Ring-substituted diaqua(1,2-diphenylethylenediamine)platinum(II) sulfate shows unusual kinetics in its reaction with salmon testis DNA. The mechanism for diaqua[meso-1,2-bis(2,6-dichloro-4- hydroxyphenyl)ethylenediamine]platinum(II) sulfate, [Pt(H2O)2(meso-6)]2+SO4(2-), a representative of this series, has been investigated and compared with that for cis-[Pt(NH3)2(H2O)2]2+. Reactions were followed by atomic absorption, analytical HPLC of Pt-DNA digests, arrest of enzymatic DNA synthesis/degradation, ultraviolet and fluorescence spectrophotometry. Except for the formation of monofunctional DNA adducts, the kinetics of the platinum(II) complexes are comparable. The pseudo-first-order rate constant for the attack of DNA by [Pt(H2O)2(meso-6)]2+ follows the concentration of DNA in a hyperbolic fashion, which is in contrast to the linear dependence for cis-[Pt(NH3)2(H2O)2]2+. The hyperbolic dependence is typical for a dissociable DNA/drug complex preceding the coordination reaction. By studying the binding of free ligand to DNA, and by correlating ligand structures and electrostatic charges with effects on adduct formation, both the phenyl residues and the positive charge of the platinum(II) complex are shown to be crucial for the stability of the dissociable complex. A non-intercalative mode of binding to the DNA backbone is suggested. At the high concentrations of DNA found in cell nuclei, the reaction of the dissociable complex can, principally, become rate-limiting in the attack of DNA and thus reduce the cytotoxic efficiency of a drug.

Animals↗

PubMed1992

Phase I-II trial of a monoclonal anti-tumor necrosis factor alpha antibody for the treatment of refractory severe acute graft-versus-host disease.

In a multicenter pilot study, 19 patients with severe acute graft-versus-host disease (aGVHD) refractory to conventional therapy and serotherapy with a monoclonal anti-interleukin-2 receptor antibody were treated by in vivo infusion of a monoclonal anti-tumor necrosis factor alpha (TNF alpha) antibody (B-C7). Ten patients were grafted from a genotypically identical sibling, five from an HLA-mismatched family member, and four from an HLA-matched unrelated donor. Before B-C7 treatment, 15 patients had grade IV and four had grade III GVHD. In all cases, patients received cyclosporine/methotrexate as aGVHD prophylaxis. Patients were administered increasing doses of antibody (from 0.1 to 0.4 mg/kg). The antibody was infused in bolus daily for 4 days and then every other day twice (6 doses). No side effects were observed during treatment regardless of the dose level used. Changes in peripheral blood cell counts occurred in 8 of the 19 patients and appeared to be unrelated to B-C7. No truly complete response was observed; eight patients achieved a very good partial response (42.6%) and six a partial response (31.5%). The treatment was ineffective in five patients (26.4%). When present, the response occurred early (less than 3 days). In the 14 responding patients, gut lesions responded best (100%), followed by skin (85%) and liver (35.7%) lesions. In 9 of 11 evaluable patients (81%), GVHD recurred when treatment was discontinued in a median delay of 3 days (range, 2 to 120 days). All except one died from aGVHD. Two patients did not experience GVHD recurrence and are still alive 13 and 18 months post-bone marrow transplantation. This pilot study shows that a monoclonal anti-TNF alpha antibody may be of benefit to some patients with severe refractory aGVHD, but is ineffective to prevent GVHD recurrence in the majority of cases.

Acute Disease↗

PubMed1992

Increased levels of tissue plasminogen activator (t-PA) and tissue plasminogen activator inhibitor (PAI) correlate with tumor necrosis factor alpha (TNF alpha)-release in patients suffering from microangiopathy following allogeneic bone marrow transplantation (BMT).

Severe microangiopathy resembling thrombotic thrombocytopenic purpura (TTP) has been reported as a complication of acute graft-versus-host disease (aGvHD) in patients receiving cyclosporin (CsA) prophylaxis following allogeneic BMT. In order to analyze the pathophysiological events involved in microangiopathy, a prospective study comparing release of von Willebrand Factor (vWF), t-PA and PAI, as well as TNF alpha and further coagulation parameters was performed in 32 patients. Endothelial damage as the central lesion was confirmed by the close association of vWF and t-PA:Antigen with severity of microangiopathy. t-PA activity, however, was neutralized by a simultaneous rise in PAI. Activation of coagulation in the course of microangiopathy was further confirmed by increased levels of DDimer (DDi), fibrinopeptide A (FPA), beta-thromboglobulin (beta TG) and platelet factor 4 (PF4). As clinical grades of microangiopathy, as well as the release of t-PA:Ag and PAI were correlated with systemic release of TNF alpha our data further support our hypothesis of cytokine induced endothelial damage in clinical complications following allogeneic BMT.

Acute Disease↗

PubMed1992

Specific inhibition of Physarum polycephalum DNA-polymerase-alpha-primase by poly(L-malate) and related polyanions.

Poly(L-malate) is an unusual polyanion found in nuclei of plasmodia of Physarum polycephalum. We have investigated, by enzymatic and fluorimetric methods, whether poly(L-malate) and structurally related polyanions can interact with DNA-polymerase-alpha-primase complex and with histones of P. polycephalum. Poly(L-malate) is found to inhibit the activities of the DNA-polymerase-alpha-primase complex and to bind to histones. The mode of inhibition is competitive with regard to DNA in elongation and noncompetitive in the priming of DNA synthesis. Spermidine, spermine, and histones from P. polycephalum and from calf thymus bind to poly(L-malate) and antagonize the inhibition. The polyanions poly(vinyl sulfate), poly(acrylate), poly(L-malate), poly(D,L-malate), poly(L-aspartate), poly(L-glutamate) have been examined for their potency to inhibit the DNA polymerase. The degree of inhibition is found to depend on the distance between neighboring charges, given by the number of atoms (N) interspaced between them. Poly(L-malate) (N = 5) and poly(D,L-malate) (N = 5) are the most efficient inhibitors, followed by poly(L-aspartate) (N = 6), poly(acrylate) (N = 3), poly(L-glutamate) (N = 8), poly(vinyl sulfate) (N = 3). It is proposed that poly(L-malate) interacts with DNA-polymerase-alpha-primase of P. polycephalum. According to its physical and biochemical properties, poly(L-malate) may alternatively function as a molecular chaperone in nucleosome assembly in the S phase and as both an inhibitor and a stock-piling agent of DNA-polymerase-alpha-primase in the G2 phase and M phase of the plasmodial cell cycle.

Animals↗

PubMed1992

Monofunctional DNA-platinum(II) adducts block frequently DNA polymerases.

The question of whether monofunctional DNA platinum(II) adducts block synthesis of DNA by purified DNA polymerases of different types and origin has been investigated by comparing the time dependence of synthesis arrest and of DNA adduct formation. Activated salmon testis DNA is used as a suitable substrate for DNA synthesis allowing to probe inhibition by platinum(II) monoadducts for the variety of inherent template-primers. Reaction amplitudes are related to defined mixtures of dichloro and chloroaqua platinum(II) complexes. It is found that (i) all investigated DNA polymerases seem arrested (100% efficiency) at bifunctional DNA adducts. (ii) human DNA polymerase beta bypasses most of the monofunctional lesions of the three platinum(II) complexes investigated. (iii) Klenow fragment is blocked by monoadducts with increasing efficiency in the order cis-diamminechloroaquaplatinum(II) (0%) less than meso-[1,2-bis(2,6- dichloro-4-hydroxyphenyl)ethylenediamine] chloroaquaplatinum(II) (50%) less than trans-diamminechloro-aquaplatinum(II) (75%). (iv) Escherichia coli DNA polymerase I, Thermus aquaticus DNA polymerase, Physarum polycephalum DNA polymerase alpha, and calf thymus DNA polymerase alpha appear to be arrested by monoadducts. According to these examples, blocking efficiencies depend on the cis/trans-stereogeometry of fixation of the carrier ligands at platinum(II) residues, on the size/chemical nature of the platin(II) carrier ligand and on the type/origin of DNA polymerase.

Animals↗

PubMed1992

Studies on the mechanism of tolerance or graft-versus-host disease in allogeneic bone marrow recipients at the level of cytotoxic T-cell precursor frequencies.

Previous studies demonstrated that the frequency of donor-versus-host-reactive cytotoxic T-cell precursors (CTL-p) before allogeneic bone marrow transplantation (BMT) from matched unrelated donors correlates with the incidence of graft-versus-host disease (GvH-D). We investigated whether clinical manifestations of GvH-D after HLA-identical sibling BMT are accompanied by an increased frequency of minor histocompatibility antigen (HA)-specific CTL-p. We further asked whether changes of third-party-reactive CTL-p as a measure of overall immunocompetence are related to infectious complications frequently seen immediately after BMT. Eighteen patients (16 with an HLA-identical bone marrow graft, two with either one HLA-A or one HLA-DR mismatch) were studied. Limiting dilution analysis (LDA) was used to assess donor CTL-p frequencies against recipient pre-BMT, donor, and third-party targets in a follow-up study. Eight cases receiving HLA-identical marrow grafts never developed signs of GvH-D. Undetectable or very low frequencies (1/131,458) of minor HA-specific CTL-p were demonstrated pre-BMT. Two recipients, one of an HLA-A- and one of an HLA-DR-mismatched graft, exhibited low frequencies of recipient-specific CTL-p (1/66,920 and 1/85,577, respectively) before transplantation, which further decreased despite mild GvH-D grade I, or decreased within 3 months after grafting in the other case. Eight patients receiving HLA-identical grafts developed GvH-D. Recipient-specific CTL-p were less than 1/300,000 in five patients during limited GvH-D (four with grade I and one with grade II disease of the skin), but were detectable in three patients presenting with extensive GvH-D grades II to III and ranged from 1/7,993 to 1/210,108. The differences in post-BMT recipient-specific CTL-p frequencies between patients with GvH-D grades 0 to I (median, less than 1/300,000) and those with GvH-D grades II to III (median, 1/111,970) were statistically significant (P less than .05). Posttransplant lymphocytes from all 18 patients contained less than 1/300,000 CTL-p with specificity for donor targets. Comparison of third-party-reactive CTL-p frequencies between donor and post-BMT recipient lymphocytes showed a severe and long-lasting depletion subsequent to BMT, which was not related to infectious complications. Again, these differences reached the level of statistical significance (median CTL-p before BMT, 1/4,417; after BMT, 1/14,289; P less than .005).(ABSTRACT TRUNCATED AT 400 WORDS)

Bone Marrow Transplantation↗

PubMed1992

DNA polymerase alpha--primase complex of Physarum polycephalum.

DNA polymerase alpha and DNA polymerase alpha--primase complex of Physarum polycephalum were purified by rapid methods, and antibodies were raised against the complex. In crude extracts, immune-reactive polypeptides of 220 kDa, 180 kDa, 150 kDa, 140 kDa, 110 kDa, 86 kDa, 57 kDa and 52 kDa were identified. The structural relationships between the 220 kDa, 110 kDa and 140 kDa (the most abundant form) was investigated by peptide mapping. The 140 kDa form was active DNA polymerase alpha. The 57 kDa and the 52 kDa polypeptides were identified as primase subunits by auto-catalytic labelling. In amoebae, the immune-reactive 140 kDa polypeptide was replaced by a 135 kDa active DNA polymerase alpha.

Animals↗

PubMed1992

[Graft versus host disease with fatal outcome after administration of filtered erythrocyte concentrates].

Transfusion-associated graft-versus-host disease (TA-GVHD) resulting from the engraftment of competent lymphocytes contained in blood products has been well described in immunocompromised patients and more recently in immunocompetent patients. Prophylactic irradiation of blood products prior to transfusion is the most efficient way to prevent TA-GVHD. Standard blood bank measures to reduce mononuclear cell contamination in red blood cell units, such as freezing, washing and filtration, may reduce the number of viable lymphocytes to prevent immunizations. However, it is unknown whether the depletion of leukocytes with these techniques would decrease the risk of TA-GVHD. In this report we describe the first case of TA-GVHD following transfusion of filtrated red blood cells given to a patient receiving cytotoxic therapy for Hodgkin's disease.

Adult↗

PubMed1992

Cancer after bone marrow transplantation. IBMTR and EBMT/EULEP Study Group on Late Effects.

Cancer may be serious late effect of marrow transplantation. Radiation, chemotherapy, immunosuppression and the original disease for which transplantation was performed may predispose to the development of cancer. 116 of 9732 patients reported to the IBMTR (International Bone Marrow Transplant Registry) have developed a new malignancy. Late effects were evaluated by the EBMT-EULEP (European Bone Marrow Transplant-European Late Effect Project) Late Effect Study Group in 147 patients surviving 6 years and 79 patients surviving more than 10 years. New malignancies developed in 11 of these patients. Lymphomas and leukemia comprised 73 cases reported to the IBMTR and one case reported to the EBMT-EULEP study. Tumors of the skin, oropharynx, vulva vagina and cervix prevailed in 41 patients with solid tumors. The distribution of malignancies is similar to that observed in organ transplant patients not given radiation or chemotherapy and suggests immunosuppression as a major contributory factor. In dogs the incidence of malignancies was studied after either chemotherapy or total body radiation in various regimens and marrow transplantation. Both chemotherapy and radiation shortened tumor-free survival in comparison to untreated dogs. Higher doses, larger fractions and shorter treatment schedules enhanced earlier tumor development. Soft tissue sarcomas and thyroid carcinoma were most frequent in treated, mammary carcinoma in untreated dogs. In treated dogs deaths from cancer were observed starting at the age of 5 years as compared to untreated dogs at the age of 9 years. The data from animal experiments indicate that the incidence of solid tumors in marrow transplant patients may still rise in the coming decades.

Animals↗

PubMed1991

Enhanced levels of cyclic AMP, adenosine(5')tetraphospho(5')adenosine and nucleoside 5'-triphosphates in mouse leukemia P388/D1 after treatment with cis-diamminedichloroplatinum(II).

As part of the exploration of the mechanism of platinum(II) complex-induced growth inhibition and/or cytotoxicity, we studied the intracellular levels of several nucleotides during treatment of mouse leukemia P388/D1 at selected concentrations of 1 microM cis-diamminedichloroplatinum(II) (cis-DDP) and 20 microM of its trans-isomer (trans-DDP). The effects and their time-dependences are correlated with those on cell growth parameters previously published (Just G and Holler E, Cancer Res 49: 7072-7078, 1989). The effects of cis-DDP are strong and irreversible, whereas those of trans-DDP are marginal and reversible, in parallel with similar effects on cell growth parameters. Concentrations of nucleoside 5'-di- and 5'-triphosphates increase in parallel with cellular DNA and protein content by three- to four-fold after 60 hr of treatment. The nucleoside monophosphates dAMP, dGMP and dTMP reveal concentration maxima during exponential cell growth that are two- to six-fold higher than in the control cultures. Levels of cyclic AMP, adenosine(5')tetraphospho(5')adenosine (Ap4A) and CDP rise three- to five-fold above those in the control cultures within a few hours of the start of treatment. The level of coenzyme NAD+ falls below that of the control, concomitantly with an arrest of cells in the G2 phase of the cell cycle and with the appearance of giant cells. Due to the high reactivity of cis-DDP and the continuous concentration increase during the treatment, purine nucleoside 5'-triphosphates provide a possibility for the acquisition of resistance to cis-DDP. The correlation of responses of metabolically and regulatory active nucleotides with biological effects suggests their function in antitumorigenesis.

Adenosine Triphosphate↗

PubMed1991

[TNF-alpha in allogeneic bone marrow transplantation. Significance for immunologic reconstitution, acute graft versus host disease and risk of complications after bone marrow transplantation].

Follow-up studies on the release of tumor necrosis factor-alpha (TNF-alpha) in 114 patients confirm that this cytokine is released early in the course of endothelial complications, acute graft-versus-host disease, and interstitial pneumonitis following allogeneic bone marrow transplantation. Possible sources of systemic TNF-alpha activity are, in particular, tissue macrophages of the recipient type that are stimulated by pretransplant conditioning and endotoxin and subsequently further activated by interferon gamma released by donor lymphocytes. Consideration of these interactions permits the early recognition of high-risk patients, and suggests new strategies for prophylaxis and treatment of complications.

Bone Marrow Transplantation↗

PubMed1991

The reaction of platinum(II) complexes with DNA. Kinetics of intrastrand crosslink formation in vitro.

The kinetics of the formation of bifunctional DNA platinum(II) adducts (DNA-crosslinks) have been investigated by endonuclease digestion and subsequent HPLC analysis of the soluble nucleotides and nucleotide platinum(II) adducts. The results indicate two waves of crosslinking [rate constants (0.2-0.3) min-1 and (0.015-0.025) min-1] that correlate with changes in ultra violet absorbance and ethidium bromide dependent fluorescence intensity, previously interpreted in terms of two consecutive, local conformational rearrangements of platinum-DNA (Schaller, W., Reisner, H., and Holler, E. (1987) Biochemistry 26, 943-950). The formation of crosslinks at sequences d(GpG) and d(GpNpG) follows identical kinetics. A minimal reaction mechanism is proposed for the binding of cis-diamminedichloroplatinum(II) to DNA under in vitro conditions. The approximately 3-fold higher rate for meso-[1,2-bis(2,6-dichloro-4- hydroxyphenyl)ethylenediamine]diaquaplatinum(II) in comparison to the rate for cis-diamminediaquaplatinum(II) indicates that crosslink formation is affected by the nature of the non-leaving platinum ligand(s).

Chromatography, High Pressure Liquid↗

PubMed1991

Investigations on the antiproliferative effects of amino acid antagonists targeting for aminoacyl-tRNA synthetases. Part III--Combination experiments.

The combined effects of amino acid antagonists with proven or potential inhibitory activities on aminoacyl-tRNA synthetases were investigated on the murine leukemic cell line P388 D1. As the best result a summation of the antiproliferative effects was observed. Combinations with established cytostatic agents like platinum complexes or other antitumor compounds also yielded partly additive effects. In experiments performed with asparaginase L-aspartic acid-beta-hydroxamate gave synergistic growth inhibition of P388 D1 cells in vitro, which was reflected by additive effects against murine leukemia P388 in vivo.

Amino Acids↗