Immune To Diseases

Mitochondria and apoptosis

Mitochondria play a central role in cellular energy metabolism and homeostasis of ion and redox potential (Cai et al., 1998). Thus, damages to mitochondria readily result in cell death. It has been reported that a group of mitochondrial proteins play central role in regulating apoptosis (Liu et al., 1996; Du et al., 2000; Li et al., 2001; Susin et al., 1999). These proteins normally reside in inner membrane space (matrix) of mitochondria, but released into cytosol or nucleus when death signals strike the cell, where they activate caspases and nucleases that mediate apoptosis.

The activation of mitochondrial (intrinsic) apoptotic pathway is dependent upon the mitochondrial permeability transition (MPT) or mitochondrial outer-membrane permeabilization (MOMP) or mitochondrial membrane permeabilization (MMP), an important early event that is considered to be the 'point of no return' results in critical alteration in mitochondrial functions during cell death process (Zoratti and Szabo, 1995; Chipuk et al., 2006). The MPT represents an abrupt increase in mitochondrial inner-membrane permeability to solutes of size <1500 Da upon opening of mega-channel that spans both inner and outer mitochondrial membranes due to death signals (Zoratti and Szabo, 1995; Kroemer et al., 1995; Lemasters et al., 1998). The mega-channels permit free influx of solutes into the matrix space of mitochondria causing rapid and complete dissipation of mitochondrial inner transmembrane potential (DYm) that ceases synthesis of ATP and increases osmotic influx of water resulting in mitochondrial damages and enzyme release.

The mitochondrial enzymes released into cytosol activate caspases and other molecules essential for induction of apoptosis (Liu et al., 1996; Lemasters et al., 1998; Kroemer et al., 1997; Green and Reed, 1998; Susin et al., 1999). Thus, activation of caspase-dependent pathway requires MPT, which is under control of Bcl2 family proteins that forms membrane-spanning pores on mitochondrial membrane through which the mitochondrial proteins are released (Zamzanii et al., 1995; Reed et al., 1998). On the other hand, MPT may also activate caspase-independent pathway, if caspase cascade is inhibited by any means.

The changes in mitochondrial transmembrane potential can be studied using several cationic fluorescent dyes that specifically stain mitochondria (Table.1). They are 3-3’ dihexycarbocyanine iodide (DiOC₆), rhodamine 123 (R123), chloromethyl X-rosamine (CMXRos), tetramethyl rhodamine ethylester (TMRM) and 5-5’, 6-6’-tetrachloro 1-1’, 3-3’-tetraethyl benzimidazole carbocyanine iodide (JC-1) (Petit et al.., 1990; Smiley, 1991; Cossariza et al., 1995; Castedo et al., 1996; Neiminen et al., 1997; Castedo et al., 2002).

For efficient staining of mitochondria, the fluorescent probes must enter into the cell and reach a critical intracellular concentration that is required for emitting adequate fluorescent signals. Thus, the probes with high lipophilic and cationic nature enter the cells easily and cytoplasmic accumulation is achieved rapidly. In this regard, JC-1 is considered to be superior among all the probes mentioned above since its lipophilic and cationic nature favor rapid cell permeabilization, where it exists monomeric form at low DYm (green fluorescence) and forms J-aggregates at high DYm (red fluorescence) (Smiley et al., 1991; Castedo et al., 2002). JC-I has high sensitivity and reliability for detection of DYm (Salvioli et al., 1997).  DiOC₆ and other probes show relatively low sensitivity because of their non-coherent behaviors. DiOC₆ has been reported to be most suitable for estimation of plasma membrane potential than DYm (Salvioli et al., 1997). Hoever, DiOC6 and TMRM offer an important advantage over other probes that they do not show any quenching effect inside the cell cytoplasm and thereby cellular concentration remains constant for long time. All these probes can be used in flow cytometer and fluorescent microscope except R123 that is not recommended for flow cytometer (Castedo et al., 2002).

    Table.1. Common fluorescent dyes used in ΔYm assay 

Caspases and apoptosis

The morphologic and biochemical changes typical of apoptosis are attributed by activation of specific proteases, called caspases. Caspases are intracellular cysteine proteases which cleave the cell skeleton proteins specifically at aspartic acid residues (Nicholson and Thornberry, 1997; Scott and Saleh, 2007; Kumar, 2007). These proteases reside in cytoplasm of animal cells as inactive zymogens called procaspases. Procaspases are converted into active proteases by caspase-cascade. Caspases initiate direct disassembling of skeletal structures essential for protection from cell injuries (Goodsell, 2000; Kumar, 2007).

A number of caspase proteins, approximately 14 caspases in human and mice, have been identified so far based on their amino acid sequence or their protease specificities. However, the classification on the basis of functional perspective is most commonly followed. For example, some caspases (e.g., caspases-2, 8 and 9) involve in activation of procaspases and are known as 'initiator' or 'signaling' or 'upstream' caspases, while other caspases carry the activation wave of signaling caspases and cleave specific cellular proteins such as PARP, DFF, a-foldrin, actin, lamins etc. and are known as 'effector' or 'downstream' caspases. Thus, caspases act through protolytic cascade mechanism, in which caspases become activated each other (Thornberry and Lazebink, 1998; Salvesen and Dixit, 1997). Most caspases (e.g. 2, 3, 6, 7, 8 and 9) are directly involved in cell death, while others (e.g. 1, 4 and 5) are involved in processing of pro-inflammatory cytokines such as IL-1a and IL-18 (Salvosen and Dixit, 1997; Reed, 1998; Scott and Saleh, 2007; Kumar, 2007). It has also been reported that the effector caspases are not involved in early nuclear morphological changes, which precedes the conventional hallmark of morphological changes associated with chemical-induced apoptosis (Johnson et al. 2000). During this caspase-independent cell death (CICD), apoptosis is mediated by either reactive oxygen and nitrogen species or loss of apoptosis inducing factor (Chipuk et al., 2006; Lamkanfi et al., 2007; Kumar, 2007).

Bcl-2 family proteins and apoptosis

The Bcl-2 family proteins play a pivotal role in regulating cell survival and death (Reed et al., 1998). Interestingly, the most decisive mitochondrial events of cell death i.e., MPT and release of cytochromeC, are governed by the Bcl-2 family proteins, which include members that facilitate (e.g., Bax, Bad or Bid) and those inhibit (e.g., Bcl-2, Bcl-X_L) these processes. Many of these proteins reside in outer mitochondrial membrane, oriented towards the cytosol. Both pro- and anti-apoptotic Bcl-2 family proteins exist in a complex network of homo- and hetrodimers (Reed, 1997; Adams and Corey, 1998). Pro-apoptotic Bcl-2 family proteins directly act on mitochondria resulting in MPT and release of cytochrome-C, while anti-apoptotic proteins prevent this process (Reed et al., 1998). Thus, Bcl-2 family proteins function as a regulatory switch between cell survival and death (Reed et al., 1998; Adams and Corey, 1998).

Free radicals and apoptosis

Oxidative stress occurs in cells when large amount of reactive oxygen species (ROS) (e.g. superoxide anion, hydroxyl radicals and hydrogen peroxide) or reactive nitrogen intermediates (RNI) (e.g. nitic oxide) produced in disease or toxic conditions, which play a major role in regulating apoptosis (Curtin et al., 2002). In a number of studies, either by addition of low level of ROS or endogenous generation of ROS, it has been reported that ROS induced apoptosis in various types of cell (Lenon et al., 1991; Suzuki et al., 1998). Similarly, nitric oxide, either delivered by NO donors or endogenously produced by nitric oxide synthase (NOS) enzyme, has been reported to induce apoptosis both in vivo and in vitro models (Albina et al., 1993; Nichikiwa et al., 1998; D'Acquisito et al., 2001).

Endogenous production of ROS and RNI in cells can be detected by several methods. Since free radicals are short-lived species with half-life less than seconds, a number of methods have been developed with varying assay sensitivity. ROS production is generally estimated by using chemiluminiscent probes such as luminol and lucigenin, cytochrome c reductase, ferrous oxidation of xylenol orange and fluorescent probe like DCFH-DA (Gyllenhammer, 1987; Suzuki et al., 1998; Nourooz-Zadeh, 1999; Dahlgren and Kalson, 1999; Carmody and Cotter, 2000; Tammariello et al., 2000). Of these, DCFH-DA is most commonly used for direct measuring of redox state of a cell. This probe readily permeable to cell and jingly sensitive to minute change in redox state of a cell. DCFH-DA is originally non-fluorescent dye that becomes fluorescent upon activation by reductase enzyme-mediated reaction in the presence of free radicals (Royall and Ischiropoulos, 1993).

A number of methods are available for detection of RNI including fluorescent probes such as DAF2-DA and DCFH-DA, chemiluminiscence of NO reacting with ozone, and Griess reagent (Archer, 1993; Nakatsubo et al., 1998; Kooy et al., 1997; Miso et al., 2000). Of these, Griess reagent is usually preferred for detection of RNI since the procedure is simple and reliable. DCFH-DA is also used commonly to detect RNI in flow cytometer.

Studying apoptosis

The execution of death program in a cell is characterized by a sequence of structural and biochemical changes including mitochondrial alterations, cell membrane damage, chromatin lysis or condensation, etc. Studying all these mechanisms, in spatial and temporal context, is a complex process. However, a number of methods have been developed by cell biologists and pathologists with certain limitations in all individual methods. The most common methods for demonstration of dead cells, either in situ or in vitro cultured cells, include the detection of cell morphology by light and electron microscopy, vital staining of nuclei and cell membrane, measurement of DNA content and chromatin organization pattern, detection of deregulated specific-proteins, and expression patterns of cell signal transducers etc. (Darzynkiewicz et al. 1997; Purnam and Boustany, 1999; Hughes and Mehmet, 2003). The most common techniques of demonstration of apoptosis are described here in details with technical procedures. However, the readers are requested to refer the specific procedures described elsewhere for detail (Purnam and Boustany, 1999; Coligan et al., 2001; Hughes and Mehmet, 2003; Sivakumar, 2007).

A) Light microscopy

Using hematoxylin and eosin stains, the apoptotic cells are identified on the basis of retraction of cytoplasm (shrunken cell) and often condensation of nuclei with dark staining and fragmentation. The sensitivity of this method is rather low and works well in sectioned tissues and embryos. However, the cultured cells can also be assessed for apoptotic changes by this method. While examining the cultured cells, the intensity of staining may slightly differ from the histological section due to the nature of loose spreading of cells in the smear.   

B) Electron microscopy

Semi-thin sections stained with toludine blue provides an excellent view of dead cells under light microscope, while ultrathin sections provide better views of the dead cells under electron microscope. The chromatin margination, condensed or often fragmented nuclei, cytoplasmic retraction and vacuolation, shrunken organelles etc. are some of the typical features of apoptotic cells observed in ultrathin sections.

C) Nuclear staining with fluorescent dyes

The cell viability is basically assessed by trypan blue dye exclusion test, in which normal or early apoptotic cells exclude trypan blue while dead or late apoptotic cells permeate it (Coligan et al., 2001). This assay is a simple, rapid and inexpensive, and most commonly used in assessing cell viability. The cell viability can be detected with high accuracy using fluorescent nuclear dyes (Puranam and Boustanu, 1999). For example, the fluorescent dyes such as ethidium bromide and propidium iodide permeate only into the dead cells and fluorescently stain their nuclei, while live cells remain unstained.

The characteristic morphologic features of apoptotic nuclei such as condensation and margination of the chromatin, formation of crescent shaped clumps at the periphery of the nuclear membrane, and fragmentation of the nuclei into round condensed apoptotic bodies are readily visualized with fluorescent dyes that specifically stain the nuclei. A number of cell membrane-permeant and impermeant fluorescent dyes are commonly used in cell death studies (Table.2). They are DNA-intercalating dyes emit fluorescence when excited by fluorescent light upon binding with DNA (McCarthy and Stevens, 1998). The fluorescent dyes such as propidium iodide (PT), acridine orange (AO) and ethidium bromide (EB) or 7-aminoactiomycin D (7-AAD) can be used with an argon laser, whereas Hoechst (HO) and diamidine-phenylindole (DAPI) require UV laser. Recently, a new series of DNA intercalating dyes (Yoyo, YOPRO, BOBO, BOPRO, TOTO, and TOPRO) have been synthesized as a replacement for EB with advantages that these impermeant dyes permit accurate analysis of apoptosis in the absence of cell permeability (Idziorek et al., 1995). The cells stained with fluorescent nuclear dyes are analyzed in fluorescent microscope and flow cytometer for visualizing the apoptotic nuclei and recording fluorescence pattern emitted by different cell populations.

Table.2. Summary of common fluorescent dyes used in cell death studies

Fluorescent nuclear staining procedures

Nuclei can be stained with different fluorescent dyes. Nuclei stained with DAPI emit blue fluorescence when visualized with a UV light, and PI emits red fluorescence. The nuclei stained with AO/EB emit green or orange fluorescence as described above in the table.

Cell fixation: prior to staining with nuclear dyes, the cells are either fixed in fixative or processed unfixed. Although, the fixation is compulsory for some specific assays, the author prefers fixation in all assays. The fixation can be achieved as follows for different type of cultured cells. 

For adherent cell lines:  Wash cells in PBS (Ph7.4), add 100 µl of 4 % paraformaldehyde (PFA) prepared in PBS pH 7.4 for 15 min at room temperature, and wash once with PBS (pH 7.4) at room temperature, and cover the cells with methanol for 5 min at room temperature before staining with fluorescent stains.

Cells grown in suspension: The cells grown in suspension (eg. Lymphocytes) are harvested by centrifuging at 1000g for 5 min and wash once with PBS and centrifuge as above. Resuspend the cells in fixative solution (100µl) directly and wash once after fixation as described above.

Protocol 1: AO/EB staining

Materials: Dye mix (AO-1mg/ml and EB-1mg/ml), PBS

Method for cultured cells (both adherent and suspension cells)

·         The cells cultured in vitro experiment are resuspended in 100 µl of PBS.

·         To the cell suspension, add 10 µl dye mix (containing equal amount of AO and EB solution) and incubate at room temperature for 10 min.

·         The stained cells are preferably washed once with PBS (optional, if you don’t have time, you can directly examine under flurescence microscope)

·         Take 20-40µl of stained solution in the glass slide and cover with clean cover slip and analyze under fluorescence microscopy

For tissue sections

·         Deparaffinize the tissue section in graded xylene and alcohol as conventional histological techniques, and bring the section to water.

·         Add 100 µl of dye mix per section, cover the section with cover slip and incubate at room temperature for 10 min. if the section is not covered thoroughly with dye mix , add additional amount as per your need and proceed further.

·         Examine under fluorescence microscope.

Interpretation

Under fluorescence microscope we could observe different fluorescence pattern. The normal and apoptotic cells are identified by the following fluorescence patterns;

·         Live cell nuclei appear green with organized nuclear structure,

·         Live-apoptotic cell nuclei appear bright-green with condensed or fragmented pattern,

·         Dead cell nuclei appear orange with organized structure, and

·         Dead-apoptotic cell nuclei appear bright-orange with condensed or fragmented pattern

·         Count a minimum of 100- 200 cells in total including normal, apoptotic and dead cells and calculate the percentage of apoptotic cells using the following formulae;

Apoptotic index = Total number apoptotic cells/ total number of cells counted x100.

Note: For further and detailed information, the readers are requested to refer the chapter (Apoptosis assay) in ‘Current protocols in Immunology’ by Coligan et al. 2001.

Protocol 2: DAPI/ PI Staining

Materials: DAPI (2mg/ml) and PI (1mg/ml)

Method:

·         The cells are prepared as above in AO and EB staining

·         The cells are resuspended in 100 µl of PBS, added with 10 µl of DAPI solution and incubated at room temperature for 2 min.

·         The cells are washed with PBS thrice, stained with 10 µl of PI and incubated at room temperature for 2 min

·         The cells are washed twice with PBS and analyzed under fluorescence microscope

Interpretation : On DAPI/PI staining, the cell morphology was categorized as follows; (i) live cells (DAPI⁺/PI^-) appeared blue with normal nuclei, (ii) live-apoptotic cells (DAPI⁺/PI^-) appeared blue with condensed and fragmented nuclei, (iii) dead cells (PI⁺) appeared red with normal nuclear structure and (iv) dead-apoptotic cells (DAPI⁺/ PI⁺) appeared red and blue with condensed and fragmented nuclei.

D) Cell biochemical assays

Cell membrane integrity is usually assessed by vital staining and lactate dehydrogenase (LDH) release. LDH is an active cytosol enzyme released abundantly upon cell damage. LDH assay is a sensitive test that gives an accurate account about cytotoxicity-mediated by oncosis (Decker and Lohmant-Mathes, 1988).

Studying cell death mechanism at its molecular level is considered to be more specific and reliable method to differentiate apoptosis from oncosis. As different specific molecules are involved in cell death process, it is essential to study the expression pattern of these molecules during apoptosis. Among them, demonstration of caspase activity is considered to be the most important step in delineation of individual enzymatic and biochemical mechanisms involved in apoptosis (McCarthy and Evans, 1998).

A number of cytoplasmic and mitochondrial proteins including death domain proteins, apoptosis-inducing factors (AIF), Bcl-2 family proteins etc. play major role in apoptosis. Both pro-(Bcl-2, Mcl-1 etc.) and anti-apoptotic (Bax, Bad etc.) proteins, and mitochondrial proteins (cytochrome C, Smac and endonuclease G) are major components in apoptosis. Expression pattern of these molecules in apoptosis can be studied by western blotting using specific monoclonal or polyclonal antibody or amplification of specific genes in reverse transcriptase and real-time PCR.

E) Assay for cell membrane changes

Loss of cell membrane integrity is a hallmark in oncosis, while maintaining membrane integrity is characteristic of apoptosis (Majno and Joris, 1995). However, translocation or externalization of the phosphatidyl serine (PS) layer of cell membrane is considered to be an early event of apoptosis, which can be detected in flow cytometer or fluorescent microscope using annexin-V that specifically binds to phosphatidyl serine layer.

Protocol 3: Annexin V assay

Materials: FITC-labeled recombinant Annexin V, Binding buffer and Propidium iodide (PI) solution

Procedure:  Wash 2x 1x10⁶ cells with PBS, dilute FITC-Annexin V at a concentration of 1 mg/ml in binding buffer and resuspend cells in 1 ml of this solution (prepare it freshly each time) and incubate 10 min in the dark at RT. Add to the cell suspension 0.1 ml of PI solution prior to analysis to give a final concentration of 1 mg/ml. 5. Analyze cells by fluorescent microscope or flow cytometer (Fig.3)

F) DNA fragmentation pattern

The endonucleases cleave the nuclear DNA into small fragments ranging from 200-300 bp that forms laddering pattern in agarose gel. Although, DNA fragmentation assay has been used frequently in the last 2 decades, the reliability of this method is disputable in late apoptosis (Puranam and Boustany, 1999). However, a highly sensitive histochemical method, called TUNEL assay, has been developed recently to visualize fragmented DNA in situ at individual nuclei, using nucleotide mix and terminal deoxynucleotidyl-transferase (TdT) enzyme that label the 3’-OH groups of DNA strand breaks.

Protocol 4: TUNEL assay

Materials: proteinase K (pK) (A2), H₂O₂ , TdT buffer (A1), TdT enzyme (A2), biotinylated dUTP (A2), TB buffer (A1), serum albumin (BSA) (A2), PBS, Extra-avidin Peroxidase (A2), AEC solution (A1), DNAse buffer (A1).

Procedure: Tissue sections are deparaffinized and rehydrated through a graded series of xylene and alchohol as in conventional histological technique. The cultured cells are harvested from the medium and smears are prepared and fixed with 4% paraformaldehyde or 10% formalin. The deparaffinized sections or cultured cells are refixed in 4% paraformaldehyde and treated with proteinase K (20 mg/ml) for 15 min at room temperature with washing in PBS at each step. Endogenous peroxidase was blocked by incubating the section with 2% H₂O₂ for 5 min at room temperature and washed in PBS. The sections are immersed with TdT equilibration buffer 5 min prior to treat the section with TdT (0.3 e.u./mL) and biotinylated dUTP in TdT buffer to facilitate the reaction in humid atmosphere at 37°C for 60 min. the reaction is stopped with SSC stop solution for 5 min and washed with PBS. Immunoreaction is developed by covering the sections with extra-avidin Peroxidase conjugate (diluted 1:10-1:20 in water) for 30 min and with AEC substrate and analysed in light microscope for brown colored nuclear staining (Fig.4).

References

Coligan, J. E., Kruisbeek, A. M., Margulies, D. H. (2001). In: Current protocols in Immunology, 4^th ed. Published by Wiley and Sons, Inc., USA

Darzynkiewicz, Z., Juan, G., Li, X., et al. (1997). Cytometry in cell necrobiolgy: analysis of apoptosis and accidental cell death (Necrosis). Cytometry, 27: 1-20.

Decker, T. and Lohmann-Mahes, M.L. (1988). A quick and simple method for the quantitation of lactate dehydrogenase (LDH) release in measurements of cellular cytotoxicity and tumor-necrosis factor activity. J. Immunol. Methods, 15: 61-70

Gao, L. and Kwaik, Y. A. (2000). Hijacking of apoptotic pathways by bacterial pathogens. Microb.Infect. 2: 1705-1719

Hughes, D. and Mehmet, H. (2003). In: Cell Proliferation and Apoptosis. Ed by D.Hughes and H Mehmet, BIOS Scientific Publishers Ltd, UK

Idziorek, T., Estaquier, J., Bels, F.D., Ameisen, J. (1995). YOPRO-1 permits cytoflourimetric analysis of programmed cell death (apoptosis) without interferring with cell viability. J. Immunol. Method, 185: 249-258.

Kerr, J, F., Wyllie, A, H., Currie, A, R. (1972). Apoptosis: a basic biological phenomenon with wide ranging implications in tissue kinetics. Br. J. Cancer. 26:239-257.

Majno, G. and Joris, I. (1995). Apoptosis, oncosis and necrosis: an overview of cell death. Am. J. Pathol., 146: 3-15.

McCarthy, N. J., and Evans, G. I. (1998). Methods for detecting and quantifying apoptosis. Curr. Top. Dev. Biol., 36: 259-278.

Purnam, K. and Boustany, R. (1999). Assessment of cell viability and histochemical methods in apoptosis. In: Apoptosis in neurobiology (ed.) Y.A. Hannun, and Boustany, R. CRC press, Washington DC.

Sivakumar P (2007). Studies on apoptosis in Mycobacterium paratuberculosis and Pasteurella multocida infections. The PhD thesis submitted to IVRI, Deemed University.