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.

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 cytometry CD45 gating for immunophenotyping of acute myeloid leukemia.

A flow cytometry method has been introduced into the routine investigation of whole bone marrow samples following red blood cell lysis on the basis of a primary CD45/side scatter (SSC) gating procedure. Blast cells were first identified by CD45/SSC gating in 74 cases of acute myeloid leukemia (AML) and the results were compared to a conventional FSC/SSC gating procedure and to MGG-staining smears. The percentages of blast cells in these samples as defined by the morphological analysis of MGG smears correlated better with the values determined by CD45/SSC gating (r = 0.94) than with the blast cell counts recorded with FSC/SSC gating (r = 0.76). These findings were not surprising because while CD45 expression was regularly lower on leukemic blasts than on normal lymphoid and monocytic cells, the FCS/SSC characteristics of these populations were overlapping. In 53 samples, the blast cell populations were also analyzed with a panel of FITC-conjugated monoclonal antibodies that were utilized in double labeling with CD45-PE. We show that the CD45/SSC gating procedure improved phenotypic determination of the blast cells in three ways: (1) by discriminating between leukemic blast cells and residual normal cells; (2) by excluding normal cells from the phenotypic analysis of leukemic blast cells; and (3) by identifying blast cell heterogeneity in many cases of leukemia on the basis of different CD45 display. Moreover, this immunophenotyping procedure on whole bone marrow samples also allowed an efficient discrimination between the various cell lineages and facilitated the analysis of leukemic blasts present in low proportions.

Incomplete stroma formation after allogeneic marrow or autologous blood stem cell transplantation.

Long term and semi-solid culture techniques were used to evaluate the quality of stroma produced by bone marrow from 33 normal subjects and 57 patients (46 allogeneic bone marrow and 11 autologous blood stem cell transplant recipients). Bone marrow from transplant recipients was capable of sustained CFU-GM and nucleated cell production in long term culture. However, only 13% of the marrow investigated developed a complete, confluent stromal layer. These stromal abnormalities were observed in spite of complete hematopoietic reconstitution after transplantation and rarely improved with time. Our results suggest that the hematopoietic microenvironment is very fragile and susceptible to long term damage as a result of chemotherapy and the conditioning regimes used prior to transplantation.

Long-term culture of peripheral blood stem cells: the effect of the addition of an irradiated stromal layer.

We have studied peripheral blood stem cells (PBSC) collected by cytapheresis following intensive chemotherapy, from 13 patients with acute leukemia, in long term culture (LTC). Peripheral blood was cultured with (n = 10) and without (n = 21) the addition of a preformed, irradiated stromal layer. In this latter LTC our results confirm that peripheral blood is capable of producing CFU-GM and nucleated cells in the absence of the formation of an adherent stromal layer. However, peripheral blood cultured in the presence of an irradiated stromal layer is capable of a significantly higher proliferative response (total production of CFU GM per flask - mean = 57529) than in the absence of an irradiated stromal layer (total production of CFU GM per flask - mean = 26739, p less than 0.03). Our results suggest that PBSC contain a primitive nonplastic adherent cell that requires the presence of a stromal layer for its expression. These findings provide further support for the use of peripheral blood stem cells for autologous transplantation.