Regulation of the cell cycle by sterols

Details Regulation of the cell cycle by sterols Regulation of the cell cycle by sterols

1999-03-08

2001-07-17

Caputa, Anthony C. (Department: 1642)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

C435S007100, C435S007200, C435S007230, C436S064000, C536S023500, C536S024100

Reexamination Certificate

active

06261760

ABSTRACT:

FIELD OF THE INVENTION
This invention relates the discovery that an increase in cellular free cholesterol is a requirement for cell division. The cellular free cholesterol concentration is regulated by ATPase-mediated selective uptake from low density lipoproteins (LDLs) and by caveolin-mediated efflux. These two processes thus provide good targets for screening of compounds that exert anti-mitotic activity through modulation of cellular free cholesterol.
BACKGROUND OF THE INVENTION
The cell division cycle is one of the most fundamental processes in biology which, in multicellular organisms, ensures the controlled generation of cells with specialized functions. Under normal growth conditions, cell proliferation is tightly regulated in response to diverse intra- and extracellular signals. This is achieved by a complex network of proto-oncogenes and tumor-suppressor genes that are components of various signal transduction pathways.
Numerous diseases are characterized by abnormal cell mitosis. For example, uncontrolled cell mitosis is a hallmark of cancer. In this instance, activation of a proto-oncogene(s) and/or a loss of a minor suppressor gene(s) can lead to the unregulated activity of the cell cycle machinery. This leads to unregulated cell proliferation and to the accumulation of genetic errors which ultimately will result in the development of cancer (Pardee (1989)
Science
246: 603-608).
Many cancers are also classified as angiogenesis dependent diseases ((i.e., those diseases which require or induce vascular growth). In addition to unregulated growth of the tumor body itself, cancers are typically characterized by the ingrowth of vasculature which provide various factors that permit continued tumor growth. Thus, both the vascular tissue as well as the cancer itself have proven to be attractive targets for anti-mitotic agents in the treatments of various neoplasias.
In addition, unregulated cell mitosis is a hallmark of a number of other pathological conditions. For example, cell mitosis is important for the normal development of the embryo, formation of the corpus luteum, wound healing, inflammatory and immune responses, and, as indicated above, angiogenesis and angiogenesis related diseases.
Consequently the use of anti-mitotic compounds to regulate cell proliferation and thereby introduce control over disease states characterized by unregulated mitotic activity has sparked considerable interest.
Cell mitosis is a multi-step process that includes cell division and replication (Alberts, et al. (1989)
In The Cell,
pp. 652-661; Stryer (1988)
Biochemistry
). Mitosis is characterized by the intracellular movement and segregation of organelles, including mitotic spindles and chromosomes. Microtubule formation is important for cell mitosis, cell locomotion, and the movement of highly specialized cell structures such as cilia and flagella.
As microtubules and microtubule-related structures are intimately involved in the mitotic process, they have provided a convenient target for putative anti-mitotic compounds. Indeed, microtubules have proven to be extremely labile structures that are sensitive to a variety of chemically unrelated anti-mitotic drugs. For example, colchicine and nocadazole are anti-mitotic drugs that bind tubulin and inhibit tubulin polymerization (Stryer (1988)
Biochemistry
). When used alone or in combination with other therapeutic drugs, colchicine has been used to treat cancer (WO9303729; J03240726-A), alter neuromuscular function, change blood pressure, increase sensitivity to compounds affecting sympathetic neuron function, depress respiration, and relieve gout (
Physician's Desk Reference,
(1993) 47: 1487).
Estradiol and estradiol metabolites such as 2-methoxyestradiol have also been reported to inhibit cell division and in particular, tubulin polymerization (Seegers et al. (1989)
J. Steroid Biochem.
32: 797-809; Lottering et al. (1992)
Cancer Res.
52: 5926-5923; Spicer et al. (1989)
Mol. and Cell. Endo.
64: 119-126; Rao et al. (1967)
J. Exp. Cell Res.
48: 71-81). However, the activity is variable and depends on a number of in vitro conditions. For example, estradiol inhibits cell division and tubulin polymerization in some in vitro settings (Spicer et al. (1989)
Mol. and Cell. Endo.
64: 119-126; Ravindra (1983)
J. Indian Sci.
64(c)), but not in others (Lottering et al. (1983)
Cancer Res.
52: 5926-5923; Ravindra, (1983)
J. Indian Sci.
64(c)).
Of most widespread and recent interest, however, are compounds with paclitaxel-like activity. These include, but are not limited to paclitaxel and paclitaxel derivatives and analogues (see, e.g., U.S. Pat. Nos: 5,569,729; 5,565,478; 5,530,020; 5,527,924; 5,508,447; 5,489,589; 5,488,116; 5,484,809; 5,478,854; 5,478,736; 5,475,120; 5,468,769; 5,461,169; 5,440,057; 5,422,364; 5,411,984; 5,405,972; and 5,296,506). Paclitaxel and its derivatives are compounds that inhibit eukaryotic cell replication by enhancing polymerization of tubulin moieties into stabilized microtubule bundles that are unable to reorganize into the proper structures for mitosis (Id.). Paclitaxel and paclitaxel-like compounds have shown significant efficacy.
While the above-identified and other anti-mitotic agents have proven useful in a variety of clinical settings, these agents operate through essentially a single modality; interference with the formation or operation of the mitotic apparatus (e.g., mitotic spindle). One would expect, however, that such compounds may show increased efficacy when coupled with anti-mitotic compounds that act through one or more different modalities.
SUMMARY OF THE INVENTION
This invention provides new methods of inhibiting the growth and/or proliferation of cells, more particularly the growth and proliferation of cancer cells, other transformed cells, or cells at other sites, such as in aortic transplant subject to restenosis. The methods of this invention are premised, in part, on the discovery that for cell division (mitosis) to proceed beyond S-phase, a doubling of cell cholesterol content must be initiated and completed. It was a discovery of this invention that this increase is dependent upon both upregulation of ATPase-dependent uptake of free cholesterol (FC) from low density lipoprotein (selective FC uptake) and the down-regulation of caveolin expression associated with a decrease in efflux. These steps represent the two termini of a novel free cholesterol (FC) recycling pathway described herein.
It was a discovery of this invention that cell division can be inhibited when free cholesterol accumulation is prevented. This can be achieved either by inhibiting free cholesterol uptake with inhibitors of cell surface H
+
-ATPase or by promoting free cholesterol (FC) efflux via caveolin overexpression. This pathway described herein thus offers two novel sites to regulate cell proliferation.
Thus, in one embodiment, this invention provides methods of identifying an anti-mitotic agent. The methods involve contacting a cell with an agent to be tested for anti-mitotic activity, and detecting the efflux of free cholesterol from said cell. An increase in efflux of free cholesterol by from the cell when contacted by said agent as compared to the efflux from the cell under the same conditions lacking the agent (e.g., a statistically significant increase, preferably at least a 10% increase, more preferably at least a 25% increase, and most preferably at least a 50%, 100% or even at least a 200% increase) indicates that the agent is has antimitotic activity (or that the agent is likely to have antimitotic activity and thus is a good candidate for further evaluation (i.e., is a good lead candidate). The assay can involve detecting the efflux of labeled (e.g., [
3
H]-labeled) free cholesterol. In addition to, and/or in alternative to this method, the detection can involve detection of the expression of a reporter gene operably linked (under the control of) a caveolin promoter. Virtually any reporter gene is suitable, however, in a preferred embodiment, the reporter gene is selected fr

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