In-vitro induction of some features of hairy cell leukemia in chronic lymphocytic leukemia and immunocytoma cells

In an attempt to induce in vitro differentiation we exposed cells from patients with chronic lymphocytic leukemia (CLL) and with immunocytoma (IC) to 12-0-tetra-decanoylphorbol-13-acetate (TPA) at 160 nM. After 3 to 5 days of culture cells became enlarged and the CLL cells developed a basophilic cytoplasm with an excentric nucleus. In some instances cells with many fine projections were seen. We then employed the monoclonal antibody (MAB) HD 6, which was generated against hairy cell leukemia (HCL) cells and reacts most strongly with such cells but is unreactive with plasmocytoma cells and with most CLL cells. TPA treatment induced a greatly enhanced HD 6 binding in CLL and IC cells compared to controls as demonstrated by indirect immunofluorescence. Cytochemical studies revealed that at the same time an acid phosphatase was induced, which was found to be tartrate-resistant in every instance tested. Thus, several features of HCL can be induced in CLL and IC demonstrating the close relatedness of these entities.     

Phorbol ester receptors and the induction of differentiation in the human T lymphoblastic cell line MOLT-3

To examine the mechanism of induction of differentiation in the human malignant T-lymphoblastic cell line, MOLT-3, by 12-O-tetradecanoylphorbol-13-acetate (TPA), the role of receptors for phorbol esters was investigated. Binding of [20-3H]-phorbol-12,13-dibutyrate to TPA-resistant subclones derived from MOLT-3 was less than 50% of that of the parental MOLT-3. Scatchard analysis showed that the concentration of phorbol ester receptors in a TPA-resistant subclone was about 50% of that in the parental MOLT-3, but affinities of binding were similar, indicating that more than a certain number of phorbol ester receptors is required to induce differentiation by TPA in this human T cell line.     

Evidence for protein kinase C--independent pathways mediating phorbol ester induced plasmacytoid differentiation of B chronic lymphocytic leukemia cells.

The effects of phorbol esters on many cell types are known to be mediated through activation of the protein kinase C (PKC) signal transduction pathway. By using the specific inhibitor of this enzyme 1-(5-isoquinolinylsulfonyl)-2-methyl-piperazine dihydrochloride (H7) we have assessed the role of PKC activation in phorbol ester (phorbol 12-myristate 13-acetate, PMA)-induced plasmacytoid differentiation of B chronic lymphocytic leukemia cells (B-CLL) as a model of terminal differentiation of human B lymphocytes. H7 affected a dose-dependent inhibition of PMA-induced thymidine and uridine uptake with ID50 values of 41 microM and 32 microM, respectively. A comparable ID50 value (34 microM) was obtained for H7 inhibition of B-CLL PKC activity in a cell-free system. PMA-induced changes in cell morphology, expression of CD20, CD37 and FMC7 surface antigens together with increased secretion of immunoglobulin were variably abrogated by H7 suggesting that PKC activation is more important in B cell activation/DNA synthesis than in the differentiative response. Consistent with this, expression of a sizable proportion of PMA-inducible genes was not significantly affected by H7. These data are consistent with the existence of a PMA-activated, PKC-independent signal transduction pathway which may be important, though by itself apparently insufficient, for eliciting full terminal differentiation in B lymphocytes.     

A human B lymphocyte antigen (P-76) shared by B-cell chronic lymphocytic leukemia cells and hairy cell leukemia cells

A membrane antigen with an apparent specificity to B lymphocytes was detected with immunochemical techniques and its properties were analyzed. Anti-B-CLL serum was raised in a rabbit by immunization with B-cell chronic lymphocytic leukemia (B-CLL) cells. This anti-B-CLL serum was absorbed with erythrocytes, liver homogenate and insolubilized immunoglobulins. After further absorption with T-CLL cells, chronic myelocytic leukemia (CML) cells and acute myelocytic leukemia (AML) cells, the anti-B-CLL serum still reacted with peripheral blood B lymphocytes, B-CLL cells and hairy cell leukemia (HCL) cells. In contrast, no reactivity was seen with peripheral blood T lymphocyte or monocytes, or leukemia cells of non-B cell origin. An immunoprecipitation of radiolabeled cell surface proteins was attempted using the anti-B-CLL serum in the presence of Staphylococcus Aureus Cowan 1 (SaCl), and the precipitates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A membrane antigen with an apparent molecular weight of 76,000 daltons (P-76) was immunoprecipitated with the anti-B-CLL serum from the lysates of normal B lymphocyte, B-CLL cells and HCL cells. The antigen (P-76) is not composed of disulfide-linked subunits and has no structural relationship with HLA-DR (Ia-like) antigens or other known antigens. These results suggest that this antigen is B-lymphocyte specific, and favour the B-lymphocyte nature of HCL cells.     

Phorbol esters specifically inhibit induction of immunoglobulin secretion in a murine B cell leukemia

We have examined the effect of tumor-promoting phorbol esters such as phorbol myristate acetate (PMA) on the murine B cell leukemia BCL-1 and its in vitro adapted derivative CW.13.20. Phorbol esters, including PMA and phorbol dibutyrate (PDBu), were potent inhibitors of BCL-1 IgM secretion induced by either LPS or lymphokines; half-maximal inhibition was obtained with 0.1 nM PMA and 0.8 nm PDBu. The inhibitory action of PDBu on BCL-1 cells was reversible for over 1 hr, but after 5 hr 70% of the inhibition was irreversible. Irreversible inhibition could be blocked by cycloheximide, suggesting a requirement for protein synthesis. The specificity of PDBu inhibition was examined by comparing the patterns of protein synthesis in PDBu-treated and control BCL-1 cells. Total incorporation of [35S]methionine into protein by BCL-1 cells cultured in the presence of PDBu was similar to that of untreated cells. Analysis of radiolabeled proteins by SDS-PAGE and autoradiography revealed no consistent changes in the pattern of protein synthesis except at those positions corresponding to the heavy and light chains of IgM. Immunoprecipitation with an affinity-purified anti-IgM indicated that PDBu inhibited the increased synthesis of heavy and light chain that follows stimulation by lymphokine but did not diminish control IgM synthesis. Induced IgM secretion from CW.13.20 cells was also inhibited by phorbol esters, indicating a direct action on B cells. Delaying the addition of phorbol ester relative to lymphokine or LPS by 24 hr significantly reduced inhibition of induced IgM secretion from both BCL-1 and CW.13.20 cells. This suggests that phorbol esters specifically interfere with the signal for induction of IgM secretion by both lymphokine and LPS.     

Relevance of stereotyped B-cell receptors in the context of the molecular, cytogenetic and clinical features of chronic lymphocytic leukemia

Highly homologous B-cell receptors, characterized by non-random combinations of immunoglobulin heavy-chain variable (IGHV) genes and heavy-chain complementarity determining region-3 (HCDR3), are expressed in a recurrent fraction of patients affected by chronic lymphocytic leukemia (CLL). We investigated the IGHV status of 1131 productive IG rearrangements from a panel of 1126 CLL patients from a multicenter Italian study group, and correlated the presence and class of HCDR3 stereotyped subsets with the major cytogenetic alterations evaluated by FISH, molecular prognostic factors, and the time to first treatment (TTFT) of patients with early stage disease (Binet A). Stereotyped HCDR3 sequences were found in 357 cases (31.7%), 231 of which (64.7%) were unmutated. In addition to the previously described subsets, 31 new putative stereotypes subsets were identified. Significant associations between different stereotyped HCDR3 sequences and molecular prognostic factors, such as CD38 and ZAP-70 expression, IGHV mutational status and genomic abnormalities were found. In particular, deletion of 17p13 was significantly represented in stereotype subset #1. Notably, subset #1 was significantly correlated with a substantially reduced TTFT compared to other CLL groups showing unmutated IGHV, ZAP-70 or CD38 positivity and unfavorable cytogenetic lesions including del(17)(p13). Moreover, subset #2 was strongly associated with deletion of 13q14, subsets #8 and #10 with trisomy 12, whereas subset #4 was characterized by the prevalent absence of the common cytogenetic abnormalities. Our data from a large and representative panel of CLL patients indicate that particular stereotyped HCDR3 sequences are associated with specific cytogenetic lesions and a distinct clinical outcome.     

Cytogenetic and molecular cytogenetic investigations in relapse of B-cell chronic lymphocytic leukemia

Сhromosomal abnormalities have been analyzed in bone marrow cells of 61 patients with relapse of B-cell chronic lymphocytic leukemia. The cytogenetic results have allowed the structural stratification of the obtained karyotypes into ten groups of clones: normal, normal/near tetraploid, abnormal/normal, abnormal/ near tetraploid/normal, evolution of clonal chromosome abnormalities; evolution of clonal chromosome abnormalities/normal, evolution of clonal chromosome abnormalities/near tetraploid/normal, independent clones, independent/normal clones; and independent/near tetraploid/normal clones. The identified structural rearrangements included translocations, deletions, insertions, and duplications; however, deletions with the involvement of bands 17p12, 13q12–q14, 11q14, and 11q23 dominated (63.8%). The application of i-FISH helped to show the presence of one to four abnormalities per karyotype. The identified cytogenetic and molecular cytogenetic rearrangements may signify a multilevel nature of the process underlying the development of resistant karyotypes. The results obtained under both methods have revealed the presence of a heterogenic cell population with possibly different levels of chemotherapy resistance.     

Surface expression of Bcl-2 in chronic lymphocytic leukemia and other B-cell leukemias and lymphomas without a breakpoint t(14;18)

Since its discovery in follicular lymphoma cells at the breakpoint t(14;18), Bcl-2 has been studied extensively in many basic and clinical science settings. Bcl-2 can locate as an integral mitochondrial membrane component, where its primary role is to block apoptosis by maintaining membrane integrity. Here we show that Bcl-2 also can position on the outer cell surface membrane of B cells from patients with chronic lymphocytic leukemia (B-CLL) and certain other leukemias that do not classically possess the chromosomal breakpoint t(14;18). Although low levels of Bcl-2 can be detected on the surface membrane of apparently healthy leukemic and normal B cells, expression of Bcl-2 correlates best with spontaneous or induced apoptosis. Notably, upon induction of apoptosis, B-CLL cells were much more efficient in upregulating surface Bcl-2 than normal B cells. It is not clear if this surface membrane expression is a passive consequence of the apoptotic process or an active attempt by the B cell to abort cell death by stabilizing the plasma membrane.     

A newly established human acute lymphoblastic leukemia cell line with characteristics of the earliest B-cell maturation

A new human acute lymphoblastic leukemia (ALL) cell line, designated HBL-3, was established from the bone marrow of a patient with non-T-ALL. The HBL-3 cell line expressed B4 (CD 19), BA-1 (CD 24) and HLA-DR antigens, but not surface immunoglobulin (SIg) or cytoplasmic immunoglobulin (CIg). The cell line lacked the common acute lymphoblastic leukemia antigen (CALLA) and antigenic markers characteristic of T-cell and myeloid cell lineages. The HBL-3 cells had structural rearrangements of both the homologous chromosome 9s, including a translocation with chromosome 1 which has been reported in a patient with common ALL. The cell line had rearranged immunoglobulin heavy chain genes but retained germ-line kappa light chain genes and germ-line T-cell receptor beta- and gamma-chain genes. The HBL-3 cell line was strongly positive for terminal deoxynucleotidyl transferase (TdT). These findings indicate that the HBL-3 cell line is derived from the earliest B-cell committed to B-cell lineage.     

A newly established human acute lymphoblastic leukemia cell line with characteristics of the earliest B-cell maturation

A new human acute lymphoblastic leukemia (ALL) cell line, designated HBL-3, was established from the bone marrow of a patient with non-T-ALL. The HBL-3 cell line expressed B4 (CD 19), BA-1 (CD 24) and HLA-DR antigens, but not surface immunoglobulin (SIg) or cytoplasmic immunoglobulin (CIg). The cell line lacked the common acute lymphoblastic leukemia antigen (CALLA) and antigenic markers characteristic of T-cell and myeloid cell lineages. The HBL-3 cells had structural rearrangements of both the homologous chromosome 9s, including a translocation with chromosome 1 which has been reported in a patient with common ALL. The cell line had rearranged immunoglobulin heavy chain genes but retained germ-line κ light chain genes and germ-line T-cell receptorβ- and γ-chain genes. The HBL-3 cell line was strongly positive for terminal deoxynucleotidyl transferase (TdT). These findings indicate that the HBL-3 cell line is derived from the earliest B-cell committed to B-cell lineage.     

Phenotypic modulation of chronic lymphocytic leukemia cells by phorbol ester: induction of IgM secretion and changes in the expression of B cell-associated surface antigens

Freshly explanted neoplastic populations from 22 cases of phenotypically well-characterized chronic type B lymphocytic leukemia were studied for their capacity to respond to the phorbol ester TPA in vitro. In all but four cases the secretion of IgM was either induced or increased, often to a high level. In contrast, the export of free immunoglobulin (Ig) light chains, an almost consistent feature of the B lymphocytic leukemias, remained relatively constant after TPA treatment. Parallel changes in leukemic cell surface phenotype were probed with both "conventional" and monoclonal antibodies, revealing some modulation of markers in every case investigated. A diminution in the level of surface Ig (preferentially IgD) and the accumulation of cytoplasmic Ig observed after phorbol ester treatment were accompanied by a corresponding reduction or loss of the B1 antigen and usually of B2 when present. The most consistent change induced by TPA was the appearance of BB-1, a marker of activated B lymphocytes, which was rarely expressed on fresh leukemic cells. Another marker of activated lymphocytes, LB-1, was also often induced or increased in its expression after exposure of the cells to TPA. The magnitude of the TPA response appeared to relate to the stage of maturation arrest of the individual leukemic clones rather than to any clinical parameter explored. The significance of the findings to normal B cell differentiation and their potential clinical utility are discussed.     

Platelet-adjusted IFN dosage in the treatment of advanced hairy cell leukemia

It has been demonstrated that hairy cell leukemia (HCL) can be efficiently treated by various preparations of alpha interferons (IFN). Nevertheless, there are several open questions, such as the route, mode and dosage of IFN application. These variables of IFN treatment may be critical since a myelosuppressive effect of IFN, which is commonly seen in the initial phase of treatment, can result in further deterioration of the already impaired platelet production in advanced HCL. The present study shows that serious side effects can be avoided and the flu-like syndromes described by others almost completely reduced by s.c. application of IFN via a portable pump during a daily 8 h period. IFN is initially given five times a week and the daily dose is adjusted according to the actual platelet count. The efficiency controls show that the increase of platelets in the peripheral blood, which is most critical in advanced HCL, may be seen earlier by this than by other protocols, which usually recommend higher daily doses of IFN and only three instead of five weekly applications.     

In vitro maturation of B cells in chronic lymphocytic leukemia. I. Synergistic action of phorbol ester and interleukin 2 in the induction of Tac antigen expression and interleukin 2 responsiveness in leukemic B cells

Recent evidence indicates that interleukin 2 (IL 2), formerly thought to serve as growth factor exclusively for activated T cells, is directly involved in human B cell differentiation. We have investigated the role of IL 2 and IL 2 receptors (as defined by monoclonal anti-Tac antibody) in the phorbol ester-induced in vitro maturation of leukemic B cells from patients with chronic lymphocytic leukemia (CLL). Peripheral blood lymphocytes from B cells from CLL patients with high (greater than 10(5)/microliters) white blood cell counts were depleted of residual T lymphocytes and low-density cells (primarily macrophages) by consecutive steps of E rosetting, complement-mediated lysis of OKT3+ and OKT4+ cells, and Percoll density gradient centrifugation. No OKT3+ T cells were detectable in these cell populations before or after culture. When incubated for 3 days with phorbol ester plus recombinant human IL 2 (rIL 2), 12 to 57% of highly purified B cells from four of five tested patients expressed Tac antigen. Both phorbol ester and rIL 2 were required for maximal Tac antigen expression. Functional studies revealed that phorbol ester-activated (but not resting) CLL B cells responded to rIL 2 with [3H]thymidine incorporation and with enhanced secretion of IgM. Tac+ B cells were isolated in two cases on a fluorescence-activated cell sorter. In one patient, stimulation of Tac+ B cells with rIL 2 resulted in enhanced [3H]thymidine incorporation but no change in IgM secretion, as compared with Tac- B cells; in the second patient, stimulation of Tac+ B cells with rIL 2 did not result in [3H]thymidine uptake, but did result in significant IgM secretion. These findings indicate that certain leukemic B lymphocytes can be induced to express IL 2 receptors and respond to IL 2. The use of resting clonal B cell populations arrested at distinct stages of differentiation may help to better define the stage(s) at which IL 2 acts directly on B cells to induce proliferation and/or terminal differentiation.     

Induction of apoptosis in chronic lymphocytic leukemia cells and its prevention by phorbol ester

Chronic lymphocytic leukemia lymphocytes were used to study mechanisms involved in apoptosis (programmed cell death). Apoptosis, which was determined by morphological changes including cell death and by internucleosomal DNA fragmentation, occurred during culture for 1 to 2 days in a portion of the cells from three of the four patients tested. Most of the cells underwent apoptosis and DNA fragmentation was greatly enhanced when cells were cultured in the presence of the microtubule inhibitor colchicine, the topoisomerase II inhibitor etoposide, or the glucocorticoid methylprednisolone. Tumor-promoting phorbol esters inhibited spontaneous DNA fragmentation and cell death including that induced by colchicine, etoposide, and methylprednisolone, indicating that they act on an event common to apoptosis caused by diverse stimuli. Phorbol esters probably act through protein phosphorylation, since they were effective at concentrations which modulated protein kinase C (PKC) and their action was prevented by H-7, which binds to and inactivates the catalytic site of PKC. In the absence of phorbol ester, H-7 itself induced some apoptosis. These findings implicate PKC in the suppression of apoptosis, but its precise role requires systematic investigation.     

Flow cytometric analysis of T-lymphocyte subpopulations in B-cell chronic lymphocytic leukemia: correlation with clinical stage

Several authors have studied the T-lymphocyte subpopulations in B-cell chronic lymphocytic leukemia (B-CLL), but previous studies were performed after preceding enrichment procedures, which are known to cause selective losses of certain subpopulations. To correct for this deficiency we used flow cytometric analysis, which enabled us to measure subpopulations directly on total blood samples. We studied T-lymphocyte subsets with OKT monoclonal antibodies in 45 patients with B-CLL. Serum levels of IgG, IgA and IgM were assayed simultaneously and findings were correlated with clinical stage (Rai classification). The absolute number of CD4-positive cells decreased in more advanced Rai stages, while the absolute number of CD8-positive cells increased, resulting in a progressive reduction in CD4/8 ratio. Results from patients in stages with equal prognosis (Rai I and II, Rai III and IV) were similar and when these results were grouped the observed differences were highly significant and clearly correlated with all prognostic groups.