The role of PBF - a novel binding partner of p53 - in differentiated thyroid cancer

Dr Chris McCabe (University of Birmingham)


Thyroid cancer has been shown to have many different causes. One gene that has been implicated in causing thyroid cancer is called PBF. This gene was shown to be highly expressed in thyroid tumours, and to induce tumour formation in mice. Very little is known however about how PBF works in the thyroid cell. Our project - generously supported by contributions to the British Thyroid Foundation - builds on preliminary data we have accrued that showed that PBF is able to attach to the extremely critical protein p53, which is a major regulator of how a cell behaves if its DNA gets damaged. One outcome of DNA damage is called genetic instability, which can arise if p53 does not function correctly. Genetic instability - literally, an unstable or mutated set of genes within a cell - is often a cause of cells escaping normal regulation and growing to form tumours.

Our novel idea, which formed the basis of our application to the BTF, is that PBF is highly expressed and attaches to p53, interfering with its ability to respond to damaged DNA. The thyroid gland is well known to be sensitive to radiation, so our experiments were designed around the irradiation of thyroid cells, which would damage their DNA. This would then allow us to compare the ability of p53 to carry out its normal function of switching other genes on and off, when it is in the presence of low levels and high levels of PBF.

Twelve months after the inception of the project, we have pushed our work into a number of exciting directions which have arisen from addressing our initial hypothesis. We have extensively irradiated human and mouse cells, and have determined the specific times and doses of radiation that induce PBF expression. We have discovered that this induction is in fact a result of the protein becoming more stable, and not through its expression being increased per se. We have examined p53 in multiple contexts, finding that increased stability of PBF in thyroid cells results in p53 itself also becoming stabilised, from as early as two hours after irradiation. We have investigated other genes related to p53 function, such as p21 and mdm2. Whilst PBF has a moderate effect on p53 induction of p21, we have characterised an alternative form of mdm2, called mdm2-A, which is over-expressed in the presence of PBF. Thus, high levels of PBF present in thyroid cancer result in p53 stabilisation and altered expression of the mdm2 isoform mdm2A.

Recent experiments have allowed us to establish repeatable murine primary thyroid cultures from wild type and our transgenic mice, which has taken time to perfect. When we irradiate these cells, we stabilise p53, as predicted from our transformed human lines. Importantly, we have also now investigated downstream gene expression changes. For example, p21 is induced in response to irradiation and p53 stabilisation, and this effect is blunted in thyroid cells over-expressing PBF. As we have also now confirmed that PBF binds p53 in human K1 and TPC1 thyroid cells through extensive co-immunoprecipitation assays in the presence and absence of irradiation, these data together firmly support our hypothesis that PBF binds p53 and interferes with its gene transcriptional activities.

To extend this observation we also performed cell survival assays in vitro. Irradiated K1 and TPC1 thyroid cells were subject to MTT assays, and cell survival monitored over time courses. These investigations revealed that over-expression of PBF was associated with increased cell number. This was an important observation which we hypothesise reflects PBF attenuating p53’s pro-apoptotic activity. We are currently addressing this by knocking down PBF in the face of irradiation and stabilised p53, and quantifying cell survival.

Another important component of this BTF-funded project has been to instigate and validate fluorescent inter simple sequence repeat (FISSR) PCR in our mouse models. Although we have published our FISSR findings in human cancers previously (e.g. Kim et al 2005 Oncogene, Kim et al 2007 Carcinogenesis), FISSR has never before been used in mice. This has required extensive calibration, given that, unexpectedly, our human approaches didn't work well in mice. However, we have now been able to achieve repeatable and robust FISSR-PCR data from wild type and murine thyroids. We are currently therefore determining the genetic instability in four murine genotypes: wild type, PBF-transgenic, PTTG-transgenic, PBF+PTTG-bi-transgenic.

In the near future we will then progress to finally uniting all of our observations. Thus, we will irradiate primary cultures from our four murine genotypes and investigate genetic instability, p21 and mdm2 expression and cell viability. Once this is complete, we will be in a position to examine the expression and function of PBF, p53 and related genes in human thyroid cells and in human thyroid tumours. This final section of work, which we anticipate being complete within the next three or four months, will thus round off what we feel has been a successful period of research supported by the BTF.

Work funded in part or wholly by this BTF grant has contributed to several conference abstracts and presentations at the American Endocrine Society meeting (Washington DC, 2009), the British Endocrine Societies conference (Harrogate, 2008 and 2009) and the British Thyroid Association meeting (London, 2009). We have also published a full-length paper in the Journal of Cell Science.


Smith VE, Read ML, Turnell AS, Watkins RJ, Watkinson JC, Lewy GD, Fong JCW, James SR, Eggo MC, Boelaert K, Franklyn JA and McCabe CJ. 2009. A novel mechanism regulating sodium iodide symporter function. Journal of Cell Science 122(18):3393-402