Cell type-dependent ROS and mitophagy response leads to apoptosis or necroptosis in neuroblastoma.

A limiting factor in the therapeutic outcome of children with high-risk neuroblastoma is the intrinsic and acquired resistance to common chemotherapeutic treatments. Here we investigated the molecular mechanisms by which the hemisynthetic cardiac glycoside UNBS1450 overcomes this limitation and induces differential cell death modalities in both neuroblastic and stromal neuroblastoma through stimulation of a cell-type-specific autophagic response eventually leading to apoptosis or necroptosis. In neuroblastic SH-SY5Y cells, we observed a time-dependent production of reactive oxygen species that affects lysosomal integrity inducing lysosome-associated membrane protein 2 degradation and cathepsin B and L activation. Subsequent mitochondrial membrane depolarization and accumulation of mitochondria in phagophores occurred after 8h of UNBS1450 treatment. Results were confirmed by mitochondrial mass analysis, electron microscopy and co-localization of mitochondria with GFP-LC3, suggesting the impaired clearance of damaged mitochondria. Thus, a stress-induced defective autophagic flux and the subsequent lack of clearance of damaged mitochondria sensitized SH-SY5Y cells to UNBS1450-induced apoptosis. Inhibition of autophagy with small inhibitory RNAs against ATG5, ATG7 and Beclin-1 protected SH-SY5Y cells against the cytotoxic effect of UNBS1450 by inhibiting apoptosis. In contrast, autophagy progression towards the catabolic state was observed in stromal SK-N-AS cells: here reactive oxygen species (ROS) generation remained undetectable preserving intact lysosomes and engulfing damaged mitochondria after UNBS1450 treatment. Moreover, autophagy inhibition determined sensitization of SK-N-AS to apoptosis. We identified efficient mitophagy as the key mechanism leading to failure of activation of the apoptotic pathway that increased resistance of SK-N-AS to UNBS1450, triggering rather necroptosis at higher doses. Altogether we characterize here the differential modulation of ROS and mitophagy as a main determinant of neuroblastoma resistance with potential relevance for personalized anticancer therapeutic approaches.

Clinical relevance of flow cytometric immunophenotyping of the cerebrospinal fluid in patients with diffuse large B-cell lymphoma.

BACKGROUND: Central nervous system (CNS) relapse is an uncommon but dramatic complication of diffuse large B-cell lymphoma (DLBCL). Several studies have demonstrated the superiority of cerebrospinal fluid (CSF) flow cytometry (FCM), as compared with conventional cytology (CC), in detecting occult leptomeningeal disease. The clinical relevance of a positive FCM still has to be clarified. PATIENTS AND METHODS: We analyzed CSF from 114 DLBCL patients at diagnosis (n = 95) or at relapse (n = 19) by FCM and CC. Most patients received meningeal prophylaxis. FCM results did not influence treatment strategies. RESULTS: Fourteen samples were FCM+, versus one CC+ (also FCM+). Within all patients without neurological symptoms (n = 101), four (4%) relapsed in the CNS, with a median time to relapse of 5.2 months. Only one-fourth (25%) was FCM+ before relapse. More than one extranodal disease site and elevated lactate dehydrogenase levels were associated with an increased risk of CNS relapse. CONCLUSIONS: FCM gives far more positive results than CC. However, a positive FCM result did not translate into a significant increase in CNS relapse rate in this histologically uniform population receiving CNS prophylaxis.

Noncoding human Y RNAs are overexpressed in tumours and required for cell proliferation.

Noncoding Y RNAs have recently been identified as essential factors for chromosomal DNA replication in human cell nuclei. Here, we investigate the expression of human Y RNAs in tumours and test their requirement for cell proliferation. Relative expression levels of all four human Y RNAs (hY1, hY3, hY4 and hY5 RNA) were determined by quantitative RT-PCR in extracts from human solid tumours, corresponding nonmalignant normal tissues and derived cultured cells. On average, all four hY RNAs are significantly overexpressed in solid tumours between 4- and 13-fold, compared to the corresponding normal tissues. In particular, hY1 and hY3 RNAs are overexpressed in carcinomas (and adenocarcinomas) of the bladder, cervix, colon, kidney, lung and prostate with extremely high statistical significance (ANOVA, between groups, P<10e-22). A functional requirement of all four hY RNAs for cell proliferation was investigated in a systematic survey for loss-of-function by RNA interference (RNAi). Degradation of hY1 and hY3 RNAs in human cell lines resulted in a significant cytostatic inhibition of cell proliferation. We conclude that noncoding hY RNAs have potential both as new cancer biomarkers and as molecular targets for anti-proliferative intervention.

A model for glioma cell migration on collagen and astrocytes.

We present a model for the migration of glioma cells on substrates of collagen and astrocytes. The model is based on a cellular automaton where the various dynamical effects are introduced through adequate evolution rules. Using our model, we investigate the role of homotype and heterotype gap junction communication and show that it is possible to reproduce the corresponding experimental migration patterns. In particular, we confirm the experimental findings that inhibition of homotype gap junctions favours migration while heterotype inhibition hinders it. Moreover, the effect of heterotype gap junction inhibition dominates that of homotype inhibition.

A cellular automaton model for the migration of glioma cells.

We present a study of in vitro cell migration in two dimensions as a first step towards understanding the mechanisms governing the motility of glioma cells. Our study is based on a cellular automaton model which aims at reproducing the kinetics of a lump of glioma cells deposited on a substrate of collagen. The dynamical effects of cell attraction and motion inertia are introduced through adequate automaton rules. We compare the density profiles given by the model to those obtained experimentally. The result of the best fit indicates a substantial cell-cell attraction due to cell-cell communication through gap junctions (or chemotaxis) and negligible inertia effects during migration. Tracking of individual migrating cells indicates highly convoluted cell trajectories.

Correlation of in vitro infiltration with glioma histological type in organotypic brain slices.

Diffuse invasion of the brain, an intrinsic property of gliomas, renders these tumours incurable, and is a principal determinant of their spatial and temporal growth. Knowledge of the invasive potential of gliomas is highly desired in order to understand their behaviour in vivo. Comprehensive ex vivo invasion studies including tumours of different histological types and grades are however lacking, mostly because reliable physiological invasion assays have been difficult to establish. Using an organotypic rodent brain slice assay, we evaluated the invasiveness of 42 grade II-IV glioma biopsy specimens, and correlated it with the histological phenotype, the absence or presence of deletions on chromosomes 1p and 19q assessed by fluorescent in situ hybridisation, and proliferation and apoptosis indices assessed by immunocytochemistry. Oligodendroglial tumours with 1p/19q loss were less invasive than astrocytic tumours of similar tumour grade. Correlation analysis of invasiveness cell proliferation and apoptosis further suggested that grade II-III oligodendroglial tumours with 1p/19q loss grow in situ as relatively circumscribed compact masses in contrast to the more infiltrative and more diffuse astrocytomas. Lower invasiveness may be an important characteristic of oligodendroglial tumours, adding to our understanding of their more indolent clinical evolution and responsiveness to therapy.

Migration pathways of human glioblastoma cells xenografted into the immunosuppressed rat brain.

Diffuse invasion of the brain by tumor cells is a hallmark of human glioblastomas and a major cause for the poor prognosis of these tumors. This phenomenon is only partially reproduced by rodent models of gliomas that display a very high rate of proliferation and limited cell migration. We have analyzed the development of human glioblastoma cells (GL15) xenografted into the brain of immunosuppressed rats, in order to define the characteristics of tumor cell invasion. As identified by the specific immunolabeling of the tumor cells for the human HLA-ABC antigen, GL15 tumors reproduced the three types of intraparenchymal invasion observed in patients. First, a majority of multipolar tumor cells intermingled rapidly and profusely with host neural cells in the margin of the injection site. This progressively enlarging area was principally responsible for the tumor growth over time. Second, in the gray matter, columns of thin bipolar tumor cells aligned along capillary walls. Third, in the white matter, elongated bipolar isolated tumor cells were observed scattered between axonal fibers. The maximum migration distances along white matter fibers remained significantly higher than the maximum migration distances along blood vessels, up to two months after injection. Development of the tumor was associated with a significant increase of vascularization in the area of tumor spread. Xenografting of human GL15 glioblastoma cells into the immunosuppressed rat brain allowed to differentiate between the three classical types of invasion identified in the clinic, to quantify precisely the distances of migration, and to evaluate cell morphology for each of these routes. The present results support the existence of host/tumor cells interactions with specific characteristics for each type of invasion.

Matrix metalloproteinase-9 is increased and correlates with severity in Guillain-Barré syndrome.

OBJECTIVE: To study the expression and activity of matrix metalloproteinases (MMPs) MMP-2 (72-kd type IV collagenase, gelatinase A), MMP-3 (58-kd stromelysin-1), and MMP-9 (92-kd type IV collagenase, gelatinase B) and tissue inhibitors of MPs (TIMP) in patients with Guillain-Barre syndrome (GBS). BACKGROUND: MMPs are able to proteolysis of basement membranes and other matrix components, promoting transmigration of inflammatory cells from circulation to nerve tissue. METHODS: Twenty-five patients with GBS were analyzed according to the phase of the disease, i.e., progression, plateau, early recovery, and late recovery. Determinations of MMP-2, MMP-3, MMP-9, and TIMP-1 were performed using ELISA, zymography, and immunocytochemistry in circulation or peripheral nerve. RESULTS: MMP-9 plasma levels were increased in 67% of patients on admission and decreased from progression to late recovery (p < 0.002). During the course of GBS, MMP-9 was progressively balanced by its inhibitor TIMP-1, as assessed by the MMP-9/TIMP-1 ratio. MMP-9 and TIMP-1 plasma levels and the MMP-9/TIMP-1 ratio correlated positively with disability. MMP-2 expression was similar to controls. MMP-3 activity was not detected, and plasma levels were not different from those in controls. Positive MMP-9 immunolabeling was 51 +/- 11% of circulating lymphocytes. It was observed in some endothelial cells and mononuclear cells adherent to the endothelium and close to myelinated fibers. CONCLUSIONS: Circulating matrix metalloproteinases (MMP-9) correlates with disease severity in Guillain-Barré syndrome (GBS). MMP-9 likely represents an important molecule in the pathogenesis of GBS and therefore could represent an interesting therapeutic target.

Primary brain lymphoma cell turnover differs in patients with and without AIDS: relationships to bcl-2 expression and host cell reaction.

Primary central nervous system lymphomas (PCNSLs) are more resistant to radiotherapy and chemotherapy in AIDS (A-PCNSLs) than in non-AIDS patients (NA-PCNSLs). We investigated 23 A-PCNSLs and 24 NA-PCNSLs. Lymphoma cell kinetics (i.e. proliferation [mitotic index, MIB-1 and PCNA labeling indices], apoptosis and turnover) were determined and compared with bcl-2 and LMP-1 expression, and to the percentage of tumor-infiltrating T-lymphocytes (T-TILs) and macrophages. A-PCNSLs showed lower proliferation (p < 0.005), less apoptosis (p < 0.0001) and slower cell-turnover (p < 0.0001) than NA-PCNSLs. LMP-1 was detected in 90% of A-PCNSLs and 5% of NA-PCNSLs, a finding correlating positively with bcl-2 expression (p < 0.0007). In contrast, T-TIL counts and CD4/CD8 T-TIL ratios were similar in A-PCNSLs and NA-PCNSLs. T-TIL counts correlated negatively with proliferation indices (from p < 0.05 to p < 0.0005) in NA-PCNSLs, but not in A-PCNSLs. Macrophage counts correlated positively with apoptosis in both groups. We concluded the following: (i) A-PCNSLs are characterized by accumulation of slow-cycling, long-lived cells that might be protected from apoptosis by LMP-1 induced bcl-2 expression, and independently from the host response; (ii) NA-PCNSLs are characterized by a faster cell turnover associated with an insufficient antiproliferative host response; and (iii) A-PCNSLs and NA-PCNSLs constitute 2 entities with distinctive morphology and different kinetic profiles that could account for different responses to therapy.

Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) and its receptor flt-1 in microcystic meningiomas.

We investigated the immunohistochemical expression of vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) and of its endothelial cell receptor flt-1 in relationship to microcyst formation in meningiomas. Expression of VPF/VEGF was studied in 60 meningiomas (6 microcystic, 38 partially microcystic and 16 with no microcystic areas) and 30 meningiomas from these three subgroups were evaluated for flt-1 expression. VPF/VEGF immunoreactivity was mainly observed in vessel endothelium. Positive vessels were present in 75% (33/44) of meningiomas with any amount of microcystic pattern and in 38% (6/16) of the solid meningiomas (P < 0.02). Densities and percentages of both VPF/VEGF-positive and flt-1-positive vessels were higher in meningiomas with microcystic areas than in solid meningiomas (P /= 0.75, P < 0.0001). A strong positive correlation between VPF/VEGF-positive vessel density and proportion of microcystic pattern in all 60 specimens was found (r = 0.75, P < 0. 0001). We conclude that accumulation of flt-1-bound VPF/VEGF on endothelial cells of meningiomas is associated with microcyst formation that leads to the histologic appearance of microcystic meningiomas.

Recurrent central neurocytoma with marked increase in MIB-1 labelling index.

Central neurocytomas (CNs) are characterized by an indolent growth and a low recurrence rate. Cell proliferation has been rarely studied in recurrent CNs despite the growing awareness of its prognostic value. We present a CN that recurred rapidly after a prolonged relapse-free interval showing a 4-fold increase of MIB-1 labelling index.

Immunohistochemical detection of vascular endothelial growth factor (VEGF) in the vasculature of oligodendrogliomas.

Vascular endothelial growth factor (VEGF) appears to be implicated in tumour angiogenesis. In the present study immunohistochemical expression of VEGF was evaluated in 34 oligodendrogliomas (13 grade II, 21 grade III [WHO]). VEGF immunoreactivity was found in 31 of 34 cases. Expression of VEGF was observed in endothelial cells and some vascular smooth muscle cells, but not in neoplastic oligodendrocytes. Vessel counts, percentages of VEGF-positive vessels and vessels with vascular endothelial proliferation were assessed. The degree of VEGF labelling and vascular-endothelial proliferation in each vessel were evaluated using a 3 degree intensity score. Expression of VEGF was higher in grade III than in grade II oligodendrogliomas as assessed by percentage of VEGF positive vessels (55.8 +/- 29.2% vs 17.0 +/- 19.0% [P < 0.001]) and by VEGF immunostaining intensity (1.90 +/- 0.60 vs 0.90 +/- 0.40 [P < 0.001]). VEGF expression did not correlate with vessel density. Intensity of VEGF expression correlated positively with that of vascular-endothelial proliferation in grade III tumours (r = +0.47 [P < 0.05]). The percentage of VEGF positive vessels showed some degree of positive correlation with the percentage of vessels showing vascular-endothelial proliferation (r = +408 [P < 0.10]). Within individual grade III tumours 67.5 +/- 29.6% of all vessels with vascular-endothelial proliferation were VEGF-positive and 31.0 +/- 20.5% of all VEGF-positive vessels showed no evidence of vascular-endothelial proliferation. We conclude that (i) expression of VEGF is observed in the vasculature of oligodendrogliomas; (ii) marked expression of VEGF is observed in grade III oligodendrogliomas; (iii) VEGF may be one of the interrelated causative stimuli acting in concert to induce vascular-endothelial proliferation.

Expression of c-erbB-2 oncogene and p53 tumor suppressor gene in benign and malignant breast tissue: correlation with proliferative activity and prognostic index.

The expression of c-erbB-2 oncogene and p53 tumor suppressor gene was studied in methacarn-fixed, paraffin-embedded biopsy specimens from 58 benign breast lesions and 129 sporadic breast carcinomas, using the supersensitive monoclonal antibodies CB 11 and BP 53-12-1 and the biotin-streptAvidin-amplified methodology. None of the benign lesions studied, which included 36 fibrocystic lesions with mild or florid epithelial hyperplasia, 12 fibrocystic lesions with ADH or ALH and 10 fibroadenomas, demonstrated membrane staining for c-erbB-2 or nuclear immunoreactivity for p53. Overall, 48.06% of primary breast carcinomas showed membrane expression of c-erbB-2, while 28.68% were p53 positive. Those showing p53 immunoreactivity displayed a nuclear and/or cytoplasmic staining type. A significant correlation was seen between c-erbB-2 and p53 expression (r = 0.213, p < 0.05), as well as between c-erbB-2 status and PSNA score (r = 0.221, p < 0.05). In addition, c-erbB-2 and p53, separately or in combination, correlated significantly with the prognostic index. In conclusion, immunohistochemistry of c-erbB-2 and p53 immunohistochemistry allows a better definition of intraductal proliferative lesions and assists in the differentiation between ADH and DCIS. It provides additional clues with regard to the biologic behavior of invasive ductal carcinomas (NOS and medullary).

Interleukin-1 expression in inflammatory myopathies: evidence of marked immunoreactivity in sarcoid granulomas and muscle fibres showing ischaemic and regenerative changes.

The most frequent autoimmune adult inflammatory myopathies are dermatomyositis, polymyositis, inclusion body myositis, and sarcoid myopathy. Interleukin-1 (IL-1) is a pleiotropic molecule, implicated in the inflammatory process, but also in tissue protection and remodelling. We evaluated the immunocytochemical expression of [L,-1alpha and beta in frozen muscle biopsy specimens from patients with dermatomyositis (15 cases), polymyositis (five cases), inclusion body myositis (five cases) and sarcoid myopathy (five cases). Positive immunoreactivities, were observed in both inflammatory cells and muscle fibres. Specificity of the immunostaining was assessed by Western blot experiments. IL-1 positive inflammatory cells were rare in polymyositis and inclusion body myositis, moderately abundant in dermatomyositis, and prominent in sarcoid myopathy granulomas. In sarcoid myopathy, 24.6 +/- 4.1% inflammatory cells were IL-1alpha-positive and 45.2 +/- 2.6% were IL-1beta-positive. IL-1 positive muscle fibres were mainly observed in dermatomyositis, usually remote from inflammatory infiltrates. Positive immunostaining for IL-1 was observed in fibres showing ischaemic punched-out vacuoles, that correspond to areas of myosinolysis, in atrophic perifascicular fibres, and in fibres located within healing microinfarcts. All NCAM-positive regenerating fibres were IL-1 positive. We conclude that: (i) IL-1 is expressed in granulomas of sarcoid myopathy, which is in keeping with the role ascribed to IL-1 in the formation of granulomas: (ii) IL-1 is expressed by muscle fibres undergoing ischaemic damage: and (iii) IL-1 expression by muscle fibres is associated with myofibrillar protein breakdown and regeneration.

Changes in the responsiveness of the colonic smooth muscle to carbachol in Hirschsprung's disease.

In vitro experiments were performed on smooth-muscle strips cut out in longitudinal and circular direction from the colon of Hirschsprung's patients who underwent an operation and from patients operated on for tumors of the sigmoid colon and rectum, serving as controls. The changes in the contractile activity of the smooth-muscle strips after carbachol applied cumulatively were examined and dose-response curves were plotted. The EC50 values for the circular strips from the aganglionic part of the colon were 6 x 10(-7) M in Hirschsprung's patients and 3.7 x 10(-8) M in control patients: the pD2 values were 6.23 and 7.43, respectively. This showed that the affinity of cholinoreceptors in the aganglionic part of the colon for carbachol was 16 times lower in Hirschsprung's patients as compared to control patients. The EC50 values for longitudinal strips from the ganglionic part of the colon were 2.6 x 10(-7) M in Hirschsprung's patients and 4 x 10(-8) M in control patients; pD2 values were 6.6 and 7.4, respectively. The affinity of cholinoreceptors in the smooth muscle of the ganglionic part of the colon for carbachol was also decreased (nearly 6.5 times) in Hirschsprung's patients as compared to controls.

[Specialized sensory perception in women with polycystic ovaries]. / Perception sensorielle spécialisée chez des femmes aux ovaires polykystiques.

Specialized sensory perception was examined in 20 women with polycystic ovary syndrome and in 20 healthy women with normal ovulatory genital cycle. None of the healthy women disclosed any anomaly in sensory perception while with no exception all patients with polycystic ovary syndrome disclosed anomalies in sensory perception of one or other sensory analyzer. Defects in smell (hypoosmis) were more common (19 cases) than those of taste (hypogeusia) (II cases), of vestibular (14 cases) or auditory (3 cases) function, although defects in two or more analyzers occurred together in most of the cases. The association of the syndrome with anomalies in the sensory perception suggested that various central nervous structures beside these controlling the releasing factors are involved in the pathomechanism of the disease and that specialized sensory perception in man has much more to do with the neuroendocrine system than previously believed.