Introduction / 8 1.1.3. Cytogenetics
Spontaneous chromosomal aberrations
Chrom osom al instability, both spontaneous or induced by ionizing radiation and radiom im etic agents such as bleom ycin, is a universal characteristic of AT. Spontaneously occurring chrom osom al abnorm alities arise at higher frequencies in peripheral blood lym phocytes as well as in fibroblasts of AT than norm al individuals (Hecht et al 1966, Oxford et al 1975, Cohen et al 1975, Taylor 1982). However, established lym phoblastoid cell lines are reported to show a norm al level of spontaneous aberrations (Cohen et al 1979, Cohen and Simpson 1980). Spontaneous chromosomal aberrations include breaks, gaps and interchanges of both chromosome- and c h ro m a tid -ty p e an d sy m m etrical ch ro m o so m al re a rra n g e m e n ts (translocations). In addition, telomeric dicentric chrom osom es, form ed by an end-to-end fusion of telomeres of two chromosomes, have been noted to occur at a high frequency in AT cells (Hayashi and Schmid 1975, Oxford et al 1975, Taylor et al 1981). The spontaneous frequency of sister chrom atid exchanges (SCE) has been found to be norm al in both lym phocytes of AT individuals (Galloway and Evans 1975, Hatcher et al 1976, Batram et al 1976, H ayashi and Schmid 1975) and AT lym phoblastoid cell lines (Cohen and Simpson 1982).
Com paring AT w ith other chromosome instability syndrom es, e.g.. Bloom's syndrom e (BS) and Fanconi's anaem ia (FA), there appears to be an obvious difference concerning the predom inant type of spontaneous aberration: BS and FA individuals show a high level of spontaneous chrom atid breaks and gaps and characteristic types of chrom atid exchanges (sym m etrical quadriradial form ed from hom ologous chrom osom es in the case of BS; triradial and quadriradial form ed from non-hom ologous chromosomes in the case of FA) (Taylor 1982). In AT, however, although on
Introduction / 9 average chrom osom al breaks are increased in com parison to norm al cells (Gropp and Flatz 1967, C ohen et al 1975, 1979), there is evidence that they often overlap w ith the norm al range (Taylor 1982). In contrast, the frequency of stable chromosomal rearrangem ents was found to be consistantly higher in all AT individuals (Kaiser-McCaw et al 1975, Aurias et al 1980, Oxford et al 1975, Taylor et al 1976,1981, O'Connor et al 1982).
The distribution of chromosomal rearrangem ents in AT patients is apparantly highly non-random , always involving chrom osom e 7 and 14 and specially involving chrom osom e bands 7pl4, 7q34, 14ql2 and 14q32 (Aurias et al 1980, O 'C onnor et al 1982, Taylor 1982). The translocations observed include paracentric inversion of chrom osom e 14 and pericentric inversion of chrom osom e 7 and the most frequent translocations observed are t(7q;14q), and t(7p;14q) (Aurias et al 1980, O 'Connor et al 1982, Taylor 1982). U sing different m itogens to stim ulate T- an d B-lym phocytes, O'Connor et al (1982) observed a high frequency of rearrangem ents in AT cells at chrom osom e 7 and 14 in both groups of lym phocytes. Although rearrangem ents of chrom osom e 7 and 14 m ay also occur in norm al individuals (Cohen and Sim pson 1982, O'Connor et al 1982), the frequency was estim ated to be 40-fold higher in AT cells than in norm al cells (Taylor 1982). The breakpoint sites of rearrangem ent have been defined to involve T-cell receptor gene loci w ithin chromosome 7 and 14 (O'Connor et al 1982, Taylor et al 1989). Furtherm ore, anomalies in chromosome 14 have shown a distinctive association w ith m alignant neoplasm s in AT patients (Keiser- McCaw and Hecht 1982).
Induced chromosomal aberrations
Frequencies of chrom osom al aberrations in d u ced by ionizing radiation and radiom im etic agents are dram atically increased in AT cells when com pared to norm al cells. Chromosomal hypersensitivity is generally
Introduction / 10 accepted as one of the hallm arks of the response of AT cells to ionizing radiation; two others are cellular hypersensitivity and radioresistant DNA synthesis. Higurashi and Conen (1973) firstly reported an elevated frequency of rings and dicentrics in y-irradiated AT lymphocytes. W hen norm al cells are irradiated at Gqphase, only chromosome-type aberrations, namely rings, dicentris and fragm ents, are usually scored in the metaphse chromosomes. While irradiation of cells in the S- or G2-phases results in chrom atid-type
aberrations, i.e., breaks, gaps an d exchanges at the first m itosis. In lymphocytes from AT individuals, however, a pronounced increase in the num ber of chrom atid-type aberrations appeared after Go irradiation and both the frequencies of chromatid breaks and gaps were observed to be m ore than 10-fold higher in AT cells w hen com pared with normal cells following
Gqirradiation (Taylor et al 1976, Taylor 1978). In contrast to the increased frequency of chrom osom e fragm ents, rings and dicentrics occured at a normal level in AT cells particularly at higher doses (4 Gy) (Taylor et al 1976). It is notable that chrom atid exchanges in these cases increased by a factor up to tw enty in AT lymphocytes following Gqirradiation (Taylor et al 1976, Taylor 1978). N atarajan and M eyer (1979) also failed to observe an increase in dicentric frequency in irradiated Go AT cells, however a 4-fold increase in chrom atid aberrations in AT lymphocytes exposed to X-rays in Gq was observed. The striking feature of the high frequency chrom atid aberrations in AT cells irradiated in Gq suggested a failure in the efficient repair of DNA dam age in AT cells following ionizing radiation and that the unrepaired or m isrepaired dam age expresses itself as chromatid aberrations at the next mitosis (Taylor et al 1976, Taylor 1978).
AT cells irradiated at G2 phase also show a higher frequency of
chromatid aberrations when com pared w ith normal G2 cells (Rary et al 1974,
Taylor et al 1976, Natarajan and Meyer 1979, M ozdorani and Bryant 1989a). Observations of Taylor (1982) suggest a ten- to tw enty-fold increase in
Introduction / I l chrom atid breaks and gaps in AT lym phocytes irradiated in G2 phase,
whereas Bender et al (1985) reported that X-ray-induced chrom atid deletions increased only 4-fold in AT lym phocytes exposed in G2 phase. The frequency
of chrom atid exchanges is also reported to be higher in X -irradiated cells from AT patients (Taylor et al 1976, Taylor 1978). Higher yields of chrom atid deletions can be observed in AT fibroblasts even at short times following X- irradiation (e.g., 1 hour including 30 m in colcemid) (Mozdarani and Bryant 1989a). Following exposure to neutrons in G2 phase, AT lym phocytes
yielded about ten times m ore chrom atid aberrations w hen com pared to norm al cells; the extent of increase being sim ilar to that follow ing X- irradiation (N atarajan et al 1982, Taylor 1982). Increased chrom osom al sensitivity to bleomycin has also been dem onstrated in AT cells (Taylor et al 1978).
It is possible to examine chromosomal damage in interphase cells by utilizing the technique of prem ature chromosome condensation (PCC). This technique visualizes chrom osom e fragm ents in interphase cells by the fusion of irradiated cells w ith unirradiated mitotic hum an or ham ster cells in which the test chrom osom es can be distinguished from those of the m itotic cells by 5-brom odeoxyuridine (BrdU) pre-labelling of the latter (Cornforth and Bedford 1983). The advantage of this technique is the possible determ ination of initial chrom osom e breaks induced by DNA damaging agents in all cells w ithout the neccessity of cells reaching mitosis. The num ber of PCC fragments m easured immediately after irradiation was found to increase linearly as a function of radiation dose (Cornforth and Bedford 1985, Pandita and Hittlem an 1992). This observation is also true for cells treated w ith bleom ycin (H ittlem an and Sen 1988). U sing the PCC technique C ornforth and Bedford (1985) found that the initial levels of chrom osom e breaks in X -irradiated Gi phase AT fibroblasts w ere no different from that in Gi norm al cells. By contrast, using an alternative
Introduction / 12 technique for investigating chrom osom al repair, nam ely G2 assay w hich
m onitor the kinetics of chrom atid aberrations in G2 cells as a function of
p o st-irrad iatio n incubation tim e, the frequency of initial chrom atid deletions was estim ated to be appoximately 2.5-fold higher in X-irradiated AT fibroblasts than norm al cells (M ozdarani and Bryant 1989a). A similar enhanced level of initial chromosomal damage in AT cells after exposure to ionizing radiation was found in the study of Pandita and H ittlem an (1992) by m easuring PCC in G i and G2 cells. They reported a 2-fold higher initial
chrom osom e breaks in G i and G2 phases AT lym phoblastoid cell lines
follow ing y-irradiation w hen com pared w ith norm al cells. An increased frequency of initial PCC breaks in G i phase AT fibroblasts after bleom ycin treatm ent was also observed (Hittleman and Sen 1988).
Modification of chromosomal sensitivity
Natarajan et al (1980a) have studied the influence of post-treatm ent of caffeine on the level of X-ray induced chromosomal aberrations in blood lymphocytes from AT and normal individuals. Caffeine is know n to release the radiation-induced G2 arrest and to increase chromosomal aberrations in
irradiated cells (Liicke-Huhle et al 1983). Post-irradiation treatm ent w ith caffeine potentiated the chromosome breaking effects of X-rays in AT to a similar extent as in normal cells (Natarajan et al 1980a). Cells in late S- or G2
phases of both AT and normal cell lines have proved to be more sensitive to caffeine p o st-irrad iatio n treatm ent than Gq cells. This indicated no
difference in the caffeine-sensitive lesions th at w ere expressed as chrom osom al aberrations in m itosis betw een AT an d norm al cells. T reatm ent of G2 AT and norm al hum an fibroblasts w ith 9 -p -D -
arabinofuranosyladenine (ara A), an inhibitor of DNA synthesis (reviewed by Cohen 1976), resulted in an increase of chromatid aberrations induced by irradiation to a similar extent betw een AT and normal cell lines (M ozdarani
Introduction / 13
and Bryant 1989a, b). A ra A did not increase the initial yields of chromosome deletions but did inhibit their repair in both AT and normal cells irradiated in G2 phase (M ozdarani and Bryant 1989a, b). Inhibition of
th e re p a ir of X -ray in d u ced ch ro m atid ab erratio n s by 9 -p -D - arabinofuranosylcytosine (ara C), w hich also inhibits DNA replication (review ed by Cohen 1976), has also been observed in G2 phase AT and
normal cells, although unlike ara A, ara C additionally caused an increase in the frequency of chrom atid deletions during the post-irradiation incubation (M ozdarani and Bryant 1988).
1.1. 4. DNA Repair in AT cells
DNA damages induced by ionizing radiation
Ionizing radiation causes a num ber of different types of lesions in DNA, these are: single-strand breaks (ssb), double-strand breaks (dsb), base dam age and crosslinks. Strand breaks can either arise from cleavage of the phosphodiester bonds or from disruption of a sugar m oiety. Two ssb occuring opposite to one another or few bases apart in distance m aybe regarded as a dsb. The majority of DNA breaks are ssb (approxim ately 1000 ssb/cell/G y) compared with relatively low production of dsb (40 dsb/cell/G y) (Taylor 1978, Bldcher and Pohlit 1982). Base dam age involves alteration of base side groups or of the ring structure and most of the modifications have not been chemically characterized. The frequency of base dam age is thought to be similar or even higher than the frequency of strand breaks. Crosslinks occur both between the strands of DNA and between DNA and protein. The frequency of D NA-protein crosslinks is estim ated to be approxim ately 133 crosslinks/ cell/ Gy, and DNA-DNA crosslinks 30/cell/G y. Of these lesions, the dsb is thought to be the lesion leading to cell death (Blocher and Pohlit 1982, Bryant 1984).
Introduction / 14
Induction and rejoining of strand breaks
M easurements of DNA strand breaks in intact cells can be achieved by use of following m ethods: 1) classical analysis of a DNA profile w ith different molecular w eights on an alkaline or a neutral sucrose gradient during velocity sedim entation (McGrath and Williams 1966, Lehmann and Stevens 1977); Dsb can be determ ined by neutral velocity sedim entation while ssb together with dsb determ ined by alkaline velocity sedim entation; 2) filter elution of DNA through a nucleopore filter in which the rate of elution is thought to depend on the m olecular weight of DNA fragm ents (Kohn and Grimeg-Ewig 1973). This m ethod can either be used under alkaline conditions for m easurem ent of the combined frequency of ssb and dsb (Kohn and Grimeg-Ewig 1973) and crosslinks (Kohn et al 1980), or at pH 7.4 or 9.6 for m easurem ent of dsb (Bradley and Kohn 1979). 3) DNA unw inding methods to determ ine the rate of unw inding from break points in the DNA helix under alkaline conditions (Ahnstrom and Erixon 1973), This method, like alkaline elution, measures a m ixture of ssb and dsb. It can be used to exam ine dsb repair on the basis of different repair kinetics between ssb and dsb (Bryant and Blocher et al 1980); 4) pulse field gel electrophoresis (PFGE) measures dsb by the migration of DNA double-strand fragments of varying molecular weight (Schwartz and Cantor 1984).
It has been found that enhanced radiosensitivities in some tum our cell lines are related to an increased induction of dsb by ionizing radiation in these cells (Peacock et al 1989). This is not true for AT cells since the induction of ssb and dsb caused by X- or y-irradiation in AT cells is identical to those in normal cells (Lehmann and Stevens 1977, Coquerelle et al 1987, Peacock et al 1989). Irradiation of AT cells by a-particles also induces a similar frequency of dsb as in normal cells (Coquerelle et al 1987). The initial num ber of PCC has been found sim ilar in AT and norm al quiescent
Introduction / 15
fibroblasts (Cornforth and Bedford 1985), while a conflicting result reporting a higher production of initial PCC breaks in GI as well as in G2 phase was
obtained in 3 AT lymphoblastoid cell lines (Pandita and Hittlem an 1992). A num ber of experim ents have dem onstrated norm al rejoining of strand breaks in AT cells by utilising various techniques to determ ine ssb or dsb. Using alkaline sucrose gradient sedim entation, AT cells have been dem onstrated to have a normal ability to rejoin ssb after irradiation (Taylor et al 1975, Vincent et al 1975, Paterson et al 1976). A normal rate and extent of ssb rejoining in AT cells has been confirmed by using procedures with higher sensitivity, e.g., alkaline unw inding (Sheridan and H uang 1979a, Thierry et al 1985) and alkaline filter elution (Fornace and Little 1980, H ariharan et al 1981). Sheridan and H uang (1979b) found no difference in the kinetics of rejoining of ssb betw een AT and norm al cells following irradiation using an alternative approach in w hich following irradiation alkali-denatured DNA is treated w ith a single strand-specific endonuclease (SI nuclease). The existence of ssb results in single-strand DNA after alkali dénaturation which are digested by the nuclease so that the num ber of ssb induced by irradiation is inversely proportional to the rem aining intact DNA.
The rejoining of dsb in AT cells has been found to be norm al by neutral velocity sedim entation (Lehmann and Stevens 1977), or by neutral filter elution (Fornace and Little 1980, Van der Schans et al 1983, Thierry et al 1985). One exception to these observations was one AT cell line (AT2BE) which, using neutral filter elution, dem onstrated that the early kinetics of dsb rejoining after irradiation were reduced in this AT strain, although the eventual extent of dsb rejoining was no different from that of norm al cells (Coquerelle and W eibezahn 1981, Coquerelle et al 1987). The ability of AT cells to rejoin dsb induced by bleom ycin (Fornace and Little 1980), neocarzinostatin (Shiloh et al 1983b), and 4-NQO (Van der Schans et al 1982)
Introduction / 16
was also found to be the same as that of normal cells. However, a higher level of residual dsb following 3 hours post-irradiation incubation was observed in AT2BE cells in com parison to norm al cells after bleom ycin treatm ent (Coquerelle et al 1987).
A lthough the rejoining of bulk ssb or dsb is norm al in AT cells, a small num ber of breaks m ay rem ain unrepaired in AT cells after a lengthy incubation time. The num ber of residual breaks m ay be undetectable by the techniques developed so far. This was postulated by Taylor (1978) from results of chrom osom e studies and also by Lehm ann and Stevens (1979). The thesis that m ore residual breaks are present in irradiated AT cells is particularly suggestive of deficient dsb rejoining, since the increased yields of chrom osom e aberrations found in AT cells following irradiation m ay arise from unrepaired dsb. W ith the PCC technique, the relationship betw een unrepaired dsb and chromosomal breaks has been investigated, X- ray induced PCC fragm ents were found to decrease during post-irradiation incubation and this was found to be m irror image of PLD repair as m easured by a clonogenic assay (Cornforth and Bedford 1983). The half tim e for disappearance of PCC has been estim ated at 2 hours (Cornforth and Bedford 1983), similar to the half time for dsb rejoining (Bryant and Blocher 1980, Blocher and Pohlit 1982), suggesting dsb as a possible origin of PCC breaks. A lthough PCC fragm ent rejoining shows norm al kinetics in AT cells, the frequency of residual breaks was found to be 5 - 6 times higher in AT than in
norm al cells (Cornforth and Bedford 1985). Unlike m easurem ents of bulk ssb or dsb, the doses of irradiation used in PCC technique are often within the dose range used for cell survival studies. The num ber of residual PCC fragments in AT cells after exposure to 6 Gy has been estim ated at 10 breaks
per cell com pared to 2 breaks per normal cell (Cornforth and Bedford 1985). O nly about 15% of initial dsb are im m ediately expressed as breaks in prem aturely condensed Gi chrom osom es (Cornforth and Bedford 1983).
Introduction / 17
This indicates that appoxim ately 4% and 0.8% of initial dsb in AT and norm al cells respectively, are probably not rejoined, and the difference of unrejoined dsb betw een AT and norm al cells are too small to detect, particularly considering the errors in measurem ents.
A part from unrepaired strand breaks, the mis-rejoined strand breaks play a large role in the determ ination of radiosensitivity. A num ber of experim ents using recom binant DNA plasm ids have been designed to investigate the fidelity of dsb rejoining in AT cells (see review of Thacker 1989). Dsb are generated by restriction endonucleases (RE) at a specific site in a selectable gene of plasm id and correct rejoining of dsb is identified by the restoration of the selectable gene function following transfection (Cox et al