Abstract
Fresh leukaemia cells from the peripheral blood of 6 patients with B-chronic lymphocytic leukaemia (CLL) were cultured in the continuous presence of the phorbolester 12-O-tetradecanoylphorbol 13-acetate (TPA) for in vitro induction of differentiation. Upon treatment with TPA the cells showed distinct morphological changes consisting of cytoplasmic and nuclear enlargement, vacuolisation and protrusion of cytoplasm, eccentric location of nuclei with perinuclear clear zones, and oval to elongated cell forms. Isoenzyme profiles of the enzymes carboxylic esterase, acid phosphatase, hexosaminidase and lactate dehydrogenase (LDH) were analysed by isoelectric focusing on polyacrylamide gels. An increase in the number and in the staining intensity of isoenzymes were observed for all 4 enzymes in the TPA-exposed cells indicating a maturation along the B cell pathway. TPA triggered the new expression of the tartrate-resistant acid phosphatase isoenzyme, a marker of hairy cell leukaemia (HCL) cells, and of the hexosaminidase I isoenzyme, a marker of multiple myeloma cells. The induced phenotypic changes are suggestive of differentiation to stages corresponding to those of HCL cells or 'pre-plasma cells'.
Highlights
In this report we describe the morphological and isoenzymatic changes in B-chronic lymphocytic leukaemia (CLL) cells which were exposed to different concentrations of tetradecanoylphorbol 13acetate (TPA)
The decrease in cell counts was less pronounced in TPA-treated cultures as compared to the controls
We noted a rapid decrease of cell number and viability in the untreated flasks containing CLL control cells; less than 5% of the originally seeded control CLL cells were viable at days 8 and 10 of in vitro culture whereas the curve of viable, TPA-treated (10-8 M) CLL cells plateaued at days 6-8 with a viability of 50% of the original cell number
Summary
The diagnosis of B-CLL was established on the basis of lymphocytosis and typical morphology of the cells and immunological surface marker analysis. Obtained peripheral blood lymphocytes were separated by Ficoll-Hypaque density gradient centrifugation. Phenotyping of the cells was performed as described in detail earlier (Drexler et al, 1985a). Goat anti-human antisera (Cappel Lab., Cochranville, PA, USA) to human immunoglobulin chains labelled with fluorescein-isothiocyanate (FITC) were used to determine the isotype of the monoclonal populations in direct immunofluorescence. The number of T lymphocytes, Ialike/HLA-Dr antigen (la) positive cells, common ALL-antigen (cALLA) positive cells and myelomonocytic cells were determined by staining
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