Genes in the RB pathway and their knockout in mice

seminars in

CANCER BIOLOGY, Vol 7, 1996: pp 279–289

Genes in the RB pathway and their knockout in mice Suh-Chin J. Lin*, Stephen X. Skapek*† and Eva Y.-H.P. Lee*

osteosarcoma, soft tissue sarcoma, and carcinomas of breast, lung, bladder and prostate. Reintroduction of wild type RB into RB-deficient tumor cell lines suppresses their neoplastic phenotype,1 consistent with a role for RB as an important tumor suppressor. The RB gene product, RB, is a member of a protein family that also includes p107 and p130.1 RB-related genes have been found in mice, chicken, Xenopus and Drosophila.1,2 Interestingly, although multiple members of the RB gene family have been identified in mice and human, only one has been found in Drosophila.2 The conservation of RB throughout evolution suggests that it is of fundamental importance for eukaryotic cell functions. Elegant studies using cell culture systems and biochemical assays have yielded much insight into the function of RB as a central cell cycle regulator (Figure 1). Cell cycle progression in mammalian cells is driven by the sequential activation of a class of kinases, the cyclin-dependent kinases (cdks).3 The activity of these cdks is controlled by (1) assembly with regulatory

The retinoblastoma susceptibility gene (RB), the first identified human tumor suppressor gene, has been shown to be directly involved in the genesis of a variety of human cancers. RB is actually one of a family of three closely related genes including p107 and p130. Many elegant biochemical studies have demonstrated that RB is a critical component of the cell cycle regulatory machinery and have characterized the downstream effectors which the RB gene product regulates. More recent advances have demonstrated that the function of RB and RB-related genes is positively and negatively regulated by an intricate network of cell cycle regulatory proteins, some of which have also been implicated as tumor suppressor genes. Despite the detailed understanding of these biochemical and genetic pathways, the full function of genes in the RB pathway in the context of a whole organism is only now being addressed. Using gene knockout technology, it is now known that RB, and RB-related proteins p107 and p130, have important functions during early mouse development. Furthermore, despite its ubiquitous expression, RB has tissue- and cell-type specific effects which account for its function as a tumor suppressor but may also be independent of its role as a cell cycle regulator. Analysis of mice lacking regulatory genes upstream of RB and effector genes downstream of RB have confirmed that other genes in this pathway have tissue-specific effects on development and tumor susceptibility in mice. Key words: cell cycle regulators / gene knockout / RB / tumor suppressor genes ©1996 Academic Press Ltd

THE RETINOBLASTOMA susceptibility gene, RB, was the first human tumor suppressor gene to be identified. RB was cloned by a positional cloning strategy based on linkage analysis of familial retinoblastoma cases.1 In addition to serving as a rate-limiting step for the development of retinoblastoma, RB mutations have also been found in other human tumors, including

Figure 1. The RB pathway and cell cycle control. Boldface type indicates those components whose knockout mouse models are available. The interplay of cyclins, cdks, CKIs, E2Fs and RB in the RB pathway controls the cell cycle progression. Middle and late G1 phase, cyclin D/cdk4 or 6 and cyclin E/cdk2 complexes phosphorylate RB sequentially. Phosphorylation of RB releases RB-bound cellular factors, such as E2F family of transcription factors, and leads to S phase entry. The negative regulatory effect of RB in cell cycle control is further enforced by CKIs. CKIs inhibit the enzymatic activity of cyclin/cdk complexes, and therefore prevent RB phosphorylation.

From the *Department of Molecular Medicine/Institute of Biotechnology, University of Texas Health Science Center at San Antonio, San Antonio, TX 78245 and †Department of Pediatric Oncology, Wilford Hall Medical Center, San Antonio, TX 78236, USA ©1996 Academic Press Ltd 1044-579X/96/050279 + 11$25.00/0

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S.-C. J. Lin et al intricate biochemical network in the context of a whole organism have been needed. This review will summarize how these important questions have been addressed recently using gene knockout technology.

subunits (cyclins), (2) specific phosphorylation and dephosphorylation of cyclins and cdks, and (3) binding by cdk inhibitors (CKIs). Cdk-mediated phosphorylation of critical cellular substrates allows cells to pass through certain cell cycle transition points. RB has been shown to be a key substrate of G1 cyclin/cdk complexes, including cyclin D/cdk4, cyclin D/cdk6 and cyclin E/cdk2, both in vitro and in vivo.1,4 In quiescent cells or cells in early G1, RB is present in a hypophosphorylated form. As G1 cyclin/ cdk complexes are activated, cells progress toward the G1/S boundary, and the overall level of RB phosphorylation increases. RB is maintained in this hyperphosphorylated form throughout S, G2, and M phases of the cell cycle. As cells emerge from M phase, RB loses multiple phosphate groups and regains a hypophosphorylated form.1,4 There are 16 potential cdk phosphorylation sites within RB. Although the phosphorylation of many of these sites can mimic the phosphorylation of RB in vivo,1,4 which specific sites (or combinations of sites) are phosphorylated and the sequence of these phosphorylation events during cell cycle progression are not fully understood. One of the key control points late in G1 phase of the cell division cycle is the restriction (R) point, after which mitogenic growth factors are no longer required to complete division (reviewed in refs 3,4). The present model for how RB suppresses cell division is that hypophosphorylated RB binds to and inhibits the activities of transcription factors, such as E2F, whose activity is required for S phase entry.5 Therefore, inactivation of the growth-suppression activity of RB by cyclin/cdk-mediated phosphorylation appears to be the molecular basis for the R point.3,4 Introduction of RB into cells by retroviral infection, transfection, or direct microinjection before this R point, slows cell growth by inhibiting the G1/S transition.1 Ectopic expression of cyclins which leads to RB hyperphosphorylation abrogates the growth suppressive effects of RB.4,6 Thus, hyperphosphorylation of RB at or near the R point releases E2F from RB-mediated repression and enables the cell cycle to progress.3-5 Despite this understanding of RB function in cell cycle regulation, key questions remain. For example, the functional importance for RB and functional differences between the closely-related members of the RB family of proteins have only recently been addressed in the context of a whole organism. Furthermore, given the aforementioned regulatory proteins ‘upstream’ of RB and effector proteins ‘downstream’ of RB, genetic studies to confirm this

RB gene family members RB, p107 and p130 are classified as members of the RB gene family based on their structural and biochemical similarities. These three proteins are closely related in the bipartite domain, termed the A/B domain, that mediates their physical interaction with certain viral oncoproteins, such as adenovirus E1A and SV40 large T antigen, and cellular factors such as members of the E2F family of transcription factors. In addition, they all share two additional regions of significant identity near their amino termini; however, the functional importance of this is unclear. All three proteins are expressed in most cells and ectopic expression of any one of them arrests many types of cells in the G1 phase.7,8 Despite the fact that RB, p107, and p130 are closely related, they appear to have distinct biochemical properties. For example, p107 and p130 show high homology in the spacer region within the bipartite domain. This spacer region mediates binding to and inactivation of cyclin E/cdk2 and cyclin A/cdk2;9,10 this activity is not present in RB, which has a distinctly shorter spacer region. Although RB, p107 and p130 are all regulators of E2F activity, they associate with specific members of the E2F family and at different times of the cell cycle.4,11 RB interacts with E2F1, E2F2 and E2F3 during late G1 to S phase. p107 preferentially interacts with E2F4 in cycling cells during late G1 and S phase. p130 binds E2F4 and E2F5 in G0 and G1 phases. Besides these biochemical differences, there are other indications that they have distinct biological functions. For example, RB is mutated in a variety of human tumors, but no specific mutations of p107 or p130 have been reported. Analysis of mice in which these RB-related genes have been inactivated reveal other distinct properties (Table 1). RB is absolutely required for normal mouse development as RB-nullizygous embryos die by the 16th embryonic day.12-14 The most prominent abnormalities in these embryos are dramatic evidence of dyserythropoiesis and ectopic mitosis and apoptosis in regions of the developing brain and spinal cord.12-15 In contrast, the RB + /– mice are developmentally normal except for pituitary tumor predisposition with nearly complete penetrance.16,17 Although it was 280

Knockout of genes in RB pathway Table 1. Phenotypes of the RB gene family knock out mice Genotype RB –/– RB +/– p107 –/– p130 –/– RB +/–; p107 –/– RB–/–; p107 –/– p107 –/–; p130 –/–

Phenotype

Major abnormality

Embryonic lethality (E13.5–E16.5) Normal except tumor predisposition Normal Normal Growth retardation and increased mortality Embryonic lethality (E11.5) Neonatal lethality

Defective neurogenesis and haematopoiesis Complete penetrance of pituitary tumors – – Pituitary tumors and retinal dysplasia Accelerated apoptosis in liver and CNS Defective endochondral bone and limb development

in which individual RB-related genes have been inactivated, in p107–/–; 130–/–, in RB + /–; p130–/– mice,21 nor in chimeric mice partly composed of RB–/– cells.22 This hypothesis of apparently overlapping function of RB and p107 in the retina predicts that the entire photoreceptor layer would be affected in RB–/–; p107–/– mice. Because RB–/–; p107–/– mice are not viable, this hypothesis might be addressed by producing mice with RB–/–; p107–/– retinal cells using tools which allow tissue-specific gene inactivation. Although data from these gene knockout models suggest overlapping functions for RB and p107 in mouse retina, it must be emphasized that there is no evidence that p107 is involved in human retinoblastoma. These differences may be related to speciesspecific differences in mouse and human retinal development or differences in effects of germ-line gene loss versus somatic cell gene loss which is thought to precipitate human retinoblastoma. Distinct roles for p130 have also been uncovered by analysis of double knockout mice. In contrast to RB + /–; p107–/– mice, p130–/–; p107–/– mice have apparently normal retina but exhibit deregulated chondrocyte growth, defective endochondral bone development, shortened limbs and neonatal lethality,20 indicating that p107 and p130 play an important role in chondrocyte proliferation. Because all three RB-related genes are ubiquitously expressed, such tissue-specific defects seen in these knockout mice may reflect either tissue-specific functions for the RB gene family members or tissue-specific susceptibility to abnormalities when these genes are inactivated. Again, possible species-specific differences in the function of these genes have not been fully addressed.

originally suggested that there was increased expression of p107 in cultured RB–/– skeletal muscle cells,18 in RB–/– embryos, as in RB-deficient tumor cells, the presence of p107 and p130 does not compensate for the absence of RB. Similarly, there appears to be no upregulation of p107 or p130 in RB knockout embryos.19 In distinct contract to the RB-deficient embryos, the p107- and p130-deficient mice are viable, healthy and fertile.20,21 Because RB-deficiency is embryonic lethal, chimeric mice partly composed of RB–/– cells have been generated to study the function of RB during later stages of development and tumorigenesis.22,23 RB–/– chimeric mice are apparently normal but susceptible to accelerated development of pituitary tumors ( ~ 4 months) when compared with RB + /– mice ( ~ 10 months). RB–/– cells contributed substantially to most tissues in adult mice, including mature erythrocytes, liver and central nervous system. However, several defects were observed in RB–/– chimeric mice, especially ectopic mitosis and apoptosis in developing retina and abnormal lens fiber cell formation. No retinoblastoma developed in these RB–/– chimeric mice. These data indicate that, despite its widespread expression, RB has distinct functions in specific cell types. Although the inactivation of RB, p107 and p130 individually suggests that these genes have distinct functions, examination of the double knockout mice shows that they may have some overlapping properties. For example, while both RB–/–; p107–/– and RB–/– embryos die during early embryogenesis, the former die two days earlier than the latter. Furthermore, loss of one RB allele alters the apparently normal phenotype of p107–/– mice. RB + /–; p107–/– mice have retarded growth with increased mortality rate, and they develop multiple retinal lesions in addition to the pituitary tumors caused by RB heterozygosity.21 Although not formally proven, the frequency and focal nature of these retinal lesions suggest that they result from the inactivation of the remaining wild-type RB allele. These retinal lesions are not found in mice

Activation and inactivation of upstream regulators of the RB protein As discussed above, specific cdks are required in the mid- to late-G1 phase to phosphorylate RB and 281

S.-C. J. Lin et al also inhibits repair DNA synthesis is controversial.29-31 The expression of p21 is induced by cellular senescence and by DNA damage in a p53-dependent manner.3 This evidence suggests that p21 may be a key component of cell cycle control, DNA repair and DNA synthesis. For example, overexpression of p21 in hepatocytes inhibits hepatocyte proliferation, resulting in a runted liver which fails to regenerate in response to partial hepatectomy.32 Interestingly, mice lacking p21 develop normally with no evidence of increased tumor predisposition.33 A major defect, however, is observed in p21–/– embryonic fibroblasts, which have impaired G1 checkpoint control in response to DNA damage and nucleotide pool perturbation, but have normal p53-dependent mitotic spindle checkpoint control and normal apoptosis in thymocytes.33 Given the fact that p21 is an important effector of p53-mediated cell cycle arrest following DNA damage, and p53-deficiency predisposes an animal to tumor formation,34 it is surprising that p21–/– mice do not develop tumors. There are several possible explanations for the lack of tumors in these mice. First, p21 function in G1 checkpoint control may be partially redundant to other CKIs. Secondly, additional genes induced by p53, such as GADD45,35 may be more important for the anti-oncogenic effects of p53. Finally, loss of p21 may diminish another function of p21, such as an anti-apoptotic effect which has been demonstrated in differentiating myoblasts and neuroblastoma cells.36,37 Thus, despite cell culture studies which demonstrate an important role for p21 in checkpoint control, loss of p21 alone is not sufficient to predispose these mice to tumor formation. p27Kip1, like p21, binds to multiple cyclin/cdk complexes to inhibit their kinase activity. p27 was first isolated from mink Mv1Lu lung epithelial cells arrested in G1 phase by contact inhibition or transforming growth factor-β (TGF-β) treatment.38 Subsequently, p27 was shown to accumulate in human fibroblasts and in HeLa cells arrested in G1 by lovastatin treatment.3 Finally, in antigen-stimulated T cells, cell cycle progression is concurrent with the elimination of p27.3 Together, these data indicate that p27 plays an important role in negatively regulating the cell cycle in a variety of cell types. Consistent with this notion, mice lacking p27 display increased body size accompanied by multiple organ hyperplasia due to enhanced cell proliferation.39-41 Similar to RB + /– mice, p27–/– mice develop tumors originating from the intermediate lobe of the pituitary gland, suggesting that RB and p27 might be in the same pathway

thereby to release RB-mediated cell cycle arrest. Of these, key regulators of G1 progression in mammalian cells are the D-type cyclins and cyclin E, which are sequentially activated when quiescent cells are stimulated with mitogens.3 There are three types of D cyclin (D1, D2 and D3), which are synthesized in a cell typespecific manner, with most cells expressing D3 and either D1 or D2.24 Despite having a very short half-life ( ~ 25 minutes), D-cyclin levels have only moderate fluctuations during the cell cycle; D-cyclins are thought to function as ‘growth factor sensors’ in mammalian cells.24 On the other hand, cyclin E is expressed periodically with peak levels achieved near the G1/S transition. The activity of these cyclin/cdk complexes is, in turn, negatively regulated by cdk inhibitors (CKIs), including the p21WAF1 and p16INK4a protein families of CKIs.3 Members of the p21WAF1 family, including p21WAF1, p27Kip1 and p57Kip2, seem to function as universal CKIs, because they can associate with and inhibit the activity of a wide range of cyclin/cdk complexes. Members of the p16INK4a family, which includes p16INK4a, p15INK4b, p18INK4c and p19INK4d, bind specifically to cdk4 and cdk6, which prevents cyclin D/cdk association. Therefore, members of the p16INK4a family of CKIs function as G1-specific CKIs. Because of this well-defined biochemical network positively and negatively regulating RB function in in-vitro systems, it has been interesting to examine manipulations of this network in the context of a whole organism. Cyclin D1 was originally identified as an oncogene, PRAD1, which is activated by chromosomal translocations in parathyroid adenoma cells.3 Cyclin D1 is also amplified or overexpressed in many human breast cancers.3 Consistent with an oncogenic role for cyclin D1, MMTV-cyclin D1 transgenic mice develop mammary hyperplasia and breast carcinoma,25 and ectopic expression of cyclin D1 and Myc in B cells leads to the rapid development of lymphoma.26,27 On the other hand, cyclin D1-deficient mice display growth retardation associated with increased mortality, severe reduction in neuronal number in the retina, and a defect of mammary epithelial cell proliferation during pregnancy.28 Thus, both knockout and transgenic studies of cyclin D1 indicate that it is a key cell cycle regulator in the RB pathway in specific cell types. p21WAF1 binds directly to cdc2, cdk2 and cdk4, and acts as a potent and universal inhibitor of cdk enzymatic activity.3 p21 also interacts with the proliferating cell nuclear antigen (PCNA), an accessory factor for DNA polymerase δ.3 This was initially reported to inhibit DNA replication, but whether it 282

Knockout of genes in RB pathway sistent with this suspected role of the INK4a locus as a tumor suppressor locus, INK4a–/– mice lacking both p16INK4a and p19ARF are viable but frequently develop tumors spontaneously and following carcinogenic treatment.49 The most common tumors in INK4adeficient mice are fibrosarcoma and B-cell lymphoma. INK4a–/– mice also display extramedullary hematopoiesis, suggesting a role for INK4a in normal hematopoietic development. This interesting observation is consistent with the notion that p16 acts in concert with RB, because RB–/– embryos also demonstrate impaired hematopoiesis. However, no pituitary tumors have been observed, implying that RB but not p16 has a distinct function in pituitary cell proliferation. Although these studies confirm the importance of the INK4a locus as a tumor suppressor in mice, the relative contribution of p16INK4a versus p19ARF to this phenotype needs to be examined using mice specifically lacking E1α and E1β, respectively.

regulating pituitary cell proliferation. Furthermore, in contrast to the increased body size in p27-deficient mice, transgenic mice overexpressing RB are smaller in size.42 These data suggest that p27-mediated regulation of the cell cycle-blocking activity of RB is inactive in p27–/– mice. However, because chimeric mice partly composed of RB–/– cells lack hyperplasia of tissues except within the pituitary gland,22 it is possible that p27 can also regulate cellular proliferation by RB-independent mechanisms. Consistent with this, it was recently reported that expression of antisense p27 in cells lacking functional RB decreases the fraction of cells in G1 phase.43 It would be interesting to see whether RB overexpression in p27–/– mice affects tissue hyperplasia and pituitary tumor development; experiments such as this might reveal whether certain functions of p27 are independent of RB. p57Kip2 is a potent inhibitor of several cyclin/cdk complexes, and its overexpression causes G1 arrest.3 Human KIP2 is located at 11p15.5,44 a region implicated in both sporadic cancers and Beckwith-Wiedemann syndrome (BWS), a familial cancer-predisposing syndrome in which a maternal carrier state and genomic imprinting are thought to be involved. Recently, p57Kip2 was found to be imprinted and only the maternal allele is expressed.45 Moreover, p57Kip2 mutations were found in BWS patients.46 These results strongly suggest a role of p57Kip2 in tumor suppression and the effect of p57Kip2 inactivation should be addressed using gene knockout models. p16INK4a specifically inhibits the kinase activity of cyclin D/cdk4 and cyclin D/cdk6 complexes and thereby blocks cell cycle progression in an RBdependent manner.3 The locus encoding p16INK4a, termed INK4a, gives rise to two distinct transcripts derived from alternative first exons, E1α and E1β.47 The transcript containing E1α encodes p16INK4a and the transcript containing E1β encodes p19ARF from an alternative reading frame. Expression of p19ARF arrests cells at both G1 and G2 phases by mechanisms which do not appear to involve inhibition of known cyclin/cdk complexes.47 Deletions or mutations of INK4a occur in many human tumors, particularly familial melanoma and pancreatic adenocarcinoma,3 suggesting that p16INK4a functions as a bona fide tumor suppressor gene. The tumor-associated mutations in the INK4a locus affect p16INK4a either alone ( ~ 50%) or together with p19ARF.47 Interestingly, inactivation of p16INK4a and RB in tumor cells appears to be mutually exclusive,48 indicating that these proteins may function in a single regulatory pathway. Con-

Activation and inactivation of downstream effectors of the RB protein The biological effects of RB are thought to be exerted through its interaction with specific effectors, the best studied of which is the transcription factor E2F. E2F was initially identified as a cellular DNA-binding activity required for the adenovirus E1A-mediated activation of the viral E2 gene.11 Biochemical studies have revealed that E2F is a heterodimer composed of two proteins, one from the E2F family (E2F1 through E2F5) and one from the DP family (DP1, DP2 and DP3). Cellular targets of E2F activity include genes required for both DNA synthesis (DNA polymerase α, thymidine kinase and dihydrofolate reductase), and cell cycle control (cyclin A, cyclin E and cdc2), and also c-Myc and B-Myb, suggesting that E2F drives the G1/S phase transition. RB interacts with E2F and inhibits its transcription activation function to block cell cycle progression. This inhibition is released upon phosphorylation of RB by cyclin/cdk complexes during late G1 phase and at the G1/S phase transition. Most RB mutations found in human tumors disrupt its interaction with E2F, thus leading to increased E2F activity and altered cell cycle control. Overexpression of E2F1 in megakaryocytes blocks differentiation during their postmitotic maturation.50 This effect may be due to the cell cycle-stimulating activity of E2F1 because these mice accumulate increased numbers of megakaryocytes in both normal and ectopic sites. However, E2F1 knockout mice 283

S.-C. J. Lin et al that dE2F is essential for the G1/S transition during Drosophila embryogenesis. Although it has not been reported, it is reasonable to speculate that RBF and dDP will also be indispensable for normal Drosophila embryogenesis.

display a somewhat surprising phenotype. They are viable, fertile and appear quite normal while they are young. This lack of an obvious developmental phenotype for such a critical cell cycle regulator may be due to functional redundancy among other E2F family members.51,52 Despite the aforementioned growthpromoting activity of E2F1, as mice lacking E2F1 age, they develop testicular atrophy, exocrine gland dysplasia and more surprisingly a broad and unusual spectrum of tumors (in 19–34% of E2F1–/– mice), including reproductive tract sarcomas, lung tumors and lymphoma. The development of tumors in E2F1–/– mice leads to the speculation that E2F1 might function to suppress proliferation in specific tissues. One plausible explanation is that E2F1 may function as a transcriptional repressor as well as a transcriptional activator. E2F-dependent repressor activity is consistent with the interesting finding that when quiescent cells are stimulated, loss of binding to an E2F site in the B-Myb promoter coincided precisely with the onset of B-Myb transcription.53 Furthermore, there are precedents for dual function transcription factors, such as YY1, which functions as an activator and a repressor.54 Therefore, loss of the putative E2Fdependent repression of growth-promoting genes could contribute to tumor development in E2F1–/– mice. Alternatively, because E2F1–/– thymocytes may also be resistant to apoptosis,51 decreased programmed cell death may predispose these cells to malignant transformation. Whichever mechanism is correct, the long latency and relatively low penetrance for these tumors are consistent with multiple steps needed for malignant transformation in these mice. Studies using dominant negative DP1 (DN DP1) mutants which retain E2F-binding but lose DNAbinding activity further demonstrate that E2F activity is necessary for cell cycle progression.55 Expression of DN DP1 mutants arrests cells in the G1 phase, while wild-type DP1 drives cells into S phase. It will be interesting to determine whether a transgenic mouse expressing these DN DP1 mutants will confirm the unexpected phenotype of the E2F1-deficient mouse. In contrast to the situation in mammalian cells, in Drosophila, only one representative of each class of genes in the RB-E2F/DP pathway has been identified — the retinoblastoma family homolog (RBF), dE2F and dDP.2,56,57 Analysis of the dE2F–/– embryos shows that as maternal dE2F activity is depleted during early cell cycles, dE2F-dependent transcription and DNA replication decline, and dE2F-deficient embryos fail to enter cell cycle 17, the first cell cycle marked by a G1/S phase transition.58 This clearly demonstrates

Primary embryonic fibroblasts derived from mice lacking genes in the RB pathway Knockout mice not only serve as invaluable models to study the function of specific genes in the context of a whole organism, mouse embryonic fibroblasts (MEFs) derived from these knockout mice provide important tools to dissect the function of these genes in more controlled settings. For example, the cell cycle regulatory effect of RB is clearly revealed using MEFs derived from RB–/– embryos. RB-deficient MEFs display shortened G1 phase, elevated and accelerated expression of cyclins, and activation of E2F-responsive genes.59,60 In contrast to RB–/– MEFs, p107–/– or p130–/– MEFs show normal growth curves, length of G1 phase, and dependency on serum-derived mitogens.20,21 These data from MEFs are consistent with results from knockout mice and reinforce the key role of RB in regulating the cell cycle. The role of the upstream regulators of the RB pathway have also been studied using MEFs. For example, p21–/– MEFs show growth properties similar to p53–/– MEFs.33 They reach a higher saturation density and, in contrast to wild-type MEFs, do not undergo crisis. As noted above, p21–/– MEFs have defective p53-mediated G1 arrest in response to DNA damage and nucleotide depletion, but have normal p53-dependent mitotic spindle checkpoint control.33 Thus, p21 only seems to mediate part of p53 function in G1 cell cycle control. Similarly, INK4a–/– MEFs grow faster and have higher colony-formation ability without a detectable senescent phase.49 Moreover, in contrast to the neoplastic transformation of other primary MEFs, which requires the cooperation of particular pairs of oncogenes, the introduction of activated Ha-ras alone into INK4a–/– MEFs causes neoplastic transformation.49 However, it is important to emphasize that information obtained from in-vitro MEF studies does not always correlate with the phenotype seen in the whole organism. For example, although p27–/– mice develop multiorgan hyperplasia and pituitary adenomas in vivo, p27–/– MEFs display normal growth properties and responses to signals causing G1 arrest, such as contact inhibition, serum depletion, DNA damage and nucleotide pool pertur284

Knockout of genes in RB pathway Table 2. Phenotypes of activation and inactivation of genes in the RB pathway Genotype

Phenotype

RB –/– *RB –RB Cyclin D1 –/– *MMTV-Cyclin D1 p21 –/– *TTR-p21 p27 –/– p16 –/– E2F1 –/–

Embryonic lethality Dwarfism Growth retardation Late onset tumors Viable and grossly normal Liver defects Giantism and multiorgan hyperplasia Normal except tumor predisposition Viable but late onset diseases

*PF4-E2F1 dE2F–/–

Thrombocytopenia Embryonic lethal

Major abnormality Defective neurogenesis and haematopoiesis Body size inversely correlated to RB dosage Retina and mammary hypoplasia Mammary carcinoma MEFs defective in G1 checkpoint control Retarded liver development and regeneration Retinal dysplasia and pituitary tumor Fibrosarcoma and lymphoma Testicular atrophy, exocrine gland dysplasia and tumor induction Proliferative megakaryocytes Cell cycle arrest at mitosis 17 in early Drosophila embryogenesis

The asterisk represents the transgenic mouse model. MMTV, mouse mammary tumor virus; TTR, transthyretin; PF4, platelet factor 4.

bation.41 Better defined studies of these MEFs will be required to recapitulate the intrinsic defect seen in vivo using in-vitro cell culture systems.

diminished expression of other neuronal genes, such as neurotrophin receptors and βII tubulin, but not Brn-3.0, neural cell adhesion molecule (NCAM) or neurotrophins.15 Whether RB directly regulates the promoters of Rig1 and other neuronal genes is presently not known.

RB activities beyond cell cycle regulation The distinct tissue-specific effects in RB knockout mice are surprising given the central role of RB in cell cycle regulation. This suggests that RB, in fact, has functions which extend beyond direct cell cycle regulation. Four cell types in which this has been studied using the RB knockout model will be briefly reviewed here.

Lens cells and photoreceptors — proliferation, differentiation and apoptosis During normal mouse lens development, posterior lens cells withdraw from the cell cycle, elongate and differentiate into lens fiber cells. In the absence of RB, ocular lens fiber cells exhibit ectopic mitoses, impaired differentiation and apoptosis.62 Interestingly, such ectopic apoptosis resulting from RBdeficiency is suppressed in the RB–/–; p53–/– embryos.62 Similar results were obtained from transgenic studies in which the expression of human papillomavirus type 16 (HPV) E7 and E6 proteins, which inactivate RB family proteins and p53, respectively, was directed to the developing lens. E7-transgenic mice demonstrated inhibition of lens fiber cell differentiation, ectopic mitosis and apoptosis.63 However, E7 3 E6 double transgenic mice showed reduced levels of apoptosis, but developed lens tumors, which were not seen in transgenic mice expressing E7 or E6 alone. Other studies in which RB family proteins in the retina are inactivated by transgenic expression of HPV E7 protein in photoreceptors showed retinal degeneration due to a photoreceptor cell apoptosis.64 When HPV E7 protein was expressed in p53 nullizygous mice, retinal tumors evolved from apoptotic retinal cells. Taken together, these data indicate that RB and p53 are important for regulation of normal lens and retina cell growth and

Neuronal cells — deregulation of gene expression Using the differential display technique, we have discovered that Rig1, a novel member of the Ig superfamily, is overexpressed in regions of the CNS in RB–/– mice (Yuan et al, unpublished). Structural analysis indicates that Rig1 may function as a cell adhesion molecule (CAM) of the Ig superfamily, members of which are thought to be important in nervous system development.61 Rig1 is normally transiently expressed at the boundary between the proliferation and the migration zones of the developing hind brain and spinal cord during days 10.5–13.5 of embryonic development. This zone is adjacent to the zone harboring the major CNS abnormalities in RBdeficient embryos. Given the observation that deregulated expression of Rig1 precedes the ectopic mitosis and apoptosis seen in this region of these RB–/– embryos, the data suggest that RB may affect neuronal development and survival by regulating gene expression independent of the cell cycle control. We have similarly shown that the absence of RB results in 285

S.-C. J. Lin et al stem cells leads to the development of hematologic malignancy.

apoptosis. Perturbation of both activities could promote tumor formation, which is consistent with the finding that many human tumors exhibit mutations in both RB and p53.65

Knockout mouse models and tumorigenesis Skeletal muscle cells — loss of cell cycle control and compromised terminal differentiation

Tumor spectrum Like human carriers, RB + /– mice are highly susceptible to tumor development.17 On the other hand, no tumors are found in p107 + /–, p130 + /–, or p107 + /–; p130 + /– mice, further confirming that RB is the only bona fide tumor suppressor gene in the RB gene family. However, the tumor spectrum in mice does not always reflect what is seen in human carriers. This could be due to species-specific differences in the number of susceptible cells and in the timing of susceptibility. For example, mouse retinal cells mature during the first two weeks after birth, and thus the second hit to inactivate the remaining wild-type allele must occur within this brief period. This difference between mouse and human retina development may explain the absence of retinoblastoma in RB + /– mice. Similarly, although the INK4a locus has been linked to familial melanoma, no melanomas have been found in the INK4a–/– mice.49 The absence of melanoma may be due to a need for a longer latency period or may reflect other environmental effects on the development of cutaneous melanona. The mechanisms of apparent species-specific differences in tumor development can be further elucidated using conditional knockout technology. For example, short latency and high mortality of some tumors may mask the development of other tumors in mice lacking a particular tumor suppressor gene. With the development of the Cre-loxP or FLP-FRT systems, conditional knockout of genes in the RB pathway can be re-evaluated in any specific tissue.68

RB has been reported to be required for MyoDinduced skeletal muscle differentiation in Saos2 osteosarcoma cells.66 However, others have shown that myocytes can apparently differentiate normally in the absence of RB (ref 18, Chen and Lee unpublished). Most importantly, in RB knockout embryos, skeletal muscle differentiation appears to be normal. More close examination of RB-deficient myocytes has, however, revealed that RB-null myocytes have a consistent defect in terminal differentiation. Specifically, when these myocytes are stimulated with serum-derived growth factors, they re-enter the cell cycle and reinitiate DNA synthesis (ref 18, Chen and Lee, unpublished), a phenomenon which is not normally observed. These data indicate that RB per se is not necessary for skeletal muscle differentiation, but it is required to insure the irreversible cell cycle withdrawal which is normally concomitant with terminal skeletal muscle differentiation.

Hematopoietic cells — mitogenic but not tumorigenic effects of RB deficiency Studies of RB chimeric mice (in either a wild-type or recombination activating gene-2 (RAG-2)-deficient background) have indicated that RB is not required for lymphoid and myeloid development, and erythrocytes in these chimeric mice are also normal.22,23,67 However, using a different system, we have recently found that lethally irradiated wild-type mice reconstituted with RB-deficient hematopoietic stem cells have abnormal hematopoiesis resulting in anemia (Hu et al, unpublished). Specifically, the erythrocyte progenitors seem to divide more rapidly and, although there is normal developmental switching from embryonic to adult hemoglobin, the expression of hemoglobin is less abundant, and immature erythrocytes are present in the peripheral blood. In addition, there is extensive marrow expansion and extramedullary hematopoiesis which may represent either primary growth dysregulation or secondary response due to increased fragility of immature erythrocytes. Most intriguingly, there is no evidence that RB deficiency in these hematopoietic

Multistep nature of tumorigenesis Tumor development is presently thought to occur after cells accumulate a series of genetic changes that contribute to the complex transformed phenotype. This multistep tumorigenesis has been documented in some human tumors, such as colorectal cancer.69 Because it is difficult to obtain human tissues at different stages of tumorigenesis in the same patient, mouse models have been established to explore the multistep nature of tumorigenesis. The apparent clonal origin and the timing of pituitary tumor development in RB + /– mice has recently confirmed 286

Knockout of genes in RB pathway mouse pituitary cells, and the cyclin D1-RB pathway might be important for breast epithelial cells. In the future, in order to confirm the direct relationship in the pathway in specific cell types, the generation of tissue-specific knockouts of different genes may be required. Moreover, it will be important to elucidate the molecular mechanisms underlying cell typespecific phenotypes seen in these knockout mice. The puzzle that inactivation of ubiquitously expressed tumor suppressor genes, such RB and p16, leads to specific tumor predisposition remains unanswered. Possible explanations for this apparent paradox may include differences in the specific time window of susceptibility and the duration of the latency period for transformation of specific cell types. In addition, it is conceivable that there are fundamental differences due to the timing of gene inactivation (germline versus somatic). For example, it is intriguing that germline inactivation of both RB alleles leads to ectopic mitosis and apoptosis in regions of the CNS whereas loss of the second RB allele in the pitutary gland in young RB + /– mice universally leads to proliferation and subsequent malignancy. Again, tissue-specific and temporallyregulated knockout of specific genes may help define the mechanisms which lead to restricted phenotypes in these knockout mice. There is growing evidence indicating that numerous genes in cell cycle regulation, cell cycle checkpoint control and DNA repair are involved in tumorigenesis. Models in which different genes in these pathways are inactivated will facilitate analysis of how these pathways collaborate during tumorigenesis. Because many of these genes are likely to have pleiotropic activities, these will be best evaluated in the animal model.

the multiple steps required for tumor formation in these mice.70 This was further demonstrated in an elegant study using a transgenic mouse model in which the expression of simian virus 40 T antigen (TAg) in the submandibular gland was controlled by a tetracycline responsive system.71 Expression of TAg, which is thought to inactivate RB, p53 and other cellular proteins, in these transgenic mice induces hyperplasia by 4 months of age. This hyperplasia is reversed when TAg is silenced after 4 months of expression but not after 7 months of expression. These data confirm that inactivation of both RB and p53 is not sufficient to irreversibly transform cells and support a time-dependent model in which cells acquire other changes that prohibit reversal of cellular transformation. The multistep nature of tumorigenesis has also been addressed by inactivating more than one gene in mouse knockouts. For example, p53 + /–; RB + /– and p53–/–; RB + /– mice develop tumors which are not seen in mice deficient only in p53 or RB at an earlier age and with faster tumor progression.65,72 Similarly, in the Wnt-1 transgenic mouse (Wnt-1 TG) model, p53–/– mice (Wnt-1 TG p53–/–) develop mammary tumors at an earlier age than Wnt-1 TG p53 + /– and Wnt-1TG p53 + / + mice.73 There are also additional histological differences and chromosomal instability seen in Wnt1TG p53–/– mice but not in the others. It is evident from these studies that tumorigenesis requires the cooperation of multiple tumor suppressor genes, oncogenes and perhaps other genes such as those involved in angiogenesis and apoptosis for the full malignant phenotype.

Conclusions and perspectives Genetic approaches using knockout mice have confirmed the role of genes in the RB pathway in cell cycle control in the context of a whole organism. These knockout mice not only provide models to test the data obtained from other systems, studies of these mice and cells derived from them have revealed additional insights into the function of these specific genes in other cellular processes. However, a direct correlation of the ‘upstream’ regulators and ‘downstream’ effectors in the RB pathway can not be easily confirmed. This may due to the potential functional redundancy among related proteins. Nevertheless, a direct relationship in the RB pathway may be established when we focus on specific cell types. For example, the p27-RB pathway may be critical for

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