SV40 infection induces telomerase activity in human ... - Nature

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Oncogene (2002) 21, 1434 ± 1442 2002 Nature Publishing Group All rights reserved 0950 ± 9232/02 $25.00 www.nature.com/onc

SV40 infection induces telomerase activity in human mesothelial cells Rudy Foddis1, Assunta De Rienzo2, Dominique Broccoli3, Maurizio Bocchetta1, Elizabeth Stekala1, Paola Rizzo1, Alessandra Tosolini2, Jennifer V Grobelny3, Suresh C Jhanwar4, Harvey I Pass5, Joseph R Testa2,3 and Michele Carbone*,1 1

Cancer Immunology Program, Department of Pathology, Cardinal Bernardin Cancer Center, Loyola University Chicago, Maywood, Illinois, IL 60153; USA; 2Human Genetics Program, Fox Chase Cancer Center, Philadelphia, Pennsylvania, PA 19111, USA; 3Tumor Cell Biology Program, Fox Chase Cancer Center, Philadelphia, Pennsylvania, PA 19111, USA; 4Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA; 5Aerodigestive Program, Karmanos Cancer Institute, Wayne State University, Detroit, Michigan, MI 48201, USA

Mesotheliomas are malignant tumors of the pleural and peritoneal membranes which are often associated with asbestos exposure and with Simian virus 40 (SV40) infection. Telomerase activity is repressed in somatic cells and tissues but is activated in immortal and malignant cells. We evaluated telomerase activity in seven primary malignant mesothelioma biopsies and matched lung specimens and 20 mesothelioma cell lines and eight corresponding primary tumor cultures. All the tumor biopsies, and nearly all primary cell mesothelioma cultures and cell lines were telomerase positive. The ®ndings in cell lines paralleled those observed in primary cultures in cases where paired samples were available. Next, we found that SV40, a DNA tumor virus present in *50% of mesothelioma biopsies in the USA, induced telomerase activity in primary human mesothelial cells, but not in primary ®broblasts. Telomerase activity became detectable as early as 72 h following wild-type (strain 776) SV40 infection, and a clear DNA ladder was detectable 1 week after infection. The amount of telomerase activity increased during passage in cell culture and appeared to parallel increases in the cellular amounts of the SV40 large T-antigen. Thus, SV40 infection leads to telomerase activity before the infected mesothelial cells become transformed and immortalized. SV40 infection of human ®broblasts did not cause detectable telomerase activity. We also determined that the SV40 small t-antigen (tag) plays an important role in inducing telomerase activity because this activity was undetectable or minimal in mesothelial cells infected and/ or transformed by SV40 tag mutants. Asbestos alone did not induce telomerase activity, and asbestos did not in¯uence telomerase activity in mesothelial cells infected with SV40. Induction of telomerase activity by SV40 may be related to the very high rate of mesothelial cell immortalization that is characteristically associated with SV40 infection of mesothelial cells.

*Correspondence: M Carbone, Loyola University Chicago, Cardinal Bernardin Cancer Center, Room 205, 2160 S. First Ave., Maywood, IL 60153, USA; E-mail: [email protected] Received 25 September 2001; revised 9 November 2001; accepted 27 November 2001

Oncogene (2002) 21, 1434 ± 1442. DOI: 10.1038/sj/ onc/1205203 Keywords: SV40; telomerase; asbestos; mesothelioma

Introduction A fundamental dierence between normal and tumor cells is that normal cells undergo a ®nite number of divisions, whereas their malignant counterparts usually have the ability to proliferate inde®nitely (reviewed in Shay et al., 2001). Shortening of the telomeric DNA at chromosome ends is known to limit the lifespan of human cells via activation of the pRb and p53 pathways and genomic instability (Shay et al., 2001). At the crisis stage, telomeres reach a critically short length, lose their ability to confer stability upon chromosome ends, and contribute to the formation of end-to-end associations and/or dicentric chromosomes. Cells that escape from crisis and give rise to immortal cell lines display stable telomeres (Counter et al., 1994). Telomerase, an eukaryotic ribonucleoprotein complex, stabilizes telomere length in human reproductive and neoplastic cells (Kim et al., 1994; Wright et al., 1996) by adding TTAGGG repeats onto telomeres using a speci®c RNA as a template. Telomerase activity is repressed in somatic cells and tissues but is activated in immortal cells and cancer cells (Shay and Wright, 1996). Some immortalized telomerase-negative cell lines can acquire very long and heterogeneous telomeres in association with immortalization throughout a dierent mechanism known as alternative lengthening of telomeres (ALT) (Bryan et al., 1995). The nature of the ALT mechanism is currently unknown but may involve non-reciprocal recombination between telomeres (Dunham et al., 2000). In addition to being telomerase negative, ALT cell lines characteristically display ultra-long telomeres (420 kb). The third characteristic feature of ALT cell lines is that a subset of the cells contain a novel form of the promyelocytic leukemia nuclear body (PML NB), in which telomeric

SV40 induces telomerase activity R Foddis et al

DNA and the telomeric proteins TRF1 and TRF2 colocalize with the PML protein (Yeager et al., 1999). These structures, called ALT-associated PML bodies (APBs), have also been found in various tumors and tumor cell lines (reviewed in Stewart and Weinberg, 2000). In most human tumors, telomerase, is activated (Shay and Wright, 1996). In addition, it has recently been demonstrated that forced expression of telomerase is sucient to generate the immortal phenotype required for tumor formation (Bodnar et al., 1998; Vaziri and Benchimol, 1998). In contrast, inhibition of telomerase limits the growth of transformed cells in vitro (Hahn et al., 1999a,b; Zhang et al., 1999). Malignant mesothelioma is an aggressive, mesodermal tumor that arises from serosal cells lining the pleural, peritoneal, and pericardial cavities. Although some benign mesothelioma exist, these are extremely rare and are histologically dierent tumors (reviewed in Carbone et al., 2002), thus the term mesothelioma will be used throughout this manuscript to indicate the classic malignant mesothelioma associated with a survival of about 9 months from diagnosis. The primary cause of this malignancy is exposure to asbestos ®bers (reviewed in Testa et al., 2001; Carbone et al., 2002). Recent studies have associated SV40 (either alone, reviewed in Jasani et al., 2001; or in association with other DNA viruses, Krynska et al., 1999) to a number of human malignancies including mesothelioma (reviewed in Klein et al., 2002). Growing experimental data suggest that simian virus 40 (SV40) and genetic factors play a role in the etiology of mesothelioma (Carbone et al., 2002). Mesothelioma is characterized by a long latency from the time of asbestos exposure to clinical diagnosis, implying that multiple somatic genetic and epigenetic changes are required for the tumorigenic conversion of a mesothelial cell (Testa et al., 2001). We have recently reported that infection of human mesothelial cells by SV40 leads to an extremely high rate of malignant transformation, which is at least 1000 times higher than that reported for other tested human cells infected by SV40 (Bocchetta et al., 2000). Furthermore, we found that 14 of 16 morphologically transformed clones that developed in cell culture 6 ± 8 weeks after SV40 infection appeared to be immortal, because these clones have been subcultured more than 100 times and have never developed a crisis (Bocchetta et al., 2000). This is very unusual for SV40 transformed cells, because it has been estimated that less than 5% of clones become established in cell culture, but earlier experiments were done mostly in ®broblasts and never with mesothelial cells. Moreover, prior works indicate that ®broblast clones develop a crisis and only rarely does a population of cells escape crisis and become immortal (reviewed in Carbone et al., 2002). In fact, in parallel experiments in which we infected primary human ®broblasts, we were unable to establish transformed immortal cell lines (Bocchetta et al., 2000). In a commentary concerning our report

(Bocchetta et al., 2000), Klein et al. (2001) wrote that it would be interesting to know whether the dierence in the immortalization of SV40-infected mesothelial cells compared to SV40-infected ®broblasts paralleled a dierence in telomerase activity. However, voluminous studies appeared to have elucidated all possible mechanisms of SV40 oncogenicity: SV40 Tag binds and inhibits a number of tumor suppressor gene products, including p53, pRb, p107, p130/Rb2, p300 and p400. Therefore, SV40 infection interferes with several cellular pathways, many of which are related to cell cycle control and DNA repair. Furthermore, SV40 infection causes IGF2 secretion and up-regulation of its receptors, and thus directly promotes cell growth. SV40 Tag also plays a role in programmed cell death, and while there are somewhat contrasting reports on this topic, it appears that low amounts of Tag have an anti-apoptotic eect, whereas high levels of Tag induce apoptosis. Finally, Tag is directly and indirectly mutagenic, and SV40-infected human cells acquire numerous chromosomal alterations (reviewed in Ali and de Caprio, 2001; Pipas and Levine, 2001; Testa and Giordano, 2001). Moreover, the other SV40 oncogene, tag, binds and inhibits phosphatase 2A (PP2A), and thus tag interferes with the de-phosphorylation of Tag and of several cellular proteins (reviewed in Rundell and Parakati, 2001). Telomerase, on the other hand, had not been previously linked to SV40 infection. The following experiments were conducted to test whether mesotheliomas, similarly to other malignancies, express telomerase activity, and whether SV40 could in¯uence this activity. To assess the relevance of telomerase activity in mesotheliomas, we ®rst veri®ed that these tumors and derived cell lines exhibited telomerase activity. Next, we tested the hypothesis that SV40 infection is related to telomerase activity. Finally, we initiated studies to establish which portion of the viral genome may induce telomerase activity.

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Results Telomerase activity is detectable in mesotheliomas, mesothelioma cell cultures, and derived cell lines Telomerase activity was detectable using the TRAP assay in seven of seven mesothelioma biopsies from dierent patients, but it was not detected in matched lung tissue from the same patients (Figure 1). Five of the seven mesothelioma biopsies contained SV40 sequences (not shown). Telomerase activity was detected in 19 of 20 (95%) established mesothelioma cell lines (all SV40 negative) and in all eight primary cultures (one positive for SV40, ®ve negative, two were not tested) (Figure 2 and data not shown). Telomerase activity or lack thereof, was found concordant in all eight primary cultures and matched established cell lines. Two cell lines had relatively low levels of telomerase activity (Figure 2, and data not shown). As expected, telomerase activity was not detected in Oncogene


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Figure 1 Telomerase activity in human pleural malignant mesotheliomas and in matching normal lung tissues. (a) Lanes 3 ± 10 correspond to four separate mesothelioma biopsies. (b) Lanes 3 ± 14 correspond to three additional mesothelioma samples with matching lung tissue, as indicated. The total protein extract was 0.5 mg per lane. Lanes 1 and 2, HeLa cell lysates (positive controls); Lanes 11 (a) and 15 (b), lysis buer (negative controls); S-IC: PCR ampli®ction control (36 bp)

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diusible inhibitor (Figure 2 and data not shown). The absence of telomerase activity in one cell line raised the possibility that telomere elongation was not always required for malignant mesothelial cell growth. Alternatively, a dierent mechanism might lead to telomere stability in these cells. It appeared important to address which one of these two hypotheses was correct. The ALT pathway provides telomere stabilization in telomerase negative mesothelioma cells

Figure 2 Representative autoradiogram of a TRAP assay used to detect telomerase activity in whole cell extracts prepared from mesothelioma cells. Numbers at top indicate mesothelioma cell line numbers. Telomerase activity was detected using 0.5 mg of extract either without (7lanes) or with (+lanes) addition of RNase to the reaction mixture. The RNase treatment con®rms that the reaction products resulted from telomerase activity. In addition, extracts from the telomerase-negative cell line Meso 10 were prepared in parallel without (10) and with (10+H) addition of HeLa cells to establish the absence of technical artifacts causing falsely negative results

any of four normal mesothelial cell cultures (data not shown). The speci®city of the TRAP reaction was established by both heat inactivation and by inclusion of RNase in the reaction mixture. As expected, heat inactivation and RNase eectively inhibited the formation of reaction products (Figures 1 and 2). Cellular extract prepared from the cell line that did not contain detectable telomerase activity was mixed with telomerase positive HeLa extract to con®rm that the absence of enzymatic activity was not due to a Oncogene

Although telomerase is the most common mechanism for maintenance of telomeric DNA in human tumors, a second mechanism, termed ALT, that apparently relies upon recombination between telomeric repeats has been described. Telomeres are ultra-long and heterogeneous in size in the telomerase-negative cell lines and tumors that utilize the ALT pathway. Therefore, we carried out Southern hybridization analysis of terminal restriction fragments to determine if the telomerasenegative mesothelioma cell line utilizes the ALT pathway. DNA analysis was performed to determine telomere length in all mesothelioma cell lines. In several cell lines, telomere length was typical of human tumor cell lines, generally being 5 ± 10 kb (Figure 3). Interestingly a number of cell lines had very short telomeres with smaller terminal restriction fragments, i.e., 55 kb (Figure 3). The telomerase-negative cell line, Meso 10, contained ultra-long telomeres characteristic of the ALT phenotype (Figure 3). To con®rm that the telomerase-negative mesothelioma cell line utilizes the ALT mechanism for telomere stabilization, we carried out indirect immuno¯uorescence to detect APBs. Consistent with previous reports in the literature, none of the three telomerase-positive cell lines contained these ALT speci®c structures (data not shown). Regarding cell line Meso 10, although


paralleling an increase in the amount of cellular SV40 Tag (not shown). In parallel experiments, three cell lines obtained by infection with SV40 tag mutant dl 884 in the presence of asbestos had barely detectable levels of telomerase activity (not shown). Telomerase activity was not detectable in the primary mesothelial cell cultures (Figures 4 and 5). Furthermore, addition of asbestos to primary cell cultures, or to SV40transformed mesothelial cells, did not in¯uence telomerase activity (not shown). These experiments suggest that telomerase activity is not merely an expression of cell division and growth. Rather, it appears that a particular malignant phenotype, i.e., mesothelial cell transformed by wild-type SV40, which expresses both, Tag and tag, is preferentially associated with telomerase activity.

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SV40 tag is required to induce telomerase activity in mesothelial cells

Figure 3 Autoradiogram of Southern blot of telomeric restriction fragments from DNA prepared from mesothelioma cell lines. Numbers at top indicate cell line numbers. Molecular weight markers in kilobases are shown at the left

occasional cells exhibited co-localization of the telomeric protein TRF2 with the PML nuclear body, these structures were not as dramatic as have been observed in other cell lines that use the ALT mechanism to maintain telomere length (not shown). Telomerase activity is detectable in human mesothelial cells transformed in vitro by SV40 In previous experiments we have shown that wild-type SV40 cause a very high rate of mesothelial cell transformation. In the same experiments, SV40 tag mutants alone, or asbestos alone were unable to transform human mesothelial cells in vitro. However, the combination of the SV40 tag mutants and asbestos caused malignant transformation, (Bocchetta et al., 2000). Here, we tested if telomerase activity was detectable in human mesothelial cells that were transformed in vitro either following SV40 infection (strain 776) or by infection with SV40 tag mutant dl 884 in the presence of asbestos. Strain 776, is the SV40 strain which has been used in almost all SV40 studies in the past 40 years. The SV40 dl 884 contains a 247 base pair deletion of tag-speci®c sequences which spans the small t donor splice site (Carbone et al., 1991). Telomerase activity was detected in six of six SV40-transformed mesothelial cell lines. Moreover, the telomerase activity increased with passage in cell culture (Figure 4a),

To test the hypothesis that SV40 directly induces telomerase activity in mesothelial cells, we infected three primary human mesothelial cell cultures, from dierent individuals, with wild-type SV40, strain 776, and with SV40 tag mutant dl 884, at a M.O.I. of 30. In parallel experiments, we infected two primary ®broblasts cell cultures with the same viruses (control). Immunostaining demonstrated that virtually all mesothelial cells express Tag within 72 h after infection (not shown). Telomerase activity became detectable in mesothelial cells 72 h after infection with wild-type SV40 (Figure 5), and a clear ladder was seen in these cells 7 days after infection (Figure 4b). Telomerase activity was not detectable in SV40-infected ®broblasts (not shown). Telomerase activity was not detected in mesothelial cells 72 h after infection with SV40 tag mutants (Figure 5). Seven days after infection, when a clear ladder was visible in cells infected with wild-type SV40, telomerase activity was barely detectable in cells infected with SV40 tag mutants (Figure 4b). Therefore, tag increases the amount of telomerase activity in SV40-infected mesothelial cells. Discussion In common with most other tumor types, the vast majority of samples analysed contained telomerase activity. Only one of our samples was telomerase negative. This cell line (i.e. Meso 10) used an alternative mechanism, i.e., ALT, to circumvent the telomeric checkpoint. Our ®nding that mesotheliomas and mesothelioma cell lines uniformly expressed telomerase activity, or used the ALT mechanism, indicate that as in other malignancies, stabilization/ elongation of telomeres is important in the pathogenesis of mesothelioma. Having con®rmed the biological relevance of telomerase activity in mesothelioma, we tested the possible link with SV40 infection. We have found that SV40 induces telomerase activity in human mesothelial cells, thus for the ®rst time Oncogene


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Figure 4 Representative experiments showing telomerase activity in SV40-transformed cells at dierent tissue culture passages (a), and in SV40-infected mesothelial cells, 1 week after infection (b). (a) Telomerase activity in HM clone 4. Lane 1 (negative control, lysis buer); lane 2, HeLa (positive control); 3, same as lane 2 after heat inactivation; lane 4, HM clone 4, passage 11; lane 5, same as lane 4, after heat inactivation; lane 6, passage 28; lane 7, same as lane 6, after heat inactivation; lane 8, passage 54; lane 9, same as lane 8 after heat inactivation. The band near the bottom of the gel in a, is the internal PCR control provided in the Intergen kit. These experiments show telomerase activity which increases during passage in tissue culture in SV40-transformed HM clone 4. (b) Telomerase activity 1 week after infection of HM with either wild-type SV-40 or SV40 tag mutant dl 884. Lane 1, HeLa (positive control); lane 2, same as lane 1, after heat inactivation; lane 3, uninfected HM; lane 4, same as lane 3, after heat inactivation; lane 5, HM 7 days after infection with SV40 tag mutant dl 884 (30 M.O.I./cell); lane 6, same as lane 5, after heat inactivation; lane 7, HM 7 days after infection with SV40 wild-type strain 776; lane 8, same as lane 7, after heat inactivation; lane 9, negative control (lysis buer). These experiments indicate that wild-type SV40 induces telomerase activity in HM and that tag plays an important role in this process. Telomerase activity was assayed using the TRAPaze kit (Intergen) according to the manufacturer's instructions, using 0.5 mg of cell extract per sample. All experiments in (b) were performed on HM from the same donor and con®rmed using HM from a second donor. The gels were resolved by silver stain. Telomerase activity without (7) and with (+) heat inactivation

directly linking SV40 infection with telomerase activation. Such activation appears to contribute to the immortalization of human mesothelial cells infected with SV40. The fact that telomerase activity became detectable as early as 72 h after SV40 infection indicates that activation of enzyme is not a consequence of malignant transformation. In fact, morphologically transformed mesothelial clones are not detectable until 2 or more weeks after SV40 infection. Thus, telomerase activity is an early event following infection of human mesothelial cells. It is unclear at this time, if SV40 induces telomerase activity in a fraction of the infected cells, and whether are some of these cells that later preferentially become transformed, or whether telomerase activity is induced in 100% of the infected cells. We did not detect such activity in ®broblasts, and in numerous infection experiments involving several dierent primary ®broblast cell cultures, we failed to establish an immortal ®broblast cell line (Bocchetta et al., 2000; Carbone M and Bocchetta M unpublished results). Other groups have also found that human ®broblasts are rarely immortalized by SV40 (reviewed in Bryan and Reddel, 1994, and in Ozer et al., 1996). The inability of SV40 to induce detectable telomerase activity in ®broblasts is probably related to the Oncogene

dierent series of events that follows infection of these cells compared to mesothelial cells. When SV40 infects ®broblasts, the virus replicates very rapidly producing numerous viral particles that ®ll both the cytoplasm and the nucleus, leading to rapid cell lysis. In contrast, in mesothelial cells, SV40 establishes mostly an episomal infection in which only a limited number of viral particles are produced, because the high levels of p53 normally present in mesothelial cells interfere with Tag-mediated viral DNA replication. The viral particles accumulate predominantly in the nucleus and do not cause signi®cant cell lysis (Bocchetta et al., 2000; Cacciotti et al., 2001; Yu et al., 2001). Thus, when some of these infected mesothelial cells become transformed because of other oncogenic activities of the SV40 Tag, these cells already have telomerase activity and, are therefore capable of continuous growth. This fact appears to explain our previous observation that 14 of 16 SV40-transformed mesothelial cell clones were rapidly immortalized and did not undergo a crisis (Bocchetta et al., 2000). The high incidence of immortal mesothelial clones we observed may appear in con¯ict with the data of Ke et al. (1989) who reported that four out of four clones of mesothelial cells transfected with an origin of replication (ori) defective SV40

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Figure 5 Representative experiment showing telomerase activity in mesothelial cells 72 h after infection with wild-type SV40 or SV40 tag mutant dl 884. Lanes 1 and 2, HeLa cell extracts (positive control); Lanes 3 ± 6 normal (non-infected) mesothelial cells (control); lanes 7 ± 8, cell extracts from HM infected with SV40 tag deletion mutant dl 884; Lanes 9 and 10, cell extract from HM infected with wild-type SV40 strain 776; lane 11, lysis buer (negative control). Telomerase activity was assayed using the TRAPaze kit, using 0.5 mg of cell extract. Telomerase activity without (-) and with (+) heat inactivation

plasmid developed a crisis, and that only one of them escaped crisis and became immortal. In fact, we obtained similar results when we tried to establish cell lines from mesothelial cells transfected with SV40 ori-minus constructs (Bocchetta et al., 2000, and Carbone et al., unpublished observations). The key factor is infection versus transfection. SV40 infections frequently cause immortal clones, transfections only rarely. The molecular mechanisms of these dierences are under investigations in our laboratory. The immortal phenotype induced by SV40 in infected mesothelial cells can account for some of the biological dierences observed between SV40 infections of mesothelial cells and ®broblasts. The latter are only rarely immortalized by SV40 (reviewed in Ozer et al., 1996). We think that the reason that SV40 does not immortalize human ®broblasts is a consequence of the technical procedure that must be used to circumvent the problem of SV40-induced ®broblast cell-lysis. To circumvent this problem, investigations in human ®broblasts were conducted using SV40 constructs in which the origin of replication (ori) of SV40 had been deleted (reviewed in Ozer et al., 1996). These experiments demonstrated that human ®broblasts transfected with ori-minus SV40 Tag+ and tag+ constructs become fully transformed because these cells have (1) an altered morphology, (2) form tridimensional foci in tissue

culture, (3) grow in low serum and in soft agar, and (4) the life-span of these cells is extended. However, these ®broblasts are not immortal and enter a well de®ned crisis period characterized by further changes in cell morphology, loss of proliferative capacity, and ultimately cell death (Shay and Wright, 1989, 1993; Klein et al., 1990). Further proliferation of Tag and tag expressing ®broblasts is dependent upon a rare event, or series of events, that must overcome the cellular restrictions on inde®nite growth (Ozer et al., 1996; Yu et al., 2001). On the other hand, in this manu-script we show that SV40 infection of mesothelial cells induces telomerase activity which appears linked to the immortalized phenotype of these cells (Bocchetta et al., 2000). Because it is well known that immortalized human cells are much more amenable to appropriate transforming stimuli, compared to nonimmortalized cells (Sager, 1984), telomerase activation in mesothelial cells may render these cells more susceptible to malignant transformation. Thus, SV40 infection of human mesothelial cells may be considered a pre-neoplastic event that increases the risk of mesothelioma development. The apparent role of tag in increasing telomerase activity is intriguing. It appears that tag mainly augments the amount of telomerase activity, and that in the absence of tag the levels of telomerase activity in SV40-infected mesothelial cells are very low, at the limit of detection. Future experiments should address the possible mechanisms used by tag to induce telomerase activity. We speculate that because tag inhibits PP2A this may cause increased levels of phosphorylated (active) AKT, which may be negatively regulated by PP2A. AKT phosphorylates the telomerase reverse transcriptase subunit and increases telomerase activity (Kang et al., 1999). Thus, SV40 tag, by inhibiting PP2A and enhancing phosphorylation of AKT, could ultimately in¯uence telomerase activity. It should be noted that we detected telomerase activity in all mesothelioma biopsies and in nearly all mesothelioma cell lines that we have tested, regardless of the presence or absence of SV40 sequences. Therefore, it could be argued that although our in vitro observations indicate that SV40 induces telomerase activity, this activation is independent of the SV40 status in mesotheliomas. Alternatively, there may be several dierent mechanisms that lead to telomerase activation in tumors. In many cancers, including many mesotheliomas, it is possible that telomerase is induced following the acquisition of numerous genetic alterations that characterize this malignancy (reviewed in Testa et al., 2001). Hahn et al. (1999a,b), proposed that malignant transformation of human cells requires a minimum of three changes: (1) a proliferative signal, e.g., the activation of an oncogene, (2) inactivation of certain tumor suppressor genes through mutation or other events, and (3) telomerase activation. When mesothelial cells harbor SV40, two of these requirements, i.e., inactivation of tumor suppressor gene products (Carbone et al., 1997; De Luca et al., 1997;

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Schrump and Waheed, 2001), and telomerase activation, may be dispensable. Furthermore, recent data indicate that SV40 activates the met oncogene (Cacciotti et al., 2001), and that met activation stimulates mesothelial cell growth. Thus, it appears that all three requirements for the malignant transformation of human cells may be ful®lled by SV40 in infected mesothelial cells. Alternatively, any additional genetic damage caused in these cells by asbestos, SV40, or other factors will occur in a cellular background prone to transformation and capable of continuous growth.

Materials and methods Specimen collection Tumor specimens were surgically resected in pleural mesotheliomas from newly diagnosed patients. Criteria for the diagnosis of mesothelioma were in accordance with established guidelines (Carbone et al., 2002). Each of the cell lines has been propagated for at least 50 passages. Southern blot analysis of telomere fragments Genomic DNA was isolated from logarithmically growing cells by standard phenol extraction methods. Digestion and quantitation of genomic DNA, agarose gel electrophoresis, and Southern transfer were as previously described (Luderus et al., 1996; van Steensel and de Lange, 1997). One hundred ng of each of the telomeric oligonucleotides (TTAGGG) and (CCCTAA) were end-labeled using g32P-ATP and T4 polynucleotide kinase and were used as probes to detect telomeric DNA. Filters were hybridized and washed as previously described (Broccoli et al., 1996). TRAP assay Cell extracts were prepared as described previously (van Steensel and de Lange, 1997). Protein concentration was determined using the Bradford Assay (Bio-Rad). Telomerase activity was detected in 0.5 mg extracts using a modi®cation of the TRAP assay, as described elsewhere (Broccoli et al., 1995; Kim et al., 1994), and/or a TRAPaze kit (Intergen) according to the manufacturer's protocol. Addition of RNase to the reaction mixture was used to con®rm that reaction products resulted from telomerase activity in mesothelioma cell lines. Addition of 0.5 mg of HeLa extract in reactions containing extract from telomerase negative cell lines was used to control for reaction conditions and to demonstrate the absence of a diusible inhibitor of the TRAP reaction in telomerase negative cell lines. Finally, extracts were prepared in parallel with and without addition of HeLa cells to establish absence of technical artifacts resulting in false negative extracts. Indirect immunofluorescence and antibody detection Cells were grown on coverslips, and analysis of immuno¯uorescence was performed as described elsewhere (Chong et al., 1995). The cells were ®xed in 3.7% formaldehyde (Fisher)/16PBS for 10 min prior to permeabilization with 0.5% NP-40/16PBS. A rabbit polyclonal antibody against a peptide contained in the amino terminal acidic domain of the Oncogene

human telomeric protein (hTRF1) was anity-puri®ed and used at a dilution of 1 : 200 in combination with a goat polyclonal antibody raised against a peptide mapping near the amino terminus of human PML (N-19; Santa Cruz), diluted 1 : 5000. The hTRF1 antibody was detected with tetramethyl rhodamine-isothiocyanate (TRITC)-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch), and the PML antibody was detected using ¯uorescein-isothiocyanate (FITC)-conjugated donkey anti-goat IgG. Cells used in the infection experiments We used three dierent primary mesothelial cell cultures: HM2, HM4, and HM5. These cells were from three separate patients who accumulated pleural ¯uid because of congestive heart failure. HM were used at passages 3 ± 7; these cells became senescent at passage 8 ± 9. The identity of HM was established morphologically and con®rmed by EM (presence of long microvilli) and positive immunostaining for cytokeratin, HBME-1, calretinin, and negative staining for LeuM1, BerEp4, B72.3, and CEA. After 2 weeks in culture, contaminating lymphocytes and epithelial cells died, and 100% of the cell stained positive for calretinin. In parallel, we used three dierent cultures of primary human diploid ®broblasts (HF) as controls: WI38 and MRC-5, both fetal lung HF, which were used in our experiments at passages 17 ± 18; and CCD1069Sk breast HF from a 70 year old woman which were used at passages 6 ± 7, all from ATCC. WI38 were chosen because they were used extensively in the past to study SV40 infection of human cells and because of their lung origin, MRC-5 because of their lung origin, and CCD1069Sk because of the early passage available and to test ®broblasts from an adult. Primary HM were established and subsequently grown in tissue culture in Dulbecco's modi®ed essential medium (DMEM) containing 20% fetal bovine serum (FBS). All ®broblast cell cultures were grown in DMEM containing 10% FBS. Infection experiments Infection experiments with wild-type (also known as nonarchetypal) SV40 were performed using SV40 strain 776. Infection experiments with SV40 tag mutant were performed using dl 884. Brie¯y, the medium was removed from the ¯asks containing either primary mesothelial cells or primary ®broblasts, and then the virus, suspended in DMEM, was added. Cells were incubated in a CO2 incubator at 378C with rocking every 30 min. Three hours after infection, the medium was removed and replaced with fresh DMEM containing FBS. At 72 h and 7 days after infection, the cells were trypsinized, suspended in DMEM, centrifuged at 48C for 5 min at 1200 r.p.m. 106 cells were washed twice with PBS to remove any remaining trypsin, and the cell pellet was snap-frozen using dry ice. Seventy-two hours after the infection, Tag immunostaining was performed on a subset of cells to verify that the cells had been infected (Bocchetta et al., 2000). SV40 testing in tumors, cell cultures and cell lines The presence or absence of SV40 in these specimens was established using both PCR analyses with primers speci®c for the SV40 DNA coding for the Rb-pocket binding domain of Tag, and using immunohistochemistry to test for SV40 Tag expression. We have described these methodologies in detail in previous publications, (for example see Carbone et al., 1997; Rizzo et al., 1998; Testa et al., 1998).


Abbreviations SV40, Simian virus 40; Tag, SV40 large tumor antigen; tag, SV40 small tumor antigen; HM, primary human mesothelial cells; HF, primary human ®broblasts; MM, malignant mesothelioma; M.O.I. multiplicity of infection; TRAP, telomeric repeat ampli®cation protocol Acknowledgments We are grateful to Dr Kathleen Rundell for the critical reading of this manuscript. This work was supported by

grants: ACS 8632 through a generous donation from Mr Dean Butchkovitz, by a grant from the Charlotte Geyer Foundation and by the Riviera Country Club, Illinois (to M Carbone); and by grants NCI CA-45745 and CA-06927, a gift from the Local #14 Mesothelioma Fund of the International Association of Heat and Frost Insulators & Asbestos Workers in memory of Hank Vaughan and Alice Haas, and an appropriation from the Commonwealth of Pennsylvania (to JR Testa).

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