Head and Neck Tumor Cells Exhibit Altered Proliferation upon Overexpression of nm23 Genes

nm23 was identified as a metastasis suppressor gene but is also appointed to a number of other biological functions. The goal of this study was to reveal the influence of ectopic expression of nm23-H1 and nm23-H2 on proliferation properties of head and neck tumor cells. The proliferation rate of transfected cells was evaluated using EGFP reporter system and flow cytometry. HEp-2 and CAL 33 cells transiently transfected with nm23 cDNA containing constructs exhibited enhanced proliferation. CAL 27 cells constitutively expressing GFP-Nm23-H2 protein, exhibited intense proliferation the first day after seeding, while the GFP-Nm23-H1 expressing clone started to proliferate after one-day lag period. The results on transiently transfected HEp-2 and CAL 33 cells generally confirmed previous findings connecting nm23 expression with altered proliferation of head and neck tumors. We speculate that the effects observed on stably transfected CAL 27 clones are due to their different attachment properties

The utilization of pEGFP reporter system in cell-cycle analysis of adherent cells

Background and Purpose: GFP (green fluorescent protein) is widely used in a variety of fluorescent methods aimed at revealing the fate of proteins in the cell, intracellular transport, transfection efficiency and is also recommended for cell-cycle analysis purposes. In our attempt to evaluate the role of nm23 genes in proliferation of head and neck tumor cells in culture we have decided to use EGFP reporter system and analyze the DNA content by flow cytometry. Materials and Methods: To optimize the method we either transiently transfected the cells with pEGFPC1-nm23 constructs or cotransfected the cells with an nm23 carrying constructs and pEGFPC1 as a reporter system. We established stable clones with pEGFPC1-nm23 constructs and analyzed them by flow cytometry, as well. Results and Conclusions:We report our experience for the use of pEGFP reporter system and flow cytometry for determining cell-cycle distribution of transiently and stably transfected adherent tumor cells. We discuss, in brief, the protocol we used and the problems that appeared during our experiments – GFP bleaching, cell clumping and degradation and insufficient number of cells to be analyzed. In conclusion, we suggest useful tips how to avoid or minimize the technical problems of this method and improve the results and analysis

Sponge non-metastatic Group I Nme gene/protein - structure and function is conserved from sponges to humans

<p>Abstract</p> <p>Background</p> <p>Nucleoside diphosphate kinases NDPK are evolutionarily conserved enzymes present in Bacteria, Archaea and Eukarya, with human Nme1 the most studied representative of the family and the first identified metastasis suppressor. Sponges (Porifera) are simple metazoans without tissues, closest to the common ancestor of all animals. They changed little during evolution and probably provide the best insight into the metazoan ancestor's genomic features. Recent studies show that sponges have a wide repertoire of genes many of which are involved in diseases in more complex metazoans. The original function of those genes and the way it has evolved in the animal lineage is largely unknown. Here we report new results on the metastasis suppressor gene/protein homolog from the marine sponge <it>Suberites domuncula</it>, NmeGp1Sd. The purpose of this study was to investigate the properties of the sponge Group I Nme gene and protein, and compare it to its human homolog in order to elucidate the evolution of the structure and function of Nme.</p> <p>Results</p> <p>We found that sponge genes coding for Group I Nme protein are intron-rich. Furthermore, we discovered that the sponge NmeGp1Sd protein has a similar level of kinase activity as its human homolog Nme1, does not cleave negatively supercoiled DNA and shows nonspecific DNA-binding activity. The sponge NmeGp1Sd forms a hexamer, like human Nme1, and all other eukaryotic Nme proteins. NmeGp1Sd interacts with human Nme1 in human cells and exhibits the same subcellular localization. Stable clones expressing sponge NmeGp1Sd inhibited the migratory potential of CAL 27 cells, as already reported for human Nme1, which suggests that Nme's function in migratory processes was engaged long before the composition of true tissues.</p> <p>Conclusions</p> <p>This study suggests that the ancestor of all animals possessed a NmeGp1 protein with properties and functions similar to evolutionarily recent versions of the protein, even before the appearance of true tissues and the origin of tumors and metastasis.</p

Head and Neck Tumor Cells Exhibit Altered Proliferation upon Overexpression of nm23 Genes

nm23 was identified as a metastasis suppressor gene but is also appointed to a number of other biological functions. The goal of this study was to reveal the influence of ectopic expression of nm23-H1 and nm23-H2 on proliferation properties of head and neck tumor cells. The proliferation rate of transfected cells was evaluated using EGFP reporter system and flow cytometry. HEp-2 and CAL 33 cells transiently transfected with nm23 cDNA containing constructs exhibited enhanced proliferation. CAL 27 cells constitutively expressing GFP-Nm23-H2 protein, exhibited intense proliferation the first day after seeding, while the GFP-Nm23-H1 expressing clone started to proliferate after one-day lag period. The results on transiently transfected HEp-2 and CAL 33 cells generally confirmed previous findings connecting nm23 expression with altered proliferation of head and neck tumors. We speculate that the effects observed on stably transfected CAL 27 clones are due to their different attachment properties

Sealing Ability of Bioactive Root-End Filling Materials in Retro Cavities Prepared with Er,Cr:YSGG Laser and Ultrasonic Techniques

The aim of this in vitro study was to compare the apical sealing ability of total fill bioceramic root repair material (BC-RRM) and mineral trioxide aggregate (MTA), regarding the retrograde preparation technique used: ultrasonic or erbium, chromium: yttrium, scandium, gallium, or garnet (Er,Cr:YSGG) laser. The study sample consisted of 48 human single-rooted teeth. After root-end resection, the samples were divided into two groups, according to the retrograde preparation technique used: Group 1: ultrasonic; Group 2: Er,Cr:YSGG laser. In each group, half of the retrograde cavities were filled with BC-RRM, and the other half were filled with MTA. The specimens were mounted in tubes and sterilized in plasma. The root canals were inoculated with Enterococcus faecalis, and the tubes were filled with fetal bovine serum, leaving the apical part of the root in the serum. After 30 days, the canals were sampled and cultured, and the colony forming units (CFUs) were counted with the additional polymerase chain reaction (PCR analysis). There was no significant difference between ultrasonic groups and the Er,Cr:YSGG-MTA group, regarding the number of CFUs (p > 0.05). The Er,Cr:YSGG-BC-RRM group showed the highest number of remaining viable bacteria (p < 0.001). Both filling materials filled in ultrasonic preparations presented similar sealing abilities. The BC-RRM showed more leakage when used in retro cavities prepared with the Er,Cr:YSGG laser