Tumor inhibiting properties of stereoisomeric [1,2-bis(3-hydroxyphenyl) ethylenediamine]dichloroplatinum(II)-complexes, Part II: Biological properties

The activity of stereoisomeric [1,2-bis(3-hydroxyphenyl)ethylenediamine] dichloroplatinum(II)-complexes (1-PtCl2,R,S; 2-PtCl2, R,R/S,S; 3-PtCl2, R,R; 4-PtCl2, S,S) on several tumor models (MDA-MB 231 breast cancer cell line; P 388 leukemia, mouse; L 1210 leukemia, mouse; L 5222 leukemia, rat; Ehrlich ascites tumor, mouse--wildtype; cisplatin-, etoposide-, cyclophosphamide-, and daunomycin-resistant, resp.) is described. For comparison the analogous [1,2-bis(4-hydroxyphenyl)ethylendiamine]dichloroplatinum (II)-complexes (5-PtCl2, R, S; 6-PtCl2, R,R/S,S; 7-PtCl2, R,R; 8-PtCl2, S,S) and cisplatin are used. 1-PtCl2 to 4-PtCl2 (OH in 3,3'-positions) show their maximum antitumor effect at lower doses than 5-PtCl2 to 8-PtCl2 (OH in 4,4'-positions). 2-PtCl2 and 6-PtCl2 (R,R/S,S) are more active than 1-PtCl2 and 5-PtCl2 (R,S). 4-PtCl2 and 8-PtCl2 (S,S) are superior to 3-PtCl2 and 7-PtCl2 (R,R). On the L 5222 leukemia 2-PtCl2 (R,R/S,S), 4-PtCl2 (S,S) and 8-PtCl2 (S,S) markedly surpass cisplatin. Strong effects are produced by 2-PtCl2 to 4-PtCl2 on the Ehrlich ascites tumor (wildtype, cisplatin-, etoposide-, cyclophosphamide-, and daunomycin-resistant, resp.). The combination of 4-PtCl2 with cisplatin results in a weakly synergistic effect.     

DNA damage and apoptosis in mononuclear cells from glucose-6-phosphate dehydrogenase-deficient patients (G6PD Aachen variant) after UV irradiation

Patients affected with X chromosome-linked, hereditary glucose-6-phosphate dehydrogenase (G6PD) deficiency suffer from life-threatening hemolytic crises after intake of certain drugs or foods. G6PD deficiency is associated with low levels of reduced glutathione. We analyzed mononuclear white blood cells (MNC) of three males suffering from the German G6PD Aachen variant, four heterozygote females of this family, one G6PD-deficient male from another family coming from Iran, and six healthy male volunteers with respect to their DNA damage in two different genes (G6PD and T-cell receptor-delta) and their propensity to enter apoptosis after UV illumination (0.08-5.28 J/cm2). As determined by PCR stop assays, there was more UV-induced DNA damage in MNC of G6PD-deficient male patients than in those of healthy subjects. MNC of G6PD-deficient patients showed a higher rate of apoptosis after UV irradiation than MNC of healthy donors. MNC of heterozygote females showed intermediate rates of DNA damage and apoptosis. It is concluded that increased DNA damage may be a result of deficient detoxification of reactive oxygen species by glutathione and may ultimately account for the higher rate of apoptosis in G6PD-deficient MNC.     

Macromolecular DNA-damage in murine and human leukemic and lymphoid cells after in vitro exposure to ASTA Z 7557 (INN mafosfamide)

Summary Cyclophosphamide (CPA) is widely used against leukemic and lymphoproliferative diseases, but in vitro studies on response to this agent so far have been limited to instable derivatives with poor galenic properties. ASTA Z 7557 is a newly synthesized activated cyclophosphamide that circumvents the need for hepatic activation and has good stability. The critical cytotoxic lesions after exposure to bifunctional alkylating agents presumably are DNA interstrand crosslinks (ISC). We have, therefore, examined the formation and apparent removal of ISC after in vitro treatment with ASTA Z 7557 by use of the highly sensitive alkaline elution technique. Survival of murine L1210 cells was determined after 1 hour in vitro exposure with a D 37 value of 5.7 g/ml (from the initial shoulder part of the survival curve) and a Do value of 1.5 g/ml (from the exponential part of the curve). Previous labelling of L1210 cells by ¹²⁵IUdR simplified the alkaline elution procedure but there was some cytotoxicity of the radiochemical itself with a reduction of cloning efficiency from 77% to 61 %. The maximum of ISC was observed at 6 h after initiation of treatment with much of the damage apparently removed at 24 h. The simultaneous presence of DNA single strand breaks (SSB), however, confounds the analysis of DNA damage at 24 h and early cytolysis and unaided death of human lymphocytes often preclude the analysis of macromolecular damage at this time. Human peripheral blood cells isolated from patients with leukemic or lymphoproliferative diseases showed a remarkable heterogeneity with regard to the formation of ISC at 3 h. Thus, analysis of macromolecular damage may become an additional prognostic factor for response to CPA beyond the morphologic classification of these diseases.     

DNA methylation changes in multiple myeloma.

Using a candidate gene approach, we analyzed the methylation status of the promoter-associated CpG islands of 11 well-characterized tumor suppressor genes by methylation-specific polymerase chain reaction in five multiple myeloma (MM) cell lines and 56 patients with malignant plasma cell disorders. The frequency of aberrant methylation among the patient samples was 46.4% for SOCS-1, 35.7% for p16, 21.4% for E-cadherin, 12.5% for DAP kinase and p73, 1.8% for p15, MGMT as well as RARbeta, and 0% for TIMP-3, RASSF1A and hMLH1. We found at least one hypermethylated gene in 80.4% of the primary patient samples, while 33.9% harbored two or more hypermethylated genes. For the first time, we show that p73 may be hypermethylated in MM and thus be involved in the pathogenesis of plasma cell disorders. Hypermethylation of p16 at diagnosis was associated with a poorer prognosis. In patients with plasma cell leukemia, we found frequent simultaneous hypermethylation of p16, E-cadherin and DAP kinase. We conclude that aberrant methylation of tumor suppressor genes is a common event in malignant plasma cell disorders and that there is a correlation between methylation patterns and clinical characteristics in MM patients.     

Post-partum acquired haemophilia after IVF without recurrence during a second pregnancy obtained by IVF

Sir, We were highly interested in the case report published by Nakauchi-Tanakaet al. (2003), reporting a factor VIII inhibitor in ovarianhyperstimulation syndrome (OHSS). Briefly, acquired haemophilia is a rare disease most often dueto the development of autoantibodies directed against factorVIII that interfere with its coagulant function. The incidenceis about 1 per million persons per year. Acquired haemophiliamay occur in association with autoimmune diseases such as systemiclupus erythematosus or rheumatoid arthritis, neoplastic diseases,drug hypersensitivity and pregnancy. However, 50% of the casesremain idiopathic. Post-partum factor VIII inhibitors most oftendisappear     

Leptin contributes to the protection of human leukemic cells from cisplatinum cytoxicity

Leptin (ob gene) and its cognate receptor (obr) are relevant for fat metabolism. Obr shares homology with the IL-6 signal transducer gp130 and is expressed in hematopoietic cells. Since cytokines and growth factors regulate both hematopoiesis and response to chemotherapy, we tested the hypothesis of whether leptin protects leukemic cells from cytotoxicity of cisplatinum. Antisense phosphorothioate oligodeoxynucleotides (ODNs) and antisense peptide nucleic acids (PNAs) complementary to the obr gene were first tested for their growth inhibitory activity in obr expressing leukemic cells. Liposome-mediated transfection of ODNs (1-2 microM) or PNAs (0.01-1 microM) inhibited growth up to 50%. Combination treatments of cisplatinum and 0.01 microM PNA reduced growth more than cisplatinum alone. Vice versa, recombinant human leptin (rhL) diminished cisplatinum-induced growth inhibition. Finally, we investigated whether rhL affects cisplatinum-induced DNA damage and repair in the housekeeping gene beta-actin by means of real time TaqMan polymerase chain reaction. RhL reduced DNA damage and increased DNA repair. The effects are, however, modest and leptin is probably not the only player in the armory of growth factors which affect drug resistance.     

Continuous infusion versus intermittent bolus injection of bleomycin in a human embryonal testicular cancer xenograft

In vivo experiments with bleomycin in conventional murine tumor systems indicate a superiority of continuous-infusion schedules over daily intermittent bolus injections. This schedule of administration has been incorporated into many clinical trials, especially in testicular cancer, on the basis of in vitro and in vivo experiments as well as favorable comparisons with historical controls. Since mechanisms linked to a superiority of continuous-infusion schedules may not be operative in every individual tumor line, we have compared both schedules of administration in a human testicular cancer xenograft. We found no significant difference with respect to antineoplastic response.     

The effect of hyperthermia on DNA repair

Summary In the past there were many individual observations on the value of hyperthermia in the treatment of human neoplasia but most of the information about the value of hyperthermia as a single agent or in the combined modality approach has come from laboratory investigations. Dose response curves for cell survival after exposure to heat are similar in shape to cell survival curves obtained after irradiation or treatment with some cytostatic agents. The shoulder in such curves suggests that repair of sublethal or potentially lethal damage takes place after hyperthermic treatment. On the level of molecular biology the process of cellular repair should correspond to repair of damage inflicted on deoxyribonucleic acid (DNA). We have shown by means of the BUdR assay that such DNA-repair synthesis does take place upon exposure to heat.Many investigations have provided evidence of a synergism between hyperthermia and ionizing irradiation or some cytostatic agents. It was suggested that such synergism might be caused by the inhibition of repair of sublethal damage by heat. After inflicting DNA damage by a strong alkylating agent (NA-AAF) we could demonstrate DNA-repair synthesis by means of the BUdR-assay during exposure to heat. At the present time results obtained by assaying DNA repair on the basis of cell survival and by means of the BUdR-assay are difficult to reconcile.

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Zur Wirkung von Hyperthermie auf DNS-Repair-Vorgänge

Zusammenfassung Bereits vor 100 Jahren war auf Grund von klinischen Beobachtungen ein nützlicher Effekt der Hyperthermie in der Krebsbehandlung postuliert worden. Über kasuistische Berichte hinaus ist jedoch der Stellenwert der Hyperthermie in der Krebsbehandlung noch nicht durch kontrollierte prospektiv angelegte klinische Studien belegt worden. Allerdings konnte in jüngster Zeit durch Versuche mit Transplantationstumoren und in Gewebekultursystemen die Wirkung der Hyperthermie auch im Zusammenhang mit Radiotherapie oder cytostatischer Chemotherapie quantifiziert werden. Die Dosis-Wirkungs-Kurven für das Überleben von Zellen in Gewebekultur nach Hyperthermiebehandlung zeigen eine Schulter im Anfangsteil, welche auch in den Dosis-Wirkungs-Kurven nach Radiotherapie und nach einigen Formen der cytostatischen Therapie gefunden wird. Man schreibt die Entstehung einer solchen Schulter in Überlebenskurven von Zellen Erholungsprozessen (repair) von subletalen oder potentiell letalen Schäden zu. Molekularbiologisch korreliert mit diesen Erholungsprozessen eine Beseitigung von Schäden am Genom, d.h. der Desoxyribonucleinsäure (DNS) im Zellkern. Wir konnten durch 5-Bromdesoxyuridin (BUdR) — Einbaustudien zeigen, daß sich, ausgelöst durch Hyperthermie, derartige Prozesse (DNA repair) an der DNS abspielen.In vitro und in vivo Untersuchungen haben einen Synergismus zwischen der Wirkung ionisierender Strahlen und gewissen Formen der Chemotherapie einerseits und der Hyperthermie andererseits aufgedeckt. Man erklärt diesen Synergismus dadurch, daß durch die Hyperthermie zelluläre Erholungsprozesse beeinträchtigt werden. Wir konnten jedoch zeigen, daß auch unter Hyperthermie DNS-Schäden behoben werden können, welche durch stark alkylierend wirkende Substanzen an der DNS hervorgerufen wurden. Weitere Untersuchungen sind erforderlich, um divergierende molekularbiologische und zellbiologische Ergebnisse befriedigend zu erklären.

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Mit Unterstützung des Landesamtes für Forschung, Ministerium für Wissenschaft und Kultur des Landes Nordrhein-Westfalen, Düsseldorf (Supported by the Landesamt für Forschung, NRW)     

Heart and muscle involvement by extra-medullary myeloid leukemia: a case report and review of the literature

Extra-medullary myeloid tumours (EMT) have been described after curative treatment for acute myeloid leukaemia (AML) in increasing numbers after allogeneic stem cell transplantation. The sites of manifestations are ubiquitous and the discovery is most frequently guided by symptoms reported by the patient or by findings on clinical examination. This study reports a case of EMT in muscles and the heart 1.5 years after allogeneic transplantation for an AML with t(8;21)(q22;23) who achieved a complete remission by use of an idarubicine-based combination chemotherapy. Pathological and imaging findings are presented and treatment options are discussed.     

Ungew?hnliche Ursache einer Erythrozytose bei einer 23-j?hrigen Erstgeb?renden in der 22. Schwangerschaftswoche

Zusammenfassung Fallbeschreibung: Eine 23-jährige Erstgebärende in der 22. Schwangerschaftswoche wurde wegen einer Erythrozytose und eines unplausibel erhöhten HbA_1c-Wertes (glykiertes Hämoglobin) internistisch vorgestellt. Familienanamnestisch ließ sich eruieren, dass ihr Vater seit zwei Jahren aufgrund einer Erythrozytose unklarer Ätiologie durch regelmäßige Aderlässe behandelt werde. Die weiterführende Familienuntersuchung erbrachte bei der Patientin und ihrem Vater eine Hämoglobinopathie vom Typ Hämoglobin (Hb) Andrew-Minneapolis. Diskussion: Die Hämoglobinvariante vom Typ Hb Andrew-Minneapolis ist in die Grupp der Hämoglobinopathien mit stark erhöhter Sauerstoffaffinität und konsekutiver kompensatorischer Erythrozytose einzuordnen. Die betroffenen Patienten sind in der Regel beschwerdefrei und normal körperlich belastbar; eine erhöhte Inzidenz von kardiovaskulären Erkrankungen konnte bisher hicht beobachtet werden. Es gibt keine sicheren Hinweise, dass eine Hämoglobinanomalie der Mutter mit deutlich erhöhter Sauerstoffaffinität mit negativen Auswirkungen auf den Fetus oder einer erhöhten Komplikationsrate unter der Geburt vergesellschaftet ist. Schlussfolgerung: Hämoglobinopathien mit deutlich erhöhter Sauerstoffaffinität sind eine seltene, aber wichtige Differentialdiagnose der Erythrozytose, deren Klärung diagnostische Irrwege für Patient und Arzt vermeidbar macht. Für die prognostische Beurteilung ist bedeutsam, dass diese Erythrozytose bei den betroffenen Patienten in weiteren Grenzen als kompensatorisch zu akzeptieren ist. Ein außergewöhnlich erhöhter oder erniedrigter HbA_1c-Wert ohne Vorliegen einer diabetischen Befundkonstellation sollte an eine Hämoglobinopathie denken lassen. Abstract Case Report: A 23-year-old pregnant woman presented with erythrocytosis and a spuriously elevated HbA_1c. Family history revealed that her father has been treated with phlebotomies for the last 2 years because of erythrocytosis of unknown cause. An examination of the family members demonstrated that the patient and her father were carriers of the hemoglobin (Hb) variant Hb Andrew-Minneapolis. Discussion: Hb Andrew-Minneapolis belongs to a group of hemoglobin variants with a high oxygen affinity resulting in compensatory erythrocytosis. The carriers of such hemoglobin variants are usually clinically asymptomatic, exercise tolerance appears unimpaired and there is no higher incidence of cardiovascular diseases. There is no clear-cut evidence that a maternal hemoglobinopathy with high oxygen affinity is accompanied by negative consequences for the fetus or a higher abortion rate. Conclusion: Hemoglobinopathies with a high oxygen affinity are a rare but important differential diagnosis of polycythemia. Under these circumstances erythrocytosis has to be accepted as the primary mode of compensation and does not require treatment, as long as blood viscosity is kept within tolerable limits. An excessively elevated or lowered HbA_1c without a history or symptoms of diabetes should lead to further investigations concerning the possibility of hemoglobinopathy.