Can a familial gastrointestinal tumour syndrome be allelic with Waardenburg syndrome?

Familial gastrointestinal stromal tumours (GISTs) are rare but otherwise well-characterized tumour syndromes, most commonly occurring on a background of germline-activating mutations in the tyrosine kinase receptor c-KIT. The associated clinical spectrum reflects the constitutive activation of this gene product across a number of cell lines, generating gain-of-function phenotypes in interstitial cells of Cajal (GIST and dysphagia), mast cells (mastocytosis) and melanocytes (hyperpigmentation). We report a three-generation kindred harbouring a c-KIT germline-activating mutation resulting in multifocal GISTs, dysphagia and a complex melanocyte hyperpigmentation and hypopigmentation disorder, the latter with features typical of those observed in Waardenburg type 2 syndrome (WS2F). Sequencing of genes known to be causative for WS [microphthalmia transcription factor (MITF), Pax3, Sox10, SNAI2 ] failed to show any candidate mutations to explain this complex cutaneous depigmentation phenotype. Our case report conclusively expands the clinical spectrum of familial GISTs and shows a hitherto unrecognized link to WS. Possible mechanisms responsible for this novel cause of WS2F will be discussed.

Quantitative bone imaging biomarkers to diagnose temporomandibular joint osteoarthritis.

Bone degradation of the condylar surface is seen in temporomandibular joint osteoarthritis (TMJ OA); however, the initial changes occur in the subchondral bone. This cross-sectional study was performed to evaluate 23 subchondral bone imaging biomarkers for TMJ OA. The sample consisted of high-resolution cone beam computed tomography scans of 84 subjects, divided into two groups: TMJ OA (45 patients with TMJ OA) and control (39 asymptomatic subjects). Six regions of each mandibular condyle scan were extracted for computation of five bone morphometric and 18 grey-level texture-based variables. The groups were compared using the Mann-Whitney U-test, and the receiver operating characteristics (ROC) curve was determined for each variable that showed a statically significance difference. The results showed statistically significant differences in the subchondral bone microstructure in the lateral and central condylar regions between the control and TMJ OA groups (P< 0.05). The area under the ROC curve (AUC) for these variables was between 0.620 and 0.710. In conclusion, 13 imaging bone biomarkers presented an acceptable diagnostic performance for the diagnosis of TMJ OA, indicating that the texture and geometry of the subchondral bone microarchitecture may be useful for quantitative grading of the disease.

Expression of a functional c-kit receptor on a subset of natural killer cells.

Natural killer (NK) cells are large granular lymphocytes thought to be important in the host's early immune response to viral infection and malignant transformation. NK cells proliferate and display enhanced cytotoxic activity in response to the T cell growth factor, interleukin 2 (IL-2). Stem cell factor or steel factor (SF) is the ligand for the c-kit receptor, and when combined with other hematopoietic growth factors, SF synergistically promotes the proliferation and differentiation of bone marrow stem cells. In the present study we show the c-kit receptor to be uniquely expressed on a subset of resting human NK cells (CD56bright) which constitutively expresses both the high affinity IL-2 receptor (IL-2R) and the intermediate affinity IL-2R. Other lymphocyte populations, including CD56dim NK cells, did not appear to express the c-kit receptor. Within the CD56bright NK cell subset, SF alone had no obvious effect on proliferation or cytotoxic activity. SF was shown to significantly augment the proliferative effect of IL-2, and caused a marked shift in the dose-response curve at IL-2 concentrations that selectively saturate the high affinity IL-2R. The potentiating effect of SF on NK cell proliferation was dependent on IL-2 binding to the high affinity IL-2R, and was blocked by a monoclonal antibody directed against the c-kit receptor. SF did not enhance proliferation at higher IL-2 concentrations that saturate the intermediate affinity IL-2R, nor did SF enhance IL-2-induced cytotoxic activity. Together, these data indicate that SF and IL-2 act synergistically to directly augment the proliferative capacity of a unique human NK cell subset constitutively expressing the high affinity IL-2R and the c-kit receptor. The implications of these findings on NK cell development and the host's early immune response to pathogen invasion are discussed.

Regulation of endothelial cell motility by complexes of tetraspan molecules CD81/TAPA-1 and CD151/PETA-3 with alpha3 beta1 integrin localized at endothelial lateral junctions.

Cell-to-cell junction structures play a key role in cell growth rate control and cell polarization. In endothelial cells (EC), these structures are also involved in regulation of vascular permeability and leukocyte extravasation. To identify novel components in EC intercellular junctions, mAbs against these cells were produced and selected using a morphological screening by immunofluorescence microscopy. Two novel mAbs, LIA1/1 and VJ1/16, specifically recognized a 25-kD protein that was selectively localized at cell-cell junctions of EC, both in the primary formation of cell monolayers and when EC reorganized in the process of wound healing. This antigen corresponded to the recently cloned platelet-endothelial tetraspan antigen CD151/PETA-3 (platelet-endothelial tetraspan antigen-3), and was consistently detected at EC cell-cell contact sites. In addition to CD151/PETA-3, two other members of the tetraspan superfamily, CD9 and CD81/ TAPA-1 (target of antiproliferative antibody-1), localized at endothelial cell-to-cell junctions. Biochemical analysis demonstrated molecular associations among tetraspan molecules themselves and those of CD151/ PETA-3 and CD9 with alpha3 beta1 integrin. Interestingly, mAbs directed to both CD151/PETA-3 and CD81/ TAPA-1 as well as mAb specific for alpha3 integrin, were able to inhibit the migration of ECs in the process of wound healing. The engagement of CD151/PETA-3 and CD81/TAPA-1 inhibited the movement of individual ECs, as determined by quantitative time-lapse video microscopy studies. Furthermore, mAbs against the CD151/PETA-3 molecule diminished the rate of EC invasion into collagen gels. In addition, these mAbs were able to increase the adhesion of EC to extracellular matrix proteins. Together these results indicate that CD81/TAPA-1 and CD151/PETA-3 tetraspan molecules are components of the endothelial lateral junctions implicated in the regulation of cell motility, either directly or by modulation of the function of the associated integrin heterodimers.

Minimally Invasive Approach for Diagnosing TMJ Osteoarthritis.

This study's objectives were to test correlations among groups of biomarkers that are associated with condylar morphology and to apply artificial intelligence to test shape analysis features in a neural network (NN) to stage condylar morphology in temporomandibular joint osteoarthritis (TMJOA). Seventeen TMJOA patients (39.9 ± 11.7 y) experiencing signs and symptoms of the disease for less than 10 y and 17 age- and sex-matched control subjects (39.4 ± 15.2 y) completed a questionnaire, had a temporomandibular joint clinical exam, had blood and saliva samples drawn, and had high-resolution cone beam computed tomography scans taken. Serum and salivary levels of 17 inflammatory biomarkers were quantified using protein microarrays. A NN was trained with 259 other condyles to detect and classify the stage of TMJOA and then compared to repeated clinical experts' classifications. Levels of the salivary biomarkers MMP-3, VE-cadherin, 6Ckine, and PAI-1 were correlated to each other in TMJOA patients and were significantly correlated with condylar morphological variability on the posterior surface of the condyle. In serum, VE-cadherin and VEGF were correlated with one another and with significant morphological variability on the anterior surface of the condyle, while MMP-3 and CXCL16 presented statistically significant associations with variability on the anterior surface, lateral pole, and superior-posterior surface of the condyle. The range of mouth opening variables were the clinical markers with the most significant associations with morphological variability at the medial and lateral condylar poles. The repeated clinician consensus classification had 97.8% agreement on degree of degeneration within 1 group difference. Predictive analytics of the NN's staging of TMJOA compared to the repeated clinicians' consensus revealed 73.5% and 91.2% accuracy. This study demonstrated significant correlations among variations in protein expression levels, clinical symptoms, and condylar surface morphology. The results suggest that 3-dimensional variability in TMJOA condylar morphology can be comprehensively phenotyped by the NN.

Association of paediatric mastocytosis with a polymorphism resulting in an amino acid substitution (M541L) in the transmembrane domain of c-KIT.

BACKGROUND: The receptor tyrosine kinase c-KIT plays a key role in normal mast cell development. Point mutations in c-KIT have been associated with sporadic or familial mastocytosis. OBJECTIVES: Two unrelated pairs of apparently identical twins affected by cutaneous mastocytosis attending the Mastocytosis Clinic at the Royal Children's Hospital, Melbourne, provided an opportunity to assess the possible contribution of c-KIT germline mutations or polymorphisms in this disease. METHODS: Tissue biopsy, blood and/or buccal swab specimens were collected from 10 children with mastocytosis. To detect germline mutations/polymorphisms in c-KIT, we studied all coding exons by denaturing high pressure liquid chromatography. Exons showing mismatches were examined by direct sequencing. The influence of the substitution identified was further examined by expressing the variant form of c-KIT in factor-dependent FDC-P1 cells. RESULTS: In both pairs of twins, a heterozygous ATG to CTG transition in codon 541 was observed, resulting in the substitution of a methionine residue in the transmembrane domain by leucine (M541L). In each case, one parent was also heterozygous for this allele. Expression of M541L KIT in FDC-P1 cells enabled them to grow in human KIT ligand (stem cell factor, SCF) but did not confer factor independence. Compared with cells expressing wild-type KIT at a similar level, M541L KIT-expressing cells displayed enhanced growth at low levels of SCF, and heightened sensitivity to the KIT inhibitor, imatinib mesylate. CONCLUSIONS: The data suggest that the single nucleotide polymorphism resulting in the substitution M541L may predispose to paediatric mastocytosis.

Interventions for learning disabled sex offenders.

BACKGROUND: The management of sex offenders is a major public concern. Behavioural and pharmacological interventions have been used for many years and more recently cognitive behavioural based interventions have become popular around the world. Programmes designed for the general population have been modified for those sex offenders with learning disability, to address their cognitive deficits. The efficacy of these modified programmes is unclear. OBJECTIVES: To determine the efficacy of interventions with learning disabled sex offenders. SEARCH STRATEGY: The reviewers searched the Cochrane Library 2006 (Issue 1), MEDLINE (1966 to Sept 2006), Embase (1980 to September 2006), CINAHL (1982 to September 2006), PsycINFO (1872 to September 2006), Biological Abstracts (1980 to September 2006). SELECTION CRITERIA: All randomised controlled trials comparing an intervention for learning disabled sex offenders to any other, or no intervention. DATA COLLECTION AND ANALYSIS: Data were independently extracted. MAIN RESULTS: No randomised controlled trial was identified. AUTHORS' CONCLUSIONS: Using the methods described the reviewers found no randomised controlled trial evidence to guide the use of interventions for learning disabled sex offenders. Until the urgent need for randomised controlled trials is met, clinical practice will continue to be guided by either extrapolation of evidence from randomised controlled trials involving sex offenders without learning disability or non-randomised trial evidence of interventions for the learning disabled sex offender.

Diversification across biomes in a continental lizard radiation.

Ecological opportunity is a powerful driver of evolutionary diversification, and predicts rapid lineage and phenotypic diversification following colonization of competitor-free habitats. Alternatively, topographic or environmental heterogeneity could be key to generating and sustaining diversity. We explore these hypotheses in a widespread lineage of Australian lizards: the Gehyra variegata group. This clade occurs across two biomes: the Australian monsoonal tropics (AMT), where it overlaps a separate, larger bodied clade of Gehyra and is largely restricted to rocks; and in the larger Australian arid zone (AAZ) where it has no congeners and occupies trees and rocks. New phylogenomic data and coalescent analyses of AAZ taxa resolve lineages and their relationships and reveal high diversity in the western AAZ (Pilbara region). The AMT and AAZ radiations represent separate radiations with no difference in speciation rates. Most taxa occur on rocks, with small geographic ranges relative to widespread generalist taxa across the vast central AAZ. Rock-dwelling and generalist taxa differ morphologically, but only the lineage-poor central AAZ taxa have accelerated evolution. This accords with increasing evidence that lineage and morphological diversity are poorly correlated, and suggests environmental heterogeneity and refugial dynamics have been more important than ecological release in elevating lineage diversity.

Identification of mutations in the coding sequence of the proto-oncogene c-kit in a human mast cell leukemia cell line causing ligand-independent activation of c-kit product.

The c-kit proto-oncogene encodes a receptor tyrosine kinase. Binding of c-kit ligand, stem cell factor (SCF) to c-kit receptor (c-kitR) is known to activate c-kitR tyrosine kinase, thereby leading to autophosphorylation of c-kitR on tyrosine and to association of c-kitR with substrates such as phosphatidylinositol 3-kinase (PI3K). In a human mast cell leukemia cell line HMC-1, c-kitR was found to be constitutively phosphorylated on tyrosine, activated, and associated with PI3K without the addition of SCF. The expression of SCF mRNA transcript in HMC-1 cells was not detectable by means of PCR after reverse transcription (RT-PCR) analysis, suggesting that the constitutive activation of c-kitR was ligand independent. Sequencing of whole coding region of c-kit cDNA revealed that c-kit genes of HMC-1 cells were composed of a normal, wild-type allele and a mutant allele with two point mutations resulting in intracellular amino acid substitutions of Gly-560 for Val and Val-816 for Asp. Amino acid sequences in the regions of the two mutations are completely conserved in all of mouse, rat, and human c-kit. In order to determine the causal role of these mutations in the constitutive activation, murine c-kit mutants encoding Gly-559 and/or Val-814, corresponding to human Gly-560 and/or Val-816, were constructed by site-directed mutagenesis and expressed in a human embryonic kidney cell line, 293T cells. In the transfected cells, both c-kitR (Gly-559, Val-814) and c-kitR (Val-814) were abundantly phosphorylated on tyrosine and activated in immune complex kinase reaction in the absence of SCF, whereas tyrosine phosphorylation and activation of c-kitR (Gly-559) or wild-type c-kitR was modest or little, respectively. These results suggest that conversion of Asp-816 to Val in human c-kitR may be an activating mutation and responsible for the constitutive activation of c-kitR in HMC-1 cells.

Small molecule inhibitors of protein kinases in cancer- how to overcome resistance.

Small molecule protein kinase inhibitors show great promise as anti-cancer agents, however, de novo and acquired resistance present problems. These are reviewed and illustrated using the receptor tyrosine kinase, KIT, as an example. Emerging solutions are presented, such as targeting active kinase conformations.

Protective effect of oral Salmonella enteritidis 11RX infection against colon tumor induction by 1,2-dimethylhydrazine in mice.

Infection of mice with Salmonella enteritidis 11RX has been shown previously to cause nonspecific immune stimulation and, consequently, resistance to subsequent challenge with a variety of transplantable tumors. The present study has examined the effect of infection with this organism in a chemical carcinogenesis system. Colonic tumors were induced in LACA and BALB/c x C57BL/6JF1 mice by weekly s.c. injection of 1,2-dimethylhydrazine (15 mg/kg) for 28 weeks. Infection of mice p.o. with live S. enteritidis 11RX at 8-week intervals during 1,2-dimethylhydrazine administration protected both strains against colon tumorigenesis. Significantly fewer infected than control BALB/c x C57BL/6JF1 mice had colonic tumors at or before termination of the experiment (34 or 40 weeks) (p less than 0.001 in all cases). Comparable results were obtained with both male and female mice. The difference in tumor incidence between control and infected LACA mice was not statistically significant, however; the number and size of the lesions was greater in control mice (p less than 0.02). Although it has not been proven that the protective effect is mediated by the immune system, the results are consistent with the operation of a macrophage-mediated surveillance system. It is suggested that enteric infections should be considered as a possible contributing factor in the epidemiology of human colonic cancer.

Isoforms of c-KIT differ in activation of signalling pathways and transformation of NIH3T3 fibroblasts.

Alternate splicing of mRNA encoding c-KIT results in isoforms which differ in the presence or absence of four amino acids (GNNK) in the juxtamembrane region of the extracellular domain of the receptor. In this study we show that these isoforms of human c-KIT, expressed at similar levels in NIH3T3 cells, display differential effects on various attributes of transformation. The GNNK- isoform strongly promoted anchorage independent growth (colony formation in semi-solid medium), loss of contact inhibition (focus formation), and led to tumorigenicity in nude mice. In contrast, the GNNK+ isoform elicited colony formation but relatively poor focus formation and no tumorigenicity. Saturation binding analysis indicated that the isoforms do not differ significantly in their affinity for the KIT ligand, Steel Factor (SLF). Negligible ligand-independent receptor phosphorylation was observed in either case but, after ligand stimulation, the GNNK- isoform displayed more rapid and extensive tyrosine autophosphorylation and faster internalization. Both isoforms recruited the p85 subunit of phosphatidylinositol 3-kinase and led to similar phosphorylation of its downstream effector c-Akt, but the GNNK- isoform gave rise to more MAP kinase phosphorylation. Thus the c-KIT isoforms display different signalling characteristics and have different transforming activity in NIH3T3 cells.

Transformation of NIH3T3 fibroblasts by the c-Kit receptor tyrosine kinase: effect of receptor density and ligand-requirement.

Ectopic expression of the normal murine receptor tyrosine kinase, c-Kit, in NIH3T3 cells induced many phenotypic changes characteristic of transformation including anchorage-independent growth, focus formation and tumorigenicity in nude mice. Although transformation was largely dependent on the presence of recombinant murine Steel Factor (SLF), the ligand to the c-Kit receptor, anchorage independent growth did occur at a low frequency in the absence of added factor, and this could not be inhibited by neutralising antibodies or by SLF anti-sense mRNA. Clones from factor-independent colonies in semi-solid agar displayed a narrow range of c-Kit surface protein levels (4.3-6.4 x 10(4) receptors/cell) which was relatively high compared with the pool from which they were derived. Analysis of a larger series of random clones derived from adherent cultures expressing different levels of c-Kit demonstrated a positive correlation between SLF-dependent, anchorage-independent growth and c-Kit protein and mRNA expression levels (respectively, Rs = 0.58, P < 0.01; and Rs = 0.53, P < 0.01) with consistent colony formation observed with clones having > 2.5 x 10(4) receptors/cell. Interestingly, two of the three clones expressing the highest levels of c-Kit protein and mRNA produced few or no colonies in the presence or absence of SLF. Sequential overexpression of human c-KIT in NIH3T3 cells using a dihydrofolate reductase (DHFR)-encoding vector and gene co-amplification through methotrexate selection, which resulted in pools expressing up to 1.5 x 10(5) receptors/cell, confirmed that high receptor densities resulted in a decrease in colony numbers. Thus, analysis of clonal and selected populations has indicated that an optimal level of c-Kit is required for transformation of NIH3T3 cells in the presence of SLF, and that some ligand-independent transformation occurs.

Enforced expression of full length c-Myb leads to density-dependent transformation of murine haemopoietic cells.

The oncogenic activation of c-myb has been associated with structural alterations to the Myb protein. Although such alterations can increase the ability of Myb to transform haemopoietic cells, it has been unresolved whether over-expression of wild type (WT) c-Myb can lead to transformation. We show here that infection with a retrovirus that expresses WT i.e. full length c-Myb leads to transformation of primary haemopoietic cells (as indicated by clonogenic assays). The transformed cells are similar to those obtained with carboxyl-truncated (CT) c-Myb in that they show phenotypic and morphological characteristics of early myeloid cells and remain dependent on exogenous growth factors. Cells expressing WTMyb form lower numbers of colonies on average and have a greater tendency to spontaneously differentiate than those expressing truncated c-Myb. Additionally, our results show that transformation by both forms of Myb is dependent on the density at which the infected cells are cultured, and that low levels of transformation can be increased by addition of conditioned medium from myb transformed cells grown at high density. This implies that transformation can be enhanced by the effects of an autocrine growth factor. Moreover, the production of, or sensitivity to, such a factor may be influenced by Myb itself, since CT Myb-infected cells cultured at low densities show higher levels of transformation than WT Myb-infected cells.

Characterisation of the mouse homologue of CD151 (PETA-3/SFA-1); genomic structure, chromosomal localisation and identification of 2 novel splice forms.

CD151 (PETA-3/SFA-1) is a member of the Transmembrane 4 Superfamily (TM4SF) of cell-surface proteins and, like other TM4SF members CD9 and CD63, is expressed by platelets, megakaryocytes and endothelial cells. The precise function of CD151 is unknown however complexes containing CD151 and beta1 integrins have been isolated from a number of cell systems and studies using anti-CD151 monoclonal antibodies have suggested a role in transmembrane signalling and cell adhesion. To further investigate the function of CD151 we have determined the genomic organisation of mouse CD151 (Cd151). Cd151 spans 4 kb and contains six coding region exons. Using 5' RACE and reverse transcriptase-polymerase chain reaction (RT-PCR) we have identified three 5' UTR splice variants which arise through alternate splicing of three exons. Splice variants were detected in a number of mouse tissues by RT-PCR. Analysis of the Cd151 genomic structure reveals a high degree of structural conservation with other TM4SF molecules supporting the theory that family members have arisen from gene duplication of a common ancestral gene. Cd151 maps to chromosome 7, in close linkage to the p gene (OCA2 in humans), and helps define a boundary in the human/mouse homology relationships.

Expression of c-Kit and functional drug efflux are correlated in de novo acute myeloid leukaemia.

P-glycoprotein (Pgp), the major mediator of multidrug resistance (MDR) has often been implicated as a poor prognostic indicator in acute myeloid leukaemia (AML). We have previously reported that high expression of the receptor tyrosine kinase c-Kit in AML is associated with poor prognosis. To determine whether the MDR phenotype is associated with high c-Kit expression, the monoclonal antibodies UIC-2 and YB5.B8, which identify Pgp and c-Kit, respectively, were used for indirect immunofluorescence labelling of 50 de novo AML specimens. Quantitative dye efflux studies using Rhodamine123 were also carried out to assess the functional drug efflux capability of these samples. Pgp expression by the majority of primary AML was comparable to that seen in subsets of cells from normal bone marrow and Spearman rank analysis showed no relationship with c-Kit expression (rs = 0.20, P = 0.16). However, c-Kit expression did show a significant correlation with Rhodamine123 efflux (rs = 0.57, P = 0.0001), suggesting that the MDR phenotype, Pgp mediated or other, may contribute to the prognostic significance of high c-Kit expression. The monoclonal antibody UIC-2 was used specifically to block Pgp activity of a limited number of leukaemic specimens and cell lines, and evidence of non-Pgp-mediated efflux was found. The existence of alternative mechanisms may explain the relatively low correlation of Pgp expression with dye efflux within the leukaemic samples (rs = 0.47, P = 0.0006) and has implications for prognosis in AML. The c-Kit ligand, stem cell factor, did not influence drug efflux activity of the nine c-Kit-positive AML specimens tested. Thus the correlation between c-Kit and the MDR phenotype in AML is likely to be a consequence of co-expression at a similar stage of differentiation, and may account for the previously observed association of high c-Kit expression with poor outcome.

Synergistic action of interleukin-2 and Steel factor (SLF) on a human T lymphoblastoid cell line.

Ten cell lines recently established from paediatric patients with acute lymphoblastic leukaemia (ALL) were examined for expression of P145c-kit, the growth factor receptor encoded by the c-kit proto-oncogene, by immunofluorescence and flow cytometry using monoclonal antibody YB5.B8. Three of five T-ALL cell lines, but none of five B lineage ALL cell lines displayed significant binding of the antibody. The cell line with the highest level of binding was PER-423 (Kees et al, Leukemia Res 1993; 17: 51-59 which has the phenotype CD7+, CD56bright, CD2-, CD4-, CD5-, CD8-, CD16-, has rearranged T cell receptor beta-chain genes, expresses cytoplasmic CD3 and is strictly dependent on interleukin 2 (IL-2) for proliferation. Recombinant to act in synergy with IL-2 to promote proliferation of PER-423 cells. In five experiments, SLF increased the maximal amount of proliferation by 105 +/- 15%, and decreased the level of IL-2 required for a half-maximal response by 43 +/- 7%. The cells constitutively express the intermediate affinity IL-2 receptor (beta/gamma), but can be induced in the presence of phorbol ester to express the alpha chain (CD25, Tac) which confers high affinity binding of IL-2. In contrast, the alpha chain was not induced by SLF. The enhancement of proliferation of PER-423 cells by SLF could be prevented by inclusion in the assay of a blocking monoclonal antibody to P145c-kit. These experiments demonstrate that SLF/P145c-kit can provide a significant growth stimulus for ALL cells, and PER-423 cells may be a useful system for investigating the mechanism of synergy between SLF and IL-2.

Increased expression of c-Kit or its ligand Steel Factor is not a common feature of adult acute myeloid leukaemia.

Cell surface levels of the receptor tyrosine kinase P145(c-kit), the product of the c-kit proto-oncogens, in a panel of 80 primary adult acute myeloid leukaemia (AML) specimens collected at presentation were quantitated by immunofluorescence and flow cytometry, and compared with levels on CD34+ bone marrow cells from normal donors. Receptor levels on AML blast cells were extremely variable and were similar to, or less than, those on normal stem and progenitor cells. In general P145(c-kit) expression was higher on cells of immature phenotype (FAB M1 and M2). c-kit mRNA was quantitated by ribonuclease protection assay (RPA) and was shown to be correlated with cell surface protein expression (r=0.76; P<0.001). This indicates that ligand-mediated receptor internalisation or other mechanisms of increased protein turnover are not responsible for variations in the level of P145(c-kit) in AML specimens. Quantitative Southern blotting was used to examine c-kit gene copy number in 25 of these specimens and was found to be normal in all but one. Thus we have found little evidence of over-expression of c-kit in adult AML. mRNA for the c-kit ligand, Steel Factor (SLF) was also quantitated by RPA in these specimens. While SLF message was detectable (limit of detection approximately 10(4) copies per 10 microgram total RNA; equivalent to 1 copy per 100 cells) in 19% of cases, these specimens in general contained low levels of c-kit mRNA. Thus, an autocrine cycle involving c-kit and SLF does not appear to be a common feature of AML.

Activating mutations of the c-kit proto-oncogene in a human mast cell leukemia cell line.

The c-kit proto-oncogene encodes a receptor tyrosine kinase that is known to play a crucial role in mast cell growth and differentiation. In a human mast cell leukemia cell line (HMC-1), KitR was found to be constitutively phosphorylated on tyrosine, activated and associated with phosphatidylinositol 3-kinase (P13K) in the absence of autocrine production of SCF. Sequencing of c-kit cDNA revealed that c-kit genes of HMC-1 cells were composed of a normal, wild-type allele and a mutant allele with two point mutations in codon 560 and codon 816, resulting in intracellular amino acid substitutions of Gly-560 for Val and Val-816 for Asp, respectively. Murine c-kit mutants encoding Gly-559 and/or Val-814, corresponding to human Gly-560 and/or Val-816, were constructed by site-directed mutagenesis and expressed in cells of a human embryonic kidney cell line (293T). In the transfected cells, KitR (Gly-559 + Val-814) and KitR (Val-814) were strikingly phosphorylated on tyrosine and activated in the absence of SCF, whereas tyrosine phosphorylation and activation of KitR (Gly-559) or wild-type KitR was modest or little, respectively. These results suggest that constitutive activation of KitR in HMC-1 results from the activating mutations of c-kit gene, and raise the possibility that the activating mutations, particularly at codon 814 of murine c-kit or at codon 816 of human c-kit, may participate in oncogenesis of mast cells.

Biotinylation: an alternative to radioiodination for the identification of cell surface antigens in immunoprecipitates.

A method has been developed in which the conventional radioiodine label is replaced by non-radioactive biotin in studies involving the immunoprecipitation and analysis of cell surface antigens. The labelling reagent, d-biotinyl-N-hydroxysulfosuccinimide ester (NHSS-biotin), reacts preferentially with lysine residues in polypeptides and possibly also with free amino-groups on carbohydrates and lipids. The reagent can be used as a cell surface label, does not interfere with antigen-antibody interactions and allows labelled molecules to be detected with high sensitivity using streptavidin-peroxidase conjugates. The target antigens of a range of monoclonal antibodies to human cell surface components have been identified using this procedure.