Cutting edge: protective effects of notch-1 on TCR-induced apoptosis.

The Notch receptor protein was originally identified in Drosophila and is known to mediate cell to cell communication and influence cell fate decisions. Members of this family have been isolated from invertebrates as well as vertebrates. We isolated mouse Notch-1 in a yeast two-hybrid screen with Nur77, which is a protein that has been shown previously to be required for apoptosis in T cell lines. The data presented below indicate that Notch-1 expression provides significant protection to T cell lines from TCR-mediated apoptosis. These data demonstrate a new antiapoptotic role for Notch-1, providing evidence that, in addition to regulating cell fate decisions, Notch-1 can play a critical role in controlling levels of cell death in T cells.

Apoptosis-associated gene expression in the corpus luteum of the rat.

The involution of the corpus luteum (CL) at parturition is an example of physiological apoptosis, a complex process involving massive vascular regression while luteal cells undergo apoptosis. In the present study, changes in gene expression associated with physiological apoptosis were examined. Three genes isolated in our laboratory because of their association with apoptotic processes in the ovary, mammary gland, and prostate served as the focus of our investigation: Y81, Gas-1, and the gene IAP encoding integrin-associated protein. Y81 is a novel gene for which three transcripts are apparent. A Y81 cDNA clone representing the longest transcript has been isolated; it shows an open reading frame exhibiting a region of very high homology with members of the frizzled family, the prototypes of which are cell autonomous polarity genes encoding seven-pass transmembrane receptor proteins, for example the receptor for Wingless. Gas-1 is known as a growth-arrest gene that inhibits DNA synthesis when microinjected into cells. Integrin-associated protein is a beta 3-integrin-binding protein for which, recently, a thrombo-spondin-binding activity has been recognized. These three genes, all sharply up-regulated in the course of physiological involution processes in the ovarian CL, in mammary gland, and in prostate, seem promising candidates-by virtue of their specific expression in distinct tissues undergoing programmed cell death-as mediators of stimuli leading to apoptosis and subsequent phagocytosis. In this study, sulfated glycoprotein-2, previously observed in many instances of physiological apoptosis, was further employed as an indicator for incipient apoptosis, and stromelysin was followed as a marker for the tissue remodeling activity that is intimately associated with apoptosis during involution.

DDC-4, an apoptosis-associated gene, is a secreted frizzled relative.

The differential display method has been used in our laboratory as a coincidence analysis to isolate genes expressed in common in each of three different rat tissues undergoing physiological apoptosis: mammary gland, ovarian corpus luteum and ventral prostate. The most interesting of these isolates, DDC-4, shows a clear association with apoptosis, its expression being confined to these three organs, and only during their involution. Using DDC-4 as probe, we screened a rat ovarian cDNA library to obtain full-length isolates. One isolate, Y81 clone 40, gives rise to a protein of approximately 40 kDa with coupled in vitro transcription/translation. Sequencing of this clone indicates an open reading frame of 1044 nucleotides encoding a protein of 39.7 kDa with a putative signal sequence. This clone exhibits a high homology with the cysteine-rich domain, i.e. the ligand-binding domain, of the fizzled gene family originally defined as tissue polarity genes in Drosophila. The homology of Y81 clone 40 is most extensive with the newly described secreted frizzled relatives, the frzb subfamily.

Apoptosis in the rat mammary gland and ventral prostate: detection of cell death-associated genes using a coincident-expression cloning approach.

Apoptosis plays a striking role in the hormone-dependent involution of the mammary gland, but it has proved difficult to distinguish between the 'cell death' associated genes and the 'tissue remodelling' genes which are expressed concurrently. To identify cell death-associated genes, we have established a 'coincidence analysis' based on the previously described 'RNA differential display' method of Liang and Pardee (1992). Coincidence analysis allows the detection of genes expressed during related processes in different organs and was employed here to identify transcripts in which expression patterns are seen to be associated with apoptosis during involution of both rat mammary- and the ventral prostate glands. That the coincidence analysis is a promising approach can be seen from the fact that while widely accepted apoptosis markers such as transglutaminase (Fesus et al, 1987; Strange et al, 1992) and sulfated glycoprotein-2 (Buttyan et al, 1989; Strange et al, 1992; Guenette et al 1994) exhibited similar expression in both regressing tissues, transcription of tissue remodelling enzymes was minimal in the involuting prostate. We describe here the characteristics of five clones isolated which show coincident expression during programmed cell death in mammary and prostate tissues. Partial sequence analysis revealed for three clones high homologies with previously described genes; the putative rat homolog of the growth arrest gene gas-1 (Schneider et al, 1988; Del Sal et al, 1992), an homolog of the mouse 'Integrin Associated Protein' (IAP) (Brown et al, 1990; Lindberg et al, 1993) and the sequence encoding for the 'Allograft Inflammatory Factor' AIF-1 (Autieri et al, 1995; Utans et al, 1995). One clone displayed homology with an expressed human sequence tag and one clone unrelated to any known DNA sequence was isolated. The expression of these genes in involuting rat mammary and ventral prostate, was correlated with that in other organs and in situ hybridization was applied to establish that the secretory epithelial cells which undergo programmed cell death are the site of elevated expression during the course of involution. Furthermore, we conclude that the coincidence analysis approach described here could be easily applied to facilitate the characterization of gene expression i.e. for the detection and comparison of hormonally regulated genes in different organs.

Isolation of cell death-associated cDNAs from involuting mouse mammary epithelium.

Although apoptosis is important in determining cell fate and maintaining tissue homeostasis, the initiation and control of apoptotic cell death in epithelium is not well understood. Post-lactationai involution of the mammary gland provides both an important developmental process and a normal physiological setting for studying apoptosis of epithelium. We used a differential screening strategy, based on previous studies correlating morphology with gene expression and nucleic acid integrity during mammary gland involution, to isolate genes involved in the regulation and execution of apoptotic cell death in regressing mammary epithelium. This screening strategy yielded a large number of genes the expression of which is significantly altered during mammary gland involution. These include genes associated with cell death processes, tissue remodelling and mesenchymal differentiation. In addition, a number of novel genes have been isolated. We have used Northern analysis and in situ hybridisation to study the expression of a selection of these putative death-associated genes during post-lactational mouse mammary gland involution.

Characterization of a novel murine testis-specific serine/threonine kinase.

Using degenerate oligos corresponding to two highly conserved motifs within the protein kinase catalytic domain and a PCR-based cloning strategy, we have isolated a cDNA fragment encoding a new member of the Ser/Thr (serine/threonine) family of protein kinases. Expression analysis revealed that the fragment recognized two transcripts (1.6 and 1.4 kb) exclusively in testis. Using this fragment as a probe, we have cloned a full-length cDNA from a mouse testis cDNA library. The sequence has a 1092-bp open reading frame encoding a protein of 364 amino acids. The N-terminally localized kinase catalytic domain has all the conserved motifs found in other Ser/Thr kinases. Northern blot analysis using the full-length sequence as a probe revealed that the cloned gene corresponds to the 1.6-kb transcript, suggesting the existence of at least two testis-specific novel Ser/Thr kinases. We propose the name testis-specific kinase-1 (TSK-1) for the gene described here. A GenEMBL databank search revealed highest homology to the human gene encoding rac protein kinase-beta and the group of yeast Ser/Thr kinases encoded by SNF-1, nim-1, KIN-1 and KIN-2.

Expression of the B cell-associated tyrosine kinase gene Lyn in primary neuroblastoma tumours and its modulation during the differentiation of neuroblastoma cell lines.

The src-related intracellular protein tyrosine kinase Lyn is a signal transducing molecule for surface immunoglobulin M and is expressed predominantly in hemopoietic cells. We report here the expression of the lyn gene in human neuroblastoma. In surgical tumour samples lyn transcripts were found preferentially at early stages whereas they were barely detectable in highly malignant tumours. In a cloned human neuroblastoma cell line, Be(2)C, lyn mRNA levels increased during neuronal differentiation induced by retinoic acid. Lyn mRNA levels were undetectable and did not respond to retinoic acid in a glial-type neuroblastoma clone, SH-EP. Retinoic acid-induced glial differentiation was associated with a reduction of lyn transcripts in a clonal I-type neuroblastoma cell line, SH-IN, which shares properties of both neuronal- and glial-type clones. Like pp60c-src Lyn may be involved in a signalling pathway of neuroblasts committed to neuronal differentiation.

Lyn, a src-like tyrosine-specific protein kinase, is expressed in HL60 cells induced to monocyte-like or granulocyte-like cells.

During the in vitro differentiation of HL60 cells tyrosine-specific kinases are activated. The expression of lyn, a src-related tyrosine kinase, was studied by analysis of the steady-state levels of its transcript during the cell differentiation process induced by retinoic acid, phorbol 12-myristate 13-acetate and 1,25-dihydroxyvitamin D3. In contrast to an earlier report we observe only a small induction of the lyn-RNA levels compared to uninduced control cells. In unstimulated HL60 cells, the level for the lyn-transcript was comparatively high. A second, minor human lyn-transcript with an estimated size of 3.7 kb which has not been previously described, was identified.