Autocrine motility factor (AMF) is a cytokine that regulates locomotion and metastasis of tumor cells. It is well known that expression levels of AMF secretion and its receptor (AMF R) are closely related to tumor malignancy and rheumatoid arthritis. We have established that AMF signaling induced anti‐apoptotic activity and that human fibrosarcoma HT‐1080 line that secreted high levels of AMF were resistant to drug‐induced apoptosis. These cells did not express the apoptotic protease activating factor‐1 (Apaf‐1) and Caspase‐9 genes that encode for the proteins that form the “apoptosome” complex. The disappearance of the Apaf‐1 and Caspase‐9 gene was recovered by a cellular signaling inhibitor of protein kinase C, phosphatidylinositol 3‐phosphate kinase and mitogen‐activated protein kinase of the in vitro cultured human fibrosarcoma HT‐1080 line. Treatment with these inhibitors favored apoptotic cell death induced by anti‐cancer drugs of the murine ascites Ehrlich line. Apoptotic resistance of tumor cells allows them to escape death from cancer chemotherapy, so an understanding of malignant anti‐apoptotic activities is important. Antibodies against AMF induced Ehrlich ascites apoptosis in vitro , and effectively aided in vivo apoptosis induced by anti‐cancer drugs. The results might indicate a novel route by which tumor cells protect themselves with products, such as AMF, and proliferate despite various stresses and chemical insults; AMF regulates expression of Apaf‐1 and caspase‐9 genes via a complex signaling pathway and indirectly regulates formation of the apoptosome. © 2003 Wiley‐Liss, Inc.

A tumour-secreted cytokine autocrine motility factor (AMF) induces in vivo invasion and metastasis, and in vitro tumour cell motility by a signal transduction through interaction with its cell surface receptor gp78. In this report, we investigated the characterization of a high-metastatic human oral squamous cell carcinoma (SCC) cell line LMF4 and low-metastatic HSC-3 in comparison with non-metastatic HSC-2 and HSC-4. Morphological and motility analyses revealed LMF4 cells to have the highest motile activity among those cells. However, LMF4 cells shared the similar features with HSC-3: high level secretion of AMF, enhancement of gp78 expression, co-expression of vimentin and cytokeratin, although LMF4 cells showed twice as high motile reactivity as HSC-3. The only difference was that LMF4 had twice as high amount of low-affinity receptor(s) as HSC-3, shown by Scatchard analysis.

Cellular migration is an integral aspect in response to extracellular stimuli, which is fundamental to numerous biological processes such as embryogenesis, inflammation, wound healing, tissue regeneration, and tumor invasion and metastasis (1,2). Abundant studies centered on the identification and characterization of factors that regulate and direct cell movement have shown that host serum components and extracellular matrix breakdown products exert a chemotactic effect on various tumor cells (3) and that basement membrane and extracellular matrix components promote cellular haptotaxis (4). Furthermore, host growth factors influence recipient cells by modulating growth and motility independently or in a coordinated manner (2). Moreover, cellular migration in vitro has been reported to be correlated with tumor invasion and metastasis in vivo. A group of motility factors has been described, the primary function of which is thought to be the regulation of cellular kinesis. Motility factors have been originally distinguished by their ability to induce the random (chemoki-netic) and directional (chemotactic) migration of the cells (5). Therefore, quantitating the cell motility is one of the most important clues to comprehend the cellular characteristics of malignancy and/or the effect and activities of motility inducing properties. Gold colloidal method was invented to measure the random motility (chemokinesis) by Albrecht-Buehler, in which area of phagokinetic track cleared by a single cell is measured (6). The Boyden chamber method, described in Chapter 5 by Brown and Bicknell, was invented to quantitate the directional motility (chemotaxis) and was modified in various ways to.

Phosphohexose isomerase (PHI) is a member of the ectoenzyme/exoenzyme family and plays a key role in both glycolysis and gluconeogenesis pathways. Upon secretion PHI acts as a cytokine with tumor autocrine motility factor (AMF), neuroleukin (NLK) and maturation factor (MF) functions. Signaling is initiated by its binding to a cell surface 78 kDa glycoprotein (gp78). However, since PHI protein is a 'leaderless' secretory protein, released from cells via a non-classical route(s), we questioned whether the molecule undergoes post-translation modification while retaining proper folding and maintaining intact enzymatic and motogenic activities. To address this, we have generated, expressed and isolated a recombinant human AMF (rhAMF). The rhAMF retained the biological activities of the native AMF, i.e., catalyzes phosphohexose isomerization and stimulated cell motility. Additionally, we show here that human PHI is phosphorylated at serine 185 by casein kinase II (CK II) and we provide experimental evidence suggesting that this phosphorylation is associated with secretion, thus providing insights for elucidating the intracellular signal transmission of cell response to stimulation by AMF/NLK/MF.

Autocrine motility factor receptor (AMFR) is a cell surface glycoprotein of molecular weight 78 000 (gp78), mediating cell motility signaling in vitro and metastasis in vivo. Here, we cloned the full‐length cDNAs for both human and mouse AMFR genes. Both genes encode a protein of 643 amino acids containing a seven transmembrane domain, a RING‐H2 motif and a leucine zipper motif and showed a 94.7% amino acid sequence identity to each other. Analysis of the amino acid sequence of AMFR with protein databases revealed no significant homology with all known seven transmembrane proteins, but a significant structural similarity to a hypothetical protein of Caenorhabditis elegans , F26E4.11. Thus, AMFR is a highly conserved gene which encodes a novel type of seven transmembrane protein.

The results obtained from fragmented protein microsequencing have suggested that autocrine motility factor (AMF), a tumor-secreted Mr 55,000 cytokine that regulates cell motility in vitro as well as invasion and metastasis in vivo, is the neuroleukin (NLK)/phosphohexose isomerase (PHI)/maturation factor (MF) polypeptide. Here, we cloned, sequenced, and studied the expression, secretion, and distribution of AMF/NLK/PHI/MF in neoplastic and their normal counterpart cells. Although both normal and neoplastic cells express the gene product, overexpression associated with selective secretion of the protein was observed only in tumor cells. The cDNA sequences of AMF/NLK/PHI/MF found in both human cancer and normal cells were found to be identical, suggesting that its secretion by neoplastic cells is independent of mutation or alternative splicing. Immunohistochemical visualization has depicted AMF/NLK/PHI/MF to be localized into tubular-like vesicles, diffusely distributed throughout the cytoplasm and not colocalized with any particular cytoskeletal network. Confocal microscopic imaging had shown a partial colocalization between AMF and its receptor (Mr 78,000 glycoprotein), especially on the malignant cell surface periphery. The results suggest that extracellular AMF activity may be a result of the product of intracellular cleavage of a precursor polypeptide, which is overexpressed and selectively secreted through a nonclassical secretory mechanism by neoplastic cells.

Tumor invasion and metastases is a complex multi-step process. In order for a tumor cell to accomplish these steps, it must have the ability to attach to extracellular matrix, degrade then and migrate through extracellular matrices. Tumor cell migration is stimulated by some glycoproteins, growth factors and motility factors including autocrine motility factor (AMF). AMF induces chemotaxis and chemokinetics via binding to its receptor, gp78. Secretion of AMF was observed in several malignant cells. Gp78 is shown to be expressed in some cancer cells in relation to poor prognosis in vivo. Hence, the autocrine loop of AMF and gp78 may regulate tumor cell invasiveness in vivo.

Autocrine motility factor (AMF) a tumor-secreted 55 kDa cytokine induces tumor cell motility by a signal transduction pathway mediated by interaction with its receptor (AMFR) a cell surface glycoprotein of 78 kDa (gp78). Here, AMF secreted by the metastatic LMF4 human oral squamous-cell carcinoma (SCC) cells, induced dose- and time-dependent morphological changes and chemotaxis of the producing cells. Expression of AMFR mRNA was associated with the metastatic ability of SCC cell variants. The data presented show for the first time that SCC cells produce AMF and express AMFR and the expression is related to their invasiveness and metastatic potentials.

Production and characterization of hemidesmosome-specific mouse monoclonal antibodies 3A1 and 8A12 raised against the oral squamous carcinoma line cells were previously reported. In this study, further investigations were carried out to characterize these antigens using those cells. Immunoaffinity-purified 3A1 antigen was identified as a 180kD glycoprotein [205kD under reduced conditions]. Acetate membrane electrophoresis of disaccharides originating from this antigen digested by chondroitinase ABC showed that this protein was associated with hyaluronate. Immunoprecipitates by 3A1 and 8A12 antibodies were composed of two bands: 180kD and 140kD [205kD and 125kD by reduction], and were similar to integrin alpha 6 beta 4 precipitated by monoclonal anti-alpha 6 subunit antibody. Immunoprecipitation-Western Blot analysis showed that the 140kD [125kD] protein precipitated by 3A1 and 8A12 was alpha 6 subunit and that the 180kD [205kD] beta 4 subunit. After the alpha 6 subunit was immunodepleted, only the 180kD [205kD] band was immunoprecipitated by 3A1 or 8A12. Both 3A1 and 8A12 blocked the adhesion of LMF5 cells to laminin. These data indicated that 3A1 and 8A12 recognized integrin beta 4 subunit and that integrin alpha 6 beta 4 functioned as a laminin receptor on these cells. It was suggested that hyaluronan detected in the 3A1 antigen was associated with two putative hyaluronan-binding motifs in the extracellular domain of the integrin beta 4 subunit.