Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-11-06
2003-07-22
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007100, C435S008000, C435S021000, C435S028000, C536S023100, C536S024300
Reexamination Certificate
active
06596480
ABSTRACT:
FIELD OF THE INVENTION
This invention relates generally to the fields of molecular biology and cell biology and, more specifically, to apoptosis and methods for detecting apoptosis in biological samples.
BACKGROUND OF THE INVENTION
Apoptosis is a process of programmed cell death by which multicellular organisms selectively delete cells. The term necrosis is used to describe the morphological changes undergone by cells that die by processes other than apoptosis. Apoptosis is characterized by a progressive condensation of the chromatin to the inner face of the nuclear membrane, cell shrinkage with consequent loss of membrane contact with neighboring cells, and fragmentation of the cells with formation of membrane-bound acidophilic globules (apoptotic bodies).
The DNA of cells that have undergone apoptosis is cleaved into fragments that are multiples of approximately 180 base pairs. These fragments can be seen after agarose gel electrophoresis as a characteristic “ladder” develops. This ladder is widely used as a biochemical marker for discriminating apoptotic cell death from necrotic cell death as the DNA of necrotic cells is randomly degraded and does not produce a ladder. The ladder develops as a result of cleavage of nuclear DNA within the linker regions between nucleosomes. Double strand cleavage results from frequent nicks on both DNA strands.
Although the endonuclease responsible for the cleavage of DNA in apoptotic cells has not been definitively identified, candidate nucleases with properties consistent with their involvement in apoptosis have been identified in apoptotic cells.
The endonucleases that have been identified in apoptotic cells are generally similar in their properties to pancreatic DNase I. Specifically, these endonucleases share the following characteristics:
(i) the DNA ends produced by DNase I cleavage (5′-phosphate and 3′-hydroxyl) are the same as those found in apoptotic nuclei;
(ii) DNase I-transfected COS cells show chromatin changes similar to those seen in apoptosis; and
(iii) DNase I cleavage of chromatin produces the same characteristic nucleosomal DNA fragments that can be isolated from apoptotic cells.
Although DNase I has been detected in cells undergoing apoptosis and the tissue distribution of DNase I is consistent with a role in apoptosis, the endonucleases partially purified from apoptotic cells were shown to be distinct from DNase I. There is less evidence for the involvement of DNase II and other endonucleases in apoptosis.
Presently, the terminal deoxynucleotidyl transferase(TdT)-mediated biotinylated dUTP nick end labeling (TUNEL) method is used to detect apoptotic cells in tissue sections. The TUNEL method utilizes TdT to incorporate a biotinylated deoxyuridine label into DNA fragments containing a 3′-hydroxyl group. The label can be detected using a variety of avidin/streptavidin based detection methodologies. Although 3′-hydroxyl groups are present in the double stranded DNA breaks in the apoptotic cells, they are also present in the DNA of cells that have undergone necrotic cell death. This method, therefore, is not suitable for distinguishing between necrotic cell death and apoptotic cell death. In addition, this methodology does not permit the simultaneous detection of 3′-hydroxyl groups and other biologically relevant molecules, such as RNA and proteins.
Presently, there is a need in the art for a methodology to specifically detect apoptotic cells. There is presently no methodology that permits the specific detection of apoptotic cell death without the simultaneous detection of necrotic cells. In the experiments reported here, we determined whether DNA double strand breaks characteristic of those produced by an endonuclease like DNase I can be detected in apoptotic cells in situ.
When DNA is bound to histones or other proteins in chromatin, it is partially protected from the action of endonucleases, which are able to cleave the DNA at approximately 10-bp intervals, the distance of a single helical turn of the DNA. Because of the helical twist of DNA, the two strands are accessible to endonucleases with production of staggered ends as well as some blunt ends. Thus DNase I cleavage of nucleosome-bound DNA gives rise to double strand cuts with 1, 2, or 3 bases of 3′ overhang.
In contrast, DNase II cleavage of DNA in chromatin yields longer 3′-overhangs of an average of 4 bases.
SUMMARY OF THE INVENTION
To detect double-stranded DNA ends in apoptotic nuclei in situ, we used several types of double-stranded, labeled DNA fragments which were ligated to DNA ends present in the nuclei of cells in sections of fixed paraffin-embedded tissues. To detect single-base 3′-overhangs, we took advantage of the fact that double-stranded DNA fragments synthesized by Taq DNA polymerase in the polymerase chain reaction have a single 3′ base extension beyond the templated sequence. Although it was originally suggested that Taq polymerase added only deoxyadenosine to the 3′-ends of double-stranded DNA, other work subsequently established that if the last templated 3′-nucleotide synthesized is deoxycytidine, Taq polymerase will add deoxyadenosine or deoxycytidine, leaving no blunt-ended DNA, thus providing a fragment that could potentially ligate to the recessed 5′-base of many of the single-base 3′-overhangs in a random DNA sequence. To prepare a fragment that can be ligated only to blunt ends, PCR was performed using Pfu DNA polymerase, because this polymerase produces blunt-ended products only.
An alternative method of preparing DNA fragments for use as ligation probes is to synthesize oligonucleotides that form ends that can be ligated. Rather than using two complementary strands in the fashion customarily employed for the production of oligonucleotide linkers, a single oligonucleotide may be synthesized such that a region on the 3′-end of the oligonucleotide is complementary to a region on the 5′-end. When the complementary regions of the oligonucleotide anneal, a hairpin structure with a stem and loop spontaneously forms.
The stem structure formed has a double-stranded end that may be ligated to a compatible end. When the regions of complementarity include the 5′-most and 3′-most nucleotides, the stem formed has a blunt end. By adding nucleotides to either the 3′-end or the 5′-end without adding the complementary nucleotide to the other end of the oligonucleotide, an overhang can be created. By including the appropriate number of nucleotides on the desired end, a 3′-overhang or a 5′-overhang of any length can be created. Additionally, any desired sequence can be incorporated into the overhanging portion of the oligonucleotide.
Using the ligation procedure and probes described, we determined that apoptotic nuclei, but not nuclei in necrotic tissue or tissue with other non-apoptotic DNA damage, have DNA ends that can be ligated to labeled DNA fragments with single-base 3′ overhangs. In addition, we found that DNA having ends characteristic of apoptosis could be detected with blunt ended DNA probes as well as with probes having two and three base 3′-overhangs.
In contrast, nuclei with all forms of DNA damage have a high concentration of 3′-hydroxyl DNA ends that are a substrate for terminal deoxynucleotidyl transferase (TdT). As TdT can extend the 3′ base of single-stranded DNA and overhanging, blunt, and recessed 3′ bases of double-stranded DNA, TdT based methodologies are not suitable to distinguish apoptotic cells from necrotic cells. In contrast, ligation based methodologies are suitable to accomplish this very desirable objective.
The present invention provides a methodology for specifically detecting the presence of apoptotic cells in tissue sections. The present invention overcomes the limitations of the prior art by employing a novel ligation methodology to detect DNA fragments that are diagnostic of apoptotic cells.
One aspect of the present invention includes a method for detecting apop
Didenko Vladimir
Hornsby Peter
Baylor College of Medicine
Fulbright & Jaworski L.L.P.
Horlick Kenneth R.
Strzelecka Teresa
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