Human cells (normal and ataxia telangiectasia) transfected with pR plasmid are hypersensitive to DNA strand-breaking agents

11

Mutation Research, DNA Repair, 255 (1991) 11-18 © 1991 ElsevierSciencePublishersB.V.0921-8777/91/$03.50 ADONIS 092187779100076P

MUTDNA 06438

Human cells (normal and ataxia telangiectasia) transfected with pR plasmid are hypersensitive to D N A strand-breaking agents Anna Antonelli, Raffaella Elli, Liana Marcucci, Roberto Bosi, DonateUa Kobal and Paola Petrinelli Dipartimento di Biopatologia Umana, Sezione di Biologia Cellulare, Unioersit~ 'La Sapienza" 00161 Rome (Italy)

(Received9 October 1990) (Revisionreceived14 January1991) (Accepted 15 January1991)

Keywords: Ataxiatelangiectasia;Plasmid transfection;Bleomycin;Vepesid

Summary Ataxia telangiectasia (AT) cells are known to be hypersensitive to ionizing radiations and to drugs such as bleomycin and epipodophyllotoxin VP16, a topoisomerase II poison. Both of these produce DNA double-strand breaks even if through different mechanisms. In this work we analyzed the sensitivity to bleomycin and to epipodophyllotoxin of AT cells after transfection with pR plasmid. This plasmid, interacting with bacterial SOS repair pathways, expresses itself in mammalian cells conferring cell resistance to the SOS inducers UV and 4NQO and cell sensitivity to different drugs such as bleomycin. This effect is presumably due to the interaction of pR products with double-strand breaks. Our findings indicate that pR plasmid, in both AT lines tested (AT5BIVA fibroblasts and ATL6 lymphoblasts), expresses itself (increasing UV protection) and amplifies the already enhanced AT cell sensitivity to both bleomycin and VP16.

Ataxia telangiectasia (AT), a human multifaceted genetic disorder, is one of the cancer-predisposing 'chromosome breakage syndromes' (McKinnon, 1987). A gene(s) involved in DNA processing or repair and possibly also involved in preventing cancer would be an appropriate candidate for this genetic disorder (Painter, 1985; Hanawalt and Sarasin, 1986). The main in vitro characteristic of AT cells is their hypersensitivity

Correspondence: Dr. A. AntoneUi,Dipartimento di Biopatologia Umana, Sezione di Biologia Cellulare, Universith 'La Sapienza', 00161 Rome(Italy).

to ionizing radiations and to drugs, such as bleomycin (BLM) (Taylor et al., 1979; Cohen et al., 1981) and epipodophyllotoxin-VP16 (Hermer and Blazka, 1986; Smith et al., 1986), known to have cellular effects similar to those of X radiation (Gupta, 1983). Indeed, BLM and VP16 (a topoisomerase II poison) both can produce DNA double-strand breaks although through different mechanisms (Hecht, 1979; Liu, 1989). It is therefore possible that the principal biochemical problem for AT cells is the repair of DNA doublestrand breaks, which have great potential to cause recombination, rearrangements and deletions. The hypersensitivity to DNA strand-breaking agents,

12 the low fidelity in DNA-strand rejoining (Cox et al., 1986) and the hypersensitivity to topoisomerase II poisons may suggest an altered topoisomerase II activity in AT cells, although conflicting observations have been published. Here we report data regarding the sensitivity to BLM and to VP16 of normal and AT cells transfected with pR plasmid. This plasmid, involved in bacterial SOS repair pathways, protects prokaryotic cells against lethal damage induced by UV and 4-nitroquinoline 1-oxide (4NQO), typical SOS inducers (Marcucci et al., 1986). When transfected into mouse cells, this plasmid is able to discriminate between different types of damage, conferring cell resistance to UV and 4NQO (Marcucci et al., 1986) and cell sensitivity to different drugs such as BLM, N-methyl-N'-nitro-Nnitrosoguanidine (MNNG) and cis-diamminedichloroplatinum (cisDDP). A possible unifying explanation for the pR-sensitizing effect is that these drugs, despite the quite different way in which they interact with DNA, induce DNA breaks as major lesions or as intermediate steps in the repair process. The pR products (coded by uvpl and uop2-mucAB regions) could interfere with specific repair pathways, which also include recombinative events (Elli et al., 1987). We used pR plasmid to transfect AT cells to see whether the potential pR-sensitizing effect to DNA strand-breaking agents could also be expressed in these already hypersensitive cells. The data reported demonstrate that pR plasmid, in both AT lines tested (AT5BIVA fibroblasts and ATL6 lymphoblasts), expresses itself through 70 generations enhancing UV protection and amplifying cellular sensitivity to both BLM and VP16.

Cell lines NL1 is an EBV-transformed human tymphoblastoid line from a normal donor (supplied by M. Fiorilli). ATL6 is an EBV-tranformed lymphoblastoid line derived from an AT homozygote donor (provided by B.A. Bridges). AT5BIVA is an SV40-transformed fibroblast line derived from strain AT5BI (group D) (supplied by C. Arlett). DNA-mediated transfection Electroporation of human cells was performed in Gene Pulser Apparatus (Biorad) using a single pulse of 500 V at 25 /~F with a rise time of 0.5 msec in PBS electroporation medium withouth Ca 2+ and Mg 2+. 1 x 107 lymphoblastoid cells or 1 x 107 fibroblasts in 0.8 ml of PBS were electroporated in the presence of 40 ttg of pR DNA a n d / o r 60 ttg of pSV2neo DNA. The cells were allowed to remain in the buffer at 0 o C for 10 rain. Subsequently 5 x 106 transfected cells were seeded in complemented medium. After 24-h incubation, G418 (Sigma) was added to the cultures every 4 days (final concentration 400 tLg/ml) in order to select cells that had acquired neomycin resistance. Independent colonies of the G418-resistant fibroblasts were picked after 14 days, expanded and maintained in a-MEM 20% FCS plus G418 (200 ttg/ml). The transfection efficiency was 1.5 x 10 4. The transfected lymphoblasts were grown in suspension in RPMI 20% FCS under continuous selection pressure (200 ttg/ml G418). The transfected lines used are shown in Table 1. DNA extraction and dot-blot hybridization The transfected lines, cloned and uncloned, were routinely tested for pR presence by dot-blot hybridization using 32p pR DNA as the probe, as

Materials and methods

Plasmids utilized for transfection The pR (Ap+UV +) plasmid is a HindIII restriction fragment (23 kb) of the TP120 plasmid. pSV2neo is an SV40 hybrid vector plasmid (Southern and Berg, 1982). It contains a Tn5 gene (neo) that confers resistance to neomycin/kanamycin antibiotics and is used as a selectable marker in eukaryotic cells grown in the presence of the aminoglycoside antibiotic G418.

TABLE 1 PLASMIDSAND TRANSFECTEDCELLS TransfectedDNA pR + pSV2neo

Line NLI(pR) ATL6(pR) AT5BIVA(pR)

pSV2neo

NLl(pneo) ATL6(pneo) AT5BIVA(pneo)

13 previously described by Marcucci et al. (1986). Only the positive lines were used for the experiments. The plasmid DNAs were isolated from E. eoli cultures as described by Humphreys et al. (1975). High-molecular-weight DNA from human cells was prepared as described by Wigler et al. (1979).

Cell viability The drugs, etoposide (VP16-213, Vepesid, Bristol Italiana) and bleomycin (BLM, Nippon Kayaku), were diluted in distilled water to the desired concentration immediately prior to use. Lymphoblastoid cells were initially seeded at a concentration of 4 × 105 cells/m1 and allowed to incubate for 24 h. BLM or VP16 was added to achieve the desired final concentration. The duration of treatment time was 30 min a n d / o r 60 min after which the cultures were washed twice with PBS and resuspended in fresh growth medium (5 × 104 cells/ml). Aliquots of cells were taken at intervals of 24 h following treatment and the viability determined by trypan blue exclusion. The results are presented as relative increase in cell number (cell number at the indicated incubation period/cell number at 0 time). In a second type of experiment, aliquots of cells were taken 6 days after the treatment and the viability was determined as the number of trypan blue-excluding cells/total seeded cells. The results are expressed as the percentage of survival demonstrated by the viability value of treated compared to untreated cultures of the same line, grown and sampled simultaneously. Fibroblasts were collected from logarithmicphase cultures and allowed to attach to 6-8 × 102/60-mm plastic dish containing growth medium at 37 °C for 24 h prior to drug treatment or UV irradiation. UV irradiation, clonigenicity evaluation and survival curve analysis were performed as previously described (Elli et al., 1983). Chromosome breakage analysis Chromosome preparations were obtained from lymphoblastoid cells by standard methods. To evaluate the induced chromosomal breakage rate, BLM (final concentration 10 or 30 /xg/ml) was added to cultured cells in the log phase of growth 4 h before harvesting, as previously described

(Fiorilli et al., 1985). Chromosome breakage, either spontaneous or induced, was blindly evaluated by 2 independent observers in almost 100 Giemsastained metaphases from 2 or 4 simultaneously grown cultures for each line and each treatment. The classification of chromosomal breakage was based on 3 different levels of damage: 1-2 breaks/cell, more than 2 breaks/cell and partial or full metaphase pulverization. Gaps were noted and scored but not included in the calculation of the frequency of breakage. The chi-square test (R × C table) was used for statistical analysis. Results

UV + phenotype in A T fibroblasts In order to verify whether pR plasmid also expresses itself in AT cells (w.t. for UV sensitivity), the parental AT5BIVA, the transfected AT5BIVApR (positive to dot-blot hybridization with pR DNA) and AT5BIVApneo lines were tested for their sensitivity to UV. The inactivation curves of the 3 fines after UV treatment (Fig. 1) showed that AT5BIVApR has a higher survival rate (D o 13.6 J / m 2) and a more shouldered survival curve than AT5BIVA (D o 3.9 J / m 2) and ATSBIVApneo (D o 5.6 j/m2). The enhancement in UV survival of AT5BIVApR was observed for about 70 passages. On the 88th passage, the line no longer showed pR presence (negative dot-blot hybridization) and the survival curve had a value of D o 4.8 J / m 2. Bleomycin sensitivity In order to establish whether the BLM-sensitizing effect of pR is also present in BLM-hypersensitive lines, 2 pR-transfected AT fines were used: SV40-immortalized fibroblasts (AT5BIVApR) and EBV-immortalized lymphoblastoid cells (ATL6pR). BLM sensitivity was evaluated on fines positive to dot-blot hybridization with pR DNA. The sensitivity was measured as cellular survival or growth kinetics and as induced chromosomal instability. This latter parameter (break rate 4 h after treatment) gives a measure of the initial BLM damage. Both AT lines when transfected with pR plasmid showed significantly enhanced BLM sensitivity. The BLM cell-killing effect in

14 100~

100,

k~

"t'X!

L"0

\f

"r

1 5> >

~

10-

>

lo,

o

2'4 BLM

1 UV d o s e

(j/

1.

(pg /ml)

Fig. 2. BLM survival curves of AT5BIVApR fibroblasts (cotransfected with pSV2neo and pR) and of the parental line AT5BIVA. O, AT5BIVA; o, ATSBIVApR. Data represent mean + SE of 3 experiments.

m2)

survival curves of AT5BIVApR fibroblasts (cotransfected with pSV2neo and pR plasmids), of AT5B1VApneo (transfected only with pSV2neo plasmid) and of the parental line AT5BIVA. Cells were irradiated at a dose rate of 1.25 J / m 2 / s . The fluences were measured with a Latarjet U V meter. O, AT5BIVA; o , AT5BIVApR; II, AT5BIVApneo. The bars represent the mean + S E o f 3 experiments. Fig.

28

dose

3°1

UV

•

o

o z z ~ 10,

z3

E c

pR-transfected and untransfected fibroblasts is shown in Fig. 2. C o m p u t e r analysis of A T 5 B I V A p R survival data yielded a D O value of 1.8 t t g / m l . The control lines A T 5 B I V A and A T 5 B I V A p n e o s h o w e d D O values of 3 . 8 / ~ g / m l and 3 . 1 / ~ g / m l respectively. The p R effect on growth kinetics of l y m p h o blasts untreated and treated with B L M is s h o w n in Fig. 3. The p R transfected cells s h o w e d a delayed entry into the plateau phase and a m o d ified doubling time in c o m p a r i s o n with the parental line. After B L M treatment, the pR-transfected line showed a marked growth decrease c o m p a r e d to the parental line. Fig. 4 shows the distribution of s p o n t a n e o u s

S

o d) L U E

0~ > t3 1

•

48 IncubQtion

i 120 p e r i o d (h)

Fig. 3. Effect of BLM (0.5 ffg/mlx60 min) on the growth kinetics of ATL6pR lymphoblasts (cotransfected with pR and pSV2neo) and of parental line ATL6. e, Untreated ATL6; A, untreated ATL6pR; o BLM-treated ATL6; zx, BLM-treated ATL6pR. Data derived from a representative experiment.

15

I

IooJNL1

~

NLlpneoNLlpR

A'rL6 ATL6pneoATL6pR

0

lO~g/ml

60

30 )Jg/ml

abed

abed

E1

abcd

abcd

abcd

abed

Fig. 4. Distribution of spontaneous and BLM-induced chromosomal breaks in untransfected and transfected normal and A T lymphoblasts. Metaphases were classified according to the n u m b e r of breaks per cell: (a) no breaks; (b) 1 - 2 breaks; (c) more than 2 breaks; (d) partially or fully pulverized. The bars represent the mean + SE of pooled data from 2 - 4 different cultures.

than in the parental line. In fact, the difference between ATL6pR and ATL6, at 30 # g / m l of BLM, is within the significance limits (0.01 < p < 0.05). This could be due to the lethal effect of the higher BLM dose on the AT cells hypersensitized by pR plasmid. This was confirmed by the observation that chromosome breakage distributions in ATL6pneo and ATL6pR cells at the higher BLM dose were not significantly different (p > 0.05); (2) the spontaneous and induced chromosome breakage of the pSV2neo-transfected lines were not significantly (p > 0.05) different from those of the respective parental lines. These data show that the BLM-sensitizing effect is really due to pR plasmid presence.

VP16 sensitivity The survival curves of AT5BIVA and AT5BIVApR following 2 different VP16 time exposures are shown in Fig. 5A and B. The AT5BIVApR 100,

B

and BLM-induced chromosome breaks in transfected and untransfected AT (ATL6) and normal (NL1) lymphoblasts. Statistical analysis of chromosome break distribution (Table 2) showed that: (1) BLM-induced chromosomal breakage both in ATL6pR and in N L l p R is significantly higher

\

10

TABLE 2 STATISTICAL A N A L Y S I S O F T H E C H R O M O S O M E B R E A K A G E D I S T R I B U T I O N (X 2 TEST) Line

BLM dose (/~g/ml) 0

10

30

NL1 NLI(pR)

p > 0.05

p < 0.01

p < 0.01

ATL6 ATL6(pR)

p > 0.05

p < 0.01

0.01 < p < 0.05

NL1 NLl(pneo)

p > 0.05

p > 0.05

ATL6 ATL6(pneo)

p > 0.05

p > 0.05

1

0.5

l

0

1 1

15

05

1

1.5

VP 16 d o s e ( pg / ml )

NLl(pneo) NLI(pR)

p > 0.05

p < 0.01

ATL6(pneo) ATL6(pR)

p > 0.05

p > 0.05

Fig. 5. VP16 survival curves of AT5BIVA and A T 5 B I V A p R after 30 min (A) and 60 rain (B) of exposure. Open symbols AT5BIVApR, closed symbols AT5BIVA. Data represent the m e a n + SE of 3 experiments.

16

cells showed a survival lower than parental cells both after 30 min (D o values 0.13 and 0.56/~g/ml respectively) and after 60 min (D o values 0.18 and 0.30/~g/ml respectively). It is interesting to note that, while AT parental cells showed lower survival after 60 rain than after 30 rain (Do ratio 1.8), the AT5BIVApR cells showed almost the same survival after the different exposure times (D o ratio 0.73). These results could be due to the fact that the accumulation of lesions reaches near saturation point after 30 rain in pR-transfected AT fibroblasts, while in the parental AT cells the VP16 toxic effect is increased with exposure time. The pR-sensitizing effect was also present in AT lymphoblastoid cells. Fig. 6 shows the inhibition of cell growth following treatment with 2 different doses of VP16 in ATL6 and ATL6pR lines and provides evidence that the growth of AT cells is abnormally depressed when harboring pR plasmid. We also compared the BLM and VP16 sensitivity of AT fibroblasts and lymphoblasts (Table 3). Despite the differences in the 2 cell types (different immortalization system and culture methods) pR plasmid conferred BLM and VP16 cytotoxicity enhancement equally affecting ATL6 and AT5BIVA cells. In pR-transfected normal lympho20. %

/

z z v

10.

•/,/ •/ /

/z/

D c m

¥ u

E

L

•/ A / / ' / / J

48 Incubation

~'/

120 per'iod

192

(h)

Fig. 6. Effect of 2 doses of VP16 on the growth kinetics of ATL6pR lymphoblasts and of parental lymphoblasts ATL6. A, ATL6 and zx, ATL6pR treated with 1 / ~ g / m l of VP16; • ATL6 and D, ATL6pR treated with 2 / t g / m l of the drug. The data are derived from a representative experiment.

TABLE 3 RELATIVE SENSITIVITY OF T H E FIBROBLASTS a A N D LYMPHOBLASTS b TO VP]6 A N D BLM Drugs

D O ratio AT5BIVA AT5BIVApR

Survival ratio (%) Dose (~g/ml)

ATL6 ATL6pR

NL1 NLlpR

BLM

2.1

0.5 5.0

1.6

2.3

VPI6

4.3

1.0 2.0 4.0

1.9 4.2

1.4 2.2

Relative sensitivity of fibroblasts is expressed as ratio of D O values. b Relative sensitivity of lymphoblasts is expressed as ratio of percentage of survival at the indicated doses (see Materials and methods).

blasts a comparable cytotoxicity enhancement was observed at higher drug concentrations (10 x for BLM and 4 x for VP16). Discussion

SV40-immortalized normal and repair-deficient human cells show a high efficiency of DNA transfection but the amount of exogenous DNA maintained in the genome is very small, especially when compared to rodent cells. In particular SV40 AT fibroblasts tend to reject plasmid DNA sequences that are not essential for survival in selective medium; furthermore, plasmid DNAs are rearranged during cell growth (Dean et al., 1989). We succeeded in stably transfecting SV40-immortalized AT fibroblasts with pR plasmid DNA. This plasmid expresses itself for about 70 passages in vitro in the absence of continuous direct selection. The observation that pR presence (tested by dot-blot hybridization) results in pR expression (evaluated in terms of enhancement of UV resistance and of BLM sensitivity) allows us to affirm that at least the pR uvpl and uup2 regions remain unmodified during cell growth. Immortalized AT cells, although retaining the major features of the AT phenotype, exhibit some variation in radiosensitivity compared to the parental line, presumably related to the different host viruses (Arlett et al., 1988; Ziv et al., 1989).

17

We therefore used 2 different immortalized AT lines to test cell radiosensitivity. We have shown that pR plasmid is able to enhance BLM cytotoxicity in SV40-immortalized fibroblasts as well as in EBV-immortalized AT lymphoblasts (see Table 3). The enhancement correlates well with the chromosome breakage rate, as shown by the analysis of chromosome break distribution in lymphoblastoid ceils. Furthermore, pR plasmid can increase VP16 sensitivity of AT5BIVA fibroblasts and ATL6 lymphoblasts to the same extent. VP16 can block cells in late S and G 2 phases of the cell cycle (Smith et al., 1986; Kalwinsky et al., 1983), inhibit DNA synthesis preventing the appearance of the large 10-kb DNA intermediate (L~Snn et al., 1989) and induce extensive chromosomal DNA fragmentation, sisterchromatid exchanges and protein cross-links (Singh and Gupta, 1983; Huang et al., 1973). The VP16-induced DNA breakage is mediated by DNA topoisomerase II. The drug interferes with the breakage-reunion reaction of DNA topoisomerase II by stabilizing the cleavable complex formed between the enzyme and DNA (Liu, 1989). However, cleavage by VP16 produces toxic free radicals (Wozniak and Ross, 1983) as does cleavage by BLM (Shiloh et al., 1983). We can therefore hypothesize that the enhancement in BLM and VP16 sensitivity conferred by pR plasmid, in both normal and AT cells, is based on the DNA-breaking ability common to these drugs. The involvement of DNA topoisomerase II in AT phenotype has found considerable support in recent literature. For this reason, many studies have been carried out on the enzyme level/activity in AT cells. A normal amount of topoisomerase II and reduced DNA unknotting activity were found in all the lymphoblastoid AT lines tested to date (Singh et al., 1988; Mohamed et al., 1987; Singh and Lavin, 1989). With regard to the AT fibroblasts tested, normal (AT4BI) or increased levels (AT5BI, AT5BIVA) of topisomerase II were reported (Davies et al., 1989; Smith and Makinson, 1989). This also makes it more difficult to explain the AT hypersensitivity to topoisomerase inhibitors such as VP16. The observation that pR plasmid determines increased sensitivity to VP16 both in AT5BIVA

fibroblasts and in ATL6 lymphoblasts, regardless of the topoisomerase II levels, further confirms that the central defect in AT cannot be due to a defect in this enzyme itself. The topoisomerase II gene and 1 AT gene (AB complementation group) have been mapped on chromosome 17 (Tsai-Pflugfelder et al., 1988) and on chromosome 11 (Gatti et al., 1988) respectively. In conclusion, enhanced sensitivity to BLM is a common phenotype of pR-transfected mammalian cells (murine and human). Furthermore, pR plasmid is able to hypersensitize AT cells to both BLM and VP16, further increasing their already enhanced sensitivity to these drugs. Therefore, pR plasmid, when transfected into mammalian cells, enhances the in vitro cytotoxicity of drugs known to act, directly or indirectly, via DNA breakage.

Acknowledgements We are very grateful to M. Fiorilli for helpful discussion and critical reading of the manuscript. We also thank M. Proietti for his skilful technical assistance. This work was partially supported by grants from the 'Associazione Italiana per la Ricerca sul Cancro', from the C.E.E. (Contract 316-E-205-I) and from the 'Consiglio Nazionale delle Ricerche' (Grant 89.02667.04).

References Arlett, C.F., M.H.L. Green, A. Priestley, S.A. Harcourt and L.W. Mayne (1988) Comparative human cellular radiosensitivity: I. The effect of SV40 transformation and immortalization on the gamma-irradiation survival of skin derived fibroblasts from normal individuals and from ataxia telangiectasia patients and heterozygotes, Int. J. Radiat. Biol., 54, 911-928. Cohen, M.M., S.J. Simpson and L. Pazos (1981) Specificity of bleomycin induced cytotoxic effects on ataxia-telangiectasia lymphoid cell lines, Cancer Res., 41, 1817-1823. Cox, R., P.G. Debenham, W.K. Masson and M.B.T. Webb (1986) Ataxia-telangiectasia: a human mutation giving high frequency misrepair of DNA double-stranded scissions, Mol. Biol. Med., 3, 229-244. Davies, S.M., A.L. Harris and I.D. Hickson (1989) Overproduction of topoisomerase II in an ataxia-telangiectasia fibroblast cell line: comparison with a topoisomerase II overproducing hamster cell mutant, Nucleic Acids Res., 17, 1337-1351. Dean, S.W., L. Kincla, H.R. Sykes, A.R. Lehmann and I.A. Wise (1989) Instability of extrachromosomal cosmid DNA

18 in SV40-transformed human (ataxia-telangiectasia) cells, Exp. Cell Res., 183, 473-483. ElK R., L. Marcucci, R. Bosi, C. Oppi, P.A. Battaglia and F. Gigliani (1983) The pR UV+ plasmid, transfected into mammalian cells, enhances their UV survival, Nucleic Acids Res., 11, 3679-3686. Elli, R., A. Antonelli, P. Petrinelli, R. Bosi and L. Marcucci (1987) The pR plasmid: a tool for discriminating between DNA lesions induced by different types of cytotoxic agents in cultured mammalian cells, Mutation Res., 191,177-181. Fiorilli, M., A. Antonelli, G. Russo, M. Crescenzi, M. Carbonari and P. Petrinelli (1985) Variant of ataxia-telangiectasia with low-level radiosensitivity, Hum. Genet., 70, 274-277. Gatti, R.A., I. Berkel, E. Boder et al. (1988) Localization of an ataxia-telangiectasia gene to chromosome 1lq22-23, Nature (London), 336, 577-580. Gupta, R.S. (1983) Genetic, biochemical and cross-resistance studies with mutants of Chinese hamster ovary cells resistant to the anticancer drugs VM-26 and VP16-213, Cancer Res., 43, 1568-1574. Hanawalt, P.C., and A. Sarasin (1986) Cancer-prone hereditary diseases with DNA processing abnormalities, Trends Genet., 2, 2124-2129. Hecht, S.M. (Ed.) (1979) Bleomycin: Biochemical and Biological Aspects, Springer, New York. Henner, W.D., and M.E. Blazka (1986) Hypersensitivity of cultured ataxia-telangiectasia cells to etoposide, J. Natl. Cancer Inst., 76, 1007-1011. Huang, C., Y. Hou and J.J. Wang (1973) Effect of a new antitumor agent, epipodophyllotoxin (VP-16), on growth and chromosomes in human hematopoietic cell fines, Cancer Res., 33, 3123-3129. Humphreys, G.O., G.A. Willshaw and E.S. Anderson (1975) A simple method for the preparation of large quantities of pure plasmid DNA, Biochim. Biophys. Acta, 383, 457-463. Kalwinsky, D.K., A.T. Look, J. Ducore and A. Fridland (1983) Effects of epipodophyllotoxin VP-16-213 on cell cycle traverse DNA synthesis and DNA strand size in cultures of human leukaemic lymphoblasts, Cancer Res., 64, 15921597. Liu, L.F. (1989) DNA topoisomerase poisons as antitumor drugs, Annu. Rev. Biochem., 58, 351-375. L/3nn, U., S. Li3nn, U. Nylen and G. Winblad (1989) Altered formation of DNA in human cells treated with inhibitors of DNA topoisomerase II (etoposide and teniposide), Cancer Res., 49, 6202-6207. Marcucci, L., F. Gigliani, P.A. Battaglia, R. Bosi, E. Sporeno and R. Elli (1986) Cellular response to DNA damage is enhanced by the pR plasmid in mouse cells and in Escheriehia coli, Mol. Cell. Biol., 6, 586-592. McKinnon, P.J. (1987) Ataxia-telangiectasia: an inherited disorder of ionizing radiation sensitivity in man, Hum. Genet., 75, 197-208. Mohamed, R., S.P. Singh, S. Kumar and M.F. Lavin (1987) A defect in DNA topoisomerase II activity in ataxia-

telangiectasia cells, Biochem. Biophys. Res. Commun., 149, 233-238. Painter, R.B. (1985) Altered DNA synthesis in irradiated and unirradiated A-T cells, in: R.A. Gatti and M. Swift (Eds.), Ataxia-telangiectasia: Genetics, Neuropathology and Immunology of a Degenerative Disease of Childhood, Liss, New York, pp. 89-100. Shiloh, Y.E., E. Tabor and Y. Becker (1983) Abnormal response of ataxia-telangiectasia cells to agents that break the deoxyribose moiety of DNA via a targeted free radical mechanism, Carcinogenesis, 4, 1317-1322. Singh, B., and R.S. Gupta (1983) Mutagenic responses of thirteen anticancer drugs on mutation induction at multiple genetic loci and on sister chromatid exchanges in Chinese hamster ovary cells, Cancer Res., 43, 577-584. Singh, S.P., and M.F. Lavin (1989) Study of DNA topoisomerase II in ataxia-telangiectasia cells. Carcinogenesis, 10, 1215-1218. Singh, S.P., R. Mohamed, C. Salmond and M.F. Lavin (1988) Reduced DNA topoisomerase II activity in ataxia-telangiectasia cells, Nucleic Acids Res., 16, 3919-3929. Smith, P.J., and T.A. Makinson (1989) Cellular consequences of overproduction of DNA topoisomerase II in an ataxiatelangiectasia cell line, Cancer Res., 49, 1118-1124. Smith, P.J., C.O. Anderson and J.V. Watson (1986) Predominant role for DNA damage in etoposide-induced cytotoxicity and cell cycle perturbation in human SV40-transformed fibroblasts, Cancer Res., 46, 5641-5645. Southern, P.J., and P. Berg (1982) Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter, J. Mol. Appl. Genet., 1,327-341. Taylor, A.M.R., C.M. Rosney and J.B. Campbell (1979) Unusual sensitivity of ataxia-telangiectasia cells to bleomycin, Cancer Res., 39, 1046-1050. Tsai-Pflugfelder, M., L.F. Liu, A.A. Liu, K.M. Tewey, J. Whang-Peng, T. Knutsen, K. Huebner, C.M. Croce and J.C. Wang (1988) Cloning and sequencing of cDNA encoding human DNA topoisomerase II and localization of the gene to chromosome region 17q21-22. Proc. Natl. Acad. Sci. (U.S.A.), 85, 7177-7181. Wigler, M., R. Sweet, G.K. Sim, R. Wold, A. Pellicer, E. Lacy, T. Maniatis, S. Silverstein and R. Axel (1979) Transformation of mammalian cells with genes from prokaryotes and eukaryotes, Cell, 16, 777-785. Wozniak, A.J., and W.E. Ross (1983) DNA damage as a basis for 4'-demethyl-epipodophyllotoxin-9-(4,6-O-ethylidene-flD-glucopyranoside) (etoposide) cytotoxicity, Cancer Res., 43, 120-124. Ziv, Y., G.J. Jaspers, S. Etkin, T_ Danieli, L. Trakhtenbrot, A. Amiel, Y. Ravia and Y. Shiloh (1989) Cellular and molecular characteristics of an immortalized ataxia-telangiectasia (group AB) cell line, Cancer Res., 49, 2495-2501.