Medizinische Fakultät der Humboldt-Universität zu Berlin

zur Erlangung der Venia legendi
für das Fach

Gynäkologie und Geburtshilfe

vorgelegt von

Dr. med. Angela Reles

Präsident: Prof. Dr. rer. nat. J. Mlynek

Dekan: Prof. Dr. med. Joachim W. Dudenhausen

Eingereicht im: 28.03.2001

Öffentlich-wissenschaftlicher Vortrag: 04.12.2001

Gutachter:
Frau Prof. Dr. med. M. Kiechle
Herr Prof. Dr. med. D. Kieback

Abbildungsverzeichnis
Fig. 1:	Structural and functional regions of the p53 protein (redrawn from May and May, 1999) Functional regions and corresponding amino acid residues are shown on top. In the middle, evolutionary highly conserved domains I-V are shown. The bottom represents the tertiary structure of the site-specific DNA-binding. L1, L2, L3 indicate loops, and LSH indicates a loop-sheet-helix structure. The tertiary structure is shown in more detail in Fig. 2.
Fig. 2:	Topological diagram of the secondary structure elements of the core domain of the p53 protein (redrawn from Cho et al. 1994). The residues at the start and the end of each secondary structure element are indicated. The DNA-binding regions of the protein (L1-S2-S‘, L2, L3, S10-H2) correspond to the conserved regions of the p53 gene and are colored yellow for region II (residue 117-142), red for region III (residue 171-181), blue for region IV (residue 234-258) and green for region V (residue 270-286). The boundaries of the two ß-sheets that make up the ß-sandwich are shaded. The scaffolding regions are white.
Fig. 3:	p53 pathways. Upstream activators are shown above in red. Multiple downstream effectors of p53 which play a role for cell cycle arrest and apoptosis are shown. Arrows indicate a positive effect, while a flat line indicates inhibition. Quadrangles indicate specific functions in p53/MDM2 interaction.
Fig. 4:	Response to cellular stresses in cells with wildtype p53 versus cells with mutated p53
Fig. 5:	Structure and functional regions of the MDM2 protein. Above functional domains with corresponding amino acid residues are shown (see Freedman et al. 1999). DNA-PK sites comprise aa 388/389, 395/396 and 407/408. The p53 binding domain and conserved region I are almost identical. Since the human mdm2 exon boundaries are not fully known yet, mouse exons 1-12 are shown with the first codon of each exon indicated. Exon 1 and 2 are noncoding (Montes de Oca Luna et al. 1996).
Fig. 6:	Autoregulatory feedback loop of the p53 tumor suppressor gene and the mdm2 gene. p53 at basal and at increased levels activates the mdm2 gene. The MDM2 protein binds to p53, inhibits its DNA binding ability, and promotes nuclear export and protein degradation.
Fig. 7:	Hypothetic model about the effects of impaired MDM2 function on p53 protein accumulation in the cell. Cellular stresses cause p53 activation, which is downregulated by functional MDM2 in an autoregulatory feedback loop. In case of nonfunctional MDM2, wildtype p53 accumulates in the nucleus.
Fig. 8a:	pcDNA3 expression vector (Invitrogen). cDNA of p53, full length mdm2 and mdm2 splice variants was ligated into the pcDNA3 expression vector.
Fig. 8b:	mdm2 splice variant cDNA after NcoI restriction enzyme digestion of the pcDNA3 expression vector. The expression vector has three NcoI restriction sites at positions 610, 1976, and 2711 and the mdm2 cDNA (654 bp) has one NcoI restriction site at position 533. The cDNA analyzed on the agarose gel shows three fragments of the vector and two fragments of the cDNA insert, one with additional vector sequence. All clones (lanes 1-6) contain the mdm2 splice variant cDNA.
Fig. 9:	Transient transfection of pcDNA constructs into vaccinia virus infected HeLa cells. The cDNA of interest was inserted into the pcDNA3 plasmid such that it came under the control of the T7 RNA polymerase promoter (pT7). Using liposome-mediated transfection, this recombinant plasmid is introduced into the cytoplasm of HeLa-cells infected with the vTF7-3 strain, a recombinant vaccinia virus encoding bacteriophage T7 RNA polymerase. During incubation, the cDNA is transcribed with high efficiency by T7 RNA polymerase. Proteins were detected by immunoprecipitation and Western blotting.
Fig. 10	A-C: p53 mutation and overexpression A: Single Strand Conformation Polymorphism (SSCP) analysis of the p53 gene, exon 5 in ovarian carcinomas. Arrows in lanes 7 and 8 indicate band shifts suspicious for mutations. B: DNA sequence analysis of exon 5 of the p53 gene in endometrioid ovarian carcinoma #2335. Missense mutation in codon 141, TGC to TGG (amino acid exchange cystein to tryptophan). C: Immunostaining shows overexpression of the p53 protein in the tumor nuclei (case #2335).
Fig. 11	A-C: p53 mutation and overexpressio A: Single Strand Conformation Polymorphism (SSCP) analysis of p53, exon 9 in ovarian carcinomas. Arrows in lanes 3 and 10 (case #3402) indicate band shifts suspicious for mutations. B: DNA sequence analysis of exon 9 of the p53 gene in endometrioid ovarian carcinoma #3402. A deletion mutation of one basepair in codon 320 causes a frameshift. C: Immunostaining shows overexpression of the p53 protein in the tumor nuclei (case #2335.)
Fig. 12:	p53 mutations in ovarian cancer. Mutations of the p53 gene were identified in 99/178 epithelial ovarian carcinomas. The majority of mutations were missense mutations.
Fig. 13:	p53 mutations in evolutionary highly conserved domains of the gene. The upper part of the figure shows the number of mutations in codons 36 through 324 of the p53 gene. Mutations in evolutionary highly conserved regions are marked in yellow, mutations in non-conserved regions in dark blue and splice site mutations in white. The lower part of the figure shows the corresponding DNA interacting regions of the p53 protein.
Fig. 14:	CGC CCC (Arg Pro) polymorphism at codon 72 of the p53 gene A: SSCP analysis of p53, exon 4 in ovarian carcinomas. DNA in lanes 2,4,5,6,and 8 shows band shifts suspicious for sequence alterations and was analyzed by DNA sequencing. B: DNA sequence analysis of p53, exon 4 shows a CGC to CCC poymorphism at codon 72, resulting in an arginine to proline amino acid exchange.
Fig. 15:	Intron 3 polymorphism of the p53 gene A: Single Strand Conformation Polymorphism (SSCP) of p53, exon 3 with the adjacent intron sequence shows a band shift in lanes 2,4,8 and 9 indicating the intron 3 polymorphism. B: Wild type (lanes 3 and 4) and the polymorphism sequence (lanes 1 and 2) of p53, intron 3 with a 16 bp insertion (lanes 1 and 2) and adjacent sequence are shown as 5‘ to 3‘: GCTggggacctggagggctGGGGGG
Fig. 16:	Intron 10 polymorphism of the p53 gene at nucleotide 17708 A novel polymorphism of the p53 gene was identified at nucleotide 17708 (a>t) of intron 10 by SSCP analysi and DNA sequencing in the ovarian cancer case #3402.
Fig. 17:	Intron 10 polymorphism of the p53 gene at nucleotide 18550 A: SSCP analysis of p53 exon 11 and flanking intron sequence in ovarian carcinomas. The band shift in lane 5 (case #3394) is suspicious for DNA sequence alterations. B: DNA sequence analysis of p53 exon 11/ intron 10 reveals a novel c>t polymorphism at nucleotide 18550 of intron 10 in the ovarian cancer cases #2660 and #3344. The polymorphism was confirmed in normal tissue DNA.
Fig. 18a:	p53 protein overexpression according to type of p53 mutation. Overexpression is found in a high percentage of cases with missense mutations, while the frequency of overexpression in nonmissense mutations is overall only 46%.
Fig. 18b:	p53 protein overexpression according to p53 mutation or wildtype p53. A high proportion of cases (38%) shows p53 protein accumulation (immunostaining in 10% of the nuclei) despite p53 wildtype sequence.
Fig. 19	A-B: Estimated probability of not progressing in epithelial ovarian cancer patients (n=74), who received platinum-based chemotherapy. (A) Ovarian carcinomas with p53 mutations versus wildtype p53. (B) Ovarian carcinomas with p53 proteinoverexpression versus normal p53 expression.
Fig. 19	C-D:: Estimated probability of (C) not progressing, and (D) overall survival respectively in epithelial ovarian cancer patients (n=74), who received platinum-based chemotherapy. Ovarian cancer with p53 alterations (mutation or overexpression or both) versus normal p53 status.
Fig. 20	A-B: Estimated probability of overall survival in patients with epithelial ovarian cancer (n=178) according to p53 alterations. (A) p53 mutations versus wildtype p53. (B) p53 mutations in evolutionary highly conserved domains versus wildtype p53 or mutations in non-conserved domains.
Fig. 20	C-D: Estimated probability of overall survival in epithelial ovarian patients (n=178) according to p53 alterations. (C) p53 mutations in DNA- interacting regions versus wildtype p53 or mutations in scaffolding regions of the p53 protein. (D) p53 protein overexpression versus normal expression of the p53 protein.
Fig. 20	E-F: Estimated probability of (E) not progressing, and (F) overall survival respectively in epithelial ovarian cancer patients (n=178). Ovarian cancer with p53 alterations (p53 mutation or p53 overexpression or both) versus normal p53 status.
Fig. 21:	Southern hybridization of genomic DNA with the mdm2 cDNA probe. The sarcoma cell line SA1 which was used as a positive control shows amplification of mdm2 DNA, while none of the ovarian cancer tissues shows DNA amplification.
Fig. 22:	Northern hybridization of total mRNA with the mdm2 cDNA probe. The sarcoma cell line SA1 which was used as a positive control shows overexpression of mdm2 RNA, while none of the ovarian cancer tissues shows mdm2 overexpression.
Fig. 23:	mdm2 RNA splice variants in ovarian carcinomas. Total RNA was ana-lyzed by RT-PCR, and PCR-products were separated on a 1.2% agarose gel. Cases in lanes 1,2,6,7,9, and 10 show splice variants of different sizes. The case in lane 3 shows no mdm2 expression. (cDNA bands on the gel contain additional 53 bp of flanking primer sequence)
Fig. 24:	A-B: RT-PCR for ß2 microglobulin as a positive control for quality and amount of RNA. (A) RNA was extracted from normal ovarian tissue. All cases show equal amplification of a 898 bp ß2M PCR product. Lane 21 shows the PCR product of cDNA from the SA1 sarcoma cell line. Lane 22 is the negative control. (B) RT-PCR for ß2 microglobulin expression in ovarian cancer RNA. Except for case #2341 in lane 7 all cases show approximately equal expression of the ß2 microglobulin RNA.
Fig. 25:	Loss of functional regions in mdm2 splice variants in comparison to the full length mdm2 gene Above, functional domains of the MDM2 protein with corresponding amino acid residues are shown (according to Freedman et al. 1999). Below, splice variants of the mdm2 gene are shown. Black lines indicate the missing part of the sequence.
Fig. 26:	A-D: Sequence overlaps in mdm2 RNA splice variants. (A-C) Splice site sequences of the 391 bp and 221 bp mdm2 splice variants show overlaps of several basepairs. The capital letters refer to transcribed bases that remain in the final mRNA products, while the lower case letters refer to untranscribed bases which are spliced out of the final mRNA product. The green boxes enclose the transcribed sequence at the donor site and the blue boxes enclose the transcribed sequence at the acceptor site. The yellow boxes indicate sequence homologies. D) As opposed to many of the aberrant splice variants which show overlapping sequences at the splice site, no such sequence homology is notable in the 654 bp mdm2-b splice variant which splices at exact exon/intron boundaries of exon 3 and exon 12.
Fig. 27:	mdm2 RNA splice variants in ovarian cystadenomas and tumors of borderline malignancy. Total RNA was analyzed by RT-PCR and PCR-products were separated on a 1.2% agarose gel. Cases in lanes 1, 2, 5-8, and 10 show splice variants of different sizes. The case in lane 3 shows no mdm2 expression despite normal expression of the ß2-microglobulin control gene. (cDNA bands on the gel contain additional 53 bp of flanking primer sequence).
Fig. 28:	mdm2 RNA splice variants in normal ovarian tissue. Total RNA of ovarian tissue was analyzed by RT-PCR and PCR-products were separated on a 1.2% agarose gel. Cases in lanes 3, 5, 7, and 8 show splice variants of different sizes. (cDNA bands on the gel contain additional 53 bp of flanking primer sequence).
Fig. 29:	Cytoplasmic expression of p53 and MDM2 proteins by transient transfection of pcDNA3 vector constructs into vaccinia virus infected HeLa cells A: Expression of the full length p53 protein (53 kDa). B: Expression of the full length MDM2 protein (90 kDa). C: Expression of a 40 kDa protein of the splice variant mdm2-b (654 bp). No protein could be expressed from the mdm2 splice variants of 351 bp and 52 bp. Proteins were analyzed by Western blotting and anti-myc respectively anti-HA immunodetection.
Fig. 30:	A-B: Clinical outcome of ovarian cancer patients dependent on mdm2 alternative RNA splicing (n=92) as analyzed by RT-PCR and DNA sequencing. A) Overall survival in patients with expression of the full length mdm2 RNA transcript, versus mdm2 splice variants, versus absence of mdm2 RNA expression. B) Overall survival in patients with aberrant splicing in the donor and acceptor or only the acceptor site, versus patients with full length mdm2 and/or alternative splicing at exon/intron boundary of exon 3 and 9 (mdm2-a, 888 bp) or exon 3 and 11 (mdm2-b, 654 bp).
Fig.30 C-D:	Clinical outcome of ovarian cancer patients dependent on mdm2 RNA alternative splicing (n=92) as analyzed by RT-PCR and DNA sequencing. C) Overall survival in patients with expression of small splice variants of <300 bp versus expression of the mdm2-b splice variant (654 bp) or full length mdm2 D) Overall survival in patients with expression of small splice variants (<300 bp) versus expression of mdm2 splice variants of >300 bp in the presence or absence of the full length mdm2 transcript. Cases with expression of the the mdm2-b splice variant (654 bp) were not included in this analysis.
Fig.30 E-F:	Clinical outcome of ovarian cancer patients dependent on mdm2 RNA alternative splicing (n=92) as analyzed by RT-PCR and DNA sequencing. E) Overall survival in patients with expression of the mdm2-b splice variant (654 bp) versus absence of the mdm2-b splice variant. F) Overall survival in patients with expression of a small splice variant of 221 bp was correlated with early stage (FIGO I/II) and marginally significant with a better clinical outcome.

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