Clonal variation in adrenocorticotropin-induced desensitization of adenylate cyclase in Y1 adrenocortical tumor cells. Association with a 68,000-dalton protein

Adrenocorticotropin(ACTH)-induced desensitization of adenylate cyclase was examined in subclones derived from the ACTH-responsive, Y1 mouse adrenocortical tumor cell line. This report describes clonal variation in ACTH-induced desensitization of adenylate cyclase and an associated variation in the level of a 68,000-dalton protein, p68. A subclone of Y1 cells with a low level of p68 (0.8% of total protein) exhibited a faster rate of desensitization and a slower rate of recovery from desensitization when compared with a clone containing a high level of p68 (10% of total protein). In three clones with low levels of p68, ACTH desensitized adenylate cyclase with ED50 values from 0.3 to 0.5 nM. In several clones with high levels of p68, the adenylate cyclase system was more resistant to ACTH-induced desensitization; the ED50 values for ACTH in these clones ranged from 2 to 12 nM. Among 11 ACTH-responsive subclones, the level of p68 correlated significantly (p less than 0.001, r = 0.87) with resistance to the desensitization induced by 1 nM ACTH. These results suggest that p68 may function in the maintenance of an ACTH-responsive adenylate cyclase system, or that the level of p68 and responsiveness to ACTH are coordinately regulated.     

Further study of aldosterone secretion-inhibitory factor and brain natriuretic peptide on cortisol production of guinea pig zona fasciculata cells

The suppressive effect of aldosterone secretion-inhibitory factor (ASIF) and brain natriuretic peptide (BNP-32) on the basal and ACTH-stimulated cortisol production in a primary culture enriched with guinea pig Zona Fasciculata (ZF) cells was further studied. The binding of 125I-labeled ACTH(1-24) and ASIF to ZF cells was found to be displaced by ACTH(1-24), [Phe2, Nle4 and Ala24]-ACTH(1-24), ASIF, and BNP in a concentration-dependent manner. The binding of 125I-labeled [Phe2, Nle4 and Ala24]-ACTH(1-24) to two transformed clones of mammalian cells expressing the guinea pig ACTH receptor was also competitively inhibited by ASIF and BNP. ASIF and BNP significantly suppressed ACTH-stimulated cAMP production in ZF cells. The 10- and 30-min cellular changes in cAMP induced by ASIF and BNP did not correlate in the rank order with the ultimate magnitude of cortisol suppression observed in ZF cells after a 24-hour treatment with these peptides. Nevertheless, the results did conform to the signaling mechanism of their action. Overall, the findings clearly demonstrated that ASIF and BNP suppressed the adrenocortical function and inhibited ACTH for their antagonistic action against ACTH primarily at the ACTH receptor site. These results support the notion that a physiological role of adrenal medulla in regulating the adrenocortical function may be mediated by the neuropeptides through a paracrine pathway.     

Relevance of c-fos proto-oncogene induction for the steroidogenic response to ACTH,dcAMP and phorbol ester in adrenocortical cells

We previously reported that ACTH, but not dibutyryl cAMP, rapidly induces the c-fos proto-oncogene in Y-1 adrenocortical cells.Here we show that PMA induces c-fos with similar kinetics when compared with ACTH (0.5–1 h peak) but reaches only 60% of the maximal ACTH induction and dcAMP is a weak c-fos inducer (15% of ACTH). However, combination of PMA and dcAMP has a synergistic effect leading to maximal c-fos induction. c-fos expression may play a role in the RNA synthesis-dependent corticosteroidogenesis response and/or growth regulation by ACTH.We also show that, in contrast to dcAMP, PMA is a poor steroidogenesis stimulator (15 to 17% of maximum ACTH-stimulated level), its activity being completely dependent on RNA synthesis. Combination of dcAMP and PMA yields an additive steroidogenesis stimulation, an effect that is also dependent on RNA synthesis. Although no strict correlation was found between c-fos induction and early steroidogenesis stimulation, particularly with respect to cAMP derivatives, the results suggest that a PKC pathway is likely to cooperate with the classical cAMP-PKA pathway in adrenal cells' RNA-dependent steroidogenesis.Abbreviations ACTH Adrenocorticotropic Hormone - PMA Phorbol-12-Myrystate-13-Acetate - dcAMP dibutyryl cyclic AMP - DME Dulbecco's Modified Eagle's minimal medium - FCS Fetal Calf Serum     

Chronic treatment with corticotropin increases the capacity of rabbit adrenocortical cells to convert pregnenolone into androgens

The present study was conducted to evaluate whether the previously demonstrated enhancement in adrenocortical androgen secretion in rabbits chronically treated with ACTH results, in addition to an increased pregnenolone production, from a more efficient conversion of this precursor of steroidogenesis into androgens. To this end, the adrenocortical cells from 14 control and 14 ACTH-treated rabbits (ACTH 1-24,200 micrograms s.c. daily for 12 days) were incubated either in the presence of different concentration of ACTH or with pregnenolone added in amounts from 0.5 to 250 micrograms. The total steroidogenic potency (maximal response to ACTH) was significantly enhanced for cells from ACTH-treated animals, as was the ACTH-induced production of dehydroepiandrosterone (DHEA), DHEA-sulfate, androstenedione and testosterone. In addition the production of these androgens from given amounts of exogenous pregnenolone was also significantly increased. The maximal capacity of adrenocortical cells to convert pregnenolone into androgens averaged (for ACTH-treated vs control group) 130 +/- 34 vs 43 +/- 10 pmol for DHEA, 138 +/- 43 vs 46 +/- 14 pmol for DHEA-sulfate, 99 +/- 31 vs 10 +/- 2 pmol for androstenedione and 8.0 +/- 2.6 vs 2.4 +/- 0.3 pmol for testosterone (P less than 0.001 for all androgens). The addition of ACTH to adrenocortical cells incubated with pregnenolone did not modify the maximal capacity of conversion of pregnenolone into androgens, which was in both experimental groups similar to that documented in the absence of ACTH. Thus, while an acute stimulatory effect of ACTH on adrenocortical steroidogenesis is devoid of any influence on the activity of the post-pregnenolone pathway of androgen synthesis, the chronic exposure of adrenocortical cells to ACTH lead to increased activity of steroidogenic pathway involved in the conversion of pregnenolone into androgens.     

Sensitivity to cell killing and the induction of cytogenetic damage following gamma irradiation in wild-type and thymidine kinase-deficient Friend mouse erythroleukaemia cells

Wild-type Friend erythroleukaemia (clone 707) cells and 2 thymidine kinase-deficient subclones, 707BUE and 707BUF, having thymidine kinase activities of 1.4% and 0.7% that of clone 707, were compared for sensitivity to killing and the induction of cytogenetic damage following irradiation. Three doses of gamma irradiation were used (150, 300 and 450 cGy), and cells were harvested for metaphase spreads after 4, 8, 12, 15, 29 and 43 h. G₂ delay was evident at 4 h following gamma irradiation in the 3 cell clones examined, and recovery of mitosis was observed to be dose-dependent. G₂ delay was found to be most prolonged in subclone 707BUE and most prompt in clone 707. Increased sensitivity to the induction of cytogenetic aberrations at all three doses was apparent in the 2 thymidine kinase-deficient subclones (as compared to wild-type cells) at 15, 29 and 43 h. Th thymidine kinase-deficient subclones also showed increased sensitivity to gamma radiation-induced cell killing. Furthermore, subclone 707BUE consistently exhibited greater to gamma irradiation than did the subclone with lower thymidine kinase activity, 707BUF. The importance of thymidine kinase levels and extended G₂ delay for DNA repair processes is discussed.     

Glucocorticoid induction of the maturation of ovine fetal adrenocortical cells

The aim of the present study was to assess whether glucocorticoids could be directly involved in the maturation of adrenocortical cells from 120-138 days old ovine fetuses. The cAMP response to ACTH1-24 of cells cultured for 24 hours in the presence of ACTH1-24 was 2 fold higher than that of control cells. However, the response of cells cultured in the presence of ACTH1-24 plus metyrapone or aminoglutethimide was lower than that of cells cultured in the presence of ACTH1-24 alone. Cells cultured for 48 hours in the presence of dexamethasone or cortisol released more cAMP than control cells when stimulated by ACTH1-24, but not in response to forskolin. However corticosteroid production stimulated by ACTH1-24, forskolin or dibutyryl cAMP was enhanced by dexamethasone treatment. These results suggest that glucocorticoids can affect the maturation of ovine fetal adrenocortical cells by an auto and/or a paracrine process, and that this effect is exerted, at least, at two different levels in the cell.     

Inhibition of aldosterone secretion by atrial natriuretic peptide in chicken adrenocortical cells

Dispersed chicken adrenocortical cells were preincubated with atrial natriuretic peptide (rANP), sodium nitroprusside (SNP) or 8-bromo cyclic GMP, followed by incubations with ACTH, chicken PTH, cholera toxin or various steroid intermediates of aldosterone production. Cyclic AMP production and aldosterone secretion were evaluated, in order to determine the sites of ANP inhibition in the sequence of events leading to aldosterone secretion. Dose-dependent inhibitory effects on ACTH-stimulated aldosterone secretion by rANP and SNP were observed. Both agents appeared to stimulate cGMP production by the particulate fraction of the avian adrenocortical cells. Aldosterone production, stimulated by cyclic AMP agonists such as ACTH, chicken PTH and cholera toxin, was significantly inhibited by ANP. On the other hand, ANP did not interfere with production or degradation of cAMP. Each of the aldosterone intermediates--pregnenolone, progesterone, 11-deoxycorticosterone and corticosterone--promoted aldosterone production when included in the incubation media. Atrial natriuretic peptide and SNP inhibited aldosterone secretion when enhanced by the intermediates, by about 40-60%, but the ACTH-stimulated secretion was inhibited by over 90%. The results suggest two sites of inhibition by ANP in the pathway of aldosterone synthesis and secretion: synthesis of cholesterol or pregnenolone, and conversion of corticosterone to aldosterone. The inhibition by 8-bromo cGMP of aldosterone secretion and the similar sites of inhibition for ANP and SNP suggest that cyclic GMP mediates the inhibition in both cases.     

Comparison of the structural features of corticotropin required for stimulation of steroidogenesis and cAMP production in rat and rabbit adrenocortical cells

The steroidogenic activities of ACTH, alpha-MSH, beta-MSH as well as analogs of the hormones have been compared in rat and rabbit adrenocortical cells. ACTH is equally active in both species and the melanotropins have very low steroidogenic potency in either species. The steroidogenic potencies of the peptide analogs are strikingly similar in the two species, suggesting that the structural requirements for eliciting steroidogenesis are the same in rat and rabbit adrenocortical cells. The analog NPS-ACTH has low, comparable steroidogenic activity in both species. NPS-ACTH is a potent antagonist of ACTH-induced cAMP production in rat adrenocortical cells but acts as a weak partial agonist in rabbit adrenocortical cells. These results suggest that steroidogenesis may be mediated by receptors different from those involved in the cAMP response observed at supraphysiological concentrations of ACTH.     

ACTH sensitivity of isolated human pathological adrenocortical cells: variability of responses in aldosterone, corticosterone, deoxycorticosterone and cortisol production

In vitro aldosterone, deoxycorticosterone, corticosterone and cortisol production of human adrenocortical cells derived from adenomas (Conn's syndrome, Cushing's syndrome), from hyperplastic adrenals (Cushing's syndrome) and from adrenals surrounding aldosteronoma are described. Cells from adenomas causing either Cushing's syndrome or Conn's syndrome harboured the highest basal and ACTH-stimulated corticosteroid production. Adrenocortical cells derived from micronodular hyperplasia causing Cushing's syndrome and cells from cortisol producing adenoma displayed predominantly cortisol and corticosterone secretion both under basal conditions and following stimulation with ACTH. Aldosteronoma cells showed highly variable aldosterone, deoxycorticosterone, corticosterone and cortisol response to ACTH. However, in aldosteronoma cell suspensions, the basal and ACTH-stimulated ratios of aldosterone to cortisol were increased when compared to ratios of steroids produced by cells from other adrenal tissues. Chronic treatment with spironolactone of patients with Conn's syndrome before surgery was associated with a decreased ratio of aldosterone to corticosterone, revealing that 18-hydroxylase in aldosteronoma cells may be inhibited during long-term therapy. Non-tumorous cells isolated from adrenals surrounding aldosteronoma displayed less aldosterone prior to and after stimulation with ACTH than aldosteronoma cells.     

X-linked control of hemoglobin production in somatic hybrids of mouse erythroleukemic cells and mouse lymphoma or bone marrow cells

Somatic cell hybrids were generated by fusion of mouse erythroleukemic cells (clone 745) to mouse lymphoma cells or mouse bone marrow cells. The erythroleukemic cells have been shown previously to have a low basal level of erythroid differentiation which is markedly amplified when the cells are grown in medium containing dimethylsulfoxide (DMSO). Hybrid cells were examined for hemoglobin production by benzidine staining. Many hybrid clones were found in which hemoglobin production in response to DMSO was either abolished or greatly reduced. From these hybrids, subclones were isolated in which hemoglobin production was restored. Karyological and enzyme analysis showed that the restoration of hemoglobin production was associated with the loss of an X chromosome contributed by the nonerythroleukemic parent. Other subclones which retained an X chromosome continued to be inhibited for hemoglobin production. Analysis of other hybrid lines capable of a limited degree of erythroid differentiation indicated a quantitative inverse correlation between the proportion of cells bearing an X chromosome and the proportion of cells able to form hemoglobin. Finally, four hybrid lines having many cells without an X chromosome were grown in medium containing DMSO. This procedure led to the selection of hybrid sublines having a nondifferentiating phenotype and concomitantly having a greatly increased proportion of cells bearing an X chromosome. Thus three lines of evidence suggest that a locus (loci) on the X chromosome is capable of inhibiting the DMSO-inducible hemoglobin production of the erythroleukemic cells.     

Adenylate cyclase activity and cyclic AMP production in the outer and inner zones of the adrenal cortex

It has been reported that cells isolated from the inner zone of the guinea pig adrenal cortex fail to have a steroidogenic response to ACTH. To further explore this, adenylate cyclase activity of membrane particles and cAMP production by cells prepared from the inner and outer adrenocortical zones were determined. The cAMP response to ACTH and forskolin was similar for cells from both zones. Basal adenylate cyclase activity was significantly higher in the inner zone; and while absolute responses to ACTH, GppNHp, GTP, NaF, and forskolin were greater for the inner zone, relative responses were similar for the two zones. These observations suggest that the inner zone of the guinea pig adrenal cortex may have a defect in ACTH action at a step(s) beyond cAMP formation.     

Enhanced 17 alpha-hydroxylation of pregnenolone and increased androgen production by rabbit adrenocortical cells stimulated chronically with corticotropin

The postulated chronic stimulatory effect of corticotropin (ACTH) on pregnenolone production and on 17 alpha-hydroxylase activity was evaluated on adrenocortical cells obtained from control and chronically ACTH-treated rabbits. The cells were incubated with various concentrations of ACTH added alone or together with trilostane, so as to inhibit further conversion of pregnenolone and dehydroepiandrosterone. The maximal steroidogenic effect of ACTH (determined in the absence of trilostane) was increased 2-fold in adrenocortical cells from ACTH-treated animals; furthermore, cortisol production was increased whereas that of corticosterone decreased. While the generation of pregnenolone was of comparable magnitude for cells from both experimental groups, chronic in vivo treatment with ACTH was followed by a 40-fold enhancement in 17-hydroxypregnenolone production. Concomitantly, maximal DHEA production documented in the presence of ACTH and trilostane was enhanced more than 200-fold, from 0.45 +/- 0.20 pmol in control rabbits to 147 +/- 67 pmol in cells from ACTH-treated animals. The corresponding values of DHEA-sulphate production were 0.86 +/- 0.12 and 432 +/- 334 pmol, respectively. Thus, a prolonged stimulatory effect of ACTH on rabbit adrenocortical cells consists in an enhancement of the capacity to generate pregnenolone, and to convert this compound into 17-hydroxylated steroids.     

Characterization of chimeric antibody producing CHO cells in the course of dihydrofolate reductase-mediated gene amplification and their stability in the absence of selective pressure

Recombinant Chinese hamster ovary (CHO) cells expressing a high-level of chimeric antibody against S surface antigen of hepatitis B virus were obtained by co-transfection of heavy and light chain cDNA expression vectors into dihydrofolate reductase (dhfr)-deficient CHO cells and subsequent gene amplification in medium containing stepwise increments in methotrexate (MTX) level such as 0.02, 0.08, 0.32, 1.0, and 4.0 microM. The highest producer (HP) subclone was isolated from each MTX level and was characterized with respect to cell growth and antibody production in the corresponding level of MTX. The specific growth rate of the HP subclone was inversely proportional to the MTX level. On the other hand, its specific antibody productivity (qAb) rapidly increased with increasing MTX level up to 0.08 microM, and thereafter, it gradually increased to 20 microg/10(6) cells/day at 4 microM MTX. Southern blot analysis showed that the enhanced qAb at higher MTX level resulted from immunoglobulin (Ig) gene amplification. The stability of the HP subclones isolated at 0.02, 0.08, 0.32, and 1.0 microM MTX in regard to antibody production was investigated during long-term culture in the absence of MTX. The qAb of all subclones significantly decreased during the culture. However, the relative extent of decrease in qAb was variable among the subclones. The HP subclone isolated at 1 microM MTX was most stable and could retain 59% of the initial qAb after 80 days of cultivation. Southern blot analysis showed that this decrease in qAb of the subclones resulted mainly from the loss of Ig gene copies during long-term culture. Despite the decreased qAb, the HP subclone isolated at 1 microM MTX could maintain high volumetric antibody productivity over three months because of improved cell growth rate during long-term culture.     

Mechanisms of insulin inhibition of ACTH-stimulated steroid secretion by cultured bovine adrenocortical cells.

Results of previous studies indicated that insulin at levels comparable to those in humans during hyperinsulinemia decreased ACTH-stimulated cortisol and androstenedione secretion by bovine adrenal fasciculata-reticularis cells in primary culture. In the present studies this inhibitory action was examined further by comparing the effects of insulin on ACTH-stimulated corticosteroid secretion with its effects on 8-(4-chlorophenylthio)-cAMP (cpt-cAMP), forskolin- and [5val]angiotensin II (Ang II)-stimulated corticosteroid secretion. Effects on corticosteroid secretion were correlated with effects on cAMP accumulation and rates of cAMP production. Monolayers were incubated for 24 h in the absence or presence of each agonist alone or in combination with insulin. Insulin (1.7 x 10(-9) or 17.5 x 10(-9) M) caused about a 50% decrease in cortisol and androstenedione secretion in response to ACTH (10(-11) or 10(-8) M). Insulin also decreased ACTH-stimulated aldosterone secretion by cultured glomerulosa cells. Cpt-cAMP (10(-4) or 10(-3) M)-stimulated increases in cortisol and androstenedione secretion were inhibited by insulin, but to a lesser extent than those in response to ACTH. The inhibition of cpt-cAMP-stimulated steroid secretion was not related to increased degradation of the cyclic nucleotide. Increases in cortisol and androstenedione secretion caused by a submaximal concentration (10(-6) M) of forskolin were decreased 50-70% by insulin. In contrast, insulin failed to significantly affect cortisol or androstenedione secretion caused by a maximal concentration (10(-5) M) of forskolin. The secretory responses to Ang II (10(-8) M) were also unaffected by insulin. The effect of insulin to inhibit ACTH-stimulated steroid secretion was accompanied by a reduction in cAMP accumulation as well as an apparent inhibition of adenylate cyclase activation. These data indicate that the effect of insulin to attenuate ACTH-stimulated corticosteroid secretion results from both an inhibition of ACTH-stimulated adenylate cyclase activity and an antagonism of the intracellular actions of cAMP.     

Different expression of alkaline phosphatase in subclones of human neoplastic salivary duct cell line

Human neoplastic salivary cell line (HSGc) and its subclones express alkaline phosphatase (AP) which is sensitive to L-phenylalanine but insensitive to L-homoarginine. Electrophoretic analysis demonstrates two distinct bands of AP, one (sb-1) is heat-stable and another (sb-2) is rather heat-labile and faster mobility than sb-1. The AP activity, especially sb-2, shows high level in less-differentiated clone (HSGc-C5) with increased growth rate but low level in nontumorigenic and well-differentiated clone (HSGc-E1) as compared to parent HSGc. This may suggest that the AP is a possible marker for identification of undifferentiated state in HSGc. This study also indicates that dibutyryl cAMP produces an increase of heat-labile AP in these clones.     

Changes in gene expression during senescence of adrenocortical cells in culture

Bovine adrenocortical cells undergo a process in which expression of steroid hydroxylases is lost progressively as a function of population doubling level (PDL) in culture. Each cytochrome P450 shows a characteristic rate of loss of expression as a function of PDL (in order of rates of loss: CYP11B > CYP21 > CYP17 > CYP11A). CYP11B and CYP21 require insulin-like growth factor I as well as cyclic AMP; these are the only factors required for induction in the primary culture. Middle- and later passage cells do not express CYP11B and CYP21 under the same conditions, but will do so when cells are grown in extracellular matrix Matrigel. In late-passage cells neither CYP17, CYP21, nor CYP11B are expressed, even in the presence of Matrigel; only CYP11A is expressed in late-passage cultures. When the different environmental factors required for induction of CYP11B and CYP21 are taken into account, induction of these genes disappears with the same kinetics as previously shown for CYP17 as a function of PDL. The primary cause of the loss of expression of these genes is likely to be a phenotypic switching event similar to that previously demonstrated for CYP17 by in situ hybridization. The mechanism of phenotypic switching is unknown. However, one HpaII site at −2.3 kb of CYP17 was methylated in the bovine adrenal cortex in vivo but showed rapid and complete demethylation when adrenocortical cells were placed in culture. This indicates a unique, reproducible, environmentally determined change in methylation, with as yet undetermined consequences. However, data from reporter constructs suggest that phenotypic switching does not result from a simple loss of regulatory factors that act within 2.5 kb of the promoter. Previous data suggested that SV40 T antigen may affect phenotypic switching, and thus that SV40 may be useful for the derivation of functional adrenocortical cell lines. Adaptation of methods previously used for bovine cells to human adrenocortical cells to produce SV40 T antigen-transfected clones yielded data indicating preservation of essential aspects of the human adrenocortical cell differentiated phenotype.     

Clonal variation for tolerance to polyethylene glycol-induced water stress in cultured tomato cells

Cell clones were isolated from a population of cultured tomato (Lycopersicon esculentum Mill cv VFNT-cherry) cells and their tolerance to polyethylene glycol (PEG)-induced water stress was measured. Considerable variation for tolerance among the clones was found. Tolerance differences between clones appeared to be spontaneous and were different from tolerance differences between adapted and unadapted cells. Unlike adapted (selected by exposure to PEG) cells, cell clones retained their relative tolerance for many generations in the absence of selection pressure, and tolerance of both relatively tolerant and intolerant clones was very dependent on growth cycle stage and inoculum density. Analysis of subclones isolated from relatively tolerant and intolerant parent clones revealed that each parent clone gives rise to progeny with tolerances near the mean tolerance of both parents. However, progeny populations of both tolerant and intolerant parents are enriched with individuals with phenotypes nearer the mean response of their respective parent populations. When exposed to PEG, relatively tolerant and intolerant clones alike become adapted to the level of PEG to which they are exposed, and have the same phenotypic level of tolerance. Thus, selection by exposure to stress is unable to discriminate (on the basis of growth) between the innately tolerant and intolerant cell types within the population. This is indicated also by the fact that clones isolated from a population of cells adjusted to growth on 25% PEG do not show an enriched frequency of tolerant phenotypes when grown in the absence of PEG compared to the nonselected normal cell population which has never been adjusted to growth on PEG.     

The additional loss of Bak and not the lack of the protein tyrosine kinase p56/Lck in one JCaM1.6 subclone caused pronounced apoptosis resistance in response to stimuli of the intrinsic pathway

Ionising radiation, hypoxia, and the cyclooxygenase-2 inhibitor Celecoxib are known agonists of the intrinsic apoptosis pathway that involves mitochondrial damage upstream of caspase activation. Mitochondrial integrity is regulated by the pro-apoptotic Bcl-2 protein family members Bak and Bax. Upstream of the mitochondria, many kinases and phosphatases control the apoptotic response. However, the role of the non-receptor tyrosine kinase p56/Lck during apoptosis is controversial. The present investigation demonstrate the existence of two JCaM1.6 subclones, one expressing and one deficient for Bak. The lack of p56/Lck expression in JCaM1.6 cells per se did hardly affect apoptosis induced by ionising radiation, hypoxia, or Celecoxib. Only the additional loss of Bak expression, as observed in one JCaM1.6 subclone, rendered the cells resistant. siRNA-mediated downregulation of Bak and p56/Lck mimicked the observed effects in the subclones. Earlier experiments performed with the Bak-negative clone might have lead to the wrong assumption that lack of p56/Lck alone, and not the additonal loss of Bak, was responsible for reduced sensitivity towards stimuli of the intrinsic apoptosis pathway.