The c-myc locus is a common integration site in type B retrovirus-induced T-cell lymphomas

Type B leukemogenic virus (TBLV) induces rapidly appearing T-cell leukemias. TBLV insertions near the c-myc gene were detectable in 2 of 30 tumors tested, whereas 80% of the tumors showed c-myc overexpression. TBLV insertions on chromosome 15 (including a newly identified locus, Pad7) may cause c-myc overexpression by cis-acting effects at a distance.     

Selection of target sites for mobile DNA integration in the human genome

DNA sequences from retroviruses, retrotransposons, DNA transposons, and parvoviruses can all become integrated into the human genome. Accumulation of such sequences accounts for at least 40% of our genome today. These integrating elements are also of interest as gene-delivery vectors for human gene therapy. Here we present a comprehensive bioinformatic analysis of integration targeting by HIV, MLV, ASLV, SFV, L1, SB, and AAV. We used a mathematical method which allowed annotation of each base pair in the human genome for its likelihood of hosting an integration event by each type of element, taking advantage of more than 200 types of genomic annotation. This bioinformatic resource documents a wealth of new associations between genomic features and integration targeting. The study also revealed that the length of genomic intervals analyzed strongly affected the conclusions drawn--thus, answering the question "What genomic features affect integration?" requires carefully specifying the length scale of interest.     

c-myc mRNA expression in non-Hodgkin’s lymphomas

Steady state c-myc mRNA levels determined by Northern blot analysis were examined in non-Hodgkin's lymphomas (NHL) of both high (n = 29) and low malignancy (n = 18), and in non-specific chronic lymphadenitis (n = 6). High grade NHL, classified according to the updated Kiel classification, revealed significantly larger amounts of c-myc mRNA compared with low grade NHL and lymphadenitis. mRNA levels in non-specific lymphadenitis were lower than in low grade NHL, but the differences were not statistically significant. No correlation between c-myc mRNA levels and the immunologic phenotype was discernible. Growth fractions of the NHL were determined by immunostaining with the monoclonal antibody Ki-67. Significant correlations between the percentages of Ki-67-positive cells, as well as the amounts of c-myc mRNA, and classification into high or low grade NHL were found. However, the percentage of Ki-67 positive cells and c-myc mRNA levels in individual cases and in the various histologic entities of NHL did not correlate. Our results indicate the overexpression of the c-myc gene in NHL, and a highly significant correlation of steady state c-myc mRNA levels with the prognosis-related histomorphologic Kiel classification of NHL into different subgroups of low and high grade malignancy.     

Differential requirement for accessory cells in polyclonal T-cell activation

Highly purified rat Ia-negative (OX-6-) and Ia-positive (OX-6+) T cells were employed to examine the requirement for accessory cells (AC) and/or soluble factors in the activation of resting T cells with Con A, PHA, sodium periodate, or antigen. A variety of cells were employed as AC, including Ia-positive and Ia-negative macrophages (M phi), gamma-irradiated (2000 rad) or non-irradiated OX-6+ T cells, and several Ia-negative adenovirus-transformed rat embryo fibroblast cell lines. Our results suggested that for the expression of IL-2 receptors (IL-2R) and proliferation of OX-6- T cells in response to Con A, PHA, or antigen, there was an obligatory requirement for the presence of AC which could not be overcome by the addition of IL-1 and/or IL-2. Activation of OX-6- T cells with antigen required the presence of Ia+ AC, while activation with mitogens could be initiated with Ia- AC. M phi were efficient in AC function in all responses tested, while the AC function of OX-6+ T cells (TAPC) proved discriminatory under different conditions. The optimal response to PHA required much higher concentrations of TAPC as AC than for the Con A response. TAPC failed to stimulate sodium periodate-treated T cells under any conditions tested. Furthermore, when TAPC were employed as AC, their antigen-presenting ability was radiosensitive, while their AC function for Con A and PHA was radioresistant. These results suggest that molecules involved in T cell-AC interactions may differ, depending on the source of AC and/or type of the proliferative stimulus provided to T cells. This data has been discussed in the context of T-cell activation.     

Rorgamma (Rorc) is a common integration site in type B leukemogenic virus-induced T-cell lymphomas

The retrovirus type B leukemogenic virus (TBLV) causes T-cell lymphomas in mice. We have identified the Rorgamma locus as an integration site in 19% of TBLV-induced tumors. Overexpression of one or more Rorgamma isoforms in >77% of the tumors tested may complement apoptotic effects of c-myc overexpression.     

Dual genotype in cutaneous T-cell lymphomas and pseudolymphomas

Summary Genomic DNA digests of skin biopsies from 20 patients with cutaneous T-cell lymphomas and pseudolymphomas were studied by hybridization, using probes for the constant region of the T-cell receptor beta chain and the joining region of the immunoglobin heavy chain gene. Skin biopsies from all 20 patients contained a monoclonal T-cell population. In addition, DNA from 5 patients contained an immunoglobulin gene rearrangement. These results demonstrate that cutaneous T-cell lymphomas are clonal T-cell malignancies that frequently express a dual genotype, which may sometimes reflect the clonotypic heterogeneity of these disorders.     

Antigen-specific murine T-cell lymphomas: functional heterogenicity

Two ovalbumin (OVA)-specific helper lymphomas (designated ROT/6.1 and ROT/6.2) were established by transformation of enriched, OVA-immune T cells with the radiation leukemia virus (RadLV). Shortly after establishment these lymphomas provided carrier (OVA)-specific help for anti-hapten antibody response. However, 5 months later ROT/6.1 lost its OVA specificity and could augment anti-hapten antibody response in the presence of an unrelated carrier. ROT/6.2 retained its antigen-specific helper function over 10 months of repeated passaging. This OVA-specific helper line inhibited anti-hapten antibody response when given together with an unrelated carrier. Cloning of ROT/6.2 by limiting dilution revealed that only 3 of 10 clones tested had OVA-specific helper activity. None of the clones could induce antigen-specific DTH reaction. The interrelationship between the functional heterogenicity, specificity, and stability of the helper lines is discussed.     

Monocyte-independent T-cell activation by polyclonal antithymocyte globulins.

The in vitro mitogenic properties of polyclonal antithymocyte and antilymphocyte globulins (ATG) on peripheral blood mononuclear cells were investigated. The ATG were mitogenic in a dose-dependent manner with maximal proliferation observed at 250 or 500 micrograms/ml. ATG activated blastogenesis of CD4+, CD8+, and CD57+ (NK cells) lymphocytes. The ATG induced interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) gene expression and lymphokine secretion in cell culture supernatant and IL-2 receptor (CD25) expression. At submitogenic concentrations ATG potentialized the effect of phorbol esters on T cell proliferation, but not that of calcium ionophore. The mitogenic effect of ATG was not abrogated by monocyte depletion indicating that like CD2 monoclonal antibodies (mAbs) ATG activate T cells via a monocyte-independent pathway. CD3 and CD2 mAbs which activate T cells without binding to B surface determinants stimulated the proliferation of B cells and their differentiation into immunoglobulin (Ig)-secreting cells. In contrast, ATG induced only a transient B cell activation, but failed to support B cell differentiation into Ig-secreting cells despite the secretion of IL-2. These properties shared by ATG obtained in horses or rabbits by immunization with different cell types appear to differ from those of other T cell mitogens.     

Cytologic and immunocytologic studies of peripheral T-cell lymphomas

Lymph node aspirates from 18 peripheral T-cell lymphomas (PTLs) were analyzed. Cytologic and immunocytologic studies were performed on Cytospin preparations using the alkaline phosphatase-antialkaline phosphatase method with a panel of monoclonal antibodies (CD3, CD4, CD8, CD19 and CD30). The cytologic diagnosis was confirmed by histologic investigation. Nine lymph node aspirates from patients with Lennert's lymphoma, angioimmunoblastic (AILD)-type PTL and pleomorphic small-cell-type PTL were composed predominantly of small-to-intermediate-sized lymphocytes. An admixture of plasma cells, eosinophils, neutrophils, lymphocytes with an irregular nucleus, granula in the cytoplasm or abundant cytoplasm was also seen. Nine lymph node aspirates from patients with T-immunoblastic lymphoma, pleomorphic large-cell-type PTL and large-cell anaplastic (Ki-1+) lymphoma showed marked cytologic heterogeneity. Immunocytologic investigation of the aspirates using the antibodies CD3, CD4, CD8, CD19 and CD30 was helpful for the differentiation of PTLs from reactive lymphadenopathy and other malignant lymphomas. A strong predominance of CD3+ cells was found in only seven cases. The aspirates expressed a helper/inducer phenotype in 11 cases and a suppressor/cytotoxic phenotype in 4 cases. A T-cell phenotype not corresponding to the normal T-cell phenotype was found in nine cases. In 15 of the 18 cases, the number of CD19+ cells was found to be less than 15%. The large cells of the large-cell anaplastic (Ki-1+) lymphoma expressed the antigens CD30 and CD45 and were negative for CD15. These findings indicate that immunocytologic studies can be used in improving the cytologic diagnosis of PTLs.     

Herpesvirus saimiri Tip gene causes T-cell lymphomas in transgenic mice

New World primates develop T-cell lymphomas on infection with Herpesvirus saimiri. To investigate the oncogenic potential of the Tip gene of Herpesvirus saimiri strain C488, we tried to establish transgenic mice that should express Tip under control of a constitutive promoter. Although transgene-positive embryos were found, lines could not be established. However, using a system in which the transgene has to be activated by a Cre recombinase-mediated deletion, we were able to obtain several Tip transgenic lines. At high expression levels, the mice developed T-cell lymphomas. Thus, Tip can induce lymphomas and is therefore very likely responsible for the oncogenicity of Herpesvirus saimiri.     

Mechanistic basis for RAG discrimination between recombination sites and the off-target sites of human lymphomas

During V(D)J recombination, RAG targeting to correct sites versus off-target sites relies on both DNA sequence features and on chromatin marks. Kinetic analysis using the first highly active full-length purified RAG1/RAG2 complexes has now allowed us to define the important catalytic features of this complex. We found that the overall rate of nicking, but not hairpinning, is critical for the discrimination between correct (optimal) versus off-target (suboptimal) sites used in human T-cell lymphomas, and we show that the C-terminal portion of RAG2 is required for this. This type of kinetic analysis permits us to analyze only the catalytically active RAG complex, in contrast to all other methods, which are unavoidably confounded by mixture with inactive RAG complexes. Moreover, we can distinguish the two major features of any enzymatic catalysis: the binding constant (K(D)) and the catalytic turnover rate, k(cat). Beyond a minimal essential threshold of heptamer quality, further suboptimal heptamer deviations primarily reduce the catalytic rate constant k(cat) for nicking. Suboptimal nonamers reduce not only the binding of the RAG complex to the recombination site (K(D)) but also the catalytic rate constant, consistent with a tight interaction between the RAG complex and substrate during catalysis. These features explain many aspects of RAG physiology and pathophysiology.     

Proteins expressed by the c-myc oncogene in lymphomas of human and avian origin

We have prepared antisera against synthetic peptides corresponding to the C-terminal region of the avian and human myc oncogene coding sequences. Immunoprecipitates from avian and human cells show two major proteins which, by the criteria of hybrid-selected translation, transfection, and peptide-blocking assays, are the c-myc protein products. These proteins are phosphorylated nuclear proteins which are tightly bound to the nuclear matrix-lamin and which have a short half-life. Analysis of avian and human lymphoma cell lines containing rearranged c-myc alleles show significant changes in the ratio of the two proteins although only the avian lymphomas have increased quantities of c-myc protein.     

Targeting Plk1 in cutaneous T-cell lymphomas (CTCLs)

Comment on: Nihal M, et al. Cell Cycle 2011; 10:1303-11.