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F Belloc

Publications and source records attributed to F Belloc.

At least 19 recordsLinked to original sources
PubMed2006

Remission induction chemotherapy induces in vivo caspase-dependent apoptosis in bone marrow acute myeloid leukemia blast cells and spares lymphocytes.

BACKGROUND: The goal of new therapeutic strategies is to adapt the treatment of acute myeloid leukemia (AML) patients to the prognostic and/or to the hematological response. METHODS: We analyzed in vivo apoptosis induction in blast cells and in lymphocytes of AML patients receiving remission induction treatment. RESULTS: We show, on 12 peripheral blood samples, that the increase of peripheral apoptotic blast cells cannot be considered as the earliest marker of the treatment efficiency, because the significant increase of apoptosis followed the white blood cell and the peripheral blast cell count reductions, probably due to an efficient clearance of circulating apoptotic cells. Furthermore, the study of 65 bone marrow samples at d15 showed that the treatment induced apoptosis of blast cells while sparing the lymphocytes. This apoptosis was evidenced both at the caspase and at the membrane levels using respectively fmk-VAD-FITC and Annexin V binding assays. We found that less than 50% of apoptosis, measured with the fmk-VAD-FITC, in the d15 residual bone marrow blast cells, correlated with lower disease-free survival probability. CONCLUSION: More studies are needed in larger series and earlier during the remission induction treatment to confirm the possible prognostic significance of in vivo apoptosis induction.

Acute Disease↗
PubMed2002

Nitric oxide induces the apoptosis of human BCR-ABL-positive myeloid leukemia cells: evidence for the chelation of intracellular iron.

Anti-leukemia activity of human macrophages involves the generation of nitric oxide (NO) derivatives. However, leukemic transformation may involve mechanisms that rescue cells from NO-mediated apoptosis. In the present work, we analyzed the effects of exogenous NO on the proliferation of BCR-ABL(+) chronic myelogenous leukemia (CML) cells. As normal leukocytes, the proliferation of leukemia cells was inhibited by SNAP (S-nitroso-N-acetyl-penicillamine), GEA (Oxatriazolium amino-chloride), and SIN-1 (Morpholino-sydnonimine), whereas SNP (sodium nitroprusside) had no effect on leukemia cell growth. SIN-1 induced higher anti-proliferation activity in BCR-ABL(+) cells, compared to normal hemopoietic cells. Inhibition of leukemia cell proliferation correlated with increased apoptosis and DEVDase activity. The simultaneous addition of exogenous iron reversed NO-mediated inhibition of cell growth, caspase activation and apoptosis in all BCR-ABL(+) cells tested. The quantification of intracellular iron levels in leukemia cells indicated that NO induced an early, dose-dependent decrease in ferric iron levels. Accordingly, elevation of intracellular iron protected leukemia cells from NO-mediated apoptosis. Together, the present work reveals the presence of an iron-dependant mechanism for leukemia cell rescue from NO-induced growth inhibition and apoptosis.

Apoptosis↗
PubMed2002

Resistance to daunorubicin-induced apoptosis is not completely reversed in CML blast cells by STI571.

The leukemogenic property of BCR-ABL in chronic myeloid leukemia (CML) is critically dependent on its protein tyrosine kinase activity. STI571 inhibits the BCR-ABL kinase activity, the growth and the viability of BCR-ABL expressing cells. In this study, we report the apoptotic effect of STI571 in combination with daunorubicin (DNR) on peripheral blood mononuclear cells from 11 CML patients and four BCR-ABL-positive cell lines: AR230, LAMA84, K562 and KCL22. Primary blast cells were identified by flow cytometry on the basis of their low CD45 expression. Nucleus fragmentation, exposure of phosphatidylserines and decrease in mitochondrial membrane potential were measured using acridine orange, FITC-annexin V and DiOC6(3), respectively, to evaluate apoptosis. On cell lines, the effect of DNR was negligible, whereas STI571 induced 10 to 35% of apoptosis in 18 h. STI571 sensitized AR230, LAMA84 and K562 cells to DNR when apoptosis was measured at the mitochondrial and membrane but not the nuclear levels. On CML blast cells, phosphatidyl serine exposure was significantly induced by both DNR and STI571 and was higher when these drugs were used in combination (P < 0.0003). However, the effects of this drug combination were only additive and no sensitization of blast cells to DNR by STI571 was observed. Interestingly, sensitization was evidenced in CML but not normal lymphocytes. These results suggest that other mechanisms additional to Bcr-Abl tyrosine kinase activity could be responsible for DNR resistance, and further investigations are needed to understand its origin.

Antineoplastic Agents↗
PubMed2001

[Physicopathologic mechanisms and methods of analysis of cellular apoptosis].

Apoptosis is a normal process occurring during development and in various tissues in humans. It appears that the mechanisms responsible for apoptosis are implicated in many aspects of human diseases. The apoptotic answer is in fact the integration of multiple different and complex signalization pathways which communicate, bifurcate and self-regulate. The mitochondria take an essential place in the description of programmed cell death and its regulation mechanisms. Caspases are the effector of apoptotic cell death. The methods of identification of the apoptosis pathways are: morphological modifications observed in microscopy, the evaluation of the difference of the mitochondrial membrane potential, the measurement of the DEVDase activity, the labelling of the phosphatidylserines by the annexin V on the cell surface, and the Western blot allowing the identification of the activated caspases. Apoptosis is implicated in many pathologies. A better understanding of the mechanisms of apoptosis and tissue specificity of the caspases make it possible to consider in the future the development of synthetic inhibitors as serious candidates for a therapeutic intervention.

Apoptosis↗
PubMed2001

[Molecular mechanisms controlling the cell cycle: fundamental aspects and implications for oncology].

INTRODUCTION: Comprehension of cell cycle regulation mechanisms has progressed very quickly these past few years and regulators of the cell cycle have gained widespread importance in cancer. This review first summarizes major advances in the understanding of the control of cell cycle mechanisms. Examples of how this control is altered in tumoral cells are then described. CURRENT KNOWLEDGE AND KEY POINTS: The typical mammalian cell cycle consists of four distinct phases occurring in a well-defined order, each of which should be completed successfully before the next begins. Progression of eukaryotic cells through major cell cycle transitions is mediated by sequential assembly and activation of a family of serine-threonine protein kinases, the cyclin dependent kinases (CDK). The timing of their activation is determined by their post-translational modifications (phosphorylations/dephosphorylations), and by the association of a protein called cyclin, which is the regulatory subunit of the kinase complex. The cyclin family is divided into two main classes. The 'G1 cyclins' include cyclins C, D1-3, and E, and their accumulation is rate-limiting for progression from the G1 to S phase. The 'mitotic or G2 cyclins', which include cyclin A and cyclin B, are involved in the control of G2/M transition and mitosis. The cyclins bind to and activate the CDK, which leads to phosphorylation (and then inhibition) of the tumor suppressor protein, pRb. pRb controls commitment to progress from the G1 to S phase, at least in part by repressing the activity of the E2F transcription factors known to promote cell proliferation. Both the D-type cyclins and their partner kinases CDK4/6 have proto-oncogenic properties, and their activity is carefully regulated at multiple levels including negative control by two families of CDK inhibitors. While members of the INK4 family (p16INK4A, p15INK4B, p18INK4C, p19INK4D) interact specifically with CDK4 and CDK6, the CIP/KIP inhibitors p21CIP1/WAF1, p27KIP1 and p57KIP2 inhibit a broader spectrum of CDK. The interplay between p16INK4A, cyclin D/CDK, and pRb/E2F together constitute a functional unit collectively known as the 'pRb pathway'. Each of the major components of this mechanism may become deregulated in cancer, and accumulating evidence points to the 'pRb pathway' as a candidate obligatory target in multistep oncogenesis of possibly all human tumor types. FUTURE PROSPECTS AND PROJECTS: Major advances in the understanding of cell cycle regulation mechanisms provided a better knowledge of the molecular interactions involved in human cancer. This progress has led to the promotion of new therapeutic agents presently in clinical trials or under development. Moreover, the components of the cell cycle are probably involved in other non-cancerous diseases and their role must be defined.

Animals↗
PubMed2000

Flow cytometry detection of caspase 3 activation in preapoptotic leukemic cells.

BACKGROUND: Procaspase 3 is a constitutive proenzyme that is activated by cleavage during apoptosis. The resulting enzyme is able to cleave several target proteins after the second aspartate of a DEVD sequence common to all the substrates of caspases 3 and 7 (DEVDase). Because active caspase 3 is a common effector in several apoptotic pathways, it may be a good marker to detect (pre-)apoptotic cells by flow cytometry (FCM). Materials and Methods Apoptosis was induced in U937 or bone marrow mononuclear cells by daunorubicin (DNR), idarubicin (IDA), or camptothecin (CAM). Viable and apoptotic cells were sorted by FCM on the basis of either fluorescein isothiocyante (FITC)-annexin V binding or DiOC6(3) accumulation. DEVDase activity was measured in sorted populations by spectrofluorometry. Cleaved caspase 3 was labeled in situ with phycoerythrin (PE)-conjugated anti-activated caspase 3 antibodies and analyzed by FCM. RESULTS: DEVDase activity was detected in sorted viable CAM- and DNR-treated U937 cells, demonstrating that a partial caspase activation occurred earlier than phosphatidyl-serine exposure and mitochondrial membrane potential dissipation. The presence of a low amount of active caspase 3 in the treated viable cells was confirmed in situ with PE-conjugated anti-active caspase 3 antibodies. The same antibody was used in combination with FITC-annexin V and CD45-PC5 to study caspase 3 activation in acute leukemia blast cells after in vitro DNR and IDA treatment. Both anthracyclines induced a caspase 3-dependent apoptosis that was more efficient in blast cells than in contaminating lymphocytes. CONCLUSIONS: These results demonstrate that anti-active caspase 3 labeling can be an alternative to fluorogenic substrates to efficiently detect early apoptosis by FCM in heterogeneous samples. They also confirm that anthracyclines induce blast cell apoptosis by a caspase 3-dependent pathway.

Adult↗
PubMed2000

Flow cytometric evaluation of fas expression in relation to response and resistance to anthracyclines in leukemic cells.

BACKGROUND: Cell chemosensitivity to cytotoxic drugs has been attributed to their ability to trigger apoptosis. The emergence of resistance in drug-exposed cells is often characterized by the appearance of drug efflux mechanisms including P-gp transport. Nevertheless, mdr1 expression may coexist with other resistance features, in particular those interfering with apoptotic signaling pathways. METHODS: Leukemic cell lines cultured in a progressively toxic environment were analyzed for Fas and P-gp expression by immunostaining and flow cytometry. Their mdr1 mRNA expression level was determined by reverse transcriptase-polymerase chain reaction (RT-PCR), and their apoptotic response was microscopically evaluated. Activation of the Fas pathway was obtained by cross-linking the Fas receptor with the 7C11 anti-Fas agonist. RESULTS: We demonstrate a dose-dependent Fas overexpression after short-term (18 h) incubation with daunorubicin. The subsequent sensitization to Fas activators led to a significant increase in the apoptotic response induced by 7C11. After long-term exposure to daunorubicin and acquisition of drug resistance, expression of P-gp was accompanied by a decrease in the number of Fas sites at the cell surface with a correlated desensitization to Fas-induced apoptosis. Additional alterations in the Fas signaling pathway can also be hypothesized in the most resistant Jurkat cell line. CONCLUSIONS: The induction of Fas expression could be one of the mechanisms of action of chemotoxic drugs and thus might enhance the cell susceptibility to Fas-mediated apoptosis. On the contrary, the emergence of the multidrug resistance phenotype is associated with a down-regulation of Fas expression and possible defects in the Fas signaling pathway.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2000

Different expression profiles of human cyclin B1 in normal PHA-stimulated T lymphocytes and leukemic T cells.

BACKGROUND: In a previous work, we demonstrated with flow cytometry (FCM) methods that accumulation of human cyclin B1 in leukemic cell lines begins during the G(1) phase of the cell cycle (Viallard et al. , Exp Cell Res 247:208-219, 1999). In the present study, FCM was used to compare the localization and the kinetic patterns of cyclin B1 expression in Jurkat leukemia cell line and phytohemagglutinin (PHA)-stimulated normal T lymphocytes. METHODS: Cell synchronization was performed in G(1) with sodium n-butyrate, at the G(1)/S transition with thymidine and at mitosis with colchicine. Cells (leukemic cell line Jurkat or PHA-stimulated human T-lymphocytes) were stained for DNA and cyclin B1 and analyzed by FCM. Western blotting was used to confirm certain results. RESULTS: Under asynchronous growing conditions and for both cell populations, cyclin B1 expression was essentially restricted to the G(2)/M transition, reaching its maximal level at mitosis. When the cells were synchronized at the G(1)/S boundary by thymidine or inside the G(1) phase by sodium n-butyrate, Jurkat cells accumulated cyclin B1 in both situations, whereas T lymphocytes expressed cyclin B1 only during the thymidine block. The cyclin B1 fluorescence kinetics of PHA-stimulated T lymphocytes was strictly similar when considering T lymphocytes blocked at the G(1)/S phase transition by thymidine and in exponentially growing conditions. These FCM results were confirmed by Western blotting. The detection of cyclin B1 by Western blot in cells sorted in the G(1) phase of the cell cycle showed that cyclin B1 was present in the G(1) phase in leukemic T cells but not in normal T lymphocytes. Cyclin B1 degradation was effective at mitosis, thus ruling out a defective cyclin B1 proteolysis. CONCLUSIONS: We found that the leukemic T cells behaved quite differently from the untransformed T lymphocytes. Our data support the notion that human cyclin B1 is present in the G(1) phase of the cell cycle in leukemic T cells but not in normal T lymphocytes. Therefore, the restriction point from which cyclin B1 can be detected is different in the two models studied. We hypothesize that after passage through a restriction point differing in T lymphocytes and in leukemic cells, the rate of cyclin B1 synthesis becomes constant in the S and G(2)/M phases and independent from the DNA replication cycle.

Blotting, Western↗
PubMed2000

Mitochondrial impairment and recovery after heat shock treatment in a human microglial cell line.

The application of a heat shock on the human microglial cell line (CHME 5) has been shown to cause cytoskeleton modifications and alterations in phosphorylated metabolite content (Macouillard-Poulletier de Gannes et al., 1998a Metabolic and cellular characterization of immortalized human microglial cells under heat stress. Neurochem. Int. 33, 61-73). In this study, we focused on the possible involvement of mitochondria in this heat stress response. The cell respiratory properties were followed during the recovering period and the possible relationships between mitochondria and the cytoskeleton were studied. We observed that the heat shock induced changes in mitochondrial activity due to protein denaturation, rather than mitochondrial loss. Furthermore, these alterations were correlated with cytoskeleton disorganization since vimentine, tubuline and mitochondria shift, simultaneously, to a perinuclear location. The perturbations of the mitochondrial distribution persisted until cytoskeleton networks had recovered. Nevertheless, the respiratory properties recovered rapidly suggesting a renaturation of mitochondrial proteins in connection with mitochondrial cytoplasmic redistribution.

Actins↗
PubMed2000

Homocysteine-thiolactone induces caspase-independent vascular endothelial cell death with apoptotic features.

OBJECTIVE: Cell death is generally classified into two large categories: apoptosis, which represents active, physiological programmed cell death, and necrosis, which represents passive cell death without underlying regulatory mechanisms. Apoptosis plays an important role in tissue homeostasis and its role in endothelium integrity can be influenced by the functional status of endothelial cells. Homocysteine, a sulfated amino-acid product of methionine demethylation, is an independent risk factor for vascular disease (arterial and venous thombosis). Our goal was to investigate the thiol-derivatives effect on the endothelial cell apoptosis. METHODS: Three parameters were measured: mitochondrial membrane potential using DiOC6(3) as the probe, DEVDase activation, and phosphatidylserine exposure on the cell surface with fluorosceinated annexin V labeling which allows apoptosis to be distinguished from necrosis. RESULTS: Homocysteine-thiolactone induced endothelial cell apoptosis in a concentration-dependent manner (range: 50-200 microM), independently of the caspase pathway. Only homocysteine-thiolactone, among the thiol derivatives tested, induced apoptosis. Apoptosis was not influenced by the serum concentration in culture medium, suggesting that the observed apoptotic process could occur in vivo. None of the inhibitors used (e.g., leupeptin, fumosinin B1, catalase, or z-VAD-fmk) was able to prevent homocysteine-induced apoptosis of vascular endothelial cells. CONCLUSION: The apoptosis of vascular endothelial cells induced by high concentration of homocysteine-thiolactone might be one step atherosclerotic cardiovascular disease, and contribute to its complication.

Apoptosis↗
PubMed2000

Early transitory rise in intracellular pH leads to Bax conformation change during ceramide-induced apoptosis.

Ceramide can induce apoptosis through a caspase independent pathway. Bax has been described as able to kill cells in the absence of caspase activity, therefore we measured Bax in situ during ceramide-induced apoptosis using anti-Bax antibodies and flow cytometry analysis. An early (<30 min) increase in Bax labeling was observed after the addition of several ceramide species to several hemopoietic-related cell types. On U937, this increase was not due to antigens synthesis or processing, but rather an increased accessibility or reactivity of Bax antigens for antibodies. This increased immuno-reactivity of Bax was not inhibited by Z-VAD-fmk nor leupeptin, and preceded nuclear fragmentation by several hours. Such an increase in immuno-reactivity was also observed after Fas ligation, but it occurred later (>2 h) accompanying nuclear apoptosis, and was inhibited by Z-VAD-fmk. Bax immuno-reactivity was found to be related to intracellular pH (pHi), and C2-Ceramide (C2-Cer) induced a very early (<10 min) transitory increase in pHi. Both Bax immunoreactivity and pHi increases were dependent on the mitochondrial permeability transition pore (PTP) status. It was concluded from these results that C2-Cer induced a transitory increase in pHi in relation to the PTP. This rise in pHi led to conformational changes in Bax which could be responsible for further apoptosis in the C2-Cer pathway while it was a consequence of caspase activation in the Fas pathway.

Apoptosis↗
PubMed2000

Study of apoptosis-related responses of leukemic blast cells to in vitro anthracycline treatment.

Anthracyclines trigger an apoptotic cell death but their molecular targets are not totally explored. We investigated the apoptotic response of blast cells and lymphocytes from medullary samples of 31 de novo acute leukemia. Mononuclear cells were treated in vitro by therapeutic concentrations of either daunorubicin (DNR) or idarubicin (IDA) for 1 h, washed and cultured for 18 h. A multivariate analysis using flow cytometry and a CD45 gating on lymphocytes and blast cells was performed. DNR and IDA induced a Fas enhancement on both leukemic and normal cells. In blast cells the DEVDases were activated and the caspase 3 was cleaved in relation to phosphatidyl serine exposure, showing a caspase-dependent pathway in anthracycline-induced apoptosis. Apoptotic percentages were always higher for blast cells than for lymphocytes, confirming that anthracycline toxicity mainly affected tumor cells. Moreover, drug-induced apoptosis was not related to spontaneous apoptosis, suggesting that variations in response intensities were due to individual variations of sensitivity rather than to programmed life span time. The apoptotic response of P-glycoprotein-expressing blast cells was not significant, giving biological argument for the poor prognosis of multidrug resistance leukemia. Finally, Fas induction and anthracycline-induced apoptosis on blast cells were significantly higher when a complete remission was achieved, thus shedding light on potential new prognostic factors in acute leukemia.

Acute Disease↗
PubMed2000

Comparative methodologic study of NFkappaB activation in cultured endothelial cells.

The transcriptional regulatory protein nuclear factor kappaB (NFkappaB) participates in the control of gene expression of many modulators of the inflammatory and immune responses. Various activators trigger NFkappaB release and nuclear translocation after phosphorylation and proteolytic degradation of IkappaB. This study evaluated the abilities of fluorescence and confocal microscopies, laser scanning cytometry (LSC), electrophoretic mobility-shift assay (EMSA), and Western blotting to detect NFkappaB activation in endothelial cells (ECs) and to investigate the role of homocysteine (Hcy) in NFkappaB activation. ECs were treated with interleukin-1B (10 ng/mL) or Hcy thiolactone (1 and 5 mmol/L) as NFkappaB activators. Hcy, a thiol-containing amino acid, has been shown to directly damage ECs in vitro. Experimental evidence suggests that the atherogenic propensity associated with hyperhomocysteinemia results from EC dysfunction. When ECs were pretreated with an inhibitor (pyrrolidine dithiocarbamate, 100 micromol/L) or with staurosporine (5 microL/mL), no NFkappaB activation was observed. NFkappaB activation in ECs could be detected with all five techniques, clearly showing NFkappaB translocation from the cytoplasm to the nuclei. Confocal microscopy was more sensitive and less subjective than immunofluorescence microscopy. LSC was even more sensitive, specific, and reproducible. EMSA, the reference method, has the disadvantages of being radioactive, expensive, and time consuming. Western blot analysis detected the NFkappaB p50 subunit implicated in NFkappaB activation. The techniques usually used to detect NFkappaB activation in ECs are immunofluorescence microscopy and confocal microscopy, LSC, EMSA, and Western blot analysis, but none of them is ready for routine use.

Cells, Cultured↗
PubMed1999

Correction of uroporphyrinogen decarboxylase deficiency (hepatoerythropoietic porphyria) in Epstein-Barr virus-transformed B-cell lines by retrovirus-mediated gene transfer: fluorescence-based selection of transduced cells.

Hepatoerythropoietic porphyria (HEP) is an inherited metabolic disorder characterized by the accumulation of porphyrins resulting from a deficiency in uroporphyrinogen decarboxylase (UROD). This autosomal recessive disorder is severe, starting early in infancy with no specific treatment. Gene therapy would represent a great therapeutic improvement. Because hematopoietic cells are the target for somatic gene therapy in this porphyria, Epstein-Barr virus-transformed B-cell lines from patients with HEP provide a model system for the disease. Thus, retrovirus-mediated expression of UROD was used to restore enzymatic activity in B-cell lines from 3 HEP patients. The potential of gene therapy for the metabolic correction of the disease was demonstrated by a reduction of porphyrin accumulation to the normal level in deficient transduced cells. Mixed culture experiments demonstrated that there is no metabolic cross-correction of deficient cells by normal cells. However, the observation of cellular expansion in vitro and in vivo in immunodeficient mice suggested that genetically corrected cells have a competitive advantage. Finally, to facilitate future human gene therapy trials, we have developed a selection system based on the expression of the therapeutic gene. Genetically corrected cells are easily separated from deficient ones by the absence of fluorescence when illuminated under UV light.

Animals↗
PubMed1999

Synthesis of Bcl-2 in response to anthracycline treatment may contribute to an apoptosis-resistant phenotype in leukemic cell lines.

BACKGROUND: Some forms of chemoresistance in leukemia may start from failure of tumour cells to successfully undergo apoptosis and Bcl-2 may play a role in this defect. Therefore, we evaluated the Bcl-2 content and synthesis in relation with the apoptotic potential in leukemic cell lines after anthracycline treatment. METHODS: U937, HL60, and K562 cells and their drug resistant (DR) variants were treated with varying concentrations of Idarubicin (IDA). Apoptosis was evaluated by fluorescence microscopy after acridine orange staining. Bcl-2 and Bax content were evaluated either by flow cytometry after indirect immunolabelling or by Western blot. RESULTS: High Bcl-2 contents were not related to a poor ability to undergo apoptosis in U937, HL60, K562 and their DR variants. IDA induced a concentration-dependent increase in Bcl-2 content in all cell lines as long as they do not perform apoptosis. Enhanced Bcl-2 expression was inhibited by cycloheximide, actinomycin D, or antisense oligonucleotide directed against bcl-2 mRNA. Bcl-2 expression was also increased in the resistant U937 variant after serum deprivation or C2-ceramide treatment. The synthesis of Bcl-2 led to an increased Bcl-2/Bax ratio solely in the cells with an apoptosis-resistance phenotype. CONCLUSIONS: These data suggest that exposure to IDA induces Bcl-2 expression in leukemic cell lines, and that this mechanism could contribute to apoptosis resistance and participate in the acquisition of chemoresistance. They also confirm that the evolution of the Bcl-2/Bax ratio reflects apoptotic ability better than the steady state level of Bcl-2 expression.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1999

Polymorphonuclear cell apoptosis in exudates generated by polymers.

Flow cytometry was used to quantify apoptotic and necrotic polymorphonuclear (PMN) cells in an exudate generated by biomaterials, and the results were compared with determinations of spontaneous apoptosis and necrosis in PMN cells from the bloodstream. The exudate formed inside cylindrical tubes subcutaneously implanted in the dorsal region of rats was collected over a 1-week period. A rapid and simple staining procedure based on the spectral properties of the bisbenzemide Hoechst 33342 was used to identify apoptotic PMN cells. Quantification of permeabilized PMN cells stained by propidium iodide was possible in the same unfixed specimens. The percentages of apoptotic and permeabilized PMN cells in peripheral rat blood were low (1.8 +/-0 0.5% and 1.7 +/- 0.7%, respectively), similar to results found in humans. In exudates generated by polyvinyl chloride (PVC), the percentages of apoptotic and permeabilized PMN cells were higher than in the blood. The percentage of PMN cells undergoing apoptosis progressively increased with time and reached a maximum at day 2 (27% +/- 6%). The percentage of permeabilized cells progressively increased with time and was much higher than the percentage of apoptotic cells on days 4 and 8. Apoptosis and necrosis of PMN cells at day 2 were inhibited when tubes were filled with 10% serum. Selective inhibition of apoptosis with a caspase inhibitor in vivo indicated that apoptosis and necrosis are two separate pathways leading to the death of PMN cells in the exudate. At day 2, polyurethane (PU) was associated with a lower rate of apoptosis than PVC or a random copolymer of trimethylene carbonate (TMC) and epsiloncaprolactone (ECL). Apoptosis was interpreted as an organized cell removal process that limits inflammation. Apoptosis was the natural route of PMN cell death at the early stage of inflammation.

Animals↗
PubMed1999

Flow cytometry study of human cyclin B1 and cyclin E expression in leukemic cell lines: cell cycle kinetics and cell localization.

Experiments by flow cytometry (FCM) after nuclei isolation have never been done to investigate cyclins. We have conducted different experiments by FCM using whole cells and isolated nuclei to study the immunolocalization and kinetic patterns of cyclin B1 and cyclin E in various leukemic cell lines. During asynchronous growth, all whole cells had a scheduled, cell cycle phase-restricted expression of cyclin B1. By using a washless immunostaining of unfixed nuclei, cyclin B1 was detected in all cell cycle phases, including G1, although to a lesser extent than in G2/M, suggesting that in whole cells the cyclin B1 epitope is masked and accessible only in isolated nuclei. When the cells were synchronized at the G1/S boundary by thymidine or in the G1 phase by sodium n-butyrate, an identical accumulation of cyclin B1 was observed. As for cyclin E, its expression was higher with thymidine treatment than with sodium n-butyrate, particularly in nuclei. The elevated cyclin B1 level in the cells arrested at the G1/S boundary may reflect the increased half-life of this protein stabilized as the result of cyclin E overexpression. However, our FCM data also support the notion that accumulation of human cyclin B1 in leukemic cell lines begins during the G1 phase of the cell cycle, probably in the nucleus. The detection of cyclin B1 by Western blot in cells sorted in the G1 phase of the cell cycle confirms this finding. It is possible, therefore, that tumor transformation or leukemic phenotype may invariably be associated with altered cyclin B1 expression.

Blotting, Western↗