Heinrich Walt

Resistance to topoisomerase poisons due to loss of DNA mismatch repair

Sporadic breast carcinomas demonstrate microsatellite instability, reflecting the presence of DNA mismatch repair‐deficient cells, in about one fourth of cases at the time of diagnosis. Loss of DNA mismatch repair has been reported to result in resistance not only to cisplatin and alkylating agents but also to the topoisomerase II poison doxorubicin, suggesting an association between DNA mismatch repair and topoisomerase II poison‐induced cytotoxicity. Our study investigates the relationship between loss of MSH2 or MLH1 function and sensitivity to the topoisomerase I and II poisons, and to the taxanes, 2 classes of cytotoxic drugs commonly used in breast cancer. Two pairs of cell lines proficient and deficient in mismatch repair due to loss of either MSH2 or MLH1 function were used. Loss of either MSH2 or MLH1 function resulted in resistance to the topoisomerase II poisons doxorubicin, epirubicin and mitoxantrone, whereas only loss of MLH1 function was associated with low‐level resistance to the topoisomerase I poisons camptothecin and topotecan. In contrast, there was no resistance to docetaxel and paclitaxel. Our data support the hypothesis that both MSH2 and MLH1 are involved in topoisomerase II poison‐mediated cytotoxicity, whereas only MLH1 is involved in topoisomerase I poison‐mediated cytotoxicity. Since our study shows that loss of DNA mismatch repair does not result in resistance to the taxanes, these drugs can be recommended for use in breast cancer deficient in mismatch repair.

Optimizing Photodynamic Therapy: In vivo Pharmacokinetics of Liposomal meta-(Tetrahydroxyphenyl)Chlorin in Feline Squamous Cell Carcinoma

Purpose: The aim of the present study was to optimize and simplify photodynamic therapy using a new liposomal formulation of the photosensitizer meta-(tetrahydroxyphenyl)chlorin [m-THPC (Foscan); liposomal m-THPC (Fospeg)] and to reduce systemic reactions to the photosensitizer. Experimental Design: To examine the pharmacokinetics of liposomal m-THPC, we determined tissue and plasma variables in feline patients with spontaneous squamous cell carcinoma. In vivo fluorescence intensity measurements of tumor and skin were done with a fiber spectrophotometer after i.v. injection of m-THPC or liposomal m-THPC in 10 cats. Blood samples, drawn at several time points after photosensitizer administration, were analyzed by high-performance liquid chromatography. Results: None of the liposomal m-THPC–treated cats showed side effects during or after drug injection. Fluorescence intensities, fluorescence ratios (tumor fluorescence divided by skin fluorescence), and bioavailability in the tumor were 2 to 4 times higher with liposomal m-THPC compared with m-THPC. Liposomal m-THPC concentration in the tumor increased constantly to reach a maximum at 4 hours after injection. Plasma concentration and bioavailability were ∼3 times higher with liposomal m-THPC compared with m-THPC measured at the time points of highest plasma concentration. The distribution half-life was shorter with liposomal m-THPC, resulting in maximal tumor accumulation up to 5.5 times earlier. Maximal tumor accumulation and maximal fluorescence ratio with liposomal m-THPC occurred at the same time point, indicating maximal selectivity. In both groups, all cats responded to therapy. Conclusions: Liposomal m-THPC was well tolerated by all cats and seems to have superior pharmacokinetic properties compared with m-THPC. The efficacy of the drug warrants further study.

Photodynamic therapy of locoregional breast cancer recurrences using a chlorin‐type photosensitizer

Chest wall recurrences are a frequent problem in patients treated by mastectomy for breast cancer. Surgery and ionizing radiation are established treatment modalities in these cases. Photodynamic therapy (PDT) provides an alternative treatment modality using a photosensitizer and laser light to induce selective tumor necrosis. PDT was performed as compassionate use in 7 patients aged 57.6 years (±12.6 SD). A total of 89 metastatic skin nodes were treated in 11 PDT sessions. As photosensitizer meta‐tetra(hydroxyphenyl)chlorin (m‐THPC) was applied intravenously. Patients (n = 3) photosensitized with a drug dose of 0.10 mg/kg bodyweight were irradiated 48 hr after drug application at a lightdose of 5 J/cm2. Patients (n = 4) were illuminated by an optical dose of 10 J/cm2 96 hr after photosensitization with 0.15 mg/kg. Laser light at a wavelength of 652 nm was generated by a diode laser and applied by a front lens light diffuser using a fluence rate of 20–25 mW/cm2. PDT using m‐THPC resulted in complete response in all patients. Response to treatment did not differ when using the 2 different drugdose protocols. Healing time depended mainly on the size of the illumination field but not on the lightdose. Pain score usually raised 1 day after PDT and lasted at higher levels for about 10 days. Healing time usually ranged between 8–10 weeks. Photodynamic technique offers a minimal‐invasive, outpatient treatment modality for recurrent breast cancer on the chest wall with few side effects, high patients satisfaction and with possible repetitive application.

Enhanced photodynamic effects using fractionated laser light

Photodynamic eradication of tumour cells depends on the presence of a photosensitizer and light delivery to the cells. The present study investigated the influence of fractionated light (on-off mode) on cell killing as documented by a colony-forming assay. Photosensitizers were m-THPC (ethanol soluble, Foscan) and m-THPC-MD (water soluble, both from Scotia Pharmaceuticals, Guildford, UK). Fractionated laser light at a wavelength of 652 nm with a light duration of 0.05 s was more effective than continuous illumination at the same power density for both photosensitizers. We propose that fractionated laser light is more toxic due to short phases of recovery during the dark intervals, probably resulting in more singlet oxygen under these conditions. By use of Foscan, for example, and fractionated laser light, a similar effect is expected for the treatment of solid tumours. In this case we expect improvements in photodynamic therapy (PDT) for patients by lowering the concentrations of photosensitizer and/or by reducing the applied light dose.

Actin and RNA are components of the chromatoid bodies in spermatids of the rat

SummaryChromatoid bodies present in spermatocytes and spermatids of the rat show directed movements around spermatid nuclei during differentiation. This transient organelle contains RNA and establishes contact to intranuclear material and to the acrosomal complex. In order to determine possible components of motility and to verify the presence of RNA, we used a recently developed low-temperature embedding resin combined with protein A-gold and enzyme-gold techniques for studies at the ultrastructural level. All chromatoid bodies analyzed display high concentrations of gold particles over the electron-dense regions when labeled with antiactin. In contrast, RNase-gold particles were localized mainly in the electron-translucent areas. Corresponding controls were always negative. The results suggest a relationship between the impressive motility of the chromatoid body and actin present in the organelle. In addition, specific localization of RNA supports earlier findings that consider the chromatoid body an essential element for differentiation during spermiogenesis.

m‐THPC‐Mediated Photodynamic Therapy (PDT) Does Not Induce Resistance to Chemotherapy, Radiotherapy or PDT on Human Breast Cancer Cells In Vitro

Photodynamic therapy (PDT) uses laser light to activate a photosensitizer that has been absorbed preferentially by cancer cells after systemic administration. A photo‐toxic reaction ensues resulting in cell death and tissue necrosis. Some cells, however, may survive PDT. This study was performed to determine if surviving human breast cancer cells (MCF‐7) can become resistant to PDT, chemotherapy or radiotherapy. The MCF‐7 cells were cultured under standard conditions prior to being exposed to the photosensitizer, 5,10,15,20‐meta‐tet‐ra(hydroxyphenyl)chlorin (zn‐THPC), for 24 h and then irradiated with laser light (652 nm). Surviving cells were allowed to regrow by allowing a 2 week interval between each additional PDT. After the third and final treatment, colony formation assays were used to evaluate the sensitivity of cultured cells to ionizing radiation and PDT and the ATP cell viability assay tested in vitro chemosen‐sitivity. Flow cytometry was used to analyze the cell cycle. No alterations in the cell cycle were observed after three cycles of PDT with m‐THPC. Similar responses to chemotherapy and ionizing radiation were seen in control and treatment groups. The m‐THPC‐sensitized PDT did not induce resistance to subsequent cycles of PDT, chemo‐ or radiotherapy. Photodynamic therapy with m‐THPC may represent a novel adjunctive treatment of breast cancer that may be combined with surgery, chemotherapy or ionizing radiation.

A human testicular germ cell tumor with borderline histology between seminoma and embryonal carcinoma secreted beta-human chorionic gonadotropin and alpha-fetoprotein only as a xenograft.

Two microscopically distinguishable components (designated soft and firm) of a human testicular germ cell tumor with borderline histology between seminoma, embryonal carcinoma and yolk sac tumor, were maintained as xenografts in nude mice for over 20 passages. Levels of beta‐human chorionic gonadotropin (β‐HCG) and of alpha‐fetoprotein (AFP) were normal in the patients serum and were undetectable by immunohistochemical studies of the surgical specimen. The xenografted soft part, however, with morphologic characteristics of an embryonal carcinoma, secreted β‐HCG and AFP during early passages. The firm variant, histologically resembling a seminoma, did not produce these markers. Chromosomal and flow cytophotometric analyses showed genetic differences between the clonally stable variants. A common origin, however, is indicated by two similar marker chromosomes present in both variants. Cancer 58:139–146, 1986.

Uptake and fate of surface modified silica nanoparticles in head and neck squamous cell carcinoma.

BackgroundHead and neck squamous cell carcinoma (HNSCC) is currently the eighth leading cause of cancer death worldwide. The often severe side effects, functional impairments and unfavorable cosmetic outcome of conventional therapies for HNSCC have prompted the quest for novel treatment strategies, including the evaluation of nanotechnology to improve e.g. drug delivery and cancer imaging. Although silica nanoparticles hold great promise for biomedical applications, they have not yet been investigated in the context of HNSCC. In the present in-vitro study we thus analyzed the cytotoxicity, uptake and intracellular fate of 200-300 nm core-shell silica nanoparticles encapsulating fluorescent dye tris(bipyridine)ruthenium(II) dichloride with hydroxyl-, aminopropyl- or PEGylated surface modifications (Ru@SiO2-OH, Ru@SiO2-NH2, Ru@SiO2-PEG) in the human HNSCC cell line UMB-SCC 745.ResultsWe found that at concentrations of 0.125 mg/ml, none of the nanoparticles used had a statistically significant effect on proliferation rates of UMB-SCC 745. Confocal and transmission electron microscopy showed an intracellular appearance of Ru@SiO2-OH and Ru@SiO2-NH2 within 30 min. They were internalized both as single nanoparticles (presumably via clathrin-coated pits) or in clusters and always localized to cytoplasmic membrane-bounded vesicles. Immunocytochemical co-localization studies indicated that only a fraction of these nanoparticles were transferred to early endosomes, while the majority accumulated in large organelles. Ru@SiO2-OH and Ru@SiO2-NH2 nanoparticles had never been observed to traffic to the lysosomal compartment and were rather propagated at cell division. Intracellular persistence of Ru@SiO2-OH and Ru@SiO2-NH2 was thus traceable over 5 cell passages, but did not result in apparent changes in cell morphology and vitality. In contrast to Ru@SiO2-OH and Ru@SiO2-NH2 uptake of Ru@SiO2-PEG was minimal even after 24 h.ConclusionsOur study is the first to provide evidence that silica-based nanoparticles may serve as useful tools for the development of novel treatment options in HNSCC. Their long intracellular persistence could be of advantage for e.g. chronic therapeutic modalities. However, their complex endocytotic pathways require further investigations.

Novel flexible light diffuser and irradiation properties for photodynamic therapy

Many current light diffusers for photodynamic therapy are inflexible, and the applied light dose is difficult to adjust during treatment, especially on complex body surfaces. A thin and flexible luminous textile is developed using plastic optical fibers as a light distributor. The textile diffuser is evaluated for flexibility, irradiance, brightness distribution, and temperature rise with a 652-nm laser set to 100 mW. The bending force of the textile diffuser resembles a defined optical film. On the textile surface, an average output power of 3.6+/-0.6 mWcm(2) is measured, corresponding to a transmission rate of 40+/-3.8% on an area of 11 cm(2). Aluminum backing enhances the irradiance to the face (treatment side). The measured brightness distribution seems to lie within a range similar to other photodynamic therapy (PDT) devices. A power setting of 100 mW increases the temperature of the textile diffuser surface of up to 27 degrees C, and 1 W raises the temperature above 40 degrees C. Results confirm that the flexible textile diffuser supplies suitable radiation for low fluence rate photodynamic therapy on an area of several cm(2).

Differentiation potential of ovarian dysgerminoma: An immunohistochemical study of 15 cases

An immunohistochemical study of 15 ovarian formalin-fixed, paraffin-embedded dysgerminomas showed positive staining of tumor cells for vimentin in all cases. Ten dysgerminomas stained for cytokeratin 18. Desmin positivity of single tumor cells was detected in four dysgerminomas. Glial fibrillary acidic protein was present in two tumors. Prominent human beta chorionic gonadotropin staining was seen in one tumor. S-100 protein was found in two and carcinoembryonic antigen in one of the dysgerminomas. Placental alkaline phosphatase was present in 12 of the 15 tumors studied. The heterogeneity of the cytoskeletal profile and of other markers showed some similarities to our previously published results on testicular seminomas. Thus, in contrast to previous concepts, dysgerminoma, as is the case with its testicular counterpart the seminoma, appears to be capable of further differentiation, albeit at a primitive level. Our observations also may help to elucidate the relationship between dysgerminoma and other nondysgerminomatous ovarian germ cell tumors, and may be of help in the differential diagnosis with poorly differentiated carcinoma, ovarian lymphoma, or other germ cell tumors.