Merlin: hanging tumor suppression on the Rac.

Cancer can result from any number of abnormalities in the control of cell-cycle progression, intracellular signaling and transduction of extracellular cues. Many insights into the crucial events that govern the regulation of cell growth have derived from studies of the gene products mutated in inherited cancer syndromes. Recent work on the neurofibromatosis 2 (NF2) tumor suppressor gene suggests that this negative growth regulator might function by modulating growth factor and extracellular matrix (ECM) signals that trigger Rac1-dependent cytoskeleton-associated processes. In this article, we propose a molecular model for NF2 protein (merlin) function in the light of these and related new findings.

CD44 enhances neuregulin signaling by Schwann cells.

We describe a key role for the CD44 transmembrane glycoprotein in Schwann cell-neuron interactions. CD44 proteins have been implicated in cell adhesion and in the presentation of growth factors to high affinity receptors. We observed high CD44 expression in early rat neonatal nerves at times when Schwann cells proliferate but low expression in adult nerves, where CD44 was found in some nonmyelinating Schwann cells and to varying extents in some myelinating fibers. CD44 constitutively associated with erbB2 and erbB3, receptor tyrosine kinases that heterodimerize and signal in Schwann cells in response to neuregulins. Moreover, CD44 significantly enhanced neuregulin-induced erbB2 phosphorylation and erbB2-erbB3 heterodimerization. Reduction of CD44 expression in vitro resulted in loss of Schwann cell-neurite adhesion and Schwann cell apoptosis. CD44 is therefore crucial for maintaining neuron-Schwann cell interactions at least partly by facilitating neuregulin-induced erbB2-erbB3 activation.

Hyaluronate-independent metastatic behavior of CD44 variant-expressing pancreatic carcinoma cells.

Several studies have demonstrated a correlation between the expression of CD44 variant isoforms and the ability of tumor cells to metastasize. The CD44 proteins carry amino acid sequence motifs that confer the ability to bind to the extracellular matrix component hyaluronate (HA). In this study, we investigated whether a CD44 variant previously shown to stimulate metastasis in a rat pancreatic carcinoma model (BSp73AS) is capable of binding to HA, and whether such binding is critical for metastasis. We show that transfection of this CD44 variant into BSp73AS cells increases the HA-binding capacity of the cells in a dose-dependent manner. Transfection of the same CD44 variant isoform into BDX2 cells also conferred strong HA-binding properties on these cells, but was insufficient to cause them to metastasize. Transfection of a surface-bound hyaluronidase into metastasizing BSp73AS cells bearing variant CD44 efficiently ablated the ability of these cells to bind to HA. However, in metastasis assays, these hyaluronidase-transfected cells showed patterns of metastasis similar to those of the parental cell line. We also show that the HA-binding capacity of a variety of tumor cells is not correlated with their metastatic proclivity, and that an antibody previously shown to block metastasis of the pancreatic carcinoma cells does not interfere with their ability to bind to HA. We conclude that although CD44 variant expression does promote metastasis formation, HA binding by tumor cells is not rate limiting for metastasis in the BSp73AS system and probably also in other metastasizing tumors. Furthermore, for metastasis by CD44 variant-expressing BSp73AS cells to occur, contact of the CD44 variant protein with a ligand other than HA Is required.

Ruffling membrane, stress fiber, cell spreading and proliferation abnormalities in human Schwannoma cells.

Schwannomas are peripheral nerve tumors that typically have mutations in the NF2 tumor suppressor gene. We compared cultured schwannoma cells with Schwann cells from normal human peripheral nerves (NHSC). Both cell types expressed specific antigenic markers, interacted with neurons, and proliferated in response to glial growth factor, confirming their identity as Schwann cells. Schwannoma cells frequently had elevated basal proliferation compared to NHSC. Schwannoma cells also showed spread areas 5-7-fold greater than NHSC, aberrant membrane ruffling and numerous, frequently disorganized stress fibers. Dominant negative Rac inhibited schwannoma cell ruffling but had no apparent effect on NHSC. Schwannoma cell stress fibers were inhibited by C3 transferase, tyrphostin A25, or dominant negative RhoA. These data suggest that the Rho and Rac pathways are abnormally activated in schwannoma cells. Levels of ezrin and moesin, proteins related to the NF2 gene product, merlin, were unchanged in schwannoma cells compared to NHSC. Our findings demonstrate for the first time that cell proliferation and actin organization are aberrant in schwannoma cells. Because NF2 is mutant in most or all human schwannomas, we postulate that loss of NF2 contributes to the cell growth and cytoskeletal dysfunction reported here.

Detection of melanocytes from uveal melanoma in peripheral blood using the polymerase chain reaction.

PURPOSE: Uveal melanoma is the most common intraocular malignancy in adults and can cause loss of vision in the affected eye and death from metastasis, usually to the liver. The techniques currently used to detect cellular dissemination from the tumor are inadequate, and lack the sensitivity required for the detection of low levels of melanocytes in the peripheral blood of patients. The detection of circulating melanocytes is important as an early indication of the possibility of metastasis. METHODS: The viability of reverse transcription/polymerase chain reaction amplification of the tyrosinase gene to detect circulating melanocytes was examined as a first sign of dissemination from uveal melanoma. RESULTS: It was shown that it is possible to detect as few as ten circulating melanocytes in 5 ml of blood. Blood-borne dissemination was also detected in three of six patients with uveal melanoma examined. Two of these patients had clinically confirmed widespread metastases. A positive result was also recorded in one patient in whom there was no other evidence for tumor dissemination. Overt metastatic disease developed in this patient 9 months after blood collection. CONCLUSIONS: The success of this technique has important implications for the detection of circulating tumor cells from uveal melanoma, as an early indication of dissemination. This may be important when considering the administration of adjuvant therapy.

Single cell Ras-GTP analysis reveals altered Ras activity in a subpopulation of neurofibroma Schwann cells but not fibroblasts.

Neurofibromatosis type 1 (NF1) is a common genetic disorder characterized by multiple neurofibromas, peripheral nerve tumors containing mainly Schwann cells and fibroblasts. The NF1 gene encodes neurofibromin, a tumor suppressor postulated to function in part as a Ras GTPase-activating protein. The roles of different cell types and of elevated Ras-GTP in neurofibroma formation are unclear. To determine which neurofibroma cell type has altered Ras-GTP regulation, we developed an immunocytochemical assay for active, GTP-bound Ras. In NIH 3T3 cells, the assay detected overexpressed, constitutively activated K-, N-, and Ha-Ras and insulin-induced endogenous Ras-GTP. In dissociated neurofibroma cells from NF1 patients, Ras-GTP was elevated in Schwann cells but not fibroblasts. Twelve to 62% of tumor Schwann cells showed elevated Ras-GTP, unexpectedly revealing neurofibroma Schwann cell heterogeneity. Increased basal Ras-GTP did not correlate with increased cell proliferation. Normal human Schwann cells, however, did not demonstrate elevated basal Ras activity. Furthermore, compared with cells from wild type littermates, Ras-GTP was elevated in all mouse Nf1(-/-) Schwann cells but never in Nf1(-/-) mouse fibroblasts. Our results indicate that Ras activity is detectably increased in only some neurofibroma Schwann cells and suggest that neurofibromin is not an essential regulator of Ras activity in fibroblasts.

Renal masses: assessment of corticomedullary-phase and nephrographic-phase CT scans.

PURPOSE: To evaluate the role of thin-section helical computed tomography (CT) performed during the corticomedullary phase (CMP) and nephrographic phase (NP) of contrast enhancement in the detection and characterization of renal masses. MATERIALS AND METHODS: Renal CT scans and medical records of 33 patients were retrospectively reviewed. In all examinations, 5-mm-thick, contiguous, helical-mode scans were obtained before and 40 seconds after initiation of dynamic bolus injection of contrast material (CMP images); 5-mm-thick, contiguous, axial-mode scans were obtained after completion of CMP scanning (NP images). RESULTS: At review of CMP, NP, and combination images, 259, 389, and 417 lesions, respectively, were identified. The greatest difference in detection occurred in the renal medulla, with 25 lesions identified on CMP images and 111 lesions identified on NP images. False-positive results occurred when CMP images were reviewed without NP images. CONCLUSION: CT scans obtained only during the CMP of contrast enhancement fail to depict many renal masses that are easily seen on NP images.

The BRG-1 subunit of the SWI/SNF complex regulates CD44 expression.

Aberrant regulation of CD44, a transmembrane glycoprotein, has been implicated in the growth and metastasis of numerous tumors. Although both CD44 overexpression and loss have been implicated in tumor progression, the mechanism of CD44 down-regulation in these tumor types is not known. By immunoblot and reverse transcription-polymerase chain reaction analysis we determined that a cervical carcinoma cell line, C33A, lacks CD44 expression. To determine how CD44 is down-regulated in C33A cells, we utilized cell fusions of C33A cells with a CD44-expressing cell line (SAOS-2). We found that SAOS-2 fusion restored CD44 expression in C33A cells, suggesting that a trans-acting factor present in SAOS-2 cells promotes CD44 production. C33A cells are BRG-1-deficient, and we found that CD44 was absent in another BRG-1-deficient tumor cell line, indicating that loss of BRG-1 may be a general mechanism by which cells lose CD44. Reintroduction of BRG-1 into these cells restored CD44 expression. Furthermore, disruption of BRG-1 function through the use of dominant-negative BRG-1 demonstrated the requirement of BRG-1 in CD44 regulation. Finally, we show that Cyclin E overexpression resulted in the attenuation of CD44 stimulation, which is consistent with previous observations that Cyclin E can abrogate BRG-1 action. Taken together, these results suggest that BRG-1 is a critical regulator of CD44 expression, thus implicating SWI/SNF components in the regulation of cellular adhesion and metastasis.

Early embryonic failure associated with uniparental disomy for human chromosome 21.

As many as 16% of all recognized pregnancies may be anembryonic, with failure of the embryo at a very early stage of development leaving only the extraembryonic components of the conceptus to proliferate. Studies in the mouse have shown that the maternal and paternal contributions to the genome of the zygote are not functionally equivalent, due to parental genomic imprinting. Uniparental disomy can reveal imprinting effects, as in this phenomenon both members of a chromosome pair are inherited from the same parent. We have carried out a systematic search for uniparental disomy in tissues from 23 cases of early embryonic failure, using variable number tandem repeat (VNTR) analysis and PCR amplification of polymorphic short sequence repeats. Two cases of maternal uniparental heterodisomy for chromosome 21 were identified. One case occurred in conjunction with trisomy for chromosomes 7 and 9, but in the other case maternal uniparental heterodisomy for chromosome 21 was the only chromosomal abnormality found. We therefore postulate that there may be developmentally important genes on human chromosome 21 which are imprinted such that both parental copies are essential for normal embryogenesis.

Detection of infection in loosened hip prostheses: efficacy of sonography.

OBJECTIVE: The purpose of this study was to determine the efficacy of sonography in the detection of infection in loosened hip prostheses. MATERIALS AND METHODS: The normal capsular morphology in 15 asymptomatic patients with total hip replacements was studied sonographically. Sonograms were then obtained in 33 patients who had pain in the hip after arthroplasty and radiologic findings of loosening of the prosthesis. These patients subsequently underwent aspiration and arthrography of the hip. Six of the 33 symptomatic patients proved to have prosthetic joint infection. RESULTS: On sonograms, the normal pseudocapsule is adherent to the proximal part of the anterior femoral cortex, and the capsule-to-bone distance is less than 3.2 mm (average, 2.6 mm). No hips with a capsule-to-bone distance less than 3.2 mm were infected. Sonograms in the six patients with infection showed marked intraarticular effusion with a mean capsule-to-bone distance of 10.2 mm. Five of these six had extracapsular fluid collections. Two patients with hip dislocations and four with aseptic loosening of the prosthesis had capsular distension on sonograms and cultures of aspirated material that showed no growth. CONCLUSION: Sonography can be used to diagnose infection around loosened hip prostheses. All patients who had an intraarticular effusion with extraarticular extension seen on sonograms had infection.

The NF2 tumor suppressor gene product, merlin, mediates contact inhibition of growth through interactions with CD44.

The neurofibromatosis-2 (NF2) gene encodes merlin, an ezrin-radixin-moesin-(ERM)-related protein that functions as a tumor suppressor. We found that merlin mediates contact inhibition of growth through signals from the extracellular matrix. At high cell density, merlin becomes hypo-phosphorylated and inhibits cell growth in response to hyaluronate (HA), a mucopolysaccharide that surrounds cells. Merlin's growth-inhibitory activity depends on specific interaction with the cytoplasmic tail of CD44, a transmembrane HA receptor. At low cell density, merlin is phosphorylated, growth permissive, and exists in a complex with ezrin, moesin, and CD44. These data indicate that merlin and CD44 form a molecular switch that specifies cell growth arrest or proliferation.

CD44 mediates constitutive type I receptor signaling in cervical carcinoma cells.

OBJECTIVE: The CD44 transmembrane glycoprotein family has been implicated in the growth and metastasis of numerous human cancers. CD44 may function in some cells through interactions with type I receptor tyrosine kinases, including erbB2. Here, we tested whether CD44 interacts with erbB2 and another type I receptor, the epidermal growth factor receptor (EGFR), in human cervical carcinoma tissues and cell lines and whether these interactions influence erbB2 signaling. METHODS: CD44, EGFR, and erbB2 colocalization were examined in 36 pT1b-pT2b cervical cancer cases and in the CaSki and SiHa cervical carcinoma cell lines by immunohistochemistry and laser scanning confocal microscopy. The role of CD44-EGFR-erbB2 interactions in erbB2 signaling was examined by immunoprecipitation and using antisense CD44 oligonucleotides. RESULTS: CD44, erbB2, and EGFR coexpression and colocalization were observed in 42% (15/36) of cervical carcinoma cases and in both cervical carcinoma cell lines. Colocalization occurred to an equivalent extent in all tumor grades examined. CD44 coimmunoprecipitated with erbB2 and EGFR in cervical carcinoma cell lysates, indicating that these proteins interact with each other. Reduction of CD44 expression inhibited constitutive erbB2 activity. High CD44 expression was linked to EGFR activity using dominant negative EGFR, suggesting that type I receptors may autoregulate their activity in these cells. CONCLUSIONS: Our data indicate that CD44 can mediate type I receptor function in cervical carcinoma cells that overexpress both CD44 and either erbB2 or EGFR and suggest a novel mechanism by which these proteins may contribute to cervical carcinoma tumor growth and metastasis.