Feb 7, 1989 - The BAL-17.7.1 (BAL-. 17) B-lymphoma cell line was a gift from William Paul ..... Ryder, K., L. F. Lau, and D. Nathans. 1988. A gene activated by.
MOLECULAR AND CELLULAR BIOLOGY, May 1989, p. 2083-2088 0270-7306/89/052083-06$02.00/0 Copyright © 1989, American Society for Microbiology
Vol. 9, No. 5
Differential Expression of a Zinc Finger-Encoding Gene in Response to Positive versus Negative Signaling through Receptor Immunoglobulin in Murine B Lymphocytes VICKI L. SEYFERT,1 VIKAS P. SUKHATME,2 AND JOHN G. MONROE'* Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104,1 and Department of Medicine, Howard Hughes Medical Institute, University of Chicago, Chicago, Illinois 606372 Received 2 December 1988/Accepted 7 February 1989
Egr-1 is a murine early growth factor-inducible gene that encodes a protein with zinc fingers and that is believed to be involved in transcriptional regulation. Expression of this gene was investigated in murine B lymphocytes stimulated through their receptor for antigen (surface immunoglobulin [sIg]) with antireceptor antibodies (anti-slg). Rapid (by 15 min) up-regulation of Egr-l mRNA expression was observed after slg cross-linking at a dose of anti-sIg sufficient to drive the majority of cells into cell cycle. Interestingly, signaling through sIg on the murine B-lymphoma cell line WEHI-231 did not up-regulate Egr-1 expression even though similar signaling pathways, including up-regulation of c-fos expression, are associated with this receptor in these cells. Importantly, cell growth and proliferation of WEHI-231 cells were inhibited by anti-slg stimulation, which suggested a relationship between Egr-1 expression and differential processing of receptor immunoglobulin signals with respect to cellular growth responses. This notion was further supported by the finding that murine B lymphomas whose proliferation was not inhibited by anti-slg showed receptor immunoglobulin-coupled Egr-l expression. In further support of this association are results showing that under conditions in which Egr-l expression was induced in WEII-231 cells in response to stimulation by anti-sIg, a concomitant change was observed in the growth response of these cells. These resultsi then, indicate a potential role for Egr-1 expression in the translation of receptor-generated signals into cellular activation or induced unresponsiveness. B lymphocytes recognize and bind foreign antigens via immunoglobulin molecules on the cell surface. Cross-linking of surface immunoglobulin (sIg) by antibodies to slg (antisIg) can cause activation typified by elevated levels of class II antigen expression (28), transition into the G1 phase of the cell cycle (5, 30), and lymphokine responsiveness (16, 31). Under some conditions, cross-linking of slg can deliver negative signals resulting in B lymphocyte unresponsiveness or tolerance (41). In several systems, receptor signaling results in a rapid induction of a class of genes termed immediate-early response genes (1, 12, 23). A subset of these genes encode putative transcriptional regulators that may subsequently regulate other genes central to the long-term cellular responses associated with a given receptor-generated signal. One such gene, designated Egr-1, has been shown to be induced in a number of cellular systems in response to growth factor or mitogen stimulation (39). Interestingly, the predicted protein product of this gene possesses three tightly clustered zinc finger regions which are believed to mediate specific protein-DNA interactions (40). Indeed, zinc finger proteins have been demonstrated to be involved in regulation of a number of genes through specific cis-acting elements (3, 18, 26, 34). P-redicting that responses to signals generated by sIg cross-linking involve induction of and regulation by genes such as Egr-1, we have undertaken studies to investigate Egr-l as a possible intermediary between slg transmembrane signaling and subsequent B-lymphocyte responses. Using unprimed murine splenic B lymphocytes, we found that sIg-generated activation signals caused the induction of
Egr-J. In contrast, the murine B-cell lymphoma cell line WEHI-231, which utilizes seemingly identical mechanisms of sIg transmembrane signal transduction (10), exhibits down-regulation of proliferation after sIg cross-linking (4, 37) and does not up-regulate Egr-J in response to slggenerated signals. These results suggest that expression of Egr-l may be involved in the translation of sIg signals into positive or negative B-lymphocyte growth responses. Consistent with this interpretation, under conditions allowing Egr-l expression in response to stimulation by anti-slg in WEHI-231 cells, we observed a concomitant change in the growth response of these cells to signaling through slg. The implications of these findings with respect to positive versus negative activation signals through the B-lymphocyte antigen receptor are discussed. MATERIALS AND METHODS Reagents and cell lines. Affinity-purified goat anti-mouse mu (anti-,u) and phorbol myristate acetate (PMA) were obtained from Sigma Chemical Co. (St. Louis, Mo.). Lipopolysaccharide (LPS) from Salmonella typhosa was obtained from Difco Laboratories (Detroit, Mich.). Nick-translated [32P]dCTP-labeled ,B-actin (7), pfos-1 (8), and Egr-J(OC68) (39) were used to probe Northern (RNA) blots. The WEHI-231 B-lymphoma clone 28, obtained from David Scott (National Institutes of Health, Bethesda, Md.), was maintained in Dulbecco modified Eagle medium supplemented with 10% fetal calf serum (Hyclone, defined), SerXtend (Hana Biologicals, Berkeley, Calif.), 100 U of penicillin per ml, 100 ,ug of streptomycin per ml, 2 mM L-glutamine, and 5 x 10-5 M 2-mercaptoethanol. The BAL-17.7.1 (BAL17) B-lymphoma cell line was a gift from William Paul
* Corresponding author. 2083
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(National Institutes of Health) and was maintained in culture in RPMI 1640 supplemented with 5% fetal calf serum (Hyclone, defined) and the other supplements mentioned above. Preparation of B lymphocytes. Murine splenic B lymphocytes were isolated, purified, and cultured exactly as described previously (29). Isolation of total cellular RNA and Northern analysis. Unprimed splenic B lymphocytes (7 x 107) and WEHI-231 or BAL-17 cells (1.2 x 107) were placed in 20 ml of medium and equilibrated for 2 to 4 h at 37°C before stimulation. Indicated stimulants were added to appropriate samples, and incubation was continued at 37°C. After incubation, samples were centrifuged to pellet the cells; control (unstimulated) samples were centrifuged immediately after equilibration unless otherwise indicated. Total cellular RNA was isolated by the guanidium isothiocyanate method with the CsCl modification (6). Equal amounts of total RNA were size fractionated by electrophoresis through 1% agarose-formaldehyde gels and transferred to GeneScreen Plus nylon membranes (Du Pont Co., Wilmington, Del.) as described previously (29). Prehybridization and hybridization in the presence of 100 ng of 32P-labeled probe were performed at 42°C in the presence of 50% formamide. Washing at high stringency was performed as described previously (29). [3HJthymidine incorporation assay. Triplicate cultures of 5 x 104 viable cells were incubated in 200 pul of medium in 96-well flat-bottom microdilution plates (Costar, Cambridge, Mass.) for 48 h. At 16 h before harvesting, the cultures were pulsed with 1 p.Ci of [3H]thymidine. Cells were harvested with a cell harvester (PHD, Cambridge, Mass.) and washed with 10% trichloroacetic acid. The radioactivity incorporated was measured by liquid scintillation spectrophotometry. RESULTS Egr-1 mRNA levels increase in resting murine B lymphocytes after stimulation through the B-cell antigen receptor. B lymphocytes were stimulated with antibodies directed to the mu heavy chain of their antigen receptor (anti-p.). Northern analysis of total RNA isolated from anti-p.-stimulated B lymphocytes showed that expression of Egr-J at the mRNA level was clearly higher by 15 min after sIg cross-linking with 10 p.g of anti-p. antibody per ml (Fig. la) than was observed in unstimulated B lymphocytes. This amount of anti-pu antibody was shown in previous studies (5, 11) to drive the majority of resting B lymphocytes into cell cycle. Further marked elevation of Egr-J expression was observed at 0.5 and 1 h after continuous anti-.. stimulation. Expression returned to basal levels by 2 h after stimulation (data not shown), consistent with observations of growth factor stimulation of other cell types (39). Since ,B-actin mRNA levels remained constant throughout the time course for these stimulated cells (Fig. lb), the observed Egr-J increase was not due to a general increase in mRNA levels. These results demonstrate that ligand-induced crosslinking of membrane-associated immunoglobulin results in rapid up-regulation of Egr-J expression. As with other receptors that are linked to the growth or proliferative response of cells (39), the B-lymphocyte antigen receptor appears to be linked to regulation of the Egr-J gene, which suggests that Egr-J is a component of the B-lymphocyte activation response generated through slg. Signaling through slg on murine B-lymphoma WEHI-231 cells does not up-regulate expression of Egr-1. The ,u-positive murine B-lymphoma cell line WEHI-231 is an interesting cell
MOL. CELL. BIOL.
a
b
.25.5 1 0 .25 .5 1 0 Time of Stimulation (hours) FIG. 1. Up-regulation of Egr-J expression after anti-p. stimulation of normal splenic B lymphocytes. Total cellular RNA was isolated from B lymphocytes stimulated for the indicated times with anti-p. (10 p.g/ml) and subjected to Northern analysis (O h, no stimulation). The same blot was analyzed for Egr-I (a) and P-actin (b) expression. Tick marks indicate positions of 28S and 18S rRNAs.
line with respect to slg signaling. Whereas ligand-induced cross-linking of normal B lymphocytes generates signals positive for entry into cell cycle and proliferation (5, 11), similar stimulation of WEHI-231 cells results in downregulation of proliferation and eventually cell death (4, 37). This drastically different response occurs despite seemingly identical transmembrane signaling such as G-protein-coupled inositol phospholipid hydrolysis (13, 19), Ca2+ fluxes (20, 27, 33), and protein phosphorylation (15). Furthermore, with respect to immediate-early gene induction, crosslinking of slg on WEHI-231 cells leads to up-regulation of both c-fos (29) and c-myc (25, 38) expression, with kinetics similar to that observed for normal B lymphocytes. The c-fos response appears to be linked to the protein kinase C (PKC) component of sIg signaling (29). On the basis of these observations, we expected Egr-J mRNA to be up-regulated in WEHI-231 cells after sIg cross-linking with anti-p. or activation of PKC by PMA. Surprisingly, no detectable expression of Egr-J was observed in WEHI-231 cells stimulated with either agent (Fig. 2a). A simultaneously hybridized blot containing identical amounts of unstimulated and anti-p.-induced RNA from normal B lymphocytes (Fig. 2b) was exposed to film for the same length of time as was the WEHI-231 blot, and marked up-regulation of Egr-J was detected. Also, the lack of Egr-J expression was not due to a lack of signaling response by WEHI-231 cells, since c-fos up-regulation (Fig. 2c) paralleled that observed previously for WEHI-231 cells and normal B lymphocytes (29). Finally, probing of the same blot for P-actin expression verified that roughly equivalent amounts of hybridizable RNA were present in all lanes. Anti-,u and PMA induce expression of Egr-1 in the Blymphoma cell line BAL-17. The inability to induce Egr-J expression in WEHI-231 cells could be explained by the fact that these cells are transformed rather than by the more interesting possibility that this inability is related to the difference in the slg-signaled proliferative response. To determine whether this effect was related to B-lymphocyte transformation in general, we examined the effects of anti-p. and PMA on another murine B-lymphoma cell line. Shown in Fig. 3 are Northern blots of BAL-17 cells stimulated with
Egr-l EXPRESSION IN sIg SIGNALING
VOL. 9, 1989
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b
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_0 W.,
.~.
_
la % t.,
c
-
41 ,. t
.'_
0.5 1 2
0.5 1 2
anti-p
PMA
hours
FIG. 3. Induction of Egr-l in murine B-lymphoma BAL-17.7.1 in response to B-lymphocyte antigen receptor stimulation. Shown are Northern blots of total cellular RNA from BAL-17.7.1 cells stimulated with anti-IL (10 ,ug/ml) or PMA (10 ng/ml) for the indicated times. Tick marks indicate positions of 28S and 18S rRNAs.
cells
We compared the effects of anti-,u cross-linking on the proliferative responses by these two transformed lines (Fig. 4). Treatment of BAL-17 and WEHI-231 cells with anti-,u antibody resulted in markedly different responses, as measured by [3H]thymidine incorporation. In contrast to WEHI-231 cells, whose proliferation was clearly abrogated by anti-,u, proliferation of BAL-17 cells remained elevated and even slightly augmented. These results, coupled with those obtained by using normal B lymphocytes, suggest a linkage between Egr-J expression and pathways leading to cellular growth and proliferation in murine B lymphocytes. Anti-> induces Egr-I expression in LPS-pretreated WEHI231 cells. Treatment of WEHI-231 cells with LPS has been shown to protect these cells from the antiproliferative effects of anti-> (17). This protection has been proposed to be analogous to the LPS-induced maturation of pre-B and immature B cells, rendering them subsequently responsive to the activation effects of antigen (14). To further explore the association of Egr-J expression and discrimination between negative versus positive signaling through slg, we evaluated the effect of LPS treatment of WEHI-231 cells on the ability to induce Egr-J expression after sIgM crosslinking. WEHI-231 cells were pretreated for 24 h with 50 ,ug of LPS per ml, followed by stimulation with anti-p. antibody. Pretreated cells clearly expressed significant levels of Egr-J in response to sIgM cross-linking (Fig. 5a). In sharp contrast to the results observed in LPS-untreated WEHI-321 cells (Fig. 2c), marked induction of Egr-J was observed by 0.5 h after sIgM cross-linking. Less but discernible expression was seen at 1 h after stimulation by anti-p.. Reprobing with ,B-actin was performed to confirm that equal amounts of mRNA were present in all lanes (not shown). Parallel [3H]thymidine incorporation assays showed that LPS pretreatment completely abrogated the antiproliferative effects of anti-p. on WEHI-231 cells (Fig. Sb). In contrast to response.
0 .5 1 2 6 0 .5 1 2 6 hours
anti-p
PMA
FIG. 2. Absence of induction of Egr-l expression in B-lymphoma WEHI-231 cells after anti-p> or PMA stimulation. Shown are Northern blots of total cellular RNA from WEHI-231 cells stimulated with anti-,u (10 jg/ml) or PMA (10 ng/ml) for the indicated times. The same blot was analyzed for expression of Egr-l (a), c-fos (c) and 3-actin (d). Panel b shows Egr-J expression by unstimulated murine splenic B lymphocytes that were either unstimulated (6) or stimulated for 1 h with 10 ,ug of anti-,u per ml. The total amount of RNA per lane and time of autoradiographic exposure were identical for experiments in panels a and b. Tick marks indicate positions of 28S and 18S rRNAs.
anti-,u or PMA. In contrast to the results for WEHI-231 cells,
Egr-J mRNA levels in BAL-17 cells were markedly upregulated, relative to levels in unstimulated cells, by both PMA and anti-IL, as has been observed for normal splenic B lymphocytes (V. Seyfert and J. Monroe, unpublished data). PMA appeared to be more effective than anti-p. in upregulating Egr-J mRNA expression, perhaps because of its direct and more prolonged activation of PKC. The blot was reprobed with 0-actin to verify that there was equal loading of RNA in all lanes (not shown). These data demonstrate that B-lymphocyte transformation itself does not account for the inability to induce Egr-J expression in WEHI-231 cells. Differential expression of Egr-J between BAL-17 and WEHI-231 cells is also important in that it suggests a correlation between Egr-l induction and a positive growth
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a
180 E BAL-17.7.1 u 160 - WEHI-231 0 ~-140 0)
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120
A
E100 a
E
4
2
80 60 40 20 0
18S -
.01
.1
1
2
5
10
[antibody] (pg/ml) FIG. 4. Proliferation of BAL-17.7.1 cells is not down-regulated by anti-p.-induced slg cross-linking. BAL-17.7.1 or WEHI-231 cells were cultured iti the presence of the indicated amounts of anti-pL antibody, pulsed with [3H]thymidine, and harvested as described in Materials and Methods. Results are expressed as percent proliferative response in counts per minute of incorporated [3H]thymidine relative to incorporation by cultures receiving no anti-,u stimulation (100%). Values represent means of three replicate cultures + standard errors of the means. Values for non-anti-,u-stimulated cultures were 171,054 ± 11,626 for BAL-17.7.1 cells and 133,072 + 9,021 for WEHI-231 cells.
-
+
+
+
+
LPS Pretreatment
-
-
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1
2
Anti-p stimulation (hrs)
b E
,,> 600 c:
U=1 No Pretreatment E_ LPS Pretreatment
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None
untreated cells, WEHI-231 cells pretreated for 24 h in the presence of 50 ,ug of LPS per ml were not inhibited in response to anti-IL stimulation. In this case, the response of LPS-pretreated WEHI-231 cells to anti-p. appeared to even be augmented (118%) over that of nonstimulated, LPSpretreated cells. Thus, under conditions permitting Egr-J expression after anti-,u stimulation, there is a concomitant change in the growth response of WEHI-231 cells in response to this signaling. These observations are consistent with a role for Egr-J expression in the translation of slg signals into distinct phenotypic responses. DISCUSSION Coupling of growth factor- or mitogen-induced stimulation to long-term adaptive cellular responses is accomplished in part by induction of a subset of immediate-early growth response genes that encode transcriptional regulatory factors. These include c-jun (21, 32, 36) and related genes (35) as well as several genes that encode zinc finger proteins (3, 17a, 18, 26, 34, 40). We have examined the expression of one member of the latter group (Egr-1) in the B-lymphocyte activation-growth response. We have shown that Egr-J is rapidly induced when resting B lymphocytes are stimulated with anti-p. under conditions that promote entry into G1. These results, combined with the prediction that the Egr-J protein product binds DNA, suggest that Egr-J plays a role in the regulation of the B-lymphocyte Go-to-G1 transition and/or proliferative responses induced by sIg cross-linking. Further support for the involvement of Egr-J in slg signaling is the observed up-regulation of Egr-J in two B-lymphocyte tumor lines, BAL-17 (Fig. 3) and FRMB.1 (J. Monroe, unpublished data), after cross-linking of their sIgs by anti-sIg or stimulation with PMA. These results are consistent with a role for Egr-J in propagating slg activation signals, since both B lymphocytes and tumor cell lines, which respond positively (activation, proliferation, or both), up-regulate
Aritit-
,
FIG. 5. Pretreatment of WEHI-231 cells with LPS allows induction of Egr-l expression by anti-IL and reverses the antiproliferative response. (a) WEHI-231 cells were cultured for 24 h in the presence of 50 jig of LPS per ml (except for the negative control, leftmost lane). After the initial pretreatment period, the cells were washed and recultured at 37°C in the presence of anti-p. (5 ,ug/ml) for the indicated times; the cells were then harvested, and total cellular RNA was isolated and fractionated as described in Materials and Methods. Unstimulated cultures were harvested at time zero. Relative Egr-J expression was determined by hybridization to the OC68 probe. (b) WEHI-231 cells were pretreated for 24 h in the presence of 50 p.g of LPS per ml. At the end of the culture, cells were washed twice to remove LPS, and equal numbers (2 x 104 per well ) of either LPS-pretreated or untreated cells were placed in culture with or without anti-p. antibody (5 jig/ml). Relative thymidine incorporation was determined at 48 h as described in Materials and Methods. Values represent means of triplicate cultures + standard errors of the means.
Egr-J after anti-p. stimulation. However, more intriguing is the observation that Egr-J is not up-regulated by slg crosslinking in WEHI-231 cells, in which the response to anti-p. stimulation is negative (inhibitory for growth). The characteristic inhibitory growth response of WEHI231 cells to slg cross-linking coupled with the phenotypic similarities of this cell line to immature B lymphocytes (4, 22, 37) has led to the notion that the WEHI-231 cell line may represent a model for B-lymphocyte tolerance induction (9, 37). Interestingly, anti-,u-stimulated WEHI-231 cells respond nearly the same as do normal B lymphocytes with respect to defined early signal transduction events which are thought to be responsible for propagating sIg activation signals (10). The observation that direct activation of PKC with PMA also causes down-regulation of proliferation in WEHI-231 cells (29) suggests that translation of these signals into a negative response occurs relatively late in the signaling process. The role of immediate-early genes in the positive or negative growth response of B lymphocytes is unknown; however, it is plausible that control over these responses is
VOL. 9, 1989
exerted at the transcriptional level by expression of regulatory growth response genes. The possibility exists, then, that translation of identical transmembrane signals into either positive or negative cellular growth responses may involve differential expression of regulatory genes. To this extent, lack of Egr-l expression in WEHI-231 cells may be indicative of or even responsible for the negative growth response to slg signaling in these cells. We hypothesize that Egr-J expression may be intimately linked to distinct growth responses after slg signaling. This notion is supported by our observations that under conditions in which the antiproliferative effects of sIg signaling are reversed by LPS, induction pathways leading to Egr-J expression are now operative. Given that WEHI-231 cells are analogous to immature B lymphocytes (9, 22) and that the antiproliferative effects of anti-p. represent a model of induction of neonatal B-cell tolerance (41), these results suggest that developmental control over Egr-J inducibility may be related to translation of sIg signals into a state of unresponsiveness or tolerance. This hypothesis is currently being tested in our laboratory through direct manipulation of the cloned Egr-l gene. Finally, another interesting aspect of Egr-l expression in WEHI-231 cells (not LPS treated) is the apparent differential regulation with respect to c-fos. In all other systems in which mitogen- or growth factor-stimulated changes in Egr-J expression have been studied, c-fos and Egr-l appear to be coregulated (40). The apparent coregulation of these genes may be explained by regulatory sequence homologies between the two genes. The 5' nontranscribed and putative regulatory region of Egr-J has several elements in common with those shown to be important in the induction of c-fos after growth factor stimulation (43). Specifically, the 5' region of the Egr-J gene contains two AP-1-binding sites and a sequence homologous to the dyad symmetry response element of c-fos (42, 43). To the extent that these elements are responsible for PMA inducibiity (presumably through PKC) of both c-fos (2, 24) and Egr-J, a lack of transactivating factors is not likely to explain the differential regulation of Egr-J in WEHI-231 cells and the other cell systems discussed. Rather, differential expression of Egr-J in WEHI-231 cells versus other B lymphocytes in which positive signaling is observed may reflect more permanent alterations in chromatin structure or active repression. Elucidation of mechanisms controlling Egr-J expression in these systems as well as the role of this gene in receptor immunoglobulin-coupled responses is currently under investigation in our laboratories. ACKNOWLEDGMENTS We thank Michael Cancro, Amy Yellen, and David Levy for critical review of the manuscript. This work was supported by Public Health Service grant A123568 from the National Institutes of Health and grant IM497 from the American Cancer Society to J.G.M. V.L.S. is a predoctoral student supported by an immunobiology training grant, and V.P.S. is supported by the Howard Hughes Medical Institute. J.G.M. is a Scholar of the Leukemia Society of America.
LITERATURE CITED 1. Almendral, J. M., D. Sommer, H. MacDonald-Bravo, J. Burckhardt, J. Perera, and R. Bravo. 1988. Complexity of the early genetic response to growth factors in mouse fibroblasts. Mol. Cell. Biol. 8:2140-2148. 2. Angel, P., M. Imagawa, R. Chiu, B. Stein, R. J. Imbra, H. J. Rahmsdorf, C. Jonat, P. Herrlich, and M. Karin. 1987. Phorbol ester-inducible genes contain a common cis element recognized
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by a TPA-modulated trans-acting factor. Cell 49:729-739. 3. Blumberg, H., A. Elsen, A. Sledziewski, D. Bader, and E. T. Young. 1987. Two zinc fingers of a yeast regulatory protein shown by genetic evidence to be essential for its function. Nature (London) 328:443-445. 4. Boyd, A., and J. Schrader. 1981. The regulation and growth of a murine B cell lymphoma. II. The inhibition of WEHI-231 by anti-immunoglobulin antibodies. J. Immunol. 126:2466-2469. 5. Cambier, J. C., and J. G. Monroe. 1984. B cell activation. V. Differential signaling of B cell membrane depolarization, increased I-A expression, GO to Gl transition, and thymidine uptake by anti-IgM and anti-IgD antibodies. J. Immunol. 133: 576-581. 6. Chirgwin, J. M., A. E. Przybyla, R. J. MacDonald, and W. J. Rutter. 1979. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18:52945298. 7. Cleveland, D. W., M. A. Lopata, R. J. MacDonald, N. J. Cowan, W. J. Rutter, and M. W. Kirschner. 1980. Number and evolutionary conservation of alpha- and beta-tubulin and cytoplasmic beta- and gamma-actin genes using specific cloned cDNA probes. Cell 20:95-105. 8. Curran, T., G. Peters, C. van Beveran, N. M. Teich, and I. M. Verma. 1982. FBJ murine osteosarcoma virus: identification and molecular cloning of biologically active proviral DNA. J. Virol.
44:674-682. 9. DeFranco, A. L., M. M. Davis, and W. E. Paul. 1982. WEHI-231 as a tumor model for tolerance induction in immature B lymphocytes. UCLA Symp. Mol. Cell. Biol. 24:445-450. 10. DeFranco, A. L., M. R. Gold, and J. P. Jakway. 1987. Blymphocyte signal transduction in response to anti-immunoglobulin and bacterial lipopolysaccharide. Immunol. Rev. 95:161176. 11. DeFranco, A. L., E. Raveche, R. Asofsky, and W. E. Paul. 1982. Frequency of B lymphocytes responsive to anti-immunoglobulin. J. Exp. Med. 155:1523-1536. 12. Denhardt, D. T., D. R. Edwards, and C. L. Parfett. 1986. Gene expression during the mammalian cell cycle. Biochim. Biophys. Acta 865:83-125. 13. Gold, M. R., J. P. Jakway, and A. L. DeFranco. 1987. Involvement of a guanine nucleotide-binding component in membrane IgM-stimulated phosphoinositide breakdown. J. Immunol. 139: 3604-3613. 14. Hammerling, U., A. F. Chin, and J. Abbott. 1976. Ontogeny of murine B lymphocytes: sequence of B-cell differentiation from surface-immunoglobulin-negative precursors to plasma cells. Proc. Natl. Acad. Sci. USA 73:2008-2012. 15. Hornbeck, P., and W. E. Paul. 1986. Anti-immunoglobulin and phorbol ester induce phosphorylation of proteins associated with the plasma membrane and cytoskeleton in murine B lymphocytes. J. Immunol. 261:14817-14824. 16. Isakson, P. 1986. Antiimmunoglobulin-treated B cells respond to a B cell differentiation factor for IgG. J. Exp. Med. 164:303-308. 17. Jakway, J. P., W. R. Usinger, M. R. Gold, R. I. Mishell, and A. L. DeFranco. 1986. Growth regulation of the B lymphoma cell line WEHI-231 by anti-immunoglobulin, lipopolysaccharide, and other bacterial products. J. Immunol. 137:2225-2231. 17a.Joseph, L. J., M. M. LeBeau, G. A. Jamieson, S. Acharya, T. B. Show, J. D. Rowley, and V. P. Sukhatme. 1988. Molecular cloning, sequencing, and mapping of EGR2: a human early growth response gene encoding a protein with zinc fingers. Proc. Natl. Acad. Sci. USA 85:7164-7168. 18. Kadonaga, J. T., K. R. Carner, F. R. Masiarz, and R. Tjian. 1987. Isolation of cDNA encoding transcription factor Spl and functional analysis of the DNA binding domain. Cell 51:10791090. 19. Klaus, G. G. B., and M. Harnett. 1988. G protein coupling of antigen receptor-stimulated polyphosphoinositide hydrolysis in B cells. 140:3135-3139. 20. LaBaer, J., R. Y. Tsien, K. A. Fahey, and A. L. DeFranco. 1986. Stimulation of the antigen receptor of WEHI-231 B lymphoma cells results in a voltage-independent increase in cytoplasmic calcium. J. Immunol. 137:1836-1844.
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SEYFERT ET AL.
21. Lamph, W. W., P. Wamsley, P. Sassone-Corsi, and I. M. Verma. 1988. Induction of proto-oncogene JUN/AP1 by serum and TPA. Nature (London) 334:629-631. 22. Lanier, L., N. Warner, J. Ledbetter, and L. Herzenberg. 1981. Quantitative immunofluorescent analysis of surface phenotypes of murine B cell lymphomas and plasmacytomas with monoclonal antibodies. J. Immunol. 127:1691-1697. 23. Lau, L. F., and D. Nathans. 1987. Expression of a set of growth-related immediate early genes in Balb/c 3T3 cells: coordinate regulation with c-fos and c-myc. Proc. Natl. Acad. Sci. USA 84:1182-1186. 24. Lee, W., P. Mitchen, and R. Tjian. 1987. Purified transcription factor AP-1 interacts with TPA-inducible enhancer elements. Cell 49:741-752. 25. McCormack, J., V. Pepe, R. Kent, M. Dean, A. MarshakRothstein, and G. Sonnenshein. 1984. Specific regulation of c-myc oncogene expression in a murine B-cell lymphoma. Proc. Natl. Acad. Sci. USA 81:5546-5550. 26. Miller, J., A. D. McLachlen, and A. Klug. 1985. Repetitive zinc-binding domains in the protein transcription factor IIIA from xenopus oocytes. EMBO J. 4:1609-1614. 27. Mizuguchi, J., W. Tsang, S. L. Morrison, M. A. Bevan, and W. E. Paul. 1986. Membrane IgM, IgD, and IgG act as signal transmission molecules in a series of B lymphomas. J. Immunol. 137:2162-2167. 28. Mond, J. J., K. Segal, J. Kung, and F. D. Finkleman. 1981. Increased expression of I-region-associated antigen (Ia) on B cells after crosslinking of surface immunoglobulin. J. Immunol. 127:881-885. 29. Monroe, J. G. 1988. Up-regulation of c-fos expression is a component of the mlg signal transduction mechanism but is not indicative of competence for proliferation. J. Immunol. 140: 1454-1460. 30. Monroe, J. G., and M. Kass. 1985. Molecular events in B cell activation. I. Signals required to stimulate GO to Gl transition of resting B lymphocytes. J. Immunol. 135:1674-1682. 31. Parker, D. C. 1980. Induction and suppression of polyclonal antibody responses by anti-Ig reagents and antigen-nonspecific helper factors: a comparison of the effects of anti-Fab, anti-IgM, and anti-IgD on murine B cells. Immunol. Rev. 52:115-139.
MOL. CELL. BIOL. 32. Quantin, B., and R. Breathnach. 1988. Epidermal growth factor stimulates transcription of the c-jun proto-oncogene in rat fibroblasts. Nature (London) 334:538-539. 33. Ransom, J., D. Digiusto, and J. C. Cambier. 1986. Single cell analysis of calcium mobilization in anti-immunoglobulin-stimulated B lymphocytes. J. Immunol. 136:54-57. 34. Rhodes, D., and A. Klug. 1986. An underlying repeat in some transcriptional control sequences corresponding to half a double helical turn of DNA. Cell 46:123-132. 35. Ryder, K., L. F. Lau, and D. Nathans. 1988. A gene activated by growth factors is related to the oncogene v-jun. Proc. Natl. Acad. Sci. USA 85:1487-1491. 36. Ryseck, R.-P., S. I. Hirai, M. Yaniv, and R. Bravo. 1988. Transcriptional activation of c-jun during the GWG1 transition in mouse fibroblasts. Nature (London) 334:535-537. 37. Scott, D. W., J. Tuttle, D. Livnat, W. Haynes, J. Cogswell, and P. Keng. 1985. Lymphoma models for B cell activation and tolerance. II. Growth inhibition by anti-,u of WEHI-231 and the selection properties of resistant mutants. Cell. Immunol. 93: 124-131. 38. Snow, E. C., J. D. Fetherston, and S. Zimmer. 1986. Induction of the c-myc protooncogene after antigen binding to hapten-specific B cells. J. Exp. Med. 164:944-949. 39. Sukhatme, V. P., S. Kartha, F. G. Toback, R. Taub, R. G. Hoover, and C. H. Tsai-Morris. 1987. A novel early growth response gene rapidly induced by fibroblast, epithelial cell and lymphocyte mitogens. Oncogene Res. 1:343-355. 40. Sukhatmne, V. P., C. Xinmin, L. C. Chang, H. M. Chon-Hwa, D. Stamenkovich, P. Ferreira, D. Cohen, S. Edwards, T. Shows, T. Curran, M. LeBeau, and E. Adamson. 1988. A zinc fingerencoding gene coregulated with c-fos during growth and differentiation, and after cellular depolarization. Cell 53:37-43. 41. Teale, J., and N. Klnman. 1980. Tolerance as an active process. Nature (London) 288:385-387. 42. Treisman, R. 1986. Identification of a protein-binding site that mediates transcriptional response of the c-fos gene to serum factors. Cell 46:567-574. 43. Tsai-Morris, C., X. Cao, and V. P. Sukhatme. 1988. 5'-Flanking sequence and genomic structure of Egr-1, a murine mitogen inducible zinc finger encoding gene. Nucleic Acids Res. 16: 8835-8846.
