Chemistry: analytical and immunological testing – Optical result – With fluorescence or luminescence
Reexamination Certificate
2000-09-19
2004-04-20
Chin, Christopher L. (Department: 1641)
Chemistry: analytical and immunological testing
Optical result
With fluorescence or luminescence
C436S063000, C436S064000, C436S517000, C436S537000, C436S546000, C436S007000, C436S007000, C436S007000, C436S008000, C436S007000, C436S069000, C436S172000, C436S172000, C436S169000, C436S169000, C436S169000, C436S169000, C436S169000
Reexamination Certificate
active
06723567
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a method for the determination of non-, anti-, or pro-apoptotic and necrotic conditions of cells, using newly designed vectors coding for marker proteins, cell lines transfected with such vector, and a method to assay the non-, pro-, or anti-apoptotic or necrotic activity of test compounds or physical stimuli.
BACKGROUND ART
Apoptosis plays an essential role in development, i.e. embryogenesis and normal cell turnover, but also in diseases such as cancer, AIDS, neurodegeneration and viral infections. Unlike necrosis, apoptosis is an active, gene-directed self-destruction process of the cell and is associated with characteristic morphological and biochemical changes
1,2
. Nuclear and cytoplasmic condensation and fragmentation of the dying cell into membrane-bound apoptotic bodies are typical characteristics of apoptosis. Another feature of apoptotic cell death is the chromosomal DNA degradation into oligonucleosomal fragments after the activation of specific nucleases
3,4
.
Apoptosis can be induced by the interaction of the cell surface molecule Fas (CD95) with Fas-Ligand (FasL), where the Fas expressing and sensitive cells undergo apoptosis. Fas is a type I membrane protein, which belongs to the tumor necrosis factor (TNF) and nerve growth factor (NGF) receptor family
5-7
. Fas expression is found on a wide variety of tissues and cells such as thymus, liver, lung, ovary, heart and myeloid cells
9-12
. FasL expression is not only found on lymphocytes but also on a wide variety of tissues and some tumors
13-19
. Both membrane bound FasL and the soluble form (sFasL) can induce apoptosis in Fas positive and sensitive cells. Other forms among a variety of apoptosis mediators include the Perforin/Granzyme system, the TRAIL/TRAIL-R system
36
, cytokine deprivation (e.g. IL-3 deprivation) and irradiation.
In contrast to apoptosis, necrosis is a non-physiological death of cells due to chemical or physical injury of the cell membrane. Morphological criteria include cell swelling and cell lysis, lysosomal leakage and loss of the cell membrane integrity.
During the last decade, it has become clear that apoptosis plays a keyrole in several diseases. Apoptosis is increased in AIDS, but decreased in cancer and certain autoimmune proliferative diseases.
Flow cytometry offers a wide variety of possibilities to measure apoptosis. Different methods have been established and implemented, some which stain on the cell surface and some which stain intracellularily.
One of the first approaches was, beside the observation that apoptotic cells shrink and have higher intracellular granularity, to stain with DNA specific fluorochromes (e.g. propidium iodide [PI], ethidium bromide [EtBr]). As soon as a lethal hit is being induced, the DNA starts. to change its profile. Apoptotic DNA not only consists of fragmented DNA (visualised as shorter bands, so called DNA ladder, in an agarose gel) but is also partially digested into single nucleotides, so that fluorochromes, like PI or EtBr, have less DNA to stain. This is typically observed by a shift to the left, called sub-G1 peak, on the specific fluorochrome detection channel in the FACScan™ (from Becton Dickinson, USA). The big disadvantage of this method is that the cell membranes have first to be permeabilized with reagents, like ethanol, in order to stain them with DNA specific dyes, like PI
20
. The treatment is time and labour consuming, and the risks of loosing cells and of handling errors are high. Furthermore, the discrimination between live and apoptotic cells cannot be standardised and requires large experience.
Another method is the terminal deoxynucleotidyl transferase (TdT)-mediated endlabeling of the DNA strand breaks (TUNEL). The TUNEL method detects DNA strand breaks in cells undergoing apoptosis. TdT is an enzyme which catalyzes the addition of deoxyribonucleotide triphosphate to the 3′-OH ends of double or single-stranded DNA. Unlike normal cells, apoptotic cell nuclei incorporate exogenous nucleotides (dUTP)-DIG in the presence of TdT. An anti-DIG antibody fragment with a conjugated fluorochrome enables the visualisation of apoptotic cells. An increase of apoptotic cells causes a higher number of DNA fragments and consequently a brighter fluorescence. An advantage of this method is the very high specificity
21
. A disadvantage of this method is that it is expensive and can only be used for a small set of samples, because it is time intensive. Therefore, it is not applicable for large screening programmes.
The loss of cell membrane polarity and the presentation of increased amounts of phosphatidyl serine (PS) on the outside of the cell membrane during the early phase of apoptosis has led to yet a new approach. Annexin V is a calcium-dependent phospholipid binding protein with high affinity for PS. Due to the fact, that the cell membrane integrity is maintained in the early and intermediate phases of apoptosis but not in necrosis, it is possible to distinguish between apoptotic and necrotic cells, when Annexin V is used concomitantly with the DNA dye PI. Early and intermediate apoptotic cells show increased binding of Annexin-FITC and are mainly negative for PI-staining. Late apoptotic stages and necrotic cells become double positive, because of PS presentation on the surface and the PI staining of intracellular nucleic acids due to disintegration of the membrane
22
. This method is also costly and labour intensive.
Green fluorescence protein (GFP) from the jellyfish
Aequorea victoria
can be used to monitor gene expression and protein localization in living organisms (in vivo) and in vitro
23-26
. GFP-fluorescence is stable, can be monitored noninvasively in living cells and persists in paraformaldehyde-fixed cells. FACS-optimized mutants of green fluorescence protein have been developed
8
. One of these mutants (GFPmut1) has been integrated into the PEGFP vectors and is commercially available (from Clontech). The big advantage of this mutant is that the maximal excitation peak of GFPmut1is 488 nm and the emission maxima is 507 nm. Conventional flow cytometers are equipped with an argon laser emitting light at 488 nm and have the suitable detection filters already installed, making the GFPmut1-protein an ideal candidate for flow cytometry studies and fluorescence microscopy.
GFP has already been used as marker for visualizing changes in cell morphology such as blebbing caused by cytotoxic agents or apoptosis, or as transfection marker (WO97/11094), or as marker for screening factors modulating gene expression (WO97/14812). GFP has also been used as a marker protein to detect the progression of the morphological changes of apoptotic cells
37
.
GFP has been used as a marker protein to detect cells transiently transfected with the commercially available plasmid pEGFP-C1 (Clontech)
34
. According to Lamm et al.
34
apoptosis was detected by reduced fluorescence of the DNA-binding dye PI in the apoptotic subpopulation. It was not recognized that GFP itself could be used as a marker for apoptosis. The great disadvantage of PI-staining is that no changes in the state of one and the same cells can be monitored but only one specific state since for PI-staining the cells have to be permeabilized and fixed.
Experiments by the present inventors have shown, that stable transfection of eukaryotic cells (e.g. A20.2J) with the pEGFP-C1 (as received from the manufacturer Clontech with the cytomegalovirus CMV promoter) used in the above cited state of the art for tansient transfection results in little or no expression of the GFPmut1gene.
For the foregoing reason there is a need for a new and improved method and tools for determining apoptosis and necrosis of cells which in particular can be used to efficiently and cheaply assay compounds on their pro-apoptotic or anti-apoptotic or necrotic activity.
DISCLOSURE OF THE INVENTION
Accordingly, objects of the invention are to provide an improved method for the determination of apoptosis and/or necrotic conditions of cells and to pr
Erb Peter
Hahn Sinuhe
Harr Thomas
Strebel Alessandro
Aponetics Ltd.
Chin Christopher L.
Gabel Gailene R.
Marshall & Gerstein & Borun LLP
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