Kevin C. Graham

Huntingtin Is Ubiquitinated and Interacts with a Specific Ubiquitin-conjugating Enzyme

Using the yeast two-hybrid system, we have identified a human ubiquitin-conjugating enzyme (hE2-25K) as a protein that interacts with the gene product for Huntington disease (HD) (Huntingtin). This protein has complete amino acid identity with the bovine E2-25K protein and has striking similarity to the UBC-1, −4 and −5 enzymes of Saccharomyces cerevisiae. This protein is highly expressed in brain and a slightly larger protein recognized by an anti-E2-25K polyclonal antibody is selectively expressed in brain regions affected in HD. The huntingtin-E2-25K interaction is not obviously modulated by CAG length. We also demonstrate that huntingtin is ubiquitinated. These findings have implications for the regulated catabolism of the gene product for HD.

Multipotent CD15+ Cancer Stem Cells in Patched-1–Deficient Mouse Medulloblastoma

Subpopulations of tumorigenic cells have been identified in many human tumors, although these cells may not be very rare in some types of cancer. Here, we report that medulloblastomas arising from Patched-1-deficient mice contain a subpopulation of cells that show a neural precursor phenotype, clonogenic and multilineage differentiation capacity, activated Hedgehog signaling, wild-type Patched-1 expression, and the ability to initiate tumors following allogeneic orthotopic transplantation. The normal neural stem cell surface antigen CD15 enriches for the in vitro proliferative and in vivo tumorigenic potential from uncultured medulloblastomas, supporting the existence of a cancer stem cell hierarchy in this clinically relevant mouse model of cancer.

Identification of proteins that interact with a protein of interest: applications of the yeast two-hybrid system.

The yeast two-hybrid system is a molecular genetic test for protein interaction. Here we describe a step by step procedure to screen for proteins that interact with a protein of interest using the two-hybrid system. This process includes, construction and testing of the bait plasmid, screening a plasmid library for interacting fusion proteins, elimination of false positives and deletion analysis of true positives. This procedure is designed to allow investigators to identify proteins and their encoding cDNAs that have a biologically significant interaction with your protein of interest.

Three-dimensional High-Frequency Ultrasound Imaging for Longitudinal Evaluation of Liver Metastases in Preclinical Models

Liver metastasis is a clinically significant contributor to the mortality associated with melanoma, colon, and breast cancer. Preclinical mouse models are essential to the study of liver metastasis, yet their utility has been limited by the inability to study this dynamic process in a noninvasive and longitudinal manner. This study shows that three-dimensional high-frequency ultrasound can be used to noninvasively track the growth of liver metastases and evaluate potential chemotherapeutics in experimental liver metastasis models. Liver metastases produced by mesenteric vein injection of B16F1 (murine melanoma), PAP2 (murine H-ras-transformed fibroblast), HT-29 (human colon carcinoma), and MDA-MB-435/HAL (human breast carcinoma) cells were identified and tracked longitudinally. Tumor size and location were verified by histologic evaluation. Tumor volumes were calculated from the three-dimensional volumetric data, with individual liver metastases showing exponential growth. The importance of volumetric imaging to reduce uncertainty in tumor volume measurement was shown by comparing three-dimensional segmented volumes with volumes estimated from diameter measurements and the assumption of an ellipsoid shape. The utility of high-frequency ultrasound imaging in the evaluation of therapeutic interventions was established with a doxorubicin treatment trial. These results show that three-dimensional high-frequency ultrasound imaging may be particularly well suited for the quantitative assessment of metastatic progression and the evaluation of chemotherapeutics in preclinical liver metastasis models.

Time-course characterization of the computed tomography contrast enhancement of an iodinated blood-pool contrast agent in mice using a volumetric flat-panel equipped computed tomography scanner.

Objective:The objective of this study was to determine the time-course of computed tomography (CT) contrast enhancement of an iodinated blood-pool contrast agent. Methods:Five C57BL/6 mice were anesthetized, imaged at baseline, and given an iodinated blood-pool contrast agent. Micro-CT scans were acquired at 0, 0.25, 0.5, 1, 2, 4, 8, and 24 hours after injection. The mean CT number was determined in a region of interest in 7 organs. Results:The CT contrast enhancement was plotted as a function of time for each organ. We identified an imaging window immediately after injection suitable for visualizing the vascular system and a second imaging window at 24 hours for visualizing liver and spleen. Conclusions:A single injection of the blood-pool contrast agent can be used for dual-phase investigations of the vasculature (t = 0 hours) and liver (t = 24 hours), which can be applied to studies of liver tumors or disease.

Noninvasive Quantification of Tumor Volume in Preclinical Liver Metastasis Models Using Contrast-enhanced X-ray Computed Tomography

Objectives:To determine a timepoint after contrast injection that yields equal liver parenchymal and vascular enhancement in micro-computed tomography images. To evaluate the utility of images acquired during this time period for the noninvasive measurement of liver-tumor volume. Materials and Methods:The imaging timepoint was determined by quantifying the enhancement kinetics of Fenestra VC (0.015 mL/g) in NIH III mice. In respiratory-gated images of tumor bearing mice, the ability to measure tumor volume was evaluated with a measurement variability study, and by comparing in vivo and histologically measured tumor volume. Results:Eight hours after contrast injection the liver parenchyma and vasculature were equally enhanced allowing for clear delineation of the unenhanced tumors. The smallest tumor detected in this study was 1.1 mm in diameter. The coefficient of variation for tumor-volume measurement ranged from 3.6% to 12.9% and from 6.3% to 25.8% for intra and interobserver variability, respectively. In vivo and histologic tumor-volume measurements were closely correlated (r = 0.98, P < 0.0001). Conclusions:Imaging at a time period of equal liver parenchyma and vascular enhancement after contrast injection allows for clear delineation of liver-tumor borders, thereby enabling quantitative tumor-volume monitoring.

Effect of anti-fibrinolytic therapy on experimental melanoma metastasis

Anti-fibrinolytic agents such as aprotinin and ε-aminocaproic acid (EACA) are used clinically to decrease peri-operative bleeding. Use of these treatments during cancer-related surgeries has led to investigation of the effect of fibrinolysis inhibition on cancer cell spread. The ability of aprotinin to reduce proteolytic activity of proteases required for metastasis suggests that it could have an anti-metastatic effect in patients undergoing tumor resection. However, many metastatic cells in the vasculature of a secondary tissue are associated with a micro-thrombus. The association of tumor cells with thrombi has been shown to increase their survival; therefore inhibition of plasmin-mediated fibrinolysis might instead increase metastatic cell survival by enhancing the association between thrombi and tumor cells. The goal of this work was to determine the effect of anti-fibrinolytic treatment on experimental metastasis and to establish the role of coagulation factors in this effect. The metastatic ability of B16F10 melanoma cells was evaluated in vivo following cell or animal pre-treatment with aprotinin or EACA. Additionally, a novel in vivo technique was developed, to permit analysis of tumor cell association with thrombi in the lung microvasculature using confocal microscopy. Aprotinin and EACA treatment of mice resulted in a significant increase in lung metastasis. Aprotinin treatment increased the size of thrombi in association with cells arrested in lung capillaries. This study suggests that clinical use of anti-fibrinolytic agents for cancer-related surgeries could result in increased metastatic ability of those cells shed immediately prior to and during surgery, and that this approach thus requires further study.

Contrast-enhanced microcomputed tomography using intraperitoneal contrast injection for the assessment of tumor-burden in liver metastasis models.

Objectives:To determine if intraperitoneally (IP) administered contrast (iohexol), used in conjunction with a liver-specific agent (Fenestra), can improve measurement precision and accuracy when quantifying tumor volume from micro-CT images of a liver metastasis model. Materials and Methods:We compared images acquired with Fenestra alone to images acquired with the combination of Fenestra and IP iohexol. The variability in tumor volume and tumor-burden measurement was evaluated for both techniques. The tumor-burden measurement accuracy of both in vivo techniques was determined by comparison with tumor-burden quantified from ex vivo images. Results:The addition of IP iohexol decreased measurement variability for individual tumors and overall tumor-burden by 4–8 fold and 2–3 fold, respectively. IP iohexol significantly improved the accuracy of tumor-burden measurement for both low and high tumor-burdened animals. Conclusions:The combination of IP iohexol with Fenestra provides superior delineation of liver tumors, in comparison to Fenestra alone. The complete tumor delineation provided by this imaging strategy allows for noninvasive quantification of liver tumor-burden.

Volume measurement variability in three-dimensional high-frequency ultrasound images of murine liver metastases

The identification and quantification of tumour volume measurement variability is imperative for proper study design of longitudinal non-invasive imaging of pre-clinical mouse models of cancer. Measurement variability will dictate the minimum detectable volume change, which in turn influences the scheduling of imaging sessions and the interpretation of observed changes in tumour volume. In this paper, variability is quantified for tumour volume measurements from 3D high-frequency ultrasound images of murine liver metastases. Experimental B16F1 liver metastases were analysed in different size ranges including less than 1 mm3, 1-4 mm3, 4-8 mm3 and 8-70 mm3. The intra- and inter-observer repeatability was high over a large range of tumour volumes, but the coefficients of variation (COV) varied over the volume ranges. The minimum and maximum intra-observer COV were 4% and 14% for the 1-4 mm3 and <1 mm3 tumours, respectively. For tumour volumes measured by segmenting parallel planes, the maximum inter-slice distance that maintained acceptable measurement variability increased from 100 to 600 microm as tumour volume increased. Comparison of free breathing versus ventilated animals demonstrated that respiratory motion did not significantly change the measured volume. These results enable design of more efficient imaging studies by using the measured variability to estimate the time required to observe a significant change in tumour volume.

Variability of three-dimensional high-frequency ultrasound measurements of small tumor volumes

The intraobserver variability in volume measurements of small (less than 2 mm/sup 3/) tumors using three-dimensional high-frequency ultrasound has been assessed in two murine liver metastasis models. The maximum coefficient of variation was 10.7% for five B16F1 murine melanoma liver metastases and 18.2% for seven HT-29 human colon carcinoma liver metastases. The intraobserver variability was small compared to the tumor growth measured at two-day intervals in the rapidly progressing B16F1 model. However, the maximum measurement variability was comparable to the four-day growth rate in the slowly progressing HT-29 model.