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Experimental Therapeutics, Preclinical Pharmacology |
Endocrine, Polypeptide and Cancer Institute, Veterans Affairs Medical Center [A. V. S., P. A.] and Section of Experimental Medicine, Department of Medicine, Tulane University School of Medicine, New Orleans, Louisiana 70112 [I. C., A. V. S., A. N., K. S., G. H.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Targeting of cytotoxic agents to cancer cells represents a modern approach to the treatment of human breast carcinomas and other cancers, which could increase inhibitory effects on tumors and reduce peripheral toxicity (4) . Targeted chemotherapy is based on the concept that by linking cytotoxic radicals to a carrier, which is able to recognize cancer cells, a selective accumulation of the chemotherapeutic agents can be achieved in the tumor while sparing healthy tissues from exposure (reviewed in Ref. 4 ).
The presence of LH-RH receptors on various human tumors, including breast, ovarian, endometrial, and prostate cancers, indicated that LH-RH analogues might be suitable carrier molecules for cytotoxic agents (3, 4, 5) . Receptors for LH-RH have been demonstrated in estrogen-independent MXT mouse mammary tumors and in several human breast cancer cell lines, including MCF-7, MX-1, and MDA-MB-231 (6, 7, 8, 9, 10, 11) . More importantly, receptor analyses of 500 primary human breast cancer specimens showed that 260 (52%) expressed binding sites for LH-RH (12) . During the past decade, several cytotoxic LH-RH analogues have been developed and tested on various tumors in our laboratory (4 , 7, 8, 9, 10) . Such hybrid molecules consisted of agonists or antagonists of LH-RH attached to various cytotoxic agents (4 , 13) . Recently, we synthesized AN-201,3 a highly active derivative of doxorubicin that is 500-1000 times more potent in vitro than the parent compound (14) . This powerful cytotoxic agent was linked covalently to agonist [D-Lys6]LH-RH to form a cytotoxic LH-RH analogue designated AN-207 (13) . We then demonstrated a high-binding affinity of AN-207 to human breast cancer specimens (8) . A high antitumor efficacy of AN-207 and a lower toxicity than its radical AN-201 was shown in studies on LH-RH receptor-positive MX-1 and MDA-MB-231 estrogen-independent human breast cancers (9 , 10) .
In the present investigation, we evaluated the effect of AN-207 on the growth and the metastatic potential of the orthotopically implanted MDA-MB-435 human estrogen-independent breast carcinoma cell line, which expresses specific high-affinity receptors for LH-RH. The mechanism of action of AN-207 on MDA-MB-435 tumors was investigated by blocking LH-RH receptors with a high dose of agonist [D-Trp6]LH-RH. The effect of treatment on the receptors for LH-RH and the expression of their mRNA in MDA-MB-435 tumors were also studied.
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MATERIALS AND METHODS |
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Animals.
Female athymic nude mice (Ncr nu/nu), 5–6 weeks of age, were obtained
from the Frederick Cancer Research Facility of the National Cancer
Institute (Frederick, MD), housed in sterile cages under laminar
airflow hoods in a temperature-controlled room with a 12-h light and
12-h dark schedule, and fed autoclaved chow and water ad
libitum. All experiments were performed according to institutional
ethical guidelines on animal care.
Cell Line and Cytotoxicity in Vitro.
The MDA-MB-435 estrogen-independent cell line, isolated from the
pleural effusions of a patient with breast carcinoma, was obtained from
American Type Culture Collection (Manassas, VA). It was maintained in
MEM (Life Technologies, Inc., Grand Island, NY) supplemented with 5%
fetal bovine serum, 2 mM L-glutamine,
nonessential amino acids, 1:50 MEM vitamins, 1 mM pyruvate,
100 units/l penicillin sodium, 100 µg/ml streptomycin sulfate, and
0.25 µg/ml amphotericin B (Life Technologies, Inc.). Cells were
cultured in Costar T-75 flasks (Corning Incorporated Science Products
Division, Acton, MA) in a humidified atmosphere of 5% carbon dioxide
and 95% air at 37°C. The cells were passaged weekly and routinely
monitored for the presence of Mycoplasma using a test kit
(Boehringer Mannheim, Indianapolis, IN). The cytotoxic activity of the
analogues was determined in vitro using colorimetric assay
based on quantitation of biomass by staining cells with crystal violet
(16)
. IC50 values, defined as the
drug concentration that inhibited cell growth by 50% compared with
untreated control cultures, were calculated from two independent
experiments, consisting of seven replicate tests each, after exposure
of MDA-MB-435 cells to cytotoxic agents for 91 h.
Experimental Protocol.
Female athymic nude mice, 8–10 weeks of age, received injections
orthotopically into the m.f.p. with MDA-MB-435 human breast carcinoma
cells. Mice were anesthetized with metoxyflurane (Metofane;
Pittman-Moore, Mundelein, IL), and a 5-mm incision was made in the skin
over the lateral thorax as described previously (17)
. The
m.f.p. was exposed, and an inoculum of 2 x
106 cells/30 µl was implanted into the tissue
through a 27-gauge needle. The skin incisions were closed with wound
clips that were removed 1 week later.
In experiment 1, when tumors reached a volume of 
40–50
mm3
, nude mice were randomly divided into seven
experimental groups of seven to eight animals each and received the
following treatment as a single i.v. injection, all of the drugs being
injected at a dose of 250 nmol/kg: group 1, control, vehicle solution
(5% mannitol); group 2, cytotoxic radical AN-201; group 3, cytotoxic
analogue of LH-RH AN-207; group 4, unconjugated mixture of cytotoxic
radical AN-201 and carrier analogue
[D-Lys6]LH-RH; group 5, carrier,
[D- Lys6]LH-RH; group 6,
pretreatment with 200 µg agonist
[D-Trp6]LH-RH 1 h before the
injection of AN-207; and group 7, AN-207, the same as in group 3. The
animals in groups 1–6 were sacrificed on day 33. In group 7, four
animals were killed on day 48 and three mice on day 55, to study LH-RH
receptor expression at different time points after the treatment with
AN-207.
In experiment 2, when tumors measured 60–65 mm3 , mice were divided into three groups of five animals each and were given i.v. injections on days 1 and 28: group 1, control, vehicle solution (5% mannitol); group 2, cytotoxic radical AN-201 at a dose of 150 nmol/kg; and group 3, cytotoxic analogue of LH-RH AN-207 at a dose of 150 nmol/kg. The experiment was terminated 50 days after the initiation of therapy.
All injections were administered through the tail vein under light methoxyflurane anesthesia. The tumors were measured once or twice a week with microcalipers, and tumor volume was calculated as length x width x height x 0.5236. Tumor growth delay was defined and calculated as T-C, where T and C are the median times (in days) of the first tumor volume doubling from the beginning of therapy in the treated and the control groups, respectively. Body weights of the animals were measured on the days of the injections and weekly thereafter. Fifteen and 22 days after treatment in experiment 1, blood samples were collected from the tail vein of the control animals and those that received cytotoxic compounds using Unopette microcollection kit (Becton Dickinson, Franklin Lakes, NJ). Total leukocyte and platelet counts were determined manually using a hemacytometer. At the end of the experiments, an autopsy was performed and the tumor weights were measured. Samples of each tumor were either fixed in formalin for histological examination or snap-frozen at -70°C for preparation of membranes for receptor assay and for extraction of RNA for RT-PCR.
Histological Procedure.
Tumor samples were processed as described previously (7)
.
Sections (6 µm thick) were cut and stained with H&E. Mitotic and
apoptotic cells were counted in 10 standard high-power microscopic
fields, and their number per 1000 cells was accepted as the mitotic and
apoptotic indices, respectively. For the demonstration of the NOR in
tumor cell nuclei, the AgNOR method was used (7)
. The
silver-stained black dots in 50 cells of each tumor were counted, and
the AgNOR number per cell was calculated (7)
. The
percentage area of necrosis was estimated in histological slides made
at the largest cross-section of the tumors by the point-counting
method.
RNA Extraction and RT-PCR.
Total RNA was extracted from frozen tissue samples by using
Rnazol B (TEL-TEST Inc., Friendswood, TX) according to the
manufacturer’s instructions. The quantity and the quality of the RNA
were assessed by spectrophotometry at 260 nm and 280 nm. One microgram
of total RNA was reverse transcribed using Gene Amp RNA PCR Kit
(Perkin-Elmer Corp., Norwalk, CT). After reverse transcription, the
cDNA was amplified using 2.5 units of Ampli-Taq DNA Polymerase
(Perkin-Elmer Corp.) and 0.4 µM of specific
oligonucleotide primers in a final reaction volume of 100 µl
containing 10 mM Tris-HCI (pH 8.3), 50 mM KCI,
2 mM MgCI2, and 0.2 mM
each of dATP, dTTP, dCTP, and dGTP (Perkin-Elmer Corp.). The primers
used for GAPDH were: sense, 5'-TCCTCTGACTTCAACAGCGACACC-3' and
antisense 5'-TCTCTCTTCCTCTTGTGCTCTTGG-3' (11)
.
For LH-RH receptors the primers were: sense,
5'-GACCTTGTCTGGAAAGATCC-3'and antisense, 5'-CAGGCTGATCACCACCATCA-3'
(11)
. Twenty-five cycles of PCR for GAPDH and 30 cycles
for LH-RH receptors were carried out using a thermal cycler
(Stratagene, La Jolla, CA) according to the step program of 94°C for
1 min (denaturation), 60°C for 1 min (annealing), and 72°C for 1
min (extension), followed by a 10-min final extension at 72°C.
Negative controls were run in parallel to confirm that the samples were
not contaminated with genomic DNA. The number of cycles was determined
in preliminary experiments to be within the exponential range of PCR
amplification. Seven microliters of the PCR products were
electrophoresed on a 1.8% agarose gel. Bands were visualized by
ethidium bromide staining and quantified with an imaging densitometer
(model GS-700; Bio-Rad Laboratories, Hercules, CA).
LH-RH Receptor Binding Studies.
Receptors for LH-RH on MDA-MB-435 tumors from all experimental
groups were characterized by the ligand competition assay. Preparation
of tumor membrane fractions and receptor binding studies of LH-RH were
performed as described previously (8)
.
[D-Trp6]LH-RH was radioiodinated by
the chloramine-T method (8)
. The LIGAND-PC computerized
curve-fitting program of Munson and Rodbard (18)
was used to determine the type of receptor binding, dissociation
constant (Kd), and maximal binding capacity of
receptors (Bmax).
Statistical Analysis.
The SigmaStat and SigmaPlot software were used for the statistical
evaluation of data and the preparation of figures. Multiple groups were
analyzed by one-way ANOVA, tumor volume changes were surveyed by
two-way repeated measures ANOVA, and the groups were examined by
Tukey’s multiple comparison test. Histological data were evaluated by
Duncan’s multiple range method, and the analysis of mRNA for LH-RH
receptors was performed by Student’s t test. Differences
were considered statistically significant when P <
0.05.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Administration of AN-207 produced a
significant growth inhibition of the MDA-MB-435 tumors. Thirty-three
days after the injection with AN-207, tumor volume was significantly
reduced (P < 0.001) to 142.4 ± 26.1
mm3
compared with controls, which measured
418.6 ± 82.8 mm3
, corresponding to a growth
inhibition of 66% (Fig. 1
a).
In contrast, in groups that received injections of equimolar doses of
AN-201, the unconjugated mixture of AN-201 and carrier
[D-Lys6]LH-RH, or the
carrier peptide alone, all tumors grew steadily (Fig. 1
a).
The growth of cancers treated with AN-207 was significantly delayed
compared with all other groups (P < 0.01–0.012; Table 1
). The weight of AN-207-treated tumors (200.0 ± 40.0 mg) was
also reduced (P = 0.051) compared with controls
(490.0 ± 90.0 mg). Tumor burden in this group was significantly
(P < 0.05) decreased as compared with controls (Table 1)
. After treatment with AN-207 only one of eight mice (12.5%) died
due to toxicity, whereas AN-201 alone or together with
[D-Lys6]LH-RH resulted in
the deaths of two of eight animals (25%; Table 1
). The carrier peptide
[D-Lys6]LH-RH was not
toxic but had no significant tumor inhibitory effects. On day 4, the
average WBC counts in the AN-207-treated group and the AN-201-treated
group were reduced markedly by 72.7% and 78%, respectively. In both
groups the leukocyte count returned to normal values within 2 weeks
(data not shown). Significant loss of body weight occurred in all
groups treated with cytotoxic agents, but body weights of the surviving
animals returned to normal by the end of the experiment (Fig. 2)
.
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and Fig. 1
b). Tumor weight and tumor burden were also reduced by
46.5% and 48%, respectively. The delay in tumor growth produced by
AN-207 was not significant in this experiment. The MDA-MB-435 tumors in
animals treated with cytotoxic radical AN-201 grew steadily even after
the administration of the second injection. No toxicity-related deaths
occurred during the experiment in any of the groups.
Effect of Pretreatment with [D-Trp6]LH-RH
on the Antitumor Activity of AN-207.
As part of experiment 1, mice bearing orthotopic MDA-MB-435 tumors were
pretreated s.c. with the agonist
[D-Trp6]LH-RH at a dose
of 200 µg per animal 1 h before a single i.v. injection of
AN-207 at 250 nmol/kg. The administration of
[D-Trp6]LH-RH at a high dose was
intended to block the LH-RH receptors on the tumor cells. As shown in
Table 1
and Fig. 1
c, pretreatment with
[D-Trp6]LH-RH attenuated
the suppressive effects of the cytotoxic agent AN-207 on the growth of
MDA-MB-435 tumors. The inhibition of tumor growth produced by AN-207
alone was 66.0%, whereas the administration of
[D-Trp6]LH-RH before
AN-207 decreased the tumor inhibition to 51.0% and increased
toxicity-related deaths to 25% (two of eight mice), as compared with
12.5% (one of eight mice) with AN-207 alone.
Effect of AN-201 and AN-207 on the Metastatic Potential of
Orthotopically Implanted MDA-MB-435 Tumors in Nude Mice.
In the first experiment, six of eight mice (75%) in the control group
and three of eight mice (37.5%) in the group given AN-201 developed
metastases in the lymph nodes (Table 2)
.
No metastatic lesions could be found in the animals treated with
AN-207. Pretreatment of nude mice with
[D-Trp6]LH-RH 1 h before
injection of AN-207 partially blocked the effect of AN-207 on the
metastases of the MDA-MB-435 tumors, as shown by the development of
lymphatic spread in two of eight mice (25%; Table 2
). Distant
hematogenous metastases were found in one of eight control animals (in
the lungs), but not in the AN-201 or AN-207-treated groups.
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. Four of five mice in the
control group (80%) and three of five mice treated with AN-201 (60%)
developed metastases in the lymph nodes. In contrast, no metastases
were found in any of the five mice that received injections of AN-207.
Lung metastasis was found in one of the control animals, but not in the
AN-201- or AN-207-treated mice.
Cytotoxicity in Vitro.
Antiproliferative activities of AN-207 and AN-201 were also evaluated
in vitro. The concentration of AN-207 that inhibited growth
of MDA-MB-435 cells by 50% as compared with control
(IC50) was 1.9 x
10-10 M, whereas the
IC50 value of the unconjugated cytotoxic radical
AN-201 was 1.7 x 10-10
M.
Histological Findings.
MDA-MB-435 mammary cancers were undifferentiated tumors consisting of
polygonal epithelial cells without any special arrangement. The nuclei
of cells were round or oval, relatively pale, and contained prominent
nucleoli. The amount of stroma was minimal. The group treated with
AN-207 had a more extensive area of necrosis than the controls
(P < 0.01; Table 3
).
Large necrosis was also found in tumors from mice given AN-207 after
pretreatment with
[D-Trp6]LH-RH
(P < 0.05). The area of necrosis in tumors from mice
treated with AN-201 was similar to that of the controls. Therapy with
AN-207 alone or after
[D-Trp6]LH-RH
pretreatment significantly increased the ratio of apoptotic to mitotic
indices to 1.55 ± 0.22 (P < 0.01) and 1.22 ± 0.19 (P < 0.01) respectively, compared with the
control group, in which the ratio was 0.64 ± 0.16. A marked
reduction of the mitotic cells (P < 0.01) was seen in
all groups treated with cytotoxic agents, whereas the number of
AgNORs/cell was significantly (P < 0.01) decreased
only in the group receiving AN-207 compared with controls (Table 3)
.
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.
Amplification with primers specific for human GAPDH produced a single
PCR product of 207 bp (Fig. 3)
. Densitometric analysis of the RT-PCR
products, after normalization of the expression of LH-RH receptor mRNA
versus the corresponding levels of mRNA for human GAPDH,
showed a 26% (P < 0.05) decrease in the expression of
mRNA for LH-RH receptor in tumors 33 days after injection of AN-207 as
compared with the controls. However, 48 days after treatment mRNA
expression returned to the level of controls.
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.
Receptors for LH-RH were also detected in tumors from animals treated
with AN-201 (Table 4)
. High-affinity receptors for LH-RH with a
Kd of 6.85 nM and a
Bmax of 273 fmol/mg were detected only in one of
three tumors from the AN-207-treated group, 33 days after the
administration of AN-207 at 250 nmol/kg. Forty-eight days after the
injection of AN-207 no specific binding for LH-RH was detected in any
of the three tumors tested, whereas after 55 days only one of two
tumors investigated showed high-affinity receptors for LH-RH with a
Kd of 8.16 nM and a
Bmax of 484.0 fmol/mg membrane protein. In the
group of animals pretreated with
[D-Trp6]LH-RH 1 h before to
the injection with AN-207, receptors for LH-RH at a concentration of
589.0 fmol/kg membrane protein (Table 4)
were detected only in one of
the three tumors tested.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In this study, we tested the effects of AN-207 on the growth of metastatic MDA-MB-435 estrogen-independent breast cancers implanted in the m.f.p. of female athymic nude mice (17) . Our results demonstrate that AN-207 inhibits significantly the growth of MDA-MB-435 tumors and totally suppresses their metastatic potential, whereas cytotoxic radical AN-201 given alone or as a mixture with the carrier peptide is more toxic and does not produce any significant tumor inhibition.
The antitumor efficacy of AN-207 was tested in two experiments
differing in the dosage regimens used: in the first experiment, AN-207
was administered as a single i.v. injection at 250 nmol/kg; and in the
second experiment, AN-207 was given twice at 150 nmol/kg. In both
experiments, AN-207 produced a similar (65%) inhibition of tumor
growth as compared with controls. However, a better tolerance to a
lower dose of AN-207 is shown by an absence of toxicity-related deaths
in mice treated twice with AN-207 at 150 nmol/kg, whereas one of eight
mice died in the group that received one injection of AN-207 at 250
nmol/kg. Thus, repeated administration of low doses of AN-207 may
represent a more efficient therapeutic option. In addition, while the
administration of a single dose of AN-207 at 250 nmol/kg resulted in
continuous, although suppressed, tumor growth, in contrast, when AN-207
was given twice at 150 nmol/kg, the second injection of AN-207 not only
arrested the tumor growth for about 12 days but also produced a
temporary tumor regression. The delay in tumor growth caused by AN-207
in the second experiment was not significantly different from controls
because the calculation was based on the first doubling time. Growth
inhibition by AN-207 in this experiment became significant only after
the second injection (Fig. 1
b).
Elimination of the lymphatic and hematogenous spread of MDA-MB-435 cells after treatment with the cytotoxic LH-RH analogue AN-207 was observed in both experimental protocols tested in this study. The most probable explanation for this finding is that the treatment with AN-207 prevented the dissemination of cancer by arresting the growth of primary tumors. It is possible, however, that AN-207 is also targeted directly to LH-RH receptor-positive metastatic lesions.
The receptor-mediated mechanism of action of AN-207 was
investigated by the administration of LH-RH agonist
[D-Trp6]LH-RH at a high dose,
before the injection of AN-207. This was expected to attenuate the
antitumor activity of AN-207. Under these conditions, AN-207 had a
weaker effect on the primary tumor and the capacity to suppress the
metastatic potential of MDA-MB-435 cells was also reduced. It is likely
that the excess of [D-Trp6]LH-RH
occupied most LH-RH receptors on the tumors, whereas the unbound
cytotoxic LH-RH analogue AN-207 was hydrolyzed by carboxylesterase
enzymes (EC.3.1.1.1) in blood, releasing unconjugated AN-201
(21)
. This hypothesis is also in accordance with the
finding that the mortality in this group was the same (25%) as in the
AN-201-treated group. A very high esterase activity in mice, which is
about 10 times higher than in humans (21)
, may be
responsible for the similar mortality and losses in body weights in all
groups treated with cytotoxic compounds (Fig. 2)
. The half-life of
AN-207 in the serum of female nude mice incubated in vitro
at 37°C in a humidified atmosphere of 95% air and 5%
CO2 is 20 min, whereas the deconjugation of
50% of cytotoxic radical AN-201 takes 120 min in human serum under
the same conditions (data not shown). Thus, in human beings a much
lower toxicity of AN-207 can be expected, as it was shown recently in
nude mice with suppressed serum esterase activity (21)
.
Some toxicity to normal tissues expressing LH-RH receptors can be
expected. However, in a recent study we found a full recovery of
pituitary function in rats within 2 weeks after treatment with the
maximum tolerated dose of AN-207 (22)
.
Histological analysis showed that treatment with AN-207 significantly increased the area of necrosis in MDA-MB-435 tumors and raised the ratio of apoptotic to mitotic indices. This suggests that tumor growth inhibition is not only caused by a decrease in the rate of cell proliferation, as indicated by the reduction in the AgNOR numbers and mitotic indices, but is also the result of cell death due to necrosis, which could be even more important (23) .
The LH-RH receptor status of MDA-MB-435 tumors was studied by RT-PCR analysis and radioreceptor assay. Using RT-PCR, we detected mRNA for LH-RH receptor in all samples tested. The levels of mRNA for LH-RH receptor decreased significantly 33 days after the injection with AN-207, but after 48 days the levels returned to controls values. Considering that the treatment consisted of a single i.v. injection of the cytotoxic analogue 33 days before mRNA analysis, the decrease in the levels of mRNA for LH-RH receptor could be a consequence of inhibition of gene transcription due to tumor suppression rather than a direct result of the interaction between the cytotoxic LH-RH analogue and the LH-RH receptor. A down-regulation of LH-RH receptor due to endocrine effects is extremely unlikely because only a single low dose of about 5 nM of the cytotoxic LH-RH analogue AN-207 was used.
Radioligand binding assays showed that treatment with AN-207 also affected the binding of LH-RH analogue to the membranes of MDA-MB-435 tumors. Although no significant difference was observed in the binding characteristics (Kd and Bmax) of MDA-MB-435 xenografts after the administration of AN-207, the number of tumors in which specific binding for LH-RH could be detected was decreased after the treatment. In contrast, RT-PCR analysis revealed the expression of mRNA for LH-RH receptor in all samples tested. A possible explanation for the results of radioligand binding studies as compared with RT-PCR is that mRNA for LH-RH receptors was not translated into functional receptor protein in some samples due to unknown mechanisms. However, a strong effect of a second injection of AN-207 on tumor growth in experiment 2 indicates that the receptors for LH-RH may still be available for targeting of AN-207 on previously treated tumors. In fact, in a recent study on the effects of AN-207 on MDA-MB-231 tumors, we demonstrated that although no receptors for LH-RH were detectable 21 days after AN-207 treatment, 39 days later high-affinity binding sites for LH-RH could be shown again on AN-207-treated tumors (10) .
In conclusion, we showed that cytotoxic analogue AN-207 can be targeted to human estrogen-independent breast cancers that express receptors for LH-RH, producing a significant tumor growth inhibition and a suppression of the metastatic potential, whereas 2-pyrrolino-DOX itself is ineffective and toxic. Our results suggest that targeted chemotherapy with cytotoxic LH-RH analogues such as AN-207 could be a promising new modality for the management of estrogen-independent breast cancer.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by the Medical Research Service
of the Veterans Affairs department (to A. V. S.) and by a grant from
ASTA Medica (Frankfurt am Main, Germany) to Tulane University School of
Medicine (to A. V. S.). 
2 To whom requests for reprints should be
addressed, at the Endocrine, Polypeptide and Cancer Institute, Veterans
Affairs Medical Center, 1601 Perdido Street, New Orleans, LA
70112-1262. Phone: (504) 589-5230; Fax: (504) 566-1625.
3 The abbreviations used are: AN-201,
2-pyrrolinodoxorubicin; LH-RH, luteinizing hormone-releasing hormone;
RT-PCR, reverse transcription-PCR; GAPDH, glyceraldehyde-3-phosphate
dehydrogenase; m.f.p., mammary fat pad; NOR, nucleolar organizer
region.
Received 4/12/00; revised 7/21/00; accepted 7/24/00.
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REFERENCES |
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