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Regular Articles |
From the Department of Molecular Genetics,*
Research
Institute for Microbial Diseases, Osaka University, Suita, Osaka; and
the Departments of Pathology,
Dermatology,
and Internal Medicine and
Molecular Science,
Osaka University Medical
School, Suita, Osaka, Japan
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Introduction |
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To study the molecular mechanisms of melanoma metastasis, various types of mouse models have been developed.12,13 Among these, the C57BL/6 mouse model using B16 melanoma cells is one of the most useful.14 From B16 cells, various sublines have been established that display distinct metastatic phenotypes.15,16 The F10 and BL6 sublines heavily metastasize to the lungs after intravenous injection.15,16 In contrast, although BL6 cells are also metastatic after subcutaneous injection, F10 cells are only poorly so.15,17 We have attempted to reveal the difference in gene expression between the two sublines by using the library subtraction method, and were able to isolate several genes that are up- or down-regulated in BL6 cells.18-22 The differentially expressed genes we analyzed had relevance to the clinical phenotypes of invasion and metastasis. The connexin 2619 and MLZE22 genes were up-regulated in BL6 cells, and were intensively expressed in the invasive descent of skin melanoma lesions. The annexin VII gene was down-regulated in BL6 cells, and its expression in human melanoma was related to a better prognosis.20 Genetic differences between F10 and BL6 cells seemed to reflect the process by which human melanomas progress to more malignant phenotypes. If there is a difference in nucleolar function between the two sublines, the comparative approach we used could provide genes that are responsible for changes in the nucleolus during the malignant progression process.
In the present study, we first compared the nucleolar size in F10 and BL6 cells, and found that BL6 cells contain nucleoli that are larger than those of F10 cells. We therefore assumed that BL6 cells express particular genes involved in nucleolar sizing at higher levels than F10 cells do. With this assumption in mind, we screened the cDNA clone pool obtained after the subtraction of a BL6 cDNA library with F10-derived mRNA. We isolated the Sik-similar protein (Sik-SP) gene, a member of the Nop5/Sik family that consists of highly conserved nucleolar proteins.23 The Nop5/Sik family members bind box C/D small nucleolar RNA.24-26 The resulting complexes, named small nucleolar ribonucleoproteins, play a pivotal role in rRNA processing necessary for ribosome assembly.27,28 In yeast, at least two members, Nop56 and Nop58, are present,29 and yeast deficient in either undergoes growth arrest because of defects in protein synthesis.29-31 In mammalian cells, the presence of counterparts to the yeast Nop5/Sik family members has already been confirmed,24,25,32 but their roles in nucleolar function are not yet well understood. Such is the case with Sik-SP: the cDNA sequence of Sik-SP is in the GenBank database, but no functional analysis has been done on this molecule yet. We assumed that Sik-SP might have a role similar to that of the yeast Nop5/Sik family members. In this study, we used transient expression experiments to show that Sik-SP is located primarily at the nucleolus, and increased the area of the nucleolus recognized by toluidine blue stain. Functional analysis indicated that Sik-SP increases rRNA synthesis and the translational efficiency of the exogenous luciferase (Luc) gene. In addition, we found that expression of a human Nop5/Sik family member correlated well with the metastatic phenotypes of clinical melanomas. The Nop5/Sik family members may therefore promote nucleolar function in metastatic melanoma cells.
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Mouse melanoma (B16, F10, BL6, and K1735-P12 ) and melanocytic (melan-a33 ) cells were kindly provided by Dr. I. J. Fidler (The University of Texas, Houston, TX) and Dr. D. C. Bennett (St. Georges Hospital Medical School, London, UK), respectively. NIH3T3 and Balb3T3 fibroblastic cells, MC/9 and P-815 mastocytoma cells, and COS-7 kidney cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA). All cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal calf serum (FCS). Two hundred nmol/L of 12-O-tetradecanoylphorbol-13-acetate was added to grow melan-a cells.
For the experiment of serum starvation and stimulation, F10 and BL6 cells (2 x 104) were plated onto 35- or 100-mm culture dishes with DMEM/10% FCS (day 0). On the next day (day 1), some dishes of F10 cells were used for transfection. On day 2, the medium was replaced with DMEM, and on day 4, the medium was replaced with DMEM/10% FCS. Cells were harvested by trypsin-ethylenediaminetetraacetic acid solution (Sigma, St. Louis, MO) from 35-mm dishes on every day from days 1 to 6, and from 100-mm dishes on each of days 7 and 8, and the cell number was counted. A portion of the cells was stained with trypan blue (Life Technologies, Inc., Gaithersburg, MD), and the proportion of dead cells was determined. It was always <0.1%. Experiments were performed in triplicate, and repeated three times. The cell-doubling time was determined by counting cells in triplicate samples at 24-, 48-, and 72-hour intervals according to the method described by Patterson.34
Isolation of cDNA Clones
The method for constructing a subtracted cDNA library has been
described previously.19,21,35
After four rounds of
subtraction, 
1400 clones were rescued and subjected to Northern blot
analysis with the RNA of F10 and BL6 cells. Clones of interest were
sequenced. The DNA sequences were used to search the National Center
for Biotechnology Information database using the BLASTN algorithm.
In Vitro Transcription and Translation
The TNT T7 quick-coupled transcription/translation system (Promega, Madison, WI) was used according to the manufacturer’s instructions.
Plasmid Construction
We previously described two modified pEGFP-C1
(Novagen, Madison, WI) vectors, termed pEGFP3B and
pEGFP3.36
Using clone 3-30 as a template, the
polymerase chain reaction (PCR) was done with two sense primers
(F-Asc-full and F-Asc-
N) containing an AscI site and two
antisense primers (R-Not-full and R-Not-C) containing a
NotI site: F-Asc-full,
5'-CATGGCGCGCCGATGGAAGGCAAAATCAATAAACAG-3'; F-Asc-N,
5'-CATGGCGCGCCGGAACAGGTAGTAGAAGAGGAGCCG-3'; R-Not-full,
5'-ATAGCGGCCGCTCACTAGCTCCCAACTGAATCGTAAGC-ATG-3';
R-Not-C, 5'-ATAGCGGCCGCTCACTATTCCTCCGCCTCCTCCTCCTCCAT-3'. Primer
sets and the amplified cDNA regions of Sik-SP are:
F-Asc-full and R-Not-full, the full-length coding region (amino acid
residues 1 to 474, Sik-SP); F-Asc-full and R-Not-C, the C-terminal
deletion form (amino acid residues 1 to 418, Sik-SPC); F-Asc-N
and R-Not-full, the N-terminal deletion form (amino acid residues 419
to 474, Sik-SPN). The PCR-amplified DNA fragments were digested with
AscI and NotI, and then ligated into the
pEGFP3B vector by way of the AscI-NotI sites
(pEGFP3B-Sik-SP, pEGFP3B-Sik-SPC, and pEGFP3B-Sik-SPN). The
plasmid constructs encode various forms of Sik-SP fused to the 3' end
of the green fluorescent protein (GFP). The pEGFP3 vector contains an
in-frame stop codon between the GFP sequence and the
AscI site, and was used as a control for the pEGFP3B vector.
The pcDNA3 expression vector (Invitrogen, San Diego, CA) was modified
to contain an AscI site as described
previously,21
and the AscI-NotI cDNA
fragment of wild-type Sik-SP was subcloned from the pEGFP3B vector into
the modified pcDNA3 vector (pcDNA3-Sik-SP).
Plasmid Transfection
The Fugene 6 transfection reagent (Roche Diagnostics Co., Indianapolis, IN) was used according to the manufacturer’s instructions. A 100-µl DMEM mixture containing 3 µl of Fugene 6 and 1 µg of plasmid DNA was added to COS-7 or F10 cells that were grown in 35-mm culture dishes at a confluency of 70%, unless otherwise indicated. For co-transfection, 1 µg of each of plasmid constructs was added to the mixture. When 100-mm culture dishes were used, the quantity of each component was scaled up. Subsequently, cells were cultured in DMEM/10% FCS.
Histocytological Examination
Fibronectin (Sigma)-coated coverslips (25
ng/cm2) were placed at the bottom of 35-mm
culture dishes, and cells were plated onto them. The next day, cells
were transfected with pEGFP3B, pEGFP3B-Sik-SP, pEGFP3B-Sik-SPC, or
pEGFP-3B-Sik-SPN. After the cells were cultured for 24 hours, the
coverslips were fixed in 3% glutaraldehyde, and mounted in an aqueous
medium. Cells were observed through an Axioplan2 microscope (Carl
Zeiss, OberKochen, Germany) equipped with epifluorescence and a
charge-coupled device camera (Power HAD; Sony, Tokyo, Japan), and
images were created by IPLab Spectrum (Scanalytics Inc., Fairfax, VA)
software on a Macintosh computer. For subsequent cell staining, the
mounting medium was washed out with distilled water.
To stain with toluidine blue, F10 and BL6 cells were cultured on fibronectin-coated coverslips (25 ng/cm2) for 3 hours, and fixed in 3% glutaraldehyde. Sections of paraffin-embedded tissues were deparaffinized with xylene, and hydrated in ethanol series. Cells and sections were stained for 10 minutes with 0.05% toluidine blue in a buffer (pH 4.1) containing 60 mmol/L citric acid and 80 mmol/L sodium phosphate. The samples were washed in water, dehydrated in ethanol, and mounted in Permount medium (Fisher Scientific, Fair Lawn, NJ). Stained cells were observed with an image processing system, which consisted of a charge-coupled device camera (HC-2500; Fuji Photo Film Co., Tokyo, Japan), a microscope (AX80; Olympus, Tokyo, Japan), a color monitor, a Macintosh computer (Power Mac G4), image processing software (Adobe Photoshop Ver 5.0; Adobe System Inc., Mountain View, CA), and a digital cursor. Images were created on the computer monitor screen at a magnification of x7250. This magnification was achieved by an intermediate zoom magnification system (Provis, Olympus). On the monitor, the nucleolar diameters were between 7 and 38 mm. A cursor was used manually to delineate the margins of the toluidine blue-stained nucleoli in at least 30 fields for each sample. The area occupied by the delineation was expressed in a pixel unit, and converted into a µm2 unit by the magnification rate on the monitor. The mean nucleolar area was calculated by observing 200 nucleoli for F10 and BL6 cells, and 50 nucleoli for COS-7 and human melanoma cells.
Luciferase Assay
The pRL-SV40, pRL-CMV, and pRL-null vectors were purchased from
Promega. The XhoI-EcoRI DNA fragment including
the modified chicken ß-actin promoter was subcloned from
the pAG-2 vector37,38
into the pRL-null by way of the
XhoI-EcoRI sites (pRL-actin). The three vectors
express the Renilla Luc gene under the control of the simian
virus 40 enhancer/early promoter, cytomegalovirus enhancer/immediate
early promoter, and chicken ß-actin promoter,
respectively. Cells were transfected with each of the three vectors. In
addition, cells were co-transfected with pcDNA3 or pcDNA3-Sik-SP. After
the cells were cultured for 24 hours, the Renilla Luc
activity was measured by using the Dual luciferase assay system
(Promega) according to the manufacturer’s instructions. The level of
Luc activity was normalized against the amount of Luc
plasmid DNA introduced by transfection, as determined by Southern blot
analysis. After the cells were extracted in the passive lysis buffer of
the Luc assay kit, a half volume of the lysate was subjected to
phenol/chloroform extraction, followed by ethanol precipitation. The
DNA obtained was blotted with an [
-32P]
dCTP-labeled NheI-XbaI DNA fragment of the
pRL-null vector, which contained the Renilla Luc gene.
Hybridized bands were quantified by measuring the peak area of the
densitometric tracing (BAS 2000 system, Fuji Photo Film Co.). Values of
Luc activity were divided by values of the band intensity, and
expressed as relative Luc activity in arbitrary units. Experiments were
performed in triplicate, and repeated three times.
Evaluation of in Vitro rRNA Synthesis
In vitro rRNA synthetic activity was evaluated
essentially as described previously.39,40
On the day
before the assay, some cells were transfected in 100-mm culture dishes
with the empty pcDNA3 vector or with pcDNA3 encoding the
Sik-SP gene. On the next day, the cells (5 x
107) were washed with phosphate-buffered saline
and resuspended in 0.25 mol/L sucrose containing 19 mmol/L Tris-HCl (pH
7.4), 10 mmol/L MgCl2, and 0.5% Triton X-100.
After homogenization, cells were centrifuged at 2500 x
g for 10 minutes. Pellets were washed twice in 0.25 mol/L
sucrose containing 10 mmol/L Tris-HCl (pH 7.4) and 10 mmol/L
MgCl2, and resuspended in 1.0 ml of 0.25 mol/L
sucrose containing 1 mmol/L MgCl2. This
suspension was considered to contain purified nuclei at concentration
of 5 x 107
nuclei/ml, given no leakage. RNA
synthetic activity in purified nuclei was measured in the absence or
presence (5 µg/ml) of -amanitin (Roche Diagnostics Co.), an
inhibitor of RNA polymerases II and III.41,42
Three
hundred µl of the assay mixture contained 50 µmol of Tris-HCl
buffer (pH 8.0); 0.2 µmol of MnCl2; 100 µmol
of
(NH4)2SO4;
0.9 µmol each of CTP, GTP, and ATP; 18 µmol of nonradioactive UTP;
0.05 µmol of [3H]UTP; and 100 µl of nuclear
suspension including 5 x 106
nuclei. The
mixture was incubated at 37°C for 10 minutes. An equal volume of 10%
(w/v) trichloroacetic acid was added and the precipitate was collected
on glass-fiber filters (Whatman GF/C; Whatman, Clifton, NJ). The
filters were washed twice with 5% trichloroacetic acid, twice with 0.1
mol/L HCl, and once with 100% ethanol. After baking at 80°C for 3
minutes, the radioactivity of the filters was measured in a liquid
scintillation counter. Experiments were performed in triplicate, and
repeated three times.
RNA and DNA Analysis
Northern and Southern blotting and hybridization were performed
using standard methods. Relative signal intensity was calculated with
the BAS 2000 system (Fuji Photo Film Co.). Total RNA was prepared from
cultured cells and mouse tissues using 3 mol/L lithium chloride and 6
mol/L urea.43
To extract total RNA from paraffin-embedded
tissues, the Paraffin block RNA isolation kit (Ambion, Austin, TX) was
used. One sheet of section was cut at a thickness of 20 µm and
processed according to the manufacturer’s instruction. Reverse
transcriptase (RT)-PCR was performed as described,35
using
a half volume of the RNA yields and two sets of primers: F-actin,
5'-AACCGCGAGAAGATGACCCAGAT-3'; R-actin, 5'-GAAGGAAGGTTGGAAGAGAGCCT-3';
and F-hNop58, 5'-GTTATGATGCTTTTGGTGAG-3'; R-hNop58,
5'-TGCTGGTACATGGTTCTTCCTCAG-3'. The PCR profile with the
ß-actin primers consisted of 20 cycles of denaturation at
94°C for 30 seconds, reannealing at 51°C for 30 seconds, and
polymerization at 72°C for 1 minute. The PCR profile with the
hNop58 primers consisted of 33 cycles of the same settings.
For probe preparation, PCR-amplified DNA fragments derived from mRNA of
BL6 cells were subcloned into the pBluescript vector (Stratagene, La
Jolla, CA). After amplification, DNA inserts of clone 3-30
(Sik-SP), ß-actin, and hNop58
were excised from the vectors, and labeled with
[-32P] dCTP by the random labeling method.
Human Surgical Samples
Melanoma samples were obtained from 15 patients with skin melanoma. The patients underwent surgical removal of the melanoma at the time of diagnosis and were followed-up for more than 5 years at the Osaka University Hospital. During the follow-up, lymph nodes with or without metastasis were excised from the patients. Several lesions with intradermal nevus, a benign melanocytic disease, were excised from individual patients in the same hospital. All patients were informed of and consented to this study. Surgical samples were fixed in 4% paraformaldehyde, and embedded in paraffin. The sections were used for diagnosis, nucleolar size measurement, and RT-PCR analysis.
Statistical Analysis
The t-test was performed for analysis of nucleolar area and Luc activity data, and the chi-square test was performed for analysis of positivity ratio data of gene expression using the StatView (Abacus Concepts Inc., Cary, NC) software on a Macintosh computer. Data are given as mean ± SD. A P value <0.05 was considered to be significant.
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Results |
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To evaluate the nucleolar size, F10 and BL6 cells were stained
with toluidine blue (Figure 1)
. Nucleoli
were often detected as separate objects in single nuclei, but the
margin of each nucleolus was clearly defined. In cells with multiple
nucleoli, the nucleolar area was measured by summing the entire
toluidine blue-stained area present in a single nucleus. The mean
nucleolar area was 1.5-fold larger in BL6 cells than in F10 cells
(Figure 1)
.
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We previously constructed a subtracted cDNA library by removing
clones complementary to the mRNA of F10 cells from a BL6 cDNA
library.18-22
After four rounds of subtraction, 1400
clones were recovered. Blots of total RNA from F10 and BL6 cells were
hybridized with each clone sequentially. One clone, 3-30, was found to
hybridize to a single 2.5-kb transcript. As shown in Figure 2
, this transcript was expressed at low
levels in F10 cells, but its levels were fivefold higher in BL6 cells.
Clone 3-30 was sequenced and found to carry a 1798-bp-long cDNA insert.
Searches of the National Center for Biotechnology Information database
using the BLASTN program revealed that clone 3-30 encompassed a large
part of the sequence designated Sik-SP (accession no. AF053232).
Structurally, Sik-SP belongs to the Nop5/Sik family,23
but
no functional analysis has been reported yet. We screened a BL6 cDNA
library by using clone 3-30 as a probe, and obtained five positive
clones that contained cDNA inserts as long as clone 3-30. Sequencing
revealed that all five clones encoded Sik-SP. When clone 3-30 was
transcribed and translated in the cell lysate of reticulocytes, the
translation product was detected as a band of 55 kd (data not shown),
consistent with the length of the open reading frame of the
Sik-SP gene.
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. The 3-30 probe
did not hybridize to any transcripts from normal mouse tissues,
including lung, intestine, liver, kidney, spleen, thymus, skeletal
muscle, heart, and brain (data not shown). In contrast, hybridized
bands were detected faintly in all mouse cell lines examined (Figure 2)
. The band intensity in nontumor cells, such as NIH3T3, Balb3T3, and
melan-a, and the intensity in K1735-P and B16 melanoma cells were
weaker than that in F10 cells. By contrast, the band intensity in P-815
and MC/9 mastocytoma cells was similar to that in F10 cells. The four
types of melanoma and mastocytoma cells examined are much less
metastatic than BL6 cells.12,13
Expression levels for the
Sik-SP gene seemed to increase in some tumor cells,
especially in highly metastatic BL6 cells. Nucleolar Localization and Sizing of Sik-SP
Recently, SAN5, another mouse member of the Nop5/Sik family, was
shown to locate to the nucleolus.44
To learn the
subcellular localization of Sik-SP, COS-7 cells were transfected with a
plasmid encoding the GFP-tagged form of Sik-SP. Because an abundance of
lysine residues in the C-terminal region is characteristic of the
Nop5/Sik family members,23-25,29,30
we wished to know
whether the C-terminal region is important for the localization of
Sik-SP. Therefore, a similar transfection was done with plasmids
encoding GFP-tagged forms of the two deletion mutants (Sik-SPC and
Sik-SPN). Sik-SPN consists of 55 C-terminal amino acid residues
of Sik-SP, while these same 55 amino acid residues are deleted in
Sik-SPC (Figure 3A)
. Expression of the
introduced genes was confirmed by the Northern blots with the clone
3-30 probe (Figure 3B)
. No transcript was detected in untreated COS-7
cells or in COS-7 cells transfected with the GFP gene alone.
On the other hand, one transcript hybridized heavily in GFP-tagged
Sik-SP-transfected cells. The larger mobility size of this transcript
as compared to the Sik-SP transcript of BL6 cells is consistent with
the message length for the gene transfected. Similarly, in GFP-tagged
Sik-SPC and Sik-SPN-transfected cells, two transcripts of
different length were detected at the expected mobility sizes.
GFP-tagged forms of Sik-SP seemed to be transcribed efficiently in
COS-7 cells.
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. GFP-tagged Sik-SP and GFP-tagged Sik-SPC showed
a well-concentrated localization within the nucleus (Figure 3C)
. We
stained the cells with toluidine blue, and found that both GFP-tagged
proteins localized to the nucleolus. In contrast, GFP-tagged Sik-SPN
was not concentrated at the nucleolus, but was found in both the
cytoplasm and nucleus (Figure 3C)
. A similar subcellular localization
was observed in F10 and BL6 cells after transfection with the
GFP-tagged forms (data not shown). These results indicated that Sik-SP
is localized to the nucleolus, and that the N-terminal region is
required for its nucleolar localization, but the C-terminal region is
not.
While observing cells, we noticed that nucleoli expressing GFP-tagged
Sik-SP were larger than nucleoli not expressing it. We compared the
nucleolar area in COS-7 cells expressing various GFP-tagged forms of
Sik-SP. After the transfection with each plasmid construct, COS-7 cells
containing GFP fluorescence were stored in an image processing system.
Then, the cells were stained with toluidine blue, and the stored cells
were identified on the computer monitor screen. The nucleolar area of
cells expressing GFP-tagged Sik-SP was larger than that of cells
expressing GFP alone and GFP-tagged Sik-SPN (Figure 3C)
. In
addition, GFP-tagged Sik-SPC did not increase the nucleolar area
despite its nucleolar localization. Sik-SP seemed to directly affect
the nucleolar size when overexpressed.
Increased Nucleolar Function in Sik-SP-Transfected COS-7 Cells
The Nop5/Sik family members are essential for ribosome biogenesis in yeast,29,30 whereas their roles are not defined precisely in mammalian cells. We examined whether Sik-SP was involved in ribosome biogenesis in COS-7 cells by the following two experiments. For fear that GFP might cause nonspecific effects, COS-7 cells were transfected with the empty pcDNA3 vector (COS-7pcDNA3), or pcDNA3 encoding the Sik-SP gene (COS-7Sik-SP).
Increased rRNA Biogenesis
Twenty-four hours after the transfection, we purified nuclei from
COS-7pcDNA3 and COS-7Sik-SP
cells, and examined the amounts of transcript that was newly
synthesized in the nuclei in vitro during a given time
period. The amount was expressed as the value of
[3H]UTP incorporated into already initiated and
elongating transcripts. COS-7Sik-SP cells showed
a remarkably higher value of [3H]UTP
incorporation than COS-7pcDNA3 cells in the
absence of -amanitin (Figure 4A)
. The
presence of -amanitin reduced the value of
[3H]UTP incorporation comparably in both cells.
Because -amanitin is an inhibitor for RNA polymerases II and
III,41,42
the results indicated that
COS-7Sik-SP cells have higher levels of rRNA
biogenesis than COS-7pcDNA3 cells, while the
synthesis of other types of RNA was comparable in both cells.
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Next, we compared translational levels of exogenous Luc
gene in COS-7pcDNA3 and
COS-7Sik-SP cells. The pRL-CMV or pRL-SV40 vector
was transfected into COS-7 cells along with pcDNA3-Sik-SP or the empty
pcDNA3 vector. Twenty-four hours after the transfection, the cells were
harvested and divided into three aliquots to examine gene dosage,
transcriptional levels, and translational levels of the Luc
gene. To measure gene dosage and transcriptional levels of the
Luc gene, DNA and total RNA were extracted from the cell
pellets and hybridized with the Luc probe (Figure 4B)
.
Translational levels of the Luc gene were evaluated by
measuring Luc activity. For the comparison between
COS-7pcDNA3 and COS-7Sik-SP
cells, the values for Luc activity were normalized to the amount of
Luc plasmid DNA introduced into each cell. The normalized
values were expressed as relative Luc activity.
COS-7Sik-SP cells showed significantly higher
levels of relative Luc activity than COS-7pcDNA3
cells (Figure 4B)
. On the other hand, mRNA expression levels for the
Luc gene were comparable in both cells (Figure 4B
,
Northern). Sik-SP seemed to up-regulate translation rather than
transcription of the exogenous Luc gene in COS-7 cells.
Nucleolar Function in F10 and BL6 Cells
RNA synthetic activity was evaluated in the nuclei isolated from
F10 and BL6 cells (Figure 5A)
. BL6 cells
showed threefold higher levels of RNA synthesis than F10 cells in the
absence of -amanitin. The presence of -amanitin reduced the
levels of RNA synthesis in both cells. Although the magnitude of
reduction by -amanitin was slightly larger in BL6 cells, BL6 cells
showed fivefold higher levels for RNA synthesis than F10 cells even in
the presence of -amanitin. This result indicated that BL6 cells
synthesize rRNA at higher levels, in preference to the other types of
RNA.
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: the Luc activity of
pcDNA3-co-transfected F10 cells was comparable to that of F10 cells,
whereas BL6 and Sik-SP-co-transfected F10 cells showed several-fold to
10-fold higher Luc activity than the other two types of F10 cells.
Because there was no difference in the mRNA levels of the
Luc gene among the four types of cells (data not shown),
these results indicate that the Luc message was translated
more efficiently in BL6 and Sik-SP-co-transfected F10 cells. In
addition, they suggested that the difference in the translational
efficiency between F10 and BL6 cells is attributable, at least in part,
to the expression level of Sik-SP.
Previous reports indicated a clear relationship between nucleolar size
and cell proliferation rate.3,4
However, there was no
difference in the mean cell-doubling time between F10 and BL6 cells
(Figure 6)
. We examined the effects of
Sik-SP on cell growth recovery after serum-starved culture conditions.
F10 and BL6 cells were plated onto culture dishes (day 0),
serum-starved for 2 days from day 2, and refed with serum on and after
day 4. Some dishes of F10 cells were used for transfection with an
empty pcDNA3 vector (F10pcDNA3), or pcDNA3-Sik-SP
(F10Sik-SP) on day 1. F10 and BL6 cells ceased to
grow actively toward the end of starvation, as did
F10pcDNA3 and F10Sik-SP
cells. After 2 days of the serum refeeding, all of the cell types
started to grow exponentially at a similar rate of cell-doubling. The
number of BL6 and F10Sik-SP cells was noticeably
larger than that of F10 and F10pcDNA3 cells on
every day after cells were refed with serum. This was primarily
attributable to the difference in the cell number on day 5, the day
after the serum refeeding. BL6 and F10Sik-SP
cells seemed to have a greater growth response to serum stimulation
than F10 and F10pcDNA3 cells. Expression of
Sik-SP mRNA was examined in F10 and BL6 cells on days 4 and 8 (Figure 6
, top). There was no significant change in the expression levels on
either day when compared with the levels under the standard culture
conditions (see Figure 2
).
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To assess the clinical relevance of the Nop5/Sik family members, we examined gene expression for human Nop58 (hNop58), a human member of the family, in human melanoma lesions. Tumor samples were obtained from 15 patients who were retrospectively divided into two groups, nonmetastatic (n = 6) and metastatic (n = 9). The primary lesions of all of the patients had been excised at the time of diagnosis. Patients of the nonmetastatic group survived at least 5 years after that time without developing lymph node metastasis. Patients of the metastatic group developed lymph node metastasis within 5 years. The nucleolar area of melanoma cells increased in the following order: primary lesions of the nonmetastatic group < primary lesions of the metastatic group < metastatic lesions of the metastatic group. This result is consistent with the previous report that nucleolar size could be a marker for poor prognosis of melanomas.7-10
Total RNA was extracted from sections of melanoma tissues, nevus
lesions, and lymph node tissues. Because melanoma lesions occupied more
than half of the area in each of the adjacent sections of melanoma
tissues (data not shown), melanoma cells were considered to be the
major source of RNA in the melanoma samples. The yield of total RNA for
each sample was <1.0 µg. All of the RNA samples were
reverse-transcribed, PCR-amplified with ß-actin-specific
primers, and subjected to Southern blot analysis. As a positive
control, total RNA (5 ng) of BL6 cells was used. The
ß-actin probe hybridized to a band of 456 bp, indicating
that ß-actin mRNA was clearly and equally amplified in all
RNA samples (Figure 7B
, top). After the
RT reaction, PCR was performed with hNop58-specific primers,
and the products were blotted with the hNop58 probe (Figure 7B
, bottom). The message levels for hNop58 were below the
limit of detection in all of the primary lesions. On the other hand, a
417-bp-long band was clearly detected in four of nine of the metastatic
lesion samples, indicating a marked amplification of hNop58
mRNA. No hNop58-specific bands were detectable in the RNA
samples from either nevus lesions or lymph node tissues, indicating a
specific expression of hNop58 mRNA in melanoma cells. When
total RNA samples were directly used as PCR templates in a negative
control experiment, no hybridized bands were detected with any probes
(data not shown), indicating little amount of genomic contamination.
The ratio of hNop58-positive to -negative samples was
significantly larger in the metastatic lesions (4:5) than in the
primary lesions (0:15) (P < 0.01 by the
chi-square test). We compared the nucleolar size between metastatic
melanoma cells expressing hNop58 (5.71 ± 1.31
µm2; n = 4) and cells not
expressing hNop58 (6.20 ± 0.953
µm2; n = 5), but found no
significant difference. The hNop58 gene expression seemed to
be closely linked to metastasis and nucleolar size, but seemed not to
be the only determinant for either phenotype.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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An abundance of lysine residues in a region of the C-terminus including
100 amino acid residues is characteristic of Nop5/Sik family
members.23-25,29,30
However, the significance of this
region is still unclear. Phylogenetic analysis divides the Nop5/Sik
family members into two subgroups, Nop56 and Nop58.24
Recently, Newman and colleagues24
reported the amino acid
sequence for mNop58 by fusing two EST clones (ATCC no. 1416459 and ATCC
no. 971387). The sequence is the most probable mouse counterpart for
yeast Nop58 (yNop58), because there is 47% identity and 71%
similarity between mNop58 and yNop58. On the other hand, Nelson and
colleagues44
isolated a novel mouse member of the Nop58
subgroup from a NIH3T3 cDNA library, and termed it SAN5. In the present
study, we isolated clones encoding Sik-SP from a BL6 cDNA library. The
genetic basis for the diversity of the mouse Nop58 subgroup remains to
be determined. Both SAN5 and Sik-SP are deletion forms of mNop58:
Sik-SP lacks 63 amino acid residues at the N-terminus, and SAN5 lacks
36 amino acid residues near the C-terminus (Figure 3A)
. The C-terminal
lysine-rich region is entirely conserved in Sik-SP, and is partially
deleted in SAN5, yet both Sik-SP (Figure 3C)
and SAN544
localize to the nucleolus. Moreover, deletion of the part of the
C-terminal region that is conserved in SAN5 did not change the
nucleolar localization of Sik-SP. The C-terminal lysine-rich region
thus does not seem to be necessary for the correct targeting of the
Nop58 subgroup members to the nucleolus, as it is in
yNop58.29
However, our results point to a possible role of
the C-terminal region in the nucleolar function of Sik-SP. Nucleolar
localization of GFP-tagged Sik-SP increased the nucleolar area stained
with toluidine blue, whereas nucleolar localization of GFP-tagged
Sik-SPC did not. Deletion of the C-terminal region likely resulted
in loss of Sik-SP function, suggesting a close relation between
nucleolar size and the function of Sik-SP in the transfected COS-7
cells. Further analysis is necessary to determine the functional
importance of the C-terminal region with more confidence.
Yeast deficient in yNop58 undergoes growth arrest because of defects in
early pre-rRNA processing events necessary for ribosome
assembly.29-31
This lethal defect in yeast ribosome
biogenesis was complemented by SAN5.44
Here we obtained
consistent results for Sik-SP in mammalian cells. Nucleolar size was
increased in COS-7 cells that expressed GFP-tagged Sik-SP, suggesting a
role for Sik-SP in the nucleolus. Transient expression of Sik-SP
increased rRNA synthesis and translational levels of an exogenous
Luc gene in COS-7 cells (Figure 4)
. The two effects of
Sik-SP were reproducible in F10 and BL6 cells (Figure 5)
. Higher levels
of rRNA synthesis and exogenous Luc gene translation were
detected in BL6 cells. Transient expression of Sik-SP in F10 cells
compensated for the inefficiency in translation. These results support
the idea that Sik-SP promotes ribosome biogenesis in mammalian cells.
In addition, BL6 cells appeared superior to F10 cells with respect to
nucleolar function including rRNA and protein synthesis, because of the
increased expression of Sik-SP.
In F10 and BL6 cells, the nucleolar size correlated well with nucleolar
function, but there was no difference in the mean cell-doubling time.
In addition, transfection of Sik-SP did not significantly shorten the
cell-doubling time of F10 cells (Figure 6)
. This was also the case with
SAN5, which did not significantly increase fibroblast
proliferation.44
Instead, we found that Sik-SP promoted
the escape of melanoma cells from starved states, and their subsequent
entrance into a logarithmic growth phase. Interestingly, SAN5 was
recently characterized as an early response gene that is induced by
multiple growth factors and links growth factor receptors to the
cellular translational machinery.44
Moreover, it has
recently been shown that Sik-SP gene expression is markedly
induced in endometrial epithelial cells by estrogen,45
which also acts as a potent inducer of proliferation in these cells.
Both Sik-SP and SAN5 are assumed to up-regulate ribosome biogenesis in
response to an increased demand for protein synthesis. By virtue of the
induction of either factor, cells could transition from a quiescent to
an actively growing state. In BL6 cells, Sik-SP gene
expression still remained high at the end of the serum-starved culture
condition, and the cells appeared ready to start active growth
immediately after serum stimulation.
Once metastasis has developed, melanoma is rarely curable and the
median survival time drops to 6 months.46
It is therefore
important to diagnose the metastatic potentials of melanoma cells. As
shown in Figure 7
, nucleolar size and hNop58 gene expression
could be markers for metastatic potentials. Although nucleolar size is
inversely related with cell-doubling time in many cancer cells,
metastatic potentials are not explained only by the rapidity of cancer
cell proliferation. In fact, there is no difference in either the
in vivo or in vitro growth rate between F10 and
BL6 cells.15
The present results suggest that increased
nucleolar function, but not rapid cell growth rate, might be involved
in the acquisition of high metastatic potentials by BL6 cells. Cancer
cells in tumors, especially in metastatic microfoci,47,48
are often exposed to various stress conditions, such as mechanical
stress, hypoxia, low pH, nutrient deprivation, and host immune
defense.49-52
Increased nucleolar function could help
cancer cells grow promptly in response to environmental changes, such
as tumor angiogenesis, which release cells from such stress conditions.
It has been reported that transient exposure to and subsequent recovery
from hypoxia, glucose starvation, and low pH enhance the metastatic
ability of several types of cancer cells, including F10
cells.53-55
This phenomenon may result from the
preferential growth of cancer cells with increased nucleolar function.
Whether nucleolar function plays a causative role in metastasis remains
unknown. However, the present study has shown that the Nop5/Sik family
members are among the factors that cause increased nucleolar size and
function in metastatic melanoma cells.
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Acknowledgements |
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Footnotes |
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Supported by grants from the Ministry of Education, Science, and Culture; the Ministry of Health and Welfare; the Osaka Cancer Society; Public Trust Haraguchi Memorial Cancer Research Fund; and the Ichiro Kanehara Foundation.
K. N. and A. I. contributed equally to this work.
Accepted for publication July 3, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-amanitin and denaturated DNA on multiple forms separated from rat liver nuclei. Ital J Biochem 1974, 23:245-256[Medline]
and -ß. J Biol Chem 2000, 275:28834-28842This article has been cited by other articles:
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