Francis Belloc

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.

Freshwater adaptation in the euryhaline teleost, Chelon labrosus. I. Effects of adaptation, prolactin, cortisol and actinomycin D on plasma osmotic balance and (Na+-K+)ATPase in gill and kidney.

Abstract Microsomal (Na + K + )ATPase activities in gills and kidneys of thick-lipped mullet, Chelon labrosus , which had been gradually acclimated to fresh water were studied using initial velocity measurements. In mullet transferred to fresh water, the branchial ATPase activity rises gradually during a 2-week period (long-term fresh water adaptation: L-TFW). The renal ATPase activity rises rapidly after the transfer to fresh water (FW). In the L-TFW mullet and in seawater (SW) mullet, cortisol injections cause no, or only slight, stimulation of renal or branchial ATPase activity; on the other hand, cortisol causes a marked decrease in ATPase activity in mullet adapted to FW for only a short period of time (S-TFW). Only branchial ATPase, and not kidney ATPase, is inhibited by prolactin, and this only in S-TFW and SW mullet. Actinomycin D does not inhibit renal ATPase activity, but the response of branchial ATPase to this antibiotic depends on the environment; SW-acclimated mullet are sensitive to actinomycin D but the longer mullet are acclimated to FW the more insensitive they become. These findings may indicate that more than one functional form of gill (Na + +K + )ATPase exists, and that these vary in activity with external salinity.

CYTOMETRIC STUDY OF INTRACELLULAR P-GP EXPRESSION AND REVERSAL OF DRUG RESISTANCE

Expression of the multidrug resistance (MDR) phenotype is responsible for chemotherapy failure in numerous cancers. This phenotype is generally due to the expression of the mdr1 gene-encoded P-gp. Modulation of P-gp activity by chemotherapy has limited possibilities because of toxicity and poor specificity. In contrast, specific transcription blockage of the mdr1 gene can be obtained by oligonucleotides forming a triple helix structure at the DNA level. We used here immunofluorescence and both flow cytometry and image analysis to evaluate surface and total P-gp content in K562 MDR cells. The mdr1 mRNA content was measured by RT-PCR. We confirm the capacity of a 27-mer oligodeoxynucleotide, targeted to an mdr1 DNA fragment, to cause a 10-fold decrease in mdr1 mRNA level. However, this specific genetic inhibition was functionally limited because cellular growth was not modified in a cytotoxic environment. We found that total P-gp content was reduced in resistant cells treated with the mdr1-targeted oligonucleotide, while it remained in high levels on the cell surface, suggesting the existence of a large cytoplasmic pool of P-gp (approximately 50% of the total cellular P-gp). Moreover, when cycloheximide was used for 72 h to suppress protein synthesis, surface P-gp expression showed no decrease, whereas total P-gp was considerably lowered. A rapid 35% decrease in surface P-gp level was reached when resistant cells were treated for 24 h with brefeldin A, an inhibitor of intracellular protein trafficking. Simultaneously, the total P-gp level remained stable, thus indicating a probable accumulation of cytoplasmic P-gp, in agreement with the interruption of protein migration. We propose that the cytoplasmic P-gp pool could be a storage pool consumed for maintaining a steady-state level of surface P-gp. Cytometry could be a useful tool to study such a mechanism of P-gp trafficking and cellular distribution, which could explain the difficulties encountered in achieving stable and rapid effects of MDR reversal with oligonucleotides.

Flow cytometric study of idarubicin and daunorubicin accumulation and the effect of verapamil in leukemic cell lines and fresh cells from patients with acute non-lymphoblastic leukemia

By using flow cytometry, the intracellular accumulation (Acc) of idarubicin (IDA) and daunorubicin (DNR) and the effect of verapamil (VRP) on both anthracycline accumulation (VRP index) were studied in leukemic cell lines (K562 and HL60 and their two DNR-resistant subclones) and fresh leukemic cells. IDA accumulated more than DNR in both parental (K562: p < 0.03 and HL60: 0.09) and resistant cell lines (p < 0.01 for both cell lines) irrespective of whether or not they were treated with VRP. VRP index was higher for DNR than for IDA (p < 0.05). Similar results were observed in fresh leukemic blasts from 25 patients with ANLL (IDA Acc superior to DNR Acc: p < 0.0001; higher VRP index for DNR than for IDA: p < 0.01). The higher Acc of IDA than DNR seen in fresh leukemic cells could explain the better clinical efficacy of IDA reported in patients with ANLL.

Freshwater adaptation in the euryhaline teleost, Chelon labrosus ☆: II. Effects of continuance of adaptation, cortisol treatment, and environmental calcium on water influx in isolated gill

Abstract The effects of freshwater adaptation duration, cortisol, and environmental calcium on water influx in isolated gill of the thick-lipped gray mullet, Chelon labrosus , were studied using an in vitro technique. An estimation of the water permeability and the osmotic permeability coefficient in this euryhaline fish was made from the rate of weight increase in isolated gills immersed in deionized water after preincubation in saline solution. The water influx in the isolated gills and/or permeability is reduced progressively with the adaptation to fresh water in C. labrosus . Cortisol increases the water influx and/or water permeability only in seawater-adapted fish. An increased calcium content of fresh water reduces the weight increase of isolated gill. These results are in accord with the osmoregulatory performance characteristic of this fish.

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.

Fluorescent in situ hybridization on flow‐sorted cells as a tool for evaluating minimal residual disease or chimerism after allogeneic bone marrow transplantation

We studied the feasibility and the sensitivity of fluorescent in situ hybridization (FISH) using leukemic or host/donor-specific probes on flow-sorted cells to assess minimal residual disease (MRD) or chimerism in transplanted patients in complete remission. We first performed experimental models of MRD and chimerism by mixing HL60 cells and normal lymphocytes in different proportions. Over 80% HL60 cells were obtained from mixtures of 5% HL60 cells in peripheral blood mononuclear cells (PBMC). We then evaluated MRD and mixed chimerism in a chronic myelogenous leukemia patient in relapse after allogeneic sex-mismatched bone marrow transplantation (BMT), who had received a donor lymphocyte infusion (DLI). Three months after DLI, mixed chimerism was observed in each bone marrow (BM)-sorted lineage (CD13+, CD14+, CD20+, and CD3+), with the highest level of recipient cells in the granulocytic lineage (CD13+). Five months after DLI, host cells were at a low level but remained detectable in the granulocytic lineage. In the same sample, the bcr-abl gene was detected in the granulocytic lineage and not in the lymphocytes. We also studied chimerism in an aplastic anemia sex-mismatched transplanted female patient. We determined the proportion of recipient total lymphocytes, CD4+ and CD8+ lymphocytes, and CD14+ monocytes under cyclosporin A therapy on five peripheral blood samples and one BM sample over 5 months. Results showed a regular decrease in recipient total lymphocytes (26.6% to 10.6%) and monocytes (20.7% to 8%). CD8(+)-recipient cells decreased rapidly, while CD4+ remained stable (17%). This work demonstrates the feasibility of FISH after cell sorting, combining the sensitivities of both flow cytometry and FISH and the specificities of both immunophenotyping and genotype analysis.

Freshwater adaptation in the euryhaline teleost, Chelon labrosus—IV. Changes of DNA and protein contents, RNA/DNA ratio and acid phosphatase activity in the branchial tissue

Abstract 1. 1. Acid phosphatase activity, as an index of lysosomal activity, of the cytoplasmic fraction of gill tissue transiently increases during the FW adaptation of the mullet, Chelon labrosus . 2. 2. A concomitant and unexpected DNA decrease occurs. Moreover, RNA/DNA ratio in the nucleimitochondria fraction increases with the continuance of FW adaptation, while the DNA renewal of gill tissue is enhanced in the fully adapted FW mullet. 3. 3. The use of histologic techniques indicates that chloride cells are probably concerned with AcPase and DNA changes. 4. 4. These results are discussed with regard to the renewal and differentiation of two chloride cell populations. In addition, a model for the control of chloride cell populations in gill epithelium of C. labrosus , is suggested.

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.

Fresh water adaptation in the euryhaline teleost, Chelon labrosus—III Biochemical characterization and increase in the acid phosphatase activity in gill

Abstract Abstrac 1. 1. In the gill of the euryhaline teleost, Chelon labrosus , the basic characteristics of acid phosphatase activity, a “lysosomal” enzyme, are similar to those of the other vertebrates. 2. 2. The pH optimum is 4.75; the enzyme activity reaches a plateau from pH 3.8 to pH 5.0; at higher pH levels, enzyme activity rapidly declines. 3. 3. Normal kinetic patterns of enzyme activity occur when substrate, enzyme concentration and time of incubation in enzyme assays are varied. The ions (Na + K + , Cl − , Ca 2+ and Mg 2+ ) and chelators (EGTA, EDTA). do not affect the enzyme activity. Decrease (12% to 25%) in in vitro enzyme activity occurs after one day at 0°-4°C. 4. 4. Acid phosphatase activity, found above all 50,000 g × 1 hr fraction and moved in slighter subcellular fraction after in vitro osmotic shock, is probably localyzed within the lysosomes. 5. 5. The specific activity increases when fish is transferred from sea water to freshwater.