GRP75 and hTERT cooperate to extend the in vitro lifespan of human fibroblasts

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Experimental Cell Research 286 (2003) 96 –101

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Overexpressed mortalin (mot-2)/mthsp70/GRP75 and hTERT cooperate to extend the in vitro lifespan of human fibroblasts Sunil C. Kaul,a,* Tomoko Yaguchi,a Kazunari Taira,a Roger R. Reddel,b and Renu Wadhwaa a

National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1, Higashi, Tsukuba, Ibaraki, 305-8566, Japan b Children’s Medical Research Institute, 214 Hawkesbury Road, Westmead, NSW 2145, Australia Received 30 July 2002, revised version received 28 November 2002

Abstract The lifespan of human foreskin fibroblasts (HFF5), cultured under standard in vitro conditions (including ambient atmospheric oxygen tension), was extended slightly by expression of exogenous mortalin (mot-2)/mthsp70/Grp75, but not by the catalytic subunit of telomerase, hTERT. Together, mot-2 and hTERT permitted bypass of senescence, a substantial extension of lifespan, and possibly immortalization. This is the first demonstration that mot-2 and telomerase can cooperate in the immortalization process. © 2003 Elsevier Science (USA). All rights reserved. Keywords: Mortalin (mot-2)/mthsp70/GRP75; Telomerase; Human skin fibroblasts; Lifespan extension; Immortalization

Introduction Normal human cells have a limited replicative lifespan. They undergo a fixed number of divisions characteristic of each cell strain and eventually enter a metabolically active state of permanent growth arrest, called senescence. This is accompanied by several changes, including increase in inhibitors of cyclin-dependent kinases, functional loss of repair mechanisms, accumulation of cellular damage, and shortening of telomeres [1–3]. Telomeres are the repetitive DNA ([TTAGGG]n) structures that, together with specific telomere binding proteins, act as protective caps preventing chromosomal end-to-end fusions [4,5]. Telomere shortening is a consistent feature of human cellular senescence. On the other hand, activation of a telomere length maintenance mechanism (telomerase or alternative lengthening of telomeres (ALT)) is a rate-limiting step in carcinogenesis and cellular immortalization [6]. The latter involves bypass of at least two mortality check-points, senescence (M1) and crisis (M2). Cells that escape senescence undergo extended pop* Corresponding author. Research Center for Glycoscience, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1, Higashi, Tsukuba, Ibaraki, 305-8566, Japan. Fax: ⫹81-298-61-6692. E-mail address: [email protected] (S.C. Kaul).

ulation doublings and encounter crisis [3,7]. During crisis, additional genetic changes, some as yet unidentified, may occur in a small minority of cells (ranging from 1 in 105 to 1 in 109 cells), permitting the activation of a telomere maintenance mechanism and resumption of proliferation [8]. Cloning of the catalytic subunit of human telomerase hTERT and reconstitution of telomerase activity enabled a molecular elucidation of the role of telomerase and of telomere length maintenance in cellular senescence and immortalization [9 –12]. Telomere-stabilization functions independent of telomere lengthening have also been proposed for telomerase [11,12]. Although telomerase is a multisubunit ribonucleoprotein enzyme, expression of hTERT in normal cells is sufficient to induce telomerase activity [12]. Many recent studies have reported the consequences of inducing telomerase activity in a variety of normal human somatic cell strains. Whereas expression of exogenous hTERT has been shown to be sufficient for telomere length maintenance and immortalization of some human cell strains (fibroblasts and endothelial cells) [13–19], some others (for example, foreskin keratinocytes and mammary epithelial cells) do not become immortalized when grown under standard cell culture conditions unless the Rb/p16INK4A pathway is inactivated [20 –25]. Complicating the situation further, some

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fibroblast and endothelial cell strains are not immortalized by expression of hTERT [24]. Culture conditions seem to influence the probability of immortalization by hTERT expression [26,27]. We have previously reported that overexpression of human mortalin (hmot-2) causes inactivation of the tumor suppressor protein p53 and extends the in vitro lifespan of normal human fetal lung fibroblasts (MRC-5) [28 –31]. Other predicted functions of hmot-2 may also contribute to lifespan extension [32]. In the present study, we report that ectopic expression of hmot-2 extends the in vitro lifespan of a human foreskin fibroblast strain and cooperates with telomerase in the immortalization process.

Material and methods Plasmids Human mortalin cDNA (hmot-2) was cloned into an expression plasmid driven by the CMV immediate– early promoter– enhancer. An expression plasmid (pCI-hTERT) encoding hTERT has been described previously [33]. Cell culture and transfections Normal human foreskin fibroblasts (HFF5, a kind gift from Ralph Boehmer, Ludwig Institute for Cancer Research, Melbourne) were cultured in Dulbecco’s modified Eagle’s minimal essential medium supplemented with 10% fetal bovine serum (FBS). Cells were serially passaged at a 1:8 ratio so that they underwent approximately three population doublings (PD) per passage with the total replicative lifespan of approximately 70 PD. Plasmid DNA was transfected into late passage HFF5 (approximately PD 55) cells using LipofectAMINE (Life Technologies, Inc.); typically, 3 and 10 ␮g DNA was used for 70% confluent 6- and 10-cm dishes, respectively. Transfected cells were selected in medium supplemented with 50 ␮g/ml G418. Colonies emerging from selection were individually trypsinized and isolated using cloning rings and cultured in a 12-well dish. Upon reaching confluence these cells were transferred into 6-cm dishes and sequentially into T25 and T75 flasks. The passage ratio was kept at 1:8 for cultures that showed slow growth or changed to 1:32 for cultures that showed faster growth. Serial passaging was continued until cells stopped dividing and entered senescence. In some cases, colonies of dividing cells appeared in a background of senescent cells; these colonies were isolated using cloning cylinders and then passaged continuously. Telomere repeat amplification protocol (TRAP) Expression of hTERT in transfected cells was determined by TRAP assay [34] with some modifications. Cells grown up to 80% confluency were harvested, cell lysates

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were prepared using the CHAPS detergent lysis method, and 2 ␮g of total protein was used in the TRAP assay. Amplification products were quantitated by Telomerase ELISA assay (Roche). Single-locus DNA fingerprinting DNA was fingerprinted with three multiallelic singlelocus probes, all of which are derived from the multilocus 33.15 probe: MS1 (locus D1S7 from chromosome 1p), g3 (locus D7S22 from chromosome 7q), and NS43 (locus D12S11 from chromosome 12) using the NICE probe kit, Cell Mark Diagnostics, UK). Genomic DNA was prepared using the Stratagene DNA extraction kit; 10 ␮g genomic DNA was incubated with 40 U HinfI restriction endonuclease in the supplied buffer at 37°C overnight. DNA fragments were separated by electrophoresis in an 0.8% agarose gel in 1 ⫻ TAE buffer at 40 V for 16 h and transferred to a Hybond N⫹ nylon membrane by Southern blotting. Hybridization was performed at 50°C for 20 min and the probe was detected by conjugated alkaline phosphatase activity. Western blotting The protein sample (10 ␮g) separated on an SDS–polyacrylamide gel was electroblotted onto a nitrocellulose membrane (BA85, Schleicher and Schuell) using a semidry transfer blotter (Biometra, Tokyo). Immunoassays were performed with anti-mortalin, anti-p16INK4A (Pharmingen), anti-SV40 TAg (Calbiochem), or anti-actin (Boehringer Mannheim) antibodies. The immunocomplexes formed were visualized with horseradish peroxidase-conjugated anti-rabbit immunoglobulin G (IgG) (ECL kit, Amersham Pharmacia Biotech). Immunostaining Cells grown at about 60% density were fixed with prechilled methanol:acetone (1:1) for 5 min on ice. These were stained with polyclonal anti-mortalin antibody and visualized by secondary staining with fluorescein isothiocyanateconjugated sheep anti-rabbit IgG. The cells were examined with a Fluoview confocal laser scanning microscope (Olympus Corp.).

Results and disussion Exogenous mortalin or telomerase alone does not lead to immortalization of human foreskin fibroblasts Human foreskin fibroblasts (HFF5) have normal diploid karyotype (46XY) and undergo approximately 70 PD under standard culture conditions. Late passage cells (approximately at PD 53–55) were transfected with expression plasmids encoding either human mortalin (hmot-2) or hTERT.

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(HFM-4 and -5) underwent 16 –18 PDs, and the other two (HFM-6 and -7) underwent 23–24 PD. HFF-hTERT derivatives (HFT-8 to -12) underwent 15–18 PDs (Fig. 1A). All 12 derivative cultures became senescent and were maintained for 6 – 8 weeks without the emergence of any continuously dividing colonies. Overexpression of mortalin leads to lifespan extension of HFF5

Fig. 1. In vitro lifespan and expression analysis of mortalin and telomerase in HFF5 and its transfected derivatives. (A) Growth curves for HFF5 cells transfected with vector (HFV-1 and HFV-2), mortalin (HFM-3 to -7), and hTERT (HFT-8 to -12). (B) Expression level of mortalin in vector- and mortalin-transfected HFF5 cells. Actin was used as a loading control and mortalin expression was normalized against relative percent units of actin using image analysis software. (C) Telomerase assay in parental HFF5, vector (HFV), and hTERT (HFT) derivatives. 293 (human embryonic kidney carcinoma) cells were used as the positive control for telomerase activity.

Colonies (sized about 200 –300 cells) were isolated after selection in G418-supplemented medium. The clones were named as HFF derivatives containing vector alone (HFV), hmot-2 (HFM), or hTERT (HFT). Since the cells were transfected at late passage, two or three colonies were pooled to obtain sufficient number of cells for subsequent passaging. Twelve such pooled HFF5-derived colonies, (HFV-1 and -2, HFM-3 to -7, and HFT-8 to -12) were isolated. All colonies were cultured without G418 after the first isolation and were passaged at 1:4 or 1:2 split ratios. HFV derivatives underwent only 10 –12 PD; this was comparable to the untransfected cells that had replicative lifespan of approximately 70 PD. Of the hmot-2-transfected clones, one culture (HFM-3) underwent 13 PD, two

HFF5 derivatives were analyzed for expression level of mortalin by Western blotting and for telomerase activity by TRAP assay. Mortalin expression level was high in four of the five HFM clones, with HFM-6 and HFM-7 showing the highest levels amongst the five (Fig. 1B). This was in agreement with the maximum lifespan extension (approximately 12 PD) of HFM-6 and HFM-7. However, only two (HFT-11 and -12) of the five HFT cultures showed TRAP activity at PD 8 and 16; the untransfected and vectortransfected cells were negative (Fig. 1C). HFT-11 and -12 cultures senesced despite the detectable level of expression of telomerase activity. It was therefore concluded that under the conditions used in this study, exogenous telomerase alone or overexpression of hmot-2 alone was not sufficient for immortalization of HFF5 cells. However, the in vitro lifespan of two of the five hmot-2 derivatives (HFM-6 and HFM-7) was extended by 10 –12 PD (Fig. 1A). Interestingly, these clones showed the highest level of expression of hmot-2 (about 1.8 to 2-fold higher than control, Fig. 1B). Similar enhancement in mortalin levels resulted in lifespan extension of human fetal lung fibroblasts, attributed to inactivation of the p53 tumor suppressor protein [28 –31]. This was consistent with the upregulation of mortalin expression in immortalized and tumor-derived cells [32].

Fig. 2. In vitro lifespan of HFF5 and its transfected derivatives. Growth curves of HFF5 cells transfected with vector (HFV), mortalin (HFM), and both mortalin and hTERT (HFMT).

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otherwise immortalized by the effect of telomerase, marker selection was not used. The transfected cells were initially passaged at 1:2 or 1:4. Colonies of small and rapidly proliferating cells appeared in a background of senescent cells in both of the cultures at PD 18 –20. These colonies were isolated and six of them were passaged continuously. Each of these clones (HFMT-13 to -18) have continued proliferation beyond 80 PD (Fig. 2). As described above, HFF5 cells have replicative lifespan of about 68 –70 population doublings. Cells transfected at 55 PD were expected to undergo remaining 15 PD. Combined effect of hmot-2 (human mortalin) and telomerase was sufficient to allow cells to undergo more than 80 PD (amounting to total lifespan of more than 145 PD). This eightfold posttransfection increase in lifespan, compared to that of control cells (about 10 PD in vector and 80 PD in hmot-2 and hTERT derivatives), along with the fact that HFMT cultures yet show no signs of slowing down or senescence suggests that they are immortalized. Characterization of HFF5 derivatives

Fig. 3. Characterization of HFF5 and its transfected derivatives. (A) Fingerprinting of HFF5 and its derivative clones transfected with mortalin and telomerase expression plasmids. Each clone had a DNA fingerprint identical to that of HFF5 cells and was distinct from HeLa cells. (B) Telomerase assay in parent HFF5, mortalin (HFM), and mortalin/hTERT (HFMT) transfectants. 293 cells were used as the positive control for telomerase activity. Telomerase activity was detected in each clone (HFMT-13 to -18) transfected with both mortalin and hTERT expression plasmids. (C) Mortalin expression level in HFF5 derivatives transfected with both mortalin and hTERT expression plasmids. Actin was used as a loading control and the expression level of mortalin was normalized against relative percentage units of actin using image analysis software.

Overexpression of mortalin and telomerase cooperate in extension of the lifespan of HFF5 cells We next used HFM-6 and -7 cultures at approximately PD 10 and transfected them secondarily with the hTERT expression plasmid. Since these cells were at late passage (had already undergone about 60 – 63 of 82 expected PDs) and were not expected to proliferate much further unless

Whenever a colony of dividing cells appears in a background of nondividing cells, it is essential to exclude culture cross-contamination. Therefore, we characterized the HFMT clones by single-locus fingerprinting using three probes, MS1 (locus D1S7 from chromosome 1p), g3 (locus D7S22 from chromosome 7q), and NS43 (locus D12S11 from chromosome 12), that detect a minimum of 80 alleles at each locus. The analysis confirmed that all six doubly transfected clones (HFMT-13 to -18) were derived from HFF5 cells (Fig. 3A). All six clones were positive for TRAP assay (Fig. 3B) and showed a higher level of mortalin expression than the parent HFF5 cells (Fig. 3C). These were also tested for SV40 LTAg as a further check for crosscontamination and were found to be negative (data not shown). Parent HFF5 cells and six HFMT derivative cultures were analyzed for transcriptional activation of p53 by single-cell microinjections of p53-dependent ␤-Gal reporter plasmid. As in mortalin transfected MRC-5 cells [29], activity of the p53-dependent reporter was weaker in HFMT derivatives than in the parent HFF5 cells (data not shown). All six HFMT clones analyzed at 30 and 60 PD underwent

Fig. 4. Mortalin staining pattern of HFF5 and its transfected derivatives. Subcellular distribution of mortalin in parent HFF5 and a derivative, HFMT-15, at PD 80 that was transfected with mortalin and telomerase expression plasmids. Both the parent and the immortalized derivative showed pancytoplasmic distribution of mortalin. Nonpancytoplasmic staining of mortalin in transformed (HT1080) cells is also shown.

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Fig. 5. p16INK4A expression in HFF5 and its transfected derivatives. Expression level of p16INK4A in parental HFF5 cells (PD 55) and five derivative clones (PD 80) transfected with both mortalin and hTERT. Actin was used as a loading control and the expression level of p16INK4A was normalized against relative units of actin using image analysis software.

density-dependent growth arrest and failed to form colonies in soft agar (data not shown). Subcellular distribution of mortalin is pancytoplasmic in normal cells and nonpancytoplasmic in transformed cells; a shift of perinuclear staining pattern to the pancytoplasmic type occurs when tumorigenic cells are induced to senesce [35–37]. HFMT clones analyzed at 30, 60, and 80 PD were found to have the pancytoplasmic staining pattern typical of normal cells (Fig. 4). We analyzed p16INK4A expression in HFF5 and HFMT clones. Each of the six clones had p16INK4A expression levels comparable to those of the parent HFF5 cells (Fig. 5). These data indicate that HFMT cells have escaped from senescence and have a substantially extended, possibly indefinite, lifespan. They have reduced p53 function, but retain normal levels of p16INK4A expression. Their growth is anchorage-dependent and they undergo density-dependent growth arrest. Thus, these lifespan-extended cells retain many properties of normal cells. Since late passage cells were used, the numbers of clones that emerged from transfections were small; two to three colonies were pooled to obtain enough cells for passaging and expression analysis. The data showed that some telomerized cultures (HFT-11 and -12) undergo senescence despite the telomerase activity and a prior enhancement of mortalin level improves the efficiency of immortalization of telomerized cells. In this regard, it is possible that the overexpression of mortalin facilitated hTERT expression and activity. Whereas only two of the six cultures transfected with hTERT alone had sufficient telomerase activity for it to be detected by the TRAP assay (Fig. 1C), when hTERT was transfected into clones that have a high level of mortalin expression (Fig. 3C), six of six clones were positive in the TRAP assay (Fig. 3B). Mortalin was shown to inactivate p53 [28 –31], which acts as a negative regulator of telomerase activity [38]. It is possible that enhanced level of mortalin helps in telomerase function by inactivation of its negative regulator, p53. The other possibilities of cooperation between mortalin and telomerase also exist and remain to be explored. The two clones transfected with hTERT only that had substantial

levels of telomerase activity were not immortalized. It has previously been observed that human fibroblasts were not immortalized directly by hTERT transduction, although in some cases they did become immortalized after a period of crisis [24], during which it is likely that additional, yet unidentified, genetic changes occurred. Various epithelial cell types grown in vitro under standard culture conditions become immortalized following hTERT transduction only if the p16INK4A/pRb pathway is inactivated [25]. In contrast, the HFF5 cells expressing both hTERT and mortalin retained normal levels of p16INK4A expression, but transactivation by p53 was attenuated. Thus, in these fibroblasts it appears that disruption of p53 functions, and not disruption of the p16INK4A/pRb pathway, cooperates with hTERT expression for escape from senescence. The HFMT cells were normal by a number of criteria examined in this study, including lack of anchorage-independent growth, presence of density-dependent growth arrest, normal subcellular distribution pattern for mortalin, expression of normal levels of p16INK4A, and retention of some p53-dependent reporter activity. Thus the expression of exogenous mortalin and hTERT seems to result in fewer abnormalities than the viral oncoproteins, such as SV40 LTAg, that have mostly been used for immortalizing human cells. Cells immortalized in this way may therefore be useful in various biotechnological applications, as well as for studies of the mechanisms of immortalization.

Acknowledgments This research was partially supported by a New Energy Development Organization (NEDO) international grant (to S.C.K. and R.R.R.), the Carcinogenesis Fellowship of the NSW Cancer Council (to R.R.R.), and grants from the Ministry of Economy, Trade and Industry (METI) of Japan. T.Y. is supported by a NEDO fellowship. Thanks also go to Nobuyoshi Chiba and Makoto Shintani of Japan Energy Corporation for useful comments on this study.

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