MutS preferentially recognizes cisplatin- over oxaliplatin-modified DNA

Loss of mismatch repair leads to tumor resistance by desensitizing cells to specific DNA-damaging agents, including the anticancer drug cisplatin. Cisplatin analogs with a diamminocyclohexane (DACH) carrier ligand, such as oxaliplatin and Pt(DACH)Cl(2), do not elicit resistance in mismatch repair-deficient cells and therefore present promising therapeutic agents. This study compared the interactions of the purified Escherichia coli mismatch repair protein MutS with DNA modified to contain cisplatin and DACH adducts. MutS recognized the cisplatin-modified DNA with 2-fold higher affinity in comparison to the DACH-modified DNA. ADP stimulated the binding of MutS to cisplatin-modified DNA, whereas it had no effect on the MutS interaction with DNA modified by DACH or EN adducts. In parallel cytotoxicity experiments, methylation-deficient E. coli dam mutants were 2-fold more sensitive to cisplatin than DACH compounds. A panel of recombination-deficient mutants showed striking sensitivity to both compounds, indicating that both types of adducts are strong replication blocks. The differential affinity of MutS for DNA modified with the different platinum analogs could provide the molecular basis for the distinctive cellular responses to cisplatin and oxaliplatin.     

Site selectivity of platinum anticancer therapeutics

X-ray crystallographic and biochemical investigation of the reaction of cisplatin and oxaliplatin with nucleosome core particle and naked DNA reveals that histone octamer association can modulate DNA platination. Adduct formation also occurs at specific histone methionine residues, which could serve as a nuclear platinum reservoir influencing adduct transfer to DNA. Our findings suggest that the nucleosome center may provide a favorable target for the design of improved platinum anticancer drugs.     

Study by HPLC-MS of the interaction of platinum antitumor complexes with potato carboxypeptidase inhibitor (PCI)

The interaction of the well-known antitumor drug cisplatin cis-[PtCl(2)(NH(3))(2)] and the compound trans-[PtCl(2)NH(3)(4-hydroxymethylpyridine)] with the small protein potato carboxypeptidase inhibitor (PCI) and a PCI mutant in which glycine-39 was substituted by methionine has been followed by HPLC/mass spectrometry. Our results showed that both Pt drugs were able to bind PCI through Met-39 and histidines in mutated PCI, whereas only the trans complex interacted significantly with wild PCI. In the cytotoxic studies, the monofunctional adduct PCI-Met-cisplatin was neither more active nor more selective than cisplatin itself when tested against three tumor cell lines with different number of EGF receptors. Those results suggested that the poor activity of the adduct could be just due to the small fraction of cisplatin which was decoordinated from the adduct and able to penetrate the tumor cells, as well as to the changes in the structure of the platinum drug after the loss of NH(3) groups upon binding PCI-Met.     

Targeting the DNA damage response (DDR) by natural compounds

Natural compounds (NC) are an important source of anticancer drugs. The genomic DNA of tumor cells is a major target of conventional anticancer therapeutics (cAT). DNA damage elicits a complex stress response programme termed DNA damage response (DDR), with the PI3-like kinase ATM and ATR being the key regulators. Since the DDR coordinates mechanisms of DNA repair and apoptosis, hence regulating the balance between death and survival, it is an attractive target of novel anticancer strategies. The aim of the study was to identify natural compounds derived from endophytic fungi, lichens, marine sponges or plants that interfere with mechanisms of the DDR. To this end, the cytotoxic and DDR modulating potency of 296 natural compounds, used alone or in combination with the cAT cisplatin (Cis) and doxorubicin (Doxo) was investigated by fluorescence-based analysis of the ATM/ATR-catalyzed S139 phosphorylation of histone 2AX (γH2AX), a surrogate marker of DNA damage-triggered DDR. After initial screening, a total of ten natural compounds were identified that were toxic in pancreatic carcinoma cells and activated the DDR on their own and/or promoted the DDR if used in combination with cAT. Their mode of action was shown to be independent of drug transport mechanisms. Based on their chemical structures, DDR modulatory activity and published data we suggest the marine NC 5-epi-nakijiquinone Q and 5-epi-ilimaquinone as well as the fungal compound secalonic acid F as most promising NC-based drug candidates for future synthesis of DDR-modulating chemical derivatives and their preclinical in vitro and in vivo testing.     

Disruption of thymidylate synthesis and glycine-serine interconversion by L-methionine and L-homocystine in Raji cells

Excessive concentrations of L-methionine inhibited the folate-dependent de novo synthesis of thymidylic acid (TMP) in Raji cells, demonstrating the usefulness of this cell line for the study of methionine-folate antagonism. The effect was also produced by L-homocystine but not by other amino acids including D-methionine and L-ethionine, suggesting that this effect is exerted by a common intermediate of methionine and homocystine metabolism. L-Methionine, L-homocysteine, S-adenosylmethionine (SAM), and S-adenosylhomocysteine (SAH) are not inhibitors of thymidylate synthase activity. On the other hand the capacity of the cells to incorporate serine 3-carbon and glycine 2-carbon into DNA is impaired by the presence of L-methionine or L-homocystine. Studies with cell-free extracts demonstrated that the glycine cleavage enzyme is inhibited by 45% by L-methionine, L-homocysteine, SAM or SAH. Serine hydroxymethylase on the other hand was slightly stimulated by these sulfur-containing compounds and this stimulation was shown to occur in the intact cell as well. These findings suggest that when levels of L-methionine metabolites are elevated, there is an increase in the use of glycine to maintain the intracellular concentration of serine, which is required for homocysteine detoxification by conversion to cystathionine. The reduction in TMP synthesis caused by excess L-methionine or L-homocystine may result from increased utilization of one-carbon units for serine synthesis.     

Cytotoxicity and DNA damage induced by a new platinum(II) complex with pyridine and dithiocarbamate

A new platinum(II) complex containing a pyridine nucleus and a dithiocarbamate moiety as ligands ([Pt(ESDT)(Py)Cl]) was evaluated for in vitro cytotoxicity in the cisplatin-sensitive human ovarian 2008 and in the isogenic-resistant C13* cell lines. In both cell types, a tumor cell growth inhibition greater than cisplatin and a complete lack of cross-resistance in C13* cells were found. Despite its molecular size, [Pt(ESDT)(Py)Cl] accumulation was much higher than cisplatin both in parent and resistant cells. Studying the mechanism of action in cell-free media, we established that [Pt(ESDT)(Py)Cl] more efficiently interacts with DNA in vitro compared to cisplatin maintaining a binding preference for GG rich sequences of DNA. On the contrary, DNA platination in vivo by [Pt(ESDT)(Py)Cl] was found lower than cisplatin. An analysis of the type of DNA lesions induced by [Pt(ESDT)(Py)Cl] suggests that the cytotoxic efficacy and the ability to overcome cisplatin resistance seem to be related to a different mechanism of interaction with DNA and/or with other key cellular components.     

Proteasome inhibition prevents cell death induced by the chemotherapeutic agent cisplatin downstream of DNA damage

Cisplatin is a highly effective chemotherapeutic drug acting as a DNA-damaging agent that induces apoptosis of rapidly proliferating cells. Unfortunately, cellular resistance still occurs. Mutations in p53 in a large fraction of tumor cells contribute to defects in apoptotic pathways and drug resistance. To uncover new strategies to eliminate tumors through a p53-independent pathway, we established a simplified model devoid of p53 to study cisplatin-induced regulated cell death, using the yeast Saccharomyces cerevisiae. We previously showed that cisplatin induces an active form of cell death accompanied by DNA condensation and fragmentation/degradation, but no significant mitochondrial dysfunction. We further demonstrated that proteasome inhibition, either with MG132 or genetically, increased resistance to cisplatin. In this study, we sought to determine how proteasome inhibition is important for cisplatin resistance by analyzing how it affects several phenotypes associated with the DNA damage response. We found MG132 does not seem to affect the activation of the DNA damage response or increase damage tolerance. Moreover, central modulators of the DNA damage response are not required for cisplatin resistance imparted by MG132. These results suggest the proteasome is involved in modulation of cisplatin toxicity downstream of DNA damage. Proteasome inhibitors can sensitize tumor cells to cisplatin, but protect others from cisplatin-induced cell death. Elucidation of this mechanism will therefore aid in the development of new strategies to increase the efficacy of chemotherapy.     

Incorporation of the sulfur of L-[35S]methionine into the biotin molecule by intact cells of Rhodotorula glutinis

The source of sulfur for biotin in microorganisms was studied. Using intact cells of Rhodotorula glutinis AKU 4847, L-methionine was much more effective for the synthesis of biotin from dethiobiotin than various other sulfur compounds tested. The reaction was carried out in the presence of L-[³⁵S]methionine. The radioactive biotin synthesized was isolated from the reaction mixture by a procedure involving cation- and anion-exchange column chromatographies, avidin treatment and membrane filtration, and then identified by radiochromatography and bioautography with Lactobacillus arabinosus. It was thus shown that the sulfur of methionine was incorporated into the biotin molecule by R. glutinis.     

Changes in the cytoskeleton pattern of tumor cells by cisplatin in vitro.

The influence of the antitumor drug cis-diamminedichloroplatinum(II) ('cisplatin') upon the structural pattern of the main cytoskeletal components, i.e. microtubules, intermediate filaments and microfilaments, was investigated in squamous carcinoma cells derived from the mouse stomach (G 22) or the human lung (L 266) and growing in vitro as monolayer cultures. The studies were performed by the indirect immunofluorescence technique using monoclonal antibodies against alpha-tubulin, type 19 cytokeratin and actin at the end of a 90-min exposure to 2.5 x 10(-6), 5 x 10(-6) or 10(-5) mol cisplatin/l and a subsequent 24-h recovery period. Under the influence of cisplatin, the cytoskeletal tubules and filaments, which were distributed in untreated cells as a finely organized network spreading through the whole cytoplasm like a spider's web, collapsed and aggregated to dense and circularly arranged bands of bright immunofluorescence around the nucleus or to cap-like structures apposing the nucleus. These phenomena developed in clear dependence upon the dose of cisplatin applied and were observable in a modified manner and to a different degree with the three structural elements of the cytoskeleton. During the subsequent 24-h interval, during which the cells were allowed to recover in drug-free growth medium, the before-mentioned collapse of the cytoskeletal network was only partially reversible following previous treatment with the medium (5 x 10(-6) mol/l) and the high (10(-5) mol/l) dose of cisplatin and restored totally to the normal structural pattern of untreated control cells when the low dose of 2.5 x 10(-6) mol cisplatin/l had been administered before. These results give evidence that the DNA cannot be the only cellular target for the antitumor drug cisplatin, but that it also effects other intracellular lesions which cause structural alterations of cellular organelles independently of the primary molecular attack at nuclear DNA strands. Probably, these additional interactions fortify the antiproliferative effect and contribute to the achievement of important biological and cytological effects of cisplatin such as growth inhibition or giant cell formation.     

Incorporation of the sulfur of L-[S]methionine into the biotin molecule by intact cells of Rhodotorula glutinis

The source of sulfur for biotin in microorganisms was studied. Using intact cells of Rhodotorula glutinis AKU 4847, L-methionine was much more effective for the synthesis of biotin from dethiobiotin than various other sulfur compounds tested. The reaction was carried out in the presence of L-[³⁵S]methionine. The radioactive biotin synthesized was isolated from the reaction mixture by a procedure involving cation- and anion-exchange column chromatographies, avidin treatment and membrane filtration, and then identified by radiochromatography and bioautography with Lactobacillus arabinosus. It was thus shown that the sulfur of methionine was incorporated into the biotin molecule by R. glutinis.     

Excision repair of uracil in higher plant cells: Uracil-DNA glycosylase and sister-chromatid exchanges

Uracil is not a normal constituent of DNA. Under natural conditions, it may appear either by deamination of cytosine residues or by incorporation of deoxyuridine monophosphate (dUMP). Visible light irradiation of BrdUrd-treated cells efficiently leads, under experimental situations, to the formation of dUMP residues in DNA. Plant cells, like other living organisms, can eliminate this potentially harmful base from DNA by an excision repair pathway, uracil-DNA glycosylase being the first enzyme acting during the incision process. Purified plant uracil-DNA glycosylase is a low molecular weight enzyme (27–29.5 kD) that specifically releases uracil present in DNA by splitting off the sugar-base bond. This enzyme is non-competitively inhibited by uracil and 6-aminouracil, but not by thymine, both in vitro and in vivo. However, other structurally related compounds do not show any inhibitory effect. This characteristic poses a number of unaswered questions regarding its mechanism of action. At the chromosome level, dUMP residues appear to be sister-chromatid exchange (SCE)-initiating events. This has been demonstrated for dUMP residue introduced either by visible light exposure of BrdUrd-treated cells or by dUMP mis-incorporation instead of dTMP in cells treated with inhibitors of thymidylate synthetase. The excision repair of uracil in plants appears to be finely regulated in different cell types depending on their proliferation rate and their development stage. Thus, high levels of uracil-DNA glycosylase do not seem to be necessarily associated with DNA replication, since non-proliferating cells, natural constituents of dormant meristems, contain enzyme levels comparable to those found in proliferating tissues, where it is modulated: the higher the cell cycle rate (and the DNA replication rate) the higher the uracil-DNA glycosylase activity. Finally, this excision repair enzyme seems to be turned off as cells enter their differentiated state.     

Cellular uptake and subcellular distribution of ruthenium-based metallodrugs under clinical investigation versus cisplatin

The cellular uptake and subcellular distribution including adduct formation with genomic DNA and uptake into mitochondria of two ruthenium(iii)-based drugs in clinical trials, KP1019 and NAMI-A, and cisplatin, was investigated in cisplatin sensitive and resistant A2780 human ovarian carcinoma cells. These data indicate that reduced metal uptake into mitochondria in combination with increased binding towards low molecular weight components involved in detoxification mechanisms is essential for cisplatin resistance. The ruthenium drugs show distinct differences with respect to cisplatin, especially in the cisplatin resistant cells; in comparison to the sensitive cells, KP1019 exhibits higher cytotoxicity and an only slightly changed metabolism of the drug, whereas NAMI-A treatment results in increased intracellular ruthenium levels and a higher number of ruthenium-DNA adducts. In addition, size exclusion-inductively coupled mass spectrometry indicates that adduct formation with high molecular weight components in the particulate and nuclear fractions is crucial for the therapeutic effect of KP1019 in both cisplatin resistant and sensitive cell lines.     

DNA binding by antitumor trans-[PtCl2(NH3)(thiazole)]. Protein recognition and nucleotide excision repair of monofunctional adducts

Antitumor effects of cis-diamminedichloroplatinum(II) (cisplatin) and the clinical inactivity of its trans isomer (transplatin) have been considered a paradigm for the classical structure-activity relationships of platinum drugs. However, several new analogues of transplatin which exhibit a different spectrum of cytostatic activity including activity in tumor cells resistant to cisplatin have been recently identified. Analogues containing the planar amine ligand of the general structure trans-[PtCl(2)(NH(3))(L)], where L = planar amine, represent an example of such compounds. DNA is believed to be the major pharmacological target of platinum compounds. To contribute to the understanding of mechanisms underlying the activation of trans geometry in transplatin analogues containing planar amine ligands, various biochemical and biophysical methods were employed in previous studies to analyze the global modifications of natural DNA by trans-[PtCl(2)(NH(3))(L)]. These initial studies have revealed some unique features of the DNA binding mode of this class of platinum drugs. As the monofunctional lesions represent a significant fraction of stable adducts formed in DNA by bifunctional antitumor trans-platinum compounds with planar ligands, we analyzed in the present work short DNA duplexes containing the single, site-specific monofunctional adduct of a representative of this class of platinum drugs, antitumor trans-[PtCl(2)(NH(3))(thiazole)]. It has been shown that, in contrast to the adducts of monodentate chlorodiethylenetriamineplatinum(II) chloride or [PtCl(NH(3))(3)]Cl, the monofunctional adduct of trans-[PtCl(2)(NH(3))(thiazole)] inhibits DNA synthesis and creates a local conformational distortion similar to that produced in DNA by the major 1,2-GG intrastrand CL of cisplatin, which is considered the lesion most responsible for its anticancer activity. In addition, the monofunctional adducts of trans-[PtCl(2)(NH(3))(thiazole)] are recognized by HMGB1 domain proteins and removed by the nucleotide excision repair system similarly as the 1,2-GG intrastrand CL of cisplatin. The results of the present work further support the view that the simple chemical modification of the structure of an inactive platinum compound alters its DNA binding mode into that of an active drug and that processing of the monofunctional DNA adducts of the trans-platinum analogues in tumor cells may be similar to that of the major bifunctional adducts of "classical" cisplatin.     

Analysis of the DNA damage produced by a platinum-acridine antitumor agent and its effects in NCI-H460 lung cancer cells

High-performance liquid chromatography in conjunction with electrospray mass spectrometry (LC-ESMS) was used to structurally characterize the adducts formed by the platinum-acridine agent [PtCl(en)(N-(2-(acridin-9-ylamino)ethyl)-N-methylpropionimidamide)](NO(3))(2) (compound 1) in cell-free DNA. Compound 1 forms monofunctional adducts exclusively with guanine, based on the fragments identified in enzymatic digests (dG*, dGMP*, dApG*, and dTpG*, where the asterisk denotes bound drug). The time course of accumulation and DNA adduct formation of compound 1 and the clinical drug cisplatin in NCI-H460 lung cancer cells at physiologically relevant drug concentrations (0.1 μM) was studied by inductively-coupled plasma mass spectrometry (ICP-MS). Compound 1 accumulates rapidly in cells and reaches intracellular levels of up to 60-fold higher than those determined for cisplatin. The hybrid agent shows unusually high DNA binding levels: while cisplatin adducts form at a maximum frequency of 5 adducts per 10(6) nucleotides, compound 1 produces 25 adducts per 10(6) nucleotides after only 3 h of continuous incubation with the lung cancer cells. The high overall levels of compound 1 in the cells and in cellular DNA over the entire 12-h treatment period translate into a rapid decrease in cell viability. Possible implications of these findings for the mechanism of action of compound 1 and the agent's potential to overcome tumor resistance to cisplatin are discussed.     

Differential recognition by the tumor suppressor protein p53 of DNA modified by the novel antitumor trinuclear platinum drug BBR3464 and cisplatin

The trinuclear platinum agent BBR3464, a representative of a new class of anticancer drugs, is more potent than conventional mononuclear cisplatin [cis-diamminedichloroplatinum(II)]. BBR3464 retains significant activity in human tumor cell lines and xenografts that are refractory or poorly responsive to cisplatin, and displays a high activity in human tumor cell lines that are characterized by both wild-type and mutant p53 gene. In contrast, on average, cells with mutant p53 are more resistant to the effect of cisplatin. It has been hypothesized that the sensitivity or resistance of tumor cells to cisplatin might be also associated with cell cycle control and repair processes that involve p53. DNA is a major pharmacological target of platinum compounds and DNA binding activity of the p53 protein is crucial for its tumor suppressor function. This study, using gel-mobility-shift assays, was undertaken to examine the interactions of active and latent p53 protein with DNA fragments and oligodeoxyribonucleotide duplexes modified by BBR3464 in a cell free medium and to compare these results with those describing the interactions of these proteins with DNA modified by cisplatin. The results indicate that structurally different DNA adducts of BBR3464 and cisplatin exhibit a different efficiency to affect the binding affinity of the modified DNA to p53 protein. It has been suggested that different structural perturbations induced in DNA by the adducts of BBR3464 and cisplatin produce a differential response to p53 protein activation and recognition and that a 'molecular approach' to control of downstream effects such as protein recognition and pathways of apoptosis induction may consist in design of structurally unique DNA adducts as cell signals.     

Molecular cloning and biochemical characterization of L-N-carbamoylase from Sinorhizobium meliloti CECT4114

An N-carbamoyl-L-amino acid amidohydrolase (L-N-carbamoylase) from Sinorhizobium meliloti CECT 4114 was cloned and expressed in Escherichia coli. The recombinant enzyme catalyzed the hydrolysis of N-carbamoyl alpha-amino acid to the corresponding free amino acid, and its purification has shown it to be strictly L-specific. The enzyme showed broad substrate specificity, and it is the first L-N-carbamoylase that hydrolyses N-carbamoyl-L-tryptophan as well as N-carbamoyl L-amino acids with aliphatic substituents. The apparent Km values for N-carbamoyl-L-methionine and tryptophan were very similar (0.65 +/- 0.09 and 0.69 +/- 0.08 mM, respectively), although the rate constant was clearly higher for the L-methionine precursor (14.46 +/- 0.30 s(-1)) than the L-tryptophan one (0.15 +/- 0.01 s(-1)). The enzyme also hydrolyzed N-formyl-L-methionine (kcat/Km = 7.10 +/- 2.52 s(-1) x mM(-1)) and N-acetyl-L-methionine (kcat/Km = 12.16 +/- 1.93 s(-1) x mM(-1)), but the rate of hydrolysis was lower than for N-carbamoyl-L-methionine (kcat/Km = 21.09 +/- 2.85). This is the first L-N-carbamoylase involved in the 'hydantoinase process' that has hydrolyzed N-carbamoyl-L-cysteine, though less efficiently than N-carbamoyl-L-methionine. The enzyme did not hydrolyze ureidosuccinic acid or 3-ureidopropionic acid. The native form of the enzyme was a homodimer with a molecular mass of 90 kDa. The optimum conditions for the enzyme were 60 degrees C and pH 8.0. Enzyme activity required the presence of divalent metal ions such as Ni2+, Mn2+, Co2+ and Fe2+, and five amino acids putatively involved in the metal binding were found in the amino acid sequence.     

Biophysical studies of the modification of DNA by antitumour platinum coordination complexes

Cisplatin (cis-diamminedichloroplatinum(II] is widely used in the treatment of various human tumours. A large body of experimental evidence indicates that the reaction of cisplatin with DNA is responsible for the cytostatic action of this drug. Several platinum-DNA adducts have been identified and their effect on the conformation of DNA has been investigated. Structural studies of platinum-DNA adducts now permit a reasonably good explanation of the biophysical properties of platinated DNA. Antitumouractive platinum compounds induce in DNA, at low levels of binding, local conformational alterations which have the character of non-denaturing distortions. It is likely that these changes occur in DNA due to the formation of intrastrand cross-links between two adjacent purine residues. On the other hand, the modification of DNA by antitumour-inactive complexes results in the formation of more severe local denaturation changes. Conformational alterations induced in DNA by antitumour-active platinum compounds may be reparable with greater difficulty than those induced by the inactive complexes. Alternatively, non-denaturation change induced in DNA by antitumour platinum drugs could represent more significant steric hindrance against DNA replication as compared with inactive complexes.     

Induction of apoptosis by chemotherapeutic drugs without generation of reactive oxygen species.

Studies in a variety of cell types have suggested that cancer chemotherapy drugs induce tumor cell apoptosis in part by inducing formation of reactive oxygen species (ROS). Using human B lymphoma cells as the targets, we have found that apoptosis can be induced in the absence of any detectable oxidative stress. Apoptosis was induced with the chemotherapy drugs VP-16 and cisplatin. To determine whether oxidants are formed as part of the drug-induced apoptotic process, intracellular markers of oxidative stress were examined. These included measurement of (1) protein carbonyl groups by Western blot immunoassay, (2) protein methionine sulfoxide residues by amino acid analysis, (3) protein sulfhydryl oxidation by Western blot immunoassay, (4) F2-isoprostanes by GC/MS, and (5) intracellular ROS production using the oxidant-sensitive dyes DCFDA and dihydrorhodamine 123. Apoptosis was quantified using fluorescence microscopy to assess nuclear morphology. The results show that VP-16 and cisplatin induce extensive apoptosis in the absence of any detectable protein or lipid oxidation, measured in both the cytosolic and mitochondrial compartments of the cell. In contrast, H2O2, which kills the cells by nonapoptotic pathways, caused increases in both protein and lipid oxidation. Three different antioxidant compounds (N-acetyl cysteine, Tempol, and MnTBAP) failed to inhibit VP-16-induced apoptosis, while inhibiting H2O2-induced cell death. Only N-acetyl cysteine inhibited cisplatin-induced cell death and this is attributed to its known ability to react directly with and inactivate cisplatin before it enters the cell. The results demonstrate that, at least in B lymphoma cells, chemotherapy-induced apoptosis occurs using a mechanism that does not involve oxidants.