Apoptosis (Programmed Cell Death) Markers

(整理 by陳怡睿)

Apoptosis is a process of deliberate life relinquishment by a cell in a multicellular organism. It is one of the main types of programmed cell death (PCD), and involves an orchestrated series of biochemical events leading to a characteristic cell morphology and death. The apoptotic process is executed in such a way as to safely dispose of cell corpses and fragments.

In contrast to necrosis, which is a form of cells death that results from acute cellular injury, apoptosis is carried out in an orderly process that generally confers advantages during an organism's life cycle. For example, the differentiation of fingers and toes in a developing human embryo requires cells between the fingers to initiate apoptosis so that the digits can separate.

TOXICOLOGICAL SCIENCES 65, 299–308 (2002)
The markers selected include several biochemical parameters (downregulation of the antiapoptotic bclXL gene, caspase-3 activation, and cytochrome C release from mitochondria), and flow cytometry determinations (analysis of the size of the nuclei, chromatin complexity, and DNA integrity). The effects of several well-known model apoptotic toxicants (galactosamine, tertiary-butyl-hydroperoxide, etoposide, campothecine, and curcumin) were analyzed in hepatocytes. The results demonstrated that (1) the apoptotic effect of 4 out of 5 compounds could be detected in low concentrations of the drugs long before cell necrosis (tertiary-butyl-hydroperoxide induced apoptosis was only detected at concentrations causing concomitant necrosis) and (2) among the markers evaluated, caspase 3 activation and nucleus and DNA analysis by flow cytometry were used to fulfil the compromise between reliability, sensitivity, and ease of performance, which are critical issues when screening for an apoptotic effect of newly developed drugs.

Cleaved PARP as a Marker for Apoptosis in Tissue Sections
Promega Notes 72
The present study supports our recent finding that a large fraction of TUNEL-positive nuclei in atherosclerotic plaques have active gene transcription, indicated by immunostaining for RNA splicing factor. As such, these cells do not appear to be apoptotic. The cell is still active and is transcribing genes that may or may not be related to the apoptotic process. These cells were negative when stained with Anti-PARP p85 Fragment pAb. However, the cells that were TUNEL-positive and RNA splicing factor-negative were positive when stained with the Anti-PARP antibody.

J Chin Med Assoc 2008;71(12):628–634
Since changes in TP53, BCL-2, BAX and c-MYC frequently occur in female genital tract sarcomas, deregulation of apoptosis appears to be involved in the pathogenesis of this group of tumors. This mechanism may occur early in tumorigenesis and include the c-MYC/BAX apoptotic pathway or BCL-2. However, TP53 mutation may play a crucial role in this process, and clinically, it could be used as a prognostic indicator.

Monitoring apoptosis using the CKChip system
The CKChip is a device designed for quantitative, imaging-based cellular assays. It enables concurrent realtime monitoring of the fluorescence emanating from multiple individual adherent or non-adherent cells, each held at a given “address” on the CKChip. Here we demonstrate the utility of the CKChip by measuring drug-induced apoptosis in heterogeneous populations of cells using various fluorescent probes. In one experiment, Annexin V staining is used to distinguish early apoptotic cells and Propidium Iodide (PI) uptake to identify necrotic cells. In the second experiment, apoptotic activity is detected using a fluorescent probe that binds only activated caspases. The results indicate that there exists considerable variation in the timing of apoptosis induction, highlighting an advantage of the CKChip platform, which allows the behavior of individual cells within a population to be evaluated over time.

Cell-Free Plasma DNA: A Marker for Apoptosis during Hemodialysis
Clinical Chemistry 52: 523-526, 2006
Plasma DNA concentrations were not significantly different between controls and patients before HD. Circulating DNA increased significantly (P <0.05) after 20 min of treatment with HD. Post-HD concentrations of DNA were significantly higher compared with pre-HD and controls (P <0.005). Agarose gel electrophoresis showed ladders typical of apoptosis in post-HD samples. Two subpopulations of CD45+ leukocytes were defined by flow cytometry: annexin V+/7AAD+ population for apoptosis, and annexin V+/7AAD– for early apoptosis. Compared with healthy controls, mean fluorescence (MF) of 7AAD+ apoptotic cells in the annexin V+/7AAD+ subpopulation before HD was not significantly increased. HD increased MF of 7AAD+ cells in the annexin V+/7AAD+ subpopulation. In this subpopulation, MF of annexin V+ cells was significantly higher (P <0.01). MF of annexin V+ cells in the annexin V+/7AAD+ subpopulation increased during HD.


Annexin V
used to detect early phases of apoptosis.
membrane staining with annexin V.

Apolipoprotein C-1
* an novel apoptotic marker that has been implicated in apoptotic human vascular smooth muscle cell death via recruiting a neutral sphingomyelinase (N-SMase)-ceramide pathway.

Bax
* a pro-apoptotic protein.
* a promoter of apoptosis.

Bcl-2
* anti-apoptotic protein with perinuclear expression.
* a marker of apoptosis control (anti-apoptosis).
* an inhibitor of apoptosis.

BM-1/JIMRO
* a marker of apoptosis.

BV2
* specifically recognizes cells undergoing developmental programmed cell death.

Caspase-1
* play a crucial role in the triggering and execution of apoptosis in a variety of cell types.

Caspase-3 (active)
* a marker of early apoptosis.
* a reliable indicator of apoptotic rate, with a favorable comparison against terminal transferase-mediated DNA nick-end labeling (TUNEL) assay.    
* main executor of apoptosis in somatic cells.

CD95 (Fas)/CD95L (FasL)
* apoptotic molecules.

cleaved cytokeratin-18 (c-CK18)
* as useful and specific as morphology for identifying apoptotic colonic epithelial cells.

Clusterin
* a protein probably related to the process of programmed cell death (apoptosis), was specifically very highly expressed in target fibers.
* clusterin is considered as a specific marker of dying cells.

Histone
* histone release from chromatin are recognized as hallmarks of apoptosis.

NAPO
* NAPO (negative in apoptosis), specifically lost during apoptosis. The anti-NAPO antibody recognizes two nuclear polypeptides of 60 and 70 kD. The antigen is maintained in quiescent and senescent cells, as well as in different phases of the cell cycle, including mitosis. Thus, immunodetection of NAPO antigen provides a specific, sensitive, and easy method for differential identification of apoptotic and nonapoptotic cells.

M30
* an early indicator of apoptosis in epithelial cells.
* M30 (cytokeratin 18 neo-epitope) specifically labels late apoptotic trophoblast cells, and is a highly reproducible marker for apoptotic trophoblast.

OX-42 IR
* may be a good indicator for measuring the cell death in hippocampal regions by KA excitotoxicity.

p41
* may serve as a marker of apoptotic cell death.

p53
* a marker of apoptosis control.

PAI-2
* cleaved plasminogen activator inhibitor 2 (PAI-2) isoform is a biochemical marker of apoptosis in the promyelocytic NB4 cell line.

PARP
* an early marker of chemotherapy-induced apoptosis.
* PARP is enzymatically cleaved during programmed cell death (apoptosis), so detection of the cleavage products is characteristic for apoptosis.

SBDP120
* 120 kDa spectrin breakdown product
* marker for neuronal apoptosis.


Single-Stranded DNA
* MAb to single-stranded DNA is a specific and sensitive cellular marker of apoptosis, which differentiates between apoptosis and necrosis and detects cells in the early stages of apoptosis.

Survivin
* a 16.5 kDa anti-apoptosis protein, inhibits the two early apoptotic enzymes caspase-3 and caspase-7, thus preventing programmed cell death.
* survivin gene is a novel apoptosis inhibitor.

TPA
* tissue polypeptide antigen (TPA), may be considered the first marker of apoptosis measured with a fully standardized quantitative method in tumor cytosol.

tTG
* tissue transglutaminase (tTG), a marker of apoptosis during treatment and progression of prostate cancer.
* tTG cleavage as a valuable biochemical marker of caspase 3 activation during the late execution phase of apoptosis.

Ubiquitin
* a protein marker of programmed cell death.

# Other Apoptosis Markers

* TUNEL Methods
* propidium iodide (PI) binding to DNA allows detection of late apoptotic/necrotic cells.
* Cell-free plasma DNA: a marker for apoptosis during hemodialysis.
* Cytosolic labile zinc: a marker for apoptosis in the developing rat brain.
* serum cytochrome c is a sensitive clinical marker of apoptosis.
* Resting membrane potential as a marker of apoptosis: studies on Xenopus oocytes microinjected with cytochrome c.
* Formation of high molecular mass DNA fragments is a marker of apoptosis in the human leukaemic cell line, U937.
* circulating DNA may be a marker of cell death, although its levels likely reflect a complex process involving the interactions of macrophages with dead and dying cells.
*plasma DNA is a cell death/tumour marker that should be taken into account in studying the cancerous process in human diseases, and could be helpful for follow-up and management of elderly patients.
* Nucleosomes in serum as a marker for cell death.

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