Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-11-28
2004-10-26
Horlick, Kenneth R. (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C536S024310, C536S024330
Reexamination Certificate
active
06808883
ABSTRACT:
The invention relates to an automatable rapid test for detection of cancer based on telomerase(hTC) mRNA, to suitable starter nucleotides and oligonucleotide probes for this test and to a corresponding detection method and a test kit.
The genetic material of eukaryotic cells is distributed on linear chromosomes. The ends of these hereditary units are termed telomers, derived from the Greek words telos (end) and meros (part or segment). Most telomers consist of repeats of short sequences which are mainly constructed from thymine and guanine (Zakian, 1995). The telomer sequences of related organisms are often similar and these sequences are even conserved between species which are more phyllogenetically remote. It is a remarkable fact that the telomers are constructed from the sequence TTAGGG in all the vertebrates which have so far been examined (Meyne et al., 1989).
The telomers exert a variety of important functions. They prevent the fusion of chromosomes (McClintock, 1941) and consequently the formation of dicentric hereditary units. Chromosomes of this nature, possessing two centromers, can lead to the development of cancer due to loss of heterozygosity or the duplication or loss of genes.
In addition, telomers serve the purpose of distinguishing intact hereditary units from damaged hereditary units. Thus, yeast cells ceased dividing when they harboured a chromosome which lacked a telomer (Sandell and Zakian, 1993).
Telomers carry out another important task in association with DNA replication in eukaryotic cells. In contrast to the circular genomes of prokaryotes, the linear chromosomes of eukaryotes cannot be completely replicated by the DNA polymerase complex. RNA primers are required for initiating DNA replication. After the RNA primers have been eliminated and the Okazaki fragments have been extended and then ligated, the newly synthesized DNA strand lacks the 5′ end because the RNA primer at that point cannot be replaced by DNA. For this reason, without special protective mechanisms, the chromosomes would shrink with every cell division (“end-replication problem”; Harley et al., 1990). The non-coding telomer sequences probably represent a buffer zone for preventing the loss of genes (Sandell and Zakian, 1993).
Over and above this, the telomers also play an important role in regulating cell ageing (Olovnikov, 1973). Human somatic cells exhibit a limited capacity to replicate in culture; after a certain time they become senescent. In this condition, the cells no longer divide even after being stimulated with growth factors; however, they do not die but remain metabolically active (Goldstein, 1990). Various observations provide support for the hypothesis that a cell determines from the length of its telomers how often it can still divide (Allsopp et al., 1992).
In summary, the telomers consequently possess central functions in the ageing of cells and in the stabilization of the genetic material and prevention of cancer.
The Enzyme Telomerase Synthesizes the Telomers
As described above, organisms possessing linear chromosomes are only able to replicate their genome incompletely in the absence of special protective mechanisms. Most eukaryotes use a special enzyme, i.e. telomerase, to regenerate the telomer sequences. Telomerase is expressed constitutively in the single-cell organisms which have so far been examined. By contrast, in humans, telomerase activity was only detected in germ cells and tumour cells whereas neighbouring somatic tissue did not contain any telomerase (Kim et al., 1994).
Activation of the Telomerase in Human Tumours
In humans, it was originally only possible to demonstrate telomerase activity in germ line cells and not in normal somatic cells (Hastie et al., 1990; Kim et al., 1994). After a more sensitive detection method had been developed (Kim et al., 1994) a low level of telomerase activity was also detected in haematopoietic cells (Broccoli et al., 1995; Counter et al., 1995; Hiyama et al., 1995). However, these cells nevertheless exhibited a reduction in the telomers (Vaziri et al., 1994: Counter et al., 1995). It has still not been clarified whether the quantity of enzyme in these cells is insufficient to compensate for the telomer loss or whether the measured telomerase activity stems from a subpopulation, e.g. of incompletely differentiated CD34
+
38
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precursor cells (Hiyama et al., 1995). In order to clarify this point, it would be necessary to detect the telomerase activity which was present in a single cell.
Interestingly enough, however, significant telomerase activity has been detected in a large number of the tumour tissues which have been tested to date (1734/2031, 85%; Shay, 1997), whereas no activity has been found in normal somatic tissue (1/196, <1%, Shay, 1997). In addition, a variety of investigations demonstrated that the telomers continued to shrink in senescent cells which were transformed with viral oncoproteins and that it was only possible to find telomerase in the subpopulation which survived the growth crisis (Counter et al., 1992). The telomers were also stable in these immortalized cells (Counter et al., 1992). Similar findings derived from investigations in mice (Blasco et al., 1996) support the assumption that reactivation of the telomerase is a late event in tumour regenesis.
Details about telomerase and in particular the human catalytic telomerase subunit and its sequence are given in WO 98/14592 (Genon Corp.) and WO 98/59040 (Bayer AG).
Detection of Telomerase mRNA for Cancer Diagnosis
Based on these results, a “telomerase hypothesis” was developed which links the loss of telomer sequences and cell ageing to telomerase activity and the genesis of cancer. In long-lived species such as humans, the shrinking of the telomers can be regarded as a tumour suppression mechanism. Differentiated cells, which do not contain any telomerase, cease dividing when the telomers have reached a particular length. If such a cell mutates, a tumour can only develop from it if the cell is able to extend its telomers. Otherwise, the cell would continue to lose telomer sequences until its chromosomes became unstable and finally die. Reactivation of the telomerase is presumably the main mechanism which tumour cells deploy in order to stabilize their telomers.
It follows from these observations and ideas that it should be possible to diagnose tumours by elevated expression of telomerase. Since telomerase activity has been detected in virtually all the tumour tissues tested to date, it would be possible to use a genetic test for diagnosing all types of cancer. This genetic test is particularly suitable for monitoring the progress of cancerous diseases, but can also be used as a prognostic test or for the early diagnosis of certain cancerous diseases.
Gene probe diagnosis, in particular in combination with amplification techniques, is a rapid, specific and highly sensitive method which permits an early identification of specific genes, gene fragments or single mutations on the DNA/RNA level. The technique can be carried out directly in the material to be examined. It is based on the DNA/RNA hybridization technique. i.e. the specific in vitro binding of complementary single-strand nucleic acid with formation of Watson-Crick base pairs. The DNA/DNA or DNA/RNA double strands formed are also referred to as DNA hybrids. For the detection of the specific DNA or RNA by the hybridization reaction, complementary sequence-specific gene probes are used. These gene probes are short, chemically synthesized oligonucleotide probes having a length of 10-200 nucleotides.
Photochemically (N. Dattagupta, P. M. M. Rae, E. D. Huguenel, E. Carlson, A. Lyga, I. S. Shapiro, J. P. Albarella, Analytical Biochem. 177, 85, 1989) or enzymatically by nick translation (Rigby, P. W. J. et al., J. Mol. Biol. 113, 237, 1977) or by random primed techniques (Feinberg and Vogelstein, Anal. Biochem. 132, 6, 1983), the gene probes can be provided with radioactive or non-radioactive labels. Suitable for this purpose is labelling with
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P NTPs or non-radioactive
Hagen Gustav
Springer Wolfgang
Wick Maresa
Zubov Dmitry
Bayer AG
Connolly Bove Lodge and Hutz LLP
Horlick Kenneth R.
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