Leena Vilpo

Synthesis, antiprotozoal and anticancer activity of substituted 2-trifluoromethyl- and 2-pentafluoroethylbenzimidazoles.

The synthesis of several halogenated benzimidazoles substituted in position 2 with trifluoromethyl, pentafluoroethyl and 2-thioethylaminodimethyl group is reported. Antiprotozoal and anticancer activity of series of newly synthesized and previously obtained compounds was studied. All of tested bezimidazoles showed remarkable antiprotozoal activity against Giardia intestinalis, Entamoeba histolytica and Trichomonas vaginalis. Of the studied collection of halogenated benzimidazoles the most anticancer-active was the 5,6-dichloro-2-pentafluoroethyl compound, particularly against breast and prostate cancer cell lines.

Frequent loss of the 11q14-24 region in chronic lymphocytic leukemia: A study by comparative genomic hybridization

The genetic basis and molecular pathogenesis of chronic lymphocytic leukemia (CLL) and the molecular mechanisms responsible for its progression remain poorly understood. Here, karyotyping techniques specifically optimized for CLL, comparative genomic hybridization (CGH), and fluorescence in situ hybridization were used to search for CLL‐specific genetic aberrations. CGH and karyotyping both revealed copy number changes in 12 of the 25 CLL cases (48%) analyzed. Loss at 11q emerged as the most common aberration (6 cases), followed by a gain of chromosome 12 (4) and loss at 13q (3). Concordance between CGH and G‐banding was found in 23 of the 25 cases (92%), which is more than reported in a recent similar CGH study of CLL. Owing to the basic differences in G‐banding and CGH, however, their simultaneous clinical application is recommended. The frequent loss of 11q14‐24 suggests that this chromosomal region deserves further attention as a candidate locus involved in the pathogenesis of CLL. Genes Chromosom. Cancer 19:286–290, 1997.

Surface antigen expression and correlation with variable heavy-chain gene mutation status in chronic lymphocytic leukemia

Abstract: Recent studies have demonstrated that B‐cell chronic lymphocytic leukemia (CLL) consists of two clinical entities with either somatically hypermutated (M‐CLL) or unmutated (UM‐CLL) immunoglobulin variable heavy‐chain (VH) regions. In view of the fact that the cellular biology of these two subsets of disease is currently unexplored, we performed an extensive analysis of the surface antigen expression and correlated this with the VH gene mutation status in a cohort of 32 CLL patients. Using polymerase chain reaction amplification and nucleotide sequencing, the VH genes were shown to be mutated in 10 cases (31%) and unmutated in 22 (69%). The expression of 27 surface membrane antigens in peripheral blood leukemic cells was analyzed by flow cytometry, measuring both the percentage of positive cells as well as the geometric mean fluorescence intensity (GMF). Most of the surface membrane antigens (CD5, CD11c, CD19, CD20, CD21, CD22, CD23, CD25, CD40, CD45, VD79b, CD80, CD95, CD122, CD124, CD126, CD130, CD154, IgM, and IgD) showed a similar expression pattern in both UM‐CLL and M‐CLL patients. The similarity of M‐CLL and UM‐CLL, as demonstrated here for the first time with many protein markers, indicates a considerably homogenous phenotype in both subsets. Furthermore, CD27 was strongly expressed in all cases, which may suggest a memory cell phenotype for both M‐CLL and UM‐CLL. More positive cells in the UM‐CLL group were observed regarding CD38, but CD38 was not a good predictor of VH gene mutation status. Seventy percent of the M‐CLL cases, but only 36% of UM‐CLL cases, were Ig‐λ+. The most striking differential expression, however, was observed in the two slicing variants of the common leukocyte antigen CD45, namely CD45RO and CD45RA. CD45RO expression was significantly associated with M‐CLL, whereas the GMF intensity of CD45RA tended to be associated with UM‐CLL. The role of these CD45 splicing variants in the pathogenesis of CLL deserves further investigation.

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

The production of the cytokines interleukin‐6 (IL‐6) and tumour necrosis factor‐alpha (TNF‐α) 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‐α 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‐α production. These results suggest that the varying capacity to produce IL‐6 and TNF‐α may play a role in B‐CLL progression and in clinical manifestations of the disease.

More extensive genetic alterations in unmutated than in hypermutated cases of chronic lymphocytic leukemia

B‐cell chronic lymphocytic leukemia (CLL) is not a uniform disease entity; approximately half of the CLL cases have undergone immunoglobulin VH gene hypermutation, whereas the other half display unmutated VH genes. We investigated genome changes in 12 hypermutated cases (M‐CLL) and 22 unmutated cases (UM‐CLL) by use of comparative genomic hybridization, G‐banding, and multicolor fluorescence in situ hybridization (m‐FISH) after optimal mitogen stimulation and FISH analysis of typical CLL aberrations: 11q deletion, 13q deletion, and trisomy 12. Very high frequencies of aberrations were found in both groups: 82% in UM‐CLL and 83% in M‐CLL. Deletions of 11q and 13q were equally distributed in M‐CLL and UM‐CLL. However, larger aberrations detectable by CGH, trisomy 12, and complex aberrations were less frequent in M‐CLL than in UM‐CLL. These observations led to a hypothesis that unmutated and mutated CLL have different biological Backgrounds, given that large and/or complex chromosomal aberrations and hypermutation of the CLL progenitor cells tend to be mutually exclusive.

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

Abstract: 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.

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.

Chemosensitivity in vitro to 2-chlorodeoxyadenosine and 9-β-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.

Mitogen induced activation, proliferation and surface antigen expression patterns in unmutated and hypermutated chronic lymphocytic leukemia cells

Abstract:  Objectives: To determine whether the immunoglobulin VH gene mutational status has an effect on the activation, proliferation and surface antigen expression of chronic lymphocytic leukemia (CLL) cells when stimulated in vitro. Methods: The proliferation and activation responses of CLL cells were studied in 22‐immunoglobulin gene VH unmutated (UM‐CLL) and 12 hypermutated (M‐CLL) CLL cases in 4‐day cultures. As the mitogen responses have been previously shown to be diverse in CLL, a case‐specific strategy based on optimized mitogen combinations (OMCs) of interleukin‐2 (IL‐2), 12‐O‐tetradecanoylphorbol 13‐acetate (TPA), Staphylococcus aureus Cowan 1 (SAC), and human recombinant tumor necrosis factor alpha (TNF) was applied in cell stimulation. The expression of 23 surface membrane antigens (CD5, CD11c, CD19, CD20, CD21, CD22, CD23, CD25, CD27, CD38, CD40, CD45, CD45RA, CD45RO, CD79b, CD80, CD95, CD124, CD126, CD130, FMC7, IgD, and IgM) was studied by flow cytometry at days 0 and 4. Results: The proliferation and activation responses were similar in UM‐CLL and M‐CLL when OMCs contained IL‐2, TPA or TNF. SAC induced faster proliferation in UM‐CLL than in M‐CLL. OMC stimulation induced preferential down‐regulation of growth‐ promoting cell surface receptors CD5, CD21, and CD124 and preferential up‐regulation of growth‐inhibiting antigen CD80 in M‐CLL. Conclusions: Difference in immunophenotypic evolution of UM‐CLL and M‐CLL can be demonstrated if appropriate matrix signals are provided. The pathways for CD5, CD21, CD124 (IL4R), and CD80 (B7‐1) regulation should be further explored in relation with somatic hypermutation and outcome of CLL.

Cryopreserved chronic lymphocytic leukemia cells analyzed by multicolor fluorescence in situ hybridization after optimized mitogen stimulation

We investigated the utility of multicolor in situ fluorescence hybridization (mFISH) on cryopreserved blood cells from 11 chronic lymphocytic leukemia (CLL) patients. The results demonstrate that an individually chosen optimized mitogen combination induces proliferation of neoplastic B‐cells after cryopreservation. Abnormal cells were detected in eight samples by mFISH, and, in six samples, the abnormality could be verified by comparative genomic hybridization or interphase FISH. In addition to typical CLL abnormalities, such as del(11q) or +12, several balanced translocations and single‐cell abnormalities were found. Thus, mFISH can reveal new prognostically relevant chromosome aberrations in CLL.