Clonal deleterious mutations in the IkappaBalpha gene in the malignant cells in Hodgkin's lymphoma.

Members of the nuclear factor (NF)-kappaB family of transcription factors play a crucial role in cellular activation, immune responses, and oncogenesis. In most cells, they are kept inactive in the cytosol by complex formation with members of the inhibitor of NF-kappaB (IkappaB) family, whose degradation activates NF-kappaB in response to diverse stimuli. In Hodgkin's lymphoma (HL), high constitutive nuclear activity of NF-kappaB is characteristic of the malignant Hodgkin and Reed-Sternberg (H/RS) cells, which occur at low number in a background of nonneoplastic inflammatory cells. In single H/RS cells micromanipulated from histological sections of HL, we detect clonal deleterious somatic mutations in the IkappaBalpha gene in two of three Epstein-Barr virus (EBV)-negative cases but not in two EBV-positive cases (in which a viral oncogene may account for NF-kappaB activation). There was no evidence for IkappaBalpha mutations in two non-HL entities or in normal germinal center B cells. This study establishes deleterious IkappaBalpha mutations as the first recurrent genetic defect found in H/RS cells, indicating a role of IkappaBalpha defects in the pathogenesis of HL and implying that IkappaBalpha is a tumor suppressor gene.

Oct-2 and Bob-1 deficiency in Hodgkin and Reed Sternberg cells.

Hodgkin and Reed Sternberg (H-RS) cells represent the malignant cells in classical Hodgkin's disease. Although derived from germinal center B cells, they do not express surface immunoglobulin. This has been explained by the presence of crippling mutations within the immunoglobulin genes in numerous cases of Hodgkin's disease. As immunoglobulin gene expression in B cells requires an interaction between octamer sites and the transactivating factors Oct-2 and Bob-1, this study addresses the expression of the transcription factors Oct-2 and Bob-1 in H-RS cells. In Hodgkin's disease-derived cell lines, low levels of Oct-2 transcripts but no Oct-2 protein were detected. Transcripts of Bob-1, a B-cell-specific co-factor of Oct-2, could not be observed in these cell lines. Absence of Oct-2 and Bob-1 protein expression in primary H-RS cells was demonstrated by performing immunohistochemistry in 20 cases of classical Hodgkin's disease. H-RS cells stained negative for both proteins in all of the cases analyzed. In conclusion, absence of functional Oct-2 and Bob-1 cells represents a novel mechanism for immunoglobulin gene deregulation in H-RS cells. Lack of Oct-2 and Bob-1 points to a defect in transcription machinery in H-RS cells and is associated with lack of immunoglobulin gene expression in these cells.

Cultivated H-RS cells are resistant to CD95L-mediated apoptosis despite expression of wild-type CD95.

OBJECTIVE: In most cases of classic Hodgkin's disease (HD), Hodgkin and Reed-Sternberg (H-RS) cells clonally derive from germinal-center B cells. Within their rearranged immunoglobulin genes, somatic mutations rendering potentially functional immunoglobulin gene rearrangements nonfunctional were detected, indicating that H-RS cells do not express a B-cell receptor. Under physiologic conditions, these cells would undergo apoptosis within the germinal center. However, H-RS cells clonally expand, disseminate, and lead to clonal relapse of HD, indicating their resistance to induced programmed cell death. The underlying mechanism remains to be elucidated. MATERIALS AND METHODS: [corrected] Analysis of receptor-ligand interactions in primary H-RS cells is difficult to perform due to their scarcity in vivo and their low proliferation rate in vitro. Therefore, two [corrected] B-cellular H-RS cell lines (L1236 and L428) were used to test for the expression of CD95 by flow cytometry and for the induction of apoptosis after incubation with CD95L obtained from retrovirally transduced murine myoblasts. Sequence analysis of CD95 cDNA obtained from these H-RS cell lines was performed. RESULTS: Expression of CD95 on the cell surface was detected in both cell lines. However, after incubation with CD95L, the cells did not undergo apoptosis. To test whether mutations within the CD95 cDNA sequence caused resistance to apoptosis in H-RS cells, sequence analysis of CD95 cDNA obtained from L1236 and L428 was performed. In both cell lines, CD95 was not affected by somatic mutations. CONCLUSIONS: Our results indicate that the two H-RS cell lines L1236 and L428 are resistant to CD95-mediated apoptosis induced via CD95L, although wild-type CD95 is expressed. For further characterization of the mechanisms leading to prevention of apoptotic cell death in H-RS cells, it is necessary to determine impairments within the signaling cascade following CD95 activation.

Lack of BCL10 mutations in Hodgkin's disease-derived cell lines.

The pathogenetic events leading to the malignant transformation of Hodgkin-Reed-Sternberg cells are unknown. As Hodgkin-Reed-Sternberg cells are resistant to CD95-mediated apoptosis and chromosomal aberrations involving the 1p22 region harbouring the proapoptotic BCL10 gene represent a recurrent event in Hodgkin's disease-derived cell lines, analysis of the BCL10 gene and its transcripts was performed. As transcription of wild-type BCL10 was detected in all Hodgkin's disease-derived cell lines analysed, alterations of the coding sequence of the BCL10 gene are unlikely to contribute to the malignant transformation of the Hodgkin-Reed-Sternberg cell.

Deregulation of immunoglobulin gene transcription in the Hodgkin-Reed Sternberg cell line L1236.

Hodgkin-Reed Sternberg (H-RS) cells harbour clonal immunoglobulin gene (Ig) rearrangements in almost all cases of classical Hodgkin's disease but lack Ig gene expression. In the H-RS cell line L1236, a somatic mutation of the Ig heavy-chain gene promoter octamer motif has been described as a putative reason for absence of Ig gene expression. We addressed transcriptional activity of this mutated promoter by performing reporter gene studies and gel retardation assays. The results showed that the mutation outside the coding region of the rearranged Ig gene in L1236 cells leads to downregulation of Ig gene expression in H-RS cells.

Class switch recombination was specifically targeted to immunoglobulin (Ig)G4 or IgA in Hodgkin's disease-derived cell lines.

In T cell-dependent immune responses, class switch recombination occurs in germinal centres. There is now evidence that Hodgkin/Reed-Sternberg cells are derived from germinal centre B cells. Cytokines specifically determine the direction of class switching, i.e the isotype of the new antibodies. We performed restriction analyses and polymerase chain reaction on the immunoglobulin heavy chain loci for five Hodgkin's disease-derived B-cell lines and one Hodgkin's disease-derived T-cell line in order to analyse class switch recombination. In all the B-cell lines, class switch recombination had been targeted to Calpha4 or Calpha1/2. This showed that cell-line precursors had undergone class switching, probably under the influence of TH2 or TH3 cell-derived cytokines. Deletions comprising several constant region genes were observed in cell lines L428, L1236, L591 and KMH2. Karyotype analyses of two of these revealed translocational breakpoints within the immunoglobulin heavy chain gene locus. Our data support the view that a chromosomal instability may occur during class switch recombination in Hodgkin/Reed-Sternberg cells causing chromosomal breaks. Thus, as in other germinal centre B cell-derived lymphomas, the immunoglobulin gene locus may be frequently involved in structural chromosomal aberrations in Hodgkin's disease.

Genetic susceptibility to Hodgkin's lymphoma and to secondary cancer: workshop report.

Although the occurrence of familial Hodgkin's lymphoma (HL) is a rare event, genetic susceptibility as a cause of HL and its influence on treatment outcome may not be rare. However, results obtained from the analysis of HL families will probably have broad implications with regard to understanding common pathogenic factors leading to the development of the disease. The description of anticipation among the affected offspring of HL patients further strengthens the view that heritable factors contribute to development of HL. Moreover, the finding that particular human leukocyte antigen (HLA) alleles are associated with susceptibility to HL may be regarded as a hint to the presence of an as yet undefined infectious agent, leading to the growth of a malignant lymphoma cell clone in those patients that are more susceptible to this agent due to their HLA genotype. In addition, since an intrinsic genomic instability was observed in a proportion of HL patients, it is plausible that these patients are not only susceptible to the causation of HL, but are also at a higher risk of developing therapy-related (TR) secondary cancers following treatment. Estimation of sister chromatid exchange was established as a tool to identify patients at higher risk of TR cancer. In this context the use of therapeutic agents known to increase genomic instability should be carefully considered prior to determing the best treatment. The future identification of heritable factors contributing to HL will be of importance both with regard to diagnosis as well as treatment of HL patients.

Detection of Epstein-Barr virus in Hodgkin-Reed-Sternberg cells : no evidence for the persistence of integrated viral fragments inLatent membrane protein-1 (LMP-1)-negative classical Hodgkin's disease.

Classical Hodgkin's disease (HD) is associated with Epstein-Barr virus (EBV) infection. Although in developing countries EBV can be demonstrated in Hodgkin-Reed-Sternberg (H-RS) cells in up to 95% of HD cases, in industrialized countries only about 50% of HD cases are associated with EBV. An open question remains whether EBV in the EBV-negative cases has escaped detection by standard screening procedures due to deletions in the viral genome associated with integration of viral fragments into the host cell genome. We, among others, recently described this phenomenon in Burkitt's lymphoma cells. To investigate whether H-RS cells in latent membrane protein-1 (LMP-1)-negative HD cases harbor fragments of the EBV genome, we combined fluorescence in situ hybridization (FISH) using a set of six overlapping DNA probes spanning the whole EBV genome with immunophenotyping of fresh frozen lymphoma sections. Results in the eight cases analyzed were as follows: in three LMP-1-positive cases, FISH analysis yielded specific signals for each EBV DNA probe in H-RS cells, which had been identified by morphology and CD30 staining. In contrast, none of the EBV DNA probes hybridized to the H-RS cells in the five LMP-1-negative cases. Thus, there is no evidence for the presence of fragments of the viral genome integrated into the host cell genome in the LMP-1-negative cases. Furthermore, in the LMP-1-positive cases analyzed, no large deletions in the viral genome were detected. These results show that, in classical HD, LMP-1-negative cases do not harbor EBV DNA within the H-RS cells. Whether, in these cases, a still unknown virus contributes to the transformation and maintenance of the malignant phenotype remains to be established.

Loss of heterozygosity in the Hodgkin-Reed Sternberg cell line L1236.

Hodgkin-Reed Sternberg cells are derived from germinal centre B-cells in most cases. Somatic mutations affecting their rearranged immunoglobulin genes were detected, rendering potential functional rearrangements non-functional. Under physiological conditions such cells would be designated to undergo apoptosis within the germinal centre. In search for the specific transforming event that prevents Hodgkin-Reed Sternberg cells from programmed cell death, cytogenetic analyses were broadly performed but did not reveal specific chromosomal aberrations. Analysis of these cells on the molecular level is difficult to perform due to the scarcity of the cells in the lymphoma tissue and the given limitations of in situ studies. To overcome these limitations, the cell line L1236, known to be derived from Hodgkin-Reed Sternberg cells in situ, was chosen for allelotype analysis. Using a panel of microsatellite loci assigned to nearly all chromosomal arms, regions of loss of heterozygosity were detected on chromosomal arms 6p, 9q and 17p. The size of lost segments was estimated by amplification of additional microsatellite loci mapped to the respective regions. Further analyses of single Hodgkin-Reed Sternberg cells will reveal whether LOH affecting these regions is a recurrent event in HD and to which extent the smallest commonly affected region can be estimated.

Suppression of the tumorigenic growth of Burkitt's lymphoma cells in immunodeficient mice by cytokine gene transfer using EBV-derived episomal expression vectors.

Epstein-Barr virus (EBV)-based expression vectors were tested for cytokine gene transfer-mediated induction of an immune response against human lymphoma cells. These vectors express the EBV latent gene EBNA 1 and carry the EBV latent origin of replication (ori P) for episomal replication in transfected cells. In addition, 3 human immunoglobulin light chain enhancer elements augment expression in B-cells. The suitability of these vectors for expression of cytokine genes in human lymphoma cells in vitro has been demonstrated. In order to extend these experiments in vivo, highly tumorigenic Burkitt's lymphoma (BL) cells were transfected with different cytokine genes of human and murine origin cloned into the EBNA 1/ori P vectors. Tumorigenicity of the transfectants was measured after inoculation into nude mice. No effect on tumorigenicity was observed after hIL 6 transfection and an inconsistent effect after hTNFalpha transfection. In contrast, complete suppression of tumor outgrowth occurred in hIL 10 transfectants. This tumor suppressive effect, however, was restricted to the IL 10 transfectants themselves and not directed against non-transfected cells. By comparison, mIL 4 transfected BL cells also were non-tumorigenic. However, co-inoculation of mIL 4 transfected and non transfected cells resulted in suppression of the tumorigenicity of the non-transfected cells. Thus, highly tumorigenic BL cells in nude mice are sensitive to immune effector mechanisms triggered by cytokine expression. In this experimental model, EBNA 1/ori P expression vectors are a suitable tool for cytokine gene transfer mediated induction of an anti-lymphoma immune response of the host.

Hodgkin disease-derived cell lines expressing ubiquitous mitochondrial creatine kinase show growth inhibition by cyclocreatine treatment independent of apoptosis.

Ubiquitous mitochondrial creatine kinase (uMtCK), a key enzyme in energy metabolism, was identified by differential display PCR to be specifically overexpressed in L1236, the first cell line of definite Hodgkin origin. RT-PCR confirmed overexpression of uMtCK in the L1236 cell line and the absence of cytosolic B-CK, which is co-expressed with MtCK physiologically. Cyclocreatine (cCr), whose phosphorylated form is a very poor substrate for CK, inhibited proliferation of the L1236 cell line nearly entirely. This inhibition by cCr was partially reversed by competition with creatine, which by itself had no effect on proliferation of the L1236 cell line. Although these results support a role of CK activity in the inhibitory action of cCr, it remains open whether the cCr effect is due to its inhibition of CK-linked energy metabolism or if alternative mechanisms have to be considered. Because the anti-proliferative effect of cCr was not due to induction of apoptosis, in contrast to most other anticancer agents, treatment with the creatine analogue cCr may represent an advantageous therapeutic approach for cells resistant to programmed cell death.