Expression of growth factor receptors and potential treatment in cholangiocarcinoma cell lines:
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| Format: | Book |
| Language: | English |
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2006
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| Subjects: | |
| Online Access: | Inhaltsverzeichnis |
| Item Description: | Regensburg, Univ., Diss., 2006 |
| Physical Description: | getr. Zählung Ill., graph. Darst. |
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| adam_text | Tables I
Tables
Table 1.1 Etiologic factors in cholangiocarcinoma 4
Table 1.2 Histological classification of cholangiocarcinoma 6
Table 1.3 TNM Staging of ICC (primary hepatocellular carcinoma) 6
Table 1.4 TNM Staging of ECC 7
Table 1.5 Selection for surgical resection - criteria for unresectibility of hilar
lesions 7
Table 1.6 EGFR-specific ligands and factors 12
Table 1.7 EGFR overexpression by tumor type 17
Table 5.1.1 The expression of mRNAs of growth factor receptors in the four cell
lines 58
Table 5.2.1 Expression of growth factor receptors in Western blot analysis 62
Table 5.3.1 The results of the expressions of EGFR, VEGFR1, VEGFR2, VEGFR3,
IGF1R, IGF2R and HGFR in four human CC cell lines by RT-PCR,
Western blot and IHC 71
Table 5.4.1.1 TFk-1 cell counting 73
Table 5.4.1.2 EGI-1 cell counting 80
Table 5.4.2.1 The values from OD value divided by cell number used for the
analysis of TFK-1 cells metabolism 86
Table 6.4.2.2 The values from OD value divided by cell number used for the
analysis of EGI-1 cells metabolism 90
Figures I
Figures
Figure 1.1 Classification of cholangiocarcinoma 2
Figure 1.2 EGFR structure 12
Figure 1.3 EGFR signaling pathways 13
Figure 1.4 The role of EGFR in the control of cellular processes 14
Figure 1.5 Cytoplasmic/traditional (A) and nuclear (B) modes of the EGFR
signaling pathway 16
Figure 1.6 Mechanism of action of cetuximab 19
Figure 1.7 The VEGF family ligands and their receptors 21
Figure 1.8 HGF as mitogen, motogen, morphogen, and as an angiogenic
factor 26
Figure 1.9 Possible contributions of HGF and cMET in cancer progression 27
Figure 4.1 6-well cell culture plate and the cetuximab concentrations 50
Figure 4.2 Procedure for cell lineTFK-1 51
Figure 4.3 Procedure for cell line EGI-1 52
Figure 5.1.1 Positive and negative controls of cDNAfrom TFK-1, EGI-1, OZ and
HuH28 cells 55
Figure 5.1.2 RT-PCR for EGFR, HGFR, IGF1R and IGF2R in TFK-1 cells 56
Figure 5.1.3 RT-PCR for EGFR, HGFR, IGFIRand IGF2R in EGI-1 cells 56
Figure 5.1.4 RT-PCR for EGFR, HGFR and IGF2R in OZ cells 57
Figure 5.1.5 RT-PCR for EGFR, HGFR, IGF1R, IGF2R and VEGFR2 in HuH28
cells 57
Figure 5.2.1 Presence of growth factor receptor proteins in the four cell lines ... 60
Figures II
Figure 5.3.1 Expression of EGFR in the four cell lines 64
Figure 5.3.2 Expression of VEGFR1 in the four cell lines 65
Figure 5.3.3 Expression of VEGFR2 in the four cell lines 66
Figure 5.3.4 Expression of VEGFR3 in the four cell lines 67
Figure 5.3.5 Expression of IGF1R in the four cell lines 68
Figure 5.3.6 Expression of IGF2R in the four cell lines 69
Figure 5.3.7 Expression of HGFR in the four cell lines 70
Figure 5.4.1.1 TFK-1 cell counting at different time points 74
Figure 5.4.1.2 The comparison of growth inhibition between primary TFK-1 cells
and subcultured cells at the same treated time point 75
Figure 5.4.1.3 The alive and dead TFK-1 cells proportion in every time point 77
Figure 5.4.1.4 EGI-1 cell counting at different time point 81
Figure 5.4.1.6 The alive and dead EGI-1 cells proportion in every time point 83
Figure 5.4.2.1 Metabolism of TFK-1 cells treated with different cetuximab
concentration at different time points 87
Figure 5.4.2.2 Metabolism of TFK-1 cells treated with different cetuximab
concentrations 88
Figure 5.4.2.3 Metabolism of TFK-1 cells 89
Figure 5.4.2.4 Metabolism of EGI-1 cells treated with different cetuximab
concentrations at different time points 91
Figure 5.4.2.5 Metabolism of EGI-1 cells treated with different cetuximab
concentrations 92
Figure 5.4.3.1 The TFK-1 cells distribution in the phase of apoptosis, G0/G1, S and
G2/M treated with different cetuximab concentration analyzed by
FACSinT4 93
Figures in
Figure 5.4.3.2 The TFK-1 cells distribution in the phase of apoptosis, G0/G1, S and
G2/M treated with different cetuximab concentration analyzed by
FACSinnewT4 94
Figure 5.4.3.3 The EGI-1 cells distribution in the phase of apoptosis, G0/G1, S and
G2/M treated with different cetuximab concentration analyzed by
FACSinT3 95
Figure 5.4.3.4 The EGI-1 cells distribution in the phase of apoptosis, G0/G1, S and
G2/M treated with different cetuximab concentration analyzed by
FACS in newT3 96
Contents I
Contents
1. Introduction 1
1.1 Cholangiocarcinoma 1
1.1.1 Epidemiology 1
1.1.2 Risk factors 3
1.1.3 Clinical presentation and diagnosis 4
1.1.4 Treatment 7
1.1.5 Prognosis 11
1.2 EGFR and anti-EGFR therapy in carcinoma 11
1.2.1 EGFR signal transduction 11
1.2.2 EGFR signaling pathways and the control of cellular
process 13
1.2.3 Nuclear EGFR signaling pathways and their function 15
1.2.4 Anti-EGFR therapy in human tumors 16
1.2.5 Cetuximab (Ertoitux®) 18
1.3 Other growth factor receptors 20
1.3.1 VEGF/VEGFR system and its role in anti-cancer
therapy 20
1.3.2 The IGF system and its role in anti-cancer therapy 23
1.3.3 HGF and its receptor in cancer 25
2. Aims 28
3. Materials 29
3.1 Instruments for cell culture 29
3.2 Other instruments 29
Contents II
3.3 Materials for cell culture 30
3.4 Other materials 30
3.5 Oligonucleotides 31
3.5.1 Oligonucleotides for qualitative PCR 31
3.5.2 Oligonucleotides for RT-PCR 32
3.6 Antibodies 33
3.6.1 Antibodies for Western blotting 33
3.6.2 Antibodies for IHC 34
3.7 Kits 35
3.8 Consumables 35
3.9 Buffer 36
3.10 Other dilutions 37
3.11 Standards 38
4. Methods 39
4.1 Cell culture 39
4.1.1 Cell thawing 39
4.1.2 Cell subculture 40
4.2 RT-PCR 40
4.2.1 Total RNA isolation 40
4.2.2 Quantification of RNA 42
4.2.3 Reverse transcription reaction 42
4.3.4 RT-PCR 43
4.2.5 PCR product sequence check 45
4.3 Western blot 45
4.3.1 Protein extraction 46
Contents III
4.3.2 Dilution of antibodies for Western blotting 46
4.3.3 Western blot analysis 47
4.4 IHC 47
4.4.1 APAAP method 48
4.4.2 ABC method 49
4.5 Experiments with cetuximab (Erbitux®) 49
4.5.1 Cell counting 52
4.5.2 Cell metabolism (XTT) 53
4.5.3 FACS analysis of cell cycle distribution 54
5. Results 55
5.1 RT-PCR 55
5.2 Western blot 59
5.3 IHC 63
5.4 Experiments with cetuximab 72
5.4.1 Cell growth 72
5.4.2 Cell metabolism 85
5.4.3 Apoptosis 93
6. Discussion 97
6.1 The expression EGFR, VEGFR1, VEGFR2, VEGFR3, IGF1R, IGF2R and
HGFR in the four human CC cell lines: TFK-1, EGI-1, OZ and
HuH28 97
6.1.1 Human CC cell lines 97
6.1.2 RT-PCR 98
6.1.3 Western blot 99
6.1.4 IHC 99
6.1.5 Summary of the expression of EGFR, VEGFR1, VEGFR2, VEGFR3,
Contents IV
IGF1R, IGF2R and HGFR in the four human CC cell lines: TFK-1,
EGI-1,OZandHuH28 99
6.2 The effect of cetuximab in the human CC cell lines TFK-1 and
EGI-1 102
6.2.1 Cetuximab and cell growth 103
6.2.2 Cetuximab and cell metabolism 106
6.2.3 Cetuximab and cell apoptosis 107
6.2.4 The different effects of cetuximab on TFK-1 and
EGI-1 108
7. Summary 109
7.1 The expression of EGFR, VEGFR1, VEGFR2, VEGFR3, IGF1R, IGF2R and
HGFR in the four human CC cell lines: TFK-1, EGI-1, OZ and
HuH28 109
7.2 The effect of cetuximab on TFK-1 and EGI-1 cell growth, metabolism and
apoptosis 109
References 111
Resume 125
Acknowledgements 127
|
| adam_txt |
Tables I
Tables
Table 1.1 Etiologic factors in cholangiocarcinoma 4
Table 1.2 Histological classification of cholangiocarcinoma 6
Table 1.3 TNM Staging of ICC (primary hepatocellular carcinoma) 6
Table 1.4 TNM Staging of ECC 7
Table 1.5 Selection for surgical resection - criteria for unresectibility of hilar
lesions 7
Table 1.6 EGFR-specific ligands and factors 12
Table 1.7 EGFR overexpression by tumor type 17
Table 5.1.1 The expression of mRNAs of growth factor receptors in the four cell
lines 58
Table 5.2.1 Expression of growth factor receptors in Western blot analysis 62
Table 5.3.1 The results of the expressions of EGFR, VEGFR1, VEGFR2, VEGFR3,
IGF1R, IGF2R and HGFR in four human CC cell lines by RT-PCR,
Western blot and IHC 71
Table 5.4.1.1 TFk-1 cell counting 73
Table 5.4.1.2 EGI-1 cell counting 80
Table 5.4.2.1 The values from OD value divided by cell number used for the
analysis of TFK-1 cells metabolism 86
Table 6.4.2.2 The values from OD value divided by cell number used for the
analysis of EGI-1 cells metabolism 90
Figures I
Figures
Figure 1.1 Classification of cholangiocarcinoma 2
Figure 1.2 EGFR structure 12
Figure 1.3 EGFR signaling pathways 13
Figure 1.4 The role of EGFR in the control of cellular processes 14
Figure 1.5 Cytoplasmic/traditional (A) and nuclear (B) modes of the EGFR
signaling pathway 16
Figure 1.6 Mechanism of action of cetuximab 19
Figure 1.7 The VEGF family ligands and their receptors 21
Figure 1.8 HGF as mitogen, motogen, morphogen, and as an angiogenic
factor 26
Figure 1.9 Possible contributions of HGF and cMET in cancer progression 27
Figure 4.1 6-well cell culture plate and the cetuximab concentrations 50
Figure 4.2 Procedure for cell lineTFK-1 51
Figure 4.3 Procedure for cell line EGI-1 52
Figure 5.1.1 Positive and negative controls of cDNAfrom TFK-1, EGI-1, OZ and
HuH28 cells 55
Figure 5.1.2 RT-PCR for EGFR, HGFR, IGF1R and IGF2R in TFK-1 cells 56
Figure 5.1.3 RT-PCR for EGFR, HGFR, IGFIRand IGF2R in EGI-1 cells 56
Figure 5.1.4 RT-PCR for EGFR, HGFR and IGF2R in OZ cells 57
Figure 5.1.5 RT-PCR for EGFR, HGFR, IGF1R, IGF2R and VEGFR2 in HuH28
cells 57
Figure 5.2.1 Presence of growth factor receptor proteins in the four cell lines . 60
Figures II
Figure 5.3.1 Expression of EGFR in the four cell lines 64
Figure 5.3.2 Expression of VEGFR1 in the four cell lines 65
Figure 5.3.3 Expression of VEGFR2 in the four cell lines 66
Figure 5.3.4 Expression of VEGFR3 in the four cell lines 67
Figure 5.3.5 Expression of IGF1R in the four cell lines 68
Figure 5.3.6 Expression of IGF2R in the four cell lines 69
Figure 5.3.7 Expression of HGFR in the four cell lines 70
Figure 5.4.1.1 TFK-1 cell counting at different time points 74
Figure 5.4.1.2 The comparison of growth inhibition between primary TFK-1 cells
and subcultured cells at the same treated time point 75
Figure 5.4.1.3 The alive and dead TFK-1 cells proportion in every time point 77
Figure 5.4.1.4 EGI-1 cell counting at different time point 81
Figure 5.4.1.6 The alive and dead EGI-1 cells proportion in every time point 83
Figure 5.4.2.1 Metabolism of TFK-1 cells treated with different cetuximab
concentration at different time points 87
Figure 5.4.2.2 Metabolism of TFK-1 cells treated with different cetuximab
concentrations 88
Figure 5.4.2.3 Metabolism of TFK-1 cells 89
Figure 5.4.2.4 Metabolism of EGI-1 cells treated with different cetuximab
concentrations at different time points 91
Figure 5.4.2.5 Metabolism of EGI-1 cells treated with different cetuximab
concentrations 92
Figure 5.4.3.1 The TFK-1 cells distribution in the phase of apoptosis, G0/G1, S and
G2/M treated with different cetuximab concentration analyzed by
FACSinT4 93
Figures in
Figure 5.4.3.2 The TFK-1 cells distribution in the phase of apoptosis, G0/G1, S and
G2/M treated with different cetuximab concentration analyzed by
FACSinnewT4 94
Figure 5.4.3.3 The EGI-1 cells distribution in the phase of apoptosis, G0/G1, S and
G2/M treated with different cetuximab concentration analyzed by
FACSinT3 95
Figure 5.4.3.4 The EGI-1 cells distribution in the phase of apoptosis, G0/G1, S and
G2/M treated with different cetuximab concentration analyzed by
FACS in newT3 96
Contents I
Contents
1. Introduction 1
1.1 Cholangiocarcinoma 1
1.1.1 Epidemiology 1
1.1.2 Risk factors 3
1.1.3 Clinical presentation and diagnosis 4
1.1.4 Treatment 7
1.1.5 Prognosis 11
1.2 EGFR and anti-EGFR therapy in carcinoma 11
1.2.1 EGFR signal transduction 11
1.2.2 EGFR signaling pathways and the control of cellular
process 13
1.2.3 Nuclear EGFR signaling pathways and their function 15
1.2.4 Anti-EGFR therapy in human tumors 16
1.2.5 Cetuximab (Ertoitux®) 18
1.3 Other growth factor receptors 20
1.3.1 VEGF/VEGFR system and its role in anti-cancer
therapy 20
1.3.2 The IGF system and its role in anti-cancer therapy 23
1.3.3 HGF and its receptor in cancer 25
2. Aims 28
3. Materials 29
3.1 Instruments for cell culture 29
3.2 Other instruments 29
Contents II
3.3 Materials for cell culture 30
3.4 Other materials 30
3.5 Oligonucleotides 31
3.5.1 Oligonucleotides for qualitative PCR 31
3.5.2 Oligonucleotides for RT-PCR 32
3.6 Antibodies 33
3.6.1 Antibodies for Western blotting 33
3.6.2 Antibodies for IHC 34
3.7 Kits 35
3.8 Consumables 35
3.9 Buffer 36
3.10 Other dilutions 37
3.11 Standards 38
4. Methods 39
4.1 Cell culture 39
4.1.1 Cell thawing 39
4.1.2 Cell subculture 40
4.2 RT-PCR 40
4.2.1 Total RNA isolation 40
4.2.2 Quantification of RNA 42
4.2.3 Reverse transcription reaction 42
4.3.4 RT-PCR 43
4.2.5 PCR product sequence check 45
4.3 Western blot 45
4.3.1 Protein extraction 46
Contents III
4.3.2 Dilution of antibodies for Western blotting 46
4.3.3 Western blot analysis 47
4.4 IHC 47
4.4.1 APAAP method 48
4.4.2 ABC method 49
4.5 Experiments with cetuximab (Erbitux®) 49
4.5.1 Cell counting 52
4.5.2 Cell metabolism (XTT) 53
4.5.3 FACS analysis of cell cycle distribution 54
5. Results 55
5.1 RT-PCR 55
5.2 Western blot 59
5.3 IHC 63
5.4 Experiments with cetuximab 72
5.4.1 Cell growth 72
5.4.2 Cell metabolism 85
5.4.3 Apoptosis 93
6. Discussion 97
6.1 The expression EGFR, VEGFR1, VEGFR2, VEGFR3, IGF1R, IGF2R and
HGFR in the four human CC cell lines: TFK-1, EGI-1, OZ and
HuH28 97
6.1.1 Human CC cell lines 97
6.1.2 RT-PCR 98
6.1.3 Western blot 99
6.1.4 IHC 99
6.1.5 Summary of the expression of EGFR, VEGFR1, VEGFR2, VEGFR3,
Contents IV
IGF1R, IGF2R and HGFR in the four human CC cell lines: TFK-1,
EGI-1,OZandHuH28 99
6.2 The effect of cetuximab in the human CC cell lines TFK-1 and
EGI-1 102
6.2.1 Cetuximab and cell growth 103
6.2.2 Cetuximab and cell metabolism 106
6.2.3 Cetuximab and cell apoptosis 107
6.2.4 The different effects of cetuximab on TFK-1 and
EGI-1 108
7. Summary 109
7.1 The expression of EGFR, VEGFR1, VEGFR2, VEGFR3, IGF1R, IGF2R and
HGFR in the four human CC cell lines: TFK-1, EGI-1, OZ and
HuH28 109
7.2 The effect of cetuximab on TFK-1 and EGI-1 cell growth, metabolism and
apoptosis 109
References 111
Resume 125
Acknowledgements 127 |
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| spelling | Xu, Ling 1976- Verfasser (DE-588)132262401 aut Expression of growth factor receptors and potential treatment in cholangiocarcinoma cell lines vorgelegt von Xu Ling 2006 getr. Zählung Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Regensburg, Univ., Diss., 2006 Epidermaler Wachstumsfaktor-Rezeptor (DE-588)4271161-7 gnd rswk-swf Gallengangkrebs (DE-588)4155847-9 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Gallengangkrebs (DE-588)4155847-9 s Epidermaler Wachstumsfaktor-Rezeptor (DE-588)4271161-7 s b DE-604 HBZ Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=014984895&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Xu, Ling 1976- Expression of growth factor receptors and potential treatment in cholangiocarcinoma cell lines Epidermaler Wachstumsfaktor-Rezeptor (DE-588)4271161-7 gnd Gallengangkrebs (DE-588)4155847-9 gnd |
| subject_GND | (DE-588)4271161-7 (DE-588)4155847-9 (DE-588)4113937-9 |
| title | Expression of growth factor receptors and potential treatment in cholangiocarcinoma cell lines |
| title_auth | Expression of growth factor receptors and potential treatment in cholangiocarcinoma cell lines |
| title_exact_search | Expression of growth factor receptors and potential treatment in cholangiocarcinoma cell lines |
| title_exact_search_txtP | Expression of growth factor receptors and potential treatment in cholangiocarcinoma cell lines |
| title_full | Expression of growth factor receptors and potential treatment in cholangiocarcinoma cell lines vorgelegt von Xu Ling |
| title_fullStr | Expression of growth factor receptors and potential treatment in cholangiocarcinoma cell lines vorgelegt von Xu Ling |
| title_full_unstemmed | Expression of growth factor receptors and potential treatment in cholangiocarcinoma cell lines vorgelegt von Xu Ling |
| title_short | Expression of growth factor receptors and potential treatment in cholangiocarcinoma cell lines |
| title_sort | expression of growth factor receptors and potential treatment in cholangiocarcinoma cell lines |
| topic | Epidermaler Wachstumsfaktor-Rezeptor (DE-588)4271161-7 gnd Gallengangkrebs (DE-588)4155847-9 gnd |
| topic_facet | Epidermaler Wachstumsfaktor-Rezeptor Gallengangkrebs Hochschulschrift |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=014984895&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT xuling expressionofgrowthfactorreceptorsandpotentialtreatmentincholangiocarcinomacelllines |