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Detection of cell death

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Publication number
WO1989006697A1
WO1989006697A1 PCT/AU1988/000015 AU8800015W WO1989006697A1 WO 1989006697 A1 WO1989006697 A1 WO 1989006697A1 AU 8800015 W AU8800015 W AU 8800015W WO 1989006697 A1 WO1989006697 A1 WO 1989006697A1
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WO
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Patent type
Prior art keywords
mrna
specific
ck
cell
actin
Prior art date
Application number
PCT/AU1988/000015
Other languages
French (fr)
Inventor
Bruce Hepher Bennetts
Cris Dos Remedios
Phillip Harris
Original Assignee
The University Of Sydney
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6802General aspects
    • C12Q1/6809Sequence identification involving differential detection
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876Hybridisation probes
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00Oligonucleotides characterized by their use
    • C12Q2600/158Expression markers

Abstract

Irreversible cell death, particularly irreversible myocardial damage, is detected by detecting the presence in the blood of mRNA specific to that cell and released following irreversible cell damage using a cDNA probe specific to the mRNA to be detected. For myocardial mRNA the cDNA preferably probes for the 3' untranslated region.

Description

DETECTION OF CELL DEATH FIELD OF THE INVENTION

The present invention relates to the detection in a patient of the irreversible death of specific cells, for example muscle cells, and in particular the detection of myocardial cell death following myocardial infarction (i.e. heart attack) . The mechanism of the detection is based on the ability to identify tissue-specific mRNAs or fragments of them using nucleic acid probes. PRIOR ART

The development of a precise and definite indicator of myocardial cell death would be a considerable advantage in the assessment of suitability of cardiac by-pass surgery and in the assessment of the viability of heart transplants. Current methods of diagnosis of acute myocardial infarction (AMI) are based on a combination of symptoms. These include changes in electrocardiogram (ECG) as well as a variety of blood-based tests such as the determination of serum creatine kinase (CK) . CK determinations are widely used and are considered to be reliable early (12 to 24 hour) indicators. However, this and other blood-based tests suffer from several shortcomings. In particular, the CK test appears to have a short (12 to 24 hour) time course and it does not seem to be a reliable predictor of the size of the myocardial infarction. It is also doubtful whether the CK tests can distinguish between reversible and irreversible myocardial damage.

The CK test is based on a quantification of serum levels of creatine kinase (CK) or its isozyme (CK-MB) . These tests are now routinely performed and generally reach their peak activity within 24 hours of the onset of chest pain, after which their titre levels fall rapidly. Creatine kinase is a dimer with a molecular weight of approximately 80,000 Daltons. The assumption that CK is too large to pass through the membrane of viable cells seems reasonable as does the assumption that its release into the circulation is probably due to the infarction of the myocardial cells. However, in a recent report of experimental AMI in dogs, elevated levels of serum CK were monitored in combination with evaluation of the extent of necrosis, and it was concluded that the application of a short (approximately 10 minutes) ligation of a coronary artery produced considerably elevated levels of serum CK without a concomitant indication of myocardial necrosis observed post mortem. This observation can be understood when we learn that when cultured myocytes are subjected to brief periods of anoxia, they release small vesicles of cytoplasm which contain high concentrations of CK. Their subsequent rupture could result in the observed rise in serum CK levels without the necessity of the death of these cells. Anoxia can produce a condition commonly referred to as the "stunned" myocardium which is unable to contract but which can recover with the return of the coronary circulation. This is a reversible phenomenon. In AMI, a portion of myocardium becomes necrotic and releases all of its cellular contents including the CK and its compliment of mRNA. AIM OF THE INVENTION

Thus, there is a need for a test which is specific to myocardial cell damage, and which is capable of distinguishing between reversible and irreversible damage i.e. to distinguish between myocardial cell destruction and anoxia.

SUMMARY OF THE INVENTION

The present invention is based on the surprising discovery that death of specific cells may be detected by detecting for quantities of mRNA specific to such cells and released on death of the cells, by the use of cDNA probes specific to that mRNA. In particular, it has been found that despite the presence of RNases in the blood the lifetime of the released mRNA is sufficient for quantitative detection.

Thus, the present invention provides a method of detection of cell death in a patient which comprises: - taking a blood serum sample from the patient,

- detecting the presence in the sample of any released cell-specific mRNA by the addition thereto of a known amount of cDNA specific to said mRNA and capable of binding thereto, and

- estimating the amount of mRNA-cDNA complex present and thus the amount of cell-specific mRNA in the blood sample, the presence of mRNA indicating death of cells of that specific type.

The patient will be a mammal, generally a human. The technique is believed to have universal application for the detection of any damaged cells by detecting release of mRNA specific to that cell. Thus, the technique has potential for detecting irreversible damage to skeletal muscle (for example due to muscular dystrophy), liver, kidneys, pancreas, stomach, intestines, uterus, brain and blood cell diseases (such as leukemia). The technique has particular application to diseases leading to muscle breakdown, and especially to heart attacks. Such irreversible damage generally involves a breakdown of at o least 1x10 cells.

In order to detect myocardial cell products, we have found it useful to detect cardiac type alpha actin mRNA using a cDNA probe specific for the 3 untranslated region of this mRNA molecule. This probe (171 base pairs) exclusively recognises the unique nucleotide sequence of cardiac actin mRNA and fails to cross-hybridize with skeletal muscle alpha actin. Other probes can be used. For example, we have one that is specific for the 3' untranslated region of the skeletal alpha actin gene. Another probe made from the coding region of the actin has also been successfully used. The cDNA probe will usually be labelled, for example using a fluorescent label, or an enzyme radio-label.

The amount of mRNA-cDNA labelled complex is easily estimated using conventional techniques for the detection of the particular label used, for example fluorescence counting, rate of enzyme reaction, radioactivity counting etc.

The amount of blood sampled is generally in the region of 1-5 ml.

DESCRIPTION OF SPECIFIC EMBODIMENTS

Embodiments of the present invention will now be described by way of example only. EXAMPLE The cardiac isoform of alpha-actin is expressed as the major (ratio approximately 80%:20%) of the two striated muscle isotypes in the left and right ventricles of non-pathological heart muscle and this level may be slightly lower (approximately 50%) in pathological specimens. Cardiac alpha actin is probably the exclusive isoform expressed greater than 95% in the atria. It is also expressed in skeletal muscle cells at a low (approximately 5%) levels. The actin represents about 30% of the myofibrillar proteins in striated muscles and therefore its mRNA can be expected to be present in high copy number relative to other proteins. Our reasoning in probing serum samples for mRNA is that their molecular weights are so large that it seemed to us extremely unlikely that it could escape from a viable cell. Therefore the probe may be an appropriate indicator of myocardial necrosis in AMI. The following procedure is carried out: - A 2-5 ml blood sample was collected into an EDTA tube and placed on ice to reduce the effect of any RNases present in the blood. 300 micro litres of plasma was added to 180 micro litres of 20 x SSC and 120 micro litres of 40% formaldehyde. The sample was mixed and placed in a 60°C water bath for 30 minutes. 5 ml of 15 x SSC was added and the sample was stored at -70°C until processed.

Samples were spun at 12,000 g for 15 minutes to remove any filamentous debris. 250 micro litres was loaded onto a dot-blot apparatus and allowed to sit on the nylon membrane (Gene-Screen Plus or Pal Biodyne) for 15 minutes prior to suction being applied to the apparatus. The nylon membrane was baked at 80°C for 2 hours under vacumm.

The nylon membranes were prehybridized in 5 x SSC, 0.5% skim milk, 1% SDS and 0.6 mg/ml sheared calf thymus DNA

(boiled prior to addition) for 6 to 18 hours at 65°C. The nylon membranes were hybridized in 5 x SSC, 0.5% skim milk, 1% SDS, 0.6 mg/ml sheared calf thymus DNA (boiled) and 30 g million counts of radiolabelled DNA (__• 1 x 10 cpm/μg of DNA) . DNA probes were labelled using random hexamer primers (Multiprime, Amersham) . Washing of the membranes was done in 2 x SSC, 0.1% SDS at 60°C for 30 minutes, followed by washing in 0.2 x SSC, 0.1% SDS at 60°C for 1 hour. The membrane was subsequently autoradiographed at -70 C for 2 to 8 days.

The cDNA probe used was 3 -untranslated cardiac actin DNA contained as a 171 base pair insert into the plasmid pHMcA-3'uτ-DB [pDB] .

The quantitation of these dot blots was achieved either from counts of 32_ activity or by scanning the dots on the autoradiograms with a densitometer (Camag) using a slit width appropriate for integrating their density. The density of the dot blots was measured by integration of the density peaks and expressed on an arbitrary scale. The time course of one patient is illustrated in Figure 1.

Figure 1 illustrates the temporal relationship between serum levels of creatine kinase (CK) , the MB isoform of CK (CK-MB) , lactic dehydrogenase (LDH) and plasma levels of mRNA detected by the 3' untranslated cardiac actin cDNA probe. Day 0 is the onset of chest pain. The ordinate units are arbitrary.

In this example, the CK-MB release generally rises to a peak at approximately 18 hours from the onset of chest pain and is closely followed by the peak in the serum CK at 24 hours. Despite the use of serum CK and LDH determinations in the diagnosis of AMI, it is clear that they are not cardiac specific. The CK-MB isozyme test is considered to be more specific for heart which contains a characteristic

SUBSTITUTE SHEET - 5a - ratio of the M and B isoform despite the fact that both isoforms are produced in other tissues.

The levels of LDH characteristically rise much later than the CK. The peak observed in Figure 1 (approximately 60 hours from the onset of chest pain) is in good agreement with the observations of others. The lateness of this serum

- 6 -

indicator has been ascribed to myocardial necrosis but this conclusion has been criticised on two grounds. Firstly, LDH is not specific to the heart and is in fact found in practically all cells. Secondly, doubts have been raised about the reliability of the test. Our cDNA probe data closely parallel the changes in serum LDH levels. However, unlike LDH, the actin DNA probes are precisely specific for the cardiac form of actin.

Specificity of the mRNA Signal for Cardiac Muscle Tissue The amino acid sequence of cardiac actin differs in only four residues (two substitutions at positions 298 and 357, and one inversion of residues 3 and 4) from skeletal muscle actin. However, cardiac actin gene sequences can be distinguished from skeletal actin gene sequences using probes made from their respective 3' untranslated regions. As stated above, both skeletal and cardiac isoforms are expressed in skeletal muscle cells although the ratio heavily favours the skeletal type. Thus, if skeletal muscle damage was suspected, a probe specific to the skeletal actin mRNA would produce a signal approximately 20 times that produced by a cardiac specific probe.

Chemical Nature of the Serum-Based Signal (RNase and RNase Inhibitor Data)

Our cDNA probes will hybridize with the appropriate sequence of either RNA or DNA. What evidence is there to suggest that the signal is in fact derived from serum mRNA? We have addressed this question in several ways. Firstly, we have added RNase to degrade mRNA. The results are shown in Figure 2. Identical volumes of a patient's serum were applied to the nylon membrane which was then treated as described above. 300 microliters of serum sampled at 3, 4, 5 and 6 days post-infarction was added to lanes A and B, Lane A was processed without delay while to sample B, 20 microliters 20 mg/ml RNase A was added and incubated at 37βC for several hours before processing. Thus, RNase rapidly destroys our signal. Secondly, we have calculated the amount of tissue death required to produce our observed - 7 -

signal amplitude if it were derived from DNA. These calculations preclude the likelihood that DNA is our signal. Finally, our conditions (particularly the presence of formaldehyde which is unlikely to allow the DNA to become single stranded and bind to the nylon membrane) are such that they do not favour DNA binding.

Claims

- 8 -CLAIMS
1. A method of detection of cell death in a patient which comprises: - taking a blood serum sample from the patient, detecting the presence in the sample of any released cell-specific mRNA by the addition thereto of a known amount of cDNA specific to said mRNA and capable of binding thereto, and - estimating the amount of mRNA-cDNA complex present and thus the amount of cell-specific mRNA in the blood sample, the presence of mRNA indicating death of cells of that specific type.
2. A method according to claim 1 applied for the detection of death of cells derived from skeletal muscle, liver, kidneys, pancreas, stomach, intestines, uterus, brain or blood.
3. A method according to claim 1 applied for the detection of irreversible myocardial muscle cell death.
4. A method according to claim 3 wherein the mRNA detected is cardiac type alpha actin mRNA.
5. A method according to claim 4 wherein the cDNA used probes for the 3' untranslated region of the mRNA.
6. A method according to claim 1 applied for the detection of irreversible skeletal muscle damage wherein the mRNA detected is skeletal muscle alpha actin mRNA, and the cDNA used probes for the 3* untranslated region thereof.
7. A method according to claim 1 wherein the cDNA is labelled.
PCT/AU1988/000015 1988-01-19 1988-01-19 Detection of cell death WO1989006697A1 (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993009437A2 (en) * 1991-10-31 1993-05-13 Matritech, Inc. Nuclear matrix protein fluid assay
US20080050783A1 (en) * 1997-03-14 2008-02-28 Oncomedx Inc. Method enabling use of extracellular RNA extracted from plasma or serum to detect, monitor or evaluate cancer

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1984002721A1 (en) * 1983-01-10 1984-07-19 Gen Probe Inc Method for detecting, identifying, and quantitating organisms and viruses
EP0186522A1 (en) * 1984-12-27 1986-07-02 Sankyo Company Limited Cancer-specific DNA

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1984002721A1 (en) * 1983-01-10 1984-07-19 Gen Probe Inc Method for detecting, identifying, and quantitating organisms and viruses
EP0186522A1 (en) * 1984-12-27 1986-07-02 Sankyo Company Limited Cancer-specific DNA

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993009437A2 (en) * 1991-10-31 1993-05-13 Matritech, Inc. Nuclear matrix protein fluid assay
WO1993009437A3 (en) * 1991-10-31 1993-06-10 Matritech Inc Nuclear matrix protein fluid assay
US5840503A (en) * 1991-10-31 1998-11-24 Matritech, Inc. Nuclear matrix protein fluid assay
US5965376A (en) * 1991-10-31 1999-10-12 Matritech, Inc. Nuclear matrix protein fluid assay
US5989826A (en) * 1991-10-31 1999-11-23 Matritech, Inc. Nuclear matrix protein fluid assay
US6162608A (en) * 1991-10-31 2000-12-19 Matritech, Inc. Nuclear matrix protein fluid assay
US6410247B1 (en) 1991-10-31 2002-06-25 Matritech, Inc. Nuclear matrix protein fluid assay
US6740494B2 (en) 1991-10-31 2004-05-25 Matritech, Inc. Nuclear matrix protein fluid assay
US20080050783A1 (en) * 1997-03-14 2008-02-28 Oncomedx Inc. Method enabling use of extracellular RNA extracted from plasma or serum to detect, monitor or evaluate cancer
US8440396B2 (en) 1997-03-14 2013-05-14 Oncomedx, Inc. Method enabling use of extracellular RNA extracted from plasma or serum to detect, monitor or evaluate cancer

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