Use of oligonucleotide telomerase inhibitors to reduce...

Details Use of oligonucleotide telomerase inhibitors to reduce... Use of oligonucleotide telomerase inhibitors to reduce...

1997-12-24

2001-02-27

Guzo, David (Department: 1635)

Chemistry: molecular biology and microbiology

Animal cell, per se ; composition thereof; process of...

Method of regulating cell metabolism or physiology

C514S04400A, C536S024310

Reexamination Certificate

active

06194206

ABSTRACT:

This invention relates to methods for therapy and diagnosis of cellular senescence and imnortalization.
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 aging, and theories or hypothesis which explain such aging and the mechanisms by which cells escape senescence and immortalize.
The finite replicative capacity of normal human cells, e.g., fibroblasts, is characterized by a cessation of proliferation in spite of the presence of serum growth factors. This cessation of replication after a maximum of 50 to 100 population doublings in vitro is referred to as cellular senescence. 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 Requirement”, 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), and is therefore suggested to reflect in vivo aging on a cellular level.
Cellular immortalization (unlimited life span) 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 replication 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 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, a free radical theory 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), somatic mutation theories propose that without genetic recombination cells lack the ability to proliferate indefinitely due to a progressive loss of genetic information (Burnet, “Intrinsic Mutagenesis—A Genetic Approach to Aging”, Wile, N.Y., 1976; Hayflick, 27
Exp. Gerontol.
363, 1992), and theories concerning genetically programmed senescence suggest that the expression of senescent-specific genes actively inhibit cell proliferation perhaps under the direction of a mitotic clock (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.
Olovnikov, 41
J. Trheoretical 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 pheno type 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 developed fetus result from 20-50 cell divisions, whereas several hundred or thousands of divisions have produced the colonic mucosa and blood cells of 60-year old individuals. Thus the degree of telomere reduction is more or less proportional to the number of cell divisions. It has been shown that the ends of Drosophila chromosomes without normal telomeres reduce in size by ~4 base pairs (bp) per cell division and that the ends of yeast chromosomes reduce by a similar degree in a mutant presumed to lack telomerase function. If we assume the same rate of reduction is occurring during somatic division in human tissues, then a reduction in TRA by 14 kb would mean that 3,500 ancestral cell divisions lead to the production of cells in the blood of a 60-year old individual; using estimates of sperm telomere length found elsewhere we obtain a value of 1,000-2,000. These values compare favourably with those postulated for mouse blood cells. Thus, we propose that telomerase is indeed lacking in somatic tissues. In this regard it is of interest to note that in maize, broken chromosomes are only healed in sporophytic (zygotic) tissues and not in endosperm (terminally differentiated), suggesting that telomerase activity is lacking in the differentiated tissues. [Citations omitted.]
The authors propose that in some tumors telomerase is reactivated, as proposed for HeLa cells in culture, which are known to contain telomerase activity. But, they state:
One alternative explanation for our observations is that in tumours the cells with shorter telomeres have a growth advantage over those with larger telomeres, a situation described for vegetative cells of tetrahymena. [Citations omitted.]
Harley, 256
Mutation Research
271, 1991, discusses observations allegedly showing that telomeres of human somatic cells act as a mitotic clock shortening with age both in vitro and in vivo in a repli

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