Introduction of the cell survival gene bcl-xL improves the viability of CTLL-2 cells without affecting their IL-2 proliferative response. Implications for the development of bioassays.

A standard method for the quantitation of cytokines is to perform a bioassay in which aliquots of samples are compared to known concentrations of a cytokine in supporting the proliferation of a cytokine-dependent cell line. In most instances however, these cell lines are dependent on the cytokine not only for proliferation but also for survival. For example, a cell line that is commonly utilized for interleukin-2 (IL-2) bioassays is the IL-2-dependent line, CTLL-2. CTLL-2 cells will die rapidly by apoptosis if withdrawn from IL-2, thus these cells can be difficult to maintain in culture for extended periods. Overexpression of the anti-apoptotic protein Bcl-x(L) can enhance CTLL-2 survival in the absence of IL-2. However, while overexpression of Bcl-x(L) can prevent CTLL-2 cells from dying in the absence of IL-2, overexpression of Bcl-x(L) does not impair the ability of CTLL-2 cells to be used for proliferation-based IL-2 bioassays. Thus the bcl-x(L)-transfected CTLL-2 cells are equivalent to the parental cell line for determination of IL-2 levels in a culture supernatant, yet are easier to maintain in culture. Introduction of Bcl-x(L) or Bcl-2 into other factor-dependent cell lines may also simplify their maintenance without significantly affecting their utility in bioassays.

Resistance to anti-IgM-induced apoptosis in a WEHI-231 subline is due to insufficient production of ceramide.

We describe the properties of a physiological cell death (PCD)-resistant subline of WEHI-231 generated from the PCD-susceptible WEHI-231.7 JM cell line maintained in our laboratory. The PCD-resistant WEHI-231.7 JMRE subline was uniquely resistant to anti-immunoglobulin (Ig)M-induced PCD but not to irradiation and etoposide. In these sublines, we compared the expression of genes implicated in regulating PCD. Northern analysis of c-myc, c-fos, egr-1, Fas, p53 and retinoblastoma revealed similar basal levels of expression in all sublines tested and comparable responses to anti-IgM treatment. Similarly, the expression of bcl-2, bcl-x, bax and IL-1 beta converting enzyme did not correlate with susceptibility to anti-IgM-induced PCD. Next, we systematically studied signal transduction events including: tyrosine phosphorylation, Ca++ flux, and ceramide production in the Jm and JMRE sublines. The tyrosine phosphorylation patterns and the Ca++ influx generated following sIgM engagement were very similar in the JM and JMRE sublines. In contrast, the generation of ceramide differed in the PCD-resistant and PCD-susceptible sublines. Ceramide is produced following cross-linking sIgM on WEHI-231.7 JM cells and causes PCD. Ceramide levels in anti-IgM-treated WEHI-231.7 JMRE cells are low and appear to be insufficient to induce PCD.

Studies on the induction of apoptosis in WEHI 231 cells by pharmacological agents and lipid mediators. Sphingosine and ceramide induce apoptosis in WEHI 231.

We report the results of a systematic study of the effects of pharmacological agents known to cause or modify physiological cell death (PCD). Using WEHI 231 cells as a model, we investigated the effects of dexamethasone, cAMP, selected growth factors/ cytokines, DNA damaging agents, metabolic inhibitors and lipid mediators. We found that WEHI 231 cells are not affected by cAMP(1-90 microM) or TGFbeta (1-50 ng/ml), both of which are known to induce PCD in other systems. We also failed to detect protection from PCD in WEHI 231 cells cultured with Zn(++), E64 and leupeptin. In contrast, dexamethasone (400 microg/ml), etoposide (10(-4)M), emetine (10(-5)M), calyculin (10(-5)M), sphingosine (8-16 microM) and ceramide (20-40 microM) all cause PCD in WEHI 231 cells. The effects of ceramide can be blocked by LPS but not by overexpression of bcl2.The role of killer lipids in PCD is discussed.

Ceramide mediates the apoptotic response of WEHI 231 cells to anti-immunoglobulin, corticosteroids and irradiation.

We demonstrate for the first time how immature B cells kill themselves. Ceramide is identified as the mediator of apoptosis in the murine B lymphoma line WEHI 231 commonly used as a model to study clonal deletion in B lymphocytes. We show that exogenous ceramide induces apoptosis in WEHI 231 cells. To maintain self tolerance, immature lymphocytes readily undergo apoptotic death in response to the cross-linking of their antigen-specific receptors. We demonstrate that endogenously produced ceramide accumulates in WEHI 231 cells exposed to anti-IgM, an antigen surrogate before the onset of apoptosis. We also show that two other inducers of apoptosis, irradiation and dexamethasone, cause intracellular accumulation of ceramide.

Physiological cell death in B lymphocytes: I. Differential susceptibility of WEHI-231 sublines to anti-Ig induced physiological cell death and lack of correlation with bcl-2 expression.

WEHI-231 is a murine lymphoma generally considered to represent an immature B cell. Cross-linking of slg on WEHI-231 leads to growth arrest and eventually physiological cell death (PCD). We characterized three sublines of WEHI-231 by flow cytometry and compared their responses with slg cross-linking. All sublines had identical expression of a series of common B cell surface markers (IgM, IgD, Fc gamma R, ICAM-1, and CD45), but one was I-A-. Despite the phenotypic similarities between these sublines, anti-IgM caused aptotosis in only two sublines, although it inhibited growth in all three. The growth arrest induced by anti-IgM was reversible by lipopolysaccharide and Th2 clones and independent of Fc gamma R engagement. Anti-IgD, unlike anti-IgM, induced neither growth arrest nor apoptosis. To further compare the sublines' susceptibility to PCD, we investigated their responses to anti-IgM by ultrastructural morphology, [3H]thymidine release, propidium iodide exclusion, and incorporation into DNA. By all these experimental criteria, two of the WEHI-231 sublines were susceptible to PCD while the third demonstrated remarkable resistance to anti-IgM, but not irradiation or Th1-induced PCD. This differential susceptibility to PCD did not correlate with either bcl-2 levels in the resting cells or to the decrease in bcl-2 expression following slg engagement. We discuss the implications of these findings for our understanding of PCD in B cells.