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P Steinbach

Publications and source records attributed to P Steinbach.

At least 19 recordsLinked to original sources
PubMed2001

Demethylation, reactivation, and destabilization of human fragile X full-mutation alleles in mouse embryocarcinoma cells.

The major causes of fragile X syndrome are mutational expansion of the CGG repeat in the FMR1 gene, hypermethylation, and transcriptional silencing. Most fragile X embryos develop somatic mosaicism of disease-causing "full" expansions of different lengths. Homogeneity of the mosaic patterns among multiple tissues in the same individual indicates that these previously unstable expansions acquire mitotic stability early in fetal life. Since mitotic stability is found strictly associated with hypermethylation in adult tissues, current theory has fixed the time of instability to developmental stages when fully expanded CGG repeats exist in an unmethylated state. We used murine embryocarcinoma (EC) cells (PC13) as a model system of pluripotent embryonic cells. Hypermethylated and unmethylated full expansions on human fragile X chromosomes were transferred from murine A9 hybrids into EC cells, by means of microcell fusion. As demonstrated in the present study for the first time, even full expansion alleles that were fully methylated and stable in the donors' fibroblasts and in A9 became demethylated, reactivated, and destabilized in undifferentiated EC hybrids. When destabilized expansions were reintroduced from EC cells into A9, instability was reversed to stability. Our results strongly support the idea that fully expanded alleles are initially unstable and unmethylated in the human embryo and gain stability upon genetic or epigenetic change of the embryonic cells.

Alleles↗
PubMed2000

Increase of FMRP expression, raised levels of FMR1 mRNA, and clonal selection in proliferating cells with unmethylated fragile X repeat expansions: a clue to the sex bias in the transmission of full mutations?

Fragile X syndrome is a triplet repeat disorder caused by expansions of a CGG repeat in the fragile X mental retardation gene (FMR1) to more than 220 triplets (full mutation) that usually coincide with hypermethylation and transcriptional silencing. The disease phenotype results from deficiency or loss of FMR1 protein (FMRP) and occurs in both sexes. The underlying full mutations arise exclusively on transmission from a mother who carries a premutation allele (60-200 CGGs). While the absolute requirement of female transmission could result from different mechanisms, current evidence favours selection or contraction processes acting at gametogenesis of pre- and full mutation males. To address these questions experimentally, we used a model system of cultured fibroblasts from a male who presented heterogeneous unmethylated expansions in the pre- and full mutation size range. On continual cell proliferation to 30 doublings we re-examined the behaviour of the expanded repeats on Southern blots and also determined the expression of the FMR1 gene by FMRP immunocytochemistry, western analysis, and RT-PCR. With increasing population doublings, expansion patterns changed and showed accumulation of shorter alleles. The FMRP levels were below normal but increased continuously while the cells that were immunoreactive for FMRP accumulated. The level of FMR1 mRNA was raised with even higher levels of mRNA measured at higher passages. Current results support the theory of a selection advantage of FMRP positive over FMRP deficient cells. During extensive proliferation of spermatogonia in fragile X males, this selection mechanism would eventually replace all full mutations by shorter alleles allowing more efficient FMRP translation. At the proliferation of oogonia of carrier females, the same mechanism would, in theory, favour transmission of any expanded FMR1 allele on inactive X chromosomes.

Base Sequence↗
PubMed1999

Tissue-specific methylation differences in a fragile X premutation carrier.

Methylation of a premutation was found in a small percentage of blood cells in a male premutation carrier for the FMR1 mutation. To investigate the inter-tissue heterogeneity and possible clinical implications of this finding, fibroblast cells from the subject were also studied. Although the premutation size was found to be the same in leukocytes and fibroblasts, the methylation pattern was different. In cultured fibroblasts, the premutation was completely unmethylated, as is typical of premutations, whereas methylation of the premutation was detected in a small percentage of lymphocytes. However, the change in methylation did not affect the FMR1 protein (FMRP) expression, as immunocytochemical analysis of FMRP performed on cultured skin fibroblasts and a blood smear revealed normal levels of expression in both tissues.

DNA↗
PubMed1998

The DMPK gene of severely affected myotonic dystrophy patients is hypermethylated proximal to the largely expanded CTG repeat.

Using methylation-sensitive restriction enzymes, we characterized the methylation pattern on the 5' side of the CTG repeat in the DMPK gene of normal individuals and of patients affected with myotonic dystrophy, showing expansions of the repetitive sequence. The gene segment analyzed corresponds to the genomic SacI-HindIII fragment carrying exons 11-15. There is constitutive methylation in intron 12 at restriction sites of SacII and HhaI, localized 1,159-1,232 bp upstream of the CTG repeat, whereas most, if not all, of the other sites of SacII, HhaI, and HpaII in this region are unmethylated, in normal individuals and most of the patients. In a number of young and severely affected patients, however, complete methylation of these restriction sites was found in the mutated allele. In most of these patients, the onset of the disease was congenital. Preliminary in vivo footprinting data gave evidence for protein-DNA contact in normal genes at an Sp1 consensus binding site upstream of the CTG repeat and for a significant reduction of this interaction in cells with a hypermethylated DMPK gene.

Adolescent↗
PubMed1998

Unusual mutations in high functioning fragile X males: apparent instability of expanded unmethylated CGG repeats.

We report on further cases of high functioning fragile X males showing decreased expression of FMR1 protein, absence of detectable methylation at the EagI site in the FMR1 gene promoter, and highly unusual patterns of fragile X mutations defined as smear of expansions extending from premutation to full mutation range. Very diffuse and therefore not easily detectable patterns of full mutations were also observed on prenatal testing using DNA from chorionic villi sampled at a time of development when full mutations were still unmethylated in this particular tissue. In the search for possible determinants of such unusual patterns, repeat expansions in the premutation and in the lower full mutation range were identified on genomic PstI blots previously prepared for fragile X DNA testing. Cases with 130 or more triplets, and a number of shorter repeats, were reinvestigated on EcoRI plus EagI digests. Among the 119 expansions, there were 22 in our sample showing either blurred bands or smears on PstI blots. This particular characteristic was strongly associated with the coincidence of a repeat size of more than 130 triplets and absence of EagI site methylation. Our data set also includes cases of mosaic patterns consisting of smears of unmethylated expansions to more than 130 CGGs and of clear bands of methylated expansions. We therefore suggest that in fragile X syndrome unusual smeared patterns of mutations result from somatic instability of larger repeats under circumstantial absence of repeat methylation.

Blotting, Southern↗
PubMed1997

Analysis of cell cycle-related Ki-67 and p120 expression by flow cytometric BrdUrd-Hoechst/7AAD and immunolabeling technique.

Flow cytometric multiparameter analysis of two proliferation associated antigens, Ki-67 and p120, was combined with cell cycle kinetic analysis, achieved by continuous labeling with 5-Bromodeoxyuridine (BrdUrd), followed by staining with Hoechst 33258 and 7-Aminoactinomycin D (7AAD). Exponential and plateau phase monolayer cultures of the human bladder carcinoma cell line J82 were examined. Resting cells, characterized by their absent BrdUrd incorporation, showed no reactivity with the MIB1 antibody, which was used for the detection of the Ki-67 antigen. Proliferating cells revealed a cell cycle phase dependent Ki-67 staining intensity, which was partially related to the time period spent in G1 after mitosis. In contrast to the Ki-67 antigen expression, no decrease in p120 immunofluorescence staining intensity of non-cycling cells could be observed. We could demonstrate that a dissection of the history of cell replication, obtained by the BrdUrd/Hoechst technique combined with a simultaneous immunofluorescence staining reveals detailed information, on a single cell level, about time dependent expression of proliferation associated antigens in all cell cycle compartments.

Antigens, Neoplasm↗
PubMed1997

Indocyanine green: intracellular uptake and phototherapeutic effects in vitro.

Indocyanine green (ICG; absorption peak in human plasma 805 nm) was investigated for ICG-mediated phototherapy in vitro. The cellular uptake of ICG (1 microM-50 microM) into HaCaT keratinocytes after an incubation period of 24 h increased up to an intracellular ICG concentration of 12.1 +/- 1.3 nmol per 10(6) cells. To examine dose dependent phototoxic effects in vitro, keratinocytes were incubated with 0 microM-50 microM ICG for 24 h and irradiated by a diode laser (805 nm) with different energy densities (0, 12, 24, 48 J cm-2). All applied ICG concentrations except for 5 microM yielded a cell killing effect in combination with irradiation depending significantly on ICG concentration and light dose. Cell viability for dark control and cells incubated with 50 microM ICG and irradiated with 48 J cm-2 was 0.82 +/- 0.15 and 0.07 +/- 0.02, respectively. Sodium azide (100 mM), a quencher of reactive oxygen species, inhibited significantly the cell killing using 50 microM ICG and 24 J cm-2. Taken together, photoactivation of ICG by irradiation with a diode laser was shown to induce effectively cell killing of HaCaT keratinocytes. Moreover, this effect was inhibited by sodium azide, thus irradiation of ICG might induce a photodynamic reaction.

Antineoplastic Agents↗
PubMed1997

Targeting of the tumor microcirculation by photodynamic therapy with a synthetic porphycene.

9-acetoxy-2,7,12,17-tetrakis-(beta-methoxyethyl)-porphycene (ATMPn) is a chemically pure substance with fast pharmacokinetics and superior photodynamic properties in vitro as compared to Photofrin. In this study the pharmacokinetics, photodynamic efficacy and tissue localization of ATMPn were investigated in vivo. Amelanotic melanomas (A-Mel-3) were implanted in dorsal skin fold chambers fitted to Syrian Golden hamsters. Fluorescence kinetics of ATMPn (1.4 mumol kg-1 b.w.i.v.; n = 8) were monitored by intravital microscopy. Quantitative measurements of fluorescence intensity were carried out by digital image analysis. For tumor growth studies 1.4 mumol kg-1 was injected 24 h (n = 3), 3 h (n = 3), 1 min (n = 6) and 2.8 mumol kg-1 1 min (n = 6) before PDT (Laser (630 nm) or lamp (600-750 nm), 100 mW cm-2, 100 J cm-2). Tumor volume was measured for 28 d. Solid tumors (n = 3) were excised 1 min after injection of ATMPn (2.8 mumol kg-1) and cryostat sections (20 mm) were analyzed by confocal laser scanning microscopy (CLSM) for tissue localization of the dye. Maximal fluorescence (mean +/- S.E.) arose in the tumor (94 +/- 7%) and surrounding host tissue (67 +/- 5%) 30 s post injection followed by a rapid decrease. Hardly any fluorescence was detectable 12 h after administration. Only PDT 1 min after injection of ATMPn was effective yielding 3/6 complete remissions (2.8 mmol kg-1, laser) and 6/6 complete remissions (2.8 mumol kg-1, lamp), respectively. One minute after injection the dye is primarily localized in the vascular wall of normal and tumor vessels as shown by CLSM. PDT at a time, when the dye is localized primarily in the tumor microcirculation, exhibits the best tumor killing effects showing that vascular targeting is effective in treating solid malignant tumors. ATMPn in liposomes makes administration and light irradiation in one session possible due to its fast pharmacokinetics. Thus, using ATMPn as a photosensitizer may provide more flexibility to perform PDT after surgical exploration and debulking as adjuvant therapy.

Animals↗
PubMed1997

Influence of a haematoporphyrin derivative on the protoporphyrin IX synthesis and photodynamic effect after 5-aminolaevulinic acid sensitization in human colon carcinoma cells.

Haematoporphyrin derivatives (HPDs) are potent sensitizers in photodynamic therapy (PDT), associated with prolonged skin photosensitivity. 5-Aminolaevulinic acid (5-ALA), a natural precusor of haem, is converted intracellularly into the photosensitive agent protoporphyrin IX (PPIX), causing direct cytotoxicity after laser light irradiation but limited skin photosensitivity over 1-2 days and higher tumour selectivity. Unfortunately, the use of 5-ALA in PDT has been shown to cause only superficial tissue necrosis. Therefore, a combination of HPD and 5-ALA could be of great clinical value in the treatment of tumours if a synergistic effect of both sensitizers on tumour cell necrosis with less skin photosensitivity could be demonstrated. Human colon adenocarcinoma cells (HT-29) were cultured with either HPD or 5-ALA alone, simultaneously for 24 h with 5-ALA and HPD or in succession with 5-ALA (18 h) followed by HPD (6 h at different concentrations. Intracellular PPIX concentrations were determined by high-performance thin-layer chromatography. Furthermore, PDT was performed with an incoherent light source (lambda = 580-740 nm) using a light dose of 30 J cm(-2) and an output power of 40 mW cm(-2). The intracellular PPIX concentration correlated well with 5-ALA drug dose and incubation time and was highest after single 5-ALA sensitization. In the presence of HPD, either simultaneously or sequentially, PPIX decreased significantly. The PDT effect after simultaneous incubation with both sensitizers for 24 h was not superior to incubation with HPD alone. If 5-ALA incubation (18 h) was followed by HPD (6 h) cytotoxicity after PDT was higher than with either single drug. 5-ALA (80 microg ml(-1)) led to a decrease in tumour cell viability by 40%. A similar effect could be observed when 5-ALA and HPD were sequentially combined allowing for a reduction of the 5-ALA dose from 80 microg ml(-1) in the absence of HPD to 60 microg ml(-1) and 5 microg ml(-1) together with 0.5 microg ml(-1) and 2 microg ml(-1) HPD respectively. We speculate that the enhanced PDT effect after the combined administration of 5-ALA and HPD to cultures of colon carcinoma cells should be even more impressive in the tumour in vivo, since HPD primarily targets the tumour microvasculature and secondarily tumour cells.

Aminolevulinic Acid↗
PubMed1997

Characterization of FMR1 promoter elements by in vivo-footprinting analysis.

Fragile X syndrome is associated with silencing of the FMR1 gene. We studied the transcriptional regulation, by analysis of the FMR1 promoter region for the presence of in vivo protein/DNA interactions and for cytosine methylation at the single-nucleotide level. Four protein-binding sites were present in the unmethylated promoter of the active FMR1 gene. In the methylated promoter of inactive genes no footprints were detected, and no evidence of active repression was found in the region investigated. We propose that the silencing of FMR1 gene transcription results from a lack of transcription-factor binding.

Adult↗
PubMed1997

Wavelength dependent photodynamic effects on chemically induced rat bladder tumors following intravesical instillation of 5-aminolevulinic acid.

PURPOSE: The aim of the study was to determine photodynamic effects in chemically induced rat bladder tumors after intravesical instillations of 5-aminolevulinic acid (5-ALA) and PDT-dependence on laser wavelength and light dose. MATERIAL AND METHODS: Following intravesical instillation of a 5-ALA solution (40 mg 5-ALA in isotonic saline and sodium-bicarbonate) rat bladder tumors have been treated with integral irradiation using laser light at wavelengths of lambda = 630 nm and lambda = 635 nm in a light dose range of 15 to 100 J/cm2. RESULTS: A significantly higher amount of necrosis including damage of normal urothelium was obtained in rat bladder tumors using laser light at lambda = 635 nm. CONCLUSIONS: For 5-ALA assisted photodynamic treatment of superficial bladder cancer in patients the irradiation wavelength of lambda = 635 nm is recommended.

Aminolevulinic Acid↗
PubMed1996

Cell-type-specific response to shock waves of suspended or pelleted cells as analysed by flow cytometry or electrical cell volume determination.

Shock-wave-induced cell damage of suspended or pelleted bladder cancer cells was analysed with the flow cytometric propidium iodide (PI)/fluorescein diacetate assay, and electrical volume determination using the CASY 1 analyser system and growth curves. The CASY system revealed a smaller fraction of suspended RT4 cells with impaired membrane integrity than the flow cytometric assay. No differences were found for pelleted RT4 cells and suspended J82 cells. The discrepancies of the two viability assays indicated a different response of the cell membrane to shock waves which was dependent on the exposure system and the cell type. Growth curves indicated delayed cell death for suspended RT4 cells and exclusively immediate cell death for pelleted RT4 cells and suspended J82 cells. PI positive suspended RT4 cells were morphologically intact while pelleted RT4 cells and suspended J82 cells were mainly disrupted. From these data it can be concluded that intracellular or membrane alterations seem to be correlated with the occurrence of cavitational effects while cell disruption can likewise occur by the direct impact of the shock wave.

Carcinoma, Transitional Cell↗
PubMed1996

9-Acetoxy-2,7,12,17-tetrakis-(beta-methoxyethyl)-porphycene (ATMPn), a novel photosensitizer for photodynamic therapy: uptake kinetics and intracellular localization.

The optimal photosensitizer for topical or systemic photodynamic therapy (PDT) has not yet been found. A promising new second-generation sensitizer is 9-acetoxy-2,7,12,17-tetrakis-(beta-methoxyethyl)-porphycene (ATMPn) whose time- and temperature-dependent uptake and intracellular localization were investigated in two human-skin-derived cell lines (HaCaT keratinocytes and dermal fibroblasts). Flow cytometry analysis (0-800 s) revealed an immediate increase in fluorescence in the cells after start of incubation with 100 ng ml-1 ATMPn (in cell culture medium). At longer incubation periods (0-24 h) a constant increase in fluorescence up to 12 h, with a steady state up to 24 h, was observed. Keratinocyte showed a faster rate of ATMPn uptake than fibroblasts within the first 12 h. Temperature-dependent ATMPn uptake was measured at 4 and 37 degrees C. An increase in fluorescence was observed even at 4 degrees C, suggesting that cellular uptake of ATMPn is partially based on passive diffusion. Confocal laser scan miscroscopy showed spotty, granular fluorescence inside the cytoplasm after incubation with ATMPn, similar to the pattern of rhodamine 123 which stains mitochondria. These results demonstrated an unusually fast intracellular, probably intramitochondrial, uptake of ATMPn in vitro. Therefore the use of ATMPn in photodynamic therapy might allow a reduction of the time span between administration of drug and irradiation.

Cell Line↗