Biomedical subjects

P Langen

Publications and source records attributed to P Langen.

At least 37 records · Page 2Linked to original sources

PubMed1990

Toxic effects of alkyl-lysophospholipids on human bone marrow and de novo leukaemias. A short overview.

Alkyl-lysophospholipids (ALPs) are reported to have an antineoplastic activity against leukaemic cells. We have tested some halogen-containing ALPs from the Central Institute of Molecular Biology (H. Brachwitz) in comparison with racemic 1-ostadecyl-2-methyl-glycero-3-phosphocholine (ET-18-OCH3) (P.G. Munder, Max-Planck-Institut für Immunobiologie, Freiburg, FRG). We found freshly dissolved ALPs to be very toxic both to human bone marrow and to leukaemic cells of patients. ALP-incubation before cryopreservation is more toxic to bone marrow (but not to AML blasts) than after cryopreservation. All experiments to test the selectivity and to establish a purging protocol should be done using 1, remission marrow including a cryopreservation step and 2, blasts of de novo leukaemias instead of cell as sensitive as HL 60 to ALP-incubation. We found direct toxicity of ALPs to be not suitable for purging lines of bone marrow from patients in remission.

Alkylation↗

PubMed1989

Effect of a mammary-derived growth inhibitor on the expression of the oncogenes c-fos, c-myc and c-ras.

A mammary-derived growth inhibitor (MDGI) inhibits the resumption of growth of stationary Ehrlich ascites carcinoma (EAC) cells in vitro. The present study shows that the resumption of growth is accompanied by a rapid increase of the steady state mRNA level of the proto-oncogenes c-fos, c-myc and c-ras, which is reduced by MDGI. EAC cells from the exponential growth phase insensitive to MDGI did not show a reduced RNA expression. The effect of MDGI represents a novel activity at the level of gene expression and suggests a link to exist between growth inhibition and the reduction of c-fos, c-myc and c-ras expression.

Animals↗

PubMed1989

Sensitivity of CFU-GM from normal human bone marrow and leukaemic clonogenic cells (CFU-L) from blood of patients with myelogenous leukaemia to 3'-deoxy-3'-fluorothymidine in comparison to 3'-azido-3'-deoxythymidine.

3'-Deoxy-3'-fluorothymidine (FT), a thymidine analogue highly effective against HIV 1 in vitro was investigated as to its in vitro effect on normal human bone marrow CFU-GM (agar colony assay) and on human peripheral myeloid leukaemic clonogenic cells (CFU-L, colony assay in methylcellulose). For comparison, 3'-azido-3'-deoxythymidine (AZT), structurally related and used in AIDS treatment, was included in the study. Both compounds inhibit the formation of clusters and colonies from bone marrow stem cells with an [IC]50 between 10(-6) and 10(-5)M. In concentrations only 5-10 times lower than the [IC]50, FT begins to stimulate cluster and colony formation. AZT and FT also inhibit the formation of clusters and colonies from CFU-L. Compared to CFU-GM, CFU-L were more sensitive to FT, and a stimulation was not seen. It is concluded that similar side effects on bone marrow could be expected for possible use of FT against AIDS as have been found for AZT and that both compounds are potential candidates for anti-leukaemic drugs.

Adolescent↗

PubMed1989

Stability and reactivation of tyrosine radicals from ribonucleotide reductase in tumor cells studied by ESR.

Naturally occurring tyrosine radicals from the M2 subunit of ribonucleotide reductase (RR) have been recorded by ESR in proliferating ordinary Ehrlich-ascites (EA) tumor cells of mice. Tyrosine radicals are stable in EA cells at room temperature for 2 h. Up to 500 mW no microwave saturation occurs. The relatively high stability and non-saturation of tyrosine radicals in EA cells suggests a suitable protein conformation in the M2 subunit enabling a close contact between the tyrosine radical and the antiferromagnetic iron complex. This facilitates an ESR study of functionally essential tyrosine radicals of RR in EA cells at low temperature and recommends this cellular system for studying such processes as inhibition and activation, which change the content of tyrosine radicals of the proliferation-linked RR. Oxygen treatment of non-proliferating (quiescent) EA cells reactivates tyrosine radicals 2-3 fold as found in strongly proliferating cells. We conclude that in quiescent cells, suffering from a lack of oxygen due to their high density in the peritoneal cavity, a reactivation of tyrosine radicals occurs by oxidation of non-radical tyrosine residues of inactive M2 subunits.

Animals↗

PubMed1989

Response of different mammary epithelial cell lines to a mammary derived growth inhibitor (MDGI).

MDGI, a 14.5-KDa protein, is a chemically defined growth inhibitor, which we have purified from lactating bovine mammary gland and characterized biologically in a mouse Ehrlich ascites mammary tumour (EAT) short term suspension culture. It has now been tested for its inhibitory activity on proliferation of four malignant mammary epithelial cell lines of human and mouse origin and normal human mammary epithelial cells. In all experiments, cells were brought to quiescence by serum or growth factor deprivation. Using [3H]TdR pulse labelling the effect of MDGI was measured on the restimulation of proliferation after medium change. MaTu and T47 D, human malignant mammary epithelial cell lines, as well as the mouse malignant mammary epithelial cell line mMaCa 20177 could be inhibited, whereas the human malignant mammary epithelial cell line MCF7 showed a slight stimulation. MDGI showed no activity on the residual DNA synthesis of all cell lines after starvation. Normal human mammary epithelial cells (HMEC) of different passages could also be inhibited. Their responsiveness seemed to be dependent on the number of passages. Cells from high passages (10-14) showed a higher sensitivity, which is also about 10 times higher than that of the malignant cell lines. Furthermore, growth factors like insulin, epidermal growth factor (EGF) and fetal calf serum (FCS), known to be potent antagonists to the MDGI activity in the EAT and, in the case of insulin, also in the MaTu culture (shown in the present study), do not abolish the inhibitory activity of MDGI on HMEC cells. These results demonstrate that the inhibitory activity of MDGI is not exclusively restricted to EAT cells studied so far.

Animals↗

PubMed1988

Phosphorylation, anti-HIV activity and cytotoxicity of 3'-fluorothymidine.

3'-fluorothymidine (FddThd) is well phosphorylated to the 5'-triphosphate in various relevant cell-lines. This results in fairly stable levels of this compound without accumulation of the 5'-monophosphate to the extent described for 3'-azidothymidine (AzT). The di- and triphosphate of FddThd seems unable to influence the ribonucleotide reductase in permeable cells. FddThd protects 50% of the MT-4 cells against the cytopathic effect of HIV at 3 nM, but reduces the number of uninfected viable cells to 50% at 1.1 microM. All other tested human cell-lines displayed a far less antiproliferative sensitivity to FddThd, comparable with that of AzT.

Animals↗

PubMed1988

Deoxycytidine, insulin and epidermal growth factor overcome the effect of two natural inhibitors of the haemopoietic system.

The effect of 2'-deoxycytidine on a pluripotent haemopoietic stem cell proliferation inhibitor, as assayed in the mouse spleen colony technique and on the synthetic haemoregulatory pentapeptide (acting predominantly on myeloid determined cells) as assayed in the agar colony technique, was studied. 2'-deoxycytidine was able to overcome the effect of both inhibitors. The inhibition was also blocked by insulin or epidermal growth factor. Since these results are in agreement with those on other cells and other inhibitors, they may describe a more general phenomenon. It is suggested that the mode of action of the inhibitors involves, probably indirectly, interaction with the activity of ribonucleotide reductase.

Animals↗

PubMed1987

Inhibition of HIV-associated reverse transcriptase by sugar-modified derivatives of thymidine 5'-triphosphate in comparison to cellular DNA polymerases alpha and beta.

The sugar-modified dTTP analogues 2',3'-didehydro-2',3'-dideoxy-thymidine 5'-triphosphate (ddeTTP), 2',3'-dideoxythymidine 5'-triphosphate (ddTTP), 3'-fluorothymidine 5'-triphosphate (FdTTP), and 3'-azidothymidine 5'-triphosphate (N3dTTP) are demonstrated to be very effective and selective inhibitors of the HIV-associated reverse transcriptase (HIV-RT). This conclusion is based on a comparison of the ID50 values of the compounds for the HIV-RT (ranging from 0.03 microM for ddeTTP to 0.1 microM for ddTTP) and the cellular DNA polymerase alpha (greater than 200 microM). DNA polymerase beta is partially affected by N3dTTP (ID50 = 31 microM) and by the other analogues (ID50 = 1-2.2 microM). FdTTP has proved as effective as N3dTTP (ID50 = 0.05 microM) in suppressing the HIV-RT activity. Kinetic analysis revealed for both dTTP analogues a competitive type of inhibition and the same K1 values (about 0.05 microM).

Cell Line↗

PubMed1987

Cytostatic activity of synthetic O-alkylglycerolipids.

A series of halogen-containing alkylglycerolipid analogs has been checked for their cytostatic activity both in vitro and in vivo. The compounds included alkyldeoxyhaloglycerols (I), alkyldeoxyhaloglycerophosphocholines (II), and alkyldeoxyhaloglycerophosphoric acids and alkyl esters (III). While compounds I and III were moderately active, compounds II were found to have a strong inhibitory effect on the proliferation of Ehrlich ascites tumor cells in vitro. Cell growth inhibition of 50% or more was found mainly in the late S- or G2-phase of the cell cycle as revealed by flow cytometry. Alkyl lysophospholipid analogs II and cholesterol form liposomes with high encapsulation efficiency and low permeability for entrapped substances. Compounds II were active against Lewis lung carcinoma in mice when applied in free form or as liposomes.

Animals↗

PubMed1987

Altered growth control by natural growth inhibitors in the mutant HD33 substrain of the Ehrlich-Lettré ascites mammary carcinoma.

The mutant substrain HD33 of the Ehrlich-Lettré ascites mammary carcinoma (EAC) was found to be altered in its ability to respond to and to produce natural growth inhibitors. Cells of this substrain did not respond to (1) a highly purified growth inhibitor from bovine mammary gland, inhibiting the proliferation of two strains of the original EAC already at concentrations of 0.5-2.0 ng/ml, (2) inhibitory activities found in the ascites fluid of these sensitive strains and partially purified. However, from the ascites fluid of the mutant substrain an inhibitory activity was partially purified, which was inhibitory towards this substrain. It also inhibited the cells from an original strain, although less effectively. This new inhibitory activity is similar in its properties to that of the others investigated in that its mol. wt is between 10,000 and 50,000 D, it is heat labile and its activity can be overcome by insulin, epidermal growth factor and 2'-deoxycytidine, the dose-response curve levels off at 35-45% inhibition. The results demonstrate that the regulation of cell proliferation by endogenous (autocrine) inhibitors can be altered by mutagen treatment.

Animals↗

PubMed1987

[(E)-5-(2-bromovinyl)-2'-desoxyuridine--a new nucleoside analog with selective inhibitory action against herpesviruses. Studies in cell culture and animal experiments].

(E)-5-(2-Bromovinyl)-2'-deoxyuridine (1; BrVUdR) inhibits the replication of herpes simplex virus type 1 (HSV-1) and of varicella-zoster virus (VZV) in vitro at concentrations of 0.01 to 0.23 mumol/l, whereas herpes simplex virus type 2 (HSV-2) is influenced only at 5.5 to 27 mumol/l. In comparison to some classical and newly developed antiherpetics, i. e. 5-iodo-2'-desoxyuridine (2; idoxuridine, IDU), 9-beta-D-arabinofuranosyladenine (4; vidarabine Ara-A), 9-(2-hydroxyethoxymethyl) guanine (5; acyclovir, ACV) and 2'-fluoro-5-iodo-1-beta-D-aracytosine (6;FIAC) the following order of decreasing activity was found:1 greater than 6 greater than 5 greater than 2 greater than 4 (against HSV-1) and 6 greater than 2 greater than 5 greater than 1 greater than 4 (against HSV-2). The high selectivity of the antiviral effect of BrVUdR towards HSV-1 and TZV is based on the fact, that proliferation of different mammalian cell lines is inhibited by 50% only at concentrations as high as 90 to 170 mumol/l, resulting in a therapeutical index of 1000 to 10,000. Successful treatment of an HSV-1 encephalitis in mice as well as an HSV-1 keratitis of rabbits confirmed the efficiency of 1 in experimental animal infections. No toxic side effects in both local and systemic applications were observed. Promising data from cell culture and animal experiments recommend 1 as a potential candidate for the local and systemic treatment of HSV-1 and VZV infections in man.

Animals↗

PubMed1987

The colony-to-cluster ratio in agar cultures of bone marrow. I. Constancy under standardized conditions and alteration in tumour-bearing mice and by various inhibitors added in vitro.

The growth of granulocyte-macrophage colony forming cells (GM-CFC) from mouse bone marrow was studied in agar cultures in glass capillaries. Under standard conditions with normal bone marrow a specific total number of cell clones (colonies + clusters) and also a specific colony-to-cluster ratio (CClR) was found for each of 4 different mouse strains. Both parameters were studied in tumour-bearing mice and found to be influenced independently and, in part, in an opposite way. Thus, for bone marrow of mice with the Ehrlich ascites carcinoma an inhibition of clone formation was found whereas the growth of those clones still formed was stimulated (only colonies present). "Natural" inhibitors as present in uraemic serum added to the cultures decreased the number of clones without altering the CClR. The same is true for the inhibitors in CSF-containing endotoxin serum. On the other hand, inhibitors such as spermine or the synthetic 3'-deoxy-3'-fluorothymidine depressed the clone formation as well as the growth rate of the clones (relative increase of clusters and decrease of colonies). It is concluded that the sensitivity of the GM-CFC against various influences may differ from that of the cells in the clones they form; therefore, the determination of the CClR will provide a more refined interpretation of influences on the haematopoietic system.

Adolescent↗

PubMed1987

The colony-to-cluster ratio in agar cultures of bone marrow. II. Influence of a GM-CFC inhibiting factor.

An inhibitory activity for the proliferation of granulocyte-macrophage colony-forming cells (GM-CFC) of mouse bone marrow in soft-agar cultures was partly purified from rat bone marrow conditioned medium by ultrafiltration and gel chromatography. It was selective in that it also inhibited the proliferation of myeloid but not that of lymphatic mouse leukaemia cells. Even at strong (greater than 60%) inhibition of the formation of cell clones (colonies + clusters) the colony-to-cluster ratio (CClR) remained as in the controls. The data point to a long-lasting and selective effect of the inhibitor on the GM-CFC without influence on the proliferation of its progeny. This conclusion is confirmed by the temporal development of the inhibitory effect in the course of culture. The determination of the CClR enables one to discriminate between proliferation modulating effects (CClR alteration) and those acting via ON/OFF-switching mechanisms (CClR constancy) influencing the clone forming efficiency at all.

Agar↗

PubMed1985

Ribonucleotide reductase in ascites tumour cells detected by electron paramagnetic resonance spectroscopy.

Tyrosine radicals localized in the M2 subunits of ribonucleotide reductase have been detected by electron paramagnetic resonance (EPR) in ordinary ascites tumour cells. The intensity of its doublet EPR spectrum is higher in rapidly proliferating cells. Hydroxyurea, a specific inhibitor of this enzyme, decreases the concentration of the tyrosine radical. Whereas in different ascites tumours the doublet EPR spectrum dominates at g = 2.004, in solid tumours another more intense EPR spectrum from nitrosyl-hemoproteins appears. In conclusion, EPR spectroscopy can be used to monitor the content and variations of active M2 subunits of ribonucleotide reductase in intact ascites tumour cells.

Animals↗

PubMed1985

Specific neutralizing antiserum against a polypeptide growth inhibitor for mammary cells purified from bovine mammary gland.

A recently published method for purification of a new inhibitor of growth of mammary cells in vitro from bovine mammary gland has been modified to yield several hundred micrograms of inhibitor per kg of glandular tissue. The inhibitory effect exerted by this preparation to Ehrlich ascites mammary carcinoma cells fulfilled all biological criteria of specificity established earlier for preparations obtained by other means, the most important being that the inhibitory effect is abolished by the epidermal growth factor and insulin. The preparation is shown to consist mainly of a protein of 13 kDa which appears to be not glycosylated. An antiserum raised in mice against the inhibitor is demonstrated to be specific for the 13 kDa bovine mammary gland protein. Neutralization of the inhibitory activity by the specific antiserum strongly supports the view that the 13 kDa protein is indeed the carrier of inhibitory activity. First data on tissue distribution obtained with an enzyme-linked immunosorbent assay revealed a high concentration of the anti-inhibitor-antiserum-reactive antigen in bovine lactating but not in non-lactating mammary gland tissue and in milk fat globule membranes. Some reactivity was also found in bovine lung. These data are interpreted with respect to a possible physiological significance of the growth inhibitor.

Animals↗

PubMed1985

3'-Fluoro-2',3'-dideoxyribonucleoside 5'-triphosphates: terminators of DNA synthesis.

It is shown that dNTP(3'F) are terminators of DNA synthesis and may serve as very effective tools for DNA sequencing with E.coli DNA polymerase I and AMV reverse transcriptase. The dNTP(3'F) are found to be chain terminator substrates for calf thymus terminal deoxyribonucleotidyl transferase but not for calf thymus DNA polymerase alpha. The optimal dNTP(3'F) concentration for DNA sequencing by DNA polymerase I is found to be an order of magnitude lower than that of ddNTPs. dNTP(3'F) produce a more clear sequence pattern than do ddNTPs.

Animals↗

PubMed1985

3'-Deoxy-3'-fluorothymidinetriphosphate: inhibitor and terminator of DNA synthesis catalysed by DNA polymerase beta, terminal deoxynucleotidyl transferase and DNA polymerase I.

3'-deoxy-3'-fluorothymidine-5'-triphosphate (dFTTP) was examined with respect to its capacity to inhibit and to terminate DNA-synthesis catalysed by DNA polymerases alpha and beta, terminal deoxynucleotidyl transferase (TdT) and E. coli DNA polymerase I (Klenow fragment). In analogy with 2', 3'-dideoxythymidine-5'-triphosphate (ddTTP) it was found that DNA polymerase beta was much more sensitive to dFTTP inhibition than DNA polymerase alpha. Moreover, the activity of TdT is strongly inhibited by both dFTTP and ddTTP, whereas the two compounds displayed a lower inhibitory potency on DNA polymerase I. Except for DNA polymerase alpha, the other investigated DNA polymerases are able to incorporate dFTTP into poly (dA).oligo (dT) and oligo (dG), respectively. The specific chain-terminating activity of dFTTP has proved for DNA sequencing to be at least equivalent to ddTTP.

Animals↗

PubMed1984

Human tissues degrade uridine much less than thymidine. Possible consequence for 5-fluorouracil therapy.

In view of clinical trials to improve FUra chemotherapy of cancer by combined application with Urd and dThd, we investigated the capacity of human tissues to split these nucleosides. All human normal and neoplastic tissues gave a uridine-splitting activity which can be inhibited by beta-L-pTdR and behaves in this respect as uridine-deoxyuridine phosphorylase (EC 2.4.2.3). dThd splitting, however, which is 2-9-fold higher than that of Urd, is insensitive towards beta-L-pTdR, confirming earlier results that it is due to thymidine phosphorylase (EC 2.4.2.4). On the other hand, tissues, e.g., spleen of rats and mice, in which dThd and Urd are split by uridine-deoxyuridine phosphorylase, degrade 2-5-fold more Urd than dThd. Thus, free pyrimidine base competing with FUra for degradation and thus prolonging the life time of the drug in the body, will be formed mainly from dThd in the human body but more so from Urd in the rat or mouse.

Animals↗