Enzyme autoinduction by mitotane supported by population pharmacokinetic modelling in a large cohort of adrenocortical carcinoma patients.

OBJECTIVE: Mitotane is used for the treatment of adrenocortical carcinoma. High oral daily doses of typically 1- 6 g are required to attain therapeutic concentrations. The drug has a narrow therapeutic index and patient management is difficult because of a high volume of distribution, very long elimination half-life, and drug interaction through induction of metabolizing enzymes. The present evaluation aimed at the development of a population pharmacokinetic model of mitotane to facilitate therapeutic drug monitoring. METHODS: Appropriate dosing information, plasma concentrations (1137 data points) and covariates were available from therapeutic drug monitoring (TDM) of 76 adrenocortical carcinoma patients treated with mitotane. Using nonlinear mixed effects modeling, a simple structural model was first developed, with subsequent introduction of metabolic autoinduction. Covariate data were analyzed to improve overall model predictability. Simulations were performed to assess the attainment of therapeutic concentrations with clinical dosing schedules. RESULTS: A one-compartment pharmacokinetic model with first order absorption was found suitable to describe the data, with an estimated central volume of distribution of 6086 L related to a high interindividual variability of 81.5%. Increase in clearance of mitotane during treatment could be modeled by a linear enzyme autoinduction process. Body mass index was found to have an influence upon disposition kinetics of mitotane. Model simulations favor a high dose regimen to rapidly attain therapeutic concentrations, with the first TDM suggested on day 16 of treatment to avoid systemic toxicity. CONCLUSION: The proposed model describes mitotane pharmacokinetics and can be used to facilitate therapy by predicting plasma concentrations.

The genetic contribution of the NO system at the glutamatergic post-synapse to schizophrenia: further evidence and meta-analysis.

NO is a pleiotropic signaling molecule and has an important role in cognition and emotion. In the brain, NO is produced by neuronal nitric oxide synthase (NOS-I, encoded by NOS1) coupled to the NMDA receptor via PDZ interactions; this protein-protein interaction is disrupted upon binding of NOS1 adapter protein (encoded by NOS1AP) to NOS-I. As both NOS1 and NOS1AP were associated with schizophrenia, we here investigated these genes in greater detail by genotyping new samples and conducting a meta-analysis of our own and published data. In doing so, we confirmed association of both genes with schizophrenia and found evidence for their interaction in increasing risk towards disease. Our strongest finding was the NOS1 promoter SNP rs41279104, yielding an odds ratio of 1.29 in the meta-analysis. As findings from heterologous cell systems have suggested that the risk allele decreases gene expression, we studied the effect of the variant on NOS1 expression in human post-mortem brain samples and found that the risk allele significantly decreases expression of NOS1 in the prefrontal cortex. Bioinformatic analyses suggest that this might be due the replacement of six transcription factor binding sites by two new binding sites as a consequence of proxy SNPs. Taken together, our data argue that genetic variance in NOS1 resulting in lower prefrontal brain expression of this gene contributes to schizophrenia liability, and that NOS1 interacts with NOS1AP in doing so. The NOS1-NOS1AP PDZ interface may thus well constitute a novel target for small molecules in at least some forms of schizophrenia.

Nuclear import of LASP-1 is regulated by phosphorylation and dynamic protein-protein interactions.

LASP-1 is a multidomain protein predominantly localized at focal contacts, where it regulates cytoskeleton dynamics and cell migration. However, in different tumor entities, a nuclear LASP-1 accumulation is observed, thought to have an important role in cancer progression. Until now, the molecular mechanisms that control LASP-1 nuclear import were not elucidated. Here, we identified a novel LASP-1-binding partner, zona occludens protein 2 (ZO-2), and established its role in the signal transduction pathway of LASP-1 nucleo-cytoplasmatic shuttling. Phosphorylation of LASP-1 by PKA at serine 146 induces translocation of the LASP-1/ZO-2 complex from the cytoplasm to the nucleus. Interaction occurs within the carboxyterminal proline-rich motif of ZO-2 and the SH3 domain in LASP-1. In situ proximity ligation assay confirmed the direct binding between LASP-1 and ZO-2 and visualized the shuttling. Nuclear export is mediated by Crm-1 and a newly identified nuclear export signal in LASP-1. Finally, dephosphorylation of LASP-1 by phosphatase PP2B is suggested to relocalize the protein back to focal contacts. In summary, we define a new pathway for LASP-1 in tumor progression.

Nuclear localisation of LASP-1 correlates with poor long-term survival in female breast cancer.

BACKGROUND: LIM and SH3 protein 1 (LASP-1) is a nucleo-cytoplasmatic signalling protein involved in cell proliferation and migration and is upregulated in breast cancer in vitro studies have shown that LASP-1 might be regulated by prostate-derived ETS factor (PDEF), p53 and/or LASP1 gene amplification. This current study analysed the prognostic significance of LASP-1 on overall survival (OS) in 177 breast cancer patients and addressed the suggested mechanisms of LASP-1-regulation. METHODS: Nucleo-cytoplasmatic LASP-1-positivity of breast carcinoma samples was correlated with long-term survival, clinicopathological parameters, Ki67-positivity and PDEF expression. Rate of LASP1 amplification was determined in micro-dissected primary breast cancer cells using quantitative RT-PCR. Cell-phase dependency of nuclear LASP-1-localisation was studied in synchronised cells. In addition, LASP-1, PDEF and p53 expression was compared in cell lines of different tumour entities to define principles for LASP-1-regulation. RESULTS: We showed that LASP-1 overexpression is not due to LASP1 gene amplification. Moreover, no correlation between p53-mutations or PDEF-expression and LASP-1-status was observed. However, nuclear LASP-1-localisation in breast carcinomas is increased during proliferation with peak in G2/M-phase and correlated significantly with Ki67-positivity and poor OS. CONCLUSION: Our results provide evidence that nuclear LASP-1-positivity may serve as a negative prognostic indicator for long-term survival of breast cancer patients.

Disruption of the FA/BRCA pathway in bladder cancer.

Bladder carcinomas frequently show extensive deletions of chromosomes 9p and/or 9q, potentially including the loci of the Fanconi anemia (FA) genes FANCC and FANCG. FA is a rare recessive disease due to defects in anyone of 13 FANC genes manifesting with genetic instability and increased risk of neoplasia. FA cells are hypersensitive towards DNA crosslinking agents such as mitomycin C and cisplatin that are commonly employed in the chemotherapy of bladder cancers. These observations suggest the possibility of disruption of the FA/BRCA DNA repair pathway in bladder tumors. However, mutations in FANCC or FANCG could not be detected in any of 23 bladder carcinoma cell lines and ten surgical tumor specimens by LOH analysis or by FANCD2 immunoblotting assessing proficiency of the pathway. Only a single cell line, BFTC909, proved defective for FANCD2 monoubiquitination and was highly sensitive towards mitomycin C. This increased sensitivity was restored specifically by transfer of the FANCF gene. Sequencing of FANCF in BFTC909 failed to identify mutations, but methylation of cytosine residues in the FANCF promoter region was demonstrated by methylation-specific PCR, HpaII restriction and bisulfite DNA sequencing. Methylation-specific PCR uncovered only a single instance of FANCF promoter hypermethylation in surgical specimens of further 41 bladder carcinomas. These low proportions suggest that in contrast to other types of tumors silencing of FANCF is a rare event in bladder cancer and that an intact FA/BRCA pathway might be advantageous for tumor progression.

FZD3 is not a risk gene for schizophrenia: a case-control study in a Caucasian sample.

BACKGROUND: Polymorphisms in the human frizzeled-3 (FZD3) gene have been associated with schizophrenia in an Asian population sample. However, this finding could not be confirmed in subsequent studies investigating other populations. Here we attempted to replicate this finding in a sample of 192 German chronically ill schizophrenic subjects. METHODS: Three single nucleotide polymorphisms in the FZD3 gene have been genotyped by primer extension and MALDI-TOF measurement. Subsequently, associations for single markers as well as haplotypes were tested. RESULTS: In German patients, neither single markers nor haplotypes in FZD3 were associated with schizophrenia. Further exploratory analyses using a different diagnostic approach did also not yield significant results. CONCLUSIONS: FZD3 is unlikely to play a role in the genetic predisposition towards schizophrenia in the Caucasian population.

A neuronal nitric oxide synthase (NOS-I) haplotype associated with schizophrenia modifies prefrontal cortex function.

Nitric oxide (NO) is a gaseous neurotransmitter thought to play important roles in several behavioral domains. On a neurobiological level, NO acts as the second messenger of the N-methyl-D-aspartate receptor and interacts with both the dopaminergic as well as the serotonergic system. Thus, NO is a promising candidate molecule in the pathogenesis of endogenous psychoses and a potential target in their treatment. Furthermore, the chromosomal locus of the gene for the NO-producing enzyme NOS-I, 12q24.2, represents a major linkage hot spot for schizophrenic and bipolar disorder. To investigate whether the gene encoding NOS-I (NOS1) conveys to the genetic risk for those diseases, five NOS1 polymorphisms as well as a NOS1 mini-haplotype, consisting of two functional polymorphisms located in the transcriptional control region of NOS1, were examined in 195 chronic schizophrenic, 72 bipolar-I patients and 286 controls. Single-marker association analysis showed that the exon 1c promoter polymorphism was linked to schizophrenia (SCZ), whereas synonymous coding region polymorphisms were not associated with disease. Long promoter alleles of the repeat polymorphism were associated with less severe psychopathology. Analysis of the mini-haplotype also revealed a significant association with SCZ. Mutational screening did not detect novel exonic polymorphisms in patients, suggesting that regulatory rather than coding variants convey the genetic risk on psychosis. Finally, promoter polymorphisms impacted on prefrontal functioning as assessed by neuropsychological testing and electrophysiological parameters elicited by a Go-Nogo paradigm in 48 patients (continuous performance test). Collectively these findings suggest that regulatory polymorphisms of NOS1 contribute to the genetic risk for SCZ, and modulate prefrontal brain functioning.

Reverse mosaicism in Fanconi anemia: natural gene therapy via molecular self-correction.

Fanconi anemia (FA) is a genetically and phenotypically heterogenous autosomal recessive disease associated with chromosomal instability and hypersensitivity to DNA crosslinkers. Prognosis is poor due to progressive bone marrow failure and increased risk of neoplasia, but revertant mosaicism may improve survival. Mechanisms of reversion include back mutation, intragenic crossover, gene conversion and compensating deletions/insertions. We describe the types of reversions found in five mosaic FA patients who are compound heterozygotes for single base mutations in FANCA or FANCC. Intragenic crossover could be shown as the mechanism of self-correction in the FANCC patient. Restoration to wildtype via back mutation or gene conversion of either the paternal or maternal allele was observed in the FANCA patients. The sequence environments of these mutations/reversions were indicative of high mutability, and selective advantage of bone marrow precursor cells carrying a completely restored FANCA allele might explain the surprisingly uniform pattern of these reversions. We also describe a first example of in vitro phenotypic reversion via the emergence of a compensating missense mutation 15 amino acids downstream of the constitutional mutation, which explains the reversion to MMC resistance of the respective lymphoblastoid cell line. With one exception, our mosaic patients showed improvement of their hematological status during a three- to six-year observation period, indicating a proliferative advantage of the reverted cell lineages. In patients with Fanconi anemia, genetic instability due to defective caretaker genes sharply increases the risk of neoplasia, but at the same time increases the chance for revertant mosaicism leading to improved bone marrow function.

Strong FANCA/FANCG but weak FANCA/FANCC interaction in the yeast 2-hybrid system.

Three of at least 8 Fanconi anemia (FA) genes have been cloned (FANCA, FANCC, FANCG), but their functions remain unknown. Using the yeast 2-hybrid system and full-length cDNA, the authors found a strong interaction between FANCA and FANCG proteins. They also obtained evidence for a weak interaction between FANCA and FANCC. Neither FANCA nor FANCC was found to interact with itself. These results support the notion of a functional association between the FA gene products. (Blood. 2000;95:719-720)

Heterogeneous spectrum of mutations in the Fanconi anaemia group A gene.

Fanconi anaemia (FA) is a genetically heterogeneous autosomal recessive disorder associated with chromosomal fragility, bone-marrow failure, congenital abnormalities and cancer. The gene for complementation group A (FAA), which accounts for 60-65% of all cases, has been cloned, and is composed of an open reading frame of 4.3 kb, which is distributed among 43 exons. We have investigated the molecular pathology of FA by screening the FAA gene for mutations in a panel of 90 patients identified by the European FA research group, EUFAR. A highly heterogeneous spectrum of mutations was identified, with 31 different mutations being detected in 34 patients. The mutations were scattered throughout the gene, and most are likely to result in the absence of the FAA protein. A surprisingly high frequency of intragenic deletions was detected, which removed between 1 and 30 exons from the gene. Most microdeletions and insertions occurred at homopolymeric tracts or direct repeats within the coding sequence. These features have not been observed in the other FA gene which has been cloned to date (FAC) and may be indicative of a higher mutation rate in FAA. This would explain why FA group A is much more common than the other complementation groups. The heterogeneity of the mutation spectrum and the frequency of intragenic deletions present a considerable challenge for the molecular diagnosis of FA. A scan of the entire coding sequence of the FAA gene may be required to detect the causative mutations, and scanning protocols will have to include methods which will detect the deletions in compound heterozygotes.

Site-specific mutagenesis in Enterobacter agglomerans: construction of nif B mutants and analysis of the gene's structure and function.

A novel technique was developed which may be generally well suited to the site-specific construction of mutations in Enterobacter agglomerans. The method is based on the observation that E. agglomerans can be cured of a plasmid of the incompatibility group IncQ by cultivation on citrate-containing medium. To test the applicability of this technique, we inserted a kanamycin cassette into the cloned nifB gene, transferred it into E. agglomerans, and selected for recombinants in which the wild-type nifB was replaced by the mutated gene by growing transformants on citrate medium with kanamycin. The nifB- mutants with the kanamycin cassette inserted in either orientation showed a nif- phenotype. Further, we determined the nucleotide sequence of nifB. A typical sigma 54-dependent promoter and a consensus NifA binding site were found upstream of nifB. Activation of this promoter by both heterologous and homologous NifA proteins was observed in vivo. The predicted amino acid sequence of the NifB protein showed strong similarity to the NifB sequences of other diazotrophic bacteria. The typical clustering of cysteine residues at the N-terminal end indicates its involvement in Fe-Mo cofactor biosynthesis.

Analysis of retroorbital T cell antigen receptor variable region gene usage in patients with Graves' ophthalmopathy.

To date, it has remained unclear whether orbit-infiltrating T cells in patients with Graves' ophthalmopathy (GO) represent a primary immune response in which a limited number of T cell clones driving the disease are activated against specific antigens, or whether they participate in a non-specific inflammatory process. To characterize these T cells at the molecular level, we examined the T cell antigen receptor (TcR) V gene repertoire in situ in retroorbital tissue specimens obtained from patients with early and late stages of clinically severe GO and from patients with non-GO orbital conditions. Ribonucleic acid extracted from orbital tissue and peripheral blood lymphocytes (PBL) was reverse transcribed and amplified using the polymerase chain reaction and 22 V alpha and 24 V beta gene-specific oligonucleotide primers. The resulting TcR V alpha and V beta transcripts were verified by Southern hybridization analysis using TcR C region-specific, digoxigenin-labeled oligonucleotide probes. Compared with matched PBL, the retroorbital TcR V alpha and V beta gene repertoire expressed was heterogeneous, but revealed marked restriction of V gene usage in samples derived from retroorbital connective tissue and extraocular muscle of all eight patients with severe GO of short duration studied. In contrast, greater diversity of the TcR V beta gene repertoire and loss of TcR V alpha gene restriction was noted in four patients with late GO undergoing reconstructive eye muscle surgery. Unrestricted TcR V gene usage was demonstrated in orbital tissue and PBL samples obtained from control subjects. These results suggest that retroorbital TcR V gene usage is variable but markedly restricted during the earlier stages of GO. With increasing disease duration, greater diversity of the TcR V gene repertoire appears to develop, and oligoclonality of the T cell response may be lost. Selection of patients with early stages of GO will be important when further dissecting TcR usage and antigen specificity of orbit-infiltrating T lymphocytes in GO.

Physiological aspects of genome variability in tissue culture. I. Growth phase-dependent differential DNA methylation of the carrot genome (Daucus carota L.) during primary culture.

Investigations were performed on growth phase-dependent EcoRII site-specific DNA methylation of the carrot genome during primary culture to elucidate physiological aspects of genome DNA variability in tissue culture. While DNA methylation of the root cambium and the secondary phloem and petioles of carrot leaves were strikingly different, the methylation level of the secondary phloem seemed to be independent of cultivar origin, the age of the plants and the extent of secondary root growth. As was shown earlier a change in the differentiated state of the secondary phloem by tissue culture leads to changes in genome modification. Whereas de novo methylation was observed during the first 2 weeks of growth initiation, the results presented demonstrate genome de-methylation during the transition to stationary growth indicating differential εnome methylation during different phases of culture. The presence of kinetin in the nutrient medium of the primary culture was found to be antagonistic to changes in genome modification in general. De novo methylation and subsequent de-methylation of the carrot genome are discussed as gross changes obviously essential to molecular genome differentiation during tissue culture.

A single-stranded DNA binding protein from S. cerevisiae specifically recognizes the T-rich strand of the core sequence of ARS elements and discriminates against mutant sequences.

A protein named ssARS-T binding protein has been purified from yeast that specifically binds to the T-rich strand of the consensus core sequence of yeast autonomously replicating sequence (ARS) elements. As assayed from gel mobility shift experiments the ssARS-T protein shows characteristics of a sequence specific single-stranded DNA binding protein. The complementary A-rich strand of the ARS core sequence is bound much more weakly and no binding can be detected for the double-stranded form of the core sequence. Three single base substitutions in the core sequence that are known to abolish ARS function in vivo also lead to weaker binding of the core sequence to the ssARS-T protein in vitro. The strong correlation between the binding of mutated sequences in vitro and the ARS properties of these sequences in vivo points to an essential function of the ssARS-T protein during replication initiation in yeast ARS elements.