Marie-Hélène Ratinaud

Recent advances of flow cytometry in fundamental and applied microbiology

Summary— This review focuses on the recent applications of flow cytometry (FCM) in microbiological research (1987‐mid 1992). It tries to give a scope of the important breakthroughs which occurred in this field during this period. The technical difficulties of microorganism analysis by flow cytometry is briefly appraised. The significance and the limits of the different microbial cell parameters attainable by flow analyses are systematically evaluated: light scatter for cell size and structure, fluorescence measurements for quantification of cellular components, microbial antigen detection and cell physiological activity estimation. Emphasis is given on the new technological advances which appeared in the last two years. The second part of the review is devoted to the analysis of the usefulness of flow cytometric approach in the different fields of microbiology: fundamental studies in microbial physiology, differentiation, microbial ecology and aquatic sciences, medical microbiology, parasitology, microbial pharmacology and biotechnology.

Assessment of fluorochromes for cellular structure and function studies by flow cytometry

Summary— Because flow cytometry permits the analysis of individual whole cells, one of the key requirements in selecting a probe is its ability to target the site of interest into cells. In addition, dyes must possess ideal properties (ie extinction coefficient, Stokes shift) rendering them appropriate for this methodology. Other characteristics, such as fluorescence quenching and energy transfer, inherent to the staining, provide numerous applications in flow cytometry. The available fluorophores used in flow cytometry are classified according to their cellular incorporation and binding. Thus, probes are presented and discussed as follows: 1) dyes of cellular components (DNA, RNA, proteins, lipids); 2) probes of membrane potential; 3) fluorophores that are sensitive to their microenvironment (pH, calcium, etc); and 4) those used for measurement of enzymatic activities into cells.

Cell cycle analysis by flow cytometry: principles and applications

Summary— Numerous flow cytometric analyses are based on DNA content studies. We have considered firstly monoparametric cell cycle analyses, which only take DNA content into account, but are sometimes of limited interest. Then, we have presented multiparametric analyses, which can be used to improve cycle phase identification by taking simultaneously into account DNA and other cellular components, or by considering some events occurring during cell cycle. Finally, we have discussed monoparametric and multiparametric cell cycle analysis interest in various application fields, particularly in pharmacology, toxicology, tumoral pathology and higher plant system studies.

Autophagy in human keratinocytes: an early step of the differentiation?

Abstract:  Studies have established that autophagy constitutes an efficient process to recycle cellular components and certain proteins. The phenomenon was demonstrated primarily in response to nutrient starvation, and there are increasing evidences that it is implied in differentiation. Keratinocyte differentiation was going along an activation of lysosomal enzymes and organelle clearance, and terminal steps are sometimes described as a specialized form of cell death leading to corneocytes. We examined whether initiation of the process in human keratinocyte HaCaT involves autophagy. The KSFM™ culture medium was substituted by M199, which contains a low glucose concentration but a high calcium level (known to induce differentiation). Metabolic stress reduced enhanced cell number in G1 phase, without apoptotic features (ΔΨmt and membrane integrity are unchanged). Morphological changes were associated with a lower integrin ß1 expression and modifications of protein levels involved in keratinocyte differentiation (involucrin, keratin K10 and ΔNp63α). Whereas autophagic signalling was supported by SIRT1 and pAMPK (T172) increase according to time kinetic, which led to the disappearance of mTOR phosphorylated on S2448 residue. The significant Bcl‐XL level reduction with stress promoted autophagy, by the release of Beclin‐1, whereas ATG5‐ATG12 and LC3‐II that are involved in autophagosome formation were enhanced significantly. Then, the level of lysosomal protein cathepsin B rose to execute autophagy. Kinetic studies established that autophagy would constitute an early signalling process required for keratinocyte commitment in differentiation pathway.

Autophagy takes place in mutated p53 neuroblastoma cells in response to hypoxia mimetic CoCl2

Solid tumors like neuroblastoma exhibit hypoxic areas, which can lead both to cell death or aggressiveness increase. Hypoxia is a known stress able to induce stabilization of p53, implicated in cell fate regulation. Recently, p53 appeared to be involved in autophagy in an opposite manner, depending on its location: when nuclear, it enhanced transcription of pro-autophagic genes whereas when cytoplasmic, it inhibited the autophagic process. Today, we used cobalt chloride, a hypoxia mimetic that inhibits proteasomal HIF-1 degradation and generates reactive oxygen species (ROS). We focused on CoCl2-induced cell death in a DNA-binding mutated p53 neuroblastoma cell line (SKNBE(2c)). An autophagic signaling was evidenced by an increase of Beclin-1, ATG 5-12, and LC3-II expression whereas the p53(mut) presence decreased with CoCl2 time exposure. Activation of the pathway seemed to protect cells from ROS production and, at least in part, from death. The autophagic inhibitors activated the apoptotic signaling and the death was enhanced. To delineate the eventual implication of the p53(mut) in the autophagic process in response to hypoxia, we monitored signaling in p53(WT)SHSY5Y cells, after either shRNA-p53 down-regulation or transcriptional activity inhibition by pifithrin alpha. We did not detect autophagy neither with p53(wt) nor when p53 was lacking whereas such a response was effective with a mutated or inactivated p53. To conclude, mutated p53 in neuroblastoma cells could be linked with the switch between apoptotic response and cell death by autophagy in response to hypoxic mimetic stress.

The cell death response to the ROS inducer, cobalt chloride, in neuroblastoma cell lines according to p53 status.

Cobalt chloride (CoCl2), a hypoxia-mimetic agent, induces reactive oxygen species (ROS) generation, leading to cell death. Divergent data have been reported concerning p53 implication in this apoptotic mechanism. In this study, we studied cobalt-induced cell death in neuroblastoma cell lines carrying wild-type (WT) p53 ( SHSY5Y) and a mutated DNA-binding domain p53 [SKNBE(2c)]. CoCl2 induced an upregulation of p53, p21 and PUMA expression in WT cells but not in SKNBE(2c). In SHSY5Y cells, p53 serine-15 phosphorylation appeared early (6 h) in the mitochondria, and also in the nucleus after 12 h. In contrast, in SKNBE(2c) cells, the slight nuclear signal disappeared with CoCl2 treatment. In SHSY5Y cells, a mitochondrial pathway dependent on caspases [collapse of mitochondrial transmembrane potential (∆Ψmt), caspase 3 and 9 activation], was activated in a time-dependent manner. SKNBE(2c) cells exhibited a delay in the cell death executive phase linked to a caspase-independent pathway, involving apoptosis inducing factor nuclear translocation, but also an autophagic process attested by LC3-II expression and cathepsin-B activation. The downregulation of p53 in SHSY5Y cells by siRNA induced a cell death pathway related to the one observed in SKNBE(2c) cells. Finally, CoCl2 induced time-dependent canonical p53 mitochondrial apoptosis in the WT p53 cell line, and caspase-independent cell death in cells with a mutated or KO p53.

Flow cytometry's contribution to the measurement of cell functions

Summary— Flow cytometry has important advantages over conventional techniques. It is rapid, highly sensitive and allows multi‐parametric analysis and cell sorting. Potential exists for the measurement of many cell functions by flow cytometry. The technique can be used to determine cell viability, intracellular calcium and pH, membrane potential, enzyme activities, membrane fluidity and endocytosis. Numerous examples are given on the applications of flow cytometry for cell functions measurements in the fundamental and biomedical fields.

Sequential gene expression of P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP) and lung resistance protein: functional activity of P-gp and MRP present in the doxorubicin-resistant human K562 cell lines.

Previous studies have reported that P-glycoprotein (P-gp), a transmembrane efflux pump involved in multidrug resistance (MDR), was overexpressed in the doxorubicin (Dox)-resistant human erythroleukemia cell line K562. Nevertheless, several results suggested that P-gp was not the only mechanism involved in these resistant cells. Sequential co-expression of other MDR-associated proteins was sometimes reported, as MDR-associated protein (MRP) and lung resistance protein (LRP), in different MDR cell lines. Thus, mRNA expression and stability of P-gp, MRP and LRP were analyzed, while their corresponding protein levels were quantified in correlation with functional assay, in the K562 cell line and two Dox-resistant variants (K562/R). Their P-gp content was in accordance with their degree of resistance, but not as much in the level of mRNA expression, suggesting a post-transcriptional regulation. On the other hand, MRP could play a minor role in MDR because of an unchanged expression in K562/R sublines. A surprising progressive disappearance of LRP in both resistant cells suggested that the original mechanism of drug redistribution may be operative, involving a negative role for LRP.

Glioblastoma, hypoxia and autophagy: a survival-prone ‘ménage-à-trois’

Glioblastoma multiforme is the most common and the most aggressive primary brain tumor. It is characterized by a high degree of hypoxia and also by a remarkable resistance to therapy because of its adaptation capabilities that include autophagy. This degradation process allows the recycling of cellular components, leading to the formation of metabolic precursors and production of adenosine triphosphate. Hypoxia can induce autophagy through the activation of several autophagy-related proteins such as BNIP3, AMPK, REDD1, PML, and the unfolded protein response-related transcription factors ATF4 and CHOP. This review summarizes the most recent data about induction of autophagy under hypoxic condition and the role of autophagy in glioblastoma.

Modifications of oxido-reductase activities in adriamycin-resistant leukaemia K562 cells

Adriamycin (ADR), a well-known antitumoral drug, interacts with DNA (nuclear and mitochondrial) and cardiolipin. Moreover, ADR induces numerous mitochondrial modifications in sensitive cells. However, no results have yet been obtained as to the repercussions of drug effects on oxido-reductase activities in ADR-resistant cells. To analyze mitochondrial damage induced by ADR treatment, we investigated lactate content, oxygen consumption, respiratory chain activities, and cytochrome content in ADR-sensitive K562 cells and two ADR-resistant variants (K562/R0.2 and K562/R0.5 cells). Biochemical investigations in ADR-resistant cells showed several mitochondrial modifications (in comparison to the parental cell line) according to the variant line and the physiologic state. More particularly, in K562/R0.5 cells cytochrome c (cyt c) oxidase (COX; EC 1.9.3.1) activity and cytochrome aa3 content dramatically decreased since cells enter into the stationary phase. Regardless of the number of multidrug-resistant cell subcultures in ADR-free medium, the cytochrome c oxidase activity in the stationary phase remained unchanged, indicating an irreversible effect of the drug. These alterations could correspond to several modifications of the nuclear and/or mitochondrial genome(s) following acquisition of the ADR resistance phenotype by K562 cells.