WRN deficiency in WS patients leads to rare premature aging syndromes associated with an excess of unusual cancer types 
including soft tissue sarcomas, thyroid cancers, and meningiomas 
. As the mechanism of how WRN
mutations lead to cancer in WS patients is still not entirely understood, researchers continue to pursue a better understanding of how these cancers evolve. We were especially interested in understanding how WRN deficient cells are able to overcome their characteristic slower proliferation rate 
and induce tumorigenicity 
We previously reported that acute silencing of WRN
significantly accelerated the growth rate of HL60 cells 
. Although an in vivo
study reported that the loss of WRN in p53 null background mice led to rapid tumorigenesis 
, this is the first time to our knowledge that the silencing of WRN
has been shown to promote cell growth in vitro
. Here, we show that the loss of WRN leads to a dramatic increase in the growth rate of p53-deficient HL60 cells (), while its loss in TK6 cells leads to a decreased proliferation rate consistent with other WRN deficient cell lines 
. Under normal culturing conditions, TK6 cells grow faster than HL60 cells, consistent with the observation that the majority of control TK6 cells were in the replication phase (S phase), while the majority of the HL60 cells were in G1 phase (). Loss of WRN in HL60 cells resulted in a significantly increased fraction of cells in S phase, suggesting that the rapid accumulation of cells in replication phase and the distinct cell cycle distribution pattern contributed to the increased proliferation rate in WRN deficient HL60 cells.
Eukaryotic cells have evolved a collection of complex networks for the regulation of the cell cycle, in which cell cycle checkpoints are crucial to preventing cells with damaged DNA from proceeding to the next phase, allowing verification of necessary phase processes, and repairing DNA damage 
. Dysregulation of the cell cycle components may cause the cell to uncontrollably multiply or lead to tumorigenesis. While there are several checkpoints within the cell cycle, the G1/S transition is the rate-limiting step that plays a central role in deciding whether the cell should divide, delay division, or enter a quiescent stage, which in turn determines the cell proliferation rate. P53 and RB are essential for the G1/S cell cycle checkpoint 
. As expected, the p53 mediated DNA repair pathway was activated in response to increased spontaneous DNA damage in WRN deficient TK6 cells, which resulted in delayed progression of cells into S phase such that slower population growth followed. In contrast, in HL60 cells, the data not only confirmed that p53 regulated G1/S checkpoint proteins were deficient, but also demonstrated that the loss of WRN confers a dramatic increase in the level of phosphorylated RB () and thus progression into S phase.
Under normal conditions, most cell types cycle through replication maintaining a consistent rate through each phase of the cycle. Of great importance in regulating this rate is the RB protein, which serves as the gatekeeper of the restriction point (R) during the late G1 phase. If this discrete check point is interrupted, the cells lose their most important control mechanism for inhibiting unwanted proliferation 
. As seen in our study, the increased levels of γRB led to the release of E2F1, a transcription factor that both induces the synthesis of S phase associated proteins and drives the G1/S phase transition, and unregulated accelerated proliferation of HL60 sh-WRN cells. We further showed that increased protein levels of cyclin D3 might be contributing to the elevated γRB level, but we are unsure if this is the sole protein responsible for the increase. Moreover, our data suggests that the presence of normal functioning p53 in TK6 cells may have compensated for the deleterious impacts resulting from the loss of WRN. This implies that WRN's effects on DNA repair and cell cycle stability may be highly modulated by the interaction with other key proteins, in particular p53 
WRN plays a role in RAD51-dependent HR DNA repair, in which WRN is believed to promote intermediate resolution and to suppress cross-over events 
. This has been thought to be critical for the role of WRN in the maintenance of genomic stability 
. It has been demonstrated previously that WRN physically and functionally interacts with RAD51 
and BLM 
, however, this is the first time that the loss of WRN has been shown to induce significant up-regulation of RAD51 and BLM protein levels in an in vitro
model. While it remains unclear how this occurs, it is likely that this dramatic increase is related to the accelerated cell proliferation rate present in WRN deficient HL60 cells as RAD51 is maximally transcribed during S phase 
. This indicates that a complex relationship exists between WRN deficiency and HR DNA repair mechanisms so as to compensate for this loss.
As one of the major DNA repair mechanisms, HR provides high-fidelity template-dependent DSB repair. If left unrepaired, DSBs could cause chromosomal loss or cell death. However, if aberrantly repaired, the result could be much worse, giving rise to mutations and chromosomal rearrangements that could potentially contribute to cancer development 
. In WS patients, increased levels of chromosomal aberrations have been reported 
, suggesting that the loss of balanced HR regulation may be associated with the development of this disease. One interesting observation from our study is that the increase in DNA damage disrepair mediated by elevated HR activity, due to the loss of WRN, is only observed in HL60 cells but not in TK6 cells. Previous reports noted that DNA DSBs stimulated HR in p53
mutant cells but not in p53
wild-type cells 
, suggesting that p53 appeared to repress HR 
. However, further studies are needed to determine whether the different status of p53 is the causal reason for the observed differences in HR activity and DNA damage disrepair between HL60 and TK6 cells. We further showed that the hyperactivity of HR resulted in a completely altered karyotype of HL60 sh-WRN cells compared to HL60 sh-NSC cells such that the characteristic abnormal double minute chromosomes and aneuploidy typical of HL60 cells were absent in WRN deficient HL60 cells (). However, the hyper-activated HR and higher repair rates resulting from the loss of WRN in HL60 cells led to a higher frequency of SCE, indicating an increased frequency of disrepair and thus genomic rearrangements and gene mutations. This is of particular importance for tumorigensis as the loss or inactivation of WRN may accelerate the mutation of other critical genes, which would thus enhance the likelihood of cancer. More importantly, this gain of function of enhanced disrepair rate confers a growth advantage in WRN deficient HL60 cells lacking sufficient DNA damage response mechanisms mediated by p53 
. In addition, previous studies have shown that WRN involved in Non-homologous end joining (NHEJ) mediated DSBs repair pathway by interacting with Ku70/80 
and XRCC proteins 
. WRN appears to play a structural role in optimizing DSBs break repair through the HR and NHEJ repair pathway 
. While it is beyond the scope of this study, further investigation of the status of NHEJ repair mechanisms in these models is warranted.
A multistep model of carcinogenesis is well accepted, in which an initial mutation in a key DNA repair or metabolism gene leads to the accumulation of somatic mutations at a higher frequency. If these somatic mutations occur in other important tumor suppressors or pro-oncogenes, the tumorigenic process is initiated and tumor cells gain the ability to start growing uncontrollably. Our study of silencing WRN
in HL60 and TK6 cells suggests that this multistep model may accurately illustrate the etiology of cancer development in WS patients as well as other diseases related to functional loss of WRN (). WRN has been proposed as a “caretaker” of the genome 
, and an increased level of chromosomal aberrations has also been reported in WS patients 
. Thus, functional loss of WRN would in turn result in the loss or gain of function of other genes due to the increased genomic instability. We thus hypothesized that the loss of WRN together with some of its interacting partners would lead to rapid proliferation through the interruption of normal cell cycle regulation, resulting in the upregulation of HR activity and the induction of more chromosomal aberrations and genomic instability that may be responsible for the transformation from precancerous cell to cancerous cell in WS patients. More importantly, this hypothesis can be applied to WRN deficiency in general, in which the combination of WRN deficiency and the functional loss of other critical genes could accelerate this transformation and subsequently promote tumor formation. This is supported by recent studies that showed association between WRN
polymorphism and risks of cancer development, including, but not limited to breast, gastric adenocarcinoma and bone and soft tissue sarcomas 
. Moreover, due to the role of WRN in protecting cells against DNA damage, functional loss of WRN in cells with chemically induced DNA damage, such as with benzene, may result in uncontrolled replication, which can result in genomic instability and disease 
A hypothetical etiological model for cancer development related with WRN deficiency.
Although this model provides a reasonable description of WRN's mechanistic role during the development of cancer, we are aware of the potential limitations of this study and the need for further studies. While HL60 and TK6 cells are both hematopoietic cell lines, p53 status is probably only one of the differences between them. Thus, while our data suggests a role of p53 status in the different responses of HL60 and TK6 cells to the silencing of WRN
, we cannot exclude the contribution of additional factors, given the wide range of interactions that occur between WRN and other cellular proteins 
. To further investigate the role of p53 in modulating the responses of HL60 and TK6 cells to WRN deficiency, we are currently conducting experiments to re-introduce p53 to HL60 sh-WRN cells and to silence p53 in TK6 sh-WRN cells. With these studies, we aim to determine if the change in p53 status will leads to a reversal of the effects observed in this study, e.g. accelerated growth rate and increased number of chromosomal aberrations induced by highly activated HR in TK6 cells with p53 and WRN double knockdown, and vice versa, in HL60 cells with a functional p53 protein. However, it is clear that additional studies using other p53 wild-type/null cell line variants or cells deficient in other key players, such as RB or its regulators, are necessary to strengthen our hypothesis.