Institute of Medical Radiobiology at the Paul Scherrer Institute and of the University of Zürich, 5232- Villigen-PSI, Switzerland1
Author for correspondence: Kurt Ballmer-Hofer.Fax +41 56 310 4417. e- mail kurt.ballmer{at}psi.ch
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The biological role of T antigens has been studied in virus-infected or -transfected cells and in transgenic animals. In primary cells, expression of both large- and middle-T is required for a fully transformed phenotype, while established, phenotypically normal cells are transformed by middle-T alone (Rassoulzadegan et al., 1982 ). These findings are in agreement with the `dual oncogene hypothesis', postulating that full transformation of primary cells requires both an immortalizing and a transforming function (Parada et al., 1984
; Land et al. , 1983
). The highly related monkey and human polyomaviruses lack the reading frame for middle-T, yet express a large-T capable of associating with p53. This protein regulates stress responses induced by various agents and is required for cell cycle surveillance and regulation of apoptosis and has therefore been termed the `guardian of the genome' (Hall & Lane, 1997
; Levine, 1997
; Teodoro & Branton, 1997
). Rodent polyomaviruses do not seem to alter p53 function (Mor et al. , 1997
).
Transformation assays with cDNAs encoding polyomavirus T antigens have been widely used in cell transfection assays. Morphological transformation, focus formation on cell monolayers, and growth in semi- solid media were taken as the end-point of the transformation process. Such assays extend over several generation cycles of the transfected cells and allow for accumulation of additional mutations in cellular genes that might obscure the role of the individual T antigens in the transformation process. Even the use of inducible expression systems only partially solves this problem, as such cell lines are derived from highly selected clones (Strauss et al., 1990 ; Raptis et al., 1985
). In addition, since efficient transfection with DNA requires cells to pass through the S phase, this approach does not address the question of how G0-arrested cells respond to the oncogenic activity of the transfected genes.
In the work described here we investigated the ability of polyomavirus T antigens to stimulate G0-arrested cells to enter the S phase. We chose to study REF52 rat fibroblasts that express normal p53 and Rb and are more resilient to transformation by oncogenes than the commonly used rodent cell lines such as 3T3 or Rat1 (Hirakawa & Ruley, 1988 ; Ishizaka et al., 1995
). Polyomavirus T antigens are difficult to produce in a biologically active form in quantities suitable for microinjection. We therefore injected growth factor-deprived cells with plasmids encoding the various T antigens. Protein expression was observed in approximately 50% of the plasmid-injected cells within 2 h. The fraction of T antigen-expressing cells capable of entering the S phase was immunohistochemically determined 1624 h later by BrdU incorporation. Control cells were injected with a ß-galactosidase expression vector that had no effect on cell growth. As shown in Fig. 1(A)
, none of the T antigens expressed alone committed cells to enter the S phase in the absence of growth factors. Surprisingly, when grown in the presence of serum, middle-T- expressing cells were unable to exit from G0. Concomitant with growth arrest, middle-T-expressing cells often assumed a flat morphology similar to senescent primary cells reaching the end of their replication potential (Fig. 2A
, B
). The morphology of these cells was maintained for up to 72 h after microinjection of the DNA, suggesting that it does not reflect an early stage of apoptosis. We did, however, observe a high rate of apoptosis in cell cultures microinjected with the middle-T expression vector (Fig. 2C
, D
). A mutant of middle-T, 1387T, unable to stimulate Ras and PI 3-kinase, did not arrest cells in G0, and neither did expression of large- or small-T (Fig. 1A
). These findings are reminiscent of earlier studies showing that non-immortalized cells transfected with an activated Ras or Raf allele were arrested in G0 (Sewing et al., 1997
; Lin et al., 1998
; Ridley et al., 1988
). In summary, these experiments show that in growth-arrested REF52 cells middle-T expression is not sufficient to stimulate S phase entry, either in the absence or presence of growth factors.
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The data presented here unravel two aspects of middle-T function in normal cells. First, middle-T alone is not sufficient to stimulate cell cycle entry in the absence of growth factors. This is in agreement with earlier published work showing that activated Ras alone is not sufficient to stimulate resting REF52 cells (Ridley et al., 1988 ; Hirakawa & Ruley, 1988
; Mor et al. , 1997
). The function(s) of polyomavirus large-T that complement middle-T in our assays are most likely linked to the ability of this protein to associate with Rb family proteins, but definitive proof for this idea is missing at present. Cell transformation and tumorigenesis by simian and human polyomaviruses is not only mediated via Rb protein interactions but depends also on the ability of large-T to subvert the function of p53. In contrast, rodent polyomavirus- transformed cells show no changes in p53 stability or expression (Mor et al., 1997
). This suggests that these viruses use either alternative pathways to prevent p53-mediated apoptosis or disable the apoptotic machinery altogether (Teodoro & Branton, 1997
). It has been shown recently that activated Ras promotes premature senescence in primary fibroblasts or REF52 cells and renders these cells unable to enter the cell cycle (Serrano et al., 1997
; Lin et al., 1998
). In these experiments, senescence was observed within a few days after introduction of Ras into randomly growing cells. In our experiments activation of Ras by middle-T caused cell cycle arrest within 1 day. Our experimental approach differs from that used by Serrano et al. (1997)
in that we used cells prearrested in G0 that are unsuitable for the currently used transfection protocols. Our work shows that normal G0-arrested cells are stimulated to enter the S phase upon expression of a combination of T antigens, even in the complete absence of growth factors. What, then, is the role of large-T in these experiments? REF52 cells are wild-type for p53 and Rb (Hirakawa & Ruley, 1988
) while most immortalized cells lack functional p53 or carry mutations in Rb or one of the cyclin-dependent kinase (CDK) inhibitor genes, contributing to an increased rate of cell transformation by Ras family oncogenes. It remains to be shown whether, similar to the data published by Serrano et al. (1997)
, the effect of large-T is mediated by CDK inhibitors such as p16Ink4 or p21CIP, which are both known to regulate progression through the cell cycle.
Our data also show that growth stimulation by serum is blocked by middle-T in REF52 cells and is restored upon coexpression of small- or large-T. It has been shown that small-T expression interferes with the function, e.g. the proper localization or the substrate specificity, of PP2A (Messerschmitt et al., 1996 ; Cayla et al., 1993
; Frost et al., 1994
). Association of PP2A with small-T is therefore expected to prevent downregulation of Ras-mediated signalling and might contribute to constitutively high MAP kinase activity that is essential for continuous cell cycle stimulation (Sontag et al., 1993
).
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References |
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Received 24 June 1999;
accepted 7 July 1999.