From the Departments of Neurological Surgery and
Laboratory Medicine and the
Graduate
Program in Neurobiology and Behavior, University of Washington School
of Medicine, Seattle, Washington 98195, the § Department of
Medical Genetics, Centre for Molecular Medicine and Therapeutics,
University of British Columbia, Vancouver, British Columbia V5Z 4H4,
Canada, the §§ Division of Human Biology, Fred
Hutchinson Cancer Research Center, Seattle, Washington 98109, and the
** Department of Molecular and Medical Pharmacology and Pediatrics, the
Brain Research Institute, University of California, Los Angeles, School
of Medicine, Los Angeles, California 90095
Received for publication, September 18, 2000, and in revised form, November 2, 2000
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ABSTRACT |
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Using a culture model of glial tumorigenesis, we
identified a novel gene that was up-regulated in malignant mouse
astrocytes following the loss of p53. The gene represents the murine
homologue of pescadillo, an uncharacterized gene that is essential for
embryonic development in zebrafish. Pescadillo is a strongly conserved
gene containing unique structural motifs such as a BRCA1 C-terminal domain, clusters of acidic amino acids and consensus motifs for post-translational modification by SUMO-1. Pescadillo displayed a
distinct spatial and temporal pattern of gene expression during brain
development, being detected in neural progenitor cells and postmitotic neurons. Although it is not expressed in differentiated astrocytes in vivo, the pescadillo protein is
dramatically elevated in malignant human astrocytomas. Yeast
strains harboring temperature-sensitive mutations in the
pescadillo gene were arrested in either G1 or G2 when grown in nonpermissive conditions, demonstrating
that pescadillo is an essential gene in yeast and is required for cell cycle progression. Consistent with the latter finding, DNA synthesis was only observed in mammalian cells expressing the pescadillo protein.
These results suggest that pescadillo plays a crucial role in cell
proliferation and may be necessary for oncogenic transformation and
tumor progression.
Astrocyte-derived neoplasms represent the most common type of
primary central nervous system tumor. Recent studies have been directed
toward defining the genes and gene products responsible for glial
tumorigenesis and progression. Mutation or loss of the tumor suppressor
gene p53 is thought to be an important event in the early neoplastic
transformation of astrocytes (1, 2). Studies of human glial tumors have
demonstrated that loss of the p53 gene is a frequent event in all
grades of astrocytoma. A subpopulation of cells will then proceed to
either mutate or delete the remaining copy of the p53 gene, leading to
uncontrolled cellular proliferation. Of interest are studies suggesting
that some high grade astrocytic malignancies result from clonal
expansion of cells that have lost or mutated their p53 (3). The genes
that may be adversely regulated following p53 alterations and that
might contribute to enhanced growth and survival are just now being
identified in different biological contexts but not yet in malignant
astrocytes (4-6).
We have previously established, using cultured astrocytes from
p53-deficient mice, an in vitro model of malignant
transformation recapitulating glial tumorigenesis (7). Early passage
p53 Glass-based high density microarray hybridization is an efficient way
to establish a detailed expression profile of up to 10,000 genes
simultaneously from a single tissue or cell type and has been
successfully used to analyze tumor- or tissue-specific patterns of gene
expression (8, 9). We have utilized cDNA arrays to identify genes
that are differentially expressed between malignant late passage
p53 The pescadillo gene was initially identified through an embryonic
mutation in zebrafish, which resulted in animals bearing small eyes,
impaired brain growth, and aberrant development of the liver and gut.
Pescadillo is widely and highly expressed during the first 3 days of
zebrafish development but was not observed in any adult tissues except
for the ovary, suggesting that its expression is principally limited to
developing tissues. In the present study, we present an analysis of the
pescadillo protein and provide evidence that pescadillo is a nuclear
cell cycle regulatory protein that is abnormally expressed in malignant
astrocytes and other transformed cell types.
Cell Culture--
COS-7, HeLa, and SW480 colon carcinoma cells
were obtained from the American Type Tissue Culture Collection. SNB-19
human glioblastoma cells (11) and p53+/+,
p53+/ Vector Construction and Cell Transfection--
Expression
constructs encoding pescadillo fused to the enhanced green fluorescent
protein (GFP)2 or to a Myc
epitope were created using a full-length human pescadillo cDNA
(derived from a Soares human fetal brain cDNA library obtained from
Research Genetics) inserted into the vector pEGFP-C3
(CLONTECH) (13) or a Myc-tagged expression vector
driven by the cytomegalovirus promoter (Cs2+Myc tag) (14),
respectively. DNA transfections were performed using a calcium
phosphate precipitation method (15) as we described previously (16).
Two days after transfection, cells were fixed with 4% paraformaldehyde
and analyzed using fluorescence microscopy, or cells were processed for immunoprecipitation.
Generation of an Anti-pescadillo Antibody--
A rabbit
polyclonal antibody was generated against a peptide derived from the
C-terminal 23 amino acids of the human pescadillo protein and used
after affinity purification. Custom antibody production service was
obtained from Quality Controlled Biochemicals (Hopkinton, MA).
Western Blotting and Immunoprecipitation--
Protein extracts
were prepared from tumor and nonmalignant brain tissue samples as
described previously (17). Cultured cells at confluency were lysed in
an extraction buffer as described previously (18). Human glioblastoma
and anaplastic astrocytoma surgical specimens, as well as brain tissue
surrounding the primary tumors (margin tissue) were generously provided
by Dr. John Silber from the Department of Neurological Surgery's Brain
Tumor Bank (University of Washington). Margin tissue is routinely
removed to minimize the chance of recurrence. All patients provided
informed consent, allowing their tissue to be used for research purposes.
Immunoblotting was performed as described previously (18) with slight
modification. The membranes were incubated overnight at 4 °C with an
affinity purified rabbit anti-pescadillo polyclonal antibody (0.5 µg/ml), an anti-SUMO-1 monoclonal antibody (1:1000, 21C7,
Zymed Laboratories Inc.), or an anti-c-Myc epitope
antibody (9E10 hybridoma culture supernatant (19)) followed by
horseradish peroxidase-conjugated secondary antibody (Santa Cruz).
Blots were subsequently reprobed with a mouse monoclonal anti-
For immunoprecipitation COS-7 cells were transfected with the
c-Myc-pescadillo construct, and protein extracts were prepared 2 days
later as described above for Western blotting. Protein extracts were
incubated with anti-c-Myc epitope antibody (9E10 hybridoma culture
supernatant) complexed with protein L agarose beads (ImmunoPure
immobilized Protein L gel; Pierce) overnight at 4 °C. Alternatively,
protein extracts were incubated with the anti-SUMO-1 antibody (5 µg)
for 3 h at 4 °C and then immunoprecipitated by using Protein L
beads for 1 h at 4 °C. The beads were washed 3× with lysis
buffer and resuspended in electrophoresis sample buffer. The
immunoprecipitates were resolved by SDS-PAGE, followed by blotting and
probing with an appropriate antibody as indicated.
Immunocytochemistry--
Cells were cultured in 4-well
multidishes (Nunc), fixed with 4% paraformaldehyde at room temperature
for 20-30 min, and then permeabilized with 0.2% Triton X-100 for 5 min. After blocking in a buffer containing 5% goat serum, 1% bovine
serum albumin, 0.1% thimerosal in phosphate-buffered saline for 30 min
at room temperature, cultures were incubated with primary antibodies
overnight at 4 °C, followed by incubation with Alexa Fluor
488-conjugated goat anti-mouse IgG and/or Alexa Fluor 594-conjugated
goat anti-rabbit IgG (both at 1:1000; Molecular Probes) for 2 h at
room temperature. Cultures were washed in phosphate-buffered saline and
examined under an inverted microscope with fluorescence optics.
Fluorescent micrographs were taken on Elite Chrome ASA 400 film (Kodak)
and scanned into Photoshop (Adobe).
Immunostaining of samples requiring a direct comparison was done in
single runs, and the subsequent processing of scanned images was
performed in an identical way for individual photographs. Anti-pescadillo antibody was used at 0.5 µg/ml, C23 monoclonal anti-nucleolin antibody (Santa Cruz) at 2 µg/ml, and G3G4 monoclonal anti-BrdU antibody (Developmental Studies Hybridoma Bank) at 1.5 µg/ml. For BrdU immunostaining, cultures were pretreated with DNase I
(10 units/ml, Life Technologies, Inc.) for 80 min at room temperature.
In Situ Hybridization Histochemistry--
In situ
hybridization was performed as described previously using
35S-labeled riboprobes (20). Briefly, animals were
sacrificed by decapitation, and their brains or whole heads were
rapidly frozen in isopentane and sectioned on a cryostat at 20-µm
thickness, post-fixed in 4% paraformaldehyde, washed, and stored at
Isolation and Characterization of Yeast Pescadillo Homologue
Temperature-sensitive Mutant Yeast Strains--
A complete deletion of
the yeast pescadillo homologue 1 (YPH1) open reading frame (YGR103w)
was generated using a PCR-mediated gene disruption method (21). HIS3
gene was amplified from pRS303 with primers
(5'-CTTGTAGAAAATAGTATAGTAACAGCGGTATCCTACTTATACAAGATTGTACTGAGAGTGCAC-3'; 5'-GAGAGGCTATTGGAAAAGAAGAGAAAACT- ATTTCTTGGAATCCTGTGCGGTATTTCACACCG-3') containing flanking sequences corresponding to the 5' and 3'
ends of YPH1 open reading frame, respectively. The HIS3 PCR product was
transformed in diploid yeast strain YPH982. Gene disruption was
confirmed in His+ colonies by PCR analysis.
His+ colonies were sporulated, and 10 tetrads were
dissected resulting in 2+:2
The temperature-sensitive mutations were generated using a PCR-mediated
procedure and integrated into the genome at the LEU2 locus (22).
Briefly, a 2-kilobase PvuII-PvuII DNA fragment
derived from a yeast genomic clone was cloned into pRS316 generating
pRS316YPH1, which was then used as a template for PCR random
mutagenesis. yph1-24 and yph1-45 mutant alleles were subcloned into
p679LEU2 vector and integrated at the LEU2 locus generating YJMF29
(Mata ura3-52 lys2-801 ade2-101 his3-
Cells growing in logarithmic phase at 25 °C were split and incubated
at either 25 or 37 °C for 3 h and subjected to DNA content analysis by fluorescence-activated cell sorting (FACS) (23) or treated
with 4', 6-diamidino-2-phenylindol and anti-tubulin antibody to monitor
nuclear and bud morphology using fluorescent microscopy.
Characterization of the Pescadillo
Protein--
p53
The human pescadillo gene is well conserved among a variety of species
and shares 40 and 79% sequence homology with yeast and zebrafish
pescadillo homologues, respectively (Fig.
1A). The human, zebrafish, and
yeast pescadillo proteins range in size from 582 to 605 amino acids,
and each contains several unique structural motifs including a BRCA1
C-terminal (BRCT) domain, two extensive acidic amino acid clusters
(glutamic and aspartic acid) in the C terminus, numerous presumptive
tyrosine/serine/threonine phosphorylation sites, and several conserved
consensus sequences for covalent attachment of SUMO-1
(small ubiquitin modification) (24)
(Fig. 1A). All three pescadillo homologues contain putative nuclear localization signals, suggesting that pescadillo may function in the nucleus.
The subcellular distribution of the pescadillo protein was evaluated by
immunostaining and by expressing a Myc or GFP fusion construct.
Endogenous pescadillo detected by an affinity purified polyclonal
antibody raised against a C-terminal peptide was typically observed as
highly localized, punctate immunofluorescence in the nucleus with faint
nucleoplasmic staining (Figs. 1B, panel A, and
4). This punctate immunofluorescence appeared to be associated with
nucleoli when viewed under phase contrast optics, which was confirmed
by double labeling with an anti-nucleolin antibody (Fig. 1B,
panels B and C). The distribution of endogenous
pescadillo protein was consistent with the pattern obtained after
expressing tagged forms of pescadillo, GFP-pescadillo (Fig.
1B, panel D) and Myc-pescadillo (Fig.
1B, panel E). This punctate nuclear distribution was reproducibly observed in every cell type transfected with the
GFP-pescadillo construct including human glioblastoma cells, HeLa
cells, normal human diploid fibroblasts, and p53
The predicted molecular mass for the human pescadillo protein is
69.2 kDa. Immunoblots of extracts prepared from COS-7 cells transfected
with human Myc-pescadillo and subjected to immunoprecipitation using an
anti-Myc antibody displayed two bands corresponding to molecular masses
of ~72 and 94 kDa (Fig. 2A).
Both bands were absent in extracts prepared from cells transfected with
the empty Myc-tagged vector. The 94-kDa protein (pes*) was a more
prominent band and was also observed in total cell extracts not
subjected to immunoprecipitation. The 72-kDa band corresponded to the
predicted size of the full-length pescadillo protein plus the Myc
epitopes contained in the expression construct. We hypothesized that
the 94-kDa band represented a post-translationally modified form of pescadillo because it was only observed in Myc-pescadillo transfected cells. We directly tested this possibility in the following study.
The pescadillo protein sequence was evaluated for the presence of
various post-translational modification motifs. We observed the
presence of several potential consensus sequences
((I/L)KXEX(K/R)) for the covalent modification by
the ubiquitin-like protein, SUMO-1 (24). The most highly conserved site
was found at position 516-521 (LKLEDK). The size of the 94-kDa band
was consistent with the covalent attachment of the SUMO-1 protein,
which has been shown to increase the apparent molecular mass of
proteins by ~20 kDa on SDS-PAGE (25). To test for the presence of the
SUMO-1 modification, immunoblots were performed using extracts prepared
from COS-7 cells transfected with Myc-pescadillo and subjected to
immunoprecipitation using an anti-SUMO-1 antibody. The blot was
subsequently probed with an anti-Myc antibody, and only the 94-kDa band
was detected (Fig. 2B). Identical results were obtained when
Myc-pescadillo was immunoprecipitated with the anti-Myc antibody, and
the blot was probed with the anti-SUMO-1 antibody (data not shown). In contrast to the results obtained from pescadillo transfection studies,
the unmodified band (70 kDa) represented the major endogenous form of
pescadillo protein in a human glioblastoma cell line, COS-7 cells and
in late passage p53 Pescadillo Expression Is Spatially and Temporally Regulated in the
Brain--
The cellular distribution and the temporal pattern of
pescadillo expression were evaluated in the mammalian brain to
determine which cell types in the brain normally express pescadillo.
Using in situ hybridization histochemistry, the pescadillo
mRNA was found to be widely and highly expressed throughout the
developing mouse brain and spinal cord at embryonic day 13, consistent
with its expression in neural progenitor cells and developing neurons (Fig. 3A). No hybridization
was detected with the sense probe or in tissue sections pretreated with
RNase A (data not shown). At the day of birth (P0; Fig. 3, B
and C), hybridization was present in the germinal zones and
developing neuronal fields, with relatively intense hybridization
detected in the developing hippocampus, piriform, and entorhinal
cortices. No hybridization signal was present within developing white
matter tracts, such as the corpus callosum (Fig. 3B).
Throughout postnatal development, the general pattern of distribution
did not appear to change (data not shown). In the adult, labeling was
detected in many, if not all, neurons of the brain, but with some
regional specificity. The most intense hybridization was present in
allocortical regions such as the hippocampus (Fig. 3, E-G),
especially in CA3 pyramidal neurons. Pescadillo mRNA was also
present within the rostral portions of the germinal zone lining the
lateral ventricles (Fig. 3D). This area is the site of
continuing neurogenesis in the adult brain. Little or no hybridization
signal was present within white matter in the adult (Fig.
3I), suggesting that normal glial cells do not express
pescadillo.
Pescadillo Protein Expression Is Elevated in Malignant
Astrocytes--
The in situ hybridization studies described
above demonstrate that pescadillo is not normally expressed by
differentiated astrocytes in vivo. Therefore, to confirm
that pescadillo is abnormally expressed in malignant astrocytes as
suggested from our cDNA microarray results,1 we
compared the expression of the pescadillo protein in normal and
transformed mouse astrocytes in culture. Low levels of pescadillo immunoreactivity were detectable in primary cultures of
p53
To demonstrate that pescadillo expression was not elevated in
established cell lines simply because of long term growth in culture,
we evaluated pescadillo expression in a primary culture of human
glioblastoma cells that was directly dissociated out of a surgically
resected tumor specimen without being passaged. The primary
glioblastoma culture expressed the same intense nuclear pescadillo
immunoreactivity (Fig. 4J) that was seen in the established cell lines (Fig. 4, I, K, and L). This
finding indicated that pescadillo expression may be elevated in
malignant astrocytes in vivo. We evaluated this possibility
by comparing the levels of pescadillo protein in matched tissue pairs
resected from individual patients consisting of malignant brain tumor
tissue and the brain tissue surrounding the tumor (margin). Pescadillo
was also evaluated in nonmalignant brain tissue obtained from patients
without neoplastic disease. Immunoblotting analyses demonstrated that
the pescadillo protein was highly expressed in malignant human glial
tumors including four glioblastomas and one anaplastic astrocytoma
(Fig. 5). When pescadillo expression
levels were normalized against actin expression levels, the malignant
tumors exhibited up to a 12-fold increase in pescadillo expression
relative to the matching margin tissue. Variations in pescadillo
expression between tumor-margin pairs may reflect the fact that margin
tissue is generally composed of normal glial cells and variable degrees
of infiltrative tumor cells. These findings clearly demonstrate that
the pescadillo protein is abnormally elevated in malignant human tumors
of astrocytic origin.
Pescadillo Regulates Cell Cycle Progression--
The BRCT (BRCA1 C
terminus) motif has been identified in a superfamily of proteins
involved in various aspects of DNA repair, recombination, and
checkpoint control (26, 27). Having identified a BRCT domain in the
pescadillo protein, we determined whether pescadillo was associated
with cell cycle progression.
Preliminary observations indicated that pescadillo expression was
suppressed when cells were maintained beyond confluency as shown for
HeLa cells (Fig. 6, A and
B), which was associated with reduced proliferation as
measured by BrdU labeling (Fig. 6C). To characterize the
relationship between cell density and pescadillo expression, HeLa cells
grown to a very high density (Fig. 6, A and B)
were replated at different densities. When plated at a low density
(Fig. 6D), 100% of the cells re-expressed pescadillo immunoreactivity (Fig. 6E), and ~20% of the cells had
incorporated BrdU (Fig. 6F) by 2.5 h after plating. In
contrast, when the cells were plated at a density that exceeded the
original density (Fig. 6G), the cells exhibited a further
reduction in pescadillo immunoreactivity (Fig. 6H) and
failed to incorporate BrdU (Fig. 6I). Alternatively, when a
strip of cells was scraped away from the confluent HeLa cell monolayer
(Fig. 6J), cells that migrated out into the denuded area
re-expressed pescadillo (Fig. 6K) with concomitant robust BrdU labeling (Fig. 6L). These results demonstrate that BrdU
labeling is only observed in cells expressing pescadillo
immunoreactivity, suggesting that pescadillo may be required for
progression through the cell cycle.
Direct evidence that pescadillo is necessary for cell cycle progression
was obtained by analyzing the yeast pescadillo homologue (YPH1). The
human pescadillo protein shares significant similarities with the yeast
Saccharomyces cerevisiae homologue, including the BRCT
domain and putative SUMOylation sites (Fig. 1A). In
addition, the yeast homologue protein localizes to the nucleus (data
not shown), suggesting that human pescadillo may subserve the same function as its yeast counterpart.
Deletion of the YPH1 open reading frame in a yeast diploid strain and
tetrad dissection demonstrated that YPH1 is an essential gene. To gain
more insight into the function of YPH1, two temperature-sensitive mutant alleles of YPH1 were generated by PCR-based mutagenesis and
integrated into the genome. At a permissive temperature (25 °C) the
two yeast mutant strains were able to grow on YPD plates (Fig.
7A). Cell cycle progression of
the two mutant strains was assessed by monitoring their DNA content by
FACS and cell morphology by bright field microscopy. At 25 °C both
wild type and mutant yeast strains exhibited the same FACS profile,
whereas at 37 °C yph1-24 (Fig. 7B) showed a
G1 phase delay, and yph1-45 (Fig. 7B) showed a
G2 phase delay. Cell morphology analysis confirmed the FACS
results obtained at the nonpermissive temperature, showing that 70% of
yph1-24 cells accumulated in the G1 phase, whereas 43% of
yph1-45 cells had a large bud with the nucleus at the neck and a short
spindle corresponding to a delay in G2/M transition (Fig.
7C). Sequence analysis indicated that both mutant strains contained a single point mutation in the BRCT domain (position 435 for
yph1-24, position 360 for yph1-45) as well as two and one additional
mutation, respectively, outside of the BRCT domain. Collectively, this
study confirms that pescadillo is necessary for cell cycle progression
and further suggests that the BRCT domain is essential for pescadillo
activity.
Loss of the tumor suppressor gene p53 has been implicated in the
genesis of many human malignancies. We attempted to define key
transcriptional changes accompanying neoplastic transformation in an
in vitro mouse model of astrocyte tumorigenesis (7). During
this process we discovered that the murine homologue of pescadillo was
highly expressed in malignant mouse astrocytes. The initial
characterization of this novel protein demonstrated: 1) that pescadillo
expression in the mouse brain is normally restricted to neural
progenitor cells and postmitotic neurons but is activated in malignant
mouse astrocytes in vitro following growth in the absence of
p53; 2) that pescadillo expression is significantly elevated in
malignant human astrocytomas in vivo; 3) that the loss of
p53 is necessary but not sufficient by itself to up-regulate pescadillo
expression in astrocytes; and 4) that pescadillo is a nuclear protein
necessary for cell cycle progression.
Pescadillo Protein Is Up-regulated in Malignant Cells--
Our
results demonstrate that a novel regulator of embryonic development in
zebrafish, pescadillo, is aberrantly expressed in a variety of
malignant mammalian cells. Our results specifically demonstrate that
pescadillo protein expression is elevated in malignant murine and
malignant human astrocytes in vitro. Moreover, the
pescadillo protein is expressed at significantly higher levels in
malignant human brain tumors relative to the adjacent tissue margins
taken from the same patient and "normal" brain tissue obtained from
patients without neoplastic disease. The tissue surrounding a brain
tumor is primarily composed of normal cells with variable degrees of
infiltrative tumor cells and inflammatory cells, suggesting that
increased pescadillo levels in the tumor extracts reflect expression by
malignant astrocytes. This is consistent with the intense pescadillo
immunoreactivity detected in primary cultures and established cell
lines derived from human glioblastomas. Interestingly, because
pescadillo expression was observed in neural progenitor cells but not
in differentiated astrocytes, it is conceivable that glial tumors arise
from pescadillo positive progenitor cells. Alternatively, glial tumors
could also develop from differentiated astrocytes with subsequent
genetic lesions activating pescadillo expression. Irrespective of the
cell of origin, our results clearly demonstrate that pescadillo is
abnormally expressed in malignant human tumors of astrocytic origin.
Pescadillo protein was also identified in a diverse array of malignant
human cell lines, including glioblastoma,
medulloblastoma,3 and colon
carcinoma cell lines. Particularly noteworthy is the significant
increase in pescadillo immunoreactivity that was detected in malignant
human breast carcinoma cells relative to normal human mammary
epithelial cells. Similarly, late passage malignant murine p53
The human pescadillo gene has been localized to chromosome 22q12.1
(28). Chromosome 22q deletions are common in human astrocytomas, but
partial deletions may occur distal to the pescadillo locus involving a
common region of deletion at 22q12.3-q13.1 (29). Amplification of the
human chromosome 22q12 locus has not been reported, suggesting that
increased levels of the pescadillo protein in malignant gliomas are not
the result of gene amplification. In addition, because the induction of
pescadillo mRNA in malignant mouse astrocytes is small relative to
increases in pescadillo protein,4 these changes may not
directly involve pescadillo gene transcription but rather
post-transcriptional mechanisms that influence pescadillo translation
or protein stability. Irrespective of the mechanism, pescadillo
induction in malignant cells is not restricted to cultured tumor cells
as demonstrated by its elevated expression in malignant human glial
tumors. These findings imply that pescadillo may subserve a function
that is essential for maintaining the malignant phenotype.
Elucidating the Function of the Pescadillo Protein--
The
biochemical function performed by pescadillo has not been defined, but
insights into its cellular role can be inferred from examining
structural motifs contained within the pescadillo protein sequence. The
most conspicuous identifiable domain in pescadillo is a protein motif
originally identified in the breast and ovarian cancer gene BRCA1 (26, 27). A unifying theme that has
evolved from studying the ~40 proteins that contain a BRCT domain is
that many of these proteins participate in DNA damage-responsive checkpoints (26, 27).
Several conserved consensus sequences for the covalent attachment of
SUMO-1 were also identified in pescadillo, and SUMO-1 modification was
demonstrated in the present study. Although the consequences associated
with SUMO-1 modification are largely unknown, there is evidence that
SUMO-1 may be important for protein targeting and protein-protein
interactions. For example, targeting of the RanGAP1 protein to the
nuclear pore is dependent upon the SUMO-1 modification (25). The SUMO-1
moiety appears to interact with important regulatory proteins such as
the death domain of the Fas/APO-1 and tumor necrosis factor receptors
(30). Overexpression of SUMO-1 confers protection against both
anti-Fas/APO-1 and tumor necrosis factor-induced cell death, suggesting
that SUMO-1 binding suppresses the activity of these receptors (30).
The SUMO-1 protein has also been shown to associate in a complex
together with human RAD52, RAD51, p53, and a ubiquitin-conjugating
enzyme, UBE2I (31). Such a complex is thought to be involved in DNA recombination and repair of double-strand breaks. These results illustrate that the SUMO-1 moiety and, by inference, proteins covalently modified by SUMO-1 can interact in protein complexes that
regulate cellular processes essential for cell proliferation and
survival. In support of this hypothesis, it has recently been shown
that SUMO conjugation is essential for viability in S. cerevisiae and is required for entry into mitosis (32).
The convergence of function observed between proteins expressing a BRCT
domain and proteins modified by SUMO-1 is consistent with the
possibility that pescadillo is involved in the regulation of a cell
cycle checkpoint. The contribution of pescadillo to the regulation of
cell growth was further substantiated by the identification and
characterization of temperature-sensitive pescadillo mutant yeast
strains. Yeast expressing mutant pescadillo displayed growth arrest in
the G1 or G2 phase of the cell cycle when
shifted to a nonpermissive temperature. Interestingly, both mutant
yeast strains contained a single point mutation in the BRCT domain, supporting the importance of this motif to pescadillo function. It is
not presently clear why the two mutant strains arrest in different
phases of the cell cycle. The yph1-24 mutant strain, which arrests in
G1, contains an additional mutation at amino acid 337 (E337G) close to the start site of the BRCT domain. This additional
mutation near the BRCT domain may further modify pescadillo binding
affinity or specificity, thus altering its activity. The results
obtained with the yeast mutants demonstrate that pescadillo is
essential for cell cycle progression, providing definitive support for
the correlation between pescadillo expression and S phase progression
observed in HeLa cells. The absence of such a critical cell cycle gene
during embryogenesis would account for the tissue abnormalities
observed in zebrafish lacking a functional pescadillo gene (10).
The results of the present study demonstrate that pescadillo, a novel
gene expressed principally in developing tissues, is inappropriately
expressed in adult human malignant brain tumors. Pescadillo is also
up-regulated in several different carcinoma cell lines, suggesting that
abnormalities in pescadillo expression may be a common feature of
malignancy. Interestingly, overexpressing pescadillo in nontransformed
cell lines, such as NIH-3T3 cells, promoted cell death instead of
inducing colony forming ability or serum-independent
growth,5 suggesting that pescadillo is not directly
oncogenic in nontransformed cells but rather may be necessary for
integrating the complex array of signals that promote progression
through the cell cycle. In contrast to nontransformed cells, pescadillo
overexpression did not affect the viability or growth of malignant cell
lines, which constitutively express high levels of the pescadillo
protein.3 Thus, the precise function of pescadillo in cell
cycle progression appears complex and requires further study.
Nevertheless, elucidation of pescadillo function may afford unique
insights into the process of neoplastic transformation and could
provide a new target for suppressing malignancy.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
astrocytes achieved higher saturation densities
than p53+/
and p53+/+ cells but did not
exhibit evidence of neoplastic transformation. With continued
passaging, p53
/
astrocytes exhibit a multistep
progression to a transformed phenotype displaying significant
aneuploidy and acquiring the ability to form large, well vascularized
tumors in nude mice. In marked contrast, p53+/+ astrocytes
fail to show a transformed phenotype and senesce after 7-10 passages.
Thus, loss of wild type p53 function promotes genomic instability,
accelerated growth, and malignant transformation in astrocytes. Using
this model system in conjunction with cDNA microarray technology,
we sought to examine the relationship between the progressive
phenotypic changes in astrocytes and alterations in gene expression.
/
astrocytes and nontransformed early passage
p53+/+ and p53+/
astrocytes.1 One expressed
sequence tag that was up-regulated in malignant p53
/
astrocytes represented the murine homologue of a novel zebrafish gene
known as pescadillo (10).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, and p53
/
astrocytes were prepared
and maintained as described previously (7). Normal human mammary
epithelial cells and MCF-7 human breast carcinoma cells were generously
provided by Dr. Karen Swisshelm (Department of Pathology, University of
Washington). A primary culture of human glioblastoma cells was
established from a primary glioblastoma resected at the University of
Washington by previously described methods (12). The procedure for
obtaining and culturing human tumor tissue received human subjects
approval from the Institutional Review Board Committee of the
University of Washington, and informed consent was obtained from the
patient prior to surgery. All cells were routinely maintained in
Dulbecco's modified Eagle's medium/Ham's F-12 medium, except for
HeLa cells, which were maintained in Dulbecco's modified Eagle's
medium, with 10% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin at 37 °C in 5% CO2.
-actin
antibody (AC-15, 1:40,000; Sigma) to normalize for protein loading. The ratio of the protein band intensities corresponding to pescadillo and
actin (pescadillo/actin) on the x-ray film was determined by
densitometric imaging using NIH Image analysis software (version 1.62).
75 °C. A partial cDNA fragment encoding the mouse pescadillo
gene was cloned into Bluescript (Stratagene). An antisense RNA probe
was prepared from the NspI- and PvuII-digested
plasmid using T7 polymerase in the presence of [35S]UTP.
Sense riboprobes transcribed with T3 polymerase and RNase pretreated
sections were used as negative controls. Tissue sections were washed,
acetylated, defatted, and incubated with the riboprobe overnight.
Following RNase A treatment, sections were washed in descending
concentrations of SSC, dried, and apposed to autoradiographic film
(
-max, Amersham Pharmacia Biotech) prior to dipping in Kodak NTB2
emulsion. The slides were then developed and counter-stained with
cresyl violet.
segregation for viability. All viable
spores were His
, indicating that YPH1 is an essential gene.
200
trp1-
63 leu2-
1 yph1::HIS3 yph1-24:LEU2) and YJMF30 (Mata ura3-52 lys2-801 ade2-101
his3-
200 trp1-
63 leu2-
1 yph1::HIS3 yph1-45:LEU2), respectively.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
astrocytes maintained in culture for
20 or more passages display the ability to form colonies in soft agar
and form large, well vascularized tumors in nude mice, in contrast to
p53+/+ astrocytes and early passage p53
/
and p53+/
astrocytes (7). We attempted to characterize
changes in gene expression that were associated with these markedly
different behavioral phenotypes using glass-based high density cDNA
arrays. We observed a reproducible series of genes whose expression
pattern was altered as a result of continuous proliferation in the
absence of p53.1 One cDNA in particular was expressed
at higher levels in late passage p53
/
astrocytes
relative to early passage p53
/
or p53+/+
astrocytes and matched the sequence of an uncharacterized gene referred
to as pescadillo (10). The pescadillo gene was selected for further
study in malignant astrocytes because its expression was largely
restricted to periods of embryonic development, and it was shown to be
essential for nervous system development in zebrafish.
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Fig. 1.
Sequence conservation among pescadillo
homologues and the subcellular distribution of the pescadillo
protein. A, schematic depicting the structural
conservation among human, zebrafish, and yeast pescadillo proteins.
Identical amino acids are shaded dark gray, and similar
amino acids are shaded light gray. This schematic
illustrates the location of several important structural motifs
including the BRCT domain, the two acidic domains, the presumptive
nuclear localization signals, and the most conserved site for
SUMOylation. The human pescadillo sequence was obtained from the
GenBankTM data base and analyzed for structural and
functional motifs using Pfam, a data base of multiple alignments of
protein domains and conserved protein regions (33). The
asterisks denote the location of the point mutations in the
BRCT domain of the yph1-24 and yph1-45 mutant yeast strains described
in Fig. 7. B, subcellular localization of pescadillo.
Panels A-C, expression of endogenous pescadillo and
nucleolin in HeLa cells. Pescadillo immunoreactivity was detected
predominantly in the nucleolus as confirmed by colocalization with
nucleolin, a nucleolar marker, with very faint nucleoplasmic staining
seen in some cells. Essentially no staining above background was
observed in the cytoplasm. Panel D, confocal microscopic
image of a COS-7 cell transfected with a GFP-pescadillo fusion
construct 2 days after transfection. Note that there is no
cytoplasmic fluorescence observed. Panel E, malignant, late
passage p53 /
mouse astrocytes (passage 40) transfected
with a Myc-tagged pescadillo construct and processed for Myc
immunoreactivity 2 days later. Bar, 50 µm (except for
D, where the bar indicates 18 µm).
/
mouse
astrocytes (data not shown). These results confirm the specificity of
the anti-pescadillo antibody as well as the normal distribution of the
epitope-tagged forms of pescadillo. Interestingly, postmitotic primary
cortical neurons in culture displayed a different distribution pattern
than that observed in the proliferating cell types; the distribution
was significantly more diffuse throughout the nucleus (data not shown).
These results demonstrate that pescadillo is a nuclear protein whose
compartmentalization within the nucleus may be regulated in a cell
type-dependent manner.
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Fig. 2.
Expression and post-translational
modification of the pescadillo protein. A, expression
of the pescadillo protein. At 24 h after plating COS-7 cells
were transfected in duplicate with a Myc-tagged pescadillo
expression vector (Myc-pes) or an empty Myc-tagged vector
(vector) using a calcium-phosphate precipitation technique
as described in the experimental procedures. Two days after
transfection cell extracts were prepared and subjected to
immunoprecipitation using the 6E10 anti-Myc antibody. The
immunoprecipitated proteins and total cell lysates were resolved by
SDS-PAGE on a 10% gel and probed with the anti-Myc antibody. Two
Myc-immunoreactive bands were detected, one with the predicted
molecular mass of pescadillo (pes) and the other with a
higher molecular mass (pes*). B, SUMO-1
modification of the pescadillo protein. Protein extracts prepared as in
A were subjected to immunoprecipitation with the 21C7
anti-SUMO-1 antibody. The immunoprecipitates were resolved by SDS-PAGE
and probed with the anti-Myc antibody. The arrow marks the
location of the SUMOylated Myc-pescadillo band that corresponds in size
to the upper Myc-immunoreactive protein band detected in A. C, pescadillo expression in malignant and immortalized cell
lines. Cell extracts were prepared from SNB19 human glioblastoma cells,
COS-7 cells, and p53 /
malignant mouse astrocytes
(passage 27), resolved by SDS-PAGE, and probed with an affinity
purified anti-human pescadillo rabbit polyclonal antibody.
/
mouse astrocytes (Fig.
2C). A band migrating at about 92 kDa, consistent with the
presence of SUMO-1, was detected in these cell lines if a film was
overexposed (data not shown). These results confirm the presence and
molecular mass of the pescadillo translation product and demonstrate
that under some circumstances the pescadillo protein may be modified by
the covalent attachment of SUMO-1.
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Fig. 3.
Pescadillo expression is spatially and
temporally regulated in developing mammalian brain.
A-E, film autoradiograms of in situ
hybridization in brain using a pescadillo antisense riboprobe. Tissue
sections from embryonic day 13 (E13) mouse embryos (A),
postnatal day 0 (P0) (B and C), and adult mice (3 months) (D and E) were processed as described
under "Experimental Procedures" and then apposed to
autoradiographic film for the same amount of time. Note the relatively
high levels of expression within the germinal zones at all ages. At
postnatal ages, expression was clearly present in numerous neurons,
such as those in the hippocampus and piriform cortex (D and
E). No hybridization was detectable within white matter
tracts at postnatal ages. Lower levels of hybridization were present in
the neostriatum. F and G, brightfield
(counter-stained with cresyl violet) and darkfield pair of
emulsion-coated slides showing cellular distribution of pescadillo
mRNA within the adult hippocampus. Note the relatively high levels
within the CA3 pyramidal cell layer. The darkfield signal was
attenuated slightly because of counter staining. H, high
power view of silver grains overlying the CA3 pyramidal cells of the
adult hippocampus. The arrow points to a pyramidal cell.
I, high power view of white matter adjacent to the CA3
pyramidal cells, demonstrating the lack of hybridization in glial cells
(arrow). Scale bar, 965 µm for A-E,
200 µm for F and G, and 10 µm in H
and I.
/
(Fig. 4,
A and C) and p53+/+ astrocytes (data
not shown). As previously reported, p53+/+ astrocytes and
early passage p53
/
astrocytes display a normal pattern
of contact-inhibited growth and fail to form tumors in nude mice (7).
Pescadillo immunoreactivity in primary cultures of p53
/
astrocytes was localized to the nucleus and in the same punctate pattern that was observed following transfection with GFP-pescadillo. Also, pescadillo immunoreactivity remained constant in these cultures independent of cell density or length of time in culture (up to 3 weeks; data not shown). In marked contrast, pescadillo immunoreactivity was significantly elevated in malignant, late passage
p53
/
mouse astrocytes (Fig. 4, B and
D), and the subcellular distribution remained the same in
malignant astrocytes. Significant up-regulation of pescadillo protein
expression was also observed in MCF-7 human breast carcinoma cells
(Fig. 4, F and H) compared with normal human
mammary epithelial cells (Fig. 4, E and G).
Additionally, significant levels of pescadillo immunoreactivity were
detected in a variety of human cancer cell lines including
glioblastoma, colon carcinoma (Fig. 4), and cervical carcinoma cells
(HeLa cells, Figs. 1 and 6).
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Fig. 4.
Pescadillo immunoreactivity is elevated in
malignant cell lines. A-D, normal and malignant
p53 /
astrocytes. E-H, normal and malignant
human mammary epithelial cells. Primary culture (7 days in culture) of
p53
/
astrocytes (A and C)
expressed a low level of pescadillo protein in contrast to late passage
(passage 27), malignant p53
/
astrocytes (B
and D), which showed significant up-regulation of pescadillo
expression. Similarly, only weak immunoreactivity was seen in a normal
human mammary epithelial cell line (E and G),
whereas robust immunostaining was detected in the MCF-7 human breast
carcinoma cell line (F and H). Thus, elevated
pescadillo expression is associated with malignant transformation.
I-L, pescadillo expression in established and malignant
cell lines and in a primary culture of human glioblastoma. Note the
strong nucleolar staining in all types of cell lines. The
insets in the upper right corner of F and H
depict cells stained with normal IgG (0.5 µg/ml) in place of the
pescadillo antibody. Bar, 50 µm.
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Fig. 5.
Pescadillo protein is abnormally expressed in
adult human glioblastomas. Pescadillo protein levels were compared
between tumor tissues (T) and margin tissues (M)
surrounding each tumor for glioblastoma multiforme and anaplastic
astrocytoma, both of which are highly malignant, as well as
nonmalignant temporal lobe cortex that was removed during the resection
of a cavernous malformation (Normal temporal cortex). Each
tumor-margin pair represents paired samples from the same patient.
Protein extracts (20 µg of protein/lane) were resolved by SDS-PAGE on
a 10% gel, and the blot was probed with the rabbit anti-pescadillo
antibody, followed by probing with an anti-actin antibody to assess
protein loading. For the four pairs of samples shown in the left
blot, pescadillo expression was normalized against actin bands and
compared between tumor and margin. The resulting fold increase in
pescadillo expression was, from left to right,
1.3, 6.8, 6.2, and 12.2, respectively. The low but significant level of
pescadillo expression in the normal cortex sample is presumed to
represent neuronal pescadillo expression as temporal lobe contains
numerous neurons that express pescadillo as shown by in situ
hybridization in Fig. 3.
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Fig. 6.
Pescadillo expression is associated with
replicating cells. HeLa cells were grown to a post-confluent
density (2 days after confluency), fixed and processed for nuclear
staining (A) with Hoechst 33258 (2.5 µg/ml) and pescadillo
immunoreactivity (B). A parallel culture was labeled with
BrdU (10 µg/ml) for the next 8 h to assess proliferative
activity at this high cell density (C). Confluent sister
cultures, as shown in A, were trypsinized and replated
either at a low density (
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Fig. 7.
The yeast pescadillo homologue is essential
for growth and regulates cell cycle checkpoints. A, the
wild type and the two YPH1 temperature-sensitive mutant strains
(yph1-24; yph1-45) were streaked on YPD plates, placed at 25 °C or
37 °C for 3 days, and photographed. B and C,
yeast strains expressing temperature-sensitive mutant pescadillo
homologues exhibit cell cycle arrest at the nonpermissive temperature.
Yph1-24 and yph1-45 cells growing in logarithmic phase at 25 °C
were split and incubated at either 25 or 37 °C for 3 h and then
subjected to DNA content analysis by fluorescence-activated cell
sorting (B). Cells that were split and incubated at 37 °C
were further analyzed for growth arrest characteristics based on
morphological hallmarks as revealed by nuclear and tubulin staining
(C).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
astrocytes express much higher levels of
pescadillo immunoreactivity than normal p53+/+ or
nontransformed early passage p53
/
astrocytes. Although
the p53 status of these various cell lines is not known, the results
obtained with nontransformed early passage p53
/
astrocytes suggest that the loss of p53 alone is not sufficient to
activate pescadillo expression.
![]() |
ACKNOWLEDGEMENTS |
---|
We gratefully acknowledge Chizuru Kinoshita for technical expertise and Joseph T. Ho for reviewing the manuscript. The G3G4 anti-BrdU antibody was obtained from the Developmental Studies Hybridoma Bank maintained by the University of Iowa (Department of Biological Sciences, Iowa City, IA 52242).
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FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grant NS35533 and a Royalty Research Fund grant (to R. S. M.), National Institutes of Health Grant CA75173 (to P. S. N.), and Department of Energy Contract DE-FC03-ER60615 (to H. I. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Present address: INSERM EPI 9906, IFRMP, Faculté de Médecine et de Pharmacie, 22 Bld Gambetta, 76183 Rouen, France.
¶¶ To whom correspondence should be addressed: Dept. of Neurological Surgery, University of Washington School of Medicine, Box 356470, Seattle, WA 98195-6470. Tel.: 206-543-9654; Fax: 206-543-8315; E-mail: yael@u.washington.edu.
Published, JBC Papers in Press, November 8, 2000, DOI 10.1074/jbc.M008536200
1 G. Foltz, J. Schuster, Y. Kinoshita, L. Hood, R. S. Morrison, and P. S. Nelson, manuscript in preparation
3 A. D. Jarell and R. S. Morrison, unpublished results.
4 J. Schuster, G. Foltz, P. S. Nelson, and R. S. Morrison, unpublished results.
5 Y. Kinoshita and R. S. Morrison, unpublished results.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: GFP, green fluorescent protein; PAGE, polyacrylamide gel electrophoresis; BrdU, bromodeoxyuridine; PCR, polymerase chain reaction; FACS, fluorescence-activated cell sorting; BRCT, BRCA1 C terminus.
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