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Research Communications |
Department of Intractable Diseases and
a Appropriate Technology Development and Transfer, International Medical Center of Japan, Shinjuku-ku, Tokyo 162-8655, Japan;
b Department of Microbiology, Tokyo Metropolitan Institute of Medical Science, Bunkyo-ku, Tokyo 113-8613, Japan;
c Department of Molecular Biology and Cellular Biology, Faculty of Pharmaceutical Sciences, Kanazawa University, Kanazawa 920-0934, Japan; and
d Jichi Medical School, Minamikawachi-machi, Kawachi-gun, Tochigi 329-0498, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: HIV-1/Vpr • multinucleation • genomic instability
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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It is now well accepted that cell cycle abnormality would serve as a molecular basis for genomic instability in tumors (20) . One of the tumor suppressor genes, p53, causes cell cycle arrest at G1/S phase when cells are treated with DNA damaging agents (21) , the dysfunction of which induces genomic instability such as gene amplification 22-24) . Recently, it was reported that p53 was also related to mitotic checkpoint control (25) . Abrogation of mitotic checkpoint control induces genomic instability 25-29) . For instance, a mutation of the human BUB1, a homologue to the yeast gene regulating mitotic spindle checkpoint control (28) , was detected in human colon tumors (29) . The expression of the mutated human BUB1 gene resulted in the abrogation of M phase checkpoint control (29) .
Tumors such as B cell lymphomas and Kaposi's sarcomas are frequently observed in AIDS patients (30 , 31 ). Although tumor development in the AIDS patients has been considered as a result of the compromised immunosurveillance mechanism 32-34) , it was recently pointed out that HIV itself was actively involved in tumorigenesis (35 , 36 ). The molecular mechanism of the AIDS-related oncogenesis remains unclear, but it is tempting to speculate that Vpr, which induces perturbation of cell cycle, works as one of the causative factors in tumor development.
We present evidence here suggesting that Vpr induces a high frequency of micronuclei (MIN) formation, a hallmark of aneuploidy 37-40) . Data suggest that Vpr serves an important role in the development of AIDS-related tumors.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Deletion mutants of Vpr, 
C2, C5, and C12, which lacked
carboxy-terminal 2, 5, and 12 amino acids, respectively, were generated
by polymerase chain reaction using Vent polymerase (Gibco BRL, Grand
Island, N.Y.) by using WT Vpr DNA fragment as a template. An
oligonucleotide, 5'-ATCGATGGAACAAGCCCCAGAA-3', was used as a common
forward primer and 5'-TTAACTGGCTCCATTTCT-3', 5'-TCAATTTCTTGTTCTCCT-3',
and 5'-TTAAATAAT-GCCTATTCT-3' were used as reverse primers for
obtaining C2, C5, and C12, respectively. Each clone was first ligated
to a TA cloning vector (Invitrogen, San Diego, Calif.). To make C18,
WT Vpr DNA was digested with SalI first, end-filled with
Klenow fragment, then ligated with XbaI linker
(5'-CTCTAGAG-3', Promega), which generated an in-frame stop codon
around the carboxyl end of LR domain (42
,
43
). Each mutant was sequenced and the deduced amino acids
were confirmed (see Fig. 4A
); then the inserts were ligated
to an expression vector, pBabepuro.
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Establishment of MIT-23
Human fibrosarcoma cell line HT1080 was obtained from the Health
Science Research Resources Bank (JCRB9113) of Japan. Cells were
maintained in Dulbecco's modified Eagle medium (Nissui, Tokyo, Japan)
supplemented with 10% heat-inactivated fetal calf serum (BioWhittaker,
Walkersville, Md.) and 2 mM glutamine (Wako, Osaka, Japan). HT1080 was
known as a pseudodiploid cell line with apparent 46XY, as described in
the ATCC catalogue. Cells transfected with pUHD172-1neo by the reported
method (44)
were selected with 400 µg/ml of G418
(Wako), then each clone was checked for the inducibility of the
tet-regulated trans-activator function using
pUHC13-3 containing luciferase gene (41)
. RTC-12, which
showed the highest fold increase of luciferase activity induced by DOX
(5 µ/ml), was further introduced by pTO/Vpr. After the second
transfection, cells were cultured in the presence of G418 and
hygromycin (25 µg/ml) (Wako). Fifty-five clones with resistance to
both drugs were picked up and tested for cell cycle abnormality with 5
µg/ml of DOX. Fourteen of these 55 clones were chosen as candidates
for DOX-inducible cell cycle abnormality, one of which was picked up as
the best clone and named MIT-23 (multinuclear cell
induced by tetracycline). As a control clone,
VPR, which was introduced with the plasmids containing the same
components except for Vpr gene, was obtained. In some
experiments, VPR-5 and –6, which were subclones from VPR cells,
were used.
FACS analysis of cell cycle and MPM-2-positive cells at M phase
Cell cycle analysis was performed by the method reported
(45)
. Propidium iodide (PI) (Sigma) at the concentration
of 50 µg/ml was used for DNA staining. Cells at M phase were stained
with MPM-2 antibody (DAKO, Bucks, U.K.) by the method described
(28
, 46
). Cells were first treated by a
double thymidine block, then further cultured for 12 h in the
presence of 0.2 µg/ml nocodazole (Sigma). The first antibody with
200-fold dilution was incubated for 1 h at 37°C in
phosphate-buffered saline (PBS) supplemented with 0.02% Tween 20
(PBST) added to 10% normal goat serum. An FITC-conjugated goat
anti-mouse IgG was used as a second antibody. After washing with PBST
several times, DNA was stained with 50 µg/ml PI, then 2-dimensional
fluorescein-activated cell sorter (FACS) analysis (Beckton Dickinson,
N.J.) was carried out.
Histone H1 kinase activity
Histone H1 kinase activity was measured according to the
method reported (47)
. Cells were treated with 0.2 µg/ml
of nocodazole for 24 h, then cell extracts of 100 µg protein
were immunoprecipitated using a polyclonal antibody to carboxy-terminal
amino acids of human CDC2. The precipitates were further incubated in
10 µl of a buffer containing 20 mM Hepes-NaCl (pH 7.5), 15 mM EGTA,
20 mM MgCl2, 1 mM dithiothreitol, 1 µg of histone H1
(Boehringer Mannheim, Mannheim, Germany), and 50 µM
-32P-ATP (1000–2000 cpm/pmol) (Amersham, Arlington
Heights, Ill.) for 10 min at 30°C. Each sample was mixed with 20 µl
of 3x sodium dodecyl sulfate (SDS) sample buffer for terminating the
reaction. It was then boiled and applied to SDS-polyacrylamide gel
electrophoresis. After electrophoresis, the signal of the
phosphorylated histone was exposed to an X-ray film and the intensity
of each band was measured.
Preparation of monoclonal antibody against Vpr and immunostaining
Recombinant Vpr protein was expressed using a yeast expression
system (Pichia Pastoris, Invitrogen) according to the
manufacturer's protocol. Western blot analysis using a polyclonal
antibody provided by the NIH AIDS Research and Reference Reagent
Program (Cat. No. 3951) confirmed that the expressed protein is Vpr.
After partial purification using hydroxyapatite column chromatography
(Seikagaku Kogyo, Tokyo, Japan), about 100 µg of the recombinant
protein was used for a single immunization. For immunostaining, cells
were fixed in 4% paraformaldehyde in PBS, followed by permeabilization
in 0.2% Triton X-100. Blocking was performed using PBST with 10%
normal goat serum. After washing with PBST, the fixed cells were
incubated with one of three antibodies, clones 2A6, 1A2, and 3B4, all
of which were IgM subclass, for 1 h at 37°C. For detecting the
centromeric component, anti-kinetochore monoclonal antibody (COSMO BIO,
Tokyo, Japan) was used. FITC-conjugated anti-mouse IgA, G, and M (Zymed
Laboratories, San Francisco, Calif.) were used as a second antibody.
DNA was stained with PI at the final concentration of 1 µg/m or 50
ng/ml of 4,6-diamidino-2-phenylindole (DAPI) (Sigma). Stained cells
were examined in an antifade solution (KPL, Gaithersburg, Md.) using a
fluorescence microscope (Olympus, Tokyo, Japan) equipped with a Sensys
CCD camera (Photometrics, Tucson, Ariz.). Image analysis was carried
out by IP lab. spectrum (Scanalytics, Fairfax, Va.). For checking the
specificity of the antibody produced by 2A6 clone, about 10 µg Vpr
protein that was purified by the BIOCAD system (PerSeptive Biosystems,
Framingham, Mass.) was added during incubation of the first antibody.
Karyotype analysis
Karyotype analysis was carried out by Biomedical Laboratories
Inc. (Japan) based on the conventional method (48)
.
Chromosomes were stained with Giemsa solution and examined by a
microscope. Twenty to 30 metaphase spreads were examined in each
sample.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In MIT-23 cells Vpr mRNA as well as its protein expression
was controlled by DOX addition. Figure 1A
shows that the transcription of Vpr mRNA was quickly induced
within 6 h after DOX addition (Fig. 1A
, lane 3) and
reached a maximum level in 24 h (Fig. 1A
, lanes 5 and
6). On the other hand, DOX removal diminished Vpr mRNA in
12 h (Fig. 1A
, lane 9). Then, Vpr expression was mostly
abolished in 48 h after DOX removal (Fig. 1A
, lane 12).
Expression of Vpr protein was induced by DOX treatment, as shown in
Fig. 1B
(panel 3). The control cell line VPR, which was
obtained by introducing the same plasmids present in MIT-23 except for
the Vpr gene (panel 1), and DOX-untreated MIT-23 cells
(panel 2) gave no positive signals. Different monoclonal antibody
commonly detected the Vpr product in the perinuclear region (Fig. 1B
, panel 4), consistent with the reports by several groups
42,43,49)
. Vpr was also detected in MIN (panel 3 in Fig. 1B
, indicated by arrowheads).
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Cell cycle abnormality was manipulated by Vpr expression, as shown in
Fig. 2
A.On day 4 after Vpr expression, about 50% and 20% of cells accumulated
in the regions of G2/M phase and hyperploidy (indicated by an arrowhead
and arrow in panel 2), respectively. When Vpr expression was switched
off by DOX removal, normal cell cycle was restored in 10 days (Fig. 2A
, panel 3). Successive DOX treatment reproduced the
Vpr-induced cell cycle perturbation (Fig. 2A
, panel 4). The
result of FACS analysis on one of the other transfectants (4b-30) is
also shown in Fig. 2A
(panels 5 and 6). This clone had G2/M
accumulation even without the presence of DOX, but DOX addition
enhanced cellular accumulation at G2/M. The striking difference after
DOX addition was that the cell population with hyperploidy was greatly
increased (panel 6). As a control, VPR cells did not show any
changes in the cell cycle with DOX treatment (panels 7 and 8).
Morphological change was observed concomitantly with cell cycle
abnormality. As shown in Fig. 2B
, Vpr caused an enlargement
of cytoplasm and multinuclear cell formation (center panel), which was
apparently reversible, as shown in Fig. 2B
(right panel).
Figure 2C
shows the effects of Vpr on cellular growth. Vpr
expression inhibited cell growth to about 10% of the control (right
panel, shown by closed triangles), but it should be noted that Vpr did
not induce complete cell cycle arrest in MIT-23 cells.
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The induction of H1 kinase activity was inhibited by Vpr
expression (Fig. 2D
), as reported by several groups
(10
, 13
, 17
). Although an
increase of H1 kinase activity after nocodazole treatment was observed
on day 1 in Vpr-expressing MIT-23 cells, this induction was gradually
reduced on day 3 and day 5 (indicated by triangles in the upper panel
of Fig. 2D
), and on day 7 it was attenuated to less than
half that of the control cells (Fig. 2D
, indicated by
triangles in upper panel and in lower panel). By contrast, VPR cells
kept high H1 kinase activity throughout the 7 days of examination
(shown by open circles in the upper panel of Fig. 2D
).
These data suggest that Vpr induces cell cycle retardation with the cellular accumulation at G2/M phase and hyperploidy, resulting in the decreased H1 kinase activity.
In MIT-23 cells, multinuclear cell formation (multinucleation) was
observed by Vpr expression. A typical morphology of multinucleated
cells stained with PI is shown in Fig. 3A
. More than 20% of cells became multinuclear in 2 days
after Vpr expression; on day 10 after Vpr expression, more than 35% of
the cells (22% for binuclear cells and 15% for cells with more than 3
nuclei) were multinuclear, as summarized in Table 1
. We also observed mononuclear cells with large nuclei and possibly
increased DNA content; some are also shown in Fig. 3A
. By
contrast, VPR and MIT-23 cells without Vpr expression did not show
any induction of multinuclear cell formation (data not shown). MIT-23
cells, even when small number of cells were plated, became multinuclear
cells by Vpr expression, suggesting that multinucleation by Vpr was not
due to cell fusion (50
, 51
).
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Figure 3A
also shows the presence of MIN, which were
resistant to RNase treatment (data not shown). We characterized the
relation between multinucleation and MIN formation. The results of an
analysis of cells with Vpr expression for 5 days are summarized in
Table 2
. The population of multinuclear cells in MIT-23 cells without Vpr
expression was only 1.7%, a quarter of which were MIN positive, making
the incidence of the MIN-positive multinuclear cells in the total
population 0.4%. By Vpr expression, the incidence of multinuclear
cells increased to 33%, 77% of which were MIN positive. Thus, the
incidence of multinuclear cells with MIN in the total population was
calculated as 25%. On the other hand, in one of two independent
control clones, VPR-6, the incidence of multinuclear cells with MIN
in the total population was 0.5%. In another clone—VPR-5—it was
not detected. When the incidence of MIN formation was normalized by the
incidence of VPR-6 as 1, the increase of Vpr-induced MIN formation
was 50-fold greater.
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To know whether MIN formation is specifically induced by Vpr or simply
due to multinucleation of the cells, we compared the incidence of MIN
formation in cytochalasin B (CCB) -treated MIT-23 cells and DOX-treated
MIT-23 cells. CCB is known as an inducer of tetraploidization
(52)
. The results are shown in Table 3
. By CCB treatment, 60% of MIT-23 cells became multinuclear (90%
binuclear) and 3.3% of these multinuclear cells were MIN positive. The
incidence of multinuclear cells with MIN induced by CCB treatment was
calculated as 2.0%. On the other hand, the incidence of multinuclear
cells with MIN induced by Vpr was 8.4%, indicating that the incidence
of MIN formation in Vpr-induced multinuclear cells was more than
fourfold higher than in CCB-induced multinuclear cells. These
observations suggest that MIN formation is not simply because of
multinucleation, but that Vpr actively induced MIN formation.
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It is important to know whether HIV infection itself induces MIN. To
answer this question, MAGI cells, a HeLa cell derivative that expresses
the CD4 containing, HIV-LTR-driven LacZ gene
(53)
, were infected with WT HIV (54)
, then
the generation of MIN formation was examined. As shown in Fig. 3B
(left panel), HIV-infected cells were identified by X-gal
staining. DAPI staining then detected MIN formation in these cells
(right panel), and 18% of HIV-infected cells were positive for MIN
formation. On the other hand, only 3% of ß-galactosidase-negative
cells contained MIN, indicating that HIV infection itself induced a
sixfold increase in MIN formation.
Next we determined the functional domain of Vpr, which is
necessary for inducing multinucleation and/or MIN formation. Since it
has been reported that the carboxy-terminal region of Vpr is important
for cell cycle abnormality (12
, 43
), deletion
mutants lacking carboxyl amino acids (Fig. 4A
)were generated and expressed in HT1080. After about 10 days of
selection with puromycin, cell cycle abnormality was analyzed. Results
representative of three independent experiments are shown (Fig. 4B, C
). Figure 4B
shows histograms of DNA content
of each transfectant and Fig 4C
represents the relative
increase of cell number at G2/M phase and 8N. The mutants of C2,
C5, and C12, which lacked carboxy-terminal 2, 5, and 12 amino
acids, respectively, could induce both G2/M accumulation and
hyperploidization, as shown in Fig. 4B
(panels 3–5). On the
other hand, the mutant of C18 did not show any changes of cell cycle
(panel 6). MIN formation was also studied in these transfectants and we
observed that it was increased in all mutants except for C18. In
Table 4
, the results of G2/M arrest, multinucleation, and MIN formation
are summarized. These data indicate that Vpr-induced MIN formation
correlates well to the activity of multinucleation as well as G2/M
arrest.
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Possible `asynchronous cytokinesis' after abolishment of Vpr
expression
Based on the observation of Vpr-induced multinucleation, cell
cycles seemed to be halted at cytokinesis. We studied the result after
DOX deprivation. Multinuclear cells started to undergo cytokinesis
after DOX removal. Typical morphology is shown in Fig. 5A
(panel 1). From a giant multinuclear cell, many nuclei seemed to be
coming out at the same time, as indicated by arrowheads. Figure 5A
(panels 2–5) also shows the time-lapse change of the
same phenotype by photographs taken serially. By 12 h after DOX
deprivation, a cell started to form a cleavage furrow (panel 2,
indicated by an arrow). A stalk containing a nucleus became elongated
(panel 3), and then a daughter cell was shown staying beside the
parental cell with a thin midbody (panel 4, indicated by an arrow).
Finally, a newly born daughter cell was seen separate from the parental
cell (panel 5). Such a phenomenon was observed in about 80% of
DOX-deprived MIT-23 cells after 10 day of Vpr expression. About 22% of
MIT-23 cells with continuous expression of Vpr also exhibited the same
phenomenon. By contrast, we never detected such a phenomenon in
DOX-treated VPR cells or MIT-23 cells without Vpr expression.
Although there is a small possibility that cells with single nuclei are
fused with a multinuclear giant cell first and then, once fused, cells
started to separate, we concluded that the phenomenon is Vpr specific
and considered it as `asynchronous cytokinesis.
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By close observation, we found the presence of cells undergoing
asynchronous cytokinesis without accompanying MIN, as shown in Fig. 5B
(nuclei just emerging from the cell and MIN are indicated
by arrows and arrowheads, respectively). Such an observation strongly
suggested that aneuploidy would be induced by transient Vpr expression.
To clarify this point, we studied the effects of transient expression
of Vpr on chromosome integrity. As the experimental protocol shows in
Fig. 6A
,the addition of DOX, followed by its removal, was repeated three times
and then the cell cycle pattern after each procedure was examined. The
populations corresponding to `4N' and `8N' gradually increased
after each cycle of transient Vpr expression (Fig. 6B
: 59,
68, and 83%, as shown in panels b, d, f). MIT-23 cells, which had
about 80% of cells located in 4N and 8N, were further cultured without
the presence of DOX. The ploidy change is shown in Fig. 7A
.The major 4N peak became more prominent and the 2N peak became very
small (panels 1–5). On day 252, the population with 2N disappeared
(panel 6). Most MIT-23 cells contained more than 4N DNA content. In
MIT-23 cells Vpr mRNA was not expressed, as examined by
Northern blot analysis (data not shown). Using a monoclonal antibody,
MPM-2, which recognizes phosphorylated proteins specifically present at
M phase (46)
, the kinetics of cell cycle was studied in
MIT-23 cells (Fig. 7B
). Cells were first synchronized at
early S phase by double thymidine block, then released in the presence
of nocodazole. MIT-23 cells just after the thymidine block contained a
few MPM-2-positive cells (Fig. 7B
, panel 1). After 12 h
treatment with nocodazole, the increase of MPM-2-positive cells was
observed in the region corresponding to cells composed of 4N DNA (Fig. 7B
, `R2' in the panel 3). The population of MPM-2-positive
cells was 9.0%. On the other hand, two kinds of MPM-2-positive
populations were recognized in the MIT-23 cells (R2 and R3 in panel
7 of Fig. 7B
). One population corresponded to an apparent
DNA content of 4N (R2 in panel 7) and the other to a possible 8N (R3).
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In two other independent experiments, we confirmed the reproducibility
of the Vpr-induced ploidy change in MIT-23 cells, whereas VPR cells
showed no changes in ploidy from the same DOX treatment (Fig. 7A
, panels 7 and 8). In addition to MIT-23 cells,
MIT-23/409, which was cultured with continuous expression of Vpr for 6
months, also became hyperploid cells (data not shown).
Karyotype analysis of MIT-23 cells detected that all of the 30
metaphase spreads studied had abnormal numbers of chromosomes. The
representative karyotypes are shown in Fig. 8
(panels 1 and 2) and the results were summarized in Table 5
. In addition, MIT-23/409 cells were also aneuploid (Fig. 8
, panel
3 and Table 5
). Before treatment of DOX, MIT-23 cells showed
pseudodiploid, as did HT1080 (Fig. 8
, panel 4 and Table 5
). We
confirmed that there were no changes in karyotype of VPR cells
exposed to DOX by the same protocol as MIT-23 cells (Fig. 8
, panels
5 and 6, and Table 5
), suggesting that aneuploidy was caused by Vpr.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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VPR cells.
Furthermore, we checked cell cycle abnormality, using more than 100
clones that were transfected with the same plasmids in which only
the Vpr fragment was replaced with other DNAs, such as SLAP
(src-like adapter protein) cDNAs (55)
. There were no
clones except for Vpr transfectants, which showed cell cycle
abnormality in response to DOX treatment. In addition to MIT-23 cells,
we observed essentially the same phenotype in other clones; the result
of one (b4-30) is presented in Fig. 2A
(panels 5 and 6).
Southern blot analysis on the DNAs extracted from clones gave different
hybridization pattern to the Vpr probe (data not shown),
indicating that the phenotype observed in the present study is not due
to the effects of integration sites of transfected Vpr gene,
but is strictly Vpr specific.
Multinucleation and G2/M arrest induced by Vpr
In MIT-23 cells, Vpr induced multinucleation as well
as G2/M arrest. It was reported that Vpr induced hyperploidy
in single nuclei when it was expressed in Jurkat cells
(13)
. There are, however, several reports showing the
formation of multinuclear giant cells as the phenotype of Vpr activity.
In these reports adherent cells such as rhabdomyosarcoma cells
(56)
, osteosarcoma cells (10)
, and HeLa cells
(43)
were used, suggesting that Vpr-induced
multinucleation is dependent on cell types. Another possible, more
likely explanation is based on our experience in the present study,
which is that multinucleation takes more time than G2/M arrest. In
HT1080 cells, G2/M accumulation was observed a few days after
transfection of Vpr gene, but it takes at least 7 days for
multinucleation (M. Shimura, unpublished results). To date, all
experiments on Vpr have been performed based on transient expression
assays, and Vpr-induced multinucleation may have passed unnoticed in
some cases. Apoptosis is not a major biological phenomenon in MIT-23
cells, although occasionally it is induced by Vpr
(57)
.
Vpr-induced MIN formation and aneuploidy
The incidence of MIN in Vpr-expressing cells was more than 50-fold
higher than in control cells (Table 2)
. Furthermore, Vpr induced a
fourfold higher incidence of MIN formation than CCB, an inhibitor of
the function of spindle fibers. Because the incidence of CCB-induced
multinucleation was twice as much as that induced by Vpr (Table 3)
, we
suggest that MIN formation induced by Vpr is not due to the secondary
effects of multinucleation, but by active function of Vpr. MIN
formation was observed in other cell lines such as V79, HeLa, and 293
cells. In V79 cells, for example, an 11-fold higher incidence of MIN
formation was found in Vpr-expressing multinuclear cells than in
Vpr-negative cells (data not shown). It is noteworthy that HIV
infection itself caused MIN formation, as presented in Fig. 2B
. These data suggest that Vpr generally induces MIN
formation.
MIN formation has been considered a hallmark of aneuploidy and is used
for assessing mutagenicity of the test compounds 37-40)
.
On the other hand, we observed MIN remaining in the cytoplasm during
asynchronous cytokinesis (Fig. 5A
), which suggests that the
transient Vpr expression easily induced genetic unbalance in parental
and daughter cells. As a consistent result, MIT-23 cells turned out to
be aneuploid after transient Vpr expression, as shown in Fig. 8
and
Table 5
. Furthermore, we found that Vpr induced chromosome breaks,
resulting in a high incidence of gene amplification (M. Shimura,
unpublished results). An apparent asymptomatic phase has been pointed
out (58)
in the natural history of HIV infection. Even
during such a period, virus is actively produced 59-61)
.
Reports that Vpr is present in the AIDS patients' sera
(62
, 63
) and that exogenously added Vpr can
enhance viral production of the cells with latent infection
(62
, 63
) suggest that Vpr would be
distributed in the whole body of the AIDS patients. Many cells are
susceptible to repeated or continuous exposure of Vpr affecting genetic
integrity. It is reasonable to speculate that Vpr serves as a molecular
basis of malignancies in AIDS patients.
Mechanism of Vpr-induced cell cycle perturbation
In the present study, we determined the functional domain of
Vpr that induced MIN formation, G2/M arrest, and multinucleation.
Deletion mutants lacking carboxy-terminal 12 amino acids could induce
all of these phenotypes (Fig. 4B, C
; Table 4
). On the other
hand, the mutant without carboxyl 18 amino acids did not show such an
abnormality, suggesting that at least the carboxyl terminus of Vpr
plays an important role for these phenotypes.
It has been reported that Tax of HTLV-1 also induced MIN formation
(64
, 65
). In an important finding, Tax
oncoprotein was recently shown to induce multinucleation by interfering
with the interaction of MAD1 and MAD2 (26)
, mammalian
homologues to yeast genes that are involved in mitotic checkpoint
control (26
, 66
). The molecular mechanism of
Tax-induced genomic instability was well studied using various mutants.
The functional domain inducing MIN formation was shown to be present
primarily in its carboxy-terminal region (65)
, whereas the
activity of multinucleation and MAD1 binding was present in the amino
half of the protein (26)
, suggesting that MIN formation
and multinucleation were perhaps regulated by different mechanisms. It
remains to be clarified whether Vpr induces cell cycle abnormality
based on the same molecular mechanism as Tax. Tax-induced
multinucleation was enhanced by the addition of nocodazole, suggesting
that the functions of Tax and nocodazole are on the same pathway of
G2/M checkpoint control (26)
. To the contrary, we observed
that Vpr-induced multinucleation was reduced by nocodazole from 26% to
18% of the population (M. Shimura, unpublished data). In addition,
immunostaining of Vpr indicated that it was not present in the midbody
(Fig. 1B
, panel 4, indicated by an arrow), where MAD1 had
been reported to be localized (26)
. The staining pattern
of MAD2 (66)
, which was reported to localize in
centrosomes, is also different from that of Vpr (M. Shimura,
unpublished results). It is likely that the mechanisms of
multinucleation by Vpr and Tax are different.
We now hypothesize that there are at least two molecular mechanisms of
Vpr-induced G2/M arrest, one occurring in cytoplasm and the other in
the nucleus. The LR domain of Vpr (42
, 43
,
67
) was reported to have 60% identity with a product of
Sac1p, a yeast gene regulating the cytoskeletal function
(68)
. Vpr, even as a whole protein, had 30% identical
amino acids with 45% similarity to SAC1P (68)
. Actually,
Vpr was reported to induce actin disruption (69)
.
Furthermore, even Vpr mutants that lacked the property of nuclear
translocation could induce G2/M arrest (43)
. This
information supports the idea that Vpr impairs cytoskeletal proteins as
well as those present in cytoplasm, which also include mitochondrial
proteins (14)
. Another possibility is that G2/M arrest
could be induced by the event evoked in nuclei. We observed a high
frequency of Vpr-induced chromosome breaks in main nuclei and in MIN
(M. Shimura, unpublished results). Vpr-induced DNA damages, in turn,
arrest cells at G2/M phase, as reported in -irradiated cells
(70
, 71
). G2/M arrest in Vpr-expressing cells
may be caused by more than a single molecular event.
Currently we are characterizing cellular proteins that interact with Vpr by comparing the peptide pattern detected in the transfectants expressing WT Vpr and mutant Vpr that fail to induce cell cycle abnormality. Studying the molecular mechanism of Vpr-induced cell cycle perturbation may uncover a novel machinery of spindle checkpoint control.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Abbreviations: CCB, cytochalasin B; DAPI,
4,6-diamidino-2-phenylindole; DOX, doxycycline; FACS,
fluorescein-activated cell sorter; HIV-1, human immunodeficiency virus
type 1; MIN, micronuclei; PBS, phosphate-buffered saline; PBST, PBS
supplemented with 0.02% Tween 20; PI, propidium
iodide.
Received for publication August 3, 1998.
Revision received November 24, 1998.
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