![3D"Journal](3D"http://jn.nutrition.org/local/img/journal_logo.gif" "3D\"Journal"){kind=logo}
(Journal =
of=20
Nutrition. 2001;131:3294-3302.)
=C2=A9 2001The American Society =
for=20
Nutritional Sciences
=C2=A9 2001
Articles
Indole-3-Carbinol and Diindolylmethane Induce =
Apoptosis=20
of Human Cervical Cancer Cells and in Murine HPV16-Transgenic =
Preneoplastic=20
Cervical Epithelium1 =
,2 =
Da-Zhi Chen*, Mei Qi*, =
Karen J.=20
Auborn* and Timothy H.=20
Carter*,=E2=80=A03,=E2=80=A0<=
/DIV>
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* ; North Shore-Long Island Jewish Research Institute, = Manhasset, NY 11030 and Department of Otolaryngology, Long Island Jewish = Medical=20 Center, The Long Island Campus of Albert Einstein College of Medicine, = New Hyde=20 Park, NY 11030; and; =E2=80=A0 ; Department of = Biological=20 Sciences, St. John=E2=80=99s University, Jamaica, NY 11439
3To whom correspondence =
should be=20
addressed. E-mail:
![3DBack=20](3D"http://jn.nutrition.org/icons/back.gif"){kind=icon}
ABSTRACT
Dietary indole-3-carbinol (I3C) has clinical benefits for both = cervical=20 cancer and laryngeal papillomatosis, and causes apoptosis of breast = cancer cells=20 in vitro. We asked whether I3C and its major acid-catalyzed condensation = product=20 diindolylmethane (DIM), which is produced in the stomach after = consumption of=20 cruciferous vegetables, could induce apoptosis of cervical cancer cell = lines. We=20 also asked whether this effect could be observed in vivo. In vitro, both = I3C and=20 DIM caused accumulation of DNA strand breaks in three cervical cancer = cell=20 lines. Induction of apoptosis was confirmed by nuclear morphology, = nucleosome=20 leakage, altered cytoplasmic membrane permeability and caspase 3 = activation.=20 Neither I3C nor DIM caused apoptotic changes in normal human = keratinocytes. In=20 C33A cervical cancer cells, DIM was more potent than I3C [dose at which = the=20 number of viable cells was 50% of that in untreated cultures = (LD50) =3D=20 50=E2=80=9360 =C2=B5mol/L for DIM and 200 =C2=B5mol/L = for I3C in a mitochondrial=20 function assay] and faster acting. Furthermore, I3C reduced Bcl-2 = protein in a=20 time- and dose-dependent manner. In HPV16-transgenic mice, which develop = cervical cancer after chronic estradiol exposure, apoptotic cells were = detected=20 in cervical epithelium by TdT-mediated dUTP nick-end labeling staining = and by=20 immunohistochemical staining of active caspase 3 only in mice exposed to = 17=C3=9F-estradiol (E2) and fed I3C. Rare apoptotic cells = were also=20 observed by hematoxylin and eosin staining in the spinous layer of the = cervical=20 epithelium in both control and transgenic mice. Estradiol reduced the = percentage=20 of these late-stage apoptotic cells in the cervical epithelium of = transgenic,=20 E2-treated mice, but this reduction was prevented by I3C. = These data=20 confirm the proapoptotic action of I3C on transformed cells in vitro, = extend the=20 observations to cervical cancer cells and to DIM and show for the first = time=20 that dietary I3C results in increased apoptosis in target tissues in = vivo.=20
KEY WORDS: =E2=80=A2 cervical cancer =E2=80=A2 = indole-3-carbinol =E2=80=A2=20 diindolylmethane =E2=80=A2 apoptosis =E2=80=A2 mice
INTRODUCTION
Indole-3-carbinol (I3C),![3DCitation=20](3D"http://jn.nutrition.org/icons/ref-arrow.gif"){kind=icon}
). A=20
diet rich in cruciferous vegetables (2 ) or=20
supplements of I3C (3 ) causes=20
regression of tumors or decreases their rate of growth or recurrence in =
two=20
thirds of patients with recurrent laryngeal papillomatosis. Clinical =
trials are=20
planned to test the efficacy of I3C as a preventive treatment for breast =
cancer=20
(4
I3C is rapidly converted in the stomach to a variety of condensation=20
products, chiefly diindolylmethane (DIM) (5 ).=20
Plasma from humans and rats fed I3C contains no detectable I3C, but =
large=20
amounts of DIM, as well as other metabolites, some of which remain=20
uncharacterized (6 ; L.=20
Bjeldanes, University of California at Berkeley, personal =
communication). Thus=20
DIM, rather than I3C, is probably the major compound initially available =
to=20
cells after ingestion of I3C. I3C is also converted slowly to DIM at =
neutral pH=20
(5 ,8 ).
We showed previously that dietary I3C prevents the appearance of =
cervical=20
cancer after chronic estrogen exposure in transgenic mice expressing the =
type 16=20
human papillomavirus (HPV) oncogenes (9 ). This=20
effect is accompanied by a shift in estrogen metabolism to favor the =
production=20
of 2-OH estrone rather than the 16=CE=B1-OH metabolite, which is =
associated with=20
prolonged estrogenic activity and carcinogenesis (our unpublished =
data)(10 ,11 ). Thus,=20
it is likely that one of the major pathways by which I3C and its =
derivatives=20
prevent the onset of cervical cancer involves alteration of estrogen =
metabolism=20
by inducing specific cytochrome P450 isoforms via the aryl=20
hydrocarbon receptor (9 ,10 ) for=20
which DIM is a weak ligand (12 ,13 ).
However, we observed that estradiol and I3C act in opposing ways on =
the=20
balance of proliferation of cervical cancer cells (unpublished data), =
and others=20
have reported that the inhibitory effect of I3C on MCF-7 breast cancer =
cells is=20
independent of estrogen signaling (14 ).=20
Therefore, at least in vitro, there must be an additional, =
estrogen-independent=20
pathway by which I3C interferes with the establishment and progression =
of=20
malignancy. Both I3C and DIM induce apoptotic changes in breast cancer =
cells in=20
vitro (7 ).=20
However, the published clinical data on therapeutic use of I3C involve =
laryngeal=20
papillomas and cervical cancer, which are both HPV-associated diseases. =
It is=20
therefore important to determine whether I3C and DIM can induce =
apoptosis in a=20
variety of cervical cancer cell lines, including those with and without =
HPV=20
genes, and whether apoptotic changes occur in target tissues in vivo. We =
looked=20
for apoptotic cells in dysplastic cervical epithelium of HPV16 =
transgenic mice=20
fed I3C under conditions that would otherwise lead to the development of =
cervical neoplasia. Furthermore, we asked whether normal cells in =
culture, as=20
well as normal cervical tissue, are affected by I3C/DIM. Our results =
indicate=20
that the ability of I3C and DIM to induce apoptosis extends to a variety =
of=20
cervical cancer cell lines and to preneoplastic cervical epithelium. =
This effect=20
appears to be specific for transformed cells, raising the possibility =
that these=20
dietary phytochemicals may be generally useful as therapeutic agents. =
MATERIALS AND METHODS
Reagents.
17=C3=9F-Estradiol (E2) and I3C were purchased from Sigma = (St. Louis,=20 MO). Diindolylmethane was a gift from Dr. M. Zeligs, BioResponse, = Boulder, CO.=20
Cell lines and cell culture.
The cervical cancer cell lines CaSki (containing multiple copies of=20
integrated HPV16 DNA) and C33A (HPV negative, mutant p53) were obtained =
from the=20
American Type Culture Collection (ATCC, Manassas, VA). C33AE6 cells =
(15 ) were=20
stably transfected with the HPV16 E6 gene (pLXSN16E6 from D. Galloway, =
Fred=20
Hutchinson Cancer Research Center, Seattle, WA) and express low levels =
of E6=20
transcripts (15 ). All=20
cells were maintained as monolayer cultures at 37=C2=B0C, 7% =
CO2. Cervical=20
cancer cells and 3T3 fibroblasts were grown in Dulbecco=E2=80=99s =
modified Eagle=E2=80=99s=20
medium (DMEM) containing 4.5 g/L glucose and bicarbonate (GIBCO-BRL,=20
Gaithersburg, MD), supplemented with 110 mg/L sodium pyruvate, 200 =
mmol/L=20
glutamine, 100 mL/L fetal bovine serum, and 1 ) as=20
described previously (16 ,17 ).
Mice.
K14-HPV16 transgenic mice were derived and described by Arbeit et al. =
(18 ), and=20
were characterized and maintained by us as described previously (9 ,17 ).=20
Control and experimental groups were as described previously (9 ).=20
Virgin normal and transgenic mice (4=E2=80=935 wk old) were implanted =
subcutaneously=20
with 0.25 mg/d release pellets of E2 and fed diets with or =
without=20
I3C as described below. Implants were repeated every 60 d until the end =
of the=20
study. Mice were housed in groups of 5/cage. All experiments involving =
mice were=20
done in strict adherence to IACUC-approved procedures.
Diet.
Mice consumed ad libitum the AIN76a diet or AIN76a diet (19 )=20
enriched with 0.1 g/kg I3C. Diets were prepared by Ziegler (Gardner, =
PA). The=20
AIN76a diet contains 5% corn oil and supplies a total of 18.5 MJ/kg, =
with 22% of=20
energy from protein, 11% from fat and 67% from carbohydrates.
Cell viability.
C33A Cells were trypsinized, seeded at 104 cells/well in = 96-well=20 plates containing 100 =C2=B5L medium/well and incubated over = night. The next=20 day, the medium was changed to 200 =C2=B5L containing either = DIM or dimethyl=20 sulfoxide (DMSO) as solvent control, employing a minimum of four = replicate wells=20 per condition. Viability was determined at indicated times by a = mitochondrial=20 function assay [reduction of=20 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)= -2H-tetrazolium=20 (MTS)] using the Cell Titer Aqueous One kit (Promega, Madison, WI) = according to=20 the manufacturer=E2=80=99s instructions. Absorbance at 595 nm of the = solution in=20 individual wells was determined with a multiwell plate reader. Data were = analyzed by plotting the mean and SD of=20 cell viability vs. DIM concentration. Protein concentration was measured = with=20 the MicroBCA kit (Pierce, Rockford, IL) using a bovine serum albumin = (BSA)=20 standard.
Nucleosomal leakage apoptosis assay.
Nucleosomal leakage was monitored with a Cell Death Detection=20 ELISAPLUS kit from Roche Molecular Biochemicals (Mannheim, = Germany)=20 that detects histones and DNA in cytoplasmic extracts. Cells were grown = in=20 96-well plates as described above in replicates of 6 wells/condition. = Cells in=20 duplicate wells were lysed and the postnuclear supernatant solution was = analyzed=20 for nucleosomal leakage according to manufacturer=E2=80=99s = instructions. Results were=20 determined by measuring absorbance at 405 nm with a microwell plate = reader as=20 above. The second set of 4 wells from the same plate was analyzed for = viability=20 using the MTS assay by absorbance at 595 nm, and the ELISA results were=20 normalized to this value (A405/A595) to correct = for=20 reduction in viable cell number after treatment with I3C and DIM. The = mean and=20 SD of the normalized data were = plotted=20 vs. DIM concentration.
Western blotting.
Cells treated with I3C, or vehicle controls, each with or without =
estradiol,=20
were lysed at room temperature in buffer containing 10 mmol/L=20
NaH2PO4, 20 g/L triton X-100, 12g/L SDS, 10g/L =
sodium=20
deoxycholate supplemented just before use with 2 =C2=B5mol/L =
aprotinin, 100=20
=C2=B5mol/L phenylmethylsulfonyl fluoride and 0.1 mmol/L EDTA, =
boiled for 2=20
min, and centrifuged for 10 min at 12,000
TdT-mediated dUTP nick-end labeling (TUNEL)=20 assay.
The assay used the in situ cell death detection kit, peroxidase = (POD), from=20 Boehringer Mannheim (Indianapolis, IN). Cells were grown for 24 h in = 8-well=20 chamber slides seeded with 105 cells/well, treated with 200=20 =C2=B5mol/L I3C and incubated at 37=C2=B0C for 48h. The slides = were washed in PBS=20 and fixed with 40 g/L paraformaldehyde for 30 min at room temperature. = Fixed=20 cells were washed in PBS, permeabilized with sodium citrate buffer = containing 1=20 g/L Triton X-100 for 2 min on ice, and then incubated with terminal=20 deoxynucleotidyl transferase for 1 h at 37=C2=B0C. After being rinsed = with PBS,=20 slides were treated with converter-POD (conjugated with HRP) at = 37=C2=B0C for 30 min=20 and mounted with a glass cover slip. At least 200 cells/well were = evaluated for=20 staining.
Assessment of DNA fragmentation by gel=20 electrophoresis.
Cells were grown for 48 h in the presence of various concentrations = of I3C or=20 DIM. After treatment, the cells were harvested, centrifuged at 500 x g for 5 min = and washed=20 with PBS. The cell pellet was lysed in 200 =C2=B5L of lysis = buffer=20 containing 50 mmol/L Tris-HCl, 20 mmol/L EDTA and 10 g/L NP-40, and = centrifuged=20 at 1600 x = g for 5=20 min. The supernatant solution was incubated with (5 mg/L) RNase A for 2 = h at=20 56=C2=B0C and then digested with proteinase K (2.5 mg/L) at 37=C2=B0C = for 16 h. DNA was=20 precipitated with an equal volume of 10 mol/L ammonium acetate and 2.5 = volumes=20 of ethanol. The precipitates were rinsed with 700 g/L ethanol, air = dried,=20 dissolved in Tris/EDTA buffer (10 mmol/L Tris-HCl, pH 7.5, 1 mmol/L = EDTA),=20 electrophoresed through a 1.5% agarose gel, stained with ethidium = bromide and=20 photographed on a UV transilluminator.
Immunohistochemistry.
Tissues were procured, fixed and processed for immunostaining as =
described=20
(9 ).
Cytologic detection of apoptosis.
Two groups of mice (n =3D 50 transgenic and 50 =
nontransgenic) were=20
treated continuously with E2 starting at 4=E2=80=935 wk of =
age. Half of each=20
group was fed a diet containing 2 g/kg I3C and the other half a control =
diet=20
without I3C. At 6 mo, when the majority of E2-treated =
transgenic mice=20
fed the control diet were found to have cervical tumors (9 ), all=20
mice were killed by CO2 inhalation and their cervical tissue =
examined=20
by H&E staining as described (9 ).=20
Samples were coded and the determination of apoptotic cells was carried =
out by a=20
single individual unaware of treatment groups (M.Q.).
Fluorescence staining of nuclei.
C33A cells and normal human keratinocytes growing in monolayer were = fixed in=20 paraformaldehyde as above, followed by 10 =C2=B5g/L=20 4,6-diamidino-2-phenylindole (DAPI) in methanol (Boehringer Mannheim, = Germany)=20 for 30 min at 37=C2=B0C. Stained cells were mounted with Aqueous = Mounting Medium=20 (Biomedia, Loomis, CA) before fluorescence microscopy.
Fluorescence-activated cell sorting = analysis.
To determine altered permeability, C33A cells were treated with or = without=20 200 =C2=B5mol/L I3C for 48 h and trypsinized. After washing = with PBS, the=20 cells were incubated in PBS containing 8 mg/L of 7-amino actinomycin D=20 (7-AAD)-fluorescein isothiocyanate (Sigma) in the dark at 4=C2=B0C for = 20 min and=20 then washed in PBS + 150 g/L BSA + 0.2 g/L NaN3 containing 20 = mg/L of=20 nonfluorescent actinomycin D (AD; Sigma). The resulting cell suspension = was then=20 analyzed cytofluorometrically on a Coulter Elite flow cytometer, set for = single=20 color, ungated fluorescence intensity.
Statistical analysis of data.
Standard deviations were calculated for all quantitative data as = indicated in=20 the figures and figure legends. Significant differences (P < = 0.05)=20 were determined using Student=E2=80=99s t test.
RESULTS
I3C and DIM induce DNA fragmentation in cervical cancer=20 cells.
After treatment with either I3C or DIM, DNA from I3C-treated C33A =
cervical=20
cancer cells accumulated double-strand breaks that generated the =
characteristic=20
apoptotic pattern of nucleosomal "laddering" (Fig.=20
1A , lanes=20
1=E2=80=935), and the effect was dose dependent because an equal amount =
of DNA from=20
cells treated with increasing concentrations of I3C gave progressively =
more=20
intense patterns of nucleosomal repeat-sized DNA fragments. A similar =
apoptotic=20
pattern was obtained when cells were treated with DIM (Fig. =
1B ). In=20
contrast, nontransformed cells such as 3T3 fibroblasts, did not =
accumulate=20
double-strand DNA breaks after DIM treatment (Fig. 1A , lanes=20
6=E2=80=9310).
= View=20 larger version (74K):
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Figure =
1.=20
Size analysis of DNA from indole-3-carbinol (I3C)- and diindolylmethane=20
(DIM)-treated human cervical cancer cells. DNA was extracted from C33A =
cells=20
grown in monolayer culture after exposure to indicated concentrations of =
I3C=20
(panel A) or DIM (panel B) for 48 h and the extracted =
DNA=20
analyzed for DNA fragmentation as described in Methods and Materials. M: =
lane=20
with DNA size markers. (A) I3C concentrations: lanes 1 and 6, 0 =
=C2=B5mol/L; lanes 2 and 7, 50 =C2=B5mol/L; lanes 3 =
and 8, 100=20
=C2=B5mol/L; lanes 4 and 9; 200 =C2=B5mol/L; lanes 5 =
and 10, 300=20
=C2=B5mol/L. (B) DIM concentrations: lane 1, 0 =
=C2=B5mol/L;=20
lane 2, 50 =C2=B5mol/L; lane 3, 100 =C2=B5mol/L.=20
We next used TUNEL to determine the fraction of cells undergoing =
apoptosis.=20
I3C treatment substantially increased the fraction of apoptotic cells in =
each of=20
three cell lines (C33A, C33AE6 and CaSki), from a 100% increase with =
CaSki cells=20
to a >200% increase with C33A (Fig. 2 ).=20
C33AE6, which express theHPV16 E6 oncogene (15 ),=20
showed an intermediate response (Fig. 2) . Thus,=20
it appeared that the induction of DNA strand breaks by I3C was a general =
phenomenon for cervical cancer cell lines and was independent of viral =
gene=20
expression because C33A cells do not express detectable viral gene =
products=20
(20 ), whereas both CaSki and C33AE6 do (15 ,21 ).
= View=20 larger version (34K):
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Figure =
2.=20
TdT-mediated dUTP nick-end labeling (TUNEL) staining of human cervical =
cancer=20
cells exposed to indole-3-carbinol (I3C). C33A, C33A/E6 and CaSki cells =
in=20
monolayer culture were exposed to 200 =C2=B5mol/L I3C for 48 h =
and then=20
fixed and stained for DNA strand breaks by TUNEL as described in Methods =
and=20
Materials. Cells from each culture and/or condition (n =3D =
2000) were=20
examined and the result expressed as a percentage of TUNEL-positive =
cells. Open=20
bars, solvent control. Gray bars, I3C. Values are means =C2=B1 SD, n =3D 3 replicate =
cultures.=20
*Untreated control and I3C-treated cultures differed, P < =
0.002.=20
Cell death induced by I3C in C33A cervical cancer cells = has the=20 characteristics of apoptosis.
If the DNA fragmentation caused by I3C were the result of late-stage=20
apoptosis, then it should be possible to detect early apoptotic changes =
in these=20
cells. We next determined whether I3C could cause the cytoplasmic =
membrane=20
changes characteristic of this process. Extroversion of =
phosphatidylserine=20
during apoptosis (22 ) is=20
accompanied by altered membrane permeability such that a fluorescent =
derivative=20
of AD, namely, 7-AAD, can enter apoptotic cells and bind to DNA in the =
nucleus=20
(23 ,24 ).=20
Fluorescence-activated cell sorting (FACS) analysis of =
7-AAD=E2=80=93stained C33A cells=20
that had been treated with 200 =C2=B5mol/L I3C confirmed that =
the compound=20
increased the percentage of stained cells from 9.8% of the total =
(Fig.=20
3A ) to 55%=20
of the total (Fig. 3B ) after=20
48 h of exposure to I3C.
= View=20 larger version (21K):
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Figure =
3.=20
Increased permeability to 7-amino actinomycin D (7-AAD) in C33A human =
cervical=20
cancer cells exposed to indole-3-carbinol (I3C). Monolayer cultures of =
C33A were=20
exposed to 200 =C2=B5mol/L I3C for 48 h, incubated with 7-AAD =
and analyzed=20
by fluorescence-activated cell sorting (FACS) as described in Methods =
and=20
Materials. (A) Untreated cells; (B) I3C-treated cells. =
Fluorescence intensity is plotted on the abscissa and number of cells on =
the=20
ordinate.=20
DIM is a more effective inducer of apoptosis than=20 I3C.
If the DIM, the dimeric adduct of I3C, were the active molecular =
form, or=20
more proximate to the active form, then DIM would be expected to act =
faster and=20
at a lower concentration than I3C because the latter is converted only =
slowly to=20
DIM in cell culture. Figure 4A shows=20
the effect of increasing concentrations of DIM and I3C on C33A cells =
after a=20
48-h exposure, using a mitochondrial function assay (MTS assay) as an =
indirect=20
measure of cell viability. We observed a reduction in the number of =
viable cells=20
in cultures treated with as little as 40 =C2=B5mol/L DIM, =
whereas I3C had no=20
observable effect at concentrations <100 =C2=B5mol/L (Fig. =
4A ). The=20
dose at which the number of viable cells is 50% of that in untreated =
cultures=20
(LD50) for DIM was =E2=88=BC60 =C2=B5mol/L, compared =
with =E2=88=BC200=20
=C2=B5mol/L for I3C. We next compared the rate of cell killing =
by DIM and=20
I3C; 100 =C2=B5mol/L DIM began to have an observable effect =
between 16 and=20
20 h after addition to growing cells, whereas 300 =C2=B5mol/L =
I3C had no=20
observable effect until =E2=88=BC36 h (Fig. 4B ). To=20
confirm that DIM was in fact inducing apoptosis, the dose-response =
experiment in=20
Figure 4A was=20
repeated, and ELISA was used to detect nucleosomal leakage into the =
cytoplasm.=20
Histones and DNA were detected in the cytoplasm of cells treated with 75 =
=C2=B5mol/L DIM, whereas release in I3C-treated cells required=20
concentrations several fold higher (Fig. 4C ).
= View=20 larger version (14K):
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Figure =
4.=20
Human C33A cell killing and nucleosomal leakage induced by =
indole-3-carbinol=20
(I3C) and diindolylmethane (DIM). Cells were exposed to varying =
concentrations=20
of I3C or DIM for indicated periods of time and the relative number of =
viable=20
cells was determined by measuring absorbance at 595 nm as described in =
Methods=20
and Materials (panels A and B). (A) Dose =
response=20
curves for 48-h treatment; (B) kinetics of cell killing. Values =
are=20
means =C2=B1 SD, n =3D =
4 independent=20
measurements. (C) Cytoplasmic extracts were assayed for DNA and =
histones by ELISA as described in Methods and Materials and expressed as =
absorbance at 405 nm. A duplicate 96-well plate was analyzed for cell =
viability=20
as in panels A and B, and results are expressed in =
panel=20
C as the ratio of absorbance at 405 and 595 nm. MTS,=20
3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)=
-2H-tetrazolium.=20
Two additional hallmarks of apoptotic cell death are nuclear =
condensation and=20
activation of caspases. Figure 5 shows an=20
immunofluorescence photomicrograph of DAPI-stained C33A cells =
(panels A=20
and B) and normal human keratinocytes (panels C and=20
D) treated with DIM. Because DAPI stains only DNA and =
chromatin, the=20
outline of the stained region indicates the relative size and shape of =
the=20
nucleus, as well as the distribution of DNA within it. At concentrations =
as low=20
as 30 =C2=B5mol/L, DIM caused observable changes in the nuclear =
morphology=20
of virtually all of the C33A cells (data not shown); at 60 =
=C2=B5mol/L DIM,=20
condensed, fragmented late-stage apoptotic nuclei were observed in C33A =
cells=20
and virtually all of the nuclei were contracted compared with those in =
untreated=20
cells (panel B, white arrows). At 100 =C2=B5mol/L, few =
C33A cells=20
remained on the cover slip after staining (not shown). In contrast, =
normal=20
keratinocytes did not exhibit these changes even at 100 =
=C2=B5mol/L DIM=20
(compare Fig. 5C and=20
D ).
= View=20 larger version (102K):
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Figure =
5.=20
Changes in nuclear morphology of human C33A cervical cancer cells =
(panels=20
A and B) and normal human keratinocytes (panels C =
and=20
D) treated with diindolylmethane (DIM). Monolayer cultures were =
treated=20
with DIM at 60 =C2=B5mol/L (panel B) and 100 =
=C2=B5mol/L (panel=20
D) for 48h, or treated with solvent alone (panels A and=20
C). The cells were then stained with =
4,6-diamidino-2-phenylindole=20
(DAPI) and photographed with a fluorescence microscope. Arrowheads =
indicate late=20
apoptotic nuclei.=20
= View=20 larger version (143K):
In= a new=20 window
Figure =
6.=20
Caspase 3 activation in human C33A cells after diindolylmethane (DIM) =
treatment.=20
Monolayer cultures were exposed to increasing concentrations of DIM for =
48 h,=20
fixed and immunostained for activated caspase 3. (A) No DIM;=20
(B) 30 =C2=B5mol/L DIM; (C) 60 =
=C2=B5mol/L DIM;=20
(D) 100 =C2=B5mol/L DIM.=20
Bcl-2 is reduced in cells treated with I3C and=20 DIM.
One of the points at which several pathways of apoptotic induction =
converge=20
is the breakdown of mitochondrial membrane integrity. This process is =
thought to=20
be controlled in part by the relative abundance of various members of =
the Bcl-2=20
family of proteins, notably BCL-2 itself and BAX, the former acting as =
an=20
antiapoptotic agent and the latter as an apoptotic inducer [reviewed in =
(15 )]. I3C=20
caused a time- and dose-dependent reduction in the amount of Bcl-2 =
protein=20
detected by Western blot (Fig. 7A and=20
B), whereas BAX was not affected by treatment with 300 =
=C2=B5mol/L=20
I3C for 72 h. DIM reduced the amount of BCL-2 to undetectable levels by =
72 h.=20
= View=20 larger version (39K):
In= a new=20 window
Figure =
7.=20
BCL-2 and BAX levels in indole-3-carbinol (I3C)-treated human C33A =
cells. C33A=20
monolayers were treated with increasing amounts of I3C for 48 h =
(panel=20
A) or with 300 =C2=B5mol/L I3C for different lengths of =
time (panel=20
B). After treatment, 30 =C2=B5g of whole-cell protein was =
analyzed for=20
BAX, BCL-2 and =CE=B1-tubulin content by Western blot.=20
Indole-3-carbinol increases apoptosis in cervical = epithelium of=20 HPV16 transgenic mice.
The observation that I3C and its major condensation product DIM can =
induce=20
apoptotic changes in cervical cancer cells in vitro led us to ask =
whether I3C=20
had the same effect in vivo. Transgenic mice expressing the human HPV E6 =
and E7=20
oncogenes under control of the keratin 14 promoter all develop cervical =
cancer=20
when exposed chronically to estradiol (9 ,18 ), but a=20
diet supplemented with I3C protects nearly all of these mice (9 ). We=20
examined sections of cervical epithelium from normal and transgenic mice =
by=20
H&E staining in a double-blind protocol, scoring for apoptotic cell =
nuclei=20
(Fig. 8A and=20
B ,=20
although the E2-treated mice showed increased cervical =
dysplasia (9 ).=20
Transgenic mice exposed to estradiol, on the other hand, had =
significantly fewer=20
apoptotic cells (Fig. 8C ). When=20
these estradiol-treated transgenic mice were also fed I3C, the number of =
apoptotic cells detected in H&E stained sections returned to normal. =
= View=20 larger version (62K):
In= a new=20 window
Figure =
8.=20
Cytologic detection of apoptosis in cervical epithelium of human =
papillomavirus=20
(HPV)16 transgenic and normal mice. 17=C3=9F-Estradiol =
(E2)-treated mice=20
were fed indole-3-carbinol (I3C) or a diet without I3C. After 6 mo, =
cervical=20
tissue was examined by hematoxylin and eosin staining as described in =
Material=20
and Methods. (A) A representative area of cervical epithelium =
from=20
E2-treated, nontransgenic (control) mice fed I3C. =
(B) An=20
area similar to that in panel A from E2-treated=20
HPV16-transgenic mice fed I3C. Arrows indicate late-stage apoptotic =
cells.=20
(C) Number of late apoptotic cells in a complete epidermal =
layer cross=20
section from each of 5 mice for each condition indicated in the figure=20
(transgenic or nontransgenic, with or without estradiol or I3C). Values =
are=20
means =C2=B1 SD, n =3D =
5. *I3C-treated=20
and untreated, transgenic mice fed I3C differed, P < 0.05.=20
We next used TUNEL as an alternative method to detect apoptotic cells =
in=20
sections of cervical tissue. Figure 9 shows=20
clear evidence of stained cells in cervical epithelium only from =
transgenic mice=20
chronically exposed to estrogen, i.e., those expected to develop =
cervical cancer=20
but also fed I3C. We did not observe TUNEL staining cells in the =
cervical=20
epithelium of normal mice or in transgenic mice exposed to amounts of =
estrogen=20
that induce epithelial hypertrophy and dysplasia.
I= n a new=20 window
Figure =
9.=20
TdT-mediated dUTP nick-end labeling (TUNEL) staining of cervical =
sections from=20
human papillomavirus (HPV)16 transgenic mice fed indole-3-carbinol =
(I3C).=20
Cervical sections from HPV16 transgenic mice treated as in Figure 8 were=20
stained by immunofluorescence for DNA strand breaks. Arrows indicate the =
cervical epithelium. (A) Non-17=C3=9F-estradiol =
(E2)-treated mice=20
fed I3C; (B) E2-treated mice fed I3C.=20
were=20
stained by immunofluorescence for DNA strand breaks. Arrows indicate the =
cervical epithelium. (A) Non-17=C3=9F-estradiol =
(E2)-treated mice=20
fed I3C; (B) E2-treated mice fed I3C.=20
Finally, we asked whether activation of caspase 3 could be detected =
in the=20
cervical epithelium of the transgenic mice fed I3C. Using the=20
immunohistochemical staining method described above for Figure =
6C ,=20
individual cells stained positively for active caspase 3 in the =
suprabasal=20
epithelium of the transgenic mice fed I3C (Fig. =
10B ), but=20
not of placebo-fed controls (Fig. 10D ).
= View=20 larger version (153K):
In= a new=20 window
Figure =
10.=20
Detection of activated caspase 3 in cervical epithelium of human =
papillomavirus=20
(HPV)16 transgenic mice fed indole-3-carbinol (I3C). Cervical sections =
from mice=20
treated as in Figures 8 and 9 were=20
immunostained for active caspase 3. Panels A and B: =
transgenic=20
mouse treated with 17=C3=9F-estradiol (E2) and fed I3C; =
panels C=20
and D: transgenic mouse treated with E2 but not fed =
I3C;=20
panels A and C: low magnification; panels B =
and=20
D: high magnification of areas of cervical epithelium =
delineated by=20
squares in panels A and B. Arrows indicate individual=20
positively stained cells.=20
and 9 were=20
immunostained for active caspase 3. Panels A and B: =
transgenic=20
mouse treated with 17=C3=9F-estradiol (E2) and fed I3C; =
panels C=20
and D: transgenic mouse treated with E2 but not fed =
I3C;=20
panels A and C: low magnification; panels B =
and=20
D: high magnification of areas of cervical epithelium =
delineated by=20
squares in panels A and B. Arrows indicate individual=20
positively stained cells.=20
DISCUSSION
The results described in this paper support the conclusion that both =
I3C and=20
DIM induce apoptotic changes specifically in transformed cervical cells. =
Many of=20
the hallmarks of apoptosis (25 ) are=20
present in I3C- and DIM-treated cells growing in culture, i.e., altered =
nuclear=20
morphology (Fig. 5) , DNA=20
fragmentation (Figs. 1 , 2) and=20
nucleosomal leakage (Fig. 4C) ,=20
changes in membrane permeability (Fig. 3) and=20
activation of caspase 3 (Fig. 6) . The=20
quantitative data from the various assays used to detect cell killing =
and=20
apoptosis were in good agreement. For example, exposure to 300 =
=C2=B5mol/L=20
I3C for 48 h caused 35% of the cells to stain positively for DNA strand =
breaks=20
in the TUNEL assay (a late apoptotic event) and induced membrane =
permeability=20
changes (an early event) in 55%. Similarly, 100 =C2=B5mol/L DIM =
for 48 h=20
killed between 80 and 90% of the cells (Fig. 4A and=20
B . In=20
contrast to cervical cancer cells, normal human foreskin keratinocytes, =
cells=20
that are similar (although clearly not identical) to those in the cervix =
that=20
most commonly undergo malignant transformation, did not exhibit major =
changes in=20
nuclear morphology associated with apoptosis upon exposure to DIM (Fig. =
5 ,=20
panels C and D).
The dose-response curves for I3C and DIM (Fig. 4) are=20
consistent with slow conversion of I3C to DIM in cell culture medium, =
assuming=20
that DIM is the active compound or is further metabolically converted to =
the=20
active compound(s). However, serum levels of DIM in rats fed I3C were =
only =E2=88=BC10%=20
of those required to induce apoptosis in vitro in our experiments (6 ). The=20
fact that relatively high concentrations of DIM (and I3C), pharmacologic =
as=20
opposed to dietary levels, were required to obtain biologic effects in =
vitro is=20
consistent with a requirement for metabolic conversion of DIM to a =
secondary=20
active compound. However, it is now widely recognized that serum levels =
of=20
bioactive compounds are often uninformative because other factors such =
as=20
carrier proteins, intracellular accumulation and localized concentration =
in=20
specific tissues may all come into play. It is therefore not surprising =
that the=20
concentrations of DIM required to produce effects in vitro are higher =
than those=20
normally attainable in vivo. Similar observations have been made for =
other=20
bioactive natural products, for example isoflavones from soy (26 ,27 ). In=20
any case, the fact that dietary I3C appeared to induce apoptosis in the =
cervical=20
epithelium of HPV16 transgenic mice suggests that this compound or its =
active=20
metabolites do indeed reach effective intracellular levels in vivo.
Detection of apoptotic cells in cervical epithelia was undoubtedly=20
facilitated by our experimental model system. In the HPV transgenic =
mouse, just=20
as in human cervical cancer, nearly every cell is expressing HPV =
oncogenes and=20
is thus initiated for transformation. If, as our results suggest, =
transformed=20
cells or cells undergoing preneoplastic conversion are differentially =
sensitive=20
to I3C/DIM, then the enrichment for these cells in cervical epithelium =
as a=20
result of VP6 and VP7 gene expression would explain our ability to =
detect what=20
in other tissues or experimental systems would be a rare event. In =
cervical=20
epithelium from HPV16 mice, chronic exposure to elevated E2 =
is=20
required for expression of the transformed phenotype (9 ,18 ). The=20
observed reduction in late-stage apoptotic cells identifiable by routine =
histopatholgy in samples from HPV16 mice exposed to E2 (Fig.=20
8C )]. By contrast, when E2-treated HPV =
transgenic mice=20
were also fed I3C, we did not observe the E2-related =
reduction in=20
apoptotic cells. This suggests that I3C either prevented transformation =
or=20
killed the cells as they became transformed. Our in vitro data are =
consistent=20
with the latter explanation.
We do not yet know either the mechanism by which these phytochemicals =
induce=20
apoptosis or what determines their apparent specificity for transformed =
cells.=20
Our data and published reports from other laboratories rule out both HPV =
and=20
estrogen effects as requirements for induction of apoptosis by I3C/DIM, =
although=20
both mechanisms may play ancillary roles in specific instances. Apart =
from sex,=20
two differences of potential relevance between cervical cancer cells, =
which are=20
sensitive to DIM, and foreskin keratinocytes, which are not, are the =
presence of=20
HPV oncogenes and estrogen receptors in the former. However C33A cells, =
unlike=20
C33AE6 and CaSki cells, do not express HPV oncogenes or any other viral =
genes at=20
detectable levels (15 );=20
therefore, it is unlikely that viral gene products account for the =
sensitivity=20
of cervical cancer cells to I3C and DIM. The reduced sensitivity of =
C33AE6 cells=20
to killing by I3C compared with the parental C33A cell line (Fig. 2) may have=20
been caused by the reported antiapoptotic effect of E6 (28 =E2=80=9330 ).=20
However, even the protection afforded by expression of this viral =
oncogene was=20
not complete.
Cell killing by DIM or I3C similarly does not seem to require =
estrogen; in=20
fact, I3C and E2 have opposing effects on cell survival. =
Although=20
C33A cells possess estrogen receptors (our unpublished data), and =
estrogen=20
induces proliferation of epithelial cells in the cervix, estradiol =
interferes=20
with induction of apoptosis caused by a number of agents, including I3C =
(our=20
unpublished observations). Conversely, I3C interferes with signaling =
from the=20
estrogen receptor (31 ). The=20
cytotoxic specificity of I3C/DIM for transformed cells also appears to =
be=20
reflected in cervical epithelium in vivo, regardless of whether mice =
were=20
exposed chronically to elevated estrogen (Figs. 8 9 10) . During=20
the course of multiple studies spanning >2 y, we have never seen =
apoptotic=20
cells in cervical epithelium or any evidence of cervical histopathology =
in=20
normal mice fed a diet containing DIM. We conclude that induction of =
apoptosis=20
in cervical cancer cells exposed to I3C or DIM both in vitro and in vivo =
occurs=20
by a pathway separate from, and independent of estrogen-responsive =
mechanisms,=20
and that it is specific for transformed cells.
How does I3C/DIM induce apoptosis of cancer cells? Several =
possibilities are=20
suggested by published work from our laboratory and others. One =
mechanism that=20
could account for the sensitivity of transformed cells is cell cycle =
inhibition.=20
I3C inhibits cdk6 expression in MCF-7 cells (32 ) by=20
interfering with transcription (33 ). Thus=20
it might be that proliferating cells are more sensitive to I3C and DIM =
due to a=20
need for cdk6 activation, which these agents prevent. Another =
possibility=20
involves the induction of proapoptotic genes, for example, via the aryl=20
hydrocarbon receptor (12 ,13 ) or=20
perhaps the sensitization of cells to the cytotoxic effects of cytokines =
or=20
other factors in the cellular microenvironment.
Our results provide further mechanistic underpinning for the = epidemiologic=20 data supporting the efficacy of a diet rich in cruciferous vegetables = for=20 reducing the incidence of cervical cancer. However, this is also one of = the=20 first reports of apoptotic changes induced by chemotherapeutic agents in = vivo,=20 particularly involving solid tumors. The fact that normal keratinocytes = did not=20 show evidence of apoptotic morphologic changes after prolonged exposure = to high=20 DIM concentrations, and that normal cervical epithelium never showed any = indication of apoptosis extensive enough to score positively in TUNEL = staining,=20 suggests that I3C/DIM may have a utility beyond dietary prevention, as a = relatively innocuous chemotherapeutic agent in the treatment of cervical = cancer=20 and, potentially, other malignancies.
FOOTNOTES
1 Presented in part =
at the=20
scientific session of the 18th International Papillomavirus Meeting, =
June 2000,=20
Barcelona, Spain [Chen, D.-Z., Carter, T. H. & Auborn, K. (2000)=20
Indole-3-carbinol and diindolylmethane induce apoptotic changes in =
cervical=20
cancer cells in vitro and in vivo. HPV 2000: 273 (abs.)] and the =
Keystone=20
Symposium on the Molecular Basis of Cancer, January, 2001, Taos, NM =
[Carter, T.=20
H., Chen, D.-Z., Qi, M. & Auborn, K. J. (2001) Indole-3-carbinol and =
diindolylmethane sensitize cancer cells to induction of apoptosis via =
the TNF=CE=B1=20
pathway. p. 122 (abs.)].
2 Supported by =
National=20
Institutes of Health, National Cancer Institute grant CA73385 (to =
K.J.A.).
4 Abbreviations =
used: 7-AAD,=20
7-amino actinomycin D; AD, actinomycin D; BSA, bovine serum albumin; =
DAPI,=20
4,6-diamidino-2-phenylindole; DIM, diindolylmethane; DMEM, =
Dulbecco=E2=80=99s modified=20
Eagle=E2=80=99s medium; DMSO, dimethyl sulfoxide; E2, =
17=C3=9F-estradiol; H&E,=20
hematoxylin and eosin; HPV, human papillomavirus; HRP, horseradish =
peroxidase;=20
I3C, indole-3-carbinol; LD50, dose at which the number of =
viable=20
cells is 50% of that in untreated cultures; MTS,=20
3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)=
-2H-tetrazolium;=20
POD, peroxidase; TUNEL, TdT-mediated dUTP nick-end =
labeling.
Manuscript received June 6, 2001. Initial review completed July 12, = 2001.=20 Revision accepted September 19, 2001.
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$mostli.each(=0A=
function(i) {=0A=
var $hdr =3D $(this).children("h4").html();=0A=
if ($(this).hasClass("most-cur-sel")) {=0A=
hasSelected =3D true;=0A=
hdrList +=3D '
';=0A=
if (hasItems) {=0A=
var header =3D $this.find("div.most-header h3");=0A=
if (header.length) {=0A=
header.after(hdrList);=0A=
}=0A=
}=0A=
mostHdrList =3D $this.find("div.most-header ul");=0A=
}=0A=
// 3) if none of the items are selected, make the first one be=0A=
if (((mostHdrList !=3D null) && mostHdrList.length) && =
(!hasSelected)) {=0A=
modClass(mostHdrList.find("li:first"), "most-cur-sel", "");=0A=
modClass($this.children("ul").children("li:first"), "most-cur-sel", =
"");=0A=
}=0A=
// 4) bind a handler to each heading li a to toggle =
class=3D"most-cur-sel" for =0A=
// that item and its corresponding item in the list below=0A=
if (((mostHdrList !=3D null) && mostHdrList.length)) {=0A=
mostHdrList.find("li a").click(=0A=
function(e) {=0A=
var curA =3D $(this);=0A=
toggleMostSelection(e, $this, curA.text());=0A=
}=0A=
);=0A=
}=0A=
}=0A=
);=0A=
if (hadMostBoxes) {=0A=
fixColHeights(-1);=0A=
}=0A=
}=0A=
=0A=
function toggleMostSelection(e, mostItemDiv, groupName) {=0A=
if (mostItemDiv.length) {=0A=
var hdrItems =3D mostItemDiv.find("div.most-header ul li");=0A=
if (hdrItems.length) {=0A=
hdrItems.each(=0A=
function() {=0A=
var hdrName =3D $(this).text();=0A=
var isChoice =3D (hdrName =3D=3D groupName);=0A=
if (isChoice) {=0A=
modClass($(this), 'most-cur-sel', '');=0A=
}=0A=
else {=0A=
modClass($(this), '', 'most-cur-sel');=0A=
}=0A=
}=0A=
);=0A=
}=0A=
var listItems =3D mostItemDiv.children("ul").find("li");=0A=
if (listItems.length) {=0A=
listItems.each(=0A=
function() {=0A=
var itemName =3D $(this).children("h4").text();=0A=
var isChoice =3D (itemName =3D=3D groupName);=0A=
if (isChoice) {=0A=
modClass($(this), 'most-cur-sel', '');=0A=
}=0A=
else {=0A=
modClass($(this), '', 'most-cur-sel');=0A=
}=0A=
}=0A=
);=0A=
}=0A=
}=0A=
fixColHeights(-1);=0A=
e.preventDefault();=0A=
}=0A=
=0A=
function toggleCollapse(collapsibleEl, hideTagExpr, modTextTagExpr, =
expandStr, contractStr) {=0A=
if (typeof(expandStr) =3D=3D "undefined") {=0A=
expandStr =3D getSiteOption('expandString','+');=0A=
}=0A=
if (typeof(contractStr) =3D=3D "undefined") {=0A=
contractStr =3D getSiteOption('contractString','-');=0A=
}=0A=
=0A=
var $this =3D collapsibleEl;=0A=
if ($this.hasClass('collapsed')) {=0A=
modClass($this, 'collapsible', 'collapsed');=0A=
}=0A=
else if ($this.hasClass('collapsible')) {=0A=
modClass($this, 'collapsed', 'collapsible');=0A=
}=0A=
var $toToggle =3D $this.find(hideTagExpr);=0A=
if ($toToggle.length) {=0A=
$toToggle.toggle();=0A=
}=0A=
var $rewrite =3D $this.find(modTextTagExpr);=0A=
if ($rewrite.length) {=0A=
$rewrite.each(=0A=
function(i, e) {=0A=
var txt =3D $(e).text();=0A=
if (txt =3D=3D expandStr) {=0A=
$(e).empty().append(contractStr);=0A=
}=0A=
else if (txt =3D=3D contractStr) {=0A=
$(e).empty().append(expandStr);=0A=
}=0A=
}=0A=
);=0A=
}=0A=
}=0A=
=0A=
function setupDockBlock(col, dockId, dockClass, contentRules) {=0A=
if ((col =3D=3D 2) || (col =3D=3D 3)) {=0A=
var navOrigHeight =3D ((col =3D=3D 2) ? gColOrigHeights.col2 : =
gColOrigHeights.col3);=0A=
var gColNum =3D col;=0A=
var gNav =3D ((col =3D=3D 2) ? gCol2 : gCol3);=0A=
var gNavHeight =3D ((gNav.length) ? navOrigHeight : 0);=0A=
var gNavTopOffset =3D (gNavHeight > 0 ? gNav.offset().top : 0);=0A=
var gScrollPos =3D 0;=0A=
var gNavDocked =3D false;=0A=
/* this won't work in IE 6, so skip it */=0A=
/* article navigation docking block */=0A=
$(window).scroll(function () {=0A=
if (gNavHeight > 0 && !isIE6) {=0A=
var offsets =3D getPageOffset();=0A=
var origHeight =3D ((gColNum =3D=3D 2) ? gColOrigHeights.col2 : =
gColOrigHeights.col3);=0A=
var navBottom =3D (gNavTopOffset + origHeight) - offsets.y;=0A=
if (gScrollPos !=3D offsets.y) {=0A=
gScrollPos =3D offsets.y;=0A=
if ((navBottom <=3D 0) && !gNavDocked) {=0A=
/* dock the nav */=0A=
gNavDocked =3D true;=0A=
addDockBlock(col, dockId, dockClass, contentRules);=0A=
addDockBlockCallback(dockId);=0A=
}=0A=
else if (gNavDocked && (navBottom > 0)) {=0A=
/* undock the nav */=0A=
gNavDocked =3D false;=0A=
removeDockBlock(col, dockId);=0A=
removeDockBlockCallback(dockId);=0A=
}=0A=
}=0A=
}=0A=
});=0A=
}=0A=
}=0A=
function addDockBlock(col, dockId, dockClass, contentRules) {=0A=
// get direct children li elements only=0A=
var colId =3D ("col-" + col);=0A=
var newDiv =3D ' 1) {=0A=
for (var i =3D 0; i < (contentRules.length - 1); i =3D i + 2) {=0A=
var addTo =3D processDockingRule(contentRules[i]);=0A=
var add =3D processDockingRule(contentRules[i + 1]);=0A=
var addTarget, addVal;=0A=
if (addTo.ruleType =3D=3D 'empty') {=0A=
addTarget =3D newDivJQuery;=0A=
}=0A=
else if (addTo.ruleType =3D=3D 'jQuery') {=0A=
addTarget =3D $(addTo.rule);=0A=
}=0A=
if (add.ruleType =3D 'jQuery') {=0A=
var addJQEl =3D $(add.rule);=0A=
if (addJQEl.length) {=0A=
addVal =3D addJQEl.clone();=0A=
}=0A=
else {=0A=
addVal =3D '';=0A=
}=0A=
}=0A=
else if (add.ruleType =3D 'html') {=0A=
addVal =3D add.rule;=0A=
}=0A=
if (addTarget.length && !(addVal =3D=3D '')) {=0A=
addTarget.append(addVal);=0A=
}=0A=
else {=0A=
}=0A=
}=0A=
}=0A=
var newDockJQrule =3D "#" + colId + " #" + dockId;=0A=
var newDock =3D $(newDockJQrule);=0A=
if (newDock.length) {=0A=
prepCollapsibles(newDockJQrule + " div.collapsible, " + newDockJQrule =
+ " div.collapsed", false);=0A=
newDock.fadeIn(250);=0A=
}=0A=
}=0A=
=0A=
function processDockingRule(rule) {=0A=
var retObj =3D new Object();=0A=
retObj.empty =3D (((rule !=3D undefined) && (!(rule =3D=3D ''))) ? =
false : true);=0A=
if (rule =3D=3D '') {=0A=
retObj.ruleType =3D 'empty';=0A=
}=0A=
else if (/^\$\(.+\)$/.test(rule)) {=0A=
retObj.ruleType =3D 'jQuery';=0A=
retObj.rule =3D rule.replace(/^\$\((.+)\)$/,"$1");=0A=
}=0A=
else {=0A=
retObj.ruleType =3D 'html';=0A=
retObj.rule =3D rule;=0A=
}=0A=
return retObj;=0A=
}=0A=
=0A=
function removeDockBlock(col, dockId) {=0A=
var dockedNav =3D $("div#" + dockId);=0A=
if(dockedNav.length) {=0A=
dockedNav.fadeOut(250, function() { dockedNav.remove(); });=0A=
}=0A=
}=0A=
// custom implementations can override this function to do something =
when a dockBlock is added=0A=
// they should return false if they want to stop any additional default =
handling=0A=
function customAddDockBlockCallback(dockId) {=0A=
return true;=0A=
}=0A=
// this function should not be overridden=0A=
function addDockBlockCallback(dockId) {=0A=
if (customAddDockBlockCallback(dockId) !=3D false) {=0A=
// no default handling, currently=0A=
}=0A=
}=0A=
// custom implementations can override this function to do something =
when a dockBlock is removed=0A=
// they should return false if they want to stop any additional default =
handling=0A=
function customRemoveDockBlockCallback(dockId) {=0A=
return true;=0A=
}=0A=
// this function should not be overridden=0A=
function removeDockBlockCallback(dockId) {=0A=
if (customRemoveDockBlockCallback(dockId) !=3D false) {=0A=
// no default handling, currently=0A=
}=0A=
}=0A=
=0A=
function updateFormInput(labelMatchString, inputMatchString, =
defaultColorString, textColorString) {=0A=
if ((defaultColorString =3D=3D null) || (defaultColorString =3D=3D '')) =
{=0A=
defaultColorString =3D "#A0A0A0";=0A=
}=0A=
if ((textColorString =3D=3D null) || (textColorString =3D=3D '')) {=0A=
textColorString =3D "black";=0A=
}=0A=
var label =3D $(labelMatchString);=0A=
var input =3D $(inputMatchString);=0A=
if (input.length) {=0A=
if ((label.length) && (input.val() =3D=3D '')) {=0A=
input.val(label.text()).css("color",defaultColorString);=0A=
}=0A=
input.focus(=0A=
function(e) {=0A=
if ((label.length) && ($(this).val() =3D=3D label.text())) {=0A=
$(this).val('').css("color",textColorString);=0A=
}=0A=
}=0A=
);=0A=
input.blur(=0A=
function(e) {=0A=
if ((label.length) && ($(this).val() =3D=3D '')) {=0A=
$(this).val(label.text()).css("color",defaultColorString);=0A=
}=0A=
}=0A=
);=0A=
}=0A=
var parentForm =3D label.parents("form").eq(0);=0A=
if (parentForm.length) {=0A=
parentForm.submit(=0A=
function() {=0A=
if ((label.length) && (input.length) && (input.val() =3D=3D =
label.text())) {=0A=
input.val('');=0A=
}=0A=
return true;=0A=
}=0A=
);=0A=
}=0A=
}=0A=
=0A=
function checkUnloadedImgs(lookupRule) {=0A=
if (lookupRule =3D=3D null) {=0A=
lookupRule =3D "img";=0A=
}=0A=
var imageNum =3D -1;=0A=
var images =3D $(lookupRule);=0A=
if (images.length) {=0A=
imageNum =3D 0;=0A=
images.each(=0A=
function() {=0A=
//if ($(this).height() =3D=3D 0) {=0A=
if (this.offsetHeight =3D=3D 0) {=0A=
imageNum++;=0A=
}=0A=
}=0A=
);=0A=
}=0A=
return imageNum;=0A=
}=0A=
=0A=
function fixHeightForImages(iter, numImagesToLoad, lookupRule) {=0A=
if (lookupRule =3D=3D null) {=0A=
lookupRule =3D "img";=0A=
}=0A=
if (numImagesToLoad > 0) {=0A=
var lastNumImagesToLoad =3D numImagesToLoad;=0A=
var newNumImagesToLoad =3D checkUnloadedImgs(lookupRule);=0A=
if (newNumImagesToLoad < lastNumImagesToLoad) {=0A=
fixColHeights(1);=0A=
if ((newNumImagesToLoad > 0) && (iter < 10)) {=0A=
setTimeout("fixHeightForImages(" + (iter+1) + "," + =
newNumImagesToLoad + ",'" + lookupRule + "')", 1000);=0A=
}=0A=
}=0A=
}=0A=
}=0A=
function getColInfo(colJQEl) {=0A=
var colInfo =3D { valid: false, height: 0, bottom: 0, lastElBottom: 0, =
extra: 0, fix: false };=0A=
if (colJQEl.length) {=0A=
var c =3D colJQEl.children();=0A=
if (c.length) {=0A=
colInfo.height =3D getObjHeight(colJQEl);=0A=
colInfo.bottom =3D colJQEl.offset().top + colInfo.height;=0A=
var last =3D c.eq(c.length - 1);=0A=
colInfo.lastElBottom =3D last.offset().top + getObjHeight(last);=0A=
colInfo.extra =3D colInfo.bottom - colInfo.lastElBottom;=0A=
colInfo.valid =3D true;=0A=
}=0A=
}=0A=
return colInfo;=0A=
}=0A=
function checkColHeights() {=0A=
var colToFix =3D 0;=0A=
if ((gIsMultiCol || gColTempResize) && ((gColOrigHeights.main + =
gColOrigHeights.col2 + gColOrigHeights.col3) > 0)) {=0A=
var colMainInfo =3D getColInfo(gColMain);=0A=
var col2Info =3D getColInfo(gCol2);=0A=
var col3Info =3D getColInfo(gCol3);=0A=
if (colMainInfo.valid && (gColLows.main =3D=3D 0)) { gColLows.main =3D =
colMainInfo.lastElBottom; }=0A=
if (col2Info.valid && (gColLows.col2 =3D=3D 0)) { gColLows.col2 =3D =
col2Info.lastElBottom; }=0A=
if (col3Info.valid && (gColLows.col3 =3D=3D 0)) { gColLows.col3 =3D =
col3Info.lastElBottom; }=0A=
// check if low point in any column has changed=0A=
if ((colMainInfo.valid) && (colMainInfo.lastElBottom !=3D =
gColLows.main)) {=0A=
colMainInfo.fix =3D true;=0A=
}=0A=
if ((col2Info.valid) && (col2Info.lastElBottom !=3D gColLows.col2)) {=0A=
col2Info.fix =3D true;=0A=
}=0A=
if ((col3Info.valid) && (col3Info.lastElBottom !=3D gColLows.col3)) {=0A=
col3Info.fix =3D true;=0A=
}=0A=
// check for columns not having same bottom points=0A=
if ((colMainInfo.valid) &&=0A=
(((col2Info.valid) && (colMainInfo.height < col2Info.height)) ||=0A=
((col3Info.valid) && (colMainInfo.height < col3Info.height)))) {=0A=
colMainInfo.fix =3D true;=0A=
}=0A=
if ((col2Info.valid) &&=0A=
(((colMainInfo.valid) && (col2Info.height < colMainInfo.height)) ||=0A=
((col3Info.valid) && (col2Info.height < col3Info.height)))) {=0A=
col2Info.fix =3D true;=0A=
}=0A=
if ((col3Info.valid) &&=0A=
(((colMainInfo.valid) && (col3Info.height < colMainInfo.height)) ||=0A=
((col2Info.valid) && (col3Info.height < col2Info.height)))) {=0A=
col3Info.fix =3D true;=0A=
}=0A=
=0A=
if (colToFix > -1) {=0A=
if (colMainInfo.valid && colMainInfo.fix) {=0A=
if ((col2Info.valid && col2Info.fix) || (col3Info.valid && =
col3Info.fix)) {=0A=
colToFix =3D -1;=0A=
}=0A=
else { colToFix =3D 1; }=0A=
}=0A=
else if (col2Info.valid && col2Info.fix) {=0A=
if (col3Info.valid && col3Info.fix) {=0A=
colToFix =3D -1;=0A=
}=0A=
else { colToFix =3D 2; }=0A=
}=0A=
else if (col3Info.valid && col3Info.fix) {=0A=
colToFix =3D 3;=0A=
}=0A=
}=0A=
=0A=
if (colToFix !=3D 0) {=0A=
fixColHeights(colToFix);=0A=
}=0A=
if (colMainInfo.valid) { gColLows.main =3D colMainInfo.lastElBottom; }=0A=
if (col2Info.valid) { gColLows.col2 =3D col2Info.lastElBottom; }=0A=
if (col3Info.valid) { gColLows.col3 =3D col3Info.lastElBottom; }=0A=
}=0A=
setTimeout("checkColHeights()", 5000);=0A=
}=0A=
function fixColHeights(colChanged) {=0A=
if ((gIsMultiCol || gColTempResize) && getSiteOption('setColSizes', =
true)) {=0A=
var setAll =3D false;=0A=
if (colChanged =3D=3D 1) {=0A=
if (gColMain.length) {=0A=
setObjHeight(gColMain,'auto');=0A=
gColOrigHeights.main =3D getObjHeight(gColMain);=0A=
}=0A=
}=0A=
else if (colChanged =3D=3D 2) {=0A=
if (gCol2.length) {=0A=
setObjHeight(gCol2,'auto');=0A=
gColOrigHeights.col2 =3D getObjHeight(gCol2);=0A=
}=0A=
}=0A=
else if (colChanged =3D=3D 3) {=0A=
if (gCol3.length) {=0A=
setObjHeight(gCol3,'auto');=0A=
gColOrigHeights.col3 =3D getObjHeight(gCol3);=0A=
}=0A=
}=0A=
else {=0A=
if (colChanged =3D=3D -1) {=0A=
// force resize all 3=0A=
if (gColMain.length) { setObjHeight(gColMain,'auto'); }=0A=
if (gCol2.length) { setObjHeight(gCol2,'auto'); }=0A=
if (gCol3.length) { setObjHeight(gCol3,'auto'); }=0A=
}=0A=
gColOrigHeights.main =3D ((gColMain.length) ? =
(getObjHeight(gColMain)) : 0);=0A=
gColOrigHeights.col2 =3D ((gCol2.length) ? (getObjHeight(gCol2)) : 0);=0A=
gColOrigHeights.col3 =3D ((gCol3.length) ? (getObjHeight(gCol3)) : 0);=0A=
setAll =3D true;=0A=
}=0A=
if ((gColOrigHeights.main + gColOrigHeights.col2 + =
gColOrigHeights.col3) > 0) {=0A=
var colInfo;=0A=
var maxH =3D Math.max(Math.max(gColOrigHeights.main, =
gColOrigHeights.col2), gColOrigHeights.col3);=0A=
if ((gColMain.length) && ((gColOrigHeights.main < maxH) || =
(getObjHeight(gColMain) > maxH) || setAll)) {=0A=
setObjHeight(gColMain, maxH);=0A=
colInfo =3D getColInfo(gColMain);=0A=
if (colInfo.valid) { gColLows.main =3D colInfo.lastElBottom; }=0A=
}=0A=
if ((gCol2.length) && ((gColOrigHeights.col2 < maxH) || =
(getObjHeight(gCol2) > maxH) || setAll)) {=0A=
setObjHeight(gCol2, maxH);=0A=
colInfo =3D getColInfo(gCol2);=0A=
if (colInfo.valid) { gColLows.col2 =3D colInfo.lastElBottom; }=0A=
}=0A=
if ((gCol3.length) && ((gColOrigHeights.col3 < maxH) || =
(getObjHeight(gCol3) > maxH) || setAll)) {=0A=
setObjHeight(gCol3, maxH);=0A=
colInfo =3D getColInfo(gCol3);=0A=
if (colInfo.valid) { gColLows.col3 =3D colInfo.lastElBottom; }=0A=
}=0A=
}=0A=
}=0A=
}=0A=
=0A=
function getObjHeight(obj) {=0A=
if (obj instanceof jQuery) {=0A=
return obj.get(0).offsetHeight;=0A=
}=0A=
else if (obj instanceof Array) {=0A=
return obj[0].offsetHeight;=0A=
}=0A=
else {=0A=
return obj.offsetHeight;=0A=
}=0A=
}=0A=
function getObjWidth(obj) {=0A=
if (obj instanceof jQuery) {=0A=
return obj.get(0).offsetWidth;=0A=
}=0A=
else if (obj instanceof Array) {=0A=
return obj[0].offsetWidth;=0A=
}=0A=
else {=0A=
return obj.offsetWidth;=0A=
}=0A=
}=0A=
function setObjHeight(obj, h) {=0A=
if (typeof(h) =3D=3D 'number') {=0A=
h =3D "" + h + "px";=0A=
}=0A=
if (obj instanceof jQuery) {=0A=
obj.get(0).style.height =3D h;=0A=
}=0A=
else if (obj instanceof Array) {=0A=
obj[0].style.height =3D h;=0A=
}=0A=
else {=0A=
obj.style.height =3D h;=0A=
}=0A=
}=0A=
function setObjWidth(obj, w) {=0A=
if (typeof(w) =3D=3D 'number') {=0A=
w =3D "" + w + "px";=0A=
}=0A=
if (obj instanceof jQuery) {=0A=
obj.get(0).style.width =3D w;=0A=
}=0A=
else if (obj instanceof Array) {=0A=
obj[0].style.width =3D w;=0A=
}=0A=
else {=0A=
obj.style.width =3D w;=0A=
}=0A=
}=0A=
=0A=
/* num pixels top of page has been scrolled offscreen */=0A=
function getPageOffset() {=0A=
var offset =3D new Object();=0A=
if( typeof( window.pageXOffset ) =3D=3D 'number' ) {=0A=
offset.x =3D window.pageXOffset;=0A=
offset.y =3D window.pageYOffset;=0A=
} else if( document.body && ( document.body.scrollTop ) ) {=0A=
offset.x =3D document.body.scrollLeft;=0A=
offset.y =3D document.body.scrollTop;=0A=
} else if( document.documentElement && ( =
document.documentElement.scrollTop ) ) {=0A=
offset.x =3D document.documentElement.scrollLeft;=0A=
offset.y =3D document.documentElement.scrollTop;=0A=
}=0A=
if (typeof(offset.x)=3D=3D"undefined") { offset.x =3D 0; }=0A=
if (typeof(offset.y)=3D=3D"undefined") { offset.y =3D 0; }=0A=
return offset;=0A=
}=0A=
function getViewportDim() {=0A=
var dim =3D new Object();=0A=
// non-IE=0A=
if (typeof window.innerWidth !=3D 'undefined') {=0A=
dim.x =3D window.innerWidth;=0A=
dim.y =3D window.innerHeight;=0A=
}=0A=
// IE6 in standards compliant mode=0A=
else if (typeof document.documentElement !=3D 'undefined'=0A=
&& typeof document.documentElement.clientWidth !=3D=0A=
'undefined' && document.documentElement.clientWidth !=3D 0) {=0A=
dim.x =3D document.documentElement.clientWidth;=0A=
dim.y =3D document.documentElement.clientHeight;=0A=
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$.ajax({=0A=
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function addFigHeaders(req) {=0A=
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setTimeout("fixHeightForImages(1" + "," + numImagesToLoad + ",'" + =
lookupRule + "')", 1000);=0A=
fixColHeights(1);=0A=
}=0A=
}=0A=
=0A=
function refLinksNewWindowTarget() {=0A=
$("div.ref-list div.cit-extra a").each(=0A=
function(i) {=0A=
var origTitle =3D $(this).attr("title");=0A=
var newTitle =3D '';=0A=
if ((origTitle =3D=3D undefined) || (!origTitle)) {=0A=
origTitle =3D '';=0A=
}=0A=
else {=0A=
newTitle =3D origTitle + ' ';=0A=
}=0A=
newTitle +=3D '[opens in a new window]';=0A=
$(this).attr("target", "_blank").attr("title", newTitle);=0A=
}=0A=
);=0A=
}=0A=
=0A=
function addRefPops() {=0A=
var numMissed =3D 0;=0A=
var maxToSkip =3D getSiteOption('refPopsMaxToSkip', 10);=0A=
var i =3D 1;=0A=
var idroot =3D "#xref-ref-";=0A=
var el =3D $(idroot + i + '-1');=0A=
/* not all refs appear in text; compensate */=0A=
while (numMissed < maxToSkip) {=0A=
if (el.length) {=0A=
numMissed =3D 0;=0A=
el.hover(dispRef, hideRef);=0A=
if ((getSiteOption("isIosRefPops") =3D=3D true)) {=0A=
//Also set the ios version of the function=0A=
el.hover(iosdispRef, hideRef);=0A=
}=0A=
var j =3D 2;=0A=
var el2 =3D $(idroot + i + "-" + j);=0A=
while (el2.length) {=0A=
el2.hover(dispRef, hideRef);=0A=
if ((getSiteOption("isIosRefPops") =3D=3D true)) {=0A=
//Also set the ios version of the function=0A=
el2.hover(iosdispRef, hideRef);=0A=
}=0A=
j++;=0A=
el2 =3D $(idroot + i + "-" + j);=0A=
}=0A=
}=0A=
else {=0A=
numMissed++;=0A=
}=0A=
i++;=0A=
el =3D $(idroot + i + "-1");=0A=
}=0A=
}=0A=
=0A=
function dispRef(e) {=0A=
var link =3D $(this).attr("href");=0A=
if($("div#hovering-ref").length) {=0A=
$("div#hovering-ref").remove();=0A=
//alert("hovering-ref div removed on new hover!");=0A=
}=0A=
var linkEl =3D $(link);=0A=
if (linkEl.length) {=0A=
var citHtml =3D linkEl.next("div").children("div.cit-metadata");=0A=
if (!(citHtml.length)) {=0A=
citHtml =3D linkEl.parent().next("div").children("div.cit-metadata");=0A=
}=0A=
if (citHtml.length) {=0A=
var newDiv =3D '' + (citHtml.clone().html()) =
+ '';=0A=
$("body").append(newDiv);=0A=
var elH =3D getObjHeight($("div#hovering-ref"));=0A=
if ((getSiteOption("isIosRefPops") =3D=3D true)) {=0A=
$("div#hovering-ref").css("left", 10).css("top", =
e.pageY-elH).css("position", "absolute");=0A=
}=0A=
else {=0A=
$("div#hovering-ref").css("left", e.pageX+10).css("top", =
e.pageY-elH).css("position", "absolute");=0A=
}=0A=
}=0A=
}=0A=
}=0A=
=0A=
function hideRef(e) {=0A=
if($("div#hovering-ref").length) {=0A=
$("div#hovering-ref").remove();=0A=
}=0A=
}=0A=
=0A=
function getHWCiting() {=0A=
var citingA =3D $("#cb-hw-citing-articles");=0A=
if (citingA.length) {=0A=
var newA =3D 'Loading citing =
article data...';=0A=
citingA.replaceWith(newA);=0A=
var href =3D citingA.attr("href");=0A=
var id =3D '';=0A=
if (href && (href.indexOf('?') > 0)) {=0A=
var args =3D href.substring(href.indexOf('?') + 1).split('&');=0A=
for (var i =3D 0; i < args.length; i++) {=0A=
if (args[i].toLowerCase().indexOf('legid=3D') =3D=3D 0) {=0A=
id =3D args[i].substring(args[i].indexOf('=3D') + 1);=0A=
if (id.indexOf('#') > 0) {=0A=
id =3D id.substring(0, id.indexOf('#'));=0A=
}=0A=
}=0A=
}=0A=
if (!(id =3D=3D '')) {=0A=
var host =3D document.location.protocol + "//" + =
document.location.host;=0A=
var ajaxUrl =3D host + '/cited-by/' + id.replace(/;/,'/');=0A=
$.ajax({=0A=
url: ajaxUrl,=0A=
dataType: "html",=0A=
type: "GET",=0A=
error: ajaxErr,=0A=
success: addHWCiting,=0A=
complete: ajaxComplete=0A=
});=0A=
=0A=
}=0A=
}=0A=
}=0A=
}=0A=
function getHWRelatedURLs() {=0A=
var relatedURLsA =3D $("#cb-related-urls");=0A=
var relatedURLsMsg =3D =
getSiteOption('relatedWebPageLoadingText','Loading related web page =
data...'); =0A=
if (relatedURLsA.length) {=0A=
var newA =3D '' + =
relatedURLsMsg + '';=0A=
relatedURLsA.replaceWith(newA);=0A=
var href =3D relatedURLsA.attr("href");=0A=
var id =3D '';=0A=
if (href && (href.indexOf('?') > 0)) {=0A=
var args =3D href.substring(href.indexOf('?') + 1).split('&');=0A=
for (var i =3D 0; i < args.length; i++) {=0A=
if (args[i].toLowerCase().indexOf('legid=3D') =3D=3D 0) {=0A=
id =3D args[i].substring(args[i].indexOf('=3D') + 1);=0A=
if (id.indexOf('#') > 0) {=0A=
id =3D id.substring(0, id.indexOf('#'));=0A=
}=0A=
}=0A=
}=0A=
if (!(id =3D=3D '')) {=0A=
var host =3D document.location.protocol + "//" + =
document.location.host;=0A=
var ajaxUrl =3D host + '/related-web-pages/' + id.replace(/;/,'/');=0A=
$.ajax({=0A=
url: ajaxUrl,=0A=
dataType: "html",=0A=
type: "GET",=0A=
error: ajaxErr,=0A=
success: addRelatedURLs,=0A=
complete: ajaxComplete=0A=
});=0A=
=0A=
}=0A=
}=0A=
}=0A=
}=0A=
function getPatientInformData() {=0A=
var pInformA =3D $("#cb-patientinform");=0A=
if (pInformA.length) {=0A=
var newA =3D 'Loading =
patientINFORMation...';=0A=
pInformA.replaceWith(newA);=0A=
var href =3D pInformA.attr("href");=0A=
var id =3D '';=0A=
if (href && (href.indexOf('?') > 0)) {=0A=
var args =3D href.substring(href.indexOf('?') + 1).split('&');=0A=
for (var i =3D 0; i < args.length; i++) {=0A=
if (args[i].toLowerCase().indexOf('legid=3D') =3D=3D 0) {=0A=
id =3D args[i].substring(args[i].indexOf('=3D') + 1);=0A=
if (id.indexOf('#') > 0) {=0A=
id =3D id.substring(0, id.indexOf('#'));=0A=
}=0A=
}=0A=
}=0A=
if (!(id =3D=3D '')) {=0A=
var host =3D document.location.protocol + "//" + =
document.location.host;=0A=
var ajaxUrl =3D host + '/related-web-pages/patientinform/' + =
id.replace(/;/,'/');=0A=
$.ajax({=0A=
url: ajaxUrl,=0A=
dataType: "html",=0A=
type: "GET",=0A=
error: ajaxErr,=0A=
success: addPatientInform,=0A=
complete: ajaxComplete=0A=
});=0A=
=0A=
}=0A=
}=0A=
}=0A=
}=0A=
function getISIRelated() {=0A=
var relatedA =3D $("#cb-isi-similar-articles");=0A=
if (relatedA.length) {=0A=
var newA =3D 'Loading Web =
of Science article data...';=0A=
relatedA.replaceWith(newA);=0A=
var href =3D relatedA.attr("href");=0A=
var id =3D '';=0A=
if (href) {=0A=
var hrefDec =3D decodeURI(href);=0A=
if ((hrefDec.indexOf('?') > 0)) {=0A=
var args =3D hrefDec.substring(hrefDec.indexOf('?') + 1).split('&');=0A=
for (var i =3D 0; i < args.length; i++) {=0A=
var argDec =3D decodeURIComponent(args[i]);=0A=
if (argDec.toLowerCase().indexOf('access_num=3D') =3D=3D 0) {=0A=
id =3D argDec.substring(argDec.indexOf('=3D') + 1);=0A=
if (id.indexOf('#') > 0) {=0A=
id =3D id.substring(0, id.indexOf('#'));=0A=
}=0A=
}=0A=
}=0A=
if (!(id =3D=3D '')) {=0A=
var host =3D document.location.protocol + "//" + =
document.location.host;=0A=
var ajaxUrl =3D host + '/isi-links/has-related/' + =
id.replace(/;/,'/');=0A=
$.ajax({=0A=
url: ajaxUrl,=0A=
dataType: "html",=0A=
type: "GET",=0A=
error: ajaxErr,=0A=
success: addISIRelated,=0A=
complete: ajaxComplete=0A=
});=0A=
}=0A=
}=0A=
}=0A=
}=0A=
}=0A=
function getCiting(service, msg, pathseg, successFn, addlParamString) {=0A=
if (typeof(addlParamString) =3D=3D "undefined") {=0A=
addlParamString =3D '';=0A=
}=0A=
var citingA =3D $("#cb-" + service + "-citing-articles");=0A=
if (citingA.length) {=0A=
var newA =3D '' =
+ msg + '';=0A=
citingA.replaceWith(newA);=0A=
var href =3D citingA.attr("href");=0A=
var id =3D '';=0A=
if (href) {=0A=
var hrefDec =3D decodeURI(href);=0A=
if ((hrefDec.indexOf('?') > 0)) {=0A=
var args =3D hrefDec.substring(hrefDec.indexOf('?') + 1).split('&');=0A=
for (var i =3D 0; i < args.length; i++) {=0A=
var argDec =3D decodeURIComponent(args[i]);=0A=
if (argDec.toLowerCase().indexOf('access_num=3D') =3D=3D 0) {=0A=
id =3D argDec.substring(argDec.indexOf('=3D') + 1);=0A=
if (id.indexOf('#') > 0) {=0A=
id =3D id.substring(0, id.indexOf('#'));=0A=
}=0A=
}=0A=
}=0A=
if (!(id =3D=3D '')) {=0A=
var host =3D document.location.protocol + "//" + =
document.location.host;=0A=
var ajaxUrl =3D host + '/' + service + '-links/' + pathseg + =
id.replace(/;/,'/') + (addlParamString !=3D '' ? '?' + addlParamString : =
'');=0A=
$.ajax({=0A=
url: ajaxUrl,=0A=
dataType: "html",=0A=
type: "GET",=0A=
error: ajaxErr,=0A=
success: successFn,=0A=
complete: ajaxComplete=0A=
});=0A=
}=0A=
}=0A=
}=0A=
}=0A=
}=0A=
=0A=
function getISICiting() {=0A=
var citingA =3D $("#cb-isi-citing-articles");=0A=
if (citingA.length) {=0A=
var newA =3D 'Loading Web of =
Science citing article data...';=0A=
citingA.replaceWith(newA);=0A=
var href =3D citingA.attr("href");=0A=
var id =3D '';=0A=
if (href) {=0A=
var hrefDec =3D decodeURI(href);=0A=
if ((hrefDec.indexOf('?') > 0)) {=0A=
var args =3D hrefDec.substring(hrefDec.indexOf('?') + 1).split('&');=0A=
for (var i =3D 0; i < args.length; i++) {=0A=
var argDec =3D decodeURIComponent(args[i]);=0A=
if (argDec.toLowerCase().indexOf('access_num=3D') =3D=3D 0) {=0A=
id =3D argDec.substring(argDec.indexOf('=3D') + 1);=0A=
if (id.indexOf('#') > 0) {=0A=
id =3D id.substring(0, id.indexOf('#'));=0A=
}=0A=
}=0A=
}=0A=
if (!(id =3D=3D '')) {=0A=
var host =3D document.location.protocol + "//" + =
document.location.host;=0A=
var ajaxUrl =3D host + '/isi-links/' + id.replace(/;/,'/');=0A=
$.ajax({=0A=
url: ajaxUrl,=0A=
dataType: "html",=0A=
type: "GET",=0A=
error: ajaxErr,=0A=
success: addISICiting,=0A=
complete: ajaxComplete=0A=
});=0A=
}=0A=
}=0A=
}=0A=
}=0A=
}=0A=
function getEntrezLinks() {=0A=
var entrezDiv =3D $("#cb-entrez-links-placeholder");=0A=
if (entrezDiv.length) {=0A=
var entrezA =3D entrezDiv.children("a");=0A=
if (entrezA) {=0A=
var host =3D document.location.protocol + "//" + =
document.location.host;=0A=
var ajaxUrl =3D host + entrezA.attr("href");=0A=
$.ajax({=0A=
url: ajaxUrl,=0A=
dataType: "html",=0A=
type: "GET",=0A=
error: ajaxErr,=0A=
success: addEntrezLinks,=0A=
complete: ajaxComplete=0A=
});=0A=
}=0A=
}=0A=
}=0A=
=0A=
function ajaxErr(req, msg, e) {=0A=
}=0A=
function ajaxComplete(req, msg) {=0A=
}=0A=
function updateCBItem(cbItem, newHTML, hasData) {=0A=
var parentItem =3D cbItem.parents("li").eq(0);=0A=
cbItem.replaceWith(newHTML);=0A=
if (!hasData) {=0A=
// hide the parent li=0A=
if (parentItem.length) {=0A=
modClass(parentItem,"nodata","");=0A=
// check if there are any siblings still being displayed=0A=
var otherItems =3D parentItem.siblings();=0A=
var allItemsEmpty;=0A=
if (otherItems.length) {=0A=
if (otherItems.length =3D=3D otherItems.filter(".nodata").length) {=0A=
allItemsEmpty =3D true=0A=
}=0A=
else {=0A=
allItemsEmpty =3D false;=0A=
}=0A=
}=0A=
else {=0A=
allItemsEmpty =3D true;=0A=
}=0A=
if (allItemsEmpty) {=0A=
var cbsection =3D parentItem.parents("div.cb-section").eq(0);=0A=
if (cbsection.length) {=0A=
modClass(cbsection,"nodata","");=0A=
}=0A=
// do we need to look further?=0A=
if (parentItem.parents("div.cb-section").length > 1) {=0A=
var cbSectionSibs =3D cbsection.siblings("div.cb-section");=0A=
if (cbSectionSibs.length) {=0A=
if (cbSectionSibs.length =3D=3D =
cbSectionSibs.filter(".nodata").length) {=0A=
allItemsEmpty =3D true=0A=
}=0A=
else {=0A=
allItemsEmpty =3D false;=0A=
}=0A=
}=0A=
else {=0A=
allItemsEmpty =3D true;=0A=
}=0A=
if (allItemsEmpty) {=0A=
var cbgrandsection =3D parentItem.parents("div.cb-section").eq(1);=0A=
if (cbgrandsection.length) {=0A=
modClass(cbgrandsection,"nodata","");=0A=
}=0A=
}=0A=
}=0A=
}=0A=
}=0A=
}=0A=
// in frameset fix targets on child links, forms=0A=
fixFrameLinks(parentItem.find("a,form"));=0A=
fixColHeights(2);=0A=
}=0A=
function fixFrameLinks(jqItems) {=0A=
if ((gIsFrameset !=3D null) && gIsFrameset) {=0A=
jqItems.each(=0A=
function(i) {=0A=
var href =3D $(this).attr("href");=0A=
var action =3D $(this).attr("action"); // if form=0A=
if ((href !=3D null) || (action !=3D null)) {=0A=
var inFrameAnchor =3D ((href !=3D null) && =
(((/frameset=3D/.test(href)) && (/#/.test(href))) || =
(href.substring(0,1) =3D=3D '#')));=0A=
if ((!inFrameAnchor) || (action !=3D null)) {=0A=
if ((navigator.userAgent.indexOf("Firefox") >=3D 0) && =
($(this).hasClass("pdf-direct-link"))) {=0A=
$(this).attr("target", "_blank");=0A=
} else {=0A=
$(this).attr("target", "_top");=0A=
}=0A=
}=0A=
}=0A=
}=0A=
);=0A=
}=0A=
}=0A=
function addRelatedURLs(xhtmlData) {=0A=
var cbA =3D $("#cb-loading-related-urls");=0A=
if (gIsFrameset) {=0A=
if (cbA.length) {=0A=
updateCBItem(cbA, '', false);=0A=
}=0A=
}=0A=
else if (xhtmlData && !(xhtmlData.indexOf('=3D 0)) {=0A=
$("#related-urls").replaceWith(xhtmlData);=0A=
var relatedWebPagesLabel =3D getSiteOption('relatedWebPagesLabel', =
'Related Web Pages');=0A=
fixColHeights(1);=0A=
if (cbA.length) {=0A=
updateCBItem(cbA, '' + relatedWebPagesLabel =
+ '', true);=0A=
}=0A=
}=0A=
else {=0A=
if (cbA.length) {=0A=
updateCBItem(cbA, '', false);=0A=
}=0A=
}=0A=
}=0A=
function addPatientInform(xhtmlData) {=0A=
var cbA =3D $("#cb-loading-patientinform");=0A=
if (gIsFrameset) {=0A=
if (cbA.length) {=0A=
updateCBItem(cbA, 'Not =
available in this view', false);=0A=
}=0A=
}=0A=
else if (xhtmlData && !(xhtmlData.indexOf('=3D 0)) {=0A=
$("#patientinform-links").replaceWith(xhtmlData);=0A=
fixColHeights(1);=0A=
if (cbA.length) {=0A=
updateCBItem(cbA, 'patientINFORMation', true);=0A=
}=0A=
}=0A=
else {=0A=
if (cbA.length) {=0A=
updateCBItem(cbA, 'No =
patientINFORMation available for this article', false);=0A=
}=0A=
}=0A=
}=0A=
function addHWCiting(xhtmlData) {=0A=
var cbA =3D $("#cb-loading-hw-cited");=0A=
if (gIsFrameset) {=0A=
if (cbA.length) {=0A=
updateCBItem(cbA, 'Not available =
in this view', false);=0A=
}=0A=
}=0A=
else if (xhtmlData) {=0A=
$("#content-block").append(xhtmlData);=0A=
var hwCitingLabel =3D getSiteOption('hwCitingLabel', 'View citing =
article information');=0A=
fixColHeights(1);=0A=
if (cbA.length) {=0A=
if (!(getSiteOption("includeHWCitingTitle") =3D=3D true)) {=0A=
updateCBItem(cbA, '' + =
hwCitingLabel + '', true);=0A=
}=0A=
else {=0A=
updateCBItem(cbA, '' + =
hwCitingLabel + '', true);=0A=
}=0A=
}=0A=
}=0A=
else {=0A=
if (cbA.length) {=0A=
updateCBItem(cbA, 'No citing =
articles', false);=0A=
}=0A=
}=0A=
}=0A=
function addISIRelated(xhtmlData) {=0A=
var cbA =3D $("#cb-isi-similar-articles");=0A=
if (xhtmlData && !(xhtmlData.indexOf('=3D 0)) {=0A=
if (cbA.length) {=0A=
updateCBItem(cbA, xhtmlData, true);=0A=
}=0A=
}=0A=
else {=0A=
if (cbA.length) {=0A=
updateCBItem(cbA, '', false);=0A=
}=0A=
}=0A=
}=0A=
function addISICiting(xhtmlData) {=0A=
var cbA =3D $("#cb-loading-isi-cited");=0A=
if (xhtmlData && !(xhtmlData.indexOf('=3D 0)) {=0A=
if (cbA.length) {=0A=
updateCBItem(cbA, xhtmlData, true);=0A=
}=0A=
}=0A=
else {=0A=
if (cbA.length) {=0A=
updateCBItem(cbA, 'No Web of =
Science citing articles', false);=0A=
}=0A=
}=0A=
}=0A=
function addScopusCiting(xhtmlData) {=0A=
var cbA =3D $("#cb-loading-scopus-cited");=0A=
if (xhtmlData && (xhtmlData.indexOf('=3D 0)) {=0A=
if (cbA.length) {=0A=
updateCBItem(cbA, xhtmlData, true);=0A=
}=0A=
}=0A=
else {=0A=
if (cbA.length) {=0A=
updateCBItem(cbA, 'No Scopus =
citing articles', false);=0A=
}=0A=
}=0A=
}=0A=
function addEntrezLinks(xhtmlData) {=0A=
var entrezDiv =3D $("#cb-entrez-links-placeholder");=0A=
=0A=
if (xhtmlData && (xhtmlData.indexOf('=3D 0)) {=0A=
if (entrezDiv.length) {=0A=
updateCBItem(entrezDiv, xhtmlData, true);=0A=
}=0A=
}=0A=
else {=0A=
if (entrezDiv.length) {=0A=
updateCBItem(entrezDiv, 'No NCBI =
links', false);=0A=
}=0A=
}=0A=
}=0A=
=0A=
function updateSBLinks() {=0A=
var fbLink =3D $("a.sb-facebook");=0A=
if (fbLink.length) {=0A=
fbLink.click(=0A=
function(e) {=0A=
window.open(this.href, 'sharer', =
'toolbar=3D0,status=3D0,width=3D626,height=3D436');=0A=
e.preventDefault();=0A=
}=0A=
);=0A=
}=0A=
}=0A=
=0A=
function addDockedNav() {=0A=
var slugEl =3D $("#col-2 #slugline");=0A=
// get direct children li elements only=0A=
var artViews =3D $("#article-cb-main .cb-section ol:has(li =
a[rel^=3D'view-']) > li").clone();=0A=
var newDiv =3D '';=0A=
$("#col-2").append(newDiv);=0A=
$("#col-2 #docked-nav").hide();=0A=
var newDivJQuery =3D $("#docked-nav");=0A=
newDivJQuery.append('This Article
');=0A=
$("#col-2 #docked-nav-views").append(artViews); /*.append(artSupp);*/=0A=
$("#col-2 #docked-nav #docked-slug li").append(slugEl.clone());=0A=
newDivJQuery.append($("#article-dyn-nav").clone());=0A=
$("#col-2 #docked-nav").fadeIn(250);=0A=
}=0A=
=0A=
=0A=
function removeDockedNav() {=0A=
var dockedNav =3D $("div#docked-nav");=0A=
if(dockedNav.length) {=0A=
dockedNav.fadeOut(250, function() { dockedNav.remove(); });=0A=
}=0A=
}=0A=
=0A=
function fixWackyReflinksMarkup() {=0A=
// There's whitespace between the
This Article
' + expandStr + ' Author Affiliations
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var ajaxUrl =3D host + '/cited-by/' + id.replace(/;/,'/');=0A=
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var citingA =3D $("#cb-isi-citing-articles");=0A=
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var host =3D document.location.protocol + "//" + =
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var ajaxUrl =3D host + '/isi-links/' + id.replace(/;/,'/');=0A=
$.ajax({=0A=
url: ajaxUrl,=0A=
dataType: "html",=0A=
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