The initiation of UV-induced G1 arrest in human cells is independent of the p53/p21/pRb pathway but can be attenuated through expression of the HPV E7 oncoprotein

Martin Loignon and Elliot A. Drobetsky1

Centre de Recherche Guy Bernier, Hôpital Maisonneuve-Rosemont, Faculty of Medecine, University of Montreal, 5415 Boulevard de l'Assomption, Montreal, Quebec HIT 2M4, Canada


    Abstract
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
It is well established that the initiation of G1 arrest in cultured cells exposed to ionizing radiation (IR) is fully dependent upon the p53/p21waf1/pRb signaling cascade. However, the extent to which this pathway regulates G1 arrest following exposure to UV is less clear. Here we demonstrate that primary human fibroblasts from either skin or lung, in which p53 has been functionally inactivated through expression of the human papillomavirus E6 oncoprotein, each undergoes a prolonged G1 arrest upon UV irradiation. This same phenomenon is also observed for UV-exposed human tumor cell strains that are genetically deficient for p53, p21waf1 and/or pRb. Furthermore, for the isogenic wild-type counterparts of these primary and tumor cell strains, the onset of UV-induced G1 arrest precedes any increase in the ratio of hypo- to hyper-phosphorylated pRb and virtually the entire period of growth arrest occurs in the absence of p21waf1 induction. The above data on UV-treated cells are in contrast to the expected situation for IR, for which G1 arrest is abolished in all deficient cell lines, and, in the wild-type counterparts, correlates precisely with p21waf1 induction and an increase in the ratio of hypo- to hyper-phosphorylated pRb. Remarkably, it was observed that both IR- and UV-induced G1 arrest are significantly attenuated in primary fibroblasts expressing the human papillomavirus E7 oncoprotein, which functionally inactivates pRb in addition to many other cellular proteins. Our findings conclusively demonstrate that the p53/p21/pRb cascade is not essential for the initiation of G1 arrest in UV-exposed human cells and, furthermore, indicate the involvement in this process of any among a number of human papillomavirus E7-interacting cellular proteins.

Abbreviations: BrdU, bromodeoxyuridine; CDKs, cyclin-dependent kinases; HPV, human papillomavirus; IR, ionizing radiation; p21, p21waf1; PBS, phosphate-buffered saline; PI, propidium iodide; pRb, retinoblastoma protein


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
During genotoxic stress the cell cycle is punctuated by actively regulated periods of growth arrest. These `checkpoints' permit more time for DNA repair mechanisms to act prior to semi-conservative replication and cytokinesis, thus facilitating the faithful transmission of hereditary material to daughter cells and, in turn, protecting against neoplastic transformation. A great deal of attention has focused on the role of the p53 tumor suppressor protein in DNA damage-inducible G1 arrest. Following exposure of human or rodent cells to a variety of mutagens, including ionizing radiation (IR) and UV light, p53 protein accumulates via post-translational stabilization and its ability to transactivate gene expression is stimulated through acetylation and phosphorylation (1). This leads to transcriptional activation of many genes involved in the cellular response to DNA-damaging agents, including that encoding p21waf1 (hereafter referred to as p21) (2). The p21 protein inhibits a broad range of cyclin-dependent kinases (CDKs) (3,4), thereby preventing phosphorylation of the retinoblastoma tumor suppressor (pRb) as well as the related `pocket' proteins p107 and p130 (5–7). In their hypophosphorylated forms all three pRb family members are able to sequester E2F-type transcription factors, thus engendering G1 arrest through repression of various E2F-modulated genes that are essential for S phase entry (8,9).

The involvement of the p53 regulatory pathway in DNA damage-inducible growth arrest following treatment with IR has been extensively studied in diverse systems. Specifically, it has been clearly established that primary or immortalized (human or rodent) cell lines genetically compromised for p53, p21 or pRb exhibit abrogation of G1 arrest upon IR exposure (3,10–14). In addition, the high risk human papillomavirus 16 (HPV) E6 oncoprotein is known to stimulate proteasomal degradation and, therefore, functional inactivation of p53 (15), while the HPV E7 gene product has been shown to similarly affect pRb (16). Thus, intracellular expression of either HPV E6 or HPV E7 also efficiently attenuates IR-induced G1 arrest in a wide variety of cell lines (5,15,17–22).

Despite the clear demonstration of an indispensable role for the p53/p21/pRb pathway in the initiation of G1 arrest in IR-exposed cells, studies of the extent to which this cascade regulates G1 arrest following treatment with UV light have yielded conflicting results. Indeed, investigations on primary human skin fibroblasts (23), human tumor cells (24,25) and mouse embryo fibroblasts (25,26) have reported p53 independence for G1 arrest after UV irradiation, while other studies employing a human squamous cell carcinoma line (27), human oral keratinocytes (28) and human epidermal carcinoma cells (29) have indicated the opposite. Yet another investigation showed that UV-induced G1 arrest was attenuated, but not completely abolished, in HPV E6-expressing primary human skin fibroblasts (30), supporting the existence of both p53-dependent and p53-independent mechanisms in such cells. On the other hand, the potential effect of intracellular HPV E7 expression on the initiation of UV-induced G1 arrest has not yet been rigorously ascertained to our knowledge. Modulation of the cellular response to UV-induced DNA damage by the HPV E6 and HPV E7 oncoproteins is of interest, since HPV has been firmly implicated in a significant proportion of sunlight-associated non-melanoma skin cancers, in addition to the well-established role of this virus in the molecular etiology of anogenital carcinomas (31–33).

The conflicting nature of the aforementioned data on UV-induced G1 arrest may reflect the possibility that p53 dependence for this process, unlike the situation for IR, varies significantly according to species, cell type and/or treatment conditions (i.e. dose, growth state, etc.). For example, it was recently demonstrated that G1 arrest in human cells is p53-dependent at relatively low doses of 254 nm UV (10 J/m2) and p53-independent at higher, much more cytotoxic doses (30 J/m2) (34). In order to comprehensively assess the role of the p53 regulatory pathway in the initiation of G1 arrest in UV-exposed human cells and the involvement of HPV-encoded oncoproteins in this process, we compared the effects of 254 nm UV (hereafter referred to as UV) versus IR on G1–S phase progression in primary human skin and lung fibroblasts, as well as in isogenic derivatives of these strains that were rendered functionally deficient in p53 or pRb via expression of the HPV E6 or HPV E7 oncoprotein, respectively. Moreover, the kinetics of G1–S progression were investigated in human tumor cell strains that are genetically deficient in p53, p21 and/or pRb, as well as in their corresponding isogenic wild-type counterparts. This latter determination using genetic mutants is crucial, since HPV E6 and HPV E7 can each bind and functionally modulate a considerable number of proteins aside from p53 and pRb (35,36; see Discussion). Finally, the timing of UV- and IR-induced G1 arrest in the wild-type primary and tumor cell derivatives was correlated with intracellular expression of p21 protein and with increases in the ratio of hypo- to hyper-phosphorylated pRb. Our results conclusively demonstrate, in complete contrast to the situation for IR, that the p53/p21/pRb cascade is not involved in the initiation of G1 arrest in human primary and tumor cells exposed to UV. Furthermore, we show for the first time that this mode of p53-independent UV-induced G1 arrest is significantly attenuated in primary human fibroblasts which express the HPV E7 oncoprotein. The overall data provide some clues concerning the mechanism of growth arrest in UV-exposed human cells and may partly explain the strong association of HPV with non-melanoma skin cancers in humans.


    Materials and methods
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 Materials and methods
 Results
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Human cell lines
CRL2097 primary diploid skin fibroblasts (p53 wild-type), SAOS-2 osteosarcoma cells (p53/pRb null) and A-253 epidermoid carcinoma cells (p53 null) were obtained from the American Type Culture Collection. The primary diploid lung fibroblast strain LF-1 was kindly provided by Dr John Sedivy (Brown University). The colon carcinoma cell lines HCT116 (p53+/+), DLD-1 (p53-/-) and their corresponding isogenic p21-knockout derivatives were a generous gift of Dr Bert Vogelstein (Johns Hopkins University). Primary skin fibroblasts were maintained in minimal essential medium with Earle's salts, containing 1 mM sodium pyruvate, 10% fetal calf serum and antibiotics (penicillin and streptomycin), while primary lung fibroblasts were grown in Ham's F10 medium containing 15% fetal calf serum plus antibiotics. SAOS-2 and colon carcinoma cell lines were grown in McCoy's 5A medium supplemented with 10% fetal calf serum plus antibiotics.

Low passage skin and lung fibroblasts were infected as previously described (37) with a replication-defective retrovirus (LXSN, obtained from the ATCC) expressing G418 resistance and either the HPV E6 or HPV E7 oncoprotein derived from the high risk HPV type 16. Briefly, culture medium was harvested from a confluent LXSN murine producer cell line and passed through a 0.22 µm filter. Fibroblasts at 50–70% confluence on 60 mm dishes were incubated for 2 h with 2 ml of this viral supernatant containing 8 µg/ml polybrene, followed by aspiration of the viral supernatant and addition of normal growth medium. After a 2 day phenotypic expression period, fibroblasts expressing the empty vector or expressing either HPV E6 or HPV E7 were selected in growth medium containing 200 µg/ml G418 (Gibco BRL).

Irradiation conditions
Cell monolayers growing in 60 mm dishes were washed with phosphate-buffered saline (PBS), covered with 2 ml of PBS, followed by irradiation with either 5 Gy IR or 10 J/m2 UV. A Philips G25T8 germicidal lamp was used to deliver UV at a fluence of 0.2 J/m2/s. Cells were treated with ionizing radiation using a 137Cs source (Gamma Cell; Atomic Energy Canada) at a dose rate of 6.3x10-2 Gy/s.

Cell cycle analysis
All strains were irradiated with either UV or IR during exponential growth at 50% confluence, followed immediately by addition of the mitotic inhibitor colcemid (1 µM final concentration) to block the re-entry of cells into G1. At various time points following addition of colcemid, irradiated or mock-treated cells were washed with PBS containing 50 mM EDTA, trypsinized, resuspended in 1 ml of PBS/EDTA and fixed by addition of 3 ml of ice-cold 100% ethanol. Fixed cells were pelleted, washed with 4 ml of PBS/EDTA and stained with modified Krishan buffer [0.05 mg/ml propidium iodide (PI) (Molecular Probes), 0.1% sodium citrate, 0.2 mg/ml RNase A and 0.3% v/v NP40]. The fraction of the population in each phase of the cell cycle was then determined as a function of DNA content using a FACScan flow cytometer equipped with CellFit software (Beckton Dickinson). The delay in the G1–S transition is expressed as the percentage of cells remaining in the G0/G1 compartment over time. Each time point represents the average of between four and six independent experiments.

In addition to analysis using the colcemid block, replicate cultures of primary CRL2097 skin fibroblasts were held at confluence for 1 week, which resulted in accumulation of 90% of the cell population in G0/G1, as determined by flow cytometry. Following irradiation of confluent cultures with either IR or UV, cells were growth stimulated by trypsinization and dilution in fresh medium and analyzed for cell cycle progression as above for colcemid-treated cells.

Bromodeoxyuridine (BrdU) incorporation assay
Determination of cellular proliferation was achieved on human skin fibroblasts at 50–70% confluence on 60 mm dishes by indirect BrdU immunofluorescence. Two hours preceding various times post-irradiation, 10 µM BrdU (Boehringer) was added to the culture medium. Cells were washed with PBS/50 mM EDTA, collected in 1 ml of PBS/EDTA and fixed by addition of 3 ml of ice-cold 100% ethanol. Fixed cells were centrifuged and resuspended in 1 ml of 2 N HCl containing 0.5% (v/v) Triton X-100 and incubated at room temperature for 30 min, followed by centrifugation. Cells were then resuspended in PBS plus 1% (w/v) BSA and 0.5% (v/v) Tween 20 and incubated at room temperature for 1 h with 10 µl of monoclonal IgG1 anti-BrdU antibody (Becton Dickinson). Cells were washed with PBS/BSA/Tween 20 and incubated with 10 µl of FITC-labeled anti-mouse antibody (Sigma) at room temperature for 30 min, centrifuged and resuspended in PBS containing 5 mg/ml PI. Bivariate analysis was performed using a FACScan flow cytometer (Becton Dickinson).

Western blotting
In parallel with analysis of cell cycle progression, replicate cultures of wild-type HCT116 and CRL2097 skin fibroblasts were processed for determination of p53, p21 and/or pRb expression by western blotting. At various time points post-irradiation cells were extracted in radioimmunoprecipitation assay buffer (10 mM Tris–HCl, pH 8, 150 mM NaCl, 1 mM EDTA, 1% v/v NP-40 and 1% sodium deoxycholate), containing 10 µg/ml aprotinin, 1 µg/ml leupeptin, 1 mM phenylmethylsulphonyl fluoride and 1 µM Na3VO4, followed by sonication and clarification by centrifugation. Protein content was determined using the bicinchoninate assay (Pierce) according to the manufacturer's specifications. Aliquots containing 100 µg protein were resolved on 7.5% acrylamide gels and electroblotted onto nitrocellulose membranes. The membranes were blocked in 5% (w/v) non-fat dry milk/PBS and incubated overnight at 4°C with primary antibodies against p53 (DO-1; Santa Cruz Biotechnology), p21 (Ab-1; Calbiochem) and pRb (Ab-5; Pharmingen), diluted 1:5000, 1:500 and 1:1000, respectively, in 1% (w/v) milk/PBS. The proteins were detected by chemiluminescence using a secondary anti-mouse antibody coupled to horseradish peroxidase (Amersham).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cell cycle progression in primary human skin and lung fibroblasts expressing HPV E6 or HPV E7
To elucidate the role of the p53 regulatory pathway and of HPV-encoded oncoproteins in UV-induced growth arrest, we compared the kinetics of G1–S phase progression following treatment with either UV or IR in primary human diploid fibroblasts derived from skin (strain CRL2097) or lung (strain LF-1) versus isogenic counterparts of these strains expressing either the HPV E6 or HPV E7 oncoprotein. In the case of exponentially growing lung or skin fibroblasts expressing HPV E6 or HPV E7, basal levels of p53 and pRb, respectively, were vastly attenuated relative to the wild-type derivatives, as determined by western blotting (data not shown).

Immediately following irradiation of each strain, colcemid was added to the growth medium to block transition of cells from mitosis to G1. At various time points post-irradiation cells were stained with PI and subjected to FACS analysis. As illustrated by the raw cell cycle profiles in Figures 1A and 4AGoGo (for primary and tumor cell lines, respectively), colcemid addition blocked cell re-entry into G1, thus allowing G1–S progression to be unambiguously monitored. The doses of radiation employed in the current study, i.e. 10 J/m2 UV or 5 Gy IR, represent equitoxic treatments in primary CRL2097 skin and LF-1 lung fibroblasts, i.e. each yielding a relative clonogenic survival of 10–15% (data not shown).



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Fig. 1. Cell cycle progression in primary human lung and skin fibroblasts. All strains were irradiated and then immediately re-fed with medium containing the mitotic inhibitor colcemid. At the indicated incubation times cells were fixed and stained with PI for analysis by flow cytometry. (A) Representative profiles of primary lung fibroblasts infected with retroviral vector alone (LXSN) at 6 h intervals following mock treatment (Control), 5 Gy IR or 10 J/m2 UV. (B–G) Graphic depictions of the G1–S phase progression for: (B) skin fibroblasts/LXSN; (C) lung fibroblasts/LXSN; (D) skin fibroblasts/HPV E6; (E) lung fibroblasts/HPV E6; (F) skin fibroblasts/HPV E7; (G) lung fibroblasts/HPV E7. •, Mock treatment; {square}, 5 Gy IR; {triangleup}, 10 J/m2 UV.

 


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Fig. 4. Cell cycle progression in human tumor cell lines. Tumor cells were treated essentially as the primary fibroblasts depicted in Figure 1Go. (A) Representative profiles of wild-type HCT116 cells at 3 h intervals following mock treatment (control), 5 Gy IR or 10 J/m2 UV. (B–G) Graphic depiction of the G1–S phase progression for: (B) HCT116 (p53+/+, p21+/+); (C) HCT116 (p53+/+, p21-/-); (D) DLD-1 (p53-/-, p21+/+); (E) DLD-1 (p53-/-, p21-/-); (F) A-253 epidermoid carcinoma cells (p53 null); (G) SAOS-2 osteosarcoma (p53/pRb null). •, Mock treatment; {square}, 5 Gy IR; {triangleup}, 10 J/m2 UV.

 
Although wild-type fibroblasts (i.e. expressing an empty LXSN vector) from either skin or lung manifested a significant G1 delay when exposed to IR or UV, different kinetics were observed for each mutagen (Figure 1B and CGo). During the first 6 h following IR treatment wild-type lung or skin fibroblasts exited G1 at a rate identical to unirradiated controls. However, after this initial period cells manifested a clear growth delay that persisted for at least 24 h post-irradiation. As previously described (38), the initial exit from G1 during the first 6 h following IR exposure in wild-type cells may partly reflect a G1 subpopulation that has passed a `restriction point' and is therefore committed to enter S phase despite the prior application of IR treatment. In contrast to the situation for their IR-exposed counterparts, however, UV-irradiated wild-type lung and skin fibroblasts displayed virtually immediate growth delay (Figure 1B and CGo). The reason for the differential kinetics observed at early time points in UV- versus IR-exposed wild-type cells remains to be clarified.

Although wild-type primary fibroblasts displayed a clear growth arrest after both IR and UV, the kinetics of G1–S progression manifested by sham- and IR-exposed HPV E6-infected skin or lung fibroblasts were indistinguishable, as expected (Figures 1D and EGo). However, in accord with our previous results using genetically p53 null Li–Fraumeni skin fibroblasts (23), UV treatment engendered a prolonged G1 delay in both lung and skin fibroblasts which express HPV E6. Moreover, expression of the HPV E7 oncoprotein in either fibroblast type resulted in complete and partial abrogation of G1 arrest, respectively, following exposure to either IR or UV (Figures 1F and GGo). It is remarkable that HPV E7-infected skin and lung fibroblasts represent the only situations among those examined in the present study where G1 arrest was significantly attenuated following UV exposure.

A potential caveat is highlighted by the use of a single method (i.e. colcemid block) to monitor growth arrest in primary and tumor cell lines. For example, it has been demonstrated that chemically induced cell cycle synchronization (e.g. via mimosine or aphidicolin treatment) can affect levels of p53 and p21 protein relative to untreated cells (39). Nonetheless, in the current study, with respect to p53-dependent growth arrest, all mock- or IR-treated strains were apparently not significantly affected by colcemid treatment, since these strains characteristically exhibited a pattern of G1–S progression and p21 induction that was fully expected based on many previous investigations. Nonetheless, to control for potential artifacts associated with colcemid treatment, the kinetics of G1–S progression in CRL2097 skin fibroblasts following treatment with IR and UV were investigated by an alternative method, i.e. cells were synchronized without chemical treatment in G0/G1 by confluent holding, followed by irradiation and release from confluence. Under these conditions, the analysis of G1–S progression using wild-type, HPV E6- and HPV E7-infected skin fibroblasts all clearly revealed the same basic conclusions as those drawn using the colcemid-based approach (compare Figures 1B, D and F and 2B–DGoGo).



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Fig. 2. Cell cycle progression in human skin fibroblasts synchronized by confluence. Monolayers of skin fibroblasts were maintained in a confluent state, irradiated and then immediately released from confluence. (A) Representative profiles of primary skin fibroblasts infected with retroviral vector alone (LXSN) at 8 h intervals following sham treatment (control), 5 Gy IR or 10 J/m2 UV. (B–D) Graphic depiction of the G1–S phase progression for: (B) skin fibroblasts/LXSN; (C) skin fibroblasts/HPV E6; (D) skin fibroblasts/HPV E7. •, Mock treatment; {square}, 5 Gy IR; {triangleup}, 10 J/m2 UV.

 
To further validate our results, cell cycle progression was measured in UV- and IR-exposed skin fibroblasts that had been double stained with BrdU and PI prior to FACS analysis (Figure 3A–CGo). Following IR treatment, the proportion of wild-type cells incorporating BrdU (relative to mock-irradiated controls) was significantly reduced by 6 h and drastically reduced by 24 h (Figure 3BGo). No such reduction was observed at either time point in IR-treated HPV E6- or E7-expressing cells. In contrast, at 6 h post-UV irradiation in normal as well as in HPV E6-expressing fibroblasts there were significant reductions in the number, and mean fluorescence, of BrdU-positive cells (Figure 3A and CGo). These latter data may reflect the initiation of G1 as well as S phase checkpoints in UV-irradiated wild-type and p53-deficient primary fibroblasts. Remarkably, however, for HPV E7-expressing fibroblasts at 6 and 24 h post-UV irradiation both the number and mean fluorescence of BrdU-positive cells were virtually identical to the control, indicating that HPV E7 expression can attenuate both G1 and S phase checkpoints after UV. At 24 h post-UV irradiation only LXSN-expressing skin fibroblasts showed a significant relative reduction in the number of cells that incorporated BrdU. We stress that the above results obtained in primary skin fibroblasts double stained with BrdU and PI are entirely consistent with those obtained using PI staining alone (as presented in Figure 1Go).



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Fig. 3. Analysis of cell cycle progression following double staining with BrdU and PI in primary skin fibroblasts. Cell cycle progression was measured in UV- or IR-exposed skin fibroblasts that had been double stained with BrdU and PI prior to FACS analysis. (A) Raw data for unirradiated LXSN-, HPV E6- and HPV E7-expressing cells (controls) and cells irrradiated with IR or UV (6 h time point only). The percentage values above each plot indicate the proportion of BrdU-positive cells in the irradiated population. (B) Numbers of BrdU-positive cells (relative to unirradiated controls) at 6 and 24 h post-IR exposure for LXSN-, HPV E6- and HPV E7-expressing cells. (C) Numbers of BrdU-positive cells (relative to unirradiated controls) at 6 and 24 h post-UV exposure for LXSN-, HPV E6- and HPV E7-expressing cells. {blacksquare}, LXSN-expressing cells; {square}, HPV E6-expressing cells; , HPV E7-expressing cells.

 
Cell cycle progression in human tumor cell lines
To ascertain the generality of our observations regarding UV-induced G1 arrest in primary human fibroblasts, we investigated the kinetics of G1–S progression after UV and IR using the well-characterized human colon carcinoma cell strains HCT116 (p53+/+, p21+/+) and DLD-1 (p53-/-, p21+/+) and their isogenic p21 null counterparts. As previously determined (11), IR-treated HCT116 wild-type cells exhibited a clear G1 arrest relative to unirradiated controls; we show here that an equitoxic dose of UV elicited a similarly prolonged delay in G1–S phase progression in these cells (Figure 4BGo). Moreover, as fully expected, the p53-deficient strain DLD-1, as well as isogenic p21 knockouts of either HCT116 or DLD-1, all manifested abrogation of G1 arrest in response to IR (Figure 4C–EGo). In complete contrast, each of these deficient tumor strains, although genetically compromised for p53 and/or p21 function, displayed an unequivocal delay in G1–S phase progression after UV exposure (Figure 4C–EGo), in agreement with our results for primary fibroblasts. These observations in colon carcinoma cells were extended and fully confirmed using the p53 null epidermoid carcinoma cell strain A-253, as well as SAOS-2 osteosarcoma cells, which carry a nullizygous deletion for pRb in addition to one for p53 (Figure 4F and GGo). It is noteworthy that strain A-253, in contrast to the results presented here, had been characterized in a previous study as deficient in UV-induced G1 arrest (27).

Expression of p21 and pRb proteins in wild-type primary and tumor cell lines
To further elucidate the G1 arrest response in UV-irradiated human cells, we used western analysis to monitor the expression of p21 and pRb proteins in wild-type primary and tumor cells following treatment with UV and IR. In IR-exposed HCT116 cells maximal induction of p53 was detected at 3 h post-irradiation and remained elevated for at least 12 h (data not shown), p21 increased gradually from 3–12 h and the ratio of hypo- to hyperphosphorylated pRb began to increase by 3 h (Figure 5AGo). These events, as expected, correlated with the occurrence of an IR-induced G1 arrest. In contrast, the observed immediate onset of G1 arrest in UV-irradiated HCT116 cells clearly preceded any apparent increase in the ratio of hypo- to hyperphosphorylated pRb (i.e. occurring at ~9–12 h post-irradiation; Figure 5AGo). Most remarkably, however, virtually the entire period of UV-induced growth arrest in HCT116 cells took place in the absence of any significant p21 induction, the latter not being detected until 24 h post-irradiation (Figure 5BGo).



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Fig. 5. Expression of p21 and pRb in human primary and tumor cells. (A) Time course of p21 protein induction and appearance of hypophosphorylated pRb in wild-type HCT116 cells treated with 5 Gy IR or 10 J/m2 UV. (B) Extended time course of p21 induction in wild-type HCT116 cells after 10 J/m2 UV. (C) Expression of p21 and pRb in wild-type primary skin fibroblasts treated with 5 Gy IR or 10 J/m2 UV. (D) Extended time course of p21 induction in primary skin fibroblasts after 10 J/m2 UV. (E) Time course of p21 protein expression in confluent wild-type versus HPV E6-infected skin fibroblasts irradiated with 5 Gy IR or 10 J/m2 UV, followed by release from confluence. (Upper) Wild-type (LXSN-expressing) skin fibroblasts. (Lower) HPV E6-expressing skin fibroblasts. pRb, hypophosphorylated form of retinoblastoma protein; pRbp, hyperphosphorylated form of retinoblastoma protein.

 
The temporal expression patterns of p21 and pRb in wild-type CRL2097 skin fibroblasts were consistent with the results in tumor cells. For CRL2097 cells expressing an empty LXSN vector maximal induction of p53 was detected at 3 h and was maintained for at least 12 h (data not shown), p21 levels peaked at 9–12 h and the ratio of hypo- to hyper-phosphorylated pRb began to increase markedly by 6 h post-irradiation (Figure 5CGo). This pattern of protein induction correlated closely with IR-induced G1 arrest. However, for UV-irradiated skin fibroblasts the ratio of hypo- to hyperphosphorylated pRb did not appreciably increase until 12 h post-irradiation (Figure 5CGo) and significant induction of p21 could not be detected during the entire period of growth arrest (Figure 5DGo). Virtually identical western blotting results to those obtained for skin fibroblasts were also obtained for primary LF-1 lung fibroblasts (data not shown). In summary, for both primary and tumor cell strains the initiation of UV-induced G1 arrest clearly precedes induction of p21 and the increase in the ratio of hypo- to hyperphosphorylated pRb.

p21 is induced by 254 nm UV independently of p53 in human diploid skin fibroblasts irradiated during confluence, but not during exponential growth
We previously reported that p21 protein and G1 arrest are concomitantly induced at early time points (within 3 h) in wild-type human skin fibroblasts as well as in p53 null Li–Fraumeni skin fibroblasts irradiated with UV, while both responses are abolished following IR treatment (23). This partially contrasts with the results presented here, where UV exposure engendered p21 induction in wild-type skin fibroblasts only after G1 arrest had clearly terminated (24 h post-irradiation). In the current investigation cell cycle progression and protein induction were monitored in parallel using exponentially growing cells synchronized with colcemid, whereas the previous study employed cells which had been irradiated during confluence (>90% of cells in G0/G1), followed by dilution in fresh medium (release from confluence). We reasoned that this methodological difference between the two studies could account for the observed discrepancy with regard to p53 dependence for p21 induction following UV exposure. To address this possibility, we studied p21 expression in wild-type CRL2097 skin fibroblasts versus their HPV E6-expressing counterparts that were irradiated either during exponential growth or at confluence. As illustrated in Figure 5EGo, in contrast to the data shown in Figure 5CGo for exponentially growing (colcemid-treated) cells, wild-type fibroblasts exposed to UV followed by release from confluence manifested significant induction of p21 protein by 3 h post-irradiation. Moreover UV-exposed, HPV E6-expressing skin fibroblasts (in agreement with our previous result on Li–Fraumeni skin fibroblasts) also displayed some up-regulation of p21 at early times following release from confluence, although p21 induction was completely abrogated subsequent to IR treatment under the same growth conditions (Figure 5EGo). In the case of exponentially growing HPV E6-expressing fibroblasts or p53-deficient tumor cell lines, no significant p21 induction could be detected up to 48 h following treatment with either IR or UV (data not shown).


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A multitude of previous studies, collectively employing a diverse array of primary and immortalized cultured cell types, have clearly demonstrated an indispensable role for the p53/p21/pRb regulatory pathway in the initiation of G1 arrest following exposure to IR. On the other hand, the role of this cascade in UV-induced G1 arrest has remained less clear. The seemingly contradictory nature of previous investigations on p53 dependence for G1 arrest after UV (see Introduction) have variously employed different doses (ranging from poorly to extremely cytotoxic) and have generally been restricted to relatively few cell types. The use of distinct UV sources, i.e. those emitting polychromatic UVB (290–320 nm) versus monochromatic 254 nm UV, may have served to further confuse the issue, as these UV wavelength regions exhibit dissimilar properties with respect to both genotoxicity and patterns of gene activation (40,41). In addition, most previous measurements of the rate of G1–S progression in UV-exposed cells and the correlation of this process with induction of critical cell cycle-related proteins have been rendered ambiguous through the use of unsynchronized populations that were monitored over insufficient periods (often only a single time point analyzed). One notable exception in terms of these latter limitations is a recent study (25) in which synchronized human tumor cell populations were carefully analyzed using multiple time points. While this investigation by Al-Mohanna et al. was able to demonstrate the existence of a p53-independent G1 arrest in human cells, only two p53-deficient human cell strains were examined and isogenic wild-type counterparts were not available. Furthermore, no data were presented regarding induction of p21 or pRb in p53-proficient cell strains following UV irradiation.

In order to comprehensively address the issue of p53 dependence for G1 arrest in UV-exposed human cells and the role of HPV in this process, we first carefully investigated the kinetics of G1–S progression in primary fibroblasts from skin and lung and in their respective counterparts expressing either the HPV E6 or HPV E7 oncoprotein. In addition to these studies on primary cells, a variety of p53-, p21- and/or pRb-deficient human tumor cell strains and their respective isogenic wild-type derivatives were similarly investigated. G1–S progression was measured by different approaches in synchronized populations using multiple time points spread over a substantial period, such that the onset of G1 arrest could be determined and precisely correlated with induction of p21 and of hypophosphorylated pRb. Moreover, in the case of every cell strain examined, G1–S progression was analyzed following irradiation with a physiologically relevant dose of UV and the ensuing response compared with that elicited by an equitoxic dose of IR. This constitutes a useful control, given the highly predictable nature of the IR response, and allows some meaningful comparisons to be made between UV and IR with respect to the mechanism of growth arrest.

Our data now conclusively demonstrate that p53 and p21 are not implicated in the initiation of G1 arrest in UV-exposed human cells. Indeed, in complete contrast to the situation for IR, UV light initiates a prolonged G1 arrest in a variety of human primary and tumor cell strains that are compromised for p53 and/or p21. In addition, the onset of G1 arrest in the isogenic wild-type counterparts of these strains correlates well with induction of p21 after IR exposure, whereas in the case of the UV-exposed wild-type counterparts p21 is not significantly up-regulated during the entire period of growth arrest. Furthermore, no induction of p21 could be detected following UV treatment in any of the exponentially growing p53-deficient cell lines examined in the current investigation. However, in apparent contrast to this latter result, we previously observed some up-regulation of p21 (and concomitant G1 arrest) in p53-deficient Li–Fraumeni skin fibroblasts at early time points (within 3 h) following exposure to UV (23). Others subsequently reported such p53-independent induction of p21 following irradiation with UV in human skin fibroblasts (42), mouse embryo fibroblasts (26) and human melanoma cells (43).

In resolving any apparent discrepancy in our own experimental system, we show here that p53-independent induction of p21 by UV may be specific for cells that are released from confluence immediately following irradiation. Indeed (in accord with our previous result using genetically p53-deficient Li–Fraumeni skin fibroblasts; 23), significant induction of p21 was observed here in UV-exposed, HPV E6-expressing skin fibroblasts when these cells were released from confluence, while no such up-regulation was observed following UV in exponentially growing (colcemid-treated) HPV E6-expressing fibroblasts (nor in any of the other p53-deficient tumor cell lines used in this study). This demonstration that p21 can be induced independently of p53 in UV-exposed skin fibroblasts released from confluence, but not in exponentially growing cells, further reinforces the need for caution when interpreting results on cellular growth arrest derived using any particular experimental approach.

Aside from demonstrating the non-involvement of p53 and p21 in UV-induced G1 arrest in human cells, our results strongly suggest that pRb also may not play a role. Firstly, a prolonged growth delay was observed in pRb null SAOS-2 human osteosarcoma cells after UV treatment, although this same strain displayed complete abolition of IR-induced G1 arrest. Secondly, in the wild-type primary and tumor cells examined here, the onset of UV-induced G1 arrest preceded any appreciable increase in the ratio of hypo- to hyper-phosphorylated pRb, in contrast to the situation for IR, where this increase correlated precisely with growth arrest. It should be noted that these data on the involvement of pRb in growth arrest are not in agreement with some previous studies. Specifically, it was shown that the timing of UVB-induced G1 arrest in primary human melanocytes correlates with induction of p21 and with pRb hypophosphorylation (40). Moreover, using a panel of murine embryo fibroblasts derived from pRb, p107 and p130 null animals, it was reported that (only) pRb plays an essential role in mediating the G1–S phase progression following treatment with a variety of DNA-damaging agents, including UV light (14).

The precise mechanism underlying p53-independent G1 arrest in UV-irradiated human cells still remains to be identified. We believe that this mechanism would largely comprise actively regulated processes, although it has been proposed that global inhibition of mRNA synthesis, due to the presence of UV-induced transcription-blocking DNA lesions (i.e. cyclobutane pyrimidine dimers) in active genes might passively contribute to UV-induced G1 arrest (44). Despite the latter possibility, our own results support a role for actively regulated mechanisms in UV-induced growth arrest, since: (i) HPV E7, which binds a considerable number of cellular growth control proteins, significantly attenuated UV-induced G1 arrest in primary lung and skin fibroblasts (see below); (ii) the UV-induced G1 arrest in human lung or skin fibroblasts observed here lasted appreciably longer (at least 24 h; Figures 1 and 2GoGo) than the time needed to completely clear transcription-blocking DNA lesions (via the transcription-coupled nucleotide excision repair pathway) from the transcribed strands of active genes in such fibroblasts, i.e. within 6–8 h (45). In addition, some very recent investigations have elucidated aspects of actively regulated mechanisms leading to the initiation of p53-independent UV-induced G1 arrest in human cells. Specifically, one study on IR- and UV-exposed breast carcinoma cells demonstrated that the initiation of G1 arrest is mediated in part through rapid degradation of cyclin D1 independently of p53, although the longer term maintenance of this arrest appears to require the p53/p21 pathway (46). In addition, it was shown that proteasome-dependent, p53-independent degradation of cdc25A phosphatase is necessary to initiate G1–S progression after both UV and IR treatment in human primary and tumor cells (47).

Our own results shed some light on a potential mechanism for the initiation of UV-induced G1 arrest. It is particularly striking that among all the cell types analyzed here, only primary skin and lung fibroblasts expressing the HPV E7 oncoprotein manifested defective G1 arrest following exposure to UV. In addition, our results on BrdU incorporation in wild-type versus HPV E7-expressing cells intriguingly suggest that HPV E7 expression may also somehow attenuate a UV-induced S phase checkpoint. HPV E7 binds all pRb family members (5,48,49) and can functionally inactivate the pRb-related `pocket proteins' p107 and p130 (48,50–52), in addition to its well-established effect on pRb. Therefore, given the implication of p53-independent effectors, including retinoblastoma phosphatases (53) and/or CDK inhibitors other than p21 (54) in the generation of hypophosphorylated pRb family members following DNA damage, our overall results may suggest that p107 and/or p130 play a role in the initiation of G1 (and possibly of S phase) arrest following UV irradiation.

Indeed, our data are in accord with a mechanism for the initiation of G1 arrest in UV-exposed human cells consisting of a p53-independent signaling pathway(s) leading to stimulation of phosphatases and/or inhibition of CDKs and subsequent hypophosphorylation of p107 and/or p130. In support of this, it was shown that p107 is rapidly dephosphorylated via the ubiquitous cellular phosphatase PP2A in NIH 3T3 fibroblasts following UV exposure, and that the minimum dose required to elicit this response (4 J/m2) corresponds precisely to that required for the initiation of G1 arrest (55). Furthermore, the dephosphorylating activity of PP2A leading to G1 arrest at low doses in NIH 3T3 cells may be specific for p107, as this phosphatase does not appear to interact with pRb and much higher doses (50 J/m2) are required to initiate dephosphorylation of p130 (56).

It should be emphasized, however, that HPV E7 regulates many cellular proteins aside from pRb family members (35), including some that are also involved in the initiation of G1–S progression following exposure to DNA-damaging agents. In particular, HPV E7 has been shown to activate cyclin A and E gene expression (57) and to inactivate CDK inhibitors such as p27kip1 (58). Further studies are therefore clearly needed to assess the participation of p107 and/or of other HPV E7-interacting protein(s) in UV-induced growth arrest.


    Notes
 
1 To whom correspondence should be addressed Email: elliot.drobetsky{at}umontreal.ca Back


    Acknowledgments
 
The authors express gratitude to Anne-Christine Goulet and Sylvana Lachance for expert technical assistance. This work was supported by operating grants from The Canadian Institutes of Health Research and The National Cancer Institute of Canada with funds from the Canadian Cancer Society. E.A.D. is a scholar of the Fonds de la Recherche en Santé du Québec (FRSQ).


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received May 11, 2001; revised August 20, 2001; accepted September 17, 2001.