Inhibitory Effects of Statins on Cytomegalovirus Production in Human Cells：Comprehensive Analysis of Gene Expression Profiles

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ORIGINAL ARTICLE

Inhibitory Effects of Statins on Cytomegalovirus Production in Human Cells:

Comprehensive Analysis of Gene Expression Pro

ﬁles

q

Tsugiya Murayama

1

, Changxiao Bi

1

, Ying Li

1

, Yasuhito Ishigaki

2

, Fumihide Takano

3

, Tsutomu Takegami

4

,

Tomihisa Ohta

3

, Hiroyuki Sumino

5

, Kimiko Ubukata

6

, Takashi Takahashi

7 *

1_{Department of Microbiology and Immunology, Faculty of Pharmaceutical Sciences, Hokuriku University, Kanazawa, Ishikawa, Japan} 2_{Division of Core Facility, Medical Research Institute, Kanazawa Medical University, Kahoku-gun, Ishikawa, Japan}

3_{Department of Pharmacognosy and Chemistry of Natural Products, Graduate School of Natural Science and Technology, Kanazawa University, Kanazawa, Ishikawa, Japan} 4_{Division of and Molecular Oncology and Virology, Medical Research Institute, Kanazawa Medical University, Kahoku-gun, Ishikawa, Japan}

5_{Department of Clinical Laboratory, Gunma University Hospital, Maebashi, Gunma, Japan}

6_{Laboratory of Molecular Epidemiology for Infectious Agents, Kitasato University, Minato-ku, Tokyo, Japan}

7_{Laboratory of Infectious Diseases, Graduate School of Infection Control Sciences, Kitasato University, Minato-ku, Tokyo, Japan}

a r t i c l e i n f o

Article history: Received: Aug 22, 2010 Revised: Oct 11, 2010 Accepted: Oct 21, 2010 KEY WORDS: DNA microarray; human cytomegalovirus; human embryonic lung; statins;

viral replication

Background/Purpose: Inhibitory effects of statins on HIV-1 or poliovirus replication were reported. Our aim was to clarify whether statins could inhibit replication of cytomegalovirus (CMV) in human cells and to determine changes of gene expression proﬁles in host cells treated with statins using a DNA microarray.

Methods: Human embryonic lung (HEL) fibroblast cells were infected with CMV (Towne strain) at a multiplicity of infection of 1 and were simultaneously treated with mevastatin, simvastatin, lovastatin, or pravastatin (0.001e10mM). HEL cells were incubated for 6 days, and progeny viral titers were quantified by plaque assay. Time-dependent effects of mevastatin or simvastatin (1mM) on CMV repli-cation were also examined. We determined effects of mevastatin or simvastatin at concentrations ranging from 0.1mM to 10mM on the expressions of CMV immediate-early (IE) 1 and late proteins using Western blotting. Comprehensive analysis of gene expression profiles in HEL cells treated with mevas-tatin (1mM) was performed with a DNA microarray at 1 day after infection.

Results: The 50% effective concentration values for the inhibition of CMV titers by mevastatin, simvas-tatin, lovassimvas-tatin, and pravastatin were 0.0006mM, 0.0055mM, 0.04mM, and 2.55mM. Inhibition of viral replication by mevastatin was observed when added 24 hours after infection, whereas that by simvas-tatin was observed when added 48 hours after infection. Mevassimvas-tatin decreased expression of the IE1 protein and simvastatin inhibited expression of the late protein. We observed signiﬁcant changes of cellular growth/differentiation-associated gene expressions (e.g., down-regulated cdk2 mRNA) in HEL cells treated with mevastatin.

Conclusion: Our data suggest that treatment with mevastatin could inhibit CMV replication at IE phase through altered expressions of cellular growth/differentiation-associated genes.

1. Introduction

Endothelial and smooth muscle cells infected with human cyto-megalovirus (CMV) may cause chronic inﬂammation in vessels, suggesting an association between arteriosclerosis and CMV infection. However, latently infected CMV is frequently activated in immunosuppressed patients, such as those with AIDS or organ

transplants, thereby causing severe morbidity and eventual mortality.1Symptomatic CMV infection has been treated success-fully with ganciclovir (GCV), but the emergence of GCV-resistant strains is a current issue in the treatment of immunocompromised patients with CMV infection. Although either foscarnet or cidofovir has been used as an alternative therapy against GCV-resistant strains, these treatments are not always successful.2There is a need for development of new or alternative anti-CMV agents.3

The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibi-tors, statins, are used as lipid-lowering agents in clinical settings. It is demonstrated that statins play a multifactorial role in mainte-nance of transplanted organs.4 Nie et al5 report that they exert q The authors had no conﬂicts of interest to declare in relation to this article.

* Corresponding author. Laboratory of Infectious Diseases, Graduate School of Infection Control Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan.

E-mail:[email protected](T. Takahashi).

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immunosuppressive effects in rats undergoing heterotopic limb allografts by inhibiting activation and proliferation of T cells. In addition to these immunomodulatory actions, statins are shown to have antiviral effects. Fluvastatin is described to inhibit CMV replication through a decrease of nuclear factor-

k

B binding activity in human endothelial cells.6However, there is very limited infor-mation regarding inhibitory actions of various statins against CMV replication in other human cell lines.

We aimed to clarify whether activities of various agents can suppress CMV production in human embryonic lung (HEL) cells and to determine signiﬁcant alterations of genetic proﬁles in cells treated with statin using a DNA microarray.

2. Methods

2.1. Virus, cells, and reagents

CMV Towne strain was applied throughout our investigation.7CMV was propagated in HEL cells, and the clariﬁed supernatant was stored in liquid nitrogen until use. Viral infectivity was titrated using a plaque assay method as previously reported.8Four statins; mevastatin, simvastatin, lovastatin, and pravastatin, were commercially available (Calbiochem Inc., San Diego, CA, USA). These reagents were dissolved in a cell culture medium.

2.2. Cell culture

HEL cells9were cultured in Dulbecco’s modiﬁed Eagle’s minimal essential medium (Nissui Pharmaceutical Co. Ltd., Tokyo, Japan) supplemented with 10% heat-inactivated fetal calf serum (Z.L. Bocknek Laboratory, Ontario, Canada), L-glutamine (0.3 mg/mL),

gentamicin (50 mg/mL), and amphotericin B (2.5 mg/mL). All cell cultures were maintained in a humidiﬁed incubator at 37_{C in 5%}

CO2/95% air.

2.3. Viral production

When HEL cells in 24-well plates (IWAKI Microplate; IWAKI Glass Co., Funahashi, Japan) reached conﬂuency, the cells were inocu-lated with CMV at a multiplicity of infection of 1. After adsorption for 1 hour, the cells were supplemented with 1 mL of Dulbecco’s modiﬁed Eagle’s minimal essential medium containing 2% fetal calf serum in presence or absence of various concentrations of statins for indicated time intervals. Production of infectious virus in culture supernatants was titrated using a plaque assay.8

2.4. Inhibitory effects of statins on CMV production

The CMV-infected cells were treated with four statins (mevastatin, simvastatin, lovastatin, or pravastatin) at different concentrations ranging from 0.001

m

M to 10

m

M. All cells were incubated for 6 days after viral inoculation, and then the CMV titers were measured. Based on curves showing relationships between relative virus titers (a percentage of the control titers) and statin concentrations used, 50% effective concentration (EC50) was calculated for each statin.

2.5. Time-dependent effects of statins on CMV production

The cells were inoculated with CMV, and treatment with mevas-tatin (at a concentration of 1

m

M) was started at indicated times (1 hour, 8 hours, 24 hours, and 48 hours) after infection. All cells were incubated for 5 or 7 days after viral inoculation, and the viral titers were quantiﬁed. Similarly, simvastatin (1

m

M) was added at various times (1 hour, 24 hours, 48 hours, and 72 hours) after infection, and the CMV titers were determined at Day 7 after infection.

2.6. Cytotoxic activity of statins

Subconﬂuent monolayers of the uninfected cells were cultured on 24-well plates in absence or presence of different concentrations ranging from 0.01

m

M to 10

m

M of mevastatin or simvastatin. They were incubated under conditions of 37C and 5% CO2for 3 or 6

days. Number of viable cells was determined using trypan-blue exclusion dye staining, and ratio of viable cells under exposure to the statins to viable negative control cells was calculated for each concentration.

2.7. Detection of viral proteins

The CMV-infected cells were treated with the indicated concen-trations ranging from 0.1

m

M to 10

m

M of mevastatin or simvastatin until 1 day after viral inoculation and were harvested for Western blot analysis to detect expression of the immediate-early (IE)-1 protein. In another trial, the cells were treated with the same concentrations of the same statins until 3 or 6 days after infection and were harvested for Western blotting analysis with the antibody against the late protein. Western blot analysis for detection of the structural IE-1 and late proteins was performed as previously described.3The

b

-actin protein was applied as an internal control. 2.8. Detection of viral mRNA

The infected cells were incubated with different concentrations ranging from 0.1

m

M to 10

m

M of mevastatin until 24 hours after infection. Total RNA was extracted using a standard method and was reversely transcribed after eliminating residual genomic DNA. The cDNA products were ampliﬁed for IE-1/IE-2 and

b

-actin gene expressions through real-time reverse transcription-polymerase chain reaction (PCR) with each speciﬁc primer set and iQ SYBR Green Supermix (Bio-Rad Laboratories Inc., Tokyo, Japan) for 34 cycles (10 seconds at 95C, 20 seconds at 55C, and 20 seconds at 72C) by a Mini Opticon real-time PCR using Gene Expression Macro software (Bio-Rad Laboratories Inc., Tokyo, Japan). The PCR primers for ampliﬁcation of IE-1/IE-2 genes were used as previously reported.10The

b

-actin gene was used as an internal control. Comparative expression levels of the IE-1/ IE-2 mRNAs were calculated according to expression levels of the

b

-actin mRNA.

2.9. Determination of gene expression proﬁles in cells by DNA microarray analysis

Total RNA was isolated from the cells using a commercially available kit (RNeasy Mini Kit; QIAGEN GmbH, Hilden, Germany). Extracted RNA was quantified by photometry at 260/280 nm, and the quality of RNA was determined by the ratio of 18S/28S ribosomal band intensities in an ethidium bromide containing 1% agarose gel through electrophoresis. Comprehensive analysis of gene expres-sion profiles in the infected cells was performed using a Human U133 Plus 2.0 GeneChip microarray system (Affymetrix Inc., Santa Clara, CA, USA) as previously reported.11The amounts of probe-speci_{fic transcripts were determined based on the average of the} differences between the perfect-match and mismatch intensities. Because replicate assays were not performed, the signal intensity of selected genes that were up- or downregulated by at least twofold changes compared with a control group were extracted by the GeneSpring GX software package version 7.3.1 (Agilent Technolo-gies Inc., Santa Clara, CA, USA). Ingenuity pathway analysis (Inge-nuity Systems Inc., Redwood City, CA, USA) was used as an additional method for evaluating functional significance of the induced gene expression profiles.

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2.10. Validation of data by DNA microarray analysis

Real-time reverse transcription-PCR analysis was performed as described regarding the detection of viral mRNA, using prepared RNA samples. Four target genes (cdk2, arf1, xrcc2, and psmb4) in the infected cells treated with mevastatin or simvastatin at different concentrations ranging from 0.1

m

M to 10

m

M until 24 hours after infection were selected among those having signi fi-cantly up- or downregulated changes based on the gene expres-sion profiles. The oligonucleotide sequences of the forward/ reverse primer set for each gene amplification were described previously.12e15 Comparative expression levels of the selected genes were calculated according to expression levels of the

b

-actin mRNA. Expression levels of the four mRNAs are expressed as fold changes relative to the baseline expression levels in the untreated control group. Together with validation of data by DNA microarray analysis, we also conﬁrmed changes of viral loads (IE-1 mRNA expression).

2.11. Data analyses

Data were analyzed using statistical analysis methods and graphing software (KaleidaGraphTM; Synergy Software, Tokyo, Japan). All data are expressed as mean standard deviation. Signiﬁcance of differences among values was determined by Dunnett’s multiple tests after one-way analysis of variance in comparison with control cultures, and values of p less than 0.05 were considered to indicate signiﬁcance. Furthermore, Fisher’s exact test was used to determine probability that biological function assigned to each network could be explained by chance alone.

3. Results

3.1. Inhibitory effects of statins on CMV production

EC50values for inhibition of CMV production by mevastatin,

sim-vastatin, losim-vastatin, and pravastatin were 0.0006

m

M, 0.0055

m

M, 0.04

m

M, and 2.55

m

M, respectively. Mevastatin showed the most pronounced suppressive effect.

3.2. Time-dependent effects of statins on CMV production

Based on the EC50data, mevastatin and simvastatin were selected

for evaluation of time-dependent actions against CMV produc-tion. Mevastatin (1

m

M) exhibited signiﬁcantly inhibitory effects on viral replication over a period of 5 or 7 days when started at 1 hour, 8 hours, and 24 hours after CMV infection but not when started at 48 hours after viral inoculation (Figure 1A). On the other hand, simvastatin (1

m

M) achieved a signiﬁcant suppression of CMV production over a period of 7 days when started 48 hours as well as 1 hour and 24 hours after the infection (Figure 1B). 3.3. Cytotoxic activity of statins

We determined cytotoxic activity of mevastatin or simvastatin without CMV infection according to the EC50data. Ratio of viable

cells under exposure to the statins (0.01e10

m

M) to viable negative control cells for 3 days were more than 90%, and those under exposure to mevastatin and simvastatin (10

m

M) for 6 days were approximately 73% and 89%, respectively. These observations suggest that decreased viral titers by these statins simply reﬂect inhibitory actions of CMV replication without direct statin cytotoxicity.

3.4. Viral protein

Because mevastatin and simvastatin showed different time-dependent effects, we selected these reagents for examination of variations in production of early and late CMV proteins during statin exposure (0.1e10

m

M). Mevastatin inhibited expression of the IE-1 protein at 1 day after viral inoculation, whereas simvastatin inhibited expressions of the late protein at 3 and 6 days after infection (Figure 2).

3.5. Viral mRNA

We determined ability of mevastatin to inhibit expressions of the IE-1/IE-2 mRNAs when incubated until 24 hours after infection, based on the protein expression data. At a concentration of 0.1

m

M, mevastatin signiﬁcantly inhibited only expression of IE-1 mRNA, whereas at 1

m

M, it signiﬁcantly inhibited expression of both IE-1 and IE-2 mRNAs (Figure 3).

3.6. Gene expression proﬁles in cells by DNA microarray analysis We performed a comprehensive analysis of gene expression pro_{ﬁles in the infected cells treated with mevastatin (1}

m

M) until 24 hours after infection. Table 1 shows mRNAs that were up- or downregulated by at least twofold compared with the control. According to ingenuity pathway analysis categories, we observed signiﬁcant changes of cellular growth/differentiation-associated gene expressions in the infected cells treated with mevastatin.

0 10 20 30 40 50 60

A

B

5 7 V irus titer , pfu/mL x 1000 Meva . (1 • M): - + + + + - + + + + Time of addition (hr): - 1 8 24 48 * * * * * * V irus titer , pfu/mL x 1000 * 0 10 20 30 40 50 60 70 80 * * * Simva. (1 • M): Time of addition (hr): - 1 24 48 72 d.p.i d.p.i

Figure 1 Time-dependent effects of (A) mevastatin or (B) simvastatin at 1mM on human cytomegalovirus replication. *Indicate signiﬁcantly different levels compared with those in the controls. Meva¼ mevastatin; Simva ¼ simvastatin; d.p.i ¼ days post infection.

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3.7. Validation of data by DNA microarray analysis

Based on the results of gene expression proﬁles in the cells, we selected four up- or downregulated genes (i.e., cdk2, arf1, xrcc2, and psmb4) in the infected cells treated with mevastatin or simvastatin (0.1e10

m

M) until 24 hours after infection. The reasons why these four genes were selected for validation of DNA microarray data were signiﬁcant levels of fold changes and estimated functions of genes. Mevastatin dose-dependently induced elevations in expressions of PSMB4 and XRCC2 mRNAs and decreased expres-sions of CDK-2, ARF-1, and IE-1 mRNAs (Figure 4A and B). Similarly, simvastatin dose-dependently increased or reduced expressions of three of the four genes except for ARF-1 mRNA, together with decreased expression of IE-1 mRNA (Figure 4C and D).

4. Discussion

Our observations suggest that treatment with mevastatin, simvas-tatin, or lovastatin at various EC50 concentrations of 0.0006

m

M,

0.0055

m

M, or 0.04

m

M could inhibit CMV production. Clinical trial

conﬁrmed achievable plasma concentration (0.10e3.92

m

M) of lovastatin administered at a dose of 25 mg/kg/d, which corresponds to the dose range that could trigger apoptosis of sensitive tumor types in vitro.16 Based onﬁndings of our experiment, the statin interventions should be further developed as novel or alternative strategies for treatment of CMV infection.

Statins are reported to play a crucial role in management of sepsis in a clinical setting. Administration of statins in patients with atherosclerosis is shown to be associated with a decreased risk of subsequent sepsis, and randomized clinical trials of statins for prevention of sepsis are warranted.17In addition to the manage-ment of sepsis, there are several descriptions concerning inhibitory effects of reagents on viral replication, including poliovirus,18 HIV-1,19and hepatitis C virus.20Furthermore, combined treatment with a statin and caffeine effectively ameliorates lung damage, inhibits viral replication, and is at least as effective as therapy with osel-tamivir and ribavirin in H5N1-, H3N2-, and H1N1-infected mice.21 This combination seems to be more effective when administered preventatively rather than as a treatment. Therefore, it will also be important to clarify antiviral actions of statins against inﬂuenza viruses in future investigations.

A potential mechanism how the viral replication in CMV-infec-ted cells could be inhibiCMV-infec-ted by mevastatin seems to be cellular arrest, similarly to statin effects on malignant cells in previous report.16 Comprehensive analysis of gene expression proﬁles in CMV-infec-ted cells revealed that mevastatin induced signiﬁcant changes of cellular growth/differentiation-associated gene expressions. In particular, this statin dose-dependently induced both reduced

0 0.1 1 10

Concentration of mevastatin (mM)

100 200 300 400 500 IE2 * * * * *

R

e

la

ti

ve

expr

essi

o

n

to

cont

ro

l (

%

)

0 IE1

Figure 3 Expressions of viral mRNAs, including IE1 and IE2 in human cytomegalo-virus-infected cells treated with mevastatin (0.1mM, 1mM, or 10mM) until 24 hours after infection. *Indicate signiﬁcantly different levels compared with those in the controls. Relative expression levels of IE1 and IE2 mRNAs were calculated according to the expression levels ofb-actin mRNA. IE¼ immediate-early.

Table 1 Gene expression proﬁles in virally infected cells treated with mevastatin by DNA microarray analysis

Upregulated mRNA Fold change Downregulated mRNA Fold change

PSMB4 4.76 RPS2 7.46

SLC25A37 4.472 CDK2 6.308

XRCC2 4.461 C7ORF11 5.224

ZNF652 4.322 HLA-B 5.066

NCRNA00094 3.972 DUT (includes EG:1854) 5.002

HELLS 3.971 ARF1 4.912

UBE2B 3.456 GADD45GIP1 4.835 RBBP6 (includes EG:5930) 3.427 IFI16 4.705

ATP6V0D2 3.408 ID1 4.697

FARP1 3.277 HSPB1 4.664

To validate the data by DNA microarray analysis, the underlined mRNAs were selected as four target genes for real-time reverse transcription-polymerase chain reaction assays.

Figure 2 Expressions of viral proteins including IE-1 and late proteins in human cytomegalovirus-infected cells treated with (A) mevastatin or (B) simvastatin at 0.1mM, 1mM, and 10mM.

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expressions of CDK-2/ARF-1 mRNAs and increased expressions of PSMB4/XRCC2 mRNAs, leading to decreased expression of CMV IE1 mRNA. CDK2 is a member of Ser/Thr protein kinase families, shows activity restricted to G1-S phase, and is essential for cell cycle G1/S phase transition. Transcriptional activation of the cdc2 is associated with Fas-induced apoptosis of human hematopoietic cells.12 Inter-estingly, mevastatin is described to inhibit cellular growth of a prostate cancer cell line through inhibition of CDK2.22Moreover, inhibition of CDK2 activity by roscovitine inhibits CMV DNA synthesis, production of infectious progeny, and viral antigen expression in infected cells in a dose-dependent manner.23ARF1, a member of RAS superfamily, is localized to Golgi apparatus and has a central role in intra-Golgi transport. This molecule regulates epidermal growth factor-dependent growth and migration of breast cancer cells through activation of phosphatidylinositol 3-kinase pathway.13 PSMB4, a member of proteasome B-type family, is distributed throughout eukaryotic cells at a high concentration and cleaves peptides in an ATP/ubiquitin-dependent process in a non-lysosomal pathway. An expression survey of a panel of glioma cell lines demonstrated expression of the PSMB4, and validity of the proteasome complex as a target for survival inhibition was conﬁrmed in a series of glioma and nonglioma cell lines.24_XRCC2, a member of RecA/Rad51-related protein family, participates in homologous recombination to maintain chromosome stability and repair DNA damage. This molecule is important to preserve or restore replication forks during rapid clonal expansion of devel-oping lymphocytes.25There are no investigations of associations between mevastatin and ARF1, PSMB4, or XRCC2. Therefore, we need to determine the detailed up- and downstream networks of CDK2, ARF1, PSMB4, XRCC2, and other molecules involved with cellular growth/differentiation in CMV-infected cells under inter-vention of mevastatin.

Effects of the four statins on blood cholesterol and lipoprotein levels are quantitatively similar when administered at equivalent

doses. We found that mevastatin, simvastatin, and lovastatin, but not pravastatin, were able to efﬁciently inhibit CMV production in HEL cells. It is unclear why pravastatin could exhibit less inhibitory effects on viral replication. Pravastatin only has hydrophilic prop-erty and no

b

-hydroxy-

d

-lactone moiety, which other three statins commonly include as chemical structures. In future basic researches, it will be necessary to conﬁrm signiﬁcant differences in the chemical structures and biochemical activities among the statin reagents in detail. Moreover, we should examine antiviral effects of mevastatin and simvastatin in vivo, using an animal model of murine CMV infection to provide experimental evidence for our hypotheses.

Acknowledgments

Our study was funded, in part, by a Grant-in-Aid for Scientiﬁc Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (grant number 21390306, to Dr T. Takahashi). References

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A

C

B

D

Figure 4 Dose-dependently altered expressions of four selected genes, that is, cdk-2, arf-1, xrcc2, and psmb4 in human cytomegalovirus-infected cells treated with (A and B) mevastatin or (C and D) simvastatin at 0.1mM, 1mM, and 10mM until 24 hours after infection. The expression levels of the four mRNAs are expressed as fold changes relative to the baseline expression levels in the untreated control group. Together with the validation of data by DNA microarray analysis, we also conﬁrmed the changes of viral loads. IE¼ immediate-early.

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