[CANCER RESEARCH 47, 6288-0293, December I, 1987]
Reciprocal Cross-Resistance in Human Rhabdomyosarcomas Selected in Vivo for
Primary Resistance to Vincristine and L-Phenylalanine Mustard1
Julie K. Morton, Peter J. Houghton,2 and Janet A. Houghton
Laboratories for Developmental Therapeutics, Department of Biochemical and Clinical Pharmacology, St. Jude Children 's Research Hospital, Memphis, Tennessee 38101
ABSTRACT
Primary resistance to vincristine (VCR) has been selected in rhabdomyosarcoma xenograft 11\Khl 2 by sequential administration of VCR at
1.5 and subsequently 3 mg/kg/passage. The resistant tumor (HxRhl2/
VCR-3) was approximately 4-fold resistant to VCR and resistance was
stable in the absence of selecting pressure (>2 yr). HxRhl2/VCR-3 was
2- to 3-fold cross-resistant to i.-phenyhlanine mustard (L-PAM) but only
slightly cross-resistant to ifosfamide. To determine whether selection for
primary resistance to L-PAM conferred cross-resistance to VCR we
selected an L-PAM-resistant subline of rhabdomyosarcoma xenograft
HxRh28 (HxRh28/L-PAM-13). This tumor was 2- to 3-fold resistant to
L-PAM
and
3-{/>-fluorophenyl)-L-alanyl-3-|m-bis-{2-chloroethyl)aminophenyl|-L-alanyl-L-methionine ethoxyhydrochloride, cross-resist
ant to cyclophosphamide and ifosfamide, and completely resistant to
VCR under in vivo conditions. Pharmacokinetic studies in HxRhl2/
VCR-3 showed decreased retention of |<V-'H]\ ( R but not alteration in
metabolism. Expression of mtlrt. a gene that encodes P-glycoprotein,
associated with the multiple drug resistance phenotype, was examined.
Expression of marl was detected in both HxRhl2 and HxRh28 tumors,
sensitive to VCR, but there was no increase in expression in tumors
selected for primary resistance to VCR or L-PAM. Data suggest that
mechanisms other than those associated with "classical" multiple drug
resistance confer resistance in these tumors.
In clinical evaluation against childhood rhabdomyosarcoma, L-PAM
has demonstrated only slight activity in patients relapsing on conventional
therapy (including VCR) but demonstrated marked activity in patients
with advanced previously untreated disease. It appears likely, therefore,
that cross-resistance between VCR and L-PAM as demonstrated in this
model may have clinical significance.
INTRODUCTION
In the treatment of soft tissue sarcomas of children, of which
RMS3 is the most common (1), combinations of alky lating
agents and natural products have proven curative in early stage
disease. In advanced disease multimodality therapy may induce
prolonged remission, with subsequent relapse. In most in
stances at relapse, RMS is resistant to agents used for induction
and other agents. Early studies on cross-resistance in animal
models indicated universal resistance to bifunctional alkylating
agents (2-4), but more recent reports showed clearly that crossresistance among alkylating agents is not universal (5-7). How
ever, certain data indicate that cells selected for primary resist
ance to an alkylating agent may exhibit cross-resistance to
natural products such as doxorubicin (8). Also, primary resist
ance to doxorubicin has been associated with cross-resistance
Received 5/1/87; revised 8/10/87; accepted 9/9/87.
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.
1Supported by Awards CA 38933 and CA 23099 from the National Cancer
Institute and by the American Lebanese Syrian Associated Charities.
1To whom requests for reprints should be addressed, at St. Jude Children's
Research Hospital, 332 N. Lauderdale, Memphis, TN 38101.
'The abbreviations used are: RMS, rhabdomyosarcoma; VCR, vincristine;
VLB, vinblastine; L-PAM, L-phenylalanine mustard; PT119, 3-(p-fluorophenyl)L-alanyl-3-[m-bis-(2-chloroethyl) aminophenylj-L-alanyl-L-methionine ethoxyhy
drochloride; Act-D, actinomycin-D; DOX, doxorubicin; CLC, colchicine; MDR,
multiple drug resistance; CHO, Chinese hamster ovary; SDS, sodium dodecyl
sulfate; cDNA, complementary DNA; SSC, 750 mM NaCI, 75 mM sodium citrate,
pH 7.O.
to L-PAM and cis dichlorodiammino platinum II (8). In con
trast, Teicher et al. (9) did not select cells cross-resistant to
DOX or VCR when primary resistance to l,3-bis(2-chloroethyl)-l-nitrosourea, Nor nitrogen mustard, or cis dichlorodiam
mino platinum II was obtained. In CHO cells, which exhibit a
MDR phenotype and in which primary resistance was selected
to colchicine, cross-resistance to L-PAM was observed (10).
Further, in a revertan! line, sensitivity to L-PAM was regained,
suggesting that these phenomena were related (11). However,
in a line of CHO cells selected for primary resistance to LPAM, the MDR phenotype was not determined (11). Thus, the
relationship between resistance to natural products (DOX,
VCR, CLC, Act-D, etc.), the MDR phenotype, and alkylating
agent resistance is complex. Presumably, factors that influence
cross-resistance are the cell line, selection procedure, degree of
resistance, and perhaps the initial definition of the sensitivity
of the wild-type cell.
The question thus arises as to whether such studies in vitro
are of value in predicting for resistance patterns in tumors
selected under conditions in vivo. For example, both P388/
ADR (selected in vivo for resistance to DOX) (12) and P388/
VCR (13) show the MDR phenotype but are not cross-resistant
to L-PAM. In contrast, P388/L-PAM is cross-resistant to
VCR, partially cross-resistant to Act-D, and not cross-resistant
to DOX (13).
Thus, whereas cross-resistance to L-PAM appears in some
instances associated with the MDR phenotype (10), there ap
pears a more specific relationship between resistance to L-PAM
and cross-resistance to VCR. Our interest in this problem was
stimulated because of data obtained using human rhabdomyosarcomas maintained in mice. Using these models (derived from
surgical specimens prior to chemotherapy), L-PAM was iden
tified as demonstrating considerable efficacy (14). In a subse
quent Phase II evaluation in patients with refractory RMS, LPAM showed only slight activity, whereas in previously un
treated patients it was clearly a highly active agent (15). Thus,
resistance to induction and maintenance therapy (VCR, DOX,
Act-D, cyclophosphamide) appeared to confer resistance to LPAM. Clearly, which agents were responsible for such clinical
cross-resistance is difficult to elucidate, although cyclophospha
mide would appear a good candidate because of its mechanism
of action. In this article we have selected for resistance to VCR
or L-PAM in two human RMS xenografts in vivo. Under these
conditions of selection there is reciprocal resistance independ
ent of the agent used for primary selection.
MATERIALS
AND METHODS
Immune Deprivation of Mice. Female CBA/CaJ mice (The Jackson
Laboratory, Bar Harbor, ME), 4 wk old, were immune deprived by
thymectomy, followed at 3 wk by i.p. administration of l-/i n-arahinofuranosylcytosine (200 mg/kg) 48 h prior to receiving whole-body
irradiation (950 rads at 170 rads/min, using a "7Cs source) (16).
Tumor Lines and Selection of Resistance. Tumors HxRhl2 and
HxRh28 have been described previously (14, 17). Briefly, HxRhl2, a
moderately differentiated embryonal RMS, was established as a xeno
graft from an untreated tumor specimen. The HxRh28 xenograft was
6288
VINCRISTINE-L-PAM
CROSS-RESISTANCE
derived from an axillary node metastasis surgically resected prior to
treatment. This tumor has alveolar histology. Both tumors grow rou
tinely in >90% of recipient mice and are human as determined by
karyotype and species-specific isoenzyme patterns (17).
The sensitivity of these RMS xenografts to VCR (16) and L-PAM
has been reported (14). Both agents given at the maximum tolerated
dose caused complete regression of advanced HxRhl2 and HxRh28
xenografts. To select for resistant lines the following strategy was
adopted. For VCR, mice bearing I IxKli 12 xenografts received a single
i.p. administration of 1.5 mg/kg (0.5 x maximum tolerated dose).
Tumor diameters were measured at 7-day intervals using Vernier cali
pers, and tumors were allowed to regrow to 4-fold their volume at
treatment. The tumor that demonstrated the poorest response was
transplanted and the process repeated. After 7 cycles, the dose of VCR
was increased to 3 mg/kg for 6 treatments. For developing an L-PAMresistant subline of HxRh28, the initial dose was 10 mg/kg for 4 cycles
and subsequently 13 mg/kg as a single administration at each passage.
Growth Inhibition Studies. Mice bearing bilateral s.c. tumors received
a single i.p. administration of agent when tumors had reached 1 cm
diameter. Response was measured by computing tumor volumes from
the measurement of two perpendicular diameters at 7-day intervals
(14). In some experiments, denoted as double-flank, parent tumor and
the resistant subline were transplanted into opposite flanks of the same
host for direct comparison of response under identical conditions of
growth and drug exposure (18).
Pharmacokinetics. Tumor-bearing mice were given i.p. injections of
[3H)VCR (3 mg/kg) or [3H]VLB (3 mg/kg; 0.1-0.2 nCi/g, body weight;
Amersham, Arlington Heights, IL, or Moravek Biochemicals, Brea,
CA), and the concentration of drug achieved in tumors was determined
between 10 min and 72 h after treatment. Tumors were rapidly excised,
washed in ice-cold 0.9% NaCl solution (saline), blotted dry, weighed,
and digested in NCS solution (5 ml/g tissue; Amersham). One ml of
solution was assayed for radioactivity. Analysis by high-performance
liquid chromatography of acidified-ethanol extracts of tumor were as
described previously (16).
Isolation of mRNA. Tumors were rapidly excised, weighed, and
homogenized (Polytron) in 4 M guanidine isothiocyanate (4 ml/g tis
sue). Extraction was performed as described by Feramisco et al. (19).
RNA was precipitated by addition of sodium acetate to 600 HIMand 3
volumes of 95% ethanol. After centrifugating the pellet was resuspended
(0.1 M Tris-HCl, pH 7.4, 50 mM NaCl, 10 mM EDTA, 0.2% SDS) and
digested with proteinase K (200 ¿tg/ml)for 2 h at 37*. Protein was
extracted twice with phenol/chloroform and a further 2 times with
chloroform at room temperature and RNA was precipitated as before.
The pellet was washed extensively with 70% ethanol. For oligodeoxythymidylate-cellulose affinity chromatography, RNA was dissolved in
loading buffer (20 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.1% SDS, 0.5
M LiCl), heated at 65* for 5 min, cooled, and applied to an oligo-dT
IN VIVO
were hybridized at 41°Cfor a minimum of 2 h in 50% formamide, 5 x
SSC, 1 x Denhardt's solution (1% Ficoll, 1% polyvinylpyrolidone, 1%
bovine serum albumin, Pentax Fraction V), 0.25 mg/ml denatured
salmon sperm DNA, 0.1% SDS, and 0.05 M sodium phosphate, pH
6.5. Prehybridization solution was replaced with fresh solution contain
ing in addition 1 x 10' cpm/ml "P-labeled MDR-1 probe. Hybridiza
tion was for a minimum of 16 h at 41V. Subsequently, filters were
washed 4 times for 5 min each in 2 x SSC, 0.1% SDS at room
temperature, then 4 times for 15 min in 0.1 x SSC, 0.1% SDS at 50'C.
Filters were dried and exposed to preflashed Kodak X-Omat AR film
using intensifying screens for 24 to 48 h. Filters were dehybridized by
boiling in water for 5 min. Complete dehybridization was confirmed by
exposing the filter to film. Filters were rehybridized using "'I' labeled
pAl as described for pMDR-1 above.
Cytotoxic Agents. L-PAM and CLC were obtained from Sigma
Chemical Co. (St. Louis, MO), VCR (Oncovin) from the Eli Lilly Co.
(Indianapolis, IN), cyclophosphamide, ifosfamide, and maytansine
from the Drug Synthesis Branch, Division of Cancer Treatment, Na
tional Cancer Institute, and PT119 was a gift from Dr. J. G. Bekesi,
Mt. Sinai Hospital, New York, NY. All agents were administered by
i.p. injection (0.1 ml/10 g body weight).
RESULTS
Selection of VCR Resistance in HxRh12 Xenografts. The
responses for groups of HxRhl2 tumors following a single
administration of VCR are presented in Fig. 1. For each curve,
the mean volume relative to that volume at the time of treatment
is shown for groups of 12 to 14 tumors. At the highest dose
level (3 mg/kg) tumors rarely regrew during the observation
period (>10 weeks). At 1.5 mg/kg VCR, tumors regressed
completely and subsequently regrew. The tumor demonstrating
the poorest response was transplanted into recipient mice and
the process repeated. Data in Fig. 1 demonstrate also the doseresponse relationship for HxRhl2. Thus, relatively low levels
of resistance would be reflected in a considerable difference in
tumor response (c.f., 3 mg/kg and 0.75 mg/kg). Growth curves
for groups of HxRh 12/VCR-3 tumors (after 6 exposures to 3
mg/kg) are presented in Fig. 2A. Although there is still some
response, tumors are approximately 4-fold resistant to VCR
(i.e., the response is equivalent to that obtained in the parent
line following 0.75 mg/kg VCR). This level of resistance has
been maintained in the absence of selection pressure for over 2
yr (data not shown). Further, this tumor response does not
column (Collaborative Research). The eluent was collected, heated at
65*, and again applied to the column an additional 2 times. After
washing with loading buffer (a20 column volumes) polyadenylatecontaining RNA was eluted in 3 ml elution buffer (10 mM Tris-HCl,
pH 7.5, 1 mM EDTA, 0.05% SDS). Aliquots of mRNA were precipi
tated, washed twice with 70% ethanol, and stored under ethanol at
—70"C.RNA was quantitated by determining the absorbance at X260/
280.
Hybridization Studies. pMDR-1, containing a genomic segment of
the mdr\ gene which encodes P-glycoprotein (20, 21), was obtained
from Dr. I. B. Roninson, Genetics Institute, Chicago, IL. The insert
was cleaved from this plasmiti using Sad and BanMl restriction
enzymes, and the digested fragments were separated on a low melting
point agarose gel. The MDR-1 fragment was extracted, precipitated
using conventional methods, and denatured by boiling. The resulting
single stranded DNA was radiolabeled using ''I'd AIT (New England
Nuclear) by the primer extension procedure of Feinberg and Vogelstein
(22). The probe was purified by Sephadex G-50 chromatography.
Plasmid pAl, containing a cDNA insert complementary to /8-actin (23),
was obtained from Dr. D. W. Cleveland, Johns Hopkins University,
Baltimore, MD. This was radiolabeled by nick translation (24). mRNA
was bound to nitrocellulose filters using standard techniques. Filters
6289
0.1
Weeks
Fig. 1. Responses of HxRh 12 xenografts to administration of single dose of
VCR •,control; O, 0.375; A, 0.75; A, 1.5; D, 3 mg/kg. Each curve represents the
mean relative tumor volume for 12 to 14 tumors ±SE. Relative volume = (VJ
Va) where VK is tumor volume on Day x and V, is tumor volume at time of
treatment (f ).
VINCRISTINE-L-PAM
IO
CROSS-RESISTANCE
IOr
O.I
Weeks
Fig. 2. A, response of HxRhl2/VCR-3 xenografts to vincristine. Mice bearing
HxRhl2/VCR-3 xenografts (selected at 3 mg/kg for 6 passages) were treated
with VCR I.S mg/kg (A); 3 mg/kg C .). or saline (•).Each curve represents the
responses of 12 or 14 tumors (mean ±SE), subsequent to a single administration
of drug. B, responses to L-PAM of HxRhl2 and HxRhl2/VCR-3 xenografts
growing in opposite flanks of recipient hosts. Mice received s.c. grafts of HxRhl2
(left flank) and HxRhl2/VCR-3
(rightflank). Mice received a single administra
tion (f ) of L-PAM (7 mg/kg) or vehicle when tumors reached 1 cm diameter.
Each curve represents the responses of 7 tumors (mean ±SE). HxRhl2: •.
control; •,L-PAM. HxRhl2/VCR-3: D, control; O, L-PAM.
Weeks
Fig. 3. Growth and responses of HxRhl2 and HxRhl2/VCR-3 tumors after
treatment with ifosfamide. HxRhl2 tumors: ».control; O, 300; A, 450 mg/kg
ifosfamide. HxRhl2/VCR-3: •.control; : I. 450 mg/kg ifosfamide. Each curve
represents the responses of 14 tumors (mean ±SE).
appear to be a consequence of drug-induced host toxicity, as
pair feeding experiments did not demonstrate any inhibition of
tumor growth.
11\Klil 2 xenografts have been shown to be very sensitive to
L-PAM (14). It was of interest therefore to compare the efficacy
of L-PAM against the parent tumor and HxRhl2/VCR-3
se
lected for resistance to VCR. Results of a representative exper
iment are shown in Fig. 2li in which HxRhl2 tumors were
implanted into the left flank and HxRhl2/VCR-3 into the right
flank of the same mice. Clearly, in such double-flank experi
ments, the parental line was considerably more sensitive to I
PAM than was HxRhl2/VCR-3. Similar results were obtained
in experiments in which mice were transplanted with only
parental or resistant tumor (data not shown). To determine
whether cross-resistance to L-PAM was a phenomenon general
to other bifunctional alkylating agents or more specific for I
PAM, ifosfamide was examined in both tumor models (Fig. 3).
Data shown depict mean responses for groups of HxRhl2
tumors treated at 450 and 300 mg/kg or HxRhl2/VCR-3
¡NVIVO
treated with 450 mg/kg ifosfamide. In the VCR-resistant tumor
this dose of ifosfamide inhibited growth by 39 days, whereas
growth inhibition for HxRhl2 tumors at 300 mg/kg tumors
was 47 days. However, with respect to the growth inhibition in
terms of the growth rate of controls (HxRhl2 doubled in 7.5
days compared to 5.5 days for HxRhl2/VCR-3),
treatment
resulted in inhibition of 7.1 and 6.3 volume doublings for VCRresistant and parent, respectively. Thus, for ifosfamide, there
appeared to be only slight cross-resistance (<1.5-fold) in
HxRhl2/VCR-3 tumors.
The responses of HxRh28 and HxRh28/L-PAM
tumors
(second exposure to 13 mg/kg) to L-PAM and VCR are shown
in Fig. 4A. In HxRh28 tumors L-PAM (13 mg/kg) caused
complete regression, with no subsequent regrowths. In contrast,
in HxRh28/L-PAM the same treatment resulted in a growth
delay of only 12 days. A similar result was obtained in "doubleflank" experiments (data not shown). This represents a 2- to 3fold increase in resistance (14). Also presented is response of
HxRh28 tumors in mice receiving a single administration of
VCR (3 mg/kg). At this dose approximately half of the tumors
regrew subsequent to complete regression. In contrast HxRh28/
L-PAM tumors are quite unresponsive to treatment with VCR
(Fig. 4B). At 1.5 and 3 mg/kg there was no significant growth
inhibition. Further studies indicated some cross-resistance to
cyclophosphamide,
ifosfamide, and the L-PAM analogue
PT119 in HxRh28/L-PAM (Fig. 5).
Cross Resistance to Natural Products. Acquired resistance to
VCR has been associated with cross-resistance to other natural
products in cells maintained in vitro. Such studies are difficult
to perform /// vivo, as agents such as colchicine, maytansine,
and actinomycin D may demonstrate only marginal therapeutic
efficacy. For HxRh28 doxorubicin demonstrates only slight
activity at maximum tolerated dose levels in the mouse, whereas
the other agents do not inhibit tumor growth in either I IxRh 12
or HxRh28 xenografts.
Biochemical Studies. Accumulation and retention of |<V-'H|VCR in HxRhl2 and HxRhl2/VCR-3
and [Q-3H]VLB in
HxRhl2 tumors are presented in Fig. 6. Acquired resistance to
VCR in HxRhl2/VCR-3 is associated with decreased retention,
such that by 72 h concentrations of VCR and VLB (in the
resistant and parent tumor, respectively) are similar. Analysis
by high-performance liquid chromatography and by mass specIOr
10r
0.1
Weeks
Fig. 4. A. Growth and response of HxRh28 and HxRh28/L-PAM-13 xeno
grafts. HxRh28: A, control; A, L-PAM 13 mg/kg; »,control; O, VCR 3 mg/kg.
HxRh28/L-PAM-13: •control; D, L-PAM 13 mg/kg. Each curve represents
the responses of 12 to 14 tumors (mean ±SE). B, response of HxRh28/L-PAM13 to vincristine. Mice bearing bilateral HxRh28/L-PAM-13 tumors received a
single administration (ÃŽ)
of VCR at •.1.5, D, 3 mg/kg, or O, saline. Each curve
represents the growth of 14 tumors [mean, SE (<10%) omitted for clarity].
6290
VINCRISTINE-L-PAM
CROSS-RESISTANCE
IN VIVO
selected for resistance in vivo. Cross-resistance to L-PAM has
been associated with the M DR phenotype in CHO cells selected
for high levels of resistance to colchicine (10). However, in
independently derived lines of P388 (P388/ADR; P388/VCR)
which show a MDR phenotype, cross-resistance to L-PAM was
not found (12, 13). Doxorubicin, an agent associated with the
MDR phenotype, may give rise to cross-resistance to L-PAM
and other alkylating agents which are not associated with the
MDR phenotype (8). Further, resistance was modulated by
decreasing reduced glutathione levels using buthionine sulfoximine. The relationship between the MDR phenotype and LPAM cross-resistance is thus tenuous, particularly where VCR
is the primary selecting agent. Of note are the data of Schabel
et al. (13) in which P388/L-PAM was significantly crossresistant to VCR but P388/VCR was not resistant to L-PAM.
These workers presented other examples of "unilateral crossWeeks
resistance." Our data suggest that, under the in vivo conditions
Fig. 5. Growth and response of A, HxRh28, and B, HxRh28/L-PAM xenografts to a single administration (at 10% lethal dose levels) <>!'•.
450 mg/kg
used, selection with either agent led to cross-resistance to the
ifosfamide; A, 150 mg/kg cyclophosphamide; T, 12.5 mg/kg PTI19; •,control.
other agent. However, to determine whether this is unilateral
Each curve represents the mean ±SE for 12 to 14 tumors.
or bilateral must await selection of HxRhl2/L-PAM
and
HxRh28/VCR-3 tumor lines in vivo. These experiments are
ongoing. Resistance to VCR in HxRhl2 tumors was significant
after the third exposure to VCR (1.5 mg/kg) and full resistance
was achieved by the second exposure to 3 mg/kg. Comparison
of responses of HxRhl2 and its VCR-resistant subline (Figs. 1
and 2) indicate that HxRhl2/VCR-3
is approximately 4-fold
resistant to VCR. Clearly, HxRhl2/VCR-3
is cross-resistant
to L-PAM, although probably only 3-fold. However, because
of the dose-response relationships for L-PAM and VCR under
these conditions, small changes in tumor sensitivity (i.e., 1- to
4-fold) have significant impact upon degree of overall responses.
We were interested to determine whether the VCR-resistant
subline of HxRhl2 was cross-resistant to other alkylating
agents and whether L-PAM resistance in HxRh28 was more
general for this class of agent. Cyclophosphamide and its isomer
ifosfamide were examined, as they are of interest for treatment
72
146
16 24
of childhood rhabdomyosarcoma and are not transported into
Hours
cells via amino acid transport systems. Consequently, crossFig. 6. Accumulation and retention of [G-'H] Vinca alkaloids in HxRhl2 and
HxRhl 2/VCR-3 xenografts. Tumor-bearing mice received a single administration
resistance between L-PAM and these agents is unlikely to be a
of VCR or VLB at 3 mg/kg. HxRhl2: •VCR; A, VLB. HxRhl2/VCR-3: •,
consequence
of altered transport. PTI 19 was chosen as this
VCR. Each point represents a mean ±SD for 6 to 12 individual tumors.
tripeptide analogue enters cells by both the L-system and ASCsystem for amino acid transport (27), hence it is similar to Ltrometry indicates that there is no difference in metabolism of
PAM. Of interest is that a slight cross-resistance of ifosfamide
VCR between the parent and resistant lines (data not shown).
was
found in HxRhl2/VCR-3.
HxRh28/L-PAM,
a subline
Expression of mdr\. Overexpression of mdr\, a gene that
encodes P-glycoprotein associated with M DR phenotype, has selected for resistance to L-PAM, was ~3- to 4-fold more
resistant to both L-PAM and PTI 19, cross-resistant to ifosfam
been reported to precede amplification (25). To examine
whether expression of mdrl was increased in VCR- and L- ide and cyclophosphamide (1.5- to 2-fold), and completely
cross-resistant to VCR.
PAM-resistant lines, mRNA was examined by dot blot analysis
Preliminary data indicate that in HxRhl2/VCR-3,
VCR is
(Fig. 7). Both parental lines have detectable levels of mdr\
mRNA, but this is not increased in tumors selected for resist
retained to a lesser extent and drug is eliminated in a manner
ance to VCR or L-PAM. To ensure similar loading of mRNA
similar to that of VLB in the parent line (16). Thus, the tv, for
onto the filter, blots were dehybridized and rehybridized to retention in HxRhl2 and HxRhl2/VCR-3 was 723 and 34 h,
pAl, complementary to /3-actin. Results indicate similar levels
respectively, and 48 h for VLB in HxRhl2 tumors. This de
of /3-actin mRNA in each lane. Similar results have been ob
creased retention is similar to that in HxRhl8/VCR-3 reported
tained using pHDRlO, a cDNA complementary to the human
mdrl gene (26)." Thus, in HxRhl2 and HxRh28, expression of previously (28) and is not associated with altered metabolism
of VCR.
mdrl is detectable but is not increased in tumors selected in
Recent studies have demonstrated that the MDR phenotype
situ for low-level resistance to VCR or L-PAM.
may be conferred by transfection of a single functional cDNA
(29). This gene (mdrl), which encodes P-glycoprotein (21), is
DISCUSSION
overexpressed in many cell lines and overexpression may pre
In this study we have identified cross-resistance between VCR
cede
amplification (25). Overexpression has also been identified
and L-PAM irrespective of the selecting agent in human tumors
in 1 of 4 clinical samples of relapsed RMS (30), and increased
4 I. Roninson, personal communication.
P-glycoprotein has been identified in some ovarian tumors (31)
6291
VINCRISTINE-L-PAM
CROSS-RESISTANCE
Rhl2- • •• •
RM2/VCR-3-
Rhl2-
• •• •
Rhl2/VCR-3-
YeasttRNARh28-
IN VIVO
•
•
Yeast tRNA• • •
Rh28-
RH28/L-PAM- •
1 • •
Yeast tRNA- •
i
•
RH28/L-PAMYeosttRNA-
Fig. 7. Doc blot analysis of mart inKNA in sensitive and resistant xenografts. niRNA was isolated and hybridized to probes as described in "Materials and
Methods." Left, serial 2-fold dilutions of mRNA from HxRhl2, HxRhl2/VCR-3, HxRh28, and HxRh28/L-PAM were probed with a fragment of pMDR-1. Right,
after dehybridization the filter was hybridized to "P-labeled pAl. Yeast tRNA was used as a negative control for hybridization.
and in «25% of adult sarcomas.5 In our RMS xenografts,
HxRhl2 and HxRh28, both sensitive to VCR, there are detect
able levels of mdr\ expression, which is of interest. Of note,
however, is that there was no apparent increase in expression
in either HxRhl2/VCR-3 or HxRh28/L-PAM (Fig. 7). Similar
results were obtained using pHDR-10, another probe for the
mdr\ gene.6 Whether this endogenous level of expression ac
counts for intrinsic resistance to DOX, CLC, or maytansine is
unknown. However, the difference in efficacy between VCR
and VLB in HxRhl2 appears to be due to the lower affinity of
binding for VLB in membrane-free extracts from tumor (32).
Several of these studies suggest that resistance to VCR in
HxRh28/L-PAM and HxRhl2/VCR-3 is not a consequence of
increased membrane efflux phenomena, (a) This mechanism
would not account for cross-resistance to cyclophosphamide,
ifosfamide, and PT119 in HxRh28/L-PAM tumors, (b) There
is no apparent increased expression of mdr\ in either resistant
line, (c) Verapamil, an agent reported to overcome VCR resist
ance (33) and to reverse the MDR phenotype in vitro (34) and
in vivo (35), does not alter the accumulation or retention of
VCR in HxRhl2/VCR-3, even when it is infused at dose levels
that exceed 10 ¿IM
for 4 days in tumor.7 Further study of the
mechanism of L-PAM selected resistance to VCR (particularly
with respect to a role for reduced glutathione) may provide
further insight into the determinants of cytotoxicity and thera
peutic selectivity for Vinca alkaloids.
The cross-resistance between agents with different chemical
structure, different cellular transport mechanisms, and appar
ently distinct biological targets (36-40) is intriguing. One pos
sibility is that by the nature of selection, from presumably
multiclonal tumors, we may have selected cells already resistant
to the other agent. This appears less likely, however, since
reciprocal cross-resistance was found for both independently
selected lines. Further, that this may be a clinically relevant
phenomenon is borne out by the clinical evaluation of L-PAM
in children with RMS.
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We wish to acknowledge the excellent technical assistance from
Ruby Cook-Tharpe and Pamela J. Lutz.
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