José-Enrique O’Connor

Intracellular coexpression of CXC- and CC- chemokine receptors and their ligands in human melanoma cell lines and dynamic variations after xenotransplantation.

BackgroundChemokines have been implicated in tumor progression and metastasis. In melanoma, chemokine receptors have been implicated in organ selective metastasis by regulating processes such as chemoattraction, adhesion and survival.MethodsIn this study we have analyzed, using flow cytometry, the systems formed by the chemokine receptors CXCR3, CXCR4, CXCR7, CCR7 and CCR10 and their ligands in thirteen human melanoma cell lines (five established from primary tumors and eight established from metastasis from different tissues). WM-115 and WM-266.4 melanoma cell lines (obtained from a primary and a metastatic melanoma respectively) were xenografted in nude mice and the tumors and cell lines derived from them were also analyzed.ResultsOur results show that the melanoma cell lines do not express or express in a low degree the chemokine receptors on their cell surface. However, melanoma cell lines show intracellular expression of all the aforementioned receptors and most of their respective ligands. When analyzing the xenografts and the cell lines obtained from them we found variations in the intracellular expression of chemokines and chemokine receptors that differed between the primary and metastatic cell lines. However, as well as in the original cell lines, minute or no expression of the chemokine receptors was observed at the cell surface.ConclusionsCoexpression of chemokine receptors and their ligands was found in human melanoma cell lines. However, this expression is intracellular and receptors are not found at the cell membrane nor chemokines are secreted to the cell medium. The levels of expressed chemokine receptors and their ligands show dynamic variations after xenotransplantation that differ depending on the origin of the cell line (from primary tumor or from metastasis).

Flow Cytometry versus Fluorescence Microscopy

Since the pioneer work of the botanist Matthias Jakob Schleiden and the zoologist Theodor Schwann in 1839, and of Rudolph Virchow in 1859, cell research progressed in two opposite directions. Cell biologists focused their increasingly more powerful microscopies into the cell structure to reveal the great morphological complexity of the cytoplasm. A growing number of subcellular organelles thus challenged the early biochemists to discover their specific molecular features and their coordination to maintain an ordered cell life. The biochemists’ answer to such a challenge consisted usually in tearing apart cells into their discrete components and obtaining information on molecules and pathways of each single part. The assembly of this jigsaw puzzle into an integrated view of a functional cell, and of such a cell within higher levels of organization, could not be achieved exclusively by pure biochemical methods. As early as in 1961, Jean Brachet wrote that “The cell biologist tries to explain in molecular terms what he sees under his microscope; he has become a molecular biologist. The biochemist in turn has become a biochemical cytologist, equally interested in the structure of the cell and the biochemical activity in which it is involved” (1).

Cytomics of Oxidative Stress: Probes and Problems

Oxidative stress has been implicated in cellular senescence and aging, as well as in the onset and progression of many diverse genetic and acquired diseases and conditions. However, reactive oxygen (ROS) and nitrogen (RNS) species initiating oxidative stress also serve important regulatory roles, mediated by intercellular and intracellular signaling, adaptation to endogenous and exogenous stress, and destruction of invading pathogens. Fluorescence-based analysis of oxidative stress and related processes is an important cytomic application; almost 4000 papers were published between 1989 and 2016. To ascertain the specific role of ROS and RNS in oxidative stress studies by cytomic methodologies, it is essential to detect and characterize these species accurately. Unfortunately, the detection and quantitation of individual intracellular ROS and RNS remains a challenge, but different, complementary cytometric strategies directed toward other endpoints of oxidative stress may also be considered. In this chapter we present and briefly discuss the limitations and perspectives of such approaches.

WITHDRAWN: Systems Biology and Immune Aging

The Publisher regrets that this article is an accidental duplication of anarticle that has already been published, http://dx.doi.org/10.1016/j.imlet.2014.09.009. The duplicate article has therefore been withdrawn.

Analysis of Subcellular Components by Fluorescent-Lectin Binding and Flow Cytometry

Because of their extensive availability and the wide spectrum of carbohydrates that may be specifically bound, lectins have become essential reagents for detection and quantitation of glycoconjugates in solution and in cell surfaces, identification and separation of cells, and functional studies based on membrane properties (1,2).