Note: Descriptions are shown in the official language in which they were submitted.
2140814
Novel use of Calreticulin in Modulating Hormone Responsiveness and
New Pharmaceuticals for Treating Cancer, Osteoporosis and Chronic
Inflammatory Disease
s BACKGROUND OF THE INVENTION
This invention relates to isolated and purified proteins, such as
calreticulin and mimetics of calreticulin, for a novel use of modulating
hormone responsiveness. These proteins are useful in gene therapy and in
manufacturing pharmaceuticals for treating a variety of diseases, including
10 cancer, osteoporosis and chronic inflammatory disease. The proteins include
or bind to an amino acid sequence KXFFYR, wherein X is either G, A or V
and Y is either K or R. This sequence is present in the DNA-binding domain,
and is critical for the DNA binding activity, of a variety of hormone receptors,including glucocorticoid receptor, minerolcorticoid receptor, androgen
receptor, progesterone receptor, estrogen receptor, retinoic acid receptor,
thyroid hormone receptor and vitamin D receptor. Proteins which bind to this
sequence may inhibit hormone receptor induced gene transcription. Proteins
which include this sequence may promote hormone receptor induced gene
transcription. The invention includes isolated DNA molecules for these
20 proteins, methods of treating diseases using these proteins, synthetic
peptides and their mimetics, and kits containing these proteins, synthetic
peptides or their mimetics.
The physiology of many organs in mammals is regulated by hormones.
These hormones include steroid hormones, thyroid hormones, metabolites of
25 vitamins, such as all trans retinoic acid, 9-cis retinoic acid, vitamin D and its
metabolite 1,25 dihydroxyvitamin D3. These hormones are proteins and bind to
intracellular receptors which regulate expression of genes (O'Malley, 1990).
There are a variety of receptors which respond to hormones.
Osteoblasts and osteoclasts respond to steroid hormones, vitamin D and
30 retinoic acid. Mammary epithelial cells and breast carcinoma cells respond to estrogens, progesterone, retinoic acid and glucocorticoids. Lymphocytes
respond to glucocorticoids.
21~0814
The response of receptors to hormones is particularly important in the
development of a number of diseases, including cancer, osteoporosis and
chronic inflammatory disease. For example, the vitamin D receptor is strongly
implicated in the evolution of osteoporosis (Morrison et al., 1994).
The hormone receptor family is called the nuclear hormone receptor
family and consists not only of receptors whose ligands are known, but also of
an increasing number of orphan receptors whose ligands are unknown
(O'Malley, 1990).
The nuclear hormone receptors can be divided into several domains
o which include the hormone (ligand) binding domain, the DNA-binding domain
and the transactivation domain (O'Malley, 1990). The DNA-binding domain
consists of two zinc fingers and is responsible for the receptor's binding to the
DNA response elements which are found in the promoter and enhancer regions
of the genes whose expression are regulated by these receptors. Once a
hormone binds to its receptor, the receptor binds to the DNA thereby inducing
gene transcription.
Proteins which modulate hormone receptor induced gene transcription
are poorly understood. Such proteins are present in the nucleus of the cell and
inhibit or promote the binding of a hormone to its receptor.
To help design pharmaceuticals and therapies for certain diseases, one
must understand the function of certain intracellular proteins and their role inmodulating hormone responsiveness. Isolation and purification of these
proteins would help in assessing whether they inhibit or promote hormone
receptor induced gene transcription. Once such proteins are isolated,
manipulation of such proteins could further inhibit or promote hormone
receptor induced gene transcription. Synthetic peptides which bind to such
proteins could be used to promote hormone receptor induced gene
transcription. Pharmaceuticals including such peptides or their mimetics
could be used to inhibit hormone receptor induced gene transcription. Gene
therapy could be used to inhibit or promote hormone receptor induced gene
transcription.
2~4081~
A need exists to identify amino acid sequences that are conserved in
hormone receptors, so that particular peptides and proteins may be designed
and used in modulating hormone responsiveness. This would lead to
improved methods of treating a variety of diseases, disorders and abnormal
5 physical states in a mammals by regulating hormone receptor induced gene
transcription in mammalian cells.
Calreticulin has been considered to be a resident protein of the
endoplasmic reticulum of a cell, where it is thought to behave as a calcium
binding protein due to its high capacity calcium binding properties (Michalak efal., 1992). It has been suspected that calreticulin is also present in the nucleus
of a cell (Opas etal., 1991), and it has been shown to have a consensus
nuclear localization sequence (Michalak, 1992) which is highly homologous to
that of histone proteins. However, before this invention, its presence in the
nucleus was unconfirmed and its function in the nucleus was unknown.
SUMMARY OF THE INVENTION
This invention relates to an isolated and purified product for use in
modulating hormone responsiveness.
In one case, the product for modulating hormone responsiveness is
20 calreticulin which inhibits hormone receptor induced gene transcription. In
another case, the product is a mimetic of calreticulin. The product binds to
the amino acid sequence KXFFYR, wherein X is G, A or V and wherein Y is K
or R.
In another case, the product for modulating hormone responsiveness
25 iS an antibody to calreticulin or a short peptide. Such an antibody or peptide
could promote hormone induced gene transcription by inhibiting calreticulin-
hormone receptor interactions. The peptide may be one selected from a
group consisting of: KGFFRR, KVFFKR, KAFFKR, KGFFKR, TGFFKR, or
modified derivatives of these peptides.
The invention described in this patent application includes an isolated
DNA molecule encoding an amino acid sequence for use in modulating
2140814
hormone responsiveness. The isolated DNA molecule may encode the
amino acid sequence for calreticulin. It may encode the amino acid sequence
for part of a mimetic of calreticulin. It may encode a first amino acid
sequence that binds to a second amino acid sequence KXFFYR, wherein X is
5 G, A or V and wherein Y is K or R.
The invention described in this patent application includes a method of
treating a disease, disorder or abnormal physical state in a mammal by
regulating hormone receptor induced gene transcription in a cell. The method
could include regulating the activity, quantity or stability of a protein for use in
hormone receptor induced gene transcription. The protein could be one that
includes or binds to the amino acid sequence KXFFYR, wherein X is G, A or
V and wherein Y is K or R. One protein which binds to such sequence is
calreticulin. The hormone receptor could be one selected from a group
consisting of: glucocorticoid receptor, minerolcorticoid receptor, androgen
receptor, progesterone receptor, estrogen receptor, retinoic acid receptor,
thyroid hormone receptor, vitamin D receptor and orphan receptors. The
disease or disorder could be one selected from a group consisting of breast
cancer, prostate cancer, promyelocytic leukemia, solid tumors, chronic
inflammatory disease, such as arthritis and osteoporosis.
The method of treating the disease could include administering to the
mammal a pharmaceutical comprising the protein, or an organic mimetic and
a carrier. A suitable carrier could be a lipid vesicle. As an alternative, the
method could include decreasing or eliminating the quantity of calreticulin
present in the cell; or decreasing the stability of calreticulin present in a cell.
The invention described in this patent application includes a kit
containing a pharmaceutical comprising a protein for use in modulating
hormone responsiveness together with a carrier. The protein included within
the kit would be one that binds to the amino acid sequence KXFFYR, wherein
X is G, A or V and wherein Y is K or R. Such a protein would include
30 calreticulin or a mimetic of calreticulin.
21~08Ig
DEFINITIONS
In this application, the following terms have the following meanings,
unless the context requires otherwise:
"A" means adenine
s "Binds" means that under given conditions of ionic strength and
temperature, a particular product binds to a substrate
"EDTA" means ethylenediaminetetraacetic acid
"EGF" means Epidermal growth factor
"ELISA" means enzyme-linked immunosorbent-assay
"F" means phenylalanine
"FGF" means Fibroblast growth factor
"G" means glysine
"HPLC" means high performance liquid chromatography
"IGF" means insulin-like growth factor
15 "IL-6" means Interleukin 6
"K" means Iysine
"KXFFYR" means an amino acid sequence, wherein X is G, A or V and
wherein Y is K or R
"p60" means a 60 kDA protein, calreticulin
20 "PAGE" means polyacrylamide gel electrophoresis
"Peptide" includes amino acids, peptides, polypeptides and proteins
"R" means arginine
"T" means Threonine
"TGF-B" means Transforming growth factor-
25 "V" means valine
21~U81~
DESCRIPTION OF DRAWINGS
FIGURE 1A shows the isolation of p60 (calreticulin) from nuclei by
afffinity chromatography on KLGFFKR-sepharose.
FIGURE 1 B shows immunofluorescent confocal images of TE-85
human osteosarcoma cell nuclei stained with an antibody
against calreticulin.
FIGURE 2A shows that preincubation of purified p60 (calreticulin) with
the recombinant receptor resulted in a dose-dependent
inhibition in the formation of the complex between the
receptor and the DNA.
B shows that recombinant calreticulin inhibits the binding of
the androgen-receptor to its response element.
C shows that co-transfection of the calreticulin-containing
plasmid resulted in a dose-dependent inhibition of
chloramphenicol acetyltransferase activity induced by the
androgen receptor.
FIGURE 3A shows that overexpression of calreticulin by calreticulin
cDNA transfection in p19EC cells dramatically
suppressed neuronal differentiation, as judged by the
expression of a specific early marker of neuronal
differentiation class lll ~-tubulin.
B shows that overexpression of calreticulin by calreticulin
cDNA transfection in p19EC cells dramatically
suppressed neuronal differentiation, as judged by the
expression of a specific early marker of neuronal
differentiation class lll ~-tubulin.
C shows the modulation of neuronal differentiation of P19EC
cells by different levels of expression of calreticulin: (D)
shows the increased levels of calreticulin inhibit neuronal
differentiation. (F) shows the decreased levels of
calreticulin enhance neuronal differentiation.
~IgO814
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
A highly homologous amino acid sequence, KXFFYR (where X is either
G, A or V and where Y is either K or R), has been found to be present in the
DNA binding domain of all known members of the steroid hormone receptor
5 family (Fuller, 1991), and amino acids in this sequence make direct contact with
nucleotides in their DNA responsive elements, and are crucial for DNA binding
(Luisi, 1991).-
By way of example, the amino acid sequence of the DNA bindingdomain of RAR is set out below:
C Y H i C Y H
S Y ~ ' . SS C
K V KD sv
D S Q A
V Zn DR Zn Q
F / \ G TH C C
K G F F R R S I Q K _ _ N MV Y EV G MS K
RXR K G F F K R
T3R~ K G F F R R
VDR K G F F R R
GR K V F F K R
MR K V F F K R
AR K V F F K R
ER K A F F K R
X G F F K R: Calreticulin Binding SequencP in Integrin a Subunits.
Naturally occurring and recombinant calreticulin, inhibit the binding of
25 receptors to DNA. Thus, calreticulin and proteins which mimic calreticulin
modulate nuclear hormone receptor regulation of gene transcription.
Calreticulin binds to nuclear hormone receptors by interacting with the
amino acid sequence KXFFYR. The interaction results in a profound inhibition
of nuclear hormone receptor DNA binding activity which can be reversed by
30 soluble competing synthetic peptides with the generic sequence KXFFYR .
The inhibition of DNA binding by calreticulin can also be reversed by an
antibody to calreticulin. Transient or stable overexpression of calreticulin by
08I I
cDNA transfection also results in the inhibition of nuclear hormone receptor
induced gene transcriptional activity. Furthermore, decreased expression of
calreticulin by stable transfection of antisense calreticulin cDNA results in
increased sensitivity of the cells to hormones due to the increased
5 transcriptional activity of the nuclear hormone receptor.
Hence, a proportion of nuclear hormone receptors may be occupied by
calreticulin in a constitutive manner, and decreased regulation of expression ofcalreticulin may therefore result in an effective increase in the number of
unoccupied receptors leading to increased transcriptional activity of these
receptors.
By this invention, hormonal sensitivity can be manipulated by (i)
increasing or decreasing the intracellular concentration of calreticulin, or (ii) by
inhibiting the interaction of calreticulin with nuclear hormone receptors by
peptides, peptide mimetics, and antibodies against calreticulin or the KXFFYR
15 sequence.
The nuclear hormone receptors that interact with calreticulin include
androgen receptor, retinoic acid receptors (RAR and RXR), glucocorticoid
receptor, and the vitamin D receptor. In all of these cases, calreticulin inhibits
receptor binding to DNA, and overexpression of calreticulin results in an
20 inhibition of receptor mediated transcriptional activity. In the case of the retinoic
acid receptor system, the decreased regulation of expression of calreticulin
results in an increased sensitivity of the cells to differentiation by retinoic acid.
For the following examples: cell lines were obtained from American
Type Culture Collection - ATCC; Chemical reagents were purchased from
25 Sigma Chemicals, St. Louis, MO; BioRad, Richmond, CA; and Amersham
Corp., Oakville, ON; Radioisotopes were purchased from Amersham Corp.,
Oakville, ON; Peptides were synthesized by HSC/Pharmacia Biotechnology
Service and Department of Clinical Biochemistry, University of Toronto;
Oligonucleotides were synthesized by University of Toronto - Carbohydrate
30 Research Group; Centrifuges used were from Beckman or Eppendorf.
, 21~U814
-
Example 1 Calreticulin is Present in Nucleus of Cells
The conservation of the KXGFFYR sequence in the a-subunits of
integrins is shown in Table 1.
A computer search of the Swiss protein data bank for the presence of
this sequence motif in other proteins revealed that a highly homologous
sequence is present in the DNA binding domain of all members of the nuclear
hormone receptors (Table 1) (Fuller, 1991; Carson-Jurica,etal., 1990).
Because amino acids in this motif have been demonstrated to be essential for
the binding of nuclear hormone receptors to their DNA responsive elements
o (Luisi et al., 1991; Haird et al., 1990), we wanted to determine whether a 60
kDa protein isolated by affinity chromatography on a KLGFFKR-sepharose
affnity matrix (Rojiani et al., 1991) could modulate DNA binding and
transcriptional activities of nuclear hormone receptors.
Table I
Conservation of an Amino Acid Sequence Motif in the Integrin Alpha-
subunit Cytoplasmic Domains and in the Steroid Hormone Receptor
Family
Integrins* Steroid Nuclear Receptors
a1 K I G F F K R RARa A C E G C KGFFRR S I Q K
a2 KLGFFKR T3Rb TCEGCKGFFRRTIQK
a3 KCGFFKR VDR TCEGCKGFFRRSMKR
a4 K A G F F K R GR T C G S C KVFFKR A V E G
a5 KLGFFKR MR TCGSCKVFFKRAVEG
a6(A) KCGFFKR AR TCGSCKVFFKRAAAG
a6(B) KCGFFKR PR TCGSCKVFFKRAMEG
a7 KLGFFKR ER SCEGCKAFFKRSIQG
a8 KCGFFDR RXR SCEGCKGFFKRTVRK
(chick)
av RMGFFKR Steroid TCEGCTGFFKRSIRK
receptor TR2
Mac-1 KLGFFKR Nerve TCEGCKGFFKRTVQK
growth factor
214~814
induced
protein 1-B
p150 KVGFFKR Early TCEGCKGFFKRTVQK
response
protein
NAK1
PS2 KCGFFNR Chorion SCEGCKGFFKRTVRK
(Drosop Factor I
hila)
In Table 1, the sequences indicated with an asterisk were obtained as
described in Rojiani etal, 1991. GR: Glucorticoid receptor; MR:
Minerolcorticoid receptor; AR: Androgen receptor; PR: Progesterone receptor;
5 ER: Estrogen receptor.
Although calreticulin contains a KDEL motif at its C-terminus and is
therefore thought to be resident in the endoplasmic reticulum (McCauliffe et al.,
1990; Fliegel et al., 1989; Michalak etal., 1992), it also has a nuclear targeting
signal (McCauliffe et al., 1990; Michalak etal., 1992; Marzluff et al., 1985),
10 raising the possibility that this protein is also present in the nucleus (Michalak et
al., 1992). The presence of p60 in nuclei was demonstrated by affinity
chromatography of human osteosarcoma cell (HOS) nuclear extracts on a
KLGFFKR-affinity column (FIGURE 1).
Nuclei were purified from HOS cells by established methods (Luisi et al.,
15 1991). The purified nuclei were either Iysed in PBS containing 1% Triton X-
100, 0.1 % SDS, 0.5% sodium deoxycholate and 1 mM PMSF, or were applied
to a glass coverslip and stained for nuclear antigen with anti-nuclear
monoclonal antibody MAB1218 obtained from Chemicon Int. Inc., Tamecula,
CA. The nuclei were visualized by indirect immunofluoresence. The total
20 cellular or nuclear extracts were subjected to affinity chromatography on a
KLGFFKR-affinity matrix, and the p60 isolated (Rojiani et al., 1991).
Cell extracts were prepared from whole cells or from purified nuclei and
applied to KLGFFKR-sepharose affinity matrix. Bound proteins were eluted
with EDTA and analyzed by SDS-polyacrylamide gel electrophoresis (Rojiani et
25 al., 1991). The separated proteins were electrophoretically transferred to
- 10-
081~
nitrocellulose filters and probed with an anti-calreticulin antibody. In FIGURE 1,
Lane 1: Total cellular extract; Lane 2: EDTA eluted material from affinity column
to which total cellular extract was applied; Lane 3: Nuclear extract; Lane 4:
EDTA eluted material from affinity column to which nuclear extract was applied.
Arrow indicates the position of p60.
Indirect immunofluoresence of HOS cells or purified nuclei with anti-
calreticulin antibody also demonstrated intranuclear calreticulin expression, asshown in FIGURE 1 B. Confocal microscopy was carried out using a BioRad
MRC 500 system. Note the non-nucleolar, intranuclear staining of the cell in (a)and (b), and the complete exclusion of intranuclear staining in the cell in (c).These data suggest that the expression of calreticulin in the nucleus is a
regulated process.
These results confirmed the presence of a calreticulin-related p60
protein in nuclei.
Example 2 The Sequence KXFFYR is rlese"l in All Known
Members of the Nuclear Receptor Family
As shown in Table 1, the sequence KXFFYR is present in all known
members of the nuclear receptor family. The region containing this sequence
in the DNA-binding domains of these receptors has been shown to play a
crucial role in DNA sequence recognition (Luisi et al., 1991). Thus calreticulin,
by binding to this common sequence, modulates the binding of all members of
the receptor family to DNA. By way of example, we have demonstrated the
inhibition by calreticulin of the interaction of the androgen receptor with its DNA
response element (see Examples 3 and 4) and of the retinoic acid receptor
heterodimer complex (RAR/RXR) with its DNA response element (see Example
5).
Example 3 Ability of Calreticulin to Modulate Binding of Nuclear
Hormone Rec~"tors In Vitro
To determine whether p60 (calreticulin) could directly modulate the
binding of nuclear hormone receptors to DNA via the KXFFYR sequence, the
214081~
interaction of the DNA binding domain of recombinant androgen receptor with
its hormone responsive element was analyzed by carrying out gel mobility shift
assays.
As described in Rennie et al., 1993, DNA binding domain of recombinant
rat androgen receptor was prepared as a GST-fusion protein using the pGEX-
3X vector and purified by glutathione-agarose afffinity chromatography. p60
(calreticulin) was purified by afffinity chromatography on KLGFFKR-sepharose,
followed by gel electrophoresis as described in detail in Rojiani et al., 1991.
Purified AR and p60 (calreticulin) were found to be greater than 90% and 95%
pure, respectively, as determined by SDS-PAGE and Coomassie Blue staining.
Recombinant calreticulin (GST-fusion protein) was prepared as described by
Baksh and Michalak, 1991. Gel retardation assays were carried out as
described by Rennie et al., 1993. To analyze the effect of p60, or recombinant
calreticulin, on receptor-DNA binding activity, the AR was pre-incubated with
p60 for 30 min at 4C. To analyze the effects of synthetic peptides, anti-
calreticulin antibody, and non-immune IgG on p60 inhibition of AR-ARE binding,
the peptide, antibody or IgG were pre-incubated with p60 for 30 min at 4C.
The androgen receptor preparation was then added to these mixtures and
further incubated for 30 min at 4C.
Afffinity purified DNA binding domain of the recombinant rat androgen
receptor (AR) was pre-incubated with or without the indicated concentrations of
purified p60 at 4C for 30 min. After this pre-incubation the reaction mixtures
were incubated with 32P-labeled 26 base pair ARE (Rennie et al., 1993)
(androgen response element), and analyzed by gel retardation assay. The
sequence of the ARE used was:
5' GTAAAGTACTCCAAGAACCTATTTgt 3'
3' CATTTCATGAGGTTCTTGGATAAAca 5'
In FIGURE 2A, the following lanes show the following results: Lane
132P-labeled ARE by itself; Lane 2: Retardation of ARE by AR; Lane 3: Effect of
pre-incubation of 0.11 mg of purified p60 with AR on AR-ARE binding; Lane 4:
Effect of the addition of a 25-fold molar excess of KLGFFKR synthetic peptide
to p60 on AR-ARE binding. Lanes 3, 5, 7, & 9: Effect of increasing
2I~081g
-
concentrations of p60 (from 0.11 mg to 0.33 mg) on AR-ARE binding; Lanes 4,
6, 8, & 10: Reversal of p60 inhibition of AR-ARE binding by KLGFFKR peptide.
Lane 11: Effect of addition of anti-calreticulin antibody to p60 inhibition of AR-
ARE binding. Lanes 12-15: Increasing amounts of p60 in the presence of anti-
calreticulin antibody. Increasing the p60 concentration overcomes the effect of
antibody on p60 inhibition of AR-ARE.
In FIGURE 2B, the following lanes show the following results: Lane 1:
32P-labelled ARE alone; Lane 2: Retardation of ARE by AR in the presence of
gluthathione-S-transferase (GST); Lane 3: Inhibition by GST-calreticulin (GST-
o calreticulin) of AR-ARE interaction; Lanes 4 and 5: Reversal of this inhibition by
KLGFFKR peptide; Lanes 6 and 7: Inability of the scrambled peptide
(KLRFGFK) in reversing the effect of calreticulin on AR-ARE interaction; Lanes
8 and 9: The peptide KVFFKR can also reverse the inhibition by calreticulin of
the AR-ARE interaction. The concentration of calreticulin used was 2 mg and
the peptides were used at a 50-fold molar excess concentration.
As shown in FIGURE 2, the migration of a 32P-labeled 26 base pair DNA
androgen responsive element residing at positions -115 to -140 of the rat
probasin gene promoter (Rennie et al.,1993) was retarded by the androgen
receptor DNA-binding domain; indicating the formation of a complex between
the receptor and the DNA (Rennie et al.,1993). Pre-incubation of purified p60
(calreticulin) with the recombinant receptor resulted in a dose-dependent
inhibition in the formation of this complex (FIGURE 2A, lanes 3, 5, 7, & 9). Thesequence specificity of this inhibition was demonstrated by the finding that theinhibition by p60 (calreticulin) of receptor-DNA binding was reversed by the
addition of competing KLGFFKR peptide (FIGURE 2A, lanes 4, 6, 8, & 10) or
KVFFKR (FIGURE 2B, lanes 8 and 9), whereas a scrambled peptide
(KLRFGFK) was much less effective (FIGURE 2B, lanes 6 and 7). An antibody
to calreticulin, which cross-reacts with p60, also reversed this inhibition by p60
(FIGURE 2A, lane 11), demonstrating p60 specificity. Non-immune IgG did not
have any effect on the inhibition of receptor-DNA interaction by p60 (FIGURE
2A, lane 15). Furthermore, neither KLGFFKR peptide, anti-calreticulin
antibody, nor non-immune IgG by themselves had any effect on the receptor-
- 13-
2140814
-
DNA interaction (data not shown). p60 did not effect the binding of AP-1 to
DNA, and other proteins of similar size (e.g. bovine serum albumin) also did nothave any effect on the nuclear receptor-DNA interaction (data not shown).
Recombinant calreticulin (obtained from Dr. Michalak, Edmonton, Alta)
(Baksh etal., 1991), in the form of a GST-fusion protein, also inhibited the
binding of the androgen-receptor to its response element (FIGURE 2B, lane 2),
and this inhibition was also reversed by KVFFKR peptide, (FIGURE 2B, lane 2),
but not by a scrambled peptide KLRFGFK (FIGURE 2B, lane 1) confirming that
the p60 purified on the KLGFFKR affinity matrix and calreticulin are functionally
similar in terms of binding to nuclear hormone receptors, and that a synthetic
peptide, KVFFKR can competitively inhibit the binding of calreticulin to the
KVFFKR sequence of the androgen receptor.
Example 4 Inhibition of Transcriptional Activity of the Androgen
s Receptor In Vivo.
To determine whether calreticulin also inhibited the transcriptional
activity of the androgen receptor in vivo, expression vectors containing full-
length calreticulin (McCauliffe et al., 1990) and androgen receptor (Rennie et
al., 1993), cDNAs were co-transfected into Vero fibroblasts together with a
chloramphenicol acetyl transferase (CAT) reporter plasmid driven by the mouse
mammary tumor virus (MMTV) long terminal repeat (LTR). MMTV-LTR
contains androgen response elements (Rennie et al., 1993).
FIGURE 2C shows inhibition of androgen-induced CAT activity by
calreticulin. Vero fibroblasts were cotransfected with an MMTV-CAT reporter
vector and various amounts of a calreticulin expression vector and the pRC-
CMV vector alone (Invitrogen) using the calcium phosphate method (Filmus et
al., 1992). In all transfections 10 mg of a ~-galactosidase expression vector
and 10 mg of an androgen receptor expression vector (Seed et al., 1988) were
included. Transfected cells were incubated in medium alone or in the presence
of 100 nM R1881 (synthetic androgen) for 12 h. Cells were then Iysed and
CAT activity measured (Seed et al., 1988). An aliquot of the cell extracts was
also assayed for ~-galactosidase activity. This activity was used to standardize
- 14-
21~0814
the measurement of CAT levels in each experiment by taking into account the
efficiency of the transfection. Every sample was tested in quadruplicate and
the average activity calculated. CAT activity induction as defined as the ratio
between the standardized CAT activity of the R1881 treated cells and the
5 corresponding untreated cultures. The Vero cells were grown in a-minimum
essential medium containing 10% charcoal-treated calf serum.
As shown in FIGURE 2C, co-transfection of the calreticulin containing
plasmid resulted in a dose dependent inhibition of CAT activity induced by the
androgen receptor. Furthermore, immunoprecipitation of calreticulin from 35S-
o methionine/cysteine labeled, androgen receptor transfected Vero cells, resultedin the co-precipitation of the 110 kDa androgen receptor, indicating a direct
interaction between calreticulin and the androgen receptor (S. Dedhar and C.
Leung-Hagesteijn, unpublished observations).
These data demonstrate that not only can calreticulin bind to the
S androgen receptor DNA binding domain and inhibit its interaction with the
androgen response elements in vifro, it can also inhibit the transcriptional
activity of the androgen receptor in vivo. Although other 59 kDa proteins have
been found in complexes with several steroid hormone receptors (Lebeau et
al., 1992; Tai et al., 1992), they are distinct from calreticulin, and none of them
20 have an effect on binding of the receptors to their DNA responsive elements.
Example 5 Regulation by Calreticulin of Hormone Receptor
Induced Gene Transcription
In order to demonstrate a physiological significance of the finding that
25 calreticulin can bind to the DNA binding domain of nuclear hormone receptors
and modulate their transcriptional activity, we utilized a retinoic acid responsive
system i.e. the induction of neuronal differentiation by retinoic acid in P19
embryonal carcinoma cells (McBurney et al., 1982). We predicted that
increased expression of calreticulin would suppress retinoic acid induced
30 neuronal differentiation, whereas decreased expression would result in the
release of calreticulin inhibition, and allow for a more rapid rate of neuronal
differentiation.
- 15-
2140814
The full length 1.9 Kb calreticulin cDNA (McCauliffe et al., 1990) was
obtained from Dr. R.D. Sontheimer, Texas and was subcloned into pRC/CMV
(Invitrogen, San Diego, CA) expression vector in the sense and antisense
orientation. pRC/CMV, pRC/CMV-Cal-1 (sense), or pRC/CMV-Cal-2
(antisense) expression plasmids were then transfected into P19 embryonal
carcinoma cells by electroporation. Neomycin-resistant transfectant cells were
then selected by growth in the presence of 600 mg/ml G418 and the resistant
cells were maintained in 100 mg/ml G418. Cal-1 and Cal-2 transfectants were
subcloned by limiting dilution, and the subclones were screened for calreticulinexpression by Western blot analysis of cell Iysates with an anti-calreticulin
antibody (Rojiani etal., 1991). Retinoic acid neuronal differentiation was
induced as described previously (McBurney et al., 1982; Dedhar et al., 1991)
and class IlI ,B-tubulin expression was analyzed by Western blotting with a class
111,B-tubulin monoclonal antibody (TuJ1). This antibody was obtained from Dr.
A. Frankfurter, University of Virginia, Charlottesville, VA, USA. The bRARE-
luciferase transient transfections in p19 (Neo), Cal-1 and Cal-2 cells were
carried out as described in Tini et al., 1993. The vector bRARE(3) tk-LUC was
constructed by linking 3 copies of the 32 base pair sequence that defines the
RARE upstream from the RAR-b gene (de The, et al., 1990; Sucov et al., 1990)
to the minimal thymidine kinase promoter and the firefly luciferase gene.
The level of calreticulin expression was estimated by Western blot
analysis of cellular Iysates (Rojiani et al.,1991) followed by densitometric
scanning. For Northern blot analysis total cellular RNA (15 mg) from the
indicated cell lines was hybridized to 32-P-labeled CRABP(II) cDNA (Giguere et
al., 1990) at 65C using Rapid Hyb buffer (Amersham Corp.). The blot was
stripped and reprobed with a mouse actin cDNA probe to check for equal
loading of RNA. Values for relative mRNA levels were derived from
quantitation of the signal in each lane using a Molecular Dynamics
Phosphorimager. CRABPII mRNA levels were normalized against the
corresponding actin mRNA signal.
The level of expression of calreticulin was modulated in P19 EC cells by
transfection with calreticulin cDNA inserted in the sense or antisense orientation
- 16-
21~0814
in the pRC/CMV (Invitrogen Corp., San Diego, CA) expression vector. P19 EC
cell subclones overexpressing calreticulin (Cal-1), or anti-sense transfectants
with reduced calreticulin expression (Cal-2), as well as control transfected cells
(Neo), were subjected to induction of neuronal differentiation by retinoic acid as
s described previously (McBurney et al., 1982; Dedhar et al., 1991). The
expression of neuron-specific class IlI ,B-tubulin (Lee etal., 1990; Alexander et
al., 1991) was then analyzed 48 hr (A) or 72 hr (B) after the addition of all-trans
retinoic acid (5 mM). Cal-1 (1A2 and 1D2) clones were transfected with
pRC/CMV containing calreticulin cDNA in the sense orientation. Cal-2 (1A4
10 and 1 B4) clones were transfected with pRC/CMV containing calreticulin cDNA
in the anti-sense orientation. (C): Effect of levels of calreticulin expression on
retinoic acid mediated neuronal differentiation.
Cells were stained with anti-class III ,B tubulin antibody (TuJi) followed by
FITC conjugated secondary antibody as described above. A and B: P19 (neo)
15 EC cells; C and D: P19-Cal-1 EC cells; E and F: P19 Cal-2 EC cells. A, C and
E; untreated cells. B, D and F: 6 days at RA (0.5 ~lM) treated cells. The cells
were visualized using a Zeiss Axioscop microscope under oil immersion and
photographed with Kodak T-Max 400 film. Magnification 100 X.
As shown in FIGURE 3A and B, overexpression of calreticulin (Cal-1), by
20 calreticulin cDNA transfection in P19 EC cells indeed dramatically suppressedneuronal differentiation, as judged by the expression of a specific early markerof neuronal differentiation, class Ill ,B.-tubulin (Lee et al., 1990; Alexander et al.,
1991). In contrast, decreased expression of calreticulin (Cal-2), by anti-sense
calreticulin cDNA transfection, resulted in markedly enhanced expression of
25 class IlI ,Q.-tubulin.
FIGURE 3C clearly shows the inhibition of neuronal differentiation by
calreticulin overexpression and enhanced differentiation by diminished
calreticulin expression.
The effect of calreticulin levels on retinoic acid induced neuronal
30 differentiation occurs via the direct regulation of retinoic acid responsive genes,
as demonstrated by an inverse relationship between calreticulin expression
level and RARE-driven luciferase gene expression (Dedhar et al., 1994).
21~081~
Furthermore, the endogenous regulation of expression of the retinoic acid
responsive genes, CRABPII (Giguere et al., 1990) and RAR-b (de The, et al.,
1990; Sucov et al., 1990) are substantially decreased in Cal-1 transfectants, but
are either unchanged or slightly increased in the calreticulin-antisense Cal-2
s transfectants (Dedhar et al., 1994).
Collectively, these results demonstrate that calreticulin, by binding to the
conserved KXFFYR sequence in the DNA binding domain of nuclear hormone
receptors (Table 1), can modulate gene expression and cellular phenotypes,
such as cell differentiation. Calreticulin may also behave as a signal modifier
o by translocating between the nucleus and the cytoplasm, where it has been
shown to bind, via an identical sequence motif, to the intracellular domains of
the a-subunits of integrin receptors (Rojiani etal., 1991).
Example 6 KLGFFKR Modulates Retinoic Acid Induced Gene
Transcription In Vivo
In order to test whether a peptide based on the calreticulin-binding
KXGFFKR sequence could modulate retinoic acid induced gene transcription in
live cells, the p19 cells were transfected with a reporter vector consisting of a
retinoic acid response element fused to the luciferase gene. Since these cells
20 contain endogenous retinoic acid receptors RAR and RXR, treatment with
retinoic acid results in an induction of the RARE driven luciferase activity (see
Table ll).
Cell culture conditions: Mouse embryonic carcinoma (P19) cell, grown in
60 mm dishes in 7.5% donor calf serum, 2.5% fetal calf serum alpha MEM
25 (Gibco/BRL) were treated with the peptides KLGFFKR or KLRFGFK for three
hours or overnight for 20 hours at 37C, 5% CO2. With few exceptions,
KLXFFKR is the peptide sequence specific within the binding domain of all
steroid receptors. KLRFGFK is the scrambled peptide of the above sequence.
Subsequently each plate was washed four times with serum free alpha MEM to
30 remove excess peptides and replenished with fresh serum containing media.
Cells were then transfected by standard calcium phosphate precipitation
method (Current Protocols in Molecular Biology 9:1 ) with 1 microgram bRARE
- 18-
, 2140814
in pTKluc, 1.5 microgram pRSV bgal, 3 microgram pKS (carrier) per 60 mm
dish. Following a 16 hour incubation at 37C 5% CO2, each plate was washed
two times with serum free alphaMEM and replenished with serum containing
media supplement with 107 M retinoic acid (Sigma R2625) and 800
s microgram/ml G418 (Gibco 1181-031). Following another24 hours incubation
cells were washed three times with PBS and each 60 mm dish of cells was
Iysed in 100 microliters of 1 % triton X100, 100 mM KPO4 pH7.8, 1 mM DTT.
Cell Iysates were stored at -70C. Prior to luciferase/bgal assays, cell debris
were spun out on an Eppendorf microfuge (5415C) at 4C full speed for 20
o minutes.
Luciferase assay: All reagents were equilibrated to room temperature
and each sample was assayed independently. Ten microliters of cell Iysate
was incubated with 50 microliters of luciferase reagent (Promega E1483) in an
Eppendorf tube. Thirty seconds later, the sample was immediately counted in a
15 Beckman scintillation counter (LS60001C) for one minute using an open
window. Standards in the range of 0.001 nanogram to 1.0 nanogram was used
to establish the linearity of the assay.
Beta-gal assay: Assay was done in a microtitre plate (Linbro 76-232-
05). Ten microliters of cell Iysate were mixed with 90 microliters of bgal reagent
20 in 88 mM phosphate buffer,11 mM KCI, 1 mM MgCI2, 55 mM 2ME, 4.4 mM
chlorophenol red b-D-galactopyranoside (BMC 884-308). Incubation period
varied from 30 minutes to 2 hours at 37C. Results were read at 570 nm in an
ELISA reader (Dynatech MR5000).
The pre-incubation of these cells with the specific KLGFFKR peptide
25 resulted in a dose-dependent increase in luciferase activity indicating a
stimulation of the retinoic acid receptor mediated gene transcription (Table ll).
The pre-incubation of a control, scrambled peptide had no such effect. The
transfection efficiency was controlled by co-transfection of the ~-galactiosidase
gene and the subsequent measurement of ,B-galactosidase activity as
30 described above.
These results provide strong evidence that such peptides based on the
KXFFYR sequence can be used to modulate hormone responsiveness by
- 19-
2140814
influencing the binding of calreticulin to the hormone receptors in live cells.
Thus in the experiment described, the KLGFFKR was able to effectively
compete for calreticulin binding with the KGFFRR sequence in the retinoic acid
receptor. This activated the calreticulin-bound receptors resulting in increased5 transcriptional activity.
Table ll
OVERNIGHT INCUBATIONS
Preincubations Conc~ dtions Luciferase x Beta-gal Corrected
micromolar 106 values luc/bgal
KLGFFKR 10 13 0.395 32.91
KLGFFKR 50 20 0.443 45.15
KLGFFKR 100 28 0.452 61.95
KLRFGFK 10 14 0.333 42.04
KLRFGFK 50 16 0.387 41.34
KLRFGFK 100 14 0.434 32.26
Controls
no retinoic acid 0 0.17 0.348 0.49
10-7 retinoic acid 0 8.3 0.323 25.7
Example 7 Peptides Having Differential Specificities for
Disrupting Different Hormone Receptor-Calreticulin
Interactions
To identify such peptides, we utilize gel mobility shift assays (see
s Example 3) in which known concentrations of purified recombinant androgen
receptor, estrogen receptor, retinoic acid receptors (RAR/RXR and RXR/RXR)
(Shago et al., 1994) and vitamin D receptor (Xu et al., 1993) are incubated withknown concentrations of either recombinant calreticulin, or calreticulin purified
by afffinity chromatography on a KLGFFKR afffinity column (see Example 3) in
20 the presence of the respective 32P-labelled DNA response elements and known
concentrations of synthetic peptides based on the KXFFYR sequence. In
addition to the linear peptides, some peptides are cyclized by adding cysteine
- 20 -
" 214081~
-
residues at either ends. These experiments result in the identification of
peptides which have distinct antagonistic specificities for the interaction of
different hormone receptors with calreticulin.
In order to derive peptides which might be specific for one receptor over
s another one, we have undertaken the synthesis of a series of peptides listed in
Table 111.
Table 111
Proposed Peptides
Proposed Peptides Variants Exploring Exploring
ProtectionHydrophobic Patch
RKFFGK Reversed X
d(CKGFFKR) D-aminoacid X
version
FGKKRK another X
scrambled
peptide
Ac-KGFFKR Acetylated X
peptide
KGLFKR X
KGFLKR X
KGYFKR X
KGFYKR X
KGPFKR X
KGFPKR X
KFGFKR Inversion X
KGDFKR X
KGFKDR X
o These peptides are being tested in gel mobility shift assays (described in
Example 3) using equivalent concentrations of various receptors: retinoic acid
receptors (RAR/RXR), vitamin D receptor (VDR), estrogen receptor (ER),
androgen receptor (AR) and glucocorticoid receptor (GR), and their respective
DNA response elements. These experiments identify specific peptides for use
against individual receptors.
Preliminary experiments using the RAR/RXR, RXR/RXR or VDR/RXR
receptors indicate that the KLGFFKR peptide is 1 O-fold more potent against the
X140814
VDR/RXR heterodimer compared to RAR/RXR heterodimer, and is 4-fold morepotent against the RXR/RXR heterodimer compared to the RAR/RXR receptor.
Our data have also identified the amino acids within this sequence which
are crucial for activity as shown in Table IV.
Table IV
Relative Ability Of Peptides To Reverse Calreticulin Inhibition Of Retinoic
Acid Receptors (RARIRXR) Binding To Dna Response Element
Peptide % Reversal
KLGFFKR 1 00%
KLRFGFK 34%
(scrambled
sequence)
GLGFFKR 45%
KLDFFKR 73%
KLGRFKR 24%
KLGFRKR 20%
KLGFFGR 65%
KLGFFKG 22%
The most critical amino acids appear to be F, F, and R in the sequence
KLGFFKR. These three amino acids are completely invariant in all steroid and
nuclear hormone receptors as well as in integrins.
Thus using the RAR/RXR system in gel mobility shift assays, the two
phenyalanines, as well as the terminal arginine, were found to be absolutely
15 essential, since substitution of these resulted in the abrogation of the peptide
activity (Table IV).
The peptides identified from these gel mobility shift assays are being
used in cellular assays described below.
Retinoic acid-fece"tor specific peptides: These are tested in the P19
20 retinoic acid induced neuronal differentiation assay described in Example 5.
Vitamin D-receptor specific peptides: These are tested in the
MC3T3-E1 osteoblastic cells which can be induced to differentiate into
osteoblasts and form a calcified matrix (mineralize) with vitamin D. The ability
2140814
to mineralize by monitoring 45Ca incorporation is determined after treatment
with peptides.
Estrogen-receptor specific peptides: These are tested for ability to
modulate estrogen-responsive breast cancer cell line proliferation. ER positive
s cells, e.g. MCF7 and T47-D are used.
Androgen-rec~,.tor specific peptides: These are tested in prostate
carcinoma LnCAP cells which are androgen responsive and express the
and rogen-receptor.
Glucocorticoid-rec~,~tor specific assays: These are tested in
10 dexamethasone treated peripheral blood Iymphocytes.
Example 8 Regulation of Endogenous Level of Expression of
Calreticulin
In murine P19 embryonal carcinoma cells, overexpression of calreticulin
inhibits all-trans retinoic acid responsiveness, whereas downregulating
calreticulin by antisense cDNA transfection results in an enhancement of
retinoic acid response (see Example 5). In order to determine whether such
modulation of calreticulin expression results in changes in the responsiveness
to other steroid hormones and vitamins, the PRC-CMV based calreticulin
20 vectors CAL-1 (sense cDNA) and CAL-2 (antisense cDNA) are used to stably
transfect mouse osteoblastic cells (MC3T3 E1), chicken osteoclast precursors,
normal rat mammary epithelial cells (Darcy et al., 1991) and chemically
transformed rat mammary adenocarcinoma cells (ATCC CRL1743), as well as
estrogen and progesterone responsive human breast carcinoma cells (MCF-7
25 and T47-D). Calreticulin expression levels in these cell types are determinedat the outset by Western blot analysis. In addition to utilizing these stable
expression vectors, we construct inducible calreticulin expression sense- and
antisense-cDNA expression vectors driven by strong metal inducible promoters
(Filmus et al., 1992). The inducible vectors allow us to turn calreticulin
30 expression on or off at will. In the MC3T3 cells, 1,25 dihydroxyvitamin D3 has a
proliferative effect on these cells at subconfluency, but when added to
confluent, mineralizing cultures, it enhances the mineralization process. The
, 21~081~
transfected cells are analyzed for the level of calreticulin expression by Western
blot analysis as described by us previously as well as by Northern blot analysisfor mRNA levels. The effect of up or down regulation of calreticulin is
determined in terms of the above mentioned responses to 1,25
5 dihydroxyvitamin D3. In addition, the effect on the expression of vitamin D
responsive genes, such as c-fos and integrin b3 subunit (Xu et al., 1993) is
determined by Northern blot and Western blot analysis. These cells are
transfected with a reporter construct consisting of a vitamin D response element(VDRE) driving the luciferase gene. The luciferase activity in mock transfected
10 versus calreticulin sense- and antisense-cDNA transfected cells (stable and
inducible) is then be determined as described by us previously (see Example
6).
Similar experiments are carried out in chicken osteoclast precursors
whose differentiation into mature osteoclasts is dependent upon vitamin D (Xu
15 et al., 1993).
The effect of modulating calreticulin levels on the glandular
differentiation of normal mammary epithelial cells is examined utilizing a cell
culture model of differentiation (Darcy, 1991). Since steroid hormones such as
estrogen and progesterone play crucial roles in this differentiation process, the
20 effect of calreticulin on this system is determined. Similarly, the effect ofmodulating calreticulin expression in mammary carcinoma cell response to
estrogen, Tamoxifen, progesterone and retinoic acid is determined. Since in
cell lines such as MCF-7, estrogen induces proliferation, whereas Tamoxifen
and all-trans retinoic acid inhibit proliferation (Pratt ef al., 1993), modulating
25 calreticulin levels results in the augmention of one response preferably overanother one. Modulation of calreticulin levels is therapeutically significant in the
control of breast cancer.
In addition to altering calreticulin levels by cDNA transfection, we
determine whether calreticulin expression is modulated by growth factors,
30 cytokines or steroid hormones and vitamins themselves. Although the
promoter of the human calreticulin gene has been cloned and characterized
(Michelak, 1992), it does not give any specific clues as to its regulation. In
- 24 -
21 10814
addition, it is conceivable that many compounds could regulate calreticulin
levels at a post-transcriptional level. Factors which are known to influence theproliferation and differentiation of the above cell types (e.g. IGF-1 and vitamin D
for osteoblasts; IL-6 for osteoclasts; EGF, FGF and TGF-b for mammary
5 epithelial cells) are evaluated initially. We have already determined that 1,25
dihydroxyvitamin D3 upregulates calreticulin mRNA levels in the MC3T3 cells.
Knowledge about the endogenous regulation of expression of calreticulin allow
in vivo manipulation of nuclear hormone receptor-calreticulin interaction.
Example 9 Modulation of Hormone Receptor-Calreticulin
Interaction by Peptides, Peptide Mimetics and
Antibodies
We have demonstrated that synthetic peptides based on the sequence
KXFFYR can behave as competitive inhibitors of calreticulin-nuclear hormone
15 receptor interaction (see Example 7). This was demonstrated by gel mobility
shift assays. When incubated with the nuclear hormone receptor and
calreticulin, the peptides can reverse the ability of calreticulin to inhibit receptor-
DNA binding in vitro. Since a scrambled peptide was completely ineffective,
this assay can distinguish peptide specificity. These data suggest that the
20 interaction of calreticulin with the nuclear hormone receptors can be
manipulated with such peptides.
We use the gel mobility shift assay and androgen receptor as well as the
retinoic acid receptors (Shago et al., 1994) to determine which amino acids
within the KXFFYR sequence are critical for the calreticulin-receptor interaction.
25 This is done by synthesizing peptides with single amino acid substitutions and
then testing them for their activity in gel mobility shift assays as described by us
previously (see Example 7). Results from these experiments identify the criticalamino acids in this sequence motif required for calreticulin-receptor interaction.
The nuclear hormone receptors can be subdivided into two categories,
30 the steroid receptors, which include the androgen receptor, glucocorticoid
receptor, mineralocorticoid receptor and estrogen receptor; and the thyroid
hormone/retinoic acid receptor group which includes the retinoic acid receptors,
- 25 -
2140814
thyroid hormone receptor and vitamin D receptor. Unlike the first category, thislatter category of receptors bind to their DNA response elements as
heterodimers with RXR. The above experiments therefore define the sequence
motif for a receptor from each of these two categories i.e. androgen receptor
5 and retinoic acid receptors (RAR/RXR; RXR/RXR). Subsequent experiments
are then be carried out with other receptors such as estrogen receptor and
vitamin D receptor.
The N-domain of calreticulin (Michalak et al, 1992) has been implicated
in the interaction with the glucocorticoid receptor. We have prepared GST-
fusion proteins in E. coli consisting of either full length human calreticulin, the N-
domain, P-domain or the C-domain. Each of these recombinant proteins is
tested in gel mobility shift assays (according to the teaching of Example 3) fortheir effectiveness in inhibiting receptor-DNA interaction. The receptors we useinitially will be the androgen receptor and the retinoic acid receptors. P19 EC
15 cells or Vero cells are also transiently transfected with expression vectors
containing the N, P or C calreticulin domains, and their effect on hormone
induced gene expression determined as described by us previously (see
Example 5). Once we have identified the calreticulin domain which interacts
with the receptors we use proteolytic fragments of the recombinant proteins
20 (generated by proteolytic cleavage and purification of peptides by HPLC) to
further define the minimal peptide(s) necessary for mediating the interaction. If
a suffficiently small peptide is found to be active, then synthetic peptides from
- within that sequence will be evaluated.
25 Example 10 Preparation and testing of delivery syste.ns fo
peptide-antagonists of calreticulin nuclear hormone
receptor interactions
After we identify peptides capable of inhibiting calreticulin-receptor
interactions in vifro, we test the effficiency of these peptides on cells. To do this,
30 the peptides are incorporated into cationic lipid vesicles (liposomes, such as
lipofectin) and incubated with the cell types described in Example 9. To assess
internalization of the peptides, some peptides are conjugated with fluorescein
- 26 -
21~081~
isothiocyanate (FITC). After incubation of the liposomes with the cells for
different time periods, the cells are examined by immunoflourescence
microscopy to assess intracellular accumulation. The biological effects of the
peptides are determined by assessing hormonal sensitivity of the target cells,
5 expression of primary response genes by Northern and Western blot analysis,
and hormone-induced expression of luciferase reporter constructs containing
various response elements, described in Example 6. The target cells and the
hormone responsive parameters to be used are described in Example 7.
These experiments determine whether the peptides defined in Example
7 are functional at the cellular level in antagonizing hormone receptor-
calreticulin interactions. Once we optimize the peptide delivery systems and
achieve the predictable cellular responses, we test these peptide-liposomes in
animal model systems such as bone formation in the mouse, and rat mammary
gland differentiation. For the former, primary osteoblasts derived from mouse
15 calvariae are njected into the gluteal muscle of recipient mice where these
osteoblasts differentiate to form mineralized nodules. The effect of local or
systemic administration of peptide-liposomes are then assessed in this model.
Similarly the effect of the peptides on normal mammary gland development
after injection of normal rat mammary epithelial cells into mammary fat pads
20 (Darcy, 1991) are assessed. If the peptides are found to be effective in
influencing these processes then their effect on animal models of osteoporosis,
and growth and differentiation of human breast and prostate cancer xenografts
in nude mice are determined.
The following examples relate to the manufacture and use of
pharmaceuticals to treat particular diseases, including cancer, osteoporosis,
and chronic inflammatory disease, using pharmaceuticals comprising a
30 protein for use in modulating hormone responsiveness and a carrier.
2140814
Example 11 Method of Treating Prostate Cancer
Prostate cancer is the most frequently diagnosed invasive cancer and
the second most common cause of cancer death in men in Western societies
(Boring,1993; Coffey,1993). At present, prostate cancer patients are
s diagnosed with either locally invasive, or disseminated disease, and the
currently available forms of treatment may prolong survival but are essentially
only palliative (Scardino,1992; Koxlowski,1991; Santer,1992).
Although primary endocrine ablation leads to an initial response in about
70% of patients with advanced disease, most patients relapse within three
years and only about 20% survive for five years (Kozlowski,1991). This rapid
progression of prostate cancer following failure of primary hormone therapy is
attributed to androgen-independent tumor growth.
In some androgen-insensitive rat as well as human prostate cancer cell
lines, androgen independence is associated with a loss or decrease in
androgen receptor (AR) mRNA and protein levels (Quarmby,1990; Tilley,
1990). However some prostate carcinoma cell lines derived from metastases
retain AR expression and androgen sensitivity (e.g. Ln CAP cell line).
Furthermore there is evidence that some prostate cancer cells which continue
to grow after initiation of anti-androgen therapy retain expression of AR (van der
Kwast,1991; Tilley,1994). Similarly, AR expression is retained by androgen-
independent mouse mammary tumors (Dabre,1987).
These observations suggest that mechanisms other than the loss of AR
expression are involved in the progression to an androgen-independent state.
One explanation could be the presence of mutations in the AR gene in a
subpopulation of tumor cells which results in aberrant regulation of growth by
steroids. Indeed mutations in the AR gene have been detected in prostate
cancer cells, although their significance in tumor progression is not yet clear.Another explanation might be alterations in the expression or function of
components which regulate AR activity and AR dependent gene expression.
As described in previous Examples, calreticulin, can bind to nuclear
hormone receptors by interacting with the KXFFYR sequence. The interaction
results in a profound inhibition of nuclear hormone receptor DNA binding
- 28 -
2140814
activity which can be reversed by soluble competing synthetic peptides with the
generic sequence KXFFYR.
The level of expression of calreticulin in prostate androgen-dependent
and independent prostate cancer cells could have significant effects on
s androgen receptor activity. Furthermore, experimental manipulation of
calreticulin levels in prostate cancer cells should also result in the modulation of
androgen-receptor activity. In addition, the interaction of androgen receptors
with calreticulin could be taken advantage of, theoretically, by utilizing
calreticulin or calreticulin-derived peptides and peptide-mimetics for the
10 inhibition of androgen receptor dependent prostate cancer cell growth. Such atherapeutic strategy might be particularly useful in recurrent, androgen-
independent prostate cancers which retain expression of AR or mutant ARs
and which may bind to DNA in the absence of androgen.
Calreticulin expression in the nuclear, cytoplasmic and microsomal
15 fractions from human prostate carcinoma cell-lines PC-3, DU-145, LnCAP, as
well as highly invasive variants of PC-3 cells (IPC31-3) (Dedjar. 1994) is
determined by Western blot analysis utilizing two different polyclonal anti-
calreticulin antibodies as described by us previously (Leung-Hagesteijn, 1994).
Expression at the level of mRNA is carried out using a 1.9 Kb calreticulin cDNA.20 Calreticulin expression in these cells is determined after treatment with
androgens (for LnCAP cells which express AR, or for PC-3 cells transfected
with AR, see below), retinoic acid, 1,25 dihydroxy vitamin D3, and growth
factors such as epidermal growth factor and insulin-like growth factors.
A subset of a large (~125) tissue bank of frozen human prostate cancers
25 (Sunnybrook Health Science Centre were treated with neo adjuvant androgen
ablative therapy prior to resection. Each frozen block has been histologically
characterized. The bank also contains hormone-resistant prostate cancer
specimens and will accrue fresh bond marrow metastases from warm autopsies
on patients dying of androgen resistant prostate cancer. These tissues are
30 freely available for the aforementioned analyses. This permits determination of
calreticulin expression in untreated, hormonally treated and hormone resistant
disease.
- 29 -
214081g
-
Calreticulin expression in cryostat sections is determined by
immunohistochemistry using the avidin-biotin complex method described by
Hsu et al., 1981 as well as by in situ hybridization using antisense cDNA
calreticulin probes as described by Naylor et a/., 1990. These procedures are
5 performed in Dr. Malik's laboratory where they are carried out on a routine
basis. Simultaneous determination of the androgen-receptor status in these
tissues on serial sections are carried out using anti-AR antibodies (Tilley, 1994).
Overexpression, or inhibition of expression of calreticulin is carried out
by stable transfection of sense (pRC-CMV-Cal1) or anti-sense (pRC-CMV-
Cal2) (Dedhar, 1994) cDNA expression vectors into LnCAP or AR expressing
PC-3 cells. AR expressing PC-3 cells are obtained from Dr. Paul Rennie,
Vancouver, B.C. We have previously described the utility of these calreticulin
expression vectors in manipulating hormone responsiveness (see Example 4).
These cells are also transfected with a tetracyclin inducible calreticulin
expression vector. This expression plasmid, pUHD10-3-CAL, has been
constructed and calreticulin sense or antisense mDNA expression is induced
via a tetracyclin-operators. Calreticulin expression levels are determined by
Western blot analysis as described above. The responsiveness of the
transfected cells to androgens, in terms of cell growth, is then determined. Cell
growth is determined by counting cell numbers as well as by 3H-thymidine
incorporation. Calreticulin overexpression makes the cells non-responsive to
androgens, whereas inhibition of calreticulin expression makes the cells more
sensitive, as was the case for retinoic acid responsiveness in ECP19 cells (see
Example 5). Stable overexpression of calreticulin does not alter intracellular
calcium concentrations, and therefore the observed effects on hormone
sensitivity are not due to effects on calcium levels.
The calreticulin transfected cells is compared with the parental or mock
transfected cells for their abilities to form tumors in nude mice upon
subcutaneous inoculation, or orthotopic inoculation into the prostate gland.
The domain of calreticulin which interacts with the KXFFYR sequence
has been identified as the globular N-domain. This domain contains a putative
ATP binding site and recombinant calreticulin can be phosphorylated in vitro on
- 30 -
214081~
a serine residue within this domain (Leung-Hagesteijn, 1994). We have
prepared GST-fusion proteins in E.Coli consisting of the full length human
calreticulin, the N-domain, P-domain, or the acidic C-domain. Each of these
recombinant peptides is tested in gel mobility shift assays for their effectiveness
s in the inhibition of androgen receptor-DNA interaction (see Example 9). These
domains are tested for their effectiveness in inhibiting androgen mediated gene
expressing by transiently expressing them in LnCAP or AR-expression PC-3
cells (described previously). Once we have identified the calreticulin domain
which interacts with the androgen receptor (although this is likely to be the N-
o domain, based on previous work, see above), we derive proteolytic fragmentsfrom these proteins (by limited proteolytic cleavage using various proteases,
and purification of peptides by high pressure liquid chromatography), and utilize
these in gel mobility shift assays to further define the minimal peptide sequence
required for interaction with the androgen receptor. If a suffficiently small
peptide is found to be active, then synthetic peptides from within that sequenceare evaluated further.
Peptides capable of inhibiting AR-DNA interactions in vitro are identified.
In order to test the effficiency of these peptides in cells, the peptides are
incorporated into cationic lipid vesicles (liposomes, such as lipofectin) and
20 incubated with the cell types (see Example 10).
These experiments determine whether the peptides defined above are
functional at the cellular level in antagonizing hormone receptor-DNA
interactions. Once we have optimized the peptide delivery systems and
achieve the predictable cellular responses, we test these peptide-liposomes in
25 animal model systems described above.
These peptides, in conjunction with current protocols of androgen
ablation, are useful in inhibiting androgen-receptor mediated prostate cancer
cell growth. This strategy is useful not only in the early treatment of androgen-
sensitive tumors, but also in more advanced androgen-resistant tumors which
30 may express normal or mutated ARs which can induce cell growth in an
androgen-independent manner. In such tumors, the maintenance of high levels
of calreticulin expression, or of adminstration of calreticulin-based peptides
- 31 -
~140814
(derived as described above), or peptide mimetics, provide a new mode of
therapeutic intervention in the inhibition of prostate cancer cell growth and
progression.
5 Example 12 Method of Treating Breast Cancer
Here. we use a similar protocol as for prostate cancer (Example 11 )
but use proteins/mimetic selective for estrogen. Transfections are made into
MCF-7, T47-D human breast cancer cells lines instead of LnCAP. For patient
treatment, the peptides or their mimetics, or calreticulin or its mimetics are
10 delivered in lipid vesicles prepared as described in Examples 10 and 11.
Example 13 Method of Treating Chronic Inflammatory Disease
The debilitating symptoms of chronic inflammatory diseases such as
arthritis arise from inadvertent immune responses. Steroidal compounds are
15 major immunosuppressive agents often used in the therapy of chronic
inflammatory diseases. The response to such therapy may be dramatically
augmented by the co-administration of calreticulin-hormone receptor
antagonists based on the KXFFYR sequence. Such peptides, or their organic
mimetics, may dramatically enhance the hormonal response by activating
20 those receptors which may be bound by calreticulin. This may also result in
the use of lower concentrations of the steroidal compounds, resulting in fewer
side effects. The mode of delivery of such peptides or organic mimetics
would be in lipid vesicles described in Examples 10 and 11.
25 Example 14 Method of Treating Osteoporosis
Osteoporosis results from an imbalance in the rate of bone resorption
versus bone formation. Specifically, in post menopausal women, the
decrease in systemic estrogen levels results in decreased bone formation in
the face of continual osteoclast mediated bone resorption. Estrogen therapy,
30 by using estrogen analogs which may specifically enhance osteoblast
function and bone formation is under intensive study. The co-administration
of K)~FFYR based peptides or mimetics specific for the antagonism of
- 32 -
21~081~
.
calreticulin-estrogen receptor interaction would be highly beneficial for this
treatment. Such peptides or mimetics may dramatically increase the efficacy
of estrogen analogs used in such therapy.
An alternative approach for the use of calreticulin based therapy might
5 be inhibition of osteoclast differentiation. The differentiation of mature
osteoclasts from osteoclast precursors is enhanced by Vitamin D3. The
specific inhibition of the vitamin D receptor by calreticulin based mimetics
could therefore result in the suppression of osteoclast mediated bone
resorption. Combined therapy of increasing bone formation and down
10 regulating bone resorption may be an effective treatment for osteoporosis.
The peptides or mimetics would once again be delivered by methods
described in Examples 10 and 11.
The present invention has been described in terms of particular
embodiments found or proposed by the present inventor to comprise
preferred modes for the practice of the invention. It will be appreciated by
those of skill in the art that, in light of the present disclosure, numerous
20 modifications and changes can be made in the particular embodiments
exemplified without departing from the intended scope of the invention. For
example, due to codon redundancy, changes can be made in the underlying
DNA sequence without affecting the protein sequence. Moreover, due to
biological functional equivalency considerations, changes can be made in
25 protein structure without affecting the biological action in kind or amount. All
such modifications are intended to be included within the scope of the
appended claims.
2140814
.
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