Research on inorganic polyphosphates: the beginning.
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Bacteria↗
Biomedical subjects
P Langen
Publications and source records attributed to P Langen.
Synthesis and antiproliferative potency of 9-beta-D-arabinofuranosyl-2-fluoroadenine phospholipid adducts.
Three novel alkylphospholipid and four novel O-alkylglycerophospholipid derivatives of fludarabine (F-ara-AMP), known as a drug for the clinical treatment of chronic lymphocytic leukemia, were synthesized. The antiproliferative activity was determined in comparison to the parent nucleoside fludarabine in an immortalized but nontumorigenic human mammary epithelial cell line (H 184 A1N4), in two human breast tumor cell lines (MaTu and MCF7), and in two leukemic cell lines (HL 60 and Daudi). Fludarabine inhibited the growth of the leucemic cell lines very effectively. The breast tumor cell lines responded with much less sensitivity. The antiproliferative potency of the new compounds strongly depended on the chemical structure of the lipid component, and derivatives with a high effectiveness against one or both of the breast tumor cell lines were described.
Antimetabolites, Antineoplastic↗
Opposite effects on cytosolic Ca2+ of antitumor phospholipids by induction of calcium influx and activation of endoplasmic reticulum Ca(2+)-ATPase.
The ability of four different antitumor phospholipids, 1-O-hexadecyl-2-chloro-2-deoxyglycero-3-phosphocholine (ET16CIPC), hexadecylphosphocholine (C16OPC), hexadecylphospho-L-serine analogs (C16OPS, C16OPS-N-Ac) and cytidine-5'-hexadecylphosphonophosphate (C16PCMP) to modulate the cytosolic Ca2+ concentration [Ca2+]i was studied in an immortalized human mammary epithelial cell line H184 A1N4. The compounds induced different modes of activity depending on their structure and concentration. ET16CIPC induced between 0.31 and 5 microM a concentration dependent transient increase which was followed by a sustained increase at 10 microM. Studies using LaCl3 and Mn2+ quench of the Fura-2 fluorescence indicated that both effects are the result of an extracellular Ca2+ influx. Low concentrations of C16OPC, C16OPS and C16OPS-N-Ac induced no, or only a small, transient increase, whereas C16PCMP caused a decrease in [Ca2+]i. Thapsigargin and cyclopiazonic acid, specific inhibitors of the endoplasmic reticulum Ca(2+)-ATPase, prolonged the transient [Ca2+]i increase following ET16CIPC concentration dependently, increased markedly the small transient increase following C16OPC and the C16-phosphoserine analogs and converted the decrease in the basal [Ca2+]i level induced by C16PCMP to an increase. The identical effects with thapsigargin and cyclopiazonic acid provide evidence that the [Ca2+]i response observed is an expression of the balance between the ability of an analog to raise [Ca2+]i and to remove Ca2+ by activation of the endoplasmic reticulum Ca(2+)-ATPase. This behaviour might contribute to the antiproliferative effectiveness of antitumor phospholipids.
Antineoplastic Agents↗
1-beta-D-Arabinofuranosylcytosine-5'-alkylphosphonophosphates and diphosphates: new orally active derivatives of ara-C.
ara-Cytidine-5'-alkylphosphonophosphates and the corresponding -diphosphates were found to be cytostatically active in vitro against the human mammary epithelial cell line H184 A1N4 and the human mammary tumor cell line MaTu. Our results indicate that the replacement of the diphosphate by the phosphonophosphate group has no influence on antiproliferative activity in this case. The compounds were more active than the corresponding cytidine phospholipid conjugates and related compounds lacking a cytostatically active nucleoside, the ara-C prodrug Cytoros, and were slightly less active than ara-C. The cytostatic effect was prevented by 2'-deoxycytidine indicating their action as prodrugs of ara-C. In contrast to ara-C, they increase [Ca2+]1 in H184 A1N4 cells, pointing to a different mechanism of action in addition to their prodrug effect. In combination with phospholipid analogs, synergistic effects could be observed. Further studies within the disease-oriented in vitro Anticancer Screening Program of the National Cancer Institute show selectivity for certain cancer cell lines. The hexadecyl derivatives revealed a significant antitumor activity in vivo against the murine lymphatic leukemia P 388 cells being equally potent or even superior to ara-C. In contrast to ara-C, they were found to be orally active. Side effects measured as leukopenia and body weight reduction were less pronounced than with the parent drug.
Animals↗
New nucleoside-5'-alkylphosphonophosphates and related compounds containing 2'-deoxycytidine, thymidine and adenosine as nucleoside component. Syntheses and their effects on tumor cell growth in vitro.
Recent studies have shown that phosphono analogs of cytidine-5'-diphosphate diacylglycerol (CDP-DAG) possessing a structurally modified lipid moiety exhibit antiproliferative activity in vitro. As an extension of our previous work we tried to elucidate whether the presence of the cytidine component is necessary for cytostatic activity. In this context we have synthesized similarly structured nucleoside-phospholipid conjugates containing nucleoside components other than cytidine, which also do not exhibit cytostatic properties as such. The compounds include 5'-alkyldiphosphates and 5'-alkylphosphonophosphates of 2'-deoxycytidine, thymidine and adenosine with different alkyl chain length as well as selected 3-hexadecyl-2-chloro-2-deoxyglycero-(1)-diphosphates and -phosphonophosphates of these nucleosides. The chemical structures of the newly synthesized nucleoside-phospholipid conjugates were confirmed by fast atom bombardment (FAB) and electrospray ionization (ESI) mass spectrometry. It was found that these compounds also inhibit the cell growth of different human cell lines, i.e. the presence of the cytidine component is not a necessary prerequisite for the antiproliferative activity of these nucleoside-phospholipid conjugates.
Adenosine↗
Modulation of the cytosolic Ca++ concentration by alkylphospho-L-serine analogs: relation to their antiproliferative action.
Antiproliferative alkyllysophospholipid (ALP) analogs produced multiple effects on the cytosolic Ca++ concentration ([Ca++]i) in an immortalized human breast epithelial cell line (H 184). The addition of small concentrations resulted in a short transient [Ca++]i response. With higher concentrations the transient rise was followed by a sustained increase. Pretreatment of cells with the ALP analogs for two minutes inhibited the transient [Ca++] response. Increases in [Ca++]i and inhibition of the transient increase were studied in relation to the dose and structure of several ALP analogs. In a series of alkylphospho-L-serine analogs with different lengths of the alkyl chain we found different dependencies of the stimulatory and inhibitory effects on the dose and the structure. The ability to increase [Ca++]i is absent with the C14 and C15 analogs, is low with the C16 and high with the C18 analog. With the exception of the C12 analog, a dose-related inhibition was observed with all derivatives but the effective concentrations differed very strongly and the maximal potency was reached with the C15 and C16 analogs. The antiproliferative action seems to correlate rather with the potency to inhibit the transient [Ca++]i response than with its stimulation.
Alkylation↗
Synthesis and antiproliferative activity of cytidine-5'-alkylphosphonophosphates and structurally related compounds.
The chemical synthesis of cytidine-5'-alkyl- and cytidine-5'-alkyl (acyl)deoxyglycerophosphonophosphates is reported. The compounds obtained represent a novel class of cytostatically active agents based on phospholipids, which inhibit the growth of various tumor cell lines in vitro. They are phosphono analogs of the cytidine-5'-diphosphate-diacylglycerol (CDP-DAG) possessing a structurally modified lipid moiety and a phospholipase C-resistant P-C bond. The antiproliferative efficacy of the cytidine-5'-alkylphosphonophosphates strongly depends on the alkyl chain length. The cytidine-5'-hexadecylphosphonophosphate was found to be the most effective compound tested in this study. Its cytostatic effect was distinctly higher than that of the alkyl(acyl) deoxyglycero derivatives and of the corresponding diphosphates. The structure of the new compounds were confirmed by fast atom bombardment mass spectrometry (FAB). The FAB fragmentation pattern is discussed.
Animals↗
Flow-cytometrically monitored chromatin in situ testing in the breast cell line H184A1N4.
Previous investigations in human precancerous and cancerous tissues identified subsets of cells that were different in respect to a heparin-induced increase in fluorescence intensity (IFI) monitored by flow cytometry. We suggested that differences in IFI were due to different chromatin types in the cells and related to different transcriptional capacities. This study is to prove our suggestion. Heparin-induced IFI was measured in the human breast cell line H184A1N4 cultured under different, defined growth conditions. Cells in quiescence obtained from a culture deprived of serum and other supplements essential for growth or from the confluent state revealed a higher IFI than cells restimulated to proliferation by addition of complete medium; here a reduced IFI was found. Changes in the magnitude of heparin-induced IFI precede changes in cell-cycle stage distribution by at least 6.5 h. We conclude that the heparin-induced effects, as revealed by flow cytometry, reflect changes in the accessibility of chromatin to transcription in different stages of proliferative activity. The results confirm our conclusions from previous findings with clinical material.
Breast Neoplasms↗
Adherent-cell-dependent stimulation of CFU-GM by nucleobases, nucleosides, their analogues, and the hemoregulatory peptide dimer.
At suboptimal concentrations of granulocyte-macrophage colony-stimulating factor (GM-CSF), nucleobases and nucleosides as well as their analogues strongly stimulated aggregate (colony and cluster) formation from murine bone marrow granulocyte-macrophage colony-forming units (CFU-GM) in agar culture. Active compounds include 2'-deoxycytidine, thymidine, 5'-deoxyarabinosyl-cytosine, 5'-deoxy-5'-fluorothymidine, uracil, 6-methyluracil, orotic acid, and also purine derivatives as adenine, guanine, adenosine, and guanosine. The stimulation was almost identical to that obtained with the dimer of the hemoregulatory pentapeptide. In the absence of colony-stimulating factor (CSF) no stimulation was seen. After separation of adherent cells from the bone marrow cells, the stimulatory effect was lost. It also decreased when the number of bone marrow cells plated was diminished. This suggests that the tested compounds induce growth factor production in adherent cells. The structure/activity relationships indicate that the active compounds are nucleosides, or they may serve for nucleoside synthesis inside the cell. However, nucleotide formation is not necessary for activity.
Animals↗
Modulation of the beta-adrenergic-response in cultured rat heart cells. II. Mammary-derived growth inhibitor (MDGI) blocks induction of beta-adrenergic supersensitivity. Dissociation from lipid-binding activity of MDGI.
'Mammary-derived growth inhibitor (MDGI)' is a 14.5 kDa polypeptide with growth-inhibitory activity for various mammary epithelial cells in vitro which is highly homologous to cardiac fatty acid-binding protein (H-FABP). Here we describe a new biological activity of MDGI: Inhibition of L(+)-lactate-, arachidonic acid- and 15-S-hydroxyeicosatetraenoic acid-induced supersensitivity of neonatal rat heart cells for beta-adrenergic stimulation, concerning particularly a small population of beta 2-receptors. Synthetic peptides corresponding to the MDGI-sequence, residue 121-131 mimic the effect of MDGI. Measurements of lipid-binding to MDGI and synthetic peptides excluded the binding of arachidonic acid, 15-S-hydroxyeicosatetraenoic acid or beta-adrenergic agonists to MDGI or the peptides as the mechanism for this effect. Also, no direct interference of MDGI and the synthetic peptides with the binding of the beta-adrenergic agent CGP 12177 to its receptor on A431 cells could be detected. We suggest that MDGI and the peptides act by interference with the function of the beta 2-adrenergic receptor and that this mechanism might also be relevant for the growth-inhibitory activity of MDGI. Furthermore, the data point to a possible function of H-FABP for the modulation of beta-adrenergic sensitivity of cardiac myocytes.
Amino Acid Sequence↗
Changes of purine nucleotide metabolism of Ehrlich ascites cells during transition of tumour growth.
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Adenine↗
Purification, biological assay, and immunoassay of mammary-derived growth inhibitor.
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Amino Acid Sequence↗
Evidence for involvement of a nuclear envelope-associated RNA helicase activity in nucleocytoplasmic RNA transport.
It seems well established that translocation of at least some mRNAs through the nuclear pore is (1) an energy-dependent process, and (2) dependent on the presence of the poly(A) segment attached to most mRNA species. We describe that RNA helicase (RNA duplex unwindase) activity is present in a nuclear envelope (NE) preparation, which also appears to be involved in nucleocytoplasmic RNA transport. This activity unwinds RNA: RNA hybrids. The helicase has a pH optimum of 7.5 and a temperature optimum of 30 degrees C. Applying the sealed NE vesicle system, it was shown that duplex RNA species are readily released from the vesicles in an unidirectional manner, in contrast to single-stranded RNA, which is much slower transported into the extravesicular space. Attachment of a poly(A) segment to the RNA duplex additionally increases the efflux rate of this RNA. Efflux of duplex RNA but not efflux of single-stranded RNA was strongly inhibited by formycin B 5'-triphosphate. Our results suggest that, besides poly(A), duplex structures, if present in a given RNA, modulate and control the export of RNA.
Animals↗
Selectivity of hemoregulatory peptide (HP5b) action in culture.
A synthetic analog of a hemoregulatory peptide associated with mature human granulocytes (HP5b) has been investigated for inhibitory effects on various cell types in culture as compared to inhibitory action on mouse and human myelopoietic colonies (CFU-gm), which occurs from 1 X 10(-13) to 1 X 10(-6) M in vitro. This includes colony formation by lymphoid T and B cells in capillary cultures, as well as mitogen activation of T, B and NK cells. At higher concentrations, i.e., above 1 X 10(-7) M, an inhibitory effect was found on colony formation. Neither the production of interleukin (IL) 3 by mitogen-activated T cells, nor the proliferation of the IL-3-dependent L/B cell line were affected by the peptide up to 1 X 10(-5) M. A slight inhibitory effect was found above 1 X 10(-9) M on mouse 3T3 fibroblasts. A series of malignant cell lines was also tested. No effect was seen between 1 X 10(-11) and 1 X 10(-7) M on human mammary carcinoma cells in culture. On Ehrlich ascites mouse mammary carcinoma cells a 30% inhibition was seen at 10(-6) M. On a human glioblastoma cell line (GaMg) no effect was seen, and on a rat glioma cell line (BT5C) an inhibitory effect was seen at 1 X 10(-7) M and above. No significant inhibition of cell growth was seen on SC1 mouse lymphoma cells from 1 X 10(-9) to 1 X 10(-5) M during 7 days of culture. The investigated normal and malignant cell types in culture were thus not inhibited in very low concentrations which act on CFU-gm. However, a variable inhibitory effect was found at higher concentrations where the inhibition of myelopoiesis was maximal and at concentrations where the inhibition is released. The hemoregulatory peptide thus seems to be a concentration-dependent selective inhibitor of myelopoiesis. The finding that various malignant cells do not respond at lower concentrations supports the possibility of using the peptide as a protector of normal cells during cancer chemotherapy.
Amino Acid Sequence↗
Halo lipids. 10. Synthesis and cytostatic activity of O-alkylglycerophospho-L-serine analogs.
The synthesis of O-alkylglycerophospho-L-serine analogs is described, which represent a new class of cytostatically active agents based on phospholipids. The new compounds were obtained by conversion of O-alkylglycerophosphoric ester analogs by means of phospholipase D and by condensing O-alkylglycerophosphoric acid analogs with protected L-serine followed by the removal of protective groups of the resulting intermediates. The structure of the O-alkylglycerophospho-L-serines was confirmed by fast atom bombardment mass spectrometry. The compounds were found to inhibit the growth of Ehrlich ascites tumor cells in vitro. Half maximum inhibition was observed at concentrations between 7 and 15 microM. For the 1-O-alkyl-2-methoxy glycerophosphoserine only a higher value (30 microM) was found. With most of the substances tested growth was completely inhibited at a concentration of 40 microM.
Animals↗
Alteration of DNA topoisomerase II activity during infection of H9 cells by human immunodeficiency virus type 1 in vitro: a target for potential therapeutic agents.
Infection of H9 cells with human immunodeficiency virus type 1 (HIV-1) was found to decrease the phosphorylation of DNA topoisomerase II during the initial phase of infection. Simultaneously, with a later overshoot of phosphorylation and the subsequent activation of DNA topoisomerase II, the production of HIV-1 started. Applying three new protein kinase C inhibitors from the class of O-alkylglycerophospholipids we demonstrated that inhibition of protein kinase C-mediated phosphorylation of DNA topoisomerase II resulted in an inhibition of HIV-1 production. Based on the differential effect of the two protein kinase C activators, phorbol ester and bryostatin, we conclude that phosphorylation of DNA topoisomerase II is mediated by the form alpha and gamma of protein kinase C. These data suggest that agents which inhibit these two forms of protein kinase C are also potential candidates for an anti-HIV therapy.
Animals↗
Quenching of tyrosine radicals of M2 subunit from ribonucleotide reductase in tumor cells by different antitumor agents: an EPR study.
The inhibition of ribonucleotide reductase (RR) of intact Ehrlich ascites tumor cells by different antitumor agents was compared using EPR spectroscopy. The inactivation of M2 subunit was measured via quenching of the functionally essential tyrosine radical. Inhibitors of different classes, for example, hydroxyurea, pyrogallol, and thiosemicarbazones, differ in their efficiency by three orders of magnitude. Most effective inhibition was found for isoquinoline-1-aldehyde-thiosemicarbazone (IQ-1) with an IC50 value of 0.18 microM. Inhibition of RR inside tumor cells is comparable with that reported for isolated enzymes.
Animals↗
Potent inhibition of hepatitis B virus production in vitro by modified pyrimidine nucleosides.
2',3'-Dideoxy-3'-fluorothymidine (FddThd), 2',3'-didehydro-2',3'- dideoxythymidine (ddeThd), and 3'-fluoro-5-methyl-deoxycytidine (FddMeCyt) are, in their triphosphate forms, selective inhibitors of human immunodeficiency virus type 1 reverse transcriptase. We report that 0.3 microM FddThd, FddMeCyt, or ddeThd as well as 3'-chloro-5-methyl-deoxycytidine (ClddMeCyt) or 3'-amino-5-methyl-deoxycytidine (AddMeCyt) almost completely blocked production of hepatitis B virus (HBV) particles by HBV DNA-transfected cell line 2.2.15 in vitro. Only at an at least 10-fold-higher concentration was a cytotoxic effect observed. These results indicate that FddThd, FddMeCyt, ClddMeCyt, AddMeCyt, and ddeThd are potent anti-HBV agents in vitro.
Antiviral Agents↗