Apoptosis may be either suppressed or enhanced with strategic combinations of antineoplastic drugs or anti-IgM.

A variety of drugs have been used to treat B-lymphocyte neoplasms, including both cell cycle-specific (CCS) and non-cell-cycle-specific drugs. Although the therapy for such cancers is complex and can include both types of drugs, the efficacy of these drugs in inducing cell death remains unclear. In this paper we have concentrated on specific CCS drugs and have examined their ability to induce programmed cell death (apoptosis) in Burkitt's lymphoma cell lines derived from patients. The CCS drugs chosen were hydroxyurea and aphidicolin (active in late G1, early S phase), the topoisomerase poisons camptothecin and etoposide (S, early G2 phase) and vincristine and Taxol (late G2, M phase). These choices allow comparison of two drugs with differing modes of action for each of the various phases of the cell cycle. Our results indicate that the variation in apoptosis between drugs that act at the same phase of the cell cycle is negligible. Both S/G2 and G2/M blockers are very potent at inducing apoptosis whereas G1/S blockers are ineffective in the induction of apoptosis. In addition, marked kinetic variations in the rate of apoptosis induction were observed, etoposide and camptothecin being more rapid in their action than the other agents. The order of effectiveness in inducing apoptosis on a kinetic basis was S/G2 agents >> G2/M agents >> G1/S agents. In this study we have also found that growth inhibition was induced by all the CCS agents chosen and by anti-IgM in various Burkitt's lymphoma lines. Furthermore c-myc was down-regulated under similar conditions. Since apoptosis was only selectively induced by some of the CCS agents, it implies c-myc expression is associated with growth regulation and c-myc down-regulation is an insufficient condition for the induction of apoptosis. In addition, cotreatments using the CCS and other agents revealed the following: Cotreatment using two CCS drugs which act at the same stage in the cell cycle showed either no change or only additivity to the effects seen with either agent alone. However, cotreatment with CCS drugs showed that an inhibitory effect is found between G1/S and G2/M drugs or S/G2 and G2/M drugs. No effect was found between G1/S and S/G2 drugs. Anti-IgM, which by itself was capable of inducing apoptosis, was observed to augment apoptosis induced by very low concentrations of G2/M-acting drugs but it has little effect on G1/S or the S/G2 drugs. The inhibitory effect of anti-CD40 or TNF-alpha on anti-IgM-induced apoptosis did not carry over to an effect on apoptosis induction by the CCS agents. Thus specific combinations of agents may lead to either enhancement, inhibition, or no interactive effect on apoptosis.

Anti-IgM-induced growth inhibition and apoptosis are independent of ornithine decarboxylase in Ramos cells.

Ornithine decarboxylase (ODC) is a key enzyme involved in polyamine production and is thought to regulate growth and apoptosis in multiple cell systems. A potential link between ODC and growth may involve the action of an oncogene c-myc which is thought to transcriptionally regulate ODC. We have examined the involvement of ODC in anti-IgM-induced growth inhibition and apoptosis in Burkitt's lymphoma cells. Inhibitors of ODC such as difluoromethylornithine (DFMO) completely blocked ODC activity, resulting in growth inhibition but not apoptosis. Addition of putrescine, the product of ODC enzymatic action, to Ramos cells had only a minor effect on growth, did not cause apoptosis, did not augment or block anti-IgM-mediated growth inhibition and apoptosis, but did reverse DFMO-mediated growth inhibition. Anti-IgM treatment of Ramos cells, which markedly decreased c-myc mRNA and protein, caused a paradoxical increase in ODC mRNA level as well as ODC enzymatic activity and increased cellular levels of putrescine. DFMO and putrescine did not alter c-myc mRNA levels directly, nor did they have any affects on anti-IgM-mediated down-regulation of c-myc mRNA. TNF-alpha, which inhibited anti-IgM-mediated apoptosis, did not inhibit either anti-IgM or DFMO-mediated inhibition of growth. These agents were without effect on ODC activity itself or on the anti-IgM-mediated increase in ODC activity. From these studies we conclude that ODC inhibition affects growth but is unrelated to the induction of apoptosis. Both anti-IgM-mediated inhibition of growth and induction of apoptosis are independent of ODC. Thus two distinct pathways for growth regulation are present: one in which ODC and polyamines are important and the other cell surface receptor-mediated (sIg) which is independent of ODC and polyamines.

Anti-IgM-mediated regulation of c-myc and its possible relationship to apoptosis.

Anti-IgM treatment of Burkitt's lymphoma cells is followed by either growth arrest or induction of apoptosis. In this study we have explored the role of c-myc in these events. Our results in Ramos cells indicate the following. (a) The decline in c-myc mRNA occurs at about 4 h; inhibition of about 80% being observed. (b) The stability of c-myc message is involved since the half-life of c-myc mRNA is decreased from about 30 min in untreated cells to about 15 min following treatment with anti-IgM. In the presence of cycloheximide, a protein synthesis inhibitor, the half-life is increased to about 50 min and was unaltered by treatment with anti-IgM. (c) By contrast, nuclear run-on experiments indicated no change in transcription rates for c-myc message due to treatment with anti-IgM. (d) A decrease in c-myc causes apoptosis since specific repression of c-myc with antisense oligonucleotides decreases the levels of c-Myc, inhibits growth rate, decreases viability, and induces apoptosis. (e) Anti-CD40 inhibition of apoptosis occurs without alteration in anti-IgM-induced down-regulation of c-myc mRNA, suggesting that it acts distally to c-myc down-regulation. Other cell lines were also investigated. In Epstein-Barr virus (EBV)-positive cell lines (Daudi, Raji, and Namalwa), anti-IgM treatment for 24 h results in growth inhibition without induction of apoptosis. In EBV-negative cell lines (ST486 and CA46, as well as Ramos), a more heterogeneous pattern of responses to anti-IgM are observed. Ramos and ST486 cells both show growth inhibition and apoptosis upon anti-IgM treatment; CA46 cells shown only growth inhibition but not apoptosis. Anti-IgM causes a decline in c-myc mRNA levels in all of these lines, as well as in c-Myc protein level in the two lines investigated, Daudi and Ramos, regardless of apoptosis. Addition of antisense c-myc oligonucleotides to the cells reduced growth in both Daudi and Ramos cells lines, however it resulted in substantial apoptosis only in Ramos cells. These results suggest that anti-IgM destabilizes c-myc mRNA by a process that involves mRNA turnover, rather than transcription rates. However anti-IgM exerts differential effects in EBV-positive and EBV-negative cell lines. EBV-positive cells are uniformly resistant to apoptosis, while EBV-negative cell lines show a tendency to apoptosis but with exceptions. Growth inhibition can be uncoupled from apoptosis in EBV-positive cell lines, but not in those EBV-negative cell lines prone to apoptosis. Furthermore, down-regulation of c-myc message correlates with growth inhibition in these cells, but is an insufficient link to apoptosis. By contrast inhibition of apoptosis by anti-CD40 occurs even though c-myc mRNA is decreased.

TNF-alpha inhibits anti-IgM-mediated apoptosis in Ramos cells.

We have examined the effects of tumor necrosis factor-alpha (TNF-alpha) as an inducer or modulator of necrosis and/or apoptosis in B cell lines. TNF-alpha does not induce either necrosis or apoptosis in EBV-positive or -negative cell lines, regardless of the culture conditions of the cells or the presence or absence of cytokines. By contrast anti-IgM induces apoptosis in two EBV-negative cell lines (Ramos and ST486) but not in EBV-positive cell lines. Since TNF receptor and CD40 belong to the TNF receptor superfamily and anti-CD40 is a known inhibitor of apoptosis, we tested for TNF-alpha's effects on the inhibition of apoptosis induced by anti-IgM. Our results indicate that TNF-alpha inhibits apoptosis induced by anti-IgM in Ramos cells but not in ST486. The effects are dose and time dependent; the degree of apoptosis achieved and the selectivity of the effect among cell lines are strikingly similar for both TNF-alpha and anti-CD40. Furthermore when both agents are tested together no additivity in the inhibition is observed. The inhibition of apoptosis is a direct effect of TNF-alpha and not a permissive effect of another cytokine, since it is observed in defined medium. Although anti-IgM induces both TNF-alpha secretion and TNF receptors in Ramos cells, the concentration of secreted TNF-alpha is too low to affect apoptosis. Inhibition does not involve perturbation of the cell cycle distribution of Ramos cells. Furthermore rapid induction of c-fos and the decrease in c-myc observed after anti-IgM treatment are both unaltered by TNF-alpha. Our results suggest that TNF-alpha is an inhibitor of apoptosis in Ramos cells, that its overall pattern of inhibition is similar to that of anti-CD40, and that both agents act at some point distal to the alteration of c-fos and c-myc by anti-IgM.

Regulation of low-density lipoprotein receptors and assessment of their functional role in Burkitt's lymphoma cells.

The status of low-density lipoprotein receptors (LDLR) in Daudi Burkitt's lymphoma (BL) cells was examined using a flow cytometric assay employing the fluorescent ligand DiI-LDL, and a radioligand-binding assay using [125I]LDL. The binding is concentration-and time-dependent; and is specific, as judged by its competitive displacement in the presence of unlabeled LDL, and inhibition by heparin, EGTA, and 4 degrees C incubation. The regulation of the receptor and its functional role were then explored. Our results suggest the following: (a) In sharp contrast to normal peripheral blood lymphocytes, the LDLR levels in BL cells are basally elevated when cultured in fetal bovine serum (FBS) medium. (b) In accord with normal peripheral blood lymphocytes, incubation in lipoprotein-deficient serum (LPDS) medium further up-regulates the level of the receptor in BL cells, and co-incubation with LDL or 25-hydroxycholesterol down-regulates the receptor level. The magnitude of the up-regulation is significantly smaller than in normal peripheral blood lymphocytes. (c) Northern blots using a plasmid-DNA probe for LDLR mRNA point to a similar pattern for message regulation as is observed in direct binding studies. (d) Although the LDLR level is constitutively high in BL cells, availability of LDL, unlike transferrin, is not a growth requirement since incubation of cells in LPDS medium does not prevent proliferation of these cells. (e) In contrast to anti-transferrin receptor antibody which results in apoptosis upon binding, anti-LDLR antibody does not inhibit growth or induce apoptosis. Our results suggest LDLR is expressed at a significantly higher level in BL cells than in normal peripheral blood lymphocytes. Although up-regulation and down-regulation of LDLR are observed, this applies only to a small population of LDLR. The bulk receptor population is significantly resistant to down-regulation. Furthermore, notable differences in the functional role of the LDLR are found relative to the transferrin receptor which is also up-regulated in the BL cells.

Dimethyl sulfoxide suppresses apoptosis in Burkitt's lymphoma cells.

Previous reports have suggested that dimethyl sulfoxide (DMSO) may be a useful reversible G1 arresting agent for synchronizing Raji Burkitt's lymphoma cells (K. Takase et al. (1992) Cell Growth Differ. 3, 515-521; M. Sawai et al. (1990) Exp. Cell Res. 187, 4-10). We have therefore critically evaluated several aspects of DMSO's effects using Daudi and Ramos Burkitt's lymphoma (BL) cells. In BL cells starved in the presence or absence of DMSO for 4 to 6 days (approximately four to six doubling times), the following observations were noted: (A) Both Daudi and Ramos cells show increased cell synchrony accompanied by apoptosis when starved in RPMI 1640 supplemented with 10% fetal calf serum (FCS). Inclusion of 1.5% DMSO causes a diminution in apoptosis with minimal effects on synchrony. (B) Lowering the FCS concentration to 5% induces apoptosis. DMSO-mediated protection from apoptosis is observed in Daudi but not in Ramos. (C) When human serum (10%) is used instead of FCS, Daudi cells show no apoptosis and DMSO is without effect on cell cycle distribution. By contrast, Ramos cells show significant apoptosis, which is prevented by the inclusion of DMSO. (D) When starved in a chemically defined medium (AIM-V), both Daudi and Ramos cells show significant apoptosis. DMSO protects Ramos from apoptosis under these conditions. (E) Upon removal of DMSO, both Daudi and Ramos cells reenter the cell cycle but with significant apoptosis. (F) The protective effect of DMSO from apoptosis is observed in a narrow range of concentration between 1 and 2%. At higher concentration, DMSO itself induces apoptosis. These results suggest that DMSO itself prevents apoptosis, an effect which may present as an apparent effect on cell synchrony.

Flow cytometric evaluation of LDL receptors using DiI-LDL uptake and its application to B and T lymphocytic cell lines.

Low density lipoprotein receptors (LDL-R) on Daudi Burkitt's lymphoma (BL) cells were assessed using fluorescent DiI (3,3'-dioctadecylindocarbocyanine iodide)-LDL and flow cytometric analyses. Receptor-specific binding of DiI-LDL is followed by internalization of the bound complex and lysosomal hydrolysis of the ligand. Increase in the fluorescence intensity per cell is hence used as a measure of DiI-LDL uptake and, implicitly, as an indication of LDL-R presence. Our results show that uptake was observed in > 98% of the Daudi cells, and the level of uptake was significant and clearly distinguishable from autofluorescence, suggesting that: (a) this assay is comparable to the iodinated LDL uptake assay, although the ED50 values for the ligands are different; (b) this assay is comparable to the flow-cytometric detection of LDL-R using a commercial antibody directed against the receptor itself, and superior to a similar assay based on an antibody directed against membrane-bound LDL; (c) LDL uptake could be monitored along with transferrin uptake, suggesting that multiple endocytic receptor activities can be concurrently studied; (d) DiI-LDL uptake can be examined along with fluorescein-conjugated anti-CD10, -CD19, and -CD71, with little cross-interference, offering the added advantage that endocytic uptake and phenotyping can be simultaneously monitored; (e) the expression of LDL-R is intrinsically elevated in diverse cell lines such as Daudi, Raji, Ramos, Jurkat, and WIL2-NS, but not in normal lymphocytes. Our results therefore indicate that flow cytometric analysis of DiI-LDL uptake has potentially useful applications in the detection and study of endocytic receptor LDL-R in B and T lymphocytic cell lines.

Multiple forms of the G protein-related beta subunit in Daudi lymphoblastoid cells.

We have explored the forms of the G protein-related beta subunit which are present in Daudi lymphoblastoid cells. Northern blotting with labeled beta-1 and beta-2 probes indicates that two messages of 3.3 kb and 1.7 kb are present for both beta-1 and beta-2, implying that multiple forms of the beta subunit are present. Antibodies were raised against two peptides of the beta subunit (residues 1-23 and 127-145). Both antibodies detected subunits at 35 kDa and 31 kDa, of which the 35-kDa form predominates in the membrane fraction and the 31-kDa one in the cell cytosol. Crosslinking of the membrane fraction with the cleavable crosslinker (DTSSP) caused a simultaneous diminution in the 31-kDa form while increasing the amount of the 35-kDa form--a pattern which was reversed upon the reduction of these crosslinks with DTT. Studies of the soluble form indicate that this is truly a soluble protein since centrifugation at 200,000 x g for 2 h did not diminish the levels of the protein in the soluble fraction. Sedimentation analysis indicates that the soluble beta-homologue is found in fractions which overlap with those which contain the mu chain of immunoglobulin at a position clearly distinct from the expected positions of free mu or free beta. Our results suggest that at least two forms of a subunit which is closely related to, or identical with, the beta subunit of G proteins are present in Daudi cells.

Induction of FMLP-mediated calcium mobilization requires crosslinking of surface immunoglobulin in Daudi cells.

We have explored the requirements for the induction of the N-formyl-methionyl-leucyl-phenylalanine (FMLP) response in Daudi cells after anti-immunoglobulin treatment. Our results indicate that (a) induction of responsiveness to FMLP was observed in Daudi only after crosslinking of surface immunoglobulin by anti-immunoglobulin; (b) this induced responsiveness was not observed in Ramos or Wil-2 cells; (c) the F(ab')2 fragment was sufficient for the induction of the FMLP response, but the Fab fragment and the Fc fragment were ineffective; (d) of the many agents active in B lymphocyte regulation which were tested, none were as effective as anti-immunoglobulin in the induction of the FMLP response; and (e) three inhibitors of calcium mobilization (W-7 (N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide), PMA (phorbol 12-myristate 13-acetate), and colchicine), acting on distinct mechanisms, inhibited both the calcium mobilization due to anti-immunoglobulin and the induction of responsiveness to FMLP. Our results suggest important determinants in the induction of a calcium-mobilizing FMLP response in cells of B lymphocyte lineage include (a) the cell type, (b) a selective requirement for activation via surface immunoglobulin, and (c) crosslinking of the surface immunoglobulin.

G-proteins and the role of second messengers in the regulation of the human neutrophil.

The ideas discussed above clearly point to increasingly complex and interactive transduction mechanisms for the regulation of the neutrophil. The central challenges to be met include the following: 1. Better assays are needed for the study of physiological parameters such as adherence, aggregation, shape change, and cytoskeletal rearrangements, assays which are not prohibitively complex and expensive while still allowing for more detailed physiological observations. 2. The neutrophil receptors need to be characterized in greater detail at the molecular level. Protein purification, sequencing, and cloning approaches are needed. Given the inherent shortcomings of working with the neutrophil system due to the presence of proteases and the problems of obtaining sufficient amounts of plasma membranes as source material for receptor purification, this is a difficult task. Advances in micropurification and sequencing may alleviate some of the difficulties here. 3. The size and complexity of the G-protein family continue to expand. However, as pointed out earlier, stimulus-responsive enzymes without G-protein-associated regulation, and G-proteins without clearly identified targets, remain. A better definition and description of the G-protein family will be required if cellular regulation is to be understood at the molecular level. In terms of second messengers and their role in cellular regulation, the main questions which remain to be answered concern identification of the precise pathways which are important to cellular regulation. In order to understand the complex cascades of arachidonate metabolism, phospholipid turnover, and calcium homeostasis, it is all the more important that the manner in which second messengers may regulate particular cell functions be better understood. An omission in this review is the role of kinases in cellular regulation. Activation of kinase C (through calcium and diacylglycerol) and kinase A (through cAMP) has been demonstrated. The substrates for these kinases have been described by various investigators. However, relating phosphorylation changes in a particular protein to the activity of the protein, and assignment of activity to particular physiological roles, has not been satisfactorily accomplished and remains a challenge for the future.

Desensitization of calcium mobilization and cell function in human neutrophils.

Neutrophils pretreated with the chemoattractant formylmethionyl-leucyl-phenylalanine become unresponsive when re-exposed to the same ligand, a process termed desensitization. We have examined whether desensitization of transduction (Ca2+ mobilization) or of other cell functions (superoxide generation, enzyme release, or aggregation) occurs synchronously. Simultaneous studies of Ca2+ mobilization and aggregation by using Fura-2-loaded cells indicate that, under conditions where the aggregation response is abolished, most of the Ca2+ mobilization is unaltered. Further studies were then carried out to ascertain whether desensitization of Ca2+ mobilization could in fact be induced. Desensitization was observed, and was dependent on the number of exposures of the cells to the ligand, the concentration of the ligand, and whether the ligand was left in the medium or was removed. The pattern of resensitization was dependent on the experimental design. Under conditions where ligand was continuously present, no recovery of the Ca2+-mobilization response was seen with subsequent challenges. In contrast, on removal of ligand, this response showed partial recovery. Whereas complete desensitization of aggregation was noted, enzyme release showed a markedly lesser degree of desensitization and required more frequent exposures to the ligand before it was observed. Little or no desensitization of superoxide generation was observed regardless of the conditions utilized. Studies using phorbol myristate acetate as the ligand showed that Ca2+ mobilization and aggregation could be simultaneously inhibited. Our results suggest that discrete mechanisms of desensitization are possible in human neutrophils, and that desensitization of one particular function (aggregation) does not imply concomitant desensitization of other functions.

Anti-immunoglobulin pretreatment induces a calcium-mobilization response to the chemotactic agent N-formylmethionylleucylphenylalanine in Daudi lymphoblastoid cells.

Anti-immunoglobulin treatment of fura-2-loaded Daudi cells induces a calcium mobilization as judged by the increase in the fluorescence of the dye fura-2, AM. No calcium mobilization by N-fMet-Leu-Phe is observed in these cells. However, exposure of the cells to N-fMet-Leu-Phe after the first hit with anti-immunoglobulin (but not after soluble IgG) shows a rapid, dose-dependent calcium mobilization by N-fMet-Leu-Phe. The expression of the calcium-mobilizing response occurs in less than 2 min and is stable. Binding of tritiated N-fMet-Leu-Phe is increased in anti-immunoglobulin-treated but not control cells. The induction is specific for N-fMet-Leu-Phe because the chemoattractant platelet-activating factor did not induce any calcium mobilization. The N-fMet-Leu-Phe antagonist t-butoxycarbonyl-L-Phe-D-Leu-L-Phe-D-Leu-L-Phe- OH did not show any calcium mobilization on its own, either before or after anti-immunoglobulin treatment, and inhibited the calcium mobilization of N-fMet-Leu-Phe at low concentrations. Treatment of the cells with phorbol 12-myristate 13-acetate or pertussis toxin prior to anti-immunoglobulin treatment caused a dose-dependent abolition of both the anti-immunoglobulin-mediated calcium mobilization and the subsequent calcium mobilization by N-fMet-Leu-Phe. Metabolic inhibitors that act predominantly by lowering the ATP levels within the cell (iodoacetate, sodium fluoride, oligomycin, and 2-deoxyglucose) all produced a greater inhibition of the N-fMet-Leu-Phe-mediated calcium mobilization than the anti-immunoglobulin-mediated response. Lowering the temperature from 37 degrees C to 22 degrees C reduced the anti-immunoglobulin response and completely inhibited the expression of the N-fMet-Leu-Phe effect. Our results indicate that activation of the calcium-mobilization pathway in B cells by crosslinking of bound surface immunoglobulin causes an induction of N-fMet-Leu-Phe-sensitive calcium mobilization.