John R K Savage
March 1999
(MRC Radiation and Genome Stability Unit, Harwell, Didcot, OX11 0RD,UK)
Introduction
Visible changes to chromosome structure and morphology have played a veryimportant part as indicators of genetic damage in both clinical and cancerstudies.
Most of the changes encountered in clinical studies are "secondary" or"derived" aberrations. This is true also in cancer studies, except that here,there is an ongoing production of aberrations, so that in some cells, a mixtureof primary and secondary changes is present, and a continuously changingkaryotype (true chromosomal instability).
To appreciate these observed secondary changes we need to understand theprimary changes from which they are derived, and it is the purpose of thisarticle to provide a brief introduction to them.
Observation
Primary aberrations are those seen at the first post-induction division,when all the parts are present and there has been no selection by passagethrough mitosis, nor any modification by subsequent chromosome duplication(Savage, 1976).
Most commonly, observation is made at metaphase, using "solid-staining" with dyes which give high-contrast chromatin staining and negligible cytoplasmic coloration. For more critical work, the chromosomes are banded in various ways, which allows chromosome identification, detection of some forms invisible with solid-staining, and offers more precise positioning of the lesion interaction points (Savage, 1977).
Recently, resolution and classification of transmissible forms has been considerably improved by the introduction of fluoresence in situ hybridisation (FISH) chromosome "painting" (Lucas et al., 1992; Savage and Tucker, 1996; Tucker et al., 1995) .
Classification of Primary changes
For purely pragmatic and diagrammatic purposes, we can regard the chromosomal changes we see down the microscope as being the result of "breaks" followed by "re-joins" of the chromosome thread. However, we must always remember that, in reality, their origin is much more complicated (Savage, 1998; Savage and Harvey, 1994).
Since the chromosome we see and score at metaphase has two (sister-)chromatids, it is convenient (and conventional) to divide all aberrations intotwo broad types:
Chromosome-type where the breaks and re-joins always affectboth sister-chromatids at any one locus. Examples in Figure 1.
Chromatid-type where the breaks and re-joins affect onlyone of the sister-chromatids at any one locus (Fig 2).
Figure 1
Figure 2
The distinction is important. For some aberration-inducing agents, likeionizing radiation, the type of aberration recovered at metaphase reflects theduplication status of the chromosomes in the treated cell. But, for themajority of chemical agents which can induce aberrations, for ultra-violetlight, and most probably all "spontaneous" (and de novo aberrations)only primary chromatid-types are recovered. When, atsubsequent interphase, the chromatids duplicate, surviving aberrations (andbits of aberrations) are converted into apparent chromosome-types, someof which are then transmitted almost indefinitely to further cell generations.These are the "derived" aberrations, and many are so modified that it isimpossible to deduce their primary origin.
Thus, following an "acute" treatment with any clastogen, surviving cellsin later generations carry only chromosome-type changes. Thepresence in such cells of chromatid-type aberrations is, therefore, anindicator of an ongoing production of primary structural changes, i.e. of someform of chromosome instability.
Nearly all the aberrations we see with solid staining appear toresult from the interaction ("re-joining") of two breaks, so we can furtherclassify them on the basis of where these breaks are situated in relation tothe chromosome arms (Savage, 1976).
- If the breaks are situated in the arms of different (non-homologous orhomologous) chromosomes we have the category of INTERCHANGES.
- If the breaks are in the opposite arms of the same chromosome, we havethe category of INTER-ARM INTRACHANGES.
- If the two breaks are both in the same arm of a chromosome, we have thecategory of INTRA-ARM INTRACHANGES.
These three categories are often referred to collectively asEXCHANGES.
- Finally, some aberrations appear to arise from a single, open break injust one arm. This category we term "BREAKS" or "DISCONTINUITIES". Many(perhaps all) of them are, in reality, intra-arm intrachanges where one end hasfailed to join up properly, though the limitations of microscopical resolutiondo not permit us to be certain that the re-joining is really incomplete.
The newer techniques, like FISH chromosome painting, are telling us that a lot of the chromosome-type aberrations we see and score as "simple" two-break interactions actually involve more than two breaks, and often more than two chromosomes, i.e. they are COMPLEX EXCHANGES (Savage and Simpson, 1994; Savage and Tucker, 1996; Savage et al., 1984; Simpson et al., 1995). These types have always been known to be fairly frequent for chromatid-types , but until the advent of FISH, were always considered to be rare for chromosome-types. However, as most complexes will be non-transmissible, and therefore rarely encountered in clinical studies (INSERTIONS, and CYCLICAL EXCHANGES involving several chromosomes, are examples of transmissible complexes), we will not look at them in any detail.
Interaction between the four ends of two breaks can obviously take place inthree ways :
- Join back to re-form the original chromosomes ("RESTITUTION") so that noaberration is produced.
- Re-join in such a way that an acentric fragment is always formed(ASYMMETRICAL RE-JOINING, A ). These forms are invariably visible withsolid staining. The fragment (which, if visible microscopically, will containmany megabases of DNA) will be lost at anaphase, and, in the case ofINTERCHANGES and INTER-ARM INTRACHANGES, there will also be mechanicalseparation problems producing "anaphase bridges". Asymmetrical aberrations aretherefore almost always cell-lethal, and so rapidly disappear from a populationof continuously dividing cells (Lea, 1946; Savage, 1989). Thus, they are rarely encountered inclinical situations where there is not an ongoing induction of aberrations.Instability in cancer cells, however, does lead to the occasional presence ofasymmetrical ("unstable") changes.
- Re-join in a way that never leads to an acentric fragment unless one of the re-joins is incomplete (SYMMETRICAL RE-JOINING, S ). Many such symmetrical chromosome-type exchanges are not visible with solid-staining, and their accurate detection requires special techniques like banding or FISH-painting (Lucas et al., 1992). In contrast, most symmetrical chromatid-type exchanges are visible with solid staining because of the retention of sister-chromatid adherence until metaphase. Because there is no loss of genetic material, and no mechanical problems at mitosis, most symmetrical forms are transmissible to future cell generations (Savage, 1976; Savage, 1995), hence they constitute the bulk of the recovered "derived" aberrations encountered in clinical and cancer cytogenetics.
As mentioned, re-joining can sometimes be (apparently) INCOMPLETE. This ismuch more frequent for chromatid-type aberrations (typically 30-50% ofinterchanges) than it is for chromosome-type aberrations (difficult tomeasure accurately, but probably around 3-5%). Incompleteness leads to geneticloss, and so to increased cell lethality.
The four basic categories discussed above are seen in their simplest formsfor chromosome-type aberrations, as shown in Figure 1. Traditionally,certain forms have specific names, as indicated. Symmetrical forms are seldomvisible with solid staining (probably less than 20% of reciprocaltranslocations lead to an obvious change in chromosome morphology), so, inthe diagrams, these forms are shown in two colours, just as they are detectedwhen using FISH chromosome (or arm) painting. There is no reliable method asyet for detecting paracentric inversions.
Because sister-chromatids tend to adhere, strongly, along their lengths,many chromatid-type symmetrical forms remain visible without recourseto special staining methods, as Figure 2 shows. Moreover, the presence ofsister-chromatids allows additional lesion interactions (inter-chromatidintra-arm intrachanges) not possible with chromosome-type changes.As a consequence, there is a much higher frequency of chromatid-typechanges, and a much greater variety of forms, compared withchromosome-types, following a given treatment.
Moreover, the interactions within chromosome arms which are now possiblemake chromatid-type aberrations a much more likely source of thecomplicated duplications/deletions etc. encountered in clinical and cancerstudies.
Combinations of the various categories are frequent, especially forchromatid-types (interchange/intra-arm intrachange particularlyso, giving rise to configurations like "triradials" (Savage and Harvey, 1994)), but these, ofcourse, constitute a type of complex exchange. Surviving remnants of suchevents are responsible for some of the curious anomalies recorded in clinicaland cancer cell studies.
Relationship to the cell cycle
Conventionally, the period between successive mitoses ("INTERPHASE") issub-divided into three phases G1, S and G2 . For critical work, furthersub-division of S is possible (Savage et al., 1984). G1 is the pre-duplication period, whenthe cell begins to prepare for DNA synthesis and the next mitosis. If the cellis not going to divide again, it passes out of cycle during this phase intoanother phase termed G0. From this phase it may, or may not, be possible tocall it back into a division cycle. Usually, however, cells pass on toirreversible differentiation with their chromosomes unduplicated.
S-phase is a discrete period of interphase of a few hours duration duringwhich the chromosomal DNA and protein is duplicated, and the new chromatinsegregated into the sister-chromatids. Each chromosome has a precise programmeof replication, closely associated with its G-band pattern.
Pale G-bands always replicate early in S-phase, dark G-bands later, andconstitutive heterochromatin tends to be among the very last regions toreplicate (Aghamohammadi and Savage, 1990; Savage et al., 1984).
During G2 , the newly replicated chromosomes undergo a rapid programme ofcondensation, packing and coiling to produce the familiar metaphase chromosomeswhere we normally identify and score aberrations. These condensed chromosomesfacilitate transport of the genetic material to the daughter cells at mitosis.This condensation and packing readily obscures, modifies and disguisesaberrations which are produced during interphase - a point that should alwaysbe borne in mind when interpreting what we see down the microscope.
Most aberration-inducing agents can introduce lesions into the chromatinat all stages of the cell cycle, but relatively few of them can produceactual structural changes in G1,( and therefore give rise to primarychromosome-type changes) or in S and G2 (producing primarychromatid-types ).
Ionising radiation, restriction endonucleases, and a few chemicals likebleomycin and some antibiotics are amongst those that can.
Almost all remaining aberration producing agents are "S-dependent" ;surviving unrepaired lesions from G1 or G2 have to pass through a scheduledS-phase to convert them into exclusively chromatid-typeaberrations.
Any interference with or abnormality in the processes of chromatinreplication also leads to chromatid-type aberrations visible at nextmitosis. It is almost certain that the vast majority of "spontaneous" andde novo aberrations arise in this way. Chromosome instability syndromesalso probably produce aberrations via defective S-phase pathways.
However they are produced, the resulting chromatid-type aberrationsare qualitatively (but not quantitatively) identical.
Meaningful quantitative work with chromatid-types is extremelydifficult because observed frequencies fluctuate with time of sample aftertreatment, and are subject to dramatic modifications as the result of mitoticperturbation and differential cell selection. This makes comparison betweendifferent treatments, or the production of sensible dose-response curves,virtually impossible (Savage and Papworth, 1991).
Aberration transmission and stability
Although there is an enormous range of primary aberration forms,very few of them are transmissible to future cell generations long term, soonly a handful of secondary ( or "derived" ) forms are recovered(Savage, 1976; Savage, 1995).
The following paragraphs list the kinds most likely to be encountered,together with comments and a note about probable primary origin.
RECIPROCAL TRANSLOCATION :
Involves no mechanical separation problems at anaphase, and usually nogenetic loss or imbalance. Problems can occur at meiosis because of multivalentformation, and degrees of sterility may arise.
At the molecular level, the re-joining points can disrupt importantgenetic sequences, leading to inactivation, mutation or position effects (e.g.the t(9;22) Ph1 chromosome of CML).
Derived directly from chromosome-type reciprocal translocations orfrom one segregation sequence of symmetrical chromatid-typeinterchanges. (Note that the alternative interchange segregation leads toimbalance and cell lethality).
PERICENTRIC INVERSION :
Very similar properties to those for reciprocal translocations given above.Large inversions lead to meiotic bridges, sterility and cell death.
Derived directly from chromosome-type or chromatid-typepericentric inversions.
PARACENTRIC INVERSION :
Very difficult to detect at the chromosome level unless they are very large(many megabases of DNA). Again the re-joining points can disrupt importantgenetic sequences, and reverse segments of the reading frame. Large inversionswill give problems at meiosis.
Derived directly from chromosome-type paracentric inversions, orfrom one form of chromatid-type intra-chromatid intra-arm intrachange(Revell-type 3 (Revell, 1959; Savage, 1976; Savage, 1989).
INTERSTITIAL DELETION :
The loss of small segments of a chromosome (usually in only one homologue)is not uncommon. Many mutations that have been genetically sequenced have beenshown to be actually small deletions.
Very occasionally, the loss of quite large segments appears to becompatible with cell survival.
Derived directly from chromosome-type interstitial deletions("double minutes") and from the alternative form of chromatid-typeintra-chromatid intra-arm intrachange (Revell-type 2(Revell, 1959; Savage, 1976; Savage, 1989)) to that whichproduces paracentric inversions. Segregation products from some complexchromatid-type interchanges can also carry deletions.
TERMINAL DELETION :
It is now questionable whether true stable terminal deletions actuallyexist. All those that have been investigated using the new fluorescenttelomere probes are found to be "capped" by telomere sequences. This eithermeans that they are disguised interstitial deletions, where one re-join pointwas almost terminal, or that survival has been rendered possible by denovo telomere synthesis. The recent development of end-specific telomereprobes should be able to solve this question(Boei and Natarajan, 1998; Boei et al., 1998).
Derivation, if genuine, from various forms of incompletechromosome-type or chromatid-type intrachanges and interchanges,followed by telomerase activity to achieve capping.
INTERSTITIAL DUPLICATION :
Segments of a chromosome repeated in tandem, sometimes in reverse sequence.This may not necessarily arise from a pre-existing structural aberration ;segment amplification and re-duplication is a well attested phenomenon undercertain conditions (e.g. HSR regions following chronic methotrexate exposure).Nevertheless, there are primary aberrations which can survive as segmentalduplication.
Most likely derived from one form chromatid-type inter-chromatidintra-arm intrachange (Revell-type 1(Revell, 1959; Savage, 1976; Savage, 1989)). Some forms of complexchromatid-type interchanges can segregate to give surviving chromosomeswith duplicated segments.
INTERSTITIAL INSERTION :
Deletion of a segment and its insertion into another chromosome within thesame cell is a fairly common transmitted aberration. Much less common is theinsertion of a segment additional to the two complete homologues within acell.
All insertions are derived from complex exchanges, since, by definition,their production requires the interaction of a minimum of 3 lesions. Eitherchromosome-type or chromatid-type complex interchanges may beinvolved, the range of inter-intrachanges in the latter being particularlyproductive of insertions.
Occasionally, a surviving dicentric may be found, usually without therelated acentric fragment. Very often, the two centromeres lie very closetogether, because, under these circumstances, only one of the centromeres isactive, so anaphase bridges do not form. Likewise, an occasional centric-ringmay survive, again usually very small so that "fall-free" separation alwayshappens. Larger rings are very unstable with respect to size, and the positiveselection pressure towards very small rings soon eliminates the bigones.
Most of the above comments apply to the situation in normal individualsand cells. When we turn to cancer-derived cells, or to transformed cell linesgrowing in culture, the situation is somewhat different. These cells areinherently chromosomally unstable. There is a continuous production ofstructural change so that new primary changes are superimposed on thealready existing background of secondary aberrations, and these newones, in their turn, become secondary.
Moreover, some of the new changes are being produced in already abnormalchromosomes, so the observed aberrations are often very complicated andbizarre.
On top of this, it is clear that most cancer cells are very tolerant ofchromosomal loss, or gain, as is evidenced by considerable numerical variationsand multiple chromosome copies. These facts make cancer cytogenetics a verydifficult and uncertain field for investigation, and considerable credit goesto those workers whose careful and painstaking efforts have produced meaningfuladvances.
Atlas of Genetics and Cytogenetics in Oncology and Haematology
An Introduction to Chromosomal Aberrations
Online version: http://atlasgeneticsoncology.org/deep-insight/20001/an-introduction-to-chromosomal-aberrations