Dipartimento di Biologia Cellulare e dello Sviluppo, Università, Viale delle Scienze, 90128 Palermo, Italy
* Author for correspondence (e-mail: clupar{at}tin.it)
Accepted 11 March 2003
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Summary |
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Key words: PTHrP, Stress proteins, Protease, Breast cancer cells, Gene expression, Invasion
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Introduction |
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In the mammary gland, PTHrP acts as a critical regulatory factor involved
in epithelial-mesenchymal cell interactions (for reviews, see
Wysolmerski and Stewart, 1998;
Dunbar and Wysolmerski, 1999
)
and its expression is essential for branching morphogenesis and sexual
dimorphism during organ development
(Wysolmerski et al., 1998
;
Dunbar et al., 1999
). In
addition, some of us have produced data demonstrating that different PTHrP
domains are also biologically active on breast tumour cells
(Luparello et al., 1995
;
Luparello et al., 1997a
;
Luparello et al., 2001
), and
that PTHrP expression by cells is drastically modulated by modifications of
the culture microenvironment (Luparello et
al., 1999
; Luparello et al.,
2000a
). In particular, a mid-region PTHrP domain, i.e.
[67-86]-amide, administered at 1 nM concentration was found able to restrain
growth and promote invasion through artificial basement membrane (matrigel) by
the 8701-BC cell line, derived from a biopsy fragment of a primary ductal
infiltrating carcinoma (DIC) (Minafra et
al., 1989
), and a highly tumorigenic clonal line of the same cells
(Luparello et al., 1997b
).
Matrigel penetration was drastically impaired by treatment of both cell lines
with anti-urokinase plasminogen activator (uPa) antibodies blocking uPa-uPa
receptor interaction; although to a lesser extent, tissue inhibitor of
metalloprotease (TIMP)-1 was also active in reducing the invasive motility of
only 8701-BC cells. By contrast, trypsin-and cystein-protease inhibitors were
poorly active in restraining cell invasion
(Luparello et al., 1995
;
Luparello et al., 1997a
).
The observation of the invasion-promoting role played by PTHrP
[67-86]-amide prompted a more detailed study of the possible effects on gene
expression arising from its interaction with breast cancer cells. In this
study we demonstrate that treatment of 8701-BC cells with this midregion PTHrP
peptide is linked to the upregulation of heat shock factor-binding protein 1
(hsbp1), coding for a factor known to interact with heat shock factor
(hsf)1 trimers and negatively regulate hsf1 activity
(Satyal et al., 1998;
Cotto and Morimoto, 1999
), and
of some members of heat shock protein (hsp) family, noticeably
hsp90
and-ß, and that such over-expression is involved in
the modulation of uPa- and matrix metalloprotease-1 (MMP-1;
interstitial collagenase) gene expression and of the invasive behaviour in
vitro of 8701-BC breast cancer cells.
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Materials and Methods |
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Treatment with PTHrP [67-86]-amide was performed as described by Luparello
et al. (Luparello et al.,
1995; Luparello et al.,
1997a
). Cells were plated at 2.5 x 104
cells/cm2 in FCS-containing RPMI 1640 medium and, after overnight
incubation to allow adhesion, cultured in serum-free medium for an additional
24 hours; subsequently, serum-free RPMI 1640 medium supplemented with 1 nM
PTHrP [67-86]-amide (Peninsula, Belmont, CA) was added to the cultures. After
24 hours, fresh medium with the same peptide supplement was added and the
cells were incubated for a further 24 hours, and stored at 80°C
until submitted to RNA extraction. Control assays were performed in the
absence of PTHrP [67-86]-amide.
Treatment with hsbp1-antisense oligo (asODN) was performed as described by
Ziegler et al. (Ziegler et al.,
1999). Essentially, cells were incubated for 24 hours with 600 nM
of phosphorothioate 5'-GTGATGTCTCAGACC-3' ODN (MWG Biotech,
Ebersberg, Germany), complementary to bases 18-32 of hsbp1 mRNA (Acc. nr.
AF068754), complexed with 18.6 µl lipofectin/ml (Life Technologies,
Gaithersburg, MD) and added to the plain or PTHrP-containing medium. Control
assays were performed in the presence of phosphorothioate
5'-ACTACCAGGTGGTTC-3' ODN (scrambled-asODN), whose sequence was
not found in EMBL-EBI human DNA database, after homology search with the
Fasta3 software available at
http://www.ebi.ac.uk/fasta33/.
The RNAdraw software (Matzura and
Wennborg, 1996
) was utilized to predict the secondary structure of
hsbp1 mRNA.
Treatment with geldanamycin was performed according to Knowlton and Sun
(Knowlton and Sun, 2001).
Essentially, cells were incubated for 24 hours with 1 µg geldanamycin/ml
(Alomone Labs, Jerusalem, Israel), added to the plain or PTHrP-containing
medium. Control assays were performed with dimethyl sulfoxide (DMSO) vector
only.
Treatment with 100 µM quercetin (USB, Cleveland, OH) was performed for
24 hours on 8701-BC cells cultured in unsupplemented RPMI 1640 medium
(Hosokawa et al., 1992).
Control assays were performed with DMSO vector only.
RNA extraction and reverse transcription
Isolation of either total or poly(A)+ mRNA from control and
treated 8701-BC cells was performed with TriPure reagent (Roche, Mannheim,
Germany) or oligo(dT)25-tailed magnetic beads (mRNA DIRECT kit,
Dynal, Oslo, Norway), respectively, following manufacturers' instructions.
Before the reverse transcription, the RNA samples were treated with RQ1
RNase-free DNase (Promega, Madison, WI) for 30 minutes at 37°C. The cDNAs
were synthesized using M-MLV RNase H- point mutant reverse
transcriptase (Promega) in the presence of 2 µg random 6-mer primers
(Promega), 50 U RNase inhibitor (Promega) and 0.5 mM each of dNTPs; reverse
transcription was performed for 60 minutes at 37°C, followed by RNase H
treatment for 20 minutes at 37°C.
Conventional qualitative polymerase chain reaction (PCR)
PCR analysis was performed using 2.5 µM of appropriate sense and
antisense primers (Table 1)
obtained from MWG Biotech, 1 U RedTaq DNA polymerase/µl (Sigma, St Louis,
MO), 250 µM each of dNTPs, and 1 µl of the cDNA template obtained from
total RNA. The thermal cycle used was a denaturation step of 94°C for 3
minutes, followed by 35-45 cycles of 94°C for 1 minute, the appropriate
annealing temperature for 1 minute, and 72°C for 1 minute. A final
extension of the product was performed for 10 minutes at 72°C. PCR
products were analysed by 2% agarose gel electrophoresis and visualized by
ethidium bromide staining under UV light.
|
Differential display-polymerase chain reaction (DD-PCR)
For differential expression analysis, DD-PCR experiments were performed
using the arbitrary 10-mer primers designed by Sokolov and Prockop
(Sokolov and Prockop, 1994),
in combinations of two. The PCR amplification was performed in an UNO
Thermoblock (Biometra, Göttingen, Germany) using 25 pmoles of each of two
primers, 1-2 µl of the cDNA template obtained from mRNA and 3.6 U of
AmpliTaq DNA Polymerase, Stoffel fragment (Perkin Elmer), as recommended by
Doss (Doss, 1996
), in 50 µl
of the appropriate reaction mixture. The thermal cycle used was a denaturation
step of 94.5°C for 3 minutes, followed by 45 cycles of 94.5°C for 1
minute, 34°C for 1 minute, 72°C for 1 minute and a final extension of
the product for 10 minutes at 72°C.
After PCR amplification, 8 µl of the amplification products were
electrophoresized in a non-denaturing 6% polyacrylamide gel in a sequencing
apparatus (21 cm x 40 cm x 0.4 mm; Sequi-Gen, Bio-Rad, Richmond,
CA) at constant 55 W and the band pattern visualized with the Silver
SequenceTM DNA staining reagents (Promega)
(Luparello et al., 2000b). For
re-amplification of differentially displayed bands, the silver-stained gel was
exhaustively washed with double-distilled water and the bands of interest
carefully scratched from the gel with a sterile syringe needle and used as
template for PCR amplifications performed as described before. Several cycles
of amplification and electrophoresis were repeated until a single pure band
was visualized in the gel and eluted using Ultrafree DA filter columns
(Millipore, Bedford, MA).
The purified PCR products were subsequently cloned using the pGEM-T Easy
vector system (Promega) and JM109 competent cells, high efficiency (Promega),
as described (Sirchia et al.,
2001); the sequence of the inserts contained in the recombinant
plasmid DNA, isolated with High Pure Plasmid Isolation kit (Roche), was
determined by MWG Biotech sequencing service. DNA sequence similarity was
searched with the BLAST algorithm (Altschul
et al., 1990
) available online at
http://www.ncbi.nlm.nih.gov.
Semi-quantitative `multiplex' polymerase-chain reaction (SM-PCR)
For SM-PCR we followed the protocol of Spencer and Christensen
(Spencer and Christensen,
1999) with minor modifications. Essentially, the cDNA species of
interest was co-amplified with ß-actin cDNA (see
Table 1 for primer sequences)
over a range of cycles, followed by 2% agarose electrophoresis and ethidium
bromide stain. Cycle profile was a denaturation step of 94.5°C for 3
minutes, followed by cycles of 94.5°C for 30 seconds, 50°C for 1
minute, 72°C for 1 minute and a final extension of the product for 5
minutes at 72°C. Cycles were limited to the minimum necessary for
detection and the intensities of the bands of interest, evaluated with
SigmaScan software (SPSS), were normalized for those of ß-actin, and
plotted as a function of cycle number. Exponential regression equations fitted
to the curves were used to calculate the number of cycles necessary to reach a
normalized intensity threshold value=1 for each sample. The relative
difference in abundance between two samples was taken as
2n where n is the difference between the numbers
of cycles required by the samples to reach the threshold. Two different RNA
preparations from each experimental condition were pooled to make the
differences, if any, between the expression levels more significant.
Western blot
8701-BC cells were seeded in FCS-containing RPMI 1640 medium, submitted to
the treatments described in the previous paragraph, scraped in 0.1%
EDTA-containing PBS and counted with a Bürker chamber. Aliquots of
106 cells were spun down in PBS and lysed with 100 µl of
pre-warmed lysis buffer (40 mM Tris-HCl, pH 6.8, containing 1% sodium dodecyl
sulphate, 1% glycerol, 1% ß-mercaptoethanol, and 0.001% bromophenol
blue). Aliquots of 20 µl of the extracts were subjected to SDS-PAGE (7.5%
acrylamide) and blotted to nitrocellulose filters by using a Bio-Rad transfer
apparatus. Following the blot, the protein gel was stained with Coomassie
Blue. After blocking for 1 hour with Tris-buffered saline/0.05% Tween-20
(TBS-T) containing 5% nonfat dry milk at room temperature, filters were
incubated for 1 hour with either anti-hsp90 from rat (Calbiochem, San Diego,
CA; final dilution 1:500), anti-hsf1 (Alexis Biochemicals; final dilution
1:10,000) or anti-hsf2 (US Biologicals, Swampscott, MA; final dilution 1:200)
in TBS-T containing 1% nonfat dry milk. After being washed six times for 5
minutes each with TBS-T, the filters were incubated for 1 hour with the
appropriate peroxidase-conjugated secondary antibody dissolved in TBS-T
containing 1% nonfat dry milk and washed six times for 5 minutes each with
TBS-T, prior to detection by the SuperSignal Chemiluminescent substrate
(Pierce), following manufacturer's recommendations.
Viability and proliferation assay
The survival and growth behaviour of 8701-BC cells in response to the
different treatments was evaluated using the colorimetric CellTiter 96
AQueous One Solution Cell Proliferation assay (Promega), in which
the MTS
[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium]
compound is reduced into soluble colored formazan by metabolically active
cells. According to manufacturers' instructions, 8701-BC cells were seeded in
FCS-containing RPMI-1640, submitted to the treatments described elsewhere in
this paper, and incubated, at the end of treatment, with CellTiter reagent for
1 hour in a humidified incubator. The absorbance of formazan produced was
recorded at 490 nm.
Invasion assay
Cell invasive behavior was evaluated using Transwell plates (Costar,
Cambridge, MA) with 6.5 mm insert diameter-and 8 µm pore diameter-filters,
separating an upper compartment of 600 µl and a lower compartment of 100
µl, following an already-published protocol
(Luparello et al., 1995;
Luparello et al., 1997a
). The
filters were coated with 8 µg of Matrigel, a reconstituted basement
membrane matrix from EHS sarcoma (Collaborative Res., Bedford, MA);
trypsinized 8701-BC cells were washed first with 10% FCS-RPMI 1640 medium for
enzyme inactivation and then twice with unsupplemented medium, and 150,000
cells were seeded in the upper compartment of each chamber. PTHrP
[67-86]-amide was dissolved at 1 nM concentration in RPMI 1640 medium and
placed in the lower compartment of the chamber.
In a first set of assays, lipofectin-complexed hsbp1-asODN was added to the cell-containing medium as described elsewhere in this paper. Control experiment was performed in the presence of lipofectin-complexed scrambled-asODN. In a second set of assays, geldanamycin was added to the cell-containing medium as previously reported in this paper. Control experiment was performed in the presence of DMSO vector only. In a third set of assays, quercetin was added to the cell-containing medium as previously reported in this paper, whereas unsupplemented RPMI 1640 medium was placed in the lower compartment of the chamber. Control experiment was made in the presence of DMSO vector only.
The invasion test was performed for 6 hours; the cells attached to the upper surface of the filter were removed mechanically, whereas those that migrated to the lower surface of the filter were fixed with ethanol, stained with toluidine blue and quantitated by counting the number of cells present in 15 random fields of the filter at a 400-fold magnification. Data are presented as mean±s.e.m. of triplicate experiments performed three times; a software-assisted one-way ANOVA was performed (SigmaStat v.2.0, SPSS) and P<0.05 was taken as the minimal level of statistical significance between treated and control samples.
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Results |
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We then checked the differential expression of hsbp1 by PCR amplification in the presence of two primers specific for this cDNA, designed using the Primer Selection software available online. As shown in Fig. 1B, in a preliminary assay we found an amplification band of the expected size after conventional PCR of all samples tested, indicating that expression of hsbp1 was switched-on in both control and PTHrP [67-86]-amide-treated cells. Subsequently, for semi-quantitative evaluation the cDNA preparations were submitted to SM-PCR as described. As shown in Fig. 1C, growth of 8701-BC cells in the presence of 1 nM PTHrP [67-86]-amide resulted in the upregulation of hsbp1 of approximately threefold with respect to control cells.
PTHrP [67-86]-amide upregulates hsp10, hsp90, hsp90ß,
hsf1 and hsf2 in 8701-BC cells
The data obtained on the upregulation of hsbp1 prompted us to
examine which was the expression pattern for other members of the stress
protein family. In a second set of experiments we submitted cDNA samples from
control and PTHrP-treated cells to conventional-and SM-PCR in the presence of
primers specific for hsp10, -27, -60, -70, -90,
-90ß, mthsp75, hsc70, grp78 and the two heat shock
factors (hsf)-1 and -2. The panel in
Fig. 2A shows that a positive
signal was found for hsp10, -60, -90
and -90ß,
mthsp75, and for the two hsfs; no amplification band was
observed for hsp27 and -70, hsc70 and grp78, at
least under the experimental conditions used. When cDNA preparations were
submitted to SM-PCR to compare the expression levels of those stress protein
genes selected on the basis of the previous results, we found that PTHrP
[67-86]-amide was able to promote the upregulation of hsp10,
hsp90
, hsp90ß, hsf1 and hsf2 by
2.6-, 2.7-, 3.1, 7-and 4.7-fold, respectively
(Fig. 2B). No statistically
significant difference was found for the expression levels of hsp60
and mthsp75 between control and PTHrP [67-86]-amide-treated cells
(not shown). Upregulation of hsf1, hsf2 and hsp90 in
PTHrP-treated cells was also confirmed by western blot
(Fig. 3).
|
|
Upregulation of hsbp1 and hsp90 influences the expression of uPa and
MMP-1 by 8701-BC cells
It is known that the human uPa and MMP-1 promoters
contain binding sites for Ets transcription factors
(Crawford and Matrisian, 1996;
Yordy and Muise-Helmericks,
2000
) (see also COMPEL database at
http://compel.bionet.nsc.ru/compel/compel.html)
and that hsf-and Ets-binding domains share a similar three-dimensional
structure of winged helix-turn-helix DNA-binding sites (see PROFILE entry:
QDOC50140 at
http://www.isrec.isb-sib.ch),
thus permitting Ets domain to be targeted by hsf1, which was proven to repress
uPa expression (Chen et al.,
1997
). The results obtained with midregion PTHrP-treated cells
indicated the upregulation of hsbp1 and also of hsp90, which
has been proven to associate with and sequester hsf1 thereby functioning as a
powerful repressor for its activation (Zou
et al., 1998
; Knowlton and
Sun, 2001
).
In consideration of present and literature data, in a third set of assays
we investigated whether upregulation of hsbp1 and hsp90
could have some consequence on the levels of expression of uPa and
MMP-1 genes, whose protein products are prominently involved in the
acquisition of an invasive phenotype by breast cancer cells. As shown by
conventional PCR analysis in Fig.
4, both uPa and MMP-1 transcripts were present
in control and PTHrP-treated cells, although the intensity of the
amplification signal for uPa was stronger in the preparation from
treated cells, conversely that for MMP-1 being much fainter.
Therefore, we examined the effect exerted on uPa and MMP-1
expression in PTHrP [67-86]-amide-treated 8701-BC cells by (1) the
downregulation of hsbp1, (2) the functional inhibition of hsp90, and
(3) the inactivation of hsfs. For (1), we designed a 15-mer ODN antisense to a
theoretically accessible hybridisation site of the mRNA, according to RNAdraw
software analysis and the suggestions of Ziegler et al.
(Ziegler et al., 1999), and
submitted cDNA preparations from mid-region PTHrP-treated cells cultured in
the presence of either hsbp1-asODN or scrambled-asODN to SM-PCR for the
evaluation of hsbp1 mRNA levels. For (2), we incubated PTHrP
[67-86]-treated cells in the presence of geldanamycin, a benzoquinoid
antibiotic from Streptomyces hygroscopicus which binds specifically
and disrupts hsp90 function preventing its interaction with hsf1 which, once
freed, can be activated (Zou et al.,
1998
; Knowlton and Sun,
2001
). For (3), we incubated untreated cells in the presence of
the flavonoid quercetin, which is known to decrease the amount of free hsf1
through downregulation of hsf1 transcriptional activation and/or inhibition of
hsf1 activation (Hosokawa et al.,
1992
; Hansen et al.,
1997
), thus exerting a negative regulatory effect similar to that
of hsbp1. Incubation with lipofectin-vehiculated hsbp1-asODN resulted in a
8.5-fold and 2-fold decrease of hsbp1 transcript amount with respect
to parallel scrambled-asODN-treated controls cultured in PTHrP-containing
medium, and to parallel untreated controls, respectively, suggesting a
prominent RNase H-recruiting capacity owned by the selected hsbp1-asODN (not
shown). The occurrence of asODN-induced cytotoxicity was also checked by
MTS-tetrazolium-based assay after 24 hours incubation; no difference in the
absorbance of produced formazan at l=490 nm was found among untreated-,
hsbp1-asODN-treated-and scrambled-asODN-treated cells (not shown), indicating
that both asODNs, and also lipofectin treatment, were unable to affect the
survival and proliferative behaviour of 8701-BC cells. Also geldanamycin-and
quercetin-treated cell cultures displayed no changes in the amount of formazan
accumulated after 24 hours' incubation with respect to controls (not shown),
indicating that these drugs did not exert any effect on cell viability and
growth. Western blot analysis indicated that hsf1, hsf2 and
hsp90 over-expression remained steady in all preparations from
PTHrP-treated 8701-BC cells, irrespective of the supplement
(Fig. 3).
|
Significantly, following addition of hsbp1-asODN we found a prominent downregulation (of approximately 6-fold) of uPa in midregion PTHrP-treated 8701 cells if compared to controls cultured in the presence of scrambled-asODN (Fig. 5, left panel); by contrast, an amplification band for MMP-1 was already detectable after 21 PCR cycles in cDNA samples from hsbp1-asODN treated cells, whereas no signal was observed in the control counterpart even if PCR cycles were increased up to 26 (Fig. 5, right panel A,B). Although to a lesser degree, a similar result was obtained when hsp90 was functionally blocked by geldanamycin: as shown in Fig. 5, left panel, following treatment, uPa was downregulated by approximately threefold, whereas a positive signal for MMP-1 was visible in cDNA samples from geldanamycin-treated cells from 44 cycles of amplification, being totally absent in control preparations (Fig. 5, right panel C,D). Interestingly, hsbp1-asODN and geldanamycin treatment were also effective in increasing MMP-1 levels of expression in untreated 8701-BC cells by approximately 6-and 3-fold, respectively, whereas no significant variation was found for uPa expression level following treatments (not shown).
|
By contrast, analogously to PTHrP [67-86]-amide treatment, albeit to a lesser extent, incubation of 8701-BC cells with quercetin resulted in the upregulation of uPa (>2-fold) and in the downregulation of MMP-1 (>3-fold) (Fig. 6), thus providing further supporting data of the involvement of hsf unavailability on the modulation of uPa and MMP-1 expression.
|
Inhibition of hsbp1 and hsp90 and inactivation of hsf1 influence the
in vitro invasive behaviour of 8701-BC cells
On the basis of the expression data obtained, in a fourth set of assays we
examined whether asODN-mediated downregulation of hsbp1 and
geldanamycin-dependent functional inactivation of hsp90 could restrain the
ability of 8701-BC cells to penetrate an artificial basement membrane in
Transwell chamber assay, as reported by Luparello et al.
(Luparello et al., 1995);
alternatively, the effect of quercetin on the invasive ability of 8701-BC
cells seeded in plain RPMI 1640 medium was also tested. As shown in
Fig. 7, hsbp1-asODN-and
geldanamycin treatments resulted in a prominent decrease of the amount of
cells able to cross the matrigel-coated filters, the number of migrated
cells/field being 7.35±0.5 and 1.5±0.04 (P<0.001)
for scrambled-asODN and hsbp1-asODN-treated 8701-BC cells, and 6.5±0.6
and 2.2±0.4 (P=0.002) for control and geldanamycin-treated
8701-BC cells (average±s.e.m.). Conversely, quercetin treatment
resulted in a remarkable increase in cell invasive activity, the number of
migrated cells/field being 1.9±0.39 and 7±0.73
(P<0.001) for control and quercetin-treated 8701-BC cells
(average±s.e.m.).
|
![]() |
Discussion |
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Further studies will be required to determine a panel of midregion
PTHrP-responsive genes in breast cancer cells and the related biological
significance; however, present results allow us to make the following
principal comments. First, the molecular data indicating a shift from a
preferential MMP-1-to a preferential uPa-expressing
phenotype obtained in the present work confirm previous postulate that
over-secretion of uPa, rather than of other extracellular proteases (i.e.
other types of MMPs, trypsin-like enzymes, cysteine proteases), was a primary
condition for the increase of invasive activity triggered by PTHrP
[67-86]-amide in vitro (Luparello et al.,
1995; Luparello et al.,
1997a
). To our knowledge, a role played by PTHrP on the regulation
of uPa activity was reported in earlier papers only for osteoblast-like cells
(Kemp et al., 1987
;
Hammonds et al., 1989
).
Significantly, in their paper, Hammonds et al.
(Hammonds et al., 1989
) had
demonstrated the more elevated efficacy of PTHrP [1-108] and [1-141] than
PTHrP [1-34] in stimulating uPa activity: in the light of our results, this
can conceivably be because of the maintenance of the midregion moiety by the
[1-108] and [1-141] fragments.
Second, our investigation further supports the concept that breast cell
differentiation state may be controlled by forms of PTHrP beyond those that
have been studied most extensively to date, i.e. N-terminal and full-length
PTHrP (e.g. Dunbar and Wysolmerski,
1999; Cataisson et al.,
2000
; Guise et al.,
2002
). Interestingly, PTHrP [67-86]-amide, which was also shown to
be biologically active on placental transport of
Ca2+/Mg2+ and on Ca2+ metabolism of squamous
carcinoma cells (Care et al.,
1990
; Kovacs et al.,
1996
; Orloff et al.,
1996
) seems not to be a physiological midregion cleavage product
of PTHrP, which according to Wu et al. (Wu
et al., 1996
) spans from aminoacid 38 to 94. We have previously
reported that PTHrP [38-94]-amide impairs 8701-BC cell invasion through
matrigel, which is the opposite to what was found with PTHrP [67-86]-amide
(Luparello et al., 2001
).
Based on our collective observation, it will be worth examining which is the
biological implication of the extra aminoacid sequence of 38-94 versus 67-86
fragment, and whether combinations of midregion PTHrPs, as well as of other
N-and C-terminal PTHrP forms, may play a role in key functional steps of
breast cancer invasion in vivo, thereby altering the net effect exerted by
PTHrP on cancer progression.
Third, the demonstration of an effect exerted on the invasive properties of
a breast tumor cell line treated with a fragment of PTHrP adds a new example
to the hitherto very limited group of biological roles attributed to hsbp1. In
fact, the only available data in literature report the influence of altered
levels of hsbp1 on the survival of Caenorabditis elegans exposed to
different stresses, both chemical and thermal
(Satyal et al., 1998). A
biophysical and biochemical characterization of human hsbp1 has come out only
very recently (Tai et al.,
2002
).
There are some questions whose answers will require further studies. First,
the degree of the specific contribution of either hsp90 or -ß to
the induction of cell invasiveness was not examined because of the absence of
specific drugs inactivating only one isoform, and the difficulty we
encountered in inhibiting hsp90
or -ß expression by asODN was
probably because of the relatively long life of the proteins (data not shown),
as also reported by Zou et al. (Zou et
al., 1998
).
Second, the selective upregulation of both hsfs tested is
intriguing. An over-expression of hsf1 has been reported in a
metastatic prostate cancer cell line and in most prostate cancer specimens
examined by Hoang et al. (Hoang et al.,
2000); no supporting data are available in the literature on the
possible explanation of the upregulation of hsf2, whose protein
product is generally acknowledged to control development and
differentiation-specific gene expression, rather than responding to stress
stimuli, by regulating genes distinct from those controlled by hsf1
(Pirkkala et al., 2001
). The
data reported here indirectly suggest that the equilibrium between bound and
unbound hsf1 in PTHrP [67-86]-amide-treated 8701-BC cells is shifted to the
former situation. Thus, whether the increase in the expression level can be
interpreted as a cell response to the possible deficit of free hsfs, being
sequestered by over-abundant hsbp1 and hsp90s, remains to be determined.
In conclusion, the results presented here have twofold significance: (1) they contribute to the knowledge of the cell biology of the diverse non-N terminal forms of PTHrP, further supporting that midregion PTHrP can be enclosed in the list of those elements potentially affecting breast cancer progression; and (2) they identify two new key protagonists in the complex scenario of DIC cell invasiveness in vitro, i.e. hsbp1 and hsp90, which deserve further and more extensive studies as potential and attractive molecular targets for the control of the malignancy of breast (and possibly other) cancer histotypes.
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Acknowledgments |
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References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Aarts, M. M., Levy, D., He, B., Stregger, S., Chen, T., Richard,
S. and Henderson, J. E. (1999). Parathyroid hormone-related
protein interacts with RNA. J. Biol. Chem.
274,4832
-4838.
Aarts, M. M., Davidson, D., Corluka, A., Petroulakis, E., Guo,
J., Bringhurst, F. R., Galipeau, J. and Henderson, J. E.
(2001). Parathyroid hormone-related protein promotes quiescence
and survival of serum-deprived chondrocytes by inhibiting rRNA synthesis.
J. Biol. Chem. 276,37934
-37943.
Aguirre Ghiso, J. A., Alonso, D. F., Farias, E. F., Gomez, D. E.
and Bal de Kier Joffè, E. (1999). Deregulation of the
signaling pathways controlling urokinase production. Its relationship with the
invasive phenotype. Eur. J. Biochem.
263,295
-304.
Altschul, S. F., Gish, W., Miller, W., Myers, E. W. and Lipman, D. J. (1990). Basic local alignment research tool. J. Mol. Biol. 215,403 -410.[CrossRef][Medline]
Amling, M., Neff, L., Tanaka, S., Inoue, D., Kuida, K., Weir,
E., Philbrick, W. M., Broadus, A. E. and Baron, R. (1997).
Bcl-2 lies downstream of parathyroid hormone-related peptide in a signaling
pathway that regulates chondrocyte maturation during skeletal development.
J. Cell Biol. 136,205
-213.
Benaud, C., Dickson, R. B. and Thompson, E. W. (1998). Roles of the matrix metalloproteinases in mammary gland development and cancer. Breast Cancer Res. Treat. 50, 97-116.[CrossRef][Medline]
Burtis, W. J. (1992). Parathyroid hormone-related protein: structure, function and measurement. Clin. Chem. 38,2171 -2183.[Abstract]
Care, A. D., Abbas, S. K., Pickard, D. W., Barri, M., Drinkhill, M., Findlay, J. B., White, I. R. and Caple, I. W. (1990). Stimulation of ovine placental transport of calcium and magnesium by mid-molecule fragments of human parathyroid hormone-related protein. Exp. Physiol. 75,605 -608.[Abstract]
Cataisson, C., Lieberherr, M., Cros, M., Gauville, C., Graulet, A. M., Cotton, J., Calvo, F., de Vernejoul, M. C., Foley, J. and Bouizar, Z. (2000). Parathyroid hormone-related peptide stimulates proliferation of highly tumorigenic human SV40-immortalized breast epithelial cells. J. Bone Miner. Res. 15,2129 -2139.[Medline]
Chen, C., Xie, Y., Stevenson, M. A., Auron, P. E. and
Calderwood, S. K. (1997). Heat shock factor 1 represses
ras-induced transcriptional activation of the c-fos gene.
J. Biol. Chem. 272,26803
-26806.
Cotto, J. J. and Morimoto, R. I. (1999). Stress-induced activation of the heat-shock response: cell and molecular biology of heat-shock factors. Biochem. Soc. Symp. 64,105 -118.[Medline]
Crawford, H. C. and Matrisian, L. M. (1996). Mechanisms controlling the transcription of matrix metalloproteinase genes in normal and neoplastic cells. Enzyme Protein 49, 20-37.[Medline]
De Miguel, F., Fiaschi-Taesch, N., Lopez-Talaverna, J. C.,
Tarane, K. K., Massfelder, T., Helwig, J. J. and Stewart, A. F.
(2001). The C-terminal region of PTHrP, in addition to the
nuclear localization signal, is essential for the intracrine stimulation of
proliferation in vascular smooth muscle cells.
Endocrinology 142,4096
-4105.
Doss, R. P. (1996). Differential display without radioactivity a modified procedure. BioTechniques 21,408 -409.[Medline]
Duffy, M. J., Maguire, T. M., Hill, A., McDermott, E. and O'Higgins, N. (2000). Metalloproteinases: role in breast carcinogenesis, invasion and metastasis. Breast Cancer Res. 2,252 -257.[CrossRef][Medline]
Dunbar, M. E. and Wysolmerski, J. J. (1999). Parathyroid hormone-related protein: a developmental regulatory molecule necessary for mammary gland development. J. Mammary Gland Biol. Neoplasia 4,21 -34.[Medline]
Dunbar, M. E., Dann, P. R., Robinson, G. W., Henninghausen, L.,
Zhang, J. P. and Wysolmerski, J. J. (1999). Parathyroid
hormone-related protein signaling is necessary for sexual dimorphism during
embryonic mammary development. Development
126,3485
-3493.
Fenton, A. J., Martin, T. J. and Nicholson, G. C. (1994). Carboxy-terminal parathyroid hormone-related protein inhibits bone resorption by isolated chicken osteoclasts. J. Bone Miner. Res. 9,515 -519.[Medline]
Guise, T. A., Yin, J. J., Thomas, R. J., Dallas, M., Cui, Y. and Gillespie, M. T. (2002). Parathyroid hormone-related protein (PTHrP)-(1-139) isoform is efficiently secreted in vitro and enhances breast cancer metastasis. Bone 30,670 -676.[CrossRef][Medline]
Hammonds, R. G. Jr, McKay, P., Winslow, G. A.,
Diefenbach-Jagger, H., Grill, V., Glatz, J., Rodda, C. P., Moseley, J. M.,
Wood, W. I., Martin, T. J. et al. (1989). Purification and
characterization of recombinant human parathyroid hormone-related protein.
J. Biol. Chem. 264,14806
-14811.
Hansen, R. K., Oesterreich, S., Lemieux, P., Sarge, K. D. and Fuqua, S. A. (1997). Quercetin inhibits heat shock protein induction but not heat shock factor DNA-binding in human breast carcinoma cells. Biochem. Biophys. Res. Commun. 239,851 -856.[CrossRef][Medline]
Henderson, J. E., Amizuka, N., Warshawsky, H., Biasotto, D., Lanske, B. M. K., Goltzman, D. and Karaplis, A. C. (1995). Nucleolar localization of parathyroid hormone-related peptide enhances survival of chondrocytes under conditions that promote apoptotic cell death. Mol. Cell. Biol. 15,4064 -4075.[Abstract]
Hoang, A. T., Huang, J., Rudra-Ganguly, N., Zheng, J., Powell,
W. C., Rabindran, S. K., Wu, C. and Roy-Burman, P. (2000). A
novel association between the human heat shock transcription factor 1 (HSF1)
and prostate adenocarcinoma. Am. J. Pathol.
156,857
-864.
Hosokawa, N., Hirayoshi, K., Kudo, H., Takechi, H., Aoike, A., Kawai, K. and Nagata, K. (1992). Inhibition of the activation of heat shock factor in vivo and in vitro by flavonoids. Mol. Cell. Biol. 12,3490 -3498.[Abstract]
Kemp, B. E., Moseley, J. M., Rodda, C. P., Ebeling, P. R., Wettenhall, R. E., Stapleton, D., Diefenbach-Jagger, H., Ure, F., Michelangeli, V. P., Simmons, H. A. et al. (1987). Parathyroid hormone-related protein of malignancy: active synthetic fragments. Science 238,1568 -1570.[Medline]
Knowlton, A. A. and Sun, L. (2001). Heat-shock
factor-1, steroid hormones, and regulation of heat-shock protein expression in
the heart. Am. J. Physiol. Heart Circ. Physiol.
280,H455
-H464.
Kovacs, C. S., Lanske, B., Hunzelman, J. L., Guo, J., Karaplis,
A. C. and Kronenberg, H. M. (1996). Parathyroid
hormone-related peptide (PTHrP) regulates fetal-placental calcium transport
through a receptor distinct from the PTH/PTHrP receptor. Proc.
Natl. Acad. Sci. USA 93,15233
-15238.
Krepela, E. (2001). Cysteine proteinases in tumor cell growth and apoptosis. Neoplasma 48,332 -349.[Medline]
Li, X. and Drucker, D. J. (1994). Parathyroid
hormone-related peptide is a downstream target for ras and src activation.
J. Biol. Chem. 269,6263
-6266.
Lam, M. H. C., Briggs, L. J., Hu, W., Martin, T. J., Gillespie,
M. T. and Jans, D. A. (1999). Importin ß recognizes
parathyroid hormone-related protein with high affinity and mediates its
nuclear import in the absence of importin . J. Biol.
Chem. 274,7391
-7398.
Lam, M. H. C., Thomas, R. J., Martin, T. J., Gillespie, M. Y. and Jans, D. A. (2000). Nuclear and nucleolar localization of parathyroid hormone-related protein. Immunol. Cell Biol. 78,395 -402.[CrossRef][Medline]
Luparello, C., Ginty, A. F., Gallagher, J. A., Pucci-Minafra, I. and Minafra, S. (1993). Transforming growth factor ß1, ß2, and ß3, urokinase, and parathyroid hormone-related peptide expression by 8701-BC cells and clones. Differentiation 55,73 -80.[Medline]
Luparello, C., Burtis, W. J., Raue, F., Birch, M. A. and Gallagher, J. A. (1995). Parathyroid hormone-related peptide and 8701-BC breast cancer cell growth and invasion in vitro. Evidence for growth-inhibiting and invasion-promoting effect. Mol. Cell. Endocrinol. 111,225 -232.[CrossRef][Medline]
Luparello, C., Birch, M. A., Gallagher, J. A. and Burtis, W. J. (1997a). Clonal heterogeneity of the growth and invasive response of a human breast carcinoma cell line to parathyroid hormone-related peptide fragments. Carcinogenesis 18, 23-29.[Abstract]
Luparello, C., Noël, A. and Pucci-Minafra, I. (1997b). Intratumoral heterogeneity for hsp90ß mRNA levels in a breast cancer cell line. DNA Cell Biol. 16,1231 -1236.[Medline]
Luparello, C., Schilling, T., Cirincione, R. and Pucci-Minafra, I. (1999). Extracellular matrix regulation of PTHrP and PTH/PTHrP receptor in a human breast cancer cell line. FEBS Lett. 463,265 -269.[CrossRef][Medline]
Luparello, C., Santamaria, F. and Schilling, T. (2000a). Regulation of PTHrP and PTH/PTHrP receptor by extracellular [Ca++] and hormones in the breast cancer cell line 8701-BC. Biol. Chem. 381,303 -308.[Medline]
Luparello, C., Chimenti, S., Santamaria, F., Sirchia, R. and Ciacciofera, V. (2000b). Use of M-MLV RT, Rnase H-, point mutant, for mRNA-differential display analysis of parathyroid hormone-related peptide (PTHrP)-treated breast carcinoma cells. Promega eNotes, http://www.promega.com/catalog/country_select.asp?/enotes/applications/a p0019_tabs.htm&ckt=2
Luparello, C., Romanotto, R., Tipa, A., Sirchia, R., Olmo, N., Lòpez de Silanes, I., Turnay, J., Lizarbe, M. A. and Stewart, A. F. (2001). Mid-region parathyroid hormone-related protein inhibits growth and invasion in vitro and tumorigenesis in vivo of human breast cancer cells. J. Bone Miner. Res. 16,2173 -2181.[Medline]
Massfelder, T., Dann, P., Wu, T. L., Vasavada, R., Helwig, J. J.
and Stewart, A. F. (1997). Opposing mitogenic and
anti-mitogenic actions of parathyroid hormone-related protein in vascular
smooth muscle cells: a critical role for nuclear targeting. Proc.
Natl. Acad. Sci. USA 94,13630
-13635.
Matzura, O. and Wennborg, A. (1996). RNAdraw: an integrated program for RNA secondary structure calculation and analysis under 32-bit Microsoft Windows. Comput. Appl. Biosci. 12,247 -249.[Abstract]
McCauley, L. K., Koh, A. J., Beecher, C. A. and Rosol, T. J.
(1997). Proto-oncogene c-fos is transcriptionally regulated by
parathyroid hormone (PTH) and PTH-related protein in a cyclic adenosine
monophosphate-dependent manner in osteoblastic cells.
Endocrinology 138,5427
-5433.
Minafra, S., Morello, V., Glorioso, F., la Fiura, A. M., Tomasino, R. M., Feo, S., McIntosh, D. and Woolley, D. E. (1989). A new cell line (8701-BC) from primary ductal infiltrating carcinoma of human breast. Br. J. Cancer 60,185 -192.[Medline]
Naito, S., Shimizu, S., Matsuu, M., Nakashima, M., Nakayama, T., Yamashita, S. and Sekine, I. (2002). Ets-1 upregulates matrix metalloproteinase-1 expression through extracellular matrix adhesion in vascular endothelial cells. Biochem. Biophys. Res. Commun. 291,130 -138.[CrossRef][Medline]
Noguchi-Takino, M., Endo, Y., Yonemura, Y. and Sasaki, T. (1996). Relationship between expression of plasminogen activator system and metastatic ability in human cancers. Int. J. Oncol. 8,97 -105.
Orloff, J. J., Ganz, M. B., Nathanson, M. H., Moyer, M. S., Kats, Y., Mitnick, M., Behal, A., Gasalla-Herraiz, J. and Isales, C. M. (1996). A midregion parathyroid hormone-related peptide mobilizes cytosolic calcium and stimulates formation of inositol triphosphate in a squamous carcinoma cell line. Endocrinology 137,5376 -5385.[Abstract]
Ozaki, I., Zhao, G., Mizuta, T., Ogawa, Y., Hara, T., Kajihara, S., Hisatomi, A., Sakai, T. and Yamamoto, K. (2002). Hepatocyte growth factor induces collagenase (matrix metalloproteinase-1) via the transcription factor Ets-1 in human hepatic stellate cell line. J. Hepatol. 36,169 -178.[Medline]
Philbrick, W. M., Wysolmerski, J. J., Galbraith, S., Holt, E., Orloff, J. J., Yang, K. H., Vasavada, R. C., Weir, E. C., Broadus, A. E. and Stewart, A. F. (1996). Defining the roles of parathyroid hormone-related protein in normal physiology. Physiol. Rev. 7,127 -173.
Pirkkala, L., Nykanen, P. and Sistonen, L.
(2001). Roles of the heat shock transcription factors in
regulation of the heat shock response and beyond. FASEB
J. 15,1118
-1131.
Satyal, S. H., Chen, D., Fox, S. G., Kramer, J. M. and Morimoto,
R. I. (1998). Negative regulation of the heat shock
transcriptional response by HSBP1. Genes Dev.
12,1962
-1974.
Sirchia, R., Ciacciofera, V. and Luparello, C. (2001). Cloning differential display-PCR products with pGEM-T Easy vector system. Promega eNotes, http://www.promega.com/catalog/country_select.asp?/enotes/applications/a p0025_tabs.htm&ckt=2.
Soifer, N. E., Dee, K. E., Insogna, K. L., Burtis, W. J.,
Matovcik, L. M. Wu, T. L., Milstone, L. M., Broadus, A. E., Philbrick, W. M.
and Stewart, A. F. (1992). Secretion of novel mid-region
fragment of parathyroid hormone-related protein by three different cell lines
in culture. J. Biol. Chem.
267,18236
-18243.
Sokolov, B. P. and Prockop, D. J. (1994). A rapid and simple PCR-based method for isolation of cDNAs from differentially expressed genes. Nucleic Acids Res. 22,4009 -4015.[Abstract]
Spencer, W. E. and Christensen, M. J. (1999). Multiplex relative RT-PCR method for verification of differential gene expression. BioTechniques 27,1044 -1052.[Medline]
Tai, L. J., McFall, S. M., Huang, K., Demeler, B., Fox, S. G.,
Brubaker, K., Radhakrishnan, I. and Morimoto, R. I. (2002).
Structure-function analysis of the heat shock factor-binding protein reveals a
protein composed solely of a highly conserved and dynamic coiled-coil
trimerization domain. J. Biol. Chem.
277,735
-745.
Valìn, A., Garcìa-Ocaña, A., de Miguel, F., Sarasa, J. L. and Esbrit, P. (1997). Antiproliferative effect of the C-terminal fragments of parathyroid hormone-related protein, PTHrP-(107-111) and (107-139), on osteoblastic osteosarcoma cells. J. Cell Physiol. 170,209 -215.[CrossRef][Medline]
Wang, S. M., Khandekar, J. D., Kaul, K. L., Winchester, D. J. and Morimoto, R. I. (1999). A method for the quantitative analysis of human heat shock gene expression using multiplex RT-PCR assay. Cell Stress Chaperones 4, 153-161.[Medline]
Westermark, J. and Kahari, V. M. (1999).
Regulation of matrix metalloprotease expression in tumour invasion.
FASEB J. 13,781
-792.
Whitfield, J. F., Isaacs, R. J., Jouishomme, H., McLean, S., Chakravarthy, B. R., Morley, P., Barisoni, D., Regalia, E. and Armato, U. (1996). C-terminal fragment of parathyroid hormone-related protein, PTHrP (107-111), stimulates membrane-associated protein kinase C activity and modulates the proliferation of human and murine skin keratinocytes. J. Cell. Physiol. 166, 1-11.[CrossRef][Medline]
Wu, T. L., Vasavada, R. C., Yang, K., Massfelder, T., Ganz, M., Abbas, S. K., Care, A. D. and Stewart, A. F. (1996). Structural and physiologic characterization of the mid-region secretory species of parathyroid hormone-related protein. J. Biol. Chem. 271,24372 -24381.
Wysolmerski, J. J., Philbrick, W. M., Dunbar, M. E., Lanske, B.,
Kronenberg, H., Karaplis, A. and Broadus, A. E. (1998).
Rescue of the parathyroid hormone-related protein knockout mouse demonstrates
that parathyroid hormone-related protein is essential for mammary gland
development. Development
125,1285
-1294.
Wysolmerski, J. J. and Stewart, A. F. (1998). The physiology of parathyroid hormone-related protein: an emerging role as a developmental factor. Annu. Rev. Physiol. 60,431 -460.[CrossRef][Medline]
Yang, K. H., de Papp, A. E., Soifer, N. E., Dreyer, B. E., Wu, T. L., Porter, S. E., Bellantoni, M., Burtis, W. J., Insogna, K. I., Broadus, A. E. et al. (1994). Parathyroid hormone-related protein: evidence for isoform-and tissue-specific posttranslational processing. Biochemistry 33,7460 -7469.[Medline]
Yordy, J. S. and Muise-Helmericks, R. C. (2000). Signal transduction and the Ets family of transcription factors. Oncogene 19,6503 -6513.[CrossRef][Medline]
Ziegler, A., Simões-Wuest, A. P. and Zangemeister-Wittke, U. (1999). Optimizing efficacy of antisense oligodeoxynucleotides targeting inhibitors of apoptosis. In Methods in Enzymology (Vol. 314), Antisense Technology Part B Applications (ed. M. I. Phillips), pp.477 -490.San Diego, CA: Academic Press.
Zou, J., Guo, Y., Guettouche, T., Smith, D. F. and Voellmy, R. (1998). Repression of heat shock transcription factor HSF1 activation by HSP90 (HSP90 complex) that forms a stress-sensitive complex with HSF1. Cell 4,471 -480.