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[Cancer Research 59, 2259-2264, May 1, 1999]
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[Cancer Research 59, 2259-2264, May 15, 1999]
© 1999 American Association for Cancer Research

Advances in Brief

Micronuclei Formation with Chromosome Breaks and Gene Amplification Caused by Vpr, an Accessory Gene of Human Immunodeficiency Virus1

Mari Shimura, Yuji Onozuka, Toshikazu Yamaguchi, Kiyohiko Hatake, Fumimaro Takaku and Yukihito Ishizaka2

Department of Intractable Diseases, International Medical Center of Japan, Tokyo 162-8655 [M. S., Y. I.]; Biomedical Laboratories, Inc., Saitama 350-1101, [Y. O., T. Y.]; and Jichi Medical School, Tochigi 329-0498 [K. H., F. T.], Japan


   ABSTRACT
Top
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 
Vpr, an accessory gene of human immunodeficiency virus, induces cell cycle abnormality by accumulating cells at the G2-M phase. We reported recently that Vpr caused both micronuclei formation and aneuploidy. Here, we show that Vpr also induced chromosome breaks and gene amplification. Expression of Vpr induced more than 10-fold increase of colonies resistant to N-(phosphonacetyl)-L-aspartate, an inhibitor of pyrimidine de novo synthesis. Fluorescence in situ hybridization analysis detected that 4 of 10 N-(phosphonacetyl)-L-aspartate resistant clones studied had intrachromosomal amplification of carbamyl-phosphate synthetase/aspartate transcarbamoylase/dihydroorotase gene. Another single clone had dicentrics. Data suggested that the Vpr-induced chromosome breaks leading to gene amplification, followed by bridge-breakage-fusion cycle, were one of the possible mechanisms of Vpr-induced genomic instability.


   Introduction
Top
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 REFERENCES
 
Vpr is an accessory gene of HIV type 1 encoding a virion-associated nuclear protein (1) , which induces cell cycle abnormality with cellular accumulation at the G2-M phase and increased ploidy (reviewed in Ref. 2 ). In Vpr-expressing cells, cyclin B-dependent p34cdc2 activity is down-regulated with an inactive form of CDC25C (3) . Recently, we have established a stable cell line (MIT-23) in which Vpr expression could be tightly regulated by DOX3, an analogue of tetracycline (4 , 5) . When Vpr is expressed in MIT-23 cells, suppressed p34cdc2 activity with a concomitant multinuclear cell formation is observed. We recognized that Vpr also induced MIN formation, a hallmark of genomic instability (5 , 6) . Consistent with such an observation, aneuploidy was observed in MIT-23 cells that experienced repeated Vpr expression (5) . In the present study, we found that Vpr-induced MIN had chromosome breaks, and here we present evidence that the transient Vpr expression promoted gene amplification. Structural analysis of the amplified gene strongly suggested that the bridge-breakage-fusion cycle was one of the molecular mechanisms of Vpr-induced gene amplification. This is the first report showing that Vpr functions as a mutagen that induces chromosome breaks with resultant genomic instability. We propose that MIT-23 provides a novel system for studying the molecular mechanism of cell cycle abnormality and the genomic instability that are often found in a variety of malignant tumors.


   Materials and Methods
Top
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 REFERENCES
 
Cells and Chemicals.
MIT-23 was established from a human fibrosarcoma cell line, HT1080 (JCRB9113; the Health Science Research Resources Bank), by tetracycline-responsive promoter combined with tetracycline-dependent transactivator (4 , 5) . A control cell line named {Delta}VPR was obtained by introducing the same plasmids present in MIT-23 except for the Vpr gene. Cells were maintained in DMEM (Nissui, Tokyo, Japan) supplemented with 10% heat-inactivated fetal calf serum (Bio Whittaker, Walkersville, MD) and 2 mM glutamine (Wako Pure Chemicals, Inc., Osaka, Japan). Cells were maintained in the presence of 400 µg/ml G418 (Wako Pure Chemicals) and 25 µg/ml hygromycin (Wako Pure Chemicals). Vpr expression was induced by the addition of DOX (Sigma Chemical Co., St. Louis, MO) at the final concentration of 5 µg/ml.

FACS Analysis.
Cell cycle analysis was performed using FACScalibur (Becton Dickinson, San Jose, CA). Cells were first fixed in ice-cold 70% ethanol and then treated with 100 µg/ml of RNase A (Sigma) for 15–30 min. After resuspending in the solution of propidium iodide (Sigma) with the concentration of 50 µg/ml, cells were subjected to fluorescence-activated cell sorter analysis. Cell cycle was analyzed based on a program of CELLQuest (Beckton Dickinson).

Immunostaining.
Cells were fixed in 4% paraformaldehyde dissolved in 10 mM phosphate buffer (pH 7.0) with 150 mM NaCl (PBS), followed by a treatment of 0.2% Triton X-100 with 0.5% Tween 20 (PBST). Blocking was performed using PBST with 10% normal goat serum. Then, cells were incubated with the first antibody (clone 3B4) for 1 h at 37°C. FITC-conjugated anti-mouse IgM (Zymed Laboratories, South San Francisco, CA) was used as a second antibody. 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, AZ). Image analysis was carried out by IP lab. spectrum (Scanalytics, Fairfax, VA).

TUNEL Method of Detecting Chromosome Breaks.
The TUNEL method (7) was carried out using a MEBSTAIN Apoptosis kit (Medical Biological Laboratories, Nagoya, Japan). The procedure was performed based on the protocol provided in the kit. Cells were fixed with 4% paraformaldehyde in PBS. DNA was stained with 50 ng/ml of DAPI (Sigma).

PALA Selection and FISH Analysis.
To assay Vpr-induced gene amplification, cells were cultured in the presence of PALA, which inhibits the activity of CAD (8) . PALA selection was done by the method described by Ishizaka et al. (9) . For selecting subpopulations with fewer preexisting PALA-resistant (PALAr) clones, about 103 cells were first plated and expanded to the 106 cell level. Then, one-half of the cells were cultured in the presence of PALA, and the number of PALAr colonies were counted. Subclones with low background of PALAr cells were further cultured in the presence of DOX for 10 days, followed by DOX-free culture for another 10 days, then 5 x 106 cells were used for PALA selection. Control MIT-23 cells or {Delta}VPR cells were directly subjected to PALA selection. Thirty µM PALA, which was a 5-fold higher dose inhibiting cell growth to 50% of control (5 x ID50), was used. Luria-Delbrück fluctuation analysis was performed according to the method reported by Tlsty (10) . Briefly, 48 small plates containing about 103 cells were first prepared, and cells were expanded to ~106 cells. During this period, DOX was added to one-half of the plates. After 10 days, DOX was removed by washing; culture was continued for another 10 days, and then cells were subjected to PALA selection.

FISH analysis of the CAD gene was performed, as described (9) . A cosmid clone of a human CAD gene was from Dr. Stark (The Cleveland Clinic Foundation, Cleveland, OH). A CAD probe was labeled with spectrum orange-dUTP (Vysis, Downers Grove, IL) using a nick translation kit (Boehringer Mannheim, Mannheim, Germany). After hybridization, chromosomes were counterstained with DAPI in an antifade solution composed of 0.1% p-phenylenediamine and 90% glycerol (Wako Pure Chemicals). Fluorescent signals were captured using a fluorescent microscope (Olympus) and Cytovision (Applied Imaging, Santa Clara, CA).


   Results
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 REFERENCES
 
Regulated Vpr Expression and Reversible Cell Cycle Abnormality in MIT-23.
Vpr expression in MIT-23 cells was tightly regulated by DOX addition, as demonstrated by immunostaining shown in Fig. 1ACitation . In 2 days after DOX addition, the induced Vpr expression was detected (panel 3), whereas DOX removal completely abolished Vpr expression in 4 days (panel 4). Control {Delta}VPR cells did not give any signals (panel 1).



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  		Fig. 1. Manipulated Vpr expression and cell cycle abnormality in MIT-23 cells. A, immunostaining of Vpr product. A monoclonal antibody (clone 3B4) was used for detecting 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). DOX of 5 µg/ml was used for induction. MIT-23 cells cultured in DOX-free culture for 4 days after transient DOX treatment (panel 4) were also shown. B, FACS analysis of MIT-23 cells with Vpr expression. A cell cycle pattern of MIT-23 cells treated with DOX for 4 days was shown (middle panel). DOX-untreated MIT-23 cells (upper panel) showed a normal cell cycle pattern. After DOX treatment for 10 days, MIT-23 cells were cultured further for 10 days in the absence of DOX, showing apparently normal cell cycle (lower panel).

 
Cell cycle abnormality was concomitantly observed by Vpr expression, as shown in Fig. 1BCitation . After 4 days of Vpr expression, about 50 and 20% of cells were accumulated in the regions of G2-M phase and hyperploidy (Fig. 1BCitation , middle panel), respectively. After DOX removal, normal cell cycle was restored in 10 days (Fig. 1BCitation , lower panel), during which time no apparent phenotype indicating apoptosis was observed. On the other hand, {Delta}VPR cells did not show any changes of cell cycle in response to DOX treatment (data not shown).

Vpr-induced Multinuclear Cells with MIN Formation.
A typical morphology of Vpr-expressing cells was shown in Fig. 2ACitation (right panel). More than 20% of the cells became multinuclear in 2 days after Vpr expression. By contrast, multinuclear cells were not observed in DOX-treated {Delta}VPR cells (left panel). The presence of MIN was shown also in Fig. 2Citation . The population of multinuclear cells with MIN in MIT-23 cells without Vpr expression was only 0.4%. Vpr expression, however, increased the incidence of MIN to 25%. On the other hand, the incidence of multinuclear cells with MIN in DOX-treated {Delta}VPR cells was only 0.5%. Data indicate that Vpr increased MIN formation up to 50-fold.



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  		Fig. 2. Vpr-induced MIN formation and chromosome breaks. A, morphology of multinuclear cells with MIN caused by Vpr. Morphology of DOX-treated {Delta}VPR cells (left panel) and DOX-treated MIT-23 cells (right panel). Cells were stained with propidium iodide and examined by the same magnification (x100). B, chromosome breaks in MIN and main nuclei. The signals positive for the TUNEL method were detected in both MIN (panels 1–3) and in main nuclei (panels 4–6). DAPI staining (green in panels 1 and 4), signals for the TUNEL method (red dots in panels 2 and 5), and merged signals (yellow in panels 3 and 6) are shown. Arrowheads in panel 6 indicate tiny yellow spots.

 
Two kinds of mutagens, aneuploidogens and clastogens, are known to generate MIN formation (11) . Aneuploidogens induce MIN that are highly positive for kinetochore, a centromeric component (~90% of frequency). On the other hand, clastogens induce kinetochore-positive MIN at low frequency (~10%; Ref. 11 ). To clarify the mutagen type of Vpr, we first carried out immunostaining using an anti-kinetochore monoclonal antibody. We detected signals positive for kinetochore only in 10% of Vpr-induced MIN (data not shown), suggesting that MIN were formed mainly by the clastogenic function of Vpr.

To obtaining direct evidence of clastogenic activity of Vpr, we next studied the presence of chromosome breaks by the TUNEL method, commonly used for detecting broken DNAs present in apoptotic cells (7) . As shown in Fig. 2BCitation , the signals positive for the TUNEL method were clearly detected as red spots both in MIN (panel 2) and main nuclei (panel 5). Totally, ~40% of MIN were positive for the TUNEL method detected as fine spots. When cells are apoptotic, homogeneous terminal deoxynucleotidyl transferase-positive signals would be observed in main nuclei4 because of an abundance of laddered DNAs in the nuclei (7) . Because FACS analysis on Vpr-expressing cells did not detect any increase of the population at the sub-G1 region (Fig. 1B)Citation , we concluded that TUNEL-positive signals meant the presence of chromosome breaks and that such cells were not apoptotic nor necrotic but had the capacity of continuous cell growth.

Gene Amplification Induced by Vpr.
It has been proposed that chromosome breaks, once generated, in turn promote gene amplification (8) . For clarifying this possibility, we studied the effects of Vpr on gene amplification using a colony assay based on the drug resistancy to PALA (9) . Because most immortalized cell lines, including HT1080, a parental cell line of MIT-23, contain preexisting PALAr clones (12) , subpopulations with low backgrounds were prepared (see procedures in "Materials and Methods"); then the effects of Vpr expression were studied. As shown in Fig. 3ACitation , the incidence of PALAr colonies was dramatically increased by the transient Vpr expression. Each subclone gave more than a 10-fold increase of PALAr colonies after Vpr expression. The frequency of PALAr colony formation was about 10-4, whereas control MIT-23 cells had the frequency of PALAr clones with about 10-5. On the other hand, the number of PALAr colonies of {Delta}VPR cells did not change in response to DOX treatment (data not shown).



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  		Fig. 3. Effects of Vpr on gene amplification. A, PALA selection on MIT-23 cells before and after Vpr expression. Three subclones of MIT-23 cells were subjected to PALA selection. After drug selection, PALAr colonies were visualized with crystal violet. B, FISH analysis of CAD gene amplified in PALAr colonies. Intrachromosomal amplification of the CAD gene (panels 1 and 2), dicentric chromosomes (panel 3), trisomy (panel 4), and an isochromosome (panel 5) is shown. Each panel shows the result of the independent clones. The FISH result on control MIT-23 cells (panel 6) is also shown.

 
To detect the precise time for the event of gene amplification, Luria-Delbrück fluctuation analysis (10) was performed. Briefly, about 103 cells were plated in small plates and expanded to about 106. During this period, DOX was added to one-half of the plates. After 10 days, DOX was washed out, and culture was continued for another 10 days. Then, cells in each well were directly subjected to PALA selection. If a single preexisting PALAr cell was present in the original 103 cells, PALA selection would give about 103 colonies in a single plate. On the other hand, if a gene amplification event had occurred during DOX treatment, there would be less than 1000 PALAr colonies. This analysis, by excluding the effects of preexisting PALAr clones, would give correct information on the time when the test compounds, Vpr in the present study, influenced gene amplification (10) . The results are shown in Table 1Citation . MIT-23 cells without Vpr expression generated only small numbers of PALAr colonies. Thirteen of 23 plates analyzed had no colonies, whereas 10 plates contained PALAr colonies with a frequency of one to four colonies per plate. By contrast, Vpr expression induced a considerable number of PALAr colonies. There were no plates that were free of PALAr colonies. Sixteen of 20 plates contained PALAr colonies with the number from 4 to 100 per plate. Furthermore, there were four plates that contained more than 100 but <1000 colonies. Expression of Vpr in these PALAr clones was examined by immunostaining, and we confirmed that Vpr was no longer expressed (data not shown). These data strongly suggested that gene amplification occurred during the time when Vpr was expressed.


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  		Table 1 Luria-Delbrück fluctuation analysis of CAD gene amplification

 
FISH analysis using a cosmid clone of the CAD gene as a probe was performed on the PALAr clones. The representative structures of the CAD gene in the PALAr clones are shown in Fig. 3BCitation . The CAD gene amplified on the same chromosome arm was observed in four PALAr clones. Two of these are shown in Fig. 3BCitation (panels 1 and 2). Dicentric chromosomes containing two copies of the CAD gene were also found in one of the PALAr clones (panel 3). Two PALAr clones contained isochromosomes (panel 5). On the other hand, aneuploidy of marker chromosomes was found in a total of seven clones with (five of seven clones) or without (two of seven clones, one shown in panel 4) other structural abnormalities.


   Discussion
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
REFERENCES
 
In the present study, we showed evidence that Vpr induced chromosome breaks and gene amplification. In the first experiment, Vpr was proved to induce MIN formation, ~40% of which were positive by the TUNEL method, suggesting the presence of chromosome breaks. We next demonstrated an increased rate of Vpr-induced gene amplification (Fig. 3Citation and Table 1Citation ). By Vpr expression, the number of PALAr colonies increased to >10-fold. FISH analysis detected the presence of amplified CAD gene on the same chromosome arms (Fig. 3BCitation , panels 1 and 2) as well as dicentrics (panel 3). HT1080, a parental cell line used for the establishment of MIT-23, gives spontaneous PALAr clones like other immortalized cell lines, and the structures of CAD genes in PALAr clones were recently analyzed in detail (12) . Intrachromosomal gene amplification was found in only 5% of HT1080-derived spontaneous PALAr clones (12) , although the same structure was often reported in rodent cell lines (13) . It is striking that 4 of 10 Vpr-induced PALAr clones studied had intrachromosomal gene amplification. Furthermore, the presence of dicentric chromosomes in a PALAr clone provides us with a theoretical basis for explaining the molecular mechanism of Vpr-induced gene amplification. Dicentrics are formed when chromosomes with recombinogenic broken ends are fused (8 , 12 , 13) . Once dicentrics are generated, the bridge-breakage-fusion cycle has been proposed to start (8 , 9 , 12 , 13) . Detection of intrachromosomal gene amplification, dicentrics, and TUNEL-positive spots in Vpr-expressing cells suggested that Vpr-induced chromosome breaks initiated the bridge-breakage-fusion cycle. This is the first report describing that Vpr functions like a mutagen, especially as a clastogen (11) .

In addition, FISH analysis also detected that 7 of 10 PALAr clones contained the increased number of marker chromosomes consistent with our previous finding that MIT-23 cells, after repeated expression or continuous expression of Vpr, became aneuploid (5) . These data suggest that Vpr induces both gene amplification and aneuploidy.

Recently, it was pointed out that cell cycle abnormality served as a molecular basis for genomic instability (14) . One of the tumor suppressor genes, p53, has been proposed to halt cells with DNA damage at the G1-S phase (9 , 15) . The relationship of cell cycle abnormality at the G1-S phase after treatment of DNA-damaging agents and genomic instability is well explained so that genetically damaged cells gain the time for repairing damaged DNA (9 , 16 , 17) . The precise mechanism of cell cycle abnormality at the G2-M phase and genomic instability, however, remains mostly unknown. Recently, several groups started to clarify the molecular mechanisms of cell cycle abnormality at the G2-M phase in relation to genomic instability (18 , 19) . As shown in the present study, MIT-23 cells can reproducibly generate gene amplification and chromosome breaks. MIT-23 cells would provide valuable information for understanding the molecular mechanism of cell cycle abnormality at the G2-M phase and genomic instability.

Recently, Tax of human T-cell leukemia virus type 1 was shown to have the function to induce chromosome breaks as well as G2-M arrest (20) . Furthermore, it was reported that Tax inhibited the interaction of MAD1 and MAD2, human homologues to yeast genes (20) controlling mitotic checkpoint control. Vpr and Tax show almost the same biological phenotypes (5 , 20) , and it is now important to question whether Vpr shares a common cellular target molecule(s) with Tax. The precise mechanism of Vpr-induced genomic instability is now under investigation.


   ACKNOWLEDGMENTS
 
We are grateful to Dr. George R. Stark (The Cleveland Clinic Foundation, Cleveland, OH) for a cosmid clone of the human CAD gene. PALA was kindly provided by the Drug Synthesis and Chemistry Branch, Developmental Therapeutics Program, Division of Cancer Treatment, National Cancer Institute. We express great thanks to Dr. Yoshifumi Takeda (Director General, Research Institute of International Medical Center of Japan, Tokyo) for his continuous support on this work.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by a grant for International Health Cooperation Research from the Ministry of Health and Welfare of Japan. Back

2 To whom requests for reprints should be addressed, at Department of Intractable Diseases, International Medical Center of Japan, 1-21-1 Toyama, Shinjuku-ku, Tokyo 162-8566, Japan. Phone: 81-3-3202-7181, extension 2887; Fax: 81-3-3202-7364; E-mail: zakay{at}ri.imcj.go.jp Back

3 The abbreviations used are: DOX, doxycycline; MIN, micronuclei; PALA, N-(phosphonacetyl)-L-aspartate; CAD, carbamyl-phosphate synthetase/aspartate transcarbamoylase/dihydroorotase; FISH, fluorescence in situ hybridization; TUNEL, terminal deoxynucleotidyl transferase-mediated nick end labeling; DAPI, 4,6-diamidino-2-phenylindole. Back

4 Y. Ishizaka, unpublished observation. Back

Received 1/25/99. Accepted 3/31/99.


   REFERENCES
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 

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