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Cancer Research 67, 10445, November 1, 2007. doi: 10.1158/0008-5472.CAN-07-1955
© 2007 American Association for Cancer Research
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Experimental Therapeutics, Molecular Targets, and Chemical Biology

Chemotherapeutic Agents Up-regulate the Cytomegalovirus Promoter: Implications for Bioluminescence Imaging of Tumor Response to Therapy

Robert U. Svensson1, J. Matthew Barnes1, Oskar W. Rokhlin2, Michael B. Cohen2 and Michael D. Henry1,2

Departments of 1 Molecular Physiology and Biophysics and 2 Pathology, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa

Requests for reprints: Michael D. Henry, Department of Molecular Physiology and Biophysics, Roy J. and Lucille A. Carver College of Medicine, 6-510 Bowen Science Building, University of Iowa, Iowa City, IA 52240. Phone: 319-335-7886; Fax: 319-335-7330; E-mail: michael-henry{at}uiowa.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Bioluminescence imaging is widely used to evaluate tumor growth and response to therapy in living animals. In cells expressing luciferase under the control of a constitutive promoter, light output in part depends on viable cell number, so that changes in bioluminescence intensity may be correlated with changes in viable tumor mass over time. We have found that treatment of cancer cell lines expressing luciferase under control of the cytomegalovirus (CMV) promoter with staurosporine, doxorubicin, and paclitaxel results in a transient increase in bioluminescence, which is positively correlated with apoptosis and inversely correlated with cell viability. In contrast, similar treatment of cell lines expressing luciferase under control of the SV40 promoter did not exhibit this result. We found that low doses of staurosporine induced bioluminescence in CMV- but not SV40-driven luciferase cell lines, whereas high doses elicited induction in both, indicating promoter-dependent and promoter-independent mechanisms of bioluminescence induction. The promoter-dependent increase in bioluminescence intensity from CMV-driven luciferase is a result of induction of luciferase mRNA and protein expression. We extended these findings in vivo; doxorubicin treatment resulted in a transient induction in bioluminescence when normalized to tumor volume in CMV- but not SV40-driven luciferase-expressing xenografts. We found that inhibition of the p38 mitogen-activated protein kinase pathway blocked bioluminescence induction by doxorubicin, paclitaxel, and staurosporine in CMV-driven luciferase-expressing cells. These findings have important implications when using bioluminescence to monitor the efficacy of anticancer therapy and underscore the complex regulation of the CMV promoter, which is widely used for high-level protein expression in mammalian cells. [Cancer Res 2007;67(21):10445–54]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The development of small animal imaging techniques, including bioluminescence imaging (BLI), has greatly impacted our ability to study the biology of cancer as well as to evaluate tumor response to therapy (1). BLI is based on the detection of photons generated from cells expressing a luciferase enzyme (2). The light-generating reaction also requires a substrate molecule (e.g., luciferin) as well as ATP, oxygen, and Mg2+, so that signal can only be generated in viable cells. Expression of luciferase may be accomplished through stable expression in cancer cell lines used for transplantable tumor models or by transgenic expression of luciferase in genetically modified cancer-prone mice (3, 4).

One application of this approach has been to use BLI to monitor response to therapy (5, 6). Although constitutive expression of luciferase can report on cytotoxic effects of therapy, expression of luciferase in various reporter strategies can show that particular biological mechanisms are engaged by drug treatment (see, for examples, refs. 7–9). Implementation of these approaches requires that the light signal is correlated with parameters, such as cell viability, pathway activation/inhibition, etc., for which photon emission is a surrogate measurement. This has been well documented in the previously cited studies. However, because this technology has emerged, there have been questions about how certain features of the tumor microenvironment may influence bioluminescence signal generation, such as hypoxia, cellular metabolism, and distribution of substrate (10–12). It is thus important to remain aware of these and other possibilities as the use of BLI in cancer models is further developed and implemented.

In xenograft and syngeneic transplant models, expression of luciferase is achieved through stable transfection or transduction of a vector for luciferase expression. Generally, strong promoters in mammalian cells such as cytomegalovirus (CMV), SV40, and others have been used for constitutive expression of luciferase (6, 13, 14). Higher luciferase expression may generate more signal, allowing for more sensitive detection of cells in vivo. However, it is important to remember that these promoters, while expressed well in many cell types, may nonetheless be regulated by various biological influences. In cells engineered to express CMV-driven luciferase, we show that treatment with chemotherapeutic agents doxorubicin and paclitaxel results in a transient increase in bioluminescence, although the cells are undergoing apoptosis and are less viable than control cells. We find that this is a result of increased steady-state luciferase mRNA and protein. Up-regulation of luciferase is dependent on the activation of the p38 mitogen-activated protein kinase (MAPK) pathway, which is known to regulate several transcription factors capable of influencing the CMV promoter. Additionally, we find that low doses of staurosporine are capable of inducing bioluminescence through the same mechanism in cells expressing CMV luciferase; however, at high doses, induction occurs in both CMV- and SV40-luciferase cell lines through a mechanism that does not require protein synthesis. We are able to recapitulate our in vitro results in vivo using a mouse xenograft model. The data presented here have important implications when using BLI to monitor the response of tumor cells to cancer therapy both in cell culture and in animal hosts.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture and generation of luciferase-expressing cell lines. All cell lines were obtained from the American Type Culture Collection. 22RV1, LNCaP, and 4T1 cells were cultured in RPMI 1640, and PC3, NIH/3T3, and MDA-MB-231 cells were cultured in DMEM. All cell culture media was supplemented with 10% fetal bovine serum (Hyclone), 1% nonessential amino acids; media for luciferase-expressing cell lines was also supplemented with 400 µg/mL Geneticin (G418). All cells were cultured at 37°C in an atmosphere containing 5% CO2. Generation of the CMV luciferase-expressing 22Rv1-CMVluc (22Rv1.Luc.PN2) cells was previously described (15). PC3-CMVluc, MDA.MB.231-CMVluc, 4T1-CMVluc, and NIH3T3-CMVluc cells were generated using the same method. Generation of SV40 luciferase-expressing 22Rv1-SV40luc (22Rv1.Luc.1.17) cells was previously described (16). Generation of CMV androgen receptor (AR)–overexpressing PC3 cells and spleen focus-forming virus (SFFV)–driven LNCaP cells were previously described (17). For transient transfection experiments, MDA.MB.231 cells were transfected with either pCDNA3.1-luc [CMV-Luc: pGEM luciferase (Promega) subcloned into pCDNA3.1 (Invitrogen)] or pGL3 (SV40 Luc-Promega) plasmids using LipofectAMINE 2000 (Invitrogen) according to manufacturer's instructions.

Drug treatments. For doxorubicin and paclitaxel treatment, cells were seeded at 2 x 105 per well in 24-well plates for caspase-3 assays or 1 x 105 per well in 48-well plates for bioluminescence and 4-[3-(4-indophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST) assays. Twenty-four hours later, media was removed and replaced with fresh media (500 µL for 24-well plates and 250 µL for 48-well plates) for nontreated controls or media containing a dose range of doxorubicin (100, 400, 750, and 1,000 nmol/L; Sigma) or paclitaxel (30, 90, 300, and 900 nmol/L; Sigma). For MDA.MB.231 cells transiently expressing luciferase, paclitaxel was added 48 h after transfection. For staurosporine treatment, cells were seeded in 48-well plates at 1.5 x 105 per well. The next day, cells were incubated for 30 min in 250 µL fresh media containing 150 µg/mL D-luciferin. About 100 µL of the appropriate dilution of STS was then added directly to cells to final concentrations of 4 µmol/L, 250 nmol/L, or 60 nmol/L, and control cells received 100 µL of 0.4% DMSO to make a total volume of 350 µL per well. For cycloheximide treatment, 22Rv1-CMVluc cells were seeded at 1 x 105 per well in a 48-well plate. The next day, cells were pretreated with 10 µg/mL cycloheximide (Sigma) or vehicle control (H2O) for 2 h. D-Luciferin was then added to cells at a final concentration of 150 µg/mL for 20 min. Cells were then treated with 4 µmol/L or 60 nmol/L staurosporine or 0.4% DMSO in a total volume of 350 µL per well. For p38 MAPK inhibition, cells were treated with a dose range of SB203580 (3, 10, or 30 µmol/L; Sigma) either alone or in combination with 400 nmol/L doxorubicin (22Rv1-CMVluc) or 90 nmol/L paclitaxel (PC3-CMVluc) for a period of 48 h. About 0.1% DMSO was used as a vehicle control. Treated cells were analyzed (see below) at various time points for bioluminescence, cell viability (WST assay), and caspase-3 activity. Media was not changed throughout any of the treatment periods. For the experiments shown in Supplementary Fig. S2, cells were either treated with 100 ng/mL Trichostatin A (TSA; Sigma) or with media for 24 h before Western blots were done as previously described (18).

Bioluminescence imaging. All BLI was done using an IVIS 100 imaging system (Caliper Life Sciences). BLI of cells was done by the addition of D-luciferin (Caliper Life Sciences) to the cell culture medium at a final concentration of 150 µg/mL. Samples were then imaged using a 15-cm field of view with exposure times varying from 1 to 15 s. Photon flux was calculated using Living Image software (version 2.5) and represented as photons/s/cm2/sr. Bioluminescence values were then represented as percent photon flux of untreated or vehicle control values.

Caspase-3 assay and cell viability assays. Assays for caspase-3 activity were done using a commercially available colorimetric caspase-3 assay kit (Sigma CASP-3-C). Cells were seeded at 2 x 105 per well in 24-well plates for 24 h at 37°C and then exposed to either apoptotic stimuli, vehicle control, or left untreated. At each time point, caspase-3 activity was measured following the manufacturer's instructions. Treated cells were compared with nontreated or vehicle control cells, and values were represented as mean ± SE percent of control value.

Cell viability was determined using a cell proliferation reagent WST-1 (Roche) according to manufacturer's instructions. Values from treated cells were divided by those from nontreated control values and represented as percent of control value. Each data point represents the mean ± SE.

Quantitative reverse transcription-PCR of luciferase mRNA. Primers are as follows: pGEM Luciferase forward primer: 5'-CCGCGTACGTGATGTTCACC-3'; pGEM Luciferase reverse primer: 5'-GAGGATGGAACCGCTGGAGA-3'. pGL3 Luciferase forward primer: 5'-CGCGGTCGGTAAAGTTGTTC-3'; pGL3 Luciferase reverse primer: 5'-CCCGGTATCCAGATCCACAA-3'. Human GAPDH forward primer: 5'-CCATGTTCGTCATGGGTGTG-3'; Human GAPDH reverse primer: 5'CAGGGGTGCTAAGCAGTTGG-3'.

Total RNA isolation was done using RNEasy tissue kit (Qiagen), and reverse transcription was carried out using iScript cDNA synthesis kit (Bio-Rad). The resulting cDNAs were used as PCR template using CYBR Green I (Invitrogen), and data were collected on iCycler thermal cycler (Bio-Rad). Experimental values were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) values. Relative expression values were calculated using a comparative Ct method (19).

Luciferase activity assay. Lysates of staurosporine-treated, doxorubicin-treated, or untreated cells were prepared by adding 500 µL of lysate buffer [250 mmol/L sucrose, 25 mmol/L HEPES, 1 mmol/L EDTA (pH, 7.2)] to monolayers growing in six-well plates and homogenizing by pipetting. Protein concentrations were calculated using a Bio-Rad Dc Protein Assay. Equal amounts of protein (averaging 37 µg per well per assay) were added at a volume of 50 µL to 96 well plates, mixed with 100 µL assay buffer [15 mmol/L MgCl2, 150 µg/mL D-luciferin, 1 µg/mL ATP, 1 mol/L Tris-HCl (pH 7.4)] and imaged in an IVIS 100.

Subcutaneous tumor model. All animal procedures were done with approval from the University of Iowa Animal Care and Use Committee. A total of 24 male athymic nu/nu mice (National Cancer Institute) were injected into the periscapular subcutis with either 2 x 106 22Rv1-CMVluc cells (12 mice) or 2 x 106 22Rv1-SV40luc cells (12 mice) while being maintained on 3% isoflurane. Mice were returned to their housing and closely monitored for body weight and general health status. After 13 days of tumor development, mice were imaged using the IVIS 100, and tumor volumes were measured using calipers and calculated using the equation (1/2)(L x W2). For each cell line, mice were randomly divided into two groups (n = 6; designated PBS and doxorubicin), each group having similar average bioluminescence intensities as determined by t test. Mice in the PBS group then received 200 µL of PBS, and mice in the doxorubicin group received 200 µL of 8 mg/kg doxorubicin (200 µL at 1 mg/mL) via i.v. tail vein injection. Mice were imaged, and tumor volumes were recorded at 1, 2, 3, 4, 5, 6, 10, and 23 days posttreatment. Body weight was measured at the same time intervals. For BLI of mice, mice were first anesthetized in a chamber with 3% isoflurane. D-Luciferin was then given to each mouse via i.p. injection at a dose of 150 mg/kg and left to incubate for 8 min while being maintained on 3% isoflurane. Mice were then imaged using a 20-cm field of view and an exposure time of 1 s. Bioluminescence values were calculated by measuring photons/s/cm2/sr in the region of interest surrounding the bioluminescence signal from the tumor, with the lower signal threshold set to 5% of the maximum signal value.

p38 MAPK Western blot. Lysates were prepared from cells treated with or without lipopolysaccharide or 400 nmol/L doxorubicin for a period of 5 min. Protein concentrations were calculated using a Bio-Rad Dc Protein Assay. Equal amounts of protein were electrophoresed in a 12% SDS poly acrylamide gel and transferred onto polyvinylidene difluoride membranes. Membranes were blotted with phospho-p38 MAPK antibody (Biosource 44-684G) or ß-actin antibody (Sigma A1978) and incubated with horseradish peroxidase–conjugated goat anti-rabbit immunoglobulin G (The Jackson Laboratory; 111-035-003), and signal was detected by enhanced chemiluminescence.

Statistical analyses. All statistical analyses were done using ANOVA with Bonferroni post-tests.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Treatment of luciferase-expressing prostate cancer cells with apoptosis-inducing agents results in a transient increase in bioluminescence. We examined the effects of chemotherapeutic agents doxorubicin, a DNA-damaging agent; paclitaxel, a microtubule-stabilizing agent; and staurosporine, a potent protein kinase inhibitor, on luciferase-expressing human prostate cancer cell lines (Figs. 1 and 2 , Table 1 , and Supplementary Fig. S1). These cell lines have been engineered to stably express luciferase either via retroviral transduction with CMV-driven luciferase (CMVluc) or stable transfection with SV40-driven luciferase (SV40luc) and are designated as 22Rv1-CMVluc, PC3-CMVluc, and 22Rv1-SV40luc. As expected, in all three cell lines, doxorubicin, paclitaxel, and staurosporine elicited apoptotic cell death and a consequent loss of cell viability as measured by caspase-3 activity and WST viability assay, respectively (Figs. 1C and 2B and Supplementary Fig. S1). High-dose staurosporine (4 µmol/L) rapidly caused significant induction of apoptosis by 2 h (Fig. 1C) and a significant loss of cell viability 6 to 8 h posttreatment (data not shown). Lower doses of staurosporine (60 and 250 nmol/L) induced a slight but significant induction of caspase-3 and reduction of cell viability by 24 h (Fig. 1D). Similarly, doxorubicin and paclitaxel treatment resulted in peak caspase-3 activity between 24 and 48 h, and cell viability significantly decreased 48 h after treatment (Fig. 2B and Supplementary Fig. S1B, C, E, F, H, and I).


Figure 1
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  		Figure 1. Staurosporine induces a transient increase in bioluminescence in luciferase-expressing prostate carcinoma cells. 22Rv1-CMVluc, 22Rv1-SV40luc, and PC3-CMVluc cells were treated with varying doses of staurosporine for up to 24 h. Y-axes in A and B represent bioluminescence signal as photon flux (photons/s/cm2/sr) expressed as % vehicle control. A, 4 µmol/L staurosporine induces a rapid increase in bioluminescence in all three cell lines after 1 h of treatment. B, 60 and 250 nmol/L staurosporine-treated 22Rv1-CMVluc and PC3-CMVluc cells exhibit an increase in bioluminescence after 24 h, which was not evident in 22Rv1-SV40luc cells. C, representative time course of 22Rv1-CMVluc cells treated with 4 µmol/L staurosporine, showing that at this high dose, the bioluminescence increase is transient and precedes induction of apoptosis (caspase-3 activity). D, representative time course of 22Rv1-CMVluc cells treated with 60 nmol/L staurosporine, showing that at this low dose, the bioluminescence increase accompanies induction of apoptosis (caspase-3 activity) and a loss in cell viability. *, P < 0.05; **, P < 0.01; and ***, P < 0.001, significant increases in photon flux relative to controls.

 

Figure 2
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  		Figure 2. Chemotherapeutics doxorubicin and paclitaxel induce a transient increase in bioluminescence in 22Rv1-CMVluc and PC3-CMVluc cells, but not 22Rv1-SV40luc cells. Luciferase-expressing cell lines were treated with chemotherapeutics doxorubicin and paclitaxel. Y-axes in A and C represent bioluminescence as photon flux (photons/s/cm2/sr) expressed as % untreated control after 48 h. A, 400 nmol/L doxorubicin- and 90 nmol/L paclitaxel-treated 22Rv1-CMVluc cells and 90 nmol/L paclitaxel-treated PC3-CMVluc cells induced a transient increase in bioluminescence, which was not evident at any dose in 22Rv1-SV40luc cells. B, representative time course of 400 nmol/L doxorubicin-treated 22Rv1-CMVluc cells showing that the bioluminescence increase is transient and accompanies an induction of apoptosis (caspase-3 activity) and loss in cell viability (WST assay). C, paclitaxel treatment induces an increase in bioluminescence in non–prostate carcinoma cell lines expressing luciferase under the control of the CMV promoter. D, paclitaxel induces bioluminescence in MDA.MB.231 cells transiently transfected with pCDNA3.1 (CMV-Luc) but not pGL3 (SV40-Luc). *, P < 0.05; **, P < 0.01; ***, P < 0.001, significant increases in photon flux relative to controls.

 

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  		Table 1. Doxorubicin and paclitaxel-induced increase in bioluminescence is accompanied by an induction of apoptosis and a loss in cell viability

 
We also measured bioluminescence from these cell lines, and surprisingly, we found increased signal upon staurosporine treatment of all three cell lines and upon doxorubicin and paclitaxel treatment of 22Rv1-CMVluc and PC3-CMVluc cells (Figs. 1A–D and 2A and B, Supplementary Fig. S1, and Table 1). The increase in bioluminescence signal in response to doxorubicin and paclitaxel occurred even as these cells were undergoing apoptotic cell death and a loss in cell viability (Fig. 2B, Table 1, and Supplementary Fig. S1B, C, and E). In contrast, we only observed an increase in bioluminescence in 22Rv1-SV40luc cells treated with a high 4-µmol/L dose of staurosporine (Fig. 1A), whereas we observed increases with 4 µmol/L and lower doses of 60 and 250 nmol/L in both 22Rv1-CMVluc and PC3-CMVluc cells (Fig. 1A–D). Bioluminescence induction in response to 4 µmol/L staurosporine was characteristically rapid, peaking within 1 h of treatment (Fig. 1C) and preceding peak induction of apoptosis, whereas the increase in bioluminescence signal from the 60-nmol/L staurosporine-treated CMVluc cell lines became maximal only after 24 h, similar to the time course for the induction of apoptosis (Fig. 1D). In 22Rv1-CMVluc cells, all three agents induced an increase in bioluminescence to levels significantly higher than control values, ranging from 150% to 250%. With the exception of doxorubicin treatment, we saw similar effects in PC3-CMVluc cells (Figs. 1A and B, and 2A). At later time points (~120 h with doxorubicin and paclitaxel), cells treated with any of the three agents exhibited decreases in bioluminescence to levels below that of untreated control values (Fig. 2B, Table 1, and Supplementary Fig. S1B, C, E, F, H, and I). Thus, each agent is able to induce a transient increase in bioluminescence, and in some cases, this occurs during a period in which these agents induce apoptosis and a loss in cell viability. We also noticed similar effects when cells were treated with the histone deacetylase inhibitor TSA. TSA was able to induce bioluminescence in both 22Rv1-CMVluc and PC3-CMVluc cell lines (Supplementary Fig. S2A). This effect was also not limited to the expression of luciferase as TSA treatment induced AR expression in PC3 cells engineered to express AR from the CMV promoter. However, TSA was unable to induce AR expression in cells engineered to express AR from the SFFV promoter (Supplementary Fig. S2B).

To determine whether the increase in bioluminescence in response to apoptotic inducing agents was specific for human prostate carcinoma cell lines, we examined human breast carcinoma (MDA.MB.231), mouse mammary carcinoma (4T1), and mouse fibroblast (NIH/3T3) cells engineered to express CMV-driven luciferase with the same retroviral expression vector as 22Rv1-CMVluc and PC3-CMVluc cells (Fig. 2C). We also examined MDA.MB.231 cells transiently transfected with either pCDNA3.1-luc (CMV-driven luciferase) or pGL3 (SV40-driven luciferase; Fig. 2D). For each of the cell lines engineered to express luciferase under the control of the CMV promoter, optimized doses of paclitaxel induced a significant increase in bioluminescence compared with nontreated controls (Fig. 2C), although the magnitude of this increase varied among these cell lines. In contrast, MDA.MB.231 cells transiently expressing luciferase under the control of the SV40 promoter did not show induction of bioluminescence signal in response to drug treatment as we observed for the 22Rv1 cell line. These data indicate that paclitaxel induces bioluminescence in cells expressing luciferase under the control of the CMV promoter, but not SV40 promoter. This occurs in multiple cell lines and is independent of the method in which the expression vector is introduced to these cells (stably integrated retroviral vector or transient transfection of a plasmid-based vector).

Bioluminescence can be induced by translation-independent and translation-dependent mechanisms. We observed a transient increase in bioluminescence in all three human prostate cancer cell lines using a high dose of staurosporine, but only 22Rv1-CMVluc and PC3-CMVluc cell lines exhibited an increase in bioluminescence in response to lower doses. To test whether the increase in bioluminescence we observed was due to new protein synthesis, possibly de novo luciferase production, we tested the effects of 4 µmol/L staurosporine and 60 nmol/L staurosporine on 22Rv1-CMVluc cells in the presence or absence of 10 µg/mL cycloheximide, a potent translation inhibitor. Figure 3A shows that cycloheximide was unable to block the induction in bioluminescence signal by 4 µmol/L staurosporine, indicating that the increase in bioluminescence from a 4-µmol/L dose of staurosporine does not result from new protein synthesis. In contrast, Fig. 3B shows that 10 µg/mL cycloheximide blocked bioluminescence induction by a 60-nmol/L dose of staurosporine at 4 and 6 h posttreatment. These data indicate that different doses of staurosporine are able to induce bioluminescence through two independent mechanisms: a translation-independent mechanism (at 4 µmol/L staurosporine) and a translation-dependent mechanism (at 60 nmol/L staurosporine).


Figure 3
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  		Figure 3. Bioluminescence can be induced by translation-independent and translation-dependent mechanisms. 22Rv1-CMVluc cells were pretreated for 2 h with 10 µg/mL cycloheximide (CHX) or fresh media and then treated with either (A) 4 µmol/L staurosporine (STS) or (B) 60 nmol/L staurosporine (STS) and analyzed for bioluminescence (photon flux; photons/s/sec/cm2/sr). Cycloheximide treatment does not block the bioluminescence increase induced using a high 4 µmol/L dose of staurosporine, but does block the low 60 nmol/L dose-induced increase in bioluminescence. ***, P < 0.001, significant increases in photon flux relative to cycloheximide-treated cells. C, 22Rv1-CMVluc and 22Rv1-SV40luc cells were treated with either 4 µmol/L staurosporine for 1 h, 60 nmol/L staurosporine for 6 h, or 0.5% DMSO (Control), and qRT-PCR was done for luciferase mRNA. About 60 nmol/L staurosporine induces an increase in luciferase mRNA in 22Rv1-CMVluc cells, but not 22Rv1-SV40luc cells. **, P < 0.01, significant increases in luciferase mRNA relative to control cells. D, lysates of treated and untreated cells were analyzed for luciferase activity (see Materials and Methods). About 60 nmol/L staurosporine significantly increased luciferase activity in 22Rv1-CMVluc cells but not 22Rv1-SV40luc cells. ***, P < 0.001, significant increases in luciferase protein levels relative to controls.

 
A previous study reported that arsenite treatment of cell lines transfected with CMV-expressing constructs induces an increase in transcription from the CMV promoter via activation of cellular stress factors (20). 22Rv1-CMVluc and PC3-CMVluc cell lines have been engineered to express luciferase through transduction with a retroviral vector that expresses luciferase from an internal CMV immediate early promoter with its proximal enhancer element (herein referred to as CMV promoter) and contains a CMV/murine sarcoma virus (MSV) hybrid promoter in the 5' long terminal repeat (pQCXIN; BD Biosciences). In contrast, 22Rv1-SV40luc cells are engineered to express luciferase through the stable integration of a vector containing SV40 promoter-driven luciferase expression. Because we did not observe an increase in bioluminescence in 22Rv1-SV40luc cells treated with varying doses of doxorubicin and paclitaxel or low doses of staurosporine, we reasoned that these agents are able to increase bioluminescence in 22Rv1-CMVluc and PC3-CMVluc cells through a promoter-dependent mechanism. To test this hypothesis, we examined whether the levels of luciferase mRNA and protein were altered upon staurosporine and doxorubicin treatment (Fig. 3C and D and Supplementary Fig. S3A). We treated both 22Rv1-CMVluc and 22Rv1-SV40luc cell lines with either 4 µmol/L staurosporine for 1 h, 60 nmol/L staurosporine for 6 h, or 400 nmol/L doxorubicin for 48 h. To evaluate the levels of luciferase mRNA, we did quantitative reverse transcription-PCR (qRT-PCR) on RNA prepared from treated and untreated 22Rv1-CMVluc and 22Rv1-SV40luc cells. Figure 3C shows that luciferase mRNA levels are significantly increased when 22Rv1-CMVluc cells are treated with 60 nmol/L staurosporine. Luciferase mRNA levels were also significantly increased when 22Rv1-CMVluc cells were treated with 400 nmol/L doxorubicin (Supplementary Fig. S3A). However, although bioluminescence intensity was significantly increased in cells treated with 4 µmol/L staurosporine, luciferase mRNA levels actually decreased relative to the control 1 h after treatment. Luciferase mRNA levels in staurosporine- and doxorubicin-treated 22Rv1-SV40luc cells were not significantly increased compared with untreated control values. These findings indicate that low doses of staurosporine and 400 nmol/L doxorubicin up-regulate the synthesis of luciferase in cells where its expression is controlled by the CMV promoter. We then prepared lysates from treated and nontreated cells at the same time points as above and analyzed luciferase activity in a cell-free assay. Figure 3D shows that the levels of luciferase protein in 22Rv1-CMVluc cells are significantly increased to ~300% of control values upon low-dose staurosporine treatment. Doxorubicin treatment also induced a significant increase in luciferase protein in 22Rv1-CMVluc cells (Supplementary Fig. S3B). Strikingly, cell-free luciferase activity was not significantly different in cells treated with 4 µmol/L of staurosporine. 22Rv1-SV40luc cells did not show a difference in the levels of luciferase activity compared with nontreated control cells. Taken together, these data indicate an increase in luciferase activity, subsequent to the up-regulation of the CMV promoter when cells are treated with low doses of staurosporine and 400 nmol/L doxorubicin. However, high doses of staurosporine are able to increase bioluminescence by a mechanism that does not involve up-regulation of the CMV promoter and is only apparent when luciferase is in intact cells.

Doxorubicin treatment of subcutaneous luciferase-expressing xenograft tumors leads to a transient increase in bioluminescence in vivo. To test whether we could extend our findings in cell-based assays to tumors in vivo, we employed a subcutaneous xenograft model. Figure 4A shows that a single dose of doxorubicin had a marked effect on tumor growth, with the average tumor volume being 4-fold lower than PBS controls 10 days after treatment. However, Fig. 4B shows that the bioluminescence signal from the doxorubicin-treated animals was comparable to PBS controls during this period. When bioluminescence is normalized to tumor volume (photons/s/cm2/sr/mm3), there is an increase in bioluminescence in the doxorubicin-treated group relative to the PBS controls over a period of 10 days following treatment (Fig. 4C). Images in Fig. 4C show examples of animals from these doxorubicin and PBS (day 6 posttreatment) groups, where it is clear that the smaller tumor (50 mm3) in the doxorubicin-treated animal is emitting a bioluminescence signal comparable to the larger tumor (220 mm3) in a PBS-treated animal. Figure 4C also shows that the bioluminescence increase is transient. Twenty-three days after treatment, both groups displayed similar normalized values. This normalized value is modestly lower than when treatment commenced, possibly due to necrosis in these tumors or decreased efficiency in light penetration as the tumors grow larger. In contrast, normalized bioluminescence signals were similar throughout the time course among doxorubicin and PBS-treated mice bearing xenografts derived from 22Rv1-SV40luc cells (Supplementary Fig. S4). Thus, these results parallel our observations of doxorubicin-induced increase in bioluminescence signal from CMV-driven luciferase in vitro.


Figure 4
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  		Figure 4. Doxorubicin treatment of 22Rv1-CMVluc subcutaneous xenograft tumors leads to a transient increase in luciferase expression in vivo. Subcutaneous 22Rv1-CMVluc xenografts were initiated in nude mice (see Materials and Methods). Groups of six randomly assigned animals were injected once via the tail vein with either 200 µL PBS or 200 µL 1 mg/mL doxorubicin (8 mg/kg). Tumor volumes were recorded, and BLI was done daily for a period of 10 d. A, doxorubicin treatment significantly delayed tumor growth. B, doxorubicin treatment had no effect on bioluminescence intensity (Photon Flux; photons/s/cm2/sr). C, when bioluminescence was normalized to tumor volume, (photons/s/cm2/sr/mm3), there was an increase in bioluminescence intensity in the doxorubicin-treated group relative to the PBS controls over a period of 10 d following treatment. At 23 d, both groups display similar normalized values, showing that the bioluminescence increase is transient. D, an example of a doxorubicin-treated and a PBS-treated animal, where it is clear that the smaller tumor (50 mm3) in the doxorubicin-treated animal has a bioluminescence intensity comparable to the larger tumor (220 mm3) PBS-treated animal. *, P < 0.05; **, P < 0.01; and ***, P < 0.001, significant increases in tumor volume and in normalized photon flux relative to controls.

 
Inhibition of the p38 MAPK pathway blocks doxorubicin- and staurosporine-induced bioluminescence in 22Rv1-CMVluc cells. The CMV promoter is complex and contains numerous binding sites for transcription factors such as Sp-1, cyclic AMP–responsive element binding protein/activating transcription factor (CREB/ATF), and nuclear factor {kappa}B (NF{kappa}B), among others, which are regulated by a variety of cell signaling pathways (21). To determine which pathway(s) might be responsible for our observations, we screened several small molecule inhibitors of the NF{kappa}B, c-jun-NH2-kinase, and p38 MAPK pathways for their ability to block doxorubicin-induced bioluminescence in 22Rv1-CMVluc cells (data not shown). Only the p38 MAPK inhibitor SB203580 was able to block bioluminescence induced by doxorubicin and staurosporine in 22Rv1-CMVluc cells. Figure 5A shows that a 30-µmol/L dose of SB203580 was able to block ~95% of the bioluminescence induction by doxorubicin (at 48 h) or low-dose staurosporine (at 24 h) in 22Rv1-CMVluc cells. We were also able to block 95% of the bioluminescence induction by paclitaxel in PC3-CMVluc cells (data not shown). Another p38 MAPK inhibitor, SB202190, also significantly blocked bioluminescence induction by doxorubicin and paclitaxel in 22Rv1-CMVluc cells, although to a lesser degree than SB203580 (data not shown). These data suggest that p38 MAPK is activated upon doxorubicin, paclitaxel, and low-dose staurosporine treatment and is able to up-regulate the CMV promoter. To determine whether SB203580 was able to block the bioluminescence induction through inhibiting the up-regulation of the CMV promoter, we also prepared RNA from the treated cells and did qRT-PCR for luciferase mRNA. Figure 5B shows that doxorubicin treatment alone induced a 5-fold increase in luciferase mRNA, but addition of the SB203580 was able to block this increase. Finally, to measure the activation of p38 MAPK, we did Western blotting for phosphorylated p38 MAPK from 22Rv1-CMVluc cells treated with doxorubicin. Figure 5C shows that p38 MAPK becomes phosphorylated when cells are treated with doxorubicin. Taken together, these results indicate that treatment with low-dose staurosporine, doxorubicin, and paclitaxel induces a p38 MAPK stress response that is responsible for the observed up-regulation of the CMV promoter and increased bioluminescence signal.


Figure 5
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  		Figure 5. Inhibition of the p38 MAPK pathway blocks the doxorubicin- and staurosporine-induced increase in bioluminescence. 22Rv1-CMVluc cells were treated with vehicle only (Control), 30 µmol/L SB203580 only (SB), 400 nmol/L doxorubicin only (DOX), 60 nmol/L staurosporine only (STS) or a combination of doxorubicin or staurosporine + 30 µmol/L SB203580 (DOX + SB and STS + SB). Bioluminescence intensity (Photon Flux; photons/s/cm2/sr) was recorded as % vehicle control after 48 h of doxorubicin treatment and 24 h of staurosporine treatment. A, SB203580 was able to block 95% of the bioluminescence induction from doxorubicin and staurosporine. B, 22Rv1-CMVluc cells were treated as above, and qRT-PCR was done using cDNA from these cells. Doxorubicin induced a significant increase in luciferase transcript levels, and SB203580 blocked this induction. ***, P < 0.001, significant changes in photon flux or luciferase mRNA levels between indicated treatments. C, 22Rv1-CMVluc cells were treated with either lipopolysaccharide, doxorubicin, or left untreated, and Western blotting for phosphorylated p38 MAPK (phospho-p38) was done. Doxorubicin treatment induces phosphorylation of p38 MAPK. Immunblotting for ß-actin confirms equal protein loading.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We found that the treatment of prostate cancer cell lines, which stably express firefly luciferase from a CMV promoter with apoptosis-inducing agents (staurosporine, doxorubicin, paclitaxel, and TSA) induced a transient increase in bioluminescence even as these cells were undergoing apoptosis. Of these three treatments, only a high dose of staurosporine (4 µmol/L) was capable of producing this same result in prostate cancer cells stably expressing luciferase under the control of the SV40 promoter. Lower doses of staurosporine (60 and 250 nmol/L) were only capable of inducing bioluminescence in CMV luciferase-expressing cells. Furthermore, cycloheximide treatment of cells expressing CMV-driven luciferase abrogated the induction of bioluminescence elicited by low doses (60 nmol/L) of staurosporine, while cotreatment of cycloheximide with high doses of staurosporine (4 µmol/L) was unable to block bioluminescence induction; thus, we concluded that the high-dose staurosporine induces bioluminescence through a promoter-independent mechanism and does not require new protein synthesis. We also observed a transient induction of bioluminescence in other, non–prostate cancer cell lines stably expressing CMV-driven luciferase. Further investigations revealed that doxorubicin treatment of 22Rv1 cells induced luciferase mRNA expression from the CMV promoter as well as increased production of luciferase protein as measured by in vitro luciferase assay, concomitant with increased bioluminescence signal. These findings in cultured cells were extended in vivo; treatment of mice bearing 22Rv1 xenografts with CMV- but not SV40-driven luciferase with doxorubicin also resulted in a transient increase in bioluminescence when normalized to tumor volume. This observation resulted from the maintenance of relatively steady mean bioluminescence values in tumors that were dramatically reduced in volume in response to doxorubicin treatment; while control tumors maintained a consistent relationship between tumor volume and bioluminescence signal.

We considered several possibilities to explain these observations: (a) these agents result in the production of reactive oxygen species that affect the redox-sensitive bioluminescence reaction; (b) apoptosis results in the temporal increase in cytosolic ATP levels, increasing bioluminescence; (c) the expression of the luciferase enzyme was increased. A previous study by Zamaraeva et al. (22) reported a transient increase in bioluminescence from staurosporine, tumor necrosis factor-{alpha}–, and etoposide-treated cell lines expressing luciferase under the control of the SV40 promoter (pGL3 control vector, Promega). This group argued that cells undergoing apoptosis exhibit an increase in cytosolic ATP, with little increase in total cellular ATP, which rapidly and transiently results in increased bioluminescence. Using the same high dose of staurosporine, we showed that the induction of bioluminescence is independent of promoter status and de novo protein synthesis. Therefore, it is possible that ATP compartmentalization or other effects on luciferase reaction components may be influencing the light-generating reaction and driving the increase in bioluminescence. We also show that lower doses of staurosporine are able to increase bioluminescence by a mechanism that is not explained by an increase in cytosolic ATP. Our data show that this alternative mechanism is dependent on de novo protein synthesis, and that the increase in bioluminescence observed is due to the up-regulation of the CMV promoter. Thus, we conclude that under the sets of conditions studied, induction of bioluminescence occurs through two independent mechanisms: (a) a promoter-independent mechanism, not requiring new protein synthesis; and (b) a promoter-dependent mechanism, which requires de novo protein synthesis and is associated with an increase in the steady-state levels of luciferase mRNA and protein.

The CMV immediate early promoter with its proximal enhancer is commonly employed to achieve high-level protein expression in mammalian cells (21). However, this element is also known to be responsive to a variety of stimuli that activate various signaling pathways, including NF{kappa}B and p38 MAPK, among others, which are involved in viral replication (23, 24). As a consequence of this biology, when the CMV promoter is used to drive expression of heterologous proteins, factors that engage those pathways such as inflammatory cytokines or cell stress-related molecules may alter the expression of proteins under its control (25). Previous studies have shown that the CMV promoter activity is increased by NF{kappa}B activation (26, 27). Because chemotherapeutic drugs have been shown to activate the NF{kappa}B pathway in some cell types, we were surprised to find that specific inhibitors of this pathway did not block CMV-driven bioluminescence induction in doxorubicin- and paclitaxel-treated prostate cancer cell lines. Rather, our studies indicated that the activation of the p38 MAPK pathway was responsible for activation of the CMV promoter. Consistent with our findings, Bruening et al. (20) reported that a p38 MAPK inhibitor completely blocked arsenite-induced up-regulation of the CMV promoter. Previous studies have shown that p38 MAPK is activated upon treatment with doxorubicin and paclitaxel (28, 29). These studies also showed that inhibition of p38 MAPK reduced doxorubicin- and paclitaxel-induced apoptosis, demonstrating an important role for p38 MAPK in the apoptotic process. More recently, Reinhardt et al. (30) also showed the importance of p38 MAPK activation in cell cycle checkpoint function after DNA damage. We were able to block ~95% of the bioluminescence induction by doxorubicin and paclitaxel in 22Rv1 cells using a specific inhibitor of p38 MAPK. p38 MAPK activates the transcription factors CREB, ATF-2, and ETS-1, all of which have binding sites within the CMV proximal enhancer and are required for optimal HCMV replication. However, paclitaxel but not doxorubicin, induced bioluminescence in PC-3 cells, indicating that this response may be cell type-specific and/or dependent on the genetic status of the cell type. Together, these results highlight the complex regulation of the CMV promoter in response to various cytokines, chemotherapeutics, and other stress-inducing stimuli and suggest the likelihood that factors other than p38 MAPK may regulate the expression from this promoter, both positively and negatively, in response to different treatments, physiologic contexts, or in a cell type–specific manner.

Given that the CMV promoter has long been known to be responsive to various stress-sensing pathways, it is not surprising that the chemotherapeutic agents employed in this study (which also engage similar pathways) would induce luciferase expression driven by the CMV promoter. Therefore, this information is highly relevant for designing and interpreting studies involving bioluminescence to assess tumor response to chemotherapeutics in mouse models. Indeed, in the original paper describing BLI, the potential problematic aspects of agents that affected constitutive promoters driving luciferase expression were noted (2). We recapitulated our in vitro observations in a subcutaneous tumor model. In response to chemotherapeutic agent doxorubicin, 22Rv1-CMVluc xenografts showed tumor regression as evaluated by traditional volume measurements. However, during a period of 3 to 10 days following a single dose of doxorubicin (8 mg/kg, i.v.), these tumors exhibited bioluminescence signals comparable to PBS-treated control tumors. Effectively, this treatment changed the relationship between viable cell number and light output following treatment. The duration of elevated bioluminescence was longer in vivo than in vitro, >10 days versus <4 days, respectively. This could be due to differences in drug exposure in tumors versus in vitro culture conditions or other mechanisms such as inflammation in the regressing tumor. In contrast, the light output from 22Rv1-SV40luc xenografts did not appreciably change as a function of tumor volume following doxorubicin treatment, although these tumors also responded by a decrease in tumor volume, arguing that the increased bioluminescence signal in 22Rv1-CMVluc xenografts did not result from a drug-induced effect on the tumor microenvironment. Thus, these in vivo findings mirrored our in vitro findings, suggesting that doxorubicin treatment induces a p38 MAPK-dependent increase in luciferase expression from the CMV-promoter in 22Rv1-CMVluc xenografts.

These findings show that caution is warranted in the design and interpretation of experiments involving BLI to assess tumor response to chemotherapeutics in mouse models. A previous study showed that paclitaxel treatment (10 mg/kg/day, i.p.) of PC-3 xenografts expressing luciferase under the control of the CMV-promoter resulted in decreased tumor weight in intramuscular primary tumors, but luciferase activity measured in homogenates from these tumors was not significantly different from control treated animals (31). This led the authors to suggest that paclitaxel was exerting its effects primarily on the tumor stroma, leading to reduced tumor weight. Our results suggest an alternative possibility that these results may be explained by paclitaxel up-regulation of luciferase expression driven by the CMV promoter in these tumors. Thus, care should be taken to evaluate the possible effects of drug treatment on luciferase expression with in vitro studies and to correlate BLI results with alternative measures of tumor growth. To the extent that specific mechanisms can be related to the regulation of the CMV promoter in tumor cells in vivo, this liability might also be useful as a pharmacodynamic marker of drug effect. Specific bioluminescent reporters have already been used successfully in mice to evaluate drug effects in mice (for example, ref. 8). Although the SV40 promoter was not influenced by some drug treatments in our experiments, we are reluctant to conclude that this will be the case with other treatments in other contexts. The SV40 promoter-driven luciferase has been used successfully to measure the response to chemotherapy in several previous studies (e.g., refs. 6, 32). In the case of studies involving the CMV-promoter, the induction of bioluminescence may not have been observed due to the cell lines or treatments involved or chosen imaging time points may not have captured a transient increase in bioluminescence signal (33). Treatments that dramatically and rapidly reduce tumor burden would be expected to mask any potential up-regulation in the CMV-promoter, although in this case, BLI may underestimate drug efficacy. Future studies involving BLI of tumor response to therapy should account for other effects on quantitation in vivo such as changes in O2 levels and pharmacokinetics of luciferin delivery to tumors, both of which may be affected by treatment and have the potential to modify the resultant signal. BLI has become an important tool for the development of sophisticated animal models of cancer and other diseases, as well as providing quantitative assessments of response to therapeutic interventions, and we expect this to continue. However, our studies underscore the fact that a biological process, the luciferase-mediated light-generating reaction, is required to generate a signal in this imaging modality. Therefore, one should carefully consider how other biological processes or agents extrinsic to the light-generating reaction may potentially affect the signal generated.


   Acknowledgments
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Dr. Hasem Habelhah for reagents, Dr. Gail Bishop and Dr. Asgar Zaheer for advice on the phospho-p38 MAPK antibody, and Dr. Mark Stinski for helpful discussions and members of the Henry laboratory for comments on the manuscript.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Received 5/25/07. Revised 8/16/07. Accepted 9/ 6/07.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Gross S, Piwnica-Worms D. Spying on cancer: molecular imaging in vivo with genetically encoded reporters. Cancer Cell 2005;7:5–15.[Medline]
  2. Contag CH, Spilman SD, Contag PR, et al. Visualizing gene expression in living mammals using a bioluminescent reporter. Photochem Photobiol 1997;66:523–31.[Medline]
  3. Edinger M, Sweeney TJ, Tucker AA, Olomu AB, Negrin RS, Contag CH. Noninvasive assessment of tumor cell proliferation in animal models. Neoplasia 1991;1:303–10.[CrossRef]
  4. Vooijs M, Jonkers J, Lyons S, Berns A. Noninvasive imaging of spontaneous retinoblastoma pathway-dependent tumors in mice. Cancer Res 2002;62:1862–7.[Abstract/Free Full Text]
  5. Sweeney TJ, Mailander V, Tucker AA, et al. Visualizing the kinetics of tumor-cell clearance in living animals. Proc Natl Acad Sci U S A 1999;96:12044–9.[Abstract/Free Full Text]
  6. Rehemtulla A, Stegman LD, Cardozo SJ, et al. Rapid and quantitative assessment of cancer treatment response using in vivo bioluminescence imaging. Neoplasia 2000;2:491–5.[CrossRef][Medline]
  7. Laxman B, Hall DE, Bhojani MS, et al. Noninvasive real-time imaging of apoptosis. Proc Natl Acad Sci U S A 2002;99:16551–5.[Abstract/Free Full Text]
  8. Gross S, Piwnica-Worms D. Real-time imaging of ligand-induced IKK activation in intact cells and in living mice. Nat Methods 2005;2:607–14.[CrossRef][Medline]
  9. Luker GD, Pica CM, Song J, Luker KE, Piwnica-Worms D. Imaging 26S proteasome activity and inhibition in living mice. Nat Med 2003;9:969–73.[CrossRef][Medline]
  10. Rudin M, Rausch M, Stoeckli M. Molecular imaging in drug discovery and development: potential and limitations of nonnuclear methods. Mol Imaging Biol 2005;7:5–13.[CrossRef][Medline]
  11. Lee KH, Byun SS, Paik JY, et al. Cell uptake and tissue distribution of radioiodine labelled D-luciferin: implications for luciferase based gene imaging. Nucl Med Commun 2003;24:1003–9.[CrossRef][Medline]
  12. Schroeder T, Yuan H, Viglianti BL, et al. Spatial heterogeneity and oxygen dependence of glucose consumption in R3230Ac and fibrosarcomas of the Fischer 344 rat. Cancer Res 2005;65:5163–71.[Abstract/Free Full Text]
  13. Contag CH. Use of reporter genes for optical measurements of neoplastic disease in vivo. Neoplasia 2000;2:41–52.[CrossRef][Medline]
  14. Scatena CD, Hepner MA, Oei YA, et al. Imaging of bioluminescent LNCaP-luc-M6 tumors: a new animal model for the study of metastatic human prostate cancer. Prostate 2004;59:292–303.[CrossRef][Medline]
  15. Drake JM, Gabriel CL, Henry MD. Assessing tumor growth and distribution in a model of prostate cancer metastasis using bioluminescence imaging. Clin Exp Metastasis 2005;22:674–84.[CrossRef][Medline]
  16. Henry MD, Wen S, Silva MD, Chandra S, Milton M, Worland PJ. A prostate-specific membrane antigen-targeted monoclonal antibody-chemotherapeutic conjugate designed for the treatment of prostate cancer. Cancer Res 2004;64:7995–8001.[Abstract/Free Full Text]
  17. Rokhlin OW, Taghiyev AF, Guseva NV, et al. Androgen regulates apoptosis induced by TNFR family ligands via multiple signaling pathways in LNCaP. Oncogene 2005;24:6773–84.[CrossRef][Medline]
  18. Rokhlin OW, Taghiyev AF, Guseva NV, Glover RA, Syrbu SI, Cohen MB. TRAIL-DISC formation is androgen-dependent in the human prostatic carcinoma cell line LNCaP. Cancer Biol Ther 2002;1:631–7.[Medline]
  19. Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 2001;29:e45.[Abstract/Free Full Text]
  20. Bruening W, Giasson B, Mushynski W, Durham HD. Activation of stress-activated MAP protein kinases up-regulates expression of transgenes driven by the cytomegalovirus immediate/early promoter. Nucleic Acids Res 1998;26:486–9.[Abstract/Free Full Text]
  21. Stinski MF. Cytomegalovirus promoter for expression in mammalian cells. In: Fernandez JF, Hoeffler JP, editors, Gene expression systems: using nature for the art of expression. San Diego (CA): Academic Press; 1999. p. 211–33.
  22. Zamaraeva MV, Sabirov RZ, Maeno E, Ando-Akatsuka Y, Bessonova SV, Okada Y. Cells die with increased cytosolic ATP during apoptosis: a bioluminescence study with intracellular luciferase. Cell Death Differ 2005;12:1390–7.[CrossRef][Medline]
  23. Chen J, Stinski MF. Role of regulatory elements and the MAPK/ERK or p38 MAPK pathways for activation of human cytomegalovirus gene expression. J Virol 2002;76:4873–85.[Abstract/Free Full Text]
  24. Johnson RA, Huong SM, Huang ES. Activation of the mitogen-activated protein kinase p38 by human cytomegalovirus infection through two distinct pathways: a novel mechanism for activation of p38. J Virol 2000;74:1158–67.[Abstract/Free Full Text]
  25. Ramanathan M, Hasko G, Leibovich SJ. Analysis of signal transduction pathways in macrophages using expression vectors with CMV promoters: a cautionary tale. Inflammation 2005;29:94–102.[CrossRef][Medline]
  26. Sambucetti LC, Cherrington JM, Wilkinson GW, Mocarski ES. NF-{kappa}B activation of the cytomegalovirus enhancer is mediated by a viral transactivator and by T cell stimulation. EMBO J 1989;8:4251–8.[Medline]
  27. Prosch S, Staak K, Stein J, et al. Stimulation of the human cytomegalovirus IE enhancer/promoter in HL-60 cells by TNF{alpha} is mediated via induction of NF-{kappa}B. Virology 1995;208:197–206.[CrossRef][Medline]
  28. Grethe S, Coltella N, Di Renzo MF, Porn-Ares MI. p38 MAPK downregulates phosphorylation of Bad in doxorubicin-induced endothelial apoptosis. Biochem Biophys Res Commun 2006;347:781–90.[CrossRef][Medline]
  29. Bacus SS, Gudkov AV, Lowe M, et al. Taxol-induced apoptosis depends on MAP kinase pathways (ERK and p38) and is independent of p53. Oncogene 2001;20:147–55.[CrossRef][Medline]
  30. Reinhardt HC, Aslanian AS, Lees JA, Yaffe MB. p53-deficient cells rely on ATM- and ATR-mediated checkpoint signaling through the p38MAPK/MK2 pathway for survival after DNA damage. Cancer Cell 2007;11:175–89.[CrossRef][Medline]
  31. El Hilali N, Rubio N, Blanco J. Different effect of paclitaxel on primary tumor mass, tumor cell contents, and metastases for four experimental human prostate tumors expressing luciferase. Clin Cancer Res 2005;11:1253–8.[Abstract/Free Full Text]
  32. Jenkins DE, Yu SF, Hornig YS, Purchio T, Contag PR. In vivo monitoring of tumor relapse and metastasis using bioluminescent PC-3M-luc-C6 cells in murine models of human prostate cancer. Clin Exp Metastasis 2003;20:745–56.[CrossRef][Medline]
  33. Buchhorn HM, Seidl C, Beck R, et al. Non-invasive visualisation of the development of peritoneal carcinomatosis and tumour regression after (213)Bi-radioimmunotherapy using bioluminescence imaging. Eur J Nucl Med Mol Imaging 2007;34:841–9.[CrossRef][Medline]



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