Aglacins A-D, first representatives of a new class of aryltetralin cyclic ether lignans from Aglaia cordata.

Four new metabolites, aglacins A-D (1-4), were identified from the methanol extract of the stem bark of Aglaia cordata. These compounds represent a new class of aryltetralin cyclic ether lignan. The structure of aglacin A (1) including the absolute configuration was elucidated by interpretation of spectral data, X-ray crystal structure determination, and employing the modified Mosher's method. In addition, three other derivatives, aglacins B-D (2-4), were isolated and identified by spectral means.

Multiple regulation of constitutive and induced interleukin 8 secretion in human myelomonocytic cell lines.

Secretion of interleukin 8 (IL-8) and its regulation was investigated in myelomonocytic leukaemia cell lines. Quantification by ELISA revealed a constitutive production in the cell lines HL-60, ML-2, MONO-MAC-6 and MUTZ-3 ranging between 1500 and ca. 5000 pg/ml IL-8 per million cells. No measurable IL-8 was detected in the culture medium of MONO-MAC-1 and THP-1. Stimulation with lipopolysaccharide (LPS) or tetradecanoyl phorbol acetate (TPA) significantly increased the IL-8 level secreted by all cell lines; the best producers were TPA-treated MONO-MAC-6 and MUTZ-3 cultures, generating more than 50 000 pg/ml IL-8. Also the calcium ionophore A-23187, IL-13, macrophage colony-stimulating factor (M-CSF), thapsigargin, an inhibitor of the Ca(2+)-ATPase, and tumour necrosis factor-alpha (TNF-alpha) strongly enhanced the IL-8 production in MONO-MAC-6 cells. The glucocorticoid dexamethasone and the protein kinase inhibitor staurosporine distinctively inhibited the IL-8 production of MONO-MAC-6 cells. Thus, our results demonstrate a strong constitutive IL-8 secretion in human myelomonocytic leukaemia cell lines; the variety of different modulators affecting IL-8 production leads to the suggestion of a multiple regulation of IL-8 expression and secretion.

Induction and secretion of the chemokines interleukin-8 and monocyte chemotactic protein-1 in human immature leukemia cell lines.

We investigated expression and secretion of the chemokines interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) in human myeloid cell lines. Quantitative determination by ELISA revealed a significant constitutive production of both chemokines in the cell lines HL-60 and NB-4 (>1000 pg/ml IL-8 and >400 pg/ml MCP-1 per million cells), while in the cell lines EOL-1, KASUMI-1 and KG-1 only 10-100 pg/ml IL-8 and MCP-1 were detected. Tetradecanoyl phorbol acetate (TPA) strongly increased the IL-8 and MCP-1 amounts in the culture supernatants of all five cell lines. The TPA-induced NB-4 produced the largest amounts of both chemokines (>40,000 pg/ml). The strongest induction was seen in EOL-1 (>100-fold increase). Besides TPA, tumor necrosis factor-alpha (TNF alpha) also distinctively enhanced IL-8 and MCP-1 production. The calcium ionophore A-23187 and thapsigargin, an inhibitor of the Ca(2+)-ATPase, differentially induced IL-8 and MCP-1 secretion in the cell lines investigated, suggesting that, at least in some cell lines, intracellular free Ca(2+) might be important for chemokine secretion. Dexamethasone significantly prevented the IL-8 and MCP-1 production of stimulated cells, emphasizing the potent anti-inflammatory property of glucocorticoids. Similarly, the protein kinase inhibitor staurosporine clearly decreased the TPA-induced chemokine secretion in NB-4 cells, indicating the involvement of protein kinases in the signal transduction pathway which leads to enhanced chemokine secretion.

Constitutive protein expression of monocyte chemotactic protein-1 (MCP-1) by myelomonocytic cell lines and regulation of the secretion by anti- and proinflammatory stimuli.

We have investigated the protein expression of the chemokine monocyte chemotactic/chemoattractant protein-1 (MCP-1) in various human myelomonocytic leukemia cell lines. Applying specific ELISA, we demonstrated that this chemokine is produced constitutively by the cell lines HL-60, ML-2, MONO-MAC-6 and MUTZ-3 ranging between 440 and 1400 pg/ml MCP-1 per million cells. In the culture medium of two other unstimulated cell lines, MONO-MAC-1 and THP-1, almost no MCP-1 was detected. Stimulation of HL-60 and MONO-MAC-6 with lipopolysaccharide (LPS), and stimulation of ML-2 and MUTZ-3 with 12-tetradecanoyl phorbol 13-acetate (TPA) dramatically increased the MCP-1 level in the culture medium. The highest amount of MCP-1 (> 80 ng/ml within 24 h) was achieved by TPA stimulation of MUTZ-3 cells. Out of 15 cytokines tested for induction or enhancement of MCP-1 secretion, interleukin-3 (IL-3), IL-6, interferon-gamma (IFN-gamma), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF) and tumor necrosis factor (TNFalpha) were able to augment (twofold to 12-fold) the MCP-1 level in the culture medium of MONO-MAC-6 cells. While the antinflammatory cytokines IL-4, IL-10 and IL-13 failed to suppress MCP-1 secretion, the glucocorticoid dexamethasone strongly inhibited the MCP-1 production of unstimulated and stimulated MONO-MAC-6 cells. Thus, several regulatory elements are involved in MCP-1 secretion. Despite the quantitative differences of MCP-1 production among the cell lines analyzed, our results demonstrated a constitutive secretion in differentiation-arrested myelomonocytic leukemia cell lines and emphasize the usefulness of these malignant cell lines as models to study MCP-1 secretion and regulation.

Structure activity relationships of antiproliferative rocaglamide derivatives from Aglaia species (Meliaceae).

Eleven rocaglamide derivatives (cyclopentatetrahydrobenzofurans) and one structurally related aglain congener all isolated from different Aglaia species (Meliaceae) were tested for growth inhibiting properties using the human cancer cell lines MONO-MAC-6 and MEL-JUSO. Proliferation of both cell lines was efficiently inhibited in a dose and compound dependent manner. Applying MTT-Assay, the IC50 of the most active compound didesmethyl-rocaglamide (1) was observed at 0.002 and 0.006 micrograms/ml (0.004 and 0.013 microM) depending on the cell line investigated. Bulky aminoacyl substituents at C-2, acetylation of the OH substituent at C-1 or insertion of a OH or OMe substituent at C-3 of the rocaglamide skeleton all diminished the activity of the compounds investigated. The aglain derivative 12 was inactive up to a concentration of 3 micrograms/ml (4.6 microM). This loss of activity is assumed to be mainly due to the presence of a pyran ring in the aglains vs. a furan ring as found in rocaglamide derivatives. Rocaglamide derivatives may act primarily by inhibition of cell proliferation as evidenced by the absence of a significant cytotoxic effect in long-term cultures of MONO-MAC-6 cells treated with high doses of didesmethylrocaglamide. Our data suggest that rocaglamide derivatives could exert a potential role in the treatment of malignant diseases and are worth to be investigated in further studies of experimental medicine and pharmacology.

Expression of receptor tyrosine kinase HTK (hepatoma transmembrane kinase) and HTK ligand by human leukemia-lymphoma cell lines.

HTK (hepatoma transmembrane kinase) is a receptor tyrosine kinase belonging to the EPH subfamily of tyrosine kinases. Binding of its ligand (HTKL) results in tyrosine phosphorylation of HTK. In the present study, we analyzed the possible involvement of this ligand-receptor signalling system in hematopoiesis by examining the expression of both HTK and HTKL in a large and comprehensive panel of 70 continuous human leukemia-lymphoma cell lines. HTK and HTKL mRNA expression were analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR). HTK mRNA was detected in 68/70 cell lines; 58/70 cell lines were positive for HTKL mRNA expression; consequently, co-expression of both receptor and ligand was demonstrated in the majority of cell lines. Collectively, the wide-spread expression suggests a role for this ligand-receptor pair in hematopoietic development and/or function. Investigation of the details of signal transduction pathway that is activated by the HTK tyrosine kinase will help to define the exact biological function of the HTK-HTKL system.

Constitutive secretion of hematopoietic cytokines by human carcinoma cell lines and its up-regulation by interleukin-1 and phorbol ester.

We investigated secretion of hematopoietic growth factors in ten human tumor cell lines, derived from 5 different tissue types. The cell lines were stimulated transiently with interleukin-1 (IL-1 ), IL-6, interferon-ç (IFN-ç), lipopolysaccharide (LPS) and tetradecanoyl phorbol 13-acetate (TPA), respectively. Conditioned media (CM) were screened in a bioassay using an indicator cell line which is growth factor-dependent and needs the supply of at least one hematopoietic cytokine. The CM of almost all tumor cell lines analyzed induced proliferation of this indicator cell line as documented by incorporation of [3H]-thymidine. The majority of constitutively cytokine-producing tumor cell lines could be induced to further increase their amounts of secreted proliferation-inducing activity. The strongest inducers were TPA and IL-1 ; weakly or not effective at all were IFN-ç, LPS and IL-6. Enzyme-linked immunosorbent assay (ELISA) identified several cytokines and macrophage-colony-stimulating factor (M-CSF) and IL-6 were secreted at the highest concentrations. Especially in kidney cell lines, the levels of granulocyte-macrophage-CSF (GM-CSF), M-CSF and IL-6 were further strongly increased by the TPA and IL-1 pretreatment. The by far highest amounts of granulocyte-CSF (G-CSF) were elaborated by the bladder cell line T-24 and could be further more than doubled by TPA. Other cell lines, derived from esophagus, lung and pancreas, responded less strongly to the pretreatment with the biomodulators. Our results demonstrating secretion of functionally active cytokines by human solid tumor cell lines suggest that these, to date so-called, hematopoietic cytokines are not entirely hematopoietic specific and might play a fundamentally important role in tumor cell biology.

1H-cyclopenta[b]benzofuran lignans from Aglaia species inhibit cell proliferation and alter cell cycle distribution in human monocytic leukemia cell lines.

Thirteen naturally occurring 1H-cyclopenta[b]benzofuran lignans of the rocaglamide type as well as one naturally occurring aglain congener all of them isolated from three Aglaia species (Aglaia duperreana, A. oligophylla and A. spectabilis) collected in Vietnam were studied for their antiproliferative effects using the human monocytic leukemia cell lines MONO-MAC-1 and MONO-MAC-6. Only rocaglamide type compounds showed significant inhibition of [3H-]thymidine incorporation and the most active compound didesmethylrocaglamide inhibited cell growth in a similar concentration range as the well-known anticancer drug vinblastine sulfate. Detailed structure-activity analysis indicated that the OH-group at C-8b which is a common structural feature of most naturally occurring rocaglamide compounds is essential for the described antiproliferative activity since replacement of this group by methylation led to a complete loss of the inhibitory activity for the resulting derivative. Rocaglamide derivatives rapidly inhibited DNA as well as protein biosynthesis of MONO-MAC-6 cells at concentrations well below those of actinomycin D or cycloheximide which were used as positive controls in the respective experiments. Didesmethylrocaglamide was furthermore able to induce growth arrest of MONO-MAC-1 cells in the G2/M and probably G0/G1-phase of the cell cycle with no morphological indication of cellular damage. Our data suggests that 1H-cyclopenta[b]benzofuran lignans of the rocaglamide type act primarily by a cytostatic mechanism.

Secretion of functional hematopoietic growth factors by human carcinoma cell lines.

We studied the constitutive production of hematopoietic cytokines in a large panel of human cell lines originating from a wide variety of solid tumors. Conditioned media (CM) from the carcinoma cell lines were collected and screened for proliferative activity using a bioassay with indicator cell lines. These indicator cell lines are dependent on hematopoietic growth factors and require the exogenous supply of at least one hematopoietic cytokine for proliferation. We found that CM of 27/70 cell lines were able to significantly and reproducibly stimulate [3H]-thymidine incorporation of the factor-dependent cell lines, indicating that the tumor cell lines secreted one or more functional cytokine(s). The CM-induced proliferation of the indicator cell lines was significantly inhibited by anti-serum against granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte-CSF (G-CSF). ELISA confirmed the presence of one or several of the following cytokines in the CM of carcinoma cell lines: GM-CSF, G-CSF, macrophage-CSF (M-CSF), stem cell factor (SCF) and IL-6. A strikingly high percentage of GM-, G- or M-CSF-secreting cell lines was found among those lines derived from carcinomas of the kidney (100%), urinary bladder (85%) and pancreas (100%). The large majority of tumor cell lines derived from breast, colon, esophagus, lung, nervous system and melanomas did not produce significant amounts of the cytokines we investigated here. The cytokines secreted have been proven to be functionally active and can support growth and viability of cytokine-dependent hematopoietic cell lines.

A new calyculin derivative from the sponge Theonella swinhoei is a novel and potent inhibitor of tumor cell proliferation.

A novel calyculin derivative was isolated from the marine sponge Theonella swinhoei. Using human and animal tumor cell lines and freshly explanted peripheral blood cells, we investigated several biological effects of this natural product (i.e. cell growth, cytotoxicity, induction of differentiation and apoptosis). The new calyculin exhibited a dose-dependent cytotoxicity against various cell lines from different species and tissues. The ID50 values ranged between 20 and 90 ng/ml. Viability of a multidrug resistant HELA subclone was not affected. Apoptosis of the Hodgkin's lymphoma cell line HDLM-2 induced by antiserum was not prevented by the substance. A reduced drug sensitivity of the monocytic cell line MONOMAC-6 could be observed after induction of differentiation of these cells by phorbol ester and lipopolysaccharide. Even so, non-dividing peripheral blood cells were also resistant to the action of the calyculin derivative, suggesting that the cytotoxin may act preferentially on proliferating cells rather than on quiescent cells. Our data introduce a new calyculin as a marine natural product with interesting features stimulating further studies as a chemotherapeutic or investigational drug.

Bladder carcinoma cell line KU-19-19-derived cytokines support proliferation of growth factor-dependent hematopoietic cell lines: modulation by phorbol ester, interferon-gamma and interleukin-1 beta.

The human bladder carcinoma cell line KU-19-19 synthesizes and secretes hematopoietic growth factors. Conditioned medium (CM) from KU-19-19 stimulated the [3H]thymidine incorporation of growth factor-dependent hematopoietic cell lines. ELISA documented high amounts of granulocyte colony-stimulating factor (G-CSF; > 5 ng/ml); also granulocyte-macrophage CSF (GM-CSF), macrophage-CSF (M-CSF), stem cell factor (SCF), IL-6, and IL-8 were detected in KU-19-19 CM. Pretreatment with phorbol ester, IL-1 beta, or IFN-gamma increased the level of G-CSF, GM-CSF, and M-CSF in KU-19-19 CM. Thus, KU-19-19 represents a reliable source for purification of G-CSF and can easily be used to support proliferation of growth factor-dependent cell lines. The ability to respond to different stimuli suggests that several regulatory pathways may be involved in cytokine production of this bladder carcinoma cell line.

In vitro culture studies of childhood myelodysplastic syndrome: establishment of the cell line MUTZ-1.

Myelodysplastic syndrome (MDS) in childhood is considered to be very rare and detailed pathobiological data are scarce. More biological information regarding MDS in children is clearly needed and in vitro culture studies provide one possibility for gaining further pathophysiological insights into this malignancy. Here, we incubated bone marrow samples from 30 children with MDS in liquid suspension culture in order to grow the transformed cells in vitro. In most cultures, the hematopoietic cells died quickly and only fibroblastic (stromal) background layers proliferated temporarily; several normal Epstein-Barr virus (EBV)-transformed B-lymphoblastoid cell lines (B-LCL) were established. Only in one instance, albeit from the peripheral blood and not from the bone marrow, could we establish a cell line, termed MUTZ-1, from the malignant cells of a 5-year-old girl with MDS (FAB subtype refractory anemia with excess of blasts). The MDS arose from a pre-existing Fanconi anemia and progressed quickly to an acute myeloid leukemia (FAB M2). Despite positivity for EBV, MUTZ-1 is not an EBV + B-LCL and further characterization of MUTZ-1 confirmed the derivation from the transformed clonal cells. Immunophenotyping showed a pre B-cell surface marker profile (CD10+ CD19+ cytoplasmic IgM+); receptor gene rearrangement analyses underlined the clonal B-cell nature of MUTZ-1 cells. MUTZ-1 cells exhibit a highly rearranged, unstable karyotype with a high frequency of spontaneous chromatid breaks and exchanges; del(5q) and additional rearrangements involving chromosome 5 [der(15)t(5;15)] were detected. The present data and results from a few other MDS-derived cell lines suggest that the transforming event in MDS seems to occur in an immature pluripotent progenitor cell. The new MDS-derived continuous cell line MUTZ-1 provides a useful in vitro model system for studies on the pathogenetic events leading to MDS.

A model system in haematology and immunology: the human monocytic cell line MONO-MAC-1.

MONO-MAC-1 is a human cell line with properties of blood monocytes, which can be used as a model system to study monocytic functions in vitro. In the present study, we prepared a karyotype of MONO-MAC-1, analysed the growth behaviour, determined the presence of differentiation-associated antigens and studied the expression and secretion of several cytokines upon stimulation with 12-O-tetradecanoyl phorbol 13-acetate (TPA) and lipopolysaccharide (LPS). The MONO-MAC-1 cells have a near diploid karyotype and contain several recurrent chromosomal rearrangements, in particular the translocation (9;11) commonly found in AML-M5. Stimulation with TPA or LPS induced changes in morphology and gene expression, especially an increase in the level of the differentiation marker CD14 and the production of monocyte-related cytokines. Both biomodulators alone were sufficient to promote TNF alpha release; however, the combination of TPA and LPS resulted in a synergistic increase of TNF alpha secretion. Northern blot analysis indicated that upregulated production of TNF alpha was due to induced synthesis of mRNA. The mRNA accumulation peaked approximately 2 h after stimulation and maximum levels of TNF alpha were found in the supernatants after 4-8 h of culture. The MONO-MAC-1 cells could not be restimulated with the same inducer to release TNF alpha when a 48 h pre-treatment was carried out with LPS or TPA. LPS induced the release of granulocyte colony-stimulating factor (G-CSF), while TPA failed to do so. Vice versa, secretion of macrophage CSF (M-CSF) could be induced by TPA, but not by LPS. However, LPS enhanced the TPA-induced M-CSF production. Similarly, incubation of MONO-MAC-1, simultaneously with TPA and LPS, led to granulocyte macrophage CSF (GM-CSF) and interleukin-1beta (IL-1beta)secretion, while both stimulators alone had almost no (TPA) or only a weak (LPS) effect on the secretion of GM-CSF and IL-1beta. Our results demonstrate that MONO-MAC-1 is a unique cell line with distinct monocytic features; certain monocytic properties can be upregulated by activation of intracellular signalling pathway(s). We suggest that, besides the LPS receptor CD14, activation of PKC participates in these process, especially in the production and secretion of cytokines by MONO-MAC-1 cells.

Expression of bcl-2 mRNA and protein in leukemia-lymphoma cell lines.

The expression of the proto-oncogene bcl-2 was examined in a panel of 75 continuous human leukemia-lymphoma cell lines originated from different hematopoietic cell types. The presence of the bcl-2 protein, as evidenced by Western blotting, and its mRNA, as determined by Northern blotting, were not restricted to cells with the chromosomal translocation t(14;18)(q32;q21), but were also detected in a large number of cell lines without t(14;18). The amount of the bcl-2 protein and mRNA in the cell lines with t(14;18) was in the same order of magnitude as in other bcl-2 expressing cell lines of the same lineage, but without the translocation. Bcl-2 was found in all types of hematopoietic cell lines which were assigned to the following lineages based on their phenotypical characteristics: pre-B, B, plasma, T, myeloid, monocytic, erythroid-megakaryocytic and Hodgkin's lymphoma derived cell lines. The levels of accumulated mRNA and protein corresponded fairly well in most of the cell lines examined. Our results suggest the notion that bcl-2 expression is widely present in hematopoietic cell lines without restriction to single lineages and, in fact, clearly independent of the chromosomal aberration t(14;18). It is conceivable that bcl-2 expression is a common feature in established hematopoietic cell lines and may contribute to their unlimited growth in vitro.

The protein kinase C activator Bryostatin-1 induces the rapid release of TNF alpha from MONO-MAC-6 cells.

Bryostatin-1 is a natural activator of protein kinase C and currently examined in phase I trials as anticancer agent. We found that Bryostatin-1 induced tumor necrosis factor alpha (TNF alpha) expression in the human cell line MONO-MAC-6. Using Northern blot analysis and a bioassay for the detection of the cytokine we observed that Bryo alone was sufficient to transiently induce mRNA synthesis and the rapid release of TNF alpha into the culture medium. However, the combination of Bryo with lipopolysaccharide resulted in a strong synergistic increase of TNF alpha secretion. The biologic activity of the secreted TNF alpha was amenable to inhibition by anti-TNF alpha antibodies. Blockade of the lipopolysaccharide receptor CD14 or inhibition of protein kinase C implied that both, CD14 and protein kinase C, are involved individually in signal transduction pathways leading to TNF alpha secretion from MONO-MAC-6 cells. The results demonstrate that Bryostatin-1 is able to induce TNF alpha secretion in human monocytes via a protein kinase C-dependent and CD14-independent pathway and by a mechanism which is most likely based on a strong increase of the TNF alpha mRNA level.

Isoenzyme analysis as a rapid method for the examination of the species identity of cell cultures.

One of the major problems in cell culturing is the misidentification or cross-contamination of authentic continuous cell lines. We applied a rapid and efficient isoelectric focusing (IEF) technique for the routine analysis to detect interspecies contamination of cell cultures and for the identification of unknown animal cell lines. The method is based on the isoelectric separation of a specific set of intracellular enzymes which can be used to distinguish between cell lines of human, murine, or other mammalian origin. By means of preformed agarose gels, standardized conditions and equipment, this technique is especially applicable for routine work and allows the analysis of a large number of unknown samples with reproducible results. One hundred seventy-seven cell lines which have been sent to the Department of Human and Animal Cell Cultures at the DSM (Deutsche Sammlung von Mikroorganismen and Zellkulturen) were analyzed for species authentication; only three cell lines were found not to be of the presumed species. Our study strongly emphasizes standardized IEF as an efficient and rapid method for routinely monitoring the authenticity of cell lines.

Different biological effects of the two protein kinase C activators bryostatin-1 and TPA on human carcinoma cell lines.

Bryostatin 1 (Bryo) is a naturally occurring macrocyclic lactone with antineoplastic activity. Like the phorbol ester 12-O-tetradecanoyl-phorbol 13-acetate (TPA) it directly activates the calcium- and phospholipid-dependent protein kinase C (PKC), thus generating a number of different cellular responses. We investigated the effects of Bryo and TPA on DNA synthesis, proliferation, viability and c-myc protooncogene expression of the human carcinoma cell lines COLO-320, MEL-HO, and KB-3-1. TPA inhibited [3H]-thymidine incorporation in all three cell lines in a dose-dependent manner, whereas Bryo only inhibited the DNA synthesis in MEL-HO, but not in KB-3-1 and COLO-320 cells. Within the concentration ranges used, TPA and Bryo were found to have a low toxicity. Counting of the cells confirmed the observed inhibition of cell proliferation. However, the enzymatic conversion of MTT, applied as a colorimetric proliferation assay, was not significantly affected by both biomodulators. Time-course experiments revealed a rapid onset of the inhibitory effect on DNA synthesis. Bryo was further able to antagonize the TPA-mediated effects on proliferation suggesting an (at least partially) different mode of action of these PKC activators. Incubation of MEL-HO and COLO-320 cells with either of the two biomodulators resulted in a rapid and strong increase of c-myc mRNA. The present study emphasizes Bryo as an interesting, natural substance for the study of PKC-mediated biological effects.

Different effects of the two protein kinase C activators bryostatin-1 and TPA on growth and differentiation of human monocytic leukemia cell lines.

The effects of bryostatin-1 (Bryo) and 12-O-tetradecanoyl-phorbol 13-acetate (TPA), both activators of protein kinase C (PKC), on proliferation and differentiation of two monocytic leukemia cell lines, JOSK-I and JOSK-M, were investigated. Treatment with TPA or Bryo inhibited cellular proliferation in a dose-dependent manner. Both drugs induced distinct phenotypic changes associated with monocytic differentiation. Although c-myc mRNA is often found to be down-regulated during biomodulator-triggered in vitro myelomonocytic differentiation, however, here the modulation of c-myc expression was less pronounced. All parameters studied were more prominently altered in TPA- than in Bryo-treated cells, and, were more distinct in JOSK-I than in JOSK-M. Since Bryo was able to antagonize the TPA-mediated effects on proliferation and morphological alterations, an (at least partially) different mode of action of these PKC activators on monocytic cell lines may be suggested.

Induction of differentiation of B-cell leukemia cell lines JVM-2 and EHEB by bryostatin 1.

The effects of bryostatin 1 (Bryo 1), a protein kinase C (PKC) activator, on proliferation, differentiation and macromolecular synthesis were investigated in the two cell lines EHEB and JVM-2, established from patients with chronic B-cell leukemia. Treatment with Bryo 1 inhibited the proliferation, DNA and RNA synthesis in a time- and dose-dependent fashion. The cells differentiated along the B-cell pathway to plasmacytoid cells as judged by morphological examination and increased their production and secretion of immunoglobulins. c-myc mRNA expression was induced in both cell lines. The phorbol ester TPA, a pharmacological PKC activator, had similar differentiation-inducing effects. The biomodulators failed to induce significant alterations in the cell surface marker profile. Except for their surface markers, all parameters studied were more strongly altered in JVM-2 than in EHEB cells. JVM-2 was established from a patient with B-prolymphocytic leukemia (PLL), whereas EHEB originated from a case of B-chronic lymphocytic leukemia (CLL). These data support the notion that PLL cells appear to be activated B-cells, in contrast to the rather quiescent CLL cells. Since Bryo 1 lacks tumor-promoting activity, this naturally occurring compound, extracted from marine animals, has a potential role in the therapy of B-cell neoplasms as a differentiating agent.

Differentiation and growth modulation of myeloid leukemia cells by the protein kinase C activating agent bryostatin-1.

Bryostatin-1 (Bryo), a macrocyclic lactone of the sea water bryozoan Bugula neritina, is a potent activator of protein kinase C and was found to exhibit antineoplastic activity in several systems. We studied the effect of Bryo on differentiation and growth modulation of human myeloid leukemia cell lines and freshly explanted blood cells from patients with myeloid leukemia. Alterations at the molecular level and phenotypic changes triggered by Bryo were similar, but not identical, to those induced by phorbol esters. Bryo was able to inhibit cellular proliferation as evidenced by [3H]-thymidine uptake and induced morphological changes associated with monocytic differentiation. In studies using continuous cell lines, the glucocorticoid dexamethasone was unable to prevent the Bryo-induced growth inhibition or the induced phenotypic changes. However, in fresh myeloid blood cells dexamethasone attenuated these Bryo-triggered effects. Our own data taken together with reports from the literature reviewed here suggest the following conclusions: (i) Bryo, while lacking tumor promoting activity, is able to induce differentiation in maturation arrested leukemia cells; (ii) it exhibits selective antiproliferative properties in normal or malignant hematopoietic cells and supports growth of multipotent stem cells. These features might qualify Bryostatin-1 as a potential candidate for promising research and possibly for future clinical applications.