Principal Investigators Meetings

Automated Measurement of Ras and Rho Activation in Cancer
Dr. Gerry Boss

Ras was first identified as the transforming principle of Harvey and Kirsten strains of rat sarcoma virus in 1964 and 1967
Ras was "rediscovered" in 1981 as the dominant oncogene in human and carcinogen-induced animal tumors; 95% pancreatic cancers and 5-10% of breast, lung, ovarian cancers
Ras is a cellular switch: in active state, it is bound to GTP and in inactive state, it is bound to GDP; Ras activation is defined as the percent of Ras molecules in the active GTP-bound state

Ras (continued)

Ras is tethered to the plasma membrane and is involved in multiple signal transduction pathways transmitting pro-proliferative signals to the nucleus
Oncogenic forms of Ras are mutationally locked in the GTP-bound state
Oncogenic Ras transforms NIH3T3 cells but in most cell types transformation requires expression of another oncogene

Rho Proteins

Rho proteins are members of the extended Ras family, and like Ras alternate between an active GTP-bound and inactive GDP-bound state
They regulate the actin cytoskeleton, and thereby affect cell morphology; they also modulate gene expression, cell cycle pro-gression, and cell proliferation and survival
Rho activation is clearly important for the development of metastases

Method to Assess Ras Activation

Original method required labeling cells with 32PO4 and then immunoprecipitate Ras; more recent method relies on Western immunoblotting and thus is semi-quantitative
Developed patented quantitative method that could be applied to animal or human tissues
Elute GTP and GDP from immunopre-cipitated Ras, and split samples in half
Measure GTP by converting it to ATP using NDPK and measure ATP by the luciferase method; assay is sensitive to 1 fmol of GTP:
GTP + ADP GDP + ATP (NDP kinase) ATP + Luciferin oxyluciferin + AMP + Light (Luciferase)
Measure sum of GTP + GDP by converting GDP to GTP using pyruvate kinase and then measure the GTP as described:
GDP + Phosphoenolpyruvate GTP + Pyruvate (Pyruvate kinase)

Method to Assess Rho Activation

Based on same assay as for measuring Ras activation except no good immunoprecipi-tating antibody for Rho
Rho-GTP is isolated using a glutathione S-transferase-tagged Rho effector protein
Sample is split in half and Rho-GDP must be converted to Rho-GTP prior to isolation

Ras and Rho Activation in Human Tumors

Methods are highly sensitive requiring only about 1 X 106 cells which can be obtained from tumor scrapings or fine needle aspirates
Have found Ras is activated in about 50% of breast cancers, 30% of neuro-fibromas/sarcomas and acute childhood lymphocytic leukemias, and 20% of astrocytomas and lung and ovarian cancers
Measurement of Ras/Rho activation could have predictive value for determining which patients would respond to Ras/Rho inhibit-ors being developed by pharmaceutical industry
Measurement of Ras/Rho activation could have prognostic value; current study on breast cancer
For assay to be used practically needs to be automated

Frontal View, ARAM

Oblique View, ARAM

Parameter Entry Screen

Tissue Extraction Device

Centrifuge Tub of TED

Immunoprecipitation/Elution Device

Needle Plate of IED

Tube Block of IED

Glass Beads Bonded to Microcentrifuge Tube

Longitudinal-Section of Tube

Cross-Linking Proteins to Glass Beads

React free Si groups on bead surface with aminopropyltriethoxysilane to yield free amino groups
Cross-link free amino groups with amines of protein A/G or glutathione using the homobifunctional imidoester dimethyl suberimidate
The immobilized proteins are stable for at least six weeks when stored at 4 C

Receiving Device

Rear of Instrument

Right Oblique of Instrument

Left Oblique of Instrument

ARAM Video

Status of Instrument

Presently assessing accuracy, reproduci-bility, specificity, and sensitivity in cultured breast cancer cells which are homogenous and can be manipulated to vary Ras and Rho activation
Compared to manual method, ARAM shows as good reproducibility and specificity, but less sensitive

Single Cell Assay For Ras and Rho Activation

Use the Ras/Rho binding domains of their effector proteins which bind only to the active GTP-bound state; detected by a GST-tag and immunofluorescence
When combined with confocal microscopy can be quantitative
Complements biochemical assay which provides a mean activation state of all cells in sample

Intensity

Credits/Collaborators