1 School of Life Sciences, University of Sussex, Brighton BN1 9QG, UK
2 Department of Dermatology, Leiden University Medical Centre, Leiden, The Netherlands
3 Cancer Research UK London Research Institute, Lincolns Inn Fields, London, UK
Correspondence
Alison J. Sinclair
a.j.sinclair{at}sussex.ac.uk
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ABSTRACT |
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MAIN TEXT |
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The human gammaherpesvirus EpsteinBarr virus (EBV) has the ability to promote cell cycle progression following the initial infection of primary resting B-lymphocytes and to cause cell cycle arrest at the onset of the viral replicative cycle (Flemington, 2001; Sinclair, 2003
; Sinclair et al., 1998
). It is therefore clear that EBV must impinge on the function of pRb at two crucial stages of its life-cycle. In considering how EBV manipulates the host cell cycle machinery, it is important to stress that it has no direct equivalent of proteins such as T-antigen or HPV E6 and E7 (Sinclair et al., 1998
), although it has been shown that one viral gene product, EBNA3C, can interact directly with pRb and confer resistance to p16INK4A overexpression in rodent fibroblasts (Parker et al., 1996
). The relevance of this interaction in virus-infected cells remains unproven, however. EBV infection of primary B-lymphocytes has been shown to cause a dramatic up-regulation of cyclin D2 (Kempkes et al., 1995
; Sinclair et al., 1994
), which is attributable to the combined actions of two viral genes EBNA-2 and EBNA-LP (Sinclair et al., 1994
). A likely outcome will be the activation of Cdk4 and Cdk6 leading to hyperphosphorylation of pRb, as is observed in these cells (Cannell et al., 1996
). Finally, there is compelling evidence that the viral LMP1 protein can down-regulate the expression of p16INK4A in primary human fibroblasts (Yang et al., 2000
). This occurs in part through effects on the localization of critical transcription factors (Ohtani et al., 2003
). However, at present it remains uncertain whether any of these three routes are sufficient to explain the ability of EBV to manipulate the host cell cycle in infected cells.
To try to gain further insight into these possibilities, we instigated a study on B-lymphocytes from an individual who is effectively p16INK4A deficient. The individual is homozygous for a 19 bp germline deletion in the INK4A/ARF locus (Gruis et al., 1995). As described in detail elsewhere and illustrated in Fig. 1
, the associated frameshift results in the production of two chimaeric proteins: p14/p16, in which the amino-terminal 88 residues of p14ARF are fused to the last 76 residues of p16INK4A, and p16/X, comprising the first 74 residues of p16INK4A followed by 64 amino acids specified by the +1 reading frame (Brookes et al., 2002
). Detailed in vitro and in vivo analyses indicate that neither protein shows any residual INK4A-associated activity whereas the p14/p16 product retains all the known functions of p14ARF (Brookes et al., 2002
).
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The phenotypes of the four LCLs were then compared using FACS analysis (Fig. 2). All displayed the characteristic phenotype for LCLs (Rickinson, 2002
); CD20+ (with a mean fluorescence intensity of 70·7±1·3 for N cells and 50·5±0·2 for L cells), with high expression of the B-lymphocyte activation marker CD23 (with a mean fluorescence intensity of 129·5±14·3 for N cells and 112·1±43·7 for L cells), coupled with low expression of the germinal centre marker CD10 (with a mean fluorescence intensity of 4·0±0·4 for N cells and 4·8±0·3 for L cells) and detectable expression of CD54 (with a mean fluorescence intensity of 6·7±1·1 for N cells and 9·1±1·1 for L cells). Expression of the EBV genes EBNA-2, EBNA-LP and LMP1 was also equivalent in the four cell lines (data not shown).
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Since apoptosis is often preceded by cell cycle arrest (Andoh, 2000; King & Cidlowski, 1998
; Meikrantz & Schlegel, 1995
; Wang et al., 2000
), we questioned whether p16INK4A status resulted in differences in the potential of the LCLs to survive in response to exposure to the pro-apoptotic agent anti-FAS (Fig. 3
). Early stages in apoptosis were detected by measuring caspase-3 activity (Fig. 3A
) (Frost & Sinclair, 2000
) and also by assaying for the cleavage and subsequent degradation of PARP (Fig. 3B
) (Frost & Sinclair, 2000
); both were altered with similar kinetics in the L and N cells. Furthermore, cell survival (Fig. 3C
) and DNA fragmentation (data not shown) were assayed to measure late stages of apoptosis. No differences were found in the response of the L and N cells to anti-FAS, or to two other pro-apoptotic agents, staurosporin or etoposide (data not shown). This clearly demonstrates that p16INK4A status has no effect on the propensity of LCLs to undergo apoptosis.
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In summary, the signal transduction events that mediate immortalization and then drive cell cycle progression in LCLs are not influenced by the expression of functional p16INK4A in LCLs. Furthermore, other events associated with cell cycle arrest, such as apoptosis and the onset of the viral lytic cycle, are similarly unaffected by the expression of p16INK4A in LCLs. Our current working model to account for this encompasses previously proposed mechanisms to regulate the function of the restriction point by EBV. (i) Infection with EBV could result in transcription of p16INK4A being down-regulated by LMP1 and thus less protein being expressed. Importantly, LMP1 does not switch-off expression of p16INK4A, since it is still detectable in LCLs. (ii) In addition, expression of cyclin D2, Cdk4 and Cdk6 are up-regulated by EBV and so could alter the stoichiometry between cyclin-bound and p16INK4A-bound forms of Cdk4 and Cdk6 within the cells. (iii) In addition, the function of pRb could be directly modulated by association with EBNA3C. This trio of routes available to EBV to modulate the function of the restriction point could be required allow EBV to regulate the proliferation of infected cells from diverse lineages.
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ACKNOWLEDGEMENTS |
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Received 20 November 2003;
accepted 11 February 2004.