«Downstream gene expression of wild type p53 tumor suppressor gene versus mutated and null p53 Master degree thesis in Molecular Biotechnology By M. ...»
Downstream gene expression of wild type p53 tumor
suppressor gene versus mutated and null p53
Master degree thesis in Molecular Biotechnology
M. Belal Al-Jabri
Department of Molecular Biotechnology
Institute of Medical Biology
University of Tromsø
Table Of Contents
P53 as a target for cancer therapy
Aim of study
Materials and Methods
The Gene switch™ system
Site-Directed Mutagenesis (SDM)
Competent cells and transformation
Mammalian Cell Culture
Isolating Total Proteins from the stably engineered cell lines after induction of gene expression
Two Dimensional Gel Electrophoresis
Site-Directed Mutagenesis (SDM)
DNA Sequence of pGene/V5-His-p53 (mutants) vectors
2D- Gel Electrophoresis
MS results of the selected Protein spots
Establishing a stable inducible p53 cell lines
Study of p53 partners
P53 and angiogenesis
P53 and intermediate filaments
P53 and glycolysis
Appendix NO. 1 Human wild type p53 sequence
Appendix NO.2 Inducible Recombinant p53wt in pGENE/V5-HIS........... 91 Appendix NO.3 DNA Sequence analysis of R249S
Appendix NO.4 Analysis from Mass Spectrometry of the identified spots. 95 III M. Belal Al-Jabri, 2006 Acknowledgement Acknowledgement This work is the result of two years of work at the Department of Molecular Biotechnlogy, Institute of Medical biology, University of Tromsø - the northernmost university in the world -, Norway.
I would like to express my gratitude to all those who gave me the possibility to complete this study.
I respectfully acknowledge the supervision, assistance, guidance and support of my supervisor Prof. Raafat El-Gewely, without whom this work could not have been performed. I express my sincere gratitude to him for providing me with the opportunity to work within the field of p53.
I am also thankful and grateful to my co-supervisor Prof. Ugo Moens for his support and help, and also to our department’s engineer Elisabeth Kjeldsen Buvang for providing technical help in the lab and for reading through this thesis.
I also express my thanks to Christian Karlsen for helping me in learning the 2D gel method, to Jack Brunn for performing the MS analysis, and to Umaer Nassir for the useful discussions and skills he has shared with me.
Many thanks to Karin Eilertsen the student consulant at the Institute of Medical Biology for her administrative help and consultancy.
And last but not least, I express my deep sense of gratitude to my parents and family and to my friends in both Norway and my homeland Syria for being very supportive and encouraging especially Khaled, Manar and my wife Maisoon for her unlimited support.
Tromsø, November 2006 M. Belal Al-Jabri
Abstract P53 is a key tumor suppressor and transcription factor protecting us from cancer. The wild type p53 protein functions as a regulatory protein, triggering a variety of cellular responses to different signals. Activation of p53 can lead to cell division arrest, DNA repair, or apoptosis. More than 60% of all human cancers contain p53 mutations. P53 is also reported in many studies to play a role in the control of other cellular important activities such as angiogenesis and glycolysis. In this study we aimed to to identify novel target genes of p53 by investigating the difference in down-stream gene expression of wt p53 in a the Saos-2 cell line which is devoid of p53 expression, in comparison to mutated form of p53 that has been reported to be associated with cancer and in relation to the lack of p53 expression. The aim was also to study protein-protein interactions between p53 and its protein partners in the different p53 variants. Two different p53 mutations (R249S and R273H), considered as hot mutations, were constructed by site-directed mutagenesis.
The GeneSwitch system was used to make stable inducible p53 cell lines. This expression is controlled by mifepristone (inducer). Total proteins were isolated from the different cell lines and separated on 2D gels. The total protein expression in Saos-2 cells containing wild type p53, R249S or R273H mutants, in addition to cells with no p53 copy were compared. The expression patterns of the different samples were similar but not identical.
Our results showed a different expression patterns in some vital proteins. Our results suggest a role of p53 in transcriptionally activating the β subunit of Prolyl 4-Hydroxylase which plays an important role in angiogenesis. Also our results show different patterns in expressing vimentin, which is the most abundant intermediate filament protein in various cell types, between the different cell lines. Also our results show a clear difference in the protein expression patterns of four proteins, which are essential in glycolysis (TIM, enolase 1, α-enolase and aldolase A.) suggesting a role of p53 in the metabolism of tumors particularly in glycolysis. Recent studies have implicated some of these proteins in cancer if not to p53 as well. Studies dealing with P53 and its partner proteins from the total protein using anti V5 antibody were attempted, but was not pursued further due to the short time. The way of doing this was proteomics.
IntroductionP53 or TP53 is a key tumor suppressor transcription factor in the cell. The level of p53 protein is reported to be very low in normal cells; however, its level increases significantly in cells under stress (1). The wild type p53 protein functions as a regulatory protein, triggering a variety of cellular responses to different signals Activation of p53 can lead to growth arrest, DNA repair, or apoptosis (2). P53 protein regulates cell responses to DNA damage to keep genomic stability by transactivation and transrepression of its downstream target genes (3). More than 60% of all human cancers contain p53 mutations (4). Mutations in p53 are frequently found in human cancers owing to the loss of tumor suppressor activities (loss of function) as well as to the gain of tumorogenic activities (gain of function) (5). Both the p53-regulated genes and interacted proteins form a large network of cell system to regulate cell division, DNA repair and apoptosis. P53 function is often inactivated or suppressed in human cancers. Thus, functional restoration of this pathway is an attractive therapeutic strategy (6).
The p53 gene is located on chromosome 17 (17p13), Figure 1. P53 gene is about 20 kb composed of 11 exons (2), Figure 2. There is a very large intron between exon 1 and exon 2. Exon 1 is untranslated region in the human p53 (7).
Figure 1: Chromosome 17. P53 gene is located on the short arm of the chromosome
Figure 2: The p53 gene consists of eleven exons. The pink region denotes the UTR (the untranslated region), the blue region denotes the coding region and the grey region denotes the internal exons within the introns. Figure is adapted after kind permission from p53 knowledgebase: http://p53.bii.a-star.edu.sg/index.php (8)
P53 protein sequence (7):
The tumor suppressor protein p53 is a 393 amino acid transcriptional enhancer phosphoprotein that reversibly associates to form tetramers. The human p53 protein
comprises of several domains, Figure 3:
1. The amino-terminus part (aa 1-44) contains the transactivation domain, which is responsible for activating downstream target genes.
2. A proline-rich domain (aa 58-101) mediates p53 response to DNA damage through apoptosis.
3. The DNA-binding domain (aa 102-292) is a core domain, which consists of a variety of structural motifs. 90% of p53 mutations found in human cancers are located in this domain, preferable as a single aa mutation.
4. The oligomerization domain (aa 325-356) consists of a β-strand, which interacts with another p53 monomer to form a dimer, followed by an α-helix which mediates the dimerization of two p53 dimers to form a tetramer.
5. Three putative nuclear localization signals (NLS) have been identified in the Cterminus, through sequence similarity and mutagenesis. The most N-terminal NLS (NLSI), which consists of 3 consecutive Lysine residues to a basic core, is the most active and conserved domain.
6. Two putative nuclear export signals (NES) have been identified. The leucine-rich C-terminal NES, found within the oligomerization domain, is highly conserved and it has been suggested that oligomerization can result in masking of the NES, resulting in p53 nuclear retention.
Figure 3: Domains of human p53 protein. Figure is adapted after kind permission from p53 knowledgebase: http://p53.bii.a-star.edu.sg/index.php (8).
The role of p53 in cell-cycle control:
The cell cycle is an ordered set of events, culminating in cell growth and division into two daughter cells. This process is composed of two basic phases: Mitosis and the Interphase, Figure 4. The Interphase consists of three phases which are G1, S and G2.
During the cell cycle, chromosomal DNAs are replicated during S phase and equally delivered into two daughter cells during the M phase.
Figure 4: The Cell cycle. It is composed: Mitotic phase (M) and interphase. Interphase consists of three phases, which are G1, S and G2. Image is adapted from Biology Corner web site http://www.biologycorner.com.
Normally the cell cycle is under tight control through three major checkpoints, especially at the transition state from G1 phase to S phase and from G2 phase to M phase. P53 protein is stabilized in response to these checkpoints in the cell cycle which are activated by DNA damage, irradiation, hypoxia, viral infection, or oncogene activation resulting in diverse biological effects, such as cell cycle arrest, apoptosis, senescence, differentiation, and antiangiogenesis (9). The p53 protein is stabilized and activated by phosphorylation, dephosphorylation, acetylation, sumoylation and ribosylation at specific sites, yielding a potent sequence-specific DNA-binding transcription factor (9).
Activation of p53 as a transcription factor causes transactivation of downstream genes, leading to cell cycle arrest in G1, before DNA replication, and in G2, before mitosis.
Also, genes involved in apoptosis are activated by p53 protein (2), Figure 5.
Figure 5: P53 role in the cell cycle. P53 is activated in response to activation of cell cycle checkpoints as a result of DNA damage or other oncogenic factors. Activation of p53 downstream genes results in cell cycle arrest, senescence or apoptosis. Image is adapted from www.humpath.com.
M. Belal Al-Jabri, 2006 Introduction
Under normal growth conditions, progression through G1 is promoted by D-type and Etype cyclins and their associated cyclin-dependent kinases (cdk2, cdk4, and cdk6) (10).
Arrest in the G1 phase of the cell cycle is critical for genomic integrity because it blocks entry into S phase and prevents replication of damaged DNA.
Upon DNA damage, p53 is activated and induces p21WAF1/CIP1, a cyclin-dependent kinase inhibitor [reviewed in (11)]. P21 sustains G1 arrest by inhibiting cdk2 and cdk4 activities.
This inhibition prevents the phosphorylation of pRb, hence the release of E2F from the pRb-E2F complex. This blocks transcription of genes required for entry into S-phase by E2F, and as a result prevents entry into S-phase since the active released E2F is the transcription factor that transactivates the target genes important for the progression of cell cycle including cyclin E. (12;13).
The G2/M checkpoint plays a role in genomic maintenance by preventing segregation of damaged chromosomes (10). In order to sustain a G2/M arrest, Cdc2-cyclinB activity must be inhibited. p53 regulates many target genes that play critical roles during G2/M arrest [reviewed in (10)]. For example, p53 regulates p21 which blocks G2/M progression by binding the Cdc2-cyclinB complex and preventing the activating phosphorylation of Cdc2 at Thr161 by CAK (14). p53 also induces 14-3-3 which blocks entry into mitosis [reviewed in (15)]. Moreover, other p53 targets, such as GADD45, BTG2, REPRIMO, B99 (GTSE-1), hematopoietic zinc finger protein (HZF), and MCG10 have been implicated in the maintenance of the G2/M checkpoint [reviewed in (10)].
The intra-S phase checkpoint is activated when DNA damage occurs during S phase (10).
Although it has yet to be confirmed, a newly identified p53 isoform called p53 may participate in the intra-S checkpoint. This p53 may promote the intra-S arrest by inducing p21 and 14-3-3 (10;16).
M. Belal Al-Jabri, 2006 Introduction
The role of p53 in DNA repair:
Various cellular insults including chemotherapeutic drugs, chemical carcinogens, gamma-irradiation, ultraviolet-irradiation (UV), reactive oxygen species (ROS), and endogenous stressors lead to DNA damage. Failure to repair damaged DNA results in cell death or oncogenic transformation, neither of which is a desired outcome for a biological system. Depending upon the type of DNA lesion, eukaryotic cells utilize multiple DNA repair pathways to mend damaged DNA including nucleotide excision repair (NER), mismatch repair (MMR), base excision repair (BER), translesion synthesis (TLS), homologous recombination (HR), and non-homologous end joining (NHEJ) pathways.