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  • Open access
  • 97 Reads
Does the modification of serine 477 of DNA mismatch repair protein MLH1 play a role in cell proliferation?

MutLα, a heterodimer consisting of MLH1 and PMS2, is a key player of the DNA mismatch repair (MMR) system and of great importance to correct incorporation errors that occur during DNA replication.

Previously, we identified that posttranslational phosphorylation of MLH1 at amino acid position serine 477 can switch off MMR activity in vitro. We also found that mutation of serine 477 prevented the posttranslational phosphorylation. Since MLH1 is involved in numerous MMR-independent cell processes, including the cell cycle control, we hypothesized that phosphorylation of MLH1 might alter the mediation of cell cycle-associated proteins and thus affects proliferation.

To investigate the impact of phosphorylation of MLH1 on proliferation an MTT-assay was used. MutLα deficient HEK293T cells were transiently cotransfected with pcDNA3.1+/MLH1 and pcDNA3.1+/PMS2 for the expression of MutLα wildtype. For the expression of the non-phosphorylatable MutLα variant cells were transiently cotransfected with pcDNA3.1+/MLH1S477A and pcDNA3.1+/PMS2. 48h after transfection cells were treated with Calyculin (50 nM), a serine-threonine-phosphatase inhibitor, to enhance the amount of phosphorylated MLH1. In parallel, cells were treated with Orthovanadate (50 µM), a competitive inhibitor of protein-phosphotyrosine phosphatases, to exclude inhibitor side effects. DMSO was used as a negative control. After a cultivation period of 15min to 3h, cells were incubated with MTT-reagent and proliferation was evaluated via ELISA reader.

In summary, significant differences of proliferation could be detected between the differently treated cells. Proliferation of Calyculin treated HEK293T cells overexpressing the non-phosphorylatable MutLα variant, however, was only weakly increased compared to cells overexpressing MutLα wildtype. Due to the fact that Calyculin and Orthovanadate are able to influence a multitude of signaling pathways, the role of MLH1 phosphorylation cannot be conclusively answered here. Further experiments are necessary to clarify the function of phosphorylated MLH1 in proliferation.

  • Open access
  • 79 Reads
Changes in gene expression of metalloproteinases-2 and -9 and their inhibitors TIMP2 and TIMP3 in human glioma cells exposed to low levels of fluoride.

Fluorine compounds are common environmental pollutants and may excessively penetrate the human body, especially the brain (fluoride penetrates the blood-brain barrier). Some of the latest studies have shown that fluoride may interfere with some of the metabolic pathways involved in the development of invasive potential in many types of cancer (eg Wnt/catenin or NF-κB). One of the stages of tumor invasion is the degradation of the extracellular matrix by metalloproteinases (MMP-2 and MMP-9), which allows the migration and metastasis of cancer cells. Taking into account the above facts, we decided to check whether low concentrations of fluoride affect the expression level of genes encoding MMP-2, MMP-9, and their TIMP-2 and TIMP-3 inhibitors in human glioblastoma cells.

U-87MG human glioblastoma cells were cultured with EMEM medium (10% FBS, 2 mM glutamine, 1% NEAA), 1 mM sodium pyruvate, 100 IU / ml penicillin, 10 μg / ml streptomycin) under optimal conditions (at 37 ° C, in an atmosphere of 5% CO2, with 95% humidity). Cells were treated with sodium fluoride (NaF; 1-5 µM) for 24, 48 and 72 hours.
The analysis of the expression level of the MMP-2, MMP-9, Timp-2, and Timp-3 genes was carried out by RT-PCR.

The results indicate that NaF (0.1-5 µM) can disrupt the expression of MMP-2, MMP-9, Timp-2, and Timp-3. In the case of MMP-2, there was an approx. 2-fold increase in expression in 48h (5 µM NaF) and about 2.5-fold increase in expression in 72h (0.1-5 µM NaF). For MMP-9, an approximately 3-fold increase in expression was observed in 24h (0.1 µM NaF) and 48h (5 µM NaF). Both Timp-2 and Timp-3 showed a significant increase in expression observed at all time points especially at the highest concentration of NaF (5 µM).

The obtained results may suggest that even low concentrations of fluorine compounds may have an undesirable influence promoting the invasive potential of human glioblastoma cells.

The project was implemented with the use of funds for science granted by the Pomeranian Medical University in Szczecin.

  • Open access
  • 106 Reads
Novel bacterial genotoxin-loaded nanoparticles for targeting therapy of radioresistant prostate cancer

Background: Prostate cancer (PCa) is one of the most commonly diagnosed cancers in men and usually becomes refractory because of recurrence and metastasis. CD44, a transmembrane glycoprotein, serves as a receptor for hyaluronic acid (HA) and has been found to be abundantly expressed in cancer stem cells (CSCs) that often exhibit a radioresistant phenotype. Cytolethal distending toxin subunit B (CdtB), produced by Campylobacter jejuni, is a genotoxin acts as a type I deoxyribonuclease (DNase I), which is responsible for creating DNA double-strand breaks (DSBs). Nanoparticles loaded with antitumor drugs and specific ligands that recognize cancerous cell receptors are promising methods to overcome the therapeutic challenges.

Results: Our results showed that administration of bacterial genotoxin significantly improved the efficacy of radiotherapy in a xenograft mouse model. We further prepared HA-decorated nanoparticles-encapsulated CdtB (HA-CdtB-NPs) and investigated the targeted therapeutic activity in radioresistant PCa cells. The results showed that HA-CdtB-NPs sensitized radioresistant PCa cells by enhancing DSB and causing G2/M cell-cycle arrest, without affecting the normal prostate epithelial cells. Our results demonstrate that HA-CdtB-NPs possess maximum target-specificity and delivery efficiency of CdtB into the nucleus, thereby enhancing the effect of radiation in radioresistant PCa cells.

Conclusions: These findings indicate that HA-loaded CdtB nanoparticles exert target-specificity accompanied with radiomimetic activity, which can be developed as an effective agent for overcoming radioresistance in PCa.