Ординатура / Офтальмология / Английские материалы / Ocular Oncology_Albert, Polans_2003

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differentiating into neuronal phenotypes [12]. Loss of Rb1 function, either by the introduction of pRB inactivating viral oncoproteins [13] or by directed mutagenesis in knockout mice, results in the activation of neuronal cell death during the period when these cells would normally be differentiating. It is not clear how loss of pRB activity leads to cell death, but this signal may be associated with elevated levels of free E2F transcription factors [10]. Independent studies indicate that overexpression of E2F1 is sufficient to activate cell death in postmitotic neurons [14,15]. In addition, studies involving chimeric mice indicate that the function of pRB in regulating cell cycle progression is cell-autonomous, but its role in differentiation is cell nonautonomous [6,16]. In chimeric embryos, Rb1 mutant neuroectoderm cells still exhibit ectopic entry into S phase, like Rb1 / embryos, but—unlike the cells in the knockout animals—they survive and differentiate into neurons. Therefore activation of cell death is likely to be modulated by several factors, such as elevated E2F concentration within the mutant cell and the level of exposure to survival (antideath) signals from neighboring wild-type cells.

Why is loss of pRB function in the developing human eye sufficient to stimulate tumorigenesis? Gallie and colleagues [17] proposed a model that encompasses many of the characteristics observed for pRB function. In their model, a developing retinoblast expresses pRB at the point of their terminal mitosis in the outer (ventricular) region of the neuroblast layer. These cells then migrate to the differentiating ganglion cell and inner nuclear layers, where extensive remodeling of the tissue is regulated by the activation of programmed cell death. Rb1 / mutant cells do not achieve terminal mitosis and are targeted for cell death through a mechanism such as that proposed above. Some cells are able to escape this signal and continue to proliferate exponentially. This model argues that a third genetic hit that disrupts the cell death signaling mechanism is required for retinoblastoma formation. One candidate is a gene with similarity to a mouse kinesin-like protein (termed RBKIN). This gene was recently cloned from a region of chromosome 6p22 that undergoes low-level amplification in a large proportion of tumors [18]. At present, however, it is not known how this gene contributes to the etiology of retinoblastoma.

II.FORMATION OF RETINOBLASTOMA TUMORS—THE CELL OF ORIGIN

Retinoblastomas occur in the developing retina, but the cell of origin is not clear. Histological analyses suggest that tumor masses can form in nearly all layers of the retina, although recent studies suggest that the majority of tumors arise from the inner nuclear layer [17]. Tumors likely develop early in the life of the patient [19–21] and the events leading to a malignancy may occur well before final specification and differentiation of either the photoreceptors or the neurons of the inner nuclear layer, leaving open the possibility that any cell in the retinal neuroblastic layer may be potentially susceptible to Knudson’s two hits and/or Gallie’s third hit. It is clear, however, that once neuroblasts acquire the Rb1 null phenotype, they have the potential to differentiate into photoreceptors. Several studies showing either morphological [22], immunogenic [23–25], or molecular [26–28] features of photoreceptor cells have prompted several investigators to suggest that retinoblas-

tomas most frequently arise from precursors of photoreceptor cells. Although this may be the case, it is also plausible that the molecular events leading to retinoblastoma occur so early in the development of the retina that affected cells are stimulated to activate a default pathway of differentiation in the absence of other clear regulatory signals. Specification of the cone cell lineage is one of the first to occur in the embryonic retina and also appears to be one of the least restricted with respect to developmental signals [29,30]. This pathway is consistent with the characteristics of the majority of differentiated retinoblastoma cells [24,26]. The incidence and consequences of differentiation of retinoblastoma cells are discussed further in the following section.

III.CELL FATE IN RETINOBLASTOMA TUMORS

Retinoblastoma tumors are composed of transformed neuroblasts and support cells. Support cells consist primarily of blood vessels derived from the retinal vasculature, and there is evidence that these tumors signal increased growth of vessels. Tumors also contain Mu¨ller-like glial cells, particularly in areas of differentiation [19]. Some observers have suggested that this glial cell type is a second form of differentiated tumor cell, based on evidence that immortalized retinoblastoma cells in culture have been found to express glial markers such as glial fibrillary acidic protein [31]. Others have refuted this speculation [19] and suggested that glial cells are derived from nonmalignant cells recruited to regions of differentiated tumor.

Tumor cells have four basic phenotypes. The primary tumor cell type contains variably sized basophilic nuclei and scanty cytoplasm [19] (Figs. 1 and 2). It is likely that these cells represent the direct progeny of the initial neuroblast that developed defects in both alleles of the Rb1 gene. There are three general fates of these cells in most retinoblastoma tumors (Figs. 1 and 2). The dominant fate is cell death [32]. In a majority of tumors, dying cells or regions of complete cell death make up more than 50% of the tumor mass. Recent studies [32] indicate that the primary mechanism of cell death is through a form of programmed cell death known as apoptosis (Fig. 3), although these regions are classically referred to as necrosis in most pathological descriptions of retinoblastoma [19]. Areas of tumors with features of necrosis are likely the result of secondary necrosis [33]. In these cases, there is such widespread apoptotic death that cellular debris is not effectively cleared by surrogate macrophage activity, leaving it to break down by a more necrotic-like mechanism.

The signal that initiates apoptosis in the primary tumor cell is not known. Some histological features of retinoblastoma suggest that cell death results from nutrient starvation. A compelling example of this is seen in the cuffs of cells that surround a central blood vessel (Fig. 3). These cuffs are sometimes referred to as ‘‘pseudorosettes’’ from their appearance in histological sections. Cells at the center of the cuff have the morphology of the primary tumor cell, while cells at the edges are actively undergoing apoptosis [32]. Burnier and colleagues [34] conducted a morphometric study of these structures and found that cell death occurred at a uniform distance from the central blood vessel (98.7 + 11.9 mm), suggesting that the reduction in nutrient supply at this region was sufficient to send cells into crisis. Although reduced access to vascular nutrition may be one of the signals that can precipitate apoptosis, there are clearly exceptions to the distance rule, and dying cells

Figure 1 Cell fates in human retinoblastoma. A flow diagram showing the potential fates of immature retinoblasts that have acquired abnormalities in both alleles of the retinoblastoma susceptibility gene (Rb1). Once these cells lose pRB function, they enter a period of unregulated division, consistent with the cell-autonomous function of pRB (16). The primary fate of proliferating Rb1 / retinoblasts is to undergo cell death (apoptosis). Studies show that this process is regulated by activation of the p53 tumor suppressor protein. Some cells escape this fate and attempt to differentiate. Morphological, immunohistochemical, and molecular studies indicate that the preferred pathway of differentiation is into a photoreceptor cone phenotype, although some differentiated tumor cells have been found to express rod photoreceptor antigens. On occasion, especially if left untreated, Rb1 / cells become invasive. This third fate may be associated with an additional malignant transformation, perhaps involving another cell cycle regulatory gene or part of the apoptotic signaling pathway. There is some evidence to suggest that this process also involves the immortalization of tumor cells.

can often be found directly adjacent to blood vessels (Fig. 3). Retinoblastoma tumor cells are already primed for cell death because of the lack of functional pRB and their inability to differentiate properly. It is possible that, after a certain number of divisions, tumor cells must make the decision to differentiate or execute the apoptotic program. In the case of the latter, cell death may be a favored fate in cells with abnormally high levels of E2Fs [10].

The second most likely fate of the primary tumor cell is differentiation (Figs. 1 and 2). A majority of retinoblastoma tumors examined in the United States exhibit some degree of differentiation, although the percentage of tumors showing extensive

Figure 2 Light and fluorescent photomicrographs of different cell phenotypes in human retinoblastoma tumors. Panels A–C are sections taken from a single tumor and stained with 4,6-diamidino-2-phenylindole (DAPI) to highlight their nuclear morphology. All micrographs are shown at the same magnification. (A) A small cluster of the primary cell type found in retinoblastoma tumors. The nuclear diameter of these cells is typically around 10 mm. Some dying cells are also evident (arrows). (B) Dying tumor cells, with brightly staining nuclear fragments. This morphology is typical of apoptosis, which is associated with nuclear condensation and subsequent fragmentation. (C) Tumor cells that have invaded the choroid. The nuclei of these cells are typically larger than the primary cell type (between 15–20 mm in diameter). No evidence of dying cells is detected. Size bar of A–C ¼ 10 mm. (D) A section stained with hematoxylin and eosin (H&E) showing the primary cell type. The cells have plump nuclei and scanty cytoplasm. Size bar ¼ 10 mm. (E) An H&E-stained section of rows of invading cells found in the choroid. These cells are tightly packed together and have a high nuclear-to-cytoplasm ratio. Pigmented cells (arrow) are choroidal melanocytes. Size bar ¼ 20 mm. (F) A section from a tumor showing cells in early stages of differentiation. The section has been stained for carbonic anhydrase activity (toluidine blue counterstain), which is present in glial cells (dark filamentous structures) and red and green cone photoreceptors (darkly stained nuclei). Unstained cells may represent primary cells that are in early stages of differentiation. It is also noteable that a high proportion of cells contain nucleoli, which are often not seen in cells undergoing rapid mitosis. (G) A section through two Flexner-Wintersteiner rosettes showing the organization typically found in differentiated tumors. The cellular structures pointing toward the interior of the rosette have features of photoreceptor outer segments. In addition, these structures are often associated with glial cells that send processes around the differentiated cells, as evidenced by the dark immunohistochemical staining for glial fibrillary acid protein (hematoxylin counterstain). (H) A section through a fleurette. These structures are often considered to represent the most highly differentiated state of cells in tumors. This section has been immunostained for a variant of S antigen (hematoxylin counterstain), which is expressed in rod and blue cone photoreceptors (arrows). Size bar (F–H) ¼ 10 mm. (D–F courtesy of Dr. T. M. Nork, University of Wisconsin. G and H, from Ref. 24.)

Figure 3 Cell death in retinoblastoma. Dying cells undergo DNA fragmentation. In apoptotic cells, these fragments form multiples of 180 base pairs that can be visualized by gel electrophoresis. Analysis of human retinoblastoma tumors shows clear evidence of these DNA ‘‘ladders’’ [32]. DNA fragmentation can also be detected histologically using methods that attach labels to the ends of fragmented sequences. (A) An H&E-stained section through a tumor showing a region of differentiated cells (note the circular structures indicative of Flexner-Wintersteiner rosettes) that contains a small pocket of ‘‘necrosis’’ (arrow). Size bar ¼ 120 mm. (B) A fluorescent micrograph of an adjacent section taken from the same region shown in (A), stained for DNA fragmentation using a method specific for apoptotic cells (30 OH overhand ligation [88]). The area of ‘‘necrosis’’ is actually a patch of cells actively undergoing apoptosis. Size bar ¼ 60 mm. (C) A section through a cuff of cells surrounding a central blood vessel (asterisk), commonly referred to as a pseudorosette. The section has been stained for DNA fragmentation using the terminal transferase deoxyUTP end-labeling method (TUNEL) [89]. Dying cells are concentrated around the edge of the cuff, although dying cells can be found throughout the mass, including directly adjacent to the central blood vessel (arrows). Size bar ¼ 30 mm. (D) A fluorescent micrograph of a pseudorosette stained with the 30 overhang method. As with TUNEL staining, dying cells are concentrated at the periphery of the cuff of cells surrounding a central vessel (arrow). Size bar ¼ 100 mm. (E). A section taken from a tumor showing a clear demarcation between differentiated cells (upper) and undifferentiated primary cells. TUNEL staining shows that dying cells are restricted to the region of primary cells. Size bar ¼ 40 mm. (C and E from Ref. 32.)

differentiation is quite small (studies in the literature, which have not been controlled for bias, range from 10–25%). Differentiated cells appear benign, with smaller, less basophilic nuclei and more cytoplasm [19]. There is little evidence of mitotic figures or the expression of proliferating antigens associated with areas of differentiated cells, and they rarely exhibit signs of cell death (Fig. 3), although regions of differentiation may contain pockets of undifferentiated and apoptotic cells. As indicated above, differentiated cells exhibit morphological and molecular features of photoreceptors, particularly cone cells. Areas of differentiated cells form either rosettes (Flexner-Wintersteiner rosettes) or fleurettes and are distinguished by cytoplasmic structures that resemble photoreceptor outer segments (Fig. 2). The level of tumor differentiation is a primary diagnostic of prognosis, with a higher survival rate being directly related to the degree of differentiation [19,20]. Interestingly, welldifferentiated tumors are more resistant to radiation therapy [19] and chemotherapeutic drugs [35]. The molecular basis for this is discussed below.

The third fate of the primary tumor cell is to undergo an additional transformation and become invasive and/or metastatic (Figs. 1 and 2). A finding of invasion is a very poor indicator of survival [19,20] and is associated with the formation of secondary metastatic tumors [36]. It is likely that some cells in nearly all retinoblastoma tumors (with the exception of highly differentiated ones) would undergo this additional transformation if left untreated. The basis for this speculation comes from several studies showing that tumors with invading cells are predominantly associated with delayed diagnosis and treatment. One study, for example, found a strong association between the misdiagnosis of retinoblastoma and an increase in mortality [37], while another found that cellular invasion was significantly higher in referral centers in rural third-world countries, where the average age of diagnosis was over a year later than at centers in developed countries [38]. Invasive tumor cells exhibit the basic morphological features of the primary cell type, although there is evidence that they have even larger nuclei and less cytoplasm (Fig. 2). These cells show no signs of either cell death or differentiation [32]. Recent evidence has linked the molecular events leading to invasion of tumor cells to a process of immortalization [39] and suggests that this fate of the primary tumor cell is associated with an additional genetic hit.

IV. THE MOLECULAR BIOLOGY OF APOPTOSIS IN

RETINOBLASTOMA TUMORS—THE ROLE OF p53

As indicated earlier, cell death, in the form of apoptosis, is the dominant fate of the primary undifferentiated cell type found in retinoblastoma tumors. Several lines of evidence suggest that p53, the tumor suppressor protein, plays an important role in regulating this process [32,40,41]. p53 is a transcription factor and one of the most important molecules found in cells, acting as a check point that controls their ability to progress through the cell cycle or enter the apoptotic pathway [42]. In many cells, sensor proteins can detect strand breaks or abnormalities in the nuclear DNA and

activate latent p53 [43]. For cell cycle control, p53 directly activates the expression of at least two genes, p21WAF-1/Cip-1 and 14–3–3s, that function to arrest the cell cycle at the G1/S and G2/M interfaces, respectively [7,8,44–47]. p21WAF-1/Cip-1 is well known

as an inhibitor of cyclin-dependent kinases. These same kinases are involved in the