Self-Renewal Inhibition in Breast Cancer Stem Cells: Moonlight Role of PEDF in Breast Cancer.

Breast cancer is the leading cause of death among females in developed countries. Although the implementation of screening tests and the development of new therapies have increased the probability of remission, relapse rates remain high. Numerous studies have indicated the connection between cancer-initiating cells and slow cellular cycle cells, identified by their capacity to retain long labeling (LT+). In this study, we perform new assays showing how stem cell self-renewal modulating proteins, such as PEDF, can modify the properties, percentage of biomarker-expressing cells, and carcinogenicity of cancer stem cells. The PEDF signaling pathway could be a useful tool for controlling cancer stem cells' self-renewal and therefore control patient relapse, as PEDF enhances resistance in breast cancer patient cells' in vitro culture. We have designed a peptide consisting of the C-terminal part of this protein, which acts by blocking endogenous PEDF in cell culture assays. We demonstrate that it is possible to interfere with the self-renewal capacity of cancer stem cells, induce anoikis in vivo, and reduce resistance against docetaxel treatment in cancer patient cells in in vitro culture. We have also demonstrated that this modified PEDF protein produces a significant decrease in the percentage of expressed cancer stem cell markers.

Case Report: Precision genetic diagnosis in a case of Dyggve-Melchior-Clausen syndrome reveals paternal isodisomy and heterodisomy of chromosome 18 with imprinting clinical implications.

A twelve-year-old patient with a previous clinical diagnosis of spondylocostal skeletal dysplasia and moderate intellectual disability was genetically analyzed through next generation sequencing of a targeted gene panel of 179 genes associated to skeletal dysplasia and mucopolysaccharidosis in order to stablish a precision diagnosis. A homozygous nonsense [c.62C>G; p.(Ser21Ter)] mutation in DYM gene was identified in the patient. Null mutations in DYM have been associated to Dyggve-Melchior-Clausen syndrome, which is a rare autosomal-recessive disorder characterized by skeletal dysplasia and mental retardation, compatible with the patient´s phenotype. To confirm the pathogenicity of this mutation, a segregation analysis was carried out, revealing that the mutation p(Ser21Ter) was solely inherited from the father, who is a carrier of the mutation, while the mother does not carry the mutation. With the suspicion that a paternal disomy could be causing the disease, a series of microsatellite markers in chromosome 18, where the DYM gene is harbored, was analyzed in all the members of the family. Haplotype analysis provided strong evidence of paternal isodisomy and heterodisomy in that chromosome, confirming the pathological effect of this mutation. Furthermore, the patient may have a compromised expression of the ELOA3 gene due to modifications in the genomic imprinting that may potentially increase the risk of digestive cancer. All these results highlight the importance of obtaining a precision diagnosis in rare diseases.

Brief Report: Evidence of Autism Spectrum Disorder Caused by a Mutation in ATRX Gene: A Case Report.

ATRX mutations are commonly associated with alpha-thalassaemia mental retardation syndrome (ATR-X syndrome) with a notable variable expressivity. This X-linked disorder is characterized by intellectual disability (ID) in a higher or lesser degree, in which the alpha-thalassaemia feature is not always present. Other phenotypic manifestations like facial dimorphism, hypotonia, microcephaly, skeletal abnormalities or urogenital malformations have been frequently observed in ATR-X syndrome. Herein, we report a missense ATRX mutation (Thr1621Met) in a patient with an autism spectrum disorder (ASD) diagnosis. Except for ID, no typical signs of ATR-X syndrome were found in the patient. These results confirm the extensive phenotypic variability associated to ATRX mutations and show the involvement of this gene in the ASD.

Effective Personality as a Protective Factor in Teachers' Occupational Health.

In recent decades, there has been a growing body of research showing the relationship between teaching work and several health problems, both physical and psychological. Some of these studies relate personal competencies and resources to teachers' occupational health. Based on the construct of Effective Personality, proposed by Martin del Buey, Martín Palacio, and Di Giusto, the aim was to analyse the relationship between the dimensions of the construct and Teachers' Occupational Health. A descriptive cross-sectional design was used. It was based on the application of the Teacher Health Questionnaire (CSD) and the Efficacy Personality Questionnaire-Adults (CPE-A). The sample consisted of 700 non-university teachers aged between 26 and 66 years, M = 47.65 SD = 8.68. Descriptive, correlational, linear regression, and structural equation analyses were carried out. The results confirmed the relationship between the Efficacy Personality construct and Teachers' Occupational Health (r = 0.45 **). In addition, the regression analysis indicated the relevance of each factor of Efficacy Personality in the factors of Teachers' Occupational Health. The variance of Self-efficacy is the most explained by the dimensions of Efficacy Personality (40.2%), with positive relationships. The structural equation analysis confirmed the influence between Efficacy Personality and the factors of Self-Efficacy and Satisfaction, explaining 55.0% of the variance. It is concluded, therefore, that Efficacy Personality has a protective function on Teacher Occupational Health; the higher the Efficacy Personality scores are, the better the results in health gain-Self-efficacy and satisfaction-and the lower the result in health loss-burnout, cognitive affections, musculoskeletal affections, and voice alterations. These results facilitate the design of prevention and intervention programmes for teachers' occupational health, which strengthen and improve personal and socio-affective competencies.

High-Performance Handball Player's Time-Motion Analysis by Playing Positions.

The purpose of this study was to analyze the on-court demands of handball players during the European Handball Federation Champions League Final Four (VELUX EHF FINAL4) 2019 to define time-motion characteristics (played time; covered distances) both in offense and defense. Furthermore; we aimed to define position-specific demands and differences among them. Forty players from three teams were analyzed during the tournament using a local positioning system (LPS) for the first time in top handball. Players covered similar distances both in offense (1388.28 ± 2627.08 m), and in defense (1305.47 ± 5059.64 m) and remained on court for a similar average time (15.69 ± 8.02 min and 15.40 ± 8.94 min respectively). When locomotion activities were normalized according to the time they spent on court; significant differences were found for defense compared to offense in walking (+20%; p < 0.000; Cohen's effect size (ES) = 1.01) and jogging (-29.6%; p = 0.000; ES = 0.90), as well as a tendency for high-intensity running (+ 25.2%; p = 0.077; ES = 0.31). Per playing position; center and left back (CB = 94.86 ± 10.98 m·min-1; LB = 96.55 ± 24.65 m·min-1) showed the highest running pace in offense and mid-left; front center defender and outside right for the defense (ML = 90.38 ± 30.16 m·min-1; FCD = 87.04 ± 14.94 m·min-1; OR = 89.64 ± 34.93 m·min-1). In conclusion; profile differences existed among players' position activity; both in offense and defense; which should be taken into account when designing specific physical training programs.

Pedf derived peptides affect colorectal cancer cell lines resistance and tumour re-growth capacity.

Relapse after chemotherapy treatment depends on the cancer initiating cells (CICs). PEDF (Pigmented Epithelium Derived Factor) is an anti-angiogenic, neurotrophic and self-renewal regulator molecule, also involved in CICs biology. Acute and chronic exposition of colon cancer cell lines to CT/CTE PEDF-derived peptides decreased drug-resistance to conventional colorectal cancer treatments, such as oxaliplatin or irinotecan. We confirmed a reduction in the irinotecan and oxaliplatin IC50 doses for all tested tumour cell lines. After xenograft transplantation, CT/CTE treatments also produced a reduction in resistance to conventional chemotherapy treatments as in culture-assays. Metastatic capacity of these treated cell lines was also depleted. The PEDF signaling pathway could be a future therapeutic tool for use as an adjuvant therapy that decreases IC50 dosis, adverse effects and treatment costs. This pathway could also be involved in an increase of the time relapse in patients, decreased tumourigenicity, and decreased capacity to produce metastasis.

Molecular and neurochemical substrates of the audiogenic seizure strains: The GASH:Sal model.

PURPOSE: Animal models of audiogenic epilepsy are useful tools to understand the mechanisms underlying human reflex epilepsies. There is accumulating evidence regarding behavioral, anatomical, electrophysiological, and genetic substrates of audiogenic seizure strains, but there are still aspects concerning their neurochemical basis that remain to be elucidated. Previous studies have shown the involved of γ-amino butyric acid (GABA) in audiogenic seizures. The aim of our research was to clarify the role of the GABAergic system in the generation of epileptic seizures in the genetic audiogenic seizure-prone hamster (GASH:Sal) strain. MATERIAL AND METHODS: We studied the K+/Cl- cotransporter KCC2 and ß2-GABAA-type receptor (GABAAR) and ß3-GABAAR subunit expressions in the GASH:Sal both at rest and after repeated sound-induced seizures in different brain regions using the Western blot technique. We also sequenced the coding region for the KCC2 gene both in wild- type and GASH:Sal hamsters. RESULTS: Lower expression of KCC2 protein was found in GASH:Sal when compared with controls at rest in several brain areas: hippocampus, cortex, cerebellum, hypothalamus, pons-medulla, and mesencephalon. Repeated induction of seizures caused a decrease in KCC2 protein content in the inferior colliculus and hippocampus and an increase in the pons-medulla. When compared to controls, the basal ß2-GABAAR subunit in the GASH:Sal was overexpressed in the inferior colliculus, rest of the mesencephalon, and cerebellum, whereas basal ß3 subunit levels were lower in the inferior colliculus and rest of the mesencephalon. Repeated seizures increased ß2 both in the inferior colliculus and in the hypothalamus and ß3 in the hypothalamus. No differences in the KCC2 gene-coding region were found between GASH:Sal and wild-type hamsters. CONCLUSIONS: These data indicate that GABAergic system functioning is impaired in the GASH:Sal strain, and repeated seizures seem to aggravate this dysfunction. These results have potential clinical relevance and support the validity of employing the GASH:Sal strain as a model to study the neurochemistry of genetic reflex epilepsy. This article is part of a Special Issue entitled "Genetic and Reflex Epilepsies, Audiogenic Seizures and Strains: From Experimental Models to the Clinic".

Immediate Implants Placed in Fresh Sockets Associated with Periapical Pathology: A Split-Mouth Design and Survival Evaluation after 1-Year Follow-Up.

PURPOSE: To compare the immediate implant success rates between sites with chronic apical lesions and healthy sites in the same patients 1 year postdelayed loading. MATERIALS AND METHODS: One hundred sixty-eight immediate implants were placed in sixty patients at upper incisor, canine, and premolar sites. A split-mouth design was used, placing a minimum of two implants, one in a fresh socket associated with chronic periapical disease, the average lesion size was larger than 4 mm and less than 8 mm (test group), and the other(s) in a healthy fresh socket (control group). Implant survival rate at 1 year postloading delayed was compared between the groups. RESULTS: The implant survival rate was 98.2% for the total sample (n = 168); out of the three implants lost, two were from the test group, and one was from the control group (in the same patient as one of the former). Among the surviving implants, five were also considered failures due to excessive bone loss (n = 3) and also because of the recurrence of the periapical lesions (n = 2). Survival rates were significantly lower in the test than control sites at 12 months postloading. CONCLUSIONS: Implant survival rates were significantly lower after the immediate implantation in postextraction sockets associated with chronic periapical disease (90.8%) than in healthy postextraction sockets (98.1%).

Hypo- and hypermorphic FOXC1 mutations in dominant glaucoma: transactivation and phenotypic variability.

Dominant glaucoma, a heterogeneous, infrequent and irreversible optic neuropathy, is often associated with elevated intraocular pressure and early-onset. The role of FOXC1 in this type of glaucoma was investigated in twelve Spanish probands via nucleotide variation screening of its proximal promoter and unique exon. Functional evaluations of the identified variants included analyses of the transcriptional activity, protein stability, DNA binding ability and subcellular localization. Four different mutations that were identified in four probands (33.3%) were associated with remarkable phenotypic variability and were functionally classified as either hypermorphic (p.Y47X, p.Q106X and p.G447_G448insDG) or hypomorphic (p.I126S) alleles. To the best of our knowledge, three of the variants are novel (p.Y47X, p.I126S and p.G447_G448insDG) and, in addition, hypermorphic FOXC1 mutations are reported herein for the first time. The presence of an intact N-terminal activation domain in the truncated proteins p.Y47X and p.Q106X may underlie their associated transactivation hyperactivity by a gain-of-function mechanism involving dysregulated protein-protein interactions. Similarly, altered molecular interactions may also lead to increased p.G447_G448insDG activity. In contrast, the partial loss-of-function associated with p.I126S was due to impaired protein stability, DNA binding, protein phosphorylation and subcellular distribution. These results support that moderate and variable FOXC1 transactivation changes are associated with moderate goniodysgenesis, dominant glaucoma and remarkable phenotypic variability.

Defects in subventricular zone pigmented epithelium-derived factor niche signaling in the senescence-accelerated mouse prone-8.

We studied potential changes in the subventricular zone (SVZ) stem cell niche of the senescence-accelerated mouse prone-8 (SAM-P8) aging model. Bromodeoxyuridine (BrdU) assays with longtime survival revealed a lower number of label-retaining stem cells in the SAM-P8 SVZ compared with the SAM-Resistant 1 (SAM-R1) control strain. We also found that in SAM-P8 niche signaling is attenuated and the stem cell pool is less responsive to the self-renewal niche factor pigmented epithelium-derived factor (PEDF). Protein analysis demonstrated stable amounts of the PEDF ligand in the SAM-P8 SVZ niche; however, SAM-P8 stem cells present a significant expression decrease of patatin-like phospholipase domain containing 2, a receptor for PEDF (PNPLA2-PEDF) receptor, but not of laminin receptor (LR), a receptor for PEDF (LR-PEDF) receptor. We observed changes in self-renewal related genes (hairy and enhancer of split 1 (Hes1), hairy and enhancer of split 1 (Hes5), Sox2] and report that although these genes are down-regulated in SAM-P8, differentiation genes (Pax6) are up-regulated and neurogenesis is increased. Finally, sheltering mammalian telomere complexes might be also involved given a down-regulation of telomeric repeat binding factor 1 (Terf1) expression was observed in SAM-P8 at young age periods. Differences between these 2 models, SAM-P8 and SAM-R1 controls, have been previously detected at more advanced ages. We now describe alterations in the PEDF signaling pathway and stem cell self-renewal at a very young age, which could be involved in the premature senescence observed in the SAM-P8 model.

GSTT1, GSTM1, and CYP1B1 gene polymorphisms and susceptibility to sporadic renal cell cancer.

PURPOSE: To estimate the prevalence and importance of GSTT1, GSTM1, and CYP1B1 genotypes in renal cell carcinoma (RCC), and to identify their value as a prognostic factor. MATERIALS AND METHODS: Cross-sectional study of a group of patients diagnosed with RCC (n = 133) and a control group (n = 208) with benign conditions and no history of tumor. Controls were selected by cumulative samples and mixed pairing. All subjects pertained to the catchment area for our hospital. Sociodemographic variables, anatomical pathology features, and presence of GSTT1, GSTM1, and CYP1B1 polymorphisms by multiplex PCR and sequencing techniques. RESULTS: There were no differences in the genotype distribution of the GSTT1 and GSTM1 genes between cases and controls. In the case of CYP1B1, the GG genotype (Ala119) was more prevalent in patients with RCC (OR = 2.08; 95% CI: 1.32-2.28) and may be implicated in 34.3% (95% CI: 16.3-52.2) of RCCs. In patients with GSTT1 deletion, TNM stages III to IV were more common (39.1%); whereas in Val432 homozygous patients in CYP1B1, Fuhrman grades 3 to 4 (54.6%) were more common. Because this was a cross-sectional study, longitudinal studies are needed in the future to confirm these data. CONCLUSIONS: No relationship between GSTT1 and GSTM1 genotypes and RCC risk was observed. Homozygous subjects with Ala119 in CYP1B1 had twice the risk of RCC as homozygous for Ser119 or heterozygotes. Patients with GSTT1 deletion had tumors of more advanced stages, and those with Val432 polymorphism in CYP1B1 had tumors of higher Fuhrman grade.

WDR36 and P53 gene variants and susceptibility to primary open-angle glaucoma: analysis of gene-gene interactions.

PURPOSE: To investigate the role of WDR36 and P53 sequence variations in POAG susceptibility. METHODS: The authors performed a case-control genetic association study in 268 unrelated Spanish patients (POAG1) and 380 control subjects matched for sex, age, and ethnicity. WDR36 sequence variations were screened by either direct DNA sequencing or denaturing high-performance liquid chromatography. P53 polymorphisms p.R72P and c.97-147ins16bp were analyzed by single-nucleotide polymorphism (SNP) genotyping and PCR, respectively. Positive SNP and haplotype associations were reanalyzed in a second sample of 211 patients and in combined cases (n = 479). RESULTS: The authors identified almost 50 WDR36 sequence variations, of which approximately two-thirds were rare and one-third were polymorphisms. Approximately half the variants were novel. Eight patients (2.9%) carried rare mutations that were not identified in the control group (P = 0.001). Six Tag SNPs were expected to be structured in three common haplotypes. Haplotype H2 was consistently associated with the disease (P = 0.0024 in combined cases). According to a dominant model, genotypes containing allele P of the P53 p.R72P SNP slightly increased glaucoma risk. Glaucoma susceptibility associated with different WDR36 genotypes also increased significantly in combination with the P53 RP risk genotype, indicating the existence of a genetic interaction. For instance, the OR of the H2 diplotype estimated for POAG1 and combined cases rose approximately 1.6 times in the two-locus genotype H2/RP. CONCLUSIONS: Rare WDR36 variants and the P53 p.R72P polymorphism behaved as moderate glaucoma risk factors in Spanish patients. The authors provide evidence for a genetic interaction between WDR36 and P53 variants in POAG susceptibility, although this finding must be confirmed in other populations.

Interaction of recombinant myocilin with the matricellular protein SPARC: functional implications.

PURPOSE: Myocilin is an extracellular glycoprotein with unknown function that is associated with glaucoma. Calpain II cleaves recombinant myocilin within the linker region of the protein, releasing the C-terminal olfactomedin domain from the N-terminal domain. The authors previously reported that myocilin interacts with the C-terminal region of hevin, a secretory glycoprotein belonging to the SPARC family of matricellular proteins. This study aims to investigate the interaction of myocilin with SPARC. METHODS: Protein-protein interactions were evaluated by the yeast two-hybrid system. The positive interactions were confirmed by solid-phase binding assays using Ni-chelating HPLC purified recombinant proteins and coexpression of recombinant proteins in HEK-293T cells. Coexpression of myocilin, SPARC, and hevin in ocular tissues was identified by immunoflorescence microscopy, Western blot, and array-based gene profiling. RESULTS: Yeast two-hybrid analyses showed that myocilin interacted with the highly conserved C-terminal extracellular calcium binding (EC) domain within SPARC and hevin. Solid-phase binding assays confirmed these interactions and showed that both myocilin and its C-terminal olfactomedin fragment interacted noncovalently with SPARC and a peptide containing the EC domain of SPARC. Full-length myocilin interacted with higher affinity with SPARC and its EC domain than the myocilin C-terminal fragment. Coexpression of the two recombinant proteins in HEK-293T cells also indicated their intracellular interaction. CONCLUSIONS: Recombinant myocilin and SPARC interact through their C-terminal domains. The data suggest that the proteolytic processing of myocilin modulates this interaction as well as the interactions of myocilin with other extracellular matrix and matricellular proteins, further supporting a functional role for this proteolytic cleavage.

Polymorphic deletions of the GSTT1 and GSTM1 genes and susceptibility to bladder cancer.

UNLABELLED: What's known on the subject? and What does the study add? Bladder cancer susceptibility may be determined by genetic differences in the activity of glutathione S-transferases, enzymes that regulate the conversion of exogenous carcinogens to excretable hydrophilic metabolites by glutathione conjugation. The discrepancy of results regarding the association of common genetic polymorphisms and complex diseases such as cancer has raised scepticism in this area of research. Although the evidence generally supports the implication of GSTM1 and GSTT1 polymorphisms in bladder cancer, there is still some debate, with some studies in favour and some against. This study shows a greater risk of bladder cancer in individuals with GSTM1 null genotype, particularly women. This relationship is less evident with GSTT1 null genotypes. Null genotypes in both genes appear to be synergistic, particularly among smokers, and to increase the predisposition to more aggressive tumours. Nevertheless, the role of GSTM1 and GSTT1 polymorphisms in predisposition to bladder cancer should be viewed with caution, due to the multifactorial genetic origin of this condition and the need for long-term longitudinal studies to confirm these results. OBJECTIVE: To estimate the prevalence and importance of GSTT1 and GSTM1 genotypes (implicated in glutathione S-transferase activity) in bladder cancer, to determine whether smoking and occupational factors influence this relationship, and to identify the value of GSTT1 and GSTM1 genotypes as prognostic factors. PATIENTS AND METHODS: A cross-sectional study was conducted with a group of patients with bladder carcinoma and a control group with benign conditions and no history of tumours. The controls were selected and paired as subjects were recruited. Sociodemographic variables, smoking, professional occupation, histological features and the presence of GSTT1 and GSTM1 polymorphisms by multiplex PCR techniques were assessed. RESULTS: GSTM1 genotypes were investigated in 201 patients and 193 controls and GSTT1 genotypes in 190 patients and 163 controls. In the patients group, GSTT1 null genotype was observed in 22.1% (not significant) and GSTM1 null genotype in 54.2% (P=0.008) (odds ratio, OR, 1.7); when considered together, 15.5% (P<0.05; OR, 3.5) of patients had both null genotypes. In the multivariate analysis, the presence of GSTM1 null genotype remained in the model (OR, 2.1) in addition to smoking and age. Subjects with bladder tumour and GSTM1 null genotype were younger than patients without gene deletion (P=0.049). Women with GSTM1 null genotype presented a higher OR than men (P=0.024). When stratified by smoking habit, smokers with both null genotypes showed an OR of 4.7. The percentage of patients with G3 tumours was higher in patients with GSTT1 null genotype (P=0.013) and in patients with both null genotypes (P=0.002). A higher percentage of infiltrating tumours was also observed in patients with both null genotypes (P=0.035). CONCLUSIONS: The data obtained in the present study suggest a higher risk of bladder cancer in individuals with the GSTM1 null genotype. This risk is twofold higher when GSTM1 and GSTT1 null genotypes are both present and is also higher in smokers. A greater predisposition for more aggressive tumours appears to exist, particularly when both null genotypes are combined. Longer-term longitudinal studies are needed to confirm these results.

Functional role of proteolytic processing of recombinant myocilin in self-aggregation.

PURPOSE: Recombinant myocilin expressed in cells in culture is endoproteolytically cleaved in the endoplasmic reticulum by calpain II, releasing an N-terminal and a C-terminal fragment. This proteolytic processing has been speculated to regulate the molecular interactions of myocilin. The main purpose of this study was to analyze the effect of the proteolytic cleavage on myocilin aggregation. METHODS: cDNAs encoding human myocilin and the N- and C-terminal fragments were transiently expressed in HEK-293T cells. Covalent interactions of recombinant myocilin were analyzed by SDS-PAGE and Western immunoblot analysis in different dissociating conditions. Noncovalent interactions were studied by solid-phase binding assays, performed with Ni-chelating HPLC-purified recombinant proteins, and by Far-Western blot analysis. RESULTS: Western blot analysis of recombinant myocilin aggregates under either increasing ionic strength or increasing concentration of reducing agent indicated that ionic interactions do not contribute to the stability of the molecular complexes linked by disulfide bridges. Disulfide myocilin homoaggregates decreased as the proteolytic processing increased. Solid-phase binding assays showed the existence of high-affinity (K(d) = 0.068 microM) noncovalent myocilin-myocilin interactions and that processed fragments bound to the full-length protein with significantly reduced affinity. Far-Western blot analysis confirmed noncovalent interactions between recombinant myocilin disulfide aggregates. CONCLUSIONS: The proteolytic processing of recombinant myocilin decreases myocilin homoaggregates. These data provide the first evidence of a functional role for this processing in myocilin aggregation and suggest that disulfide complexes of myocilin could organize into a dynamic extracellular network sustained by noncovalent N-terminal interactions.

An update on the biology of cancer stem cells in breast cancer.

Breast cancer stem cells are defined as cancer cells with self-renewal capacity. These cells represent a small subpopulation endowed with the ability to form new tumours when injected in nude mice. Markers of differentiation have been used to identify these cancer cells. In the case of breast cancer, CD44+/CD24- select a population with stem cell properties. The fact that these cells have self-renewal ability has suggested that this population could be responsible for new tumour formation and cancer relapse. These cells have been shown to be more resistant to chemotherapy and radiotherapy than normal cancer cells. The identification of the molecular druggable alterations responsible for the initiation and maintenance of cancer stem cells is an important goal. In this article we will review all these points with special emphasis on the possible role of new drugs designed to interact with molecular pathways of cancer stem cells.

Heterozygous expression of myocilin glaucoma mutants increases secretion of the mutant forms and reduces extracellular processed myocilin.

PURPOSE: Heterozygous mutations in the myocilin gene (MYOC) cause glaucoma by an unknown mechanism. MYOC encodes an extracellular protein of unidentified function that undergoes intracellular endoproteolytic processing in the secretory pathway. It has been described that co-expression of wild-type/mutant myocilin reduces the secretion of the wild-type protein and that single expression of glaucoma myocilin mutants reduces its proteolytic processing. However, the effect of wild-type myocilin on mutant myocilin secretion and how mutant myocilin affects the proteolytic processing of wild-type myocilin have not been investigated. We herein analyze these two issues. METHODS: We modeled the heterozygous state for 4 missense (E323K, R346T, P370L, D380A) and 1 nonsense (Q368X) myocilin mutants by transiently co-expressing each mutant with the wild-type protein in HEK-293T cells. Recombinant mutant and wild-type myocilin in both culture media and cellular fractions were quantified by western immunoblot and densitometry. RESULTS: A 24 h transient co-expression of each myocilin mutant with the wild-type protein elicited an augmented secretion of the mutant forms from 1.5 fold (D380A) to 5.4 fold (E323K). Under such conditions, extracellular mutant myocilin represented up to 20% of the total mutant protein. Other than this effect, secreted wild-type myocilin significantly decreased from 2.6 fold (E323K) to 36 fold (Q368X). When myocilin proteolytic processing was enhanced (96 hour co-expression) the extracellular amount of wild-type processed myocilin diminished from approximately 2.1 fold (E323K) to 6.3 fold (P370L). Nonreducing SDS-PAGE indicated that extracellular myocilin resulting from 24 h co-expression of wild-type myocilin and each of the 4 missense mutants forms hetero-oligomers and that glaucoma mutations do not increase the size of myocilin aggregates. CONCLUSIONS: Increased extracellular levels of mutant myocilin expressed in heterozygosis may play a relevant role in glaucoma pathogenesis. This effect is likely the result of intracellular mutant/wild-type myocilin hetero-oligomerization.

Nonsense-mediated decay: paving the road for genome diversification.

The expression of protein-encoding genes is a complex process culminating in the production of mature mRNA and its translation by the ribosomes. The production of a mature mRNA involves an intricate series of processing steps. The majority of eukaryotic protein-encoding genes contain intron sequences that disrupt the protein-encoding frame, and hence have to be removed from immature mRNA prior to translation into protein. The mechanism involved in the selection of correct splice sites is incompletely understood. A considerable body of evidence suggests that the splicing machinery has suboptimal efficiency and fidelity leading to substantial processing inaccuracy. Here we discuss a recently published article that extends observations that cells rely on nonsense-mediated mRNA decay (NMD) to compensate for such suboptimal processing accuracy. Intriguingly these authors provide evidence for a strong selective pressure in favour of premature termination of mRNA translation in the event of intron retention. The analysis presented implies a positive role of NMD in transcript diversification through alternative splicing and suggest that this ancient surveillance mechanism may have co-evolved with intron acquisition born from the need for quality control of splicing patterns.

Implications of p53 gene mutations on patient survival in transitional cell carcinoma of the bladder: a long-term study.

OBJECTIVE: To determine the prognostic value of p53 gene mutations and P53 overexpression for predicting the incidence of recurrence, progression and long-term survival of patients with transitional cell carcinoma (TCC) of the bladder. METHODS: Prospective cohort study with 94 consecutive patients diagnosed and treated for TCC. DNA was obtained from tumor tissue to perform PCR-SSCP of p53 exons 5-9, with automatic sequencing of any mutated samples. Immunohistochemistry using anti-human P53 monoclonal antibody was also performed. Survival was analyzed and the survival curves compared (Mantel-Haenszel). Lastly, a Cox proportional hazards model was constructed. RESULTS: Mutations were found in 46.8% of samples, with 61.8% in infiltrating tumors. Exon 8 was involved in 42.3%. P53 overexpression (cutoff > or =20%) was found in 52.1%. Mean follow-up was 44.1 months; 43.6% had died by the end of this period. Mean survival was lower in patients with exon 8 mutations (38.4 months), compared with patients without this exon mutated (P = 0.016). There were no differences in patient survival based on positive or negative immunohistochemistry (cutoff > or =20%), although survival was lower in patients with a percentage higher than 50% of antibody-stained cells (P = 0.02). In the Cox analysis, tumor stage, pM stage, and interaction between stage > or =pT2 and mutated p53 gene were independent risk factors, with a 6.13-fold risk of death in these patients (P = 0.019). The number of tumors, nuclear grade, pTa stage, and the interaction between GI degree and nonmutated p53 gene remained in the Cox model for superficial tumors, such that these patients had a lower risk of recurrence or progression (P = 0.008). CONCLUSIONS: Alterations in the p53 gene may be indicative of poorer prognosis and greater recurrence in patients with urothelial bladder tumor, in particular, the presence of mutations in exon 8 and a greater percentage of stained cells in the immunohistochemistry. Nevertheless, the classic prognostic factors (primarily, pTNM stage) should still be considered the most useful factors for follow-up of these patients.

Implications of mismatch repair genes hMLH1 and hMSH2 in patients with sporadic renal cell carcinoma.

OBJECTIVES: To analyse the implications of DNA mismatch repair genes hMLH1 and hMSH2 in sporadic renal cell carcinoma (RCC). MATERIALS AND METHODS: Specimens of tumour and healthy renal tissue were collected from 89 patients treated for sporadic RCC. Another 95 blood samples taken from individuals with no history of cancer were also analysed. After DNA extraction and PCR amplification, microsatellite instability (MSI) was determined using the Bethesda microsatellite panel, two exonic microsatellites of the TGFbRII and BAX genes, and the microsatellite D3S1611. The promoter methylation status of hMLH1 was investigated using the HpaII and MspI restriction enzymes. In addition, a sequencing analysis of complete coding region of hMLH1 and hMSH2 genes was performed. RESULTS: MSI and promoter hypermethylation of hMLH1 were not detected. Interestingly, loss of heterozygosity (LOH) was common among patients with RCC, particularly in microsatellite D3S1611 (34.9%). Mutations were identified in eight patients: K618A and V716M in gene hMLH1; and I145V, G322D, and the novel mutation P349A, in gene hMSH2. The mutations also appeared in healthy renal tissue and therefore, were considered as germline DNA sequence variations. There were G322D and K618A changes in >1% of the healthy control subjects, suggesting that they are DNA polymorphisms. CONCLUSIONS: Our data show that loss of function of both hMLH1 and hMSH2 is not involved in sporadic RCC, either by promoter methylation or mutation in their exons. However, LOH indicated that chromosomal instability affecting large fragments of DNA was the main genetic alteration we detected associated with RCC.