Cellular stress-induced temporal alterations—i.e., dynamics—are typically exemplified by the dynamics of p53 that serve as a master to determine cell fate. p53 dynamics were initially identified as the variations of p53 protein levels. However, a growing number of studies have shown that p53 dynamics are also manifested in variations in the activity, spatial location, and posttranslational modifications of p53 proteins, as well as the interplay among all p53 dynamical features. These are essential in determining a specific outcome of cell fate. In this review, we discuss the importance of the multifaceted features of p53 dynamics and their roles in the cell fate decision process, as well as their potential applications in p53-based cancer therapy. The review provides new insights into p53 signaling pathways and their potentials in the development of new strategies in p53-based cancer therapy. Cells are constantly bombarded by a variety of endogenous and environmental stress that results in cellular damage. This usually leads to cell cycle arrest allowing the damage to be repaired by cellular repair mechanisms. However, in an occasion of severe damage, cells have to directly activate a “suicide” apoptotic death program to induce cell death, preventing initiation of oncogenic processes. Thus, cells have two possible fates—to survive or die. Failure in making the right decision in determining cell fate can ultimately lead to the development of diseases such as cancer. The cell fate decision machinery is composed of multiple complex signaling pathways, in which p53, a key tumor suppressor, plays a central role in coordinating the multiple cellular signaling pathways as well as determining cell fate .P21 gene and pro-apoptotic genes such as BAX, BAK, PUMA, and P53AIP1 [As a transcription factor, p53 is activated by various types of stimuli. This selectively regulates the transcription of p53 target genes that subsequently mediate cellular responses to stress. In the past two decades, hundreds of p53 target genes have been identified. These include the cyclin-dependent kinase inhibitor  P53AIP1 . The pro P53AIP1 . While tIn this review, we discuss the progress in an emerging new field in cell biology that explores a p53-mediated cellular signaling network via the dynamics of key signaling molecules in the network ,8, whereIn the 1990s, it was observed that treatment of rat neuronal precursors with epidermal growth factor (EGF) and neural growth factor (NGF) can lead to cell proliferation and differentiation, respectively. Further investigation revealed that both stimuli activated the same protein kinase, the extracellular signal-regulated kinase (ERK), with a distinct temporal feature—i.e., EGF induces a transient ERK activation—whereas NGF triggers a sustained ERK activation ,11,12. TFurthermore, several studies support the notion that cells can translate different p53 dynamical patterns into a specific type of downstream cellular responses. It has been found that IR-induced transient p53 pulses are associated with activation of pro-cell cycle arrest genes that result in cell-cycle arrest, whereas prolonged p53 pulses induced by UV are involved in upregulation of pro-apoptotic genes that can lead to apoptosis . This isAs a transcription factor, p53 can activate pro-cell cycle arrest and pro-apoptotic genes as well as induce the expression of genes that regulate its own gene expression and the stability of its protein. This results in the formation of a feedback loop leading to the pulsatile dynamics of p53 protein level.The first identified feedback loop in the p53 network is the p53 mouse double minute 2 (Mdm2) negative feedback loop. Discovered in 1992, Mdm2 was initially proposed as an oncoprotein because it functions as a p53-specific E3 ubiquitin ligase that promotes ubiquitination and subsequent proteasomal degradation of p53 . Later iWIP1 gene can result in the dynamics induced by UV-like stimuli [p53 can be activated by a variety of stimuli including IR, UV radiation, activated oncogenes, hypoxia, and chemotherapeutic drugs. However, our current understanding about the p53-mediated stress response mainly results from the response to double-strand DNA breaks (DSBs) induced by IR and single-strand DNA breaks (SSBs) induced by UV radiation ,24,28. T stimuli . It has  stimuli . Thus, tBecause IR-induced p53 pulses are fixed in amplitude and duration, it was initially proposed that the decision of cell fate is governed by the frequency of p53 pulses . It is cWith the discovery of multiple functional feedback loops that result in different p53 dynamics (transient or sustained pulse), an emerging question is how the p53 dynamics determine cell fate in a coordinated manner? To address this, several computational models that integrate transient and sustained p53 pulses have been developed to understand the cell fate decision process. The models have shown that in response to UV radiation, p53 exhibits a single prolonged pulse that is associated with the irreversible cell fate—apoptosis . In contIt is noteworthy that the majority of studies on p53 dynamics focus on how p53 responds to stimuli, i.e., exogenous DNA damaging agents such as IR and UV radiation. Few studies explore how p53 reacts to intrinsic or spontaneous DNA damage caused by normal physiological processes. With quantitative time-lapse microscopy of individual human cells (MCF7 cell line), a recent study showed that p53 exhibits excitable spontaneous transient pulses in non-stressed cells to respond to spontaneous DNA damage with a dynamic pattern similar to that in response to IR . This fiIn general, p53 dynamics are designated as the variation in p53 protein level in response to stimuli. However, this notion has been challenged by the fact that the maintenance of a steady level of p53 protein induced by IR with a p53-stabilizing agent only can switch cell fate from survival to irreversible senescence . This isAs a transcription factor, p53 is continuously transported among the nucleus, cytosol, and mitochondria. Export of p53 from the nucleus to mitochondria is thought to be a negative regulation of p53 transcription activity by excluding the protein from its target genes. Yet, recent studies have shown that non-transcriptional mitochondrial p53 proteins also play an important role in p53 dynamics at the spatial level of the protein ,37,38.Because of its regulatory roles in gene transcription, p53 proteins are mainly located in the nucleus, where they bind to the promoter and/or enhancer of the target genes to regulate gene expression. Yet, a small portion of p53 protein is located in mitochondria to perform its non-transcriptional function. It appears that such a small amount of p53 in mitochondria is sufficient to mediate mitochondrial apoptosis effectively ,39. UponIt appears that nuclear and mitochondrial p53 are orchestrated to determine the fate of cells that are damaged by IR . This haThe posttranslational modifications of p53—including ubiquitinylation, phosphorylation, methylation, and acetylation —play impDiscovery of the dynamics of p53 ubiquitinylation also led to the discovery of the dynamics of other types of post-translational modifications of p53 . Thus faAcetylation of p53 occurs on several lysine residues at the C-terminal domain of p53 including K370, K372, K373, K381, and K382, and this is carried out by a p53 co-factor, p300/CBP. The modification mainly results in increased DNA binding of p53, promoting the activation of its target gene. In contrast, methylation of p53 by methyltransferases at histidines, arginines, and lysines, suppresses transcriptional activity of p53 . InteresRecent studies have shown that besides its crucial role in regulating gene expression, p53 can also activate its target miRNAs, which include miR-192, miR-194, miR-215, miR-107, and miR-605 ,52. InteOur current understanding of p53 dynamics is limited to the pulsatile switches of its protein level. However, the interplay among different types of p53 dynamics is critical in governing a specific cellular damage response. Based on this, p53 dynamics can be designated as a multi-faceted dynamic network comprised of all variables that can be measured over time. These include p53 protein level, its activity, subcellular localization and posttranslational modifications. We therefore propose a model that illustrates the roles of multiple dynamic features of p53 in governing cell fate . In non-The central role of p53 in governing the cell fate decision and preventing tumorigenesis makes it a promising target for cancer therapy. Over the past decades, p53-based cancer therapeutic strategies have mainly focused on p53 gene-transfer therapy with an adenovirus vector, or on restoring p53 activity using small molecule drugs that interact with mutant p53 protein and alter the conformation of the protein . These tHowever, it should be noted that such cancer therapeutic strategies may be complicated by the multiple roles of Mdm2. This is because Mdm2 can mediate translocation of p53 from the nucleus to mitochondria to initiate rapid apoptosis ,37. ThisIn a scenario where the mitochondrial p53-mediated apoptotic pathway fails to be initiated, a nuclear p53-mediated apoptosis can be initiated as an alternative pathway to promote cell death. A critical step of this process is to promote p53 transactivation of pro-apoptosis genes. Based on the fact that p53 transcriptional activity is determined by both its protein level and posttranslational modifications, two strategies can be employed to alter p53 transcriptional activity. One is to switch p53 pulses from a transient mode to a sustained mode. Defects in this switch have been identified in human cancers in a tissue-specific manner. For example, Wip1, a phosphatase, is essential for the initiation of transient p53 pulses. Thus, it acts as an oncogene in many cancers such as breast cancer, glioma, and colon carcinoma . In contThe dynamics of p53 have emerged recently as a new field of cell signaling. However, its biological significance remains to be elucidated. With the development of fluorescent labeling and time-lapse technology, studies on p53 dynamical profiles in protein level, spatial location, and posttranslational modifications will provide new insights into the biological function and implications of p53 dynamics in cancer treatment. Because p53 dynamics have been mainly studied in IR-induced DNA damage models, many of which are based on computational and mathematic approaches, additional experimental results that support these models are in urgent need. These include the results of p53 dynamics induced by other stimuli such as ROS, heavy metals, and chemotherapeutic drugs. In addition, because the measurements of p53 dynamics are currently restricted in cultured cells, the dynamic profiles of this protein need to be further verified in a biological system such as fruit flies, worms, zebra fish, mice, and humans, if it is feasible. Nevertheless, our knowledge about dynamic interactions among various cell signaling networks and the dynamics of the components of the network will provide valuable guidance to manipulate and control cell signaling pathways for achieving therapeutic purposes in both cancer and other human diseases such as neurodegenerative diseases.


N = 201) were assessed on the BCET and a number of other neuropsychological tests that have been demonstrated to rely on aspects of executive processing  and the Repeatable Battery for the Assessment of Neuropsychological Status (RBANS). Internal consistency of the total BCET was good; however, interpretable solutions for existing subscales were not discerned. The BCET total score demonstrated positive associations with tests of executive functioning; however, it was also significantly associated with more general aspects of neuropsychological functioning suggesting that it does not solely assess executive processes in ABI patients undergoing rehabilitation. Hierarchical multiple regression suggested that the BCET accounted for significant additional variance in community integration after severity of disability, executive functioning, and more general aspects of neuropsychological status were statistically controlled. While the subscale structure of the BCET may be somewhat inconsistent, the total scale score accounts for some unique variance in pragmatic rehabilitation outcome and may be a useful tool in postacute rehabilitation assessment protocols.The purpose of the present study was to examine the convergent and divergent validity of the Biber Cognitive Estimation Test (BCET) in individuals with ABI undergoing postacute rehabilitation and to assess the measure's ability to account for unique variance in community integration following rehabilitation. Participants with ABI referred for postacute rehabilitation ( The ability to provide reasonable numerical estimates from available but incomplete information is an ability not normally acquired directly through formal education, yet it is an important aspect of problem solving that is regularly called upon in everyday life. Previously acquired information may not always have direct relevance to a particular estimation problem and, as such, problems in providing reasonable or plausible cognitive estimations are related not simply to an abstract problem in processing numbers, but also to probabilistic judgements (mapping components) arising from domain-specific reasoning and general heuristics , 2. FailThe first formal examination and detailed assessment of estimation abilities were performed on individuals with lesions to the frontal lobes . This laCommunity integration of people with ABI rests on the premise that such individuals should have the same rights and be involved in community life like any other member of their community. It is generally considered to represent a person's ability to carry out everyday activities in their home and community, enjoy interaction with its members, and participate in some aspect of productivity in the community. While community integration is also the ultimate goal of rehabilitation following ABI, formal examination of the construct and its possible relationship to variation in community integration outcomes in the context of rehabilitation have not been undertaken to date. This mandates a comprehensive examination of a widely used measure of cognitive estimation and its influence on more distal community integration outcomes.There is no clear consensus on the use of a particular measure of cognitive estimation that has been rigorously developed and standardised. In terms of measurement of the construct, it has been observed  that theThe aims of the study were as follows: firstly, to examine the latent variable structure of the BCET in relation to the subscale assessment domains of quantity, time/age, length, and weight in a sample of people referred for postacute rehabilitation following acquired brain injury; secondly, given the question posed by previous research as to whether estimation is associated more with performance on executive or more general cognitive function assessments, to investigate the convergent and divergent validity of cognitive estimation using established neuropsychological tests that are considered to represent executive and more general measures of cognitive function; thirdly, to assess what aspects of cognitive function measured by the battery of neuropsychological tests may be responded to differently in an impaired estimation group relative to an unimpaired estimation group; and finally, to investigate whether the BCET adds any unique variance to prediction of community integration, given that community integration is the ultimate goal of rehabilitation, and impairments in estimation ability may have real-world consequences in terms of such community integration aims .A total of 201 participants who had sustained an acquired brain injury and who were referred for postacute neurorehabilitation services in the South and Mid-West of Ireland participated in the study. All participants engaged in a programme of accredited and individualized Home and Community Based Rehabilitation (HCBR). HCBR is defined by the Commission of Accreditation of Rehabilitation Facilities (CARF), as a programme of postacute rehabilitation that provides integrated, case managed, and outcome focused rehabilitation. In the HCBR model, services are developed from a comprehensive needs assessment and focus on the expectations and outcomes identified in treatment planning with the person served by the programme. The positive effects of such individualized community based programmes are well established .Inclusion criteria were participants accessing ABI-specific neurorehabilitation services (home and community rehabilitation), confirmed medical diagnosis of ABI, age over 18 years and not older than 65 years at study end, and English as a first language. Exclusion criteria were the presence of aphasia/communication, comprehension, or significant visual difficulty that necessitated an alternative assessment protocol.The principal purpose of recruiting ABI rather than a single cause of brain injury  was that it would reflect the possible ecological or real-world rehabilitation usage of the measure of cognitive estimation and permit an examination of its ability to predict the ultimate goal of rehabilitation (community integration) over and above the standard assessments undertaken in this group.n = 150; 74.6%; women n = 51; 25.4%) and were aged between 19 and 63 years . Age at onset of brain injury ranged from 1 to 60 years , and duration of injury ranged from 2 years to 20 years . Twenty percent of participants (n = 40) completed more than 14 years of education, 33% (n = 67) 12 to 14 years, and 38% (n = 76) 8 to 12 years, and 9% of participants (n = 18) completed less than 8 years of formal education. Participants' functional disability was assessed on the Mayo Portland Adaptability Index [All participants gave informed consent. Participants were predominantly male , 35% had stroke (n = 71), and 15.5% (n = 31) had other diagnoses including neoplasm , anoxia , encephalitis , and aboulia . Due to the small number of participants in some subcategories of brain injury, 3 categories were created for subsequent analysis: traumatic brain injury, stroke, and a third category comprising neoplasm, anoxia, aboulia, and encephalitis.49.5% of participants sustained a TBI . T-scores were used as an assessment of the severity of functional limitations such that scores less than 50 indicated mild to moderate limitations, while scores above 60 were suggestive of more severe limitations arising from ABI. One hundred and four participants (51.7%) had disability in the moderate to severe range, while 97 participants (48.3%) had disability scores in the mild to moderate category.Mean MPAI The study was approved by the local research ethics committee  and all participants provided informed consent commensurate with the declaration of Helsinki. The study adhered to standardised reporting guidelines for cohort studies (STROBE). All participants completed the following assessment measures.The BCET  is a 20-The RBANS  is a briParticipants were required to generate as many words as possible that either begin with a particular letter, for example, F, A, and S (phonemic fluency), or that belong to a specific semantic category, fruits, vegetables, and animals (semantic fluency) within a specified time interval. These measures are commonly used to assess the organisation and integrity of lexical and semantic memory representations and executive functions. Phonemic and semantic verbal fluency relies on a partially different network of brain regions , and theThe Modified Six Elements Test (MSET)  consistsThe TMT  was usedCommunity Integration of participants was assessed by use of the Community Integration Questionnaire (CIQ) . The CIQp's > 0.11). Fourteen cases had some missing data from their assessments. No participant had missing data in excess of 7% on the assessment measures, and no participant had missing data on the neuropsychological assessments. To assess whether the data was Missing Completely at Random (MCAR) or whether there was an underlying pattern to this missing data, Little's MCAR test was utilised. MCAR values ranged from χ2 = 6.399, p = 0.93, to χ2 = 78.016, p = 0.97, which suggested that the small amount of missing data was randomly missing. Therefore, these cases were retained and the standard expectation-maximisation procedure was utilised to maximise power. Means and standard deviations were calculated for the main measures and subscales as appropriate. Analysis of Variance with Scheffe post hoc test was used to examine the effects of educational attainment and cause of ABI on BCET scores. Independent samples t-test was used to examine mean differences between scores on the neuropsychological measures and disability total score as a function of BCET cut-off scores, and Chi Square analysis examined the association between the BCET cut-off and levels of disability severity. Relationships between cognitive estimation and demographic variables, neuropsychological measures, disability, and community integration were undertaken by Pearson correlation coefficient. Latent variable structure of the BCET was undertaken via the Categorical Principal Components Analysis (CatPCA) program. Logistic regression analysis was used to examine neuropsychological factors associated with cognitive estimation scores that were below threshold, and hierarchical multiple regression was used to explore whether the BCET accounted for unique variance in community integration. All data were analysed using SPSS 24.Q-Q plots and Shapiro-Wilk's test were used to examine the distribution of data. Because of the nonnormal distribution of trail-making scores in both raw  and derived measures  and in the Modified Six Elements Test (MSET), transformation was applied before further analysis to render this data suitable for parametric analysis. Observed data showed no significant deviation from normality , with subscale scores being acceptable for estimates of distance/length (α = 0.73) and weight (α = 0.69) and somewhat lower for quantity (α = 0.57) and time/age (α = 0.62). Correlations between each of the four domains and the total scale score were as follows: quantity (r = 0.62), weight (r = 0.60), time/age (r = 0.55), and distance/length (r = 0.71).Descriptive statistics (means and standard deviations) for the BCET, TMT, verbal fluency, MSET, and the six indices of the RBANS are presented in In order to appropriately examine the latent variable structure of the BCET, categorical PCA (CatPCA) was utilised. CatPCA is a relatively new algorithmic model and can be considered the nonlinear equivalent of PCA. It is especially suitable for the dimension reduction problem with categorical variables, as it accounts for the nature of items, the different role of items in determining the measure, and the possible multidimensionality of the underlying concept , 55. TheA scree plot was firstly examined to determine the most parsimonious number of components to retain in the analysis. Nonlinear PCA solutions are not nested, so the position of the scree plot “elbow” can move with the number of components. In the present analysis, different dimensionalities consistently placed the elbow at the fourth component.Component loadings are presented in Figures 1Examination of the biplots suggest that, graphically, the variables do not seem to form distinct or particularly meaningful groups/clusters that might conform to the original 4 estimation domains or that represent some new groupings with clinical meaning.Following examination of the biplots, item analysis was used to examine the distribution of responses to each item of the BCET. Examination of the percentage of correct and incorrect responses  shows thRotation options are not available in the CatPCA program and given the well-documented difficulty in interpretation of unrotated solutions, the transformed variables were additionally saved and utilised as input variables to permit a rotated solution . The comAcross the subscale assessment dimensions of quantity, weight, time, and distance, individual compatible items did not consistently fall into these established dimensions. Moreover, the factors produced were also not interpretable in any obvious manner . Given the difficulty in obtaining a fully interpretable solution in relation to the proposed subscales  as suggested in Figures 1F, 6.84, p = 0.001) and Scheffe post hoc tests suggested that the effect was largely monotonic, with individuals who had attained a postgraduate education scoring significantly higher on the BCET (BCET mean (SD) for primary school leaver 13.29 (3.17), secondary school leaver 14.36 (2.79), and postgraduate 16.33 (2.49)).Mean total BCET scores rose with educational attainment. This represented a significant effect  and with an older age at onset , but not with duration with ABI .Higher (better) BCET scores were associated with increasing age of the participant , 4.73, p = 0.01).One-way ANOVA suggested that people who had sustained a TBI scored more poorly on the BCET, mean (SD) TBI = 14.04 (2.44), than people who had sustained a stroke, BCET mean SD = 15.44 (3.19), or other causes, mean SD = 15.9, 3.10 (T score (mean (SD) for mild/moderate disability = 15.09 (2.97); mean (SD) for severe disability = 14.25 (2.81); t = 1.58, p = 0.11).BCET total score did not significantly differ as a function of the severity of participant's disability as assessed by MPAI-4 r = .42, p = 0.001), phonemic fluency , TMT B , and the TMT difference score  but not with the TMT ratio score (r = −.07). Additionally, the BCET was also highly correlated with more general indices of neuropsychological functioning including the TMT A  and the 6 scales of the RBANS . Higher BCET scores were also associated with increased community integration .t = −4.62, p = 0.001), on verbal fluency (mean (SD) = 21.45 (11.34) versus 26.07 (11.85); t = −2.68, p = 0.006) and semantic fluency (mean (SD) = 13.40 (4.25) versus 15.63 (4.71); t = −3.28, p = 0.001) and on RBANS indices of immediate memory (mean SD = 67.04 (18.34) versus 79.14 (21.27); t = −4.05, p = 0.001), visuospatial functioning (mean SD = 79.60 (19.03) versus 88.96 (23.20); t = −2.91, p = 0.004), language (mean SD = 77.91 (13.57) versus 88.96 (23.20); t = −2.95, p = 0.004), attention (mean SD = 72.24 (17.56) versus 84.56 (17.41); t = −4.57, p = 0.001), and delayed memory (mean SD = 66.34 (21.67) versus 79.15 (18.48); t = −4.09, p = 0.001). The BCET cut-off was not significantly associated with scores on the TMT A mean SD = 69.34 (49.88) versus 57.06 (43.21) and TMT B (mean SD = 204.44 (157.36) versus 184.39 (143.60); t = −1.76, p = 0.07) or its difference (mean SD = 149.01 (102.11) versus 110.87 (107.04); t = −1.19, p = 0.23) or ratio score (mean SD = 3.32 (2.33) versus 2.56 (2.20); t = −1.29, p = 0.19). The cut-off score was not significantly affected by severity of disability . Participants with a younger age at onset were more likely to score above the BCET cut-off (mean SD = 29.43 (13.85) versus 37.83 (13.91); t = −4.08, p = 0.001). A forward conditional logistic regression analysis including the statistically significant univariate results above suggested that BCET cut-off scores were predicted by a younger age at onset of ABI (odds ratio (OR) = 1.04, 95% CI = 1.01–1.06, Wald = 10.49, p = 0.001) poorer scores on the MSET  and poorer scores on RBANS attention index . The model discriminated the unimpaired cognitive estimation group from the unimpaired estimation group with an accuracy of 76.8%.Using the established cut-off for the BCET (scores below 15.6 indicating impairment in estimation abilities), 55.7% of participants had scores in the unimpaired range, with 44.3% scoring in the impaired range. The estimation-impaired group scored significantly lower on the MSET (mean (SD) = 1.93 (1.1) versus 2.76 (1.2); Given the BCET's apparent lack of specificity in assessment in terms of performance based cognitive functioning, the BCET total score was regressed onto participants' community integration scores to examine if the functions assessed by the BCET may explain unique variance in more pragmatic social integration outcomes when entered on the final step in the model. Therefore, a hierarchical multiple regression analysis was undertaken with variables that correlated with community integration as IVs. Thus, severity of disability as assessed by MPAI was entered on step (1), RBANS total score on step (2), MSET on step (3), semantic fluency on step (4), TMT B (as it was the most highly correlated of the TMT measures) on step (5), and BCET on step (6).Results  suggesteThe current study comprehensively examined the convergent and divergent validity of estimation abilities operationalised through the Biber Cognitive Estimation Test (BCET) in a large sample of individuals with acquired brain injury undergoing postacute rehabilitation. The study examined the latent variable structure of the BCET, reported an item analysis of responses, and addressed whether performance on executive or more general assessments of neuropsychological functioning were more strongly associated with the BCET total score and with its impairment cut-off score. This is also the first study to date to examine the effects of estimation abilities on what is usually considered to be the end point of rehabilitation, namely, the person's level of integration into their community.The latent variable structure of the BCET was examined by Categorical Principal Components Analysis, a relatively new algorithmic model which was considered to be more appropriate than more traditional models given that CatPCA can reveal nonlinear relationships by quantifying categorical or nonlinearly related variables to reveal relational structures among the observed variables. Despite examining the BCET with a number of procedures including CatPCA variance accounted for, examining the coordinates for each item on each dimension in relation to the centroid, the use of biplots, and utilising rotation procedures, none of these represented the information in the data sufficiently closely such that it might be reduced in a meaningful way to the smaller number of summary variables . Neither were there any obvious theoretically informed justifications for interpretation of the factors, such as components representing a process of estimation that may have relied upon direct procedural experience  versus a process that may have relied on more remote declarative/semantic procedures to effect an estimate . While it was judged prudent from a psychometric perspective to restrain further analysis of the BCET in the current study to its total score, the prospect that the areas of estimation targeted by the subscales would be isomorphic in clinical practice with rehabilitation patients may not represent a sensible assumption in any case . For exaThe current study found a relationship between BCET estimation performance, education, and age, in that more years of formal education, being older, and sustaining their injury at an older age tended to be associated with better estimation performance. While the original development and validation study for the BCET  did not r = .28). Poorer MSET performance suggests a difficulty in alerting the individual to changes that should be taken into account during the task and to faulty reevaluation of goals and monitoring tasks in progress [r = 0.42 and r = 0.39, resp.). Of the RBANS subscales, immediate memory and attention could be considered more legitimately to require aspects of executive function in their performance, particularly updating semantic working memory. Similarly, in terms of discriminating an impaired estimation group from an unimpaired group, patients whose BCET scores placed them in the impaired category were significantly more likely to have a younger age at onset of their brain injury, lower MSET scores, and poorer RBANS attention index scores.In terms of the relationship between the BCET and the more established tests of executive function, the BCET showed a stronger relationship with tests involving planning and set shifting  than with the verbal fluency component of executive functions. Both the MSET and the TMT could be considered to map onto the response inhibition and set shifting components of Miyake et al.'s  model ofprogress , which wThe distal goal of postacute rehabilitation is to integrate the person into their community. While the assessed model accounted for a relatively modest proportion of the variance in community integration (21%), some important findings were uncovered. Namely, that the RBANS total score accounted for the majority of the variance in community integration, with severity of ABI-induced disability, MSET, TMT B, and semantic fluency not making any significant contribution to the model. The BCET however did account for unique variance in community integration after it was entered on the last step of the model accounting for an additional 4% of the variance. While modest, it nonetheless suggests that the BCET may be assessing something distinctive and of potential value in this area. Further work needs to be undertaken to develop the assessment protocol for cognitive estimation including various forms of presentation that may involve visual cueing as an attention orientation strategy. Similarly, goal management interventions  may provThe study has some limitations. Firstly neuroimaging data was not available on participants and therefore no detailed formal information can be provided from a structural-functional perspective on anterior versus posterior injuries or in terms of issues of laterality and their possible effects on BCET performance. However, since brain injury patients with moderate to severe injuries typically have large lesions affecting fairly large tracts of frontal and temporoparietal cortex, it is also possible that multiple functional systems may be affected which would limit the ability of the BCET to localise function in such samples in any case. Secondly, variability in the ages at which participants sustained their brain injury, while reflecting the nature of routine referrals to ABI postacute rehabilitation, may have influenced the results given issues of brain plasticity in more established versus more recent injury. Thirdly the sample comprised people who had sustained brain injuries of varying causes; thus, standard severity measures which might be utilised in TBI cases were not sensible to be used  and that criteria for severity in CVA such as Rankin criteria are not routinely utilised in the Republic of Ireland. Therefore, a valid and reliable measure of disability arising from acquired brain injury (MPAI-4) was used in order to provide a sympathetic measure that would have relevance to all types of ABI. The presence of clinical severity information from the time the person sustained their injury in addition to specific and contemporary lesion location information would have been a helpful adjunct to the study.This is the first study to examine the effects of estimation abilities on community integration in a rehabilitation sample of people with acquired brain injury. The study has also provided detailed information on estimation assessment in this patient group. While the latent structure of the BCET in the current study did not readily map onto the four subscale indices of quantity, weight, distance/length, and time/age and to describe the BCET solely as a test of executive functioning in the current rehabilitation sample would not be sensible, the measure has some merit in terms of its association with pragmatic outcomes and may have a continued role in assessment protocols in postacute rehabilitation.


Diffuse large B cell lymphoma (DLBCL), currently the most common type of non-Hodgkin lymphoma (NHL), is an aggressive B cell neoplasm that typically presents in older adults as a rapidly enlarging mass. The enlarging mass typically represents a lymph node, although extranodal disease can occur in a significant percentage (40%) of cases. The most common extranodal sites of involvement include the gastrointestinal tract and skin; primary bladder lymphoma represents only 0.2% of extranodal non-Hodgkin lymphomas. We report a case of diffuse large B cell lymphoma occurring in the bladder of an 83-year-old gentleman with an initial presentation of hematuria. This neoplasm displayed large, atypical cells with vesicular chromatin and prominent nucleoli that involved the bladder mucosa with invasion into muscularis propria, prostate, and urethra. Positive staining for p63 initially raised suspicion for poorly differentiated urothelial carcinoma; however, lack of staining for pancytokeratin and positive staining for LCA, CD20, CD79a, and PAX-5 confirmed the diagnosis of diffuse large B cell lymphoma. Though it does not occur in all cases, p63 can be positive in a significant percentage of cases of DLBCL; therefore, a diagnosis of lymphoma remains an important entity on the differential diagnosis of aggressive and particularly poorly differentiated neoplasms. Diffuse large B cell lymphoma (DLBCL) represents the most common type of non-Hodgkin lymphoma (30–40% of NHL cases) . This agWe present a case of an 83-year-old gentleman with a past medical history of prostate cancer status after radiation treatment, hypertension, BPH, hyperlipidemia, and renal impairment who presented with gross hematuria. A transurethral resection revealed an infiltrating, poorly differentiated tumor with abundant apoptosis and mitotic activity showing invasion into the muscularis propria. Due to the patient's history of prostate carcinoma, immunohistochemical stains were performed. The malignant cells were positive for p63 and negative for PSA, cytokeratin 7 (CK7), and cytokeratin 20 (CK20); therefore, a diagnosis of poorly differentiated urothelial carcinoma was favored. A CT scan of the abdomen and pelvis was remarkable for a remarkably thickened bladder wall with ill-defined boundaries and dilation of the distal right ureter. Small pelvic lymph nodes were also visualized.Subsequent sampling revealed an aggressive neoplasm with invasion to bladder muscularis propria, prostate, and urethra. The tumor displayed large malignant cells with vesicular chromatin and prominent nucleoli arranged in a mildly cohesive pattern . Based oDiffuse large B cell lymphoma may present in extranodal sites. This case illustrates the importance of considering a diagnosis of lymphoma when confronted with a poorly differentiated neoplasm. Expression of p63 by lymphoma cells presents a potential diagnostic pitfall. Review of the literature reveals several reports of p63 expression in high-risk diffuse large B cell lymphoma. P63 is positive in a significant amount of DLBCL cases, with percentages ranging from 15 to 70% depending upon the study examined [ p63 gene, homolog of the p53 tumor suppressor gene, is located on chromosomes 3q27-3q28 and encodes proteins responsible for p53 activation and apoptosis [The p53 is widely acknowledged as a tumor suppressor gene and its mutations are responsible for tumor progression, the role of p63 as a cancer oncogene continues to be studied. Two particular groups of p63 isoforms, TAp63 and ΔNp63, have received further study in the development of neoplasia.P63 comprises a group of six different proteins produced through alternative splicing of a single mRNA transcript . These i p53 target genes and inducing apoptosis. The ΔNp63 isoform lacks this N-terminal domain and likely has a dominant-negative effect on p53 [The TAp63 isoform contains an N-terminal transactivation domain with sequence homology to the p53 transactivation domain. This isoform is responsible for transactivatingt on p53 . RT-PCR t on p53 . This imRecent studies , 10 have p63 gene responsible for creating these various protein isoforms is located on chromosomes 3q27-3q28. Interestingly, the Bcl-6 gene is also located on chromosome 3q27, adjacent to the p63 locus. Given that rearrangements in the Bcl-6 gene are seen in approximately 30% of diffuse large B cell lymphomas [ p63 to Bcl-6 may contribute to its potential involvement in DLBCL tumor progression [As previously noted, theymphomas , some auSeveral studies have adopted the task of analyzing p63 expression in diffuse large B cell lymphoma and its prognostic impact, fueled by an earlier study by Di Como et al. that showed high levels of p63 in a subset of DLBCL 25%) and follicular lymphoma (22%) . These s% and fol P63, homolog of the tumor suppressor p53, has an important role in the development and differentiation of stratified epithelia; as such, it is usually interpreted as a marker of epithelial differentiation/carcinoma. However, as illustrated by this case, p63 can be positive in a significant subset of cases of diffuse large B cell lymphoma. Of particular note, the isoform TAp63 appears to be involved in the development of lymphoma, while the ΔNp63 isoform is typically seen in cases of squamous cell carcinoma. Numerous studies support this phenomenon and correlate p63 expression with DLBCL tumor progression; its impact on prognosis and survival remains to be seen.This case illustrates the importance of considering hematologic malignancies in the differential diagnosis of poorly differentiated neoplasms and serves as a reminder that no single immunohistochemical marker is entirely specific for a certain tumor type. Incorporation of morphology and immunophenotype is vital to final diagnosis.


The p53 tumor suppressor continues to be distinguished as the most frequently mutated gene in human cancer. It is widely believed that the ability of p53 to induce senescence and programmed cell death underlies the tumor suppressor functions of p53. However, p53 has a number of other functions that recent data strongly implicate in tumor suppression, particularly with regard to the control of metabolism and ferroptosis (iron- and lipid-peroxide-mediated cell death) by p53. As reviewed here, the roles of p53 in the control of metabolism and ferroptosis are complex. Wild-type (WT) p53 negatively regulates lipid synthesis and glycolysis in normal and tumor cells, and positively regulates oxidative phosphorylation and lipid catabolism. Mutant p53 in tumor cells does the converse, positively regulating lipid synthesis and glycolysis. The role of p53 in ferroptosis is even more complex: in normal tissues, WT p53 appears to positively regulate ferroptosis, and this pathway appears to play a role in the ability of basal, unstressed p53 to suppress tumor initiation and development. In tumors, other regulators of ferroptosis supersede p53’s role, and WT p53 appears to play a limited role; instead, mutant p53 sensitizes tumor cells to ferroptosis. By clearly elucidating the roles of WT and mutant p53 in metabolism and ferroptosis, and establishing these roles in tumor suppression, emerging research promises to yield new therapeutic avenues for cancer and metabolic diseases. TP53 has been the most heavily studied human gene since its discovery nearly 40 years ago (The tumor suppressor gene ears ago . The maiears ago . Howeverears ago . A summavia the transactivation of SCO2 (cytochrome c oxidase assembly), which plays a direct role in oxidative phosphorylation (GLS2 (Glutaminase 2); this enzyme allows glutamine usage as an energy source for the mitochondria  or arginine (R72). This amino acid variation can impact p53 function with regard to cell fate after stress. In response to DNA damage, the P72 variant of p53 predominantly triggers cell cycle arrest, while the R72 variant predominantly induces cell death, or apoptosis , 19. DesR72 mice . InteresR72 mice . These fR72 mice .Cpt1c-deficient mice display delayed tumor development and higher survival rates (LPIN1) is another p53 target gene; LPIN1 is necessary for proper adipocyte development and is induced under low nutrient conditions , a critical p53 transcriptional target. The mechanism through which p21 delays ferroptosis has yet to be elucidated, but it is believed that the conservation of intracellular glutathione may be a contributing factor for reduced ferroptosis sensitivity. The authors conclude that the p53–p21 axis enables cancer cells to survive under conditions of metabolic stress, such as cystine deprivation, by suppressing the onset of ferroptosis (A study recently published by Tarangelo and colleagues shows that p53 negatively regulates ferroptosis in cancer cells . This grroptosis . A recenroptosis . The bidMDM2 and MDM4). The Pro47Ser variant (hereafter S47) is the second most common SNP found in the p53 coding region (after Pro72Arg) that alters the amino acid sequence of the protein. To better elucidate the impact of this variant on p53 function and cancer risk, the Murphy group generated a humanized p53 knock-in mouse model, in which exons 4–9 of murine p53 were replaced by human p53 exons containing either the wild type or the S47 variant (Gls2 (glutaminase 2) and Sco2  in the TP53 gene and other proteins known to regulate this pathway (such as  variant –55. The  variant . In mousand Sco2 . Consistand RSL3 . This deSLC7A11, which inhibits sensitivity to ferroptosis (SLC7A11 appear to predominate in the regulation of this gene. For example, the master antioxidant transcription factor NRF2 can also regulate the expression of SLC7A11 at the transcriptional level, and NRF2 has been implicated as a key player in protecting cancer cells against ferroptosis. For example, inhibition of NRF2 in hepatocellular cancer cells increases the anti-cancer activity of Erastin and Sorafenib in vivo (SLC7A11, and thus show increased sensitivity to ferroptosis. Notably, overexpression of SLC7A11 in mutant p53 models led to drug resistance, suggesting that levels of SLC7A11 expression must be considered when targeting mutant p53 driven cancers with ferroptosis-inducing compounds (Wild-type p53 negatively regulates the expression of the cystine importer  in vivo . Mutant ompounds . In suppompounds . These dThe role of p53 in metabolism is quite clear and possibly even intuitively obvious: WT p53 limits glucose metabolism and lipid synthesis, while mutant p53 appears to do the opposite. The contribution of its metabolic role to tumor suppression by p53, and to the ability of mutant p53 to drive tumor progression, remains to be unequivocally proven. The role of p53 in the regulation of ferroptosis, and the contribution of this function, to tumor suppression is even less clear. While compelling data from mouse models supports the premise that p53 regulates the sensitivity of cells to ferroptosis, this may be restricted to the ability of basal p53 to suppress spontaneous tumor development, and in oncogene-stressed mouse models, it is clear that senescence and apoptosis play the predominant role. Similarly, p53 may regulate ferroptosis sensitivity in a cell type-specific manner. More studies in animal models, with attention to ferroptosis in different tissues, need to be done to more fully understand the role of p53 in ferroptosis and ferroptosis in tumor suppression. Additionally, a clearer idea of what p53-target genes play a role in sensitivity to ferroptosis needs to be attained. Resolution of these questions should provide for much needed novel avenues to combat tumors with mutant p53.KG, SB, TB, AB-K, C-PK, and MM each wrote one to two paragraphs of this article. KG and SB did the figure. KG and MM outlined the chapter.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer OAF and handling Editor declared their shared affiliation.


Cell Death & Differentiation leading scientists in the p53 field elucidate some of these questions taking us a step ahead towards our understanding of this versatile protein.1TP53 is the most frequently mutated gene in cancer and its study helped to define fundamental principles underlying tumorigenesis. However, despite over three decades of accumulating knowledge , we stilCDKN1A) or association with MDM2 – the main p53 regulator – which acts as a repressor by its own.2 In particular, as shown by Engeland in this Issue, p21 connects the p53 pathway with the DREAM transcriptional repressor complex which includes members of the retinoblastoma family of tumor suppressors – whose inclusion within the DREAM is regulated by the cyclin-dependent kinases that are inhibited by p21. The DREAM controls hundreds of cell cycle-associated targets, further expanding the number of genes affected by p53 modulation, and connecting p53 to the regulation of all cell cycle checkpoints.3 Espinosa also discusses findings further detailing the functions of p53 trans-activation and carboxy terminal domains required for its transcriptional activator role.2 Interestingly, p53 emerged as a pioneer transcription factor able to bind a core set of enhancers even when the chromatin is closed, overriding epigenetic regulatory landscapes. Not all of the p53 binding, however, seems productive: rather, it becomes functional upon changes of the chromatin environment, which are time and context dependent. This explains why, although p53 binding to chromatin is largely invariable, the p53-regulated programme is extremely variegated.2P53 controls the expression of wide gene sets owing to its transcriptional activity leading to both mRNA up- and downregulation. However, its ability to act as a direct repressor of transcription has been long disputed. Here Espinosa and colleagues provide evidence that such repressive effects are mainly attributable to downstream effectors such as microRNAs or p21 . So, they suggest that these two types of mechanisms are the main contributors to the loss of function phenotype characterized by impaired DNA binding and transcriptional ability, which blunt p53 tumor suppressor activities.4 Some of the most common missense mutations are likely due to the effect of environmental mutagens, such as UV rays or cigarette smoke, which act in a tissue-specific manner, whereas methylation of CpG residues within the TP53 sequence seems a more general mechanisms increasing p53 mutational rate in most tissues, which also explains while arginine residues (encoded by four CG-containing codons) are among the top most frequently mutated. The reason why only specific mutations, over all the methylated CpG residues within the TP53 sequence, are selected could depend on the structural and biological relevance of the affected residues and their contribution in fostering tumorigenesis.4Differently from other tumor suppressors that are mostly inactivated by deletions or truncating mutations, 5 Also, p53 mutants that acquire oncogenic activity through a gain-of-function (GOF) mechanism have an increasingly recognized role. Kim and Lozano in this issue explore a wide set of p53 mutants and the mechanisms whereby they gain new oncogenic functions. Such mutants mostly act through protein-protein interactions with other transcription or chromatin remodelling factors affecting vast downstream gene networks. These interactions, which so far have been mostly described in cell lines, are highly context dependent determining various degrees of GOF activity in different tumor types with mutated p53. Interestingly, it seems that some p53 mutants acquire similar GOF activities binding common transcription factors and transactivating the same gene targets. This could have translational relevance because it would ideally limit the set of targets which should be tackled to suppress the new oncogenic GOFs.5 Overall, determining the mechanisms whereby p53 loss of function, DN and GOF activity drive tumor development and progression is crucial to definitively establish the tumor suppressor function of p53.Beyond loss of function, p53 mutants can drive tumorigenesis through other mechanisms: in heterozygosity, when both the wt and mutant alleles coexist, the mutant proteins can act as dominant negative (DN) over the wt, forming heterotetramers with impaired transcriptional ability.6 In particular, as discussed in this Issue by Strasser and colleagues, the ability of p53 to induce apoptosis in transforming cells through the direct activation of pro-apoptotic proteins (such as PUMA and NOXA) was initially regarded as the main cancer preventing function of p53. However, in contrast with Trp53 null mice that develop tumors with complete penetrance within the first year of life, mice depleted of all the (known) main downstream effectors of p53-mediated apoptosis, do not develop spontaneous tumors, even when coupled to the loss of p53 G1/S cell cycle arrest and senescence effectors, suggesting that full p53 antitumoral potential relies on further capabilities.7Induction of apoptosis, cell cycle arrest and senescence are the ‘classical’ outputs of p53 activation that seem to guarantee tumor suppression.6So, how does p53 really protect from cancer development? Kaiser and Attardi, provide some clues derived from the analysis of genetically engineered mice modelling p53 alterations, which help to reconcile older and new studies. The type of stress that challenges p53 action seems to be the key: whereas the above-mentioned classical outputs of p53 activation are needed to respond to acute DNA damage cues, incipient cancer cells are more likely exposed to other stresses such as nutrient and oxygen deprivation, oncogenic signalling, replication or oxidative stress, and also low levels of genotoxic, chronic DNA damage. To face these, p53 might induce common outcomes  through alternative routes – triggering metabolic changes, autophagy, promoting DNA repair – in a context-dependent manner. Here the authors analyze both cell autonomous and non-autonomous emerging functions of p53 and its downstream targets, which also play a part in the p53 antitumoral response.8But p53 does not act alone as commander in chief when it comes to cell fate decisions. The fine tuning of the cellular responses to a wide range of stimuli is achieved through the cross-talk between complex gene networks and signalling pathways. As an example, Oren and colleagues here discuss the cross talk between the p53 and the Hippo tumor suppressor pathway, which – similarly to p53 – regulates a variety of cellular processes. The authors highlight how the direct interaction between core components of both p53 family and Hippo pathways, and their regulation of common targets, is coordinated to modulate the response to different stresses and maintain homeostasis between stemness and differentiation, cell death and proliferation, anabolism and catabolism. Alterations of either pathway, as occurring in cancer cells, disrupt this balance with profound effects on cell fate.9Regardless of the mechanism, p53 tumor suppressor functions protect organisms against cancer development by ensuring genome stability through DNA repair or eliminating damaged cells that might undergo neoplastic transformation. However, factors that limit indefinite proliferation protect from cancer but foster aging, the gradual process of cell and tissue deterioration. Depletion of functional cells, stem cell exhaustion and the induction of a chronic inflammatory microenvironment by senescent cells, all feature aging. Although intimately related, the link between p53 and aging is still unclear with p53 activity being found associated to both premature aging and increased longevity. To clarify this conundrum Wu and Prives analyzed the mechanisms whereby p53, and its main regulator MDM2, could affect aging by regulation of genomic stability, senescence, cross talk with metabolic pathways (such as the insulin/IGF1 and mTOR pathways), mitochondrial dysfunction and regulation of reactive oxygen species, at the cellular level. Also, based on the analysis of mouse models with modulated p53-MDM2 axis, they propose a model to infer the relation between lifespan and cancer resistance as a function of p53 stability achieved through the loss of MDM2 regulation or chronic stress. Hyperactivation of p53 could provide greater cancer resistance but at the cost of accelerated ageing. Therefore, the underlying mechanisms of this delicate balance will have to be carefully determined when considering anti-aging approaches as well p53-reactivating strategies against cancer.Another layer of complexity, hampering our understanding of p53 physiological and pathological functions is added by the roles, overlapping or disjointed, of the other family members p63 and p73 and their respective isoforms. The presence of an highly conserved DNA binding domain shared by the three members of the p53 family explains some functional redundancy in their regulation of gene expression. However, p63 and p73 can also forms heterotetramers that are more stable than individual tetramers. Indeed both proteins are found to co-occupy DNA target sites throughout the genome, as discussed here by Melino and colleagues, who argue that studying these genes singularly could provide only a restricted view of their global functions. Melino and colleagues also report on latest findings implicating p63 and p73 in the regulation of cell metabolism which also contributes to the p53-family-mediated flexible response to stress. Besides tumor-related functions the authors present recent evidence establishing p73 as the master regulator of ciliogenesis whose defects underlie various human diseases. This overlooked function finally explains disparate multi-organ defects previously observed in p73 knock out models providing a unifying view that will help to better characterize other non-oncogenic functions of p73 such as infertility neurodevelopment and inflammation.


However, we provide evidence for a preservation of the corresponding genes in two animals unable to synthesize cholesterol (auxotrophs): Drosophila melanogaster and Caenorhabditis elegans.It is known that primary sequences of enzymes involved in sterol biosynthesis are well conserved in organisms that produce sterols bona fide orthologs of several ERG genes in both organisms using a series of complementary approaches. We have detected strong sequence divergence between the orthologs of the nematode and of the fruitfly; they are also very divergent with respect to the orthologs in organisms able to synthesize sterols de novo (prototrophs). Interestingly, the orthologs in both the nematode and the fruitfly are still under selective pressure. It is possible that these genes, which are not involved in cholesterol synthesis anymore, have been recruited to perform different new functions. We propose a more parsimonious way to explain their accelerated evolution and subsequent stabilization. The products of ERG genes in prototrophs might be involved in several biological roles, in addition to sterol synthesis. In the case of the nematode and the fruitfly, the relevant genes would have lost their ancestral function in cholesterogenesis but would have retained the other function(s), which keep them under pressure.We have been able to detect ERG24 and ERG25 in D. melanogaster and genes encoding factors involved in intracellular protein trafficking and folding and with Start1 involved in ecdysteroid synthesis. These potential functional connections are worth being explored not only in Drosophila, but also in Caenorhabditis as well as in sterol prototrophs.By exploiting microarray data we have noticed a strong expressional correlation between the orthologs of  DrosophilaCaenorhabditis elegans possesses several genes encoding proteins with regions similar to Hh and potentially undergoing cholesterylation Cholesterol and other sterols such as ergosterol and phytosterols are universal components of eukaryotic plasma membranes and are absent from the membranes of prokaryotes. These sterols  modulate membrane fluidity Saccharomyces cerevisiae, which involve ERG (i.e. from ERGosterol) genes and the respective ERG proteins.Yeast, plants and mammals synthesize sterols through a series of complex reactions that occur in the endoplasmic reticulum (ER) and, therefore, most of the involved enzymes have transmembrane domains . We outlERG25, ERG26, and ERG27, in cooperation with ERG28p. Finally, ERG6p (C-24 methylase) converts zymosterol to fecosterol, which is further transformed into ergosterol by ERG2p, ERG3p, ERG5p and ERG4p The first three steps of sterol biosynthesis are catalized by ERG9p , ERG1p  and ERG7p (lanosterol synthase), respectively. These proteins are essential for aerobic viability and their absence results in an inability to synthesize ergosterol. The third enzyme, ERG7p, converts squalene epoxide into lanosterol, the first cyclic component of the cholesterol biosynthesis cascade (i.e. the first sterol). The remaining enzymes of the pathway metabolize lanosterol into ergosterol. The sequential action of ERG11p (lanosterol demethylase) and ERG24p (C-14 reductase) leads to 4,4-dimethylzymosterol. They are also essential in aerobic conditions Recent results show that, in yeast, ergosterol biosynthetic enzymes display specific protein-protein interactions and form a functional complex called ergosome. Proteins ERG11p, ERG25p, ERG27p and ERG28p, appear to form a core center of the complex and would interact with other enzymes of the pathway D. melanogaster and C. elegans cannot synthesize sterols de novoC. elegans takes sterols from animal feces or yeast and plant remnants Drosophila obtains sterols from the diet: ergosterol from yeast and phytosterols from plants Most animals synthesize cholesterol. However, some animals such as dauer larva formation and molting depend on sterols. Cholesterol, or more likely its derivatives, seem to act as hormones. Indeed, recent papers report the identification of natural steroid ligands for the DAF-12 nuclear receptor As already noted, sterols in these animals are required as hormone precursors and/or developmental effectors C. elegans, the distribution and transport of cholesterol in vivo has been studied by using dehydroergosterol (DHE), a fluorescent analog which mimics many cholesterol properties C. elegans might be involved in the structural and functional organization of the plasma membrane cell types that are richer in this lipid In D. melanogaster with Anopheles gambiae and prototrophs has suggested that these insects have lost most of the genes involved in sterol synthesis Drosophila is unable to synthesize cholesterol. However, in a previous work we have shown that Drosophila contains an ERG28 ortholog that has undergone a process of acceleration in its evolution, and is undetectable using the current techniques for ortholog detection by sequence homology C. elegans and D. melanogaster, not only for ERG28, but most of the genes/enzymes involved in the sterol synthesis pathway, in the light of new genomic and functional data.A previous comparative genome analysis of ERG gene orthologs in C. elegans and D. melanogaster following their order in the sterol synthesis cascade . This protein, which also exists in C. elegans, contains two functional domains: monooxygenase and UbiH (2-polyprenyl-6-methoxyphenol hydroxylase and FAD-dependent oxidase). This is also the case in yeast ERG1p. However, in a reverse BLAST, CBG19254 recognized with very high score yeast Coq6 and only marginally ERG1p . A similar behavior was observed for GA20231-RA from D. pseudoobscura, so we did not proceed to a further analysis of these sequences.We failed to detect the squalene synthase (ERG9p) homolog, which catalyzes the first committed step in cholesterogenesis. In the search for squalene epoxidase (ERG1p) in a BLAST using ERG1p from yeast, we detected the gene CBG19254 in For lanosterol synthase (ERG7p), which follows in the classical pathway, we could not gather any convincing evidence for the existence of orthologs in the fruitfly and the nematode.Drosophila . However, the Drosophila genes are likely to belong to other Cyp subfamilies (not Cyp51). Cyp51 is probably missing, which is in agreement with the results of Tijet, Helvig & Feyereisen P450 gene superfamily. Cyp51 is also absent in C. elegansThe following enzyme in the pathway is a sterol 14α-demethylase, ERG11p/Cyp51, involved in the biosynthesis of cholesterol, phytosterols and ergosterol. Thus, it is the only cytochrome P450 having an ortholog common to animals, plant and fungi ERG24 orthologs in D. melanogaster (CG17952) and in C. elegans (B0250.9) were easily found. The corresponding proteins contain the ERG4-24 domain. D. melanogaster produces three isoforms that are longer than the yeast ortholog, a peculiarity that they share with the human ortholog. The ortholog of ERG24 in mammals encodes the Lamin B receptor (LBR), a nuclear envelope protein first described in vertebrates. LBR bears extensive structural similarities with the members of the sterol reductase family (ERG24p and ERG4p). Human LBR (hLBR) cannot restore ergosterol biosynthesis in an ERG4 yeast mutant, whereas it is able to restore ergosterol prototrophy in an ERG24 mutant. This strongly suggests that hLBR is a sterol C14-reductase hLBR gene causes an autosomal recessive disease called hydrops-ectopic calcification-‘moth-eaten’ (HEM). This mutation leads to high levels of cholesta-8,14-dien-3-beta-ol in cultured skin fibroblasts, which is compatible with a deficiency of the cholesterol biosynthetic enzyme 3-beta-hydroxysterol delta(14)-reductase Potential LBR gene arose from an ancestral gene coding for a soluble nuclear protein  and that the rest of the protein evolved from another gene, similar to yeast ERG24. Indeed, the C-terminal hydrophobic domain of LBR can be retained in the endoplasmic reticulum when expressed in transfected cells, as expected for the ortholog of ERG24 in mammals. In turn, the N-terminal domain is transported to the nucleus The hLBR contains two major domains: a ∼220-amino-acid N-terminal segment highly charged, and a hydrophobic C-terminal half with eight putative transmembrane segments Drosophila CG17952 gene is the ortholog of vertebrate LBRDrosophila LBR (dLBR) possesses a highly charged N-terminal domain of 307 amino acids followed by eight transmembrane segments. Transmembrane segments 1–6 are similar in length and position to the transmembrane domains 1–6 of hLBR. However, the putative membrane domains 7 and 8 of dLBR are shorter than those of hLBR. Thus, dLBR is expected to have a topological organization similar to that of its vertebrate orthologs Drosophila lamin B, a function residing in the N-terminal domain. Not unexpectedly, dLBR does not display sterol C14 reductase activity when expressed in the yeast ERG24 mutant. This shows that, during insect evolution, although the enzymatic activity of this protein has been lost, its capacity to bind lamin B has not. However, depletion of dLBR by RNA interference does not lead to any obvious effect on nuclear architecture, or viability, of treated cells and embryos. Thus, although dLBR might be important, it is not a limiting component of the nuclear architecture in Drosophila cells, at least during the first days of development ERG24 ortholog in C. elegans. It would be interesting to experimentally assess if it has kept LBR activity.Recent functional studies show that the D. melanogaster (CG1998/dERG25A and CG11162/dERG25B) and C. elegans (F49E12.9/ERG25A and F49E12.10/ERG25B) using the sequence of yeast ERG25p as a starting point. In both organisms the duplicated copies of ERG25 are located in the same chromosome (chromosome II for C. elegans and chromosome X for D. melanogaster). The two paralogs in D. melanogaster are separated by 0.25 Mb and contain a different number of predicted exons. Namely, CG1998 contain 6 exons, while CG11162 contains only 2. However, the last intron of both genes interrupts the coding sequence at very similar positions  were also easily found in both C. elegans (ZC8.1) and D. melanogaster (CG7724) was obtained in BLAST searches taking the sequence from yeast as the starting point  orthologs. However, a BLAST with C32D5.12 detected as the first hits NSDHL in man and ERG26 in yeast (ERG26p (C-3 dehydrogenase) belongs to the 3β-hydroxysteroid dehydrogenase family and convincing evidence for the presence of orthologs in in yeast . Thus, iet al.Drosophila and C. elegans, although several oxidoreductases were detected.In agreement with Breitling C. elegans (C14C10.6) was hardly detectable by BLAST starting with yeast sequences. This precludes the use of standard phylogenetics methods to show orthology. However, further evidence of sequence relatedness was gathered using Psi-BLAST with the yeast sequence against the Metazoa division of Genbank D. melanogaster, which is the ortholog of ERG28p according to our previous results −27). Reverse Psi-BLAST also suggested orthology (i.e. significant scores). The sequence of C14C10.6 is so divergent that it had no match in the conserved domain database (CDD). Moreover, we also computed the hydrophobic profiles for some of the orthologs As outlined above, ERG28p might tether many other ERG proteins to the ER. The ERG28p ortholog of C. elegans (H14E04.1) was easily detected by BLAST with protein sequences from either yeast or A. thaliana (SMT1). We used the plant sequence since no clear ERG6 homolog could be detected in human. The issue with Drosophila turned out to be more complex because, when starting the search with the yeast sequence, we detected CG8067 marginally (E>1). This was even worse when starting with the sequence of A. thaliana. However, when using the C. elegans sequence as a starting point, the first significant hit in Drosophila (E<10−6) was CG2453, which proved to be the ortholog of yeast Coq5, but not of ERG6p. Finally, considering i) the similar lengths of the previously marginally detected CG8067, of H14E04.1 and of SMT1 proteins and ii) their similar pI, we have preferred CG8067 as the most likely ortholog. Indeed, the hydrophobic profiles of SMT1 and the protein encoded by CG8067 displayed a strong correlation. Namely, we obtained an R = 0.43 with a p-value 10−16.The ortholog of ERG6p in C. elegans (W08F4.3) was marginally detected by BLAST with the yeast and the human protein . However, W08F4.3 was found to contain a sigma1-receptor domain when compared with the CDD. This strengthens the idea that this gene is the ortholog of OPRS1 and ERG2. The situation in Drosophila was more complex. When starting our BLAST with either yeast or human sequences, we detected the sequence HDC14735 (DAA04220) very marginally (E≫1) and no conserved domain was found. However, when starting with the C. elegans sequence, it came as the best hit with E = 0.002 . Although not significant, this result was taken as suggestive of similarity. The results were improved using Psi-BLAST. Again, we computed the hydrophobic profiles for the various potential orthologs and we found strong correlations . Therefore, we propose that the orthologs of ERG3 are potentially missing in both organisms.In BLAST searches with yeast ERG3p, we detected again CG1998 and CG11162 in For ERG5p, which belongs to the big Cyp protein family, no clear orthologs could be established. However, three potential candidates were found: CG4321-PA (Cyp4d8), CG3540-PA (Cyp4d14) and CG8859-PA (Cyp6g2). In the reverse BLAST, they all matched ERG5p as the best scoring hit in yeast.Finally, the search for ERG4p orthologs led to the same ERG24 orthologs in the nematode and the fruitfly. Moreover, with ERG4p the BLAST scores were worse than with ERG24p. Thus, either ERG4 orthologs are missing or they have been replaced by ERG24.ERG orthologs mentioned above undergo a selective pressure, we have examined the ratio of the number of non-synonymous substitutions per non-synonymous site (Ka) and the number of synonymous substitutions per synonymous site (Ks). The ratio Ka/Ks is indicative of the mode of evolution operating on the sequences. If selection is dominantly purifying, then we expect few non-synonymous substitutions per background synonymous changes and hence, a low ratio. If selection is absent, then a ratio of unity is expected  ratios show that these genes are under selective pressure . This property is important for enzymes because protein-protein and enzyme-substrate interactions are often electrostatic in nature. Thus, we should expect the pI of an enzyme to be similar in different organisms if protein-protein and/or enzyme-substrate interactions are conserved. In order to test this idea we gathered the protein sequences of the orthologs of ERG2, 6, 24, 25, 26 and 28. Next, we asked whether the corresponding vergence . SimilarERG orthologs that we have described above for both D. melanogaster and C. elegans. Co-expression can be assessed by determining the correlation coefficient. The correlation coefficient can be artificially inflated by flat profiles (no changes in the expression of the relevant genes). To avoid this, we focused on experiments where the genes of interest display strong variation  and it was not possible to proceed further with the analysis.Co-expression is indicative of i) physical interaction between proteins and ii) of membership to the same complex or molecular process tion see . Thus, wD. melanogaster ERG orthologs were correlated (dLBR (ERG24) and CG1998 (ERG25) with an associated p-value of 10−16 (after a Bonferroni correction). Such a p-value means that only one correlation coefficient out of 1016 is expected to be as high as 0.89 just by chance . Considering the maximum number of possible correlations for the 14000 transcripts in the microarrays (representing the Drosophila genome), such a high R cannot be found by chance. The behavior of dLBR and CG1998 might be reminiscent of the situation in yeast because ERG24p and ERG25p are supposed to interact, according to Epistatic MiniArray Profiling experiments First, we asked whether the expression profiles of the rrelated . The strERG orthologs in Drosophila. However, it seems that only dLBR and CG1998 still “remember” their ancestral belonging to the sterol biosynthesis pathway. Poor expressional correlation among the rest of the ERG orthologs also suggests that the corresponding proteins either have lost their ability to physically interact in order to form stable complexes, or they do so in conditions/moments not covered by the microarray experiments explored here. Then, we focused our attention on dLBR and CG1998 by determining which other genes were expressionally correlated with them. For this, we gathered 84 genes displaying R≥0.875 with respect to both genes. For a correlation involving 51 data points, this R cut-off is associated with a safe p-value of 10−13 after correction , chaperone cofactors or unfolded protein-binding factors. A similar analysis conducted using g∶profiler −5). The existence of expressional correlation does not imply any causality. In fact, from this exploration it is not possible to determine whether dLBR and CG1998 somehow interact with other partners to participate in intracellular protein trafficking or folding, or on the contrary, they undergo the action of the latter. Since dLBR has been shown to be a nuclear protein ERG orthologs. However, no unifying theme emerged from this analysis . All in all, the strong expressional correlation between dLBR and CG1998 with proteins involved in intracellular protein trafficking or folding, and the absence of such correlation with other Erg orthologs  suggest that the involvement of dLBR and CG1998 in both processes is worth exploring.In the analysis using DAVID, the most overrepresented class included genes encoding membrane proteins, often targeted to the ER, where sterol biosynthesis takes place in prototrophs  was found for Start1. Interestingly, Start1, which is involved in intra-mitochondrial sterol transport, is expressed ubiquitously. However, in situ hybridization demonstrates a stronger expression in the prothoracic gland, where ecdysteroids are synthesized from cholesterol. These and other observations are consistent with the idea that Start1 plays a key role in the regulation of ecdysteroid synthesis In a previous paper, given the structural similarity between cholesterol and ecdysteroids, we had proposed that divergent ERGp orthologs might somehow participate in the synthesis of the latter ERG genes in Drosophila melanogaster and Caenorhabditis elegans. In spite of their sequence divergence with respect to the corresponding orthologs in sterol prototrophs, they still are under selective pressure. Since insects are unable to synthesize cholesterol de novo, an appealing way to explain this evolutionary acceleration is that ERGp orthologs have other biological functions in addition to sterol synthesis. This is clearly the case of the LBR, which is also a reductase in sterol prototrophs. Shut-down of cholesterogenesis in insects and nematodes would have allowed these proteins to evolve as much as their other functions were not compromised ERG24 and ERG25 in D. melanogaster and genes encoding factors involved in intracellular protein trafficking and folding. This is compatible with our idea that ERGp might be involved in other biological roles in addition to sterol synthesis. The potential link between ERG proteins and intracellular protein trafficking and folding deserves experimental exploration not only in Drosophila and Caenorhabditis but also in sterol prototrophs. Moreover, the potential link between dLBR, CG1998 and Start1 is to be explored in D. melanogaster. This is compatible with our previous idea of a potential implication of these proteins in the synthesis of ecdysteroids. We hope that this genomic exploration and the hypotheses prompted here might open new avenues of experimental research.In conclusion, we detected a preservation of We used BLASTp For some distant homologues we computed the hydrophobic profiles and calculated the Pearson correlation coefficient “R” for various pairs of profiles. The values of R range between 1 (perfect match between the profiles) and −1 (profiles are mirror images). Strong positive correlation is not a proof of orthology but strengthens the idea of structural relationship at the protein level.The protein-coding nucleotide sequences were first translated into amino acid sequences and aligned. Then, this alignment was used to define the alignment of the corresponding nucleotide sequences to avoid frame-shifting indels as alignment artifacts http://www.expasy.ch/tools/pi_tool.html). Then, we asked whether the corresponding Drosophila and Caenorhabditis sequences displayed outlier pI values. In short, we performed an extreme studentized deviate test to determine whether one of the values in the pI list was a significant outlier from the rest.We have investigated whether the pI of presumably orthologous proteins were similar. In order to test this, we gathered the protein sequences of the orthologs of ERG2, 6, 24, 25, 26 and 28 listed in the HomoloGene division of the NCBI database and estimated their pI (


Due to the extent of functional overlap within the Rb family, it has been difficult to assess which functions are exclusive to individual members and which are shared. Like its family members, p107 can bind a variety of cellular proteins to affect the expression of many target genes during cell cycle progression. Unlike Rb and p130, p107 is most highly expressed during the G1 to S phase transition of the cell cycle in actively dividing cells and accumulating evidence suggests a role for p107 during DNA replication. The specific roles for p107 during differentiation and development are less clear, although emerging studies suggest that it can cooperate with other Rb family members to control differentiation in multiple cell lineages. As a tumor suppressor, p107 is not as potent as Rb, yet studies in knockout mice have revealed some tumor suppressor functions in mice, depending on the context. In this review, we identify the unique and overlapping functions of p107 during the cell cycle, differentiation, and tumorigenesis. Rb tumor suppressor was first identified as the gene whose loss causes hereditary retinoblastoma in children . HoweveRb-related genes can be found across multiple species, including humans, mice, chickens, reptiles, flies, and even some plants. Most unicellular and lower organisms have only one Rb-related gene, while higher organisms tend to have two or three family members, perhaps reflecting an increasing complexity of cell cycle control in these species. For example, the unicellular alga Chlamydomonas reinhardtii only contains one Rb-like gene (mat3), whose loss leads to a deregulation of proliferation and a reduced cell size [Whi5 appears to play a functionally similar role to Rb, despite a lack of sequence homology [Rb-related gene, although recently a second Rb-related gene was identified in maize and rice [Caenorhabditis elegans also contains one Rb-like gene, lin-35, which in sequence homology is more similar to p107 than to Rb [Rb-related genes in Drosophila, RBF1 and RBF2 [lin35 in C. elegans, these two genes are more similar to mammalian p107 and p130 than they are to Rb itself. Interestingly, the gene duplication events in plants and flies are only two examples of many duplication events within the Rb-family over the course of evolution as additional duplication events have occurred in Gallus gallus (chicken), Danio rerio (zebrafish), and Anolis carolinensis (lizard)  as it was originally named, was identified through its interaction with SV40 Large T antigen and adenovirus E1A . StructuUnlike Rb and p130, p107 levels are generally, but not always, low in quiescent and differentiated cells and higher when cells proliferate -37. p107Of the three Rb family members, p107 is thought to be the most heavily regulated at the transcriptional level ,36. p107p107, like p130 and Rb, is a substrate for Cyclin/Cdk kinase activity during cell cycle progression, which is thought to cause p107 to release E2F transcription factors and relieve repression of target gene promoters -54. In qin vivo, p107 preferentially binds to E2F4 and E2F5 at the promoters of E2F target genes in cycling cells [in vivo. Future experiments should aim to identify more extensive sets of target genes bound by individual Rb family members in various cellular contexts. Experiments such as genome-wide chromatin immunoprecipitation followed by sequencing (ChIP-Seq) with antibodies specific to p107, p130, and Rb in different cell types or at different phases of the cell cycle may help to shed light on this question.Once bound to E2Fs, Rb family members repress transcription through a variety of methods. The Rb family can directly recruit chromatin modifying enzymes such as histone deacetylase HDAC1  derived from these animals display no significant cell cycle defects [p107-deficient mice in a Balb/c background show a severe postnatal growth deficiency, as well as myeloid hyperplasia in the spleen and liver. MEFs and myoblasts derived from these animals exhibit increased proliferation that was associated with constitutive expression of Cyclin E [-/- p107pre-adipocytes could not upregulate Rb, which is required to initiate differentiation in vivo through interaction with Pgc1α [ defects . InteresCyclin E . These mth Pgc1α .-/- p107mouse may be due to compensation from p130 or Rb. -/-;p130-/- p107embryos die at birth with multiple defects in tissue development. Interfollicular keratinocytes from -/-;p130-/- p107newborns show impaired terminal differentiation in the epidermis, decreased numbers of hair follicles, and a developmental delay in hair, whisker, and tooth formation [p107 and p130 seem to play overlapping roles during embryonic development in the mouse, and one reason for the lack of severe phenotypes in the ormation . These d-/-;p130-/- p107embryos as well as -/-;p27-/- p107embryos display defects in ossification of the long bones and chondrocyte proliferation [p107 has also been implicated in the control of bone and cartilage development. Double knockout feration ,103. Durferation . Overexpferation . Biochemin vitro and in vivo [In the adult mouse brain, p107 expression is unique from the other Rb family members in that it is restricted to cycling progenitor cells in the ventricular zone, and its expression decreases as these cells begin to differentiate into cortical neurons ,105. Rb  in vivo . This apHes1, a key downstream target of the Notch signaling pathway, since p107-deficient animals display increased Hes1 signaling  is increased in neural progenitors of p107-deficient brains. Recently, p107 was shown to repress E2F3 activity at the promoter of Fgf2, an essential growth factor that modulates the population of neural precursors in the developing brain [-/- p107brains, as increased FGF2 would be expected to promote survival. Therefore, the mechanisms driving apoptosis in -/- p107progenitors may be independent of FGF2 signaling, and have yet to be identified.p107 may also be acting through the FGF growth factor signaling pathway to control the numbers of neural precursors in embryonic and adult brains Figure . FGF is ng brain . HoweverThe two examples in chondrocytes and neurons suggest a scenario in which FGF signaling can indirectly activate p107, which could then repress FGF signaling through direct promoter binding. Although this feedback loop between p107 and FGF has yet to be identified within one specific cell type, it suggests the potential for a complex regulatory system for p107 and growth signaling during development.p107 shortened the lifespan of -/- Rbembryos from birth to E14.5, and further increased the abnormal levels of proliferation and apoptosis that are present in the central nervous system and the ocular lens of Rb-deficient embryos. Mutation of p107 in an Rb-deficient background also caused heart development defects that were not seen in the Rb-deficient or p107-deficient embryos alone. These heart defects are likely the result of blood vessel endothelium and endocardial cell proliferation in the absence of both Rb and p107. Additionally, the cerebellar architecture is severely disrupted in adult mice lacking both Rb and p107 in the dorsal mid-hind brain junction, and these mice exhibit impaired terminal differentiation and migration defects in granule cell precursors, in addition to increased granule cell apoptosis upon maturation [Recent studies have uncovered functions of p107 that overlap with Rb during development. Additional loss of turation . While tturation , a basicturation . Thus, pRb is mutated in a variety of sporadic and familiar human cancers, most notably in pediatric retinoblastoma and osteosarcoma. Mutations in p107 itself have not been observed in human tumors [p107 in human tumors, it still may play a role in tumorigenesis, as mutations in upstream regulators of the Rb family are common [n tumors . So far,n tumors -118. Dese common ,120. Inae common . This fip107 do not develop spontaneous tumors [+/-;p130-/- p107and -/-;p130+/- p107mice revealed no spontaneous loss of either p107 or p130 allele and no obvious tumor phenotypes [-/-;p130-/- p107mice has not been described, these studies suggest that p107 and p130 do not by themselves have tumor suppressor functions in the mouse. However, these observations do not exclude the possibility that p107 and/or p130 may act as tumor suppressors in other contexts.It is clear that p107 is not a strong tumor suppressor by itself, as mice with mutations in s tumors . Since penotypes . While cRb loss, p107 can contribute some tumor suppressor functions within a cell  of the retina showed that Rb expression was redundant with p130 . In the  retinas . Thus, iigenesis . The mecRb-heterozygosity results in retinoblastoma with 100% penetrance in humans but is not sufficient to cause retinoblastoma in mice. This difference in mice may be due to the ability for other family members, namely p107, to compensate for the loss of Rb in this context [p107 in response to Rb loss, whereas mouse retinal cells do [p107 promoters contain two tandem E2F binding sites. The mouse promoter has a single point mutation in the proximal 3' E2F site that may affect the binding of E2Fs or other transcriptional machinery that is recruited there [p107 in response to Rb loss in specific contexts, while the other cannot. These differences in p107 transcriptional regulation may also be seen within the different tissues of the same organism. For example, if deregulation of the Rb pathway can be found in almost all human tumors, why, then, do Rb-heterozygous patients primarily only develop a narrow spectrum of tumors, mainly retinoblastoma and osteosarcomas? This discrepancy may be explained by the fact that some tissues can upregulate p107 in response to Rb loss, whereas others cannot. Indeed upregulation of p107 is seen in several cell types upon Rb loss, including mouse retinal progenitors [p130 is generally not seen in response to Rb or p107 or both [ context . Interescells do . This died there . Alternagenitors , keratingenitors , hepatocgenitors , and lungenitors ,126. Int or both .To fully understand the tumor suppressor functions of Rb in human tumors, it is important to understand the functions of each of the family members, both individually and as a group. In particular, p107 functions in cell cycle control and tumor suppression have remained elusive.During cell cycle progression, p107 function may be divided into two categories; those that require E2F and those that do not. ChIP-Seq for Rb family members and E2F family members in normal cells would shed light on the normal binding patterns of these proteins and may identify promoters that are regulated by individual Rb family members or by several at once. Expanding upon this, one could then compare the binding profiles of the Rb family members in normal cells to that of tumor cells, cells in different phases of the cell cycle, or cells from different tissues. p107 can also interact with several other key transcription factors such as Sp1, B-myb, c-myc, and Smad3. The significance of these interactions is not well understood, and several questions remain. For example, is p107's ability to regulate the cell cycle mainly exerted through E2F repression or can the interaction with other transcription factors also arrest the cell cycle independently of E2Fα One way to answer these questions would be to take advantage of the fact that E2Fs bind different regions of p107 than do the other transcription factors.Evidence for p107 function during S phase suggests that it may play a critical role outside of the control of G1. Rb and p130 have both been implicated in the control of G0 and G2 -136, so p107 mutations to mice carrying mutations in other known cancer causing genes outside of the Rb pathway. Furthermore, no studies have examined whether p107 mutation and DNA damaging agents can contribute to faster or more aggressive tumors. Future studies should clarify the specific contexts in which p107 can act as a tumor suppressor, with or without compound mutation of Rb.It has yet to be determined whether p107 can serve as a tumor suppressor in the context of other mutations outside of the Rb pathway. To date, there have been no studies crossing mice with Although much has been uncovered since the discovery of the Rb family, the complexities in functional overlap, regulation, and tumor suppressor abilities of each of the Rb family members is only just beginning to be explored. The use of transgenic, knock-in, and knock-out mouse studies, as well as in vitro cell culture systems will be critical to increase our understanding of the role of these genes during multiple cellular functions, and these techniques will continue to reveal the subtle and distinct ways in which these proteins can interact with each other as well as the hundreds of other proteins known to associate with them. More analysis of how the Rb family normally functions is needed to understand their functions within a single cell, in addition to their tumor suppressor capabilities.Rb: Retinoblastoma; Cdk: Cyclin-dependent kinase; HDAC: histone deacetylase; DHFR: dihydrofolate reductase; BMP: bone morphogenetic protein; FGF: fibroblast growth factor; VZ: ventricular zone; ChIP: chromatin immunoprecipitation.The authors declare that they have no competing interests.SW and JS designed and wrote the manuscript together. Both authors have read and approved the final manuscript.


Several studies have focused on the role of p53 inactivation in cervical cancer, either by inactivating mutations in the TP53 gene or by degradation of the p53 protein by human papillomavirus (HPV). In this study, primary cervical carcinomas from 365 patients were analysed for presence of HPV using both consensus primer-sets and type-specific primer-sets. Nineteen samples were determined to have no or low virus load, and were selected for further analyses: mutation screening of the TP53 gene using constant denaturant gel electrophoresis (CDGE) followed by sequencing, and protein expression of p53, MDM2 and p21 using immunohistochemistry (IHC). Mutations in the TP53 gene were found in eight samples (42%). Elevated p53 protein expression was significantly associated with presence of a mutation (P < 0.007). P21 protein expression was detected in 16 of the 19 carcinomas. No p21 expression was seen in normal cervical tissue. Two samples, both with wild-type p53, had elevated MDM2 expression. Compared with a previous study from our group, of mainly HPV-positive cervical carcinomas, in which only one sample was found to contain a TP53 mutation, a significantly higher mutation frequency (P < 0.001) was found among the carcinomas with no or low virus load. Although p53 inactivation pathways are not detected in every tumour, our study supports the hypothesis that p53 inactivation, either by binding to cellular or viral proteins or by mutation, is essential in the development of cervical carcinomas.