BJ fibroblasts display high antioxidant capacity and slow telomere shortening independent of hTERT transfection

Free Radical Biology & Medicine, Vol. 31, No. 6, pp. 824 – 831, 2001 Copyright © 2001 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/01/$–see front matter

PII S0891-5849(01)00664-5

Original Contribution BJ FIBROBLASTS DISPLAY HIGH ANTIOXIDANT CAPACITY AND SLOW TELOMERE SHORTENING INDEPENDENT OF hTERT TRANSFECTION MARIO LORENZ,* GABRIELE SARETZKI,† NICOLLE SITTE,‡ SUSANNE METZKOW,*

and

THOMAS

VON

ZGLINICKI†

*Institute of Pathology, Charite, Humboldt University, Berlin, Germany; †Department of Gerontology, University of Newcastle, Newcastle upon Tyne, UK; and ‡University Clinics Benjamin Franklin, Free University, Berlin, Germany (Received 13 April 2001; Accepted 19 July 2001)

Abstract—Human foreskin BJ fibroblasts are well protected against oxidative stress as shown by their low intracellular peroxide content, low levels of protein carbonyls, and low steady-state lipofuscin content as compared to other primary human fibroblasts. This correlates with a long replicative life span of the parental cells of about 90 population doublings and a telomere-shortening rate of only 15–20 bp/PD. This value might define the upper limit of a telomere-shortening rate that can still be explained by the end replication problem alone. In BJ clones immortalized by transfection with hTERT, the catalytic subunit of telomerase, the same telomere-shortening rate as in parental cells is observed over a long time despite strong telomerase activity. Hyperoxia, which induces oxidative stress and accelerates telomere shortening in a variety of human fibroblast strains, does not do so in BJ cells. It is possible that the high antioxidative capacity of BJ cells, by minimizing the accumulation of genomic damage, is instrumental in the successful immortalization of these cells by telomerase. © 2001 Elsevier Science Inc. Keywords—Fibroblasts, Hyperoxia, Telomeres, Telomerase, Senescence, Immortality, Free radicals

INTRODUCTION

There are data to suggest that the interaction of telomerase with telomeres in human cells is far from simple: hemagglutinin-antigen-tagged versions of hTERT are clearly able to elongate oligonucleotide primers and are active in the TRAP assay, but do not act upon telomeres in human embryonic kidney cells [9]. Telomere shortening with age in human T cells was not slower than in neutrophils, despite the fact that T cells are positive for telomerase activity and neutrophils are not [10]. Transfection of BJ fibroblasts with the pZeoSVhTERT [5], which contains the full-length cDNA of hTERT including wild-type 5⬘- and 3⬘-untranslated regions under control of the SV40 promoter, results in clones with elongated telomeres after selection. In this system, telomerase activity (probably as a result of increasing promoter methylation) and telomere length fell over time. The remaining low telomerase activity appeared to stabilize predominantly the shortest telomeres, allowing the growth of these clones despite the average telomere length being significantly shorter than in parental fibroblasts at senescence [11]. So, it appears that telomerase, even if highly active on an artificial substrate, is not necessarily equally active on telomeres in cells.

Telomeres in human fibroblasts and other somatic human cells shorten with each cell division. The amount of this loss varies between about 20 and 200 bp per population doubling (PD) under standard cell culture conditions. There is good experimental evidence that eventually telomere shortening triggers replicative senescence. Although there are additional, often cell-type specific, possibilities to induce a senescence-like arrest [1–3], maintenance of telomeres has been established as an essential prerequisite for cellular immortality. Transfection of human BJ foreskin fibroblasts with hTERT, the catalytic subunit of human telomerase, has restored telomerase activity in these cells [4] and allowed essentially unlimited growth of telomerase-expressing clones [5,6] without obvious genotype changes, interruptions of cell cycle control mechanisms or induction of anchorage-independent growth properties [7,8]. Address correspondence to: Dr. Thomas von Zglinicki, Department of Gerontology, Wolfson Research Centre, Newcastle General Hospital, Westgate Road, Newcastle upon Tyne NE4 6BE, UK; Tel: ⫹44 (191) 256-3310; Fax: ⫹44 (191) 219-5074; E-Mail: t.vonzglinicki@ ncl.ac.uk. 824

Telomere shortening in fibroblasts

To learn more about the interaction of telomerase with telomeres, we decided to follow changes in telomere length in BJ cells immortalized by transfection with pGRN145 [4], containing a hTERT consensus Kozak sequence downstream of the MPSV promoter. Two clones were recovered which showed high and stable telomerase activity and greatly elongated telomeres following selection [5]. We report here that despite high telomerase activity, telomere length decreases in these clones for a long time at the same rate as in parental, telomerase-negative BJ fibroblasts. Ultimately, telomere length is stabilized without a major change in telomerase activity and without any telomeres becoming as short as thought to be necessary for induction of senescence. Telomerase-immortalized cells are an interesting model to examine the role of free radical-mediated stress, especially oxidative stress, and the resultant damage to DNA for senescence and ageing. Increased oxidative stress induces premature cell cycle arrest very similar to senescence [12–16]. This stress-induced premature senescence (SIPS) is undistinguishable from “normal” senescence by many parameters (for review, see [17]). Importantly, a reduction of stress levels in cell culture, for instance by low oxygen concentration in the medium [15,18] or by using free radical scavengers [13,19], delayed replicative senescence. Oxidative stress has a profound effect on telomeres in human fibroblasts [14,19 – 21]: because of a specifically low efficiency of single strand break repair, telomeres accumulate breaks under stress before DNA replication [22], and this is a major cause of telomere shortening [19,23] in addition to the well-known “end replication problem[24].” These data suggest that telomeres act as “sentinel” for oxidative damage to the genome in telomerase-negative cells [25]. Replicative senescence might be triggered by telomeres as a consequence of DNA damage, which is reasonably well repaired in the bulk of the genome but is accumulated and/or transferred into shortening of the telomeres. Telomerase would override the sentinel function of the telomere by restoring its integrity. The question is then whether the oxidative defense and repair mechanism of the cells would be efficient enough to avoid the build-up of damage elsewhere in the genome to sustain immortal growth. To examine this, we subjected BJ fibroblasts to chronic mild hyperoxia, a condition that leads to SIPS in a variety of human fibroblast strains. Interestingly, both parental and immortalized cultures were highly resistant to this type of treatment. In contrast to other fibroblast strains, peroxides were not formed at a higher rate under hyperoxia, telomere shortening was not accelerated, and neither was senescence induced prematurely in parental BJ cultures, nor was the growth of immortalized BJ clones affected. We conclude that an especially efficient antioxidant defense system is one

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prerequisite for the successful immortalization of BJ fibroblasts by telomerase. MATERIALS AND METHODS

Cells Parental BJ human foreskin fibroblasts and telomerase-positive clones BJ5T and BJ6T were obtained from Geron Corporation (Menlo Park, CA, USA) at a PD of 40 (BJ), 127 (BJ5T), and 123 (BJ6T). The clones were produced by transfection of the parental cells with the plasmid pGRN145 containing the hTERT Kozak consensus sequence under control of the MPSV promoter as described [4,5]. If compared to human tumor cells, these cells expressed very high levels of hTERT mRNA and displayed high telomerase activity in the TRAP assay [26]. MRC-5 fibroblasts (human fetal lung) were obtained from the European Collection of Cell Culture (Salisbury, UK) at passage 18. Because the supplier could not provide the PD for this culture, the initial PD was arbitrarily set at 20. Cells were grown in DMEM plus 10% FCS under either normoxia (air plus 5% CO2) or mild normobaric hyperoxia (40% oxygen, 5% CO2) in a three-gas incubator (Zapf Instruments, Sarstedt, Germany). Growing cell cultures were counted and replated weekly to estimate the PD. Cultures approaching senescence were fed weekly but were replated only when reaching confluence. BJ6T subclones were generated at a PD of 320 by limited dilution. Telomeres and telomerase For isolation of genomic DNA with minimum induction of artificial strand breaks, standard procedures for pulsed field gel electrophoresis were followed. Cells were trypsinized and embedded in 0.65% low-melting agarose in 80 ␮l plugs at a density of 107 cells/ml before deproteination by proteinase K treatment [22,23]. DNA was digested until completion with HinfI (60 units per plug, Boehringer Mannheim, Germany) at 37°C. The DNA as embedded in the plugs was analyzed in a 1.0% agarose gel by pulsed field gel electrophoresis (BioRad, Hercules, CA, USA). Gels were blotted to Hybond N⫹membranes and hybridized with the telomeric probe (TTAGGG)4, conjugated directly to alkaline phosphatase (Promega, Madison, WI, USA). A chemiluminescence signal was recorded on film within the linear range and analyzed in an imaging densitometer (BioRad). The average telomere length was calculated as weighted mean of the optical density as described [22]. Telomerase activity was studied by TRAP assay (Intergen Inc., Purchase, NY, USA) according to the recommendations of the manufacturer.

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Oxidative stress indicators Fibroblast cultures were grown for at least 3 weeks in parallel under either normoxia (control) or 40% normobaric hyperoxia. To measure the content of intracellular peroxides, cells were stained for 30 min at 37°C in the dark with 80 ␮M dihydrochlorofluorescein-diacetate, which became trapped and was oxidized by intracellular peroxides into the fluorescent dye, dihydrochlorofluorescin (DCF). Cells were than trypsinized, stained with 2 ␮g/ml propidium iodide for 5 min on ice in the dark, and the fluorescence of 2 ⫻ 104 cells was measured in a flow cytometer (PARTEC, Munster, Germany) using blue excitation. Cells showing red propidium iodide fluorescence, indicative of a damaged plasma membrane, were gated out (1–2% of all cells). The average fluorescence intensity was measured in arbitrary units. Oxidative modification of proteins was measured as protein carbonyl content in cell lysates (4 mg/ml) by the ELISA of Buss [27] with modifications as described [28]. Measurements were performed in triplicate at four different PD within the period of logarithmic growth (phase II) of fibroblasts. The cellular lipofuscin content was measured as the autofluorescence intensity in the yellow/green range of the spectrum of unfixed cells by flow cytometry. About 2 ⫻ 104 BJ or MRC-5 cells in log growth phase were examined using a Becton-Dickinson FACScan with blue excitation. Emission was measured in the range of 563 to 607 nm. Measurements were performed in triplicate at two different PD within the logarithmic growth phase.

RESULTS

Initial growth rates of telomerase-positive BJ clones under standard culture conditions were very similar to that in parental cultures. However, growth of parental cells slowed and the cultures reached senescence at a PD of 87– 88. On the contrary, BJ5T and BJ6T cells grew for more than 400 PD without any change in growth rate (Fig. 1). Cells were also subjected to growth under 40% chronic hyperoxia. These conditions induced significant oxidative stress in different strains of human fibroblasts, including WI-38 and MRC-5, leading to the induction of SIPS after less than five PD [12–14,19]. Surprisingly, growth of BJ fibroblasts was hampered only slightly under hyperoxia. The growth rate in parental cultures decreased only insignificantly, and the cultures senesced eventually at nearly the same PD as under normoxia. Telomerase-positive clones adapted after a short while to hyperoxia and showed the same growth rate as under normoxia (Fig. 1). These results suggested that both parental BJ fibroblasts and their telomerase-positive de-

Fig. 1. Growth of BJ fibroblasts (cumulative population doublings, PD) under basal conditions (normoxia, N) and under chronic 40% hyperoxia (H). BJpar: parental cells; BJ5T and BJ6T: clones of hTERT-immortalized fibroblasts. Experiments were terminated at the times shown in the figure.

rivatives are better protected against hyperoxia-induced oxidative stress than many other human cell strains. To test this suggestion, we measured the amount of intracellular peroxides using DCF fluorescence (Fig. 2). Basal levels of DCF fluorescence under normoxia were similar for all BJ cultures. MRC-5 fibroblasts showed a significantly higher fluorescence level already under

Fig. 2. Intracellular peroxide content (measured as DCF fluorescence intensity in arbitrary units) in parental BJ fibroblasts, immortalized clones BJ5T and BJ6T and in human MRC-5 primary fibroblasts. Data are mean ⫾ SEM from three independent experiments. Cells were grown for 3– 6 weeks under hyperoxia before measurement. Significant differences (p ⬍ .05) between normoxia and hyperoxia are indicated by an asterisk, and those between BJpar and any other cell type by a ⫹ sign

Telomere shortening in fibroblasts Table 1. Oxidation Products in BJ and MRC-5 Fibroblasts

Protein carbonyls (nmol/ mg protein) Yellow/green autofluorescence intensity (arbitrary units) Lipofuscin accumulation rate

BJpar

MRC-5

p

0.31 ⫾ 0.01

1.24 ⫾ 0.16

0.001

150 ⫾ 5

203 ⫾ 16

0.028

28 ⫾ 2

102 ⫾ 10

0.001

Protein carbonyls were measured by ELISA and are averages from independent measurements at PD 45, 55, 63, and 71 in BJpar [29] and at PD 28, 37, 46, and 56 in MRC-5 [30]. The lipofuscin content was measured as autofluorescence at two different PD during the logarithmic growth phase of BJpar and MRC-5. The lipofuscin accumulation rate was calculated as described [31]. Data are mean ⫾ SEM. The differences are significant with an error probability p as given in the table.

basal conditions. Exposure of MRC-5 cells to hyperoxia resulted in a sustained and significant increase of the cellular peroxide content. All BJ cell cultures, on the contrary, were able to maintain their basal peroxide levels even under chronic hyperoxia. These results were confirmed by measurements of the cellular peroxidation products, lipofuscin and protein carbonyl groups (Table 1). Protein-bound carbonyl moieties are an established marker of protein oxidation. Their concentration is four times higher in MRC-5 fibroblasts as compared to BJ cells. Accumulation of lipofuscin is dependent on the rate of formation of autofluores-

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cent, nondegradable material, and on the dilution of lipofuscin by cell proliferation. The rate of lipofuscin accumulation is a good measure of peroxidation processes, and it can easily be estimated under steady-state conditions of cell growth [31]. This estimation results again in a 3– 4-fold lower level of oxidative stress in BJ fibroblasts. These data confirm that there is a very low level of oxidative stress in BJ fibroblasts, notably under chronic hyperoxia. The measurement of telomere length (Fig. 3) confirms that telomerase-immortalized clones not only have much longer telomeres than parental BJ cells, but also that the variation of telomere length (expressed in percent of average length) is less in these clones than in the parental culture (see [5]). Moreover, telomeres in BJ5T and BJ6T clones shorten gradually over more than 100 PD. Only after nearly 1 year in culture, i.e., at a PD of about 270 in BJ5T and about 320 in BJ6T, telomere shortening is compensated for and no further shortening was seen for at least a further 100 PD. This changed telomere maintenance is not correlated to the activity of telomerase as measured by TRAP assay (Fig. 4): there is no significant difference in telomerase activity in both clones over time. Moreover, there is also no change in the growth rate of cells (see Fig. 1). The average telomere length in BJ5T and BJ6T clones even at their lowest level (i.e., before telomere length becomes stabilized) is still significantly higher than that in parental BJ cells. Moreover, there was no evident

Fig. 3. Telomere Southern blots of parental BJ fibroblasts (left), BJ5T (middle), and BJ6T (right) clones. DNA size markers (in kbp) are indicated, and the PD for each lane is given on top of the figure.

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Fig. 4. Telomerase activity of the BJ5T and BJ6T clones at three different PD each. One typical gel out of three measurements is shown. Differences between PDs were not significant. NC ⫽ negative control (no lysate); arrowhead ⫽ internal standard.

increase in the percentage of short telomeres at that point. In fact, no telomeric signal at all could be seen around 5 kbp, while there is a significant fraction of

telomeres of about this length in parental BJ cells (Fig. 3). It might be possible that the sensitivity of the Southern blot is too low to pick out a few short telomeres in the mass culture. Therefore, we generated subclones from the BJ6T clone at a PD of 320 by limited dilution of cells. If there would be cells in the BJ6T culture with short telomeres, one would expect to find them enriched in at least one of the subclones. This was not the case. Although there was some variation in telomere length in the subclones (Fig. 5), not a single one out of 25 examined showed telomeres nearly as short as in the parental culture. A quantitative comparison of telomere shortening rates measured in hTERT-positive BJ clones during the period of telomere loss and in parental BJ cells between PD 45 and 80 reveals the same rate of shortening in all three cultures under normoxia (Fig. 6). Telomeres tend to shorten slightly faster under hyperoxia. However, this increase is only significant in one of the clones (BJ6T). Even in this clone, the telomere-shortening rate under hyperoxia is lower than in many other fibroblast cultures (compare [32]) including MRC-5 (Fig. 6). In fact, telomere-shortening rates in parental and hTERT-transfected BJ cells both under basal and under hyperoxic conditions are at the lower limit of all those reported for human somatic cells so far. DISCUSSION

In this study, we found a very low rate of telomere shortening in BJ fibroblasts of about 15–20 bp/PD. This

Fig. 5. Telomere length in 25 different subclones of BJ6T. Subclones were generated at a PD of 320 and measured at a PD of approximately 332. DNA size markers (in kbp) are indicated, and the clone numbers for each lane are given on top of the figure.

Telomere shortening in fibroblasts

Fig. 6. Telomere shortening in BJ fibroblasts. (A) Average telomere length as measured in parental fibroblasts (BJpar, data from four independent lifespan experiments with 5– 8 samples each; points are averages of 2– 4 independent estimates on different gels) and in immortalized clones BJ5T and BJ6T (one lifespan experiment each, three different gels) under normoxia. (B) Average telomere shortening rate in human BJ and MRC-5 primary fibroblasts under normoxia and 40% hyperoxia. Data are mean ⫾ SEM from four (BJpar normoxia) or two independent experiments. Ranges considered were between PD 45 and 80 in BJpar, PD 145 and 273 in BJ5T, and PD 134 and 328 in BJ6T. Significant differences (p ⬍ .05) between normoxia and hyperoxia are indicated by an asterisk, and those between BJpar and any other cell type by a ⫹ sign.

corroborates our earlier findings [31,32] and is in accordance with shortening rates seen in pZeoSV-hTERT immortalized BJ fibroblasts during the period of loss of their telomerase activity [11]. However, others have reported a telomere-shortening rate of nearly 50 bp/PD for parental BJ fibroblasts [33,34]. This might be due to

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intrinsic differences in batches of cells, use of different media, or differences in serum composition. For instance, it has been shown that trace amounts of selenium, which are present in some media but absent in others, can make a major difference with respect to cellular antioxidative defense [35]. To our knowledge, this is about the lowest rate of telomere loss measured in human fibroblasts so far. Interestingly, this rate does not increase significantly if the cells are cultured under chronic hyperoxia. We had shown before that hyperoxia generates oxidative stress in different human cell lines and that this stress accelerates telomere shortening and induces SIPS [14,17,21]. We had also shown a positive correlation between telomere-shortening rates and antioxidative capacity in a number of human fibroblast strains [32]. Most importantly, long-term treatment with ␣-phenyl-t-butylnitrone, a free radical scavenger, was shown to decrease intracellular DCF levels, slow down telomere shortening, and prolong replicative lifespan of MRC-5 fibroblasts [19]. Similarly, enhancement of intracellular vitamin C levels decreased the peroxide content and decelerated telomere shortening in HUVEC [36], and copper chelation protected telomeres from copper/ascorbic acid-mediated premature shortening [37]. The low telomereshortening rate in BJ fibroblasts, therefore, suggested an uncommonly high degree of antioxidant protection. This suggestion was confirmed: in BJ fibroblasts, in comparison to other human primary fibroblast strains, for which MRC-5 is an example, we measured a significantly lower level of intracellular peroxides, of oxidized proteins as estimated by the concentration of protein-bound carbonyl groups, and of the steady-state lipofuscin content. We can conclude that excellent antioxidant protection in BJ cells allows a long replicative lifespan of 80 –90 PD as compared to the more common 40 – 60 PD in other human fibroblast strains, prevents the induction of premature senescence by chronic mild hyperoxia, and enables a low telomere-shortening rate. There are different mechanisms contributing to telomere shortening. The so-called end-replication problem, i.e., the loss of the most distal RNA primer for DNA replication, does contribute to telomere shortening, but possibly by no more than 2– 6 nucleotides per division, assuming that the most distal primer is accurately positioned at the very end of the telomere. Nuclease processing of the 5⬘ end of the chromosome might form the 3⬘G-rich overhang and contribute to telomere shortening [38]. In fact, an overhang size of about 150 nt has been reported for BJ fibroblasts [33] and a proportionality between overhang length and telomere-shortening rate in four human cell strains, including BJ fibroblasts, has been shown [34]. However, published data are contradictory both with respect to the frequency of G-rich overhangs [33,38] and to the variation of their length

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with telomere shortening [19,34]. It might be possible that the overhang length is much more heterogeneous than originally thought [39]. Whatever the impact of these mechanisms might be, our data indicate that their combined contribution to telomere shortening is less than 20 bp/PD in BJ fibroblasts (and less than about 35 bp/PD in antioxidant-protected MRC-5 fibroblasts [19]). Each faster telomere shortening should be indicative of additional processes acting on the telomeres, with telomerespecific accumulation of oxidative damage being a prominent one [19]. It was interesting to note that over long periods of growth the rate of telomere shortening in telomeraseimmortalized BJ clones was the same as in the parental cells devoid of telomerase activity. In contrast to clones immortalized using the pZeoSV-hTERT vector [11], this was not due to low levels and/or complete loss of hTERT expression and telomerase activity. Steady-state levels of hTERT mRNA were at least one order of magnitude higher in our clones than in breast tumor cells, so that they could be clearly detected by RNAse protection assay. Telomerase activity levels in the TRAP assay were similar to that in breast tumor cells [26]. While we cannot exclude minor changes in telomerase activity over time, there was clearly no major decrease with time, and telomerase activity was present in the TRAP assay at all times (Fig. 4). Still, for 9 –11 months of continuous culture, corresponding to 130 –190 PD, the active telomerase did not seem to counteract telomere shortening. Only afterwards a stable maintenance length was reached. Possibly, this telomere dynamics could be explained by clonal evolution and overgrowth of subclones with relatively short telomeres. However, this is not very plausible given the constant growth rate of the cultures over time. On the other hand, the strict parallelism between telomere-shortening rates in parental and immortalized fibroblasts suggests that telomerase, although present and active in the TRAP assay, did not act upon telomeres for a long while. This is comparable to the telomere dynamics observed in telomerase-positive tumor cells, where the existence of a rather stable maintenance length, to which subclones would tend to return, has been clearly demonstrated [40,41]. We show here that such a return can happen with the same telomereshortening rate as in the telomerase-negative parental line. While factors such as TRF1 have been identified as negative regulators of telomere length in human cells [42], those that determine the maintenance length remain obscure. Interestingly, the maintenance length reached in the immortalized clones was still much longer than the telomere length in the parental strain, even at low PD. Especially, we could not see any telomeres in the range around and below 5 kb, which are thought to trigger the

cell cycle arrest pathways. So, it is not clear at all how stabilization of telomere length was signaled. It appears that under conditions of limited telomerase activity and/or very short telomeres (as present in many tumor cells), telomerase is preferentially recruited to stabilize the shortest telomeres [11]. In the extreme, this might be due to a simple selection process, by which cells with unstabilized telomeres (or uncapped telomeres [43]) are lost from the proliferating pool. This is clearly different from the case described here of long telomeres and abundant telomerase activity. Further work will be necessary to understand the regulation of telomere maintenance. Our data indicate a high degree of antioxidant protection not only in parental BJ fibroblasts, but also in hTERT-immortalised clones (Fig. 2). Not only 40% oxygen, but even “normoxic” oxygen partial pressure is considerably higher than the oxygen partial pressure that cells in the human body experience, and hence induces oxidative stress in a wide variety of human cell strains. This is not so in BJ fibroblasts. There is no doubt that this efficient antioxidant defense system minimizes the accumulation of genomic damage during the long-term growth of immortalized clones. It appears possible that a good antioxidative defense is a prerequisite for the successful immortalization of human cells by telomerase. Acknowledgements — The work was supported by grants from VERUM Foundation for Behaviour and Environment, Munich, and from the Deutsche Forschungsgemeinschaft (ZG2/4-3). Cells used in this study were gratefully obtained from Geron Corp., Menlo Park, CA, USA.

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