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Herpes simplex virus induces unscheduled DNA synthesis in virus-infected cervical cancer cell lines
Res. ViroL 1992, 143, 351-359
~) INSTITUTPASTEuR/ELSEVIER Paris 1992
Herpes simplex virus induces unscheduled D N A synthesis in virus-infected cervical cancer cell lines P. Kulomaa (z), j. Paavonen (2) and M. Lehtinen (1) (*) (/) Institute o f Biomedical Sciences, University o f Tampere, Tampere (Finland) and c2)Department of Obstetrics and Gynecology, University Central Hospital, Helsinki
SUMMARY We evaluated herpes-simplex-virus-type-2(HSV2)-induced unscheduled DNA synthesis in virus-infected cervical cancer (HeLa, CaSki, C-33A, and SiHa) cells. HSV2 replication was approximately 100-fold more efficient in the HeLa cells than in less susceptible C-33A and SiHa cells. In dual parameter flow cytometric analysis of bromodeoxyuridine (BrdU} incorporation, HSV2-infected HeLa cells showed a rapid increase in the proportions of DNA-synthesizing G1- and S-phase cells, whereas in C-33A and SiHa cells, the proportions of DNA-synthesizing G 1- and early S-phase cells were increased late in the infection. Blocking of HSV2 replication by phosphonoformate inhibited virus-induced changes in HeLa cells, but not in C-33A and SiHa cells. Anti-BrdU antibodies exhibited a coarse globular nuclear staining pattern in the C-33A cells, while the other cells showed speckled and/or fine globular nuclear fluorescence. Anti-ICP8 (HSV-specified major DNA-binding protein) antibodies revealed that, in C-33A cells, ICP8 remained in the cytoplasm, whereas in the other cells, speckled or globular nuclear fluorescence was found. Our results showed that HSV2 induced the unscheduled synthesis of cellular DNA, which was host-cell-dependent, and in virus infected C-33A cells, it may be attributable to both viral and cellular proteins. Key-words: HSVY, DNA, Cervical cancer; Cell lines C-33A, HeLa, CaSki and SiHa, Unscheduled DNA synthesis, Flow cytometry.
INTRODUCTION Herpes simplex virus (HSV) causes formation of double minute chromosomes and induces DNA amplification and mutations in human cells (Chenet-Monte et al., 1986; Heilbronn and zur Hausen, 1989; Galloway and McDougall, 1990). These changes may play a role in HSV-
mediated carcinogenesis, and therefore the requirements of underlying cell-virus interaction are of interest. Replication of episomal D N A is the key mechanism of D N A amplification (Stark et al., 1989). In addition, unscheduled DNA synthesis, i.e., repair replication of chromosomal DNA ac-
Submitted November 12, 1991, accepted September 3, 1992. (*) Correspondence:Dr M. Lehtinen,Inst. of Biomed.Sciences,Univ. of Tampere, POB 607, SF-33101 Tampere(Finland).
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P. K U L O M A A E T A L .
cording to an anion skin model is involved (Stark and Wahl, 1984; Wahl, 1989). Following heavy carcinogen exposure or HSV infection, the intensities o f BrdU-labeUing in G l-phase cells (unscheduled D N A synthesis) and S-phase cells (semi-conservative D N A replication) are about equal and can be detected by dual p a r a m e t e r flow cytometry (Bose and Allison, 1987; Beisker and Hittelman, 1988; Lehtinen et al., 1989). HSV is extremely efficient in amplifying episomal D N A (Danovich and Frenkel, 1988; Heilbronn and zur Hausen, 1989), but also induces up to 10-fold amplification o f integrated papovavirus D N A (Brandt et al., 1987; Gerspach and Matz, 1988). The present study was u n d e r t a k e n to answer the question: is it possible that HSV infection could result in D N A amplification during cervical carcinogenesis?
ogy, University of Helsinki and Department of Microbiology, University of Leeds, respectively). Fluorescein isothiocyanate(FITC)-labelled antimouse and anti-rabbit conjugates were from Dakopatts a/s (Denmark). BrdU and RNase A were purchased from Sigma (USA), phosphonoformate (PFA) was kindly provided by Dr. B. Oberg (Astra Research and Developmental Laboratories, Sweden). Propidium iodide (PI) was from Boehringer Mannheim GMBH (Germany). Labelling o f infected cells
After adsorption (1 h), the monolayers were washed twice with growth medium. For one set of cells, growth medium supplemented with 2 °70 FCS was added; for the other set of cells, growth medium was supplemented with 2 07o FCS and PFA (500 raM). The cells were labelled with 10 mM BrdU for 1 h at I, 7, 13 and 19 h post-infection (p.i.) as previously described (Dolbeare et al., 1983). The labelling was stopped by pouring off the medium and washing the cells twice with ice-cold PBS and fixing in ice-cold ethanol (70 % v/v).
MATERIALS A N D METHODS Cells and viruses
A n a l y s i s o f D N A synthesis
The cervical cancer cell lines CaSki (ATCC CRLI550, USA), C-33A (ATCC HTB31), SiHa (ATCC HTB35), HeLa (ATCC CCL2), and a Burkitt lymphoma cell line Raji were cultured in RPMI-1640 medium supplemented with 10 070 foetal calf serum (FCS) and gentamicin as described (Lehtinen et al., 1987, 1989). HSV2 (strain G) obtained from Dr. L. Aurelian (Virology/Immunology Laboratories, University of Maryland) was passaged in Vero cells and used at its early passages in Vero cells. Unless otherwise indicated, nearly confluent cell monolayers were infected with HSV2 at a multiplicity of infection (m.o.i.) of 2 PFU/ceI1. Infectious virus was titrated by a routine plaque formation method.
The proportion of DNA-synthesizing cells was determined by staining the cells for both newly synthesized DNA and total DNA as described in detail elsewhere (Lehtinen et ai., 1989). Briefly, cells were analysed by an "Epics C R'' (Coulter Electronics Inc., USA) flow cytometer using an excitation wavelength of 488 nm. Green fluorescence was measured between 515 and 530 nm. Ceils were gated on both forward angle light scatter and PI fluorescence to exclude cell debris and large aggregates from the analysis. The dual parameter BrdU-DNA distributions were analysed using the Coulter Software program "Quadstat", which allows the identification of DNA-synthesizing cells in different cell cycle compartments : G1, S and G2 phase, and mitosis (fig. 1). Relative proportions of cells in the G1 and early S phases were recorded in repeated experiments.
Antisera and c h e m i c a l s
Monoclonal anti-bromodeoxyuridine fBrdU) antibodies (IU2) and monospecific rabbit anti-ICP8 (ICSPI1/12) antibodies were generous gifts of Drs. S. Nordling and K.L. Powell (Dept. of Pathol-
BrdU FCS F1TC
= bromodeoxyuridine. = foetal calf serum. = fluoresceinisothiocyanate.
HPV HSV
= human papillomavirus. = herpes simplex virus.
Immunofluorescence
For immunofluorescence analysis, cells were grown on "Nunclon" 9 cm 2 slide flasks (Nunc,
m.o.i.
= multiplicity of infection.
PCNA = proliferatingcell nuclearantigen. PFA = phosphonoformate. PI = propidiumiodide. p.i.
= post-infection.
HSV2, UNSCHEDULED DNA SYNTHESIS A N D CERVICAL CANCER CELLS
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Fig. 1. Dual-parameter flow cytometric analysis of the relative proportions of DNA-synthesizingGland S-phase cells. BrdU-labelled cervical carcinoma (C-33A) cells were stained with monoclonal anti-BrdU + FITC rabbit anti-mouse antibodies (LGFL) and propidium iodine (RFL). The result of the analysis of 20,000 cells is shown as a three 3-dimensional histogram. X-axis: LGFL = intensity of green fluorescence (logarithmic scale); Y-axis: RFL -- intensity of red fluorescence (linear scale); Z-axis = number of cells (linear scale). Shaded area: BrdU-labeUed G r and early S-phase cells; long arrow: GI-phase cells; short arrow: G2-phase cells.
Denmark). The cells were fixed for 20 min with a Carnoy solution (l part v/v of methanol and 2 parts v/v of acetone) at - 20°C. The cells were first stained with anti-ICP8 (I/100) for l h and then with FITCconjugated anti-rabbit antibodies for 30 min as described (Lehtinen et al., 1987).
RESULTS
Following HSV2 infection, all cell lines (ex-. cept for HeLa) showed an initial fail in the proportions o f DNA-synthesizing G 1- and early S-phase cells. In H e L a and CaSki ceils, the proportions o f these cells grew exponentially f r o m 2 to 20 h p.i. (fig. 2). In contrast, the
proportions o f DNA-synthesizing C-33A and SiHa cells that were in the G l- or early S-phase were increased only late in the infection (14 to 20 h p.i. ; fig. 3). Addition o f a virus-specific D N A synthesis inhibitor, P F A at 500 M 2 h before infecting the ceils significantly reduced or totally inhibited an HSV2-induced increase in the proportions o f DNA-synthesizing H e L a and CaSki cells, respectively. In C-33A and SiHa cells, the late induction o f increase in the proportions o f DNA-synthesizing G I- and early S-phase cells was PFA-resistant. The a m o u n t o f o u t p u t virus was m o n i t o r e d during the first replicative cycle at various doses
P. K U L O M A A E T A L .
354
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Fig. 2. Kinetics of the relative proportions of cellssynthesizing DNA during HSV2 replication in virus-infected cervical cancer cells.
Fig. 3. Kinetics of the relativeproportions of cellssynthesizing D N A during H S V 2 replication in virus-infected cervical cancer ceils.
HeLa (left) and CaSki (right) cells. Data on G : and early S-phase cells only are given as mean values (+ standard errors) of three to five separate analyses. O = PFA-treated cells; I = PFA-untreated cell.
C-33A (left)and SiHa (right) ceils. Data on the G : and early S-phase cells only are given as mean values (+standard errors) of three to five separate analyses. (O) = PFA-treated cells, ()) = PFA-untreated ceils.
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Fig. 4. Growth of H S V 2 in cervical cancer cellsat various multiplicities(I0, I, 0. I and 0.01 PFU/cell) of infecting virus. CaSki ceils (A), HeLa cells (B), C-33A cells (C), SiHa cells (D). The amount of output virus is given as log of PFU/ml of tissue culture medium.
h
HSV2, U N S C H E D U L E D D N A S Y N T H E S I S A N D C E R V I C A L C A N C E R C E L L S
o f input virus (10 to 0.01 PFU/celI). In C-33A and SiHa ceils, the amounts o f intracellular infectious virus were continuously about 10-fold lower than in corresponding CaSki cells and about 100-fold lower than in corresponding HeLa cells (fig. 4). At an a m o u n t of input virus as high as 10 PFU/cell, no infectious virus was detected in the PFA-treated cells 20 h p.i. (data not shown). The location of ICP8 and the uptake of BrdU by the virus-infected cells was compared by immunofluorescence microscopy at 20 h p.i. Mockinfected cells showed a speckled immunofluorescence staining pattern when anti-BrdU antibodies were used (fig. 5). Following HSV2 infection,
355
three cell lines (CaSki, SiHa and HeLa) showed a speckled or free globular staining pattern with the anti-BrdU antibodies, whereas in the HSV2 infected C-33A cells,staining appeared to be restricted to a few large globular structures only (fig. 5). In general, after HSV2 infection at a m.o.i. of 2 PFU/cell, about half of the cellswere positive for ICP8. In addition to a speckled nuclear fluorescence sccn in SiHa cells,CaSki and HeLa cells also exhibited a globular staining pattern with anti-ICP8 antibodies (fig. 6). In the C-33A cells, perinuclear and cytoplasmic staining predominated, resulting in an appearance similar to that seen in persistently HSV2-infected Raji cells (fig. 6).
G
Im Fig. 5. Indirectimmunofluorescence of BrdU-labelled cervicalcancer cells. Cells were stained with monoclonal anti-BrdU antibodies 20 h p.i. Mock-infected CaSki cells (A), HSV2-infected CaSki cells (B), mock-infected C-33A cells (C), and HSV2-infected C-33A cells (D). HSV2-infected HeLa and SiHa cells showed a fluorescence pattern similar to that seen in CasKi ceils.
P. K U L O M A A E T AL.
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Fig. 6. Indirect immunofluorescence of HSV2-infected cancer cells. The cells were stained with monospecific rabbit anti-ICP8 antiserum 20 h p.i. SiHa cells (A), CaSki cells (B), Raji cells (C), and C-33A cells (D). HSV2-infected HeLa cells showed a fluorescence pattern similar to that seen in the CaSki cells.
DISCUSSION
Replication of episomal DNA has been suggested to be one of the key mechanisms of DNA amplification (Stark et aL, 1989) but cellular replication machinery seems to replicate episomal virus DNA only at a constant level (Adams et ai., 1989; Stanley et al., 1989; Crook et aL, 1990). Integration of HPV DNA was recently reported to result in amplification of both viral and cellular flanking sequences 0aopescu and DiPaolo, 1990; Wagatsuma et al., 1990). However, integration of HPV DNA occurs in only about half of cervical tumours (Cullen et al., 1991 ; Stoler et al., 1990), yet amplified HPV and c-myc DNA are frequently found in cervi-
cal cancer (Riou et al., 1984; Ocadiz et al., 1987). Thus, factors other than cellular replication machinery and integration of viral DNA might also contribute to the amplification of HPV DNA in cervical cancer. HSV2 and HPV16 (human papillomavirus) sequences have been demonstrated concomitantly in cervical cancer (DiLuca et al., 1989). Although, in productive infection, HSV replication machinery is able to amplify both episomal and most likely also integrated HPV DNA (Brandt et aL, 1987; Gerspach and Matz, 1988; Heilbronn and zur Hausen, 1989), as a consequence of this kind of an infection the cell would die. Unscheduled DNA synthesis is
HSV2. UNSCHEDULED DNA S Y N T H E S I S A N D CERVICAL C A N C E R CELLS
another key mechanism of DNA amplification (Wahl, 1989). In the present study, we demonstrated significant differences in the rate of HSVinduced unscheduled DNA synthesis in HPVDNA + (CaSki, HeLa and SiHa) and HPVDNA- (C-33A) cervical cancer cell lines. HSV2-infected C-33A and SiHa cells proved to be relatively unsusceptible to the infection, but exhibited PFA-resistant DNA synthesis late in the infection. In the C-33A (and SiHa) cells DNA synthesis was detected predominantly in G1 cells; thus it represents unscheduled DNA synthesis, a prerequisite of chromosomal DNA amplification. Late in the infection, C-33A cells stained with anti-BrdU antibodies showed globular staining which, in HSV susceptible cells corresponds to virus repilcation compartments (Gao and Knipe, 1989; Knipe, 1989). However, HSV replication was relatively inefficient in the C-33A cells. The cytoplasmic location of ICP8 in the C-33A ceils resembled that seen in persistently HSV-infected Raji cells (Lehtinen et al., 1987), as if ICP8 could not properly enter the nucleus. Furthermore, the measured DNA synthesis was PFA-resistant, i.e. not due to HSV DNApol. These findings suggest that, in C-33A cells, ICP8 and HSV DNApol are at least partially inactive or can be inactivated without an effect upon unscheduled DNA synthesis. This is important because the normal performance of ICP8 and DNApol would most likely kill the virus-infected cell. Six HSV-specific genes, UL5, UL8, UL29 (ICP8), UL30 (DNApol), UL42 and UL52, are involved in the amplification of episomal DNA (Heilbronn and zur Hausen, 1989). Cellular DNA polymerases alpha and delta, as well as cyclin, take part in the replication of episomal DNA (Zuber et al., 1989), but may also be involved in repair repilcation and amplification of integrated viral DNA (Ceils and Madsen, 1986; Heilbronn et al., 1985). HSV DNApol has 3' to 5' exonuclease activity analogous to DNA polymerase delta (O'Donnell et al., 1987). HSV DNApol and ICP8 have considerable sequence homology with DNA polymerase alpha and proliferating cell nuclear antigen (PCNA) (Wong et al., 1988; Matsumoto et al., 1987). Further-
357
more, ICP8 and PCNA exhibit similar fluorescence patterns (Bravo et al., 1986; Lehtinen et aL, 1987). Thus, the mode of action of HSV and cellular replication machineries in DNA amplification may be similar. What then is their role in the described unscheduled DNA synthesis in C-33A cells ? ICP8 may not be actively involved. HSV DNApol has exonuclease activity and HSV specifies an additional alkaline exonuclease which is normally complexed with ICP8 and HSV DNApol (Vaughan et al., 1984). Thus, relative dysfunction of the HSV replication machinery in the C-33A cells might set the stage for unscheduled DNA synthesis by causing DNA damage. Nishiyama and Rapp (1981) showed that HSV2 is able to induce repair replication of cellular DNA under non-permissive conditions. We could not find unscheduled DNA synthesis in highly HSV-susceptible CaSki and HeLa cells, even when virus DNA synthesis wasblocked by PFA. In fact, HSV-induced repair replication of cellular DNA has been previously described in non-permissive rat XC cells, but not in virussusceptible cell hybrids (Epstein and Jacquemont, 1980; Epstein et al., 1980, 1985). This further suggests that specific cellular factors might be involved. How and to what extent the cellular replication machinery (especially, PCNA and DNA-polymerases alpha and delta) takes part in HSV-induced unscheduled DNA synthesis warrants further investigation.
Acknowledgements
The authors wishto thank Mrs. LeenaPankko for excellent technicalassistance. This workwas supportedby grants fromthe EmilAaltonen foundation and FinnishCancer Organizations.
Le virus de I'herp~s simplex induit la synth~e d'ADN ~ hors programme)~ dans les l i g h t s ceilulaires de cancer infectC~es par le virus
Darts cette ~tude nous avons observ~ la synth~se d'ADN ~ hors programme, dans des lign~es cellu-
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P. K U L O M A A
laires de cancer cervical infectc~espar le virus de l'herp~s simplex type 2 (HSV2). Les lign~es cellulaires HeLa, CaSki, C-33A et SiHa ont ~t~ infect~es par I'HSV2 et marquees par la bromod~oxyuridine (BrdU). Le double param&re de I'analyse cytom~trique du flux de l'incorporation du BrdU montre l'existence d'une rapide augmentation des proportions de ceIlules en phase S e t Gl synth~tisant de I'ADN. Dans [es cellules C-33A et SiHa les proportions de cellules synth~tisant I'ADN ont augment~ seulement tardivement lors de rinfection. Si la r~plication de I'HSV2 est bloqu~e par [e phosphonoformate, les changements induits par le virus se produisent dans les ceIlules C-33A et SiHa mais ne sont pas observ6es dans les cellules HeLa et CaSki. La r~plication de I'HSV2 est environ 100 fois plus efficace dans les cellules HeLa que dans les cellules C-33A et SiHa. Les diff&ences sont aussi considc~rables entre les images de l'immunofluorescence des cellu[es infect~es. Les anticorps anti-BrdU forment des zones de marquage nucl~aire grossi&ement globuleuses dans les cellules C-33A alors que les images du marquage nucI~aire dans les autres cellules sont form~es par des petites taches et elles sont tlnement globuleuses. Les anticorps anti-ICP8 (prot~ine majeure de I'HSV liant I'ADND r~v~lent que I'ICP8 reste darts le cytoplasme des cellules C-33A alors que dans les autres ceIlules on observe une fluorescence sous forme de petites taches ou de noyau globuleux. Ces r~sultats indiquent que (1) I'HSV2 induit la synth~se d'ADN cellulaire hors programme, (2) l'induction est dc~pendante de l'h6te, (3) dans les cellules C-33A infect6es ce ph~nom~ne peut ~tre attribu~ aux m~anismes conjoints de r~plication virale et cellulaire. Connartre le r61e des ADN-polym&ases ceIIulaires ,, et 8 et celui de la cycline qui partage des s6quences homologues d'acides amines et/ou/l des fonctions communes avec l'ADNpol de I'HSV et I'ICP8, justitle les futures recherches. Mots-clc~s: HSV2, ADN, Cancer du col; Lign~es cellulaires C-33A, HeLa, CaSki and SiHa, Synth~se ~ hors programme >>de I'ADN, Cytom6trie du flux.
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HSV2, UNSCHEDULED DNA SYNTHESIS AND CERVICAL CANCER CELLS
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~) INSTITUTPASTEuR/ELSEVIER Paris 1992
Herpes simplex virus induces unscheduled D N A synthesis in virus-infected cervical cancer cell lines P. Kulomaa (z), j. Paavonen (2) and M. Lehtinen (1) (*) (/) Institute o f Biomedical Sciences, University o f Tampere, Tampere (Finland) and c2)Department of Obstetrics and Gynecology, University Central Hospital, Helsinki
SUMMARY We evaluated herpes-simplex-virus-type-2(HSV2)-induced unscheduled DNA synthesis in virus-infected cervical cancer (HeLa, CaSki, C-33A, and SiHa) cells. HSV2 replication was approximately 100-fold more efficient in the HeLa cells than in less susceptible C-33A and SiHa cells. In dual parameter flow cytometric analysis of bromodeoxyuridine (BrdU} incorporation, HSV2-infected HeLa cells showed a rapid increase in the proportions of DNA-synthesizing G1- and S-phase cells, whereas in C-33A and SiHa cells, the proportions of DNA-synthesizing G 1- and early S-phase cells were increased late in the infection. Blocking of HSV2 replication by phosphonoformate inhibited virus-induced changes in HeLa cells, but not in C-33A and SiHa cells. Anti-BrdU antibodies exhibited a coarse globular nuclear staining pattern in the C-33A cells, while the other cells showed speckled and/or fine globular nuclear fluorescence. Anti-ICP8 (HSV-specified major DNA-binding protein) antibodies revealed that, in C-33A cells, ICP8 remained in the cytoplasm, whereas in the other cells, speckled or globular nuclear fluorescence was found. Our results showed that HSV2 induced the unscheduled synthesis of cellular DNA, which was host-cell-dependent, and in virus infected C-33A cells, it may be attributable to both viral and cellular proteins. Key-words: HSVY, DNA, Cervical cancer; Cell lines C-33A, HeLa, CaSki and SiHa, Unscheduled DNA synthesis, Flow cytometry.
INTRODUCTION Herpes simplex virus (HSV) causes formation of double minute chromosomes and induces DNA amplification and mutations in human cells (Chenet-Monte et al., 1986; Heilbronn and zur Hausen, 1989; Galloway and McDougall, 1990). These changes may play a role in HSV-
mediated carcinogenesis, and therefore the requirements of underlying cell-virus interaction are of interest. Replication of episomal D N A is the key mechanism of D N A amplification (Stark et al., 1989). In addition, unscheduled DNA synthesis, i.e., repair replication of chromosomal DNA ac-
Submitted November 12, 1991, accepted September 3, 1992. (*) Correspondence:Dr M. Lehtinen,Inst. of Biomed.Sciences,Univ. of Tampere, POB 607, SF-33101 Tampere(Finland).
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cording to an anion skin model is involved (Stark and Wahl, 1984; Wahl, 1989). Following heavy carcinogen exposure or HSV infection, the intensities o f BrdU-labeUing in G l-phase cells (unscheduled D N A synthesis) and S-phase cells (semi-conservative D N A replication) are about equal and can be detected by dual p a r a m e t e r flow cytometry (Bose and Allison, 1987; Beisker and Hittelman, 1988; Lehtinen et al., 1989). HSV is extremely efficient in amplifying episomal D N A (Danovich and Frenkel, 1988; Heilbronn and zur Hausen, 1989), but also induces up to 10-fold amplification o f integrated papovavirus D N A (Brandt et al., 1987; Gerspach and Matz, 1988). The present study was u n d e r t a k e n to answer the question: is it possible that HSV infection could result in D N A amplification during cervical carcinogenesis?
ogy, University of Helsinki and Department of Microbiology, University of Leeds, respectively). Fluorescein isothiocyanate(FITC)-labelled antimouse and anti-rabbit conjugates were from Dakopatts a/s (Denmark). BrdU and RNase A were purchased from Sigma (USA), phosphonoformate (PFA) was kindly provided by Dr. B. Oberg (Astra Research and Developmental Laboratories, Sweden). Propidium iodide (PI) was from Boehringer Mannheim GMBH (Germany). Labelling o f infected cells
After adsorption (1 h), the monolayers were washed twice with growth medium. For one set of cells, growth medium supplemented with 2 °70 FCS was added; for the other set of cells, growth medium was supplemented with 2 07o FCS and PFA (500 raM). The cells were labelled with 10 mM BrdU for 1 h at I, 7, 13 and 19 h post-infection (p.i.) as previously described (Dolbeare et al., 1983). The labelling was stopped by pouring off the medium and washing the cells twice with ice-cold PBS and fixing in ice-cold ethanol (70 % v/v).
MATERIALS A N D METHODS Cells and viruses
A n a l y s i s o f D N A synthesis
The cervical cancer cell lines CaSki (ATCC CRLI550, USA), C-33A (ATCC HTB31), SiHa (ATCC HTB35), HeLa (ATCC CCL2), and a Burkitt lymphoma cell line Raji were cultured in RPMI-1640 medium supplemented with 10 070 foetal calf serum (FCS) and gentamicin as described (Lehtinen et al., 1987, 1989). HSV2 (strain G) obtained from Dr. L. Aurelian (Virology/Immunology Laboratories, University of Maryland) was passaged in Vero cells and used at its early passages in Vero cells. Unless otherwise indicated, nearly confluent cell monolayers were infected with HSV2 at a multiplicity of infection (m.o.i.) of 2 PFU/ceI1. Infectious virus was titrated by a routine plaque formation method.
The proportion of DNA-synthesizing cells was determined by staining the cells for both newly synthesized DNA and total DNA as described in detail elsewhere (Lehtinen et ai., 1989). Briefly, cells were analysed by an "Epics C R'' (Coulter Electronics Inc., USA) flow cytometer using an excitation wavelength of 488 nm. Green fluorescence was measured between 515 and 530 nm. Ceils were gated on both forward angle light scatter and PI fluorescence to exclude cell debris and large aggregates from the analysis. The dual parameter BrdU-DNA distributions were analysed using the Coulter Software program "Quadstat", which allows the identification of DNA-synthesizing cells in different cell cycle compartments : G1, S and G2 phase, and mitosis (fig. 1). Relative proportions of cells in the G1 and early S phases were recorded in repeated experiments.
Antisera and c h e m i c a l s
Monoclonal anti-bromodeoxyuridine fBrdU) antibodies (IU2) and monospecific rabbit anti-ICP8 (ICSPI1/12) antibodies were generous gifts of Drs. S. Nordling and K.L. Powell (Dept. of Pathol-
BrdU FCS F1TC
= bromodeoxyuridine. = foetal calf serum. = fluoresceinisothiocyanate.
HPV HSV
= human papillomavirus. = herpes simplex virus.
Immunofluorescence
For immunofluorescence analysis, cells were grown on "Nunclon" 9 cm 2 slide flasks (Nunc,
m.o.i.
= multiplicity of infection.
PCNA = proliferatingcell nuclearantigen. PFA = phosphonoformate. PI = propidiumiodide. p.i.
= post-infection.
HSV2, UNSCHEDULED DNA SYNTHESIS A N D CERVICAL CANCER CELLS
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Fig. 1. Dual-parameter flow cytometric analysis of the relative proportions of DNA-synthesizingGland S-phase cells. BrdU-labelled cervical carcinoma (C-33A) cells were stained with monoclonal anti-BrdU + FITC rabbit anti-mouse antibodies (LGFL) and propidium iodine (RFL). The result of the analysis of 20,000 cells is shown as a three 3-dimensional histogram. X-axis: LGFL = intensity of green fluorescence (logarithmic scale); Y-axis: RFL -- intensity of red fluorescence (linear scale); Z-axis = number of cells (linear scale). Shaded area: BrdU-labeUed G r and early S-phase cells; long arrow: GI-phase cells; short arrow: G2-phase cells.
Denmark). The cells were fixed for 20 min with a Carnoy solution (l part v/v of methanol and 2 parts v/v of acetone) at - 20°C. The cells were first stained with anti-ICP8 (I/100) for l h and then with FITCconjugated anti-rabbit antibodies for 30 min as described (Lehtinen et al., 1987).
RESULTS
Following HSV2 infection, all cell lines (ex-. cept for HeLa) showed an initial fail in the proportions o f DNA-synthesizing G 1- and early S-phase cells. In H e L a and CaSki ceils, the proportions o f these cells grew exponentially f r o m 2 to 20 h p.i. (fig. 2). In contrast, the
proportions o f DNA-synthesizing C-33A and SiHa cells that were in the G l- or early S-phase were increased only late in the infection (14 to 20 h p.i. ; fig. 3). Addition o f a virus-specific D N A synthesis inhibitor, P F A at 500 M 2 h before infecting the ceils significantly reduced or totally inhibited an HSV2-induced increase in the proportions o f DNA-synthesizing H e L a and CaSki cells, respectively. In C-33A and SiHa cells, the late induction o f increase in the proportions o f DNA-synthesizing G I- and early S-phase cells was PFA-resistant. The a m o u n t o f o u t p u t virus was m o n i t o r e d during the first replicative cycle at various doses
P. K U L O M A A E T A L .
354
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Fig. 2. Kinetics of the relative proportions of cellssynthesizing DNA during HSV2 replication in virus-infected cervical cancer cells.
Fig. 3. Kinetics of the relativeproportions of cellssynthesizing D N A during H S V 2 replication in virus-infected cervical cancer ceils.
HeLa (left) and CaSki (right) cells. Data on G : and early S-phase cells only are given as mean values (+ standard errors) of three to five separate analyses. O = PFA-treated cells; I = PFA-untreated cell.
C-33A (left)and SiHa (right) ceils. Data on the G : and early S-phase cells only are given as mean values (+standard errors) of three to five separate analyses. (O) = PFA-treated cells, ()) = PFA-untreated ceils.
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Fig. 4. Growth of H S V 2 in cervical cancer cellsat various multiplicities(I0, I, 0. I and 0.01 PFU/cell) of infecting virus. CaSki ceils (A), HeLa cells (B), C-33A cells (C), SiHa cells (D). The amount of output virus is given as log of PFU/ml of tissue culture medium.
h
HSV2, U N S C H E D U L E D D N A S Y N T H E S I S A N D C E R V I C A L C A N C E R C E L L S
o f input virus (10 to 0.01 PFU/celI). In C-33A and SiHa ceils, the amounts o f intracellular infectious virus were continuously about 10-fold lower than in corresponding CaSki cells and about 100-fold lower than in corresponding HeLa cells (fig. 4). At an a m o u n t of input virus as high as 10 PFU/cell, no infectious virus was detected in the PFA-treated cells 20 h p.i. (data not shown). The location of ICP8 and the uptake of BrdU by the virus-infected cells was compared by immunofluorescence microscopy at 20 h p.i. Mockinfected cells showed a speckled immunofluorescence staining pattern when anti-BrdU antibodies were used (fig. 5). Following HSV2 infection,
355
three cell lines (CaSki, SiHa and HeLa) showed a speckled or free globular staining pattern with the anti-BrdU antibodies, whereas in the HSV2 infected C-33A cells,staining appeared to be restricted to a few large globular structures only (fig. 5). In general, after HSV2 infection at a m.o.i. of 2 PFU/cell, about half of the cellswere positive for ICP8. In addition to a speckled nuclear fluorescence sccn in SiHa cells,CaSki and HeLa cells also exhibited a globular staining pattern with anti-ICP8 antibodies (fig. 6). In the C-33A cells, perinuclear and cytoplasmic staining predominated, resulting in an appearance similar to that seen in persistently HSV2-infected Raji cells (fig. 6).
G
Im Fig. 5. Indirectimmunofluorescence of BrdU-labelled cervicalcancer cells. Cells were stained with monoclonal anti-BrdU antibodies 20 h p.i. Mock-infected CaSki cells (A), HSV2-infected CaSki cells (B), mock-infected C-33A cells (C), and HSV2-infected C-33A cells (D). HSV2-infected HeLa and SiHa cells showed a fluorescence pattern similar to that seen in CasKi ceils.
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Fig. 6. Indirect immunofluorescence of HSV2-infected cancer cells. The cells were stained with monospecific rabbit anti-ICP8 antiserum 20 h p.i. SiHa cells (A), CaSki cells (B), Raji cells (C), and C-33A cells (D). HSV2-infected HeLa cells showed a fluorescence pattern similar to that seen in the CaSki cells.
DISCUSSION
Replication of episomal DNA has been suggested to be one of the key mechanisms of DNA amplification (Stark et aL, 1989) but cellular replication machinery seems to replicate episomal virus DNA only at a constant level (Adams et ai., 1989; Stanley et al., 1989; Crook et aL, 1990). Integration of HPV DNA was recently reported to result in amplification of both viral and cellular flanking sequences 0aopescu and DiPaolo, 1990; Wagatsuma et al., 1990). However, integration of HPV DNA occurs in only about half of cervical tumours (Cullen et al., 1991 ; Stoler et al., 1990), yet amplified HPV and c-myc DNA are frequently found in cervi-
cal cancer (Riou et al., 1984; Ocadiz et al., 1987). Thus, factors other than cellular replication machinery and integration of viral DNA might also contribute to the amplification of HPV DNA in cervical cancer. HSV2 and HPV16 (human papillomavirus) sequences have been demonstrated concomitantly in cervical cancer (DiLuca et al., 1989). Although, in productive infection, HSV replication machinery is able to amplify both episomal and most likely also integrated HPV DNA (Brandt et aL, 1987; Gerspach and Matz, 1988; Heilbronn and zur Hausen, 1989), as a consequence of this kind of an infection the cell would die. Unscheduled DNA synthesis is
HSV2. UNSCHEDULED DNA S Y N T H E S I S A N D CERVICAL C A N C E R CELLS
another key mechanism of DNA amplification (Wahl, 1989). In the present study, we demonstrated significant differences in the rate of HSVinduced unscheduled DNA synthesis in HPVDNA + (CaSki, HeLa and SiHa) and HPVDNA- (C-33A) cervical cancer cell lines. HSV2-infected C-33A and SiHa cells proved to be relatively unsusceptible to the infection, but exhibited PFA-resistant DNA synthesis late in the infection. In the C-33A (and SiHa) cells DNA synthesis was detected predominantly in G1 cells; thus it represents unscheduled DNA synthesis, a prerequisite of chromosomal DNA amplification. Late in the infection, C-33A cells stained with anti-BrdU antibodies showed globular staining which, in HSV susceptible cells corresponds to virus repilcation compartments (Gao and Knipe, 1989; Knipe, 1989). However, HSV replication was relatively inefficient in the C-33A cells. The cytoplasmic location of ICP8 in the C-33A ceils resembled that seen in persistently HSV-infected Raji cells (Lehtinen et al., 1987), as if ICP8 could not properly enter the nucleus. Furthermore, the measured DNA synthesis was PFA-resistant, i.e. not due to HSV DNApol. These findings suggest that, in C-33A cells, ICP8 and HSV DNApol are at least partially inactive or can be inactivated without an effect upon unscheduled DNA synthesis. This is important because the normal performance of ICP8 and DNApol would most likely kill the virus-infected cell. Six HSV-specific genes, UL5, UL8, UL29 (ICP8), UL30 (DNApol), UL42 and UL52, are involved in the amplification of episomal DNA (Heilbronn and zur Hausen, 1989). Cellular DNA polymerases alpha and delta, as well as cyclin, take part in the replication of episomal DNA (Zuber et al., 1989), but may also be involved in repair repilcation and amplification of integrated viral DNA (Ceils and Madsen, 1986; Heilbronn et al., 1985). HSV DNApol has 3' to 5' exonuclease activity analogous to DNA polymerase delta (O'Donnell et al., 1987). HSV DNApol and ICP8 have considerable sequence homology with DNA polymerase alpha and proliferating cell nuclear antigen (PCNA) (Wong et al., 1988; Matsumoto et al., 1987). Further-
357
more, ICP8 and PCNA exhibit similar fluorescence patterns (Bravo et al., 1986; Lehtinen et aL, 1987). Thus, the mode of action of HSV and cellular replication machineries in DNA amplification may be similar. What then is their role in the described unscheduled DNA synthesis in C-33A cells ? ICP8 may not be actively involved. HSV DNApol has exonuclease activity and HSV specifies an additional alkaline exonuclease which is normally complexed with ICP8 and HSV DNApol (Vaughan et al., 1984). Thus, relative dysfunction of the HSV replication machinery in the C-33A cells might set the stage for unscheduled DNA synthesis by causing DNA damage. Nishiyama and Rapp (1981) showed that HSV2 is able to induce repair replication of cellular DNA under non-permissive conditions. We could not find unscheduled DNA synthesis in highly HSV-susceptible CaSki and HeLa cells, even when virus DNA synthesis wasblocked by PFA. In fact, HSV-induced repair replication of cellular DNA has been previously described in non-permissive rat XC cells, but not in virussusceptible cell hybrids (Epstein and Jacquemont, 1980; Epstein et al., 1980, 1985). This further suggests that specific cellular factors might be involved. How and to what extent the cellular replication machinery (especially, PCNA and DNA-polymerases alpha and delta) takes part in HSV-induced unscheduled DNA synthesis warrants further investigation.
Acknowledgements
The authors wishto thank Mrs. LeenaPankko for excellent technicalassistance. This workwas supportedby grants fromthe EmilAaltonen foundation and FinnishCancer Organizations.
Le virus de I'herp~s simplex induit la synth~e d'ADN ~ hors programme)~ dans les l i g h t s ceilulaires de cancer infectC~es par le virus
Darts cette ~tude nous avons observ~ la synth~se d'ADN ~ hors programme, dans des lign~es cellu-
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P. K U L O M A A
laires de cancer cervical infectc~espar le virus de l'herp~s simplex type 2 (HSV2). Les lign~es cellulaires HeLa, CaSki, C-33A et SiHa ont ~t~ infect~es par I'HSV2 et marquees par la bromod~oxyuridine (BrdU). Le double param&re de I'analyse cytom~trique du flux de l'incorporation du BrdU montre l'existence d'une rapide augmentation des proportions de ceIlules en phase S e t Gl synth~tisant de I'ADN. Dans [es cellules C-33A et SiHa les proportions de cellules synth~tisant I'ADN ont augment~ seulement tardivement lors de rinfection. Si la r~plication de I'HSV2 est bloqu~e par [e phosphonoformate, les changements induits par le virus se produisent dans les ceIlules C-33A et SiHa mais ne sont pas observ6es dans les cellules HeLa et CaSki. La r~plication de I'HSV2 est environ 100 fois plus efficace dans les cellules HeLa que dans les cellules C-33A et SiHa. Les diff&ences sont aussi considc~rables entre les images de l'immunofluorescence des cellu[es infect~es. Les anticorps anti-BrdU forment des zones de marquage nucl~aire grossi&ement globuleuses dans les cellules C-33A alors que les images du marquage nucI~aire dans les autres cellules sont form~es par des petites taches et elles sont tlnement globuleuses. Les anticorps anti-ICP8 (prot~ine majeure de I'HSV liant I'ADND r~v~lent que I'ICP8 reste darts le cytoplasme des cellules C-33A alors que dans les autres ceIlules on observe une fluorescence sous forme de petites taches ou de noyau globuleux. Ces r~sultats indiquent que (1) I'HSV2 induit la synth~se d'ADN cellulaire hors programme, (2) l'induction est dc~pendante de l'h6te, (3) dans les cellules C-33A infect6es ce ph~nom~ne peut ~tre attribu~ aux m~anismes conjoints de r~plication virale et cellulaire. Connartre le r61e des ADN-polym&ases ceIIulaires ,, et 8 et celui de la cycline qui partage des s6quences homologues d'acides amines et/ou/l des fonctions communes avec l'ADNpol de I'HSV et I'ICP8, justitle les futures recherches. Mots-clc~s: HSV2, ADN, Cancer du col; Lign~es cellulaires C-33A, HeLa, CaSki and SiHa, Synth~se ~ hors programme >>de I'ADN, Cytom6trie du flux.
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HSV2, UNSCHEDULED DNA SYNTHESIS AND CERVICAL CANCER CELLS
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