Non-random radial higher-order chromatin arrangements in nuclei of diploid human cells.

A quantitative comparison of higher-order chromatin arrangements was performed in human cell types with three-dimensionally (3D) preserved, differently shaped nuclei. These cell types included flat-ellipsoid nuclei of diploid amniotic fluid cells and fibroblasts and spherical nuclei of B and T lymphocytes from peripheral human blood. Fluorescence in-situ hybridization (FISH) was performed with chromosome paint probes for large (#1-5) and small (#17-20) autosomes, and for the two sex chromosomes. Other probes delineated heterochromatin blocks of numerous larger and smaller human chromosomes. Shape differences correlated with distinct differences in higher order chromatin arrangements: in the spherically shaped lymphocyte nuclei we noted the preferential positioning of the small, gene dense #17, 19 and 20 chromosome territories (CTs) in the 3D nuclear interior--typically without any apparent connection to the nuclear envelope. In contrast, CTs of the gene-poor small chromosomes #18 and Y were apparently attached at the nuclear envelope. CTs of large chromosomes were also preferentially located towards the nuclear periphery. In the ellipsoid nuclei of amniotic fluid cells and fibroblasts, all tested CTs showed attachments to the upper and/or lower part of the nuclear envelope: CTs of small chromosomes, including #18 and Y, were located towards the centre of the nuclear projection (CNP), while the large chromosomes were positioned towards the 2D nuclear rim. In contrast to these highly reproducible radial arrangements, 2D distances measured between heterochromatin blocks of homologous and heterologous CTs were strikingly variable. These results as well as CT painting let us conclude that nuclear functions in the studied cell types may not require reproducible side-by-side arrangements of specific homologous or non-homologous CTs. 3D-modelling of statistical arrangements of 46 human CTs in spherical nuclei was performed under the assumption of a linear correlation between DNA content of each chromosome and its CT volume. In a set of modelled nuclei, we noted the preferential localization of smaller CTs towards the 3D periphery and of larger CTs towards the 3D centre. This distribution is in clear contrast to the experimentally observed distribution in lymphocyte nuclei. We conclude that presently unknown factors (other than topological constraints) may play a decisive role to enforce the different radial arrangements of large and small CTs observed in ellipsoid and spherical human cell nuclei.

Bladder carcinoma associated with cyclophosphamide therapy for ovarian cancer occurring with a latency of 20 years.

BACKGROUND: Cyclophosphamide-induced urothelium cancer of the bladder is a well-known entity. The risk for inducing such cancer grows with duration and dosage of cyclophosphamide therapy. The lag time between termination of treatment and development of urothelial cancer has been observed to be between 9 months and 11 years. Single cases have been reported 14, 16, 17, and 21 years after cyclophosphamide treatment. CASE: We report a case of a bladder cancer occurring after a lag time of 20 years after oral therapy with cyclophosphamide for ovarian cancer. The bladder cancer was detected due to gross hematuria. CONCLUSION: It is of great importance for gynecologists to continue to care for patients who have received long-term cyclophosphamide treatment, even if the treatment was completed decades ago. One possible early symptom of cyclophosphamide-induced bladder cancer is painless hematuria. This can easily be used to detect bladder cancer in women at risk, even after a very long latency period.

Long time therapy with topotecan in patients with recurrence of ovarian carcinoma.

BACKGROUND: In several phase II and III topotecan studies the large number of patients with stable disease is striking. Since no severe organ toxicity has been described for topotecan, long-term therapy with topotecan seems to be reasonable. In this summary we present evidence, that long-term topotecan therapy can be managed without cumulative hematological and non-hematological toxicity. PATIENTS AND METHODS: Thirty-three patients with recurrent ovarian cancer, who were treated with at least 7 courses of topotecan, were evaluated in this retrospective study (patient database from SmithKline Beecham, Germany). Most of the patients had more than 2 previous courses of chemotherapy. The starting dose was between 1.0 and 1.5 mg/m2 topotecan administered for 5 days every 3 weeks as an i.v. infusion. All patients were evaluated for toxicity, 32 patients for response. RESULTS: The 33 patients received 343 courses of topotecan, an average of more than 10 courses per patient. The highest number of courses given to a single patient was 33. The hematotoxicity was in the expected range, but toxicity was not cumulative. The number of interventions for growth factors and blood cell transfusions were constant over the whole therapy. Dose reductions were conducted in more than 75% of the patients as early as in course two. There was no grade 3 or 4 non-hematological toxicity. Alopecia was the only toxicity to be cumulative. Remissions were observed in 12 out of 32 eligible patients. The remissions were achieved after an average of 4.3 courses (range 2-7). The median time to progression was 33 weeks. CONCLUSION: Long-term therapy with topotecan is reasonable and can be conducted without cumulative hematological toxicity.

Induced micronucleus frequencies in peripheral lymphocytes as a screening test for carriers of a BRCA1 mutation in breast cancer families.

Enhanced sensitivity to the chromosome-damaging effects of ionizing radiation is a feature of many cancer-predisposing conditions. It has been suggested that women with breast cancer are deficient in the repair of radiation-induced DNA damage. We have now investigated whether mutagen sensitivity is related to mutations in the breast cancer gene BRCA1. We studied the induction and repair of DNA damage in lymphocytes of women from families with familial breast cancer and breast and ovarian cancer. The mutagens used were gamma-irradiation and hydrogen peroxide and the DNA effects were determined with the micronucleus test and the comet assay. Women with a BRCA1 mutation (n = 12) and relatives without the familial mutation (n = 10) were compared to controls (i.e., healthy women without family history of breast or ovarian cancer; n = 17). Our results indicate a close relationship between the presence of a BRCA1 mutation and sensitivity for the induction of micronuclei. Compared to a concurrent control, 10 of 11 women with a BRCA1 mutation showed elevated radiation sensitivity. Of the 10 related women without the familial mutation, only 2 had clearly enhanced micronucleus frequencies. In addition to the sensitivity toward gamma-irradiation, hypersensitivity toward hydrogen peroxide was also observed, indicating that the mutagen sensitivity is not solely due to a defect in the repair of DNA double strand breaks. In contrast to the results with the micronucleus assay, we found no significant difference between women with and without a BRCA1 mutation with respect to the induction and repair of DNA damage in the comet assay. This finding suggests a normal rate of damage removal and points to a disturbed fidelity of DNA repair as a direct or indirect consequence of a BRCA1 mutation. Our results support the usefulness of induced micronucleus frequencies as a biomarker for cancer predisposition and suggest its application as a screening test for carriers of a BRCA1 mutation in breast cancer families.

Review: biocatalytic transformations of ferulic acid: an abundant aromatic natural product.

In this review we examine the fascinating array of microbial and enzymatic transformations of ferulic acid. Ferulic acid is an extremely abundant, preformed phenolic aromatic chemical found widely in nature. Ferulic acid is viewed as a commodity scale, renewable chemical feedstock for biocatalytic conversion to other useful aromatic chemicals. Most attention is focused on bioconversions of ferulic acid itself. Topics covered include cinnamoyl side-chain cleavage; nonoxidative decarboxylation; mechanistic details of styrene formation; purification and characterization of ferulic acid decarboxylase; conversion of ferulic acid to vanillin; O-demethylation; and reduction reactions. Biotransformations of vinylguaiacol are discussed, and selected biotransformations of vanillic acid including oxidative and nonoxidative decarboxylation are surveyed. Finally, enzymatic oxidative dimerization and polymerization reactions are reviewed.