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Int. J. Cancer: 68,5 14-5 19 ( 1996)
0 1996 Wiley-Liss, Inc.
Publlcatlon of the lnternatlonal Un on Against Cancer
PmI,cat on de I unlon memationale b n t r e le Cancer
ESTABLISHMENT, CHARACTERIZATION AND DRUG SENSITIVITY
OF FOUR NEW HUMAN SOFT TISSUE SARCOMA CELL LINES
Wei-Wei LI', Carlos CORDON-CARDO~,
Quanguang cHEN3, Suresh C. J H A N W A R
and
~ Joseph R. BERTrN01,4
Laboratories of 'MolecularPharmacology, 'Molecular Immunopathology and 3Solid Tumor Genetics,
Memorial Sloan-KetteringCancer Center, New York, M: USA.
Four new cell lines were established from patients with soft
tissue sarcomas. Drug sensitivity as well as genotypic characterization, which may be related to drug sensitiviky in these cell
lines, was determined. Karyotype, H-ras, c-myc and mutant p53
gene expression, Rb, GI- and S-phase cyclins, E2F and major
cyclin/CDK inhibitors such as p16 and p21 and p-glycoprotein
were analyzed usingcytogenetic, Northern blot and immunological methods. Drug sensitivity was determined using growth
inhibition tests.These cell lines differed in their morphologyand
growth rates, forming colonies in soft agar with a cloning
efficiency of 4.3-13.4%. and 3 of the 4 cell lines grew in nude
mice. Cytogenetic analysis of cell lines revealed highly aneuploid
karyotypes. Deletion and/or translocation of chromosome 17
was seen in HS-16, HS-18 and HS-30 cells, and both copies of
chromosome I 3 were lost or marranged in the HS- I8 cell line.
Mutant p53 protein was present in all 4 cell lines. HS- I8 cells
showed no expression of the Rb protein and high levels of
expression of EZF, cyclin A, cyclin E and CDKZ. HS- I 6 expressed
a higher level of cyclin D than the other 3 cell lines. p2Id1
expression was seen in all cell lines, but p16'*' was expressed
only in HS-30 and HS-42 cell lines. These cell lines were
sensitive to tax01 and relatively resistant to methotrexate,
vinblastine and 5-fluorouracil when compared with the fib-coma cell line HT-1080. These new cell lines should provide a
useful model for the study of soft tissue sarcomas and for
evaluating new drugs or treatments.
c 1996 Wiley-Liss,Inc.
The soft tissue sarcomas are a heterogeneous group of
neoplasms composed of various histological subtypes and
remain one of the most chemotherapy-refractory human malignancies (Greenall et al., 1986). A better understanding of the
biology and reasons for drug failure in this disease could lead
to improved treatment. As detailed investigations are difficult
to perform in fresh samples from patients, we sought to
establish new cell lines that would reflect the phenotypic and
genotypic properties of these tumors.
We describe herein the establishment and characterization
of 4 new soft tissue sarcoma cell lines, including growth
properties, tumorigenicity in nude mice, cytogcnetics, determination of some oncogenes and tumor-suppression genes,
cyclins and cyelin-dependent kinase inhibitors. In addition, the
sensitivity of these cell lines to different anti-cancer drugs was
determined. These cell lines should provide a useful model for
further studies in exploring the biology of these tumors and
response to chemotherapeutic agents.
MATERIAL AND METHODS
Tumor samples
Tumor specimens of surgically excised soft tissue sarcomas
were obtained from the Surgery Department, Memorial SloanKettering Cancer Center. Histopathological analysis o f the 4
tumor samples revealed them to be mesenchymal chondrosarcoma (HS-16), myxoid liposarcoma (HS-18), malignant hemangiopericytoma (HS-30) and malignant mesenchymoma composed of liposarcoma and rhabdomyosarcoma (I IS-42). HS-16,
HS-18 and 11s-30 were isolated from primary tumors from the
leg of an 18-year-old-male, the retroperitoneum of a 42-yearold female and the pelvis of a 46-year-old malc, respectively.
HS-42 was isolated from a metastasis in thc right ovary of a
67-year-old female. None of the patients had received prior
chemotherapy or radiotherapy at the time of surgery.
Cell culture
Tumor samples were trimmed free of connective tissue,
washed several times with RPMI-1640 media, finely minced
and incubated in RPMI-1640 medium with 10% FBS, 0.6%
collagenase I1 and 0.002% DNAse I at 37°C for 2 hr, then
passed through a 100 mesh screen, and cell suspensions
obtained were placed in RPMI-1640 medium enriched with
10% FBS, 2 mM L-glutamine, 0.2 mM sodium pyruvate, 0.4
mM 1.-serine and 0.3 mM ascorbic acid at a density of 2.5 to 5 x
l@ cells/cm2. After 3-7 days of incubation at 37°C in a
humidified atmosphere of 5% C o t , the cell monolayer was
subcultured after detachment with 0.2% trypsin plus 2 mM
EDTA. Cells were maintained in RPMI-1640 medium containing 10% FBS, 2 mM glutamine and 0.2 mM sodium pyruvate.
With the use of this procedure, we have been able to establish
12 cell lines from 30 tumors, i.e., a success rate of 40%.
In vitro doubling time
Triplicate T-25 tissue culture flasks were seeded with 5 X
lo4cells in 5 ml of medium/flask. At 24 hr intervals for 10 days,
the cell monolayer was trypsinized and cells were counted.
These data were plottcd, and population doubling times were
calculated.
Colony-formingefficiency
Colony-forming efficiency was determined both in monolayer culture on plastic and in soft agar. In monolayer cultures,
500-2,000 cells were plated in a 60 mm dish. For soft agar
cloning, assays were performed using the method of Hamburger and Salmon (1977). One milliliter of underlayer consisting of RPMI-1640 medium, 10% FBS and 0.5% Noble agar
was plated in 24-well plates; 5 to 10 x lo3 cells/ml were
suspended in RPMI-1640 with 10% FBS and 0.3% agar, and 1
ml of the suspension was plated on the gelled underlayer.
After incubation at 37°C in 5% C 0 2 for 12 days, colonies of
greater than 50 cells were counted under a microscope.
Growth in nude mice
Five-week-old nude mice (Swiss nulnu) were purchased
from Charles River (Wilmington, MA) and maintained under
pathogen-free conditions. The ability of cultured cells to form
tumors was assessed by injecting 3 x lo6cells S.C. in a volume
of 0.2 ml in the right flank region. Three to 5 micc were used
for each tumor. Tumors were allowed to grow to a volume of
approximately 1 cm3 before they were removed a t the time
animals were killed.
Cytogeneticanalysis
Exponentially growing cells were incubated with Colcemid
(0.2 kg/ml) for 4-6 hr at 37°C. Cells were then trypsinized,
washed and processed for chromosome preparations following
4Towhom correspondence should be addressed, at the Laboratory
of Molecular Pharmacology, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA. Fax: (212) 639-2767.
Received: Junc lY, 1996 and in revised form August 5. 1996.
ESTAHI.ISHMEN1 OF HUMAN SOFI TISSUE SARCOMA CELL LINES
conventional methods using 0.075 M potassium chloride as the
hypotonic solution and 3:l methanol acetic acid as the fixative.
Air-dried metaphase spreads were stained to reveal G-banding
patterns. Clonal chromosome abnormalities wcre defined and
karyotypes described according to the international system for
human cytogenctic nomenclature (Mitelman, 1991).
Southern blot analysis
To test c-myc amplification, genomic DNA was isolated
from frozen cells. A 10 pg aliquot of each sample was digested
by EcoR I and elcctrophoresed on 0.8% agarose gels. After
staining with ethidium bromide, DNA was transferred to nylon
filters. Pre-hybridization, hybridization and washing were performed. The probe used was a fragment (3rd cxon) of c-myc
cDNA (Oncogene Science, Uniondale, NY). Aftcr removal of
the probe, filters were rehybridized with a human P-rnicroglobin cDNA as an internal control. Quantification of amplification was performed with a blot analyzer (Betascopc 603).
Northern blot analysis
Total RNA was extracted from sarcoma cells with RNAzol
(Biotecx, Houston, TX); 20 pg RNA were subjected to
electrophoresis in a 1.0% agarose gel containing 1.5% formaldehyde. After staining with ethidium bromide, RNA was
transfcrred to a nylon membrane. Pre-hybridization, hybridization and wash were performed. Oncogene probes used were
C-myc exon 3 (pHSR-1; ATCC, Rockville, MD) and H-ras
exon 1 (PCR-generated fragment).
Immunological analysis of expression of cell-cycle regulators
Mutant p53, Rb, cyclin A, cyclin D1, cyclin E, CDK2, E2F,
p16 and p21 were detected following incubation with the
appropriate antibodies: monoclonal mouse anti-human p53
(pAb 1801), monoclonal mouse anti-human Rb (IF8), polyclonal rabbit anti-human cyclin A (BF1683), monoclonal
mousc anti-human cyclin D1 (HDI l), polyclonal rabbit antihuman cyclin E (HE1 1l), polyclonal rabbit anti-human CDK2
(M2), polyclonal rabbit anti-human E2F (KH95), polyclonal
rabbit anti-human p16 (C20) and polyclonal rabbit anti-human
p21 (C19) (all from Santa Cruz Biotechnology, Santa Cruz,
CA). Immunohistochemical analysis was performed after cytospin using a standard avidin-biotin-peroxidasetechnique (Cordon-Cardo et al., 1987). Expression of the various proteins in
tumor cells was classified in 1 of 3 categories by estimating the
percentage of cultured cell nuclei staining: (i) negative ( < lo%),
(ii) heterogeneous ( 10-70%), (iii) homogeneous ( > 70%). For
immunoblotting, 100 pg samples of total cell lysates were
size-fractionated by SDS-PAGE and transferred onto nitrocellulose membranes. Protein expression was detected using ECL
detection reagents (Amersham, Arlington Heights, IL).
Cell growth inhibition
Monolayer cells in 6-well plates (10 x 104 cclls/well) were
cxposcd to various concentrations o f drugs, including methotrexate (MTX), 10-ethyl-10-dezaaminopterin (10-EDAM), trimetrexate (TMTX), taxol, doxorubicin (DOX), actinomycin D
(ACD), vinblastine (VLB) and 5-fluorouracil (5-FUra). After
24 hr, the media were removed and the cell layer was washed
twice with cold PBS. Fresh drug-frcc medium was added, and
growth was followed for an additional 96 hr. Cells were
counted after appropriate dilutions of the cell suspension
using a model ZB Coulter counter (Hialeah, FL). Percentage
of growth inhibition and EDSOvalues were determined as
previously described (Li et al., 1992).
Determination of the multidrug resistance gene product
(p-glycoprotein)
Determination of p-glycoprotein in these cell line was made
using a method previously described (Cordon-Cardo et al.,
1987). Mouse monoclonal antibodies (MAbs) HYB-241 (a gift
515
from Dr. L Grauer of Hybritech, San Diego, CA) and C-219
(from Centocor, Malvern, PA) wcre used at a concentration of
20 pg/ml. As a negative control, purified mouse MAb directed
against the cell surface antigen anthranylate synthase of
Escherichia coli was used at a concentration of 20 pg/ml. As a
positive control, purified MAbs against cytokeratins and other
intermediate filaments wcre also used at the same coneentration. Secondary antibodies used were biotinylated horse antimouse IgG affinity-purified antibodies (1:lOO dilution in PBS).
Immunocytochemical analysis was performed as described
above.
RESULTS
In vitro growth characteristics
All cell lines grew as monolayer cultures with loss of contact
inhibition at confluence. Each cell line showed different
morphological features with considerable variation in the size
and shape of cells (Fig. 1). Doubling times varied from 33 to 40
hr HS-18 cells proliferated more rapidly than the other cell
lines (Table I). Plating efficiencies (PE) were measured at
passages 80,66,68 and 58 for HS-16, HS-18, HS-30 and HS-42,
respectively, and ranged from 16.4% for HS-16 to 6.4% for
HS-18. Cloning efficiencies in soft agar were similar for HS-18
and HS-30 but lower for HS-16 and HS-42 cell lines (Table I).
Tumorigeniciv of cultured cells in nude mice
Cultured HS-16, HS-18 and HS-30 cells (3 x lo6) injected
S.C. formed detectable tumors at the injection site within 5, 9
and 5 weeks, rcspectively, and grew to reach 1 cm in size in
about 12 wccks for the HS-16 and HS-30 cell lines and in about
20 weeks for the HS-18 cell line. Histopathological characteristics of tumors from nude mice were similar to that from the
patients' initial tumors (not shown). The cells obtained from
nude mousc tumors were plated in vitro and immediately
showed outgrowth. No tumor growth was observed after
inoculation of HS-42 cells, even at 26 weeks (Table I).
Karyotype
Cytogenetic analysis of the 4 cell lines showed highly
aneuploid karyotypes (Fig. 2; Table 11). Chromosome numbers
ranged 72-77, 67-72, 69-79 and 82-85 for HS-16, HS-18,
HS-30 and HS-42 cell lines, respectively. Chromosomal alterations included trisomics, monosomies, translocations, deletions, derivative chromosomes, additional or missing whole
chromosomes and marker chromosomes, but no double minutes or homogeneously staining regions were seen in any of the
cell lines (Table 11). Deletion and/or translocation of chromosome 17 was seen in HS-16, HS-18 and HS-30 cells, and both
copies of chromosome 13 were lost or re-arranged in the HS-18
cell line. A representative karyotype from the cell line HS-18 is
presented in Figure 2 to dcmonstratc the complexity of
chromosomal abnormalities seen.
Oncogene amplification and expression
DNA and RNA from normal human lymphocytes and from
a non-malignant lymphoblastoid cell line (RPMI-1788) were
used for comparison with the 4 sarcoma cell lines. Southern
blot analysis showed no significant increase of c-myc gene copy
number in any of the cell lines. Over-expression of c-myc and
H-ras mRNA was also not observed in these cells (data not
shown).
Expression of cell-cycleregulatoryproteins
Mutated p53 and Rb protein expressions were analyzed
immunohistochemically using MAbs. PAB-1801, an antibody
that recognizes p53, showed intense diffuse nuclear staining in
>70% of cells in all 4 sarcoma cell lines. In HT-1080, a
fibrosarcoma obtained from the ATCC, nuclear staining was
observed in about 20% of cells. HS-16, IIS-30 and HS-42 cell
LI ETAL.
516
FIGURE
1 - Morphology of the soft tissue sarcoma cell lines by phase contrast microscopy (original magnification ~ 2 0 0 )(n)
. HS-16. (b)
HS-18, (c) HS-30, (d) HS-42.
TABLE I - GROWTIi PROPERTIES OF4 KEWLY ESTABLISHED IiUMAN
SOFT TISSUE SARCOMA CELL LlXES
Cell
line
HS-16
HS-18
HS-30
HS-42
Pacsage
number
-
198
187
186
165
Doubling
(hr)
% Plating
o/, Cloning
efficiency
efficiency
40
33
39
34
16.4
6.4
11.6
5.9
5.2
12.4
2.3
7.0
Growth in
nude mice
1
+
+
2
'
3
4
5
-I-
-
6
7
\
9
10
11
12
lines expressed pRb in the nuclei of all cells. However, there
was no dctectable Rb protein in the HS-18 cell line. All cell
lines expressed E2F-1, cyclin A, cyclin E and CDK2, with the
highest expression of these proteins observed in HS-18 cells.
Immunoblotting was used to determine expression of cyclin
D1, p16Ink4and p2lWaf1and to confirm the absence of Rb
protein in HS-18 cells. HS-16 cells expressed a higher levcl of
cyclin D1 than the other 4 cell lines. There was no significant
difference of p2Iwaf*expression observed in thc cell lines, while
~
1 expression
6
~ was~not detected
~
~ in HT-1080, HS-16 and
HS-18 cell lines. Higher cyclin A expression and absence of Rb
protein were confirmed in HS-18 cells. These results are
summarized in Figure 3 and Table 111.
FIGURE2 - A representative karyotype of HS-18 cells. Structural
chromosomal alteration such as translocations and deletions
(arrows). Note loss of both copies of chromosome 13 and the
presence of marker chromosomes.
Drug sensithiy
The sensitivity of the 4 new sarcoma cell lines and the
HT-1080 cell line to several chemotherapeutic agents was
determined by exposure to different concentrations of drugs
for 24 hr (Table IV). All cell lines were very sensitive to taxol
(ED5"values were 3.0 nM for HS-18 cells and less than 1.0 nM
for the other 3 cell lines). Sensitivity to ACD and DOX was
also observed (EDso values ranged 1-9 and 6 1 6 nM, respectively, for different cell lines). Sensitivity of cells to VLB varied
more markedly. The EDSOvalues for the 4 new cell lines were
3- to 17-fold higher than that for HT-1080 eells I1 (6.0 nM).
-
!?
2 1 ' 2 2
ic
x x
ESTABLISHMENT OF HUMAN SOFT TISSUE SARCOMA CEI.1. LINES
TABLE II - cmoGENmc ANALYSIS
517
lines are composed of malignant cells: ( i ) the cells grew rapidly
to a high saturation density in vitro and showed a lack of
Cell
Numberof
Chromoxmal
contact inhibition after slow proliferation during the first few
line
chromosomes
alterations
passages (generally passages 1-5). (ii) all 4 cell lines have been
cultured in vitro for at least 160 passages, (iii) all 4 cell lines
HS-16 50
72-77
XX,-X(3n? , +1 +11 +21
form colonies in soft agar, (iv) cytogcnetic analysis displayed
+22 2X deI)9)(q22 or 3L),
+2X de (22Ma12).
marked aneuploidy and (v) the cell lines were found to be
+del(5)($ljj,bef(l7)(pll)
tumorigenic in nude mice (with the exception of HS-42).
random monosomies and triMutant p53 protein, as measured immunohistochemically,
somies, random mars
was observed in all 4 new cell lines. As the presence of a
XX, -X (3nc), -2, +3, -4,
HS-18 50
67-72
mutant p53 was not determined in the fresh tumors from which
-7, -9, -10, -13, -13, -14,
-16, -17, -18, +19, -21,
the lines were derived, it is not clear whether the p53
-22, -22, del 1)( 13 32),
mutations were present in the fresh tumor from patients
del(6)(q16 2!9, Jel(~(plS),
before these cell lines were established or grew in culture
2Xdel 11)( \3), del(l7)(pll),
selected for cells with p53 mutations. p53 mutations have been
+der( ), + Xder (22)t(9;
found frequently in a wide variety of tumors, including soft
22)(qll; 13), +der(17)t(17;
tissue sarcomas (Toguchida et af., 1992) and are associated
?)(pll;?p, +mar 1, +mar
with tumorigenesis, cellular transformation, proliferation and
2 + random mars
69-79
protection of cells from apoptosis and drug cytotoxicity
HS-30 50
XX, -Y, (3nz), de1(2)(p21),
der(2)t(2;17) q l l ;
(Zambetti and Levine, 1993). Abnormalities of thep53 and the
pW,der( 13) tq32)
Rb genes may occur together in primary human tumors
der(19)t(7;19)(qll~pll).
(Stratton et al., 1990), as found in the HS-18 cell line. Thep53
+mar 1, +mar 2
andpRb
genes reside on the short arm of chromosome 17 and
82-85 XX, -X, (3nz), der(l)t(l;
HS-42 50
the long arm of chromosome 13, respectively. Abnormalities of
l)(p22;q32), de1(7)( 13,
these genes may result from the structural alteration of these
der(7)t( 7;?)(q11.2;?7
chromosomes. Structural changes of chromosome 17 were
present in HS-16 (del(l7)(pl l)), HS-18 (del(l7)(pll),
dcr(17)t(17;?)(pll;?)) and HS-30 (der(2)t(2;17)(qll;p13)),
1
2
3
4
5
respectively. In addition, we observed loss of both copies of
chromosome 13 in the HS-18 cell line.
H-ras expression and amplification of the c-rnyc gene are
frequently present in a wide spectrum of human tumors and
are involved in tumorigenesis, transformation, cellular growth
---mmwcyclin D and signal transduction, but information is limited for soft
tissue sarcomas. Activated c-myc or H-ras could co-operate
with mutant p53 protein in the oncogenic development of cells
(Toguchida et af., 1992), and alteration of these oncogenes may
-pRb
result in anti-cancer drug resistance in tumor cells (Denis et al.,
1991; Niimi et al., 1991). However, significant amplification or
over-expression of these oncogenes was not observed in these
cell lines.
p16
Cyclin D1, cyclin A and cyclin E and associated CDKs are
regarded for GI- to S-phase progression and are linked to
tumor development (Grana and Rcddy, 1995). E2F-1, and its
related family mcmbers, is a crucial transcription factor in
cellular proliferation and activates the transcription of growthFIGURE
3 - Protein expression of cyclin A, cyclin D1, Rb, plfPkk4 associated genes, including DHFR, TS and DNA polymerase
and p21”afI in soft tissue sarcoma cell lines as shown by Western (Johnson et al., 1994). E2F over-expression may induce cellular
blotting. One hundred micrograms of protein extract o f each cell transformation, tumor formation in nude mice and even drug
line were separated on SDS/PAGE and clectroblotted to a resistance (Johnson et al., 1994; Li ef al., 1995). However,
nitrocellulose membrane. Proteins were detected with the various E2F-mediated transactivation can be normally inhibited by
antibodies as described in “Material and Methods”. Lane 1, pRb (Helin et af., 1993). ~ 1 6 ’and
” ~ p2lWaf1,
~
as major cyclin/
HT-1080; lane 2, 11s-16; lane 3, HS-18; lane 4, HS-30; lane 5, CDK inhibitors, play an important role in G I arrest and
HS-42.
suppression of cellular transformation (Scrrano et al., 1995;
Michieli et al., 1996). These proteins interact with p53 and pRb
to control cell-cycle progression (Slebos et af., 1994). Intensive
TMTX and 10-EDAM were more inhibitory than MTX in the expression of E2F was observed in all new cell lines; cyclin A
new cell lines compared with HT-I080 cells. 5-FUra was 2-5 was highly expresscd in HS-18 cells, cyclin D levels were higher
time less inhibitory to growth in the new cell lines cornpared in HS-16 cells and p16 was not detected in the HS-16 and
with HT-1080.
HS-18 cells. These results suggest that oncogenic transformation of some soft tissue sarcomas may be due to abnormalities
Expression ofp-glycoprotein
of cell-cycle regulators rather than changes in expression of ras
None of the cell lines had detectable p-glycoprotein.
or c-rnyc genes.
An important aspect of our results is the use of these cell
lines to determine drug sensitivity. The success of chemothcrDISCIJSSION
apy is limited in soft tissue sarcomas, and in vitro drug
Four new cell lines were established from primary soft tissue sensitivity or resistance information may bc of value to guide
sarcomas. The following evidence establishes that these cell individual treatment or new drug development. Taxol, a potent
$ 1
-
--
LI E T A I .
518
TABLE 111 -MUTANT p53, pRb, CYCLIN A CYCLIN E,CDKZ, E2F AND OLYCOPROTEIN EXPRESSION
I N SOFT +ISSUE SARCOMA CELL LiNiH
Cell line
HS-16
HS-18
HS-30
HS-42
HT-1080
Mutant p53
Rh
Cyclin A
loo++
1(K)+++
70++
100+++
20++
100+
loo+++
20++
60+++
100+++
20+
ND
100+
Staining (70)’
.~
Cyclin E
CDKZ
10+
40+
70+++
50++
70++
ND
70++
80+++
80++
50++
ND
-
E2F
P-glycoprotein
loo+++
lo()+++
-
70+++
70+++
-
ND
-2
I % indicates the number of positive cells; + indicates the degree of intensity.-2From Slovak et al.
(1991). See “Material and Methods” for details. ND, not done.
TABLE IV - GROWTH-INHIBITORY LFFEClS OF 8 ANTI-CANCER
SARCOMA CEI.1. LINES
Cell line
~~
HS-16
HS- 18
HS-30
HS-42
HT-1080
DRUGS O N SOFT TISSUE
ICCO
(nM)
MTX
-
TMTX
10-EDAM
DOX
ACD
VLB
Taxol
5-FUra
1,990
1,740
600
2,830
170
100
2,100
150
220
230
250
80
13.0
12.0
16.0
7.0
6.0
1.0
2.0
9.0
2.0
2.0
18.0
100.0
70.0
30.0
6.0
0.1
3.0
0.2
0.3
0.1
9,750
9,330
22,230
7,620
4,900
170
30
180
Cells were exposed to drugs for 24 hr, then washed for an additional 96 hr and counted. Values
are the averagc of 2-4 different experiments.
inhibitor of tumor cell replication isolated from the plant
Tarus brevifolia, has been used in the clinic to treat solid
tumors, including breast, ovary and colon cancer. Phase I1
trials showed that the response rate to this drug in a small
group of soft tissue sarcomas was only 18% (5/28) (Kaye,
1995). However, this compound is a potent inhibitor of growth
of these sarcoma cell lines. Based on these studies, this drug
should be further evaluated in this disease. DOX and ACD
have some activity against soft tissue sarcomas clinically
(Greenall et al., 1986), and resistance to these drugs may be
mediated by increased cxpression of p-glycoprotein. Overexpression of p-glycoprotein is present in approximately 20%
of some soft tissue sarcomas (Gerlach et al., 1987). The
sarcoma cell lines we studied were sensitive to DOX and ACD,
consistent with the lack of expression of p-glycoprotein.
The 4 new cell lines were relatively resistant to MTX and
VLB compared with HT-1080, a fibrosarcoma ccll line. Resistance to MTX in HS-16, HS-30 and HS-42 cells was partially
explained by the inability of these cell lines to accumulate
long-chain polyglutamates of MTX (Li et al., 1992). The
mechanism of resistance of the HS-18 cells to MTX was
associated with increased expression of DHFR, linked to
increased free E2F due to the absence of pRb (Li et al., 1995).
Alteration of the other cell-cycle regulators in these cell lines
may also contribute to drug resistance.
The establishment and phenotypic and genotypic characterization of these cell lines should provide a useful model for the
study of soft tissue sarcomas and for evaluating new drugs or
treatments.
ACKNOWLEDGEMENTS
The authors thank Drs. M. Brennan, L. Freedman, E.
Casper and J. Woodruff for their continued support and
co-operation. J.R.B. is an American Cancer Society Professor
of Medicine and Pharmacology. This work was supported by
NIH grant CA 47179.
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