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Cancer Patent Abstract
The expression and subcellular localization of peripheral-type benzodiazepine
receptors (PBR) is shown in this application to correlate with the
metastatic potential of cells, and increased cell proliferation.
Inhibition of PBR expression, function or stability results in a
decrease in cell proliferation. Compositions and methods for regulating
and/or monitoring PBR and its expression are useful for the detection,
diagnosis, prognosis and treatment of solid tumors, in particular,
breast cancer.
Cancer Patent Claims
What is claimed is:
1. An isolated nucleic acid consisting of SEQ ID NO:1, SEQ ID NO:2,
or the complete complement thereof.
2. An isolated nucleic acid that comprises a nucleotide sequence
that is the complete complement of SEQ ID NO:1; wherein said nucleic
acid, when introduced into a cell line that expresses a polynucleotide
comprising SEQ ID NO:1 or which encodes a peripheral-type benzodiazepine
receptor protein having a mutant threonine residue at position 147,
inhibits the expression of the polynucleotide.
3. An isolated nucleic acid consisting of SEQ ID NO:1 or the complete
compliment thereof.
4. An isolated nucleic acid comprising the complete complement
of SEQ ID NO:1.
5. A composition comprising the isolated nucleic acid of claim
1.
6. The composition of claim 5, wherein the nucleic acid is present
in a vector and is synthesized in a mammalian cell in vitro following
introduction of said vector into said cell.
7. The composition of claim 6, wherein the nucleic acid is synthesized
in a mammary gland cell in vitro following introduction of said
vector into said mammary gland cell.
8. The nucleic acid of claim 2, which is comprised in a proteoliposome
containing viral envelope receptor proteins.
9. The nucleic acid of claim 2, which is present in a vector.
10. The nucleic acid of claim 2, which is contained in a carrier.
11. The nucleic acid of claim 10 wherein said carrier is a protein
selected from the group consisting of a cytokine or polylysine-glycoprotein
carrier.
12. The nucleic acid of claim 2, which is comprised in a microbead.
13. The nucleic acid of claim 9, which is synthesized in a mammalian
cell in vitro following introduction of said vector into said cell.
Cancer Patent Description
INTRODUCTION
Tumor progression is a multi-step process in which normal cells
gradually acquire more malignant phenotypes, including the ability
to invade tissues and form metastases, the primary cause of mortality
in breast cancer. During this process, the "aberrant"
expression of a number of gene products may be the cause or the
result of tumorigenesis. Considering that the first step of tumor
progression is cell proliferation, it can be proposed that tumorigenesis
and malignancy are related to the proliferative potential of tumoral
cells.
Studies in a number of tumors such as rat brain containing glioma
tumors [Richfield, E. K. et al. (1988) Neurology 38:1255-1262],
colonic adenocarcinoma and ovarian carcinoma [Katz, Y. et al. (1988)
Eur. J. Pharmacol. 148: 483-484 and Katz, Y. et al. (1990) Clinical
Sci. 78:155-158] have shown an abundance of peripheral-type benzodiazepine
receptors (PBR) compared to normal tissue. All documents cited herein
infra and supra are hereby incorporated in their entirety by reference
thereto. Moreover, a 12-fold increase in PBR density relative to
normal parenchyma, was found in human brain glioma or astrocytoma
[Cornu, P. et al. (1992) Acta Neurochir. 119:146-152]. The authors
suggested that PBR densities may reflect the proliferative activity
of the receptor in these tissues. Recently, the involvement of PBR
in cell proliferation was further shown [Neary, J. T. et al. (1995)
Brain Research 675:27-30; Miettinen, H. et al. (1995) Cancer Research
55:2691-2695], and its expression in human astrocytic tumors was
found to be associated with tumor malignancey and proliferative
index [Miettinen, H. et al. supra; Alho, H. (1994) Cell Growth Different.
5:1005-1014].
PBR is an 18-kDa protein discovered as a class of binding sites
for benzodiazepines distinct from the GABA neurotransmitter receptor
(Papadopoulos, V. (1993) Endocr. Rev. 14:222-240]. PBR are extremely
abundant in steroidogenic cells and found primarily on outer mitochondrial
membranes [Anholt, R. et al. (1986) J. Biol. Chem. 261:576-583].
PBR is thought to be associated with a multimeric complex composed
of the 18-kDa isoquinoline-binding protein and the 34-kDa pore-forming
voltage-dependent anion channel protein, preferentially located
on the outer/inner mitochondrial membrane contact sites [McEnery,
M. W. et al. Proc. Natl. Acad. Sci. U.S.A. 89:3170-3174; Garnier,
M. et al. (1994) Mol. Pharmacol. 45:201-211; Papadopoulos, V. et
al. (1994) Mol. Cel. Endocr. 104:R5-R9]. Drug ligands of PBR, upon
binding to the receptor, simulate steroid synthesis in steroidogenic
cells in vitro [Papadopoulos, V. et al. (1990) J. Biol. Chem. 265:3772-3779;
Ritta, M. N. et al. (1989) Neuroendocrinology 49: 262-266; Barnea,
E. R. et al. (1989) Mol. Cell. Endocr. 64:155-159; Amsterdam, A.
and Suh, B. S. (1991) Endocrinology 128:503-510; Yanagibashi, K.
et al. (1989) J. Biochem. (Tokyo) 106: 1026-1029]. Likewise, in
vivo studies showed that high affinity PBR ligands increase steroid
plasma levels in hypophysectomized rats [Amri, H. et al. (1996)
Endocrinology 137:5707-5718]. Further in vitro studies on isolated
mitochondria provided evidence that PBR ligands, drug ligands, or
the endogenous PBR ligand, the polypeptide diazepam-binding inhibitor
(BDI) [Papadopoulos, V. et al. (1997) Steroids 62:21-28], stimulate
pregnenolone formation by increasing the rate of cholesterol transfer
from the outer to the inner mitochondrial membrane [Krueger, K.
E. and Papadopoulos, V. (1990) J. Biol. Chem. 265:15015-15022; Yanagibashi,
K. et al. (1988) Endocrinology 123: 2075-2082; Besman, M. J. et
al. (1989) Proc. Natl. Acad. Sci. U.S.A. 86: 4897-4901; Papadopoulos,
V. et al. (1991) Endocrinology 129: 1481-1488].
Based on the amino acid sequence of the 18-kDa PBR, a three dimensional
model was developed [Papadopoulos, V. (1996) In: The Leydig Cell.
Payne, A. H. et al. (eds) Cache River Press, IL, pp 596-628]. This
model was shown to accomodate a cholesterol molecule and function
as a channel, supporting the role of PBR in cholesterol transport.
Recently we demonstrated the role of PBR in steroidogenesis by generating
PBR negative cells by homologous recombination [Papadopoulos, V.
et al. (1997) J. Biol. Chem. 272:32129-32135] that failed to produce
steroids. However, addition of the hydrosoluble analogue of cholesterol,
22R-hydroxycholesterol, recovered steroid production by these cells,
indicating that the cholesterol transport mechanism was impaired.
Further cholesterol transport experiments in bacteria expressing
the 18-kDa PBR protein provided definitive evidence for a function
as a cholesterol channel/transporter [Papadopoulos, V. et al. (1997)
supra].
Diazepam has been shown to induce murine Friend erythroleukemia
cell differentiation and inhibit 3T3 cell proliferation. Moreover,
benzodiazepines (BZs) inhibited thymoma cell proliferation at micromolar
concentrations [Clarke, G. D. and Ryan, P. J. (1980) Nature 287:160-161;
Wang, J. K. T. et al. (1984) Proc. Natl. Acad. Sci. U.S.A. 81: 753-756].
Since the cells used do not express GABA receptor, these studies
supported an effect by BZs on cell proliferation acting through
a GABA receptor-independent mechanism. Then stimulation of glioma,
astrocytoma, and V79 Chinese Hamster lung cell proliferation was
shown to occur with treatment with nanomolar concentrations of PBR
ligands Ro5-4864 or PK11195, while micromolar amounts of these compounds
inhibited proliferation [Ikezaki, K. and Black K. L. (1990) Cancer
Letters 49:115-120; Bruce, J. H. et al. (1991) Brain Research 564:
167-170; Camins, A. et al. (1995) Eur. J. Pharm. 272:289-292]. The
use of PK11195 (an exclusive PBR ligand) provided unequivocal evidence
that the effects seen were mediated by PBR. In addition, micromolar
amount of PBR ligands were shown to inhibit growth factor-induced
cell proliferation in both astrocytes and lymphoma cells [Laird
II, H. E. et al. (1989) Eur. J. Pharm. 171:25-35; Neary, J. T. et
al. (1995) Brain Research 675:27-30].
We hypothesized that the peripheral-type benzodiazepine receptor
is part of the changes in cellular and molecular functions that
account for the increased aggressive behavior in cancer, and we
chose to examine this hypothesis in human breast cancer. Breast
cancer is the most common neoplasm and the leading cause of cancer-related
deaths for women in most developing countries [Lippman, M. E. (1993)
Science 259:631-632], affecting nearly 184,000 women, with over
46,000 deaths annually in the U.S. alone (American Cancer Society,
1996). Human breast cells are unlike brain and gonadal cells and
cannot produce steroids, but like many other cells in the body,
are able to metabolize steroids. Initial results indicated that
invasive and non-aggressive human breast cancer cell lines most
commonly used for modeling human breast cancer bound the PBR-specific
ligand to amounts similar to normal breast tissue. Only when aggressive
breast cancer cell lines were assayed was a dramatic increase in
PBR binding relative to invasive but non-aggressive cell lines evident.
Applicants believe that involvement of PBR in aggressive human breast
cancer was not previously discovered because these aggressive cell
lines are not the standard cell lines used for studying aberrant
behavior in human breast cancer.
In view of these initial results using aggressive human breast
cancer cell lines, further characterization of PBR in human breast
cancer biopsies, led to the discovery that the invasive and metastatic
ability of human breast tumor cells is proportional to the level
of PBR expressed, and correlates with the subcellular localization
of PBR in these cells in that PBR is found primarily in the nucleus
in aggressive tumor cells whereas PBR is found primarily in the
cytoplasm of invasive but non-aggressive cells. These changes in
PBR expression can be used as a tool for detection, diagnosis, prevention
and treatment in breast cancer patients, in particular, and in aggressive
solid tumors in general.
SUMMARY OF THE INVENTION
In this application is described a novel cellular and molecular
indicator for the detection, diagnosis, treatment and prognosis
of aggressive tumors, in particular, breast cancer.
We used a battery of breast cancer cell lines that differ in their
invasive and metastatic abilities in order to determine whether
PBR expression correlates with the metastatic potential of these
cells. In addition, we used biopsies from normal breast tissue and
metastatic breast tumors to study PBR expression. Our results demonstrate
that the expression of PBR correlates with the expression of breast
cancer cell aggressive phenotype. In addition, and in agreement
with the well documented function of PBR in steroid synthesizing
tissues, cholesterol transport into mitochondria, the function identified
in aggressive breast tumor cells is cholesterol uptake by the nucleus
which may lead to increased cell proliferation and metastasis. Moreover,
inhibition of the expression of the receptor in tumor cells, using
targeted disruption of the PBR gene, resulted in a decrease in cell
proliferation.
Therefore, it is a purpose of this invention to provide a method
for detecting the level of metastatic ability of cells by measuring
the level of peripheral benzodiazepine receptors (PBR) in tumor
cells and comparing it to the level of PBR in normal cells. This
method is applicable to any solid tumor cells, in particular, breast
cancer cells, cells from gonadal tumors, and cells from brain tumors.
It is a further object of the invention to provide a composition
effective for detecting peripheral-type benzodiazepine receptors
such as an anti-PBR antibody or a natural or synthetic ligand of
PBR including natural ligands, meaning ligands derived from a natural
source such as a plant extract or ligands naturally present in the
body or cell, or synthetic ligands such as chemically synthesized
ligands or synthesized derivatives of natural ligands of PBR for
prognosis of breast cancer, monitoring response to anticancer therapy,
and detecting recurrence of metastatic breast cancer.
It is another purpose of the present invention to provide a method
for determining the phenotype of a tumor by detecting the location
of PBR in cells whereby localization of PBR in the cytoplasm indicates
a non-aggressive phenotype and localization of PBR in the nucleus
indicates an aggressive phenotype.
It is a further object of the present invention to provide a diagnostic
kit comprising ligands or antibodies suitable for detecting PBR
and ancillary reagents required for such a detection.
It is yet another object of the present invention to provide a
method for detecting the level of PBR in tumor cells using the polymerase
chain reaction said method comprising:
(i) extracting RNA from a sample;
(ii) reverse transcribing said RNA into cDNA
(ii) contacting said cDNA with (a) at least four nucleotide triphosphates,
(b) a primer that hybridizes to PBR cDNA, and (c) an enzyme with
polynucleotide synthetic activity,
under conditions suitable for the hybridization and extension of
said first primer by said enzyme, whereby a first DNA product is
synthesized with said DNA as a template therefor, such that a duplex
molecule is formed;
(iii) denaturing said duplex to release said first DNA product
from said DNA;
(iv) contacting said first DNA product with a reaction mixture
comprising: (a) at least four nucleotide triphosphates, (b) a second
primer that hybridizes to said first DNA, and (c) an enzyme with
polynucleotide synthetic activity,
under conditions suitable for the hybridization and extension of
said second primer by said enzyme, whereby a second DNA product
is synthesized with said first DNA as a template therefor, such
that a duplex molecule is formed;
(v) denaturing said second DNA product from said first DNA product;
(vi) repeating steps iii-vi for a sufficient number of times to
achieve linear production of said first and second DNA products;
(vii) fractionating said first and second DNA products generated
from said PBR cDNA; and
(viii) comparing the level of PBR cDNA with the level of PBR cDNA
from a normal cell;
wherein, an increase in PBR level over normal cells indicates the
progression of the tumor cell to an aggressive phenotype.
It is yet another object of the present invention to provide a
composition suitable for detecting the level of PBR RNA in a cell,
such as oligonucleotide probes specific for PBR cDNA or RNA for
use in methods to detect PBR expression such as in situ hybridization
of tissue samples, or northern hybridization assays, or PCR assays.
It is a further object of the present invention to provide a diagnostic
kit comprising primers or oligonucleotides specific for PBR RNA
suitable for hybridization to PBR RNA and/or amplification of PBR
sequences and ancillary reagents suitable for use in detecting PBR
RNA in mammalian tissue.
It is another object of the invention to provide a composition
effective for inhibiting the binding of PBR ligands, for the purpose
of reducing the function of PBR in cells.
It is yet an object of the invention to provide a method for reducing
human breast cancer cell proliferation, the method comprising administering
to a cell a compound which reduces or inhibits PBR function or expression
such that cell proliferation is reduced.
It is yet another object of the invention to provide a composition
effective for reducing or inhibiting peripheral-type benzodiazepine
receptor expression or function in metastatic breast tumor cells
for use as a treatment for metastatic breast cancer.
It is further another object of the present invention to provide
a therapeutic method for the treatment or amelioration of symptoms
of metastatic breast cancer, said method comprising providing to
an individual in need of such treatment an effective amount of anti-PBR
composition in a pharmaceutically acceptable excipient such that
PBR expression or function is reduced in said breast cancer cells,
or entry of PBR into the nucleus of said breast cancer cells is
reduced.
It is yet a further object of the present invention to provide
a cDNA sequence encoding PBR found in invasive cells and vectors
incorporating all or a fragment of said sequence, and cells, prokaryotic
and eukaryotic, transformed or transfected with said vectors, for
use in screening agents and drugs which inhibit expression of PBR
in such cells.
It is another object of the present invention to provide cells,
such as R12, wherein the PBR gene has been interrupted for use in
screening agents and drugs which alter PBR expression.
BRIEF DESCRIPTION OF THE DRAWINGS
These and other features, aspects, and advantages of the present
invention will become better understood with reference to the following
description and appended claims, and accompanying drawings where:
FIG. 1 demonstrates specific PBR binding characteristics of various
human breast cancer cell lines. PK11195 specific binding was determined
using increasing concentrations of cellular protein (figure shows
specific PBR binding at 50 ug of protein) for each of the indicated
cell lines described in Table 1. ***, p<0.05; **, p<0.01;
NS, not significant.
FIG. 2 represents Scatchard plots and saturation isotherms for
MDA-231 and ADR human breast cancer cell lines. [.sup.3H]PK11195
binding studies were carried out for ADR (A), MDA-231 (B), and MCF-7
cell lines as described [Papadopoulos, V. et al. (1990) J. Biol.
Chem 265: 3772-3779]. Saturation isotherms and Scatchard plot analyses
for MDA-231 (B) and ADR (A) cells are shown. Although specific binding
could be detected in MCF-7 cells, an accurate Scatchard plot analysis
of the data generated could not be performed.
FIG. 3 shows PBR mRNA expression in MDA-231, ADR, and MCF-7 cell
lines. Total RNA was isolated from MDA-231, ADR, and MCF-7 cells
and loaded onto a 1% formaldehyde gel at a concentration of 10 ug/lane.
Northern blots were incubated with .sup.32P-labeled hPBR probe and
exposed to XOMAT Kodak film. Top, 28S and 18S rRNA visualized by
ethidium bromide staining. Middle, autoradiogram of the blot. PBR
migrates at 0.9 Kb. Bottom, relative intensity of the PBR mRNA/28S
ribosomal RNA.
FIG. 4 shows subcellular localization of PBR using the compound
4 PBR fluorescent probe. MA-10 (a), MCF-7(b), and MDA-231 (c,d)
cells were cultured on coverslips and incubated with compound 4
(1 uM) for 45 min at 37.degree. C. MDA-231 cells were incubated
with compound 4 (1 uM) for 45 min at 37.degree. C. in the presence
of 100 uM of FGIN-27 (e), the non-fluorescent PBR ligand used to
develop compound 4 [Kozikowski, A. P. et al. (1997) J. Med. Chem.
40: 2435-2439]. At the end of the incubation time, the cells were
washed, and PBR was localized by fluorescence microscopy. (f), phase-contrast
of the same image as shown in e.
FIG. 5 demonstrates the binding specificity of MDA-231 PBR. Specific
binding of [.sup.3H]PK11195 (2 nM) to MDA-231 cells was measured
in the presence of the indicated concentrations of each competing
ligand [Papadopoulos, V. et al. (1990) supra]. 100% binding corresponds
to 21 fmol [.sup.3H]PK11195. All data are expressed as the means
of quadruplicate assays.
FIG. 6 represents cholesterol uptake by MDA-231 and MCF-7 nuclei.
Uptake of [.sup.3H] cholesterol by nuclei isolated from MDA-231
and MCF-7 cells was measured in response to varying doses of PK11195.
Data is expressed as % cholesterol uptake into MCF-7 nuclei in the
absence of any PK11195. Data points represent the mean.+-.S. E.
of five (MDA-231) or four (MCF-7) independent experiments carried
out in quadruplicate.
FIG. 7 demonstrates the effect of PK11195 on MDA-231 cell proliferation.
MDA-231 cells grown in 96-well plates were washed with PBS and cultured
in media supplemented with 0.1% FBS 24 h prior to any treatment.
The indicated concentrations of PK11195 were added to the cells
cultured in DMEM supplemented with 0.1% FBS and incubated for 24
h at 37.degree. C. 4 h prior to the end of incubation, bromodeoxyuridine
(BrdU) was added to each well. Incorporation of BrdU was measured
at 450 nm (reference=700 nm). Data points represent the mean.+-.S.E.
of three independent experiments carried out in quadruplicate. ***,
p<0.05.
FIG. 8 shows PBR mediated nuclear cholesterol uptake correlates
with the proliferation rate of MDA-231 cells. The means of all data
points for 0, 10-10, 10-8, and 10-6 M PK11195 from the previously
described cell proliferation assay were plotted against the corresponding
means from the previously described cholesterol uptake assay. A
regression line drawn for all plotted data gives a coefficient of
correlation of 0.99. Numeric values in (n) indicate the number data
points taken for each mean.+-.S. E.
FIG. 9 shows PBR expression in normal human breast tissue. Paraffin
embedded sections of normal breast tissue were immunostained with
an anti-PBR antiserum at 1:500 dilution and counterstained with
hematoxylin as previously described [Oke, B. O. et al. (1992) Mol.
Cell. Endocr. 87:R1-R6; Garnier, M. et al. (1993) Endocrinology
132:444-458].
(a) localization of PBR in the epithelium of human breast ducts
(horseradish peroxidase staining)[Garnier, M. et al. (1994) J. Biol.
Chem. 269: 22105-22112].
(b) The hematoxylin counterstaining was omitted in order to examine
whether the nucleus of the cells contained immunoreactive PBR protein.
(c) Localization of immunoreactive PBR protein using an FITC-coupled
secondary antibody.
(d) Phase contrast microscopy of the same tissue area.
(e) Detection of PBR ligand binding protein using the fluorescent
PBR derivative compound 4 [Kozikowski, A. P. et al. (1997) supra].
A filter was used to enhance the detection of low fluorescence levels.
(f) Displacement of the fluorescence with 1000 fold excess of the
competitive ligand PK11195 [Kozikowski, A. P. et al. (1997) supra].
FIG. 10 shows PBR expression in aggressive metastatic human breast
carcinoma tissue. All biopsies were obtained from the Lombardi Cancer
Center at Georgetown University Medical Center. Biopsies were histologically
characterized by the pathologist. Paraffin embedded sections of
normal breast tissue were immunostained with an anti-PBR antiserum
at 1:500 dilution and counterstained with hematoxylin as previously
described [Oke, B. O. et al. (1992) supra; Garnier, M. et al. (1993)
supra].
(a) Localization of PBR in the epithelium of aggressive metastatic
human breast carcinoma (horseradish peroxidase staining) [Garnier,
M. et al. (1994), supra].
(b) Hematoxylin counterstaining was omitted in order to examine
whether the nucleus of the cells contained immunoreactive PBR protein.
(c) Localization of immunoreactive PBR protein using an FITC-coupled
secondary antibody.
(d) Phase contrast microscopy of the same tissue area.
(e) Detection of PBR ligand binding protein using the fluorescent
PBR derivative compound 4 [Kozikowski, A. P. et al. (1997) supra].
(f) Displacement of the fluorescence with 1000 fold excess of the
competitive ligand PK11195 [Kozikowski, A. P. et al. (1997) supra].
FIG. 11 shows cell proliferation rates of wild-type and mutant
R2C tumor cells. Cell proliferation rates of wild type R2C tumor
cells and PBR mutant R12 cells. The rate of cell proliferation was
determined using the MTT proliferation assay (Boehringer Mannheim).
Results shown represent the mean.+-.S.E. of two independent experiments
carried out in triplicate.
DETAILED DESCRIPTION
In one embodiment, the present invention provides compositions
and methods for detecting peripheral-benzodiazepine receptors (PBR)
for the determination of the metastatic potential of a tumor. As
discussed above, increased PBR expression correlates with increased
aggressive behavior of tumor cells. Invasive tumors invade and grow
locally but they do not metastasize. However, the aggressive tumors
have the ability to invade and metastasize through the blood vessels
to different places of the human body. Tumor metastasis into vital
organs (such as lungs) is the most common cause of death.
The correlation between high levels of expression of PBR and metastatic
potential is shown in this application for human breast cancer.
However, due to the involvement of PBR in cell proliferation, and
the expression of PBR in all cells, it is likely that this correlation
would exist for other solid tumors and cancers such as prostate
cancer, colon cancer, brain tumors, and tumors in steroid producing
tissues such as gonadal tumors, to name a few.
The level of expression of PBR, for the purposes of diagnosis or
prognosis of a cancer or tumor, can be detected at several levels.
Using standard methodology well known in the art, assays for the
detection and quantitation of PBR RNA can be designed, and include
northern hybridization assays, in situ hybridization assays, and
PCR assays, among others. Please see e.g., Maniatis, Fitsch and
Sambrook, Molecular Cloning; A Laboratory Manual (1982) or DNA Cloning,
Volumes I and II (D. N. Glover ed. 1985), or Current Protocols in
Molecular Biology, Ausubel, F. M. et al. (Eds), Wiley & Sons,
Inc. for general description of methods for nucleic acid hybridization.
Polynucleotides probes for the detection of PBR RNA can be designed
from the sequence available at accession number L21950 for the human
PBR sequence [Riond, J. et al. (1991) Eur. J. Biochem. 195:305-311;
Chang, Y. J. et al. (1992) DNA and Cell Biol. 11:471-480]. The sequence
of PBR from other sources such as bovine [Parola, A. L. et al. (1991)
J. Biol. Chem 266: 14082-14087] and mouse [Garnier, M. et al. (1994)
Mol. Pharm. 45:201-211] are also known. In addition, in this application
is disclosed a partial DNA sequence of the PBR gene found in invasive
cells. Partial cDNA sequences were obtained for both MDA-231 PBR
identified as SEQ ID NO:1, and MCF-7 PBR identified as SEQ ID NO:2.
The nucleotide sequences obtained revealed four mutations at the
DNA level for the gene from MCF-7 and MDA-231, namely, an N to adenine
change at nucleotide 83, a guanine to adenine change at nucleotide
362, an adenine to guanine change at nucleotide 408 and a thymine
to guanine change at nucleotide 573. An additional change at nucleotide
10 of PBR from MDA-231 was found which was a substitution of guanine
for adenine. The changes in the PBR gene encoded by the cDNA of
MCF-7 and MDA-231 result in two changes at the amino acid level,
a replacement of histidine 162 with arginine and replacement of
alanine 147 with a threonine. The amino acids encoded by SEQ ID
NO:1 and SEQ ID NO:2 are specified in SEQ ID NO:3. The region surrounding
the translation site, and 5' to the translation site has not yet
been obtained but may provide key evidence for the differential
localization (cytoplasmic versus nuclear) of PBR between the two
cell lines. In particular, the PBR sequence derived from MCF-7 or
MDA-231 can be used to construct vectors, and produce cell lines
which express the altered PBR. Since tumorigenesis is considered
to be a multi-step process, it is possible that the changes between
the normal PBR and PBR from MCF-7 and MDA-231 represent the initial
steps in this process. With this in mind, these cell lines expressing
the aberrant PBR can be used to identify what agents would result
in a second step towards tumorigenesis, and what drugs would reduce
of alter PBR expression. Vector design is known in the art. Transformed
cells would include prokaryotic and eukaryotic cells, such as bacteria,
most of which do not express PBR, and yeast and mammalian cells.
Methods for transforming bacteria and transfecting cells are known
in the art. In addition, the sequence of SEQ ID NO:1 or SEQ ID NO:2
can be used to clone the remainder of the PBR sequence of MCF-7
and MDA-231 around the translation start site.
The complete sequence of the PBR, normal or mutant, can be used
for a probe to detect RNA expression. Alternatively, a portion or
portions of the sequence can be used. Methods for designing probes
are known in the art. Polynucleotide sequences are preferably homologous
to or complementary to a region of the PBR gene, preferably, the
sequence of the region from which the polynucleotide is derived
is homologous to or complementary to a sequence which is unique
to the PBR gene. Whether or not a sequence is unique to the PBR
gene can be determined by techniques known to those of skill in
the art. For example, the sequence can be compared to sequences
in databanks, e.g., GenBank. Regions from which typical DNA sequences
may be derived include but are not limited to, for example, regions
encoding specific epitopes, as well as non-transcribed and/or non-translated
regions.
For example, RNA isolated from samples can be coated onto a surface
such as a nitrocellulose membrane and prepared for northern hybridization.
In the case of in situ hybridization of biopsy samples for example,
the tissue sample can be prepared for hybridization by standard
methods known in the art and hybridized with polynucleotide sequences
which specifically recognize PBR RNA. The presence of a hybrid formed
between the sample RNA and the polynucleotide can be detected by
any method known in the art such as radiochemistry, or immunochemistry,
to name a few.
One of skill in the art may find it desirable to prepare probes
that are fairly long and/or encompass regions of the amino acid
sequence which would have a high degree of redundancy in the corresponding
nucleic acid sequences. In other cases, it may be desirable to use
two sets of probes simultaneously, each to a different region of
the gene. While the exact length of any probe employed is not critical,
typical probe sequences are no greater than 500 nucleotides, even
more typically they are no greater than 250 nucleotides; they may
be no greater than 100 nucleotides, and also may be no greater than
75 nucleotides in length. Longer probe sequences may be necessary
to encompass unique polynucleotide regions with differences sufficient
to allow related target sequences to be distinguished. For this
reason, probes are preferably from about 10 to about 100 nucleotides
in length and more preferably from about 20 to about 50 nucleotides.
The DNA sequence of PBR can be used to design primers for use in
the detection of PBR using the polymerase chain reaction (PCR) or
reverse transciption PCR (RT-PCR). The primers can specifically
bind to the PBR cDNA produced by reverse transcription of PBR RNA,
for the purpose of detecting the presence, absence, or quantifying
the amount of PBR by comparison to a standard. The primers can be
any length ranging from 7-40 nucleotides, preferably 10-15 nucleotides,
most preferably 18-25 nucleotides homologous or complementary to
a region of the PBR sequence. Reagents and controls necessary for
PCR or RT-PCR reactions are well known in the art. The amplified
products can then be analyzed for the presence or absence of PBR
sequences, for example by gel fractionation, by radiochemistry,
and immunochemical techniques. This method is advantageous since
it requires a small number of cells. Once PBR is detected, a determination
whether the cell is an aggressive tumor phenotype can be made by
comparison to the results obtained from a normal cell using the
same method. The level of aggressiveness can be determined by comparing
PBR expression in sample cells to PBR expression of cells with varying
levels of aggressive phenotypes since the level of PBR expression
correlates with the level of aggressive phenotype of a cell. Increased
PBR RNA levels correlate with increased aggressive behavior in a
cell.
In another embodiment, the present invention relates to a diagnostic
kit for the detection of PBR RNA in cells, said kit comprising a
package unit having one or more containers of PBR oligonucleotide
primers for detection of PBR by PCR or RT-PCR or PBR polynucleotides
for the detection of PBR RNA in cells by in situ hybridization or
northern analysis, and in some kits including containers of various
reagents used for the method desired. The kit may also contain one
or more of the following items: polymerization enzymes, buffers,
instructions, controls, detection labels. Kits may include containers
of reagents mixed together in suitable proportions for performing
the methods in accordance with the invention. Reagent containers
preferably contain reagents in unit quantities that obviate measuring
steps when performing the subject methods.
In a further embodiment, the present invention provides a method
for identifying and quantifying the level of PBR present in a particular
biological sample. Any of a variety of methods which are capable
of identifying (or quantifying) the level of PBR in a sample can
be used for this purpose.
Diagnostic assays to detect PBR may comprise a biopsy or in situ
assay of cells from an organ or tissue sections, as well as an aspirate
of cells from a tumour or normal tissue. In addition, assays may
be conducted upon cellular extracts from organs, tissues, cells,
urine, or serum or blood or any other body fluid or extract.
When assaying a biopsy, the assay will comprise, contacting the
sample to be assayed with a PBR ligand, natural or synthetic, or
an antibody, polyclonal or monoclonal, which recognizes PBR, or
antiserum capable of detecting PBR, and detecting the complex formed
between PBR present in the sample and the PBR ligand or antibody
added.
PBR ligands include the natural ligand diazepan-binding inhibitor
(DBI), in addition to natural and synthetic classes of ligands and
their derivatives which can be derived from natural sources such
as animal or plant extracts. PBR ligands include benzodiazepines
such as Ro-4864, diazepam, flunitrazepam, clonazepam, isoquinoline;
carboxamides such as PK 11195, PK 14105, PK14067/8 (stereoisomers);
imidazopyridines, such as alpidem and zolpidem; 2-aryl-3-idoleacetamides
such as FGIN-1-27 and its fluorescent derivative compound 4, and
porphyrins such as protophorphyrin IX. In addition to the PBR ligands
mentioned above, there is a list of other compounds, essentially
those containing aromatic rings, that appear to bind to PBR with
different affinities. This list includes dipyridamole, thiazide
diuretics, pyrethroid insecticides, carbamazepine, lidocaine, certain
steroids, and dihydropyridines. For a review of PBR ligands, please
see Papadopoulos, V. (1993) Endocrine Reviews 14: 222-240, incorporated
in its entirety by reference thereto.
Monoclonal or polyclonal antibodies which recognize PBR can be
generated against the complete PBR or against a portion thereof.
Persons with ordinary skill in the art using standard methodology
can raise monoclonal and polyclonal antibodies to PBR protein (or
polypeptide) of the present invention. Polyclonal antibodies are
available from the present inventors and commercially available
from Sanofi, Inc., France. Materials and methods for producing antibodies
are well known in the art (see for example Goding, in, Monoclonal
Antibodies: Principles and Practice, Chapter 4, 1986). In addition,
the protein or polypeptide can be fused to other proteins or polypeptides
which increase its antigenicity, thereby producing higher titers
of antibodies. Examples of such proteins or polypeptides include
any adjuvants or carriers, such as aluminum hydroxide. These antibodies
can be used in passive antibody therapy wherein antibodies can be
employed to modulate PBR dependent processes such as cell proliferation,
and cholesterol transport.
PBR ligands or anti-PBR antibodies, or fragments of ligand and
antibodies capable of detecting PBR may be labeled using any of
a variety of labels and methods of labeling for use in diagnosis
and prognosis of disease, such as breast cancer, particularly for
assays such as Positron Emission Tomography and magnetic resonance
imaging [Leong, D. et al. (1996) Alcohol Clin. Exp. Res. 20:601-605].
Examples of types of labels which can be used in the present invention
include, but are not limited to, enzyme labels, radioisotopic labels,
non-radioactive isotopic labels, and chemiluminescent labels.
Examples of suitable enzyme labels include malate dehydrogenase,
staphylococcal nuclease, delta-5-steroid isomerase, yeast-alcohol
dehydrogenase, alpha-glycerol phosphate dehydrogenase, triose phosphate
isomerase, peroxidase, alkaline phosphatase, asparaginase, glucose
oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate
dehydrogenase, glucoamylase, acetylcholine esterase, etc.
Examples of suitable radioisotopic labels include .sup.3H, .sup.111In,
.sup.125I, .sup.32P, .sup.35S, .sup.14C, .sup.57To, .sup.58Co, .sup.59Fe,
.sup.75Se, .sup.152Eu, .sup.90Y, .sup.67Cu, .sup.21Ci, .sup.211At,
.sup.212Pb, .sup.47Sc, .sup.109Pd, .sup.11C, .sup.19F, .sup.123I,
etc.
Examples of suitable non-radioactive isotopic labels include .sup.157Gd,
.sup.55Mn, .sup.162Dy, .sup.52Tr, .sup.46Fe, etc.
Examples of suitable fluorescent labels include a .sup.152Eu label,
a fluorescein label, an isothiocyanate label, a rhodamine label,
a phycoerythrin label, a phycodyanin label, an allophycocyanin label,
a fluorescamine label, etc.
Examples of chemiluminescent labels include a luminal label, an
isoluminal label, an aromatic acridinium ester label, an imidazole
label, an acridinium salt label, an oxalate ester label, a luciferin
label, a luciferase label, etc.
Those of ordinary skill in the art will know of other suitable
labels which may be employed in accordance with the present invention.
The binding of these labels to ligands and to antibodies or fragments
thereof can be accomplished using standard techniques commonly known
to those of ordinary skill in the art. Typical techniques are described
by Kennedy, J. H., et al., 1976 (Clin. Chim. Acta 70:1-31), and
Schurs, A. H. W. M., et al. 1977 (Clin. Chim Acta 81:1-40). Coupling
techniques mentioned in the latter are the glutaraldehyde method,
the periodate method, the dimaleimide method, and others, all of
which are incorporated by reference herein.
The detection of the antibodies (or fragments of antibodies) of
the present invention can be improved through the use of carriers.
Well-known carriers include glass, polystyrene, polypropylene, polyethylene,
dextran, nylon, amylases, natural and modified celluloses, polyacrylamides,
agaroses, and magnetite. The nature of the carrier can be either
soluble to some extent or insoluble for the purposes of the present
invention. The support material may have virtually any possible
structural configuration so long as the coupled molecule is capable
of binding to PBR. Thus, the support configuration may be spherical,
as in a bead, or cylindrical, as in the inside surface of a test
tube, or the external surface of a rod. Alternatively, the surface
may be flat such as a sheet, test strip, etc. Those skilled in the
art will note many other suitable carriers for binding monoclonal
antibody, or will be able to ascertain the same by use of routine
experimentation.
The ligands or antibodies, or fragments of antibodies or ligands
of PBR discussed above may be used to quantitatively or qualitatively
detect the presence of PBR. Such detection may be accomplished using
any of a variety of immunoassays known to persons of ordinary skill
in the art such as radioimmunoassays, immunometic assays, etc. Using
standard methodology well known in the art, a diagnostic assay can
be constucted by coating on a surface (i.e. a solid support) for
example, a microtitration plate or a membrane (e.g. nitrocelluolose
membrane), antibodies specific for PBR or a portion of PBR, and
contacting it with a sample from a person suspected of having a
PBR related disease. The presence of a resulting complex formed
between PBR in the sample and antibodies specific therefor can be
detected by any of the known detection methods common in the art
such as fluorescent antibody spectroscopy or colorimetry. A good
description of a radioimmune assay may be found in Laboratory Techniques
and Biochemistry in Molecular Biolocgy. by Work, T. S., et al. North
Holland Publishing Company, N.Y. (1978), incorporated by reference
herein. Sandwich assays are described by Wide at pages 199-206 of
Radioimmune Assay Method, edited by Kirkham and Hunter, E. &
S. Livingstone, Edinburgh, 1970.
The determination of elevated levels of PBR is done relative to
a sample with no detectable tumor. This may be from the same patient
or a different patient. For example, a first sample may be collected
immediately following surgical removal of a solid tumor. Subsequent
samples may be taken to monitor recurrence of tumor growth and/or
tumor cell proliferation. Additionally, other standards may include
cells of varying aggressive phenotype such that an increase or decrease
in aggressive phenotype can be assessed.
The distinct subcellular localization of PBR in the cytoplasm of
epithelial cells of normal breast ducts and the absence of staining
in the nucleus, in contrast with the localization of PBR in aggressive
carcinomas in the nucleus and the perinuclear area of the aggressive
tumor cells provides a simple method for diagnosing the aggressive
phenotype of a tumor cell. Immunostaining using labeled PBR ligand
or labeled PBR antibody or fragment of ligand or antibody capable
of binding to PBR and determining the subcellular location of PBR
in the cellular samples provides yet another diagnostic assay of
the present invention. In addition, antiserum which recognizes PBR
can also be used along with a secondary antibody reactive with the
primary antibody. Immunostaining assays are well known in the art,
and are additionally described in the Examples below with respect
to breast cancer cells and biopsies.
The diagnostic methods of this invention are predictive of proliferation
and metastatic potential in patients suffering from breast cancinomas
including lobular and duct carcinomas, and other solid tumors, carcinomas,
sarcomas, and cancers including carcinomas of the lung like small
cell carcinoma, large cell carcinoma, squamous carcinoma, and adenocarcinoma,
stomach carcinoma, prostatic adenocarcinoma, ovarian carcinoma such
as serous cystadenocarcinoma and mucinous cytadenocarcinoma, ovarian
germ cell tumors, testicular carcinomas, and germ cell tumors, pancreatic
adenocarcinoma, biliary adenocarcinoma, heptacellular carcinoma,
renal cell adenocarcinoma, endometrial carcinoma including adenocarcinomas
and mixed Mullerian tumors (carcinosarcomas), carcinomas of the
endocervix, ectocervix, and vagina such as adenocarcinoma and squamous
carcinoma, basal cell carcinoma, melanoma, and skin appendage tumors,
esophageal carcinoma, carcinomas of the nasopharyns and oropharynx
including squamous carcinoma and adenocarcinomas, salivary gland
carcinomas, brain and central nervous system tumors including tumors
of glial, neuronal, and meningeal origin, tumors of peripheral nerve,
soft tissue sarcomas and sarcoms of bone and cartilage. Cells of
these tumors which express increased levels of PBR RNA or PBR protein,
and/or PBR which localizes to the nucleus are considered acquiring
the aggressive tumor phenotype and can result in increased metastasis.
Agents which decrease the level of PBR (i.e. in a human or an animal)
or reduce or inhibit PBR activity may be used in the therapy of
any disease associated with the elevated levels of PBR such as metastatic
cancer, for example breast cancer, or diseases associated with increased
cell proliferation or increased cholesterol transport into the cell.
An increase in the level of PBR is determined when the level of
PBR in a tumor cell is about 2-3 times the level of PBR in the normal
cell, up to about 10-100 times the amount of PBR in a normal cell.
Agents which decrease PBR RNA include, but are not limited to, one
or more ribozymes capable of digesting PBR RNA, or antisense oligonucleotides
capable of hybridizing to PBR RNA such that the translation of PBR
is inhibited or reduced resulting in a decrease in the level of
PBR. These antisense oligonucleotides can be administered as DNA,
as DNA entrapped in proteoliposomes containing viral envelope receptor
proteins [Kanoda, Y. et al. (1989) Science 243:375] or as part of
a vector which can be expressed in the target cell such that the
antisence DNA or RNA is made. Vectors which are expressed in particular
cell types are known in the art, for example, for the mammary gland,
please see Furth, (1997) (J. Mammary Gland Biol. Neopl. 2:373) for
examples of conditional control of gene expression in the mammary
gland. Alternatively, the DNA can be injected along with a carrier.
A carrier can be a protein such as a cytokine, for example interleukin
2, or polylysine-glycoprotein carrier. Such carrier proteins and
vectors and methods of using same are known in the art. In addition,
the DNA could be coated onto tiny gold beads and said beads introduced
into the skin with, for example, a gene gun [Ulmer, J. B. et al.
(1993) Science 259:1745].
Alternatively, antibodies, or compounds capable of reducing or
inhibiting PBR, that is reducing or inhibiting either the expression,
production or activity of PBR, such as antagonists, can be provided
as an isolated and substantially purified protein, or as part of
an expression vector capable of being expressed in the target cell
such that the PBR-reducing or inhibiting agent is produced. In addition,
co-factors such as various ions, i.e. Ca2+[Calvo, D. J. and Medina,
J. H. (1993) J. Recept. Res. 13:975-987], or anions, such as halides
or anion channel blockers such as DIDS (4,4'diisothiocyanostilbene-2,2'-disulfonic
acid), an ion transport blocker [Skolnick, P. (1987) Eur. J. Pharmacol.
133:205-214], or factors which affect the stability of the receptor
such as lipids, for example, the phospholipids phosphatidylserine
and phosphatidylinositol whereby the presence of the phospholipids
is required for receptor activity [Moynagh, P. N. and Williams,
D. C. (1992) Biochem. Pharmacol. 43:1939-1945] can be administered
to modulate the expression and function of the receptor. These formulations
can be administered by standard routes. In general, the combinations
may be administered by the topical, transdermal, intraperitoneal,
oral, rectal, or parenteral (e.g. intravenous, subcutaneous, or
intramuscular) route. In addition, PBR-inhibiting compounds may
be incorporated into biodegradable polymers being implanted in the
vicinity of where drug delivery is desired, for example, at the
site of a tumor or implanted so that the PBR-inhibiting compound
is slowly released systemically. The biodegradable polymers and
their use are described, for example, in detail in Brem et al.(1991)
J. Neurosurg. 74:441-446.
These compounds are intended to be provided to recipient subjects
in an amount sufficient to effect the inhibition of PBR. Similarly,
agents which are capable of negatively affecting the expression,
production, stability or function of PBR, are intended to be provided
to recipient subjects in an amount sufficient to effect the inhibition
of PBR. An amount is said to be sufficient to "effect"
the inhibition or induction of PBR if the dosage, route of administration,
etc. of the agent are sufficient to influence such a response.
In line with the function of PBR in cell proliferation, agents
which stimulate the level of PBR, such as agonists of PBR, may be
used in the therapy of any disease associated with a decrease of
PBR, or a decrease in cell proliferation, wherein PBR is capable
of increasing such proliferation, e.g. developmental retardation.
PBR has also been shown to be involved in cholesterol transport,
therefore, an agent or drug which results in an increase in expression,
function, or stability of PBR can be used to increase cholesterol
transport into cells. Diseases where cholesterol transport is deficient
include lipoidal adrenal hyperplasia, and diseases where there is
a requirement for increased production of compounds requiring cholesterol
such as myelin and myelination including Alzheimer's disease, spinal
chord injury, and brain development neuropathy [Snipes, G. and Suter,
U. (1997) Cholesterol and Myelin. In: Subcellular Biochemistry,
Robert Bittman (ed.), vol. 28, pp. 173-204, Plenum Press, New York],
to name a few.
In providing a patient with antibodies, or fragments thereof, capable
of binding to PBR, or an agent capable of inhibiting PBR expression
or function to a recipient patient, the dosage of administered agent
will vary depending upon such factors as the patient's age, weight,
height, sex, general medical condition, previous medical history,
etc. Similarly, when providing a patient with an agent or agonist
capable of inducing or increasing expression or function of PBR,
the dosage will vary depending upon such factors as the patient's
age, weight, height, medical history, etc. In general, it is desirable
to provide the recipient with a dosage of agent which is in the
range of from about 1 pg/kg to 10 mg/kg (body weight of patient),
although a lower or higher dosage may be administered.
A composition is said to be "pharmacologically acceptable"
if its administration can be tolerated by a recipient patient. Such
an agent is said to be administered in a "therapeutically effective
amount" if the amount administered is physiologically significant.
An agent is physiologically significant if its presence results
in a detectable change in the physiology of a recipient patient.
The compounds of the present invention can be formulated according
to known methods to prepare pharmaceutically useful compositions,
whereby these materials, or their functional derivatives, are combined
in admixture with a pharmaceutically acceptable carrier vehicle.
Suitable vehicles and their formulation, inclusive of other human
proteins, e.g., human serum albumin, are described, for example,
in Remington's Pharmaceutical Sciences [16th ed., Osol, A. ed.,
Mack Easton Pa. (1980)]. In order to form a pharmaceutically acceptable
composition suitable for effective administration, such compositions
will contain an effective amount of the above-described compounds
together with a suitable amount of carrier vehicle.
Additional pharmaceutical methods may be employed to control the
duration of action. Control release preparations may be achieved
through the use of polymers to complex or absorb the compounds.
The controlled delivery may be exercised by selecting appropriate
macromolecules (for example polyesters, polyamino acids, polyvinyl,
pyrrolidone, ethylenevinylacetate, methylcellulose, carboxymethylcellulose,
or protamine sulfate) and the concentration of macromolecules as
well as the method of incorporation in order to control release.
Another possible method to control the duration of action by controlled
release preparations is to incorporate the compounds of the present
invention into particles of a polymeric material such as polyesters,
polyamino acids, hydrogels, poly(lactic acid) or ethylene vinylacetate
copolymers. Alternatively, instead of incorporating these agents
into polymeric particles, it is possible to entrap these materials
in microcapsules prepared, for example, interfacial polymerization,
for example, hydroxymethylcellulose or gelatin-microcapsules and
poly(methylmethacrylate)microcapsules, respectively, or in colloidal
drug delivery systems, for example, liposomes, albumin microspheres,
microemulsions, nanoparticles, and nanocapsules or in macroemulsions.
Such techniques are disclosed in Remington's Pharmaceutical Sciences
(1980).
Having now generally described the invention, the same will be
more readily understood through reference to the following examples
which are provided by way of illustration, and are not intended
to be limiting to the present invention, unless specified.
The following MATERIALS AND METHODS were used in the examples that
follow.
Cell Culture--Human breast cancer cell lines (BT549, HS-578-T,
MCF-7, MDA-231, MDA-435, MDA-468, T47D, and ZR-75-1) were obtained
from the Lombardi Cancer Center, Georgetown University Medical Center.
The U937 human histiocytic lymphoma cell line was obtained from
the American Type Culture Collection (Rockville, Md.). MA-10 mouse
Leydig tumor cells were a gift from Dr. Mario Ascoli (University
of Iowa) and were maintained in Waymouth's MB752/1 medium supplemented
with 15% horse serum as previously described [Papadopoulos et al.,
(1990) J. Biol. Chem 265:3772-3778]. All cell lines were cultured
on polystyrene culture dishes (Corning) and, with the exception
of the U937 cell line, grown in Dulbecco's modified Eagle medium
(DMEM) supplemented with 10% fetal bovine serum (FBS). The U937
cell line were grown in RPMI medium (Gibco) supplemented with 10%
FBS.
Radioligand Binding Assays--Cells were scraped from 150 mm culture
dishes into 5 ml phosphate buffered saline (PBS), dispersed by trituration,
and centrifuged at 500.times.g for 15 min. Cell pellets were resuspended
in PBS and assayed for protein concentration. [.sup.3H]PK11195 binding
studies on 50?g of protein from cell suspensions were performed
as previously described [Papadopoulos et al 1990, supra; Garnier
et al., (1994) Molecular Pharmacology 45:201-211]. Scatchard plots
were analyzed by the LIGAND program [Munson, (1980) Anal. Biochem.
107:220]. Specific binding of [.sup.3H]PK11195 (2.0 nM) to MDA-231
cells was measured in the presence or absence of the indicated concentrations
of competing PBR ligands as previously described (Garnier, 1994,
supra). IC50 estimation was performed using the LIGAND program (Munson,
1980, supra).
Protein Measurement--Protein levels were measured by the Bradford
method [Bradford (1976) Anal. Biochem. 72:248-2554] using the Bio-Rad
Protein Assay kit (Bio-Rad Laboratories) with bovine serum albumin
as a standard.
Transmission Electron Microscopy--MDA-231, MCF-7ADR, and MCF-7
cells cultured on 25 cm.sup.2 culture dishes (Corning) were first
washed with PBS for 5 min three times. The cells were then fixed
with a solution of 1% paraformaldehyde, 2% gluteraldehyde, and 0.1M
PBS for 15 min at room temperature and then washed three times with
PBS. The cells were then embedded in Epon-araldite and further processed
as previously described [Li et al. (1997) Endocrinology 138:1289-1298].
Northern Analysis--The levels of hPBR mRNA from MDA-231, MCF-7,
ADR, and U937 cells were compared by Northern Blot analysis. Total
cellular RNA was isolated from cells grown on 150 mm culture dishes
by the addition of 4.5 ml RNAzol B (TEL-TEST, Inc.) and 0.45 ml
chloroform. After vigorous shaking and centrifugation at 9,000.times.g
for 30 min, the aqueous phase was transferred to a fresh tube and
mixed 1:1 with isopropanol (v:v), stored at -20.degree. C. for 2
hr, and centrifuged at 9,000.times.g for 30 min. The RNA pellet
was then washed with 75% ethanol and centrifuged 7,500.times.g for
8 min. The pellet was then air dried and resuspended in formazol.
RNA concentrations and purity were determined at 260/280 nm.
20 ug of total RNA from each cell line were run on 1% agarose gels
containing 1.times.MOPS and 5.3% formaldehyde using the 0.24 to
9.5 kb RNA Ladder (GIBCO) as a size marker. Gels were then transferred
overnight to nylon membranes (S&S Nytran, Schleicher & Schuell,
Keene, N.H.) (Maniatis, 1989). A 0.2 kb human PBR (hPBR) cDNA fragment
(derived from the pCMV5-PBR plasmid vector containing the full length
hPBR kindly given by Dr. Jerome Strauss, University of Pennsylvania,
Pa.) was radiolabeled with [?-.sup.32P]dCTP using a random primers
DNA labeling system (Life Technologies, Gaithersburg, Md.). The
filter was first prehybridized overnight at 68.degree. C. in 6.times.SSC,
0.5% SDS, and 100 ug/ml denatured, fragmented, salmon sperm DNA.
After hybridization, the membrane was washed twice with 2.times.SSC,
0.5% SDS for 10 min, once with 0.2.times.SSC, 0.5% SDS for 30-60
min at room temperature, and once with 0.2.times.SSC, 0.5% SDS for
30 min at 60.degree. C. Autoradiography was performed by exposing
the blots to X-OMAT AR film (Kodak, Rochester, N.Y.) at -70.degree.
C. for 4-48 hr. Quantification of PBR mRNA was carried out using
the SigmaGel software (Jandel Scientific, San Rafael, Calif.).
Partial cDNA Sequencing--PBR cDNAs were prepared from total MDA-231
and MCF-7 RNA using the Perkin Elmer RT-PCR Kit (Branchburg, N.J.).
PCR was performed on cDNAs using primers designed from the known
human sequence (Riond, 1991, supra). Labeling of PCR products was
performed using the ABI PRISM Dye Terminator Cycle Sequencing Ready
Reaction Kit (Perkin Elmer, Branchburg, N.J.). Labeled PCR product
was then given to the Lombardi Sequencing Core Facility (Georgetown
University Medical Center, Washington, D.C.) for sequence analysis.
Fluorescent Microscopy with the compound 4 fluorescent PBR Ligand--MA-10,
MDA-231, MCF-7, and ADR cells were grown on glass coverslips as
previously described [Kozikowski et al. (1997) J. Med. Chem. 40:
2435-2439]. Cells were then washed twice with sterile PBS and incubated
for 45 min with 1 uM compound 4, a fluorescent derivative of the
PBR ligand FGIN-27, with or without a competing PBR ligand, FGIN-27,
at a concentration of 100 uM. After the incubation period, the cells
were washed with PBS and examined by fluorescent microscopy using
an Olympus BH-2 fluorescence microscope.
Immunocytochemistrv of MDA-231 Cells--MDA-231 cells were cultured
overnight on 8-chambered SuperCell Culture Slides (Fisher Scientific,
Pittsburgh, Pa.) at a concentration of approximately 50,000 cells/chamber.
Cells were then fixed in 70% EtOH for 15 min at 4.degree. C. After
washing 3.times. in distilled H.sub.2O for 2 min each, the fixed
cells were incubated overnight at 4.degree. C. with either PBR [Amri
et al. (1996) Endocrinology 137:5707-5708] or DBI [Garnier et al.
(1993) Endocrinology 132: 444-458] polyclonal antisera at concentrations
of 1:100, 1:200, 1:500, or 1:1,000. After incubation with primary
antiserum, slides were washed 3.times. in PBS for 2 min each. Slides
were then incubated at room temperature for 1 h with horseradish
peroxidase-coupled goat anti-rabbit secondary antibody diluted 1:1,000
in PBS supplemented with 10% calf serum. After washing slides 3.times.
in PBS for 2 min each, fresh H.sub.2O.sub.2 diluted 1:1,000 with
3-amino-9-ethyl carbazole (AEC) was added and slides were incubated
for 1 h at 37.degree. C. Slides were then rinsed in distilled H20
and counterstained with hematoxylin for 2 min, washed with tap H.sub.2O
and left in PBS until cells turn blue (approximately 30 s), and
rinsed in distilled H.sub.2O before mounting with Crystal/Mount.
Nuclear Uptake of .sup.3H-Cholesterol--Nuclei were isolated from
MDA-231, MCF-7, and as described by Elango et al (1997). Isolated
nuclei were resuspended in 1 ml ice-cold PBS. .sup.3H-cholesterol
uptake in MDA-231 and MCF-7 nuclei was examined using the indicated
concentrations of PK11195 incubated in 0.3 ml final volume in the
presence of 6.7 nM [1,2].sup.3H-cholesterol (50.0 Ci/mmol) and 3
ug nuclear protein (determined using the Bradford method as previously
described) for 60 min at 37.degree. C. Samples were then centrifuged
at 500.times.g for 30 min and pellets were washed in 500 ml ice-cold
PBS. After a second centrifugation at 500.times.g for 30 min, 200
ul 1.0 N NaOH was then added to the pellets and incubated overnight
at 37.degree. C. After incubation, 200 ul 1.0 N HCl was added and
samples were vigorously vortexed. 3 ml scintillation cocktail (Eco-Lite)
was then added prior to reading radioactivity on a Wallac 1409 Liquid
Scintillation Counter.
BrdU Cell Proliferation Assays and BrdU-labeling of MDA-231 Cells--MDA-231
cells were plated on 96-well plates (Corning) at a concentration
of approximately 10,000 cells/well (24 h incubation) or approximately
5,000 cells/well (48 h incubation) in DMEM supplemented with 0.1%
FBS. The cells were then incubated in either 0.1% or 10% FBS with
various concentrations of PK11195 (10-10, 10-9, 10-8, 10-7, 10-6,
10-5, or 10-4 M) for both 24 h or 48 h. Differences in cell proliferation
were analyzed by measuring the amount of 5-bromo-2'deoxyuridine
(BrdU) incorporation as determined by the BrdU ELISA (Boehringer
Mannheim).
EXAMPLE 1
Increased Expression of the Peripheral-Type Benzodiazepine Receptor
Corresponds With Increased Aggressive Phenotype in Human Breast
Cancer Cell Lines
In order to establish a correlation between PBR expression and
increased aggressive behavior in cancer we chose to examine this
proposed phenomenon in human breast cancer. To this end, binding
studies were initially performed on nine human breast cancer cell
lines using the PBR-specific high affinity ligand PK11195. The results
from these early experiments indicate that those cell lines with
a more invasive and chemotactic potential such as HS-578-T and MDA-231
display dramatically increased levels of PBR binding relative to
non-aggressive cell lines such as ZR-75-1, T47D, and MCF-7 (Table
1 and FIG. 1).
TABLE-US-00001 TABLE 1 Comparison of Invasive Characteristics of
Human Breast Cancer Cell Lines to PBR Expression. Cell Estrogen
Line Receptor Vimentin Invasion Chemotaxis CD44 PBR ZR-75-1 + -
+ + - - T47D + - + + - + MCF-7 + - + + + + + + MDA-435 - + + + +
+ + + + + + ADR - + + + + + + + + + + BT549 - + + + + + + + + +
+ + + + + + + + + MDA-468 - .+-. + + + + + + + + + + + + HS578-T
- .+-. + + + + + + + + + + + + + + + + + + + MDA-231 - + + + + +
+ + + + + + + + + + + + + + The various characteristics of the human
breast cancer cell lines described above are from Culty et al, 1993,
J. Cell Phys. 160: 275 286. The presence or absence of estrogen
receptor and vimentin are indicated by either a + or =, respectively.
The invasive and chemotaxis assays were determined by quantifying
the migration of cells in Boyden chamber assays using fibroblast
conditioned medium as the chemo-attactant. Chemoinvation was studied
using polycarbonate filters coated with a uniformlayer of matrigel
constituting a barrier that the cells had to degrade in order to
reach the filters and migrate through them. To determine the chemotactic
behavior of the cells, the filters were coated with a thin layer
of collagen IV that promotes cell attachment and allows the free
migration of the cells toward the gradient of fibroblast conditioned
medium. Invasion, chemotactic, and PBR binding were graded as %
of MDA-231 values (-, not detectable, +, 0 20%; + +, 20 40%, + +
+, 40 60%, + + + +, 60 8%, + + + + +, >80%). The relative amounts
of PBR were determined with binding assays in which increasing concentrations
of cellular protein were incubated with a constant level of [.sup.3H]PK11195
(6 nM). Non-specific binding was determined in the presence of cold
PK11195.
Further, the MCF-7 adriamycin-resistent derivative cell line, MCF-7ADR
(ADR), which expresses medium invasive and chemotactic potential
as well as intermediate levels of CD44, expressed approximately
20 to 40% PBR binding relative to the MDA-231 cell line (Table 1).
Scatchard analysis of PBR binding in the MDA-231 and ADR cell lines
further shows each to have a Bmax of 8.7.+-.1.4 and 1.3.+-.0.23
pmol/mg protein, respectively (Table 2 and FIGS. 2a and 2b). Despite
obtaining specific PK11195 binding, the low levels of binding were
inadequate for estimating the binding characteristics using Scatchard
plot analysis (Table 2). RNA (Northern) blot analysis was performed
in order to determine if the differences shown in PBR binding between
the cell lines reflects differential expression of PBR MRNA. As
shown in FIG. 3, MDA-231 cells express approximately 20-fold more
PBR mRNA than MCF-7 cells. This result fits with the correlation
between PBR expression and increased aggressive behavior between
these cell lines. The amount of PBR mRNA expressed in the ADR cell
line does not conform to this, however. In fact, ADR cells express
almost 1.5-fold more PBR mRNA than MDA-231 cells (FIG. 3). This
seemingly anomalous result will be discussed later.
TABLE-US-00002 TABLE 2 PBR Binding Characteristics of MDA-231,
ADR, and MCF-7 Cells PK11195 K.sub.D B.sub.max Cell Line (nM) (pmol/mg
protein) MDA-231 7.8 .+-. 1.8 8.7 .+-. 1.4 ADR 1.9 .+-. 0.47 1.3
.+-. 0.23 MCF-7 ND ND Ligand binding studies on MDA-231, ADR, and
MCF-7 cells (50 .mu.g) were performed using [3H]PK11195 as we described
[Papadopoulos et al. (1990) J. Biol. Chem. 265: 3772 3779]. The
results were analyzed by Scatchard plot carried out using the LIGAND
program (Munson, 1980, supra). ND, not detectable because Scatchard
plot analysis of the binding data could not be performed although
low levels of specific binding could be seen, indicating the presence
of PBR but at extremely low levels.
Previous studies demonstrated that, in most tissues, PBR is primarily
localized to the mitochondria (Papadopoulos, 1993, supra). In order
to rule out the possibility that the differences between aggressive
and non-aggressive human breast cancer cell lines is not due to
differences in mitochondrial content morphometry analysis was performed
on transmission electron micrographs on two of the extreme cell
lines, MDA-231 and MCF-7 (Data not shown). Numerous morphological
differences between the two cell lines, including differences in
vacuole content and the presence of mysterious dark bodies, that
may reflect their differences in metabolic activity. Morphometric
analysis indicates that the larger MCF-7 mitochondria cover the
same surface area/cell in the micrographs as do the MDA-231 mitochondria.
In order to further characterize the differences between these
human breast cancer cell lines, subcellular localization was carried
out using compound 4, the fluorescent derivative of FGIN-27, a specific
PBR ligand (Kozikowski et al., 1997, supra). PBR has previously
been shown to localize primarily to the outer mitochondrial membrane
in MA-10 mouse tumor Leydig cells, the cell line used to characterize
the only known PBR function (Papadopoulos, 1993, supra). In MA-10
cells, compound 4 fluorescent labeling is localized to the cytoplasm,
presumably to the mitochondria (FIG. 4a). Similar to MA-10 cells,
PBR is localized almost exclusively to the cytoplasm in MCF-7 cells
(FIG. 4b). Strikingly however, PBR localizes primarily to the nucleus
in MDA-231 cells (FIG. 4c,d). This fluorescence indicates localization
to either the nucleoplasm (FIG. 4c) or the peri-nuclear envelope
(FIG. 5d). The displacement of fluorescent binding by 100 uM FGIN-27
indicates that compound 4 labeling is specific for PBR (FIG. 4e).
Scatchard analysis of [.sup.3H] PK11195 binding to nuclei isolated
from MDA-231 cells revealed a KD of 10.3.+-.8.4 nM and a Bmax of
6.9.+-.4.8 pmol/mg nuclear protein (Table 3). Similar analysis of
nuclei isolated from MCF-7 cells yielded a KD of 7.6.+-.4.6 nM and
a Bmax of 0.4.+-.0.2 pmol/mg nuclear protein (Table 3). While not
shown, in ADR cells, PBR localizes chiefly to the cytoplasm, although
nuclear fluorescence is also seen. Further, anti-PBR immunostaining
of MDA-231 cells supports the nuclear localization of the receptor
seen with the fluorescent compound 4 (data not shown).
TABLE-US-00003 TABLE 3 PBR-binding Characteristics of MDA-231 and
MCF-7 Nuclei PK11195 K.sub.D B.sub.max Cell Line (nM) (pmol/mg protein)
MDA-231 10.3 .+-. 8.4 6.9 .+-. 4,8 MCF-7 7.6 .+-. 4.6 0.4 .+-. 0.2
Intact nuclei were isolated from MDA-231 and MCF-7 cells. Ligand
binding studies were performed and analysed as described in Table
2.
EXAMPLE 2
PBR Found in the MDA-231 Human Breast Cancer Cell Line is Similar
to PBR Found in Other Human Tissues
Given the numerous differences between both the expression and
localization of PBR in MDA-231 cells and the other human breast
cancer cell lines studied, as well as previous published reports,
it became important to determine if we were dealing with the same
receptor. The first step towards this end was to establish a pharmacological
profile for MDA-231 PBR. Displacement of [.sup.3H] PK11195 by increasing
concentrations of various PBR ligands is similar to the pharmacological
profile previously reported for human PBR (FIG. 5) (Chang et al.
(1992), supra). Next we obtained partial PBR cDNA sequences for
both MDA-231 and MCF-7 PBR. The nucleotide sequences obtained revealed
several point mutations resulting in two amino acid replacements
replacing alanine 147 with a threonine and a replacing of histidine
162 with arginine in both MDA-231 and MCF-7. Given that this mutation
occurs in both cell lines it is unsure what role it plays in cancer
pathogenesis. Despite many efforts, a sequence could not be obtained
for the region immediately surrounding the translation start sight.
The region 5' to the start sight may provide key evidence for the
differential localization (cytoplasmic versus nuclear) of PBR between
these two cell lines.
EXAMPLE 3
A Functional Role for PBR in Human Breast Cancer
Previous studies from this laboratory have shown that PBR plays
a key role in steroidogenesis by mediating the translocation of
cholesterol from the outer mitochondrial membrane to the inner mitochondrial
membrane (Krueger and Papadopoulos, 1990, supra). More recently,
we have shown that PBR mediates cholesterol uptake even in non-mitochondrial
membranes (Papadopoulos et al., 1997, supra). To test whether or
not PBR may play a similar role in MDA-231 nuclear membranes, intact
nuclei were isolated from both MDA-231 and MCF-7 cells. Isolated
nuclei were incubated with 100 nM [.sup.3H] cholesterol in the absence
or presence of increasing concentrations of PK11195 (FIG. 6). MDA-231
nuclei demonstrated the ability to uptake 30% more cholesterol relative
to MCF-7 nuclei. In MDA-231 nuclei, -8 to -6M PK11195 resulted in
roughly a 20% decrease in the amount of cholesterol uptake, levels
comparable to both stimulated and unstimulated MCF-7 cholesterol
uptake. MCF-7 nuclei failed to respond to the PK11195 dose-response
(FIG. 6).
Numerous studies performed in the early 1980's showed that Ro5-4864
and PK11195, specific PBR ligands, regulate cell proliferation in
a number of cancer models [Clarke and Ryan (1980) Nature 287: 160-161;
Wang (1984) PNAS U.S.A. 81:753-756; Laird (1989) Eur. J. Pharm.
171:25-35; Ikezaki and Black (1990) Cancer Letters 49:115-120; Bruce
(1991) Brain Res. 564:167-170; Garnier et al. (1993) Endocrinology
132:444-458; Camins (1995) Eur. J. Phar. 272: 289-292; Neary (1995)
Brain Research 675:27-30). Using the Bromodeoxyuridine (BrdU) Cell
Proliferation ELISA (Boehringer-Mannheim, Indianapolis, Ind.), we
examined the effects of PK11195 on MDA-231 cell proliferation (FIG.
7). After 24 h, low nanomolar PK11195 (-10 and -9M) showed no effect
on MDA-231 cell proliferation. However, -8M PK11195 stimulated MDA-231
cell proliferation between 20% to 25%, an increase similar to earlier
reports (Ikezaki and Black, 1990, supra). Stimulation of MDA-231
cell proliferation was maximal (40%) at -5M PK11195. After 48 h,
the dose-response curve shifted to the left (data not shown). Cell
proliferation was stimulated 40% by -8M PK 11195, although no stimulation
was seen at any of the micromolar concentrations.
EXAMPLE 3
A Decrease in Cholesterol Uptake Into MDA-231 Nuclei Correlates
With an Increase in Cell Proliferation
We have shown that PK11195 inhibits the uptake of cholesterol into
the nucleus at nanomolar and low micromolar concentrations. We have
also shown that PK11195 also stimulates cell proliferation at these
concentrations. We were then interested in determining whether or
not the regulation of nuclear cholesterol uptake correlates with
the PBR-mediated regulation of cell proliferation. In order to determine
such a relationship, all of the cholesterol data for given concentrations
of PK11195 was plotted against all of the proliferation data at
the same PK11195 concentrations. A regression line for all points
gave a coefficient of correlation (r) of 0.75. Considering that
-4M PK11195 is a toxic concentration, removal of the data from -4M
PK11195 yields a coefficient of correlation (r) of 0.99 (FIG. 8).
EXAMPLE 4
MDA-231 Cells Express DBI, the Endogenous PBR Ligand
Given the ability of exogenous PBR ligands to regulate nuclear
cholesterol uptake and cell proliferation in MDA-231 cells, we then
examined whether or not MDA-231 cells express the endogenous PBR
ligand the polypeptide diazepam binding inhibitor (DBI). The presence
of DBI in an aggressive human breast cancer cell line would give
support to the hypothesis that PBR is involved in the advancement
of human breast cancer. Indeed, immunocytochemistry of MDA-231 cells
with anti-DBI antiserum reveals that this cell line possesses cytoplasmic
DBI (data not shown).
EXAMPLE 5
Localization of PBR in Human Breast Tissue Biopsies From Normal
Tissue
Paraffin embedded sections of normal breast tissue were immunostained
with an anti-PBR antiserum at 1:500 dilution and counterstained
with hematoxylin as previously described [Oke, B. O. et al. (1992)
Mol. Cel. Endocr. 87: R1-R6; Garnier, 1993, supra]. Please note
the distinct localization of PBR in the cytoplasm of the epithelial
cells of normal human breast ducts (a). Obviously there is a low
level of expression of PBR. In some samples, the hematoxylin counterstaining
was omitted in order to examine whether the nucleus of the cells
contained immunoreactive PBR protein (b). FIG. 9c shows also the
localization of PBR in normal breast tissue cells. In this experiment
an FITC-coupled secondary antibody was used to localize the immunoreactive
PBR protein. FIG. 9d shows the phase contrast of the same tissue
area. PBR ligand binding activity was determined using the fluorescent
PBR derivative compound 4 [Munson, 1980, supra] (FIG. 9e). Ligand
binding activity could be detected in the cytoplasm of the cells
and at low levels, in agreement with the protein localization studies.
Use of 1000 fold excess of the competitive ligand PK 11195 completely
displaced the fluorescence, demonstrating the specificity of the
labeling.
EXAMPLE 6
Nuclear Localization of High Levels of PBR in Human Breast Tissue
Biopsies From Invasive/Metastatic Carcinomas
Histologically breast carcinomas are classified into ductal and
lobular types. Each type is further divided into in situ, invasive
and aggressive these being the metastatic form of the cancer. All
biopsies were obtained from the Lombardi Cancer Center at Georgetown
University Medical Center. Biopsies were histologically characterized
by the pathologist. In order to determine whether the results obtained
using the invasive and aggressive human breast cancer cell lines
are not an artifact of the cell culture system we used biopsies
from in situ, invasive and aggressive breast carcinomas. FIG. 10
shows PBR expression and localization in aggressive carcinomas.
Please note the distinct localization of PBR in the nucleus and
the perinuclear area of the aggressive tumor cells (a). In some
samples, the hematoxylin counterstaining was omitted in order to
confirm the PBR positive staining of the nuclei of breast carcinoma
cells (b). FIG. 11c also shows the localization of PBR in normal
breast tissue cells. In this experiment an FITC-coupled secondary
antibody was used to localize the immunoreactive PBR protein. A
strong nuclear immunostaining could be observed. FIG. 10d shows
the phase contrast of the same tissue area. PBR ligand binding activity
was also determined in the aggressive breast carcinomas using the
fluorescent PBR derivative compound 4 (FIG. 10e). Strong ligand
binding activity could be detected in the nucleus of the cells,
in agreement with the protein localization studies. Use of 1000
fold excess of the competitive ligand PK 11195 completely displaced
the fluorescence, demonstrating the specificity of the labeling.
It should be noted that data from in situ and invasive breast carcinoma
closely resembles the data obtained using the normal breast tissue.
These findings clearly indicate that increase expression of PBR
and nuclear localization is a characteristic of the aggressive phenotype
of the tumor. Invasive breast tumors invade and grow locally but
they do not metastasize. However, the aggressive tumors have the
ability to invade and metastasize through the blood vessels to different
places of the human body. Tumor metastasis into vital organs (such
as lungs) is the most common cause of death.
EXAMPLE 7
Inhibition of PBR Expression Results in Reduced Rate of Cell Proliferation
of Tumor Cells
To evaluate the role of PBR in cell function, we developed a molecular
approach based on the disruption of PBR gene, by homologous recombination,
in the constitutive steroid producing R2C rat Leydig tumor cell
line [Papadopoulos, V. et al. (1997) J. Biol. Chem. 272:32129-32135].
Inactivation of one allele of the PBR gene resulted in the suppression
of PBR mRNA and ligand binding expression. Immunoblot and electron
expression of PBR and nuclear localization is a characteristic of
the aggressive phenotype of the tumor. Invasive breast tumors invade
and grow locally but they do not metastasize microscopic immunogold
labeling analyses confirmed the absence of the 18 kDa PBR protein
in the selected mutant clones. The rate of cell proliferation was
determined using the MTT proliferation assay (Boehringer Mannheim).
FIG. 11 clearly shows that the rate of cell proliferation in the
PBR mutant cell was reduced compared to the wild type cell suggesting
a role of the receptor in cell proliferation.
DISCUSSION
In this report, we examined the role of PBR in human breast cancer
through a model system comprising a series of human breast cancer
cell lines. Through the course of this study we describe a strong
correlation between the expression of PBR ligand binding activity
and the invasive and chemotactic potential, as well as the expression
of the breast cancer marker CD44, among the cell lines. Further,
we show that PBR is differentially localized between highly aggressive
and non-aggressive cell lines. Characterization of breast cancer
PBR reveals that it is similar to the PBR studied in other human
tissues with the exception of several point mutations that lead
to the replacement of an alanine residue at position 147 with a
threonine residue and a replacement of histidine 162 with arginine.
Functionally, we find that PBR is responsible for the increased
uptake of cholesterol by the nuclei of a highly aggressive cell
line, MDA-231, relative to a non-aggressive cell line, MCF-7. Also,
we find that PBR regulates cell proliferation of MDA-231 and, moreover,
that this regulation is strongly linked to the ability of PBR to
regulate cholesterol uptake into MDA-231 nuclei. The fact that nanomolar
and low micromolar concentrations, and not high micromolar concentrations,
of PK11195 are responsible for both of these actions indicates that
these events are the result of specific interactions, of the drugs
used, with PBR and not some non-specific activity of the ligand.
The expression of PBR protein levels in the model system studied
in this paper mirrors that seen in other human cancer studies. Cornu
et al (1992, Acta Neurochir. 119:146-152) have shown that PBR site
densities are as much as 12-fold higher in high grade astrocytomas
and glioblastomas relative to normal brain tissue. A study by Miettinen
et al (1995, supra) also indicates that PBR is highly upregulated
in high grade human astrocytic tumors relative to low grade tumors.
Further, a Positron Emission Tomography study by Pappata et al (1991,
J. Nuclear Med. 32:1608-1610) revealed that binding of PK11195,
the PBR-specific ligand utilized throughout the current study, is
two-fold greater in glioblastomas than in normal human gray matter.
Our data supports these previous studies by showing that PBR binding
in MDA-231 cells is approximately seven-fold higher than the mildly
aggressive ADR cell line and infinitely greater than in the non-aggressive
MCF-7 cell line.
At the transcriptional level, however, this correlation does not
appear to be as tight. While MDA-231 cells express 17 to 20-fold
higher PBR cDNA than MCF-7 cells, PBR cDNA expression is almost
1.5-fold greater in the ADR cell line compared to MDA-231 cells.
This result appears to be anomalous, however, considering that ADR
cells apparently localize PBR to the cytoplasm and the nuclear envelope,
it may represent a transition phase between the non-aggressive state
to a more aggressive state in the context of the battery of human
breast cancer cell lines examined in this paper. It is difficult
to rectify, however, because little is known about the regulation
of PBR expression.
Partial sequence analysis revealed that a point mutation in both
MDA-231 and MCF-7 cells results in the replacement of alanine 147
with a threonine residue. Molecular modeling of the receptor indicates
that this residue lies within the cholesterol pore region of the
receptor (Papadopoulos, 1997, supra). Currently, it is not apparent
whether or not this mutation has a resulting phenotype. It appears
that it does not alter the ability of cholesterol to move through
the pore since cholesterol is incorporated into MDA-231 nuclei.
The fact that it is present in both the MDA-231, a highly aggressive
breast cancer cell line, and in MCF-7, a non-aggressive cell line,
indicates that this mutation may represent an early event in the
progression of this disease.
PBR is primarily targeted to the outer mitochondrial membrane in
tissues in which it is expressed in great abundance (Papadopoulos,
1993, supra). It has also been found, however, in other cellular
organelles such as the plasma membrane as well as the peroxisome
[Papadopoulos, 1993, supra; Woods et al. (1996) Biochemical Pharmacol.
51: 1283-1292]. The lack of a distinct mitochondrial target sequence
and the largely hydrophobic nature of PBR makes it feasible that
PBR could exist in a variety of membranes. Differential localization
of PBR may also be possible through the existence of chaperone proteins
and PBR-associated proteins that may direct PBR to the membranes
of specific organelles and may influence PBR's functioning [Papadopoulos,
V. (1998) Proc. Exp. Biol. Med.217: 130-142]. The significance of
such differential localization, however, has not been investigated
and is currently unknown. It will be necessary to distinguish whether
the nuclear localization of PBR in MDA-231 cells is the result of
a specific amino acid sequence present in the yet undetermined amino-terminus
of the protein or the shuttling of PBR to the nucleus via association
with another protein.
The data presented in this application suggests that nuclear PBR
is responsible for regulating movement of cholesterol into the nuclear
membrane and that this regulation is related to its modulation of
cell proliferation. Cholesterol is a major lipid component of every
membrane and has been suggested to play a role in the advancement
of a variety of pathologies including breast cancer [Coleman et
al. (1997) In: Cholesterol: Its Functions and Metabolism in Biology
and Medicine. R. Bittman (Ed.). Plenum Press, New York, pp. 363-435;
Kokogleu et al. (1994) Cancer Letters 82: 175-178]. Further, reports
on animal dietary, cellular, and enzyme-specific studies implicate
a role for cellular cholesterol in the regulation of cell proliferation
(Coleman, 1997, supra). Cholesterol has been shown to tightly regulate
the activity of the sterol regulatory element binding proteins (SREBP)
found in the nuclear membrane and the endoplasmic reticulum [Brown
and Goldstein, 1997) Cell 89:331-340]. In the presence of excessive
cholesterol, premature SREBP is not fully cleaved and, therefore,
the mature form is not released and cannot enter the nucleus to
carry out transcription (Brown and Goldstein, 1997, supra). SREBPs
are responsible for the transcriptional regulation of the enzymes
involved in the cholesterol biosynthetic pathway as well as the
enzymes involved in fatty acid synthesis and uptake (Brown and Goldstein,
1997, supra). One possible outcome of concentrating cellular cholesterol
to the nuclear membrane may be to inhibit the activation of nuclear
membrane SREBPs. With the tight correlation between nuclear uptake
of cholesterol in MDA-231 and PBR's regulation of MDA-231 cell proliferation,
the SREBP pathway may shed some light as to how PBR is regulating
cell proliferation in these cells and should be the target of future
research in this area.
It is distinctly possible that the correlation between PBR expression
and aggressive phenotype, as well as the nuclear localization of
PBR in a highly aggressive breast cancer cell line, may be due to
the overall differential metabolism and cellular activity between
the cell lines studied. The functionality of PBR in the MDA-231
cell line, namely the ability to regulate both nuclear cholesterol
uptake and cell proliferation, as well as the strong correlation
between these two seemingly separate events, suggests that indeed
PBR is playing a role in the progression of breast malignancies.
The presence of the putative endogenous PBR ligand, DBI in the cytoplasm
of MDA-231 cells further suggests the likelihood that PBR is fully
functional in these cells.
Malignant breast tumors are primarily characterized by aberrant
cell proliferation, tumor invasion and metastasis. Several molecular
and cellular mechanisms have been proposed to account for these
phenomena and a number of prognostic indicators have been identified.
While these markers have been useful in helping clinicians develop
prognoses, they have failed to provide adequate enough information
about the mechanisms responsible for tumor malignancy so that effective
anti-cancer therapies may be developed. Given the data presented
in this report, we believe that PBR is a major component of the
progression of breast cancer. While a great deal more needs to be
learned about PBR and its ability to regulate cell proliferation
and cholesterol movement, we believe it is a major step in understanding
this disease. Our data as well as previous studies implies that
PBR may serve well as a prognostic marker indicating that higher
levels of PBR in cancerous tissues implying advancement of disease.
Further, a great number of PBR ligands are known, including benzodiazepines
and isoquinoline carboxamides, whose PBR-binding and pharmacological
characteristics are well documented. Many of these ligands have
been shown to act either agonists or as antagonists to PBR action
and may be potential targets for anti-cancer therapies. In addition,
the availability of radiolabeled and fluorescent ligands may be
useful in the diagnosis and prognosis of the disease. |