Inhibition of the androgen receptor by antisense oligonucleotides regulates the biological activity of androgens in SZ95 sebocytes.

One novel strategy for the blockade of the androgen receptor could be the selective inhibition of androgen receptor by antisense oligonucleotides or small interfering RNA molecules. Here we describe the down regulation of the androgen receptor in cultured human SZ95 sebocytes with antisense oligonucleotides modified with phosphorothioates and 2'- O-methylribosyl residues. The ability of antisense oligonucleotides to cross the cellular membrane was enhanced by establishing a transient transfection system based on cationic lipid vesicles. Both antisense oligonucleotide types administered caused assumedly translational arrest. Dose-dependent inhibition of androgen receptor protein expression was observed after SZ95 sebocyte transfection with modified phosphorothioate oligonucleotides and modified 2'- O-methylribonucleotides which were directed against the translational start of the androgen receptor mRNA. The strongest transient inhibition of androgen receptor expression was detected after 14 hours with 1.0 muM antisense 2'- O-methylribonucleotides (88+/-1.3%, p<0.001). With longer recovery times than 24 hours, androgen receptor protein expression returned to the native control levels. Inhibition of the expression of androgen receptor by antisense oligonucleotides, reduced the enhanced proliferation of SZ95 sebocytes challenged by testosterone and 5alpha-dihydrotestosterone. This administration opens new therapeutic possibilities in androgen-associated skin diseases, since we could also show androgen inhibition with these antisense oligonucleotides in a reconstituted human epidermis model (Horm Metab Res 2007; 39:157-165).

[Unilateral acral necrosis as a minor form of hand-foot syndrome. Patient with unilateral acral necrosis secondary to capecitabine therapy for metastatic breast cancer]. / Unilaterale akrale Nekrosen als Abortivform einer palmoplantaren Erythrodysästhesie. Patientin mit unilateralen akralen Nekrosen unter Capecitabine-Therapie bei metastasiertem Mammakarzinom.

Hand-foot syndrome is a common toxic side-effect, particularly during chemotherapy, with an incidence of 6-64% overall and 10-24% for capecitabine (Xeloda) monotherapy. Consequently, differential diagnostic considerations should include hand-foot syndrome when acral skin lesions appear, particularly during capecitabine monotherapy. The dosage should be reduced or therapeutic regimen changed. We present a unique patient who while on capecitabine developed unilateral acral necrosis which should be classified as a minor form of hand-foot syndrome.

Zileuton, an oral 5-lipoxygenase inhibitor, directly reduces sebum production.

BACKGROUND: Zileuton, a 5-lipoxygenase inhibitor, reduces the number of inflammatory lesions in moderate acne and inhibits the synthesis of sebaceous lipids. OBJECTIVE: To detect whether zileuton directly reduces sebum synthesis. METHODS: A 40-year-old female with mild disseminated sebaceous gland hyperplasia and seborrhea was treated with zileuton 4 x 600 mg/day over 2 weeks, was followed-up for 6 weeks after discontinuation of zileuton and was re-treated with low-dose isotretinoin 10 mg/2nd day over 5 weeks. Casual skin surface lipids and sebum synthesis were determined. RESULTS: Under treatment with zileuton increased casual skin surface lipids were normalized and synthesis of facial sebum was decreased. Six weeks after discontinuation of treatment casual skin surface lipids were increased again and synthesis of sebum returned to baseline. Subsequent low-dose isotretinoin treatment led to similar changes of casual skin surface lipids and sebum synthesis with zileuton already after 2 weeks. CONCLUSION: Zileuton directly inhibits sebum synthesis in a transient manner with a potency similar to low-dose isotretinoin at least in our patient.

Fluorescent 5'-exonuclease assay for the absolute quantification of Wilms' tumour gene (WT1) mRNA: implications for monitoring human leukaemias.

The Wilms' tumour gene (WT1) has been suggested as a powerful parameter for molecular monitoring of minimal residual disease (MRD) in leukaemias. However, molecular monitoring via WT1 RNA levels is far from being routinely performed, which is possibly owing to the complex and inaccurate quantitative reverse transcription polymerase chain reaction (RT-PCR) procedures. Using a newly-developed quantitative real time RT-PCR, we measured WT1 transcripts in peripheral blood leucocytes of patients with acute myeloid (AML), acute lymphoid (ALL) and chronic myeloid leukaemia (CML). While healthy blood donors did not show measurable amounts of WT1 transcripts, WT1 RNA levels were detectable in all types of leukaemia. Furthermore, intraindividual WT1 transcript kinetics were exclusively dependent on disease progression, treatment and subsequent disease outcome. Using this approach, we could distinguish between treatment response and failure within the first days of therapeutic intervention. Moreover, gradually rising WT1 levels over a period of weeks and months paralleled long-term disease progression and appeared to be a prognostic indicator for subsequent clinical relapse. A linear correlation between quantities of WT1 and bcr/abl fusion transcripts could be seen in CML. We conclude that quantitative assessment of WT1 transcripts using real-time PCR is an appropriate method for molecular monitoring of AML, ALL and CML, and can be used independently for both short- and long-term monitoring of leukaemia patients.

Development of efficient transient transfection systems for introducing antisense oligonucleotides into human epithelial skin cells.

Systemic treatment with antisense oligonucleotides is confounded by the dual problems of potential cytotoxicity of antisense oligonucleotides and carrier molecules such as cationic lipids. Treatment of pathologic conditions affecting the skin may avoid these problems to a large degree due to local application. The success of antisense strategies has been limited by the poor uptake of the transfection reagent and inadequate intracellular compartmentalization. Human skin epithelial cells, therefore, are attractive experimental tools for testing both in vitro and in vivo antisense therapies. In the present study, we determined commercially available liposomes which reproducibly induced a nontoxic increase of oligonucleotide uptake in cultured SZ95 sebocytes and keratinocytes. The final protocol for SZ95 sebocytes was a 4-hour incubation with DOTAP in a 2:1 (w/w) lipid/oligonucleotide ratio in serum-free medium. The fluorescein-labeled (ATCG)(5) random oligonucleotide molecules were detected within the nucleus. The optimum transfection system for primary keratinocytes was poly-L-ornithine (12 microg/ml) in a medium without bovine pituitary extract over 4 hours. The uptake of the oligonucleotide increased in the presence of the polycation and oligonucleotide molecules were localized in the cytoplasm of keratinocytes. Oligonucleotide transfection with the help of cationic lipids did not affect the expression of androgen receptor and of the house-keeping gene beta-actin. Thus, cationic lipids are useful for delivery of antisense oligonucleotides into skin cells in vitro and may be used for topical application on animal and human skin.