EXPERIMENTAL
CELL RESEARCH
194,56-61 (1991)
Involvement of Protein Kinase C in the Regulation of Ornithine Decarboxylase mRNA by Phorbol Esters in Rat Hepatoma Cells’ ANDREWP.BUTLER,‘PENNY K.MAR,FRANCES F. MCDONALD,ANDRAECHELLEL.RAMSAY University
of Texas M.D. Anderson
Cancer Center, Department
of Carcinogenesis,
The tumor promoter 12-O-tetradecanoylphorbol13-acetate (TPA) stimulates a rapid increase in ornithine decarboxylase (EC 4.1.1.1’7; ODC) activity in target cells. Here we demonstrate that this process involves a rapid accumulation of ODC mRNA, which is maximal 3 h after treatment (three- to eightfold greater than control cells) and decays to control levels within 18 h. Stimulation of ODC mRNA by TPA is blocked by phorbol dibutyrate down-regulation of protein kinase C (PKC). ODC mRNA was also induced by the PKC activators, phospholipase C and 1-oleoyl-2-acetyl-rac-glycerol, and blocked by kinase inhibitors (trifluoroperazine, H7, and palmitoyl+carnitine), consistent with a requirement for PKC activation in the induction mechanism. However, the non-PKC-specific protein kinase inhibitor HA1004 also suppressed expression of ODC mRNA in response to TPA, under conditions where it did not inhibit PKC, suggesting that additional kinases may be involved in the intracellular signalling process. The stability of the ODC mRNA (control value = 6.2 +- 1.6 h) is not significantly changed by either TPA (6.7 f. 0.8 h) or by cycloheximide (6.0 h). These results are inconsistent with any contribution from altered mRNA half-life towards the accumulation of ODC mRNA following treatment with phorbol ester tumor promoters. Q 1981 Academic Press, Inc.
INTRODUCTION
Phorbol esters, such as 12-O-tetradecanoylphorbol13-acetate (TPA), are effective tumor promoters in the mouse skin and elicit pleiotropic responses from target cells [l-3]. Many of the effects of TPA on cultured cells are reminiscent of those caused by the binding of polypeptide growth factors to cell-surface receptors, including rapid intracellular calcium and monovalent ion fluxes; phosphorylation of a number of membrane, cytosolic, and nuclear proteins; and both induction and re’ Work sponsored by National Institutes of Health Grant CA46629 by SIG-14 from the American Cancer Society. 2 To whom correspondence and reprint requests should be addressed.
(A.P.B.)and
0014-4827&l $3.00 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
Science Park-Research
Division,
Smithville,
Texas 78957
pression of specific gene products. The demonstration that TPA binds tightly to protein kinase C (PKC), causing translocation of the kinase to the cell membrane and subsequent activation, offers a potential mechanism by which many of the actions of TPA may be mediated [4-61. Furthermore, the existence of multiple genes encoding several proteins related to PKC [ 7-101 suggests a possible basis for differential effects of TPA and growth factors on various cell types. However, the mechanistic steps by which activation of PKC, at the cell surface, is able to modulate gene expression within the nucleus remain obscure. One of the earliest changes in gene expression caused by TPA is an increase in ornithine decarboxylase (ODC) activity. This enzyme is rapidly induced by TPA treatment in viuo in mouse skin and several rat tissues [ll, 121 and in many types of cultured cells. ODC is an attractive model for study of the activation of specific genes by TPA. ODC is the major control point in the biosynthesis of the polyamines putrescine, spermidine, and spermine, which are required for growth of both normal and transformed cells [13,14]. The transition of quiescent cells from G, to G, is accompanied by a substantial increase in ODC enzyme activity and polyamine synthesis which coincides with increased transcription of a number of specific genes, including c-myc [15, 161. H35 rat hepatoma cells have proven to be an excellent model for the study of the biological effects of TPA. Treatment of these cells with TPA leads to a rapid increase in ODC enzyme activity [17]. Recently, we demonstrated that this increase is due in part to an accumulation of ODC mRNA [ 181.This increase occurred in the absence of on-going protein synthesis and was, in fact, superinduced by inhibition of protein synthesis with cycloheximide. Furthermore, the induction was abolished by treatment with actinomycin D, suggesting that phorbol ester regulation of ODC could occur at the transcriptional level. Novel patterns of nuclear protein phosphorylation have been observed following treatment of H35 cells with TPA [19], and both a ribosomal protein S6 kinase and CAMP-dependent kinase are also activated [20, 211. In this paper, we demonstrate that stimulation of ODC mRNA accumulation by TPA in H35 hepatoma cells is mediated by PKC. Evidence is 56
ODC MRNA
EXPRESSION
IN RAT
HEPATOMA
57
CELLS
also presented suggesting the involvement of at least one additional protein kinase in the accumulation of ODC mRNA following stimulation of PKC. MATERLALS AND METHODS Muteriaki. TPA was obtained from LC Services (Woburn, MA). Cell culture media and serum were from GIBCO. H7 [1-(5-isoquinolinesulfonyl)-2-metbylpiperazine] and HA1004 [N-(2-guanidinoethyl)-5-isoquinolinesulfonamide hydrochloride] were obtained from Seigakaku America (St. Petersburg, FL). Phorbol dibutyrate, phospholipase C, lipids and other inhibitors were from Sigma Chemical (St. Louis, MO). The pOD48 plasmid was a gift from Dr. Phillip Coffino, University of California, San Francisco. Restriction enzymes were purchased from Boehringer-Mannheim (Indianapolis, IN) and Bethesda Research Laboratories (Gaithereburg, MD). Cell culture. Reuber H35 rat hepatoma cells were maintained in Dulbecco’s modified Eagle’s medium containing 5% fetal calf serum, 5% newborn calf serum. Growth of cells and serum depletion were described previously [19]. Serum-starved cells were treated with TPA (0.8 p&f final concentration, unless otherwise specified) and dissolved in dimethyl sulfoxide (DMSO). The final concentration of DMSO in the medium was never more than 0.1%. Diacylglycerols were dissolved in DMSO under N,, diluted into medium containing 100 pglml BSA, and sonicated before addition to cells. RNA purifiotion. After treatment, culture medium was decanted and the cell monolayer was rinsed with ice-cold diethylpyrocarbonate-treated phosphate-buffered saline (PBS). Cells were scraped from the dishes and centrifuged at 8OOg for 5 min at 4°C. The cell pellets were lysed by homogenization in guanidine isothiocyanate [22]. Total RNA was collected by centrifugation through a 5.7 M CsCl block gradient and purified by organic extractions and ethanol precipitation as described [18]. RNA recovery was quantitated by absorbance at 258 nm, using E,,,, = 25lmglml. Nucleic acid hybridization. Dot blot and Northern hybridizations were conducted using standard procedures [23]. For dot blot analysis, RNA was denatured in 50% formamide at 65°C and applied to nitrocellulose (Schleicher and Schuell, BA85) in 10X SSC. For Northern analysis, RNA was electrophoresed through 1% agarose gels containing 2.2 M formaldehyde. The RNA was transferred to nitrocellulose in 20X SSC (1X SSC = 0.15 M NaCl, 0.15 M sodium citrate, pH 7.0). Filters were prehybridized and hybridized as previously described, using a nick-translated Hind111 fragment of pOD48 [18, 24). The filters were washed to a final stringency of 0.1X SSC at 52°C. Autoradiography was performed at -70°C using Kodak XAR film and DuPont Cronex intensifying screens. Autoradiograms of Northern blots were quantitated by scanning densitometry, using a Biomed Instruments soft laser densitometer. Northern blots were washed for rehybridization as described by Meinkoth and Wahl[25]; equal loading of samples was verified by rehybridization to dihydrofolate reductase cDNA [26]. Dot blots were quantitated by liquid scintillation counting of the excised radioactive spots. The least squares linear regression slope of a dilution series of each sample (cpm hybridized vs pg of RNA) was determined and used as a measure of the specific ODC mRNA content of the samples. Protein kinase C extraction and assay. Cells were homogenized in Buffer I [20 mM Tris-HCl (pH 8.0), 2 mh4 EDTA, 2 mM EGTA, 1 n&f DTT] containing 0.5% Triton X-100, 1 mAf phenylmethylsulfonyl fluoride (PMSF), 10 gglml aprotinin, 10 pg/ml leupeptin, and 5 pg/ml pepstatin A. The extract was incubated on ice with occasional mixing for 30 min prior to centrifugation at 541,000g for 10 min in a Beckman TLlOO ultracentrifuge. The supernatant was applied to a DEAE Sephadex A50 column equilibrated in Buffer I containing 0.1 mhf PMSF. The column was washed with the same buffer, and a step gradient of 120 n&f NaCl in Buffer I plus 0.1 r&f PMSF was applied to elute the PKC activity. Initial experiments demonstrated that vir-
4
8
12
16
Hours of TPA treatment FIG. 1. Kinetics of ODC mRNA accumulation following TPA treatment of quiescent H35 cells. Cells were grown and treated with 0.8 &f TPA as described under Materials and Methods. Total RNA was isolated at various times following treatment with TPA, as indicated. Serial dilutions of RNA from each time point were applied to nitrocellulose in 20x SSC using a vacuum manifold. The dot blots were hybridized, washed, and autoradiographed as described. Following X-ray exposure, the samples were excised and quantitated by scintillation counting. The slopes of linear regression lines (cpm vs RNA applied) were determined and used as a measure of specific ODC mRNA accumulation.
tually all of the calcium-, phospholipid-dependent protein kinase ac- , tivity eluted in the 120-d step; additional kinase activity eluting at higher salt concentrations was independent of calcium and phospholipid. Kinase activity was assayed using a variation of published procedures [6]. The assay solution contained 20 m.M Tris-HCl (pH 8.0), 10 pg/ml phosphatidyl serine, 10 mM MgCl,, 2 mA4 CaCl,, 0.5 mM EDTA, 0.5 m&f EGTA, 0.5 mg/ml histone (Sigma Type IIIs), and 20 pM Y~~P-ATP (ICN; =400-500 Bq/pmol) in a volume of 100 ~1. Nonspecific kinase activity was determined in the absence of calcium and phosphatidyl serine, in mixtures containing 1.5 mAf EGTA. Following 10 min incubation at 3O”C, reactions were terminated by pipeting the mixture onto pieces of P81 phosphocellulose paper (Whatman) which were immediately washed once in 75 mM phosphoric acid, three times in water, air-dried, and counted in a Beckman LS1801 liquid scintillation spectrophotometer. Samples were assayedin triplicate, and PKC activity was stated as the difference between the incorporation in the presence and absence of Caa’ and phosphatidyl serine. Protein concentration was determined using a Coomassie blue dyebinding assay [27] with bovine serum albumin as a standard. Enzyme activity is reported as picomoles of 3ZP transferred to histone per milligrams protein per minute. Preliminary experiments demonstrated that the assay was linear in time for at least 20 min under these conditions and throughout the range of protein concentrations employed.
RESULTS
Induction of ODC mRNA by TPA. Addition of TPA to H35 hepatoma cells growing in serum-free medium leads to a rapid increase in ODC enzyme activity [28]. Recently, we showed that this increase is due, at least in part, to an increased accumulation of mRNA hybridizing to a murine ODC cDNA probe [ 181.Figure 1 shows a detailed time course for the appearance of ODC mRNA in TPA-treated H35 cells, as determined by dot hybrid-
58
BUTLER
TABLE Effect
of Protein
Kinase
Compound DMSO (0.1%) TPA (0.8 PM) OAG (20 PM) OAG (100 PM) PLC (0.01 U/ml) PLC (0.1 U/ml) ’ ODC mRNA hybridization
ET AL.
1
C Activators
12345 on ODC! mRNA
ODC mRNA
Levels”
(% of control)
100 405 + 80 681 2 70 567 + 45 242 + 45 160 + 17 + SD of three experiments.
ization. The maximum level of ODC mRNA accumulation occurred about 3 h after treatment of the cells with TPA; expression returned to control values by 18-24 h. Two major species of ODC mRNA are observed, with sizes of 2.8 and 2.4 kb, and these bands intensify coordinately (Fig. 2). TPA consistently stimulated a three- to eightfold increase in ODC mRNA under these conditions, as determined by scintillation counting of dot blots. The variability observed in the increase is due to significant differences observed in the basal expression of ODC as a function of small differences in cell confluence. Similar differences have been reported for ODC enzyme activity as a function of the growth state in H35 cells [17]. The biologically inactive parent compound, phorbol (at concentrations up to 1.6 PM), did not cause a significant change in ODC mRNA levels [18]. Induction of ODC mRNA by PKC activators. To determine the potential role of PKC in the induction of ODC, cells were treated with several diacylglycerols which have been reported to be activators of PKC. Total RNA was isolated as described, applied to nitrocellulose, and hybridized to nick-translated pOD48 DNA. Results are given in Table 1. The synthetic diacylglycerol OAG stimulated expression of ODC mRNA by approximately sixfold, while treatment with monodecanoylglycerol had no effect. In addition, treatment of the cells with C. perfringens phospholipase C (PLC) caused a similar accumulation of ODC mRNA, presumably through the release of endogenous diacylglycerols. High doses of both phospholipase C (at >0.5 units/ml) and the short chain (ClO) mono- and d&glycerides (at >50 PM) were cytotoxic. However, at the doses and treatment times for PLC and 1-oleoyl-2-acetyl-rat-glycerol (OAG) used in these studies, no evidence of toxicity was observed, as judged by cell morphology and trypan blue staining. Examination of Northern hybridizations indicates that the ODC mRNA species expressed following treatment with either OAG or phospholipase C is the same size as that observed in TPA-treated cells (Fig. 2). Thus, other known activators of PKC produce a qualitatively and semiquantitatively similar accumulation of ODC mRNA as does TPA. Effect of protein kinase inhibitors on ODC mRNA accumulation. The results above support the idea that
FIG. 2. Northern blot analysis of ODC mRNA. Quiescent H35 cells were treated with DMSO, TPA, C. perfringens phospholipase C or OAG for 3 h. Cells were lysed in 4 M guanidine isothiocyanate and the RNA was isolated as described in the text. Samples (10 pg) were electrophoresed on a 1% agarose gel in the presence of 2.2 Mformaldehyde and transferred to nitrocellulose by capillary blotting. The membrane was hybridized to nick-translated murine ODC cDNA, washed to a final stringency of 0.1X SSC, 0.1% SDS at 55”C, and exposed to Kodak XAR film. (Lane 1) control, 0.1% DMSO, (lane 2) TPA (0.8 a); (lane 3) control (duplicate of lane 1); (lane 4) C. perfringens phospholipase C (0.01 U/ml); (lane 5) OAG (20 p&f).
the accumulation of ODC mRNA caused by TPA is mediated by protein kinase C. To further test this model, H35 cells were stimulated with TPA in the presence of one of several protein kinase inhibitors of varying specificity for PKC. Total cellular RNA was isolated and analyzed by hybridization as described above. The results demonstrate that these agents are potent inhibitors of the effect of TPA on ODC messenger levels (Table 2). Of these inhibitors, trifluoroperazine is the most effective. Approximately 95% of the increase in ODC mRNA is blocked by 10 PM trifluoroperazine, while a concentration of 50 PM virtually abolishes the response. Palmitoyl-L-carnitine at 10 FM blocked about 70% of the induction, while acetyl-DL-carnitine was ineffective at this concentration. At higher doses (50 PM; data not
TABLE
2
Effects of Protein Kinase Inhibitors on ODC mRNA Levelsa Treatment Control TPA TPA + TPA + TPA + TPA + TPA +
Palmitoyl carnitine Acetyl camitine Trifluoroperazine H7 HA1004
ODC mRNA
(% of control)
100 627 + 86 242 k 26 6OOk53 128 + 42 437 + 84 184+42
a TPA concentration was 0.8 PM; inhibitors were at 10 pM when present. Control = 0.1% DMSO. ODC mRNA hybridization * SD of three experiments.
ODC MRNA
..I..
0
10
20
30
A--
EXPRESSION
H7
40
7
50
60
Concentration, PM FIG. 3. Effects of ODC mRNA by TPA, or with TPA After 3 h, cells were by dot hybridization text.
of kinase inhibitors H7 and HA1004 on induction TPA. Quiescent cells were treated with 0.8 &f and the indicated concentrations of inhibitors. lysed and total RNA was prepared and analyzed and scintillation counting as described in the
response, however actetyl-DL-carnitine also inhibited about 70% of the increase in ODC mRNA at this concentration, suggesting the possible involvement of nonspecific effects in the inhibition by these compounds at the higher concentrations. The isoquinolinesulfonamide derivitives, H7 and HA1004, are effective protein kinase inhibitors. H7 has been reported to be the most potent and selective PKC inhibitor of the series, with a Ki of 6 NM, while HA1004 inhibits PKC only weakly with a Ki of 40 PM [29]. Unexpectedly, both H7 and HA1004 blocked the increase in ODC mRNA caused by TPA treatment of H35 cells (Table 2). Indeed, HA1004 proved to be more effective than the reported PKC-specific inhibitor H7. Preliminary studies confirm that the Ki of HA1004 for DEAE Sephadex-purified PKC from H35 cells is approximately 40 PM, and the Ki of H7 is -10 PM. Although both H7 and HA1004 inhibit TPA-induced ODC mRNA accumulation, H7 also inhibits the basal expression to a degree (Fig. 3). To eliminate cytotoxicity towards H35 cells as a possible mechanism for the observed effect, quiescent cells were incubated for 12 h in the presence of each inhibitor. No gross morphological differences were noted, and the viable cell count (determined by trypan blue exclusion) was the same (-13%) for cells treated with H7 or HA1004 and control cultures. Down-regulation of PKC byphorbol dibutyrate. Cells may be functionally depleted of protein kinase C by prolonged exposure to biologically active, phorbol esters [30, 311. Under these conditions, PKC activity and immunoreactive protein virtually disappear, and cells are no longer sensitive to subsequent challenge by TPA. In order to determine the effect of such PKC down-regulation on the expression of ODC mRNA, H35 cells were
IN RAT
HEPATOMA
59
CELLS
grown for 2 days in the absence of serum, then treated with 1 PM phorbol dibutyrate for 20 h. Protein kinase C (cytosolic plus membrane bound) was extracted from cells and assayed as described under Materials and Methods. Under these conditions, PKC activity was at least 92% less than that observed in control (DMSOtreated) cultures (Fig. 4). At this time, cells were treated with TPA (0.8 PM) or OAG (20 a) for an additional 3 h. As shown in Fig. 5, cells treated continuously with phorbol dibutyrate express levels of ODC mRNA slightly above control (40 f 40% induction). Although this small increase is not statistically significant, it is reproducible. Cells which were not preincubated with phorbol dibutyrate responded to TPA in the typical fashion, with a significant increase (550 + 60% over control) in ODC mRNA. Pretreatment with phorbol dibutyrate caused a nearly complete loss of response to TPA (80 _+60% induction), as well as a significant decrease in the response to OAG. Effects of TPA on ODC mRNA stability. The accumulation of ODC mRNA in the presence of TPA could be due to increased transcription of the ODC gene, increased stability of the message, or both. In order to determine if mRNA stabilization is a factor in ODC induction, we analyzed the half-life of ODC mRNA from control and TPA-treated cells. Cultures were treated with TPA (1.6 PM) or 0.1% DMSO for 3 h. They were then exposed to 1 PM actinomycin D. Under these conditions, total RNA synthesis is inhibited by >90% in H35 hepatoma cells (data not
n + calcium, ps 0 - Calcium,-PS q netC-kinase activity
control PdBu pretreatment (20 hr) FIG. 4. Protein kinase C activity in H35 cells pretreated with phorbol dibutyrate. Quiescent cells were pretreated for 20 h with either 0.1% DMSO (control) or 2 crM phorbol dibutyrate. PKC was extracted and analyzed as described under Materials and Methods. Kinase activity was assayed in the presence (+) or absence (-) of calcium and phosphatidyl serine; the difierence represents PKC-specific activity (*SD of triplicate determinations).
60
BUTLER
shown and [32]). RNA was isolated at various times during the actinomycin D chase and analyzed by hybridization to labeled ODC cDNA. The amount of hybridization observed with samples taken at 0 h actinomycin D chase was considered lOO%, and data from subsequent time points was correspondingly normalized. Stability of the ODC mRNA was assessed for control cells, TPAtreated cells, and cycloheximide-treated cells. The results of a representative experiment are shown in Fig. 6. The mean half-life of ODC mRNA (determined from regression coefficients of three independent experiments) was 6.2 + 1.6 h in control cells and 5.7 f 0.8 h in TPA-treated cells. A similar analysis for cells treated with 20 PM cycloheximide resulted in a measured halflife for ODC mRNA of -6 h (Fig. 6).
ET AL.
50
/ lo"
0 .
control TPA
A
CHX
.
.
0 DISCUSSION
Three major conclusions can be drawn from the data presented above. First, the results presented are consistent with an essential role for PKC activation in the induction of ODC mRNA by TPA treatment of H35 cells. However, the effects of the isoquinolinesulfonamide inhibitors H7 and HA1004 suggests that additional protein kinases (or other factors) are required in subsequent steps of the signal transduction process. Finally, these results imply that alterations in the half-life of ODC mRNA play little or no role in the induction of ODC by TPA treatment H35 hepatoma cells. Three lines of evidence support an essential role for PKC activation in the induction of ODC by phorbol esters in H35 cells. First, the induction can be mimicked by either the addition of the exogenous diacylglycerol OAG, or by release of endogenous diacylglycerols gener-
Treatment FIG. 5. Effect of PKC down-regulation on induction of ODC mRNA. Cells were pretreated exactly as described in the legend to Fig. 4. After 20 h in the presence of PdBu, cultures were challenged with either 0.1% DMSO (control) or with 0.8 fl TPA. Three hours later, the cells were harvested and RNA was analyzed by hybridization to ODC cDNA. Error bars, +SD of triplicate experiments.
Hours
' 4
\ *
-
*
of Actinomycin
'
a
*
*
*
12
D chase
FIG. 6. Stability of ODC mRNA. Quiescent H35 cells were treated with TPA, 20 pM cycloheximide, or 0.1% DMSO (control) for 3 h. At this time, the cultures were treated with 1 &f actinomycin D. Cells were harvested at the indicated times and total RNA was isolated as described. Samples were analyzed by dot hybridization with labeled ODC probe followed by scintillation counting. Open circles, control cultures; closed circles, TPA-treated (1.6 &f) cultures; open triangles, cycloheximide-treated (20 pM).
ated by treatment with phospholipase C (Table 1). Thus, other activators of PKC have qualitatively and quantitatively similar effects as those of TPA on ODC mRNA levels. The fact that the response of ODC mRNA to TPA can be almost completely blocked by depletion of PKC (Figs. 4 and 5) offers strong evidence that PKC activation is a necessary step in the intracellular signaling mechanism. In addition, the potent effects of the protein kinase inhibitors (Table 2) on expression of ODC is consistent with a role for PKC. The unexpected results obtained with the inhibitors H7 and HA1004 are of considerable interest. Under the conditions of our experiments, both H7 and HA1004 were effective inhibitors of ODC expression. These results have been repeated in several independent experiments (using three separate lots of inhibitors), and control experiments demonstrate that the effects are not due to cytotoxicity. If PKC were the only kinase required for ODC expression in response to TPA, then HA1004 would not be expected to inhibit the process, at least at concentrations below 20 @f, since the measured ki for this compound is 40 pM. However, HA1004 actually appeared to be slightly more potent than H7 in terms of inhibiting the induction of ODC mRNA in H35 cells. This suggests that the activity of other factors in addition to PKC may be required for ODC induction. Because HA1004 is a potent inhibitor of both CAMP-dependent protein kinase [29] and casein kinase II (unpublished data), it is possible that additional protein kinases transmit the PKC signal to the nucleus. A second explanation consistent with the present data is that
ODC MRNA
EXPRESSION
a minor isozyme of PKC, which is preferentially inhibited by HA1004, might be responsible for the major mediation of TPA effects on ODC induction in H35 cells. Both of these possible mechanisms are currently under investigation. Recent results of Coffin0 and co-workers suggest that a major portion of the induction of ODC by mitogens may involve post-translational effects on protein turnover [33]. However, data from this and other laboratories [18, 34-381 continue to support the idea that one component of ODC induction by mitogens involves increased amounts of ODC mRNA and nascent transcripts. The data presented in Fig. 6 demonstrate that the accumulation of ODC mRNA in TPA-treated H35 cells is not due to stabilization of the ODC mRNA. Conclusions similar to our own have recently been obtained in another epithelial cell, the murine keratinocyte [39]. During the review of this manuscript, evidence was published demonstrating transcriptional regulation of ODC mRNA in Swiss 3T3 cells and post-transcriptional regulation in T lymphocytes [40]. Sequence data on rodent ODC genes suggest the presence of several potential binding sites for TPA-inducible transcriptional factors within the 5’ flanking regions and the first intron [41441. We are currently investigating the possible role of these sequences in the regulation of ODC in rat H35 hepatoma cells.
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41. 42. 43. 44.
M., and Coffino,
P. (1989)
CELL RESEARCH
194,56-61 (1991)
Involvement of Protein Kinase C in the Regulation of Ornithine Decarboxylase mRNA by Phorbol Esters in Rat Hepatoma Cells’ ANDREWP.BUTLER,‘PENNY K.MAR,FRANCES F. MCDONALD,ANDRAECHELLEL.RAMSAY University
of Texas M.D. Anderson
Cancer Center, Department
of Carcinogenesis,
The tumor promoter 12-O-tetradecanoylphorbol13-acetate (TPA) stimulates a rapid increase in ornithine decarboxylase (EC 4.1.1.1’7; ODC) activity in target cells. Here we demonstrate that this process involves a rapid accumulation of ODC mRNA, which is maximal 3 h after treatment (three- to eightfold greater than control cells) and decays to control levels within 18 h. Stimulation of ODC mRNA by TPA is blocked by phorbol dibutyrate down-regulation of protein kinase C (PKC). ODC mRNA was also induced by the PKC activators, phospholipase C and 1-oleoyl-2-acetyl-rac-glycerol, and blocked by kinase inhibitors (trifluoroperazine, H7, and palmitoyl+carnitine), consistent with a requirement for PKC activation in the induction mechanism. However, the non-PKC-specific protein kinase inhibitor HA1004 also suppressed expression of ODC mRNA in response to TPA, under conditions where it did not inhibit PKC, suggesting that additional kinases may be involved in the intracellular signalling process. The stability of the ODC mRNA (control value = 6.2 +- 1.6 h) is not significantly changed by either TPA (6.7 f. 0.8 h) or by cycloheximide (6.0 h). These results are inconsistent with any contribution from altered mRNA half-life towards the accumulation of ODC mRNA following treatment with phorbol ester tumor promoters. Q 1981 Academic Press, Inc.
INTRODUCTION
Phorbol esters, such as 12-O-tetradecanoylphorbol13-acetate (TPA), are effective tumor promoters in the mouse skin and elicit pleiotropic responses from target cells [l-3]. Many of the effects of TPA on cultured cells are reminiscent of those caused by the binding of polypeptide growth factors to cell-surface receptors, including rapid intracellular calcium and monovalent ion fluxes; phosphorylation of a number of membrane, cytosolic, and nuclear proteins; and both induction and re’ Work sponsored by National Institutes of Health Grant CA46629 by SIG-14 from the American Cancer Society. 2 To whom correspondence and reprint requests should be addressed.
(A.P.B.)and
0014-4827&l $3.00 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
Science Park-Research
Division,
Smithville,
Texas 78957
pression of specific gene products. The demonstration that TPA binds tightly to protein kinase C (PKC), causing translocation of the kinase to the cell membrane and subsequent activation, offers a potential mechanism by which many of the actions of TPA may be mediated [4-61. Furthermore, the existence of multiple genes encoding several proteins related to PKC [ 7-101 suggests a possible basis for differential effects of TPA and growth factors on various cell types. However, the mechanistic steps by which activation of PKC, at the cell surface, is able to modulate gene expression within the nucleus remain obscure. One of the earliest changes in gene expression caused by TPA is an increase in ornithine decarboxylase (ODC) activity. This enzyme is rapidly induced by TPA treatment in viuo in mouse skin and several rat tissues [ll, 121 and in many types of cultured cells. ODC is an attractive model for study of the activation of specific genes by TPA. ODC is the major control point in the biosynthesis of the polyamines putrescine, spermidine, and spermine, which are required for growth of both normal and transformed cells [13,14]. The transition of quiescent cells from G, to G, is accompanied by a substantial increase in ODC enzyme activity and polyamine synthesis which coincides with increased transcription of a number of specific genes, including c-myc [15, 161. H35 rat hepatoma cells have proven to be an excellent model for the study of the biological effects of TPA. Treatment of these cells with TPA leads to a rapid increase in ODC enzyme activity [17]. Recently, we demonstrated that this increase is due in part to an accumulation of ODC mRNA [ 181.This increase occurred in the absence of on-going protein synthesis and was, in fact, superinduced by inhibition of protein synthesis with cycloheximide. Furthermore, the induction was abolished by treatment with actinomycin D, suggesting that phorbol ester regulation of ODC could occur at the transcriptional level. Novel patterns of nuclear protein phosphorylation have been observed following treatment of H35 cells with TPA [19], and both a ribosomal protein S6 kinase and CAMP-dependent kinase are also activated [20, 211. In this paper, we demonstrate that stimulation of ODC mRNA accumulation by TPA in H35 hepatoma cells is mediated by PKC. Evidence is 56
ODC MRNA
EXPRESSION
IN RAT
HEPATOMA
57
CELLS
also presented suggesting the involvement of at least one additional protein kinase in the accumulation of ODC mRNA following stimulation of PKC. MATERLALS AND METHODS Muteriaki. TPA was obtained from LC Services (Woburn, MA). Cell culture media and serum were from GIBCO. H7 [1-(5-isoquinolinesulfonyl)-2-metbylpiperazine] and HA1004 [N-(2-guanidinoethyl)-5-isoquinolinesulfonamide hydrochloride] were obtained from Seigakaku America (St. Petersburg, FL). Phorbol dibutyrate, phospholipase C, lipids and other inhibitors were from Sigma Chemical (St. Louis, MO). The pOD48 plasmid was a gift from Dr. Phillip Coffino, University of California, San Francisco. Restriction enzymes were purchased from Boehringer-Mannheim (Indianapolis, IN) and Bethesda Research Laboratories (Gaithereburg, MD). Cell culture. Reuber H35 rat hepatoma cells were maintained in Dulbecco’s modified Eagle’s medium containing 5% fetal calf serum, 5% newborn calf serum. Growth of cells and serum depletion were described previously [19]. Serum-starved cells were treated with TPA (0.8 p&f final concentration, unless otherwise specified) and dissolved in dimethyl sulfoxide (DMSO). The final concentration of DMSO in the medium was never more than 0.1%. Diacylglycerols were dissolved in DMSO under N,, diluted into medium containing 100 pglml BSA, and sonicated before addition to cells. RNA purifiotion. After treatment, culture medium was decanted and the cell monolayer was rinsed with ice-cold diethylpyrocarbonate-treated phosphate-buffered saline (PBS). Cells were scraped from the dishes and centrifuged at 8OOg for 5 min at 4°C. The cell pellets were lysed by homogenization in guanidine isothiocyanate [22]. Total RNA was collected by centrifugation through a 5.7 M CsCl block gradient and purified by organic extractions and ethanol precipitation as described [18]. RNA recovery was quantitated by absorbance at 258 nm, using E,,,, = 25lmglml. Nucleic acid hybridization. Dot blot and Northern hybridizations were conducted using standard procedures [23]. For dot blot analysis, RNA was denatured in 50% formamide at 65°C and applied to nitrocellulose (Schleicher and Schuell, BA85) in 10X SSC. For Northern analysis, RNA was electrophoresed through 1% agarose gels containing 2.2 M formaldehyde. The RNA was transferred to nitrocellulose in 20X SSC (1X SSC = 0.15 M NaCl, 0.15 M sodium citrate, pH 7.0). Filters were prehybridized and hybridized as previously described, using a nick-translated Hind111 fragment of pOD48 [18, 24). The filters were washed to a final stringency of 0.1X SSC at 52°C. Autoradiography was performed at -70°C using Kodak XAR film and DuPont Cronex intensifying screens. Autoradiograms of Northern blots were quantitated by scanning densitometry, using a Biomed Instruments soft laser densitometer. Northern blots were washed for rehybridization as described by Meinkoth and Wahl[25]; equal loading of samples was verified by rehybridization to dihydrofolate reductase cDNA [26]. Dot blots were quantitated by liquid scintillation counting of the excised radioactive spots. The least squares linear regression slope of a dilution series of each sample (cpm hybridized vs pg of RNA) was determined and used as a measure of the specific ODC mRNA content of the samples. Protein kinase C extraction and assay. Cells were homogenized in Buffer I [20 mM Tris-HCl (pH 8.0), 2 mh4 EDTA, 2 mM EGTA, 1 n&f DTT] containing 0.5% Triton X-100, 1 mAf phenylmethylsulfonyl fluoride (PMSF), 10 gglml aprotinin, 10 pg/ml leupeptin, and 5 pg/ml pepstatin A. The extract was incubated on ice with occasional mixing for 30 min prior to centrifugation at 541,000g for 10 min in a Beckman TLlOO ultracentrifuge. The supernatant was applied to a DEAE Sephadex A50 column equilibrated in Buffer I containing 0.1 mhf PMSF. The column was washed with the same buffer, and a step gradient of 120 n&f NaCl in Buffer I plus 0.1 r&f PMSF was applied to elute the PKC activity. Initial experiments demonstrated that vir-
4
8
12
16
Hours of TPA treatment FIG. 1. Kinetics of ODC mRNA accumulation following TPA treatment of quiescent H35 cells. Cells were grown and treated with 0.8 &f TPA as described under Materials and Methods. Total RNA was isolated at various times following treatment with TPA, as indicated. Serial dilutions of RNA from each time point were applied to nitrocellulose in 20x SSC using a vacuum manifold. The dot blots were hybridized, washed, and autoradiographed as described. Following X-ray exposure, the samples were excised and quantitated by scintillation counting. The slopes of linear regression lines (cpm vs RNA applied) were determined and used as a measure of specific ODC mRNA accumulation.
tually all of the calcium-, phospholipid-dependent protein kinase ac- , tivity eluted in the 120-d step; additional kinase activity eluting at higher salt concentrations was independent of calcium and phospholipid. Kinase activity was assayed using a variation of published procedures [6]. The assay solution contained 20 m.M Tris-HCl (pH 8.0), 10 pg/ml phosphatidyl serine, 10 mM MgCl,, 2 mA4 CaCl,, 0.5 mM EDTA, 0.5 m&f EGTA, 0.5 mg/ml histone (Sigma Type IIIs), and 20 pM Y~~P-ATP (ICN; =400-500 Bq/pmol) in a volume of 100 ~1. Nonspecific kinase activity was determined in the absence of calcium and phosphatidyl serine, in mixtures containing 1.5 mAf EGTA. Following 10 min incubation at 3O”C, reactions were terminated by pipeting the mixture onto pieces of P81 phosphocellulose paper (Whatman) which were immediately washed once in 75 mM phosphoric acid, three times in water, air-dried, and counted in a Beckman LS1801 liquid scintillation spectrophotometer. Samples were assayedin triplicate, and PKC activity was stated as the difference between the incorporation in the presence and absence of Caa’ and phosphatidyl serine. Protein concentration was determined using a Coomassie blue dyebinding assay [27] with bovine serum albumin as a standard. Enzyme activity is reported as picomoles of 3ZP transferred to histone per milligrams protein per minute. Preliminary experiments demonstrated that the assay was linear in time for at least 20 min under these conditions and throughout the range of protein concentrations employed.
RESULTS
Induction of ODC mRNA by TPA. Addition of TPA to H35 hepatoma cells growing in serum-free medium leads to a rapid increase in ODC enzyme activity [28]. Recently, we showed that this increase is due, at least in part, to an increased accumulation of mRNA hybridizing to a murine ODC cDNA probe [ 181.Figure 1 shows a detailed time course for the appearance of ODC mRNA in TPA-treated H35 cells, as determined by dot hybrid-
58
BUTLER
TABLE Effect
of Protein
Kinase
Compound DMSO (0.1%) TPA (0.8 PM) OAG (20 PM) OAG (100 PM) PLC (0.01 U/ml) PLC (0.1 U/ml) ’ ODC mRNA hybridization
ET AL.
1
C Activators
12345 on ODC! mRNA
ODC mRNA
Levels”
(% of control)
100 405 + 80 681 2 70 567 + 45 242 + 45 160 + 17 + SD of three experiments.
ization. The maximum level of ODC mRNA accumulation occurred about 3 h after treatment of the cells with TPA; expression returned to control values by 18-24 h. Two major species of ODC mRNA are observed, with sizes of 2.8 and 2.4 kb, and these bands intensify coordinately (Fig. 2). TPA consistently stimulated a three- to eightfold increase in ODC mRNA under these conditions, as determined by scintillation counting of dot blots. The variability observed in the increase is due to significant differences observed in the basal expression of ODC as a function of small differences in cell confluence. Similar differences have been reported for ODC enzyme activity as a function of the growth state in H35 cells [17]. The biologically inactive parent compound, phorbol (at concentrations up to 1.6 PM), did not cause a significant change in ODC mRNA levels [18]. Induction of ODC mRNA by PKC activators. To determine the potential role of PKC in the induction of ODC, cells were treated with several diacylglycerols which have been reported to be activators of PKC. Total RNA was isolated as described, applied to nitrocellulose, and hybridized to nick-translated pOD48 DNA. Results are given in Table 1. The synthetic diacylglycerol OAG stimulated expression of ODC mRNA by approximately sixfold, while treatment with monodecanoylglycerol had no effect. In addition, treatment of the cells with C. perfringens phospholipase C (PLC) caused a similar accumulation of ODC mRNA, presumably through the release of endogenous diacylglycerols. High doses of both phospholipase C (at >0.5 units/ml) and the short chain (ClO) mono- and d&glycerides (at >50 PM) were cytotoxic. However, at the doses and treatment times for PLC and 1-oleoyl-2-acetyl-rat-glycerol (OAG) used in these studies, no evidence of toxicity was observed, as judged by cell morphology and trypan blue staining. Examination of Northern hybridizations indicates that the ODC mRNA species expressed following treatment with either OAG or phospholipase C is the same size as that observed in TPA-treated cells (Fig. 2). Thus, other known activators of PKC produce a qualitatively and semiquantitatively similar accumulation of ODC mRNA as does TPA. Effect of protein kinase inhibitors on ODC mRNA accumulation. The results above support the idea that
FIG. 2. Northern blot analysis of ODC mRNA. Quiescent H35 cells were treated with DMSO, TPA, C. perfringens phospholipase C or OAG for 3 h. Cells were lysed in 4 M guanidine isothiocyanate and the RNA was isolated as described in the text. Samples (10 pg) were electrophoresed on a 1% agarose gel in the presence of 2.2 Mformaldehyde and transferred to nitrocellulose by capillary blotting. The membrane was hybridized to nick-translated murine ODC cDNA, washed to a final stringency of 0.1X SSC, 0.1% SDS at 55”C, and exposed to Kodak XAR film. (Lane 1) control, 0.1% DMSO, (lane 2) TPA (0.8 a); (lane 3) control (duplicate of lane 1); (lane 4) C. perfringens phospholipase C (0.01 U/ml); (lane 5) OAG (20 p&f).
the accumulation of ODC mRNA caused by TPA is mediated by protein kinase C. To further test this model, H35 cells were stimulated with TPA in the presence of one of several protein kinase inhibitors of varying specificity for PKC. Total cellular RNA was isolated and analyzed by hybridization as described above. The results demonstrate that these agents are potent inhibitors of the effect of TPA on ODC messenger levels (Table 2). Of these inhibitors, trifluoroperazine is the most effective. Approximately 95% of the increase in ODC mRNA is blocked by 10 PM trifluoroperazine, while a concentration of 50 PM virtually abolishes the response. Palmitoyl-L-carnitine at 10 FM blocked about 70% of the induction, while acetyl-DL-carnitine was ineffective at this concentration. At higher doses (50 PM; data not
TABLE
2
Effects of Protein Kinase Inhibitors on ODC mRNA Levelsa Treatment Control TPA TPA + TPA + TPA + TPA + TPA +
Palmitoyl carnitine Acetyl camitine Trifluoroperazine H7 HA1004
ODC mRNA
(% of control)
100 627 + 86 242 k 26 6OOk53 128 + 42 437 + 84 184+42
a TPA concentration was 0.8 PM; inhibitors were at 10 pM when present. Control = 0.1% DMSO. ODC mRNA hybridization * SD of three experiments.
ODC MRNA
..I..
0
10
20
30
A--
EXPRESSION
H7
40
7
50
60
Concentration, PM FIG. 3. Effects of ODC mRNA by TPA, or with TPA After 3 h, cells were by dot hybridization text.
of kinase inhibitors H7 and HA1004 on induction TPA. Quiescent cells were treated with 0.8 &f and the indicated concentrations of inhibitors. lysed and total RNA was prepared and analyzed and scintillation counting as described in the
response, however actetyl-DL-carnitine also inhibited about 70% of the increase in ODC mRNA at this concentration, suggesting the possible involvement of nonspecific effects in the inhibition by these compounds at the higher concentrations. The isoquinolinesulfonamide derivitives, H7 and HA1004, are effective protein kinase inhibitors. H7 has been reported to be the most potent and selective PKC inhibitor of the series, with a Ki of 6 NM, while HA1004 inhibits PKC only weakly with a Ki of 40 PM [29]. Unexpectedly, both H7 and HA1004 blocked the increase in ODC mRNA caused by TPA treatment of H35 cells (Table 2). Indeed, HA1004 proved to be more effective than the reported PKC-specific inhibitor H7. Preliminary studies confirm that the Ki of HA1004 for DEAE Sephadex-purified PKC from H35 cells is approximately 40 PM, and the Ki of H7 is -10 PM. Although both H7 and HA1004 inhibit TPA-induced ODC mRNA accumulation, H7 also inhibits the basal expression to a degree (Fig. 3). To eliminate cytotoxicity towards H35 cells as a possible mechanism for the observed effect, quiescent cells were incubated for 12 h in the presence of each inhibitor. No gross morphological differences were noted, and the viable cell count (determined by trypan blue exclusion) was the same (-13%) for cells treated with H7 or HA1004 and control cultures. Down-regulation of PKC byphorbol dibutyrate. Cells may be functionally depleted of protein kinase C by prolonged exposure to biologically active, phorbol esters [30, 311. Under these conditions, PKC activity and immunoreactive protein virtually disappear, and cells are no longer sensitive to subsequent challenge by TPA. In order to determine the effect of such PKC down-regulation on the expression of ODC mRNA, H35 cells were
IN RAT
HEPATOMA
59
CELLS
grown for 2 days in the absence of serum, then treated with 1 PM phorbol dibutyrate for 20 h. Protein kinase C (cytosolic plus membrane bound) was extracted from cells and assayed as described under Materials and Methods. Under these conditions, PKC activity was at least 92% less than that observed in control (DMSOtreated) cultures (Fig. 4). At this time, cells were treated with TPA (0.8 PM) or OAG (20 a) for an additional 3 h. As shown in Fig. 5, cells treated continuously with phorbol dibutyrate express levels of ODC mRNA slightly above control (40 f 40% induction). Although this small increase is not statistically significant, it is reproducible. Cells which were not preincubated with phorbol dibutyrate responded to TPA in the typical fashion, with a significant increase (550 + 60% over control) in ODC mRNA. Pretreatment with phorbol dibutyrate caused a nearly complete loss of response to TPA (80 _+60% induction), as well as a significant decrease in the response to OAG. Effects of TPA on ODC mRNA stability. The accumulation of ODC mRNA in the presence of TPA could be due to increased transcription of the ODC gene, increased stability of the message, or both. In order to determine if mRNA stabilization is a factor in ODC induction, we analyzed the half-life of ODC mRNA from control and TPA-treated cells. Cultures were treated with TPA (1.6 PM) or 0.1% DMSO for 3 h. They were then exposed to 1 PM actinomycin D. Under these conditions, total RNA synthesis is inhibited by >90% in H35 hepatoma cells (data not
n + calcium, ps 0 - Calcium,-PS q netC-kinase activity
control PdBu pretreatment (20 hr) FIG. 4. Protein kinase C activity in H35 cells pretreated with phorbol dibutyrate. Quiescent cells were pretreated for 20 h with either 0.1% DMSO (control) or 2 crM phorbol dibutyrate. PKC was extracted and analyzed as described under Materials and Methods. Kinase activity was assayed in the presence (+) or absence (-) of calcium and phosphatidyl serine; the difierence represents PKC-specific activity (*SD of triplicate determinations).
60
BUTLER
shown and [32]). RNA was isolated at various times during the actinomycin D chase and analyzed by hybridization to labeled ODC cDNA. The amount of hybridization observed with samples taken at 0 h actinomycin D chase was considered lOO%, and data from subsequent time points was correspondingly normalized. Stability of the ODC mRNA was assessed for control cells, TPAtreated cells, and cycloheximide-treated cells. The results of a representative experiment are shown in Fig. 6. The mean half-life of ODC mRNA (determined from regression coefficients of three independent experiments) was 6.2 + 1.6 h in control cells and 5.7 f 0.8 h in TPA-treated cells. A similar analysis for cells treated with 20 PM cycloheximide resulted in a measured halflife for ODC mRNA of -6 h (Fig. 6).
ET AL.
50
/ lo"
0 .
control TPA
A
CHX
.
.
0 DISCUSSION
Three major conclusions can be drawn from the data presented above. First, the results presented are consistent with an essential role for PKC activation in the induction of ODC mRNA by TPA treatment of H35 cells. However, the effects of the isoquinolinesulfonamide inhibitors H7 and HA1004 suggests that additional protein kinases (or other factors) are required in subsequent steps of the signal transduction process. Finally, these results imply that alterations in the half-life of ODC mRNA play little or no role in the induction of ODC by TPA treatment H35 hepatoma cells. Three lines of evidence support an essential role for PKC activation in the induction of ODC by phorbol esters in H35 cells. First, the induction can be mimicked by either the addition of the exogenous diacylglycerol OAG, or by release of endogenous diacylglycerols gener-
Treatment FIG. 5. Effect of PKC down-regulation on induction of ODC mRNA. Cells were pretreated exactly as described in the legend to Fig. 4. After 20 h in the presence of PdBu, cultures were challenged with either 0.1% DMSO (control) or with 0.8 fl TPA. Three hours later, the cells were harvested and RNA was analyzed by hybridization to ODC cDNA. Error bars, +SD of triplicate experiments.
Hours
' 4
\ *
-
*
of Actinomycin
'
a
*
*
*
12
D chase
FIG. 6. Stability of ODC mRNA. Quiescent H35 cells were treated with TPA, 20 pM cycloheximide, or 0.1% DMSO (control) for 3 h. At this time, the cultures were treated with 1 &f actinomycin D. Cells were harvested at the indicated times and total RNA was isolated as described. Samples were analyzed by dot hybridization with labeled ODC probe followed by scintillation counting. Open circles, control cultures; closed circles, TPA-treated (1.6 &f) cultures; open triangles, cycloheximide-treated (20 pM).
ated by treatment with phospholipase C (Table 1). Thus, other activators of PKC have qualitatively and quantitatively similar effects as those of TPA on ODC mRNA levels. The fact that the response of ODC mRNA to TPA can be almost completely blocked by depletion of PKC (Figs. 4 and 5) offers strong evidence that PKC activation is a necessary step in the intracellular signaling mechanism. In addition, the potent effects of the protein kinase inhibitors (Table 2) on expression of ODC is consistent with a role for PKC. The unexpected results obtained with the inhibitors H7 and HA1004 are of considerable interest. Under the conditions of our experiments, both H7 and HA1004 were effective inhibitors of ODC expression. These results have been repeated in several independent experiments (using three separate lots of inhibitors), and control experiments demonstrate that the effects are not due to cytotoxicity. If PKC were the only kinase required for ODC expression in response to TPA, then HA1004 would not be expected to inhibit the process, at least at concentrations below 20 @f, since the measured ki for this compound is 40 pM. However, HA1004 actually appeared to be slightly more potent than H7 in terms of inhibiting the induction of ODC mRNA in H35 cells. This suggests that the activity of other factors in addition to PKC may be required for ODC induction. Because HA1004 is a potent inhibitor of both CAMP-dependent protein kinase [29] and casein kinase II (unpublished data), it is possible that additional protein kinases transmit the PKC signal to the nucleus. A second explanation consistent with the present data is that
ODC MRNA
EXPRESSION
a minor isozyme of PKC, which is preferentially inhibited by HA1004, might be responsible for the major mediation of TPA effects on ODC induction in H35 cells. Both of these possible mechanisms are currently under investigation. Recent results of Coffin0 and co-workers suggest that a major portion of the induction of ODC by mitogens may involve post-translational effects on protein turnover [33]. However, data from this and other laboratories [18, 34-381 continue to support the idea that one component of ODC induction by mitogens involves increased amounts of ODC mRNA and nascent transcripts. The data presented in Fig. 6 demonstrate that the accumulation of ODC mRNA in TPA-treated H35 cells is not due to stabilization of the ODC mRNA. Conclusions similar to our own have recently been obtained in another epithelial cell, the murine keratinocyte [39]. During the review of this manuscript, evidence was published demonstrating transcriptional regulation of ODC mRNA in Swiss 3T3 cells and post-transcriptional regulation in T lymphocytes [40]. Sequence data on rodent ODC genes suggest the presence of several potential binding sites for TPA-inducible transcriptional factors within the 5’ flanking regions and the first intron [41441. We are currently investigating the possible role of these sequences in the regulation of ODC in rat H35 hepatoma cells.
IN RAT
Pegg, A. E., and McCann, c212-c221.
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3. 4. 5. 6. 7.
8.
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