Search

Background: Standard tools are not sensitive enough for hepatocellular carcinoma (HCC) early detection. This study aimed to evaluate the accuracy of dickkopf-1 (DKK1) and soluble Axl (sAxl) and their combined for early differentiating of HCC from premalignant benign liver diseases., Methods: A total of 210 chronic hepatitis C (CHC) patients (55 fibrotic, 45 cirrhotic and 110 HCC) were enrolled. Both DKK1 and sAxl were tested using ELISA for all participants., Results: HCC patients were accompanied by a significant increase (P<0.05) in DKK1 (5.38±2.05 ng/mL) and sAxl (178.02±49.39 ng/mL) compared to patients with fibrosis (2.16±0.6, 97.63±19.71 ng/mL, respectively) and cirrhosis (2.62±0.8, 121.84±34.66 ng/mL, respectively). Both DKK1 (AUC=0.852) and sAxl (AUC=0.882) had a good diagnostic accuracy in separating HCC from all non-HCC patients. Multiplying DKK1 with sAXL yielded values that significantly (P=0.0001) increased in patients who developed HCC (674.3 (434.2-1413.9)) versus fibrotic (204.9 (161.7-262)) and cirrhotic (254.4 (205.4-343.7)) patients. This model improves HCC diagnostic performances [AUC=0.921; sensitivity 90.9%, specificity 87%, PPV 88.5%, NPV 89.7% and efficiency 89.1%]. Elevated DKK1×sAxl values were associated with aggressive tumor features including multiple nodules, large size, Child-Pugh and BCLC late stages., Conclusions: combined use of DKK1×sAxl is simple and feasible HCC diagnostic model that could enhance HCC diagnostic accuracy and could replace AFP in follow up of patients with premalignant diseases.

The TAM family of receptor tyrosine kinases is implicated in multiple distinct oncogenic signaling pathways. However, to date, there are no FDA-approved small molecule inhibitors for the TAM kinases. Inhibitor design and screening rely on tools to study the kinase activity. Our goal was to address this gap by designing a set of synthetic peptide substrates for each of the TAM family members: Tyro3, Axl, and Mer. We used an in vitro phosphoproteomics workflow to determine the substrate profile of each TAM kinase and input the identified substrates into our data processing pipeline, KINATEST-ID, producing a position-specific scoring matrix for each target kinase and generating a list of candidate synthetic peptide substrates. We synthesized and characterized a set of those substrate candidates, systematically measuring their initial phosphorylation rate with each TAM kinase by LC-MS. We also used the multimer modeling function of AlphaFold2 (AF2) to predict peptide-kinase interactions at the active site for each of the novel candidate peptide sequences against each of the TAM family kinases and observed that, remarkably, every sequence for which it predicted a putative catalytically competent interaction was also demonstrated biochemically to be a substrate for one or more of the TAM kinases. This work shows that kinase substrate design can be achieved using a combination of preference motifs and structural modeling, and it provides the first demonstration of peptide-protein interaction modeling with AF2 for predicting the likelihood of constructive catalytic interactions.

Post-acute conditions after coronavirus disease 2019 (COVID-19) are quite common, although the underlying pathogenetic mechanisms leading to these conditions are not yet completely understood. In this prospective observational study, we aimed to test the hypothesis that Growth Arrest-Specific 6 (Gas6) and its soluble receptors, Axl (sAxl) and MerTK (sMer), might be implicated. A total of 263 subjects underwent a structured clinical evaluation one year after their hospital discharge for COVID-19, and they consented to donate a blood sample to measure their circulating Gas6, sAxl, and sMer levels. A total of 98 (37.3%) post-COVID-19 subjects complained of at least one residual physical symptom one year after their hospital discharge. Univariate analysis revealed that sAxl was marginally associated with residual symptoms, but at the level of logistic regression analysis, only the diffusing capacity of the lungs for carbon monoxide (DLCO) (OR 0.98, CI 95%: 0.96-0.99; p = 0.007) and the female sex (OR 2.49, CI 95%: 1.45-4.28; p = 0.001) were independently associated with long-lasting symptoms. A total of 69 (26.2%) subjects had hair loss. At the level of univariate analysis, Gas6, sAxl, DLCO, and the female gender were associated with its development. In a logistic regression analysis model, Gas6 (OR 0.96, CI 95%: 0.92-0.99; p = 0.015) and sAxl (OR 0.98, CI 95%; 0.97-1.0; p = 0.014), along with the female sex (OR 6.58, CI 95%: 3.39-12.78; p = 0.0001), were independent predictors of hair loss. Decreased levels of Gas6 and sAxl were associated with a history of hair loss following COVID-19. This was resolved spontaneously in most patients, although 23.7% complained of persistent hair loss one year after hospital discharge.

RON is a receptor tyrosine kinase (RTK) of the MET receptor family that is canonically involved in mediating growth and inflammatory signaling. RON is expressed at low levels in a variety of tissues, but its overexpression and activation have been associated with malignancies in multiple tissue types and worse patient outcomes. RON and its ligand HGFL demonstrate cross-talk with other growth receptors and, consequentially, positions RON at the intersection of numerous tumorigenic signaling programs. For this reason, RON is an attractive therapeutic target in cancer research. A better understanding of homeostatic and oncogenic RON activity serves to enhance clinical insights in treating RON-expressing cancers., Competing Interests: The authors declare that they have no conflict of interests.

Receptor tyrosine kinase AXL exerts pivotal roles in cancer cell survival, metastasis, and drug resistance. Pharmacologic or genetic targeting of the aberrant AXL signaling has proven preferable antitumor efficacies in both preclinical and clinical studies, which highlights AXL as an attractive antitumor drug target. By conformational restriction of the anilinopyrimidine 10e and systematic structure-activity relationship (SAR) exploration, we discovered 10 H -benzo[ b ]pyrido[2,3- e ][1,4]oxazine 16j as a potent and orally bioavailable AXL inhibitor. As a type II AXL inhibitor, compound 16j displayed about 15-fold selectivity for AXL over its highly homologous kinase c-Met. And it significantly blocked cellular AXL signaling, inhibited AXL-mediated cell proliferation, and impaired growth arrest-specific protein 6 (Gas6)/AXL-stimulated cell migration and invasion. Moreover, 16j exhibited significant antitumor efficacy in AXL-driven xenograft model at a well-tolerant dosage, causing tumor stasis or regression.

The receptor tyrosine kinase (RTK) anexelekto (AXL) is mutated and/or overexpressed in various malignancies, and plays a central role in tumor development and acquired drug resistance. Although highly selective inhibitors have been developed in recent years, direct inhibition of AXL may block its ubiquitination, eventually leading to surface accumulation of the protein. Herein, we designed and synthesized a series of AXL degraders with high selectivity and without compensatory increase of AXL. In particular, compounds 20 and 22 showed significant AXL degradation capacity, which inhibited the proliferation and migration of cancer cells in vitro. In addition, these compounds induced the formation of cytoplasmic vacuoles and triggered methuosis, a new type of non-apoptotic cell death, by stimulating excessive production of macropinosomes. Vacuole formation was mediated via H-Ras activation, and was attenuated upon inhibition of its downstream regulatory factor Rac1. Furthermore, compound 20 inhibited the growth of tumor cell xenografts in vivo, and prolonged the survival of the tumor-bearing mice., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Masson SAS. All rights reserved.)

To investigate the role of miR-BART4-3p in EBV-associated gastric cancer (EBVaGC) and its regulation of cell proliferation, apoptosis, and migration by targeting AXL in GC. Quantitative real-time PCR and western blot were used to detect the expression of AXL. The methylation status of AXL gene promoter region was determined by bisulfite sequencing PCR. Luciferase reporter assay was used to detect whether miR-BART4-3p targets AXL. The key molecules of EMT and PI3K/AKT pathway were used to examine by western blot. CCK8, Transwell, and flow cytometry were used to detect the phenotypic gastric cancer cells after interference with AXL and miR-BART4-3p. EBV infection inhibited the expression of AXL in GC cells and the inhibition was not caused by the change of promoter methylation status. MiR-BART4-3p directly targeted AXL. Moreover, both inhibition of miR-BART4-3p and AXL inhibited cell proliferation and migration and promoted cell apoptosis. In addition, E-cadherin, Vimentin, ZEB1, and p-AKT were found to be the downstream molecules of the miR-BART4-3p/AXL pathway. The change of promoter methylation status was not the reason for the downregulation of AXL expression in EBV-positive cells. MiR-BART4-3p may inhibit the proliferation and migration and promote apoptosis of GC cells by directly targeting AXL., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Pediatric high-grade glioma is a rare tumor characterized by high mortality. The authors report the first case of a high-grade glioma associated with a GOPC(FIG)-ROS1 fusion in a pediatric patient. The patient underwent gross-total resection at the age of 4 years, followed by adjuvant high-dose chemotherapy and autologous hematopoietic stem cell rescue. At 30 months after transplantation, she remains disease free.

Hepatocyte growth factor (HGF) is a well-known multifunctional growth factor, and evidence has accumulated indicating that the HGF/MET (HGF receptor) signaling axis is involved in the progression of cancer. Macrophage-stimulating protein (MSP) is also known as a growth factor which activates not only macrophages but also cancer cells and osteoclasts through the activation of the specific Receptor d'origine nantais (RON). Pro-HGF and pro-MSP lack biological activity and, therefore, require proteolytic activation for conversion to an active two-chain form by HGF activator (HGFA). Although, there are several studies on HGF/MET signaling with castration-resistant prostate cancer (CRPC) and bone metastasis, reports on plasma protein are rare. In addition, the MSP/RON signaling axis in PC is not well understood. Here, we analyzed associations between PC progression and plasma HGF and MSP levels. We tested plasma samples from 58 patients with PC: 36 with castration-resistant (CR) PC and 22 with pretreatment for PC as control. We used enzyme-linked immunosorbent assay (ELISA) kit to determine plasma levels of HGF, MSP and HGFA, and examined correlations with clinicopathological characteristics such as Gleason grade and bone metastasis. PCR was used to evaluate HGF and MSP-related molecules in PC cell lines. Plasma levels of HGF, MSP and HGFA in the CRPC group were higher than in the control group (HGF: P < 0.001; MSP: P = 0.008; HGFA: P < 0.001). HGF and MSP levels were significantly correlated (P = 0.003). In the CRPC group, plasma HGF and MSP levels and Gleason score were not correlated; however, high plasma MSP level correlated with bone metastasis. (P = 0.016). In cell lines, PC3 expressed significantly more HGF, MET and RON than did LNCaP (P < 0.001), and both cell lines expressed MSP. Plasma concentrations of HGF, MSP and HGFA are significantly elevated in patients with CRPC. Also, as plasma MSP levels are significantly associated with bone metastasis in CRPC patients, MSP may be a candidate for serum marker of bone metastasis. Our results show the importance of the HGF/MET and MSP/RON signaling systems in CRPC.

Purpose: Anaplastic lymphoma kinase (ALK) gene rearrangements define a distinct molecular subset of non-small cell lung cancer (NSCLC). Recently, several case reports and small series have reported that ALK rearrangements can overlap with other oncogenic drivers in NSCLC in crizotinib-naïve and crizotinib-resistant cancers., Experimental Design: We reviewed clinical genotyping data from 1,683 patients with NSCLC and investigated the prevalence of concomitant EGFR or KRAS mutations among patients with ALK-positive NSCLC. We also examined biopsy specimens from 34 patients with ALK-positive NSCLC after the development of resistance to crizotinib., Results: Screening identified 301 (17.8%) EGFR mutations, 465 (27.6%) KRAS mutations, and 75 (4.4%) ALK rearrangements. EGFR mutations and ALK rearrangements were mutually exclusive. Four patients with KRAS mutations were found to have abnormal ALK FISH patterns, most commonly involving isolated 5' green probes. Sufficient tissue was available for confirmatory ALK immunohistochemistry in 3 cases, all of which were negative for ALK expression. Among patients with ALK-positive NSCLC who acquired resistance to crizotinib, repeat biopsy specimens were ALK FISH positive in 29 of 29 (100%) cases. Secondary mutations in the ALK kinase domain and ALK gene amplification were observed in 7 of 34 (20.6%) and 3 of 29 (10.3%) cases, respectively. No EGFR or KRAS mutations were identified among any of the 25 crizotinib-resistant, ALK-positive patients with sufficient tissue for testing., Conclusions: Functional ALK rearrangements were mutually exclusive with EGFR and KRAS mutations in a large Western patient population. This lack of overlap was also observed in ALK-positive cancers with acquired resistance to crizotinib., (©2013 AACR.)

In patients with metastatic colorectal cancer, the predictive value of KRAS mutational status in the selection of patients for treatment with anti-epidermal growth factor (EGFR) monoclonal antibodies is established. In patients with non-small-cell lung cancer (NSCLC), the utility of determining KRAS mutational status to predict clinical benefit to anti-EGFR therapies remains unclear. This review will provide a brief description of Ras biology, provide an overview of aberrant Ras signaling in NSCLC, and summarize the clinical data for using KRAS mutational status as a negative predictive biomarker to anti-EGFR therapies. Retrospective investigations of KRAS mutational status as a negative predictor of clinical benefit from anti-EGFR therapies in NSCLC have been performed; however, small samples sizes as a result of low prevalence of KRAS mutations and the low rate of tumor sample collection have limited the strength of these analyses. Although an association between the presence of KRAS mutation and lack of response to EGFR tyrosine kinase inhibitors (TKIs) has been observed, it remains unclear whether there is an association between KRAS mutation and EGFR TKI progression-free and overall survival. Unlike colorectal cancer, KRAS mutations do not seem to identify patients who do not benefit from anti-EGFR monoclonal antibodies in NSCLC. The future value of testing for KRAS mutational status may be to exclude the possibility of an EGFR mutation or anaplastic lymphoma kinase translocation or to identify a molecular subset of patients with NSCLC in whom to pursue a drug development strategy that targets the KRAS pathway.

Menin upregulates transcription of cell-cycle inhibitors to suppress endocrine tumors, but it is poorly understood how menin suppresses non-endocrine tumors such as lung cancer. Here, we show that menin inhibits proliferation of human lung cancer cells and growth of lung cancer in mice. The menin-mediated tumor suppression requires repression of growth factor pleiotrophin (PTN), which binds to its cell surface receptor, anaplastic lymphoma kinase (ALK) that is activated in certain lung adenocarcinomas. Menin represses PTN transcription and PTN-induced proliferation of human lung cancer cells, and menin expression is substantially reduced in primary human lung adenocarcinomas. Notably, menin binds the PTN locus and enhances Polycomb gene Enhancer of Zeste homolog 2 (EZH2)-mediated histone H3 lysine 27 trimethylation (H3K27m3), a negative mark for gene transcription but does not affect histone H3K4 methylation that is usually upregulated by menin in endocrine cells. Together, our findings indicate that menin suppresses lung cancer partly through increasing Polycomb gene-mediated H3K27 methylation and repressing PTN transcription, unraveling a novel, epigenetically regulated PTN-ALK signaling pathway in suppressing lung cancer.

As in HIV-1 infection in humans, SIVsm infection of rhesus macaques causes a slow progressive loss of CD4 T-cells followed by the onset of AIDS. In addition, there is a loss of dendritic cells (DC) in peripheral blood, peripheral lymphoid tissues, and the skin. Increasing the number of CD4 T cells and DC may be an important step in restoring immune competence and thus delay disease progression. Recently, progenipoietins (ProGP), a new family of chimeric Flt3 and G-CSF receptor agonists, were demonstrated to possess the capacity to mobilize hematopoietic progenitor cells in normal rhesus monkeys. In addition, these molecules induced increased numbers of myeloid cells, including dendritic cells, in the blood. Here we demonstrate that SIVsm-infected macaques, treated with ProGP-1, developed increased numbers of both plasmacytoid (CD123+, CD11c-) and myeloid (both CD11b+, CD11c+, and CD123-, CD11c+ subsets) DC and CD4 and CD8 T cells in peripheral blood. Importantly, during treatment, no changes in plasma virus load were observed. After 14 to 20 days of treatment, antibodies were formed against ProGP in all animals. As a consequence, white blood cell levels returned to baseline in several animals. In other animals values only returned to baseline after termination of ProGP treatment. In conclusion, ProGP-1 may be used to generate a transient increase in DC as well as CD4 T-cell numbers, thereby creating a window of opportunity for immunotherapeutic intervention.

Aims: The pathogenic mechanism and predictive indicators of biological behaviour of inflammatory myofibroblastic tumour are poorly understood. We investigated molecular abnormalities of p53 and MDM2 in order to assess whether these play an important role in pathogenesis, and whether they also contribute to clinicopathological aggressive phenotype in inflammatory myofibroblastic tumour., Methods and Results: We compared the immunohistochemical expression of calponin, h-caldesmon, ALK, and p53 gene mutation and MDM2 gene amplification with clinicopathological findings in 15 cases of inflammatory myofibroblastic tumour. Histologically, cellular atypia was observed in five (33.3%) out of 15 cases. Local recurrences were observed in two (14.3%) of 14 informative cases, but no distant metastasis was observed. The expression of calponin (9/14; 64%) but not h-caldesmon (0/14; 0%) was seen, which suggested myofibroblastic differentiation. ALK expression was seen in eight (53.3%) out of 15 cases, particularly in patients under 40 years old. Nuclear expression of p53 protein was recognized in only one (6.7%) of 15 cases, and polymerase chain reaction single-strand conformation polymorphism followed by direct sequencing revealed p53 gene missense mutations in two (13.3%) of 15 cases. Nuclear expression of MDM2 was seen in four (26.7%) of 15 cases, and the MDM2 gene amplification was observed in two of the four cases., Conclusion: Inflammatory myofibroblastic tumour shows a wide spectrum of cellular atypia and biological behaviour with p53 and MDM2 expression. However, the alterations in the p53 pathway seem not to play a major role in the pathogenesis of inflammatory myofibroblastic tumour.

Purpose: To determine whether mice that are homozygous for a targeted disruption of the Mer receptor tyrosine kinase gene (mer(kd)) manifest a retinal dystrophy phenotype similar to RCS rats, which carry a mutation in the orthologous gene MERTK:, Methods: Eyes of mer(kd) and C57BL/6 wild-type (WT) mice were examined by light and electron microscopy, whole-eye rhodopsin measurement, and Ganzfeld electroretinography (ERG)., Results: The mer(kd) mice showed rapid, progressive degeneration of the photoreceptors (PRs). Features of the phenotype common to mer(kd) mice and RCS rats included the absence or near absence of phagosomes in the retinal pigment epithelium (RPE) at the peak of outer segment (OS) disc shedding, accumulation of debris and whorls of membranes at the RPE-OS interface, transient supernormal rhodopsin content and OS lengths, the presence of OS vacuoles beginning at early ages, and a relatively slow removal of pyknotic PR nuclei. Most PRs were missing, and OS debris was removed by approximately postnatal day (P)45. Scotopic ERG responses were lower than age-matched WT responses and declined with PR loss. Photopic responses were preserved better than scotopic responses, corresponding with preferential cone preservation as judged histologically. ERG amplitudes were usually unmeasurable beyond P40, although a small-amplitude scotopic threshold response (STR) could still be elicited at P253 in some mice when only scattered PR nuclei remained., Conclusions: Ablation of Mer function in mer(kd) mice results in a retinal phenotype almost identical with that of RCS rats. The similarity in phenotypes between the two rodent models suggests that an RPE phagocytic defect is a feature of all types of retinal degeneration caused by loss of function of Mer tyrosine kinase, perhaps including mutations in human MERTK.

Background: Progenipoietin-4 (ProGP-4) is an E. coli derived chimeric growth factor that activates the human Flt3 and G-CSF receptors. ProGP-4 possesses cross-species activity and treatment of mice with ProGP-4 results in increases in the number of WBC and Class II+/CD11c+ cells in both spleen and peripheral blood. Herein, we report morphologic, phenotypic and functional evaluation of Class II+/CD11c+ cells generated by in vivo administration of ProGP-4., Material/methods: C57BL/6 mice were injected daily with ProGP for 7 to 18 days. Leukocytes from spleen and peripheral blood were analyzed by flow cytometry to enumerate and characterize changes in DC populations. Spleens from ProGP treated mice were evaluated by immunocytochemistry and enriched CD11c+ populations were functionally assessed in a mixed lymphocyte assay and in an antigen dependent CTL assay., Results: Administration of this dual receptor agonist to mice resulted in dose-dependent increases in the numbers of total white blood cells and Class II+/CD11c+ cells in spleen and peripheral blood. CD11c+ cells from ProGP-4 treated mice co-expressed DEC205 and also expressed CD80, CD86 and CD40, albeit at lower levels as compared to Class II+/CD11c+ cells from untreated animals. Despite lower co-stimulatory molecule expression, ProGP-4-generated Class II+/CD11c+ cells stimulated proliferation of allogeneic T cells and an antigen-specific T cell hybridoma as efficiently as bone marrow derived dendritic cells from untreated mice., Conclusions: The data presented in this report highlight the ability of E. coli derived ProGP-4 to expand large numbers of functional DC in the peripheral blood and lymphoid organs in vivo using a rodent model of hematopoiesis. E. coli derived chimeric receptor agonists such as ProGP-4 may enable further investigations of immunotherapeutic approaches to the treatment of diseases such as cancer and autoimmunity.

Mice lacking the membrane tyrosine kinase c-mer have been shown to have altered macro-phage cytokine production and defective phagocytosis of apoptotic cells despite normal phagocytosis of other particles. We show here that c-mer-deficient mice have impaired clearance of infused apoptotic cells and that they develop progressive lupus-like autoimmunity, with antibodies to chromatin, DNA, and IgG. The autoimmunity appears to be driven by endogenous antigens, with little polyclonal B cell activation. These mice should be an excellent model for studying the role of apoptotic debris as an immunogenic stimulus for systemic autoimmunity.

Apoptosis is fundamental to the development and maintenance of animal tissues and the immune system. Rapid clearance of apoptotic cells by macrophages is important to inhibit inflammation and autoimmune responses against intracellular antigens. Here we report a new function for Mer, a member of the Axl/Mer/Tyro3 receptor tyrosine kinase family. mer(kd) mice with a cytoplasmic truncation of Mer had macrophages deficient in the clearance of apoptotic thymocytes. This was corrected in chimaeric mice reconstituted with bone marrow from wild-type animals. Primary macrophages isolated from mer(kd) mice showed that the phagocytic deficiency was restricted to apoptotic cells and was independent of Fc receptor-mediated phagocytosis or ingestion of other particles. The inability to clear apoptotic cells adequately may be linked to an increased number of nuclear autoantibodies in mer(kd) mice. Thus, the Mer receptor tyrosine kinase seems to be critical for the engulfment and efficient clearance of apoptotic cells. This has implications for inflammation and autoimmune diseases such as systemic lupus erythematosus.

Sprouty (SPRY) was first identified in a genetic screen in Drosophila as an antagonist of fibroblast and epidermal growth factor receptors and Sevenless signaling, seemingly by inhibiting the receptor tyrosine kinase (RTK)/Ras/MAPK pathway. To date, four mammalian Sprouty genes have been identified; the primary sequences of the gene products share a well conserved cysteine-rich C-terminal domain with their Drosophila counterpart. The N-terminal regions do not, however, exhibit a large degree of homology. This study was aimed at identifying proteins with which human SPRY2 (hSPRY2) interacts in an attempt to understand the mechanism by which Sprouty proteins exert their down-regulatory effects. Here, we demonstrate that hSPRY2 associates directly with c-Cbl, a known down-regulator of RTK signaling. A short sequence in the N terminus of hSPRY2 was found to bind directly to the Ring finger domain of c-Cbl. Parallel binding was apparent between the Drosophila homologs of Sprouty and Cbl, with cross-species associations occurring at least in vitro. Coexpression of hSPRY2 abrogated an increase in the rate of epidermal growth factor receptor internalization induced by c-Cbl, whereas a mutant hSPRY2 protein unable to bind c-Cbl showed no such effect. Our results suggest that one function of hSPRY2 in signaling processes downstream of RTKs may be to modulate c-Cbl physiological function such as that seen with receptor-mediated endocytosis.

Male "viable motheaten" (me(v)) mice, with a naturally occurring mutation in the gene of the SH2 domain protein tyrosine phosphatase SHP-1, are sterile. Known defects in sperm maturation in these mice correlate with an impaired differentiation of the epididymis, which has similarities to the phenotype of mice with a targeted inactivation of the Ros receptor tyrosine kinase. Ros and SHP-1 are coexpressed in epididymal epithelium, and elevated phosphorylation of Ros in the epididymis of me(v) mice suggests that Ros signaling is under control of SHP-1 in vivo. Phosphorylated Ros strongly and directly associates with SHP-1 in yeast two-hybrid, glutathione S-transferase pull-down, and coimmunoprecipitation experiments. Strong binding of SHP-1 to Ros is selective compared to six other receptor tyrosine kinases. The interaction is mediated by the SHP-1 NH(2)-terminal SH2 domain and Ros phosphotyrosine 2267. Overexpression of SHP-1 results in Ros dephosphorylation and effectively downregulates Ros-dependent proliferation and transformation. We propose that SHP-1 is an important downstream regulator of Ros signaling.

The RCS rat presents an autosomal recessive retinal pigment epithelium dystrophy characterized by the outer segments of photoreceptors being phagocytosis-deficient. A systematic genetic study allowed us to restrict the interval containing the rdy locus to that between the markers D3Mit13 and D3Rat256. We report the chromosomal localization of the rat c-mer gene in the cytogenetic bands 3q35-36, based on genetic analysis and radiation hybrid mapping. Using a systematic biocomputing analysis, we identified two strong related candidate genes encoding protein tyrosine kinase receptors of the AXL subfamily. The comparison of their expression patterns in human and mice tissues suggested that the c-mer gene was the best gene to screen for mutations. RCS rdy- and RCS rdy+ cDNAs were sequenced. The RCS rdy- cDNAs carried a significant deletion in the 5' part of the coding sequence of the c-mer gene resulting in a shortened aberrant transcript encoding a 20 amino acid peptide. The c-mer gene contains characteristic motifs of neural cell adhesion. A ligand of the c-mer receptor, Gas6, exhibits antiapoptotic properties., (Copyright 2000 Academic Press.)

The c-Mer receptor tyrosine kinase (RTK) is most closely related to chicken c-Eyk and belongs to the Axl RTK subfamily. Although not detected in normal lymphocytes, c-Mer is expressed in B- and T-cell leukemia cell lines, suggesting an association with lymphoid malignancies. To gain an understanding of the role of this receptor in lymphoid cells, we expressed in murine interleukin-3 (IL-3)-dependent Ba/F3 pro-B-lymphocyte cells a constitutively active receptor, CDMer, formed from the CD8 extracellular domain and the c-Mer intracellular domain. Cells transfected with a plasmid encoding the CDMer receptor became IL-3 independent. When tyrosine (Y)-to-phenylalanine (F) mutations were introduced into c-Mer, only the Y867 change significantly reduced the IL-3-independent cell proliferation. The Y867 residue in the CDMer receptor mediated the binding of Grb2, which recruited the p85 phosphatidylinositol 3-kinase (PI 3-kinase). Despite the difference in promotion of proliferation, both the CDMer and mutant F867 receptors activated Erk in transfected cells. On the other hand, we found that both transcriptional activation of NF-kappaB and activation of PI 3-kinase were significantly suppressed with the F867 mutant receptor, suggesting that the activation of antiapoptotic pathways is the major mechanism for the observed phenotypic difference. Consistent with this notion, apoptosis induced by IL-3 withdrawal was strongly prevented by CDMer but not by the F867 mutant receptor.

The processes of endocytosis and recycling have been well characterized in renal proximal tubule and yolk sac epithelia. We utilized a yolk sac teratocarcinoma cell line, BN/MSV, which expresses two glycoproteins, megalin/gp330 and gp280, also detected in renal proximal tubule and yolk sac epithelial cells. In this study, we further define the localization, internalization and intracellular trafficking of both proteins in BN/MSV cells. For this purpose, double indirect immunofluorescence and immunoelectron microscopy were performed on BN/MSV cells. In addition, antibodies against gp280 and gp330, coupled to colloidal gold particles, were used as tracers to follow the endocytosis and recycling of the two glycoproteins in BN/MSV cells. BSA and MOPC21 (a nonspecific monoclonal antibody) coupled to gold particles were used as controls. We have previously shown that gp280 and megalin/gp330 were localized in clathrin-coated pits; both proteins can also be detected in noncoated areas. Vesicular labeling has previously been seen in the cytoplasm of permeabilized BN/ MSV cells. The results of the present study revealed that the glycoproteins were colocalized in the same cells. Ultrastructural analysis of ultracryosections of BN/MSV cells revealed a localization of both proteins in coated invaginations and small and large endocytic vacuoles. In addition, gp280 and megalin/gp330 were found in the Golgi apparatus and in the granular endoplasmic reticulum. Furthermore, incubation of BN/MSV cells in the presence of colloidal gold particles labeled with antibodies to gp280 and gp330 demonstrated an internalization from the apical membrane through coated pits into small and large endocytic vacuoles. While anti-gp330 is predominantly localized in large endocytic vacuoles, the anti-gp280 gold is mainly concentrated in the tubulovesicular structures, which probably correspond to dense tubules known to enable membrane recycling in the epithelial cells of renal proximal tubules. Moreover, anti-gp330 gold particles are also found in lysosomes, but to a lesser extent than BSA and MOPC21 gold particles, which were highly concentrated in lysosomes. In conclusion, our results show that gp280 is internalized in BN/MSV cells and that anti-gp280 gold is accumulated in a recycling compartment. Thus, we suggest that gp280 is a receptor for endocytosis.

The luminal plasma membrane of the epithelial cells lining the visceral layer of the yolk sac and the renal proximal tubule display a well developed brush border defining numerous clathrin-coated intermicrovillar areas which are further characterized by expressing two glycoproteins, gp280 and gp330, the latter also known as the Heymann nephritis antigen. The present study analyzes the distribution, the internalization and intracellular trafficking of gp280 and gp330 in yolk sac epithelium by immunoultrastructural and cell surface labeling techniques. Immunocytochemistry revealed that gp280 and gp330 were distributed very similarly in the endocytic pathway including dense apical tubules, with the exception that gp330 was found in lysosomes to a much greater extent than gp280. To demonstrate internalization of gp280, apical cell membrane proteins of paired yolk sacs were labeled at 4 degrees C with biotin, linked via a disulfide bond cleavable under mild reducing conditions by glutathione, and either kept at 4 degrees C or incubated at 37 degrees C. These experiments showed that biotin could be cleaved from gp280 by glutathione in yolk sacs kept at 4 degrees C, whereas it became inaccessible to glutathione after incubation at 37 degrees C, suggesting internalization of gp280. Furthermore, incubation of yolk sacs in the presence of colloidal gold-labeled antibodies to gp280 demonstrated that gp280, initially expressed on the cell membrane, was translocated into endocytic vacuoles and accumulated in dense apical tubules, whereas only a small fraction reached the lysosomes. Under similar conditions, gold-labeled antibodies to gp330 were also internalized and followed a similar intracellular routing, but lysosomal accumulation was also found. Bovine serum albumin-labeled gold particles accumulated in lysosomes but were virtually absent from dense apical tubules. These observations suggest that gp280 and gp330, visualized by anti-gp280 and anti-gp330 antibodies coupled to gold particles, returned to the cell surface via dense apical tubules, whereas albumin gold particles dissociated from a potential binding protein in the early endocytic compartment and were subsequently accumulated in lysosomes. Since gp280 is internalized and apparently translocated to a recycling compartment as is gp330, our results suggest that gp280 may play a role as a receptor for endocytosis of as yet unknown ligands.

After the onset of pupation, sensory organ precursors, the progenitors of the interommatidial bristles, are selected in the developing Drosophila eye. We have found that wingless, when expressed ectopically in the eye via the sevenless promoter, blocks this process. Transgenic eyes have reduced expression of acheate, suggesting that wingless acts at the level of the proneural genes to block bristle development. This is in contrast to the wing, where wingless positively regulates acheate to promote bristle formation. The sevenless promoter is not active in the acheate-positive cells, indicating that the wingless is acting in a paracrine manner. Clonal analysis revealed a requirement for the genes porcupine, dishevelled and armadillo in mediating the wingless effect. Overexpression of zeste white-3 partially blocks the ability of wingless to inhibit bristle formation, consistent with the notion that wingless acts in opposition to zeste white-3. Thus the wingless signaling pathway in the eye appears to be very similar to that described in the embryo and wing. The Notch gene product has also been suggested to play a role in wingless signaling (J. P. Couso and A. M. Martinez Arias (1994) Cell 79, 259-72). Because Notch has many functions during eye development, including its role in inhibiting bristle formation through the neurogenic pathway, it is difficult to assess the relationship of Notch to wingless in the eye. However, we present evidence that wingless signaling still occurs normally in the complete absence of Notch protein in the embryonic epidermis. Thus, in the simplest model for wingless signalling, a direct role for Notch is unlikely.

Fgf6 is the only known member of the FGF family whose expression is restricted to the muscle cell lineage during development, suggesting it may have a role in myogenesis. Muscle satellite cells but not C2 myoblast cells were found to express Fgf6. We have used purified recombinant FGF6 protein to explore the effect of this factor on C2 cells in culture. FGF6 stimulated the proliferation of C2 myoblasts and, in combination with heparin, induced their morphological transformation. FGF6, added at 5 ng/ml and in the presence of heparin, increased the expression of a subset of muscle cell differentiation markers. In contrast, at 25 ng/ml, it down-regulated the expression of myogenic markers and myogenic transcription factors examined and delayed differentiation into myotubes of C2 cells. It also up-regulated the expression of FgfR1 and had an opposite effect on FgfR4. These results suggest that intramuscular FGF6 concentrations could influence the proliferation and differentiation processes taking place during development.

The sevenless gene encodes a receptor tyrosine kinase which is required for the development of the R7 photoreceptor cell in each ommatidium of the Drosophila eye. We have previously used a sensitized genetic screen to identify mutations, designated Enhancers of sevenless (E(sev)), which affect genes that encode components of the sevenless signaling pathway. Here, we report that one of these mutations, E(sev)1Ae0P is a dominantly inhibiting allele of corkscrew, which encodes an SH2 domain-containing protein tyrosine phosphatase (Perkins et al., 1992). We show that corkscrew function is essential for sevenless signaling and that expression of a membrane-targeted form of corkscrew can drive R7 photoreceptor development in the absence of sevenless function. Furthermore, we have used the dominantly inhibiting corkscrew allele to examine the role of corkscrew during signaling by activated forms of Ras1 and Raf. Our analysis indicates that corkscrew function is still required during signaling by activated forms Ras1 and Raf proteins. These results define a function for corkscrew that is either downstream of Ras1 activation or in a parallel pathway that acts with activated Ras1/Raf to specify R7 photoreceptor development.

R7 photoreceptor fate in the Drosophila eye induced by the activation of the Sevenless receptor tyrosine kinase and the RAS/MAP kinase signal transduction pathway. We show that expression of a constitutively activated JUN isoform in ommatidial precursor cells is sufficient to induce R7 fate independent of upstream signals normally required for photoreceptor determination. We present evidence that JUN interacts with the ETS domain protein Pointed to promote R7 formation. This interaction is cooperative when both proteins are targeted to the same promoter and is antagonized by another ETS domain protein, YAN, a negative regulator of R7 development. Furthermore, phyllopod, a putative transcriptional target of RAS pathway activation during R7 induction, behaves as a suppressor of activated JUN. Taken together, these data suggest that JUN and Pointed act on common target genes to promote neuronal differentiation in the Drosophila eye, and that phyllopod might be such a common target.

The family of protein kinases includes many oncogenes and growth factor receptors, many of which have been linked to the pathogenesis and progression of cancer. Protein tyrosine kinases such as HER-2/c-erbB-2 and the epidermal growth factor receptor (EGFR) have been linked specifically to breast cancer, and perturbations of HER-2 affect response to chemotherapy. We have reviewed the biology of protein kinases in human breast cancer, as well as their translational applications to breast cancer patients. We have studied the spectrum of protein kinases expressed in human breast cancer cells and have identified four protein kinases with potentially important functions in breast cancer: rak (src-related), TK5 (which we now designate JAK3), the focal adhesion kinase (FAK), and STK1 (human M015/CAK). We describe the potential significance of these genes in breast cancer, as well as our methodology for identifying and characterizing novel genes in breast cancer.

We have cloned the putative mouse homologue of the human c-mer receptor tyrosine kinase proto-oncogene. Comparison of the mouse and human c-mer amino acid sequences reveals an overall identity of 88%. Similar to the human, the extracellular region of the murine c-mer protein possesses two amino terminal immunoglobulin-like domains and two membrane proximal fibronectin type III domains, which places it in the Axl family of tyrosine kinases. Our analysis of the Axl family identifies eight different regions of amino acid consensus that have residues characteristic of this and no other tyrosine kinase family; six of the eight are within tyrosine kinase subdomains. The homology within the Axl family is highest between c-mer and c-eyk, the chicken proto-oncogene of the tumor virus gene product v-eyk. Northern analysis of adult tissues suggests that the mouse c-mer, although expressed in many tissues, has an expression pattern unique among Axl family members. In normal adult hematopoietic cells c-mer seems to be expressed predominantly if not exclusively in the monocytic lineage. Mouse c-mer is expressed during most, if not all, stages of embryological development beginning in the morula and blastocyst and progressing through the yolk sac and fetal liver stages. This early and consistent expression of c-mer was confirmed during in vitro differentiation of embryonic stem cells. The embryonic expression profile of c-mer suggests that this tyrosine kinase may play an important function in the developing mouse.

A human B-lymphoblastoid lambda gt11 expression library was screened using anti-phosphotyrosine antibodies yielding complementary DNAs encoding active tyrosine kinases. The resulting clones were used to obtain the sequence of a novel 984 amino acid transmembrane tyrosine kinase. Analysis of the complementary DNA revealed extracellular immunoglobulin and fibronectin type III domains and the unusual kinase signature sequence KWIAIES; all are characteristic of the axl family of tyrosine kinases. The novel tyrosine kinase was not expressed in normal B- and T-lymphocytes but, unlike axl, was expressed in numerous neoplastic B- and T-cell lines. Transcripts for the novel receptor-like tyrosine kinase were detected in normal peripheral blood monocytes and bone marrow. One alternatively spliced transcript was detected which contained an insert in the membrane proximal region that could encode for a truncated, soluble receptor. Sequence comparison shows that the kinase may be the human protooncogene for the recently isolated chicken retroviral oncogene v-ryk (recently renamed v-eyk), a truncated tyrosine kinase whose expression by retroviral infection produced sarcomas in chickens. The intracellular domain of the human kinase shows 83% similarity and 71% identity to v-ryk. Since the ryk designation has been used to name another tyrosine kinase and an analysis of RNA expression demonstrated that this novel human kinase is expressed in monocytes and tissues of epithelial and reproductive origin, we have designated our novel protooncogene c-mer.

We examined the effect of Steel factor (SLF) on the development of human mast cells in hematopoietic colonies from cord blood mononuclear cells in methylcellulose culture. When cord blood cells were cultured for 3 weeks, SLF increased the cellular tryptase levels detected in total cultured cells. It also stimulated the formation of small-cell colonies consisting mainly of polymorphonuclear granulocytes and immature blastoid cells in a concentration-dependent manner but not the formation of colonies consisting of large macrophagic cells. A low percentage of tryptase-positive mast-cell-like cells was found in 39 out of 100 granulocyte/blastoid cell colonies. Four of the 100 colonies contained 10-20% tryptase-positive cells, but we failed to observe colonies consisting of > 20% of tryptase-positive cells. These results suggest that the effect of SLF on mast cell growth is brought on by stimulating the growth of primitive hematopoietic progenitors.

The transforming gene of avian sarcoma virus UR2, v-ros, encodes a receptor-like protein tyrosine kinase and differs from its proto-oncogene, c-ros, in its 5' truncation and fusion to viral gag, a three-amino-acid (aa) insertion in the transmembrane (TM) domain, and changes in the carboxyl region. To explore the basis for activation of the c-ros transforming potential, various c-ros retroviral vectors containing those changes were constructed and studied for their biological and biochemical properties. Ufcros codes for the full-length c-ros protein of 2,311 aa, Uppcros has 1,661-aa internal deletion in the extracellular domain, CCros contains the 3' c-ros cDNA fused 150 aa upstream of the TM domain to the UR2 gag, CVros is the same as CCros except that the 3' region is replaced by that of v-ros, and VCros is the same as CCros except that the 5' region is replaced by that of v-ros. The Ufcros, Uppcros, CCros, and CVros are inactive in transforming chicken embryo fibroblasts, whereas VCros is as potent as UR2 in cell-transforming and tumorigenic activities. Upon passages of CCros and CVros viruses, the additional extracellular sequence in comparison with that of v-ros was delected; concurrently, both viruses (named CC5d and CV5d, respectively) attained moderate transforming activity, albeit significantly lower than that of UR2 or VCros. The native c-ros protein has a very low protein tyrosine kinase activity, whereas the ppcros protein is constitutively activated in kinase activity. The inability of CCros and CVros to transform chicken embryo fibroblasts is consistent with the inefficient membrane association, instability, and low kinase activity of their encoded proteins. The CC5d and CV5d proteins are indistinguishable in kinase activity, membrane association, and stability from the v-ros protein. The reduced transforming potency of CC5d and CV5d proteins can be attributed only to their differential substrate interaction, notably the failure to phosphorylate a 88-kDa protein. We conclude that the 5' rather than the 3' modification of c-ros is essential for its oncogenic activation; the sequence upstream of the TM domain has a negative effect on the transforming activity of CCros and CVros and needs to be deleted to activate their biological activity.

A genomic contig of the human ret protooncogene was created with four overlapping cosmid clones isolated from two libraries. After southern analysis with portions of the ret cDNA, eight cosmid fragments were analysed in detail for the presence of ret exons using exon trapping. PCR products corresponding to spliced exons were isolated and subcloned. Exon boundaries were delineated by comparison of the PCR product sequence and the published ret cDNA sequence. The exons were initially positioned on a genomic map defined by BamHI, EcoRI and HindIII restriction sites. The positions of the exons were then refined by amplifying genomic DNA using primer pairs derived from one or more exons along the ret gene, the length of the PCR product indicating the approximate genomic distance between the exon sequences. The ret proto-oncogene is composed of at least 20 exons, ranging in size from 60 bp to 287 bp, distributed along 30 kb of genomic DNA. The extracellular domain is encoded by 10 exons and the cytoplasmic domain by 9 exons. The transmembrane domain is encoded by a single exon.

We examined the effect of herbimycin A, a potent inhibitor of tyrosine kinases, on NIH(ret) cells and TPC-1 papillary thyroid carcinoma cells, both of which express the active ret genes. Herbimycin A reversed the morphology of NIH(ret) cells to flat cells with a concomitant reassembly of microfilament bundles. On the other hand, it did not induce a significant change in cell shape of TPC-1 cells. When tyrosine kinase activities of the active ret gene products in herbimycin A-treated NIH(ret) and TPC-1 cells were examined in immunocomplex kinase assays, they drastically decreased in both cells as compared with untreated cells. In addition, herbimycin A strongly inhibited tyrosine phosphorylation of 40 kDa and 31 kDa proteins present in the immunoprecipitates of both cells, suggesting that these proteins could associate with the Ret proteins.

The human FLT3 cDNA was cloned from a pre-B cell line and characterized. The deduced amino acid sequence shows that FLT3 codes for a receptor-type tyrosine kinase of 993 residues, presenting a strong similarity with the corresponding mouse FLT3/FLK2 protein as well as with the receptors for colony-stimulating factor 1 (CSF1R/FMS) and steel locus factor (SLFR/KIT). An analysis of the expression of the gene using amplification of reverse transcribed FLT3 mRNA by polymerase chain reaction shows that FLT3 is expressed in various lymphohematopoietic cells and tissues, including a series of immature cell lines and leukemias of lymphocytic origin.

Mouse pluripotent hematopoietic stem cells (PHSC) were fractionated based on size and density using counterflow centrifugal elutriation (CCE). These heterogeneous PHSC populations were further enriched by subtraction of cells with lineage-specific markers (Lin-) followed by positive sorting for c-kit expression. The cells were characterized for their functional and biochemical properties. We defined a subpopulation of c-kit-positive cells that expressed high numbers of c-kit receptors (c-kitBR). One hundred c-kitBR cells from either low- or higher-density fractions were sufficient to repopulate the lymphohematopoietic system in WBB6F1-W/Wv (W/Wv) recipients, whereas no PHSC were found in cells with low (c-kitDULL) or no (c-kitNEG) c-kit expression. Lin- c-kitBR cells were separated into RhoDULL and RhoBR subsets based on their ability to efflux rhodamine 123 (Rho). The PHSC were concentrated in Lin- c-kitBR RhoDULL cells and the number of Lin- c-kitBR RhoBR cells correlated directly with the number of day 12 colony-forming unit-spleen (CFU-S12) in each fraction. We were not able to enrich further for PHSC using monoclonal antibodies to the cell-surface markers AA4.1 or CD4, which have been used by others to isolate PHSC. The small, low-density Lin- c-kitBR subset contained PHSC and few CFU-S12. This enabled us to assay PHSC for expression of the flk-2 gene, which encodes a tyrosine kinase receptor present on fetal liver PHSC. Purified RNA from the low-density Lin- c-kitBR subset did not contain flk-2 mRNA. We suggest that AA4.1, CD4 and flk-2 are expressed as stage-specific markers on PHSC in cell cycle.

Elevated risk of thyroid cancers among the atomic bomb survivors as compared to the nonexposed population suggests that some genetic events related to thyroid cancer must be caused by ionizing radiation. Accordingly, inducibility of RET oncogene rearrangements, i.e., the generation of the RET-PTC oncogene, specific for thyroid cancer, was investigated among human undifferentiated thyroid carcinoma cells (8505C), which do not have RET oncogene rearrangement, after 0, 10, 50, and 100 Gy of in vitro X-irradiation by means of reverse transcription polymerase chain reaction. After testing 10(8) cells at each dose point, 3 independent samples obtained with 50 Gy of X-irradiation and 6 independent samples obtained with 100 Gy of X-irradiation showed a rearranged RET oncogene amplified band. No rearranged transcripts were obtained from cells irradiated with 0 or 10 Gy. All of the transcripts were sequenced and found to contain the D10S170 and RET sequence. Interestingly, two types of rearrangements were included in these transcripts: one is specific for thyroid cancer and the other, which contains a 150-base pair insert, is atypical, not usually seen in vivo. This insert was found to be the exon of D10S170. Furthermore, in fibrosarcoma cells (HT1080), X-irradiation also induced RET oncogene rearrangements, which included the same two types of rearrangements observed in the X-irradiated thyroid cells (8505C). These results are in favor of the hypothesis that some radiation-induced thyroid cancers, including those among atomic bomb survivors, might have developed when a growth advantage was obtained through a specific form of RET oncogene rearrangement induced by radiation exposure.

Forty-three fresh tumor specimens of human neuroblastoma belonging to different clinical stages were analyzed for the expression of 2 proto-oncogenes: trk, which encodes a tyrosine-kinase receptor for nerve growth factor (NGF) and ret, another receptor-type tyrosine kinase whose ligand is unknown. The mRNA expression of the trk gene was detected in 67.4% of cases, with increased frequency in I, II and IVs Evans' stages and in patients with favorable prognosis according to the Shimada classification. Moreover, trk expression inversely correlated with Nmyc-gene amplification. ret mRNA was found in 36.8% of cases and equally distributed in the different stages. In addition, ngfR (low-affinity NGF receptor)-gene expression was present in 9 out of 25 cases. The simultaneous presence of mRNA related to both forms of the NGF receptor, while not proving the presence of a functional receptor, indicates the existence of a sub-set of neuroblastoma cells potentially responsive to NGF.

We investigated tyrosine kinase activity of the ret proto-oncogene products (proto-Ret proteins), using a cell lysate of NB-39-nu neuroblastoma cells. The 150 kDa and 170 kDa proto-Ret proteins immunoprecipitated with antibodies against their carboxy-terminal 20 amino acids were shown to be phosphorylated predominantly on tyrosine residues in immunocomplex kinase assay. The level of tyrosine phosphorylation of the 150 kDa proto-Ret protein was approximately 10-fold higher than that of the 170 kDa proto-Ret protein, although both proteins were expressed at similar levels in neuroblastoma cells. This result was confirmed by using a lysate of SK-N-MC human primitive neuroectodermal tumor cells transfected with the ret proto-oncogene. The kinase activity of proto-Ret proteins was significantly inhibited by antibodies against their kinase domain, indicating that these antibodies recognize crucial epitopes for the enzymatic activity. On the other hand, the proto-Ret proteins were not phosphorylated in vivo in NB-39-nu cells and SK-N-MC transfectants.

Multiple endocrine neoplasia type 2A (MEN 2A) is a dominantly inherited cancer syndrome that affects tissues derived from neural ectoderm. It is characterized by medullary thyroid carcinoma (MTC) and phaeochromocytoma. The MEN2A gene has recently been localized by a combination of genetic and physical mapping techniques to a 480-kilobase region in chromosome 10q11.2 (refs 2,3). The DNA segment encompasses the RET proto-oncogene, a receptor tyrosine kinase gene expressed in MTC and phaeochromocytoma and at lower levels in normal human thyroid. This suggested RET as a candidate for the MEN2A gene. We have identified missense mutations of the RET proto-oncogene in 20 of 23 apparently distinct MEN 2A families, but not in 23 normal controls. Further, 19 of these 20 mutations affect the same conserved cysteine residue at the boundary of the RET extracellular and transmembrane domains.

The K-FGF/HST (FGF-4) growth factor is a member of the FGF family which is efficiently secreted and contains a single N-linked glycosylation signal. To study the role of glycosylation in the secretion of K-FGF, we mutated the human K-fgf cDNA to eliminate the glycosylation signal and the mutated cDNA was cloned into a mammalian expression vector. Studies of immunoprecipitation from the conditioned medium of cells expressing this plasmid revealed that the lack of glycosylation did not impair secretion, however the unglycosylated protein was immediately cleaved into two NH2-terminally truncated peptides of 13 and 15 kD, which appeared to be more biologically active than the wild-type protein. These two proteins also showed higher heparin binding affinity than that of wt K-FGF. We have expressed in bacteria the larger of these two proteins (K140), in which the NH2-terminal 36 amino acids present in the mature form of K-FGF have been deleted. Mitogenicity assays on several cell lines showed that purified recombinant K140 had approximately five times higher biological activity than wild-type recombinant K-FGF. Studies of receptor binding showed that K140 had higher affinity than wt K-FGF for two of the four members of FGF receptor's family, specifically for FGFR-1 (flg) and FGFR-2 (bek). K140 also had increased heparin binding ability, but this property does not appear to be responsible for the increased affinity for FGF receptors. Thus removal of the NH2-terminal 36 amino acids from the mature K-FGF produces growth factor molecules with an altered conformation, resulting in higher heparin affinity, and more efficient binding to FGF receptors. Although it is not clear whether cleavage of K-FGF to generate K140 occurs in vivo, this could represent a novel mechanism of modulation of growth factor activity.

By successive screenings of cDNA libraries prepared from human tumours and from human foreskin keratinocytes, we have isolated overlapping cDNAs coding for a novel protein which we call Ron, with sequence characteristics of a receptor protein tyrosine kinase. Ron is a 1400 amino acid protein structurally similar to the 1408 amino acid product of the C-MET proto-oncogene, the receptor for hepatocyte growth factor and scatter factor. The two proteins have 63% overall sequence identity in their intracellular regions. We have localised the RON gene to human chromosome region 3p21, a region frequently deleted in small cell carcinoma of the lung and in renal cell carcinoma, and which is believed to harbour unidentified tumour suppressor genes. Interestingly, normal lung tissue contains transcripts of the RON gene.

Immunoglobulin (Ig)-, fibronectin type III (FN-III)-, and cadherin-related sequences are often found in multiple repeats in the extracellular regions of various cell adhesion molecules. The amino acid sequences of 82 different cadherin-like repeats from 13 known members of the cadherin superfamily were compared for six highly conserved regions, and a frequency matrix represented as amino acids versus position matrix was calculated based on the alignment. With the frequency matrix, further members of the cadherin superfamily were searched for in the protein data base. It was found that the ret protein, a receptor-type-protein tyrosine kinase, contains cadherin-like repeats in the extracellular region. A similar analysis was also carried out for the FN-III superfamily. Nine receptor-type-protein tyrosine kinases were shown to exhibit significant similarities, in terms of sequence, with known FN-III-like repeats. Several receptor-type-protein tyrosine kinases have already been reported to have Ig-like repeats in their extracellular regions. Thus these receptor-type-protein tyrosine kinases--together with the remaining receptors, whose structures are not yet characterized--may be classified into at least four distinct groups based on the structural differences in the extracellular domains. A molecular phylogenetic tree inferred from the shared kinase domains of these receptor-type-protein tyrosine kinases revealed a close relationship between the branching patterns and the grouping based on the structural differences of the extracellular region.

We report the nucleotide sequence of the mouse ret proto-oncogene (proto-ret) and the deduced amino acid sequence. It encodes a transmembrane tyrosine kinase of 1115 amino acids that shows 83% homology with the human proto-Ret protein. The amino acid sequence revealed that the structures of the extracellular domain as well as the tyrosine kinase domain are similar in human and mouse proto-Ret proteins. Interestingly, the extracellular domains of both human and mouse proto-Ret proteins contain a cadherin-related sequence that is known to be important for Ca(2+)-dependent homophilic binding of the cadherins. When we examined transcription of the proto-ret gene in a variety of mouse tissues, it was detected in lymph nodes of C3H/HeJ-gld/gld mice and in normal mouse spinal cord. Furthermore, its transcription was found in the Neuro-2a mouse neuroblastoma cell line but not in 13 other rodent cell lines surveyed. Western blot analysis showed that proto-Ret proteins are expressed as 140-kDa and 160-kDa glycoproteins in Neuro-2a cells.

We recently cloned an additional member of the receptor type tyrosine kinase class III. This new gene, called Flt3 by our group [Rosnet, O., Matteï, M.G., Marchetto, S. & Birnbaum, D. (1991). Genomics, 9, 380-385; Rosnet, O., Marchetto, S., deLapeyriere, O. & Birnbaum, D. (1991). Oncogene, 6, 1641-1650] and Flk2 by others [Matthews, W., Jordan, C.T., Wieg, G.W., Pardoll, D. & Lemischka, I.R. (1991). Cell, 65, 1143-1152] is strongly related to the important developmental genes Kit, Fms and Pdgfr. The murine 3.2-kb full-length cDNA, when introduced into COS-1 cells, shows the expression of two polypeptides with apparent molecular weights of 155 kDa and 132 kDa. Treatment of cells with N-linked glycosylation inhibitors results in the expression of a 110-kDa protein. We have shown that FLT3 contains an intrinsic tyrosine kinase activity. A point mutation in a highly conserved residue within the phosphoryltransferase domain inactivates the catalytic function of this receptor, whereas activation by way of a chimeric molecule between the ligand-binding domain of colony-stimulating factor type 1 (CSF-1) receptor (CSF-1R) and the kinase domain of FLT3 results, in the presence of CSF-1, in the development of the transforming activity of this receptor as shown by anchorage-independent cell growth. Finally, expression analysis of the FLT3 protein shows that, in addition to the hematopoietic system, FLT3 is strongly expressed in neural, gonadal, hepatic and placental tissues in the mouse.

We have developed rabbit polyclonal antibodies to the C-terminus of the flt3-encoded protein, which is a member of the receptor tyrosine kinase family. Immunoprecipitation using this antiserum brings down two protein bands, a major band of 143 kDa and a less abundant, more diffuse, band of 158 kDa. Pulse-chase analysis of flt3 protein from transfected COS-7 cells shows that the larger band is derived from the smaller one and presumably represents maturation of the protein from a glycosylated high-mannose form to a complex carbohydrate form. N-glycosidase F digestion confirmed the presence of N-linked carbohydrates, and cell-surface labeling of flt3-transfected cells indicated that the 158-kDa glycoprotein is the species found on the cell surface. A mutated form of the flt3 protein that was defective in its glycosylational processing was identified. Western blotting of the immunoprecipitated flt3 protein showed that it is heavily phosphorylated on tyrosine, and that this phosphorylation probably occurs in the absence of ligand. In this regard, the flt3 protein resembles the c-erbB2 protein, which is also highly phosphorylated in the absence of ligand. These data suggest that the flt3 receptor regulates the growth and differentiation of cells via an as yet unknown ligand.

We have recently succeeded in producing transgenic mice carrying a hybrid gene consisting of mouse metallothionein promoter-enhancer and the ret oncogene (MT/ret). (Iwamoto et al., 1991b). A retroperitoneal tumour developed in one of 17 MT/ret transgenic founder mice. Histological analysis revealed that the tumour consisted of undifferentiated neuroblasts and differentiated ganglion cells, the latter of which were strongly positive for neuron specific enolase. Expression of the ret transgene was observed at high levels in RNA from the tumour, but not in those of other normal tissues. In addition, a 100kDa ret protein was detected in the cell lysate of the tumour. Taken together with our previous data, these results suggest a possible role for the ret oncogene in the proliferation of neural crest cells.