Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1996-11-15
2001-03-20
Hutzell, Paula K. (Department: 1642)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S018000, C435S023000, C435S025000
Reexamination Certificate
active
06204001
ABSTRACT:
BACKGROUND OF THE INVENTION
Throughout this application, various reference are referred to by abbreviation. Disclosures of these publication in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains. Full bibliographic citations for these references may be found at the end of this application, preceding the claim.
A model system is described that allows an analysis of the molecular and biochemical changes associated with expression and suppression of the oncogenic and metastatic phenotype of cloned rat embryo fibroblast (CREF) cells. Ha-ras-transformed CREF cells are morphologically transformed, anchorage-independent and both tumorigenic and metastatic in thymic nude mice and syngeneic Fischer rats. Coexpression of the Ha-ras oncogene and Krev-1 tumor suppressor gene in CREF cells results in suppression of in vitro transformation. In contrast, Ha-ras/Krev-1 transformed CREF cells retain, with greatly extended latency periods, both tumorigenic and metastatic capabilities in thymic nude mice. The present study investigates changes in the Ha-ras suppressor gene, rrg (lysyl oxidase), during expression and suppression of the oncogenic phenotype in CREF cells. Nontumorigenic CREF cells and CREF cells transformed by the Ha-ras and Krev-1 gene that express a suppression in in vitro transformation contain elevated levels of lysyl oxidase mRNA and protein. In contrast, Ha-ras and Ha-ras/Krev-1 nude mouse tumor- and nude mouse lung metastasis-derived CREF cells contain reduced levels of lysyl oxidase mRNA and protein. Nuclear run-on assays indicate that suppression of lysyl oxidase expression in transformed subclones of CREF cells correlates with a reduction in transcription of the lysyl oxidase gene. Taken together, the current studies support a transcriptional switching model in which lysyl oxidase expression correlates directly with suppression of the Ha-ras-induced transformation phenotype and escape from oncogenic suppression correlates with a transcriptional silencing of the lysyl oxidase gene and decreased lysyl oxidase mRNA and protein.
Cancer is a multistep process involving a complex interplay between genes that promote the cancer phenotype (oncogenes) and genes that normally function as inhibitors of oncogenesis (tumor suppressor genes) (1-4). A genetic change seen in a high percentage of human cancers is the mutational activation of the cellular ras gene (5,6). The ras gene encodes a 21,000 M
r
(ras p21) GTP-binding protein with intrinsic GTPase activity (5-7). In its cellular and mutated forms, ras p21 impinges on a number of signal transduction pathways resulting in growth stimulation, transformation and differentiation (5-9). Over expression of the mutant Ha-ras or Ki-ras oncogene in rodent fibroblasts results in morphological transformation and acquisition of oncogenic potential (5-9). By transfecting a human expression vector cDNA library into a v-Ki-ras-transformed NIH 3T3 cell line (DT), Noda et al (10,11] identified a gene, Krev-1, capable of suppressing the ras-induced transformation phenotype in DT cells. Krev-1 encodes a p21 protein with approximately 50% homology to the ras p21 oncogene-encoded gene product (11). Prominent areas of similarity between Krev-1 p21 and ras p21 are in the functional domains of these proteins, including regions involved in GTP binding and GTPase activity, the isoprenylation signal sequence and the ras/GTPase-activating protein (GAP) effector binding domain (11). Studies using chimeric ras-Krev-1 genes suggest that Krev-1 suppresses ras-induced transformation by directly binding with and sequestering ras p21 effector target molecules necessary for cellular transformation (12-14).
Transfection of a Ha-ras oncogene into cloned rat embryo fibroblast (CREF) cells results in morphological transformation, anchorage-independence and acquisition of tumorigenic and metastatic potential (15,16). Ha-ras-transformed CREF cells exhibit major changes in the transcription and steady-state levels of genes involved in suppression and induction of oncogenesis (15). These include: a reduction in the level of expression of nm23 (a putative metastasis suppressing gene) and TIMP-1 (tissue inhibitor of metalloproteinase-1); and an increase in the levels of cripto, 92-kDa gelatinase/type IV collagenase (92-kDa GEL), osteopontin (OPN) and transin/stomelysin (15). Simultaneous overexpression of Krev-1 in Ha-ras-transformed CREF cells results in morphological reversion, suppression of agar growth capacity and a delay in in vivo oncogenesis (15,16). Reversion of transformation in Ha-ras/Krev-1-transformed CREF cells correlates with a reversion in the transcriptional and steady-state mRNA profile to that of nontransformed CREF cells (15). Following long latency times, Ha-ras/Krev-1 transformed CREF cells form both tumors and metastases in a thymic nude mice (15,16). In the present study, applicants have determined the level of expression of the ras recision gene (rrg, which is lysyl oxidase) (17-19) as a function of expression and suppression of the transformed and oncogenic phenotype. Applicants demonstrate that lysyl oxidase expression (mRNA and protein levels) correlates with suppression of the oncogenic phenotype. As tumors form following escape from Krev-1 induced suppression, both lysyl oxidase and TIMP-1 expression are transcriptionally extinguished. When metastases develop from Krev-1 suppressed CREF cells, both lysyl oxidase and TIMP-1 expression remain suppressed and transcription of the 92-kDa GEL and transin is initiated. These results indicate that transcriptional switching, involving extinction of specific suppressor genes and induction of specific cancer promoting genes, occurs in a defined sequence during expression, suppression and escape from suppression of transformation and oncogenesis.
SUMMARY OF THE INVENTION
This invention provides a method for determining whether a compound is capable of suppressing ras functions comprising: (a) contacting an effective amount of the compound with Ha-ras transformed cloned rat embryo fibroblast cells under conditions permitting the compound to suppress the ras functions in the cells; and (b) determining the expression of lysyl oxidase, the expression of lysyl oxidase indicating that the compound is capable of suppressing the ras function.
This invention also provides a method for determining whether a compound is capable of suppressing the ras functions comprising:(a) contacting an effective amount of the compound with Ha-ras transformed cloned rat embryo fibroblast cells under conditions permitting the compound to suppress the ras functions in the cells; and (b) determining the expression of TIMP-1, the expression of TIMP-1 indicating that the compound is capable of suppressing the ras functions.
This invention also provides a method for determining whether a compound is capable of suppressing the ras functions comprising: (a) contacting an effective amount of the compound with Ha-ras transformed cloned rat embryo fibroblast cells under conditions permitting the compound to suppress the ras functions in the cells; and (b) determining the expression of 92-kDa gelatinase, the inhibition of the expression of 92-kDa gelatinase indicating that the compound is capable of suppressing the ras functions.
This invention also provides a method for determining whether a compound is capable of suppressing ras functions comprising:(a) contacting an effective amount of the compound with Ha-ras transformed cloned rat embryo fibroblast cells under conditions permitting the compound to suppress the ras functions in the cells; and (b) determining the expression of transin, the inhibition of the expression of transin indicating that the compound is capable of suppressing the ras functions.
In an embodiment, the tested compounds were not previously known. This invention also provides compounds which are determined to be capable of suppressing the ras functions by the above methods.
This invention also provides a pharmaceutical
Fisher Paul B.
Su Zao-zhong
Bansal Geetha P.
Cooper & Dunham LLP
Hutzell Paula K.
The Trustees of Columbia University in the City of New York
White John P.
LandOfFree
Methods for determining compounds capable of inhibiting the... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Methods for determining compounds capable of inhibiting the..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Methods for determining compounds capable of inhibiting the... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2508378