[]~EVIEWS 3 2 Goutte, C. and Johnson, A. (1988) Cell 52, 875-882 3 3 Dranginis, A.M. (1990) Nature 347, 682-685 3 4 Landschulz, W.H., Johnson, P.F. and McKnight, S.L. (1988) Science 240, 1759-1764

1 6 Yamnae, H.K. et al. (1990) Proc. Natl Acad. Sci. USA 87,

17 18 19

20 21 22 23 24 25 26 27 28 29 30 31

5868-5872 Goodman, L.E. et al. (1990) Proc. Natl Acad. Sci. USA 87, 9665-9669 Sakagami, Y. et al. (1979) Agric. Biol. Chem. 43, 2643-2645 Sakagami, Y., Yoshida, M., Isogai, M. and Suzuki, A. (1981) Science 212, 1525-1527 Kamiya, Y. et al. (1979) Agric. Biol. Chem. 43, 363-369 Kuchler, K., Sterne, R.E. and Thorner, J. (1989) EMBOJ. 8, 3973-3984 Nakayama, M., Miyajima, A. and Arai, K. (1985) ~ B O J . 4, 2643-2648 Marsh, L., Neiman, A.M. and Herskowitz, I. (1991) A n n u . Rev. Cell Biol. 7, 699-728 Teague, M.A., Chaleff, D.T. and Errede, B. (1986) Proc. Natl Acad. Sci. USA 83, 7371-7375 Puhalla, J. (1968) Genetics 60, 461-474 Puhalla, J. (1970) Genet. Res. 16, 229-232 Kronstad, J. and Leong, S. (1989) Proc. NatlAcad. Sci. USA 86, 978-982 Schulz, B. etal. (1990) Cell60, 295-306 Kronstad, J. and Leong, S. (1990) Genes Dev. 4, 1384-1395 Kissinger, C.R. el al. (1990) Ce1163, 579-590 Gillissen, B. etal. (1992) Cel168, 6474557

35 O'Shea, E.K., Klemm, J.D., Kim, P.S. and Alber, T. (1991) Science 254, 539-544 36 Murre, C. el al. (1989) Cell 58, 537-544 37 Lassar, A.B. et al. (1991) Cel166, 305-315 3 8 Casselton, L.A. (1978) in The Filamentous Fungi (Vol. 3) (Smith, J.E. and Berry, D.R., eds), pp. 275-297, Arnold 3 9 Novotny, C.P. et al. (1991) in More Gene Manipulations in Fungi (Bennett, J.W. and Lasure, L.L., eds), pp. 234-257, Academic Press 40 Banuett, F. (1991) Proc. Natl Acad. Sci. USA 88, 3922-3926 41 Day, P. and Anagnostakis, S. (1971) Nature New Biol. 231, 19-20 42 Kenaga, C.B., Williams, E.B. and Green, R.J. (1971) Plant Disease Syllabus, Bait Publishers, Lafayette 43 Scott, M.P., Tamkun, J.W. and Hartzell, G.W., Ill (1989) Biochim. Biophys. Acta 989, 25--48 44 Wolberger, C. et al. (1991) Cell 67, 517-528

F. BANUEITIS IN THE DEPARTMENTOF BIOCHEMISTRYAND BIOPHY$1C~ SCHOOLOFMEDICINE, UNIVERSITYOF CALIFORNIA SAN FRANaSC~ SAN FRANaSC¢~ CA 94143-0448~ USA.

I

T h e initiation and progression of malignant disease are generally thought to involve the accumulation of mutations in several genes (proto-oncogenes) with roles in the control of cellular proliferation 1. While much progress has been made in the past several years in unraveling the complex interactions among proto-oncogene products and other cellular proteins, particularly in various signal transduction pathways, a detailed understanding of how mutations cause cancer remains to be elucidated. One reason for the intense recent interest in the retinoblastoma gene (RB1) on human chromosome 13 is that mutations in both alleles of this gene induce tumor formation in the retina. The apparent simplicity of this model system suggests that understanding the function of the RB1 gene may provide important insights into the regulation of cellular proliferation. Young children with a germ-line mutation in one RB1 allele have a 95% chance of developing a retinoblastoma tumor in their eyes; in most instances, multiple tumors develop, affecting both eyes 2. Germ-line mutation in RB1 also predisposes to a discrete set of other tumors, and approximately 10% of patients develop osteosarcomas or fibrosarcomas. Analysis of epidemiological and clinical data led Knudson to recognize in 1971 that initiation of retinoblastoma tumors was dependent on only two mutations, the first of which, in hereditary cases, is a germ-line RB1 mutation3. Nonhereditary, unilateral retinoblastoma tumors arise when two somatic mutations occur in the same retinal cell. The predisposition to retinoblastoma was linked to chromosome 13ql4,,and restriction fragment length polymorphism (RFLP) analysis of chromosomes in retinoblastoma tumors revealed homozygous loss of function in both alleles of the RB1 locus 4. Since the TIC, MAY 1 9 9 2 ~1992 Elsc~ icr Science Publishers Ltd (I'K)

The retin0blast0ma protein and cell cycle regulation P.A. HAMEL, B.L. GAIHE AND R.A. pHIl.liPS Although the precise function of the retinoblastoma gene product, p l lO P~1, remains unknown, recent data suggest that it plays a role in the control of ceUular proliferation by regulating transcription of genes required f o r a cell to enter or stay in a quiescent or GO state, or f o r progression through the G1 phase of the cell cycle. However, it is difficult to rationalize the expression of p I 10 T M in a wide range of tissues with the fact that mutations in the RBI gene initiate cancers in a limited number of tissues.

presence of the gene product prevents the formation of retinoblastoma tumors, the RB1 gene has been called a 'tumor suppressor gene' or a 'recessive oncogene'. In 1986 Dryja, Friend and Weinberg cloned a cDNA corresponding to the RB1 geneS,6. The 4.7 kb transcript detected by their cloned cDNA is derived from the 27 exons of the RB1 gene, which spans 180 kb on chromosome 13 and contains two very large introns, of 35 kb and 70 kb. Using the cloned cDNA, several investigators confirmed the Knudson hypothesis by showing that all retinoblastoma tumors had mutations in both RB1 alleles ('-1°. Individuals with heritable retinoblastoma had a germ-line mutation in the RB1 gene, affecting either the coding region9 or the promoter region n. The open reading frame of 928 amino acids (Fig. 1) predicted a protein of 110 kDa, but sequence analysis provided no clues to the function of RB1. Analysis of VOL. 8 NO. 5

18(

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FIGH The human retinoblastoma protein. The 4.7 kb transcript of RB1 encodes a protein of 928 amino acids; specific exons of RB1 are indicated for reference. Binding of pll0 Rs~ by viral transforming proteins, Tag or E1A, requires two domains (indicated by brackets), one of 180 (large T-binding domain 1; amino acids 393--572) and another of 128 amino acids (large T-binding domain 2; amino acids 646-773) 3~. As pointed out by Hu et al., all naturally occurring mutations in RB1 disrupt one of these two domains 38. Considering conserved sequences between the human and murine proteins, the human protein has ten Thr or Ser residues contained in the general p34 caC2consensus sequence, Thr/Ser-Pro-X-Basic. Seven are preceded by basic or polar residues (Thr 252, Thr 356, Thr 373; Ser 612, Ser 788, Ser 795 and Ser 811) and three by nonpolar residues (Ser 608, Ser 807 and Thr 821). Five phosphorylation sites have been positively identified and are indicated by an asterisk (*); four of these sites have the p34 cdc2 consensus sequences, while the additional site (Ser 249) is followed by a proline residue but is not contained within the general p34 ~dc2motif ~s. It is interesting that all these sites are outside the T~g/EIA-binding domains.

RB1 mutations in retinoblastoma tumors also gave no clues about functional domains, since most mutations were predicted to lead to premature termination of translation and a truncated protein product. Most mutations that did not alter the reading frame involved substantial deletions of coding sequence. Surprisingly, compared with other proto-oncogenes, the number of RB1 missense mutations in retinoblastoma tumors is very small. p l l 0 ~ 1 , t h e RB1 g e n e p r o d u c t Using antibodies prepared against a fusion protein, Lee and his colleagues showed that the RB1 gene product is a nuclear phosphoprotein ( p l l 0 m l ) t2. A major surprise in studies of the RB1 gene product was the finding that most normal proliferating cells express easily detectable amounts of p l l 0 RB1 (Refs 6, 7, 13). Given the small number of tissues susceptible to malignant transformation in individuals with germ-line mutations in the RB1 gene, most investigators expected RB1 to be developmentally regulated and expressed predominantly in those tissues susceptible to tumor formation by mutations in this gene - retina, bone and connective tissue. The phosphorylation state of p l l 0 TM seems to be important for its function 12. First, the number of 32p_ labeled tryptic peptides ~4 indicates that there are at least ten phosphorylated amino acids. Most of the phosphorylation sites" have not yet been located, although Lees et aL have recently identified five phosphorylation sites (see Fig. 1) 15. Second, phosphorylation occurs exclusively on serine and threonine; there is no detectable tyrosine phosphorylation 12. Third, phosphorylation appears to be tightly regulated during the cell cycle, p110 RB1 is relatively hypophosphorylated in quiescent cells and during G1, but becomes hyperphosphorylated in late G1 and S phase. In cells in G2, only the hyperphosphorylated form of p l l 0 ~1 has been detected ~s-m. The cell c y c l e and RB1 Since RB1 mutations lead to uncontrolled growth and tumor formation in some tissues, and since p110 RBI is subjected to cell cycle-dependent phosphorylation,

it seems likely that RBI plays a role in regulation of the cell cycle. The cell cycle Recent studies indicate that similar mechanisms control the cell cycle in all eukaryotic organisms, from yeast to humans (Fig. 2) 19,20. There appear to be two major points for control of the cell cycle: 'Start', a point in G1 before the onset of DNA synthesis (S phase), and the onset of mitosis. In yeast, both control points seem to involve a unique kinase - p34 ~ & e - whose activity is modulated by association with other regulatory proteins called cyclins. In both yeast and humans, the onset of mitosis (the G2-M transition) is regulated by p34 cdc2 complexed with cyclin B. Genetic studies have shown that in budding and fission yeast, p34 c&a also regulates Start2t; in vertebrates, different but related kinases appear to regulate the control point in G1 (Ref. 22). Exposure of budding yeast to external stimuli such as pheromones (mating factors) before Start inhibits proliferation. Start depends on the p34 c&e kinase and a discrete set of cyclins (CLN1, CLN2 and CLN3) 23. The functions of these cyclins appear to be redundant, since inhibition of S phase requires deletion of all three genes. FAR1 is another important gene that acts at Start e4. FARI mediates the pheromone-induced exit from the cell cycle, probably by turning off CLN2 expression, and f a r l mutants are resistant to cell cycle arrest by pheromones because the CLN2 gene is unregulated. Thus, studies in yeast provide a framework for thinking about the genetic regulation of proliferation in animal cells and for investigating how various growth regulatory genes may function. For example, yeast f a r i mutations, like RB1 mutations, are recessive, loss-offunction mutations leading to uncontrolled growtheL Several recently identified cyclins are probably involved in the G0/G1 to S transition in animal cells. Matsushime et al. 2~ identified three new cyclins in mouse, two of which are expressed in G1. One of these cyclin genes, CYLI, was also isolated from human cells by two other groups using different cloning strategies; it has been named CYCDF 6 and P R A D F by the different groups. Lew et aL es have also isolated

TIG MAY 1 9 9 2 VOL. 8 NO. 5

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FIGE The activity of pll0 ml during the cell cycle. A linear representation of the cell cycle is presented with G0 or the quiescent state considered to be out of cycle, pll0 T M is in a hypophosphorylated form in GO and G1. Just before entering S phase, pll0 ml becomes highly phosphorylated and remains in this hyperphosphorylated state throughout S phase, G2 and into mitosis. During or immediately after mitosis, pll0 ml is dephosphorylated 6°, returning the protein to its presumably active, hypophosphorylated state. While p34-cyclin complexes can phosphorylate pll0 ml in vitro on the same tryptic peptides identified on pll0 ml hyperphosphorylated in vivo 14, it is likely that a related kinase-cyclin complex normally acts on the pll0 ~* before S phase. The phosphatase responsible for dephosphorylation of p110RB1after mitosis has yet to be identified, although protein phosphatase 1 or 2A are likely candidates.

three human G1 cyclins, cyclins C, D and E. The D cyclin is identical to the PRAD1 (CYCD1, CYL1) gene product; cyclin C has a homologue in Drosophila, but there are no reports of cyclins homologous to cyclin E in other species 28.

Interaction of p110RB1with viral transforming proteins

p l lO ~ 1 a n d the GO~G1 to S p h a s e transition Animal cells are thought to follow a regulatory program similar to Start, having an opportunity to enter a quiescent or GO state each time they pass through mitosis into G1. Entry into a GO state is required to control cell proliferation and to allow the differentiation of cells along specific cellular lineages. For example, the differentiated cells of the retina - the photoreceptors and other neurons - cannot achieve their specialized status until they enter GO. Several investigators have suggested that p l l 0 ml may be involved in the control of this transition, or it may act at a restriction point in G1 as cells progress towards S phase 16-18,29. The latter role for p l l 0 ~1 is supported by the recent observation that injection of a bacterially expressed form of p l l 0 *ml truncated at the amino terminus, which mimics the activity of the hypophosphorylated protein, prevented a susceptible osteosarcoma cell line that lacks endogenous p l l 0 RB1 from entering S phase30. In this case, the cells were arrested in G1 several hours before the onset of S phase. It should be noted, however, that a number of other cell lines lacking endogenous p l l 0 RB1, including several malignant cell types with multiple oncogenic mutations, still proliferated unchecked when reconstituted with a retinoblastoma protein that was normally phosphorylated in the cell cycle3L As a normal cell passes through a cell cycle, inactivation of p110 ml is thought to.accompany progression towards S phase. The observed phosphorylation of p110 RBz probably reflects the normal cellular mechanism that inactivates the protein. The existence of many

Several DNA tumor viruses, such as adenovirus, polyoma virus, simian virus 40 (SV40) and papillomavirus, produce proteins that bind to p l l 0 RB1during infection and transformation of cells, and it has been proposed that this binding inhibits the function of p l l 0 ml (Refs 33-35). Such inhibition would be consistent with the idea that the RB1 gene product plays a role in maintaining cells in a quiescent state, since these tumor viruses act to stimulate the proliferation of resting or GO cells. The best studied of such viral proteins ai'e the adenovirus E1A protein and the SV40 large T antigen (Tag) , for which the precise regions required for binding to p l l 0 m* have been identified 36,37. Both of these proteins interact with the same regions of p l l 0 m31 (Ref. 38). Mutation of the p l l 0 RB1binding domains in E1A and Tag usually eliminates the ability of these viruses to transform cells3435; however, p l l 0 Rnl is not the only cellular target for the viral proteins and recent data indicate that, at least for Tag, binding to proteins such as the tumor suppressor p53 may be more important than binding to p l l 0 RB1 (Refs 39-41). The proposal that viral proteins inhibit p l l 0 ml implied that the form of p l l 0 ml that is bound should be the functional form. Ludlow et al. 42 found that Tag preferentially binds the hypophosphorylated form of p110 RB1, consistent with the hypothesis that the active form of p110 ml is hypophosphorylated and that the hyperphosphorylated form is nonfunctional, at least in the regulation of G0/G1. It is interesting that mutations in RB1 that prevent binding to E1A or Tag also prevent phosphorylation of p l l 0 RB1 (Ref. 43). An appealing interpretation of this

tumors deleted for both copies of the RB1 gene32 indicates that progression through the cell cycle, at least in tumor cells, does not require any p110 m~.

TIG MAY1992 VOL.8 NO. 5

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~'~EVIEWS observation is that p110 RB1 is phosphorylated in the cell while bound to other cellular proteins through the same domains recognized by E1A and Tag. Consistent with this hypothesis, during S phase, the p107 protein - a relative of p l l 0 Rm that also binds to Tag and EtA - binds cyclin A in the region between the two Tag-binding domains 44. Complexes containing p107 and cyclin A have detectable associated kinase activity45, suggesting a cyclin-mediated mechanism for the observed phosphorylation 46 of p107. On the basis of these observations, it will not be surprising if one or more G1 cyclins can bind to p l l 0 ml. In this connection, Pines ~9 has suggested that specificity of phosphorylation of substrates during the cell cycle is determined by various complexes of cyclins and p34CaC2-related kinases.

p llO ~1 interacts with important transcription factors It was reasoned that the binding of E1A o r Tag to p l l 0 Rul might prevent p l l 0 m~ from interacting with other cellular proteins. Kaelin et al. demonstrated the existence of a number of cellular binding partners for p l l 0 RB1 by showing that a fusion protein, comprising approximately the carboxy-terminal half of the p l l 0 RB~ protein, including the two Tag-binding domains (see Fig. 1), physically associated with at least eight different cellular proteins 47. Subsequently, Huang et al. characterized a specific 46 kDa protein that interacted with the Tag/E1A-binding domain on p110 ml (Ref. 48). Recently, several groups have presented evidence that p l l 0 RB* interacts with cellular proteins to regulate the transcription of genes implicated in growth control. Robbins et al. demonstrated that p l l 0 ml negatively regulates the c-fos gene 49. They identified a short sequence, which they called the retinoblastoma control element or RCE, in the human c-fos promoter which was sufficient for this negative regulatory effect. Subsequently, Kim et al. proposed that the regulation of c-fos, c-myc and the transforming growth factor-J3 (TGF-~) gene are all influenced by p l l 0 ml acting through RCE elements in the promoters of these genes 5°. They also made the unexpected discovery that p l l 0 m~l induced c-myc and c-fos transcription in some cells but inhibited it in others. Several aspects of the RCE, however, raise questions concerning its proposed role in the regulation of gene expression. First, the observations have proven difficult to replicate; for example, we have found that repression of c-fos by p l l 0 ml has been difficult to reproduce consistently and we have observed only slight repression of c-fos in a cell line reported to allow induction of c-fos by p110 ml (P.A. Hamel et al., submitted). Second, the RCE sequence is not conserved in the mouse promoter; if RB1 is an important regulator of c-fos transcription, one would expect the regulatory elements to be conserved between species. Third, if the RCE is an important cis regulatory element, specific proteins or complexes, probably including p l l 0 m31, should bind to the RCE; no such complexes have yet been described. Clearer evidence for a transcriptional role for p l l 0 ~eu~ has emerged from a series of recent studies showing that p l l 0 RB* interacts with E2F - a cellular transcription factors~-53 first identified as essential for

transcription of the E2 gene of adenovirus 54 - and with another related transcription factor, DRTF155; these factors are involved in the regulation of cellular genes such as c-myc and DHFR (encoding dihydrofolate reductase). Since the interactions between p l l 0 ~eB/and these factors appear to be identical, we will focus our discussion on E2F, for which there are more data. Hypophosphorylated p l l 0 ~ / physically associates with E2F52; following adenovirus infection, E1A has been shown to bind p l l 0 m~l, releasing free E2F. The interpretation suggested for these results is that E2F is inactive when bound to other cellular proteins such as p l l 0 RB1 and, when released from these complexes by EIA, can initiate transcription of viral and cellular genes. Interactions between E2F and other cellular proteins have been shown to change throughout the cell cycle. During G1, E2F binds to pll0m~; in late G1, this complex dissociates after p l l 0 m~ is phosphorylated52, 53,56. During early S phase, most of the E2F appears in a free form, as in adenovirus-infected cells. However, later in S phase E2F appears in a complex s(' that contains p107, cyclin A and p33 oak2, a p34 c"c2related kinase found in mammalian cells 45,57. No p107 was detected in E2F complexes in G1 phase 56. The sequences involved in the regulation of c-myc expression by p l l 0 ~1 are unclear at present. As mentioned above, Kim et al. claimed that c-myc is regulated through an RCE5°. Recent results of Pietenpol et al. imply that another element 5' of the first promoter (P1), which they call the TGF-~ control element (TCE), is essential for the effects of p l l 0 Rp~ on c-myc expressionS~; the TCE, like the RCE, is not highly conserved between mice and humans. Finally, we have found that the E2 element 5' of the second transcrip tion initiation site (P2) in c-myc is essential for its high-level expression in differentiated embryonal carcinoma cells and that the E2 element is required for the transcriptional repression of c-myc by p110 m~ (Ref. 43), presumably mediated through E2F (P.A. Hamel et al., submitted).

A model for the role of pllO ~* during the cell cycle Most of the available evidence suggests that p110 R•1 plays a role in transcriptional regulation. However, as recently stressed by Devoto et al. 57, transcription factors have been implicated in the function of several origins of DNA replication 59 and, at this stage, the possibility that p110 ~/participates directly in DNA replication cannot be eliminated. In fact, given the large number of proteins that interact with p l l 0 Rm, it may participate in multiple protein complexes, playing different roles in different cells. Nevertheless, because of the abundance of data suggesting a role in regulation of growth-controlling genes, we will describe a model (Fig. 3) in which p l l 0 ~ l is involved in the regulation of proliferation either at a switch point after mitosis where cells can enter GO (Ref. 29), or at a restriction point in late G1 (Ref. 30); conceptually, there is little difference between these two sites of action. Entry into a stable GO state or prolongation of G1 phase occurs when hypophosphorylated p110 R'~1 represses transcription of growth-stimulating genes. Exit from GO or passage through G1 requires activation of these genes by

TIG MAY1992 VOL. 8 NO. 5

~"~EVIEWS

(inactive) (inactive)

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,~TGIII Model for p l l 0 TM interactions in controlling cellular proliferation. The model depicts both normal pathways of control (dark tinted arrows) and the parallel, but abnormal, pathway in cells infected with adenovirus (light tinted arrows). Starting at the end of G1 and until the end of G2, p l l 0 ~1 is hyperphosphorylated and in an inactive state (ppll0R•0. During or immediately after mitosis, a phosphatase returns p l l 0 eel to an active hypophosphorylated form capable of binding to various transcription factors and allowing the cells an opportunity to enter a GO state or prolonging the length of G1. As an example, pll0 ~1 is shown interacting with the E2F transcription factor to turn off essential genes. In addition, p l l 0 "~1 may interact with different factors (pX) to activate transcription of other genes. Cells can leave GO and progress through G1 by exposure to growth factors or other mitogenic stimuli that activate kinases, allowing phosphorylation of pll0 RBI, activation of E2F (or inactivation of pX), and transcription of growth-regulating genes such as c-myc. This inactivation is indicated by displacement of p110 RB* from the transcription factors. As cells enter late G1 and S phase, p110 ~1 becomes hyperphosphorylated and E2F becomes associated with p107, cyclin A (Cy A) and p33cak2; the activity of this latter complex is unknown. Cells infected with adenovirus (top) follow a parallel but slightly different pathway out of G0. The initial activation of essential genes occurs by a. viral protein, E1A, binding p l l 0 e*3* and inactivating it. EIA also binds to p107 and will result in modification of E2F activity in S phase in virus-infected cells, but since the function of the p107 complex is unknown, it is not possible to predict the effect of this modification.

transcription factors such as E2F. In GO and in early G1, h y p o p h o s p h o r y l a t e d p l l 0 RS~ binds and inactivates E2F. Presumably, growth-stimulating signals in G1 activate kinases that phosphorylate p l l 0 ~ , releasing active E2F. Mtematively, virus infection causes exit from GO or G1 progression through the binding of viral proteins to p110 ~1, activating E2F in the process. In addition, it is possible that p l l 0 RB* may bind to other factors, pX, to activate transcription of antiproliferation factors, such as TGF-~ (Ref. 50); in this case, phosphorylation of p l l 0 ~ 1 releases and thus inactivates pX. The p l l 0 '~B~ complex, like the p l 0 7 - E 2 F complex, p r o b a b l y also contains a cyclin and a kinase. As mentioned above, mutational analysis is consistent with the idea that p l l 0 ~ 1 is p h o s p h o r y l a t e d as a c o m p l e x and not as a free molecule. If this hypothesis is correct, the binding of cyclin and kinase to the E 2 F - p l l 0 RB* complex may result in phosphorylation of p l l 0 ~ and

activation of E2F. In addition, with such a mechanism, phosphorylation of a small proportion of the total p110 k~l in a cell could have a marked effect on transcription without producing a detectable effect on the overall level of p h o s p h o r y l a t k m of p110a%

The puzzle of tissue specificity The m o d e l described a b o v e suggests possible explanations for the puzzling tissue specifici W of the effects of mutations in the RB1 gene. Individuals having a germ-line mutation in one RB1 allele are predisposed to d e v e l o p tumors in a limited n u m b e r of tissues, primarily retina, b o n e and connective tissue. Since initiation of tumors requires loss of the remaining normal allele, one might expect that all cells that normally express p l l 0 RB1 w o u l d be susceptible to malignant transformation if both copies of the RB1 gene were lost or mutated. However, the incidence of leukemia, colon cancer and skin cancer is not elevated

TIG MAY 1 9 9 2 VOL. 8 NO. 5

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~]~E,VIEWS

above the normal population in patients with germ-line RB1 mutations 2, suggesting that rapidly proliferating tissues such as the blood-forming cells, skin and gut are not susceptible to transformation by RB1 mutations, even though they normally express high levels of p l l 0 ml. On the basis of the k n o w n properties of the RB1 gene and its product, we can envisage several potential explanations for the tissue specificity. (1) Since multiple proteins bind to p l l 0 RR~, the protein may have multiple and/or unique functions in different tissues: a c o m m o n function in all proliferating cells, and a specialized function in differentiating cells. Tissues such as retina that normally enter a terminal, irreversible GO as they develop a highly differentiated phenotype may be critically dependent on the differentiation function of p l l 0 Ru~. (2) Redundant mechanisms, suggested by studies of yeast 23, may be present in some rapidly proliferating tissues, making them less susceptible to transformation when p l l 0 m~ is lost. (3) Loss of p l l 0 ml, in tissues other than those susceptible to transformation in the absence of p l l 0 m~, may be lethal. Detailed analyses of the interactions of pll0/eBI with cyclins, kinases, transcription factors and other proteins may clarify the role of RB1 in cancer and the cell cycle.

Acknowledgements We acknowledge helpful discussions with all of the members of the Toronto Retinoblastoma Group and with Brenda Andrews, Mike Drebot and Phil Branton. We appreciate the unpublished data provided by Joe Nevins. We apologize to our colleagues whose work was not cited because of space limitations. This work was supported through the National Cancer Institute of Canada with funds raised by the Canadian Cancer Society and the Terry Fox Marathon of Hope, the Medical Research Council of Canada, the Canadian Human Genetic Disease Network, and the Retinoblastoma Family Association. B.L.G. is a research associate of the Ontario Cancer Treatment and Research Foundation; P.A.H. is supported by the National Cancer Institute of Canada.

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1197-1206 29 Cooper, J.A. and Whyte, P. (1989) Ce1158, 1009-1011 30 Goodrich, D.W. et al. (1991) Cell67, 293-302 31 Muncaster, M., Cohen, B., Phillips, R.A. and Gallie, B. (1992) CancerRes. 52,654-661 32 Horowitz, J.M. et al. (1990) Proc. Natl Acad. Sci. USA 87,

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4, 383-388 3 6 Egan, C. et al. (1988) Mol. Cell. Biol. 8, 3955-3959 3 7 Cherington, V. et al. (1988) Mol. Cell. Biol. 8, 1380-1384 38 Hu, Q.J., Dyson, N. and Harlow, E. (1990) EMBOJ. 9,

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IP.A. HAME** IlL. GALLIE AND R.A. PHILLIPS AU~ IN THE 1}EPARTMENI$ OF MEDICAL GENETIC~s OPHTHALMOLOGY AND IMMUNOLOGY, UNIVERSITY OF TORONTO, AND DIVISION OF IMMUNOLOGY AND CANCER RESEARCH, ThE HOSPITAL FOR SICK CHILDREN, 5 5 5 UNIVERSITY AVE, TORONT~ CANADA

M5G IX8.

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