Principal Investigators Meetings
Automated Measurement of Ras and Rho Activation in CancerDr. Gerry Boss
Table of Contents:
- Title and Author
- The Ras Proto-Oncogene
- Ras (continued)
- Rho Proteins
- Method to Assess Ras Activation
- Method to Assess Rho Activation
- Ras and Rho Activation in Human Tumors
- 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
- Receiving Device
- Rear of Instrument
- Right Oblique of Instrument
- Left Oblique of Instrument
- ARAM Video
- Status of Instrument
- Single Cell Assay For Ras and Rho Activation
- Intensities
- Credits/Collaborators
Automated Measurement of Ras and Rho Activation in Cancer
Gerry R. Boss
University of California, San Diego
La Jolla, CA
The Ras Proto-Oncogene
- 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
- David Gough, Biomedical Engineer
- Stephen Jones, Electronics Technician
- Jeffrey Chen, Research Technician
- James Feramisco, Director UCSD Digital Imaging Lab
- Anne Wallace, Surgical Oncologist
- Linda Wasserman, Pathologist
- Stephen Qualman, Gynecologic Oncology Tissue Bank, Columbus, Ohio