SUBSTRATE-ATTACHED GLYCOPROTEINS MEDIATING ADHESION OF NORMAL AND VIRUS-TRANSFORMED MOUSE FIBROBLASTS

When BALB/c 3T3, simian virus 40 (SV40)-transformed 3T3 (SVT2), and revertant variants of the transformed cells are removed by EGTA treatment from the substrate on which they were grown, they leave behind a layer of glycoprotein which has been characterized biochemically (Terry, A. H. and L. A. Culp. 1974. Biochemistry. 13:414.)—substrate-attached material (SAM). The influence of SAM from normal and from transformed cells on cellular attachment to the substrate, morphology, movement, and growth has been examined. All three cell types displayed a 30% higher plating efficiency when grown on 3T3 SAM. The morphology of SVT2 colonies and of individual SVT2 cells was dramatically affected by growth on 3T3 SAM—the cells (a) were more highly spread on the substrate, (b) resisted crawling over neighboring cells, and (c) resisted movement away from the edge of colonies; SVT2 SAM was not effective in causing these changes. A cell-to-substrate attachment assay using thymidine-radiolabeled cells and untreated or SAM-coated cover slips was developed. SVT2 cells attached to 3T3 SAM- or SVT2 SAM-coated cover slips with a faster initial rate and to a higher saturation level than to untreated substrate, whereas 3T3 and revertant cells exhibited no preference; there was no species specificity in these cell-substrate attachment phenomena. Trypsin-released cells attached much more slowly than EGTA-released cells. 3T3 SAM, however, was not effective in lowering the saturation density of mass cultures of virus-transformed cells. These experiments suggest that the substrate-attached glycoproteins of normal cells affect the cellular adhesivity, morphology, movement, and perhaps growth patterns of virus-transformed cells—i.e., causing partial reversion of these properties of transformed cells to those found in contact-inhibited fibroblasts. A model for the involvement of substrate-attached glycoproteins in cell-to-substrate adhesion, and possibly cell-to-cell adhesion, has been proposed

CONTACT-INHIBITED REVERTANT CELL LINES ISOLATED FROM SV40-TRANSFORMED CELLS : IV. Microfilament Distribution and Cell Shape in Untransformed, Transformed, and Revertant Balb/c 3T3 Cells

A comparison is made of the ultrastructure of the cell periphery in three cloned cell lines: untransformed Balb/c 3T3 cells, SV40-transformed Balb/c 3T3 cells, and revertant cells obtained from the transformed cell line by a selection technique utilizing concanavalin A. Both thin-section and surface replication techniques are used for in situ examination of the cell lines. Microfilaments, 70 Å in diameter (called alpha filaments), are abundant in untransformed and revertant cell lines, particularly in the anterior expansions of the cells, which tend to have many microvilli and small pseudopodia. Alpha filaments are diminished in the anterior expansions of transformed cells, which contain large blunt pseudopodia and relatively few microvilli. Surface replicas confirm the impression gained from thin sections that transformed cells have a greater proportion of their cell surface involved in bulging pseudopodia than either untransformed or revertant cells. Since alpha filaments are shown to bind heavy meromyosin and are similar to F-actin, these filaments are thought to be important in cell motility. These observations suggest that a close relationship exists between decreased alpha filaments, bulging pseudopodia, and loss of contact inhibition of movement in transformed cells

Robust Biomarkers: Methodologically Tracking Causal Processes in Alzheimer’s Measurement

In biomedical measurement, biomarkers are used to achieve reliable prediction of, and useful causal information about patient outcomes while minimizing complexity of measurement, resources, and invasiveness. A biomarker is an assayable metric that discloses the status of a biological process of interest, be it normative, pathophysiological, or in response to intervention. The greatest utility from biomarkers comes from their ability to help clinicians (and researchers) make and evaluate clinical decisions. In this paper we discuss a specific methodological use of clinical biomarkers in pharmacological measurement: Some biomarkers, called ‘surrogate markers’, are used to substitute for a clinically meaningful endpoint corresponding to events and their penultimate risk factors. We confront the reliability of clinical biomarkers that are used to gather information about clinically meaningful endpoints. Our aim is to present a systematic methodology for assessing the reliability of multiple surrogate markers (and biomarkers in general). To do this we draw upon the robustness analysis literature in the philosophy of science and the empirical use of clinical biomarkers. After introducing robustness analysis we present two problems with biomarkers in relation to reliability. Next, we propose an intervention-based robustness methodology for organizing the reliability of biomarkers in general. We propose three relevant conditions for a robust methodology for biomarkers: (R1) Intervention-based demonstration of partial independence of modes: In biomarkers partial independence can be demonstrated through exogenous interventions that modify a process some number of “steps” removed from each of the markers. (R2) Comparison of diverging and converging results across biomarkers: By systematically comparing partially-independent biomarkers we can track under what conditions markers fail to converge in results, and under which conditions they successfully converge. (R3) Information within the context of theory: Through a systematic cross-comparison of the markers we can make causal conclusions as well as eliminate competing theories. We apply our robust methodology to currently developing Alzheimer’s research to show its usefulness for making causal conclusions

Tagged tumor cells reveal regulatory steps during earliest stages of tumor progression and micrometastasis

Histochemical marker genes were used to "tag" mouse fibrosarcoma or human neuroblastoma cells, providing a better understanding of their subsequent progression and metastasis mechanisms in nude mice. Micrometastases in the lung were initiated from clusters of 2-6 cells rather than single cells in most cases; tumor cells were also visualized binding to the endothelium of small blood vessels to initiate these micrometastases. Shortterm, these mechanisms relied heavily on fluidity of cell surface proteins, rather than nuclear events. Micrometastases in some organs were transient and never became established. Angiogenesis was visualized in both primary tumor systems via "fixation" of the animal's circulation; very small microvessels were growing toward the primary tumor as soon as 48-72 hours post-injection. Marker genes were also valuable for quantitating genetic instability of specific tumor cell populations and potential gene regulatory mechanisms operating in specific organ sites. These latter studies have direct relevance to the significance of N-myc oncogene amplification in neuroblastoma during progression and CD44 gene plasticity of expression in fibrosarcoma during metastasis. Marker gene-tagged single tumor cells can now be analyzed for gene regulatory events in virtually any organ and in combination with laser capture microdissection and other high-resolution methodologies, providing insight into the very earliest gene-regulatory events during micrometastasis