Paclitaxel and vincristine potentiate adenoviral ... - Semantic Scholar

Cancer Gene Therapy (2006) 13, 1105–1114 r

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ORIGINAL ARTICLE

Paclitaxel and vincristine potentiate adenoviral oncolysis that is associated with cell cycle and apoptosis modulation, whereas they differentially affect the viral life cycle in non-small-cell lung cancer cells MAI AbouEl Hassan1, SR Braam and FAE Kruyt Department of Medical Oncology, VU University Medical Center, Amsterdam, The Netherlands Chemotherapy, including microtubule (MT)-interacting agents, can enhance the tumor-eradicating activity of replication-competent adenoviruses. The purpose of this study was to obtain more insight into the mechanism underlying this enhancement that may be exploited for the development of improved therapy. Two MT-interacting agents with opposite activity, paclitaxel (PTX) that stabilizes and vincristine (VCR) that destabilizes MTs, were found to synergistically enhance adenoviral oncolysis in non-small-cell lung cancer (NSCLC) cells. To explore the possibility that these drugs affect the viral life cycle by modulating adenoviral gene expression, we used a quantitative reverse transcription-polymerase chain reaction assay and found that PTX, but not VCR, increased the expression of E1A13S, ADP and Penton genes, which correlated with an increase in viral particle assembly and release. Next, the effect of combined treatment on cell-cycle progression was studied. Both drugs suppressed adenovirus-induced S-phase arrest and instead caused G2/M arrest, which was accompanied by an increase in apoptotic cells. Taken together, the enhancement of oncolysis by MT-interacting drugs appears not to require specific MT transport or scaffold functions. Our findings suggest that MT-interacting drug-induced cellular signals that modulate cell-cycle arrest and apoptosis are primarily on the basis of their oncolysis-enhancing activity. Cancer Gene Therapy (2006) 13, 1105–1114. doi:10.1038/sj.cgt.7700984; published online 14 July 2006 Keywords: adenovirus; gene expression; microtubules; chemotherapy; lung cancer

Introduction

Conditionally replicating adenoviruses (CRAds) represent a promising class of biologic agents that selectively replicate in and lyse cancer cells (for reviews see Kirn, 2000; Alemany et al.,2000; and McCormik 2001).1–3 In animal studies and in the clinic, CRAds appear to be safe and well tolerated; however, their anti-tumor activity as a single agent is modest and improvement of the therapy is required.4–6 Strategies currently explored to enhance the efficacy of these agents include the combined use of CRAds with conventional chemotherapeutic agents, which showed synergistic anti-tumor activity.7–10 The molecular mechanism underlying this synergy is largely unknown and its elucidation may lead to the design of improved adenoviral vectors and/or treatment regimens. Correspondence: Dr FAE Kruyt, Department of Medical Oncology, VU University Medical Center, PO Box 7057, 1007 MB Amsterdam, The Netherlands. E-mail: [email protected] 1 Current address: Department of Cancer Biology, National Cancer Institute, Cairo University, Cairo, Egypt. Received 27 November 2005; revised 10 April 2006; accepted 12 June 2006; published online 14 July 2006

Several studies have been set out in order to understand the mechanism of synergy between adenoviruses (Ads) and chemotherapy and the role of a number of cellular proteins that are known to influence the sensitivity for chemotherapeutic agents has been extensively investigated. These include MDR, p53 and other proteins involved in apoptosis regulation.11–14 On the other hand, the effect of chemotherapy on Ad genes has to our knowledge hardly been explored. The life cycle of the adenovirus is rather complex and different stages can be recognized, which include the internalization and disassembly of the virus, the take over of host gene expression by early genes and the assembly of progeny virus with structural components provided by the late Ad genes followed by lysis of the infected cells and release of progeny.15,16 Recently, we have developed a quantitative reverse transcription-polymerase chain reaction (RT-PCR) assay to monitor the expression of several Ad type 5 genes, including the immediate-early gene E1A13S, the E2 region gene DNA polymerase (Pol) and the late region genes encoding the adenoviral death protein (ADP) and Penton (Pent), as a means to study the effect of host cell factors or exogenous treatments on Ad gene expression.17

Adenoviruses and microtubule-interacting agents MAI AbouEl Hassan et al

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In the present study, we examined the effects of two drugs with opposite activity on the microtubule (MT) network, paclitaxel (PTX) that stabilizes MT polymerization and vincristine (VCR) that prevents the assembly of MT molecules,18,19 on Ad-dependent oncolysis of nonsmall-cell lung cancer (NSCLC) cells. Both drugs strongly enhanced the oncolytic effect of Ad5, although by different mechanisms with respect to their effect on Ad gene expression and viral particle (VP) assembly and release. However, PTX and VCR had similar effects on cell-cycle arrest and induction of cell death. Considering the overlap in effects, we propose that MT-interacting drugs predominantly enhance Ad oncolytic activity by modulating the cell cycle that is associated with cell death activation. Our findings implicate that specific MT functions are not required for the potentiation of oncolysis.

Materials and methods

Cell culture and treatment NSCLC NCI-H460 cells were grown in Rosewell Park Memorial Institute medium supplemented with 10% fetal bovine serum (FBS), 100 U penicillin/ml and 100 mg of streptomycin/ml and incubated at 371C in humidified 5% CO2. H460 cells grown to confluence in six-well plates were incubated with Ad5 at multiplicity of infection (MOI) of 100 with(out) 10 or 50 nM PTX or VCR. Two hours post–infection, cells were refreshed with virus-free medium containing 10 or 50 nM PTX or VCR. MTT assay Two and 5 days after treatment with Ad5 alone or in combination with different concentrations of PTX or VCR, the viability of H460 cells was measured by 3-[4,5dimethylthiazol]-2,5-diphenyltetrazolium bromide (MTT) assays as described previously.20 The percentage survival (taking the blank as 100% survival) was plotted as a function of the MOI and the lethal concentration (LC)50 values of Ad5 alone or in combination with PTX or VCR were determined being the MOI of Ad5 required to kill 50% of the cultured cells. Possible synergistic effects of the combined treatments were evaluated by calculating the combination index (CI) according to Chou and Talalay21 using the commercially available CalcuSyn software version 1.1.1 from Biosoft (Cambridge, UK). Strict criteria were adopted for the interpretation of CI, whereby CI values p0.7 indicate moderate to strong synergy, and CI values X1.2 indicate antagonism. CI values within the range of 0.7–1.2 represent either weak synergism or additivity. Quantitative RT-PCR Ad5 gene expression assay H460 cells were harvested at 0, 6, 12, 24, 30, 36 and 48 h post-infection and were dissolved in 1 ml Trizol for the isolation of total RNA as described by the manufacturer (Invitrogen, Breda, The Netherlands). RNA quality and quantity were evaluated by agarose gel electrophoresis

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and with UV spectrometry optical density (OD260/OD280 ratio is generally X1.7).22 Total RNA (1mg) was reverse transcribed with 300 U Moloney murine leukemia virus reverse transcriptase (M-MLV RT) (Invitrogen) in a total volume of 40 ml containing 2 mM dithiothreitol, 0.4 nM deoxynucleoside triphosphates mix (Roche, Basel, Switzerland), 0.2 mg/ml Hexas (Amersham Biosciences, Uppsala, Sweden) and 40 U RNAsin (HT-Biotechnology Ltd, Cambridge, UK) and incubated at 371C for 2 h followed by 10 min at 651C The expression levels of Ad5 E1A13S, Pol, ADP and Pent genes were monitored in time using real-time RT-PCR with specific primer sets and standard samples prepared by a twofold serial dilution of the cDNA derived from H460 cells infected with Ad5 alone as we described previously.17 The expression levels were normalized by arbitrarily setting the adenoviral mRNA levels from Ad5-alone infected cells at 24 h time point to one. Negative controls, samples without RT or cDNA template were included with every PCR run and were always negative (not shown).

Determination of replication and release indices Medium and H460 cell pellets were collected at 6 and 32 h post-infection with MOI 100 with(out) 10 or 50 nM PTX or VCR. The media and cell pellets (suspended in 0.5 ml PBS) were subjected to four cycles of freeze/thawing in dry ice–ethanol bath in order to obtain all VPs from the infected cells (Ad crude supernatants). The Ad5 VPs and total DNA was estimated according to Ma et al.23 with minor modification. In brief, Ad VP were estimated from the amount of encapsidated Ad DNA in one portion of the Ad crude supernatants by removing non-encapsidated Ad DNA with DNase I (Roche Diagnostics GmbH, Benzberg, Germany) followed by proteinase-K to remove virus-bound proteins. Another portion of Ad crude supernatants was used to estimate the total Ad DNA by direct treatment of the Ad crude supernatants with proteinase-K, thereby pooling the non-encapsidated Ad DNA together with the encapsidated Ad DNA for PCR reaction using hexon-specific primers: 50 -ATGATGCCG CAGTGGTCTTA-30 and 50 -GTCAAAGTCCGTGGAA GCCAT-30 . Real-time PCR was performed by mixing 2 ml of the previously mentioned different Ad DNA preparations and 18 ml of the reaction mix of the LightCycler FastStart DNA Master SYBR Green I (Roche) according to the manufacturer’s protocol and as described before.17 Fluorescence was measured at an acquisition temperature of 871C allowing the construction of the amplification plot and determination of the crossing point (Cp). The amount of DNA copies (total and encapsidated DNA (VP)) were estimated by interpolating the Cp of each sample on standard curves previously constructed from serially diluted proteinase-Ktreated Ad5 CsCl-purified stocks with known DNA copy number. The PCR was carried out using a LightCycler System (Roche) and the data were analyzed with the provided LightCycler software. The replication index was calculated in order to correct for a possible unequal


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uptake of the virus at 6 h (equations below):

Replication index ðVPÞ VP ðcell pellet þ mediumÞ at 32h time point ¼ VP ðcell pellet þ mediumÞ at 6h time point Comparing the replication index calculated from the VP to that calculated from the total DNA indicates the efficiency of Ad assembly. In order to calculate the release of the assembled virus and discriminate it from the nonspecific release of naked Ad DNA, which could be released owing to the cytotoxic effects of chemotherapy, we have calculated the release index from both total DNA and VP: Release index ðTotal DNAÞ Total DNA in medium at 32h time point ¼ 100 Total DNA ðcell pellet þ mediumÞ at 32h time point

Release index ðVPÞ VP in medium at 32h time point 100 ¼ VP ðcell pellet þ mediumÞ at 32h time point

Cell-cycle analysis In order to determine the effects of Ad5 (MOI 100) alone or in combination with 10 or 50 nM of PTX or VCR on cell-cycle progression and apoptosis activation, H460 cells were incubated with Nicoletti buffer (50 mg/ml propidium iodide (Sigma Chemicals, St Louis, MO), 0.1% sodium citrate, 0.1% Triton X-100 and 1 mg/ml RNA-seA (Roche, Basel, Switzerland) in PBS), as described earlier,24 at 6 and 24 h post-treatment and analyzed by FACScan (Becton Dickinson, Mountainview, CA). The fraction of cells in the sub-G1 phase, representing apoptotic cells, was assessed using the CellQuest program (version 3.2.1f1, Becton Dickinson). The fluorescenceactivated cell sorter (FACS) data were further analyzed using ModFitLT software (version 3.0, Verity Software House Inc., Topsham, ME) to calculate separately the fraction of cells in G1, S and G2 phases.

Results

PTX augments the oncolytic effect of Ad5 First, we studied the effect of PTX on Ad5-induced cell killing in NSCLC H460 cells, a cell culture model that in our laboratory is extensively studied with respect to mechanisms of cancer therapy-induced cell death. Cells were infected with Ad5 alone (MOI 100) or in combination with 10 or 50 nM of PTX and viability was determined by MTT assays at 2 and 5 days post-infection (Figure 1 and Table 1). We found a concentration of

% Control

100 75 50 25 0

Ad5 Ad5 + 10 nM PTX Ad5 + 50 nM PTX

b 125 MOI

100 % Control

Replication index ðTotal DNAÞ Total Ad DNA ðcell pellet þ mediumÞ at 32h time point ¼ Total Ad DNA ðcell pellet þ mediumÞ at 6h time point

75 50 25 0 0.0001 0.001

0.01

0.1

1

10

100

MOI Figure 1 PTX augments the oncolytic effect of Ad5. The viability of H460 cells was measured by MTT assay at 2 (a) and 5 (b) days postinfection with different MOI of Ad5 with(out) different concentrations of PTX. The depicted effect of Ad5 in combination with PTX was corrected for the cytotoxic effect of PTX alone at all time points by taking cells treated with the single agent as 100% viability. Values are the mean (n ¼ 3)7s.d.

10 nM PTX to have minimal toxicity on H460 cells, whereas 50 nM PTX induced about 30% killing of treated cells, comparable to our previous report (and results not shown).24 Ad5 induced an MOI-dependent cell killing with an LC50 of 30 and 0.17 PFU (plaque-forming unit)/ cell at 2 and 5 days post-infection, respectively. PTX greatly enhanced the oncolytic effect of Ad5 at 5 days post-infection with a sensitization factor (SF) of 6.8- and 34-fold for 10 and 50 nM PTX (Po0.01), respectively (Table 1). In order to study whether this PTX-induced sensitization of cells to Ad5 is synergistic, the CIs were calculated from the same set of data and found to range between 0.3 and 0.8, indicative of strong and moderate synergistic effects between Ad5 and PTX, respectively.

Expression of E1A13S, ADP and Pent is enhanced by PTX In order to investigate the mechanism underlying the synergistic effect of PTX and Ad5, we studied the effect of PTX on the expression level of several Ad5 genes representing early and late genes that are active at different stages of the viral reproductive cycle. For this, H460 cells infected with an MOI of 100 were simultaneously treated with 10 and 50 nM PTX and the expression levels of E1A13S, Pol, ADP and Pent were measured by real-time RT-PCR at different time points for 48 h, the approximate duration of one round of replication. Figure 2 shows that co-treatment with 50 nM

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Adenoviruses and microtubule-interacting agents MAI AbouEl Hassan et al Table 1 PTX and VCR enhance the oncolytic effect of Ad5 in H460 cells in MTT assays LC50 (PFU./ml)a 2 days

Mean SF

Mean SF

5 days

Ad

Ad+10PTX

Ad+50PTX

Ad

Ad+10PTX

Ad+50PTX

30.0 1.0

28.0 1.1

20.0 1.5

0.17 1.0

0.025 6.8

0.005 34.0

Ad

Ad+10VCR

Ad+50VCR

Ad

Ad+10VCR

Ad+50VCR

28.5 1.0

25.0 1.1

6.0 4.8

0.17 1.0

0.02 8.5

0.004 42.5

Abbreviations: MOI, multiplicity of infection; MTT, 3-[4,5-dimethylthiazol]-2,5-diphenyltetrazolium bromide; PTX, paclitaxel; SF, sensitization factor; VCR, vincristine. Results obtained at different MOI and combined with 10 or 50 nM drug after 2 and 5 days post-infection are indicated. a Values are the mean (n ¼ 3)7s.d., which was always less than 10% of the mean.

E1A13S

5

ADP

(*: p < 0.05)

12

Ad5 Ad5 + 10PTX Ad5 + 50PTX 2.5 Relative expression (arbitrary units)

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**

**

** **

6

**

**

0

0 Pol

5

Pent

10

** **

5

2.5

0

**

0 0

12

24 Time (h)

36

48

0

12

24 Time (h)

36

48

Figure 2 PTX enhances adenoviral gene expression in a concentration-dependent manner. The levels of expression of adenoviral E1A13S, Pol, ADP and Pent were measured within 48 h after Ad5 infection at an MOI of 100, solely or in combination with different concentration of PTX. Values are the mean (n ¼ 3)7s.d. The P-values were calculated by comparing the expression of each gene in cells infected with Ad5 alone or in combination with different concentrations of PTX at a given time point using Student’s t-test.

PTX significantly enhanced the expression levels of a number of Ad genes at different times after infection; E1A13S, maximally fivefold at 36 h post-infection (Po0.05); ADP, approximately a threefold increase between 30 and 36 h post-infection (Po0.05); and Pent, a maximal fivefold increase at 24 h post-infection (Po0.05). The expression of the DNA Pol gene was not significantly altered by PTX and also indicates that the

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enhancing effect of PTX on Ad gene expression is not a general phenomenon. Moreover, it was noted that the variability in Ad gene expression was larger in experiments where 50 nm PTX was added. This is likely caused by the strong effects of high concentrations of PTX on cell-cycle progression, which influences Ad gene expression that is known to vary in a cell cycle-dependent manner (see also below).25 Overall, co-treatment with


10 nM PTX was not as potent as 50 nM PTX in stimulating Ad gene expression. It mainly significantly affected ADP and Pent with an approximately twofold (Po0.05) increase at 36 h post-infection. The smaller effect of a low concentration of PTX on Ad gene expression correlated with a lower SF observed at 10 nM PTX when compared to the higher SF values at 50 nm drug (Table 1).

PTX increases the assembly and release of VPs We next studied whether PTX has an influence on the replication, assembly and release of Ads. For that purpose, the replication and release indices were calculated from the total amount of adenoviral DNA and VP (see also Materials and methods), which were determined in Ad5-infected H460 cells and in the overlaying medium at 6 and 32 h post-infection. The replication indices of Ad5 DNA and VP were around 2500- and 1100-fold of the initial amount of internalized virus, respectively, indicating a 3log increase in Ad5 virions. The release indices of DNA and particles were comparable and represented about 2% of the total intra- and extracellular viral DNA or particles, respectively. Figure 3a shows that PTX did not significantly affect the DNA replication index. However, determination of viral assembly clearly showed a 6.8-fold increase (P ¼ 0.01) of the VP replication index. In line with this, Figure 3b shows that the release

Folds replication index

a 10 8

(p = 0.01)

Ad5 DNA Ad5 VP

6

index of Ad5 was not affected by the combined treatment with PTX, whereas it was significantly increased when calculated from the VP release (2.4-fold at 50 nm PTX, P ¼ 0.016). Thus, in particular, the high concentration of PTX enhances the amount of VP assembly and release and does not influence viral DNA replication, which is in agreement with the RT-PCR results showing that 50 nM PTX increases the expression of Pent and ADP but not of Pol. These data suggest that the increased expression levels of Pent and ADP translate in elevated levels of VP production and perhaps more efficient cell lyses.

Effect of PTX on cell-cycle progression and apoptosis activation in Ad5-infected cells Both PTX and Ad5 are known to trigger cell-cycle arrest at the G2/M and S phase, respectively. To study their combined effects on cell-cycle progression, we co-treated H460 cells with Ad5 (MOI 100) and different concentrations of PTX and performed FACS analyses at 6 and 24 h post-infection. As expected, the sole treatment of H460 cells with PTX caused a time- and concentrationdependent increase in cells in the G2/M fraction, up to approximately 50% at 24 h post-treatment with 50 nm PTX (Figure 4). In addition, cells infected with Ad5 alone did not noticeably alter the cell-cycle profile 6 h after infection, whereas after 24 h, a strong accumulation of cells (approximately 70%) in the S phase was observed. The cell-cycle profile of H460 cells exposed to both agents at 6 h post-infection was comparable to the profiles observed after treatment with PTX alone. However, interestingly, after 24 h a strong accumulation of cells occurred in the G2/M fraction (approximately 70%) and with almost no cells detectable in the S phase.

4 2

a 100

0

G1 S G2 / M

60 40 20

2

0 1

b 100 80

0P T A d5 +5

0P T d5 +1

X

X

0 d5

Figure 3 PTX co-exposure stimulates adenoviral assembly and release independent from Ad5 DNA replication. The replication (a) and release (b) indices were calculated from the Ad5 genome copy number or from Ad5 VP in the medium and pellet of H460 cells at 6 and 32 h post-infection with Ad5 (MOI 100) alone or simultaneously treated with different concentrations of PTX. Values are the mean (n ¼ 3)7s.d. The P-values were calculated by comparing the fold replication/release index of Ad5 alone or in combination with different concentrations of PTX using Student’s t-test.

20

A

d5

A

A

d5

A

40

X

0 +5

0 +1

60

50 PT

A

X PT

X

X PT

10 PT

d5

B

0

% Cells

Folds release index

(p = 0.016)

3

% Cells

80

b

Figure 4 H460 cells co-exposed to PTX and Ad5 arrest in the G2/M phase. Fractions of H460 cells in G1, S and G2 phases were determined by FACS analysis of propidium iodide (PI) -stained cells 6 h (a) and 24 h (b) post-treatment with MOI 100 and/or 10 or 50 nM PTX. Values are the mean (n ¼ 3)7s.d.

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1110 20

6h

P = 0.02

24 h

% Cells

15

P = 0.01

P = 0.02

P = 0.01

10 5

X PT

X d5 + A

A

d5 +

10

50

PT

d5 A

PT X 50

10 PT X

B

0

Figure 5 Effect of Ad5 on PTX-induced apoptosis. The fraction of sub-G1 cells was measured by FACS analysis of propidium iodide (PI)-stained cells at 6 and 24 h post-infection at an MOI of 100 with(out) 10 or 50 nM PTX. Values are the mean (n ¼ 3)7s.d. The P-values were calculated by comparing the fraction of apoptotic cells after infection with Ad5 alone or in combination with different concentrations of PTX at a certain time point using Student’s t-test.

addition, co-treatment of H460 cells with VCR appeared not to enhance either the replication or the release indices of Ad5 (Figure 6d and e) that corresponds with the lack of VCR-dependent increases, found on the level of capsid protein (Pent) and oncolysis-mediating ADP. Interestingly, the effects after combining VCR and Ad5 on cellcycle progression were highly similar as earlier observed for PTX (Figures 4, 6f and g). VCR-dependent accumulation of cells in G2/M was confirmed, in particular enforced by 50 nm VCR. Ad5 alone evoked accumulation of cells in the S phase and co-treatment with VCR prevented this and caused a strong accumulation of cells in the G2/M phase. In addition, VCR triggered a concentration- and time-dependent increase in hypodiploid (sub-G1) cells up to around 20% at 50 nM and 24 h after treatment (Figure 6h). The combined treatment with Ad5 did not further enhance the apoptotic cell fraction, but instead reduced apoptosis more clearly than PTX. Discussion

PTX is known to trigger apoptosis and we next assessed the sub-G1 cell fraction representing cells with a hypodiploid genome caused by DNA degradation during apoptosis. PTX caused a concentration-dependent increase in the sub-G1 fraction detectable 24 h after treatment with around 10% at 10 nm and 20% at 50 nm, as shown in Figure 5. Cells co-exposed to Ad5 and PTX showed similar levels of apoptotic cells when compared to PTX alone. Cells infected with Ad5 alone did not induce the accumulation of cells in sub-G1. Taken together, these results suggest that PTX overrides Ad5-induced S-phase arrest and instead causes G2/M arrest that is accompanied by apoptosis activation.

Mechanism of VCR-dependent enhancement of Ad5-induced cell killing The experiments above were performed with PTX that is known to stabilize MTs. In order to examine further a possible role of the MT network in mediating the synergistic activity between Ad5 and PTX, we performed similar experiments with VCR, a potent anticancer drug known to destabilize MTs. In MTT assays (Figure 6a and b), VCR combined with Ad5 demonstrated at least equal or even slightly higher synergistic cell killing in H460 cells when compared to the PTX/Ad5 combination, with an SF of 4.8 at 2 days after treatment and with 50 nm PTX (see Table 1). At 5 days post–infection, VCR greatly sensitised H460 cells to Ad5 oncolysis with an SF of 8.5- and 42.5fold for 10 and 50 nM VCR, respectively (Table 1). Evaluation of the effect of VCR on adenoviral gene expression revealed less dramatic changes as observed for PTX. Figure 6c shows only a modest VCR-dependent increase on E1A13S expression at time points later than 24 h post-infection and a small increase on Pol transcript levels. However, ADP and Pent expression were not affected by VCR treatment. The somewhat lower levels of Ad5-only expression obtained in Figure 6c in comparison with Figure 2 represents some experimental variation. In

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Here, we studied the effect of PTX and VCR on Adinduced oncolysis of NSCLC cells and found that both stabilization of MT polymerization and depolymerization enhances tumor cell killing. The native Ad5 virus was used as a representative oncolytic agent rather than specific CRAds that differ considerably depending on the genetic modifications made. Nonetheless, our unpublished results also demonstrated synergistic enhancement of CRAds by PTX, including infectivity-enhanced D24RGD,26 in line with a previously demonstrated augmentation of ONYX-015 by PTX.10 The sequence of administration of Ads and chemotherapeutic agents is known to influence synergistic activity. For example, Heise et al.27 showed that the treatment with the virus followed by cisplatin-based chemotherapy, or concurrent therapy, were superior to the treatment with cisplatin followed by the virus. In our studies, Ads and drugs were simultaneously added, which also gave optimal enhancement of oncolysis when compared to schedules were PTX or VCR were added prior or after Ad infection (data not shown). Taking the standpoint of an adenovirus, several mechanisms could account for the increased oncolytic activity after drug treatment, such as increased infection efficiency, improved MT-dependent intracellular transport of the adenovirus, a MT-dependent stimulatory effect on VP assembly or improvement of viral release. In this context, the use of an MT-stabilizing and -destabilizing agent allowed us to evaluate the involvement of the MT network. As both drugs enhanced the efficacy of Adinduced cell killing to a similar extent while imposing opposite effects on MTs, we conclude that MT-dependent functions are unlikely to be main determinants of synergistic activity. In addition, PTX has been reported to facilitate the nuclear transport of adenoviral as well as herpes simplex virus capsids after endosomal escape.28,29 If this phenomenon has a significant enhancing effect on the lytic cycle of Ads is questionable, as in our RT-PCR


1111

a 125

c Relative expression (arbitrary units)

50 Ad5 Ad5 + 10 nM VCR Ad5 + 50 nM VCR

75 50 25

2.5

1

10

0

0.0 12

24 Time (h)

100

3

Pent

5

0 0.1 MOI

10

Pol

36

48

0

12

24 Time (h)

f 100

Total DNA VP

% Cells

80

2 1

36

48

G1 S G2/M

60 40 20 0

0 3

g 100

R VC

R

50 d5

P = 0.005

h 20

6h

P = 0.02

24 h

P = 0.04

15 % Cells

A

A

d5

+

Ad5 + 10VCR5 Ad5 + 50VCR

+

R VC

Ad5

10

0

VC

1

80 60 40 20 0 10

2

B

Folds release index

e

5.0

% Cells

Folds replication index

d

0

d5

0 0.0001 0.001 0.01

0.0

A

% Control

100

6

R

0 125

2.5

VC

25

12

Ad5 Ad5+10VCR Ad5+50VCR

50

% Control

b

75

ADP

E1A13S

5.0

100

P = 0.01

10 5

R

d5 A

A

d5

+

+

10 V

50 V

C

C

R

d5 A

R VC

R VC 10

50

B

0

Figure 6 VCR enhances Ad5-dependent oncolysis and does not affect Ad assembly and release. The effect of VCR on Ad5-dependent oncolysis in H460 cells was examined by MTT assays (a, 2 days; b, 5 days post-infection), RT-PCR analyses of Ad genes expression (c), determination of replication and release indices (d and e, respectively), and analyses of propidium iodide (PI)-stained cells for cell-cycle progression (f, 6 h; g, 24 h) and apoptosis induction (h). The P-value was calculated by comparing the values of apoptotic cells after Ad5 infection or in combination with different concentrations of VCR at a certain time point using Student’s t-test.

experiments this did not lead to a noticeable earlier onset of Ad gene expression. These findings suggest that PTX treatment does not reduce the transport time of the virus into the nucleus.

Our study provides evidence for a PTX-induced increase in adenoviral assembly in infected cells. PTX enhanced the amount of intracellular VP at later stages after infection in the absence of a parallel increase in the

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total viral DNA, which indicates that Ad5 assembly increased independent of DNA replication. This correlates with the observed PTX-induced stimulation of Pent gene expression, whereas expression of the Pol gene remains the same. Our data are in line with a report by Yu et al.,9 who showed that PTX increased the burst size of a prostate-specific variant of replicating Ads as a result of the enhancement of the amount of the intracellular titer of the virus. The authors attributed this increase in viral titer to an enhancement of the replication efficiency of the adenovirus. As an alternative and supported by our data, the increase in burst size could be caused by an enhancement of viral assembly and consequently viral release, leaving DNA synthesis unaffected. PTX may also assist Ad5 assembly in an MT-dependent manner. MT-stabilizing agents have been reported to increase the probability of the binding of Ad capsid to the MT,30 and therefore it could be envisioned that MT stabilization by PTX will provide a stable matrix of MT that facilitates the assembly of the capsid proteins. In accordance with this, we found increased Ad5 assembly after PTX as illustrated by an increase of the VP in the medium that can be explained by enhanced VP assembly. Moreover, these phenomena were not observed after VCR treatment, indicating that they are associated with MT stabilization. Finally, we consider a possible drug-induced enhancement of Ad5 infection/uptake unlikely as we found no enhanced expression of CAR after PTX treatment on H460 cells (our unpublished data). Our data are the first to show an effect of chemotherapeutic agents on adenoviral gene expression. In our experiments, Ad gene expression was followed during one round of replication, thereby using an MOI of 100. Although these conditions do not reflect the conditions that gave optimal augmentation of oncolysis, this experimental setup was chosen for the following reasons. At low MOIs, multiple rounds of replication are required for the eradication of tumor cells. In a background of nonsynchronized host cells and asynchronously replicating Ads, the detection of PTX-dependent effects on Ad gene expression would be nearly impossible. Moreover, high infection rates results in higher Ad transcript levels and thus improves the sensitivity of the assay, allowing the detection of more subtle effects of the drugs on gene expression. Regardless of this, PTX clearly selectively enhanced the expression of E1A13S, ADP and Pent but not of Pol. It is presently not clear how this is achieved. It may involve direct effects of PTX on Ad gene expression, perhaps via signal-transduction mechanisms know to be activated by PTX, such as cyclin-dependent kinases and jun kinases.31–34 Alternatively, the observed MT-interacting agent-dependent changes in cell-cycle arrest in Adinfected cells may indirectly influence Ad genes expression by, for example, cell cycle-specific factors. As we also found, Ad infection is known to induce arrest in the S phase at which the adenoviral gene expression occurs.15 Co-treatment with the MT-interacting drugs prevented the accumulation of cells in the S phase and instead elicited the accumulation of cells in G2/M. The situation is reminiscent to the observations reported by Thierry

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et al.35, who showed that G2/M arrest activates gene transcription of the human immunodeficiency virus. In fact, we found alterations in Ad5 gene expression to occur predominantly 24 h after treatment, which is concomitant with the majority of cells being arrested in the G2/M phase. However, the found differences in kinetics between the PTX-responsive Ad genes likely reflect the involvement of multiple PTX-inducible factors that modulate Ad gene expression by either direct or indirect ways. The MT-interacting agents could also act by enhancing viral release as mentioned earlier. The observed enhancement of viral assembly will subsequently lead to an increased production of functional viruses causing enhanced oncolysis and the release of more virions in the infection medium. Interestingly, we also found PTX to enhance ADP transcript levels that may lead to more efficient oncolysis as ADP is known to facilitate cell lysis.36 The found increase in E1A13S transcripts at later stages of viral infection may also account for enhanced cell death as previously also reported by others.37,38 Presently, we cannot discriminate whether one or all of these phenomena contribute to the enhanced cytotoxicity observed in PTX co-treated cells when compared to cells infected with Ad5 alone. However, although this may be relevant for PTX, this is unlikely to mediate the effect of VCR that caused only a modest increase of E1A13S and Pent gene expression and without affecting ADP gene expression. In line with this, hardly any stimulatory effect of VCR on Ad5 assembly and release was detected. PTX and VCR could also enhance oncolysis by stimulating the disruption of the cells in the last stage of the viral cycle. Previously, we showed that Ad5 induces necrosis-like programmed cell death, which is triggered independent from the basic apoptotic machinery.20 It is conceivable that drug-induced activation of other cell death route, such as caspase activation, will enhance cell killing and viral release. Taken together, the finding that both VCR and PTX act synergistically on Ad-induced oncolysis in vitro indicates that the functionality of the MT network itself is apparently not essential for mediating this effect. The found overlap in the investigated parameters between the two drugs, being the suppression of S-phase arrest and the accumulation of infected cells in G2/M, suggests that these events play an important role in mediating synergistic activity. Elucidation of the cellular signaling pathways responsible for these effects are likely to provide clues for the development of new or perhaps the use of existing specific biologicals that are equally – or perhaps more potent than the MT-interacting drugs in enhancing adenoviral oncolysis. Furthermore, it can be predicted that the combination of CRAds with PTX will be more effective than when combined with VCR in killing tumor cells in animal models or in patients, as PTX also stimulates viral assembly and release. Clearly, more work is required to further elucidate MT-interacting agent – adenovirus interactions in tumor cells that could lead to the development of improved treatment strategies.


Acknowledgements

We thank Guus van der Sluis for his contribution and Dr HM Pinedo and Dr G Giaccone for their support.

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