Malcolm A. King

Detection of dead cells and measurement of cell killing by flow cytometry.

A flow cytometer can quickly perform numerous quantitative, sensitive measurements on each individual cell within a large, heterogeneous population. The modern commercially available analytical instruments, which can be found in most hospitals, pathology laboratories, and cell biology research laboratories in the industrially developed countries, can now routinely measure fluorescence simultaneously at four different wavelengths, in addition to light scatter in two directions, at rates of thousands of cells per second. Thus, flow cytometry provides a powerful and versatile approach to the measurement of cell death and cell killing. However, it is important to stress that the biological nature of the phenomenon under investigation will determine which assay is appropriate, and at what stage in the process of cell killing it should be applied. This consideration is particularly important in the study of drug- or cell-mediated cytotoxicity.

Antimycin A–induced killing of HL-60 cells: Apoptosis initiated from within mitochondria does not necessarily proceed via caspase 9

Antimycin A (AMA) inhibits mitochondrial electron transport, collapses the mitochondrial membrane potential, and causes the production of reactive oxygen species. Previous work by me and my colleagues has demonstrated that AMA causes an array of typical apoptotic phenomena in HL‐60 cells. The hypothesis that AMA causes HL‐60 apoptosis by the intrinsic apoptotic pathway has now been tested.

Natural killer cells and CD56+ T cells in the blood of multiple myeloma patients: Analysis by 4-colour flow cytometry

The blood of multiple myeloma patients was examined for non-MHC-restricted cytotoxic lymphocytes. Four colour flow cytometry was used to phenotype the cells within a light scatter gate large enough to include all lymphocytes. NK and T cells were identified using CD16, CD56, and CD3 antibodies, and myeloid cells with CD13 and CD14 antibodies. Three subpopulations of NK cells and 3 subpopulations of CD16+ or CD56+ T cells were enumerated. The CD56+ NK and T cells were also examined with CD69, CD25, and anti-HLA-DR antibodies to assess their activation state. We found no evidence that either the percentage or the absolute number of any subpopulation of the NK cells or CD56+ T cells correlated with disease activity. Neither did we find any significant abnormalities in the numbers of activated CD56+ NK or T cells. We conclude that it is unlikely that circulating non-MHC-restricted cytotoxic lymphocytes are responsible for maintaining disease stability in myeloma patients with indolent disease.

Atypical multidrug resistance in CCRF-CEM cells selected for high level methotrexate resistance: reactivity to monoclonal antibody C219 in the absence of P-glycoprotein expression

A series of CCRF-CEM sublines selected for extreme resistance to methotrexate has been shown previously to exhibit cross resistance to a number of agents belonging to the multidrug resistance phenotype (J.Natl.Cancer Inst.1989; 81, 1250-1254). The role of the mdr1 gene and its product (P-glycoprotein) in this atypical pattern of multidrug resistance has now been investigated. Southern and Northern analyses failed to demonstrate any amplification, rearrangement or over-expression of the mdr1 gene in the drug-resistant cells. Similarly, monoclonal antibodies MRK16 and JSB1 revealed no increase in the amount of P-glycoprotein present. By contrast, monoclonal antibody C219 detected a 170 kDa protein in all sublines, and in highest concentration in the most resistant cells. The results raise the possibility that a novel, C219-reactive protein may mediate resistance to both methotrexate and members of the multidrug resistance family.

A comparison of three flow cytometry methods for evaluating mitochondrial damage during staurosporine-induced apoptosis in Jurkat cells

Measuring cytochrome c release during apoptosis provides valuable information about the nature and extent of apoptosis. Several years ago a flow cytometric method (based on selective permeabilization of the plasma membrane with digitonin) was developed that has advantages over other techniques. These experiments describe a comprehensive evaluation of that method. Apoptosis was triggered in Jurkat cells with staurosporine and then flow cytometry was used to measure three aspects of mitochondrial damage: (1) cytochrome c release (with the digitonin assay and a commercially available kit based on the same principle), using a DNA‐binding dye to define cell cycle stage; (2) loss of mitochondrial cardiolipin, assessed by a decrease in 10 N‐nonyl acridine orange (NAO) binding; and (3) loss of mitochondrial membrane potential, assessed by a decrease in tetramethylrhodamineethylester (TMRE) binding. The results from these three assays were compared with an antibody‐based assay for cleaved caspase 3. The digitonin assay and the commercially available kit gave comparable results, showing that staurosporine caused cytochrome c release in all phases of the cell cycle and clearly defining those cells that had lost DNA due to internucleosomal DNA fragmentation. The pattern of fluorescence demonstrated that the mitochondrial apoptotic pathway was either the sole or the predominant pathway to be activated and that cytochrome c release in an individual cell was all‐or‐nothing. However, comparison with the other assays showed that the cytochrome c release assay underestimated the true extent of apoptosis. This was caused by the selective loss of some digitonin‐treated apoptotic cells. The flow cytometry assay for cytochrome c release provides valuable information but it underestimates the percentage of apoptotic cells.

FLOW CYTOMETRIC ANALYSIS OF CELL KILLING BY THE JUMPER ANT VENOM PEPTIDE PILOSULIN 1

Pilosulin 1 is a synthetic 56-amino acid residue polypeptide that corresponds to the largest allergenic polypeptide found in the venom of the jumper ant Myrmecia pilosula. Initial experiments showed that pilosulin 1 lysed erythrocytes and killed proliferating B cells. Herein, we describe how flow cytometry was used to investigate the cytotoxicity of the peptide for human white blood cells. Cells were labeled with fluorochrome-conjugated antibodies, incubated with the peptide and 7-aminoactinomycin D (7-AAD), and then analyzed. The effects of varying the peptide concentration, serum concentration, incubation time, and incubation temperature were measured, and the cytotoxicity of pilosulin 1 was compared with that of the bee venom peptide melittin. The antibodies and the 7-AAD enabled the identification of cell subpopulations and dead cells, respectively. It was possible, using the appropriate mix of antibodies and four-color analysis, to monitor the killing of three or more cell subpopulations simultaneously. We found that 1) pilosulin 1 killed cells within minutes, with kinetics similar to those of melittin; 2) pilosulin 1 was a slightly more potent cytotoxic agent than melittin; 3) both pilosulin 1 and melittin were more potent against mononuclear leukocytes than against granulocytes; and 4) serum inhibited killing by either peptide.

Use of four-colour flow cytometry to evaluate conjugate formation between human peripheral blood mononuclear cells and tumour target cells.

A four-colour flow cytometry technique is described for the determination of the number and phenotype of conjugate-forming mononuclear effector cells from human blood. The discriminatory power of previously described techniques was improved by labelling the effector cells with antibodies that simultaneously identified natural killer (NK), T, and myeloid cells and by labelling the tumour target cells with fluorescein octadecyl ester (FOE), so that cell conjugates could be differentiated from nonbinding effector cells more effectively than by the use of light scatter. The simultaneous characterisation of the three classes of conjugate-forming cells within a heterogeneous human peripheral blood mononuclear cell (PBMC) population made it possible to investigate conjugate formation by each subpopulation without purification, or semipurification (e.g., by monocyte removal), of the subpopulations. The binding of PBMCs to K562 and RPMI 1788 targets was examined. Binding for all three PBMC subpopulations was optimal at 37 degrees C and was negligible at 0 degree C (except for some T cell binding to RPMI 1788 cells). At 37 degrees C, maximum binding was essentially achieved by 10 min. Sodium azide inhibited the majority of the conjugation by NK and T cells, and that inhibition could be removed by washing the cells prior to conjugation, whereas azide had a negligible effect on the binding by monocytes. It appears that effective conjugation by human peripheral blood NK and T lymphocytes requires the operation of an energy-dependent process, differentiating it from conjugation by monocytes.

Monitoring circulating B cells in patients with multiple myeloma at diagnosis or in plateau phase: how prevalent is light chain isotype suppression?

Peripheral blood lymphocytes from 25 patients with multiple myeloma at diagnosis or in plateau phase, and from 18 healthy subjects, were analysed for the surface expression of CD19 and immunoglobulin (Ig) light chain isotypes by dual parameter flow cytometry. The aim was to test for the existence of light chain isotype suppression (LCIS). LCIS is a hypothetical suppressor mechanism which supposedly operates in myeloma patients with stable disease, causing a drop in the number of circulating B cells bearing the same Ig light chain isotype as the myeloma paraprotein. Our experiments emphasized that the use of a non‐Ig, pan‐B cell marker, combined with dual antigen analysis, is necessary to distinguish Ig on B cells from (cytophilic) Ig on some non‐B cells. We found good correlation between results obtained using polyclonal versus monoclonal anti‐light chain antibodies, and washed blood versus peripheral blood mononuclear cells. There was no evidence of an abnormal ratio of CD19+, K+ to CD19+, λ+ cells in any of the patients. Thus, these patients did not manifest LCIS, and these experiments raise doubts about the existence of this phenomenon. The results also illustrate the problem that may arise in the interpretation of one parameter flow cytometry.