LEAD ARTICLE Genetics and Cytogenetics of Retinoblastoma John K. Cowell and Annette Hogg INTRODUCTION Cancer cells are referred to as " i m m o r t a l i z e d , " because, for the most part, they no longer r e s p o n d to intra- and extracellular signals preventing their proliferation or causing t h e m to differentiate. In some cases the b r e a k d o w n of ordered cell division m a y be due to either the inappropriate expression (in the wrong cell or at the wrong time) of a particular protein(s) or the overproduction or stable expression of a growth-promoting gene product. In other tumors, particularly those inherited as a d o m i n a n t trait, it is the absence of a functional gene product, at an important stage of cellular development, that is the rate-limiting step. Because it is the normal function of these genes that prevents tumorigenesis they have been referred to as " t u m o r suppressor genes" or "recessive oncogenes" or, perhaps inappropriately, "antioncogenes" [1]. The study of one particular tumor suppressor gene, responsible for the d e v e l o p m e n t of retinoblastoma, is allowing us to investigate and understand f u n d a m e n t a l processes required for m a n y aspects of tumorigenesis and is the subject of this review. Retinoblastoma Genetics Retinoblastoma is an intraocular t u m o r occurring almost exclusively in young c h i l d r e n and has both hereditary and sporadic forms. In the familial form, w h i c h represents 1 0 % - 1 2 % of all cases, the t u m o r p h e n o t y p e segregates as an autosomal d o m i n a n t trait with 90% penetrance. This means that inheritance of a single mutant gene will result in the tumor p h e n o t y p e in 9 of 10 cases. Transmitting individuals, therefore, are heterozygous for the mutation, their c h i l d r e n having a 50 : 50 chance of inheriting it. Knudson [2], in a mathematical treatise of the subject, showed that, in those i n d i v i d u a l s carrying a predisposing mutation, a single a d d i t i o n a l event (which was later proved to be at the homologous normal locus) is sufficient for tumorigenesis. Thus, in familial cases, it is a p r e d i s p o s i t i o n to cancer that is inherited, and only a single r a n d o m mutational event is required for tumor initiation. Because the chances of this single a d d i t i o n event occurring in the precursor retinal cell p o p u l a t i o n is relatively high, gene carriers develop multiple, bilateral tumors with an early age of onset. In contrast, because in sporadic cases, both mutations must occur in homologous genes in the same retinal precursor

From the ICRF Oncology Group, Institute of Child Health,

London. Address reprint requests to: Dr. John K. Cowell, ICRF Oncology Group, Institute of Child Health, 30 Guilford Street, London WCINIEH, UK. Received March 11, 1992; accepted June 12, 1992.

© 1992 Elsevier Science Publishing Co.. Inc. 655 Avenue of the Americas, New York, NY 10010

cell, they are u s u a l l y unilateral, unifocal, and have a later age of onset. The age of onset m a y be s o m e w h a t m i s l e a d i n g because, histopathologically, Rb consists of e m b r y o n i c - l i k e cells that have failed to differentiate. Because differentiation of the retina is c o m p l e t e soon after birth, it is likely that all Rb tumors have begun d e v e l o p i n g during embryogenesis, and presentation d e p e n d s on m a n y factors, including the stage of d e v e l o p m e n t at w h i c h the second mutation occurred, tumor growth rate, and identification of the symptoms. Because the second m u t a t i o n in hereditary cases occurs r a n d o m l y , the n u m b e r of tumors that develop follow a Poisson distribution that accounts for some of the heterogeneity seen in the p h e n o t y p e , but not all of it. Matsunaga [3] suggested in a series of papers that other factors m a y affect the occurrence of Rb, i n c l u d i n g host resistance. These genetic factors m a y also serve to m o d i f y the expression of the RB1 gene [4]. Although the vast majority of familial cases develop multifocal tumors (3-4, on average) in both eyes, occasional unilaterally affected i n d i v i d u a l s occur w i t h i n these families. In our own series, a p p r o x i m a t e l y 1 0 - 1 5 % of families have at least one unilaterally affected i n d i v i d u a l . However, there are other families where all affected i n d i v i d u a l s only develop either a single, unilateral t u m o r or no t u m o r at all [5]. In some families, a p p a r e n t l y unaffected i n d i v i d u a l s have been seen to have retinal scars that resemble successfully treated tumors. These have been described as benign t u m o r s - - r e t i n o m a s [ 6 ] - - o r as regressed tumors. In family linkage studies, it has been confirmed that these patients with scars carry the m u t a n t gene. Many of these various " m i l d " forms of the tumor can occur in different i n d i v i d u als in the same family [5]. It is not clear w h e t h e r these u n u s u a l p h e n o t y p e s are just part of a c o n t i n u o u s s p e c t r u m of the p h e n o t y p e or w h e t h e r specific m u t a t i o n s w i t h i n the gene are responsible (see later). Occasionally, several affected children can be born to unaffected parents w i t h no prior family history. W h i l e this m a y be due to the segregation of u n u s u a l insertional translocations (see later), it is also possible that it is related to tissue m o s a i c i s m (see ref. 7 for review). A major challenge in the genetic analysis of Rb will be to try to rationalize all this p h e n o t y p i c heterogeneity in terms of m o l e c u l a r events w i t h i n the retinoblastoma gene, RB1. Localization of the RB1 Gene The first steps toward the isolation of the RB1 gene came from cytogenetic analysis of rare Rb patients w i t h other congenital abnormalities, especially mental retardation [8]. These patients had constitutional deletions from one copy of c h r o m o s o m e 13 [9]. A l t h o u g h these deletions vary in size, part of c h r o m o s o m e band 13q14 was always lost, indi1 Cancer Genet Cytogenet 64:1-11 (1992) 0165-4608/92/$05.00

2 cating the location of the predisposition gene. Usually, the larger the deletion, the greater is the range of associated congenital abnormalities [10]. The esterase-D gene (ESD) is also located in 13q14, and those patients with constitutional deletions had reduced cellular ESD enzyme levels [11]. This assay allowed population-based studies demonstrating that approximately 3% of Rb patients carry 13q14 deletions [10, 12]. The position of the ESD gene within 13q14 was demonstrated by Mitchell and Cowell [13], who analyzed an Rb patient with a 13q14-q31 deletion [14] and who had normal ESD levels. When the deletion chromosome was isolated in a somatic cell hybrid, the ESD gene was present, but RB1 was not, indicating that ESD lay proximal to Rb. The suggested location of RB1, within 13q14, varies depending on the particular study. Sparkes et al. [15] favored a 13q14.1 location based on the analysis of the breakpoints in one deletion patient. Yunis and Ramsay [16], using high-resolution cytogenetics, suggested the 13 q14.2-14.3 region as the site of the Rb gene from studying a microdeletion, and we [14] and others [17] favor a more distal location in 13q14.3. Although not of fundamental importance in the cloning of the gene, it may be important in siting the position of other genes and chromosomal breakpoints within 13q14. The dominantly inherited form of Rb was also shown to be due to a gene located in 13q14 by virtue of its linkage to the ESD gene in Rb families [18, 19]. Finally, evidence was presented [20-22] that the same gene was likely involved in sporadic tumors because they frequently underwent loss of heterozygosity for the 13q14 region. The suggestion here was that in retinal precursor cells, an acquired mutation was duplicated at the expense of its normal homologue, rendering the cell homozygous for the initial loss of function RB1 mutation. The mechanisms most frequently involved were due to chromosome nondisjunction and mitotic recombination [21]. This assumption was confirmed because in hereditary Rb, the chromosome retained in the tumor was that transmitted by the affected parent [23]. It now appears that up to 70% of tumors experience this loss of heterozygosity (LOH) [21, 24, 25]. These kind of analyses allowed the origin of the parental mutation to be determined [26]. In sporadic cases there was no differential susceptibility to somatic mutation between the homologous copies of the gene. However, for new germline mutations, the heritable mutation arose on the paternally derived chromosome. These findings and those of Zhu et al. [24] argue against genomic imprinting being important in Rb tumorigenesis but point to new mutational events arising predominantly during spermatogenesis. It has not, however, been possible to attribute these to a paternal age effect [27].

Cytogenetic Analysis of Rb Tumors The mechanisms leading to LOH can clearly occur without structural chromosome rearrangements. It was hardly surprising, therefore, that chromosome 13 abnormalities were found infrequently in tumor cells, although several interesting observations have come out of these cytogenetic analyses. Occasionally, tumor cells from different histologic sites

J.K. Cowell and A. Hogg reveal structural chromosome abnormalities that are specific to that tumor and, therefore, potentially important in the malignant process. However, because this analysis is usually performed on advanced stage tumors, it is difficult to detemine whether these changes are causal in tumorigenesis or consquences of it. This is particularly true for Rb because, in countries where adequate treatment is available, tumors are only removed when too large to treat in situ. Other difficulties encountered in the cytogenetic analysis of Rb is their low mitotic index and the relatively poor quality of the chromosomes derived from them. Despite these limitations several groups have presented interesting observations. Much of the early work has been collated by Potluri et al. [28], who reviewed cytogenetic analysis from 82 cases prepared directly from tumor tissue or cultured cells and cell lines from patients with both sporadic and hereditary Rb. By far the most consistent finding was the presence of (usually two copies of) an isochromosome 6p [i(6p)] and trisomy for all, or part, of the long arm of chromosome 1 (lq+) in 45% and 44% of tumors, respectively. Chromosome abnormalities involving chromosome 13, usually resulting in monosomy 13, was only found in 20% of cases. Similar observations were reported independently by Squire et al. [29] from a single-center study, with i(6p) present in 56% of tumors and trisomy for 1q23-qter in 75%. In this study only 11% of tumors had abnormalities involving chromosome 13. Abnormalities involving lq are the most commonly quoted in all tumor cells. By contrast i(6p) is less frequently observed, being restricted largely to Rb and malignant melanomas [30]. The presence of i(6p) has also been described by others [31-33] but its exact significance in tumorigenesis is still unclear. One possibility is that duplication of certain genes on the short arm of chromosome 6 may be important in tumor progression. Trent and colleagues [34] presented evidence for a tumor suppressor gene on chromosome 6 by introducing this chromosome into melanoma cells and suppressing the malig~ nant phenotype. Other, less frequently reported chromosome abnormalities [28] in Rb tumors include monosomy 16, lp ÷ and the presence of double minutes (DM) and homogeneously staining regions (HSR). DM and HSR represent the cytologic manifestation of gene amplification [35]. In Rb, amplification of the N-myc oncogene has been reported in a few tumors [36-38]. Another cellular oncogene, INT-1, was also shown to the amplified in three tumors [39]. Both of these oncogenes appear to be expressed in cells of neurogenic origin, although, again, their role in tumorigenesis is unclear. Chromosome analysis of independent tumor foci from a bilaterally affected patient showed that each had distinct abnormalities, suggesting an independent origin for them [29]. Tien et al. [31] analyzed a large, apparently unilateral tumor and found cytogenetically distinct clones. This was interpreted to mean that the tumor probably arose as the result of fusion of several foci. This has important implications for counseling, because unilateral tumors are thought to be associated predominantly with sporadic events. Multifocal tumors, however, even in only one eye, probably identify that patient as a hereditary case, especially if these tumors have a relatively early age of onset.

Genetics and Cytogenetics of Retinoblastoma

Constitutional Chromosome Abnormalities In addition to deletions of 13q14, individuals predisposed to Rb may also carry constitutional chromosome translocations with breakpoints in 13q14. In some cases inheritance of an unbalanced form of the rearrangement results in deletion of the 13q14 region [40-45]. In other cases the translocation appears to be reciprocal, and molecular analysis of the breakpoint junctions, so far, has shown that the 13q14 breakpoint interrupts the RB1 gene )46-48]. In one case, at least, this rearrangement is associated with a small deletion within the RB1 gene [47]. A few cases have been reported where the translocation partner chromosome is the X [49-51]. Because random X-activation occurs in females, when the derivative chromosome is inactivated in retinal precursor cells, the position of the breakpoint on 13 is not important because the whole chromosome experiences "genetic silencing," thereby constituting the first hit. In two families in our series, with t(1;13)(q22;q14) and t(13;20)(q14;p12) translocations, the patient with Rb inherited the rearrangement from a parent who was not affected (B. Gibbons, personal communication). A similar situation was discussed by Dryja et al. [22] for 13q14 deletions, in that deletion carriers often only have unilateral, unifocal tumors. In our survey [10], only half of the 16 cases reported had unilateral tumors. There are also reports of deletion patients who have never developed tumors [52-54]. The age of onset of tumors from patients with chromosome 13 abnormalities also appears to be later than those with germinal mutations [55]. The explanation for the low penetrance in deletion patients is not clear, although one proposal is that these deletions expose lethal mutations that are deleterious to the rapidly growing tumor cells [22]. This does not explain the low penetrance af the reciprocal translacatian carriers, unless large deletions are associated with the rearrangement. In one case of a t(1;13) rearrangement [47], although a deletion was associated with the translocation, it was maximally 8 kb long and confined to the RB1 gene. Keith and Webb [56] described a patient with a 13q14 deletion with a single tumor in one eye and a retinoma in the other, both of which are considered to be "mild" forms of the disease. Isolation and Structure of the RB1 Gene The cloning of the RB 1 gene proceeded very rapidly following the construction of a chromosome 13 specific (DNA) library from flow-sorted chromosomes [57]. Of 12 probes isolated at random, one was shown to lie in the 13q14 region, and adjacent sequences identified a highly conserved region [58]. In fact, this probe lay within the RB1 gene and was used to isolate a 4.7-kb cDNA from a fetal retinal cell library [59]. Several other groups [60, 61] repeated this procedure and collectively showed that structural rearrangements of the RB1 gene were present in approximately 20% of tumors. Abnormal mRNAs were seen to a greater [60] or lesser [62] degree. Often the mRNA product was completely missing in tumors, reflecting the loss of function nature of many of the mutations. RB1 is a relatively large and complex gene, approximately 180 kb long and composed of 27 exons ranging in length from 31 bp to 1873 bp [63, 64]. The intervening

3

intron sequences vary in size from 80 bp to 70,500 bp. The exons are clustered into three groups, separated by the two largest introns [65]. In human cells, the gene encodes an mRNA transcript of 4.7 kb [59, 60], which is expressed in the majority of cell types studied. The RB 1 promoter region is unusual in that it lacks a TATA box or CCAAT motif [65], typically found in eukaryotic promoters and involved in the positioning of RNA polymerase II and binding of the CAT/enhancer binding protein (C/EBP), respectively. The lack of a typical TATA box element may account for the variable transcription start sites observed in $1 nuclease experiments [66]. The 5' promotor region is GC-rich and has characteristics of a CpG island [65, 67], whereas the 3' end of the gene, involved with transcription termination, contains a hexanucleotide with a consensus signal for polyadenylation. Several studies have defined regions of the RB1 promotor required for transcription. One such region, between 185 and 206 bp upstream of the initiating methionine residue, has been located by the analysis of deletion mutants and appears to be essential for transcription [64, 66, 68]. This area contains a sequence showing homology to the binding site for the SP1 transcription factor [64], suggesting it might contain the RB1 promotor, although SP1 itself shows only weak binding. This binding site associates with another nuclear factor, referred to as RBF1, whose function is not known [68]. A sequence showing homology to the binding sites for the ATF/CREB family of transcription factors is also located within the putative RB 1 promotor region and occurs adjacent to the site where RBF-1 and SP1 bind. Potential zinc-finger motifs, associated with DNA binding ability, were found in exons 17, 18, 20, and 21 and putative leucine zipper motif, associated with protein-protein interaction, was entirely encoded for by exon 20 [66]. Experimental evidence that these regions function as such is still lacking.

Family Linkage Analysis With the cloning of the RB1 gene, the expectation was that the cDNA would identify restriction fragment length polymorphisms (RFLPs) within the population that, in turn, could be used in linkage analyses. However, despite extensive studies in several laboratories, no useful RFLPs were discovered. To overcome this limitation, Wiggs et al. [69] isolated five unique DNA sequences from the introns of RB1 that, in combination, were useful in gene carrier detection. The principles of "gene tracking" have been presented elsewhere (see ref. 70), but essentially the transmitting individual must be heterozygous at the locus in question. Despite the theoretical probability of recombination within the RB1 gene, no recombinants have yet been reported in Rb families [25, 69, 71, 72]. The most useful single copy probe isolated by Wiggs et al. [69] is RS2.0, which identifies a variable number tandem repeat (VNTR) based on a 53-bp repeat with eight different alleles. In our own study [(72) and unpublished observations), 60% of patients were heterozygous--"informative"--at this locus, and 80-85% of all families were informative using a combination of all five probes. Using this type of linkage analysis, one can now determine which individuals in Rb families

4 have inherited the predisposing mutation and, equally important, which have not. For those who, unequivocally, have not inherited the mutant gene, repeat ophthalmologic screening of subsequent generations is not necessary. For those patients identified as gene carriers (affected and unaffected), prenatal screening is possible. The first report of this kind [73] used chorionic villus (CV) sampling after only 9 weeks of pregnancy and demonstrated the child would be unaffected. This patient is now 55 months old and tumor free. Since that report, we have performed nine more pre- and postnatal predictions, most of which have been followed for at least 2 years, and all of which have proved correct [74]. Since the first report by Wiggs et al. [69], it has become possible to analyze two of the polymorphic sites within RB1 using the polymerase chain reaction [75, 76]. This means that the analysis can be completed in 2 days compared with 5-7 days for probes requiring analysis by Southern blotting techniques [74]. In addition, several other RFLPs have been added to the armory of tests available for family studies, some of which are single base pair polymorphisms identified by sequencing [77], and another is a VNTR based on a small (1-4 bp) repeat unit, which must be resolved on sequencing gels. This latter RFLP, called Rbl.20, has approximately 28 different alleles and is informative in 85-95% of families [74, 77]. Thus, it is now possible to offer family linkage analysis and prenatal screening to virtually all Rb families with the realistic expectation that all gene carriers can be identified. In addition to the obvious advantages to the patients and their families, the screening load for ophthalmology departments will be greatly reduced in the future. The application of the polymerase chain reaction (PCR) to the analysis of polymorphic sites means that DNA isolated from formalin fixed, histopathologically processed materials can now also be used. We have been able to use this strategy to establish linkage phase in a family where the key member had died [78]. For the 85% of Rb patients without a family history, many will carry "new" germline mutations. The problem for them is that linkage analysis will not be available for their firstborn children. It is necessary, therefore, to identify the causative mutation in these cases that will demonstrate, unequivocally, who is at risk to tumor development. The characterization of the structure of the RB1 gene has made this possible. The RB Protein The RB1 gene is widely expressed in all human tissues examined to date [59, 60, 79]. A messenger ribonucleic acid (mRNA), 4.7 kb long, is transcribed in human tissues and encodes a protein of 928 amino acids. In all but a few exceptions, rodent and murine tissues also express a similar-sized transcript. A shorter 2.3-kb transcript has been reported in rat fetal brain [60], and a 2.8-kb transcript was found in adult mouse testes [80]. In the latter case, the onset of expression of the shorter mRNA coincided with the appearance of spermatids in the testes. The significance of these shorter transcripts has not yet been determined but may result from differential processing of the RB1 gene. The protein product of the RB1 gene (pRB) is generally thought to suppress cell growth because the absence of a

J.K. Cowell and A. Hogg normal functional product is associated with tumor formation [60, 81]. Reintroduction of a functional pRB into cell lines lacking the RB1 gene apparently suppresses tumorigenicity in immunosuppressed mice [82-85]. Others, however, have failed to repeat this observation and cast doubt on the infallability of the nude mouse assay systems used for this kind of analysis [86, 87]. Early analysis of pRB showed that it could be phosphorylated, mainly on serine and threonine residues [88, 89]. A number of species of pRB, ranging from approximately 110 to 115 kd, were detected using Western blotting techniques [88, 90]. The higher molecular weight species represented more highly phosphorylated proteins, those with lower molecular size being less phosphorylated [89, 91]. Subsequently, the phosphorylation status of pRB was shown to be cell cycle dependent [92-94]. The underphosphorylated forms of pRB were found in quiescent cells, whereas the phosphorylated forms appeared toward the onset of DNA synthesis [93, 94]. Moreover, in synchronized cells, the appearance of highly phosphorylated forms of pRB coincided with the transition from G1 --~ S-phase of the cell cycle [93, 94]. The more highly phosphorylated forms were still present in G2 and M, although the underphosphorylated forms began to predominate [91, 94, 95]. These observations provided strong circumstantial evidence that pRB was involved in the control of the cell cycle, pRB, however, does not have DNA sequence-specific binding motifs so must apparently exert any regulatory controls as a result of interactions with other cellular proteins. Association of pRB with Viral Oncoproteins Several DNA tumor viruses produce proteins that can override the normal growth regulation of the cell, driving infected cells through S-phase where viral DNA can be synthesized.This is apparently activated by binding to host cellular proteins. During studies aimed at characterizing these cellular proteins, pRB was found to interact with the Ela protein of adenovirus [90] as well as with the transforming proteins of two members of the papovavirus family; the Simian virus 40 large T (LT) antigen [95] and the E7 protein of human papilloma virus [96]. All of the viral transforming proteins share conserved regions that are necessary for their transforming function. Mutations in these conserved regions, which prevent cellular transformation, also prevent binding to pRB [97]. LT binds specifically to the underphosphorylated form of pRB [89], and LT-pRB complexes are found only in G1 when the underphosphorylated form of pRB is present [89]. These observations suggested that the underphosphorylated pRB is active in the suppression of cell growth and that phosphorylation, or sequestration by LT, allowed the cells to enter S-phase. Furthermore, entry of cells into S-phase could be blocked by microinjection, into cells lacking pRB function, of either the full length or a truncated protein lacking the LT binding domain [98]. By selective deletion of parts of the RB1 gene, two noncontiguous regions were identified that were necessary for Ela and LT binding [99-101). These protein binding domains comprised amino acids 393-572 and 646-772, respectively, which have collectively been called the RB

Genetics and Cytogenetics of Retinoblastoma "pocket." Deletion of the spacer region, located between these two noncontiguous sequences, prevents Ela/LT binding [99], but replacement with nonspecific sequences restored that function. It appears, therefore, that, by sequestering pRB from the cell during G1, the viral transforming proteins allow the cell to enter S-phase. In fact, it has emerged that pRB participates in the establishment of protein complexes that associate and dissociate during the cell cycle. Many of these processes are under tight control, and the whole system appears to be regulated by the biochemical modification of the participants in these complexes and their availability to join the complex.

Involvement of pRB in the Cell Cycle The Ela protein is thought to transform cells by altering the activity of cellular transcription factors. One such protein is E2F, which has been shown to be involved as a transcription regulator of several cellular genes [102]. Ordinarily E2F is complexed with specific cellular proteins that effectively suppress its function [103]. Ela, however, can dissociate E2F from its protein complexes, releasing free E2F. To exert its transcriptional regulation, E2F must form a stable complex with other proteins in order to bind to specific DNA sequences in the promotor regions of the genes it controls. The conserved regions of Ela facilitate E2F binding; the same regions are also responsible for binding pRB. It was not surprising, therefore, to find that pRB also complexes with E2F and that Ela can dissociate them [103-106]. E2F associates with the form of pRB found primarily in G1 [106]. The E2F/pRB complex ordinarily dissociates near the G1-S boundary, before S phase, releasing free E2F. The suggestion is that pRB can control the transcriptional activities of E2F by binding to it. Dissociation of this complex allows free E2F to activate responsive cellular promotors that contribute to the release of cells from their proliferative suppression. pRb has also been shown to complex with a developmentally regulated cellular transcription factor, DRTF-1, which binds to the same sequence motif as E2F, although the relationship between them is unclear [103, 105, 107, 108]. The DRTF-la complex associates with pRB [107], and the complex can be dissociated by Ela. The suggestion is that, by binding with a sequence-specific transcription factor, pRB can control transcription of target genes. Because DRTF is developmentally regulated and its expression is tissue dependent, this association suggests a role for pRB in differentiation [105, 107, 108]. Two other Rbbinding cellular proteins, called RBF-1 and RBF-2, have recently been cloned and sequenced. They contain amino acid sequences homologous to the Ela/LT/E7 Rb binding domains [109]. E2F binding sites are present in the promoters of a number of cellular genes, implicating pRB in transcriptional regulation. Sequences resembling the E2F binding site are present in the c - m y c promoter [110] and pRb can suppress c - m y c transcription. Robbins et al. [111] showed pRB could interact with the c-los promotor region to repress transcription as well as down regulating Apl, a transcription factor thought to be involved in regulating a set of early response genes required for cell growth. The m y c and los gene prod-

5 ucts are part of the early response genes following growth stimulation and the demonstration that pRB can bind to their promoters in vitro suggests a cooperation in the control of cell proliferation [110]. The same c/s-acting element is found in the promoter of the transforming growth factorfll (TGFB) gene [112]. TGFB molecules are potent growthinhibiting polypeptides and act in G1, although the exact mechanism is not known. Transcriptional initiation of growth related genes such as c - m y c is inhibited by TGFB. Treatment of cells with TGFB prevents phosphorylation of pRB and maintains it in its growth suppressive state [113]. Because TGFB also inhibits cells lacking pRB their interaction, however, is not obligatory for suppression of cell growth [114]. From the preceeding discussion, it is clear that pRB may be involved in a variety of cell-cycle-regulated activities. The cyclins constitute a family of related, cell-cycle-dependent, proteins [115] that associated with cellular protein kinases, including the protein products of cdc2 and cdk2 [116, 117], which are thought to be key regulators of cell cycle progression [115]. Both cyclin A and pRB locate to DNA by virtue of the ability to complex with DRTF and cyclin A functions to assemble pRB into the transcription factor complex. It is the underphosphorylated form of pRB that joins the complex [107]. Cyclin A, therefore, may serve to direct cdc2 kinase to dephosphorylate pRB releasing it from the complex. These observations further support that pRB is involved in the regulation of the cell cycle, pRB can be phosphorylated at all potential sites in vitro by cdc-2, at least [116]. Further evidence supporting an in vivo role for cdc 2 phosphorylation of pRB is currently lacking. Cyclin A also associates with the form of E2F that appears once cells have entered S-phase [118]. In constrast, E2F has been found to interact with the unphosphorylated form of pRB, implying that this complex occurs in G1 [106]. Another protein, p107, is associated with the cyclin A/ E2F/cdk2 complex [117, 119-121]. It has been proposed that the E2F/p107 containing S-phase complex and the E2F/ pRB containing Gl-phase complex may each serve specific functions relevant to the maintenance of normal cell growth. Both complexes were virtually absent in G2/M [120]. The p107 protein is highly homologous to pRB and was known to be a cellular target for Ela [90, 95-97,121]. p107 has been cloned and has two viral oncoprotein binding domains, homologous to those forming the Rb pocket in pRB, but separated by a much larger "spacer element" (121). It is the spacer element that binds cyclin A. The ability of cyclins to activate cellular kinases raises many possibilities for their role in these complexes. The p107 and pRB proteins may themselves be phosphorylated through these interactions, or the E2F protein may be modified as part of its cell cycle specific activity. It is thought that this may result in the release of free E2F, which can then activate transcription of other genes. It is possible, however, that the mechanism is more indirect and that other, as yet unknown, intermediary factors are involved. The p107 protein is found in cell lines with homozygous deletions of RB1 [121]. If one assumes p107 is normal in these cells, it clearly cannot compensate for the lack of pRB. The p107 protein is found in a cyclin A/E2F complex in S-phase

6 [117], which also contains cdk2, suggesting pRB and p107 cooperate in regulating E2F activity at different stages of the cell cycle. RB Inactivation in Other Tumors

Individuals carrying constitutional mutations in the RB1 gene are at significantly higher risk than the general population to the development of second, nonocular tumors later in life (122-124). These tumors are usually osteosarcomas (OS) and soft-tissue sarcomas. For some patients who have received radiation therapy for their retinoblastoma(s), these second tumors arise within the irradiated field, but a significant proportion occur in unirradiated tissues. These observations suggest that the RB1 gene also plays a role in tumorigenesis in other tissues, the frequency of which can be raised by 7 irradiation. Although the risk of the development of OS is highest in adolescence, the risk to other cancers persists throughout the lives of mutant gene carriers [125]. Structural rearrangements of the RB1 gene have been identified in osteosarcomas [81,126,127] and in soft-tissue sarcomas [63,128], supporting its role in the development of these tumors. Loss of heterozygosity (LOH) for RB 1 infers that a recessive mutation is exposed and has been demonstrated in a wide range of human malignancies. A combination of LOH studies, analysis of structural rearrangements by Southern blotting, and absence of pRB using immnnofluorescence and Western blotting has implicated the RB1 gene in tumorigenesis in breast cancer [129, 130], small cell lung carcinoma [131-133], bladder cancer (134-136), esophageal cancers (137), hepatocarcinomas (138), prostate cancer (83), and a range of leukemias (139). However, only a proportion of these tumors have RB1 abnormalities and it is unlikely that RB1 mutations are the rate limiting step in these tumors. Instead, these mutations probably contribute to a multistep process of tumorigenesis in these tissues. One interesting tumor associated with hereditary Rb is pineoblastoma. Because the pineal is thought to be a vestigial photoreceptor organ, patients with retinal and pineal tumors have been described as having "trilateral" retinoblastoma [140, 141]. The chances of two such rare tumors occurring coincidentally is negligible and suggests that the RB1 gene also contributes to the development of pineoblastoma. Mutations in RB1

Early data, derived mostly from Southern blot analysis, identified structural abnormalities of RB1 in 10%-40% of hereditary and sporadic Rb tumors. The majority of these involved deletions of all or part of the gene [59-61]. It appears that deletion breakpoints can occur throughout the length of the gene [48, 142,143]. In the study by Canning and Dryja [142] 2 of 12 deletions had breakpoints in the region containing exons 13-17, and 7 of 12 deletions included that region. Others also found this region was involved in deletions and rearrangements [47, 61] possibly indicating the location of a breakpoint cluster region. A clustering of dyad symmetrical elements was found in exons 16 and 17 and an inverted repeat sequence in exon 17 [64]. Whether this is related to the chromosome abnormali-

J.K. Cowell and A. Hogg ties is not known, but these DNA sequences can form stem and loop structures that possibly promote rearrangement. In 6 of 8 deletions reported by Canning and Dryja, there was a direct (sometimes imperfect) repeat at the breakpoint site, and one of these was always lost. We (Hogg, unpublished) and others [144] have also found short, direct repeats associated with small deletions within RB1. This observation is consistent with a slipped mispairing during DNA replication. In this model, pairing on one DNA strand occurs with the downstream sequence on the other strand creating a loop. When the replication loop is resolved, one copy of the repeat, plus the intervening sequence, is deleted. The majority of mutations, however, appear to be more subtle sequence changes and have been found in both Rb tumors and normal cells from predisposed individuals [145-148]. Analysis of mRNA from tumors is not often possible, because the majority of them are successfully treated in situ. Of those that are removed, some do not produce RB1 and mRNA, and from many others the samples are too small to analyze. Depsite this limitation, Dunn and colleagues [145,146] were able to use tumor cell lines to isolate RNA and, with the use of RNase protection, identify mutations in 50%-65% of cases. They found that small deletions were the most common abnormality but suggested this may reflect limitations in the RNase protection procedure. Small deletions were also found most frequently in a proportion of SCLC cell lines studied by Mori et al. [149]. Unfortunately, it appears that, in constitutional cells heterozygous for the causative RB 1 mutation, the normal transcript dominates over the abnormal one [146]. This effectively excludes the possibility of using RNase protection, or sequencing from mRNA, to identify mutations in constitutional cells from Rb patients. To overcome these problems, Yandell et al. [147] sequenced the RB1 gene, exon by exon, following amplification using (PCR). Although this approach proved useful in identifying mutations, it was very time-consuming. The introduction of methods to prescreen the amplified products, such as the single strand conformation polymorphism (SSCP) technique [150] and chemical cleavage mismatch methods such as the HOT technique [151], have made the analysis more rapid. In combination, the available data shows that there is no apparent hot spot for mutations within RB 1, the majority of which are insertions, deletions, or single base pair substitutions that result in the production of premature stop codons. These mutations would be predicted to result in a structurally grossly abnormal protein and are consistent with studies where Rb tumors mostly had no detectable pRB [81]. Approximately 10% of mutations affect splice junction sites. Bookstein et al. [152], analyzing mRNA from a prostate carcinoma cell line, identified an in-frame deletion of exon 21, but, surprisingly, the splice junction sequences were normal. A single-point mutation within exon 20 had resulted in removal of the exon. A similar situation was reported by Mori et al. [149], who showed that the mRNA from an SCLC cell line was missing exon 22. A two base pair mutation GC --~ TT, within the exon, apparently converted a glutamine to a stop codon but, in fact, resulted in the entire exon being spliced out. Presumably these muta-

Genetics and Cytogenetics of Retinoblastoma

tions generate cryptic splice sites that result in the endogenous splice sites being ignored. A n o t h e r e x a m p l e was described in an SCLC that occurred as a second tumor in a patient with hereditary Rb [151]. A single base pair substitution w i t h i n exon 21, b e y o n d the splice site, results in excision of that exon from the mRNA. In some cases, however, [134, 146], mutations actually occur in the splice junction and affect splicing accordingly. It is clear, therefore, that the consequences of p o i n t mutations found by sequencing the DNA m a y not be so obvious, and, without access to the mRNA, h o w processing will be affected cannot always be predicted. Missence mutations, s i m p l y substituting one amino acid for another, appear to be less common. In our o w n survey of hereditary Rb patients, one example was found in exon 20, w h i c h was associated with a "lowp e n e t r a n c e " p h e n o t y p e [153]. It is tempting to speculate that the substitution of a single amino acid only compromises the function of the protein, and, unless the second m u t a t i o n in the tumor precursor cell causes loss of RB1 function, d u p l i c a t i o n of the " w e a k " mutation allows sufficient functional pRB to be produced, so preventing tumorigenesis. This is consistent with our observation in this particular family, because m a n y of the mutant gene carriers were either unaffected or have regressed tumors. Sakai et al. [68] also investigated low-penetrance families and found mutations in recognition sequences for different transcription factors in the promoter region of RB1. Again, the suggestion was that, as a result, a quantitative decrease in transcription occurs rather than complete inactivity. Sufficient pRB is p r o d u c e d , however, and any p h e n o t y p i c consequences are mild. Whether single amino acid changes will generally be found in patients with m i l d phenotypes is still not clear. Against this, Yandell et al [147] reported a ser--~ leu substitution in exon 14 in a tumor, and we have found a met --~ arg change in one t u m o r in the same exon (Hogg, unpublished). A cys ~ phe amino acid substitution in exon 21 was reported that resulted in a protein unable to b i n d to the viral oncoproteins and unable to complex with the set of Rb-associated cellular proteins [104, 154]. CONCLUSIONS The d e v e l o p m e n t of techniques to analyze the RB1 gene exon by exon means that heterozygous mutations can n o w be detected in constitutional cells in Rb patients. This means that, for the 85% of cases without a family history, it will be possible to identify mutant gene carriers that will greatly i m p r o v e the clinical m a n a g e m e n t of the disease. Too few naturally occurring mutations in Rb patients have been identified, so far, to allow correlations to be d r a w n b e t w e e n genotype and phenotype. Depending on the patterns that might emerge, it m a y eventually be possible, however, to predict the course of the disease and perhaps even to determine who is susceptible to the d e v e l o p m e n t of second tumors. Analysis of mutations in RB1 has also h e l p e d our understanding of the function of the gene. In those tumor cells where a nonfunctional pRB is produced, the mutations u s u a l l y prevent Ela, LT, and E7 b i n d i n g [90, 95, 96]. The same is true for m a n y of the other proteins that b i n d to pRB

7

[104], i n c l u d i n g transcription factors DRTF1 a n d RBP-1, and early growth response genes such as c-myc a n d N-myc [110]. This analysis has d e m o n s t r a t e d i m p o r t a n t functional domains of pRB and highlighted w h i c h interactions are important for normal cell growth control. The attachment of pRB to nuclear structures w i t h i n the cell [155] is also affected by mutations in the Rb pocket. One issue that is still v i r t u a l l y u n a d d r e s s e d is h o w pRB controls the differentiation of i m m a t u r e retinal cells into mature photoreceptors. Presumably, there are other, as yet undiscovered, proteins (growth factors?) that m u s t bind pRB to carry out their function but are o n l y available in particular cells at a particular stage of development. These interactions, promoting terminal differentiation, m u s t be critical, because loss of function of RB1 alone is responsible for tumorigenesis. The challenge is still to find these proteins and resolve one of the f u n d a m e n t a l processes of embryonic development.

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