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PubMedDate not supplied

Cytokines and Epstein Barr virus (EBV) genes expression in blood chronic lymphocytic leukaemia (CLL) cells and their immortalised CLL cell lines.

We have encountered two unique chronic lymphocytic leukaemia (CLL) patients, PG and NN. Some blood CLL cells of these patients have been infected and carry Epstein Barr virus (EBV) in vivo. In spite of their early-activated G0/G1 stage of post germinal center (GC) memory cells, ex vivo EBV-carrying blood CLL cells of PG clone expressed LMPs and used specific QUK splice for their EBNA1 expression, similar to the EBV-carrying cells of non-B origin. Interestingly, EBV-carrying CLL cells of NN clone expressed LMP2a and used UK-splice for their EBNA1 expression, similar to the in vivo EBV-carrying high density normal B cells in the blood of healthy individuals. The CLL-derived lines but not normal lymphoblastoid cell line (LCL) used QUK- and YUK-splice for their EBNA1 expression. As expected, LCL and their permanent CLL-derived lines used Cp promoter and up-regulated their EBNA2 expression. Blood CLL cells and the CLL-derived cell lines of these patients spontaneously produced cytokines as shown by microarray assay. The types and quantities of cytokines might relate to their CLL origin and viral strain in the given CLL cells. Neither blood CLL nor their CLL-derived cell lines express any detectable apoptosis-inducer ligands, CD95L or Apo 3L. As a consequence of cell cycle progression, CLL-derived cell lines up-regulated their co-stimulator molecules CD80 and apoptosis-related receptor CD95. Since only the rare EBV-carrying CLL cells grew in vitro, the combination of viral genome and cytokines seems to be critical for the outgrowth of EBV-carrying CLL cells over their EBV-negative counterpart in vitro but not in vivo.

Cell Cycle↗
PubMed2001

Chronic lymphocytic leukemia (CLL) is rare, but the proportion of T-CLL is high in Japan.

Chronic lymphocytic leukemia (CLL) is a rare disease in Japan. Recent advances in molecular biology, diagnostic criteria and classification of CLL have reinforced the concept of each category of CLL as a distinct entity. Since there have been no recent studies on the incidence and prevalence of CLL in Japan, the Kyushu Hematology Organization for Treatment (K-HOT) Study Group conducted two studies of CLL. One study is a prospective registration of newly diagnosed hematological disorders, which gave us some idea of the incidence of CLL in our region (Kyushu island) where adult T-cell leukemia is endemic. A total of 677 patients with hematological disorders were registered over a 6-month period and 11 patients were diagnosed as having CLL among 182 leukemia patients. This amounts to 6% of all leukemias, which is twice as frequent as previously reported in Japan. The other study is a retrospective analysis of CLL. Eleven institutions of the K-HOT Group analysed their diagnostic records of chronic lymphoid leukemia, and 145 patients with CLL were found over a period of 3-12 yr. After the data were reviewed 11 patients were excluded through having a different type of leukemia. The proportion of chronic B-cell lymphoid leukemia was 73% (98/134), while that of T-cell leukemia was 18% (24/134). The proportion of T-cell chronic leukemia was 5-6 times higher than that in Western countries. Two institutions had a complete database on hematological disorders. From this database, the annual incidence of CLL was estimated to be 0.48 per 100 000. Thus, the incidence of CLL in Japan is at least 4-5 times lower than that in Western countries, suggesting that chronic B-cell leukemia is really rare, but chronic leukemia of T-cell lineage develops in Japan as frequently as in Western societies. Further investigation is required to delineate why the incidence of B-CLL is so low in Japan.

Adult↗
PubMed1990

Distinct characteristics of lymphokine-activated killer (LAK) cells derived from patients with B-cell chronic lymphocytic leukemia (B-CLL). A factor in B-CLL serum promotes natural killer cell-like LAK cell growth.

We show that lymphokine-activated killer (LAK) cell precursors derived from patients with B-cell chronic lymphocytic leukemia (B-CLL) and cultured in the presence of recombinant interleukin-2 and normal human serum (NHS), develop into primarily NK cell-like (CD 57+) LAK cells, whereas identically prepared LAK cell precursors from normal subjects develop into mainly T cell-like (CD 3+, CD 8+) LAK cells. B-CLL LAK cells exhibited greater proliferative capacity than did normal LAK cells. When normal LAK cells were grown in B-CLL serum instead of NHS, their proliferation increased; NK cell levels also increased to those found in B-CLL LAK cells, suggesting that B-CLL serum contains a factor that promotes NK cell-like growth, LAK cells derived from normal or B-CLL patients demonstrated similar lytic activity toward K562 and Raji cells. Growth in B-CLL serum did not reduce their lytic potential. Thus, the altered phenotype and growth exhibited by B-CLL LAK cells and normal LAK cells grown in B-CLL serum does not lead to abnormalities in their cytolytic functions. We propose instead that the predominance of NK-like cells in B-CLL LAK cell populations and the presence of an NK cell-like growth factor in B-CLL serum reflect abnormalities related to NK cell-mediated B-cell regulation; ie, either inhibition of normal B-cell growth and/or growth stimulation of the leukemic clone in B-CLL.

Antigens, CD↗
PubMed2002

Differentiation of monoclonal B lymphocytosis of undetermined significance (MLUS) and chronic lymphocytic leukemia (CLL) with weak CD5 expression from CD5(-) CLL.

Chronic lymphocytic leukemia (CLL) is recognized as a unique lymphoproliferative disorder of CD5(+) B cells. However, many published series of CLL included a subgroup of CD5(-) cases. CD5(-) B cell CLL is a topic of controversy and its nature and true incidence remain unclear. We in this study performed a retrospective analysis of a total of 128 consecutive patients with a diagnosis of CLL and available immunophenotypic record. Of these, 14 cases were previously considered CD5(-) CLL. From a further analysis of clinical, hematological and immunophenotypic results, we have reclassified seven of the patients as having weak or dim expression of CD5 and four patients as being monoclonal B lymphocytosis of undetermined significance (MLUS). The remaining three cases had clinical and morphological features consistent with prolymphocytic leukemia (PLL) or mixed CLL/PLL. Our results suggest that the CD5(-) phenotype probably does not qualify for CLL. Previous CD5(-) CLL may include false negatives due to heterogeneity of the intensity of CD5 expression, CD5(-) MLUS and variant CLL; the latter likely represents CLL in transformation. All the patients with MLUS were found to have a mild and non-progressing lymphocytosis with CD5(-) phenotype. These features may be used to differentiate them from CLL.

Adult↗
PubMed1998

Similarities between the sensitivity to 2-chlorodeoxyadenosine of lymphocytes from CLL patients and bryostatin 1-treated WSU-CLL cells: an infrared spectroscopic study.

Infrared (IR) spectroscopy was used to compare the drug resistance mechanism of cells from chronic lymphocytic leukemia (CLL) patients with that of WSU-CLL cells. Bryostatin 1 (Bryo 1), a macrocyclic lactone and protein kinase C activator, was used to render WSU-CLL cells more susceptible to 2-chlorodeoxyadenosine (2-CdA). The IR spectroscopic analysis revealed some changes in protein and DNA content in Bryo 1-treated WSU-CLL cells, however, the most significant alterations were observed in the membrane lipids, which resemble those found between 2-CdA-sensitive and 2-CdA-resistant cells from CLL patients. In addition, Bryo 1 treatment induced WSU-CLL cells to become CD11c, CD25 and tartrate-resistant acid phosphatase-positive, specific markers for hairy cell leukemia, a disease exquisitely sensitive to 2-CdA. Our results suggest that 2-CdA-sensitive CLL cells have cellular characteristics resembling the hairy cell stage. The similarity between the membrane lipids in 2-CdA-sensitive CLL cells and the Bryo 1-treated WSU-CLL cell line supports the suggestion that membrane lipid alteration might be an important step in the drug resistance mechanism of CLL cells.

Adult↗
PubMed2004

Clonal lymphocytes in persons without known chronic lymphocytic leukemia (CLL): implications of recent findings in family members of CLL patients.

Several genetic abnormalities have been characterized in chronic lymphocytic leukemia (CLL) but these are predominantly secondary events and the initiating phenomena in the etiology of the disease are yet to be established. Studies of inherited susceptibility have identified the early oncogenic events in both familial and "sporadic" forms of several malignant disorders, and this may also be possible in CLL. However, the utility of linkage analysis in identifying a predisposition locus for the disease is limited because large multigenerational families segregating CLL are rare, while the more frequent small nuclear CLL families contain insufficient numbers of affected individuals. The power to detect predisposition gene(s) could be greatly increased by extending the number of affected individuals within a particular family, for example, by identifying family members with subclinical levels of disease. High-sensitivity flow cytometry techniques, developed to monitor disease in CLL patients undergoing treatment, have allowed accurate enumeration of subclinical levels of CLL cells in healthy individuals from the general population and CLL families. Emerging evidence confirms the phenotypic, genotypic, and clinical associations between the aberrant cells in healthy individuals and those in CLL patients. The data suggest that inherited factors increase the susceptibility to both indolent and aggressive CLL, and they provide unbiased demonstration that the age of onset in CLL families is younger than in the general population.

Age Factors↗
PubMed2005

Potential approach to immunotherapy of chronic lymphocytic leukemia (CLL): enhanced immunogenicity of CLL cells via infection with vectors encoding for multiple costimulatory molecules.

Chronic lymphocytic leukemia (CLL) is a disease of CD5(+) B lymphocytes (designated as CLL cells) that are inefficient antigen-presenting cells. Their poor ability to present antigens to the T cells, largely due to an inadequate costimulatory capacity, is manifested as a failure to stimulate proliferation of both allogeneic and autologous T cells. We have investigated the ability of in vitro manipulated CLL cells, via hyperexpression of a triad of costimulatory molecules (B7-1, intercellular adhesion molecule 1 [ICAM-1], and leukocyte-function-associated antigen 3 [LFA-3], designated TRICOM), to stimulate effective antitumor T-cell responses. A recombinant modified vaccinia virus strain Ankara (MVA), which is a highly attenuated, replication-impaired virus variant, was successfully used to infect and deliver the simultaneous expression of the 3 human costimulatory molecules in TRICOM on the surface of the CLL cells. Proliferation of allogeneic and autologous T cells was observed when MVA-TRICOM-infected CLL cells were used as stimulators in proliferation assays. Cytotoxic T lymphocytes, generated in vitro by stimulation of autologous T cells with MVA-TRICOM-infected CLL cells, showed cytotoxicity against unmodified/uninfected CLL cells. Therefore, our findings suggest that the use of CLL cells infected ex vivo with MVA-TRICOM or direct injection of MVA-TRICOM in patients with CLL has potential for the immunotherapy of CLL.

Aged↗
PubMed2001

Broad alterations of self-reactive antibody-repertoires of plasma IgM and IgG in B-cell chronic lymphocytic leukemia (B-CLL) and B-CLL related target-restricted autoimmunity.

B-cell chronic lymphocytic leukemia (B-CLL) is characterized by a malignant CD5+ B-cell clone. The leukemic clone commonly expresses IgM antibodies exhibiting reactivity toward a wide range of self-antigens. However, B-CLL associated autoimmunity is typically restricted to self-antigens expressed by blood cells, and mediated by IgG autoantibodies of polyclonal origin. In the present study, we addressed the question whether self-reactive antibody repertoires of plasma IgM and IgG are disturbed by monoclonal immunoglobulins of B-CLL patients, and whether antibody repertoires of patients exhibiting B-CLL associated target-restricted autoimmune disease (AID) differ from those of B-CLL patients without AID. We investigated antibody repertoires at a global level, using a technique of quantitative immunoblotting that allows for the quantitative screening of antibody reactivities in complex antibody mixtures toward a large panel of antigens derived from homologous tissue extracts, followed by multiparametric statistical analysis of the data. We demonstrate that self-reactive antibody repertoires of plasma IgM and IgG are broadly altered in patients with B-CLL, that alterations in self-reactive antibody repertoires are not restricted to B-CLL patients exhibiting AID, and that target-restricted autoimmunity in B-CLL patients is associated with altered antibody repertoires not restricted to the target organ. We conclude that monoclonal alterations of immunoglobulin production in B-CLL are associated with broad defects of self-reactive antibody repertoires. Our observations suggest that the application of therapeutic IVIg preparations might influence B-CLL by restoring normal self-reactive antibody repertoires in plasma.

Adult↗
PubMed2004

Generation of B-cell chronic lymphocytic leukemia (B-CLL)-reactive T-cell lines and clones from HLA class I-matched donors using modified B-CLL cells as stimulators: implications for adoptive immunotherapy.

Allogeneic stem cell transplantation following reduced-intensity conditioning is being evaluated in patients with advanced B-cell chronic lymphocytic leukemia (B-CLL). The curative potential of this procedure is mediated by donor-derived alloreactive T cells, resulting in a graft-versus-leukemia effect. However, B-CLL may escape T-cell-mediated immune reactivity since these cells lack expression of costimulatory molecules. We examined the most optimal method to transform B-CLL cells into efficient antigen-presenting cells (APC) using activating cytokines, by triggering toll-like receptors (TLRs) using microbial pathogens and by CD40 stimulation with CD40L-transfected fibroblasts. CD40 activation in the presence of IL-4 induced strongest upregulation of costimulatory and adhesion molecules on B-CLL cells and induced the production of high amounts of IL-12 by the leukemic cells. In contrast to primary B-CLL cells as stimulator cells, these malignant APCs were capable of inducing the generation of B-CLL-reactive CD8(+) CTL lines and clones from HLA class I-matched donors. These CTL lines and clones recognized and killed primary B-CLL as well as patient-derived lymphoblasts, but not donor cells. These results show the feasibility of ex vivo generation of B-CLL-reactive CD8(+) CTLs. This opens new perspectives for adoptive immunotherapy, following allogeneic stem cell transplantation in patients with advanced B-CLL.

Antigen-Presenting Cells↗
PubMed1995

A scoring system for the classification of CD5-B CLL versus CD5+ B CLL and B PLL.

B CLL is a monoclonal proliferation of lymphocytes which express the CD5 antigen (CD5+ CLL). Rare exceptions (less than 10%) are CD5-, as are the majority of B PLL. We have studied the clinical, cytological and immunophenotypic characteristics of a series of 12 CD5-CLL and have established a score which allows the distinction between CD5+ CLL, CD5- CLL and PLL. Among the CD5- CLL, there were significantly more cases with advanced stage (Rai and Binet) and splenomegaly. The cytological study found more mixed CLL according to FAB classification (more prolymphocytes). There were significantly more CD23-, FMC7+, SIg strong positive cases. A score from 0 to 6 was established based on clinical, cytological and immunophenotypic criteria. Typical CD5+ CLL was scored 0, score 6 corresponded to typical PLL. There were significantly more higher scores amongst CD5- CLL. It therefore appears that CD5- CLLs share certain features with B PLL. The use of this scoring system will allow determination of prognosis within these different categories, thus identifying groups which require specific therapy.

Adult↗
PubMed1984

Prolymphocytic leukaemia: surface morphology in 21 cases as seen by scanning electron microscopy and comparison with B-type CLL and CLL in 'prolymphocytoid' transformation.

The surface architecture of leukaemic cells obtained from 21 cases of proven prolymphocytic leukaemia (PLL) and eight cases of chronic lymphocytic leukaemia (CLL) with 'prolymphocytoid' transformation (PL-CLL) was compared with the cell surface morphology of leukaemic cells obtained from 46 cases of B-type CLL, using the scanning electron microscope (SEM). All cases were defined by cytochemistry, immunological markers and transmission electron microscopy prior to SEM examination. B-CLL cells showed the well-recognized spectrum of surface architecture described in earlier studies. The majority of cells had moderate numbers of short microvilli, although in a minority, cells with relatively smooth surfaces predominated. In seven of the eight cases of PL-CLL, cells were villous in nature and in this respect similar to CLL cells; however, more cells with dense microvilli were seen. The prolymphocytic cells were recognized by their larger size and in 18 of the 19 cases of B-derived PLL, villous cells predominated. Two cases of T-derived PLL showed variable cell surface morphology ranging from smooth to moderately villous. It appears that B-PLL cells are most frequently villous and display more surface microvilli than B-CLL cells. B-prolymphocytes display the surface features regarded as characteristic for neoplastic B-cells as seen in patients with B-type lymphoma and leukaemia.

B-Lymphocytes↗
PubMed1993

Confirmation of Rb gene defects in B-CLL clones and evidence for variable predominance of the Rb defective cells within the CLL clone.

In 70 B-CLL patients, deletion or translocation, at or near the retinoblastoma (Rb) site, was detected in 20 by cytogenetic analysis. Purified B cell clones from 13 of these B-CLL patients were isolated and studied for Rb gene status, Rb mRNA and the Rb protein product. Southern blot analysis of the Rb site detected internal deletions (N = 1) or a single allele loss (N = 2) in five patients. Northern blots detected reduced Rb mRNA in four patients. Immunoblot of whole cell lysate revealed reduced levels of unphosphorylated Rb protein in six CLL patients. No CLL B cell clone contained phosphorylated Rb species. These molecular studies have confirmed the cytogenetic alteration of 13q12-14 sites in B-CLL cells. In addition, cytogenetic and molecular biologic analysis suggest heterogeneity in the B cell clone for Rb gene abnormality. B-CLL patients with abnormalities in both cytogenetic and Rb DNA/RNA analysis will have a dominance of B cells with an Rb abnormality (N = 5). In patients whose Rb defective CLL cells constitute only a minor subpopulation of the total B cell clone, only cytogenetic defects would likely be detected (N = 7).

Blotting, Northern↗
PubMed1995

Frequent somatic mutations in D and/or JH segments of Ig gene in Waldenström's macroglobulinemia and chronic lymphocytic leukemia (CLL) with Richter's syndrome but not in common CLL.

V(D)J recombination and somatic hypermutations are developmentally regulated during B-cell differentiation; therefore, DNA analysis of the Ig gene delineates the cellular origin of B-cell neoplasms. We analyzed the third complementarity-determining region and adjacent regions of the Ig heavy-chain gene of tumor cells from 7 patients with Waldenström's macroglobulinemia (WM) and from 10 patients with B-cell chronic lymphocytic leukemia (CLL), 2 of whom progressed to high-grade non-Hodgkin's lymphoma (NHL), ie, Richter's syndrome (RS). There were no intraclonal variations resulting from VH replacements or ongoing somatic mutations in both WM and CLL. We found replacement mutations in the D and/or JH segments in all patients with WM and in 4 of the 10 patients with CLL, including the 2 RS patients. Replacement mutations were clustered in codon 102 of the JH segment. Preferential utilization of the JH4 gene was found in WM (5 of 7 [71.4%]) and in CLL (7 of 10 [70.0%]), and DXP family genes in CLL (5 of 10 [50.0%]). In conclusion, WM and CLL with RS are generated under the influence of antigenic stimulation and selection. However, the majority of CLL may arise from a distinct subpopulation that has the restricted repertoire of nonmutated Ig genes.

Adult↗
PubMed2001

Interleukin 4 content in chronic lymphocytic leukaemia (CLL) B cells and blood CD8+ T cells from B-CLL patients: impact on clonal B-cell apoptosis.

B-chronic lymphocytic leukaemia (CLL) clonal B cells are characterized by resistance to apoptosis. We evaluated clonal B cells and blood T cells for interleukin 4 (IL-4) content as IL-4 is able to increase CLL cell resistance to apoptosis. The content of IL-4 in CD8+ T cells of CLL patients (n = 9) ranged from 37% to 63% of the total CD8+ T cells (mean level of 49% +/- 3.4) compared with a range of 5-10% for control CD8+ T cells. Clonal B cells positive for cytoplasmic IL-4 ranged from 1% to 97% (mean value 57.8 +/- 6.9%). CD8+ T cells and clonal B cells secreted detectable levels of IL-4, but only clonal CLL B cells (n = 4) secreted IL-4 in association with increasing cell numbers. Fludarabine (F-ara-AMP, 0.1-100 micromol/ml) was able to downregulate the IL-4 content of CD8+ T cells, but not clonal B-cell IL-4. Culture supernatant from CLL CD8+ T cells decreased the spontaneous apoptotic rate of clonal B cells that was reversed with anti-IL-4 and soluble IL-4 receptor. These findings show that IL-4 is present in the microenvironment of B-CLL. In addition, use of agents that can interfere with IL-4 presentation to clonal B cells can be effective in increasing clonal B-cell apoptosis.

Antineoplastic Agents↗
PubMedDate not supplied

Alkylator resistance in human B lymphoid cell lines: (1). Melphalan accumulation, cytotoxicity, interstrand-DNA-crosslinks, cell cycle analysis, and glutathione content in the melphalan-sensitive B-lymphocytic cell line (WIL2) and in the melphalan-resistant B-CLL cell line (WSU-CLL).

Two human B lymphoid cell lines WIL2 (melphalan sensitive. ***IC50:8.57 +/- 1.08 mM) and WSU-CLL (melphalan resistant, ***IC50:223.18 +/- 6.45 mM) were used as models to study alkylator resistance in human lymphoid cells. Melphalan transport studies demonstrated decreased initial melphalan accumulation in WSU-CLL cells as compared to WIL2 cells. Lineweaver-Burk plots of the rate of initial melphalan uptake showed an approximately 3.5-fold decrease of Vmax in WSU-CLL cells as compared to WIL2 cells. Melphalan transport was inhibited by 2-amino-bicyclo[2,2,1] heptane-2-carboxylic acid(BCH) in both cell lines, indicating that the amino acid transport (System L, which is sodium independent and inhibited by BCH) is functional in these two cell lines. Only a minor degree of inhibition of melphalan transport was noted after sodium depletion (System ASC, which is sodium dependent and unaffected by BCH). Interstrand-DNA-cross-link formation showed a highly significant correlation with in-vitro cytotoxicity in both two cell lines. However, the melphalan concentration at which such interstrand DNA cross-linking occurred differed significantly when WIL2 cells and WSU-CLL cells were compared. The kinetics of interstrand-DNA-cross-link formation and removal following treatment with melphalan also differed significantly, with WSU-CLL cells, showing a much more rapid rate of removal of interstand DNA cross-links as compared to WIL2 cells. Cell cycle analysis showed that melphalan treatment resulted in the progressive arrest of the WSU cells in G1 and G2 phases. But WIL2 cells failed to enter G1 or G2 arrest after melphalan treatment, suggesting an increased rate of DNA repair occurring in melphalan-resistant WSU-CLL cells. There was no significant difference between the two cell lines in regard to either glutathione content or glutathione-S transferase activity. These findings indicate that multiple factors are associated with alkylator resistance in lymphoid cells including alteration of uptake, DNA repair and cell cycle progression. However no evidence for alteration in glutathione content and glutathione-S-transferase activity was found.

Antineoplastic Agents, Alkylating↗
PubMed2002

Do CLL B cells correspond to naive or memory B-lymphocytes? Evidence for an active Ig switch unrelated to phenotype expression and Ig mutational pattern in B-CLL cells.

Recent work suggests that chronic lymphocytic leukemia (B-CLL) expressing unmutated immunoglobulin V genes could correspond to the proliferation of naive B cells whereas those expressing mutated genes, may correspond to the proliferation of post-germinal center B cells. Current data from gene profiling expression have failed to demonstrate a clear-cut distinction between these two forms of B-CLL disease. In the present study, we have investigated the complete V(H) nucleotide sequence and the presence of RNA transcripts from different C(H) domains in 25 B-CLL patients. Our results demonstrate that: (1) expression of IgD is not related to the mutational frequency and activation of the isotype switch pathway; (2) isotype switch, leading to simultaneous expression at the transcriptional and protein level of IgM, IgD, IgG and IgA, occurs in a small percentage of patients, and (3) different mechanisms such as VDJ duplication and trans-splicing or RNA splicing of long nuclear transcript, could be involved in isotype switch. Our results highlight the difficulty in assigning a normal counterpart to B-CLL cells and raise the possibility that a different B cell development pathway, independent from classical germinal centers, might exist in B-CLL.

Adult↗
PubMed2002

Benzylamide sulindac analogues induce changes in cell shape, loss of microtubules and G(2)-M arrest in a chronic lymphocytic leukemia (CLL) cell line and apoptosis in primary CLL cells.

Given our interest in cyclic nucleotide phosphodiesterase inhibitors in chronic lymphocytic leukemia (CLL), we studied the effects of sulindac sulfone (exisulind), a non- cyclooxygenase-inhibitory end metabolite of the NSAID sulindac that has been reported to inhibit cGMP phosphodiesterases. We focused on a novel benzylamide analogue of sulindac sulfone, CP461, which is in clinical trials as a chemotherapeutic agent. As previously reported for colon carcinoma cell lines, we found that CP461 induced a rise in cGMP levels and blocked cell proliferation in the CLL cell line WSU-CLL. Surprisingly, however, cell cycle analysis revealed that CP461 caused G(2)-M arrest with an EC(50) of 1.1 micro M. G(2)-M arrest was associated with phosphorylation of Bcl2 (but not BAD, Bax, or Bcl-XL): both of these end points were abrogated by treatment with a calcium chelator. Although CP461 induces p53 up-regulation, G(2)-M arrest and Bcl2 phosphorylation were independent of p53. Because microtubule-active drugs such as vincristine also induced G(2)-M arrest and Bcl2 phosphorylation in WSU-CLL, whereas the genotoxic drugs etoposide and doxorubicin did not, we examined the effect of CP461 on microtubules by indirect immunofluoresence microscopy. CP461 eliminated microtubules rapidly, with reduction detected within 30 min of drug treatment. CP461 also induced marked changes in cell shape. Neither sulindac sulfide (a cyclooxygenase inhibitor) nor sulindac sulfone induced G(2)-M arrest, Bcl2 phosphorylation, microtubule disassembly, or cell shape changes. Treatment with 30 micro M CP461 induced greater than 50% apoptosis in 10 of 10 primary CLL leukemic cell samples, whereas the same drug concentration had only marginal effects (14% apoptosis) on whole mononuclear cells. Our work demonstrates that addition of a benzylamide moiety to sulindac compounds results in markedly altered pharmacological properties that may be of use in the therapy of lymphoid malignancies.

Antineoplastic Agents↗