Jürgen Thomale

Elevated urinary excretion of RNA catabolites as an early signal of tumor development in mice.

Determination of the urinary excretion rates of 12 modified nucleosides and bases in mice after tumor induction by application of a single dose of 3-methylcholanthrene revealed that mice bearing tumors in advanced stages excrete many-fold amounts of these nucleic acid catabolites compared with the control values. The excretion rate of several of these nucleic acid constituents like ac4C, m1A, PsU, and m2Gua increased before the tumor was diagnosable. Untreated control mice as well as mice having received the carcinogen, but not developing a tumor, did not show an alteration in the excretion values of any of the modified nucleosides and bases determined.

Non-coordinate regulation of enzymes involved in transfer RNA metabolism in Escherichia coli.

During different steady state growth conditions in Escherichia coli the level of the three tRNA-modifying enzymes, the tRNA(m5Urd)-, tRNA(m1Guo)- and tRNA(mam5s2Urd)methyltransferase and of five aminoacyl-tRNA synthetases, the leucyl-, valyl-, isoleucyl-, arginyl- and threonyl-tRNA-synthetase, has been determined. It is shown that those two classes of tRNA affecting enzymes are not coordinately regulated and that even within these two groups of enzymes the constituents are regulated independently of each other. Furthermore it is demonstrated that none of the aminoacyl-tRNA synthetases and only one of the three tRNA-methyltransferases, the tRNA(m5Urd)methyltransferase, is under control of the relA+-gene.

Platinum-induced kidney damage: Unraveling the DNA damage response (DDR) of renal tubular epithelial and glomerular endothelial cells following platinum injury

BACKGROUND Platinum compounds are potent anticancer drugs but also evoke considerable normal tissue damage. Here, we elucidate the molecular mechanisms contributing to the nephrotoxic effects of cisplatin. METHODS We comparatively investigated the stress responses of rat kidney tubular (NRK-52E) and glomerular cells (RGE) following treatment with cisplatin (CisPt), oxaliplatin (OxaliPt) and carboplatin (CarboPt). To this end, cell viability, apoptosis, cell cycle progression, DNA damage response (DDR) and repair of DNA adducts were investigated. RESULTS CisPt reduced the viability of tubular NRK-52E and glomerular RGE cells most efficiently. Cytotoxicity evoked by CarboPt occurred with a delay, which might be related to a retarded formation of Pt-(GpG) intrastrand crosslinks. RGE cells were more sensitive towards all platinum compounds than NRK-52E cells. Platinum drugs efficiently induced caspase-mediated apoptosis in tubular cells, while RGE cells favored G2/M arrest when treated with equitoxic platinum doses. Mitotic index of NKR-52E and RGE cells was worst affected by OxaliPt. Activation of the DDR was strikingly agent- and cell type-specific. Most comprehensive and substantial stimulation of DDR mechanisms was provoked by CisPt. Repair of Pt-(GpG) intrastrand crosslinks was best in RGE, which was reflected by high mRNA expression of nucleotide excision repair (NER) factors. CONCLUSIONS There are substantial differences regarding the cause of sensitivity and mechanisms of DDR between tubular and glomerular cells following platinum injury. CisPt is the most potent stimulator of the DDR. We hypothesize that specific DNA adducts and thereby forcefully activated pro-toxic DDR mechanisms contribute to the exceptionally high acute nephrotoxicity of CisPt.

Alteration of structure or level of threonyl-tRNA-synthetase in borrelidin resistant mutants of E. Coli

Borrelidin inhibits the growth of E. coli effectively [ 1, 21. Since no other biochemical reaction is known to be attacked directly by Borrelidin than the enzymatic activity of the threonyl-tRNA-synthetase (ThRS) [2, 31, we isolated Borrelidin-resistant mutants of E. coli K12 and E. coli B with the aim of finding mutants with an altered structure or level of ThRS. The ThRS of fifteen Borrelidin-resistant mutants of each E. coli strain was investigated. The properties of the ThRS of two Borrelidin-resistant mutants of E. coli K12 has been analysed extensively [4] . Here now we describe the characterization of the ThRS of the remaining Borrelidin-resistant mutants. By means of determination of enzyme constants and antibody neutralisation curves it is shown that the Borrelidinresistant mutants can be divided into three groups: one group of mutants exhibits constitutively increased levels of wildtype ThRS, the second group structurally altered ThRS, and in a third group of mutants no alteration of the structure or level of ThRS could be detected. This suggests that Borrelidin-resistance in the latter mutants is due to some other reason than alteration of ThRS activity. Since it is known that the ThRS participates in the regulation of formation of the threonine biosynthetic enzymes [2, 51, the level of aspartokinase was also determined in the Borrelidin-resistant mutants grown in the presence and absence of Borrelidin.

Induction of malignant transformation by various chemicals in Balb/3T3 clone A31-1-1 cells and biological characterization of some transformants

Balb/c A31-1-1 cells were used for the study of transformation induction by chemicals with different mutagenic specificities. We show that survival of these cells and therefore the calculated transformation frequency per cells at risk is dependent upon the cell density at the time of treatment. It is suggested that equal cell densities should be used for measuring survival values and transformation induction. The quantitative results obtained are discussed in the light of the known mutagenic mechanisms of the chemicals tested. We also characterized morphologically transformed foci induced by different chemicals with respect to some biological properties. Anchorage independence was determined by testing growth in soft agar, loss of contact inhibition was quantitated by measuring maximum cell densities and malignancy was tested by tumor induction in nude mice. Although no very close correlation between these parameters and morphology was observed, the most malignant clones are also the ones with the highest values in the other tests. Our data make one or few genetical targets for transformation induction likely. We therefore speculate that the diverse phenotypes obtained might be due to differential activation of one or very few transforming genes in these cells.

Excretion of modified nucleosides during development of malignant lymphomas in mice after whode body irradiation

SummaryDuring x-ray-induced development of malignant lymphomas in mice their urinary excretion of eight modified nucleosides was monitored and the values were compared to the results of the histological examination of the animals at time of their sacrifice.It was found that the pathologically augmented excretion of modified nucleosides begins as much as several weeks before the malignant lymphomas can be diagnosed clinically. Thus some mice had increased levels of modified nucleosides even 10 weeks before sacrifice, though at the time of sacrifice the histological investigation reveled only some small foci of reticulum cell neoplasm in their spleen.It is therefore stressed that the usefulness of the determination of urinary modified nucleosides as an early noninvasive screening test for cancer in man and as an in vivo carcinogenicity test should be evaluated.

Lovastatin prevents cisplatin-induced activation of pro-apoptotic DNA damage response (DDR) of renal tubular epithelial cells.

The platinating agent cisplatin (CisPt) is commonly used in the therapy of various types of solid tumors. The anticancer efficacy of CisPt largely depends on the formation of bivalent DNA intrastrand crosslinks, which stimulate mechanisms of the DNA damage response (DDR), thereby triggering checkpoint activation, gene expression and cell death. The clinically most relevant adverse effect associated with CisPt treatment is nephrotoxicity that results from damage to renal tubular epithelial cells. Here, we addressed the question whether the HMG-CoA-reductase inhibitor lovastatin affects the DDR of renal cells by employing rat renal proximal tubular epithelial (NRK-52E) cells as in vitro model. The data show that lovastatin has extensive inhibitory effects on CisPt-stimulated DDR of NRK-52E cells as reflected on the levels of phosphorylated ATM, Chk1, Chk2, p53 and Kap1. Mitigation of CisPt-induced DDR by lovastatin was independent of the formation of DNA damage as demonstrated by (i) the analysis of Pt-(GpG) intrastrand crosslink formation by Southwestern blot analyses and (ii) the generation of DNA strand breaks as analyzed on the level of nuclear γH2AX foci and employing the alkaline comet assay. Lovastatin protected NRK-52E cells from the cytotoxicity of high CisPt doses as shown by measuring cell viability, cellular impedance and flow cytometry-based analyses of cell death. Importantly, the statin also reduced the level of kidney DNA damage and apoptosis triggered by CisPt treatment of mice. The data show that the lipid-lowering drug lovastatin extensively counteracts pro-apoptotic signal mechanisms of the DDR of tubular epithelial cells following CisPt injury.

Change of isoaccepting threonyl-tRNA constitutively increased level of threonyl-tRNA-synthetase in E. coli

The level of an aminoacyl-tRNA-synthetase in bacteria can be increased reactively in answer to specific environmental conditions or constitutively due to mutation (cf. [ 1,2] and [3--61). In neither case the molecular reactions determining the intracellular level of an aminoacyl-tRNA-synthetase are fully understood. We have described mutants of E. coli K12, in which the level of wildtype threonyl-tRNA-synthetase (ThrRS) is increased constitutively by a factor of three to ten, depending on the mutant [4,7]. Since in many respects aminoacyl-tRNA-synthetases and tRNA can be looked at as a functional unit, we investigated the tRNAThr in one of our mutants, which has a five-fold increased level of ThrRS. Here we show that this intracellular increase of wildtype ThrRS is accompanied by a specific change in the amount of the three threonine isoaccepting tRNA’s.

ΔNp63 activates the Fanconi anemia DNA repair pathway and limits the efficacy of cisplatin treatment in squamous cell carcinoma

TP63, a member of the p53 gene family gene, encodes the ΔNp63 protein and is one of the most frequently amplified genes in squamous cell carcinomas (SCC) of the head and neck (HNSCC) and lungs (LUSC). Using an epiallelic series of siRNAs with intrinsically different knockdown abilities, we show that the complete loss of ΔNp63 strongly impaired cell proliferation, whereas partial ΔNp63 depletion rendered cells hypersensitive to cisplatin accompanied by an accumulation of DNA damage. Expression profiling revealed wide-spread transcriptional regulation of DNA repair genes and in particular Fanconi anemia (FA) pathway components such as FANCD2 and RAD18 - known to be crucial for the repair of cisplatin-induced interstrand crosslinks. In SCC patients ΔNp63 levels significantly correlate with FANCD2 and RAD18 expression confirming ΔNp63 as a key activator of the FA pathway in vivo. Mechanistically, ΔNp63 bound an upstream enhancer of FANCD2 inactive in primary keratinocytes but aberrantly activated by ΔNp63 in SCC. Consistently, depletion of FANCD2 sensitized to cisplatin similar to depletion of ΔNp63. Together, our results demonstrate that ΔNp63 directly activates the FA pathway in SCC and limits the efficacy of cisplatin treatment. Targeting ΔNp63 therefore would not only inhibit SCC proliferation but also sensitize tumors to chemotherapy.