nucleic acid fingerprinting by pcr-based methods: applications to problems in aging and mutagenesis

ELSEVIER

DNAging Genetic Instability and Aging

Mutation Research 338 (19YS) 215-229

Nucleic acid fingerprinting by PCR-based methods: applications to problems in aging and mutagenesis

John Welsh *, Nick Rampino, Michael McClelland, Manuel Perucho * California Instmlte of Btologtcal Research, 11099 North Torrey Pines Road, La Jolla, CA 92037, USA

Accepted I5 May 1995

Abstract

There are many methods of inference in common use in biology that arc based on population sampling, including such diverse areas as sampling organisms to determine the population structure of an ecosystem, sampling a set of DNA sequences to infer evolutionary history, sampling genetic loci to build a genetic map, sampling differentially expressed genes to find phenotypic markers, and many others. Recently developed PCR-based methods for nucleic acid fingerprinting can be used as sampling tools with general applicability in molecular biology, evolution and genetics. These methods include arbitrarily primed PCR (AP-PCR; Welsh and McClelland, 1990) and random amplified polymorphic DNA (RAPD; Williams et al.. 1990) for the fingerprinting of DNA, and RNA arbitrarily primed PCR (RAP-PCR; Welsh et al., 1992a) and differential display (DD; Liang and Pardee, 1992) for the fingerprinting of RNA. Novel ways of looking at genetic control are facilitated by the high data-acquisition capabilities of the fingerprinting methods. In this article, we review some of the applications of DNA fingerprinting to the study of mutagenesis, and of RNA fingerprinting to the study of normal and abnormal signal transduction. We propose that these fingerprinting approaches may also have applications in the study of senescence and aging.

Keywords: DNA and RNA fingerprinting; Arbitrarily primed PCR; Somatic mutation; Cancer; Senescence

1. Introduction

DNA fingerprinting by AP-PCR or RAPD provide information-rich and highly reproducible patterns of DNA fragments that reflect differ-

Abbreviations: PCR, polymerase chain reaction; AP-PCR. Arbitrarily primed polymerase chain reaction; RAP-PCR, RNA arbitrarily primed polymerase chain reaction; DD, differential display; RAF’D. randomly amplified polymorphic DNA.

* Corresponding authors. Tel.: l-(619) S35-S478, 5477. 5471: Fax: l(619) 5355472.

ences in template sequence or relative abundance. DNA fingerprinting can be achieved by PCR under conditions where low specificity priming is encouraged (i.e. high divalent cation and low temperature). The sequence of the primer is chosen arbitrarily and the primer interacts with the template at sites where the interaction is moderately stable. Under these conditions, up to about 100 arbitrarily sampled sequences per reaction are amplified to detection levels. When displayed on a denaturing polyacrylamide gel, the resulting pattern of products reveals mutations that have accumulated either somatically or over

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216 J. W&h ct al. /Muration Research 338 (1995) 215-229

evolutionary time, or differences in the relative abundances of the corresponding sequences. DNA fingerprinting by AP-PCR is straightfor- ward and reproducible. Several thousand repro- ducibly selected DNA fragments can be exam- ined simultaneously, making DNA fingerprinting a powerful tool for the detection and evaluation of certain phylogenetic and ontogenetic phenomena. The versatility of AP-PCR stems from the simple nature of the information present in a fingerprint, the high throughput of the method, and the ease with which the fingerprints can be generated (reviewed in Welsh and Ralph, 1994; McClelland and Welsh, 1994).

genetic machinery accompanying different phenotypic phenomena are dramatic, e.g. telomere shortening or the lower expression of certain genes in senescent fibroblasts, but many are subtle in experimental terms, and difficult to detect. The high data acquisition capabilities of DNA and RNA fingerprinting methods allow for the relatively efficient detection of DNA structural alterations and signal transduction modulation associated with phenotypic variation. In this sense, fingerprinting methods may be useful tools for the characterization of molecular events associated with normal and abnormal development.

RNA fingerprinting methods are useful for the identification of differentially regulated genes (Liang and Pardee, 1992; Welsh et al., 1992). RNA fingerprinting by RAP-PCR, such as AP- PCR, is based on the fortuitous presence of good matches between a nucleic acid template population and an arbitrarily chosen primer, and therefore provides a sample that is not biased with respect to sequence. The relatively large number of bands in a few dozen fingerprints represents a sizable arbitrary sample of the total complexity of the message population of the cell. Many problems in biology are related to differential gene expression, including development and the differential response of cells or organisms to environ- mental stimuli. RNA fingerprinting can be used to address many of the problems previously ap- proachable only through subtraction methods, or differential screening. RNA fingerprinting is semi-quantitative, and can be used to scan mRNA populations for differentially regulated genes, based on message abundance (reviewed in Welsh and Ralph, 1994).

2. DNA fingerprinting by arbitrarily primed PCR

2.1. Somatic genetic alterations

From the detection point of view, DNA fingerprinting is hypothesis-neutral and does not distin- guish between normal (i.e. reflecting normal development) and abnormal (i.e. reflecting damage accumulation) genetic alterations. Hypotheses for development, for example, fall into both categories. Quantitative and qualitative somatic changes have been detected in DNA sequences in a number of developmental situations. For example, programmed changes may play a functional role in controlling segregation of germline and somatic cells (Boveri, 1887; Tobler et al., 1985). Sequences that show germline and to a lesser extent somatic variation include the highly vari- able minisatellites and mono, di- and trinu- cleotide repeated sequences or microsatellites (reviewed in Pardue, 1991; Weber and Wong, 1993).

Development, aging, cellular senescence, and Some aspects of aging and cellular senescence oncogenesis are all complex processes accompa- may reflect normal developmental pathways. The nied by changes in genomic structure and gene shortening of telomeres has been associated with expression. Some of these changes behave as aging in vivo (Hastie et al., 1990) and with cellu- cell-autonomous developmental cascades, such as lar senescence in vitro (Harley et al., 1990). The telomere shortening or negative pleiotropy in ag- loss of telomerase activity has been postulated as ing, while others reflect error accumulation at the cause of telomere shortening in aging somatic either the cellular or organismal levels, such as cells, with the consequent loss of functions essen- mutational damage leading to oncogenesis, nega- tial for cell survival (reviewed in Greider, 1990; tively pleiotropic whole organism effects, e.g. hor- Blackburn, 1991). Other structural genomic alter- monal changes, etc. Some of the changes in the ations associated with aging and senescence are a

J. Welsh et ul. /Mufarmn Research .f.?K ClYYS) 215-229 217

variety of karyotypic abnormalities in senescent cells (Sherwood et al., 1988) and deletions in mitochondrial DNA in old individuals (Corto- passi and Arnheim, 1990). A connection between mitochondrial DNA damage, mitochondrial mor- phological disorganization in postmitotic cells and free radical activity has been postulated (Corto- passi and Arnheim, 1990). Pleiotropic interaction between DNA replication/repair mechanisms and proper mitochondrial function suggests the possibility of an error catastrophe mechanism, where the impedance of normal DNA replication and repair results in the progressive accumulation of damage in the DNA replication/repair mechanism, itself, by feedforward error propagation (Orgel, 1963).

Outside of normal development, many phenomena are associated with genomic structural alteration. In cancer biology, AP-PCR has re- sulted in the discovery of the ‘microsatellite mutator phenotype’ (Ionov et al., 1993) characteristic of some sporadic and hereditary non-polyposis colon carcinomas (HNPCC) and other gastroin- testinal and urogenital tumors. Particularly interesting disruptions of the normal genetic machinery are changes in ploidy and the loss of het- erozygosity. Changes in ploidy and/or heterozy- gosity have been associated with cancer and, to a minor extent, with senescent cells, and with various experimental systems such as somatic cell hybrids. AP-PCR is an excellent tool for molecular cytogenetics (Peinado et al., 1992). We discuss these findings in greater detail, later. First, we discuss some technical aspects bearing on the sensitivity of AP-PCR in the detection of mutations.

2.2. The detection sensitirity of AP-PCR

The usefulness of AP-PCR in addressing problems associated with genetic structural alterations depends on the nature of the alteration and its extent, vis-a-vis the amount of sequence complexity involved in the alteration. It would be futile, for example, to use AP-PCR to detect a single chromosomal break point. On the other hand, the loss of a chromosome arm would be easy to detect. Next, we discuss some of the general

principles connecting AP-PCR genomic fingerprinting to the detection of genetic alterations and review its application to the detection and characterization of somatic mutations during neoplastic transformation. Many of these principles also hold for RNA fingerprinting strategies, to be presented in the second half of this article.

Mutations can affect the fingerprint in four ways: by altering the ability of the primer to anneal, by altering the distance between the two primers, by altering the ability of the polymerase to extend, or by altering the relative amounts of the targets of amplification. Of these four different types of mutations, only the third does not have solid experimental support. The other three types of mutations have been extensively studied by AP-PCR DNA fingerprinting and can be also classified by their localization relative to the annealing arbitrary primers: at the annealing sites, between the annealing sites and outside of the annealing sites.

2.2.1. Mutations at the primer-template interaction sites

Mutations which underlie the primer interaction site result in the gain or loss of a band or a change in its intensity. For a fingerprint gel with 2000 bands generated with lo-mer primers, about 40000 nucleotides are scanned for polymorphisms underlying the primer binding sites. Poly- morphisms of this kind are very useful in population, evolutionary and genetic mapping studies (reviewed in McClelland and Welsh, 1994). The precise nature of the mutation cannot be assessed from the sequence of the AP-PCR product because the sequence underlying the primer is always altered to exactly match the primer sequence during AP-PCR. Therefore, the PCR product will contain at both ends the arbitrary primer sequences, rather than their genomic tar- get sequences. The exact sequence differences at the primer binding sites underlying these polymorphic bands need to be determined by an independent approach.

The impact of a mutation on the stability or kinetics of primer-template interaction is difficult to predict. A single base change is more likely to have a profound effect on primer stability for

shorter primers, but intermediate band intensities are commonplace. In our experience, the 5-6 nucleotides at the 3’ end of the primer are critical for the specificity of the fingerprint. Deletions or insertions of just a few bases underlying a short primer are likely to entirely eliminate the product from the fingerprint.

2.2.2. Mutations between the primer-template interaction sites

Small deletions or insertions between two primer-template interaction sites result in a mobility shift in sequencing-style, denaturing polyacrylamide gels, which can usually resolve up to a 0.2% difference in molecular weight. Conse- quently, all deletions and insertions in products smaller than about 500 bases can be detected. The average size of visible products in AP-PCR is usually around 400 nt, although this can vary. Some protocols, for example, yield a handful of products in the 500 to 3kb range. In general, about 20000 base pairs of DNA can be displayed in a single lane on a denaturing sequencing-style gel. With two concentrations of each DNA sample, a side-by-side comparison of two templates and 100 lanes per sequencing gel, 5 X 10” base pairs can be scanned for deletions or insertions on a single gel. Deletions or insertions of a single base pair become difficult to detect for the largest fingerprint products.

Single base substitutions however, cannot be detected by this method. In principle, single base substitutions present in the AP-PCR amplified genomic sequences could be detected by single strand conformational polymorphism (SSCP) analysis (Hayashi, 1991; McClelland et al., 1994b). SSCP is based on the resolution of single stranded molecules according to their secondary structure. Single base substitutions can result in an altered set of structural possibilities, and therefore altered mobilities. Approximately 50% of all single base substitutions can be detected, although higher sensitivity has been reported (Hayashi. 1991).

The use of 20 different single primers, both singly and in pairwise combination, generates about 7000 different genomic sequences ([(20!/2!(18)!1 X (20 + 50)/2 = 7350}, or one sam-

ple every 400000 bp on average. Assuming an average size of 400 bp per band, the cumulative size of the sequence sampled using 20 primers is about 3 000 000 bp, which represents about l/1000 of the haploid genome. Because there is no apparent bias as to their chromosomal derivation (Perucho et al., in press), the size of the window that AP-PCR provides to look at the genome in an unbiased manner is larger than provided by other molecular techniques. SSCP gels afford a second method of analysis that increases the spectrum of mutations that can be detected by AP-PCR DNA fingerprinting. Given that SSCP can detect 50% of all single base mutations in molecules smaller than 500 bp, 25% of the point mutations in the 5 x 10’ base pairs could, in principle, be detected. In agreement with this prediction, Okano et al. detected somatic single base substitutions in liver cancer by the AP-PCR/SSCP combined approach (Okano et al., in preparation).

2.2.3. Mutations outside the region between the primer-template interaction sites

The quantitative nature of the amplification levels achieved by AP-PCR permits an additional and important application of the method for the detection of somatic genetic alterations. When two fingerprints are compared, the ratio of intensities of any particular band depends on the relative representation of its corresponding template sequence in the population. Thus, when an amplified fingerprint band originates from a region of the genome where ploidy changes, the intensity of the band will change to reflect an alteration in allelic composition (Peinado et al., 1992). In heterozygotes for a length-polymorphic band, loss of a region of a chromosome can result in the complete loss of the band otherwise contributed by the lost allele. In homozygotes for a non-polymorphic band, it will result in a change of intensity of the band up to 50%. Conversely, gains of chromosomes or chromosomal regions repre- sented in the fingerprints will result in increases of intensity of the corresponding bands, propor- tionally to the number of gained copies.

Given that 2500 bands can be displayed on a single sequencing gel (with a control and dupli-

cates), quantitative changes affecting chrornoso- ma1 regions as small as 10’ base pairs can, in principle, be detected on a single gel with 95% confidence. Although such alterations would each comprise a very minor fraction of the total genome, they have been successfully detected. For example, using 62 arbitrary primers, Yokota’s group achieved an unbiased scanning of the genome at megabase intervals and detected 6 different fingerprint bands amplified in a lung carcinoma cell line. All these amplified bands were localized in the chromosome 8 amplicon containing the c-myc protooncogene (Okazaki et al., submitted).

In addition, fluctuations in chromosome copy number can be readily detected by the use of a single or a few arbitrary primers, because there is no apparent bias for the chromosomal origins of the fingerprint bands. The chromosomal locations of many of the bands can be determined simultaneously by AP-PCR fingerprinting of rodent/human monochromosome cell hybrids. Therefore, a single fingerprinting gel and every one of the chromosomes in multiple samples (Perucho et al., in press; Malkhosyan et al, in preparation).

2.3. DNA fingerprinting by AP-PCR in cancer research

DNA fingerprinting by AP-PCR is a powerful tool for the detection and characterization of somatic genetic alterations during tumorigenesis (Peinado et al., 1992; Ionov et al., 1993). The applications of the technique in cancer research can be divided into two main areas: the detection of qualitative (structural) and quantitative (an- euploid) genetic alterations, corresponding to the previous sections B ii and B iii, respectively. We describe separately these applications below.

2.3.1. The detection of allelic losses and gains in cancer cells

As described above, AP-PCR can identity regions of the genome that have lost their diploid state in tumor cells. The differences in the intensities of the AP-PCR bands from tumor DNA, compared to those from the normal diploid genome from the same individual, provide an

estimation of the tumor cell aneuploidy. Because of the unbiased chromosomal origins of the fingerprint bands, and because of the possibility of simultaneously identifying their chromosomal derivation, DNA fingerprinting by AP-PCR provides a molecular approach for cancer cytogenet- its (Perucho et al., 1994b). DNA sequences from known chromosomal origins, undergoing consis- tent gains and losses in particular types of tumors, can be readily detected in this manner.

For instance, we have identified moderate gains of sequences from chromosomes 7,8 and 13 in a majority of colorectal carcinomas at late stages of tumor progression (Malkhosyan et al., in preparation). The ability of AP-PCR DNA fingerprinting to detect moderate gains of genetic material (in the trisomy/tetrasomy range) represents a considerable technical advance because such genomic changes cannot be readily identified by conventional RFLP or microsatellite allelotyping.

2.3.2. The detection of small deletions and insertions in cancer cells

The high resolution of sequencing gels used in DNA fingerprinting by AP-PCR permits the detection of small deletions or insertions that can pass undetected in other analytical methods such as Southern blots. This allowed the discovery in some colorectal tumors of the microsatellite mutator phenotype for cancer (lonov et al., 1993). In these studies, the finding of somatic mutations in simple repeated sequences (SRS) could be ex- trapolated to the existence of hundreds of thou- sands of such mutations in the genome of these cancer cells. These mutations are likely due to the failure to repair replication errors that accu- mulate due to slippage by strand misalignment of these highly repetitive sequences (Streisinger et al., 1966). Defects in long patch mismatch repair (reviewed in Modrich, 1991) have been impli- cated in the accumulation of these ubiquitous somatic mutations (USM) at SRS in colon and other tumors, which also are the genetic defects underlying some hereditary cancer syndromes (reviewed in Marra and Boland, in press). Unbi- ased DNA fingerprinting by AP-PCR was crucial to the recognition of this genome-wide instability

which made possible the subsequent correct in- terpretation of the mobility shifts of microsatellite sequences sporadically encountered during allelotyping analysis.

2.4. Future directions

2.4. I. A possible clock for the biological age oj clones

SRS or microsatellites are unstable sequences with high spontaneous mutation rates due to slippage by strand misalignment (Levinson and Gut- man, 1987; Weber and Wong, 1993). The frequency of mutations at microsatellites is inversely proportional to the number of mutated repeat units (i.e. deletions or insertions of one repeat unit are more frequent than those of two units, and so on). Therefore, microsatellite mutations are, in principle, suitable as molecular clocks not only during phylogeny. but also to estimate the biological age (i.e. the number of cell divisions, as opposed to the chronological age) of somatic cells. Unfortunately, due to the ‘reversible’ nature of slippage mutations. the number of cell replications cannot be estimated from the size of the mutation. For instance. an insertion followed by a deletion in the same tandem repeat will cancel each other, and two insertions plus one deletion (three mutational events) will appear as the same ‘mutational age’ as a single insertion.

An exception, however, is the striking unidi- rectionality in the mutations at monotonic runs of deoxyadenosines (we have never found insertions) in colon tumors of the microsatellite mutator phenotype. Therefore, the size of the mutation (number of deleted basepairs) is a measure of the number of consecutive mutational events each deleting a single basepair (Shibata et al.. 1994). In this scenario, the accumulation of deletions in these poly A tracts can be used to estimate the approximate number of cell replications undergone by the tumor cell, if the mutation frequency is known. For instance, a tumor with an average number of four deleted As would be twice as ‘old’ than a tumor with only an average of two deleted As in the same microsatellite sequences (lonov et al.. 1993; Perucho et al.. 1994). However, this is an oversimplification be-

cause different mutator genes induce different frequency (and spectrum) of mutations at microsatellites (Malkhosyan et al., in preparation).

One of the peculiar characteristics of this mutator pathway for cancer is that the initial mutator mutation is not immediately accompanied by a growth advantage or a territorial expansion capability (Perucho et al., 1994). The unfolding of the mutator phenotype is also a very early event in tumorigenesis (Shibata et al., 1994). Therefore, the expression of the microsatellite mutator phenotype may occur before neoplastic transformation. In this case, it might be possible eventually to extrapolate these molecular clocks, not only to the tumor historical mitotic activity, but also to the number of cell replications of the normal stem cells of the colon crypts precursors of the tumors.

2.4.2. The detection of somatic genetic variation by A P- PCR DNA fingerprin tins

In principle, DNA fingerprinting could also be applied to the detection, during somatic deveiop- ment and aging, of similar quantitative (confined or global genomic losses or gains) and qualitative (small deletions/insertions) changes described before, as long as the clonality intrinsic to tumor formation could be naturally achieved by organ differentiation during ontogeny in vivo or artifi- cially reproduced by cloning experiments in vitro. Single base substitutions could be also included by the AP-PCR/SSCP combination approach. However, the only evidence to support this hypothesis is the observation of fingerprint differences among single cell clones of immortalized rodent cell lines and human tumor cell lines (M.P., unpublished observations). No changes have been observed in the DNA fingerprints between different organs (liver, spleen, kidney, etc.) of rodent and humans, in the few cases analyzed. Nevertheless, the use of many arbitrary primers could reveal low-level somatic variation in clones. This approach would be comparable to the search and detection of rare homozygous deletions (Kohno et al., 1994) and amplicons (Okazaki et al., submitted) in tumors. Also in this line, AP- PCR fingerprinting has been successful in the detection of germline mutations occurring de novo

during gametogenesis. Thus. Kubota et al., (1992) reported the identification by AP-PCR of X-ray- induced genetic damage in fish embryos.

3. RNA fingerprinting by arbitrarily primed PCR

3. I. RNA fingerprinting strutegies

There are many strategies for RNA fingerprinting. In one method (Liang and Pardee, 1992), first strand synthesis is primed with an oligo dT based primer having one or more of the bases A. G, or C at the 3’ end. These additional bases anchor the primer at the junction between the poly A tail and the 3’-non-coding region of the RNA. Second strand synthesis is initiated by arbitrarily primed PCR. When every possible combination of two extra 3’ bases are used to anchor the oligo dT primer, the RNA population is divided into 12 non-overlapping groups, which are then independently sampled in the second strand synthesis step. However, this does not affect the number of fingerprints that must be generated to achieve full coverage of the population. This strategy preferentially amplifies 3’ non-coding parts of the message. which restricts direct database sequence comparisons to phylogeneti- tally close organisms.

The approach to RNA fingerprinting developed by Welsh et al. ( 1992), RAP-PCR (for RNA arbitrarily primed PCR), involves the use of arbitrary priming for both first and second strand synthesis. Operationally, RAP-PCR is almost identical to DNA fingerprinting by AP-PCR, except that reverse transcriptase is used for first strand synthesis. First strand synthesis is performed with an arbitrary primer. then second strand synthesis is performed using many possible second arbitrary primers. An anchored oligo d7 primer would be expected to sample only a fraction of all RNAs. The same is expected for a single arbitrary primer, except that, for an arbitrary primer, the sampled population might be expected to be even more restricted. This leads us to the important and unresolved problem of the abundance normalization of sampling.

3. I. 1. Abundance normalized sampling Abundance normalized sampling is sampling

that is independent of abundance (Ralph et al., 1993). RNA populations exhibit wide distribu- tions of abundances, and the problem with fingerprinting is that the more abundant messages tend to dominate. This problem is similar, in a manner of speaking, to the problem with two-dimensional protein electrophoresis, where only the few thousand most abundant proteins are easily visible. A systematic analysis of the limitations of RNA fingerprinting methods vis-8-vis abundance normalization is lacking. However, the problem is coming into focus.

In principle, the intensity of a band in an RNA fingerprint depends on (1) the likelihood of the appearance of adequate priming sites that are closely spaced and on opposite strands, (2) the stability of the primer-template interaction, (3) the abundance of the template, and (4) the ease with which the particular sequence can be extended in all of the steps (vis. secondary structure). Clearly, sequence complexity favors the first two factors, but members of the complex class of RNA are the least abundant. Thus, in RAP-PCR, both primers preferentially interact with mem- bcrs of the complex class (where a good sequence match is more likely). For rare messages to appear in the fingerprint, however, selectivity due to primer match must be on the order of 10” to 10”. Otherwise. messages that interact only poorly with the primer but are present in several hundred thousand copies per cell will dominate. There is some qualitative data that RAP-PCR has high selectivity based on the complexity argument. First, we have sequenced several dozen randomly chosen low intensity bands from RAP-PCR fingerprints, and have not yet encountered a eukary- otic ribosomal sequence, which are usually thou- sands of times more abundant than the most abundant transcripts. Second, in products that match something in the database. a minimum of 6 out of the 10 nucleotides at the 3’ for each primer match, setting a lower limit on selectivity.

One strategy we have worked with has been to reamplify the products of a primary RAP-PCR fingerprinting reaction with nested primers (Ralph et al., 1993). These nested primers are

222 .I. M’&h CI al. / Mttution kwcrrch 338 (1995) 215-229

identical to those used to generate the primary fingerprint, but have one or more arbitrary nucleotides added to the 3’ end. The result is a secondary fingerprint where the predominant bands are selected from the background of the primary fingerprint. The most intense bands in the secondary fingerprint come from a population of molecules that had complexity sufficient to contain molecules having, by chance, the additional arbitrarily chosen nucleotide immediately interior to the 3’ end of the original primer. In theory, nesting of this sort may solve the abundance normalization problem, but there are many experimental details remaining to be solved.

As a cautionary reminder, as abundance normalized sampling improves. new technical problems become more important. For example, hn- RNA has been estimated to contain as high as 100 times the sequence complexity of messenger RNA. As abundance normalization improves, which we imagine will require increasing the complexity component of the selectivity equation, hnRNA will become preferentially amplified. Se- lection of poly A plus RNA will be necessary to overcome this difficulty. Priming with oligo dT- based primers may reduce the impact of this problem for total RNA, but it is likely that any primer, regardless of sequence, will participate in some arbitrary priming.

3.1.2. The resolr,ing power of RNA fingerprinting The use of RNA fingerprinting as a molecular

phenotype derives much of its resolving power from in-parallel comparisons of multiple treatment or developmental scenarios. Each experimental treatment, for example, parses genes roughly into three response classes, ‘up-regulated’, ‘down-regulated’ or ‘unaffected’. When two treatments are used in every possible combination, Y response classes result; when three treatments are used, 27 possible response classes are produced, and so on. Thus, a manageably small number of experimental treatments can yield an enormous number of possible response classes, only a few of which will be occupied. Treatments may include hormones, vitamins, etc., or may include developmental phenomena, such as different cell or tissue types. When genes parse

repeatedly into the same group, i.e. when their message abundances covary, this can be taken as provisional evidence of coordinate regulation. The meaningfulness of the coordinately regulated group depends on the criteria used for parsing, e.g. which hormones are chosen.

For example, an investigator may be interested in the genes that are controlled by a family of hormones. It may be of interest to determine to what extent the different family members produce different effects, and to obtain examples of genes that are characteristic of some family members but not others. Such data may be useful in testing hypotheses that relate molecular features shared by only some members of the family. It may turn out that a certain set of genes are differentially regulated by all members of the family that contain for instance, some structural motif.

3.2. An RNA fingerprint is a molecular phenotype

RNA fingerprinting can provide interesting information about the physiological state of the cell. Due to the arbitrary nature of sampling by RAP-PCR, a fingerprint can be thought of as a ‘molecular phenotype’ reflecting the current physiological state of the cell (McClelland et al., lYY4al. As a molecular phenotype, an RNA fingerprint can reveal patterns in genetic control. Much of molecular biology is dedicated to ex- plaining observable phenotypic behavior in molecular terms. For a phenotype to be useful in this regard, it must be observable and quantifi- able. The vast majority of cellular responses are outside this rather small window. Thus, RNA fingerprinting by RAP-PCR provides (if we disre- gard the possible skew for abundant transcripts), an unbiased molecular phenotype characteristic of the current state of signal transduction of the cell as it bears on gene expression.

In this context, RNA fingerprinting can define several system parameters, including additivity, synergism or antagonism. In principle, fingerprints provide kinetic data for multiple concur- rent pathways, which can therefore be studied simultaneously. Even some rather complex (i.e. highly branched, and feedback or feedforward

regulated) pathways display only a limited array of all possible regulatory behaviors. We have shown, for example, that most of the genes that respond to Transforming Growth Factor b (TGFb), cycloheximide (Cx) or both fall into only 8 of the possible 27 bipolar response categories (i.e. + l/O/ - 1. corresponding to up-regulated, unchanged, down-regulated) (McClelland et al., 1994a). Most of the observed classes can be mod- eled as outcomes of simple monolinear pathways, but many of the sparsely populated or empty classes apparently require branching. For example, no gene that could be induced (or message stabilized) by either TGFb or Cx was repressed (or message destabilized) by the combination of both TGFb and Cx. Such genes may be uncov- ered through more extensive fingerprinting, and would imply a more complex pathway structure.

Genes that respond to a particular experimental treatment are assumed to be under the control of that treatment. Finer levels of parsing can be achieved by using multiple treatments. Genes that respond in a similar way to a large number of treatments are expected to be controlled by overlapping or sometimes identical signal transduction pathways. Because similarity in response is a qualitative matter, covariation in response is suggestive but not conclusive, so more focused experiments must be performed, such as pro- moter analysis. However. the coordinated gene expression is often a good starting point for further analysis of the molecular basis of cellular phenotype.

3.21. RNA fingerprinting in the study of derv+p- ment

The developmental importance of some genes is implied by the time course of their expression. In one set of experiments, we identified a gene that is highly expressed in the neocortex of the prenatal mouse, and difficult to detect in the adult. Because of the timing of expression of this gene, developmental significance is implied (Dalal et al., 1995). Further experiments will be necessary to further elucidate the role it may play. A possible application of RNA fingerprinting involves the identification of genes whose expression depends on cell lineage vs. other develop-

mental cues. Because cells divide, cell fate in a developing organism can always be superimposed on a bifurcating tree structure. Developmental decisions that are lineage specific will occur at branch-points in the tree and will be inherited by some of the downstream lineages. Decisions based on other factors, such as position, will be independent of the branching structure. RNA fingerprinting is an effective way to obtain many examples of genes in both categories.

.3.3. A brief reliew of the literature

Applications of RNA fingerprinting to developmental biology include the cloning of genes differentially expressed in prenatal and neonatal mammalian brain (Dalal et al., 1995; Joseph et al., 1994) and in preimplantation embryos (Zimmerman and Schultz, 1994). Applications of RNA fingerprinting to cancer biology include the identification of genes differentially expressed between normal versus tumor in mammary epithe- ha1 cells (Liang et al., 1992) and ovarian epithelia (Wang et al., 1993; Mok et al., 19941. Note that if non-isogenic materials are being compared, such as clinical samples from different individuals, it is very important to fingerprint samples from many individuals. Any sequence polymorphisms between individuals are thereby eliminated as can- didate differentially expressed genes.

Other genes cloned by the RNA fingerprinting methods have included mouse mammary tumor markers (Zhang and Medina, 19931, a vitamin induced gene in osteosarcoma (Kumar and Hau- gen, 19941, genes induced by radiation exposure in a squamous carcinoma cell line (Jung ct al., 19941, genes differentially expressed between megakaryoblast proliferation and megakaryocyte differentiation to platelets (Darn et al., 19941, acidic FGF-induced gene expression in murine NIH 3T3 cells (Donahue et al.. 19941, glucose induced genes in aortic smooth muscle cells and retinal pericytes (Nishio et al., 1994; Aiello et al., 19941, and genes regulated by TGFP and cycloheximide in epithelial cells (Ralph et al., 1993; McClelland et al.. 1994a). One interesting application to a whole organ was the identification of several genes altered during chronic cardiac re-

jection in allogenic rat cardiac transplant (Utans et al., 1994).

3.4. Future directions

3.4.1. Gene expression and aging RAP-PCR is a powerful tool for the study of

coordinately regulated differential gene expression and may allow the detection of changes in messenger RNA that accompany aging or cellular senescence. With age, closely related cell types show quantitative and qualitative differences during a lifespan. For example, in liver tissue differential increases (e.g. SMP-2) and decreases (e.g. a2,-globulin) in mRNA levels correlate with age. In most organs. however, a range of cell functions appear to remain unaltered throughout life. Gen- eral alterations are found in cells that become deprived of trophic support, such as steroid tar- get cells after ovarian secretions cease. These examples illustrate that subtle aspects of differentiation influence specific cellular functions strongly and selectively during cellular senescence and organismal aging. Some events in senescence may represent endpoints in an onto- genie cascade of gene regulation (Finch, 1976).

RNA fingerprinting seems particularly well suited for following slowly evolving cascades of gene regulation in the progression toward aging or cellular senescence. Using RNA fingerprinting, one might be able to identify genes involved in negative pleiotropy, predicted by population genetics. This hypothesis suggests that the advan- tageous expression of some genes early in life may have adverse consequences later in life. Changes in gene expression during the transition to mid-life, where a superior pre-mature ability gives way to a predisposed disability, may be of great interest in this regard. The many changes in hormones and other regulatory factors that begin shortly after maturation (e.g. changes in dehy- droepiandrosterone sulfate or pituitary go- nadotropins levels) appear to induce altered gene activity, and thus may help us correlate gene expression cascades via RNA fingerprinting to the concentrations of trophic factors. Studies on cellular senescence in vitro by comparative analysis of RNA fingerprints during longitudinal studies of aging in vivo would be informative. Due to

the semiquantitative nature of RNA fingerprinting by AP-PCR, not only all or nothing changes may be detected, but also fluctuations in the levels of expression of senescence-associated genes.

3.4.2. Finite proliferative life span of cells in culture Human diploid fibroblast cell lines are a con-

venient systems for studying gene expression in the context of the finite proliferative life span of cells in culture (Hayflick, 1965). Much of our current understanding of in vitro cellular senescence is derived from the WI-38 cell line, estab- lished by Hayflick, or the IMR-90 cell line. These cell lines appear well suited for RNA fingerprint studies, as they are well characterized and gene expression by Northern analysis, in young and senescent cells, has been measured (for a review see Christofalo and Pignolo, 1993). Senescent fibroblasts exhibit alterations in gene expression quite distinct from differential expression associated with the reversible growth arrest state at- tainable by contact-inhibited young cells. Relative to their younger counterparts, senescent cells overexpress some genes (e.g. fibronectin, IGF- BP3), and underexpress others (e.g. PCNA, cdc-2, c-fos), while many genes (e.g. /3-actin, ~53, c-myc) are expressed at levels similar to those in young cells. As a cell population ages, the interdivision time increases, and the cell cycle displays an extended G, interval. Transitions to the senescent phenotype occur asynchronously within a primary culture of fibroblasts, but ultimately all cells reach a proliferative arrest state. The changes that occur during this type of cellular senescence, while somewhat uncoordinated, appear to reflect a defined genetic program. RNA fingerprinting may provide an effective way of surveying the genes that are controlled by this program. The impact of other physiological agents, such as DNA damaging agents, on the same set of genes may help to better define which signal transduction subroutines participate in senescence, and what their normal functions are in younger cells.

3.4.3. Longitudinal studies Longitudinal studies, which track subjects

through time, are well suited for fingerprint anal-

J. U'dsh et al. />Mututron Krsrurch 33X 11YY.T) 215-229 225

ysis. RNA fingerprinting could be used to reveal evolving cascades of differential gene expression in either a longitudinal study of inbred mice, where sequence polymorphism is not a factor, or a longitudinal study of humans where RNA has been isolated from the same individual at different ages. A good source of such material resides in the National Institutes of Health sponsored Baltimore Longitudinal Study on Aging (BSLA), which makes a collection of retrospective and prospective patient material available to re- searchers.

5. DNA repair, mutagenesis and aging

Aging and diseases like cancer clearly have a genetic character, which may be substantially modified by increasing genetic alterations brought on by genotoxic agents. For example, young pro- liferating human diploid fibroblast can be induced to suddenly take on a senescent morphol- ogy by exposure to hydrogen peroxide (Chen and Ames, 1994). While in cancer. an accumulation of genetic defects appears to cause normal cells to become cancerous, and cancerous cells to become increasingly aggressive (Cavenee and White, 1995).

The somatic mutation theory of organismic senescence was one of the earliest theories put forward to address senescence at a molecular level. The theory arose from attempts to explain the shortened lifespan of mammals exposed to sublethal doses of radiation. These ‘late effects’ of radiation continue to be important in the molecular biology of aging, as many humans are chronically exposed to low levels of radiation. There are many variations of the somatic mutation theory. Szilard’s (1959) proposal was that genes on chromosomes of somatic cells are inacti- vated by a random ‘aging hit’, and with the accumulation of such damage dysfunctional cells arise. Such damages may arise not only from radiation, but more importantly from endogenous agents that produce reactive oxygen species which attack DNA.

With respect to the aging process, two types of experimental evidence have been used to support

a role for DNA repair. First, attempts have been made to correlate the life span of an organism with the overall efficiency of DNA repair. In these studies, unscheduled DNA synthesis following UV irradiation has been used as a measure of the DNA repair capacity. It was initially shown that there was a linear relationship between the logarithm of the life span and the degree of unscheduled DNA synthesis (Hart and Setlow, 1974). However, subsequent studies with additional mammalian species have weakened the high correlation that was originally found (Kato et al., 1980). The second approach has been to look for an attenuation of DNA repair efficiency in cells from aging individuals, or in senescent cultures. The results from many such studies have provided no clear consensus for a correlation between overall genome repair, and the aging process.

These previous studies, however, have not been gene specific. In such studies of DNA repair over the total genome, major changes in select, important genes would be missed, since the genes con- stitute about only 3% of the total cellular DNA. It should be noted that DNA repair of UV dimers in telomeres has recently been assessed and found to decrease with age (Kruk et al., 1995). With respect to human diseases clinically classified under syndromes of premature aging, there is Cock- ayne’s syndrome, in which there appears to be a selective deficiency in the preferential repair of the transcribed strand in expressed genes without any decrease in overall genome repair (Venema et al., 1990).

Recently, oxidative damage and preferential repair in young and senescent human diploid fibroblasts has been measured by a new assay (Rampino, 1992; 1995). In this assay, single- stranded DNA, capable of hybridizing to gene specific probes, is generated enzymatically by the 3’-5’ exonucleolytic procession of T4 DNA polymerase. DNA lesions inhibit the processivity of this enzyme, and decrease the amount of comple- mentary sequence produced, when assayed by gene specific probe hybridization. With the progression of repair, increasing quantities of single stranded DNA become available for probe hybridization. The assay appears applicable for de-

tection of gent specific and strand specific DNA repair (Rampino and Bohr. lY941. For a lesion to be detected in this assay it must inhibit the exonucleolytic activity of T4 DNA polymerase. Se- quencing analysis has shown that the 3’-5’ exonuclease procession of T4 DNA polymerase is blocked by a wide variety of lesions in DNA produced by such agents as ultraviolet light (Doetsch et al., 19851. and N’-guanine adduct of 4-nitroquinoline l-oxide (Panigrahi and Walker, 1990).

Using this T4 DNA polymerase assay, oxidative damage and repair in DUG, a human mutS mismatch repair homolog, was measured in young and senescent human diploid fibroblasts exposed to 50% killing level hydrogen peroxide. It was found that: (I) After a 50 PM hydrogen peroxide exposure, senescent human diploid fibroblasts suffer a higher level of DNA oxidation, and repair their DUG gene less efficiently, (II) Without any hydrogen peroxide treatment, DNA from senescent cells contains more T4 DNA polymerase blocking lesions. This difference in DNA oxidation, and lower level of repair agrees with the finding that senescent cells are more sensitive to oxidative stress than young, based on cell via- bility by trypan blue exclusion. This is the first evidence of decreased gene specific repair with age. Whether this lower level of repair found in the senescent human diploid fibroblasts is due to a loss of preferential repair of the transcribed strand in expressed genes. which would parallel what has been found in Cockayne’s syndrome (Leadon and Copper, 19931. is currently being investigated.

The importance of replication and DNA repair in aging and cancer is revealed by the associ- ation of various biochemical defects in these processes that are found in a number of rare hereditary diseases of premature aging and cancer prone syndromes. DNA repair, specifically transcription coupled repair, which mediates the preferential repair of the transcribed strand in expressed genes, has been shown to be defective in Cock- ayne’s syndrome (CS), and in xeroderma pigmen- tosum (XPI. The numerous complementation groups for these diseases are indicative of the number of gene products involved in normal DNA

repair. For XP patients, which suffer a predispo- sition to cancer in sun-exposed skin as a conse- quence of their DNA-repair defects, there are currently seven complementation groups (XPA through XPG) each carrying a mutation in a different gene. In CS patients, which exhibit symptoms of premature aging along with growth retardation, neurological deficiencies and skeletal abnormalities, complementation group B (CSB) has been associated with the ERCC6 gene product, while for complementation group A (CSA) a defect in the transcription elongation factor SII appears to be involved (Hanawalt, 19941.

There are also complementation groups in humans associated with defects in the mismatch repair gene homologs hMSH2, hPMS1, hPMS2, and hMLH1 (Modrich, 1991; Kumar et al., 1994; Casares et al., 1995). In addition, there appears to be a mutator phenotype associated with genetic alterations in polymerase 6 (da Costa et al., 1995). The microsatellite mutator phenotype for cancer, associated with mismatch repair, is gener- ally recessive and may act through diverse cell growth-related genes (suppressor genes and oncogenes) as targets for its mutagenic action (Casares et al., 1995). Somatic genetic alterations (in the two wild type allelles in sporadic cancer cases, and in the remaining wild type allele in hereditary cases), unfolds the expression of a mutator phenotype that eventually precipitates the disastrous genetic program of cancer. If with aging there are decreased levels of gene repair, as is the case in tissue cultured human diploid fibroblasts exposed to hydrogen peroxide, then genetic alterations in selected genes may be expected to also play a role in deteriorative aging. In this regard, it is anticipated that RAP-PCR and possibly AP-PCR will provide information about the molecular phenotypes of known gene products and the, as of yet, undiscovered factors involved in genome maintenance, mutagenesis, and aging.

Acknowledgments

This work has been supported by NIH grants CA 63585, CA 38579 Al 32644.

.I. W&h et al. /Mutation Research 338 (199.5) 215-229 227

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