Synthesis and anticancer activity of 5'-phthaloylnucleosides.

A series of novel 5'-phthaloylnucleosides were synthesized via Mitsunobu reaction starting from AZT or 2'-deoxyadenosine and numerous phthalimides and their sulphur analogues-thioimides. Some of them showed significant anticancer activity.

Surface membrane antigen expression changes induced in vitro by exogenous growth factors in chronic lymphocytic leukemia cells.

The factors determining the growth and survival of cells in B chronic lymphocytic leukemia (CLL) have remained poorly understood. We investigated the effects of optimal mitogen combinations (OMCs) on the expression of 26 surface membrane antigens among 33 CLL patients. The seven OMCs used were selected after pre-testing 14 combinations of (1) S. aureus Cowan I (SAC), (2) interleukin-2 (IL-2), (3) tumor necrosis factor alpha (TNF-alpha) and (4) 12-O-tetradecanoylphorbol 13-acetate (TPA; also known as phorbol 12-myristate 13-acetate or PMA). In flow cytometry we revealed that OMCs induced statistically highly significant upregulation of the expression of CD5, CD11c, CD19, CD22, CD23, CD25, CD38, CD40, CD45, CD45RO, CD95, CD126, CD130 and FMC7, and downregulation of CD20 and CD124 expression. Interestingly, the expression of CD27, CD45RA, CD79b, CD80, CD122 and that of the immunoglobulin gene superfamily members CD21, Ig-kappa, Ig-lambda, Ig-delta and Ig-micro were not significantly affected under similar conditions. The expression of several antigens was co-regulated, suggesting common regulatory pathways. These antigens include CD11c/CD5, CD11c/CD22, CD11c/CD126, CD11c/FMC7 as well as CD27/CD45, CD27/CD45RA and CD27/CD79b. Upregulation of surface antigen expression, induced by OMCs, should be applicable in antibody therapy in vitro and in vivo, and in negative stem cell selection for autotransplantation. Furthermore, the current strategy to enhance cell surface antigen expression may be a versatile tool to raise humoral and cell-mediated host defense against CLL cells. Upregulation of proteins mediating positive growth signals (eg CD25, CD40) and negative signals or apoptosis (eg CD95) may be used to sensitize cells to chemotherapy and programmed cell death.

Surface antigen expression in chronic lymphocytic leukemia: clustering analysis, interrelationships and effects of chromosomal abnormalities.

Chronic lymphocytic leukemia (CLL) is a phenotypically distinguishable form of B-lymphoid leukemias. The regularity of surface membrane antigen expression patterns, their interrelationships as well as the effects of the three frequent chromosomal aberrations, ie 11q deletion, 13q deletion and trisomy 12, were investigated in 35 classic CLL cases by flow cytometry. The two-way cluster analysis of 31 individual antigens revealed three expression patterns: (1) most cells in most cases positive (CD5, CD19, CD20, CD23, CD27, CD40, CD45, CD45RA); (2) most cells in most cases negative (CD10, CD14, CD34, CD122, CD154, mIgG); and (3) a mixed pattern with a variable number of positive cases and a variable percentage of positive cells in individual cases (CD11c, CD21, CD22, CD25, CD38, CD45RO, CD79b, CD80, CD95, CD124, CD126, CD130, FMC7, mIgD, mIgkappa, mIglambda, mIgM). The expressions of several antigens were strongly interdependent, even when antigens belonged to entirely different gene families. Such antigen pairs were: CD11c/CD21; CD19/CD45; CD19/CD79b; CD22/CD45RA; CD23/Igkappa; CD25/mIgM; CD27/CD45; CD45/CD79b; CD45RA/Igkappa. In contrast, the expression of some antigens was mutually exclusive, the best examples being CD45RA/CD45RO, CD38/CD80 and CD45RA/CD80. Deletion of chromosome arm 11q attenuated expression of splicing variant CD45RA, but enhanced CD45RO expression. In contrast, cases of trisomy 12 were associated with enhanced CD45RA and attenuated CD45RO expression. Similarly, trisomy 12 was associated with enhanced CD27 and mIgkappa expression. The variable levels of signaling surface membrane antigens, their interactions and interference by genetic aberrations are likely to affect the clinical progression and drug response of CLL.

Distinct gene expression profiling in chronic lymphocytic leukemia with 11q23 deletion.

Chronic lymphocytic leukemia (CLL) is a heterogeneous disease with regard to its clinical course. The limitations of the methods currently available for prognostic assessment in CLL do not allow accurate prediction of the risk of disease progression in individual patients. The recently developed cDNA array technique provides a unique opportunity to study gene expression in various malignancies. To identify new molecular markers for prognostication of CLL patients, we analyzed cDNA arrays by using hierarchical clustering and standard statistic t-test on 34 CLL patients. We found significant expression differences in 78 genes compared to the reference tonsillar B lymphocytes. A cluster of genes, LCP1, PARP, BLR1, DEK, NPM, MCL1, SLP76, STAM, HIVEP1, EVI2B, CD25, HTLF, HIVEP2, BCL2, MNDA, PBX3, EB12, TCF1, CGRP, CD14, ILB, GZMK, GPR17 and CD79B, was associated (P < 0.05) with the unfavorable 11q deletion and also with the unfavorable Binet stages B and C. We present here gene expression profiling that is associated with CLL patients with the 11q23 deletion. Many of the genes in the cluster have not previously been shown to be related to the initiation or progression of CLL. These novel findings provide fundamental information for further attempts to understand the interaction of the clustered genes in the leukomogenesis of CLL in order to better design treatments aimed at specific molecular target(s).

Cyclosporin A-induced cell kill in vitro in various clinical-hematological types of B-cell chronic lymphocytic leukemia.

We investigated whether a known T cell modulator, Cyclosporin A (CyA) is also toxic to chronic lymphocytic leukemia (B-CLL), in vitro. In contrast to seven other drugs and two types of irradiation the dose-response curves for CyA were very steep among the 36 CLL patients investigated, and the intraindividual variation of ID(80) values was remarkably lower. The mode of CyA-induced cell death was 'apoptotic-like' as indicated by flow cytometric analysis, revealing cell condensation and annexin positivity. The partially smeary DNA fragmentation pattern together with the relatively slow process of cell death revealed a distinctive pattern of cell death in CLL. Leukemia cells from patients at an advanced clinical stage, of a diffuse histologic type and showing fast progression were the most sensitive to CyA. These new observations may have therapeutic implications in CLL.

Calcium antagonists potentiate P-glycoprotein-independent anticancer drugs in chronic lymphocytic leukemia cells in vitro.

BACKGROUND AND OBJECTIVES: A major obstacle to the successful use of chemotherapy in the treatment of leukemia and other cancers is the emergence of drug resistance. One of the most studied resistance mechanisms is mediated by P-glycoprotein, which can be modulated by calcium channel blockers. Here we investigated whether the Ca(2+) channel blockers verapamil and nifedipine are toxic alone and in combination with P-glycoprotein-independent anticancer drugs against chronic lymphocytic leukemia (CLL) cells in vitro. DESIGN AND METHODS: Verapamil cytotoxicity was investigated in peripheral blood samples of 35 patients with B-cell CLL and 10 healthy control subjects. Cytotoxicity was assessed in in vitro 4-day cultures using 14C-leucine incorporation as an indicator of cell viability. Interactions were tested with Ca2+ channel blockers and cyclosporine or 7 anticancer drugs: (i) chlorambucil, (ii) 2-chlorodeoxyadenosine, (iii) cisplatin, (iv) fludarabine, (v) prednisolone, (vi) adriamycin, and (vii) vincristine. The mode of cell death was assessed by annexin binding and DNA ladder formation. RESULTS: Verapamil induced dose- and time -dependent death of CLL cells in vitro. A statistically significant effect (p = 0.0085) was noted with as little as 4 microM verapamil. The mode of cell death was apoptotic as determined by annexin positivity and condensation of verapamil-treated cells. Verapamil effectively potentiated the toxicity of cyclosporine and all the anticancer drugs mentioned above. Furthermore, nifedipine, a more specific L-type calcium channel antagonist, significantly potentiated the effect of chlorambucil against CLL cells. Interpretation and Conclusions. Calcium channel blockers enhance the effect of P-glycoprotein-independent anticancer drugs remarkably. This indicates that the death signals initiated by calcium depletion and anticancer drugs together facilitate cell death. This novel finding opens a new avenue to modulate, by using calcium channel antagonists, the effect of traditional anticancer drugs having different mechanisms of P-glycoprotein-independent action.

Complex chromosomal aberrations in chronic lymphocytic leukemia are associated with cellular drug and irradiation resistance.

Drug resistance is a major problem in chemotherapy of chronic lymphocytic leukemia (CLL). The genetic basis and molecular pathogenesis of drug resistance in CLL remain poorly understood. Here, we have investigated the association between chromosomal aberrations and cellular resistance of CLL cells against seven drugs, gamma and ultraviolet irradiation. Samples were obtained from 35 patients having a classical form of B-CLL. Chromosomal aberrations were first analyzed by traditional karyotyping improved by using optimized mitogen combinations. DNA sequence copy number changes throughout the genome were next screened by comparative genomic hybridization. Finally, fluorescence in situ hybridization was used to detect trisomy 12 and loss of Rb and deletions at chromosome 11. The cellular sensitivity in vitro was assessed by the reduction of macromolecular protein synthesis measured as incorporation of radioactive L-leucine as an endpoint. The overall analysis disclosed a statistically highly significant difference in cellular drug resistance between patients having at least three aberrations compared with patients with fewer or no aberrations. This strongly indicates that complex rather than simple molecular mechanisms are responsible for the drug and irradiation resistance in CLL. According to published results, complex aberrations are constantly associated with poor prognosis in CLL. We demonstrated here that complex chromosomal aberrations were associated with cellular irradiation and drug resistance, which, on the other hand, may be responsible for the poor clinical outcome in CLL.

Interleukin-1 beta, interleukin-1 receptor antagonist and interleukin-6 plasma levels and cytokine gene polymorphisms in chronic lymphocytic leukemia: correlation with prognostic parameters.

BACKGROUND AND OBJECTIVE: The growth of B-cell chronic lymphocytic leukemia (B-CLL) cells has been shown to be dependent on exogenous growth factors in vitro. We wanted to evaluate the clinical relevance of interleukin (IL)-6, IL-1 beta and interleukin-1 receptor antagonist (IL-1Ra) in B-CLL. As the plasma levels of IL-6, IL-1 beta and IL-1Ra have been suggested to be partly dependent on gene polymorphism, the previously described polymorphisms of the IL-1 complex genes and the IL-6 gene were also studied. DESIGN AND METHODS: The plasma levels of these cytokines were measured in a cohort of 36 patients with B-CLL and in 400 healthy subjects. The previously described polymorphisms of the IL-1 complex genes and the IL-6 gene were studied using PCR and RFLP. These data was correlated with other parameters associated with severity and prognosis of B-CLL and a number of clinical and laboratory findings. RESULTS: The plasma concentrations of IL-1 beta and IL-1Ra were lower in B-CLL patients than in normal controls (p < 0.001). The IL-1 beta plasma levels were dependent on the cell immunophenotype score and state of progression of the disease. Moreover, plasma concentrations of IL-6 were elevated in B-CLL patients compared with healthy subjects (p < 0.005) and correlated with disease stage, hemoglobin levels, anemia and erythrocyte sedimentation rate in the patients. The allele frequencies of the analyzed genes were similar in patients and controls. INTERPRETATION AND CONCLUSIONS: Our data demonstrate that in B-CLL, plasma levels of IL-1 beta, IL-1Ra and IL-6 differ from normal, and mechanisms other than allelic imbalance of their genes account for the distinct cytokine profiles observed in this disease.

Selective toxicity of vincristine against chronic lymphocytic leukemia cells in vitro.

The cytotoxicity of vincristine in vitro was investigated in B chronic lymphocytic leukemia (CLL) cells and in normal peripheral blood mononuclear cells. An approximately 25-fold selectivity towards leukemic vs. normal lymphocytes was demonstrated. Cells from patients having a mature subtype (CLL or CLL/mix) or a slowly progressing form of CLL were significantly more sensitive to vincristine in vitro than the cases with a CLL/PL phenotype or faster-progressing disease. Depending on the vincristine dose, the number of dead CLL cells accumulated slowly during the 4-d observation period. Our data indicate a marked individual variation in vincristine susceptibility among individual CLL cells. Vincristine induced annexin positivity, nuclear blebbing and DNA fragmentation in CLL cells. These indicate an "apoptosis-like" cell death. Since CLL cells are in the G0/G1 phase of the cell cycle, the only known mode of anticancer action of vinca alkaloids, i.e. anti-mitotic action, cannot explain the death of CLL cells. Furthermore, similar cellular uptake and efflux of vincristine by normal and CLL cells excluded pharmacokinetic differences as a cause of selectivity of vincristine towards leukemic lymphocytes. Immunostaining of filamentous structures of CLL cells revealed that vincristine brings about selective changes in alpha-tubulin but not in beta-actin or vimentin. Although the antitubulin action of vinca alkaloids in the biochemical sense is well demonstrated, this kind of anticancer effect has not previously been shown. Vincristine is used in several regimens for CLL, but its efficacy in CLL has never been demonstrated in a clinical context and its value in routine CLL chemotherapy has been questioned. The present data strongly support the need for further evaluation of the role and mode of action of vincristine in chemotherapy of CLL and other cancers as well.

Kinetics of excision repair of UV-induced DNA damage, measured using the comet assay.

The kinetics of UV- (254 nm) irradiation-induced DNA single-strand breaks (SSBs), generated during the excision repair of UV-induced DNA damage, in leukemic lymphocytes and in normal blood mononuclear cells (MNCs) were studied using the alkaline comet assay. The cells were isolated by density gradient centrifugation from peripheral blood of patients with chronic lymphocytic leukemia (CLL) and from healthy study subjects. The cytotoxicity of UV irradiation was determined in vitro in peripheral blood mononuclear lymphocytes from 36 CLL patients and from eight healthy donors using the incorporation of radioactive leucine in 4-day cultures. A remarkable difference in excision repair capability was observed between normal and leukemic lymphocytes. In contrast to normal lymphocytes, there was always a subpopulation of CLL cells that did not complete the repair of UV-induced DNA damage during the 24-h repair period. Furthermore, differences were also recorded between UV-sensitive and UV-resistant CLL cases. The differences in DNA migration between the maximum increase (59-77 microm) and that at 24 h after irradiation (21-66 microm) was statistically significant in two of three patients exhibiting UV-resistance. Correspondingly, only in one of three patients exhibiting UV-sensitivity was the difference in DNA migration statistically significant (maximum increase: 44-107 microm, vs. 24 h after: 42-100 microm). Our results confirm an abnormal pattern of the CLL cell response to UV irradiation. Furthermore, we identified defective processing of UV-induced DNA damage in CLL versus normal lymphocytes, particularly in UV-sensitive cases.

Induction of beta-2-microglobulin release in vitro by chronic lymphocytic leukaemia cells: relation to total protein synthesis.

The in vitro production of beta2-M by B-CLL cells from 27 patients was investigated. In all cases, low spontaneous beta2-M release was observed. The production of beta2-M was enhanced to various extents when induced with 13 different stimulants and their combinations including IL-2, TNFalpha, SAC and TPA. Beta2-M release was 3.8-fold (range from 1.9 to 9.2-fold) in cultures stimulated with TPA (10 ng/ml), compared with the spontaneous release, and even faster if TNFalpha or IL-2 were added. A strong correlation was revealed between beta2-M production and the total protein synthesis of leukaemic cells when the latter was assessed using 14C-L-leucine incorporation. Hence, both beta2-M release and leucine incorporation are promising activation markers for CLL B-lymphocytes.

Opsonising immunoglobulins and mannan-binding lectin in chronic lymphocytic leukemia.

We investigated the influence of plasma concentrations of immunoglobulin G (IgG), IgA, IgM, IgG subclasses and mannan-binding lectin (MBL) on susceptibility to infection in patients with chronic lymphocytic leukemia (CLL). Of 28 patients with CLL, increased susceptibility to infection was recorded in nine. Four of them (44%) had hypogammaglobulinemia as opposed to only one (5%) of the 19 patients without increased susceptibility to infection (OR 14.4; 95% CI, 1.6-130). When the effect of IgG subclasses contributing to hypogammaglobulinemia was studied, only the decreased concentrations of IgG4 and IgG2 were associated with increased susceptibility to infection. They, in turn, were intercorrelated and also highly correlated with the concentration of IgA. In fact, when these parameters were studied by a multivariable model, only the decreased concentration of IgA was shown as an independent risk factor for infection (P = 0.03). The mean concentration of MBL was significantly higher in patients with infections than in those without (6.54 mg/l and 2.75 mg/l, respectively; P = 0.001). The monitoring of its concentrations might be useful in the follow-up of infectious morbidity in CLL.

Gamma-irradiation-induced DNA single- and double-strand breaks and their repair in chronic lymphocytic leukemia cells of variable radiosensitivity.

Gamma-irradiation-induced DNA single- and double-strand break (SSB and DSB) formation and their repair kinetics in normal hematopoietic cells and in leukemic lymphocytes was investigated using alkaline and neutral comet assays. The cells were isolated by density gradient centrifugation from peripheral blood of patients with chronic lymphocytic leukemia (CLL) and from healthy study subjects. Furthermore, CD34+ progenitor cells isolated with immunomagnetic beads from bone marrow of non-leukemic persons were investigated. The cytotoxicity of 137Cs irradiation was determined in vitro in peripheral blood mononuclear lymphocytes from 36 CLL patients and from 8 healthy donors using radioactive leucine incorporation assay in 4-day culture. A dose-dependent increase in DNA migration was observed in alkaline (SSBs) and neutral (DSBs) gel electrophoresis when the cells were exposed to gamma-irradiation doses up to 10.4 Gy. After irradiation with doses of 2.4 and 5.4 Gy, the cells repaired their single- and double-strand breaks almost completely. The formation and repair of DNA strand breaks were essentially similar in all normal cell populations investigated and in CLL cells. The gamma-irradiation-induced cytotoxicity did not correlate with DNA strand break formation and repair capacity. According to these results, the differences of gamma-irradiation tolerance among individual CLL cases and among healthy persons are explicable in terms other than DNA strand break formation or repair.

Discontinuous deletions at 11q23 in B cell chronic lymphocytic leukemia.

Loss of genomic material in 11q is one of the most common structural chromosome aberrations in B cell chronic lymphocytic leukemia (B-CLL). In order to characterize the deletions of 11q23 in B-CLL, we performed fluorescence in situ hybridization (FISH) with eight YAC (yeast artificial chromosome) probes on peripheral leukocytes of 30 patients. These YACs form a contig spanning 7.8 Mb at 11q23.1-q23.3. We found deletions in nine out of 30 cases (30%) and five of them had discontinuous deletions in this region. The region represented by YAC 755b11 (1.6 Mb in size) was involved in all cases with deletions, supporting the hypothesis that this region might contain a novel gene of pathogenic importance to B-CLL. A more distal region represented by YAC 785e12 (760 kb in size) was deleted frequently and specifically. Whether there is another novel gene of pathogenic importance to B-CLL and what is its potential relationship to the deletions in the region represented by YAC 755b11, are issues that require further studies.

Chemosensitivity in vitro to 2-chlorodeoxyadenosine and 9-beta-D-arabinofuranosyl-2-fluoroadenine in previously unexposed cases of chronic lymphocytic leukemia.

The chemosensitivity of leukemia cells, obtained from the peripheral blood of 35 patients with B-cell chronic lymphocytic leukemia, was determined by a leucine-incorporation assay in vitro. There was good correlation between the sensitivities to two purine analogs, 2-chlorodeoxyadenosine and 9-beta-D-arabinofuranosyl-2-fluoroadenine among previously untreated patients when tested at the 80% inhibition level. Previous exposure to chlorambucil did not affect the sensitivity to these compounds.

Optimized mitogen stimulation induces proliferation of neoplastic B cells in chronic lymphocytic leukemia: significance for cytogenetic analysis.The Tampere Chronic Lympocytic Leukemia group.

We tested the effects of interleukin-2 (IL-2), human recombinant tumor necrosis factor alpha (TNF-alpha), Staphylococcus aureus Cowan I (SAC), TPA, and their combinations, using a standard thymidine incorporation assay, in order to identify an optimal mitogen combination (OMC) for 24 consecutive patients with B-cell chronic lymphocytic leukemia (B-CLL). The combination that induced the highest thymidine incorporation was chosen as the OMC for each patient. Among 14 mitogen combinations tested, there were six different OMCs, of which the most frequent was TNF-alpha + IL-2. It was the OMC in 9 of 24 cases. The other OMCs were TNF-alpha + TPA1 (5/24), SAC + IL-2 (5/24), TPA1 + IL-2 (3/24), TPA10 + IL-2 (1/24), and TNF-alpha + TPA10 + IL-1 (1/24). The mitogenic power of the selected OMC in each case was then evaluated both by the combination of immunophenotyping and molecular cytogenetic techniques known as MAC (Morphology, Antibody, Chromosomes) and standard chromosome analysis. After OMC stimulation, the levels of DNA synthesis and B-cell proliferation (mitotic index) were, on average, 10-fold higher than those observed after standard TPA stimulation (P < 0. 0001). The proportion of mitotic B cells exceeded the proportion of mitotic T cells in 70.1% of the cases after OMC stimulation. After TPA stimulation, 7.7% +/- 2.5% of all mitoses were B-cell mitoses, whereas after OMC stimulation this proportion rose to 57.9% +/- 5.3%. The frequency of clonal chromosomal aberrations increased from 46% after TPA stimulation to 79% after OMC stimulation. The clonal aberrations del(6q), del(11q), and/or del(13q) were observed in 26%, 32%, and 42% of the patients with the respective clonal chromosomal aberrations, whereas the corresponding frequencies after TPA stimulation were only 4%, 21%, and 17%. When the lineage involvement of cells with clonal chromosomal aberrations from three patients was analyzed, the aberrations were found to be restricted to B cells only, and in one patient to a minor subset of B cells. The results demonstrate that an individually chosen OMC induces a high rate of proliferation in neoplastic B cells. We found deletions in 6q, 11q, and 13q at higher frequencies than reported previously, most probably as a result of an improved mitogenic response. The identification of an optimal mitogen stimulation for each patient, prior to chromosome analysis, can well be expected to reduce the rate of false-normal results in the future. This is essential for accurate evaluation of the prognostic significance of chromosomal aberrations in B-CLL.

Diminished production of interleukin-6 in chronic lymphocytic leukaemia (B-CLL) cells from patients at advanced stages of disease. Tampere CLL Group.

The production of the cytokines interleukin-6 (IL-6) and tumour necrosis factor-alpha (TNF-alpha) in B-CLL cells from 24 patients at different stages of chronic lymphocytic B-cell leukaemia (B-CLL) was investigated in vitro. In the majority of these cases, low spontaneous IL-6 production was measured. Mitogenic stimulation with phorbol 12-myristate 13-acetate (PMA) or PMA plus interleukin-2 (IL-2) resulted in a tremendous increase in TNF-alpha and IL-6 production in cells representing early stage (Binet A) disease. In contrast, very little, if any, production took place in cells from patients with advanced stage (Binet C) B-CLL. The results from stage B patients were intermediate. The most remarkable difference was recorded in PMA-stimulated (1 ng/ml) IL-6 production. In stimulated 72 h cultures, IL-6 concentrations were 1280 +/- 1080 pg/ml for Binet A (n = 11), 757 +/- 597 pg/ml for Binet B (n = 8) and 46.0 +/- 84.0 pg/ml for Binet C (n = 5). The differences in IL-6 production between stage C v B and stage C v A were both statistically significant (P=0.025). Similar effects, but to a lesser extent, were observed in TNF-alpha production. These results suggest that the varying capacity to produce IL-6 and TNF-alpha may play a role in B-CLL progression and in clinical manifestations of the disease.