L. C. J. M. Oomen

The FYVE domain in Smad anchor for receptor activation (SARA) is sufficient for localization of SARA in early endosomes and regulates TGF-beta/Smad signalling.

Abstract Background: Transforming growth factor‐β (TGF‐β) initiates intracellular signalling by inducing the formation of a heteromeric complex between TGF‐β type I (TβR‐I) and TGF‐β type II serine/threonine kinase receptors (TβR‐II). After the activation of TβR‐I kinase by TβR‐II kinase, specific receptor‐regulated Smads (R‐Smads) are phosphorylated by TβR‐I kinase. Smad anchor for receptor activation (SARA), which contains a FYVE finger domain, regulates the subcellular localization of R‐Smads and presents them to TβR‐I. However, it is unclear where SARA is localized in the cell and which phospholipid(s) interacts with its FYVE domain.

Interaction of the Type Iα PIPkinase with phospholipase D: a role for the local generation of phosphatidylinositol 4,5‐bisphosphate in the regulation of PLD2 activity

Phosphoinositides are localized in various intracellular compartments and can regulate a number of intracellular functions, such as cytoskeletal dynamics and membrane trafficking. Phospholipase Ds (PLDs) are regulated enzymes that hydrolyse phosphatidylcholine (PtdCho) to generate the putative second messenger phosphatidic acid (PtdOH). In vitro, PLDs have an absolute requirement for higher phosphorylated inositides, such as phosphatidylinositol 4,5‐bisphosphate [PtdIns(4,5)P2]. Whether this lipid is able to regulate the activity of PLD in vivo is contentious. To examine this hypothesis we studied the relationship between PLD and an enzyme critical for the intracellular synthesis of PtdIns(4,5)P2: phosphatidylinositol 4‐phosphate 5‐kinase α (Type Iα PIPkinase). We find that both PLD1 and PLD2 interact with the Type Iα PIPkinase and that PLD2 activity in vivo can be regulated solely by the expression of this lipid kinase. Moreover, PLD2 is able to recruit the Type Iα PIPkinase to its intracellular location. We show that the physiological requirement of PLD enzymes for PtdIns(4,5)P2 is critical and that PLD2 activity can be regulated solely by the levels of this key intracellular lipid.

Localization in situ of costimulatory molecules and cytokines in B-cell non-Hodgkin's lymphoma.

Costimulatory molecules are essential in cognate interactions between T and B lymphocytes. To study the prerequisites of functional interactions between malignant B cells and intermingled T cells in B‐cell non‐Hodgkin’s lymphomas (B‐NHL), we examined the expression of CD40, CD80 and CD86 and their ligands CD40 ligand (CD40L, CD154), CD28 and CTLA4 (CD152) using immunohistochemistry and confocal laser scanning microscopy. Almost all mucosa‐associated lymphoid tissue (MALT) NHL were positive for CD40 and CD80 and in nine out of 14 cases were positive for CD86. The majority of follicle centre cell lymphomas (FCCL) expressed CD40, but were heterogeneous in their expression of CD80 and CD86. Most diffuse large cell lymphomas (DLCL) were CD80+, but lacked expression of CD86. These patterns reflect the differences in phenotype of normal marginal‐zone B cells (as counterparts of MALT NHL) and germinal centre cells (as counterparts of FCCL and DLCL). Counter‐receptors on T cells were detectable in 13 of 14 MALT NHL, 12 of 16 FCCL but only occasionally in DLCL (three of 12 cases). A subgroup of FCCL was identified with T‐cell expression of CD40L, CD28 and CTLA4 simultaneously with strong expression of CD40 and CD86 on the tumour B cells. These results indicate that MALT NHL and a subset of FCCL are most optimally equipped for functional interactions with T cells. This may be supported by the demonstration of cytokine production – mainly in T cells – in MALT NHL [interleukin‐2 (IL‐2), interferon‐γ (IFN‐γ), IL‐10] and FCCL (IL‐2, IFN‐γ) and to a lesser extent in DLCL.

A susceptibility gene for alveolar lung tumors in the mouse maps between Hsp70.3 and G7 within the H2 complex

Lung tumor susceptibility (LTS) in the mouse is influenced by multiple loci within the H2 complex. We compared the LTS of two H2 congenic strains with intra H2 recombinations, B10.A(1R) and B10.A(2R), whose genetic difference has been reduced to a region of approximately 50 kilobases within the C4-H2D interval, between Hsp70.3 and G7. After transplacental induction with N-ethyl-N-nitrosourea the load of alveolar lung tumors in strain B10.A(2R) is significantly higher than in strain B10.A(1R) (P <0.001). For papillary tumors no significant differences were observed. We conclude that the alveolar lung tumor load is influenced by an LTS gene located within the Hsp70.3-G7 interval.

MHC and Non-MHC Genes in Lung Tumor Susceptibility in the Mouse: Implications for the Study of the Different Lung Tumor Types and Their Cell of Origin

Lung tumorigenesis in the mouse is controlled by multiple genes, which until now largely escaped detection because of the limitations of the available genetic tools. Therefore, we have developed the Recombinant Congenic Strains (RCS), which can be used to separate and map individual tumor susceptibility genes. The two strains, B10.O20/Dem and O20/A (used to produce the RCS series O20.c.B10.O20/Dem) differ in number, type, and degree of malignancy of lung tumors. Thus the genetic control of the different aspects of lung tumorigenesis can now be analyzed using RCS. The tests on the role of the Major Histocompatibility Complex (MHC; H-2 in mice) in lung tumorigenesis show that the H-2 influence is different for alveolar tumors and papillary tumors. In addition, only the development of papillary tumors is influenced by glucocorticoid administration concomitant with the transplacental carcinogen treatment. The MHC influence on lung tumor development is probably related to H-2 effects on the regulation of lung differentiation. In the H-2 congenic strains used papillary tumors developed early, whereas alveolar tumors appeared later, if at all. This differential impact of genetic and hormonal factors on the development of alveolar and papillary tumors suggests that they arise either from different cell types or from a common cell type at different stages of differentiation.

Multidrug resistance-associated protein 9 (ABCC12) is present in mouse and boar sperm

The human and murine genes for MRP9 (multidrug resistance-associated protein 9; ABCC12) yield many alternatively spliced RNAs. Using a panel of monoclonal antibodies, we detected full-length Mrp9 only in testicular germ cells and mouse sperm; we obtained no evidence for the existence of the truncated 100 kDa MRP9 protein reported previously. In contrast with other MRPs, neither murine Mrp9 nor the human MRP9 produced in MRP9-transfected HEK-293 cells (human embryonic kidney cells) appears to contain N-linked carbohydrates. In mouse and boar sperm, Mrp9 localizes to the midpiece, a structure containing all sperm mitochondria. However, immunolocalization microscopy and cell fractionation studies with transfected HEK-293 cells and mouse testis show that MRP9/Mrp9 does not localize to mitochondria. In HEK-293 cells, it is predominantly localized in the endoplasmic reticulum. We have been unable to demonstrate transport by MRP9 of substrates transported by other MRPs, such as drug conjugates and other organic anions.

Alkyl-lysophospholipid 1-O-octadecyl-2-O-methyl- glycerophosphocholine induces invasion through episialin-mediated neutralization of E-cadherin in human mammary MCF-7 cells in vitro.

1‐O‐octadecyl‐2‐O‐methyl‐glycerophosphocholine (ET‐18‐OMe) is an analogue of the naturally occurring 2‐lysophosphatidylcholine belonging to the class of antitumor lipids. Previously, we demonstrated that ET‐18‐OMe modulates cell‐cell adhesion of human breast cancer MCF‐7 cells. In the present study, we tested the effect of ET‐18‐OMe on adhesion, invasion and localisation of episialin and E‐cadherin in MCF‐7/AZ cells expressing a functional E‐cadherin/catenin complex. The MCF‐7/6 human breast cancer cells were used as negative control since their E‐cadherin/catenin complex is functional in cells grown on solid substrate but not in suspension. The function of E‐cadherin, a calcium‐dependent transmembrane cell‐cell adhesion and signal‐transducing molecule, is disturbed in invasive cancers by mutation, loss of mRNA stability, proteolytic degradation, tyrosine phosphorylation of associated proteins and large cell‐associated proteoglycans or mucin‐like molecules such as episialin. Episialin, also called MUC1, is an anti‐adhesion molecule that by its large number of glycosylated tandem repeats can sterically hinder the adhesive properties of other glycoproteins. ET‐18‐OMe inhibited the E‐cadherin functions of MCF‐7/AZ cells as measured by inhibition of fast and slow aggregation and by the induction of collagen invasion. These effects were enhanced by MB2, an antibody against E‐cadherin and blocked by monoclonal antibodies (MAbs) 214D4 or M8 against episialin. ET‐18‐OMe had no influence on tyrosine phosphorylation of β‐catenin and the E‐cadherin/catenin complex remained intact. Transcription, translation, protein turnover and cell surface localisation of episialin were not altered. ET‐18‐OMe induced finger‐like extensions with clustering of episialin together with E‐cadherin and carcinoembryonic antigen but not with occludin. In cells in suspension, ET‐18‐OMe caused a shift in the flow‐cytometric profile of episialin toward a lower intensity for MCF‐7/AZ cells. In contrast with MCF‐7/AZ cells, the adhesion‐deficient and noninvasive MCF‐7/6 cells showed neither morphotypic changes nor induction of aggregation nor invasion in collagen I upon treatment with ET‐18‐OMe. Co‐localisation of episialin with E‐cadherin was rarely observed. We conclude that in the human breast cancer cells MCF‐7/AZ, E‐cadherin and episialin are key molecular players in the regulation of promotion and suppression of cell‐cell adhesion and invasion.

Stem cell carcinoma in the small intestine of mice treated transplacentally with N-ethyl-N-nitrosourea: Some quantitative and histological aspects

A high incidence of tumours of the small intestine occurred in mice of the B10.A/SgSnA strain treated transplacentally with N-ethyl-N-nitrosourea (ENU). In these adenocarcinomas, histologically different tumour cells which resembled the 4 cell types of the normal intestinal epithelium were present. Since normal intestinal epithelial cells are thought to originate from common stem cells, the tumours seem to be derived from these stem cells.

Influence of MHC and Non-MHC Genes on Tumorigenesis and the Use of the Recombinant Congenic Strains as a Novel Tool for the Genetic Analysis of Tumor Susceptibility

One of the main developments in the contemporary biology is the rapid progress in the structural analysis of the mammalian genome, concentrated mainly on two species - the human and the mouse. A specific potential of the analysis of the mouse genome resides in the possibility of combining the structural analysis with the study of the genetic determination of various functional traits. This contribution of mouse studies is especially valuable in view of the limited possibilities of direct biological experiments in humans.