Targeting the cell cycle in breast cancer: towards the next phase

Deregulation of the cell cycle is a hallmark of cancer that enables limitless cell division. To support this malignant phenotype, cells acquire molecular alterations that abrogate or bypass control mechanisms in signaling pathways and cellular checkpoints that normally function to prevent genomic instability and uncontrolled cell proliferation. Consequently, therapeutic targeting of the cell cycle has long been viewed as a promising anti-cancer strategy. Until recently, attempts to target the cell cycle for cancer therapy using selective inhibitors have proven unsuccessful due to intolerable toxicities and a lack of target specificity. However, improvements in our understanding of malignant cell-specific vulnerabilities has revealed a therapeutic window for preferential targeting of the cell cycle in cancer cells, and has led to the development of agents now in the clinic. In this review, we discuss the latest generation of cell cycle targeting anti-cancer agents for breast cancer, including approved CDK4/6 inhibitors, and investigational TTK and PLK4 inhibitors that are currently in clinical trials. In recognition of the emerging population of ER+ breast cancers with acquired resistance to CDK4/6 inhibitors we suggest new therapeutic avenues to treat these patients. We also offer our perspective on the direction of future research to address the problem of drug resistance, and discuss the mechanistic insights required for the successful implementation of these strategies.     

Potassium as a signal for both proliferation and differentiation of rabbit retinal (Müller) glia growing in cell culture

Retinal glial (Müller) cells were grown from explants of early postnatal rabbit retinae. The resulting monolayers of flat cells were exposed to control media (containing 5.85 mM K+), and to media with enhanced K+ concentrations (10 and 20 mM) or arginine-vasopressin (AVP, 20 micrograms/ml) or epithelial growth factor (EGF, 10 ng/ml). Autoradiographically, protein synthesis was quantified as L-[3H]-lysine incorporation, and DNA synthesis as [3H]-thymidine incorporation. Furthermore, the activity of Na+,K(+)-ATPase was measured radiochemically. Short exposure to either moderately enhanced K+ concentrations (10 mM) or to AVP, stimulated L-[3H]-lysine incorporation into the cells. Long-lasting exposure to either high K+ concentrations (20 mM) or to EGF stimulated [3H]-uptake. The Na+,K(+)-ATPase activity of cell cultures increased with increasing K+ concentration of the media. It is suggested that release of K+ by active neuronal compartments stimulates local protein synthesis of glial cells, resulting in the formation of glial sheaths with active K+ uptake capacity. Strong K+ release may even induce glial proliferation.     

Effect of sodium chloride on growth of heterotrophic marine bacteria

The effect of NaCl on the growth rates and yields of 31 gram-negative, heterotrophic, marine bacteria was determined. The strains used were representative of aerobic genera (Alteromonas, Pseudomonas, Alcaligenes, Bdellovibrio) as well as genera comprised of facultative anaerobes (Beneckea, Photobacterium). Two media were used-the first, a medium designed for the cultivation of marine bacteria and, the second, a medium used for the cultivation of terrestrial strains. These two media differed in the concentrations of divalent cations; the terrestrial medium (TM) contained 2 mM Mg⁺⁺ and 0.55 mM Ca⁺⁺ while the marine medium (MM) contained 50 mM Mg⁺⁺ and 10 mM Ca⁺⁺. The amount of NaCl necessary for optimal growth varied in different strains and was usually considerably higher in TM (100 to 460 mM) than in MM (70 to 300 mM). Many strains which grew in MM and TM had a shorter generation time in the former than in the latter medium. In addition, four strains which grew well in MM usually failed to grow in TM. These results show that higher levels of divalent cations are either essential for growth or stimulate growth rate, indicating that for many marine strains a terrestrial medium modified by the addition of NaCl cannot support optimal growth. Fourteen terrestrial strains of the genera Pseudomonas, Alcaligenes, Acinetobacter, Salmonella, Aeromonas, and Vibrio did not have ionic requirements comparable to those of the marine strains. All of the terrestrial organisms grew in TM without added NaCl (0.068 mM Na⁺ was present as a contaminant). In some terrestrial organisms, growth was stimulated by the addition of NaCl, the highest stimulation being found in Vibrio cholerae. The optimal growth rates and yields for four strains of this species were observed at 2.5 to 5.0 mM NaCl while the growth rates and yields in TM with no added NaCl were 40 to 50% of the optimum.     

ISOLATION OF AN ACID-SOLUBLE PROTEIN WITH LOW MOLECULAR WEIGHT FROM MONKEY BRAIN

Abstract— The isolation of a perchloric acid-soluble low molecular weight protein from brain of Macaca irus is reported. Sodium dodecyl sulphate polyacrylamide gel electrophoresis and gel isoelectric focusing indicate that the protein is free of impurities. The molecular weight, as determined by gel filtration and sodium dodecyl sulphate gel electrophoresis, is shown to be 10,400 and 9900, respectively. This is in agreement with the value of 10,700 obtained from amino acid analysis. The protein contains 27 per cent acid amino acids and 15 per cent basic amino acids. However, the relatively high amide content gives the protein a neutral nature as shown by isoelectric point determination using gel isoelectric focusing.     

Isolation and structure of an epidermal mitosis inhibiting pentapeptide

A mitosis inhibiting peptide pyroGlu-Glu-Asp-Ser-GlyOH has been isolated from mouse skin extracts. Both the biological and a synthetic peptide with the same structure reversibly inhibit epidermal mitoses in a curvilinear fashion after intraperitoneal injection. The two compounds are chromatographically identical.     

MASDET—A fast and user-friendly multiplatform software for mass determination by dark-field electron microscopy

Electron microscopy has been used to measure the mass of biological nanoparticles since the early 60s, and is the only way to obtain the mass of large structures or parameters such as the mass-per-length of filaments. The ability of this method to sort heterogeneous samples both in terms of mass and shape promises to make it a key tool for proteomics down to the single cell level. A new multiplatform software package, MASDET, that can be run under MATLAB or as a standalone program is described. Based on a user-friendly graphical interface MASDET streamlines mass evaluation and greatly increases the speed of required optimisation procedures. Importantly, the immediate application of Monte-Carlo simulations to describe multiple scattering is possible, allowing the mass analysis of thicker samples and the generation of mass thickness maps.     

Antiproliferative Effect of the Tripeptide PyroGlu-Phe-GlyNH2on Murine Melanocytes: Transitory Delay of Cell Growthin Vitroand the Cell Cycle Specificity

Cell growth and differentiation in melanocyte cell populations are regulated by a wide range of bioactive substances. Recently, the tripeptide pyroGlu-Phe-GlyNH₂which inhibits melanocyte growthin vitrowas identified in both murine nontransformed melanocytes and malignant melanoma cells. The present study was undertaken to investigate the cell cycle specificity as well as the growth inhibitory profile of the tripeptide after a single or repeated administration to melanocyte cultures. Dose-related effects of the peptide were studied using three different bioassay systems: estimation of cell number, DNA synthesis, and cell flux into mitosis. Growth of melanocyte cultures as well as melanocyte mitotic activity were found to be reduced significantly by the tripeptide at two separate dose levels (10⁻¹¹and 10⁻¹⁴–10⁻¹⁵M). Growth inhibition of melanocyte population did not last long: less than 36 h after the first and less than 24 h after the second peptide addition to the cultures. The level of DNA synthesis in melanocytes remained unchanged after a single peptide administration. The findings indicate that the tripeptide pyroGlu-Phe-GlyNH₂causes transitory delay of cell growth in cultured melanocyte population resulting from a reversible inhibition of melanocyte transition from the G₂-phase of the cell cycle into mitosis.     

Novel Quantitative Autophagy Analysis by Organelle Flow Cytometry after Cell Sonication

Autophagy is a dynamic process of bulk degradation of cellular proteins and organelles in lysosomes. Current methods of autophagy measurement include microscopy-based counting of autophagic vacuoles (AVs) in cells. We have developed a novel method to quantitatively analyze individual AVs using flow cytometry. This method, OFACS (organelle flow after cell sonication), takes advantage of efficient cell disruption with a brief sonication, generating cell homogenates with fluorescently labeled AVs that retain their integrity as confirmed with light and electron microscopy analysis. These AVs could be detected directly in the sonicated cell homogenates on a flow cytometer as a distinct population of expected organelle size on a cytometry plot. Treatment of cells with inhibitors of autophagic flux, such as chloroquine or lysosomal protease inhibitors, increased the number of particles in this population under autophagy inducing conditions, while inhibition of autophagy induction with 3-methyladenine or knockdown of ATG proteins prevented this accumulation. This assay can be easily performed in a high-throughput format and opens up previously unexplored avenues for autophagy analysis.