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The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 58:B873-B878 (2003)
© 2003 The Gerontological Society of America

Increased Expression of the Huntingtin Interacting Protein-1 Gene in Cells From Hutchinson Gilford Syndrome (Progeria) Patients and Aged Donors

Shinichi Chigira1, Katsuo Sugita4, Kazuko Kita2, Shigeru Sugaya2, Yoshiko Arase2, Masaharu Ichinose1, Hiroshi Shirasawa3 and Nobuo Suzuki2

1 Department of Plastic Surgery
2 Department of Environmental Biochemistry
3 Department of Molecular Virology, Graduate School of Medicine, Chiba University, Japan.
4 Faculty of Education, Chiba University, Japan.


   Abstract
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 Abstract
Methods
 Results
 Discussion
 References
 
Hutchinson Gilford syndrome (progeria [PG]) is a human disease associated with accelerated aging. To elucidate the acceleration mechanism, we first tried to transform a PG-derived cell line by infection of a recombinant adenovirus expressing HPV (human papilloma virus)-E6 and HPV-E7 genes. The transfected PG cells had a greater number of population doublings (PD) (>80), faster doubling time, and less staining of senescence-associated ß-galactosidase than the nontransfected PG cells. The transfected cells also showed markedly more detectable telomerase activity than the nontransformed cells. The expression levels of the genes in the E6-transduced and E7-transduced cell line were then compared with those of the nontransfected cell line using an mRNA differential display method, following reverse-transcriptase polymerase chain reaction analysis. Expression of huntingtin interacting protein-1 (HIP-1) gene was found to be increased not only in PG cells but also in fibroblast cells from aged healthy donors. Thus, HIP-1 might be a molecular assistant in the pathogenesis of the cellular senescent process in the human cells tested.

HUMAN aging, consisting of complex processes, is a highly conserved phenomenon with a finite replicative life span. There is no explainable theory for overall cellular aging. However, Hayflick originally reported "replicative senescence" in which normal human fibroblasts can be maintained only for a finite period (1,2). Human fibroblasts isolated from old donors have a much shorter life span in vitro than those from younger donors (3–5), showing a correlation between cell aging and individual aging. This relation is marked in hereditary premature aging syndrome, as cells from humans with the syndrome show more rapid onset of senescence than cells from age-matched controls (6,7).

Aging is a dominant phenomenon at the cellular level. In cell fusion experiments, senescence overrode the immortalization or proliferation potential (8). The microinjection of mRNA from senescent cells resulted in growth arrest of the injected proliferating cells (9). Several genes, such as telomerase, p53, or p21, have been proposed to have a pivotal role in cellular aging (10). Thus, molecular changes occurring with extension of cell life need to be identified in order to define senescence markers and targets for study.

In the present study, we prepared fibroblast cells derived from two progeria (PG) patients. PG is one of the typical diseases showing premature senescence, and is considered a human disease model of accelerated aging (11,12). PG patients show aging signs, such as low height, bone malformation, and arteriosclerosis. PG cells show a slow division potential and a short replicative life span in vitro.

We here tried to establish PG-derived fibroblast cells that extend the replicative life span. In vitro transformation of fibroblast cells with oncogenes or with carcinogens has proven successful means to establish cell lines that show accelerated rates in cell growth and extended life span. Such cell lines escaping from senescence may be valuable in the analysis of the aging process and clarification of genes responsible for senescence. We therefore compared gene expression profiles of the E6-transduced and E7-transduced cells with those of the nontransfected parent fibroblasts using a polymerase chain reaction (PCR)-based mRNA differential display method.


   METHODS
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 Abstract
 Methods
Results
 Discussion
 References
 
Cells and Culture Conditions
PG2OS and PG3KT fibroblasts were established from PG patients (13). FF cells were obtained from fetus-derived fibroblast cells, as described elsewhere (14). CUPS-13, CUPS-12, and PC-78 cells were fibroblasts obtained from normal controls aged 6 years, 15 years, and 78 years, respectively. All controls and patients gave informed consent to the protocol, which was approved by the committee for the protection of human subjects in research at Chiba University. Cells were cultured with Dulbecco's modified Eagle's medium (Nissui Pharmaceutical, Tokyo, Japan) containing 10% calf serum (CS) (GIBCO/BRL, Grand Island, NY) and antibiotics at 37°C in a humidified atmosphere containing 5% CO2. Cells were washed with 0.05% trypsin-EDTA (edetic acid) to release cells from the tissue culture surface.

PG2OS+AxE67 cells were derived from PG2OS cells by infecting them with recombinant adenovirus AxCACSHPV16E67 (AxE67), encoding E6/E7 genes of HPV 16 (15). PG2OS cells were infected with an empty adenovirus vector devoid of E6 and E7, and the transfected cells were used as a control for PG2OS+AxE67 cells. Briefly, PG2OS cells were infected with the virus containing or not containing E6 and E7 at 100 multiplicity of infection (MOI). Next, 6 ml of fresh medium was added to 60 mm dishes, and thereafter the cells were maintained for 1 week without the exchange of the medium. Cells were serially passaged by trypsinization at a split ratio of 1:2, and the population doubling (PD) for each passage was estimated assuming 1 PD length per passage.

Senescence-Associated (SA) ß-Galactosidase Staining
SA ß-galactosidase-positive cells were detected according to the method of Dimri and colleagues (16). After the staining, cells were observed by phase contrast microscopy and photographed; the staining positive and negative cells were counted on the photograph.

Telomerase Activity
Cells were lysed in a buffer containing 10 mM Tris-Cl (pH 7.5), 1 mM MgCl2, 1 mM EGTA, 0.1 mM phenylmethylsulfonyl fluoride, 5 mM ß-mercaptoethanol, 0.5% Chaps, and 10% glycerol. Telomerase activity of the cell lysates was measured by the telomere repeat amplification protocol (TRAP) using TRAPEZE (Intergen, NY) with minor modifications. The products of the telomerase reaction were amplified by a three-step PCR (94°C for 30 s, 52°C for 30 s, and 72°C for 30 s) with 29 cycles in the presence of fluorescence-labeled TS primer (5'-AATCCGTCGAGCAGAGTT-3') according to the instructions. The PCR products were electrophoresed on a 10% polyacrylamide gel, blotted onto a Hybond-N nylon membrane (Amersham Life Science Inc., Arlington Heights, UK), cross-linked to the membrane with ultraviolet light, and then detected using ECL Direct Nucleic Acid Labeling and Detection Systems (Amersham Pharmacia Biotech, UK). Telomerase activity was shown as the ratio of the intensity of the telomere ladder to that of the 36-bp internal standard with the TS primer-annealing site.

RNA Extraction
Total cellular RNA was isolated from cells by guanidine isothiocyanate-phenol-chloroform extraction (17) using TRIZOL reagent (GIBCO/BRL), as described elsewhere. Total RNA was freed from contaminating chromosomal DNA with RNase-free DNase (GeneHunter, Nashville, TN) and used as total RNA.

Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) and Differential Display
Differential display was performed as described previously (18,19) using a Differential Display Kit (Display Systems Biotech, CA). Briefly, 250 ng of total RNA was used in a reverse transcription (RT) reaction using 2 different anchored downstream primers (DW6: 5'-TTTTTTTTTTTCG-3' and DW7: 5'-TTTTTTTTTTTGA-3'). The resulting cDNA was subsequently amplified by PCR using 3 upstream primers (AP15: 5'-GATCAATCGC-3', AP16: 5'-TCGGTCATAG-3', and AP18: 5'-TCGATACAGGUP-3') and the matching downstream primer with 12.5 µCi of [{alpha}-35S] dATP. The PCR products were electrophoresed on a 5% denaturing polyacrylamide gel. After electrophoresis, gels were dried and exposed directly to Fuji LI-FM X-ray film (Fuji Photo Film Co., Kanagawa, Japan). Fragments of cDNA corresponding to bands with higher intensities in PG2OS cells than in FF and PG2OS+AxE67 cells were excised from the dried gel and eluted. The eluted cDNA fragments were reamplified using the same set of primers as described above and subsequently cloned into pCR 2.1 vector using the Original TA cloning kit (Invitrogen, CA).

Sequencing of cDNAs
Nucleotide sequences of cloned cDNAs were determined by the dideoxy-mediated chain termination method (20) using BigDye Terminator Cycle Sequencing Kit (PE Applied Biosystems, Foster City, CA) with the M13 primer according to the supplier's instruction. The resulting products were electrophoresed and analyzed on an ABI PRISM 310 Genetic Analyzer (PE Applied Biosystems). DNA sequences from the cloned cDNAs were compared with known nucleotide sequences in the Genebank and DNA Data Base of Japan databases (DDBJ) using the Basic Local Alignment Tool (BLAST) program.

Semiquantitative RT-PCR
To examine mRNA expression levels for huntingtin interacting protein-1 (HIP-1) and 2 clones with sequences corresponding to the human expressed sequence tag (HSP), HSPC311, and HSPC269, in cells, we carried out RT-PCR. Total RNA was first reverse-transcribed using oligo (dT) primer, and then the resulting cDNA was amplified with specific primers for each gene. The primer pairs were designed to span introns so that we could distinguish between products amplified from cDNA and those amplified from contaminating genomic DNA. The sequences of specific primers were as follows; 5'-AAGCCAATGAACAGCGATAT-3' and 5'-TCCAGCTCTTTTTTCTCTCGT-3' for HIP-1 mRNA, 5'-CCTATTCTGCAGGTGTAGCTTCG-3' and 5'-TCGAAGTAATAGTAGACGTCGCC-3' for HSPC311 mRNA, 5'-TGATTCCTTGTACTCTTACGTGG-3' and 5'-GATGAGGGAATGAAGGGTACC-3' for HSPC269 mRNA, and 5'- GGTGA AGGTC GGAGT CAACG-3' and 5'- CAAAG TTGTC ATGGA TGACC-3' for GAPDH mRNA, respectively. The primer pairs for HIP-1 mRNA, HSPC311, HSPC269, and GAPDH gave PCR products with lengths corresponding to those expected from the respective mRNA, 126 bp, 120 bp, 132 bp, and 225 bp, respectively. Furthermore, the nucleotide sequences of the PCR products were identical to those of the corresponding parts of the respective mRNAs.

The RT reaction was performed at 42°C for 30 minutes in the presence of 1 µg of oligo (dT) primer and 5 U of reverse transcriptase. PCR was performed in a 30 µl mixture consisting of 1 mM MgCl2, 40 µM dNTP mixture, 1.5 U Ampli Taq Gold, and 1.65 µM specific primers, and PCR parameters were run at 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 30 seconds for one cycle. Amplification for each cDNA was performed in 32 cycles for HIP-1, 34 cycles for HSPC311, 32 cycles for HSPC269, and 32 cycles for GAPDH, because the optimal number of amplification cycles was determined at the exponential reaction points. The PCR products were electrophoresed on a 2% agarose gel and stained with ethidium bromide, and then the product amounts were quantified by using a LAS-100 luminoimage analyzer (Fujifilm, Tokyo, Japan). The mRNA expression levels were normalized by dividing those of GAPDH mRNA.


   RESULTS
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 Abstract
 Methods
 Results
Discussion
 References
 
Cell Transformation
Figure 1 shows the growth curve of PG2OS cells and PG2OS+AxE67 cells. PG2OS cells stopped dividing at 8 PDs (52 days), while PG2OS+AxE67 cells continued to divide for more than 70 PDs. The cells transfected with the empty adenovirus devoid of E6 and E7 grew slower than PG2OS+AxE67 cells. The doubling times within 50 days of both the empty adenovirus-transfected cells and PG cells and that of PG2OS+AxE67 cells were about 7 days and 2 days, respectively.



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  		Figure 1. Growth curve of nontransfected and transfected progeria cells. Population doublings of nontransfected progeria cells, PG2OS (•), and transfected progeria cells, PG2OS+AxE67 ({blacksquare}), as estimated in the Methods section

 
As an assessment of senescence, we first analyzed the SA ß-galactosidase activity of the two cell lines. Very few SA ß-galactosidase-staining positive cells were detected in PG2OS+AxE67 cells, while they were abundantly detected in PG2OS cells, as shown in Figure 2. The staining-positive cells were 10.4% (7 cells/67 cells) of the formercells and 74.1% (23 cells/31 cells) of the latter cells, respectively.



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  		Figure 2. SA ß-galactosidase staining of PG2OS and PG2OS+AxE67 cells. PG2OS and PG2OS+AxE67 cells were stained at approximately 10 population doublings (PDs) and 50 PDs, respectively, as described in Methods, and the cells were observed by phase contrast microscopy

 
Telomerase activity was comparatively examined between the two cell lines. The sample containing telomerase, as a positive control, showed a telomere ladder, but the sample containing buffer alone, as a negative control, did not show the ladder (Figure 3). PG2OS+AxE67 cells expressed the telomerase activity as shown by heat labile generation of the telomere ladder, while the activity was not detected in PG2OS cells.



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  		Figure 3. Telomerase activities of PG2OS and PG2OS+AxE67 cell lysates. Telomerase activity of cell lysates without (Heat –) and with (Heat +) heat treatment (85°C, 10 min) were measured as described in Methods. Samples containing human telomerase reverse transcriptase (hTERT) and buffer alone were used as positive and negative controls, respectively

 
Differential Display
RT-PCR was performed using 6 different primer combinations. The series of differential display reactions yielded 68 cDNA bands ranging from 100 to 500 bp in size (data not shown). From these cDNA bands, 11 bands were selected, because their intensity was higher in PG2OS cells than in FF and PG2OS+AxE67 cells (Table 1).


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  		Table 1. cDNAs Up-Regulated in PG2OS Cells Compared With PG2OS+AxE67 and FF Cells, in mRNA Differential Display.

 
Table 1 also shows the results of the homology search of the nucleotide sequences of all cDNAs. Four clones showed greater than 90% nucleotide identity with known human mRNAs. The nucleotide sequence of clone A7 was homologous to a part of the sequence of mRNA for HIP-1 (GenBank accession Nos. U79734) except for one nucleotide substitution (Table 1). Sequences of 2 clones, C2 and C8, were highly homologous to human expressed sequence tags (ESTs), HSPC311 (346 bp/356 bp matched), and HSPC269 (239 bp/245 bp matched), respectively. HSPC311 and HSPC269 were both cloned from cd34+ stem cells, and those lengths were 454 bp and 1384 bp, respectively. The remaining 7 clones showed no significant homology to any sequence.

Semiquantitative RT-PCR Analysis
mRNA expression levels of HIP-1, HSPC311, and HSPC269 were examined in nontransfected progeria cells (PG3KT, PG2OS), transfected PG2OS+AxE67 cells, and FF cells by semiquantitative RT-PCR analysis. All specific primer pairs could amplify the products (Figure 4A) whose lengths were concordant with the expected one. The altered expression for the 3 clones observed in the differential display analysis was confirmed by the results of semiquantitative RT-PCR analysis (Figure 4A). The expression levels of HIP-1 and HSPC311 mRNAs relative to those of GAPDH mRNA were markedly higher in PG3KT and PG2OS cells than in PG2OS+AxE67 and FFcells (Figure 4B and 4C). On the other hand, the mRNA levels of HSPC269 were slightly higher in PG3KT and PG2OS than in PG2OS+AxE67 and FF cells (Figure 4D).



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  		Figure 4. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of huntingtin interacting protein-1 (HIP-1), HSPC269, and HSPC311 mRNA expression levels in cells. Total RNA was prepared from PG3KT, PG2OS, PG2OS+AxE67, and FF cells, and the mRNA expression levels of the cloned cDNA and GAPDH were analyzed by semiquantitative RT-PCR as described in Methods (A). The mRNA levels were divided by GAPDH mRNA levels. The levels of HIP-1 (B), HSPC311 (C), and HSPC269 (D) in each cell line are shown as a relative expression ratio to those in FF cells; i.e., the levels of FF cells are assigned an arbitrary value of 1. The mRNA expression levels of the cells were analyzed independently three times and the data are presented as means ±SD (standard deviation)

 
To examine whether the gene expression of HIP-1 is affected by aging, the HIP-1 mRNA levels of fibroblast cells from normal volunteers of different ages were examined using RT-PCR. The levels were the lowest in the fetus, and tended to increase with aging (Figure 5).



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  		Figure 5. Huntingtin interacting protein-1 (HIP-1) mRNA expression levels in cells from different-aged donors. Total RNA was prepared from CUPS-13, CUPS-12 and PC-78 cells were fibroblasts obtained from normal controls (aged 6 years, 15 years, and 78 years, respectively), and HIP-1 mRNA expression levels were analyzed by reverse transcriptase polymerase chain reaction (A). The mRNA levels were divided by GAPDH mRNA levels, and the levels in each cell line are shown as a relative expression ratio to those in FF cells (B). The mRNA expression levels of the cells were analyzed independently three times, and the data are presented as means ±SD (standard deviation)

 

   DISCUSSION
Top
 Abstract
 Methods
 Results
 Discussion
References
 
In an attempt to define the molecular basis of cellular senescence of progeria, we tried to establish the transformed PG cells, which show significantly more elongated PD than the parental cells by infection with recombinant adenovirus containing E6 and E7. The E6- and E7-transduced PG cells grew much faster than the cells infected with the empty adenovirus vector devoid of E6 and E7 and the nontransfected cells. It appears likely that the E6- and E7-transduced cells overcome crises. These results were in accordance with those of primary human keratinocytes transfected with E6 and E7 (21,22). In addition, we confirmed increased levels of telomerase activity in the E6-transduced and E7-transduced cells but not in nontransfected cells.

SA ß-galactosidase staining was performed as a marker of replicative senescence in vitro. Cultures established from the E6-transduced and E7-transduced PG cells stained much fewer in number than nontransfected cells. Dimri and colleagues reported that SA ß-galactosidase was undetectable in immortal cells, including large tumor antigen-immortalized WI-38 cells and HeLa cells (16). Many cell lines derived from tumor tissues contain immortal cells without detectable SA ß-galactosidase activity or cells with positive telomerase acitivity and an extended replicative life span (23). According to the analysis of tumor cell development, Rb and P53, tumor suppressors that commonly suffer loss-of-function mutations in human cancers, are critical for cellular senescence. On the other hand, it is conceived that E6 and E7 inhibit the effects of Rb and P53 (15,24). Thus, the inhibition may have led to the extension of the cellular replicative life span in the E6-transduced and E7-transduced PG cells.

This study also demonstrated that HIP-1 mRNA expression levels were increased in senescent PG cells, PG3KT, and PG2OS, compared with those in the E6-transduced and E7-transduced PG cells, PG2OS+AxE67, and furthermore that the levels tended to increase with aging in normal fibroblast cells. HIP-1 was identified by its altered interaction with mutant huntingtin, which cause Huntington disease with onset at age 20 years or older. HIP-1 is a 116-kDa cytosolic protein that is ubiquitously expressed and highly enriched in human and mouse brain tissues (25,26). The biological role of the protein was reported to be involved in inducing apoptosis via a novel caspase-dependent death effecter domain (27). Moreover, Hackam and colleagues reported that HIP-1-induced cell death involves the intrinsic apoptosis pathway (27). Therefore, it is suggested that the E6- and E7-transduced PG cells may be saved from cellular death due to HIP-1-induced apoptosis.

It was previously reported that the aging process might alter the regulation of the apoptotic response (28). For example, hepatocytes from old rats appeared to be blunt to apoptotic response, compared with those from young rats (29). On the other hand, aging was reported to sensitize hepatocytes to apoptosis (30) and to increase the basal rate at which chondrocytes undergo apoptosis (31). Taken together with these reports, the present results represent HIP-1 as a candidate for a senescence-associated gene.

It remains unknown how HIP-1 functions are related to those of E6 and E7 and how HIP-1 mRNA expression levels are decreased in E6- and E7-transfected PG cells. Recently, Defilippis and colleagues reported that, in HeLa cells, repression of the E7 protein activated the Rb pathway but not the p53 pathway and triggered senescence, whereas repression of the E6 protein activated the p53 pathway but not the Rb pathway and triggered both senescence and apoptosis (24). Thus, HIP-1 functions may be related to the p53 pathway.

In general, neurodegenerative diseases have their peculiar onset, although each responsible gene has been acquired congenitally. Biological aging, an inevitable process in multicellular organisms, features an exponential role in the incidence of neurodegenerative diseases. The present findings suggest that HIP-1 may be a molecular accomplice in the pathogenesis of not only Huntington disease but of other senescence-associated diseases. Messenger RNA expression of HSPC311 was also much higher in senescent PG cells than in the transfected PG cells. HSPC311 was reported to be an expressed sequence tag clone, and the cellular function remains unknown. The present results also suggest the candidature of HSPC311 as a senescence-associated gene in PG cells. Further analyses of the clones are currently in progress.


   Acknowledgments
 
This work was supported in part by grants-in-aid from the following foundations: the Smoking Research Foundation, "Ground Research for Space Utilization" by NASDA and the Japan Space Forum, the Nissan Science Foundation, the Japan Atomic Energy Research Institute (by contract with the Nuclear Safety Research Association), the Nakatomi Foundation, the Tokyu Foundation for Better Environment, the REIMEI Research Resources of Japan Atomic Energy Research Institute, and the Ministry of Education, Science and Culture (Japan).

Address correspondence to Nobuo Suzuki, MD, PhD, Department of Environmental Biochemistry, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba City, Chiba, 260-8670, Japan. E-mail: nobuo{at}med.m.chiba-u.ac.jp


   Footnotes
 
Decision Editor: James R. Smith, PhD

Received December 6, 2002

Accepted June 20, 2003


   References
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