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Limited predictive value of achieving beneficial plasma (Z)-endoxifen threshold level by CYP2D6 genotyping in tamoxifen-treated Polish women with breast cancer
Get PDFBackground
Tamoxifen, the most frequently used drug for treating estrogen receptor-positive breast cancer, must be converted into active metabolites to exert its therapeutic efficacy, mainly through CYP2D6 enzymes. The objective of this study was to investigate the impact of CYP2D6 polymorphisms on (Z)-endoxifen-directed tamoxifen metabolism and to assess the usefulness of CYP2D6 genotyping for identifying patients who are likely to have insufficient (Z)-endoxifen concentrations to benefit from standard therapy.
Methods
Blood samples from 279 Polish women with breast cancer receiving tamoxifen 20 mg daily were analyzed for CYP2D6 genotype and drug metabolite concentration. Steady-state plasma levels of tamoxifen and its 14 metabolites were measured by using the ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method.
Results
In nearly 60 % of patients, including over 30 % of patients with fully functional CYP2D6, (Z)-endoxifen concentration was below the predefined threshold of therapeutic efficacy. The most frequently observed CYP2D6 genotype was EM/PM (34.8 %), among which 83.5 % of patients had a combination of wild-type and *4 alleles. Plasma concentration of five metabolites was significantly correlated with CYP2D6 genotype. For the first time, we identified an association between decreased (E/Z)-4-OH-N-desmethyl-tamoxifen-β-D-glucuronide levels (r 2 = 0.23; p < 10 −16 ) and increased CYP2D6 functional impairment. The strongest correlation was observed for (Z)-endoxifen, whose concentration was significantly lower in groups of patients carrying at least one CYP2D6 null allele, compared with EM/EM patients. The CYP2D6 genotype accounted for plasma level variability of (Z)-endoxifen by 27 % (p < 10 −16 ) and for the variability of metabolic ratio indicating (Z)-endoxifen-directed metabolism of tamoxifen by 51 % (p < 10 −43 ).
Conclusions
The majority of breast cancer patients in Poland may not achieve a therapeutic level of (Z)-endoxifen upon receiving a standard dose of tamoxifen. This finding emphasizes the limited value of CYP2D6 genotyping in routine clinical practice for identifying patients who might not benefit from the therapy. In its place, direct monitoring of plasma steady-state (Z)-endoxifen concentration should be performed to personalize and optimize the treatment
Bicyclic cytarabine analogues: synthesis and investigation of antitumor properties of novel, 6-aryl- and 6-alkyl-3H-pyrrolo[2,3-d] pyrimidin-2(7H)-one arabinosides.
Get PDFA series of sixteen hitherto unknown Cytarabine analogues bearing a bicyclic 3H-pyrrolo[2,3-d]pyrimidin-2(7H)-one base modified with aryl, pyridyl, benzyl and alkyl substituents was prepared in a straightforward approach. This is the one of the few examples of the synthesis of pyrrolo[2,3-d]pyrimidin-2(7H)-one nucleosides and the first example of pyrrolo[2,3-d]pyrimidin-2(7H)-one nucleosides possessing arabinose moiety. For the first time, the conversion of the furopyrimidine arabinoside products to a series of novel pyrrolopyrimidines by ammonolysis reaction was thoroughly investigated using aqueous and methanolic reaction conditions under classical and micro-wave heating. This approach resulted in a small library of compounds, which were evaluated for their antiproliferative properties against HL-60 and Jurkat E6.1 cell lines. All synthesised compounds exhibited a weaker cytotoxic effect in comparision to the mother compound. Of all the tested compounds, the derivative bearing a 4-n-pentylphenyl substituent exhibited the highest antiproliferative activity
Novel Mutations in the IRF6 Gene on the Background of Known Polymorphisms in Polish Patients With Orofacial Clefting.
Get PDFOBJECTIVE
To examine the role of the IRF6 mutations in Polish families with Van der Woude syndrome and popliteal pterygium syndrome and to determine the effect of IRF6 single nucleotide polymorphisms (rs7552506, rs2013162, and rs2235375) on cleft lip and/or palate susceptibility.
DESIGN
IRF6 mutation screening was performed by direct sequencing of all coding exons of the gene and their flanking intronic regions. Cosegregation analysis was performed to establish the relation of single nucleotide polymorphisms and cleft lip and/or palate phenotypes.
PATIENTS
We screened the IRF6 gene in eight families with clinical recognition of Van der Woude syndrome and popliteal pterygium syndrome.
RESULTS
In five families we identified pathogenic mutations, all affecting the DNA-binding or the protein-binding domain of IRF6. Two of the mutations were novel-a missense mutation Arg31Thr and a small deletion Trp40Glyfs*23. In most cases we found also a haplotype of three single nucleotide polymorphisms-rs7552506, rs2013162, and rs2235375. The association of the single nucleotide polymorphisms and cleft lip and/or palate susceptibility has been previously published. The variants did not cosegregate with phenotype in examined families nor did they cosegregate with pathogenic mutations. The single nucleotide polymorphisms were deemed not causative, due to their presence in unaffected family members.
CONCLUSIONS
Two novel mutations (Arg31Thr and Trp40Glyfs*23) in the IRF6 gene were identified to be causative for Van der Woude and popliteal pterygium syndromes. In the present study no association between the single nucleotide polymorphisms rs7552506, rs2013162, and rs2235375 and the cleft lip and/or palate phenotype was found. The hypothesis, whether the haplotype of the three single nucleotide polymorphisms was correlated with IRF6 expression level, demands further investigation
Fluorophosphate Nucleotide Analogs and Their Characterization as Tools for 19F NMR Studies
No full textTo broaden the scope of existing methods based on 19F nucleotide labeling, we developed a new method for the
synthesis of fluorophosphate (oligo)nucleotide analogues containing an O to F substitution at the terminal position of the(oligo)phosphate moiety and evaluated them as tools for 19F NMR studies. Using three efficient and comprehensive synthetic approaches based on phosphorimidazolide chemistry and tetra-n-butylammonium fluoride, fluoromonophosphate, or fluorophosphate imidazolide as fluorine sources, we prepared over 30 fluorophosphate-containing nucleotides, varying in
nucleobase type (A, G, C, U, m7G), phosphate chain length (from mono to tetra), and presence of additional phosphate
modifications (thio, borano, imido, methylene). Using fluorophosphate imidazolide as fluorophosphorylating reagent for 5′-phosphorylated oligos we also synthesized oligonucleotide 5′-(2-fluorodiphosphates), which are potentially useful as 19F NMR hybridization probes. The compounds were characterized by 19F NMR and evaluated as 19F NMR molecular probes. We found that fluorophosphate nucleotide analogues can be used to monitor activity of enzymes with various specificities and metal ion
requirements, including human DcpS enzyme, a therapeutic target for spinal muscular atrophy. The compounds can also serve as reporter ligands for protein binding studies, as exemplified by studying interaction of fluorophosphate mRNA cap analogues with eukaryotic translation initiation factor (eIF4E)
The Quest for Simplicity: Remarks on the Free-Approach Models
No full textNuclear magnetic relaxation provides a powerful method giving insight into molecular motions at the atomic resolution on a broad time scale. Dynamics of biological macromolecules has been widely exploited by measuring 15N and 13C relaxation data. Interpretation of these data relies almost exclusively on the use of the model-free approach (MFA) and its extended version (EMFA) which requires no particular physical model of motion and a small number of parameters. It is shown that EMFA is often unable to cope with three different time scales and fails to describe slow internal motions properly. In contrast to EMFA, genuine MFA with two time scales can reproduce internal motions slower than the overall tumbling. It is also shown that MFA and simplified EMFA are equivalent with respect to the values of the N-H bond length and chemical shift anisotropy. Therefore, the vast majority of 15N relaxation data for proteins can be satisfactorily interpreted solely with MFA
Genetic variation of Colobanthus quitensis from King George Island (Antarctica)
Get PDFAbstract: Antarctic pearlwort (Colobanthus quitensis) is one of the flowering plant species
considered native to maritime Antarctica. Although the species was intensively analyzed
towards its morphological, anatomical and physiological adaptation to local environment,
its genetic variability is still poorly studied. In the presented study, a recently developed
retrotransposon−based DNA marker system (iPBS – inter Primer Binding Site) was applied
to assess the genetic diversity and differentiation of C. quitensis populations from King
George Island (South Shetland Islands, West Antarctic). A total of 143 scoreable bands
were detected using 7 iPBS primers among 122 plant specimens representing 8 populations.
55 (38.5%) bands were found polymorphic, with an average of 14.3% polymorphic frag−
ments per primer. Nine of all observed fragments were represented as a private bands de−
ployed unevenly among populations. Low genetic diversity (on average He = 0.040 and
I = 0.061) and moderate population differentiation (FST = 0.164) characterize the analyzed
material. Clustering based on PCoA revealed, that the populations located on the edges of
the study area diverge from the central populations. The pattern of population differentia−
tion corresponds well with their geographic location and the characteristics of the sampling
sites. Due to the character of iPBS markers, the observed genetic variation of populations
may be explained by the genome rearrangements caused by mobilization of mobile genetic
elements in the response to various stress factors. Additionally, this study demonstrates the
usefulness of iPBS markers for genetic diversity studies in wild species
Dominance from the perspective of gene-gene and gene-chemical interactions.
Get PDFIn this study, we used genetic interaction (GI) and gene-chemical interaction (GCI) data to compare mutations with different dominance phenotypes. Our analysis focused primarily on Saccharomyces cerevisiae, where haploinsufficient genes (HI; genes with dominant loss-of-function mutations) were found to be participating in gene expression processes, namely, the translation and regulation of gene transcription. Non-ribosomal HI genes (mainly regulators of gene transcription) were found to have more GIs and GCIs than haplosufficient (HS) genes. Several properties seem to lead to the enrichment of interactions, most notably, the following: importance, pleiotropy, gene expression level and gene expression variation. Importantly, after these properties were appropriately considered in the analysis, the correlation between dominance and GI/GCI degrees was still observed. Strikingly, for the GCIs of heterozygous strains, haploinsufficiency was the only property significantly correlated with the number of GCIs. We found ribosomal HI genes to be depleted in GIs/GCIs. This finding can be explained by their high variation in gene expression under different genetic backgrounds and environmental conditions. We observed the same distributions of GIs among non-ribosomal HI, ribosomal HI and HS genes in three other species: Schizosaccharomyces pombe, Drosophila melanogaster and Homo sapiens. One potentially interesting exception was the lack of significant differences in the degree of GIs between non-ribosomal HI and HS genes in Schizosaccharomyces pombe
The pentameric nucleoplasmin fold is present in Drosophila FKBP39 and a large number of chromatin-related proteins.
Get PDFNucleoplasmin is a histone chaperone that consists of a pentameric N-terminal domain and an unstructured C-terminal tail. The pentameric core domain, a doughnut-like structure with a central pore, is only found in the nucleoplasmin family. Here, we report the first structure of a nucleoplasmin-like domain (NPL) from the unrelated Drosophila protein, FKBP39, and we present evidence that this protein associates with chromatin. Furthermore, we show that two other chromatin proteins, Arabidopsis thaliana histone deacetylase type 2 (HD2) and Saccharomyces cerevisiae Fpr4, share the NPL fold and form pentamers, or a dimer of pentamers in the case of HD2. Thus, we propose a new family of proteins that share the pentameric nucleoplasmin-like NPL domain and are found in protists, fungi, plants and animals
Effects of flavonoids on expression of genes involved in cell cycle regulation and DNA replication in human fibroblasts
Get PDFFlavonoids have been studied as potential agents in medicine for many years. Among them, genistein was found to be active in various biological systems, mainly in prevention of cancer. Our recent work supported the idea that genistein also impacts multiple cellular processes in healthy fibroblasts; however, its effects on cell cycle related pathways remained to be elucidated. Thus, in this work, high throughput screening with microarrays coupled to real-time quantitative Reverse Transcription PCR analyses was employed to study the changes in expression of key genes associated with cell cycle regulation and/or DNA replication in response to genistein, kaempferol, daidzein, and mixtures of genistein and either kaempferol or daidzein. Among them, genistein was found as the most significantly modulating, in a time- and dosedependent manner, compound of activity of studied genes, whose products are involved in different phases of the cell cycle and/or in regulatory processes important for DNA replication and cell growth. It considerably reduced the efficiency of expression of genes coding for MCM2-7 and MCM10 helicases, as well as some other proteins involved in the S phase control. In addition, genistein caused cell cycle arrest in the G2/M phase, which was accompanied by activation of CDKN1A, CDKN1C, CDKN2A, CDKN2B, CDKN2C, and GADD45A genes, as well as down-regulation of several mRNAs specific for this stage, demonstrated by transcriptomic assessments. We believe that studies described in this paper will be helpful in elucidating molecular mechanisms of action of genistein as modulator of cell cycle and inhibitor of DNA replication in humans
Unusual Zn(II) Affinities of Zinc Fingers of Poly(ADP-ribose) Polymerase 1 (PARP-1) Nuclear Protein
No full textPoly(ADP-ribose) polymerase 1 (PARP-1) is a key eukaryotic enzyme, catalyzing the NAD+ dependent poly(ADP-ribosyl)ation of protein substrates, crucial for major DNA repair pathways, and involved in other fundamental cellular processes, such as transcription, cell cycle control, and apoptosis. Its ability to bind DNA depends on two CCHC zinc finger domains, in short, PARPzf1 and PARPzf2. Using spectroscopic methods and competitive titrations with Zn(II), Co(II) and Ni(II) ions we determined conditional dissociation constants for Zn(II) complexes of PARPzf1 and PARPzf2 at pH 7.4 (HEPES buffer), as 26 ± 4 nM and 4 ± 1 pM, respectively. The former value indicates an extremely low affinity of PARPzf1 towards metal ions, meaning that under cellular conditions PARP1zf might be largely present in a “metal-free” state. This finding provides a clue for high susceptibility of PARP-1 to oxidative stress, but also raises questions regarding activation of PARPzf1 under cellular conditions. We also determined conditional dissociation constants for Ni(II) complexes of PARPzf1 and PARPzf2 under the same conditions, as 0.78 ± 0.04 µM and 0.26 ± 0.05 nM, respectively