The standard therapeutic regimen for treating ovarian cancer is a combinatorial treatment of platinum (cisplatin or carboplatin) and taxol (paclitaxel) based drugs often followed by de-bulking surgery 
. Both these drugs induce apoptotic pathways either by forming DNA adducts or by inducing cell cycle arrest and subsequent cell death 
. Activation of p53 is a central molecular event that guides the cells to follow either a survival or an apoptotic route after the genotoxic insult. Activation of p53 might down-regulate the PIK3CA/Akt signalling as indirectly evidenced by association of PIK3CA
gene amplification with p53 mutations in ovarian carcinoma 
p110α, the catalytic subunit of class I PI3K and encoded by PIK3CA
gene is tightly regulated in normal cells. PI3KCA activation either by mutation or gene amplification initiates a signal transduction pathway that promotes growth, metabolism, and survival in cancer cells 
. The putative ~900 bp PIK3CA
promoter carries several important binding sites for p53, NF-κβ, HIF, and AP1 transcription factors 
. While direct binding of p53 attenuates the PIK3CA
promoter activity, inhibition of NF-kβ degradation or treatment with TNF-α results in moderate induction. However, none of these studies have attempted to find the effect of a chemotherapeutic drug on this promoter.
Non-invasive imaging of molecular events in small animals has become a standard practice to evaluate new drugs. Reporter genes or combination of reporter genes which can be used with multiple imaging devices add the advantage of collecting multiple signals from deep inside the body, with higher sensitivity and specificity over reporter genes suitable for only a single imaging modality 
. Thus fusion reporter genes have gained popularity in preclinical imaging. Over the years we have built a small library of fusion reporter vectors and have been applying to monitor tumor metastasis, cell/stem cell trafficking, stem cell therapy, and other areas 
. These fusion vectors comprise of a bioluminescent (either fluc
and their mutants), a fluorescent (either gfp
or red fluorescent proteins and their mutants) and a PET reporter (sr39
mutant thymidine kinase or wild type thymidine kinase) gene joined by small peptide linkers 
. Our previous fusion reporters using a monomeric red fluorescent protein1 emitting light at 608 nm wavelength have suffered at sensitivity due to poor quantum yield and low photostability of the protein. Recently Tsien’s group at UCSD developed several mutant red fluorescent proteins of which tdTomato is the most optimal for in vivo
imaging. Even a low number of breast cancer cells expressing tdTomato can be imaged noninvasively from living mice including metastasis to lymph nodes 
. The mCherry protein though having lower quantum yield has excitation spectra at 613 nm, a far red region optimal for in vivo
imaging. When both these RFP mutants (tdTomato and mCherry) were tried as triple fusion partners, only tdTomato fusions could retain significantly higher fluorescence activity 
. Among the bi-fusions and triple fusions carrying tdTomato, the bi-fusions are the brightest.
In parallel to improvement of fluorescent proteins, the luciferase genes were also attempted for enhancement of light output by mutagenesis, deletion of cryptic transcription factor binding sites and codon optimization for improved mammalian expression. The optimized version of fluc (fluc2) from Promega is able to generate 10-fold higher signals than fluc gene and this fluc2 was used to generate the third generation fusion reporters by replacing the mutated thermostable fluc (mtfl). Interestingly, the newly developed fluc2 containing fusion reporter (fluc2-tdt) show higher fluorescence along with higher luciferase activity than the previous fusions. This apparent increase in overall fluorescent activity after introduction of fluc2 protein maybe due to the change in the quaternary structure resulting in better exposure of the flurophores present in the fluorescent proteins.
To understand the regulation of PIK3CA
promoter in ovarian cancer cells, we utilized this optimized fluc2-tdt
fusion reporter to monitor the effects of cisplatin and paclitaxel from intact cells to living animals. To our best of knowledge this is the first report of understanding the kinetics of drug induced PIK3CA
promoter modulation. Our newly constructed PIK3CA (PIK3CA-fluc2-tdt)
sensor exhibited significant attenuation after treatment with three chemotherapeutic drugs (cisplatin, paclitaxel and adriamycin) commonly used for ovarian cancer patients in two different cancer cells. The similar treatments did not affect the TK
(Thymidine kinase) promoter in PA1 and A2780 cells. The level of attenuation varied between the cell lines with PA1 cells being more sensitive (2.3 fold reduction in promoter activity) than A2780 cells (1.3 fold) at same concentrations of cisplatin or paclitaxel (5 µg/ml) reflecting their respective clinical behaviour. All these drugs are known to induce cell death directly or indirectly via p53 mediated apoptotic pathways 
. Reduction in promoter activity but no detectable variation in the endogenous p110α level after cisplatin or paclitaxel treatments indicates the strength of fluc2-tdt
fusion reporter and reporter assay technique in measuring subtle changes of PIK3CA
at molecular level. The endogenous p53 protein level, however, was significantly induced by the drugs. Activation of p53 by these drugs thereby leads to increased binding of p53 to the PIK3CA
promoter and its suppression. This drug mediated attenuation of PIK3CA
activity due to increased binding of activated p53 was not detected in a p53 deficient EOC (SKOV3) cell line. Both cisplatin and paclitaxel with increasing concentrations were not able to attenuate the PIK3CA
promoter in these cells. The in vivo
imaging kinetics showed a decrease in bioluminescence signal in both PA1 and A2780 tumors (expressing PIK3CA-fluc2-tdt
) after cisplatin treatment. While a single treatment of cisplatin caused measurable reduction in luminescence activity in PA1 tumors at 14th
day, it did not effectively reduce the PIK3CA promoter activity in A2780 tumors. Two successive treatments were required to achieve significant reduction in luminescence activity in A2780 tumors. This differential effect of cisplatin on two different tumor types correlates well with their origin as ovarian germline tumors are known to be more sensitive to cisplatin in comparison to epithelial ovarian tumors. In corroboration with the in vitro
results, cisplatin treatment in vivo
also did not reduce the bioluminescence signals of SKOV3 tumor xenografts stably expressing the PIK3CA sensor indicating that presence of p53 protein is essential for PIK3CA
regulation in ovarian cancer.
Surprisingly, sequential deletion of the p53 binding sites exhibited a gradual increase in the normal promoter activity indicating a temporary relief of p53 mediated suppression. Cisplatin induced attenuation of these mutant promoters were abolished. Inability to down regulate the mutant PIK3CA promoter by cisplatin was also reflected through non-invasive imaging of tumor xenografts stably expressing MPFT3, the promoter carrying three mutated p53 binding sites. Surprisingly, MPFT4 carrying mutations at all the four p53 binding sites showed an overall attenuation which might occur due to destabilization of co-operative bindings of other transcription factors required for PIK3CA expression. Indeed after careful analysis of PIK3CA promoter, site 2 was found to contain overlapping binding sites for NF-kβ and Hypoxia Inducible Factor ancillary sequence (). We are currently analyzing the role of these two factors in regulation of this MPFT-4 promoter. The PIK3CA pathway is one of the most crucial cellular defence mechanisms and therefore requires tight regulation at transcriptional and translational level. These newly developed PIK3CA-fluc2-tdt and the mutant reporter sensors could act as screening tools for potential new drugs. The power of these vectors from translating such information from single cells to the organism level will facilitate wide application in functional assessment of PIK3CA pathway for future therapeutic evaluation.