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2427
A Unique Human Ovarian Carcinoma Cell Line
Expressing CD34 in Association with Selection for
Multidrug Resistance
Hans Minderman, Ph.D.'
Udo Vanhoefer, M.D., Ph.D.*
Karoly Toth, M.D., Ph.D.'
M. Denice Minderman, B.SC.'
YOUCef M. Rustum, Ph.D.'
' Department of Experimental Therapeutics, Roswell Park Cancer Institute, Buffalo, New York.
* Departmenit of Internal Medicine, West German
Cancer Cent'er, University of Essen, Essen, Germany.
Presented in part at the 85th Annual Meeting of
the American Association for Cancer Research, San
Francisco, California, April 10-13, 1994.
Supported in part by Grant DHP-115 from the
American Cancer Society, Cancer Center Core
Grant CA16056, and NIH program project grant
CAI 3038.
Dr. R. J. Scheper of the Department of Pathology, Free University Hospital, Amsterdam, The
Netherlands, provided the LRP (LRP-56) and
MRP (MRPm6) monoclonal antibodies.
Dr. Udo Vanhoefer was sponsored by a special
research fund of the West German Cancer Foundation.
Dr. M. L. Slovak of the Department of Cytogenetics, City of Hope National Medical Center,
Duarte, California, provided the HT1080 and the
HT1080Dr4 Ice11 lines.
Dr. W. R. Greco and Mr. J. C. Parsons of the
Biomathematics Department provided statistical
assistance with the curve-fitting of the doseresponse curves.
Dr. M. R. Bat?rof the Division of Medicine, Ros-
C? 1996 American Cancer Society
BACKGROUND. The role of P-glycoprotein (Pgp) in multidrug resjstance (MDR) is
uncontested. Expression of Pgp on hematopoietic cells has been correlated with
CD34 expression. For acute myeloid leukemia, the prognostic value of Pgp for
clinical response is at best equivalent to that of CD34. The current study investigated whether expression of CD34 can be associated with selection for drug resistance.
METHODS. Several established MDR cell lines were screened by flow cytometry for
expression of CD34. Human ovarian carcinoma cells (A27801, which simultaneously expressed CD34 and Pgp, were identified. Subsequent cloning resulted in a
new cell line (A2780-Dx5c)that expressed CD34 in the absence of Pgp. Involvement
of non-Pgp-mediated MDR mechanisms was assessed by immunohistochemistry
(MRP and LRP), enzyme activity studies (glutathione pathway), cross-resistance
patterns, and Northern blot (type IIcv topoisomerase).
RESULTS. A2780-Dx5c was cross-resistant to doxorubicin, daunorubicin, idarubicin, and W-16. However, unlike the Pgp-expressing cells, it was not cross-resistant to vincristine or amsacrine. The drug resistance was correlated with a decreased level of type IIa topoisomerase in the A2780-Dx5c cell line compared with
the parental cell line. No evidence was found of involvement of MRP, LRP, or the
glutathione pathway with drug resistance in this cell line.
CONCLUSIONS. A new cell line of nonhematopoietic and nonvascular endothelial
origin that expresses CD34 in association with selection for MDR was cloned. A
study of MDR mechanisms in this cell line revealed that reduced type IIcv topoisomerase levels were likely responsible for the MDR observed. A study of the causal
relation between the selection of drug resistance and the expression of CD34 may
provide insight into why CD34 correlates with poor clinical response in patients
with acute myeloid leukemia. Cancer 1996; 78:2427-36.
0 1996 American Cancer Society.
KEYWORDS: CD34, multidrug resistance, P-glycoprotein, MRP, LRP, topoisomeraseIl-a.
T
he CD34 antigen is commonly known for its association with
normal huinan hematopoietic progenitor cells' and acute leukeCD34 is expressed in vascular endothelial cells5
m i a ~ . In
~ addition,
-~
and vascular neoplasms.6 Increasing evidence supports that CD34
well Park Cancer Institute, provided the bone
marrow samples.
cer Drug Center, Roswell ParkCancer Institute, Elm
and Carlton Streets, Buffalo, NY 14263.
Address for reprints: Hans Minderman, Ph.D., Department of Experimental Therapeutics, Grace Can-
Received June 18, 1996; revision received August 8, 1996; accepted August 8, 1996.
2428
CANCER December 1, 1996 I Volume 78 / Number 11
is expressed in a limited number of other cell types.
Screening of 357 neoplasms of nonvascular derivation6 showed CD34 expression in epithelioid sarcoma (8 of 16 cases) and rare cases of leiomyosarcoma (3 of 221, whereas carcinomas were found to
be negative with the exception of 1 case of squamous
cell carcinoma. Notably, CD34 was found not to be
expressed in ovarian carcinoma. Recently, CD34 expression was also described on a fibrohistiocytic tumor (dermatofibrosarcoma p r o t ~ b e r a n s )The
. ~ human CD34 gene has been isolated and characterized
at a molecular l e ~ e l . Two
~ . ~ alternative forms of
cDNA encoding human CD34 were characterized"
and expression of a full length and a truncated form
of CD34 on human KMT2 and KGla cells was recently described," but their functional significance
has yet to be determined.
In the context of leukemia and predictors for clinical response, expression of CD34 on hematopoietic
progenitors correlates closely with expression of P-glycoprotein ( P ~ P ) , ' ~a- 'marker
~
associated with multidrug resistance (MDR).l6,I7 A multicenter trial conducted by the Southwestern Oncology Group involving
155 patients with de novo acute myeloid leukemia
(AML) correlated clinical response with expression of
Pgp and CD34.l' From this study, CD34 appeared to be
a better single prognosticator for decreased complete
remission rate than MDR-1 gene expression (encoding
for Pgp).
To further evaluate the association of CD34 with
drug resistance, several MDR cell lines and their parental lines were screened for expression of Pgp and
CD34. CD34 was found to be expressed on a MDR cell
line of nonhematopoietic and nonendothelial origin.
This article describes cloning and characterization of
this human ovarian carcinoma cell line with regard
to drug sensitivity, Pgp, multidrug resistance protein
(MRP), lung resistance protein (LRP), and CD34 expression, activities of chemical detoxification enzymes
of the glutathione pathway and levels of the key target
enzyme of topoisomerase-I1 inhibitors: topoisomerase-11-a.
MATERIALS AND METHODS
Cell Lines
The cell lines used were A2780 (human ovarian carcinoma), MCF7 (human breast carcinoma), KB 3-1 (human cervical carcinoma), HL60 (human promyelocytic
leukemia), HT1080 (human fibrosarcoma), and MDR
sublines derived from these cell lines (A2780-Dxl, Dx3, -Dx5, MCF7-Adr, KB 8-5, HLGO/Adr, and HT1080/
Dr4) and KG1 (human acute myelogenous leukemia).
Detailed procedures for culturing and selection of the
MDR strains have been published p r e v i o ~ s l y . ' Cell
~-~~
cloning was performed applying standard limiting dilution techniques in 96-well microculture plates.
Bone Marrow Samples
After informed consent, bone marrow aspirates were
obtained from patients presenting with AML as part
of an Institutional Review Board-approved protocol.
Nucleated cells obtained after lysis with ammonium
chloride buffer (8.29 g NH,Cl, 1 g KHC03, 37 mg
K,EDTA per liter, pH 7.3) were cryopreserved and
thawed using standard cryopreservation procedures.
Total Growth Assay
Total growth was assessed by measuring total protein
(sulforhodamine-B [SRBI assay). Three hundred exponentially growing cells were seeded per well into 96well tissue culture plates (Falcon #3072, Becton Dickinson, Lincoln Park, NJ). The cells were allowed to
attach for 24 hours at 37 "C, 5% CO, in air, in a fully
humidified atmosphere. Doxorubicin, daunorubicin,
and idarubicin (Adria Laboratories, Columbus, OH),
and vincristine (Sigma, St. Louis, MO) were added to
the desired final concentrations from stocks made in
phosphate-buffered saline (PBS). Stock solutions of
amsacrine (m-AMSA) (Lot no. MK5-27-1, gift from the
Drug Synthesis and Chemistry Branch, National Cancer Institute, Bethesda MD) and W-16 (Bristol-Myers,
Syracuse, NY;freshly prepared) were prepared in 100%
dimethylsulfoxide (DMSO) so that the final DMSO
concentration in the cultures was < 0.25%, which was
determined to have no effect on cell growth. After 2hour drug exposure, cells were washed twice with PBS,
resuspended in RPMI1640 + 10% fetal bovine serum
(Gibco, Grand Island, NY), and cultured for an additional 4 cell doubling times, except for cells exposed
to vincristine, in which case the drug was present continuously. Cell monolayers were fixed in trichloroacetic acid for at least 1 hour, and washed with distilled
water using an automated microplate washer (model
EL403; Bio-Tek Instruments, Winooski, VT), after
which SRB (0.4%weightlvolume [wlv] in 1%volume/
volume acetic acid) was added for 10 minutes at room
temperature. Plates were subsequently washed with
1% acetic acid and air dried. Protein-bound dye was
solubilized in 10 mM Tris base. Absorbance at 570 nm
was measured by an automated Bio Kinetics reader
[model EL340; Bio-Tek Instruments). All cytotoxicity
assays were performed at least in triplicate. Curve fitting to assess ID50 concentrations was performed as
described p r e v i o ~ s l y . ~ ~
lmmunohistochemical Staining for von Willebrand Factor
(Factor Vlll)
Paraffin sections of cell pellets were incubated with a
primary antibody against factor VIII (Lipshaw Immu-
CD34 Expression on Drug Resistant Cells/Minderrnan et al.
non, Pittsburgh, PA) and antibody binding was visualized with a universal streptavidin/biotin staining kit
(Lipshaw Omnitags, Pittsburgh, PA). The staining procedure was performed according to the manufacturer's instructions. Endothelial cells in paraffin sections
of normal human brain served as a positive control.
lmmunoperoxidase Staining for MDR Markers
Paraffin sections of cell pellets were stained for Pgp
expression using the JSB-1 monoclonal antibody
(MoAb) (Accurate Chemical and Scientific Corp, Westbury, NYI applying an immunoperoxidase sandwich
Staining of cytospin
method i1S described previo~sly.~'
preparations with LRP-563Land MRPm632monoclonal
antibodies (MoAbs) (gift from Dr. Scheper) was performed after air drying overnight and 10-minute fixation in acetone. Isotype controls (mouse immunoglobulin IIgIG2b for LRP-56 and mouse IgGl for MRPm6)
were used in place of and at the same concentration
as the primary antibodies. A polyvalent biotinylated
secondary antibody and streptavidin-peroxidase reagent (Shandon-Lipshaw, Pittsburgh, PA) were used
to visualize the binding of the primary antibodies.
lmmunofluorescent Labeling
The MRKl6 MoAb (1 pg/106 cells) (gift from Dr.
Tsuruo) was used to detect Pgp expression. An indirect
labeling method using goat antimouse F(ab'), conjugated with phycoerythrin (PE) or fluorescein (Caltag,
San Francisco, CA) was used to visualize MRK16 binding.
Expression of CD34 was visualized with the PEconjugated HPCA-2 MoAb (Becton Dickinson, San
Jose, CA), the fluorescein isothiocyanate-conjugated
QBEND-10 MoAb (AMAC, Inc), or the biotinylated 128 MoAb33(CellPro, Inc., Bothell, WA). All MoAbs were
used according to the manufacturer's instructions.
The biotiriylated MoAb was visualized using streptavidin-RED613 (Gibco, Gaitersburg, MD).
Flow Cytometry
Flow cytometry was performed with a FACSCAN (Becton Dickinson, San Jose, CA) using the 488-nm line of
a 50 mW argon laser running at 15 mW output for
excitation. Fluorescein emission was collected through
a 530130 band-pass filter whereas phycoerythrin emission was collected through a 585/42 band-pass filter.
RED613 emission was collected through a 650 longpass filter. Analysis was performed using the Win List
software (Verity Software House Inc., Topsham, MN).
Flow cytometric cell sorting was performed with a
FACSTAR (Becton Dickinson).
2429
Enzyme Assays
Cells in exponential growth were harvested with 1 mM
EDTA in PBS, washed twice with ice-cold PBS, and
lysed by sonication in 5 mM dipotassium hydrogen
phosphate buffer. The enzyme activities of the glutathione-S-transferases (substrate: l-chloro-2,4-dinitrobenzene), glutathione peroxidase (substrate: butyl hydroperoxide), glutathione reductase, and glucose 6phosphate-dehydrogenase were determined at 25 "C
in the 120,000 g supernatant fluid as described previou~ly."~~'
Results are expressed as nmol min- ' per
mg protein extract.
Reverse Transcriptase Polymerase Chain Reaction
Amplification and Northern Blot Hybridization for
Topoisomerase-11-a Levels
Total RNA was extracted by a single-step guanidine
isothiocyanate-phenol-chloroform extraction.38 One
yg total RNA of HL60 cells was used for topoisomerase-11-a cDNA probe synthesis with an RNA polymerase chain reaction (PCR) kit (Perkin-Elmer, Branchburg, NJ). Reverse transcriptase was performed using
random hexamers as primer according to the instructions of the manufacturer. The PCR reaction was performed in a GeneAmp 2400 PCR system (Perkin-Elmer,
Oak Brook, IL) for 30 cycles of denaturation (95 "C, 60
seconds), annealing (52 "C, 60 seconds), and extension
(72 "C, 90 seconds). The topoisomerase-11-a specific
PCR primers (position 12-59939were 5'-ACCATTGCAGCCTGTA-3' (forward) and 5'-GCTCTTCCCATATTATCC-3' (reverse), respectively.4o The PCR products
were gel purified and the band of the expected size of
587 base pairs was isolated.
Northern blots containing 15 pg total RNA of untreated A2780 and A2780/DX5c cells were prehybridized at 65 "C (1 hour) in 0.5 M dipotassium hydrogen
phosphate buffer containing 7% (w/v) sodium dodecyl
sulfate, 1% (w/v) bovine serum albumin, and 1 mM
EDTA. Hybridization was performed overnight at 65
"C with a [a-"PIdATP random prime labeled topoisomerase-11-a cDNA fragment corresponding to nucleotides 12 to 599. After autoradiography, blots were
stripped and reprobed with a ["PI-labeled cDNA for
G3PDH (Clontech Laboratories Inc., Palo Alto, CA) to
determine variation in RNA loading.
RESULTS
Screening of MDR Cell Lines for Pgp And 0 3 4
Expression
Several MDR cell lines and their parental cell lines
were analyzed for Pgp expression and CD34 expression by flow cytometry. Positive cells were determined
by comparing the signal intensity with that of an IgG
isotype control. The KG-1 cell line served as a positive
CANCER December 1,1996 I Volume 78 I Number 11
2430
110
,
I
A2780 wt
100
90
-0
8
80
70
9 60
s
-%me!!
50
,S
40
5
30
O
20
+A2780wt
10
-0-A-
A2780-Dx1
0
0
-10
A2780-DXl
A2780-DX3
U A2780-DX5
A2780-Dx3
0.001
L
0.01
0.1
A2780-Dx5
L
1
10
h
h,
L!%%l!L
I
Doxorubicin pglml
FIGURE 1, Total growth of A2780 cells after 2-hour exposure to doxorubicin assessed by the sulphorhodamine-B assay (mean 2 95% confidence
interval).
control for CD34 expression. Pgp expression could be
demonstrated in the MDR substrains of the A2780,
MCF-7, and KB cells. As expected," no Pgp expression
was detectable in the HL60 cell line. In addition to the
KG-1 cell line, CD34 expression was only detected on
the A2780 cell lines. Because CD34 and Pgp expression
were only detected simultaneously on the A2780 cell
lines, subsequent studies were focused on these cells.
Characterization of MDR A2780 Cell Lines
Dmg resistance
Sensitivity of the A2780 cell lines to doxorubicin at 2hour exposure was evaluated by the SRB assay. Compared with the A2780-wt, the A2780-Dxl, A2780-Dx3,
and A2780-Dx5 are 4-fold, 8-fold, and 30-fold resistant
respectively, (Fig. 1) (Table 1).
Pgp expression
Figure 2 and Table 1 show the flow cytometric characterization of the A2780 cell lines in more detail. A heterogenous expression of Pgp was observed for all three
resistant substrains, represented by the existence of
two major subpopulations. The average level of expression (intensity of the fluorescence signal) for each
individual subpopulation was similar for A2780-Dxl,
A2780-Dx3, and A2780-Dx5, but their relative contribution to the total cell population was different for
A2780-Dx1 compared with A2780-Dx3 and A2780-Dx5
(Table 1).
MRKl6 +
FIGURE 2. Flow cytometric evaluation of P-glycoprotein expression (left
column) and CD34 expression (right column) on A2780-wt (first row),
A2780-Dx1 (second row), A2780-Dx3 (third row), and A2780-Dx5 (fourth
row). Dotted lines: isotype control (immunoglobulin [Ig]G2a for MRKl6
and IgG1 for HPCA-2); solid lines: specific monoclonal antibodies.
GO34 expression
The A2780-wt cell line showed a very low expression
of CD34 with HPCA-2 (Fig. 2, right column), evident
from the tailing to the right of the histogram. Similar
low expression compared with the A2780-wt was observed for A2780-Dx1 and A2780-Dx3 but significant
increase of expression was detectable in the A2780Dx5 cells (Fig. 2, right column, fourth row (Table 1).
Comparing reactivity of the A2780-Dx5 cell line
with the HPCA-2, QBEND-10, and 12-8 MoAbs revealed high reactivity with HPCA-2, low reactivity with
QBEND-10, and no reactivity with 12-8 (Fig. 3). To
confirm reactivity of all three MoAbs with CD34 expressed on hematopoietic cells, the human myelogenous leukemia cell line KG1 and leukemic blasts from
a patient with AML were used as positive controls.
Although the pattern of reactivity and resolution was
different, possibly related to the use of different fluorochromes and recognition of different epitopes, all
three MoAbs showed high reactivity with these controls (Fig. 3).
Flow Cytometric Screening of AML Bone Marrow
Samples for CD34 Expression
To determine the occurrence of the HPCA-2+, 12.8CD34 phenotype in clinical material, five bone marrow
CD34 Expression on Drug Resistant CellslMinderman et al.
2431
TABLE 1
Relation between Sensitivity of A2780 Cells to 2-Hour Exposure to Doxorubicin and Immunophenotype with Regard to CD34
and P-GlycoproteinExpression
A2780-wt
A2780-Dxl
A2780-Dx3
A2780-Dx5
%Pat
WPgjtt
R CD34
% I&
IC50 (PglmL)
0
4
0
29
65
80
8
9
10
42
1
0.05
0.2
0.4
1.5
12
9
Pm:p-dycaprotein; I&: immunodobulin
1
0
0
Resistance factor
1
4
8
30
G; t:weak; tt:strone;
KG-1
A2780-Dx5
F-QBEND10 3
PE-HPCA2 3
810-12.8/
STREPTlAVlDlN .)
FIGURE 3. Flow cytometric evaluation of three different anti-CD34 monoclonal antibodies (MoAbs) on acute myeloid leukemia blasts (top row), KG1
cells (middle row), and A2780-Dx5 (bottom row). The anti-CD34 MoAbs used were QBEND-10 (left column), HPCA-2 (middle column), and SP12.8
(right column). Dotted line: auto fluorescence.
samples fiom AML patients were compared for their
reactivity with the HPCA-2 and 12.8 MoAbs (Table 2).
The samples were selected for high expression of CD34
(assessed by HPCA-2). Both concordance and discordance were observed, but when discordance was observed (Patients 1 and 2) the percentage of CD34 positive
cells was higher when HPCA-2 was used. These findings
suggest the existence of two phenotypes of CD34, one
being the HPCA-2+,12.8' phenotype expressed on hematopoietic cells, and the other the HPCA-2+,12.8- phenotype expressed on the A2780 cell lines and some hematopoietic cells.
lmmunohistochemical Staining for von Willebrand Factor
(Factor VIM)
To exclude the possibility of admixture of the A2780
cell lines with cells of endothelial origin, the cells were
screened for the presence of factor VIII. All cell lines
proved to be negative (data not shown).
Cloning and Sorting of A2780-Dx5 Cells
One hundred ninety-two wells were seeded at a dilution of 0.1 cell per well. Microscopic evaluation for
cell number per well limited the number of potential
clones to 32. From these 32 wells, 17 potential clones
2432
CANCER December 1, 1996 I Volume 78 / Number 11
MRKl6
+
HPCA-2 .)
FIGURE 4. Flow cytometric irnrnunophenotyping of A2780Dx5 clones with regard to P-glycoprotein (left column)
and CD34 (right column) expression. Clone A2780-Dx5a (top row), A2780-Dx5b (second row), and A2780-Dx5c (bottom
row). The third row represents the cell line from which A2780 Dx5c was derived through flow cytometric cell sorting.
Dotted line: auto fluorescence
TABLE 2
Concordance and Discordance between Assessment of the Fraction of
CD34-ExpressingCells in Acute Myeloid Leukemia Bone Marrow
Samples by HPCA-2 and 12.8 Staining
~~~~~
Parient 1
Patient 2
Patient 3
Patient 4
Patient 5
% CD34 (HPCA-2)
% CD34 (12.8)
85
81
65
50
87
64
33
ti0
50
91
Discordant
Discordant
Concordant
Concordant
Condordant
could be expanded to cell numbers large enough to
allow flow cytometric screening for Pgp and CD34 expression. Three clones of interest were obtained, of
which the flow cytometric profiles are displayed in
Figure 4. Unlike the parent A2780-Dx5 cell line, clone
A2780-Dx5a (Fig. 4 top row) displayed a single population of Pgp-expressing cells that coexpressed CD34,
whereas clone A2780-Dx5b (Fig. 4, second row) displayed a single population of Pgp-expressing cells in
the absence of CD34.
No clones were obtained displaying CD34 expression
in the absence of Pgp. Therefore, the cell population displayed in Figure 4, third row, was chosen for flow cytometric cell sorting because this population showed an almost
equal distribution of Pgp-expressing and nodlow Pgpexpressing cells in the presence of CD34. Sorting was
performed based on Pgp expression and the cells were
subsequently analyzed again for CD34 and Pgp expression (Fig. 4, bottom row). Thus, a cell line was obtained
displaying CD34 expression in the absence of Pgp, which
was designated A278O-Dx5c. The absence of Pgp expression was confirmed by immunohistochemical staining
with the JSB-1 MoAb applying the immunoperoxidase
sandwich method (data not shown).3"Nomenclature and
phenotype of the newly derived clones are summarized
in Table 3.
CD34 Expression on Drug Resistant Cells/Minderman et al.
TABLE 3
Nomenclature and Phenotype of A2780 Cell Lines
P-glycoprotein
A2780-Dx5
(parental cell line)
A2780-Dx5a
A2780-Dx5b
A2780-Dx5~
TABLE 4
Sensitivity of A2780 Cell Lines to Doxorubicin, Daunorubicin,
Idarubicin, Vincristine, VP-16,and Amsacrine
CD34 (HPCA-2)
t, Heterogenous
t , Homogenous
t
t
t, Homogenous
-
-
t
PgP
CD34
Dox, 2h
t
positive,
~
2433
negdtive
Cloning and Sorting of A2780-wt Cells
Because the initial flow cytometric profile of the
A2780-wt cells revealed a low expression of CD34 (Fig.
2, second column) an attempt was made to subclone
an A2780-Wt cell line that would not show the low
CD34 expression. Both limiting dilution and flow cytometric cell sorting failed to produce a cell line with a
CD34 staining pattern distinct from the original A2780wt cell line. Therefore, the A2780-wt cells should be
considered a clonal cell population with an intrinsic
low reactivity with the HPCA-2 MoAb.
Dnr, 2h
Ida, 2h
Vcr, cont.
VP-16, 2h
M-AMSA, 2h
A2780 wt
~ 2 7 8 0D
-
-It
-1tt
tt
tt
Fold resistance
Fold resistance
Fold resistance
1
1
1
1
1
1
47.1=
2.V
2.P
Activity of Glutathione Pathway Enzymes
No significant differences were found in the activities
of glutathione peroxidase, glutathione reductase, or
glutathione-S-transferases between A2780-wt and
A2780-DX!ic cells. In addition, the activity of glucose6-phosphate dehydrogenase, which is related to the
-
ND
1.4
0.8
2.Y
1.1
6a
209.6
6.5a
4.V
TABLE 5
Antioxidant Enzyme Activities in Cell Lines A2780-wt and A2780Dx5c
nmol m i d per mg proteina
Enzymes
lmmunohistochemical Screening for Pgp, LRP, and MRP
Expression
Using the A2780-Dx5 as a positive control for Pgp expression, the HLGO/Adr cell line as a positive control
for MRP expression, and the HT1080Dr4 cell line as
a positive control for LRP and MRP expression, no
evidence was found for the expression of these MDR
markers on the A2780Dx5c cell line.
A2780 Dx5c
Pgp: p-glycoprotein; dox: doxorubicin; Dnr: daunoruhicin: Ida: idarubicin, Vcr: vincristine, 2 h 2-hour;
cont: continuous; M-AMSA: amsacrine.
a Significanily different from A2780 wt in 2 3, P < 0.05).
~
Cross-Resistance of A2780-Dx5c Cell Line
The sensitivity of the newly obtained cloned cell lines
to doxorubicin, daunorubicin, idarubicin, vincristine,
m-AMSA, and VP-16, was assessed by a total growth
assay (SRB). As expected, because of the presence of
Pgp, the A2780-Dx5a and A2780-Dx5b cell lines
showed cross-resistance to all drugs tested (data not
shown). The A2780-DX5c cell line displayed a low (2.6fold) but significant ( P < 0.05) resistance to doxorubicin compared with the A2780-wt cell line and only
displayed cross-resistance to daunorubicin, idarubicin, and VP-16. The results are summarized in Table 4.
X ~
~-
~27no-w
A278ODx5c
44.8 (212.5)
50.4 (k9.2)
13.5 (24.8)
72.5 (24.2)
43.9 (i1.4)
48.5 (1-11.2)
10.8 (23.6)
54.6 (214.3)
-~
Glutathione-S-transferasesb
Glutathione reductase
Glutathione peroxidase'
Glucose 6-phosphate dehydrogenase
a The results are presented as the mean values (standard deviation) from two independent experiments
in duplicate.
l-chloro-2,4-dinitrobenzene
as substrate.
' Butvl hvdrooeroxide as substrate.
reduced nicotinamide adenine dinucleotide phosphate generation by the pentose phosphate pathway,
was not altered. The activities of the glutathione-dependent enzymes are summarized in Table 5.
Northern Blot for Topoisomerase-ll-cu Levels
The A2780-DX5c and A2780-wt cell lines were compared for differences in RNA levels of topoisomerase11-a. Blots of total cellular RNA extracts were hybridized with a topoisomerase-11-a specific cDNA probe
using a G3PDH probe for RNA loading control. The
radiolabeled topoisomerase-11-a probe hybridized
with a -6.3-kilobase mRNA in both cell lines, a transcript size comparable to that previously described for
topoisomerase-11-a mRNA.40However, a lower level of
the topoisomerase-11-a transcripts was detected in the
A2780-DX5c cell line compared with the A2780-wt cell
line. (Fig 5).
2434
CANCER December 1,1996 I Volume 78 / Number 11
FIGURE 5. Northern blot analysis of cellular RNA extracts from A2780wt and A2780-DX5c cell lines. The blots were hybridized with a topoisomerase-ll-cl specific cDNA probe while a G3PDH probe served as a loading
control.
DISCUSSION
Studies designed for determining which (immuno)phenotypes predict the clinical response of AML demonstrated that both Pgp e x p r e ~ s i o n ' ~and
~ ' ~CD34 expressi0n14,
18,41-43 are negatively associated with clinical
response. The combination of both has shown to be
the best prognostic parameter.14 However, as single
prognosticators, CD34 expression is at least of equalI4
or better value than Pgp.'* Although the CD34 protein
has been characterized at the molecular level, its function has not been fully revealed. Most data reported
so far indicate a role in cell adhesion with a possibility
that different glycosylated forms of the protein might
serve different adhesive functions.44It is not clear how
this function correlates unfavorably with (clinical)
drug response.
The current study is the first to describe overexpression of CD34 associated with selection of MDR
cells of nonhematopoietic and nonvascular origin. Using a panel of three anti-CD34MoAbs different reactivity of the A2780 cell lines was observed compared with
established CD34-expressing cells (KG-1). The most
notable difference was observed with the 12.8 MoAb,
which showed no reactivity with A2780 cells, whereas
KG-1 cells were clearly positive. It is verified that the
existence of the HPCA-2+, 12.8- CD34 phenotype is
relevant in bone marrow samples from AML patients
by determining discordance between staining with
HPCA-2 and 12.8, which is in agreement with reports
by others.45
It has further been demonstrated that the A2780Dx5c clone obtained from the A2780-Dx5 cell line expresses CD34 in the absence of Pgp and displays a
low but significant level of MDH compared with the
sensitive wild type cell line. The drug sensitivity studies showed that clone A2780-Dx5c was cross-resistant
to the anthracyclines and VP-16. This cross-resistance
pattern, together with the demonstration of a decreased level of the primary target enzyme of topoisomerase-I1 inhibitors, strongly suggests that the
MDR observed in this cell line is related to the decreased topoisomerase-11-a levels. Involvement of
other established non-Pgp-mediated resistance mechanisms (MRP, LRP, and glutathione pathway) was
ruled out.
The current data confirmed that in vitro selection
of drug resistance by continuous drug exposure can
result in a collateral expression of multiple resistance
mechanisms in the resultant resistant cell population.
Evidence was presented that selection of drug resistance can lead to a stable induction of CD34 expression. However, the results did not rule out that for
the induction to occur, a low level of unstable CD34
expression (like that observed in the A2780-wt cell line)
is a prerequisite. Because decreased topoisomerase11-(Ylevels were detected in the resistant cell line, an
active role of CD34 in drug resistance in this cell line
model is not likely. However, it will be useful to determine the causal relationship between selection of drug
resistance and induction of CD34 overexpression because expression of CD34 may have been collaterally
induced with expression of a marker that is actively
involved in drug resistance. In this regard, the c-myc
protooncogene is of particular interest because it is
known that c-myc can play a role in development of
drug r e ~ i s t a n c ethat
, ~ ~ mRNA turnover is reduced in
AML,47and that c-myc expression is highest in the
CD34-expressing cells.48The presence of c-myc-like
motifs upstream of the CD34 encoding regions may
suggest a possible regulatory role of c-myc in the expression of CD34.8
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