Heterogeneity in a mouse model of histiocytosis: transformation of Langerin+ dendritic cells, macrophages, and precursors.

Neoplastic diseases of macrophages (M phi) and dendritic cells (DC), collectively called histiocytoses, are relatively rare. The etiology of most forms of histiocytosis is poorly understood, and the development of animal models is crucial for further research in this field. Previously, an animal model for malignant histiocytosis (MH), involving transformed histiocytic cells, has been generated by infecting mice with malignant histiocytosis sarcoma virus (MHSV). However, increased insight into the heterogeneity of M phi and DC, and the associated reappraisal of human proliferative diseases involving these cells inspired us to re-evaluate the mouse model. We analyzed spleen, bone marrow, and lymph nodes of susceptible mice at various time points after infection. From day 11 onwards, a heterogeneous population of cells, consisting of CD8 alpha(+) Langerin(+) DC, ER-MP58(+) CD11b(+) myeloid precursor cells, CD169(+) metallophilic M phi, and CD71(hi) erythroblasts, was affected by viral transformation. In different mice, these subsets expanded at different rates in different organs, causing a variable disease profile in terminal stages. Cell lines, which were generated from MHSV-transformed tumors, showed a DC-like morphology and phenotype, and appeared to be arrested in different stages of maturation. Upon injection into healthy mice, different preferential homing patterns were observed for the various cell lines, and the cells acquired distinct phenotypes depending on the organ of homing. This indicates that these transformed cells adapt to their microenvironment by switching between precursor, DC/Langerhans cell, and M phi phenotypes. Our results demonstrate that the MHSV model represents a heterogeneous neoplastic disease with characteristics of M phi/DC sarcomas.

Homing and invasiveness of MLL/ENL leukemic cells is regulated by MEF2C.

Acute myelogenous leukemia is driven by leukemic stem cells (LSCs) generated by mutations that confer (or maintain) self-renewal potential coupled to an aberrant differentiation program. Using retroviral mutagenesis, we identified genes that generate LSCs in collaboration with genetic disruption of the gene encoding interferon response factor 8 (Irf8), which induces a myeloproliferation in vivo. Among the targeted genes, we identified Mef2c, encoding a MCM1-agamous-deficiens-serum response factor transcription factor, and confirmed that overexpression induced a myelomonocytic leukemia in cooperation with Irf8 deficiency. Strikingly, several of the genes identified in our screen have been reported to be up-regulated in the mixed-lineage leukemia (MLL) subtype. High MEF2C expression levels were confirmed in acute myelogenous leukemia patient samples with MLL gene disruptions, prompting an investigation of the causal interplay. Using a conditional mouse strain, we demonstrated that Mef2c deficiency does not impair the establishment or maintenance of LSCs generated in vitro by MLL/ENL fusion proteins; however, its loss led to compromised homing and invasiveness of the tumor cells. Mef2c-dependent targets included several genes encoding matrix metalloproteinases and chemokine ligands and receptors, providing a mechanistic link to increased homing and motility. Thus, MEF2C up-regulation may be responsible for the aggressive nature of this leukemia subtype.

Immunohistochemical characterisation of cell-type specific expression of CK1delta in various tissues of young adult BALB/c mice.

BACKGROUND: Casein kinase 1 delta (CK1delta) phosphorylates many key proteins playing important roles in such biological processes as cell growth, differentiation, apoptosis, circadian rhythm and vesicle transport. Furthermore, deregulation of CK1delta has been linked to neurodegenerative diseases and cancer. In this study, the cell specific distribution of CK1delta in various tissues and organs of young adult BALB/c mice was analysed by immunohistochemistry. METHODOLOGY/PRINCIPAL FINDINGS: Immunohistochemical staining of CK1delta was performed using three different antibodies against CK1delta. A high expression of CK1delta was found in a variety of tissues and organ systems and in several cell types of endodermal, mesodermal and ectodermal origin. CONCLUSIONS: These results give an overview of the cell-type specific expression of CK1delta in different organs under normal conditions. Thus, they provide evidence for possible cell-type specific functions of CK1delta, where CK1delta can interact with and modulate the activity of key regulator proteins by site directed phosphorylation. Furthermore, they provide the basis for future analyses of CK1delta in these tissues.

HLA-DR alpha 2 mediates negative signalling via binding to Tirc7 leading to anti-inflammatory and apoptotic effects in lymphocytes in vitro and in vivo.

Classically, HLA-DR expressed on antigen presenting cells (APC) initiates lymphocyte activation via presentation of peptides to TCR bearing CD4+ T-Cells. Here we demonstrate that HLA-DR alpha 2 domain (sHLA-DRalpha2) also induces negative signals by engaging TIRC7 on lymphocytes. This interaction inhibits proliferation and induces apoptosis in CD4+ and CD8+ T-cells via activation of the intrinsic pathway. Proliferation inhibition is associated with SHP-1 recruitment by TIRC7, decreased phosphorylation of STAT4, TCR-zeta chain & ZAP70, and inhibition of IFN-gamma and FasL expression. HLA-DRalpha2 and TIRC7 co-localize at the APC-T cell interaction site. Triggering HLA-DR - TIRC7 pathway demonstrates that sHLA-DRalpha2 treatment inhibits proinflammatory-inflammatory cytokine expression in APC & T cells after lipopolysaccaride (LPS) stimulation in vitro and induces apoptosis in vivo. These results suggest a novel antiproliferative role for HLA-DR mediated via TIRC7, revise the notion of an exclusive stimulatory interaction of HLA-DR with CD4+ T cells and highlights a novel physiologically relevant regulatory pathway.

Mutant p53(R270H) gain of function phenotype in a mouse model for oncogene-induced mammary carcinogenesis.

In human breast cancer, mutations in the p53 gene are associated with poor prognosis. However, analysis of patient data so far did not clarify, whether missense point mutations in the p53 gene, in addition to causing loss of wild-type p53 function, also confer a gain of function phenotype to the encoded mutant p53. As heterogeneity of patient material and data might obscure a clear answer, we studied the effects of a coexpressed mutant p53(R270H) in transgenic mice in which SV40 early proteins initiate the development of mammary adenocarcinoma (WAP-T mice). In such tumors the endogenous wild-type p53 is functionally compromised by complex formation with SV40 T-antigen, thereby constituting a loss of wild-type p53 function situation that allowed analysis of the postulated gain of function effects of mutant p53(R270H). We found that mutant p53(R270H) in bi-transgenic mice enhanced the transition from intraepithelial neoplasia to invasive carcinoma, resulting in a higher frequency of invasive carcinoma per gland and per mouse, a more severe tumor phenotype, and more frequent pulmonary metastasis. Surprisingly, mutant p53(R270H) in this system does not increase genomic instability. Therefore, other postulated gain of function activities of mutant p53 must be responsible for the effects described here.

Interferon-gamma induces chronic active myocarditis and cardiomyopathy in transgenic mice.

Chronic heart failure is associated with an activation of the immune system characterized among other factors by the cardiac synthesis and serum expression of proinflammatory cytokines. There is unequivocal clinical and experimental evidence that the cytokine tumor necrosis factor-alpha is involved in the development of chronic heart failure, but a putative cardiotoxic potential of the proinflammatory cytokine interferon (IFN)-gamma remains primarily unknown. To investigate this issue we analyzed the cardiac phenotype of SAP-IFN-gamma transgenic mice, which constitutively express IFN-gamma in their livers and hence exhibit high circulating serum levels of this cytokine. SAP-IFN-gamma mice spontaneously developed chronic active myocarditis, characterized by the infiltration of not only CD4(+) and CD8(+) T cells but also Mac2(+) (galectin 3(+)) macrophages and CD11c(+) dendritic cells, eventually culminating in cardiomyopathy. Echocardiographic analyses exhibited a left ventricular dilation and impaired systolic function induced by IFN-gamma overexpression. IFN-gamma-mediated cardiotoxicity was associated with high-level cardiac transcription of the proinflammatory cytokines tumor necrosis factor-alpha and interleukin-12 and the macrophage-attracting chemokines MCP1 and MIP1-alpha. Myotoxic IFN-gamma effects could not be detected in smooth or striated muscle tissue, suggesting cardiomyocellular specificity of the toxic IFN-gamma effect. The precise mechanism of IFN-gamma cardiotoxicity remains to be elucidated.

Essential role of ubiquitin-specific protease 8 for receptor tyrosine kinase stability and endocytic trafficking in vivo.

Posttranslational modification by ubiquitin controls multiple cellular functions and is counteracted by the activities of deubiquitinating enzymes. UBPy (USP8) is a growth-regulated ubiquitin isopeptidase that interacts with the HRS-STAM complex. Using Cre-loxP-mediated gene targeting in mice, we show that lack of UBPy results in embryonic lethality, whereas its conditional inactivation in adults causes fatal liver failure. The defect is accompanied by a strong reduction or absence of several growth factor receptor tyrosine kinases (RTKs), like epidermal growth factor receptor, hepatocyte growth factor receptor (c-met), and ERBB3. UBPy-deficient cells exhibit aberrantly enlarged early endosomes colocalizing with enhanced ubiquitination and have reduced levels of HRS and STAM2. Congruently immortalized cells gradually stop proliferation upon induced deletion of UBPy. These results unveil a central and nonredundant role of UBPy in growth regulation, endosomal sorting, and the control of RTKs in vivo.

RUNX1 DNA-binding mutants, associated with minimally differentiated acute myelogenous leukemia, disrupt myeloid differentiation.

Mutations in the RUNX1 gene are found at high frequencies in minimally differentiated acute myelogenous leukemia. In addition to null mutations, many of the mutations generate Runx1 DNA-binding (RDB) mutants. To determine if these mutants antagonize wild-type protein activity, cDNAs were transduced into murine bone marrow or human cord blood cells using retroviral vectors. Significantly, the RDB mutants did not act in a transdominant fashion in vivo to disrupt Runx1 activity in either T-cell or platelet development, which are highly sensitive to Runx1 dosage. However, RDB mutant expression impaired expansion and differentiation of the erythroid compartment in which Runx1 expression is normally down-regulated, showing that a RDB-independent function is incompatible with erythroid differentiation. Significantly, both bone marrow progenitors expressing RDB mutants or deficient for Runx1 showed increased replating efficiencies in vitro, accompanied by the accumulation of myeloblasts and dysplastic progenitors, but the effect was more pronounced in RDB cultures. Disruption of the interface that binds CBFbeta, an important cofactor of Runx1, did not impair RDB mutant replating activity, arguing against inactivation of Runx1 function by CBFbeta sequestration. We propose that RDB mutants antagonize Runx1 function in early progenitors by disrupting a critical balance between DNA-binding-independent and DNA-binding-dependent signaling.

Importance of receptor usage, Fli1 activation, and mouse strain for the stem cell specificity of 10A1 murine leukemia virus leukemogenicity.

Murine leukemia viruses (MuLV) induce leukemia through a multistage process, a critical step being the activation of oncogenes through provirus integration. Transcription elements within the long terminal repeats (LTR) are prime determinants of cell lineage specificity; however, the influence of other factors, including the Env protein that modulates cell tropism through receptor recognition, has not been rigorously addressed. The ability of 10A1-MuLV to use both PiT1 and PiT2 receptors has been implicated in its induction of blast cell leukemia. Here we show that restricting receptor usage of 10A1-MuLV to PiT2 results in loss of blast cell transformation capacity. However, the pathogenicity was unaltered when the env gene is exchanged with Moloney MuLV, which uses the Cat1 receptor. Significantly, the leukemic blasts express erythroid markers and consistently contain proviral integrations in the Fli1 locus, a target of Friend MuLV (F-MuLV) during erythroleukemia induction. Furthermore, an NB-tropic variant of 10A1 was unable to induce blast cell leukemia in C57BL/6 mice, which are also resistant to F-MuLV transformation. We propose that 10A1- and F-MuLV actually induce identical (erythro)blastic leukemia by a mechanism involving Fli1 activation and cooperation with inherent genetic mutations in susceptible mouse strains. Furthermore, we demonstrate that deletion of the Icsbp tumor suppressor gene in C57BL/6 mice is sufficient to confer susceptibility to 10A1-MuLV leukemia induction but with altered specificity. In summary, we validate the significance of the env gene in leukemia specificity and underline the importance of a complex interplay of cooperating oncogenes and/or tumor suppressors in determining the pathogenicity of MuLV variants.

TIRC7 is induced in rejected human kidneys and anti-TIRC7 mAb with FK506 prolongs survival of kidney allografts in rats.

TIRC7 delivers essential signals during immune activation as antibodies targeting TIRC7 inhibit lymphocyte proliferation and Th1 cytokine expression in vitro and prolonged kidney and heart allograft survival in vivo. Immunohistochemical analysis of biopsy specimens from human renal allografts undergoing rejection despite treatment with Calcineurin inhibitors (CI) showed elevated TIRC7 expression. Accordingly, with a view to clinical application, we evaluated the therapeutic effect of a chimerized anti-TIRC7 mAb in combination with Tacrolimus (FK506) using a rat kidney transplantation model (DA to Lewis). The combination of sub-therapeutic doses of both compounds significantly (p<0.05) prolonged the median graft survival to 19.5 days compared to monotherapy with FK506 (median survival, 7d) or mAb against TIRC7 (7d). These results suggest a potential synergism of anti-TIRC7 mAb and FK506 action, which could be developed into a novel combination therapy in the clinic by lowering side effects of present CI treatment. Moreover, the identification of TIRC7 in graft infiltrating lymphocytes might serve as a diagnostic marker to detect allograft rejection.

TIRC7 inhibits T cell proliferation by modulation of CTLA-4 expression.

Ab targeting of TIRC7 has been shown previously to inhibit T cell proliferation and Th1 lymphocyte-associated cytokine production. In this study, we demonstrate that Ab targeting of TIRC7 induces early cell surface expression of CTLA-4. The majority of stimulated CD4+ and CD8+ human T cells coexpress CTLA-4 and TIRC7. Similar to CTLA-4, TIRC7 rapidly accumulates at the site of Ag adhesion upon T cell activation. TIRC7 seems to colocalize with CTLA-4 in human T cells, and both molecules are associated with clathrin-coated vesicles, indicating they share intracellular transport systems. Moreover, Ab targeting of TIRC7 results in an early activation of CTLA-4 transcription. The inhibition of cell proliferation mediated by TIRC7 is dependent on CTLA-4 expression because the TIRC7-mediated inhibitory effects on cell proliferation and cytokine expression are abolished by Ab blockade of CTLA-4. Splenocytes obtained from CTLA-4-deficient mice are not responsive to TIRC7 Ab targeting. Thus, TIRC7 acts as an upstream regulatory molecule of CTLA-4 expression.

IFNgamma expression inhibits LHBs storage disease and ground glass hepatocyte appearance, but exacerbates inflammation and apoptosis in HBV surface protein-accumulating transgenic livers.

BACKGROUND/AIMS: Interferon gamma (IFNgamma) controls hepatitis B virus replication. As systemic application may cause severe adverse effects, approaches of liver-directed IFNgamma gene therapy may represent an attractive alternative for treatment of chronic viral hepatitis B and thus needs testing in vivo in suitable animal models. METHODS: We therefore crossbred Alb-1HBV transgenic mice overexpressing the large HBV surface protein (LHBs) in their livers and developing LHBs storage disease and ground glass hepatocyte appearance with SAP-IFNgamma transgenic animals previously shown to exhibit constitutive hepatic IFNgamma expression, and analyzed the resulting double-transgenic offspring. RESULTS: We found that IFNgamma coexpression significantly reduced hepatic LHBs expression and thereby inhibited hepatocellular LHBs storage disease and ground glass hepatocyte appearance. The beneficial antiviral IFNgamma effects as observed in Alb1-HBV SAP-IFNgamma double-transgenic livers were associated with significantly elevated serum ALT concentrations, massive mononuclear cell infiltrates, appearance of Councilman bodies, and increased alpha-PARP (poly(ADP-ribose) polymerase cleavage). CONCLUSIONS: Exacerbation of hepatic necroinflammation and increased hepatocellular apoptosis rate in IFNgamma-expressing Alb1-HBV transgenic livers suggest that special precautions be taken for testing approaches of liver-specific IFNgamma expression in patients with chronic hepatitis B.

Defining the oncogenic function of the TEL/AML1 (ETV6/RUNX1) fusion protein in a mouse model.

The t(12;21) translocation, generating the TEL/AML1 fusion protein, is the most common genetic lesion in childhood cancer. Using a bone marrow transplantation model, we demonstrate that TEL/AML1 expression impinges on normal hematopoietic differentiation, leading to the in vivo accumulation and persistence of an early progenitor compartment with a Sca1(+)/Kit(hi)/CD11b(+) phenotype and an increased self-renewal capacity, as documented by replating assays in vitro. Differentiation of these cells is not blocked, but the frequency of mature blood cells arising from TEL/AML1-transduced progenitors is low. Impaired differentiation is prominently observed in the pro-B-cell compartment, resulting in an proportional increase in early progenitors in vivo, consistent with the t(12;21) ALL phenotype. Despite the accumulation of both multipotent and B-cell progenitors in vivo, no leukemia induction was observed during an observation period of over 1 year. These results are consistent with findings in twins with concordant ALL, showing that TEL/AML1 generates a preleukemic clone in utero that persists for several years in a clinically covert fashion. Furthermore, our studies showed that the pointed domain of TEL/AML1, which recruits transcriptional repressors and directs oligomerization with either TEL/AML1 or wild-type TEL, was essential for the observed differentiation impairment and could not be replaced with another oligomerization domain.

Inhibition of casein kinase I delta alters mitotic spindle formation and induces apoptosis in trophoblast cells.

The serine/threonine-specific casein kinase I delta (CKIdelta) is ubiquitously expressed in all tissues, is p53 dependently induced in stress situations and plays an important role in various cellular processes. Our immunohistochemical analysis of the human placenta revealed strongest expression of CKIdelta in extravillous trophoblast cells and in choriocarcinomas. Investigation of the functional role of CKIdelta in an extravillous trophoblast hybrid cell line revealed that CKIdelta was constitutively localized at the centrosomes and the mitotic spindle. Inhibition of CKIdelta with the CKI-specific inhibitor IC261 led to structural alterations of the centrosomes, the formation of multipolar spindles, the inhibition of mitosis and, in contrast to other cell lines, the induction of apoptosis. Our findings indicate that CKIdelta plays an important role in the mitotic progression and in the survival of cells of trophoblast origin. Therefore, IC261 could provide a new tool in treating choriocarcinomas.

Epigenetic mechanisms affect mutant p53 transgene expression in WAP-mutp53 transgenic mice.

We describe the construction and phenotypic characterization of 23 whey acidic protein (WAP)-mutp53 transgenic mouse lines. The mutp53-expressing lines showed a mosaic expression pattern for the transgenes, leading to a heterogeneous yet mouse line-specific expression pattern for mutp53 upon induction. Only few lines were obtained, in which the majority of the induced mammary epithelial cells expressed the mutp53 transgene, most of the transgenic lines did not express mutp53, or expressed the transgene in less than 2% of the induced mammary epithelial cells. Hormone requirements for mutp53 transgene expression from the WAP-promoter differed in high and low expressing lines, being low in high expressing lines, and even lower in multiparous mutp53 mice, where persistent expression of the transgene occurred. Repeated induction of mutp53 expression through repeated parturition resulted in the formation of expanding mutp53-expressing foci within the mammary alveolar epithelium. The data suggest that epigenetic mechanisms play a role in modulating the expression of the mutp53 transgene. To support this idea, we crossed a nonexpressing WAP-mutp53 line with a strongly SV40 T-antigen-expressing WAP-T mouse line. In the bitransgenic mice, T-antigen-induced chromatin remodeling led to re-expression of epigenetically silenced mutp53 transgene(s). In these mice, mutp53 expression was much more variable compared to SV40 T-antigen expression, and seemed to depend on the coexpression of SV40 T-antigen. Mutp53 expression in this system thus resembles the situation in many human tumors, where one can observe a heterogeneous expression of mutp53, despite a homogeneous distribution of the p53 mutation in the tumor cells.

The casein kinase 1 family: participation in multiple cellular processes in eukaryotes.

Phosphorylation of serine, threonine and tyrosine residues by cellular protein kinases plays an important role in the regulation of various cellular processes. The serine/threonine specific casein kinase 1 and 2 protein kinase families--(CK1 and CK2)--were among the first protein kinases that had been described. In recent years our knowledge of the regulation and function of mammalian CK1 kinase family members has rapidly increased. Extracellular stimuli, the subcellular localization of CK1 isoforms, their interaction with various cellular structures and proteins, as well as autophosphorylation and proteolytic cleavage of their C-terminal regulatory domains influence CK1 kinase activity. Mammalian CK1 isoforms phosphorylate many different substrates among them key regulatory proteins involved in the control of cell differentiation, proliferation, chromosome segregation and circadian rhythms. Deregulation and/or the incidence of mutations in the coding sequence of CK1 isoforms have been linked to neurodegenerative diseases and cancer. This review will summarize our current knowledge about the function and regulation of mammalian CK1 isoforms.

A dominant-negative mutant of C/EBPalpha, associated with acute myeloid leukemias, inhibits differentiation of myeloid and erythroid progenitors of man but not mouse.

The CCAAT/enhancer binding protein alpha (C/EBPalpha) is an essential transcription factor for granulocytic differentiation. C/EBPalpha mutations are found in approximately 8% of acute myeloid leukemia (AML) patients. Most of these mutations occur in the N-terminal coding region, resulting in a frame shift and the enhanced translation of a dominant-negative 30-kDa protein, which may be responsible for the differentiation block observed in AML. To test this hypothesis, we introduced a cDNA encoding an N-terminal mutated C/EBPalpha (mut10) into primary hematopoietic progenitors using a retroviral vector. Expression of mut10 in human CD34+ cord blood cells dramatically inhibited differentiation of both myeloid and erythroid lineages. Immunohistochemical analysis demonstrated coexpression of both myeloid and erythroid markers in the immature transformed cells. Surprisingly, mut10 did not block myelocytic differentiation in murine progenitors but did alter their differentiation kinetics and clonogenicity. Experiments were performed to confirm that the differential effect of mut10 on murine and human progenitors was not due to species-specific differences in C/EBPalpha protein sequences, expression levels, or inefficient targeting of relevant cells. Taken together, our results underline the intrinsic differences between hematopoietic controls in mouse and human and support the hypothesis that mutations in CEBPA are critical events in the disruption of myeloid differentiation in AMLs.

10A1-MuLV but not the related amphotropic 4070A MuLV is highly neurovirulent: importance of sequences upstream of the structural Gag coding region.

Recombinants of Moloney murine leukemia virus (MoMuLV) with either an amphotropic (MoAmphoV) or 10A1-tropic host range (Mo10A1V) induce a spongiform neurodegenerative disease in susceptible mice. To test whether MoMuLV -derived sequences are required for induction of neuropathology, mice were inoculated with either the original 10A1 or the amphotropic (4070A) MuLV isolate. Strikingly, wild-type 10A1 was more neurovirulent than Mo10A1V, inducing severe neurological clinical symptoms with a median latency of 99 days in 100% of infected mice. In contrast, no motor disturbances were detected in any of the 4070A-infected mice, although limited central nervous system lesions were observed. A viral determinant conferring high neurovirulence to 10A1 was mapped to a region encompassing the first 676 bases of the viral genome, including the U5 LTR and encoding the amino-terminus of glycosylated Gag (glycoGag). In contrast to studies with the highly neurovirulent CasFr(KP) virus, an inverse correlation between surface expression levels of glycoGag and neurovirulence was not observed; however, this does not rule out a common underlying mechanism regulating virus pathogenicity.

Casein kinase I delta (CKIdelta) is involved in lymphocyte physiology.

The casein kinase I isoform delta (CKIdelta) plays an important role in vesicular trafficking, chromosome segregation, cell cycle progression, cytokinesis, developmental processes, and circadian rhythm. In this study we examined the distribution pattern of CKIdelta and quantified its kinase activity in various tissues of BALB/c mice. Whereas CKIdelta is ubiquitously expressed, differences in the kinase activity were detected in organs with comparable CKIdelta protein levels. To elucidate the role of CKIdelta in splenocytes, which displayed the highest kinase activity, the cell type-specific distribution of CKIdelta within the spleen was investigated. Immunohistochemical analysis revealed a strong CKIdelta immunolabeling in lymphoid cells of the white pulp, while in the red pulp CKIdelta immunoreactivity was found in cells of various haematopoietic lineages. Furthermore, high CKIdelta kinase acitivity was observed in isolated lymphocytes and granulocytes of young BALB/c mice. In lymphocytes the CKIdelta activity increased upon mitogenic stimulation, whereas upon gamma-irradiation CKIdelta protein and activity levels were diminished. Interestingly, the comparison of CKIdelta activity in p53+/+ and p53-/- lymphocytes revealed a higher activity in p53+/+ lymphocytes. In addition, we observed an increased immunostaining in cells of hyperplastic B follicles and advanced B-cell lymphomas in p53-deficient mice. Thus, our results indicate that CKIdelta plays several roles in lymphocyte physiology.

Tenascin-N: characterization of a novel member of the tenascin family that mediates neurite repulsion from hippocampal explants.

Tenascin-N, a novel member of the tenascin family, was identified and shown to encode characteristic structural motifs of a cysteine-rich stretch, 3.5 epidermal growth factor-like repeats, 12 fibronectin type III homologous domains, and a fibrinogen-like domain. The third fibronectin type III homologous domain is altered by RNA splicing. Characterization of the expression of tenascin-N by in situ hybridization analysis assigned transcripts to many types of neurons in the central nervous system, to the medullary region in the kidney, and to resident macrophages of the T-cell zone in the splenic white pulp. By immunohistochemistry, tenascin-N expression is detectable in all brain regions, with a characteristic staining pattern in the hippocampus demarcating the CA3 region. Recombinantly expressed protein fragments of the alternatively spliced isoforms were presented in choice assays on patterned substrates to neurites and migrating neurons from hippocampal CA3 region explant cultures. The smaller splice variant inhibited neurite outgrowth or cell migration, whereas the longer splice form did not inhibit these functions. These observations suggest that the novel tenascin family member mediates specific repulsive properties on neurites and neurons by generating splice isoforms.