ExperimentalGerontology, Vol. 27, pp. 403-407, 1992 0531-5565/92 $5.00 + .00 Printed in the USA. All fights reserved. Copyright © 1992 Pergamon Press Ltd.

A L T E R E D E X P R E S S I O N O F C E L L C Y C L E D E P E N D E N T G E N E S I N S E N E S C E N T W l - 3 8 C E L L S

PAUL D. PHILLIPS a n d ROBERT J. PIGNOLO

The Center for Gerontological Research and the Department of Physiology/Biochemistry, Medical College of Pennsylvania, Philadelphia, Pennsylvania 19129

Key Words: c-los, cell aging, transcription, c-jun, jun B

ThE HALLMARK of cell aging in culture is the reduced and ultimate loss of the ability to initiate DNA synthesis in response to serum mitogens or purified growth factors (Hayflick and Moorhead, 1961; Hayflick, 1965; Ohno, 1979; Plisko and Gilchrest, 1983; Phillips et al., 1984). The final failure in the proliferative response is preceded by a lengthening in the cell cycle time primarily at the expense of the G~ phase (Grove and Cristofalo, 1977). Since it is the G0/GI period which is believed to be most critical in regulating cell proliferation, many laboratories have focused on this prereplicative phase in order to try and understand the process of cellular senescence. Our own studies (Carlin et al., 1983; Phillips et al., 1983; Brooks et al., 1987; Phillips et al., 1987; Gerhard et al., 1991) as well as those of other groups (Chua et al., 1986; Paulsson et al., 1986) have examined growth factor-receptor interactions and have found that the number of epidermal growth factor (EGF), platelet- derived growth factor (PDGF), and insulin-like growth factor-I (IGF-I) receptors (per cm 2 of cell surface area) are the same in young and senescent cells, as are the three ligand-recep- tor affinities. It has also been observed in situ that growth factor-stimulated tyrosine phos- phorylation of the EGF and PDGF receptors is unaffected in senescent cells (Gerhard et al., 1991). Since there are no apparent changes in these initial growth factor-receptor interactions, researchers in several laboratories examined the expression and regulation of genes expressed in the G0/Gt phase of the cell cycle. Most of the genes studied are expressed normally at the mRNA level in senescent cells (Rittling et al., 1986; Seshadri and Campisi, 1990). These studies have included several of the protooncogenes, with the finding that some (myc and ras) are expressed normally (Rittling et al., 1986; Seshadri and Campisi, 1990), while at least one (fos) is not expressed in senescent WI-38 cells (Seshadri and Cam- pisi, 1990). These genes are widely believed to play critical roles in regulating cell prolif- eration. In this article we present the results of experiments designed to measure the expression of the early response genes c-jun and jun B, the effect of the forced expression

Correspondence to: P.D. Phillips, Ph.D., Center for Gerontological Research, Medical College of Pennsylva- nia, 3300 Henry Avenue, Philadelphia, PA 19129.

403

404 P.D. PHILLIPS AND R.J. P|GhlOLO

of c'fos in young and senescent cells, and preliminary data on the nature of the altered regulation of the c-los gene in senescent cells.

Seshadri and Campisi (1990) have demonstrated that c-fos is barely detectable in serum- stimulated senescent WI-38 cells, as opposed to serum-stimulated young cells. We have examined the expression of mRNAs for other early response genes c-jun and jun B. We found similar expression ofc-jun andjun B in both young and senescent cells. This was of interest because the protein products of c-jun and jun B genes are known to form transactivating AP-1 complexes with the c-fos gene product. This raises the possibility of "fine tuning" in the formation of various transactivating complexes and thus the regula- tion of differential gene expression.

In view of the fact that c-fos is expressed at very low levels in senescent WI-38 cells (Ses- hadri and Campisi, 1990), we undertook a series of transient transfection experiments (in collaboration with K. Nishikura of the Wistar Institute) in which a c-fos containing con- struct was expressed in young and senescent WI-38 cells. The mouse c-fos gene was placed in a plasmid under the control of the sheep metallothionein promoter. Following trans- fection the gene was induced with Zn ÷ + or not induced, and [3H]-TdR was added to all cultures. After 24 h the cells were fixed and prepared for autoradiography, and the percent labeled nuclei were scored for each culture. All of the experiments included mock trans- fected control cultures.

The data are summarized in Fig. 1 and are expressed as the percent increase in labeled nuclei in the transfected Zn + +-induced cultures versus the transfected, uninduced cul- tures. In the youngest cultures tested (44% life span completed) there was no increase in

6 0 0 - Zn Induced vs. Uninduced Control

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FIG. 1. T h e p e r c e n t i n c r e a s e in l abe led nuc le i in t r a n s f e c t e d c u l t u r e s i n d u c e d w i th Z n + + as c o m -

p a r e d to t r a n s f e c t e d u n i n d u c e d cu l tu re s . *Differs a t t he < 0 . 0 1 level.

GENE EXPRESSION AND CELL AGING 405

the percent of cells in DNA synthesis in the induced cultures. This was presumably due to the fact that the uninduced cultures were near their maximum level of DNA synthesis (approximately 80% labeled nuclei in a 24-h period). As the cultures became older and began to enter what we consider the senescent phase, a marked stimulation was seen. This peaked at 94% life span completed with approximately a fivefold increase in the number of cells entering DNA synthesis in the induced (c-fos expressing) cultures. It is important to note that this stimulation declined to an insignificant level at the end of the life span (100% life span completed). In BrdU-selected cultures (in which no control or uninduced cells enter DNA synthesis) none of the Zn + + induced cells entered DNA synthesis. Thus, at the very end of the proliferative life span the forced expression of c-fos could not drive these cells to enter DNA synthesis. The only way known, thus far, to stimulate these cells to enter DNA synthesis is by infection with the simian virus 40 (SV40) (Gorman and Cris- tofalo, 1985). However, as cells go through the process of senescence, there appears to be a period in which they can be stimulated to enter DNA synthesis if the c-los gene can be expressed, at least under the experimental conditions used in these studies. Those senes- cent cells that respond to the c-fos induction may otherwise be refractory to serum stim- ulation. In this case a second event may not yet have occurred, and c-fos expression along with serum would be sufficient for the cells to enter DNA synthesis. Alternately, the responsive cells may be representative of those senescent cells with an extended G~ phase. In this case higher levels of c-fos expression may be enough to shorten the senescent cells' G~ transit time and thus allow them to enter DNA synthesis during the labeling period. In either case the data are consistent with a model of senescence in which a series of pheno- typic changes occurs that ultimately leads to the inability of the cell to enter DNA synthesis (except in the case of SV40 infection).

Seshadri and Campisi (1990) carried out nuclear run-on assays in which they measured the rate of c-fos transcription. Their results indicate that the c-fos gene is transcriptionally repressed in senescent cells. In preliminary experiments we have recently taken a different approach to studying the regulation of the c-fos gene in young and senescent cells. We used the reverse transcription-polymerase chain reaction (RT-PCR). This technique allows the detection of short lived mRNA precursors (heterogeneous nuclear RNA [hnRNA]). We have used it in past experiments to detect a posttranscriptional block in the expression of the proliferating cell nuclear antigen (PCNA), a subunit ofDNA polymerase delta (Change et al., 1991).

This approach allows us to measure the steady state levels ofc-fos hnRNA derived from both young and senescent cells. The amplimers chosen for this reaction were from the first intron and the first exon of the human c-fos gene, and controls were run to exclude the possibility of DNA contamination. Following amplification and Southern blotting, the fil- ters were hybridized with a probe derived from a sequence 5' from the internal intron l amplimer.

Our preliminary data show essentially the same steady state levels of c-fos hnRNA in young and senescent cells following serum stimulation. This is in contrast to the very low levels of c-fos mRNA in senescent cells under the same conditions. Initially this appears to be at variance with the nuclear run-on experiments previously reported (Seshadri and Campisi, 1990); however, the assays do not measure the same thing. The nuclear run-on assay measures the rate of transcription, and assuming that the transcripts are not differ- entially terminated, it can be used as a reliable indicator of the rate of transcription initi- ation. The RT-PCR assay measures the steady state levels ofhnRNA. If the initial rate of transcription is the same but the transcripts are prematurely and differentially terminated

406 P.D. PHILLIPS AND R.J. PIGNOLO

in senescent cells, then the early 5' regions of the transcripts will appear equivalent in both age groups. In this case the nuclear run-on assay is actually measuring the rate of transcript elongation, and the young cells will have a greater rate since the transcripts will be longer and have a higher specific activity. Thus, the two assays complement each other and the data need not be contradictory. We are currently pursuing both approaches to try and understand the changes in c-fos expression in senescent WI-38 cells.

In conclusion we have found that the c-jun andjun B mRNAs are transcribed normally in senescent cells. This is important since their gene products form transactivating com- plexes with the product of the c-fos gene. The forced expression of c-los in serum-stimu- lated senescent cells that have not yet reached the end of their proliferative life span is sufficient to drive them into DNA synthesis. However, at the end of the proliferative life span c-los expression is not sufficient to cause cells to enter DNA synthesis. These data are consistent with a multistep process through which senescent cells ultimately lose their abil- ity to enter DNA synthesis. Finally, our preliminary data suggest that expression of the c- los gene may be regulated, at least in part, by a posttranscriptional block shortly after the initiation of transcription.

Acknowledgments - - We wish to recognize the technical contributions made to this work by Wei Chou and Man Wang. RJP is a graduate student in the Graduate Group in Molecular Biology at the University of Pennsylvania. This work was supported by PHS grants R01 AG09778 and P01 AG000378.

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GENE EXPRESSION AND CELL AGING 407

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