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 ester tumor promoters block the transition from the early to the heme-dependent late program of friend cell differentiation

In this study, the mechanism of inhibition of differentiation of Friend erythroleukemia cells by the phorbol ester tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), has been examined. These studies indicate that some early events associated with Friend cell differentiation, including an early change in ⁸⁶Rb⁺ transport and a decrease in cell volume, still occur in the presence of TPA. However, several late events in the program of Friend cell differentiation, including the induction of heme synthesis and the loss of proliferative capacity, are inhibited by TPA. These effects of TPA can be reversed by hemin, which alone does not induce Friend cells to differentiate. The addition of hemin to cultures grown in the presence of inducer plus TPA for several days results in the rapid restoration of hemoglobin synthesis, and also causes a parallel decrease in colony-forming ability. These results suggest that tumor promoters may inhibit only heme-dependent events, rather than the entire program of Friend cell differentiation.     

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.     

Phorbol ester induction of rat hepatic metallothionein in vivo and in vitro.

1. The induction of metallothionein (MT) protein by TPA (O-tetradecanoyl phorbol acetate), a protein kinase C activator, was demonstrated in vivo in rat liver and in vitro in rat hepatocytes in primary culture. In vivo half maximal induction at 25 hr was seen at 26 nmol TPA/kg body wt. Five- to seven-fold inductions were seen in vivo. De novo protein synthesis was required for this induction as demonstrated by cycloheximide inhibition of [35S]cysteine incorporation into MT protein. 2. TPA induction of MT protein in primary cultures of rat hepatocytes reached levels of 2.6-4.1-fold, as assessed by [35S]cysteine incorporation, 1.34-2.20-fold, as assessed by 109Cd binding in a metal displacement/HPLC assay, and 2.5-5-fold, as assessed by 109Cd binding in a metal displacement/Sephadex G-75 Superfine assay. 3. The induction of MT mRNA by TPA was demonstrated in vivo in rat liver and in vitro in 2 rat hepatoma cell lines, EC3 and 2M. MT mRNA was quantitated using dot blot and Northern gel assays. In vivo TPA induced hepatic MT mRNA 2.36-5.88-fold (dot blot) and 7.4-22-fold (Northern gels). In vitro TPA induced MT mRNA 1.71-15.26-fold in EC3 cells and 2.23-8.43-fold in 2M cells. MT mRNA was 0.54 kb, and alpha-tubulin mRNA was 1.62 kb in size on Northern gels. 4. TPA induction of MT protein and mRNA in vivo and in vitro is rapid and persistent and occurs at low concentrations. The 2 rat hepatoma cell lines provide a useful system in which to study MT induction in vitro without confounding secondary effects which can occur in vivo.     

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.     

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.     

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.     

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.     

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.     

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.