Screening methods to identify inhibitors of telomerase activity

Details Screening methods to identify inhibitors of telomerase activity Screening methods to identify inhibitors of telomerase activity

1995-06-05

2002-04-09

Brusca, John S. (Department: 1631)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S007210, C435S007220, C435S007400, C435S007720, C435S091310

Reexamination Certificate

active

06368789

ABSTRACT:

This invention relates to methods for therapy and diagnosis of cellular senescence and immortalization.
BACKGROUND OF THE INVENTION
The following is a general description of art relevant to the present invention. None is admitted to be prior art to the invention. Generally, this art relates to observations relating to cellular senescence, and theories or hypothesis which explain such aging and the mechanisms by which cells escape senescence and immortalize.
Normal human somatic cells (e.g., fibroblasts, endothelial, and epithelial cells) display a finite replicative capacity of 50-100 population doubling characterized by a cessation of proliferation in spite of the presence of abundant growth factors. This cessation of replication in vitro is variously referred to as cellular senescence or cellular aging, See, Goldstein, 249
Science
1129, 1990; Hayflick and Moorehead, 25
Exp. Cell Res.
585, 1961; Hayflick,
ibid.,
37:614, 1985; Ohno, 11
Mech. Aging Dev.
179, 1979; Ham and McKeehan, (1979) “Media and Growth Requirements”, W. B. Jacoby and I. M. Pastan (eds), in:
Methods in Enzymology,
Academic Press, NY, 58:44-93. The replicative life span of cells is inversely proportional to the in vivo age of the donor (Martin et al., 23
Lab. Invest.
86, 1979; Goldstein et al., 64
Proc. Natl. Acad. Sci. USA
155, 1969; and Schneider and Mitsui,
ibid.,
73:3584, 1976), therefore cellular senescence is suggested to play an important role in aging in vivo.
Cellular immortalization (the acquisition of unlimited replicative capacity) may be thought of as an abnormal escape from cellular senescence, Shay et al., 196
Exp. Cell Res.
33, 1991. Normal human somatic cells appear to be mortal, i.e., have finite replicative potential. In contrast, the germ line and malignant tumor cells are immortal (have indefinite proliferative potential). Human cells cultured in vitro appear to require the aid of transforming viral oncoproteins to become immortal and even then the frequency of immortalization is 10
−6
to 10
−7
. Shay and Wright, 184
Exp. Cell Res.
109, 1989. A variety of hypotheses have been advanced over the years to explain the causes of cellular senescence. While examples of such hypotheses are provided below, there appears to be no consensus or universally accepted hypothesis.
For example, the free radical theory of aging suggests that free radical-mediated damage to DNA and other macromolecules is causative in critical loss of cell function (Harmon, 11
J. Gerontol.
298, 1956; Harmon, 16
J. Gerontol.
247, 1961). Harman says (Harman, 78
Proc. Natl. Acad. Sci.
7124, 1981) “aging is largely due to free radical reaction damage . . . .”
Waste-product accumulation theories propose that the progressive accumulation of pigmented inclusion bodies (frequently referred to as lipofuscin) in aging cells gradually interferes with normal cell function (Strehler, 1
Adv. Geront. Res.
343, 1964; Bourne, 40
Prog. Brain Res.
187, 1973; Hayflick, 20
Exp. Gerontol.
145, 1985).
The gradual somatic mutation theories propose that the progressive accumulation of genetic damage to somatic cells by radiation and other means impairs cell function and that without the genetic recombination that occurs, for instance, during meiosis in the germ line cells, somatic cells lack the ability to proliferate indefinitely (Burnet, “Intrinsic Mutagenesis—A Genetic Approach to Aging”, Wile, N.Y., 1976; Hayflick, 27
Exp. Gerontol.
363, 1992). Theories concerning genetically programmed senescence suggest that the expression of senescent-specific genes actively inhibit cell proliferation (Martin et al., 74
Am. J. Pathol.
137, 1974; Goldstein, 249
Science
1129, 1990).
Smith and Whitney, 207
Science
82, 1980, discuss a mechanism for cellular aging and state that their data is:
“compatible with the process of genetically controlled terminal differentiation . . . . The gradual decrease in proliferation potential would also be compatible with a continuous build up of damage or errors, a process that has been theorized. However, the wide variability in doubling potentials, especially in mitotic pairs, suggests an unequalled partitioning of damage or errors at division.”
Shay et al., 27
Experimental Gerontology
477, 1992, and 196
Exp. Cell Res.
33, 1991 describe a two-stage model for human cell mortality to explain the ability of Simian Virus 40 T-antigen to immortalize human cells. The mortality stage 1 mechanism (M1) is the target of certain tumor virus proteins, and an independent mortality stage 2 mechanism (M2) produces crisis and prevents these tumor viruses from directly immortalizing human cells. The authors utilized T-antigen driven by a mouse mammary tumor virus promoter to cause reversible immortalization of cells. The Simian Virus 40 T-antigen is said to extend the replicative life span of human fibroblast by an additional 40-60%. The authors postulate that the M1 mechanism is overcome by T-antigen binding to various cellular proteins, or inducing new activities to repress the M1 mortality mechanism. The M2 mechanism then causes cessation of proliferation, even though the M1 mechanism is blocked. Immortality is achieved only when the M2 mortality mechanism is also disrupted.
It has also been proposed that the finite replicative capacity of cells may reflect the work of a “clock” liked to DNA synthesis in the telomere (end part) of the chromosomes. Olovnikov, 41
J. Theoretical Biology
181, 1973, describes the theory of marginotomy to explain the limitations of cell doubling potential in somatic cells. He states that an:
“informative oligonucleotide, built into DNA after a telogene and controlling synthesis of a repressor of differentiation, might serve as a means of counting mitosis performed in the course of morphogenesis. Marginotomic elimination of such an oligonucleotide would present an appropriate signal for the beginning of further differentiation. Lengthening of the telogene would increase the number of possible mitoses in differentiation.”
Harley et al., 345
Nature
458, 1990, state that the amount and length of telomeric DNA in human fibroblasts decreases as a function of serial passage during aging in vitro, and possibly in vivo, but do not know whether this loss of DNA has a causal role in senescence. They also state:
“Tumour cells are also characterized by shortened telomeres and increased frequency of aneuploidy, including telomeric associations. If loss of telomeric DNA ultimately causes cell-cycle arrest in normal cells, the final steps in this process may be blocked in immortalized cells. Whereas normal cells with relatively long telomeres and a senescent phenotype may contain little or no telomerase activity, tumour cells with short telomeres may have significant telomerase activity. Telomerase may therefore be an effective target for anti-tumour drugs.
There are a number of possible mechanisms for loss of telomeric DNA during ageing, including incomplete replication, degradation of termini (specific or nonspecific), and unequal recombination coupled to selection of cells with shorter telomeres. Two features of our data are relevant to this question. First, the decrease in mean telomere length is about 50 bp per mean population doubling and, second, the distribution does not change substantially with growth state or cell arrest. These data are most easily explained by incomplete copying of the template strands at their 3′ termini. But the absence of detailed information about the mode of replication or degree of recombination at telomeres means that none of these mechanisms can be ruled out. Further research is required to determine the mechanism of telomere shortening in human fibroblasts and its significance to cellular senescence.” [Citations omitted.]
Hastie et al., 346
Nature
866, 1990, while discussing colon tumor cells, state that:
“[T]here is a reduction in the length of telomere repeat arrays relative to the normal colonic mucosa from the same patient.
Firm figures are not available, but it is likely that the tissues of a deve

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