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. Author manuscript; available in PMC: 2011 Feb 5.
Published in final edited form as: Virology. 2009 Nov 27;397(1):139. doi: 10.1016/j.virol.2009.10.051

The human papillomavirus type 58 E7 oncoprotein modulates cell cycle regulatory proteins and abrogates cell cycle checkpoints

Weifang Zhang 1,2, Jing Li 2, Sriramana Kanginakudru 1, Weiming Zhao 2, Xiuping Yu 2,3, Jason J Chen 1,3
PMCID: PMC2818596  NIHMSID: NIHMS163372  PMID: 19945133

Abstract

HPV type 58 (HPV-58) is the third most common HPV type in cervical cancer from Eastern Asia, yet little is known about how it promotes carcinogenesis. In this study, we demonstrated that HPV-58 E7 significantly promoted the proliferation and extended the lifespan of primary human keratinocytes (PHKs). HPV-58 E7 abrogated the G1 postmitotic checkpoints, although less efficiently than HPV-16 E7. Consistent with these observations, HPV-58 E7 down-regulated the cellular tumor suppressor pRb to a lesser extent than HPV-16 E7. Similar to HPV-16 E7 expressing PHKs, Cdk2 remained active in HPV-58 E7 expressing PHKs despite the presence of elevated levels of p53 and p21. Interestingly, HPV-58 E7 down-regulated p130 more efficiently than HPV-16 E7. Our study demonstrates a correlation between the ability of down-regulating pRb/p130 and abrogating cell cycle checkpoints by HPV-58 E7, which also correlates with the biological risks of cervical cancer progression associated with HPV-58 infection.

Keywords: HPV-58, E7, checkpoint, Rb, p130, Cdk2, p53, p21

Introduction

The high-risk HPV types are commonly associated with lesions that can progress into cervical carcinoma, which is one of the leading causes of cancer death in women, worldwide (zur Hausen, 2002). The oncogenic properties of high-risk HPVs primarily reside in the E6 and E7 genes. The ability of high-risk HPV E6 and E7 proteins to promote the degradation of tumor suppressors p53 and pRb, respectively, has been suggested as a mechanism by which HPV induces cellular transformation (reviewed in (Fan and Chen, 2004)). The E7 proteins from high-risk HPVs also bind and target other Rb family members, p107 and p130, for degradation of (Dyson et al., 1992; Gonzalez et al., 2001; Jones and Munger, 1997).

p53 and pRb family members regulate cell cycle checkpoints and maintain genomic stability (Burkhart and Sage, 2008; Stewart and Pietenpol, 2001). p53 controls these processes mainly by regulating transcription of target genes (Levrero et al., 2000). One of the most important targets of p53 for cell cycle regulation is p21, which is a universal inhibitor of cyclin and cyclin-dependent kinase (Cdk) (Xiong et al., 1993). pRb binds to E2F and inhibits its transcriptional activation of genes, including cyclin A and cyclin E, which are important for the G1-to-S phase transition (Cobrinik, 2005). Inactivation of p53 and Rb family members by HPV E6 and E7 abrogates cell cycle checkpoints and induces genomic instability, which is believed to be important for cervical carcinogenesis (reviewed in (Fan and Chen, 2004)).

Worldwide, HPV types 16 and 18 (HPV-16 and -18) together account for approximate 70% of all cervical cancers (Clifford et al., 2006). Although we have already learnt a lot about the mechanism by which several high-risk HPV types such as HPV-16 and -18 induce cellular transformation, little is known about how other high-risk, cervical cancer-related HPV types promote carcinogenesis. With the availability of HPV vaccines for HPV-16 and -18, it is important to devote some of our effort to study other high-risk HPV types. HPV-58 is the third most common HPV type in cervical cancer from Eastern Asia, accounting for 9.8% of cervical cancer (Parkin, Louie, and Clifford, 2008). The regional importance of HPV-58 in Eastern Asia can also been seen with their prevalence associated with normal cytology, low-grade squamous intraepithelial lesion (LSIL), and high-grade squamous intraepithelial lesion (HSIL) (Parkin, Louie, and Clifford, 2008). Specifically, in Eastern Asia, HPV-58 is the third most common HPV type in women with normal cytology or LSIL, and the second most common HPV type in women with HSIL. The propensity of HPV-58 infected individuals to progress to cervical cancer is lower than those of HPV-16 infected patients (Parkin, Louie, and Clifford, 2008). A recent study demonstrated the ability of HPV-58 to immortalize PHKs, the E6 protein of HPV-58 to bind PDZ proteins and to promote the degradation of p53 (Hiller et al., 2006; Muench et al., 2009). However, these HPV functions do not seem to be reliable predictors for carcinogenic behavior of HPV in the cervix. So far, we know nothing about the biological activities of HPV-58 E7.

In the present study, we explored the biological activities of HPV-58 E7 in PHKs. Our results demonstrate that HPV-58 E7 promotes cell proliferation, extends lifespan of PHKs, and abrogates cell cycle checkpoints, though does so less efficiently than HPV-16 E7. We also found that Cdk2 remained active in HPV-58 E7 expressing PHKs despite the presence of elevated levels of p53 and p21. Notably, HPV-58 E7 down-regulates both pRb and p130. Thus, there is a correlation between the propensity of HPV-58-induced cervical cancer progression and the ability of HPV-58 E7 to target pRb/p130 leading to abrogation of cell cycle checkpoints.

Results

HPV-58 E7 promotes proliferation and extends the lifespan of PHKs

The HPV-58 E7 (GI No. 222386) and HPV-16 E7 (GI N0. 9627100) are quite similar in their amino acids composition (60% identify and 75% similarity) (Fig. 1A). Like HPV-16 E7, HPV-58 E7 contains the LXCXE pRb-binding motif, a casein kinase II (CKII) phosphorylation motif, and two Cys-X-X-Cys motifs. Stable cell lines were created by infection of PHKs with retroviruses containing HPV-58 E7 (PHK-58 E7) or an empty vector (PHK-Vector). PHKs expressing HPV-16 E7 (PHK-16 E7) were included in the study as a control. HPV-58 E7 expression in the stable cell lines was confirmed by RT-PCR (Fig. 1B).

Figure 1.

Figure 1

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Sequence, expression, proliferation, and lifespan extension of PHK-58 E7. (A). Sequence alignment of HPV-58 E7 and HPV-16 E7 proteins. Similar residues are shaded in black. Amino acid sequences corresponding to Conserved regions (CR1, CR2 and CR3), Retinoblastoma protein binding domain (pRB), Casein Kinase II phosphorylation domain (CKII) and Zinc Finger (ZF) domains are indicated. (B) Total RNA isolated from PHK-58 E7 and PHK-vector was subjected to RT-PCR. β-actin was used as a control. (C) PHK-16 E7, -58 E7, and -Vector were seeded in 6-well plate at 5×104. Cell numbers were counted every 24 hours. Error bars represent standard deviations of the means. Data from a representative experiment of two are shown. (D) HPV E7 expressing PHKs or control PHKs were passaged continuously until they cease to grow or 5 months. The number of cell doublings was plotted against days in culture.

HPV-58 E7 promoted the proliferation of PHKs. As shown in Figure 1C, within four days, PHK-58 E7 proliferated as efficiently as PHK-16 E7 and significantly more rapidly than the vector control cells (p < 0.001). HPV-58 E7 also significantly extended the lifespan of PHKs (Fig. 1D). While the vector control PHKs stopped dividing at passage 5 or after approximate 22 population doublings (PDs), PHKs expressing both HPV-58 E7 and HPV-16 E7 continued to proliferate and they did not show any crisis period. After 5 months in culture, PHK-58 E7 multiplied 82 PDs while PHK-16 E7 multiplied for 90 PDs) (Fig. 1D). The E7 expressing PHKs are still proliferating after 7 months in culture and so far have multiplied beyond 100 PDs.

HPV-58 E7 abrogates cell cycle checkpoints

It is well established that HPV-16 E7 abrogates the G1 cell cycle checkpoint (Reviewed in (McLaughlin-Drubin and Munger, 2009)). To examine the ability of HPV-58 E7 to modulate the G1 checkpoint, we treated PHKs expressing HPV-58 E7 with bleomycin, a radiomimetic agent capable of inducing both single-stranded and double-stranded DNA breaks that lead to cell cycle arrest at G1 and G2 checkpoints (Chen and Stubbe, 2005). As expected, upon bleomycin treatment, a reduction in the number of S phase cells occurred in the vector control PHKs (Fig. 2). Other than that, there were no major changes in cell cycle profile for these cells, suggesting that PHK-Vector arrested at both G1 and G2 checkpoints upon bleomycin treatment. In contrast, PHK-58 E7 showed a decreased number of cells in the G1 phase of the cell cycle (39% in PHK-58 E7vs. 52.1% in PHK-Vector) and an increase in the number of S phase cells (20.2% vs. 14.2%). These results demonstrate that HPV-58 E7 can abrogate the DNA damage-induced G1 checkpoint. Similarly, PHK-16 E7 also showed decreased number of cells in the G1 phase and increased number of cells in S phase (Fig. 2). HPV-16 E7 appears to be more efficient in alleviating the G1 checkpoint.

Figure 2.

Figure 2

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HPV-58 E7 abrogates cell cycle checkpoints and induces polyploidy in PHKs. Asynchronous cultures of PHK-16 E7, -58 E7 and -Vector were treated with DMSO, bleomycin (10 µg/mL for twenty-four hours) or nocodazole (50 ng/mL for sixty hours). After the feeder cells were removed, PHKs were collected, fixed, stained with PI, and analyzed by flow cytometry. G1 phase, S phase, G2/M phase and polyploidy cells are indicated. Data are from a representative experiment of two.

We have recently shown that in response to mitotic stress, HPV-16 E7 induces polyploidy by abrogation of the postmitotic checkpoint (Heilman et al., 2009), which could functionally prevent tetraploid cells from entering S phase. Polyploidy has been shown to occur as an early event in cervical carcinogenesis and to predispose the cells to aneuploidy (Olaharski et al., 2006). Accordingly, we treated cells with the microtubule poison nocodazole, which activates the spindle checkpoint and subsequently the postmitotic checkpoint. After treating with nocodazole, PHK-58 E7 showed an increase in polyploidy compared to vector controls (26.2% vs. 10.6%, Figure 2). However, HPV-58 E7 is less efficient than HPV-16 E7 in inducing polyploidy (26.2% vs. 44.2%). Taken together, our results suggest that HPV-58 E7 abrogates the postmitotic checkpoint and induces S phase entry in cells containing tetraploid genome.

HPV-58 E7 up-regulates Cdk2 activity in PHKs

Cdk2 is a major regulator in S phase entry, although its function can be compensated by other Cdks in its absence (Reviewed by (Satyanarayana and Kaldis, 2009)). To explore the mechanism by which HPV-58 E7 abrogates cell cycle checkpoint, we examined the expression of Cdk2 and Cdk2-associated cyclins as well as its kinase activity in PHK-58 E7. As shown in Fig. 3A, the steady-state levels of Cdk2 were up-regulated for approximately 10-fold in PHK-58 E7 or PHK-16 E7. The regulatory partners of Cdk2, cyclin A and cyclin E, were also increased in these cells (Fig. 3A). These results are consistent with the previous observations of E7 from HPV-16 and some other potential high-risk HPV types ((Mansour et al., 2007) and references therein). In contrast, there was no significant difference in the steady-state levels of Cdk6 in E7 expressing PHKs and the vector control PHKs. Cdk6, or structurally and functionally similar Cdk4, is the partner of D-type cyclins and an early G1 checkpoint regulator. Because pRb, the E7 degradation target, seems to be the only essential substrate for Cdk6 or Cdk4, they are not expected to be up-regulated by HPV E7.

Figure 3.

Figure 3

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Expression and activities of cyclins and Cdks in PHK-58 E7. (A) 50 µg of whole cell extracts of PHK-58 E7, -16 E7, and -Vector were resolved by SDS-PAGE and examined by Western blot with anti-Cdk2, -cyclin E, -cyclin A, and -Cdk6 antibodies as indicated. A representative β-actin blot is shown as a loading control. (B) Elevated Cdk2 activity in PHK-58 E7. 100µg of total protein extracts of PHK-58 E7, -16 E7, and -Vector were immunoprecipitated with anti-Cdk2 and anti-Cdk6 antibodies respectively, in vitro kinase assay were performed with full length Rb protein as a substrate. Data are from a representative experiment of three.

What is more important to note is the up-regulation of the Cdk2-associated kinase activity in E7-expresing PHKs (for more than 2-fold) as compared with the vector control cells (Fig. 3B). There were no significant differences between HPV-16 E7 and HPV-58 E7 expressing PHKs in Cdk2-associated kinase activities. In contrast and consistent with the expression levels, the kinase activity of Cdk6 were similar in E7 and vector containing PHKs (Fig. 3B). These results suggest a role of Cdk2 and its associated cyclins in abrogation of cell cycle checkpoint induced by HPV-58 E7. However, since HPV-58 E7 is less efficient in abrogating cell cycle checkpoints but maintains similar level of Cdk2-kinase activity in PHKs as compared with HPV-16 E7, Cdk2 does not appears to be the limiting factor.

Up-regulation of p53 and p21 in HPV-58 E7 expressing PHKs

p53 plays a key role in mediating both G1 and the postmitotic checkpoint (Andreassen et al., 2001; Lanni and Jacks, 1998; Sablina et al., 1999). p21 is responsible for at least part of p53-mediated G1 and postmitotic checkpoints (Khan and Wahl, 1998; Lanni and Jacks, 1998; Stewart, Leach, and Pietenpol, 1999). Previous studies demonstrated that HPV-16 E7 overrides the tumor suppressive activities of p53 and p21 and proliferate efficiently in the presence of elevated levels of p53 and p21 (Demers et al., 1994; Funk et al., 1997; Jones, Alani, and Munger, 1997). Furthermore, HPV-16 E7 overrides the tumor suppressor activity of p21 in a transgenic model of cervical carcinogenesis (Shin et al., 2009). We therefore examined the expression of p53 and p21 in HPV-58 E7 expressing PHKs. As shown in Fig. 4A, the steady-state levels of both p53 and p21 were increased (for more than 3-fold) in E7 expressing PHKs, despite the fact that the G1 and postmitotic checkpoints are abrogated in these cells (Fig. 2). These results are consistent with previous observation with HPV-16 E7 (Demers et al., 1994; Funk et al., 1997; Jones, Alani, and Munger, 1997) and suggest that HPV-58 E7, like HPV-16 E7, overrides the tumor suppressor activity of p53 and p21.

Figure 4.

Figure 4

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Expression of p53 and pRb family members in PHK-58 E7. 50 µg of whole cell extracts of PHK-58 E7, -16 E7, and -Vector were resolved by SDS-PAGE and examined by Western blot analysis with (A) anti-p53 and anti p21 antibodies. (B) anti-pRb family member antibodies. For the p130 blot, the upper band is believed to be a nonspecific cross-reaction from an unknown protein and is marked with a star. A representative β-actin blot is shown as a loading control. Data are from a representative experiment of at least three.

Down-regulation of pRb and p130 in HPV-58 E7 expressing PHKs

It has been suggested that the ability of high-risk HPV E7 proteins to bind and destabilize pRb family members is necessary for abrogating the G1 checkpoint (McLaughlin-Drubin and Munger, 2009). Our recent study suggests a role of Rb inactivation in abrogating the postmitotic checkpoint by HPV-16 E7 (Heilman et al., 2009). We therefore examined the steady-state levels of pRb family members in HPV-58 E7 expressing PHKs by Western blot. As shown in Fig. 4B, the overall steady-state levels of pRb were reduced (for more than 1-fold) in PHK-58 E7 as compared with the PHK-vector. Notably, the levels of pRb were consistently higher in PHK-58 E7 than in PHK-16 E7. Thus, the steady-state levels of pRb in E7 expressing PHKs correlate with the ability of E7 to abrogate cell cycle checkpoints. These results suggest that degradation of pRb is a mechanism by which HPV-58 E7 regulate cell cycle checkpoints.

Surprisingly, the steady-state levels of p107 did not change significantly in PHKs expressing both HPV E7s as compared with the vector control PHKs (Fig. 4B). These results indicate that destabilization of p107 does not play an important role in checkpoint abrogation by HPV-58 E7, at least under our experimental conditions. On the other hand, the steady-state levels of p130 were significantly reduced (for more than 2-fold) in PHK-58 E7 (Fig. 4B). Notably, the steady-state levels of p130 in PHK-58 E7 were consistently and significantly lower than that of PHK-16 E7, despite the fact that HPV-58 E7 is less efficient in abrogating cell cycle checkpoints than HPV-16 E7 (Fig. 2). The significance of more efficient p130 down-regulation by HPV-58 E7 remains to be explored. Taken together, the ability of HPV-58 E7 to reduce the steady-state levels of pRb and p130, but not p107, correlated with its ability to abrogate cell cycle checkpoints.

Discussion

A meta-analysis indicates that HPV-16 is more prevalent in squamous cell carcinoma of the cervix (SCC) than high-grade squamous intraepithelial lesions (HSIL), whereas the reverse is true for other oncogenic types including HPV-58 (Clifford et al., 2003). These data suggest that HSILs infected with HPV16 preferentially progress to SCC while those infected with HPV-58 do not. The mechanism for the difference is not well understood. More efficient down-regulation of pRb and abrogation of the cell cycle checkpoints by HPV-16 E7 as compared with HPV-58 E7, as shown in this study, may be a factor for the differential progression.

In contrast to what was previously observed, we did not detect a significant change in the steady-state levels of p107 in HPV-16 E7 expressing PHKs (Fig. 4B). We noticed that most of the previous studies did not express HPV-16 E7 in keratinocytes and the results are therefore not comparable. In the two studies where the steady-state levels of p107 were examined in PHKs (Helt and Galloway, 2001; Zhang, Chen, and Roman, 2006), serum-free keratinocyte medium was used. Keratinocyte culture condition may therefore play a role in destabilization of p107 by E7. In support of this notion, it was shown that HPV-16 E7 destabilized p107 in keratinocytes cultured in serum-free but not in serum-containing medium (Zhang, Chen, and Roman, 2006).

We have demonstrated in the present study that HPV-58 E7 is more efficient than HPV-16 E7 in down-regulation of p130. The significance of this observation is yet to be known. Two recent studies have demonstrated that destabilization of p130 is the shared activity of both low-risk and high-risk HPV E7s and plays a role in decreasing keratinocytes differentiation or inducing S-phase reentry (Genovese et al., 2008; Zhang, Chen, and Roman, 2006). It is quite likely that down-regulation of p130 contributes to the life cycle of HPV-58. It also remains possible that down-regulation of p130 contributes to the ability of HPV-58 E7 to activate Cdk2, whose activity has been shown to be inhibited by p130 ((Lacy and Whyte, 1997) and references therein). This may explain why Cdk2 is equally active in PHKs expressing HPV-58 E7 or HPV-16 E7, though they express different amount of pRb (Fig. 3B and Fig. 4A). A recent study showed that p130 blocks telomerase-independent telomere lengthening (or alternative lengthening of telomeres (ALT)) (Kong, Meloni, and Nevins, 2006). HPV-16 E7 extended the lifespan of PHKs in the absence of telomerase (Stoppler et al., 1997). In normal human embryonic fibroblasts expressing HPV-16 E7, Yamamoto et al demonstrated that telomere lengths were maintained by the alternative lengthening of telomeres (ALT) mechanism (Yamamoto et al., 2003). Here we demonstrated that HPV-58 E7 significantly extend the lifespan of PHKs. It is therefore possible that down-regulation of p130 by HPV-58 E7 contributes to its ability to induce the extension of PHKs lifespan.

HPV-16 E7 overrides the tumor suppressor activity of p21 in a transgenic model of cervical carcinogenesis (Shin et al., 2009). Several mechanisms have been proposed for activation of Cdk2 by E7 in HPV-infected tissues upon differentiation or DNA damage, where p21 expression is elevated (Zehbe et al., 1999). E7 was shown to associate with cyclin/Cdk2 complexes, either directly or through pRb family members ((McIntyre, Ruesch, and Laimins, 1996; Nguyen and Munger, 2008; Tommasino et al., 1993) and references therein). It was suggested and subsequently shown in vitro that the association of E7 with cyclin/Cdk2 led to their activation (He et al., 2003). It was also suggested that E7 bind and inhibit the functions of p21 (Funk et al., 1997; Jones, Alani, and Munger, 1997; Mansour et al., 2007).

However, others were unable to show efficient E7/p21 interactions (Hickman, Bates, and Vousden, 1997; Ruesch and Laimins, 1997; Westbrook et al., 2002). Induction of S phase in differentiated human keratinocytes in the presence of p21 does not appear to be a frequent event and only occurs when high levels of E7 protein is induced (Banerjee et al., 2006). Some studies suggest that E7 does not prevent p21-mediated inhibition of cyclins/Cdk2 activity but work down-stream by activating E2F1 (Morozov et al., 1997; Ruesch and Laimins, 1997). While p21 is capable of inhibiting E2F activity in the absence of Rb (Dimri et al., 1996), E7 can bind E2F1 and enhance E2F1 mediated transcription (Hwang et al., 2002). On the other hand, E7 can also bind E2F6 and abrogates the ability of E2F6 to repress transcription (McLaughlin-Drubin, Huh, and Munger, 2008). Notably, an E7-associated kinase activity, which is not inhibited following DNA damage, has been detected and shown to phosphorylate pRb (Hickman, Bates, and Vousden, 1997). The identity of the E7-associated kinase remains to be characterized. Another study showed that E7 prevents p21 nuclear accumulation to retain cyclin E-Cdk2 activity in mouse NIH 3T3 cells (Westbrook et al., 2002). The levels of cyclins A and E as well as Cdk2 are higher in E7 expressing cells ((Mansour et al., 2007) and references therein), which may potentially increase the pool of p21-free Cdk2-associated kinase. It remains to be determined the mechanism by which HPV-58 E7 activates Cdk2 in the presence of high levels of p21.

In summary, we observed several biological activities for HPV-58 E7 in PHKs. Down-regulation of pRb and p130 correlated with the ability of HPV-58 E7 to abrogate cell cycle checkpoints and cervical cancer progression. The mechanism by which HPV-58 E7 activates Cdk2 in the presence of p21 remains to be explored.

Materials and methods

Cell culture

PHKs were derived from one neonatal human foreskin epithelium obtained from the University of Massachusetts Memorial Hospital as described (Liu et al.). PHKs were maintained on mitomycin C-treated J2-3T3 feeder cells in F-medium composed of 3 parts Ham’s F12 medium and 1 part Dulbecco’s modified Eagle medium (DMEM) plus 5% fetal bovine serum (FBS) with all supplements as previously described (Flores et al., 2000). Amphotrophic packaging cell line PA317 and J2-3T3 cells were maintained in DMEM plus 10% FBS and antibiotics. To analyze the ability of E7 to extend the lifespan of PHKs, 5 × 104 E7 expressing PHKs were seeded on 10-cm dishes at each split. After they reached 80% confluent, cells were split and cell numbers were counted for calculating PDs versus time.

Retroviral infections

PHKs expressing HPV-16 E7 were described previously (Liu et al., 2007). PHKs expressing HPV-58 E7 were established by retrovirus-mediated infection using the pBabe Puro-based retroviral construct. After puromycin selection, populations of infected cells were pooled and maintained in puromycin-containing medium. HPV-58 E7 expression was confirmed by RT-PCR using the following oligos:

  • HPV-58 E7, sense, 5’- ATGAGAGGAAACAACCCAAC-3’

  • HPV-58 E7, antisense, 5’-AGCTAGGGCACACAATGGTA-3’

  • β-Actin, sense, 5’-TGGCATTGCCGACAGGATGCAGAA-3’

  • β-Actin, antisense, 5’-CTCGTCATACTCCTGCTTGCTGAT-3’.

Immunoblotting and kinase assay

Protein extraction was performed in RIPA lysis buffer (150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCl, pH7.5, 5mM EDTA, protease inhibitors (Complete EDTA-free, Roche). Protein concentrations were determined by bicinchoninic acid (BCA) analysis (Pierce). Equal amounts of protein from each cell lysate were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a PVDF membrane. The membranes were probed with antibodies against pRb (BD Biosciences, 554136), p130 (BD Biosciences, 610261), p107 (Santa Cruz, sc-318), p53 (Santa Cruz, sc-126), p21 (BD Biosciences, 610234), Cdk2 (Santa Cruz, sc-6248), Cdk6 (Santa Cruz, sc-177), cyclin A (Santa Cruz, sc-751), cyclin E (Santa Cruz, sc-198), and β-actin (Sigma, A2066). Immunoreactive proteins were visualized with SuperSignal® West Pico chemiluminescent substrate (Pierce). The membranes were scanned with an LAS-4000 Image Reader (Fuji Photo Film Inc.).

For kinase assay, cells were lysed in lysis buffer (50mM HEPES, pH7.5, 150mM NaCl, 1mM EDTA, 2.5mM EGTA, 0.1%Tween 20, 1mM dithiothreitol, 10% glycerol, 0.1mM phenylmethylsulfony fluoride, 1mM sodium fluoride, 0.1mM sodium orthovanadate and protease inhibitor). 100 µg of total protein extract were immunoprecipitated with appropriate antibodies and protein A/G plus-agarose (Santa Cruz). After incubation for 4 hours at 4¬, the beads were washed three times with lysis buffer without glycerol and twice with kinase buffer (50mM HEPES, pH7.5, 10mM MgCl2, 1mM dithothreitol, 2.5mM EGTA, 0.1mM sodium orthovanadate, 1mM sodium fluoride and protease inhibitor). Assays were performed in the presence of 10 µCi of [γ-32P] ATP (3000 Ci/mmol, PerkeinElmer Life Sciences) and 20µM ATP for 30min at 30J. 1 µg of full length Rb (QED Bioscience Inc.) was used as substrate in each reaction. Following the kinase reaction, samples were boiled in loading buffer and separated by 8% SDS-PAGE. Phosphorylated proteins were visualized by autoradiography.

Flow cytometry

Asynchronous cultures of cells were treated with DMSO (Sigma), Bleomycin (Sigma, 10 µg/mL) or nocodazole (Sigma, 50 ng/mL). The cells were harvested, fixed in 90% ethanol overnight, and resuspended in a PBS-propidium iodide (PI, Sigma, 50 µg/mL)-RNase A (Sigma, 70 µg/mL) solution. The PI stained cells were analyzed by flow cytometry. Cell cycle analysis was performed using FlowJo software (Becton Dickinson).

Statistical analysis

Data were expressed as mean ± standard deviation. Differences between means were assessed by Student's t-test. p ≤ 0.05 was considered significant.

Acknowledgements

We thank Dr. Elliot Androphy for helpful discussions, Dr. Xueli Fan for help preparing PHK-Vector. The project described was supported by Grant Number R01CA119134 from the National Cancer Institute (NCI) and Grant Number R21AI070772 from the National Institute of Allergy and Infectious Diseases (NIAID). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NCI, NIAID, or NIH.

Footnotes

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