B-type Chronic Lymphocytic Leukemia Research Articles

The influence of naturally occurring flavonoids, chalcones and statins on the activity of voltage-gated potassium channels Kv1.3 and viability of Kv1.3 channel-expressing cancer cells

Voltage-gated potassium channels Kv1.3 are widely expressed in many cell types, both in the plasma membrane and in the inner mitochondrial membrane (mito Kv1.3 channels). An inhibition of the channels may putatively find clinical application in therapy of some cancers characterised by an over-expression of Kv1.3 channels, such as melanoma, pancreatic ductal adenocarcinoma (PDAC), multiple myeloma and B-type chronic lymphocytic leukaemia (B-CLL). An ability to inhibit Kv1.3 channels in cancer cells is shared by some plant-derived polycyclic compounds from the groups of flavonoids, chalcones and statins. The inhibitory effect on the channels may be significantly augmented when the flavonoids and the chalcones are co-applied with the statins: simvastatin and mevastatin. The inhibition of Kv1.3 channels, presumably mito Kv1.3 channels, may be involved in a pro-apoptotic activity of these compounds on Kv1.3 channel-expressing cancer cells. This pro-apoptotic activity may significantly be improved when the selected flavonoids and chalcones are co-applied with the statins. Therefore, these naturally occurring compounds may putatively find clinical application in therapy of above-mentioned cancers, especially when applied in a combination.

Voltage-Gated Potassium Channel Kv1.3 as a Target in Therapy of Cancer.

Voltage-gated potassium channel Kv1.3 is an integral membrane protein, which is selectively permeable for potassium ions and is activated upon a change of membrane potential. Channel activation enables transportation of potassium ions down their electrochemical gradient. Kv1.3 channel is expressed in many cell types, both normal and cancer. Activity of the channel plays an important role in cell proliferation and apoptosis. Inhibition of Kv1.3 channel may be beneficial in therapy of several diseases including some cancer disorders. This review focuses on Kv1.3 channel as a new potentially attractive molecular target in cancer therapy. In the first part, changes in the channel expression in selected cancer disorders are described. Then, the role of the channel activity in cancer cell proliferation and apoptosis is presented. Finally, it is shown that some low molecular weight organic inhibitors of the channel including selected biologically active plant-derived polycyclic compounds may selectively induce apoptosis of Kv1.3-expressing cancer cells while sparing normal cells and healthy organs. These compounds may be promising candidates for putative application in therapy of some cancer disorders, such as melanoma, pancreatic ductal adenocarcinoma (PDAC), or B-type chronic lymphocytic leukemia (B-CLL).

Urinary excretion of CD23 antigen in normal individuals and patients with chronic lymphocytic leukaemia (CLL)

A soluble form of CD23 (sCD23) was found in the urine from 12 normal individuals but was not present in 20 normal sera, suggesting that sCD23 produced by cells in tissues is eliminated in the urine. The sCD23 from urine differed in physicochemical properties from the sCD23 found in supernates from B-lymphoblastoid cell lines (B-LCL) and in the sera of patients with B type chronic lymphocytic leukaemia (B-CLL). On SDS-PAGE analysis under reducing conditions urinary sCD23 showed two bands corresponding to molecular weights of 45-60 kD and 28-35 kD indicating that sCD23 may be excreted in combination with another molecule. When subjected to gel filtration in its native state, sCD23 from urine showed a major peak at approximately 150 kD and a minor peak (probably a breakdown product) at 21 kD. Urinary sCD23 was more strongly held by DEAE-cellulose and required 0.5 M buffer pH 8.0 for elution, suggesting that it is more anionic than sCD23 from culture supernates. Five MoAbs recognizing different epitopes on sCD23 from B-LCL supernates were tested on urinary sCD23. Four of the MoAbs were reactive but one (EBVCS-1) was not. Urinary sCD23 did not bind to IgE. The level of sCD23 found in normal urine (approximately 0.02-0.05 micrograms/ml) was exceeded in 17 of 24 cases of B-CLL. In one case with a high cell count and a serum concentration of 10 micrograms/ml, the urine contained 80 micrograms/ml sCD23. In another case a high serum sCD23 was not matched by a high urinary level. In this case the gel filtration pattern was closer to that found with urine sCD23 rather than the B-LCL pattern found with sera of other B-CLL patients.

Analysis of apoptosome dysregulation in pancreatic cancer and of its role in chemoresistance

The apoptosome is a multiprotein complex mediating the mithochondrial pathway of cell death. Its importance during development has been clearly demonstrated by knocking out key genes in mouse. APAF1 is the core protein of the apoptosome and its dosage is also critical in various cancer types, i.e. melanoma, germ line tumor, gastrointestinal cancer and B-type chronic lymphocytic leukemia. This is generally due to inactivation of the APAF1 locus by epigenetic phenomena or by activity of promoter regulators. We investigated the putative roles of the apoptosome in pancreatic ductal adenocarcinoma (PDAC). We found that both APAF1 mRNA and protein are dysregulated in human PDAC samples. Similarly, several PDAC cell lines exhibited variable levels of both APAF1 protein and mRNA. The response to cell death induction and its biochemical features were assessed by treatment of each line with commonly used chemotherapeutic agents. We found that the apoptosome pathway was not functional in most cell lines upon cytochrome c release from mitochondria. In addition, we restored APAF1 and Caspase-9 dosage in Panc-1 cells, where the apoptosome is downregulated, by overexpressing the murine cDNA of the two molecules, and we improved the death response to chemotherapeutic agents.

Silencing of APAF‐1 in B‐CLL results in poor prognosis in the case of concomitant p53 mutation

Apoptosis protease-activating factor 1 (APAF-1), a transcriptional target of p53, is a cytosolic adaptor protein that links the mitochondrial apoptosis pathway to the caspase cascade. Here, we aimed to study the impact of APAF-1 expression levels on cell death induced by anticancer drugs or ionizing irradiation (IR) and disease prognosis in B-type chronic lymphocytic leukemia (B-CLL) patients. Samples from 138 patients with B-CLL were investigated for APAF-1 expression and p53 mutations. The results were related to survival data, in vitro cytotoxicity of various cytotoxic drugs and IR and clinico-pathological data. Variable APAF-1 expression was observed in all investigated B-CLL samples. Reduction in APAF-1 expression was observed at both mRNA and protein level indicating transcriptional silencing whereas mutation of p53 or the immunoglobulin heavy chain variable genes (IgH(V)) had no impact on APAF-1 expression. Surprisingly, APAF-1 loss did not result in resistance to cytotoxic therapies. Likewise, APAF-1 downregulation on its own showed no impact on disease prognosis. Nevertheless, a poor prognosis was observed in patients with loss of APAF-1 expression and additional p53 mutation. Thus, loss of APAF-1 may become relevant when additional core apoptosis signaling components are disrupted.

In B-CLL, the codon 72 polymorphic variants of p53 are not related to drug resistance and disease prognosis

BackgroundA common sequence polymorphism at codon 72 of the p53 gene encoding either arginine or proline was recently shown to be functionally relevant for apoptosis induction in vitro. In B-type chronic lymphocytic leukemia (B-CLL), p53 gene mutations occur in a subset of patients and are associated with impaired survival and drug resistance. Here, we address the functional relevance of the codon 72 single nucleotide (SNP) polymorphism for cell death sensitivity following exposure to clinically employed cytotoxic drugs and γ-irradiation.Methods138 B-CLL samples were analysed by SSCP-PCR and sequencing for single nucleotide polymorphism at codon 72 of the p53 gene. The in vitro cytotoxicity assay (DiSC-assay) was performed with 7 drugs (chlorambucil, mafosfamide, fludarabine phosphate, methylprednisolone, doxorubicin, vincristine) or γ-irradiation.ResultsOf the138 B-CLL samples, 9 samples were homozygous for proline (Pro/Pro), 78 samples homozygous for arginine (Arg/Arg), and 49 samples heterozygous (Arg/Pro). No differences were found for patient survival and cell death triggered by 7 cytotoxic drugs or γ-irradiation.ConclusionThese data indicate that polymorphic variants of p53 codon 72 are not clinically relevant for apoptosis induction or patient survival in B-CLL.

Sensitivity of B-CLL to Cell Death Induction by Anticancer Drugs Is Independent of the Immunoglobulin Heavy Chain Variable Genes (IgHv) Mutation Status.

B-type chronic lymphocytic leukemia (B-CLL) is a heterogeneous disease. Unmutated immunoglobulin (Ig) VH genes, i.e. a post-germinal center genotype, are associated with a more aggressive natural course of disease. To explore the impact of Ig variable region hypermutation on response to cytotoxic therapies we analyzed the sensitivities to a panel of cytotoxic modalities by use of the DiSC assay that measures cell death. These data were compared to the Ig VH mutation status in 113 B-CLL patients. 29 patients exhibited IgHV hypermutation (age 66.3±1.8 years, 18 male, 11 female) whereas 84 patients (age 62.2±1.1, 66 male, 18 female) were of pre-germinal center B-cell origin. Hypermutated samples were from Binet stage A in 11 cases (unmutated 12), Binet stage B in 5 cases (14), Binet stage C in 10 cases (44), and in stage A/B in 2 cases (10 cases), Chi-square test p=0.02. 11/29 patients with Ig VH hypermutation and 54 of the 84 of the patients with unmutated B-CLL had received prior chemotherapy. Ig VH hypermutation had no impact in the percentage of surviving cells after 92 hours for ionizing irradiation (IR, 2 Gy by a 137Cs source) (hypermutated, 35.3%±8.2, unmutated 34.2%±4.5). Furthermore, there was no significant difference for the log LC90 values for chlorambucil (Mann-Whitney U test, p=0.22), doxorubicin (p=0.86), fludarabine (p=0.36), and mafosfamide (p=0.49). Interestingly, the log LC90 value for methylprednisolone showed a significant difference and was lower in the unmutated (n=83, mean±SEM 0.8±0.13) as compared to the hypermutated samples(n=29, 1.36±0.2; p=0.038). Conclusions: The mutation status of the IgHV gene segment does not appear to be responsible for differences in the response to various drug treatments in B-CLL. Vice versa, differences in gene expression that were described in hypermutated versus unmutated B-CLL do not seem to impact on drug sensitivity profiles. Thus, both DiSC assay result and VH Gene hypermutation are valuable independent prognostic markers.

Dendritic Cells from Chronic Lymphocytic Leukemia Patients Are Normal Regardless of Ig V Gene Mutation Status

Patients with B-type chronic lymphocytic leukemia (B-CLL) segregate into 2 subgroups based on the mutational status of the immunoglobulin (Ig) V genes and the patients in these subgroups follow very different clinical courses. To examine whether dendritic cells (DCs) generated from CLL patients can be candidates for immune therapy, we compared the phenotypic and functional capacities of DCs generated from patients of the 2 CLL subgroups (normal age-matched subjects [normal-DCs]). Our data show that immature DCs from B-CLL patients (B-CLL-DCs) have the same capacity to take up antigen as those from normal controls. Furthermore, B-CLL-DCs generated from the 2 CLL subgroups up-regulated MHC-II, CD80, CD86, CD83, CD40, and CD54 and down-regulated CD206 in response to stimulation with a cocktail of cytokines (CyC) and secreted increased levels of tumor necrosis factor alpha, interleukin (IL)-8, IL-6, IL-12 (p70), and RANTES in a manner typical of mature normal-DCs. Interestingly, CD54 was significantly more up-regulated by CyC in B-CLL-DCs compared with normal-DCs. Except for CD54, no significant differences in surface molecule expression were observed between normal-DCs and B-CLL-DCs. B-CLL-DCs from both subgroups, including 6 patients with VH1-69, that usually fare poorly, presented tetanus toxoid to autologous T cells in vitro similar to normal- DCs. Our data show that DCs generated from the B-CLL subgroup with unmutated Ig V genes are functionally normal. These results are very promising for the use of DCs from patients with poor prognosis for immunotherapy.

Effect of chlorambucil on B-lymphocytes in the peripheral blood of patients with chronic lymphocytic leukemia--study of cellular ultrastructure

B type Chronic Lymphocytic Leukemia (B-CLL) is a malignant disease characterized by the progressive accumulation of morphologically mature, but immunologically dysfunctional CD 5+ lymphocytes in the blood, bone marrow and lymphatic organs in the early phase of the cell cycle. B-CLL is an example of human malignancy caused by alternations in the pathways of programmed cell death--apoptosis. Recent investigations showed a probable role of apoptosis as a prognostic parameter in B-CLL patients. Since the introduction of chlorambucil in the therapy in 1952, besides all the achievements in modern oncology, chlorambucil remained the most common antineoplastic agent in the treatment of CLL. Numerous experimental studies, both in vitro and in vivo, showed the capability of antineoplastic agents to induce the process of apoptosis of neoplastically transformed cells. In this study the effect of chlorambucil on B lymphocytes was monitored in 16 samples of peripheral blood taken from B-CLL diagnosed patients. According to the investigations performed in this study by ultrastructure analysis of B-CLL cells, it was concluded that chlorambucil either induced apoptosis in B-CLL cells, or activated cell response to the stress.

Thioredoxin prolongs survival of B-type chronic lymphocytic leukemia cells

Thioredoxin (Trx) is a ubiquitous protein disulfide oxidoreductase with antioxidant, cytokine, and chemotactic properties. Previously, we showed that Trx, in synergy with interleukin 1 (IL-1), IL-2, IL-4, tumor necrosis factor alpha (TNF-alpha), and CD40-ligation induced S-phase entry and mitosis in normal B cells and B-type chronic lymphocytic leukemia (B-CLL) cells. The viability of B-CLL cells stimulated by these protocols is high, and it has been hypothesized that the overexpression of Bcl-2 found in B-CLL protects the cells from apoptosis in vitro and in vivo. In this study, we have analyzed the response of cells derived from 12 samples of patients with B-CLL to recombinant human Trx in spontaneous apoptosis, with special reference to the Bcl-2 expression. Long-term cultures of B-CLL clones showed significantly higher viability when supplemented with human Trx (P =.031), also exemplified with clones surviving more than 2 months. Short-term cultures of B-CLL cells exposed to 1 microg/mL of Trx for 1, 5, or 12 days maintained expression or delayed down-regulation of Bcl-2 compared with control cultures containing RPMI 1640 medium and 10% fetal calf serum only (P =.032,. 002,.026, respectively). All B-CLL cells expressed constitutive Trx at varying but low levels, in contrast to adult T-cell leukemias, which overexpress Trx, as previously reported. We found that Trx added to B-CLL cells increased in a dose-dependent fashion the release of TNF-alpha, which has been suggested to be an autocrine growth factor for these cells. In conclusion, we have found that human recombinant Trx induced TNF-alpha secretion, maintained Bcl-2, and reduced apoptosis in B-CLL cells. (Blood. 2000;95:1420-1426)

Modulation of Costimulatory Molecules on Follicular Lymphoma Cells by TNF and CD40

TNF has recently been implicated in the formation of germinal center cells in lymphoid organs. Follicular lymphoma (FL) is thought to represent the pathological counterpart of germinal center B-cell. High levels of TNF and its soluble receptors were found in the plasma of FL patients whereas the transcripts of these molecules were previously found to be present in FL patients lymph nodes. We therefore studied here the effects of TNF on the expression of costimulatory molecules implicated in the cytotoxic T cell response on purified FL cells. In contrast to results described with B-type chronic lymphocytic leukemia, also characterized by high levels of circulating TNF, none of the tested samples showed a regulation of CD80, CD86, CD27 and CD70 in response to TNF. To confirm that the lack of regulation of these molecules was not due to the FL cells inability to modulate their expression, we therefore analyzed costimulatory molecules expression after CD40 pathway stimulation. After culture with human CD40L-transfected L-cells, an up-regulation of CD80, CD86 and CD70 expression was observed, while TNF addition in this model did not influence these changes. In this context, the CD27 molecule was down-regulated except in a single case, where its expression was increased.Taken together, this data demonstrates that in vitro expression of costimulatory molecules such as CD80, CD86, CD27 and CD70, which are implicated in the anti-tumoral response, can be regulated by CD40 ligand but not by TNF.

Chronic lymphocytic leukemia B cells express restricted sets of mutated and unmutated antigen receptors.

To better understand the stage(s) of differentiation reached by B-type chronic lymphocytic leukemia (B-CLL) cells and to gain insight into the potential role of antigenic stimulation in the development and diversification of these cells, we analyzed the rearranged VH genes expressed by 83 B-CLL cells (64 IgM+ and 19 non-IgM+). Our results confirm and extend the observations of a bias in the use of certain VH, D, and JH genes among B-CLL cells. In addition, they indicate that the VH genes of approximately 50% of the IgM+ B-CLL cells and approximately 75% of the non-IgM+ B-CLL cells can exhibit somatic mutations. The presence of mutation varies according to the VH family expressed by the B-CLL cell (VH3 expressers displaying more mutation than VH1 and VH4 expressers). In addition, the extent of mutation can be sizeable with approximately 32% of the IgM+ cases and approximately 68% of the non-IgM+ cases differing by > 5% from the most similar germline gene. Approximately 20% of the mutated VH genes display replacement mutations in a pattern consistent with antigen selection. However, CDR3 characteristics (D and JH gene use and association and HCDR3 length, composition, and charge) suggest that selection for distinct B cell receptors (BCR) occurs in many more B-CLL cells. Based on these data, we suggest three prototypic BCR, representing the VH genes most frequently encountered in our study. These data suggest that many B-CLL cells have been previously stimulated, placing them in the "experienced" or "memory" CD5(+) B cell subset.

Examples of in vivo isotype class switching in IgM+ chronic lymphocytic leukemia B cells.

Chronic lymphocytic leukemia (CLL) usually involves the expansion of a clone of CD5+ B cells synthesizing IgM antibodies. These B cells appear to be blocked at the antigen receptor-expressing stage of B cell differentiation and are thought not to undergo an isotype class switch to IgG or IgA production. In vivo and in vitro studies suggest, however, that in some instances terminal differentiation and isotype switching can occur. To test the hypothesis that in vivo isotype class switching occurs in IgM+ B-type CLL cells, we analyzed the PBMC of 19 CLL patients for the presence of transcripts encoding the rearranged CLL V(H)DJ(H) associated with either gamma or alpha H chains. The molecular data indicate that approximately 50% of B-CLL patients have amplifications of IgM+ B cells that undergo an isotype class switch. Switching to IgA appears to occur more often than to IgG; also, switching can involve different IgG subclasses in individual patients. In many instances, these CLL-related gamma and alpha transcripts are much more plentiful than those of normal B cells that produce the same isotype. These switched transcripts do not reveal evidence for the accumulation of significant numbers of new V(H) gene mutations. The cellular data indicate that B cells with lesser amounts of surface membrane IgD and higher IgM/IgD ratios are more likely to undergo this switching process. Furthermore, B cells expressing IgG and IgA of the same idiotype or V(H) family and the same CDR3 length as those of the CLL IgM+ clone can be identified in the blood of patients studied using multiparameter immunofluorescence analyses. Collectively, these data suggest that not all members of a B-CLL clone are frozen at the surface membrane Ig-expressing stage of B cell maturation, and that some members can switch to the production of non-IgM isotypes. The occurrence of switching without the accumulation of V gene mutations indicates that the processes of differentiation and diversification are not linked.

Evidence for Progenitors of Chronic Lymphocytic Leukemia B Cells That Undergo Intraclonal Differentiation and Diversification

Peripheral blood mononuclear cells from five patients with IgG+ B-type chronic lymphocytic leukemia (B-CLL) were analyzed for the presence of clone-specific Ig H chain variable region gene mRNA transcripts linked to C mu and/or C alpha. This was assessed by (1) comparing the lengths of portions of the VHDJH of the IgG+ CLL clones with those of the mu and alpha isotype-expressing B cells, (2) performing clone-specific endonuclease digestion studies, and (3) determining the DNA sequences of the mu and alpha isotype-expressing cDNA. Thus, when B-cell mRNA from these five patients were reverse transcribed with C gamma-specific primers and then amplified by polymerase chain reaction, dominant cDNA were found with lengths corresponding to those of the IgG+ CLL B cell. In addition, in four cases, cDNA of lengths identical to those of the CLL B cell were detected when mRNA was reverse transcribed and amplified using c mu- and/or C alpha-specific primers, strongly suggesting clonal relatedness. These CLL-related mu- and alpha-expressing cDNA were present in greater amounts that unrelated (non-CLL) mu- and alpha-expressing cDNA from normal B cells that used genes of the same VH family. When the sequences of these CLL-related C mu- and C alpha-expressing cDNA were compared with those of the IgG+ CLL clones, it was clear that they were derived from the same ancestral gene as the IgG-expressing CLL B cell, thus documenting their common origin. Finally, nucleotide point mutations were observed in the mu- and alpha-expressing cDNA of certain patients, indicating divergence with the CLL. These data suggest that IgM+ B cells, which are precursors of the leukemic B cells, exist in increased numbers in the blood of most patients with IgG+ B-CELL and that these cells may differentiate, accumulate V genes mutations, and undergo isotype switching in vivo. In addition, the data are consistent with a sequential-hit model for the evolution of CLL.

Selective cytotoxic activity of cyclosporins against tumor cells from patients with B cell chronic lymphocytic leukemia

A fluorometric microculture cytotoxicity assay was employed for the study of cyclosporin A induced cytotoxicity in tumor samples from patients with B type chronic lymphocytic leukemia (B-CLL). Tumor cells from patients with B-CLL were found to be significantly more sensitive to the cytotoxic actions of cyclosporin A than normal blood mononuclear cells and tumor cells obtained from patients with different types of acute leukemia and solid tumors. The effect of cyclosporin A on B-CLL samples could be reproduced by a non-immunosuppressive cyclosporin A analogue. One B-CLL patient treated with cyclosporin A responded with a significant decrease in tumor mass and alleviation of anemia and B symptoms. The results show that cyclosporin A and its non-immunosuppressive analogues appear selectively toxic to B-CLL cells, an observation which may have clinical implications.

168. Induction of growth and differentiation in B-type chronic lymphocytic leukemia (B-CLL) cells by cytokines and thioredoxin

168. Induction of growth and differentiation in B-type chronic lymphocytic leukemia (B-CLL) cells by cytokines and thioredoxin

On the role of endogenously produced TNF-alpha and IL-6 as regulators of growth and differentiation of B-type chronic lymphocytic leukemia cells in vitro.

Chronic lymphocytic leukemia of CD5+ B cells is clinically a heterogeneous disease [1]. This heterogeneity is also easily demonstrable in vitro [2]. In the vast majority of the cases B-CLL is a malignancy of CD5+ B cells with a very low capacity for proliferation in peripheral blood and with a phenotype resembling that of virgin [2] and mantle zone memory [3] B cells. In some cases the stage of differentiation is more advanced, as reflected by the capacity of the leukemic cells to secrete immunoglobulin [Ig]. In other rare cases Ig production may be very low and undetectable by immunofluorescence. The possibility that B-CLL might be a disease of differentiating leukemic B cells has not been extensively studied, but our own unpublished observations suggest that the stage of differentiation of the B-CLL cells may be different in peripheral lymphoid organs and in the bone marrow of the patient than in the peripheral blood, and the capacity for proliferation seems often to be about a 10-fold higher in these tissues.KeywordsChronic Lymphocytic LeukemiaPhorbol Myristic AcetateHairy Cell LeukemiaChronic Lymphocytic Leukemia CellPeripheral Lymphoid OrganThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Expression of the c-myc protein is down-regulated at the terminal stages during in vitro differentiation of B-type chronic lymphocytic leukemia cells

The translocated c-myc oncogene in Burkitt's lymphoma (BL) and murine plasmacytoma (MPC) has been proposed to be expressed at a stage of differentiation at which the gene is normally silent, resulting in a continuous proliferation and an inhibited terminal differentiation. To determine whether c-myc is differently expressed at the various stages of the differentiation pathway, we used B-type chronic lymphocytic leukemia (B-CLL) cells, representing resting B lymphocytes, inducible to proliferation and/or differentiation in vitro. The c-myc protein, and Ig lambda-light chain and PCA-1 antigen as markers of B-cell maturation, were analyzed in single, morphologically defined cells by immunocytochemical double-staining. The proliferation of individual cells was determined by 3H-thymidine incorporation and by analysis of Ki-67 antigen expression. The results show that the level of c-myc expression correlates to the stage of differentiation and to the proliferative activity. Uninduced resting cells did not express c-myc. The c-myc protein was observed in the highest amount at the proliferative B-lymphoblast stage of maturation and was reduced in plasmablasts and undetectable in plasma cells. The results suggest that maturation of B cells into nonproliferative, terminally differentiated plasma cells is associated with a downregulated c-myc expression and thus support the view that the deregulated c-myc gene in BL and MPC is expressed at an inappropriate stage of maturation and thereby inhibits terminal differentiation.

Expression of the c-myc Protein Is Down-Regulated at the Terminal Stages During In Vitro Differentiation of B-Type Chronic Lymphocytic Leukemia Cells

The translocated c-myc oncogene in Burkitt's lymphoma (BL) and murine plasmacytoma (MPC) has been proposed to be expressed at a stage of differentiation at which the gene is normally silent, resulting in a continuous proliferation and an inhibited terminal differentiation. To determine whether c-myc is differently expressed at the various stages of the differentiation pathway, we used B-type chronic lymphocytic leukemia (B-CLL) cells, representing resting B lymphocytes, inducible to proliferation and/or differentiation in vitro. The c-myc protein, and Ig lambda-light chain and PCA-1 antigen as markers of B-cell maturation, were analyzed in single, morphologically defined cells by immunocytochemical double-staining. The proliferation of individual cells was determined by 3H-thymidine incorporation and by analysis of Ki-67 antigen expression. The results show that the level of c-myc expression correlates to the stage of differentiation and to the proliferative activity. Uninduced resting cells did not express c-myc. The c-myc protein was observed in the highest amount at the proliferative B-lymphoblast stage of maturation and was reduced in plasmablasts and undetectable in plasma cells. The results suggest that maturation of B cells into nonproliferative, terminally differentiated plasma cells is associated with a downregulated c-myc expression and thus support the view that the deregulated c-myc gene in BL and MPC is expressed at an inappropriate stage of maturation and thereby inhibits terminal differentiation.

Human b cells express membrane-bound and soluble forms of the cd 14 myeloid antigen

The expression of the myeloid differentiation antigen CD14 on the B lineage was analyzed. A CD14-specific monoclonal antibody was used to isolate the antigen from normal B, B-type chronic lymphocytic leukemia cells, and a representative Epstein-Barr virus-transformed B lymphoblastoid cell line (EBVLCL). A soluble form of this protein was detected in the culture supernatant of all the B cell types tested. The molecule expressed in the normal B and B-type chronic lymphocytic leukemia cells was identical in size to the 52,000 mol. wt monocyte-isolated CD14 glycoprotein. A 64,000 mol. wt antigen was isolated from the lymphoblastoid cell line. Similar 2-D gel electrophoretic patterns to that of the monocyte-derived CD 14 were obtained from the normal B and B-type chronic lymphocytic leukemia cell-isolated molecules. These similarities were reflected in minor isoelectric point (p I) differences between the polypeptide spots (p I4.8), in the first dimension, and identical molecular weight (52,000) in the second dimension. The EBVLCL-isolated polypeptide, when analyzed by 2-D gel electrophoresis, showed a p I identical to that of the myeloid antigen (p I 4.6). The isolated soluble form was of smaller (47,000 mol. wt, normal B and B-type chronic lymphocytic leukemia cells) or similar size (64,000 mol. wt, lymphoblastoid cell line) compared with their corresponding membrane-bound forms. Interestingly, two-colour immunofluorescence analysis showed that only two out of four CD14-speciflc mAb tested bound to the B cells. We conclude that the CD14 antigen is, in fact, expressed in the B lineage. Its cell surface expression and serum level in the prognosis of B-type chronic lymphocytic leukemia patients needs to be evaluated.