KLF6 has also been implicated as a candidate susceptibility gene for prostate tumourigenesis (Narla, et al., 2001). KLF6 is a Kruppel-like transcription factor and maps to 10p15, a region frequently deleted in prostate carcinomas (Bar-Shira, et al., 2006). Wild type KLF6 suppresses cell growth through a p53-independent transactivation of p21 (Narla, et al., 2005). Deletions and mutations in this gene result in lack of suppression of cell growth and hence has been characterised as a tumour suppressor gene (Narla, et al., 2005). A large scale population study of 300 Jewish prostate cancer patients were screened for mutations in KLF6 gene and results showed that only 17 patients had KLF6 mutations (Bar-Shra, et al., 2006). This indicates that mutations in KLF6 are associated with prostate cancer risk but in a minority of prostate carcinomas in Jewish men.
A tri-institutional study of 3,411 men showed that a single nucleotide polymorphism in the KLF6 region of 10p15 was significantly associated with increased risk of prostate cancer (Narla, et al., 2005). The mutant KLF6 protein isoforms mislocalise to the cytoplasm and antagonise wild type KLF6 function which results in decreased p21 expression which leads to increased unregulated
although further study would warrant the general population frequency of KLF6 mutations and prostate carcinoma frequency.
1.9.2.3 ANX7
ANX7 (also known as ANXA7) gene is localised on chromosome 10q21, a region that is most frequently deleted in prostate cancers, and codes for a calcium ion activated GTPase (Srivastava,
et al., 2001; Dong, 2006). DU145 and LNCaP prostate cancer cell lines show marked decrease in tumour cell growth and colony formation when ANX7 gene is up-regulated in these cells (Srivastava, et al., 2001). This along with the evidence of (ANX7+/-) mice models develop many cancers including prostate cancer and microarray analysis of these tumours showed a reduction of expression of several other tumour suppressor genes, indicates that ANX7 is a potential prostate tumour suppressor gene. However more investigation into ANX7 gene deletions in human prostate cancer would give more detailed analysis into this gene.
1.9.3 Chromosome 13q
Chromosome 13 is one of the most frequently altered chromosomes in cancer including prostate cancer (Hyytinen, et al., 1999). Studies have shown that the most frequently deleted regions on chromosomes 13 are 13q14, 13q21-22, and 13q33 in prostate cancer (Hyytinen, et al., 1999; Brookman-Amissah, et al., 2007; Dong, et al., 2001).
1.9.3.1 BRCA2
The tumour suppressor gene BRCA2 is located at 13q12.2 and has been shown to be deleted in many cancers including prostate cancer (Moro, et al., 2008) and males carrying a BRCA2 mutation have a more aggressive prostate cancer phenotype (Mitra, et al., 2008) but this gene has not been found to be frequently altered in primary prostatic neoplasms (Hyytinen, et al., 1999). This suggests that this deletion of BRCA2 is a late event in prostate carcinoma
development and there is a strong association with hereditary loss of BRCA2 linking to loss of heterozygosity of BRCA2 in men who develop hereditary prostate cancer (Willems, et al., 2008). BRCA2 plays critical roles in DNA repair, transcription, and cell proliferation (Moro et al., 2008). It has recently been reported that loss of BRCA2 promotes cell migration and invasion through a reconstructed Matrigel basement membrane and BRCA2 overexpression decreases both migration and invasion (Moro et al., 2008).
1.9.3.2 RB1
RB1 is involved in cell cycle arrest and like many proteins involved in inactivation of cell growth, it is often lost or mutated in cancer (Bettendorf et al., 2008). It is considered to play an important role in carcinogenesis and inactivation is one of the necessary steps to malignant transformation (Bettendorf, et al., 2008). RB1 is located on chromosome 13q14 and loss of heterozygosity of this region is reported in one of three prostate cancer cases (Cooney, et al., 1996). RB1 codes for retinoblastoma protein (pRB) and is so named as loss of both alleles results in cancer of the eye (Lai, et al., 2003). The protein binds to transcription factors during G1 of the cell cycle and prevents the cell from entering S phase (Lai, et al., 2003). If correct cyclin dependent kinases have bound their respective cyclins it is phosphorylated and deactivated (Lai, et al., 2003). Hence, if RB1 is not expressed there would be less pRB in the cell cycle and less regulation of cell growth.
Studies have been undertaken to research into genes in the loci that are affected to see if candidate tumour suppressor genes can be discovered. This will lead to a greater understanding into how cells turn cancerous and will eventually lead to a mechanism or drug to reverse or prevent it.
1.10 Aim
The aim of this study is to derive a reliable, stable, representable human cell model of prostate tissue. As previously discussed the current human prostate models were derived by expression of viral oncogenes which disrupt progression in the normal cell cycle and results in un-representable models.
Here I report successful immortalisation of human prostate cells from two different regions of the prostate, superficial and deep, and are denoted S and D respectively, with over-expression of two human genes.
Transformed clones of P21s and P21d were created so that I had available for study the immortalised normal cells and the transformed cancer cell lines for comparison. All cell lines were fully characterised with growth curves, radiation survival curves, Single Nucleotide Polymorphism (SNP) and Spectral Karyotyping (SKY) analysis, Matrigel invasion assays and Anchorage Independent colony assays.
The cell lines were used to investigate their responses to strawberry polyphenols and define whether a differential effect on cancer as compared to normal cells exists. Further, whether polyphenols exerted a protective effect against radiation induced damage was also investigated.
Finally, the expression and association of metabolic enzymes within the cells cytosol was investigated to define whether the cancer cells were potentially metabolising ATP in accordance to the Warburg effect.