Influence of different noxa on DNA repair capacity of primary human skin cells

(1)

Influence of different noxa on DNA repair capacity of primary human skin cells

Dissertation

zur Erlangung des akademischen Grades des Doktors der Naturwissenschaften (Dr. rer. nat.) an der Universität Konstanz (Fachbereich Biologie)

vorgelegt von

Katharina Burger

Tag der mündlichen Prüfung: 14.07.2011

Referent: Prof. Dr. A. Bürkle Referent: Prof. Dr. J. Bergemann

Konstanzer Online-Publikations-System (KOPS) URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-146886

(2)

It is not the mountain we conquer but ourselves - Nicht der Berg ist es, den man bezwingt, sondern das eigene Ich.

Edmund Hillary

Meinem Vater

der leider nur ein viel zu kurzes Stück meines Lebenswegs begleitet hat

Meiner Mutter

die ich für ihren Mut und ihre Ausdauer zutiefst bewundere

(3)

Page | I It is a pleasure to thank those who made this thesis possible!

I would like to express my deep and sincere gratitude to my supervisor, Prof. Dr. Jörg Bergemann, Head of the department of Biomedical Engineering, University of applied

sciences Sigmaringen. Without his encouragement, patience, guidance and support from the initial to the final level I wouldn’t have been able to manage this.

I’ am deeply grateful to my supervisor, Prof. Dr. Albrecht Wendel, initial speaker of the research training group 1331 at the University of Konstanz, for the possibility and his encouragement to do this Ph. D. thesis and to Prof. Dr. Alexander Bürkle who adopted the supervision during the final stage of my work.

I am grateful for the excellent working facilities and support provided at the university of applied sciences, Sigmaringen. In this context, I owe my most sincere gratitude to Prof. Dr.

Günther Rexer, Rector, and Prof. Dr. Volker Riethmüller, Head of the faculty of Life Sciences.

My sincere thanks are due to my thesis committee and the members of the RTG 1331 for hard discussions, constructive critics and the possibility to look outside of the box.

The courses I attended during my membership enabled me to acquire consolidated knowledge beyond the topic of my Ph. D. study.

I warmly thank my colleagues from the Department of Biomedical Engineering: Sylvie, Betty, Maya, Sven and especially the Ph. D. students Katja und Daniel.

(4)

Acknowledgement

Page | II I also wish to thank Prof. Dr. Katja Wegner for her guidance in statistical analysis and for her essential assistance in preparing my manuscripts.

Last but not least, I owe my deepest gratitude to my mother, Sarah and Kai and especially Heiko. Without their encouragement and sympathy it would have been impossible for me to finish this work!

(5)

Page | III 6,4-PP pyrimidin-(6,4)-photoproduct

CAT chlormaphenicol acetyl transferase

CPD cyclobutan pyrimidin dimer

CsA cyclosporin A

DNA desoxyribonucleic acid

DRC DNA repair capacity

FACS fluorescence activated cell sorting

GGR global genome repair

GFP green fluorescent protein

HCRA host cell reactivation assay

HDAC histon-deacetylase

NER nucleotide excision repair

OECD organization for economic co-operation and development

RNA ribonucleic acid

ROS reactive oxygen species

SCCNFP scientific committee on cosmetic products and non-food products TCR transcription coupled repair

UVA ultra violet A (320 nm-380 nm)[1]

UVB ultra violet B (280 nm-320 nm)[1]

UVC ultra violet C (100 nm-280 nm)[1]

XP xeroderma pigmentosum

Abbreviations used in the result part were listed in the respective subchapters

(6)

Table of content

Page | IV

Table of content

1 Introduction ... 1

1.1 DNA damage and repair ... 1

1.2 Induction and repair of UV induced DNA damage in human skin ... 3

1.2.1 Interactions of UV radiation and human skin ... 3

1.2.2 UV induced DNA damages ... 5

1.2.3 Nucleotide excision repair ... 6

1.3 Measuring DNA Repair Capacity ... 8

1.3.1 Methods in general ... 8

1.3.2 Host cell reactivation assay ... 10

1.3.3 Host cell reactivation assay using fluorescent proteins as reporter ... 10

1.4 Agents influencing the repair capacity of human cells ... 12

1.4.1 Folic Acid ... 12

1.4.3 UV Radiation ... 15

1.4.4 Other Substances ... 16

1.5 Aims of the study ... 17

2 Publication A A modified fluorimetric host cell reactivation assay to determine the repair capacity of primary keratinocytes, melanocytes and fibroblasts ... 18

2.1 Abstract ... 19

2.2 Background ... 69

2.3 Results and Discussion ... 20

2.4 Conclusions ... 24

2.5 Methods... 69

2.6 Authors Contribution ... 69

2.7 References ... 69

3 Publication B The influence of folic acid depletion on the nucleotide excision repair capacity of human dermal fibroblasts measured by a modified host cell reactivation assayA modified flourimetric host cell reactivation assay ... 30

3.1 Abstract ... 31

3.2 Introduction ... 31

3.3 Materials and Methods ... 32

3.4 Results ... 35

3.5 Discussion ... 69

(7)

Page | V 4 Publication C

Low doses of UVB enhance reactivation of UV damaged reporter gene in dermal

fibroblasts ... 41

4.1 Abstract ... 42

4.2 Background ... 43

4.3 Results and Discussion ... 45

4.4 Conclusions ... 53

4.5 Materials and Methods ... 54

4.6 Authors Contribution ... 60

4.7 References ... 62

5 Summarizing discussion ... 68

5.1 Measuring DNA repair capacity using host cell reactivation assay ... 68

5.2 Application of primary skin cells for host cell reactivation assay ... 69

5.3 Development of dermatologic ingredients using host cell reactivation assay ... 70

5.4 Detection of potentially carcinogenic properties using host cell reactivation assay . 71 5.5 Detection of interindividual differences using host cell reactivation assay ... 72

5.6 Population based studies on DRC and detection of influences caused by UV radiation using Host Cell Reactivation assay ... 73

6 Summary ... 74

7 Zusammenfassung ... 75

8 Declaration of authors contributions ... 76

9 References ... 77

(8)

Introduction

Page | 1

1 Introduction

1.1 DNA damage and repair

The human genome provides the basis for reproduction and proper development as well as for functioning of the whole organism. Nevertheless, DNA is constantly under attack from environmental agents, either physical agents such as UV light and ionizing radiation, or chemical pollutants found in food and air that can result in crosslinks, bulky adducts, base alkylations, and other DNA alterations [2]. Structure and integrity of DNA molecules can also be affected by spontaneously arising alterations through intrinsic instability of chemical bonds in DNA (e. g. deamination, depurination, etc)[3]. It is estimated, that a human cell faces over 10.000 spontaneous depurination events every day, together with hundreds of deaminations and depyrimidinations [4].

Indeed, the load of base damage from natural occurring and environmentally related sources would be incompatible with life unless cells were endowed with specific mechanisms for repairing DNA damage and for maintaining mutations at reasonable levels[5]. Therefore, a variety of specialized cellular responses have evolved. Depending on the appearance of the base damage, different pathways are activated that enable cells either to eliminate the damage and restore lost information or to activate programmed cell death process [6].

Figure 1: Consequences of an imbalance between DNA damage and DNA repair

Healthy cells show equal rates of DNA damage and DNA repair. When DNA damage is beyond repair, cells acquire mutations leading to cancer apoptosis and senescence. Image adapted from [7]

(9)

Page | 2

Figure 2: DNA repair mechanisms[2]

Base excision repair (figure A) repairs damage to a single base caused by oxidation, alkylation, hydrolysis, or

deamination

Nucleotide excision repair (figure B) recognizes bulky helix distorting lesion such as cyclobutan pyrimidine dimers (CPD) and pyrimidin-(6,4)-

photoproducts as well as multitude of DNA damages induced by environmental sources by a copy-and-paste mechanism.

Single strand break repair (figure C) directly removes nicks induced spontenously by endogenous and exogenous influences.

Direct damage reversal (figure D) removes damages directly without base excision

Double-strand break repair (figure E) which both strands in the double helix are severed, are particularly

hazardous to the cell because they can lead to genome rearrangements.

Double strand breaks are repaired either by nonhomologous endjoining or by homologous recombination.

Mismatch repair (figure F) corrects errors of and that result in mispaired (but undamaged) nucleotides.

(10)

Introduction

Page | 3 The four types of pathways elicited by DNA damage which are known or presumed to

ameliorate harmful damage effects are [6]:

 removal of DNA damage and restoration of the continuity of the DNA duplex;

 activation of a DNA damage checkpoint, which arrests cell cycle progression so as to allow for repair and prevention of the transmission of damaged or incompletely replicated chromosomes;

 transcriptional response, which causes changes in the transcription profile that may be beneficial to the cell;

and

 apoptosis, which eliminates heavily damaged or seriously deregulated cells.

The term DNA repair refers to the removal of DNA damage and the restoration of the continuity of DNA duplex. DNA repair processes could be subdivided in the pathways direct repair, base excision repair, nucleotide excision repair, double-strand break repair, and repair of interstrand cross-links [2] as shown in figure 2.

1.2 Induction and repair of UV induced DNA damage in human skin

1.2.1 Interactions of UV radiation and human skin

On the one hand, without sunlight containing ultraviolet radiation, life wouldn’t be possible on earth-on the other hand UV radiation can result in serious health problems depending on wavelength and residence time.

Only parts of the solar spectrum can be detected by the human eye. The UV radiation at the short-wave end cannot be detected, though it is visible to some insects [8]. This part of the

Figure 3: Interactions of UV radiation and ozone layer [1]:

Parts of solar radiation are absorbed by UV radiation and therefore cannot reach the earth’s surface.

(11)

Page | 4 specific interactions with the ozone layer as described by Jendritzky [1] and shown in figure 3. UV radiation is estimated to be one of the most effective and carcinogenic exogenous agents interacting with DNA and threatening the integrity of the genome which can result in alterations of life processes (reviewed in [9])

The effects on human skin and the penetration depth differ from spectral range to spectral range [11]:

 Given that UVC doesn’t reach the earth surface, only the interactions of UVB and UVA are of interest, both absorbed in different skin layers

 UVB is almost completely absorbed by the epidermis (only 10-20 % of UVB energy reaches the epidermal stratum basale and the dermal stratum papillare).

 UVA penetrates deeper into the dermis and deposits 30-50% of its energy in the dermal stratum papillare.

While the effects of UVB on the skin are mediated predominantly by direct DNA damage, the effects of UVA (320–400 nm) are dominated by indirect damage caused by reactive oxygen species (ROS) such as singlet oxygen [12].

Figure 4: Cross-section of human skin, adapted from [10]

(12)

Introduction

Page | 5 1.2.2 UV induced DNA damages

The possibility that sunlight and artificial sources of UV radiation in general might be harmful to animals did not arise in force until the 20^th century [13] and nothing was known about the influences of different wavelengths on biological materials. In 1936, Funding et al. described for the first time that 290-320 nm (UVB) was the region of sunlight most responsible for inducing tumours in experimental animals (cited in[13]). In 1963, Deering and Setlow discovered the molecular mechanisms of UV induced dimers between adjacent tymines and described the wavelength dependent formation, breakage and steady state fraction [14]. One decade later Setlow proposed that changes in DNA, such as formation of pyrimidine dimers and other photoproducts could be responsible for the biological effects of UV, although the detailed molecular mechanism by which the biological effects of UV are produced weren’t already known[15].

Nowadays it is generally accepted, that UV light in the UVB range causes predominantly two types of DNA photoproducts, the Cyclobutan pyrimidin dimer (CPD) and the 6,4-photoproduct ( 6,4-PP) [16]. A study by Yang et al however demonstrated that CPDs can also be directly induced by UVA irradiation[17].

Whereas a CPD is formed if the absorption of a photon by DNA opens two adjacent 5-6 double bond of pyrimidines and these open structures form a stable ring structure, a 6,4-PP results, when a 5-6 double bond in a pyrimidin opens and reacts with the exocyclic moiety of the adjacent 3’ pyrimidine to form the covalent 6-4 linkage[18].

The development of the first monoclonal antibody against CPDs by Strickland and Boyle 1981 [19] and against 6,4-PPs by Mori [20] et al. in 1988 set new standards in the field of photochemical and photobiological studies and facilitated the first explant culture elimination studies[21].

After UV irradiation, the CPDs are the most abundant and probably most cyctotoxic lesions but the 6,4- PPs may have more serious, potentially lethal, mutagenic effects [22]. In

consequence of UV radiation 75% of the damages are CPDs, only 25% of the damages are 6,4-PPs [22].

Figure 5: Cyclobutan pyrimin dimer and (6,4)- photoproduct

CPDs and (6,4)-PPs arise in consequence of UVB irradiation and are removed by nucleotide excision repair

(13)

Page | 6 they interfere with transcription and replication and may result in mutation and cell death. As already described in chapter 1.1, an intricate network of repair pathways has evolved to protect the integrity and functionality of the genome. NER is a highly versatile and

sophisticated DNA damage removal pathway that counteracts beside CPDs and 6,4-PPs the deleterious effects a multitude of DNA damages induced by environmental sources [12,23].

The degree of helix distortion caused by different DNA damaging agents seems to have an important impact on the induction of NER [24,25].It is possible that the ability to counteract such big variety of damages has evolved solely to repair sporadic lesions caused by the influence of environmental mutagenic substances [26].

Setlow and Hanawalt firstly described in 1960 a recovery mechanism eventually able to restore the DNA synthesis rate following UV radiation nearly to that of uniradiated control [27]

providing a first evidence for the existence of a repair mechanism yet before thymidine dimers were discovered. 1963, the first model of NER was introduced (for review see [28]).

Later on it was improved that NER employs essentially the same steps that were

hypothesized in the original model: recognition of a lesion in DNA, introduction of incisions in the damaged strands, one on each side of the lesion; removal of the oligonucleotide

containing the lesion; resynthesis of the deleted nucleotide sequence using the

complementary DNA strand as template; and finally, ligation of the newly synthesized repair patch to the pre-existing strand[29]. Nowadays, the complete NER pathway is well

understood and was reconstructed in vitro in prokaryotes [30] as well as in eukaryotes (reviewed in[31]). In spite of everything there are currently unresolved issues concerning e.g. the regulation of NER (for review see [32]). All together NER uses the products of around 30 genes to remove the damage containing oligonucleotide and restore the original state [32].

As figure 6 shows, NER consist of the two subpathways global genome repair (GGR), removing damages in the genome overall and transcription-coupled repair (TCR) which specifically repairs the transcribed strand of active genes. This sub-division of NER was revealed by the analysis of the repair of UV –induced CPDs in transcriptional active versus inactive regions of the genome, and within transcribed and non –transcribed strands of expressed genes (for review see [29]).

DNA damages like CPDs and 6,4- PPs alter the base-pairing ability of the involved bases and can be cytotoxic due to the blockage of transcription and replication[18].

(14)

Introduction

Page | 7 Replication:

The importance of the functionality of NER in genome maintenance is shown clearly by the example of the rare hereditary disease Xeroderma Pigmentosum (XP) which was linked by Cleaver in 1969 with the dysfunction of DNA repair [33]. XP is characterized by extreme sun sensitivity and an extremely increased risk for the development of skin cancer.

Transcription:

DNA damages located in transcribed regions may affect transcription in a number of ways.

Transcriptional mutagenesis or accumulation of p53 and subsequent induction of apoptosis in case of RNA polymerase-arrest are just two examples out of many others (for review see [35]). TCR is characterized by more rapid removal of certain modified bases from the transcribed strand of actively expressed genes when compared to silent DNA [36]. It has been proposed that TCR might exist to ensure that transcription can readily continue following a genotoxic assault, thus providing a means for producing transcripts essential for continued cell survival [37].

Figure 6: Two subpathways of mammalian NER [34]

(A) Damage/distortion recognition in GGR and TCR: While GGR is initialized by the recognition and binding of XPC-RAD23B and UV-DBB, TCR is triggered by DNA damage mediated blockage of RNAPIIo. In the next steps the two sub-pathways converge.

(B) Gap filling and ligation After dual incision around the lesion, the single strand gap is filled by polymerase δ;

PCNA and RFC. And finally sealed by DNA ligase III-

(15)

Page | 8 be repaired by NER. However the reasons therefore could not be conclusively ascertained until now.

In this case, the only recourse to survive is a lesion bypass[38]. Most mutagenesis resulting from damage by UV radiation, ionizing radiation or various chemicals seems to be due to a process of translesion synthesis, in which a polymerase or replicative assembly encounters a noncoding or miscoding lesion, inserts an incorrect nucleotide opposite the lesion and then continues elongation [39].

Corresponding to the multistep hypothesis of cancer incidence introduced by Hanahan and Weinberg [40], accumulation of mutation provides the basis for the development of (skin) malignancies including cancer.

1.3 Measuring DNA Repair Capacity

1.3.1 Methods in general

According to the guidance’s of the Scientific Committee on Cosmetic Products and Non-Food Products (SCCNFP) to realize the requirements of the cosmetic directive 2003/15/EC, the toxicological areas shown in figure 7 may need to be addressed for the safety of cosmetic products. Influences on the repair capacity are not needed to be addressed; although there is an increasing body of evidence for the existence of substances without direct genotoxic impact on cells but with inhibitory effects on the DRC [41,42] . Nevertheless, the OECD recommends the usage of a method of proof for DRC in context of genetic toxicology [43].

Repair processes play a prominent role in context of carcinogenesis and are therefore subject of intensive research in the area of cancer therapy and prevention. Special attend is paid to apparently existing donor variations: Given a common carcinogenic insult, some individuals develop neoplasm while others remain clinically free of effects- even

Figure 7: Toxological areas that may need to be addressed for assessing the safety of cosmetic products

(16)

Introduction

Page | 9

Figure 8: Principle of UDS and CFS.

Labelled nucleotides were provided and build in during repair synthesis

within specific cancer populations, age of onset, extend and severity vary between patients [44]. These observations refer strongly to an involvement of repair processes.The methods to determine DNA repair capacity (DRC) can be sub-divided in to detection methods for specific damages and methods to detect the activity of a specific repair pathway. However methods appreciating the DRC via the detection of DNA damages aren’t methods of proof for the activity of DRC in the classical meaning. Specific damages like thymine dimers can be

detected via T4- Endonuclease- digestion for example. One further possibility is the detection of CPDs or 6-4 PPs via incubation with specific antibodies. Breaks and break inducing lesions can be detected by comet assay (single cell gel electrophoresis). The comet assay is a simple method to detect strand breaks in eukaryotic cells based on the detection of DNA fragments forming cometlike structures after cell lyses. The intensity of the comet tail relative to the head reflects the number of DNA breaks [45]. The interpretation of these data is challenging and error prone, even if any analysis programs were used. To detect damages removed by NER, damages have to be converted enzymatically into strand breaks (modified single cell gel electrophoresis). Another possibility to detect specific damages is PCR screening of defined regions for crosslinks and adducts. Also micronucleus test which is often used to determine genotoxicity, isn’t a DRC assay in classical meaning.

Methods to detect the activity of specific repair pathways are more sophisticated and experimentally challenging.

The cell free system (CFS) was introduced by Wood and his colleagues in the late 1980s [46]. Using this method the activity of repair synthesis could be determined. In brief, the cell population to be tested is converted into a cell free but repair competent extract which is introduced to in vitro damaged DNA. This test batch is supplemented with radiolabelled nucleotides build into the DNA strand (e. g. plasmids) during repair synthesis. The amount of integrated radiolabelled nucleotides is directly proportional to DRC.

The unscheduled DNA synthesis assay (UDS) was first described by Rasmussen and Painter [47]. This method works on the same principle such as the CFS: the activity of a repair pathway is quantified via incorporation of labelled nucleotides. In contrast to CFS, the cell population to be tested is damaged with UV light. The cells are allowed to repair

(17)

Page | 10 Both CFS and UDS detect only excision and repair synthesis but not ligation and functional restoration. Furthermore, the handling of radioactive substances demands a great deal of laboratory equipment and the interpretation via autoradiography is very demanding.

Additionally, the entry of the cells into the S-phase has to be blocked prior to the experiment.

UDS is described in the OECD testguideline 482 in context of the determination of genetic toxicology.

1.3.2 Host cell reactivation assay

The Host Cell Reactivation Assay (HCRA) is a further technique to measure the repair capacity of a cell population. Cell populations are transfected with a reporter construct which has been deactivated in vitro due to the influence of DNA damaging substances. The ability of the cell population to repair the damage in the plasmid allows the reporter gene to be reactivated. Due to the reactivation, the cell starts to produce its reporting product, which can be detected in different ways. The amount of reporting product is directly proportional to the DNA repair capacity. The plasmid reactivation assay indirectly monitors cellular repair of transcriptional activity by measuring transient enzyme activity associated with the transfected marker gene. The measurement of the ability to completely restore a (reporter-) gene was first described by Protic-Sablic in 1985 [48]. This rather complicated assay was modified by Athas et al. to measure the inter-individual variation in DRC in a large number of subjects [44]. For the first time DRC could be used as a kind of biomarker to monitor increased risk for the development of long-term health consequences.

1.3.3 Host cell reactivation assay using fluorescent proteins as reporter

In 2000, Roguev and Russev presented a modified HCRA using green fluorescent protein (GFP) and Yellow Fluorescent Protein as reporter genes, where luminescence was the marker for the restoration of the plasmid [49]. Using two instead of one reporter gene provides the advantage of an internal control. Therefore, the assay is independent from transfection efficiencies. Using luminometer-technology to determine the amount of reporting product as described by Roguev and Russev implicates the disadvantage of not being suitable for primary cells that don’t transfect well. To reach higher sensitivities, population based interpretation has to be replaced by single cell based analysis

(18)

Introduction

Page | 11

Figure 9: Host Cell Reactivation Assay

(A) HCRA using one single reporter: The cell population is transfected with a damaged reporter. Cells able to restore it will express the reporter protein. The result depends on the efficacy of transfection.

(B) Dual reporter HCRA: The cell population is transfected with a mixture of a damaged and an undamaged reporter. Repaircompetent cells express the reporterprotein and the controlprotein while repairdeficient cells only express the controlprotein. The result is independend of the efficacy of transfection.

methods. In principle, fluorescence microscopy as described by Atanassov [50] and FACS technology are in consideration, whereupon FACS technology was not used in context of host cell reactivation, so far.

As already described in chapter 1.3.1, DRC plays a prominent role in context of

carcinogenesis and is therefore subject of intensive research in the area of cancer therapy and prevention. Especially skin cancer is at the focus: Since the UV component of solar radiation exists as the predominant environmental risk factor for skin cancer, a causal association between UV exposure, defective repair of UV-induced DNA photoproducts and skin cancer is inferred [44].

In default of suitable transfection procedures for primary skin cells, most of the studies concerning the association between skin cancer susceptibility and DRC were done with primary lymphocytes [44], cell lines [50] or in exceptional cases with fibroblasts [51].

However, it is known that DRC varies considerably between different cell types and tissues.

[52]. Assays resorting to primary dermal and epidermal cells therefore provide a real

alternative to conventional systems in context of skin and could enhance the significance of DRC assays especially with regard to dermal carcinogenesis.

(19)

Page | 12 Influencing DRC is interesting from various points of aspect.

Firstly, DRC is a promising target in the context of cancer prevention. In conjunction with cosmetic products stimulation of DRC is -beside the prevention of skin cancer- interesting in the context of photoageing [53].

With regard to the development of antitumoral drugs, DRC has to be taken into

consideration: Whereas DNA repair is essential for a healthy cell on the one hand, DNA repair enzymes affect the performance of antitumoral drugs targeting the integrity of DNA and therefore play a prominent role in connection with drug design [26]. Thus it has been shown that mismatch repair deficient colon cancer cells are more sensitive in vitro to camptothecin that inhibits topoisomerase 2 [54]. Furthermore, Munshi and college have shown in 2005 that HDAC-inhibitors radiosensitize human melanoma cells by suppressing DRC [55]. Additionally, correlations between lifestyle, DRC and carcinogenesis are

interesting from an epidemiologic point of view. DRC has been reported to be a promising predictive biomarker in context of anticancer therapies and cancer risk [56].

1.4.1 Folic Acid

Epidemiologic studies have observed that diminished folate status is associated with cancer of the cervix, colorectum, lung, esophagus, brain, pancreas and breast [57,58]. Candidate mechanisms for folate associated carcinogenesis are altered DNA methylation, altered RNA methylation, disruption of DNA integrity and disruption of DNA repair (for review see[58]). A study of Wei and colleagues involving 559 individuals provided a distinct clue to the validity of the hypothesis of an association between low dietary folate intake and suboptimal DRC [59]. It has been known for a long time, that folate deficiency causes uracil misincorporation into human DNA and chromosomal breakage [58].Folic acid exists in a number of different forms, occurring naturally as food folates concentrated in selected food such as orange juice, dark green leafy vegetables, dried beans and peas, asparagus, strawberries and peanuts

Figure 10: Principle components of the folate biochemical cycle [56]

Reactions: ❶ Biosynthesis of nucleotides for

incurporation into DNA and RNA; ❷ Remethylation of homocysteineto form methionine (vitamin B12 serves as a coenzyme in this reaction);❸ Methylation of

substrates, including DNA, RNA, phospholipids, and proteins; ❹ MTHFR, which catalyzes the formation of 5- methyltetrahydrofolate needed for methylation

reactions; ❺ Dihydrofolate reductase enzyme.

Abbreviations:

DHFR - dihydrofolate reductase; MTHFR - methylenetetrahydrofolate reductase

(20)

Introduction

Page | 13 [60].

Folate coenzymes participate in biochemical processes involvingsingle carbon group transfers, including the metabolism of aminoacids and synthesis of (for review see [61]).

Folates are integrallyinvolved in the remethylation of homocysteine to form methionine, essential for the synthesis of proteins and polyamines, which is subsequently converted to S- adenosylmethionine, serving as donor of metylgroups,in aconsiderable number of chemical reactions (for review see [61]). The link between DRC and folate uptake is promising in context of cosmetical ingredients, also DNA damage to both genomic and mitochondrial DNA and subsequent DNA repair is known to contribute greatly to age associated skin changes and carcinogenesis [62]. With respect to normal skin, however, there is only very little known regarding the role of folates, although there are several studies on folate status and skin disease [63].

Furthermore, photodegradation of folate and some other carotinoids has been well documented [64]. A study by Branda et al. indicated that fair-skinned patients undergoing photochemotherapy for dermatological conditions have low serum folate concentrations, suggesting that photolysis may occur also in vivo [65]. In their study published in 2007 Knott et al. could show that dermal applicated folic acid penetrates into human skin and is uptaken by the cells [63], providing a novel treatment option for photoaged skin. In this regard, Knott et al. could also show that cultured full thickness epidermal skin models supplemented with folic acid and creatine showed accelerated skin regeneration compared to untreated controls [66].

1.4.2 Cyclosporin A

Cyclosporin A (CsA) is a product of Novartis Pharma and is in Germany commercially

available under the registered trademarks Sandimmun and Sandimmun Optoral as well as in form of generica. Licensed indications for CsA are transplantations of kidneys, liver,

pancreas, heart, heart and lung and bone marrow as well as prophylaxis and therapy of graft versus host disease [67]. Further indications are severe psoriasis and the treatment of atopic dermatitis. The discovery of CsA in the 1970s revolutionized the field of transplant medicine and the area of modern immunosuppressive therapy was heralded by the clinical

development and use of the first Calcineurin-Inhibitor CsA. Till this day CsA is often described as key drug leveraging transplant medicine into an established method of treatment.

(21)

Page | 14 trials were done using CsA in all forms of clinical

transplantations. Almost all of the studies arrived at the conclusion that CsA leads to a considerably enhanced 1-year graft survival compared to standard procedures [67].

But one of the prices to pay for the prevention of graft rejection is appearance of de novo cancer [68]. It was reported, that

immunsppressed organ allograft recipients have a 3- to 4- fold increased risk of developing tumors, but the risk of developing certain cancers is increased several hundredfold [69]. With the exception of skin and lip cancers, most of the common malignancies seen in the general population are not increased in incidence [69]. Up to 50% of the patients that receive an immunosuppressive treatment will develop malignancies [70].

A study of Jensen and colleagues published in 1999 dealt with the question whether renal transplant recipients on CsA based immunosuppressive regiments are more likely to develop skin cancer than those on other immunosuppressive regiments. Investigating a cohort of 2561 cases of kidney and heart transplantations they found that transplant recipients had an increased risk for squamous cell carcinoma (65-fold), malignant melanoma (3-fold) and Kaposi’s Sarkoma (84-fold) compared with general population. After adjustment for age, kidney transplant recipient receiving a combination of CsA, Azathioprine and Prednisolone had a significantly (2.8 times) higher risk of squamous cell carcinoma relative to those

receiving an adjunction without CsA [71]. Hollenback et al. showed an increased incidence of melanoma in transplant recipients (3.6 fold) too [72].

The skin cancers arise in CsA treated individuals on sun exposed skin and have high frequency of signature UV mutations in p53 [73]. In 2004 André, Roquelaure and Conrath reviewed the different molecular and cellular effects of CsA and hypothesized that cancer arises from a complex interplay of immunosuppression, synthesis of TGF beta, influences on proapoptotic or inhibitory effects on apoptosis and inhibition of DRC [68]. Especially the association between inhibition of DRC and calicneurin inhibitor was clearly shown [74–76].

Figure 11: Therapeutic mechanism of Cyclosporin [67]

After entering the cytosolic compartment, Cyclosporin A binds to Cyclophilin. The Cyclosporin/Cyclophilin inhibits Calicneurinphosphatase and interrupts the signaling cascade leading to the activation of NFAT.

Additionally, members of the map-kinase famlily are inhibited interrupting the signaling cascade leading to the activation of transcription factor AP-1.

Activation of T-Cells didn’t take place, the cell stay in dormancy.

T cell

Nucleus

(22)

Introduction

Page | 15 Ahlers et al. suggested the inhibition of Polymerase beta to be responsible for the inhibition of DRC [77]. Sugie et al. provided a clue that the p53 dependend repair synthesis and apoptosis are impaired by CsA [78,79]. Using luciferase based HCRA, Thoms as well

Kuschal demonstrated negative effects of CsA onto the DRC of fibroblasts and lymphoblasts.

1.4.3 UV Radiation

UVR is the main carcinogen in conjunction with the development of non melanoma skin cancer [80–84]. Furthermore, chronic photodamage of the skin manifests itself as extrinsic skin ageing [85,86]. The UVB range has almost been at the center of attention because of its well established role in carcinogenesis [87]. Therefore protection against UVR is of particular importance. Current methods of exogenous photoprotection are sun avoidance, protective clothing, sunscreens and chemopreventive agents [88]. The main endogenous mechanisms to protect against, reduce and/or repair UV damages are increasing epidermal thickness, antioxidant enzymes and, last but not least, DNA repair mechanisms and apoptosis [89]. A possible chemopreventive agent to influence DRC is folic acid, as already described in chapter 1.4.1. Further possible chemopreventives will be described in chapter 1.4.4.

Very little is known about molecular photoadaptive mechanisms-especially in context with nucleotide excision repair. However, induced radioresitance is well documented in

conjunction with ionizing radiation (for review see [90]). The term adaptive response usually means, that a relatively small “conditioning” radiation dose induces increased radioresistance when the cells are irradiated with higher doses several hours’ later [91]. However there are a few reports suggesting that excision repair in human cells is enhanced by low dose UV irradiation.

Francis, Bennett and Jeeves described a UV enhanced reactivation of a UV-damages reporter gene using HCRA [51,92–95]. Francis also observed that UVR-cell survival is enhanced following very low doses of UVR [51]. Something similar was reported by Liu et al.

2004: The pretreatment of Chinese hamster ovary cells enhanced host cell reactivation of a UV damaged reporter gene [96]. Germanier et al. reported in 2000, that prior low dose irradiation clearly enhanced the rate of CDP removal in transcribed strand [97]. In contrast to Francis and Rainbow, they used a technique allowing quantification of CPDs at the level of a specific strand.

In 2005, Decrane et al. reported an adaptive response of dermal keratinocytes via p53- dependend upregulation of the cell-cycle-arrested associated gene p21/WAF1 and the repair –associated-gen p21 [98].

Using skin equivalents Chouinard et al. demonstrated in 2001 that repeated exposure to low doses of UVB lead to the accumulation of CPDs in the epidermis [99], arguing against a photoadaptive response.

(23)

Page | 16 the damaging agent [51]. HCRA is the method of choice to determine possible influences on DRC in context of UVR.

1.4.4 Other Substances

Beside the already mentioned substances some other compounds are up for discussion to influence DRC.

Metal ions provide the best investigated group in this context. In a review published by Hartwig [100], compounds of chrome (VI), nickel, cadmium, cobalt and arsenic are described to be essential for genome integrity but also not to be genotoxic in animal experiment and/or for humans. The underlying mechanisms are described to be still unclear, especially since the mutagen potency of these substances except for chromates is rather low. However, considerably low doses of metal ions being not cytotoxic in other respects influence different repair systems [100].

Selene, a micronutrient and an environmental, a chemical and an industrial agent in any products, can have genotoxic effects as well as antimutagenic and/or anticarcinogenic properties; depending on its concentration and oxidation state [42]. Inhibitory effects on DRC are also reported to be at least in parts responsible for the enhanced cytotoxicity [42].

The administration of chemotherapeutic is linked with influences of DRC, too:

The TNFalpha neutralizing antibody infliximab directly affects the cell cycle and repair in premalignant human keratinocytes after UVB irradiation [101]. In contrast, p33ING2, a novel candidate tumor suppressor which has been shown to be involved in the regulation of gene transcription, cell cycle arrest and apoptosis enhances NER [102].

Furthermore, hypoxia and low pH are described to diminish DRC and elevate mutagenesis in mammalian cells [103] whereas tobacco smoke is associated with enhanced DRC [104].

Forskolin is known to increase DRC of human keratinocytes [105].

In context of cosmetic ingredients repair enzymes and oligonucleotides are under discussion.

Topical application of lotions containing the bacterial repair enzyme T4 endonuclease has been reported to reduce ultraviolet induced skin cancer [106,107].

Additionally oligonucleotides mimicking excised DNA fragments containing UV damages have been shown to increase DRC [108–110] as well as thymine dinucleotides [111,112].

(24)

Introduction

Page | 17

1.5 Aims of the study

As described in chapter 1.1 the genome is constantly attacked by various damaging influences of environmental or endogenous origin. However, repair mechanisms have evolved to counteract the deleterious effects of DNA damages and prevent the formation of mutations to safeguard the integrity of the genome. Mutations arise, if there is an imbalance between DNA damage and repair causing over the years apoptosis, senescence and cancer [7]. Corresponding to the multistep hypothesis of cancer incidence presented by Hanahan and Weinberg in 2000, tumorigenesis is characterized by the accumulation of six hallmark capabilities acquired in a multistep process [40]. As shown in chapter 1.4, in principle, repair processes can be influenced by various agents. In this context positive as well as negative influences are of interest. The OECD recommends in testguideline 482 to use the

unscheduled DNA synthesis assay to determine potentially repair-inhibiting properties of chemicals within the framework of genetic toxicity studies. As described in chapter 1.3 this assay, however, bears a couple of disadvantages. HCRA provides an alternative solution to determine the DRC of cells but is limited with regard to the cell types being examined. As described in chapter 1.2 skin cells are notably affected by DNA damaging agents.

An exogenous source gaining in significance amongst others due to the depletion of the ozone layer is doubtlessly UV radiation. Epidemiological, clinical and laboratory studies have implicated solar ultraviolet radiation as a tumour initiator, tumour promoter and complete carcinogen, and excessive exposure to UV radiation can lead to the development of various skin disorders including melanoma and nonmelanoma skin cancers [113]. One of the most important autologous defence mechanisms protecting DNA from UV fingerprint mutations is nucleotide excision repair. Therefore this mechanism provides important point of attack in connection with the prevention of skin cancer.

Aims of the study are:

 to establish a host cell reactivation based DNA repair assay to determine influences on the NER DNA repair capacity of primary human dermal skin cells.

o to establish a procedure for the simultaneous isolation of kreatinocytes, melanocytes and fibroblasts of one donor

o to establish transfection procedures suitable for all three main skin cell types o to adapt existing protocols to FACS-Technology to enhance the cellnumbers

and therefore sensitivity of the testsystem

 to identify substances influencing DNA repair capacity

 to examine underlying mechanisms and inter-individual differences.

(25)

Page | 18

2 A modified fluorimetric host cell reactivation assay to determine the repair capacity of primary keratinocytes, melanocytes and fibroblasts

Katharina Burger, Katja Matt, Nicole Kieser, Daniel Gebhard and Jörg Bergemann

Department of Biomedical Engineering, University of applied sciences, Anton-Günther- Strasse 51, 72488 Sigmaringen, Germany

Corresponding Author Prof. Dr. Jörg Bergemann

University of applied sciences Sigmaringen Anton-Günther-Str 51

72488 Sigmaringen Germany

Phone: +49 7571 732 273

Mail: bergemann@hs-albsig.de

(26)

A modified fluorimetric host cell reactivation assay to determine the repair capacity of primary keratinocytes, melanocytes and fibroblasts

Page | 19

(27)

Page | 20

(28)

Page | 21

(29)

Page | 22

(30)

Page | 23

(31)

Page | 24

(32)

Page | 25

(33)

Page | 26

(34)

Page | 27

(35)

Page | 28

(36)

Page | 29

(37)

Page | 30

3 The influence of folic acid depletion on the nucleotide excision repair capacity of human dermal fibroblasts measured by a modified host cell reactivation assayA modified flourimetric host cell reactivation assay

Katharina Burgerâ, Nicole Kieserâ, S. Gallinat^b, H. Mielke^b, S. Knott^b and Jörg Bergemannâ

Department of Biomedical Engineering, University of applied sciences, Anton-Günther-

aStrasse 51, 72488 Sigmaringen, Germany

bBeiersdorf AG, Hamburg, Germany

Corresponding Author Prof. Dr. Jörg Bergemann

University of applied sciences Sigmaringen Anton-Günther-Str 51

72488 Sigmaringen Germany

Phone: +49 7571 732 273

Mail: bergemann@hs-albsig.de

(38)

The influence of folic acid depletion on the Nucleotide Excision Repair capacity of human dermal fibroblasts measured by a modified Host Cell Reactivation Assay

Page | 31

(39)

Page | 32

(40)

Page | 33

(41)

Page | 34

(42)

Page | 35

(43)

Page | 36

(44)

Page | 37

(45)

Page | 38

(46)

Page | 39

(47)

Page | 40

(48)

Low doses of UVB enhance reactivation of UV damaged reporter gene in dermal fibroblasts

Page | 41

4 Low doses of UVB enhance reactivation of UV damaged reporter gene in dermal fibroblasts

Katharina Burger, Sonja Schneider, Patrick Maier, Achim Buck, Katja Matt, Daniel Gebhard and Jörg Bergemann

Department of Biomedical Engineering, University of applied sciences, Anton- Günther-Strasse 51, 72488 Sigmaringen, Germany

Corresponding Author:

Prof. Dr. Jörg Bergemann

Department of Biomedical Engineering Albstadt-Sigmaringen University

Anton-Günther-Strasse 51 72488 Sigmaringen

Germany

phone: 07571-732-8273

mail: bergemann@hs-albsig.de

e-mail addresses of the further authors:

BK:

burger@hs-albsig.de

SS:

sonyschneider@gmx.de

PM:

patrick.maier@pharma.uzh.ch

BA:

buckachi@hs-albsig.de

MK:

matt@hs-albsig.de

GD:

gebhard@hs-albsig.de

(49)

Page | 42

chronic exposure to UV radiation; thereby UVB is known to cause the most harmful effects in this context. Skin adapts to UV radiation in order to reduce DNA damage from subsequent exposures by melanin synthesis and epidermal thickening. Very little is known about molecular adaptive responses following low dose UV radiation, although an adaptive response is well documented in the field of ionizing radiation. A possible target of adaptive responses is nucleotide excision repair, dealing mainly with the UV induced photoproducts.

Results: We have studied DNA repair capacity (DRC) in human dermal fibroblasts from different donors following low dose UVB irradiation. Using a modified

fluorescence based host cell reactivation assay, we found that DNA repair capacity is significantly enhanced by pre-irradiation with 100 J/m

²

and 50 J/m². A pre-irradiation with 30 J/m² caused a slight but statistically not significant change of DRC. Using mtt- assay, the used doses were shown to cause no statistically significant cytotoxic effects. All over, gene expression pattern of 7 core nucleotide excision repair genes was only slightly modified after low dose UV irradiation. The expression of XPC was enhanced the clearest, suggesting an involvement of GGR.

Conclusions: Or study indicates that very low doses of UV radiation benefit DNA

repair capacity. The results give further advice for the existence of a molecular

adaption to sunlight providing a new aspect in the context of photoprotective

concepts.

(50)

Page | 43

Background

At present, it is scientifically accepted that solar UV-exposure represents the most important environmental risk factor for the development of non-melanoma skin cancer [1–5] representing more than one third of all malignant neoplasias among the Caucasian population [6]. Furthermore, UVR has been clearly linked with

photoageing [7]. UVB is significantly more powerful than UVA [6], although there are hints for a possible role for UVA in human skin carcinogenesis [8]. Therefore, in the last decades exogenous photoprotection from UVB and UVA deserves more and more emphasis. Current methods of photoprotection are sun avoidance, protective clothing, using sunscreens and the use of chemoprotective agents [9]. However, endogenous mechanisms to protect against reduce and/or repair damages certainly exceed exogenous mechanisms many times. Melanin is generally acknowledged to be the most important endogenous defense mechanism facing photocarcinogenesis [10]. Melanin inhibits the formation of UV induced DNA damages acting as a kind of shield for basal cells

[11,12]. In addition, epidermal thickening increases the light path and consequently

decreases the transmission of UV radiation to the vulnerable cells of the basal and

suprabasal layers [13]. Further, endogenous mechanisms facing UV- induced DNA

damages are –beside antioxidant enzymes- DNA repair and finally apoptosis when

cell damage is beyond repair [14]. UVB induced damages are mainly repaired by

nucleotide excision repair (NER), a highly versatile and sophisticated DNA damage

removal pathway that counteracts beside UV induced CPDs and 6-4 PPs the

deleterious effects a multitude of DNA damages induced by environmental sources

[15]. The important function of NER to protect against skin cancer becomes obvious

by the rare genetic disease Xeroderma Pigmentosum in which diverse NER genes

(51)

Page | 44

progressive degenerative changes of sun-exposed portions of the skin and eyes, often leading to neoplasia, with a frequency about 1000 times higher than seen in the general population under 20 years of age [16].

While epidermal thickening und skin pigmentation are photoadaptive mechanisms that protect the skin from further damage after subsequent UV exposures [17], very little is known about molecular adaptive responses, especially NER. In conjunction with ionizing radiation, an adaptive response is well known (for review see [18]). The term “adaptive response” usually means that relatively small “conditioning” radiation doses induce increased radioresistance when the cells are irradiated with higher doses several hours later [19]. However there are a few reports suggesting that excision repair in human cells is enhanced by low dose UV irradiation.

Within the scope of this study, we analyzed the influence of low dose UVB radiation

onto the DRC using a modified host cell reactivation assay HCRA. Additionally, we

investigated the expression of 7 core NER genes following low dose UV radiation in

order to reveal possible analogies.

(52)

Page | 45

Figure 1: Impact of low dose UVB pre-irradiation on DRC of human skin cells

Primary human skin fibroblasts obtained from apparently normal Caucasian donors of different age were pre-irradiated with 30 J/m², 50 J/m² and 100 J/m² UVB 24 h before determination of DRC using a modified HCRA. Shown are mean values of three parallel determinations. The DRC of cells irradiated with 50 J/m² UVB as well as DRC of the cells pre-irradiation with 100 J/m² were statistically significant different from untreated controls.

Results and discussion

DNA repair is enhanced following low dose UVB pre-irradiation

DRC was determined using HCRA. In contrast to other repair assays, HCRA can detect alterations in all sectors of NER. Obstructions of damage recognition process can be detected as well as incorrect ligations after repair synthesis because the expression of the in vitro damaged reporter protein results only from the correct chronology of all repair steps.

HCRA demonstrated a concentration dependent enhancement of DRC following low dose pre-irradiations. Doses of 100 J/m² and 50 J/m² resulted in statistically

significant enhanced DRC 24 h after irradiation. Additionally, DRC seems to be slightly but statistically not significantly enhanced following a single 30 J/m² dose (Figure 1).

These findings are consistent with the results of Francis, Bennett and Jeeves who

described a UV enhanced reactivation of a UV-damaged reporter gene using host

cell reactivation, too [20–24]. Francis also observed that D

37

of cells is enhanced

following low dose UV pretreatment [20]. Something similar was reported by Liu et al.

(53)

Page | 46

reactivation of a UV damaged reporter gene [25]. Germanier et al. reported in 2000, that prior low dose irradiation clearly enhanced the rate of CDP removal in

transcribed strand [26]. In contrast to Francis and Rainbow, they used a technique allowing quantification of CPDs at the level of a specific strand.

The used doses are not cytotoxic

As described earlier, the cytoxicity of UV radiation shows a huge spread among different skin cell types [27]. Additionally, the source of radiation has a bearing on the cytotoxicity. Thereby broad band UV radiation seems to be more cytotoxic than narrow band UV radiation as described by Cho [27].

To identify irradiation doses for further experiments, we irradiated primary dermal human fibroblasts with increasing doses of UVB. These experiments were carried out using a BLX-312 UV crosslinker (Vilber Lourmat, Paris, France), emitting a broad band UVB spectrum with a peak at 312 nm, just like all other experiments discribed here. Because we expected a donor variability, we examined cells from 6 different donors (5 caucasian and 1 afro-american individual).

Using mtt-assay, we haven’t found negative influences on the reductase activity at

doses lower than 200 J/m². As shown in figure 2a/b, the resistance against UV

radiation revealed a big donor variety. Whereas the obliviously most sensitive cells

showed already significantly decreased reductase activity following 200 J/m², the

most resistant cells showed decreased reductase activity not until 350 J/m². Using

mtt-assay, we haven’t found negative influences on the reductase activity at doses

lower than 200 J/m². As shown in figure 2a/b, the resistance against UV radiation

revealed a big donor variety. Whereas the obliviously most sensitive cells showed

already significantly decreased reductase activity following 200 J/m², the most

(54)

Page | 47

Figure 2: Dose response curve for viability of human fibroblasts and UV dose

Primary human foreskin fibroblasts obtained from apparently normal Caucasian donors and one Afro- american donor were treated with increasing doses of UVB irradiation 24 h prior to the determination of cell viability using mtt- assay. Minimal cytotoxic doses varied markedly among the different donors. No donor showed significant cytotoxic reactions following irradiation lower than 200 J/m² as shown in figure 2b.

resistant cells showed decreased reductase activity not until 350 J/m². Surprisingly, the cells of the afro-american individual showed the highest tolerance against UV irradiation.

Surprisingly, the cells of the afro-american individual showed the highest tolerance against UV irradiation.

These findings are consistent with the results of Cho et al. [27], showing first significant cytotoxic effects of broad band UV irradiation between 250 J/m² and

A

B

(55)

Page | 48

was determined to be the minimal cytotoxic dose of broad band UVB.

Mtt- as well as mts-assay is based upon the detection of active reductase, often used as a measure of living cells. Nevertheless, changes in metabolic activity can

influence reductase activity, limiting the capabilities of these assays to determine cytotoxic influences. Therefore we did an additional cell counting experiment using a ViCell (Beckman Coulter, Brea, USA) approving the results shown in figure 2.

Interestingly, 5 out of six donors showed slightly enhanced viabilities following low dose UV irradiation. This finding could be validated by cell counting, too. We

speculate that this mitogenic effect is due to the activation of EGF receptor which to been shown earlier to be inducible by UV irradiation [29]

Taken together, the results show that broad band UVB doses beneath 200J/m² have most likely no cytotoxic influences onto human dermal fibroblasts, within the

limitations of mtt-assay.

Expression of XPC is clearly enhanced following low dose UVB irradiation

While host cell reactivation assay gives information about DNA repair capacity and

therewith about the repair process in general, gene expression analysis allows a

more detailed analysis of single steps of nucleotide excision repair. 24 h post low

dose UV irradiation, we analyzed the expression of 7 NER genes. We have chosen

XPC which is known to be the initiator of GGR [30], CSB which is essential for

TRCusing the ΔΔC

t

method, we found XPC, XPG, XPB and Polymerase δ

significantly enhanced, whereupon XPC was enhanced the clearest as shown in

figure 3. The other examined genes did not show significantly changed expression

levels.

(56)

Page | 49

Figure 3: Induction of 7 core NER Genes following low dose UVB irradiation Primary human skin fibroblasts obtained from apparently normal Caucasian donors of different age were pre- irradiated with 100 J/m² UVB 24 h before collection of total RNA. After cDNA synthesis, gene expression was determined by realtime PCR analysis.

The variability of the XPC response is well illustrated by the standard deviation values. The transcriptional induction of p48 and XPC in response to UV has been shown earlier and it has been supposed that the induction may help to maintain a critical cellular level of these proteins that have been depleted by proteasomal

degradation at sites of DNA damage [37]. The basal levels of p48 and XPC appear to be regulated by p53 independent of UV-inducible responses, maintaining higher levels of these gene products in wt cells for immediate use in the early steps of NER [38–40]. Decraene et al. reported in 2005 an adaptive response of dermal

keratinocytes via p53-dependend upregulation of the cell-cycle-arrested associated gene p21/WAF1 and the repair –associated gen p53R2 [41]. p53 is known to play an important role in the adaptive response of the UVB to DNA damage [42]. The

mediating role of p53 in cell cycle arrest, repair and apoptosis predominantly but probably not exclusively, occurs through its ability to transactivate genes with an active role in cell cycle arrest, global genomic repair and apoptosis [43]. Fitch et al.

as well as Ford have demonstrated that loss of the tumor suppressor protein p53

(57)

Page | 50

CPD repair much more than 6-4 PP repair [37,44,45].

Therefore, the enhanced expression of XPC following low dose UVB irradiation is consistent with the findings of others. Contemplating the result of gene expression analysis in the context of the HCRA results, one’s would expect an upregulation of CSB rather than an upregulation of XPC in the first instance because the transfected plasmid should have been repaired primarily by TCR. That reveals that probably the induction of the incision step genes isn’t determining for the induction of DRC

measured by HCRA. However, beside XPC, XPG, XPE and polymerase δ are significantly enhanced following low dose irradiation suggesting an induction on the gene expression level concerning unwinding, dual incision and repair synthesis.

Therefore the results we obtained argue that TCR as well as GGR are involved in the photoprotective effect.

The influence of low dose UV irradiation on the expression of XPC and DRC is more pronounced in younger donors

In respect of DRC we found an excessive donor variability being consistent with the findings of others [46].However, we didn’t observe an age depended decline in DRC as described by others [47].

The enhancement of DRC seems to be slightly more pronounced in older donors as shown in figure 4. In contrast, the change of XPC expression following low dose irradiation is considerably larger in younger than in older donors (see figure 4).

Interestingly, in older donors TFIIH is markedly impaired following low dose

irradiation whereas younger donors didn’t show any change.

(58)

Page | 51

Figure 4: Induction of 7 core NER Genes following low dose UVB irradiation in young and old donors Primary human skin fibroblasts obtained from apparently normal Caucasian donors of different age were pre- irradiated with 100 J/m² UVB 24 h before collection of total RNA. After cDNA synthesis, gene expression was determined by realtime PCR analysis.

Repeated exposures only boost the effect of the 30 J/m² preirradtion

In contrast to the application of one single pre-irradiation, a series of three 100 J/m² irradiations at intervals of 24 h hours resulted in a decrease of DNA repair capacity.

However, a series of three 30 J/m² irradiations resulted in a moderate increase of DNA repair capacity (Figure 5). It has been shown earlier [41] that an adaptive response is only achieved if the interval between subsequent UVB insults allows sufficient time. In context of the findings by Decraene et al., a period of 24 h is considered to be sufficient for keratinocytes.

Because cytotoxic effects of UV are distinctly different in melanocytes, fibroblasts

and keratinocytes [30], UV doses and time intervals can hardly be compared.

(59)

Page | 52

Figure 5: Average change of DRC in consequence of repeated low dose UV irradiations

Primary human skin fibroblasts obtained from apparently normal Caucasian donors of different age were irradiated 3 times at intervals of 24 h. DRC was determined using HCRA

intervals were examined.

Given that one single pre-irradiation results in an increased DRC, we speculate, that

the negative effect of a serial irradiation with 100 J/m² could be traced back to the

fact that the interval between the irradiations was not sufficient. It had been shown

earlier, that only 30—50 % of solar simulator induced DNA damages were found to

be repaired within 2 h [44,45,48,49]. The effects of the pre-irradiations seem to be

more pronounced in younger donors. These findings suggest a possible protective

effect of low dose pre-irradiations.

(60)

Page | 53

Conclusions

In this study we compared DNA repair capacity and gene expression pattern in irradiated and non-irradiated human dermal fibroblasts. Our results confirm the existence of a photoadaptive and protective response following a low dose UVB irradiation. The existence of a protective effect has already been proven by multiple approaches. Especially the in vivo results of Wassberg et al.[42] are particularly impressive in this context. In their study, they could show that UV photoproducts are removed more effectively in chronically sun exposed skin compared to non-sun- exposed skin in vivo. Elwood and Gallagher reported in 1998 that the rate of melanoma on the usually heavily sun exposed hands is remarkably low [50].

However, there is no doubt that UV-radiation is mutagenic and is the main reason for the development of non-melanoma skin cancer and therefore excessive sun