The aim of the study was to determine whether the combination of p53 over-expression and p53 low-expression associated with Ki67 over-expression (high Ki67/p53 ratio) could lead to a more sensitive parameter. Immunohistochemical expression of Ki67 and p53 was measured in 54 specimens from OSCC; 27 specimens from moderate/severe epithelial dysplasia; 32 specimens from oral leukoplakias without epithelial dysplasia, and 13 specimens with normal epithelium. p53 over-expression was found in 31 (53%) samples from OSCC, in 10 (37%) samples from severe dysplasias, and in 5 (15%) samples from non-dysplastic lesions, while the combination of high p53 values with high Ki67/p53 ratio was observed in 93% of OSCC, in 81% of dysplastic lesions, and in 50% of non-dysplastic lesions. This parameter may have a clinical implication to detect early lesions with an impairment of p53 pathway, and probably at risk of progress to OSCC. It has been widely demonstrated that most oral squamous cell carcinomas (OSCC) develop through their precancerous steps which are defined as potentially malignant lesions and are histologically characterized by increasing grades of epithelial dysplasia -4.So far, the only accepted method to quantify the risk of progression to OSCC of a potentially malignant lesion, is the presence/absence of histological findings of dysplasia -6, althoThese considerations have stimulated intense research in this area, and many studies have been conducted so far, in an attempt to find out molecular markers that are associated with OSCC and that can predict malignant transformation when found in epithelial precursor lesions, especially in cases without signs of dysplasia -13.The immunohistochemical analysis of p53 protein is a simple and inexpensive method that has been widely used for this purpose, and many studies have shown that it is involved in oral carcinogenesis and its alteration occurs early in the process of neoplastic transformation, often preceding recognizable histological alterations -14-16.p53 over-expression has been widely demonstrated to be a reliable predictor of progression to OSCC, either if we consider the over-expression as a physiologic response to induce cell cycle arrest in genetically altered hyper-proliferating cells or as a primary genetic defect leading to the production of high levels of mutated non-functional p53 protein; this is why the most utilized antibodies in the literature stain the total form of p53 protein and can not discriminate between wild or mutated form -18.However, p53 over-expression as a marker of progression to OSCC has not been considered highly sensitive, probably because only about 50% of OSCC are associated with over-expression of p53 protein -22.It follows that if the occurrence of p53 over-expression can be considered specific in detecting oral lesions at risk of developing p53-positive tumours, the presence of low p53 values should be considered as non-informative for prognostic purposes, since it can not distinguish between lesions with a genetically reduced production of p53 at high risk of developing p53-negative tumours, and lesions with a physiological low p53 activity or lesions that follow different carcinogenic pathways in which the p53 abrogation does not play a role ,11-22.It is well known that in non-neoplastic cells the expression of p53 is usually related to the cell proliferating rate, since p53 is considered as having an “oncogene checkpoint” function that guards cells against hyperproliferative signals .It follows that in non-neoplastic cells that respond to proliferative signals from inflammatory changes, traumas or hypoxia, a proportional increase in p53 activity should be expected -25.Thus, we may assume that the finding of an increased proliferating rate associated to a depressed p53 expression (increased mitotic/apoptosis ratio) in potentially malignant lesions might possibly identify genetically damaged lesions lacking of p53 protective role during cell cycling and may be at risk of progressing to p53-negative cancer.In the present study, the Ki67 protein has been utilized as a immunohistochemical marker of proliferating cells , and samA total of 126 biopsy samples from 126 consecutive patients who have referred to the Department of Oral Sciences of the University of Bologna, Bologna  between January 2004 to September 2006 were analyzed: 54 specimens from infiltrating OSCC, 27 from moderate/severe epithelial dysplasias , 32 from oral leukoplakias with epithelial hyperplasias and no signs of dysplasia, and 13 from normal epithelium oral mucosa obtained during third molar removal or as part of the excision of benign conditions. All samples underwent histological and immunohistochemical analysis.Histological diagnoses were performed at the Department of Pathology of the University of Bologna, at Bellaria Hospital , following the criteria describe in the WHO book. Specifically moderate\severe dysplasia  was characterized by increasing atypia, loss of polarity, and frequent mitoses, involving more than two-third of the epithelium while lacking infiltrative growth. Squamous cell Hyperplasia  was characterized by increased basal-parabasal layers, acanthosis, in absence of architectural alterations . No case1) shows an example of positive and negative staining of p53 protein.Immunostaining was performed on 2-µm thick sections serially cut from the selected blocks. The following antibodies were employed: monoclonal anti-Ki67  and monoclonal anti-p53 . The processing was performed in an automatic stainer . All the cases contained an internal control as basal cells of the oral epithelium show nuclear positivity for the two markers under study. Negative controls consisted in omitting the primary antibody. Counting the percentage of positive nuclei in 400 consecutive epithelial cells of selected areas representative of the lesion gave a semi-quantitative evaluation of the immunohistochemical results. Fig.  were considered when equal or more than 20%, while high Ki67/p53 ratio was considered when equal or more than 3.A multiple logistic regression model was fitted to describe the relationship between the presence/absence of dysplasia (dependent variables) or between dysplasia and OSCC (dependent variables)and 2 independent variables: p53 over-expression versus p53 low expression, and the combination of samples with high p53 and samples with high Ki67/p53 ratio versus low p53 and low Ki67/p53 ratio.One-way ANOVA was used to compare p53 mean values (continuous dependent variable) between samples from OSCC, epithelial hyperplasia, epithelial dysplasia and controls . Fisher’s least significant difference procedure (LSD) was the method to discriminate among the means at 95.0% confidence level.2). The wide Standard Deviation in samples from OSCC and oral lesions with dysplasia is the consequence of the contemporary presence of high and low p53 values in these oral lesions as documented by the density trace diagrams in Fig. (3).One-way ANOVA showed that the mean p53 value in OSCC (38.4±34.9) was significantly higher  than those found in controls and in oral lesions either with or without dysplasia . Fisher’s least significant difference procedure did not show any difference between the last three groups .Normal mucosa: the density trace diagram from normal samples showed a shape of distribution of p53 values characterized by a single peak with a median of 10% (range 5-15), and values always less or equal to 15%. The sample come from a normal distribution with a standardized skewness (.74) and kurtosis (-.17) values within a range expected from a normal distribution .OSCC: the density trace diagram from OSCC showed a shape of distribution of p53 values characterized by two well defined peaks that distinguished two very different population where high or low p53 values were expressed respectively in 31 (57%) and 23 (43%) samples . All samples with high p53 values had low Ki67/p53 ratios.A Ki67/p53 ratio equal or more than 3 was found in 19 patients out of 23 (83%) showing low p53 values (Table 3c). High Ki67/p53 ratio was found in 12 out of 17 (71%) samples with low p53 values of which 9 (82%) from margins of OSCC (Table 1). All samples with high p53 values had low Ki67/p53 ratios.Epithelial Dysplasia: similarly to OSCC the shape of distribution of p53 values showed two well defined peaks where high values are expressed respectively in 10 (37%) samples of which 8 (42%) from non-tumoral areas adjacent to OSCC , and high Ki67/p53 ratio was found in 11 out of 27 (37%) samples with low p53 values (Table 1). Again, all samples with high p53 values had low Ki67/p53 ratios.Epithelial Hyperplasia: high p53 values were found in 5 (15%) samples  of OSCC and in 81% (22 out of 27) of epithelial dysplasias, of which 89% (17 out of 19) from non-tumoral areas adjacent to OSCC, and in 50% (16 out of 32) of epithelial hyperplasias, while it was never observed in any sample from normal mucosa. Table The results of fitting a multiple logistic regression model to describe the relationship between the presence/absence of dysplasia and the 2 independent variables  showed that the only parameter related to the presence/absence of dysplasia and reaching the significance  was the presence/absence of samples with high p53 associated with samples with high Ki67/p53 ratio.No significant difference was found between OSCC and epithelial dysplasia when this combination was utilized .It is widely accepted that OSCC develops as a result of accumulation of genetic errors in the same tissue ,29, and TP53 mutations may result in a over-production of p53 inactive proteins which accumulate in the epithelium either due to blocking by another protein or due to partial degradation , or instThe shape of distribution of p53 values from OSCC in the present study, is in line with this statement, clearly showing the presence of two well defined peaks that distinguish two very different OSCC population, where high or low p53 values were expressed respectively in about 57% and 43% of cases. The prevalence of higher p53 values in the entire population explains the statistical difference in the mean p53 values between malignant samples and samples from normal mucosa.The evidence that the p53 pathway is very important in the biology of oral cancer has led to the application of such immunohistochemical analysis as a simple rapid and inexpensive method to potentially malignant lesions in an attempt to find a useful marker to predict progression to OSCC.Higher mean p53 values with respect to normal mucosa has been demonstrated in lesions with and without signs of dysplasia and it has been documented that lesions progressed to OSCC showed p53 over-expression early during the process, although most of the studies in the literature have utilized antibodies that can not discriminate between wild or mutated form ,35-36. NIn our study we did not find significantly higher p53 mean values in potentially malignant lesions with respect to normal mucosa although samples from lesions with severe dysplastic alterations showed higher mean p53 value than normal mucosa, but did not reach statistical significance. The limited number of controls may have contributed to hide a significant difference between p53 mean values from potentially malignant lesions and normal mucosa, although all p53 values from normal mucosa kept lower than the cut-off considered in the present and in a previously published study .However, p53 analysis did reveal differences between our potentially malignant lesions and normal mucosa since about 40% of sample in the group of dysplastic lesions and 15% in the group of leukoplakias without dysplastic alterations showed p53 values higher than the maximum value found in normal mucosa. We suppose that the prevalence of normal or low values in dysplastic lesions can explain the lack of statistical difference in the mean p53 values between pre-malignant lesions and normal mucosa.When concerning lesions with low p53 expression, the present data showed that low staining was present in more than 40% of OSCC, and even in about 60% of lesions with severe dysplasia, most of them at margins of OSCC and probably precursors of p53-negative tumours .If we take the p53 over-expression as a predictive marker, the occurrence of positive samples in our population is probably too low to discriminate lesions at “high risk” of malignancy, also considering that not all p53-positive lesions will progress to cancer .Furthermore, if we consider samples with low p53 expression as lesions “not at risk” of developing cancer, the occurrence of p53-negative lesions in our population of severe epithelial dysplasias is too high, considering that most of them were situated at margins of OSCC and thus at very high risk of malignant changes -39.These data underline the low predictive value of p53 when used as a single marker, since the high occurrence of low p53 staining in pre-malignant lesions is very difficult to be interpreted and not enough to rule out the progression to OSCC.This is in agreement with many reports that have stated that p53 has a good specificity, i.e the likelihood of malignant transformation is high when it is over-expressed, but a low sensitivity since the absence of p53 staining did not preclude malignant transformation in a considerable proportion of pre-malignant lesions ,40-41.Instead, in the presence of a lesion in predicate to develop cancer, the avoidance of false-negative results takes priority, so as not to miss patients who actually have a disease at risk of developing OSCC; in this situation, the sensitivity is most important to maximize negative-predictive accuracy, i.e. the likelihood that a negative test relates to the absence of any risk of malignancy .To this direction have gone our results obtained by the combination of samples with high p53 values and samples with low p53 values but high Ki67 values, i.e. a high Ki67/p53 ratio.We utilized this approach to see whether the presence of an increased mitotic activity not associated to an increased p53 activity might discriminate lesions with high proliferative activity where the presence of a genetic damage prevents a reactive stimulation of p53.The rationale was that in non-neoplastic cells, an increased cell proliferation due to a series of factors such inflammation, trauma etc, usually elicits a p53 protein accumulation that is considered to be a physiologic response to the increased proliferation rate of keratinocytes as a guardian of epithelial growth -44.In the present study, high Ki67/p53 ratios were found in 83% of OSCC with low p53 expression and even in 71% of severe epithelial dysplasias with low p53 values, and the combination of samples with p53 over-expression and samples with low p53 values but high Ki67/p53 ratio, led to a very high association with OSCC and epithelial dysplasias. About 90% of both OSCC and epithelial dysplasias were positive to this combination, while normal epithelium never was.Dysplasia is still now the most important predictive factor providing a surgical or non-surgical therapeutic approach. However histological assessment of dysplasia is extremely subjective , and somIn the present study, the combination of p53-over-expression and high Ki67/p53 ratio was present in about 50% of leukoplakias without signs of dysplasia.This percentage surely overestimates the risk of developing cancer, but could be useful to increase the predictive sensitivity and to rule out at least 50% of lesions with a presumably very low risk of developing OSCC.p53 immunostaining as a single marker can not be considered a sensitive marker that would help to assess the degree of cell alteration in oral lesions at risk of developing cancer.While it could be accepted that p53 over-expression in potentially malignant lesions may indicate a risk of developing cancer, the occurrence of low p53 staining is very difficult to be interpreted and does not rule out a possible link to oral carcinogenesis.In the present study the combination of p53 over-expression and high Ki67/p53 ratio seemed to better associate with the occurrence of OSCC and severe epithelial dysplasia, and may have a routine implication as an easy method to early detect oral lesions with an impairment of p53 pathway, and probably at risk of developing OSCC.


Overexpression of p53 correlates with neoplasia in many cytological specimens. To test the specificity of overexpressed p53 as a tumour marker for the detection of pancreatic cancer, we analysed cytological specimens of pancreatic juice samples from patients with pancreatitis or pancreatic carcinoma (n = 42) for p53 protein overexpression. p53 protein overexpression was found in 59% of patients with pancreatitis and 67% of patients with pancreatic carcinoma. Thus, the assessment of p53 protein overexpression is not useful in the diagnosis of pancreatic cancer. Overexpressed p53 during pancreatitis appears to be wild-type p53. Overexpression of p53 may result from DNA damage occurring during chronic inflammation. It is well established that p53 can induce apoptosis upon DNA damage. Consequently, we found apoptotic cell death in five out of five tested cytological preparations from patients with pancreatitis as well as in one out of one pancreatic carcinoma specimen.


Loss of p53 function because of mutation of the p53 gene is associated with over half all human cancers. Certain human p53 mutants are conformationally flexible in vitro and are temperature sensitive, with partial or complete recovery of wild-type (wt) properties at 32 degrees C. We have now tested the functional capacities of selected p53 mutants in vivo, by transfection into established human cell lines. Unexpectedly, we found that wt p53 can be temperature sensitive for transactivation of a co-transfected target gene in vivo. Flexible mutants retained varying degrees of functional capacity in transfected cells, and the recipient cell line appeared to be a significant determinant of both wt and mutant p53 function; importantly, two p53 null cell lines commonly used to study p53 function (Saos-2 and Hep3B) differed markedly in this latter respect. We also show that the p53 mutant V272M, which exhibits sequence-specific DNA binding in vitro, is nonetheless defective for transactivation and is unable to induce apoptosis in vivo. The valine 272 residue may thus be crucial for properties (other than sequence-specific DNA binding) that are important for p53 function(s) in vivo.


Understanding                        the molecular mechanisms and biological consequences of genetic changes                        occurring during bypass of cellular senescence spans a broad area of                        medical research from the cancer field to regenerative medicine. Senescence                        escape and immortalisation have been intensively studied in murine                        embryonic fibroblasts as a model system, and are known to occur when the                        p53/ARF tumour suppressor pathway is disrupted. We showed recently that murine                        fibroblasts with a humanised p53 gene  first senesce, and then become immortalised in the same way as their                        homologues with normal murine p53. In both cell types, immortalised cultures                        frequently sustain either a p53 gene mutation matching a human tumour mutation and                        resulting in loss of p53 transcriptional transactivation, or a biallelic deletion                        at the p19/ARF locus.  Whilst these genetic events were not unexpected, we were                        surprised to find that a significant proportion of immortalised cell cultures                        apparently had neither a p53 mutation nor loss of p19/ARF.  Here we consider                        various routes to p53/ARF disruption in senescence bypass, and dysfunction of                        other tumour suppressor networks that may contribute to release from tenacious                        cell cycle arrest in senescent cultures.                  Cellular senescence is an important                            defence mechanism against tumour metastasis, growth and progression ,2.     Our                            MEF cell line library comprises immortalised cell lines from cultures of                            fibroblasts derived from (a) embryos of a standard laboratory wild-type mouse                            strain (129/Sv), and (b) embryos from human p53 knock-in (Hupki) mice, which we                            constructed for our research on p53 biology -17.  TheWhilst                            the prevalence of cell lines immortalised by p19/ARF biallelic deletion or p53                            mutation was not unexpected , we were surprised to find that a significant                            fraction of cell lines (derived both from WT and Hupki primary cells) appeared                            to have retained WT p53 and p19/ARF expression, as examined by DNA sequencing                            or PCR amplification and promoter methylation analyses,  respectively ( and    Some                            of the MEF cell lines from (homozygous) WT or Hupki mice that we have examined                            and tentatively classified as p53 and p19/ARF wild-type thus may in fact have                            only one normal allele of these suppressor genes, having suffered loss of one                            copy in vitro allowing unlimited growth in culture.                                        Given                            that at least half of the spontaneously arising >100 MEF cell lines we                            examined for p53 and p19/ARF aberrations appeared to have overcome senescence                            block upon explanting in vitro by mechanisms other than the 2 canonical genetic                            events , there could well be various                            alternative pathways to immortalisation not directly involving damage to the immediate                            p53/p19ARF axis.                                        Considerable effort is underway to                            identify the key regulators of senescence and immortalisation. Since the                            process of senescence bypass provides an intrinsic phenotypic readout of                            functionality and automatically generates cell lines amenable to subsequent                            analysis, reverse genetics is a powerful approach to deciphering the important                            molecular events involved in senescence control . SeveralThe                            role of p53 mutation/p19ARF deletion in senescence bypass in MEFs reflects the                            importance of the p53/p19ARF axis as a master regulatory pathway of senescence                            in these cells.  What is apparent from genetic screening, as well as from other                            complementary work, is that many of the novel senescence-associated genes                            identified can also impact on this key pathway, both upstream and downstream of                            p53 ,26,28-31An entirely new aspect to the importance of cellular                            senescence has recently surfaced from experiments to produce iPS (induced                            pluripotent stem) cells from embryonic fibroblasts.  It has been shown that                            senescence provides a progressive barrier to conversion of primary MEFs (and                            indeed other types of differentiated cells) to pluripotency.  Crucially,                            disruption of the p53/p19ARF signalling axis greatly increased efficiency of                            their conversion ,41.  Fur


The p53 tumor suppressor protein is arguably the most                        important guardian of the mammalian genome. p53 promotes longevity by reducing                        somatic mutations or the survival and proliferation of mutant cells. Under                        normal conditions, this proteins role is inconsequential because of the                        rapidity of its degradation. But stress signals of almost any form halt the                        degradation of p53, unleashing an active protein that triggers transient cell                        cycle arrest, apoptosis, or cellular senescence . Both apAlmost all human cancers contain                        impairments in the p53 signaling pathway . MDM2  Senescence is a complex genetic program and a cell                        fate decision that establishes permanent growth arrest, which is a theurapeutic                        goal in cancer treatment. Although there is no direct link between cellular                        senescence and aging, a correlation does exist, as the number of senescent                        cells increases in mammals as they age . ActivatSenescent cells are characterized by highly compacted                        heterochromatin, termed "senescence-associated hetero-chromatin foci", or SAHF                        . These SHuang et al. conclude that the decline in                        p53 functionality in senescent cells is due to changes in upstream signaling                        that ultimately leads to destabilization of p53 and abrogated signaling.  They                        point to the possibility that the pro-apoptotic functions of p53 may be                        redundant in senescent cells, which have already lost their ability to                        proliferate and become cancerous.  These findings add to a recent publication                        by this group revealing that nutlin-3 can induce a senescence-like state in                        several epithelial cancer cell lines , but it The study by Huang and coworkers highlights several                        important issues in cancer therapy development. In addition to p53-mediated                        apoptosis, nutlins may induce and maintain a permanent dormancy in cancer                        cells. The ability for nutlin-3 to activate p53 without the genotoxic stresses                        incurred by most chemotherapeutic agents is of tremendous clinical importance.                        It may be of value to note that acetylation of p53 also inhibits p53-MDM2                        interactions ,12. Seve


The p53 signalling pathway has hundreds of inputs and outputs. It can trigger cellular senescence, cell-cycle arrest and apoptosis in response to diverse stress conditions, including DNA damage, hypoxia and nutrient deprivation. Signals from all these inputs are channeled through a single node, the transcription factor p53. Yet, the pathway is flexible enough to produce different downstream gene expression patterns in response to different stresses.We construct a mathematical model of the negative feedback loop involving p53 and its inhibitor, Mdm2, at the core of this pathway, and use it to examine the effect of different stresses that trigger p53. In response to DNA damage, hypoxia, etc., the model exhibits a wide variety of specific output behaviour - steady states with low or high levels of p53 and Mdm2, as well as spiky oscillations with low or high average p53 levels.We show that even a simple negative feedback loop is capable of exhibiting the kind of flexible stress-specific response observed in the p53 system. Further, our model provides a framework for predicting the differences in p53 response to different stresses and single nucleotide polymorphisms. The tumor suppressor protein, p53, is a transcription factor that regulates the activity of hundreds of genes involved in cell growth and death ,2. Over both activity and stability of p53. Mdm2 is an E3 ligase that binds to p53. Mono-ubiquitination of p53 by Mdm2 inhibits its transcriptional activity, while poly-ubiquitination triggers its degradation [mdm2 gene is activated by p53, thus forming a negative feedback loop [mdm2) on the p53 response.How does the regulatory network around p53 retain this exibility even though all inputs converge at a single node? We argue in this paper that the particular design of the p53-Mdm2 feedback loop at the core of this network could be the source of this flexibility. p53 is regulated by other proteins at two levels: its stability , and radation . In turnack loop . We use p; Mdm2, m; Mdm2 mRNA, mm; and the p53-Mdm2 complex, c. The temporal dynamics of these components of the model are specified by four differential equations:Our model focuses on the following four concentrations: nuclear-p53, http://www.ebi.ac.uk/biomodels-main, model number 1006280000.The model is provided in SBML format as additional file δ), activation of mdm2 by p53 (kt)). Others are "effective" parameters which model the combined action of several proteins that all affect p53 or Mdm2. For example, α models all Mdm2-independent processes which result in the reduction of the active p53 concentration in the nucleus: spontaneous degradation, export out of the nucleus, physical interaction with other proteins, sequestration in the cytoplasm (e.g. by Pirh2), modifications which prevent activity, etc. We also assume that activation of mdm2 by p53 has an associated Hill coefficient of 2 , and thpromotor  shows thMost previous models have used an explicit time delay to model transcription and translation . In This model system of a negative feedback loop shows plenty of variety in output behaviour. Depending on the values of parameters, the system is capable of steady state solutions with any combination of high or low p53 and Mdm2, as well as oscillations with high or low average p53. Figure Ideally, we would like to correlate each of these output states of the pathway to specific physiological responses like cell cycle arrest or apoptosis. Clearly, the level of p53 is an important determinant of the response ,22,23, aIn the absence of stress, p53 levels are typically maintained quite low. For this, a sufficient Mdm2 level is required to keep the half-life of p53 short. Thus, in a typical "resting" state there is a fairly high turnover of p53. The area shaded in green and blue in Figures δ and kt than to the p53-Mdm2 dissociation constant kD = kb/kf. Most stresses, however, affect more than one parameter.The system can be triggered by numerous stresses. We model different stresses as affecting different parameter combinations, as shown in Table Nutlin. Nutlin reduces the binding of Mdm2 to p53, while leaving its other properties unchanged. Nutlin treatment can trigger cell cycle arrest, but not apoptosis [kD (weakening the binding) alone causes a very modest increase in p53 levels  increased auto-ubiquitination of Mdm2, (b) decreased ubiquitination of p53 by Mdm2 and (c) weaker binding of p53-Mdm2 [γ, decreasing δ, and increasing kD. Single-cell experiments have found that irradiation of various types triggers oscillations in p53 levels with a period of 5-6 hours. The parameter changes used to mimic DNA damage stress were chosen such that the response matches the observations of Ref. [kD and γ, while lowering δ, corresponding to the molecular processes described above, our model produces an oscillatory solution in accordance with the experimental observations  when we apply a hypoxic stress when compared to other stresses with similar fold-changes in parameter values  results in an increased expression of mdm2 compared to the T allele [kt , as illustrated in Figure Our model can also be used to examine the behaviour of certain mutant cell lines. For instance, the G allele of the T allele . This conM, corresponds to 50,000 molecules per cell (assuming the cell is a sphere of radius around 6 μm). Such high numbers mean that the noise due to stochasticity in production and degradation of molecules is very small. Thus, the result of stochastic simulations of our model using the standard Gillespie algorithm [Single-cell measurements of p53 oscillations in response to various types of irradiation exhibit a fair amount of variability in the response across different cells ,31. The lgorithm  are indilgorithm  also see1. The variability in p53 response observed in ,31 must 2. Increasing amounts of noise are likely to introduce more variability in the position of later p53 peaks than in earlier peaks, as observed.However, a proper analysis of these hypotheses requires a better knowledge of which sources of noise underly the variability observed, so that they can be modelled accurately.γ result in large changes in peak p53 levels but hardly any change in average p53 levels.The sensitivity analysis in Figure mdm2 SNP309 - a prediction that is confirmed by observations. In addition, if the increase in kt due to the SNP is sufficient, then although p53 will be upregulated in response to stress, oscillations will not occur  between the first and second peaks.Our model could eventually be extended to cover other stresses that trigger a p53 response as more data becomes available. Nitric oxide (NO) is a free radical produced in inflamed tissue which can trigger the p53 pathway by phosphorylating p53 and thereby inhibiting its Mdm2-mediated degradation . AnotherκB signalling in the immune system [κB is a transcription factor that controls hundreds of downstream genes. It activates production of IκBα, which binds to and inhibits the action of NF-κB [Overall, we have shown that this kind of negative feedback loop, consisting of a relatively slow transciptional activation on one leg of the loop, and an inhibition based on fast complex formation on the other, can be designed to respond specifically to a number of different input triggers. This kind of negative feedback loop also occurs in another important signalling pathway that is triggered by hundreds of input signals, namely NF-e system . NF-κB iof NF-κB . The resof NF-κB ,47 similAH, SK and MHJ designed, implemented and analyzed the model, and wrote the paper. All authors read and approved the final manuscript.Model in SBML format. This file provides the p53-Mdm2 model we use in SBML format.Click here for fileSupplementary information. This file provides additional information and figures to supplement the main text.Click here for file


The p73 protein shows structural and functional homology to p53. For these reasons, p73 was considered as a positional and functional candidate tumour suppressor gene. Thus far, mutation analysis has provided no evidence for involvement of p73 in oligodendrogliomas, lung carcinoma, oesophageal carcinoma, prostatic carcinoma and hepatocellular carcinoma. In neuroblastoma, two mutations have been observed in a series of 140 tumours. In view of the occurrence of 1p deletions in Merkel cell carcinoma (MCC) and the location of p73 we decided to search for mutations in the p73 gene in five MCC cell lines and ten MCC tumours to test potential tumour suppressor function for this gene in MCC. In view of the possible complementary functions of p73 and TP53 we also examined the status of the TP53 gene. Sequence analysis of the entire coding region of the p73 gene revealed previously reported polymorphisms in four MCCs. In one MCC tumour, a mis-sense mutation located in the NH2-terminal transactivation region of the p73 gene was found. These results show that p73, analogous to neuroblastoma, is infrequently mutated in MCC. This is also the first report in which the role of TP53 in MCC has been investigated by sequencing the entire coding region of TP53. TP53 mis-sense mutations and one non-sense mutation were detected in three of 15 examined MCCs, suggesting that TP53 mutations may play a role in the pathogenesis or progression of a subset of MCCs. Moreover, typical UVB induced C to T mutations were found in one MCC cell line thus providing further evidence for sun-exposure in the aetiology of this rare skin cancer. © 2000 Cancer Research CampaignThe


Purpose. Rhabdomyosarcoma (RMS) is an embryonal tumor thought to arise from skeletal muscle cells that fail todifferentiate terminally. The majority of RMSs express MyoD, a protein essential to the differentiation of skeletal muscle.It was recently shown that during myogenesis, MyoD activates the expression of the cyclin-dependent kinase inhibitor(CDKi), p21, which itself plays a critical role in normal muscle development. To investigate the integrity of the MyoD/p21pathway in RMS, we analyzed p21 and its relationship to MyoD expression in RMS.            Methods. A panel of RMS samples was assembled from primary biopsies and from cell lines. Integrity of p21 was analyzedby single-strand conformation polymorphism (SSCP) and sequencing. Expression of p21 and MyoD was determined byNorthern blot analysis, and the ability of exogenous p21 to arrest the cell cycle of RMS cell line was determined bytransfection studies.            Results. Our analysis indicates that although p21 is wild type in RMS, there is an inverse correlation between the levelsof p21 and MyoD in these tumors. Tumors that express significant amounts of MyoD fail to express p21. This does notappear to be the result of mutations within the potential CACGTG sites present in the p21 promoter region or in thecoding region of p21. An additional group of RMSs express very high levels of p21 but express little, if any, MyoD.Furthermore, RD, a RMS cell line which expresses high levels of endogenous p21, undergoes withdrawal from the cellcycle following forced expression of p21, suggesting that the pathway which would lead to G1 arrest from endogenous p21 activity is defective.            Discussion. These data suggest that the interaction between p21 and MyoD is defective in RMS although the precisenature of the defect remains to be elucidated.


BMC Genomics has uncovered the early transcriptional responses to three types of stress and has demonstrated a central role for p38 MAPK in mediating these responses.The mitogen-activated protein kinase p38 (p38 MAPK) is activated by a number of stresses. A recent study in http://www.biomedcentral.com/1471-2164/11/144See research article  BMC Genomics by Ferreiro et al. [When cells are subjected to stress, such as osmotic stress, heat shock or infection, a number of intracellular signaling pathways are activated, leading to a coordinated cellular response. This may include the cessation of the cell cycle, commitment to apoptosis, the activation of DNA-repair pathways, regulation of protein translation, and the initiation of immune responses. A large body of evidence over the past 15 years has demonstrated that the p38 mitogen-activated protein kinase (MAPK) pathway is an important mediator of these stress responses . What isThe p38 pathway, like other MAPK pathways, features a cascade of protein kinases, culminating in the phosphorylation of p38 MAPK on specific threonine and tyrosine residues within its activation loop by the dual-specificity MAPK kinases MKK3 and MKK6, thereby leading to its activation ,2. ThereSaccharomyces cerevisiae p38 homolog Hog1 is primarily activated by osmotic stress and mediates a specific program of gene expression to combat and adapt to high osmolarity [Caenorhabditis elegans, genetic experiments have established a role for p38 in innate immunity and transcription profiling has demonstrated that p38 is required for upregulation of pathogen-response genes [Studies in yeast and nematodes, where the p38 pathway is conserved, have shed some light on how it regulates the transcriptional response to stress. The molarity . This ismolarity . In the se genes . In mammse genes ,8).et al. [To uncover the immediate p38-dependent transcriptional response to stress, Ferreiro et al. [Which genes are upregulated by the stimuli and how do they function in the cellular response to stress? To begin to address this important question, Ferreiro et al.  analyzedet al. [et al. and there is little overlap in the pool of genes upregulated [To obtain a fuller picture of how changes in transcription direct the cellular response to stress, Ferreiro et al.  analyzedegulated ,7,8. ThiThe temporal changes in the transcriptional profiles in response to osmotic stress can be linked to specific cellular responses. For example, short exposures to osmotic stress (45 minutes) led to the upregulation of solute carriers and ion transporters to allow cells to rapidly counteract the differences in osmotic pressure between the inside of the cell and the environment. Longer exposures (2 hours) results in the upregulation of genes encoding transcription factors and those involved in ribosome biogenesis, indicating a requirement for new protein synthesis, whereas 8 hours of exposure promotes the expression of genes involved in regulating cell survival and cell growth as well as cytokine and chemokine genes, suggesting a strong defense response.jun transcription via MEF2 and ATF2, two direct substrates of p38, whereas fos and egr-1 can be regulated by other p38 substrates, such as ternary complex factors (TCFs) [One of the significant findings of the study was that the most highly enriched functional grouping across all three stresses was for genes encoding transcription factors Figure . This was (TCFs) . The enret al. [et al. [For the future it will be important to determine how the p38-mediated gene-expression profiles differ between cell types and in response to other types of stress. The prediction, based on the study by Ferreiro ChIP: chromatin immunoprecipitation; GO: Gene Ontology; IPA: Ingenuity Pathway Analysis; MAPK: mitogen-activated protein kinase; MEFs: mouse embryonic fibroblasts; MKK: MAPK kinase; TNFα: tumor necrosis factor-α.


Carcinoma is more commonly found within the confines of benign PTs; whereas it is more often found surrounding the PT or in the contralateral breast in malignant PTs. Previous radiotherapy treatment is reported in only two cases. The aetiology of co-existing carcinoma is unclear but the rarity of previous radiotherapy treatment suggests that it is incidental. This case highlights the diverse pathology that can occur with PTs, which should be considered when evaluating pathology specimens as they may impact on patient management.A 43-year-old woman presented with a right breast lump that had enlarged over 5 months. She had chemoradiotherapy for non-Hodgkin's lymphoma in 1989. Histology revealed a malignant phyllodes tumour (PT) with liposarcomatous differentiation and ductal carcinoma in situ (DCIS) within the tumour with invasive tubular carcinoma, DCIS, and lobular carcinoma in situ in the surrounding breast. She had surgery and adjuvant radiotherapy. One year follow-up showed no recurrence or metastatic disease. Liposarcomatous differentiation is uncommon in PTs, and coexisting carcinoma is rare with 38 cases in 31 reports in the literature. Carcinoma is reported in malignant ( Phyllodes tumors (PTs) of the breast are uncommon biphasic fibroepithelial neoplasms that account for <1% of all breast tumours. Most PTs are benign and carry a risk of local recurrence whereas malignant PTs have a 13% risk of haematogenous metastasis .PTs are believed to arise from intralobular or periductal stroma and may arise de novo or from pre-existing fibroadenomas . Up to 3A 43-year-old woman presented with a palpable lump in the central aspect of the left breast below the nipple. The lump was present for five months. It had increased in size over that time and was tender. Mammography showed a relatively well-circumscribed 3.4 cm mass in the lower central aspect of the breast . An ultrA wide local excision of the left breast was performed which was followed by a wider cavity excision and latissimus dorsi flap reconstruction. The patient was given adjuvant radiotherapy. The breast received 50.4 Gy at 1.8 Gy per fraction with a cone-down boost to the tumour bed, giving a total dose of 63 Gy to the area considered at the greatest risk of recurrence. Follow-up after one year showed no evidence of recurrence or metastasis. Axillary sampling was not performed but no axillary lymphadenopathy was identifiedat one year follow-up.per 10 high power fields) were seen  included skeletal muscle and showed no residual PT. A single focus of DCIS, high-nuclear grade with comedo-necrosis and microcalcification, 3 mm in size, was present within 1 mm to the medial margin . MultiplMetaplastic change within PTs is uncommon. In the largest series of PTs reported, stromal metaplasia was present in only 11 of 335 cases, and malignant transformation of epithelium in the form of DCIS and LCIS was seen in only two cases . Stromaln = 19) in malignant PTs. In the malignant PT category, carcinoma occurred more frequently in the ipsilateral [n = 7) [n = 6; DCIS, n = 10) than lobular phenotype  [n = 8, 47%) [Malignant epithelial elements are reported in all categories of PT. In situ and invasive carcinoma may involve the tumour itself and/or coexist with a PT elsewhere in the same or contralateral breast. Carcinoma is most commonly reported  , 28, 33., n = 4) , 19–33.  8, 47%) , 30, 33. 8, 47%) , 25, 27,n = 10) [n = 5) [n = 1) [n = 10) were invasive, with or without an in situ component and most were ductal NST type (n = 6) [Carcinoma is less common in benign PTs compared with malignant PTs, given the higher prevalence of the former. Coexistent carcinoma is reported in 16 benign PTs  , 13, 17 n = 10) , 14, 17  [n = 5) , 15–17 o (n = 6) , 13, 15  (n = 6) , and tub (n = 6) , 15, oneThere are only three reports of carcinoma (DCIS) arising in association with borderline PT. Two patients had DCIS both within the PT and in the ipsilateral breast , 18, oneThe molecular events involved in transformation and progression of PTs are largely unknown, and the rarity of coexisting epithelial malignancy makes it difficult to draw conclusions about the aetiological association between the carcinoma and PT in these cases. Genetic aberrations have been consistently demonstrated in PTs with increasing frequency from benign to borderline to malignant PTs. Most studies to date focused on stromal alterations and showed recurrent copy number gains and losses at +1q, −13q, −6q, +5, and −10p –39. WherThe present case taken together with those in the literature demonstrates the diverse pathology that can be found with PTs, both within the tumour and in the surrounding and/or contralateral breast. The distinction between a malignant PT with coexisting carcinoma and a metaplastic carcinoma or carcinosarcoma should be considered in the diagnosis as these entities are managed differently and the distinction affects patient outcome. Carcinosarcomas have mixed malignant epithelial and stromal components with the latter showing no reactivity for epithelial immunohistochemical markers. While the present case fulfils these criteria, the characteristic leaf-like structure with malignant heterologous differentiation favours a diagnosis of malignant PT with coexisting carcinoma rather than a true carcinosarcoma. Carcinomas coexisting with PTs are generally diagnosed incidentally on the wide local excision, and the prognosis is dictated by the category of PT. In the present case, the malignant PT and not the carcinomatous elements will have the greatest impact on prognosis. However, the presence of coexistent carcinoma must be taken into account in management decisions, especially for benign and borderline PTs. Wider adequacy of excision of carcinoma, as in this case, and lymph node sampling should be considered.


Bronchial epithelial dysplasia is thought to be a premalignant stage in the evolution of lung cancers. Using the CM-1 polyclonal antibody, we have examined the expression of the p53 protein in a larger series of bronchial dysplasias (n = 60) than hitherto investigated. The p53 protein was detected in 14% of mild, 25% of moderate and 59% of severe dysplasias; increased p53 expression correlated with the severity of dysplasia. p53-positive dysplasias had greater PCNA indices than p53-negative dysplasias. p53 expression in dysplastic tissues was compared with that in two groups of histologically normal epithelium: 14 bronchial biopsies from non-cancer patients of which all but one were negative and 32 bronchial margins from resected carcinomas, of which 17 showed infrequent solitary cells with p53-positive nuclei in predominantly basal locations scattered throughout the epithelium. These results for resection margins were confirmed by use of a second antibody, DO-1. Sixty-nine per cent of the corresponding carcinomas were p53 positive, but in 15 cases the p53 reactivity differed from resection margins. No correlation between p53 expression and any of the clinicopathological characteristics of these tumours was found. This study supports the observation that abnormal p53 expression may be an early but not obligatory event in malignant transformation in lung.


Many cancer patients develop antibodies that recognize the overexpressed p53 protein. The presence of these antibodies is, in some tumour types, associated with poor prognosis. Gastric cancer is a highly prevalent disease associated with a high rate of mortality, there is a need for improved clinical and biological markers for disease behaviour. To investigate the clinical relevance of serum anti-p53 antibodies in patients with gastric adenocarcinoma, we have examined the sera of 501 gastric cancer patients for the presence of serum antibodies against the p53 protein. By immunoblotting analysis using a cell lysate containing overexpressed p53 protein as well as affinity-purified recombinant p53 protein as antigens, we have detected anti-p53 antibodies in 11.2% (61 of 501) of gastric cancer patients, but in none of 46 cancer-free individuals. The presence of anti-p53 antibodies was tightly associated with tumours of higher nuclear grade and lymph node metastasis, and a negative association was found between the presence of anti-p53 antibodies and survival. These results suggest that a preoperative test of serum anti-p53 antibodies in gastric cancer patients can be useful to identify subset of patients who may need gastrectomy with lymph node dissection and post-operative adjuvant therapy. © 1999 Cancer Research CampaignMutation of the


Malignant pleural mesothelioma (MPM) is a rare, aggressive tumor for which no effective therapy exists despite the discovery of many possible molecular and genetic targets. Many risk factors for MPM development have been recognized including environmental exposures, genetic susceptibility, viral contamination, and radiation. However, the late stage of MPM diagnosis and the long latency that exists between some exposures and diagnosis have made it difficult to comprehensively evaluate the role of risk factors and their downstream molecular effects. In this review, we discuss the current molecular and genetic contributors in MPM pathogenesis and the risk factors associated with these carcinogenic processes. Malignant pleural mesothelioma (MPM) is a solid, locally aggressive tumor of the pleura that encases and invades the lung parenchyma in late stages of the disease  at 22q12 are frequently altered TSGs that account for some of these deleted sites in MPMs [p16/CDKN2A in MPM tumor samples and cell lines, and for another TSG candidate RASSF1A located at 3p21 in cell lines [Tumor suppressor genes (TSGs) play vital roles in regulating the cell cycle in response to DNA damage and other stressors. The loss of TGS function is one of the fundamental events in tumorigenesis . Loss of in MPMs -22. Epigll lines ,24. Wilm+/- mice with crocidolite asbestos fibers, the mice that had lost the functioning allele of p53 had shorter latent periods and more aggressive tumors than the mice that still maintained one copy. Outside of this model, p53 function is more commonly affected by mutations in upstream and downstream members of the p53 pathways [A surprising finding in mesothelioma research is the lack of frequent mutations in the two most notorious TSGs: p53 and pRb. Although p53 mutations have been found in MPM cell lines ,25, therpathways ,25. The pathways . With thp16/CDKN2A is mutated. In brief, p16/CDKN2A can no longer inhibit cyclin-dependent kinases 4 and 6, which are responsible for the G1-S phase transition of the cell cycle. Left unchecked, these kinases can phosphorylate and thereby inactivate pRb, which allows an uncontrolled entry into S phase [p16/CDKN2A has been reported in >70% of malignant mesotheliomas, and has been associated with poor prognosis [The loss of pRb function occurs in a similar manner to p53. This time  S phase . Homozygrognosis ,31. In arognosis -37. HoweNF2, may help mesothelioma cells acquire this ability. Through the use of in vitro assays, Poulikakos et al. [in vivo.Not only must malignant tumors continue their growth, but they must also be able to move and invade. A recent paper by Poulikakos et al.  proposess et al.  found thWhile the loss of TSGs allows the cell to grow in light of aberrant changes in cellular DNA and function, it is the activation of oncogenes that inspires cell growth and proliferation. AP-1 and β-catenin transcription factors are implicated in MPM pathogenesis. The AP-1 family of transcription factors is known for mediating a wide range of processes including proliferation, apoptosis and transformation in response to a variety of stimuli . The actThe transcription factor β-catenin is regulated by an ubiquitin ligase pathway. In the absence of Wnt signaling, glycogen synthase kinase 3B (GSK3β) phosphorylates adenomatous polyposis coli (APC) and axin, which increases their affinity for β-catenin. GSK3β can then phosphorylate β-catenin, marking it for destruction. When Wnt is present, GSK3β phosphorylation is inhibited, allowing β-catenin to escape ubiquination, and enter the nucleus where it can bind to T-cell factor/lymphoid-enhancer factor (Tcf/Lef) transcription factor proteins thereby activating transcription of downstream effectors . The rolThe role of growth factors in carcinogenesis has already been suggested above in the case of MAPK pathway activation by EGF. Growth factors can stimulate proliferative pathways through their contact with membrane receptors. They may also play a role in the process of tumor invasion and metastasis, which has been demonstrated by their ability to stimulate chemotactic and/or chemokinetic motility in mesothelioma cell lines . MoreoveMany growth factors, such as insulin-like growth factor-1 (IGF-1) ,59, hepain vitro [VEGF is an angiogenic peptide, which is an independent prognostic factor in MPM . Higher in vitro . The usein vitro . One mecin vitro . It appein vitro .HGF has many possible roles in MPM pathogenesis. It may stimulate morphological changes , promotein vitro that asbestos induces the expression of TNF-α and its receptor in human mesothelial cells, and that cell survival may be mediated by the NF-κB pathway downstream of receptor activation. They also propose that other cells, macrophages in particular, may be important contributors of TNF-α in vivo [TNF-α may help to explain the survival of mesothelial cells after exposure to asbestos . Human m in vivo . More reApoptosis frequently occurs in response to signals from outside the cell. For example, TNF-related apoptosis-inducing ligand (TRAIL), Fas ligand and a lack of growth factor stimulation may result in programmed cell death . HoweverMany risk factors have been identified as contributors to MPM pathogenesis. Above, we began the discussion surrounding the contribution of genetics, asbestos, and SV40 infection. Here we will complete our discussion of these factors and further introduce concerns surrounding the relationship of MPM to radiation and to the pediatric population.Asbestos' contribution to the pathogenesis of MPM is multifaceted with effects ranging from direct to indirect, genetic to molecular. Asbestos induces mutations in mesothelial cells. The more direct mechanism of injury includes deposition of asbestos fiber in the pleura. Longer fibers can penetrate deeply into parietal pleura and have a high likelihood of causing cancer . AsbestoThe response of mesothelial cells to asbestos and ROS is an important factor in MPM pathogenesis. Above, we described the possible role of TNF-α in protecting mesothelial cells from death after asbestos exposure . The abiThe use of the single term "asbestos" to describe at least five unique fibrous silicate minerals . A causal relationship between malignant mesothelioma and asbestos exposure, radiation, and/or isoniazid could not be established. As far as asbestos is concerned, the long latency of asbestos-related mesothelioma makes a role for this risk factor in pediatric MPM unlikely [Mesothelioma is very rare in childhood with an estimated 2%–5% of all cases occurring in the first two decades of life. The diagnosis may be challenging because of its rarity and its pathologic similarities to other papillary or spindle cell neoplasms in the pediatric age group ,138. To unlikely ,141 unleunlikely .WT1 as a TSG in MPM pathogenesis. However, this picture is complicated by data that reports a lack of inactivating mutations in MPM [WT1 in a 45-year old woman with a perotineal mesothelioma [Pediatric and familial cases may open an avenue for the study of genetic contributions to MPM. Small case studies have found that pediatric patients with Wilm's tumor who were treated with radiation may be at an increased risk for mesothelioma ,142. This in MPM -145. Alsthelioma . It may Familial cases also support the role of genetic predisposition to MPM ,147. Howin vitro studies, and epidemiology, we may be able to gain a better understanding of which risk factors and molecular targets are the most important for future preventative and therapeutic measures.MPM has a complex etiology in which asbestos, ionizing radiation, viruses, genetic factors, and even diet  may act The authors declare that they have no competing interests.SJW and SNM both contributed to the research and writing. All authors read and approved the final manuscript.


Recent reports suggest that the product of hepatitis B virus (HBV) interacts with p53 and that the hepatitis C virus (HCV) core protein reduces p53 expression. A novel p73 gene, which is related to p53, has recently been identified and mapped to chromosome 1p36.3, which is a locus of multiple tumour-suppressor genes for many cancers, including hepatocellular carcinoma (HCC) and neuroblastoma. Here, we investigated mRNA expression, allelotype and mutation of p73 in 48 HCCs obtained from untreated patients. Reverse transcriptase polymerase chain reaction (RT-PCR) revealed that p73 mRNA was expressed ubiquitously at low levels in all the tumour tissues, as well as in the adjacent normal liver tissues. The frequency of p73 loss of heterozygosity was observed in 20% of HCCs, but PCR-single strand conformation polymorphism (SSCP) analysis showed no mutations in the 48 tumours except for three types of polymorphisms. These results suggest that p73 may play a role in hepatocellular carcinogenesis in a different manner from a Knudson two-hit model. The regulatory mechanism of interaction between p73 and hepatitis viruses remains to be determined. © 1999 Cancer Research CampaignAccumulating evidence has demonstrated that aberration of the


This review is intended to summarize the risk factors, classification, diagnosis, and treatment of heterotopic ossification (HO) of previously published studies. Heterotopic ossification is a common complication of total hip arthroplasty. Its prevalence is not the same in all of the patient groups. Frequency of HO varies from 15 to 90%. Hip ankylosis, male gender, and previous history of HO are said to be risk factors with a significant level. Diagnosis is based on a single AP radiograph: the Brooker classification that divides HO into four grades is the most commonly used. The confirmation test that can be used is a bone scan. A great amount of bone metabolic turnover markers have been tested, but none of them seems to be relevant in case of prevention or diagnosis of HO. The most effective prophylactic treatment is radiotherapy or administration of nonsteroidal anti-inflammatory drugs. Over the years a lot of different RT protocols have been tested. Nowadays the most often used regimen is 7 Gy given postoperatively in a single dose. The most commonly prescribed drug in prophylaxis of HO is indomethacin. Also, the efficacy of ibuprofen and diclofenac was proven. Recently researchers focused on selective COX-2 inhibitors. They appear to be as effective as nonselective NSAIDs having less side effects. The one and only treatment of HO is a revision arthroplasty. Heterotopic ossification (HO)  is a relThis is an extremely important challenge, as ossifications may destroy the healing effect of surgery, limiting to a large extent of range of motions in the hip joint and additionally exposing the patient to subsequent surgical treatment, revision arthroplasty.A search was performed on PubMed, Embase, and Cochrane Central Register. It was conducted using key word “heterotopic ossification” combined with either “THR”, “total hip replacement”, “THA”, “total hip arthroplasty”, “NSAIDs”, “risk factors”, “radiotherapy”, “radiation”, “marker”, or “markers”. Also, the search of references in included studies was performed. There were no restrictions in language or date of publication. Studies were excluded from our analysis if there were abstracts, letters, case reports, case series, guidelines, or irrelevant significance for our study topic. We identified 4628 articles. Of these articles, 836 were removed due to duplicate reportage, and 3675 were excluded based on the titles and abstracts. The remaining 117 articles were accessed for the full text and screened for further assessment. Finally, we included 63 articles in our review.A number of studies demonstrating the existence of potential risk factors for heterotopic ossifications after hip arthroplasty have been found in the literature , 19–24.In the recent meta-analysis it was estimated that male sex, cemented prosthesis, bilateral hip joint arthroplasty procedure, ankylosing spondylitis, and hip joint ankylosis are associated with increased risk of nonarticular ossification . In addiAccording to the same study, the only factor that is said to decrease HO is rheumatoid arthritis .In addition, it has been demonstrated that surgeries due to femoral neck fracture, degenerative disease, previous hip fracture, hypertrophic type of osteoarthritis, and age are not associated with an increased risk of heterotopic ossification.There have been studies evaluating the assessment of the impact of revision surgery, lateral approach in hip arthroplasty, BMI, bone grafting, and trochanteric osteotomy on the potential occurrence of ossification but even if they reported a positive correlation they require further research .The most commonly used method of classification is Brooker classification based only on a single X-ray image in the anteroposterior (AP) projection , 25, 26.Usually, ossifications that develop after surgery are visible in the radiological examination after 4-6 weeks , 27–30.The Brooker classification divides the heterotopic ossifications into four grades :I - described as islands of bone within the soft tissues about the hip;II - consisting of bone spurs originating from the pelvis or proximal end of the femur, leaving at least 1 cm between opposing bone surfaces;III - consisting of bone spurs originating from the pelvis or proximal end of the femur, reducing the space between opposing bone surfaces to less than 1 cm;IV - hip joint ankylosis , 26.Types III and IV are referred to as clinically relevant , 30, 31.The level of alkaline phosphatase in serum increases during tissue injuries; therefore its predictive value in heterotopic ossifications after hip arthroplasty is doubtful . HoweverWilkinson et al. tried to find alternative bone metabolic turnover markers that could be used in the diagnosis of heterotopic ossifications in patients after hip arthroplasty. According to their study, C telopeptide of type I-collagen (CTX-1) is potentially useful marker, which increases its concentration in the first postoperative week by 42% and shows a sensitivity of 89% and specificity of 82% in the prediction of the diagnosis of ossifications in the 26th week after surgery . UnfortuBased on Wilkinson's research, we can reject N-terminal propeptide of type I-procollagen (PINP) and osteocalcin (OC) from potential testings, as their increased correlation with HO occur too late to implement effective prophylaxis. Moreover, the levels of N-telopeptide of type I-collagen (NTX-1) and deoxypyridinoline (DPD) were not correlated at all with the presence of ossifications .Bone metabolic turnover markers have also general diagnostic limitations. Their level fluctuates in relation to gender, coexisting diseases and some are prone to daily or seasonal fluctuations, which makes them difficult to use as a simple diagnostic tool .In papers heterotopic ossification symptoms include swelling, redness, and pain as subjective symptoms. Therefore, symptoms of ossification may imitate deep vein thrombosis in the lower limbs or cellulitis. Unfortunately, none of these studies are supported by the relevant figures , 39–41.Scintigraphy is the most sensitive method in diagnosing HO , 36, 42.The formerly used radiopharmaceutical was strontium, currently 99mTc; pyrophosphate is used , 41, 42.Scintigraphy is based on the administration of a radiopharmaceutical and the subsequent production of a series of scans that will provide information during three phases: a dynamic blood flow, blood accumulation, and the accumulation of a radiopharmaceutical in the bone .Scintigraphy is particularly effective in determining the maturity of heterotopic ossifications and planning their operational removal , 42. TheSchurch et al. conducted a study to evaluate the usefulness of prostaglandin E2 (PGE-2) measurements in urine to predict the formation of HO. They reported increased level of PGE-2 in urine in patients with developing ossification. The authors recommended examining 24-hour PGE-2 level once a week for a period of 3 to 4 months. The increase in this excretion was expected to indicate the development of heterotopic ossification .Orzel et al., in their retrospective analysis of 24 patients' ossifications, showed that calcium levels in serum had decreased below the reference level in 23 cases. The reduction occurred on average during fifth day and lasted on average ten days . It seemProphylaxis of Heterotopic Ossification: Methods and Their Effectiveness(1) Radiotherapy (RT). It is estimated that the prevalence of HO should not be greater than 10% after the radiation prophylactic treatment [reatment , 44.Over the years many RT regimens have been tested. The first observation was published by a team from Mayo Clinic. They used fractionated RT (2 x 10 Gy) . Then reIn a meta-analysis Milakovic et al. found no significant differences in a HO prevalence between the patients treated with banana equivalent dose (BED)>2500 cGy and the patients treated with BED doses <2500 cGy .The difference in HO occurrence was so significant that the researchers advised not to use that protocol again. It seems that Finegorth et al. found the boundary of an effective radiation protocol with a 6 Gy given postoperatively .Nowadays the most often prescribed is a single dose radiation of 700 Gy .Also, the effectiveness of preoperative radiation has been measured , 27, 43.The most important factor in a prophylactic radiation treatment is time. Seegenschmiedt concluded that it must not be given earlier than 8 hours before the procedure and later than 72 hours after the procedure . The othFinally, the fractionated radiation has been compared with a single dose radiation. Konsi et al. in a randomized study showed the same efficacy of a single dose radiation compared to a fractioned one . MilakovRT is not only effective, but safe. Sheybani et al. analyzed records of over 3500 patients who underwent a THR [(2) Relevance of Using Medications(a) Nonsteroidal Anti-Inflammatory Drugs (NSAIDs). The most commonly used drug in HO prophylaxis is indomethacin. It is probable mechanism of stopping the bone formation is by inhibiting the inflammation process [ process .Schmidt et al. in a prospective, double-blind, randomized study proved the effectiveness of indomethacin in HO prevention . It was Another drug whose efficacy has been proved is ibuprofen. Elmsted et al. showed its effectiveness compared to a placebo . SodemanIt appears that the shortest possible treatment time with nonselective oral NSAIDs is one week . It shouPritchett studied the efficacy of ketorolac in a prospective, double-blind, randomized study . It was It appears that there are no big differences between the two most studied prophylactic treatments. Kienapfel et al. conducted a study that compared indomethacin given in a dose of 2x 50 mg for 42 days with a single dose 600 Gy postoperative radiation. The treatments were equally effective in preventing HO [Knells et al. conducted prospective study comparing indomethacin with acetylsalicylic acid and radiotherapy. They concluded that the most effective prophylactic treatment for the general population is indomethacin prescribed in a dose of 250 mg daily for 14 days starting on the first postoperative day. The patients with a higher chance of developing HO or with the contradictions for nonselective NSAIDs should be qualified for RT .Kolbl et al. compared preoperative radiation (7 Gy 16-20h before the procedure) and diclofenac (2x75mg for 14 days starting at the 1st postoperative day). They showed no difference in lowering the clinically significant HO occurrence, although the overall prevalence of HO was lower in diclofenac group .The only prospective study evaluating efficacy of combined therapy conducted Pakos et al. . They coSelective cyclooxygenase-2 (COX-2) inhibitors are a very attractive form of treatment, because they show less gastrointestinal side effects and lack interaction with platelets aggregation , 23, 57.The elevated cardiovascular risk was observed after 6 months of treatment , period way longer than prophylactic treatment time after total hip replacement (THR) .Rofecoxib and celecoxib has been the most studied agents and proved reliable .Lavernia et al. proved effectiveness of etoricoxib compared to historical group that did not take any prophylaxis at all . PatientFinally, the very conclusive results came with both Grohs and van der Heide studies , 58. TheOni et al. conducted prospective study with celecoxib . PatientSaudan et al. compared celecoxib (2x200 mg) and ibuprofen (3x400 mg). Celecoxib turned out to be more effective in preventing HO than ibuprofen .Xue et al. conducted a meta-analysis of randomized studies that compared NSAIDs with selective COX-2 inhibitors . They foAnother meta-analysis by Shun Lin et al. showed better outcomes in HO prevention after NSAIDs prophylaxis treatment compared with placebo. In this study, researchers also measured the discontinuation of the therapy due to gastrointestinal side effects and compared it between groups treated with nonselective NSAIDs and selective COX-2 inhibitors. The discontinuation was more frequent in the nonselective NSAIDs group.(b) Diphosphonates. The prophylaxis of HO that was proven to be ineffective is treatment with diphosphonates [phonates , 47, 49.phonates , 47, 59.phonates , 47, 59.phonates . The firBijovet et al. also proved inefficacy of diphosphonates in HO prophylaxis by conducting a prospective study . PatientGrades I and II HO may not need any treatment unless they become symptomatic or functionally disabling. The gold standard therapy for grades III and IV HO is a revision arthroplasty and surgical resection of the ossification , 42, 46.HO is one of the most common complications after THA. Until now, no algorithm for detection and treatment has been developed. A series of papers agree on risk factors and we collected those in


LPA, which encodes for apolipoprotein(a) [apo(a)], is causally associated with CAVD. Apo(a) is the protein component of Lp(a), a LDL-like particle, which transports oxidized phospholipids (OxPLs). Autotaxin (ATX), which is encoded by ENPP2, is a member of the ecto-nucleotidase family of enzymes, which is, however, a lysophospholipase. As such, ATX converts phospholipids into lysophosphatidic acid (LysoPA), a metabolite with potent and diverse biological properties. Studies have recently underlined that ATX is enriched in the Lp(a) lipid fraction. Functional experiments and data obtained in mouse models suggest that ATX mediates inflammation and mineralization of the aortic valve. Recent findings also indicate that epigenetically-driven processes lower the expression of phospholipid phosphatase 3 (PLPP3) and increased LysoPA signaling and inflammation in the aortic valve during CAVD. These recent data thus provide novel insights about how lipoproteins mediate the development of CAVD. Herein, we review the implication of lipoproteins in CAVD and examine the role of ATX in promoting the osteogenic transition of valve interstitial cells (VICs).Calcific aortic valve disease (CAVD) is a complex trait disorder characterized by calcific remodeling of leaflets. Genome-wide association (GWA) study and Mendelian randomization (MR) have highlighted that  Il1rn), a natural circulating inhibitor of interleukin 1 beta (Il1b), develop a thickening of the aortic valve and a rise of transaortic velocities  is a prevalent cardiovascular disorder. Fibro-calcific remodeling is one key process involved in the development of CAVD. During CAVD, fibrosis and mineralization of the aortic valve (AV) cusps progressively lead to a narrowing of the aortic orifice, a clinical condition referred to as aortic valve stenosis . Differelocities . VIC, a locities . As suchLPA, which encodes for apoliprotein(a) [apo(a)], is causally associated with CAVD  and traits/disorders, have underlined the genetic architecture of these traits and have fuelled the development of novel therapies. Though the genetic architecture of CAVD is just emerging, recent findings have transformed the field and have helped to pinpoint causal pathways  in the region encoding for kringle IV type 2 domain (KIV2), which is inversely related to the circulating level of Lp(a) (LPA locus rs10455872 is associated with the number of KIV2 repeats and with plasma level of Lp(a). It is worth highlighting that plasma level of Lp(a) is largely determined by genetic factors and heritability may explain up to 90% of the variance  . The indvariance . In a MR in CAVD . However in CAVD . In a la in CAVD . Also, g in CAVD . Among s in CAVD . These f10 domain of apo(a) is attached covalently to OxPL (Apo(a) is a highly polymorphic lipoprotein, which is linked to apolipoprotein B(apoB) moiety of low-density lipoprotein (LDL) by a disulfide bridge . Lp(a) i to OxPL . Of note to OxPL . Also, t to OxPL . OxPL is to OxPL , 30.ACTA2) and secrete ECM components such as collagen and remodeling enzymes (TGFB1) is expressed during the early stages of CAVD and promotes the transformation of quiescent cells into activated VICs . ALPL along with other ecto-nucleotidases, such as ENPP1, are overexpressed in surgically explanted mineralized aortic valves  . Of intePLPP3 was decreased by 49% in mineralized AVs. These data were corroborated by measuring PLPP activity, which was also reduced by 31% in mineralized AVs  was increased by 1.5-fold. Mapping provided evidence that a dysregulated epigenetic process was impinging on the expression of PLPP3 during the mineralization of cusps. DNA methylation mapping showed that CpG methylation in intron 1 (cg02468627) of PLPP3 was inversely associated with the expression of this gene and was increased in mineralized portion of explanted aortic valves  are membrane-associated enzymes involved in the degradation of LysoPA. As such, PLPPs, which reside at the cell membrane along with LysoPA receptors (LPARs), are key modulator of LysoPA signaling in cells. Transcriptomic analysis of control non-mineralized and mineralized aortic valves showed that among the PLPP enzymes PLPP3 was significantly downregulated during CAVD . Analysiized AVs . Of notec valves . This Cpof PLPP3 . Epigenen by 38% . These fCOL1A1 and the mineralization of VIC cultures through a nuclear factor kappa B (NFκB) pathway. In VICs, LPAR1 activated RhoA and Rho kinase (ROCK), which promoted a robust expression of NFκB-dependent genes such as IL6 and IL8. In addition, we found that BMP2, a morphogenetic protein that drives osteogenesis, was overexpressed downstream of LPAR1-NFκB. Analysis showed that promoter region of BMP2 contained κB responsive elements and that LysoPA promoted the phosphorylation of p65 on serine 536 (p65 S536) (BMP2 in chromatin immunoprecipitation (ChIP) assay. During NFκB signaling the inhibitor of κB (IκB) is rapidly induced and transits to the nucleus where it competes with p65 for κB sites. This process ensures a negative feedback loop to control the inflammatory response mediated by the NFκB pathway. However, phosphorylation of p65 S536 is not sensitive to the nuclear inhibition provided by IκB. Hence, genes dependent on phosphorylated p65 S536 are less sensitive to negative retro-feedback and experienced sustained expression. Experiments carried out with constitutively active mutant IκB super repressor (IκBα SS32-36AA) showed that LysoPA-induced expression of BMP2 was not sensitive to the repression  . Of intepression . These dve cusps . In thisLPA in CAVD, which has been validated in different cohorts, has provided considerable interest to understand the role of lipoproteins in CAVD. MR studies showed that Lp(a) is causally associated with CAVD. Thus, these data strongly suggest that interventions aimed to modify the level of Lp(a) could provide therapeutic benefit   conducted with statins have been negative in CAVD and have questioned the role of lipoproteins as causal factors in CAVD. However, the strong genetic signal for  benefit . The fai benefit . For ins benefit . Recentl benefit . Also, i benefit . In addiPL (EO6) . FurtherPL (EO6) .LPAR1 and PLPP3 respectively. LPAR1 promotes a signaling cascade that culminate in the phosphorylation of p65 S536 and a sustained expression of BMP2, a key driver of osteogenic transformation in VICs. On the other hand, epigenetically reprogrammed VICs at a transposon-based enhancer is a proximal event that contributes to lower PLPP3 and to promote/exacerbate LysoPA signaling in VICs. Together, these data suggest that lipoprotein metabolism during CAVD is perturbed and promotes inflammation and mineralization. Further work and elucidation of key processes in lipoprotein metabolism during CAVD could lead to the development of novel therapies.Genetic studies have underscored that Lp(a) is causally associated with CAVD. Molecular analyses have provided evidence that interaction between ATX and Lp(a) is promoting inflammation and osteogenic reprograming in VICs. Dysregulation of LysoPA signaling in mineralized aortic valve includes a higher and a lower expression of The datasets for this manuscript are not publicly available. Requests to access the datasets should be directed to Patrick Mathieu.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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.