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Cell Growth & Differentiation Vol. 12, 233-242, May 2001
© 2001 American Association for Cancer Research

Preservation of the Myofibroblastic Phenotype of Human Papilloma Virus 16 E6/E7 Immortalized Human Bone Marrow Cells Using the Lineage Limited {alpha}-Smooth Muscle Actin Promoter1

Corinne Loeuillet2,, 3, Luc Douay, Patrick Hervé and David E. Chalmers

EFS de Bourgogne-Franche Comté, Besançon 25000, France [C. L., P. H., D. E. C.], and INSERM U417, Hôpitaux St Antoine et Trousseau, Paris 75012, France [L. D.]


   Abstract
TOP
 Abstract
Introduction
 Results
 Discussion
 Materials and Methods
 References
 
The in vitro study of mammalian hematopoiesis is hindered by the lack of immortalized human stromal cell lines that support hematopoiesis. We have immortalized human stromal vascular smooth muscle cells characterized by the expression of the {alpha}-smooth muscle ({alpha}-SM) actin. This marker is usually down-regulated as a result of oncogenic transformation. To correct this dedifferentiation, we placed the expression of human papilloma virus 16 E6/E7 oncogenes under the control of the tissue-specific {alpha}-SM actin promoter. The immortalization event is rare and requires polyclonal culture, but the corresponding established line retains {alpha}-SM actin expression. Moreover, when compared with other lines derived from the same cells from vectors made with the same oncogenes but driven by either an internal SV40 promoter or the viral long terminal repeat, this line is less transformed as shown by anchorage-independent growth assay. We show therefore that the use of a physiological promoter allows the production of human cell lines with a conserved phenotype.


   Introduction
TOP
 Abstract
 Introduction
Results
 Discussion
 Materials and Methods
 References
 
The influence of the stromal microenvironment on hematopoiesis is usually studied in vitro using a LTBMC4 system (1 , 2) . The adherent layer is comprised mainly of myofibroblastic cells that differentiate along a vascular smooth muscle pathway characterized by the expression of the {alpha}-SM actin gene (3) . This feeder layer supports the expansion and differentiation of various blood cell lineages, depending on the culture conditions used (4) .

However, these layers are complex, short lived in culture, and difficult to expand; they vary considerably between samples and are virtually impossible to freeze and thaw. Therefore many groups have tried to establish supportive stromal cell lines to perform reproducible studies (for review see, Ref. 5 ). Due to the ease of transforming rodent cells, many murine stromal lines are available, and some, such as MS5, maintain human hematopoiesis (6) . This latter approach is clearly limited, and species conformity is an essential requirement of cellular interaction studies. For example, CD34 is clearly a key molecule in hematopoiesis, yet the murine thymocytes that express it adhere to human stromal cells but not to layers from their own species (7) . Furthermore, the use of murine stromal cell lines to study human hematopoiesis often requires the addition of human cytokines (8) . Moreover, the description of the establishment of human stromal cell lines remains scant. Historically, human cell lines and, in particular, that of bone marrow have been difficult to establish compared with murine cells due to the different mechanistic immortalization stages that have to be overcome between the two species (for review see, Ref. 9 ).

The greatest success in terms of transformation has been achieved using combinations of highly disruptive oncogenes such as large T and small t of the SV40 virus. However, these cell lines usually fail to support hematopoiesis (10, 11, 12, 13) . On the other hand, one report has shown that it is possible to produce human stromal cell lines that are both supportive and phenotypically less altered using the HPV-16 E6/E7 oncogenes controlled by a LTR promoter (14) . Such functional preservation was also observed in other cell types such as aortic smooth muscle, vascular endothelial cells, and human corneal fibroblasts immortalized by E6 and E7 proteins (15, 16, 17) . For this reason, we chose to use these oncogenes, despite the fact that immortalization occurs by the same mechanism, i.e., the E6 and E7 proteins bind, like the SV40 large T and small t, respectively, to the tumor suppressor transcription factor p53 and the retinoblastoma gene product pRB (18 , 19) . These interactions lead to the degradation of the tumor suppressor proteins via the ubiquitin-proteasome pathway, provoking cell cycle progression that favors the passage through the first stage of mortality (for review see, Ref. 9 ). Other modifications in human cells, such as telomerase reactivation, are required for crossing the subsequent mortality stages and reaching complete immortalization (for review see, Ref. 20 ).

Simmons and Torok-Storb (21) have shown that hematopoiesis is preferentially supported by a subpopulation of stromal cells that stains with the Stro1 antibody. Therefore, we targeted our different retroviruses to stromal cells separated by this antibody. Three promoters were used for controlling oncogene expression: a retroviral LTR or internal promoters SV40 or tissue-specific {alpha}-SM actin. We hypothesized that the use of the {alpha}-SM actin promoter might result in continuous expression of the corresponding protein, which has been previously demonstrated to be down-regulated in ras- or E7-transfected cells (22 , 23) .

All constructs resulted in the immortalization of a human bone marrow cell population. We analyzed the influence of the promoter type on cell growth and transformation. When the LTR promoter drove the oncogene expression, immortalization always occurred; however, when oncogene expression was driven by an internal promoter, immortalization was infrequent and dependent on the culture conditions. The highest level of {alpha}-SM actin expression was observed in cells immortalized by using the {alpha}-SM actin promoter to express the oncogenes. Moreover, using this promoter, the phenotype of the resulting line was more conserved, especially in comparison with those produced by the LTR-E6/E7 combination. This study therefore raises the possibility of using alternative lineage limited promoters to immortalize specific cell types that retain many characteristics of their normal counterparts.


   Results
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 Abstract
 Introduction
 Results
Discussion
 Materials and Methods
 References
 
Retroviral Vectors Used for Cell Immortalization.
The retroviral vectors were based on the pMP1N and pSF1N constructs as described in "Materials and Methods." Internal SV40 or {alpha}-SM actin promoters and a MCS were added before the cloning of the E6/E7 genes. Vectors pMP/SV40/E6/E7, pMP/{alpha}SM/E6/E7, and pSF/LTR/E6/E7 are shown in Fig. 1Citation . Infected cells were selected in G418 using the neor gene, and oncogene expression was confirmed by IF.



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  		Fig. 1. Retroviral vectors used for cell immortalization. Proviral forms after genomic integration are shown. The E6/E7 genes were cloned between a 5' HindIII site and a 3' XhoI site. A, E6/E7 expression is driven by the SV40 internal promoter. A tissue-specific internal promoter of the aortic {alpha}-SM actin gene (B) or under the retroviral promoter LTR (C) is shown. The neor gene expression was placed under the retroviral promoter or under the internal promoter.

 
Generation of Stromal Cell Lines.
pStro1+ layers were infected with viral supernatants from {Psi}CRIP/pMP/{alpha}SM/E6/E7, {Psi}CRIP/pMP/SV40/E6/E7, or {Psi}CRIP/pSF/LTR/E6/E7 cells (Table 1)Citation . Noninfected pStro1+-derived cultures were passaged only four times before senescence. Only NCCs were immortalized when the pMP/SV40/E6/E7 corresponding viral supernatant was used. These NCCs reached 97 passages in 16 months for the first layer infected (derived from the cells of a 56-year-old patient; BM 56 years) and 91 passages after 1 year for the second layer infected (derived from the cells of a 9-year-old patient; BM 9 years). Among the 133 clones isolated from 3 experiments, only 1 reached 30 passages and then entered senescence (as determined by ß-galactosidase staining; data not shown). The other clones did not survive after eight passages (Table 1)Citation .


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  		Table 1 Establishment of human bone marrow stromal lines

 
As with pMP/SV40/E6/E7, only NCCs were immortalized when the pMP/{alpha}SM/E6/E7 construction was used. For the first layer (BM 56 years), no clone was isolated from the culture. However, this bulk culture has reached 100 passages. The NCC of the second infection (BM 9 years) was passaged 11 times before senescence. Among the 101 clones isolated from 3 separate infections, no clone has survived >9 passages. The BM 9 years was also infected with the pSF/LTR/E6/E7 construction. With this construct, both clones and NCCs were immortalized. Thirty-six clones reached 40 passages, on average, after 5 months. Four clones were kept in culture for 14 months and passaged approximately 80 times (Table 1)Citation . The others were cryopreserved. Simultaneously, a NCC continues to proliferate after more than 110 passages.

We also infected pStro1+ cells derived from two other adult bone marrows (BM 27 years and BM 46 years) and one fetal bone marrow with the three constructs. All of the nonclonal layers grew independently of the promoter used. They have been frozen between passages 15 and 43. Notably, the LTR/E6/E7-derived cultures expanded rapidly.

Differentiation markers ({alpha}-SM actin, fibronectin, laminin, vimentin, and collagen IV) have been examined in the nonclonal lines: the SV40 and {alpha}-SM of the BM 56 (subsequently called SV40-56 and {alpha}SM-56) and the LTR of BM 9 (LTR-9). All studies were performed on cultures after 100 passages. Early passages (<30) were also used for RT-PCR studies, telomerase detection, oncogene and {alpha}-SM actin expression by IF.

Genomic Integration of Retroviral DNA, E6/E7 Expression, and Protein Localization.
HPV-16 E6/E7 vector sequences were detected in the infected cells by Southern blotting at late passage. Single integration was observed in each case showing that bulk cultures became clonal.

The E6 and E7 oncogenes were expressed in both early and late passage cell lines as determined by RT-PCR (Fig. 2)Citation . We observed only one product after RT-PCR, corresponding to an unspliced message of both E6 and E7 genes. Splicing from this type of construct was less efficient than from other retroviral vectors.5



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  		Fig. 2. HPV-16 E6/E7 expression. RT-PCR results are shown for late passage cell lines; a similar profile was detected in early passage cells. A 1-kb ladder (A) and cDNA originating in {alpha}SM-56 cells (B), SV40-56 cells (C), LTR-9 cells (D), pStro1+ nonimmortalized cells (E), and plasmid control (F) are shown. The expected band of 748 bp was detected in all cell lines.

 
The E6 oncoprotein was found in both a cytoplasmic and a perinuclear location for the SV40-56 and LTR-9 lines at early passage by IF (Fig. 3ACitation , SV40-56). E6 and {alpha}-SM actin were coexpressed in the {alpha}SM-56 line at an early passage (Fig. 3, C and DCitation , respectively). The E7 oncoprotein was also observed in the cytoplasm of all lines at an early passage (Fig. 3ECitation , SV40-56). No oncogene expression was found in pStro1+ cells (data not shown). The {alpha}-SM actin formed a network of thin filaments in pStro1+ cells (Fig. 3G)Citation , as it did in an early passage line (Fig. 3DCitation , {alpha}SM-56).



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  		Fig. 3. Oncoprotein localization. Confluent layers were fixed with methanol and incubated with the appropriate antibody (see "Materials and Methods"). A and B, SV40-56/E6 label (x160) and the corresponding phase-contrast; C and D, {alpha}SM-56 line E6 label (x400) and the corresponding {alpha}-SM actin label; E and F, SV40-56 line E7 label (x100) and the corresponding phase-contrast; G and H, pStro1+ cells/{alpha}-SM actin label (x250) and the corresponding phase-contrast.

 
Telomerase Expression Was Detected in the E6/E7 Lines.
Telomerase activity was analyzed in all three cultures at late passage. There were 6–10 bands in established cell lines, corresponding to the presence of 9–13 telomere repeats (Fig. 4Citation , Lanes E, G, and I). In contrast, no activity was detected in early passages (Fig. 4Citation , Lanes D, F, and H) or in pStro1+ cells (Fig. 4Citation , Lane B) or in heat-inactivated cell line extracts (data not shown).



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  		Fig. 4. Telomerase expression. Lane A, TSR8 quantitative control; Lane B, pStro1+ cells; Lane C, telomerase-positive control; Lane D, LTR-9 line P18; Lane E, LTR-9 line P115; Lane F, SV40–56 line P17; Lane G, SV40-56 line P105; Lane H, {alpha}SM-56 line P30; Lane I, {alpha}SM-56 line P105. These experiments were performed three times with similar results. A representative gel is shown. pStro1+ nontransformed cells were used as a negative control (B), as well as the heat-treated extract (data not shown).

 
Anchorage-independent Growth of the E6/E7 Lines.
Because transformed cells grow in an anchorage-independent manner, we used this parameter as an indicator of transformation. Uninfected pStro1+ cells did not form colonies in soft agar, in contrast to established virally transduced lines. The number and diameter of the colonies were used to estimate the degree of transformation (Table 2)Citation . {alpha}SM-56 formed fewer and smaller (diameters of 100 or 250 µm) colonies than SV40-56 (P < 0.01 and P < 0.05, respectively) and fewer 100-µm-diameter colonies than LTR-9 (P < 0.01). The total colony number was similar for the SV40-56 and the LTR-9 cells, but only the LTR-9 cells produced large (500-µm-diameter) colonies (P < 0.05, compared with {alpha}SM-56 and SV40-56).


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  		Table 2 Number of colonies in soft agar

 
The Myofibroblastic Phenotype Is More Preserved in the {alpha}SM-56 Line.
{alpha}-SM actin level expression, determined by flow cytometry at late passage, was higher in {alpha}SM-56 (53.8± 10.5%) than in SV40-56 (27.9± 6.4%; P < 0.05) or LTR-9 (15.6± 2.9%; P < 0.01) cells. {alpha}-SM actin was detected in 69.3± 10.1% of pStro1+ cells after 14 days in culture.

As seen for {alpha}-SM actin, a dense vimentin network filled the cytoplasm of primary cells (Fig. 5A)Citation and lines (Fig. 5BCitation , {alpha}SM-56 cell line; Fig. 5CCitation , SV40-56 cell line; Fig. 5DCitation , LTR-9 cell line). The network was mainly perinuclear for the {alpha}SM-56 line (Fig. 5B)Citation and for the LTR-9 line (Fig. 5D)Citation .



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  		Fig. 5. IF studies of cytoskeletal and extracellular matrix proteins. Vimentin staining in the nontransformed pStro1+ cells (A, x250), the {alpha}SM-56 line (B, x200), the SV40-56 line (C, x200), and the LTR-9 line (D, x200) is shown. Fibronectin staining in the nontransformed pStro1+ cells (E, x200), the {alpha}SM-56 line (F, x800), the SV40–56 line (G, x800), and the LTR-9 line (H, x250) is shown. Laminin staining in the nontransformed pStro1+ cells (I, x250), the {alpha}SM-56 line (J, x200), the SV40-56 line (K, x200), and the LTR-9 line (D, x800) is shown. Collagen IV staining in the nontransformed pStro1+ cells (M, x300), the {alpha}SM-56 line (N, x200), the SV40-56 line (O, x300), and the LTR-9 line (P, x800) is shown.

 


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  		Fig. 5A. Continued.

 
The fibronectin was organized as a filamentous envelope around the pStro1+ cells (Fig. 5E)Citation . The fibers followed the cell surface and formed prolongations throughout the cell. The {alpha}SM-56 cells are ensheathed in a very similar fibronectin matrix but with a slight decrease in fiber density (Fig. 5F)Citation , as seen in the SV40-56 cell line (Fig. 5G)Citation . LTR-9 cells have a significantly less dense network as compared with primary cells and SV40-56 cells (Fig. 5H)Citation .

The primary cells contained abundant cytoplasmic laminin that is also visible at the cell surface (Fig. 5I)Citation . The adipocytic-like cells within the culture were more intensively stained for both locations. The {alpha}SM-56 (Fig. 5J)Citation and SV40-56 (Fig. 5K)Citation cells contained similar amounts of laminin, but slightly less than that seen in primary cells, and some fibrils were also detected in the extracellular compartment between cells. Only intracellular laminin was observed for LTR-9 cells (Fig. 5L)Citation .

Collagen IV was assembled as a veil covering the primary cells and formed folds in some places (Fig. 5M)Citation . A similar observation was made for the {alpha}SM-56 cells (Fig. 5N)Citation . Collagen IV only formed deposits where SV40-56 cell density was maximal (Fig. 5O)Citation ; very little collagen IV was seen in LTR-9 cells, in which it was only visible at the cell border (Fig. 5P)Citation .


   Discussion
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 Abstract
 Introduction
 Results
 Discussion
Materials and Methods
 References
 
Little is known about the interactions between the human stromal microenvironment and hematopoietic stem cells that expand and differentiate during normal hematopoiesis or during reconstitution of the bone marrow after transplantation. This gap in our knowledge is due in part to the complexity of the process and in part to the lack of human stromal cell lines available that still bear some phenotypic resemblance to their normal nonimmortalized counterparts. Such cell lines would enable a species-specific reproducible analysis of the process to be carried out.

In this report, we describe the use of the tissue-specific {alpha}-SM actin promoter in combination with the HPV-16 E6/E7 oncogenes that results in a stromal cell line with a quite well preserved phenotype. We adopted this approach because (a) there have been several reports that the use of these oncogenes can immortalize certain cell types that retain most of their original phenotype (15, 16, 17) and (b) oncogenes such as ras and E7 itself have been shown down-regulate the {alpha}-SM actin promoter (22 , 23) . This may be detrimental when trying to preserve a functional phenotype because the {alpha}-SM actin protein is the major marker of stromal cell differentiation that follows a vascular smooth muscle pathway (24) . We reasoned that by placing the immortalizing agents in this situation, we could perhaps partially uncouple the immortalization/differentiation pathways because only cells with an active {alpha}-SM actin promoter should escape senescence and become immortal. Our strategy would thus create a loop in which immortalized cells retain {alpha}-SM actin expression and, we hope, a conserved phenotype. We have shown the feasibility of this approach by comparing this type of retroviral construct with more conventional vectors that express the oncogenes from either an internal broad-range SV40 promoter or directly from the LTR.

When we placed the E6/E7 oncogenes immediately downstream of the strong retroviral LTR promoter, we were able to directly clone immortalized stromal lines after infection. Very few of these lines actually undergo senescence, indicating the efficacy of this type of vector. These cells are characterized by very rapid growth and by their ability to form numerous large colonies in soft agar. They show a reduced expression of {alpha}-SM actin, and expression of other proteins (fibronectin, collagen IV, and laminin) is also disturbed.

In contrast, when we used the {alpha}-SM actin promoter, we were not able to directly clone cell lines despite successful infection. We always obtained cell expansion compared to uninfected controls, but only the lines that were kept as a nonclonal culture were able to bypass senescence. These cells formed slow-growing colonies in soft agar, reflecting a less "aggressive" transformation. Therefore, polyclonal culture conditions seemed necessary here to bypass the M1 stage of immortalization (for review, see Ref. 9 ). We can hypothesize that this may be due to the influence of paracrine growth factors and adherence molecules differentially expressed by complementary clones. This may allow favorable conditions for initial viability during which other mutational events occur that eventually lead to the emergence of a single dominant clone. Certain mutational events are shared by all cell lines that emerged from our experiments. For example, telomerase was not expressed in any early passage cells but was reactivated in all cultures that became immortal, in keeping with other reports (25) . Despite this, the line produced using this {alpha}-SM actin promoter retains a significant level of {alpha}-SM actin expression, and the expression of other proteins such as vimentin, fibronectin, laminin, and collagen IV is less disturbed.

Intermediate results between the LTR and the {alpha}-SM actin promoters were obtained using the SV40 internal promoter. Cells were only immortalized from polyclonal cultures, but these produced larger colonies in soft agar than did the {alpha}SM-56 cell line; the expression of {alpha}-SM actin was also reduced, and the network of fibronectin and collagen IV was disturbed.

Some reports indicate that certain human cell types may be immortalized by using the E6 or E7 gene alone (26 , 27) or by using the human telomerase catalytic component (28) . The latter, in particular, has been shown to be less disruptive than conventional oncogenes. We have tried these strategies on fetal and adult stromal cells without success (data not shown). One report has suggested that it may be necessary to use a combination of E7 and telomerase (29) .

This lack of success with human bone marrow stromal cells may also be due to their resistance to immortalization, as reflected by the paucity of functional human cell lines described in the literature. This can be overcome by our approach and by the sledgehammer approach of Foster and Galloway (30) of expressing E6 and E7 from the strong retroviral LTR promoter. We demonstrate that this disturbs the expression of proteins that characterize their normal counterparts. However, we also show that under certain conditions, it is possible to immortalize cells that retain a differentiated phenotype by using the {alpha}-SM actin promoter. These two lines both support hematopoiesis with different kinetics (31) and are superior to previously published human stromal cell lines (14) . We have also shown that they differ in their cytokine profiles, and this should allow for manipulation of the lines by introducing further constructs that express hematopoietic supporting cytokines. These results suggest that other tissue-specific promoters corresponding to protein down-regulated during immortalization of other cell types could be used in the same way.


   Materials and Methods
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 Abstract
 Introduction
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References
 
Plasmid Construction
Amplification of E6/E7 Genes.
The E6/E7 coding sequences were obtained by PCR amplification of DNA from plasmid pLXSN16/E6/E7 [provided by Dr. A. D. Galloway (Fred Hutchinson Cancer Center, Seattle, WA); Ref. 30 ] with the appropriate primers containing a HindIII extension on the sense primer (5'-AGG-AAG-CTT-ACC-GGT-TAG-TAT-AAA-AGC-3') and a XhoI extension on the antisense primer (5'-CCC-CTC-GAG-GGT-AGA-TTA-TGG-TTT-CTG-3') to facilitate cloning into retroviral vectors (italics indicate enzyme restriction sites). PCR was performed using 5 pg of the plasmid, 5.3 µM each primer (Eurogentec, Seraing, Belgium), and 12 units of Taq polymerase (Boehringer Mannheim, Mannheim, Germany). Forty cycles of 1 min at 94°C, 1 min at 55°C, and 1 min at 72°C were performed. The amplification product was visualized on a 0.8% agarose electrophoresis gel followed by ethidium bromide staining, and a 826-bp band was then purified by electroelution. DNA was digested by HindIII and XhoI, cloned into plasmid pBCSK+ (Stratagene, St. Quentin en Yuelines, France), and sequenced to verify the E6/E7 PCR product using an Abi Prism Dye Primer kit (Perkin-Elmer, Paris, France).

Construction of pMP/SV40/E6/E7.
pMP/SV40/E6/E7 was based on pMN1N vector [provided by Dr. C. Baum (Heinrich-Pette-Institut für Experimentele Virologie und Immunologie, Universität Hamburg, Hamburg, Germany); Ref. 32 )] carrying a neomycin resistance (neor) gene under the control of myeloproliferative sarcoma virus LTR. The SV40 promoter and the MCS were removed from pZeoSV plasmid (Invitrogen) by cutting with BamHI and PvuII. Vector pMP1N was cut by HindIII, klenowed to generate a compatible blunt end, and then cut by BamHI for ligation of the promoter and the MCS. The resulting vector backbone, pMP/SV40, was digested with HindIII and XhoI to clone the PCR product E6/E7 (Fig. 1A)Citation .

Construction of pMP/{alpha}SM/E6/E7.
The {alpha}-SM promoter (891 bp) was amplified from plasmid pHSMA891-CAT [provided by Dr. T. Miwa (Osaka University, Osaka, Japan); Ref. 33 ]. The primers included enzyme restriction site extensions: sense primer 5'-CCCGAATCCGAGACGAGA-3' contained a BamHI site; and the antisense primer 5'-TTTAAGCTTTGAAGGGTTATATAGCCC-3' contained a HindIII site (italics indicate enzyme restriction sites). PCR was performed as described previously. The PCR product was cloned into pBSCK+ and verified by sequencing. pMP/SV40 was digested with BamHI and HindIII to remove the SV40 promoter, which was then replaced by the {alpha}-SM promoter to generate pMP/{alpha}SM. This was then digested again by HindIII and XhoI for cloning of the E6/E7 genes (Fig. 1B)Citation .

Construction of pSF/LTR/E6/E7.
This vector was based on pSF1N vector (provided by Dr. C. Baum; Ref. 31 ), a vector similar to pMP1N vector but with a SFFVp LTR (polycythemic strain of the spleen focus-forming virus) controlling the neor gene after infection.

pSF1N was cut with NotI and BamHI to remove the neor gene. The insert was blunt-ended and cloned into the EcoRV site of pBCSK+ (Stratagene) to give pBC/neo.This vector was then digested with HindIII and EcoRI, and the neor gene was cloned into pZeo/SV to give pZeo/SV/neo.

This was then cut with BamHI and PvuII, and the neor cassette was cloned into pSF1N cut previously with HindIII, klenowed, and cut by BamHI to give pSF/neo/SV40/neo.The polylinker of pZeo/SV was amplified by PCR with the appropriate extension primers and then added to the preceding vector digested by NotI and BamHI, which resulted in a vector with a polylinker downstream of the LTR and the superfluous neor sequence removed.

This vector was then finally digested by HindIII and XhoI to add the E6/E7 genes to produce pSF/LTR/E6/E7 vector (Fig. 1C)Citation .

All constructs have the pCMV LTR [a derivative of the myeloproliferative sarcoma virus LTR passaged through the PCC4 cell line (34) ].

Virus-producing Cells
The {Psi}CRIP packaging cell line [provided by Dr. J-M. Heard (Pasteur Institut, Paris, France)] was maintained in DMEM supplemented with 10% newborn calf serum and a 1% penicillin-streptomycin solution (Life Technologies, Inc., Gaithersburg, MD). The different vectors were integrated into {Psi}CRIP cells by the calcium phosphate precipitation method (35) . The transfected cells were selected 48 h later in medium containing 0.4 mg/ml Geneticin (G418; Life Technologies, Inc.), and clones were picked using cloning cylinders and amplified. Producer clones were selected after titration on Mus dunni cells [provided by Dr. C. Bagnis, Institut Paoli-Calmettes, INSERM U119, Marseilles, France); Ref. 35 ]. Viral titers were in the range of 3 x 104 to 1 x 105 colony-forming unit/ml. The viral supernatants were filtered (0.45 µm, pore size) and stored at -80°C before stromal cell infection. No helper virus was detected using Mus dunni cells as targets (35) .

LTBMC
Sternal bone marrow aspirates were obtained from patients undergoing cardiac surgery and collected in heparinized glass bottles. Six-week-old fetal stromal cells were provided by the laboratory of Dr. B. Peault (INSERM U506, Villejuif, France). LTBMC and pStro1+ cell isolation was performed as described previously (21 , 36) .

Infection and Selection of Stromal Cells
After 7 days, adherent layers of pStro1+ cells were exposed to retroviral supernatant with Polybrene (8 µg/ml; Sigma Chemical Co., St. Louis, MO) for 16 h at 37°C. Normal growth medium was renewed for 8 h, and two subsequent identical steps were performed. The cultures were selected 48 h after the final infection in LTBMC medium containing 0.4 mg/ml G418.

After 12 days of selection, total polyclonal cultures were maintained, or clones were picked using standard tissue culture techniques and grown in LTBMC medium. After 20 passages, the cells were switched into LTBMC medium without basic fibroblast growth factor. The cells were always passaged at confluence.

The absence of replication-competent retrovirus was repeated as described above.

ß-Galactosidase Senescence Assay
Determination of senescent cells was done as described previously (37) .

E6/E7 Expression
Expression of the E6/E7 genes was demonstrated at the RNA level by RT-PCR. Polyadenylated RNAs were isolated using oligo-dT-coated beads (Dynabeads mRNA DIRECT kit; Dynal). Corresponding cDNA was obtained using Moloney murine leukemia virus reverse transcriptase (Promega, Madison, WI) at 42°C for 60 min, followed by 3 min at 95°C. RNA purity was verified in a Raf PCR as described previously (38) . Primers were 5'-ATG-CAC-CAA-AAG-AGA-ACT-GCA-3' (upstream) and 5'-CAC-ACA-ATT-CCT-AGT-GTG-CCC-3' (downstream). The PCR mixture contained 1 unit of Taq polymerase (Promega) and 50 pM each primer. Forty cycles of 1 min each at 94°C, 55°C, and 72°C were performed. The amplification product was visualized on a 0.8% agarose ethidium bromide-stained electrophoresis gel.

Protein expression was demonstrated using IF as described below.

Telomerase Expression
Telomerase expression was measured using the Oncor (Gaithersburg, MD) TRAPeze Telomerase Detection Kit according to the manufacturer’s instructions.

Soft Agar Assay
Cell (104) were plated in growth medium containing 0.3% (w/v) noble agar in a 60-mm-diameter Petri dish as described previously (39) . Only colonies greater than 100 µm in diameter were counted after 14 days. Values are expressed as the mean ± SE of three independent experiments. Statistically significant differences between series were assessed by ANOVA using the Statview software (Abacus Concepts, Berkeley, CA).

Determination of {alpha}-SM Actin Expression by Flow Cytometry
Cell suspensions of pStro1+ and lines were washed twice in PBS (Life Technologies, Inc.), pelleted, and fixed in 1% (v/v) formaldehyde in PBS (30 min, 37°C; Sigma Chemical Co.). After centrifugation, cells were resuspended and permeablized with 0.5% (v/v) Triton in PBS for 5 min at room temperature (Sigma Chemical Co.). After two washes in PBS, the cells were resuspended in PBS with 0.5% (w/v) BSA (BSA; Sigma) and incubated with a monoclonal anti-{alpha}SM actin antibody (1:25; Dako, Glostrup, Denmark) or a nonspecific isotype for 30 min at 4°C. After two washes in PBS, cells were incubated with the secondary antibody (30 min, 4°C). Cells were analyzed using a FACSort flow cytometer (Becton Dickinson); data acquisition and analysis were performed with Cell Quest software for 10,000 events. Positive events were counted for a fluorescence peak superior to that of isotypic control. The mean fluorescence intensity ratio (antigen:isotype) was always greater than 2. {alpha}-SM actin was also visualized by IF.

IF Studies
Confluent adherent layers were treated with trypsin and seeded at 5 x 103 cells/well in a Labtek culture chamber (8-well permanox slides; Nunc, IL) for cell lines and at 1 x 104 cells/well for the control pStro1+ cells. At confluence, cells were washed once and fixed for 30 min at 4°C with 3.7% (v/v) formaldehyde in PBS for extracellular or plasma membrane labeling or permeabilized and fixed using cold methanol for intracellular labeling. The primary [polyclonal rabbit anti-E6 (Transgene, Strasbourg, France); MoAb anti-E7, 8C9 (Zymed, San Francisco, CA); MoAb anti-{alpha}SM actin, 1A4 (Dako); polyclonal rabbit anti-laminin (Pasteur Institut); MoAb anti-vimentin, VIM 13.2 (Sigma Chemical Co.); and polyclonal goat anti-collagen IV (Southern Biotechnology Associates, Birmingham, AL)] and secondary antibodies (antimouse polyvalent IgG-FITC conjugate and antirabbit IgG FITC conjugate, both from Sigma Chemical Co.) were incubated for 30 min at room temperature, and each step was followed by two PBS washes. Irrelevant antibody was used as a control. Slides were examined with an Aristoplan microscope (Wild Leitz, Wetzlar, Germany).


   Acknowledgments
 
We thank Dr. A. D. Galloway for providing the pLXSN16/E6/E7 vector, Dr. C. Baum for the pMN1N and pSF1N vectors, Dr. T. Miwa for the pHSMA891-CAT construct, Dr. J-M. Heard for the {Psi}CRIP packaging cell line, Dr. C. Bagnis for the Mus dunni cells, and Dr. C. Harley (Geron Corp.) for human telomerase reverse transcriptase.


   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 Supported by Association pour la Recherche contre le Cancer Grant 9217 (to D. E. C.). Back

2 C. L. was supported by a grant from the educational ministry of France. Back

3 To whom requests for reprints should be addressed, at EFS de Bourgogne-Franche Comté, Laboratoire d’étude de l’hématopoïèse, 1, Bd A. Fleming BP 1937, 25020 Besançon Cedex, France. Phone: 33-381-615-615; Fax: 33-381-615-617; E-mail: c.loeuillet{at}voila.fr Back

4 The abbreviations used are: LTBMC, long-term bone marrow culture; {alpha}-SM, {alpha}-smooth muscle; HPV, human papilloma virus; LTR, long terminal repeat; MCS, multiple cloning site; pStro1+, primary Stro1+; NCC, nonclonal culture; RT-PCR, reverse transcription-PCR; IF, immunofluorescence; MoAb, monoclonal antibody. Back

5 D. E. Chalmers, C. Ferrand, I. Newton, S. Ebling, A. Haagnbeck, P. T. Tiberghien, J. A. Apperley, J. V. Melo, E. Garrett, and M. Garin. Elimination of the truncated message from the human HSV-Tk suicide gene, submitted for publication. Back

Received for publication 12/ 4/00. Revision received 4/ 2/01. Accepted for publication 4/ 9/01.


   References
TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

  1. Dexter T. M., Allen T. G., Lajtha L. G. Conditions controlling the proliferation of hemopoietic stem cells in vitro. J. Cell. Physiol., 91: 335-344, 1976.
  2. Gartner S., Kaplan H. S. Long term culture of human bone marrow cells. Proc. Natl. Acad. Sci. USA, 77: 4756-4759, 1980.[Abstract/Free Full Text]
  3. Galmiche M. C., Koteliansky V. E., Brère J., Hervé P., Charbord P. Stromal cells from human long-term marrow cultures are mesenchymal cells that differentiate following a vascular smooth muscle differentiate pathway. Blood, 82: 66-76, 1993.[Abstract/Free Full Text]
  4. Novotny J. R., Duehersen U., Welch K., Layton J. E., Cebon J. S., Boyd A. W. Cloned stromal cell lines derived from human Whitlock/Witte-type long-term bone marrow culture. Exp. Hematol., 18: 775-784, 1990.[Medline]
  5. Deryugina E. I., Müller-Sieburg C. E. Stromal cells in long-term culture: keys to the elucidation of hematopoietic development?. Crit. Rev. Immunol., 13: 115-150, 1993.[Medline]
  6. Isaad C., Croisille L., Katz A., Vainchenker W., Coulombel L. A murine stromal cell line allows the proliferation of very primitive human CD34++/CD38- progenitor cells in long-term cultures and semisolid assay. Blood, 81: 2916-2924, 1993.[Abstract/Free Full Text]
  7. Krause D. S., Fackler M. J., Civin C. I., May W. S. CD34: structure, biology, and clinical utility. Blood, 87: 1-13, 1996.[Free Full Text]
  8. Tsuji T., Nishimura-Morita Y., Watanabe Y., Hirano D., Nakanishi S., Mori K. J., Yatsunami K. A murine stromal cell line promotes the expansion of CD34high+-primitive progenitor cells isolated from human umbilical cord blood in combination with human cytokines. Growth Factors, 16: 225-240, 1999.[Medline]
  9. Shay J. W., Woodring E., Werbin H. Defining the molecular mechanisms of human cell immortalization. Biochim. Biophys. Acta, 1072: 1-7, 1991.[Medline]
  10. Singer J. W., Charbord P., Keating A., Nemuniatis J., Raugi G., Wight T. N., Lopez A. J., Roth G. J., Dow L. W., Fialkow P. J. Simian virus 40-transformed adherent cells from human long-term marrow cultures: cloned cell lines produced cells with stromal and haemopoietic characteristics. Blood, 70: 464-474, 1987.[Abstract/Free Full Text]
  11. Thalmeier K., Meibner P., Reisbach G., Falk M., Brechtel A., Dörmer P. Establishment of two permanent human bone marrow stromal cell lines with long-term post irradiation feeder capacity. Blood, 83: 1799-1807, 1994.[Abstract/Free Full Text]
  12. Paul S. R., Yang Y-C., Donahue R. E., Goldring S., Williams D. A. Stromal cell-associated hematopoiesis: immortalization and characterization of a primate bone marrow-derived stromal cell line. Blood, 77: 1723-1733, 1991.[Abstract/Free Full Text]
  13. Cicuttini F. M., Martin M., Salvaris E., Ashman L., Begley C. G., Novotny J., Maher D., Boyd A. W. Support of human cord blood progenitor cells on human stromal cell lines transformed by SV40 large T antigen under the influence of an inducible (metallothionein) promoter. Blood, 80: 102-112, 1992.[Abstract/Free Full Text]
  14. Roecklein B. A., Torok-Storb B. Functionally distinct human marrow stromal cell lines immortalized by transduction with the human papilloma virus E6/E7 genes. Blood, 85: 997-1005, 1995.[Abstract/Free Full Text]
  15. Perez-Reyes N., Halbert C. L., Smith P. P., Benditt E. P., McDougall J. K. Immortalization of primary human smooth muscle cells. Proc. Natl. Acad. Sci. USA, 89: 1224-1228, 1992.[Abstract/Free Full Text]
  16. Fontjin R., Hop C., Brinkman H-J., Slater R., Westerveld A., van Mourik J. A., Pannekoek H. Maintenance of vascular endothelial cell-specific properties after immortalization with an amphotropic replication-deficient retrovirus containing human papilloma virus 16 E6/E7 DNA. Exp. Cell Res., 216: 199-207, 1995.[Medline]
  17. Peters D. M., Dowd N., Brandt C., Compton T. Human papilloma virus E6/E7 genes can expand the lifespan of human corneal fibroblasts. In Vitro Cell Dev. Biol., 33: 279-284, 1996.
  18. Werness B. A., Levine A. J., Howley P. M. Association of human papillomavirus types 16 and 18 E6 proteins with p53. Science (Wash. DC), 248: 76-79, 1990.[Abstract/Free Full Text]
  19. Dyson N., Howley P. M., Münger K., Harlow E. The human papilloma virus-16 E7 oncoprotein is able to bind to the retinoblastoma gene product. Science (Wash. DC), 243: 934-936, 1989.[Abstract/Free Full Text]
  20. Yeager T. R., Reddel R. R. Constructing immortalized human cell lines. Curr. Opin. Biotechnol., 10: 465-469, 1999.[Medline]
  21. Simmons P. J., Torok-Storb Identification of stromal cell precursors in human bone marrow by a novel monoclonal antibody, STRO-1. Blood, 78: 55-62, 1991.[Abstract/Free Full Text]
  22. Buschel P., Kim J. H., Chang W., Catino J. J., Ruley H. E., Kumar C. C. Two serum response elements mediate transcriptional repression of human smooth muscle {alpha}-actin promoter in ras-transformed cells. Oncogene, 10: 1361-1370, 1995.[Medline]
  23. Nishida M., Miyamoto S., Kato H., Miwa T., Imamura T., Miwa K., Yasumoto S., Barrett J. C., Wake N. Transcriptional repression of smooth-muscle {alpha}-actin gene association with human papillomavirus type 16 E7 expression. Mol. Carcinog., 13: 157-165, 1995.[Medline]
  24. Owens G. K. Regulation of differentiation of vascular smooth muscle cells. Physiol. Rev., 75: 487-517, 1995.[Abstract/Free Full Text]
  25. Steenbergen R., Walboomers J., Meijer C., Van der Raaji-Helmer E., Parker J., Chow L., Broker T., Snijders P. Transition of human papillomavirus type 16 and 18 transfected human foreskin keratinocytes towards immortality: activation of telomerase and allele losses at 3p, 10p, 11q and/or 18q. Oncogene, 13: 1249-1257, 1996.[Medline]
  26. Wazer D. E., Liu X-L., Chu Q., Gao Q., Band V. Immortalization of distinct human mammary epithelial cell types by human papilloma virus 16 E6 or E7. Proc. Natl. Acad. Sci. USA, 92: 3687-3691, 1995.[Abstract/Free Full Text]
  27. Halbert C. L., Demers G. W., Galloway D. A. The E7 gene of human papillomavirus type 16 is sufficient for immortalization of human epithelial cells. J. Virol., 65: 473-478, 1991.[Abstract/Free Full Text]
  28. Morales C. P., Holt S. E., Ouellette M., Kaur K. J., Yan Y., Wilson K. S., White M. A., Wright W. E., Shay J. W. Absence of cancer-associated changes in human fibroblasts immortalized with telomerase. Nat. Genet., 21: 115-118, 1999.[Medline]
  29. Kiyono T., Foster S. A., Koop J. I., McDougall J. K., Galloway D. A., Klingelhultz A. J. Both Rb/p16INK4a inactivation and telomerase activity are required to immortalize human epithelial cells. Nature (Lond.), 396: 84-88, 1998.[Medline]
  30. Foster A. S., Galloway A. D. Human papillomavirus type 16 E7 alleviates a proliferation block in early passage human mammary epithelial cells. Oncogene, 12: 1773-1779, 1996.[Medline]
  31. Loeuillet, C., Bernard, G., Rémy-Martin, J-P., Saas, P., Hervé, P., Douay, L., and Chalmers, D. Distinct hematopoietic support by two human stromal cell lines. Exp. Hematol. in press, 2001.
  32. Eckert H-G., Stockschläder M., Just U., Hegewish-Becker S., Grez M., Uhde A., Zander A., Ostertag W., Baum C. High-dose multidrug resistance in primary human hematopoietic progenitor cells transduced with optimized retroviral vectors. Blood, 88: 3407-3415, 1996.[Abstract/Free Full Text]
  33. Nakano Y., Nishihar T., Sasayama S., Miwa T., Kamada S., Kakunaga T. Transcriptional regulatory elements in the 5' upstream and first intron regions of the human smooth muscle (aortic type) {alpha}-actin-encoding gene. Gene (Amst.), 99: 285-289, 1991.[Medline]
  34. Baum C., Hegewisch-Becker S., Eckert H-G., Stocking C., Ostertag W. Novel retroviral vectors for efficient expression of the multidrug resistance (mdr-1) gene in early hematopoietic cells. J. Virol., 69: 7541-7547, 1995.[Abstract]
  35. Riviere I., Sadelain M. Methods for construction of retroviral vectors and the generation of high-titer producers Robbins P. D. eds. . Gene Therapy Protocols, 59-78, Humana Press Totowa, NJ 1997.
  36. Tamayo E., Charbord P., Li J., Herve P. A quantitative assay that evaluates the capacity of human stromal cells to support granulomonopoiesis in situ. Stem Cells (Basel), 12: 304-315, 1994.[Medline]
  37. Dimri G. P., Lee X., Basile G., Meileen A., Scott G., Oskelley C., Medranos E. E., Linskens M., Rubelj I., Pereira-Smith O., Peacocke M., Campisi J. A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc. Natl. Acad. Sci. USA, 92: 9363-9367, 1995.[Abstract/Free Full Text]
  38. Bonner T., Kerby S. B., Sutrave P., Gunnel M. A., Mark G., Rapp U. Structure and biological activity of the human homologs of Raf/miloncogene. Mol. Cell. Biol., 5: 1400-1407, 1985.[Abstract/Free Full Text]
  39. Bouvard V., Massimi P., Banks L. Characterisation of cellular changes which influence progression of human papillomavirus type-16 immortalized keratinocytes to anchorage-independent phenotype. Int. J. Oncol., 8: 159-167, 1996.




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