Lothar Böhm

Hyperprolactinemia in acute myeloid leukemia and indication of ectopic expression of human prolactin in blast cells of a patient of subtype M4

The sera of 28 patients with acute myeloid leukemia AML--subtypes M1 to M6 were screened for human prolactin (h-PRL). Serum TSH (thyroid stimulating hormone), LH (luteinizing hormone), FSH (follicle stimulating hormone), beta-estradiol, free T4 (tetra-iodotyronine) and testosterone were also determined. It was found that in 16 of the 28 patients h-PRL was significantly elevated while all other endocrine values were normal. In a patient subtype M4 with elevated serum h-PRL we demonstrate by immunoblotting that the hormone and its dimer are present in the blast cells. This may reflect ectopic synthesis due to altered expression of homeobox genes and a requirement of h-PRL as a growth stimulant of the leukemic myeloblast.

Cytotoxicity of azadirachtin A in human glioblastoma cell lines.

The neem toxin azadirachtin A exhibits selective toxicity on insects. Despite its well-proven efficacy, the mode of action of this toxin remains obscure. The toxicity on vertebrate cells compared to insect cells is also not well characterized. We have cultivated six human glioblastoma cell lines G-28, G-112, G-60 (TP53 mutant) and G-44, G-62, G-120 (TP53 wild-type) in the presence of 28 microM of azadirachtin. This toxin concentration was chosen because it represents the 25 to 50% lethal dose in the glioma cells. Toxicity was measured in terms of cell proliferation (binucleation index), formation of micronuclei and cell survival. In the TP53 mutant cell lines, azadirachtin reduced the proportion of dividing cells and induced formation of micronuclei. Except for G-44 which showed a decrease in binucleation index, proliferation in the TP53 wild-type cell lines was unaffected by azadirachtin. In the TP53 wild-type cell lines, the decrease in micronuclei frequency is attributed to fewer cells entering mitosis to produce micronuclei. This is also apparent from the low surviving fractions. Cell survival was suppressed by 25-69% in all cell lines. The reduction of cell survival is a clear indication that azadirachtin affects reproductive integrity and cell division. The induction of micronuclei reflects DNA damage. Similar studies on damage induction in insect cell lines could elucidate the processes which precede the antifeedant and antimoulting effects of azadirachtin and other neem toxins in insects.

H1-mediated Repression of Transcription Factor Binding to a Stably Positioned Nucleosome

Previously, we reported that histone H1 binding to nucleosome cores results in the repression of binding of the basic helix-loop-helix upstream stimulatory factor (USF) (Juan, L.-J., Utley, R. T., Adams, C. C., Vettese-Dadey, M., and Workman, J. L. (1994) EMBO J. 13, 6031-6040). We have tested whether this inhibition resulted from H1-mediated changes in nucleosome positioning (Ura, K., Hayes, J. J., and Wolffe, A. P. (1995) EMBO J. 14, 3752-3765) forcing the USF recognition sequence into less accessible locations within the nucleosome. Nucleosome boundaries were determined by assays combining micrococcal nuclease and restriction endonuclease digestion. A unique pair of boundaries were observed, indicating a single nucleosome translational position. This nucleosome position did not change on H1 or USF binding. Thus, H1 repression of USF binding was independent of nucleosome mobility, indicating an alternative mechanism of H1 repression. H1 repressed USF binding at a site 35 base pairs into the nucleosome core more effectively than at a site near the “linker” DNA, suggesting that inhibition by H1 was not simply due to steric occlusion. Instead, these data are consistent with a model by which H1 binding reduces transient dynamic exposure of the DNA from the histone octamer surface (Polach, K. L., and Widom, J. (1995) J. Mol. Biol. 254, 130-149).

The role of G2-block abrogation, DNA double-strand break repair and apoptosis in the radiosensitization of melanoma and squamous cell carcinoma cell lines by pentoxifylline

Purpose : To examine the role of G2-block abrogation, DNA repair inhibition and apoptosis in the enhancement of radiotoxicity by pentoxifylline. Materials and methods : The influence of pentoxifylline on radiotoxicity was assessed by colony assay in TP53 wild-type Be11 and mutant MeWo melanoma, and in TP53 wild-type 4197 and mutant 4451 squamous cell carcinoma (SCC) cell lines. G2-block abrogation was assessed by flow cytometry. Induction of DNA damage and repair was measured over a dose range of 0-100Gy by constant field gel electrophoresis (CFGE). The Annexin-V binding assay was used to identify apoptotic cells. Results : Pentoxifylline, when combined with irradiation, significantly increased radiotoxicity in the TP53 mutant MeWo and 4451 cell lines by radiotoxicity enhancement factors of 3 and 14.5 respectively. No radiosensitization was seen in the TP53 wild-type Be11 and 4197 cells. When the drug was added after irradiation at the time of maximum G2-block expression, no radiosensitization was seen in any of the four cell lines. CFGE analyses showed that pentoxifylline effectively suppressed DNA double-strand break (DSB) repair in all four cell lines, as indicated by 20 h repair inhibition factors of 1.4-2.4. Pentoxifylline did not increase apoptosis in any of the four cell lines. Conclusion : These data suggest that radiosensitization by pentoxifylline is not a consequence of G2-block abrogation alone, but that inhibition of DSB repair plays a role in certain cell types.PURPOSE To examine the role of G2-block abrogation, DNA repair inhibition and apoptosis in the enhancement of radiotoxicity by pentoxifylline. MATERIALS AND METHODS The influence of pentoxifylline on radiotoxicity was assessed by colony assay in TP53 wild-type Bell and mutant MeWo melanoma, and in TP53 wild-type 4197 and mutant 4451 squamous cell carcinoma (SCC) cell lines. G2-block abrogation was assessed by flow cytometry. Induction of DNA damage and repair was measured over a dose range of 0-100 Gy by constant field gel electrophoresis (CFGE). The Annexin-V binding assay was used to identify apoptotic cells. RESULTS Pentoxifylline, when combined with irradiation, significantly increased radiotoxicity in the TP53 mutant MeWo and 4451 cell lines by radiotoxicity enhancement factors of 3 and 14.5 respectively. No radiosensitization was seen in the TP53 wild-type Be11 and 4197 cells. When the drug was added after irradiation at the time of maximum G2-block expression, no radiosensitization was seen in any of the four cell lines. CFGE analyses showed that pentoxifylline effectively suppressed DNA double-strand break (DSB) repair in all four cell lines, as indicated by 20 h repair inhibition factors of 1.4-2.4. Pentoxifylline did not increase apoptosis in any of the four cell lines. CONCLUSION These data suggest that radiosensitization by pentoxifylline is not a consequence of G2-block abrogation alone, but that inhibition of DSB repair plays a role in certain cell types.

Induction of morphological differentiation in the human leukemic cell line K562 by exposure to thalidomide metabolites.

The lineage and state of differentiation of cells in the mammalian haemopoietic compartment is associated with specific patterns of homeobox gene expression (EMBO J. 7, 2131, 1988). Agents which influence homeobox gene expression are thus of great interest in the study of human leukemias. Retinoic acid has direct regulatory actions on homeobox gene transcription (TIBS 158, 52, 1989; Differentiation 37, 773, 1988) and can induce select human leukemia cell lines to undergo terminal differentiation in vitro (Proc. natl Acad. Sci. U.S.A. 77, 2936, 1980). Retinoic acid is also a known teratogen for vertebrate foetal limb-bud development. Some of the teratogenic effects are duplicated by the drug Thalidomide (Embryopathic Activity of Drugs, Little Brown, Boston, p. 167, 1965; Haematological Cytology, Wolf Med. Pub. Ltd, London, p. 118, 1982). To investigate Thalidomide for other retinoid-like effects, we exposed cultures of human leukemia K562 cells to the metabolites generated in a Thalidomide hepatic-microsomal enzyme drug metabolizing system (Proc. natl Acad. Sci. U.S.A. 78, 2545, 1981). Here we report evidence that a single 2 h pulse-exposure to Thalidomide metabolites, induces K562 cells to undergo morphological differentiation in vitro. We also demonstrate a significant cytotoxic effect for these metabolites.

Inhibition of DNA repair by pentoxifylline and related methylxanthine derivatives

The methylxanthine drug Pentoxifylline is reviewed for new properties which have emerged only relatively recently and for which clinical applications can be expected. After a summary on the established systemic effects of Pentoxifylline on the microcirculation and reduction of tumour anoxia, the role of the drug in the treatment of vasoocclusive disorders, cerebral ischemia, infectious diseases, septic shock and acute respiratory distress, the review focuses on another level of drug action which is based on in vitro observations in a variety of cell lines. Pentoxifylline and the related drug Caffeine are known radiosensitizers especially in p53 mutant cells. The explanation that the drug abrogates the G2 block and shortens repair in G2 by promoting early entry into mitosis is not anymore tenable because enhancement of radiotoxicity requires presence of the drug during irradiation and fails when the drug is added after irradiation at the G2 maximum. Repair assays by measurement of recovery ratios and by delayed plating experiments indeed strongly suggested a role in repair which is now confirmed for Pentoxifylline by constant field gel electrophoresis (CFGE) measurements and for Pentoxifylline and for Caffeine by use of a variety of repair mutants. The picture now emerging shows that Caffeine and Pentoxifylline inhibit homologous recombination by targeting members of the PIK kinase family (ATM and ATR) which facilitate repair in G2. Pentoxifylline induced repair inhibition between irradiation dose fractions to counter interfraction repair has been successfully applied in a model for stereotactic surgery. Another realistic avenue of application of Pentoxifylline in tumour therapy comes from experiments which show that repair events in G2 can be targeted directly by addition of cytotoxic drugs and Pentoxifylline at the G2 maximum. Under these conditions massive dose enhancement factors of up to 80 have been observed suggesting that it may be possible to realise dramatic improvements to tumour growth control in the clinic.

Determination of the initial DNA damage and residual DNA damage remaining after 12 hours of repair in eleven cell lines at low doses of irradiation

Purpose : To determine the relationship between DNA damage and radiosensitivity at low doses (1-10Gy) for the initial DNA damage and residual DNA damage remaining after 12-h repair. Materials and methods : Eleven cell lines, normal human lung epithelial L132, HT29 human colon carcinoma, ATs4 human ataxia telangiectasia, normal CHO-K1 hamster, repair-deficient xrs1 and xrs5 mutants, repair-deficient SCID rodent cell line, the human normal fibroblast 1BR.3, human ataxia telangiectasia fibroblast AT1BR and the repair-deficient fibroblasts 180BR.B and 46BR.1 were irradiated with 60 Co γ-rays. Radiosensitivity was measured by clonogenic survival assay. DNA damage was measured by fluorometric analysis of DNA unwinding (FADU). Results : The radiosensitivity in the 11 cell lines ranged from SF2 of 0.02-0.61. By FADU assay, the undamaged DNA at 5-Gy ranged from 56 to 93%. The initial DNA damage and radiosensitivity were highly correlated (r 2 =0.81). After 5-Gy irradiation and 12-h repair, two groups of cell lines emerged. Group 1 restored undamaged DNA to a level ranging from 94 to 98%. Group 2 restored the undamaged DNA to a level ranging from 77 to 82%. No correlation was seen between residual DNA damage remaining after 12-h repair and radiosensitivity. Conclusion : It is shown that the initial DNA damage correlates with radiosensitivity at low doses of irradiation. This suggests that the initial DNA damage must be considered as a determinant for radiosensitivity.PURPOSE To determine the relationship between DNA damage and radiosensitivity at low doses (1-10 Gy) for the initial DNA damage and residual DNA damage remaining after 12-h repair. MATERIALS AND METHODS Eleven cell lines, normal human lung epithelial L132, HT29 human colon carcinoma, ATs4 human ataxia telangiectasia, normal CHO-K1 hamster, repair-deficient xrs1 and xrs5 mutants, repair-deficient SCID rodent cell line, the human normal fibroblast 1BR.3, human ataxia telangiectasia fibroblast AT1BR and the repair-deficient fibroblasts 180BR.B and 46BR.1 were irradiated with 60Co gamma-rays. Radiosensitivity was measured by clonogenic survival assay. DNA damage was measured by fluorometric analysis of DNA unwinding (FADU). RESULTS The radiosensitivity in the 11 cell lines ranged from SF2 of 0.02-0.61. By FADU assay, the undamaged DNA at 5-Gy ranged from 56 to 93%. The initial DNA damage and radiosensitivity were highly correlated (r2 = 0.81). After 5-Gy irradiation and 12-h repair, two groups of cell lines emerged. Group 1 restored undamaged DNA to a level ranging from 94 to 98%. Group 2 restored the undamaged DNA to a level ranging from 77 to 82%. No correlation was seen between residual DNA damage remaining after 12-h repair and radiosensitivity. CONCLUSION It is shown that the initial DNA damage correlates with radiosensitivity at low doses of irradiation. This suggests that the initial DNA damage must be considered as a determinant for radiosensitivity.

RBE variation as a function of depth in the 200-MeV proton beam produced at the National Accelerator Centre in Faure (South Africa).

BACKGROUND AND PURPOSE Thorough knowledge of the RBE of clinical proton beams is indispensable for exploiting their full ballistic advantage. Therefore, the RBE of the 200-MeV clinical proton beam produced at the National Accelerator Centre of Faure (South Africa) was measured at different critical points of the depth-dose distribution. MATERIAL AND METHODS RBEs were determined at the initial plateau of the unmodulated and modulated beam (depth in Perspex = 43.5 mm), and at the beginning, middle and end of a 7-cm spread-out Bragg peak (SOBP) (depths in Perspex = 144.5, 165.5 and 191.5 mm, respectively). The biological system was the regeneration of intestinal crypts in mice after irradiation with a single fraction. RESULTS Using 60Co gamma-rays as the reference, the RBE values (for a gamma-dose of 14.38 Gy corresponding to 10 regenerated crypts) were found equal to 1.16 +/- 0.04, 1.10 +/- 0.03, 1.18 +/- 0.04, 1.12 +/- 0.03 and 1.23 +/- 0.03, respectively. At all depths, RBEs were found to increase slightly (about 4%) with decreasing dose, in the investigated dose range (12-17 Gy). No significant RBE variation with depth was observed, although RBEs in the SOBP were found to average a higher value (1.18 +/- 0.06) than in the entrance plateau (1.13 +/- 0.04). CONCLUSION An RBE value slightly larger than the current value of 1.10 should be adopted for clinical application with a 200-MeV proton beam.

Monoclonal anti-histone H1 autoantibodies from MRL lpr/lpr mice

Hybridomas producing anti-histone monoclonal antibodies were generated from a 2-month old MRL lpr/lpr mouse. Five IgM antibodies showed binding to histone H3 and three of these IgM antibodies also bound histone H1. Two IgG2a antibodies (MRA3 and MRA12) were specific for histone H1 and their binding was further characterized. Both reacted strongly with mouse and calf thymus histones H1, but showed limited (if any) binding to H1 from non-mammalian species such as duck, trout, or sea urchin sperm. Histone H1 is composed of three domains: N (N-terminal), G (globular) and C (C-terminal). The binding of MRA3 and MRA12 antibodies to the N, G and C domains of the histone H1 molecule was also investigated, using purified H1 fragments. Significant binding was observed only with the GC fragment but not with isolated NG, G or C fragments. Moreover, the integrity of most, if not all, of the G domain (residues 33-122) was necessary for antibody binding, since cleavage of the H1 molecule either at residue 72 or 106 abolished the binding to MRA3 and MRA12. Taken together, these results could indicate that MRA3 and MRA12 antibodies recognize a conformational determinant of the H1 molecule.

Influence of apoptosis on the enhancement of radiotoxicity by ouabain.

Background: The Na+, K+-ATPase inhibitor ouabain enhances the toxocity of irradiation and we have previously demonstrated that the drug suppresses repair capacity. The influence of ouabain on apoptosis is not known and is examined in this study. Materials and Methods: Seven human cell lines of defined TP53 status were irradiated with 60Co-γ irradiation in the presence and absence of 10−10 M ouabain. Cell survival was determined by the clonogenic assay, apoptosis by acridine orange staining and cell cycle delays by flow cytometry. Results: The ouabain-induced enhancement of radiotoxicity, expressed as the ratio of SF2s, is independent of TP53 status and ranges from 1.1 to 2.8 depending upon cell line. Ouabain prolongs the irradiation-induced G2 delay in TP53 mutant tumor cell lines by a factor greater than 2, but not in the normal lung fibroblase L132, where the cell recovery is not altered in the presence of ouabain. Twenty hours postirradiation, ouabain enhances apoptosis induced by irradiation by factors of 1.3 to 1.7 depending on the cell line. Conclusion: Ouabain preferentially enhances the radiotoxocity in tumor cells irrespective of TP53 status. In the pattern of DNA damage responses which are influenced by ouabain we show that the G2 cell cycle delay is prolonged and that early apoptosis events are upregulated in TP53 wild type and TP53 mutant cells. It is concluded that apoptosis plays a significant role in the enhancement of radiotoxocity by ouabain.Hintergrund: Die Gegenwart des Na+-K+-ATPase-Inhibitors Ouabain erhöht die Strahlentoxizität. Wir haben bereits an anderer Stelle gezeigt, dass dieser Effekt bevorzugt in Tumorzellen auftritt und auf Unterdrückung der Reparaturkapazität beruht. Die Rolle der Apoptose ist in diesem Zusammenhang nicht bekannt und wurde hier untersucht. Material und Methodik: Sieben humane Zellinien mit bekanntem TP53-Status wurden mit 60Co-γ-Strahlen in Gegenwart von Ouabain bestrahlt. Zellüberleben wurde durch den Koloniebildungstest, Apoptose durch Acridine-Orange-Färbung und Zellzyklusänderungen mit Hilfe der Durchflusszytometrie untersucht. Ergebnisse: Die Erhöhung der Strahlentoxitiztät durch Ouabain, berechnet aus dem SF2-Verhältnis gegenüber Kontrollen, liegt im Bereich von 1,1 bis 2,8 und ist abhänging von der jeweils benutzten Zelllinie. Ein Einfluss des TP53-Status konnte nicht festgestellt werden. In TP53-mutanten Tumorzellen verlängert Ouabain den strahleninduzierten G2-Block um mindestens ein bis zwei Zellzyklusrunden. 20 Stunden nach Bestrahlung bewirkt Ouabain je nach Zelllinie eine Verstärkung der strahleninduzierten frühen Apoptoseereignisse um den Faktor 1,3 bis 1,7. Schlussfolgerungen: Zugabe von Ouabain bei der Bestrahlung bewirkt eine markante Erhöhung der Strahlentoxizität besonders in Tumorzellen, unabhängig vom TP53-Status. Im Muster der DNA-Schadensreaktionen zeigen wir, dass Ouabain den strahleninduzierten G2-Block drastisch verlängert und die frühen Apoptoseereignisse deutlich erhöht, und zwar sowohl in TP53-Wildtypen als auch in TP53-Mutanten. Wir folgern, dass Apoptose in der durch Ouabain ausgelösten Verstärkung der Strahlentoxizität eine wichtige Rolle spielt.