From: Subject: Persistence of a small subpopulation of cancer stem-like cells in the C6 glioma cell line -- Kondo et al. 101 (3): 781 -- Proceedings of the National Academy of Sciences Date: Fri, 17 Nov 2006 15:06:37 +0100 MIME-Version: 1.0 Content-Type: multipart/related; type="text/html"; boundary="----=_NextPart_000_0002_01C70A59.FAEAAFC0" X-MimeOLE: Produced By Microsoft MimeOLE V6.00.2900.2962 This is a multi-part message in MIME format. ------=_NextPart_000_0002_01C70A59.FAEAAFC0 Content-Type: text/html; charset="iso-8859-1" Content-Transfer-Encoding: quoted-printable Content-Location: http://www.pnas.org/cgi/content/full/101/3/781 Persistence of a small subpopulation of cancer = stem-like cells in the C6 glioma cell line -- Kondo et al. 101 (3): 781 = -- Proceedings of the National Academy of Sciences
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Published online before print January = 7, 2004,=20 10.1073/pnas.0307618100
PNAS | = January 20,=20 2004 | vol. 101 | no. 3 | 781-786


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DEVELOPMENTAL=20 BIOLOGY
Persistence of a = small=20 subpopulation of cancer stem-like cells in the C6 glioma cell line=20

Toru Kondo * 3D{dagger}=20 3D{ddagger}=20 3D=A7=20,=20 Takao Setoguchi 3D{dagger}=20 3D{ddagger}=20 =B6, and Tetsuya=20 Taga *

*Department of Cell Fate Modulation, Institute of = Molecular=20 Embryology and Genetics, Kumamoto University, 2-2-1 Honjo, Kumamoto = 860-0811,=20 Japan;3D{dagger}Centre for Brain Repair, University of Cambridge, E. D. = Adrian=20 Building, Forvie Site, Robinson Way, Cambridge CB2 2PY, United Kingdom; = and=20 =B6Department of Orthopaedic Surgery, Kagoshima Graduate = School of=20 Medicine and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8520, = Japan=20

Communicated by Martin C. Raff, University College London, London, = United=20 Kingdom, November 20, 2003 (received for review September 1, 2003)


3D"   =20 Abstract
3D"Top
3D"Abstract
3D"Materials and Methods
3D"Results
3D"Discussion
3D"References
 
B= oth stem=20 cells and cancer cells are thought to be capable of unlimited = proliferation. Paradoxically, however, some cancers seem to = contain=20 stem-like cells (cancer stem cells). To help resolve this = paradox, we=20 investigated whether established malignant cell lines, which = have=20 been maintained for years in culture, contain a subpopulation = of stem=20 cells. In this article, we show that many cancer cell lines = contain a=20 small side population (SP), which, in many normal tissues, is = thought=20 to contain the stem cells of the tissue. We demonstrate that = in the=20 absence of serum the combination of basic fibroblast growth = factor=20 and platelet-derived growth factor maintains SP cells in the = C6=20 glioma cell line. Moreover, we show that C6 SP cells, but not = non-SP cells, can generate both SP and non-SP cells in = culture=20 and are largely responsible for the in vivo malignancy of = this=20 cell line. Finally, we provide evidence that C6 SP cells can=20 produce both neurons and glial cells in vitro and in = vivo.=20 We propose that many cancer cell lines contain a minor=20 subpopulation of stem cells that is enriched in an SP, can be = maintained indefinitely in culture, and is crucial for their=20 malignancy.

Like stem cells, cancer cells are widely thought to be able = to=20 proliferate indefinitely, and yet it is notoriously difficult = to=20 establish immortal cell lines in culture from primary cancers. = When=20 such cell lines have been established, however, they retain = the=20 ability to reform the original cancer in vivo. One = possibility=20 is that cancers arise in cells with the characteristics of = stem=20 cells, which, by definition, can self-renew indefinitely. = Another=20 possibility is that cancer cells acquire the property of = unlimited=20 proliferation by mutation, which might be essential for = malignancy,=20 as well as for primary human cells in culture to pass through = the Hayflick limit (1).=

There is increasing evidence that cancers might contain their = own=20 stem cells. Many cancers, like normal organs, seem to be = maintained=20 by a hierarchical organization that includes slowly dividing = stem=20 cells, rapidly dividing transit amplifying cells (precursor = cells),=20 and differentiated cells (2=966).=20 Malignant gliomas, for example, often contain both = undifferentiated=20 and differentiated cells and sometimes contain cells that=20 express neuronal markers as well as cells that express glial=20 markers, suggesting that they may contain multipotent neural = stem=20 cell (NSC)-like cells (7=969). = Such mixed=20 gliomas may develop from NSCs or from more restricted glial = lineage=20 cells that acquire multipotential stem-cell-like properties = by=20 mutation (10,=20 11).=20 Yet, even normal oligodendrocyte precursor cells can be = induced=20 by extracellular signals in vitro to acquire = stem-cell-like=20 properties, and some NSCs in vivo express the astrocyte = marker=20 glial fibrillary acidic protein (GFAP) (12=9617).

The presence of a small subpopulation of slowly dividing cancer=20 stem cells might explain why so many cancers recur after = treatment=20 with irradiation or cytotoxic drugs, even when most of the = cancer=20 cells seem to be killed by the therapy. Usually, some cancer=20 cells survive the treatment, and these surviving cells may be = cancer stem cells, which may be not only resistant to the = therapy=20 but also essential for the malignancy of the cancer. It has=20 been shown that various types of ATP-binding cassette (ABC)=20 transporters, including those encoded by the = multidrug-resistant=20 (MDR) gene 1, the MDR protein (MRP), and the breast = cancer-resistant=20 protein 1 (BCRP1), contribute to drug resistance in many = cancers=20 by pumping the drugs out of the cell (18).=20 Interestingly, some of these transporters are expressed also = by many=20 kinds of stem cells. BCRP1, for example, pumps out the = fluorescent=20 dye Hoechst 33342, identifying an unlabeled side population = (SP),=20 which is enriched for stem cells (19=9622). = Taken=20 together, these findings suggest that cancers might contain = an SP=20 with the characteristics of stem cells.

Here we show that many established cancer cell lines contain = SP=20 cells, which are apparently maintained in normal serum-containing=20 cultures over decades. We demonstrate that platelet-derived=20 growth factor (PDGF) and basic fibroblast growth factor = (bFGF)=20 can maintain the SP cells in the C6 glioma cell line in the=20 absence of serum and that the SP cells can produce both = neuronal=20 and glial cells. Finally, we show that FACS-sorted C6 SP = cells,=20 but not non-SP C6 cells, can produce both SP and non-SP cells = in culture and form tumors in multiple tissues in nude mice,=20 which contain both neurons and glia, indicating that these = cells=20 have the characteristics of multipotent cancer stem cells. = Our=20 findings suggest that the SP may be a general source of = cancer=20 stem cells, which need to be targeted for effective cancer = therapy.=20


3D"   =20 Materials and Methods
3D"Top
3D"Abstract
3D"Materials and Methods
3D"Results
3D"Discussion
3D"References
 
<= B>Chemicals.=20 Chemicals were purchased from Sigma unless indicated = otherwise.=20 Recombinant cytokines were purchased from PeproTech (Rocky = Hill, NJ)=20 unless indicated otherwise.

Cell Culture. Various cancer cell lines were studied, = including=20 the rat glioma line C6, the human breast cancer line MCF-7, = the=20 human osteosarcoma lines U-20S and SaOS-2, the rat neuroblastoma = line=20 B104, and the human adenocarcinoma line HeLa. The cells were = cultured=20 in DMEM, supplemented with 10% FCS, 100 units/ml penicillin = G, and=20 100 =B5g/ml streptomycin (GIBCO). In some experiments, C6 = cells were=20 cultured in serum-free DMEM containing 10 =B5g/ml bovine = insulin, 100=20 =B5g/ml human transferrin, 100 =B5g/ml BSA, 60 ng/ml = progesterone, 16=20 =B5g/ml putrescine, 40 ng/ml sodium selenite, 63 =B5g/ml=20 N-acetylcysteine, 5 =B5M forskolin, 50 units/ml = penicillin, and=20 50 =B5g/ml streptomycin (GIBCO), as well as 10 ng/ml bFGF, 10 = ng/ml=20 PDGF, or both. In all experiments, cells were maintained in=20 100-mm culture dishes (Nunc) at 37=B0C in a humidified 5%=20 CO2/95% air atmosphere.

Flow Cytometry. To identify and isolate the SP cells in = the=20 cancer cell lines, we cultured the lines, as described above, = in either FCS or serum-free culture medium with bFGF, PDGF, = or=20 both. The cells were removed from the culture dish with trypsin = and=20 EDTA (GIBCO BRL), washed, suspended at 106 cells per ml = in=20 DMEM containing 2% FCS (staining medium), and preincubated in = a=20 1.5-ml Eppendorf tube at 37=B0C for 10 min. The cells were = then labeled=20 in the same medium at 37=B0C for 90 min with 2.5 =B5g/ml = Hoechst 33342=20 dye (Molecular Probes), either alone or in combination with = 50 =B5M=20 verapamil (Sigma), which is an inhibitor of some=20 (verapamil-sensitive) ABC transporters (19). = Finally,=20 the cells were counterstained with 1 =B5g/ml propidium iodide = to label=20 dead cells.

Then, 3=965 x 104 = cells were=20 analyzed in a FACSVantage fluorescence-activated cell sorter = (Becton=20 Dickinson) by using a dual-wavelength analysis (blue, = 424=96444 nm;=20 red, 675 nm) after excitation with 350-nm UV light. Propidium = iodide-positive dead cells (<15%) were excluded from the = analysis.=20 In the case of C6, the SP cells or non-SP cells were sorted = and=20 cultured in serum-free culture medium with bFGF and = PDGF.

RNA Extraction and RT-PCR Assay. Cells were harvested, and=20 poly(A)+ RNA was prepared by using a QuickPrep = Micro mRNA=20 purification kit (Amersham Biosciences) and reverse = transcribed by=20 using a First-Strand cDNA synthesis kit (Amersham = Biosciences),=20 as described (13). = The RT-PCR=20 was carried out in a 20-=B5l reaction mixture that contained = 1 =B5l of=20 cDNA as template, 1 =B5M specific oligonucleotide primer = pair, and 0.5=20 unit of Taq DNA polymerase (Takara Shuzo, Kyoto). = Cycle=20 parameters for bcrp1, mdr1, or g3pdh cDNAs were = 30 sec=20 at 94=B0C, 30 sec at 60=B0C, and 60 sec at 72=B0C for 33, 32, = and 25=20 cycles, respectively. The identity of the amplified products = was=20 checked by digestion with appropriate restriction enzymes.=20 Oligonucleotide DNA primers were synthesized as follows. For = rat=20 bcrp1, we used sequences conserved between human and = mouse:=20 5', 5'-CCAGTTCCATGGCACTGGCCATA-3' and 3',=20 5'-CAGGGCCACATGATTCTTCCACA-3'. For rat mdr1, we used = sequences=20 conserved between human and mouse: 5', = 5'-GCAAAGCTGGAGAGATCCTCACCA-3'=20 and 3', 5'-CAACATTTTCATTTCAACAACTCCTGC-3'. For rat g3pdh, = the=20 following sequences were used: 5', 5'-ACCACAGTCCATGCCATCAC-3' = and 3', 5'-TCCACCACCCTGTTGCTGTA-3'.

Immunostaining of Cultured Cells. To examine the = expression=20 of neuronal and glial markers in C6 SP cells cultured for 1 = or=20 10 d, the cells were cultured overnight in chamber slides = (Nunc)=20 precoated with 1 =B5g/ml fibronectin (Invitrogen) and 15 = =B5g/ml=20 ornithine (Sigma). The cells were fixed with 2% = paraformaldehyde for=20 10 min at room temperature, treated with 20% FCS, and then = stained=20 with the following mouse monoclonal antibodies: anti-GFAP = (1:200;=20 Sigma), anti-3D{beta}-III tubulin (1:200; Sigma), = anti-microtubule-associated=20 protein 2 (MAP2; 1:500; Sigma), and anti-nestin (1:200; = Pharmingen).=20 The primary antibodies were detected with Texas = red-conjugated goat=20 anti-mouse IgM or IgG (1:100; Jackson ImmunoResearch) as = described=20 (13).=20 The cells were counterstained with Hoechst 33342 to identify = all=20 nuclei.

Transplantation into Nude Mice. KSL/slc nude mice were = purchased=20 from SLC (Shizuoka, Japan). FACS-sorted C6 SP and non-SP = cells=20 were cultured for 7 d in bFGF plus PDGF, and 105 cells = of=20 each type were injected i.p. into three 4-wk-old female nude=20 mice. The same experiment was repeated twice with similar=20 results. The mice were killed 18 d after injection and = examined for=20 tumors, as described below. Mice were treated according to = the=20 guidelines of the Kumamoto University Animal Committee. =

Hematocrit Analysis. Blood was collected in EDTA at a = final=20 concentration of 1 mM. Glass microcapillary tubes = (Hirschmann)=20 were filled with blood, capped with Parafilm, and centrifuged = at 3,000 x g for 1 min, = and the=20 hematocrit was calculated as the proportion of the tube = containing=20 erythrocytes.

Immunostaining of Tissue Sections. Tumor-bearing tissues = were=20 fixed in 4% paraformaldehyde, embedded in Tissue-Tek OCT = (optimal=20 cutting temperature) compound, and then frozen at =9620=B0C.=20 Cryostat sections (12 =B5m) were cut, mounted on poly-L-lysine-coated slides, and air-dried for 24 = h. To=20 characterize the cells in tumors, the sections were treated = with 10%=20 normal goat serum (DAKO) for 30 min at room temperature and = then=20 stained with the following mouse monoclonal antibodies: = anti-nestin=20 antibody, anti-GFAP antibody, and anti-low molecular weight=20 neurofilament antibody (1:200; Sigma). The primary antibodies = were=20 detected with Alexa 594-conjugated goat anti-mouse IgG = (1:200;=20 Molecular Probes), as described (13). = The cells=20 were counterstained with Hoechst 33342 to identify all = nuclei. The=20 stained sections were examined and photographed in an AX70=20 fluorescence microscope (Olympus, Orangeburg, NY). =


3D"   =20 Results
3D"Top
3D"Abstract
3D"Materials and Methods
3D"Results
3D"Discussion
3D"References
 
<= B>Many=20 Cancer Cell Lines Contain a Small SP. To determine whether = any of=20 the six established cancer cell lines in our collection (see=20 Materials and Methods) contained SP cells, we removed the=20 cells from the culture dishes with trypsin and EDTA and = stained=20 them with the fluorescent dye Hoechst 33342, which has been=20 shown to be extruded actively by the SP cells in various = tissues=20 by means of verapamil-sensitive ABC transporters. We then = analyzed=20 them by flow cytometry. As shown in Fig. 1, = the C6=20 glioma cells contained 0.4% SP cells (A), the MCF7 = breast=20 cancer cells contained 2.0% SP cells (B), the B104=20 neuroblastoma cells contained 0.4% SP cells (C), and = the HeLa=20 carcinoma cells contained 1.2% SP cells (D). In each = case, the=20 SP population was decreased greatly by treatment with = verapamil (Fig. = 1),=20 indicating that the populations were bona fide SPs. Thus, = some cancer=20 cell lines contain a small SP, despite having been maintained = in=20 culture for many years. We could not detect SP cells in the = two human=20 osteosarcoma lines (U-2OS and SaOS-2) that we tested (data = not=20 shown), suggesting that not all cancer cell lines have an SP. = The=20 rest of our experiments were focused on C6 cells.



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  		Fig. 1. = Existence=20 of SP cells in established cancer cell lines. Cells of the = rat C6=20 glioma (A), human MCF7 breast carcinoma (B), = rat B104=20 neuroblastoma (C), and human HeLa carcinoma = (D) cell=20 lines were labeled with the Hoechst 33342 and then analyzed = by flow=20 cytometry. (Lower) Results when the cells were = treated with=20 50 =B5M verapamil during the labeling procedure. The SP, = which=20 disappears in the presence of verapamil, is outlined and = shown as a=20 percentage of the total cell population. These experiments = were=20 repeated at least three times with similar results. =

=

C6 SP Cells Can Be Expanded in bFGF Plus PDGF. We then=20 investigated which factor(s) can maintain C6 SP cells in = serum-free=20 culture media. We tested PDGF and bFGF (FGF-2) as possible=20 candidates. PDGF is the main mitogen for oligodendrocyte = precursor=20 cells (23=9625), = whereas=20 bFGF is a major mitogen for NSCs (26,=20 27). = We=20 first cultured unfractionated C6 cells on uncoated dishes in = 10% FCS=20 or in serum-free culture medium with PDGF, bFGF, or both.=20 Surprisingly, the morphology of the cells was very different = in the=20 different culture conditions. In FCS or bFGF alone, the cells = had a=20 flat, fibroblast-like shape (Fig. 2 = A=20 and B). In PDGF, they had a round body but were still=20 attached to the dish (Fig. = 2C).=20 In the presence of both bFGF and PDGF, by contrast, they = formed=20 floating, neurosphere-like cell aggregates after 10 d (Fig. = 2D),=20 just as CNS NSCs do under similar conditions (28). = We then=20 stained the cells with Hoechst 33342 and analyzed them by = flow=20 cytometry. When cultured in serum-free medium with both PDGF = and=20 bFGF, SP cells were maintained, and their proportion = increased with=20 time (Fig. 2 = E,=20 H, and I). By contrast, when cultured in = either bFGF=20 or PDGF alone, the C6 cells survived and proliferated, but by = 3 wk=20 few SP cells could be detected (Fig. 2 = F, G,=20 and I). These findings suggest that C6 SP cells = can expand=20 in a combination of bFGF and PDGF but cannot be maintained in = either=20 growth factor alone.



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  		Fig. 2. = The roles=20 of PDGF and bFGF on C6 SP cells. C6 cells were cultured in = FCS=20 (A) or serum-free medium with bFGF (B), PDGF = (C), or=20 bFGF plus PDGF (D) for 3 wk and were then = photographed while=20 alive in an inverted phase-contrast microscope. C6 SP cells = were=20 cultured for 3 wk in FCS (E) or serum-free medium = with bFGF=20 (F), PDGF (G), or both (H) and were = analyzed by=20 flow cytometry as shown in Fig.=20 1. (I) C6 cells were cultured in FCS (3D{circ}=20), bFGF = (), PDGF (3D{blacksquare}=20), or = bFGF plus=20 PDGF (3D{bullet}) for the indicated times, and the proportion of = SP cells=20 was analyzed by flow cytometry. (J) C6 cells were = cultured as=20 in A=96D for 3 wk, and then the expression of=20 bcrp1, mdr1, or g3pdh mRNAs was = analyzed by=20 RT-PCR. All experiments were repeated at least three times = with=20 similar results. (Scale bar in D, 100 =B5m.) =

=

It was shown previously that BCRP1, which is a = verapamil-sensitive=20 ABC transporter, exports the Hoechst 33342 from some types of = SP cells (21, 22, 29). = We,=20 therefore, examined the expression of bcrp1 mRNA, as = well as=20 mdr1 mRNA, which encodes another ABC transporter, in = C6 cells=20 cultured in the four conditions described above. As shown in = Fig. 2 = J,=20 bcrp1 mRNA expression was detected in the presence of = both=20 PDGF and bFGF but not in FCS or in bFGF or PDGF alone. In = contrast,=20 mdr1 mRNA was expressed in FCS, bFGF, or bFGF plus = PDGF,=20 although it was not expressed in PDGF alone. Together, these = data=20 suggest that BCRP1, but not MDR1, is responsible for the SP = character=20 of some C6 cells, as reported for some other types of SP = cells (21, 22, 29).

To investigate the individual roles of PDGF and bFGF in = expanding=20 C6 SP cells, we cultured C6 cells in either bFGF or PDGF for = 2=20 wk and then in bFGF plus PDGF for an additional 2 wk. We then = stained=20 the cells with Hoechst 33342 and analyzed them by flow = cytometry. As=20 shown in Fig.=20 3B, 1.8% of the cells cultured in bFGF and then in = bFGF=20 plus PDGF were SP cells. By contrast, although there seemed = to be SP=20 cells after culturing in PDGF and then in bFGF plus PDGF, = these cells=20 were still seen when stained in the presence of verapamil (Fig. = 3A=20 Lower), indicating that they were not bona fide SP = cells. We=20 also examined the expression of both bcrp1 and = mdr1=20 mRNA in C6 cells cultured under these two conditions. As = shown in Fig. = 3D,=20 we detected bcrp1 mRNA in the cells that were cultured = in bFGF=20 and then in PDGF plus bFGF, but we could not do so in the = cells=20 cultured in PDGF and then in PDGF plus bFGF; by contrast, we = could=20 detect mdr1 mRNA in both conditions. It is possible,=20 therefore, that bFGF on its own maintains C6 SP cells at an=20 undetectable low level and that PDGF stimulates the = proliferation of=20 these cells.



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  		Fig. 3. = Different=20 roles of bFGF and PDGF on C6 SP cells. C6 cells were = cultured in=20 PDGF (A), bFGF (B), or both (C) for 2 = wk and=20 then expanded in bFGF plus PDGF for an additional 2 wk. They = were=20 then analyzed by flow cytometry as described for Fig.=20 1. (D) C6 cells were cultured as shown in=20 A=96C, and the expression of bcrp1, = mdr1,=20 or g3pdh mRNAs was analyzed by RT-PCR. All = experiments were=20 repeated at least three times with similar results. =

=

C6 SP Cells Can Repopulate both SP and Non-SP C6 Cells. To=20 compare the ability of C6 SP cells with the ability of non-SP = cells to produce SP cells, we cultured C6 cells without FCS = and=20 in PDGF plus bFGF for 2 wk, stained them with Hoechst 33342, = and=20 sorted them into SP and non-SP fractions by flow cytometry, = and=20 then we expanded the SP and non-SP cells separately in the = same=20 medium for an additional 2 wk. As they proliferated, the = cells in the=20 two populations had different morphologies. The cells in the = SP=20 cultures formed floating spheres (Fig.=20 4A), whereas the cells in the non-SP cultures = remained=20 attached to the culture dishes and had a fibroblast-like = morphology=20 (Fig.=20 4B). When we restained the cells with Hoechst 33342 and = reanalyzed them by flow cytometry, we found that the cultures = initiated with SP cells contained both SP and non-SP cells, = whereas=20 the cultures initiated with non-SP cells contained only = non-SP=20 cells (Fig. = 4C).=20 These data are consistent with previous findings that only = the SP=20 cells in primary neurospheres can produce both SP and non-SP = cells in=20 culture (30).=20 Furthermore, when single FACS-sorted SP cells were cultured = alone in=20 the same medium in a well of a 98-well culture plate, 70% of = the cells=20 proliferated and reformed floating spheres; in contrast, = single=20 non-SP cells cultured in the same way proliferated much more = slowly,=20 and almost all of the cells died by 3 wk (data not shown). = Thus,=20 apparently C6 SP cells, but not non-SP cells, can form = floating=20 spheres, proliferate extensively, and produce C6 SP cells in=20 culture.



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  		Fig. 4. = SP cells=20 in the C6 glioma cell line can generate both SP and non-SP = C6 cells.=20 C6 cells were cultured in bFGF plus PDGF for 2 wk and then = sorted by=20 flow cytometry as described for Fig.=20 1. The SP (A) and non-SP (B) cells were = then=20 cultured separately in the same conditions for an additional = 2 wk.=20 The cells were then photographed while alive and analyzed by = flow=20 cytometry (C) as shown in Fig.=20 1. (Scale bar in B, 100 =B5m.) =

=

C6SP Cells Can Produce both Neurons and Glia in Culture. = To=20 explore the developmental potential of C6 SP cells, we sorted = SP cells, cultured them for 1 or 10 d in PDGF plus bFGF on=20 ornithine/fibronectin-coated chamber slides, and then = immunolabeled=20 them for neuronal and glial markers. After 1 d, >90% of = the cells=20 were labeled for the NSC marker nestin (92 =B1 2%), but none = were=20 labeled for the neuronal markers MAP2 or 3D{beta}=20-III tubulin or the = astrocyte marker GFAP (Fig. = 5A),=20 suggesting that C6 SP cells might be undifferentiated = NSC-like cells.=20 After 10 d, however, 3D{approx}70% were immunolabeled for 3D{beta}=20-III tubulin, 3D{approx}=205% for MAP2, and 3D{approx}7% for = GFAP=20 (Fig.=20 5B). Thus, C6 SP cells can apparently generate both = neurons=20 and glial cells in culture.



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  		Fig. 5. = C6 SP=20 cells can differentiate into both neurons and glia. C6 SP = cells were=20 prepared as described for Fig.=20 4A and then cultured in serum-free medium with = bFGF plus=20 PDGF on ornithine/fibronectin-coated chamber slides for 1 = (A)=20 or 10 (B) d. Cells were then immunolabeled for = neuronal and=20 glial markers. (Scale bar in B, 30 =B5m.) =

=

The Malignancy of C6 Cells in Vivo Is Largely Dependent on = the=20 SP Cells. To address whether SP and non-SP C6 cells differ = in=20 their malignancy, we sorted C6 cells growing in PDGF plus = bFGF=20 into SP and non-SP cells, expanded them in the same medium = for=20 1 wk, and then injected 105 cells from either = population=20 i.p. into nude mice. After 18 d, all mice injected with cells = from the SP cultures showed intraabdominal hemorrhages (Fig. 6 = A=20 and B) and tumor invasion into the mesentery (Fig. = 6C,=20 right), uterus (Fig. = 6D,=20 right) and lymph nodes. In four of six mice, there was also = tumor=20 invasion into the lungs (Fig. = 6E,=20 right). In contrast, after the same period, the cells from = non-SP=20 cultures had not formed tumors that invaded into these = tissues,=20 although we detected one s.c. tumor (Fig. = 6A)=20 and an occasional small metastasis in mesenteric lymph nodes = (Fig. = 6C,=20 left, arrow). Thus, much of the malignancy of the C6 line = apparently=20 depends on SP cells.



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  		Fig. 6. = Evidence=20 of malignancy of C6 SP cells in vivo. SP or non-SP C6 = cells=20 (105) were injected i.p. into 4-wk-old female = nude mice=20 (A, right and left mice, respectively). The mice were = killed=20 18 d later. (B=96E) The hematocrit was = measured=20 (B), and tumor tissue in the mesentery (C), = uterus=20 (D), and lung (E) was photographed (right, = SP-injected=20 tissue; left, non-SP-injected tissue). Frozen sections of = the tumors=20 in the mesentery in SP-injected mice were immunolabeled for = nestin=20 (F), GFAP (G), or low molecular weight = neurofilament=20 (NF-L; H). The sections were labeled also with = Hoechst 33342=20 (blue) to identify nuclei. (Scale bar in H, 50 =B5m.) =

=

To determine whether C6 cells could produce neurons and glia = in=20 vivo, we fixed the tumor-bearing tissues in 4% = paraformaldehyde,=20 cut frozen sections, and immunolabeled them for neuronal and=20 glial markers. As shown in Fig. 6, = 35% of = the cells in=20 the tumors were immunolabeled for nestin (Fig. = 6F),=20 3D{approx}30% for GFAP=20 (Fig.=20 6G), and 3D{approx}=2015% for the neuronal = marker NF-L=20 (low molecular weight neurofilament) (Fig. = 6H),=20 suggesting that C6 SP cells can differentiate into both = neurons and=20 glia in vivo.


3D"   =20 Discussion
3D"Top
3D"Abstract
3D"Materials and Methods
3D"Results
3D"Discussion
3D"References
 
A= lthough=20 the concept of cancer stem cells has been around for many = years (31=9633), = there has=20 been surprisingly little attention paid to them in recent = years. Now,=20 however, there is increasing evidence that at least some = cancers=20 contain stem cells that are largely responsible for the = malignancy of=20 the cancer. This possibility has been shown, for example, for = acute myeloid leukemia (34) = and breast=20 carcinoma (35). = Cancer=20 stem cells are potentially extremely important because it is=20 likely that they are often responsible for recurrences that = occur=20 after treatment. Thus, for cancer therapy to be curative, it=20 probably must eliminate these cells, which is why it is = important=20 to identify and study cancer stem cells.

Our findings suggest that cancer stem cells may be present also = in=20 many cancer cell lines in culture, even when they have been=20 maintained for many years. We can detect an SP in four of the = six cancer cell lines that we have tested, and in most normal = tissues, the stem cells are found in the SP. We find that the = SP of the C6 glioma line, which is only 0.4% of the cells = maintained=20 in serum, has a number of characteristics that are expected = of=20 cancer stem cells. The SP cells in culture can self-renew and = produce=20 both SP and non-SP C6 cells, whereas the non-SP cells under = the same=20 culture conditions can produce non-SP cells only. Also, the = C6 SP=20 cells in culture can form neurospheres and produce neurons as = well as=20 glial cells, suggesting that they have NSC-like properties. = Finally,=20 they produce tumors in nude mice with high efficiency, = whereas the=20 non-SP C6 cells do not.

Besides providing further evidence for the existence of cancer=20 stem cells, our finding that cancer stem cells can be = enriched=20 by isolating the SP in some cancer cell lines has a number of = important implications. It may, for example, provide a simple = and general strategy for isolating cancer stem cells. Because = stem cells in any tissue are usually present in very small = numbers,=20 they are difficult to identify and even more difficult to = isolate.=20 There is a pressing need for methods to identify and isolate=20 cancer stem cells so that their properties, including their=20 sensitivity to various anti-cancer therapeutic agents, may be = characterized.

Our findings that cancer SP cells can be maintained indefinitely = in permanent cancer cell lines raise the possibility that = these=20 cell lines may provide attractive models for studying the = molecular=20 basis that defines stem cells. It seems likely that most of = the=20 stem cells and the other cells in a particular cancer cell = line=20 contain the same mutations, including the activation of = specific=20 protooncogenes and the loss of specific tumor-suppressor = genes, but=20 these changes are apparently not enough to make every cancer = cell a=20 stem cell. The difference between the stem cells and all of = the other=20 cells in a cancer is likely to be epigenetic, and cancer cell = lines=20 should be useful in trying to identify what these epigenetic=20 differences are, which is a fundamental question in stem cell = biology.

We find that the combination of PDGF and bFGF, but not either=20 alone, allows C6 SP cells to expand in culture, as it has = been=20 shown previously that both mitogens can contribute to glial=20 oncogenesis. For example, inhibition of bFGF signaling was = shown=20 not only to block cell proliferation but also to induce = apoptosis=20 in glioma cells (36, 37). = Enforced=20 expression of PDGF in either NSCs or GFAP-positive astrocytes = was=20 found to induce malignant gliomas (10, 11). = We find=20 that the two growth factors seem to have different roles in = promoting=20 the proliferation of C6 SP cells in culture. bFGF on its own = seems to=20 be able to maintain these cells but not to allow their = expansion,=20 whereas PDGF on its own apparently cannot maintain them, but = in the=20 presence of bFGF, helps to stimulate their = proliferation.

It is of interest that C6 SP cells can generate neurons as well = as=20 glial cells; we and others (13, 16, 17) = showed that=20 oligodendrocyte precursor cells can be induced by = extracellular=20 signals to convert to an NSC-like phenotype and generate = neurons and=20 astrocytes, as well as oligodendrocytes. The molecular = mechanisms=20 involved in this conversion are unknown. C6 cells might = provide an=20 attractive model to study the molecular basis of this type of = plasticity.

In summary, our studies underline the importance of cancer stem=20 cells and suggest a simple strategy for isolating them. They=20 also suggest that cancer cell lines may be important models = for=20 studying the basic biology of stem cells.


3D"   =20 Acknowledgements
 
We = thank Martin Raff=20 for editing the manuscript and Yuko Saiki for technical = assistance=20 with flow cytometry. This work was supported in part by a=20 grant-in-aid from the Japan Ministry of Education, Science, = Sports,=20 and Culture; the Nakajima Foundation; the Uehara Memorial = Foundation;=20 the Japan Brain Foundation; the Higo Foundation; the Yamada = Science=20 Foundation; and Merck Sharp and Dohme.


3D"   =20 Footnotes
 
Abbreviations: SP, side population; PDGF,=20 platelet-derived growth factor; bFGF, basic fibroblast growth = factor;=20 NSC, neural stem cell; GFAP, glial fibrillary acidic protein; = ABC,=20 ATP-binding cassette; MDR, multidrug-resistant; BCRP1, breast = cancer-resistant protein 1; MAP2, microtubule-associated = protein=20 2.

3D{ddagger}=20 T.K. and = T.S.=20 contributed equally to this work. 3DBack

3D=A7=20 To whom = correspondence=20 should be sent at the 3D{dagger}=20address. = E-mail: tk294{at}cam.ac.uk .=20


3D"   =20 References
3D"Top
3D"Abstract
3D"Materials and Methods
3D"Results
3D"Discussion
3D"References
&nb= sp;

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Opera = requires=0A= the Sun Java Runtime Environment .=0A= =0A= by Andrew Gregory=0A= http://www.scss.com.au/family/andrew/webdesign/xmlhttprequest/=0A= =0A= This work is licensed under the Creative Commons Attribution License. To = view a=0A= copy of this license, visit = http://creativecommons.org/licenses/by-sa/2.5/ or=0A= send a letter to Creative Commons, 559 Nathan Abbott Way, Stanford, = California=0A= 94305, USA.=0A= =0A= Attribution: Leave my name and web address in this script intact.=0A= =0A= Not Supported in Opera=0A= ----------------------=0A= * user/password authentication=0A= * responseXML data member=0A= =0A= Not Fully Supported in Opera=0A= ----------------------------=0A= * async requests=0A= * abort()=0A= * getAllResponseHeaders(), getAllResponseHeader(header)=0A= =0A= */=0A= // IE support=0A= if (window.ActiveXObject && !window.XMLHttpRequest) {=0A= window.XMLHttpRequest =3D function() {=0A= var msxmls =3D new Array(=0A= 'Msxml2.XMLHTTP.5.0',=0A= 'Msxml2.XMLHTTP.4.0',=0A= 'Msxml2.XMLHTTP.3.0',=0A= 'Msxml2.XMLHTTP',=0A= 'Microsoft.XMLHTTP');=0A= for (var i =3D 0; i < msxmls.length; i++) {=0A= try {=0A= return new ActiveXObject(msxmls[i]);=0A= } catch (e) {=0A= }=0A= }=0A= return null;=0A= };=0A= }=0A= // Gecko support=0A= /* ;-) */=0A= // Opera support=0A= if (window.opera && !window.XMLHttpRequest) {=0A= window.XMLHttpRequest =3D function() {=0A= this.readyState =3D 0; // = 0=3Duninitialized,1=3Dloading,2=3Dloaded,3=3Dinteractive,4=3Dcomplete=0A= this.status =3D 0; // HTTP status codes=0A= this.statusText =3D '';=0A= this._headers =3D [];=0A= this._aborted =3D false;=0A= this._async =3D true;=0A= this._defaultCharset =3D 'ISO-8859-1';=0A= this._getCharset =3D function() {=0A= var charset =3D _defaultCharset;=0A= var contentType =3D = this.getResponseHeader('Content-type').toUpperCase();=0A= val =3D contentType.indexOf('CHARSET=3D');=0A= if (val !=3D -1) {=0A= charset =3D contentType.substring(val);=0A= }=0A= val =3D charset.indexOf(';');=0A= if (val !=3D -1) {=0A= charset =3D charset.substring(0, val);=0A= }=0A= val =3D charset.indexOf(',');=0A= if (val !=3D -1) {=0A= charset =3D charset.substring(0, val);=0A= }=0A= return charset;=0A= };=0A= this.abort =3D function() {=0A= this._aborted =3D true;=0A= };=0A= this.getAllResponseHeaders =3D function() {=0A= return this.getAllResponseHeader('*');=0A= };=0A= this.getAllResponseHeader =3D function(header) {=0A= var ret =3D '';=0A= for (var i =3D 0; i < this._headers.length; i++) {=0A= if (header =3D=3D '*' || this._headers[i].h =3D=3D header) {=0A= ret +=3D this._headers[i].h + ': ' + this._headers[i].v + '\n';=0A= }=0A= }=0A= return ret;=0A= };=0A= this.getResponseHeader =3D function(header) {=0A= var ret =3D getAllResponseHeader(header);=0A= var i =3D ret.indexOf('\n');=0A= if (i !=3D -1) {=0A= ret =3D ret.substring(0, i);=0A= }=0A= return ret;=0A= };=0A= this.setRequestHeader =3D function(header, value) {=0A= this._headers[this._headers.length] =3D {h:header, v:value};=0A= };=0A= this.open =3D function(method, url, async, user, password) {=0A= this.method =3D method;=0A= this.url =3D url;=0A= this._async =3D true;=0A= this._aborted =3D false;=0A= this._headers =3D [];=0A= if (arguments.length >=3D 3) {=0A= this._async =3D async;=0A= }=0A= if (arguments.length > 3) {=0A= opera.postError('XMLHttpRequest.open() - user/password not = supported');=0A= }=0A= this.readyState =3D 1;=0A= if (this.onreadystatechange) {=0A= this.onreadystatechange();=0A= }=0A= };=0A= this.send =3D function(data) {=0A= if (!navigator.javaEnabled()) {=0A= alert("XMLHttpRequest.send() - Java must be installed and = enabled.");=0A= return;=0A= }=0A= if (this._async) {=0A= setTimeout(this._sendasync, 0, this, data);=0A= // this is not really asynchronous and won't execute until the = current=0A= // execution context ends=0A= } else {=0A= this._sendsync(data);=0A= }=0A= }=0A= this._sendasync =3D function(req, data) {=0A= if (!req._aborted) {=0A= req._sendsync(data);=0A= }=0A= };=0A= this._sendsync =3D function(data) {=0A= this.readyState =3D 2;=0A= if (this.onreadystatechange) {=0A= this.onreadystatechange();=0A= }=0A= // open connection=0A= var url =3D new java.net.URL(new = java.net.URL(window.location.href), this.url);=0A= var conn =3D url.openConnection();=0A= for (var i =3D 0; i < this._headers.length; i++) {=0A= conn.setRequestProperty(this._headers[i].h, this._headers[i].v);=0A= }=0A= this._headers =3D [];=0A= if (this.method =3D=3D 'POST') {=0A= // POST data=0A= conn.setDoOutput(true);=0A= var wr =3D new = java.io.OutputStreamWriter(conn.getOutputStream(), this._getCharset());=0A= wr.write(data);=0A= wr.flush();=0A= wr.close();=0A= }=0A= // read response headers=0A= // NOTE: the getHeaderField() methods always return nulls for me :(=0A= var gotContentEncoding =3D false;=0A= var gotContentLength =3D false;=0A= var gotContentType =3D false;=0A= var gotDate =3D false;=0A= var gotExpiration =3D false;=0A= var gotLastModified =3D false;=0A= for (var i =3D 0; ; i++) {=0A= var hdrName =3D conn.getHeaderFieldKey(i);=0A= var hdrValue =3D conn.getHeaderField(i);=0A= if (hdrName =3D=3D null && hdrValue =3D=3D null) {=0A= break;=0A= }=0A= if (hdrName !=3D null) {=0A= this._headers[this._headers.length] =3D {h:hdrName, = v:hdrValue};=0A= switch (hdrName.toLowerCase()) {=0A= case 'content-encoding': gotContentEncoding =3D true; break;=0A= case 'content-length' : gotContentLength =3D true; break;=0A= case 'content-type' : gotContentType =3D true; break;=0A= case 'date' : gotDate =3D true; break;=0A= case 'expires' : gotExpiration =3D true; break;=0A= case 'last-modified' : gotLastModified =3D true; break;=0A= }=0A= }=0A= }=0A= // try to fill in any missing header information=0A= var val;=0A= val =3D conn.getContentEncoding();=0A= if (val !=3D null && !gotContentEncoding) = this._headers[this._headers.length] =3D {h:'Content-encoding', v:val};=0A= val =3D conn.getContentLength();=0A= if (val !=3D -1 && !gotContentLength) = this._headers[this._headers.length] =3D {h:'Content-length', v:val};=0A= val =3D conn.getContentType();=0A= if (val !=3D null && !gotContentType) = this._headers[this._headers.length] =3D {h:'Content-type', v:val};=0A= val =3D conn.getDate();=0A= if (val !=3D 0 && !gotDate) this._headers[this._headers.length] = =3D {h:'Date', v:(new Date(val)).toUTCString()};=0A= val =3D conn.getExpiration();=0A= if (val !=3D 0 && !gotExpiration) = this._headers[this._headers.length] =3D {h:'Expires', v:(new = Date(val)).toUTCString()};=0A= val =3D conn.getLastModified();=0A= if (val !=3D 0 && !gotLastModified) = this._headers[this._headers.length] =3D {h:'Last-modified', v:(new = Date(val)).toUTCString()};=0A= // read response data=0A= var reqdata =3D '';=0A= var stream =3D conn.getInputStream();=0A= if (stream) {=0A= var reader =3D new java.io.BufferedReader(new = java.io.InputStreamReader(stream, this._getCharset()));=0A= var line;=0A= while ((line =3D reader.readLine()) !=3D null) {=0A= if (this.readyState =3D=3D 2) {=0A= this.readyState =3D 3;=0A= if (this.onreadystatechange) {=0A= this.onreadystatechange();=0A= }=0A= }=0A= reqdata +=3D line + '\n';=0A= }=0A= reader.close();=0A= this.status =3D 200;=0A= this.statusText =3D 'OK';=0A= this.responseText =3D reqdata;=0A= this.readyState =3D 4;=0A= if (this.onreadystatechange) {=0A= this.onreadystatechange();=0A= }=0A= if (this.onload) {=0A= this.onload();=0A= }=0A= } else {=0A= // error=0A= this.status =3D 404;=0A= this.statusText =3D 'Not Found';=0A= this.responseText =3D '';=0A= this.readyState =3D 4;=0A= if (this.onreadystatechange) {=0A= this.onreadystatechange();=0A= }=0A= if (this.onerror) {=0A= this.onerror();=0A= }=0A= }=0A= };=0A= };=0A= }=0A= // ActiveXObject emulation=0A= if (!window.ActiveXObject && window.XMLHttpRequest) {=0A= window.ActiveXObject =3D function(type) {=0A= switch (type.toLowerCase()) {=0A= case 'microsoft.xmlhttp':=0A= case 'msxml2.xmlhttp':=0A= case 'msxml2.xmlhttp.3.0':=0A= case 'msxml2.xmlhttp.4.0':=0A= case 'msxml2.xmlhttp.5.0':=0A= return new XMLHttpRequest();=0A= }=0A= return null;=0A= };=0A= }=0A= ------=_NextPart_000_0002_01C70A59.FAEAAFC0 Content-Type: application/octet-stream Content-Transfer-Encoding: quoted-printable Content-Location: http://www.pnas.org/javascript/ajax/utility.js /************************************************************************= *****=0A= * javascript/ajax/utility.js=0A= *=0A= * Utility functions for working with XMLHttpRequest data.=0A= *=0A= * Copyright 2006 Board of Trustees of the Leland Stanford Junior = University.=0A= = *************************************************************************= ***/=0A= =0A= /*=0A= * Copy XML nodes into an HTMLElement. This effectively=0A= * clones XML markup which uses XHTML naming conventions=0A= * into an HTML DOM.=0A= */=0A= function copy_xml_to_html(src, dst) {=0A= if (src.nodeType =3D=3D 1) { /* Node.ELEMENT_NODE */=0A= var e =3D document.createElement(src.nodeName);=0A= for (var i =3D 0; i < src.childNodes.length; i++) {=0A= copy_xml_to_html(src.childNodes[i], e);=0A= }=0A= for (var i =3D 0; i < src.attributes.length; i++) {=0A= var n =3D src.attributes[i].name;=0A= var v =3D unescape_xml_string(src.attributes[i].value); =0A= e.setAttribute(n, v);=0A= if (n =3D=3D "class") {=0A= e.className =3D v;=0A= }=0A= else if (n =3D=3D "style") {=0A= set_css_style(v, e, "");=0A= }=0A= }=0A= dst.appendChild(e);=0A= }=0A= else if (src.nodeType =3D=3D 3) { /* Node.TEXT_NODE */=0A= dst.appendChild(document.createTextNode(src.nodeValue));=0A= }=0A= }=0A= =0A= /* =0A= * It is unclear that this is the right thing to be calling=0A= * from copy_xml_to_html, but it appears that Safari decides=0A= * to convert & to the NCR #, and then encodes that=0A= * NCR to &%26%2338;. So, I'm going to treat the DOM Attr=0A= * value as a plain string, and run our XML string input=0A= * through the decoding routine below.=0A= */=0A= function unescape_xml_string(s) {=0A= return s.replace(/'/g, "'")=0A= .replace(/'/g, "'")=0A= .replace(/"/g, "\"")=0A= .replace(/"/g, "\"")=0A= .replace(/>/g, ">")=0A= .replace(/>/g, ">")=0A= .replace(/</g, "<")=0A= .replace(/</g, "<")=0A= .replace(/&/g, "&")=0A= .replace(/&/g, "&");=0A= }=0A= =0A= /*=0A= * Parse set of CSS rules and apply them to an element.=0A= * This is quite horrifying, but I'm unable to determine=0A= * how else to handle this with IE 6. FireFox and other=0A= * sane browsers let you simply set the style attribute=0A= * or use e.style.setProperty(rule, value, priority),=0A= * IE 6 appears to have neither of these capabilities..=0A= */=0A= function set_css_style(css, e, priority) {=0A= var rules =3D css.split(";");=0A= for (var i =3D 0; i < rules.length; i++) {=0A= var nvpair =3D rules[i].split(":");=0A= if (nvpair.length =3D=3D 2) {=0A= try {=0A= var name =3D nvpair[0]; /* style attribute */=0A= var value =3D nvpair[1]; /* attribute value */=0A= =0A= /*=0A= * For each possible style attribute, set the=0A= * appropriate style property in the element.=0A= */=0A= if (name =3D=3D "background") {=0A= e.style.background =3D value;=0A= }=0A= else if (name =3D=3D "background-attachment") {=0A= e.style.backgroundAttachment =3D value;=0A= }=0A= else if (name =3D=3D "background-color") {=0A= e.style.backgroundColor =3D value;=0A= }=0A= else if (name =3D=3D "background-image") {=0A= e.style.backgroundImage =3D value;=0A= }=0A= else if (name =3D=3D "background-position") {=0A= e.style.backgroundPosition =3D value;=0A= }=0A= else if (name =3D=3D "background-position-x") {=0A= e.style.backgroundPositionX =3D value;=0A= }=0A= else if (name =3D=3D "background-position-y") {=0A= e.style.backgroundPositionY =3D value;=0A= }=0A= else if (name =3D=3D "background-repeat") {=0A= e.style.backgroundRepeat =3D value;=0A= }=0A= else if (name =3D=3D "behavior") {=0A= e.style.behavior =3D value;=0A= }=0A= else if (name =3D=3D "border") {=0A= e.style.border =3D value;=0A= }=0A= else if (name =3D=3D "border-bottom") {=0A= e.style.borderBottom =3D value;=0A= }=0A= else if (name =3D=3D "border-bottom-color") {=0A= e.style.borderBottomColor =3D value;=0A= }=0A= else if (name =3D=3D "border-bottom-style") {=0A= e.style.borderBottomStyle =3D value;=0A= }=0A= else if (name =3D=3D "border-bottom-width") {=0A= e.style.borderBottomWidth =3D value;=0A= }=0A= else if (name =3D=3D "border-collapse") {=0A= e.style.borderCollapse =3D value;=0A= }=0A= else if (name =3D=3D "border-color") {=0A= e.style.borderColor =3D value;=0A= }=0A= else if (name =3D=3D "border-left") {=0A= e.style.borderLeft =3D value;=0A= }=0A= else if (name =3D=3D "border-left-color") {=0A= e.style.borderLeftColor =3D value;=0A= }=0A= else if (name =3D=3D "border-left-style") {=0A= e.style.borderLeftStyle =3D value;=0A= }=0A= else if (name =3D=3D "border-left-width") {=0A= e.style.borderLeftWidth =3D value;=0A= }=0A= else if (name =3D=3D "border-right") {=0A= e.style.borderRight =3D value;=0A= }=0A= else if (name =3D=3D "border-right-color") {=0A= e.style.borderRightColor =3D value;=0A= }=0A= else if (name =3D=3D "border-right-style") {=0A= e.style.borderRightStyle =3D value;=0A= }=0A= else if (name =3D=3D "border-right-width") {=0A= e.style.borderRightWidth =3D value;=0A= }=0A= else if (name =3D=3D "border-style") {=0A= e.style.borderStyle =3D value;=0A= }=0A= else if (name =3D=3D "border-top") {=0A= e.style.borderTop =3D value;=0A= }=0A= else if (name =3D=3D "border-top-color") {=0A= e.style.borderTopColor =3D value;=0A= }=0A= else if (name =3D=3D "border-top-style") {=0A= e.style.borderTopStyle =3D value;=0A= }=0A= else if (name =3D=3D "border-top-width") {=0A= e.style.borderTopWidth =3D value;=0A= }=0A= else if (name =3D=3D "border-width") {=0A= e.style.borderWidth =3D value;=0A= }=0A= else if (name =3D=3D "bottom") {=0A= e.style.bottom =3D value;=0A= }=0A= else if (name =3D=3D "clear") {=0A= e.style.clear =3D value;=0A= }=0A= else if (name =3D=3D "clip") {=0A= e.style.clip =3D value;=0A= }=0A= else if (name =3D=3D "color") {=0A= e.style.color =3D value;=0A= }=0A= else if (name =3D=3D "cssText") {=0A= e.style.Sets =3D value;=0A= }=0A= else if (name =3D=3D "cursor") {=0A= e.style.cursor =3D value;=0A= }=0A= else if (name =3D=3D "direction") {=0A= e.style.direction =3D value;=0A= }=0A= else if (name =3D=3D "display") {=0A= e.style.display =3D value;=0A= }=0A= else if (name =3D=3D "font") {=0A= e.style.font =3D value;=0A= }=0A= else if (name =3D=3D "font-family") {=0A= e.style.fontFamily =3D value;=0A= }=0A= else if (name =3D=3D "font-size") {=0A= e.style.fontSize =3D value;=0A= }=0A= else if (name =3D=3D "font-style") {=0A= e.style.fontStyle =3D value;=0A= }=0A= else if (name =3D=3D "font-variant") {=0A= e.style.fontVariant =3D value;=0A= }=0A= else if (name =3D=3D "font-weight") {=0A= e.style.fontWeight =3D value;=0A= }=0A= else if (name =3D=3D "height") {=0A= e.style.height =3D value;=0A= }=0A= else if (name =3D=3D "ime-mode") {=0A= e.style.imeMode =3D value;=0A= }=0A= else if (name =3D=3D "layout-flow") {=0A= e.style.layoutFlow =3D value;=0A= }=0A= else if (name =3D=3D "layout-grid") {=0A= e.style.layoutGrid =3D value;=0A= }=0A= else if (name =3D=3D "layout-grid-char") {=0A= e.style.layoutGridChar =3D value;=0A= }=0A= else if (name =3D=3D "layout-grid-line") {=0A= e.style.layoutGridLine =3D value;=0A= }=0A= else if (name =3D=3D "layout-grid-mode") {=0A= e.style.layoutGridMode =3D value;=0A= }=0A= else if (name =3D=3D "layout-grid-type") {=0A= e.style.layoutGridType =3D value;=0A= }=0A= else if (name =3D=3D "left") {=0A= e.style.left =3D value;=0A= }=0A= else if (name =3D=3D "letter-spacing") {=0A= e.style.letterSpacing =3D value;=0A= }=0A= else if (name =3D=3D "line-break") {=0A= e.style.lineBreak =3D value;=0A= }=0A= else if (name =3D=3D "line-height") {=0A= e.style.lineHeight =3D value;=0A= }=0A= else if (name =3D=3D "list-style") {=0A= e.style.listStyle =3D value;=0A= }=0A= else if (name =3D=3D "list-style-image") {=0A= e.style.listStyleImage =3D value;=0A= }=0A= else if (name =3D=3D "list-style-position") {=0A= e.style.listStylePosition =3D value;=0A= }=0A= else if (name =3D=3D "list-style-type") {=0A= e.style.listStyleType =3D value;=0A= }=0A= else if (name =3D=3D "margin") {=0A= e.style.margin =3D value;=0A= }=0A= else if (name =3D=3D "margin-bottom") {=0A= e.style.marginBottom =3D value;=0A= }=0A= else if (name =3D=3D "margin-left") {=0A= e.style.marginLeft =3D value;=0A= }=0A= else if (name =3D=3D "margin-right") {=0A= e.style.marginRight =3D value;=0A= }=0A= else if (name =3D=3D "margin-top") {=0A= e.style.marginTop =3D value;=0A= }=0A= else if (name =3D=3D "min-height") {=0A= e.style.minHeight =3D value;=0A= }=0A= else if (name =3D=3D "overflow") {=0A= e.style.overflow =3D value;=0A= }=0A= else if (name =3D=3D "overflow-x") {=0A= e.style.overflowX =3D value;=0A= }=0A= else if (name =3D=3D "overflow-y") {=0A= e.style.overflowY =3D value;=0A= }=0A= else if (name =3D=3D "padding") {=0A= e.style.padding =3D value;=0A= }=0A= else if (name =3D=3D "padding-bottom") {=0A= e.style.paddingBottom =3D value;=0A= }=0A= else if (name =3D=3D "padding-left") {=0A= e.style.paddingLeft =3D value;=0A= }=0A= else if (name =3D=3D "padding-right") {=0A= e.style.paddingRight =3D value;=0A= }=0A= else if (name =3D=3D "padding-top") {=0A= e.style.paddingTop =3D value;=0A= }=0A= else if (name =3D=3D "page-break-after") {=0A= e.style.pageBreakAfter =3D value;=0A= }=0A= else if (name =3D=3D "page-break-before") {=0A= e.style.pageBreakBefore =3D value;=0A= }=0A= else if (name =3D=3D "pixelBottom") {=0A= e.style.pixelBottom =3D value;=0A= }=0A= else if (name =3D=3D "pixelHeight") {=0A= e.style.pixelHeight =3D value;=0A= }=0A= else if (name =3D=3D "pixelLeft") {=0A= e.style.pixelLeft =3D value;=0A= }=0A= else if (name =3D=3D "pixelRight") {=0A= e.style.pixelRight =3D value;=0A= }=0A= else if (name =3D=3D "pixelTop") {=0A= e.style.pixelTop =3D value;=0A= }=0A= else if (name =3D=3D "pixelWidth") {=0A= e.style.pixelWidth =3D value;=0A= }=0A= else if (name =3D=3D "posBottom") {=0A= e.style.posBottom =3D value;=0A= }=0A= else if (name =3D=3D "posHeight") {=0A= e.style.posHeight =3D value;=0A= }=0A= else if (name =3D=3D "position") {=0A= e.style.position =3D value;=0A= }=0A= else if (name =3D=3D "posLeft") {=0A= e.style.posLeft =3D value;=0A= }=0A= else if (name =3D=3D "posRight") {=0A= e.style.posRight =3D value;=0A= }=0A= else if (name =3D=3D "posTop") {=0A= e.style.posTop =3D value;=0A= }=0A= else if (name =3D=3D "posWidth") {=0A= e.style.posWidth =3D value;=0A= }=0A= else if (name =3D=3D "right") {=0A= e.style.right =3D value;=0A= }=0A= else if (name =3D=3D "ruby-align") {=0A= e.style.rubyAlign =3D value;=0A= }=0A= else if (name =3D=3D "ruby-overhang") {=0A= e.style.rubyOverhang =3D value;=0A= }=0A= else if (name =3D=3D "ruby-position") {=0A= e.style.rubyPosition =3D value;=0A= }=0A= else if (name =3D=3D "scrollbar-3dlight-color") {=0A= e.style.scrollbar3dLightColor =3D value;=0A= }=0A= else if (name =3D=3D "scrollbar-arrow-color") {=0A= e.style.scrollbarArrowColor =3D value;=0A= }=0A= else if (name =3D=3D "scrollbar-base-color") {=0A= e.style.scrollbarBaseColor =3D value;=0A= }=0A= else if (name =3D=3D "scrollbar-darkshadow-color") {=0A= e.style.scrollbarDarkShadowColor =3D value;=0A= }=0A= else if (name =3D=3D "scrollbar-face-color") {=0A= e.style.scrollbarFaceColor =3D value;=0A= }=0A= else if (name =3D=3D "scrollbar-highlight-color") {=0A= e.style.scrollbarHighlightColor =3D value;=0A= }=0A= else if (name =3D=3D "scrollbar-shadow-color") {=0A= e.style.scrollbarShadowColor =3D value;=0A= }=0A= else if (name =3D=3D "scrollbar-track-color") {=0A= e.style.scrollbarTrackColor =3D value;=0A= }=0A= else if (name =3D=3D "float") {=0A= e.style.styleFloat =3D value;=0A= }=0A= else if (name =3D=3D "table-layout") {=0A= e.style.tableLayout =3D value;=0A= }=0A= else if (name =3D=3D "text-align") {=0A= e.style.textAlign =3D value;=0A= }=0A= else if (name =3D=3D "text-align-last") {=0A= e.style.textAlignLast =3D value;=0A= }=0A= else if (name =3D=3D "text-autospace") {=0A= e.style.textAutospace =3D value;=0A= }=0A= else if (name =3D=3D "text-decoration") {=0A= e.style.textDecoration =3D value;=0A= }=0A= else if (name =3D=3D "textDecorationBlink") {=0A= e.style.textDecorationBlink =3D value;=0A= }=0A= else if (name =3D=3D "textDecorationLineThrough") {=0A= e.style.textDecorationLineThrough =3D value;=0A= }=0A= else if (name =3D=3D "textDecorationNone") {=0A= e.style.textDecorationNone =3D value;=0A= }=0A= else if (name =3D=3D "textDecorationOverline") {=0A= e.style.textDecorationOverline =3D value;=0A= }=0A= else if (name =3D=3D "textDecorationUnderline") {=0A= e.style.textDecorationUnderline =3D value;=0A= }=0A= else if (name =3D=3D "text-indent") {=0A= e.style.textIndent =3D value;=0A= }=0A= else if (name =3D=3D "text-justify") {=0A= e.style.textJustify =3D value;=0A= }=0A= else if (name =3D=3D "text-kashida-space") {=0A= e.style.textKashidaSpace =3D value;=0A= }=0A= else if (name =3D=3D "text-overflow") {=0A= e.style.textOverflow =3D value;=0A= }=0A= else if (name =3D=3D "text-transform") {=0A= e.style.textTransform =3D value;=0A= }=0A= else if (name =3D=3D "text-underline-position") {=0A= e.style.textUnderlinePosition =3D value;=0A= }=0A= else if (name =3D=3D "top") {=0A= e.style.top =3D value;=0A= }=0A= else if (name =3D=3D "unicode-bidi") {=0A= e.style.unicodeBidi =3D value;=0A= }=0A= else if (name =3D=3D "vertical-align") {=0A= e.style.verticalAlign =3D value;=0A= }=0A= else if (name =3D=3D "visibility") {=0A= e.style.visibility =3D value;=0A= }=0A= else if (name =3D=3D "white-space") {=0A= e.style.whiteSpace =3D value;=0A= }=0A= else if (name =3D=3D "width") {=0A= e.style.width =3D value;=0A= }=0A= else if (name =3D=3D "word-break") {=0A= e.style.wordBreak =3D value;=0A= }=0A= else if (name =3D=3D "word-spacing") {=0A= e.style.wordSpacing =3D value;=0A= }=0A= else if (name =3D=3D "word-wrap") {=0A= e.style.wordWrap =3D value;=0A= }=0A= else if (name =3D=3D "writing-mode") {=0A= e.style.writingMode =3D value;=0A= }=0A= else if (name =3D=3D "z-index") {=0A= e.style.zIndex =3D value;=0A= }=0A= else if (name =3D=3D "zoom") {=0A= e.style.zoom =3D value;=0A= }=0A= }=0A= catch (e) {=0A= /* ignore error on attempt to set e.style.[property] */=0A= }=0A= }=0A= }=0A= }=0A= ------=_NextPart_000_0002_01C70A59.FAEAAFC0 Content-Type: application/octet-stream Content-Transfer-Encoding: quoted-printable Content-Location: http://www.pnas.org/javascript/entrez/callback.js /************************************************************************= *****=0A= * javascript/entrez/callback.js=0A= *=0A= * Entrez Linking callback to populate content box.=0A= *=0A= * Copyright 2006 Board of Trustees of the Leland Stanford Junior = University.=0A= = *************************************************************************= ***/=0A= =0A= /*=0A= * Execute callback to fill content box with Entrez Linking information.=0A= */=0A= function entrez_callback(pmid, callback_url) {=0A= /*=0A= * MSIE 5.5 and below have issues with the JavaScript=0A= * used for Entrez Linking. For now we have to disable=0A= * the callback until we can track down a proper fix=0A= * (or everybody sanely upgrades to version 6 or 7!).=0A= */=0A= if (navigator) {=0A= var appname =3D navigator.appName;=0A= if (appname =3D=3D "Microsoft Internet Explorer") {=0A= var userAgent =3D navigator["userAgent"];=0A= var s =3D "MSIE ";=0A= var n =3D -1; =0A= if ((n =3D userAgent.indexOf(s)) !=3D -1) {=0A= var v =3D parseFloat(userAgent.substring(n+s.length));=0A= if (v < 6) {=0A= return;=0A= }=0A= }=0A= }=0A= }=0A= =0A= /*=0A= * Acquire table row element to update, initiate callback=0A= * to update table with Entrez Links.=0A= */=0A= var tr =3D document.getElementById('entrez_callback_'+pmid);=0A= if (!tr) {=0A= return;=0A= }=0A= var req =3D new XMLHttpRequest();=0A= if (!req) {=0A= return;=0A= }=0A= req.onreadystatechange =3D function() {=0A= if (req.readyState =3D=3D 4 && (req.status =3D=3D 200 || req.status = =3D=3D 304)) {=0A= var src =3D req.responseXML.documentElement;=0A= var dst =3D document.createDocumentFragment();=0A= for (var i =3D 0; i < src.childNodes.length; i++) {=0A= copy_xml_to_html(src.childNodes[i], dst);=0A= }=0A= var tbl =3D tr.parentNode;=0A= tbl.replaceChild(dst, tr);=0A= }=0A= }=0A= req.open('GET', callback_url, true);=0A= req.send(null);=0A= }=0A= ------=_NextPart_000_0002_01C70A59.FAEAAFC0 Content-Type: application/octet-stream Content-Transfer-Encoding: quoted-printable Content-Location: http://www.google-analytics.com/urchin.js //-- Google Analytics Urchin Module=0A= //-- Copyright 2005 Google, All Rights Reserved.=0A= =0A= //-- Urchin On Demand Settings ONLY=0A= var _uacct=3D""; // set up the Urchin Account=0A= var _userv=3D1; // service mode (0=3Dlocal,1=3Dremote,2=3Dboth)=0A= =0A= //-- UTM User Settings=0A= var _ufsc=3D1; // set client info flag (1=3Don|0=3Doff)=0A= var _udn=3D"auto"; // (auto|none|domain) set the domain name for cookies=0A= var _uhash=3D"on"; // (on|off) unique domain hash for cookies=0A= var _utimeout=3D"1800"; // set the inactive session timeout in seconds=0A= var _ugifpath=3D"/__utm.gif"; // set the web path to the __utm.gif file=0A= var _utsp=3D"|"; // transaction field separator=0A= var _uflash=3D1; // set flash version detect option (1=3Don|0=3Doff)=0A= var _utitle=3D1; // set the document title detect option = (1=3Don|0=3Doff)=0A= var _ulink=3D0; // enable linker functionality (1=3Don|0=3Doff)=0A= var _uanchor=3D0; // enable use of anchors for campaign = (1=3Don|0=3Doff)=0A= var _utcp=3D"/"; // the cookie path for tracking=0A= var _usample=3D100; // The sampling % of visitors to track (1-100).=0A= =0A= //-- UTM Campaign Tracking Settings=0A= var _uctm=3D1; // set campaign tracking module (1=3Don|0=3Doff)=0A= var _ucto=3D"15768000"; // set timeout in seconds (6 month default)=0A= var _uccn=3D"utm_campaign"; // name=0A= var _ucmd=3D"utm_medium"; // medium (cpc|cpm|link|email|organic)=0A= var _ucsr=3D"utm_source"; // source=0A= var _uctr=3D"utm_term"; // term/keyword=0A= var _ucct=3D"utm_content"; // content=0A= var _ucid=3D"utm_id"; // id number=0A= var _ucno=3D"utm_nooverride"; // don't override=0A= =0A= //-- Auto/Organic Sources and Keywords=0A= var _uOsr=3Dnew Array();=0A= var _uOkw=3Dnew Array();=0A= _uOsr[0]=3D"google"; _uOkw[0]=3D"q";=0A= _uOsr[1]=3D"yahoo"; _uOkw[1]=3D"p";=0A= _uOsr[2]=3D"msn"; _uOkw[2]=3D"q";=0A= _uOsr[3]=3D"aol"; _uOkw[3]=3D"query";=0A= _uOsr[4]=3D"aol"; _uOkw[4]=3D"encquery";=0A= _uOsr[5]=3D"lycos"; _uOkw[5]=3D"query";=0A= _uOsr[6]=3D"ask"; _uOkw[6]=3D"q";=0A= _uOsr[7]=3D"altavista"; _uOkw[7]=3D"q";=0A= _uOsr[8]=3D"search"; _uOkw[8]=3D"q";=0A= _uOsr[9]=3D"netscape"; _uOkw[9]=3D"s";=0A= _uOsr[10]=3D"cnn"; _uOkw[10]=3D"query";=0A= _uOsr[11]=3D"looksmart"; _uOkw[11]=3D"qt";=0A= _uOsr[12]=3D"about"; _uOkw[12]=3D"terms";=0A= _uOsr[13]=3D"mamma"; _uOkw[13]=3D"query";=0A= _uOsr[14]=3D"alltheweb"; _uOkw[14]=3D"q";=0A= _uOsr[15]=3D"gigablast"; _uOkw[15]=3D"q";=0A= _uOsr[16]=3D"voila"; _uOkw[16]=3D"kw";=0A= _uOsr[17]=3D"virgilio"; _uOkw[17]=3D"qs";=0A= _uOsr[18]=3D"live"; _uOkw[18]=3D"q";=0A= _uOsr[19]=3D"baidu"; _uOkw[19]=3D"wd";=0A= _uOsr[20]=3D"alice"; 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}=0A= else {=0A= if (iq=3D=3D-1 && ih=3D=3D-1) f.action+=3D"?"+p;=0A= else if (ih=3D=3D-1) f.action+=3D"&"+p;=0A= else if (iq=3D=3D-1) = f.action=3Df.action.substring(0,ih-1)+"?"+p+f.action.substring(ih);=0A= else = f.action=3Df.action.substring(0,ih-1)+"&"+p+f.action.substring(ih);=0A= }=0A= }=0A= return;=0A= }=0A= function __utmSetVar(v) {=0A= if (!v || v=3D=3D"") return;=0A= if (!_udo || _udo =3D=3D "") {=0A= _udh=3D_uDomain();=0A= if (_udn && _udn!=3D"") { _udo=3D" domain=3D"+_udn+";"; }=0A= }=0A= if (!_uVG()) return;=0A= var r=3DMath.round(Math.random() * 2147483647);=0A= _ubd.cookie=3D"__utmv=3D"+_udh+"."+_uES(v)+"; path=3D"+_utcp+"; = expires=3DSun, 18 Jan 2038 00:00:00 GMT;"+_udo;=0A= var s=3D"&utmt=3Dvar&utmn=3D"+r;=0A= if ((_userv=3D=3D0 || _userv=3D=3D2) && _uSP()) {=0A= var i=3Dnew Image(1,1);=0A= i.src=3D_ugifpath+"?"+"utmwv=3D"+_uwv+s;=0A= i.onload=3Dfunction() { _uVoid(); }=0A= }=0A= if ((_userv=3D=3D1 || _userv=3D=3D2) && _uSP()) {=0A= var i2=3Dnew Image(1,1);=0A= = i2.src=3D_ugifpath2+"?"+"utmwv=3D"+_uwv+s+"&utmac=3D"+_uacct+"&utmcc=3D"+= _uGCS();=0A= i2.onload=3Dfunction() { _uVoid(); }=0A= }=0A= }=0A= function _uGCS() {=0A= var t,c=3D"",dc=3D_ubd.cookie;=0A= if ((t=3D_uGC(dc,"__utma=3D"+_udh,";"))!=3D"-") = c+=3D_uES("__utma=3D"+t+";+");=0A= if ((t=3D_uGC(dc,"__utmb=3D"+_udh,";"))!=3D"-") = c+=3D_uES("__utmb=3D"+t+";+");=0A= if ((t=3D_uGC(dc,"__utmc=3D"+_udh,";"))!=3D"-") = c+=3D_uES("__utmc=3D"+t+";+");=0A= if ((t=3D_uGC(dc,"__utmx=3D"+_udh,";"))!=3D"-") = c+=3D_uES("__utmx=3D"+t+";+");=0A= if ((t=3D_uGC(dc,"__utmz=3D"+_udh,";"))!=3D"-") = c+=3D_uES("__utmz=3D"+t+";+");=0A= if ((t=3D_uGC(dc,"__utmv=3D"+_udh,";"))!=3D"-") = c+=3D_uES("__utmv=3D"+t+";");=0A= if (c.charAt(c.length-1)=3D=3D"+") c=3Dc.substring(0,c.length-1);=0A= return c;=0A= }=0A= function _uGC(l,n,s) {=0A= if (!l || l=3D=3D"" || !n || n=3D=3D"" || !s || s=3D=3D"") return "-";=0A= var i,i2,i3,c=3D"-";=0A= i=3Dl.indexOf(n);=0A= i3=3Dn.indexOf("=3D")+1;=0A= if (i > -1) {=0A= i2=3Dl.indexOf(s,i); if (i2 < 0) { i2=3Dl.length; }=0A= c=3Dl.substring((i+i3),i2);=0A= }=0A= return c;=0A= }=0A= function _uDomain() {=0A= if (!_udn || _udn=3D=3D"" || _udn=3D=3D"none") { _udn=3D""; return 1; }=0A= if (_udn=3D=3D"auto") {=0A= var d=3D_ubd.domain;=0A= if (d.substring(0,4)=3D=3D"www.") {=0A= d=3Dd.substring(4,d.length);=0A= }=0A= _udn=3Dd;=0A= }=0A= if (_uhash=3D=3D"off") return 1;=0A= return _uHash(_udn);=0A= }=0A= function _uHash(d) {=0A= if (!d || d=3D=3D"") return 1;=0A= var h=3D0,g=3D0;=0A= for (var i=3Dd.length-1;i>=3D0;i--) {=0A= var c=3DparseInt(d.charCodeAt(i));=0A= h=3D((h << 6) & 0xfffffff) + c + (c << 14);=0A= if ((g=3Dh & 0xfe00000)!=3D0) h=3D(h ^ (g >> 21));=0A= }=0A= return h;=0A= }=0A= function _uFixA(c,s,t) {=0A= if (!c || c=3D=3D"" || !s || s=3D=3D"" || !t || t=3D=3D"") return "-";=0A= var a=3D_uGC(c,"__utma=3D"+_udh,s);=0A= var lt=3D0,i=3D0;=0A= if ((i=3Da.lastIndexOf(".")) > 9) {=0A= _uns=3Da.substring(i+1,a.length);=0A= _uns=3D(_uns*1)+1;=0A= a=3Da.substring(0,i);=0A= if ((i=3Da.lastIndexOf(".")) > 7) {=0A= lt=3Da.substring(i+1,a.length);=0A= a=3Da.substring(0,i);=0A= }=0A= if ((i=3Da.lastIndexOf(".")) > 5) {=0A= a=3Da.substring(0,i);=0A= }=0A= a+=3D"."+lt+"."+t+"."+_uns;=0A= }=0A= return a;=0A= }=0A= function _uTrim(s) {=0A= if (!s || s=3D=3D"") return "";=0A= while ((s.charAt(0)=3D=3D' ') || (s.charAt(0)=3D=3D'\n') || = (s.charAt(0,1)=3D=3D'\r')) s=3Ds.substring(1,s.length);=0A= while ((s.charAt(s.length-1)=3D=3D' ') || = (s.charAt(s.length-1)=3D=3D'\n') || (s.charAt(s.length-1)=3D=3D'\r')) = s=3Ds.substring(0,s.length-1);=0A= return s;=0A= }=0A= function _uEC(s) {=0A= var n=3D"";=0A= if (!s || s=3D=3D"") return "";=0A= for (var i=3D0;i0) r=3Da.substring(i+1,i2); else return = ""; =0A= if ((i=3Da.indexOf(".",i2+1))>0) t=3Da.substring(i2+1,i); else return = ""; =0A= if (f) {=0A= return r;=0A= } else {=0A= var c=3Dnew = Array('A','B','C','D','E','F','G','H','J','K','L','M','N','P','R','S','T'= ,'U','V','W','X','Y','Z','1','2','3','4','5','6','7','8','9');=0A= return = c[r>>28&m]+c[r>>23&m]+c[r>>18&m]+c[r>>13&m]+"-"+c[r>>8&m]+c[r>>3&m]+c[((r= &7)<<2)+(t>>30&3)]+c[t>>25&m]+c[t>>20&m]+"-"+c[t>>15&m]+c[t>>10&m]+c[t>>5= &m]+c[t&m];=0A= }=0A= }=0A= function _uIN(n) {=0A= if (!n) return false;=0A= for (var i=3D0;i"9") && (c!=3D".")) return false;=0A= }=0A= return true;=0A= }=0A= function _uES(s,u) {=0A= if (typeof(encodeURIComponent) =3D=3D 'function') {=0A= if (u) return encodeURI(s);=0A= else return encodeURIComponent(s);=0A= } else {=0A= return escape(s);=0A= }=0A= }=0A= function _uUES(s) {=0A= if (typeof(decodeURIComponent) =3D=3D 'function') {=0A= return decodeURIComponent(s);=0A= } else {=0A= return unescape(s);=0A= }=0A= }=0A= function _uVG() {=0A= if((_udn.indexOf("www.google.") =3D=3D 0 || _udn.indexOf(".google.") = =3D=3D 0 || _udn.indexOf("google.") =3D=3D 0) && _utcp=3D=3D'/') {=0A= return false;=0A= }=0A= return true;=0A= }=0A= function _uSP() {=0A= var s=3D100;=0A= if (_usample) s=3D_usample;=0A= if(s>=3D100 || s<=3D0) return true;=0A= return ((__utmVisitorCode(1)%10000)<(s*100));=0A= }=0A= ------=_NextPart_000_0002_01C70A59.FAEAAFC0--