THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1988 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 263, No. 23, Issue of August 15, pp. 11431-11435,1988 Printed in U.S.A.

Regulation of Ornithine Decarboxylase and Other Cell Cycle-dependent Genes during Senescence of IMR-90 Human Diploid Fibroblasts*

(Received for publication, March 11,1988)

Zee-Fen Chang and Kuang Yu ChenS From the Departments of Biochemistry and Chemistry, Rutgers, The State University of New Jersey, New Brunswick, New Jersey 08903

Aging of IMR-90 human diploid fibroblasts in vitro is accompanied by significant changes of polyamine metabolism, most notably, a 5-fold decrease of serum- induced activity of ornithine decarboxylase, the key enzyme in the biosynthesis of polyamines (Chen, K. Y., Chang, Z. F., and Liu, A. Y.-C. (1986) J. Cell. Physiol. 129, 142-146). In this paper, we employed Northern blot hybridization and affinity radiolabeling tech- niques to investigate the molecular basis of this age- associated change of ornithine decarboxylase activity. Since the induction of ornithine decarboxylase by serum is a mid-GI event, we also examined expressions of other cell cycle-dependent genes that are induced before and after the mid-G1 phase to determine if their expressions may also be age-dependent. Our results demonstrated a %fold decrease of the amount of active ornithine decarboxylase molecules that can be labeled by a-difluoromethyl[’H]ornithine in senescent IMR-90 cells (population doubling level (PDL) = 52) as com- pared to young cells (PDL = 22). However, the levels and kinetics of induction of ornithine decarboxylase mRNA in both young and senescent IMR-90 cells were found to be identical throughout a 24-h time period after serum stimulation. The time course and the mag- nitude of the expression of c-myc, an early GI gene, were quite similar in young and senescent IMR-90 cells and appeared to be PDL-independent. In contrast, the expression of thymidine kinase, a late G1/S gene, was significantly reduced in senescent IMR-90 cells. Levels of thymidine kinase mRNA and thymidine kinase ac- tivity in senescent IMR-90 cells were 6- and 8-fold less than those in young cells, respectively. Based on these data, we proposed that impairment of cell cycling in senescent IMR-90 cells may occur at the late G1/S phase and that decreases of ornithine decarboxylase activity and putrescine accumulation during cell senescence may contribute to this impairment.

Normal diploid fibroblasts have a limited doubling potential in tissue culture (1,2). The remarkable consistency of the life span of these cells in culture, which is inversely related to the age of the donor, and the species specificity of the life span

* This work was supported by National Institutes of Health Grant AGO3578 and a grant from The Charles and Johanna Busch Memorial Fund. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ To whom correspondence should be addressed Dept. of Chem- istry, P. 0. Box 939, Rutgers, The State University of New Jersey, Piscataway, NJ 08855-0939.

(3-5) have made t,hem a useful model to study the biochemical basis of cellular aging.

Polyamines (putrescine, spermidine, and spermine) are nat- urally occurring organic cations widely distributed in living organisms (6, 7). The induction of ornithine decarboxylase (EC 4.1.1.17), the key enzyme for polyamine biosynthesis, and the subsequent accumulation of putrescine (or spermi- dine) have been implicated in the initiation of DNA synthesis (8,9). Inhibition of the induction of ornithine decarboxylase by a-difluoromethylornithine leads to a decrease of DNA synthesis and growth cessation in rat ventral prostate (lo), EMT6 tumor cells ( l l ) , and regenerating rat liver (12). That polyamines may be linked to DNA synthesis is also suggested by the observation that exogenously added putrescine or spermidine can reverse the inhibition of DNA synthesis by a- difluoromethylornithine (12). We have recently reported that aging of IMR-90 human diploid fibroblasts i n vitro is accom- panied by a 5-fold decrease in the serum-induced ornithine decarboxylase activity and a lack of putrescine accumulation (13). Since the hallmark of cellular aging is the failure of senescent cells to initiate DNA synthesis (14), it is possible that the altered regulation of ornithine decarboxylase in se- nescent cells may contribute to the loss of dividing potential in these cells.

Ornithine decarboxylase has a very short half-life i n uitro and i n vivo and can be modulated by a variety of agents that affect growth (15, 16). Recent developments of methods of labeling specifically the ornithine decarboxylase molecules with a-difl~oromethyl[~H]ornithine, an enzyme-activated ir- reversible inhibitor of ornithine decarboxylase (17, 18), and the construction of cDNA probes (19-22) have made it pos- sible to investigate the cause for the alteration of ornithine decarboxylase activity during senescence of IMR-90 human diploid fibroblasts at molecular levels. In this study, we have compared the time course and levels of induction of ornithine decarboxylase mRNA in young (PDL’ = 22) and senescent (PDL = 52) IMRQO cells after serum stimulation. We have also compared the amount of ornithine decarboxylase mole- cules in young and senescent IMR-90 cells using an affinity labeling technique. Since ornithine decarboxylase activity is cell cycle-dependent, reaching maximal value at the mid-G1 phase of the cell cycle, it is of interest to determine whether other cell cycle-dependent genes in IMR-90 cells also exhibit age-dependent changes in expression during senescence. For this purpose, we have chosen an early G1 gene, c-myc, and a late Gl/S gene, thymidine kinase, and compared levels and time course of induction of these genes in young and senescent cells after serum stimulation. Our data suggest that the func-

The abbreviations used are: PDL, population doubling time; kb, kilobase pair; MOPS, 3-(N-morpholino)propanesulfonic acid.

11431

11432 Gene Expression in Cellular Aging

tional integrity of cell cycle-dependent genes in senescent IMR-90 cells is not compromised until the cells reach the GI/ S boundary.

EXPERIMENTAL PROCEDURES

Cell Culture-Low passage IMR-90 human embryonic lung diploid fibroblasts (passage 5, PDL = 12) were obtained from the Institute for Medical Research (Camden, NJ). Cells were grown in Dulbecco's modified Eagle's medium (with 4500 mg of glucose/liter, without pyruvate) supplemented with 10% fetal bovine serum at 37 "C in a Forma water-jacketed CO:! incubator (95% air, 5% Con). The low passage cultures of IMR-90 cells were expanded through subculturing at a 1:4 or 1:2 split ratio to obtain cultures at higher population doubling levels as previously described (13). Under our experimental conditions, IMR-90 cultures routinely achieved a PDL value of 52 2 3 before entering phase 111, defined as the passage at which cultures could not become confluent 7 days after a 1:2 split subculturing.

Growth Stimulation-Confluent cultures of young (PDL = 22) and senescent (PDL = 52) IMR-90 cells were serum-deprived in fresh Dulbecco's medium for 48 h to ensure that cells were in a quiescent state. Growth stimulation was initiated by replenishing the serum- deprived cultures with fresh Dulbecco's medium supplemented with 10% fetal bovine serum. Cells were harvested at various times after serum stimulation for the preparation of poly(A)+ RNA and enzyme assays.

Isolation of Poly(A)+ RNA and Northern Blot Hybridization-Total cellular RNA was prepared according to the procedure of Chirgwin et al. (23). Poly(A)+ RNA was isolated from total RNA using an oligo(dT)-cellulose column as described (24). Poly(A)+ RNA was incubated for 10 min at 65 "C in a buffer (pH 7.0) containing 20 mM MOPS, 5 mM sodium acetate, 1 mM EDTA, 50% formamide, and 6% formaldehyde and then subjected to electrophoresis on a 1% agarose gel. Gels were blotted onto a nylon paper (Genescreen). The blot was incubated first with denatured salmon sperm DNA (250 pg/ml) at 42 "C in a prehybridization buffer containing 5 X Denhardt's solution, 5 X SSC (0.75 M NaCl, 0.75 M sodium citrate), 50% formamide, and 50 mM sodium phosphate (pH 6.5) for 20 h. The treated blot was then hybridized with nick-translated probe in the prehybridization buffer containing 10% dextran sulfate. The blot was washed and then exposed to Kodak XAR-5 film. For rehybridization, the same blot was washed in 5 mM Tris-HC1 (pH 8.0) containing 0.2 mM EDTA, 0.05% sodium pyrophosphate, and 0.002% each bovine serum albu- min, Ficoll, and polyvinylpyrrolidone for 2 h a t 65 "C and then rehybridized with the appropriate probe. The probes used in this study were (i) the 1.3-kb SstIISstI fragment of hu-myc isolated from the hu-myc 12.7-kb EcoRI insert cloned in pBR327 (25), (ii) plasmid pODC54 (20), (iii) plasmid pTKl l containing the 1.5-kb human thymidine kinase cDNA insert (26), and (iv) cDNA of @-actin (27).

Enzyme Assays-The activity of thymidine kinase was determined according to procedures described by Bradshaw (28). Briefly, cells were harvested into a Tris buffer (50 mM, pH 7.8) containing 1 mM 6-mercaptoethanol and homogenized by sonication. The supernatants obtained from the cell homogenates after brief centrifugation were used as a source of enzyme. The reaction was carried out in 50 mM Tris-HC1 (pH 7.8) containing 1 mM @-mercaptoethanol, 5 mM MgC12, 5 mM ATP, 10 mg/ml bovine serum albumin, and 74 p~ ["C] thymidine at 37 "C for 30 min. The reaction was terminated by adding 10 pl of a solution containing 200 pM ammonium formate and 10 mM thymidine. Aliquots of reaction mixture were spotted onto a 24-mm Whatman DE81 disc, and the amount of radioactivity retained on the disc after washings was determined. Protein concentration was determined by the method of Lowry et al. (29).

Quuntitation ofornithine Decarboxylase-The method used to label ornithine decarboxylase in SV-3T3 cells by Erwin et al. (18) was employed in this study. Briefly, quiescent cultures of young and senescent IMR-90 cells were serum-stimulated for 8 h in fresh Dul- becco's medium containing 10% fetal bovine serum. Cells were lysed in a hypotonic buffer consisting of 25 mM Tris-HC1 (pH 7.5), 0.1 mM EDTA, 25 mM dithiothreitol, and 40 p M pyridoxal phosphate, and cytosolic lysates were prepared by centrifugation at 17,000 X g for 30 min. The labeling was carried out by incubating the cytosolic lysates with 3.6 p~ c~-difluoromethyl[~H]ornithine (26.5 Ci/mmol, 10 pCi/ ml) at 37 "C for 5 h. Ornithine decarboxylase was completely inhibited under this condition. Monospecific rabbit antiserum against ornithine decarboxylase (1:lOO) was added to the cell extracts, followed by the addition of Staphylococcus aureus protein A adsorbent. Proteins bound to protein A were eluted with a buffer containing 2.5% sodium

dodecyl sulfate, 5% @-mercaptoethanol, 20% glycerol, and 62.5 mM Tris-HC1 (pH 6.8) and subjected to sodium dodecyl sulfate-polyacryl- amide gel electrophoresis and fluorography.

Materials-All tissue culture media and sera were obtained from GIBCO. [ ~ Y - ~ * P ] ~ C T P (>3000 Ci/mmol), [2-14C]thymidine (60 mCi/ mmol), and ~~-c~-difluoromethyl[5-'~C]ornithine (>50 mCi/mmol) were purchased from Amersham Corp. The cloned human c-myc gene was obtained from Dr. P. Leder (Harvard Medical School). Dr. P. L. Deininger (Louisiana State University Medical Center, New Orleans, LA) provided plasmid pTK11, and Dr. Olle Janne (Rockefeller Uni- versity) provided plasmid pODC54. A potent rabbit antiserum against mouse ornithine decarboxylase was obtained from Dr. Lo Persson (University of Lund).

RESULTS

Regulation of Ornithine Decarboxylase Gene Expresswn- Fig. 1 shows the expression of ornithine decarboxylase mRNA in young (PDL = 22) and senescent (PDL = 52) cells after serum stimulation. The size of the ornithine decarboxylase mRNA in young and senescent IMR-90 cells was 2.3 kb, similar to that reported in other mammalian systems (19,20). The time course of the increase of levels of ornithine decar- boxylase mRNA in young IMR-90 cells correlated well with the initial increase of ornithine decarboxylase activity in these cells (13); both reached maximal values about 7 h after serum stimulation. Whereas ornithine decarboxylase activity de- clines thereafter (13), the level of ornithine decarboxylase mRNA remained elevated (Fig. l) , suggesting that the decline in serum-induced ornithine decarboxylase activity was not regulated at transcriptional level. In contrast to previous findings (13,30) that serum-induced ornithine decarboxylase activity in senescent cells is low compared to that in young cells, the levels of ornithine decarboxylase mRNA in senes- cent IMR-90 cells were comparable to those in young cells

2.3Kb-

PDL 22 I PDL 52

FIG. 1. Effect of serum stimulation on induction of orni- thine decarboxylase mRNA in young (PDL = 22) and senes- cent (PDL = 52) IMR-90 cells. Quiescent cultures of young and old cells were serum-stimulated for various time periods. Poly(A)+ RNA was isolated and fractionated on a formaldehyde-agarose gel, blotted, and hybridized with 32*P-labeled plasmid pODC54 as de- scribed under "Experimental Procedures." The specific activity of the labeled probe was about 1 X 10' cpmlpg of DNA. The radioactivity was visualized by autoradiography.

Gene Expression in Cellular Aging 11433

92K -

68K -

Tlme(hr ) 0 1 2 7 12 24 0 1 2 7 12 24

c 2.4kb

43K - 30K -

20K - 14K -

FIG. 2. Polyacrylamide gel electrophoresis of protein radi- olabeled by a-difl~oromethyl[5-~HIornithine in young (PDL = 20) and senescent (PDL = 51) IMR-90 cells. Confluent cul- tures of young and old cells were serum-deprived for 36 h and then stimulated by 10% fetal bovine serum for another 8 h. Cells were then harvested, and cytosolic extracts (450 pg of proteins) were prepared for the labeling experiment as described under “Experimen- tal Procedures.” Lane A , IMR-90 cells at PDL = 20; Lzne B, IMR-90 cells a t PDL = 51; lane C, partially purified mouse kidney ornithine decarboxylase.

throughout the time course studied (Fig. 1). These data indi- cated that expression of the ornithine decarboxylase gene at the mRNA level in IMR-90 cells was not affected by aging and that the age-associated decrease in serum-induced orni- thine decarboxylase activity was due to changes at sites other than transcription. Studies from other laboratories (31, 32) have shown that exogenously added polyamines (1 mM) cause a striking decrease of ornithine decarboxylase activity without affecting the existing level of ornithine decarboxylase mRNA, also indicating that ornithine decarboxylase activity can be regulated at sites other than transcriptional control.

To identify further the cause for the alteration of ornithine decarboxylase activity in IMR-90 cells during senescence, we took advantage of the fact that a-difluoromethylornithine, a suicidal inhibitor, binds stoichiometrically to ornithine decar- boxylase and thus can be used to quantify the amount of active ornithine decarboxylase molecules (17, 18). When the same number of young (PDL = 20) and senescent (PDL = 51) cells were processed for radiolabeling by a-difluoromethyl- [3H]ornithine, the amount of labeled ornithine decarboxylase molecules in young IMR-90 cells was 3-fold higher than that in senescent cells (Fig. 2). This ratio correlated well with that of the enzyme activities, suggesting that the difference in the number of ornithine decarboxylase molecules may account, at least in part, for the difference in enzyme activities between young and senescent cells.

Induction of c-myc Expression-The expression of c-myc mRNA is responsive to growth stimulation and occurs early in the GI phase of the cell cycle (33, 34). Fig. 3 shows that addition of serum to quiescent IMR-90 cultures of both young

PDL = 22 I PDL=52

FIG. 3. Effect of serum stimulation on c-myc mRNA expres- sion in young (PDL = 22) and senescent (PDL = 52) IMR-90 cells. Quiescent cultures of both young and senescent cells were serum-stimulated for various times, and poly(A)+ RNA was isolated for Northern blot hybridization as described under “Experimental Procedures.” The 1.3-kb SstI fragment of hu-myc was used as the probe. The specific radioactivity of the nick-translated probe was about 1 X 10’ cpmlpg of DNA.

1 -

J7- 0 6 12 18 24 30 36

TIME ( HOUR)

FIG. 4. Time course of induction of thymidine kinase activ- ity in IMR-90 cells at different PDLs and in AG3198 cells. Confluent cultures of young (PDL = 22; 0), middle-age (PDL = 34; A), senescent (PDL = 53; O), and AG3198 (progeria; X) cells were serum-deprived for 48 h and then stimulated by 10% fetal bovine serum. A t various times thereafter, cells were harvested for the assay of thymidine kinase activity as described under “Experimental Pro- cedures.” Inset, plot of the logarithm of peak thymidine kinase activity as a function of the PDL of the culture.

(PDL = 22) and senescent (PDL = 52) cells caused an increase of the c-myc mRNA level, which reached peak value within 2 h after stimulation. The magnitudes of the induced mRNA levels in young and old cells were comparable throughout the time course examined.

Thymidine Kinase-Thymidine kinase (EC 2.7.1.21), al- though a salvage pathway enzyme, has been reported to be closely associated with cell proliferation and is an integral component of the DNA synthetic apparatus (35, 36). Many studies (35, 36) have shown that growth stimulation is corre- lated with an induction of thymidine kinase activity which

11434

FIG. 5. A , effect of serum stimulation on induction of thymidine kinase mRNA in young (PDL = 22) and senescent (PDL = 52) IMR-90 cells. Quiescent cells were serum-stimulated for various times and then harvested for poly(A)+ RNA isolation. Poly(A)+ RNA was frac- tionated on a formaldehyde-agarose gel, and Northern blot hybridization was car- ried out as described under “Experimen- tal Procedures.” The 1.5-kb BamHI frag- ment of pTK11, containing most of the human thymidine kinase cDNA insert, was used as the probe. B, the same blot was washed and rehybridized with the “P-labeled @-actin probe. The specific activities of both probes were about 1 X 10’ cpm/pg of DNA.

285-

PDL 22 I coincides with the initiation of DNA synthesis. Fig. 4 shows that the kinetics for the induction of thymidine kinase activity in IMR-90 cells appeared to be independent of PDL values; the magnitude of thymidine kinase activity in senescent IMR- 90 cells, however, was significantly reduced. When the loga- rithm of the peak thymidine kinase activity was plotted as a function of PDL, an inverse linear relationship was observed (Fig. 4, inset), suggesting that thymidine kinase activity is closely associated with the “age” of the cultures. It is also of interest to note that serum-induced thymidine kinase activity in AG3198 human fibroblasts, a progeria strain, was extremely low, even compared to that in senescent IMR-90 cells.

The decrease of thymidine kinase activity in senescent IMR-90 cells appeared to be due to alteration of thymidine kinase gene expression at the transcriptional level, as indi- cated in Fig. 5A. The level of thymidine kinase mRNA of young (PDL = 22) IMR-90 cells was more than 8-fold higher than that in senescent (PDL = 52) cells 24 h after serum stimulation. The same experiment was repeated two more times with similar results. The same blot was also rehy- bridized with a cloned cDNA probe of a cell cycle-independent gene, @-actin (27), to ensure an internal control. As can be seen in Fig. 5B, no difference in the levels of @-actin mRNA can be observed in young and senescent IMR-90 cells.

DISCUSSION

The original discovery by Hayflick and Moorhead (1, 3) that human diploid fibroblasts, unlike transformed tumor cell lines, have a limited life span in tissue culture has greatly stimulated interests in using human diploid fibroblasts as a model to investigate the biochemical basis of aging at the cellular level (2). Since the hallmark of cellular aging is the failure of senescent cells to enter or to complete the S phase of the cell cycle (2, 14), it is reasonable to speculate that the underlying cause of aging may be related to alterations of factors or gene expressions which are involved in DNA rep- lication.

Our interest in studying the control of gene expression in the G1 phase of the cell cycle is also enhanced by the finding that senescence of IMR-90 cells is accompanied by significant changes of polyamine metabolism (13, 37), most notably, a decrease of ornithine decarboxylase activity (13). That the induction of ornithine decarboxylase activity during the mid- GI phase may be needed for the initiation of DNA synthesis in animal cells has been previously suggested (10-12).

Mammalian ornithine decarboxylase activity can be regu-

PDL 52

28s -

Y

PDL 22 I PDL 52

lated at many levels of gene expression. Both transcriptional/ post-transcriptional and translational control of the induction of ornithine decarboxylase activity in cultured cells have been reported (18, 31). In addition, there has been some evidence that post-translational modification such as phosphorylation (38) and association with a specific inhibitory protein, anti- zyme (39), may also be involved in controlling ornithine decarboxylase activity. In this study, we detected a difference neither in the levels nor in the time course of serum-induced ornithine decarboxylase mRNA between young and senescent IMR-90 cells (Fig. 1). Quantitative affinity labeling of cyto- solic ornithine decarboxylase with a-difluoromethyl [5-3H]- ornithine supported the notion that the age-associated de- crease of ornithine decarboxylase activity was not regulated at the transcriptional level (Fig. 2). The more precise mech- anism of this age-associated alteration remains to be eluci- dated. For example, the translatability of ornithine decarbox- ylase mRNA in senescent IMR-90 cells may decrease; alter- natively, there may be an age-associated change of distribution of ornithine decarboxylase mRNA between mRNA particles and polysomes.

The temporal relationship between the expression of the c- myc and ornithine decarboxylase genes is well documented (40). Mitogens such as lipopolysaccharides, concanavalin A, and growth factors that induce c-myc expression (33, 34) inevitably induce ornithine decarboxylase activity (15, 16). It has been demonstrated (41), that DNA synthesis in 3T3 fibroblasts can be initiated by microinjecting c-myc protein in the presence of platelet-poor plasma, suggesting that c-myc expression is needed for cells to enter the S phase. Both young and senescent IMR-90 cells responded equally well to serum stimulation with an early induction of c-myc mRNA that precedes the induction of ornithine decarboxylase mRNA (Fig. 3). Although expressions of the c-myc and ornithine decarboxylase genes may not be causally related, the fact that these two genes were expressed equally well at the mRNA level in both young and senescent IMR-90 cells suggested that senescent IMR-90 cells were as functionally active as young cells in responding to serum stimulation and in tra- versing through the first half of the G1 phase of the cell cycle.

In view of the recognized importance of polyamines in growth regulation and DNA replication (8-12), it is tempting to speculate that the decrease in ornithine decarboxylase activity and the lack of putrescine accumulation in senescent cells (13) may affect biochemical events that occur in the late GI and/or S phase. Our data on thymidine kinase gene expres-

Gene Expression in Cellular Aging 11435

sion (Figs. 4 and 5A) support this notion. Although the causal relationship between polyamine biosynthesis and thymidine kinase induction remains to be established, it is of interest to note that Cheetham and Bellett (42) have reported that a- methylornithine and methylglyoxal bis(guanylhydrazone), both inhibitors of polyamine biosynthesis, inhibit serum- induced thymidine kinase activity in quiescent rodent cells. The apparent inverse relationship between thymidine kinase activity and PDL (Fig. 4) suggests that the inducibility of thymidine kinase may be tightly coupled to the aging process.

It has been proposed (43) that the assembly of a multien- zyme complex, termed replitase for DNA replication, may signal the initiation of the S phase of this cell cycle. The functional integrity of this complex will be compromised if one if its components is inactivated or absent (43). Thymidine kinase has been suggested to be one of the components comprising this complex (43). If this is the case, our data indicate that the thymidine kinase gene plays a critical role in aging of IMR-90 cells. Alternatively, thymidine kinase gene expression and the onset of DNA synthesis may be coordi- nated by a common cellular signal which is altered in cells during senescence. This notion implies that expressions of other genes located at the late G1 and/or G,/S boundary of the cell cycle may also be altered during senescence. Thus, it is possible that alteration of expressions of any one or two genes will be sufficient to impair DNA synthesis. In this regard, it is relevant to note a recent study by Rittling et al. (44). Although they suggested that alterations of gene expres- sion should occur at the G1/S phase or deep in the S phase in senescent cells, they did not find significant differences in levels of thymidine kinase mRNA between young and old WI- 38 human fibroblasts. One possible explanation for this dis- crepancy is that the defect of gene expressions in the late G1 or Gl/S phase of the cell cycle during cell senescence may be different for different cell strains. We are currently investi- gating whether alteration of gene expression during senes- cence of IMR-90 cells is unique for thymidine kinase or is a more general phenomenon involving other Gl/S genes.

Based on the results presented above and on our previous findings (13,37), we have formulated a working hypothesis to illustrate the possible mechanism of cellular aging (45). In this model, we propose that changes of gene expression during the late GI or Gl/S phase (e.g. thymidine kinase) may cause a defect in the DNA replication apparatus and hence prevent senescent IMR-90 cells from entering or completing the S phase. Whether decreases of ornithine decarboxylase activity and putrescine accumulation are directly involved in the alteration of late GI/S gene expression will be investigated.

Acknowledgments-We thank Drs. P. Leder, 0. Janne, P. L. Dein- inger, and L. Persson for kindly providing us with cloned human c- myc gene, plasmid pTK11, plasmid pODC54, and monospecific anti- ornithine decarboxylase antiserum, respectively. We also thank Dr. A. Y.-C. Liu for critical reading of the manuscript.

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