Peptabody-EGF: a novel apoptosis inducer targeting ErbB1 receptor overexpressing cancer cells.

The epidermal growth factor receptor (EGFR) plays a central role in cell life by controlling processes such as growth or proliferation. This receptor is commonly overexpressed in a number of epithelial malignancies and its upregulation is often associated with an aggressive phenotype of the tumor. Thus, targeting of EGFR represents a very promising challenge in oncology, and antibodies raised against this receptor have been investigated as potential antitumor agents. Various putative mechanisms of action were proposed for such antibodies, including decreased proliferation, induction of apoptosis, stimulation of the immunological response against targeted cancer cells or combinations thereof. We report here the development of an alternative high affinity molecule that is directed against EGFR. Production of this pentameric protein, named peptabody-EGF, includes expression in a bacterial expression system and subsequent refolding and multimerization of peptabody monomers. The protein complex contains 5 human EGF ligand domains, which confer specific binding towards the extracellular portion of EGFR. Receptor binding of the peptabody-EGF had a strong antiproliferative effect on different cancer cell lines overexpressing EGFR. However, cells expressing constitutive levels of the target receptor were barely affected. Peptabody-EGF treated cancer cells exhibited typical characteristics of apoptosis, which was found to be induced within 30 min after the addition of the peptabody-EGF. In vitro experiments demonstrated a significantly higher binding activity for peptabody-EGF than for the therapeutic monoclonal EGFR antibody Mab-425. Furthermore, the antitumor action provoked by the peptabody-EGF was greatly superior than antibody mediated effects when tested on EGFR overexpressing cancer cell lines. These findings suggest a potential application of this high affinity molecule as a novel tool for anti-EGFR therapy.

Cutting edge: apoptosis of superantigen-activated T cells occurs preferentially after a discrete number of cell divisions in vivo.

Staphylococcal enterotoxins are bacterial products that display superantigen activity in vitro as well as in vivo. For instance, staphylococcal enterotoxin B (SEB) polyclonally activates T cells that bear the Vbeta8 gene segment of the TCR. SEB-activated T cells undergo a burst of proliferation that is followed by apoptosis. Using an in vivo adaptation of a fluorescent cell division monitoring technique, we show here that SEB-activated T cells divide asynchronously, and that apoptosis of superantigen-activated T cells is preferentially restricted to cells which have undergone a discrete number of cell divisions. Collectively, our data suggest that superantigen-activated T cells are programmed to undergo a fixed number of cell divisions before undergoing apoptosis. A delayed death program may provide a mechanistic compromise between effector functions and homeostasis of activated T cells.

Macrophages induce neutrophil apoptosis through membrane TNF, a process amplified by Leishmania major.

Neutrophils are recruited to the site of parasite inoculation within a few hours of infection with the protozoan parasite Leishmania major. In C57BL/6 mice, which are resistant to infection, neutrophils are cleared from the site of s.c. infection within 3 days, whereas they persist for at least 10 days in susceptible BALB/c mice. In the present study, we investigated the role of macrophages (MPhi) in regulating neutrophil number. Inflammatory cells were recruited by i.p. injection of either 2% starch or L. major promastigotes. Neutrophils were isolated and cultured in the presence of increasing numbers of MPhi. Extent of neutrophil apoptosis positively correlated with the number of MPhi added. This process was strictly dependent on TNF because MPhi from TNF-deficient mice failed to induce neutrophil apoptosis. Assays using MPhi derived from membrane TNF knock-in mice or cultures in Transwell chambers revealed that contact with MPhi was necessary to induce neutrophil apoptosis, a process requiring expression of membrane TNF. L. major was shown to exacerbate MPhi-induced apoptosis of neutrophils, but BALB/c MPhi were not as potent as C57BL/6 MPhi in this induction. Our results emphasize the importance of MPhi-induced neutrophil apoptosis, and membrane TNF in the early control of inflammation.

A modified assay for measuring thymocyte co-stimulatory activity

The observation that murine thymocytes increase their proliferation to interleukin 1 (IL-1) in the presence of phytohemagglutinin (PHA) when pre-incubated with interleukin 2 (IL-2) allowed the introduction of a modified assay for the measurement of IL-1 or the search of thymocyte-inducing proliferative activities in biological samples. Pre-incubation of thymocytes for 24 hr with 50 u/ml IL-2, followed by washings, elicited their maximal response to IL-1 in the usual lymphocyte activating factor (LAF) assay. This suggests that sequential events lead to thymocyte activation. The responsiveness is three to five fold greater than, and the total time of assay is the same as that of the LAF assay. Interestingly, pre-incubation with IL-2 renders thymocytes more sensitive than responsive to crude monocyte conditioned media. The use of the MTT colorimetric method for the assessment of thymocyte proliferation, and of the lectin jacalin as a co-mitogen are suggested as alternatives to be used in co-stimulatory assays.

Dominant negative MyD88 proteins inhibit interleukin-1beta /interferon-gamma -mediated induction of nuclear factor kappa B-dependent nitrite production and apoptosis in beta cells.

Insulin-dependent diabetes mellitus is an autoimmune disease in which pancreatic islet beta cells are destroyed by a combination of immunological and inflammatory mechanisms. In particular, cytokine-induced production of nitric oxide has been shown to correlate with beta cell apoptosis and/or inhibition of insulin secretion. In the present study, we investigated whether the interleukin (IL)-1beta intracellular signal transduction pathway could be blocked by overexpression of dominant negative forms of the IL-1 receptor interacting protein MyD88. We show that overexpression of the Toll domain or the lpr mutant of MyD88 in betaTc-Tet cells decreased nuclear factor kappaB (NF-kappaB) activation upon IL-1beta and IL-1beta/interferon (IFN)-gamma stimulation. Inducible nitric oxide synthase mRNA accumulation and nitrite production, which required the simultaneous presence of IL-1beta and IFN-gamma, were also suppressed by approximately 70%, and these cells were more resistant to cytokine-induced apoptosis as compared with parental cells. The decrease in glucose-stimulated insulin secretion induced by IL-1beta and IFN-gamma was however not prevented. This was because these dysfunctions were induced by IFN-gamma alone, which decreased cellular insulin content and stimulated insulin exocytosis. These results demonstrate that IL-1beta is involved in inducible nitric oxide synthase gene expression and induction of apoptosis in mouse beta cells but does not contribute to impaired glucose-stimulated insulin secretion. Furthermore, our data show that IL-1beta cellular actions can be blocked by expression of MyD88 dominant negative proteins and, finally, that cytokine-induced beta cell secretory dysfunctions are due to the action of IFN-gamma.

Persistent inhibition of FLIP(L) expression by lentiviral small hairpin RNA delivery restores death-receptor-induced apoptosis in neuroblastoma cells.

Neuroblastoma represents the most common and deadly solid tumour of childhood, which disparate biological and clinical behaviour can be explained by differential regulation of apoptosis. To understand mechanisms underlying death resistance in neuroblastoma cells, we developed small hairpin of RNA produced by lentiviral vectors as tools to selectively interfere with FLIP(L), a major negative regulator of death receptor-induced apoptosis. Such tools revealed highly efficient in interfering with FLIP(L) expression and function as they almost completely repressed endogenous and/or exogenously overexpressed FLIP(L) protein and fully reversed FLIP(L)-mediated TRAIL resistance. Moreover, interference with endogenous FLIP(L) and FLIP(S) significantly restored FasL sensitivity in SH-EP neuroblastoma cell line. These results reveal the ability of lentivirus-mediated shRNAs to specifically and persistently interfere with FLIP expression and support involvement of FLIP in the regulation of death receptor-mediated apoptosis in neuroblastoma cells. Combining such tools with other therapeutic modalities may improve treatment of resistant tumours such as neuroblastoma.

Ectopic expression of the serine protease inhibitor PI9 modulates death receptor-mediated apoptosis.

Apoptosis is a highly controlled process, whose triggering is associated with the activation of caspases. Apoptosis can be induced via a subgroup of the tumor necrosis factor (TNF) receptor superfamily, which recruit and activate pro-caspase-8 and -10. Regulation of apoptosis is achieved by several inhibitors, including c-FLICE-inhibitory protein, which prevents apoptosis by inhibiting the pro-apoptotic activation of upstream caspases. Here we show that the human intracellular serine protease inhibitor (serpin), protease inhibitor 9 (PI9), inhibits TNF-, TNF-related apoptosis-inducing ligand- and Fas ligand-mediated apoptosis in certain TNF-sensitive cell lines. The reactive center P1 residue of PI9 was required for this inhibition since PI9 harboring a Glu --> Ala mutation in its reactive center failed to impair death receptor-induced cell death. This suggests a classical serpin-protease interaction. Indeed, PI9 inhibited apoptotic death by directly interacting with the intermediate active forms of caspase-8 and -10. This indicates that PI9 can regulate pro-apoptotic apical caspases.

TRAIL receptors 1 (DR4) and 2 (DR5) signal FADD-dependent apoptosis and activate NF-kappaB.

TRAIL induces apoptosis through two closely related receptors, TRAIL-R1 (DR4) and TRAIL-R2 (DR5). Here we show that TRAIL-R1 can associate with TRAIL-R2, suggesting that TRAIL may signal through heteroreceptor signaling complexes. Both TRAIL receptors bind the adaptor molecules FADD and TRADD, and both death signals are interrupted by a dominant negative form of FADD and by the FLICE-inhibitory protein FLIP. The recruitment of TRADD may explain the potent activation of NF-kappaB observed by TRAIL receptors. Thus, TRAIL receptors can signal both death and gene transcription, functions reminiscent of those of TNFR1 and TRAMP, two other members of the death receptor family.

MAP kinase pathways in UV-induced apoptosis of retinal pigment epithelium ARPE19 cells.

The retinal pigment epithelium (RPE) is constantly exposed to external injuries which lead to degeneration, dysfunction or loss of RPE cells. The balance between RPE cells death and proliferation may be responsible for several diseases of the underlying retina, including age-related macular degeneration (AMD) and proliferative vitreoretinopathy (PVR). Signaling pathways able to control cells proliferation or death usually involve the MAPK (mitogen-activated protein kinases) pathways, which modulate the activity of transcription factors by phosphorylation. UV exposure induces DNA breakdown and causes cellular damage through the production of reactive oxygen species (ROS) leading to programmed cell death. In this study, human retinal pigment epithelial cells ARPE19 were exposed to 100 J/m(2) of UV-C and MAPK pathways were studied. We first showed the expression of the three major MAPK pathways. Then we showed that activator protein-1 (AP-1) was activated through phosphorylation of cJun and cFos, induced by JNK and p38, respectively. Specific inhibitors of both kinases decreased their respective activities and phosphorylation of their nuclear targets (cJun and cFos) and reduced UV-induced cell death. The use of specific kinases inhibitors may provide excellent tools to prevent RPE apoptosis specifically in RPE diseases involving ROS and other stress-related compounds such as in AMD.

Oncogenic Ras inhibits Fas ligand-mediated apoptosis by downregulating the expression of Fas.

Tumor growth is the result of deregulated tissue homeostasis which is maintained through the delicate balance of cell growth and apoptosis. One of the most efficient inducers of apoptosis is the death receptor Fas. We report here that oncogenic Ras (H-Ras) downregulates Fas expression and renders cells of fibroblastic and epitheloid origin resistant to Fas ligand-induced apoptosis. In Ras-transformed cells, Fas mRNA is absent. Inhibition of DNA methylation restores Fas expression. H-Ras signals via the PI 3-kinase pathway to downregulate Fas, suggesting that the known anti-apoptotic effect of the downstream PKB/Akt kinase may be mediated, at least in part, by the repression of Fas expression. Thus, the oncogenic potential of H-ras may reside on its capacity not only to promote cellular proliferation, but also to simultaneously inhibit Fas-triggered apoptosis.

Effect of antiretroviral therapy on apoptosis markers and morphology in peripheral lymph nodes of HIV-infected individuals.

BACKGROUND: CD4+ T cell depletion and destruction and the involution of the lymphoid tissue are hallmarks of HIV infection. Although the underlying mechanisms are still unclear, apoptosis appears to play a central role. The objective of this study was to investigate the effect of antiretroviral therapy on the lymph node tissue, particularly with respect to morphology and apoptosis. PATIENTS AND METHODS: Between 1997 and 1999, two inguinal lymph nodes were excised from 31 previously untreated individuals who were in an early stage of HIV infection, the first one prior to treatment and the second after 16 to 20 months of treatment. Paraffin sections were investigated for lymph node architecture, distribution of cellular and viral markers, apoptosis, and expression of apoptotic key molecules which indirectly reflect apoptotic processes. RESULTS: After 16-20 months of antiretroviral therapy, a significant decrease in highly activated HIV-driven immune response was observed in the lymph node tissue as a marked reduction in follicular hyperplasia, a normalization of the follicular dendritic cell network, a significant increase in the number of CD4+ T cells, and a significant decrease in the number of CD8+ T cells. The expression of several proapoptotic (Fas, TRAIL, and active caspase 3) and antiapoptotic (Bcl-2 and IL-7Ralpha) molecules that were reconstituted in the tissues during therapy resembled their expression in lymph nodes of HIV-negative individuals. Limitations of the study are (a) the lack of untreated patients in the late stages, (b) for ethical reasons, the lack of a control group with untreated patients, and (c) for methodological reasons, the restriction of sequential measurements of apotpotic markers to one-third of the patients. CONCLUSION: Antiretroviral therapy initiated in the early stages in HIV infection may halt the irreversible destruction of the lymph node tissue and may partially normalize apoptotic processes.

Peripheral T cells undergoing superantigen-induced apoptosis in vivo express B220 and upregulate Fas and Fas ligand.

Staphylococcal enterotoxin B (SEB) is a bacterial superantigen (SAg) that predominantly interacts with V(beta)8+ T cells. In vivo treatment of mice with SEB leads to an initial increase in the percentage of V(beta)8+ T cells, followed by a decrease in the numbers of these cells, eventually reaching lower levels than those found before treatment with the SAg. This decrease is due to apoptosis of the SEB-responding cells. In the present study, we use the distinct light scattering characteristics of apoptotic cells to characterize T cells that are being deleted in response to SEB in vivo. We show that dying, SEB-reactive T cells express high levels of Fas and Fas ligand (Fas-L), which are implicated in apoptotic cell death. In addition, the B cell marker B220 is upregulated on apoptotic cells. Moreover, we show that the generation of cells with an apoptotic phenotype is severely impaired in response to SEB in functional Fas-L-deficient mutant gld mice, confirming the role of the Fas pathway in SAg mediated peripheral deletion in vivo.

Apoptosis as a target for gene therapy in rheumatoid arthritis

Rheumatoid arthritis (RA) is characterized by chronic inflammation of the synovial joints resulting from hyperplasia of synovial fibroblasts and infiltration of lymphocytes, macrophages and plasma cells, all of which manifest signs of activation. All these cells proliferate abnormally, invade bone and cartilage, produce an elevated amount of pro-inflammatory cytokines, metalloproteinases and trigger osteoclast formation and activation. Some of the pathophysiological consequences of the disease may be explained by the inadequate apoptosis, which may promote the survival of autoreactive T cells, macrophages or synovial fibroblasts. Although RA does not result from single genetic mutations, elucidation of the molecular mechanisms implicated in joint destruction has revealed novel targets for gene therapy. Gene transfer strategies include inhibition of pro-inflammatory cytokines, blockade of cartilage-degrading metalloproteinases, inhibition of synovial cell activation and manipulation of the Th1-Th2 cytokine balance. Recent findings have iluminated the idea that induction of apoptosis in the rheumatoid joint can be also used to gain therapeutic advantage in the disease. In the present review we will discuss different strategies used for gene transfer in RA and chronic inflammation. Particularly, we will highlight the importance of programmed cell death as a novel target for gene therapy using endogenous biological mediators, such as galectin-1, a beta-galactoside-binding protein that induces apoptosis of activated T cells and immature thymocytes.