EXPERIMENTAL
CELL
RESEARCH
203,
285-288
(1992)
SHORT NOTE The El a Gene Prevents Inhibition of Keratinocyte Proliferation by Dexamethasone M. FLORIN-CHRISTENSEN,* Departments
of *Internal
Medicine
C. MISSRRo,t
and TPathology,
G. P. DOTTO,?’
Yale University
School of Medicine,
Glucocorticoids have inhibitory effects on the proliferation of several cell types. In this study, we found that dexamethasone, a synthetic steroid with glucocorticoid activity, inhibits proliferation of established mouse Pam 212 keratinocytes. Transfection with the adenoviral early region la (Ela) gene confers a strong resistance to the inhibition by dexamethasone. Two deletion Ela mutants, one whose product lacks the ability to bind the cellular proteins p6O/p105/p107 and another that is unable to bind ~300, were shown to induce a resistance similar to that associated with the intact Ela gene. These results differ from those previously observed with two other growth inhibitory signals, transforming growth factor p1 and adenosine 3’,5’-cyclic monophosphate, in which the mutated E la genes confer only partial or no resistance, indicating that a different mechanism mediates resistance against glucocorti0 1992 Academic Press, Inc. coids.
INTRODUCTION It is now recognized that cell proliferation is regulated not only by growth factors, but also by inhibitory signals. Among them, the transforming growth factors /3 (TGF-P) have been found to have strong inhibitory effects on keratinocyte growth [l]. In addition, similar effect,s have been described for agents that elevate CAMP intracellular levels [2,3]. Another line of investigation has disclosed the existence of several genes, which either positively or negatively affect cell proliferation [4,5]. Among the first are classical protooncogenes, like c-ras, c-myc, and c-fos [6]. Those in the second group are known as tumor suppressor genes. They include the gene encoding the retinoblastoma protein
1 To whom correspondence should he addressed. Fax: (203)7857467. ’ Present address: University of Cincinnati, Department of Biological Sciences, Cincinnati, OH 45221-0006. 285
AND J. FLORIN-CHRISTENSEN~ 310 Cedar Street, New Hauen, Connecticut
06510
~105, which is probably the best characterized, and the one encoding the ~53 nuclear protein [4,5]. Recent work has pointed out possible connections between negatively acting extracellular signals and the function of tumor supressor genes [7, 81. The adenoviral early region la (Ela) gene encodes a series of related proteins with multiple effects, including the immortalization of primary culture cells [9], the trans-activation of a variety of genes, and the repression of others [lo]. Immunoprecipitation experiments have shown that the proteins encoded by Ela form stable complexes with a number of cellular proteins. These include the retinoblastoma ~105; a related protein ~107; ~60, now identified as cyclin A; and ~300, a nuclear protein which may be involved in the control of DNA synthesis as well as in gene transcription [ll-161. In addition, at least five other proteins are known to associate with Ela [ 121. It has been shown that the binding to the cellular proteins ~105, ~107, and ~300 is critical to the Ela transforming effects [17, 181. Transfection of keratinocytes with the Ela gene renders them completely resistant to the growth inhibition induced by TGF-/3, [ 191. If cells are transfected with mutated versions of the Ela gene whose products are unable to bind either the proteins p6O/p105/p107 or ~300, the resistance to TGF-P, is only partial in each case, indicating that both regions of the gene are important to prevent inhibition [ 191. We have recently found that Ela also induces resistance to adenosine 3’,5’-cyclic monophosphate (CAMP) growth inhibition. In this case, the effect appears to involve binding by Ela to p60/ pIO5/pIO7 but not ~300 1201. A third type of growth inhibitory signals are the glucocorticoid steroids. Their inhibitory action has been known for many years [Zl] but the mechanism involved is still unknown [22]. They affect selectively certain tissues in whole animals [21]. Keratinocytes are susceptible to inhibition [23], and effects on other cell types have been recently described [24, 251. In this study, we examine the effect of Ela transformation on the suscepnn14-4827/92 $5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
286
SHORT
‘*Oc
NOTE assays were carried out in triplicate condition differed by less than 10%. RESULTS
I
-
1L .l
1
FM DEX
FIG. 1. Dose effect of DEX on the proliferation of Pam 212 keratinocytes. Subconfluent cultures of Pam 212 were exposed to different doses of DEX (IO-’ to 1Om6M) for 18 h after which [3H]TdR incorporation was measured as described under Materials and Methods. Values are expressed as percentages of the control (0) that received no DEX. In each case triplicate wells were tested and the results are representative of three other independent experiments.
tibility to corticosteroid 212 keratinocytes. MATERIALS
inhibition
AND
in established
Pam
METHODS
Cells and cultures. The cells used in this study were: (i) mouse Pam 212 keratinocytes [26], (ii) Pam 212 keratinocytes infected with a retrovirus (MD-Ela 12s) transducing an Ela 12 S cDNA together with a G-418 resistance gene (Ela cells) or with a virus (pCGM-neo) carrying the G-418 resistance gene alone (neo cells) [19], and (iii) Pam 212 keratinocytes transfected via lipofection (GIBCO BRL, Gaithersburg, MD) with different plasmids. These plasmids contain mutated versions of the Ela gene (d1787N, Ntd1598, d1922/947) [17, 181 plus a G-418 resistance gene, or the G-418 resistance gene only (SVXneo, NE0 cells), for use as control for plasmid alone [19]. The Ela mutant d1787N has an internal deletion that leaves intact the ability to bind to the cellular proteins ~60, ~105, ~107, and ~300 (Ela 787 cells). The Ela mutant Ntd1598 has lost the ability to bind to the ~300 protein, without losing that for ~60, ~105, and ~107 (Ela 598 cells), while the mutant Ela d1922/947 has retained the ability to bind to ~300 but has lost that for ~60, ~105, and ~107 (Ela 922 cells). All cells were cultured in Dulbecco’s modified essential medium (DMEM; GIBCO)/lO% calf serum (Sigma Chemical Co., St. Louis, MO) at 37”C, in a 5% CO, humidified atmosphere. Drugs. Dexamethasone (phosphate salt; DEX; Sigma) was prepared in a 1 mM stock solution in DMEM. Forskolin (ICN, Costa Mesa, CA) was dissolved in dimethyl sulfoxide (Sigma) in a 20 mM stock solution. 8-Br-CAMP (Sigma) and 8-Cl-CAMP (gift from Dr. Y. S. Cho Chung, NIH, Bethesda, MD) were prepared in DMEM in 100.mM stock solutions. Measurement of proliferation rates. Cells were plated onto 24.well trays (lo5 cells per well) and tested 1 day later. At the time of the experiments the cells were in a subconfluent state. After exposure to different concentrations of the drugs for 18 h, 1 PCi of [methyl-sH]thymidine ([3H]TdR), 40-60 Ci/mmol (Amersham, Arlington Heights, IL), was added to each well and the cells were further incubated for 1 h. The pulses were terminated by trichloroacetic acid precipitation which was performed directly on the dishes, and the precipitates were dissolved in NaOH and counted by liquid scintillation. All
AND
and values obtained
for each
DISCUSSION
We have investigated the effect of different doses of the synthetic glucocorticoid DEX on Pam 212 keratinocyte proliferation as measured by [3H]TdR incorporation (Fig. 1). The inhibitory effects we observed are in good agreement with previous studies in which primary neonatal murine keratinocytes were used [23]. They point out that in Pam 212 keratinocytes, an established murine cell line [26], glucocorticoids can still act as a growth inhibitory signal. To examine the effects of the Ela oncogene on the response to DEX, we employed two cell lines derived from Pam 212 keratinocytes. The first was transformed with the Ela 12 S gene using a retroviral vector (Ela cells) and the second harbors the same vector without Ela and serves as control (neo cells) [19]. The results of the proliferation assays performed after 18 h of exposure of these cells to DEX are shown in Fig. 2. While the control cell line shows considerable growth inhibition at the doses tested, almost no inhibition is observed in the Ela cells. These results are the first to demonstrate that Ela can antagonize glucocorticoid growth inhibition. In another set of experiments, we used cells carrying intact or variously mutated Ela genes [ 191. Figure 3 shows the effects of 0.1 pA4 DEX on the proliferation of these cell lines. An Ela gene with an intact transforming region (d1787) induces resistance to the steroid as observed with the Ela 12s virus. This demonstrates that the retroviral framework is not required for the effect, as the d1787 gene in a plasmid vector has the same property. We also tested Pam 212 cells which carry the mutated Ela d1922/947 or Ela Ntd1598 genes. The d1922/947
120 1OnMDEX
100 nM DEX
1
t
0
I
_J
neo Ela
neo Ela
FIG. 2. Effect of DEX on Ela-transformed and control keratinocytes. Cultures of Ela and neo cells were exposed to 10 and 100 nM DEX and their proliferative rates were tested by [3H]TdR incorporation as described in the legend to Fig. 1. The results are representative of three other independent experiments.
SHORT
deletion prevents binding of Ela to ~60, ~105, and ~107 and the Ntd1598 mutation impairs binding to ~300 [ 18, 191. These two mutants induce essentially the same resistance to DEX as Ela 787 cells (Fig. 3). This result suggests that both p6O/p105/p107 and ~300 may be involved in transmission of the DEX growth inhibitory signal and that their binding by Ela is sufficient to block their function. An alternative and equally possible explanation is that neither of these groups of proteins is involved in transmission of the DEX signal. In either case it is clear that the mechanism by which Ela induces resistance to DEX inhibition can be genetically distinguished from that by which it induces resistance to TGF-P and CAMP growth inhibitory signals. In fact, as pointed out before, both mutant versions of Ela analyzed here provide partial resistance against TGF-(3, [I91 and only the mutated Ela whose product can bind to p6O/p105/p107 confers resistance to CAMP [20]. The suggestion that growth inhibition by CAMP and DEX acts through different mechanisms is supported by the results shown in Fig. 4. In this experiment, we found that Pam 212 keratinocytes exposed to the CAMP enhancing agent forskolin together with DEX show considerably stronger growth inhibition than those exposed to either agent separately. We obtained similar results using 1 mM 8Br-CAMP or 1 mA4 8-Cl-CAMP instead of 10 pM forskolin (data not shown). Glucocorticoids act mostly by affecting gene transcription [27]. An attractive possibility is that they could enhance expression of proteins with tumor supressor activity to inhibit proliferation. The Ela gene products could prevent this process or neutralize the effects of these mediators of glucocorticoid action. In conclusion, this work adds glucocorticoids to the
FIG. 4. Effects of simultaneous exposure to DEX and forskolin on growth inhibition of Pam 212 keratinocytes. Cultures of Pam 212 were exposed to 0.1 @f DEX (DEX), 10 pM forskolin (FSK), or 0.1 pM DEX + 10 WM forskolin (DEX + FSK) and tested for proliferation as in the legend to Fig. 1. Results are representative of two independent experiments.
group of growth inhibitory signals that are counteracted by Ela gene product. Different domains appear to act in each case, which emphasizes the pleiotropic nature of this gene. Such a feature is not surprising in view of the number of cellular proteins that associate with the Ela gene product [ 111. This molecule appears to be remarkably shaped to sustain proliferative rates under a wide variety of conditions. M.F.C. and J.F.C. were supported by NIH-Fogarty Fellowships. C.M. is a recipient of the Leslie H. Warner Postdoctoral Fellowship (Cancer Center, Yale University).
REFERENCES 1.
3 3 3 E g 8
-IL 90
-Ii
J-
2. 3. 4. 5. 6.
60
7.
8.
i
0 LNE0
Ela 787
Ela 598
Ela 922
FIG. 3. Proliferation rates of different Ela mutants exposed to DEX. Pam 212 keratinocytes transfected with the plasmids SV2neo (NEO), d1787N (Ela 787), Ntd1598 (Ela 598), or d1922/947 (Ela 922) were exposed to 0.1 &f DEX and their proliferative rates were tested as in the legend to Fig. 1. These results are representative of two other independent experiments.
287
NOTE
9. 10. 11. 12.
Pittelkow, M. R., Coffey, R. J., Jr., and Moses, H. L. (1988) Ann. N. Y. Acad. Sci. 548, 211-224. Cho-Chung, Y. (1990) Cancer Res. 50,7093-7100. Zendegui, J. G., Inman, W. H., and Carpenter, G. (1988) J. Cell. Physiol. 136, 257-265. Weinberg, R. A. (1989) Biochemistry 28,8263-8267. Weinberg, R. A. (1991) Science 254, 1138-1146. Marshall, C. J. (1989) in Oncogenes (Glover, D. M., and Hames, B. D., Eds.), pp. 1-21, IRL Press, Oxford. Pietenpol, J. A., Stein, R. W., Moran, E., Yaciuk, P., Schiegel, R., Lyons, R. M., Pittelkow, M. R., Munger, K., and Moses, H. L. (1990) Cell 61, 777-785. Laiho, M., De Caprio, J. A., Ludlow, J. W., Livingston, D. M., and Massague, J. (1990) Cell 62, 175-185. Ruley, H. E. (1983) Nature 304, 602-606. Berk, A. J. (1986) Cancer Suru. 5, 367-387. Harlow, E., Whyte, P., Franza, B. R., Jr., and Schley, C. (1986) Mol. Cell. Biol. 6, 1579-1589. Whyte, P., Buchkovich, K. J., Horowitz, J. M., Friend, S. H., Raybuck, M., Weinberg, R. A., and Harlow, E. (1988) Nature 334,124-129.
288 13. 14. 15. 16. 17. 18. 19. 20.
SHORT Ewen, M. E., Faha, B., Harlow, E., and Livingston, D. M. (1992) Science 256,85-87. Stein, R. W., Corrigan, M., Yaciuk, P., Whelan, J., and Moran, E. (1990) J. Virol. 64, 4421-4427. Faha, B., Ewen, M. E., Tsai, L.-H., Livingston, D. M., and Harlow, E. (1992) Science 255,87-90. Pines, J., and Hunter, T. (1990) Nature 346, 760-763. Whyte, P., Ruley, H. E., and Harlow, E. (1988) J. Virol. 62, 257-265. Whyte, P., Williamson, N. M., and Harlow, E. (1989) Cell 56, 67-75. Missero, C., Filvaroff, E., and Dotto G. P. (1991) Proc. Natl. Acad. Sci. USA 88,3489-3493. Florin-Christensen, M., Missero, C., Florin-Christensen, J.,
Received June 19, 1992
NOTE Tranque, P., Ramon y Cajal, S., and Dotto, G. P. (1992), suhmitted for publication. 21. 22.
23. 24. 25. 26. 27.
Loeb, J. N. (1976) N. Engl. J. Med. 295, 547-552. Haynes, R. C., Jr. (1990) inThe Pharmacological Basis ofTherapeutics (Goodman Gilman, A., Rall, T. W., Nies, A. S., and Taylor, P., Eds.), pp. 1431-1462, Pergamon, New York. Marcelo, C. L., and Duell, E. A. (1978) J. Invest. Dermatol. 70, 211. Xu, X., and Bjorntorp, P. (1990) Exp. Cell Res. 189, 247-252. Gregoire, F., Genart, C., Hauser, N., and Remacle, C. (1991) Exp. CellRes. 196, 270-278. Yuspa, S. H., Hawley-Nelson, P., Koehler, B., and Stanley, J. R. (1980) Cancer Res. 40,4694-4703. Evans, R. M. (1988) Science 240,889-895.
CELL
RESEARCH
203,
285-288
(1992)
SHORT NOTE The El a Gene Prevents Inhibition of Keratinocyte Proliferation by Dexamethasone M. FLORIN-CHRISTENSEN,* Departments
of *Internal
Medicine
C. MISSRRo,t
and TPathology,
G. P. DOTTO,?’
Yale University
School of Medicine,
Glucocorticoids have inhibitory effects on the proliferation of several cell types. In this study, we found that dexamethasone, a synthetic steroid with glucocorticoid activity, inhibits proliferation of established mouse Pam 212 keratinocytes. Transfection with the adenoviral early region la (Ela) gene confers a strong resistance to the inhibition by dexamethasone. Two deletion Ela mutants, one whose product lacks the ability to bind the cellular proteins p6O/p105/p107 and another that is unable to bind ~300, were shown to induce a resistance similar to that associated with the intact Ela gene. These results differ from those previously observed with two other growth inhibitory signals, transforming growth factor p1 and adenosine 3’,5’-cyclic monophosphate, in which the mutated E la genes confer only partial or no resistance, indicating that a different mechanism mediates resistance against glucocorti0 1992 Academic Press, Inc. coids.
INTRODUCTION It is now recognized that cell proliferation is regulated not only by growth factors, but also by inhibitory signals. Among them, the transforming growth factors /3 (TGF-P) have been found to have strong inhibitory effects on keratinocyte growth [l]. In addition, similar effect,s have been described for agents that elevate CAMP intracellular levels [2,3]. Another line of investigation has disclosed the existence of several genes, which either positively or negatively affect cell proliferation [4,5]. Among the first are classical protooncogenes, like c-ras, c-myc, and c-fos [6]. Those in the second group are known as tumor suppressor genes. They include the gene encoding the retinoblastoma protein
1 To whom correspondence should he addressed. Fax: (203)7857467. ’ Present address: University of Cincinnati, Department of Biological Sciences, Cincinnati, OH 45221-0006. 285
AND J. FLORIN-CHRISTENSEN~ 310 Cedar Street, New Hauen, Connecticut
06510
~105, which is probably the best characterized, and the one encoding the ~53 nuclear protein [4,5]. Recent work has pointed out possible connections between negatively acting extracellular signals and the function of tumor supressor genes [7, 81. The adenoviral early region la (Ela) gene encodes a series of related proteins with multiple effects, including the immortalization of primary culture cells [9], the trans-activation of a variety of genes, and the repression of others [lo]. Immunoprecipitation experiments have shown that the proteins encoded by Ela form stable complexes with a number of cellular proteins. These include the retinoblastoma ~105; a related protein ~107; ~60, now identified as cyclin A; and ~300, a nuclear protein which may be involved in the control of DNA synthesis as well as in gene transcription [ll-161. In addition, at least five other proteins are known to associate with Ela [ 121. It has been shown that the binding to the cellular proteins ~105, ~107, and ~300 is critical to the Ela transforming effects [17, 181. Transfection of keratinocytes with the Ela gene renders them completely resistant to the growth inhibition induced by TGF-/3, [ 191. If cells are transfected with mutated versions of the Ela gene whose products are unable to bind either the proteins p6O/p105/p107 or ~300, the resistance to TGF-P, is only partial in each case, indicating that both regions of the gene are important to prevent inhibition [ 191. We have recently found that Ela also induces resistance to adenosine 3’,5’-cyclic monophosphate (CAMP) growth inhibition. In this case, the effect appears to involve binding by Ela to p60/ pIO5/pIO7 but not ~300 1201. A third type of growth inhibitory signals are the glucocorticoid steroids. Their inhibitory action has been known for many years [Zl] but the mechanism involved is still unknown [22]. They affect selectively certain tissues in whole animals [21]. Keratinocytes are susceptible to inhibition [23], and effects on other cell types have been recently described [24, 251. In this study, we examine the effect of Ela transformation on the suscepnn14-4827/92 $5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
286
SHORT
‘*Oc
NOTE assays were carried out in triplicate condition differed by less than 10%. RESULTS
I
-
1L .l
1
FM DEX
FIG. 1. Dose effect of DEX on the proliferation of Pam 212 keratinocytes. Subconfluent cultures of Pam 212 were exposed to different doses of DEX (IO-’ to 1Om6M) for 18 h after which [3H]TdR incorporation was measured as described under Materials and Methods. Values are expressed as percentages of the control (0) that received no DEX. In each case triplicate wells were tested and the results are representative of three other independent experiments.
tibility to corticosteroid 212 keratinocytes. MATERIALS
inhibition
AND
in established
Pam
METHODS
Cells and cultures. The cells used in this study were: (i) mouse Pam 212 keratinocytes [26], (ii) Pam 212 keratinocytes infected with a retrovirus (MD-Ela 12s) transducing an Ela 12 S cDNA together with a G-418 resistance gene (Ela cells) or with a virus (pCGM-neo) carrying the G-418 resistance gene alone (neo cells) [19], and (iii) Pam 212 keratinocytes transfected via lipofection (GIBCO BRL, Gaithersburg, MD) with different plasmids. These plasmids contain mutated versions of the Ela gene (d1787N, Ntd1598, d1922/947) [17, 181 plus a G-418 resistance gene, or the G-418 resistance gene only (SVXneo, NE0 cells), for use as control for plasmid alone [19]. The Ela mutant d1787N has an internal deletion that leaves intact the ability to bind to the cellular proteins ~60, ~105, ~107, and ~300 (Ela 787 cells). The Ela mutant Ntd1598 has lost the ability to bind to the ~300 protein, without losing that for ~60, ~105, and ~107 (Ela 598 cells), while the mutant Ela d1922/947 has retained the ability to bind to ~300 but has lost that for ~60, ~105, and ~107 (Ela 922 cells). All cells were cultured in Dulbecco’s modified essential medium (DMEM; GIBCO)/lO% calf serum (Sigma Chemical Co., St. Louis, MO) at 37”C, in a 5% CO, humidified atmosphere. Drugs. Dexamethasone (phosphate salt; DEX; Sigma) was prepared in a 1 mM stock solution in DMEM. Forskolin (ICN, Costa Mesa, CA) was dissolved in dimethyl sulfoxide (Sigma) in a 20 mM stock solution. 8-Br-CAMP (Sigma) and 8-Cl-CAMP (gift from Dr. Y. S. Cho Chung, NIH, Bethesda, MD) were prepared in DMEM in 100.mM stock solutions. Measurement of proliferation rates. Cells were plated onto 24.well trays (lo5 cells per well) and tested 1 day later. At the time of the experiments the cells were in a subconfluent state. After exposure to different concentrations of the drugs for 18 h, 1 PCi of [methyl-sH]thymidine ([3H]TdR), 40-60 Ci/mmol (Amersham, Arlington Heights, IL), was added to each well and the cells were further incubated for 1 h. The pulses were terminated by trichloroacetic acid precipitation which was performed directly on the dishes, and the precipitates were dissolved in NaOH and counted by liquid scintillation. All
AND
and values obtained
for each
DISCUSSION
We have investigated the effect of different doses of the synthetic glucocorticoid DEX on Pam 212 keratinocyte proliferation as measured by [3H]TdR incorporation (Fig. 1). The inhibitory effects we observed are in good agreement with previous studies in which primary neonatal murine keratinocytes were used [23]. They point out that in Pam 212 keratinocytes, an established murine cell line [26], glucocorticoids can still act as a growth inhibitory signal. To examine the effects of the Ela oncogene on the response to DEX, we employed two cell lines derived from Pam 212 keratinocytes. The first was transformed with the Ela 12 S gene using a retroviral vector (Ela cells) and the second harbors the same vector without Ela and serves as control (neo cells) [19]. The results of the proliferation assays performed after 18 h of exposure of these cells to DEX are shown in Fig. 2. While the control cell line shows considerable growth inhibition at the doses tested, almost no inhibition is observed in the Ela cells. These results are the first to demonstrate that Ela can antagonize glucocorticoid growth inhibition. In another set of experiments, we used cells carrying intact or variously mutated Ela genes [ 191. Figure 3 shows the effects of 0.1 pA4 DEX on the proliferation of these cell lines. An Ela gene with an intact transforming region (d1787) induces resistance to the steroid as observed with the Ela 12s virus. This demonstrates that the retroviral framework is not required for the effect, as the d1787 gene in a plasmid vector has the same property. We also tested Pam 212 cells which carry the mutated Ela d1922/947 or Ela Ntd1598 genes. The d1922/947
120 1OnMDEX
100 nM DEX
1
t
0
I
_J
neo Ela
neo Ela
FIG. 2. Effect of DEX on Ela-transformed and control keratinocytes. Cultures of Ela and neo cells were exposed to 10 and 100 nM DEX and their proliferative rates were tested by [3H]TdR incorporation as described in the legend to Fig. 1. The results are representative of three other independent experiments.
SHORT
deletion prevents binding of Ela to ~60, ~105, and ~107 and the Ntd1598 mutation impairs binding to ~300 [ 18, 191. These two mutants induce essentially the same resistance to DEX as Ela 787 cells (Fig. 3). This result suggests that both p6O/p105/p107 and ~300 may be involved in transmission of the DEX growth inhibitory signal and that their binding by Ela is sufficient to block their function. An alternative and equally possible explanation is that neither of these groups of proteins is involved in transmission of the DEX signal. In either case it is clear that the mechanism by which Ela induces resistance to DEX inhibition can be genetically distinguished from that by which it induces resistance to TGF-P and CAMP growth inhibitory signals. In fact, as pointed out before, both mutant versions of Ela analyzed here provide partial resistance against TGF-(3, [I91 and only the mutated Ela whose product can bind to p6O/p105/p107 confers resistance to CAMP [20]. The suggestion that growth inhibition by CAMP and DEX acts through different mechanisms is supported by the results shown in Fig. 4. In this experiment, we found that Pam 212 keratinocytes exposed to the CAMP enhancing agent forskolin together with DEX show considerably stronger growth inhibition than those exposed to either agent separately. We obtained similar results using 1 mM 8Br-CAMP or 1 mA4 8-Cl-CAMP instead of 10 pM forskolin (data not shown). Glucocorticoids act mostly by affecting gene transcription [27]. An attractive possibility is that they could enhance expression of proteins with tumor supressor activity to inhibit proliferation. The Ela gene products could prevent this process or neutralize the effects of these mediators of glucocorticoid action. In conclusion, this work adds glucocorticoids to the
FIG. 4. Effects of simultaneous exposure to DEX and forskolin on growth inhibition of Pam 212 keratinocytes. Cultures of Pam 212 were exposed to 0.1 @f DEX (DEX), 10 pM forskolin (FSK), or 0.1 pM DEX + 10 WM forskolin (DEX + FSK) and tested for proliferation as in the legend to Fig. 1. Results are representative of two independent experiments.
group of growth inhibitory signals that are counteracted by Ela gene product. Different domains appear to act in each case, which emphasizes the pleiotropic nature of this gene. Such a feature is not surprising in view of the number of cellular proteins that associate with the Ela gene product [ 111. This molecule appears to be remarkably shaped to sustain proliferative rates under a wide variety of conditions. M.F.C. and J.F.C. were supported by NIH-Fogarty Fellowships. C.M. is a recipient of the Leslie H. Warner Postdoctoral Fellowship (Cancer Center, Yale University).
REFERENCES 1.
3 3 3 E g 8
-IL 90
-Ii
J-
2. 3. 4. 5. 6.
60
7.
8.
i
0 LNE0
Ela 787
Ela 598
Ela 922
FIG. 3. Proliferation rates of different Ela mutants exposed to DEX. Pam 212 keratinocytes transfected with the plasmids SV2neo (NEO), d1787N (Ela 787), Ntd1598 (Ela 598), or d1922/947 (Ela 922) were exposed to 0.1 &f DEX and their proliferative rates were tested as in the legend to Fig. 1. These results are representative of two other independent experiments.
287
NOTE
9. 10. 11. 12.
Pittelkow, M. R., Coffey, R. J., Jr., and Moses, H. L. (1988) Ann. N. Y. Acad. Sci. 548, 211-224. Cho-Chung, Y. (1990) Cancer Res. 50,7093-7100. Zendegui, J. G., Inman, W. H., and Carpenter, G. (1988) J. Cell. Physiol. 136, 257-265. Weinberg, R. A. (1989) Biochemistry 28,8263-8267. Weinberg, R. A. (1991) Science 254, 1138-1146. Marshall, C. J. (1989) in Oncogenes (Glover, D. M., and Hames, B. D., Eds.), pp. 1-21, IRL Press, Oxford. Pietenpol, J. A., Stein, R. W., Moran, E., Yaciuk, P., Schiegel, R., Lyons, R. M., Pittelkow, M. R., Munger, K., and Moses, H. L. (1990) Cell 61, 777-785. Laiho, M., De Caprio, J. A., Ludlow, J. W., Livingston, D. M., and Massague, J. (1990) Cell 62, 175-185. Ruley, H. E. (1983) Nature 304, 602-606. Berk, A. J. (1986) Cancer Suru. 5, 367-387. Harlow, E., Whyte, P., Franza, B. R., Jr., and Schley, C. (1986) Mol. Cell. Biol. 6, 1579-1589. Whyte, P., Buchkovich, K. J., Horowitz, J. M., Friend, S. H., Raybuck, M., Weinberg, R. A., and Harlow, E. (1988) Nature 334,124-129.
288 13. 14. 15. 16. 17. 18. 19. 20.
SHORT Ewen, M. E., Faha, B., Harlow, E., and Livingston, D. M. (1992) Science 256,85-87. Stein, R. W., Corrigan, M., Yaciuk, P., Whelan, J., and Moran, E. (1990) J. Virol. 64, 4421-4427. Faha, B., Ewen, M. E., Tsai, L.-H., Livingston, D. M., and Harlow, E. (1992) Science 255,87-90. Pines, J., and Hunter, T. (1990) Nature 346, 760-763. Whyte, P., Ruley, H. E., and Harlow, E. (1988) J. Virol. 62, 257-265. Whyte, P., Williamson, N. M., and Harlow, E. (1989) Cell 56, 67-75. Missero, C., Filvaroff, E., and Dotto G. P. (1991) Proc. Natl. Acad. Sci. USA 88,3489-3493. Florin-Christensen, M., Missero, C., Florin-Christensen, J.,
Received June 19, 1992
NOTE Tranque, P., Ramon y Cajal, S., and Dotto, G. P. (1992), suhmitted for publication. 21. 22.
23. 24. 25. 26. 27.
Loeb, J. N. (1976) N. Engl. J. Med. 295, 547-552. Haynes, R. C., Jr. (1990) inThe Pharmacological Basis ofTherapeutics (Goodman Gilman, A., Rall, T. W., Nies, A. S., and Taylor, P., Eds.), pp. 1431-1462, Pergamon, New York. Marcelo, C. L., and Duell, E. A. (1978) J. Invest. Dermatol. 70, 211. Xu, X., and Bjorntorp, P. (1990) Exp. Cell Res. 189, 247-252. Gregoire, F., Genart, C., Hauser, N., and Remacle, C. (1991) Exp. CellRes. 196, 270-278. Yuspa, S. H., Hawley-Nelson, P., Koehler, B., and Stanley, J. R. (1980) Cancer Res. 40,4694-4703. Evans, R. M. (1988) Science 240,889-895.
