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Clin Cancer Res 2003 Wall 2487 96

Clin Cancer Res 2003 Wall 2487 96

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2003;9:2487-2496. Published online July 1, 2003.
Clin Cancer Res 
Lucy Wall, Frances Burke, Caroline Barton, et al.
in Vitro 
and
in Vivo 
Induces Apoptosis in Ovarian Cancer Cells
γ 
IFN-
Updated Version
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.permissions@aacr.orgDepartment atTo request permission to re-use all orpart of this article, contact the AACR Publications American Association for Cancer ResearchCopyright © 2003  on July 5, 2011clincancerres.aacrjournals.orgDownloaded from
 
IFN-
Induces Apoptosis in Ovarian Cancer Cells
in Vivo
and
in Vitro
1
Lucy Wall,
2
Frances Burke,
2
Caroline Barton,John Smyth, and Fran Balkwill
3
Cancer Research UK, University Department of Oncology, WesternGeneral Hospital, Edinburgh EH4 2XU, United Kingdom [L. W.,J. S.], and Cancer Research UK Translational Oncology Laboratory,Barts and The London, Queen Mary’s School of Medicine andDentistry, London EC1M 6BQ, United Kingdom [F. Bu., C. B.,F. Ba.]
ABSTRACT
 Purpose:
The purpose of this study was to compare
invitro
and
in vivo
responses of primary human tumor cells toIFN-
.
 Experimental Design:
IFN-
may have therapeutic ac-tivity in patients with ovarian cancer. We showed previouslythat this cytokine had direct antiproliferative activityagainst human ovarian cancer cell lines and xenografts innude mice. To further understand the role of IFN-
inovarian cancer, we compared its action on 8 ovarian cancercell lines with the response of 14 primary cultures of ovariantumor cells isolated from patients with ascitic disease. Apilot clinical study was then conducted to see whether IFN-
would also induce apoptosis in human tumor cells
in vivo
.Six patients with ascites and advanced disease were givenIFN-
by i.p. injection, and sequential samples of asciteswere analyzed.
 Results:
IFN-
had antiproliferative activity in 8 of 8ovarian cancer cell lines and 11 of 14 primary cultures. Thisactivity was dose related, and cleaved poly(ADP-ribose) po-lymerase in protein isolates provided evidence of apoptosis.In the clinical study, there was a 3 log
10
pharmacokineticadvantage in peritoneal compared with plasma levels of IFN-
. In two of six patients, there was a 90% reduction intumor cells in ascites after IFN-
treatment, and this wasrelated to clinical benefit as assessed by intervals betweenparacentesis. In all six patients, there were increasedamounts of cleaved poly(ADP-ribose) polymerase in proteinextracts of ascitic cells sampled during IFN-
treatment.
Conclusions:
IFN-
induces apoptosis
in vitro
and
invivo
in human epithelial ovarian cancer.
INTRODUCTION
IFN-
is a potent immunomodulatory, antiviral, and anti-proliferative cytokine that has anticancer activity. IFN-
directlyinhibits human tumor cell growth and induces apoptosis. Exten-sive experiments in a range of animal cancer models suggestthat endogenous IFN-
may be involved in immune surveillanceof tumors via a combination of lymphocyte-mediated responses,direct actions on tumor cells, and inhibition of tumor angiogen-esis (reviewed in Ref. 1). We demonstrated previously thathuman IFN-
can directly inhibit the growth of human ovariantumor cells
in vitro
and human ovarian cancer xenografts grow-ing in nude mice. IFN-
had antiproliferative activity in three of four ovarian cancer cell lines, and this was associated with theinduction of apoptosis (2). The effects were not immediate butrequired 2–3 days of exposure to IFN-
for an irreversible effecton cell survival. Treatment of two of three human ovariancancer xenografts with IFN-
at clinically relevant doses in-duced tumor cell growth arrest and apoptosis. Likewise, thiseffect was not immediate but required 14–21 days of continuoustreatment to significantly increase mouse survival (3).IFN-
has been used in the treatment of patients withadvanced ovarian cancer. In a Phase II trial, 108 patients withresidual disease documented at second-look laparotomy afterfirst-line cisplatin-based chemotherapy were treated with i.p.IFN-
twice a week for 3–4 months (4). Ninety-eight patientswere assessable, with 23 patients achieving a complete re-sponse, and 8 patients achieving a partial response. Age andtumor size were the only factors affecting response to IFN-
.The probability of IFN-
response was independent of previousresponse to first-line chemotherapy. In a randomized Phase IIIstudy, 148 women treated with cisplatin and cyclophosphamideas first-line chemotherapy for ovarian cancer were randomlyallocated to receive additional IFN-
three times weekly onalternate weeks (5). IFN-
administration was associated with asignificant increase in progression-free survival, but an observedincrease in overall survival was not statistically significant.Response to IFN-
was not related to volume of disease.The mechanisms of action of IFN-
treatment in patientswith ovarian cancer are not known. Because we had evidencefrom ovarian cancer cell lines and xenografts that direct actionof IFN-
on tumor cells might be important, the aim of the work described in this paper was to investigate the direct actions of IFN-
on primary ovarian epithelial cancer cells, both
in vitro
and
in vivo
in patients with advanced ascitic disease.
MATERIALS AND METHODS
Ovarian Cancer Cell Lines.
The OAW42 cell line wasderived from the ascites of a patient with serous adenocarci-noma treated previously with cisplatin (Ref. 6; obtained from
Received 10/15/02; revised 3/5/03; accepted 3/5/03.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked
advertisement 
in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.
1
Supported by Cancer Research UK.
2
Both authors contributed equally to this work.
3
To whom requests for reprints should be addressed, at Cancer Re-search UK Translational Oncology Laboratory, Barts and The London,Queen Mary’s School of Medicine and Dentistry, Charterhouse Square,London EC1M 6BQ, United Kingdom. Phone: 020-7882-6106; Fax:020-7882-6110; E-mail: frances.balkwill@cancer.org.uk.
2487
Vol. 9, 2487–2496, July 2003
Clinical Cancer Research
 
the European Tissue Culture Collection, Porton Down, UnitedKingdom). Ovcar-3 originated from the ascites of a patient withpoorly differentiated papillary adenocarcinoma treated previ-ously with chemotherapy (7). Ovcar-4 originated from the as-cites of a patient with adenocarcinoma treated previously withchemotherapy (8). Ovcar-5 was derived from the ascites of apatient with untreated adenocarcinoma (9). The Ovcar cell lineswere obtained from T. Hamilton (Fox Chase Cancer Institute,PA). PEO1 was derived from ascites in a patient with poorlydifferentiated adenocarcinoma. The PEO1 (CDDP) cell line wasobtained by continuous exposure of the PEO1 cell line toincreasing concentrations of cisplatin. PEO14 was derived fromthe ascites of a patient with a well-differentiated serous adeno-carcinoma who had not received prior drug therapy. PEO16originated from the ascites of a patient with serous adeno-carcinoma who had been treated previously with radiother-apy. All of the PEO cell lines were raised in the CancerResearch UK Laboratories in Edinburgh (10). SW626 is anadenocarcinoma cell line initially described in Ref. 11. Thiscell line was obtained from the American Type CultureCollection (Manassas, VA).
rhIFN-
.
4
rhIFN-
for the
in vitro
experiments was pro-vided by Roussel UCLAF as a lyophilized preparation (20
10
6
units/vial) and was
95% pure. It was diluted in water andstored in aliquots at
70
°
C until use. Endotoxin levels were lessthan 0.24 endotoxin units/vial, and the specific activity was 2
10
7
units/mg protein. rhIFN-
1b (200
g/ml; specific activity,2
10
7
units/mg protein) was used for the clinical trial (Boeh-ringer Ingelheim, Bracknell, United Kingdom). It was suppliedas an isotonic solution, stored at
4
°
C until use and diluted to50 ml in normal saline immediately before administration. TheIFN-
was administered by slow i.p. injection on the oppositeside of the abdomen to the ascitic drain at a site that had beenmarked by ultrasound.
Ascites Sample Collection and Preparation.
Sampleswere collected from patients with advanced ovarian cancer atthe time of therapeutic paracentesis or surgery. Written consentwas obtained from all patients. Cells were collected by centrif-ugation at 500
g
for 5 min. If necessary, red cells wereremoved by centrifugation through Ficoll according to the man-ufacturer
s instructions (Sigma). Cells were resuspended in 20%FCS with 10% DMSO and stored in liquid nitrogen.Malignant cells were purified from the ascitic cell suspen-sion according to the method of Hurteau
et al.
(12). In brief,cells were layered onto discontinuous density gradients of Per-coll (Pharmacia, Uppsala, Sweden) and centrifuged at 850
g
,and the top layer of cells was harvested. Cells were then mixedwith CD45-coated Dynabeads (Dynal Ltd.) at 4
°
C for 30 min.The beads were magnetically removed. Cytospins were per-formed on the purified samples to assess composition. Sampleswere discarded unless
90% of cells stained with epithelialmarkers (cytokeratin antibodies used were AUA1, BerEP4,and EMA).
Cytospins.
A small aliquot of cells was removed beforefreezing and passed 5
10 times through a 19-gauge needle tobreak up clumped cells. Cells were suspended at a concentrationof 5
7
10
5
cells/ml. One hundred
l of the cell suspensionwere spun onto electrostatically charged slides (
Superfrost;
BDH Laboratories) at 500 rpm for 5 min using a cytocentrifuge(Shandon, Runcorn, United Kingdom). Slides were air dried,fixed in formal saline, rinsed in distilled water, and stored atroom temperature. Alternatively, the cytospins used for cleavedPARP were fixed in 4% paraformaldehyde (Sigma), and theimmunohistochemistry was performed at once.
Tissue Culture.
Cell lines were grown in a humidifiedatmosphere in 5% CO
2
at 37
°
C under pyrogen-free conditions.Pipettes were plastic and individually wrapped. Tissue culturemedia and fetal calf serum (Life Technologies, Inc.) were cho-sen for their low endotoxin content, and plastic tubes and flaskswere used at all times. Cell lines were grown in RPMI 1640 withglutamine (Life Technologies, Inc.), FCS (10%), penicillin (100units/ml), and streptomycin (100
g/ml; Sigma). Primary cellswere cultured in RPMI 1640 with glutamine FCS (20%), pen-icillin (100 units/ml), streptomycin (100
g/ml), and amphoter-icin B (0.625
g/ml; Sigma).Primary cells were seeded at 2
10
6
cells/ml into
Primaria
-coated tissue culture dishes (Marathon LaboratorySupplies, London, United Kingdom). After 24
48 h of culture,nonadherent cells and tissue culture medium were aspirated, andadherent cells were washed in RPMI 1640 and treated withIFN-
. At the time that the experiment was harvested, cells werescraped from an untreated well of the dish, and cytospins wereprepared.
Crystal Violet Proliferation Assay.
Cell lines wereseeded at 5
10
3
cells/well into 96-well tissue culture dishes.Primary cells were seeded at 5
10
4
cells/well. Six to 12 wellswere treated per concentration time point. Cells were allowed tosettle overnight in the dishes and then treated with IFN-
-containing medium. After 8 days, the medium was discarded,and adherent cells were washed in PBS and fixed in methanol.A 1% aqueous solution of crystal violet was added for 20 min.Cells were washed again, and then the remainder of the dye wasresolubilized in methanol. The transmission of each well wasread at 600 nm (Bio-Rad 2550 EIA Reader).
Protein Extraction.
Proteins were extracted accordingto the method supplied by Enzyme System Products (EnzymeSystem Products, Livermore, CA). Briefly, adherent cells werescraped from one well of a 6-well plate, and adherent andfloating cells were collected by centrifugation. A small aliquotof cells was removed for protein estimation, which was per-formed using the Bio-Rad protein Assay System (Bio-Rad Lab-oratories, Munich, Germany) according to the manufacturer
sinstructions. The remainder of the cells were suspended inloading buffer [62.5 m
M
Tris (pH 6.8), 6
M
urea, 10% glycerol,2% SDS, 0.003% bromphenol blue, and 5% 2-mercaptoethanolfreshly added]. Cells were sonicated on ice and stored at
40
°
Cuntil analysis.
PAGE.
Electrophoresis was performed using standardconditions as described previously (13). Ten percent gels wereused, and 25
g of protein were loaded per sample. Proteinlysates were run at 110 V for 2 h using 1
running buffer (25m
M
Tris, 192 m
M
glycine, and 0.1% SDS). Full-range Rainbow
4
The abbreviations used are: PARP, poly(ADP-ribose) polymerase;rhIFN, recombinant human IFN; FAM, carboxyfluorescein; FMK, flu-oromethyl ketone; NK, natural killer.
2488
Effects of IFN-
on Ovarian Cancer Cells

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