Caspase-dependent immunogenicity of doxorubicin-induced tumor cell death.

Systemic anticancer chemotherapy is immunosuppressive and mostly induces nonimmunogenic tumor cell death. Here, we show that even in the absence of any adjuvant, tumor cells dying in response to anthracyclins can elicit an effective antitumor immune response that suppresses the growth of inoculated tumors or leads to the regression of established neoplasia. Although both antracyclins and mitomycin C induced apoptosis with caspase activation, only anthracyclin-induced immunogenic cell death was immunogenic. Caspase inhibition by Z-VAD-fmk or transfection with the baculovirus inhibitor p35 did not inhibit doxorubicin (DX)-induced cell death, yet suppressed the immunogenicity of dying tumor cells in several rodent models of neoplasia. Depletion of dendritic cells (DCs) or CD8+T cells abolished the immune response against DX-treated apoptotic tumor cells in vivo. Caspase inhibition suppressed the capacity of DX-killed cells to be phagocytosed by DCs, yet had no effect on their capacity to elicit DC maturation. Freshly excised tumors became immunogenic upon DX treatment in vitro, and intratumoral inoculation of DX could trigger the regression of established tumors in immunocompetent mice. These results delineate a procedure for the generation of cancer vaccines and the stimulation of anti-neoplastic immune responses in vivo.

Use of protein biotinylation in vivo for immunoelectron microscopic localization of a specific protein isoform.

Tagging of proteins in vivo by covalent attachment of a biotin moiety has emerged as a new prospective tool for protein detection and purification. Previously, we established a strategy for expression of in vivo biotinylated proteins in mammalian cells. It is based on coexpression of the protein of interest fused to a short biotin acceptor peptide and biotin ligase BirA cloned in the same vector. We show here that the in vivo biotinylation can be used for immunogold postembedding labeling in immunoelectron microscopy experiments. We show that immunoelectron microscopy with biotinylated nuclear proteins is compatible with a wide range of postembedding methods, facilitating combination of morphological and localization studies in a single experiment. We also show that the method works in both transient transfection and stable cell line expression protocols and can be used for colocalization studies. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

Participation of kin17 protein in replication factories and in other DNA transactions mediated by high molecular weight nuclear complexes.

The Homo sapiens kin17 ((HSA)kin17) protein is a chromatin-associated protein conserved during evolution and overproduced in certain human tumor cell lines. For the first time, immunoelectron microscopy analysis of endogenous (HSA)kin17 protein revealed an ultrastructural co-localization of (HSA)kin17 and bromodeoxyuridine (BrdUrd) at sites of DNA replication after either short (15 min) or long (120 min) pulses of BrdUrd labeling. After hydroxyurea (HU) or L-mimosine (Mimo) block and withdrawal, we observed that (HSA)kin17 was recruited onto the chromatin during the re-entry and the progression in the S phase. These results are consistent with a major role of (HSA)kin17 protein in DNA replication factories. Other treatments hampering replication fork progression and/or inducing double-strand breaks also triggered an accumulation and a concentration of the chromatin-bound (HSA)kin17 protein into large intranuclear foci 24 h post-treatment. Moreover, HU- and Mimo-induced (HSA)kin17 foci were retained in the nucleus after detergent extraction, suggesting a strong association with nuclear structures. Gel filtration analyses of cellular extracts showed that endogenous (HSA)kin17 protein co-eluted with both replication proteins RPA32 and RPA70 in a fraction containing complexes of M(r) 600,000. Interestingly, HU-induced G(1)-S arrest triggered an increase in the molecular weight of complexes containing (HSA)kin17 protein. Hence, treatments interfering with either initiation and/or elongation of DNA replication also recruited chromatin-bound (HSA)kin17 protein. We hypothesize that in the presence of unrepaired DNA damage, (HSA)kin17 protein concentrated into high molecular weight complexes probably to create a bridge that contributes to the harmonization of DNA replication and repair.

Overexpression of galectin-7 in mouse epidermis leads to loss of cell junctions and defective skin repair.

BACKGROUND: The proteins of the galectin family are implicated in many cellular processes, including cell interactions, polarity, intracellular trafficking, and signal transduction. In human and mouse, galectin-7 is almost exclusively expressed in stratified epithelia, notably in the epidermis. Galectin-7 expression is also altered in several human tumors of epithelial origin. This study aimed at dissecting the consequences of galectin-7 overexpression on epidermis structure and functions in vivo. METHODS: We established transgenic mice specifically overexpressing galectin-7 in the basal epidermal keratinocytes and analyzed the consequences on untreated skin and after UVB irradiation or mechanical injury. RESULTS: The intercellular cohesion of the epidermis is impaired in transgenic animals, with gaps developing between adjacent keratinocytes, associated with loss of adherens junctions. The epidermal architecture is aberrant with perturbations in the multilayered cellular organisation of the tissue, and structural defects in the basement membrane. These transgenic animals displayed a reduced re-epithelialisation potential following superficial wound, due to a defective collective migration of keratinocytes. Finally, a single mild dose of UVB induced an abnormal apoptotic response in the transgenic epidermis. CONCLUSION: These results indicate that an excess of galectin-7 leads to a destabilisation of adherens junctions associated with defects in epidermal repair. As this phenotype shares similarities with that of galectin-7 null mutant mice, we conclude that a critical level of this protein is required for maintaining proper epidermal homeostasis. This study brings new insight into the mode of action of galectins in normal and pathological situations.

Adenovirus infection targets the cellular protein kinase CK2 and RNA-activated protein kinase (PKR) into viral inclusions of the cell nucleus.

The effects of the adenovirus infection on the distribution of the cellular protein kinase CK2 and double-stranded RNA-activated protein kinase (PKR) were examined at the ultrastructural level. Immunogold labeling revealed the redistribution of CK2 subunits and PKR to morphologically distinct structures of the cell nucleus. The electron-clear amorphous structures, designated pIX nuclear bodies in our previous work (Rosa-Calatrava et al., 2001), contained CK2 alpha and PKR. The protein crystals, which result from the regular assembly of hexon, penton base, and fiber proteins [Boulanger et al. (1970) J Gen Virol 6:329-332], contained CK2 beta and PKR. Both viral structures were devoid of viral RNA, including the PKR-inhibitor VA1 RNA generated by the RNA polymerase III. Instead, VA1 RNA accumulated in PKR-free viral compact rings in which the viral RNA generated by the RNA polymerase II was excluded.

[What's new in the treatment of cancer pain?]. / Actualités dans les traitements des douleurs d'origine cancéreuse.

Improvements have been made recently in the treatment of cancer pain. First of all, this symptom is better recognized and evaluated in cancer patients. Then new therapeutic options have became available in France : tramadol, WHO level II analgesic, for intermediate to severe pain; gabapentine, a new anticonvulsivant drug, for neuropathic pain; oral transmucosal fentanyl citrate for breakthrough pain; hydromorphone and oxycodone, morphine agonists, as an alternative to morphine; development of patient controlled analgesia via portable pump; better evaluation of alternative therapeutics.

[2003 clinical practice guideline: Standards, Options and Recommendations for pain assessment in adult and children with cancer (summary report)]. / Recommandations pour la pratique clinique: Standards, Options et Recommandations pour l'évaluation de la douleur chez l'adulte et l'enfant atteints d'un cancer (mise à jour)--rapport abrégé.

CONTEXT: The "Standards, Options and Recommendations" (SOR) project, which started in 1993, is a collaboration between the French Federation of Cancer Centers (FNCLCC), the 20 French Regional Cancer Centers, and specialists from French public universities, general hospitals and private clinics. The main objective is the development of clinical practice guidelines to improve the quality of health care and the outcome of cancer patients. OBJECTIVE: To update clinical practice guidelines for the assessment of pain in adult or children with cancer in collaboration with the French society for pain study and treatment. METHOD: The methodology is based on a literature review and critical appraisal by a multidisciplinary group of experts who define the CPGs according to the definitions of the Standards, Options and Recommendations project. Once the guideline has been defined, the document is submitted for review by independent reviewers. RESULTS: This article is a summary version of the full document presenting the clinical practice guidelines with algorithms. The main recommendations concern the means used to evaluate pain and its consequences and their use in specific cases (acute or chronic pain, patients able to communicate or not, children under or over 6 years old). Others recommendations were also established concerning the evaluation ofpsychological, social and family context, the evaluation of pain in hospital or at home, in terminal phase patients and for the establishment of a therapeutic strategy and follow-up of patient with pain.

[Emergency management of acute pain in oncology]. / La prise en charge en urgence des douleurs cancérologiques.

Acute pains requiring emergency management in oncology can be considered as physiopathological, somatic or visceral nociceptive pains. They are linked: to the tumour, indicating a modification of the tumoural evolution (necrosis, haemorrhage, fracture, acute obstruction of hollow organs or canals, occlusion, hydronephrosis); to the treatment: (inflammation of mucosal membranes, anusitis, post PL syndromes); and to invasive investigations. They are equally neuropathic, revealing an underlying threatened or confirmed medullary compression, or induced by neurotoxic chemotherapy. They are also analysed according to their mode of apparition: mechanical, arising as acute on chronic pain (the pre-fracture pain of metastases); insufficiency of the duration of therapeutic efficacity; an acute episode of neuropathic pain that is often lancing, unpredictable and inevitable. In all cases, it needs to be quantitatively and qualitatively analysed: evaluation, flavour of the symptoms; in order to choose one or a combination of adapted molecules, true antalgics or co-antalgics, antidepressants and anticonvulsants. To counteract this pain, medications with a short onset of action and a short half-life should be used to avoid side effects. These are administered in an intercurrent manner, initially starting at a low dose, modified daily according to the utilisation of supplementary doses. It is necessary to anticipate pain provoked by physical examinations or nursing care as much in the timing as the pharmacology, in using antalgics and/or anxiolytics with a short duration of action. Acutely emerging pains, whatever be their type, arising in the context of cancer and long-term pain are sensitising elements to all further pains, as they imprint in the memory, and are very negatively conditioned by the anguishing context of the illness.

Basal foot MTOC organizes pillar MTs required for coordination of beating cilia.

Coordination of ciliary beating is essential to ensure mucus clearance in the airway tract. The orientation and synchronization of ciliary motion responds in part to the organization of the underlying cytoskeletal networks. Using electron tomography on mouse trachea, we show that basal bodies are collectively hooked at the cortex by a regular microtubule array composed of 4-5 microtubules. Removal of galectin-3, one of basal-body components, provokes misrecruitment of γ-tubulin, disorganization of this microtubule framework emanating from the basal-foot cap, together with loss of basal-body alignment and cilium orientation, defects in cilium organization and reduced fluid flow in the tracheal lumen. We conclude that galectin-3 plays a crucial role in the maintenance of the microtubule-organizing centre of the cilium and the 'pillar' microtubules, and that this network is instrumental for the coordinated orientation and stabilization of motile cilia.

Signal peptide peptidase-catalyzed cleavage of hepatitis C virus core protein is dispensable for virus budding but destabilizes the viral capsid.

The capsid of hepatitis C virus (HCV) particles is considered to be composed of the mature form (p21) of core protein. Maturation to p21 involves cleavage of the transmembrane domain of the precursor form (p23) of core protein by signal peptide peptidase (SPP), a cellular protease embedded in the endoplasmic reticulum membrane. Here we have addressed whether SPP-catalyzed maturation to p21 is a prerequisite for HCV particle morphogenesis in the endoplasmic reticulum. HCV structural proteins were expressed by using recombinant Semliki Forest virus replicon in mammalian cells or recombinant baculovirus in insect cells, because these systems have been shown to allow the visualization of HCV budding events and the isolation of HCV-like particles, respectively. Inhibition of SPP-catalyzed cleavage of core protein by either an SPP inhibitor or HCV core mutations not only did not prevent but instead tended to facilitate the observation of viral buds and the recovery of virus-like particles. Remarkably, although maturation to p21 was only partially inhibited by mutations in insect cells, p23 was the only form of core protein found in HCV-like particles. Finally, newly developed assays demonstrated that p23 capsids are more stable than p21 capsids. These results show that SPP-catalyzed cleavage of core protein is dispensable for HCV budding but decreases the stability of the viral capsid. We propose a model in which p23 is the form of HCV core protein committed to virus assembly, and cleavage by SPP occurs during and/or after virus budding to predispose the capsid to subsequent disassembly in a new cell.