Carl T. McGary

Metastasis suppressed, but tumorigenicity and local invasiveness unaffected, in the human melanoma cell line MelJuSo after introduction of human chromosomes 1 or 6

Progression of human melanoma toward increasingly malignant behavior is associated with several nonrandom chromosomal aberrations, most commonly involving chromosomes 1, 6, 7, 9, and 10. We previously showed that introduction of human chromosome 6 into the highly metastatic human malignant melanoma cell line C8161 completely suppressed metastasis without altering tumorigenicity (Welch DR, Chen P, Miele ME, et al., Oncogene 9:255–262, 1994). Alterations of chromosome 1 are the most frequent chromosome abnormality observed in melanomas, and they frequently arise late in tumor progression. The purpose of the study presented here was to compare the effects of chromosomes 1 and 6 on malignant melanoma metastasis. By using microcell‐mediated chromosome transfer, single copies of neo‐tagged human chromosomes 1 or 6 were introduced into the human melanoma cell line MelJuSo. The presence of the added chromosome was verified by G banding of karyotypes, fluorescence in situ hybridization, and screening for polymorphic markers on each chromosome. The incidence and number of metastases per lung after intravenous or intradermal injection of parental MelJuSo cells was significantly (P < 0.01) greater than those of hybrids containing either chromosome 1 or chromosome 6, although chromosome 1 was a less potent inhibitor of metastasis than chromosome 6. Cultures established from primary tumors and metastases remained neomycin resistant, suggesting that portions of the added chromosomes were retained. These results strengthen the evidence for the presence of a melanoma metastasis suppressor gene on chromosome 6. neo6/MelJuSo hybrids expressed 2.4‐ to 3.4‐fold more of the melanoma differentiation‐associated gene mda‐6 (previously shown to be identical to WAF1/CIP1/Sdi1/CAP20) than parental metastatic cells. mda‐6/WAF1 is among the candidate genes on chromosome 6. These results also demonstrate, for the first time, the existence of metastasis suppressor genes on human chromosome 1, although these genes appear to be less potent than the one encoded on chromosome 6.

Study of Hyaluronan-Binding Proteins and Receptors Using Iodinated Hyaluronan Derivatives

This chapter detailed methodology for the purification of high molecular weight HA, as well as procedures to fragment the HA to prepare large oligosaccharides in the range of 40,000-80,000 Da. The aforementioned procedures used to prepare HA-alkylamine and HA-Bolton-Hunter adducts, as well as 125I-labeled HA, have been very reproducible, and the latter preparations are of adequate length to retain high-affinity interactions and specific binding, e.g., to human fibrinogen and HARE. For example, we were able to isolate, characterize, and clone the rat HARE using 125I-labeled HA initially with the dot blot assay to monitor solubilization and partial purification, and later with the ligand blot assay, to identify the protein after SDS-PAGE. The ligand blot assay enabled us to determine that HARE is actually present as two discrete isoreceptors of different molecular masses. These techniques should provide a means to analyze purification strategies and to characterize additional HA receptors and binding proteins involved in a variety of physiologic processes.

Hyaluronan and its receptors in mucoepidermoid carcinoma

Hyaluronan (HA) is a prominent extracellular matrix component undergoing continuous production and degradation. Increased HA levels have been described in a variety of tumors. The objective of this study was to examine the staining patterns of HA and two of its associated receptors (CD44 and HARE) in relation to the metastatic potential of mucoepidermoid carcinoma (MC). Immunohistochemical staining of preserved surgical specimens was used.

Specific intracellular hyaluronic acid binding to isolated rat hepatocytes is membrane-associated.

Intact isolated rat hepatocytes show a small amount of specific 125I-labeled hyaluronic acid (HA) binding. However, in the presence of digitonin, a very large increase in the specific binding of 125I-HA is observed. Chondroitin sulfate, heparin and dextran sulfate were as effective as unlabeled HA in competing for 125I-HA binding to permeabilized hepatocytes, indicating that the binding sites may have a general specificity for glycosaminoglycans. After rat hepatocytes had been homogenized in a hypotonic buffer, more than 98% of the 125I-HA binding activity could be pelleted by centrifugation at 100,000 x g for 1 h. Mild alkaline treatment of hepatocyte membranes did not release 125I-HA binding activity, suggesting that the HA binding site is an integral membrane molecule. Furthermore, trypsin treatment of deoxycholate-extracted membranes destroyed the binding activity, as assessed by a dot-blot assay. This suggests that a protein component in the membrane is necessary for 125I-HA binding activity. Rat fibrinogen could be a possible candidate for the HA binding activity because HA binds specifically to human fibrinogen (LeBoeuf et al. (1986) J. Biol. Chem. 261, 12 586). Also, fibrinogen can be found in a quasi-crystalline form in rat hepatocytes and could be pelleted with the membranes. Rat fibrinogen was not responsible for the 125I-HA binding activity, since (1) purified rat fibrinogen did not bind to 125I-HA, and (2) immunoprecipitation of rat fibrinogen from hepatocyte extracts did not decrease the 125I-HA binding of these extracts. We conclude that the internal HA binding sites are membrane- or cytoskeleton-associated proteins and are neither cytosolic proteins nor fibrinogen.

PURIFICATION AND CHARACTERIZATION OF THE HYALURONAN RECEPTOR FOR ENDOCYTOSIS (HARE)

ABSTRACT Liver sinusoidal endothelial cells (LECs) express two HARE proteins of 175 kDa and ∼300 kDa that endocytically clear hyaluronan (HA) from the circulation. We have characterized eight monoclonal antibodies (mAbs) raised against the partially purified 175 kDa HARE. Seven mAbs recognize the 175 kDa HARE after nonreducing SDS-PAGE and in all cases also crossreact with the ∼300 kDa HARE. Two of the mAbs inhibit 125I-HA binding and endocytosis by LECs at 37°C. We purified these two HAREs and showed that the 175 kDa HARE is a single protein, whereas the ∼300 kDa species contains three subunits at 260, 230 and 97 kDa (Zhou, et al. J. Biol. Chem. 274, 33831–33834, 1999). Two mAbs recognized both the two nonreduced HARE species and three of the four proteins present after reduction. The 97 kDa subunit was not recognized by any mAbs in Western blots. Based on their cross-reactivity with the mAbs against the 175 kDa HARE, the 175, 230 and 260 kDa proteins are related to each other. Immunocytochemistry using these mAbs shows high protein expression levels in rat liver sinusoids, the venous sinuses of the red pulp in spleen, and the medullary sinuses of lymph nodes. Little or no HARE expression was observed in muscle, heart, lung, kidney, brain, skin, eye, pancreas, thymus, testis, adipose, salivary gland, adrenal, thyroid, larynx, tongue, stomach or intestine. We propose the name HARE (HA Receptor for Endocytosis) because this receptor mediates the very rapid endocytosis of HA and its tissue distribution is not unique to liver.

Use of Iodinated Hyaluronan Derivatives to Study Hyaluronan Binding, Endocytosis, and Metabolism by Cultured Cells

We have described several simple procedures to determine whether cells have specific receptors or binding proteins for HA. The advantage of using the uniquely modified 125I-labeled HA as a probe is that low levels of binding can be detected. It has been straightforward and productive to use 125I-labeled HA to quantitate cell surface and intracellular binding sites to determine affinity constants for HA binding and to determine whether the cell type being studied can efficiently endocytose and degrade the HA. The 125I-labeled HA preparations and assays described here and in Chapter 26 provide a very useful array of methods for studying the biochemistry and cell biology of HA.