Notch-regulation upon Dll4-stimulation of TGFb-induced apoptosis and gene expression in human B-cell non-Hodgkin lymphomas.

Notch-signalling has been implicated as a pathogenetic factor and a therapeutical target in T-cell leukaemias and in some lymphomas of B-cell origin. Our aim was to investigate the role of Notch-signalling in apoptosis regulation in human non-Hodgkin B-cell lymphoma (B-NHL) cell lines and in primary chronic lymhocytic leukaemia (CLL) cells using Delta-like 4 (Dll4) ligand mediated Notch activation and gamma-secretase inhibitor (GSI) mediated Notch inhibition in vitro. The potential cross-talk of Notch with the transforming growth factor-beta (TGFb) pathway in apoptosis induction was also explored, and the effect of GSI on drug-induced apoptosis was assessed. Modulation of Notch-signalling by itself did not change the rate of apoptosis in B-NHL cell lines and in CLL cells. TGFb-induced apoptosis was decreased - but not completely abolished - by GSI in TGFb-sensitive cell lines, but resistance to the apoptotic effects of TGFb were not reversed by Notch activation or inhibition. Drug-induced apoptosis was not modified by GSI. We identified Hairy/Enhancer of Split (HES)-1 as a TGFb target gene in selected - TGFb-sensitive - B-NHL cell lines. TGFb-induced HES-1 was only partially Notch-dependent in later phases. Apoptosis regulation by TGFb and GSI was not dependent on the transcriptional regulation of c-myc. In conclusion, our data does not support a unifying role of Notch in regulating apoptosis in B-NHL, but warns that gamma-secretase inhibitors may actually counteract apoptosis in some cases.

Activity of the notch-signalling pathway in circulating human chronic lymphocytic leukaemia cells.

Dysregulation of the Notch-pathway has been implicated in the pathogenesis of chronic lymphocytic leukaemia (B-CLL). We characterized the mRNA expression of Notch pathway elements in circulating normal B- and B-CLL cells, and compared expression profiles with clinical and prognostic data. Similar expression profiles were found in normal B-cells and B-CLL cells, however, most B-CLL samples showed lower Hairy/Enhancer of Split-1 expression than normal B-cells, which suggests that the pathway is not over-activated in B-CLL. The expression of Notch-pathway genes did not correlate with other prognostic factors of B-CLL. The importance of Notch-signalling in CLL cells in lymphatic tissue microenvironments remains to be determined.

Richter's and prolymphocytic transformation of chronic lymphocytic leukemia are associated with high mRNA expression of activation-induced cytidine deaminase and aberrant somatic hypermutation.

Chronic lymphocytic leukemia (CLL) is an indolent B-cell non-Hodgkin's lymphoma that may transform into higher-grade lymphoma. The transformation involves an increased number of prolymphocytic cells, termed prolymphocytic transformation (PLT) or the development of diffuse large B-cell lymphoma (DLBL), also referred to as Richter's transformation (RT). To analyze whether activation-induced cytidine deaminase (AID), which is essential for somatic hypermutation (SHM) of normal B-cells, and malfunction of SHM termed aberrant somatic hypermutation (ASHM) are associated with higher-grade transformation of CLL, AID mRNA expression and the mutation pattern of c-MYC, PAX-5 and RhoH genes were analyzed in eight cases of CLL without transformation and in 21 cases that showed RT or PLT. Chronic lymphocytic leukemia cases, which showed no transformation or eventually transformed into higher-grade lymphoma, showed low levels of AID mRNA expression and low frequency of mutations of c-MYC, PAX-5 and RhoH genes. In both RT and PLT, high-levels of AID mRNA expression and high-frequency mutations of c-MYC, PAX-5 and RhoH genes were detected. These results indicate that AID expression and ASHM are associated with higher-grade transformation of CLL and provide further evidences that AID expression and ASHM may be activated during the clonal history of B-cell lymphomas.

Clonal selection in the bone marrow involvement of follicular lymphoma.

To characterize the pathways of bone marrow (BM) involvement of follicular lymphoma (FL), we performed morphological and immunophenotypical analysis of tumor cells from lymph nodes (LNs) and corresponding BMs in 21 patients with FL. In three cases, genealogical trees were constructed based on the immunoglobulin variable region heavy chain (IgV(H)) gene sequences of tumor clones from LNs and BMs. Results showed that FLs within the BMs display identical or lower cytological grades than in the LNs. In the majority of cases, different proportions of tumor cells expressed bcl-2, CD10 and Ki67 in LNs and BMs. Tumor cells in the BM showed ongoing somatic hypermutation of the IgV(H) genes; the distribution of these mutations was highly consistent with antigen selection. The topology of the genealogical trees revealed that different subclones populate the LN and BM and BM infiltration may occur at different points of the clonal evolution of FL. Early descendants of the original tumor clone and derivatives of diversified tumor clones may invade the BM. These results suggest that the BM involvement of FL is associated with intensive clonal selection of tumor cells, and the BM provides a microenvironment similar to the germinal centers of LNs, where tumor cells retain their biological nature.

Relationship between the mutational status of VH genes and pathogenesis of diffuse large B-cell lymphoma in Richter's syndrome.

Patients with chronic lymphocytic leukemia (CLL) may develop diffuse large B-cell lymphoma (DLBL), also known as Richter's syndrome. Mutational status of immunoglobulin (Ig) heavy-chain variable region (VH) genes have prognostic impact in CLL. Patients with mutated VH genes have a stable disease, whereas patients with unmutated VH gene have more aggressive disease. The mutational status of CLLs that transform to DLBL is unknown. To reveal whether Richter's syndrome occurs in CLLs with mutated or unmutated VH genes, we have performed mutational analysis on serial specimens from eight patients. CLL and DLBL tumorclones were identical in five cases and they were different in three cases. Six CLLs expressed unmutated and two cases expressed mutated VH genes. In five of the six unmutated CLLs, the DLBL clones evolved from CLL tumorclones and the VH genes expressed by DLBLs were also unmutated. In one unmutated and two mutated CLLs, the DLBLs expressed mutated VH genes, but in these three cases the DLBL tumorclones developed as independent secondary neoplasm. These results suggest that Richter's syndrome may develop in both mutated or unmutated CLLs, but clonal transformation of CLL to DLBL occur only in the unmutated subgroup of CLL.

Hypermethylation of the gene promoter and enhancer region can regulate Fas expression and sensitivity in colon carcinoma.

Expression of the cell surface receptor Fas is frequently lost or decreased during tumor progression in human colon carcinomas. The methylation status of a 583 bp CpG-rich region within the Fas promoter (-575 to +8) containing 28 CpG sites was determined in human colon carcinoma cell lines. In Caco(2) (no Fas expression), 82-93% of CpG sites were methylated, whereas none were methylated in GC(3)/c1 (high Fas expression). In RKO (intermediate level of Fas), a single CpG site, located at -548, was 100% methylated. The inhibitor of methylation, 5-aza-2'-deoxycytidine (5-azadC), upregulated Fas expression in four of eight cell lines, and sensitized RKO cells to recombinant FasL-induced apoptosis. The p53-binding region in the first intron of the Fas gene was partially methylated in Caco(2), and 5-azadC potentiated Ad-wtp53-induced upregulation of Fas expression. Methylation-specific PCR of the first intron detected partial methylation in four out of 10 colon carcinoma tumor samples in vivo. The data suggest that DNA hypermethylation is one mechanism that contributes to the downregulation of Fas expression and subsequent loss of sensitivity to Fas-induced apoptosis in colon carcinoma cells.

Molecular pathology of tumor metastasis. I. Predictive pathology.

Millennium reviews of oncology agreed that the last century produced major developments mainly in the management of the primary tumor, but despite all of these results, cancer still remains among the leading causes of death due to the failure of clinical management of disseminated disease. This failure is primarily due to the lack of detailed information on the molecular mechanisms of tumor metastasis. Therefore, one of the hottest fields in experimental oncology is metastasis research, which provides more and more information about the molecular mechanisms. However, this information is fragmented and is not yet exploited in clinical practice. A new field of diagnostic pathology recently emerged, which translates basic research data to diagnostic practice to provide clinically relevant information on the biological potential (in this case metastatic potential) of the malignant tumors. Since tumor cell-extracellular matrix interactions are key features of tumor dissemination, expression of genes responsible for them can define the metastatic potential of malignant tumors. This review summarizes our recent knowledge on the metastatic geno- and phenotype of major human solid tumors: lung, colon, breast, prostate cancers and malignant melanoma.

TGF beta 1 induces caspase-dependent but death-receptor independent apoptosis in lymphoid cells.

Transforming growth factor beta 1 (TGF beta 1) is an antiproliferative and proapoptotic cytokine for normal B-cells, however many B-cell lymphomas have lost their response to TGF beta 1. The aim of this study was to identify the sequence of events in apoptosis induced by TGF beta 1 in an EBV negative, human B-cell lymphoma line (HT58). The proportion of apoptotic cells increased gradually (up to 72 hr) at an optimal dose range of 0.5-1.0 ng/ml. The induced cell death required the action of downstream caspases. Caspase activation was accompanied by an increase in the permeability of mitochondrial membranes, but there was no change in the expression of certain members of Bcl-2 family (Bcl-2, Bax, Bcl-XL). Similarly, none of the death receptors or ligands were involved in apoptosis induction. Further study will include the participation of TGF beta 1 target genes in the pore formation of mitochondrial membranes and/or the elimination of a putative survival signal.

Syndecan-1 expression in different soft tissue tumours.

BACKGROUND: In vitro studies have suggested that expression of syndecan-1 (CD138) is correlated with morphologic phenotype (epithelioid or spindle) of cultured tumour cells. MATERIALS AND METHODS: Fifty-seven different soft tissue tumours were selected to analyse their syndecan-1 (CD138) reactivity. Immunohistochemical staining of paraffin sections, following a high temperature unmasking technique, was performed. The intensity and the pattern of staining was studied. RESULTS: Cell membrane positivity was observed in epithelioid sarcomas and epithelial elements of synovial sarcomas. GISTs, some malignant epithelioid schwannoma and some fibromatosis showed intracytoplasmatic reaction, while pyogenic granuloma, Kaposi's sarcoma, fibrosarcoma and dermatofibrosarcoma protuberans were negative. CONCLUSION: At first it seemed that the cell membrane positivity of syndecan-1 accompanied true epithelial differentiation in soft tissue sarcomas, but the results further highlighted the non specific nature of this expression. Therefore the heterogeneity in the appearance of syndecan-1 in various soft tissue tumours is not simply associated with the phenotype, suggesting more complex functions.

[The role of syndecans in lymphoid systems] / Syndecan-expresszió és a lymphoid rendszer.

Syndecans, transmembrane heparan sulfate proteoglycans, play an important role in cell-cell and cell-matrix interactions, as receptors/co-receptors of matrix elements, cytokines, growth factors. These functions are partly non-specific and due to the heparan sulfate chains attached to the ectodomain, and partly specific related to the transmembrane and cytoplasmic domains of the core protein. In hemopoietic cells syndecan-1 is expressed in certain B cells, in pre-B cells and plasma cells. In lymphoproliferative diseases this normal syndecan-1 expression of plasma cells is retained in myelomas/plasmocytomas, other lymphoplasmocytic NHL subtypes and primary effusional lymphomas. Syndecan-1 expression is probably gained in B-CLL, and lost in other NHLs of pre- or post-follicular origin. These results suggest that the expression of syndecan is essential for some NHLs, probably ensuring the required connections to the microenvironment. From a diagnostic point of view, syndecan-1 is a very useful phenotypic marker to identify cells with plasmocytic differentiation. The importance of syndecan expression in CLL and Hodgkin-lymphoma still requires further studies.

Syndecan-1-dependent homotypic cell adhesion in HT58 lymphoma cells.

OBJECTIVES: Many cellular functions are controlled by cell-cell and cell-matrix interactions. It has recently been found that syndecans, transmembrane heparan sulphate (HS) proteoglycans, can act as receptors or co-receptors and modulate cell adhesion. Our aim was to study the role of syndecan-1 in the aggregation of human lymphoma cells, and to investigate its effect on cell survival. METHODS: Immunocytochemistry, confocal laser scanning microscopy, flow cytometry and aggregation/reaggregation bio-assays were used on HT58, BL41/95 and Raji lymphoma cell lines. RESULTS: Bio-assays showed that the aggregation of HT58 cells was inhibited by heparin, HS, removal of the HS chain and binding of the anti-syndecan-1 monoclonal antibody. In the search for a counter-receptor of syndecan-1, several adhesion molecules were tested, but none of them proved to be the adhesion partner. In the case of heparitinase/trypsin digestion with long-term inhibition of HS synthesis (sodium chlorate treatment), the inhibited aggregation was accompanied by cell cycle arrest and the induction of apoptosis. CONCLUSIONS: The results obtained showed that surface syndecan-1 expression contributes to homotypic adhesion. In addition, HS chains, including those on syndecan-1, take part in the regulation of cell proliferation and active cell death in HT58 lymphoma cells.

Syndecans and the lymphoid system.

Syndecans, transmembrane proteoglycans, play an important role in cell-matrix and cell-cell interactions, as well as modulators in receptor activation. These functions are partly non-specific and related to the heparan sulfate chains attached to the ectodomain, and partly specific due to the transmembrane and cytoplasmic domains of the core protein. In hemopoietic cells syndecan-1 is expressed only in B cells at certain differentiation stages (pre-B and plasma cells). In lymphoproliferative conditions this selective expression is retained in myelomas/plasmacytomas and other lymphoplasmacytic NHL subtypes, and primary effusional lymphomas. It is probably gained in B-CLL, and lost in other NHLs of pre- or post-follicular origin. It is concluded from these empiric results that the expression of syndecan is essential for some NHLs, probably ensuring the required connections to the microenvironment. From a diagnostic point of view, syndecan-1 is a very useful phenotypic marker to indentify cells with plasmacytic differentiation. The importance of syndecan expression in CLL and Hodgkin's lymphoma still requires further studies.

IgE-containing cells in gastric mucosa with and without Helicobacter pylori infection.

Gastric mucosa responds with inflammation to Helicobacter pylori (H. pylori) infection. While numerous reports have shown that the immune system produces specific IgG, IgA, and IgM isotype anti H. pylori antibodies, IgE-mediated pathways of H. pylori-associated gastritis are mostly unknown. Our aim was to evaluate whether an increased presence of IgE in the antral gastric mucosa is responsible for the severity of the H. pylori-associated gastritis. The number of IgE-containing cells was estimated in formalin-fixed, paraffin-embedded antral gastric biopsy specimens using immunohistochemistry in three groups of patients: (i) 20 H. pylori-positive cases with moderate inflammation, (ii) 19 H. pylori-negative cases with moderate inflammation, and (iii) 19 H. pylori-negative cases with normal mucosa. In chronic gastritis, the number of IgE-positive cells increased significantly as compared to normal mucosa. In gastritic patients, H. pylori positivity was accompanied by a significant accumulation of IgE-positive cells, mainly plasma cells. These data suggest that IgE-mediated immune response probably plays an important role in the development of H. pylori-associated gastritis.

Interaction of cytocidal drugs and the inhibition of caspase-3 by 3-nitrosobenzamide.

The effect of 3-nitrosobenzamide (NOBA) on the etoposide, staurosporine and dexamethason induced rapid (4-6 hr), caspase-dependent apoptosis was investigated in thymocytes and lymphoma cells by flow cytometric assay of DNA fragmentation. When NOBA (ED(50) = 4 microM) was added to these cell systems, the rapid onset of apoptosis was prevented. Such apparent protection by NOBA was related to the inactivation of caspase-3, by s-nitrosylation of 1.3 mol -SH per enzyme molecule out of 7 -SH groups. Since NOBA by itself induces DNA fragmentation within 18 hr in lymphoma cells, our results indicate that at least two active cell death pathways exist with apparent dissimilar kinetics and molecular mechanisms.

[Cell death and tumor formation]. / A sejt halála és a daganat keletkezése.

Proliferation and death (apoptosis) are the main cellular functions in the maintenance of the homeostasis of the organism. Disturbances in the regulation of these two basic phenomena may results in the continuous and irreversible accumulation of cells in time and space. This process which could become autonomous without any obvious biological "advantage". Both the overproduction of anti-apoptotic factors (e.g. bcl-2) or the failure of pro-apoptotic signals (e.g. p53) can contribute to tumorigenesis. Authors review the different phases of apoptosis with emphasis on the most important decision making elements in cell death, i.e. caspases and mitochondria, as well as on the interactions of the regulatory pathways. The better understanding of these coordinated and interdependent actions may help to achieve improvements in many fields of oncology.

Syndecan-1 (CD138) expression in human non-Hodgkin lymphomas.

Syndecan-1, an important transmembrane heparan sulphate proteoglycan is expressed in distinct stages of differentiation of normal lymphoid cells: in pre-B cells and Ig-producing plasma cells; however, its normal function, or presence in lymphoid malignancies, is still largely unknown. The expression of syndecan-1 (CD138) was studied in 57 human non-Hodgkin lymphomas using immunocytochemistry and immunohistochemistry. Positive expression of syndecan-1 was found in the plasma cells in chronic lymphoblastic leukaemia (B-CLL) cases, in different plasmocytoid lymphomas as well as in myeloma. All normal and malignant Tcells, or CD5+ cells other than B-CLL proved to be negative. These results strongly suggest that syndecan-1 expression is a characteristic phenotypic marker for B-CLL and lymphoplasmocytoid lymphomas and could be used for diagnostic purposes.

[The effect of trace elements--especially zinc--on metastasizing of Lewis lung tumor]. / Nyomelemek--elsösorban a cink--hatása a Lewis lung tumor áttétképzésére.

The effect of a widely used trace element preparation (Béres-Drop Plus, BDP) on the liver metastasis forming potential of 3LL-HH tumor was studied. The daily, oral administration of BDP caused a decrease in the number of liver metastases, irrespective of the presence or absence of the primary spleen tumor. It is suggested, that the zinc component of BDP is responsible, at least partly--for the inhibitory action.

Oral administration of a trace element preparation and zinc inhibit liver metastasis of 3LL-HH murine tumor cells.

A trace element preparation (TEP-Beres Drops Plus) was given to liver metastasizing 3LL-HH tumor bearing mice in the presence and after the removal of the primary spleen tumor. In both models when TEP was provided daily and orally in a dose range of 100-5,000 microgram/kg, the number of liver metastasis decreased at an end-point of day 14 and TEP also had inhibitory effect on the primary tumor. The antitumor action proved to be dependent on tumor burden. There was no change in body weight, size distribution of metastases and in the life span of mice. Zn++ could be one of the antimetastatic components among the trace elements. This is the first report on the antimetastatic effect of trace elements in an experimental tumor model.

DNA content of parathyroid tumors.

BACKGROUND: The significance of DNA ploidy in indicating the benign or malignant character of parathyroid and other endocrin tumors is controversial. MATERIALS AND METHODS: DNA content of paraffin embedded parathyroid samples from 25 patients was measured with flow cytometry. RESULTS: The DNA index (DI) was 1.0 in all nonneoplastic samples as well as in 50% of adenomas (10/20) and in one carcinoma (1/2). The remaining 45% of the adenoma cases (9/20) and the other carcinoma showed doubled DNA content (DI = 1.9-2.0). The increased DI did not correlate with either clinical data (sex, age, tumor size, preoperative serum Ca++ or parathormone, primary or secondary hyperparathyroidism) or morphology. CONCLUSIONS: These results indicate that the DI has no value in deciding the benign or malignant character of a given sample. However, the prognostic value of tetraploidization (the potentially increased risk for malignancy) could not be ruled out.