Ординатура / Офтальмология / Английские материалы / Ocular Oncology_Albert, Polans_2003

The underlying nature of those weak alleles causing low penetrance retinoblastoma involves a mutational process that either induce a reduction in the amount of pRB synthesized or the production of a partially nonfunctional pRB [70]. Otterson et al. proposed the classification of those two kinds of mutational mechanisms into two classes [71]: class 1 comprising mutations leading to a reduction of pRB expression and class 2 including mutations leading to the production of a partially inactivated pRB. Harbour reviewed all the mutations described in low-penetrance retinoblastoma, classified them according to these two groups, and provided an insight into their consequences for tumorigenesis [72]. Class 1 mutations are less common and either affect the promoter region, disturbing binding with transcription factors required for an efficient pRB expression (e.g., SP1 or ATF), or involve splice-site sequences. Class 2 mutations affect the coding region of RB1. They consist mainly of small in-frame deletions or point mutations affecting amino acid residues critical for pRB structure and function (localization in the LXCXE or non-LXCXE binding site, E2F binding in particular). Thus, the identification of these mutations provides further information on pRB.

F.Genetic Anomalies Associated to pRB Inactivation and Concept of a Third Hit

RNB often displays other genetic changes in association with RB1 inactivation [73]. This observation has led to the concept of a third hit, which would be involved in tumorigenesis, tumor progression, and resistance to treatment [74]. A third additional mutation could disrupt signals that would normally lead to apoptosis in the absence of a functional pRB, giving rise to a tumor. This could be the case if the chormosome imbalance resulted in an additional copy of a portion of a chromosome at an oncogene location or a monosomy at a tumor suppressor gene locus, thus providing a growth advantage. The most frequently described genetic anomaly is the gain of one or two extra copies of the short arm of chomosome 6, often as an isochromosome i6p, which is unique to RNB. This anomaly is found in 60% of the tumors [75] and has been shown to be associated with a worse prognosis and undifferentiated histological forms [76]. The other most common chromosomal imbalances found in RNB are a partial or complete trisomy of 1q (50% of RNB), monosomy 16 (46%), and extra copies of 2p (37.5%) [77]. Interestingly, Herzog et al. found more common and complex chromosomal imbalances on older patients at the age of surgery, which suggests that mutational abnormalities leading to the progression of RNB might be different in younger versus older patients [78]. Different authors are focusing on identifying potential candidates—oncogene or tumor suppressor gene—localized in those portions of chromosomal imbalance. Among the candidates are Notch, a receptor involved in neuronal development [74]; the oncogene MYCN, located in 2p [77]; glioblastoma amplification on chromosome 1; the GAC1 gene and the renin gene, REN, located in 1q32 [78]; and the kinesin-like gene, RBKIN, in 6p22 [79]. The identification of these potential candidates will give us a better understanding of the genesis of retinoblastoma.

V.CONCLUSION

Since the two-hit hypothesis was announced by Knudson, our understanding of the molecular genetics underlying the development of retinoblastoma has increased dramatically. This has led to a better evaluation of patient risks, enabling a more informed choice of appropriate treatment and follow-up. Our increased knowledge of the structure and function of pRB and its implication in the development of retinoblastoma also gives us a better understanding of its involvement in cell proliferation and of tumorigenesis in other tissues.

REFERENCES

1.Tamboli A, Podgor MJ, Horm JW. The incidence of RNB in the United States: 1974 through 1985. Arch Ophthalmol 1990; 108:128–132.

2.Knudson AG Jr. Mutation and cancer: Statistical study of retinoblastoma. Proc Nat Acad Sci USA 1971; 68:820–823.

3.Yunis JJ, Ramsy N. Retinoblastoma and subband deletion of chromosome 13. Am J Dis Child 1978; 132:161–163.

4.Sparkes RS, Sparkes MC, Wilson MG, Towner JW, Benedict W, Murphree AL, Yunis JJ. Regional assignment of genes for human esterase D and retinoblastoma to chromosome band 13q14. Science 1980; 208:1042–1044.

5.Sparkes RS, Murphree AL, Lingua RW, Sparkes MC, Field LL, Funderburk SJ, Benedict WF. Gene for hereditary retinoblastoma assigned to human chromosome 13 by linkage to esterase D. Science 1983; 219(4587):971–973.

6.Connolly MJ, Payne RH, Johnson G, Gallie BL, Alderdice PW, Marshall WH, Lawton RD. Familial, EsD-linked, retinoblastoma with reduced penetranceand variable expressivity. Hum Genet 1983; 65:122–124.

7.Harbour JW, Lai SL, Whang-Peng J, Gazdar AF, Minna JD, Kaye FJ. Abnormalities in structure and expression of the human retinoblastoma gene in SCLC. Science 1988; 241:353–357.

8.Lee EY, To H, Shew JY, Bookstein R, Scully P, Lee WH. Inactivation of the retinoblastoma susceptibility gene in human breast cancers. Science 1988; 241:218–221.

9.Smith SM, Sorsby A. Retinoblastoma: some genetic aspects. Ann Hum Genet 1958; 23:50–58

10.Ashley DJ. The two ‘‘hit’’ and multiple ‘‘hit’’ theories of carcinogenesis. Br J Cancer 1969; 23(2):313–328.

11.Vogel F. Genetics of retinoblastoma. Hum Genet 1979; 52:1–54.

12.Commings DE. A general theory of carcinogenesis. Proc Natl Acad Sci USA 1973; 70:3324–3328.

13.Benedict WF, Murphree AL, Banerjee A, Spina CA, Sparkes MC, Sparkes RS. Patient with 13 chromosome deletion: Evidence that retinoblastoma gene is a recessive cancer genes. Science 1983; 219:973–975.

14.Cavenee WK, Dryja TP, Phillips RA, Benedict WF, Godbout R, Gallie BL, Murphree AL, Strong LC, White RL. Expression of recessive alleles by chromosomal mechanisms in retinoblastoma. Nature 1983; 305:779–784.

15.Friend SH, Bernards R, Rogelj S, Weinberg RA, Rapaport JM, Albert DM, Dryja TP. A human DNA segment with properties of the gene that predisposes to retinoblastoma and osteosarcoma. Nature 1986; 323:643–646.

16.Lee WH, Bookstein R, Hong F, Young LJ, Shew JY, Lee EY. Human retinoblastoma susceptibility gene: Cloning, identification, and sequence. Science 1987; 235:1394–1399.

17.Fung Y-KT, Murphree AL, T’Ang A, Qian J, Hinrichs SH, Benedict WF. Structural evidence for the authenticity of the human retinoblastoma gene. Science 1987; 236:1657– 1661.

18.Goodrich DW, Lee W-H. Molecular characterization of the retinoblastoma susceptibility gene. Biochem Biophys Acta 1993; 1155:43–61.

19.Kaelin WG, Jr. Functions of the retinoblastoma protein. Bioessays 1999; 21:950–958.

20.DiCiommo D, Gallie BL, Bremner R. Retinoblastoma: The disease, gene and protein provide critical leads to understand cancer. Cancer Biol 2000; 10:255–269.

21.Nevins JR. The Rb/E2F pathway and cancer. Hum Mol Genet 2001; 10:699–703.

22.Harbour JW, Dean DC. The RB/E2F pathway: Expanding roles and emerging paradigms. Genes Dev 2000; 14:2393–2409.

23.Zheng L, Lee W-H. The retinoblastoma gene: A prototypic and multifunctional tumor suppressor. Exp Cell Res 2001; 264:2–18.

24.Sears RC, Nevins JR. Signaling networks that link cell proliferation and cell fate. J Biol Chem 2002; 277:11617–11620.

25.Lipinski MM, Jacks T. The retinoblastoma gene family in differentiation and development. Oncogene 1999; 18:7873–7882.

26.Ferguson KL, Slack RS. The Rb pathway in neurogenesis. Neuroreport 2001; 12:55–62.

27.Toguchida J, McGee TL, Paterson JC, Eagle JR, Tucker S, Yandell DW, Dryja TP. Complete genomic sequence of human retinoblastoma susceptibility gene. Genomics 1993; 17:535–543.

28.Ohtani-Fujita N, Fujita T, Aoike A, Ofsifchin NE, Robbins PD, Sakai T. CpG methylation inactivates the promoter activity of the human retinoblastoma tumor suppressor gene. Oncogene 1993; 8:1063–1067.

29.Sakai T, Othani N, McGee TL, Robbins PD, Dryja TP. Oncogenic germ-line mutations in Sp1 and ATF sites in the human retinoblastoma gene. Nature 1991; 353:83–86.

30.Sakai T, Toguchida J, Ohtani N, Yandell DW, Rapaport JM, Dryja T. Allelic-specific hypermethylation of the retinoblastoma tumor-suppressor gene. Am J Hum Genet 1991; 48:880–888.

31.T’Ang A, Varley JM, Chakraborty S, Murphee AL, Fung YKT. Structural rearrangement of the retinoblastoma gene in human breast carcinoma. Science 1988; 241:263–266.

32.Classon M, Dyson N. p107 and p130: versatile proteins with interesting pockets. Exp Cell Res 2001; 264:135–147.

33.DeCaprio JA, Ludlow JW, Lynch D, Furukawa Y, Griffin J, Piwnica-Worms H, Huang CM, Livingston DM. The product of the retinoblastoma susceptibility gene has properties of a cell cycle regulatory element. Cell 1989; 58:1085–1095.

34.Harbour JW, Dean DC. Chromatin remodeling and Rb activity. Curr Opin Cell Biol 2000; 12:685–689.

35.Ferreira R, Naguibneva I, Pritchard LL, Ait-Si-Ali S, Harel-Bellan A. The Rb/ chromatin connection and epigenetic control: Opinion. Oncogene 2001; 20:3128–3133.

36.Lee JO, Russo AA, Pavlvetich NP. Structure of the retinoblastoma tumour-suppressor pocket domain bound to a peptide form HPV E7. Nature 1998; 391:859–865.

37.Kaye FJ, Kratze RA, Gerster JL, Horowitz JM. A single amino acid substitution results in a retinoblastoma protein defective in phosphorylation and oncoprotein binding. Proc Natl Acad Sci USA 1990; 87:6922–6926.

38.Paggi MG, Martelli F, Fanciulli M, et al. Defective human retinoblastoma protein identified by lack of interaction with E1A oncoprotein. Cancer Res 1994; 54:1098–2104.

39.Zhu XP, Dunn JM, Phillips RA, Goddard AD, Paton KE, Becker A, Gallie BL. Preferential germline mutation of the paternal allele in retinoblastoma. Nature 1989; 340:312–313.

40.Dryja TP, Mukai S, Petersen R, Rapaport JM, Walton D, Yandell DW. Parental origin of mutations of the retinoblastoma gene. Nature 1989; 339:556–558.

41.Dryja TP, Morrow JF, Rapaport JM. Quantification of the paternal allele bias for new germline mutations in the retinoblastoma gene. Hum Genet 1997; 100:446–449.

42.Bader JL, Meadows AT, Zimmerman LE, Rorke LB, Voute PA, Champion LA, Miller RW. Bilateral retinoblastoma with ectopic intracranial retinoblastoma: Trilateral retinoblastoma. Cancer Genet Cytogenet 1982; 5:203–213.

43.Wiggs JL, Dryja TP. Predicting the risk of hereditary retinoblastoma. Am J Opthalmol 1988; 106:346–351.

44.Mukai S. Molecular genetic diagnosis of retinoblastoma. Semin Ophthalmol 1993; 8:292–299.

45.Bunin GR, Emanuel BS, Meadows AT, Buckley JD, Woods WG, Hammond GD. Frequency of 13q abnormalities among 203 patients with retinoblastoma. J Natl Cancer Inst 1989; 81:370–374.

46.Kloss K, Wa¨hrisch P, Greger V, Messmer E, Fritze H, Ho¨pping W, Passarge E, Horsthemke B. Characterization of deletions at the retinoblastoma locus in patients with bilateral retinoblastoma. Am J Med Genet 1991; 39:196–200.

47.Blanquet V, Creau-Goldberg N, de Grouchy J, Turleau C. Molecular detection of constitutional deletions in patients with retinoblastoma. Am J Med Genet 1991; 39:355– 361.

48.Dunn JM, Phillips RA, Zhu X, Becker A, Gallie BL. Mutations in the RB1 gene and their effects on transcription. Mol Cell Biol 1989; 9:4596–604.

49.Yandell DW, Campbell TA, Dayton SH, Petersen R, Walton D, Little JB, McConkieRosell A, Buckley EG, Dryja TP. Oncogenic point mutations in the human retinoblastoma gene: Their application to genetic counseling. N Engl J Med 1989; 321:1689–1695.

50.Yandell DW, Dryja TP. Detection of DNA sequence polymorphisms by enzymatic amplification and direct genomic sequencing. Am J Hum Genet 1989; 45:547–555.

51.Lohmann D, Horsthemke B, Gillessen-Kaesbach G, Stefani FH, Hofler H. Detection of small RB1 gene deletions in retinoblastoma by multiplex PCR and high-resolution gel electrophoresis. Hum Genet 1992; 89:49–53.

52.Hogg A, Onadim Z, Baird PN, Cowell JK. Detection of heterozygous mutations in the RB1 gene in retinoblastoma patients using single-strand conformation polymorphism analysis and polymerase chain reaction sequencing. Oncogene 1992; 7:1445–1451.

53.Shimizu T, Toguchida J, Kato MV, Kaneko A, Ishizaki K, Sasaki MS. Detection of mutation of the RB1 gene in retinoblastoma patients by using exon-by-exon PCR-SSCP analysis. Am J Hum Genet 1994; 54:793–800.

54.Mastrangelo D, Squitieri N, Bruni S, Hadjistilianou T, Frezzotti R. The polymerase chain reaction (PCR) in the routine genetic characterization of retinoblastoma: A tool for the clinical laboratory. Surv Ophthalmol 1997; 41:331–340.

55.Noorani HZ, Khan HZ, Gallie BL, Destky AS. Cost comparison of molecular versus conventional screening of relatives at risk for retinoblastoma. Am J Hum Genet 1996; 59:301–307.

56.Musarella MA, Gallie BL. A simplified scheme for genetic counseling in retinoblastoma. J Pediatr Ophthalmol Strabismus 1987; 24:124–125.

57.Lohmann DR, Brandt B, Ho¨pping W, Passarge E, Horsthemke B. The spectrum of the RB1 germ line mutations in hereditary retinoblastoma. Am J Human Genet 1996; 58:940–949.

58.Greger V, Passarge E, Horsthemke B. Somatic mosaicism in patient with bilateral retinoblastoma. Am J Hum Genet 1990; 46:1187–1193.

59.Sippel KC, Fraioli RE, Smith GD, Schalkoff ME, Sutherland J, Gallie BL, Dryja TP. Frequency of somatic and germ-line mosaicism in retinobalstoma: Implications for genetic counseling. Am J Hum Genet 1998; 62:610–619.

60.Blanquet V, Turleau C, Gross-Morand MS, Senamaud-Beaufort C, Doz F, Besmond C. Spectrum of germline mutations in the RB1 gene: A study of 232 patients with hereditary and non hereditary retinoblastoma. Hum Mol Genet 1995; 4:383–388.

61.Harbour JW. Overview of RB gene mutations in patients with retinoblastoma. Implications for clinical genetic screening. Ophthalmology 1998; 105:1442–1447.

62.Mancini D, Singh S, Ainsworth P, Rodenhiser D. Constitutively methylated CpG dinucleotides as mutation hot spots in the retinoblastoma gene (RB1). Am J Hum Genet 1997; 61:80–87.

63.Hogg A, Bia B, Onadim Z, Cowell JK. Molecular mechanisms of oncogenic mutations in tumors from patients with bilateral and unilateral retinoblastoma. Proc Natl Acad Sci USA 1993; 90:7351–7355.

64.Lohmann DR. RB1 gene mutations in retinoblastoma. Hum Mutat 1999; 14:283–288.

65.Dryja TP, Cavenee W, White R, Rapaport JM, Petersen R, Albert DM, Bruns GAP. Homozygosity of chromosome 13 in retinoblastoma. N Engl J Med 1984; 310:550–553.

66.Knudson AG. Antioncogenes and human cancer. Proc Natl Acad Sci USA 1993; 90:10914–10921.

67.Hagstrom SA, Dryja TP. Mitotic recombination map of 13cen-13q14 derived from a an investigation of loss of heterozygosity in retinoblastomas. Proc Natl Acad Sci USA 1999; 96:2952–2957.

68.Matsunaga E. Hereditary retinoblastoma: penetrance, expressivity and age of onset. Hum Genet 1976; 33:1–15.

69.Lohmann DR, Brandt B, Hopping W, Passarge E, Horsthemke B. Distinct RB1 gene mutations in patients with low penetrance in hereditary retinoblastoma. Hum Genet 1994; 94:349–354.

70.Sakai T, Othani N, McGee TL, Robbins PD, Dryja TP. Oncogenic germ-line mutations in Sp1 and ATF sites in the human retinoblastoma gene. Nature 1991; 353:83–86.

71.Otterson GA, Chen WD, Coxon AB, Khleif SN, Kaye FJ. Incomplete penetrance of familial retinoblastoma linked to germ-line mutation that result in partial loss of RB function. Proc Natl Acad Sci USA 1997; 94:12036–12040.

72.William Harbour JW. Molecular basis of low-penetrance retinoblastoma. Arch Ophthalmol 2001; 119:1699–1704.

73.Squire J, Gallie BL, Phillips RA. A detailed analysis of chromosomal changes in heritable and non-heritable retinoblastoma. Hum Genet 1985; 70:291–301.

74.Gallie BL, Campbell C, Devlin H, Duckett A, Squire JA. Developmental basis of retinalspecific induction of cancer by RB mutation. Cancer Res 1999; 59:1731s–1735s.

75.Squire J, Phillips RA, Boyce S, Godbout R, Rogers B, Gallie BL. Isochromosome 6p, a unique chromosomal abnormality in retinoblastoma: Verification by standard staining techniques, new densitometric methods, and somatic hybridization. Hum Genet 1984; 66:46–53.

76.Cano J, Oliveros O, Yunis E. Phenotype variants, malignacy, and additional copies of 6p in retinoblastoma. Cancer Genet Cytogenet 1994; 76:112–115.

77.Mairal A, Pinglier E, Gilbert E, Peter M, Validire P, Desjardins L, Doz F, Aurias A, Couturier J. Detection of chormosome imbalances in retinoblastoma by parallel karyotype and CGH analyses. Genes Chromosomes Cancer 2000; 28:370–379.

78.Herzog S, Lohmann DR, Buitingt K, Schu¨ler A, Horsthemke B, Rehder H, Rieder H. Marked differences in unilateral isolated retinoblastomas from young and older children studied by comparative genomic hybridization. Hum Genet 2001; 108:98–104.

79.Chen D, Pajovic S, Duckett A, Brown VD, Squire JA, Gallie BL. Genomic amplification in retinoblastoma narrowed to 0.6 megabase on chromosome 6p containing a kinesinlike gene, RBKIN. Cancer Res 2002; 62:967–971.

The development of technologies for introducing specific mutations into the germline of mice has led to the creation of several new models of retinoblastoma, which have been employed both for studying the molecular genetics of retinoblastoma and for evaluating novel therapeutic strategies. The basic methods for creating these mice are briefly described below, followed by a description of various mouse models and their application to the study of retinoblastoma.

II.METHODS FOR THE CREATION OF GENETICALLY ENGINEERED MICE

In the past two decades, several related methods have been developed for introducing specific genetic modifications into the mouse genome [9,10], allowing for the creation of a wide range of mouse models of human disease, including cancer. The most straightforward of these methods involves the introduction of new genes into the mouse genome to create ‘‘transgenic mice.’’ In cancer research, this approach is well suited for creating models involving expression of dominantly acting oncogenes. Alternatively, targeted mutations that specifically inactivate genes normally present in the mouse genome can be introduced to create ‘‘knockout’’ mice. This approach allows one to model loss-of-function mutations, such as the heterozygous or homozygous inactivation of tumor suppressor genes. More recently, a variety of refinements of these strategies have been developed that allow for conditional transgene expression or gene inactivation, greatly amplifying the power of these technologies for modeling human disease [11,12].

A.Production of Transgenic Mice

Although the term transgenic can strictly be applied to any mouse carrying experimentally introduced DNA, including knockout mice, the term is generally used to refer to mice in which a new gene or segment of DNA is experimentally introduced into the germline by nonhomologous recombination. Most often, this involves microinjection of a solution containing experimentally modified DNA into one of the two pronuclei of fertilized mouse eggs and reimplantation of the microinjected eggs into the oviducts of recipient females for subsequent development (Fig. 1). The resulting pups are screened for the presence of the ‘‘transgene’’ in their genome by biopsying a small piece of tail or other tissue at the time of weaning and preparing genomic DNA for polymerase chain reaction (PCR) or Southern blot analysis. In order for the transgene to be retained in the cells of the fully developed mouse, it must physically integrate into one of the chromosomes of the microinjected pronucleus, an event that occurs in approximately 10–20% of injected eggs on average. When the transgene does integrate into a chromosome, it becomes part of the genetic makeup of the resulting ‘‘founder’’ mouse and is subsequently transmitted to offspring in a Mendelian fashion. It should be noted that the site of chromosomal integration is random and can profoundly influence expression of the transgene. In addition, transgenes can integrate in single or multiple copies (usually in a tandem head-to-tail array at a single locus); copy number also influences transgene expression level. Thus, there can be considerable variability in transgene expression levels and resulting phenotype among lines of mice established

Figure 1 Production of transgenic mice by pronuclear injection.

from different founders carrying the same transgene. However, within any given line of mice, the patterns of transgene expression and phenotype are generally relatively consistent and stable over multiple generations.

A major consideration in the creation of transgenic mice is the design of the transgene to be introduced. Although full-length unmodified genes or even larger segments of chromosomes can be introduced, more often the transgene consists of an experimentally created gene containing two critical elements, a protein-coding region and a regulatory region. The protein-coding region (often derived from a cDNA) dictates the gene product to be produced by the transgene, while the promoter or regulatory region of the transgene determines the cell-type specificity, developmental timing, and level of transgene expression. For the creation of transgenic mouse models of cancer, the protein-coding region often encodes a known dominantly acting oncogene or gene whose expression can contribute to the development of cancer.

B.Production of Knockout Mice

In contrast to the genetic modification in transgenic mice, which involves the random integration of the transgene DNA into the genome, the genetic modification in knockout mice involves the introduction of a defined mutation into a specific gene in the mouse genome by a much less frequent homologous recombination event (Fig. 2). Thus, it becomes necessary to screen a large number of cells to identify those in which the desired homologous recombination event has occurred among a much larger number of cells in which the foreign DNA has randomly integrated into the genome. This is accomplished by introducing the ‘‘targeting

Figure 2 Production of knockout mice by gene targeting in ES cells.

vector’’ by electroporation into cultured embryonic stem (ES) cells, which are totipotent cells derived from the inner cell mass of day E3.5 mouse blastocysts [13]. The targeting vector usually contains several kilobases of DNA homologous to the gene being targeted for mutation as well as a gene encoding a drug-resistance enzyme (e.g., a bacterial neomycin resistance gene), which serves both to disrupt the coding region of the targeted gene, and to allow for the selection of cells that have incorporated the foreign DNA. Individual surviving clones of ES cells are then molecularly screened to identify the homologous recombinants. Once clones of correctly targeted ES cells are identified, these are microinjected into recipient blastocysts, where they commingle with cells of the endogenous inner cell mass. The blastocysts are then reimplanted into the uterus of foster females and allowed to develop to birth. The resulting mice are referred to as chimeras, since many or all of their tissues are derived from a mix of both normal and genetically modified ES cells. If any of the germline cells (e.g., sperm) of the chimeric mice are derived from the genetically modified ES cells, their offspring can inherit the mutation, which will subsequently be transmitted to future generations in a Mendelian fashion. The first generation offspring from the founding chimeras are heterozygous for the mutated gene and are usually either phenotypically normal or display only modest phenotypes. To observe the phenotype resulting from complete absence of expression of the targeted gene, heterozygotes must be interbred to generate homozygous offspring.