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91345: Biochemistry, Genes and Disease Department of Medical & Molecular Biosciences, Spring 2011

Jennifer Wright: 10878508

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91345: Biochemistry, Genes and Disease Department of Medical & Molecular Biosciences, Spring 2011

Jennifer Wright: 10878508

Cell Proliferation Rate: The Effect of Three PTEN Gene Mutations on a Mammalian Host Cell Model. The eukaryotic cell cycle consists of two main phases, the S and M phases which are separated by Gap phases G1, G2 and G0. The duplications of chromosomes and mitosis along with cytokinesis occur in the S and the M phases respectively (Morgan, 2007). The cell cycle is a complex biological process and must therefore be under strict control carried out during the Gap phases of the cycle. Phases G1 and G2 are often referred to as DNA checkpoints as their function in the cell cycle is to confirm that the chromosomes are intact and induce DNA repair or apoptosis if necessary (Kianian & Phillips, 2007). During G1 cells must either continue to divide or exit from cell cycle to the prolonged non-dividing quiescent state known as G0 as dictated by the presence of factors such as inhibitory signals or unfavourable growth conditions. Exit from G0 is the result of growth factors stimulating the cell to continue to divide (Morgan, 2007). Mutations in the genes involved in these checkpoints often result in human cancers. Uncorrected damage to DNA sequences of proteins that are vital in the normal function of cells, such as control of cell growth, may cause defective gene products thus resulting in cellular abnormalities and neoplasia (Kianian & Phillips, 2007). Oncogene products function in signal transduction pathways that cause uncontrolled cellular proliferation which can result in invasive neoplasia (Stansfeild et al, 2004). Tumour suppressor gene deactivation by mutation causes the loss of function as negative regulators of cell proliferation or positive regulators of apoptosis, consequently advancing the formation of cancer (Hodgeson, 2010). Colorectal cancer (CRC) is the second most frequent cause of cancer related deaths in the developed world (Nassif et al, 2004) with more than 14,000 new cases each year (Cancer Council Australia, 2011). CRC is the result of a number of genetic and epigenetic changes brought on by the presence of carcinogens or lifestyle factors such as poor diet or lack of exercise (Nassif et al, 2004). This causes genetic heterogeneity that may result in the inactivation of tumour-suppressor genes or activation of oncogenes to promote colorectal carcinogenesis (Nassif et al, 2004). Germ-line mutations of the tumour-suppressor gene PTEN have linked to multitude of cancers in various tissues throughout the body, such as breast, brain (Li et al, 1997) and more recently colorectal cancers(Nassif et al, 2004). PTEN is a phosphatase and tensin homolog located on chromosome 10 with both lipid phosphatase and a protein tyrosine phosphatase function. The N-terminal phosphatase domain has shown to contain the CKAGKGR phosphatase catalytic core motif at codons 124130 (Jin et al, 2010) encoded by exon 5 (Nassif et al, 2004). In its function as a lipid phosphatase, PTEN acts to antagonise the phosphoinositol-3-kinase/ Protein kinase AKT (PIP3/AKT) signalling pathway in which AKT is activated by phosphorylation. This PIP3/AKT pathway is described as the major physiological pathway of PTEN (Yin & Shen, 2008). The activated AKT exerts anti-apoptotic effects on an unhealthy cell, and antagonism of this activity by PTEN promotes apoptosis and so inhibits excess cell proliferation (Yin & Shen, 2 of 10

91345: Biochemistry, Genes and Disease Department of Medical & Molecular Biosciences, Spring 2011

Jennifer Wright: 10878508

2008). Thus, the regulation of apoptosis and cell proliferation is carried out by PTEN and it can be seen that the PTEN gene plays a vital role in the prevention of sporadic cancers (Yin & Shen, 2008). PTEN is the second most frequently mutated gene in human cancer (Yin & Shen, 2008). Homozygous deletion of PTEN is embryonically lethal and mutations have been detected in as high as 80% of patients suffering from cancer susceptibility diseases, such as Cowden syndrome (Yin & Shen, 2008), indicative of PTENs vital role in embryonic development as well as the maintenance of healthy tissue. The essential role PTEN plays within our cells, the mutations found within the PTEN gene and their effects on the mechanisms of tumour suppression is under constant investigation. The PTEN mutants C124S, C124X and Y65C were transfected using the PTEN null plasmid vector pCR4TOPO into cancer cells of the U87MG human glioblastoma cell line in order to determine the effect of theses mutations on the cell proliferation rate in comparison to the PTEN wild-type (WT) and plasmid vector-only transformed cell lines. Transversional point mutation C124S and frame-shift mutation C124X are located at amino acid 124 in exon 5 and point mutation Y65C is located at amino acid 65 in exon 3. As C124S and C124X occur in the CKAGKGR phosphatase catalytic core motif, they are predicted to alter the lipid phosphatase activity and therefore the tumour suppressor function of the PTEN gene. The expected outcome for these PTEN mutations is uncontrolled cell proliferation. Method and Materials The details of materials used and methods undertaken for this experiment can be found on pages 39-83 of 91345 Biochemistry, Genes and Disease Subject and Practical Manual (University of Technology Sydney, 2011). The three mutants and the wild-type PTEN gene were isolated and amplified from the plasmid DNA template by the use of specific primers and PCR. The PCR product DNA was analysed by agarose gel electrophoresis to determine the size and concentration of the sample. The DNA was purified using PureLink PCR Purification Kit and the sequencing reaction was prepared. Automated sequencing analysis was carried out at the Australian Genome Research Facility (AGRF, QLD). Electropherograms provided by AGRF were analysed to determine the location and identity of the mutations. Human glioblastoma cells were transiently transfected via Lipofectamine with the pCR4TOPO containing four PTEN types and a vector-only by technical staff. The proliferation rates of these cells and an untransformed cell line were measured and compared spectrophotometrically. It is important to note the change of reading the proliferation assays at an Absorbance of 540nm as listed in the practical manual (p 52). It was more appropriate for us to read the Absorbance at 500nm based on the use of the XXT/ Formazan reagent. Data mining software 'Clustalw' (European Bioinformatics Institute, 2011) was used to align nucleotide and protein sequences. The T-test statistical analysis was then performed on 3 of 10

91345: Biochemistry, Genes and Disease Department of Medical & Molecular Biosciences, Spring 2011

Jennifer Wright: 10878508

the results using Microsoft Excel. Results The stained agarose slab gel (Fig.1) determined the size (bp) and concentration (ng/band) of four unknown PTEN sequence PCR amplicons though comparison to a hyperladder standard. The size of amplicons T1-T3 are determined to be 1200bp and T4 to be 400bp, therefore being identified as truncated PTEN mutant C124X. The concentration read from the gel indicates all templates are 40ng/BAND. As 10l of PCR product was used, the concentration is determined to be 4ng/l. The assumption is made that the purified PCR sample contains 80% of the starting DNA amount; therefore the starting DNA concentration is determined to be 5ng/l. No DNA is found in lanes C1-C4 and, as lanes T1-T4 only contain a single band each, the template is confirmed to be pure. The PCR products were aligned to determine any nucleotide (Table 1) and amino acid (Table 2) sequence divergence between the wild-type PTEN (WT) and the 3 mutants. Nucleotide sequencing showed that C124S differed from WT at base 370, showing the point mutation transversion of thymine to adenine. Similarly, mutation Y65C showed the point mutation transition from adenine to guanine at base 194 when aligned with the PTEN wild-type. Mutation C124X demonstrated a frame-shift mutation from the deletion of a guanine at base 370 when compared to the wild-type. Comparison of mutant amino acid sequences to the PTEN wild-type showed the change from cystein to serine at amino acid 124 within catalytic core motif in C124S. Mutant C124X shows the complete loss of the catalytic core motif, CKAGKGR at amino acid 124. Mutant Y65C shows a change from tyrosine to cystein at amino acid 65, outside of the catalytic core motif. The absorbance of untransformed cells and cells transformed with WT PTEN, PTEN mutants, and vector-only at 500nm, with a standard deviation, showed an increase with time (fig 2). There are two distinct groups with one trend-line allocated per group to avoid confusion due to overlap. A greater trend-line gradient is observed in group containing untransformed host cells, C124X, C124S, and vector-only transformed cells. In comparison, the trend-line gradient of the group comprising of WT PTEN and Y65C is seen to be shallower. The degree of the absorption gradient is proportional to the proliferation rate the cells. Therefore, the group with the greater gradient has a higher proliferation rate. When the T-test was carried out between the proliferation rate (slope) of the vector-only transformed and untransformed cells, no significant difference was observed nor was there indication of significant difference when the proliferation rate of the vector-only transformed glioblastoma cell line was compared to mutants C124S and C124X (Table 3).When the proliferation rate vector-only transformed cells was compared to WT PTEN and Y65C, the proliferation of WT and Y65C was seen to be significantly lower than that of the vector-only cells (Table 3). When the T-test was carried out between the slope of WT PTEN and the slope of the 4 of 10

91345: Biochemistry, Genes and Disease Department of Medical & Molecular Biosciences, Spring 2011

Jennifer Wright: 10878508

mutant PTEN cell lines, no significant difference is observed between the proliferation rate of WT PTEN and Y65C (Table 3). However, it was observed that both C124S and C124X proliferation rates are significantly higher than that of the WT PTEN (Table 3).

Figure 1: A stained agarose gel image of template size (bp) and concentration (ng/band) of unknown PTEN amplicons as determined by electrophoresis and comparison to standard hyperladder1 with control samples to detect contamination. Amplicons T1-T3 are 1200bp as read from the hyperladder standard. The size of T4 corresponds with 400bp and can be identified as truncated PTEN mutant C124X. The concentration read from the gel indicates all templates are 40ng/BAND which is determined to be 4ng/l. The PCR sample contains 80% of the starting DNA amount; therefore the starting DNA concentration is determined to be 5ng/l. As no DNA is found in control lanes C1-C4 and lanes T1-T4 only contain a single band each, the template is concluded to be pure.

Table 1: The comparison of DNA sequences sections of C124S, Y65C, and C124X mutants to wild-type (WT) PTEN as determined by sequence analysis to observe sequence divergence. a
DNA nucleotide sequence WT C124S WT Y65C WT b C124X 361 GCAATTCACTGTAAAGCTGGAAAGGGACGAACTGGTGTAATGATATGTGCATATTTATTA 420 361 GCAATTCACAGTAAAGCTGGAAAGGGACGAACTGGTGTAATGATATGTGCATATTTATTA 420 ********* ************************************************** 181 CATAAAAACCATTACAAGATATACAATCTTTGTGCTGAAAGACATTATGACACCGCCAAA 240 181 CATAAAAACCATTGCAAGATATACAATCTTTGTGCTGAAAGACATTATGACACCGCCAAA 240 ************* **********************************************

361 GCAATTCACTGTAAAGCTGGAAAGGGACGAACTGGTGTAATGATATGTGCATATTTATTA 420 361 GCAATTCACT-TAA---------------------------------------------- 373 ********** a Homology between sequences is indicated by an asterisk (*), divergence is indicated by a dash (-). b Attached affinity flag-tag sequence not included.

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91345: Biochemistry, Genes and Disease Department of Medical & Molecular Biosciences, Spring 2011

Jennifer Wright: 10878508

Table 2: The comparison of amino acid sequences of C124S, Y65C, and C124X mutants to wildtype (WT) PTEN as determined by sequence analysis to determine sequence divergence. a b
Amino Acid Sequence WT C124S WT Y65C WT c C124X 121 AIHCKAGKGRTGVMICAYLLHRGKFLKAQEALDFYGEVRTRDKKGVTIPSQRRYVYYYSY 180 121 AIHSKAGKGRTGVMICAYLLHRGKFLKAQEALDFYGEVRTRDKKGVTIPSQRRYVYYYSY 180 *** ******************************************************** 61 HKNHYKIYNLCAERHYDTAKFNCRVAQYPFEDHNPPQLELIKPFCEDLDQWLSEDDNHVA 120 61 HKNHCKIYNLCAERHYDTAKFNCRVAQYPFEDHNPPQLELIKPFCEDLDQWLSEDDNHVA 120 **** *******************************************************

121 AIHCKAGKGRTGVMICAYLLHRGKFLKAQEALDFYGEVRTRDKKGVTIPSQRRYVYYYSY 180 121 AIH--------------------------------------------------------- 123 *** a Homology between sequences is indicated by an asterisk (*), divergence is indicated by a dash (-). b The CKAGKGR phosphatase catalytic core motif is indicated in bold underline. c Attached affinity flag-tag sequence not included.

Figure 2: The absorbance of untransformed cells and cells transformed with WT PTEN, PTEN mutants, and vector-only at 500nm over a set time period with standard deviation and trend-lines to determine the rate of cell growth. In all cell lines, the absorbance is seen to increase with time. Two distinct groups are observed, with one trend-line per group to avoid confusion due to overlap. The group containing untransformed host cells, C124X, C124S, and vector-only transformed cells shows a steeper gradient when compared to the group containing WT PTEN and Y65C. The steeper gradient indicates that this group has a higher proliferation rate. Error bars indicate the standard deviation on every data-point.

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91345: Biochemistry, Genes and Disease Department of Medical & Molecular Biosciences, Spring 2011

Jennifer Wright: 10878508

Table 3: The average proliferation rate and standard deviation of various cell lines with T-test P values from comparison to vector-only and WT PTEN proliferation rates to determine significant difference.
Cell Line Average Proliferation Rate (Slope) Untransformed Host Vector-only WT Y65C C124S C124X
* Indicates a significant decrease. + Indicates significant increase.

Standard Deviation

P Value (vs. vector-only)

P Value (vs. Wild-type)

0.3661 0.3579 0.1788 0.1892 0.3655 0.3839

0.01696 0.00304 0.01097 0.01749 0.0115 0.01540

p >0.05 p <0.05* p <0.05* p >0.05 p >0.05

p >0.05 p <0.05 p <0.05


+

Discussion: The PTEN protein has been shown to have lipid phosphatase activity that plays significantly on the normal function of cells. The phosphoinositol-3-kinase/ Protein kinase AKT (PIP3/AKT) transduction pathway, in which AKT is activated by phosphorylation, is regarded as the major physiological target of PTEN. PTENs lipid phosphatase activity antagonises the PIP3/AKT signalling pathway to prevent anti-apoptotic effects on unhealthy cells and inhibition of excess cell proliferation. The N-terminal phosphatase domain (amino acids 7185), containing the CKAGKGR phosphatase catalytic core motif (Jin et al, 2010) encoded by exon 5 (Nassif et al, 2004), has an important biological impact on the lipid phosphatase function of PTEN and therefore in the prevention of cancer formation. The effect of the WT and mutant PTEN genes were investigated by determining the proliferation rate following transfection into PTEN null U87MG human glioblastoma cells. The results indicated no significant difference between the proliferation rate of untransformed host cells and vector-only transformed cells (Table 3), allowing the assumption to be made that transfection has no effect on cell proliferation rate. When compared to the functioning WT PTEN, vectoronly cells show a significantly high proliferation rate (Table 3). Due to the absence of PTEN antagonism of the PIP3/AKT transduction pathway the vector-only cell line has the presence of anti-apoptotic effects resulting excess cell proliferation. The CKAGKGR phosphatase catalytic core motif is located at amino acids 124130, therefore any mutations in these residues will result in loss of lipid phosphatase function of PTEN. Many mutations in this region have been found in primary human cancers, for instance H123Y detected in endometrial cancer (Yin & Shen, 2008). DNA mutations in both C124S and C124X 7 of 10

91345: Biochemistry, Genes and Disease Department of Medical & Molecular Biosciences, Spring 2011

Jennifer Wright: 10878508

were found within this vital motif (Table 1). This resulted in divergence from the normal codon sequence (Table 2), and in the case of C124X, truncation. Given these are not a silent mutations; they would result in the conformational change in the phosphatase catalytic core motif and would inhibit the lipid phosphatase functions of this domain. This would result in an inability to suppress active AKT levels in the glioblastoma cells, impinging on the normal cell cycle and thus promoting excess growth in C124S and C124X cell lines. It was therefore was determined that the cell lines will exhibit PTEN null behaviour. This was confirmed by measurement of proliferation rates (figure 2) and statistical analysis (Table 3) indicating a significant difference from wild-type PTEN and no significant difference from PTEN null vector cells. The mutation C124S has previously been indicated as a likely candidate for promotion of tumorigenesis (Takashima et al, 2009). The mutation Y65C, located on exon 3, did not occur in the phosphatase catalytic core motif (Table 1; Table 2) although it was located in the proteins N-terminal phosphatase domain (Nassif et al, 2004). Literature has associated the heterozygous mutation of Y65C with primary sporadic colorectal cancers where the biallelic mutations of PTEN have occurred (Nassif et al, 2004). However, the results of this experiment indicated no excessive cell growth or potential neoplastic formation. The statistical analysis indicated that the proliferation rate of Y65C transformed cells was significantly lower when compared to the PTEN null vector cell line and that no significant difference in proliferation rate of Y65C cells when compared to the wild-type PTEN cell line and (Table 3). This indicates that the level of antagonism of the PIP3/AKT signalling pathway is approximately equivalent to the level of the PTEN wild-type. Y65C is not located in the region of the PTEN protein that is responsible for the lipid phosphatase functions of the cell and consequently did not affect the suppression of the levels of phosphorylated AKT in the glioblastoma cell model. As well as understanding and cataloguing the mutations in PTEN, it is also be of use to understand cancers in context with other mutations occurring in the PIP3/AKT signalling pathway. The enzyme responsible for the formation of PIP3, PIP3K, has shown to have gene mutations in the p110 catalytic subunit which has been associated with human cancer (Chaloub & Baker, 2009). Mutations of the encoding gene, PIK3CA, show increased activity of PIP3K, leading to increased phosphorylation, activation of AKT resulting in cell proliferation and survival (Chaloub & Baker, 2009). Similarly, mutants of the AKT1 gene have been shown to exhibit the ability to transform cells independent of PIP3 with AKT1 gene amplification reported in a multitude of human cancers, including breast, colorectal and ovarian cancers (Chaloub & Baker, 2009). Understanding the precise way in which the phosphatase catalytic core motif in PTEN acts to antagonise the PIP3/AKT signalling pathway is of great importance for the study of potential cancer therapies. It is clear that the PTEN protein itself is under complex regulation, and thus research into the regulation of PTEN as well as its physiological functions can supply knowledge 8 of 10

91345: Biochemistry, Genes and Disease Department of Medical & Molecular Biosciences, Spring 2011

Jennifer Wright: 10878508

for the development of drugs to target and suppress the PIP3/AKT signalling pathway (Wang & Jiang, 2008). Through the exploration of three PTEN gene mutations, it was shown the PTEN protein is vital for maintenance of normal replication, cell proliferation and prevention of sporadic cancers. The expected outcome of the analysis of PTEN gene mutations was achieved. Mutations C124S and C124X, occurring in the CKAGKGR phosphatase catalytic core motif, resulted in excess cell proliferation attributable to the loss of lipid phosphatase activity and the resultant loss of regulation of the PIP3/AKT transduction pathway. Mutation Y65C did not occur in the catalytic core motif and therefore did not exhibit loss of lipid phosphatase activity. Investigation of these mutations and regulatory mechanisms allows better appreciation of the complexity of the human cell cycle. Coupled with the comprehension of the physiological outcome of mutations, this allows for development of cancer therapies to aid in battle against cancer and potentially save lives.

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91345: Biochemistry, Genes and Disease Department of Medical & Molecular Biosciences, Spring 2011

Jennifer Wright: 10878508

References: Cancer Council Australia, 2011, Colorectal cancer, Last Accessed: 8th October 2011. <http://www.cancer.org.au/aboutcancer/cancertypes/colorectalcancer.htm>

Chaloub N & Baker S, 2009, PTEN and the PI3-Kinase Pathway in Cancer in Annual Review of Pathology: Mechanisms of Disease, Vol. 4 pp 127-150. European
Bioinformatics Institute, 2011, ClustalW2 - Multiple Sequence Alignment. Last accessed: 8th October, 2011 <http://www.ebi.ac.uk/Tools/msa/clustalw2/> Hodgson E, 2010, "General Aspects of Cancer." A Textbook of Modern Toxicology. 4th ed., NJ: John Wiley & Sons, New Jersey, p. 239 Jin G, Kim MJ, Jeon H, Choi JE, Kim DS, Lee EB, Cha SI, Yoon GS, Kim CH, Jung TH, Park JY, 2010, PTEN mutations and relationship to EGFR, ERBB2, KRAS, and TP53 mutations in non-small cell lung cancers in Lung Cancer, Vol. 69 no. 3 pp 279-283 Kianian M & Philips R, 2007, Investigation of the Plant Cell Cycle in Reference to Human Cancer, Faculty of the Graduate school of the University of Minnesota, Minnesota USA, pp. 6-8 Li, J, Yen C, Liaw D, Podsypanina K, Bose S, Wang S, Puc J, Miliaresis C, Rodgers L, McCombie R, Bigner S, Giovanella B, Ittmann M, Tycko B, Hibshoosh H, Wigler M & Parsons R, 1997, 'PTEN, a Putative Protein Tyrosine Phosphatase Gene Mutated in Human Brain, Breast, and Prostate Cancer ' in Science, Vol. 275 no. 5308 pp. 1943-1947 Morgan D, 2007, The Cell Cycle, Principles of Control. New Science Press Ltd, Sunderland USA, pp. 4-5 Nassif NT, Lobo GP, Wu X, Henderson C, Morrison C, Eng C, Jalaludin B, Segelov E, 2004, PTEN mutations are common in sporadic microsatellite stable colorectal cancer in Oncogene Vol. 23, pp. 617628. Stansfield , Colom J, & Cano , 2004, Eucaryotic Cells and Their iruses. Schaum's Outline of Theory and Problems of Molecular and Cell Biology. McGraw-Hill, New Delhi p. 242 Takashima M, Parsons C, Ikejima K, Watanabe S, White E, & Rippe R, 2009,'The tumor suppressor protein PTEN inhibits rat hepatic stellate cell activation' in Journal of Gastroenterology Vol. 44, No. 8, pp.847-855 University of Technology Sydney, 2011, 91345: Biochemistry, Genes and Disease Subject and Practical Manual, UTS Department of Medical & Molecular Biosciences, Sydney. pp. 39-62 Yin Y & Shen H, 2008, PTEN: a new guardian of the genome in Oncogene Vol. 27, pp. 54435453 Wang X & Jiang X, 2008, Post-translational regulation of PTEN in Oncogene Vol. 27, pp. 54545463 TO DO: Conclusion COLORECTAL CANCER STAT AND PTEN RE READ FOR NEED FOR REFERENCES

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