Ординатура / Офтальмология / Английские материалы / Ocular Oncology_Albert, Polans_2003

Figure 5 A diagram showing the relationship between p21WAF-1/Cip-1 and p53 expression in cells of the pseudorosette. Cells in zone I express high levels of p21WAF-1/Cip-1 and low levels of

p53. This may be indicative of efforts by the cells to restrict uncontrolled proliferation in this zone. Since p21WAF-1/Cip-1 is unable to affect cell division in the absence of pRb function, the

cells continue to proliferate until they reach a point of crisis (zone II). The actual signal for crisis is not well understood but may be related to the distance these cells get from the central blood vessel leading to a lack of vascular nutrients. It may also be related to the accumulation of unbound E2Fs (see text) and thus a function of the number of divisions the cells have undergone. Whatever the signal, cells in crisis downregulate p21WAF-1/Cip-1 and upregulate p53 expression, which leads to apoptotic cell death (zone III).

activate cell death of an immortalized retinoblastoma cell line in gene transfer experiments [40], underlying the molecular differences between these two cell phenotypes.

The other role of p53 is to stimulate cell death, which it can do by directly activating the expression of a gene called Bax [48]. Bax is a member of the Bcl2 gene family and plays a crucial role in the committed step of apoptosis [49]. Its actual function is not known, but there is strong evidence that it accelerates mitochondrial dysfunction leading to the activation of cysteine proteases called caspases [50–55]. In the sequence of events associated with apoptotic cell death, caspases participate in an activation cascade that marks one of the last steps in this cellular suicide program [56,57]. Their role is to systematically digest the dying cell from within, leaving little or no cellular debris that could otherwise elicit an inflammatory response in the affected organ or tissue. Recent evidence indicates that caspases play an important role in retinoblastoma cell death, both in vitro [58] and in vivo (G. Poulsen and R. Nickells, unpublished results).

Because one of the principal mechanisms of activating p53 is the formation of DNA strand breaks, treatments that elicit DNA damage, such as ionizing radiation

or chemotherapy with alkylating agents, invariably stimulate p53-dependent cell death. Conversely, tumor cells that carry a p53 mutation and/or a loss of heterozygosity of the p53 allele are much more resistant to these treatments [59,60]. Retinoblastoma tumor cells likely fall into the former category. Cells of these tumors are wild-type for p53 (see below), and immunocytochemical studies indicate that the p53 protein is expressed at varying levels in the primary cell type of most tumors [32,39]. These same studies have also noted that cells appear to accumulate higher levels of p53 just prior to cell death [32]. This phenomenon is particularly evident in cells surrounding blood vessels (pseudorosettes), in a zone just adjacent to dying cells present at the periphery of the cuff [32,41] (Figs 4 and 5). More direct evidence of p53-mediated cell death has come from the study of immortalized retinoblastoma cell lines. Transfection of WERI-Rb1 cells with a temperaturesensitive variant of p53 activated cell death, but only at the permissive temperature [32] (Fig. 6). Similarly, Kondo and coworkers [40] found that Y79 cells were sensitive

Figure 6 Activation of cell death in immortalized retinoblastoma cells by deregulated expression of an exogenous p53 gene. (A–C) Phase contrast photomicrographs of WERI-Rb1 cells transfected with a control plasmid (pBK CMV). (D–F) A plasmid containing a temperature sensitive mutant of mouse p53 (pCMV XV-D). Transfected cells were selected for antibiotic resistance (G418) for 8 days at 388C, the temperature at which mouse p53 exhibits a mutant phenotype. After this period, transfected cells in both conditions appear relatively normal (A, D). After 6 days at 32.58C (B, E), at which the mouse p53 assumes a wild-type phenotype, cells transfected with pCMV XV-D show signs of dying that are consistent with apoptosis, such as the formation of pyknotic nuclei (arrow in E). After 11 days at 32.58C (C, F), cells transfected with pBK CMV still appear normal. Cells transfected with pCMV XV-D are almost all dead. Size bar ¼ 10 mm. (Modified from Ref. 32.)

to ionizing gamma irradiation, which stimulated an increase in both p53 and p53dependent gene expression, while ultimately leading to cell death.

It is noteworthy that p53 expression has not been detected in differentiated cells of retinoblastoma tumors [32]. This is consistent with the evidence that apoptotic cells are only rarely found in differentiated regions (Fig. 3) and the observation that these cells are resistant to therapies that primarily stimulate cell death by the activation of p53.

V.THE ROLE OF p53 IN THE DEVELOPMENT OF INVASIVE RETINOBLASTOMA

As discussed above, p53 plays a role in regulating cell death in retinoblastoma tumors. Ironically, some of the best tools for studying this phenomenon are immortalized retinoblastoma cell lines, raising the question of why these cells are not susceptible to the expression of their endogenous p53 genes. One possible explanation is that the immortalization process of retinoblastoma cells is associated with a third mutation that disables either p53 or the p53 response pathway. The fact that these cells are receptive to exogenous p53 expression or treatments that upregulate endogenous p53 indicates that immortalized cells have a functional response pathway. Examination of the p53 genes in both tumor specimens and a variety of tumor derived immortalized cell lines have invariably revealed wild type alleles for this tumor suppressor gene [17,39,41,61,62]. Immunocytochemical analyses of six different lines, however, showed that the majority of them had abnormal localization of wild-type protein, with it being concentrated in the cytoplasm [39] (Fig. 7). Nuclear exclusion of p53 is not uncommon in neuroblastoma cells [42,63,64]. Since the primary function of p53 is to act as a transcription factor, it is not unreasonable to assume that cells with this localization pattern have a p53-null phenotype and are thus more likely to exhibit aggressive malignant behavior. In neuroblastomas, nuclear exclusion of p53 is found only in poorly differentiated cells rather than differentiated ones [63], while this localization pattern for p53 is associated with a reduced survival rate of individuals with either colorectal adenocarcinoma or stage II breast cancer [65,66]. Nuclear exclusion of p53 is only rarely observed in retinoblastoma cells found within an ocular tumor mass, but it has been detected more frequently in cells that have invaded other ocular tissues such as the choroid and ciliary body [39] (Fig. 7). Based on their similarities of p53 immunolocalization, invasive cells may be the most likely source of cells for developing cell lines, possibly because they have already acquired a genetic makeup that effectively immortalizes them.

The mechanism of nuclear exclusion is not yet defined, but a study using murine erythroleukemia cells suggested that increased expression of both c-myc and Bcl2 genes can alter the subcellular trafficking of p53 leading to cytoplasmic accumulation [67]. Similar molecular conditions have been reported in retinoblastoma cells. Immortalized culture cells express high levels of the Bcl2 homologue, BclX [39], while very little of this gene product is detected in primary tumors themselves (Fig. 8). In addition, several observations of elevated N-myc expression, including amplification of this gene, have been reported in both immortalized cells

Figure 7 Nuclear exclusion of p53 in retinoblastoma. (A) Immunofluorescent localization of p53 in the Y79 immortalized retinoblastoma cell line. The majority of the p53 in these cells is in the cytoplasm ringing the nuclei. Several other retinoblastoma cell lines exhibit a similar pattern of localization [39]. Size bar ¼ 10 mm. (B) Western blot analysis of p53 levels in wholecell lysates and soluble cytosolic fractions of Y79 and WERI-Rb1 cells. As a control, cells from a small cell lung carcinoma (H510A cells), which exhibit nuclear accumulation of a mutant form of p53, were included in this fractionation experiment. All three cell lines contain p53 in the whole-cell lysates, but only the retinoblastoma cell lines have soluble p53 in the cytoplasmic fraction. (C, D) Immunohistochemical localization of p53 in a human tumor specimen. (C) Cells in the tumor mass found inside the globe exhibit nuclear localization of p53 (arrows). (D) In a region from the same eye, cells that have invaded the choroid exhibit cytoplasmic localization of p53 (arrows). Size bar (C, D) ¼ 10 mm. (C and D from Ref. 39.)

and primary tumors [68–70]. Both BCL-X and N-myc proteins have functions similar to BCL-2 and c-myc, respectively.

Nuclear exclusion may not be the only mechanism of p53 inactivation leading to metastasis. In a study of 25 retinoblastoma tumors (23 primary, 1 recurring, and 1 that had metastasized to the lung), loss of heterozygosity for the chromosomal location of the p53 allele was detected in only 1 primary tumor and the single recurring tumor, while the metastatic tumor showed both a loss of heterozygosity and a mutation in p53 [62]. Whether the loss of p53 function is common for all invasive/metastatic retinoblastomas is a question that remains to be examined.

Figure 8 BCL-X is highly expressed in immortalized retinoblastoma. (A) Immunofluorescent photomicrograph of BCL-X localization in Y79 cells. BCL-X is normally localized to mitochondria and exhibits a typical punctate staining pattern as seen in these cells. Size bar ¼ 10 mm. (B) Western blot of equal amounts (50 mg) of total protein lysates isolated from control retina (C), whole retinoblastoma tumors tissue taken from inside the globe of an affected eye (Rb), and Y79 cells. The Western blot membrane was challenged with a polyclonal antibody against human BCL-X. Y79 cells contain significantly more BCL-X than either the normal retina or an ocular retinoblastoma tumor.

VI. WHAT TRANSGENIC MOUSE MODELS OF RETINOBLASTOMA TELL US ABOUT THE MOLECULAR BIOLOGY OF THE HUMAN DISEASE?

A dramatic surge in the study of retinoblastoma has come from the development of transgenic mouse models of this ocular tumor. A detailed description of the various mouse models is found in Chap. 6 of this volume. Essentially, these models have revealed information on both the developmental timing of tumor formation and the molecular events associated with malignant transformation and possibly the evolution of the invasive cell phenotype.

Like all other mammals, mice do not naturally develop retinoblastoma tumors [71,72]. The reason for this phenomenon has not yet been elucidated. Knockout mice, completely lacking a functional Rb1 gene, are unable to survive beyond 13–15 days of gestation because of abnormalities in both erythroid and neuronal development [73,74]. Complete loss of pRB throughout the organism, however, is not analogous to the development of Knudson’s two hits in the human eye. A more accurate model comes from the study of chimeric mice, composed of both wild-type and Rb1 / cells. These studies have shown that pRB.-deficient cells do not form tumors but instead survive and differentiate normally when surrounded by cells with a normal genotype [16,75]. Similarly, mouse cells that are heterozygous for Rb1 (Rb1þ/ ) do not exhibit an increased incidence of eye tumors, although they are significantly more susceptible to acquiring pituitary and thyroid tumors [76].

More aggressive strategies have been employed to stimulate retinoblastoma tumor growth in mice, and all of them require inactivation of both Rb1 and p53. These strategies have utilized the expression of viral oncoproteins in select target cells in transgenic mice. The primary models employ the simian virus 40 (SV40) large T-

antigen controlled by promoters that are expressed in the developing retina. This oncoprotein binds to and inactivates both pRB and p53 [77,78], so typically its ability to transform cells has been attributed to this dual role. It is important to note, however, that this protein also appears to have a variety of other transforming functions not yet fully understood [79].

Tumor formation in the mouse eye using the SV40 T antigen requires early expression in retinal progenitor cells present in the developing neuroblast layer. One of the most useful promoters for this requirement is from the gene for interphotoreceptor retinoid binding protein (IRBP) [80], which is expressed in developing mouse photoreceptors prior to postnatal day 5 [81]. A similar transgenic line, with the SV40 T antigen under the control of the rod opsin promoter failed to develop tumors and instead exhibited widespread cell death [82]. The reason for this failure is likely due to the timing of expression of the opsin-controlled transgene, which is activated quite late in the differentiation sequence of photoreceptors [81]. A second successful line expressing the SV40 T antigen was fortuitously generated using the promoter for lutenizing hormone b (LHb) [83]. These mice, which were originally developed for the study of pituitary gonadotropin-derived tumors, showed ectopic expression in a cell lineage present in the retinal inner nuclear layer, making these tumors possibly more analogous to human retinoblastoma with respect to the actual cell of origin [17]. In similar experiments, ectopic expression of the human papillomavirus (HPV) E7 and E6 oncoproteins in presumptive bipolor neurons (these genes were being expressed by the lens a-A crystalline promoter) also caused the formation of a retinoblastoma tumor, but only in certain genetic backgrounds [84]. The E7 protein binds to and inactivates pRB and the E6 protein binds to p53 and targets it for degradation [85]. Subsequent lines generated by using HPV E7 under the control of the IRBP promoter to specifically inactivate pRB did not develop tumors but instead exhibited pronounced cell death at the period coinciding with the rod photoreceptor terminal differentiation [86]. Conversely, some retinal cells in the IRBP-HPV E7 transgenic mice were able to give rise to retinoblastoma tumors when placed on a p53-null genetic background.

VII. SUMMARY

Rb1 / cells in both mice and men appear to activate a p53-dependent pathway of cell death, but tumors can arise in mice only if both pRB and p53 are disabled. This is in direct contrast to findings in humans, in which wild-type p53 is always detected in these tumors, leading some to question whether these mice represent an accurate model of this eye tumor [87]. Rather than being contradictory, however, it is possible that tumor cells in mice are more similar to the immortalized retinoblastoma cells found in some human tumors. Mouse tumors never exhibit signs of differentiation and only seldom show histological evidence of cell death [72]. Typically, cells in these tumors initially organize into Homer-Wright rosettes, which in human tumors are composed of undifferentiated primary cells and have been associated with cytoplasmic localization of p53 (T. M. Nork, C. L. Schlamp, and R. W. Nickells, unpublished observations). Lastly, mouse tumor cells are also highly aggressive and often metastasize within a few months [72,80]. In this context, mouse models argue

that more aggressive behavior of cells in human tumors is associated with additional genetic damage involving p53.

ACKNOWLEDGMENTS

The authors would like to thank Dr. T. Michael Nork for helpful discussions and for providing several photomicrographs used in this chapter and Dr. Isabelle Audo for her help in researching the literature. Some of this work was supported by funding from Research to Prevent Blindness and by an institutional grant from the American Cancer Society to the University of Wisconsin.

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