Immunostaining of Cultured Cells. Anit-MISRII rabbit polyclonal
antibodies (153p兾MISRII) were developed for Western blot
analysis in the Pediatric Surgical Research Laboratories (35).
Immunofluorescence was performed on MOVCAR 7 and 4306
cells by using 153p as described (25). Images were obtained by
using either epifluorescent (Nikon Eclipse E400 microscope,
SPOT camera, and
SPOT ADVANCE software) or confocal mi-
crosc opy (Leica TCS NT confocal microscope,
C ONFOCAL soft-
ware Version 2.5 Build 1227, and krypton 568-nm laser; Leica,
For BCRP1 immunostaining, cells were double-labeled in sus-
pension with Hoechst 33342 and BCRP1 antibody as described in
ref. 5 and as detailed in Supporting Methods.
Reverse Transcriptase PCR. Total RNA from MOVCAR 7 and 4306
SP and NSP cells was extracted by using the Qiagen (Valencia, CA)
RNeasy Mini Kit (catalog no. 74104) according to the manufac-
turer’s instructions, and 0.5
g of RNA was reverse transcribed into
cDNA by using Superscript II reverse transcriptase as directed by
the manufacturer (Invitrogen). All RT-PCRs were run for 30 cycles
with an annealing temperature of 57°C and separated on 2%
agarose gels. Mouse PCR primers are as in Table 4, which is
published as supporting information on the PNAS web site.
Growth Inhibition by MIS
. MTT assay was used to assess
proliferation inhibition. MOVCAR 7 and 4306 cells were har-
vested, sorted for SPs and NSPs, and plated in the inner wells of
96-well plates at 1,000 cells per well in 200
l of medium per well.
Twenty-four hours after plating, each set of 10 wells of SP or NSP
cells was treated with 10
g兾ml recombinant human MIS (25), 4 nM
paclitaxel (6 mg兾ml; NovaPlus, Irving, TX), a 4-nM doxorubicin
hydrochloride injection (2 mg兾ml; NovaPlus), or media alone. At
day 5 or 7 of incubation, cell viability was quantified by measuring
mitochondrial activity (38) on an ELISA plate reader at an absor-
bance of 550 nm to generate an OD proportional to the relative
abundance of live cells in a given well.
Growth of MOVCAR 7 SP Cells
. MOVCAR 7 SP and NSP cells
were sorted and injected into T and B cell-deficient 6-week-old
female Swiss nude mice in equal numbers (first experiment, 5.0 ⫻
; second experiment, 7.5 ⫻ 10
) into the dorsal fat pad between
the scapulae. Mice were housed in the Edwin L. Steele Laboratory
for Tumor Biology under American Association for Laboratory
Animal Science guidelines with the approval of the MGH Animal
Care and Use Committee (protocol no. 2005N000384).
Purification of Recombinant Human MIS. The human MIS gene was
transfected into CHO cells, amplified, purified, and maintained in
a dedicated facility in the Pediatric Surgical Research Laboratories
for use in this study as described in ref. 39. MIS levels were
measured by using human MIS-specific ELISA (40). MIS was
purified by a combination of lectin affinity chromatography and
FPLC anion-exchange chromatography (39). The MIS purified by
this method causes regression in the organ culture bioassay for MIS
Statistical Analysis. In MTT assays, MIS-, doxor ubicin-, and
paclit axel-treated and untreated samples were analyzed by using
the univariant two-tailed Student t test, with P ⱕ 0.05 c onferring
st atistical significance. All experiments were performed in
We thank Dr. Rakesh K. Jain for per mitting us to perform the nude mice
experiments in the Edwin L. Steele L aboratory for Tumor Biology; Dr.
Thomas Flotte and Ms. Margaret E. Sher wood for guidance in perfor m-
ing confocal microscopy; Dr. Tyler Jacks for access to the 4306 cell line
developed by D.M.D.; and Drs. Michael Seiden, Jose Teixeira, and David
Scadden for many suggestions and an internal review of the manuscript.
Human tissues were provided through the Ovarian Cancer Tumor Bank
at the Massachusetts General Hospit al (MGH), supported by the
Dana–Farber Harvard Cancer Center Ovarian SPORE (1P50CA105009)
and grants from the OCEAN Foundation at MGH and the Harvard Stem
Cell Institute. P.P.S. is supported by Ruth L. Kirchstein National
Research Service Award T32 Training Grant in Cancer Biology
5T32CA071345-10. D.M.D. is supported by the Burroughs Wellcome
Fund Career Award. D.C. is supported by National Institutes of Health
(NIH) Grants P50 CA083638 and U01 CA084242. P.K.D. and D.T.M.
are supported by NIH Grants CA17393 and HD32112. D.T.M. is also
supported by the Edmund C. Lynch, Jr. Cancer Fund through Dr. Jeff rey
Gelfand. This work was also supported by contributions to the Pediatric
Surgical Research Laboratories from the McBride Family and the W.
Gerald Austen Funds.
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July 25, 2006