Three‐dimensional cell culture model utilization in cancer stem cell research

Three‐dimensional (3D) cell culture models are becoming increasingly popular in contemporary cancer research and drug resistance studies. Recently, scientists have begun incorporating cancer stem cells (CSCs) into 3D models and modifying culture components in order to mimic in vivo conditions better. Currently, the global cell culture market is primarily focused on either 3D cancer cell cultures or stem cell cultures, with less focus on CSCs. This is evident in the low product availability officially indicated for 3D CSC model research. This review discusses the currently available commercial products for CSC 3D culture model research. Additionally, we discuss different culture media and components that result in higher levels of stem cell subpopulations while better recreating the tumor microenvironment. In summary, although progress has been made applying 3D technology to CSC research, this technology could be further utilized and a greater number of 3D kits dedicated specifically to CSCs should be implemented.     

Monoclonal antibodies against the presynaptic calcium channel antagonist omega-conotoxin GVI A from cone snail poison

Monoclonal antibodies have been prepared against omega-conotoxin GVI A, a peptide isolated from marine snails of the genus Conus (Conus geographus and Conus magus). This toxin is a blocker of select presynaptic Ca2+ channels in the central nervous system. Antigenic omega-conotoxin GVI A was synthesized as a covalent conjugate with bovine serum albumin and injected s.c. An ELISA assay combined with a competitive inhibition assay was used to select and characterize monoclonal antibodies able to recognize and bind the free toxin. Several of the antibodies were found to block omega-conotoxin GVI A inhibition of 45Ca transport into rat brain synaptosomes and to block omega-conotoxin GVI A binding to membranes from the same preparation. The antibodies recognize native, synthetic toxin, and are useful for analysis of toxin in biological fluids.     

Use of Drosophila mutants to investigate the effect of disuse on the maintenance of muscle

The effect of continued muscular inactivity and prolonged paralysis on the structure and function of muscles was investigated in Drosophila melanogaster. A number of flightless mutants was examined to see whether their flight muscles degenerated as a result of disuse. No sign of progressive deterioration was observed in any of these mutants. Further, by producing mosaic flies in which part of the body expressed the temperature-sensitive paralytic mutation shibire^ST139, reversible local paralysis was obtained, and maintained for prolonged periods. Flies in which parts of the leg or flight musculature had been paralysed for several days were examined; no effect of such inactivity on the structure and function of the muscles was observed in any of the flies. These results indicate that in Drosophila continued muscular inactivity does not result in extensive degeneration of the musculature.     

In vivo and in vitro phosphorylation studies of numatrin, a cell cycle regulated nuclear protein, in insulin-stimulated NIH 3T3 HIR cells

Numatrin, a nuclear matrix protein has been implicated to be involved in mitogenesis of normal and malignant cells (Feuerstein and Mond, J. Biol. Chem. 262, 11389, 1987) and was later found to be identical to the nuclear phosphoprotein, B23. To study whether phosphorylation of numatrin is regulated by mitogenic stimulation, we examined the effect of phosphorylation of numatrin in the insulin-responsive cells, NIH 3T3 HIR. We found that an increase in phosphorylation of numatrin was associated with stimulation of the cells with insulin for 4 h and that the level of phosphorylation remained elevated after 8 h. By this time there was no increase in numatrin abundance as shown by Coom-massie blue stain and Western blot analysis. The induction in phosphorylation of numatrin could not be detected after 30 min stimulation with insulin, thus, indicating that the increase in phosphorylation of numatrin is not a rapid event. Analysis of the phosphopeptides by thin layer chromatography indicated four peptides that were phosphorylated in numatrin (one major and three minor). Stimulation with insulin was associated primarily with an increase in phosphorylation of the minor phosphopeptides. The phosphopeptide map of numatrin was identical after 4, 8, 17, 24, and 32 h stimulation with insulin, indicating that identical sites are phosphorylated at different phases of the cell cycle. In a search for the protein kinase which is involved in phosphorylation of numatrin we found that numatrin is a most prominent substrate for the cell cycle regulated cdc2 (p 34) kinase. However, the major phosphopeptides which were phosphorylated by this kinase did not comigrate with either of the phosphopeptides phosphorylated in insulin-stimulated intact cells. This may indicate that it is unlikely that cdc2 kinase may account for the mechanism(s) associated with phosphorylation of numatrin by insulin under physiological conditions.     

Release factor regulating termination of complete protein in a eukaryotic organism

Polypeptide release reaction was studied using a protein release factor and a physiological substrate containing a complete polypeptide chain attached to monosomes of the insect Tenebrio molitor. The intermediate substrate used for the release reaction was synthesized using a cell-free protein synthesizing system from Tenebrio capable of polypeptide synthesis but not release of the completed chain. This system synthesized predominantly adult cuticular protein. The released product was characterized by chromatography after tryptic digestion; many of the tryptic peptides corresponded to those of cuticule labeled in vivo. The protein release factor was obtained as microsomal wash and was further purified by ammonium sulfate precipitation and column chromatography. It released about 30% of the monosome-bound peptide in the absence of GTP. The remaining 70% of peptidyl-tRNA was released as peptidyl-puromycin in the absence of release factor, but required transferase II and GTP. The peptidyl-puromycin varied in size from dipeptide to almost complete protein. The puromycin reaction was inhibited by diphtheria toxin and NAD and was dependent on GTP, while the release of completed peptide was independent of GTP and not affected by diphtheria toxin and NAD. The release factor was capable of releasing formylmethionine as formylmethionine-puromycin from ribosomes in response to poly(A₃,U). Hence it is suggested that the release factor is responding to UAA as terminating codon.     

Function from within: Autophagy induction by HPSE/heparanase—new possibilities for intervention

HPSE (heparanase) is the predominant enzyme in mammals capable of cleaving heparan sulfate, an activity highly implicated in cellular invasion and tumor metastasis. HPSE expression is induced in many types of cancer and increased HPSE levels are most often associated with increased tumor metastasis and reduced patient survival post operation. In addition, HPSE induction is associated with progression of the primary tumors but the mechanism(s) underlying tumor expansion by HPSE have not been sufficiently resolved. Our results establish a role for heparanase in modulating autophagy in normal and malignant cells, thereby conferring growth advantages as well as resistance to chemotherapy.