[Circadian and septadian variation in sudden cardiac death : Autopsy registry of the Tunisian North]. / Variation circadienne et hebdomadaire de la mort subite d'origine cardiaque : registre autopsique du nord de la Tunisie.

INTRODUCTION: Several studies have suggested a circadian and septadian pattern of incidence of sudden cardiac death with a morning peak and a Monday peak. OBJECTIVE: To analyze the circadian and septadian pattern of occurrence of sudden cardiac death in the eight northern Tunisian governorates. METHODS: We prospectively collected epidemiological and autopsy data of sudden cardiac death victims occurring in the northern region of Tunisia between January 2013 and December 2019. RESULTS: The population included 1834 men (79.6%) and 468 women (20.4%) with a mean age of 56.5 ± 14 years. Smoking (53.9%) was the most prevalent cardiovascular risk factor. One-fifth (20.9%) of victims had known heart disease, and 3% had a family history of sudden death. ischemic heart disease was the leading cause of sudden death (46.8% of cases). One- fourth (25.7%) of autopsies were negative. Analysis of the circadian pattern of occurrence of sudden cardiac death identified a peak (36.1%, p < 0.001) between midnight and 6 am. This nocturnal excess mortality was significant (p < 0.001) and independent of sex (34.1 % in men and 43.8 % in women) and cause of death (39.3 % of cases of sudden ischemic death and 33.3 % of cases of nonischemic death). Moreover, there was a significant septadian variability in the occurrence of sudden death (p: 0.0015), with a peak on Friday (15.8 %, p: 0.042). CONCLUSION: This study showed a peak of sudden death between midnight and 6 am, and on Fridays, confirming the modification of the classic circadian and septadian pattern of sudden death occurrence. These results may help optimize the deployment of emergency mobile teams and structures during the most vulnerable periods.

Patterns of unintentional fatal drowning among children in North Tunisia : A 10-year study.

AIMS: This study aims to better understand patterns of unintentional fatal drowning among children in North Tunisia. METHODS: A cross-sectional retrospective study including all unintentional fatal drowning among children was conducted in the Legal and Forensic Medicine department in the Charles Nicolle Hospital, Tunis, between January 2010 and December 2019. Socio-demographic variables, as well as death circumstances, were documented for each victim and analyzed. RESULTS: A total of 200 casualties were included in this study. The highest rate of deaths was observed in the summer (N=44). Most of the victims were males and 55.5% were aged between 13 and 18 years. The drowning occurred in a canal or the sea in 33.5 % and 29.5 % of the cases respectively. The distribution of drowning sites varied significantly by season and place of living: drowning in the sea was more likely to occur in the summer and in urban areas (p < 0.05). In the first years of life, drowning occurred mostly in buckets and wells (N=9 and N=10, respectively) while between 7 and 18 years, it was more frequent in a canal or the sea. Swimming was the leading activity before death in 50% of the cases. CONCLUSIONS: Unintentional fatal drowning among children remains an underestimated major health problem in Tunisia especially in the population aged from 7 to 18 years. Effective prevention measures should be implemented nationwide, especially around seas and canals.

A cadaveric anatomical study of the adrenals: Relationship with the posterior abdominal wall muscles revisited.

AIM OF THE STUDY: The main difficulties during retroperitoneal laparoscopic adrenalectomies are due to its location. Our objective was to define the relationship of the adrenals with the diaphragm and the psoas muscle. METHODS: Our work is an anatomical dissection of 80 fresh cadavers' adrenals. To study the right adrenal, we performed a right nephrectomy and adrenal remained attached to the Inferior vena cava by its main vein. On the left, the edges of the adrenal have been identified by needles and the adrenal was reclined to study its projection on the posterior muscular wall. RESULTS: The right adrenal is located higher, 13mm [4-20mm] above the medial arcuate ligament (MAL) in 16 cases (40%). Its lower border was at the same level as the MAL in 18 cases (45%) and 11mm [10-17mm] below the MAL in 6 cases (15%). The posterior support of the right adrenal was the right crus of the diaphragm (Right-CD) in 34 cases (85%) and straddling the Right-CD and the psoas in 6 cases (15%). The study of the relationships of the left adrenal with the MAL showed that the lower edge of the gland was at its same level in 16 cases (40%) and below in 24 cases (60%) by 14mm [8-24mm]. The posterior support of the left adrenal was the left crus of the diaphragm (Left-CD) in 16 cases (40%) and straddling the Left-CD and the psoas in 24 cases (60%). CONCLUSIONS: Our results showed that the right adrenal is higher. The MAL is an important posterior element to the adrenal gland that could serve as an anatomical landmark to identify the adrenal during laparoscopic adrenalectomy.

[Sudden cardiac death in women, data from the northern Tunisian sudden cardiac-death registry]. / La mort subite cardiaque chez la femme, registre du nord de la Tunisie.

BACKGROUND: Sudden cardiac death is a major public health problem. Epidemiological and clinical differences according to gender have been described in sudden cardiac death. The aim of this study was to examine the gender differences between autopsy findings and circumstance of occurrence associated with sudden cardiac death. METHODS: We prospectively collected epidemiological and autopsy data of victims of sudden cardiac death occurring in the northern governorates of Tunisia between January 2013 and December 2019. Symptoms preceding death, circadian, weekly and seasonal variations of sudden death were also analyzed. RESULTS: The study population included 1834 men and 468 women with a mean age of 56.5±14.2 years. All cardiovascular risk factors except smoking were significantly more frequent among women but ischemic heart disease was the most common cause of death in men (51.3 %, versus 28 %, P<0.001). Women were more likely to have a negative macroscopic autopsy than men (34 % versus 23.6 %, P<0.001). Chest pain preceding sudden death was more frequent in male (24 % versus 13.2 %, P<0.001). In contrast, women were more likely to have dyspnea (8.1 % versus 15.6 %, P<0.001). Sudden death in women occurred indoors more often than in men (63.9 % versus 54.5 %, P<0.001) and also more often during night (midnight to 6 am). We also recorded an excess cardiac mortality in winter in both sexes. CONCLUSIONS: Women had considerably more cardiovascular risk factors and more commonly negative macroscopic autopsy. Death occurred indoors and during night more often than in men.

Peptides derived from the dependence receptor ALK are proapoptotic for ALK-positive tumors.

ALK is a receptor tyrosine kinase with an oncogenic role in various types of human malignancies. Despite constitutive activation of the kinase through gene alterations, such as chromosomal translocation, gene amplification or mutation, treatments with kinase inhibitors invariably lead to the development of resistance. Aiming to develop new tools for ALK targeting, we took advantage of our previous demonstration identifying ALK as a dependence receptor, implying that in the absence of ligand the kinase-inactive ALK triggers or enhances apoptosis. Here, we synthesized peptides mimicking the proapoptotic domain of ALK and investigated their biological effects on tumor cells. We found that an ALK-derived peptide of 36 amino acids (P36) was cytotoxic for ALK-positive anaplastic large-cell lymphoma and neuroblastoma cell lines. In contrast, ALK-negative tumor cells and normal peripheral blood mononuclear cells were insensitive to P36. The cytotoxic effect was due to caspase-dependent apoptosis and required N-myristoylation of the peptide. Two P36-derived shorter peptides as well as a cyclic peptide also induced apoptosis. Surface plasmon resonance and mass spectrometry analysis of P36-interacting proteins from two responsive cell lines, Cost lymphoma and SH-SY5Y neuroblastoma, uncovered partners that could involve p53-dependent signaling and pre-mRNA splicing. Furthermore, siRNA-mediated knockdown of p53 rescued these cells from P36-induced apoptosis. Finally, we observed that a treatment combining P36 with the ALK-specific inhibitor crizotinib resulted in additive cytotoxicity. Therefore, ALK-derived peptides could represent a novel targeted therapy for ALK-positive tumors.

Stability of a flow down an incline with respect to two-dimensional and three-dimensional disturbances for Newtonian and non-Newtonian fluids.

Squire's theorem, which states that the two-dimensional instabilities are more dangerous than the three-dimensional instabilities, is revisited here for a flow down an incline, making use of numerical stability analysis and Squire relationships when available. For flows down inclined planes, one of these Squire relationships involves the slopes of the inclines. This means that the Reynolds number associated with a two-dimensional wave can be shown to be smaller than that for an oblique wave, but this oblique wave being obtained for a larger slope. Physically speaking, this prevents the possibility to directly compare the thresholds at a given slope. The goal of the paper is then to reach a conclusion about the predominance or not of two-dimensional instabilities at a given slope, which is of practical interest for industrial or environmental applications. For a Newtonian fluid, it is shown that, for a given slope, oblique wave instabilities are never the dominant instabilities. Both the Squire relationships and the particular variations of the two-dimensional wave critical curve with regard to the inclination angle are involved in the proof of this result. For a generalized Newtonian fluid, a similar result can only be obtained for a reduced stability problem where some term connected to the perturbation of viscosity is neglected. For the general stability problem, however, no Squire relationships can be derived and the numerical stability results show that the thresholds for oblique waves can be smaller than the thresholds for two-dimensional waves at a given slope, particularly for large obliquity angles and strong shear-thinning behaviors. The conclusion is then completely different in that case: the dominant instability for a generalized Newtonian fluid flowing down an inclined plane with a given slope can be three dimensional.

Wnt signaling is involved in human articular chondrocyte de-differentiation in vitro.

Osteoarthritis is the most prevalent form of arthritis in the world. Certain signaling pathways, such as the wnt pathway, are involved in cartilage pathology. Osteoarthritic chondrocytes undergo morphological and biochemical changes that lead to chondrocyte de-differentiation. We investigated whether the Wnt pathway is involved in de-differentiation of human articular chondrocytes in vitro. Human articular chondrocytes were cultured for four passages in the presence or absence of IL-1 in monolayer or micromass culture. Changes in cell morphology were monitored by light microscopy. Protein and gene expression of chondrocyte markers and Wnt pathway components were determined by Western blotting and qPCR after culture. After culturing for four passages, chondrocytes exhibited a fibroblast-like morphology. Collagen type II and aggrecan protein and gene expression decreased, while collagen type I, matrix metalloproteinase 13, and nitric oxide synthase expressions increased. Wnt molecule expression profiles changed; Wnt5a protein expression, the Wnt target gene, c-jun, and in Wnt pathway regulator, sFRP4 increased. Treatment with IL-1 caused chondrocyte morphology to become more filament-like. This change in morphology was accompanied by extinction of col II expression and increased col I, MMP13 and eNOS expression. Changes in expression of the Wnt pathway components also were observed. Wnt7a decreased significantly, while Wnt5a, LRP5, ß-catenin and c-jun expressions increased. Culture of human articular chondrocytes with or without IL-1 not only induced chondrocyte de-differentiation, but also changed the expression profiles of Wnt components, which suggests that the Wnt pathway is involved in chondrocyte de-differentiation in vitro.

The roles of canonical and non-canonical Wnt signaling in human de-differentiated articular chondrocytes.

Osteoarthritis is the most prevalent form of arthritis in the world and it is becoming a major public health problem. Osteoarthritic chondrocytes undergo morphological and biochemical changes that lead to de-differentiation. The involvement of signaling pathways, such as the Wnt pathway, during cartilage pathology has been reported. Wnt signaling regulates critical biological processes. Wnt signals are transduced through at least three intracellular signaling pathways including the canonical Wnt/ß-catenin pathway, the Wnt/Ca2 + pathway and the Wnt/planar cell polarity pathway. We investigated the involvement of the Wnt canonical and non-canonical pathways in human articular chondrocyte de-differentiation in vitro. Human articular chondrocytes were cultured through four passages with no treatment, or with sFRP3 treatment, an inhibitor of Wnt pathways, or with DKK1 treatment, an inhibitor of the canonical pathway. Chondrocyte-secreted markers and Wnt pathway components were analyzed using western blotting and qPCR. Inhibition of the Wnt pathway showed that the canonical Wnt signaling probably is responsible for inhibition of collagen II expression, activation of metalloproteinase 13 expression and regulation of Wnt7a and c-jun expression during chondrocyte de-differentiation in vitro. Our results also suggest that expressions of eNOS, Wnt5a and cyclinE1 are regulated by non-canonical Wnt signaling.

[Sudden death during sport activity in Tunisia: autopsy study in 32 cases]. / La mort subite au cours d'une activité sportive en Tunisie : à propos d'une série autopsique de 32 cas.

PURPOSE: To develop profile of the victims and to study circumstances, causes of death and autopsy findings. METHODS: Retrospective study of cases of sudden death in sport activity whose autopsy was performed in forensic department of Tunis, between January 2005 and December 2009. RESULTS: During study period, 32 cases of SD in sport activity were recorded. These are amateur athletes predominantly male (84% of cases). Victims are aged between 15 and 79 years with an average age of about 33.16 years. Young subjects whose age is less than 35 years representing 68.7% of cases. 9.3% of victims had a family history of SD and 18.7% of cases had a known cardiac history. The sports most involved are running (40.6% of cases), football (31.3% of cases) and dance (12.5% of cases). Sixty-nine percent of victims died during sports activities. Presence of witnesses was noted in all cases; however, none of these witnesses has begun resuscitation. Cause of death was cardiac in 84.4% of cases. In young athletes, hypertrophic cardiomyopathy is the leading cause (nine cases), followed by arrhythmogenic right ventricular dysplasia (three cases). Among other causes, there is the myocardial bridge, congenital anomalies of the coronary arteries, aortic dissection and dilated cardiomyopathy. Beyond 35 years, coronary artery diseases represent the cause of death (nine cases). Only case of death secondary to non-cardiac disease occurred after a severe asthma attack. In four cases (12.5%), no cause of death was identified after a complete autopsy accompanied by further investigations. The cause of the death was imputed to a rhythmic pathology. CONCLUSION: This is the first study dealing with autopsy in SD in sport have provided a specific profile of victims. Other studies on larger samples and using standardized autopsy protocols are needed.

Expression patterns of Notch receptors and their ligands in human osteoarthritic and healthy articular cartilage.

Notch pathway plays a pivotal role in cell fate determination. There is much interest surrounding its therapeutic potential, in osteoarthritis, but the expression profile of Notch-related molecules, as well as their relation with cartilage pathological parameters, remains unclear. The purpose of our study is to analyze the expression pattern of Notch family members, type II and type I collagen, in normal (healthy) and osteoarthritic human knee cartilage. Osteoarthritic cartilages were obtained from 3 patients undergoing a total knee replacement. Macroscopically normal cartilage was dissected from 3 human knees at the time of autopsy or surgery. Immunohistochemical staining was performed using Notch1,2,3 and 4, Delta, Jagged, type II collagen and type I collagen antibodies. In healthy cartilage, type II collagen was abundantly expressed while type I was absent. This latter increased proportionally to the osteoarthritic grade. Type II collagen expression remained intense in osteoarthritic cartilage. In healthy cartilage as well as in cartilage with minor lesions, Notch family member's proteins were not or just weakly expressed at the surface and in the cells. However, Notch molecules were over-expressed in osteoarthritic cartilage compared to healthy one. This expression pattern was different according to the cartilage zone and the severity of OA. Our data suggest that Notch signaling is activated in osteoarthritic cartilage, compared to healthy cartilage, with a much more abundant expression in the most damaged areas.

[Rudimentary horn pregnancy: an unusual cause of spontaneous hemoperitoneum during the second-trimester of pregnancy]. / Grossesse dans une corne utérine rudimentaire : une cause rare d'hémopéritoine spontané au deuxième trimestre de grossesse.

Spontaneous hemoperitoneum during pregnancy is an unusual but serious event. We report a case of profuse spontaneous hemoperitoneum resulting from the rupture of a non-communicating rudimentary uterine horn at 26 weeks of gestation. An emergency laparotomy was performed and a 900 g birth weight safe infant was delivered. We performed a hemi-hysterectomy in the same time. Fetal and maternal outcomes resulted without complication. Causes of spontaneous hemoperitoneum are discussed.

Genetic identification of decomposed cadavers using nails as DNA source.

Blood or muscle can be used as a DNA source for the genetic identification of recently deceased persons. If the post mortem interval increases, bones and teeth are used. In this case, collection and DNA isolation will be more difficult and time consuming. The aim of this study was to evaluate the use of nails as an alternative DNA source for the genetic identification of decomposed cadavers. DNA extraction from 5mg of fingernails from 7 volunteers using 1h cell lysis in a standard buffer and a DNA purification on QIAamp DNA mini kit columns allowed to acquire a mean quantity of 100 ng DNA/mg nail. This was unexpected, as blood and muscle contain comparable amounts of DNA. Our protocol allowed to obtain full PowerPlex 16 DNA profiles from 10 cadavers characterized by post mortem intervals ranging from 5 days to more than 6 months. The good quality of these profiles indicated that DNA from nail is well preserved. In conclusion, nails are very easy to collect and contain large amounts of good quality DNA that can be extracted within a few hours. They may therefore represent an attractive DNA source not only for routine, but also for urgent genetic identification of decomposed cadavers.

[Anxiety and depressive disorders in 4,425 long term benzodiazepine users in general practice]. / Troubles anxieux et dépressifs chez 4 425 patients consommateurs de benzodiazépines au long cours en médecine générale.

Consumption rates of anxiolytic drugs, and especially of benzodiazepines, remain very high in France compared to other Western countries, whereas clinical guidelines limit their indications to short term treatments and only for some precise anxiety disorders. Recent epidemiologic surveys in the community indicated that more than 15% of people used once or more an anxiolytic drug in the past year. The issue of chronic treatments is particularly crucial because of their poor benefit/risk ratio in most anxiety disorders (limited efficacy, cognitive side effects, withdrawal and dependence problems). To address this important public health issue, and knowing that, in France, benzodiazepines are prescribed mainly by general physicians, our aims were to explore psychiatric diagnoses in GP's patients with chronic use of anxiolytic benzodiazepines. We included 4 425 patients consuming such drugs regularly for six months or more, and assessed their anxiety and depression symptoms through various clinical scales (Hospital Anxiety and Depressive scale - HAD, Clinical Global Impression scale - CGI, Sheehan Disability Scale - SDS, Cognitive Dependence to Benzodiazepines scale - CDB) and with the Mini International Neuropsychiatric Interview for DSM IV criteria. Only 2.2% of the subjects had neither anxious nor depressive symptoms as indicated by low scores on both subscores (less than 8) of the HAD scale, used as a screener. Nearly three quarters of the 4,257 subjects (73.2%), had CGI scores of at least 5 (markedly ill to extremely ill). Social and familial disability was also high in more than 40% of the sample (marked to extreme disruption according to SDS scores). About half of the sample had CDB scores suggesting a benzodiazepine dependence. According to the MINI, 85.1% of the patients had at least one current DSM IV diagnosis of affective disorder. The most frequent diagnoses were major depressive episode (60%), generalized anxiety disorder (61.2%), and panic disorder (22.5%). An anxiety and depressive comorbidity wad found in 41.9% of the subjects. Some methodological limitations must be taken into account in the discussion of our results, and especially the fact that the included patients were not supposed to be totally representative of all patients consuming anxiolytic benzodiazepines in general practice. However, the size of our sample is sufficiently large to limit possible biases in patient selection. The main result of this study is that a great majority of the patients had significant symptomatology, in particular major depressive episodes and generalized anxiety disorder, often with marked severity and disability. These data are in line with the knowledge of a lack of efficacy of benzodiazepines in depressive and most anxiety disorders, despite long term treatment. They also confirm the current guidelines which recommend prescribing serotoninergic antidepressants, and not benzodiazepines, when long term treatments are needed for severe and chronic affective disorders. This epidemiologic study leads to the conclusion that a specific and attentive diagnostic assessment should be done in all patients receiving benzodiazepines for more than three months, in order to purpose in many cases other long term therapeutic strategies.

A crosstalk between the Wnt and the adhesion-dependent signaling pathways governs the chemosensitivity of acute myeloid leukemia.

Relapses following chemotherapy are a major hindrance to patients' survival in acute myeloid leukemia (AML). To investigate the role of the hematopoietic niche in the chemoresistance of leukemic cells, we examined two pathways: one mediated by adhesion molecules/integrins, and the other by soluble factors of the morphogen Wnt pathway. In our study, both the adhesion of leukemic blasts to fibronectin and the addition of Wnt antagonists induced, independently, resistance of AML cells to daunorubicin in a cell survival assay. Using pharmacological inhibitors and siRNA, we showed that both resistance pathways required the activity of the glycogen synthase kinase 3beta (GSK3beta). Moreover, the AML cell protection downstream of GSK3beta was mediated by NF-kappaB. A link between the adhesion and the Wnt pathway was found, as adhesion of U937 on human osteoblasts, a component of the hematopoietic niche, triggered the secretion of the Wnt antagonist sFRP-1 and supported resistance to daunorubicin. The osteoblast-conditioned medium could also confer chemoresistance to U937 cells cultured in suspension, and this cell protective effect was abrogated after depletion of sFRP-1. In the context of this potential double in vivo resistance, modulators of the common signal GSK3beta and of its target NF-kappaB could represent important novel therapeutic tools.

Establishment of a novel anaplastic large-cell lymphoma-cell line (COST) from a 'small-cell variant' of ALCL.

Anaplastic large-cell lymphoma (ALCL) is a distinct biological and cytogenetic entity with a broad spectrum of morphological features (common type, small-cell variant and lymphohistiocytic variant). Few cell lines of ALCL are available and they all originate from primary tumors demonstrating the common type morphology (ie large-sized lymphoma cells). We established a new ALCL cell line (COST) from the peripheral blood of a patient with a small-cell variant of ALCL, at diagnosis. Cells growing in vitro and in SCID mice consisted of two populations, that is, small- and large-sized cells as seen in the patient's tumor. Both large and small malignant cells were positive for CD43/MT1 T-cell associated antigen, perforin, granzyme B and TIA-1, but negative for CD2, CD3, CD5, CD7, CD4 and CD8 antigens. Standard cytogenetic studies as well as multiplex FISH confirmed the presence of the canonical t(2;5)(p23;q35) translocation, but also revealed additional numerical and structural abnormalities. The COST cell line is the first ALCL small-cell variant cell line, and thus provides a potentially useful tool for further functional and molecular studies that should improve our understanding of the small-cell variant of ALCL, which is more frequently complicated by a leukemic phase.

Expression of the oncogenic NPM-ALK chimeric protein in human lymphoid T-cells inhibits drug-induced, but not Fas-induced apoptosis.

Anaplastic large cell lymphomas (ALCLs) are frequently associated with the t(2;5)(p23;q35) translocation, leading to the expression of NPM-ALK, a fusion protein linking nucleophosmin and anaplastic lymphoma kinase, a receptor tyrosine kinase. In ALCLs, dimerization of NPM-ALK leads to constitutive autophosphorylation and activation of the kinase, necessary for NPM-ALK oncogenicity. To investigate whether NPM-ALK, like other oncogenic tyrosine kinases, can inhibit drug-induced apoptosis, we permanently transfected NPM-ALK into Jurkat T-cells. As in ALCLs, NPM-ALK was expressed as a constitutively kinase-active 80 kDa protein, and could be detected by immunocytochemistry in nucleoli, nuclei and cytoplasm. Doxorubicin-induced apoptosis (assessed by cell morphology and annexin V-FITC binding) was significantly inhibited in two independent NPM-ALK-expressing clones (5.2+/-1.8 and 7.5+/-0.8% apoptosis), compared to control vector-transduced cells (36+/-6.7%). Similar results were observed with etoposide. In contrast, Fas-induced apoptosis was not inhibited. Cytochrome c release into the cytosol was delayed in doxorubicin-, but not anti-Fas-treated transfectant cells, indicating that apoptosis inhibition occurred upstream of mitochondrial events. Using NPM-ALK mutants, we demonstrated that inhibition of drug-induced apoptosis: (1) requires functional kinase activity, (2) does not involve phospholipase C-gamma, essential for NPM-ALK-mediated mitogenicity and (3) appears to be phosphoinositide 3-kinase independent, despite a strong Akt/PKB activation observed in wild type NPM-ALK-expressing cells. These results suggest that the NPM-ALK antiapoptotic and mitogenic pathways are distinct.

Ligation of the CD44 adhesion molecule inhibits drug-induced apoptosis in human myeloid leukemia cells.

Adhesion molecules can improve hematopoietic cell survival; however, their role in leukemic cell resistance to drug-induced apoptosis is poorly documented. The CD44 adhesion molecule is strongly expressed on acute myeloid leukemia (AML) blasts. Using 2 myeloid cell lines, HL60 and NB4, evidence is presented that prior incubation with the CD44-specific monoclonal antibody (mAb) A3D8, reported to induce differentiation of AML blasts, significantly decreases apoptosis induced by 3 drugs used in AML chemotherapy: daunorubicin (DNR), mitoxantrone, and etoposide. In addition, in HL60 cells, CD44 ligation with A3D8 mAb fully abrogates the DNR-triggered generation of ceramide, a lipid second messenger involved in the DNR apoptotic signaling pathway. Moreover, results show that the A3D8 mAb and Bcl-2 additively inhibit DNR-induced apoptosis in HL60 cells overexpressing Bcl-2. These results suggest that, to eradicate AML blasts, the differentiation-inducing anti-CD44 mAb A3D8 should not be administered prior to apoptosis-inducing drugs.

Influence of Bcl-2 overexpression on the ceramide pathway in daunorubicin-induced apoptosis of leukemic cells.

We have previously demonstrated that daunorubicin (DNR) induces apoptosis in some leukemic myeloid cell lines. We investigated a potential protective role for Bcl-2 in apoptosis induced by DNR in two leukemic cell lines, one myeloid and one lymphoid, overexpressing the anti-apoptotic gene Bcl-2. Parental cells treated with DNR exhibited classical features of apoptosis 6 h after drug exposure, all the cells being dead after 30-48 h. In contrast, overexpression of Bcl-2 significantly delayed, but did not prevent the occurrence of DNR-induced apoptosis, with no surviving cells 96 h after drug exposure. To elucidate the mechanism of the protection mediated by Bcl-2, we explored the signaling pathway which initiates DNR-induced apoptosis. In this report, we show that, in both the myeloid and lymphoid parental cell lines, DNR triggered a sphingomyelin (SM) hydrolysis after 10-15 min with a concomitant ceramide generation. Moreover, exogenous ceramide induced DNA fragmentation in these cells, with levels similar to those observed with DNR treatment. In contrast, Bcl-2 overexpression protected the cells against apoptosis induced by ceramide treatment, without preventing the early SM hydrolysis nor the ceramide generation in these cells. Our results strongly suggest that Bcl-2-mediated protection of DNR-induced apoptosis is effected downstream of the SM-ceramide signaling pathway.

Increased gadd153 messenger RNA level is associated with apoptosis in human leukemic cells treated with etoposide.

Treatment of leukemic cells with topoisomerase inhibitors can lead to growth arrest and subsequent apoptotic cell death. The relationships between cell cycle regulation and apoptosis triggering remain poorly understood. The gadd153 gene encodes the nuclear protein CHOP 10 that acts as a negative modulator of CCAAT/enhancer binding protein transcriptional factors and inhibits cell cycle progression. We have investigated the relationships between gadd153 gene expression and apoptosis induction in four human leukemic cell lines with different sensitivities to apoptosis induced by etoposide (VP-16), a topoisomerase II inhibitor. The gadd153 gene was constitutively expressed in the four studied cell lines. In U937 and HL-60 cells that were very sensitive to apoptosis induction by the drug, VP-16 induced a time- and dose-dependent increase of gadd153 gene mRNA expression. Using agarose gel electrophoresis and a quantitative filter elution assay, apoptotic DNA fragmentation was observed to begin when gadd153 gene expression increased. Equitoxic doses of VP-16 (as defined using a 96-h 3-4,5-dimethylthiazol-2,5-diphenyltetrazolium bromide assay) did not increase the gadd153 mRNA level in K562 and KCL22 cell lines that were more resistant to apoptosis induction by the drug. Nuclear run-on and mRNA stability experiments demonstrated that VP-16 treatment increased gadd153 gene transcription in the sensitive U937 cells. Cycloheximide did not prevent gadd153 expression increase. Both gadd153 mRNA level increase and internucleosomal DNA fragmentation were inhibited by N-tosyl-L-phenylalanine chloromethylketone, a serine threonine protease inhibitor, N-acetyl-leucyl-leucyl-norleucinal, an inhibitor of calpain, N-acetylcysteine, an inhibitor of oxidative metabolism, and overexpression of Bcl-2. Z-VAD and Z-DEVD peptides that inhibit interleukin 1beta-converting enzyme-like proteases suppressed DNA fragmentation without preventing gadd153 mRNA increase in VP-16-treated U937 cells. These results indicate that gadd153 gene expression increase occurs downstream of events sensitive to N-tosyl-L-phenylalanine chloromethylketone, calpain inhibitor I, and Bcl-2 and upstream of interleukin 1beta-converting enzyme-related proteases activation in leukemic cells in which treatment with VP-16 induces rapid apoptosis.

Autocrine IL-2-dependent growth of a newly established CD3+, CD16-, CD56+, CD57+, J(H)-, TCRbeta-, TCRgamma- leukemia cell line (NOI-90).

Leukemic cells from a 45-year-old male patient with a CD3+, CD56+, CD57+, CD7+ acute lymphoblastic leukemia were cultured in vitro in the absence of any added growth factor for up to 6 years and a continuous lymphoblastoid cell line (NOI-90) was established. NOI-90 cells have the same phenotype and karyotype as initial leukemic cells. Southern blot of DNA from NOI-90 cells showed that TCRbeta, TCRgamma, and J(H) were in germ line. Two and 25% of NOI-90 cells were positive when stained with the IOT14 and 7G7/B6 moAbs, which recognize the CD25 molecule (IL-2R alpha chain); moreover, 4% and 13% of the cells were positive when stained with the TU-27 and mik beta3 moAbs which recognize the CD122 molecule (IL-2Rbeta chain). Equilibrium binding experiments with radiolabelled IL-2 revealed the presence of a small number of high affinity IL-2R on both fresh and continuously growing cells. Media conditioned by NOI-90 cells could induce proliferation of an IL-2-dependent cell line and this IL-2 activity could be detected by a sensitive immunoenzymatic assay using antibodies recognizing distinct epitopes of IL-2. Moreover, IL-2 activity could be adsorbed by immunoaffinity on anti-IL-2 polyclonal purified IgG and the retained molecule displayed a m.w. of 14.5 kDa in SDS-PAGE. In addition, IL-2 immunoreactive molecules could be revealed in the cytoplasm of the cells. Finally, IL-2 fixed on the cell membrane could be detected by indirect immunofluorescence. Although added IL-2 could not induce cell proliferation, monoclonal antibodies against CD25, CD122 and IL-2 could specifically inhibit spontaneous cell proliferation in a dose-dependent manner. NOI-90 cells failed to demonstrate any cytotoxic activity against the K-562, Raji or Daudi cells. These findings indicate that NOI-90 cells are of non-T, non-B, origin lacking NK activity but proliferate under an autocrine pathway which involves, at least partly, the IL-2/IL-2R system.