Influence of Vitamin D - Metabolites and -Analogs on Growth and Apoptosis of C6-Rat-Glioma-Cells

TABLE OF CONTENTS

1. INTRODUCTION
1.1. General aspects of Vitamin D
1.1.1. Synthesis and Metabolism
1.1.2. Biological Role of Vitamin D
1.2. Mechanisms of Cell Death: Apoptosis versus Necrosis
1.3. Vitamin D and Apoptosis of Malignant Cells
1.3.1. Vitamin D as an Anti-cancer Agent
1.3.2. Anti-tumor Actions of Vitamin D on Glioma
1.3.3. C6-Rat Glioma Cells: Model to Study Vitamin D Actions
1.4. Aim of diploma thesis

2. MATERIALS AND METHODS
2.1. Used devices and reagents
2.1.1. Cell culture
2.1.2. Compounds used in Incubations
2.1.2.1. Etoposide: solution of 1,2 mg/ml (2 mmol/l) in ethanol (96 vol.%);
2.1.2.2. Vitamin D-analogues or -metabolites : (were kindly provided by S. Reddy (Brown University, Rhode Island, USA))
2.1.3. Materials used in Analytical procedures Materials for DNA-Isolation
2.1.5. Materials for Capillary electrophoresis
2.2. Methods
2.2.1. Cell Culture
2.2.1.1. Thawing of frozen samples of C6-cells and cultivation
2.2.1.2. Subcultivation of C6-cells ("splitting")
2.2.2. Incubations
2.2.3. Analytical methods
2.2.3.1. Neutral Red Assay
2.2.3.2. Trypan-blue-assay and cell-counting
2.2.3.3. Staining of cell nuclei with Hoechst No
2.2.4. DNA-Isolation
2.2.5. Capillary gel electrophoresis
2.2.6. Evaluations, Calculations

3. RESULTS
3.1. Effect of 1α,25(OH)2D3 on growth of C6-rat-glioma-cells
3.1.1. Growth of C6-rat-glioma-cells in serum-containing culture medium
3.1.2. Growth of C6-rat-glioma-cells in the absence of serum
3.2. Viability of C6-rat-glioma-cells and induction of apoptosis by 1,25(OH)2D3 and 1,25(OH)2-3-epi-D3
3.2.1. Dose-dependent effects on viability
3.2.2. Time course of apoptosis and dose-dependent effects
3.3. Influence of various Vitamin D - metabolites and and - analogs on growth and apoptosis of C6-rat-glioma-cells
3.3.1. Effects of natural metabolites on growth and apoptosis
3.3.2. Effects of synthetic analogs on growth and apoptosis
3.4. Detection of DNA-fragmentation in C6-rat-glioma cells by Capillary electrophoresis (CE)
3.4.1. Cell numbers and yield of DNA
3.4.2. Separation of DNA by CE

4. DISCUSSION
4.1. Anti-cancer effects of 1α,25-(OH)2-D3 on glioma
4.2. Can 3-epi-vitamin D-analogues offer advantages over 1α,25-(OH)2-D3 in the treatment of gliomas?
4.2.1. C-3 epimerization is a metabolic pathway of 1α,25-(OH)2-D3
4.2.2. Established biological activities of 3α-vitamin D-analogues
4.3. Vitamin D-metabolites and -analogs tested in this study: actions on growth and apoptosis of C6-glioma-cells
4.3.1. Relevance of applied methods
4.3.2. Effects of 1α,25-(OH)2-D3 on cell growth in the presence and in absence of serum
4.3.3. Comparison: 1α,25-(OH)2-D3 and 1α,25-(OH)2-3-epi-D3
4.3.4. Effects of other vitamin D-metabolites
4.3.5. Vitamin D-analogues : Comparison 3β- and 3α-epimers
4.4. Availability of vitamin D-metabolites/-analogs at the target site
4.4.1. Uptake into the brain
4.4.2. Availability at the tumor site
4.5. Conclusions

5. REFERENCES

1. INTRODUCTION

1.1. General aspects of Vitamin D

1.1.1. Synthesis and Metabolism

Vitamin D in the human body exists in two forms, derived from two sources. In the form of Vitamin D3, it is generated from 7-dehydrocholesterol in the skin by exposure to ultraviolet light (270-300 nm range), and in the form of Vitamin D2 or Vitamin D3 (Fig. 1), it can be derived from diet.

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Fig. 1. Chemical structures of vitamin D3 and vitamin D2

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Fig. 2. Electron transport chain for mitochondrial steroid hydroxylases. Concept for 3-dimensional arrangement of components of mitochondrial cytochrome P-450-containing hydroxylases is shown (Jones et al. (1998)).

Both forms of Vitamin D are precursor of functionally active hormones and undergo the same two-step activation process leading to 1α,25-(OH)2-D2 or 1α,25-(OH)2-D3, respectively. These metabolic activations of Vitamin D3 are carried out a by specific P-450-containing enzymes, the vitamin D3-25- hydroxylase (CYP27A) and the 25-hydroxyvitamin D-1α-hydroxylase (CYP27B1). Both hydroxylases are located in the inner mitochondrial membrane of cells (Fig. 2).There is little evidence that the two hormones differ in their mode of action (Jones et al. (1998)).

In contrast to earlier assumptions, which strictly localized 25- hydroxylation in the liver and sequential 1α−hydroxylation in the kidney (Blunt et al. (1968)), many different cell types were shown to be capable of the two-step activation process too. Regulation of both enzymes appears to be tissue- specific: CYP27A is only loosely regulated (Bhattacharyya et DeLuca (1973)) or constitutively expressed (Schüssler et al. (2001)). 1α-Hydroxylase in the kidney is tightly regulated by the levels of plasma 1,25-(OH)2D3 and calcium (via the parathyroid hormone (PTH)) (reviewed by Jones et al. (1998)), however constitutively expressed in skin (Schüssler et al. (2001)). A third vitamin D- related mitochondrial cytochrome P-450-containing enzyme is the 25- hydroxyvitamin D-24-hydroxylase (CYP24). This enzyme is strongly inducible by 1α,25-(OH)2-D3 in practically all cell types of the body, prefers 1α,25-(OH)2- D3 as a substrate over 25-OH-D3, and catalyzes several steps of 1α,25-(OH)2- D3 - metabolism, collectively known as the C-24 oxidation pathway (Fig.3., Reddy and Tserng, 1989; Makin et al., 1989).

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Fig. 3. Vitamin D Cascade:Synthesis and metabolism of hormonally active vitamin D.

Besides catalyzing three successive oxidations, two at C-24 and one at C-23, there is strong evidence that CYP24 cleaves the resulting intermediate to give the C-23 alcohol/acid (Beckman et al., 1996; Schuster et al., 2001b). Although the terminal product of this pathway, calcitroic acid, is less active than 1α,25-(OH)2-D3 in vivo, most intermediates still retain substantial biological activity (Schuster et al. 1997, Jones et al. (1998)).

Recently, a novel tissue specific pathway was found, namely the C-3 epimerization pathway, in which 1α,25-(OH)2-3-epi-D3 is formed as a result of the change in the orientation of the hydroxyl group at the C-3 position from β to α (see also Fig. 3) (Reddy et al. (1997, 2001), Astecker et al. (2000)). 1α,25- (OH)2-3-epi-D3 also undergoes 24-oxidation, however at a much slower rate than 1α,25-(OH)2-D3 (Astecker et al. 2000, Schuster et al. 2001b).

1.1.2. Biological Role of Vitamin D

1α,25-(OH)2-D3 is believed to be the major active form of Vitamin D Its mode of action in generating biological responses is analogous to that of the classical steroid hormones like estradiol, testosterone, progesterone, cortisol and aldosterone (Evans (1988), Haussler et al. (1988)).

Genomic mode of action of 1 α ,25-(OH)2-D3. 1α,25-(OH)2-D3 exerts effects on transcription of so called vitamin D-target genes by means of a nuclear receptor protein, the vitamin D-receptor (VDR). 1α,25-(OH)2-D3 (and its analogues) are lipophilic molecules, that easily pass cellular membranes and enter the nucleus, where they bind with high affinity to the VDR. The hormone- receptor-complex then binds to specific DNA sequences termed response elements (vitamin D responsive elements, VDRE) in the promoter regions of these genes, thereby up- or downregulating them, respectively (reviewed by Jones et al. (1998)). This process includes interaction of the VDR with other nuclear receptors (like the retinoid X receptor, RXR) for the formation of dimeric complexes and contact with cofactors, such as coactivators and corepressors, for modulation of transcriptional activities (Carlberg et al. (2001)). Thus, the presence of the VDR in a cell confers it to become a target cell which is producing the biological response (Stumpf et al. (1979), Reichel et al. (1989a)).

Actions of 1 α ,25-(OH)2-D3. 1α,25-(OH)2-D3 regulates the expression of a very wide variety of gene, resulting in pleiotropic activities. Besides its central role in calcium homeostasis and, therefore in bone formation (DeLuca et al. (1990)), 1α,25-(OH)2-D3 is also crucially involved in controlling cellular growth, differentiation and apoptosis in normal as well as in malignant cells (Walters (1992)). Moreover, important immunoregulatory activities have been demonstrated, in particular, inhibition of the expression of Th1-cytokines (interferon γ, interleukin 2) and modulation of antigen expressing cells (reviewed by Mathieu and Adorini, 2002). Key functions have been confirmed in the n]ervous system, including regulation of synthesis of neurotrophic factors and neurotransmitters (reviewed by Garcion et al. 2002) Table 1 displays a selection of genes that have been shown to be responsive to Vitamin D.

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Table 1. Examples for established transactivation by the 1α,25-(OH)2-D3 - receptorcomplex in the transcription of various genes/proteins (Minghetti et Norman (1988), Reichel et Norman (1989b), Darwish et DeLuca (1996), Demay et al. (1992), Naveilhan et al. (1996), Neveu et al. (1994a), Neveu et al. (1994b)).

1.2. Mechanisms of Cell Death: Apoptosis versus Necrosis

Cell death can occur by either of two distinct mechanisms, necrosis or apoptosis. Necrosis, an “accidental” form of cell death, can be briefly defined as a pathological process, which occurs when cells are exposed to a serious physical or chemical insult. Apoptosis, on the other hand, is a tightly regulated process and thus also called “programmed” cell death. It is the physiological process by which unwanted or useless cells are eliminated during development and other normal biological processes (reviewed by Wyllie et al. (1998)). There are many observable morphological (Fig. 4, Fig. 5 and Table 2) and biochemical differences (Table 2) between necrosis and apoptosis.

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Fig. 4. Illustration of the morphological features of necrosis (Wyllie et al. (1998)).

Necrosis occurs when cells are exposed to severe changes from physiological conditions (e. g. hypothermia, hypoxia), which may result in damage to the plasma membrane. It begins with an impairment of the cell’s ability to maintain osmotic homeostasis, leading to an influx of water and extracellular ions. Intracellular organelles and the entire cell swell and rupture (cell lysis). As a consequence, the cytosolic contents including lysosomal enzymes are released into the extracellular fluid. Therefore, in vivo, necrotic cell death is often associated with extensive tissue damage resulting in an intense inflammatory response (reviewed by Wyllie et al. (1998)).

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Fig. 5. Illustration of the morphological features of apoptosis (Wyllie et al. (1998)).

Apoptosis, in contrast, is a mode of cell death that occurs under normal physiological conditions and the cell is an active participant in its own demise (“cellular suicide”). It is most often found during normal cell turnover and tissue homeostasis, embryogenesis, induction and maintenance of immune tolerance, development of the nervous system and endocrine-dependent tissue atrophy. Cells undergoing apoptosis show chromatin aggregation, nuclear and cytosolic condensation, and partition of the cytoplasm and nucleus into membrane bound-vesicles (apoptotic bodies). These are rapidly recognized and phagocytized by either macrophages or adjacent epithelial cells in vivo, therefore no inflammatory response is elicited in this situation. In vitro, however, the apoptotic bodies as well as the remaining cell fragments ultimately swell and finally lyse (“secondary necrosis”, Fig. 5) (reviewed by Wyllie et al. (1998)).

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Table 2. Differential features and significance of necrosis and apoptosis (Wyllie et al., (1998)).

1.3. Vitamin D and Apoptosis of Malignant Cells

1.3.1. Vitamin D as an Anti-cancer Agent

Apoptosis, which has been described in the previous section (1. 2.), is a process that counteracts the development and progression of cancer. Cancer is often associated with cells that fail to undergo apoptosis. This leads to the survival of aberrant cells, which would normally die, and, in turn, to malignant overgrowth.

1α,25-(OH)2-D3 is a potent anti-cancer agent, as demonstrated by abundant in vitro and in vivo investigations, ranging from studies on representative cell-lines to tumor models and clinical studies (reviewed by Hansen et al. (2001)). In vivo, 1α,25-(OH)2-D3 possesses the ability to suppress the formation of chemically induced tumors, causes regression of tumors, prevents the development of metastases, inhibits angiogenesis and prolongs survival time in tumor-bearing animals (reviewed by van den Bemd et al. (2000)) Moreover, a protective role of vitamin D against cancer becomes evident from epidemiological investigations, which show an inverse relationship between vitamin D deficiency and cancer (Schwartz et Hulka (1990), Garland et Garland (1980), Studzinski et Moore (1995), Garland et al. (1989), Garland et al. (1991), Newmark (1994)).

Hormonally active vitamin D may cause its anti-tumor by several distinct mechanisms. Vitamin D can

A) Directly inhibit growth of tumor cells and
B) Stimulate their terminal differentiation: Since the early studies by Colston et al. (1981) and Abe et al. (1981), numerous in vitro investigations have demonstrated that 1α,25-(OH)2-D3 is able to affect growth and differentiation in most cancer cell types posessing VDRs (Feldman et al. (1997), MacDonald et al. (2001), Hansen et. al. (2000), van den Bemd et al. (2000)).
C) The fact that vitamin D is able to induce apoptosis in a number of different cancer cell types suggests that this is likely a major mechanism by which vitamin D exerts its anti-cancer effects. This theory has recently been supported by in vivo studies demonstrating large areas of apoptotic cells in mammary tumors from rats and mice, which have been treated with the vitamin D-analogue Seocalcitol (EB 1089) (James et al. (1998), VanWeelden et al. (1998)). Induction of apoptotic characteristics in response to vitamin D has been demonstrated in breast, colon, and prostate cancer cells as well as in melanoma, myeloma, and glioblastoma cells in vitro. However, the effect may vary between the different subtypes of these cells (reviewed by: Hansen et al. (2000), van den Bemd et al. (2000), Blutt et Weige (1999) and Feldman et al. (2000)).

In spite of the encouraging effects of 1α,25-(OH)2-D3 on cancer cells, clinical trials revealed that the therapeutic window of this substance is extremely narrow, i.e. effective doses cannot be administered without inducing hypercalcemia (reviewed by Hansen et al. (2000)). Therefore much effort has been directed to identify new analogs with potent cell regulatory effects but with decreased risk of calcemic adverse effects. Several hundred new vitamin D analogs, most of which show chemical modifications in the C17 side chain structure, have been synthesized with the aim of separating the growth regulating effects from the calcemic adverse effects. Some of these compounds have quite promising biological profiles (Hansen et al. (2001), Bouillon et al. (1995), Binderup et al. (1997)). So far, only few reports exist concerning analogs bearing a 3α-hydroxyl group in the A-ring.

In 1994, Reddy et al. reported that 1α,25-(OH)2-D3 is metabolized into 1α,25-(OH)2-3-epi-D3 in neonatal human keratinocyte cell culture. Meanwhile, epimerization of the hydroxyl group at C-3 of the A-ring is established as a new metabolic pathway of 1α,25-(OH)2-D3 in several vitamin D-target cells in vitro (Astecker et al. (2000), Masuda et al. (2000)) and in vivo (Sekimoto et al (1999)). Regarding calcemic activities, Norman et al. (1990) reported that 1α,25-(OH)2-3-epi-D3 was less potent in stimulating intestinal calcium absorption, calcium mobilization from bone, induction of calbindin D28K, and VDR binding affinity than 1α,25-(OH)2-D3. A most recent report from Nakagawa et. al. (2001) discusses the impact of the stereochemistry at the C-3 position on differentiation and apoptosis of HL-60 cells: Whereas a representative series of 3α-hydroxy analogues exerted far weaker stimulation of the VDR-mediated cell differentiation than their corresponding 3β-hydroxy analogues, 3α-hydroxy analogues were found to be more potent inducers of apoptosis. In summary, these findings suggest that 3α-hydroxy analogues might have an interesting potential in the treatment of cancer, since they could combine reduced VDR- mediated actions with substantial apoptotic capacity.

1.3.2. Anti-tumor Actions of Vitamin D on Glioma

Malignant gliomas account for approximately 2,5 % of deaths due to cancer. In spite of multimodality therapies, which include neurosurgical resection of the tumor and radiotherapy and/or chemotherapy, the median survival of patients with glioblastoma is less than 20 months following diagnosis (Ushio (1991), Weingart et Brem (1992)).

In recent years several investigations indicated that 1α,25-(OH)2-D3 or its analogues alone or in combination with other therapeutic approaches could be of interest in the treatment of brain glial tumors. In 1994, Naveilhan et al. provided the first evidence for a cytotoxic action of 1α,25-(OH)2-D3 on glioma cells from rat (C6-cells) and man (GHD-cells). Since this toxic effect of 1α,25- (OH)2-D3 was obtained in vitro in malignant glioma cells, but not in primary cultures of normal glial cells, they concluded that this specificity of action could offer a new strategy for the inhibition of glioma growth in vivo. One year later, the group investigated the effects of several vitamin D3-analogues, which had previously been shown to be less calcemic than 1α,25-(OH)2-D3, on C6.9 (rat glioma) cells (Baudet et al. (1996)). The study not only revealed that several of the tested substances were able to induce cell death of C6.9 cells, but also that the toxic effect was accompanied by several of the biochemical features of apoptosis, such as DNA fragmentation and induction of the c-myc protooncogene.

Recently, the usefulness of the vitamin D-analogue alfacalcidol was evaluated in a clinical trial, in the indication glioblastoma (Trouillas et al. (2001)). In combination with surgery or biopsy, radiotherapy and chemotherapy, the substance was safe to use and able to induce in some patients regression of the tumor.

1.3.3. C6-Rat Glioma Cells: Model to Study Vitamin D Actions

In this work, the rat glioma cell-line C6 - a validated model of glial tumors - was used. It was first cloned in 1968 from a glial tumor induced by N- nitrosomethylurea by Benda et al. in the species Rattus norvegicus after a series of alternate culture and animal passages (Fig. 6).

C6-cells are known to undergo a cell death program in response to 1α,25-(OH)2-D3 treatment (Naveilhan et al. (1994)). In search for therapeutic approaches for the treatment of brain glial tumors this cell-line has already been used by various groups as an in vitro experimental model to study hormoneinduced apoptosis (Canova et al. (1997), Canova et al. (1998)) or for screening of 1α,25-(OH)2-D3 analogues (Baudet et al. (1996)).

Investigations by Neveu et al. (1994a) have revealed that C6-cells express the vitamin D receptor (VDR) and that it is upregulated by 1α,25-(OH)2- D3 at the mRNA level.

In this work, C6-rat-glioma-cells generally were grown in (serum- containing) nutrient medium, described in section 2.1.1.

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Fig. 6. C6 glioma cells in culture.

1.4. Aim of diploma thesis

Hormonally active vitamin D is a potent anti-cancer agent, which induces apoptosis in a wide variety of tumor cell types. However, for severe calcemic side effects, clinical use of the compound is strongly limited. In spite of enormous effort, acceptable vitamins D-analogs with potent cell regulatory effects, but reduced calcemic adverse effects have not been identified so far. Recent in vitro studies in the HL-60 cell line, point to an improved profile of novel analogues, which bear the C-3-hydroxyl group in the α−position instead of the β-position found in 1α,25-(OH)2-D3 (Nakagawa et al. (2001)). If validated in relevant in vitro and in vivo models, such compounds could initiate beneficial new strategies in cancer treatment, in particular in tumors that resist currently available therapies, such as e.g. glioblastomas, which have a very poor prognosis.

Goal 1: The present study aims at evaluating the impact of the stereochemistry at the C-3 position on the capacity of vitamin D- metabolites and analogues to reduce growth and induce apoptosis in a malignant glioma cell line. The following pairs of vitamin D-metabolites and analogues differing in their stereochemistry at C-3 were selected (for chemical structures see 2. 1. 2.).

- 1α,25-(OH)2-D3 and 1α,25-(OH)2-3-epi-D3,
- 1α,25-(OH)2-16-ene-D3 and 1α,25-(OH)2-16-ene-3-epi-D3,
- 1α,25-(OH)2-16-ene-23-yne-D3 and 1α,25-(OH)2-16-ene-23-yne-3-epi-D3,
- 1α,25-(OH)2-16-ene-23-yne-26,27-F6-D3 and 1α,25-(OH)2-16-ene-23-yne- 26,27-F6-3-epi-D3
- Side chain modified forms Gemini and Gemini-3-epi

Goal 2: The study compares 1α,25-(OH)2-D3 with major natural vitamin D- metabolites for their capacity to induce apoptosis. Since glioma cells possess inducible 24-hydroxylase activity, we describe the apoptotic potential of 1α,24,25(OH)3D3 and 24,25(OH)2D3, the primary 24-hydroxylated products of hormone and its precursor 25(OH)D3.

Goal 3: Aiming at an improved method to monitor apoptosis, we tested whether capillary gel electrophoresis could be used to quantify internucleosomal DNA-fragmentation.

Test protocol: As a validated model to study effects of vitamin D on apoptosis, C6-rat glioma cell cultures were used throughout the study. We incubated these cells with the compounds at a range of concentrations, covering achievable levels on therapy (0.3 to 90 nM). During incubation periods up to one week, growth of the cells was checked by their capacity to incorporate Neutral Red. Induction of apoptosis was evaluated by staining the cell nuclei with the fluorescent dye Hoechst No. 33258 and counting condensed bodies. Furthermore, extracted DNA was subjected to capillary gel electrophoresis.

This study is to our knowledge the first investigation of the apoptotic effects of 3α-vitamin D-analogues in C6-rat glioma cells. Our data should indicate whether 3-epi-conformers of vitamin D metabolites/analogs might have a reasonable therapeutic potential of in the treatment of brain glial tumors.

2. MATERIALS AND METHODS

2.1. Used devices and reagents

2.1.1. Cell culture

Autoclave: KS113

Centrifuge: HERAEUS Instruments Biofuge Stratos

Cryo-medium: composed of 93 vol.% Nutrient Mixture F-12 (HAM) (GIBCOBRL®, Cat. No. 21765-029) and of 7 vol. % dimethyl sulfoxide (DMSO); used for storage of C6-cells at -58°C

Cryovial® - tubes: from Bibby Sterilin Ltd., 2CRIR, volume: 2 ml; used for storage of C6-cells at -58°C

Disinfectant: B. Braun Melsungen AG, Softa-Man®, Cat. No. 03865290 (Composition: 52,3% ethanol; 20,9% propan-1-ol)

Incubator: from EHRET (Labor- und Pharmatechnik), CO2 6000 NAPCO; temperature: 37 °C, atmosphere: 5 Vol.% CO2, saturated with water

Insulin-Transferrin-Sodium Selenite stock solution: Insulin-Transferrin- Sodium Selenite Media Supplement (Sigma-Aldrich Product No. I1884; contains 25 mg insulin, 25 mg transferrin and 25 µg sodium selenite) solved in 50 ml DPBS solution (Sigma-Aldrich Product No. D8662); aliquots of 5 ml were stored at -20 °C.

Laminar air flow workbench: EHRET "Biosafe 3"

Latex Examination Gloves: lightly powdered, Health Line®, Hand Safe®, REF GN01NC; Lot. 39170096

Micro Test Tubes (eppendorf® Safe-Lock 1,5 ml; Order-No. 0030 120.086) Microscope: inverted tissue culture microscope NIKON® TMS-F

Nutrient medium (for C6-cells), sterile, composition: 89 vol.% Nutrient Mixture F-12 (HAM) (GIBCOBRL®, Cat. No. 21765-029), 10 vol.% Newborn Calf Serum (GIBCOBRL®, Cat. No. 26010-033), 1 vol.% Penicillin-Streptomycin solution (GIBCOBRL®, Cat. No. 15140-122)

PBS - solution: Dulbecco's Phosphate Buffered Saline (Sigma-Aldrich Product No. D8662)

Pipette tips (sterile): AHN Biotechnologie GmbH; volume range 0,1 - 10 µl (Cat. No. 1-001-96-0), volume range 1 - 200 µl (Cat. No. 1-111-96-0), volume range 100 - 1000 µl (Cat. No. 1-201-96-0)

Pipettes (sterile): from Bibby Sterilin Ltd., Sterile Disposable Plastic Pipettes; with 2 ml volume (Code: 40102, Lot. No. 128412), with 10 ml volume (Code: 47110, Lot. No. 145410), with 25 ml volume (Code: 40125, Lot. No. 141422)

Pipettors: from Corning Inc., Corning® LamdaTM Single-Channel Pipettor; volume range 0,5 - 10 µl (Cat. No. 4960), volume range 10 - 100 µl (Cat. No. 4962), volume range 100 - 1000 µl (Cat. No. 4964)

Portable Mechanical Pipetter: Becton-Dickinson & Co., FALCON® ExpressTM Pipet-AidTM; Cat. No. 357591

Serum-free nutrient medium (for C6-cells), sterile, composition: 98 vol.% Nutrient Mixture F-12 (HAM) (GIBCOBRL®, Cat. No. 21765-029), 1 vol.% Insulin-Transferrin-Sodium Selenite stock solution (mentioned above), 1 vol.% Penicillin-Streptomycin solution (GIBCOBRL®, Cat. No. 15140-122)

Sterile-filter: Millipore Co., StericupTM, presterilized, 500 ml Vacuum Driven Disposable Filtration System (Filter: 0,45 µm; HA Mixed Esters of Cellulose Membrane), Cat. No. SCHAU05RE, Lot. No. H1EN96311

Test-tubes: Greiner bio-one, CELLSTAR®, PP-Test-tubes, sterile; volume 15 ml (Cat. No. 188271), volume 50 ml (Cat. No. 227270)

Tissue culture flasks: with 75 cm² growth area and 250 ml volume (from Falcon®, Cat. No. 353135), with 175 cm² growth area and 750 ml volume (from BD FalconTM, Cat. No. 353028)

Tissue culture well plates: from Greiner Labortechnik, CELLSTAR®; TC-Plate 6 Well (Cat. No. 657160), TC-Plate 24 Well (Cat. No. 662160), TC-Plate 96 Well (Cat. No. 655180)

Trypsin solution, composition: 10 vol.% Trypsin solution 2,5 % (GIBCOBRL®, Cat. No. 25090-028); 10 vol.% HBSS (10x) (GIBCOBRL®, Cat. No. 14180- 038) ; 1 vol.% HEPES buffer 1M (GIBCOBRL®, Cat. No. 15630-049) ; 0,5 vol.% NaHCO3 7,5 % solution (GIBCOBRL®, Cat. No. 25080-052); 0,5 vol.% Penicillin-Streptomycin solution (GIBCOBRL®, Cat. No. 15140-122); 78 vol.% sterile water

Trypsin-EDTA solution, composition: 10 vol.% Trypsin/EDTA solution (10x) (GIBCOBRL®, Cat. No. 35400-027); 10 vol.% HBSS (10x) (GIBCOBRL®, Cat. No. 14180-038) ; 1 vol.% HEPES buffer 1M (GIBCOBRL®, Cat. No. 15630- 049) ; 0,5 vol.% NaHCO3 7,5 % solution (GIBCOBRL®, Cat. No. 25080-052); 0,5 vol.% Penicillin-Streptomycin solution (GIBCOBRL®, Cat. No. 15140-122); 78 vol.% sterile water

2.1.2. Compounds used in Incubations

2.1.2.1. Etoposide:

solution of 1,2 mg/ml (2 mmol/l) in ethanol (96 vol.%); Etoposide: 4'-Desmethylepipodophyllotoxin 9-(4,6-O-ethylidene-beta-D- glucopyranoside); C29H32O13, molecular weight: 588.6, CAS No.: 33419- 42-0; melting point: 236-251°C, white powder; Sigma-Aldrich Product No. E-1383; Antitumor agent that complexes with topoisomerase II and DNA to enhance double-strand and single-strand cleavage of DNA and reversibly inhibit religation (Hande (1998), Burden et. al. (1996), Sehested and Jensen (1996)). Blocks the cell cycle in in S-phase and G2-phase of the cell cycle (Chow and Ross (1987), Inaba et al. (1994)); induces apoptosis in normal and tumor cell lines (Droin et al. (1998), Kaufmann (1998)).

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2.1.2.2. Vitamin D-analogues or -metabolites : (were kindly provided by S. Reddy (Brown University, Rhode Island, USA))

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2.1.3. Materials used in Analytical procedures

Geltol; preparation of 100 ml: 6 g Glycerol (Merck Cat. No. 4095) and 2.4 g Mowiol® (Calbiochem No. 475904) are mixed with 6 ml bidistilled water and allowed to swell for 2 hours at room temperature. Thereafter, 12 ml 0.2 M Tris- buffer (Tris(hydroxymethyl)-aminomethan; Merck Cat. No. 8382) is added, pH is adjusted to 8.5 with HCl and the solution is diluted with bidistilled water to 100 ml. Then, it is stirred at 50 °C in a waterbath for 10 minutes and centrifuged at 5000 g for 15 minutes. Storage: at -20 °C tenable approximately for 1 year; at 4 °C tenable for 1-3 months.

Hoechst No. 33258 - solution: c = 0.8 µg/ml in PBS-solution (section 2. 1. 1.); Hoechst No. 33258 (bis-Benzimide Trihydrochloride): C25H24N6 . 3HCl; molecular weight 533.9; soluble in water (10 mg/ml); solid at room temperature; CAS No. 23491-45-4; formula of base:

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Membrane-permeable, fluorescent DNA stain with low cytotoxicity that intercalate in A-T regions of DNA. Useful for staining DNA, chromosomes and nuclei. May be used for fluorecence microscopy or flow cytometry. Excitation max. = 346 nm; Emission max. = 460 nm

Lysing solution; composition: mixture of 70 vol.% ethanol (in deionized water) and acetic acid 99+1

Neutral Red (3-Amino-7-dimethylamino-2-methylphenazine hydrochloride): molecular formula: C15H16N4 . HCl, molecular weight: 288.8; solution in water (10mg/ml), pH range 6.8 (red) - 8.0 (yellow); CAS No. 553-24-2; formula:

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