Alf Grandien

Phosphorylation by Cdk2 is required for Myc to repress Ras-induced senescence in cotransformation

The MYC and RAS oncogenes are frequently activated in cancer and, together, are sufficient to transform rodent cells. The basis for this cooperativity remains unclear. We found that although Ras interfered with Myc-induced apoptosis, Myc repressed Ras-induced senescence, together abrogating two main barriers of tumorigenesis. Inhibition of cellular senescence required phosphorylation of Myc at Ser-62 by cyclin E/cyclin-dependent kinase (Cdk) 2. Cdk2 interacted with Myc at promoters, where it affected Myc-dependent regulation of genes, including Bmi-1, p16, p21, and hTERT, which encode proteins known to control senescence. Repression of senescence by Myc was abrogated by the Cdk inhibitor p27Kip1, which is induced by antiproliferative signals like IFN-γ or by pharmacological inhibitors of Cdk2 but not by inhibitors of other Cdks. In contrast, a phospho-mimicking Myc-S62D mutant was resistant to these manipulations. Inhibition of cyclin E/Cdk2 reversed the senescence-associated gene expression pattern imposed by Myc/cyclin E/Cdk2. This indicates a role of Cdk2 as a transcriptional cofactor and activator of the antisenescence function of Myc and provides mechanistic insight into the Myc-p27Kip1 antagonism. Finally, our findings highlight that pharmacological inhibition of Cdk2 activity is a potential therapeutical principle for cancer therapy, in particular for tumors with activated Myc or Ras.

Functional diversity and clonal frequencies of reactivity in the available antibody repertoire

The present experiments address functional antibody diversity and clonal distribution in murine available repertoires. IgM‐containing supernatants were prepared by unbiased, polyclonal stimulation of resting splenic B cells from C57BL/6 mice, to ensure similar numbers of responding clones/culture and equivalent growth and maturation of all clones. The repertoires of clones and clonal mixtures were quantitatively assayed by limiting dilution analysis (LDA) on immunoblots of sodium dodecylsulfate polyacrylamide gel electrophoresis of homologous liver extracts, allowing to determine specific clonal frequencies towards the many hundred blotted antigens. The clonal frequency of reactivity of B cells with the extract was shown to be a bi‐modal distribution of specific frequencies between 1/220 and 1/100u2009000. Cross‐correlation analysis of reactivity to different bands in individual supernatants revealed low levels of cross‐reactivity, suggesting that the blotted extract provides a very diverse set of antigens. Investigation of the affinity/concentration thresholds for detection of antigen‐antibody interactions of our assay supports the notion that global repertoire analyses on immunoblots were highly discriminative and non‐degenerate. Furthermore, reactivity patterns obtained with complex antibody mixtures correlated with the frequency of clonal reactivities as determined by LDA. The results demonstrate a large functional diversity of resting B lymphocytes, indicating a minimal repertoire size that is orders of magnitude higher than previous theoretical proposals suggested, and extensively heterogeneous in the size of clonal specificities.

Technical advance: live-imaging analysis of human dendritic cell migrating behavior under the influence of immune-stimulating reagents in an organotypic model of lung.

This manuscript describes technical advances allowing manipulation and quantitative analyses of human DC migratory behavior in lung epithelial tissue. DCs are hematopoietic cells essential for the maintenance of tissue homeostasis and the induction of tissue‐specific immune responses. Important functions include cytokine production and migration in response to infection for the induction of proper immune responses. To design appropriate strategies to exploit human DC functional properties in lung tissue for the purpose of clinical evaluation, e.g., candidate vaccination and immunotherapy strategies, we have developed a live‐imaging assay based on our previously described organotypic model of the human lung. This assay allows provocations and subsequent quantitative investigations of DC functional properties under conditions mimicking morphological and functional features of the in vivo parental tissue. We present protocols to set up and prepare tissue models for 4D (x, y, z, time) fluorescence‐imaging analysis that allow spatial and temporal studies of human DCs in live epithelial tissue, followed by flow cytometry analysis of DCs retrieved from digested tissue models. This model system can be useful for elucidating incompletely defined pathways controlling DC functional responses to infection and inflammation in lung epithelial tissue, as well as the efficacy of locally administered candidate interventions.

Coelomic and Bone Marrow‐Derived B Cells

Models of B-cell development and function usually consider a common linear pathway with branch points, giving rise to the various populations and compartments. The postulated branch points in ontogenic commitment of B cells, however, are yet to be identified. The debate surrounding CD5 B lymphocytes should be placed in this context. After several years of intensive work, we continue to discuss whether this population is defined already in the differentiation pathways of pre-B cells (before the emergence of immunocompetent B cells) or whether CD5 B cells merely represent a (se1f)Ag-selected, activated, and persistent compartment of a unique B-cell system.* In other words, CD5 B cells may either represent evolutionary selection fixed in a specific program of development or the manifestation of Ig-dependent and activation-related somatic selection, requiring defined local conditions, or both.3 The view preferring the notion of a CD5 B-cell lineage bears the problem that a B-cell precursor that gives rise to CD5 B cells exclusively has not been isolated thus far.4s5 The alternative is somewhat vague as to the selective ligands and mechanisms and has difficulties explaining both the population dynamics of a persistent and yet noncycling set of lymphocytes that maintain an activated cell morphology6 and the similarity of V,/V, repertoire patterns with other B cells.

Transformation of mouse T cells requires MYC and AKT activity in conjunction with inhibition of intrinsic apoptosis

Peripheral T-cell lymphoma is an aggressive non-Hodgkins lymphoma characterized by excessive proliferation of transformed mature T cells. The number and nature of genetic aberrations required and sufficient for transformation of normal T cells into lymphomas is unknown. Here, using a combinatorial in vitro-approach, we demonstrate that overexpression of MYC together with activated AKT in conditions of inhibition of intrinsic apoptosis rapidly resulted in transformation of mature mouse T cells with a frequency approaching 100%. Injection of transformed cells into mice resulted in rapid development of aggressive T cell lymphoma, characterized by spread to several organs, destruction of tissue architecture and rapid death of the animals. TcR-sequencing revealed a polyclonal repertoire of tumor cells indicating that co-expression of MYC, activated AKT and BCLXL is sufficient for tumor transformation and do not require acquisition of additional genetic events. When analyzing cells with inducible expression we found that proliferation of transformed T cells required sustained expression of both MYC and AKT. AKT exerted a dual function as it inhibited induction of, and promoted exit from, cellular quiescence and contributed to inhibion of apoptosis. Downregulation of AKT and/or MYC together with BCLXL resulted in rapid and complete elimination of cells through induction of apoptotic cell death.