, 6(5H)-phenanthridinone, to modulate the antiproliferative activity of bleomycin, carmustin and doxorubicin in a murine (RDM4) and a human (U937) lymphoma cell lines. 6(5H)-phenanthridinone was shown to suppress PARP activity with the same potency in both cell lines. At 25 microM, this compound potentiated the activity of carmustin in RDM4 but not in U937 cells. In contrast, 6(5H)-phenanthridinone failed to affect the doxorubicin toxicity in murine lymphoma cells, whereas it prevented the cytotoxicity of this drug in the human cell line. Altogether, these findings indicated that 6(5H)-phenanthridinone modulates the cytotoxicity of anticancer agents differently according to the cell type and the drug. Therefore, this PARP inhibitor could be considered as the prototype of a new class of adjuncts in cancer chemotherapy.

Ex vivo determination of the effect of whole-body exposure to fast neutrons on murine spleen cell viability and apoptosis.

The effects of high-linear energy transfer (LET) radiations on lymphoid tissues and lymphocytes are not well understood. As a first approach to delineate these effects, the present work was conducted to assess the effects of high-LET radiations on murine spleen cells ex vivo and in vitro. BALB/c mice were irradiated whole-body with 65 MeV neutrons or 15 MV X rays at doses ranging from 0.2 to 3 Gy. Spleens were removed 1 day postirradiation and weighed, and single cell suspensions were prepared and cultured for several days. Apoptosis occurring in vitro was determined at different times by flow cytometry analysis of cells labeled with propidium iodide. It was found that irradiation with fast neutrons reduced spleen weight and cellularity to a greater extent than photons. Considering the spleen cellularity as end point, the relative biological effectiveness (RBE) of fast neutrons was 2. However, for both modes of irradiation, apoptosis of recovered spleen cells in vitro increased as a function of dose and the duration of culture. The level of apoptosis occurring at various times postirradiation was found to be identical for high- and low-LET radiations. Taken together, these results suggest that external as well as cellular factors might differentially modulate the sensitivity of lymphocytes to fast neutrons and photons.

Evaluation of the antitumor activity of 1-(3-C-ethynyl-beta-D-ribofuranosyl) (PJ272), a recent ribonucleoside analogue.

The antiproliferative properties of a new ribonucleoside derivative, 1-(3'-C-ethynyl-beta-D-ribofuranosyl)uracil (PJ 272) that we synthesized a few years ago, were investigated in vitro on a variety of tumor cell lines from human and murine origins and in vivo, in tumor bearing mice. Using the 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, we showed the ability of this compound to depress, at nanomolar concentrations, the growth of leukemia and lymphoma cultured cells. In 7 out of 8 tumor cell lines tested concentration of 50% inhibition (IC50) was found to be less than 25 nM. PJ 272 was also shown to present the same cytotoxicity against K562 Adriamycin-resistant cell line, which express a multi-drug resistance (MDR) phenotype, and its Adriamycin-sensitive parent cell line. Moreover, when injected intraperitoneally at 20 mg/kg every three days, PJ 272 was found to significantly increase the survival rate (T/C = 149%) of DBA/2 mice injected intraperitoneally with L1210 leukemic cells. Taken together, these results suggest that PJ 272 could be considered as a potentially very active drug against lymphoma and leukemia.

Caspase-3-like activity determines the type of cell death following ionizing radiation in MOLT-4 human leukaemia cells.

Caspases, a family of cysteine proteases, play a central role in the pathways leading to apoptosis. Recently, it has been reported that a broad spectrum inhibitor of caspases, the tripeptide Z-VAD-fmk, induced a switch from apoptosis to necrosis in dexamethasone-treated B lymphocytes and thymocytes. As such a cell death conversion could increase the efficiency of radiation therapy and in order to identify the caspases involved in this cell death transition, we investigated the effects of caspase-3-related proteases inhibition in irradiated MOLT-4 cells. Cells were pretreated with Ac-DEVD-CHO, an inhibitor of caspase-3-like activity, and submitted to X-rays at doses ranging from 1 to 4 Gy. Our results show that the inhibition of caspase-3-like activity prevents completely the appearance of the classical hallmarks of apoptosis such as internucleosomal DNA fragmentation or hypodiploid particles formation and partially the externalization of phosphatidylserine. However, this was not accompanied by any persistent increase in cell survival. Instead, irradiated cells treated by this inhibitor exhibited characteristics of a necrotic cell death. Therefore, functional caspase-3-subfamily not only appears as key proteases in the execution of the apoptotic process, but their activity may also influence the type of cell death following an exposure to ionizing radiation.

Apoptosis at the interface of immunosuppressive and anticancer activities: the examples of two classes of chemical inducers, oxysterols and alkylating agents.

Recent progresses in the understanding of molecular and biochemical pathways involved in apoptotic cell death offer novel perspectives for therapeutic interventions, in particular in immunosuppressive and anti-cancer therapies. In this review, we examine some chemical, biological, and mechanistic aspects of two classes of apoptosis chemical inducers: oxysterols and alkylating agents. Oxysterols represent a vast family of oxygenated derivatives of sterols. Found in both animal and vegetal kingdoms, they can be considered as ultimate products of an oxidative stress, and are chemically inert. Some of them (7beta-hydroxycholesterol, 25-hydroxycholesterol and 7, 25-dihydroxycholesterol) are cytotoxic at micromolar concentrations towards normal and tumor cells in culture, particularly lymphocytes, and reduce the growth of murine transplanted tumors. Thus, possible applications of oxysterols in medicine as immunosuppressants or as anticancer agents may be considered. Alkylating agents, on the other hand, have been widely used in cancer chemotherapy for decades. There toxicity results from their high chemical reactivity, causing lesions to macromolecules through covalent linkage. Some representative members of this class, mainly bifunctional derivatives which possess dichloroethyl groups, such as Chlormethine, Cyclophosphamide and Chlorabucil, express a pronounced cytotoxicity against lymphoid cells, and have therefore potent immunosuppressive properties. Because they triggers apoptosis via both common and distinct mechanisms, oxysterols and alkylating agents provide unique tools for exploring the initiation of this phenomenon in lymphoid cells, and may help design novel pharmacological approaches based on apoptotic modulation of these cells.

Annexin V delays apoptosis while exerting an external constraint preventing the release of CD4+ and PrPc+ membrane particles in a human T lymphocyte model.

Phosphatidylserine exposure in the exoplasmic leaflet of the plasma membrane is one of the early hallmarks of cells undergoing apoptosis. The shedding of membrane particles carrying Ags testifying to their tissue origin is another characteristic feature. Annexin V, a protein of as yet unknown specific physiologic function, presents a high Ca2+-dependent affinity for phosphatidylserine and forms two-dimensional arrays at the membrane surface. In this study, we report the delaying action of annexin V on apoptosis in the CEM human T cell line expressing CD4 and the normal cellular prion protein (PrPc), two Ags of particular relevance to cell degeneration and with different attachments to the membrane. The effect of annexin V was additive to that of z-Val-Ala-Asp-fluoromethyl ketone, a potent caspase inhibitor. Annexin V significantly reduced the degree of proteolytic activation of caspase-3, and totally blocked the release of CD4+ and PrPc+ membrane particles. z-Val-Ala-Asp-fluoromethyl ketone was a more powerful antagonist of caspase-3 processing, but prevented the shedding of CD4+ vesicles only partially and had no effect on that of PrPc+ ones. These results suggest that an external membrane constraint, such as that exerted by annexin V, has important consequences on the course of programmed cell death and on the dissemination of particular Ags. In vivo, annexin V had a significant protective effect against spleen weight loss in mice treated by an alkylating agent previously shown to induce lymphocyte apoptosis.

Assessment of apoptosis occurring in spleen cells from nitrogen mustard-treated or gamma-irradiated mice.

The short-term consequences on spleen cells of the intraperitoneal administration of nitrogen mustard (HN-2) to mice or of a whole-body gamma irradiation have been evaluated. Experiments were designed to assess the induction of apoptosis in spleen cells following exposure to these agents. The occurrence of this type of cell death was analysed by several methods, in particular the quantification in the blood of phosphotidylserine-bearing microparticles shed by apoptotic cells. In response to HN-2 or radiations, spleens undergo a rapid involution of their weight and cellularity. Ex vivo apoptosis occurs within 24 hours in cultured lymphocytes in a dose-dependent manner after both treatments. As compared with untreated controls, circulating microparticles increased 3-fold after the injection of 5 mg/kg of HN-2.

Cytotoxic properties of a phosphoglycoconjugated derivative of 7 beta-hydroxycholesterol upon normal and tumor cells in culture.

The cytotoxic activity of a new hydrosoluble axysterol derivative, a phosphoric acid diester of 7 beta-hydroxycholesterol (7 beta-OHC, one of the most toxic oxysterol) and of galactose has been evaluated using cultured tumor cells of various origins, and compared with 7 beta-OHC. As its parent compound, XG-142 exhibits a significant cytotoxic activity against all the cell lines tested, but the IC50's were higher than those obtained with 7 beta-OHC. Moreover, the cytotoxicity was slower to appear than after a 7 beta-OHC treatment. Cell phase distribution was analysed, and revealed some differences between the two compounds. Both oxysterols induced apoptosis at micromolar concentrations, as evidenced by several methods including agarose gel electrophoresis of fragmented DNA and flow cytometry of propidium iodide labeled cells. Apoptosis was also obtained when 7 beta-OHC and XG-142 were combined at concentrations unable to induce this type of cell death when used separately. Upon normal murine spleen cells, XG-142 was found to be less toxic than 7 beta-OHC, and the capacity to respond to Con A stimulation was preserved. Therefore, XG-142 can be considered as a promising soluble analogue of 7 beta-OHC, and its application as anticancer agent should be considered.

Effect of 6(5H)-phenanthridinone, a poly (ADP-ribose)polymerase inhibitor, and ionizing radiation on the growth of cultured lymphoma cells.

The ability of 6(5H)-phenanthridinone (Phen), a new potent poly(ADP-ribose)polymerase (PARP) inhibitor, to potentiate the effect of ionizing radiation on tumour cells was evaluated. RDM4 murine lymphoma cells were irradiated using a 60Co panoramic source and then examined for their growth, cell cycle distribution and apoptosis. Phen (100 microM) was found to inhibit more than 90% of the PARP activity in control and irradiated cells. Cell proliferation was assessed using Alamar Blue, a new fluorometric assay. Phen was found to sharply increase the radiation-induced inhibition of cell proliferation. Indeed, at 2.5 Gy the relative cell number of Phen-treated cells was 60% below control levels. At the same radiation dose, the G2M arrest was also significantly reinforced by the addition of Phen. Furthermore, this PARP inhibitor was shown to significantly increase the amount of DNA fragmentation as revealed by the DNA migration pattern in agarose gel electrophoresis. Comparable results were obtained with 3-aminobenzamide, another PARP inhibitor, but at concentrations 200-fold higher. Taken together, these results indicate the potential interest of Phen as a valuable pharmacological probe for investigating the role of PARP in cellular responses to radiation. They also suggest a possible use of Phen as an adjuvant in radiotherapy.