Gallium(III) and iron(III) complexes of alpha-N-heterocyclic thiosemicarbazones: synthesis, characterization, cytotoxicity, and interaction with ribonucleotide reductase.

A series of gallium(III) and iron(III) complexes with five different 4N-substituted alpha-N-heterocyclic thiosemicarbazones, viz., 2-acetylpyridine N,N-dimethylthiosemicarbazone (1), 2-acetylpyridine N-pyrrolidinylthiosemicarbazone (2), acetylpyrazine N,N-dimethylthiosemicarbazone (3), acetylpyrazine N-pyrrolidinylthiosemicarbazone (4), and acetylpyrazine N-piperidinylthiosemicarbazone (5), with the general formula [GaLCl2] (HL = 1 and 2) and [ML2][Y] (M = Ga, HL = 1-5, Y = PF6; M = Fe, HL = 1-5, Y = FeCl4 and PF6) were synthesized and characterized by elemental analysis, a number of spectroscopic methods (NMR, IR, UV-vis), mass spectrometry, and X-ray crystallography. The in vitro antitumor potency was studied in two human cancer cell lines (41M and SK-BR-3). The central metal ions exert pronounced effects in a divergent manner: gallium(III) enhances, whereas iron(III) weakens the cytotoxicity of the ligands. The capacity of ligand 1 and its Ga(III) and Fe(III) complexes to destroy the tyrosyl radical of the presumed target ribonucleotide reductase is reported.

Anticancer drug sensitivity and expression of multidrug resistance markers in early passage human sarcomas.

We have established new human sarcoma lines and examined their sensitivity to common antitumor drugs and expression of putative multidrug resistance (MDR) proteins. Eighty-two sarcoma samples were transplanted in nude mice. Fourteen of these sarcomas were established as tumor cell lines. We determined a chemosensitivity profile to antitumor drugs (MDR drugs = doxorubicin, mitoxantrone, and vincristine; non-MDR drugs = cisplatin, ifosfamide, and bleomycin) for each tumor line in vivo. Response to chemotherapy with doxorubicin and ifosfamide was observed in 30-50% of these tumor lines. Our results obtained with xenotransplants are similar to the results documented in clinical trials in which doxorubicin and ifosfamide are effective in 30-50% of the patients. Furthermore, we examined expression of MDR-relevant markers like P-glycoprotein, MDR-associated protein, lung resistance protein, and mdr1 mRNA in these xenotransplants. A relationship between mdr1 mRNA expression and response to doxorubicin was demonstrated in >90% of our tumor lines. In six sarcomas with mdr1 mRNA expression, five were resistant against doxorubicin and cross-resistant against several other drugs, whereas from eight sarcomas, which lacked detectable mdr1 mRNA, seven were sensitive to doxorubicin and other drugs. We found lung resistance protein or MDR-associated protein expressed in three resistant and mdr1 mRNA-positive sarcomas. These results demonstrate that mdr1 mRNA expression is a putative marker for drug resistance in our sarcoma lines. We conclude, therefore, that inherent P-glycoprotein expression might be also responsible for drug resistance occurring in treatment of patients with sarcomas. The established tumor lines are useful for additional investigations on mechanisms of drug resistance in sarcomas and as models for preclinical screening of new antitumor drugs.

Expression of the mdr1 gene in bone and soft tissue sarcomas of adult patients.

The expression of the mdr1 gene was evaluated at the RNA level by northern and slot blot analysis, and at the protein level by immunohistochemistry, in a total of 29 bone and 32 soft tissue sarcomas. All patients, mainly adults, had not received previous chemotherapy. Of the tumours investigated, 69% were mdr1-positive. An intermediate mdr1 expression was observed most frequently, with the exception of osteosarcomas (high) and malignant fibrous histiocytomas (low). Detection of P-glycoprotein in selected tumours revealed consistent results. However, no conclusion can be drawn as yet regarding correlation of mdr1 expression and drug resistance in patients.

CT evaluation of osseous tumors.

Computed tomography was performed on 153 patients with histologically proved bone tumors. CT has been found to be important for demonstrating the extent of the lesion, in aiding decisions about the operative approach, especially in tumors of the shoulder girdle and pelvis, and also for planning of localized surgery to maintain extremities. CT is of limited value in determining the nature of tumors. CT has proved efficacious in the detection or ruling out of tumor recurrences and in the assessment after chemotherapy or radiation therapy.

[The value of computed tomography in monitoring the preoperative chemotherapy of osteosarcoma]. / Zum Wert der Computertomographie für das Monitoring der präoperativen Chemotherapie des Osteosarkoms.

Computed tomography was performed in 45 patients with osteosarcomas before and after preoperative chemotherapy. Changes of bone alterations, intramedullary tumour spread and the size of soft tissue masses are computer tomographic criteria for tumour response after chemotherapy. The computed tomography decision of responder or non-responder was correct in 37/45 patients. In comparison with conventional x-rays CT was superior. CT is a reliable method for preoperative extension diagnosis and for the decision-marking for limb-saving operations.

[Malignant fibrous histiocytoma of soft tissue. Possibilities and limitations of computed tomography]. / Das maligne fibröse Histiozytom (MFH) der Weichteile. Möglichkeiten und Grenzen der Computertomographie.

Computed tomography was performed in 27 patients with confirmed malignant fibrous histiocytoma of the soft tissue. Compared with the CT results obtained in other tumours of the soft tissues. CT accuracy in malignant fibrous histiocytoma was only 74%. Non-typical attenuation values, diffuse tumour growth and non-typical contrast enhancement are discussed as reasons for the diagnostic understaging or overstaging of these soft tissue tumours. A higher accuracy was found on ultrasound examination with regard to tumour extension. Bone destruction was better visualised by CT. A base-line CT is recommended postoperatively for the earlier detection of recurrent tumours in further investigations.

Homologous expression of the nrdF gene of Corynebacterium ammoniagenes strain ATCC 6872 generates a manganese-metallocofactor (R2F) and a stable tyrosyl radical (Y˙) involved in ribonucleotide reduction.

Ribonucleotide reduction, the unique step in the pathway to DNA synthesis, is catalyzed by enzymes via radical-dependent redox chemistry involving an array of diverse metallocofactors. The nucleotide reduction gene (nrdF) encoding the metallocofactor containing small subunit (R2F) of the Corynebacterium ammoniagenes ribonucleotide reductase was reintroduced into strain C. ammoniagenes ATCC 6872. Efficient homologous expression from plasmid pOCA2 using the tac-promotor enabled purification of R2F to homogeneity. The chromatographic protocol provided native R2F with a high ratio of manganese to iron (30:1), high activity (69 µmol 2'-deoxyribonucleotide·mg⁻¹ ·min⁻¹) and distinct absorption at 408 nm, characteristic of a tyrosyl radical (Y˙), which is sensitive to the radical scavenger hydroxyurea. A novel enzyme assay revealed the direct involvement of Y˙ in ribonucleotide reduction because 0.2 nmol 2'-deoxyribonucleotide was formed, driven by 0.4 nmol Y˙ located on R2F. X-band electron paramagnetic resonance spectroscopy demonstrated a tyrosyl radical at an effective g-value of 2.004. Temperature dependent X/Q-band EPR studies revealed that this radical is coupled to a metallocofactor. Similarities of the native C. ammoniagenes ribonucleotide reductase to the in vitro activated Escherichia coli class Ib enzyme containing a dimanganese(III)-tyrosyl metallocofactor are discussed.

PELDOR study on the tyrosyl radicals in the R2 protein of mouse ribonucleotide reductase.

Pulse electron-electron double resonance (PELDOR) has been employed to measure the distance between the putative tyrosyl radicals in the two halves of the R2 subunit from mouse ribonucleotide reductase. The results provide experimental evidence that the active, tyrosyl radical containing mouse R2 subunit forms a homodimeric form in solution. The distance between the two tyrosyl radicals present in the dimer was determined to be 3.25 +/- 0.05 nm.

[Malignant bone tumors--possibilities and limits of surgical treatment]. / Bösartige Knochentumoren--Möglichkeiten und Grenzen der operativen Behandlung.

Surgical treatment of patients for malignant bone tumours, its possibilities and limits, is expounded in this paper on the basis of results and experience obtained from more than 330 cases. Reference is made to adjuvant chemotherapy which today is an indispensable component of complex treatment at least for osteosarcoma and Ewing's sarcoma. Described are ablative procedures, ranging from amputation to hemicorporectomy, and limb-saving operations, including implantation of tumour-specific endoprotheses which has assumed growing importance in recent years.

The development of osteosarcoma in four-fold selected group of patients.

Patients with osteosarcoma have been submitted to four-fold selection using parameters previously shown to be associated factors. These are sex, age at onset, haptoglobins and histological type. Further data have been added to the final resulting sequence of patients. In the four-fold selected group there is an age-dependent AB0 distribution which confirms the relevance of the criteria used and, consequently, the influence on sarcoma development. It can be assumed from these investigations that the occurrence of osteosarcoma is modified by several genetically fixed factors (HP type, sex, AB0 group).

[Diagnostic imaging of peripheral soft tissue tumors with special reference to computed tomography]. / Bildgebende Diagnostik peripherer Weichteiltumoren unter besonderer Berücksichtigung der Computertomographie.

The further development of modern therapeutical procedures in the treatment of soft tissue tumors requires an improvement of radiological diagnostics. The diagnostic values of X-rays, angiography, sonography and computed tomography is estimated critically. The results of own CT examinations in 120 patients with soft tissue tumors and of 50 ultrasound examinations show the importance of these methods in the extent diagnosis of soft tissue tumors. Ultrasound or CT- guided biopsy are helpful to prove the definitive diagnosis. These methods have reduced the use of angiography.

[Are expanded tumor resection and reconstruction of soft tissue sarcoma justified by recurrence rate and survival time?]. / Rechtfertigen sich erweiterte Tumorresektion und Rekonstruktion beim Weichgewebesarkom aus Rezidivrate und Uberlebenszeit?

The outcome of two groups of patients treated for soft tissue sarcoma either by a "limited resection" or by a radical procedure, including reconstructive techniques to vessels and soft tissues coverage, was compared. The latter group showed a higher rate of R0 resections, prolonged time to recurrence and a significantly decreased recurrence rate. Also, fewer patients developed distant metastases. From these data all efforts to achieve a radical resection seem to be worthwhile.

Stability and transmetallation of the magnetic resonance contrast agent MnDPDP measured by EPR.

MnDPDP [manganese(II) N, N'-dipyridoxylethylenediamine- N, N'-diacetate-5,5'-bis(phosphate)] is the active component of Teslascan, a contrast medium for magnetic resonance imaging of the liver. It has previously been shown that MnDPDP is rapidly dephosphorylated to the monophosphate MnDPMP and the non-phosphorylated MnPLED, and that all these substances are rapidly transmetallated to the corresponding Zn complexes. In the present study we used EPR at 9 and 230 GHz to show that no free Mn(2+) ions can be detected in the product or in a mixture of MnDPDP and human serum. Competition experiments between MnDPDP and Zn(2+), Ca(2+), and Mg(2+) ions revealed approximately 15% transmetallation with Zn(2+) in a buffer system containing metal ion concentrations similar to that in serum, whereas approximately 10% transmetallation was obtained with Ca(2+) and only negligible transmetallation was obtained with Mg(2+) under these conditions. Binding experiments with Mn(2+) added to human albumin and human serum indicate that albumin accounts for most of the protein-bound Mn(2+) in serum.

Examples of high-frequency EPR studies in bioinorganic chemistry.

Low-temperature EPR spectroscopy with frequencies between 95 and 345 GHz and magnetic fields up to 12 T has been used to study metal sites in proteins or inorganic complexes and free radicals. The high-field EPR method was used to resolve g-value anisotropy by separating it from overlapping hyperfine couplings. The presence of hydrogen bonding interactions to the tyrosyl radical oxygens in ribonucleotide reductases were detected. At 285 GHz the g-value anisotropy from the rhombic type 2 Cu(II) signal in the enzyme laccase has its g-value anisotropy clearly resolved from slightly different overlapping axial species. Simple metal site systems with S>1/2 undergo a zero-field splitting, which can be described by the spin Hamiltonian. From high-frequency EPR, the D values that are small compared to the frequency (high-field limit) can be determined directly by measuring the distance of the outermost signal to the center of the spectrum, which corresponds to (2 S-1)* mid R: Dmid R: For example, D values of 0.8 and 0.3 cm(-1) are observed for S=5/2 Fe(III)-EDTA and transferrin, respectively. When D values are larger compared to the frequency and in the case of half-integer spin systems, they can be obtained from the frequency dependence of the shifts of g(eff), as observed for myoglobin in the presence ( D=5 cm(-1)) or absence ( D=9.5 cm(-1)) of fluoride. The 285 and 345 GHz spectra of the Fe(II)-NO-EDTA complex show that it is best described as a S=3/2 system with D=11.5 cm(-1), E=0.1 cm(-1), and g(x)= g(y)= g(z)=2.0. Finally, the effects of HF-EPR on X-band EPR silent states and weak magnetic interactions are demonstrated.