Klaus G. Steube

Detection of pharmacologically active natural products using ecology : selected examples from indopacific marine invertebrates and sponge-derived fungi

This review article presents our groups recent research findings with regard to bioactive natural products from marine sponges and tunicates, as well as from sponge derived fungi. The organisms discussed originate in the Indopacific region, which has an exceptionally rich marine biodiversity. Major topics that are covered in our review include the chemical ecology of sponges, focusing on defense against fishes, as well as the isolation and identification of new bioactive constituents from sponges and tunicates. Sponge derived fungi are introduced as an emerging source for new bioactive metabolites, reflecting the currently growing interest in natural products from marine microorganisms.

Bioactive natural products from marine invertebrates and associated fungi.

Marine natural products with their unique structural features and pronounced biological activities continue to provide lead structures in the search for new drugs from nature. Invertebrates such as sponges, tunicates, mollusks and others that are either sessile or slow moving and mostly lack morphological defense structures have so far provided the largest number of marine-derived secondary constituents including some of the most interesting drug candidates. This review highlights recent research findings of our group related to natural products from marine invertebrates. Areas that are covered include ecological functions of secondary constituents from sponges against predatory fish, the search for new pharmacologically active constituents from sponges and tunicates, and sponge-associated fungi as an evolving source for new bioactive natural products.

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.

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 MONOMAC- 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/Gl-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.

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 a MTT-Assay, the IC50 of the most active compound didesmethyl-rocaglamide (1) was observed at 0.002 and 0.006 μg/ml (0.004 and 0.013 μM) 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 μg/ml (4.6 μᴍ) . 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

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.

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. Int. J. Cancer 78:120–124, 1998.© 1998 Wiley‐Liss, Inc.

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.

Multiparameter Approach in the Identification of Cross-Contaminated Leukemia Cell Lines

A common problem in cell culturing is cross-contamination with other cells or misidentification of cells. An effective cell culture quality and identity control is required in order to avoid inter- and intraspecies contamination of cell lines and their further propagation and dissemination. We present evidence that supposedly unrelated cell lines that we received from the original investigators are in fact related to the chronic myeloid leukemia cell line K-562. The sister cell lines SPI-801 and SPI-802 were originally established from a patient with T-cell acute lymphoblastic leukemia and displayed T-cell associated features. However, data from morphological evaluation, immunophenotyping, bcr-abl gene rearrangement analysis, DNA fingerprinting, Northern blot analysis of globin gene expression and esterase isoenzyme analysis clearly established that the three cell lines are related. Cytogenetic examination while not proving the common identity of the cells provided further evidence for the suspected common origin of all three cell lines. Chromosome banding, DNA fingerprinting and bcr-abl genotyping suggested further evolution of these clones during long-term cultivation. Quality and identity control is an essential feature of cell culture technique. Only regular monitoring for purity and integrity of cell lines will significantly reduce the incidence of cell line contamination and misidentification.

Treatment of mycoplasma-contaminated continuous cell lines with mycoplasma removal agent (MRA)

Thirty-nine continuous adherent or suspension cell lines were treated with a quinolone antibiotic, Mycoplasma Removal Agent (MRA), for the elimination of chronic mycoplasma contamination. In preliminary experiments MRA did not show any cytostatic or cytotoxic effects on mycoplasma-free cell cultures in concentrations up to ten-fold the concentration used for mycoplasma eradication. Twenty-eight cell lines (72%) were effectively cleansed of the mycoplasma contaminants by MRA treatment. The persistent removal of the mycoplasma infection was monitored by three mycoplasma detection assays. In seven cell lines (18%) the mycoplasmas were resistant to treatment with MRA. The resistant species was mainly M. arginini followed by M. orale and A. laidlawii; however, other cell lines harboring these species were cured. Four cell lines (10%) which prior to treatment presented with decreased viability and poor or no cell growth were lost during or shortly after the exposure to the antibiotic. If an antibiotic elimination is attempted it is imperative to closely examine the effectiveness of treatment and possible eukaryotic cytotoxicity. The treated mycoplasma-free cells may also no longer express the original features as a result of treatment or the absence of mycoplasma.