Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Reexamination Certificate
2000-04-18
2003-05-20
Kunz, Gary (Department: 1647)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
C435S325000, C435S375000, C530S300000, C530S350000
Reexamination Certificate
active
06566047
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to methods for identifying and using compounds that inhibit cancer cell metastasis or promote neurite growth and regeneration.
BACKGROUND OF THE INVENTION
Amoeboid locomotion requires a quasi-metastable state of the adhesion sites, i.e., of the interactions between (i) the cell's contractile apparatus, (ii) adhesion molecules and other plasma membrane components, and (iii) the growth substratum.
FIG. 1
plots in principle the relationship between the strength of cell adhesion to the substratum and cell motility. At the extreme ends of the curve, locomotion is reduced to zero. At these ends, the cell is either too tightly adherent, or it is so loosely attached that force generation against the substratum is impossible. Optimum motility requires an intermediate, dynamic state that facilitates the making and breaking of adhesions as the cell moves. In other words, a factor that increases motility would not be expected to increase or decrease attachment but to facilitate assembly and disassembly of adhesion sites. Knowledge of the attachment mechanism has increased significantly in recent years, but knowledge about detachment has been rudimentary until recently. Both processes must be elucidated to understand the motility of metastatic cancer cells, and the influence of motility factors.
For example, chemorepellents provide important guidance cues for growth cones during nervous system development. These repellents cause developing neurites to change their course of outgrowth away from the repellent source and, thus, play a critical role in pathfinding for growing or regenerating nerves. However, the mechanisms of action of the repellents are not well understood.
The establishment of metastases is a complex, multi-step phenomenon that begins with dissociation of cancer cells from the primary tumor and invasion of surrounding tissues. Although understanding of the metastatic process is incomplete, at least four elements (in addition to continued cell proliferation) have been identified in recent years: (i) changes in cell adhesion molecules, (ii) secretion/surface expression of proteases, (iii) increased cell motility, and (iv) vascularization of primary and secondary tumors. In addition, factors that promote metastatic progression include genetic instability and defects in cell-cell signaling. For example, loss of the NDP kinase-like protein encoded by nm23, a putative metastasis-suppressor gene, seems to result in altered signaling responsiveness, e.g., in motility assays described in MacDonald et al.,
J. Biol. Chem
. 268:25780-25789 (1993) and Kantor et al.,
Cancer Res
. 53:1971-1973 (1993). Motility has been correlated with metastatic potential as reported in Guirguis et al.,
Nature
, 329:261-263 (1987); Partin et al.,
Cancer Res
, 48:6050-6053 (1988); Partin et al.,
Proc Natl Acad Sci. USA
, 86:1254-1258 (1989); Mohler, supra.; and Stearns & Steams,
Cancer Metastasis Rev
. 12:39-52 (1993).
A growing number of factors contributing to metastastic progression are being identified. The recently identified KAI 1 gene encodes a putative cell adhesion molecule whose expression reduces prostate carcinoma cell motility and metastasis. However, a universal prognostic marker of prostate carcinoma has not been identified to date. Therefore, a thorough understanding of the mechanisms that trigger invasive cancer cell behavior is particularly important for prostate carcinoma.
Vertebrate amoeboid cell systems, such as polymorphonuclear leukocytes, platelets and the nerve growth cone (the pseudopodal, enlarged leading edge of the growing nerve fiber) have been studied in some detail. Pseudopods of locomoting cells are filled with actin microfilaments, and there is considerable knowledge of the components involved in the regulation of polymerization and of force generation in the actin-based cytoskeleton. At so-called focal adhesion sites, the cytoskeleton interacts with the plasma membrane and, via adhesion molecules, with the extracellular matrix or adhesion molecules on neighboring cells. To make locomotion possible, attachment of adhesion molecules to the growth substratum has to be regulated coordinately with the binding of these adhesion molecules, via linker proteins, to the actin cytoskeleton. Numerous proteins are involved in the intracellular interactions. They include, among others, talin, vinculin, Src family non-receptor tyrosine kinases, focal adhesion kinase, certain types of protein kinase C, and the protein kinase substrate, myristoylated alanine-rich C-kinase substrate (MARCKS) (Burridge et al.,
Ann Rev Cell Biol
. 4:487-525 (1988); Jaken et al.,
J. Cell Biol
., 109:697-704 (1989); Luna and Hitt,
Science
258:955-964 (1992); Blackshear,
J. Biol. Chem
., 268:1501-1504 (1993); Schaller and Parsons,
Trends Cell Biol
. 3:258-262 (1993)).
There are several classes of adhesion molecules. At least in the case of integrins and cadherins, there is evidence that they function not only as adhesion molecules, but also as receptors that signal ligand binding across the membrane. Conversely, external ligand affinity can be modulated by integrin or cadherin phosphorylation on the inside of the cell. Outside-in signaling triggers focal adhesion assembly by a process known to require tyrosine kinase activity. In summary, adhesion sites are distinctive cell organelles comprised of protein assemblies that regulate cell attachment and play an important role in amoeboid motility.
A variety of amoeboid systems, including macrophages, platelets and nerve growth cones, exhibit high activity of cytosolic phospholipase A
2
(cPLA
2
) and generate high levels of cytosolic arachidonic acid (AA). In platelets, cPLA
2
is activated via the thrombin receptor. Also, ras-transfected cancer cells with increased motility exhibit increased cPLA
2
activity. This suggests a role for cPLA
2
and its product, AA, in the regulation of cell motility. The eicosanoid, 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE) has long been known to affect leukocyte motility and has been implicated in cancer cell attachment. 12-lipoxygenase (12-LOX), is the enzyme that converts AA into 12-hydroperoxyeicosatetraenoic acid, which is reduced spontaneously to 12(S)-HETE. A correlation between metastatic potential and expression levels of 12-LOX has been reported in Honn et al.,
Cancer Metastasis Rev
. 13:365-396 (1994). These observations also implicate AA and HETEs in the regulation of cell attachment and/or motility.
There is considerable interest in eicosanoids as they relate to the prostate because unsaturated fatty acids inhibit steroid 5&agr; reductase and lowered AA levels have been correlated with increased malignancy of prostate carcinoma cells. In addition, 12-LOX is elevated in advanced-stage human prostate carcinoma.
Results obtained by other laboratories and discussed above were generated in isolation and never synthesized in the manner described herein. Furthermore, the signaling pathway mediating cellular shape and motility responses to thrombin had not been elucidated. In fact, prior to the present invention, functional assays were performed in vivo or in culture with intact cells and monitored a combination of cellular behaviors such as cell adhesion, detachment and motile behavior.
Accordingly, a need exists for assays that can quickly and selectively identify agents that modulate cell adhesion and detachment. The present invention satisfies this need and provides related advantages as well.
SUMMARY OF THE INVENTION
The present invention generally relates to methods of identifying agents that modulate the motility of cells. In one aspect, the methods are accomplished by (a) attaching pseudopods, preferably cancer cells or neurite growth cones, on a substratum, (b) exposing the attached pseudopods to a putative agent, and (c) determining the effect of the putative agent on the pseudopods, wherein a significant change in pseudopod attachment indicates the putative agent is an effective repellent agent. The pseudopods arc preferably attached to the
De La Houssaye Becky
Drabkin Harry
Helmke Steve
Mikule Keith
Pfenninger Karl
Bunner Bridget E.
Kunz Gary
The Regents of the University of Colorado
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