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 wildtype. 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 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.

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.

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. Wildtype)

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 phosophorylation, 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, 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, vector only cells show a signficantly high proliferation rate (Table 3). Due to the absence of PTEN antagonism of thePIP3/AKT transduction pathway occurs and thus 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 at these codons 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 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 (amino acids 7185) (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 potenital 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 was 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 cells. As well as understanding and cataloguing the mutations in PTEN, it is also be of use to understand cancers in context with other mutations occuring 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 phorphorylation, activation of AKT with cell proliferation and survival (Chaloub & Baker, 2009). Similarly, mutants of the AKT1 gene have been shown to exhibit the ability to transform cells independant 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 in antagonism 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 for the development of drugs to target and suppress the PIP3/AKT signalling pathway and aid in battle against cancer (Wang & Jiang, 2008).