Malignant and reactive cells from human lymphomas frequently express Fas ligand but display a different sensitivity to Fas-mediated apoptosis.

Fas ligand (FasL) is capable of inducing apoptosis of lymphoid cells by cross-linking with its natural receptor, Fas. We aimed to investigate the possible role of the Fas/FasL-mediated apoptosis in the development of human lymphomas. FasL mRNA was detected by reverse transcriptase-polymerase chain reaction in 38 out of 63 lymphoma biopsy specimens representative of various subtypes of non-Hodgkin's lymphoma (NHL) and Hodgkin's disease. FasL was co-expressed with Fas mRNA in most cases. Flow cytometry (FACS) analysis showed a bright FasL staining in 31% to up to 75% of the total cell population from 14 out of 16 samples; the presence of the FasL protein was confirmed by Western blotting. Dual-color FACS analysis showed that FasL was expressed by T cells in B-NHLs and T-NHLs. A significant percentage of B cells in various B-NHLs also stained positively for FasL. Freshly separated neoplastic B cells from three FasL+ and one FasL- B-NHLs displayed a relative resistance to Fas-mediated apoptosis, when compared to reactive T cells isolated from the same tissue samples. In contrast, the sensitivity to Fas-mediated killing of the T cells isolated from two FasL+ T-NHLs was not uniform. These data show that (1) FasL is expressed in both neoplastic and reactive cells from a significant proportion of lymphoma cases, and (2) that the intratumoral FasL+/Fas+ reactive T cells are more sensitive to Fas-induced apoptosis than the neoplastic FasL+/Fas+ malignant B cells. A putative defect in the Fas/FasL pathway may thus favor the development of malignant B cell populations.

Localization of the human CD40 gene to chromosome 20, bands q12-q13.2.

CD40 is a surface glycoprotein, member of the nerve growth factor receptor family, which is expressed on B cells and plays an important role in their development, growth, and differentiation. Using chromosomal in situ hybridization, we localized the CD40 gene to the long arm of chromosome 20, bands q12-q13.2. This localization correlates well with the mapping of the murine CD40 gene to the distal region of chromosome 2, syntenic to the human 20q11-q13 region.

Quantitative analysis detects ubiquitous expression of apoptotic regulators in B cell non-Hodgkin's lymphomas.

To determine whether the expression levels of Bcl-2 family apoptotic regulators are correlated with the histopathological heterogeneity of B cell non-Hodgkin's lymphomas (NHL), we quantified their expression in malignant B cell populations isolated from 33 biopsy samples, including small lymphocytic lymphoma (SLL, n = 9), mantle cell lymphoma (MCL, n = 8), follicular lymphoma (FL, n = 8), and diffuse large cell lymphoma (DLCL, n = 8). Normal B cells purified from reactive lymph nodes and tonsil (n = 3) were used as controls. Cell lysates were analyzed by Western blotting, and signals quantified by densitometry. Expression of Bcl-2 and its homologues, Bcl-xL, Bcl-xS, Bax, Bad, Bak and Bag-1, was detected in all NHL cases, with wide variations between histological subtypes and within each subtype. Statistically significant differences were: (1) a higher level of Bad expression in DLCL compared to FL and MCL; (2) a lower level of Bak expression in FL compared to DLCL, SLL and MCL; and (3) a higher Bag-1 expression level in FL compared to SLL. When compared to NHL cells, normal B cells showed a higher level of Bax expression, and a lower level of Bcl-xL expression. Thus, quantitative analysis shows ubiquitous expression of Bcl-2 family proteins in normal and neoplastic B cells; the variations in expression levels may contribute to both the B-NHL clinicopathological diversity and the different apoptotic sensitivities of normal B cells vs B-NHL cells.

c-fms expression in acute leukemias with complex phenotypes.

The c-fms proto-oncogene product, which is the receptor for the macrophage colony-stimulating factor CSF-1, is always found expressed in acute myeloid leukemia cells, irrespective of their stage of differentiation according to the FAB classification (Dubreuil P, Torrès H, Courcoul M, Birg F, Mannoni P. Blood 1988;72:1081-1085). We have extended this study and looked for c-fms expression in poorly differentiated myeloid leukemias, in a series of acute leukemias of either T or B origin and in biphenotypic leukemias. We now report that expression of c-fms is still related to the myeloid origin of the leukemic proliferation, but that it can also be found in some acute leukemias presenting clonal rearrangements of the T cell receptor gene. Thus expression of the c-fms/CSF-1 receptor may not be exclusively a marker for myeloid proliferations.

Atypical response to all-trans retinoic acid in a der(5)t(5;17) acute promyelocytic leukemia.

Typical acute promyelocytic leukemia (APL) is associated with the t(15;17) translocation, expression of a PML/RARA fusion transcript, and responsiveness to all-trans retinoic acid (ATRA). Rare APL cases implicating the RARA but not the PML gene have been reported. Cases with t(11;17)(q23;q21) which fuses the PLZF and RARA genes do not respond to ATRA. In contrast, cases with t(11;17)(q13;q21) and t(5;17)(q35;q21) which fuse RARA with NuMA and NPM, respectively, were reported to be sensitive to ATRA. We described previously an APL case with an unbalanced t(5;17) implicating RARA but neither PML nor PLZF. Here, we show that in this case: (1) the NPM gene is not involved, as demonstrated by RT-PCR and Southern blot; (2) response to ATRA in vitro is atypical, as demonstrated by morphological and functional maturation assays; and (3) PML nuclear bodies are not disrupted, as evidenced by immunofluorescence staining.

Coexpression of the genes for interleukin 6 and its receptor without apparent involvement in the proliferation of acute myeloid leukemia cells.

Leukemic cells isolated from patients with either acute myeloid leukemia (AML) or acute lymphoid leukemia (ALL) were screened for their capacity to express the interleukin 6 (IL-6) and IL-6 receptor genes, both at the RNA and protein levels. Variable levels (10 to greater than 600 U/ml) of an IL-6 activity, inhibited by neutralizing anti-IL-6 antibodies, were detected in AML cell supernatants using the B9 cell bioassay. High levels (greater than 100 U/ml) were observed in differentiated (M4 and M5 stages) AML, as well as in less mature (M1 and M2 stages) AML. Detection of the IL-6 transcript correlated with the biological activity. In addition, both IL-6 activity and IL-6 mRNA were detected in "fresh" leukemic cells, indicating that the glycoprotein was actually synthesized in vivo. In contrast, the IL-6 gene was less frequently expressed in ALL. The IL-6 receptor gene was transcribed in both AML and ALL; binding experiments showed that the protein was present at the cell surface. The spontaneous in vitro proliferation of leukemic cells coexpressing the transcripts for IL-6 and its receptor was not significantly inhibited by a neutralizing anti-IL-6 antibody, suggesting that IL-6 is not primarily implicated in the proliferation of the leukemic clone via an autocrine loop. Synthesis of IL-6 could, however, confer on leukemic cells a selective growth advantage through activation of the cytokine cascade.

Complementary DNA characterization and chromosomal localization of a human gene related to the poliovirus receptor-encoding gene.

The human poliovirus (PV) receptor (PVR) is a member of the immunoglobulin (Ig) superfamily with unknown cellular function. We have isolated a human PVR-related (PRR) cDNA. The deduced amino acid (aa) sequence of PRR showed, in the extracellular region, 51.7 and 54.3% similarity with human PVR and with the murine PVR homolog, respectively. The cDNA coding sequence is 1.6-kb long and encodes a deduced 57-kDa protein; this protein has a structural organization analogous to that of PVR, that is, one V- and two C-set Ig domains, with a conserved number of aa. Northern blot analysis indicated that a major 5.9-kb transcript is present in all normal human tissues tested. In situ hybridization showed that the PRR gene is located at bands q23-q24 of human chromosome 11.

A defective retroviral vector encoding human interferon-alpha2 can transduce human leukemic cell lines.

Using the LXSN backbone, a defective retroviral vector (LISN) was constructed that encodes the human interferon (IFN)-alpha2 (hIFN-alpha2) gene and the neomycin resistance gene; the hIFN-alpha2 gene was cloned from human placental genomic DNA. High titers of the LISN retrovirus were produced by the amphotropic packaging cell line GP+envAM12. LISN is able to infect three human hematopoietic and leukemic cell lines: K562, LAMA-84, and TF-1. G418-resistant cells were detected in a similar proportion after infection with either the LISN retroviral vector or the LnLSN retroviral vector (encoding the nlsLacZ gene instead of hIFN-alpha2), suggesting that hIFN-alpha2 does not inhibit (or only partially inhibits) the production of retroviral particles by the packaging cell line and the infection of human cells. LISN-infected cells express and secrete hIFN-alpha2 as demonstrated by Northern blot analysis of poly(A)+ RNA, detection of the intracellular protein by fluorescence-activated cell sorter analysis, and detection of secreted hIFN-alpha in cell supernatants using an enzyme-linked immunosorbent assay. Retrovirally produced hIFN-alpha2 is biologically active, as demonstrated by the partial inhibition of the growth of K562 and TF-1, the modulation of the expression of cell surface antigens, the induction of the (2'-5') oligoadenylate synthetase, and, for LAMA-84, the down-modulation of the BCR-ABL protein. We conclude that the infection of human leukemic cell lines with a retroviral vector encoding hIFN-alpha2 is feasible and induces the expected biological effects. This experimental model will be useful in investigating the possibility of transducing normal and leukemic cells and hematopoietic progenitors and in determining the consequences of the autocrine production of hIFN-alpha2 on the behavior of these cells.

Ancestrally-duplicated paraHOX gene clusters in humans.

A paraHox gene cluster has been described recently in Amphioxus. We show here using bioinformatics and cytogenetics that, as the probable result of the duplication of an ancestral paraHox gene cluster, human paraHOX genes are located in four paralogous regions of the genome, on chromosomes 4, 5, 13 and X. By analogy with the four HOX gene clusters, we propose to designate the four paraHOX gene clusters as paraHOX-A to D clusters. We also propose a scenario for the evolution of HOX and paraHOX genes. Several chromosomal translocation breakpoints of hemopathies are located in the paralogous regions that contain the paraHOX genes. Two of the paraHOX genes are involved in these rearrangements.

Caspase 7 downregulation as an immunohistochemical marker of colonic carcinoma.

Caspases play a crucial role as apoptotic effectors; their potential implication in tumorigenesis remains to be clarified. We investigated the expression and function of caspases 7, 8, and 9 in colon cancer tissues and cell lines. Immunohistochemistry (IHC) showed downregulation of caspase 7 (22 of 26 cases) and caspase 9 (12 of 26 cases) in colonic cancer samples compared with normal mucosa on the same tissue section. Caspase 8 expression was unchanged or slightly upregulated (19 of 27 cases). The combination of IHC and Western blot analysis showed expression of the proforms of caspases 7, 8, and 9 in HT29-19A and HT29-16E colonic carcinoma cell lines. Apoptosis could be induced by staurosporine in both HT29 cell lines, with a sensitivity similar to that of the HGT cell line, but lower than that of the DAUDI cell line. Apoptosis induction in HT29 cells was concomitant with processing of caspases 3, 7, 8, and 9 and was inhibited by the caspase inhibitor ZVAD. Our data show that (1) human colon cancer cells downregulate caspase 7 and, to a smaller extent, caspase 9 in vivo and (2) in vitro staurosporine-induced apoptosis of colonic cancer cells involves caspases 7 and 9. Caspase 7 deficiency thus appears as a new immunohistochemical marker of colonic neoplasia; its correction represents a potential basis for new therapies.

Cysteine protease CPP32, but not Ich1-L, is expressed in germinal center B cells and their neoplastic counterparts.

Ich-1/Nedd2 and CPP32/YAMA are cysteine proteases related to interleukin 1-beta-converting enzyme (ICE), which act as apoptosis effectors. Both molecules are expressed in T- and B-cell lines. The authors investigated their in vivo cellular distribution in normal and neoplastic human lymphoid tissues. Sixty-eight representative non-Hodgkin's lymphomas (NHL) and Hodgkin's disease (HD) samples, normal lymphoid organs, and nonlymphoid tumors were analyzed by immunohistochemistry (IHC). CPP32 expression in benign tissues was restricted to germinal center B cells, plasma cells, and a few interfollicular immunoblasts. All follicular NHLs and most diffuse large cell NHLs were CPP32 positive. Among T-cell NHLs, CPP32 expression was mainly observed in anaplastic large cell NHLs, whereas the other subtypes were less frequently positive. In contrast, lymphoid organs displayed only weak Ich1-L expression, located in sinusal histiocytes and thymic epithelial cells. Lymphomas were Ich1-L negative, except for T-cell-rich B-cell NHLs, and about half of the HD samples, in which Reed-Sternberg cells (RSC) were usually Ich1-L positive/CPP32 negative. Extralymphoid Ich1-L reactivity was found in particular organs like the kidney and various tumors. Western blot analysis confirmed the specificity of immunostaining. Neither CPP32 nor Ich1-L expression were correlated with intratumoral DNA fragmentation, as determined by the TUNEL assay. Altogether, these results indicate that CPP32 is preferentially expressed in germinal centers and thus could be involved in B-cell maturation. The differential expression of CPP32 and Ich1-L suggests that cysteine proteases differ in substrate specificities and carry out functions unrelated to apoptosis.

Search for rearrangements and/or allelic loss of the fas/APO-1 gene in 101 human lymphomas.

The authors analyzed the possible occurrence of rearrangements and/or allelic loss of the fas/APO-1 gene in a representative series of human lymphomas, including 101 cases of Hodgkin's disease (HD) and non-Hodgkin's lymphomas (NHL). The rationale for this study was double. Chromosome 10 alterations, frequently observed in lymphoma subtypes, encompass the chromosomal localization of fas/APO-1. In addition, Ipr mouse mutants, which present with a generalized lymphoproliferative disease, were shown to exhibit alterations of fas/APO-1 structure and expression. In this retrospective study, the authors performed gene dosage of fas/APO-1 by Southern blots. No fas/APO-1 alterations were observed in the 31 HD cases. Among 70 T-cell and B-cell NHL, allelic loss of fas/APO-1 was observed in three cases. Two cases with different clinical, phenotypic, and histologic presentations showed a rearrangement of fas/APO-1. A third case showed amplification. Thus fas/APO-1 alterations do occur in human lymphomas, although at a relatively low frequency.

Heterogeneity of rearranged T-cell receptor V-alpha and V-beta transcripts in tumor-infiltrating lymphocytes from Hodgkin's disease and non-Hodgkin's lymphoma.

Hodgkin's disease is histologically characterized by the presence of Reed-Sternberg cells (RSC) and reactive cells, including numerous T lymphocytes. Some forms of B cell-non-Hodgkin's lymphoma also contain a rich T-cell population. Previous studies have suggested that the T-cell receptor repertoire of tumor-infiltrating T lymphocytes (TITL) that act specifically against tumor-related antigens, should be restricted. The authors in this study used the polymerase chain reaction to explore the possible existence of such an immunologic response of TITL against RSC or neoplastic B cells. They studied variable (v) region genes of T-cell receptor alpha and beta chains expressed by infiltrating lymphocytes in biopsy specimens from seven patients with Hodgkin's disease and three with B cell-non-Hodgkin's lymphoma. These latter samples were selected based on a rich T-cell content. Primers specific for 18 different V-alpha and 21 V-beta families were used. In every case, TITL showed an unrestricted pattern of expression similar to the repertoire observed in peripheral blood lymphocytes. Although such experiments are limited, the apparent lack of selection of a single or a limited number of T-cell subsets in the affected tissues did not support the existence of an in vivo immunologic interaction between TITL and antigens related to RSC or neoplastic B cells.

The expression of FMS, KIT and FLT3 in hematopoietic malignancies.

Three receptor molecules, belonging to the class III of receptor tyrosine kinases, namely the receptors for colony-stimulating factor 1, CSF1R (product of the FMS proto-oncogene) and Steel factor, SLFR (product of the KIT proto-oncogene), as well as the recently identified FLT3/FLK2 gene product, appear to play distinct roles in normal hematopoietic differentiation. Their potential role in leukemic hematopoiesis has been approached by expression studies in hematopoietic malignancies, especially in acute leukemias of the myeloid and lymphoid lineages. We present here a review of available data, and discuss the possible significance and potential applications of these results.

BCL-X and the apoptotic machinery of lymphoma cells.

The BCL-X gene belongs to the family of BCL-2 homologues and plays an important role in the regulation of programmed cell death (PCD) in normal lymphoid tissues. BCL-X is transcribed into 2 mRNAs through alternative splicing. The protein product of the larger BCL-X mRNA (BCL-XL) functions as a PCD repressor. The second mRNA species, BCL-XS, encodes a protein capable of accelerating cell death. BCL-XL is a potential contributor to the pathogenesis of malignant lymphomas because the BCL-XL isoform is predominantly expressed by the neoplastic cells in the majority of lymphoma cases. This review is focused on the possible influence of BCL-X and other PCD regulatory agents on lymphomagenesis.

Caspases: conductors of the cell death machinery in lymphoma cells.

The present review focuses on recent insights into the regulation of caspases by other components of the apoptotic pathway, including the mechanisms by which caspase activation influence the death of lymphoma cells. In the light of our recent findings and similar observations of other investigators, it is likely that lymphoma cells possess the complete caspase machinery required for the apoptotic process. Inhibition of caspases activation appears as a potential mechanism to explain apoptotic defects of malignant B-cells, and thus may constitute the basis for new cancer therapies.

Variant and masked translocations in acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL) is characterized by a unique hemorrhagic syndrome, disseminated intravascular coagulation, and the association with the specific (15;17 chi q22-23:q12-21) translocation, which disrupts the retinoic acid receptor alpha (RARA) and the promyelocytic leukemia (PML) genes. The t(15;17) leads to the formation of two reciprocal fusion genes, PML/RARA on chromosome 15 and RARA/PML on chromosome 17; it is responsible for the unique response of the disease to retinoic acid (ATRA) treatment. As was described for chronic myeloid leukemia and its associated t(9;22) [Philadelphia chromosome], variant translocations have been reported in APL, which are either complex translocations involving additional chromosome(s), or simple variant translocations involving only either one chromosome 15 or 17 and any of several chromosomes. Rearrangements of RARA and PML were documented in some of these variant translocations. In contrast, recent molecular analysis of APL cases with cytogenetically normal chromosomes 15 and 17 revealed the occurrence of submicroscopic translocations, leading to the formation of non reciprocal fusion genes, either PML/RARA or RARA/PML only. Detailed analysis of such cases may shed light on the mechanisms of translocation, on the selection of oncogenic products, and on the respective role(s) of the products of the translocation. Demonstration of the existence, in some APL-like leukemias, of masked translocations with involvement of PML and RARA, thus allows to (i) confirm the diagnosis of APL, (ii) adapt the treatment and (iii) monitor the residual disease. Finally APL-like leukemias were recently reported, with either a t(11;17) or t(5;17), resulting in the fusion of RARA to genes other than PML; these patients do not appear to respond to ATRA treatment. Altogether, these results emphasize the usefulness of a molecular definition of APL.

Analysis of BAX expression in human tissues using the anti-BAX, 4F11 monoclonal antibody on paraffin sections.

BAX, a heterodimer partner of BCL-2, is an apoptosis inducer. We aimed to characterize the distribution of the BAX protein in normal adult human tissues using immunohistochemistry (IHC). The monoclonal antibody anti-BAX 4F11 was used on paraffin sections: immunodetection of BCL-2 was performed simultaneously on serial sections. The specificity of BAX IHC staining was verified by Western blot analysis. IHC positivity was correlated with the detection of a specific 21 kDa band on Western blots. BAX immunostaining was mainly cytosolic and occasionally on the nuclear membrane. Amounts of BAX protein were high in liver, renal tubules, endocrine islets of the pancreas, gastric glands, cardiac muscle, epididymis, lymph node germinal centers, and neurons; intermediate in the colon, stomach, bronchus. Fallopian tube, salivary gland, breast, thymus, spleen, and testis; low or undetectable in the other tissues. BAX IHC positivity correlated with apoptotic features in neurons and germinal center lymphocytes. There was no strict correlation between the IHC profiles of BAX and BCL-2 expression, although a reciprocal pattern of staining was observed in lymph node and colon. This report shows the usefulness the monoclonal antibody anti-BAX 4F11 on paraffin sections and demonstrates that the human BAX tissular distribution is close to, but not similar, to the profile observed in the mouse. The widespread BAX expression suggests that BAX alone is insufficient to trigger cell death in human tissues. BAX may either modulate the role of other regulators of apoptosis or carry out functions unrelated to apoptosis.

[Use of high-density filters ("DNA arrays") for gene expression analysis in human tissues]. / Utilisation de filtres à haute densité (<>) pour l'analyse de l'expression génique dans les tissus humains.

Most technical strategies for the analysis of gene expression in tissues are able to study only one protein or RNA product at the same time. A new recent method referred to as <> or <> is able to analyze simultaneously several hundreds of different genes. The DNA array is a nylon membrane on which are spotted equal amounts of cDNAs corresponding to different genes. This filter is hybridized with a <> probe synthesized with mRNA derived from the tissue analyzed. The result gives a global profile of gene expression within the tissue and allows quantitative and comparative analysis between different tissues or cell types.

[DNA-array analysis of the expression profile of apoptosis gene regulators in malignant lymphoma]. / Analyse par "biopuces" du profil d'expression des gènes régulateurs de l'apoptose des lymphomes malins.

Microarray technology has recently led to the identification of molecular prognostic subgroups in non Hodgkin's lymphomas. In order to determine the usefulness of ready-made macroarrays as routine diagnosis tools in haemato-pathology, we have analysed lymph node biopsies using a cDNA macroarray containing genes involved in apoptosis, including caspases. Nine biopsy specimens were analysed on total frozen tissues: 4 samples of B-cell follicular lymphoma (FL), two of B-cell diffuse large cell lymphoma (DLCL), and three of non-neoplastic lymph nodes from benign lymphadenitis. Eight cell populations were sorted from fresh tissues: malignant B-cells from 2 FL cases and 2 DLCL cases, reactive B-cells from 1 benign lymph nodes, reactive T-cells from 1 benign lymph node, virgin (mantle zone) B-cells and germinal center (GC) B-cells from benign tonsils. Immunohistochemistry (IHC) on paraffin sections was performed for localization of caspases 2, 3, 4, 7, 8, and 9. In the clustered array data, sorted cells from samples sharing common histological lesions grouped together, whereas the array/histology correlation was less satisfactory for tissues. The expression profiles of both array and IHC methods were correlated for most caspases and samples. Variations in array profiles of sorted cell populations can be statistically associated with specific histological features, suggesting a possible diagnostic application of ready-made "Apoptosis macroarrays" in haematopathology.