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Cell Growth & Differentiation Vol. 13, 59-67, February 2002
© 2002 American Association for Cancer Research

SV40 T Antigen and Telomerase Are Required to Obtain Immortalized Human Adult Bone Cells without Loss of the Differentiated Phenotype

Christian Darimont, Ornella Avanti, Yvonne Tromvoukis, Patricia Vautravers-Leone, Nori Kurihara, G. David Roodman, Lorel M. Colgin, Heide Tullberg-Reinert, Andrea M. A. Pfeifer, Elizabeth A. Offord and Katherine Mace1

Nestle Research Center, 1000 Lausanne 26, Switzerland [C. D., O. A., Y. T., P. V-L., A. M. A. P.]; University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania 15260 [N. K., G. D. R.]; Children’s Medical Research Institute, Westmead 2145, New South Wales, Australia [L. M. C.]; and Institute of Pathology, University Hospital Basel, 4003 Basel, Switzerland [H. T-R.]


    Abstract
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
In most human primary bone cells, SV40 T-antigen expression was able to expand life span for a few passages before cells undergo growth arrest, described as crisis. In this study, telomerase activity was reconstituted in human osteoblast precursors (hPOB cells) and marrow stromal cells (Saka cells) transformed with the SV40 T antigen. Bone cells with telomerase activity were able to bypass crisis and show unlimited life span. Despite chromosomal aberrations observed in hPOB-tert cells, these immortalized precursors were able to differentiate into osteoblasts like precrisis hPOB cells. Saka-tert cells enhanced the formation of human osteoclast-like cells in a similar manner as Saka cells. These results demonstrate that reconstitution of telomerase activity in transformed SV40 T-antigen human osteoblast precursors or marrow stromal cells leads to the generation of immortalized cells with a preserved phenotype.


    Introduction
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Primary cells, derived from normal rodent or human bone, present a reliable phenotype but have a limited proliferative life span before reaching a permanent growth arrest, known as replicative senescence (1 , 2) . This restricted proliferative capacity renders their use, in screening assays, limited and labor intensive. Osteosarcoma constitutes an unlimited source of cells with some osteoblastic features. However, because of large genetic transformations, these tumorigenic cell lines may display a modified differentiation pattern and respond abnormally to hormonal treatments (3 , 4) . Immortalization of cells by overexpression of oncogenes, such as the SV40 T-Ag,2 is an interesting approach to obtain relevant human cell lines. Transformation of human bone precursors with SV40 T-Ag was shown to extend their life span and to preserve their phenotype (5, 6, 7, 8, 9, 10) . However, in most of these reports (6, 7, 8) cells were able to overcome replicative senescence for only few PDs before entering into a nonreplicative phase called "crisis" (11) . In most cell types, rare cellular clones are able to escape from crisis and to proliferate indefinitely (12) . Nevertheless, these immortalized cell lines present important genomic modifications, which usually induce phenotypic alterations (13 , 14) .

Functional telomeres are essential for chromosomal integrity, and recent reports suggest that critical telomere shortening is associated with the onset of cellular senescence (15) . Therefore, ectopic expression of the hTERT gene, which catalyzes the addition of telomeric DNA repeats on the ends of chromosomes, has been proposed as an alternative method for human cell immortalization (15) . Immortalization of human fibroblasts and epithelial cells by reconstitution of telomerase activity alone is nevertheless still controversial (16, 17, 18, 19) . In human osteoblasts, hTERT gene expression was shown recently to delay the replicative senescence for ~20 more PDs than telomerase-negative cells but did not lead to cellular immortalization (20) . Telomerase activity reconstitution in SV40 T-Ag-transformed human fibroblasts, myoblasts, and pancreatic and kidney cells was shown to allow cells to escape from crisis and to become immortal (21, 22, 23, 24) . Although the association of SV40 T-Ag and hTERT gene expression appears as a promising approach for human cell immortalization, it nevertheless remains to be shown that this process of immortalization is effective in human bone cells and avoids phenotypic drift of the cells.

For this purpose, human osteoblast precursors of periosteal origin were infected with a SV40 T-Ag-recombinant retrovirus. SV40 T-Ag-expressing cells (designated hPOB cells) showed extended life span when compared with non infected primary cells but entered rapidly into crisis. Ectopic expression of the hTERT gene allowed hPOB cells to bypass crisis and to acquire indefinite life span. Similar results were obtained when telomerase activity was reconstituted in the SV40 T-Ag-transformed human marrow stromal Saka cell line described previously (10) . Both immortalized cell lines, named hPOB-tert and Saka-tert, present the same characteristics as precrisis cells, i.e., capacity to differentiate into osteoblast and to support osteoclast differentiation, respectively. These human cell lines overcome many of the limitations of existing human cellular models and represent valuable tools to gain additional insights into the process of bone remodeling.


    Results
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Establishment of the SV40 T-Ag-expressing Osteoblast Precursors.
Primary human osteoblast precursors, prepared from a periosteal specimen of femur, were infected with the pLHXSD retroviral vector carrying the SV40 T-Ag gene. After three to four passages, noninfected primary cells stopped growing, whereas infected cells continued to proliferate. Proliferative cells, designated hPOB cells, were expanded, and the homogeneity of the SV40 T-Ag-expressing cell population was checked by immunostaining. Fig. 1Citation shows that the totality of the hPOB cell nucleus strongly reacted with the antibody directed against the SV40 T-Ag. These cells were able to grow until passages 12–15, corresponding to a maximum of 89 PDs before entering in crisis. Several attempts were made to allow the cells to escape crisis; however, no survivor cells arose.



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Fig. 1. Immunofluorescent staining of hPOB cells with anti-SV40 T-Ag antibody. Cells, seeded on glass chamber slides, were fixed and immunostained with a SV40 T-Ag-specific antibody. Magnification of the photograph x 200. No fluorescence was observed in cells incubated with the second antibody alone (not shown).

 
Reconstitution of Telomerase Activity in Bone Cells.
To try to bypass crisis and immortalize the cells, hPOB cells were infected at a low passage (passage 9; 54 PD) with a pLHXSD retroviral vector carrying the hTERT gene. Infected hPOB cells, designated hPOB-tert cells, were able to grow, without any modification in cell proliferation, for at least 428 PDs (passage 62) and were considered as immortal. To demonstrate that the hTERT gene was active in hPOB-tert cells, telomerase activity was measured. Fig. 2ACitation shows that as early as passage 20, corresponding to 138 PDs, hPOB-tert cells were able to express telomerase activity, whereas no activity was detected in hPOB cells. As expected, telomerase activity reconstitution in hPOB-tert cells was correlated with an increased in telomere length. The mean average telomere length measured in hPOB-tert cells between 214 and 228 PDs was 18.3 kb, which is similar to what has been observed previously in transformed SV40-T Ag cells expressing hTERT (24) .



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Fig. 2. Telomerase activity in precisis and hTERT-expressing human bone cells. Telomerase activities were measured in: A, hPOB cells at 60 and 72 PDs, and in hPOB-tert cells from 138 to 256 PDs; and B, Saka cells at 34 and 36 PDs, and in Saka-tert cells from 124 to 242 PDs using the TRAP assay.

 
The Saka cell line is a human marrow stromal cell line transformed with the SV40 T-Ag and described for its capacity to enhance human osteoclast formation (10) . Like most of the SV40 T-Ag-transformed human bone cells, proliferation of Saka cells was strongly reduced at about passage 14 (43 PDs). To confirm that association of telomerase activity and SV40 T-Ag allows immortalization of human bone cells, Saka cells, at passage 11, were infected with a recombinant virus carrying the hTERT gene. Infected cells displayed telomerase activity (Fig. 2B)Citation and were able to bypass crisis and to grow until at least 319 PDs (passage 54) with no decrease in cell proliferation.

Karyotype Analysis.
To define the chromosome patterns during the immortalization process, karyotype analysis was performed in hPOB cells at passage 10 (60 PDs) and in hPOB-tert cells at passage 39 (269 PDs). The data from 100 metaphases indicate that 68% of hPOB cells were diploid, 27% tetraploid, and 5% higher. A significant shift in ploidy was observed in hPOB-tert cells. Indeed, 97% of cells were tetraploid with no diploid cells. Consequently, the modal number increased from 45 in hPOB cells to 85 in hPOB-tert cells. Two marker chromosomes were detected in hPOB-tert cells, whereas none of these chromosomes were found in the hPOB cells karyotype. The isoenzyme phenotype patterns in hPOB-tert cells were concordant with those of hPOB cells, identifying hPOB-tert cells as a derivative of hPOB cells.

Phenotypic Characterization.
Experiments designed for the determination of cell phenotype were performed in hPOB and in hPOB-tert cells cultured from passage 8 to 12 (48 to 72 PDs) and from passage 33 to 40 (228 to 276 PDs), respectively.

Cell proliferation was assessed in both cell types. hPOB-tert cells presented a slightly higher proliferation rate than hPOB cells with a doubling time of 20.8 h and 24.1 h for hPOB-tert and hPOB cells, respectively. Saka cell proliferation was strongly increased after hTERT gene expression. The doubling time raised from ~60 h in Saka cells (passages 10–14) to 28.4 h in Saka-tert cells (passages 20–35). Similar observations have been reported previously in SV40 T-Ag/hTERT-transformed human myoblast and pancreatic cells (22 , 24) .

The ability of hPOB and hPOB-tert cells to differentiate into osteoblast-like cells was then tested. ALP activity was measured at different times during differentiation under osteogenic culture conditions. From day 0 to 9 after confluence, cells were cultured in the basal medium alone or supplemented with 0.1 µM of Dex, 10 nM of 1,25-(OH)2D3, or with the combination of both agents. ALP activity did not change significantly in hPOB cells incubated from day 0 to 9 with the basal medium (Fig. 3A)Citation . However, when 1,25-(OH)2D3 was added to the culture medium, ALP activity increased from day 4 after confluence and reached a plateau at day 6. Dex alone increased the enzyme activity slightly but not significantly (320.1 ± 36.0 nmol pNP/mg/h with 1,25-(OH)2D3 versus 221.5 ± 49.5 nmol pNP/mg/h with Dex at day 9). No additive effect was observed when Dex and 1,25-(OH)2D3 were added simultaneously to the culture medium. In hPOB-tert cells the basal activity of ALP was ~2-fold higher as compared with the basal activity of hPOB cells (Fig. 3BCitation ; hPOB cells: 117.9 ± 19.6 nmol pNP/mg/h; hPOB-tert cells: 231.8 ± 22.7 nmol pNP/mg/h at day 0). As early as day 2, 1,25-(OH)2D3 enhanced ALP activity, which reached a plateau at day 4. The fold increase of ALP activity stimulated by 1,25-(OH)2D3 at day 9, as compared with untreated cells, was similar in both cell types (2.5-fold in hPOB cells versus 2.4-fold in hPOB-tert cells). Dex did not increase significantly ALP activity in hPOB-tert cells, and no additive effects of 1,25-(OH)2D3 plus Dex were observed. These results show that hPOB and hPOB-tert cells cultured with 1,25-(OH)2D3 had a maximal ALP activity at day 6, indicating that, under this appropriate osteogenic culture condition, both cell types reached a differentiated status 6 days after confluence. Interestingly, under the same culture conditions primary culture of human periosteal cells displayed a similar stimulation of ALP activity than hPOB cells (4-fold increase versus 3-fold in hPOB cells).



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Fig. 3. ALP activity in hPOB and hPOB-tert cells during differentiation. hPOB (A) and hPOB-tert cells (B) were cultured in the presence of the basal medium containing 1 mM of ß-glycerophosphate and 50 µg/ml ascorbate alone (control; {square}) or supplemented with 10 nM dexamethasone ({triangleup}), 10 nM 1,25-(OH)2D3 ({diamond}), or both dexamethasone and 1,25-(OH)2D3 ({circ}). ALP activity was measured in cell homogenates of both cell types at days 0, 2, 4, 6, and 9 after confluence. Data are the means of values obtained in at least three separate experiments; bars, ±SE. Values statistically different from the control condition are indicated by * (P < 0.05) or ** (P < 0.01).

 
To determine whether osteoblast markers were present in differentiated cells, expression levels of osteocalcin, Cbfa-1, and osteonectin were measured by semiquantitative RT-PCR at day 0 and 6 after confluence. Osteocalcin, the most abundant noncollagenous proteins in bone, was hardly detectable in both cell types at day 0 and day 6 without any anabolic agents (Fig. 4)Citation . However, 1,25-(OH)2D3 induced a large and significant increase in mRNA levels of hPOB (2.3-fold) and hPOB-tert cells (3.3-fold). In hPOB cells, the addition of Dex plus 1,25-(OH)2D3 induced an additive effect (2-fold), whereas in hPOB-tert cells Dex did not change the effect of 1,25-(OH)2D3 (Fig. 4)Citation .



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Fig. 4. Expression of osteocalcin in confluent and differentiated hPOB and hPOB-tert cells. Osteocalcin (OC) mRNA expression was analyzed by RT-PCR at day 0 (confluent cells) and in cells incubated 6 days under the following conditions: in the presence of the basal medium containing 1 mM ß-glycerophosphate and 50 µg/ml ascorbate alone (Cont), pr supplemented with 10 nM Dex, 10 nM 1,25-(OH)2D3 (Vit D), or both effectors (Dex + Vit D). Bar graphs represent the ratio of osteocalcin to actin mRNA levels. Data are the means of values obtained at least in three separate experiments;; bars, ±SE. Values statistically different from the control condition at day 6 (Cont) are indicated by * (P < 0.05) or ** (P < 0.01).

 
Cbfa-1 was expressed at day 0 in both cell types (Fig. 5)Citation . At day 6, Dex significantly increased Cbfa-1 mRNA in hPOB cells, whereas it was only slightly enhanced in hPOB-tert cells (Fig. 5)Citation . Osteonectin was expressed in hPOB cells at a lower level at day 6 (Cont) than day 0 (Fig. 6)Citation . Dex and 1,25-(OH)2D3 slightly stimulated its expression at day 6, but no additive effect was observed. In hPOB-tert cells osteonectin expression was slightly decreased during differentiation but not affected by any effectors (Fig. 6)Citation . A lack of stimulation of osteonectin expression was also observed in primary human osteoblast precursors cultured with Dex and 1,25-(OH)2D3 for 6 days (not shown). However, under the same culture conditions, Cbfa-1 was increased by 1.7-fold in these primary cell preparation.



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Fig. 5. Expression of bone-specific transcription factor Cbfa-1 in confluent and differentiated hPOB and hPOB-tert cells. Cbfa-1 mRNA expression was analyzed by RT-PCR at day 0 (confluent cells) and in cells incubated for 6 days under the following conditions: in the presence of the basal medium containing 1 mM ß-glycerophosphate and 50 µg/ml ascorbate alone (Cont) or supplemented with 10 nM Dex, 10 nM 1,25-(OH)2D3 (Vit D), or both effectors (Dex + Vit D). Bar graphs represent the ratio of Cbfa-1 to actin mRNA levels. Data are the means of values obtained in at least three separate experiments; bars, ±SE. Values statistically different from the control condition at day 6 (Cont) are indicated by * (P < 0.05).

 


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Fig. 6. Expression of osteonectin in confluent and differentiated hPOB and hPOB-tert cells. Osteonectin (ON) mRNA expression was analyzed by RT-PCR at day 0 (confluent cells) and in cells incubated 6 days under the following conditions: in the presence of the basal medium containing 1 mM ß-glycerophosphate and 50 µg/ml ascorbate alone (Cont) or supplemented with 10 nM Dex, 10 nM 1,25-(OH)2D3 (Vit D), or both effectors Dex and 1,25-(OH)2D3 (Dex + Vit D). Bar graphs represent the ratio of osteonectin:actin mRNA levels. Data are the means of values obtained in at least three separate experiments; bars, ±SE. Values statistically different from the control condition at day 6 (Cont) are indicated by * (P < 0.05) or * (P < 0.01).

 
The formation of in vitro mineralization was studied in hPOB and hPOB-tert cells cultured for 21 days in the presence of 1,25-(OH)2D3 and Dex. Calcium deposition was visualized with the Alzarin Red-S-based colorimetric reaction. As shown in Fig. 7Citation , both cell types presented in vitro mineralization.



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Fig. 7. Mineralization of the extracellular matrix by hPOB and hPOB-tert cells. hPOB (A) and hPOB-tert cells (B) were cultured in the basal medium supplemented with 1 mM ß-glycerophosphate, 50 µg/ml ascorbate, 10 nM dexamethasone, and 10 nM 1,25-(OH)2D3. At Day 21 cells were stained by Alizarin Red as described in "Materials and Methods." Magnification of the photographs, x 200.

 
To estimate whether reconstitution of telomerase activity in Saka cells has modified their phenotypic characteristics, both precrisis cells and Saka-tert cells were evaluated and compared for their capacity to enhance human osteoclast formation. The number of 23c6-positive multinuclear cells was highly increased when Saka-tert cells were cocultured with human marrow mononuclear cells in the absence of osteotropic agents (Fig. 8)Citation . Saka-tert cells appeared twice more efficient than Saka cells to enhance osteoclast formation (Fig. 8)Citation .



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Fig. 8. Effects of Saka and Saka-tert cells on human bone marrow multinuclear cells formation. Human bone marrow nuclear cells were overlaid onto Saka or Saka-tert cells plated 2 days before without osteotropic agents. Data are the means of values obtained in four separate experiments; bars, ±SE. Values statistically different from the control condition (without Saka or saka-tert cells) are indicated by ** (P < 0.01).

 

    Discussion
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
The aim of the present study was to determine whether reconstitution of telomerase activity in SV40 T-Ag-transformed human bone cells allows them to overcome crisis and to maintain their phenotype. The results clearly show that expression of the hTERT gene in SV40 T-Ag-transformed human osteoblast precursors (hPOB cells) or marrow stromal cells (Saka cells) allowed cells to escape from crisis and to maintain cell growth until at least 428 and 319 PDs, respectively. Similar observations were reported previously for SV40 T-Ag-transformed fibroblasts, myoblasts, and pancreatic and kidney cells (21, 22, 23, 24) , but to our knowledge, it has never been demonstrated in human bone cells. In our hands, stable expression of the SV40 T-Ag gene alone in primary osteoblast precursors did not lead to immortalization, because after 89 PDs (p15) a growth arrest was observed with no spontaneous survivors of crisis. Similarly, Saka cells stopped growing after ~45 PDs (p14). Several reports have described the onset of crisis in SV40 T-Ag-expressing bone cell lines (6, 7, 8) . To our knowledge, no postcrisis cellular clones aroused in these studies. All together, these results suggest that the ability to bypass crisis is a very rare event for SV40 T-Ag-transformed human bone precursors. Only two studies have reported the absence of growth reduction of human SV40 T-Ag-expressing bone cells. In the first study, cell proliferation was measured until at least 100 PDs (9) , which approximately corresponds to the precrisis period in hPOB cells. The second cell line cultured after >70 passages was established with cells derived from fetus, which may explain the difference in cell behavior during the immortalization process (5) . Because reconstitution of telomerase activity alone does not allow immortalization of adult bone cells (20) , the present study proposes that association of both SV40 T-Ag gene expression and telomerase activity is required to obtain an immortal stage of these cells.

It remained to be demonstrated that this immortalization procedure maintains the initial phenotype of precrisis cells. Marrow stromal cells and osteoblast precursors are directly involved in the modeling and remodeling of periosteum and endosteum bone mineral mass (25) . Both cell types can differentiate into osteoblasts and support osteoclast differentiation. Saka cells were described previously to display this former characteristic (10) . hTERT gene expression in Saka-tert cells did not affect the capacity of these cells to enhance the formation of osteoclastic-like multinuclear cells. Saka-tert cells appear even to be more efficient than Saka cells to support osteoclast differentiation. This difference may be because of the increasing proportion of Saka cells entering in crisis and which, during passages, could express less factors involved in osteoclast differentiation such as the osteoclast differentiation factor.

As reported previously in other SV40 T-Ag-expressing human osteoblast cell lines (5, 6, 7, 8, 9, 10 , 26) , expression of this oncogene in hPOB cells did not affect their capacity to differentiate into osteoblast-like cells. Under osteogenic culture conditions [1,25-(OH)2D3 and Dex], we observed that ALP activity was stimulated with a similar magnitude in human precursor cells, prepared from periost (3.4-fold increase) or from trabecular bone (1.5-fold increase; Ref. 27 ) than in hPOB cells (2.4-fold increase). Siggelkow et al. (27) showed a 50-fold increase of osteocalcin RNA induction by 1,25-(OH)2D3 in trabecular bone cells. The lower induction of this gene observed in hPOB cells (4.3-fold increase) might be related to a difference of kinetic of differentiation because of the origin of the cells or the culture conditions. The two other osteoblast-specific markers measured in this study, osteonectin and Cbfa-1, were detected in differentiated hPOB cells. Interestingly, their expression was not or slightly affected during differentiation. The constitutive expression of osteonectin RNA, with even a slight decline during hPOB cell maturation, is similar to what we observed in primary culture of periostal cells. This result is also in agreement with previous reports on primary human osteoblast precursors (27) and on human SV40 T-Ag-transformed bone cells (28) . The absence of clear induction of Cbfa-1 RNA in hPOB and hPOB-tert cells is in agreement with data showing a constitutive expression of this gene during differentiation of several T-Ag-transformed bone cells (28) .

Under the same culture conditions, hPOB-tert cells presented a very similar pattern of osteoblast markers expression than hPOB cells indicating that telomerase reconstitution did not affect the capacity of cell to differentiate into osteoblasts. Furthermore, visualization of calcium deposition showed that hPOB-tert cells, like hPOB cells, were able to mineralize. Altogether, these data contrast with the phenotypic instability usually observed during spontaneous emergence of cells from crisis (14 , 29) .

Nevertheless, to compare genomic stability between precrisis and immortalized cell lines, karyotyping analysis was performed in both hPOB and hPOB-tert cells. The results showed a doubling of the chromosome number in hPOB-tert cells at passage 39 (269 PD) as compared with hPOB cells at passage 10 (60 PD), with a majority of tetraploid cells. Similarly, an increase of marker chromosomes was observed in hPOB-tert cells. Because postcrisis hPOB cells were not obtained, direct comparison of chromosome integrity in both cell lines at high passages could not be performed. Therefore, multiplication of passage number rather than expression of hTERT could directly account for the increased chromosomal abnormalities observed in hPOB-tert versus hPOB cells. Although, to our knowledge, karyotyping analysis has never been reported in SV40 T-Ag-transformed human bone cells, increased number of both chromosomes and marker chromosomes is commonly observed in other types of cells expressing SV40 T-Ag (12, 13, 14 , 30, 31, 32, 33) . Indeed, SV40 T-Ag-transformed epithelial (passage 35) and uroepithelial cells (passage 50) escaped from crisis, presented 6 and 10 marker chromosomes, respectively, whereas untransformed cells had no chromosomal abnormalities (32 , 33) . In comparison with these previous reports, three to five times fewer marker chromosomes were observed in hPOB-tert cells at a similar passage (passage 39). Altogether these results tend to indicate that telomerase activity could limit rather than increase chromosome aberrations induced by SV40 T-Ag.

In conclusion, this study shows that reconstitution of telomerase activity in SV40 T-Ag-transformed human bone cells allowed them to overcome crisis and to become immortal. Despite altered chromosome numbers and the presence of few chromosome markers observed in the human periosteal model, these cells exhibit the main characteristics of precrisis cells. Associated with inactivation of p53 and retinoblastoma protein, telomere stabilization appears to be a powerful way to generate human bone cells with indefinite life span and preserved phenotype.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Materials.
Cell culture materials, medium, and FBS were purchased from Life Technologies, Inc. (Basel, Switzerland). Alizarin Red S and 1,25-(OH)2D3 were purchased from Sigma Chemical Co. (Buchs, Switzerland) and Calbiochem (Lucerne, Switzerland), respectively. Ascorbic acid and ß-glycerophosphate were obtained from Merck (Darmstadt, Germany).

Recombinant Virus Preparation.
A recombinant retroviral vector carrying the 3.4-kb hTERT cDNA or the 2.5-kb BglI/HpaI fragment of the SV40, which contains the large T- but not the small t-antigen gene, were constructed by insertion, with standard recombinant DNA techniques, into the BamHI site of the pLHXSD retroviral vector (34) . This vector contains the Moloney murine leukemia virus promoter controlling SV40 T-Ag or hTERT gene expression and the histidinol gene as selection marker. Infectious recombinant virus particles were generated through transfection of the recombinant retroviral vector into the amphotropic packaging cell line Phoenix (Clontech, Basel, Switzerland), followed by coculturing with the ecotropic packaging cell line, Psi2 (American Type Culture Collection), to allow "ping-pong" infection (35) .

Preparation and Infection of the Human Bone Cells.
For the establishment of hPOB cells, osteoblast precursors from the femoral periosteum of a 13-year-old female patient were prepared as described previously (36) . At 70–80% confluence, primary cells were incubated for 3 h at 37°C (90% humidity) with recombinant virus containing the SV40 T-Ag, prepared as described above, in the presence of 20 µg/ml DEAE-dextran. After infection, the culture medium was changed to {alpha}-MEM supplemented with 10% FBS and penicillin/streptomycin. The polyclonal population of SV40 T-Ag-expressing cells overcoming senescence were infected at passage 9, as described above, with recombinant virus carrying the hTERT gene. The same procedure was used for the infection of precrisis Saka cells with the hTERT gene.

Immunodetection of the SV40-T-Ag Protein in hPOB Cells.
Immunodetection of the T antigen in hPOB cells, grown at 90% confluence on eight-well chambered glass slides, was performed with a specific monoclonal antibody (Oncogene; 1:30 dilution), as described previously (37) .

TRAP Assay and TRF Analysis.
The TRAP protocol assay was performed on cell extracts as described previously (38) . PCR product was separated in a 10% acrylamide gel and visualized by SYBR gold gel staining (Molecular Probes, Basel, Switzerland) on a UV transilluminator.

The TRF analysis was performed according to the protocol described previously (38) . Telomere length was estimated by calculating the band size observed on the scanned TRF gel using a linear curve generated by regression analysis of molecular weight marker positions.

Karyotype and Isoenzyme Analysis.
Semiconfluent cultures were sent to the Cell Culture Laboratory (Children’s Hospital of Michigan, Detroit, Michigan) for karyotyping analysis. For the chromosome study, exponentially growing cultures were treated with 0.04 µg/ml of Colcemid for 1–2 h, trypsinized, treated with 37.5 mM of KCl for 9 min, and fixed in 3:1 methanol:glacial acetic acid mixture. The suspension was centrifuged and washed twice with fixative, and finally dropped on cold, wet slides as reported previously (39) . Slides were air dried and stained with 4% Giemsa solution. Giemsa-stained slides were used for ploidy distribution, counts, and constitutional aberrations. For trypsin Giemsa banding, karyotypes were prepared by a modified procedure of Seabright (40) . The slides were dried at 60°C on a slide warmer for 16–20 h, immersed in 0.025% trypsin for 1–2 s, stained with 4% Giemsa solution for 11 min, washed in buffer (phosphate buffered saline), and then dried and mounted in permount. Well-banded metaphases were karyotyped using the AKSII Image Analysis system. A minimum of seven karyotypes were prepared from these prints and arranged according to standard human karyotype. The karyotypes were described according to standard nomenclature. An isozyme analysis was carried out by methods described by Ottenbreit et al. (41) .

Proliferation and Differentiation of hPOB and hPOB-Tert Cells.
hPOB and hPOB-tert cells were cultured in {alpha}-MEM supplemented with 10% FBS and penicillin/streptomycin. This medium is referred to as the basal medium. The proliferation rate of hPOB, hPOB-tert, and Saka-tert cells was analyzed on cells seeded at 715 cells/cm2 and cultured in the basal medium. Cells were counted each day for 8 days. The number of PDs at every passage was calculated as ln N/ln 2 where N is the number of cells at the time of passage divided by the number of cells initially seeded.

To differentiate hPOB and hPOB-tert cells into osteoblasts, cells were seeded on collagen I (30 µg/ml; bovine skin-type I collagen; Roche Biomedical, Basel, Switzerland) -coated dishes at a density of 12,000 cells/cm2 in the basal medium. Confluent cells were incubated for 2–21 days in the basal medium supplemented with 1 mM of ß-glycerophosphate and 50 µg/ml ascorbate supplemented with 10 nM of Dex or 10 nM of 1,25-(OH)2D3.

Mineralized matrix formation was followed in cells cultured at day 0 and 21 after confluence under the differentiation conditions. After cell fixation by incubation with ice-cold 70% ethanol for 1 h, the mineralized matrix was stained by the Alzarin Red-S-based colorimetric reaction.

ALP Activity Measurement.
hPOB and hPOB-tert cells cultured under the differentiation conditions were harvested at days 0, 2, 4, 6, and 9 after confluence, and homogenized in a lysis buffer containing 10 mM Tris (pH 7.5), 0.5 mM MgCl2, and 0.1% Triton X-100. ALP activity was measured on cell homogenates using a commercially available kit (Sigma Chemical Co., Buchs, Switzerland), and the results were normalized to total protein content, as measured by the Bradford assay method.

RNA Preparation and Expression Analysis by RT-PCR.
At day 6 after confluence, hPOB and hPOB-tert cells cultured under the differentiation conditions were washed with HBSS and stored at -80°C. RNA was extracted using the RNeasy Total RNA Purification System (Qiagen AG, Basel, Switzerland).

Reverse transcription was performed with an input of 10 µg of total RNA using the 1st strand cDNA synthesis kit for RT-PCR (AMV; Roche Biomedical, Basel, Switzerland) with oligo(dT)15 as primer. Primers used for the amplification of cDNAs of interest were synthesized by Mycrosynth (Windisch, Switzerland). The sequences of the forward and reverse primers were, respectively: 5'-GTTGCTATCCAGGCTGTG-3' and 5'-CATAGTCCGCCTAGAAAGC-3' for the actin gene; 5'-ATGAGAGCCCTCACACTCCT-3' and 5'-GATGTGGTCAGCCAACTCGT-3', for the osteocalcin gene; 5'-AGAGGTGGTGGAAGAAACTG-3' and 5'-GCTTCTGCTTCTCAGTCAGA-3' for the osteonectin gene; and 5'-CAGTGATTTAGGGCGCATTC-3' and 5'-GAAATGCGCCTAGGCACATC-3' for the Cbfa-1 gene. The PCR reaction was heated for 2 cycles to 98°C for 1 min, 60°C for 2 min, and 72°C for 2 min, and then cycled 28 times through a 1-min denaturation step at 94°C, a 1-min annealing step at 60°C, and a 2-min extension step at 72°C in a DNA thermal cycler apparatus (Bioconcept, Allschwill, Switzerland). Actin primers were included in the reaction as an internal control. PCR products (10 µl) were separated on a 2% agarose gel and visualized by ethidium bromide staining. Quantification of the PCR products was performed using the densitometric NIH Imager Program.

Detection of Osteoclast Differentiation.
Saka and Saka-tert cells were grown in {alpha}-MEM supplemented with 10% FBS and penicillin/streptomycin as described previously (10) . To detect the capacity of Saka and Saka-tert cells to support osteoclast differentiation, cells were cocultured in the presence of human bone marrow mononuclear cells without 1,25-(OH)2D3, as described previously (10) . After 3 weeks of coculture, the cells were fixed and stained with the 23c6 antibody to detect osteoclast-like cells, as reported previously (10) .

Statistical Analysis.
All of the statistical analysis were performed on absolute values using the two-tailed paired t test.


    Acknowledgments
 
We thank Dr. R. Reddel (Children’s Medical Research Institute, Westmead, Australia) and E. Federici (Nestlé Research Center, Lausanne, Switzerland) for helpful discussions, suggestions, and encouragement.


    Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 To whom requests for reprints should be addressed, at Nestlé Research Center, Vers-Chez-Les-Blanc, 1000 Lausanne 26, Switzerland. Phone: 41-21-785-84-02; Fax: 41-21-785-85-44; E-mail: catherine.mace{at}rdls.nestle.com Back

2 The abbreviations used are: SV40 T-Ag, SV40 T antigen; hTERT, human telomerase reverse transcriptase; PD, population doubling; Dex, dexamethazone; 1,25-(OH)2D3, 1,25-dihydroxyvitamin D3; FBS, fetal bovine serum; TRAP, telomerase repeat amplification; TRF, terminal restriction fragment; ALP, alkaline phosphatase; RT-PCR, reverse transcription-PCR. Back

Received for publication 8/28/01. Revision received 11/ 8/01. Accepted for publication 12/17/01.


    References
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

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