Antisense modulation of human Rho family gene expression

Details Antisense modulation of human Rho family gene expression Antisense modulation of human Rho family gene expression

1999-08-31

2002-06-25

LeGuyader, John L. (Department: 1635)

Chemistry: molecular biology and microbiology

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

Method of regulating cell metabolism or physiology

C435S091100, C435S006120, C435S325000, C435S366000, C536S023100, C536S024310, C536S024500

Reexamination Certificate

active

06410323

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to compositions and methods for modulating expression of members of the human Rho gene family, which encode low molecular weight GTPases that act as molecular switches in signal transduction. This invention is also directed to methods for inhibiting hyperproliferation of cells; these methods can be used diagnostically or therapeutically. Furthermore, this invention is directed to treatment of conditions associated with expression of the human Rho family member genes.
BACKGROUND OF THE INVENTION
The Rho family of genes are a sub-family of low molecular weight GTPases and are related to each other based on sequence homology and function (Vojtek, A. B., and Cooper, J. A.,
Cell
1995, 82, 527-529). Other sub-families include Ras, Rab, Arf, and Ran. As GTPases, these proteins bind and hydrolyze GTP. In an active state, they bind to GTP and transduce signals of other proteins in signal transduction pathways. In their inactive state, they are bound to GDP. Members of the Rho family are typically involved in regulation of the actin cytoskeleton. Members of the Rho family include RhoA, RhoB, RhoC, RhoD, RhoE, RhoG, Rac1, Rac2, Rac3 and Cdc42.
Each class appears to have a unique function in actin reorganization. Rho has been shown to be essential for the formation of stress fibers and focal adhesions (Ridley, A. J. and Hall, A.,
Cell
1992, 70, 389-399). Focal adhesions are an area of the cell where integrin receptors cluster and extracellular matrix proteins such as fibronectin and collagen are bound. Stress fibers attach at these focal adhesions within a cell. Rac has been shown to be essential for the formation of membrane ruffles, which results from the formation of large vesicles within the cell (Ridley, A. J., et al.,
Cell
1992, 70, 401-410). Cdc42 (also known as Cdc42Hs and G25K) regulates the formation of filopodia, short bundles of actin filaments that protrude from a cell (Nobes, C. D. and Hall, A.,
Cell
1995, 81, 53-62). Such activities on cell morphology may play an important role in cell motility, cytokinesis, and endocytosis.
Additional functions for the Rho family have begun to be elucidated. Rac and Rho have been found to promote cadherin-based cell-cell adhesion (Takaishi, K., et al.,
J. Cell Biol.
1997, 139, 1047-1059). Rac1 and Cdc42 play a critical role in the c-jun amino-terminal kinase (JNK)/stress-activated protein kinase (SAPK) signaling pathway, thereby, potentially having an important role in gene transcription (Coso, O. A. et al.,
Cell
1995, 81, 1137-1146). RhoA, Rac1 and Cdc42 also regulate transcription through JNK-independent pathways by binding to either serum response factor (SRF; Hill, C. S., et al.,
Cell
1995, 81, 1159-1170) or NF-&kgr;B (Perona, R., et al.,
Genes and Develop.
1997, 11, 463-475).
Members of the Rac subfamily have also been found to regulate oxygen radical production. Both Rac
1
(Sundaresan, M., et al.,
Biochem. J.
1996, 318, 379-382) and Rac2 (Knaus, U. G., et al.,
Science
1991, 254, 1512-1515) are involved in this process.
Members of the Rho family are thought to be involved in various disease processes, including cancer. Rho, Rac and Cdc42 all play a role in Ras transformation. Rac was found to essential for transformation by Ras, but not RafCAAX, a modified Raf kinase with a localization signal from K-ras (Qiu, R.-G., et al.,
Nature
1995 374, 457-459). Rho is not essential for Ras transformation, but acts cooperatively in transformation by Ras and RafCAAX (Qiu, R.-G., et al.,
Proc. Natl. Acad. Sci. USA
1995, 92, 11781-11785). Cdc42 was also found to be essential for Ras transformation, but its role is distinct from that of Rac (Qiu, R.-G., et al.,
Mol. Cell Biol.
1997, 17, 3449-3458). In addition to transformation, members to of the Rho family may also play a role in invasion and metastasis. Michiels, F. et al. (
Nature
1995, 375, 338-340) demonstrated that T-lymphoma cells that constitutively expressed Rac1 became invasive. Yoshioka, K. et al. (
J. Biol. Chem.
1998, 273, 5146-5154) found that cells stably transfected with RhoA were also invasive. The RhoB gene has been classified as an immediate-early gene, which means that its transcription is rapidly activated upon exposure to certain growth factors or mitogens. The factors shown to activate RhoD transcription include epidermal growth factor (EGF), platelet-derived growth factor (PDGF), genotoxic stress from UV light, alkylating xenobiotics and the retroviral oncogene v-fps. Each of these stimuli triggers DNA synthesis in cultures of high cell density (Engel et al.,
J. Biol. Chem.,
1998, 273, 9921-9926). The response of RhoB to these factors implies a role for RhoB in wound repair and tissue regeneration upon growth factor stimulation and tumorigenesis upon mitogen stimulation.
The involvement of Rho family proteins in ras-mediated transformation and tumor cell invasion suggests that they could be novel targets for cancer treatment (Ridley, A. J.,
Int. J. Biochem. Cell Biol.
1997, 29, 1225-1229). In particular, overexpression of the RhoC gene has been associated with pancreatic cancer. Suwa, H. et al. (
Br. J. Cancer,
1998, 77, 147-152) looked for a role of RhoA, RhoB and RhoC genes in ductal adenocarcinoma of the pancreas. They found that expression levels of RhoC were higher in tumors than in normal tissue and that metastatic tumors expressed RhoC at higher levels than primary tumors. Rho C expression is also elevated in a megakaryocytic leukemia cell line, CMK. Takada et al.,
Exp. Hematol.,
1996, 24, 524-530. Manifestations of altered RhoB regulation also appear in disease states, including the development of cancer. Cellular transformation and acquisition of the metastatic phenotype are the two main changes normal cells undergo during the progression to cancer. Expression of constitutively activated forms of RhoB have been shown to cause tumorigenic transformation of NIH 3T3 and Rat1 rodent fibroblasts (Khosravi-Far et al.,
Adv. Cancer Res.,
1998, 72, 57-107). RhoB has also been shown to be overexpressed in human breast cancer tissues (Zalcman et al.,
Oncogene,
1995, 10, 1935-1945). RhoA is also believed to be involved in the development of cancer. Cellular transformation and acquisition of the metastatic phenotype are the two main changes normal cells undergo during the progression to cancer. Recent studies demonstrate that RhoA-regulated pathways can induce both changes in cells. Injecting cells transformed with rhoA genes directly into the bloodstream of mice produced metastasis, or tumor growth, in distant organs (del Peso et al.,
Oncogene,
1997, 15, 3047-3057).
It has also been suggested that inhibition of Rac genes may be useful for preventing reoxygenation injury as it occurs when ischemic cells undergo reperfusion (Kim, K.-S., et al.,
J. Clin. Invest.
1998, 101, 1821-826). With reoxygenation, reactive oxygen species are presented to the cell, greatly augmenting cell death. Kim, K.-S., et al. showed that adenoviral-mediated transfer of a dominant negative Rac1 could inhibit the formation of reactive oxygen species and protect cells against hypoxia/reoxygenation-induced cell death. They suggest that inhibition of rac
1
would be useful, clinically, in treatment in cases where there is the possibility of reperfusion injury.
Manifestations of altered RhoA regulation also appear in both injury and disease states. It has been proposed that acute central nervous system trauma may contribute to the development of Alzheimer's disease. Findings that show a high concentration of thrombin, a serine-protease in the blood clotting cascade, localized to the plaques of Alzheimer's disease brains support this claim. An excess of thrombin has been shown to stimulate Rho A activity with a concomitant increase in apoptosis (programmed cell death) (Donovan et al.,
J. Neurosci.,
1997, 17, 5316-5326). These studies also imply a role for RhoA in wound repair and clotting disorders.
Although members of the Rho family have been implicated in various disease processes including cancer and reoxyge

Affiliated with

Also associated with

Rating

Antisense modulation of human Rho family gene expression does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Antisense modulation of human Rho family gene expression, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Antisense modulation of human Rho family gene expression will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2918022