Loss of p21 increases sensitivity to ionizing radiation and delays the onset of lymphoma in atm-deficient mice

Ataxia telangiectasia (AT) is an autosomal recessive disorder characterized by growth retardation, cerebellar ataxia, oculocutaneous telangiectasias, and a high incidence of lymphomas and leukemias. In addition, AT patients are sensitive to ionizing radiation. Atm-deficient mice recapitulate most of the AT phenotype. p21cip1/waf1 (p21 hereafter), an inhibitor of cyclin-dependent kinases, has been implicated in cellular senescence and response to γ-radiation-induced DNA damage. To study the role of p21 in ATM-mediated signal transduction pathways, we examined the combined effect of the genetic loss of atm and p21 on growth control, radiation sensitivity, and tumorigenesis. As might have been expected, our data provide evidence that p21 modifies the in vitro senescent response seen in AT fibroblasts. Further, it is a downstream effector of ATM-mediated growth control. In addition, however, we find that loss of p21 in the context of an atm-deficient mouse leads to a delay in thymic lymphomagenesis and an increase in acute radiation sensitivity in vivo (the latter principally because of effects on the gut epithelium). Modification of these two crucial aspects of the ATM phenotype can be related to an apparent increase in spontaneous apoptosis seen in tumor cells and in the irradiated intestinal epithelium of mice doubly null for atm and p21. Thus, loss of p21 seems to contribute to tumor suppression by a mechanism that operates via a sensitized apoptotic response. These results have implications for cancer therapy in general and AT patients in particular.

The IgG Fc receptor, FcγRIIB, is a target for deregulation by chromosomal translocation in malignant lymphoma

Rearrangement of chromosomal bands 1q21–23 is one of the most frequent chromosomal aberrations observed in hematological malignancy. The genes affected by these rearrangements remain poorly characterized. Typically, 1q21–23 rearrangements arise during tumor evolution and accompany disease-specific chromosomal rearrangements such as t(14;18) (BCL2) and t(8;14) (MYC), where they are thus thought to play an important role in tumor progression. The pathogenetic basis of this 1q21–23-associated disease progression is currently unknown. In this setting, we surveyed our series of follicular lymphoma for evidence of recurring 1q21–23 breaks and identified three cases in which a t(14;18)(q32;q21) was accompanied by a novel balanced t(1;22)(q22;q11). Molecular cloning of the t(1;22) in a cell line (B593) derived from one of these cases and detailed fluorescent in situ hybridization mapping in the two remaining cases identified the FCGR2B gene, which encodes the immunoreceptor tyrosine-based inhibition motif-bearing IgG Fc receptor, FcγRIIB, as the target gene of the t(1;22)(q22;q11). We demonstrate deregulation of FCGR2B leading to hyperexpression of FcγRIIb2 as the principal consequence of the t(1;22). This is evidence that IgG Fc receptors can be targets for deregulation through chromosomal translocation in lymphoma. It suggests that dysregulation of FCGR2B may play a role in tumor progression in follicular lymphoma.

Proliferation of adult T cell leukemia/lymphoma cells is associated with the constitutive activation of JAK/STAT proteins

Human T cell leukemia/lymphotropic virus type I (HTLV-I) induces adult T cell leukemia/lymphoma (ATLL). The mechanism of HTLV-I oncogenesis in T cells remains partly elusive. In vitro, HTLV-I induces ligand-independent transformation of human CD4+ T cells, an event that correlates with acquisition of constitutive phosphorylation of Janus kinases (JAK) and signal transducers and activators of transcription (STAT) proteins. However, it is unclear whether the in vitro model of HTLV-I transformation has relevance to viral leukemogenesis in vivo. Here we tested the status of JAK/STAT phosphorylation and DNA-binding activity of STAT proteins in cell extracts of uncultured leukemic cells from 12 patients with ATLL by either DNA-binding assays, using DNA oligonucleotides specific for STAT-1 and STAT-3, STAT-5 and STAT-6 or, more directly, by immunoprecipitation and immunoblotting with anti-phosphotyrosine antibody for JAK and STAT proteins. Leukemic cells from 8 of 12 patients studied displayed constitutive DNA-binding activity of one or more STAT proteins, and the constitutive activation of the JAK/STAT pathway was found to persist over time in the 2 patients followed longitudinally. Furthermore, an association between JAK3 and STAT-1, STAT-3, and STAT-5 activation and cell-cycle progression was demonstrated by both propidium iodide staining and bromodeoxyuridine incorporation in cells of four patients tested. These results imply that JAK/STAT activation is associated with replication of leukemic cells and that therapeutic approaches aimed at JAK/STAT inhibition may be considered to halt neoplastic growth.

Screening for human T cell leukaemia/lymphoma virus among blood donors in Sweden: cost effectiveness analysis

Objective: To analyse the cost effectiveness of a national programme to screen blood donors for infection with the human T cell leukaemia/lymphoma virus.

Nitric oxide production in SJL mice bearing the RcsX lymphoma: a model for in vivo toxicological evaluation of NO.

SJL mice spontaneously develop pre-B-cell lymphoma that we hypothesized might stimulate macrophages to produce nitric oxide (NO.). Transplantation of an aggressive lymphoma (RcsX) was used to induce tumor formation. Urinary nitrate excretion was measured as an index of NO. production and was found to increase 50-fold by 13 days after tumor injection. NO. production was prevented by the addition of a nitric oxide synthase (NOS) inhibitor. The expression of inducible NOS (iNOS) in various tissues was estimated by Western blot analysis and localized by immunohistochemistry. The synthase was detected in the spleen, lymph nodes, and liver of treated but not control mice. To assess whether the iNOS-staining cells were macrophages, spleen sections from ResX-bearing animals were costained with anti-iNOS antibody and the anti-macrophage antibody moma-2. Expression of iNOS was found to be limited to a subset of the macrophage population. The concentration of gamma-interferon, a cytokine known to induce NO. production by macrophages, in the serum of tumor-bearing mice, was measured and found to be elevated 25-fold above untreated mice. The ability of ResX-activated macrophages to inhibit splenocyte growth in primary culture was estimated and macrophage-derived NO. was found to inhibit cell division 10-fold. Our findings demonstrate that ResX cells stimulate NO. production by macrophages in the spleen and lymph nodes of SJL mice, and we believe this experimental model will prove useful for study of the toxicological effects of NO. under physiological conditions.

Vaccination with syngeneic, lymphoma-derived immunoglobulin idiotype combined with granulocyte/macrophage colony-stimulating factor primes mice for a protective T-cell response.

The idiotype of the Ig expressed by a B-cell malignancy (Id) can serve as a unique tumor-specific antigen and as a model for cancer vaccine development. In murine models of Id vaccination, formulation of syngeneic Id with carrier proteins or adjuvants induces an anti-idiotypic antibody response. However, inducing a potent cell-mediated response to this weak antigen instead would be highly desirable. In the 38C13 lymphoma model, we observed that low doses of free granulocyte/macrophage colony-stimulating factor (GM-CSF) 10,000 units i.p. or locally s.c. daily for 4 days significantly enhanced protective antitumor immunity induced by s.c. Id-keyhole limpet hemocyanin (KLH) immunization. This effect was critically dependent upon effector CD4+ and CD8+ T cells and was not associated with any increased anti-idiotypic antibody production. Lymphocytes from spleens and draining lymph nodes of mice primed with Id-KLH plus GM-CSF, but not with Id-KLH alone, demonstrated significant proliferation to Id in vitro without any biased production of interferon gamma or interleukin 4 protein or mRNA. As a further demonstration of potency, 50% of mice immunized with Id-KLH plus GM-CSF on the same day as challenge with a large s.c. tumor inoculum remained tumor-free at day 80, compared with 17% for Id-KLH alone, when immunization was combined with cyclophosphamide. Taken together, these results demonstrate that GM-CSF can significantly enhance the immunogenicity of a defined self-antigen and that this effect is mediated exclusively by activating the T-cell arm of the immune response.

Activation of Jak/STAT proteins involved in signal transduction pathway mediated by receptor for interleukin 2 in malignant T lymphocytes derived from cutaneous anaplastic large T-cell lymphoma and Sezary syndrome.

Signaling through the interleukin 2 receptor (IL-2R) involves phosphorylation of several proteins including Jak3, STAT5, and, in preactivated cells, STAT3. In the present study, we examined the functional status of the IL-2R-associated Jak/STAT pathway in malignant T lymphocytes from advanced skin-based lymphomas: anaplastic large T-cell lymphoma (ALCL) and Sezary syndrome (SzS). Proliferation of three ALCL cell lines (PB-1, 2A, and 2B) was partially inhibited by rapamycin, a blocker of some of the signals mediated by IL-2R, but not by cyclosporin A, FK-506, and prednisone, which suppress signals mediated by the T-cell receptor. All the cell lines expressed on their surface the high-affinity IL-2R (alpha, beta, and gamma c chains). They showed basal, constitutive phosphorylation, and coassociation of Jak3, STAT5, and STAT3. Weak basal phosphorylation of IL-2R gamma c was also detected. In regard to SzS, peripheral blood mononuclear cells from 10 of 14 patients showed basal phosphorylation of Jak3, accompanied by phosphorylation of STAT5 in 9 patients, and STAT3 in 4 patients. However, in vitro overnight culture of SzS cells without exogenous cytokines resulted in markedly decreased Jak3 and STAT5 phosphorylation, which could be reversed by stimulation with IL-2. This indicates that the basal phosphorylation of Jak3 and STAT5 in freshly isolated SzS cells is induced rather than constitutive. The basal activation of the Jak/STAT pathway involved in IL-2R signal transduction in ALCL and SzS cells reported here suggests that this pathway may play a role in the pathogenesis of cutaneous T-cell lymphomas, although the mechanism (induced versus constitutive) may vary between different lymphoma types.

Deregulation of PAX-5 by translocation of the Emu enhancer of the IgH locus adjacent to two alternative PAX-5 promoters in a diffuse large-cell lymphoma.

Analyses of the human PAX-5 locus and of the 5' region of the mouse Pax-5 gene revealed that transcription from two distinct promoters results in splicing of two alternative 5' exons to the common coding sequences of exons 2-10. Transcription from the upstream promoter initiates downstream of a TATA box and occurs predominantly in B-lymphocytes, whereas the TATA-less downstream promoter is active in all Pax-5-expressing tissues. The human PAX-5 gene is located on chromosome 9 in region p13, which is involved in t(9;14)(pl3;q32) translocations recurring in small lymphocytic lymphomas of the plasmacytoid subtype and in derived large-cell lymphomas. A previous molecular analysis of a t(9;14) breakpoint from a diffuse large-cell lymphoma (KIS-1) demonstrated that the immunoglobulin heavy-chain (IgH) locus on 14q32 was juxtaposed to chromosome 9p13 sequences of unknown function [Ohno, H., Furukawa, T., Fukuhara, S., Zong, S. Q., Kamesaki, H., Shows, T. B., Le Beau, M. M., McKeithan, T. W., Kawakami, T. & Honjo, T. (1990) Proc. Natl. Acad. Sci. USA 87,628-632]. Here we show that the KIS-1 translocation breakpoint is located 1807 base pairs upstream of exon 1A of PAX-5, thus bringing the potent Emu enhancer of the IgH gene into close proximity of the PAX-5 promoters. These data suggest that deregulation of PAX-5 gene transcription by the t(9;14)(pl3;q32) translocation contributes to the pathogenesis of small lymphocytic lymphomas with plasmacytoid differentiation.

Predominant role of alpha 4-integrins for distinct steps of lymphoma metastasis.

To analyze the role of alpha4-integrins in lymphoma metastasis, sublines of the T-cell lymphoma LB were generated by retrovirus-mediated gene transfer that differ exclusively in the expression of alpha4-integrins. Using LB-alpha4 and control LB-NTK cells, we demonstrate that expression of alpha4-integrins strongly suppresses metastasis formation of LB lymphoma cells in secondary lymphoid organs such as spleen, mesenteric and peripheral lymph nodes, or Peyer's patches after i.v. injection into syngeneic BALB/c mice. Moreover, alpha4-integrin expression inhibited development of metastatic tumors in liver, lung, and kidney. Expansion of LB lymphoma cells in bone marrow was not affected by alpha4-integrin expression. In vivo migration assays using 51Cr-labeled lymphoma cells demonstrated that low-metastatic LB-alpha4 cells accumulated with the same efficiency as high-metastatic LB-NTK cells in all target organs examined and were even enriched in mucosal lymphoid organs. Collectively, these results indicate that alpha4-integrins inhibit metastasis formation of lymphoma cells at a stage subsequent to the invasion of target organs.

Characterization of the transforming activity of p80, a hyperphosphorylated protein in a Ki-1 lymphoma cell line with chromosomal translocation t(2;5).

We have molecularly cloned a cDNA encoding a protein uniquely expressed and hyperphosphorylated at tyrosine residues in a Ki-1 lymphoma cell that contained chromosomal translocation t(2;5). The encoded protein p80 was shown to be generated by fusion of a protein-tyrosine kinase and a nucleolar protein B23/nucleophosmin (NPM). The coding sequence of this cDNA turned out to be virtually identical to that of the fusion cDNA for NPM-anaplastic lymphoma kinase (ALK) previously cloned from the transcript of the gene at the breakpoint of the same translocation. Overexpression of p80 in NIH 3T3 cells induced neoplastic transformation, suggesting that the p80 kinase is aberrantly activated. The normal form of p80 was predicted to be a receptor-type tyrosine kinase on the basis of its sequence similarity to the insulin receptor family of kinases. However, an immunofluorescence study using COS cells revealed that p80 was localized to the cytoplasm. Thus, subcellular translocation and activation of the tyrosine kinase presumably by its structural alteration would cause the malignant transformation. We also showed that a mutant p80 lacking the NPM portion was unable to transform NIH 3T3 cells. Thus, the NPM sequence is essential for the transforming activity, suggesting that the chromosomal translocation is responsible for the oncogenesis. Finally, Shc and insulin receptor substrate 1 (IRS-1) were tyrosine-phosphorylated and bound to p80 in p80-transformed cells. However, mutants of p80 that were defective for binding to and phosphorylation of Shc and insulin receptor substrate 1 could transform NIH 3T3 cells. Association of these mutants with GRB2 was still observed, suggesting that interaction of p80 with GRB2 but not with Shc or IRS-1 was relevant for cell transformation.

Tumor dormancy and cell signaling: anti-mu-induced apoptosis in human B-lymphoma cells is not caused by an APO-1-APO-1 ligand interaction.

Signal transduction initiated by crosslinking of antigen-specific receptors on T- and B-lymphoma cells induces apoptosis. In T-lymphoma cells, such crosslinking results in upregulation of the APO-1 ligand, which then interacts with induced or constitutively expressed APO-1, thereby triggering apoptosis. Here we show that crosslinking the membrane immunoglobulin on human lymphoma cells (Daudi) (that constitutively express APO-1) does not induce synthesis of APO-1 ligand. Further, a noncytotoxic fragment of anti-APO-1 antibody that blocks T-cell-receptor-mediated apoptosis in T-lymphoma cells does not block anti-mu-induced apoptosis. Hence, in B-lymphoma cells, apoptosis induced by signaling via membrane IgM is not mediated by the APO-1 ligand.