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REVIEW ARTICLE
Year : 2013  |  Volume : 50  |  Issue : 3  |  Page : 274-283
 

Various methods available for detection of apoptotic cells- A review


1 Department of Oral and Maxillofacial Pathology, Royal Dental College, Kerala, India
2 Department of Oral and Maxillofacial Pathology, MAHE Institute of Dental Sciences, Kerala, India
3 Rajiv Gandhi Institute of Dental Sciences, Bangalore, India
4 Department of Oral and Maxillofacial Pathology, Farooquia Dental College, Bangalore, India

Date of Web Publication23-Sep-2013

Correspondence Address:
K L Kumaraswamy
Department of Oral and Maxillofacial Pathology, Farooquia Dental College, Bangalore
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0019-509X.118720

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 » Abstract 

Apoptosis is a process of programmed cell death occurring in multicellular organisms in whom development, maintenance and sculpturing organs and tissues. Taken together, apoptotic processes are of widespread biological significance; being involved in e.g. development, differentiation, proliferation/homoeostasis, regulation and function of the immune system and in the removal of defected harmful cells. Dys regulation of apoptosis can play a primary or secondary role leading to cancer whereas excessive apoptosis contributes to neuro degeneration, autoimmunity, AIDS, and ischemia. Gaining insight into the techniques for detecting apoptotic cells will allow the development of more effective, higher specific and therefore better-tolerable therapeutic approaches. The goal of this review article is to provide a general overview of current knowledge, on the various technical approaches for detecting apoptotic cells.


Keywords: Annexin V, apoptosis, apoptotic index, caspase 3, M 30, p53, TUNEL


How to cite this article:
Archana M, Bastian, Yogesh T L, Kumaraswamy K L. Various methods available for detection of apoptotic cells- A review. Indian J Cancer 2013;50:274-83

How to cite this URL:
Archana M, Bastian, Yogesh T L, Kumaraswamy K L. Various methods available for detection of apoptotic cells- A review. Indian J Cancer [serial online] 2013 [cited 2017 Mar 24];50:274-83. Available from: http://www.indianjcancer.com/text.asp?2013/50/3/274/118720



 » Introduction Top


Development of all multicellular organisms depends on cell division, differentiation, maturation and cell death and an exquisite balance of these keeps the cell viable. [1],[2] However, death is integral part of life. The term programmed cell death was introduced in 1964, which stated that cell death during development is not accidental in nature but follows a sequence of controlled steps leading to self-destruction. [3] Apoptosis is one of the main types of programmed cell death in multicellular organisms. Tremendous progress has been made regarding understanding; up regulation of apoptosis could help fight autoimmune disease and cancer, its inhibition could help control events ranging from aging to ischemic heart disease to brain disease. [4] Various means of detecting apoptotic cells have been explored and made available over the time.

Historical Review: Over the years apoptosis has been discovered and rediscovered several times.

  • The German scientist Carl Vogt was first to describe the principle of apoptosis in 1842.
  • Walther Flemming (1885) delivered precise description of programmed cell death. John Foxton Ross Kerr distinguished Apoptosis from traumatic cell death. [4]
  • John F. Kerrand colleagues (1972) coined the term "apoptosis" (a-po-toe-sis; second P is silent), Greek word for the process of leaves falling from trees or petals falling from flowers. [4]
  • Wyllie described the first and the most dramatic DNA fragmentation in 1980.
  • David L. Vaux and colleagues (1988) described the anti-apoptotic and tumorigenic role of bcl-2. [5]
  • Horvitz (1980) gave a better process of apoptosis in mammalian cells on the nematode Caenorhabditis elegans. [6]
  • Norbury and Hickson (2001) showed apoptosis as a defence mechanism in immune reactions and cell damage by disease or noxious agents
  • Hirsch, 1997 and Zeiss, 2003 distinguished apoptosis from necrosis
Various technical approaches available for detection of apoptotic cells are discussed below

Light Microscopy

Light Microscopy has aided in identifying the various morphological changes that occur during apoptosis. During the early process of apoptosis, cell shrinkage and pyknosis are visible by routinely stained light microscopy. [4],[7],[8] With haematoxylin and eosin stain, apoptotic cell appears as a round or oval mass with dark eosinophilic cytoplasm and dense purple nuclear chromatin fragments [Figure 1].[9],[10],[11],[12] With cell shrinkage, the cells are smaller in size, the cytoplasm is dense and the organelles are more tightly packed. Pyknosis is the result of chromatin condensation and this is the most characteristic feature of apoptosis. The shrunken cell fragments as apoptotic bodies, which are phagocytized phagocytosed by adjacent cells or degraded or extruded out of the lumen. There is essentially no inflammatory reaction associated neither with the process of apoptosis nor with the removal of apoptotic cells because: (a) apoptotic cells do not release their cellular constituents into the surrounding interstitial tissue; (b) they are quickly phagocytised phagocytosed by surrounding cells thus likely preventing secondary necrosis; and, (c) the engulfing cells do not produce anti-inflammatory cytokines. [3],[11],[13],[14]
Figure 1: H and E stained liver specimen showing hepatocyte arrow points out at an apoptotic cell. www.bioscience.org/2005/v10/af/1765/ fig2.jpg

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Advantages

It is fairly a reliable and inexpensive method for detection of apoptotic cells.

Disadvantages

Quantitative measurement lacks objectivity and reproducibility

Less sensitive and prone to lot of errors, which can be avoided if 20 fields of 1000 × magnification (containing an average of 1550 cells each) and so time consuming. [14]

At low magnification fewer apoptotic cells are detected and there is an increase in inter observer variability; therefore a high-power lens should be used.

Lowest numbers of apoptotic cells are usually scored in light microscopy based solely on morphology hence, revealing only tip of the iceberg.

Electron Microscopy


Defines the subcellular changes better. It shows most conspicuous changes like chromatin condensation phase and electron-dense nuclear material aggregating peripherally under the nuclear membrane; [9],[15] there can also be uniformly dense nuclei. [16] Extensive plasma membrane blabbing blebbing occurs followed by karyorrhexis and separation of cell fragments into apoptotic bodies by "budding." Apoptotic bodies consist of cytoplasm with tightly packed organelles with or without a nuclear fragment [Figure 2]. The organelle integrity is still maintained and all of this is enclosed within an intact plasma membrane. Apoptosis can be studied either under scanning electron microscope SEM or transmission electron microscope TEM.

  • TEM shows chromatin condensation around the nuclear membrane and convolutions in the nuclear membrane preceding nuclear fragmentation. [17],[18],[19] Simultaneously with these nuclear alterations, the cytoplasm condenses, microvilli disappear, blebs are formed in the cellular surface; cells separate and junctional cell junctions are lost. Several biochemical features have been identified as associated to apoptotic cell death. Among these, cleavage of genomic DNA into multiple fragments of 180-200 bp is the most typical feature. SEM shows cell surface alterations like smoothening, loss of microvillus structures, blabbing blebbing, shrinking etc [Figure 2].[18] However, these are important signs of cell injury and not considered as specific markers of apoptosis. [13]
Figure 2: Scanning electron microscopic picture (a) Cell shrinks following the cleavage of lamina lamina and actin filaments in the cytoskeleton (b) Breakdown of chromatin in the nucleus leading to nuclear condensation (c) Continued cell shrinkage that permits removal by macrophages (d) Appearance of membrane blebs (e) www.reading. ac.uk/.../apoptosis/apoptosis.jpg

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TEM analysis is essentially qualitative whereas, SEM studies can provide information of the cell surface, cell-cell and cell-substrates interactions but it is very difficult to evaluate apoptotic features by SEM.

Advantages

The plethora of information provided is wide, may be useful for subsequent biochemical or molecular studies furnishing important controls for experimental. TEM is preferred for analyzing tissues.

Disadvantages

  • The procedure is time consuming (TEM consumes 5-6 days; SEM requires 24hrs) Expensive
  • Many samples cannot be analysed it requires laborious preparation
  • Because only a small area can be visualized, quantification of the extent of apoptosis is also difficult.
Gel Electrophoresis

DNA fragmentation during apoptosis occurs in two stages. [20] There is sequential degradation of DNA initially to High molecular weight (HMW) DNA fragments of approximately 300 kb, which are detected by gel electrophoresis ([Figure 3]). [21] Characteristic DNA feature of apoptotic DNA fragmentation is that both single and double stranded breaks are produced. HMW DNA fragments are considered more reliable biochemical marker for apoptosis. [22]
Figure 3: Gel electrophoresis showing typical DNA ladder pattern JPEG - www.chinaphar.com/1671-4083/25/figs/1509f6.jpg

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Principle

DNA from apoptotic cell is extracted from the culture and is precipitated with polyethylene glycol (PEG) or agarose or polyarcylamide. The fragmented DNA remains in the supernatant and can be easily subjected for gel electrophoresis or quantification using fluorescent dyes. DNA fragmentation into oligonucleosomal ladders is characteristically seen in early events of apoptosis in the range of 20-300kb. But recent evidence indicates that all apoptotic cells need not show extensive DNA fragmentation. When DNA from apoptotically dying cells was subjected to agarose gel electrophoresis, ladders with ~200 bp repeats were observed, corresponding histone protection in the nucleosomes of native chromatin. Subsequent pulsed field gel techniques have revealed earlier DNA cleavage patterns into larger fragments. Since even a few double stranded DNA breaks will render the cell unable to undergo mitosis successfully, such DNA fragmentation can be regarded as a biochemical definition of death. Fragmented DNA in cells undergoing apoptosis can be studied by various gel electrophoresis techniques like

Conventional gel electrophoresis : is used to separate low molecular weight DNA appears as characteristic "ladder" pattern of discontinuous DNA fragments, which is a hallmark of apoptosis. [23] Such a pattern of DNA degradation generally serves as a marker of the apoptotic mode of cell death.

Pulse field gel electrophoresis is a specialized technique for resolving DNA molecules in the range of kilo to mega bases i.e., 50kb with length up to 10 Mbp. [21] By alternating the electric field between the pair of electrodes, HMW DNA and chromosome size DNA from 200 to over 12000 kb can be separated because they are able to reorient and move differentially through the pores of an agarose gel.

Field-inversion gel electrophoresis (FIGE) is a method, which employs periodic inversion of the electric field essentially in one dimension, which results in net migration by using a longer time or higher voltage in one direction than in the opposite direction. FIGE permits separation of DNA or protein mixtures in size ranges not accessible to ordinary electrophoresis. This technique allows analysis of the integrity of DNA of molecular weights of up to 2 Mbp, whereas conventional gel electrophoresis of DNA is restricted to 20 kbp fragments and below. [24]

Single cell gel electrophoresis (SCGE) : visualizes DNA damage measured at the level of individual cells. It is a more sophisticated and precise method of cell death measurement at the single cell level compared with classical cell morphology assays. It is known as comet assay as the degraded DNA resembles comet shaped image on the electrophoregrams ([Figure 4]). [25] The comet produced can characterizes the amount of DNA in the nucleus or "head" and the amount and pattern of DNA that has migrated away from the nucleus forming the tail embedded in the thin-layer agarose gels during the electrophoretic separation. [25] It can detect various forms of DNA strand breakage dependent on the pH of electrophoresis. [26] Under alkaline conditions (pH >13) it detects single-strand breakage, double-strand breakage, excision repair site, and alkaline-labile sites. [27],[28] Under neutral conditions, it mainly detects double-strand DNA breakage and is therefore considered to be suitable for detection of apoptosis. [27],[28],[29] It's useful in assessing viability of cell: dead or living and cell death type: apoptosis or necrosis.
Figure 4: Single cell gel electrophoresis comet assay showing apoptotic cells with small head and large tail. www.kpk.gov.pl/.../ coe/z/d/alert_3d.jpg

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Apoptotic cell appear comet-like structures with large tails and small heads. Viable cell display a large head with only minute tail. Necrotic cells display large nuclear remnants and almost invisible tails [Table 1]. Comet assay has been introduced to many different cell models as a convenient method of DNA damage and repair screening, toxicity of anti-cancer agents and apoptosis studies.
Table 1: Showing description of normal, apoptotic and Necrotic cells in single cell gel electrophoresis

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Advantages of Comet assay: [29]

  • Easy, sensitive, quantitative
  • Precise in the determination of cell death and DNA damage
  • Comet assay has higher sensitivity than DNA ladder assay and TUNNEL staining
  • It can provide more specific information about the extent and heterogeneity of DNA damage compared to TUNEL staining
  • More accessible and feasible than EM
Disadvantages

  • These techniques are often tedious; might damage the cell membrane changing the distribution of cell population of live, apoptotic and or necrotic cells.
  • Provide qualitative rather than quantitative results
  • These procedures have multiple steps and require more time
Flow Cytometry (FCM)

Appears to be a choice technique for the accurate quantification of apoptosis and is a method which distinguishes apoptotic from non-apoptotic cells by means of DNA staining. It is a technique for counting, examining, and sorting microscopic particles suspended in a stream of fluid. It allows simultaneous multi parametric analysis of the physical and or chemical characteristics of single cells flowing through an optical and or electronic detection apparatus.

Principle

Apoptotic cells is stained with fluorescent dye and passed through beam of light of single wavelength. Each cell passing through this light scatters light to some extent. Such forward scatter [FSC] versus side scatter [SSC] distinguishes apoptotic cells from others and allows determination of the immunophenotype of cells undergoing apoptosis. [30]

Apoptosis is marked by altered cell morphology while plasma membrane excludes uptake of DNA-specific fluorochromes like propidium iodide [PI], Trypan blue, DAPI, acridine orange, Hoechst dyes (HO]. Staining methods for flow cytometry use either fixed cells or treat cells with a hypotonic solution to permit DNA staining by non-vital dyes. The apoptotic cells with degraded DNA appear as cells with hypo diploid DNA content and are represented in so-called "sub-G1" peaks on DNA histograms. [31],[32] Initially it was not completely clear whether this finding was due to reduced DNA content or to altered conformation of chromatin now less accessible to staining. It was later demonstrated that activation of an endonuclease in apoptotic cells resulted in extraction of the low molecular weight DNA following cell permeabilization, permeability which in turn, led to their decreased staining ability with DNA-specific fluorochromes. [32],[33],[34]

FCM DNA analysis also allows definition of the relationship between the induction of apoptosis by different agents and their specific cell cycle phase.

In early apoptosis, PI does not enter in to the cell as the integrity of the cell plasma membrane is preserved. On the other hand, following exposure to dyes such as Hoechst 33342, apoptotic cells appear brighter than controls. Hence, simultaneous staining with these two dyes provides a means of identification, measurement and discriminates apoptotic from both living and necrotic cells as per the principle of light scatter [Table 2]. It is shown that higher the nuclear cytoplasmic ratio of a given cell; better is the distinction between apoptotic, necrotic and healthy cells. [30]
Table 2: Staining intensity of normal healthy cell, apoptotic cell and necrotic cell with Hoechst dye and propidium iodide stains

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Advantages

Easy, rapid and accurate quantitation of apoptosis in both viable and fixed single cells.

Explain the relationship between induction of apoptosis by different agents and their cell cycle phase specificity.

Disadvantages

Very time consuming as it has multiple steps and is quantitative. Therefore, intact tissues usually require pre-treatment with an enzyme to release the individual cells for analysis.

In situ3 -end labelling method (ISEL): These techniques make use of radioactive or non-radioactive labelling of the free ends of the DNA, allowing accurate identification of single apoptotic cells ([Figure 5]). [35] Fragmentation of DNA into 180-200bp fragments is used in the morphological analysis of apoptosis. It comprises of two variants, namely
Figure 5: Apoptotic cells detected by TUNEL and fluoresce green; while necrotic cells are stained with red-fluorescent propidium iodide. - tools.invitrogen.com/.../A23210_TF_080828.jpg

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  • DNA polymerase or its Klenow fragments is used to incorporate labelled nucleotides into fragmented DNA by in situ nick translation. [36] Quantification of apoptosis by ISEL can be done on a cell-to-cell basis with preservation of topological information. [36] This method is particularly valuable when apoptotic cells are present in low frequencies. Therefore, is applied in the quantification of tumors, in which apoptosis is otherwise difficult to detect.
  • On the other hand terminal transferase is used to add labelled nucleotides into the 3-end of the DNA. This is terminal deoxy transferase transferase-mediated dUTP nick end labelling (TUNEL). [37],[38]
TUNEL is more sensitive in comparison to in situ nick translation. [39],[40] This is at least at least partially due to the ability of terminal transferase (used in TUNEL) to label both double and single-stranded DNA breaks, whereas polymerase I (of in situ nick translation) label only single-stranded breaks. Also, the kinetics of the enzymes is different; DNA polymerase-I is slower than terminal transferase in incorporating nucleotides [Table 3]. It enables in situ visualization of the process at the single cell level [Figure 5]. TUNEL staining precedes (and therefore, does not depend on) the appearance of the nucleosomal ladder in gel electrophoresis and leads to the explanation of ultra-structural aspects of the process.
Table 3: Differences between in situ nick labelling and TUNEL

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Advantage [39]

  • The reactions are based on the direct labelling of 3′ -hydroxyl termini of DNA breaks, and thus the lesions measured are identifiable at the molecular level.
  • The DNA breaks occur very early in apoptosis, prior to changes in cell morphology the method thus detects apoptotic cells, which cannot yet be recognized based on changes in morphology. These assays can be applied therefore, to study the very early events of apoptosis.
  • Since DNA content is measured in addition to DNA breaks, apoptosis can be related to the cell's position in the cycle present in the sample. However, the sensitivities and specificities of these techniques depend on fixative used, pre-treatment and concentration of terminal transferase enzyme.
A new assay for non-radioactive in situ nick translation has been reported and employed to detect DNA strand breaks in apoptotic cells. Combining this assay with PI-stained DNA enables to use FCM to reveal the cell cycle phase specificity of DNA breaks.

Immunohistochemistry (IHC) : The latest in the trend is immunohistochemcial detection of apoptotic cells using antibodies against a wide range of substrates most importantly Caspases 3, p53, Annexin V, and M30.

Caspases 3: belongs to the family of cysteine proteases that cleave their target proteins at aspartic acid residues in a defined sequence, hence the name. [41],[42] Caspase-3 is a critical executioner of apoptosis, as it is either partially or totally responsible for the proteolytic cleavage of many key proteins such as the nuclear enzyme poly (ADP-ribose) polymerase (PARP). [42] Among the group of 11 caspases, caspase-3 has been recognized as a central player in mediating apoptosis and hence is studied most widely. [43] Cleavage of caspase-3 requires aspartic acid at the P1 position; active caspase 3 then activates other caspase. Enzymatic activation of pro-caspases results in generation of neo-epitopes. Neo-epitopes are used as antigens for generating antibodies specific for immunodetection of the cleaved product without recognition of the intact substrate.

Detection of active caspase 3 in situ may be a more unique, direct and sensitive indicator of apoptosis than detection of secondary process such as DNA fragmentation or cleavage of caspase substrate. [44] Activated caspase-3 IHC is an easy, sensitive, and reliable method for detecting and quantifying apoptosis. [45] Inhibition of caspase activities has been considered to be a novel therapeutic strategy for a variety of apoptosis- related diseases involving nervous system defects, retinal degeneration, liver injury, stunned myocardium, and sepsis, by reducing apoptosis and thereby improving organ function.

p53: It belongs to small family of related proteins; located on chromosome 17pl3. It has evolved in higher organisms and prevents tumor development. It regulates the cell cycle and has the ability to eliminate excess, damaged or infected cells by apoptosis. It is a major obstruction to tumorigenesis, hence is a tumor suppressor gene. p53 is a transcription factor activates vital damage to restrict aberrant cell growth in response to DNA damage, oncogene activation, hypoxia and the loss of normal cell contacts. It restricts cellular growth by inducing senescence, cell cycle arrest (at G1 and/or G2 phase) or apoptosis. [46]

p53 function in cancers can be lost by various mechanisms, including lesions that prevent activation of p53, mutations within the TP53 gene itself or mutations of mediators of p53 function. [47] The presence of mutant p53 in the tumor specimens often predicts an adverse therapeutic outcome ([Figure 6]). A wide range of clinical possibilities is available both for diagnosis and treatment, rendering p53 an ideal target for anti-cancer drug design. Safety and efficacy of newly designed peptides or small molecules capable of modulating mutant p53 is been used successfully in a number of clinical trials. Hence, most of the attention on p53 has focused on its role in cancer, in suppressing tumorigenesis and chemo resistance and chemotherapy induced cell death. [48],[49] Exogenous p53 has been used in chemo- or radiation-resistant advanced cancers, which resulted in substantial improvement of symptoms. [50] Recent advances in gene therapy and combined viral and gene therapy (though is still in its infancy) have many correlations with the status of p53. [50]
Figure 6: P53 abundant immuno expression of p53 in nuclei whereas cytoplasmic staining in occasional neoplastic cells. - www.scielo.br/.../ rimtsp/v46n1/46n1a05f01.gif

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Annexin V : It is a 35-36 kDa, calcium dependent, phospholipid-binding protein with a high affinity for phospholipid phosphatidylserine [PS]. They are a family of proteins first described in 1990, all of which share the property of binding calcium and phospholipids. [51],[52] As established, one of the earliest indications of apoptosis is the translocation of the membrane PS from the inner to the outer leaflet of the plasma membrane. This precedes other apoptotic processes such as loss of plasma membrane integrity, DNA fragmentation, and chromatin condensation. Annexin V binds to these PS exposing membranes in a calcium dependant manner. It is often used in conjunction with vital dyes such as 7-amino-actinomysin (7-AAD) or propidium iodide (PI), which bind to nucleic acids, but can only penetrate the plasma membrane when membrane integrity is breached, as occurs in the later stages of apoptosis or in necrosis. [53]

Annexin V behaves as an extrinsic membrane, hence an excellent tool to detect cell surface exposed to PS in vitro and in vivo and is by far the most sensitive technique to detect ongoing apoptosis. [54] A potential drawback is that annexin V preferably binds to apoptotic cells even in conditions of excess necrosis. This can be resolved by using combining annexin V assay with DNA marker such as propidium iodide, this can also be used to monitor the progression of apoptosis: from cell viability, to early-stage apoptosis, and finally to late-stage apoptosis and cell death ([Table 4] and [Figure 7]). [52]
Table 4: Showing difference between normal cell, early apoptosis and late apoptosis using annexin v staining along with a vital dye

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Figure 7: Lymphocytes stained with annexin V and PI showing early stages of apoptosis with increasing membrane permeability from top to bottom.www.compucyte.com/images/apopart.gif

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Radioactive 99mTc-labeled annexin V used in vivo is tested extensively in animals. Alternative non-invasive near-infrared fluorescent imaging can detect annexin V in apoptotic cells and tumor cells affected by chemotherapy in vivo.[55]

M30 : A neoepitope in cytokeratin 18 (CK18), termed M30 neoantigen, becomes available at an early caspase cleavage event during apoptosis of epithelium-derived cells, and is not detectable in vital or necrotic epithelial cells. A monoclonal antibody, M30, specifically recognizes a fragment of CK18 cleaved at Asp396 (M30 neoantigen) ([Figure 8]). [56] M30 antibody is specific to this CK18 cleavage site and does not react with viable or necrotic cells. [57] It is an early indicator of apoptosis in epithelial cells. [57],[58] M30 is a marker validated both in vitro and in vivo on trophoblastic tissue in human placenta, endometrium, colon, and salivary glands. [59] M30 remains immuno reactive in paraffin embedded tissue and is absent in non-apoptotic cells hence can be used in the study of apoptosis in clinical and experimental materials.
Figure 8: Apoptotic cells of human colon cancer confined to cytoplasmic staining of CK 18 using M30. - static.enzolifesciences. com/fileadmin/thumbs/

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Advantage of M30

  • Easy to interpret than in situ end labelling
  • Procedure for M30 IHC is technically simpler, easier to handle more specific for apoptosis
  • Less prone to artifacts and not expressed in necrotic cells.
Disadvantage

There is high expression of M30 in most cancer cells, but not in non-epithelial (lymphoid) cells. This makes M30 neo-epitope a specific marker of apoptosis in only epithelial cells.

The major limitation of IHC is that, all these current methods require detaching, washing and transferring the cells. These procedures might damage the cell membranes changing the cell population distribution of live, apoptotic and/or necrotic cells. These multiple steps also consume more time and more materials allowing for loss of the cells through the procedures.

Apoptosis provides a conceptual framework to link cancer genetics, tumor initiation progression or metastasis with cancer therapy and prognosis. It is now well documented that most cytotoxic anti-cancer agents induce apoptosis, raising the intriguing possibility that defects in apoptotic programs contribute to treatment failure. Apoptotic index is defined as a percentage of apoptotic cells and bodies per all tumor cells. Some authors however, use it to denote the number of apoptotic cells per 1000 tumor cells. Furthermore, in some investigations, apoptosis is measured as number of apoptotic cells per 10 high-power fields. [60] Apoptotic index is the most accurate index to reflect apoptosis and is applied during and after chemotherapy. [61] Tumors, which display high levels of cell death after one cycle of chemotherapy is more likely to achieve pathological regression. High apoptotic index after chemotherapy predicts that patient may have a good pathological response. [62] Though contradictory data have also been accumulated in the past on the relationship between apoptosis and prognosis, it is now known to be the most important predictor of prognosis and lymph node metastasis and was found be a better predictor than conventional tumor grade. [63] Apoptotic index has been shown to be of clinical and biological relevance in breast carcinomas, hepatocellular carcinoma, renal cell carcinoma, prostatic carcinoma, laryngeal carcinoma, and cervical carcinoma. It has been hypothesized that high apoptotic index predicts metastatic phenotype and poor survival. [64]


 » Summary Top


The morpho-functional state of apoptotic cell can be detected; studied and extensive data can be obtained by various technical approaches. It should be kept in mind that the method of detection employed should allow

  • Quantification
  • Analyze qualitative differences between experimental conditions
  • Determine the different stages
  • Validate observations obtained with other approaches etc.
In general, the light microscopy approach can provide both qualitative and quantitative data, TEM analysis is essentially qualitative, and SEM studies can provide information of the cell surface, cell-cell and cell-substrates interactions. The cytochemical and immunocytochemical techniques allow investigating morpho-functional correlates of the various apoptotic pathways. Double staining procedures can be often used to correlate both apoptotic cells to their phenotype. Moreover, in situ immunocytochemical techniques are useful to investigate the distribution of the various cell proteins both at the single cell level and at the cell-to-cell contact sites. Accurate detection of apoptosis in various stages help in assessing apoptotic index, which is known to be an indicator of prognosis and metastasis thereby predicts outcome of the treatment.

 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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