Role of Bag-1 in the Survival and Proliferation of the Cytokine-Dependent Lymphocyte Lines, Ba/F3 and Nb2

Charles V. Clevenger, Karen Thickman, Winnie Ngo, Wan-Pin Chang, Shinichi Takayama and John C. Reed

Department of Pathology and Laboratory Medicine (C.C., K.T., W.N., W.P.C.) University of Pennsylvania Medical Center Philadelphia, Pennsylvania 19104
The Burnham Institute (S.T., J.R.) La Jolla, California 92037


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The expression and function of the newly identified Bcl-2- and Raf-1- binding protein, Bag-1, during the cytokine-regulated growth of B and T cell lines was examined. Immunoblot analysis of lysates from the interleukin-3 (IL-3)-dependent B cell line Ba/F3, and the PRL-dependent T cell line Nb2, revealed that variations in Bag-1 levels paralleled alterations in cellular proliferation, viability, and apoptosis induced by the presence or absence of growth factor. To test whether up-regulation of Bag-1 levels altered cellular survival and proliferation, Ba/F3 cells were transfected with a Bag-1 expression construct. The overexpression of Bag-1 in transfected Ba/F3 cells induced an IL-3-independent state. Such transfectants demonstrated sustained viability and proliferation, with minimal apoptosis, in the complete absence of exogenous IL-3. Bag-1 expression was also compared in glucocorticoid-sensitive Nb2 cells and a PRL-independent, glucocorticoid-resistant subline, SFJCD1, during culture of these lines in dexamethasone (Dex). Bag-1 levels were profoundly decreased by the addition of Dex to Nb2 cells, precedent to the onset of apoptotic cell death. In contrast, Dex treatment or PRL withdrawal had no effect on levels of Bag-1 within the SFJCD1 line. These findings establish that the overexpression of Bag-1 in the appropriate cellular context promotes cellular survival and growth, events that may result from the juxtaposition of this protein with mitogenic and antiapoptotic signaling pathways.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The expansion of hematopoietic progenitors and effectors, mediated by peptide growth factors, is required during an effective immune response. This selective growth within the hematopoietic system occurs as the summation of increased cellular proliferation and decreased programmed cell death or apoptosis (1, 2, 3). Two peptide growth factors that control such growth are interleukin-3 (IL-3) and PRL. IL-3 has been found to increase the proliferation of most hematopoietic precursors (4) and improve cellular survival by inhibiting apoptosis (1, 5, 6). IL-3 acts as an immune stress factor, released by activated T lymphocytes and mast cells, thereby mediating a paracrine up-regulation of the pool of hematopoietic progenitors (4). Like IL-3, PRL is also a peptide stress factor, whose secretion from the pituitary and activated T cells is increased during an immune response. PRL is necessary for both in vitro (7, 8, 9, 10) and in vivo (11, 12, 13) lymphocyte proliferation. PRL is required for IL-2-driven T cell proliferation; specifically, signals supplied from the PRL receptor (PRLR) complex drive progression from G1 to S phase of the cell cycle (14, 15). In addition, PRL has been found to inhibit dexamethasone (Dex)-driven apoptosis of lymphocyte progenitors (16, 17, 18). Thus, IL-3 and PRL may contribute to the overall immmunoresponsiveness through their regulation of lymphocyte apoptosis.

The regulatory role of apoptosis extends to many other aspects of the immune response. The elimination of autoreactive lymphocyte precursors, the lysis of target cells by cytotoxic T lymphocytes, and the loss of CD4+ T cells during infection with human immunodeficiency virus are mediated, in part, by apoptosis (19, 20). Although the intracellular mechanisms that effect such cell death are incompletely characterized, members of the Bcl-2 (for B cell lymphoma-2) family of genes are thought to serve as central regulators of these phenomena (20, 21, 22). Overexpression of Bcl-2 inhibits lymphocyte apoptosis initiated by several disparate stimuli including the Fas ligand (23, 24, 25), T cell cytolysis (26), glucocorticoids (27, 28, 29, 30), chemotherapy (31), and growth factor withdrawal (32, 33, 34). Although the linkages between Bcl-2 and the downstream factors that directly effect apoptosis are incompletely characterized (35), Bcl-2 function is significantly modulated by heterodimerization with other Bcl-2 family members (36, 37, 38). For example, the extent of Bcl-2 and Bax homo- and heterodimerization is thought to contribute to the control of cellular survival. In addition to Bcl-2 family members, other proteins, most notably Bag-1, have been found to interact with Bcl-2 and modulate its function. Bag-1 (for Bcl-2-associated anti-death gene 1) was identified by the screening of a murine embryo cDNA library with recombinant Bcl-2 (39). The significance of the in vitro and in vivo interaction of Bag-1 with Bcl-2 was confirmed by overexpression of Bag-1 in Jurkat and NIH3T3 cells. Overexpression of Bag-1 promoted the survival of these transfectants to a variety of apoptotic stimuli. Thus, the multimeric interactions of the Bcl-2/Bag-1/Bax complex, as regulated by the stoichiometry of its constituents, may significantly affect cellular survival.

Despite the linkage between Bcl-2 family overexpression and apoptosis, little is known of the regulation of these gene products in nontransfected cell lines experiencing stimuli that trigger cell death. If the regulation of such protein levels is central to the control of apoptosis, such changes, at the protein level, should be detectable by biochemical analysis either preceding and/or during entry into the apoptotic pathway. In this study, we tested whether the expression of the Bcl-2-binding protein, Bag-1, was associated with apoptosis induced by growth factor withdrawal and/or dexamethasone treatment of cytokine-dependent lymphocyte lines Ba/F3 and Nb2. These data revealed a direct association between Bag-1 levels and cellular growth. To confirm that increased levels of Bag-1 could enhance cellular proliferation and viability during growth factor deprivation, a Bag-1 expression construct was transfected into Ba/F3. Examination of several independently obtained transfectants has revealed that overexpression of Bag-1 confers growth factor independence to the Ba/F3 line.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Bag-1 Expression Is Regulated during IL-3-Driven Ba/F3 Proliferation and Apoptosis
To determine whether an association existed between the levels of the Bcl-2-binding protein, Bag-1, and apoptosis induced by cytokine withdrawal, immunoblot analysis was performed on lysates obtained from the lymphocyte lines, Ba/F3 and Nb2. Significant decreases in Bag-1 levels were noted in both cell lines during withdrawal of IL-3 and PRL, respectively. To expand upon these preliminary findings, the dependence of Ba/F3 on IL-3 for cell growth (Fig. 1AGo) and viability (Fig. 1BGo) was correlated with Bag-1 expression with respect to dose and time (Fig. 1Go, C and D). Ba/F3 cells cultured in the absence of IL-3 expressed 6- to 8-fold less Bag-1 than parallel cultures incubated in the presence of IL-3. This was due to a modest (50%) increase in Bag-1 levels in the IL-3-stimulated cultures and a 4-fold decrease in Bag-1 levels in IL-3-deprived cultures. As described below, the loss of cellular viability, as measured by trypan blue uptake, was found to occur shortly after the apoptosis-associated phenomena of cleavage and loss of cellular DNA (Fig. 3Go). The temporal decreases in Bag-1 expression were noted to occur in rough approximation with the initiation of endonucleolytic DNA cleavage. Taken together, these data indicate an association between decreased Bag-1 expression and the onset of apoptosis.



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Figure 1. Association between Bag-1 Expression and IL-3-Dependent Growth and Survival of the Murine B Cell Lymphoma Ba/F3

Ba/F3 cells were cultured in the presence of varying concentrations of murine IL-3; at various time intervals, cell growth (A) and survival (B) were measured using trypan blue exclusion. C, Parallel measurement of Bag-1 levels in IL-3-stimulated Ba/F3 were performed by immunoblot analysis using an anti-Bag-1 antiserum. Probing of a duplicate blot with preimmune serum demonstrated the specificity of these results (data not shown). Molecular mass standards indicated at left are in kilodaltons. Bag-1 levels below blot represent densitometric quantification of the Bag-1 protein normalized to total cellular protein. D, Temporal decrease of Bag-1 levels during IL-3 deprivation as measured by scanning densitometry of anti-Bag-1 immunoblots. As in Fig. 1CGo, these values in arbitrary units (au) were normalized for total cellular protein.

 


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Figure 3. The Dependence of Ba/F3 Cells on IL-3 for Cell Growth and Viability Is Abrogated by the Transfection of Bag-1

Parental and transfected Ba/F3 cells were cultured at various times in the absence of IL-3. Cell growth (A) and viability (B) were assessed as per Fig. 1Go. Quantitative assessment of apoptosis during IL-3 deprivation was performed by two independent assays. C, Cells with endonucleolytic cleavage were labeled with ddUTP in the presence (or absence as control) of TdT; the incorporated ddUTP was subsequently detected with a fluorescein-conjugated anti-digoxigenin antibody. Incorporation of this fluorescent marker of DNA cleavage was then assessed by flow cytometry. D, Loss of cellular DNA, as manifested by a hypodiploid DNA content, was assessed by flow cytometric analysis of propidium iodide-stained cells.

 
Overexpression of Bag-1 Permits Sustained Ba/F3 Viability and Proliferation in the Absence of IL-3
To explore whether up-regulation of Bag-1 levels could inhibit cellular death induced by cytokine withdrawal, stable transfectants of Ba/F3 that constitutively expressed Bag-1 were generated. High levels of Bag-1 were observed in clones containing the Bag-1 expression construct (Fig. 2AGo), in comparison to either the parental line or control transfectants that received the same vector lacking Bag-1. Similar to the parental Ba/F3, control transfectants demonstrated significant decreases in Bag-1 levels as a function of IL-3 deprivation (Fig. 2BGo). When IL-3 was withdrawn from these subclones, sustained proliferation and viability were observed in only those transfectants that overexpressed Bag-1 (Fig. 3Go, A and B). Indeed, the viability of the Bag-1 transfectants in IL-3-deficient medium paralleled that of the parental line raised in medium containing IL-3 (see Fig. 1BGo). Concurrent with the enhanced survival, the Bag-1 transfectants continued to proliferate in the absence of IL-3 (Fig. 3AGo); however, a cell density of approximately 2-fold greater than the parental line was reached (see Fig. 1AGo). Additional studies revealed that if the IL-3-deficient medium was not changed between 48–60 h after culture initiation, the Bag-1 transfectants would demonstrate a decrease in viability and proliferation. However, thrice-weekly refeeding with medium lacking IL-3 permitted continued growth and viability (>95%) for at least 2 months.



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Figure 2. Expression of Bag-1 in Parental and Transfected Ba/F3 as a Function of IL-3 Deprivation

Ba/F3 cells were stably transfected with Bag1-pCMV or Neo-pCMV and cultured for various times without IL-3 as indicated (A). Cell lysates from these cultures were prepared and subjected to sequential SDS-PAGE and immunoblot analysis using an anti-Bag-1 antiserum. Bag-1 levels in both the Ba/F3 parent and control Neo-pCMV transfectants decreased temporally during IL-3 deprivation (B). Levels of Bag-1 were determined by immunoblot analysis of cell lysates using an anti-Bag-1 antibody as above; the control blot represents parallel blot probed with preimmune serum. Results similar to the control clone Neo-pCMV-1 were obtained for Neo-pCMV-3 (data not shown).

 
To confirm that the improvement in cell viability of the Bag-1 transfectants was secondary to a decrease in the percentage of cells undergoing apoptosis, three independent measures of programmed cell death were performed. Two of these measures, presented in Fig. 3Go, C and D, were flow cytometry-based assays that respectively measured the percentage of cells undergoing endonucleolytic DNA cleavage [as measured by the incorporation of digoxigenin labeled dUTP (ddUTP) by terminal deoxynucleotide transferase (TdT)] or DNA degradation (as measured by hypodiploid DNA content of propidium iodide-stained cells). These analyses demonstrated that, in contrast to the transfected controls and the parental line, the Bag-1 transfectants had a significant reduction in the percentage of cells undergoing apoptosis as a result of IL-3 withdrawal. Further analysis of these DNA content histograms revealed an increase in the %S-phase fraction of the Bag-1 transfectants by approximately 2-fold (data not shown). Taken together, these data indicate that constitutive overexpression of Bag-1 in the Ba/F3 cell line induces an immortalized, IL-3-independent state that prevents apoptosis upon withdrawal of IL-3 and permits continued proliferation under such conditions.

Effects of PRL and Dex on Nb2 Cell Growth Viability and Apoptosis
To extend the data obtained with the Ba/F3 cells, the growth, viability, and apoptosis of PRL and Dex-treated Nb2 cells were ascertained (Fig. 4Go) at concentrations of PRL (5 ng/ml, 0.2 nM) and Dex (100 nM) previously determined to induce maximal proliferation or apoptosis (16, 17), respectively (Fig. 4Go, A and B) The addition of Dex to Nb2 cells in the absence of exogenous PRL was found to inhibit proliferation and induce a profound decrease in viable cell number. The extent of Dex-induced apoptosis in Nb2 cells was examined with the same methodology used for Ba/F3 (Fig. 4Go, C and D) and confirmed that a significant increase in apoptosis (also confirmed morphologically) occurred in Dex-treated cultures.



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Figure 4. Effects of PRL and Dex on Rat T Cell Lymphoma Nb2 Growth and Survival

Cell growth (A) and viability (B) of Nb2 cells were measured at various times in the presence or absence of 5 ng PRL/ml or 100 nM Dex, as per Fig. 1Go. Apoptosis was assessed by flow cytometric measurement of endonucleolytic cleavage (C) or loss of cellular DNA content (D), as per Fig. 3Go.

 
In contrast to Dex-treated Nb2 cultures, the viable cell number of Nb2 cells cultured in the presence of both exogenous PRL and Dex was relatively stationary. Concomitantly, the incidence of apoptotic death within these cultures was less marked than in cultures that received Dex only. Of note, parallel growth and viability curves were observed between cultures that received both or neither of the two hormones.

The Regulated Expression of Bag-1 Found in Nb2 Is Absent in the PRL-Independent Nb2 Subline, SFJCD1
The expression of Bag-1 in Nb2 cells was measured by immunoblot analysis (Fig. 5Go). For these experiments, Nb2 cells were deprived of PRL for 24 h and then restimulated with various concentrations of PRL or Dex before cell lysates were prepared at various time intervals and immunoblot analysis of Bag-1 was performed. The regulation of Bag-1 expression in Nb2 cells by PRL was dose-dependent (Fig. 5AGo), whereas a 5-fold decrease in Bag-1 levels was observed in PRL-deficient cultures. In contrast, an inverse relationship between Bag-1 levels and Dex concentration was noted (Fig. 5BGo), with an approximately 15-fold decrease in Bag-1 levels in Nb2 cultures treated with 1–3 µM Dex. Further delineation of the temporal regulation of Bag-1 levels in response to concomitant PRL (5 ng/ml) and/or Dex (100 nM) stimulation is presented in Fig. 5CGo. These data revealed that treatment with only Dex induced a rapid decline (after 6 h) in Bag-1, with 10-fold reduction in levels by 24 h. Dex also induced decreases in Bag-1 protein levels in PRL-stimulated Nb2 cells, but the effects were less striking than in cultures that received Dex alone. Taken together, these data indicate that Bag-1 levels correlate with cell growth and apoptosis in Nb2 cells during treatment with Dex, PRL, or a combination of these reagents.



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Figure 5. The Regulated Expression of Bag-1 in Nb2 Cells Is Absent in the Nb2-Derived, PRL-Independent SFJCD1 Subclone

Measurement of Bag-1 levels by immunoblot analysis in Nb2 cells stimulated with PRL as a function of dose and time (A). The concentrations of PRL used were either submitogenic (0.05 ng/ml), or induced half-maximal (0.5 ng/ml) or maximal (5 ng/ml) growth. Bag-1 levels below blot represent densitometric quantitation of the Bag-1 protein normalized to total cellular protein. B, Measurement of Bag-1 levels by immunoblot analysis of lysates from Nb2 cells treated with Dex as a function of dose. Reported Bag-1 levels were quantitated as per Fig. 5BGo. C, Temporal measurement of Bag-1 levels in lysates from Nb2 cells treated with 5 ng PRL/ml and/or 100 nM Dex as determined by immunoblot analysis with an anti-Bag-1 antiserum. The relative Bag-1 levels reported here are normalized to total cellular protein. D, Bag-1 expression in Nb2 or SFJCD1 cells treated with 100 nM Dex or PRL-deprived for 48 h.

 
To extend these correlations further, Bag-1 levels were examined in the PRL-independent SFJCD1 T-cell line during PRL withdrawal or Dex treatment. Unlike Nb2, this PRL-independent subline is resistant to Dex-induced apoptosis (40, 41). As seen in Fig. 5DGo, PRL withdrawal or treatment with Dex (100 nM) had little effect on Bag-1 levels or on the viability of the SFJCD1 subline, in contrast to the parental Nb2 line. These data, therefore, further confirm an association between Bag-1 levels and Nb2 growth and survival, suggesting a regulatory role for this protein in Nb2 cells, similar to that seen in Ba/F3.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The receptors for IL-3 and PRL belong to the growth factor/cytokine receptor superfamily that is widely expressed within the hematopoietic system (42, 43, 44). Ligand stimulation of progenitor cells promotes proliferation, survival, and differentiation of select cell lineages, resulting in a coordinated immune response. A common structure/function aspect of the growth factor receptor superfamily is its lack of intrinsic kinase activity (42). Thus, signaling via these receptors is mediated by associated transduction factors. Signals supplied by receptor-associated kinase cascades, such as the JAK/STAT and the Ras/Raf pathways (45, 46, 47, 48, 49, 50), are believed in part to transmit signals from this receptor superfamily that initiate cell cycle progression. In contrast, the structure/function basis for the immediate antiapoptotic signals supplied by these receptors is incompletely understood. Nevertheless, alterations in the levels of the Bcl-2 family, induced by overexpression of transfected gene constructs (i.e. Bcl-2 and Bax), clearly alters the survival, but not the proliferation, of cytokine-dependent cell lines (33, 36, 51). Overexpression of the Bcl-2 gene in transgenic animals also inhibits the in vivo apoptosis of select lymphocyte progenitor populations (27, 52, 53). Taken together, these data suggest that alterations in the levels of members of the Bcl-2 family may control apoptosis by several stimuli, including growth factor/cytokine withdrawal. Initial studies performed in our laboratory, however, revealed no significant changes in the levels of Bcl-2, Bax, or Bcl-XL proteins during stimulation with or withdrawal of growth factor from the Ba/F3 or Nb2 lines (not shown). These observations largely agree with a previous report (54), which demonstrated modest changes in the levels of Bcl-2 and Bax protein during stimulation of Nb2 with PRL, despite the significant induction of Bcl-2 and Bax mRNA during such treatment.

In contrast to Bcl-2, Bax, and Bcl-XL, these studies indicate that the level of the Bcl-2-associated protein, Bag-1, was closely associated with the growth (i.e. the proliferation and inhibition of apoptosis) of both the Ba/F3 and Nb2 lymphocyte lines. Coexpression of Bag-1 and Bcl-2 in Jurkat T cells resulted in a synergistic improvement in cellular viability secondary to apoptotic stimuli induced by Fas, staurosporine, or cytotoxic T lymphocytes (39). These effects were not noted in Jurkat transfectants that overexpressed Bag-1 alone. In contrast, the overexpression of Bag-1 alone in 3T3 fibroblasts was sufficient to inhibit staurosporine-induced apoptosis. These data suggest that the function of Bag-1 is influenced by its cellular environment and the nature of the apoptotic challenge. This hypothesis was further confirmed in the present study, which demonstrated that overexpression of the Bag-1 in Ba/F3 resulted in the induction of IL-3 independence. Thus, in the absence of IL-3, Bag-1 transfectants maintained high viability and continued to proliferate at a rate that exceeded that of the IL-3-stimulated parental line. Growth factor independence has been conferred on IL-3-dependent lines previously by the cotransfection of Bcl-2 and Myc (51), an effect not observed when either of these gene products was individually transfected (33, 34). Thus, these data suggest that the functional effects of Bag-1 may extend beyond the downstream effectors of Bcl-2 action.

The ability of Bag-1 to induce factor-independent cell growth may be related to its interaction with signaling factors intimately associated with apoptosis and mitogenesis, namely Bcl-2 and Raf-1. As part of a signaling cascade widely used by the growth factor receptor superfamily, Raf is a central serine/threonine kinase that controls cell cycle-dependent gene expression via a kinase cascade that induces the phosphorylation of specific transcription factors (i.e. Myc, Jun, and p62TCF) (55). These transcription factors are substrates for mitogen-activated protein kinase (56), which in turn is a substrate for mitogen-activated kinase kinase (MEK), the only high-affinity substrate of Raf-1 known to date (57, 58, 59). Raf-1 activity is allosterically modulated by the signaling proteins Ras, 14–3-3, and as most recently identified, Bag-1 (60, 61, 62, 63, 64). Like 14–3-3, Bag-1 binding occurs in the carboxy terminus of Raf-1 (60). Although this binding site represents the kinase domain of Raf-1, significant phosphorylation of Bag-1 does not occur. In vitro and in vivo data indicate, however, that this interaction up-regulates Raf-1 kinase activity (60). Recent studies indicate that Bcl-2 and Raf-1 can be coimmunoprecipitated (32, 65, 66) and that the conserved BH4 domain within Bcl-2 is required for interaction with both Raf-1 and Bag-1. Taken together, these data suggest that the Bcl-2/Raf-1 interaction may be facilitated by Bag-1. It remains to be determined whether the Bcl-2/Raf-1/Bag-1 complex is targeted to intracellular membranes where Bcl-2 resides (67), such as the mitochondrial outer membranes, nuclear membrane, endoplasmic reticulum, etc. Furthermore, the stoichiometric relationships and functional significance of each member of the Bax/Bcl-2/Bag-1/Raf-1 vis-a-vis apoptosis requires further elaboration. It is interesting to note that the overexpression of Raf-1 in growth factor-dependent cells can induce a spectrum of changes related to cell type, ranging from growth factor independence to apoptosis (65, 66). Thus, alterations in Bag-1 levels may directly effect the coupling and composition of the Bcl-2/Bag-1/Raf-1 complex, regulating its function during cellular proliferation and apoptosis. Decreases in Bag-1 levels (as seen in growth factor-deprived cells), therefore, may lead to an uncoupling of the complex and decreased viable cell growth. Conversely, the increased levels of Bag-1 may support the association of this signaling complex and stimulate the increased proliferative rates and cell densities observed in the Bag-1 transfectants. Alternatively, increases in Bag-1 levels may lessen the dependence on other rate-limiting nutrients or growth factors within the medium, thereby enabling enhanced rates of growth.

Like the IL-3-dependent Ba/F3 B-cell line, the PRL-dependent Nb2 T cell line has served as an excellent model for examining growth factor signal transduction. Previous studies have shown that treatment of this line with PRL or Dex results in mitogenesis or apoptosis, respectively (16, 17, 40). Addition of both hormones simultaneously to Nb2 cells inhibited proliferation and diminished, but did not entirely prevent, apoptosis. These in vitro data support several lines of in vivo research, indicating that one mechanism through which the body may regulate its immunoresponsiveness during periods of stress is by alterations in the PRL/Dex balance (11, 12, 13, 68, 69, 70). As evidenced by the SFJCD1 line, escape from these regulatory mechanisms can impart growth factor independence (17). The SFJCD1 subline was derived from Nb2; karyotypic analysis has demonstrated that a de novo translocation involving chromosomes 14 and 17 occurred within the SFJCD1 cells (41). Significant differences in PRLR-associated signaling also exist between these PRL-dependent and independent lines. During PRL stimulation of Nb2 cells, the activation of a cascade involving guanine nucleotide exchange factors (Sos- and Vav-associated activity), Ras, Raf, and mitogen-activated protein kinase has been documented (71, 72, 73). In contrast, both the PRL-independent Sp (71) and SFJCD1 cell lines contain a constitutively activated Raf-1, irrespective of the presence of PRL. Thus, these data indicate that some alteration in the inducible PRLR-associated kinase cascade has occurred in SFJCD1 cells. Although many possibilities exist, this up-regulation of Raf-1 kinase activity may result from its allosteric activation secondary to the constitutively elevated levels of Bag-1 found in SFJCD1. Studies to evaluate this hypothesis are currently underway in our laboratory. Nevertheless, these data, coupled with those from the Ba/F3 system, suggest that Bag-1 is involved in the integration of the mitogenic and apoptotic signaling cascades within the hematopoietic and the immune systems.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cell Culture
The mouse IL-3-dependent pro-B cell line, Ba/F3 (74), was maintained in RPMI 1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin in the presence of 1 ng IL-3/ml (Prepro-Tech, Rocky Hill, NJ). For experimental purposes this medium was used with varying concentrations of IL-3. The rat pre T cell lymphoma line Nb2/11 (hereafter referred to as Nb2) and the SFJCD1 cell lines (both obtained from Dr. Peter Gout) were cultured in Fisher’s medium containing 10% FBS, 10% gelding horse serum, 10-4 M ß-mercaptoethanol, and penicillin/streptomycin (40, 71). For experimental purposes, both the Nb2 and SFJCD1 cells were stimulated with varying concentrations of PRL (NIDDK) and Dex (Sigma, St. Louis, MO) in a chemically defined medium consisting of DMEM supplemented with sodium selenite, linoleic acid, insulin, transferrin (ITS+ supplement, Becton-Dickinson, San Jose, CA), ß-mercaptoethanol, and penicillin/streptomycin. For some studies, 5 ng PRL/ml and/or 100 nM Dex were used; these concentrations, respectively, have been found to induce maximal cell proliferation (71) or apoptosis (16). The rates of cell death induced by cytokine withdrawal for Ba/F3 and Nb2 differ and have been well documented (40, 71, 74); loss of exogenous IL-3 from the culture medium of Ba/F3 induces 90% cell death by 24–40 h, while removal of PRL from the Nb2 medium induces a similar level of death by 40–60 h. Cell density and viability were assessed by cell labeling with trypan blue; all measurements were performed in triplicate by hemocytometry on at least 200 cells per point.

Apoptosis Measurements
Cell apoptosis was measured by three independent methodologies, as follows: 1) Endonucleolytic DNA cleavage was detected in the Ba/F3 and Nb2 using a commercially available kit (Apoptag, Oncor, Gaithersburg, MD), as described previously (75). Briefly, 2 x 106 washed cells were sequentially fixed in 2% paraformaldehyde and 70% ethanol; DNA cleavage was then detected through the incorporation of ddUTP by terminal deoxynucleotide transferase. To determine the background level of ddUTP incorporation, parallel samples were incubated in the absence of terminal deoxynucleotidyl transferase as a control. Incorporated ddUTP was then subsequently labeled with a fluorescein-conjugated antidigoxigenin antibody. 2) Loss of cellular DNA, as manifest by a hypodiploid DNA content, was measured a described previously (76). Briefly, 2 x 106 washed cells were fixed in 70% ethanol, treated for 20 min with 150 units RNAseA (Worthington Biochemical Corp., Freehold, NJ)/ml, and labeled with 50 µg propidium iodide (Calbiochem, San Diego, CA)/ml. 3) Alteration in cellular morphology, as manifest by chromatin condensation, nuclear blebbing, and fragmentation, and cellular shrinkage were assessed by microscopy of Cytospin (Shandon, Sewickley, PA) preparations of 5 x 104 cells treated with a DiffQuick stain (3). Although the data from the morphological assessments for apoptosis are not presented here, they confirmed in all cases the data obtained by flow cytometry.

Flow Cytometry
For quantification of both endonucleolytic DNA cleavage and loss of DNA content, 1 x 104 cells were analyzed at 488 nm with a FACSTAR flow cytometer (Becton Dickinson) using appropriate filters, as described previously (77). For ddUTP labeling, control specimens (see above) were used to determine the cut-off below which 98% of the negative cells fell.

Immunoblot Analysis
Immunoblot analysis was performed as described previously (39) with modifications. Approximately 5 x 105 washed cells resuspended in 2x Laemmli buffer were subjected to 10% SDS-PAGE and transferred to nitrocellulose. The blot was blocked with a TN-TBM solution that consisted of 10 mM Tris (pH 7.7), 150 mM NaCl, 0.01% Triton X-100, 2% BSA, fraction V (Sigma), 5% non-fat dried skim milk, and 0.01% NaN3. Antigen was detected with a rabbit anti-mouse Bag-1 antiserum (39) at a 1:1000 (vol/vol) dilution in the TN-TBM buffer containing 1 µl normal goat serum and 50 ng/ml ovalbumin (Sigma). Parallel control blots were probed with preimmune rabbit serum. Antigen-antibody complexes were then detected using enhanced chemiluminescence (ECL kit, Amersham, Arlington Heights, IL).

Cell Transfection
For constitutive overexpression of Bag-1, full-length murine Bag-1 cDNA was subcloned into the BamHI site of Neo-pCMV, which contains the neomycin resistance gene and a CMV promoter/enhancer element upstream of the BamHI insertion site. Ba/F3 cells (1.5 x 107) were transfected with 50 µg of linearized empty vector (Neo-pCMV) or vector containing Bag-1 (Bag1-pCMV) by electroporation (78). Stable transfectants were generated by selection with G418, and clones were obtained by limiting dilution. Five independent clones from each transfection were selected on the basis of highest levels of Bag-1 expression as determined by immunoblot analysis. Although the data shown here are from two independent clones, comparable results were obtained for each of the five transfectants overexpressing Bag-1.

Statistics
Quantitative analyses of Bag-1 levels were obtained by scanning densitometry (Molecular Dynamics, Sunnyvale, CA) of anti-Bag-1 immunoblots. Unless otherwise indicated, all experiments were performed in triplicate and reported as mean values; where not visible, error bars SEM) lie within the symbol’s diameter.


    ACKNOWLEDGMENTS
 
We thank Mr. Seong-Joo Jeong and the Lucille Markey Flow Cytometry Unit at the University of Pennsylvania for their excellent support.


    FOOTNOTES
 
Address requests for reprints to: Charles V. Clevenger, M.D., Ph.D., Department of Pathology and Laboratory Medicine, University of Pennsylvania Medical Center, 509 Stellar-Chance Laboratories, 422 Curie Boulevard, Philadelphia, Pennsylvania 19104.

This work was supported by American Cancer Society Grant BE-250 (to C.C.) and NIH Grants AI-33510 (to C.C.) and CA-67329 (to J.R.). C. Clevenger is a recipient of an American Cancer Society Junior Faculty Research Award (JFRA-588).

Received for publication October 10, 1996. Revision received January 14, 1997. Accepted for publication February 13, 1997.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Williams GT, Smith CA, Spooncer E, Dexter TM, Taylor DR 1990 Hemopoietic colony stimulating factors promote cell survival by suppressing apoptosis. Nature 343:76–79[CrossRef][Medline]
  2. Wyllie AH 1980 Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature 284:555–556[Medline]
  3. Kerr JFR, Wyllie AH, Currie AR 1972 Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 26:239–257[Medline]
  4. Schrader JW, Thomson A (eds) 1994 Interleukin 3. In: The Cytokine Handbook, ed 2. Academic Press, San Diego, chapter 5, pp 81–98
  5. Iscove NN, Shaw AR, Keller G 1989 Net increase of pluripotential hematopoietic precursors in suspension culture in response to IL1 and IL3. J Immunol 142:2332–2337[Abstract/Free Full Text]
  6. Yanagida M, Fukamachi H, Ohgami K, Kuwaki T, Ishii H, Uzamaki H, Amano K, Tokiwa T, Mitsui H, Saito H, et al. 1995 Effects of T-helper 2-type cytokines, interleukin-3 (IL3), IL4, IL5, and IL6 on the survival of cultured human mast cells. Blood 86:3705–3714[Abstract/Free Full Text]
  7. Pelligrini I, Lebrun J-J, Ali S, Kelly PA 1992 Expression of prolactin and its receptor in human lymphoid cells. Mol Endocrinol 6:1023–1031[Abstract]
  8. Hartmann DP, Holoday JW, Bernton EW 1989 Inhibition of lymphocyte proliferation by antibodies to prolactin. FASEB J 3:2194–2202[Abstract/Free Full Text]
  9. Clevenger CV, Russell DH, Appasamy PM, Prystowsky MB 1990 Regulation of IL2-driven T-lymphocyte proliferation by prolactin. Proc Natl Acad Sci USA 87:6460–6464[Abstract]
  10. Clevenger CV, Altmann SW, Prystowsky MB 1991 Requirement of nuclear prolactin for interleukin-2-stimulated proliferation of T lymphocytes. Science 253:77–79[Medline]
  11. Bernton EW, Meltzer MS, Holaday JW 1988 Suppression of macrophage activation and T-lymphocyte function in hypoprolactinemic mice. Science 239:401–404[Medline]
  12. Benedetto N, Folgore A, Galdiero M, Meli R, DiCarol R 1995 Effect of prolactin, rIFN-gamma or rTNF-alpha in murine toxoplasmosis. Pathol Biol 43:395–400[Medline]
  13. Di Carlo R, Meli R, Galdiero M, Nuzzo I, Bentivoglio C, Carratelli CR 1993 Prolactin protection against lethal effects of salmonella typhimurium. Life Sci 53:981–989[CrossRef][Medline]
  14. Clevenger CV, Sillman AL, Hanley-Hyde J, Prystowsky MB 1992 Requirement for prolactin during cell cycle regulated gene expression in cloned T-lymphocytes. Endocrinology 130:3216–3222[Abstract]
  15. Schwarz LA, Stevens AM, Hrachovy JA, Yu-Lee L-Y 1992 Interferon regulatory factor-1 is inducible by prolactin, interleukin-2 and concanavalin A in T cells. Mol Cell Endocrinol 86:103–110[CrossRef][Medline]
  16. Fletcher-Chiappini SE, Comptom MM, Lavoie HA, Day EB, Witorsch RJ 1993 Glucocorticoid-prolactin interactions in Nb2 lymphoma cells: antiproliferative vs. anticytolytic effects. Proc Soc Exptl Biol Med 202:345–352[Abstract]
  17. Witorsch RJ, Day EB, Lavoie HA, Hashemi N, Taylor JK 1993 Comparison of glucocorticoid-induced effects in prolactin-dependent and autonomous rat Nb2 lymphoma cells. Proc Soc Exp Biol Med 203:454–460[Abstract]
  18. Lavoie HA, Witorsch RJ 1995 Investigation of intracellular signals mediating the anti-apoptotic action of prolactin in Nb2 lymphoma cells. Proc Soc Exp Biol Med 209:257–269[Abstract]
  19. Alderson MR, Tough TW, Davis-Smith T, Braddy S, Falk B, Schooley KA, Goodwin RG, Smith CA, Tamsdell F, Lynch DH 1995 Fas ligand mediates activation-induced cell death in human T-lymphocytes. J Exp Med 181:71–77[Abstract]
  20. Linette GP, Korsmeyer SJ 1994 Differentiation and cell death: lessons from the immune system. Curr Opin Cell Biol 6:809–815[Medline]
  21. Reed JC 1994 Bcl-2 and the regulation of programmed cell death. J Cell Biol 124:1–6[Medline]
  22. Boise LH, Gottschalk AR, Quintans J, Thompson CB 1995 Bcl-2 and Bcl-2-related proteins in apoptosis regulation. Curr Top Microbiol Immunol 200:107–121[Medline]
  23. Dhein J, Salczak H, Baumler C, Debatin K-M, Krammer PH 1995 Autocrine T-cell suicide mediated by APO-1/(Fas/CD95). Nature 373:438–441[CrossRef][Medline]
  24. Ju ST, Panka DJ, Cui H, Ettinger R, El-Khatib M, Sherr DH, Stanger BZ, Marshak-Rothstein A 1995 Fas(CD95)/FasL interaction required for programmed cell death after T-cell activation. Nature 373:444–448[CrossRef][Medline]
  25. Liu YJ, Joshua DE, Williams GT, Smith CA, Gordon J, Maclennan ICM 1989 Mechanism of antigen-driven selection in germinal centers. Nature 342:929–931[CrossRef][Medline]
  26. Smyth MJ 1995 Dual mechanisms of lymphocyte-mediated cytotoxicity serve to control and deliver the immune response. Bioessays 17:891–898[Medline]
  27. Merino R, Ding L, Veis DJ, Korsmeyer SJ, Nunez G 1994 Developmental regulation of the Bcl-2 protein and susceptibility to cell death in B lymphocytes. EMBO J 13:683–691[Abstract]
  28. Zubiaga AM, Munoz E, Huber BT 1992 IL4 and IL2 selectively rescue Th cell subsets from glucocorticoid-induced apoptosis. J Immunol 149:107–112[Abstract/Free Full Text]
  29. Thompson EB 1994 Apoptosis and steroid hormones. Mol Endocrinol 8:665–673[Medline]
  30. Migliorati G, Nicoletti I, Pagliacci MC, D’Adamio L, Riccardi C 1993 Interleukin-4 protects double-negative and CD4 single-positive thymocytes from dexamethasone-induced apoptosis. Blood 81:1352–1358[Abstract]
  31. Miyashita T, Reed JC 1992 Bcl-2 gene transfer increases relative resistance of S49.1 and WEHI7.2 lymphoid cells to cell death and DNA fragmentation induced by glucocorticoids and multiple chemotherapeutic drugs. Cancer Res 52:5407–5411[Abstract]
  32. Wang H-G, Millan JA, Cox A, Der CJ, Rapp UR, Beck T, Zha H, Reed JC 1996 R-ras promotes apoptosis caused by growth factor deprivation via a Bcl-2 suppressible mechanism. J Cell Biol 129:1103–1114[Abstract]
  33. Nunez G, London L, Hockenberry D, Alexander M, McKearn JP, Korsemeyer SJ 1990 Deregulated Bcl-2 gene expression selectively prolongs survival of growth factor-deprived hemopoietic cell lines. J Immunol 144:3602–3610[Abstract/Free Full Text]
  34. Marvel J, Perkins GR, Rivas AL, Collins MKL 1994 Growth factor starvation of Bcl-2 overexpressing murine bone marrow cells induce refractoriness to IL-3 stimulation of proliferation. Oncogene 9:1117–1122[Medline]
  35. Stellar H 1995 Mechanisms and genes of cellular suicide. Science 267:1445–1449[Medline]
  36. Oltvai ZN, Millman CL, Korsmeyer SJ 1993 Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programed cell death. Cell 74:609–619[Medline]
  37. Hanada M, Aime-Sempe C, Sato T, Reed JC 1995 Structure-function analysis of Bcl-2 protein. J Biol Chem 270:11962–11969[Abstract/Free Full Text]
  38. Sedlak TW, Oltvai ZN, Yang E, Wang K, Boise LH, Thomspon CB, Korsmeyer SJ 1995 Multiple Bcl-2 family members demonstrate selective dimerizations with Bax. Proc Natl Acad Sci USA 92:7834–7838[Abstract]
  39. Takayama S, Sato T, Krajewski S, Kochei K, Irie S, Millan JA, Reed JC 1995 Cloning and functional analysis of BAG-1: a novel Bcl-2-binding protein with anti-cell death activity. Cell 80:279–284[Medline]
  40. Gout PW, Beer CT, Noble RL 1980 Prolactin-stimulated growth of cell cultures established from malignant Nb rat lymphomas. Cancer Res 40:2433–2436[Abstract]
  41. Horsman DE, Masui S, Gout PW 1991 Karyotypic changes associated with loss of prolactin dependency of rat Nb2 node lymphoma cell cultures. Cancer Res 51:282–287[Abstract]
  42. Bazan JF 1990 Structural design and molecular evolution of a cytokine receptor superfamily. Proc Natl Acad Sci USA 87:6934–6938[Abstract]
  43. Ihle JN 1995 Cytokine receptor signaling. Nature 377:591–594[CrossRef][Medline]
  44. Horseman ND, Yu-Lee L-Y 1994 Transcriptional regulation by the helix bundle peptide hormones: growth hormone, prolactin, and hematopoietic cytokines. Endocr Rev 15:627–649[Medline]
  45. Witthuhn BA, Quelle FW, Silvennoinen O, Yi T, Tang B, Miura O, Ihle JN 1993 JAK2 associates with the erythropoietin receptor and is tyrosine phosphorylated and activated following stimulation with erythropoietin. Cell 74:227–236[Medline]
  46. Silvennoinen O, Witthuhn BA, Quelle FW, Cleveland JL, Yi T, Ihle JN 1993 Structure of the murine Jak2 protein tyrosine-kinase and its role in interleukin 3 signal transduction. Proc Natl Acad Sci USA 90:8429–8433[Abstract/Free Full Text]
  47. Quelle FW, Sato N, Witthuhn BA, Inhorn RC, Eder M, Miyajima A, Griffen JD, Ihle JN 1994 JAK2 associates with the bc chain of the receptor for granulocyte-macrophage colony-stimulating factor, and its activation requires the membrane-proximal region. Mol Cell Biol 14:4335–4341[Abstract]
  48. Sato N, Sakamaki K, Terada N, Arai K, Miyajima A 1993 Signal transduction by the high-affinity GM-CSF receptor: two distinct regions of the common beta subunit responsible for different signaling. EMBO J 12:4181–4189[Abstract]
  49. Satoh T, Nakafuku M, Miyajima A, Kaziro Y 1991 Involvement of ras p21 protein in signal-transduction pathways for interleukin2, interleukin3, and granulocyte/macrophage colony-stimulating factor, but not from interleukin 4. Proc Natl Acad Sci USA 88:3314–3318[Abstract]
  50. Welham MJ, Duronio V, Sanghera JS, Pelech SL, Schrader JW 1992 Multiple hemopoietic growth factors stimulate activation of mitogen-activated protein kinase family members. J Immunol 149:1683–1693[Abstract/Free Full Text]
  51. Vaux DL, Cory S, Adams JM 1988 Bcl-2 gene promotes hemopoietic survival and cooperates with c-myc to immortalize pre-B cells. Nature 335:440–442[CrossRef][Medline]
  52. Katsumata M, Siegel RM, Louie DC, Miyashita T, Tsujimoto Y, Nowell PC, Greene MI, Reed JC 1992 Differential effects of Bcl-2 on T and B cells in transgenic mice. Proc Natl Acad Sci USA 89:11376–11380[Abstract]
  53. McDonnell TJ, Deane N, Platt FM, Nunez G, Jaeger U, McKeran JP, Korsmeyer SJ 1989 Bcl-2-immunoglobulin transgenic mice demonstrate extended B cell survival and follicular lymphoproliferation. Cell 57:79–88[Medline]
  54. Leff MA, Buckley DJ, Krumenacker JS, Reed JC, Miyashita T, Buckley AR 1996 Rapid modulation of the apoptosis regulatory genes, bcl-2 and bax by prolactin in rat Nb2 lymphoma cells. Endocrinology 137:5456–5462[Abstract]
  55. Seth A, Gonzalez FA, Gupta S, Raden DL, Davis RJ 1992 Signal transduction within the nucleus by mitogen-activated protein kinase. J Biol Chem 267:24796–24804[Abstract/Free Full Text]
  56. Lange-Carter CA, Pleiman CM, Gardner AM, Blumer KJ, Johnson GL 1993 A divergence in the MAP kinase regula-tory network defined by MEK kinase and Raf. Science 260:315–319[Medline]
  57. Kyriakis JM, App H, Zhang X-F, Banerjee P, Brautigan DL, Rapp UR, Avruch J 1992 Raf-1 activates MAP kinase-kinase. Nature 358:417–421[CrossRef][Medline]
  58. Huang W, Alessandrini A, Crews CM, Erikson RL 1993 Raf-1 forms a stable complex with Mek1 and activates Mek1 by serine phosphorylation. Proc Natl Acad Sci USA 90:10947–51[Abstract]
  59. Force T, Bonventure JV, Heidecker G, Rapp U, Avruch J, Kyriakis JM 1994 Enzymatic characteristics of the c-Raf-1 protein kinase. Proc Natl Acad Sci USA 91:1270–4[Abstract]
  60. Wang H-G, Takayama S, Rapp UR, Reed JC 1996 Bcl-2 interacting protein, Bag-1, binds to and activates the kinase Raf-1. Proc Natl Acad Sci USA 93:7063–7068[Abstract/Free Full Text]
  61. Williams NG, Paradis H, Agarwal S, Charest DL, Pelech SL, Roberts TM 1993 Raf-1 and p21v-ras cooperate in the activation of mitogen-activated protein kinase. Proc Natl Acad Sci USA 90:5772–5776[Abstract]
  62. Dickson B, Sprenger F, Morrison D, Hafen E 1992 Raf functions downstream of Ras1 in the Sevenless signal transduction pathway. Nature 360:600–603[CrossRef][Medline]
  63. Van Aelst L, Barr M, Marcus S, Polverino A, Wigler M 1993 Complex formation between RAS and RAF and other protein kinases. Proc Natl Acad Sci USA 90:6213–6217[Abstract]
  64. Leevers SJ, Paterson HF, Marshall CJ 1994 Requirement for Ras in Raf activation is overcome by targeting Raf to the plasma membrane. Nature 369:411–414[CrossRef][Medline]
  65. Wang H-G, Miyashita T, Takayama S, Sato T, Torigoe T, Krajewski S, Tanaka S, Hovey L, Troppmair J, Rapp U, et al. 1994 Apoptosis regulation by interaction of Bcl-2 protein and Raf-1 kinase. Oncogene 9:2751–2756[Medline]
  66. Troppmair J, Cleveland JL, Askew DS, Rapp UR 1992 v-raf/v-myc synergism in abrogation of IL-3 dependence: v-raf suppresses apoptosis. Curr Top Microbiol Immunol 182:453–459[Medline]
  67. Krajewski S, Tanaka S, Takayama S, Schibler MJ, Fenton W, Reed JC 1993 Investigation of the subcellular distribution of the bcl-2 oncoprotein: residence in the nuclear envelope, endoplasmic reticulum, and outer mitochondrial membranes. Cancer Res 53:4701–14[Abstract]
  68. Kant GJ, Bauman RA, Anderson SM, Mougey EH 1992 Effects of controllable vs. uncontrollable chronic stress on stress-responsive plasma hormones. Physiol Behav 51:1285–1288[CrossRef][Medline]
  69. Prystowsky MB, Clevenger CV 1994 Prolactin as a second messenger for interleukin 2. Immunomethods 5:49–55[CrossRef][Medline]
  70. Zellweger R, Wichman MW, Ayala A, DeMaso CM, Chaudry IH 1996 Prolactin: a novel and safe immunomodulating hormone for the treatment of immunodepression following sever hemorrhage. J Surg Res 63:53–58[CrossRef][Medline]
  71. Clevenger CV, Torigoe T, Reed JC 1994 Prolactin induces rapid phosphorylation and activation of prolactin receptor associated Raf-1 kinase in a T-cell line. J Biol Chem 269:5559–5565[Abstract/Free Full Text]
  72. Erwin RA, Kirken RA, Malbarba MG, Farrar WL, Rui H 1995 Prolactin activates Ras via signaling proteins SHC, growth factor receptor bound 2, and son of sevenless. Endocrinology 136:3512–3518[Abstract]
  73. Clevenger CV, Ngo W, Luger SM, Gewirtz AM 1995 Vav is necessary for prolactin-stimulated proliferation and is translocated into the nucleus of a T-cell line. J Biol Chem 270:13246–13253[Abstract/Free Full Text]
  74. Chang W-P, Clevenger CV 1996 Modulation of growth factor receptor function by isoform heterodimerization. Proc Natl Acad Sci USA 93:5947–5952[Abstract/Free Full Text]
  75. Li X, Traganos F, Melamed MR, Darzynkeiwicz Z 1995 Single-step procedure for labeling DNA strand breaks with fluorescein- or BODIPY-conjugated deoxynucleo-tides: detection of apoptosis and bromodeoxyuridine incorporation. Cytometry 20:172–180[Medline]
  76. Brunetti M, Martelli N, Colasante A, Piantelli M, Musiani P, Aiello F 1995 Spontaneous and glucocorticoid-induced apoptosis in human mature T lymphocytes. Blood 86:4199–4205[Abstract/Free Full Text]
  77. Clevenger CV, Epstein AL, Bauer KD 1985 A method for simultaneous nuclear immunofluorescence and DNA content quantitation using monoclonal antibodies and flow cytometry. Cytometry 6:208–214[Medline]
  78. Baker SJ, Markowitz S, Fearon ER, Willson JKV, Vogelstein B 1990 Suppression of human colorectal carcinoma cell growth by wild-type p53. Science 249:912–915[Medline]