Search

Cellular senescence has evolved as a protective mechanism to arrest growth of cells with oncogenic potential but is accompanied by the often pathologically deleterious senescence-associated secretory phenotype (SASP). Here we demonstrate an H2O2-dependent functional disruption controlling senescence-associated Ca2+ homeostasis and the SASP. Senescent cells fail to respond to H2O2-dependent plasma lamellar Ca2+ entry when compared to pre-senescent cells. Limiting exposure to senescence-associated H2O2 restores H2O2-dependent Ca2+ entry as well as transient receptor potential cation channel subfamily C member 6 (TRPC6) function. SA-TRPC6 and SASP expression is blocked by restoring Ca2+ entry with the TRP channel antagonist SKF-96365 or by the mTOR inhibitors rapamycin and Ku0063794. Together, our findings provide compelling evidence that redox and mTOR-mediated regulation of Ca2+ entry through TRPC6 modulates SASP gene expression and approaches which preserve normal Ca2+ homeostasis may prove useful in disrupting SASP activity. Impact statement Through its ability to evoke responses from cells in a paracrine fashion, the senescence-associated secretory phenotype (SASP) has been linked to numerous age-associated disease pathologies including tumor invasion, cardiovascular dysfunction, neuroinflammation, osteoarthritis, and renal disease. Strategies which limit the amplitude and duration of SASP serve to delay age-related degenerative decline. Here we demonstrate that the SASP regulation is linked to shifts in intracellular Ca2+ homeostasis and strategies which rescue redox-dependent calcium entry including enzymatic H2O2 scavenging, TRP modulation, or mTOR inhibition block SASP and TRPC6 gene expression. As Ca2+ is indispensable for secretion from both secretory and non-secretory cells, it is exciting to speculate that the expression of plasma lamellar TRP channels critical for the maintenance of intracellular Ca2+ homeostasis may be coordinately regulated with the SASP.

Iron is an essential element required for human life. Iron is highly regulated in the body, as iron deficiency leads to many adverse health effects, such as anemias. Ferric iron (Fe3+) bound to serum transferrin (Tf) is internalized into cells via the transferrin receptor (TfR). Since the exact mechanisms of iron release in cells are not well known, a technique that allows detection of Tf bound iron inside intact human cells has been developed. Methods to determine when and where Tf releases iron inside a cell are required to better understand disease progression, including cancer. We have previously shown that Raman micro-spectroscopy is able to detect and quantify the Tf-bound iron in epithelial cells. In this work, we applied hyperspectral Raman imaging to visualize the spatial distribution of Tf-bound iron in human breast cancer T47D cells internalized with iron-loaded Tf, oxalate-Tf, a chemical Tf mutant unable to release iron, and iron-depleted Tf. We have also shown that Raman imaging can quantify the amount of iron under different times of Tf internalization (Tf uptake time), prior to fixation. Raman micro-spectroscopy provides a unique way to determine the amount of iron under different Tf internalization times by employing the Raman metric, which was used to quantify iron content in oxa- , apo-, and holo-Tf samples. Importantly, Raman microspectroscopy can be used to follow iron release from Tf in breast cancer cells. Determining the kinetics and location of iron release in cancer cells is key to further our understanding of iron metabolism during cancer progression.

Methamphetamine (METH) is a drug of abuse, the acute and chronic use of which induces neurotoxic responses in the human brain, ultimately leading to neurocognitive disorders. Our goals were to understand the impact of METH on microglial mitochondrial respiration and to determine whether METH induces the activation of the mitochondrial-dependent intrinsic apoptosis pathway in microglia. We assessed the expression of pro- apoptosis genes using qPCR of RNA extracted from a human microglial cell line (HTHU). We examined the apoptosis-inducing effects of METH on microglial cells using digital holographic microscopy (DHM) to quantify real-time apoptotic volume decrease (AVD) in microglia in a noninvasive manner. METH treatment significantly increased AVD, activated Caspase 3/7, increased the gene expression levels of the pro- apoptosis proteins, APAF-1 and BAX, and decreased mitochondrial DNA content. Using immunofluorescence analysis, we found that METH increased the expression of the mitochondrial proteins cytochrome c and MCL-1, supporting the activation of mitochondrion-dependent (intrinsic) apoptosis pathway. Cellular bio-energetic flux analysis by Agilent Seahorse XF Analyzer revealed that METH treatment increased both oxidative and glycolytic respiration after 3 h, which was sustained for at least 24 h. Several events, such as oxidative stress, neuro-inflammatory responses, and mitochondrial dysfunction, may converge to mediate METH-induced apoptosis of microglia that may contribute to neurotoxicity of the CNS. Our study has important implications for therapeutic strategies aimed at preserving mitochondrial function in METH abusing patients.

Non-invasive live cell measurements are an important tool in biomedical research. We present a combined digital holography/Raman spectroscopy technique to study live cell cultures during apoptosis. Digital holographic microscopy records an interference pattern between object and reference waves, so that the computationally reconstructed holographic image contains both amplitude and phase information about the sample. When the phase is mapped across the sample and converted into height information for each pixel, a three dimensional image is obtained. The measurement of live cell cultures by digital holographic microscopy yields information about cell shape and volume, changes to which are reflective of alterations in cell cycle and initiation of cell death mechanisms. Raman spectroscopy, on the other hand, is sensitive to rotational and vibrational molecular transitions, as well as intermolecular vibrations. Therefore, Raman spectroscopy provides complementary information about cells, such as protein, lipid and nucleic acid content, and, particularly, the spectral signatures associated with structural changes in molecules. The cell cultures are kept in the temperature-controlled environmental chamber during the experiment, which allows monitoring over multiple cell cycles. The DHM system combines a visible (red) laser source with conventional microscope base, and LabVIEW-run data processing. We analyzed and compared cell culture information obtained by these two methods.

We used phase microscopy and Raman spectroscopic measurements to assess the response of in vitro rat C6 glial cells following methamphetamine treatment in real time. Digital holographic microscopy (DHM) and three-dimensional (3-D) tomographic nanoscopy allow measurements of live cell cultures, which yield information about cell volume changes. Tomographic phase imaging provides 3-D information about the refractive index distribution associated with the morphology of biological samples. DHM provides similar information, but for a larger population of cells. Morphological changes in cells are associated with alterations in cell cycle and initiation of cell death mechanisms. Raman spectroscopy measurements provide information about chemical changes within the cells. Our Raman data indicate that the chemical changes in proteins preceded morphological changes, which were seen with DHM. Our study also emphasizes that tomographic phase imaging, DHM, and Raman spectroscopy are imaging tools that can be utilized for noninvasive simultaneous monitoring of morphological and chemical changes in cells during apoptosis and can also be used to monitor other dynamic cell processes.

We present a three-dimensional microscopic technique based on digital holographic imaging, which allows highly accurate axial localization of features inside of a three dimensional sample. When a light wave is propagating through, or reflecting from, a microscopic object, the phase changes can be converted into intensity variations using the existing digital microscopic techniques. The phase change indicates the change in the optical path length, which can be then converted to physical thickness, providing the sample height information. This property of holograms is used in phasecontrast techniques, and can also be used for quantitative 3D imaging. However, if the sample contains features with different indices of refraction, this method can only provide the overall optical thickness, and cannot determine where in the axial direction the particular feature is located. As a result, the application of Digital Holographic Microscopy to imaging of organelles within live cells, or defects within semiconductor substrates, is limited to overall morphology of the sample. To determine the axial location of features inside of a three dimensional sample, we developed a phase image processing method based on analyzing images taken from non-zero incident angles. When compared, these images can discriminate between various axial depths of features, while still retaining the information about the overall thickness profile of the sample.

Methamphetamine induces neurotoxic responses in human brain that can further deteriorate into neural damage and cellular apoptosis. We examined the apoptosis inducing effects of Methamphetamine in real time in glial cells by digital holographic microscopy.