Research Use Only

Ras p21 ELISA

Intended Use
The Ras p21 ELISA is an enzyme-linked immunoassay used for the quantitation of circulating
ras p21 in human serum and plasma. FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.

Background
Ras p21 is a GDP/GTP binding G protein that acts as a molecular switch, converting signals from the cell membrane to the nucleus to regulate cell proliferation and differentiation and to synthesize protein [1]. Other G-protein family members include the Rho/Rac, Rab, Arf, and Ran
subfamilies. The ras gene family is a group of three closely related genes (H-, K-, and N-) encoding 21-kDa proteins of 189 amino acids which are found in all eukaryotic cells on the inner surface of the plasma membrane. The three main ras p21 isoforms (H-ras, N-ras, and K-ras [4A and 4B]) share a high degree of homology (> 90%) as well as common downstream effectors and upstream guanine-nucleotide exchange factors (GEFs). The first 165 amino acids of the different ras p21 isoforms are largely homologous and include the effector, the exchange factor, and the nucleotide-binding sites at the amino-terminal conserved domain. The carboxy-terminal region after amino acid 165 is known as the hypervariable region (HVR) and is comprised of the linker domain and the membrane-targeting domain. The membrane-targeting domain includes the carboxy-terminal CAAX box shared by all ras p21 proteins and a secondary membrane-targeting domain that determines the route taken by the different ras p21 proteins out of the endoplasmic reticulum to the plasma membrane. H-ras follows the classical secretory pathway through the Golgi to caveolae and lipid rafts while K-ras follows a Golgi-independent route to the plasma membrane. Recent studies report that H- and N-ras but not K-ras can also signal from the Golgi and the endoplasmic reticulum [2]. Microlocalization within the membrane differs between the ras p21 isoforms and may influence activation and downstream effectors. Recent studies are now investigating the consequences of the different plasma membrane anchors of each isoform and how the spatial orientation of different ras p21 isoforms within the plasma membrane may account for some of the biological differences now being identified between them [3]. Other ras p21 proteins include R-ras and M-ras, both sharing approximately 55% homology with the major isoforms. R-ras can promote malignant transformation but its mechanism of action may be different. It activates the PI-3K/Akt pathway but not the Raf/MEK pathway. M-ras only weakly activates the MAP kinase pathway. It may also signal through alternate pathways [2].

Normally, the binding of ras p21 to GTP is triggered by a signal initiated by growth factor
binding to plasma membrane receptors with a tyrosine kinase activity. Guanine nucleotide
exchange factors (GEFS, SOS1/2, ras GRF1/2, ras GRP, and CNrasGEF) facilitate activation. The GTP-bound form of ras p21 is capable of interacting directly with RasGAP, neurofibromin, and the Raf kinases. The reversal of active ras-GTP back to inactive ras-GDP is facilitated by GTPase activating proteins (GAPs) [4]. Activation of ras p21 oncogenes has been identified in a variety of cancers as well as precursor lesions. Ras p21 proteins activated by substitutions at amino acid positions 12, 13, or 61 can act as oncogenes with an increased transforming capacity [5,6]. Mutated forms of ras p21 lock ras p21 in a constitutively active state by reducing GTP hydrolysis, which results in uncontrolled signals for cell growth.

Activated ras genes are uncommon in stomach, esophagus, ovary, prostate, and breast tumors [7], but are reported in 20–40% of lung carcinomas, 30% of acute myelogenous leukemia, 40–50% of colorectal tumors and thyroid tumors, and > 80% of pancreatic tumors. Overall, it is reported that approximately 30% of all human neoplasms have a ras gene mutation [8,9]. H-ras mutations are common in bladder, kidney, and thyroid carcinomas; K-ras mutations occur in non-small-cell lung, colorectal, and pancreatic carcinomas; and N-ras mutations are found in melanoma, hepatocellular carcinoma, and hematologic malignancies [1]. Transformation of cells by oncogenic ras p21 mutants can increase the expression of metalloproteinases such as gelatinase and stromelysin which can enhance tumor metastasis. The expression of vascular endothelial growth factor (VEGF) is increased in K-ras- and H-ras-transformed epithelial cells through the Raf pathway.

Because of its important role in tumorigenesis, the ras p21 signaling pathways have become
intensely studied targets for anticancer therapy [10,11,12]. One approach to therapy is the
inhibition of ras p21 protein expression through ribozymes, antisense oligonucleotides, or RNA.
Another approach is the prevention of membrane localization (e.g., by inhibition of ras p21
farnesylation) which is essential for ras p21 activation [13,14,15]. The third strategy is the inhibition of downstream effectors of ras p21 function including the Raf serine/threonine kinases (A-Raf, B-Raf, and C-Raf-1) which then prevents the constitutive activation of the MAPK (Mitogen-activated protein kinase) signal cascade [16]. The Raf-MAPK pathway activates the ETS family of transcription factors that affect tumor growth, invasion, and stroma formation. Other effectors include Rac, Rho, Ral-GDS, Rg1, Rlf, and PI-3K (phosphoinositide 3-kinase) of the PI-3K/AKT pathway. AKT, also known as protein kinase B, phosphorylates and regulates the function of many cellular proteins, including those involved in metabolism, apoptosis, and proliferation, through regulation of cell cycle progression [17].

Assay Principle
The Ras p21 ELISA is a sandwich-type immunoassay that uses a mouse monoclonal Capture Antibody and a biotinylated mouse monoclonal antibody as detector. The Capture Antibody has been immobilized on the interior surface of the microtiter plate wells. To perform the test, an appropriate volume of specimen is incubated in the wells to allow binding of the antigen by the Capture Antibody. The immobilized antigen is then exposed to the biotinylated Detector Antibody. A streptavidin-HRP Conjugate is then added. Addition of Substrate to the wells allows the catalysis of a chromogen into a colored product, the intensity of which is proportional to the amount of ras p21 that is bound to the plate.

Standards are provided in the kit that allow accurate, quantitative determinations of ras p21
in suitable samples. Using a microtiter plate reader, one can measure simultaneously the
absorbance of the colored product in the Standards and sample wells. Correlating the absorbance values of samples with the Standards allows the investigator to determine the levels of ras p21 in a sample. Samples may be assigned a quantitative value of ras p21 in picograms per milliliter (pg/mL) of serum or plasma.


Selected References

1. Pruitt K, Der Channing J. Ras and Rho regulation of the cell cycle and oncogenesis. Cancer Letters 2002;171:1–10.
2. Brunner TB, Hahn S, Gupta AK, et al. Farnesyltransferase inhibitors: An overview of the results of preclinical and clinical investigations. Cancer Research 2003;63:5656–5668.
3. Prior IA, Hancock JF. Compartmentalization of Ras proteins. Journal of Cell Science;114:1603–1608.
4. Wittinghofer A, Scheffzek K, Ahmadian MR. The interaction of Ras with GTPase-activating proteins. FEBS Letters 1997;410:63–67.
5. Carney WP, Petit D, Hamer P, et al. Monoclonal antibody specific for an activated RAS protein. Proc Natl Acad Sci USA 1986;83:7485–7489.
6. Hamer PJ, La Vecchio J, Ng S, et al. Activated Val-12 ras p21 in cell culture fluids and mouse plasma. Oncogene 1991;6(9):1609–1615.
7. Rundle A, Tang D, Brandt-Rauf P, et al. Association between the ras p21 oncoprotein in blood samples and breast cancer. Cancer Lett 2002;185(1):71–78.
8. Bos JL. The ras gene family and human carcinogenesis. Mutation Research 1988;195:255–271.
9. Carney WP. Oncogenes and non-oncogene related human tumor antigens. The Cancer Journal 1991;4:156–161.
10. Adjei AA. Blocking Ras signaling for cancer therapy. Journal of National Cancer Institute 2001;93(14): 1062–1074.
11. Bos JL. Ras oncogenes in human cancer; a review. Cancer Research 1989;49:4682–4689.
12. Cox AD, Der Channing J. Ras family signaling: Therapeutic targeting. Cancer Biol Ther 2002;1(6):599–606.
13. Lerner EC, Qian Y, Blaskovich MA, et al. Ras CAAX peptidomimetic FTI-277 selectively blocks oncogenic Ras signaling by inducing cytoplasmic accumulation of inactive Ras-Raf complexes. J Biol Chem 1995;270(45):26802–26806.
14. Kohl NE, Conner MW, Gibbs JB, et al. Development of inhibitors of protein farnesylation as potential chemotherapeutic agents. J Cell Biochem Suppl 1995;22:145–150.
15. Gibbs JB, Kohl NE, Koblan KS, et al. Farnesyltransferase inhibitors and anti Ras therapy. Breast Cancer Res Treat 1996;38(1):75–83.
16. Chang F, Steelman LS, Lee JT, et al. Signal transduction mediated by the Ras/Raf/MEK/ERK pathway from cytokine receptors to transcription factors: Potential targeting for therapeutic intervention. Leukemia 2003;17(7):1263–1293.
17. Dancey JE. Agents targeting ras signaling pathway. Curr Pharm Des 2002;8(25):2259–2267.