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INTRODUCTION |
The human MAT1 gene (ménage
à trois 1) was discovered as the third
subunit of cyclin-dependent kinase
(CDK)1-activating kinase
(CAK) (1-3). CAK exists in either of two forms: (a) the
major ternary CAK containing CDK7, cyclin H, and MAT1, which is
assembled by MAT1 in the absence of activating phosphorylation of the
T-loop of CDK7; and (b) the minor binary CAK lacking MAT1, which requires the phosphorylation of the T-loop of CDK7 for CDK7 binding to cyclin H in the absence of MAT1 (1, 4, 5). MAT1 is
associated with both a free form of CAK and a transcription factor IIH
(TFIIH)-bound form of CAK (1, 4, 6-10). Up to the present, the known
functions of MAT1 have been associated with CAK. CAK was originally
implicated in cell cycle control by its ability to phosphorylate and
activate CDK1, CDK2, and CDK4 (11-16). Subsequently, CAK was found to
associate with TFIIH and to phosphorylate the carboxyl-terminal domain
of RNA polymerase II (1, 4, 7-10). TFIIH is required for both
initiation of RNA polymerase II-catalyzed transcription and nucleotide
excision repair (17-19). Thus, the concept that CAK functions in the
regulation of the cell cycle, DNA repair, and transcription has emerged.
Interestingly, all of the functions that link CAK to the cell cycle,
DNA repair, and transcription are mediated by MAT1, i.e. MAT1 activation of CAK by stabilizing the association of CDK7 with
cyclin H (1, 4, 6) and MAT1 determination of substrate specificity of
CAK through (a) switching the substrate preference of CAK to
the carboxyl-terminal domain over CDK in the presence of MAT1 (20) and
(b) the requirement of MAT1 for efficient phosphorylation of
the tumor suppressor protein p53 by CDK7-cyclin H (21). The efficiency
of bipartite CDK7-cyclin H in CDK2 phosphorylation is not affected by
the addition of MAT1 (20, 21), suggesting that MAT1 more likely acts as
a targeting subunit of CAK than significantly influencing CDK7-cyclin H
phosphorylation of CDKs. TFIIH lacking the CAK subcomplex completely
recovers its transcriptional activity in the presence of free ternary
CAK; MAT1 interacts with essential components of the DNA repair
machineries XPB (ERCC3) and XPD (ERCC2), which are two helicase
subunits of TFIIH that mediate the association of CAK with core TFIIH
(10, 22, 23). However, these studies have provided little information
about the cell cycle phase specificity and the corresponding regulatory mechanisms of MAT1 itself in the control of cell proliferation.
It is known that the fundamental feature of vascular occlusive disease,
including atherosclerosis, hypertension, restenosis, and transplant
arteriopathy, is an accumulation of cells and extracellular matrixes in
the intima, which consequently results in the narrowing of the vascular
lumen. The vascular smooth muscle cell (SMC) represents the cell type
most often implicated in the process of luminal narrowing (24-31). In
recent years, rapid progress has been made in identifying and
understanding the functions of some cell cycle regulators in
controlling SMC proliferation. Many important cell cycle factors,
including the catalytic subunit CDC2 (32), the negative cell cycle
regulator retinoblastoma protein (pRb) (29, 33), and the CDK inhibitor
p21 (34), have shown to play important roles in regulating SMC
proliferation, even though the molecular mechanisms still remain to be
further studied. Considering that eukaryotic cell proliferation is a
process that is highly regulated by the ordered assembly and determined
substrate specificity of CDKs (35, 36), the functions of MAT1 in
assembling and determining substrate specificity of CAK (20, 21) may be
directly involved in the regulation of a specific cell cycle phase.
Given that MAT1 functions in the cell cycle as an assembly factor and a
targeting subunit of CAK, we intended to determine the cell cycle phase specificity of MAT1 functions and the corresponding mechanism of the
regulation of SMC proliferation. Our studies show that MAT1 is required
for G1 exit. A loss of MAT1 arrests SMCs in G1 phase and induces apoptosis to kill these arrested cells.
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EXPERIMENTAL PROCEDURES |
Retroviral Vectors--
An N-terminal 462-bp fragment of the
MAT1 gene starting from the 5'-end, 40 bp upstream of the
ATG initiation codon and extending to +422 bp of the coding region, was
cloned into retroviral vector G1xSvNa (Genetic Therapy Inc./Novartis)
in the antisense orientation (G1AsMatSvNa). G1AsMatSvNa indicates the
order of the promoter and coding region of the constructs (G1, Moloney
murine leukemia virus long terminal repeat sequences; AsMat, antisense
MAT1 RNA fragment; Sv, SV40 early enhancer and promoter; Na,
neomycin phosphotransferase). The G1AsMatSvNa construct was confirmed
by sequencing as described previously (37, 38) using primers
complementary to the vector-flanking sequence upstream of the
MAT1 antisense fragment and a Sequenase reaction kit (U. S.
Biochemical Corp.). G1nBgSvNa bearing a nuclear targeting
-galactosidase was used for testing gene transfer efficiency.
Production of High Titer Amphotropic Replication-defective
Retrovirus--
Retroviral supernatants were prepared as described
(39). Briefly, murine amphotropic PA317 packaging cells (American Type Culture Collection) were plated at 20% confluent density (5 × 105 cells) in 100-mm plates with Dulbecco's modified
Eagle's medium (Life Technologies, Inc.) containing 10% fetal bovine
serum (Hyclone Laboratories) and incubated at 37 °C for 16 h.
Cells were transfected with 20 µg of DNA from G1xSvNa, G1AsMatSvNa,
or G1nBgSvNa using calcium phosphate precipitation. 12 h after
transfection, the cells were washed, and the medium was replaced. After
an additional 48-h post-transfection period, cells were split 1:5 and
selected with 0.6 mg/ml G418 (Life Technologies, Inc.) for 10 days. The colonies formed by G418-resistant cells were pooled as producer cells,
and 4 × 106 cells/175-cm flask were plated for virus
production. The supernatants containing amphotropic
replication-defective retrovirus were collected after the cells reached
90% confluence and were clarified by centrifugation at 550 × g for 10 min at 4 °C. For determining the viral titer, NIH 3T3 cells were plated at 2.5 × 104 cells/6-well
plate and transduced with viral supernatants in the presence of 8 µg/ml Polybrene for 2 h. After a 48-h post-transduction incubation, the cells were selected with 0.6 mg/ml G418 for 10 days.
The colonies formed by G418-resistant cells were stained with 0.1%
methylene blue (Sigma).
Cells, Retrovirus-mediated Transduction, and Maintenance of
Stable Clones--
Rat aortic SMCs (A-10 cell line obtained from
American Type Culture Collection) were grown in Dulbecco's modified
Eagle's medium supplemented with 15% fetal bovine serum, 200 mM L-glutamine, 100 units/ml penicillin, and
100 µg/ml streptomycin at 37 °C in a 5% CO2 and 95%
air atmosphere. The G418 concentration for selection of stable clones
was determined by a 7-day lethal dose test. SMCs were plated in 100-mm
plates at a 20% confluent density for overnight attachment and
transduced with either G1AsMatSvNa (MAT1-AS) or G1xSvNa
(vector) at a multiplicity of infection of 10 in the presence of
Polybrene (8 µg/ml) for 2 h. After a 48-h post-transduction incubation, the cells were selected for 7 days with 0.25 mg/ml G418.
About 20 single stable clones were picked from antisense MAT1-transduced SMCs and expanded for detection of the
MAT1 expression phenotype. G418-resistant colonies from
G1xSvNa (vector)-transduced SMCs were pooled as a control. Our working
model was that G1AsMatSvNa (MAT1-AS)-transduced cells, the
MAT1 expression of which has been inhibited, served as a
testing sample, whereas G1xSvNa (vector)-transduced cells and
additional nontransduced (blank) cells served as controls. The same
dose of G418 used for selection was added to the medium for maintenance
of stable clones. MAT1 expression in stable clones was
tested before and after experiments by Western blotting.
Western Analysis of MAT1 Expression--
Western analysis was
performed as described previously (3, 40) with minor modifications.
Briefly, cells from G1AsMatSvNa (MAT1-AS)-transduced,
G1xSvNa (vector)-transduced, and nontransduced (blank) cultures were
grown to
80% confluence in 6-well plates. The cells in each well
were lysed with 80 µl of lysis buffer (50 mM Tris-HCl,
150 mM NaCl, 2 mM EDTA, 2 mM EGTA,
25 mM sodium fluoride, 25 mM
-glycerol
phosphate, pH 7.5, 0.1 mM sodium orthovanadate, 0.1 mM phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin,
0.2% (v/v) Triton X-100, and 0.5% (v/v) Nonidet P-40) on ice for 10 min. Cell lysate proteins were centrifuged at 14,000 × g for 15 min at 4 °C. The same amount of protein was
denatured in SDS sample buffer at 95 °C for 3 min, separated by
electrophoresis on 16% SDS-polyacrylamide gels, and
electrophoretically transferred to a polyvinylidene difluoride membrane
(Millipore Corp.) in Tris/glycine buffer containing methanol. The MAT1
protein was detected by polyclonal rabbit anti-human MAT1 antibodies
(Santa Cruz Biotechnology) and enhanced by chemiluminescence
(Western-star, Tropix Inc.) using alkaline phosphatase-conjugated
secondary goat anti-rabbit IgG (Sigma) as recommended by the
manufacturer. A 40-kDa recombinant His-MAT1 protein expressed from the
pET23b construct as described (37) was used as a positive control. The
expression level of MAT1 was determined by scanning MAT1 content using
a ScanJet IICX/T densitometer (Hewlett-Packard Co.).
Cell Proliferation Analysis--
Cells from G1AsMatSvNa
(MAT1-AS)-transduced, G1xSvNa (vector)-transduced, and
nontransduced (blank) cultures were used for determining the ratio of
cell duplication. The same number of cells were plated in 24-well
plates. 24 h after plating, the cells were counted for 3 consecutive days before reaching confluence.
To determine the number of living cells in culture, a cell
proliferation assay kit (Promega) was used as recommended by the manufacturer. Briefly, the same number of cells were plated in 96-well
plates and cultured for 72 h to
90% confluence. Cells were
incubated with MTS tetrazolium compound (which is bioreduced to soluble
formazan by dehydrogenase enzymes in metabolically active cells) for
1 h at 37 °C. The quantity of the formazan product as measured
by the absorbance at 490 nm using a Kinetic Microplate Reader
(Molecular Devices) is directly proportional to the number of living
cells in culture.
The activation of SMC proliferation from a nonproliferative to
proliferative state in an "in vitro injured tissue" was
determined as described (41) with minor modifications. Cells were grown in 12-well plates to confluence (when they exhibited contact
inhibition) and then scraped with a 200-µl pipette tip to create a
1-mm track devoid of cells in the central area of the wells. The
"wound" tracks were immediately washed to remove the detached
cells, and fresh medium was added. 24 h after the wound tracks
were created, G1nBgSvNa retroviral constructs bearing a nuclear
targeting
-galactosidase were added at a multiplicity of infection
of 10 in the presence of Polybrene (8 µg/ml) for 2 h. 48 h
post-transduction, gene transfer efficiency was measured by determining
the percentage of
-galactosidase-positive cells upon exposure to 1 ml of staining solution (5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 2 mM
MgCl2, and 400 µg/ml X-gal (GBT Inc.)). The number of
blue cells, which reflected the activation of SMC proliferation and the
time for closing the wound track, was assessed under a phase-contrast
microscope either at ×40 or ×100 magnification.
Flow Cytometric Analysis of Cell Cycle Status and
Apoptosis--
Cell cycle status and cell cycle phase specificity of
apoptosis were determined by simultaneously measuring DNA content and DNA breaks as described (42). An APO-DIRECT detection kit (Pharmingen), which works by "tail labeling" dUTP to 3'-OH termini of the DNA breaks by exogenous terminal deoxynucleotidyltransferase and
counterstaining integral DNA by propidium iodide, was used following
the manufacturer's instructions with some modifications. Briefly,
~1.5 × 106 cells (
80% confluent) each from
G1AsMatSvNa (MAT1-AS)-transduced, G1xSvNa
(vector)-transduced, and nontransduced (blank) cultures were collected;
centrifuged at 300 × g for 5 min; washed twice with
ice-cold PBS; fixed in 1% paraformaldehyde on ice for 30 min; washed
twice again; and post-fixed in 70% ethanol for
4 h at
20 °C.
The pellet was collected by centrifugation at 1000 × g
for 10 min, washed twice with 2 ml of PBS, resuspended in 50 µl of
FITC-dUTP staining solution, and incubated for 1 h at 37 °C.
The negative control for the blank was incubated with reaction buffer
lacking terminal deoxynucleotidyltransferase. Labeled cells were washed
twice with PBS and stained with 1 ml of the propidium iodide/RNase
solution for 30 min at 37 °C. Flow cytometric analysis was performed
on a FACScalibur flow cytometer (Becton Dickinson). The cell cycle
status was analyzed using ModFit LT software, and a dual parameter
display for FITC-dUTP labeling was created with CellQuest Version 3.1 software (BDIS, San Jose, CA).
Assessment of Cellular and Nuclear Apoptotic
Morphologies--
Apoptotic morphologic changes of cells from
G1AsMatSvNa (MAT1-AS)-transduced, G1xSvNa
(vector)-transduced, and nontransduced (blank) SMC cultures were
determined with or without propidium iodide staining. For propidium
iodide staining of nuclei, cells were grown on 8-well plastic chamber
slides for 72 h to
80% confluence, fixed in 4%
paraformaldehyde for 30 min at room temperature, permeabilized in 0.1%
Triton X-100 and 0.1% sodium citrate on ice for 2 min; stained with
0.015% propidium iodide (Sigma) at room temperature for 30 min; and
examined under epifluorescence illumination at ×400 magnification. The
morphologic apoptotic changes of cells without propidium iodide
staining were monitored under a phase-contrast microscope at ×100 magnification.
In Situ Detection of Apoptosis Using Terminal
Deoxynucleotidyltransferase-mediated dUTP Nick End Labeling
(TUNEL)--
In situ DNA fragmentation was detected using a
TUNEL reaction kit (Boehringer Mannheim) following the manufacturer's
instructions with some modifications. In brief, cells from G1AsMatSvNa
(MAT1-AS)-transduced, G1xSvNa (vector)-transduced, and
nontransduced (blank) cultures were grown to
80% confluence on
8-well chamber slides, fixed in 4% paraformaldehyde solution for 30 min at room temperature, permeabilized in 0.1% Triton X-100 and 0.1%
sodium citrate for 2 min on ice, washed twice with PBS, and incubated
with 50 µl of TUNEL reaction mixture in a humidified chamber for
1 h at 37 °C. Negative controls for blank, vector, and
MAT1-AS cells were incubated with reaction buffer lacking
terminal deoxynucleotidyltransferase. The slides were washed three time
with PBS and stained with 0.015% propidium iodide for 30 min at room
temperature. After a final wash with PBS, the slides were air-dried and
mounted under glass coverslips with aqueous medium. The apoptotic cells
in each field were examined with a Zeiss epifluorescence microscope
(Axioplan/MC100) at ×400 magnification using three different filters
for co-locating apoptotic nuclei: rhodamine filter
(red-stained total DNA), FITC-rhodamine dual filter
(yellow green-stained DNA breaks and red-stained
integral DNA), and FITC filter (green-stained DNA breaks).
Photomicrographs were obtained using Kodak Ektachrome Tungsten 160T film.
Analysis of DNA Fragmentation--
Low molecular weight DNA of
the soluble cytoplasmic fraction was isolated as described (43-45).
~1.5 × 106 cells from G1AsMatSvNa
(MAT1-AS)-transduced, G1xSvNa (vector)-transduced, and
nontransduced (blank) SMC cultures were lysed in 0.5 ml of hypotonic
buffer containing 10 mM Tris, pH 7.5, 1 mM
EDTA, and 0.5% (v/v) Triton X-100 on ice for 30 min and centrifuged at
27,000 × g for 15 min at 4 °C. RNA in the
supernatant was degraded by incubation with DNase-free RNase (50 µg/ml) at 37 °C for 1 h, followed by proteinase K digestion.
DNA was precipitated with 0.1 volume of 3 M sodium acetate,
pH 5.2, 20 µg/ml glycogen, and 2 volumes of anhydrous ethanol at
20 °C for 20 min. The same amount of DNA (10 µg) was
electrophoresed through agarose gel, stained with ethidium bromide, and
visualized under UV light. A 100-bp DNA ladder was used as a molecular
size marker.
 |
RESULTS |
MAT1 Expression Is Inhibited by RNA Antisense Sequence--
To
examine whether retrovirus-mediated gene transfer of antisense
MAT1 RNA inhibits endogenous MAT1 expression, the
cellular MAT1 content from G1AsMatSvNa (MAT1-AS)-transduced,
G1xSvNa (vector)-transduced, and nontransduced (blank) cultures
was determined by Western blotting. The results show that the MAT1
content was reduced ~70% in MAT1-AS-transduced cells
(Fig. 1A). To ensure that the
phenotype of MAT1 expression was consistently maintained
during the experimental period, MAT1 content was examined again 5 months later, after finishing all of the testing (Fig.
1B). The data confirmed that >50% decreased MAT1 expression in MAT1-AS cells was maintained
during the experimental period.

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Fig. 1.
MAT1 expression is inhibited by
RNA antisense sequence. Aortic SMCs were either sham-transduced or
transduced with G1xSvNa (vector) or G1AsMatSvNa (MAT1-AS)
retroviral vectors. The MAT1 content in the transduced stable clones
was determined by Western analysis. 10 µg of cell lysate proteins
from stable clones as well as from nontransduced (blank) cells were
separated on 16% SDS-polyacrylamide gel. The expression level of
37-kDa MAT1 protein was detected by polyclonal anti-MAT1 antibodies.
The 40-kDa recombinant His-MAT1 protein (Rec.MAT1) served as
a positive control. A, MAT1 expression level
before the experiments; B, MAT1 content 5 months later after
a series of experiments.
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Abrogation of MAT1 Inhibits Aortic SMC Proliferation and
Activation--
To examine whether the abrogation of MAT1 affects SMC
proliferation, we used three different approaches. First, we sought to
test whether abrogation of MAT1 inhibits cell duplication. This was
accomplished by monitoring cell numbers over a 3-day culture period.
The same number of cells from G1AsMatSvNa
(MAT1-AS)-transduced, G1xSvNa (vector)-transduced, and
nontransduced (blank) cultures were plated at t = 0 h and counted at 24, 48, and 72 h. The cell number from all
groups declined ~20% at 24 h because of natural attachment of
the cells. The data show that cell duplication was at least 50%
inhibited in the MAT1-AS culture compared with the blank and
vector controls (Fig. 2A).

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Fig. 2.
Abrogation of MAT1 inhibits aortic SMC
proliferation. The same number of cells from G1AsMatSvNa
(MAT1-AS)-transduced, G1xSvNa (vector)-transduced, and
nontransduced (blank) cultures were plated for proliferation assays.
A, SMC proliferation was monitored by counting the cell
number up to 3 days before confluence. The growth curves represent the
mean ± S.D. of cells from triplicate wells. B,
subconfluent cells were incubated with MTS tetrazolium compound and
quantified at 490 nm absorbance to determine the proportion of living
cells in culture. The data represent the mean ± S.D. of
triplicate wells.
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Our second approach was to determine the proliferating cell ratio in
culture. The same number of cells from G1AsMatSvNa
(MAT1-AS)-transduced, G1xSvNa (vector)-transduced, and
nontransduced (blank) cultures were plated and grown for 72 h for
proliferation assay. The results show that the number of living cells,
represented by the amount of bioreduced formazan, was reduced ~50%
in the MAT1-AS culture compared with the blank and vector
controls (Fig. 2B).
Arterial SMCs in vivo are normally maintained in a
nonproliferative state within the tunica media. Upon arterial injury,
activated SMCs migrate into the intimal layer of the arterial wall,
where they proliferate and produce extracellular matrix components, which leads to neointima formation and causes angioplasty failure (25, 28, 46). Our third approach was to test whether the abrogation of
MAT1 inhibits the activation of SMC proliferation from a
nonproliferative state. Cells from G1AsMatSvNa
(MAT1-AS)-transduced, G1xSvNa (vector)-transduced, and
nontransduced (blank) cultures were grown to confluence and then
scraped to release cells from contact inhibition. 24 h after the
wound tracks were created, retrovirus-mediated
-galactosidase gene
transfer was performed to test the activation of SMC proliferation.
Cell proliferation along the wound margin was markedly inhibited (Fig.
3A), as evidenced by the fact
that only one positive X-gal-stained cell was detected in the
MAT1-AS culture. Also, the closure of the wound track was significantly inhibited in the MAT1-AS cultures (Fig.
3B, MAT1-AS panel IV) compared with
the blank (Blank panel IV) and vector (Vector panel IV) cultures, in which the wound
tracks were completely closed at t = 60 h. In
contrast, the time for closing the wound track in the
MAT1-AS culture was t = 120 h (data not
shown). These results show that proliferation and migration were
inhibited in MAT1-AS cells by ~50%. Our data strongly
suggest that MAT1 expression is required for the activation
of SMC proliferation and migration from a nonproliferative state.

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Fig. 3.
Abrogation of MAT1 inhibits the activation of
aortic SMCs from a nonproliferative state in an in vitro
injured tissue. Confluent SMCs from G1AsMatSvNa
(MAT1-AS)-transduced, G1xSvNa (vector)-transduced, and
nontransduced (blank) cultures were scraped to release cells from
contact inhibition. A, analysis of cell activation
efficiency. 24 h after the wound tracks were created,
proliferation activation was tested by transduction efficiency of
retroviral vector G1nBgSvNa. Activated SMC proliferation was measured
at the margin of the wound track by counting the
-galactosidase-positive cells upon exposure to X-gal under a
phase-contrast microscope. B, the time for closure of the
wound track. Blank panel I,
Vector panel I, and MAT1-AS
panel I are photographs (magnification × 40) showing
that the same size wound tracks were created. Blank
panel II, Vector panel
II, and MAT1-AS panel II
are photographs (magnification × 100) showing the appearance of
the wound margin immediately upon scraping and washing to remove the
detached cells. Blank panel III,
Vector panel III, and
MAT1-AS panel III show proliferation
and migration of cells into the wound track at t = 24 h. Blank panel IV,
Vector panel IV, and
MAT1-AS panel IV show the closure of
the wound track at t = 60 h, in which time the
wound tracks in the blank and vector cells were completely closed. The
time for closure of the wound track in MAT1-AS cells was
t = 120 h (data not shown).
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Abrogation of MAT1 Arrests Cells in G1 Phase and
Triggers Apoptosis--
Since our preliminary data (data not shown)
and some previous studies (20, 21) indicate that CDK7-cyclin H
phosphorylation of CDKs may be MAT1-independent, we linked the cell
cycle function of MAT1 using cell cycle analysis rather than testing
whether the reduced MAT1 expression affects CAK-mediated
phosphorylation of CDKs in these studies. Our initial studies showed
that RNA antisense abrogation of MAT1 inhibited cell proliferation and activation; we sought to determine whether this inhibition was due to
disruption of the normal cell cycle regulation and/or induction of
apoptosis. To answer these questions, we quantified the effect of MAT1
abrogation on the cell cycle and apoptosis in relation to their
position in the cell cycle. The cell cycle status and apoptosis of
G1AsMatSvNa (MAT1-AS)-transduced SMCs versus
controls of G1xSvNa (vector)-transduced and nontransduced (blank) SMCs were measured by flow cytometry (Fig. 4).
As shown in Fig. 4 (A-C), the negative controls for blank
(lacking terminal deoxynucleotidyltransferase), blank, and vector cells
showed normal cell cycle profiles with 32-35% of the cells in
G0/G1 phase, 39-46% in S phase, and 22-27% in G2/M phase, and the total of replicating and dividing
cells was 61-73%. However, the cells from the MAT1-AS
culture showed 66% of the cells in G0/G1
phase, 21% in S phase, and 13% in G2/M phase (Fig.
4D), and the total of replicating and dividing cells was
34%. Also, a peak of less than 2N DNA content falling in front of
G0/G1 phase represented DNA degradation, an
event reminiscent of apoptosis in the MAT1-AS culture (Fig.
4D). By comparing MAT1-AS cells with controls,
the data show that in MAT1-AS cells, 1) twice as many cells
were arrested in G1 phase, 2) replicating and dividing cells decreased ~50%, and 3) DNA degradation occurred during the cell cycle. We further analyzed the apoptotic cell ratio and its position in the cell cycle using the dual parameter display method (Fig. 4, E-H). The results show apoptotic cell death to be
undetectable in the negative controls for blank (lacking terminal
deoxynucleotidyltransferase), blank, or vector cultures (Fig. 4,
E-G). In contrast, apoptotic cell death occurred ~42% of
the cells in MAT1-AS cultures (Fig. 4H), and the
majority of these cell deaths occurred in arrested G1
cells. These results strongly suggest that MAT1 is required for
G1 exit and that apoptosis is triggered by G1
arrest.

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Fig. 4.
Abrogation of MAT1 arrests cells in
G1 phase and induces apoptosis. Nuclei of cells from
G1AsMatSvNa (MAT1-AS)-transduced, G1xSvNa
(vector)-transduced, and nontransduced (blank) cells were analyzed for
DNA content and DNA breaks by flow cytometry. The estimated proportion
of G0/G1 phase cells in the negative control
for blank (lacking terminal deoxynucleotidyltransferase), blank, and
vector cultures (A-C, respectively) ranged from 32 to 35%.
In contrast, 66% of the MAT1-AS cells were in
G0/G1 phase (D). DNA breaks were not
detectable in the blank and vector cultures (F and G,
respectively). An estimated 42% DNA breaks occurred in the
MAT1-AS cells, and the majority of DNA breaks consisted of
arrested G1 phase cells (H).
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Apoptotic Morphologic Criteria in Cells Transduced with Antisense
MAT1 RNA--
G1AsMatSvNa (MAT1-AS)-transduced cells not
only showed inhibited cell proliferation, but also had a visible
apoptotic morphology (Fig. 5). The cells
became rounded and developed vesiculations around the cell borders
(Fig. 5C) compared with nontransduced (blank) and G1xSvNa
(vector)-transduced cells (Fig. 5, A and B). We
also used the propidium iodide stain to analyze the apoptotic morphologic changes in the nuclei of the cells. Under an
epifluorescence microscope, we found condensation of nuclear chromatin
and nuclear fragmentation in MAT1-AS cells (Fig.
5F). Similar apoptotic changes were undetectable in blank
and vector cultures (Fig. 5, D and E). These
results show that the retarded SMC proliferation is associated with
apoptosis.

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Fig. 5.
Apoptotic changes of cellular and nuclear
morphologies. Cells from G1AsMatSvNa
(MAT1-AS)-transduced, G1xSvNa (vector)-transduced, and
nontransduced (blank) cultures were photographed for cellular and
nuclear morphologies. A-C were photographed under a
phase-contrast microscope. MAT1-AS cells (C)
displayed cell shrinkage, the blebbing of plasma membranes, nuclear
condensation, and nuclear fragmentation. The blank (A) and
vector (B) cultures showed normal cell morphology.
D-F are propidium iodide-stained nuclei and were
photographed under an epifluorescence microscope. MAT1-AS
cells (F) showed condensation of nuclear chromatin and
nuclear fragmentation, whereas the blank (D) and vector
(E) cultures showed normal nuclei.
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Detection of Apoptotic Cell Death--
The biochemical hallmark of
apoptosis is the cleavage of chromosomal DNA into oligonucleosomal
units (47-49). To characterize this cell death process, the TUNEL
assay was used to detect in situ apoptotic cell death. As
shown in Fig. 6, the nuclear
fragmentation was easily identified by co-locating apoptotic nuclei of
G1AsMatSvNa (MAT1-AS)-transduced cells under an
epifluorescence microscope using three different filters (Fig. 6,
C, F, and I), and this was not seen in
either the blank (Fig. 6, A, D, and G)
or vector (Fig. 6, B, E, and H)
culture. We also used an alternative method, agarose gel
electrophoresis, to detect DNA fragmentation (Fig. 7). The low molecular weight DNA of the
soluble cytoplasmic fraction isolated from G1AsMatSvNa
(MAT1-AS)-transduced cells displayed a characteristic
oligonucleosomal fragmentation ~200 bp in length, which was
undetectable in nontransduced (blank) and G1xSvNa (vector)-transduced cells.

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Fig. 6.
In situ detection of apoptotic
cell death. Cells from G1AsMatSvNa
(MAT1-AS)-transduced, G1xSvNa (vector)-transduced, and
nontransduced (blank) cultures were analyzed in situ for
apoptosis using the TUNEL assay. DNA breaks of cells were labeled with
FITC-dUTP, followed by counterstaining with propidium iodide. The
nuclei of cells were co-located under an epifluorescence microscopy
using three different filters: red for total DNA stain
(A-C), yellow green for apoptotic nuclei stain
and red for integral DNA stain (D-F), and
green for apoptotic nuclei stain (G-I). The
apoptotic nuclei were identified in the MAT1-AS culture
(C, F, and I) (arrowhead;
bar = 30 µm), but were undetectable in the blank
(A, D, and G) and vector
(B, E, and H) cells. Negative controls
for blank, vector, and MAT1-AS cells were incubated with
reaction buffer lacking terminal deoxynucleotidyltransferase (data not
shown).
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Fig. 7.
Analysis of oligonucleosomal
fragmentation. Lanes 2-4 contain 10 µg of low
molecular weight DNA isolated from nontransduced (blank), G1xSvNa
(vector)-transduced, and G1AsMatSvNa (MAT1-AS)-transduced
cells. Lane 1 contains molecular size markers.
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DISCUSSION |
Cell cycle transitions are processes that are highly regulated by
ordered assembly and timing-activated CDK complexes, and inhibition of
these kinases will cause cell cycle arrest (35, 36). CAK regulates cell
cycle progression by activating CDK complexes through phosphorylation
of a critical threonine residue in their T-loop domain (50, 51),
whereas MAT1 functions in the cell cycle by stabilizing the association
of CDK7 with cyclin H (1, 4, 6) and determining substrate specificity
of CDK7-cyclin H (20, 21, 23). In contrast to the detailed mechanisms
of regulatory subunit cyclins and catalytic subunit CDKs, whose
functions have been relatively defined in a certain period of the cell
cycle, little is known about the cell cycle phase specificity of the
targeting subunit MAT1 in cell cycle regulation. In this study, we have
shown that abrogation of MAT1 arrests SMCs in G1 phase,
triggers apoptosis, and retards SMC proliferation. Thus, our data
suggest a novel mechanism by which MAT1 controls cell proliferation
through regulating the G1 exit and mediating apoptosis.
MAT1 Regulates G1 Exit to Control Cell
Proliferation--
G1, S, G2, and M phases are
major cell cycle states. Identifying the factors that trigger the
transitions between cell cycle phases is one of the major goals in cell
cycle research (35, 36, 50-53). In our studies using RNA antisense
abrogation of MAT1, we found that the proportion of G1
phase cells is doubled and that cells in S and M phases decrease 50%
(Fig. 4D) compared with controls (Fig. 4, A-C).
These data, showing that abrogation of MAT1 causes G1
arrest, reduces proliferating cells, and induces DNA degradation (Fig.
4, A-D), are consistent with the results of cell
proliferation assays. For example, SMC proliferation is decreased
~50% in antisense MAT1-transduced cells (Fig. 2); SMC proliferation efficiency from a nonproliferative to proliferative state
is inhibited at least 50% (Fig. 3). It is known that cell cycle
transition through the G1 restriction point and entry into S phase are controlled by the activities of CDK complexes (35, 36, 51).
Our results strongly suggest that abrogation of MAT1 may cause failure
of assembly and/or determination of substrate specificity of CAK or
other CDK complexes at the G1 exit, so that the cells are
arrested in G1 phase. Currently, a CDK4-cyclin D complex
has been depicted downstream of CAK (11, 12, 16). The CDK4-cyclin D
complex phosphorylates and inactivates pRb for G1 exit (54,
55). A loss of cyclin D-dependent kinase activity prevents
many cultured cell lines from entering S phase (56), whereas
overexpression of cyclin D shortens the G1 phase (57). Besides the CDK4-cyclin D complex, CDK2-cyclin E and CDK2-cyclin A
complexes are also involved in G1/S transition (58, 59). Given that a loss of MAT1 causes G1 phase arrest, our data
support the view that MAT1 functions in G1 exit to control
G1/S transition. The challenging question that awaits to be
answered is how the G1 exit is regulated by MAT1:
(a) by determining substrate specificity of CAK at
G1/S transition through directly interacting with those known CDK complexes of G1/S transition, e.g.
cyclin D-, A-, or E-associated CDK complexes; (b) by
regulating other CDK complex assembly and interaction; or
(c) by modification of substrate specificity before the
restriction point or at G1/S transition, e.g.
whether pRb or p53 is one such target.
Abrogation of MAT1 Triggers Apoptosis--
Apoptosis removes
damaged, virus-infected, and unwanted cells for (i) development and
homeostasis, (ii) defense, and (iii) aging (60-62). Under normal
circumstances, if any damage or block is irreparable, most cells
initiate a sequence of biochemical events leading to programmed cell
death or apoptosis (63). In our studies, apoptotic cell death was
induced by G1 phase arrest (Fig. 4D), and the
majority of apoptosis occurred in the arrested G1 cells
(Fig. 4H). Our data also show that the retarded cell proliferation (Figs. 2 and 3) is the consequent incident following G1 phase arrest and apoptotic cell death. Control of cell
number is determined by an intricate balance of cell death and cell
proliferation. Our data not only demonstrate that the inhibited SMC
proliferation was due to G1 arrest and apoptosis, but also
indicate a link between cell cycle control mechanisms and apoptosis. A
recent report that an antitumor agent, Noscapine, arrests cells at
mitosis and induces apoptosis is an example that apoptosis machinery
responds to disregulated cell cycle events and kills dividing cells
(45). Some cell cycle components, e.g. p34CDC2-,
cyclin B-, and cyclin A-associated CDKs, may participate, directly or
indirectly, in part of the apoptotic pathway under certain conditions
(64-67). Our results, together with other studies, support the view
that the MAT1 gene, as a G1 exit factor to
trigger G1/S transition, may also act as an upstream
regulator of apoptosis machinery for homeostatic balance of cell
population. Determining how the abrogation of MAT1 causes
G1 phase arrest and induces apoptosis may elucidate the
mechanisms that link cell cycle control with apoptosis and allow the
design of therapies that prevent cells from replication and that
enhance cell death in the treatment of luminal narrowing in vascular
occlusive diseases and proliferation disorders in cancer.