Histone deacetylase inhibitors in plasma cell leukemia treatment: effect of bone marrow microenvironment.

In the presented study we analysed the effect of histone deacetylase inhibitors (HDACi) suberoylanilide hydroxamic acid (SAHA) and valproic acid (VPA) on human plasma cell leukemia (PCL) cell line UHKT-944 in the presence of bone marrow microenvironment (BMM). For the analysis, the cells were cultured alone, with bone marrow stromal cells (BMSCs), with extracellular matrix (ECM) components or with interleukin-6, and treated with varied concentrations of SAHA and VPA for 24/48 hours. To study the effect of HDACi, we investigated cell proliferation, apoptosis, cell cycle and changes in selected signalling pathways. We found that both SAHA and VPA induced apoptosis, but had no effect on the cell cycle distribution of UHKT-944 cells. Investigation of the antiproliferative effect of SAHA and VPA revealed that BMSCs and high concentration of interleukin-6 had partial protective effect against SAHA or both inhibitors, respectively. No effect of ECM components on the efficiency of HDACi was observed. We further revealed that VPA down-regulated STAT3 phosphorylation while both inhibitors decreased Akt phosphorylation. In conclusion, VPA and SAHA might represent an additional therapeutic strategy in the PCL treatment. Protective effect of BMM should be taken into account when investigating prospective therapeutic agents against plasma cell disorders.

Epigenetic modulation of gene expression of human leukemia cell lines - induction of cell death and senescence.

Histone deacetylase inhibitors (HDACi) are emerging new class of anticancer agents that act by inhibiting cell growth, inducing cell cycle arrest and apoptosis of various cancer cells. However, in some conditions, apoptosis can be blocked and non apoptotic cell death and irreversible growth arrest, namely senescence, can be activated as potential tumor-suppressor mechanism. Here we evaluated the dosage effects of HDAC inhibitors suberoylanilide hydroxamic acid (SAHA) and valproic acid (VPA) in a series of human leukaemia cell lines. We investigated, what concentration of SAHA and VPA can optimally induce apoptosis, growth inhibition or stress-induced premature senescence. We have found that SAHA inhibited proliferation and induced apoptosis in concentration 1000x lower than VPA. The senescence phenotype was preferentially induced by lower dosage of HDACi and required longer incubation time (5 days) while apoptosis was induced by higher dosage and appeared already after 24h. The optimal doses for the induction of cell death are 2,5-5 µM of SAHA and 2,5-5 mM of VPA. These doses of HDACi induce both apoptosis and senescence of studied leukemia cell lines.

Growth inhibitory effect of the antibody to hematopoietic stem cell antigen CD34 in leukemic cell lines.

Growth-inhibitory and proapoptotic effects of the monoclonal antibody to CD34 molecule, clone 4H11, were tested in CD34+ leukemic cell lines (MOLM-9, JURL-MK1, HEL) and CD34- cell lines (PS-1, ML-2 and CTV-1). We have found that the monoclonal antibody to CD34 inhibited the proliferation and induced apoptosis of all CD34+ cell lines. We did not observe induction of differentiation by anti-CD34 antibody, but a growth arrest of cells in the G0/G1 phase of the cell cycle was detected in all the cell lines studied. Combinations of anti-CD34 antibody with both type I (alpha, beta) or type II (gamma) interferons did not enhance the effects on the cell growth or inhibition of cellular proliferation of the antibody alone. Our data suggest that the monoclonal antibody to CD34 molecule prepared from clone 4H11, after sufficient experimental and preclinical testing on laboratory animals, may provide a new basis for targeted antibody therapy of acute or chronic myeloid leukemia.

Semiquantitative RT-PCR evaluation of the MDR1 gene expression in patients with acute myeloid leukemia.

Resistance to chemotherapy is one of the major obstacles to effective treatment in acute myeloid leukemia (AML). The most extensively studied protein involved in multidrug resistance (MDR) is the transmembrane glycoprotein P (P-gp), the product of the multidrug resistance gene 1 (MDR1). MDR1/P-gp overexpression is frequently observed in hematological malignancies, especially in acute leukemia, and has been reported to correlate with poor prognosis in acute myeloid leukemia (AML). The aim of this study was to evaluate the level of MDR1 gene expression in bone marrow and/or peripheral blood samples in 92 AML patients in relation to their prognosis. The analyzed group was stratified according to presence or absence of prognostically favorable aberrations (PFAs), such as t(15;17) with PML/RARalpha fusion gene, t(8;21) with AML1/ETO fusion gene or inv(16)/ t(16;16) with CBFbeta/MYH11 fusion gene. These prognostically favorable aberrations were detected by RT-PCR and/or standard cytogenetic techniques. MDR1 expression was detected by semiquantitative comparative RT-PCR using software-based evaluation. The levels of MDR1 expression in the bone marrow predicted induction of complete remission in the whole group of analyzed patients (P = 0.032). They were significantly lower in PFA negative patients who achieved complete remission compared to those who failed to achieve complete remission (P = 0.008). In PFA negative patients, MDR1 expression was higher when compared to PFA positive patients (P = 0.055). No such difference was found when analyzing peripheral blood samples. Our experiments showed no impact of MDR1 expression in bone marrow or peripheral blood cells on overall survival (P = 1.000 and P = 0.903 respectively). In summary, the present study shows the prognostic impact of MDR1 expression on induction of complete remission in AML patients. We confirmed that MDR1 overexpression is an unfavorable prognostic factor in AML, which may help to stratify the risk rate of PFA negative patients. In future studies, quantitative detection of MDR1 expression might be a valuable tool to predict prognosis in this patient subset.

Intranucleolar translocation of AgNORs in early granulocytic precursors in chronic myeloid leukaemia and K 562 cells.

The present study was undertaken to provide missing information on the distribution of AgNORs in large nucleoli of human leukaemic early granulocytic precursors in vivo as well as in vitro. In vivo, the distribution of AgNORs was studied in early granulocytic precursors of patients suffering from chronic myeloid leukaemia who were both untreated and treated with imatinib mesylate. AgNORs were visualized by silver reaction under conditions which facilitated to see their distribution by light microscopy. In vitro, the distribution of AgNORs was studied in proliferating and ageing K 562 cells which originated from chronic myeloid leukaemia. In vitro, the ageing of K 562 cells produced intranucleolar translocation of AgNORs to the nucleolar periphery. Such translocation was also observed in some leukaemic early granulocytic precursors in vivo, e.g. in bone marrow myeloblasts and promyelocytes of leukaemic patients. As was expected, the intranucleolar translocation of AgNORs in early granulocytic precursors was more frequent in patients treated with the cytostatic therapy--imanitib mesylate. The abovementioned findings suggest that myeloblasts and promyelocytes with AgNORs translocated to the periphery of large nucleoli might be in the ageing state, similarly as blastic cells of leukaemic myeloid origin (K 562 cells) in ageing cultures. Thus, the translocation of AgNORs might be a useful marker of premature ageing in the future and might contribute to the evaluation of the single cell state under various experimental as well as clinical conditions. However, more clinically oriented studies are required in this direction.

An antigen recognized on cells in apoptosis detected by monoclonal antibody 2E12.

Monoclonal antibody 2E12 was prepared by immunization of mice with cells of a chronic myeloid leukemia cell line MOLM-7. Human hematopoietic cell lines JURKAT, HPB-ALL, RC2A and MOLM-7 were induced to receptor mediated apoptosis by the treatment with anti-Fas monoclonal antibody 7C11 and subsequently tested for reactivity with 2E12 antibody in comparison to staining with annexin V-FITC and PI in the two-color immunofluorescence and flow cytometry. After 2, 5, 24, and 48 hours of induction, a gradual increase of the percentage of 2E12 positive cells in all cell lines was observed, which partially correlated with an increase of annexin V-FITC binding with a delay of about 12 hours. In the two- color fluorescence microscopy the 2E12 antibody positivity was restricted to the annexin V positive cells, but their number was lower. The binding of 2E12 did not induce apoptosis nor influenced the binding of annexin V. We suppose that the antibody 2E12 detects an antigen expressed on a subpopulation of cells in death. Therefore it can be useful as a new marker for further dissection between living, apoptotic and necrotic cellular populations in vitro.

[Hematopoietic cell lines and their importance in studies of leukemia and lymphoma etiopathogenesis]. / Hematopoetické bunecné linie a jejich význam pro studium etiopatogeneze leukémií a lymfomu.

Hematopoietic cell lines phenotypically and genotypically corresponding to original patient's leukemic or lymphoma cells are the powerful tools for studies of many aspects of malignant process and also for studies of normal and malignant hematopoesis. Until recently, permanent cell lines were derived from nearly all types of hematopoietic malignancies--leukemias and lymphomas. Availability of leukemia and lymphoma cell lines enabled the development of hybridoma technology for production of monoclonal antibodies and also discovery of the first human retroviruses HTLV-1,2 and HIV. Last but not least, leukemic cell lines are also used as standards for molecular diagnostics of leukemias and lymphomas. Leukemic and lymphoma cell lines have fully proved and extended their importance for modeling of malignant process ex vivo.

[Long-term culture of plasma cells from patients with myeloma and establishment of a permanent cell line]. / Dlouhodobá kultivace plazmatických bunek nemocných s myelomem a moznosti získání permanentních bunecných linií.

UNLABELLED: Multiple myeloma plasma cells are actively dividing cells with the long surviving ability in the ex-vivo culture. In the effort for better understanding of the proliferative potential of malignant myeloma cells and establishment of permanent myeloma cell lines we performed long term cultures of human myeloma cells ex-vivo. During the last two years we cultured 41 bone marrow samples from 39 patients with multiple myeloma. Cells were cultured in the RPMI 1640 culture medium with 15% fetal calf serum at 37 degrees C in 5% CO2 and approximately one third of the culture medium was changed regularly twice a week. Most of the marrows cultures died by apoptosis within 30 days. Four bone marrow samples were cultured for more than 11 months, however, no culture can be qualified as an established cell line. In three cases permanent B-lymphoblastoid cell lines were established (UHKT-55, UHKT-56 a UHKT-57) but secondary immortalization by Epstein-Barr virus was suggested. CONCLUSIONS: Presented results suggest that myeloma plasma cells can survive and are able to proliferate in the ex-vivo culture for several months up to one year independently on the addition of any external growth factor without spontaneous apoptosis or necrosis. The probability of the establishment of a permanent cell line of plasma cell origin is, however, low. Presence of accessory bone marrow cells was the most important factor for the long-term survival of myeloma cells.

Aleukemic granulocytic sarcoma with AML1/ETO fusion gene expression and clonal T cell populations.

We report a unique case of aleukemic granulocytic sarcoma of the neck, originally misdiagnosed as non-Hodgkin's lymphoma (NHL), though chloroma was also suspected due to a greenish macroscopic appearance and the presence of myeloid chloroacetate esterase (CAE)+ cells. The proof of clonal T cell receptor gamma chain (TcRgamma) gene rearrangements in the recurring tumor was deemed to confirm the initial diagnosis of T cell NHL. Altogether five distinct types of clonal TcRgamma gene rearrangements were found in the tumor, bone marrow and peripheral blood. Only retrospectively, using RT-PCR, did we detect the acute myeloid leukemia subset-specific fusion gene AML1/ETO in the frozen samples of the relapsed tumor, as well as in the otherwise unaffected bone marrow and peripheral blood (representing 'minimal initial disease' in the latter two samples). Simultaneous staining verified that the neoplastic CAE+ cells and CD45RO+ T cells were different populations.

Antitumor action of bovine seminal ribonuclease.

Unlike the bovine pancreatic ribonuclease (RNaseA), bovine seminal ribonuclease (BS RNase) displays various biological activities, including antitumor activity, immunosuppressivity, spermatogenicity and embryotoxicity. To learn more about its antitumor effect we tested BS RNase on the growth of 16 cell lines derived from patients with various hematological malignancies. The cells of lymphoid origin were generally more susceptible to BS RNase, administered in the range of concentrations from 2 to 100 micrograms/ml, than the myeloid ones. RNaseA used at the same concentrations did not exert any inhibitory effect. The inhibitory effect of BS RNase persisted in cultured cells after three times wash in complete medium and cell recultivation in fresh medium free of BS RNase. Four cell lines were very little sensitive (KG-1 and U-937) or resistant (JOK and NAMALWA) to BS RNase regardless of their origin. The in vivo antitumor effect of BS RNase was tested on human prostate carcinoma transplanted to athymic nude mice. The daily dose of BS RNase (0.25 mg/20 g) was administered for three weeks except weekends (15 doses) by three different ways (intraperitoneally-i.p., subcutaneously-s.c. and intratumorally-i.t.). Whereas i.p. administration was ineffective, s.c. administration significantly reduced size of the tumors and i.t. administration abolished half of the tumors in treated mice. The average weight of treated mice decreased during the experiment by 10-15%.

Establishment and characterization of a novel immature T-ALL cell line, UHKT-42, with TCR beta/delta expression.

A novel immature human T-ALL cell line, UHKT-42, was established from a 12 year old male patient with acute undifferentiated leukemia. The cell line expressed surface CD7, CD5 and cytoplasmic CD3 antigens. All other T-lymphocytic antigens were undetectable on the surface or in the cytoplasm of cultured cells. Expression of the T-cell receptor (TCR) beta, TCR delta, CD3 delta and CD3 epsilon genes was detected by Northern blotting in total cellular RNA extracts, however, the expression of TCR alpha and TCR gamma was undetectable. After stimulation by TPA for 3 days, only the appearance of CD25 (Tac antigen) was detected by immunofluorescence and flow cytometry. Secretion of interleukin-2 (IL-2) into the culture media was also detected after stimulation by PHA or TPA, but not in unstimulated cells. These results suggest that UHKT-42 cells are early precursors of T cells, with TCR beta/delta expression.

Suppression of basal, PMA- and IFN-alpha-, but not IFN-gamma-induced expression of HLA class I in v-myc-transformed U-937 monoblasts.

Recent studies have suggested that certain oncogenes, in particular members of the myc family, may be involved in the down-regulation of HLA class-I antigen expression observed in many types of tumor. We report that constitutive expression of an OK10 v-myc gene in human monoblastic U-937 cells results in a reduced expression of HLA class-I cell-surface expression and decreased levels of HLA class-I protein and mRNA. All class-I alleles, with the possible exception of HLA A3, were affected, as shown by one-dimensional isoelectric focusing (ID-IEF). Basal expression of the beta 2m chain was also reduced, although to a lesser extent. In addition, we show that the PMA-, and at least partially the IFN-alpha-induced increase in HLA class-I antigen expression, was inhibited in U-937-myc cells both at the protein and the mRNA level. In contrast, the response to IFN-gamma was normal. Another important difference in the response to IFN-gamma and alpha was that, while IFN-gamma abrogated the v-myc block of PMA-induced differentiation of U-937 cells, as previously reported, IFN-alpha did not. Our data show that v-myc negatively affects the regulation of both basal and inducible HLA class-I antigen expression.

Monoclonal antibodies against human antithrombin III.

Three monoclonal antibodies identified as D8, B11 and C5 of different specificities have been produced against human antithrombin III (AT). The apparent dissociation constants (Kd app) of the AT-antibody interaction were determined by ELISA method: Kd app (D8) = 2.4 nmole, Kd app (B11) = 13 nmole, Kd app (C5) = 24 nmole. All three antibodies reacted with isolated AT on immunoblots obtained with "native" PAGE. The D8 antibody also reacted with plasma and serum AT while B11 antibody reacted with serum thrombin-antithrombin (TAT) complexes as well.

[Isolation of monoclonal antibodies from mouse ascitic fluid using chromatography on "blue" DEAE cellulose]. / Izolace monoklonálních protilátek z mysích ascitických tekutin chromatografií na "modré" DEAE celulóze.

A method is described of the isolation of monoclonal antibodies form mouse ascites fluids by chromatography on beaded DEAE cellulose with covalently bound Reactive Blue-2 ("Blue" DEAE cellulose). The method yields antibodies of 80-90% purity, free from protease activity.

Features of immaturity in cells derived from granulocytic differentiation inducer treated human myeloid leukaemia (ML-1) cells.

Cells of the human myeloid leukaemia cell line ML-1 were exposed to differentiation inducing doses of dimethylsulfoxide (DMSO) and retinoic acid (RA). DMSO (but not RA) caused an inhibition of cell growth which was reversible. Some granulocytic maturation associated changes were induced by both agents: nuclear segmentation in 10-20% of cells, B43.4 antigen positivity. In contrast, several other markers were not induced: nucleoli persisted in almost 100% cells including the segmented ones, the nuclear membrane regions did not stain with Victoria blue B (which stains these regions in normal neutrophils), the expression of other antigens of mature neutrophils was also not induced. Maturation asynchrony and non-physiological segmentation of round and oval nuclei were observed. Examination of nucleoli on a single cell level revealed a reversible decrease of pre-rRNA synthesis in 28-48% of the induced cells. These results indicate that terminal differentiation did not occur and confirm the dissociation in induction of various differentiation markers.

Derivatives of benzo(c)fluorene. XXIV. Cytostatic effect of benfluron on human leukemic cells in vitro.

Benfluoron (BF), a new cytostatic drug, synthesized at the Institute of Pharmacy and Biochemistry in Prague, was tested for its cytostatic and cytotoxic effect. The concentrations of BF ranging from 0.1-2 micrograms/ml had a significant cytostatic effect on nine stabilized human leukemic cell lines. This effect was demonstrated by cell counts, cell viability and 3H-thymidine incorporation. The BF concentrations of 2 micrograms/ml and higher were considerably cytostatic causing the cell death and cell degradation. The effect of BF on the cell growth being irreversible could not be eliminated by washing the cells and recultivating them in fresh medium. The BF concentrations halving the total number of viable cells (EC50 value) induced a higher cytotoxicity in lymphoblastoid cell lines then in myeloid ones. BF did not influence the binding of monoclonal antibodies with membrane markers of different cell subpopulations. Prospective application of BF as a cytostatic and immunosuppressive drug is discussed.

Distribution of T-lymphocytic subpopulations detected by monoclonal antibodies in peripheral blood of patients with chronic lymphatic leukaemia.

This paper reports on 93 patients with chronic lymphatic leukaemia (CLL) where CLL of B-type (E-, T-, B+, Ig+/-) was defined in 84 cases (90%), CLL of T-type (E+, T+, B-, Ig-) in 3 cases (3.2%) and in the remaining 6 cases (6.4%) neither T nor B were clearly detected. In 18 patients exhibiting lower total leukocyte count in peripheral blood T-lymphocytic population (OKT-3+), helper/inducer (OKT-4+) and suppressor/cytotoxic subpopulations (OKT-8+) were analysed using monoclonal antibodies in an indirect immunofluorescence test. The results show that the mean T-lymphocytic population counts in patients with CLL of B-type amounted to 1.7 X 10(9)/1 which is the value somewhat increased compared with the mean values observed in normal donors (1.5 X 10(9)/1). The counts of helper/inducer T-lymphocytic subpopulations (OKT-4+) were 1.7 X 10(9)/1 corresponding to those of healthy donors. In contrast, the counts of suppressor/cytotoxic T-lymphocytic subpopulations (OKT-8+) in patients with B-CLL were increased (0.7 X 10(9)/1). The immunoregulation index in patients with CLL decreased to 1.4 compared with that of a control 1.96).

Changes in cell surface glycoproteins and antigens during differentiation of the human myeloid leukemia cell lines ML-1, ML-2, and HL-60.

Two recently derived human myeloid leukemia cell lines, ML-1 and ML-2, were induced to differentiate by treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) or with retinoic acid for 5 to 12 days. They were then compared with similarly treated promyelocytic HL-60 cells. TPA-treated ML-1 and ML-2 cells became firmly surface adhesive with a fibroblastoid morphology, while TPA-treated HL-60 cells adhered as rounded macrophages. In contrast, retinoic acid induced only slight morphological changes in all three cell lines. The differentiation-related alterations of the surface membrane glycoproteins were followed by polyacrylamide gel electrophoresis after surface labeling by the periodate-NaB3H4 or galactose oxidase-NaB3H4 methods. The expression of surface membrane differentiation antigens was analyzed with a panel of monoclonal antibodies against myeloid, myelomonocytic, monocytic, and granulocytic determinants using FACS IV flow cytometry. The acquisition of surface adhesiveness by TPA-treated ML-1 and ML-2 cells coincided with the appearance of membrane surface proteins of varying molecular weights, ranging between 90,000 and 155,000, which were not labeled in untreated ML-1 and ML-2 cells. These findings and the results obtained by monoclonal antibody staining and FACS analysis indicate that treatment of the myeloid lines ML-1, ML-2, and HL-60 by TPA induced the expression of antigenic and membrane molecular features compatible with a monocytic-macrophage phenotype, while treatment by retinoic acid induced granulocytic differentiation. The ML-1 and ML-2 cells offer interesting models for studies on the membrane molecular events occurring when nonadherent, monocytic cells become surface adherent.