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Advances in Brief |
San Raffaele Scientific Institute, 20132 Milan [F. S., F. Vi., S. G., G. S., M. C., E. V., F. Ve., A. D., P. C. M.], and Department of Medical Sciences, University of Eastern Piedmont, 28100 Novara [S. B.], Italy
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ABSTRACT |
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Introduction |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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Prominent nuclear changes, including changes in size and shape, enlarged nucleoli, and abnormal heterochromatin distribution, traditionally are recorded by pathologists during diagnostic procedures. Indeed, nuclear changes reflect the relationships between extracellular matrix, cell adhesion, cytoskeleton, and nuclear matrix (2 , 3) that are in a broad network of events that control gene expression and contribute to cancer evolution (4) .
Recently, a highly conserved protein, p27BBP, which interacts with the cytodomain of integrin subunit ß4, was identified and cloned (5) . Characterization studies showed that p27BBP was expressed ubiquitously, but the highest levels were found in renewing epithelia, including the intestinal mucosa. Biochemical and morphological analyses showed that p27BBP was found in both the cytoplasm of epithelial cells, in association with intermediate filaments associated with adhesion structures, and the nucleus, as a nuclear matrix-associated protein accumulating in the nucleolus (6) . The latter location was also found in non-epithelial cells that do not express ß4. Depletion of the p27BBP homologue in the budding yeast, Saccharomyces cerevisiae, caused cells to arrest in G1 as a result of reduced levels of free 60S ribosomal subunits, indicating that this molecule is essential for cell growth. The effects of this loss of yeast p27BBP were reversed by transfection with the human gene (6) . These data strongly suggested that p27BBP has a very conserved function in ribosome biogenesis and became linked to epithelial adhesion much later in evolution.
Because of its nucleolar location and its putative adhesion role
involving 
6ß4, we assumed that p27BBP might
represent a link between the environment of epithelial cells,
e.g., the basement membrane, and the nuclear machinery
controlling ribosome assembly and that this connection might be altered
in colorectal neoplasias, where transformed cells lose proper
relationship with their environment and concurrently proliferate and
migrate. Indeed, biological connections between cell adhesion and
altered nuclear signaling have been described in the E-cadherin,
ß-catenin, LEF-1 signaling pathway (7)
, and nucleolar
alterations have also been described in colorectal carcinomas (8
, 9)
. Therefore, we studied the expression of
p27BBP in a group of controlled colorectal
neoplasias.
Our study showed that the nuclear matrix-associated p27BBP is overexpressed in colorectal neoplasms and is particularly high in carcinomas. The mRNA that codes for p27BBP is also up-regulated in carcinomas versus normal mucosa, but to date, no gross alteration of its gene has been detected.
Although the molecular mechanisms underlying its overexpression are still largely unknown, our data suggest that changes in p27BBP expression may play a role in the processes governing colorectal malignant transformation. In addition, p27BBP could represent a useful marker for diagnostic purposes.
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Materials and Methods |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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, -
smooth, - cytoplasmic actins
(AAL20) antiserum was a generous gift from G. Gabbiani, University of
Geneva, Geneva, Switzerland. Goat anti-lactate dehydrogenase polyclonal
antibody and antigoat horseradish peroxidase-conjugated IgG were
purchased from Chemicon International Inc., and Santa Cruz
Biotechnology, respectively.
Immunohistochemical and Histochemical Staining.
Five-µm cryosections were collected on polylysine-coated slides and
fixed in 3% paraformaldehyde in PBS for 10 min at room temperature.
Sections were treated with 0.5% Triton X-100 for 10 min, followed by
0.3% hydrogen peroxide in PBS for 30 min, and then blocked with normal
goat serum for 70 min and incubated for 2 h at room temperature
with the anti-p27BBP antibody at a 1:200
dilution. After being washed with PBS and incubated for 80 min
at room temperature with the biotin-conjugated secondary antirabbit at
a 1:200 dilution, the sections were labeled using the avidin-biotin
amplification method (ABC kit Vectastain; Vector Laboratories) and
revealed by horseradish peroxidase, using 3,3'-diaminobenzidine as
chromogen (Biogenex, San Ramon, CA). The slides were slightly
counterstained with Harris’ hematoxylin. Staining for ß4 integrin
was performed according to the above procedure, except for the
treatment with Triton X-100. Anti-ß4 monoclonal antibody was used at
a 1:200 dilution, labeled with the biotin-conjugated antimouse IgG1 at
a 1:100 dilution, and revealed by three-amino-9-ethylcarbazole.
Silver staining was performed on cryosections fixed in a methanol-acetic acid (3:1, v/v) solution for 30 min at 4°C. The sections were treated for 30 min at room temperature with the following mixture: 2 volumes of 50% silver nitrate and 1 volume of formic acid-jelly mixture (2% of gelatin in 1% aqueous formic acid). The sections then were washed in hot distilled water. The specimens were treated briefly with thiosulfate, washed with distilled water, and counterstained with methyl green.
Western Blot and Fractionation Analysis.
Total protein extracts were obtained by homogenizing tissues in boiling
lysis buffer (2.5% SDS in 0.125 M Tris-HCl, pH 6.8),
boiled 20 min, sonicated, and centrifuged at 14,000 rpm. All samples
were resuspended in Laemmli buffer, and equal amounts of protein (30
µg) were electrophoresed on a denaturing 12% SDS-acrylamide gel and
transferred to Immobilon P membranes (Millipore). Filters were
blotted with the rabbit anti-p27BBP antiserum, at
a 1:1000 dilution, and then with horseradish peroxidase-conjugated
protein A (Amersham Pharmacia Biotech). Specific bands were revealed by
enhanced chemiluminescence (ECL Western blotting analysis system;
Amersham Pharmacia Biotech). Filters were stripped in 0.3 M
NaOH for 5 min; reblotted with the rabbit antiactins (AAL20), diluted
1:1000; and revealed as described.
Subcellular fractionation for nuclei and cytoplasm was performed on the HT-29 colorectal adenocarcinoma-derived cell line (ATCC no. HTB-38) and normal fibroblasts, isolated from human foreskin (generous gift of E. Bianchi, San Raffaele Scientific Institute, Milan, Italy). Cells were washed with PBS and lysed by incubation with STM buffer (250 mM sucrose, 10 mM Tris, pH 8, 10 mM MgCl2, 10 µg/ml aprotinin) at 4°C. Cells were disrupted with 40 strokes in a Dounce homogenizer and centrifuged at 500 x g. The pellet (nuclear fraction) was resuspended, sonicated in lysis buffer containing 2.5% SDS, and cleared by centrifugation. Fractions (20 µg) were analyzed for p27BBP and lactate dehydrogenase by 12% SDS-PAGE.
Isolation of RNA and Northern Blot Hybridization.
Total cellular RNA was isolated from snap-frozen human tissues,
homogenized in guanidinium thiocyanate solution and extracted in
phenol-chloroform. Ten µg of denatured total RNA samples were run on
a 1% denaturing agarose gel and transferred to Hybond-N filters
(Amersham Pharmacia Biotech). Hybridization was carried out with
1 x 106 cpm of random
primer-labeled full-length p27BBP cDNA probe in
50% formamide at 42°C. The expression of the
p27BBP transcripts was quantified by
densitometric analysis of X-ray films (Kodak) and normalized for human
glyceraldehyde-3-phosphate dehydrogenase mRNA.
Southern Blotting.
The human 745-nucleotide cDNA encompassing the entire
p27BBP open reading frame was labeled with
[32P]dCTP and used as probe (Prime-a-Gene
Labeling System; Promega). Five µg of high-molecular weight DNA
derived from tumor tissues of 28 patients were digested with
PstI and BamHI and then run on a 0.8% agarose
gel, followed by transfer to a nylon membrane (Hybond-N+; Amersham).
The membrane was sequentially hybridized with a cDNA probe at 65°C
for 16 h in 0.25 M sodium phosphate buffer
(pH 6.8; made by diluting a stock containing 2 M
NaH2PO4 and 2
M
Na2HPO4), containing
7% SDS, and 200 µg/ml denatured sonicated salmon sperm DNA. After
hybridization, the membrane was washed once in 2x
SSPE3
-0.1% SDS for 20 min at room temperature and then, for high stringency,
washed sequentially with 1x SSPE-0.1% SDS, 0.5x SSPE-0.1%
SDS, and 0.1x SSPE-0.1% SDS for 20 min at 65°C.
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Results |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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).
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). This pattern was also observed elsewhere in the mouse
as well as human gut (not shown) and was specifically localized in the
nucleolus. The same staining was also found in human colonic epithelium
(Fig. 1, F and H)
, and was abolished by
incubating the sections with rabbit pre-immune serum or with the
anti-p27BBP antiserum pre-adsorbed with the
recombinant protein (Fig. 1G)
. Some cytoplasmic staining was
preserved, particularly in goblet cells, and was attributed to
background unspecific reaction (Fig. 1, F–H
).
In gut epithelial cells, p27BBP was concentrated
in one or more regularly shaped, small nucleolar dots, whereas in
stromal cells, p27BBP usually was in a single,
barely visible dot (Fig. 1, A, F, and H)
. The signal generated by p27BBP was
enriched in rapidly cycling crypt cells and decreased in the villus,
where shedding of cells undergoing apoptosis occurs progressively
(10)
. In human colonic epithelium,
p27BBP was likewise observed in the nuclei of
glandular crypt cells but was mostly decreased in cells moving toward
the surface [Fig. 2A, B, and C
(*)].
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Distribution of p27BBP and ß4 in Adenomas and
Carcinomas.
Distribution of p27BBP in normal intestinal
epithelium prompted us to investigate its expression in 9 colorectal
adenomas and 48 carcinomas by immunohistochemistry.
In all cases of tubulo-villous adenomas, irrespective of their degree
of dysplasia, p27BBP was localized in the nucleus
as regularly shaped, intensely stained multiple dots (Fig. 2D)
. This pattern was detected in most dysplastic cells, but
some were similar to frankly malignant cells as described below.
In all carcinoma cells [Fig. 2, C
(arrowhead),
E–I], p27BBP always appeared
enriched in large, irregularly shaped dots; some had aberrantly large
accumulations of p27BBP that may reflect
polyploidy or any cell atypia and may redistribute with chromosomes at
mitosis (Fig. 2, E, G, and H
,
indicated by arrows). In contrast, the
p27BBP content in stromal cells was much lower
and occasionally was structured in tiny dots (Fig. 2C
versus Fig. 1H
).
The expression of ß4 was investigated in most samples by
immunohistochemistry, and showed a change during malignant
transformation. In normal colorectal glands, ß4 was located basally
in contact with the basement membrane and facing the stroma (Fig. 3A)
. In adenomatous dysplastic cells, ß4 was redistributed
pericellularly but was still present at the basal aspect (Fig. 3B)
. The distribution and the intensity of immunostaining
for ß4 was heterogeneous in colorectal tumors. In moderately
differentiated cancers, ß4 expression at the carcinoma-stroma
boundary was weak and discontinuous and clearly associated with a
subverted redistribution within the cell membrane. The same phenomenon
was observed in poorly differentiated tumors in association with a
strong and diffuse pericellular and cytoplasmic positivity (Fig. 3, C and D)
.
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p27BBP Distribution Is Similar to That of AgNORs and Is
Associated with the Nuclear Matrix.
The cellular localization of p27BBP was strongly
reminiscent of a group of nucleolar proteins, AgNORs, that are
selectively stained by silver methods and specifically associated with
transcriptionally active sites of ribosomal DNA. Sections from
colorectal carcinomas (Fig. 4, C and D)
or the associated normal mucosa (Fig. 4, A and B)
were stained using the
silver-staining technique, revealing brown nuclear AgNOR dots. In both
normal (Fig. 4B)
and neoplastic (Fig. 4D)
cells,
AgNORs were localized in the nucleus in a pattern similar to that shown
by p27BBP antibodies (Fig. 4, A and C)
.
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, thus suggesting that the abnormally accumulated protein
in transformed cells is associated with the nuclear matrix. Taken together, these data indicate that p27BBP may also be part of a complex involved in 60S ribosomal subunit assembly in in situ tumor cells, as was shown previously in cultured neoplastic cells (6) . The accumulation of p27BBP may also modify the process of ribosomal assembly in transformed cells of solid tumors.
p27BBP Is Overexpressed in Adenomas and Carcinomas.
As suggested by immunohistochemistry and to quantify the accumulation
of p27BBP in carcinoma cells, protein levels were
analyzed by Western blot for 34 carcinomas and 8 tubulo-villous
adenomas. In the latter, paired analysis of blotted samples (ratio of
normal mucosa versus dysplastic adenomatous sample, ± SE) yielded 3.33 ± 0.56, which roughly matched
the immunohistochemical data.
In 30 carcinomas, densitometric analysis of paired blotted samples (ratio of normal mucosa versus dysplastic adenomatous sample, ± SE) gave a rough increase of 5.6 ± 0.9. From this group, four cases did not show any increase but rather a slight decrease in p27BBP expression (0.8 ± 0.1).
The biochemical analysis showed a progressive increase of
p27BBP from normal mucosa to dysplastic adenomas
and carcinomas (Fig. 5A)
. Moreover, p27BBP accumulated in
carcinomas proportionally to the Astler-Coller modified Duke’s
malignancy stages (Fig. 5B)
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1.5 (Fig. 5C)
. These data demonstrate that the increased levels of p27BBP in dysplastic adenomas and carcinomas match the abnormal pattern observed in the nuclei of transformed cells. p27BBP accumulation may be detected in early precancerous lesions and markedly increases in carcinomas according to progression in Duke’s tumor stage, suggesting that the alteration of p27BBP is an early event in the process of transformation and might eventually be linked to other malignancy parameters. In addition, these results show that the ratio of cytoplasmic to nuclear p27BBP is altered in cancer cells.
p27BBP Is Up-Regulated at the Transcriptional Level.
To understand the mechanisms underlying the overexpression of
p27BBP protein, its mRNA was investigated by
Northern blotting in five carcinomas and their normal mucosa. The
filters were hybridized with the human p27BBP
cDNA probe, which revealed a 1100-bp transcript in both normal and
neoplastic tissue. A significant up-regulation of
p27BBP mRNA was observed in all cases of
carcinomas analyzed (e.g., Fig. 5D
) in which the
transcript levels ranged between 2- and 11-fold those of the normal
counterparts.
To establish whether the up-regulation of p27BBP resulted from rearrangement or amplification of its gene, 28 carcinoma cases were screened by Southern blot analysis. No obvious amplification of the gene was identified, as indicated by identical band patterns (not shown).
These results support the idea that the major mechanism responsible for p27BBP increase in adenomas and carcinomas stems from transcriptional regulation that may also involve increased messenger stability.
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Discussion |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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Here we report that p27BBP is extraordinarily overexpressed in colorectal neoplasms and in notably higher concentrations in carcinomas. Its tissue and cellular distributions match those of AgNORs, and we suggest that p27BBP may indeed be one molecule responsible for AgNOR-based diagnostic procedures that have been related to malignancy by surgical pathologists (8) . The present availability of controlled reagents also allows immunochemical measurement of its expression by morphology and Western blotting; we therefore suggest that p27BBP may become one additional and powerful tool in the hands of surgical pathologists, particularly for the fine detection of micrometastases in lymph nodes. In addition, the mRNA coding for p27BBP is highly increased in tumor versus normal colorectal tissues, suggesting that malignancy is accompanied by an up-regulation of its genetic expression.
The role of the increase in p27BBP in colorectal
tumors and also in other tumor cell
types4
may be interpreted as a simple consequence of increased
protein synthesis in rapidly proliferating cells on the basis of its
reported major role in ribosomal assembly (6)
. However,
its chromosomal location at 20q11.2 (14)
, a site of high
genetic instability in many cancer cell types (15, 16, 17)
,
suggests a more subtle role of the p27BBP gene in
the progression of epithelial cancers. To date, however, we have not
detected any obvious gene alterations that account for
p27BBP overexpression. On the basis of its
original identification as a molecular interactor of the cytodomain of
ß4 (5)
, p27BBP may be related to
the interaction to the integrin 6ß4, whose surface expression is
strongly subverted in colorectal as well as in epidermal carcinomas
(18)
. This phenomenon is more marked in poorly
differentiated malignant tumors and also involves the accumulation of
p27BBP in the nucleus. In addition, the
association of increased 6ß4 and p27BBP
levels with poor prognosis in colorectal carcinomas and evidence of the
direct involvement of ß4 integrin in the aggressiveness of colorectal
cancer cells have been reported previously (19, 20, 21)
.
One further potential possibility is that p27BBP may be involved in the protection of colorectal tumor cells against apoptosis. To date, we do not have any precise indication except the observation that, in gut mucosa, p27BBP is high in nonapoptotic and rapidly proliferating crypt cells and is progressively down-regulated in the cells of the villus that are sloughed off in the lumen from gut epithelia as a consequence of apoptosis (10) . In contrast, p27BBP remains steadily overexpressed in these tumors where no apoptosis obviously occurs.
The overexpression of p27BBP is tumor-specific. In fact, stromal cells around the tumor did not show any difference from those surrounding normal mucosa. Moreover, the increased expression of p27BBP is a very early event in tumor progression: it is already clearly observed in adenomas, suggesting that p27BBP gene activation occurs concurrently with the early onset of transformation.
The involvement of p27BBP in tumor progression is unclear. This protein is a component of the nuclear matrix, which is connected to the cytoskeleton and is increasingly recognized as playing a major cell function in DNA transcription and/or replication, and RNA processing and transport (2) . Although it is definitely a nonribosomal protein, p27BBP is involved in the assembly of these organelles, where plays an essential role (6) , probably connected to those of proteins such as nucleolin, which is involved in chromatin decondensation (22) , or B23, which is involved in RNA transport (23) . The latter proteins have been recognized as part of the AgNOR complex (24) . Finally, only the nucleolar fraction of p27BBP undergoes major changes in tumors.
In summary, the results of the present study indicate that p27BBP is a nuclear matrix protein largely increased in precancerous and cancerous lesions of the human colon, where it accumulates in nucleoli and is likely to be involved in essential processes that govern cell growth and cancer evolution. It is also a major marker of transformed cells and may become a powerful tool for diagnostic purposes.
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FOOTNOTES |
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1 These studies were supported by Grants 156/98
and 163/99 (to P. C. M.) from the Associazione Italiana per
la Ricerca sul Cancro. Alessandra Donadini is a fellow of Dystrophic
Epidermolysis Bullosa Research Association. 
2 To whom requests for reprints should be
addressed, at Istologia Molecolare, Department of Biological and
Technological Research, San Raffaele Scientific Institute, Via
Olgettina 58, 20132 Milan, Italy. Phone: 39-0226434857; Fax:
39-0226434855; E-mail: sanvito.francesca{at}hsr.it
3 The abbreviations used are: SSPE, saline-sodium
phosphate-EDTA; AgNOR, argyrophylic nucleolar organizing
region-associated protein.
4 P. Rosso, F. Sanvito, G. Cortesina, S. Biffo,
and P. C. Marchisio, "Expression of p27BBP in head and neck
cancer," manuscript in preparation.
Received 7/21/99. Accepted 12/15/99.
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REFERENCES |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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6 ß4 and BM-600/nicein. J. Investig. Dermatol., 101: 352-358, 1993.[Medline]
6ß4 in the invasive properties of colorectal carcinoma cells. Cancer Res., 56: 4811-4819, 1996.This article has been cited by other articles:
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  S. Zhu, M.-L. Si, H. Wu, and Y.-Y. Mo MicroRNA-21 Targets the Tumor Suppressor Gene Tropomyosin 1 (TPM1) J. Biol. Chem., May 11, 2007; 282(19): 14328 - 14336. [Abstract] [Full Text] [PDF]
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 S. Mika and B. Rost NMPdb: Database of Nuclear Matrix Proteins Nucleic Acids Res., January 1, 2005; 33(suppl_1): D160 - D163. [Abstract] [Full Text] [PDF]
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