Effect of irradiation of head and neck tumours on the proteins
139 pages
English

Effect of irradiation of head and neck tumours on the proteins' composition of the in vivo salivary pellicle [Elektronische Ressource] / vorgelegt von Evangelia Dounis

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Aus der Universitätsklinik für Zahn-, Mund- und Kieferheilkunde der Albert-Ludwigs-Universität Freiburg i. Br. Abteilung Poliklinik für Zahnerhaltungskunde und Parodontologie. Ärztlicher Direktor: Prof. Dr. E. Hellwig. Effect of irradiation of head and neck tumours on the proteins’ composition of the in vivo salivary pellicle INAUGURAL – DISSERTATION zur Erlangung des Zahnmedizinischen Doktorgrades der Medizinischen Fakultät der Albert-Ludwigs-Universität Freiburg i. Br. Vorgelegt 2003 von Evangelia Dounis geboren in Bristol/ Großbritannien Dekan: Prof. Dr. M. Schumacher 1. Gutachter: Prof. Dr. M. Hannig 2. Gutachter: Prof. Dr. Dr. N-C Gellrich Jahr der Promotion: 2003 To my parents CONTENTS 1 INTRODUCTION ........................................................................................ 1 2 LITERATURE REVIEW............. 3 2.1 The radiobiological basis for tissue reactions after irradiation .................... 3 2.2 Radiation type and method used for the tumours of the head and neck region......................................................................................................................... 4 2.3 The clinical effect of therapeutic irradiation on the tissues of the head and neck region..............

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Publié le 01 janvier 2004
Nombre de lectures 8
Langue English



Aus der Universitätsklinik für Zahn-, Mund- und Kieferheilkunde
der Albert-Ludwigs-Universität Freiburg i. Br.

Abteilung Poliklinik für Zahnerhaltungskunde und Parodontologie.
Ärztlicher Direktor: Prof. Dr. E. Hellwig.







Effect of irradiation of head and neck tumours on the
proteins’ composition of the in vivo salivary pellicle





















INAUGURAL – DISSERTATION
zur
Erlangung des Zahnmedizinischen Doktorgrades

der Medizinischen Fakultät
der Albert-Ludwigs-Universität
Freiburg i. Br.


Vorgelegt 2003
von Evangelia Dounis
geboren in Bristol/ Großbritannien


































Dekan: Prof. Dr. M. Schumacher
1. Gutachter: Prof. Dr. M. Hannig
2. Gutachter: Prof. Dr. Dr. N-C Gellrich
Jahr der Promotion: 2003







































To my parents




CONTENTS
1 INTRODUCTION ........................................................................................ 1
2 LITERATURE REVIEW............. 3
2.1 The radiobiological basis for tissue reactions after irradiation .................... 3
2.2 Radiation type and method used for the tumours of the head and neck
region......................................................................................................................... 4
2.3 The clinical effect of therapeutic irradiation on the tissues of the head and
neck region................ 5
2.3.1 Mucosa.................................................................................................................5
2.3.2 Taste....................7
2.3.3 Bone.....................8
2.3.4 Muscles and joints ..............................................................................................11
2.3.5 Salivary glands and saliva...................11
2.3.6 Teeth and periodontium......................20
2.4 Pellicle........................................................................................................... 25
2.4.1 Function.............25
2.4.2 Formation...........27
2.4.3 Ultrastructure......................................................................29
2.4.4 Chemical Composition........................30
3 PLAN OF THE STUDY............................................. 42
4 PATIENTS, MATERIALS, METHODS.................... 44
4.1 Patients......................................................................... 44
4.2 Pellicle samples............................. 45
4.3 Electrophoretical Separation....................................... 47
4.4 Western Blotting and Immunodetection ...................................................... 51
4.5 Gel’s Imaging and Analysis.......................................... 54
4.6 Statistic analysis of data............... 55
5 RESULTS................................................................... 58
5.1 Silver stained gels......................................................................................... 58
5.1.1 Electrophoretical profile......................58
5.1.2 Percentage of each band in the lane.....61
5.1.3 Total protein volume...........................62
5.2 Blots .............................................................................................................. 63
5.2.1 Amylase.............63
5.2.2 IgA.....................64
5.2.3 Acidic PRPs .......................................................................................................66
6 DISCUSSION............................ 68
6.1 Discussion of the patients’ selection............................. 68
6.2 Discussion of the method.............................................................................. 69
6.2.1 In vivo formation of the samples.........69
6.2.2 Pellicle formation ...............................................................................................71
6.2.3 Collection and processing of the samples............................72
6.2.4 Analysis of the samples.......................74
6.3 Discussion of the Results.............................................................................. 77
6.3.1 Polyacrylamide gel electrophoresis (PAGE)........................77
6.3.2 Immunoblots ......................................................................83
6.4 Conclusions................................... 87
7 SUMMARY ................................ 88
8 LITERATURE........................................................... 89
9 TABLES................................... 117
10 CURRICULUM VITAE........................................ 132
11 Acknowledgements................................................ 133 1 Introduction
1 INTRODUCTION
About 600.000 new cancer cases of the head and neck region are reported
every year worldwide. From these patients about the half will recieve
irradiation as main or adjuvant therapy (PARKIN et al., 1988). The
overwhelming majority of these patients experience post-radiation
problems, which affect dramatically the quality of their life in terms of
health, comfort and function.
The effect of irradiation on the tissues of the head and neck region has
been thoroughly investigated in many studies (RUBIN and DOKOU, 1976;
MARX, 1983; HAMMANS, 1993; ATTKINSON and AVA, 1994;
GUCHELAAR et al., 1997). Especially xerostomia and the high incidence
of carious lesions are the most frequent side-effects of irradiation therapy.
Even though the reduced flow-rate of saliva during and after radiotherapy
is without doubt the major factor that leads to the rapid increase in the
incidence of caries, it seems that it is not the only one. The altered
composition of saliva after irradiation could possibly play a significant
role in the loss of tooth integrity.
Acquired salivary pellicle represents the interface between saliva and
tooth surface. The formation and role of the acquired enamel pellicle have
already been investigated in previous studies (HANNIG et al., 1994a, b;
AMAECHI et al., 1999; YAO et al., 1999; LAMKIN et al., 2001). This
organic layer derives from saliva and can, therefore, be affected by any
changes in its physical and chemical properties. At the same time, pellicle
protects the integrity of dental surfaces and regulates the microbial
colonization. Hence, it can be assumed that the radiation-induced changes
of saliva can affect the composition of acquired enamel pellicle and this
could lead to the destruction of dental tissues observed during and after 2 Introduction
irradiation therapy. This assumption, however, has not yet been
investigated in any study.
Thus, aim of this comparative study was to investigate the effect of
irradiation of head and neck tumours on the proteins’ composition of in
vivo salivary pellicle. For this reason, salivary pellicle was collected from
oral cancer patients who were treated with radiation therapy and equal
number of patients treated with chemotherapy and/or surgery. The
samples were studied and compared by means of electrophoretical
separation and immunodetection.
3 Literature review
2 LITERATURE REVIEW
2.1 Radiobiological basis for the tissue reactions after
irradiation
The clinical manifestation of radiation injury is the result of a complex
pathomechanism, which starts at the time that the radiation energy is
absorbed by a cell. The first physicochemical alterations happen within the
-16 -13 first 10 to 10 s and damage the organic molecules that comprise the
cells (SCHERER and SACK, 1996). This damage can result either directly
from the irradiation, or indirectly. The latter takes place when irradiation
encounters water molecules in the cells, as the free radicals produced are
especially harmful to the organic molecules (MORGENROTH, 1967). The
destruction of DNA, proteins, enzymes or other macromolecules effect the
physiology and function of the cells and can lead to their death. In the case
that the affected cells are somatic, the clinical manifestations are expressed
at the individuals who received the irradiation either as early or chronic
not malignant tissue injury or as malignant processes. On the contrary,
when the irradiation encounters germ cells, then the clinical results will
affect the next generation (GRÖTZ, 2001).
The radiosensitivity of the cells is determined as their remaining capability
to duplicate after irradiation. It depends on the generation cycle’s phase,
during which the cell received the radiation. The phases in which the cells
are especially radiosensitive are the M-phase in which the mitosis takes
place, the early S-phase which is characterized of the reduplication of the
chromosomes and the G2-phase during which the cell is preparing for the
mitosis (STREFFER, 1996). Consequently, tissues with a high
proliferation’s rate demonstrate also a high radiosensitivity. This fact is 4 Literature review
responsible for the therapeutic effect of radiation due to the elimination of
the rapidly proliferating cells of a tumour. On the other hand, this also
leads to numerous adverse effects due to the destruction of adjacent
healthy tissues that have a rapid turnover cell renewal system (e.g. the
epithelial tissue) (GRÖTZ, 2001).
2.2 Radiation type and method used for tumours of
the head and neck region
The method and dose of therapeutic irradiation depends on the type,
stage, grade, size and localisation of the tumour (DECLOS, 1965).
Generally, tumours that react well to radiotherapy are the small and
exophytic ones, which have good vascularisation and oxygenation. On the
contrary, in ulcerated, infected, nodular and infiltrating tumours the
radiotherapy does not have the desirable results (ENGELMEIER and
KING, 1983).
The type of irradiation that is mostly used today for the treatment of
tumours of head and neck region is the linear accelerated irradiation
therapy, where gamma irradiation is applied (SCHERER and SACK, 1996).
The goal of radiation therapy is to eliminate the neoplastic cells, while
causing tolerable side effects. Therefore, the total dose of irradiation is set
under the tolerance limit of the adjacent healthy tissues. The most usual
total dose for the carcinomas of head and neck region is 60 Gy.
Significantly higher doses (70 Gy) are used for the adenocystic carcinoma
which has a low radiosensitivity, whereas the malignant lymphomas of
the region are irradiated with low doses (40 Gy) (RUBIN, 1989).
The irradiation method used today is the protracted-fractionated radiation
and is accumulated for a period of 4 to 6 weeks. According to the protocol
followed currently by the most therapeutic centres the total dose is 5 Literature review
fractionated to 2 Gy per day, 5 days per week. This prolonged time
increases the possibility that a neoplastic cell will receive the radiation
during the radiosensitive phase of its regeneration cycle (MARX and
JOHNSON, 1987). Additionally, these time intervals allow the tumour to
“shrink” slowly, enabling better oxygenation of the tumour cells and
therefore more efficient treatment, while the healthy tissues can also
regenerate to some extent (RUBIN and DOKU, 1976). On the other hand,
the risk of repopulation of the tumour in this time period, even though
small, is not insignificant (TROTT and KUMMERMER, 1985).
2.3 Clinical effect of therapeutic irradiation on the
tissues of the head and neck region
2.3.1 Mucosa
The radiation-induced mucositis is a painful inflammatory process of the
moderately radiosensitive mucosal epithelium (RUBIN and DOKOU,
1976). It is one of the most frequent symptoms following radiation, as it
appears in 74-81% of the cases (KALZ, 1990). Mucositis is of great clinical
importance, as it is -with 86%- the most common reason that leads to an
interruption of the radiation therapy (HERRMANN et al., 1994). The
mucosal breakdown may have substantial consequences, such as the
limiting of food and fluid intake, whereas it can also be a potential portal
for infectious processes (EPSTEIN et al., 1986). The dose of irradiation, the
general health condition of the patient, as well as the status of his oral
hygiene, seem to be factors that affect the grade of irradiation injury seen
in the oral mucosa (HAMMANS, 1993).
The acute response of the epithelial tissue is representative of the response
of a rapid turnover cell renewal system (RUBIN and CASARETT, 1968).