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Kufe DW, Pollock RE, Weichselbaum RR, et al., editors. Holland-Frei Cancer Medicine. 6th edition. Hamilton (ON): BC Decker; 2003.

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Holland-Frei Cancer Medicine. 6th edition.

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Retinoblastoma—A Paradigm for Tumor-Suppressor Gene Function

, MD, PhD and , MD.

Essentially concurrent with the initial cell fusion experiments of Harris and colleagues, Knudson's analysis of the age-specific incidence of retinoblastoma led him to propose that two “hits” or mutagenic events were necessary for retinoblastoma development.15 Retinoblastoma occurs sporadically in most cases, but in some families, it displays autosomal dominant inheritance. In an individual with the inherited form of the disease, Knudson proposed that the first hit is present in the germ line, and thus in all cells of the body. However, the presence of a mutation at the susceptibility locus was argued to be insufficient for tumor formation, and a second somatic mutation was hypothesized to be necessary for promoting tumor formation. Given the high likelihood of a somatic mutation occurring in at least one retinal cell during development, the dominant inheritance pattern of retinoblastoma in some families could be explained. In the nonhereditary form of retinoblastoma, both mutations were proposed to arise somatically within the same cell. Although each of the two hits could theoretically have been in different genes, subsequent studies (see below) led to the conclusion that both hits were at the same genetic locus, ultimately inactivating both alleles of the retinoblastoma (RB1) susceptibility gene. Knudson's hypothesis served not only to illustrate mechanisms through which inherited and somatic genetic changes might collaborate in tumorigenesis, but it also linked the notion of recessive genetic determinants for human cancer to somatic cell genetic findings on the recessive nature of tumorigenesis.

The first clue to the location of a putative gene responsible for inherited retinoblastoma was obtained from karyotypic analyses of patients with retinoblastoma. Constitutional deletions of chromosome 13 were observed in some cases.16 Subsequent cytogenetic studies of patients with retinoblastoma identified detectable germ line deletions of chromosome 13 in only about 5% of all patients. However, in cases where deletions were observed, the common region of deletion was centered around chromosome band 13q14.17 Levels of esterase D, an enzyme of unknown physiologic function, were found to be reduced in patients with deletions of 13q14, when compared with karyotypically normal family members.18 This finding implied that the esterase D gene might be contained within chromosome band 13q14. Indeed, analysis of the segregation patterns of esterase D isozymes and retinoblastoma development in families with inherited retinoblastoma established that the esterase D and RB1 loci were genetically linked.19

Subsequently, a child with inherited retinoblastoma was found to have esterase D levels approximately one-half of normal, although no deletion of chromosome 13 was seen in karyotype studies of his blood cells and skin fibroblasts.20 Interestingly, tumor cells from this patient had a complete absence of esterase D activity, despite harboring one apparently intact copy of chromosome 13. Based on these findings, it was proposed that the copy of chromosome 13 retained in the tumor cells had a submicroscopic deletion of both the esterase D and RB1 loci. Moreover, it was concluded that the initial RB1 mutation in the child was recessive at the cellular level (ie, cells with inactivation of one RB1 allele had a normal phenotype). The effect of the predisposing mutation, however, could be unmasked in the tumor cells by a second event, such as the loss of the chromosome 13 carrying the wild-type RB1 allele. This proposal was entirely consistent with Knudson's two-hit hypothesis.15, 21

To establish the generality of these observations, Cavenee, White, and their colleagues undertook studies of retinoblastomas, both inherited and sporadic types, by using DNA probes from chromosome 13. Probes detecting DNA polymorphisms were used, so that the two parental copies of chromosome 13 in the cells of the patient's normal and tumor tissues could be distinguished from one another. By using such markers to compare paired normal and tumor samples from each patient, they were able to demonstrate that loss of heterozygosity (ie, the loss of one parental set of markers) for chromosome 13 alleles had occurred during tumorigenesis in more than 60% of the cases studied.22 Loss of heterozygosity (LOH) for chromosome 13, and specifically for the region of chromosome 13 containing the RB1 gene, occurred via a number of different mechanisms (Figure 7-2). In addition, through study of inherited cases, it was shown that the copy of chromosome 13 retained in the tumor cells was derived from the affected parent and that the chromosome carrying the wild-type RB1 allele had been lost.22, 23 These data established that the unmasking of a predisposing mutation at the RB1 gene, whether the initial mutation had been inherited or had arisen somatically in a single developing retinoblast, occurred by the same chromosomal mechanisms.

Figure 7-2. Chromosomal mechanisms that result in loss of heterozygosity for alleles at the retinoblastoma predisposition (RB1) locus at chromosomal band 13q14.

Figure 7-2

Chromosomal mechanisms that result in loss of heterozygosity for alleles at the retinoblastoma predisposition (RB1) locus at chromosomal band 13q14. In the inherited form of the disease (top left), the affected daughter inherits a mutant RB1 allele ( (more...)

Patients with the inherited form of retinoblastoma were known to be at an increased risk for the development of a few other cancer types, particularly osteosarcomas. LOH for the chromosome 13q region containing the RB1 locus was seen in osteosarcomas arising in patients with the inherited form of retinoblastoma, suggesting that inactivation of both RB1 alleles was critical to the development of osteosarcomas in those with inherited retinoblastoma.24, 25 Chromosome 13q LOH was also frequently observed in sporadic osteosarcomas. These molecular studies of retinoblastomas and osteosarcomas provided strong support for Knudson's two-hit hypothesis, and suggested that a variety of tumors might arise from the unmasking of recessive mutations at different tumor suppressor loci.11, 21, 23 In addition, the studies demonstrated that both the inherited and sporadic forms of a tumor appeared to arise as a result of similar genetic alterations. Moreover, osteosarcoma, a common second primary neoplasm in patients with inherited retinoblastoma, was found to have pathologic genetic mechanisms in common with retinoblastoma.

Cloning and Analysis of the RB1 Gene

The molecular cloning of the RB1 gene was facilitated by the identification of an anonymous DNA marker from the chromosome 13q14 region that detected DNA rearrangements in retinoblastomas.26 Through the analysis of the DNA sequences flanking this DNA marker, a gene with the properties expected of RB1 was identified.27–29 The RB1 gene has a complex organization with 27 exons, spanning greater than 200 kilobases (kb) of DNA, and an RNA transcript of about 4.7 kb.30 The RB1 gene appears to be expressed ubiquitously rather than being restricted to retinoblasts and osteoblasts.

Cloning of RB1 allowed study of mutations that inactivate the gene. Although gross deletions of RB1 sequences have been observed in a small subset of retinoblastomas and osteosarcomas, most tumors appear to express full-length RB1 transcripts and do not have detectable gene rearrangements when analyzed by Southern blotting.31–35 Hence, the detection of inherited and somatic mutations in the RB1 gene in most cases has required detailed characterization of its sequence. Mutant RB1 alleles from both constitutional cells of individuals with the inherited form of the disease and from retinoblastomas of both inherited and sporadic types have now been quite extensively analyzed.35, 36 This analysis has provided definitive molecular evidence supporting Knudson's two-hit model. As predicted, patients with inherited retinoblastoma have been found to have one mutated and one normal allele in their constitutional (blood) cells. In retinoblastomas of such individuals, the remaining RB1 allele has been found to be inactivated by somatic mutation, usually by loss of the normal allele through a gross chromosomal event (see Figure 7-2), but in some cases by point mutation. Multiple tumors arising in an individual patient with inherited retinoblastoma all were found to contain the same germ line mutation but had different somatic mutations affecting the remaining RB1 allele. The vast majority of patients with a single retinoblastoma and no family history of the disease have two somatic mutations in their tumors and two normal alleles in their constitutional cells.

Although the identification of mutations in both alleles of the RB1 gene in retinoblastomas and osteosarcomas provides strong support for the proposal that the cloned gene is, indeed, the gene whose inactivation is a crucial and likely rate-determining step in tumor formation, additional support for the critical growth regulatory function of the gene was provided by the demonstration that restoration of RB1 function could suppress some aspects of retinoblastoma tumorigenesis. The transfer of a cloned copy of wild-type RB1 to retinoblastoma and other tumor cells in culture affects a number of cellular properties, including morphology and differentiated phenotype, growth rate in culture, and the ability of the cells to form colonies in soft agar and progressive tumors in nude mice.37–39 However, such studies generally involve expression of an exogenous gene at nonphysiologic levels, and the significance of the phenotypes produced is questionable. Indeed, many genes that have little or no role in tumorigenesis can inhibit the growth of transfected cells when expressed at high levels.

The observation that RB1 is ubiquitously expressed is rather puzzling, given the spectrum of tumors that develop in patients with germ line RB1 mutations. Patients with germ line mutations of RB1 are at elevated risk for the development of only a rather limited number of tumor types, including retinoblastomas in childhood, osteosarcomas, soft-tissue sarcomas, and melanomas later in life. RB1 germ line mutations fail to provide a strong predisposition to most common cancers, despite the fact that somatic RB1 mutations have been observed in a wide variety of other cancer types, including breast, small cell lung, bladder, pancreas, and prostate cancers.40 It is possible that retinoblastoma functions slightly differently in retinal epithelial cells than in other cell types, so that the RB1 gene acts as a “gatekeeper” in retinal cells but not in other cell types.

Function of the Retinoblastoma Protein (p105-RB)

The protein product of the RB1 gene is a nuclear phosphoprotein with a molecular weight of about 105,000 Daltons known as p105-Rb or, more commonly, as pRb.39 Harlow and colleagues' studies provided the first critical insights into pRb function. They demonstrated that pRb formed a complex with the E1A oncoprotein encoded by the murine DNA tumor virus adenovirus type 5.41 Prior studies of E1A had established that it had many effects on cell growth, including cell immortalization and cooperation with other oncogenes (eg, mutated Ras oncogene alleles) in neoplastic transformation. It was thus hypothesized that functional inactivation of pRb through its interaction with E1A might contribute to some of E1A's transforming functions. Additional support for this proposal was provided by data establishing that mutations inactivating the ability of E1A to bind to pRb also inactivated E1A's transforming function.42, 43

The significance of physical interaction between pRb and a DNA tumor virus oncoprotein was further supported by the subsequent demonstration that other DNA tumor virus oncoproteins also formed complexes with pRb, including SV40 T antigen and the E7 proteins of human papillomavirus (HPV) types 16 and 18 (Figure 7-3).44, 45 Many of the mutations inactivating the transforming activities of these oncoproteins also inactivated their ability to interact with pRb. Furthermore, E7 proteins from “high-risk” HPVs (ie, those linked to cancer development), such as HPV 16 and 18, formed complexes more tightly with pRb than did E7 proteins of “low-risk” viruses (eg, HPV types 6 and 11). These studies of pRb provided compelling evidence that DNA tumor viruses might transform cells, at least in part, by inactivating tumor-suppressor gene products. In addition, given the critical dependence of DNA tumor viruses on harnessing the cell's machinery for replication of the viral genome, the studies also provided support for the hypothesis that pRb might normally control cell growth by interacting with cellular proteins that regulated the cell's decision to enter into the DNA synthesis (S) phase of the cell cycle.

Figure 7-3. Representation of interactions between tumor-suppressor gene products and proteins encoded by DNA tumor viruses.

Figure 7-3

Representation of interactions between tumor-suppressor gene products and proteins encoded by DNA tumor viruses. Large T antigen from polyomaviruses (such as simian virus 40 [SV40]) binds both the retinoblastoma (pRb) and p53 proteins. For the adenoviruses (more...)

The functional activity of pRb is regulated by phosphorylation during normal progression through the cell cycle.39, 46–48 Accordingly, pRb appears to be predominantly unphosphorylated or hypophosphorylated in the G1 phase of the cell cycle and maximally phosphorylated in G2 (Figure 7-4). The critical phosphorylation events regulating the function of pRb are likely to be mediated at the boundary between the G1 and S phases of the cell cycle by cyclin and cyclin-dependent kinase (cdk) protein complexes.39, 40 Presumably, phosphorylation of pRb, particularly at the G1-S boundary, inactivates its ability to interact with cellular proteins that regulate entry into S phase. For example, when it is not phosphorylated, pRb forms complexes with proteins in the E2F family and inhibits transcription by recruiting proteins involved in transcriptional repression.39 When phosphorylated, pRb can no longer efficiently form complexes with E2Fs (see Figure 7-4). The E2F proteins, when dimerized with their DP (differentiation-regulated transcription factor) partner proteins, are then capable of activating the expression of a number of genes that are likely to regulate/promote entry into S phase, including DNA polymerase α, thymidylate synthase, ribonucleotide reductase, cyclin E, and dihydrofolate reductase.39 That E2F family members directly affect cellular proliferation was recently shown in conditional mouse knockout models.49 Several other cellular proteins that bind to pRb have been identified, but their functions and the significance of their interactions with pRb remain less-well characterized than pRb's interactions with E2Fs.

Figure 7-4. Phosphorylation regulates the function of pRb during the cell cycle.

Figure 7-4

Phosphorylation regulates the function of pRb during the cell cycle. The pRb protein is hypophosphorylated in the G1 phase of the cell cycle, and phosphorylation (P) of specific sites appears to increase during progression through the cell cycle. A protein (more...)

The retinoblastoma protein shares significant similarity with two proteins known as p107 and p130. Like Rb, these proteins have been found to form complexes with certain DNA tumor virus proteins.50–52 Because of their similarity to pRb, the p107 and p130 proteins have been termed pRb “cousins.” Although all three proteins may have related cellular functions, there is only rather limited evidence indicating that mutations in the p107 or p130 genes contribute to cancer development. Germ line mutations in p107 and p130 have not been reported in humans, somatic mutations in the p130 gene have been seen in only a small fraction of small cell lung and nasopharyngeal cancers,53, 54 and somatic mutations in p107 appear to be even rarer or absent in cancer. Furthermore, whereas germ line inactivation of the mouse pRb gene predisposes the animals to pituitary adenomas and carcinomas as well as thyroid tumors, germ line inactivation of the murine homologs of the p130 and p107 genes appears to have no effect on tumor predisposition.55, 56 Future studies will undoubtedly shed further light on the means by which loss of pRb function, but not that of p107 or p130, contributes to cancer development. Nevertheless, a reasonable hypothesis is that pRb, via its apparently selective interactions with certain E2F family members, such as E2F1, may regulate expression of cellular genes distinct from those regulated by p107 and p130.

By agreement with the publisher, this book is accessible by the search feature, but cannot be browsed.

Copyright © 2003, BC Decker Inc.
Bookshelf ID: NBK13944

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