The vaccinia-related kinase (VRK) proteins are a new family with three members in the human kinome. The VRK1 protein phosphorylates several transcription factors and has been postulated to be involved in regulation of cell proliferation. In normal squamous epithelium, VRK1 is expressed in the proliferation area. Because VRK1 can stabilize p53, the expression of the VRK1 protein was analyzed in the context of the p53 pathway and the proliferation phenotype in a series of 73 head and neck squamous cell carcinomas. VRK1 protein level positively correlated with p53 response proteins, particularly hdm2 and p21. The VRK1 protein also correlated positively with several proteins associated with proliferation, such as cyclin-dependent kinase 2 (CDK2), CDK6, cdc2, cyclins B1 and A, topoisomerase II, survivin, and Ki67. The level of VRK1 protein behaves like a proliferation marker in this series of head and neck squamous cell carcinomas. To identify a possible regulatory role for VRK1 and because it regulates gene transcription, the promoters of two genes were studied, CDK2 and SURVIVIN, whose proteins correlated positively with VRK1. VRK1 increases the activity of both the CDK2 and SURVIVIN gene promoters. The expression of VRK1 was analyzed in the context of regulators of the G1-S transition. VRK1 protein levels increase in response to E2F1 and are reduced by retinoblastoma and p16. These data suggest that VRK1 might play a role in cell cycle regulation and is likely to represent the beginning of a new control mechanism of cell cycle, particularly late in the G1-S phase. (Mol Cancer Res 2006;4(3):177–85)

Head and neck squamous cell carcinoma (HNSCC) constitutes the sixth most common type of cancer worldwide. In the United States, it accounts for ∼3% of new cases and 2% of the deaths annually. Pathogenically, the development of these tumors has been associated with the mutagenic role of tobacco carcinogens (1), which can induce specific mutations in different genes, including p53 (2-5). The strong association with carcinogens suggests that pathways related with the cellular response to genotoxic damage might be implicated in HNSCC; in addition, there might be a genetic susceptibility affecting genes that are implicated in DNA repair processes or metabolism of carcinogens (6). There are also ethnic differences in its incidence, with the Black population presenting higher rates, whereas the Hispanic and Asian populations have lower rates (1). A role for certain types of human papillomaviruses has also been suggested (7, 8).

The vaccinia-related kinase 1 (VRK1) protein belongs to a new family of serine/threonine kinases in the human kinome (9, 10). This family has three members in mammals, but Drosophila and Caenorhabditis elegans have only one homologue gene. The inactivation of the C. elegans homologue causes embryonic lethality (11). Inactivation of the distant homologue in yeast, both Schizosaccharomyces pombe and Saccharomyces cerevisiae, has been shown to be implicated in the response to DNA damage (12, 13). VRK1 is highly expressed in human tumor cell lines (14) and murine embryos (15), suggesting it might be associated with cell proliferation. This association was further supported by experiments where VRK1 inactivation with small interfering RNA resulted in a block of cell division (16). VRK1 is expressed at very high level in the retina neurons, and its expression drops dramatically the first day after birth (17). In chronic myelogenous leukemia, the expression of VRK1 can differentiate responders from nonresponders cases to treatment with imatinib (18). In B cells, analysis by quantitative mass spectrometry indicates that it is down-regulated when Myc expression is induced (19). VRK1 is also regulated in the response to peroxisome proliferators in murine hepatocytes (20). VRK1 expression is also activated by E2F and inhibited by p16 and nonphosphorylated retinoblastoma (Rb; ref. 21). VRK1 has a serine/threonine kinase activity and phosphorylates several transcription factors, including human p53 (22), and can also cooperate with the c-Jun NH2-terminal kinase pathway by phosphorylation of c-Jun (23) and ATF2 (24). All these proteins phosphorylated by VRK1 have been associated with cellular responses to stress (25-27). VRK1 contributes to p53 stability by two mechanisms, one of them dependent on Thr18 phosphorylation, and also seems to be implicated in the control of normal proliferation in the absence of cellular stress (16). The loss of VRK1 also affects the endocytic transport with a phenotype similar to that induced by silencing of mitogen-activated protein kinase (28). The other VRK members are not well known. VRK2 is located in the cytoplasm and is membrane bound; it is catalytically active, but nothing is known regarding its substrates (29). VRK3 is catalytically inactive and probably functions as a scaffold protein (29).

Among the sensor mechanisms of cellular response to DNA damage is the p53 molecule that has been dubbed as the guardian of the genome (30). The p53 reaction to cellular damage triggers several types of responses, ranging from stopping the cell cycle to induction of apoptosis (31, 32). Thus, the p53 pathway plays a central role in cancer biology (32, 33), being at the center of all processes implicated in the cellular response to genotoxic stress (25). The p53 protein contributes to proliferation by its participation in the checkpoints that controls the cell cycle (34-36). In the control of these processes, phosphorylation of p53 is a major regulatory mechanism, and several kinases phosphorylating p53 have been implicated in cancer and in its therapeutic responses, such as ATM, ATR, CHK1, CHK2, and c-Jun NH2-terminal kinase among others (37-40). The p53 protein also can modulate in some circumstances cellular sensitivity to radiotherapy (41) and chemotherapeutic drugs (42-44).

VRK1, a new regulator of p53, phosphorylates p53 in Thr18 (22, 45), resulting in its stabilization and favoring its interaction with the transcriptional coactivator p300 (16). Phosphorylated Thr18 affects the interaction of p53 with hdm2 (46, 47), and it is acquiring more relevance lately (48, 49), particularly because phosphorylation in other better known residues, such as Ser15 or Ser20, seems to be dispensable for p53 activity (49-51). The phosphorylation of p53 Thr18 has been associated with cellular senescence (52) and with the response to Taxol (53). These observations led to the proposal that VRK1 might be a component of a novel mechanism that controls basal p53 levels during normal proliferation, or suboptimal stress situations, and thus permits the cell to respond when situations of severe stress arise (16).

In this work, we have studied in a series of squamous head and neck carcinomas the correlation between VRK1 expression at the protein level with regard to the p53 pathway and other proteins related with the proliferation phenotype, as well as identified the role of VRK1 as a regulatory factor of genes implicated in cell cycle control, such as cyclin-dependent kinase 2 (CDK2) and survivin. It is postulated that VRK1 plays a role in both G1-S and G2-M progression of the cell cycle.

Expression of VRK1 in Normal Squamous Epithelium

The expression of human VRK1 was determined in normal epithelium. The determination was done in a tonsil that has a well-characterized squamous epithelium. VRK1 was detected as present particularly near the basal layer where cellular proliferation takes place, and as the epithelial cells differentiate, the signal for VRK1 is lost (Fig. 1, top). VRK1 was also detected in many lymphocytes within the follicles. The same sample was analyzed for the expression of the Ki67 antigen, a typical proliferation marker; this antigen is also present in the same compartment as VRK1, although fewer cells were stained (Fig. 1, bottom). We conclude from these observations that both VRK1 and Ki67 seem to be located within the proliferation area of squamous epithelia.

FIGURE 1.

Expression of human VRK1 in normal squamous epithelium. A biopsy from a human tonsil was stained for immunohistochemical detection of the VRK1 (top) and Ki67 (bottom) proteins. In the section, the normal squamous epithelium and part of the follicle with lymphocytes are clearly identified. The staining in the epithelium is located in the zone of proliferation. VRK1 was detected with a rabbit polyclonal antibody (VE1). The Ki67 antigen was detected with MIB1 antibody.

FIGURE 1.

Expression of human VRK1 in normal squamous epithelium. A biopsy from a human tonsil was stained for immunohistochemical detection of the VRK1 (top) and Ki67 (bottom) proteins. In the section, the normal squamous epithelium and part of the follicle with lymphocytes are clearly identified. The staining in the epithelium is located in the zone of proliferation. VRK1 was detected with a rabbit polyclonal antibody (VE1). The Ki67 antigen was detected with MIB1 antibody.

Close modal

VRK1/p53 Pathway in Head and Neck Tumors

The substrates thus far identified regarding the enzymatic activity of VRK1 are transcription factors (23, 24) and include p53 (16, 22). Therefore, it was decided to study if a correlation could be detected between the levels of VRK1 protein and those of p53 (Table 1A) and p53 response proteins, such as hdm2 or p21. For these four proteins, a panel of 64 HNSCCs was available for study in a tissue array. Different levels of VRK1 protein could be detected by immunohistochemistry in different tumors, ranging from almost no expression to very high level expression (Fig. 2). As shown in Table 1, the immunostaining for hdm2 and p21 was grouped into two categories and compared with the levels of VRK protein. As can be observed, as the level of VRK1 increased, so was the level of the p53 targets, such as hdm2 and p21, indicating an augmented activity of p53-dependent transcriptional regulation. The correlation reached statistical significance between VRK1 and Hdm2 (P < 0.02; Table 1B) but not between p21 (P < 0.6) and p53 (P < 0.2; Table 1C), although a positive correlation trend was clearly detectable with these two proteins. In the positive case shown in Fig. 1, p53 is functional because the proteins hdm2 and p21 coded by p53-dependent genes are also increased. However, and although there seemed to be an association between levels of p53 and VRK1 (Table 1A), it is possible that determination of p53 gene status, instead of only protein immunostaining, would lead to more definitive results. This was not possible in the present study because there was no DNA available to determine if p53 was mutated and thus stabilized. The inverse correlation between p53 and hdm2 is also detectable (Table 1D).

Table 1.

Correlations of VRK1 with p53, hdm2, and p21 in Head and Neck Tumors with Wild-type p53

Antigen levels (score)VRK1 (0)VRK1 (1)VRK1 (2)Totals
A.      
    p53 (−) 10 (71%) 30 (85%) 8 (53%) 48  
    p53 (+) 4 (29%) 5 (15%) 7 (47%) 16  
    Totals 14 35 15 64  
B.      
    hdm2 (−) 12 (86%) 24 (68%) 9 (60%) 45  
    hdm2 (+) 2 (14%) 11 (32%) 6 (40%) 19  
    Totals 14 35 15 64  
C.      
    p21 (−) 8 (57%) 21 (60%) 5 (33%) 34  
    p21 (+) 6 (53%) 14 (40%) 10 (67%) 30  
    Totals 14 35 15 64  
      
Antigen levels (score)
 
p53 (0)
 
p53 (1)
 
p53 (2)
 
p53 (3)
 
Totals
 
D.      
    hdm2 (−) 24 (89%) 7 (77%) 8 (66%) 6 (37%) 45 
    hdm2 (+) 3 (11%) 2 (13%) 4 (33%) 10 (63%) 19 
    Totals 27 12 16 64 
Antigen levels (score)VRK1 (0)VRK1 (1)VRK1 (2)Totals
A.      
    p53 (−) 10 (71%) 30 (85%) 8 (53%) 48  
    p53 (+) 4 (29%) 5 (15%) 7 (47%) 16  
    Totals 14 35 15 64  
B.      
    hdm2 (−) 12 (86%) 24 (68%) 9 (60%) 45  
    hdm2 (+) 2 (14%) 11 (32%) 6 (40%) 19  
    Totals 14 35 15 64  
C.      
    p21 (−) 8 (57%) 21 (60%) 5 (33%) 34  
    p21 (+) 6 (53%) 14 (40%) 10 (67%) 30  
    Totals 14 35 15 64  
      
Antigen levels (score)
 
p53 (0)
 
p53 (1)
 
p53 (2)
 
p53 (3)
 
Totals
 
D.      
    hdm2 (−) 24 (89%) 7 (77%) 8 (66%) 6 (37%) 45 
    hdm2 (+) 3 (11%) 2 (13%) 4 (33%) 10 (63%) 19 
    Totals 27 12 16 64 
FIGURE 2.

VRK1/p53/hdm2 pathway in head and neck squamous cell carcinomas. Immunohistochemical detection of the VRK1 protein and its relation with p53 and p53 response proteins hdm2 and p21. Negative (left) and positive (right) cases. The four proteins were analyzed in the same tumor present in a tissue microarray to show the range and variation of the signal. In the positive case, p53 appears to be functional because the levels of two proteins (p53 and hdm2) expressed from p53-dependent genes are also high.

FIGURE 2.

VRK1/p53/hdm2 pathway in head and neck squamous cell carcinomas. Immunohistochemical detection of the VRK1 protein and its relation with p53 and p53 response proteins hdm2 and p21. Negative (left) and positive (right) cases. The four proteins were analyzed in the same tumor present in a tissue microarray to show the range and variation of the signal. In the positive case, p53 appears to be functional because the levels of two proteins (p53 and hdm2) expressed from p53-dependent genes are also high.

Close modal

No correlation between VRK1 levels and survival could be detected in this series of patients (data not shown), probably due to the advanced stage of the disease at the time of diagnosis.

Relation between Proteins Implicated in the Proliferation Phenotype and VRK1

The proliferation phenotype is a hallmark of the cancer phenotype resulting from alterations in growth regulation (54). The p53 protein is one of the main regulatory proteins of the cell cycle, participating in several checkpoints. Because p53 is a target of VRK1 and because of the role of p53 in cell cycle regulation, several proteins associated with cell cycle progression and cellular proliferation phenotype were analyzed in these HNSCC cases. The markers determined by immunohistochemistry in addition to VRK1 were bcl2, p21, p53, p27, p16, hdm2, cdk2, cdk6, cdc2, cyclin A, cyclin B1 (nuclear and cytosolic), cyclin D1, cyclin D3, topoisomerase II α, survivin, chk2, Ki67, Rb, and phosphorylated Rb (Rb-P). All these markers were quantified and analyzed with respect to VRK1 protein level. Nine of them presented a positive correlation with VRK1 that reached statistical significance (Table 2). Interestingly, the correlation was associated with several markers related with the proliferation and cell cycle progression phenotype. No statistically significant correlation was found with the remaining proteins studied, although it is likely that with a larger sample, some of them might also be significant. Interestingly, it did not correlate with cyclins D1 and D3 that are required early in the G1 phase.

Table 2.

Positive Correlation of VRK1 with Proteins Implicated in the Proliferation Phenotype in HNSCC

ProteinP (P < 0.05)
hdm2 0.02 
CDK2 0.01 
CDK6 0.009 
CB1N (cyclin B1 nuclear) 0.02 
Topoisomerase II 0.01 
Survivin 0.01 
Cdc2 0.001 
Ki67 0.03 
Cyclin A 0.008 
ProteinP (P < 0.05)
hdm2 0.02 
CDK2 0.01 
CDK6 0.009 
CB1N (cyclin B1 nuclear) 0.02 
Topoisomerase II 0.01 
Survivin 0.01 
Cdc2 0.001 
Ki67 0.03 
Cyclin A 0.008 

NOTE: The statistical analysis is described in Materials and Methods. Only the proteins with P < 0.05 are shown.

The staining of some of these antigens that positively correlated with VRK1 is shown in Fig. 3. The Rb-P protein (P < 0.09), an important protein for cell cycle progression, CDK2, and survivin staining are shown (Fig. 3), because they were selected for further study to try to establish a link between them and VRK1.

FIGURE 3.

Immunohistochemical detection of the expression of some proliferation markers Rb, CDK2, and survivin that positively correlates with VRK1 in HNSCC. Example of negative (−) and positive (+) cases for the antigens to illustrate the difference between cases.

FIGURE 3.

Immunohistochemical detection of the expression of some proliferation markers Rb, CDK2, and survivin that positively correlates with VRK1 in HNSCC. Example of negative (−) and positive (+) cases for the antigens to illustrate the difference between cases.

Close modal

VRK1 Activates the CDK2 Gene Promoter

Next, the potential connection between VRK1 levels and other proteins that positively correlated in this series of HNSCC was determined. Based on the tumor expression data, and the fact that VRK1 can activate transcription factors, it was decided to test if VRK1 could be an activator of genes coding for proteins with which VRK1 was detected as having a positive correlation in these HNSCC cases. For this aim, it was tested if VRK1 expression could affect the transcriptional activity of the CDK2 and SURVIVIN gene promoters. The association between VRK1 and the CDK2 and/or survivin proteins might be due to two different possibilities. One is that because VRK1 can activate transcription factors, both CDK2 and SURVIVIN genes are located in the response pathway of VRK1 activation and thus the observation of their common overexpression. Alternatively, the three protein (VRK1, CDK2, and survivin) expression levels might be a common component of the same phenotype and respond perhaps to another common signal.

To attempt to discriminate between the two possibilities, the effect of VRK1 overexpression on the human CDK2 gene promoter activity was first determined. The human CDK2 gene plays a role mainly late during G1-S and G2-M phases of the cell cycle (55-57). The CDK2 gene promoter was analyzed in Cos1 cells that were transfected with a plasmid pCEFL-HA-VRK1 expressing VRK1 and different constructs of the CDK2 gene promoter linked to the luciferase reporter gene (58). The CDK2 promoter has some activity by itself but was further activated by VRK1 (Fig. 4A), and an additional increase in activity was identified as mediated by an element located between nucleotides −68 and −440 in the promoter sequence. This effect is a potentiation of the response already observed in the absence of VRK1 (Fig. 4A). When the −2400 promoter region was included, the increase in activity was not detected when VRK1 was overexpressed, suggesting that the VRK1 role in the regulation of the CDK2 gene promoter is complex.

FIGURE 4.

VRK1 activates the human CDK2 gene promoter. A. Cos1 cells line were transfected with 1 μg of luciferase constructs containing several fragments of the CDK2 gene promoter in the pGL2 plasmid and the pRL-tk-luciferase as internal control with and without 2 μg of pCEFL-HA-VRK1. The dual luciferase was determined as described in Materials and Methods. The specific promoter-luciferase activity was normalized with Renilla luciferase used as internal control and shown as a relative value. Columns, mean of three independent experiments determined each by triplicate; bars, SD. As controls for the detection system, the plasmids pGL2-basic and pGL2-control (SV40 enhancer/promoter) were used. B. VRK1 dose response of the CDK2 gene promoter construct up to the −683 nucleotide. Cos1 cells were transfected with 1 μg of pCDK2(-683)-luc and the indicated amount of plasmid pCEFL-HA-VRK1. This experiment was done three times, and the activity was determined in triplicate. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIGURE 4.

VRK1 activates the human CDK2 gene promoter. A. Cos1 cells line were transfected with 1 μg of luciferase constructs containing several fragments of the CDK2 gene promoter in the pGL2 plasmid and the pRL-tk-luciferase as internal control with and without 2 μg of pCEFL-HA-VRK1. The dual luciferase was determined as described in Materials and Methods. The specific promoter-luciferase activity was normalized with Renilla luciferase used as internal control and shown as a relative value. Columns, mean of three independent experiments determined each by triplicate; bars, SD. As controls for the detection system, the plasmids pGL2-basic and pGL2-control (SV40 enhancer/promoter) were used. B. VRK1 dose response of the CDK2 gene promoter construct up to the −683 nucleotide. Cos1 cells were transfected with 1 μg of pCDK2(-683)-luc and the indicated amount of plasmid pCEFL-HA-VRK1. This experiment was done three times, and the activity was determined in triplicate. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Close modal

To further confirm the effect of VRK1 on the −68 to −440 region of the CDK2 gene promoter, a dose response experiment was done. Cos1 cells were transfected with the CDK2 luciferase construct containing from the −683 position in combination with increasing amounts of the plasmid expressing VRK1. As shown in Fig. 4B, as the amount of VRK1 was increased, so did the activity of this CDK2 promoter region, thus confirming the existence of a VRK1 response element in this promoter.

VRK1 Activates the SURVIVIN Gene Promoter

Survivin is a protein required for cell cycle progression (59), and in some tumors, such as meningiomas, it also varies in correlation with the Ki67 antigen (60). Survivin participates in a mitotic arrest checkpoint regulated by p53 (61), protects the cells from apoptosis, and is usually an indicator of bad prognosis (62). These biological effects are consistent with the role of VRK1 as a regulator of p53. Because there was a positive correlation between VRK1 and survivin protein levels in these biopsies, it was determined if VRK1 could also be implicated in SURVIVIN gene regulation. To study this effect, Cos1 cells were transfected with plasmid pCEFL-HA-VRK1 and several constructs of the SURVIVIN gene promoter with a luciferase reporter. In these experiments, VRK1 induced an up-regulation of SURVIVIN gene expression (Fig. 5A). The response element in the SURVIVIN gene promoter is located between residues −1430 and −649. The survivin promoter has a basal activity level, and its magnitude was increased ∼3-fold in the presence of VRK1, an effect similar to that detected with the CDK2 promoter. The SURVIVIN gene promoter also increased its transcriptional activity in a dose-dependent manner with respect to VRK1 (Fig. 5B).

FIGURE 5.

VRK1 activates the human SURVIVIN promoter. A. The Cos1 cell line was transfected with 0.8 μg luciferase constructs containing the SURVIVIN promoter in the pGL2 plasmid and the pRL-tk-luciferase as internal control with and without 2 μg of pCEFL-HA-VRK1. The dual luciferase was determined as described in Materials and Methods. The specific promoter-luciferase activity was normalized with Renilla luciferase used as internal control and shown as a relative value. As controls for the detection system, the plasmids pGL2-basic and pGL2-control (SV40 enhancer/promoter) were used. Columns, means from three experiments determined in triplicate; bars, SD. B. VRK1 dose response of the SURVIVIN promoter using the construct p-2480-SURV-Luc and the indicated amount of plasmid pCEFL-HA-VRK1. This experiment was done three times, and the activity was determined in triplicate. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIGURE 5.

VRK1 activates the human SURVIVIN promoter. A. The Cos1 cell line was transfected with 0.8 μg luciferase constructs containing the SURVIVIN promoter in the pGL2 plasmid and the pRL-tk-luciferase as internal control with and without 2 μg of pCEFL-HA-VRK1. The dual luciferase was determined as described in Materials and Methods. The specific promoter-luciferase activity was normalized with Renilla luciferase used as internal control and shown as a relative value. As controls for the detection system, the plasmids pGL2-basic and pGL2-control (SV40 enhancer/promoter) were used. Columns, means from three experiments determined in triplicate; bars, SD. B. VRK1 dose response of the SURVIVIN promoter using the construct p-2480-SURV-Luc and the indicated amount of plasmid pCEFL-HA-VRK1. This experiment was done three times, and the activity was determined in triplicate. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Close modal

E2F1 Activates VRK1 Expression

Up to now, all the evidence points to a role of VRK1 in proliferation. Therefore, it was attempted to determine in what phase of the cell cycle VRK1 might be implicated. The transcription factor E2F is one of the main inducers of cell cycle progression through the G1-S phase. Earlier experiments using microarray expression analysis have been shown that expression of E2F1 increases the level of VRK1 message (21). E2F is activated by release from the Rb-E2F complex when Rb is phosphorylated by cyclin D/CDK complexes. In addition, in this series of HNSCC cases, there was a positive correlation between Rb-P protein and VRK1 (P < 0.09). Based on these two lines of evidence, it was decided to determine if there was a sequential connection between the release of free E2F1 by its overexpression and the level of VRK1 protein. For this aim, U2OS cells (p16−/−), which have low levels of endogenous E2F1, were transfected with a plasmid expressing E2F1, or a combination of plasmids expressing the cell cycle inhibitors p16 and Rb as negative control. The level of endogenous VRK1 protein was determined with a specific monoclonal antibody (1F6). As shown in Fig. 6, the E2F1 protein induces a 2-fold increase in VRK1 protein, and the inhibitory combination of p16 plus Rb, used as a negative control, somewhat reduces the endogenous VRK1 basal level. These data suggest that the E2F1 protein required for G1-S transition increases the level of VRK1, an observation consistent with the potential role of VRK1 as a control mechanism during cell cycle and proliferation.

FIGURE 6.

Effect of overexpression of E2F1 on VRK1 protein level in U2OS cells (p16−/−). A. Immunoblot detection of the effects of E2F1 or the combination of p16 and Rb on VRK1 levels. This Western blot is a representative experiment. B. Quantification of the effects relative of E2F1 or the combination of p16 and Rb on VRK1 levels. Columns, mean of three experiments; bars, SD. *, P < 0.01. The cells were transfected with the indicated amount of plasmids, pCMV-E2F1 or the combination of pX-p16 and pCMV-Rb, expressing the proteins indicated in the graph, and the VRK1 protein was determined with a specific monoclonal antibody (clone 1F6) 48 hours after transfection in whole-cell lysates. The signal was quantified in the linear response range and was normalized with respect to the β-actin level. As control for proteins that inhibit cell proliferation, a transfection was done using a combination of p16 and Rb expression plasmids (in these transfections, equal amounts of each plasmid were used, and the amount indicated corresponds to the total specific plasmid DNA used). Immunoblot from a representative experiment. The details of the experiment are described in Materials and Methods.

FIGURE 6.

Effect of overexpression of E2F1 on VRK1 protein level in U2OS cells (p16−/−). A. Immunoblot detection of the effects of E2F1 or the combination of p16 and Rb on VRK1 levels. This Western blot is a representative experiment. B. Quantification of the effects relative of E2F1 or the combination of p16 and Rb on VRK1 levels. Columns, mean of three experiments; bars, SD. *, P < 0.01. The cells were transfected with the indicated amount of plasmids, pCMV-E2F1 or the combination of pX-p16 and pCMV-Rb, expressing the proteins indicated in the graph, and the VRK1 protein was determined with a specific monoclonal antibody (clone 1F6) 48 hours after transfection in whole-cell lysates. The signal was quantified in the linear response range and was normalized with respect to the β-actin level. As control for proteins that inhibit cell proliferation, a transfection was done using a combination of p16 and Rb expression plasmids (in these transfections, equal amounts of each plasmid were used, and the amount indicated corresponds to the total specific plasmid DNA used). Immunoblot from a representative experiment. The details of the experiment are described in Materials and Methods.

Close modal

Different types of evidence suggested that VRK1 is likely to play a role in cell proliferation. In this work, a positive correlation has been found between VRK1 and a large number of proliferation-related proteins, suggesting that this VRK1 correlation is consistent with its implication in different aspects of this phenotype and cell cycle regulation in the context of HNSCC. The advanced stage of the tumors analyzed is better suited to find a correlation with phenotypic aspects of the dominant tumor cell population than with their clinical characteristics.

It has been postulated that the role of VRK1 in normal proliferation is to maintain the p53 molecule in a readiness state that will permit cell cycle progression under nonstress situation (16). Regarding the p53 pathway, the accumulation of some p53 response proteins, such as hdm2, was expected, because the phosphorylation of p53 in Thr18 by VRK1 partially stabilizes a transcriptionally active p53 molecule that can not interact with hdm2; consequently, hdm2 expression was increased as a result of the transcription potential of p53 (16). However, the correlation with the level of p53 is less clear, although a positive trend was observed. This may be due in part to the occurrence of p53 mutations that might behave differently (63-65); in these patients, the mutational status of p53 could not be determined; and also to the existence of an autoregulatory loop between VRK1 and p53.6

6

Unpublished results.

However, in most cases of this series where p53 correlated with VRK1, it also correlated with p21 and hdm2, which suggests that at least in this group, p53 must be wild type because it seemed to be functional.

The correlation with several proliferation markers, such as some cdks and cyclins, is consistent with a role in the context of cell cycle regulation, notwithstanding other functions that remain to be identified. Several of the proteins that positively correlated with VRK1 in this series were already known to be prognostic markers in HNSCC; these include cdc2 (66), cyclin A (67), and survivin (68), among others (69). Another protein, Ki67, is a generally accepted proliferation marker and is widely used for this purpose in tumor analysis (70). However, in this series, VRK1 could not be used for a prognostic correlation because of the already advanced stage of the tumors.

The expression of cdk2 and cdk6 clearly indicates a potential role associated with cell cycle progression. The positive correlation with some cdk, such as cdk2, cdk6, or cdc2, indicates that VRK1 might be required for a function that is necessary at the same time that these proteins, probably late in G1 to the G2-M phase. CDK6 phosphorylates Rb in the G1-S transition, thus releasing the transcription factor E2F1, and increased CDK2 activity is a consequence of this activation and cell cycle progression. This role is further supported by the positive correlation with phosphorylated Rb and with the accumulation of nuclear cyclin B1 required for progression through the G2-M phase of the cell cycle. In this context, survivin also promotes progression by relieving the inhibition of cdk2 complexes (59). However, this may not be the only role for VRK1 within correct cell cycle progression. The positive correlation with markers, such as survivin, a protein required for stable checkpoint activation (71) that also participates in apoptosis protection and correct cell division (72), might indicate additional effects for VRK1. It is interesting to note that survivin has been associated with the response to Taxol (73), which is able to induce the unique phosphorylation of p53 in Thr18 (53), a phosphorylation that can be done by VRK1 (16, 22).

Because VRK1 can regulate transcription factors, it is very likely that it will modulate some of the genes whose proteins have been found associated with proliferation and perhaps provide a clue to what phase of the cell cycle VRK1 might participate. The activation by VRK1 of the gene promoters of two of the proteins with which it correlates positively, CDK2 and SURVIVIN, which are important for different aspects of cell cycle regulation, further supports a role for VRK1 in regulation of genes implicated in cell proliferation. Each gene, CDK2 and SURVIVIN, must have a in their upstream regulatory region a sequence recognized by a transcription factor regulated by VRK1. The activation of the human SURVIVIN gene promoter by VRK1 locates the presence of a functional element between nucleotides −1430 and −649, an active region initially detected in HeLa cells (74). In Cos1 cells, the same response region has been detected, but in the presence of VRK1, there is a significant increase in transcriptional activity. The human CDK2 gene promoter has a response element located between −440 and −68, which is important for basal expression in NIH3T3 cells (58). This element is also functional in Cos1 cells, but this region also increases its transcriptional activity in the presence of VRK1.

Microarrays studies have shown that VRK1 gene expression is up-regulated by activation of the Rb pathway (21) and thus provided additional evidence for a role in proliferation. This has been experimentally confirmed in this report because overexpression of E2F1, a general transcription factor released by activated (phosphorylated) Rb and required for G1-S progression, was able to induce an increase in the level of VRK1 protein. Members of the E2F family have predictive value for lymph node metastasis in HNSCC (75). These data places VRK1 expression late in the G1-S transition of the cell cycle, because after activation of Rb, the E2F1 transcription factor up-regulates VRK1, which is consistent with the lack of correlation between early cyclins D1 and D2 and VRK1. The implication of E2F1 has also been reported to be required for the expression of the cyclin A gene (76, 77); overexpression of cyclin A correlated positively with VRK1 in this study. Furthermore, E2F1 induces phosphorylation and accumulation of p53 (78), an observation consistent with the effects of VRK1 on p53 (16, 22).

All the evidence points to a role for VRK1 required throughout the progression of the cell cycle and perhaps with more than one role. If that is the case, it is likely to be placed at some point high in the pathway so that more than one control point might be affected. This generic role for VRK1, or the existence of other proteins with such a role, must exist in higher eukaryotes because other proteins, such as cdk2 or cdk4, seem to be dispensable in mammalian cell cycle progression as shown in the corresponding knock-out mice (79).

HNSCCs have a poor prognosis and are very resistant to radiotherapy and chemotherapy. Because of that, survival has not improved significantly in the last 20 years. In this context, the identification of new pathways that may be of potential use as diagnostic marker or therapeutic target represents a major aim of cancer research. In this context, the characterization of the VRK1 pathway might be an important new step in the better knowledge and future control of HNSCC.

Samples

The 73 cases of HNSCCs were diagnosed and obtained with written informed consent between 1998 and 2000 in several Spanish hospitals according to the institutional guidelines and ethics review committees. All tumors were in stages III and IV according the WHO classification. The median age of the patients was 56 years; the median overall survival for the cohort was 37.7 months; the median to progression was 29.1 months. The characteristics of these cases have already been reported (67). Tumor biopsies were taken at diagnosis before initiation of treatment. Biopsies were fixed in formalin and embedded in paraffin. These biopsies were used to prepare a tissue array made with a tissue arrayer (Beecher Instruments, Silver Spring, MD) as previously reported (67). The biopsies were examined by two pathologists who selected tumor areas, avoiding those with necrosis, inflammation, and keratinization. To assess reproducibility from each tumor, two separate areas were taken from the cylinders (0.6-mm diameter). Sections (3 μm thick) were cut and transferred to positively charged surface glass slides. Sections were dried for 16 hours at 56°C followed by dewaxing and rehydration through a graded ethanol series and washed with PBS. To retrieve antigens, the slides were treated in a pressure cooker for 2 minutes in 10 mmol/L citrate buffer (pH 6.5). The tissue array slides were then used for immunochemical analysis.

Antibodies

The antibody specific for human VRK1 was a rabbit polyclonal (VE1) previously described and prepared with a GST-VRK1 fusion protein (16). The proteins p53, survivin, cdk2, cdk6, cyclin B1, topoisomerase II, cdc2, ki67, Rb-P, and cyclin A were detected with the following antibodies. CDK2 was detected with antibody clone 8D4 (Neomarkers, Freemont, CA). The p53 was detected with clone DO-7 (Novocastra, Newcastle upon Tyne, United Kingdom); cdc2 with antibody 1 (Transduction Laboratory, Lexington, KY); CDK6 with antibody BD (BD PharMingen, San Diego, CA); cyclin A with antibody GEG (Novocastra); hdm2 with clone 1F2 (Oncogene, La Jolla, CA); and Ki67 with clone M1B1 (DAKO, Glostrup, Denmark). The processing of the biopsies, the dilutions used for primary antibodies, and the secondary antibodies for detection have all been described (67). The cutoff criteria has been previously reported (67). Briefly, hdm2 immunostaining was scored as negative when <5% of tumor cells presented immunoreactivity. Mesenchymal and endothelial cells were used as the internal positive control. For p21, staining was scored as negative when <10% of tumor cells showed immunoreactivity. Nuclear immunostaining of some lymphoid and granulocytic cells was taken as the internal positive control. For p53, immunostaining was scored as negative when <10% of tumor cells showed nuclear staining. In the case of topoisomerase, <5% was considered negative, between 5% and 30% was moderate (level 1), and >30% was high expression (level 2). For VRK1 and survivin, the criteria was 0 when the number of positive cells were below 5%, level 1 when between 5% and 50%, and level 2 with >50% of the population (68, 80).

Statistical Analysis

The statistical analysis and criteria for quantification has been previously described (67). Briefly, the frequencies were compared either by the Fisher's exact test or the χ2 contingency test using the SPSS program version 10.0.5 (SPSS, Inc., Chicago, IL), and differences with a P < 0.05 were considered as statistically significant.

Plasmids, Transfections, Transcription Reporter Assays, and Immunoblots

Plasmid pCEFL-HA-VRK1 has already been described (16). pX-p16, pCMV-E2F1, and pCMV-Rb were from M. Malumbres (Centro Nacional de Investigaciones Oncológicas, Madrid, Spain). Plasmid p-68-CDK2-Luc, p-440-CDK2-Luc, p-683-CDK2-Luc, and p-2400-CDK2-Luc have been previously described (58). The survivin gene promoter constructs pLuc-1430c, pLuc-649c, pLuc-441c, pLuc-230c, and pLuc-42c were previously reported (74). U2OS (p16−/−) and Cos1 cells grown in DMEM supplemented with 10% FCS.

For transfections of Cos1, 300,0000 cells were plated in P35 dishes. The cells were transfected with 0.8 μg of pSurvivin-luc constructs and 50 ng of pRL-tk-luc as internal control, or 1 μg of pCDK-luc constructs and 20 ng of pRL-TK-luc using 6 μL of the JetPEI reagent (Polytransfection, Illkirch, France) according to manufacturer's instructions. Luciferase activity was determined with a Dual luciferase system from Promega (Madison, WI) following the manufacturer's instructions.

For transfections, 500,000 U2OS cells were plated in P35 dishes. After 24 hours, cells were transfected with the indicated amounts of pX-p16, pCMV-E2F1, or pCMV-Rb using 10 μL of the JetPEI reagent from Polytransfection according to manufacturer's instructions. Forty-eight hours after transfection, cells were washed twice with ice-cold PBS; harvested in radioimmunoprecipitation assay buffer [150 mmol/L NaCl, 1.5 mmol/L MgCl2, 10 mmol/L NaF, 10% glycerol, 4 mmol/L EDTA, 1% Triton X-100, 0.1% SDS, 1% deoxycholate, 50 mmol/L HEPES (pH 7.4)], plus 1 mmol/L Na3VO4, 10 μg/mL leupeptin, 10 μg/ml aprotinin, and 1 mmol/L phenylmethylsulfonyl fluoride; incubated on ice for 20 minutes; and precleared by centrifugation at 13,200 rpm for 20 minutes at 4°C in an Eppendorf 5415D minifuge. Thirty micrograms of the extracts were analyzed by SDS-PAGE under denaturing conditions and transferred onto Immobilon-P membranes (Millipore, Bedford, MA) for 1 hour at 90 V. The membranes were blocked with 5% skimmed milk in TBS-T and then incubated with the specific antibody. As secondary antibody, a sheep/anti-mouse/horseradish peroxidase (Amersham Biosciences, Little Chalfont, United Kingdom) was used at a 1:5,000 dilution. The chemiluminescence in the blots was detected using the with the enhanced chemiluminescence reagent from Amersham Biosciences.

Grant support: Fondo de Investigación Sanitaria grant FIS02/0585 (P.A. Lazo); Ministerio de Educación y Ciencia grant SAF2004-02900 (P.A. Lazo); Junta de Castilla y León grants SAN/SA-01/04, SAN/SA-04/05, and CSI05A05 (P.A. Lazo); Fundación de Investigación Medica MM (P.A. Lazo); Ministerio de Ciencia y Tecnología grant SAF2002-01595 (M. Sánchez-Céspedes); Comunidad de Madrid grant CAM 08.1/0032/2003 (M. Sánchez-Céspedes); and Fundación Científica de la Asociación Española contra el Cáncer (F.M. Vega), Fundação para a Ciência e a Tecnologia Portugal (C.R. Santos), Ministerio de Educación y Ciencia (S. Blanco and A. Valbuena), and Consejo Superior de Investigaciones Cientificas predoctoral fellowships (A. Sevilla).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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