Apoptotic cells overexpress vinculin and induce vinculin-specific cytotoxic T-cell cross-priming.

Here we show that apoptotic cells overexpress vinculin and are ingested by dendritic cells, which subsequently cross-prime vinculin-specific cytotoxic T lymphocytes (CTLs). Successful cross-priming requires that the apoptotic cells provide maturation signals to dendritic cells through CD40-CD40 ligand (CD40L) interactions. If apoptotic cells are CD40L-, the help of a third T cell is needed for priming, indicating a regulatory role for apoptotic cells in determining priming or tolerance. Vinculin-specific CTL priming is also related to apoptosis in vivo, given that in HIV-seropositive individuals, the frequency of specific CTLs depends on the proportion of peripheral CD40L+ apoptotic cells.

Transfection of the c-myc oncogene into normal Epstein-Barr virus-harboring B cells results in new phenotypic and functional features resembling those of Burkitt lymphoma cells and normal centroblasts.

Activated c-myc gene was introduced into the cells of three normal Epstein-Barr virus (EBV)-positive lymphoblastoid B cell lines (LCL). The cells were monitored for the appearance of new phenotypic and functional features compared with the control LCL cells transfected with plasmid that did not contain the c-myc gene. The LCL-expressing c-myc constitutively did not arrest growth in low serum concentration. However, the cell number in the cultures failed to increase because of substantial cell death. Death was due to apoptosis as demonstrated by flow cytometric analysis of propidium iodide-stained cells, by typical DNA laddering in gel electrophoresis, and by the inspection of Giemsa-stained cell smears. Apoptosis was also induced by exposing the transfected cells to antibodies directed to the immunoglobulin mu chain (a-mu-ab) irrespective of the serum concentration in the culture. Exposure of the cells to CD40 ligand (CD40L) or CD40 monoclonal antibody prevented cell apoptosis. Upon transfection with c-myc, the LCL cells acquired a vacuolated morphology that was never observed in control cells. Moreover, the expression of CD10 and CD38 was upregulated, while that of CD39 and especially CD23 was downregulated. Unlike that observed in certain Burkitt lymphoma (BL) cell lines that share the same surface phenotype (CD10+CD38+CD23-CD39-), the c-myc-transfected cells expressed lymphocyte function-associated (LFA) 1, LFA-3, and intercellular adhesion molecule 1 and grew in large clumps rather than single-cell layers. Expression of CD10 and CD38 was particularly evident on the cells undergoing apoptosis, thus suggesting a correlation between the presence of these markers and the apoptotic process. Cells placed in conditions favoring in vitro apoptosis displayed downregulation of Bcl-2 protein. Bcl-2 expression was, however, upregulated when the cells were exposed to CD40L. These data indicate that the B cells expressing c-myc constitutively acquire some of the features of normal centroblasts and of BL cells, including the expression of CD10 and CD38, and the propensity to undergo apoptosis, which can be prevented by exposure to CD40L. Therefore, these cells can serve as a model system to study both BL lymphomagenesis as well as the process of B cell selection occurring in the germinal centers.

Inhibition of Burkitt's lymphoma cells growth in SCID mice by a PNA specific for a regulatory sequence of the translocated c-myc.

In Burkitt's lymphoma (BL) cells due to a t(8;14) chromosomal translocation c-myc is often placed in proximity to the Emu enhancer of the Ig locus and upregulated. We demonstrated that in BL cells a peptide nucleic acid (PNA), complementary to intronic Emu sequences (PNAEmuwt), specifically blocks the expression of the c-myc oncogene under the Emu enhancer control and inhibits BL cell growth in culture. Here, we investigated whether PNAEmuwt was also able to block tumor growth in SCID mice inoculated with human BL cell lines. After subcutaneous inoculum in mice BL cells reproducibly form tumors. Both pre-treatment of BL cells with PNAEmuwt before inoculum and chronic intravenous administration of PNAEmuwt to mice already inoculated with BL cells selectively caused increased latency of tumor appearance and decreased final tumor size. Tumors from PNAEmuwt-treated animals showed substantial areas of cell necrosis and of c-myc downregulation. Inhibition of tumor growth was specific and was not observed with PNAEmumut carrying sequence mutations and in BL cell lines where the translocated c-myc is not under the control of the Emu enhancer. These data confirm the potential therapeutic value of PNA targeted to regulatory non-coding regions.

Lack of mutagenicity and clastogenicity of PNAEmu-NLS targeted to a regulatory sequence of the translocated c-myc oncogene in Burkitt's lymphoma.

Peptide nucleic acids (PNAs) are synthetic homolog of nucleic acids in which the phosphate-sugar polynucleotide backbone is replaced by a flexible polyamide. They bind complementary polynucleotide sequences with higher affinity and specificity than their natural counterparts. PNAs linked to the appropriate nuclear localization signal (NLS) peptide have been used to selectively down-regulate the expression of several genes in viable cells. For example in Burkitt's lymphoma (BL) cells the c-myc oncogene is translocated in proximity to the Emu enhancer of the Ig gene locus and upregulated. PNAs complementary to the second exon of c-myc or to the Emu enhancer sequence (PNAEmu-NLS), selectively and specifically block the expression of the c-myc oncogene and inhibit cell growth in vitro and in vivo. PNAEmu-NLS administration to mice did not exhibit toxic effects even at the highest concentration allowed by the experimental conditions. Because of the accumulating data confirming PNAEmu-NLS potential therapeutic value, PNAEmu-NLS was evaluated for the inability to induce mutations in tester strains of Salmonella typhimurium, Escherichia coli, and at the hprt locus in Chinese hamster ovary cells (CHO). Moreover, the induction of chromosomal aberrations in CHO cells and of micronuclei in human lymphocytes were investigated. We may conclude that PNAEmu-NLS neither induces mutations nor has clastogenic effects as detectable by treatment under the standard test conditions.

Effects in live cells of a c-myc anti-gene PNA linked to a nuclear localization signal.

Peptide nucleic acids (PNA) are synthetic homologs of nucleic acids in which the phosphate-sugar polynucleotide backbone is replaced by a flexible polyamide. In this study, a PNA construct was employed as an anti-gene agent in intact cells in culture. The cell lines studied were derived from Burkitt's lymphomas (BL) that presented a translocated and hyperexpressed c-myc oncogene. A 17-mer anti-myc PNA, complementary to a unique sequence located at the beginning of the second exon of the oncogene, and was covalently linked at its N terminus to a nuclear localization signal (NLS) (PNA-myc(wt)-NLS). When BL cells were exposed to PNA-myc(wt)-NLS, the anti-gene construct was localized predominantly in the cell nuclei and a rapid consequent downregulation of c-myc expression occurred. Under these conditions, both completion of a productive cell cycle and apoptosis were inhibited.

Survival and Immunosuppression Induced by Hepatocyte Growth Factor in Chronic Lymphocytic Leukemia.

Chronic lymphocytic leukemia (CLL), the most common leukemia among adults in the western world, is characterized by a progressive accumulation of relatively mature CD5+ B cells in peripheral blood, lymph nodes and bone marrow. Despite much recent advancement in therapy, CLL is still incurable. Lymph nodes and bone marrow represent sanctuary sites preserving leukemic cells from spontaneous or drug-induced apoptosis, and infiltration of leukemic cells in these districts correlates with clinical stages and prognosis. The central role played by the microenvironment in the disease has become increasingly clear. Different chemokines (CXCL12, CXCL13, CCL19, CCL21) may in fact participate in attracting CLL cells into bone marrow and lymph nodes, where various factors, such as IL-15 and CXCL12, enhance leukemic cells survival. Recently, we have suggested that hepatocyte growth factor (HGF), produced by microenvironmental stromal cells, can contribute to CLL pathogenesis. We have demonstrated that HGF exerts a double effect on CLL B cells through the interaction with its receptor c- MET; HGF, infact, protects CLL B cells, which are c-MET+, from apoptosis, and also polarizes mono/macrophages towards the M2 phenotype, thus facilitating the evasion of the CLL clone from immune control. This double effect appears mediated by the activation of two major signaling pathways: STAT3TYR705 and AKT. The aim of this review is to summarize data on HGF and c-MET expression in normal B cells and in B cell malignancies, with a particular emphasis on our results obtained in CLL. Altogether, the observations described here suggest that the HGF/c-MET axis may have a prominent role in malignancy progression further indicating novel potential therapeutic options aimed to block HGF-induced signaling pathways in B lymphoproliferative disorders.

Effects of miRNA-15 and miRNA-16 expression replacement in chronic lymphocytic leukemia: implication for therapy.

Chronic lymphocytic leukemia (CLL) clones are characterized by loss of a critical region in 13q14.3, (del(13)(q14)) involving the microRNA (miRNA) cluster miR-15a and miR-16-1. We have investigated the effects of replacement of miR-15a and miR-16-1. CLL cells transfected with these miRNA mimics exhibited a decrease in cell viability in vitro and impaired capacity for engraftment and growth in NOD/Shi-scid,γcnull (NSG) mice. No synergistic effects were observed when the two miRNA mimics were combined. The phenomena were not restricted to CLL with the del(13)(q14) lesion. Similar effects induced by miRNA mimics were seen in cells with additional chromosomal abnormalities with the exception of certain CLL clones harboring TP53 alterations. Administration of miRNA mimics to NSG mice previously engrafted with CLL clones resulted in substantial tumor regression. CLL cell transfection with miR-15a and miR-16-1-specific inhibitors resulted in increased cell viability in vitro and in an enhanced capacity of the engrafted cells to grow in NSG mice generating larger splenic nodules. These data demonstrate that the strong control by miR-15a and miR-16-1 on CLL clonal expansion is exerted also at the level of full-blown leukemia and provide indications for a miRNA-based therapeutic strategy.

lncRNA profiling in early-stage chronic lymphocytic leukemia identifies transcriptional fingerprints with relevance in clinical outcome.

Long non-coding RNAs (lncRNAs) represent a novel class of functional RNA molecules with an important emerging role in cancer. To elucidate their potential pathogenetic role in chronic lymphocytic leukemia (CLL), a biologically and clinically heterogeneous neoplasia, we investigated lncRNAs expression in a prospective series of 217 early-stage Binet A CLL patients and 26 different subpopulations of normal B-cells, through a custom annotation pipeline of microarray data. Our study identified a 24-lncRNA-signature specifically deregulated in CLL compared with the normal B-cell counterpart. Importantly, this classifier was validated on an independent data set of CLL samples. Belonging to the lncRNA signature characterizing distinct molecular CLL subgroups, we identified lncRNAs recurrently associated with adverse prognostic markers, such as unmutated IGHV status, CD38 expression, 11q and 17p deletions, and NOTCH1 mutations. In addition, correlation analyses predicted a putative lncRNAs interplay with genes and miRNAs expression. Finally, we generated a 2-lncRNA independent risk model, based on lnc-IRF2-3 and lnc-KIAA1755-4 expression, able to distinguish three different prognostic groups in our series of early-stage patients. Overall, our study provides an important resource for future studies on the functions of lncRNAs in CLL, and contributes to the discovery of novel molecular markers with clinical relevance associated with the disease.

Characterization of EBV-positive lymphoblastoid cell lines obtained from HIV seropositive patients with or without lymphomas.

Epstein-Barr-virus- (EBV-) positive lymphoblastoid cell lines (LCLs) spontaneously arising in vitro were obtained from the peripheral blood of six HIV-seropositive patients and from the peripheral blood and the bone marrow of one patient (LAM) with AIDS and lymphoma. The LCLs from HIV-seropositive patients had phenotypic, cytogenetic, and biological characteristics indistinguishable from those of normal LCLs obtained by infecting B cells with EBV in vitro. The LCLs from LAM patient comprised composite cell populations. Cloning analysis and cell fractionation procedures showed that, beside normal EBV-infected cells, these lines contained a malignant subset population characterized by c-myc rearrangement, abnormal karyotype, and a surface phenotype similar to that of Burkitt's lymphoma cells. Analyses of Ig heavy chain and c-myc oncogene loci showed that these malignant cells were the progeny of a single precursor. Nevertheless, these cells had heterogeneous EBV-fused termini, a finding which indicates that EBV infection followed c-myc rearrangement.

Analysis of K-ras, p53, bcl-2 and Rb expression in non-small cell lung cancer cell lines.

Six non-small cell lung cancer (NSCLC) cell lines (A-549, Ca-Lu-6, SK-Lu-1, Ca-Lu-1, SK-Mes-1 and LX-1) were studied to assess the presence of multiple concomitant alterations of different oncogenes (K-ras, bcl-2) and tumor suppressor genes (p53, Rb) in NSCLC. K-ras (exon 1) and p53 (exons 5-8) gene mutations were determined via a PCR-based-DGGE (Denaturing Gradient Gel Electro-phoresis) and by sequencing approach. Different mutations were found in the Ist exon of K-ras gene in 5 of 6 cell lines examined. Five of six cell lines contained K-ras mutations at codon 12 (A-549, SK-Lu-1, LX-1) or codon 13 (SK-Mes-1, Ca-Lu-1). In addition, 5 of 6 cell lines showed p53 mutations of exon 8 (SK-Mes-1, Ca-Lu-1 cod. 280; LX-1 cod. 273) or exon 6 (Ca-Lu-6 cod. 196; SK-Lu-1 cod. 193). In 4 of these cell lines, p53 protein nuclear expression was also confirmed with DO-7 mAb immunocytochemistry. Expression of cytoplasmic bcl-2 protein, by anti-bcl-2 mAb flow cytometric analysis, was found in A-549, Ca-Lu-1, SK-Lu-1, SK-Mes-1 cell lines. In contrast, RT-PCR analysis of Rb gene could not identify any change in the cell lines examined. In conclusion, most NSCLC cell lines tested displayed concomitant multiple oncogene/tumor suppressor gene alterations.

A retinoic acid resistant HL-60 cell clone sensitive to N-(4-hydroxyphenyl) retinamide-mediated clonal growth inhibition.

Among the Retinoic Acid (RA) derivatives, retinamides, and in particular N-(4-hydroxyphenyl) retinamide (4-HPR), are currently being investigated in selected cases of cancer chemoprevention. The cellular target range, however, seems to be limited, as cells of hemopoietic origin are virtually incapable of terminal differentiation upon addition of the compound. We have reconsidered the effect of 4-HPR on HL-60 cells by taking advantage of a mutant clone, generated in our laboratory, unresponsive to RA but highly responsive to dimethylsulfoxide (DMSO). We show here that this clone, upon addition of 4-HPR, although unable of undergoing full differentiation, shows considerable reduction of clonal growth. Moreover, the combination of 4-HPR and RA resulted in a much greater effect than the administration of 4-HPR alone. We suggest that 4-HPR and RA, at least in terms of mediating growth inhibition, may follow different metabolic pathways.

H and L ferritin gene expression in U937 cells induced to macrophage differentiation.

Ferritin is an ubiquitous protein that has been shown to regulate cell differentiation in several experimental systems. In this study we have investigated the expression of ferritin genes encoding the heavy (H) and light (L) chains in t'B U937 cell line, induced to differentiate to macrophage-like cells by 12-O-tetradecanoylphorbol-13-acetate (TPA), retinoic acid (RA) or 1-beta-D-arabinofuranosylcytosine (Ara-C). An increase in the level of H ferritin mRNA was detected in U937 cells that had been incubated with Ara-C. Treatment of U937 cells with Actinomycin D suggested that the H ferritin mRNA increase was mediated by post-transcriptional mechanisms. The L ferritin mRNA level increased only following stimulation of U937 cells with RA. Immunophenotypic and cytochemical analyses showed that Ara-C was the strongest inducer of the macrophagic differentiation of U937 cells. These results suggest that the increase of H ferritin mRNA expression may represent a sensitive marker of myeloid cells differentiating along the monocyte-macrophage lineage.

Cellular and Molecular Characterization of Two Cases of Castleman's Disease, Plasma Cell Variant.

We report the cellular and molecular characterization of two cases of Castleman's disease, plasma cell variant, that differed in their clinical presentation and course. Patient 1 had Castleman's disease in association with Kaposis's sarcoma unrelated to human immunodeficiency virus (HIV) infection and died while he was receiving an aggressive chemotherapeutic regimen for Kaposi's sarcoma. Patient 2 had an isolated retroperitoneal lymphoid mass with an adjacent enlarged limph nodes and his symptoms disappeared completely following the surgical removal of both. Pathologic and immunohistochemical analyses in both cases, revealed that there was a massive infiltration of polyclonal plasma cells in the interfollicular areas of the lymph nodes. Immunoglobulin gene rearrangement studies confirmed the polyclonal nature of B-lineage cells in the involved lymph nodes. Southern blot experiments failed to demonstrate the presence of EBV genome copies in the same lymph nodes. These paradigmatic cases lend further support to the notion that Castleman's disease is an extremely heterogeneous entity.

Cytogenetic rearrangement of C-MYC oncogene occurs prior to infection with Epstein-Barr virus in the monoclonal malignant B cells from an AIDS patient.

Two cell lines were originated from the peripheral blood (PB-LAM) and bone-marrow (BM-LAM) of a patient with Burkitt-type acute lymphoblastic leukemia and AIDS. 26 and 7 clones were isolated from PB-LAM and BM-LAM respectively by limiting dilution. All of these had surface IgM lambda and the CD10 marker with low to absent CD23, CD30, CD39 and surface adhesion molecules. Furthermore, they shared the same chromosomal abnormalities (trisomy 7 and t(8;14) translocation) and the same rearrangements of immunoglobulin L and H chain and of c-myc gene loci. These features are those most frequently found in Burkitt's lymphoma (BL) cells and were different from those of the parental cell lines, which, besides cells identical to those of the malignant clones, also contained normal lymphoblastoid cells. Therefore, the cloning procedure used selected for the growth of cells with malignant features. EBV latent antigens were detected in all clones by Western blotting and their pattern of expression resembled that usually observed in BL cells. All the clones were positive for the EBV genome by Southern blotting and had monomorphic EBV-fused termini as determined by using cDNA probes specific for sequences at either end of the viral genome. However, the clones derived from PB-LAM had EBV fused termini of a different size from that of the clones derived from BM-LAM. The presence of different EBV-fused termini in otherwise monoclonal malignant cells indicate that EBV infection was possibly a late event in lymphomagenesis following rearrangement of the c-myc and the Ig gene loci.

Prognostic factors in CLL.

Chronic lymphocytic leukemia (CLL) is a clinically heterogeneous disease, as some patients progress rapidly toward the more advanced studies, whereas others survive for a long period without the need for treatment. This heterogeneity of clinical course was somehow unexplained until studies on the CLL cell features disclosed that the CLL clones were heterogeneous and were characterized by different phenotypic and genotypic features in the different patients. On the basis of these observations, it was determined in retrospective studies that clones characterized by unmutated IGHV genes, and/or CD38 and/or ZAP-70 expression conferred a more severe prognosis to the CLL patients. Here, we present data on prospective studies carried out on Binet A-stage patients, in whom the markers were determined at diagnosis and their predictive value was assessed in comparison with chromosomal abnormalities and gene expression or micro RNA profiles. In addition, hypothesis on the potential pathogenetic role of these markers will be presented.

PNAEmu can significantly reduce Burkitt's lymphoma tumor burden in a SCID mice model: cells dissemination similar to the human disease.

In human Burkitt's Lymphoma (BL) BRG cells, a t(8;14) translocation, placing c-myc near the Emu enhancer of the H chain locus, causes tumor expansion. Earlier, we showed that a peptide nucleic acid complementary to the Emu sequence (PNAEmu), specifically inhibited the expression of translocated c-myc and impaired the growth of BRG cells-induced subcutaneous tumors in mice suffering from severe combined immunodeficiency (SCID). In this study, the therapeutic potential of PNAEmu was evaluated in a systemic mouse model. BRG-BL cells transfected with the luciferase gene were inoculated intravenously into SCID mice resulting in a preferential expansion, similar to the one of human adult patients, in the abdominal cavity, central nervous system and bone marrow. The mice were chronically injected intraperitoneally either with PNAEmu or with control PNA. The treatment was stopped when the control animals developed severe neurological symptoms. As detected both by inspection at necropsy and imaging, overall tumor growth in PNAEmu-treated mice decreased by >80%. Histological and immunohistochemical studies showed, only in PNAEmu-treated mice, a substantially reduced BL cell growth at the major sites of invasion and vast areas of necrosis in the lymphomatous tissues, with concomitant c-myc expression downregulation. Altogether, the data support the therapeutic potential of PNAEmu in human adult BL.

Transforming growth factor beta-1 (TGF-beta 1) released by an Epstein-Barr virus (EBV) positive spontaneous lymphoblastoid cell line from a patient with Kostmann's congenital neutropenia inhibits the growth of normal committed haemopoietic progenitors in vitro.

This study reports the characterization of a spontaneous lymphoblastoid cell line (LCL) raised from the peripheral blood of a patient with Kostmann's congenital neutropenia. The LCL was composed of EBV-infected polyclonal B cells and displayed surface markers and pattern of growth in vitro typical of normal LCLs. The supernatant of the LCL contained a colony inhibiting activity (CIA) that decreased the cloning efficiency of normal committed haemopoietic progenitors and was identified as immunoreactive transforming growth factor beta 1 (TGF-beta 1) by neutralization experiments with a specific antiserum. Control studies with a panel of LCLs spontaneously derived from the peripheral blood of patients seropositive for Epstein-Barr virus (EBV) infections showed that 5/30 LCLs produced a CIA. This CIA was not identifiable as TGF-beta 1 but rather was due to the combined effects of tumour necrosis factor alpha (TNF alpha), tumour necrosis factor beta (TNF beta) and interferon alpha (IFN alpha), that were present in the LCL supernatants. The hypothesis that the B cells latently infected by EBV in vivo and possibly expanded as a consequence of the infection may have contributed to the inhibition of the patient granulopoiesis by releasing TGF-beta 1 will be discussed.