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(The FASEB Journal. 2005;19:521-532.)
© 2005 FASEB

Expression of the Leo1-like domain of replicative senescence down-regulated Leo1-like (RDL) protein promotes senescence of 2BS fibroblasts

Liang Zhao*,{dagger}, Tanjun Tong*,{dagger},1 and Zongyu Zhang*,{dagger},1

* Peking University Research Center on Aging, Peking University Health Science Center, and
{dagger} Department of Biochemistry & Molecular Biology, Peking University Health Science Center, Beijing, People’s Republic of China

1Correspondence: Department of Biochemistry & Molecular Biology, Peking University Health Science Center, 38 Xueyuan Rd., Beijing 100083, P. R. of China. E-mail: ttj{at}bjmu.edu.cn


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Replicative senescence is thought to relate to aging in vivo and tumor suppression. In this report, we isolated a gene and designated it as RDL (replicative senescence down-regulated Leo1-like gene). RDL’s expression decreased upon replicative senescence of human diploid 2BS fibroblasts. Overexpression of RDL slightly delayed 2BS fibroblast senescence, whereas suppression of RDL expression imposed no obvious effects on senescence. However, introduction of cDNA fragment encoding the Leo1-like domain of RDLp (Leo) alone shortened the replicative life span of 2BS fibroblasts and promoted several senescent features; the introduction of truncated RDL cDNA fragment resulting from deletion of Leo (RDL-Leo–) significantly prolonged 2BS life span and caused a noticeable delay of these senescent features. We demonstrated that introduction of Leo obviously increased the expression of p16INK4a, p21WAF1, and PTEN, whereas introduction of RDL-Leo– distinctly decreased p16INK4a expression. Taken together, our results suggest that the Leo1-like domain of RDLp is a senescence-associated domain that accelerates the senescence of 2BS fibroblasts and that there should be another counteractive domain in the remaining part of RDLp.—Zhao, L., Tong, T., Zhang, Z. Expression of the Leo1-like domain of replicative senescence down-regulated Leo1-like (RDL) protein promotes senescence of 2BS fibroblasts.


Key Words: replicative senescence • subtractive hybridization • RDL • Leo1-like domain


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
MAMMALIAN CELLS undergo a limited number of cell divisions, or population doublings (PD), in culture, then irreversibly arrest at G1 phase of the cell cycle. This is defined as replicative senescence (1) . It is thought to underlie the organismal aging in vivo and relate to tumor suppression and age-related diseases (2 3 4 5) . Although viable and metabolically active, the senescent cells are no longer sensitive to growth factor stimulation (6) . After serial passaging, cultures of mammalian cells display the features of senescent cells (7) : suppression of proliferation capacity, cell cycle arrest at G1 phase (1) , an enlarged, flat morphology (8) , and expression of a neutral senescence-associated ß-galactosidase (SA-ß-Gal) (9) .

Although epigenetic factors may play an important role during aging, senescence is dominant over immortality at the cellular level, according to cell fusion and microinjection studies (10) , which revealed that replicative senescence might rely on a genetic background.

Replicative senescence is associated with a complex change of gene expression. There are extensive reports about changes in the pattern of gene expression during the process of replicative senescence of human fibroblasts using subtractive hybridization (11 12 13 14 15) , microarray (16 , 17) , and other methods (18) . However, the principal mechanisms and the hierarchy of regulatory events during senescence are not yet fully understood. More research on differential gene expression patterns should be conducted, and more senescence-associated genes need to be identified.

Initial studies have proposed that negative regulators whose expressions are up-regulated upon senescence may play important roles. Do genes with down-regulated expression upon senescence also affect induction of the senescence? To find the answer, we identified genes with down-regulated expression in human diploid 2BS fibroblasts entering replicative senescence using suppression subtractive hybridization and differential screening, and chose one candidate gene to analyze its function in 2BS senescence.

A novel cDNA was first identified and named replicative senescence down-regulated Leo1-like (RDL) gene. To further characterize RDL expression, its tissue distribution pattern was examined and the subcellular localization of RDLp was determined to provide hints of its function. To analyze RDL’s effects on 2BS senescence, sense and antisense RDL expression vectors were constructed and stably transfected into 2BS cells. According to bioinformatics analysis, which suggested there was a Leo1-like domain at the C terminus of RDLp, expression vectors that contained cDNA fragment coding for the Leo1-like domain of RDLp (Leo) and truncated RDL cDNA fragment resulting from deletion of Leo (RDL-Leo–) were constructed and stably transfected into 2BS cells. After transfection, several markers of replicative senescence, including cell growth rate, G1 DNA content, morphological phenotype, SA-ß-Gal activity, and replicative life span, were observed to assess the senescence from a comprehensive perspective. The relationship between RDL and its domains and three known important genetic pathways (p16INK4a/Rb, p53/p21WAF1, and PTEN/p27KIP1) (19) has been analyzed.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell line and cell culture
The 2BS cell line from human female embryo lung was established at the National Institute of Biological Products (Beijing, China) and has been fully characterized (20 21 22 23) . The expected replicative life span of 2BS cells is 55–60 PD. 2BS cells were considered to be young at PD 30 or below and fully senescent at PD 55 or above. The cells were maintained in Dulbecco’s modified Eagle’s medium (Life Technologies, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT, USA), 100 units/mL penicillin, and 100 µg/mL streptomycin, at 37°C in 5% CO2. 80% confluent cultures were detached by PBS solution containing 0.25% trypsin and 0.02% EDTA, then split at a ratio of 1:2.

RNA isolation and cDNA synthesis
Cells were harvested by trypsin-EDTA treatment at 70–80% confluence. Total RNA was isolated using RNeasy Mini Kit (Qiagen, Chatsworth, CA, USA). cDNA was synthesized from 1 µg total RNA with SMART PCR cDNA Synthesis Kit (Clontech, Palo Alto, CA, USA) using a modified oligo (dT) primer. After PCR amplification, the product was digested with RsaI (Promega, Madison, WI, USA) and extracted using QIAEX II Gel Extraction System (Qiagen).

Suppression subtractive hybridization
Suppression subtractive hybridization was performed with a PCR-Select cDNA Subtraction Kit (Clontech) according to the manufacturer’s protocol. cDNA of young 2BS cells (PD 25) was used as tester and cDNA of senescent 2BS cells (PD 56) as driver. Briefly, tester cDNA was divided in two, then each part was ligated with two different adapters. Each sample was mixed with an excess amount of driver cDNA, heat denatured, then allowed to reassociate. The resulting two samples were mixed to allow the remaining complementary single strands to anneal, and suppression PCR was performed. The subtracted cDNA was ligated to pGEM-T easy (Promega) and transformed into Escherichia coli strain JM109 to create a subtractive cDNA library Y-S.

To create a subtracted probe for differential screening, a reverse subtractive hybridization was performed simultaneously, using cDNA of senescent 2BS cells as tester and cDNA of young cells as driver. The resulting subtracted product was designated S-Y.

Differential screening
Since the subtracted sample may still contain cDNAs that correspond to mRNAs common to tester and driver samples, a differential screening step was performed. Single colonies were picked from the subtracted library and cDNA inserts were amplified by PCR. Differential screening was performed using the PCR-Select Differential Screening Kit (Clontech) following the manufacturer’s protocol: 3 µL of each PCR product was spotted onto four nylon membranes (Biodyne B, Pall); the blots were hybridized with the [{alpha}-32P] dCTP-labeled unsubtracted (young and senescent cDNA) and subtracted (forward Y-S and reverse S-Y subtracted cDNA pool) probes separately. All probes were labeled with Prime-a-Gene Labeling System (Promega) and hybridized to the blots in ExpressHyb Hybridization Solution (Clontech). Inserts showing a signal with Y-S probe 3-fold stronger than that with S-Y probe were considered to be differentially expressed and were sent to Sangon Company (Shanghai, China) for sequencing.

Virtual Northern and Northern blot analyses
Total cDNA was synthesized as described above: 1 µg of each total cDNA was electrophoresed on 1% agarose/EtBr gel, blotted to a nylon membrane, and hybridized with [{alpha}-32P] dCTP-labeled subtracted cDNA products. Hybridization with GAPDH (glyceraldehyde-3-phosphate dehydrogenase) served as a loading control.

Northern blot analysis was performed to further confirm the decreased expression of clone Y114. Total RNAs were isolated from young and senescent 2BS cells. Approximately 20 µg of each RNA sample was separated on a 1% agarose/5% formaldehyde gel and blotted to a nylon membrane. PCR-generated fragments of clone Y114 and GAPDH were labeled with [{alpha}-32P] dCTP, then hybridized to the filters.

Rapid amplification of cDNA ends (RACE) of Y114
cDNA from young 2BS cells was subjected to rapid amplification of cDNA ends using the 5' and 3' RACE System (Life Technologies) according to the manufacturer’s instructions. The primers used were: 5'GSP1, 5'-TGCGTTGGCATTCAGG-3'; 5'GSP2, 5'-CCTATCCGCAAGTGACAGAGT-3'; 5'GSP3, 5'-TGTCCGTAGAGTGAGGTCTGAAG-3'; 3'GSP1, 5'-CAGCAGCGCCGAATGAGAG-3' and 3'GSP2, 5'-GATCAGCGATGAAGAGGAAGA-3'. Amplification products were cloned into pGEM-T easy vector and sequenced.

Tissue distribution pattern analysis
The tissue distribution pattern was determined by using a human multiple tissue Northern blot (Clontech) and [{alpha}-32P] dCTP-labeled Y114 cDNA fragment as the probe.

Determination of subcellular localization
The entire RDL coding region was amplified by PCR [30 cycles, annealing temperature 58°C, and primers 5'-GATAATGGCGGATATGGAG-3' (forward) and 5'-CAATCAAAATAACTCATGTTTACAT-3' (reverse)]. The amplified products were ligated into the pT-Adv (Clontech) and subcloned into KpnI and ApaI sites of pEGFP C3 (Clontech) to yield the construct pEGFP C3-RDL (Fig. 1 B). After sequencing to ensure no errors were introduced into the RDL coding region by PCR, the construct pEGFP C3-RDL and empty vector pEGFP C3 were transiently transfected into young 2BS cells (PD 29) and HeLa cells using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s instructions. After 36 h the cells were fixed with 4% paraformaldehyde, stained in 2 µg/mL DAPI (4',6-diamidino-2-phenylindole) for 1 min at room temperature according to established method (24) , then observed by fluorescence confocal microscopy.



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  		Figure 1. Strategy for creation of Leo and RDL-Leo– (A) and summary of all the constructs in this paper (B).

Bicistronic expression vectors constructions and stable transfection
The RDL coding region was ligated into pGEM-T easy, then cloned into the NotI site of bicistronic vector pIRESneo2 (Clontech) to generate pIRES-RDL (sense) and pIRES-RDLas (antisense) recombinant vectors, which were selected by restriction enzyme analysis.

cDNA fragment encoding the Leo1-like domain of RDLp (Leo) was amplified by PCR (Fig. 1A ) [30 cycles, annealing temperature 66°C, and primers 5'-TATGGACACTGAGGTGCCAAAAGA-3' (forward, underlined letters indicate mutated bases) and 5'-TCACCTTCCTCATCACTGGTAAGT-3' (reverse)]. The forward primer introduced an in-frame start codon 5' of Leo.

Truncated RDL cDNA fragment resulting from deletion of Leo (RDL-Leo–) was yielded by PCR with the ligation product of the RDL-Leo– 5' fragment and RDL-Leo– 3' fragment as the amplification template (Fig. 1A ). The RDL-Leo–- 5' fragment was amplified [30 cycles, annealing temperature 56°C, and primers 5'-GATAATGGCGGATATGGAG-3' (forward) and 5'-TATCCGCTCCGGAATCAGATGC-3' (reverse, underlined letters indicate two mutated bases and italic letters indicate a Kpn2I site)]. The RDL-Leo–- 3' fragment was amplified [30 cycles, annealing temperature 56°C, and primers 5'-TGAACCTTCCGGAAAGAGAAAAG-3' (forward, italic letters indicate a Kpn2I site) and 5'-CAATCAAAATAACTCATGTTTACAT-3' (reverse)]. The reverse primer for amplifying RDL-Leo– 5' fragment introduced a Kpn2I site 3' of the fragment. Correspondingly, there was a Kpn2I site 5' of RDL-Leo– 3' fragment. Both fragments were digested with Kpn2I and ligated together. The resulting product was used as PCR template to amplify RDL-Leo– by using the forward primer of RDL-Leo– 5' fragment and the reverse primer of RDL-Leo– 3' fragment.

The Leo and RDL-Leo– fragments were ligated into the pGEM-T easy respectively and fully sequenced. The inserts were subsequently released with EcoRI (pGEM-T-Leo) and NotI (pGEM-T-RDL-Leo–) and cloned into the corresponding sites of pIRESneo2 separately to yield pIRES-Leo and pIRES-RDL-Leo–. Sense vectors were selected by restriction enzyme analysis.

pIRES-RDL, pIRES-RDLas, pIRES-Leo, pIRES-RDL-Leo– (Fig. 1B ), and the empty plasmid pIRESneo2 were transfected into young 2BS cells (PD 25) with Lipofectamine 2000. After 48 h, the cells were selected by G418 (300 µg/mL; Life Technologies). Colonies of stable transfectants were isolated 3–4 wk later and propagated accordingly in complete medium containing 50 µg/mL G418. The resulting transfectants were termed 2BS/RDL, 2BS/RDLas, 2BS/Leo, 2BS/RDL-Leo–, and 2BS/neo, respectively.

Expression of 6xHis-tagging proteins
cDNA fragments encoding RDL, Leo, and RDL-Leo– with in-frame 6xHis-Tag at the C terminus (RDL-His, Leo-His, and RDL-Leo–-His) were amplified by PCR using RDL, Leo, and RDL-Leo– cDNA as template, respectively. The primers used were: RDL-His: forward 5'-GATAATGGCGGATATGGAG-3', reverse 5'- GTGATGGTGATGGTGATGATCATCATCTTCTTCCTCTTC-3' (30 cycles, annealing temperature 56°C); Leo-His: forward 5'- TAGTGACACTGAGGTGCCAAAAGA-3', reverse 5'-TCAGTGATGGTGATGGTGATGTTCACCTTCCTCATCACTGGTAAGT-3' (30 cycles, annealing temperature 60°C); RDL-Leo–-His: forward 5'-GATAATGGCGGATATGGAG-3', reverse 5'-GTGATGGTGATGGTGATGATCATCATCTTCTTCCTCTTC-3' (30 cycles, annealing temperature 56°C).

The RDL-His, Leo-His, and RDL-Leo–-His fragments were ligated into the pGEM-T easy respectively and fully sequenced. The inserts were subsequently released with EcoRI (pGEM-T-Leo-His) or NotI (pGEM-T-RDL-His and pGEM-T-RDL-Leo–-His), then cloned into the corresponding sites of pIRESneo2 separately to yield pIRES-RDL-His, pIRES-Leo-His, and pIRES-RDL-Leo–-His. Sense vectors were selected by restriction enzyme analysis.

The pIRES-RDL-His, pIRES-Leo-His, pIRES-RDL-Leo–-His (Fig. 1B ) and the empty plasmid pIRESneo2 were transiently transfected into 2BS cells (PD 30) with Lipofectamine 2000 according to the manufacturer’s instructions. After 36 h, cells were lysed in modified RIPA buffer (50 mM Tris-HCl (pH 7.4), 1% NP-40, 0.25% Na-deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 mg/mL Aprotinin, 1 mg/mL leupeptin, 1 mg/mL pepstatin, 1 mM Na3VO4 and 1 mM NaF, according to Pierce). The quantity of protein in cell lysates was determined through Lowry method. 20 µg of proteins were subjected to 12% SDS-PAGE under reducing conditions and blotted to PVDF membrane (Millipore). The membrane was blocked in Tris-buffered saline containing 0.05% Tween-20 and 5% nonfat dry milk for 1 h at room temperature. Immunodetection was performed by incubating with a His-Tag (27E8) antibody (Cell Signaling) followed by a goat anti-mouse horseradish peroxidase (HRP) -conjugated secondary antibody (Zhongshan, China). Immunoreactive proteins were visualized using SuperSignal WestPico Chemiluminescent Substrate (Pierce).

Measurement of PD of transfectants
The PD number of a single cell growing to ~106 cells (70–80% confluence of a 25 cm2 culture flask) is ~20. Therefore, the actual PD number of the transfectants should be increased by 20 PD when compared with that of the untransfected cells.

Cell proliferation assay
Cell proliferation was assayed using the MTT method (25) . Cells were plated at a density of 2 x 103 cells/well into 96-well plate and cultured for periods ranging from 1 to 6 days. The medium was replaced at 24 h intervals. At each time point, 10 µL MTT (10 mg/mL) was added to each well. After 3 h of incubation at 37°C, cells were lysed using 10% SDS/50% N, N-dimethylformamide and the absorbance at 490 nm was recorded using an ELISA plate reader. Duplicate measurements were performed on three independent wells at each time point.

Cell cycle analysis
Cells at 70–80% confluence were harvested, fixed with 75% ethanol overnight, then stained with propidium iodide (Sigma, St. Louis, MO, USA) in the dark for 30 min. The DNA content was measured with a Becton Dickinson FACScan flow cytometer and the data were analyzed with CellFIT software.

SA-ß-Galactosidase activity at pH 6.0
Cells were washed twice in PBS, fixed in 3% formaldehyde, and washed again in PBS. The cells were incubated overnight at 37°C (without CO2) with freshly prepared SA-ß-Gal staining solution as described (9) .

Western blot analysis
Cells were lysed in modified RIPA buffer as described before: 20 µg of proteins were subjected to 12% SDS-PAGE under reducing conditions and blotted to PVDF membrane (Millipore). Immunodetection was performed by incubating with primary antibodies [anti-p16INK4a (sc-9968) (Santa Cruz); anti-Cip1/Waf1/p21 (Upstate); anti-PTEN (Upstate); anti-actin(sc-7320) (Santa Cruz)] at room temperature for 1 h, followed by rinses and addition of HRP-conjugated secondary antibodies (Zhongshan, China). Incubation with anti-actin served as a loading control. Immunoreactive proteins were visualized using SuperSignal WestPico Chemiluminescent Substrate (Pierce) and the optical density of each band was analyzed by ImageMaster VDS software.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Identification of genes down-regulated upon replicative senescence of 2BS fibroblasts
mRNAs from young (PD25) and senescent (PD56) 2BS fibroblasts were isolated and subjected to suppression subtractive hybridization. Subsequent differential screening of 140 randomly picked clones revealed that 30 clones were down-regulated on transcriptional level. The cDNA inserts were sequenced followed by homology search against NCBI nr and dbEST databases (http://ncbi.nlm.nih.gov/blast) (Table 1 ). These down-regulated genes encode proteins involved in cell signaling, cell cycle regulation, transportation, cytoskeleton, DNA synthesis, RNA processing, and metabolism.


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  		Table 1. Identified genes down-regulated upon replicative senescence of 2BS fibroblastsa

Virtual Northern blot and Northern blot analyses
Seven clones (Y31, Y114, Y120, Y138, Y152, Y167, and Y168) corresponding to unknown or poorly characterized genes were chosen for virtual Northern blot analysis (26) , and their down-regulated expression upon senescence of 2BS cells was confirmed (Fig. 2 A), consistent with the results of differential screening analysis.



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  		Figure 2. Virtual Northern blot and Northern blot analysis showed the down-regulation of transcripts upon 2BS senescence. A) Down-regulated expression of several chosen clones upon 2BS senescence was verified using virtual Northern blot analysis. B) The transcriptional down-regulation of clone Y114 upon 2BS senescence was confirmed by Northern blot analysis. All results were normalized to the housekeeping gene GAPDH.

Clone Y114 was chosen for further study. Transcriptional down-regulation of the corresponding gene of clone Y114 was confirmed through Northern blot analysis (Fig. 2B ).

Molecular cloning of replicative senescence down-regulated Leo1-like (RDL) gene
The length of the senescence down-regulated clone Y114 is 610 bp. RACE was used to extend 5'- and 3'-ends of the transcript by an additional 1468 bp at 5' and 130 bp at 3', to a final length of 2208 bp (GenBank accession no. AY302186) (Fig. 3 ). An open reading frame was identified that started at ATG (+41) within the consensus Kozak translation initiation sequence (ATAATGG) (27) and coded for a polypeptide of 666 amino acids (Fig. 3) . The 3'-untranslated region contained a canonical polyadenylation signal (AATAAA) near the poly (A) tail.



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  		Figure 3. Nucleotide and deduced amino acid sequence of RDL cDNA. The polyadenylation signal is underlined.

The corresponding gene of clone Y114 is located on 15q15.3, i.e., LOC123169 by NCBI annotation project (human genome sequence database, http://www.ncbi.nlm.nih.gov/genome/guide/human/). According to the human genome BLAST result, the gene is 33.7 kb long, with 12 exons. All of the predicted splice sites match the "GT-AG" splicing consensus sequence. The homologene database at NCBI (http://www.ncbi.nlm.nih.gov/HomoloGene/) shows that this gene has significant homology to genes from species ranging from yeast to rat (Fig. 4 ). As the yeast homologue is called Leo1, the corresponding gene of clone Y114 was designated replicative senescence down-regulated Leo1-like, or RDL, gene. The existence of homologous genes in various species from yeast to human indicates that RDLp is a well-conserved protein and may have important physiological functions.



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  		Figure 4. Alignment of deduced amino acid sequences of RDLp and its homologues in S. cerevisiae, C. elegans, D. melanogaster, M. musculus, and R. norvegicus through Clustal W program. Amino acid residues with the same consensus sequence are shown on a black background; those that are identical are shown on a gray background. Gaps were introduced for maximal alignment. The solid black bar represents the Leo1-like domain; the gray bar indicates the AAA domain.

The calculated molecular mass of RDLp is 75.4 kDa. The estimated isoelectric point of the protein is pH 4.23. ScanProsite analysis (http://us.expasy.org/tools/scanprosite/) revealed that RDLp contains 1 tyrosine kinase phosphorylation site (aa. 387-394), 4 cAMP- and cGMP-dependent protein kinase phosphorylation sites (aa. 18-21, aa. 502-505, aa. 548-551, aa. 626-629), several protein kinase C phosphorylation sites (aa. 57-59, aa. 81-83, etc.), and up to 46 casein kinase II phosphorylation sites (aa. 10-13, aa. 14-17, etc.). The putative amino acid sequence does not contain any signal sequence, transmembrane domain, ER retention motif, mitochondrial targeting sequence, peroxisomal targeting signal, or DNA and RNA binding motifs based on PSORT II program (http://psort.nibb.ac.jp/form2.html) analysis. However, RDLp was assumed to function as a nuclear protein because its amino acid sequence contains several putative nuclear localization signals residing in the C terminus. Protein structure analysis using Pfam program (http://pfam.wustl.edu/hmmsearch.shtml) showed that there is a Leo1-like domain at aa. 301-631 of RDLp. The same result was obtained using SMART program (http://smart.embl-heidelberg.de/). Moreover, an AAA (ATPases Associated with a variety of cellular Activities) domain at aa. 56-253 was suggested by SMART program.

The Saccharomyces cerevisiae Leo1-like structure can be found in homologues from other species, like Caenorhabditis elegans, Drosophila melanogaster, Mus musculus, D. norvegicus, and Homo sapiens. The most striking difference among the primary sequences of the Leo1 homologous proteins is that proteins from S. cerevisiae, C. elegans, and D. melanogaster do not have an AAA domain whereas those from M. musculus, Rattus norvegicus, and H. sapiens do.

Distribution pattern of RDL in multiple tissues
By examining the distribution pattern of RDL in multiple human tissues using Northern blot analysis with a Y114 cDNA probe, a single band of ~2.4 kb was seen at high intensities in skeletal muscle and heart, and at lower intensities in placenta, liver, and other tissues (Fig. 5 ). It remains to be investigated that whether RDL is involved in the aging of these tissues.



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  		Figure 5. Multiple-tissue Northern blot analysis of RDL. The human multiple-tissue RNA gel blot (Clontech) was hybridized with a [{alpha}-32P]-labeled Y114 cDNA probe, washed, and autoradiographed.

RDLp is localized predominantly in the nucleus
Localization of RDLp in cultured cell lines was examined by generating an EGFP-RDL fusion protein with EGFP positioned at the N terminus. As expected, the EGFP-RDL protein was localized predominantly in the nucleus of 2BS cells, with only a little in the cytoplasm (Fig. 6 ). A similar localization pattern was observed when pEGFP C3-RDL, which encodes EGFP-RDL, was transfected into HeLa cells.



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  		Figure 6. Predominant nuclear localization of EGFP-RDL fusion protein in 2BS (A) and HeLa (B) cells. Cells were transiently transfected with pEGFP C3-RDL and pEGFP C3 as a control, fixed, then stained with DAPI to allow visualization of the nucleus. The green fluorescence signals from the EGFP-RDL fusion protein show a predominant (A) and almost exclusive (B) nuclear pattern of localization.

Expression of 6xHis-tagging RDL, Leo, and RDL-Leo– in 2BS cells
To confirm that RDL, Leo, and RDL-Leo– cDNAs can be expressed in 2BS cells, pIRESneo2 vectors were constructed that contained cDNA fragments encoding RDL, Leo, and RDL-Leo– with 6xHis-Tag at the C terminus. Vectors pIRES-RDL-His, pIRES-Leo-His, and pIRES-RDL-Leo–-His were transfected into 2BS cells. The corresponding His-Tagging protein bands were detected at estimated sizes by using His-tag antibody (Fig. 7 ). Thus, it was demonstrated that the full-length RDL, Leo, and RDL-Leo– could be translated in 2BS cells.



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  		Figure 7. Expression of 6xHis-Tagging RDL, Leo, and RDL-Leo–. Cells were transiently transfected with pIRES-RDL-His (lane 1), pIRES-Leo-His (lane 2), pIRES-RDL-Leo– (lane 3), and the control pIRESneo2 (lane 4). After 36 h, lysates of the transfected cells together with untransfected control (lane 5) were subject to Western blot analysis using a His-Tag antibody.

To avoid any possible influence of 6xHis-Tag (28) , constructs without 6xHis-Tag were used in other experiments of stable transfection and functional analysis.

Exogenous Leo expression induces cell growth arrest and premature senescence whereas RDL-Leo– expression induces senescence delay and life span extension
To investigate RDL’s effects on senescence of 2BS cells, the coding sequence of RDL cDNA was inserted in both orientations into the bicistronic vector pIRESneo2 (pIRES-RDL and pIRES-RDLas). To determine the function of the conserved Leo1-like domain in RDLp, pIRES vectors that contained cDNA fragment coding for the Leo1-like domain of RDLp (Leo) and truncated RDL cDNA fragment resulting from deletion of Leo (RDL-Leo–) were constructed and designated pIRES-Leo and pIRES-RDL-Leo–, respectively. These vectors were then stably transfected into young 2BS cells. Several senescent features of the transfectants were analyzed along with pIRESneo2 empty vector-transfected cell control and untransfected 2BS cell controls, all of which came from the same batch of young cells.

Leo inhibits cell proliferation greatly whereas RDL-Leo– promotes proliferation
To observe the proliferative changes after transfection, the growth rate of the transfectants (PD 48) was compared with those of the controls (Fig. 8 ). 2BS/Leo cells showed complete growth inhibition similar to senescent cells (PD56), whereas the growth of 2BS/RDL-Leo– was significantly promoted. 2BS/RDL cells grew faster than did the untransfected control cells, whereas 2BS/RDLas cells grew a little slower. However, the effects of RDL and RDLas were smaller than those of RDL-Leo– and Leo. The control empty pIRESneo2 had no significant effect on the cell growth rate.



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  		Figure 8. Replication characteristics of transfected and control cells. Growth curves of 2BS/Leo, 2BS/RDL-Leo–, 2BS/RDL, 2BS/RDLas, 2BS/neo (all at PD48), young (PD 27), middle-aged (PD 48), and senescent (PD 56) 2BS cells were determined by the MTT method. Duplicate measurements were performed on 3 independent wells at each time point indicated. Value: X ±SE; n = 6.

G1 cell cycle arrest is hastened by Leo but postponed by RDL-Leo–
The cell cycle profile of transfectants (all at PD48) and untransfected controls were analyzed by fluorescence-activated cell sorting (Fig. 9 ). In contrast to the significantly increased G1 DNA content of 2BS/Leo, which was somewhat like senescent cells, RDL-Leo– reduced the G1 content of transfected cells. Introduction of RDL decreased G1 DNA content slightly and introduction of RDLas increased it a little. The effects of RDL and RDLas were tiny. Control 2BS/neo cells had a similar G1 DNA content as untransfected middle-aged 2BS cells did. These changes were consistent with the changes in cell proliferation rate. Thus, Leo/RDL-Leo– expression may influence cell proliferation and cell senescence by influencing cell cycle progress.



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  		Figure 9. Fluorescence-activated cell sorting analysis of 2BS/RDL-Leo– (A), 2BS/RDL (B), 2BS/neo (D), 2BS/Leo (F), and 2BS/RDLas (all at PD48) (G) compared with untransfected young (PD27) (C), middle-aged (PD48) (E), and senescent (PD56) (H) 2BS cells.

Leo causes a senescence-like cell morphology and increases SA-ß-Gal activity whereas RDL-Leo– expression inhibits SA-ß-Gal activity
Morphological changes of transfectants were monitored (Fig. 10 ). 2BS/Leo cells showed increasing gross enlargement, flattening, and accumulation of granular cytoplasmic inclusions, like senescent cells (PD56); whereas 2BS/RDL-Leo– cells retained a refractive cytoplasm with thin and long projections, like young control cells (PD27). However, no significant morphologic changes were observed in 2BS/RDL and 2BS/RDLas cells (PD 48).



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  		Figure 10. Morphology and SA-ß-Gal staining for 2BS/RDL-Leo– (A), 2BS/RDL (B), 2BS/neo (D), 2BS/Leo (F), 2BS/RDLas (all above at PD 48) (G), untransfected young (PD 27) (C), middle-aged (PD 48) (E), and senescent (PD 56) (H) 2BS cells. Cells were fixed in 3% formaldehyde and stained with X-gal (x100).

An unspecific (29 , 30) but generally accepted senescence-associated marker, pH 6.0 optimum ß-galactosidase (SA-ß-Gal), was assayed by X-gal staining (Fig. 10) . No SA-ß-Gal activity was observed in 2BS/RDL-Leo– (PD 48) and young (PD 27) cells, whereas almost all 2BS/Leo (PD 48) cells were strongly stained, as were senescent 2BS control cells (PD 56). Only sporadic SA-ß-Gal positive cells were observed in 2BS/RDL (PD 48). However, compared with SA-ß-Gal activity in 2BS/neo and untransfected cells, the changes were not obvious in 2BS/RDLas

RDL-Leo– results in a finite extension of 2BS replicative life span whereas Leo causes a reduction of life span
The replicative senescence of normal human diploid cells is directly correlated with their PD number rather than with their growth and metabolic time (1) . The replicative life spans, i.e., cumulative PD numbers (CPD) of the 2BS/Leo, 2BS/RDL-Leo–, 2BS/RDL, 2BS/RDLas, 2BS/neo, and untransfected 2BS cells, were observed. The results revealed that the life span of 2BS/RDL-Leo– (CPD 67-70) was ~10 to 15 PD longer than those of 2BS/neo (CPD 54-56) and normal 2BS cells (CPD 55-60). In contrast, 2BS/Leo ceased cell division ~7–12 PD (CPD 48-50) earlier than did normal cells. More impressively, the life span of 2BS/RDL-Leo– was ~17–22 PD longer than that of 2BS/Leo. Introduction of RDL slightly extended the life span of 2BS cells (CPD 59-62), whereas introduction of RDLas hardly affected it (CPD 53-55). These results all indicate that introduction of RDL-Leo– enhances the replicative capacity, extends the replicative life span, and finally delays the onset of senescence of 2BS cells; introduction of Leo does the opposite.

Both Leo and RDL-Leo– affect known important senescence-associated genetic pathways
To provide hints of molecular mechanisms of RDL and its domains, protein levels of p16INK4a, p21WAF1, and PTEN in each transfectant were examined (Fig. 11 ). The results showed that introduction of Leo into 2BS cells affected all three pathways by increasing the expression of p16INK4a, p21WAF1, and PTEN. Besides, the increases of p16INK4a and p21WAF1 were more significant than that of PTEN. Introduction of RDL-Leo– reduced p16INK4a expression to an undetectable level, like the level in young 2BS cells. However, RDL-Leo– also caused an increase of p21WAF1. The introduction of RDL and RDLas into 2BS cells led to no significant changes in expression of these three genes.



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  		Figure 11. Leo and RDL-Leo– have effects on known important senescence-associated genes: p16INK4a, p21WAF1, and PTEN. Introduction of Leo into 2BS cells increased the expression of p16INK4a, p21WAF1, and PTEN significantly. In contrast, introduction of RDL-Leo– mainly reduced p16INK4a expression. However, RDL-Leo– caused an increase of p21WAF1 expression. A) Western blot analyses of p16INK4a, p21WAF1, and PTEN in transfected 2BS cells along with untransfected control cells. Control with an actin antibody verified the amount of protein loaded. Band intensities were determined by densitometric scanning. B) Ratios of p16INK4a, p21WAF1, and PTEN levels to actin level.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We have successfully cloned a novel replicative senescence-associated gene RDL that encodes an uncharacterized protein of 666 amino acids.

RDL is an evolutionarily conserved gene, which suggests it may have an important physiological function. Its homologues can be found in various species, from yeast and worm to fruit fly, mouse, and rat. Analysis shows RDLp contains a Leo1-like domain at the C terminus, and possibly an AAA domain at the N terminus of the protein as well. The Leo1-like domain exists in all the other homologues, including C. elegans 4M94, D. melanogaster Atu, M. musculus LOC235497, and R. norvegicus LOC300837. However, the AAA domain does not exist in homologues of yeast, worm, and fruit fly but in homologues of mouse, rat, and human.

RDL is down-regulated in mRNA level upon senescence of 2BS fibroblasts. The question is whether RDL’s down-regulation influences the senescence of 2BS cells and to what extent. Our data shows that overexpression or inhibition of RDL causes only mild changes in 2BS senescence, although the trends of the effects are in accordance with RDL’s down-regulation upon the senescence of 2BS cells: overexpression of RDL slightly postpones the onset of senescence whereas inhibition of RDL by introduction of RDLas promotes some senescence markers.

Many genes have been associated with replicative senescence in recent years, like p16INK4a, p21WAF1, and PTEN (19) . However, it remains unclear what kinds of domains are involved in senescence. Therefore, we explored the relationship between the conserved Leo1-like domain in RDLp and the senescence of 2BS cells. Surprisingly, exogenous expression of the Leo1-like domain (Leo) strongly accelerated senescence and shortened the replicative life span of 2BS fibroblasts; expression of the remaining part of RDL (RDL-Leo–) did the reverse.

Due to their nonspecificity for the irreversible cell growth arrest, expressions of those proposed "senescence-associated genes," such as p16INK4a, p21WAF1, and PTEN (they are also linked with other cellular events like quiescence, differentiation, or tumorigenesis) are not suitable as senescence markers (29) . However, it would be valuable to explore the relationships between RDL/its domains and these tumor suppressors to provide clues to the molecular mechanisms of RDL/its domains. Our data show that Leo increases the expression of p16INK4a, p21WAF1, and PTEN whereas RDL-Leo– decreases the expression of p16INK4a. They suggest that Leo and RDL-Leo– may affect the 2BS senescence through these known senescence pathways. It remains to be clarified how and why RDL-Leo– causes an increase of p21WAF1 expression, although this increase seems less important than does the decrease of p16INK4a.

The yeast homologue of RDLp–Leo1p is a part of the Paf1/RNA polymerase II complex (31 , 32) . The Paf1 complex (33) is essential for the expression of a series of yeast genes involved in cell cycle regulation, protein synthesis, etc., and plays an important role in transcription elongation (33 34 35) . Some researchers reported that Leo1p might be less important in this complex as its deletion did not result in any obvious phenotype (36) . However, deletion of Leo1 partly rescued the severe phenotype of a paf1{Delta} strain (31) . Others reported that leo1{Delta} strain exhibited a marked impairment in transcription elongation (34) . RDL homologue-C. elegans 4M94, a Leo1 like protein, is an essential gene involved in larval arrest, embryonic lethal, sterile progeny, various morphological defects, and slow growth (37 38 39 40) . Other RDL homologues in fruit fly, mouse, and rat are still uncharacterized. It remains to be clarified whether RDLp has the ability to form a complex with RNA polymerase II and other proteins through its Leo1-like domain and subsequently play a role in transcriptional elongation of certain genes and affects the aging program, although we have shown it is predominantly localized in the nucleus.

The fact that both RDL and RDL-Leo– extend the life span of 2BS suggests that the activity of RDL-Leo– as a part of RDLp is stronger than that of Leo. Therefore, in RDL-Leo– there must be another domain strongly associated with senescence. We suppose that this domain, which inhibits senescence, may be the putative AAA domain suggested by SMART analysis, as no other known domains were proposed in this part of RDLp. The N-terminal sequence of RDLp (mainly the AAA domain) shows a low homology with yeast MDN1p (identities: 22%, positives: 39%). MDN1p is a member of the AAA ATPase family. The proteins of this family are unified by their sharing of a common structural organization that is based on a conserved AAA ATPase domain of ~225 residues. In contrast to their shared structure, the AAA proteins participate in diverse cellular activities (41) . MDN1p may function as a nuclear chaperone and be involved in the assembly/disassembly of macromolecular complexes in the nucleus (42) . It is not clear by what mechanism of RDL-Leo–, or the AAA domain in it, inhibits p16INK4a expression and prolongs the life span of 2BS fibroblasts.

It is of great interest to find out why intact RDL has no apparent effect on senescence while Leo and RDL-Leo– has clear and opposite effects, and why such two counteractive domains (Leo1-like domain and AAA domain) would be integrated into one molecule during evolution. There are several hypotheses that need to be verified in our subsequent work. The regulation of RDL might be post-transcriptional. According to Aceview analysis of RDL-related sequences (http://www.ncbi.nlm.nih.gov/IEB/Research/Acembly), the gene might have six different transcripts produced by alternative splicing. The difference among these proposed transcripts includes truncation of the 3' end, presence or absence of five cassette exons, and common exons with different boundaries. The sequence presented in this paper is one of the six isoforms. A transcription factor of the T-box gene family, TBX3 is able to immortalize mouse embryo fibroblast cells whereas TBX3 + 2a, a TBX3 isoform produced by alternative splicing, shows an acceleration of senescence (43) . It is possible that different RDL isoforms might be expressed at certain stages of replicative senescence and play different or even opposite roles, just like the instance of TBX3/TBX3 + 2a. It may provide a reasonable answer to why intact RDLp has no apparent function whereas its truncated forms do.

It is possible that, as we did artificially to create Leo and RDL-Leo–, RDLp could be post-translationally processed into two parts containing the Leo1-like domain and the AAA domain, respectively. The preproglucagon gene (44) provides such an example. It has different post-translational products: glucagon, glucagon-like peptide 1 (GLP-1), and glucagon-like peptide 2 (GLP-2). Glucagon and GLP-1 have opposite effects on regulation of blood sugar (45) . Alternatively, the two domains in Leo and RDL-Leo– may antagonize each other directly, providing a refined control point for senescence regulation. Analysis suggests there are many potential protein kinase phosphorylation sites on the protein. It is likely that through specific phosphorylation or dephosphorylation of these sites under different conditions, the function of the Leo1-like domain and/or the AAA domain is blocked or activated. Further research will be conducted to elucidate these possibilities and to define precisely how the RDL is regulated.

This study is the first to identify a senescence-associated domain. Association of Leo1-like domain with replicative senescence has not only informed us on the function of Leo1 family, but advanced our knowledge of the aging program at the protein structure level.


   ACKNOWLEDGMENTS
 
This work was supported by Grant G2000057001 from the Major State Basic Research Development Program of China and Grant 30271432, 30371561 from the National Natural Science Foundation of China. We thank Dr. Shuzhen Guo and Dr. Zebin Mao for helpful discussions and Dr. Jason Wong for critical reading of this manuscript.

Received for publication August 18, 2004. Accepted for publication November 19, 2004.


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 DISCUSSION
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