Expression of the receptor tyrosine kinase Ret on the plasma membrane is dependent on calcium.

Mutations in the Ret receptor tyrosine kinase are responsible for a variety of human syndromes, including multiple endocrine neoplasia 2 and Hirschsprung's disease. Ret is expressed as a 150-kDa precursor form in the endoplasmic reticulum and a 170-kDa mature form at the plasma membrane. Here we show that expression of p170(ret) is dependent on calcium. Depletion of extracellular calcium completely blocks p170(ret) expression, which is not caused by a decrease in half-life of p170(ret) at the plasma membrane but by a defect in processing of p150(ret) into p170(ret). This processing defect can be mimicked by treating the cells with thapsigargin, a drug that releases calcium from internal stores, indicating that reduction in luminal calcium is responsible for the processing defect. We propose that a relatively high concentration of luminal calcium is necessary for the proper folding of Ret in the endoplasmic reticulum.

Protein kinase B activation and lamellipodium formation are independent phosphoinositide 3-kinase-mediated events differentially regulated by endogenous Ras.

Regulation of phosphoinositide 3-kinase (PI 3-kinase) can occur by binding of the regulatory p85 subunit to tyrosine-phosphorylated proteins and by binding of the p110 catalytic subunit to activated Ras. However, the way in which these regulatory mechanisms act to regulate PI 3-kinase in vivo is unclear. Here we show that several growth factors (basic fibroblast growth factor [bFGF], platelet-derived growth factor [PDGF], and epidermal growth factor [EGF; to activate an EGF receptor-Ret chimeric receptor]) all activate PI 3-kinase in vivo in the neuroectoderm-derived cell line SKF5. However, these growth factors differ in their ability to activate PI 3-kinase-dependent signaling. PDGF and EGF(Ret) treatment induced PI 3-kinase-dependent lamellipodium formation and protein kinase B (PKB) activation. In contrast, bFGF did not induce lamellipodium formation but activated PKB, albeit to a small extent. PDGF and EGF(Ret) stimulation resulted in binding of p85 to tyrosine-phosphorylated proteins and strong Ras activation. bFGF, however, induced only strong activation of Ras. In addition, while RasAsn17 abolished bFGF activation of PKB, PDGF- and EGF(Ret)-induced PKB activation was only partially inhibited and lamellipodium formation was unaffected. Interestingly, in contrast to activation of only endogenous Ras (bFGF), ectopic expression of activated Ras did result in lamellipodium formation. From this we conclude that, in vivo, p85 and Ras synergize to activate PI 3-kinase and that strong activation of only endogenous Ras exerts a small effect on PI 3-kinase activity, sufficient for PKB activation but not lamellipodium formation. This differential sensitivity to PI 3-kinase activation could be explained by our finding that PKB activation and lamellipodium formation are independent PI 3-kinase-induced events.

Signal transduction by the receptor tyrosine kinase Ret.

Ret is the receptor for glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN). Defects in this receptor underlie several genetic syndromes. The receptor is a transmembrane tyrosine kinase which transduces Ret-mediated signaling to a variety of signaling pathways, most notably the Ras signaling pathway and the phosphatidylinositol-3 kinase pathway. These pathways are activated through the interaction of adaptor proteins to tyrosine phosphorylated receptor. The ultimate biological effects, depending on the cell type, include morphological changes in the cytoskeleton, cell scattering, proliferation, and differentiation.

CD44 is involved in tumor angiogenesis; an activation antigen on human endothelial cells.

CD44 is described to be an activation molecule in a number of different cell types. We investigated the role of CD44 on human endothelial cells (EC) and in tumor angiogenesis. Using flow cytometry we showed that EC from the vasculature of human solid tumors display an enhanced expression of CD44 as compared to EC from normal tissue. This finding was confirmed by immunohistochemical studies on frozen tissue sections. Because tumors are dependent on angiogenesis, the role of angiogenic stimuli in the enhanced CD44 expression was investigated. We found that basic fibroblast growth factor (bFGF) and vascular endothelial growth factor were able to efficiently upregulate CD44 expression on cultured human EC. The upregulation reached maximal levels after treatment for 3 days with 10 ng/mL bFGF. The physiological impact of this upregulation was shown by the enhanced binding of EC to hyaluronate after pretreatment with bFGF. In a next set of studies that were designed to unravel the regulation of CD44 expression on EC we concluded that CD44 is an activation antigen on human EC since (1) human umbilical vein derived endothelial cells, which in vivo do not express CD44, begin to express CD44 when plated and cultured, (2) CD44 expression is enhanced after subculture of confluent cultures, (3) CD44 is predominantly expressed on the BrdU incorporating subset of cultured EC. The specific expression of CD44 on activated and tumor EC prompted us to study the usefulness of CD44 as an endothelial target for therapy with immunotoxins. In vitro experiments showed that EC are efficiently killed after targeting immunotoxin to CD44.

Cell scattering of SK-N-MC neuroepithelioma cells in response to Ret and FGF receptor tyrosine kinase activation is correlated with sustained ERK2 activation.

The c-ret proto-oncogene encodes a receptor tyrosine kinase which plays an important role in kidney and enteric nervous system development. Germline mutations in c-ret are responsible for the dominantly inherited cancer syndromes, multiple endocrine neoplasia types 2A and 2B and familial medullary thyroid carcinoma as well as the developmental disorder Hirschsprung's disease. Using SK-N-MC neuroepithelioma cells stably transfected with an EGFR/Ret chimeric receptor, we have studied cellular consequences and signalling events following activation of exogenous EGFR/Ret and endogenous FGF and PDGF receptor tyrosine kinases in cells of neuroectodermal origin. Here we report that Ret activation led to cell scattering, growth inhibition and loss of anchorage-independent growth. Basic FGF, but not PDGF, evoked similar responses in those cells. Nevertheless, activation of all three receptor tyrosine kinases led to ERK2 activation. Analysis of the kinetics of ERK2 activation and downstream events revealed that Ret and FGF receptor activation led to sustained ERK2 activation and SRE transactivation, while PDGF treatment led to transient ERK2 activation and failed to induce SRE transactivation. Our results suggest that sustained, but not transient ERK2 activation may be involved in cell scattering.

Glial cell line-derived neurotrophic factor induces Ret-mediated lamellipodia formation.

Ret is a receptor tyrosine kinase required during embryogenesis for the survival of enteric and sympathetic neuroblasts. Recently, glial cell line-derived neurotrophic factor (GDNF) has been identified as a ligand for Ret. We investigated early events in GDNF-induced signal transduction. We show that GDNF activates the Ras-ERK2 signaling pathway in SK-N-MC cells stably transfected with a full-length Ret construct. In addition, activation of Ret tyrosine kinase activity, either via GDNF stimulation of full-length Ret or via epidermal growth factor stimulation of an epidermal growth factor receptor-Ret chimeric receptor, results in phosphatidylinositol 3-kinase activation and phosphatidylinositol 3-kinase-dependent formation of large lamellipodia. Our results indicate that GDNF can serve as a genuine ligand for Ret. In addition, we show that GDNF can induce Ret-mediated formation of lamellipodia, cell surface protrusions that are implicated in neuritogenesis.

GLUT4 vesicle dynamics in living 3T3 L1 adipocytes visualized with green-fluorescent protein.

Insulin stimulates glucose uptake into its target cells by a process which involves the translocation of the GLUT4 isoform of glucose transporter from an intracellular vesicular compartment(s) to the plasma membrane. The step(s) at which insulin acts in the vesicle trafficking pathway (e.g. vesicle movement or fusion with the plasma membrane) is not known. We expressed a green-fluorescent protein-GLUT4 (GFP-GLUT4) chimaera in 3T3 L1 adipocytes. The chimaera was expressed in vesicles located throughout the cytoplasm and also close to the plasma membrane. Insulin promoted a substantial translocation of GFP-GLUT4 to the plasma membrane. Time-lapse confocal microscopy demonstrated that the majority of GFP-GLUT4-containing vesicles in the basal state were relatively static, as if tethered (or attached) to an intracellular structure. A proportion (approx. 5%) of the vesicles spontaneously lost their tether, and were observed to move rapidly within the cell. Other vesicles appear to be tethered only on one edge and were observed in a rapid stretching motion. The data support a model in which GLUT4-containing vesicles are tightly tethered to an intracellular structure(s), and indicate that a primary site of insulin action must be to release these vesicles, allowing them to then translocate to and fuse with the plasma membrane.

Ret receptor tyrosine kinase activates extracellular signal-regulated kinase 2 in SK-N-MC cells.

Ret is a receptor tyrosine kinase predominantly expressed in tissue derived from the neuroectoderm and is involved in multiple endocrine neoplasia type 2A and 2B, familiar medullary thyroid carcinoma, and Hirschsprung's disease. The ligand for the receptor is still unknown. Previously, using a human epidermal growth factor receptor - Ret chimaeric receptor (HERRet) stably transfected into fibroblasts, it was shown that Ret activation induces the activation of p21ras, but, surprisingly, activation of extracellular signal-regulated kinase 2 (ERK2) was not observed (Santoro et al. (1994) Mol. Cell. Biol., 14, 663). In this report we describe early signaling events induced by the activated HERRet fusion receptor in a cell line derived from neuroectodermal tissue, SK-N-MC. In these cells, activated HERRet induces tyrosine phosphorylation of Shc, complex formation of Shc with Grb2 and Sos and activation of p21ras. Importantly, also ERK2 is activated. This activation was strong and sustained for at least 2 h. Activation was abolished by the dominant negative p21rasasn17 mutant, showing that activation of ERK2 is mediated by p21ras. These results suggest that Ret can induce ERK2 activation in a p21ras dependent manner in cells derived from tissue where Ret is endogenously expressed.

A PCR-based method for the analysis of human CD44 splice products.

CD44 is a transmembrane glycoprotein involved in the interaction between cells and extracellular matrix. Several variant forms of CD44 exist, which differ from each other in the composition of both the intra- and extracellular domain of the protein. Post-translational modification and alternative RNA processing are responsible for this variation. Recently, it was found that certain variant CD44 proteins, containing extra sequences in the extracellular domain of the protein, are involved in metastatic spread of tumor cells. Variant CD44 proteins are also involved in immunological functions of T and B cells. A large variety of alternatively spliced CD44 mRNAs can be expressed by cells. We have developed a method for the analysis of CD44 mRNAs present in the cell. This reverse transcription-polymerase chain reaction (RT-PCR)-based method can be used to analyze the exon composition of each major CD44 mRNA species present in the cell. In this study we describe the analysis of CD44 mRNAs isolated from six different human cell lines.

K-ras oncogene activation in adenocarcinoma of the human pancreas. A study of 82 carcinomas using a combination of mutant-enriched polymerase chain reaction analysis and allele-specific oligonucleotide hybridization.

We examined 82 surgically resected or biopsied, formalin-fixed, paraffin-embedded primary adenocarcinomas of the pancreas for the presence of activating point mutations in codon 12 of the K-ras oncogene. Mutations were detected using primer-mediated, mutant-enriched, polymerase chain reaction-restriction fragment length polymorphism analysis and characterized further by allele-specific oligonucleotide hybridization. This combination of mutant-enriched polymerase chain reaction-restriction fragment length polymorphism analysis and allele-specific oligonucleotide hybridization results in a rapid and sensitive characterization of the mutations in codon 12 of K-ras. Sixty-eight (83%) of the 82 carcinomas examined harbored a point mutation. Of the 68 mutations, 33 (49%) were guanine to adenine transitions, 27 (39%) were guanine to thymine transversions, and eight (12%) were guanine to cytosine transversions. Mutations were found in carcinomas of the head (61 of 75, 81%) as well as in carcinomas of the body or tail (seven of seven, 100%) of the pancreas. The overall prevalence of K-ras point mutations in adenocarcinomas of the pancreas obtained from patients who smoked cigarettes at some point during their lives (88%; 86% in current smokers and 89% in ex-smokers) was greater than that seen in pancreatic adenocarcinomas from patients who never smoked cigarettes (68%, P = 0.046). The presence of K-ras point mutations did not correlate with tumor ploidy, tumor proliferating index, or patient survival. These results demonstrate that primer-mediated, mutant-enriched polymerase chain reaction-restriction fragment length polymorphism analysis combined with allele-specific oligonucleotide hybridization can be used to detect and characterize mutations in codon 12 of the K-ras oncogene in formalin-fixed, paraffin-embedded tissues, and the results confirm that activating point mutations in codon 12 of the K-ras oncogene occur frequently in adenocarcinomas of the pancreas.

Intratumoral low-dose interleukin-2 induces rejection of distant solid tumour.

This study shows that local tumor treatment with low-dose recombinant interleukin-2 (IL-2) can mediate rejection of a large distant solid tumour. When SL2 lymphoma cells were injected intraperitoneally (i.p.) in syngeneic DBA/2 mice on day 0.70% of these mice were cured by daily i.p. injections with 20,000 units IL-2 on days 10-14. After injecting mice with SL2 both i.p. and subcutaneously (s.c.) on the flank. 50% of the mice treated i.p. with low-dose IL-2 rejected both the i.p. tumour and the large distant s.c. tumour. In contrast, i.p. IL-2 treatment on days 10-14 cured fewer than 10% of the mice bearing only a s.c. SL2 tumour. The described IL-2 immunotherapy also caused systemic tumour rejection in mice bearing both ascitic and solid P815 mastocytoma. Thus it was shown that low-dose IL-2 can induce systemic tumour rejection, when injected at a site of tumour growth. Interleukin-2-induced rejection of s.c. SL2 tumour was highly specific, as mice that were rejecting i.p. and solid s.c. SL2 lymphoma did not reject solid P815 mastocytoma, which was injected s.c. simultaneously on the other flank. Furthermore, solid s.c. tumours consisting of mixtures of SL2 and P815 were not rejected in mice that rejected i.p. SL2 or P815. We conclude that intratumoral injections of low-dose IL-2 can enhance an ongoing weak immune reaction against the tumour resulting in systemic tumour rejection.