Autocrine Motility Factor Receptor Research Articles

Autocrine Motility Factor Receptor as a Possible Urine Marker for Transitional Cell Carcinoma of the Bladder

Purpose To determine whether autocrine motility factor receptor (AMFR) is detectable in the urine of patients with transitional cell carcinoma (TCC) of the bladder. Materials and Methods We assayed the urine of 89 patients with bladder pathology and 28 normal controls for AMFR. A monoclonal antibody to AMFR was used. Results All patients with muscle-invasive TCC tested positive for AMFR. Autocrine motility factor receptor was detectable for 80 percent of superficial tumors, with a correlation between AMFR and tumor grade. Seventy-five percent of control urines tested negative. Conclusions Autocrine motility factor receptor is detectable in the urine of patients with TCC. Long-term follow-up and refinements in the assay should define the marker's utility for detection and prognosis.

Autocrine Motility Factor Receptor as a Possible Urine Marker for Transitional Cell Carcinoma of the Bladder

Autocrine Motility Factor Receptor as a Possible Urine Marker for Transitional Cell Carcinoma of the Bladder

Differential expression of autocrine motility factor receptor (AMFR) mRNA in normal and cancer cells detected by in situ hybridization

The receptor for autocrine motility factor(AMFR) is known to be involved in the process of MF-mediated cell migration and metastasis.This paper describes the procedures of non-radioactive in situ hybridization(ISH) detection of AMFR mRNA in both paraffin-embedded surgical sections and cultured cells using either biotinylated oligonucleotide probes of digoxigenin-labeled RNA probes.The results showed that the AMFR mRNA was expressed at an enhanced level in hyperplastic and malignant tissues of breast and prostate cancer patient surgical specimens,indicating that the elevated AMFR expression was associated with the tissue malignancy.Moreover,AMFR mRNA was detected in both normal and carcinoma cells when cultured at a subconfluent density.However,AMFR expression was inhibited in confluent normal(3T3-A31 murine fibroblast and FHs 738 BL huamn bladder)cells while it continued to express in carcinoma(J82 human bladder) and metastatic(3T3-M murine fibroblast) cells irrespective of cell density.This suggested a cell-cell contact down-regulation of AMFR mRNA expression in normal but not in cancer cells.The ISH data obtained in this study are closely consistent with the AMFR protein expression pattern previously reported,implying that the differential expression of AMFR gene may be rgeualted and controlled at the transcriptional level.

Loss of cell‐contact regulation and altered responses to autocrine motility factor correlate with increased malignancy in prostate cancer cells

Tumor cell migration and proliferation in new organ environments are critical steps in cancer progression and can be modulated by tumor- and host-secreted molecules. Autocrine motility factor (AMF) is a tumor-secreted cytokine which regulates growth and motility by a receptor-mediated pathway. The AMF receptor, a 78-kDa cell surface glycoprotein (gp78), is regulated by cell contact in normal fibroblastic and bladder cells; however, this mechanism is disrupted during tumor progression. A prostatic carcinoma cell line which is low- to non-metastatic in nude mice (PC-3) and a derived metastatic variant (PC-3M) were examined to determine if gp78 cell density regulation is involved in prostate cancer progression. Both cell lines expressed gp78 and, although the basal migration of the parental PC-3 cells was higher than that of the metastatic variant, only the PC-3M cells were capable of responding to tumor-derived AMF with increased motility. Furthermore, these cells exhibited differential patterns of wound closure in an experimental system whereby the low-metastatic PC-3 cells migrated primarily along the wound edge while individual high-metastatic PC-3M cells entered the cell-free wound area directly. Cell surface gp78 distribution distinguished the cell populations with a markedly concentrated display of gp78 in polarized capped regions on the surface of the metastatic cells. Cell-cell contact down-regulated gp78 expression in the parental, but not the metastatic, cells, and mitogenic responses to exogenous AMF differed between these cell lines as well. In this model, metastasis appears to be associated with aberrant regulation of gp78 expression and distribution, coupled with enhanced exploitation of AMF's locomotory and proliferative effects.

Identification of an Upstream Region That Controls the Transcription of the Human Autocrine Motility Factor Receptor

We have isolated from a human placenta cosmid library a 0.7 kb genomic clone that contains the 5′ terminal portion of the autocrine motility factor receptor (hAMFR) coding region. Chloramphenicol Acetyl Transferase (CAT) reporter gene assays have identified this region as the promoter of the hAMFR gene. A single transcription initiation site (+1) has been mapped to 129 bp upstream of the ATG start codon by primer extension. DNA sequence analysis and CAT assay revealed a TATA element at the position -485/-468 which was able to conduct only a marginal transcription (less than 5% of the total activity). The majority of the hAMFR promoter′s activity is contributed by a transcription initiator (Inr) element overlapping the initiation site (+1) which independently controls the transcription of the hAMFR gene.

Autocrine motility factor receptor is a marker for a distinct membranous tubular organelle.

Autocrine motility factor (AMF) is secreted by tumor cells and is capable of stimulating the motility of the secreting cells. In addition to being expressed on the cell surface, its receptor, AMF-R, is found within a Triton X-100 extractable intracellular tubular compartment. AMF-R tubules can be distinguished by double immunofluorescence microscopy from endosomes labeled with the transferrin receptor, lysosomes labeled with LAMP-2, and the Golgi apparatus labeled with beta-COP. AMF-R can also be separated from a LAMP-2 containing lysosomal fraction by differential centrifugation of MDCK cells and is found within a 100,000 g membrane pellet. By electron microscopic immunocytochemistry, AMF-R is localized predominantly to smooth vesicular and tubular membranous organelles as well as to a lesser extent to the plasma membrane and rough endoplasmic reticulum. AMF-R tubules have a variable diameter of 50-250 nm and can acquire an elaborate branched morphology. By immunofluorescence microscopy, AMF-R tubules are clearly distinguished from the calnexin labeled rough endoplasmic reticulum and AMF-R tubule expression is stable to extended cycloheximide treatment. The AMF-R tubule is therefore not a biosynthetic subcompartment of the endoplasmic reticulum. The tubular morphology of the AMF-R tubule is modulated by both the actin and microtubule cytoskeletons. In a similar fashion to that described previously for the tubular lysosome and endoplasmic reticulum, the linear extension and peripheral cellular orientation of the AMF-R tubule are dependent on the integrity of the microtubule cytoskeleton. The AMF-R tubule may thus form part of a family of microtubule-associated tubular organelles.

Expression and function of autocrine motility factor receptor in human choriocarcinoma cells

Expression and function of autocrine motility factor receptor in human choriocarcinoma cells

Expression of autocrine motility factor receptor in colorectal cancer as a predictor for disease recurrence

New biologic prognostic factors are needed to guide the treatment of patients with colorectal cancer. The prognostic value of the altered expression of the cell surface glycoprotein gp78, which has been implicated in tumor-cell invasion and metastasis as an autocrine motility factor (AMF) receptor, was evaluated. The level of expression of AMF receptor gp78 was analyzed immunohistochemically in tumor specimens from 118 patients who had undergone surgery for colorectal cancer. Multivariate analysis was used to determine whether gp78 expression status was correlated with patient prognosis. Among the 118 patients studied, the 5-year survival rate for the 51 patients who had gp78-positive tumors was significantly poorer that that of the 67 patients who had gp78-negative tumors (49.8% vs. 89%). The incidence of gp78 positivity was correlated with tumor progression as reflected by histologic type, depth of invasion, lymph node metastasis, vessel invasion, and tumor stage. Among the 101 patients who underwent curative resection, the difference in disease free survival between patients with gp78-positive tumors and those with gp78-negative tumors was significant. This significant difference remained among patients who had Stage II tumors. Multivariate analysis with the Cox regression model indicated that gp78 positivity was a good predictor of disease recurrence, ranking with extent of tumor invasion (T classification) and lymph node status (N classification). Increased expression of gp78 is correlated with a high incidence of recurrence and, consequently, with decreased survival of patients with colorectal cancer. Expression of gp78 might be of prognostic value in these patients.

A Lipoxygenase Metabolite, 12-(S)-HETE, Stimulates Protein Kinase C-Mediated Release of Cathepsin B from Malignant Cells

The process of tumor cell invasion of the basement membrane is proposed to consist of three steps: attachment, local proteolysis and migration. 12-(S)-HETE, a 12-lipoxygenase metabolite of arachidonic acid, upregulates surface expression of integrin cytoadhesins and an autocrine motility factor receptor, suggesting that this metabolite may play an important regulatory function in tumor cell invasion. In the present study, we determined whether 12-(S)-HETE affects surface expression and/or release of cathepsin B, a cysteine protease that has been implicated in focal degradation of basement membrane. Secretion and distribution of cathepsin B was evaluated in two model systems for various stages of neoplastic progression: (i) murine B16 melanoma lines of low (B16-F1) and high (B16a) lung colonization potential, and (ii) immortalized and ras-transfected MCF-10 human breast epithelial cells that differ in their invasive capacities in vitro. In the B16a cells, 12-(S)-HETE induced release of native and latent cathepsin B activity and concomitantly reduced cell-associated cathepsin B immunoreactivity. In contrast, 12(S)-HETE did not induce the release of cathepsin B from B16-F1 cells, suggesting that there may be an enhanced response to 12-(S)-HETE in more malignant cells. This was confirmed in the MCF-10 system, in which 12-(S)-HETE was able to induce the release of cathepsin B from the ras-transfected cells, but not from the immortal cells. A simultaneous reduction in staining for cathepsin β was observed in the ras-transfected cells, but not in their immortal counterparts. The release of cathepsin B may be mediated by PKC as pretreatment of B16a cells with the selective PKC inhibitor calphostin C, but not with the PKA inhibitor H8, prevented the stimulated release of cathepsin B. In B16a cells, the release of cathepsin B was accompanied by a translocation toward the cell periphery of vesicles staining for cathepsin B, resulting in focal areas of accumulation of cathepsin B. After 12-(S)-HETE stimulation of the ras-transfected MCF-10 cells, cathepsin B was distributed homogeneously on the apical surface. Thus, 12-(S)-HETE can upregulate the surface expression on tumor cells of proteins able to mediate each of the three steps of tumor cell invasion: adhesion, degradation, and migration.

Regulation of melanoma-cell motility by the lipoxygenase metabolite 12-(S)-HETE.

Cellular motility, a prerequisite for metastasis of tumor cells, is affected by a 55-kDa tumor-cell-secreted cytokine which influences the migration of the producing cells and is called autocrine motility factor (AMF). Previous studies indicated that AMF stimulates motility by binding to its receptor, a cell-surface glycoprotein of 78 kDa (gp78), inducing its phosphorylation, activating a pertussis toxin (PT)-sensitive G-protein, and stimulating inositol metabolism. However, the intracellular signaling mechanisms which transduce and regulate the AMF motility response remain largely unknown. 12-(S)-HETE, a lipoxygenase metabolite of arachidonic acid which affects the cytoskeletal architecture of murine melanoma cells, also stimulates cell motility independently of PT-sensitive G-proteins and up-regulates gp78 surface expression. 12-(S)-HETE induces the phosphorylation of gp78 in a manner analogous to AMF and the motility response of these murine melanoma cells to both AMF and 12-(S)-HETE is inhibited by protein kinase C inhibitors. Furthermore, perturbation of the AMF receptor stimulated endogenous biosynthesis of 12(S)HETE. These results suggest the existence of an "autocrine motility cycle" which influences melanoma cell motility by gp78 activation, and production of second messengers which affect the cytoskeletal architecture and expression of the AMF receptor itself.

AUTOCRINE MOTILITY FACTOR-RECEPTOR IN HUMAN BLADDER-CARCINOMA - GENE-EXPRESSION, LOSS OF CELL-CONTACT REGULATION AND CHROMOSOMAL MAPPING

Cell lines derived from normal (FHs738BL), papilloma (RT4) and carcinoma (J82) human urinary bladder tissue were characterized for motility and for expression of autocrine motility factor (AMF) receptor, i.e. the 78 kDa cell surface glycoprotein, gp78. Further, the human gene for gp78 was mapped to the long arm of chromosome 16, band q21 just distal to 16q13. Although the papilloma cells were immotile, the normal and carcinoma cells exhibited similar basal migration, while only the carcinoma cells were capable of responding to tumor-derived AMF. No difference in gene structure or copy number was found among the three cell lines and all expressed gp78. Cell surface distribution and level of expression distinguished the three cell populations with a markedly concentrated display of gp78 in focal regions on the surface of the carcinoma cells. Cell contact down-regulated gp78 expression in the normal but not the carcinoma cells. In this model for bladder carcinoma, malignancy is apparently associated in part with the spatial localization of gp78 on the cell surface, coupled with a loss of contact inhibition-mediated gp78 downregulation, the result of which is an AMF-receptive signal transduction motility response.

Expression of autocrine motility factor receptor in serum- and protein-independent fibrosarcoma cells: implications for autonomy in tumor-cell motility and metastasis.

The motile response of serum-dependent (Gc-4 SD) and protein-independent (Gc-4 PF) murine fibrosarcoma cells to monoclonal antibody (MAb) that binds to gp78 a cell-surface receptor (M(r) 78,000) for an autocrine motility factor (AMF) was analyzed. The Gc-4 PF cells responded to the anti-gp78 by increased motility in vitro (3-fold) and increased lung colonization in vivo (8- to 20-fold), while the serum-dependent counterpart failed to respond to motile stimulation both in vitro and in vivo. Immuno-analysis of cell-surface expression and cell extracts revealed a smaller amount of gp78 in Gc-4 SD cells than in Gc-4 PF cells. Both cell lines secrete an equal amount of AMF to the culture media. Our results suggest that protein-free culture of Gc-4 PF cells is associated with high response to AMF and with high expression of its receptor, and that autonomous motile regulation may play a role in tumor dissemination.

Purification of human tumor cell autocrine motility factor and molecular cloning of its receptor

Tumor autocrine motility factor (AMF) has been detected in and purified from serum-free conditioned medium of human HT-1080 fibrosarcoma cells. Under nonreducing conditions, AMF migrates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a single band of 55 kDa but under reducing conditions as a band of 64 kDa. Two-dimensional polyacrylamide gel electrophoresis of the purified AMF resolved two groups of polypeptides with isoelectric points of 6.1 and 6.2 (majors), 6.35 and 6.4 (minors). Purified AMF stimulated HT-1080 cell migration in a dose-dependent fashion. The motility stimulation of the fibrosarcoma cells with AMF is associated with the phosphorylation of the AMF receptor, a 78-kDa cell surface glycoprotein (gp78), suggesting protein kinase participation in migratory signal transduction. The gene encoding gp78 was cloned from an HT-1080 fibrosarcoma complementary DNA library. The deduced sequence encodes a polypeptide of 323 amino acids. The nucleotide and predicted amino acid sequence of the gp78 reveals significant homology with the human suppressor/oncogene p53 protein.

Tumor cell autocrine motility factor receptor.

The ability to locomote and migrate is fundamental to the acquisition of invasive and metastatic properties by tumor cells. Autocrine motility factor (AMF) is a cytokine produced by various tumor cells which stimulates their in vitro motility and in vivo lung-colonizing ability. AMF stimulates cell motility via a receptor-mediated signalling pathway. Signal transduction following binding of AMF to its receptor, a cell surface glycoprotein of 78 kD (gp78), is mediated by a pertussis toxin sensitive G protein, inositol phosphate production and the phosphorylation of gp78. AMF induces gp78 internalization to intracellular tubulovesicles and transport to the leading edge stimulating pseudopodial protrusion and cell motility.