Sister Chromatid Exchange in Cancer*

Transcription

1 ANNALS OF CLINICAL AND LABORATORY SCIENCE, Vol. 13, No. 4 Copyright 1983, Institute for Clinical Science, Inc. Sister Chromatid Exchange in Cancer* PETER H. KOHN, P h.d. Department of Pediatrics University of Florida College of Medicine Gainesville, FL ABSTRACT Sister chrom atid exchange (SCE) techniques have been shown to have great potential for use in the in vitro screening of suspected mutagens and carcinogens. Application of these techniques to bone marrow cells and/or lym phocytes from patients w ith various hem atologic m alignancies has demonstrated significant abnormalities in SCE frequencies in some of these diseases, as well as showing drug treatm ent effects. Interpretation of abnorm al SCE findings in cancer patients is currently hindered by the lack of a clear understanding of the basic molecular and biochemical mechanisms involved in SCE formation. The potential practical use of SCE techniques in the diagnosis and treatm ent of cancer can not adequately be appreciated until this basic understanding is achieved. Introduction though it appears to involve an interaction between the stains employed and the molecular structure of the BrdU-substituted chromosomes. W hether SCE occurs spontaneously or is induced by BrdU that is incorporated is unknow n. It is clear, how ever, that large increm ents in SCE can be induced either in vivo or in vitro by a wide range of physical, chem ical, and biological agents, including m utagens, carcinogens, and therapeutic drugs.3840 Many of these agents have been causally implicated in the etiology of various neoplasias. As such, SCE has been advocated as an extrem ely sensitive test for detecting the presence of potentially m u tagenic and/or carcinogenic substances.7-38 A pplication of SCE techniques to the study of cancer has been a logical outcome from these findings. It is the intent of this review to summarize data pertinent to SCE and neoplasia /83/ $01.20 Institute for Clinical Science, Inc. Sister chrom atid exchange (SCE) re fers to the reciprocal, apparently equal exchange of deoxyribonucleic acid (DNA) betw een sister chrom atids from individual chromosomes. This phenom enon can be observed in m etaphase preparations from cells that have been exposed to the thym idine analogue, 5-bromodeoxyuridine (BrdU), for two synthetic (S) phases of the cell cycle, followed by the application of appropriate staining techniques.3039 Sister chromatid exchanges are visible as sharp discontinuties in staining intensity along a chromatid (figure 1). The m echanism involved in th e differential staining of sister chrom atids by BrdU-labeling methods is poorly understood, al * Address reprint requests to Peter H. Kohn, Ph. D., Division of Genetics, Department of Pediatrics, Box J296, University of Florida, Gainesville, FL

2 268 KOHN a. b. f n- produce effects similar to those of x-ray; 0 SCE I SCE I SCE F i g u r e 1. Diagrammatic and pictorial representation of differential staining and SCE obtained following two rounds of replication in the presence of BrdU. (a) Diagrams showing differential staining of chromatids, and from left: to right, 0 and one SCE, respectively, (b) Photograph showing one SCE. Arrows and dotted lines indicate the location of SCE. Shaded chromatid material consists of one thyminesubstituted (parental) and one bromouracil-substituted DNA strand; unshaded chromatid material consists of two bromouracil-substituted strands. Sister C hrom atid Exchange in Normal Cells Sister chrom atid exchange frequencies in phytohem agglutinin (PHA)-stimulated lymphocytes from normal individuals vary widely from laboratory to laboratory (table I). These differences can primarily be attributed to such factors as concentration of BrdU, concentration and components of sera, conditions of culture, and processing of cells. Large differences betw een norm al donors are also obtained w ithin individual laboratories, m aking interpretation of abnormal SCE levels in peripheral blood lymphocytes somewhat difficult. Bone marrow cells have an advantage over lymphocytes in that SCE frequencies are lower and have a narrow er range. A recent report11 suggests that baseline SCE frequencies follow a multifactorial mode of inheritance. Effects of Various Agents on Sister Chrom atid Exchange E levations of SCE above baselin e levels can b e o b serv ed follow ing th e treatm ent of cells with a wide variety of exogenous agents. In general, substances can be divided into two or perhaps three groups according to their efficiency at inducing SCE and chromosome breakage. O ne group consists of substances that that is, m inimal increases in SCE frequency are obtained at dose levels sufficient to cause significant increases in chromosome breakage.3-25'28 Among those agents reported to have this type of effect are bleom ycin, caffeine, 8-ethoxycaffeine, and nitrosoguanidine. A second group of agents shows effects similar to those found following the treatm ent of cells with ultraviolet (UV) light Even at very low doses, these substances are capable of producing large increm ents in SCE and, in addition, some are also capable of producing significant levels of chromosome aberrations. Within this UVlike category can be placed most alkylating agents and some DNA intercalating agents, such as acrid in e orange and ethidium brom ide. Bifunctional alkylating agents, such as mitomycin C and chlorambucil are generally much b etter in d u cers o f S C E 38 and chrom osom e breakage23 than are m onofunctional alkylating agents. Even within the class of m onofunctional ag en ts, differen ces in SCE response can be observed, m ethylated sulfonates (e.g., m ethyl m ethanesulfonate) being more potent SCE inducers than their ethylated counterparts TABLE I Selected Reports on Sister Chromatid Exchange in Normal Human Lymphocytes SCE/ M e ta p h a s e No. o f s u b j e c t s N o. o f M e ta p h a s e s BrdUt \ig / m l~ l R e f e r e n c e N.D.* , , *N.D. «no data f5-bromodeoxyuridine

3 (e.g., ethyl m ethanesulfonate).38 Clearly, a wide variety of DNA-damaging agents possesses the capability of inducing SCE; fu rther screening of chem icals is w arranted in order to attem pt to clarify the relationship of SCE to different types of DNA damage. Most of the SCE-inducing compounds thus far tested have been proven to be mutagenic and/or carcinogenic.4 In combination w ith analysis for chrom osom e breakage, SCE appears to be a sensitive, specific, and accurate test for potential carcinogenicity, although only a minority of the 465 suspected carcinogens41 have to date been adequately tested for carcinogenicity.50 A final decision as to the ultim ate applicability of the SCE test to carcinogen detection m ust await results from further testing. Sister C hrom atid Exchange in Hematologic Diseases The ability of SCE techniques to detect DNA-damaging agents and carcinogens with great sensitivity in vitro has led to the application of these techniques in the study of human cancer. The majority of studies have been perform ed using peripheral blood lymphocytes, and in most instances SCE frequencies have b een found to fall w ith in th e norm al range.12 Findings of increased SCE have generally b een a ttrib u ted to th e action of chem otherapeutic treatm en t of patie n ts.40 R elatively few a tte m p ts have been m ade to test the SCE response of actual tum or cells. D etection of those individuals with preleukem ic conditions that will go on to develop a frank leukem ia could allow for early, and perhaps more effective, treatment. The potential value of SCE analysis for this purpose has not yet been adequately determined. In the single known p u b lish ed stu d y,26 b o n e m arrow SCE levels from five untreated patients diagnosed as having preleukemia were found SIST E R C H R O M A T ID E X C H A N G E IN C A N C E R 2 69 to be not significantly different from those of normals. However, one of the patients did show a significant increase in SCE in cells from a pseudodiploid (5q-) clone as com pared w ith the patient s diploid bone marrow cells. Chronic myelogenous leukemia (CML) p rovides an ex cellen t o p p o rtu n ity to study SCE in tum or cells, as the leukemic cells can readily be identified by virtue of th e ir having th e Philadelphia (Ph1) chrom osom e. To date, four such studies have been perform ed (table II). Two groups of investigators, Kakati et al21 and Knuutila et al,26 found SCE levels in bone marrow from treated chronic phase CM L patients not to be significantly diffe re n t from th o se of norm al controls. These same investigators observed significant increases in SCE in treated blastic phase CM L patients, although the total n u m b e r of indiv id u als exam ined was quite small. In contrast, Becher et al5 and Stoll et al48 both found a significantly decreased freq u en cy of SCE in bone m arrow from u n tre a te d chronic phase CM L patients (table II). In a follow-up of th eir original 16 patients, Stoll et al48 found a fu rth er highly significant d e crease in SCE at blast crisis. In CM L, it therefore appears that SCE levels are reduced below those normally found, although the reason for this is not clear. In TABLE II Bone Marrow Sister Chromatid Exchange in Patients with Chronic Myelogenous Leukemia D i s e a s e N o. o f T r e a t SCE R e f e r P h a s e P a t i e n t s m e n t F r e q u e n c y * e n c e Chronic 8 + NC 21 Blastic Chronic 3 + NC 26 Blastic Chronic Chronic Blastic *NC = not significantly different from controls; + = significantly increased; + = significantly decreased

4 2 70 K O H N those chronic phase cases w here normal SCE levels have been obtained,2126 it can be hypothesized that chem otherapeutic treatm ent of these patients induced this SCE response. O ther data are needed in order to establish the relationship of SCE in treated and untreated CML. Bone marrow SCE studies on patients with acute myelocytic leukem ia (AML) (table III) have shown normal SCE frequencies at diagnosis1-2 and elevations of SCE following treatm ent at rem ission12'26 and at relapse,126although a tendency toward low SCE in untreated patients was noted in one rep o rt.1in a study designed to examine SCE in both nonleukemic and leukemic cell populations of patients with AML (table III), Abe and Sanberg2 analyzed diploid and aneuploid cells from 10 such patients. No differences w ere found at diagnosis betw een these two cell types or betw een either cell type and controls. Only the diploid cells showed a significant increase in SCE at remission, indicating to the authors that the response of SCE to chemotherapy was lower in aneuploid (possibly leukem ic) cells than in diploid (possibly nonleukem ic) cells. TABLE III Bone Marrow Sister Chromatid Exchange in Patients with Acute Myelocytic Leukemia D i s e a s e P h a s e N o. o f P a t i e n t s T r e a t m e n t SCE F r e q u e n c y R e f e r e n c e Diagnosis (diploid) 6 NC 1 Remission (diploid) Relapse (diploid) Diagnosis _ (diploid) 10 NC 2 Diagnosis _ (aneuploid) 10 NC 2 Remission (diploid) Remission (aneuploid) 1 + NC 2 Remission Relapse *NC = not significantly different from controls; + = significantly increased TABLE IV Sister Chromatid Exchange in Childhood Acute Lymphocytic Leukemia at Diagnosis N u m b e r o f P a t i e n t s : C o n t r o l s : P a t i e n t s S p e c i SCE/ S C E / R e f e r ( C o n t r o l s ) m en C hrom osom e C hrom osom e e n c e 16 (14) Stimulated blood 8 (4) Stimulated marrow 4 (2) Unstimu-lated marrow These SCE increases obtained following treatm ent can again likely be ascribed to th e action of th e ch em o th e ra p eu tic agents. Several reports exist in which SCE increases in peripheral blood lymphocytes of patients with various hematologic m a lignancies have been shown. In some diseases, sim ilar findings have been obta in e d using b o n e m arrow cells. In a study of p erip h eral blood lym phocytes from 16 untreated childhood acute lymphocytic leukem ic (ALL) patients, O tter et al35 showed a highly significant SCE elevation at the tim e of diagnosis (table IV). Seven of these patients were restudied as long-term survivors. Levels of SCE rem ained elevated in three patients from this group who had been on continuous chem otherapy for at least eight months prior to the tim e samples w ere obtained. Their SCE frequencies w ere found to be not significantly different from those of normal controls. It has been suggested42 that the SCE elevations observed in the untreated patients may reflect viral infection and possible systemic effects of the leukemic process, although other interpretations are equally plausible.35 The data from survivors in this study strongly im p licate c h e m o th e ra p eu tic agents as SCE inducers. In a follow-up study of these ALL lymphocyte findings, Heerem a et al20 exam

5 ined bone m arow cells from untreated childhood ALL patients. Frequencies of SCE were found to be significantly elevated over controls in both PHA-stimulated and unstim ulated marrow cultures (table IV). Values of SCE for both ALL patients and controls w ere significantly higher in stim ulated as com pared with unstim ulated samples, indicating either a direct effect of the PHA or PHA selection of a cell type with elevated SCE. Levels of SCE in stim ulated m arrows w ere similar to those previously obtained35 in stim ulated lymphocytes (table IV). The finding of increased SCE freq u en cies in lym phocytes and bone m arrow from u n tre a te d ALL p atien ts raises the question of the possible use of SCE analysis as an adjunctive diagnostic test in this disease; however, further confirmational studies are necessary. Sister chrom atid exchange levels have been found to be significantly elevated in lymphocytes from patients with chronic lymphocytic leukemia (CLL) who had received chem otherapy w hen com pared with untreated patients and normal controls.32 Increased SCE levels were interp reted as being a function of therapy and not of the leukemic process. K nuutila et al27 examined SCE in bone marrow cells and lymphocytes from eight u n treated patients w ith m egaloblastic anem ia. A lthough significant SCE elevations w ere found in both lymphocytes and bone marrow cells from these patients, th e resu lt was m ore m arked in bone marrow. This increase was attributed to the presence of small populations of bone marrow cells which had as much as a tenfold increase in SCE. The authors speculated that this finding may have been caused by im pairm ent of DNA synthesis or increased incorporation of BrdU into the DNA of the cells. In a study involving 47 patients with m alignant lym phom a, K urvink et al31 found more than a doubling of SCE in perip h eral blood lym phocytes from 13 SIST ER C H R O M A T ID E X C H A N G E IN C A N C E R 271 untreated patients and a further increase in SCE in 11 patients who had received cyclophospham ide w ithin four w eeks prior to study. Seven patients treated with radiotherapy alone showed SCE values not significantly different from those of norm al controls. These data suggest that untreated patients with malignant lymphom a have elevated SCE frequencies which may be further increased by treatm en t w ith c e rta in c h e m o th e ra p eu tic agents. Abnormal SCE frequencies appear to be associated with at least some hum an neoplastic states. D ecreased SCE levels have been dem onstrated in CM L patients,548 and similar findings have been suggested in A M L.1 Increases in SCE have been shown to occur in childhood ALL,20-35 megaloblastic anemia,27 and malignant lymphoma.31 The ability of various chem otherapeutic agents to induce high levels of SCE both in vitro38 and in cells from patients with various malign an cies40 has b e e n w ell estab lish ed. W h eth er the aberrant SCE responses observed in untreated patients with these malignancies reflect basic neoplastic processes, or represent viral involvement, secondary physiologic changes, or other related phenom ena can not be d e te r m ined at the present time. Informative interpretation of these data is prevented by a lack of understanding of the basic m echanism (s) involved in SCE form a tion. Sister C hrom atid Exchange in the Chromosome Instability Syndromes In c re a s e d le v e ls o f c h ro m o so m e breakage are found constitutionally in Bloom syndrom e (BS), ataxia telangiectasia (AT), and Fanconi anem ia (FA).15 This phenom enon is also observed in xero d erm a p ig m en to su m (XP), b u t only after exposing cells from these patients to UV lig h t.37 Increased chrom osom e

6 272 K O H N breakage in these four chromosome instability syndromes has been thought to be a manifestation of defects in DNA repair, as has their commonly shared increased susceptibility to cancer.43 Since a significant num ber of mutagens and carcinogens w ith know n D N A -dam aging cap ab ilities also p ro d u c e increases in chromosome breakage and, in addition, elevations in SC E,3-7'38 the chromosome instability syndromes have been selected for intensive study in an attem pt to explain the basic nature of SCE. In untreated cells from patients with three of these diseases (FA,19 AT,29 and XP9) with dem onstrated DNA repair deficien cies,43 th e freq u en cy o f SC E is normal. In BS cells, however, there is a 10 to 15 fold in crease in spontaneous SCE.8 This frequency can be raised further by chemicals such as mitomycin C (M M C ).46 Bloom sy n d ro m e cells have been shown to be normal in terms of their capacity to carry out all known forms of DNA repair,51 although DNA replication has been rep o rted to proceed abnormally. 16 Thus, available evidence from the chromosome instability syndromes does not suggest any direction connection betw een SCE and know n D N A re p air mechanisms. Proposed M odels for Sister Chrom atid Exchange A num ber of hypotheses have been proposed to explain th e existence and significance of SCE. Theories attem pting to correlate SCE induction with particular forms of DNA damage have failed to provide a unifying concept to account for all that is known about SCE formation. O ther models have focused not on DNA repair itself, b u t rather replication and disturbances thereof. O ne such model is the replication bypass m odel of Shafer.44 In this model it is proposed that a SCE is generated w hen bidirectional replicating processes encounter an intact or p artially re p a ire d D N A lesion, w hich serves to block further replication fork progress. Parental strand incisions effected by repair endonucleases and tortional tw isting w ould produce parental strand transfers and allow replication to bypass the lesion and proceed normally. T he site of th e DNA strand exchange process w ould th en b e visualized as a SCE. This m odel has b een criticized47 owing to its perceived inability to explain certain SCE data. Modifications of this m odel45 involving several alternate, but not mutually exclusive, methods of replication bypass appear to overcome these objections and warrant the need for further study. A second proposed replication model36 states that blockage of replication in some, b u t not all, replication clusters (areas) leads to juxtaposition of unreplicated and replicated segm ents of DNA for extended periods of time. D uring this tim e double strand breakage, perhaps enzymatically catalyzed, occurs at the junctio n s b e tw e e n th e s e c lu s te rs w ith subsequent reannealing. Occasionally, instead of normal reannealing, the break may b e sealed by th e jo in in g of th e daughter strands of the replicated molecule to the parental strands of the unreplicated molecule, creating at this juncture what will ultim ately be visualized as an SCE. This m odel predicts that substances that block replication will induce SCE, regardless of their ability to produce various DNA lesions, and that these effects would be dose dependent. Unfortunately, there is no direct evidence to support this m odel as yet, although it appears to be consistent with SCE data accumulated thus far. The m ajor im pedim ent to more w idespread application of SCE techniques in the study of carcinogenesis is the rather poor current understanding of the SCE mechanism itself. Until the molecular and biochemical details of SCE formation are

7 clarified, th e biological significance of SCE will remain unresolved. R eferences 1. A b e, S., K a k a t i, S., and S a n d b e r g, A. A.: Growth rate and sister chromatid exchange (SCE) incidence of bone marrow cells in acute myeloblastic leukemia (AML). Cancer Genet. Cytogenet. 1: , A b e, S. and S a n d b e r g, A. A.: Sister-chromatid exchange and growth kinetics of marrow cells in aneuploid acute nonlymphocytic leukemias. Cancer Res. 40: , A b e, S. and S a s a k i, M.: Chromosome aberrations and sister chromatid exchanges in Chinese hamster cells exposed to various chemicals, J. Natl. Cancer Inst. 5S , A b e, S. and S a s a k i, M. : SCE as an index of mutagenesis and/or carcinogenesis. Sister Chromatid Exchange. Sandberg, A. A., ed. New York, Alan R. Liss, 1982, pp B e c h e r, R., S c h m i d t, C. G., T h e i s, G., and H o s s f e l d, D. K.: Sister chromatid exchange in Ph1-positive chronic myelocytic leukemia. Internat. J. 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