E4orf6 Paper (for current biology)

Revised Manuscript (M4:09934) Hart et al

The adenovirus E4orf6 protein inhibits DNA double strand break repair and

radiosensitizes human tumor cells in an E1B-55K-independent manner.

Lori S. Hart§¶, Steven M. Yannone#, Christine Naczki§, Joseph S. Orlando�, Stephen B. Waters¶,

Steven A. Akman¶, David J. Chen#, David Ornelles¶�, and Constantinos Koumenis§¶�*

Departments of §Radiation Oncology, ¶Cancer Biology, �Neurosurgery, and �Microbiology and

Immunology, Wake Forest University School of Medicine, Winston-Salem, NC 27157, USA.

#Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.

Corresponding Author: *Constantinos Koumenis, Section of Radiation Biology, NRC 412, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157. Phone: (336) 713-7637. Fax: (336) 713-7639. Email: [email protected]. Running Title: Ad5 E4orf6 Radiosensitizes Human Tumor Cells

Keywords. E4orf6, DNA-PK, DNA repair, radiosensitization, gene therapy

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JBC Papers in Press. Published on October 26, 2004 as Manuscript M409934200

Copyright 2004 by The American Society for Biochemistry and Molecular Biology, Inc.

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Summary

The adenoviral protein E4orf6 has been shown to inhibit both in vitro V(D)J recombination and

adenoviral DNA concatenation, two processes that rely on cellular DNA double strand break

repair (DSBR) proteins. Most of the known activities of E4orf6 during adenoviral infection

require its interaction with another adenoviral protein, E1B-55K. Here we report that E4orf6,

stably expressed in RKO human colorectal carcinoma cells or transiently expressed by

adenoviral vector in U251 human glioblastoma cells inhibits DSBR and induces significant

radiosensitization in the absence of E1B-55K. Expression of a mutant form of E4orf6 (L245P),

failed to radiosensitize RKO cells. E4orf6 reduced DSBR capacity in transfected and infected

cells, as measured by sublethal DNA damage repair assay and phosphorylated H2AX (γ-H2AX)

levels, respectively. Consistent with the inhibitory effect of E4orf6 on DSBR, expression of

wild-type but not mutant E4orf6 reduced recovery of a transfected, replicating reporter plasmid

(pSP189) in 293 cells, but did not increase the mutation frequency measured in the reporter

plasmid. The kinase activity of DNA-PKcs towards heterologous substrates was not affected by

expression of E4orf6; however, autophosphorylation of DNA-PKcs at Thr2609 following IR was

prolonged in the presence of E4orf6 compared to control-infected cells. Our results demonstrate

for the first time that E4orf6 expression hinders the cellular DNA repair process in mammalian

cells in the absence of E1B-55K or other adenoviral genes, and suggest that viral-mediated

delivery of E4orf6, combined with localized external beam radiation could be a useful approach

for the treatment of radioresistant solid tumors such as glioblastomas.

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Introduction

DNA double strand breaks (DSBs1) occur naturally during DNA replication and V(D)J

recombination, but are also produced during the treatment of human malignancies with ionizing

radiation (IR) and genotoxic drugs. In mammalian cells, the predominant pathway for repairing

DSBs is non-homologous end joining (NHEJ). The DNA-dependent protein kinase (DNA-PK)

complex is required for NHEJ. This complex, which includes Ku70, Ku80 and the 450kDa

DNA-PK catalytic subunit (DNA-PKcs), recruits several other repair proteins including the

MRE11/Rad50/NBS1 (MRN) complex, XRCC4, and Ligase IV (1). Despite the large number of

proteins and seemingly redundant pathways involved in the processing of DNA damage, targeted

gene deletions or mutations of any of the NHEJ proteins in mice results in growth deficiency,

immune deficiency from defective V(D)J recombination, hypersensitivity to IR, neuronal

apoptosis, and in some cases tumorigenesis due to increased genomic instability (1,2). These

consequences of defective NHEJ demonstrate the importance of DSBR proteins in maintaining

genomic integrity and cellular viability. Conversely, the hypersensitivity of DSBR-deficient

cells to IR makes repair proteins an attractive target for radiosensitization of tumor cells with

localized beam radiation approaches (3).

Interestingly, adenoviral proteins have recently been shown to interfere with the NHEJ

pathway to prevent host cell-mediated ligation of the linear adenoviral DNA, which would

otherwise lead to viral DNA concatemer formation (4,5). During adenoviral infection, E4orf6

cooperates with E1B-55K to target p53 and MRE11 for degradation, preventing a p53-mediated

anti-viral response and inhibiting the NHEJ-dependent process of concatemer formation (4-6). It 1The abbreviations used are: DSBR, DNA double-strand break repair; DSBs, DNA double-strand breaks; NHEJ, non-homologous end joining; DNA-PK, DNA-dependent protein kinase; DNA-PKcs, DNA-PK catalytic subunit; MRN, MRE11/Rad50/Nbs1 complex; IR, ionizing radiation; SLDR, sublethal damage repair.

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is likely that the ability of E4orf6 and E1B-55K to cooperate in suppressing concatemer

formation may have evolved as a means of bypassing host cell defenses against viral DNA

replication and productive infection. E4orf6, a 34kDa protein encoded by open reading frame 6

of the E4 gene, has been reported to interact with DNA-PKcs and to inhibit V(D)J recombination

in vitro (7), an NHEJ-dependent process that generates immunological diversity through DNA

rearrangements. Since these experiments were performed in 293 cells, which express E1B-55K,

it is unknown if E4orf6 alone can inhibit cellular DSBR or DNA-PKcs activity. The inhibition

of NHEJ proteins in suppressing concatemer formation and V(D)J recombination makes E4orf6

an attractive tool for inhibiting cellular NHEJ. Considering that radiation therapy is a frequent

treatment for solid tumors and proficient DSBR limits tumor cell kill in response to IR treatment

(8,9), the inhibition of DSBR proteins is an attractive approach for tumor radiosensitization

(10,11). Therefore, we examined the ability of E4orf6 to inhibit NHEJ and to radiosensitize

tumor cells.

Experimental Procedures

Cell Culture- All cell lines (American Type Culture Collection, Manassas, VA) were

incubated at 37ºC with 5%CO2. U251 cells were maintained with RPMI-1640 media, RKO cells

with McCoys5A, and M059 J and K cell lines with DMEM/F12, each supplemented with 10%

fetal bovine serum, L-glutamine, and penicillin/streptomycin. The M059 cell lines were also

supplemented with sodium bicarbonate (1.5g/L), nonessential amino acids, and sodium pyruvate.

RKO stable clones were established by transfection of plasmids (LipofectAMINE PLUS,

Invitrogen, Carlsbad, CA) and selection with neomycin (300µg/ml). Harvested RKO clones

were maintained in media supplemented with 100µg/ml neomycin.

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Clonogenic Survival Assay- Clonogenic survival assays were performed as previously

described (12). For survival assays involving infections, U251 cells were plated in 35mm plates

in triplicate at equal density, infected the next day at an MOI predetermined to yield maximal

infectivity, and irradiated 48 hours later with a 137Cs source (dose rate of 4.41Gy/min) at a range

of IR doses. When unirradiated control plates were near confluency (at or near day 7 post-IR),

the cells were fixed and stained with crystal violet as previously described (12). Crystal violet

was solubilized in 33% acetic acid and the absorbance at 540nm was measured in triplicate for

each well as described by Bernardi et al (13).

Immunoblotting- RKO clones and infected cells were harvested as previously described

(12). For E4orf6, E1B-55K, p53, MRE11, and β-actin immunoblots, lysates (60µg total protein)

were resolved on 12% SDS-PAGE gels, transferred to PVDF membrane, blocked for 30 minutes

at room temperature, and incubated for one hour at room temperature with the following

antibodies: anti-E4orf6 antibody (MAb#3 at 1:100), anti-E1B-55K (9C10 at 1:4), anti-p53 (DO-1

at 1:5000; Santa Cruz Biotechnology, Santa Cruz, CA), anti-MRE11 (12-D7 at 1:5000;

GeneTex, San Antonio, TX), anti-β-actin (at 1:50,000; Sigma, St. Louis, MO). DNA-PKcs

lysates were resolved on 6% SDS-PAGE gels, transferred to nitrocellulose overnight at 25V, and

blotted with anti-DNA-PKcs monoclonal antibody (25-4 at 1:5000; NeoMarkers, Fremont, CA)

or anti-Thr2609 polyclonal DNA-PKcs antibody (1:1000) (14). Cells used in γ�H2AX

immunoblots were directly lysed in 1.5X SDS sample buffer, boiled for 10 minutes, and passed

through a 28 gauge syringe 3 times. Equal volumes were loaded on 15% SDS-PAGE gels,

transferred to PVDF, and membranes were incubated overnight at 4ºC with anti-γ-H2AX

antibody (1:5000; Upstate, Charlottesville, VA). All membranes were incubated with HRP-

conjugated anti-mouse or anti-rabbit secondary antibodies (Santa Cruz Biotechnology, Santa

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Cruz, CA) and immunoreactive bands were detected using ECL Plus chemiluminescence

(Amersham Biosciences, Piscataway, NJ). Optical density of reactive bands was measured by

image analysis software (Scion Image; Scion Corporation, Frederick, MD) as previously

described (12).

Immunocytochemistry- Indirect immunofluorescence microscopy of RKO and U251

cells was performed as previously described (15) at 48 hours post-transfection or post-infection.

The cells were fixed in either methanol:acetone (1:1) or 2% paraformaldehyde, blocked with

TBS-BGT [137mM NaCl, 3mM KCl, 20 mM Tris, pH 7.6, 5.0mg/ml BSA, 1.0mg/ml glycine,

0.05% Tween-20, 0.02% sodium azide], and incubated with mouse monoclonal anti-E4orf6

antibody (MAb#3 at 1:100 in TBS-BGT) or rat monoclonal anti-E1B-55K (9C10 at 1:100 in

TBS-BGT) for 20 minutes at 37ºC. Primary antibody binding was detected with either Alexa

Fluor 488-conjugated anti-mouse or Alexa Fluor 568-conjugated goat-anti-rat secondary

antibodies (Molecular Probes, Eugene, OR). U251 cells were counterstained with Hoescht33342

(1µg/ml in TBS-BGT) or 4',6-diamidino-2-phenylindole (DAPI).

Mutagenicity Assay- Mutation frequency was measured in a replicating reporter plasmid

pSP189 originally described by Seidman and associates (16). The pSP189 shuttle vector was co-

transfected with a plasmid to express the wild-type E4orf6 or mutant L245P gene in 293 cells.

After two days, the shuttle vector was purified by the protocol of Hirt (17). Plasmid DNA was

purified and mutations within the 158 bp supF gene were identified by transforming the E. coli

strain MBL-50. Bacteria containing a mutant supF gene were counted after 48 hours of growth

on minimal media containing L-arabinose and ampicillin. The total number of transformants

was determined by plating appropriate dilutions of the cells on Luria broth plates containing

ampicillin. The amount of material used for each transformation was empirically adjusted so

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that approximately one-half of 16 independent transformations yielded no supF-mutant colonies.

Mutation frequencies were calculated by application of the Poisson distribution. To quantify the

amount of replicated plasmid, the Hirt-purified DNA was treated with Dpn I, purified, and

limiting amounts of the Dpn I-treated DNA used to transform MBL-50 bacteria. The number of

transformants was enumerated after plating serial dilutions on Luria broth bacterial plates

supplemented with ampicillin. No kanamycin-resistant bacteria were generated from Dpn I-

treated DNA, confirming that the non-replicating plasmids were inactivated.

In Vitro DNA-PKcs Kinase Assays- This protocol is adapted from previously published

reports (18) and the SignaTECT DNA-Dependent Protein Kinase Assay System manufacturer

protocol (Promega, Madison, WI). Briefly, cell pellets were resuspended in extraction buffer,

lysed by 3 consecutive cycles of freeze/thawing, and cleared by centrifugation. The high-salt

lysates (50µL) were diluted by adding four volumes of dilution buffer. Equilibrated dsDNA-

cellulose (Sigma, St. Louis, MO) diluted 1:2 in wash buffer was added to each sample and

incubated for at least 30minutes on a rocker at 4ºC. Cellulose was pelleted and the extracted

supernatant was re-extracted with 40µL cellulose slurry. The combined pellets were resuspended

in wash buffer. The cellulose (10µL) was used in place of the enzyme samples in the

SignaTECT kinase reaction (Promega, Madison, WI). Terminated reactions were spotted on

biotinylated membrane (SAM2®; Promega, Madison, WI) and the membranes were washed as

described in the protocol. Measurement of radioactivity was performed with a scintillation

counter (LS5000CE; Beckman, Fullerton, CA).

Statistical Analysis- Where necessary, data was statistically analyzed to generate S.E.

values and determine level of significance using the two-tailed Student�s t-test.

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Results

Stable expression of E4orf6 radiosensitizes RKO cells- To examine the effects of E4orf6

on human tumor cell radiosensitivity, we first established clones of RKO human colorectal

carcinoma cells stably expressing wild-type E4orf6, a control neomycin resistance gene, or

E4orf6-L245P, a non-functional variant during adenovirus infection (15). The wild-type E4orf6

expressed in the RKO cells directed E1B-55K to the nucleus (Figure 1A) and cooperated with

E1B-55K to target p53 and MRE11 for degradation (Figure 1B), indicating that functional

protein was expressed. In contrast, the E4orf6-L245P mutant failed to direct E1B-55K to the

nucleus and failed to cooperate with E1B-55K to target p53 and MRE11 for degradation (Figure

1). Interestingly, reduced levels of E1B-55K protein were observed in the control (neo) and

L245P cell lines infected with the E4-mutant virus dl1014 (Figure 1B). Because all the cells

were equally infected (data not shown), it seems likely that the decreased E1B-55K protein

levels may have resulted from diminished stability of the E1B-55K protein in the absence of a

functional E4orf6 protein. To our knowledge this is the first reported case of stable E4orf6

expression in mammalian cells.

In order to determine if E4orf6 increased the radiosensitivity of the RKO cells,

clonogenic survival was analyzed after exposure to a range of IR doses. Two independently

isolated clones expressing wild-type E4orf6 were significantly more radiosensitive (10-fold at

8Gy) than either a vector-transfected clone or an E4orf6-L245P mutant clone (Figure 2). E1B-

55K is not expressed in the clones, indicating that E4orf6 radiosensitizes RKO cells without

affecting p53 or MRE11 protein levels. The radiosensitization effect was quantified as a dose-

enhancement ratio (DER) of approximately 1.4 over the range of IR doses.

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Transient expression of E4orf6 radiosensitizes U251 cells- Although E4orf6

radiosensitized two independent RKO clones, it remained possible that random integration of the

foreign DNA in the RKO clones, rather than E4orf6 expression contributed to the

radiosensitization effect (19). To test this, we measured the ability of E4orf6 to radiosensitize

human tumor cells following transient expression from a non-replicating and non-integrating

adenovirus, a viral vector that is frequently used in cancer gene therapy modalities. Both control

virus (vCMV-null) and E4orf6-expressing virus (vCMV-E4orf6) lack the E1 gene, which renders

them non-replicative (Figure 3A) (20). Although the vCMV-null virus contains an intact E4

promoter and gene, expression of the E4orf6 protein from the endogenous gene was not detected

by either immunoblotting (Figure 3B) or immunofluorescent microscopy (Figures 3C), as

expected in the absence of the E1 gene products. The vCMV-E4orf6 virus expresses E4orf6

from a CMV promoter and lacks the endogenous viral E4 gene (20), and therefore, lacks

expression of the other E4 gene products. U251 glioblastoma cells infected with vCMV-E4orf6

were significantly more radiosensitive than those infected with vCMV-null virus (Figure 4).

Thus, transient expression of E4orf6 from a non-integrating, non-replicating adenoviral vector

significantly radiosensitizes U251 cells, indicating that E4orf6 expression and not stable

integration is responsible for radiosensitization by E4orf6.

E4orf6 inhibits cellular DNA DSBR- The inhibition of clonogenic survival (Figures 2 and

4) and the reported interaction between E4orf6 and DNA-PKcs (7) led us to hypothesize that

E4orf6 radiosensitizes RKO and U251 cells by preventing repair of IR-induced DNA breaks. To

test this hypothesis, we measured sublethal damage repair (SLDR) in the RKO clones. The

SLDR assay is a variation of the standard clonogenic survival assay based on the fact that cell

survival increases with increasing amounts of time between two doses of sublethal IR, providing

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that the cells can repair the initial IR damage prior to the second dose (21-23). RKO cells

expressing E4orf6 did not benefit from increased time between two radiation treatments of 3Gy

(Figure 5A). In contrast, the neomycin-transfected control cells demonstrated a two-fold

increase in survival when the two doses were separated by 6 hours. This result strongly suggests

that the E4orf6-expressing RKO clones have a decreased capacity for DSBR.

Recently, is was shown that γ�H2AX can serve as a quantitative measure of DSBs

because H2AX is phosphorylated at serine 139 in the region of the DSB and is subsequently

dephosphorylated following repair of the DNA break (24-27). DSBR in infected U251 cells was

assayed by measuring the levels of phosphorylation of histone 2AX at Ser139 (termed γ�H2AX)

by immunoblotting at various time points post-IR (28-31). U251 cells were infected with either

vCMV-null or vCMV-E4orf6 and irradiated with 4Gy at 48 hours post-infection. At several

time points post-irradiation, cells were harvested and analyzed for γ�H2AX by SDS-PAGE and

immunoblotting. Figure 5B shows that at 6 hours post-IR, a time at which more than 90% of

DSBs should have been repaired, the cells expressing E4orf6 contained substantially higher

levels of γ�H2AX compared with cells infected with control virus, suggesting that unrepaired

DSBs accumulate in the presence of E4orf6. This experiment was repeated with similar results

and immunoreactive bands were quantified, normalized to β�actin levels, and expressed as fold-

increase compared with the unirradiated control for each virus (Figure 5C). In addition, we have

analyzed the loss of phosphorylation of H2AX at Ser139 (termed �percent repair�) by examining

the difference between the 30 minute post-IR timepoint (consistently the maximum level) and

the 6 hour timepoint (the point at which most DSBR has occurred) (Figure 5D). In both cases,

E4orf6-expressing U251 cells retain significantly high levels of γ�H2AX indicative of

unrepaired DSBs.

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E4orf6 expression does not increase mutation frequency- We have demonstrated that

E4orf6 radiosensitizes by inhibiting DSBR (measured by SLDR assay) leading to the

accumulation of DSBs (measured by γ-H2AX levels); however, it has been previously suggested

that expression of E4orf6 itself may be mutagenic based on the hprt test for mutagenicity (32).

To determine if the decreased capacity for DSBR was due to increased cellular mutation

frequency, 293 cells were transfected with empty expression vector, or a vector expressing the

wild-type E4orf6 or the mutant L245P gene and a replication-competent reporter vector, pSP189.

In a representative experiment (Table I), 293 cells transfected with the empty expression vector

and the pSP189 reporter vector exhibited a mutation frequency of 3.5×10-7 mutations per bp,

consistent with the background mutation frequency reported for this cell line (33). Expression of

the wild-type E4orf6 or mutant L245P variants did not increase the mutation frequency. By

contrast, exposure of vector-transfected cells to mutagenic levels of UV light (50 J/m2) increased

the mutation frequency by nearly two orders of magnitude. Similar results were obtained in two

independent experiments. Thus, although E4orf6 reportedly increases the mutation frequency

two-fold at the chromosomal hypoxanthine guanine phosphoribosyl transferase (hprt) locus in

the D422 Chinese hamster ovary cell line (32), it does not elicit point or frameshift mutations in

a replicating plasmid. However, the amount of reporter plasmid recovered from 293 cells

expressing the E4orf6 protein was significantly reduced. The results of two independent

experiments indicated that expression of E4orf6 in 293 cells for 48 hours reduced the amount of

recovered pSP189 reporter by 50% compared to that recovered from cells transfected with the

empty expression vector or the L245P construct (Table I). This result is consistent with the

ability of the E4orf6 but not L245P protein to block DSBR, as naturally occurring DNA breaks

are formed and repaired during plasmid replication.

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E4orf6 does not inhibit the kinase activity of DNA-PKcs, but prolongs its

autophosphorylation following IR- Our results described thus far support a model in which

E4orf6 radiosensitizes cells by inhibiting DSBR, perhaps by inhibiting the DNA-PK complex

through a direct interaction with DNA-PKcs (7). The effect of E4orf6 on DNA-PK activity was

assessed by measuring the ability of DNA-PK to phosphorylate a p53-related peptide in vitro.

When expressed in U251 cells by an adenoviral vector, E4orf6 did not affect DNA-PK kinase

activity towards a heterologous substrate (Figure 6A). However, it remains controversial

whether p53-phosphorylation is an appropriate measure of DNA-PK activity with regards to

DNA repair and radioresistance because DNA-PK-dependent end-joining activity and radiation

resistance can be compromised while DNA-PK is still able to phosphorylate p53 (34,35).

Recently, the autophosphorylation of DNA-PKcs has been identified as a critical factor in

maintaining radioresistance, with either hypo- or hyper-autophosphorylation of DNA-PKcs

resulting in radiosensitization (14,36,37). Regulation of the autophosphorylation of DNA-PKcs

within a cluster of six serine and threonine sites is important in maintaining radioresistance (14).

Thus, we hypothesized that E4orf6 may affect DNA-PK autophosphorylation, which in

conjunction with subsequent dephosphorylation, is necessary for radioresistance.

Following treatment of infected U251s with IR, autophosphorylation of DNA-PKcs at

Thr2609 was measured over time by immunoblotting with a phospho-specific antibody.

Comparable amounts of Thr2609-phosphorylated DNA-PKcs accumulated in U251 cells

infected with either vCMV-E4orf6 or vCMV-null at early time points post-irradiation (Figure

6B). However, in contrast to vCMV-null infected cells, in which dephosphorylation was near

completion 6 hours post-irradiation, Thr2609-phosphorylated DNA-PKcs remained upregulated

in E4orf6-expressing cells, which is consistent with a radiosensitive phenotype. The experiment

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was repeated two more times with similar results. Reactive bands from all three experiments

were quantified and normalized to total DNA-PKcs levels (Figure 6C).

Discussion

Our data identifies the adenoviral protein E4orf6 as a radiosensitizer of human tumor

cells. We have demonstrated both stable and transient expression of E4orf6 in two different

tumor cell lines. This radiosensitization is occurring in the absence of E1B-55K, the adenoviral

protein that interacts with E4orf6, an interaction that is required for the degradation of MRE11

and p53 and the inhibition of adenoviral DNA concatemer formation (5,6). Our data reveals that

E4orf6 is able to radiosensitize tumor cells by inhibiting cellular DNA DSBR in the absence of

E1B-55K, and therefore, independent of MRE11 and p53 protein levels.

A recent study reported that transient transfection of E4orf6 in a variety of tumor cell

lines, including RKO cells, did not result in significant radiosensitization (19). We believe that

this discrepancy may result from the fact that following transient transfection not all of the cells

express E4orf6, which would be important in order to accurately assess clonogenic survival

following treatment with clinically relevant doses of IR. Furthermore, the lack of

radiosensitization by the E4orf6-L245P mutant suggests that the wild-type activity or the proper

conformation of E4orf6 is necessary for its radiosensitizing capabilities (the L245P mutation was

introduced to disrupt a critical amphipathic alpha helix in the E4orf6 protein) (15). Finally, the

similar radiosensitization effect measured in two independently selected wild-type E4orf6 clones

and the ability of adenoviral-expressed non-integrated E4orf6 to produce a similar effect,

suggests that the radiosensitizing property was not the result of positional effects from genomic

integration.

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Our results also argue against the possibility that E4orf6 is mutagenic. The levels of γ�

H2AX (DSBs) are similar between vCMV-null and vCMV-E4orf6 infected cells in the absence

of IR and E4orf6 does not increase the frequency of mutations (point or frameshift) in 293 cells.

However, E4orf6 plasmid recovery is reduced when compared with control plasmids, which is

consistent with our theory of an inhibition of cellular DSBR by E4orf6. More specifically,

during episomal DNA replication, type II topoisomerases transiently introduce double-strand

breaks in the nascent molecules (38). If any of the plasmids acquire a double-strand break

during this process, the inhibition of DSBR by the E4orf6 protein may prevent rejoining of the

DNA, thereby reducing the number of intact, replicated plasmids recovered.

We have found that expression of E4orf6, while not affecting the kinase activity of DNA-

PKcs with regards to heterologous substrates, prolongs the autophosphorylation of DNA-PKcs at

Thr2609. These data lead us to propose a model (Figure 7) in which E4orf6 inhibits the late

stages of repair as DNA-PKcs remains able to phosphorylate itself in response to DNA damage

and repair, but dephosphorylation of DNA-PKcs at Thr2609 is prevented. DNA-PKcs

autophosphorylation is Ku-dependent (14) and is proposed to occur as a signal for the repair

complex to undergo a conformational change conducive to ligation and/or post-ligation

dissociation from the DNA (34,35). Two possible modes of action for E4orf6 include either A) a

physical interaction with DNA-PKcs, or B) an inhibition of protein phosphatase 5, the

phosphatase implicated in the dephosphorylation of DNA-PKcs at Thr2609 (36). Although

E4orf6 has been shown to require E1B-55K to degrade MRE11 and prevent concatemer

formation (5), our model is consistent with these findings in that regardless of the state of DSB

ligation, damage-induced signaling is sustained by phosphorylation of H2AX at Ser139 and

DNA-PKcs at Thr2609 in the presence of E4orf6 alone. It has been recently proposed that

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sustained autophosphorylation of DNA-PKcs results in a radiosensitive phenotype (14,34)

possibly by signaling incomplete DNA repair and initiating growth arrest or cell death. Our data

is in agreement with this model.

In summary, we have demonstrated that the adenovirus E4orf6 protein radiosensitizes

human tumor cells by inhibiting DSBR without affecting p53 or MRE11 protein levels. E4orf6

promotes the accumulation of DSBs as observed by prolonged phosphorylation of both H2AX at

Ser139 and DNA-PKcs at Thr2609. Our results show for the first time that an adenoviral protein

has radiosensitizing properties by inhibiting the repair of damaged host cell DNA in vivo. A

model consistent with our data and that of other investigators is that E4orf6 promotes viral DNA

replication by inhibiting cellular NHEJ in an effort to prevent concatenation of viral DNA

(4,5,7). Consequently, overexpression of E4orf6 is sufficient to inhibit the NHEJ response to IR-

induced cellular DSBs thereby radiosensitizing the host cell. The ability of E4orf6 to inhibit the

activity of repair proteins other than DNA-PK has not yet been explored. It remains possible that

E4orf6 is able to inhibit MRE11 function without degrading it (a process that requires E1B-

55K). Future experiments will test such possibilities.

The significant radiosensitization achieved at clinically relevant doses (2-8Gy) by E4orf6

has promising implications for improving radiation therapy, especially in radioresistant tumors

such as glioblastomas. It is conceivable that E4orf6 delivered by a viral vector or by non-viral

methods could substantially improve the cytotoxic effects of localized beam radiation therapy,

such as gamma knife radiosurgery.

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Acknowledgements. We would like to thank C.S. Young for the generous donation of viruses

and Drs. Karin Drotschmann, Doug Lyles, and Heidi Chial for critical reading of the manuscript.

This work was supported in part by grants ARG0037 (CK) from the North Carolina

Biotechnology Center and CA77342 (DO) from the National Cancer Institute. JSO was

supported in part by NIH training grant T32 AI07401 to the Department of Microbiology and

Immunology. LSH would like to personally thank Drs. Pizzorno and Chernin for their guidance.

Note: J. S. Orlando is currently at the Department of Medicine, Harvard Medical School, 99 Brookline Ave. RN132, Boston MA, 02215. S. B. Waters is currently at the Curriculum in Toxicology, The University of North Carolina, Chapel Hill NC 27599.

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Figure Legends

Figure 1. Stable expression of functional E4orf6 in RKO cells. (A) Wild-type E4orf6 stably

expressed in RKO cells is functional as measured by localization of E1B-55K to the nucleus. The

stable RKO clones (neomycin vector control, E4orf6, E4orf6-L245P) were transfected with a

plasmid expressing E1B-55K and analyzed for expression and localization of E4orf6 and E1B-

55K by indirect immunofluorescence microscopy. Total cells in the field are represented by DIC

and counterstained with 4',6-diamidino-2-phenylindole (DAPI). (B) Stable RKO clones

expressing wild-type E4orf6 exhibit E1B-55K-dependent degradation of p53 and MRE11. The

RKO stable clones were infected with one of three adenoviruses in order to supply the clones

with E1B-55K and/or E4orf6: dl309 (wild-type Ad), expresses both E1B-55K and E4orf6;

dl1014, expresses wild-type E1B-55K but lacks E4orf6 expression; dl1017, deficient at

expressing both E1B-55K and E4orf6. The infected clones were harvested for immunoblot to

analyze the stability of p53 and MRE11 protein levels. Early and late passage numbers of wild-

type E4orf6 clone 4 were used to demonstrate that E4orf6 expression remains functional at late

passage.

Figure 2. Stable expression of functional E4orf6 radiosensitizes RKO cells. The RKO clones

(neomycin vector, closed circles; E4orf6 clone 4, open circle; E4orf6 clone 1, closed triangle;

and E4orf6-L245P clone 2, open triangle) were plated at low density and treated with ionizing

radiation. Surviving fraction was normalized by plating efficiency and plotted on a semi-log

scale. Error bars represent SEM values.

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Figure 3. Transient expression of E4orf6 via infection with a non-replicating adenoviral

vector. (A) Diagram of E4orf6-expressing and control adenovirus genomes. Both viruses lack

the E1 region rendering them non-replicative. (B and C) E4orf6 expression from vCMV-E4orf6

and not vCMV-null virus. U251 glioblastoma cells were infected with either vCMV-null or

vCMV-E4orf6 at an MOI sufficient to infect more than 95% of the cells. (B) At 48 hrs post-

infection, the cells were harvested for immunoblotting for E4orf6 protein levels. β�actin was

used as a loading control. (C) At 48 hrs post-infection the cells were fixed for indirect

immunofluorescence microscopy to identify E4orf6-expressing cells. Cells were counterstained

with Hoescht33342.

Figure 4. Transient expression of E4orf6 via infection with a non-replicating adenoviral

vector radiosensitizes U251 glioblastoma cells. U251 cells were plated at equal density, mock

infected (closed circles) or infected with either vCMV-null (open triangles) or vCMV-E4orf6

(closed squares), and scored for long-term cell survival by crystal violet staining. Absorbance

values were normalized to survival at 0 Gy for each infection condition. Significant

radiosensitization as determined by a two-tailed Student�s t test (n=3; *p<0.05, **p<0.005) is

marked with the appropriate asterisk.

Figure 5. Both stable and transient expression of E4orf6 inhibits DSBR in response to

ionizing radiation. (A) A sublethal DNA damage repair assay (SLDR) was performed by

plating the RKO clones at low density and irradiating with two 3 Gy doses of gamma radiation

separated by the indicated time points. Surviving colonies were normalized to T0 (no time

between doses). Values are represented by fold-increase in survival with time and each value

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represents the average of three plates. The asterisk represents a significant difference in survival

as determined by Student�s t test (n=3, *p<0.05). (B) E4orf6 inhibits DSBR as measured by γ�

H2AX levels. U251 cells were irradiated with 4Gy at 48 hrs post-infection. At the given time

points post-irradiation, the cells were harvested for immunoblotting with anti- γ-H2AX

antibodies. Immunoblotting with β-actin was used as a loading control. (C) The immunoreactive

bands from three independent γ-H2AX immunoblots were quantified using Scion Image

software, normalized to β-actin, and expressed as -fold increase in γ-H2AX compared with the

unirradiated control for each virus (*p<0.01). (D) The loss of phosphorylation from 30 minutes

to 6 hours was analyzed from the data in (C) to yield percent repair values (*p<0.005).

Table I. E4orf6 decreases the recovery of plasmid without affecting mutation frequency.

The average amount of pSP189 plasmid recovered is expressed as a percentage of the plasmid

recovered from cells transfected with the empty expression vector. The results of two

independent experiments are shown in parentheses.

Figure 6. E4orf6 does not inhibit the kinase activity of DNA-PKcs towards heterologous

substrates, but prolongs autophosphorylation of DNA-PKcs at Thr2609. (A) Mock, vCMV-

null, or vCMV-E4orf6 infected U251 cells were tested for in vitro kinase activity on a p53

peptide. Wortmannin (30µM for 3 hours) treated U251 cells served as a positive control for

kinase inhibition and M059J and M059K cell lysates served as negative and positive controls,

respectively, for the function of the assay. (B) U251 cells infected with either vCMV-null or

vCMV-E4orf6 were irradiated with 20Gy and harvested at the given time points post-irradiation

for immunoblotting with a Thr2609 phospho-specific antibody of DNA-PKcs. A 20Gy IR dose

was chosen for this experiment, due to the relatively low affinity of the phospho-specific

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23

antibody. An in vitro kinase reaction sample was loaded as a positive control for P-Thr2609 and

total DNA-PKcs was used as a loading control. (C) The immunoreactive bands from three

independent experiments were quantified using Scion Image software and normalized to total

DNA-PKcs. Consistent with previously published reports (36), in some cases P-Thr2609 became

partially degraded before 360 minutes post-IR, in which case we included both bands (450 kDa

and 250 kDa) in our analysis of both viruses. The data was analyzed for significance by a two-

tailed Student�s t test (n=3, *p<0.001).

Figure 7. Model of the inhibition of the late stages of DNA repair by E4orf6. In the presence

of E4orf6, cells retain their ability to phosphorylate H2AX at Ser139 (an early event) and

undergo Ku-dependent DNA-PKcs autophosphorylation at Thr2609 (a late event). The

autophosphorylation step is thought to induce a conformational change in the repair complex

preparing it for ligation and dissociation of the proteins from the DNA (34,35). However, these

proteins remain phosphorylated at times when efficient repair would be complete and H2AX and

DNA-PKcs would otherwise be dephosphorylated. Therefore, E4orf6 is most likely inhibiting

the repair process at the late stages, by preventing ligation and/or inhibiting the

dephosphorylation which would otherwise signal complete repair and result in cell survival. Our

model is in agreement with and partially adapted from Block et al (35).


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control wild type E4orf6 E4orf6-L245PA Clone 1 Clone 1 Clone 4 Clone 2

DIC

DAPI

E4orf6

E1B-55K

3X inset

Red/Green Merge

neo neo L245P

E4orf6

.4 ea

rly

E4orf6

.4 lat

e

Cell line

Virus dl309 dl1014 dl1017

E4orf6

p53

MRE11

ß-actin

E1B-55kD

neo L245P

E4orf6

.4ea

rly

E4orf6

.4 lat

e

B

Figure 1


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IR Dose (Gy)

0 2 4 6 8

Sur

vivi

ng F

ract

ion

0.001

0.01

0.1

1 RKO.neo.1RKO.E4orf6.4RKO.E4orf6.1RKO.L245P.2

Figure 2


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AvCMV-null

CMVp

E4p∆E1A ∆E1B

CMVp

∆E1A ∆E1B

∆E4E4orf6vCMV-E4orf6

E4orf6

dl309Moc

kvC

MV-null

β-Actin

vCMV-E4orf6

B

C

vCMV-null

vCMV-E4orf6

Hoescht33342 E4orf6 Combined

Figure 3


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Dose (Gy)

0 2 4 6 8

Sur

vivi

ng F

ract

ion

(Nor

mal

ized

)

0.01

0.1

1MockNullE4orf6

**

*

Figure 4


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A

B

γ-H2AX

β-actin

vCMV-null vCMV-E4orf60Gyt post-IR (min) 5 30 360 5 30 3600Gy

0.5

1.5

2.5

0 2 4 6Time (h) between 3Gy Doses

E4orf6.1Neo.1

*Fo

ld In

crea

se in

Sur

viva

l

C D

0

2

4

6

8

10

0Gy 5 30 360

Time Post-IR (min)

Fold

Incr

ease

in p

hos/

n (E

/C)

vCMV-nullvCMV-E4orf6 *

0

20

40

60

80

100

6hr

Time Post-IR

% R

epai

r (no

rmal

ized

) vCMV-nullvCMV-E4orf6

*

Figure 5


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0no plasmid

100Vector

108 (118.9, 97.1)E4orf6-L245P

49 (46.1, 51.9)E4orf6

Average (%)PlasmidPlasmid Recovery

2.5x10-5Vector/UV

2.3x10-7E4orf6-L245P

4.3x10-7E4orf6

3.5x10-7Vector

Mut Frequency (mut/bp)Plasmid

Mutation Frequency

Table I


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P-Thr2609

DNA-PKcs

IR 20Gy - + + - + +t post-IR (min) - 30 360 - 30 360

In vi

tro vCMV-null vCMV-E4orf6B

0

0.2

0.4

0.6

0.8

1

1.2

mock

vCMV-null

vCMV-orf6

wortman

n.

M059J

M059K

Kin

ase

activ

ity

(Nor

m.)

A

00.20.40.60.8

11.21.41.61.8

0Gy 30 360

Time Post-IR (min)

OD

(nor

mal

ized

to to

tal

DN

A-P

K)

vCMV-nullvCMV-E4orf6

*C

Figure 6


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IRIR

H2AX H2AX

P

PPP P

H2AX

P

H2AX

PDNA-PK

PDNA-PK

E4orf6E4orf6

Figure 7


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Waters, Steven A. Akman, David J. Chen, David Ornelles and Constantinos KoumenisLori S. Hart, Steven M. Yannone, Christine C. Naczki, Joseph S. Orlando, Stephen B.

radiosensitizes human tumor cells in an E1B-55K-independent mannerThe adenovirus E4orf6 protein inhibits DNA double strand break repair and

published online October 26, 2004J. Biol. Chem.

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