Priority Reports

Stem and Progenitor-Like Cells Contribute to the Aggressive

Behavior of Human Epithelial Ovarian Cancer
1 1 2 1
Sharmila A. Bapat, Avinash M. Mali, Chaitanyananda B. Koppikar, and Nawneet K. Kurrey
1
National Centre for Cell Science, Ganeshkhind, and 2Jehangir Hospital and Medical Centre, Pune, India

Abstract symptoms, rapid progression to peritoneal metastases, and poor

The cellular mechanisms underlying the increasing aggres- prognosis for patients. At advanced stages of the disease, the
siveness associated with ovarian cancer progression are ascites gets enriched with tumor cells that form multilayered
poorly understood. Coupled with a lack of identification of spheroids—the cellular nature of which has not yet been defined
specific markers that could aid early diagnoses, the disease (8). The present study constitutes the first report of the
becomes a major cause of cancer-related mortality in women. establishment, isolation, cloning, and propagation of the cellular
Here we present direct evidence that the aggressiveness of content of ovarian multilayered spheroids. We further show that
human ovarian cancer may be a result of transformation and the multilayered spheroid cells display heterogeneity in their
dysfunction of stem cells in the ovary. A single tumorigenic clonogenic, tumorigenic, and invasive properties and also exhibit
clone was isolated among a mixed population of cells derived stem and progenitor-like qualities.
from the ascites of a patient with advanced ovarian cancer.
During the course of the study, yet another clone underwent Materials and Methods
spontaneous transformation in culture, providing a model of Cell culture and clone derivation. The present study was approved by
disease progression. Both the transformed clones possess the Bio-Ethics Committee of National Centre for Cell Science and informed
stem cell–like characteristics and differentiate to grow in consent was obtained from the patients. Cells from the ascites sample from
an anchorage-independent manner in vitro as spheroids, a 63-year-old patient undergoing surgery for the removal of ovarian tumor
although further maturation and tissue-specific differentia- diagnosed as a malignant grade IV serous adenocarcinoma were isolated
tion was arrested. Significantly, tumors established from these using standard procedures. The scheme developed for clone isolation is
depicted in Supplementary Fig. 1A. Cultures were incubated in a humidified
clones in animal models are similar to those in the human
tissue culture incubator at 37jC, 5% CO2 atmosphere and were fed with
disease in their histopathology and cell architecture. Further- fresh medium-MEM (E) supplemented with 5% fetal bovine serum and 1%
more, the tumorigenic clones, even on serial transplantation nonessential amino acids every 4 days. Initially, the multilayered spheroids
continue to establish tumors, thereby confirming their did not adhere to the surface of culture dishes and were collected at each
identity as tumor stem cells. These findings suggest that: (a) passage by centrifugation, resuspended in fresh medium and plated out,
stem cell transformation can be the underlying cause of continuing until substrate adherence was achieved. Sixty-five individual
ovarian cancer and (b) continuing stochastic events of stem sublines were isolated from an attached spheroid (Supplementary Fig. 1B),
and progenitor cell transformation define the increasing following a 1:25 split ratio for marking single cells and picking up the clones
aggression that is characteristically associated with the developed with cloning cylinders.
disease. (Cancer Res 2005; 65(8): 3025-9) Determination of capacity for anchorage-independent growth
in vitro. Standard clonogenicity and spheroid generation assay procedures
were followed. Briefly, for colony formation, subconfluent cultures were
Introduction harvested, suspended in 0.5% agarose at a concentration of 5  103 cells per
A hypothesis based on the similarities between normal and milliliter, and seeded in 35 mm plates containing a basal layer of 1%
tumor stem cells postulates that the former could themselves be agarose. Colonies were counted after 2 weeks under a microscope at 10
targets of stochastic transforming mutations or give rise to a magnification. For sphere formation, 106 cells were suspended in 3 mL
medium (plain MEM without fetal bovine serum), and plated in
hierarchy of progenitors and differentiated cells that make up the
bacteriologic dishes to prevent adherence. Spheres were counted at 3 and
tumor mass (1). Such approaches have been extensively investi- 6 days after plating. All experiments were done in triplicate.
gated in leukemia (2, 3) and indicate that along with the Evaluation of tumorigenicity in nude mice by subcutaneous and
establishment of a hierarchy, the transformed leukemic blast stem metastases by intraperitoneal injections. Animal experimentation was
cells may be differentiation arrested (4). done in accordance with the rules and regulations of the National Centre
An understanding of the emergence and organization of solid for Cell Science Animal Ethics Committee. Tumorigenicity was assessed by
cancers at the cellular level has only recently been addressed, and injecting A2 and A4-T cells (1  106 cells per milliliter in PBS) into nude
has resulted in the prospective isolation and characterization of mice, either s.c. into the thigh or i.p. Animals were kept in pathogen-free
cancer stem cells in breast (5) and brain (6, 7). Ovarian cancer is an conditions and tumor growth was assessed every 3 days for 2 months or
extremely aggressive disease associated with a lack of early until the tumor diameter was 1 cm, whereupon animals were sacrificed to
dissect out tumors. Mice given i.p. injections were observed for lethargy and
poor appetite, and were sacrificed when their movements were compro-
mised due to accumulation of ascites in the abdomen. To determine the
Note: A.M. Mali and N.K. Kurrey contributed equally to this paper. extent of metastases and infiltration of the cells, samples of the omentum,
Supplementary data for this article are available at Cancer Research Online (http:// stomach, intestine, liver, lungs, kidney, and heart from the experimental
cancerres.aacrjournals.org/). animals were collected and fixed for histologic examination (H&E staining),
Requests for reprints: Sharmila A. Bapat, National Centre for Cell Science, Lab 4,
or collagenase digested, washed thrice with PBS, filtered through 40 Am cell
NCCS Complex, Ganeshkhind, Pune, Maharashtra 411007, India. Phone: 91-202-569-
0922; Fax: 91-202-569-2259; E-mail: sabapat@nccs.res.in. strainers and reinjected into nude mice (1  106 cells per milliliter in PBS)
I2005 American Association for Cancer Research. to determine sequential tumorigenicity.

www.aacrjournals.org 3025 Cancer Res 2005; 65: (8). April 15, 2005

Downloaded from cancerres.aacrjournals.org on February 8, 2022. © 2005 American Association for Cancer
Research.
Cancer Research

Immunofluorescence staining. Cells were grown on glass coverslips for attributed to either, their being derived from committed non-
48 hours, fixed in ice-cold methanol or 4% paraformaldehyde at 4jC for 10 immortalized progenitors, or due to the selective pressure imposed
minutes, washed with PBS and blocked at room temperature for 30 minutes by an unvarying culture regime.
with 1% normal goat serum. Primary antibody incubation followed at room The immortalized clones showed variations in morphology and
temperature for 30 minutes. After washing thoroughly with PBS, cells were
growth rates (Fig. 1A). The A2 clone had the shortest doubling time
further probed with FITC/PE-tagged secondary antibody. Coverslips were
of 18 hours, whereas the others displayed doubling times ranging
mounted in antifade solution and viewed under confocal microscope (Carl
Zeiss, Jena, Germany). The list of primary antibodies used and their specific
from 34 to 47 hours. A2 cells grew as small, spindle-shaped cells
dilutions can be provided on request. that were not contact-inhibited, could form foci, and grow to high
RNA isolation and reverse transcription-PCR. Total RNA was densities in culture. A4 cells were similar in morphology to A2 cells,
extracted from cells using TRIzol. RNA was reverse-transcribed using but lacked their growth potential at early passages. Since all cell
cDNA Synthesis Kit, (Invitrogen, Carlsbad, CA). cDNA was amplified using cultures were maintained at identical growth conditions in the
1 AL of the reverse transcriptase reaction products in 25 AL with 10 pmol of same culture milieu, their varying growth kinetics could be
the primers for 35 cycles. Each cycle consisted of 30 seconds of denaturation attributed to cell intrinsic mechanisms.
at 94jC, 30 seconds of annealing and 60 seconds of extension at 72jC. The Semiquantitative reverse transcription (RT)-PCR was carried out
PCR products were electrophoresed on a 1% agarose gel and visualized by in 10 representative clones in order to identify the nature of the
ethidium bromide staining. The primer sequences used for cDNA isolated cells. Coexpression of cytokeratin 18 and vimentin was
amplification ( forward and reverse) can be provided on request. h-Actin evident in all the clones (Fig. 1B). Similarly, the growth factor
was used as the internal control in all reactions.
receptors c-met and epidermal growth factor receptor were up-
Statistical analysis. Results of experimental points obtained from
regulated as was the surface adhesion molecule CD44. Cells also
multiple experiments are reported as mean F SD. The significance of
differences in mean values were determined using Student’s t test. expressed E-cadherin; an exception being the A4 clone in which
E-cadherin transcripts were not detected (Fig. 1C). Correspondingly,
Snail, a known mediator of epithelial-mesenchymal transition
Results and Discussion
through transcriptional repression of E-cadherin in ovarian cancer
The stringent, low-density culture system gave rise to 65 clones, 19 (9), was expressed in the A4 cells and to a lesser extent in the B7
of which were spontaneously immortalized (designated A2, A3, A4, clone. These expression patterns indicate the mesothelial nature of
A5, B2, B7, C4, D4, F1, F2, F3, F4, F5, E1, E2, G5, G7, H1, and H4). the cells (10)—which is in agreement with the current understanding
Further work with these clones was carried out with cultures of epithelial ovarian carcinoma resulting from the transformation of
between the 8th and 12th passages after cloning. The remaining a primitive mesothelium, i.e., the ovarian surface epithelium (11).
clones underwent senescence within 4 to 5 weeks of cloning, in a Another member of the Snail family—Slug—was also seen to be
manner similar to that of the adherent populations initially obtained expressed at comparable levels in the clones without the expected
from the sample (Supplementary Fig. 1A). This senescence could be association of E-cadherin down-regulation (considered a marker

Figure 1. Isolation and characterization of tumorigenic clones from a heterogeneous population in the ovarian multilayered spheroids. A, growth kinetics of the 19
immortalized clones isolated from the multilayered spheroids. Cell proliferation assays were carried out over a period of 7 days. Figures at the top of each set of
columns indicates the doubling time of that clone. The experiment was conducted in triplicate and average number of cells was plotted against time (days); B, RT-PCR
analyses of expression of cytokeratin 18, vimentin, c-met, epidermal growth factor receptor, CD44, and mRNA in 10 selected clones that indicate the mesothelial nature
of the isolated clones; C , RT-PCR analyses of expression of E-cadherin, Snail, SCF, c-kit, and Slug mRNA in the 10 clones that indicate heterogeneity of
expression of epithelial-mesenchymal transition and survival-associated molecules. h-Actin mRNA expression was used as an internal control in (B) and (C ).

Cancer Res 2005; 65: (8). April 15, 2005 3026 www.aacrjournals.org

Downloaded from cancerres.aacrjournals.org on February 8, 2022. © 2005 American Association for Cancer
Research.
 Human Ovarian Cancer Stem Cells

Figure 2. Spheroid and tumor characterization. A, (i, top ) phase contrast image of typical spheroids generated in culture; (bottom ) Ki-67 expression
(immunofluorescence) indicating self-renewing A2 spheroids (A2Sp-X100) and A4-T spheroids (A4TSp-60X); (ii ) RT-PCR expression of Nanog, Oct-4, and Nestin
mRNA derived from monolayers (A2 and A4-T) and spheroids (A2Sp and A4-TSp). h-Actin mRNA expression used as an internal control; (iii ) Nestin expression
(immunofluorescence) in monolayers (M-X10) and spheroids (Sp-X20); B (i) RT-PCR analyses of expression of cytokeratin 18, vimentin, and E-cadherin mRNA in
monolayers and spheroids. h-Actin mRNA expression used as an internal control; (ii ) immunofluorescent staining of cell-specific markers (60). Ck8/18 (red) and
vimentin (green ), alkaline phosphatase expression, stage-specific embryonal antigen 1 and 4 expression; C, representative photomicrographs of H&E stained sections
(4) of patient’s tumor (top ) and s.c. tumors derived from A2 and A4-T cells in nude mice (bottom ). A4-T tumors seem to be more differentiated than A2 tumors initiated
at the same time; D, representative photomicrographs of H&E stained sections (4) tumor metastases to omentum, stomach, intestine, liver, pancreas, and heart.

for epithelial-mesenchymal transition). This indicated that Slug, in 593 F 72.6 (mean F SD) colonies in soft agar within 14 days of
ovarian cancer, may have alternative function(s). Recently, Slug has culture. After f20 passages, A4 cells (thenceforth termed as A4-T
been described as an important mediator of hematopoietic stem cells) gave indications of continuing mutagenesis, evinced through
cell survival through the c-kit-SCF pathway (12). Our analysis an increased proliferation rate and clonogenicity-forming 1,915 F
revealed that c-kit and SCF are differentially expressed in the 10 142.66 soft agar colonies within 10 days by the 25th passage with an
clones—ranging from total absence to high-level expression
average larger colony size than that of the A2 colonies in soft agar
(Fig. 1C) from which a specific definitive role for Slug through
the same pathway cannot be arrived at. Nevertheless, the varying (Supplementary Figs. 2A, B, and C, respectively). When grown in
expression patterns extend the earlier observation of differences in suspension, only cells of the tumorigenic clones, A2 and A4-T gave
growth patterns of the clones to their differential levels of rise to organized spheroids (Fig. 2A, i, top) with A4-T showing a
expression of molecules known to be associated with epithelial- much higher spheroid-forming capacity than A2 (data not shown).
mesenchymal transition and cell survival. The spheroids are self-renewing and have been maintained for over
In exploring the capacity for anchorage-independent growth of 15 to 20 passages in vitro—a capacity that correlates with a high
the 19 clones, only the A2 cells expressed this potential and formed level expression of Ki-67, a proliferation marker (Fig. 2 A, i, bottom).

www.aacrjournals.org 3027 Cancer Res 2005; 65: (8). April 15, 2005

Downloaded from cancerres.aacrjournals.org on February 8, 2022. © 2005 American Association for Cancer
Research.
Cancer Research

Table 1. In vivo tumorigenicity of A2, A4, and A4-T cells

Cells Site of injection Tumor formation Ascites formation Metastases Mortality Tumor formation on
serial transplantation

A2 (P8-P10) s.c. 9/9 — — — 3/3

i.p. 4/4 2/4 4/4 1/4 —
A4 (P8) s.c. 0/2 — — — —
A4-T (P25-P28) s.c. 6/6 — — — 3/3
i.p. 5/5 3/5 4/5 2/5 3/3

The A2 and A4-T cells were further analyzed for expression of serous adenocarcinomas in both the clones. In A4-T tumors, tumor
specific markers known to be associated with stem and/or infiltration to the other layers was also evident, whereas abnormal
progenitor cells including Nestin, a progenitor marker (13), and mitoses were more frequent in the A2 intestinal tumors. The extent
Oct4 and Nanog, transcriptional determinants essential for the of tumor infiltration was higher in the pancreas and liver, which
maintenance of an undifferentiated state (14). Nestin and Nanog showed moderate differentiation. Tumors formed in the heart were
were distinctly expressed in A2 as well as A4-T monolayers, and relatively solid and undifferentiated, with growth being observed
were reduced on differentiation into spheroids (Fig. 2 A, ii and iii), within the lumen and attached to the endocardium. No signs of
whereas Oct4 was expressed in A2 monolayers but was totally metastases to the kidneys, lungs, or spleen could be detected.
absent in spheroids. The expression of these three markers To conclusively show that the clones A2 and A4-T are
indicates a possible multipotent nature of the A2 and A4-T clones. indisputably tumorigenic stem cells, s.c. tumor–derived cells were
Tissue-specific differentiation is a unique characteristic of adult further reinjected into mice. Successful serial tumor formation
stem cells (15). Unfortunately, the development of cell lineages from confirmed the existence of cells in both the clones that were able to
adult stem cells in the ovary is not as well delineated as in other tissues propagate the tumor even in consecutive generations. Such cells in
like bone marrow. Although the existence of mesothelial and germ the tumor mass are retained due to stem cell renewal mechanisms,
line stem cells in the postnatal adult mammalian ovary have been despite overall tumor differentiation and progression, and provide
implied (16, 17), their isolation and characterization has not been strong evidence of the existence of a small fraction in the tumor
achieved as yet. Since spheroid formation itself represents a capable of driving tumorigenesis.
differentiation event, we probed for the expression of markers in In conclusion, the two transformed clones, A2 and A4-T,
spheroids that could indicate either differentiation into ovarian represent tumor stem cells because (a) they self-renew and are
surface epithelium (cytokeratin 18 and vimentin), granulosa (cytoke- clonogenic, (b) differentiate in vitro to form organized spheroids in
ratin 18 and E-cadherin), or germ cells (alkaline phosphatase, stage- suspension, (c) express multipotency and tissue-specific differen-
specific embryonal antigens 1, 3, and 4, and tumor rejection antigen tiation markers, (d) express self-renewal mechanisms in vivo
1-60 and 1-81). Indeed, differentiation along these three ovarian (sequential tumorigenicity), and (e) undergo in vivo differentiation
lineages could be identified (Fig. 2 B, i and ii), albeit the fact that germ to produce a disease similar to that in the patient.
cell differentiation was aberrant (alkaline phosphatase, stage-specific The data thus provides evidence of the origin of ovarian cancer
embryonal antigens 1 and 4 were expressed, whereas stage-specific from the transformation of a stem cell that has extensive self-
embryonal antigen 3 and tumor rejection antigens 1-60 and 1-81 could renewal capabilities and also undergoes differentiation (A2 clone).
not be detected). Tissue-specific differentiation thus seems to be Continuing stochastic mutagenic events in other stem/progenitor
incomplete and may be blocked in a manner akin to the maturation populations (evinced in the spontaneous transformation of A4 to
arrest during blast crisis (18) or in mammary tumor spheroids (19). A4-T), further suggests a mechanism for the increased aggressive-
The in vivo correlate of the in vitro clonogenic potential of the ness during tumor progression that is usually associated with
candidate tumor stem cells was further assessed. Both clones formed ovarian cancer. Further characterization of the tumorigenic
tumors and underwent metastasis in nude mice, with A4-T cells populations will allow for the identification of molecules expressed
being more aggressive than A2 cells (Table 1). The average latency in these cells that could serve as targets to eliminate this fraction of
periods in case of A2 cells for s.c. tumor formation (f1 cm diameter) cancer cells that can rapidly develop the critical tumor cell mass.
and i.p. ascites formation were 28 and 55 days, respectively, whereas Consequently, defining the unique properties of these tumor stem
those for A4-T cells were 7 and 18 days, respectively. The tumors were cells remains a high priority for developing early diagnostic and
classified as serous adenocarcinomas, and exhibited a high degree of effective therapeutic strategies.
similarity with the primary tumor in the patient. An appreciable
difference was that A2 tumors were relatively undifferentiated as
Acknowledgments
seen from their solid pattern and limited gland formation in
comparison with the A4-T tumors; from these differences in the cell Received 3/11/2004; revised 2/9/2005; accepted 2/11/2005.
Grant support: Funded by the Department of Biotechnology, Ministry of Science
architecture, it was inferred that the A4-T tumors were more similar and Technology, Government of India, New Delhi. N.K. Kurrey received a research
to those in the human disease (Fig. 2C). fellowship from the Council of Scientific and Industrial Research.
In the i.p. mode of introduction into nude mice, along with The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
ascites formation, omental growth and peritoneal metastases were 18 U.S.C. Section 1734 solely to indicate this fact.
evident in both clones. The pathology of the omental mass was We thank Dr. G.C. Mishra, Director, National Centre for Cell Science (Pune, India) for
similar to that of the s.c. tumors (Fig. 2D). In the stomach and encouragement and support, Ms. A.N. Atre for assistance in capturing images on the
confocal microscope, the staff of the Experimental Animal Facility at the National
intestine, tumors developed mainly on the serosal surface, showed Centre for Cell Science, and Dr. Avanti Golwilkar and late Dr. J. Abhyankar for the
atypical nuclei at high frequency and progressed to form papillary histopathologic analyses and discussions.

Cancer Res 2005; 65: (8). April 15, 2005 3028 www.aacrjournals.org

Downloaded from cancerres.aacrjournals.org on February 8, 2022. © 2005 American Association for Cancer
Research.
 Human Ovarian Cancer Stem Cells

References 7. Galli R, Binda E, Orfanelli U, et al. Isolation and 13. Pessina A, Eletti B, Croera C, Savalli N, Diodovich C,
characterization of tumorigenic, stem-like neural pre- Gribaldo L. Pancreas developing markers expressed on
1. Reya T, Morrison SJ, Clarke MF, Weissman IL. Stem cursors from human glioblastoma. Cancer Res 2004;64: human mononucleated umbilical cord blood cells.
cells, cancer, and cancer stem cells. Nature 2001; 7011–21. Biochem Biophys Res Commun 2004;323:315–22.
414:105–11. 8. Burleson KM, Casey RC, Skubitz KM, Pambuccian SE, 14. Chambers I, Smith A. Self-renewal of teratocarcinoma
2. Hope KJ, Jin L, Dick JE. Acute myeloid leukemia Oegema TR Jr, Skubitz AP. Ovarian carcinoma ascites and embryonic stem cells. Oncogene 2004;23:7160.
originates from a hierarchy of leukemic stem cell spheroids adhere to extracellular matrix components 15. Morrison SJ, Shah NM, Anderson DJ. Regulatory
classes that differ in self-renewal capacity. Nat Immunol and mesothelial cell monolayers. Gynecol Oncol 2004; mechanisms in stem cell biology. Cell 1997;88:287–98.
2004;5:738–43. 93:170–81. 16. Bukovsky A, Bukovsky A, Candle MR, Swetlikova M,
3. Bonnet D, Dick JE. Human acute myeloid leukemia is 9. Kurrey NK, Amit K, Bapat SA. Snail and Slug are major Upadhyaya NB. Formation of new primary follicles in
organized as a hierarchy that originates from a determinants of ovarian cancer invasiveness at the adult human ovaries. Reprod Biol Endocrinol 2004;2:20.
primitive hematopoietic cell. Nat Med 1997;3:730–7. transcription level. Gynecol Oncol. In press 2005. 17. Johnson J, Canning J, Kaneko T, Pru JK, Tilly JL.
4. Lessard J, Sauvageau G. Bmi-1 determines the 10. Herrick S, Mutsaers SE. Mesothelial progenitor cells Germline stem cells and follicular renewal in the
proliferative capacity of normal and leukaemic stem and their potential in tissue engineering. Int J Biochem postnatal mammalian ovary. Nature 2004;428:145–50.
cells. Nature 2003;423:255–60. and Cell Biol 2004;36:621–42. 18. Jamieson CH, Ailles LE, Dylla SJ, et al. Granulocyte-
5. Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison 11. Auersperg N, Wong AS, Choi KC, Kang SK, Leung PC. macrophage progenitors as candidate leukemic stem
SJ, Clarke MF. Prospective identification of tumorigenic Ovarian surface epithelium: biology, endocrinology and cells in blast-crisis CML. N Engl J Med 2004;351:657–67.
breast cancer cells. Proc Natl Acad Sci U S A 2003; pathology. Endocr Rev 2001;22:255–88. 19. Petersen OW, Ronnov-Jessen L, Howlett AR, Bissell
100:3983–8. 12. Perez-Losada J, Sanchez-Martin M, Rodriguez-Garcia MJ. Interaction with basement membrane serves to
6. Singh SK, Clarke ID, Terasaki M, et al. Identification of A, et al. Zinc-finger transcription factor Slug contributes rapidly distinguish growth and differentiation pattern
a cancer stem cell in human brain tumour. Cancer Res to the function of the stem cell factor c-kit signaling of normal and malignant human breast epithelial cells.
2003;63:5821–8. pathway. Blood 2003;100:1274–86. Proc Natl Acad Sci U S A 1992;89:9064–8.

www.aacrjournals.org 3029 Cancer Res 2005; 65: (8). April 15, 2005

Downloaded from cancerres.aacrjournals.org on February 8, 2022. © 2005 American Association for Cancer
Research.
Stem and Progenitor-Like Cells Contribute to the Aggressive
Behavior of Human Epithelial Ovarian Cancer
Sharmila A. Bapat, Avinash M. Mali, Chaitanyananda B. Koppikar, et al.

Cancer Res 2005;65:3025-3029.

Updated version Access the most recent version of this article at:
http://cancerres.aacrjournals.org/content/65/8/3025

Supplementary Access the most recent supplemental material at:

Material http://cancerres.aacrjournals.org/content/suppl/2005/04/18/65.8.3025.DC1

Cited articles This article cites 16 articles, 4 of which you can access for free at:
http://cancerres.aacrjournals.org/content/65/8/3025.full#ref-list-1

Citing articles This article has been cited by 47 HighWire-hosted articles. Access the articles at:
http://cancerres.aacrjournals.org/content/65/8/3025.full#related-urls

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Subscriptions Department at pubs@aacr.org.

Permissions To request permission to re-use all or part of this article, use this link
http://cancerres.aacrjournals.org/content/65/8/3025.
Click on "Request Permissions" which will take you to the Copyright Clearance Center's
(CCC)
Rightslink site.

Downloaded from cancerres.aacrjournals.org on February 8, 2022. © 2005 American Association for Cancer
Research.