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Principal Investigators Meetings

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


Table of Contents:
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 symbol 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 symbol 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

Frontal view of ARAM



Oblique View, ARAM

Oblique view of ARAM



Parameter Entry Screen

Parameter Entry Screen



Tissue Extraction Device

Tissue Extraction Device



Centrifuge Tub of TED

Centrifuge Tub of TED



Immunoprecipitation/Elution Device

Immunoprecipitation/Elution Device



Needle Plate of IED

Needle Plate of IED



Tube Block of IED

Tube Block of IED




Glass Beads Bonded to Microcentrifuge Tube

Glass Beads Bonded to Microcentrifuge Tube



Longitudinal-Section of 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

Receiving Device



Rear of Instrument

Rear of Instrument



Right Oblique of Instrument

Right Oblique of Instrument



Left Oblique of Instrument

Left Oblique of Instrument



ARAM Video

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

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