cox-2 gene expression in chemically induced skin papillomas cannot predict subsequent tumor fate
TRANSCRIPT
M O L E C U L A R O N C O L O G Y 4 ( 2 0 1 0 ) 3 4 7e3 5 6
ava i lab le at www.sc ienced i rec t . com
www.e lsev ie r . com/ loca te /molonc
Cox-2 gene expression in chemically induced skin papillomas cannot
predict subsequent tumor fate
Tomo-o Ishikawaa, Naveen K. Jaina, Harvey R. Herschmana,b,c,*aDepartment of Molecular and Medical Pharmacology, David Geffen School of Medicine, UCLA, Los Angeles, CA, USAbDepartment of Biological Chemistry, David Geffen School of Medicine, UCLA, Los Angeles, CA, USAcMolecular Biology Institute, UCLA, Los Angeles, CA, USA
A R T I C L E I N F O
Article history:
Received 27 February 2010
Received in revised form
9 June 2010
Accepted 9 June 2010
Available online 16 June 2010
Keywords:
Cyclooxygenase
Luciferase
Knock-in mouse
Gene expression
Skin cancer
DMBA/TPA
* Corresponding author at: 341 Boyer Hall, Ufax: þ1 310 825 1447.
E-mail address: [email protected]/$ e see front matter ª 2010 Federdoi:10.1016/j.molonc.2010.06.004
A B S T R A C T
Elevated cyclooxygenase-2 (COX-2) expression is observed in a variety of premalignant
neoplastic tissues, suggesting COX-2 expression might serve as a potential indicator of sub-
sequent tumor development. However, it has not been possible to compare the relation-
ship between Cox-2 gene expression in premalignant lesions and their subsequent fate,
because conventional studies require tissue destruction for analysis of gene expression.
To monitor COX-2 expression non-invasively during tumor development, we created
a Cox-2 luciferase knock-in mouse, Cox-2luc, in which the firefly luciferase coding region re-
places the Cox-2 coding region. Luciferase activity was non-invasively, quantitatively and
repeatedly monitored in Cox-2luc/þ mice subjected to DMBA/TPA multistage skin tumor in-
duction. Luciferase activity is significantly higher in all papillomas than in surrounding
skin. However, the magnitude of Cox-2 promoter-driven luciferase activity in small papil-
lomas cannot predict subsequent papilloma regression or growth. Elevated Cox-2 pro-
moter-driven luciferase signal can be detected when papillomas first become visible, but
not before this time.
ª 2010 Federation of European Biochemical Societies.
Published by Elsevier B.V. All rights reserved.
1. Introduction and Cox-2 knock-out mice (Muller-Decker et al., 1998; Fischer
In human skin tumors, cyclooxygenase-2 (COX-2) is overex-
pressed in premalignant actinic keratoses and keratoacantho-
mas, as well as in squamous cell carcinomas (Muller-Decker
et al., 1999; An et al., 2002). Similarly, COX-2 expression in-
creases both in UVB-induced murine skin cancer induction
and in the 7,12-dimethylbenz[a]anthracene (DMBA)/12-O-tet-
radecanoylphorbol-13-acetate (TPA) multistage skin carcino-
genesis model (Muller-Decker et al., 1995). A requirement for
COX-2 function during induction of skin tumors in murine
models is suggested by studies using both COX-2 inhibitors
CLA, 611 Charles E. Youn
la.edu (H.R. Herschman).ation of European Bioche
et al., 1999, 2007; Tiano et al., 2002).
The DMBA/TPA multistage skin carcinogenesis model is
among the most intensively studied mouse cancer models
(Kemp, 2005). Tumor induction is initiated on the dorsal skin
with a single, low “initiator” dose of a carcinogen, most com-
monly DMBA, unable by itself to elicit tumor formation. “Initi-
ation” is followed by multiple applications of a tumor
promoter, TPA, also unable by itself to elicit tumor formation.
The initiationepromotion combination causes the develop-
mentofpapillomas; benignneoplastic lesions consistingofhy-
perplastic keratinocytes and supporting stromal cells. A small
percentage of DMBA-initiated, TPA-promoted papillomas
g Drive East, Los Angeles, CA 90095, USA. Tel.: þ1 310 825 8735;
mical Societies. Published by Elsevier B.V. All rights reserved.
M O L E C U L A R O N C O L O G Y 4 ( 2 0 1 0 ) 3 4 7e3 5 6348
“progress” to malignant, invasive squamous cell carcinomas.
In contrast, some papillomas remain as non-invasive benign
tumors, while many papillomas regress and disappear.
Non-steroidal anti-inflammatory drugs (NSAIDs), which
block the activity of cyclooxygenases, prevent or reduce
DMBA/TPApapillomaformation (Fischeretal., 1987).COX-2se-
lective inhibitors are also effective in blocking multistage skin
carcinogenesis (Muller-Decker et al., 1998; Brecher, 2002;Howe
andDannenberg, 2002). Moreover, knock-out of the Cox-2 gene
reduces DMBA/TPA-induced skin papilloma production (Tiano
et al., 2002). These pharmacologic and genetic studies suggest
thatCOX-2playsarequisite role inDMBA/TPA-inducedskin tu-
mor development.
COX-2 protein is overexpressed in all DMBA/TPA-induced
epidermal carcinomas. In contrast, COX-2 protein levels in
papillomas vary from barely detectable to equal in magnitude
to that observed in carcinomas (Muller-Decker et al., 1995).
These data raise the question of whether COX-2 expression
in papillomas is correlated with and/or predictive of papillo-
mas that will regress, papillomas that will remain in the be-
nign, non-malignant state, or papillomas that will progress
from benign to malignant tumors. However, until recently
there has been no way to measure the level of Cox-2 gene ex-
pression in papillomas and also to examine the subsequent
fates of those same papillomas.
Wepreviously generatedamurine “knock-in” allele,Cox-2luc,
in which the firefly luciferase reporter enzyme is expressed at
the start site of translation of the endogenous Cox-2 gene
(Ishikawa et al., 2006). Using heterozygous Cox-2luc/þ mice, Cox-
2 transcriptional activity can be monitored non-invasively,
quantitatively and repeatedly in living animals by biolumines-
cence imaging; Cox-2 promoter-driven luciferase activity from
the Cox-2luc allele accurately reflects the level of COX-2
80
60
40
20
00 5 10 15 20 25
Weeks
DMBA TPA
week 0 1 2 3 1 2 3 4 5 6 7 1 2 3 4 5 6 7 Day 1 2 3 4 5 6 7 1 2 3 4 5 6 7
A
B C
Tum
or in
cide
nce
(%)
Figure 1 e Papilloma development in DMBA-initiated, TPA-promoted Co
treatment and bioluminescent imaging. Cox-2luc/þ 129/C57Bl6 mice (n [
promoted with two applications per week of TPA (10 mg). Bioluminescent
TPA treatment for each cycle and immediately prior to the start of the next T
Tumor multiplicity (average number of tumors per mouse).
expression from the Cox-2wild-type allele in response to a vari-
ety of stimuli in cultured murine embryo fibroblasts and in
mouse organs in vivo, following both local and systemic inflam-
matory stimuli (Ishikawa et al., 2006). In this study, we use Cox-
2luc/þ heterozygous mice to monitor longitudinally expression
from the Cox-2 gene during tumor development induced by
DMBA/TPAmultistep carcinogenesis.
2. Materials and methods
2.1. Mice
The Cox-2luc/þ mouse, in which the firefly luciferase coding re-
gionisknockedintotheCox-2geneat thestart siteof translation,
was described previously (Ishikawa et al., 2006). HRS/J hairless
mice were obtained from Jackson Laboratory (Bar Harbor, ME,
USA). To obtain hairless mice, Cox-2luc/þ (129/C57Bl6) mice
were crossedwithHRS/Jmice twiceandCox-2luc/þprogenywith-
out hair were selected for study. Animal experiments were car-
riedoutwithapprovalof theUCLAAnimalResearchCommittee.
2.2. DMBA/TPA treatment
The dorsal surfaces of 13 female Cox-2luc/þ 129/C57Bl6 mixed
genetic background mice were shaved, and 50 mg of DMBA
(Sigma, Saint Louis, MO, USA) in 200 ml of acetone was applied.
Mice were 7e12 weeks old when treated with DMBA. Starting
one week later, mice were treated with 10 mg of TPA (Sigma) in
200 ml of acetone twice-weekly (Figure 1A). Papilloma numbers
were counted weekly. A similar protocol was followed for fe-
male Cox-2luc/þ 129/C57Bl6/HRS mice for tumor promotion/re-
gression experiments.
8
6
4
2
0
Tum
or m
ultip
licity
Weeks0 5 10 15 20 25
5 6 7 8
imagingimaging
1 2 3 4 5 6 7 1 2 3 4 5 6 7 1 2 3 4 5 6 7 1 2 3 4 5 6 7
x-2luc/þ 129/C57Bl6 mice. (A) Time line for DMBA treatment, TPA
13) were initiated with a single DMBA (50 mg) treatment and then
images were taken at two-week intervals, three days after the second
PA cycle. (B) Tumor incidence (percentage of mice with tumors). (C)
M O L E C U L A R O N C O L O G Y 4 ( 2 0 1 0 ) 3 4 7e3 5 6 349
2.3. In vivo imaging of luciferase activity
Mice were anesthetized by intraperitoneal administration of
a ketamine (80 mg/kg, Phoenix Pharmaceutical, St. Joseph,
MO, USA) and xylazine (4 mg/kg, Phoenix Pharmaceutical)
mixture. Anesthetized mice were injected intraperitoneally
with D-luciferin (125 mg/kg, Caliper Life Sciences, Hopkinton,
MA, USA) and placed in the light-tight box of the IVIS imaging
system (Caliper Life Sciences). Whole body one-minute im-
ages were acquired repeatedly until the maximum peak of
photon number was confirmed during 1-min scans. Data at
the time point that gave the highest photon number during
1min of scanning timewere used for quantification. Collected
photon number and images were analyzed using LIVING IM-
AGE software (Caliper Life Sciences). Photon numbers are
shown as maximum number of radiance from one of the
pixels in ROI (region of interest) or average radiance of entire
ROI area. Radiance is shown as photons/second/cm2/stera-
dian (p/s/cm2/sr).
2.4. Measurement of tumor size
Length and width of each tumor was measured on the imag-
ing photos of eachmouse. The tumor volume inmm3 was cal-
culated using the formula: tumor volume ¼ length � (width)2/
2 (McCawley et al., 2008).
2.5. Statistical analysis
Data were analyzed using t-test or ANOVA where applicable.
3. Results
The use of Cox-2luc/þ knock-in mice, in which luciferase re-
porter gene expression can be monitored repeatedly, quanti-
tatively and non-invasively in living animals (Ishikawa et al.,
2006), provides an opportunity to analyze longitudinal
changes of Cox-2 transcriptional activities (Ishikawa et al.,
2006, 2009). To investigate the correlation between Cox-2 ex-
pression and skin papilloma fate, skin tumors were initiated
in Cox-2luc/þ mice with DMBA and then promoted with TPA
in the standard skin tumor induction regimen (Figure 1A).
3.1. Papilloma fate and Cox-2 promoter-drivenluciferase activity in Cox-2luc/D 129/C57Bl6 mice
Cox-2luc/þ mice were generated from 129 ES cells and are in
a mixed genetic 129/C57Bl6 background (Ishikawa et al.,
2006). Because mouse strains are not uniform with regard to
DMBA/TPA skin tumor induction, it is difficult to predict the
number of tumors expected. Furthermore, Cox-2 deficiency re-
duces papilloma formation; tumor number (multiplicity) is re-
duced in heterozygous and homozygous Cox-2 knock-outmice
to 30% and 60% respectively when compared with wild-type
mice (Tiano et al., 2002). Cox-2luc/þ mice are functionally het-
erozygous at the Cox-2 locus; the Cox-2luc knock-in allele is
also a Cox-2 knock-out allele, since the Cox-2 coding region is
replaced with the luciferase reporter gene. The Cox-2luc/þ het-
erozygous mice used for in vivo imaging would, therefore, be
expected to show a reduced number of DMBA/TPA-induced
tumors. However, Cox-2luc/þ mice in 129/C57Bl6 mixed genetic
background receiving a single DMBA (50 mg) initiator dose and
subsequent TPA (10 mg) promoter treatments developed papil-
lomas in sufficient number (Figure 1B and C) to carry out tu-
mor promotion/regression studies for in vivo imaging.
In vivo imaging of luciferase activity was performed every
other week on DMBA/TPA-treated Cox-2luc/þ mice (Figure 1A).
To investigate the Cox-2 promoter-driven luciferase activity
regulated by tumor formation, and not by transient activation
by TPA, each image was taken 72 h after the last TPA applica-
tion in each twice-weekly TPA regimen (Figure 1A); transient
TPA induction of luciferase activity in the skin of DMBA/
TPA-treated Cox-2luc/þ mice returns to baseline by this time
(data not shown). Initial images were taken at 14 weeks and
continued every two weeks for 20e48 weeks, or until total tu-
mor burden reached themaximal size permitted for the proto-
col, depending on the individual mouse.
Following their initial appearance, papillomas either re-
main relatively small (<30 mm3), grow in size, or regress and
disappear during the TPA-mediated promotion phase
(Figure 2A, top panels; photos of a DMBA/TPA-treated Cox-
2luc/þ 129/C57Bl6 mouse). All morphologically distinguishable
tumors have significantly higher luciferase activity compared
with other areas of the skin (Figure 2A, bottom panels; over-
lapping photos and pseudocolor patterns of photon emission
detected following luciferin injection and bioluminescent im-
aging). These results demonstrate that Cox-2 transcriptional
activity in DMBA/TPA-induced skin tumors can be monitored
repeatedly in vivo in living Cox-2luc/þ mice by bioluminescent
imaging.
For the mouse shown in Figure 2A, 21 tumors were identi-
fied from successive photos taken every two weeks (with one
exception, for technical reasons) between weeks 14 and 48 af-
ter initiation by DMBA treatment. The tumor sizes and Cox-2
promoter-driven luciferase activities of each tumor were
quantified from all papillomas at each 2-week interval. The
sizes were measured from the photo images. To quantify the
luciferase activity from each tumor, regions of interest (ROI)
that includes the entire tumor area were defined manually
from the photo. To compare luciferase activities of the tu-
mors, we used both the maximum radiance value (Figure 2C)
and the average value of photon emission within each ROI
(Figure 2D).
The specific question we wished to address from this ex-
periment is “Can the magnitude of Cox-2 promoter-driven lu-
ciferase activity in small papillomas predict whether these
papillomas will subsequently regress or will continue to
grow?” To answer this question, at the end of the 48-week ex-
periment we scored the fates of the 21 tumors into three cat-
egories, based on the photographic record (Figure 2A); tumors
that “regressed” during the experiment, tumors that
“remained small” (<30 mm3) during the experiment and tu-
mors that “grew” (>30 mm3) during the experiment
(Figure 2B). For each papilloma, we considered the Cox-2 pro-
moter-driven luciferase activity during the time period when
the papillomas were between 3 and 30 mm3.
For tumors that regressed, the average of the maximum bio-
luminescent radiance values (i.e., the maximum pixel value) for
Cox-2 promoter-driven luciferase was 7,024,788 � 1,100,788 p/s/
Figure 2 e Bioluminescent in vivo luciferase imaging of a Cox-2luc/þ 129/C57Bl6 mouse during DMBA/TPA papilloma induction. (A) Images of
Cox-2 promoter-driven luciferase expression in papillomas of a Cox-2luc/þ 129/C57Bl6 mouse. At least 72 h after each TPA application, the mouse
was injected with luciferin, photographed (top panel) and imaged for luciferase activity. Overlaid images with pseudocolor patterns of photon
emission are shown in the bottom panel. A pseudocolor scale of photon numbers (p/sec/cm2/sr) is also shown. (B) Fate of papillomas shown in the
upper panel of Figure 2A. Each line represents the continued presence of an individual papilloma. Lines begin when papillomas are distinguishable
M O L E C U L A R O N C O L O G Y 4 ( 2 0 1 0 ) 3 4 7e3 5 6350
M O L E C U L A R O N C O L O G Y 4 ( 2 0 1 0 ) 3 4 7e3 5 6 351
cm2/sr (Figure2C).Theaverageofvalues forpapillomasthatgrew
beyond30mm3was14,985,611� 2,682,171p/s/cm2/sr, anaverage
value significantly different from that of papillomas that
regressed ( p < 0.01). However, the luciferase values ranged be-
tween375,700and62,050,000p/s/cm2/srfortumorsthatregressed
and between 656,900 and 46,370,000 p/s/cm2/sr for tumors that
grewbeyond 30mm3. The ranges of theseCox-2 promoter-driven
luciferase values overlap, without a clear threshold that permits
one to distinguishbetween individual tumorswithdifferent fates
in these two groups (Figure 2C). For papillomas that “remained
small” (i.e., papillomas that never exceeded 30 mm3, but did not
disappear during the course of the experiment), the average of
Cox-2 promoter-driven luciferase values did not show significant
differenceswhencomparedwiththeothergroups (Figure2C),and
ranged from 500,400 to 48,600,000 p/s/cm2/sr. Thus, the magni-
tude of Cox-2 promoter-driven luciferase activity for papillomas
smaller than 30 mm3 cannot distinguish papillomas that will re-
gress from those that will remain small and/or from those that
grow to substantially larger size.
Comparisons of DMBA/TPA-induced tumors present on ad-
ditionalCox-2luc/þ129/C57Bl6micealsodemonstratednocorre-
lation between Cox-2 transcriptional activity, as measured by
Cox-2 promoter-driven luciferase activity, and papilloma fate.
When we analyzed the values of average photon emissions
(Figure 2D) from tumors that regressed, tumors that remained
small but did not regress, or tumors that grew in size beyond
30 mm3, we obtained essentially the same results.
Examples of longitudinal analyses of bioluminescence and
tumor size for several individual papillomas are shown in
Figure 2E. Tumor 11 reached 30 mm3 at 20 weeks. It reached
its size 32e36 weeks after the DMBA treatment, then started to
regress and was no longer morphologically distinguishable at
48 weeks. Tumor 7 did not reach 30mm3 at 32 weeks, however
it continued to growafter reaching30mm3andwasnot regress-
ing when the mouse was euthanized. Tumor 18, which
appeared after both tumor 7 and tumor 11, reached 30 mm3,
remained relatively small compared to tumors 7 or 11, and
obtained a stable size until the mouse had to be euthanized.
Cox-2 promoter-driven luciferase activity did not correlate well
with tumor size in any of these tumors. Even after the 30 mm3
sizewas obtained and surpassed, neither the pattern of lucifer-
aseactivitynor themagnitudeof luciferase activityatany single
time point could suggest the fate of the papillomas.
3.2. Appearance of DMBA/TPA-induced papillomas andCox-2 promoter-driven luciferase activity in Cox-2luc/D
129/C57Bl6/HRS hairless mice
In the experiment above, our impression was that elevated
Cox-2 promoter-driven luciferase activity was apparent as
morphologically, and end when the papillomas are no longer visible. Thin li
>30 mm3. Black lines, papillomas that regressed (“regressed”); red lines, p
lines, papillomas that exceeded 30 mm3 (“grew”). (C) Luciferase activity from
tumors are between 3 and 30 mm3 (i.e., for imaging values during the perio
drawn manually outside the tumor boundary on the photo for each image, at
bioluminescent radiance in each ROI was analyzed and plotted. (D) Avera
bioluminescent radiance values, for the same ROIs used in panel C. (E) Lon
(tumors number 11, 7 and 18 shown in Panel B). Red lines indicates lucife
references to colour in this figure legend, the reader is referred to the web
soon as we could detect a morphologically identifiable
DMBA/TPA-induced papilloma. We could not, however, deter-
mine whether Cox-2 promoter-driven luciferase activity could
indicate the presence of initiated/promoted cells prior to a vis-
ible, morphologically apparent papilloma.
Skin pigments and hair can significantly reduce detectable
photon emission from mouse skin and tumors. Moreover, in-
jury caused by shaving the Cox-2luc/þ mice may affect expres-
sion from the Cox-2 promoter. To exclude these factors from
influencing in vivo imaging of Cox-2 promoter-driven lucifer-
ase expression, Cox-2luc/þ mice were crossed with HRS/J
mice. HRS/J mice, albino mice that are immunocompetent
(in contrast to nude mice), are particularly valuable for in
vivo imaging procedures that utilize optical detection
(Collaco and Geusz, 2003).
Cox-2luc/þ 129/C57Bl6/HRS mice were subjected to the same
DMBA/TPA skin tumor induction protocol described above.
Initial non-invasive bioluminescence images were taken at 6
weeks, before tumors first became clearly visible. To analyze
the induction of Cox-2 expression in early papillomas, images
of individual animals compared before and after visible tu-
mors can be identified. In the example shown in Figure 3, tu-
mors could not be observed visually at week eight
(Figure 3A). An arbitrary grid placed on the mouse was used
to define a set of ROIs covering the dorsal surface. Maximum
photon emission was measured from each grid every 2 weeks
and plotted (Figure 3A, graphs). The first papilloma appeared
on this mouse at week 10 (Figure 3A, red arrow). Luciferase ac-
tivity was elevated only in grid 11, which includes this single
papilloma. At week 12, a second papilloma (yellow arrow)
appeared on the mouse; grid eight, containing this tumor,
now shows elevated Cox-2 promoter-driven luciferase activity
when compared with adjacent grids. At week 14, a third pap-
illoma (orange arrow) is visible. Grid 12, which contains this
papilloma, now shows an elevated luciferase activity. This
analysis was performed on 7 tumors, present on four different
DMBA/TPA-treated Cox-2luc/þ mice.
For each papilloma, the photon emission of the grid con-
taining the papilloma at the time of first morphological detec-
tion was compared to photon emission of the same grid, on
the same mouse, from the images taken two weeks earlier
(Figure 3B). The grids in which papillomas were visible at their
respective “week 0” had elevated bioluminescent signals rela-
tive to the same grid two weeks earlier (“week 2”) ( p < 0.01 by
t-test). In contrast, comparisons of regions of skin that do not
have papillomas at “week 0” with their same skin regions at
“week 2” show no significant change in photon emission
( p > 0.05). Moreover, the luciferase activities of grids contain-
ing a visible papilloma are significantly higher than that of
surrounding grids without tumor, for all the tumors analyzed
nes are for papillomas 3e30 mm3 in size, thick lines are for papillomas
apillomas that did not grow beyond 30 mm3 (“remained small”); green
all papillomas illustrated in panel 2B, for all time points when these
ds when they are represented by thin lines in Figure 2B). ROIs were
each time point when tumors were between 3 and 30 mm3. Maximum
ge bioluminescent radiance values, rather than maximum
gitudinal analyses of the size and luciferase activity from three tumors
rase activity, blue lines show tumor size. (For interpretation of the
version of this article.)
Figure 3 e Papilloma formation and Cox-2 promoter-driven luciferase expression during the early phase of tumor development in a DMBA/TPA-
treated Cox-2luc/þ 129/C57Bl6/HRS mouse. (A) Photos of tumor morphology and in vivo bioluminescence imaging and quantification of Cox-2
promoter-driven luciferase activity during papilloma appearance from 8 to 14 weeks following DMBA application. Photos on the far left show
tumor morphology. The middle photos overlay the morphological image with pseudocolor representations (see bar scale, p/sec/cm2/sr) of Cox-2
promoter-driven luciferase activity. Photos on the right illustrate the grid identification numbers used in the quantitative analysis. Morphologically
M O L E C U L A R O N C O L O G Y 4 ( 2 0 1 0 ) 3 4 7e3 5 6352
M O L E C U L A R O N C O L O G Y 4 ( 2 0 1 0 ) 3 4 7e3 5 6 353
(Figure 3B; p < 0.01). The data demonstrate that, although ele-
vated Cox-2 transcriptional activity is detectable when papillo-
mas are first visible, the appearance of a papilloma cannot be
predicted from Cox-2 promoter-driven luciferase activity two
weeks prior to morphological tumor identification.
3.3. Papilloma fate and Cox-2 promoter-drivenluciferase activity in Cox-2luc/D 129/C57Bl6/HRS hairlessmice
Both tumor incidence and tumor multiplicity are reduced in
Cox-2luc/þ 129/C57Bl6/HRS mice when compared to Cox-2luc/þ
129/C57Bl6 mice (compare Figure 4 with Figure 1). However,
we were also able to examine the relationship of Cox-2 pro-
moter-driven luciferase expression e a surrogate for Cox-2
gene expression and COX-2 protein levels (Ishikawa et al.,
2006, 2009) e and papilloma fate in Cox-2luc/þ 129/C57Bl6/HRS
hairless mice. As in Cox-2luc/þ 129/C57Bl6 mice, Cox-2 pro-
moter-driven luciferase activity in DMBA/TPA-induced papil-
lomas on the hairless strain could not distinguish
papillomas that regressed from those that did not regress.
An example of Cox-2 promoter-driven luciferase expression
in regressing (yellow arrow) and non-regressing (orange ar-
row) papillomas is shown in Figure 5.
4. Discussion
COX-2 overexpression is observed in a variety of premalignant
neoplastic tissues (Subbaramaiah and Dannenberg, 2003). Ge-
netic, epidemiologic and pharmacologic studies demonstrate
that COX-2 expression is involved in and, in some cases, es-
sential in the development of epithelial cancers. For example,
deletion of the Cox-2 gene reduced substantially the number
and the size of intestinal polyps in Apc knock-out mice
(Oshima et al., 1996). Epidemiologic studies suggest a link be-
tween the use of NSAIDs and reduction of both colon and
breast cancer in humans (Smalley and DuBois, 1997; Johnson
et al., 2002). Furthermore, COX-2 specific inhibitors (COXIBs)
are effective chemopreventive agents for colon cancer
(Steinbach et al., 2000). These data, andmany additional stud-
ies, suggest that COX-2 plays an important role(s) in early de-
velopment and progression of cancer. One might, therefore,
expect that variations in COX-2 expression during the early
stages of tumor formationmight serve as a potential indicator
of subsequent tumor development, if we could analyze COX-2
expression non-invasively.
In conventional studies, expression from the Cox-2 gene is de-
termined by ex vivo analyses which require destruction of tissue
samples (e.g. Northern blotting, Western blotting, immunohisto-
chemistry); consequently one cannot analyze, for an individual
tumor, bothCOX-2expressionatagiven timeandthesubsequent
identified tumors are indicated by the colored arrows. Maximum radiances
containing the three tumors are identified with the same colours as the arr
Quantification of Cox-2 promoter-driven luciferase activity during early tu
papillomas are quantified for the week when the papillomas were first morph
grids taken two weeks earlier (week 2). Data are for seven tumors from three
at week 0 and week 2.
fate of that tumor. A non-invasive imaging method to monitor
COX-2 expression would overcome this problem. We previously
generatedaknock-inmouseinwhichthecodingregionfora lucif-
erase reporter gene is placedat thebeginningof the coding region
of the endogenous Cox-2 gene (Ishikawa et al., 2006) and showed,
in inflammationmodels, thatCox-2 gene expression can bemon-
itored quantitatively, non-invasively and repeatedly (Ishikawa
et al., 2006). In this study, we demonstrate that Cox-2 expression
can be monitored repeatedly in developing skin tumors of living
mice.Using this technology,wefind thatCox-2 expressioncannot
distinguishbetweenDMBA/TPApapillomasthatsubsequently re-
gress and papillomas that do not regress.
TPA-induced COX-2 expression in skin is transient, peak-
ing at six hours and subsequently decreasing (Scholz et al.,
1995). Cox-2 promoter activity monitored by luciferase expres-
sion in Cox-2luc/þ skin is consistent with previously reported
COX-2 expression for TPA-treated mice (data not shown). Al-
though repeated TPA treatment causes hyperplasia of the
skin, this hyperplasia does not lead to substantial alteration
of COX-2 accumulation. In contrast, DMBA/TPA-induced pap-
illomas and carcinomas constitutively express COX-2 (Muller-
Decker et al., 1995). The Cox-2 promoter-driven luciferase
signals from DMBA/TPA-induced tumors on Cox-2luc/þ mice
can also be clearly distinguished from surrounding areas of
skin and can easily be monitored longitudinally.
Bioluminescence imaging is not as quantitatively accurate as
radionuclide technologies such as positron emission tomogra-
phy, due to the nature of light scattering and absorption in tis-
sues. However, in these experiments the tumors from which
luciferase activity is quantified are early stage skin papillomas
that are superficial and are small in size (<30 mm3). Conse-
quently, there are no superficial tissues or other structures that
could significantly complicate the propagation of light towards
the camera lying above these tumors, and the small (30mm3) tu-
mors themselveshaveminimalself-absorptionandscatteringof
the emitted light. Therefore, DMBA/TPA skin cancer presents
perhaps the best possible model for quantification of non-inva-
sive, repeated optical imaging of tumor progression.
We initially used the maximum bioluminescent radiance
(maximum pixel value) to analyze the luciferase activity from
each ROI (Figure 2C). We think that this value is the most reli-
able representative of the tissue emission, the most reproduc-
ible, and the most consistent measurement least subject to
investigator bias. We also compared the maximum biolumi-
nescent radiance values with the average bioluminescence
values for ROIs for tumors <30 mm3; the two analyses exhibit
essentially same results (Figure 2D, compare with Figure 2C).
The use of total photonnumber for eachROI is less reliable, be-
cause tumor size and shape vary. To detect the early appear-
ance of luciferase signal (Figure 3), we only used maximum
radiance because this signal must be confined in small area
of ROI and average calculation would dilute the signal.
in each grid are plotted in the graphs. The values for the grids
ows that identify the individual papillomas in the photos. (B)
mor development. Luciferase activities from ROIs of grids with
ologically identified (week 0) and from the previous images of the same
mice. For controls, adjacent grids without papillomas were compared
0 5 10 15 20 25
Tum
or m
ultip
licity
Weeks
8
6
4
2
0
80
60
40
20
00 5 10 15 20 25
Weeks
A
Tum
or in
cide
nce
(%)
B
Figure 4 e Papilloma development in DMBA-initiated, TPA-promoted Cox-2luc/þ 129/C57Bl6/HRS hairless mice. (A) Tumor incidence. (B)
Tumor multiplicity.
M O L E C U L A R O N C O L O G Y 4 ( 2 0 1 0 ) 3 4 7e3 5 6354
In our experiments,malignant squamous carcinomaswere
not observed by histological examination of hematoxylin-eo-
sin stained sections of tumors (data not shown), for either
the Cox-2luc/þ 129/C57Bl6 or the Cox-2luc/þ HRS/J hairless
mice. A low frequency of papilloma to carcinoma progression
may be the result of a Cox-2 gene dosage effect (Tiano et al.,
2002); Cox-2luc/þ mice are functionally heterozygous at the
Cox-2 locus. One way to overcome this limitation might be to
perform similar skin tumor induction experiments with amu-
rine strain in which the progression of DMBA/TPA-induced
Figure 5 e Bioluminescent in vivo luciferase imaging of a Cox-2luc/þ 129/C
images of Cox-2 promoter-driven luciferase expression in papillomas of a C
Overlaid images with pseudocolor patterns of photon emission are shown in
24 weeks that regresses and is no longer visible at 38 weeks. The orange arr
period.
papillomas to carcinomas is greater than that observed in
the 129/C57Bl6 genetic background (Hennings et al., 1993). Al-
ternatively, mice homozygous for the wild-type Cox-2 gene,
but in which a Cox-2luc transgene (rather than a knock-in al-
lele) is present, might be useful in monitoring Cox-2 gene ex-
pression in the papilloma to carcinoma conversion.
Nevertheless, our results suggest that the Cox-2 expression
level early in the process of tumorigenesis is not a good surro-
gatemarkerwithwhich to predict the fate of papillomas in the
DMBA/TPA multistage tumor induction model.
57Bl6/HRS hairless mouse during DMBA/TPA treatment. Sample
ox-2luc/þ 129/B6/HRS hairless mouse are shown in the upper panel.
the bottom panel. The yellow arrow indicates a papilloma present at
ow indicates a papilloma that is present throughout the 24e40 weeks
M O L E C U L A R O N C O L O G Y 4 ( 2 0 1 0 ) 3 4 7e3 5 6 355
The “initiation” event caused by DMBA in DMBA/TPA skin tu-
mor induction isnearly alwaysanoncogenicH-rasgenemutation
(Quintanilla et al., 1986).A single initiatedcellwithanH-rasmuta-
tion is thought to be “promoted” by the TPA stimulus and, in re-
sponse, to expand into a papilloma. Increased Cox-2 gene
transcriptioninskincancer isproposedtoresult fromanactivated
Ras-MAP kinase cascade, as a result of activated signaling from
the oncogenic H-ras gene (Marks et al., 1998). In the experiments
described here, all morphologically distinguishable tumors ex-
hibit elevated Cox-2 promoter-driven luciferase activity when
compared with adjacent areas of skin. This result is consistent
with the hypothesis that DMBA-induced H-ras mutations cause
the constitutive Cox-2 expression observed in all papillomas.
Elevated Cox-2 promoter-driven luciferase activity is not
detected two weeks before tumors become visible (Figure 3),
although cells with activated H-ras genes must be present.
Our inability to detect these cells may be due to limiting sen-
sitivity of the current in vivo reporter gene imaging system of
the Cox-2luc/þ mouse; we are currently developing more sensi-
tive and cell-type specific non-invasive reporter genes to pur-
sue this question. Alternatively, additional biological events
could be required for elevated Cox-2 expression only at the
time of (or after) a DMBA-initiated, TPA-promoted tumor cell
population becomes visible. In contrast, non-invasive biolu-
minescence from a transgenic luciferase reporter has been ob-
served prior to morphological tumor detection of
subcutaneous NK peripheral lymphomas (Rauch et al., 2009).
We conclude that Cox-2 promoter activity is observed in all
DMBA/TPA-induced skin papillomas, coincident with the abil-
ity to identify themmorphologically, but not before. Although
COX-2 expression is essential for appearance of DMBA/TPA-
induced papillomas (Tiano et al., 2002), and present in these
papillomas (Muller-Decker et al., 1995), the level of Cox-2
gene expression in papillomas is not informative with regard
to their subsequent fate.
Tumor cells often have elevated rates of glucose uptake
that can be measured quantitatively and non-invasively by
positron emission tomography (PET), using 2-deoxy-2 [18F]-
fluoro-D-glucose (18F-FDG). Because DMBA/TPA-induced
skin tumors are easily identified at a very early stage, we
asked whether tumor that will regress, progress to squamous
cell carcinomas, or remain as papillomas can be distin-
guished by microPET/18F-FDG analysis (Ishikawa et al.,
2010). We observed elevated 18F-FDG uptake in all papillomas,
as soon as these tumors are visible. However, 18F-FDG uptake
could not distinguish papillomas destined to regress, to prog-
ress to squamous cell carcinomas, or to remain as papillo-
mas. In conclusion, neither non-invasive monitoring of
glucose metabolism nor non-invasive monitoring of Cox-2
gene expression can predict the fate of DMBA/TPA-induced
skin papillomas.
Acknowledgements
These studieswere supported by NIH grants R01CA123055 and
R01CA084572 to HRH. We thank Arion Chatziioannou and Da-
vid Stout for discussions and for advice in analysis of biolumi-
nescence data, and Art Catapang for technical assistance.
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