2nd Principal Investigators Meeting

Automated Measurement of Ras and Rho Activation in Cancer
Gerry Boss, Anna Dreilinger, David Gough, James Harrell, Stephen Jones, Stephen Qualman, Anne Wallace, and Linda Wasserman

Automated Measurement of Ras and Rho Activation in Cancer
Gerry Boss (1), Anna Dreilinger (2), David Gough (3), James Harrell (1), Stephen Jones (4), Stephen Qualman (5), Anne Wallace (6), and Linda Wasserman (2). Departments of Medicine (1), Pathology (2), Bioengineering (3), and Surgery (6), and Electronics Shop (4), University of California, San Diego, La Jolla, CA, and Department of Pathology (5), Ohio State University, Columbus, OH.

Abstract
Ras and Rho transmit pro-proliferative and cellular transforming signals when appropriate ligands bind to growth factor receptors on the plasma membrane; both proteins cycle between an active GTP-bound state and an inactive GDP-bound state. We devised a method to measure Ras activation (the ratio of Ras-bound GTP over Ras-bound GTP plus GDP) in human tumors and found that Ras is highly activated in a significant number of neuronal tumors and in breast, lung, and ovarian cancers, even in the absence of a genetic mutation in the ras gene. Assessing Ras activation provides information not only about the basic biology of a tumor, but it may also have therapeutic importance because a large number of Ras inhibitors are under development or are already in clinical trials; in cell culture and animal models, these drugs are most effective when Ras is in an activated state. Some of the agents designed to disrupt Ras function have been found to exert their inhibitory effects on cell growth by inhibiting Rho, and Rho may be involved in the development of metastases. It is likely, therefore, to be of clinical value to assess the activation states of Ras and Rho as part of developing specific chemotherapeutic regimens based on the molecular alterations found in a tumor. The methods for assessing Ras and Rho activation require a large amount of technician time and could not be expected to be used in clinical laboratories. We, therefore, built an instrument, referred to as an Automated Ras/Rho Activation Measurement (ARAM) device, that fully automates the method for measuring Ras and Rho activation starting with either a small piece of tissue or a cell suspension. The ARAM consists of three separate devices all housed and linked together in one unit: a Tissue Extraction Device, an Isolation/Elution Device, and a Receiving Device. The complete unit can process up to four separate samples simultaneously and has a minimal throughput time of 90 min. The entire ARAM is under control of a microcontroller which monitors all unit functions; each step of the process can be varied independently and the software has been written so that the unit is user-friendly with the operator prompted to input appropriate parameters. When designing each device, we considered not only efficiency and performance, but also reliability and durability. We are now in the process of determining the accuracy, reproducibility, sensitivity, and specificity of the instrument by measuring Ras and Rho activation in cultured breast cancer cells and in approximately 40 samples each of breast cancers, non-small cell lung cancers, and epithelial ovarian cancers. For all samples, results from the ARAM will be compared to results using established manual procedures..

Introduction
Ras and Rho transmit pro-proliferative and cellular transforming signals when appropriate ligands bind to growth factor receptors on the plasma membrane; both proteins cycle between an active GTP-bound state and an inactive GDP-bound state. We devised a method to measure Ras activation (the ratio of Ras-bound GTP over Ras-bound GTP plus GDP) in human tumors and found that Ras is highly activated in a significant number of neuronal tumors and in breast, lung, and ovarian cancers, even in the absence of a genetic mutation in the ras gene. Assessing Ras activation provides information not only about the basic biology of a tumor, but it may also have therapeutic importance because a large number of Ras inhibitors are under development or are already in clinical trials; in cell culture and animal models, these drugs are most effective when Ras is in an activated state. Some of the agents designed to disrupt Ras function have been found to exert their inhibitory effects on cell growth by inhibiting Rho, and Rho may be involved in the development of metastases. It is likely, therefore, to be of clinical value to assess the activation states of Ras and Rho as part of developing specific chemothera-peutic regimens based on the molecular alterations found in a tumor. The methods for assessing Ras and Rho activation require a large amount of technician time and could not be expected to be used in clinical laboratories.

We, therefore, built an instrument, referred to as an Automated Ras/Rho Activation Measurement (ARAM) device, that fully automates the method for measuring Ras and Rho activation starting with either a small piece of tissue or a cell suspension. The ARAM consists of three separate devices all housed and linked together in one unit: a Tissue Extraction Device, an Isolation/ Elution Device, and a Receiving Device. The complete unit can process up to four separate samples simultaneously and has a minimal throughput time of 90 min. The entire ARAM is under control of a microcontroller which monitors all unit functions; each step of the process can be varied independently and the software has been written so that the unit is user-friendly with the operator prompted to input appropriate parameters. When designing each device, we considered not only efficiency and performance, but also reliability and durability. We are now in the process of determining the accuracy, reproducibility, sensitivity, and specificity of the instrument by measuring Ras and Rho activation in cultured breast cancer cells and in approximately 40 samples each of breast cancers, non-small cell lung cancers, and epithelial ovarian cancers. For all samples, results from the ARAM will be compared to results using established manual procedures.

Discussion
Measuring the activation states of Ras and/or Rho in human cancer could be of both prognostic value, i.e., provide information concerning the natural history of a cancer, and of predictive value, i.e., provide information concerning the patient's response to a particular therapy. From a small study of 20 breast cancers, we have preliminary evidence that increased Ras activation may portend a poor prognosis, but much more work needs to be done to address this issue. It seems possible that cancers exhibiting high levels of Ras and/or Rho activation should respond to farnesyl transferase inhibitors, or other drugs targeted to Ras and/or Rho function. Clearly, target-based chemotherapy has come of age, and because of the central roles Ras and Rho play in several signal transduction pathways, they are ideal chemotherapeutic targets. Once the ARAM instrument described in this work has been fully tested and validated, it should provide automated measurements of Ras and Rho activation from biopsies or fine needle aspirates of human tumors. Thus, even in the absence of an surgical operation, one could predict a priori, prior to initiating therapy, whether a specific patient is likely to respond to a particular therapeutic regimen. Because the ARAM is user-friendly, it could ultimately be used by technicians in clinical laboratories.

Fig. 1. Frontal View of ARAM
The three major units of the ARAM are shown: the TED, or Tissue Extraction Device; the IED, or Isolation/Elution Device; and the RED, or Receiving Device. All three units are housed within a plastic casing to maintain a constant temperature of 4ºC. Mounted above the IED and the RED are the microcontroller of the unit with touchpad and LED display, and process controllers which regulate temperatures of the TED and IED. Operational parameters are entered via the touchpad including, for example, in the TED, the duration of homogenization and sonication, and the speed of centrifugation, and in the IED, the duration of incubation with antibody, and the temperature during elution.

Fig. 2. Oblique View of ARAM
As in Fig 1, the TED, IED, and RED can be seen as well as the microcon [missing text] and process controllers. The refrigeration fan for cooling the TED, IED, and RED [missing text] below the on/off switch; the condensing coils can be seen in the rear of the instru [missing text] because the back casing has been removed.

Fig. 3. Close-Up View of TED
The four major components of the TED are easily visualized, i.e., the homogenizer, the probe sonicator, the aspiration needle assembly, and the eight-place swinging bucket centrifuge. Two wash basins for cleaning the homogenizer, the sonicator probe, and the aspiration needle are in the rear of the TED. The homogenizer, sonicator probe, and aspiration needle move vertically via linear actuators under control of stepper motors.

Fig. 4. Close-Up View of IED
The needle plate contains both a long aspiration needle which reaches to the bottom of the microcentrifuge tubes, and a short dispensing needle for adding wash buffer to the tubes. During inclubation of samples with antibody, the sample block is shaken gently by both rotary and vertical movement.

Fig. 5. Close-Up View of RED
The RED consists of a needle plate that moves vertically via a linear actuator, and a microtiter plate tray that moves the precise distance of one column width of a microtiter plate. Movement of the microtiter plate allows different wells of the microtiter plate to be positioned under the needle plate.

2nd Principal Investigators Meeting

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