Treatment of Cells with Detergent Activates Caspases and Induces Apoptotic Cell Death

Due to their amphiphilic properties, detergents readily disrupt cellular membranes and cause rapid cytolysis. In this study we demonstrate that treatment of cells with sublytic concentrations of detergents such as Triton X-100, Nonidet P-40, n-octylglucoside and the bile salt sodium deoxycholate induce typical signs of apoptosis including DNA fragmentation and cleavage of poly(ADP-ribose) polymerase molecules. The detergent concentration required for apoptosis was below the critical micellar concentration. Induction of apoptosis was not restricted to human cells but similarly occurred in a variety of other vertebrate cell lines. Unstimulated peripheral blood mononuclear cells were susceptible to apoptosis induction by detergent suggesting that apoptosis in this circumstance is not mediated by CD95. Cell death was not due to influx of calcium from the medium. Apoptosis was blocked and cytolysis prevented by treatment with peptide inhibitors of caspases. These findings suggest a process of apoptosis that is initiated upon nonspecific alterations at the cell membrane level. Physiologic correlates of this process still have to be defined. Received: 12 November 1999/Revised: 6 March 2000     

Use of cyclosporin a in establishing epstein-barr virus-transformed human lymphoblastoid cell lines

Summary We have investigated the potential for using cyclosporin A to increase the efficiency with which Epstein-Barr virus-transformed human lymphoblast lines can be prepared. Use of this immunosuppressive drug has permitted the development of a procedure with success rates exceeding 95% despite the processing of very large numbers of samples. This work was supported by National Institute of Neurological and Communicative Disorders and Stroke Grants NS16367 (Huntington's Disease Center Without Walls) and NS 20012.     

Demonstration of a functional receptor for vasoactive intestinal polypeptide on Molt 4b T lymphoblasts

Viable human T lymphoblasts derived from the "Molt 4b" cell line have been shown to possess functional plasma membrane receptors for vasoactive intestinal polypeptide (VIP). Specific binding of 125I-VIP to these lymphoblasts is rapid, reversible and linearly dependent on the number of cells present. Analysis of binding at 17 degrees C reveals a single class of high affinity binding sites over the concentration range of 10(-7) to 10(-11) M VIP (KD = 7.3 +/- 1.3 nM). The Bmax of 0.24 +/- 0.07 nM extrapolates to 15 000 +/- 4000 sites/cell. The binding of 125I-VIP to T lymphoblasts is highly specific; secretin and glucagon, peptides of similar molecular weight which show sequence homology with VIP, are unable to competitively inhibit binding of 125I-VIP to Molt 4b lymphoblasts. VIP activates adenylate cyclase in membrane preparations from Molt 4b lymphoblasts and increases cAMP in intact cells. Half maximal activation in both membrane preparations and intact cells occurs at 5 nM VIP. This demonstration of a functional receptor for VIP suggests that the Molt 4b lymphoblastic cell line may be a useful model system in which to study neuropeptide modulation of T lymphocyte function.     

Identification of a cadmium-binding protein from a cadmium-resistant variant of human lymphoblastoid cells (WI-L2)

Cadmium-binding protein synthesis and induction by cadmium chloride were studied in the human lymphoblastoid cell line WI-L2. Lymphoblasts were adapted to growth in 5 μM cadmium chloride (Cd^r) and these cells were 2.5-fold more resistant to cadmium than the parental line. There was no difference in the cellular protein profile between the parental line and lymphoblasts grown for a short period, less than 10 days, in cadmium chloride as measured by [³⁵S]cysteine labelling and SDS-polyacrylamide gel electrophoresis. A basal level of cadmium binding protein was apparent, however, by gel filtration. The Cd^r lymphoblasts were found to synthesize a substantial amount of cadmium-binding protein, approximately 25-fold more than the parental line. The cadmium-binding protein has the following properties which are consistent with its being a metallothionein: (1) [³⁵S]Cysteine-labelled protein eluted at a on a Sephadex G-75 column; (2) the molecular weight was estimated as 11 kDa on 7–17% SDS polyacrylamide gels; (3) the protein was heat-stable; (4) the unlabelled protein bound ¹⁰⁹Cd²⁺.