Prior research has identified the lateral occipital complex (LOC) as a critical cortical region for the representation of object shape in humans. However, little is known about the nature of the representations contained in the LOC and their relationship to the perceptual experience of shape. We used human functional MRI to measure the physical, behavioral, and neural similarity between pairs of novel shapes to ask whether the representations of shape contained in subregions of the LOC more closely reflect the physical stimuli themselves, or the perceptual experience of those stimuli. Perceptual similarity measures for each pair of shapes were obtained from a psychophysical same-different task; physical similarity measures were based on stimulus parameters; and neural similarity measures were obtained from multivoxel pattern analysis methods applied to anterior LOC (pFs) and posterior LOC (LO). We found that the pattern of pairwise shape similarities in LO most closely matched physical shape similarities, whereas shape similarities in pFs most closely matched perceptual shape similarities. Further, shape representations were similar across participants in LO but highly variable across participants in pFs. Together, these findings indicate that activation patterns in subregions of object-selective cortex encode objects according to a hierarchy, with stimulus-based representations in posterior regions and subjective and observer-specific representations in anterior regions. a.d., the Arab scholar Ibn al-Haytham suggested that visual experience was not veridical, but inherently subjective. During the last few decades, this observation has given rise to one of the core questions in visual neuroscience: how does the subjective experience of visual stimuli relate to their neural representations in the brain? It is well-known that visual shape is represented in a brain region called lateral occipital complex (LOC). However, do these representations reflect physical or perceptual stimulus characteristics? We presented observers with a set of complex visual stimuli and obtained three measures of similarity for these stimuli: a physical similarity measure based on stimulus parameters; a behavioral similarity measure based on discrimination performance; and finally a neural similarity measure based on multivariate pattern analyses in LOC. We found that in anterior LOC, neural stimulus similarities correlated with subjective perceptual similarities, but not with physical stimulus similarities; the reverse was true in posterior LOC. In addition, neural similarities were consistent across participants in posterior LOC, but highly variable across participants in anterior LOC. Together these findings suggest a two-part answer to the question of how cortical object representations relate to subjective experience: anterior regions appear to contain subjective, individually variable shape representations, whereas posterior regions contain stimulus-based shape representations.As early as 1031  How does the subjective experience of visual shapes relate to the neural representations of these shapes in the brain? Using psychophysics, functional MRI, and multivariate pattern analysis methods, this study shows that activation patterns in anterior, shape-selective brain regions reflect perceptual shape similarities, whereas patterns in posterior regions reflect physical similarities. What is the neural code for object shape? This question has been at the core of systems neuroscience for decades. In monkeys, inferotemporal (IT) cortex has been shown to contain cells selective for complex shapes ; in humaA number of previous studies suggest that the coding of objects in high-level visual cortex may reflect subjective perceptual experience of shapes. For instance, LOC adapts across changes in low-level physical stimulus properties that leave perceived shape unaltered, but not across changes that affect perceived shape ,7. FurthFMRI studies of visual processing have traditionally focused on mean activation levels, looking for brain regions showing a difference in activation between different stimulus conditions. More recent studies, in contrast, have illustrated the importance of the distributed pattern of activation in representing information about stimulus conditions –14. HaxbWe used a combination of human fMRI and psychophysics to test the hypothesis that distributed activation patterns in LOC reflect perceived shape. We created a novel artificial shape space, in which physical similarity was controlled by gradual, parametric changes in aspect ratio and skew. Perceptual similarity was measured by psychophysical discrimination performance between the shapes, and neural similarity was measured by the correlations between the fMRI activation patterns of these shapes in LOC.  We founWe created a novel artificial stimulus space consisting of four complex objects A. Each sTo study the relationship between perceptual, physical, and neural similarities in pFs and LO, we obtained three similarity measures for each pair of stimuli as follows. First, for each of the six possible pairs of nonidentical stimuli, physical similarity was measured by the inverse pairwise distances of the four shapes in the aspect ratio/skew space , followed by a 1,500 ms response period. The proportion of trials on which a particular participant responded “identical” to a pair of stimuli that were in fact different was used as a measure of the perceptual similarity of that pair of stimuli. An example of a perceptual similarity matrix is shown in p < 10−4). LOC can be subdivided into a posterior portion, LO, on the lateral surface of occipitotemporal cortex; and an anterior portion, pFs, on the fusiform gyrus of the temporal lobe [Finally, to obtain a measure of neural similarity, we scanned the brains of eight participants using fMRI. Since we had a specific hypothesis about neural coding in object-selective cortex, we first identified the human object-selective region LOC in an independent localizer scan, using the standard comparison of intact versus scrambled everyday objects  . The purpose of this task was to keep participants' attention focused on the stimuli. We then extracted the spatially distributed activation patterns of each individual stimulus from the two LOC ROIs, on a voxel-by-voxel basis. Thus, in each participant and for each ROI, we obtained four vectors, each representing the voxelwise activation pattern of one particular shape in that ROI. We then computed the correlations between each of the six pairs of activation patterns for nonidentical stimuli, separately for pFs and LO. This resulted in six correlation coefficients for each ROI, one for each possible pair of the four shapes. An example of a neural similarity matrix is shown in Thus, we obtained physical, perceptual, and neural similarity measures for each possible pair of stimuli. We next compared these six-element similarity matrices to one another, by computing their correlation coefficients within participants and ROIs B. A highThe results confirmed this hypothesis, with an interesting twist. Neural and perceptual similarities were positively correlated in pFs, with an average correlation of 0.35 across participants, whereas in LO the average neural-perceptual correlation was only 0.001 . Converst(5) = 5.66, p < 0.001), but not greater than zero in LO (t(5) = 0.10, p = 0.38). Conversely, the correlations between neural and physical similarities were significantly greater than zero in LO (t(5) = 2.66, p < 0.05), but not in pFs (t(5) = 0.70, p = 0.29). A two-way analysis of variance (ANOVA) with region of interest (pFs versus LO) and correlation type  as factors revealed a significant interaction of ROI and correlation type  = 13.79, p < 0.005), confirming the dissociation between these ROIs: neural pattern similarities in pFs correspond to subjective shape similarities, while neural pattern similarities in LO correspond to physical shape similarities.To quantify these results, we initially applied the Fisher z transformation to all correlation coefficients. This method transforms the non-normally distributed correlation coefficients into normally distributed variables, which allows the use of standard analysis of variance methods  for det. StatistF = 1.86, p = 0.17; LO: F = 0.17, p = 0.92). Moreover, these results cannot be due to task performance, since critically the perceptual similarity measure was obtained in a separate testing session, while in the scanner participants performed an easy one-back task. These results were not due to differential mean signal levels for any of our stimuli, for two reasons: first, the correlation analysis does not take into account mean levels of activation; second, there were no differences in mean signal between the four stimuli in either region of interest  = 0.92, p = 0.26; t(25) = 7.83, p < 0.00000005; t(25) = −2.80, p < 0.05).As predicted, inter-participant reliability was low in pFs  does not depend on whether they correlate with the behavioral similarities within participants (tested above).p < 10−4); and the parahippocampal place area, PPA [p < 10−4). In none of these regions did the neural similarities exhibit significant correlations with either perceptual or physical similarity =2.77, p < 0.05), showing that pairs of stimuli that a particular participant confused with high probability were also perceptually similar for the other participants. However, at the same time the fact that this correlation was not extremely high leaves room for subjective, observer-specific patterns of perceptual similarities.The psychophysical same-different task used outside the scanner to obtain a measure of perceptual similarity was made as difficult as possible by presenting stimuli for extremely short durations (17 ms each) and using both forward and backward high-energy noise masks (50 ms each). Nevertheless, the performance level was high, with an average of 93% ± 1% correct performance on the same-different task. However, this level of performance corresponds to an average of 44 ± 9 errors over the course of the psychophysical experiment; this number of errors, distributed over the four stimuli used, proved sufficient to obtain a reliable measure of perceptual similarity. In support of this claim, the inter-participant reliability of perceptual similarity was on average r = 0.38; this value was significantly different from zero (t(5) = 2.75, p < 0.05). Thus, perceptual similarity correlated with physical similarity, but not perfectly, again leaving room for the neural similarity measures to correlate with either the perceptual or the physical similarities without necessarily also correlating with the other.The average correlation between the physical and perceptual similarity measures across participants was t(7) = 8.11, p < 0.001), and stimuli 1 and 2  = 2.88, p < 0.05). To assess the effect of this difference, we used the reaction time differences among the four stimuli during the task in the scanner as a new behavioral similarity measure within each participant; e.g., if a particular participant took an average of 635 ms to respond to stimulus 1, and 649 ms for stimulus 2, the new behavioral similarity of these stimuli would be the negative reaction time difference, i.e. −14 ms (negative to turn the measure from a “difference” into a “similarity” measure). We then re-computed the correlations between the neural similarities and this new behavioral similarity measure. None of the resulting correlations were significant in any of our ROIs, although there was a nonsignificant trend towards a correlation between neural similarities and the new reaction time similarities in LO  = 1.75, p = 0.09).The one-back task participants performed in the scanner was designed purely to keep participants' attention focused on the stimuli and was very easy to perform; none of our participants made a single mistake on this task. However, to nevertheless control for any possible effects of performance in the scanner, we analyzed the reaction times during the task in the scanner as follows. Since the order of stimulus presentation was randomized using m-sequences, each stimulus was preceded equally often by each other stimulus; therefore, each of the six possible discriminations among the four stimuli entered the neural pattern an equal number of times, and differential performance on any of these conditions could therefore not influence the neural pattern. However, it is possible that the task was easier for some stimuli in general; such a difference would enter the neural pattern of those stimuli and could thus potentially bias our results. Indeed, we observed a significant difference in the reaction times of stimuli 1 and 3  = 6.32, p < 0.001; t(5) = 0.56, p = 0.32); in contrast, LO exhibited a significant correlation between neural and physical similarities  = 2.33, p < 0.05; r = 0.06, t(5) = 0.56, p = 0.32). The interaction was again significant  = 12.00, p < 0.005). Similarly, the inter-participant reliability was again high in LO  = 2.82, p < 0.05), but low in pFs  = 0.60, p = 0.33; t(25) = 2.04, p = 0.05). Note, however, that subsampling reduced the inter-participant reliability in LO by a factor of one-half (mean r = 0.42 to mean r = 0.21). In light of this change, and the fact that the average sizes of pFs and LO differed by a factor greater than two, we repeated the subsampling for the inter-participant reliability analysis using not 50% of voxels, but instead equalizing voxel numbers across the two ROIs. Specifically, we excluded random subsets of voxels from the larger ROI, until its size matched that of the smaller ROI, again with 100-fold bootstrapping. The results were comparable to that of the initial analysis: the inter-participant reliability was high in LO  = 7.79, p < 0.0001), but low in pFs  = 0.91, p = 0.26), with a significant difference between LO and pFs (t(25) = 2.65, p < 0.05). Thus, our results are independent of the size of our ROIs.As pointed out above, it is conceivable in principle that differential numbers of voxels in different ROIs affect the likelihood of detecting correlations. Indeed, LO contained more voxels on average than pFs . We therefore wished to test whether the results described above depend on the number of voxels in each ROI. To this end, we conducted a control in which we randomly excluded 50% of the voxels of each ROI. This procedure was repeated 100 times, and an average correlation estimate was obtained by averaging over the 100 bootstrapping iterations. The results were the same as in the main analysis reported above  and 6: tr = 0.67) with the physical similarity measure reported above, i.e., closeness of the stimuli in parameter space. However, the neural similarities of pFs and LO showed no correlation with this V1-type physical similarity measure: in pFs, the mean correlation of the neural similarities with the mean Gabor similarity measure was r = 0.06 (not different from zero: t(5) = 0.42, p = 0.34); in LO, it was r = 0.07 (t(5) = 0.45, p = 0.34). In addition, we repeated this analysis for each individual Gabor filter ; none of the resulting 20 correlations between neural and physical similarities were significantly different from zero across participants in either pFs  or LO . Thus, the correlation of neural and physical similarities in LO appears to be specific to the case when the physical similarities are described in terms of aspect ratio and skew [The physical similarity measure reported above was based on the distances of the stimuli from each other in terms of aspect ratio and skew parameters. The high correlation of these aspect ratio/skew distances with neural similarities in LO is consistent with the proposal that LO encodes stimuli in terms of aspect ratio and skew, as has been reported previously for high-level visual cortex in monkeys ,24. Thisr = 0.15, which did not differ from zero across participants (t(5) = 0.76, p = 0.27). Moreover, none of the correlations were significant when the individual physical similarity matrices resulting from each of the 20 Gabor filters were correlated one-by-one with neural similarities . This result is probably due to the fact that the images were presented with a random jitter of ∼2 degrees during scanning, which likely resulted in sufficiently nonoverlapping activations in retinotopic cortex to disrupt the neural similarity estimates in this region, and therefore also any correlation between neural and physical similarities. In support of this hypothesis, the stimuli were not distinguishable in retinotopic cortex using Haxby's pattern discrimination method [The neural patterns in retinotopic cortex only showed a weak correlation with the physical stimulus distances based on this V1-type similarity measure; the mean correlation between neural and Gabor similarities in retinotopic cortex was F = 3.67, p < 0.05, two-way ANOVA with ROI and within/between as factors). As a further test of pattern discriminability, we computed the “Haxby Index” [t(7) = 4.20, p < 0.005; LO: t(7) = 4.55, p < 0.005). Thus, the patterns in both ROIs were stable enough across the split halves to successfully discriminate between our stimuli.The results reported above indicate that the neural patterns in our regions of interest contain fine-grained information about perceptual and physical stimulus similarity. These analyses were based on correlations between the neural activation patterns of pairs of stimuli; this measure controls for noise in the data because the formula for the correlation coefficient includes a division by the standard deviations of the data vectors. However, we additionally wished to confirm with a conventional analysis method that these neural patterns were indeed stable and contained information about stimulus identity. To this end, we applied the widely used technique of Haxby et al. : we extry Index” ,12,13,26In sum, we have found that distributed activation patterns in human object-selective cortex contain information about the subjective perceptual similarities between complex visual stimuli. Specifically, we show a dissociation between neural coding of perceptual versus physical similarities within LOC: using independent measures of neural, perceptual, and physical similarity on our set of novel artificial shapes, we find that the neural similarities of shapes in anterior LOC (pFs) correlate with their perceptual similarities. Conversely, the neural similarities in posterior LOC (LO) correlate with the physical similarity of the shapes in the stimulus space. Furthermore, the agreement across participants of the neural similarities is high in LO, but low in pFs, consistent with a physically based representation in LO and a representation based on observer-specific subjective shape experience in pFs.These results are specific to object-selective cortex, i.e., the regions LO and pFs; additional ROIs including retinotopic cortex, FFA, OFA, and PPA did not show significant correlations between either neural and perceptual or physical similarities. Moreover, the results did not depend on the number of voxels in each ROI.Our findings confirm previous studies showing that object representations in the ventral stream reflect subjective perception –9,11,27,Similarly, the finding that posterior LOC shows a correlation between neural and physical stimulus similarity confirms previous studies that have shown selectivity for physical shape features of moderate complexity in high-level visual cortex. In area V4, single-cell and fMRI studies have demonstrated tuning to contour curvature in monkeys ,28,29 anPutting the results from pFs and LO together, our findings are consistent with previous evidence regarding an anterior-posterior functional subdivision within LOC: Grill-Spector et al.  showed tHowever, it should be noted that other recent studies have found evidence highlighting the informativeness and behavioral relevance of neural activation patterns in LO: Eger et al.  showed tThree previous studies have shown correlations between pattern information and subjective perception in object-selective cortex. First, Edelman et al.  used mulSecond, Op de Beeck et al.  showed iThird, Williams et al.  used a pIn conclusion, our results indicate that object shape may be coded in terms of physical features in posterior LOC, and in terms of subjective shape experience in anterior LOC. Of course, this claim simply replaces one puzzle with another: “What is the neural code for object shape?” is transformed into the equally difficult question, “What are the determinants of subjective shape experience?” However, the advantage of this new question is that it has been the subject of intensive study since the time of the Gestalt psychologists , and theWe recruited eight participants from the MIT Human Subject Pool. Each participant was compensated US$60. The study was approved by the MIT Committee on the Use of Humans as Experimental Subjects (COUHES). All participants gave informed consent.Localizer scans: The LOC was localized as the region that responded more strongly to grayscale images of intact objects than to images of scrambled objects (p < 10−4), as described previously [p < 10−4). The PPA [p < 10−4). The retinotopic ROI was defined based on activation at the occipital pole in a contrast between all stimulus conditions versus baseline in the localizer scans .Experimental scans and behavioral experiment: Four novel stimuli, each measuring 10 degrees across, were used for the experimental scans. The use of novel stimuli ensured that correlations were not due to semantic associations with the stimuli; this was a potential confound in previous pattern similarity studies [y = na x. The parameters a and n could be used to vary the skew and aspect ratio of each protrusion parametrically. In doing so, the total area of the stimuli was always kept constant to avoid low-level confounds. We defined aspect ratio as the ratio of the height to the base width of each protrusion, and skew as the position of the vertex with respect to the center of the base; for instance, 0% skew indicates a vertex directly above the center of the base, skew of 100% indicates a vertex directly above the right end of the base, and skew of −100% indicates a vertex directly above the left end of the base. From the left to the right end of the stimulus spectrum, the aspect ratio of the second and fourth protrusions  decreased by 1.4 and 1.6 on each morph step, respectively; the aspect ratio of the first and third protrusions was fixed. For skew, the first, second, and fourth protrusions moved counterclockwise by 60%, 24%, and 24% for each step, respectively (where a cumulative skew change greater than 100% simply meant moving the vertex of the protrusion beyond its base); the skew of the third protrusion changed in the clockwise direction by 25% on each step. Thus, the four stimuli used were equidistant in terms of aspect ratio and skew, forming a straight line in the stimulus space. The magnitude of the parametric distances between the stimuli was chosen based on informal testing to be at the same time discriminable and not too obvious. The stimuli were filled with random dots, with a mean luminance of 50%, to ensure activation throughout the ventral visual stream. In addition, a chair and a face were included in the stimulus set, to prevent adaptation in ventral visual cortex due to the high similarity among the novel shapes. studies . Further studies ; we therfMRI experiment: Each participant was run in one session of about 2 h, consisting of eight experimental scans and four LOC localizer scans. Stimuli were presented using the Psychophysics Toolbox [ Toolbox  and MatlThe localizer scans were run as described previously ,20,23.The experimental scans were event-related, and each scan contained 144 stimulus trials and 36 fixation trials. On each trial, one of the six possible stimuli  was presented at the center of the screen for 300 ms, followed by a 1,700-ms response period during which participants indicated whether the current stimulus was identical or different from the previous one. The purpose of this task was to keep participants' attention focused on the stimuli. The order of stimulus presentation was optimized using m-sequences (Optseq). Each stimulus occurred 24 times per scan, resulting in a total of 192 times for the whole experiment.Behavioral experiment: In a separate behavioral session that followed the fMRI experiment with a delay of at least 1 wk, each of the original participants performed a same-different task on pairs of the same four shapes, plus the face and the chair, that were presented in the fMRI experiment. On each trial, two stimuli were shown sequentially, for 17 ms each, with a forward and a backward mask (consisting of a full screen noise field of random letters with high density and overlap) of 50 ms each, followed by a 1,500-ms response period. Perceptual similarities were obtained by computing the proportion of trials on which a particular pair of different stimuli was erroneously considered “identical”. Participants performed 630 trials total. Each stimulus appeared with equal probability on each trial, and with equal probability as the first and second stimulus of each pair.fMRI scanning was performed on a 3T Siemens Trio Scanner (Siemens) at the Athinoula A. Martinos Center for Biomedical Imaging at the McGovern Institute for Brain Research at MIT. A Gradient Echo single-shot pulse sequence was used . Twenty-five slices were collected with a 12-channel head coil. Slices were oriented roughly perpendicular to the calcarine sulcus and covered most of the occipital and posterior temporal lobes, as well as some of the inferior parietal lobes. Slices were 2 mm thick, with a 10% gap, and had an in-plane resolution of 1.6 × 1.6 mm.http://surfer.nmr.mgh.harvard.edu), fROI (http://froi.sourceforge.net), and custom-written software. Before statistical analysis, images were motion corrected [Data analysis was performed using FS-FAST  to intact objects than to scrambled versions of the same objects [p < 10−4); the PPA [p < 10−4); and a retinotopic ROI based on activation at the occipital pole in a contrast between all stimulus conditions versus baseline in the localizer scans . The FFFor the blocked localizer scans, statistical maps were calculated by correlating the signal time course with a gamma function  for each voxel convolved with the block timecourse. For the event-related scans, the hemodynamic response was extracted using a deconvolution analysis, without any assumptions about the shape of the response. The peaks of the fMRI responses of each of the four novel shapes were extracted from each ROI, for all voxels separately. This resulted in four patterns per ROI, each representing the distributed activation pattern to a particular stimulus in that ROI. Neural similarities were obtained by computing the Pearson correlation coefficient between these patterns, as described above.t-tests and ANOVAs. This transformation is necessary because correlation coefficients do not follow a normal distribution, and are therefore strictly not amenable to analysis of variance statistics [r, the Fisher z is given byTo assess the statistical significance of the correlation matrices results, we first applied the Fisher z transformation to the data and then performed atistics . The FisWe took care to use the standard error formula specific to the Fisher z:This formula has fewer degrees of freedom and is therefore more conservative than the conventional standard error.r = 0.67) with our other physical similarity measure, i.e. distance of the stimuli in parameter space.To obtain a V1-like physical similarity measure, we applied a set of Gabor filters to the images and then computed pixelwise similarities between the images. This analysis was motivated by the fact that V1 cells exhibit tuning profiles that are well-described by Gabor filters ,43. Each


In this study we investigated tumour growth in relation to the immunohistochemical expression of p53 and bcl-2 and to patient survival data in 33 operated hepatocellular carcinomas (HCCs). In order to estimate the growth, a growth index, based on the degree of cell proliferation, apoptosis and necrosis, was calculated for each tumour. Cell proliferation was determined immunohistochemically by the number of proliferating cell nuclear antigen (PCNA)-positive cells in tumours, the extent of apoptosis was determined by counting the number of cells labelled by the in situ 3'-end labelling technique and tumour necrosis was estimated as the percentage of necrotic areas in haematoxylin--eosin-stained tissue sections. In our analysis we found that the survival of patients with HCCs showing a high growth index (i.e. tumours showing a high proliferation and simultaneously a low degree of apoptosis and necrosis) was significantly shorter than with other patients . When analysed separately, cell proliferation, apoptosis or necrosis did not show any significant association with survival. p53 positivity was found in 8/33 (24%) of tumours. There were significantly more p53-positive cases in tumours with a high growth index  suggesting that dysfunction of the p53 gene may affect tumour growth. p53-positive cases did not, however, have a significantly shorter survival time than p53-negative cases . bcl-2 positivity was found in only 1/33 (3%) of the HCCs. Thus bcl-2 overexpression does not seem to play an important role in hepatocellular carcinogenesis. In summary, our results suggest that in HCCs a compound score based on the evaluation of the degree of cell proliferation, apoptosis and necrosis is a biologically more relevant prognostic indicator than any of its composite parameters alone.


A total of 169 colorectal adenocarcinomas, obtained from patients with a median follow-up of 6.5 years, were studied with immunohistochemical staining on cryosections using a monoclonal anti-tenascin antibody to evaluate the possible association between the staining patterns and tumour stage, tumour differentiation and survival. We found two different staining patterns in the tumour stroma--a diffuse stromal fibrillar staining in 92 out of 169 (54%) tumours and a subglandular staining in the remaining 77 tumours. When the entire group of patients (P < 0.01) and the group of potentially cured patients (P < 0.03) were analysed univariately, it was found that diffuse stromal fibrillar staining was associated with a shorter survival time than subglandular staining. In a multivariate analysis, the Dukes' stage and age were independent prognostic factors, whereas the tenascin expression did not retain a clear independent relationship to survival (P = 0.06). Hence, it appears that the tumour expression of tenascin may be a potential prognostic marker in colorectal cancer, in so far as a diffuse stromal fibrillar staining pattern seems to indicate an increased risk of poor outcome. However, after adjustment for age and Dukes' stage, the additional prognostic value of tenascin remains to be established in further analyses.


The fine mapping and deciphering of specific cancer phenotypes is taking advantage of molecular-profiling studies based on genome-wide approaches. Currently, high-throughput methods such as array-based comparative genomic hybridization (CGH array), single nucleotide polymorphism array (SNP array), expression arrays and ChIP-on-chip arrays are available to study mutant p53-associated alterations in human cancers. Here we will mainly focus on the integration of the results raised through oncogenomic platforms that aim to shed light on the molecular mechanisms underlying mutant p53 gain of function activities and to provide useful information on the molecular stratification of tumor patients.Cancer is caused by the spatial and temporal accumulation of alterations in the genome of a given cell. This leads to the deregulation of key signalling pathways that play a pivotal role in the control of cell proliferation and cell fate. The p53 tumor suppressor gene is the most frequent target in genetic alterations in human cancers. The primary selective advantage of such mutations is the elimination of cellular wild type p53 activity. In addition, many evidences  Structural studies have allowed the classification of p53 mutant proteins in two major categories [in vitro and in vivo studies that show how mutant p53 proteins confer transformed properties to cell cultures and increased tumorigenicity in mice [The p53 tumor suppressor protein is a sequence-specific transcription factor that is present in a latent form under normal conditions and becomes activated by a variety of stress signals, such as DNA damage, oncogene activation and improper mitogenic stimulation. p53 acts as a potent transcriptional activator through the binding as a homotetramer to specific sequences, named p53 responsive elements (p53RE). Activated p53 modulates the expression of a large set of target genes involved in many cellular processes, including cell cycle arrest, DNA repair, apoptosis and senescence. Growth arrest or cell death prevents damaged DNA from being replicated; thus suggesting a role for p53 in the maintenance of genome integrity -5. p53 itegories ,12: a) D in mice ,14. It h in mice ,16. p53- in mice ,17. Inde(a) mutant p53 can bind to DNA through the association with DNA binding proteins and transcriptionally activate specific target genes using its functional transactivation domain (TAD). In support of this molecular mechanism, it has been reported that human tumor-derived p53 mutants, whose TAD was inactivated by site-directed mutagenesis, lost the ability to increase tumorigenicity in vitro and in vivo [(b) recent work by Di Agostino et al. has shown that mutant p53 proteins physically interact in vivo with the transcription factor NF-Y, whose DNA binding consensus are present in the regulatory regions of many key genes involved in the regulation of the cell cycle. Mutant p53 can be recruited in vivo onto the promoter of NF-Y target genes, as a member of a large transcriptional complex that also includes histone acetyltransferases (HAT). Of note, mutant p53 favours the selective recruitment of HAT in response to DNA damage, thereby dictating the transcriptional activation of its target genes [ (c) mutant p53 binds to and sequesters proteins whose function is required for anti-tumor effects such as apoptosis or growth inhibition. Interestingly, it has been reported that human tumor-derived p53 mutants can associate with p73 and p63 and interfere with their transcriptional activity and ability to induce apoptosis when co-expressed in transient transfection assays  [To date, we can depict the three following molecular scenarios to explain gain of function of mutant p53 proteins.  in vivo , 19-21; et genes . (c) mut -26.in vitro and knock-in models in vivo. However, as mentioned above, mutations can occur within the entire DBD of the protein. The activity of a large number of p53 mutants has been analyzed by Kato et al. [Many of the studies exploring the molecular mechanisms that underlie gain of function of mutant p53 proteins have mainly characterized the activities of p53 hot spot mutations. These mutants display an oncogenic potential by themselves, as demonstrated by transformation assays o et al. . This ano et al. ,17,28 ano et al. .Since p53 mutations of the DBD are selected in cancer, it is reasonable to hypothesize that hot spot mutations are directly selected because they represent the driving force during the neoplastic transformation. The remaining p53 mutations are selected following a second mutational event that might be permissive for mutant p53 gain of function activities. Cell-type specificity for gain of function of mutant p53 might explain why certain p53 mutants do not display any activity when exogenously overexpressed in cell systems different from those where the mutation was originated. et al. [Since the first description of a p53 mutation in human cancers in 1989, several thousands of papers have described clinical studies in which p53 has been tentatively linked to the response to treatment (predictive power) or patient survival (prognostic power). The usefulness of these studies was questioned in terms of strategy  and methodology ,31. Indeet al.  have shoet al. -36. Now in situ hybridization, Sigurdsson et al. [et al. [et al. [et al. [The genome of cancer cells is thought to be unstable as they often harbour many chromosomal abnormalities. Due to the role of TP53 in maintaining genomic stability, its inactivation profoundly impairs repair of DNA damage and could promote genomic alterations. Several research groups have reported a correlation between genomic instability and TP53 mutations in breast cancer tissues. Using fluorescence n et al.  have sho [et al.  combined [et al. . They fo [et al.  and Klei [et al.  performe3). Currently, high throughput methods such as array-based comparative genomic hybridization (CGH array) and single nucleotide polymorphism array (SNP array) are available to study mutant p53-associated alterations in a genome-wide fashion. Array-CGH is an established high-resolution method to study the whole genome for chromosomal amplifications and deletions . One lima) cell lines overexpressing different mutant forms of p53 have been generated to analyze the impact of mutant p53 on cell transcriptome; b) tumor samples with characterized p53 status (mutated or wild-type) have been examined by microarray analysis for their global gene expression. The latter has been compared with that of normal counterparts to define  clusters of genes associated with the mutated status of p53.Two different approaches have been followed to identify mutant p53-associated gene-expression signatures: et al. report that the most obvious difference related to the ectopic expression of two diverse p53 mutants (R175H and A138P) is that R175H retained less wild-type transcriptional regulatory events than A138P. A long list of mutant p53-regulated genes has been obtained from microarray analyses, suggesting the involvement of mutant p53 in several cellular processes ranging from transcriptional and translational regulation, signal transduction, cell motility, invasion and metastatization. This list obviously includes some of the genes previously characterized as mutant p53 targets. Importantly, all the identified genes provide evidence for the potential disclosure of new molecular targets and for substantial insights into the molecular mechanisms underlying gain of function activities of mutant p53.A number of studies employed microarrays to evaluate global gene expression in H1299 lung cancer cells expressing p53-175H, p53-273H and p53-281G -47, p53-in vitro models of cell lines overexpressing p53 mutants, the impact of a mutated p53 on the transcriptome has also been evaluated through in vivo microarray analyses. As mentioned above, the mutational status of p53 is prognostic in many malignancies. In breast cancer, p53 mutations are associated with worse overall and disease-free survival rates and have been implicated in resistance to anticancer therapies. Miller et al. [In addition to the r et al.  reportedr et al. ) where pin vivo analyses in identifying gene expression signatures associated with wild type or mutant p53, unfortunately these studies are unable to assess whether the characterized genes are really dependent on the presence of mutant p53 or whether they merely represent the result of a loss of wild-type p53 activity. The data sets derived from microarray analyses on cell lines overexpressing p53 mutants, enclosing the putative genes responsible for gain of function, need to be matched with those derived from in vivo analyses to define which of the transcripts are truly expressed in cancer tissues carrying p53 mutation. Up until now few p53 mutations have been analyzed in vitro for their ability to transcriptionally affect gene expression. Further microarray analyses employing additional p53 mutants to be compared with those performed on tumor specimens with known p53 status might unveil important molecular details on the gain of function properties of the different mutant p53 proteins present in human cancers.Despite the efficacy of these via interaction with other transcription factors is strongly supported by the work of Di Agostino et al. [Little is known about the mechanisms through which mutant p53 proteins, that have lost the ability to bind DNA in a sequence-specific manner, can achieve the specificity in gene regulation. Chromatin Immunoprecipitation experiments have shown that mutant p53 can be found onto the regulatory regions of putative target genes, but despite extensive efforts, the identification of the specific DNA binding consensus of mutant p53 has not yet been found. It has been proposed that mutant p53 can be recruited to its putative target promoters through the physical interaction with sequence specific transcription factors whose specific DNA binding consensus are present on those regulatory regions. Sp1 and Ets, which were shown to cooperate with mutant p53 in transcriptional regulation -57, are o et al.  which hain vivo binding sites are outside the predicted promoter regions of given genes. It has been shown that the binding locations of NFkB, cMyc, Sp1, p53, CREB are found within both coding and non-coding regions. More binding sites than those predicted were found and of those only a relatively small fraction of the sites occurred in genomic regions that would typically be considered “promoters”. The use of tiling arrays in the study of mutant p53 transcriptional activity will overcome the difficulties existing in the prediction of the binding sites for this oncogenic transcription factor. In addition, mutant p53 has been reported to bind a wide range of DNA secondary structures. For example, mutant p53 was shown to preferentially bind matrix attachment regions (MARs) [Due to the absence of a canonical consensus sequence for mutant p53, the only approach that seems truly promising for the precise deciphering of its transcriptional code is the use of whole-genome tiling arrays, designed to interrogate an entire genome in an unbiased fashion, for ChIP-chip analysis. ChIP-chip (Chromatin Immunoprecipitation-chip) permits a simultaneous analysis of the occupancy of a specific transcription factor on thousands of its target genes. Briefly, crosslinked chromatin (DNA/protein) complexes are extracted from the analyzed cell culture or from a tissue and sheared down to relatively short fragments (500-1000 bp). The chromatin fragments containing the transcription factor of interest are then immunoprecipitated using a specific antibody and the target sequences are subsequently identified through hybridization to DNA microarrays. At a high probe tiling resolution multiple overlapping probes may contain the actual transcription factor binding motif and thus enable a fine mapping of the binding site to a resolution of less than 25 bp. Apart from the most recent generated tiling arrays, different kinds of microarrays have been generated for ChIP-chip analysis. Initial studies were performed using slides carrying spotted oligonucleotides or PCR products from selected predicted promoters. However these arrays are unable to interrogate the entire genome and are unable to address the possibility that transcription factors might bind to other locations. Recent studies using tiling arrays interrogating entire chromosomes show that a large fraction of s (MARs) . MARs ar4).The ChIP-chip analysis of chromatin initially immunoprecipitated with antibodies against mutant p53 and successively with antibodies directed to the various transcription factors that mediate mutant p53’s binding to its target genes (Re-ChIP) is likely to lead to the identification of subgroups of target genes controlled by different mutant p53-TF protein complexes (Fig. Genome wide analyses are turning out to be powerful instruments in the molecular stratification of cancer patients. The complexity of the data raised through the oncogenomic approaches calls for the integration of multidisciplinary expertises and potentially accounts for the fine deciphering and molecular characterization of specific cancer patient phenotypes. An understanding of the molecular events regulating and/or governed by gain of function mutant p53 proteins might lead to the disclosure of cancer signalling pathways to be tackled with novel anticancer therapies.


Science magazine , p53 may also function in a                            sexually antagonistic manner, limiting the life span of adult female flies                            while promoting the longevity of adult males.                                        New insights into the roles that p53 plays in animal                            aging are provided by Waskar et al. in this issue of Drosophila life span                            studies have primarily been concerned with the effects of reducing p53 function                            using DN p53 alleles , to dietary restriction                            [reviewed in 20], to mitochondrial gene manipulations ,21.  WesThe slight elevation in p53 may help maintain genomic                            fidelity during early life, resulting in healthier, longer-lived adults, at the                            cost of developmental speed and elevated energy requirements.  In addition,                            increased p53 activity during development may have resulted in a decrease in                            body mass/size of the animals.   Although the relationship between body size                            and longevity is complicated , this maIt has also not escaped our attention that p53                            regulates mitochondrial electron transport chain (ETC) activity .  Our owA final thought worthy of consideration is that it is at least possible that the effects                            observed with neuronal expression of DN p53 transgenes could be due to the                            transgenic p53 having effects distinct from inhibition of endogenous p53.  The                            DN p53 has either a mutation in the DNA binding domain, or it is a truncated                            version of the protein, but these can presumably still carry out some of the                            functions of p53, such as localization to the mitochondria,                            or direct regulation of autophagy.  In addition, while the adult fly is usually                            referred to as a ‘post-mitotic' system, there are dividing stem cells and                            progeny cells in the gonad, gut, and malphigian tubule -28.  Cha


Survival analysis of 144 histologically confirmed cases of colon carcinoma diagnosed in a 12 year period (1971-82) at the Tampere University Hospital was performed to test the hypothesis that p53 overexpression is associated with a poor clinical outcome. Immunohistochemical staining of paraffin-embedded sections using a polyclonal antibody CM-1 against p53 protein was performed to identify aberrant expression of the p53 tumour-suppressor gene. Sixty-nine per cent of the tumours (100/144) showed overexpression of the p53 protein. The prevalence of p53 overexpression was independent of age and sex of the patient and subsite of the tumour, but was slightly, although not statistically significantly, higher in advanced than in localised tumours. Overexpression was associated with a higher S-phase fraction. Some indication of a larger proportion of aneuploid tumours among those with overexpression was also observed, although this finding did not reach statistical significance. Significantly reduced patient survival for tumours with p53 overexpression was found. Patients with p53-overexpressing tumours had a corrected 5 year survival rate of 37% compared with 58% among patients whose tumours had normal expression of p53. The corresponding 10 year rates were 34% and 54%. In the multivariate analysis using a Cox model, the survival difference was independent of age and sex of the patient, as well as of subsite and stage of the tumour. Furthermore, the effect of p53 overexpression remained after controlling for flow cytometric parameters in an analysis of a subset of tumours. Thus, p53 overexpression appears to be a useful prognostic indicator in colon carcinoma.


Rb gene deletion in concert with inactivation of Rb family members have recently been developed. These new Rb knockout models of retinoblastoma provide excellent tools for pre-clinical studies and for the exploration of the genetics of tumorigenesis driven by RB inactivation. This review focuses on the developmental consequences of Rb deletion in the retina and the genetic interactions between Rb and the two other members of the pocket protein family, p107 (Rbl1) and p130 (Rbl2). There is increasing appreciation that homozygous RB mutations are insufficient for human retinoblastoma. Identifying and understanding secondary gene alterations that cooperate with RB inactivation in tumorigenesis may be facilitated by mouse models. Recent investigation of the p53 pathway in retinoblastoma, and evidence of spatial topology to early murine retinoblastoma are also discussed in this review.Novel murine models of retinoblastoma based on  The RB gene was the first tumor suppressor gene cloned [RB is now known to be mutated not only in retinoblastoma but in many cancers, including osteosarcoma, soft tissue sarcoma, small cell lung, breast, brain, prostate, leukemia, and other sporadic cancers [RB mutation are at increased risk for many of these tumor types and for melanoma [RB is mutated widely in various human tumor types, the retina is exquisitely sensitive to cancer upon RB loss and the reason for this is tissue sensitivity is still poorly understood. Our current knowledge of RB function places this gene in pathways central to growth control not only in the retina, but in all tissues. RB is a prototypic tumor suppressor gene that encodes pRB, a nuclear phosphoprotein implicated in cell cycle control, differentiation, apoptosis, and many other biological processes. Despite intensive study of this tumor suppressor gene since its cloning, the critical functions of pRB important for tumor suppression are still being unraveled.Retinoblastoma is the most common pediatric primary intraocular tumor. Study of retinoblastoma, a malignant cancer that occurs unilateral or a bilateral form, led to the "two-hit hypothesis" now applicable to other inherited cancer syndromes . In bilamutation ,3. In spe cloned , and RB  cancers -9. Patiemelanoma -12. Theymelanoma ,14. EvenC elegans SynMuvB genes  and Drosophila melanogaster dREAM  complex members. There is also evidence that pRB may bind pro- differentiation factors, such as CBFA1, MITF, C/EPBs and MyoD to promote cell type specific differentiation [RB family members that contribute to retinoblastoma are not well understood. Here, we focus on insights that study of the murine Rb family function in the retina in vivo have for understanding human retinoblastoma.pRB is one member of the "pocket proteins", a family that also includes p107 and p130. These proteins are named owing to the presence of a conserved pocket domain, a region that binds E2F transcription factors and various critical protein interactors, many containing an LxCxE motif. The three pocket proteins exhibit similarities, particularly in the A and B pocket, but there are also clear differences. p107 and p130 have much closer similarity to each other than either to pRB. The pocket proteins differ in their expression patterns, with p107 highly expressed in cycling cells, and p130 expressed at higher levels in cells that have exited the cell cycle . Criticantiation -25. Datain vivo using plasmid electroporation, or, if stable expression over many cell divisions is needed, viral vectors. These properties make the retina an ideal system to study not only development, but also cancer initiation. For childhood cancers these processes are clearly intertwined.The neural retina is composed of seven classes of cell types derived from a common population of progenitors, with cell bodies organized into three nuclear layers. The outer nuclear layer contains cell bodies for rod and cone photoreceptors; the inner nuclear layer contains bipolar, amacrine, horizontal and Müller glia cell bodies, and the ganglion cell layer is made up of ganglion and amacrine cells Fig . DevelopRetinoblastoma is a pediatric cancer that is typically detected in the first few years of life but can arise during embryonic retinal development . The ideLineage tracing studies in the normal rodent retina do not support the existence of specific progenitors that generate only rod and cone photoreceptors . InsteadThere are limitations to using human tumor samples to identify the tumor cell of origin. In the establishment and long term passaging of cell lines, genetic alterations may occur that alter the cell's properties. Also, accessing early retinoblastomas is rarely possible, limiting analysis to late stage tumors that may have quite different characteristics compared to the earliest lesions. Use of model systems can help in assessing the early events in tumorigenesis.LH-beta T-Ag model expresses the SV40 early region under control of the Luteinizing hormone beta promotor [LH-beta T-Ag line was in part a consequence of the genomic site of integration directing transgene expression to cells in the retina, a tissue in which luteinizing hormone beta is not normally expressed. Both Large-T and small-t oncoproteins are encoded by the SV40 early region and may contribute to tumorigenesis. Large-T Ag binds the pRB family, p53, and other proteins, such as CBP/p300. Small-t Ag also contributes to transformation in cell culture, in part through binding the phosphatase pp2A [LH-beta T-Ag model, retinoblastoma with neuronal characteristics and histological similarities to human retinoblastomas emerged. Transgenic mice expressing T-Ag/t-Ag from the IRBP promoter have also been generated and develop retinoblastoma [PNMT-T-Ag) transgenic mice develop retinal tumors that arise at the retina periphery. This spatial localization of retinal tumors in mice occurred despite T-Ag/t-Ag being expressed in amacrine and horizontal cells in both central and peripheral retina [A number of models of retinoblastoma have been developed that have made use of transgenic expression of specific viral oncoproteins. The most widely used such model, the promotor . The devase pp2A . In the blastoma -51. IRBPblastoma , raisingl retina . The autl retina  but it iIRBP-E7 mice underwent photoreceptor degeneration, while IRBP-E7; p53-/- animals were prone to tumors of the retina that arise in the photoreceptor layer [p53 loss suppressed E7-dependent apoptosis, leading to retinoblastoma, p53 loss may contribute in other ways, as these tumors are now recognized to emerge in the midst of high levels of apoptosis and photoreceptor degeneration even upon p53 deficiency [p53 independent of apoptosis are critical for tumor suppressor ability [p53 in retinoblastoma discussed further on in this review. Whether the tumors in the IRBP-E7;p53-/-, LH-beta T-Ag and PNMT-Tag models exhibit a similar cell of origin or if these models represent distinct tumors of different cell types remains to be determined.Papillomavirus E7 protein binds to the pocket protein family, and other effectors, but does not inactivate p53. or layer . While ificiency . This di ability . This isRb but not other effectors of viral oncoproteins has been a clear goal for improvement of mouse retinoblastoma models. The main disadvantage of transgenic models expressing viral oncoproteins concerns the pleiotropic effects of the oncoproteins, which impact upon multiple signaling pathways. These oncoproteins may also have important targets not yet identified. Furthermore, tumors in which p53 is functionally ablated through T-Ag expression are not ideal for testing therapies, whose effectiveness may depend on the p53 pathway being intact [Use of transgenic retinoblastoma models has been valuable for the testing of preclinical therapeutics; however, the development of models that target g intact ,59.Rb deletion took a number of years. Three groups inactivated Rb in the mouse germline, resulting in embryonic lethality in Rb-/- animals and the development of pituitary and thyroid tumors in Rb+/- animals [Rb heterozygotes, in stark contrast to the high retinoblastoma incidence in humans with germline RB mutation. The Jacks and the Berns groups both generated chimeric animals in which Rb-/- cells contributed to the retina, but retinoblastomas still did not form [Rb and p107 came the realization that mice were capable of developing retinoblastoma dependent on Rb deletion [Rb-/-; p107-/- cells to the developing embryo.The generation of mouse models that, like human retinoblastoma, exhibit  animals -62. Retinot form ,64. Withdeletion . HoweverCre-lox technology and Cre-expressing transgenic animals with expression in the developing retina paved the way for the generation of breedable Rb-knockout models of retinoblastoma. The first attempt, based upon the previous descriptions of photoreceptor characteristics in retinoblastoma used Cre regulated by the IRBP promoter together with RbLox alleles to drive Rb deletion in the photoreceptor compartment and in other tissues [p107 and p53 deficiency, indicating that the tumor-initiating cells in Rb/p107 DKO chimeras were not being targeted when these genes were deleted in developing photoreceptors. Breedable Rb-knockout models of retinoblastoma were independently generated using Cre transgenics with expression driven by Nestin, Chx10 and Pax-6 promoter in progenitor and other cells [p107 first revealed in chimera studies. Mouse models have now revealed that inactivation of the third pocket protein, p130, also cooperates very strongly with Rb deletion to promote retinoblastomas in both Rb knockout and chimeric mice [Rb-knockout and chimeric retinoblastoma models is provided in Table Rb knockout models of retinoblastoma now provide excellent tools to hone in on the cell of origin and interrogate the molecular genetics of retinoblastoma progression.Nevertheless, the advent of  tissues . These mer cells -70. New ric mice ,69,71. ARb in the retina. Homozygous germline Rb deletion in mice leads to embryonic lethality in midgestation, caused by placental deficiency [Rb has been inactivated in the retina using a number of different systems that bypass the lethality associated with systemic Rb deletion. Rb-/-;+/+ chimeric animals and various Cre-expressing transgenics, including Chx10-cre, Pax6 α-enhancer Cre (α-Cre) and NesCre1, in concert with an Rb lox/lox allele exhibit Rb deletion in the retina [Rb at different times and in different cells. Chx10-Cre is expressed in a mosaic pattern in progenitor cells, and in adults is expressed in most bipolar cells and in some Müller cells [NesCre1 is active in the optic vesicle by E9.5 and exhibits different expression upon paternal vs. maternal inheritance of the transgene [Cre transmission and is accompanied by lethality at birth in Rblox/lox animals. A mosaic pattern of Cre expression is observed with maternal NesCre1 inheritance allowing for the survival of some animals on an Rblox/lox background. α-Cre is expressed by E10.5 in distal progenitor cells, with a dorsal gap in expression [α-Cre at late stages both in central and peripheral retina. A synthesis of the data from many groups using different systems has revealed common phenotypes that have implicated Rb in control of proliferation, apoptosis and differentiation in the developing retina  to proliferate inappropriately [Rb loss [Rb-deficient retina ultimately exited the cell cycle or died such that by PND21, Rb-/- cells no longer proliferate, and retinoblastoma never develops [Rb deletion differentially affects specific cell types. Bipolar cells, ganglion cells as well as the majority of rod photoreceptors are lost in α-Cre Rblox/lox mice. Photoreceptor apoptosis is specific to rods, as cells expressing cone markers were present in normal numbers, revealing specificity to rod cell death [Rb -/-; +/+ chimeras, in which there was a low, but not null, contribution of Rb-/- cells to the adult retina [In embryonic stages of retinal development, the first indication that there were cell cycle defects upon to E18.5 . Ectopic mutants . Ectopicre1 mice ,69. Normextended -69. Notapriately . However[Rb loss . Importadevelops ,68. Intell death . The celt retina .Rb loss has been associated with differentiation defects. Rod photoreceptors in Chx10Cre Rblox/loxmice have been reported to exhibit an altered pattern of heterochromatin suggestive of rod maturation defects [Rb loss, but it is difficult to distinguish between downstream effects of Rb loss on cell cycle control vs. direct effects on differentiation. Interestingly, defects in rod development were not found when IRBP-Cre was used to drive Rb deletion in photoreceptor transition cells [Rb-/- explant cultures or in Chx10Cre Rblox/lox retinas [Chx10Cre Rblox/lox animals [NesCre1, Chx10Cre and α-Cre Rblox/lox mice all exhibit the presence of ectopic cells in the outer plexiform layer that extend to the photoreceptor layer [Rb-mutant retinas have also been noted. Rb-deficient starburst amacrine cells were found to lack the expression of markers associated with their maturation [Rb-deficient horizontal cells exhibit enlarged abnormally shaped nuclei, reminiscent of polyploid cells that have undergone endoreduplication in other Rb-deficient cell types, such as skeletal muscle and Purkinje cells [Rb-deficient horizontal cells involving ectopic processes that extend into the outer nuclear layer have also been described [Finally,  defects ,77,78. Ion cells . One expor layer ,78, and turation . Also, aje cells ,69. DefeRb deletion using chimera systems and different Cre-transgenic lines . A simiRb loss are quite similar across studies of chimeras, α-Cre and NesCre1 mice, with milder phenotypes observed upon Chx10Cre-mediated Rb inactivation. Differences highlight the need to assess the consequences of Rb deletion at different times across different cell types, and the need to better understand the extent to which phenotypes are cell autonomous. Some of these aims may be achieved by expressing Cre in a temporally and dosage controllable fashion using either viral delivery or tamoxifen-inducible Cre-ER transgenics with Cre-ER expression driven by cell-type specific promoters.The phenotypes upon E2f family members in mediating Rb dependent phenotypes is of great debate. Analyses of E2f contribution to Rb-mutant developmental phenotypes have been performed in the whole animal through compound mutant studies, but the non-cell autonomous consequences of placental dysfunction in Rb-/- embryos complicate the interpretation of these results. Compound mutant analysis specifically in the developing retina provides an excellent system to probe these interactions. The importance of E2f1 in mediating the effects of Rb loss was nicely shown by the Bremner group as compound mutation of Rb and E2f1 rescued much of the proliferation and apoptosis associated with Rb deletion using the α-Cre Rblox/lox system, and even restored response on the electroretinogram, a measure of retinal electrical activity in response to light [E2f1 loss did not rescue differentiation defects in Rb-deficient starburst amacrine cells. In contrast, compound mutation of Rb and E2F3a led to a rescue in starburst amacrine cell development suggesting that Rb/E2F3a interactions promote the differentiation of these cells. While pro-differentiation effects of pRB are often thought E2f-independent, this is one example of pRB promoting the differentiation of a cell type mediated through E2F.The importance of specific to light . InteresRb loss and apoptosis in the developing retina. The above study showed that apoptosis is E2f-dependent, but the E2f effectors that contribute to apoptosis remain to be identified. We were interested in whether p53 was downstream of Rb inactivation in the retina [NesCre1 Rblox/lox p53-/- double mutants did not exhibit rescue of apoptosis in the embryonic retinal ganglion cell layer. Also, the loss of photoreceptors was not rescued in NesCre1 Rblox/lox p53-/- animals exhibiting deletion of Rb in a mosaic pattern. Rb loss has been linked to apoptosis as pRB is an anti-apoptotic protein and many E2F target genes are apoptosis effectors [Rb deletion is abrogated [p53-independent apoptosis downstream of Rb loss in the retina will be an important goal for broadly understanding the events that synergize with Rb loss to promote cancer.It will be critical to dissect the pathway between e retina . Compounffectors . Whetherbrogated . ElucidaRb as well as p107 or p130 loss clearly illustrate tumor suppressive activity of p107 or p130 in the context of Rb deficiency. It is not clear why retinoblastoma manifests itself in mice only if p107 or p130 is inactivated in addition to pRB. p107 expression is upregulated in murine retinas lacking Rb but not in human retina explants with knockdown of RB expression, raising the possibility that p107 upregulation prevents retinoblastoma in mice. [Rb-/- murine retinal cells has been reported only at postnatal stages of retinal development, or in explant cultures differentiated in vitro to recapitulate postnatal stages of retinal development and not in embryonic Rb-/- retina [Rb-/- retinas at late time points in retinal development [p107 is an E2f target gene typically expressed in proliferating but not post-mitotic cells, the p107 increase has not been uncoupled from the cell cycle phenotypes associated with Rb loss at late stages in retinal development. Other Rb targets, such as Cyclin E are found strongly upregulated at both embryonic and postnatal stages of retinal develoment in Rb-deficient retinas (D.M. unpublished). It is also possible that functional overlap among the pocket proteins without increased p107 or p130 expression is critical for suppressing retinoblastoma in mice. Indeed, p130 is a stronger suppressor of retinoblastoma than p107 despite no alterations in p130 overall levels upon Rb loss [Rb loss) and many upstream regulators of pocket proteins are known to be important for cell cycle control in the retina. Cyclin D1-/- retinas are hypoplastic, corresponding to altered pocket protein phosphorylation [Rb family inactivation and this decrease may contribute to retinoblastoma suppression in mice through regulation of pocket protein activity [Rb or both Rb and p130 compared to controls. p27-/- retinas exhibit an extension in the period of retinogenesis, implicating p27 in cell cycle exit in a subset of cells [(Ink4d) loss also extends the normal period of retinal proliferation in the retina [p107 or p130 being intact will be important to elucidate. It is likely that complex differences between mice and humans in the levels and activity of multiple regulators of the RB family contributes to the species difference in retinoblastoma susceptibility. Thus, a broader understanding of the regulation of pRB, p107 and p130 in the cell from which retinoblastoma is derived is needed.Data from chimeras and conditional mutants exhibiting inactivation of in mice. . However- retina ,84. Impoelopment -69. As p Rb loss ,69. Factrylation . High cyof cells . p19 that must be overcome.Long latency of tumorigenesis and incomplete penetrance suggests that Rb and p130 in the retina have revealed roles for p130 in tumor suppression and retinal development in α-Cre Rblox/lox p130-/-, NesCre1 Rblox/lox p130-/- and in Rb-/-p130-/- chimeras [NesCre1 Rb/p107 DKO retinas, histological defects were not found in NesCre1 Rb/p130 DKO embryonic retinas [α-Cre Rb/p130DKOs, relative to α-Cre Rblox/lox single mutants, revealing cooperation between Rb and p130 in controlling cell cycle exit in late stages of retinal development. By PND21, Rb-/- cells have all exited the cell cycle, but cells in α-Cre Rblox/lox p130-/- retinas continue to inappropriately undergo S-phase entry and exhibit the presence of cells with large aberrant nuclei [α-Cre Rblox/lox p130-/- retina. The extent to which this cell death is cell autonomous has not yet been determined and it is possible that effects of Rb and p130 loss on certain cell types may lead to the death of other cells through secondary effects. Cell death is more pronounced in these animals than α-Cre Rblox/lox p107-/- retinas, although some cells, including horizontal cells, Müller cells and rare amacrine cells survive at PND21. The survival and increase in horizontal cells at this stage is a specific effect of Rb and p130 loss not observed upon Rb and p107 deletion.Interactions between chimeras ,69,71. It nuclei . ProlifeRb and p130, NesCre1 Rblox/lox p130-/- and α-Cre Rblox/lox p130-/- mice all develop retinoblastomas in which marker expression is heterogeneous and includes the expression of amacrine markers [α-Cre Rblox/lox p130-/- and NesCre1 Rblox/lox p130-/- retinoblastomas . Finally, secondary alteration analysis reveals that tumors in both models undergo N-myc amplification, suggestive of a common pathway to transformation [Despite differences in kinetics and cell type composition, abundant data supports a model of a common cell of origin in rosettes . Rb/p107ormation .Rb and p130 deletion leads to an increase in Calbindin-positive horizontal cells [Chx10Cre drives Rblox/lox deletion in p130-/-, p107+/- animals, termed "p107 single" [PNMT-Tag mice, functional pRB family ablation in horizontal cells led to distinct effects depending on retinal topology [PNMT-Tag expression led to tumors in peripheral retina but not in central retina, where horizontal cell death eventually occurred; it will be important to carefully examine whether peripheral vs. central horizontal cells also respond differently in p107-single mice. While Dyer et al (2007) conclude that the cell of origin is a horizontal cell, the possibility of another cell type that generates the late-stage tumors cannot be ruled out. The metastatic cells in p107-single retinoblastomas did not resemble horizontal cells either in ultrastructure or in cell marker expression, suggesting that the retinoblastomas could derive from cells that can produce both differentiated horizontal cells and non-horizontal metastatic cells or that there may be a mixed tumor phenotype with differing cells of origin. This issue is also complicated by the labeling of amacrine cell subsets by horizontal cell markers such as Calbindin and Prox1 [p107-single studies clearly show that horizontal cells can divide and raise a very interesting hypothesis regarding a possible cell of origin to the tumors, deletion of these genes specifically in differentiated horizontal cells will be necessary to prove this hypothesis.While al cells , the hor single" . The auttopology . PNMT-Tand Prox1 . OverallRB is much more likely to occur in a proliferating cell than a post-mitotic cell as DNA replication and cell division can promote an initial gene mutation or loss of heterozygosity. Despite differences in the cell type composition of murine vs. human retinoblastomas, the cell of origin in human and mouse retinoblastoma may both be a similar multipotent cell. That is, the requirement to inactivate p107 or p130 in addition to Rb in the mouse would be expected to mask potential for the murine retinoblastoma cells to exhibit photoreceptor character, as there is widespread death of both rods and cones in the absence of Rb and p107 or Rb and p130 when deleted in early progenitors using Pax6 alpha Cre [Rb and p107 or Rb and p130 deletion, i.e. amacrine, Müller and horizontal cells. The specific composition of tumor cell types may be a result of the effects of p107 or p130 on the survival, proliferation and differentiation of the retinoblastoma cell of origin and its daughters.Differentiated cells are not strong candidates for the cell of origin of human retinoblastoma, given the evidence of multipotent potential to human retinoblastoma cells , althouglpha Cre ,68. Instpatched heterozygous mice, persistent proliferation of cells expressing the progenitor marker nestin was found at the extreme retina periphery in adults, suggesting that the hedgehog pathway may be important for the late proliferating cells [α-Cre Rblox/lox p130-/- or α-Cre Rblox/lox p107-/- retinas [Observations of early lesions at the extreme retina periphery in mouse retinoblastoma models raised the question of whether spatial differences in the localization of human retinoblastomas occur. Careful examination of retinoblastoma topology has revealed that retinoblastomas that arise in older children tend to occur at the periphery of the retina, while those that occur in younger children are typically central ,98. In hng cells . Furtherng cells -105. The retinas . Potenti retinas ). It wilRb family loss in mouse models suggests a model in which inactivation of RB allows the proliferative period of retinal development to be extended in the mutant cells. Upon mutation of both Rb and p107 or Rb and p130, this phenotype is exacerbated in mice. This model proposes that homozygous inactivation of RB must occur during the normal proliferative period of retinal development, and that this event increases the chance of subsequent mutations by creating a proliferative lesion balanced by cell cycle exit and differentiation, or cell death. The loss of both copies of RB may increase the rate of subsequent mutations, as was found in embryonic stem (ES) cells, in which loss of Rb led to a dramatic increase in the rate of chromosomal alterations that result in loss of a selectable marker [Rb can lead to chromosome segregation defects through misregulation of the expression of genes important for processes such as centrosome duplication [RB deletion during development undergoes the secondary mutations needed for tumor formation. In some cases, retinoblastoma may form from a benign precursor lesion, retinoma that exhibits some features of senescence and has recently been shown to exhibit less complex patterns of genetic alteration than adjacent retinoblastoma [RB inactivation. Specifically, is this a differentiation block, or must cell death or other processes be overcome? Examination of the nature of secondary alterations will be needed to shed light on this question.An examination of the effects of lication ,109, mitlication  or throulication . In humablastoma ,113. Intblastoma . One of RB mutation [RB inactivation is not sufficient for retinoblastoma, as cytogenetic studies have revealed consistent chromosomal abnormalities in addition to RB deletion [Rb and p130, array CGH revealed recurrent whole chromosomal gains at chromosome 1  and 12  [α-Cre Rb/p130 DKO mouse model. While alterations in regions syntenic to human 6p and 16q were not observed in α-Cre Rb/p130 DKO retinoblastomas, larger studies are needed, and it is possible that loss of p130 in the murine model may negate selection for 6p gain or 16q loss. Indeed, RBL2 (p130) lies at 16q12 and is a good candidate tumor suppressor in this region in humans [DEK as well as an interesting candidate, E2F3, which conceivably could antagonize p130 upon overexpression. Use of mouse models will certainly help in defining the critical genes in these regions, as mouse/human comparisons have revealed highly concordant genomic alterations in other tumor types and have allowed for the identification and validation critical cancer-contributing genes [The two-hit hypothesis suggests that the first few years following birth reflects the time-interval for homozygous mutation . Howeverdeletion . Early cdeletion -120, revdeletion . The vasan 2p24) . Such fin humans . Genes ing genes ,123. As ng genes , here, IN-MYC locus [N-MYC is the gene in the amplicon selected for increased copy number, as other genes are co-amplified, including the DEAD box gene DDX1. However, in α-Cre Rblox/lox p130-/- metastatic retinoblastomas (3 of 16 metastases), amplicons containing N-myc were also observed, ranging from 450 kb to 3.3 MB, and the only known gene in the minimal region of amplification was N-myc [N-myc/N-MYC is found more frequently that high level amplification. The fact that N-Myc amplification is enriched in metastatic α-Cre Rblox/lox p130-/- retinoblastoma suggests that this event contributes to formation of more aggressive tumors but how this is mediated is unknown. Genetic inactivation of N-myc in the developing retina has revealed a critical role for this gene in progenitor cell proliferation; this is in part mediated by regulation of p27 levels [Rb and N-myc in the developing retina and in retinoblastoma models should help us to understand why these mutations synergize in tumorigenesis and to identify the critical N-myc target genes.The most well characterized secondary alteration in human retinoblastoma is gain or amplification of a region of 2p that includes the YC locus ,124-126.as N-myc . This re7 levels . ExaminaMDM4 and GAC1, and they also identified a second common minimal region of gain at 1q22 [MDM4 is related to MDM2 and both genes cooperate to inhibit p53 function, with MDM4 an inhibitor of p53 transcriptional activity [MDM4 undergoes copy number increase and is overexpressed in retinoblastomas. They report that 65% of human retinoblastomas had extra copies of MDM4 and 10% exhibited extra copies of MDM2. Furthermore, transfection of MDM4 plasmid DNA into newborn retinas in vivo led to more rapid tumorigenesis in the α-Cre Rb/p107 DKO model. p53 is a good candidate effector downstream of MDM4 as homozygous p53 loss accelerated retinoblastoma development in vivo in Chx10-Cre; Rblox/lox p53-/-; p107-/- animals compared to animals with wild-type p53 [Rb on a p53-/- background, (NesCre1 Rb lox/lox p53-/- mice) retinoblastomas did not result [p53 loss can impact upon retinoblastomas that have already been initiated, but that inactivation of a second pocket protein in addition to Rb is still critical. Based on experiments in culture using retinal explants, the authors suggest that suppression of p53-dependent apoptosis is crucial [Rb exhibit very low levels of apoptosis [Rb-/- retinas in vivo . Importse model ,59. It ipatients . The lonRb and p130 have revealed secondary alterations similar to human retinoblastoma, suggesting that the steps to tumorigenesis in the murine and human retina are similar. Mouse models will be particularly powerful for testing which candidate genes are important for retinoblastoma progression. The novel models provide valuable tools to help us understand the in vivo function of pRB, to elucidate the properties of a cancer cell of origin and to test preclinical therapies. We are now poised to gain important insights into the steps between RB inactivation in a developing retinal cell and the progression towards retinoblastoma.Research into retinoblastoma has revealed a great deal about a malignant eye cancer, and has also led to many general insights into the general mechanisms of tumor suppression. Early genomic analyses of murine retinoblastoma lacking


We have previously shown that mutations in the N-ras gene occur more frequently in melanomas originating from sun-exposed body sites, indicating that these mutations are UV induced. To investigate whether sun exposure also affects p53 in melanoma, we analysed 81 melanoma specimens for mutations in the p53 gene. The mutation frequency is higher than thus far reported: 17 specimens (21%) harbour one or more p53 mutations. Strikingly, 17 out of 22 mutations in p53 are of the C:G to T:A or CC:GG to TT:AA transitional type, strongly suggesting an aetiology involving UV exposure. Interestingly, the p53 mutation frequency in metastases was much lower than in primary tumours. In the case of metastases, a role for sun exposure was indicated by the finding that the mutations are present exclusively in skin metastases and not in internal metastases. Together with a relatively frequent occurrence of silent third-base pair mutations in primary melanomas, this indicates that the p53 mutations, at least in these tumours, have not contributed to melanomagenesis and may have originated after establishment of the primary tumour. 1999 Cancer Research CampaignIn melanoma, the relationship between sun exposure and the origin of mutations in either the N-


Gene lesions of both ras(0%) and p53 (3%) were rare. While bcl-2 expression was not observed in any sample, bax expression was noted in 76% of samples and was associated with a significantly worse patient prognosis both overall (P< 0.005) and specifically in Chinese patients (P< 0.02). Apoptotic cells were found in 61% of samples, and were significantly associated with the presence of bax expression (P= 0.002), but not patient survival. These results suggest that prostate tumours from non-Japanese Asians are genetically distinct from prostate tumour found in both Japanese and Caucasian patients, and that treatment modalities may need to be tailored for specific population groups. © 2000 Cancer Research CampaignMolecular studies have suggested that ethnicity may play a significant role in prostate tumorigenesis, but no information exists for groups other than Caucasian or Japanese patients. We examined 62 archival samples of prostate tumours from Asians of non-Japanese origin for the over-expression of p53, for the possible presence of mutated


Gadd45 genes have been implicated in stress signaling in response to physiological or environmental stressors, which results in cell cycle arrest, DNA repair, cell survival and senescence, or apoptosis. Evidence accumulated implies that Gadd45 proteins function as stress sensors is mediated by a complex interplay of physical interactions with other cellular proteins that are implicated in cell cycle regulation and the response of cells to stress. These include PCNA, p21, cdc2/cyclinB1, and the p38 and JNK stress response kinases. What deterministic factors dictate whether Gadd45 and partner proteins function in either cell survival or apoptosis remains to be determined. An attractive working model to consider is that the extent of cellular/DNA damage, in a given cell type, dictates the association of different Gadd45 proteins with particular partner proteins, which determines the outcome. The cellular response to environmental and physiological stress is very complex, encompassing a myriad of molecular pathways, with a plethora of regulators and effectors. Both environment agents  and physAberrations in DNA repair pathways are the major molecular pathology in several cancer prone hereditary diseases . DefectsGrowth Arrest DNA Damage (Gadd) 45 genes play as sensors of cellular stress.Mammalian cells have evolved an elaborate defense mechanism to maintain genomic integrity by preventing the fixation of permanent damage from genotoxic stress. This includes activation of cell cycle arrest checkpoints at the G1/S and G2/M transitions -14 and aGadd45 genes were cloned in this laboratory ,25, in tEvidence accumulated in recent years implies that Gadd45 proteins function as stress sensors, is mediated by a complex interplay of physical interactions with other cellular proteins that are implicated in cell cycle regulation and the response of cells to stress Fig. .It was shown that all three Gadd45 proteins interact with PCNA -43, a nuAll three Gadd45 proteins were observed to interact with p21 ,43, a p5In normal cells p21 and PCNA are found in quaternary complexes that also include cyclins and cyclin dependent kinases (cdks) -52. It wInteraction of Gadd45 proteins with MEKK4, an upstream activator of the stress induced p38/JNK kinases, and the resulting stimulation of its kinase activity, has been implicated in stress induced p38 and JNK activation . EvidencGadd45 family members are rapidly induced by genotoxic stress agents ,64,65, acell cycle arrest[DNA repair[cell survival[apoptosis[senescence[susceptibility of cells for transformation in vitro and in tumor development in vivo[As detailed below, gadd45 genes have been implicated in the control of le arrest,28,38,58NA repair-41, cell survival,76-79, aapoptosis,43,80-82enescence, and sust in vivo,83.Inhibiting endogenous expression of gadd45a, gadd45b, or gadd45g in human cells by antisense gadd45 constructs was found to impair the G2/M checkpoint following exposure to UV radiation or MMS ,38,58, aEvidence was obtained that Gadd45a and Gadd45b function in DNA excision repair through their interactions with PCNA -41. FurtAmple evidence exists that Gadd45 proteins have a pro-apoptotic function. For example, it was observed that blocking MyD118 (Gadd45b) by antisense expression in M1 myeloblastic leukemia cells impaired TGFb-induced cell death, thereby implicating Gadd45b as a positive modulator of TGFb-induced apoptosis ,69. ConsIntriguingly, in apparent contradiction to the role Gadd45 proteins play in cell death, many observations are consistent with a role in cell survival. Clonogenic survival assays with gadd45a-/- MEFs  and RKO ARF, which is encoded by a partially overlapping alternative reading frame at the p16INK4A locus, has been implicated as a major mediator of replicative senescence. p19ARF binds directly to and sequesters MDM2, thereby inhibiting the ability of MDM2 to induce degradation of the p53 tumor suppressor protein [INK4A/pRB and p19ARF/p53 pathways is essential for oncogene-induced senescence. Studies with MEFs deficient for one or more gadd45 genes, notably gadd45a, have provided evidence that gadd45 genes play important roles in both replicative and oncogene mediated senescence [INK4A/pRB and p19ARF/p53 pathways remains to be determined.Senescence represents a physiological stress response associated with cellular aging (90). When primary mammalian cells are cultured in vitro they undergo a limited number of cell divisions and then arrest in a state known as replicative senescence. Such cells are irreversibly arrested in the G1 phase of the cell cycle and are no longer sensitive to growth factor stimulation ,86 & the & the86 nescence ,83. The The complex role of stress sensors in monitoring oncogenic stress in tumor development is not fully understood, the best example being the multiple functions attributed to p53 in tumor development and suppression. Recent observations have implicated Gadd45 proteins as important sensors of oncogenic stress, both in vitro and in vivo.It is known that whereas primary mouse cells require introduction of two activated oncogenes for transformation, disruption of certain key growth control genes allows single oncogene transformation ,86. It wEvidence was obtained that Gadd45 proteins also play a role in modulation of tumor development in vivo. Gadd45a-/- and gadd45b-/- mice were observed to display increased mutation frequency, and susceptibility to ionizing radiation (IR) and chemical carcinogenesis. Also, it has been documented that NF-kB-mediated repression of gadd45a and gadd45g is essential for cancer cell survival. More recently, evidence was obtained that loss of Gadd45a accelerates ras-driven mammary tumor formation. Ras-driven tumor formation in the absence of Gadd45a resulted in both a decrease in apoptosis, linked to a decrease in JNK activation, and a decrease in senescence, correlated with a decrease in p38 kinase activation . AltogetGadd45 genes have been implicated in stress signaling, which results in cell cycle arrest, DNA repair and cell survival, or apoptosis Fig. . What deThe authors declare that they have no competing interests.DAL participated in the sequence alignment and drafted the manuscript. BH participated in drafting the manuscript. All authors read and approved the final manuscript.