Ординатура / Офтальмология / Английские материалы / Ocular Oncology_Albert, Polans_2003

in the first 3 weeks of life. Of the mice that survive to adulthood, the majority develop intermediate lobe tumors like the Rbþ/ mice; no additional tumor types are observed. The Rbþ/ :p107 / mice do develop multiple focal retinal lesions characterized as retinal dysplasias or degeneration of the photoreceptor cell layer, but retinoblastomas are not observed [60]. These studies demonstrate that there is functional overlap between p107 and pRb in several tissues of the developing and adult mouse, including the retina, but Rb heterozygous mutants nevertheless fail to develop retinoblastoma even in the complete absence of p107.

Because retinal development could not be assessed in the Rb / :p107 / mice due to embryonic lethality, chimeric mice have been generated with ES cells that are deficient in both Rb and p107 (Rb / :p107 / ) in a wild-type background [61]. In contrast to each of the previous knockout approaches described, Rb / :p107 / chimeric mice develop retinoblastomas, and lesions can be observed as early as day E17.5. Although these tumors are often located between the photoreceptor cell layer and the RPE, they express markers consistent with amacrine rather than photoreceptor cell differentiation. Rb / :p107 / retinoblasts expressing IRBP are detectable at day E17.5 but are completely absent by postnatal day 15, indicating that Rb / :p107 / retinoblasts committed to photoreceptor cell differentiation are eliminated during this developmental window. This cell loss is not p53-dependent, since the same fate is also observed when chimeras are generated with Rb / :p107 / ES cells also expressing a dominantnegative p53 gene under the control of the IRBP promoter [61]. Thus, simultaneous inactivation of both Rb and p107 strongly predisposes mice to the development of retinoblastomas of amacrine cell origin, but even the additional inactivation of p53 does not contribute to tumorigenesis of photoreceptor precursors.

D.Conditional Rb Knockout Mice

One additional approach that has been employed to address the effect of Rb inactivation in the mouse retina is the creation of cell type–specific Rb knockout mice using the Cre/loxP system. Conditional Rb knockout mice with two loxP sites flanking exon 19 (RbF19) were generated and interbred to IRBP-Cre transgenic mice, to create mice in which the Rb gene is inactivated only in IRBP-expressing cell types [62]. As noted above, the IRBP gene is expressed both in photoreceptor cells and the

pineal gland, beginning at approximately day E12 [33]. As expected from the results of the previous studies, the IRBP-Cre:RbF19/F19 mice display completely normal

retinal development and fail to develop either retinoblastomas or pinealomas, despite the presence of large numbers of Rb-deficient cells in these cell types. Rather, like the Rb heterozygous knockout mice, they develop pituitary tumors, although in this case the tumors are of both intermediate and anterior lobe origin [62]. The development of pituitary tumors in this model is particularly surprising, since Cre is not expected to be expressed in this cell type. However, the pituitary tumor cells

show exon 19 deletion of the Rb gene, indicating that Cre recombinase must be ectopically expressed in the pituitary of the IRBP-Cre mice. The IRBP-Cre:RbF19/F19 mice were also bred to p107 / mice to generate IRBP-Cre:RbF19/F19:p107 /

mice, and these mice also displayed normal retinal development. Although this might seem surprising in light of the fact that Rb / :p107 / chimeras develop retinoblastomas, it should be recalled that the Rb / :p107 / cells gave rise

only to tumors of amacrine cell origin and not photoreceptor origin in the chimeras,

while Rb inactivation in this model is restricted to photoreceptor cell precursors. Finally, IRBP-Cre:RbF19/F19 mice were also bred to p53 knockout mice to generate both IRBP-Cre:RbF19/F19:p53þ/ and IRBP-Cre:RbF19/F19:p53 / mice, and a small number of IRBP-Cre:RbF19/F19:p107þ/ :p53 / and IRBPCre:RbF19/F19:p107 / :p53 / mice were also generated. Remarkably, none of

these mice developed retinoblastoma; rather, they displayed accelerated pineal and pituitary tumorigenesis [62]. Thus, it must be concluded that inactivation of Rb in the mouse is insufficient to induce tumors of photoreceptor origin, and neither p107 nor p53 inactivation can cooperate with Rb loss to induce tumors of this cell type.

E.Implications from the Rb Knockout Mice

An obvious question arises from these studies: Why does germline Rb mutation cause retinoblastoma in humans but pituitary tumors in mice? The intermediate lobe of the pituitary gland is present in only vestigial form in humans, which could account for the failure of patients with germline Rb mutations to develop tumors arising in this cell type. However, it is less clear why Rb heterozygous mice fail to develop retinoblastomas [63–66]. One possibility is that somatic loss of the remaining normal Rb allele (or acquisition of other required mutations) in developing retinoblasts has a much lower probability of occurring in mice, since they presumably have a significantly smaller population of susceptible cells and a much shorter developmental time frame during which this event can occur. The fact that Rb / cells in chimeric mice fail to form retinoblastomas argues against this explanation if loss of the second Rb allele is the only rate-limiting step in retinoblastoma development. However, if additional genetic events are required, then the number of target cells and timing of susceptibility may still be contributing factors. This leads to a second possibility, which is that retinoblasts of mice and humans may differ subtly in the mechanisms controling cell growth, differentiation, and apoptosis, such that mouse retinoblasts have additional genetic safeguards against oncogenic transformation that are lacking in human retinoblasts. For example, the fact that Rb / :p107 / cells but not Rb / cells can give rise to retinal tumors in mouse chimeras suggests a degree of redundancy between pRb and p107 in mice, such that functional p107 can protect against transformation of Rbdeficient retinoblasts. In contrast, there is no evidence for mutation of the p107 gene in human retinoblastoma [67]. It should be noted, however, that loss of both pRb and p107 function is still not sufficient to transform mouse retinoblasts, since not all Rb / :p107 / chimeric retinas develop retinoblastomas. It seems likely that an additional mutational event required for full transformation would be one that suppresses apoptosis, since the normal fate of Rb-deficient cells in the developing retina and in many other cell types is apoptosis. Although the p53 gene might seem to be an obvious candidate, there is little evidence from the mouse models that p53 mutation contributes significantly to retinoblastoma development. It has similarly been suggested that the development of retinoblastoma in humans requires not only loss of both Rb alleles but also at least one other genetic event, which could potentially alter the apoptotic susceptibility of the tumor cells (see this volume, Chapters 7 and 10). Therefore, identification of additional mutations that contribute

to retinoblastoma in the mouse may have direct implications for a better molecular understanding of human retinoblastoma.

V.TRANSGENIC MOUSE MODELS OF RETINOBLASTOMA IN THERAPEUTIC STUDIES

In addition to their applications in the study of the molecular genetics of cancer, genetically engineered mouse models of cancer are increasingly being used for the in vivo evalution of novel therapeutic approaches. The LHbeta-Tag mice in particular have been extensively used for evaluating a wide range of therapeutic approaches for the treatment of retinoblastoma, including local delivery of standard chemotherapeutic agents, external-beam radiation, a variety of combined modality approaches, and several novel anticancer agents.

A.Chemotherapeutic Agents

Chemotherapy is the most widely used modality for the treatment of invasive and metastatic retinoblastoma and is also an important component in the primary treatment of intraocular tumors [68,69]. However, systemic administration of cytotoxic agents at the doses required to achieve a sufficient intraocular drug concentration for effective tumor control is often associated with significant toxicity. Local delivery of carboplatin directly to the eye, either by intravitreal or subconjunctival injection, has therefore been explored in the LHbeta-Tag mice [70,71]. These studies demonstrated effective tumor growth inhibition in a dosedependent manner, but with a relatively narrow therapeutic window and dosedependent retinal toxicity. Local carboplatin administration has also been evaluated in combination with external-beam radiation therapy (EBRT) or cryotherapy. Although no benefit was gained with the addition of cryotherapy in this model [72], significant enhancement of tumor control was achieved by the addition of radiation therapy [73].

B.External Beam Radiation Therapy

In addition to its combination with carboplatin, EBRT has been explored as a single modality and in combination with hyperthermia in the LHbeta-Tag transgenic mice. In a study evaluating dose and schedule of EBRT administration, hyperfractionated EBRT (administered twice daily) was compared to standard daily EBRT and was found to significantly increase tumor control, allowing for a reduction in the total radiation delivered dose [73]. EBRT was also evaluated in combination with ferromagnetic hyperthermia in this model. Significant synergy was observed with this combination, suggesting that hyperthermia may reduce the total radiation dose necessary for tumor control [74].

C.Vitamin D Analogues

The presence of calcification as a consistent feature of retinoblastoma has led to the suggestion that vitamin D analogues may have activity as chemotherapeutic agents

in this tumor type. Systemic administration of vitamin D3 to LHbeta-Tag mice inhibited the growth and local extension of tumors in a dose-dependent manner [75] and was further shown to inhibit tumor angiogenesis in this model [76]. However, vitamin D3 treatment was associated with significant toxicity, including hypercalcemia, weight loss, and death [75]. A number of vitamin D analogues have been developed in recent years, several of which appear to have greater antitumor activity with reduced toxicity [77]. One such analogue, 1,25-dihydroxy-16-ene-23-yne- vitamin D3, has also been evaluated in the LHbeta-Tag mice, and this compound showed comparable antitumor activity with significantly reduced systemic toxicity [78,79] compared to vitamin D3. The evaluation of novel vitamin D analogs in LHbeta-Tag transgenic mice is described in greater detail in Chapter 14.

D.Attenuated Herpes Simplex Virus

A number of strategies are currently being developed to employ cytolytic viruses as anticancer agents [80]. Of particular interest are naturally occurring or experimentally modified viruses that retain their ability lyse rapidly growing cells, including tumor cells, but are attenuated in their ability to replicate in normal cells. Herpes simplex virus (HSV) has a natural tropism for neuronal cells, and several attenuated mutant viruses have been characterized, making HSV an attractive virus for the treatment of neuronal malignancies [81]. One such HSV mutant, RE6, was evaluated in the LHbeta-Tag mouse model and was shown to significantly reduce tumor growth following intravitreal injection, although complete tumor control was not obtained [82].

VI. CONCLUDING REMARKS

The development of methodologies for introducing defined genetic alterations into the mouse genome have allowed for the creation of a number of mouse models of retinoblastoma that, like human retinoblastoma, involve loss of pRb function and to varying degrees resemble the human tumor histologically and ultrastructurally. It is somewhat ironic that the majority of these models are transgenic mouse lines that express dominantly acting oncogenes (SV40 T antigen or HPV E7), rather than knockout mice in which the Rb gene has been inactivated, since the latter approach would be expected to yield more genetically accurate models of human tumor types originating from loss of tumor suppressor genes. Although none of the approaches that have been employed for inactivating the Rb gene in the mouse have yielded mouse models of retinoblastoma (with the exception of the Rb / :p107 / chimeras), these experiments have raised important questions relating to the molecular genetics of retinoblastoma that are likely to contribute to a better understanding of the human disease. On the other hand, the transgenic mouse models of retinoblastoma have been particularly useful for the evaluation of novel therapeutic strategies, many of which have significant clinical potential, and it is hoped that these studies will lead to improved treatment strategies for retinoblastoma.

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