Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1997-11-14
2001-10-09
Huff, Sheela (Department: 1642)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S004000, C435S007210, C435S007230, C435S030000, C435S320100, C424S130100, C424S133100, C424S134100, C436S536000, C530S350000, C530S380000, C530S385000, C530S386000, C530S387100, C530S389100
Reexamination Certificate
active
06300081
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a method for detecting activated ras proteins.
BACKGROUND OF THE INTENTION
Harvey, Kirsten and N ras proteins (termed H-ras, K-ras, and N-ras, respectively) are immunologically related proteins and are collectively termed p21. They are products of the ras family of cellular genes which are found in a wide variety of nucleated mammalian cells. The ras genes appear to be frequent targets of genetic alterations that can lead normal cells along the pathway to malignancy. Ras oncogenes have been identified in a wide array of premalignant and malignant cells.
The p21 proteins consist of about 188-189 amino acids having a molecular weight of about 21,000 daltons. Viral and cellular ras genes encode membrane bound proteins (Willingham et al.,
Cell
19;1005 (1980)) which bind guanine nucleotides (Schlonick et al.,
PNAS
(
USA
) 76:5355 (1979); Papageorge et al.,
J. Virol
. 44:509 (1982); and Fine et al.,
Cell
37:151(1984)) and possess intrinsic GTPase activity (McGrath et al.,
Nature
301:644 (1984); Sweet et al.,
Nature
311:273 (1984); Gibbs et al.,
PNAS
(
USA
) 81:5704 (1984); and Manne et al.,
PNAS
82:376 (1985)).
DNA mediated transfection experiments using NIH3T3 cells as recipients have led to the identification of a family of activated transforming genes homologous to the ras genes of the H-ras and K-ras sarcoma viruses. A third member of the ras family designated N-ras has been identified but has not been found to have a retroviral counterpart. Activated (mutated) ras genes are structurally distinct from their normal homologs, having amino acid substitutions in the protein at positions 12, 13, or 61. (Tabin et al.,
Nature
300:143 (1982); Reddy et al.,
Nature
300:149 (1982); Bos et al.,
Nature
315:716 (1985); Yuasa et al.,
Nature
, 303:775-779 (1983); Der et al., cell 44:167-176 (Jan. 17, 1986)). Taparowsky et al.,
Banbury Report
, 14:123-133 (1983) cited in
Chem. Abstracts CA
100(1):1425n, teaches that the change at residue 12 from N-terminus of the H ras p21 from glycine to valine is sufficient to convert the normal protein to a transforming protein.
Shimizu et al.,
Nature
304:497-500 (1983) cited in
Chem. Abstracts
99(19):1530936, teaches the presence of a cysteine residue at amino acid 12 in the human lung cancer cell line calu-1 homolog of the v-Ki-ras gene. Fasano et al.,
J. Mol. Appl. Genet
., 2(2):173-180, cited in
Chem. Abstracts CA
99(19):153080v, teaches that the T24 H-ras-1 gene product is nearly identical to the v-H-ras p21 transforming protein encoded by Harvey sarcoma virus. Recent reports have shown the presence of activated ras p21 proteins in 40-50% of human colorectal cancers and preneoplastic lesions of the colon termed adenomas (Bos et al.,
Nature
327:293 (1987), Forrester et al.,
Nature
327:299 (1987) and Volgelstein et al.,
NEJM
319:525 (September 1988)). Recent studies have also shown expression of activated ras genes and mutated ras p21 proteins in 20-30% of lung carcinomas (Rodenhuis et al.,
Cancer Res
., 48:5738 (1988)) and over 90% of pancreatic carcinomas (Almoguera et al.,
Cell
53:549 (1988)). In certain forms of leukemia, such as acute myelogeneous leukemia and in certain preleukemic states, activated ras p21 proteins have been described.
These activated ras genes and mutated proteins have also been found in established cell lines as well as primary and metastatic tumors. Gambke et al.,
Nature
307:476, (1984), demonstrated a transforming N-ras gene in bone marrow cells from a patient aith acute myeloblastic leukemia (“AML”). In contrast, DNA from fibroblast cells from the same patient was not transforming.
The p21 ras protein in its normal nonactivated form contains the glycine amino acid at positions 12 and 13 and the glutamine amino acid at position 61. The p21 protein found in normal cells has the following primary amino acid structure for the amino acid sequence 5 to 19:
5
Lysine-leucine-valine-valine-valine-glycine-alanine-glycine-glycine-valine-glycine-lysine-serine-alanine-leucine
19
.
Ras proteins act as molecular switches relaying proliferative signals from cell surface receptors to the nucleus and cytoskeleton. Activation of these receptors leads to the activation of a guanines nucleotide exchange factor, which induces the exchange of guanine diphosphate (“GDP”) for guanine triphosphate (“GTP”). Specifically, the activation of Ras by the binding of GTP is required for the ability of many growth factors and cytokines to induce non-proliferating cells to enter G1 phase of the cell cycle. Activation of membrane-bound Ras by growth factor and cytokine receptors is generally achieved by the recruitment of Grb2-Sos complexes to the receptors themselves or to adaptor proteins such as Shc.
A primary target of activated Ras during growth factor stimulation is Raf, which is the first component of a protein kinase cascade that leads to activation of the MAP kinases Erk1 and Erk2 (Avruch et al., “Raf Meets Ras: Completing the Framework of a Signal Transduction Pathway,”
Trends Biochem. Sci
., 19:279-83 (1994)). The phosphorylation of transcription factors by these MAP kinases results in the expression of immediate early response genes, such as c-fos, that are required for early G1 progression. Although these signalling events occur within minutes of growth factor stimulation, microinjection of neutralizing anti-Ras antibodies in late G1 phase blocks progression of fibroblasts into S phase (Mulcahy, et al., “Requirement for Ras Proto-oncogene Function During Serum-Stimulated Growth of NIH 3T3 Cells,
Nature
, 313:214-43 (1985)). Furthermore, studies using combinations of cell cycle inhibitors and anti-Ras microinjection clearly demonstrate multiple points of Ras requirement in early and late G1 phase (Dobrowolski et al., “Cellular Ras Activity Is Required for Passage Through Multiple Points of the G-0-G-1 Phase in BALB-c 3T3 Cells,”
Molecular and Cellular Biology
, 14:5441-49 (1994). These findings, together with the observations that expression of oncogenic Ras increases cyclin D1 levels and shortens G1 phase (Liu, et al., “Ras Transformation Results in an Elevated Level of Cyclin D1 and Acceleration of G1 Progression in NIH 3T3 Cells,”
Mol. Cell Biol
., 15:3654-63 (1995); Winston et al., “Regulation of the Cell Cycle Machinery by Oncogenic Ras,”
Oncogene
, 12:127-34 (1996)) and that Ras and cyclin D1 cooperate in cellular transformation assays (Hinds et al., “Function of a Human Cyclin Gene as an Oncogene,”
Proc. Natl. Sci. USA
, 91:709-13 (1994); Lovec et al., “Oncogenic Activity Cyclin D1 Revealed Through Cooperation with Ha-ras; Link Between Cell Cycle Control and Malignant Transformation,”
Oncocene
, 9:323-26 (1994)) point to an important role for Ras in regulating progression from G1 into S phase.
However, important questions remain as to whether Ras controls signalling everts during cell cycle progression, and, if so, at which point in the cell cycle it is activated. The Ras proteins function by cycling between active and inactive forms; in the active form Ras binds to GTP and is converted to the inactive form by conversion of GTP to GDP. Activation of Ras is promoted by numerous extracellular signals such as growth factors, and, when activated, Ras specifically interacts with intracellular targets to transduce growth stimulatory signals from the cell's exterior to the nucleus. One such target of activated Ras in the Raf-1 proto-oncoprotein, a protein kinase involved in signalling to the nucleus.
The mutations of ras gene., that occur in human cancer, as discussed above, cause a constitutive activation of the Ras protein, and the resulting deregulation of growth control is believed to contribute to the cancer process. Furthermore, it is known that ras gene mutation occurs at a particular: stage in the multi-step process of colon cancer progression, and it is likely that ras mutations might occur at defined, perhaps early, stages in other types of cancer. Accordingly, the detection of ras activation in human tumors might be of great diagn
Shalloway David
Taylor Stephen J.
Cornell Research Foundation Inc.
Harris Alana M.
Huff Sheela
Nixon & Peabody LLP
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