Hypertonicity-induced cation channels in HepG2 cells: architecture and role in proliferation vs. apoptosis.

KEY POINTS: Na+ conducting hypertonicity-induced cation channels (HICCs) are key players in the volume restoration of osmotically shrunken cells and, under isotonic conditions, considered as mediators of proliferation - thereby opposing apoptosis. In an siRNA screen of ion channels and transporters in HepG2 cells, with the regulatory volume increase (RVI) as read-out, δENaC, TRPM2 and TRPM5 were identified as HICCs. Subsequently, all permutations of these channels were tested in RVI and patch-clamp recordings and, at first sight, HICCs were found to operate in an independent mode. However, there was synergy in the siRNA perturbations of HICC currents. Accordingly, proximity ligation assays showed that δENaC was located in proximity to TRPM2 and TRPM5 suggesting a physical interaction. Furthermore, δENaC, TRPM2 and TRPM5 were identified as mediators of HepG2 proliferation - their silencing enhanced apoptosis. Our study defines the architecture of HICCs in human hepatocytes as well as their molecular functions. ABSTRACT: Hypertonicity-induced cation channels (HICCs) are a substantial element in the regulatory volume increase (RVI) of osmotically shrunken cells. Under isotonic conditions, they are key effectors in the volume gain preceding proliferation; HICC repression, in turn, significantly increases apoptosis rates. Despite these fundamental roles of HICCs in cell physiology, very little is known concerning the actual molecular architecture of these channels. Here, an siRNA screening of putative ion channels and transporters was performed, in HepG2 cells, with the velocity of RVI as the read-out; in this first run, δENaC, TRPM2 and TRPM5 could be identified as HICCs. In the second run, all permutations of these channels were tested in RVI and patch-clamp recordings, with special emphasis on the non-additivity and additivity of siRNAs - which would indicate molecular interactions or independent ways of channel functioning. At first sight, the HICCs in HepG2 cells appeared to operate rather independently. However, a proximity ligation assay revealed that δENaC was located in proximity to both TRPM2 and TRPM5. Furthermore, a clear synergy of HICC current knock-downs (KDs) was observed. δENaC, TRPM2 and TRPM5 were defined as mediators of HepG2 cell proliferation and their silencing increased the rates of apoptosis. This study provides a molecular characterization of the HICCs in human hepatocytes and of their role in RVI, cell proliferation and apoptosis.

Reversible Cryo-arrests of Living Cells to Pause Molecular Movements for High-resolution Imaging.

Fluorescence live-cell imaging by single molecule localization microscopy (SMLM) or fluorescence lifetime imaging microscopy (FLIM) in principle allows for the spatio-temporal observation of molecular patterns in individual, living cells. However, the dynamics of molecules within cells hamper their precise observation. We present here a detailed protocol for consecutive cycles of reversible cryo-arrest of living cells on a microscope that allows for a precise determination of the evolution of molecular patterns within individual living cells. The usefulness of this approach has been demonstrated by observing ligand-induced clustering of receptor tyrosine kinases as well as their activity patterns by SMLM and FLIM (Masip et al., 2016).

Reversible cryo-arrest for imaging molecules in living cells at high spatial resolution.

The dynamics of molecules in living cells hampers precise imaging of molecular patterns by functional and super-resolution microscopy. We developed a method that circumvents lethal chemical fixation and allows on-stage cryo-arrest for consecutive imaging of molecular patterns within the same living, but arrested, cells. The reversibility of consecutive cryo-arrests was demonstrated by the high survival rate of different cell lines and by intact growth factor signaling that was not perturbed by stress response. Reversible cryo-arrest was applied to study the evolution of ligand-induced receptor tyrosine kinase activation at different scales. The nanoscale clustering of epidermal growth factor receptor (EGFR) in the plasma membrane was assessed by single-molecule localization microscopy, and endosomal microscale activity patterns of ephrin receptor A2 (EphA2) were assessed by fluorescence lifetime imaging microscopy. Reversible cryo-arrest allows the precise determination of molecular patterns while conserving the dynamic capabilities of living cells.

Adaptive responses of cell hydration to a low temperature arrest.

Slow cooling leads to a passive dehydration of cells, whereas rehydration during warming reflects the active regain of functionality. The ability to modulate such an energy demanding process could be instrumental in optimizing the cryo-arrest of living systems. In the present study, various levels of hypertonic stress were used to disturb the water content of cells and to define the energy profiles of aquaporins and (Na(+) conducting) cation channels during rehydration. Na(+) import was found to be the rate-limiting step in water restoration, whereas aquaporins merely played a permissive role. Indeed, regulated Na(+) import was increased 2-fold following cryo-arrests, thus facilitating the osmotic rehydration of cells. Freezing temperatures increased cell viscosity with a remarkable hysteresis and viscosity was a trigger of cation channels. The peptide hormone vasopressin was a further activator of channels, increasing the viability of post-cryo cells considerably. Hence, the hormone opens the path for a novel class of cryo-protectants with an intrinsic biological activity.

Mass-Spectrometry-Based Proteomics Reveals Organ-Specific Expression Patterns To Be Used as Forensic Evidence.

Standard forensic procedures to examine bullets after an exchange of fire include a mechanical or ballistic reconstruction of the event. While this is routine to identify which projectile hit a subject by DNA analysis of biological material on the surface of the projectile, it is rather difficult to determine which projectile caused the lethal injury--often the crucial point with regard to legal proceedings. With respect to fundamental law it is the duty of the public authority to make every endeavor to solve every homicide case. To improve forensic examinations, we present a forensic proteomic method to investigate biological material from a projectile's surface and determine the tissues traversed by it. To obtain a range of relevant samples, different major bovine organs were penetrated with projectiles experimentally. After tryptic "on-surface" digestion, mass-spectrometry-based proteome analysis, and statistical data analysis, we were able to achieve a cross-validated organ classification accuracy of >99%. Different types of anticipated external variables exhibited no prominent influence on the findings. In addition, shooting experiments were performed to validate the results. Finally, we show that these concepts could be applied to a real case of murder to substantially improve the forensic reconstruction.

The ΔC splice-variant of TRPM2 is the hypertonicity-induced cation channel in HeLa cells, and the ecto-enzyme CD38 mediates its activation.

Hypertonicity-induced cation channels (HICCs) are key-players in proliferation and apoptosis but their molecular correlate remains obscure. Furthermore, the activation profile of HICCs is not well defined yet. We report here that, in HeLa cells, intracellular adenosine diphosphate ribose (ADPr) and cyclic ADPr (cADPr), as supposed activators of TRPM2, elicited cation currents that were virtually identical to the osmotic activation of HICCs. Silencing of the expression of TRPM2 and of the ecto-enzyme CD38 (as a likely source of ADPr and cADPr) inhibited HICC as well as nucleotide-induced currents and, in parallel, the hypertonic volume response of cells (the regulatory volume increase, RVI) was attenuated. Quantification of intracellular cADPr levels and the systematic application of extra- vs. intracellular nucleotides indicate that the outwardly directed gradient rather than the cellular activity of ADPr and cADPr triggers TRPM2 activation, probably along with a simultaneous biotransformation of nucleotides.Cloning of TRPM2 identified the ΔC-splice variant as the molecular correlate of the HICC, which could be strongly supported by a direct comparison of the respective Ca²âº selectivity. Finally, immunoprecipitation and high-resolution FRET/FLIM imaging revealed the interaction of TRPM2 and CD38 in the native as well as in a heterologous (HEK293T) expression system. We propose transport-related nucleotide export via CD38 as a novel mechanism of TRPM2/HICC activation. With the biotransformation of nucleotides running in parallel, continuous zero trans-conditions are achieved which will render the system infinitely sensitive.

A new approach to the investigation of sexual offenses-cytoskeleton analysis reveals the origin of cells found on forensic swabs.

There are forensic inquiries in which an identification of epithelial cell types would provide important probative evidence. In cancer diagnosis, this information is yielded by histological examination of cytokeratin (Ck). Therefore, we tested 19 antibodies against different Cks (Ck1, Ck2e, Ck4, Ck5-6, Ck7, Ck8, Ck9, CK10, Ck13, Ck14, Ck15, Ck16, Ck17, Ck18, Ck19, Ck20, Ck903, PanCkAE1_3, and CAM5-2) on histological sections of epidermis, buccal mucosa, vaginal mucosa, penis, urogenital tract, and rectum and could identify two antigens unique to buccal-cell and vaginal-cell (Ck4) and skin epithelial-cell (Ck10) cytokeratin. Subsequently, we developed an immunocytological technique for distinguishing swabbed skin and mucosal cells up to at least 1 year after sampling. By the detection of the Ck4 and Ck10 mRNAs in biopsy and laser capture microdissection collected samples via quantitative real-time polymerase chain reaction, we were able to confirm our immunological findings. Hence, this study offers techniques to discriminate between skin and mucosal cells (buccal and vaginal) in forensic casework.

[Reconstruction of a fire homicide after pouring denatured alcohol on the victim]. / Rekonstruktion eines Brandmordes nach Ubergiessen mit Spiritus.

A 48-year-old man died from extensive burns suffered especially on the upper part of the body during a dispute with his ex-wife, who had poured spirit or a spirit-water mixture over him. For initially unknown reasons, the man's clothing caught fire. Whereas the public prosecutor assumed that the woman had intentionally poured a larger amount of spirit over her ex-husband before setting fire to him, the defendant first claimed to have poured the rest of a water-spirit mixture left over from cleaning the windows over the man and that his clothing caught fire on lighting a cigarette. To clarify the course of events, fire tests with spirit in various dilutions were conducted, which showed that even with undiluted spirit a direct contact with the flame of at least 1 second is necessary to start a fire. There is no deflagration, if spirit is used as a fire accelerant. In the trial, the defendant made a confession and admitted to have poured a mixture of 75% spirit and 25% water over her ex-husband and set fire to his right sleeve with the intention to kill him.

[Lethal intravenous infusion of a wound antiseptic containing polyhexanide]. / Tödliche intravenöse Infusion eines polyhexanidhaltigen Wundantiseptikums.

Polyhexamethylene biguanide (PHMB) is considered to be highly histocompatible and is one of the most frequently used wound antiseptics. Only one case of intoxication has been reported so far. The present case of a lethal intoxication is the first fatal incident described where causality is substantiated by a temporal coincidence between application and ascertainable organ damage. The laboratory-chemical and histological investigations verified the toxicity of this substance after intravenous application with the main findings being severe hepatic and pancreatic damage.

alpha-ENaC is a functional element of the hypertonicity-induced cation channel in HepG2 cells and it mediates proliferation.

The molecular correlate of hypertonicity-induced cation channels (HICCs) and their role in proliferation vs. apoptosis is a matter of debate. We report in this paper that, in whole-cell patch-clamp recordings, hypertonic stress (340-->450 mosM) reversibly increased the Na(+) conductance of HepG2 cells from 0.8 to 5.8 nS. The effect was dose-dependently inhibited by flufenamate and amiloride, known blockers of HICCs, with some 50% efficiency at 300 muM. In parallel, both drugs decreased HepG2 cell proliferation [in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays and with automatic cell counting]. Small interfering RNA (siRNA) silencing of the alpha-subunit of the epithelial Na(+) channel (ENaC) reduced hypertonicity-induced Na(+) currents to 60%, whereas the rate of HepG2 cell proliferation was approximately half of that of the control. Moreover, alpha-ENaC siRNA inhibited the regulatory volume increase of HepG2 cells (measured with scanning acoustic microscopy) by 60%. In florescence-activated cell sorting measurements, silencing of alpha-ENaC led to a significant decrease in the G1 and an increase in the G2/M phase of the cell cycle, whereas the S phase was not changing. Finally (determined by a caspase 3/7 assay), HICC inhibition by flufenamate and silencing of alpha-ENaC increased the rate of apoptosis in HepG2 cells. It is concluded that alpha-ENaC is one functional element of the HICC in HepG2 cells and that the channel is an important mediator of cell proliferation; likewise, HICC blockage shifts the system from a proliferative into a rather apoptotic one. This is the first report of a role of alpha-ENaC in cell proliferation.

Immunocytochemical examinations of biological traces on expanding bullets (QD-PEP).

When a crime victim has been injured with several different objects, it is of central importance for the forensic investigation to be able to show which object caused which injury, especially if one of the injuries was lethal. In cases of bullet penetration wounds it is often not possible to find such evidence. However, immunocytochemical investigations can accurately match a victim's injury to a particular bullet path through the body. In cases where expanding bullets have been used and the heart or liver has been struck by a projectile, it can be shown that the cells remaining on the bullet stem from those particular organs. In this case the specific cytological evidence was established by means of marking heart- and liver-specific tissue proteins with appropriate antibodies (cardiac troponin I and HepPar 1) followed by disclosure with an appropriate chromogen. Thus, in principle, cells can be used as evidence after being extracted from the projectiles by either damp cotton-wool swabs or adhesive trace evidence tape. Because of the specificity of the used immunocytochemical antibodies, finding evidence of an antigen on a particular projectile proves that it was the object that injured the organs.

Hypertonicity-induced cation channels rescue cells from staurosporine-elicited apoptosis.

Cell shrinkage is one of the earliest events during apoptosis. Cell shrinkage also occurs upon hypertonic stress, and previous work has shown that hypertonicity-induced cation channels (HICCs) underlie a highly efficient mechanism of recovery from cell shrinkage, called the regulatory volume increase (RVI), in many cell types. Here, the effects of HICC activation on staurosporine-induced apoptotic volume decrease (AVD) and apoptosis were studied in HeLa cells by means of electronic cell sizing and whole-cell patch-clamp recording. It was found that hypertonic stress reduces staurosporine-induced AVD and cell death (associated with caspase-3/7 activation and DNA fragmentation), and that this effect was actually due to activation of the HICC. On the other hand, staurosporine was found to significantly reduce osmotic HICC activation. It is concluded that AVD and RVI reflect two fundamentally distinct functional modes in terms of the activity and role of the HICC, in a shrunken cell. Our results also demonstrate, for the first time, the ability of the HICC to rescue cells from the process of programmed cell death.

Indoloquinolizidine derivatives as novel and potent apoptosis inducers and cell-cycle blockers.

A collection of approximately 11 000 natural-product derived and inspired compounds was screened for potential apoptosis inducers in the human tumour cell lines HepG2 (liver), HeLa (cervix) and MCF-7 (breast) by means of MTT and ATP-luminescence assays, automated cell counting, caspase 3/7 assay as well as by fluorescence activated cell sorting (FACS) analysis. A group of seven indoloquinolizidine derivatives was identified that exhibited IC(50) values for cell proliferation as low as 2 mumol L(-1), with no major necrosis of cells detectable. At the same time, an increase in the rate of apoptosis of up to 600 % relative to the reference level was observed. FACS analysis indicated that these effects are related to an arrest of cells in the G(2)M phase of the cell cycle.

Subunits alpha, beta and gamma of the epithelial Na+ channel (ENaC) are functionally related to the hypertonicity-induced cation channel (HICC) in rat hepatocytes.

Specific small interfering RNA (siRNA) constructs were used to test for the functional relation of subunits alpha, beta, and gamma of the epithelial Na(+) channel (ENaC) to the hypertonicity-induced cation channel (HICC) in confluent rat hepatocytes. In current-clamp recordings, hypertonic stress (300 --> 400 mosM) increased membrane conductance from 75.4 +/- 9.4 to 91.1 +/- 11.2 pS (p < 0.001). The effect was completely blocked by 100 microM amiloride and reduced to 46, 30, and 45% of the control value by anti-alpha-, anti-beta-, and anti-gamma-rENaC siRNA, respectively. Scanning acoustic microscopy revealed an initial shrinkage of cells from 6.98 +/- 0.45 to 6.03 +/- 0.43 pl within 2 min. This passive response was then followed by a regulatory volume increase (RVI) by 0.42 +/- 0.05 pl (p < 0.001). With anti-alpha-, anti-beta-, and anti-gamma-rENaC siRNA, the volume response was reduced to 31, 31, and 36% of the reference level, respectively. It is concluded that all three subunits of the ENaC are functionally related to RVI and HICC activation in rat hepatocytes.

[Sudden death in the presence of systemic mastocytosis and urticaria pigmentosa]. / Plötzlicher Tod bei systemischer Mastozytose mit Urticaria pigmentosa.

Systemic mastocytosis is an extremely rare disease characterised by abnormal mast cell production and accumulation of mast cells in the bone marrow or organs, and to some extent also in the skin (urticaria pigmentosa). To date there have been no descriptions of death caused by systemic mastocytosis. The present first description of such a fatality is based upon a case of anaphylactic histamine shock, probably caused by the consumption of peanuts. The affected person suffered from urticaria pigmentosa and developed a fairly typical pruritus prior to death. In the serum sample taken post-mortem the tryptase concentration was markedly elevated. The diagnosis of systemic mastocytosis was established on the basis of two primary and three secondary criteria according to the WHO classification, which underlines the importance of histological investigations in cases where the cause of death is unclear.

The epsilon-isoform of PKC mediates the hypertonic activation of cation channels in confluent monolayers of rat hepatocytes.

We were interested whether PKC alpha, delta, epsilon or zeta is the isoform actually employed in the activation of hypertonicity-induced cation channels (HICCs) in primary cultures of rat hepatocytes. Quantitative SDS-page and Western-blot experiments revealed that PKC alpha, delta and epsilon were stimulated by Indolactam V (as a DAG substitute for activation of c and nPKCs) but that only PKC delta and epsilon did respond to hypertonic stress. Furthermore, chelation of intracellular Ca(++) by BAPTA-AM did not alter HICC activation in cable-analysis experiments whereas Indolactam V as well as V8 (an Indolactam derivative specific for PKC delta and epsilon) activated HICC currents under isotonic conditions. Finally, by use of Rottlerin (as an inhibitor exhibiting a slight preference for PKC delta over epsilon) PKC epsilon could be identified as the most likely isoform responsible for the activation of the HICC.

[Visualization of biological traces on evidence by ninhydrin]. / Visualisierung von biologischem Spurenmaterial auf Spurenträgern mit Hilfe von Ninhydrin.

Because of possible contamination of samples with PCR inhibitors and to avoid the typing of mixed profiles the source material for forensic DNA investigations should be collected as directly and securely as possible from the evidence. This approach requires a detectability of the source material which is often not given. The procedure introduced here using selected cases enables visualization of DNA-containing materials on evidence and hence controlled analysis. For this purpose the specimen is treated with ninhydrin. A following dye reaction verifies the presence of biological material, which possibly contains DNA. An impact on subsequent STR-analysis was not observed.

[Immunohistochemical proof of amelogenin in teeth--a contribution to the evaluation of the age in the identification of unknown corpses]. / Immunhistochemischer Nachweis von Amelogenin an Zähnen--ein Beitrag zur Abschätzung des Lebensalters bei der Identifikation unbekannter Leichen.

When a corpse is found that cannot be identified, one of the forensic tasks is to determine how old the person was when she or he died. To solve this frequently asked question in forensic practice, the enamel protein amelogenin was marked immunohistochemically in 249 extracted teeth. Amelogenin is already produced during prenatal development and is gradually used up throughout life into old age. Hence, the demonstrability decreases with age. The extent of the labelling can be quantified by measuring the mean optical density (MOD). While it is not possible to determine the age on the basis of the mean optical density measurements alone, logistic regression of the MOD together with dichotomisation of the teeth according to age allows statements as to whether the person was younger or older than 30 years.

[Immunocytochemical examination of biological traces on knife blades]. / Immunzytochemische Untersuchungen biologischer Spuren an Tatklingen.

In cases of penetrating stab wounds by different knives it is highly relevant to prove which knife caused which injury, especially if one of the injuries was lethal. This is possible by immunocytochemical examination of cellular material remaining on the injuring blade because some organs have organ-specific antigen determinants such as alpha-l-fetoprotein in the liver cells or cardiac troponin I in the heart muscle cells, to which antibodies can bind. Even when penetrations occur through several layers of clothing, enough cells from the injured organ remain on the blade of a knife to allow immunohistochemical examination. These cells can be collected by means of adhesive film or wiping the blade and can be stained immunocytochemically. The organ specificity of the examined proteins allows proof of their origin. The present study shows that immunocytochemical alpha-l-fetoprotein and cardiac troponin I staining of the cells remaining on a knife blade enables proof of whether the knife blade injured the heart or the liver, or both.

A novel inhibitor of hypertonicity-induced cation channels in HeLa cells.

Despite the paramount significance of hypertonicity-induced cation channels (HICCs) in cell proliferation and apoptosis, a detailed analysis of these processes is hindered by the very limited number of HICC blockers available. Here, 2-aminoethoxydiphenyl borate (2-APB) is introduced as a novel and effective inhibitor of the HICC in HeLa cells. Its efficiency is defined with reference to flufenamic acid (as the most potent blocker so far), both, in whole-cell patch-clamp recordings and in measurements of cell volume regulation.