Kuang Yu Chen

Department of Chemistry and The Cancer Institute of New Jersey, Rutgers - The State University of New Jersey, Piscataway, NJ. 08855-0939

Received 8/19/97 Accepted 8/25/97

3 CHANGES OF GENE EXPRESSION DURING SENESCENCE

3.1 Global attenuation of G1/S genes

Normal human diploid fibroblasts at quiescent state can be stimulated by serum or appropriate growth factors to enter the cell cycle. We and others have compared the changes of gene expression during cell cycling between young (low PDL) and senescent (high PDL) human cells. The senescent cells can enter the cell cycle and are capable to fully express many cell cycle-dependent genes all the way to mid-G1 phase (6-9). However, the cell cycling journey of senescent cells appear to be blocked at the G1/S boundary. The results from these studies are summarized in Figure 1. All of the G1/S genes studied thus far show attenuation of gene expression during senescence (6-9). These include genes encoding TK, PCNA, TS, RNR, DHFR, histone proteins H1, H2A, H2B, H3, and H4, cdc2, cyclin A and cyclin B (18). In contrast to G1/S genes, all mid-G1 genes that we and others have examined, including ornithine decarboxylase (ODC) and eIF-5A, appear to be fully induced by serum in senescent cells.. However, it should be noted that although expressions of mid-G1 genes such as eIF-5A and ODC do not show age-dependent attenuation at mRNA levels, a significant difference exists in the translation and/or post-translational modification of their gene products (6, 7, 19).

Figure 1. Schematic diagram of the expression of various cell cycle-dependent genes in serum-stimulated senescent human diploid fibroblasts. Among the early- and mid-G1 genes examined, only c-fos appears to be suppressed in senescent cells. In contrast, all the late G1/S genes examined appear to be suppressed in serum-stimulated senescent IMR-90 cells. Genes whose expressions show age-dependent attenuation are indicated in the box.

3.2 Molecular basis for the global attenuation of G1/S genes

The fact that G1/S genes that we and others have studied all exhibit age-dependent down-regulation in senescent cells raises a possibility that these genes may share a common or similar regulatory mechanism during cell senescence. To investigate this possibility, one needs to examine the regulatory mechanism of each G1/S gene in normal cells during senescence. The general strategy of this approach includes (i) performing nuclear run-on and promoter activity assay to determine whether the gene is transcriptionally regulated during the progression of cell cycle, (ii) performing nuclease protection assay to identify the promoter region that binds nuclear proteins, (iii) performing gel mobility shift assay to define the cis-elements which exhibit the cell cycle- and age-dependent binding activity, and finally (iv) using the cis-element as the probe or affinity media to isolate and characterize the trans-acting factor responsible for the age-dependent attenuation of the particular G1/S gene. Figure 2 shows that using this approach we have identified the promoter region and cis-elements responsible for the TK and DHFR gene during the progression of cell cycle and during senescence. The age-dependent CBP/tk binding activity is confined in the promoter region containing Y-box sequence. E2F1 is likely to be the key transcription factor for the age-dependent binding activity in DHFR promoter region containing E2F sequence (TTTCGCGC). Both CBP/tk and E2F binding activities are serum-responsive and age-dependent. Transcription factors such as CBP/tk and E2F1, may represent a new class of trans-acting factors which control gene expression not only during the cell growth but also during cell senescence.

Figure 2. Gel mobility shift assay of the nuclear protein binding to the human TK and DHFR gene promoter in young (PDL=21) and old (PDL=50) IMR-90 cells following serum stimulation. The CBP/tk sequence used to perform the binding studies was the 28-bp fragment (-155/-128). The E2F sequence used was a 33-bp fragment spanning -77/ -45 bp relative to ATG codon

3.3 Binding Activity as measured by gel mobility shift assay

Another approach to assess the role of transcription factors and their binding sites in cell aging is to use a synthetic oligonucleotide probe to examine whether a particular transcription factor may exhibit age-dependent binding activity. The probe contains the consensus binding sequence for that transcription factor. Figure 3 illustrates how this approach could lead to the identification of CREBP and CTF as putative age- dependent transcription factors. CREBP recognizes the sequence TGACGTCA whereas CTF recognizes GCCAAT. Using the synthetic probe containing tandem CRE or CTF, we show here that the CREBP and CTF binding activities in human IMR-90 cells are both serum-responsive and age-dependent. Similar results have been reported by Dimri and Campisi for WI38 cells (20). This approach, however, would not allow us to identify the target genes of these transcription factors. Thus, the physiological significance of this finding remains to be established.

Figure 3. Gel mobility shift assay of the the CRE and CTF probes. The synthetic oligonucleotide probes with sequences CRE [TGACGTCA]5'-GATCTGACGTCATGACTGACGTCATGACTGACGTCATCA-3',CTF[GCCAAT]5'-GATCGCCAATGAT CGCCAAT GATC GCCAATGATC-3' were purchased from GIBCO-BRL. The nuclear extracts obtained from young (PDL=21) and old (PDL=50) IMR-90 cells at designated time points after serum stimulation were used for gel mobility shift assays with an oligonucleotide probe.

3.4.The regulation of transcription factor activity during senescence

Once an age-dependent transcription factor is identified, it will be of interest to study as how this transcription factor is regulated during senescence. Studies have already shown that E2F1 is controlled at transcriptional level by E2F proteins during the progression of cell cycle (21). Since the E2F1 message is greatly reduced in senescent cells (11), it is likely that the E2F binding sites will be responsible for the down-regulation of E2F1 gene during senescence. With regards to the transcription factor specific for TK gene regulation, CBP/tk, it has been suggested that CBP/tk is identical to NF-Y, the binding protein for the Y box within E alpha gene promoter (12). However, we note that (i) the half-life of NF-Y is much longer than that of CBP/tk in IMR-90 cells; (ii) the effect of serum is more pronounced with CBP/tk (>10-fold induction) than with A subunit of NF-Y (~2-to 3-fold induction) in IMR-90 cells; (iii) B subunit of NF-Y is not serum responsive in IMR-90 cells; and (iv) the increase in NF-Y does not correlate with the increase in CBP/tk binding activity in HeLa cells (13). These discrepancies prompt us to speculate that either CBP/tk is similar but not identical to NF-Y or there exist other factors in CBP/tk binding complex. Recently, a third component of NF-Y, CBF-C (NF-YC), has been isolated and cloned (14). Thus, it remains to be seen whether NF-YC or other unidentified factor may be responsible for the unique age-dependent features of CBP/tk.