Mutagenesis, Vol. 15, No. 4, 367-374,
July 2000
© 2000 UK Environmental Mutagen Society/Oxford University Press
Isolation of camptothecin-sensitive Chinese hamster cell mutants: phenotypic heterogeneity within the ataxia telangiectasia-like XRCC8 (irs2) complementation group
School of Biological Sciences, Donnan Laboratories, University of Liverpool, Liverpool L69 7ZD and 1 School of Biomedical Sciences, University of St Andrews, St Andrews KY16 9TS, UK
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Using a replica microwell method, four Chinese hamster lines which exhibit hypersensitivity to the topoisomerase I inhibitor camptothecin, designated CM1, CM2, CM3 and CM6, have been isolated. Their sensitivity towards camptothecin varied from 3.5- to 8.2-fold with relative sensitivity as follows: CM2 < CM3 < CM6 < CM1. Genetic analysis of the CM mutants has established that CM1, CM3 and CM6 fail to complement each other and can each be assigned to the irs2 (XRCC8) complementation group. The mutant CM2 could not be definitively assigned to a complementation group because it presented a semi-dominant phenotype. In contrast to their sensitivity to camptothecin, the four CM mutants were less sensitive (1.1- to 2.2-fold) to the topoisomerase II inhibitors etoposide and adriamycin, although CM1, CM3 and CM6 were more sensitive (2.5- to 3.8-fold) to streptonigrin (a free radical generator and a topoisomerase II inhibitor). All four mutant lines displayed an increased sensitivity to the bifunctional alkylating agent mitomycin C (2.4- to 5.1-fold). Surprisingly, given their assignment to the irs2 (XRCC8) complementation group, CM1, CM3 and CM6 displayed only a minor increase in sensitivity to ionizing radiation (1.6-fold or less). Similar sensitivity of these CM mutants was observed for the radiomimetic compound bleomycin (1.7-fold sensitive or less). This study indicates that XRCC8 mutants are isolated at high frequency from the parent line V79 and that phenotypic heterogeneity amongst the irs2 (XRCC8) complementation group is greater than previously encountered. Mutations in different regions of the XRCC8 gene may be responsible for the differing cellular phenotypes. Hamster XRCC8 mutants show phenotypic similarities to cultured cells from ataxia telangiectasia and Nijmegen break syndrome (NBS) patients and are likely to be defective in the same pathway in which the ATM (ataxia telangiectasia-mutated) and the NBS genes operate.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Chinese hamster cell mutants isolated on the basis of their hypersensitivity to DNA-damaging agents have proved excellent models for the genetic and biochemical analysis of human DNA repair and recombination processes (Thompson, 1998
). The availability of Chinese hamster (CH) mutants hypersensitive to UV radiation enabled molecular cloning of a number of human repair genes following functional complementation of the CH mutants by transfection of genomic DNA or cDNA expression libraries (Thompson, 1998). Many of these genes, termed ERCC for excision repair cross-complementing, correspond to those defective in the cancer-prone syndrome xeroderma pigmentosum, including ERCC2/XPD, ERCC3/XPB, ERCC4/XPF and ERCC5/XPG (Thompson, 1998; DeLaat et al., 1999). A number of ionizing radiation-sensitive XRCC (X-ray repair cross-complementing) Chinese hamster mutants (XRCC4, XRCC5 and XRCC7) are defective in DNA double-strand break repair and were subsequently found to be defective in V(D)J recombination (Jeggo, 1997), whilst other XRCC mutants are defective in homologous recombination (Tebbs et al., 1995; Liu et al., 1998; Johnson et al., 1999). Many of the XRCC Chinese hamster mutants are cross-sensitive to either or both topoisomerase (topo) I or topo II inhibitors such as camptothecin (topo I inhibitor) and etoposide (topo II inhibitor), which produce DNA strand breaks via inhibition of the resealing step of the topoisomerase enzyme. For example, Chinese hamster XRCC4, XRCC5 and XRCC7 mutants are hypersensitive to etoposide because they are defective in non-homologous DNA double-strand break rejoining and are unable to repair etoposide/topo II-mediated DNA breaks (Jeggo, 1997; Johnson and Jones, 1999). Sensitivity to camptothecin in other XRCC mutants is less well understood and, to date, no Chinese hamster cell mutants have been directly isolated on the basis of sensitivity to this drug.
Camptothecin, a plant alkaloid purified from Camptotheca acuminata, is an experimental anticancer agent that has been shown to be a potent inducer of DNA strand breaks via its inhibition of the enzyme topo I (Horwitz et al., 1971; Hsiang et al., 1985, 1989; Hsiang and Liu, 1988; Holm et al., 1989). Camptothecin belongs to the class of topo I poisons that trap the topo I enzyme once it has produced a nick in the DNA strand and prevent religation of the nick (Froelich-Ammon and Osheroff, 1995). Topo I progressively unwinds supercoiled DNA by introducing single-strand breaks at specific sites, passing the intact DNA strand through the gap before ligating the ends (Wang, 1985). It functions during transcription and replication to reduce torsional strain in DNA (Crumplin, 1981; Fleischmann et al., 1984; Trask and Muller, 1988; Zhang,H. et al., 1988; Bendixen et al., 1990; Kaufmann et al., 1991). Topo I is associated with transcriptionally active chromatin and is a cofactor for RNA polymerase II-mediated transcription (Kretzschmar et al., 1993; Merino et al., 1993). A role in DNA repair processes for topo I, and topoisomerases in general, has been proposed (Boothman et al., 1989, 1994; Hickson et al., 1990; Pommier and Bertrand, 1993; Theilmann et al., 1993) and recently it was shown that UV-induced DNA damage stimulates topo I–DNA complex formation in vitro (Gobert et al., 1996). In addition, p53 binds to topo I and activates its catalytic DNA relaxation and SR kinase activities in vivo (Subramanian et al., 1998). Stabilization of the topo I–DNA complex by camptothecin generates single-strand DNA breaks that may lead to more complex breaks by interaction of transcription machinery or the DNA replication fork (Degrassi et al., 1989; Ryan et al., 1991; Froelich-Ammon and Osheroff, 1995; Nitiss and Wang, 1996).
A number of DNA repair mutants in Chinese hamster cells that are assigned to one of the XRCC complementation groups exhibit hypersensitivity to camptothecin (Collins, 1993; Thompson and Jeggo, 1995). These mutants include EM9 and EM7 (mutated in the XRCC1 gene), irs1 (XRCC2), irs1SF (XRCC3) and irs2, V-C4, V-E5 and V-G8 (XRCC8). The cloning of XRCC1, XRCC2 and XRCC3 has provided some insight into the processes involved with the repair of camptothecin-induced DNA damage. XRCC1 encodes a predicted polypeptide of 69.5 kDa, which is not homologous to any known protein and has been shown to complex with DNA ligase III (Caldecott et al., 1994). It is thought to be required for repair of X-ray-induced single-strand breaks and those generated during base excision repair (Caldecott et al., 1994). XRCC2 and XRCC3 both represent new human Rad51 family members which promote chromosome stability and protect against DNA crosslinks and other forms of damage, including topoisomerase poisons, and are defective in the repair of DNA double-strand breaks by homologous recombination (Tebbs et al., 1995; Liu et al., 1998; Johnson et al., 1999). XRCC8 is represented by four Chinese hamster mutants [irs2 (Jones et al., 1987); V-C4, V-E5 and V-G8 (Zdzienicka and Simons, 1987)] and has yet to be cloned. The phenotype of the XRCC8 group mutants show similarities to those of cultured cells from the human cancer-prone syndrome ataxia telangiectasia (AT) (Meyn, 1999). Chinese hamster XRCC8 mutants and AT cell lines display hypersensitivity to ionizing radiation and camptothecin, have no apparent inability to rejoin single- or double-strand breaks and display normal V(D)J recombination (Smith et al., 1989; Zdzienicka et al., 1989; Jones et al., 1990; Thacker and Ganesh, 1990). A gene that corrects the defect in AT cells, termed ATM (AT-mutated), has been identified and cloned (Savitsky et al., 1995; Zhang,N. et al., 1997). ATM encodes a 350 kDa protein which shows homology through a C-terminal domain to the PI-3-K protein family (Savitsky et al., 1995; Meyn, 1999). Other proteins that share such an homology with PI-3-K in their C-terminal domain (e.g. DNA PKcs, mTOR and FRAP) are involved in control of the cell cycle or in the response to DNA damage (Hoekstra, 1997).
Here we describe four new Chinese hamster cell mutants isolated on the basis of their camptothecin sensitivity. Three of these new mutants are members of the irs2 (XRCC8) complementation group, demonstrating a greater phenotypic heterogeneity than previously encountered within this group. These new mutants are likely to prove valuable in future studies aimed at understanding the function and elucidating the role played by XRCC8 in DNA repair, the cell cycle and DNA damage response mechanisms.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Cells and culture conditions
The parental wild-type cell line V79 and the mutants irs1 and irs2 have been described previously (Jones et al., 1987
). EM9 (Thompson et al., 1980) was kindly provided by Dr David Busch. Cells were routinely maintained in Dulbecco's modified Eagle's medium (D-MEM) (Gibco) with Glutamax, supplemented with 10% fetal calf serum, 100 U/ml penicillin and 100 mg/ml streptomycin sulfate (Gibco). Cells were grown at 37°C under 5% CO2. Trypsinization was performed with 0.12% trypsin and 0.008% EDTA (TE).
Mutant isolation
Isolation of camptothecin-sensitive mutants of V79 was performed using a modification of a replica microwell system previously described by Jones et al. (1987). This improved technique was designed to be less labour intensive and less subjective than the original method and has been described in detail elsewhere (Johnson and Jones, 1999).
Briefly, V79 cells were mutagenized with 10 mM ENU for 30 min at 37°C. Upon trypsinization, independent populations of ~1000 survivors were set up, grown for 5–6 days for mutant expression and aliquots stored in liquid nitrogen. Mutagenesis was assessed at the hypoxanthine phosphoribosyltransferase (HPRT) locus by determining the frequency of 6-thioguanine-resistant mutants (HPRT–) and was >1 mutant/103 survivors.
For each screening experiment mutagenized cells were spread into Petri dishes, incubated for 7 days, colonies picked in 20 µl medium using an auto-pipette and transferred to the wells of a 96-well microtitre plate (Nunc), followed by addition of 180 µl of fresh medium and incubation overnight. Colonies were then dispersed in 40 µl of trypsin/EDTA with mechanical agitation using an eight channel pipetter and reincubated in fresh medium. Once confluent, wells were trypsinized with 200 µl TE and the cells dispersed and 25 µl of the cell suspension transferred to the corresponding wells of two further microwell plates, one of which acted as a control (no chemical added) whilst the other replica was treated with 40 nM camptothecin (a dose determined not to significantly inhibit growth of the parental line V79). Plates were incubated and wells were scored for growth on day 3 and then again on day 5, with one final check on day 7, before candidate clones were selected for re-testing. Cells with no growth or strongly reduced growth in camptothecin, when compared with the control microwell, were considered as potential sensitive clones. These were recovered from the control plate and re-tested. In the re-test potentially mutant clones were tested with a range of camptothecin doses alongside wild-type V79 cultures (Johnson and Jones, 1999).
Survival curves and drug treatments
These were performed as previously described (Johnson and Jones, 1999). Exponentially growing cells were plated into 9 cm Petri dishes and, following 2 h for attachment, chemical added. Five control dishes without chemical were prepared at 200 cells/dish, those with chemical were prepared in triplicate with increasing cell numbers used at higher doses. Camptothecin, etoposide, streptonigrin adriamycin, bleomycin and mitomycin C (MMC) were obtained from Sigma. After 7–10 days to allow for colony formation, dishes were fixed, stained and colony numbers determined. Each survival curve represents the mean of a minimum of three experiments and the data were fitted to a semi-log plot and sensitivity quantified by determining D37 (dose required to reduce survival to 37% of control) for each mutant.
-Ray treatments
Cells were trypsinized, resuspended and irradiated in growth medium in plastic bijou bottles with 137Cs -rays in a blood irradiator (CIS UK Bio-International, High Wycombe, UK), to produce a dose rate of ~4 Gy/min. The cell suspension was diluted appropriately to give ~100 survivors/plate and seeded into 6 cm diameter tissue culture Petri dishes with 5 ml medium. Radiation doses were checked by a modified ferrous sulfate method (Frankenberg, 1969). Control (unirradiated) samples were similarly diluted and seeded in dishes. Cells were incubated at 37°C in a humidified incubator with 5% CO2 for 7–10 days.
Cell fusion, hybrid formation and selection of TOR clones
Hybrids were formed between pairs of cell lines using the thioguanine/ouabain-resistant (TOR) hybridization and hypoxanthine/azaserine/thymidine (HAT)/ouabain selection system previously described (Thacker, 1981; Jones et al., 1988; Jones, 1994). TOR clones of camptothecin-sensitive cell lines were isolated by selecting spontaneous mutants as previously described (Jones, 1994), whilst irs1TOR and irs2TOR were isolated by Jones et al. (1988). All TOR cell lines were shown to exhibit a response to camptothecin which was similar to its respective unmarked parent.
Cell fusion was performed using polyethylene glycol (PEG) 1000 at 50% (w/v) in Hank's balanced salt solution in 0.15 M HEPES, pH 7.6. One million cells of each cell line to be fused (one TOR line, one unmarked) were seeded in a 90 mm dish (self-cross controls were seeded with 2 000 000 cells) and incubated for 24 h. Following incubation, dishes were treated with 5 ml PEG for 1 min, rinsed three times with Hank's balanced salt solution and replaced with fresh growth medium for another 24 h to allow recovery and hybrid formation. Cells were trypsinized, counted and re-spread at 2x105 in a 90 mm dish (three replicates) and an 80 cm2 flask in growth medium containing HAT/ouabain (100 µM hypoxanthine, 40 µM azaserine, 16 µM thymidine and 1 mM ouabain). Only hybrid cells formed between a TOR cell line and an unmarked line are able to survive in this selective medium. The self-crosses will not survive in HAT/ouabain and will ensure that mutations to ouabain resistance (in unmarked cell lines) or reverse mutation to HAT resistance/6-thioguanine sensitivity (in TOR lines) occur at very low frequency, when compared with hybrid formation, and ensure cell line status. Dishes/flasks were incubated for 7–10 days to allow for colony formation. Dishes were fixed, stained and colony number determined. Colonies in the 80 cm2 flask were trypsinized to form a pooled population of hybrids and grown for a further 2–3 days in HAT/ouabain medium. Survival responses of the hybrid populations were then determined as described above.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Isolation of mutants
In total, 732 clones from the mutagenized V79 populations were screened for their sensitivity to camptothecin. Twenty-three putative mutants were re-tested for camptothecin sensitivity as described above. Survival curves were constructed for six of these, which were designated CM1–CM6. In Figure 1
, the responses to camptothecin of V79 and four of the mutants are shown. CM1, CM2, CM3 and CM6 exhibit 8.2-, 3.4-, 4.3- and 7.4-fold greater sensitivity to camptothecin, respectively, compared with V79. CM4 and CM5 exhibited only slight sensitivity to camptothecin (1.6- and 1.8-fold, respectively) and were not studied in any further detail (data not shown).
|
Hybridization and complementation analysis
The responses to camptothecin in the form of D37 values for all cell lines used for complementation analysis are summarized in Table I
. These sensitivities were established from full survival curves. Responses of TOR cell lines were very similar to their unmarked counterparts.
|
The frequency with which hybrids were generated was between 5.1x10–4 and 1.5x10–3 and is consistent with previous reports (Jones et al., 1988
; Jones, 1994). Reverse mutation to HAT resistance or mutation to ouabain resistance was not measured directly, however, no revertant/mutant colonies were seen on the self-cross control plates in any of the hybridization experiments conducted.
The ionizing radiation-sensitive phenotypes of irs1, irs2 and EM9 have previously been shown to be recessive in nature (Jones et al., 1988). The camptothecin sensitivities of irs2, V-C4 and V-E5 have also been shown to be recessive in nature (Jones et al., 1993). The recessiveness of the CM mutants was not formally checked by crossing them with V79, although it can be inferred that CM1, CM3 and CM6 are recessive since crosses between irs1TOR and CM1, CM3 and CM6 all produced hybrids which exhibited a survival response to camptothecin similar to that of the wild-type parent V79 (Table I
and Figure 2a for irs1TORxCM6). CM1TOR was crossed with CM3 and CM6 to determine the genetic relationship between the mutants. In all hybrid populations (i.e. CM1TORxCM3 and CM1TORxCM6) a clear mutant phenotype was observed with respect to camptothecin sensitivity (Table I), therefore, CM1, CM3 and CM6 belong to the same complementation group. However, it should be noted that CM1, CM3 and CM6 were isolated from three separate independent populations of mutagenized V79 cells and therefore represent three independent mutations.
|
To determine whether CM1, CM3 and CM6 belong to an existing complementation group or represent a new group of camptothecin-sensitive mutants they were crossed with irs1, irs2 and EM9. The hybrids irs1TORxCM1, irs1TORxCM3 (Figure 2a
) and irs1TORxCM6 all displayed a response to camptothecin similar to that of V79, indicating genetic complementation (Table I). The marked cell line CM1TOR was fused with the camptothecin-sensitive line EM9 and the survival response to camptothecin determined. Again, the response was similar to that of V79, establishing genetic complementation between the mutants (Table I). Finally, the mutants CM3 and CM6 were crossed with irs2TOR and CM1TOR crossed with irs2. All three hybrid populations exhibited a mutant phenotype with respect to camptothecin sensitivity (Table I and Figure 2b for irs2TORxCM3), clearly defining CM1, CM3 and CM6 as new members of the irs2 (XRCC8) complementation group.
The results of crosses between CM2 and irs1TOR and CM2 and CM1TOR (Table I and Figure 2c for irs1TORxCM2) indicated that the camptothecin-sensitive phenotype seen in CM2 may not be recessive in nature. To determine if CM2 displayed a semi-dominant phenotype, CM2TOR was crossed with the parental line V79 and the survival response to camptothecin tested. The hybrid population CM2TORxV79 exhibited an intermediate survival response to camptothecin with respect to V79 and CM2TOR (Figure 2c), establishing that CM2 does indeed possess a semi-dominant phenotype.
Figure 2 shows examples of the camptothecin response of a complementing hybrid (irs1TORxCM6), a non-complementing hybrid (irs2TORxCM3) and hybrids with intermediate sensitivity (irs1TORxCM2 and CM2TORxV79).
Sensitivity to topo II inhibitors and other genotoxic agents
The four CM mutants exhibited much less sensitivity to topo II inhibitors than they did to camptothecin (Table II). CM6 was the most sensitive of the mutants to the intercalating anthracycline adriamycin (2.1-fold), whilst CM2 exhibited the greatest sensitivity to the non-intercalating epipodophyllotoxin etoposide (2.2-fold). In contrast, CM1, CM3 (both 3.8-fold) and CM6 (2.5-fold) exhibited marked sensitivity to streptonigrin (Figure 3a). Streptonigrin is a phenyl pyridylquinoline that can undergo bioactivation by DT-diaphorase to generate hydroxyl radicals that have also been shown to be an inhibitor of topo II, causing cleavage at sites different from other topo II inhibitors (Capranico et al., 1994; Beall et al., 1996). Each of the four cell lines displayed increased sensitivity to MMC. CM3 exhibited the most pronounced increase, being 5.1-fold more sensitive than wild-type, whilst CM1, CM2 and CM6 were all between 2.4- and 3.5-fold more sensitive (Figure 3b). Given that CM1, CM3 and CM6 were assigned to the XRCC8 (irs2) complementation group, their response to ionizing radiation was unexpected. All three showed only a marginal increase in sensitivity to -irradiation (Figure 4). The survival responses of CM1 and CM6 were similar, whilst the CM3 curve had a larger shoulder than these two mutants. On the basis of D37 values only, CM1 was 1.6-fold and CM6 was 1.3-fold sensitive, whilst the D37 of CM3 was the same as V79 due to the pronounced shoulder in the response of this mutant (Table II). Similar small increases in sensitivity were observed for the radiomimetic drug bleomycin (Table II) for mutants CM1 (1.7-fold sensitive), CM3 (1.3-fold) and CM6 (1.5-fold).
|
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
In this study we describe the isolation and initial characterization of camptothecin-sensitive mutants of V79 Chinese hamster cells. Extensive genetic analysis of the mutants CM1, CM2, CM3 and CM6 was undertaken in order to establish the genetic relationships both within these mutants and with existing camptothecin-sensitive Chinese hamster cell mutants. CM1, CM3 and CM6 were shown to belong to the same complementation group and each of the three complemented the XRCC2 mutant irs1, which is cross-sensitive to camptothecin (Jones et al., 1987
, 1993). Hybrid populations between CM1, CM3 or CM6 and irs1TOR all displayed a response to camptothecin similar to wild-type (Table I and Figure 2a), as did a hybrid formed between CM1TOR and the XRCC1 mutant EM9 (Thompson et al., 1980), indicating genetic complementation. CM1, CM3 and CM6 were crossed with the ionizing radiation- and camptothecin-sensitive mutant irs2 (Jones et al., 1987, 1993; Thacker and Ganesh, 1990). All three hybrid populations exhibited a camptothecin-sensitive phenotype and thus a lack of genetic complementation (Table I and Figure 2b), establishing CM1, CM3 and CM6 as new members of the irs2 (XRCC8) complementation group.
The irs2 (XRCC8) group also contains three other Chinese hamster cell mutants, designated V-C4, V-E5 and V-G8, which were isolated on the basis of sensitivity to ionizing radiation (Zdzienicka and Simons, 1987; Zdzienicka et al., 1989; Thacker and Wilkinson, 1991). Camptothecin sensitivity appears to be a general feature of the irs2 complementation group (Thacker and Ganesh, 1990; Jones et al., 1993). It was previously suggested that the same genetic alteration that confers ionizing radiation sensitivity also confers sensitivity to camptothecin in these mutants as there is a lack of complementation in hybrids formed between irs2 and both V-C4 and V-E5 when tested for camptothecin sensitivity (Jones et al., 1993). The current study, with its isolation of three new mutants, confirms that XRCC8 mutants exhibit a high degree of camptothecin sensitivity. Given the isolation of CM1, CM3 and CM6 here and the previous isolation of irs2, V-C4, V-E5 and V-C8 from V79 cells, it is evident that XRCC8 mutants are obtained at very high frequency from this cell line. This most likely reflects the degree of hemizygosity displayed by the V79 Chinese hamster cell line (Thacker, 1981; Zdzienicka and Simons, 1987).
Genetic analysis of the mutant line CM2 produced ambiguous results. In crosses with irs1TOR and CM1TOR (mutants established to be in different complementation groups in this study) hybrids showed intermediate sensitivity (Table I and Figure 2c). The lack of full complementation between CM2 and mutants from distinct complementation groups indicate that CM2 may possess a semi-dominant phenotype. This was confirmed in a CM2TORxV79 hybrid in which an intermediate result was obtained (Figure 2c), contrasting with the expected full complementation between a recessive mutant and its parent line (Jones et al., 1988; Thacker and Wilkinson, 1991; Jones, 1994). Semi-dominance has previously been observed in Chinese hamster mutants. It was shown that irs1SF (XRCC3) when crossed with EM7 (XRCC1) or the parental line AA8 exhibited a semi-dominant phenotype with respect to ionizing radiation sensitivity (Thacker and Wilkinson, 1991). More importantly, the phenomenon of semi-dominance in Chinese hamster DNA repair mutants has been shown in the XRCC8 mutants V-C4, V-E5 and V-G8 with respect to radiation-resistant DNA synthesis (RDS), although this did not extend to radiation sensitivity (Verhaegh et al., 1993). Therefore, the possibility exists that the CM2 line may represent another XRCC8 group member which exhibits a semi-dominant phenotype, but in this instance with respect to camptothecin sensitivity. This is further suggested by the observation that the hybrid irs2TORxCM2 displays a sensitivity to camptothecin (D37 = 15 nM) very similar to the CM2 phenotype (D37 = 15.6 nM). This possibility is at least consistent with the high frequency of XRCC8 mutants obtained from V79.
The newly identified XRCC8 mutants CM1, CM3 and CM6 exhibit a pleiotropic response to a range of DNA-damaging agents. A significant observation was the relatively minor increase in ionizing radiation sensitivity and bleomycin sensitivity in CM1, CM3 and CM6 when compared with other XRCC8 mutants like irs2 (Table III). Marked sensitivity to ionizing radiation was considered to be central to the XRCC8 group, as all the existing mutants had been isolated on this basis (Jones et al., 1987; Zdzienicka and Simons, 1987). Also, it has been proposed that XRCC8 mutants may represent hamster homologues of the human genetic disorder AT, whose cultured cells always display significant ionizing radiation and bleomycin sensitivity.
|
In response to genotoxic agents other than ionizing radiation, CM1, CM3 and CM6 are similar to the other XRCC8 mutants in that they exhibit a somewhat varied sensitivity. Table III
summarizes the response of all the XRCC8 group mutants to the seven DNA-damaging agents used in this study. For example, CM3 shows a greater sensitivity to MMC than any other XRCC8 mutant and in this respect CM3, together with CM1 and CM6, are like V-C4 and V-E5, whereas irs2 and V-G8 show no MMC sensitivity (Jones et al., 1987; Zdzienicka and Simons, 1987). However, the responses to the topo II inhibitors adriamycin and etoposide shown by CM1, CM3 and CM6 are similar to irs2, whereas V-C4, V-E5 and V-G8 display a significant hypersensitivity to these agents (Jongmans et al., 1993a; Table III). All XRCC8 mutants appear to possess a streptonigrin-sensitive phenotype (Jones et al., 1993; Jongmans et al., 1993a), although V-E5 does appear to be unusually sensitive to this agent in comparison with the other mutants (Table III). Therefore, there is clear phenotypic heterogeneity both within the CM mutants and within the XRCC8 group. However, all the mutants display significant sensitivity to camptothecin (
4-fold) regardless of whether they were isolated as X-ray-sensitive or camptothecin-sensitive, and perhaps this should now be regarded as the defining characteristic of XRCC8 mutants.
In certain respects XRCC8 mutants exhibit characteristics similar to human AT cell lines, including the chromosomal instability, normal strand break repair and radiation-resistant DNA synthesis observed in irs2, V-C4, V-E5 and V-G8 (Zdzienicka et al., 1989; Jones et al., 1990, 1993; Thacker and Ganesh, 1990; Bryant et al., 1993). Nevertheless, Jongmans et al. (1993b) demonstrated that human chromosome 11 did not complement V-E5 or V-G8, although it was later shown that mouse chromosome 9, carrying the murine ATM gene, did complement these mutants (Jongmans et al., 1996). Recent cloning of the ATM gene (Savitsky et al., 1995; Zhang,N. et al., 1997) will allow the XRCC8 mutants to be checked using a number of biological end points for molecular complementation by expression of ATM cDNA (Zhang,N. et al., 1997). Another possibility is that the gene defective in XRCC8 mutants may be homologous to the gene defective in the phenotypically similar Nijmegen break syndrome (NBS) or may operate in the same pathway as the ATM and NBS genes. Like AT cells and hamster XRCC8 cells, NBS cells are X-ray sensitive (2-fold), display RDS and were recently reported to be 3-fold sensitive to camptothecin (KraakmanvanderZwet et al., 1999). The availability of the ATM gene and other genes such as NBS and Mre11 should now allow one of these possibilities to be confirmed or eliminated, either by complementation analyses following cDNA transfection or northern analysis in the mutants. It is interesting to note that AT cell lines have been reported to have mixed responses to DNA-damaging agents other than ionizing radiation and that a semi-dominant/dominant phenotype has been proposed for AT (Lehmann, 1982; Morgan et al., 1997; Meyn, 1999). Molecular cloning of the XRCC8 gene is vital to a further understanding of the complex AT/NBS-like phenotype exhibited by the XRCC8 mutants. The XRCC8 gene may be novel or it may be a new member of the PI-3-K proteins, like ATM. Nevertheless, understanding how genotype determines the cellular phenotype in these phenotypically varied mutants can only be achieved through molecular analysis of both the nature and position of the mutations in the XRCC8 gene for each of the mutants. Thus, the three, possibly four, new XRCC8 mutants reported here, together with the four previously isolated mutants, represent a valuable resource with which to gain insight into one of the most important aspects of the mammalian cell response to different DNA-damaging agents.
|
Acknowledgments |
|---|
We are grateful to Kim Krishnan for performing the bleomycin survivals and to Professor Steve Kemp for his comments on the manuscript. This work was supported by project grants from the North West Cancer Research Fund (NWCRF-CR386 and NWCRF-CR527) to N.J.J., the award of a BBSRC research studentship to M.A.J. and a Liverpool University Research Development Fund grant.
|
Notes |
|---|
2 Present address: MRC Laboratory of Molecular Biology, Cambridge CB2 2QH, UK
3 To whom correspondence should be addressed. Tel: +44 151 794 3628; Fax: +44 151 794 3655; Email: njjones{at}liv.ac.uk
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
-
Beall,H.D., Liu,Y., Siegel,D., Bolton,E.M., Gibson,N.W. and Ross,D. (1996) Role of NAD(P)H:quinone reductase (DT-diaphorase) in cytotoxicity and induction of DNA damage by streptonigrin. Biochem. Pharmacol., 51, 645–652.[Web of Science][Medline]
Bendixen,C., Thomsen,B., Alsner,J. and Westergaard,O. (1990) Camptothecin-stabilized topoisomerase I-DNA adducts cause premature termination of transcription. Biochemistry, 29, 5613–5619.[Medline]
Boothman,D.A., Trask,D.K and Pardee,A.B. (1989) Inhibition of potentially lethal DNA damage repair in human tumor cells by ß-lapachone, an activator of topoisomerase I. Cancer Res., 49, 605–612.
Boothman,D.A., Fukunaga,N. and Wang,M. (1994) Down-regulation of topoisomerase I in mammalian cells following ionizing radiation. Cancer Res., 54, 4618–4626.
Bryant,P.E., Jones,N.J. and Liu,N. (1993) Radiosensitive Chinese hamster irs2 cells show enhanced chromosomal sensitivity to ionizing radiation and restriction endonuclease induced blunt-ended double-strand breaks. Mutagenesis, 8, 141–147.
Caldecott,K.W., Mekeown,C.K., Tucker,J.D., Ljungquist,S. and Thompson,L.H. (1994) An interaction between the mammalian DNA repair protein XRCC1 and DNA ligase III. Mol. Cell. Biol., 14, 68–76.
Capranico,G., Palumbo,M., Tinelli,S. and Zunino,F. (1994) Unique sequence specificity of topoisomerase II cleavage stimulation and DNA binding mode of streptonigrin. J. Biol. Chem., 269, 25004–25009.
Collins,A.R. (1993) Mutant rodent cell lines sensitive to ultraviolet light, ionizing radiation and cross-linking agents: a comprehensive survey of genetic and biochemical characteristics. Mutat. Res., 293, 99–118[Web of Science][Medline]
Crumplin,G.C. (1981) The involvement of DNA topoisomerase in DNA repair and mutagenesis. Carcinogenesis, 2, 157–160.
Degrassi,F., De Salvia,R., Tanzarella,C. and Palitti,F. (1989) Induction of chromosomal aberrations and SCE by camptothecin, an inhibitor of mammalian topoisomerase I. Mutat. Res., 211, 125–130.[Web of Science][Medline]
DeLaat,W.C., Jaspers,N.G.J. and Hoeijmakers,J.H.J. (1999) Molecular mechanism of nucleotide excision repair. Genes Dev., 7, 768–785.
Del Bino,G., Lassota,P. and Darzynkiewicz,Z. (1991) The S-phase cytotoxicity of camptothecin. Exp. Cell Res., 193, 27–35.[Web of Science][Medline]
Fleischmann,G., Pflugfelder,G., Steiner,E.K., Javaherian,K., Howard,G.C., Wang,J.C. and Elgin,S.C.R. (1984) Drosophila DNA topoisomerase I is associated with transcriptionally active regions of the genome. Proc. Natl Acad. Sci. USA, 81, 6958–6962.
Frankenberg,D. (1969) A ferrous sulphate dosemeter independent of photon energy in the range from 25 keV up to 59 MeV. Phys. Med. Biol., 14, 597–605.[Web of Science][Medline]
Froelich-Ammon,S.J. and Osheroff,N. (1995) Topoisomerase poisons—harnessing the dark side of enzyme mechanism. J. Biol. Chem., 270, 21429–21432.
Gobert,C., Bracco,L., Rossi,F., Olivier,M., Tazi,J., Lavelle,F., Larsen,A.K. and Riou,J.F. (1996) Modulation of DNA topoisomerase I activity by p53. Biochemistry, 35, 5778–5786.[Medline]
Hickson,I.D., Davies,S.L., Davies,S.M. and Robson,C.N. (1990) DNA repair in radiation sensitive mutants of mammalian cells: possible involvement of DNA topoisomerases. Int. J. Radiat. Biol., 58, 561–568.[Web of Science][Medline]
Hoekstra,M.F. (1997) Responses to DNA damage and regulation of cell-cycle checkpoints by the ATM protein kinase family. Curr. Opin. Genet. Dev., 7, 170–175.[Web of Science][Medline]
Holm,C., Covey,J.M., Kerrigan,D. and Pommier,Y. (1989) Differential requirement of DNA replication for the cytotoxicity of DNA topoisomerase I and II inhibitors in Chinese hamster DC3F cells. Cancer Res., 49, 6365–6368.
Horwitz,S.B., Chang,C.K. and Grollman,A.P. (1971) Studies on camptothecin. I. Effects of nucleic acid and protein synthesis. Mol. Pharmacol., 7, 632–644.
Hsiang,Y.H. and Liu,L.F. (1988) Identification of mammalian DNA topoisomerase I as an intracellular target of the anticancer drug camptothecin. Cancer Res., 48, 1722–1726.
Hsiang,Y.H., Hertzberg,R., Hecht,S. and Liu,L.F. (1985) Camptothecin induces protein-linked DNA-breaks via mammalian DNA topoisomerase I. J. Biol. Chem., 260, 14873–14878.
Hsiang,Y.H., Lihou,M.G. and Liu,L.F. (1989) Arrest of replication fork by drug-stabilized topoisomerase I-DNA cleavable complexes as a mechanism of cell killing by camptothecin. Cancer Res., 49, 5077–5082.
Jeggo,P.A. (1997) DNA-PK: at the cross-roads of biochemistry and genetics. Mutat. Res. DNA Repair, 384, 1–14.
Johnson,M.A. and Jones,N.J. (1999) The isolation and genetic analysis of V79-derived etoposide sensitive Chinese hamster cell mutants: two new complementation groups of etoposide sensitive mutants. Mutat. Res. DNA Repair, 435, 271–282.
Johnson,R.D., Liu,N. and Jasin,M. (1999) Mammalian XRCC2 promotes the repair of DNA double-strand breaks by homologous recombination. Nature, 401, 397–399.[Medline]
Jones,N.J. (1994) Genetic analysis of mitomycin C hypersensitive Chinese hamster ovary cell mutants. Mutagenesis, 9, 477–482.
Jones,N.J., Cox,R. and Thacker,J. (1987) Isolation and cross-sensitivity of X-ray-sensitive mutants of V79-4 hamster cells. Mutat. Res., 254, 125–133.
Jones,N.J., Cox,R. and Thacker,J. (1988) Six complementation groups for ionizing-radiation sensitivity in Chinese hamster cells. Mutat. Res., 193, 139–144.[Web of Science][Medline]
Jones,N.J., Stewart,S.A. and Thompson,L.H. (1990) Biochemical and genetic analysis of the Chinese hamster mutants irs1 and irs2 and their comparison to cultured ataxia telangiectasia cells. Mutagenesis, 5, 15–23.
Jones,N.J., Ellard,S., Waters,R. and Parry,E.M. (1993) Cellular and chromosomal hypersensitivity to DNA crosslinking agents and topoisomerase inhibitors in the radiosensitive Chinese hamster irs mutants: phenotypic similarities to ataxia telangiectasia and Fanconi's anaemia cells. Carcinogenesis, 14, 2487–2494.
Jongmans,W., Verhaegh,G.W.C.T., Sankaranarayanan,K., Lohman,P.H.M. and Zdzienicka,M.Z. (1993a) Cellular characteristics of Chinese hamster cell mutants resembling ataxia telangiectasia cells. Mutat. Res., 294, 207–214.[Web of Science][Medline]
Jongmans,W., Wiegant,J., Oshimura,M., James,M.R., Lohman,P.H.M. and Zdzienicka,M.Z. (1993b) Human chromosome 11 complements ataxia telangiectasia cells but does not complement the defect in AT-like Chinese hamster cell mutants. Hum. Genet., 92, 259–264.[Web of Science][Medline]
Jongmans,W., Verhaegh,G.W.C.T., Jaspers,N.G.J., Demant,P., Natarajan,A.T., Shiloh,Y., Oshimura,M., Stanbridge,E.J., Athwal,R.S., Cuthbert,A.P., Newbold,R.F., Lohman,P.H.M. and Zdzienicka,M.Z. (1996) The defect in the AT-like hamster cell mutants is complemented by mouse chromosome 9 but not by any of the human chromosomes. Mutat. Res. DNA Repair, 364, 91–102.
Kaufmann,W.K., Boyer,J.C., Estabrooks,L.L. and Wilson,S.J. (1991) Inhibition of replicon initiation in human cells following stabilization of topoisomerase-DNA cleavable complexes. Mol. Cell. Biol., 11, 3711–3718.
KraakmanvanderZwet,M., Overkamp,W.J.I., Friedl,A.A., Klein,B., Verhaegh, G.W.C.T., Jaspers,N.G.T., Midro,A.T., EckardtSchupp,F., Lohman,P.H.M. and Zdzienicka,M.Z. (1999) Immortalization and characterization of Nijmegen Breakage Syndrome fibroblasts. Mutat. Res. DNA Repair, 434, 17–27.
Kretzschmar,M., Meisterernst,M. and Roeder,R.G. (1993) Identification of human DNA topoisomerase I as a cofactor for activator-dependent transcription by RNA polymerase II. Proc. Natl Acad. Sci. USA, 90, 11508–11512.
Lehmann,A.R. (1982) The cellular and molecular responses of ataxia-telangiectasia cells to DNA damage. In Bridges,B.A. and Harnden,D.G. (eds) Ataxia-Telangiectasia. Wiley, New York, NY, pp. 83–101.
Liu,N., Lamerdin,J.E., Tebbs,R.S., Schild,D., Tucker,J.D., Shen,R., Brookman,K.W., Siciliano,M.J., Fan,C.A., Narayana,L.S., Zhou,Z.Q., Anderson,A.W., Sorensen,K.J., Chen,D.J., Jones,N.J. and Thompson,L.H. (1998) XRCC2 and XRCC3, new members of the Rad51 family, promote chromosome stability and protect against DNA cross-links and other damages. Mol. Cell, 1, 783–793.[Web of Science][Medline]
Merino,A., Madden,K.R., Lane,W.S., Champoux,J.J. and Reinberg,D. (1993) DNA topoisomerase I is involved in both repression and activation of transcription. Nature, 365, 227–232.[Medline]
Meyn,M.S. (1999) Ataxia-telangiectasia, cancer and the pathobiology of the ATM gene. Clin. Genet., 55, 289–304.[Web of Science][Medline]
Morgan,S.E., Lovly,C., Pandita,T.K., Shiloh,Y. and Kastan,M.B. (1997) Fragments of ATM which have dominant-negative or complementing activity. Mol. Cell. Biol., 17, 2020–2029.[Abstract]
Nitiss,J.L. and Wang,J.C. (1996) Mechanisms of cell killing by drugs that trap covalent complexes between DNA topoisomerases and DNA. Mol. Pharmacol., 50, 1095–1102.[Abstract]
Pommier,Y. and Bertrand,R. (1993) The mechanisms of formation of chromosomal aberrations: role of eukaryotic DNA topoisomerases. In Kirsch,I.R. (ed.) The Causes and Consequences of Chromosomal Aberrations. CRC Press, London, UK, pp. 277–297.
Ryan,A.J., Squires,S., Strutt,H. and Johnson,R.T. (1991) Camptothecin cytotoxicity in mammalian cells is associated with the induction of persistent double-strand breaks in replicating DNA. Nucleic Acids Res., 19, 3295–3300.
Savitsky,K., Barshira,A., Gilad,S., Rotman,G., Ziv,Y., Vanagaite,L., Tagle,D.A, Smith,S., Uziel,T., Sfez,S., Ahkenazi,M., Pecker,I., Frydman,M., Harnik,R., Patanjali,S.R., Simmons,A., Clines,G.A., Sartiel,A., Gatti,R.A., Chessa, L, Sanal,O., Lavin,M.F., Jaspers,N.G.J., Malcolm,A., Taylor,R., Arlett,C.F., Miki,T., Weissman,S.M., Lovett,M., Collins,F.S. and Shiloh,Y. (1995) A single ataxia telangiectasia gene with a product similar to PI 3-kinase. Science, 268, 1749–1753.
Smith,P.J., Makinson,T.A. and Watson,J.V. (1989) Enhanced sensitivity to camptothecin in ataxia-telangiectasia cells and its relationship with the expression of DNA topoisomerase I. Int. J. Radiat. Biol., 55, 217–231.[Web of Science][Medline]
Subramanian,D., Rosenstein,B.S. and Muller,M.T. (1998) Ultraviolet-induced DNA damage stimulates topoisomerase I-DNA complex formation in vivo: possible relationship with DNA repair. Cancer Res., 58, 976–984.
Tebbs,A.M.R., Zhao,Y., Tucker,J.D., Scheerer,J.B., Siciliano,M.J., Hwang,N., Liu,N., Legerski,R.J. and Thompson,L.H. (1995) Correction of chromosomal instability and sensitivity to diverse mutagens by cDNA of the XRCC3 DNA repair gene. Proc. Natl Acad. Sci. USA, 92, 6354–6358.
Thacker,J. (1981) The chromosomes of a V79 hamster line and a mutant subline lacking HPRT activity. Cytogenet. Cell Genet., 29, 16–25.[Web of Science][Medline]
Thacker,J. and Ganesh,A.N. (1990) DNA-break repair, radioresistance of DNA synthesis and camptothecin sensitivity in the radiation-sensitive irs mutants: comparisons to ataxia telangiectasia cells. Mutat. Res., 235, 49–58.[Web of Science][Medline]
Thacker,J. and Wilkinson,R.E. (1991) The genetic basis of resistance to ionizing radiation damage in cultured mammalian cells. Mutat. Res., 254, 135–142.[Web of Science][Medline]
Theilmann,H.W., Popanda,O., Gersbach,H. and Gilberg,F. (1993) Various inhibitors of DNA topoisomerases diminish repair-specific DNA incision in UV-irradiated human fibroblasts. Carcinogenesis, 14, 2341–2351.
Thompson,L.H. (1998) Chinese hamster cells meet DNA repair: an entirely acceptable affair. Bioessays, 20, 589–597.[Web of Science][Medline]
Thompson,L.H. and Jeggo,P.A. (1995) Nomenclature of human genes involved in ionizing radiation sensitivity. Mutat. Res., 337, 131–134.[Web of Science][Medline]
Thompson,L.H., Rubin,J.S., Cleaver,J.E., Whitmore,G.F. and Brookman,K. (1980) A screening method for isolating DNA repair-deficient mutants of CHO cells. Somat. Cell Genet., 6, 391–405.
Trask,D.K. and Muller,M.T. (1988) Stabilization of type I topoisomerase-DNA covalent complexes by actinomycin D. Proc. Natl Acad. Sci. USA, 85, 1417–1421.
Verhaegh,G.W.C.T., Jaspers,N.G.J., Lohman,P.H.M. and Zdzienicka,M.Z. (1993) Co-dominance of radioresistant DNA synthesis in a group of AT-like Chinese hamster cell mutants. Cytogenet. Cell Genet., 63, 176–180.[Web of Science][Medline]
Wang,J.C. (1985) DNA topoisomerases. Annu. Rev. Biochem., 54, 665–697.[Web of Science][Medline]
Zdzienicka,M.Z. and Simons,J.W.I.M. (1987) Mutagen-sensitive cell lines are obtained with a high frequency in V79 Chinese hamster cells. Mutat. Res., 178, 235–244.[Web of Science][Medline]
Zdzienicka,M.Z., Jaspers,N.G.J., Van der Schans,G.P., Natarajan,A.T. and Simons,J.W.I.M. (1989) Ataxia telangiectasia-like Chinese hamster cell mutants with radioresistant DNA synthesis, chromosomal instability and normal DNA strand break repair. Cancer Res., 49, 1481–1485.
Zhang,H., Wang,J.C. and Liu,L.F. (1988) Involvement of DNA topoisomerase I in transcription of human ribosomal RNA genes. Proc. Natl Acad. Sci. USA, 85, 1060–1064.
Zhang,N., Chen,P., Khanna,K.K., Scott,S., Gatei,M., Kozlov,S., Watters,D., Spring,K., Yen,T. and Lavin,M.F. (1997) Isolation of full-length ATM cDNA and correction of the ataxia-telangiectasia cellular phenotype. Proc. Natl Acad. Sci. USA, 94, 8021–8026.
Received on March 20, 2000; accepted on April 3, 2000.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  C. Liu, J. J. Pouliot, and H. A. Nash Repair of topoisomerase I covalent complexes in the absence of the tyrosyl-DNA phosphodiesterase Tdp1 PNAS, November 12, 2002; 99(23): 14970 - 14975. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. B. Wilson, M. A. Johnson, A. P. Stuckert, K. L. Trueman, S. May, P. E. Bryant, R. E. Meyn, A. D. D'Andrea, and N. J. Jones The Chinese hamster FANCG/XRCC9 mutant NM3 fails to express the monoubiquitinated form of the FANCD2 protein, is hypersensitive to a range of DNA damaging agents and exhibits a normal level of spontaneous sister chromatid exchange Carcinogenesis, December 1, 2001; 22(12): 1939 - 1946. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||