The cyclin D1
gene amplification has been observed in cancers of the breast, head and neck or larynx 
. Chromosomal rearrangement is another cause of Cyclin D1 over-expression associated with centrocytic lymphomas 
, parathyroid adenomas 
and mantle cell lymphoma 
. The obvious association of Cyclin D1 with cancer has led the investigators to uncover its oncogenic properties. In fact, Cyclin D1 was shown to cooperate with the Ras oncoprotein for cell transformation 
Earlier reports have suggested that immortalization of primary B-lymphocytes by EBV is accompanied by transcriptional activation of the cyclin D2
gene but not cyclin D1 
. However, a number of studies showed noticeable changes in Cyclin D1 protein levels in both EBV positive LCLs 
and EBV positive SCID mice lymphomas 
. Despite the controversy regarding the Cyclin D1 expression in EBV positive B-lymphoma cells, it is clear that in order to deregulate the entire mammalian cell-cycle, EBNA3C manipulates G1 restriction point through disruption of Cyclin/CDK-pRb-E2F pathway 
Cyclin D1 is over-expressed in a variety of human cancers that do not exhibit cyclin D1
gene amplification or structural abnormalities of the cyclin D1
locus, which suggests that increased Cyclin D1 stability is a potential mechanism. Mutations of cyclin D1
at T286 and P287 have been found in human tumors 
and alter Cyclin D1 nuclear localization as well as stability. Our data showed that both EBV infection in primary B-cells and EBV persistence in cancer cell lines increased protein stability. However the cyclin D1
mRNA level was unchanged. Similar to virus infection, EBNA3C gene expression increased Cyclin D1 levels without altering mRNA levels. It is important to determine if these effects also occur in vivo
The results presented here also demonstrated that the expression of Cyclin D2 and D3 were up-regulated in quiescent cells infected with EBV probably through distinctly different mechanisms. EBV infection or its transforming protein latent membrane protein 1 (LMP1) up-regulates Cyclin D2 expression in primary B-lymphocytes and Burkitt's lymphoma cells 
. None of the studies have shown an important role for Cyclin D3 in EBV-mediated cell transformation. Studies have suggested that the D-type cyclins may have non-overlapping functions at specific steps in B-cell differentiation 
, and that the expression of different D-type cyclins may be influenced by EBV infection through distinctive pathways. Thus, a potential mechanism which involves the contribution of Cyclin D1 in EBV-mediated B-cell transformation is the constitutive induction of these key cell-cycle regulators which leads to pRb hyper-phosphorylation and uncontrolled cell proliferation.
Several lines of evidence suggest that Cyclin D1 is targeted by the E3 ligase, SCFFBX4-αB crystallin
for degradation 
. Elevated expression of FBX4 and αB crystallin is also found to trigger the destruction of wild-type Cyclin D1, but not the phosphorylation-deficient Cyclin D1 mutant, D1T286A 
. Thus, impairment of SCFFBX4-αB crystallin
function may also account for Cyclin D1 overexpression. Data from the ubiquitination assay showed that EBNA3C efficiently inhibits Cyclin D1 poly-ubiquitination, which led us to speculate that EBNA3C may interact with this particular E3 ligase and inhibit its ability to ubiquitinate Cyclin D1. The SCFSkp2
ligase has also been shown to be involved in the degradation of Cyclin D1 
, and knockdown of Skp2 molecule promoted marked accumulation of Cyclin D1 
. EBNA3C interacts with SCF components to regulate the stability of p27KIP1
and pRb 
. It is likely EBNA3C inhibition of SCFSkp2
reduces Cyclin D1 ubiquitination. EBNA3C may be a deubiquitinase or associate with one to regulate the stability of Mdm2 
and likely Cyclin D1. Since the expression level of Cyclin D1 is related to the potential for malignancy and the prognosis of a variety of cancers 
, revealing the mechanisms governing the ubiquitin-proteasome mediated degradation of Cyclin D1 is of importance in designing therapeutic interventions. Conceivably, this approach could amplify the therapeutic window using Cyclin D1 as a target and enhance the efficacy of conventional drugs against EBV mediated oncogenesis.
We have shown earlier that EBNA3C can interact with Cyclin D1 using an in vitro
GST-pulldown experiment 
. Here, we examined the molecular association between EBNA3C and Cyclin D1 complexes to obtain a more in-depth understanding of the different domains of EBNA3C that modulate the activity of Cyclin D1 which will lead to further understanding the basic mechanism by which EBV regulates the mammalian cell-cycle. EBNA3C associates with Cyclin D1 via the same N-terminal domain, residues 130-190, that has been shown to bind many critical cell-cycle regulators 
including other Cyclins - A and E 
. In addition, a recent genetic study using recombinant EBV expressing conditionally active EBNA3C showed that deletion of this particular domain could not support cell proliferation of EBV transformed LCLs, signifying the importance of this domain within EBNA3C 
. The association of EBNA3C with different Cyclins suggests is perhaps cell-cycle dependent and one may replace another depending on the stage in the cell-cycle, which ultimately leads to aberrant cell proliferation in EBV transformed cells. The previously published data and the data herein were generated using asynchronously growing cells; therefore it would be interesting to further elucidate these interactions in a cell-cycle dependent manner. However, using chemical synchronization is likely to distort the true activities underlying EBNA3C function with Cyclin complexes. Nevertheless, we will be undertaking this line of experimentation in the near future.
To promote G1-S phase transition, nuclear localization of Cyclin D1 is critical and it occurs either via decreased proteolysis in cytoplasm which facilitates nuclear import or through inhibition of GSK-3β function which triggers nuclear export via phosphorylation at T286 
. Immunofluorescent studies showed that EBNA3C expression enforces nuclear localization of Cyclin D1. Increased stability and nuclear accumulation of Cyclin D1 in the presence of EBNA3C prompted us to examine whether EBNA3C can also negatively regulate GSK-3β function linked to the stability of Cyclin D1. Indeed, our data show that EBNA3C forms a complex with GSK-3β and significantly reduces its kinase activity toward Cyclin D1, thereby enhancing the nuclear retention of Cyclin D1. Altogether, these data suggest that either by increasing nuclear import by blocking the poly-ubiquitination level of Cyclin D1 or inhibiting nuclear export of Cyclin D1 via inhibiting the kinase activity of its negative regulator GSK-3β, EBNA3C would likely ensure the efficient nuclear accumulation of Cyclin D1 during G1-phase. However, we cannot eliminate the possibility that EBNA3C may also facilitate Cyclin D1 nuclear accumulation through additional strategies.
Cyclin D1 is central to the coordination of the cell-cycle progression at the G1 to S phase transition by integrating the control of pRb phosphorylation with the transcriptional activity of E2F 
. Cyclin D1 in association with its binding partner, CDK4 or 6 phosphorylates pRb to facilitate S phase entry 
. Previously we have shown that EBNA3C enhances the kinase activity of Cyclin A/CDK2 complex 
and recruits an E3 ligase SCFSkp2
to degrade pRb 
. Similarly, here we show that by an in vitro
kinase assay EBNA3C increases the activity of Cyclin D1/CDK6 complex toward both Histone H1 and a truncated mutant of pRb. Moreover, EBNA3C in conjunction with Cyclin D1/CDK6 complex increases pRb poly-ubiquitination and thereby enhances its degradation process. In addition, we show EBNA3C coupled with Cyclin D1/CDK6 complex significantly abolishes the growth suppressive function of pRb in Saos-2 cells.
Studies using serum starved conditions have shown that both EBV and its essential nuclear antigen, EBNA3C can bypass G1 restriction point probably through disruption of Cyclin/CDK-pRb-E2F pathway 
. LMP1 has also been shown to be associated with resistance to G1 arrest during serum starvation 
. Taking advantage of these findings, together with the use of sh-RNA mediated gene knockdown strategies, we have generated knockdown lymphoblastoid cell-lines targeting both EBNA3C
and cyclin D1
transcripts and assayed for cellular proliferation to carefully determine the plausible role of both of these viral and cellular oncoproteins. Indeed, our results show that both EBNA3C and Cyclin D1 are critical for efficient proliferation of EBV infected B-cells. Moreover, the results point out that upon knockdown of these gene products, cells undergo significant apoptosis, probably through induction of the activities of the tumor suppressors – p53 and pRb. Earlier results 
and the data herein adequately show that EBNA3C critically regulates the growth suppressive properties of both p53 and pRb.
Overall, we have shown in this report that the essential EBV latent antigen, EBNA3C physically interacts with and stabilizes Cyclin D1 by blocking nuclear export or inhibiting the poly-ubiquitination. In addition, EBNA3C alters pRb phosphorylation as well as stability by enhancing Cyclin D1/CDK6 kinase activity, thereby nullifying pRb mediated growth suppressive activity (). Furthermore, knockdown of both EBNA3C and Cyclin D1 expression by lentivirus-delivered sh-RNA demonstrated that both EBNA3C and Cyclin D1 play a critical role in cell proliferation in EBV transformed cells. Thus, the present study provides an insight into the mechanisms linked to the development of EBV-associated B-cell lymphomas through the enhancement of a major cell-cycle component, Cyclin D1, which is known to orchestrate the activities of a vast range of cellular networks that are important in the development of human cancers.
A schematic illustration of how EBNA3C regulates Cyclin D1 stability and functions to facilitate G1 to S phase transition in EBV positive cells.