ABSTRACT
Selected microenvironmental stimuli confer to leukemic cells a growth advantage and an extended survival. We aimed at dissecting the differential support provided by the different cellular components of the microenvironment where CLL cells accumulate. To this end we cultured purified CLL cells in vitro in the presence or absence of different accessory cells (stromal cells, autologous T lymphocytes) and/or soluble molecules (IL-4, sCD40L) and assessed the leukemic cell response in terms of cell viability and chemoattracting capacity. The results indicate that both T lymphocytes and stromal cells are involved in sustaining the survival of leukemic B cells, but indicate that their support is different in terms of time of onset and duration. T cells have a short-term support activity while stromal cells provide long-term support.
Subject(s)
Leukemia, Lymphocytic, Chronic, B-Cell/pathology , B-Lymphocytes/immunology , B-Lymphocytes/pathology , Cell Communication , Cell Culture Techniques , Cell Survival , Chemokines/biosynthesis , Coculture Techniques , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/immunology , Models, Biological , Stromal Cells/immunology , Stromal Cells/pathology , T-Lymphocytes/immunology , T-Lymphocytes/pathology , Tumor Cells, CulturedABSTRACT
In B-cell chronic lymphocytic leukemia (B-CLL), defective apoptosis causes the accumulation of mature CD5(+) B cells in lymphoid organs, bone marrow (BM), and peripheral blood (PB). These cells are the progeny of a proliferating pool that feeds the accumulating compartment. The authors sought to determine which molecular mechanisms govern the proliferating pool, how they relate to apoptosis, and what the role is of the microenvironment. To begin to resolve these problems, the expression and modulation of the family of inhibitor of apoptosis proteins (IAPs) were investigated, with consideration given to the possibility that physiological stimuli, such as CD40 ligand (CD40L), available to B cells in the microenvironment, might modulate IAP expression. The in vitro data on mononuclear cells from PB or BM of 30 patients demonstrate that B-CLL cells on CD40 stimulation express Survivin and that Survivin is the only IAP whose expression is induced by CD40L. Through immunohistochemistry, in vivo Survivin expression in lymph node (LN) and BM biopsies was evaluated. In reactive LN, Survivin was detected only in highly proliferating germinal center cells. In LN from patients with B-CLL, Survivin was detected only in pseudofollicles. Pseudofollicle Survivin(+) cells were actively proliferating and, in contrast to Survivin(+) B cells found in normal GC, were Bcl-2(+). In B-CLL BM biopsies, CD5(+), Survivin(+) cells were observed in clusters interspersed with T cells. These findings establish that Survivin controls the B-CLL proliferative pool interfacing apoptosis and that its expression may be modulated by microenvironmental stimuli.
Subject(s)
Apoptosis , Leukemia, Lymphocytic, Chronic, B-Cell/immunology , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Microtubule-Associated Proteins , Proteins/immunology , Aged , Aged, 80 and over , Apoptosis/immunology , CD40 Antigens/immunology , Cell Division/immunology , Female , Humans , Inhibitor of Apoptosis Proteins , Leukemia, Lymphocytic, Chronic, B-Cell/metabolism , Male , Neoplasm Proteins , Protein Biosynthesis , SurvivinSubject(s)
Antigens, CD/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/immunology , Receptors, Antigen, B-Cell/genetics , Alternative Splicing , CD79 Antigens , Humans , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Neoplasm/genetics , RNA, Neoplasm/metabolism , Tumor Cells, CulturedABSTRACT
Several functional anomalies of B-chronic lymphocytic leukemia (B-CLL) cells may be explained by abnormalities of the B-cell receptor (BCR), a multimeric complex formed by the sIg homodimer and the noncovalently bound heterodimer Igalpha/Igbeta (CD79a/CD79b). Because the expression of the extracellular Ig-like domain of CD79b has been reported to be absent in the cells of most CLL cases, we have investigated the molecular mechanisms that may account for this defect. Peripheral blood lymphocytes (PBL) from 50 patients and two cell lines (MEC1, MEC2) obtained from the PBL of one of them were studied. MEC1, MEC2, and 75% of CLL cases did not express detectable levels of the extracellular Ig-like domain of CD79b, which was nevertheless present in greater than 80% CD19(+) cells from normal donors. In healthy subjects the expression of CD79b was equally distributed in CD5(+) and CD5(-) B-cell subsets. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of CD79b RNA from all patients and from MEC1 and MEC2 cell lines consistently yielded two fragments of different size (709 bp and 397 bp). The 709-bp band corresponds to CD79b entire transcript; the 397-bp band corresponds to an alternatively spliced form lacking exon 3 that encodes the extracellular Ig-like domain. Both fragments were also visible in normal PBL. The expression of the 397-bp fragment was increased in normal activated B cells, while no difference was seen between CD5(+) and CD5(-) B cells. To obtain a more accurate estimate of the relative proportions of the two spliced forms, a radioactive PCR was performed in 13 normal and 22 B-CLL samples and the results analyzed using a digital imager. The mean value of the CD79b to the CD79b internally deleted ratio was 0.64 +/- 0.20 SD in normal donors and 0.44 +/- 0.27 SD in B-CLL (P =.01). Direct sequencing of 397-bp RT-PCR products and of genomic DNA corresponding to exon 3 from MEC1, MEC2, their parental cells, and five fresh B-CLL samples did not show any causal mutation. Single-strand conformation polymorphism analysis of exon 3 performed in 18 additional B-CLL cases showed a single abnormal shift corresponding to a TGT --> TGC polymorphic change at amino acid 122. We propose a role for the alternative splicing of CD79b gene in causing the reduced expression of BCR on the surface of B-CLL cells. As normal B cells also present this variant, the mechanism of CD79b posttranscriptional regulation might reflect the activation stage of the normal B cell from which B-CLL derives.
Subject(s)
Antigens, CD/genetics , Antigens, Neoplasm/genetics , B-Lymphocyte Subsets/metabolism , Gene Expression Regulation, Leukemic , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Neoplastic Stem Cells/metabolism , Protein Isoforms/genetics , RNA Splicing , Receptors, Antigen, B-Cell/deficiency , Antigens, CD/biosynthesis , Antigens, CD/immunology , Antigens, Neoplasm/biosynthesis , Antigens, Neoplasm/immunology , B-Lymphocyte Subsets/immunology , B-Lymphocyte Subsets/pathology , CD5 Antigens/analysis , CD79 Antigens , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/immunology , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Lymphocyte Activation , Macromolecular Substances , Neoplastic Stem Cells/immunology , Neoplastic Stem Cells/pathology , Protein Isoforms/biosynthesis , RNA, Messenger/biosynthesis , RNA, Neoplasm/biosynthesis , Receptors, Antigen, B-Cell/biosynthesis , Receptors, Antigen, B-Cell/chemistry , Tumor Cells, CulturedABSTRACT
We report the establishment and characterization of two cell lines, MEC1 and MEC2, that grew spontaneously on two subsequent occasions from the peripheral blood (PB) of a patient with B-chronic lymphocytic leukemia (B-CLL) in prolymphocytoid transformation. The patient was EBV-seropositive, his leukemic cells were EBNA negative, but the spontaneously grown cell lines are EBNA-2 positive. In liquid culture MEC1 cells grow adherent to the vessel wall and as tiny clumps; MEC2 cells do not adhere and form large clumps. The doubling time of MEC1 is 40h and of MEC2 is 31h. Both cell lines express the same light (kappa) and heavy chains (mu, delta) as the fresh parental B-CLL cells at the same high intensity, share the expression of mature B cell markers (CD19, CD20, CD21, CD22), differ in the expression of CD23 and FMC7, are CD11a+, CD18+, CD44+, CD49d+, CD54+ and express at high levels both CD80 and CD86. CD5 is negative on MEC1 cells (as on the vast majority of parental cells) and it has been lost by MEC2 cells after several months of culture. The cells have a complex karyotype. The tumour origin of MEC1 and MEC2 has been demonstrated by Southern blot analysis of the IgH loci and by Ig gene DNA sequencing. They use the VH4 Ig family and have not undergone somatic mutations (94.8% homology with germline Ig gene 4-59). Cytofluorographic analysis and RT-PCR reveal that MEC1 and MEC2 overexpress Bcl-2 together with Bax, express large amounts of Bcl-xL and trace amounts of Bcl-xS.
Subject(s)
Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Leukemia, Prolymphocytic/pathology , Amino Acid Sequence , Apoptosis , Base Sequence , Chromosome Aberrations , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/metabolism , Male , Middle Aged , Molecular Sequence Data , Proto-Oncogene Proteins/analysis , Proto-Oncogene Proteins c-bcl-2/analysis , Tumor Cells, Cultured , bcl-2-Associated X ProteinABSTRACT
CD5+ B-chronic lymphocytic leukaemia (B-CLL) and mantle cell lymphoma (MCL) in leukaemic phase are characterized by defects in cell death induction that primarily involves the Bcl-2 family of genes. Fludarabine (9-beta-D-arabinofuranosyl-2-fluoradenine, F-ara-A) is a potent inducer of apoptosis in CLL cells. This study aimed to determine whether F-ara-A-induced apoptosis might be related to Bcl-2 modifications and to evaluate in vitro/in vivo correlations. Peripheral blood lymphocytes from eight B-CLL and four leukaemic MCL were cultured in the presence of different concentrations of F-ara-A +/- methylprednisolone (MP). F-ara-A down-regulated the expression of Bcl-2 in 5/12 cases. mRNA down-regulation was maximal at 48 h; protein down-regulation was prominent after 48 h. Both events were dose-dependent. The amount of apoptosis was significantly higher in the samples treated with F-ara-A than in those exposed to MP alone. In the seven remaining cases, no Bcl-2 down-regulation was observed after exposure to F-ara-A and the degree of F-ara-A-induced apoptosis overlapped that induced by MP. The in vivo outcome after treatment with three to six courses of F-ara-A was evaluable in 10 patients: 4/5 cases, whose cells had shown in vitro Bcl-2 down-regulation and prominent apoptosis after exposure to F-ara-A, had a complete response (CR) and a partial response (PR) was observed in the remaining patient. Of the five patients whose cells had shown no in vitro Bcl-2 modulation after exposure to F-ara-A, two had a PR, but the other three did not show any in vivo clinical response.