Malignant cells often display defects in autophagy, an evolutionarily conserved pathway for degrading long-lived proteins and cytoplasmic organelles. However, as yet, there is no genetic evidence for a role of autophagy genes in tumor suppression. The beclin 1 autophagy gene is monoallelically deleted in 40-75% of cases of human sporadic breast, ovarian, and prostate cancer. Therefore, we used a targeted mutant mouse model to test the hypothesis that monoallelic deletion of beclin 1 promotes tumorigenesis. Here we show that heterozygous disruption of beclin 1 increases the frequency of spontaneous malignancies and accelerates the development of hepatitis B virus-induced premalignant lesions. Molecular analyses of tumors in beclin 1 heterozygous mice show that the remaining wild-type allele is neither mutated nor silenced. Furthermore, beclin 1 heterozygous disruption results in increased cellular proliferation and reduced autophagy in vivo. These findings demonstrate that beclin 1 is a haplo-insufficient tumor-suppressor gene and provide genetic evidence that autophagy is a novel mechanism of cell-growth control and tumor suppression. Thus, mutation of beclin 1 or other autophagy genes may contribute to the pathogenesis of human cancers.

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Promotion of tumorigenesis by heterozygous disruption of the beclin 1 autophagy gene

To investigate the role of TNF alpha in the development of in vivo immune response we have generated TNF alpha-deficient mice by gene targeting. Homozygous mutant mice are viable and fertile, develop lymph nodes and Peyer's patches and show no apparent phenotypic abnormalities, indicating that TNF alpha is not required for normal mouse development. In the absence of TNF alpha mice readily succumb to L. monocytogenes infections and show reduced contact hypersensitivity responses. Furthermore, TNF alpha knockout mice are resistant to the systemic toxicity of LPS upon D-galactosamine sensitization, yet they remain sensitive to high doses of LPS alone. Most interestingly, TNF alpha knockout mice completely lack splenic primary B cell follicles and cannot form organized follicular dendritic cell (FDC) networks and germinal centers. However, despite the absence of B cell follicles, Ig class-switching can still occur, yet deregulated humoral immune responses against either thymus-dependent (TD) or thymus-independent (TI) antigens are observed. Complementation of TNF alpha functioning by the expression of either human or murine TNF alpha transgenes is sufficient to reconstitute these defects, establishing a physiological role for TNF alpha in regulating the development and organization of splenic follicular architecture and in the maturation of the humoral immune response.

/pdf/immune-and-inflammatory-responses-in-tnf-alpha-deficient-4l1tij7lf4.pdf

Immune and inflammatory responses in TNF alpha-deficient mice: a critical requirement for TNF alpha in the formation of primary B cell follicles, follicular dendritic cell networks and germinal centers, and in the maturation of the humoral immune response.

A Putative Chemokine Receptor, BLR1, Directs B Cell Migration to Defined Lymphoid Organs and Specific Anatomic Compartments of the Spleen

The generation and selection of somatic antibody mutants are key elements of acquired immunity, essential for the affinity maturation of antibody responses dependent on T cells. The mutants are generated through a mechanism that introduces point mutations at high rate into rearranged variable (V) region genes in the course of cell proliferation. Their appearance coincides with the generation of germinal centres, which are characterized by oligoclonal B-cell proliferation and have been suggested to be the microenvironment in which antibody mutants are generated. We report here direct evidence for this hypothesis. Rearranged V-region genes were amplified from the genomic DNA of cells picked from individual germinal centres. The sequence analysis of these genes revealed that most represent cells of distinct B-cell clones which expanded locally, generating somatic antibody mutants at high rate. By contrast, antigen-induced proliferation of B cells at another site, periarteriolar lymphocyte sheath-associated foci, was not associated with somatic hypermutation.

Intraclonal generation of antibody mutants in germinal centres

Maturation of the immune response in germinal centers

Peanut (Arachis hypogaea) lectin (PNL) has been shown to agglutinate the 90% of cells from murine thymus which are supposed to be immature cortical thymocytes. Further studies on the numbers of thymocytes binding fluorescein isothiocyanate conjugated PNL (FITC-PNL) confirmed the large proportion of PNL binding cells. In other organs such as bone marrow, spleen and peripheral lymph nodes, smaller proportions of PNL positive cells have been recorded. PNL-positive cells outside the thymus have been reported to be either Thy 1-positive or null cells. It has also been suggested that PNL binding may be a marker for immaturity not only in relation to T lymphocytes but also amongst haematopoietic stem cells. Thus PNL binding as an aspect of lymphocyte differentiation is a matter of considerable interest. The current study describes the distribution of horseradish peroxidase-conjugated PNL (HRP-PNL) on frozen sections of mouse lymphoid organs. It seems that PNL binds to cells in germinal centres but not to those in some other areas containing activated lymphocytes. There is good correlation between the presence of PNL-binding germinal centres in frozen sections of lymphoid organs and the number of PNL-binding cells counted in cell suspensions from the same organs.

Peanut lectin binding properties of germinal centres of mouse lymphoid tissue.

A METHOD of obtaining large numbers of fixed cells in metaphase arrested by colchicine, from a population of cells growing on a glass surface, by a method dependent on the attenuated adhesion of the cells to the surface has been described1. A modification of this method was suggested to obtain a viable population of synchronous cells. This was demonstrated2 and a method for obtaining large numbers of HeLa cells of which 80 per cent to 95 per cent showed mitotic figures has been described3. The decrease in attachment of the mitotic cells was ascribed to a decrease in the area over which the adhesion occurred1. It is, however, possible that there is a change in the intrinsic strength of the cell-glass adhesions arising from an alteration in the physical properties of the surface of the cells which round up during mitosis. One of the most easily determined of these properties is the ξ-potential arising from the cell surface charge, and accordingly we have made determinations of the electrophoretic mobility of parasynchronous populations of HeLa cells at various stages in the cell cycle.

Changes in surface charge of HeLa cells during the cell cycle.

SINCE their discovery anti-lymphocyte antisera (ALS) have been widely investigated in order to clarify their mode of action and to determine their usefulness in clinical practice. Briefly, it seems that the efficacy of a particular ALS depends on its mode of production and on the method adopted for testing its immunosuppressive effect. It also seems likely that many ALS have a harmful effect on a population of thymus-dependent (T) lymphocytes1. It is the intention of this paper to confirm this notion and to indicate that other lymphocytes (B cells) are likely to be less affected by ALS.

Differential Response of B and T Cells to ALS revealed by Electrophoretic Mobility Determinations

CHANGES in cell surface charge density have been reported several times in various types of malignancy1–4. One of the problems of applying the cell electrophoretic method to such problems is the need to obtain a quantity of single cells in suspension in a medium of known composition. The spleen, however, can be dispersed mechanically with ease, and we have chosen to investigate such spleen cell suspensions electrophoretically during the course of Friend virus disease, where gross pathological changes occur in the spleen.

Cell Electrophoretic Mobilities in Friend Virus Disease

Swelling of nuclei in Amoeba proteus is quite independent of the ionic strength of the medium or the osmolarity. Rather, it seems that surface charge is important in determining the configuration of a biological membrane which could give rise to large and specific changes in its permeability.

J. A. Forrester

Papers

Peanut lectin binding properties of germinal centres of mouse lymphoid tissue.

Changes in surface charge of HeLa cells during the cell cycle.

Differential Response of B and T Cells to ALS revealed by Electrophoretic Mobility Determinations

Cell Electrophoretic Mobilities in Friend Virus Disease

Size changes in isolated nuclei of Amoeba proteus on treatment with polyionic substances.