Retinoblastoma

ABSTRACT

The expression from a reporter construct driven by a cytomegalovirus (CMV) immediate early (IE) promoter is strongly inducible by UV in human fibroblasts. This response is induced at lower UV fluences in transcription-coupled repair (TCR)-deficient fibroblasts compared with normal fibroblasts and is absent in their simian virus 40-transformed counterparts. In this study we demonstrate that expression of human papilloma virus (HPV) E7 (but not of HPV E6) can attenuate UV-induced expression from the human CMV-IE-driven reporter construct in human fibroblasts. Furthermore, UV-induced expression from the reporter construct appears impaired in murine fibroblasts harboring inactivating mutations in the retinoblastoma (Rb) gene family members p107 and pRb but not in fibroblasts harboring such mutations in the p53 gene. Taken together, these data suggest that one or more members of the pRb family (but not p53) play an essential role in mediating UV-induced expression from the CMV-IE promoter. In this study we report normal UV-upregulation of reporter expression in xeroderma pigmento-sum (XP) group E fibroblasts, consistent with normal TCR. Because XP-E cells deficient in the p48 subunit of the damaged DNA-binding protein are impaired in E2F-1-activated transcription, these results also suggest that the (pRb-regulated) transcription factor E2F-1 does not play an essential role in UV-enhanced expression from the CMV-IE promoter.

INTRODUCTION

UV irradiation induces expression from a number of cellular and viral promoters (reviewed in Sachsenmaier et al. [1] and Bender et al. [2]). This response can be divided into two components (reviewed in Bender et al. [2]). The "immediate" response arises at the cell membrane with the activation of cell surface receptors and their corresponding signaling cascades. This leads to a rapid induction of expression for a subset of UV-responsive genes. These genes primarily encode additional transcription factors, although several immediate-responsive genes (such as the genes for mitogen-activated protein kinase phosphalases [3,4]) provide a negative feedback for the immediate response.

Whereas the strength of the immediate response is directly proportional to the strength of the initial UV exposure, the strength and duration of the "delayed" (or secondary) response is inversely related to the cell's ability to repair transcriptionally active genes (5-7), which is mediated by a process known as transcription-coupled repair (TCR) (8,9). Expression of the delayed-responsive genes is not observed for several hours after UV exposure and is dependent on de novo protein synthesis (presumably of the transcription factors upregulated by the immediate response). Delayed-responsive genes include the genes encoding collagenase, metallothionein, interleukin-1, basic fibroblast growth factor and numerous viral promoters (2,5,6,10-12). Similarly, we have demonstrated that treatment of primary human fibroblasts with UV results in increased expression from a reporter driven by either the human or the murine cytomegalovirus (CMV) immediate early (IE) promoters (13). The enhancement of reporter activity after lower UV exposures in TCR-deficient fibroblasts, compared with their TCR-proficient counterparts, indicates that persistent damage in active genes plays a role in this induction.

In contrast to results with primary cell strains, no UV-induced expression of reporter activity was observed in any simian virus 40 (SV40)-transformed cell line (TCR-proficient or TCR-deficient) examined (13). Cellular transformation by SV40 results in a number of alterations of cellular metabolism, including the binding and inactivation of p53 and members of the pRb family (Rb stands for retinoblastoma) by the SV40 large tumor antigen (LT) (14-19). Both p53 and pRb accumulate in response to UV exposure (20,21) and can modulate the transcription of a large number of cellular genes (reviewed in Lakin and Jackson [22], el-Deiry [23] and Mayol and Grana [24]). Because we have previously demonstrated a role for p53 in enhanced reactivation of a UV-damaged reporter (25), it was of interest to examine the possible roles of these proteins in mediating enhanced expression from Ad5HCMVsp1 lacZ in response to UV (Ad stands for adenovirus and HCMV for human CMV). However, experiments with Li-Fraumeni syndrome cell lines, both heterozygous and hemizygous for two distinct mutations in the p53 gene, showed normal levels of reporter activity in UV-irradiated cells (13), suggesting that p53 is not essential for this response.

Like SV40, other viruses also encode proteins that target p53, pRb family members or both. The polyoma LT has considerable homology to SV40LT and binds pRb family members (26,27) but has not been observed to bind to or interfere with the transactivation activity of p53 (28). The human papilloma virus (HPV) E6 protein forms a specific complex with p53 and promotes its degradation in vitro (29,30), whereas the HPV E7 protein complexes with pRb family members (31,32). The human cervical carcinoma cell line HeLa expresses the HPV early genes (33) and has very low activity of both p53 and pRb family members.

Unlike SV40-transformed cells, HeLa cells exposed to UV showed increased [beta]-galactosidase ([beta]-gal) activity from Ad5HCMVsp1lacZ (13). This suggests that (if HPV E6 and E7 are as efficient as SV40LT at inactivating p53 and pRb), neither p53 nor pRb is essential for UV-enhanced expression of the reporter. However, it has been reported that although HPV E7 binds pRb and its family members p107 and p130, only pRb is targeted for degradation, whereas levels of the other two proteins are not significantly altered by E7 expression (34). Furthermore, even pRb remains at significant levels and accumulates still more in HeLa cells after UV exposure (35). Thus, it is possible that sufficient levels of one or more of these proteins remain to induce expression of reporter activity. To examine this possibility, we have examined UV-induced expression from a reporter construct in human fibroblasts constitutively expressing the HPV E6 and E7 proteins and in murine embryonic fibroblasts (MEF) containing knockouts in each of the p53, pRb and p107 genes. Results suggest a role for one or more pRb family members, but not for p53, in mediating UV-induced expression from the HCMV-IE promoter.

MATERIALS AND METHODS

Cells. Repair-deficient human fibroblasts XP12BE (XP-A, GM 5509), XPCS2BA (XP-B, GM13026), XP2BE (XP-C, GM 677), XP2RO (XP-E, GM 2415), CS3BE (CS-A, GM 1856) and CS1AN (CS-B, GM 739) and repair-proficient human fibroblasts strains GM 38 and GM 9503 were obtained from the Human Genetic Cell Repository: National Institute of General Medical Sciences (Camden, NJ) (XP stands for xeroderma pigmentosum and CS for Cockayne syndrome). A neonatal foreskin fibroblast strain (established by Dr. D. A. Galloway, Fred Hutchinson Cancer Research Center, Seattle, WA), the E6- and E7-expressing transformants of this normal strain, the E6-expressing transformant of XP12BE and the E7-expressing transformant of XPCS2BA (36) were all obtained from Dr. B. C. McKay, Centre for Cancer Therapeutics, Ottawa Regional Cancer Centre, Ottawa, Ontario, Canada. MEF with specific knockouts in the p53 (p53^sup tm1Tyj^, originally obtained from Jackson Laboratories, Bar Harbor, ME), pRb (R20) and p107 (p107-/-) (37) genes as well as their parental strains were obtained from Dr. M. A. Rudnicki, Centre for Cancer Therapeutics, Ottawa Regional Cancer Centre, Ottawa, Ontario, Canada.

All cell cultures were grown at 37[degrees]C in a humidified incubator in 5% CO2 and cultured in Eagle's [alpha]-minimal essential media ([alpha]-MEM) supplemented with 10% fetal bovine serum and antimycotic-antibiotic 100 [mu]g/mL penicillin, 100 [mu]g/mL streptomycin and 250 ng/mL amphotericin B (Gibco BRL, Grand Island, NY). Media for the E6 and E7 transformants were supplemented with 100 [mu]g/mL geneticin (G418; Sigma-Aldrich Canada, Oakville, Ontario, Canada).

Viruses. The replication-deficient recombinant Ad, Ad5HCMVsp1lacZ (38), was obtained from Dr. F. L. Graham, McMaster University, Hamilton, Ontario, Canada. Ad5HCMVsp1lacZ carries the bacterial lacZ gene under the control of the HCMV-IE promoter (-299 to +72 relative to the transcription start site) inserted into the E1 deleted region of Ad. The deletion in the E1 region of the genome renders the virus unable to replicate in most mammalian cells. The virus was propagated, collected and titered as described previously (39).

Treatment of cells with UV. Fibroblasts were seeded in 96-well plates (Falcon, Lincoln Park, NJ) at a density of 1.9 x 10^sup 4^ cells per well in supplemented [alpha]-MEM. In the interest of maintaining identical conditions for all cell strains, the media in this and all subsequent stages of the experiment were not supplemented with any selective agents (i.e. G418). Once seeded, cells were incubated for 18-24 h at 37[degrees]C in a humidified incubator in 5% CO2. After the appropriate incubation, the medium was aspirated from the wells, and cells were overlaid with 40 [mu]L per well of warmed phosphate-buffered saline (140 mM NaCl, 2.5 mM KCl, 10 mM Na^sub 2^HPO^sub 4^, 1.75 mM KH^sub 2^PO^sub 4^ [pH 7.4]) and UV-irradiated (or mock irradiated) using a General Electric germicidal lamp emitting predominantly at 254 nm, at an incident fluence rate of 1 W/m^sup 2^. UV exposures used were corrected for irradiation in 96-well plates as reported previously (40). Immediately after treatment (or mock treatment) with UV, cells were infected with Ad5HCMVsp1lacZ in a volume of 40 [mu]L at a multiplicity of infection of 10-20 pfu/cell. After incubation at 37[degrees]C for 90 min, cells were refed with warmed supplemented media and further incubated to allow for reporter gene expression.

Quantitation of [beta]-gal activity. After the appropriate period of incubation (see the Results section and figure legends), infected cell layers were incubated with 1 mM chlorophenolred [beta]-D-galactopyranoside (CPRG; Boehringer-Mannheim, Indianapolis, IN) in 0.01% Triton X-100, 1 mM MgCl^sub 2^ and 100 mM phosphate buffer at pH 8.3. The absorbance at 570 nm was determined at several time points after the addition of the CPRG solution using a 96-well plate reader (Biotek Instruments EL340 Bio Kinetics Reader).

RESULTS

UV-induced reporter expression is reduced in fibroblasts constitutively expressing HPV E7 but not in those constitutively expressing HPV E6

The absence of UV-induced expression of reporter activity in SV40-transformed celts (13) suggested some involvement of p53 or members of the pRb family of proteins in UV-enhanced expression of the CMV-driven reporter gene. To explore this possibility more directly, we examined UV-induced expression from Ad5HCMVsp1lacZ in human fibroblasts that have been transformed by retroviral vectors to express either the HPV E6 or the HPV E7 proteins. Reporter expression was examined in a normal human fibroblast strain and in DNA repair-deficient strains belonging to the human genetic disorder XP complementation groups A and B (XP-A and XP-B, respectively) expressing either E6 or E7. The corresponding parental cell strains were examined in parallel.

A summary plot for values observed at 44 h after exposure of the cells to UV radiation and immediate infection with Ad5HCMVsp1lacZ is presented in Fig. 1. Similar values were obtained when reporter activity was quantitated at 24 and 36 h after UV exposure (data not shown). Expression from Ad5HCMVsp1lacZ does not significantly differ between cell strains expressing HPV E6 and their corresponding parental strains. In contrast, UV-induced reporter expression is greatly reduced in cell strains expressing HPV E7, relative to the parental strains. Because HPV E7 specifically binds pRb family members and HPV E6 binds p53, these data support a role for a pRb family member, but not for p53, in the induction of activity from the HCMV-IE promoter in response to UV.

Examination of UV-induced reporter expression in MEF strains with specific gene knockouts

To assess further the role of p53 and individual pRb family members in UV-induced reporter expression from Ad5HCMVsp1lacZ, we obtained MEF lines with specific knockouts in each of the p53, pRb and p107 genes. UV-induced expression from Ad5 HCMVsp1lacZ was assessed in the MEF lines 24 h after UV exposure and infection with the reporter construct. Although the MEF line lacking functional p107 still showed an enhancement of reporter activity in response to UV, the activity was reduced relative to that of parental controls (Fig. 2). In contrast, values for either the p53- or the pRb-knockout lines were not significantly different from those for normal controls at 24 h (Fig. 3).

To examine whether abrogation of either p53 or pRb affected the time course of induction, samples were collected at various times after UV exposure and infection with Ad5HCMVsp1lacZ (Fig. 3). Although UV-enhanced expression from Ad5HCMVsp1lacZ generally appears to be slightly lower in pRb-null MEF than in the parental controls in samples collected 12-48 h after cellular irradiation, the difference is not significant at most points. However, combined with the observations in p107-null MEF, this suggests the possibility that complete inhibition of UV-enhanced expression from Ad5HCMVsp1lacZ may require inactivation of multiple members of the pRb family.

Increased expression from the reporter construct was observed in p53-null cells as early as 12 h after UV exposures of 6 J/m^sup 2^ or more. However, in samples collected at later times after UV exposure, it appears that higher UV exposures are required to elicit a similar enhancement of reporter expression. By 48 h, an increase in reporter expression is observed in cells exposed to 15 J/m^sup 2^ but not in cells exposed to 9 J/m^sup 2^ . Neither the pRb-null MEF nor the parental MEF line shows an enhancement of reporter activity by 12 h after UV exposure. As in human cells, it is possible that this response is indicative of a deficiency in a p53-dependent repair pathway. In several experiments, an enhancement of reporter activity in TCR-deficient human cells has been noted at earlier times than in their repair-proficient counterparts. Furthermore, the apparent shift of the UV-induced induction to higher UV exposures at later times may represent the repair of DNA damage induced by lower exposures over this time period.

Taken together, these data suggest that p53 does not play an essential role in the induction of reporter expression from Ad5HCMVsp1lacZ in response to UV irradiation. However, a possible role in this response is indicated for the pRb family member p7107. Furthermore, we consider it likely that multiple members of the pRb family are involved in mediating this response and that loss of one of the family members can be compensated for (at least in part) by its remaining relatives.

UV-enhanced reporter expression in XP-E fibroblasts does not differ from that in normal controls

We have previously reported that UV-enhanced expression of the reporter gene is induced at lower UV fluences in TCR-deficient fibroblasts compared with normal human fibroblasts (13), suggesting that unrepaired damage in active genes triggers increased reporter activity from constructs driven by the CMV promoter in human fibroblasts. Recent reports indicate that the p48 gene, which is deficient in XP patients from complementation group E, is involved in global genome repair (41,42), suggesting that XP-E cells have normal TCR. Therefore, examination of UV-enhanced expression of the reporter gene in XP-E cells was considered to be of interest. Reporter activity was assessed in XP-E fibroblasts at 44-48 h after UV exposure and immediate infection with Ad5HCMVsp1lacZ. Whereas expression was induced by significantly lower UV exposures in a TCR-deficient CS cell strain as reported previously (13), there was no significant difference between the values observed for XP-E fibroblasts and those for identically treated normal controls (Fig. 4). These results are consistent with a normal TCR capacity in XP-E cells.

The members of the Rb family interact with (and affect the activity of) a large number of transcription factors (reviewed in Mayol and Grana [24] and Sellers and Kaelin [43]). Probably the best characterized of these interactions is that between the pRb family members and the members of the E2F transcription factor family. DNA damage induced by UV (and gamma) irradiation has been shown to result in increased E2F-1 DNA-binding activity (44). The heterodimer damaged DNA-binding (DDB) protein functions as a transcriptional partner of E2F-1 (45), and naturally occurring mutants of DDB (which fall into the genetic repair disorder XP group E or XP-E) are impaired in E2F-1-activated transcription (46). The p48 subunit of the DDB protein is also strongly upregulated in response to both UV and ionizing radiation (41,42). Consequently, the normal activation of reporter expression in XP-E cells shown in Fig. 4 also suggests that the transcriptional activity of E2F-1 is not essential for UV-enhanced expression from Ad5HCMVsp1lacZ.

DISCUSSION

We have demonstrated that UV-enhanced expression from Ad5HCMVsp1lacZ can be attenuated by constitutive expression of HPV E7 but not of HPV E6 in primary human fibroblasts. Furthermore, specific knockouts of the pRb and p107 genes (but not of the p53 gene) result in an impaired enhancement of reporter activity (at some time points) after cellular exposure to UV. Taken together, these data suggest that UV-enhanced expression from Ad5HCMVsp1lacZ requires one or more members of the pRb family. Additionally, because the p107 and pRb gene knock-outs resulted in small but incomplete inhibitions of UV-enhanced reporter expression from Ad5HCMVsp1lacZ, we suggest that multiple members of the pRb family may participate in the response and that the loss of one family member may be partly compensated for by the remaining members. Similar overlapping of functions between pRb family members has been reported (reviewed in Mulligan and Jacks [47]).

UV activation of pRb-related transcription factors

pRb and its family members interact with a number of transcription factors and affect the expression of a wide range of genes (reviewed in Mayol and Grana [24], Sellers and Kaelin [43] and Rohde et al. [48]). Transcription factors affected by pRb family members include E2F (49-51), Sp1 (52,53), ATF-2 (54,55), AP-1 (56) and NF-[kappa]B (57). There is evidence linking each of these factors to the cellular response to UV exposure. AP-1 (58,59), ATF-2 (60,61), CREB (62) and NF-[kappa]B (63-65) are all activated in response to UV irradiation. The DNA-binding activity of E2F-1 is increased after UV exposure (44), and SP1 consensus binding sites have been linked to UV-responsive regulation of p21^sup waf1^ (66).

Activation of the transcription factors listed above correlates well with the immediate UV response (i.e. rapid, relatively short-lived and independent of DNA damage) (reviewed in Bender et al. [2]). Unfortunately, the possible roles of specific transcription factors in mediating the delayed UV response (with which UV-enhanced expression from the CMV-IE promoters corresponds) have not been extensively examined. One exception is NF-[kappa]B, which has recently been shown to be activated both immediately in a DNA damage-independent manner and later in a DNA damage-dependent manner (64). Additionally, JNK (a kinase that is rapidly activated by UV [67,68] and in turn activates both AP1 [67,68] and ATF-2 [60,61] activity) has also been reported to be activated by DNA damage (69).

Normal reporter expression from Ad5HCMVsp1lacZ in XP-E fibroblasts

Expression from Ad5HCMVsplacZ is induced in TCR-deficient cells after significantly lower UV exposures compared with similarly treated normal controls (Fig. 4; Francis and Rainbow [13]). The similarity between the reporter-activation profiles in XP-E and normal cells is consistent with reports that XP-E cells retain normal TCR capacity (41).

Because XP-E cells (including the specific strain examined in this study) have been shown to be impaired in stimulating E2F-1-activated transcription (46), the observation that Ad5HCMVsp1lacZ reporter activity after cellular exposure to UV in this XP-E strain is indistinguishable from that of normal controls suggests that E2F-1 activity is not essential for this response.

Comparison of promoter elements in CMV-IE-driven reporter constructs

The published sequence of the promoter elements in the CMV-IE-driven construct was examined for the consensus sequence of several transcription factor-binding sites. Consistent with the results presented in this article, we found no consensus sequences in the CMV-IE promoter for p53 (70), E2F-1 (71) or the "ultraviolet-responsive element" (72). However, the fragment of the HCMV-IE promoter used in our reporter construct (-299 to +72) did contain two NF-[kappa]B-binding sites with the consensus sequence GGGACTTTCC (73-75) and a third with the sequence GGGATTTCC (74,75); one AP1 site (74,75); two CRE/ATF sites (74,75); and two putative SP1 sites (75). This suggests the possible involvement of one or more of these factors in mediating the enhanced expression from the reporter construct after UV exposure.

Acknowledgements-We thank Dr. Bruce C. McKay for providing the E6- and E7-expressing human fibroblasts and Dr. Michael Rudnicki for providing the various knockout MEF used in this study. We thank Dr. Frank L. Graham for providing the recombinant Ad used in this study. This work was supported by the National Cancer Institute of Canada with funds from the Canadian Cancer Society.

[para]Posted on the website on 7 April 2003.

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Murray A. Francis[dagger] and Andrew J. Rainbow*

Department of Biology, McMaster University, Hamilton, Ontario, Canada

Received 19 November 2002; accepted 14 March 2003

*To whom correspondence should be addressed at: Department of Biology, McMaster University, Hamilton, Ontario, Canada L8S 4K1. Fax: 905-522-6066; e-mail: rainbow@mcmaster.ca

[dagger]Current address: 8129 Elm Wing, Hospital for Sick Children, Toronto, Ontario, Canada.

Abbreviations: Ad, adenovirus; CMV, cytomegalovirus; CPRG, chlorophenolred [beta]-D-galactopyranoside; CS, Cockayne syndrome; DDB, damaged DNA-binding; [beta]-gal, [beta]-galactosidase; HCMV, human CMV; HPV, human papilloma virus; IE, immediate early; LT, large tumor antigen; MEF, murine embryonic fibroblast; [alpha]-MEM, [alpha]-minimal essential media; Rb, retinoblastoma; SV40, simian virus 40; TCR, transcription-coupled repair; XP, xeroderma pigmentosum.

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