MDM2 regulates RB levels during genotoxic stress.

The retinoblastoma tumour suppressor protein (RB) regulates a number of diverse cellular functions including differentiation, angiogenesis, chromatin remodelling, senescence and apoptosis. The best-characterised function of RB is cell cycle regulation, and it has been considered a phosphoprotein regulated by cyclin-dependent kinases. In its hypophosphorylated form, RB binds the transcription factor E2F1, arresting the cell cycle in the G1 phase. Here, we show that MDM2 controls the cell cycle through synthesis and degradation of RB protein in a cell cycle condition-dependent fashion. MDM2 induces G1 cell cycle arrest by enhancing the translation of the RB mRNA under genotoxic stress. Translation requires direct interaction between the RB mRNA and the MDM2 protein that accompanies the RB mRNA to the polysomes. However, MDM2 ubiquitinates and degrades RB protein at the G2/M phase under genotoxic stress. The ATM phosphomimetic mutant MDM2(S395D) corroborates that the effect on the RB levels is dependent on the DNA damage. These results provide the basis of a dual regulatory mechanism by which MDM2 controls cell cycle progression during DNA damage.

Expression and purification of the recombinant full-length retinoblastoma protein and characterisation of its interaction with the oncoprotein HDM2.

Retinoblastoma (Rb) was the first tumour suppressor factor described, and it is dysfunctional in several types of cancers. Structurally, Rb is a very large, multifunctional protein organized in different domains connected by intrinsically disordered regions. Due to the complex structure of Rb, biochemical manipulation is difficult. The Rb protein has been implicated in many different cellular processes, such as the cell cycle control, senescence and even apoptosis. The activity of Rb is regulated by phosphorylation, and many different sites of phosphorylation have been described. However, the oncoprotein HDM2, can promote Rb degradation by the proteasome. This form of Rb regulation is largely unknown. Here we report the expression and purification of the full-length Rb protein and its phosphomimetic form, Rb(S567D), in a recombinant system. We also produced and purified the HDM2 protein and its phosphomimetic mutant, HDM2(S395D). The proteins interacted strongly when we used the phosphomimetic mutants, mimicking damaged DNA conditions. The expression of the proteins in E. coli allowed us to control the phosphorylation status of the proteins.

Targeting miR-21-3p inhibits proliferation and invasion of ovarian cancer cells.

MicroRNA-21 is overexpressed in most cancers and has been implicated in tumorigenesis. Accumulating evidence supports a central role for the miR-21 guide strand (miR-21-5p) in ovarian cancer initiation, progression, and chemoresistance. However, there is limited information regarding the biological role of the miR-21 passenger strand (miR-21-3p) in ovarian cancer cells. The aim of this study was to investigate the role of miR-21-3p and its target genes in cisplatin-resistant ovarian cancer cells. Expression profiling of miR-21-5p and miR-21-3p was performed in a panel of cancer cells by qPCR. Colony formation and invasion assays were carried out on ovarian and prostate cancer cells transfected with miR-21-5p and miR-21-3p inhibitors. Dual luciferase reporter assays were used to identify the miR-21-3p target genes in ovarian cancer cells. Our results show that miR-21-5p had higher expression levels compared to miR-21-3p on a panel of cancer cells. Moreover, inhibition of miR-21-5p or miR-21-3p resulted in a significant decrease in ovarian and prostate cancer cell proliferation and invasion. Luciferase reporter assays identify RNA Binding Protein with Multiple Splicing (RBPMS), Regulator of Chromosome Condensation and POZ Domain Containing Protein 1 (RCBTB1), and Zinc Finger protein 608 (ZNF608) as miR-21-3p target genes. SiRNA-induced RBPMS silencing reduced the sensitivity of ovarian cancer cells to cisplatin treatment. Immunohistochemical analyses of serous ovarian cancer patient samples suggest a significant decrease of RBMPS levels when compared to normal ovarian epithelium. Taken together, the data generated in this study suggests a functional role for miR-21-3p in ovarian cancer and other solid tumors.

Dual function of MDM2 and MDMX toward the tumor suppressors p53 and RB.

The orchestrated crosstalk between the retinoblastoma (RB) and p53 pathways contributes to preserving proper homeostasis within the cell. The deregulation of one or both pathways is a common factor in the development of most types of human cancer. The proto-oncoproteins MDMX and MDM2 are the main regulators of the well- known tumor suppressor p53 protein. Under normal conditions, MDM2 and MDMX inhibit p53, either via repression of its transcriptional activity by protein-protein interaction, or via polyubiquitination as a result of MDM2-E3 ubiquitin ligase activity, for which MDM2 needs to dimerize with MDMX. Under genotoxic stress conditions, both become positive regulators of p53. The ATM-dependent phosphorylation of MDM2 and MDMX allow them to bind p53 mRNA, these interactions promote p53 translation. MDM2 and MDMX are also being revealed as effective regulators of the RB protein. MDM2 is able to degrade RB by two different mechanisms, that is, by ubiquitin dependent and independent pathways. MDMX enhances the ability of MDM2 to bind and degrade RB protein. However, MDMX also seems to stabilize RB through interaction and competition with MDM2. Here, we will contextualize the findings that suggest that the MDM2 and MDMX proteins have a dual function on both p53 and RB.

Consenso Argentino intersociedades de Infección Urinaria 2018-2019 - Parte II / Argentine Intersociety Consensus on Urinary Infection 2018-2019 - Part II

La segunda parte del Consenso Argentino Intersociedades de Infección Urinaria incluye el análisis de situaciones especiales. En pacientes con sonda vesical se debe solicitar urocultivo solo cuando hay signo-sintomatología de infección del tracto urinario, antes de instrumentaciones de la vía urinaria o como control en pacientes post-trasplante renal. El tratamiento empírico recomendado en pacientes sin factores de riesgo es cefalosporinas de tercera generación o aminoglucósidos. Las infecciones del tracto urinario asociadas a cálculos son siempre consideradas complicadas. En caso de obstrucción con urosepsis, deberá realizarse drenaje de urgencia por vía percutánea o ureteral. En pacientes con stents o prótesis ureterales, como catéteres doble J, el tratamiento empírico deberá basarse en la epidemiología, los antibióticos previos y el estado clínico. Antes del procedimiento de litotricia extracorpórea se recomienda pesquisar la bacteriuria y, si es positiva, administrar profilaxis antibiótica según el antibiograma. Cefalosporinas de primera generación o aminoglúcosidos son opciones válidas. Se recomienda aplicar profilaxis antibiótica con cefalosporinas de primera generación o aminoglúcosidos antes de la nefrolitotomía percutánea. La biopsia prostática trans-rectal puede asociarse a complicaciones infecciosas, como infecciones del tracto urinario o prostatitis aguda, principalmente por Escherichia coli u otras enterobacterias. En pacientes sin factores de riesgo para gérmenes multirresistentes y urocultivo negativo se recomienda realizar profilaxis con amikacina o ceftriaxona endovenosas. En pacientes con urocultivo positivo, se realizará profilaxis según antibiograma, 24 horas previas a 24 horas post-procedimiento. Para el tratamiento dirigido de la prostatitis post-biopsia trans-rectal, los carbapenémicos durante 3-4 semanas son el tratamiento de elección.

The second part of the Inter-Society Argentine Consensus on Urinary Tract Infection (UTI) includes the analysis of special situations. In patients with urinary catheter, urine culture should be requested only in the presence of UTI symptomatology, before instrumentation of the urinary tract, or as a post-transplant control. The antibiotics recommended for empirical treatment in patients without risk factors are third-generation cephalosporins or aminoglycosides. UTIs associated with stones are always considered complicated. In case of obstruction with urosepsis, an emergency drainage should be performed via a percutaneous nefrostomy or ureteral stenting. In patients with stents or ureteral prostheses, such as double J catheters, empirical treatment should be based on epidemiology, prior antibiotics, and clinical status. Before the extracorporeal lithotripsy procedure, bacteriuria should be investigated and antibiotic prophylaxis should be administered in case of positive result, according to the antibiogram. First generation cephalosporins or aminoglycosides are valid alternatives. The use of antibiotic prophylaxis with first-generation cephalosporins or aminoglycosides before percutaneous nephrolithotomy is recommended. Transrectal prostatic biopsy can be associated with infectious complications, such as UTI or acute prostatitis, mainly due to Escherichia coli or other enterobacteria. In patients without risk factors for multiresistant bacteria and negative urine culture, prophylaxis with intravenous amikacin or ceftriaxone is recommended. In patients with positive urine culture, prophylaxis will be performed according to the antibiogram, from 24 hours before to 24 hours post-procedure. For the targeted treatment of post-transrectal biopsy prostatitis, carbapenems for 3-4 weeks are the treatment of choice.