Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1996-01-16
2003-01-07
Arthur, Lisa B. (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S023500, C536S024310
Reexamination Certificate
active
06503706
ABSTRACT:
BACKGROUND OF THE INVENTION
The invention relates to DNA protein complexes, proteins, DNA sequences, and antibodies that are suitable for the detection of cells having an unlimited proliferation and tumor-formation potential; methods for obtaining such DNA protein complexes, proteins or DNA sequences and their use to identify animal and human cells having an unlimited proliferation and tumor-formation potential.
All differentiated human and animal cells have a limited in vivo and in vitro proliferation potential before they undergo aging (senescence) and cell death. The number of cell divisions that are possible at a given time depend on the degree of differentiation of a cell, its age, and the species of the donor from which the cell was obtained, and on the duration of the cell cultures (Goldstein, S.: Replicative Senescence, Science 249 (1990), 1129-1133). Unlimited proliferation, however, is frequently found in neoplastically transformed cells. They thus form constantly growing symplasms leading to the formation of a tumor and, finally, together with the altered characteristics of other properties of these cells to tumor diseases. As compared to benign tumors, these malignant tumors are clinically characterized by their rapid growth and frequent formation of metastases. A neoplastic transformation of the cell is characterized by a heterogeneous picture of numerous alterations of the cellular morphology and physiology which also depend on the degree of differentiation of the cells. To date, only few molecularly definable parameters of neoplastic transformation are known; they include, for example, an altered degree of methylation of certain genes (W. Doerfler et al., Eukaryotic DNA methylation: facts and problems, FEBS Letters 268 (1990), 329-330), modified gene expression, or altered phosphorylation of certain gene products.
What is common to most tumor cells is the capability of unlimited in-vivo proliferation. Based on various observation, investigators assume that the proliferation of tumor cells which is independent of growth factors is, indeed, a frequently occurring characterisitic of tumor-forming cells, but not an absolutely necessary one (M. Strauss, B. E. Griffin, Cellular Immortalization, Cancer Cells 2 (1990), 360-365). However, for many questions in the field of diagnostics and therapy, it is of critical importance to identify those cells which no longer obey normal proliferation control and, hence, have acquired the potential for unlimited proliferation.
The problem involved in this matter is to distinguish this property of unlimited proliferation of tumor cells from the capability to regenerate that is found in many tissues. Regeneration is a controlled, short (transient) cell replication and only a intermittent suppression of programmed cell death as a response to a physiological stimulation. The transient proliferation of normal cells is again inhibited after regeneration of the tissue by still unidentified signals.
Known methods for the detection of malignant tumor cells are based on clinical, histological, and cytological observations, and on altered physiological measurements. Based on histological examinations, diagnostic routine methods are usually done with tissue sections (W. A. D. Anderson and J. M. Kissane, Pathology I, II, Mosby, Saint Louis 1977, 7th edition; C. S. Herrington and J. O. D. McGee, Diagnostic Molecular Pathology, Oxford University Press, Vol. I, II, 1st ed., 1992). As opposed to benign growth, the group of malignant cells is only vaguely separated from adjacent tissue, and the cells grow into surrounding tissue which they infiltrate and destroy. Most cases are triggered by a perifocal inflammation. Frequently, a great number of mitoses indicate increased proliferation activities of the tumor cells.
In addition to these histological examinations, it has become more common to use antibodies to detect proliferating malignant cells which recognize antigens that are preferably expressed by proliferating tumor cells such as Ki67 (D. C. Matthews, F. O. Smith, I. D. Bernstein, Monoclonal antibodies in the study and therapy of hematopoietic cancers, Curr. Opinion Immunol. 4 (1992), 641-646; M. Schwouzen, V. Diehl, M. Pfreundschuh, Immunozytologische Phänotypisierung von Leukämien und Lymphomen, Med. Klinik 85 (1990), 533-547). However, these methods are based on the determination of indirect parameters without detecting the molecular processes that are linked to unlimited proliferation.
SUMMARY OF THE INVENTION
It is, hence, an object of the invention to identify cells with a potential of unlimited proliferation, especially malignant tumor cells, in a rapid and reliable manner without cultivating these cells in vitro or propagating them after injection in laboratory animals. It should be possible to distinguish between cells having the potential for unlimited proliferation and cells with transient proliferation in regenerating tissue.
This object is accomplished with a DNA protein complex (hereinafter referred to as complex) which is suitable for detecting human or animal having an unlimited proliferation and tumor-formation potential. Said complex can be obtained by isolating a mitochondria-free fraction of the cytoplasm from human or animal cells which can permanently divide and have a density of approx. 1.82-1.89 g/cm
3
in a cesium chloride gradient; this is followed by isolating the complex from this fraction by extraction with phenol and precipitation with ethanol.
Experience has surprisingly shown that cells having an unlimited proliferation and tumor-formation potential, as opposed to normal resting cells, senescent cells, or cells with transient proliferation do have such complexes in their cytoplasm. With these complexes in accordance with the invention it is possible to detect such cells, which exhibit unlimited growth potential as a consequence of malignantant growth. In order to accomplish this, one uses cytoplasm or a suitable cytoplasm fraction in a complex in accordance with the invention as a standard in a gel electrophoresis by determining the DNA contents or reaction with a specific antibody.
In order to isolate complexes of the invention, cytoplasts of human or animal cells that are permanently capable of dividing are obtained according to known methods (for example EP-B-0 093 436, EP-B-0 256 512 and Proc. Natl. Acad. Sci. 85 (1988), 468-472 and lysed for example using detergents like NP40 or SDS, Wigler and Weistein, Biochem. Biphys. Res. Commn. 63 (1975) 669-674). The mitochondria are separated from these cytoplast fractions, preferably with the aid of a sucrose gradient (J. Biol. Chem. 249 (1974) 7991-7995). Those fractions who do not contain any mitochondria are incubated with RNase, preferably RNase A and RNase T1 as well as with proteinase, e.g. proteinase K or pronase. A fraction from this mixture with a density of approx. 1.82-1.89 g/cm
3
in a cesium chloride gradient is enriched, and from this fraction, a complex is isolated by extraction with phenol and precipitation with ethanol. A fraction which has a density of approx. 1.82-1.89 g/cm
3
in a cesium chloride gradient can be obtained both by means of centrifugation with a cesium chloride density gradient and by means of electrophoretic separation; these methods are known to the expert in the field (Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory, 2nd edition, 1989).
The mitochondria-free fraction of the cytoplasm that is used for isolating the complexes can also be obtained by repeated freezing and thawing of the cells or by obtaining a cell fraction which is free of nuclear DNA. Such a fraction is preferably obtained by lysis of the cell with sodium chloride and SDS (“Hirt extraction”, J. Mol. Biol. 26 (1967) 365-369) followed by centrifugation. The complexes can then be isolated from the supernatant.
In a preferred method, the complexes are isolated via sedimentation from a cytoplasmatic lysate of the cells to be examined using a salt gradient. The cells are lysed in a Mg
2+
-containing buffer (50 mmol/l Tris-HCl, pH 7.2, 10 mmol/l E
Abken Hinrich Johann
Albert Winfried
Jungfer Herbert
Arthur Lisa B.
Roche Diagnostics GmbH
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