10TH ANNIVERSARY ARTICLE Molecular and Biochemical Aspects of Bloom's Syndrome Thomas M. Nicotera

A B S T R A C T : B l o o m ' s syndrome (BS) is an uutosomal r e c e s s i v e disorder, characterized bv a h i g h incidence of cancer at a y o u n g age. Cytogenetically, BS ee,lls exhibit a high fr,equency of chromo-

samal damage and sister chromatid exchanges. Thus, BS provides one af the best correlations af u human genetic disorder exhibiting both chromosomal instability and a high incidence a~ cancer. It is increasingly evident that a spautaneotJs mutagenic event may be responsible for the inherent chromosomal instability. Oxidative s t r e s s is now s h o w n to o c c u r in BS ceils and may be responsible fl~r the observed chromosomal instability, Furthermore. treatment with antioxidunts decreases the level of s i s t e r ehromatid e x c h a n g e s . The combination of a mutagenie event and an elevated rate of recombination eauld potentiall 5" lead to harnozygosity of tunlar s u p p r e s s o r g e n e function. Hypomethylation and expression of an activated c-myc gene are now demanstrated in BS lymphoblastoid cells, ldentif}'ing the mechanism(s) of the o n g o i n g cellular and DNA damage is impartant in understanding the etiology of this complex d i s o r d e r . This article reviews the recent biochemical and molecular advances in the s t u d y a f BS.

INTRODUCTION Bloom's syndrome (BS) is a rare autosomal recessive disorder initially identified in Ashkenazi Jews and later found to be widespread in the general population [1]. The distinguishing clinical features of this disorder are numerous and include sew~.re growth deficiency, sun-sensitive facial skin lesions, areas of hypo- and hyperpiginenration of the skin and immunodeficiency, leading to infections of the respiratory tract and chronic lung disease. A newly recognized coInplication is the development of diabetes mellitus resembling late-onset diabetes [21. The most prominent feature of BS is a marked predisposition toward the development of cancer I1]. The age of onset of cancer is considerably earlier than that of the general population. To date, 57 neoplasms have been detected in the 130 clinically verified cases in the BS registry with a mean age of 25 years at the time of diagnosis. Nearly one third of the surviving cancer patients develop multiple primary tumors. There is no consistent pattern in the occurrence of the cancer type or its location. It has been suggested that the actual number of patients may be nnderreported because of the c o m p l e x clinical nature and the premature death associated with this disorder. It has been assumed over the years that BS, along with other chrolnosomal instability syndroines, is defective in some aspect of its DNA repair capacity that leads to spontaneous chromosomal damage. This viewpoint is based on the discovery by

From Roswell Park Cancer Instilut(;. Biophysics Department, Buffalo, Ne,w York.

Address reprint requests to: Dr. Thomas M. Nicotera, Roswell P a r k Cancer Institute. B i o p h y s ics Department, Elm and Carlton Streets, Buffalo, N Y 14263. Received May I5, 1990; accepted May 17. 1990.

((') 1991 Elsevier Science Publishing Co., inc. 655 A v e n u e of the Americas. New York. NY 10010

C a n c e r G e n e t Cytogenet 53:1 13 (1991] 0165-4608'911S03,50

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f . M . Nicotera Cleaver 13] that another ctlromosomal instability syndrome, xeroderma pigmentostun, is deficient in the repair of ultraviolet-induced DNA damage. [o this regard, several potential DNA repair and processing defects are proposed to explain the inherent chromosomal instability of BS. Regardless of which mechanism is responsible for this disorder, BS represents one of the best correlations of a genetic disorder exhibiting both c h r o m o s o m a l instability and an increased risk of malignant tumor formation [4 I. Cytogenetic characterization of BS chromosomes has failed to identify a specific aberrant locus that could potentially lead to the identification of the gene responsibb~ for this disorder. Nonetheless, differential staining of BS chromosomes has been useful in the identification of the high rate of sister chromatid exchanges (SCEs) between homologous chromosomes, which serves as a clinical marker for this disorder [41. Despite the lack of specific genetic information, there has been considerable excitement in recent years in the study of BS. The cytogenetic observations and SCEs in particular have led to the d e v e l o p m e n t of new strategies at the biochemical and molecular level. It is in this area that this review focuses. Comprehensive reviews have otherwise been provided [1, 2, 4 61.

SISTER CHROMATID EXCHANGES

Since the discovery by Chaganti et al. [71 of an abnormally high SCE rate in cells of BS origin, this p h e n o m e n o n has been regarded as the most prominent cytogenetic characteristic and has been used extensively in the phenotypic verification of this disorder [8]. The abnormal illcidence of SCEs is considered to be a consequence of the genetic defect in these cells. However, it is reported that 2-bromo-5-deoxy uridine (BrdU), used in the detection of SCEs, causes most if not all the SCEs observed in BS u p o n replication of the BrdU-substituted DNA template [9]. Ira contrast, Tsuji and Kojima [10] report that SCE frequencies remain constant at BrdU concentrations below 10/zg/ml. Above that concentration, SCEs are substantially induced. In two-way or three-way differential staining of M3 metaphases at 5 /zg/ml BrdU of BS cells, an a b n o r m a l l y high incidence of SCE is observed in each cell cycle. When DNA replicates on a template strand containing little incorporated BrdU, little or no SCE induction takes place [11]. Analysis of SCEs at very low BrdU substitution levels {0.9%-4.5%) using an anti-BrdU antibody into DNA shows a residual count of 35 40 SCEs per BS metaphase c o m p a r e d to 5 SCEs per normal metaphase. At higher BrdU substitution rates, the SCE frequency rises sharply, thus confirming the hypersensitivity of BS cells to replication on a BrdU-substituted template. This result is compatible with the early observation showing increased quadriradial formation ira BS cells in the absence of BrdU [12}. Shiraishi [13] also used a similar anti-BrdU antibody technique to show a residual 2 5 - 3 0 SCEs per metaphase in two BS cell lines at low BrdU concentrations. On further exainination of endomitotic chromosomes, this rate falls to 3 6 SCEs per metaphase. While it is unclear why this should be the case, it is possible that the short duration between the first and second replication cycles of the e n d o r e d u p l i c a t e d chromosornes may not subject the DNA to potential endogenous nmtagenic factors present in BS cells or possibly that an additional round of DNA repair may a c c o m p a n y the e n d o m i t o t i c replication process. COMPLEMENTATION A N A L Y S I S

L y m p h o c y t e s from most BS patients exhibit a high SCE frequency; however, in some cases the coexistence of a small s u b p o p u l a t i o n of cells exhibiting a relatively normal SCE frequency has been reported by German et al. [14]. No case of BS fibroblasts exhibiting normal SCE has been reported. The heterogeneous SCE population in

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lymphocytes, in conjunction with the variability in the response of BS fibroblasts to DNA-damaging agents, has led to the speculation that genetic heterogeneity may account for these differences. A d d i t i o n a l l y . BS cells with the high SCE p h e n o t y p e are corrected by cocultivation with normal h u m a n fibroblasts [41, although this is not a universal finding. Hybridization experiments have been extensively used to test for potential genetic diversity within the BS population and rely on the SCE frequency as a measure of compleinentation. Fusion of BS fibroblasts exhibiting a high SCE frequency with control h u m a n cells or with CHO cells results in the correction of the SCE levels [15 171. Complementation, however, is not observed in hybrids of fibroblasts from two Japanese BS patients [18]. In a comprehensive analysis of lymphoblast hybrids derived from BS patients of various national origins, no BS cell line is able to correct the characteristically high SCE level of any other BS cell line [19]. Thus, conlplementation analysis indicates that a single gene locus is responsible for the BS phenotype. This study a d d i t i o n a l l y demonstrates that cell lines with the Iow-SCE p h e n o t y p e were capable of correcting the high-SCE phenotype in BS lymphoblasts. Low-SCE BS l y m p h o i d cells are also shown to have normal levels of spontaneous c h r o m o s o m a l breakage [14], spontaneous mutation rates [20], and SOD activities 1211. It is suggested that Iow-SCE l y m p h o i d lines arise by reversion to the normal p h e n o t y p e [20]. The BS l y m p h o i d cell line GM4408 and fibroblast strain GM3498, originating from the same individual, were analyzed by DNA bybridization with hyperwtriable probes to determine whether they were still of the same origin. It is determined that patterns of hybridized DNA fragments from both cell types are identical and unique c o m p a r e d to several controls [22]. Therefore, it is c o n c l u d e d that these cells originated from the same i n d i v i d u a l and that this l y m p h o i d cell line is capable of p h e n o t y p i c reversion. Recent evidence indicates that fusion of BS l y m p h o b l a s t o i d cells with malignant cell lines derived from chronic l y m p h o c y t i c leukemia results in partial normalization of SCEs, yielding 1 5 - 3 0 SCEs per cell [23]. This result is interpreted as support for the notion that elevated SCEs may be indicative of the malignant state.

DNA LIGASE I ACTIVITY

Several reports p u b l i s h e d within the last several years demonstrate the existence of a r e d u c e d DNA ligase I activity, assayed by diverse methodologies, ill BS cell extracts [24-28]. It is suggested that a mutation in the ligase I gene and the concomitant reduction in ligase I activity, could be responsible for the BS p h e n o t y p e [24]. Ligase I activity is associated with DNA replication and elutes chromatographically with a protein of a p p r o x i m a t e l y 200 kDa. The DNA ligase I defect is s u b d i v i d e d into two categories, type I-1, w h i c h is a heat labile enzyme of normal size and, type I-2, which is found in dimeric form and is heat stabile [26]. DNA ligase II is found to be normal in BS extracts, has a molecular mass of 80 kDa, and is associated with DNA repair. The fibroblast cell strain GM5289, which is c l a s s i f e d as having a type I-2 defect, demonstrates normal ligase I activity in one study [24] and an apparently elevated total activity that is associated with two distinct enzyme fractions in a second study [271. Unfortunately, total ligase specific activity is not reported. Fusion of cells with the two distinct ligase defects does not demonstrate complementation, whereas fusion of BS with normal cells results in suppression of the elevated SCE ]19]. Moreover, two BS l y m p h o i d cell lines that exhibit a low SCE frequency both retain the DNA ligase I defect [28]. This same group reports that i m m u n o b l o t analysis using polyclonal antibodies does not discriminate between normal and BS ligase I protein and also finds the BS protein content to be normal while demonstrating a reduced specific

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T . M . Nicotera activity. Hence, the composite data in this line of investigation is not entirely selfconsistent. Most recently, Mezzina et al. [29] have shown that total ligase activity is equal to or greater than in control cells whether the activity is measured in crude cell extracts or in purified form. These results are attainable only when considerable exogenous DNA (50/xg/ml) is a d d e d to the assay mixture. Under these conditions, BS cell extracts exhibit elevated DNA ligase activity (1.5-4-fold higher] compared to control extracts w h i l e e x h i b i t i n g no increased heat sensitivity. Upon analysis of DNA ligase I by FPLC, crude cell extracts from a l y m p h o b l a s t o i d control and a BS l y m p h o b l a s t o i d cell line each demonstrate a single major enzyme fraction with a molecular mass of 180 kDa. These authors c o n c l u d e that spontaneous, free radical-induced, single-strand breaks may occur in BS, which requires a constitutively increased DNA ligase activity necessary for joining the broken DNA strands. Based on these results, the presence of a markedly elevated DNase activity is demonstrated in virus-transformed cells, which hydrolyses the DNA to a greater extent than in untransformed cells. This nuclease activity is associated with a 42-kDa protein and is specific for 5'-P termini in doublestranded DNA [301. The reaction mechanism is similar to that of DNase IV, hut it is not yet k n o w n whether it is a repair or a recombinogenic enzyme. Among all untransformed fibroblasts analyzed, BS strain GM1492 possesses the highest enzyme activity. This cell strain exhibits a preneoplastic phenotype because of its partial ability to grow in methylcellulose. However. the DNase activity does not appear to correspond to a general enhailcement of DNA processing enzymes that are related to the proliferative state. Since this activity is elevated in tumorigenic cells, the possibility is raised that it may be required for neoplastic progression [29].

ALTERED DNA EXCISION REPAIR Uracil-DNA Glycosylase Previous studies demonstrate several DNA repair enzymes in the base excision repair p a t h w a y are enhanced prior to the induction of DNA replication [31,32]. The enhancement of repair enzyme activities may serve as a proofreading of the DNA to remove aberrant bases prior to their fixation in the genome. BS cells are deficient in this capacity. In particular, uracil DNA glycosylase [31, 32] is induced simultaneously with replication, rather than several hours prior, as occurs in control cells. One caveat in this line of investigation, however, is that only relative activities are measured. Fujiwara and Yamamoto [32], on the other hand, tested two BS cell lines and found the m a x i m u m levels of both glycosylase activity and DNA synthesis to be comparable to the control cell line. Defective regulation of uracil-DNA glycosylase in synchronous cell p o p u l a t i o n s is observed in only one of two BS cell lines examined. Interestingly, the cell line showing aberrant regulation also demonstrates atypically high glycosylase activity in e x p o n e n t i a l l y growing cells. In this case neither BS nor control glycosylase function are heat labile. In yet another investigation, high nracil-DNA glycosylase activity is present in two BS cell lines; however, cell cycle d e p e n d e n c y is not examined [33]. An altered distribution within the cell cycle is also observed. Further investigation of BS uracil-DNA glycosylase indicates an altered enzyme structure as defined by monoclonal antibody reactivity [341. One of the three monoclonal antibodies obtained fails to recognize any of five BS fibroblast strains tested yet still retains specificity for the enzyme derived from control cell strains [35]. This antibody also fails to inhibit uracil DNA glycosylase activity from any of five BS cell strains. However, the denatnred enzyme is recognized. These same characteristics are also observed in BS l y m p h o b l a s t o i d cell lines [36]. The authors speculate that this evidence may reflect a germline mutation in individuals with BS.

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It is difficult to reconcile that such a purportedly abnormal enzyme structure c a n result in the relatively normal cellular activity previously described [32, 33]. Several possible explanations for these apparently anomalous results are proposed. Based on the newly cloned c-DNA encoding, the uracil-DNA glycosylase gene, a molecular weight of 33.8 kDa was predicted for the encoded protein [37]. This differs from the 37-kD polypeptide iminunoprecipitated by the uracil-DNA glycosylase monoclonal antibody [38]. A peptide of altered size may result from either posttranslational modification of the protein or possibly from the presence of diverse uracil-DNA glycosylases in placental extracts. The existence of two distinct genetic defects (i.e., the proposed altered temporal regulation of base excision repair and an abnormal structural defect in uracil-DNA glycosylase) further presents a conceptual problem in view of the fact that only one complementation group is established in this autosomal recessive disorder.

Hypoxanthine-DNA Glycosylase Hypoxanthine-DNA glycosylase excises hypoxanthine residues from DNA produced by the deamination of adenine. Normal human cells enhance the base excision enzyme prior to DNA replication; however, BS cells are reported to induce the enzyme concomitant with DNA replication [39]. This altered temporal sequence was identical to that observed for uracil DNA glycosylase.

O6-Methylguanine-DNA Methyltransferase In experiments similar to those discussed above, a third base excision repair enzyme O~Lmethylguanine-DNA methyltransferase is completely incapable of induction at any interval in the cell cycle in two BS cell lines tested [40]. Again, control cells demonstrate enhancement of methyltransferase activity just prior to cell proliferation. Nonenzymatic methylation of nucleotides in DNA by s-adenosyhnethionine may contribute to the observed spontaneous mutagenesis if not repaired prior to replication. An intrinsic genetic defect in BS is suggested, which results in altered regulation of base excision repair enzymes during cell proliferation [31-33, 40]. Failure of BS cells to enhance repair activity during cell proliferation could contribute to the spontaneous mutation frequency of these cells by compromising their ability to repair endogenously mutated sites. Alternatively, it is possible that an ongoing DNA damaging process could be responsible for delaying the repair processes well into the replication stage.

OXIDATIVE STRESS Superoxide Dismutase Activity The eukaryotic superoxide dismutase enzymes {CuZn- and Mn-SOD) have been extensively investigated and found to catalyze the disproportionation of 02" to H202 [41]. Catalase and peroxidases then converts H202 to H20. The maintenance of low concentrations of 02. and H202 prevents the formation of the more highly reactive OH-, which is capable of extensive cellular damage. Enzymatic activity is readily induced by endogenous 02. or a byproduct of 02. . In fact, SOD has been used as a relative measure of intracellular 02. concentration [42]. Measurement of SOD in BS cell strains and cell lines of various origin are shown to posses elevated activities

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T . M . Nicotera for both CuZn- and Mn-SOD compared to controls 121]. Elevated SOD activity may therefore be indicative of elevated O2 in cells of BS origin. In prokaryotic cells, MnSOD is a member of the soxR regulon that is specifically i n d u c e d by 02. and includes glucose-6-phosphate dehydrogenase, endonuclease IV, and 32 other unidentified proteins [43,441. It is of particular interest that the DNA repair enzyme, e n d o n u c l e a s e IV, is considered to be significant in mitigating against O e. - i n d u c e d DNA damage [43]. The soxR regulon genes do not correspond with the oxyR regulon genes, which are H2Oz-inducible. The oxyR gene product is a transcriptional activator of the pertinent genes and is activated by direct oxidation [45[. Consequently, the oxyR protein acts as both a sensor and transducer of an oxidative stress signal. How closely these systems are paralleled in eukaryotic cells is not known.

Induction of SOD and SCE

Treatment of a h u m a n control cell strain with paraquat resulted in a d o s e - d e p e n d e n t i n d u c t i o n of both CuZn- and Mn-SODs [21]. Paraquat is enzymatically reduced, and the reduced paraquat consequently reduces O~ to generate O z. . The level of SOD activity i n d u c e d by 1.0 mM paraquat is comparable to the SOD activity in untreated BS cells. Induction of SCE occurs in the same range of paraquat concentration used to induce SOD in control cells [21]. The level of SCE induction in control cells does not attain that of BS cells: however, it provides a further indication that O z- plays a role in SCE formation. In the same study, another redox cycling c o m p o u n d , menadione, and an inhibitor of CuZnSOD, diethyldithiocarbamate, are used to induce SCE in a dose d e p e n d e n t fashion.

Modulation of SCE in BS Cells

Treatment of cells with the SOD mimetic, Copper(lI)-(3-5-diisopropyl salicylate)~ (CuDIPS), results in a d o s e - d e p e n d e n t increase in SCE to a level double that of untreated BS ceils [21]. Copper alone in the same concentration as CuDIPS does not affect the SCE response. This result was interpreted as an indication that the high concentration of H202 conceivably present in BS cells is further increased with the a d d i t i o n of CuDIPS that results in still higher rates of H202 formation and further promotes the formation of Fenton-derived OH.. The indirect nature of these observations necessitates their verification by alternative means. It is likely that OH. formation is ultimately responsible for tile DNA damage leading to SCE formation. Significantly, m o d u l a t i o n of the SCE phenotype, which is used as the distinguishing feature of BS, is a c c o m p l i s h e d through the control of the O2' metabolic pathway in BS cells. Generation of elevated amounts of oxy radicals in cells leads to a variety of cellular abnormalities. Lipid peroxidation is one component of free radical-mediated events that has been c o m p r e h e n s i v e l y studied and shown to cause extensive cellular damage. One product of lipid peroxidation, 4-hydroxynonenal, generates SCEs in cells 146]. It is therefore not surprising that treatment of a BS cell line with ~-tocopherol, which is an inhibitor of lipid peroxidation, causes up to a 30% reduction in SCEs [21]. The fact that only a portion of the SCEs are eliminated may be indicative of m u l t i p l e pathways for free radical-mediated DNA damage may be occurring. Peroxidation of lipids nonspecifically activates p h o s p h o l i p a s e A., which is responsible for the cleavage of arachidonic acid from membrane triglycerides and activates the prostaglandin cascade [47[. Preliminary evidence shows that SCEs can also be reduced by treatment of BS cells with inhibitors of the prostaglandin pathway. The cyclooxygenase inhibitor, indomethacin, and the lipoxygenase inhibitor, norhydrogu-

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aretic acid, each r e d u c e d the SCE rate by a p p r o x i m a t e l y 20% (unpublished observations).

Enzyme Inactivation It is well established that free radical inactivation of the enzymes involved in the metabolism of active oxygen species can occur. SOD, catalase, and glutathione reductase [48-50] are inhibited by elevated concentrations of oxy radicals. Of these enzymes, catalase activity was shown to be 60% lower in one BS cell line compared to controls [51]. Whether catalase activity is d i m i n i s h e d in all BS cell lines remains to be demonstrated. The possibility that DNA processing enzymes are susceptible to attack by either free radicals or their free radical byproducts (:an therefore be raised. Unsaturated a l d e h y d e s such as 4 - h y d r o x y n o n e n a l markedly inhibited O~-methylguanine-DNA methyltransferase, an enzyme known to have a cysteine residue in its active site [521. Dialysis of the enzyme does not restore enzymatic activity. In contrast, uracil-DNA glycosylase is insensitive to sulfhydryl reagents and is also unaffected by 4-hydroxynonenal. This p h e n o m e n o n may explain the experiments described earlier whereby O~-methylguanine methyltransferase was found to be uninducible (luring the proliferative stage of the cell cycle in BS cells [39].

Altered Cell Cycle Regulation Cell cycle kinetics studies were performed in BS and control (:ells by e m p l o y i n g a flow cytometric technique [53]. BS fibroblasts show arrest in the G2 phase of the (:ell cycle along with a prolongation of the G1 phase. This pattern of perturbation is similar to that elicited in normal fibroblasts by 4-hydroxynonenal. Treatment of BS (:ells with a - t o c o p h e r o l improves the growth response at twice the rate of control (:ells. BS l y m p h o b l a s t o i d cells experience only a minor arrest in G2 phase after one round of BrdU incorporation but are more strongly inhibited during and after the second S phase. Thus, the second cell cycle delay in lymphocytes may be d e p e n d e n t on BrdU incorporation.

Decreased 5-methylcytosine Preliminary HPLC analysis of digested DNA extracted from two BS l y m p h o b l a s t o i d cell lines reveals little or no detectable 5-methylcytosine content [54]. Control lymphoblastoid cell lines have between 3.5%-4.0% of the total cytosine as 5-methylcytosine (m5cyt). BS fibroblasts have an a p p a r e n t l y normal m'~cyt content; however, a comprehensive analysis has not as yet been accomplished. H y p o m e t h y l a t i o n is associated with altered differentiation, altered gene expression, and carcinogenesis [55]. In order to test whether oxygen free radicals may be responsible for the h y p o m e t h y l ation taking place in BS cells, a control l y m p h o b l a s t o i d cell line was treated with paraquat and the DNA of treated cells was extracted, digested, and analyzed by HPLC for mScyt content. A dose and time d e p e n d e n t reduction in mScyt is observed with a m a x i m u m decrease of 35% after 4 days of treatment at 10 ~ M paraquat [54]. This represents the first correlation between paraquat-generated oxygen radicals and DNA h y p o m e t h y l a t i o n . The free radical m e c h a n i s m for h y p o m e t h y l a t i o n is not known: however, two plausible m e c h a n i s m s are 1) inactivation of cytosine methyltransferase and 2) h y d r o x y l a t i o n of 5-methylcytosine to yield 5-hydroxymethylcytosine or other oxidized products. Repair of modified mScyt and its rernethylation is probable except in cases where damage occurs on both DNA strands and the methylation signal is lost. This is the first epigenetic effect reported for oxygen radicals. Thus, h y p o m e t h y l a -

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tion of BS l y m p h o b l a s t o i d cell lines may represent yet another biochemical consequence of oxygen radi(:al overprodu(:tion. TOPOISOMERASE II

DNA replication is closely associated with induction of SCE in BS 1561. Analysis of enzymes involved in the replication process may be instrumental in understanding the SCE pro(:ess. DNA polymerase activity is examined in BS lymphoblasts and found to be normal [57[. Induction of SCEs is delnonstrated by either elevated BrdU concentrations Illl or by inhibitors of topoisomerase l1158]. This correlation has led to the speculation for a potential role of topoisomerase 1I in the mediation of SCE formation in BS. Growth of fibroblasts in the presence of BrdU specifically reduces salt-extractable topoisoinerase II activity to a mu(:h greater extent in BS (:ells than in control cells [59]. It is speculated that tighter binding of topoisomerase II to Brd~!substituted DNA might lead to an increased frequency of protein-linked, doublestrand breaks and SCEs. This hypothesis implies the existence of either a defective topoisomerase 1I or a protein complexed with topoisomerase 1I. The possibility that BrdU induces topoisomerase If-mediated DNA breaks was tested by alkaline elution of DNA extracted from normal and BS fibroblasts 1601. Exposure to BrdU indu(:es similar amounts of single-strand breaks in both (:ell lines. However, these breaks were not protein associated. Topoisomerase lI inhibition by amsacrine and etoposide produced similar DNA strand breaks in normal and Bloom's svndrome (:ells. In contrast, the topoisomerase I inhibitor, camptothecin, yields a higher level of protein-linked DNA single-strand breaks in BS than in normal fibroblasts, which indicates an elevated topoisomerase I activity in BS cells 161}. The relevance of the elevated topoisomerase I activity remains unclear. ERROR-PRONE RECOMBINATION

Altered rates of mitotic recombination have been proposed for BS [12]. A novel approa(:h used to address the problem of in vivo DNA end-joi ning activity incorporates the use of linearized plasnfids [62]. Overlapping or blunt end plasmids are constructed and transfe(:ted into BS and (:ontrol (:ells. The extent of ligation is measured as a function of the repli(:ated p l a s m i d ' s ability to transform bacteria. The results show that either plasmid type demonstrates a lower joining efficien(:v in BS cell lines c o m p a r e d to controls. A wide array of mutations are observed at the plasmid joining site, w h i c h include deletions, insertions of both plasmid as well as some unidentified sequences, reverse insertions of plasmid DNA, and other complex rearrangements, in addition, the spontaneous mutation frequency of the (:ircular plasmid is 2-21-fold higher in BS cell lines compared with controls. The authors suggest that the redu(:ed joining of free ends (:ould Ire due to either a decreased ligase activity or to an elevated exonuclease activity. In light of the more recent report demonstrating the presence of a elevated DNAse activity in vitally transformed BS cells [291, the latter explanation is more likely. It is possible that the high frequency of large nmtations could also be explained by an elevated DNAse activity, but the high spontaneous rate of point nmtations cannot be explained by this mechanism. Point mutations can occur through a defective excision repair, spontaneous damage, or a combination of both. The glycophorin A {GPA) assay is also used to estimate the somatic recombination frequency in BS-derived hemopoietic cells by measuring the incidence of mutant p h e n o t y p e s [631. This is done through the use of fluorescently tagged monoclonal antibodies specific for the M and N allelic forms of the GPA protein. Fluorescent sorting of labeled erythrocytes detects altered segregation of the specific genetic locus to produce homozygous variants at a 50-fold higher frequency in BS than in normal

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individuals. Hemizygous and partial-loss variants are also markedly elevated. Unequal recombination between homologous chromosomes could explain these observations and result in an error-prone SCE response. However, it is also reported that the GPA gene is u n u s u a l l y susceptible to unequal meiotic crossing over between the GPA gene and the partially homologous GPB gene that results in a mutant glycophorin protein. Hence, interpretation of these results may be tenuous. Furthermore, unequal recombination of d u p l i c a t e d neo gene fragments inserted into CHO cells is a rare event and may be dissociated from the SCE response [64]. A second group [65] utilizing the same glycophorin-A assay confirms the results of Langlois et al. [63] but considers an alternative explanation for their results. They propose that an endogenous mutagenic process during erythroid stem cell differentiation could result in the high level of GPA variants, This bias is explained by the GPA analysis of atomic bomb survivors in which leukemia occurred in 3% of the people receiving 2 - 3 Gy of radiation, while in BS patients about 30%-40% develop various types of cancers by age 30. The authors further speculate that, in Bddition to somatic recombination, the possibility that oxygen radicals play a role in the spontaneous mutagenic frequency and in the developlnent of neoplasia. In a d d i t i o n to the elevated DNase activity, an elevated RecA-like activity is reported in BS fibroblast strain GM 1492 [66]. RecA activity is measured by the presence of Dloop structures from supercoiled RFI&x174 DNA and homologous double-stranded DNA fragments retained to a nitrocellulose filter. This activity is not specifically related to SCE formation as it is also detected in CHO cells that do not exhibit high SCE levels. Whether there is any relationship between the 42-kD endonuclease described earlier [301 and the recombinogenic activity is not known. CONCLUSION

A two-hit paradigm has been postulated by Knudson [67} in which sequential loss of both alleles of a single relevant gene has been associated with malignant transformation. Heterozygotes inherit a cancer-related nmtant gene from one parent and a normal gene from the other parent. Mutation or loss of the normal gene nmst occur for homozygosity to develop. This model is best exemplified by retinoblastoma where the loss of the RB gene, located at 13q14. involves two distinct steps for malignant transformation. Constitutional chromosome 13 deletions in retinoblastolna patients share a loss of this region, as do tumor cells. Recent advances in the m a p p i n g of W i h n s ' tumor suppressor genes indicate that more than one suppressor gene may be involved in this pediatric tumor 168]. Analogous observations have been made with other tumors [69]. Therefore, loss of i n d i v i d u a l suppressor genes may follow the twoevent paradigm, but this paradigm may not be appropriate in describing all the genetic changes in adult tumors. Knudson (70) hypothesizes that BS is highly oncogenic because of its increased rates of both mutation and recombination. The combination of events e n c o m p a s s e d first by somatic mutation to render a cell heterozygous for a relevant gene, followed by a second event resulting from either mutation or recombination, fits the multistage model for oncogenesis. This hypothesis is consistent with the notion that a random mutagenic process is occurring and may explain the activation or deletion of relevant genes in their respective target cells. The wide spectrum of adult and pediatric tumors that characterize BS, including Wihns' tumor, lends support for this concept. In vitro carcinogenesis models demonstrate that cancer is a multistep process in w h i c h tumor promoter genes, as well as tumor suppressor genes, play essential roles [711. The nature of these gene types and their interactions will likely yield significant information in the d e v e l o p m e n t of neoplasia. The recent demonstration of an activated c-myc gene in BS B-lymphoblastoid cells is consistent with the nmltistep oncogenesis

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m o d e l [72]. T h e cause of its a c t i v a t i o n is not k n o w n , h o w e v e r , the c-myc and c-los p r o t o - o n c o g e n e s can be i n d u c e d in vitro by e x o g e n o u s free radical g e n e r a t i o n [73, 74]. In a d d i t i o n to the genetic e v e n t s discussed, e p i g e n e t i c m e c h a n i s m s s u c h as h y p o m e t h y l a t i o n can play a role in m u l t i s t e p c a r c i n o g e n e s i s through the a b n o r m a l a c t i v a t i o n of genes. It is clear at this t i m e that there is no c o n s e n s u s gene that can e x p l a i n the n m l t i p l e array of p h e n o t y p e s associated w i t h this genetic disorder. Several genes have been p r o p o s e d to cause the h y p e r m u t a b i l i t y in BS, but c o n c l u s i v e proof at the m o l e c u l a r level has not b e e n p r e s e n t e d . Recent c l o n i n g of the uracil-DNA glycosylase gene will s o o n p r o v i d e an a n s w e r as to w h e t h e r this gene is i n d e e d altered in BS cells [371. I d e n t i f i c a t i o n of s o m e other gene r e s p o n s i b l e for the BS p h e n o t y p e is possible. A n o v e l a p p r o a c h c o u l d rely on i d e n t i f y i n g a gene r e s p o n s i b l e for the g e n e r a t i o n of the i n t r a c e l l u l a r O 2 . . O v e r p r o d u c t i o n of Q ' is the only p r o p o s e d b i o c h e m i c a l defect. w h o s e control results in the m o d u l a t i o n of SCEs directly in BS cells. T h e large n u m b e r of p o t e n t i a l c a n d i d a t e s makes this an u n a t t r a c t i v e approach. F u r t h e r m o r e , w h e t h e r O2" g e n e r a t i o n is a c o n s e q u e n c e of s o m e other b i o c h e m i c a l aberration is not clear. In any event, the u n d e r s t a n d i n g of the n m t a g e n i c p a t h w a y s in BS will u n d o u b t a b l y lead to a better u n d e r s t a n d i n g of the m u l t i s t e p processes of neoplasia.

REFERENCES 1. German J, Passarge E (1989): Bloom's syndrome, XII. Report from tile Registry for 1987. Clin Genet 35:57-69. 2. German ], Takebe H (1989): Bloom's syndrome. XIV. The disorder in Japan. Clin Cenet 35:93-110, 3. Cleaver JE (1968J: Defective repair re,plication of DNA in xeroderma pigmentosum. Nature 218:652-656. 4. Passarge E, (1983): Bloom's syndrmne. In: Chromosome Mutation and Neoplasia, J German, ed, Alan R. lass, Inc.. New York, pp. 11-21. 5. Cohen MM, Levy HP (1989): Chromosome instability syndromes. In: Advances in Hunran Genetics, Vol. 18, H Harris, K Hirschhorn, eds. Plenum Press, New York, pp. 4:1-149. 6. Kuhn EM, Therman E (1956) Cytogenetics of Bloom's Syndrome. (lancer Genet Cytogenet 22:1-18. 7. Chaganti RSK, Schonberg S, German J (1974): A manyfold increase in sister chromatid exchanges in Bloom's syndrome lymphocytes. Proc Natl Acad Sci USA 71:4508-4512. 8. Ray JH, German J (1983): The cytogenetics of the chromosomes breakage syndromes. In: (]hromosome Mutation and Neoplasia, ] German, ed. Alan R. Liss, Inc., New York, I)P. 135-167. 9. Shiraishi Y, Yosida TH, Sandberg AA (1983): Analyses of Bromodeoxyuridine-associated sister chromatid exchanges (SCEs) in Bloom's syndrome based on cell fusion: Single and twin SCEs in endoreduplication. Proc NatlAcad Sci USA 80:4369 4373. 10. Tsuji H, Kojima T (1955/: Presence of abnormally high incidences of sister chromatid exchanges in three successive cell cycles in Bloom's syndrome ]ymphocytes. Chromosoma 93:87 93. 11. Tsuji H, Heartline MW, Latt SA (1958): Disparate effects of 5-bromodeoxyuridine on sisterchromatid exchanges and chromosomal aberrations in Bloom syndrome fibroblasts. Mutat Res 195:241 253. 12, German J. (1964): Cytological evidence for crossing-over in vitro in human lymphoid cells. Science 144:298-301. 13. Shiraishi Y, Ohtsuki Y {1987): SCE levels in Bloom-syndrome cells at very low bromodeoxyuridine (BrdU) concentrations: Monoclonal anti-BrdU antibody. Mutat Res 176:157-164. 14. German J, Schonberg S, Louie E, Chaganti RSK (1977): Bloom's syndrome. IV. Sister chromatid exchanges in lymphocyts. Am JHum Genet 29:248 255. 15. Bryant EM, Hoehn H, Martin GM (19791: Normalisation of sister chromatid exchange frequencies in Bloom's syndrome by euploid cell hybridisation. Nature 279:795-796.

A s p e c t s of B l o o m ' s S y n d r o m e

11

16. Aldaheff B, Velivasakis M, Pagan-Charry 1, Wright WC, Siniscalco M (1980): High rate of sister chromatid exchanges of Bloom's syndrome chromosomes is corrected in rodent human somatic cell hybrids, Cytogenet Cell Genet 27:8-23. 17. Shiraishi Y, Matsui S, Sandberg AA (1981): Normalization by cell fusion of sister chromatid exchange in Bloom syndrome lymphocytes. Science 212:82(I-822. 18. Yoshida H (1981): Complementation analysis of Bloom's syndrome by means of somatic cell hybridization. Proc ]pn Assoc Cancer Res 154. 19. Weksberg R, Smith C, Anson-Cartwright L, Maloney K [1988): Bloom Syndrome: A single complementation group defines patients of diverse ethnic origin, Am I Hum Genet 42:816-824. 20. Ray ]H, German J (1984): Sister chromatid exchange in chromosome breakage syndromes. In: Sister Chromatid Exchange, AA Sandberg, ed. Alan R. l,iss, inc., New York, pp. 553-577. 21. Nicotera TM, Notaro J, Notaro S, Schumer I, Sandberg AA (1989): Elevated superoxide disnmtase activity in Bloom's syndrome: A genetic condition of oxidative stress. Cancer Res 49:5239-5243. 22. Willis AE, Spurr NK, Lindahl T {1989): Concomitant reversiol/of the characteristic phenotypic properties of a cell line of Bloom's syndrome origin. Carcinogenesis 10:217-219. 23. Shiraishi Y, Kobuchi H, Utsumi K, Minowada J (1990): Levels of sister-chroinatid exchanges in hybrids between bloom syndrome B-lymphohlastoid (:ells and various cell lines with lymphoid malignancy. Mutat Res 243:13-20. 24. Willis AE, Lindahl T (1987}: DNA Ligase deficiency in Bloom's syndrome. Nature ;125:355-357. 25. Chan JYH, Becker FF, German J, Ray JH {19871: Altered I)NA Ligase 1 activity in Bloom's syndrome cells. Nature 325:357-359. 26. Willis AE, Weksberg R, Tomlinson S, Lindahl T {1987): Structural alterations of DNA ligase ! in Bloom syndrome. Proc Natl Acad Sci USA 84:8016-8020. 27. Lehman AR, Willis AE. Broughton BC, James MR, Steingrimsdottir H, Harcourt, Arlett CF, Lindahl T (1988): Relation between the human fibroblast strain 46BR and cell lines representative of Bloom's syndrome. Cancer Res 48:6343-6347. 28. Chan ]YH, Becker FF (1988): l)efective DNA Ligase I in Bloom's syndrome cells, l Biol Cbem 263:18231-18235. 29. Mezzina M, Nardelli ], Nocentini S, Renault (L Sarasin A (19891: I)NA ligase activity in h u m a n ceils from normal donors and Bloom's syndrome patients. Nucleic Acid Res 17:3091 3106. 3(I. Mezzina M, Nocentini S, Nardelli J, Renault G, Moustacchi E, Sarasin A (19891: Enhanced deoxyribonuclease activity in Human transformed ceils and in Bloom's syndrome. Mol Carcinogenesis 2:179-183. 31. Gupta P, Sirover MA {1984): Altered temporal expression of DNA repair in hypermutable Bloom's syndrome cells. Proc Natl Acad Sci t1SA 81:757-761. 32. Fujiwara Y, Yamamoto Y {19861: Abnormal regulation of uracil-DNA glycosylase induclion during cell cycle and cell passage in Bloom's syndrome fibroblasts, Carcinogenesis 7:305-310, 33. Vilpo ]A, Vilpo LM {1989): Normal uracil-DNA glycosylase activity in Bloom's syndrome cells. Mutat Res 210:59-62. 34. Vollberg TM, Seal G, Sirover MA (1987): Monoclonal antibodies detect conformational abnormality of uracil I)NA glycosylase in Bloom's syndrome cells. Carcinogenesis 8:1725-1729. 35. Seal G, Brech K, Karp S], Cool BL, Sirover MA {1988): Imnmnological lesions in human uracil DNA glycosylase: Association with Bloom syndrome, Proc Natl Acad Sci USA 85:2339-2343, 36. Seal G. Henderson EE, Sirover MA (1990): Immunological alteration of the Bloonfs syndrome uracil DNA glycosylase in Epstein Barr virus-transfected h u m a n lymphoblastoid cells. Murat Res 243:59-62. 37. Olsen LC, Aasland R, Wittwer CU, Krokan HE, Helland DE (1989): Molecular cloning of h u m a n uracil-DNA glycosylase, a highly conserved DNA repair enzyme. EMBO 1 8:3121-3125.

12

T.M. Nicotera

38. Vollberg TM, Cool BL, Sirover MA (1987): Biosynthesis of the h u m a n excision repair enzyme uracil-DNA glycosylase. Cancer Res 47:123-128. 39. Dehazya P, Sirover MA (1986): Regulation of hypoxanthine DNA glycosylase in normal h u m a n and Bloom's syndrome fibroblasts. Cancer Res 46:3756-3761. 40. Kim S, Vollberg TM, Ro J-Y, Kim M, Sirover MA (1986}: (OCMethylguanine methyltransferase increases before S phase in normal h u m a n cells but does not increase in hypermutable Bloom's syndrome cells, Mutat Res 173:141-145. 41. Hassan H (1989): Microbial superoxide dismutases. Adv Genet 26:43-97. 42. Hassan HM, Fridovich I (1979): Intracellular production of superoxide radical and hydrogen peroxide by redox active compounds. Arch Biochem Biophys 196:385-395. 43. Chan E, Weiss B (1987): Endonuclease IV of Escheriehia coli is induced by paraquat. Proc Natl Acad Sci USA 84:3189-3193. 44. Greenberg JT, Demple B (1989): A global response is induced in Escherichia eoli by redoxcycling agents overlaps with that induced by peroxide stress. I Bacteriol 171:3933-3939. 45. Storz G, Tartaglia LA, Ames BN (1990): Transcriptional regulator of oxidative stress-inducible genes: Direct activation by oxidation. Science 248:189 194. 46. Brambilla G, Sciaba L, Faggin P, Maura A, Mariani UM, Ferro M, Esterbauer H (1986): Cytotoxicity, DNA fragmentation, and sister chromatid-exchange in chinese hamster ovary cells exposed to lipid peroxidation product 4-hydroxynonenal and homologous aldehydes. Murat Res 171:169-176. 47. Hemler ME, Lands WEM (1980): Evidence for a peroxide-initiated free radical mechanism of prostaglandin biosynthesis. J Biol Chem 255:6253-6261. 48. Bray RC, Cockle SA. Fielden EM, Roberts PB, Rotilio C, Calabrese L (1974): Reduction and inactivation of superoxide dismutase by hydrogen peroxide. Biochem J 58:1174-1184. 49. Kono Y, Fridovich I (1982): Superoxide radical inhibits catalase. J Biol Chem 257:5751-5754. 50. Blum J, Fridovich I (1985): Inactivation of glutathione peroxidase by superoxide radicals. Arch Biochem Biophys 240:500-508. 51. Vuillaume M, Calvayrac R, Best-Belpomme M, Tarraoux P, Hubert M, Decroix Y, Sarasin A (1986): Deficiency in catalase activity of Xeroderma pigmentosum cells and in simian virus 40-transformed h u m a n cell extracts. Cancer Res 46:538-544. 52. Krokan H, Crafstrom RC, Sundqvist K, Esterbauer H, Harris, CC {1985): Cytotoxicity, thiol depletion and OCmethylguanine-DNA methyltransferase by various aldehydes in cultured h u m a n bronchial fibroblasts. Carcinogenesis 6:1755-175!}. 53. Poot M, Hoehn H, Nicotera TM, Rudiger HW {1989): (Jell kinetic evidence suggests oxidative stress in cultured cells of Bloom's syndrome. Free Rad ResComms 7:179 187. 54. Nicotera TM, Christopher R, Beehler B {1990): Lack of 5-methyh:ytosine in Bloom's syndrome lymphoblast DNA. Am Assoc ('ancer Res 31:68 55. Riggs AD, Jones PA (1983): 5-Methylcytosine, gene regulation and cancer. Adv (lancer Res 40:1 30. 56. Kato H (1974): Possible role of DNA synthesis in formation of sister chromatid exchanges. Nature 252:739-741. 57. Spanos A, Holliday R, German I {1986): Bloom's syndrome XII. DNA-polymerase activity of cultured lymphoblastoid cells. Hum Genet 7:1:119-122. 58. Pommier Y, Zwelling, Kao-Shan C-S. Whang-Peng J, Bradley MO (1985): Correlations between intercalator induced DNA strand breaks and sister chromatid exchanges, nmtations, and cytotoxicity in Chinese hamster cells. Cancer Res 45:3143 3149. 59. Heartline MW, Tsuji H, Latt SA (1987]: 5-Bromodeoxyuridine-dependent increase in sister chromatid exchange formation in Bloom' syndrome is associated with reduction in topoisomerase 11 activity. Exp Cell Res 169:245-254. 60. Ponrmier Y, Runger TM, Kerrigan D, Kraemer KH (1988): Relation of topoisomerase lI activity to DNA single-strand breaks induced by 5-bromodeoxyuridine, topoisomerase lI inhibitors, or 313nm ultraviolet light in Bloom's syndrome fibroblasts. ] Cell Biochem 12A:296. 61. Pommier Y, Runger TM, Kerrigan D, Kraemer K (1988): Abnormal topoisomerase activity in Bloom's syndrome fibroblasts. J Cell Biochem 12A:191. 62. Runger RM, Kraemer KH (1989): Joining of linear plasmid DNA is reduced and error-prone in Bloom's syndrome cells. EMBO I 8:1419-1425.

A s p e c t s of B l o o m ' s S y n d r o m e

13

63. l,anglois RG, Bigbee WL, lensen RH, German I (1989): Evidence for increased in vivo mntation and somatic recombination in Bloom's syndrome, Proc Natl Acad Sci USA 86:670-674, 64. Hellgren D. Sahlen S, Bamberg B {1990): Unequal S(]E is a rare event in homologous recombination between duplicated neo gene fragments in CHO ceils. Mutat Res 243:75 80. 65. Kvoizumi S, Nakamura N, Takebe H, Tatsumi K, German 1, Akivama K (1989): Frequency of variant ervthrocytes at the glycophorin-A locus in two Bloom's svndronle patients. Mutat Res 214:215-222, 66. Kcnnc K, Ljungquist S {1987]: RecA-like acth'ity in mammalian cell extracts of different origin. Mutat Res 184:229-236. 67. Knudson AG (1985): Hereditary cancer, oncogenes and antioncogenes. (]am:er Res 45:1437-1443. 68, Grundy P, Koufos A. Morgan K, l,i, FP, Meadows AT, CavaneeW(1988): Familial predisposition to Wihn's tumor does not map to the short arm of chromosome 11. Nature 336:374-376. 69. Vogelstein B, Fearon ER, Baker SI, Nigro ]M, Kern SE, Hamilton SR, Bos ], Leppert M, Nakamura Y, White R (1989): Genetic alterations accumulate during colorectal tumorigenesis. In: Recessive Oncogenes and Tumor Suppression, W Cavanee, N Hastle, E Stanbridge, ed., (]old Spring Harbor Laboratory, Cold Spring Harbor, NY. 70. Knudson AG (1989): The genetic predisposition to cancc'r, in: Geneti~ Susceptibility to Environmental Mutagens and Carcinogens, Monograph No, 2 AD Bloom, L Spatz, N\\,' Paul. eds. March of Dimes, White Plains, New York, pp. 15-27. 71. Oshimura M, (Elmer T, Barrett j(2 {1985): Nonrandom loss of chromosome 15 in Syrian hamster tumours induced by v-Ha-ras plus v-myc oncogenes. Nature 316:636-639. 72. Sullivan NF, Willis AE, Moore JP, l,indahl T (1989): High levels of the C-lnV(; protein in cell lines of Bloom's syndrome origin. Oncogene 4:1509-1511. 73. Crawford D, Zbinden I, Amstad P, Cerutti P (1988): Oxidant stress induces tile protooncogene.s c-fos and c-myc in mouse epidermal ceils. Oncogene 3:27-32. 74. Shibanuma M, Kuroki T, Nose K (19881: Induction of DNA replication and expression of proto-oncogene c-myc and c-los in quiescent Balb/3T3 cells by xanthine oxidase. Oncogene 3:17 21.