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(The FASEB Journal. 1999;13:621-637.)
© 1999 FASEB

Research Communications

Micronuclei formation and aneuploidy induced by Vpr, an accessory gene of human immunodeficiency virus type 1

MARI SHIMURA, YOSHIKAZU TANAKAb, SATOSHI NAKAMURAa, YUZURU MINEMOTOc, KATSUMI YAMASHITAc, KIYOHIKO HATAKEd, FUMIMARO TAKAKUd and YUKIHITO ISHIZAKA*

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


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Vpr, an accessory gene of HIV-1, induces cell cycle abnormality with accumulation at G2/M phase and increased ploidy. Since abnormality of mitotic checkpoint control provides a molecular basis of genomic instability, we studied the effects of Vpr on genetic integrity using a stable clone, named MIT-23, in which Vpr expression is controlled by the tetracycline-responsive promoter. Treatment of MIT-23 cells with doxycycline (DOX) induced Vpr expression with a giant multinuclear cell formation. Increased micronuclei (MIN) formation was also detected in these cells. Abolishment of Vpr expression by DOX removal induced numerous asynchronous cytokinesis in the multinuclear cells with leaving MIN in cytoplasm, suggesting that the transient Vpr expression could cause genetic unbalance. Consistent with this expectation, MIT-23 cells, originally pseudodiploid cells, became aneuploid after repeated expression of Vpr. Experiments using deletion mutants of Vpr revealed that the domain inducing MIN formation as well as multinucleation was located in the carboxy-terminal region of Vpr protein. These results suggest that Vpr induces genomic instability, implicating the possible role in the development of AIDS-related malignancies.—Shimura, M., Tanaka, Y., Nakamura, S., Minemoto, Y., Yamashita, K., Hatake, K., Takaku, F., Ishizaka, Y. Micronuclei formation and aneuploidy induced by Vpr, an accessory gene of human immunodeficiency virus type 1.


Key Words: HIV-1/Vpr • multinucleation • genomic instability


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
VPR IS AN ACCESSORY GENE of human immunodeficiency virus type 1 (HIV-1)1 encoding a virion-associated nuclear protein of about 15 kDa in size 1-3) . It is a conserved gene present in HIV-2 as well as in the simian immunodeficiency virus (4 , 5 ), crucial for productive infection to macrophages (6 , 7 ). Recently, several groups have reported that the Vpr of HIV-1 induces cell cycle abnormality (see review, ref 8 ), causing cell accumulation at G2/M phase and increased ploidy. Such a biological function of Vpr is conserved in primate lentiviruses (9) and in a variety of cells including mammalian cells 10-13) , yeast (14 , 15 ), and bacteria (16) . In Vpr-expressing cells, cyclin B-dependent p34cdc2 activity is down-regulated with a nonphosphorylated inactive form of CDC25C (17) . It was recently reported that the viral replication increased more than twofold in the cells arrested by Vpr (18) , which could explain why the virus with wild-type (WT) Vpr would have a replication advantage in vivo (18 , 19 ).

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.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmids and reagents
Plasmids for the reverse-tet system (41) were from Dr. Bujard (Heidelberg University, Germany). A plasmid, pUHD172-1neo containing a neomycin-resistant gene as a selective marker, encodes a tet-responsive trans-activator, the activity of which is switched on by addition of doxycycline (DOX) (Sigma, St. Louis, Mo.). The plasmid with a tet-responsive DNA element, pUHG16-3 (40) , which was inserted with a hygromycin-resistant gene, was named pTO. An effector plasmid, pTO/Vpr, was constructed by ligating a BamHI fragment of Vpr gene, which was excised from pBABE-vpr (10) to the same sites of pTO. The Vpr gene was originally cloned from HIV-1 NL4-3. The sense orientation of the constructs was confirmed by restriction mapping.

Deletion mutants of Vpr, {Delta}C2, {Delta}C5, and {Delta}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 {Delta}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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  		Figure 4. Functional domain inducing cell cycle abnormality and MIN formation. A) Amino acid sequence of carboxy-terminal region Vpr expressed in HT1080 cells. Amino acids in wild-type (WT) Vpr and its mutants based on the nucleotide sequence data are shown. Dots indicate the position at 20 or 10 amino acids from carboxyl terminus. Single letters are used to describe amino acids. Some mutations were present in the described region, but the sequence in other regions (especially for {Delta}C18) was identical to that of WT Vpr. B) Cell cycle analysis of transfectants. FACS analysis of transfectants with the vector (panel 1), WT Vpr (panel 2), Vpr mutants of {Delta}C2 (panel 3), {Delta}C5 (panel 4), {Delta}C12 (panel 5), and {Delta}C18 (panel 6) was performed after puromycin selection for about 10 days. Cell population in the transfectants with {Delta}C18 showed almost the same cell cycle pattern as that of the control vector. C) Increased cell population at G2/M and 8N in the transfectants with WT Vpr, {Delta}C2, {Delta}C5, and {Delta}C12. Based on the results shown in panel B, the number of cells located at G1/S, G2/M, and 8N in each sample was subtracted from those of control cell populations, which were transfected with the vector. The cell cycle pattern was then plotted.

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, {Delta}VPR, which was introduced with the plasmids containing the same components except for Vpr gene, was obtained. In some experiments, {Delta}VPR-5 and –6, which were subclones from {Delta}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 {gamma}-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.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
We first established MIT-23 in which Vpr expression was regulated by DOX addition (see Materials and Methods).

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 {Delta}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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  		Figure 1. Establishment of MIT-23 cells. A) Northern blot analysis of Vpr mRNA expression regulated by DOX. RNA was prepared at 0 (lane 2), 6 (lane 3), 12 (lane 4), 24 (lane 5), and 120 (lane 6) h at DOX addition. As a control, RNA was extracted from cells of {Delta}VPR (lanes 1 and 7), which was transfected with the same plasmids as those in MIT-23 except for the Vpr gene. After preincubation of MIT-23 cells with DOX for 24 h (lane 8), cells were washed and RNA was prepared after 12 (lane 9), 24 (lane 10), 36 (lane 11), and 48 (lane 12) h. As an internal control, human GAPDH cDNA was used as a probe. B) Immunostaining of the Vpr product. One of the monoclonal antibodies (2A6) was used to detect the product in DOX-treated {Delta}VPR cells (panel 1), DOX-untreated MIT-23 cells (panel 2), and DOX-treated MIT-23 cells (panel 3). Another monoclonal antibody (1A2) was also used to detect Vpr in DOX-treated MIT-23 (panel 4). When recombinant Vpr protein was included at the time of the first antibody treatment, the signal was completely abolished (data not shown). Panels 3 and 4 show MIN and a midbody, indicated by arrowheads and an arrow, respectively (see text).

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, {Delta}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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  		Figure 2. Characterization of Vpr-induced cell cycle abnormality. A) FACS analysis of MIT-23 cells with Vpr expression. MIT-23 cells treated with DOX (5 µg/ml) for 4 days is shown in panel 2. As a control, a cell cycle pattern of DOX-untreated MIT-23 cells is also shown in panel 1. After 4 day of DOX treatment, cells were washed and further cultured in the absence of DOX for another 10 days (panel 3), followed by a second DOX treatment (panel 4). Another clone (b4-30) also responded to the addition of DOX, leading to cellular accumulation at G2/M phase and hyperploidy (panels 5 and 6). Control {Delta}VPR cells without or with DOX (panels 7 and 8, respectively) are also shown. In panel 2, 4N and 8N peaks are marked by an arrowhead and an arrow, respectively. B) Morphological changes of MIT-23 cells without (left) or with (center) Vpr expression. Cellular morphology after 4 days with DOX, followed by 10 days of DOX-free culture, are shown (right panel). Each photograph was taken at the same magnification (x100). C) Cell growth inhibition by Vpr. Each group of 103 cells of MIT-23 and control RTC-12 was plated to 35 mm plates in the presence of 1 µg/ml of DOX. Cell count was plotted every 3 days up to day 10. RTC-12 is a subline of HT1080 cells transfected with a pUHG172-1neo. D) H1 kinase activity. The activity was measured by the method reported (47). MIT-23 and {Delta}VPR cells were cultured in the presence of DOX for the periods indicated. After nocodazole treatment for 24 h, each cell extract was subjected to kinase assay. The signal intensity from each band, shown in the lower panel, was measured and the relative activity was plotted (see upper panel). The samples are {Delta}VPR cells without nocodazole (indicated by squares), with nocodazole (circles), MIT-23 cells without nocodazole (diamonds), and MIT-23 cells with nocodazole (triangles).

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, {Delta}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, {Delta}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 3. Multinuclear cell formation and MIN formation by Vpr. A) Morphology of MIT-23 cells with Vpr expression. MIT-23 cells with Vpr expression for 4 days presented typical giant multinuclear cells. Bizarre-shaped MIN are also observed in MIT-23 with Vpr expression. B) MIN formation after HIV infection. MAGI cells were infected with HIV; on day 3 after HIV infection, ß-galactosidase-positive cells were examined for MIN formation. Cell morphology after X-gal staining (left panel) and DAPI staining (right panel) are shown.


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  		Table 1. Time course of multinuclear cell formation in MIT-23 cellsa

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, {Delta}VPR-6, the incidence of multinuclear cells with MIN in the total population was 0.5%. In another clone—{Delta}VPR-5—it was not detected. When the incidence of MIN formation was normalized by the incidence of {Delta}VPR-6 as 1, the increase of Vpr-induced MIN formation was 50-fold greater.


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  		Table 2. Incidence of MIN formation in Vpr-induced multinuclear cellsa

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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  		Table 3. Incidence of MIN in multinuclear cells by Vpr and cytochalasin Ba

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 {Delta}C2, {Delta}C5, and {Delta}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 {Delta}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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  		Table 4. Activity of Vpr mutants for inducing cell cycle perturbationa

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 {Delta}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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  		Figure 5. Morphological change after turning off the Vpr expression. A) Asynchronous cytokinesis. As a typical morphology, a giant multinuclear cell with multiple numbers of nuclei (indicated by arrowheads) emerging is shown (panel 1). Such a unique cell morphology was obtained on day 4 after DOX removal from the MIT-23 cells that had expressed Vpr for 10 days. In panels 2–5, photographs taken serially are shown. These photographs were taken 12 h after DOX removal from the cells pretreated with DOX for 4 days. B) Asynchronous cytokinesis with MIN being retained in the cytoplasm. A photograph shows multinuclear cells with asynchronous cytokinesis in which MIN continued to be present. Cells were stained with PI. Arrows and arrowheads indicate nuclei and MIN, respectively.

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{gamma} 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{gamma} cells contained more than 4N DNA content. In MIT-23{gamma} 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{gamma} 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{gamma} 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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  		Figure 6. The effects of repeated expression of Vpr on ploidy control. A) Experimental protocol. DOX treatment was repeated three times. One cycle was composed of DOX treatment for 10 days followed by DOX-free culture for another 10 days. Cell cycle analysis was performed each time before and after treatment of DOX. Sublines after the first, second, and third cycles of Vpr expression were named MIT-23{alpha}, ß, and {gamma}, respectively, For obtaining MIT-23/409, MIT-23 cells were cultured in the presence of DOX for 6 months and the culture was continued in the absence of DOX. B) FACS analysis on MIT-23{alpha}, ß, and {gamma}. Each panel (a–f) corresponds to the cells shown in the experimental protocol.




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  		Figure 7. Chronological ploidy change after repeated Vpr expression. A) FACS analysis of MIT-23{gamma}. Ploidy of MIT-23{gamma} cells were monitored during DOX-free culture for the periods indicated (panels 1–6). {Delta}VPR cells treated with DOX three times were also analyzed (panels 7 and 8). B) MPM-2 staining on MIT-23{gamma}. MIT-23 and MIT-23{gamma} were first treated with double thymidine block (2.5 mM) (panels 1, 2, 5, and 6), then transferred to the culture with the presence of nocodazole (2.5 µg/ml). After 12 h, cells were harvested (panels 3, 4, 7, and 8) and subjected to analysis. Cell cycle analysis (panels 2, 4, 6, and 8) and 2-dimensional analysis of DNA content and MPM-2 (panels 1, 3, 5, and 7) are shown. The population of R2 region in panel 3 was 9%. The populations corresponding to R2 and R3 region in panel 7 were 3% and 15%, respectively.

In two other independent experiments, we confirmed the reproducibility of the Vpr-induced ploidy change in MIT-23 cells, whereas {Delta}VPR cells showed no changes in ploidy from the same DOX treatment (Fig. 7A , panels 7 and 8). In addition to MIT-23{gamma} 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{gamma} 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 {Delta}VPR cells exposed to DOX by the same protocol as MIT-23{gamma} cells (Fig. 8 , panels 5 and 6, and Table 5 ), suggesting that aneuploidy was caused by Vpr.



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  		Figure 8. Karyotype analysis of MIT-23{gamma} cells. Karyotypes of MIT-23{gamma} cells (panels 1 and 2) and MIT-23/409 cells (panel 3) are shown. As a control, karyotypes of MIT-23 cells (panel 4) and {Delta}VPR/d-5 before and after DOX treatment (panels 5 and 6) are also shown. Number of chromosomes shown in panels 1–6 are 89, 69, 89, 46, 46, and 46.


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  		Table 5. Karyotype analysis of MIT-23 cells after Vpr expression


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Vpr-specific cell cycle abnormality in MIT-23 cells
In the present study, we established a cell line in which Vpr expression was tightly regulated by DOX treatment. Cell cycle abnormality was never observed in DOX-treated control {Delta}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 {gamma}-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.


   ACKNOWLEDGMENTS
 
We are grateful to Ms Mari Takizawa (National Institute of Infectious Diseases) for FACS analysis and Mrs. Mika Taniguchi for preparation of monoclonal antibodies. Dr. Testuya Tanaka (International Medical Center of Japan) kindly gave us advice on monoclonal antibody preparation. We thank Dr. Michiyuki Matsuda (International Medical Center of Japan) for infecting HIV to MAGI cells, Dr. H. Gossen (Heidelberg University) for providing plasmids for the tet-inducible expression system, and Dr. Nathaniel R. Landau (Aaron Diamond AIDS Research Center) for pBABE-vpr plasmid. A polyclonal antibody to Vpr was kindly provided by the NIH AIDS Research and Reference Reagents Program. We also express heartfelt thanks to Dr. Yoshifumi Takeda (Director General, Research Institute of International Medical Center of Japan) for his continuous support of this work. This work was supported by a Grant for International Health Cooperation Research from the Ministry of Health and Welfare of Japan.


   FOOTNOTES
 
* Correspondence: Department of Intractable Diseases International Medical Center of Japan, 1-21-1 Toyama, Shinjuku-ku, Tokyo 162-8655, Japan. E-mail: zakay{at}ri.imcj.go.jp

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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 DISCUSSION
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