Kersti Alm

Non-invasive, label-free cell counting and quantitative analysis of adherent cells using digital holography

Manual cell counting is time consuming and requires a high degree of skill on behalf of the person performing the count. Here we use a technique that utilizes digital holography, allowing label‐free and completely non‐invasive cell counting directly in cell culture vessels with adherent viable cells. The images produced can provide both quantitative and qualitative phase information from a single hologram. The recently constructed microscope Holomonitor™ (Phase Holographic Imaging AB, Lund, Sweden) combines the commonly used phase contrast microscope with digital holography, the latter giving us the possibility of achieving quantitative information on cellular shape, area, confluence and optical thickness. This project aimed at determining the accuracy and repeatability of cell counting measurements using digital holography compared to the conventional manual cell counting method using a haemocytometer. The collected data were also used to determine cell size and cellular optical thickness. The results show that digital holography can be used for non‐invasive automatic cell counting as precisely as conventional manual cell counting

Cell guidance by magnetic nanowires.

The phenomenon of contact guidance on thin fibers has been known since the beginning of the 20th century when Harrison studied cells growing on fibers from spiders web. Since then many studies have been performed on structured surfaces and fibers. Here we present a new way to induce guidance of cells or cell processes using magnetic nanowires. We have manufactured magnetic Ni-nanowires (200 nm in diameter and 40 microm long) with a template-based electro-deposition method. Drops of a nanowire/ethanol suspension were placed on glass cover slips. The nanowires were aligned in an external magnetic field and adhered to the cover slips after evaporation of the ethanol. When the wires had adhered, the magnetic field was removed. L929 fibroblasts and dissociated dorsal root ganglia (DRG) neurons from mice were cultured on the nanowire-coated cover slips for 24 h and 72 h respectively. The fibroblasts were affected by the aligned nanowires and displayed contact guidance. Regenerated axons also displayed contact guidance on the wires. There were no overt signs of toxicity caused by Ni-wires. Aligned magnetic nanowires can be useful for lab-on-a-chip devices and medical nerve grafts.

Cells and polyamines do it cyclically

Cell-cycle progression is a one-way journey where the cell grows in size to be able to divide into two equally sized daughter cells. The cell cycle is divided into distinct consecutive phases defined as G(1) (first gap), S (synthesis), G(2) (second gap) and M (mitosis). A non-proliferating cell, which has retained the ability to enter the cell cycle when it receives appropriate signals, is in G(0) phase, and cycling cells that do not receive proper signals leave the cell cycle from G(1) into G(0). One of the major events of the cell cycle is the duplication of DNA during S-phase. A group of molecules that are important for proper cell-cycle progression is the polyamines. Polyamine biosynthesis occurs cyclically during the cell cycle with peaks in activity in conjunction with the G(1)/S transition and at the end of S-phase and during G(2)-phase. The negative regulator of polyamine biosynthesis, antizyme, shows an inverse activity compared with the polyamine biosynthetic activity. The levels of the polyamines, putrescine, spermidine and spermine, double during the cell cycle and show a certain degree of cyclic variation in accordance with the biosynthetic activity. When cells in G(0)/G(1) -phase are seeded in the presence of compounds that prevent the cell-cycle-related increases in the polyamine pools, the S-phase of the first cell cycle is prolonged, whereas the other phases are initially unaffected. The results point to an important role for polyamines with regard to the ability of the cell to attain optimal rates of DNA replication.

Topoisomerase II is nonfunctional in polyamine‐depleted cells

The polyamines—putrescine, spermidine, and spermine—are essential for normal cell proliferation. Polyamine depletion affects DNA structure and synthesis. Topoisomerase II (topo II) is also necessary for normal cell proliferation, and it has been shown in vitro that polyamines may affect topo II activity. In order to investigate the effect of polyamine depletion on topo II activity, we treated Chinese hamster ovary cells with either α‐difluoromethylornithine (DFMO) or 4‐amidinoindan‐1‐one‐2′‐amidinohydrazone (CGP 48664), which are polyamine biosynthesis inhibitors. Treatment with the topo II inhibitor etoposide results in DNA strand breaks only if there is active topo II in the cells. By quantitating DNA strand breaks after etoposide treatment using single cell gel electrophoresis, we were able to estimate intracellular topo II activity. We also quantitated topo II activity in crude nuclear extracts from control and polyamine biosynthesis inhibitor‐treated cells. Using single cell gel electrophoresis, we noted a clear decrease in the function of topo II in polyamine biosynthesis inhibitor‐treated cells, as compared with untreated control cells. However, the topo II activity in crude nuclear extracts did not differ significantly in control versus polyamine biosynthesis inhibitor‐treated cells. Taken together, these results indicate that although the function of topo II in polyamine‐depleted cells was impaired, topo II remained functional in an in vitro assay. Using the single cell gel electrophoresis assay, we also found that spermine depletion itself caused DNA strand breaks. J. Cell. Biochem. 75:46–55, 1999.

Inhibition of cell proliferation and induction of apoptosis by N(1),N(11)-diethylnorspermine-induced polyamine pool reduction.

Reduction of cellular polyamine pools results in inhibition of cell proliferation and sometimes in induction of cell death. Reduction of cellular polyamine pools can be achieved by several strategies involving all the mechanisms of polyamine homoeostasis, i.e. biosynthesis, catabolism and transport across the cell membrane. In the present paper, we concentrate on results achieved using the polyamine analogue DENSPM (N(1),N(11)-diethylnorspermine) on different cell lines. We discuss polyamine levels in DENSPM-treated cells in relation to effects on cell cycle kinetics and induction of apoptosis. To really understand the role of polyamines in cell cycle regulation and apoptosis, we believe it is now time to go through the vast polyamine literature in a meta-analysis-based manner. This short review does not claim to be such a study, but it is our hope to stimulate such studies in the polyamine field. Such work is especially important from the viewpoint of introducing drugs that affect polyamine homoeostasis in the treatment of various diseases such as cancer.

Digital Holography and Cell Studies

Digital holographic microscopy (DHM) is a novel high-resolution imaging technique that offers real-time imaging and quantitative measurements of physiological parameters. It has developed into a broad field, and one of many interesting applications is to study cells without staining or labeling them and without affecting them in any way. Digital holography makes it possible to easily measure cell properties that previously have been very difficult to study in living cells, such as cell thickness, volume, and cell refractive index (Marquet et al., 2005; Rappaz et al. 2005; Molder et al., 2008; El-Schish et al., in press; Persson et al., in press). Living, dying or dead cells as well as fixed cells can be studied. The first DHM images showing living cells were published in 2003 and 2004 (You et al., 2003; Carl et al., 2004), making this field of research rather new. Two of the most interesting functions of DHM is 3-D imaging of objects and to make in-focus measurements over time. Digital holography has been used to study a wide range of cells, e.g. protozoa, bacteria and plant cells as well as several types of mammalian cells such as nerve cells and tumor cells (Emery et al., 2007; Kemper et al., 2006; Moon and Javidi 2007). It has also been applied for studies of cell proliferation, cell movement and cell morphology (Kemper et al., 2009; Yu et al., 2009). Movement in both 2-D and 3-D has been studied (Langehanenberg et al., 2009; Persson et al., in press). In addition, cell viability status can be determined using DHM (Kemper et al., 2006; Kemmler et al., 2007). Interestingly, it is possible to study both single cells and entire populations simultaneously, allowing for very detailed studies. In this chapter we will compare DHM with previously used techniques and discuss the benefits and drawbacks of digital holography cell measurements. We will also present cell studies made possible by DHM.

Different cell cycle kinetic effects of N1,N11-diethylnorspermine-induced polyamine depletion in four human breast cancer cell lines.

Polyamine analogues are presently undergoing clinical evaluation in the treatment of cancer. To better understand under what circumstances treatment with a polyamine analogue will yield beneficial results, we have investigated the effect of N1,N11-diethylnorspermine (DENSPM) on cell cycle kinetics of the human breast cancer cell lines SK-BR-3, MCF-7, HCC1937, and L56Br-C1. A bromodeoxyuridine–DNA flow cytometry method was used to evaluate the treatment with 10 μmol/l DENSPM on cell cycle kinetics. A correlation between polyamine pool size after DENSPM treatment and cell cycle kinetic effects was found. The most sensitive cell cycle phase was the S phase, followed by an effect on the G2+M phase and then the G1/S transition. The levels of a number of cell cycle regulatory proteins such as cyclin E1, cyclin A2, and cyclin B1 were lowered by DENSPM treatment, which may explain the effects on cell cycle kinetics. The two cell lines that were most sensitive to DENSPM treatment belong to the basal-like subtype of breast cancer and they were deficient with respect to p53, BRCA1, and RB1.

Cells and Holograms – Holograms and Digital Holographic Microscopy as a Tool to Study the Morphology of Living Cells

Digital holographic microscopy (DHM) is an emerging high-resolution imaging technique that offers real-time imaging and quantitative measurements of physiological parameters without any staining or labeling of cells. The first DHM images of living cells were obtained 8-10 years ago [1, 2]. Analysis of human hepatocytes showed that DHM was a versatile tool for in vivo cell analysis by using quantitative amplitude and phase-contrast imaging with very high resolution [1]. Another study showed that the quantitative distribution of the opti‐ cal path length created by transparent specimens contained information concerning both morphology and refractive index of the observed mouse cortical neurons [2]. This could on‐ ly be measured by DHM and not by phase contrast and Nomarski’s differential interference contrast (DIC) microscopy. In addition, the high sensitivity of these phase-shift measure‐ ments enables sub-wavelength axial accuracy, offering attractive possibilities for the visuali‐ zation of cellular dynamics.

Novel anti-apoptotic effect of Bcl-2: Prevention of polyamine depletion-induced cell death

The spermine analogue N1,N11‐diethylnorspermine (DENSPM) efficiently depletes the polyamine pools in the breast cancer cell line L56Br‐C1 and induces apoptotic cell death via the mitochondrial pathway. In this study, we have over‐expressed the anti‐apoptotic protein Bcl‐2 in L56Br‐C1 cells and investigated the effect of DENSPM treatment. DENSPM‐induced cell death was significantly reduced in Bcl‐2 over‐expressing cells. Bcl‐2 over‐expression reduced DENSPM‐induced release of the pro‐apoptotic proteins AIF, cytochrome c, and Smac/DIABLO from the mitochondria. Bcl‐2 over‐expression reduced the DENSPM‐induced activation of caspase‐3. Bcl‐2 over‐expression also prevented DENSPM‐induced Bax cleavage and reduction of Bcl‐XL and survivin levels. The DENSPM‐induced activation of the polyamine catabolic enzyme spermidine/spermine N1‐acetyltransferase was reduced by Bcl‐2 over‐expression, partly preventing polyamine depletion. Thus, Bcl‐2 over‐expression prevented a number of DENSPM‐induced apoptotic effects.

Effect of polyamine deficiency on proteins involved in Okazaki fragment maturation

Polyamine depletion causes S phase prolongation, and earlier studies indicate that the elongation step of DNA replication is affected. This led us to investigate the effects of polyamine depletion on enzymes crucial for Okazaki fragment maturation in the two breast cancer cell lines MCF‐7 and L56Br‐C1. In MCF‐7 cells, treatment with N1,N11‐diethylnorspermine (DENSPM) causes S phase prolongation. In L56Br‐C1 cells the prolongation is followed by massive apoptosis. In the present study we show that L56Br‐C1 cells have substantially lower basal expressions of two Okazaki fragment maturation key proteins, DNA ligase I and FEN1, than MCF‐7 cells. Thus, these two proteins might be promising markers for prediction of polyamine depletion sensitivity, something that can be useful for cancer treatment with polyamine analogues. DENSPM treatment affects the cellular distribution of FEN1 in L56Br‐C1 cells, but not in MCF‐7 cells, implying that FEN1 is affected by or involved in DENSPM‐induced apoptosis.