Download Characterization of a Human Squamous

[CANCER RESEARCH 47, 388-393, January 15, 1987]
Characterization of a Human Squamous Carcinoma Cell Line Resistant to
m-Diamminedichloroplatinum(II)1
Beverly A. Teicher,2 Sylvia A. Holden, Michael J. Kelley, Thomas C. Shea, Carol A. Cucchi, Andre Rosowsky,
W. David Henner, and Emil Frei III
Division of Cancer Pharmacology, Dana-Farber Cancer Institute, Boston, MA 02115
ABSTRACT
We have developed a human head and neck squamous cell carcinoma
cell line (SCC-25/CP) which is relatively stably resistant to m-diamminedichloroplatinum(II) (('1)1)1') after repeated exposure to escalating
doses of the drug. The studies reported elucidate the mechanism(s) by
which the SCC-25/CP cell line is resistant to CDDP. The SCC-25/CP
cell line is approximately 30-fold resistant to CDDP, approximately 10fold resistant to carboplatin, and about 9-fold resistant to iproplatin.
Using ["*"Pt]CDDP, we examined the levels of platinum in whole cells
and cellular fractions of both the SCC-25 and SCC-25/CP cells after l h
exposure to 100 iiM drug. The SCC-25 cells took up 30 pmol of platinum/
10* cells in I h; 64% of the drug was in the nucleus and 21% in the
cytosol. The SCC-25/CP cells took up 7 pmol of platinum/10' cells; of
this, 41% was in the nucleus and 33% in the cytosol. The SCC-25 cell
nuclei contained 331 pmol of platinum/mg protein and the cytosol 21
pmol of platinum/mg protein, whereas the SCC-25/CP cell nuclei con
tained 47 pmol of platinum/mg protein and the cytosol 8.1 pmol/mg
protein. The release of drug from both cell lines followed a very similar
course and was most rapid over the first 6 h.
There was no difference in the non-protein sulfhydryl content of the
cell lines. The protein sulfhydryl content, as measured by Ellman's
procedure, indicated that the SCC-25/CP cell line has approximately a
2-fold increase in protein sulfhydryl content compared to the SCC-25 cell
line. The SCC-25/CP cell line is about 2-fold resistant to cadmium
chloride at 50% cell kill and about 2.5-fold resistant at 1 log kill compared
to the SCC-25 cell line. Glutathione transferase activity in crude cytoplasmic extracts was measured and found to be approximately 2- to 3fold higher in the CDDP resistant cells. The isoelectric point of the
glutathione transferase isozyme was 4.8 in both the sensitive and resistant
cell lines, suggesting induction of the predominant isozyme present in the
parent cell line. By alkaline elution there was greater cross-link formation
by CDDP in the SCC-25 cell line than in the SCC-25/CP cell line at the
same drug concentrations. In conclusion, the mechanism of resistance of
the SCC-25/CP cell line to CDDP is multifactorial, involving plasma
membrane changes, increased cytosolic binding, and decreased DNA
cross-linking.
INTRODUCTION
CDDP3 has demonstrated a broad range of activity against
several malignancies in humans (1). CDDP is classified with
the antitumor alkylating agents because it forms bidentate
adducts with DNA (2-4). It is believed that DNA is the critical
intracellular target of CDDP and that DNA cross-linking is the
lethal lesion caused by this drug (2, 5, 6). Like many antineoplastic agents, CDDP is a potent mutagen, inducing frame shift
and base substitution mutations in both bacterial and human
cells (7-10). CDDP-sensitive and -resistant LI210 cell lines
have been examined by several laboratories. These studies have
Received 5/19/86; revised 9/12/86; accepted 10/14/86.
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 18 U.S.C. Section 1734 solely to indicate this fact.
1This work is supported by National Cancer Institute grants 1RO1-CA36508,
lPOl-CA38493,and
5F32-CA07821.
2To whom requests for reprints should be addressed.
3The abbreviations used are: CDDP, cú-diamminedichloroplatinum(II); GSH,
reduced glutathione; GST, glutathione transferase; PAM, L-phenylalanine mus
tard; FBS, fetal bovine serum; PBS, phosphate-buffered 0.9% saline solution;
OPT, ortho-phthaldialdehyde; BSO, D,L-buthionine-S,A-sulfoxime.
described changes in the plasma membrane (11-13) and
changes at the level of DNA adduct formation (14) in the
resistant cell lines.
Experimental studies of other alkylating agents have indi
cated that resistance to these drugs may occur by a variety of
mechanisms. Goldenberg and Begleiter (IS) and Goldenberg et
al. (16) found resistance to nitrogen mustard to be associated
with a transport defect in the choline carrier impeding uptake
of nitrogen mustard into the cells. Resistance to PAM has also
been attributed to a transport defect, probably involving the
leucine carrier (17, 18). In other studies, PAM resistance was
associated with an elevation in intracellular glutathione (1922). Multifactorial resistance to PAM involving rate of drug
efflux, sulfhydryl levels, and DNA interstrand cross-link for
mation and repair has been reported in several cell lines (23,
24). Hilton and Colvin (25) have reported that cyclophosphamide-resistant human and rodent cell lines have increased levels
of an aldehyde dehydrogenase that inactivates the aldophosphamide metabolite of the drug. Increased repair of DNA
monoadducts via the action of guanine 06-methyltransferase
has been described as the mechanism of resistance in l,3-bis(2chloroethyl)-l-nitrosourea-resistant
bacterial and human cell
lines (26-29).
We have developed a human head and neck squamous cell
carcinoma line (SCC-25/CP) which exhibits relatively stable
resistance to cis-diannninedichloroplatinum(II)
after repeated
exposure to escalating doses of the drug (30). The studies
reported here were designed to elucidate the mechanism(s) by
which the SCC-25/CP cell line are resistant to CDDP.
MATERIALS
AND METHODS
Drugs. CDDP, diammine [l,l-cyclobutanedicarboxylato(2)]-O,O'platinum(II)
(carboplatin),
and
cw-dichloro-/ra/w-dihydroxobis(isopropylamine)platinum(IV) (iproplatin) were gifts from Johnson
Matthey, Inc. (West Chester, PA). ["5mPt]cw-diamminedichloroplatinum(II) in isotonic saline was made available by Drs. J. D. I loes
chele and F. F. Knapp, Jr. at Oak Ridge National Laboratories (Oak
Ridge, TN) (31, 32). CdCl2 was purchased from Aldrich Chemical Co.
(Milwaukee, WI).
Cell Lines. SCC-25 and SCC-25/CP human squamous carcinoma of
the head and neck cells grow as monolayers in Dulbecco-Vogt modified
Eagle's minimum essential medium supplemented with antibiotics and
5% FBS (30). These cell lines have a plating efficiency of 10-30% and
a doubling time of 48-50 h in vitro (33). For cloning, SCC-25 and
SCC-25/CP cells were suspended by trypsinization, diluted in complete
growth medium, and plated into 60- x 15-mm tissue culture dishes
containing 5 ml of complete growth medium. Colonies grow to a
countable size (>50 cells) in 2 weeks.
The SCC-25/CP cell line has been maintained for 9 months in the
absence of exposure to CDDP and the resistance of this cell line to
CDDP has remained stable for that period.
Survival Studies. SCC-25 and SCC-25/CP cells in exponential
growth were exposed for 1 h to concentrations of CDDP, carboplatin,
iproplatin, or CdCl2 ranging from 1-1000 /¿M
in media without sera.
The cells were then washed three times with PBS and plated for colony
formation as described above. Each survival curve was determined in
three independent experiments.
388
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CELLULAR
RESISTANCE
Cellular Fractionation. [195mPt]CDDP was received with a specific
activity of 145-165 mCi/mmol. The half-life of "5mPt is 4.02 days, and
TO CDDP
niques. The cells were washed three times with PBS and suspended in
50 HIMsodium phosphate buffer, pH 6.5. The cell suspensions were
kept at 4"( ', disrupted by sonication (Sonifier 200; Bronson, Inc.), and
therefore experiments were carried out immediately upon receipt of the
drug. Two million SCC-25 and SCC-25/CP cells/plate were incubated
centrifuged at 16,000 x g for 30 min. GST activity in the supernatant
with 100 /iM [l95mPt]CDDP in media without sera for 60 min at 37°C was measured by the method of Habig et al. (42) using 1 mM 1-chloro2,4-dinitrobenzene as the electrophilic substrate. GST activity is ex
in humidified air with 8% CO2. Cells were washed six times with PBS,
pressed as nanomoles of GSH-l-chloro-2,4-dinitrobenzene
conjugate
fresh medium containing serum was added, and the plates were replaced
in a 37°C/8%CO2 incubator for 0, 3, 6, or 24 h. At the end of each
formed per min per mg protein. The results presented are from cells
harvested on three separate occasions.
time period, cells were suspended using 0.5% EDTA/0.125% trypsin,
centrifugea at 500 x g, resuspended in water, and lysed by sonication
Cell extracts were analyzed by isoelectric focusing on 0.5 mm 5%
polyacrylamide gels through a pH 3-10 ampholine gradient (Serva
on ice.
Biochemicals). After focusing was complete, the gels were divided
The fractionation procedure of Sharma and Edwards (34) was per
formed at 4°Cand the final pellets were resuspended in water. Lysed
according to pH into 0.75-cm wide segments from which GST activity
cells were centrifuged at 1000 x g for 10 min and the pellet resuspended
was eluted and measured as described above (42). The isoelectric points
in 0.25 M sucrose/1.8 HIMCaCl2/l% Triton X-100 solution. An equal
of the GST isoenzymes were determined by use of marker proteins
(Serva Biochemicals) and pH measurement of adjacent gel segments.
volume of 0.34 M sucrose/0.18 imi CaCl2 solution was added to the
bottom of the tube, pushing up the lighter solution. After cent rifugat ion The major isozyme was defined as containing more than 70% of the
total GST activity recovered from the gel.
at 600 x g for 10 min, the nuclear pellet was obtained. The supernatant
Alkaline Elation. Alkaline elution was performed by standard pro
from the first 1000 x g spin was centrifuged at 3500 x g for 10 min to
cedures (5, 6, 43-45). SCC-25 or SCC-25/CP cells (2.75 x IO5)were
obtain the mitochondria! pellet. The second supernatant was centri
labeled with 1.25 nCi of [l4C]thymidine (New England Nuclear, Boston,
fuged at 16,000 x g for 20 min to obtain the h soso mal pellet. The
MA) in Dulbecco-Vogt modified Eagle's minimum essential medium
third supernatant was centrifuged at 100,000 x g for 60 min to obtain
supplemented with 5% FBS for approximately three doublings, after
the microsomal pellet. This final supernatant contained the cytosol.
which the [uC]thymidine was removed and the cells washed. Drugs
The purity of the subcellular fractions from each cell line was checked
by electron microscopy. 195mPtcontent was determined using a Beckman
were added to the cells in fresh medium without serum and then
incubated for 60 min at 37°C.The cells were washed with PBS, fresh
Gamma 4000 counter. The amounts of ["5mPt]CDDP were determined
medium plus 5% FBS was added, and incubation at 37°Cwas resumed.
by comparison to standards. Each point is the mean of duplicate
At
various time intervals cells were removed with 0.1% EDTA. Onedeterminations in two independent experiments. The range of duplicate
half of each group of cells was irradiated on ice with 600 rads using a
determinations was ±3%(SE) and the range of experimental values
Gammacell 40 (Atomic Energy of Canada, Ltd.). Approximately 5 x
was ±8%.
IO5cells/group were placed onto an alkaline elution filter (2.0 nm pore
Protein Determinations. Protein was measured using a modified
size)
(Millipore, Bedford, MA). As an internal control, the same number
biuret assay (Sigma Diagnostics, Sigma Chemical Co., St. Louis, MO)
of'l'H |thy midine-labeled LI 210 cells irradiated with 150 rads were also
following the cellular fractionation procedure described above.
placed onto each filter. Cells were first washed with cold PBS, then
Sulfhydryl Measurements. SCC-25 and SCC-25/CP cells in expo
nential growth (4 x 10') were lysed in 2 ml of 5% perchloric acid. The
lysed with 3 ml of 0.2% sodium dodecylsarkosine/2 M NaCl/0.04 M
EDTA, pH 10, which was allowed to flow through by gravity. To study
protein was removed with 5 M potassium phosphate by centrifugation
DNA-DNA interactions, 0.5 mg/ml proteinase K (Sigma) was added
at 1000 x g for 3 min at 5°C.The supernatant was neutralized then
to the lysis solution, and incubation on filters was allowed to proceed
passed through a 0.2-itm filter before derivatization. Glutathione was
for 60 min at room temperature. Alkaline elution was carried out using
assayed at four dilutions of the supernatants.
2% tetrapropylammonium
hydroxide (Fisher Scientifïc)/0.025 M
For the fluorescence assay, 0.1 ml of the sample was added to 3 ml
EDTA at a rate of 2.4 ml/h in the dark. Fractions were collected at 90of 0. l M potassium phosphate buffer containing 5 HIMEDTA (pH 8),
min intervals and were assayed for radioactivity after adding 12 ml of
then 0.15 ml of an OPT (Aldrich Chemical Co., Milwaukee, WI)
Aquasol
(Dupont/New England Nuclear). The remaining DNA on the
solution (1 mg/ml OPT in methanol) was added. The derivatization
filters was removed by treatment with 0.4 ml of l N HC1 for 60 min at
was allowed to continue for 15 min at room temperature in the dark.
65°C,and the solution was neutralized with 2.5 ml of 0.4 N NaOH
Fluorescence was measured with an excitation wavelength of 350 nm
before Aquasol addition. Samples were counted on an LS 7000 Beckand an emission wavelength of 420 nm. The calibration curve was linear
man scintillation counter. Each point was measured in three independ
from 0.05-50 nmol of GSH/ml (35, 36). The data shown are the mean
ent experiments. Cross-linking factor was calculated as
of five determinations.
Glutathione was also determined by the method of Fahey et al. (37).
/irradiated
L°g(—....control\ 1
Glutathione was derivatized by monobromobimane and quantitated by
control
V
peak height on high-performance liquid chromatography. Non-protein
and total sulfhydryl contents were determined using a modification of
/irradiated drug \
the Ellman method (38-41). SCC-25 and SCC-25/CP cells (IO7) were
L0gl
control
j
lysed in 0.02 M EDTA (6 ml). For non-protein sulfhydryl determina
tion, the cell lysate (5 ml) was treated with 10% trichloroacetic acid (5
ml), then centrifuged at 500 x g for 5 min at 5°C.The supernatant (4
RESULTS
ml) was adjusted to pH 8.9 with 0.2 M Tris buffer. Ellman's reagent
[0.01 M 5,5'-dithiobis(2-nitrobenzoic acid)] in methanol (Aldrich) (12.5
The SCC-25/CP cell line was developed by repeated treat
Ml/ml sample) was added and color was allowed to develop for 30 min
at room temperature. Total sulfhydryl content was determined by
adding cell lysate (0.5 ml) to 0.2 M Tris buffer ( 1.5 ml, pH 8.2) followed
by 0.1 ml of 0.01 M 5,5'-dithiobis(2-nitrobenzoic
acid) in methanol.
ment with escalating concentrations of CDDP and is generally
not cross-resistant to alkylating agents of the nitrogen mustard
and nitrosourea classes as determined by median inhibitory
concentration ratios (30). However, it was not known whether
After bringing the sample volume to 3 ml with methanol, the color was
the SCC-25/CP line would be resistant to other platinum
allowed to develop for 30 min at room temperature. All of the samples
complexes. The survival of SCC-25 and SCC-25/CP cells ex
were filtered (0.45 nm) and absorbance was read at 412 nm. Protein
posed to various concentrations of CDDP and two secondsulfhydryl content was determined from the difference between the total
generation platinum complexes, carboplatin and ipropiatin, is
sulfhydryl content and non-protein sulfhydryl content. The measure
shown in Fig. 1. A 30-fold higher concentration of CDDP is
ment was repeated three times.
required to kill 90% of SCC-25/CP cells compared to SCC-25
Glutathione Transferase Measurements. SCC-25 and SCC-25/CP
cells were maintained in cell culture and harvested by standard tech
cells. Carboplatin and ipropiatin are less potent cytotoxic agents
389
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CELLULAR RESISTANCE TO CDDP
100 300 500
1000 100 300 500
1000 100 300 500
DRUG CONCENTRATION,
1000
yU
Fig. 1. Survival of SCC-25 (•)and SCC-25/CP (O) cells treated with various
doses of CDDP (A), carboplatin (/(), or iproplatin (('). Points, means of three
independent determinations ±SE (bars).
30.0
0.03
036
24 0 3 6
Time (hrs)
Fig. 2. Subcellular distribution of "5mPt in SCC-25 and SCC-25/CP cells.
Cells were exposed to 100 ^M ("5"Pt]CDDP for 1 h. The zero time is immediately
after drug removal. A, cytosol; •,nuclei; O, mitochondria; •,lysosomes; D,
microsomes.
SCC-25/CP cells as indicated by the levels of platinum in these
cells after 1 h exposure to the drug provides these cells with
one mechanism of resistance. However, the amount of platinum
which is found in the nuclei of SCC-25/CP cells is lower than
expected on the basis of the plasma membrane barrier alone.
Therefore, we examined the cytosol of both cell lines for pos
sible differences which could account for the increased differ
ential in nuclear platinum content. The non-protein sulfhydryl
content of both cell lines was assayed using OPT derivatization
and fluorescence measurement for total non-protein sulfhydryls
and monobromobimane derivatization, high-performance liq
uid chromatography, and fluorescence detection for GSH (Ta
ble 1) (35-37). There was no difference in the non-protein
sulfhydryl content between the cell lines, and the ratio of GSH
levels between the cell lines was 1.11 ±0.13. However, the
protein sulfhydryl content, as measured by Ellman's procedure,
indicated that the SCC-25/CP cell line has approximately a 2fold increase in protein sulfhydryl content compared to the
SCC-25 cell line (38-41). Metallothioncins, which contain
about 30% cysteine, can comprise a large percentage of cellular
protein sulfhydryl content. Resistance to the cytotoxic effects
of cadmium chloride has been used as an indicator for an
increased metallothionein content (46-49). The survival of
SCC-25 and SCC-25/CP cells exposed to various concentra
tions of cadmium chloride for 1 h is shown in Fig. 3. The SCC25/CP cell line is about 2-fold resistant to cadmium chloride
at 50% cell kill and about 2.5-fold resistant at 1 log kill
compared to the SCC-25 cell line. This finding correlates very
well with the increase in protein sulfhydryl content of the SCC25/CP cells.
In addition to metallothionein binding, conjugation of CDDP
to GSH by glutathione-S-transferase could account for the
reduced nuclear content of CDDP. GST activity in crude cytoplasmic extracts of both cell lines was measured and GST
activity was found to be 2- to 3-fold higher in the SCC-25/CP
cells. The isoelectric point of the principal GST isozyme was
4.8 in both the SCC-25 and SCC-25/CP cell lines. This suggests
induction of the predominant isozyme present in the parent cell
line rather than induction of a new or previously undetectable
isozyme.
It is believed that the major mechanism of the cytotoxicity
of CDDP is the formation of cross-links in DNA (2, 5, 6). The
formation of DNA cross-links by CDDP in the SCC-25 and
SCC-25/CP cells was assessed using alkaline elution (45) at
three concentrations (50, 10, and 2 /IM)after 1 h exposure to
the drug (Fig. 4). The formation of DNA cross-links was
followed for 48 h. Overall, there was greater cross-link forma
tion in the SCC-25 than in the SCC-25/CP cell line. Although
the formation of cross-links in the SCC-25/CP cell line appears
to plateau after 24 h, there was no evidence of removal of the
cross-links formed up to 48 h. When the levels of cross-links
formed at the various drug concentrations are compared, the
cross-linking factor in SCC-25/CP cells is approximately 2fold lower than that of the SCC-25 cell line at the same drug
concentration.
than CDDP. The SCC-25/CP cell line is approximately 10fold resistant to carboplatin and 9-fold resistant to iproplatin.
We conclude, therefore, that SCC-25/CP cells are cross-resist
ant to these platinum complexes which are structurally related
to CDDP.
Using [l95mPt]CDDP,we examined the levels of platinum in
whole cells and cellular fractions of both the SCC-25 and SCC25/CP cells after l h exposure to 100 /¿M
drug (Fig. 2) (34).
Measurements of platinum levels in the various cellular frac
tions were made both immediately after removal of drug and at
various times over the following 24 h. The SCC-25 cells took
up 30 pmol of platinum/IO6 cells in 1 h; 64% of the drug was
in the nucleus and 21% in the cytosol. Each of the other cellular
fractions (lysosomes, microsomes, and mitochondria) contained
5% of the total platinum taken up. The SCC-25/CP cells took
up 7 pmol of platinum/10* cells; of this, 41% was in the nucleus
and 33% in the cytosol. Each of the other cellular fractions
from this cell line contained 8-9% of the platinum taken up.
The SCC-25/CP cells (0.391 mg protein/106 cells) are some
what smaller than the SCC-25 cells (0.457 mg/106 cells); how
ever the nuclei in both cell lines contain 0.058 mg protein/IO6
cells. Therefore, the SCC-25 cell nuclei contained 331 pmol of
DISCUSSION
platinum/mg protein and the cytosol 21 pmol of platinum/mg
protein, whereas the SCC-25/CP cell nuclei contained 47 pmol
CDDP has gained wide clinical use as an antineoplastic
of platinum/mg protein and the cytosol 8.1 pmol/mg protein. alkylating agent (1, 50). The goal of this study is to understand
The release of drug from both cell lines followed a similar the mechanism(s) by which the SCC-25/CP human squamous
course and was most rapid over the first 6 h. After 24 h, the carcinoma cell line is resistant to CDDP. These mechanisms
SCC-25 nuclei retained 26 pmol of platinum/mg protein. The may also account for the cross-resistance to carboplatin and
iproplatin in this cell line. Cross-resistance with CDDP ana
SCC-25/CP nuclei retained 5.5 pmol of platinum/mg protein.
logues has previously been reported in a human ovarian cancer
Reduced uptake of CDDP through the plasma membrane of
390
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CELLULAR
RESISTANCE
TO CDDP
Table 1 Protein and non-protein sulfliydryl content and glutathione transferase specific activity in SCC-25 and SCC-25/CP cells
nmol/106 cells
Glutathione
transferase
activity*
Non-protein
Protein
Isoelectric
sulfhydryl"
sulfhydryl*
point**
(nmol/min)
Cell line
25±2<(55±5/
231
±
25
(503
±
55)*
SCC-25
23 ±3 (48 ±7)
4.8
SCC-25/CP
447 ±73(1145 ±186)
21 ±3 (54 ±7)
39 ±2 (100 ±5)
4.8
•
Measured by fluorescence emission at 420 nm of an OPT derivative.
0 Measured by the difference between total sulfhydryl content and non-protein sulfhydryl content using Ellman's method. Absorbance was measured at 412 nm.
'' Activity determined by absorption at 340 nm of product formed by the reaction of GSH with l-chloro-2,4-dinhroben/ene.
d For the principal glutathione transferase isozyme present in these cells.
' Mean ±S.E.
^Numbers in parentheses, nmol per 10* cells per mg protein.
* Numbers in parentheses, nmol per mg protein.
1.0
CdCI2
0.1
0.01
0.001
50100
500
1000
Drug Concentration uMolar
Fig. 3. Survival of SCC-25 (•)and SCC-25/CP (O) cells treated with various
doses of cadmium chloride. Points, means of three independent determinations ±
SE (bars).
14
SCC-25
,-12
o
o
«10
O)
-E 8
JÃC
m
S 4j
0 ,
t-
C
3 12
24
48
Time, Hrs.
Fig. 4. DNA cross-linking in SCC-25 and SCC-25/CP cells at various times
after drug exposure. The concentrations of CDDP used were: •,50; O, 10; and
•.2 /IM. The cross-linking factor was calculated according to the method of Kohn
et al. (43). Data were derived from three independent experiments ±SE (bars).
cell line made resistant to CDDP (51). The cellular distribution
of platinum following treatment with CDDP has been described
for cells in culture (52, 53) and in several tissues in vivo (34,
54, 55). Using analytical electron microscopy, Khan and Sadler
(52) found that CDDP was predominantly localized in the
nucleolus and the inner side of the nuclear membrane. In
nucleosomes, using a fluorescent probe, histone H3 as well as
DNA were found to be targets for CDDP (53). The intracellular
localization of platinum in liver, kidney, and other tissues has
been measured by atomic absorption (34, 55) and scanning
transmission electron microscopy in conjunction with X-ray
probe microanalysis (54). In these tissues, high percentages of
platinum were found in the cytosol bound to metallothioneinlike proteins and in lysosomes in the kidney tubules. Studies of
L1210 cells resistant to CDDP have provided evidence that the
drug is transported into the cells by an amino acid transport
system (11-13). Plasma membrane alteration is a common
mechanism of cellular resistance to al kviating agents (11-13,
15-18) and to a wide variety of other drugs and toxins (56-58).
We have found that both SCC-25 and SCC-25/CP cells con
centrate platinum in the nuclei to some degree; however, there
is a difference in platinum levels in the parent line compared
to the CDDP resistant line, implying an alteration in the plasma
membrane of the SCC-25/CP cells leading to decreased plati
num levels in that cell line under the same exposure conditions
as the parent line.
There also appears to be cytosolic changes in the SCC-25/
CP cell line. Although the levels of the non-protein sulfhydryl,
GSH, are the same in both cell lines, there is an increase in
protein sulfhydryl content and an increase in GST in the SCC25/CP cell line. When the SCC-25 and SCC-25/CP cell lines
were treated with BSO, the levels of GSH in both cell lines
decreased similarly. The response of both cell lines to the
cytotoxic actions of CDDP remained unaffected by the BSO
treatment.4 Similar results with BSO have been reported re
cently in two human ovarian carcinoma cell lines 2.5- to 3-fold
resistant to CDDP (59). Increased protein sulfhydryl content
and cadmium chloride resistance indicate that the SCC-25/CP
cell line may have increased levels of metallothionein-like pro
teins. These proteins have been induced acutely by exposure to
cadmium, zinc, and glucocorticoid hormones through increased
transcription (60,61) or chronically through gene amplification
(46). Although CDDP has not been shown to induce metallothionein (47, 48), cells which have a high content of metallothionein are resistant to CDDP (49), and CDDP has been
shown to bind to metallothioneins both in vitro and in vivo (62,
63).
In conclusion, the mechanism of resistance of the SCC-25/
CP cell line to CDDP is multifactorial: (a) there are reduced
intracellular levels of platinum in the SCC-25/CP compared to
SCC-25 cells, implying altered plasma membrane properties in
the resistant line; (b) there is a 2-fold increase in protein
sulfhydryl content, a 2-fold increase in GST, and a 2-fold
reduction in sensitivity to CdCl2, implying altered cytosolic
binding and metabolism of CDDP in the resistant line; and (c)
there is a reduced level of DNA cross-linking by CDDP in the
SCC-25/CP line compared to the parent line. In addition to
these changes which we have measured, there may be other
alterations in the SCC-25/CP cell line which result in the 30fold resistance observed.
' E. Frei III, B. A. Teicher, and C. A. Cucchi, upublished observations.
391
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CELLULAR RESISTANCE TO CDDP
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Characterization of a Human Squamous Carcinoma Cell Line
Resistant to cis-Diamminedichloroplatinum(II)
Beverly A. Teicher, Sylvia A. Holden, Michael J. Kelley, et al.
Cancer Res 1987;47:388-393.
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