The role of hypoxia in the regulation of membrane transporters

ATP binding cassette (ABC) and solute carrier (SLC) transporters are responsible for the majority of the transcellular movement of various substrates, including drugs, among epithelial cells. Despite the well characterized regulation of influx (SLC) and efflux (ABC) transporters by endogenous mediators, such as inflammatory cytokines, little is known about how changes in oxygen levels may affect expression of these transporters. In this study we showed that the expression of SLC22A4, SLC22A5, SLC22A1, SLC02B1, SLC10A2, ABCC2 and ABCC3 transporters is upregulated by hypoxia in HT29 colon carcinoma cells, but not in HepG2 hepatocarcinoma cells. Moreover, OCTN1 (SLC22A4), OCT1 (SLC22A1) and OATP-B (SLC02B1) transporter expression is also induced by inflammatory cytokines but in a smaller extent than in hypoxia. Furthermore our experiments indicate that there is no cross talk between HIF-1 and NF-κB pathways in HT-29 cells, but these two pathways act simultaneously activating common genes, such as, some SLC and ABC transporters. Our preliminary results from studies with an in vivo murine model of colitis, suggest that HIF-1is stabilized and OCTN1 is strongly induced during severe inflammation, which can be relevant for a recovery from the inflammatory process. We have also been interested in the distribution of HIF-1α variants among different ethnic groups as well as their contribution for cancer risk. Thus, we have demonstrated that there is an ethnicity-related variation in the frequency of the C1772T (P582S) single nucleotide polymorphism (SNP) in the HIF-1α gene. Furthermore, we performed a case-control study in a breast cancer population and our results suggest that there is no association between this SNP or the rare G1790A (A588T) SNP and the incidence of breast cancer. Taken together, the results obtained in this study contribute to a better knowledge of drug influx and efflux during hypoxia and inflammation as well as to the understanding of the pharmacogenetic variability of the HIF-1.

Development of a Piggybac based direct reprogramming system for derivation of integration free induced pluripotent stem cells

Induced pluripotent stem cells (iPSc) have great potential for applications in regenerative medicine, disease modeling and basic research. Several methods have been developed for their derivation. The original method of Takahashi and Yamanaka involved the use of retroviral vectors which result in insertional mutagenesis, presence in the genome of potential oncogenes and effects of residual transgene expression on differentiation bias of each particular iPSc line. Other methods have been developed, using different viral vectors (adenovirus and Sendai virus), transient plasmid transfection, mRNA transduction, protein transduction and use of small molecules. However, these methods suffer from low efficiencies; can be extremely labor intensive, or both. An additional method makes use of the piggybac transposon, which has the advantage of inserting its payload into the host genome and being perfectly excised upon re-expression of the transposon transposase. Briefly, a policistronic cassette expressing Oct4, Sox2, Klf4 and C-Myc flanked by piggybac terminal repeats is delivered to the cells along with a plasmid transiently expressing piggybac transposase. Once reprogramming occurs, the cells are re-transfected with transposase and subclones free of tranposon integrations screened for. The procedure is therefore very labor intensive, requiring multiple manipulations and successive rounds of cloning and screening. The original method for reprogramming with the the PiggyBac transposon was created by Woltjen et al in 2009 (schematized here) and describes a process with which it is possible to obtain insert-free iPSc. Insert-free iPSc enables the establishment of better cellular models of iPS and adds a new level of security to the use of these cells in regenerative medicine. Due to the fact that it was based on several low efficiency steps, the overall efficiency of the method is very low (<1%). Moreover, the stochastic transfection, integration, excision and the inexistence of an active way of selection leaves this method in need of extensive characterization and screening of the final clones. In this work we aime to develop a non-integrative iPSc derivation system in which integration and excision of the transgenes can be controlled by simple media manipulations, avoiding labor intensive and potentially mutagenic procedures. To reach our goal we developed a two vector system which is simultaneously delivered to original population of fibroblasts. The first vector, Remo I, carries the reprogramming cassette and GFP under the regulation of a constitutive promoter (CAG). The second vector, Eneas, carries the piggybac transposase associated with an estrogen receptor fragment (ERT2), regulated in a TET-OFF fashion, and its equivalent reverse trans-activator associated with a positive-negative selection cassette under a constitutive promoter. We tested its functionality in HEK 293T cells. The protocol is divided in two the following steps: 1) Obtaining acceptable transfection efficiency into human fibroblasts. 2) Testing the functionality of the construct 3) Determining the ideal concentration of DOX for repressing mPB-ERT2 expression 4) Determining the ideal concentration of TM for transposition into the genome 5) Determining the ideal Windows of no DOX/TM pulse for transposition into the genome 6) 3, 4 and 5) for transposition out of the genome 7) Determination of the ideal concentration of GCV for negative selection We successfully demonstrated that ENEAS behaved as expected in terms of DOX regulation of the expression of mPB-ERT2. We also demonstrated that by delivering the plasmid into 293T HEK cells and manipulating the levels of DOX and TM in the medium, we could obtain puromycin resistant lines. The number of puromycin resistant colonies obtained was significantly higher when DOX as absent, suggesting that the colonies resulted from transposition events. Presence of TM added an extra layer of regulation, albeit weaker. Our PCR analysis, while not a clean as would be desired, suggested that transposition was indeed occurring, although a background level of random integration could not be ruled out. Finally, our attempt to determine whether we could use GVC to select clones that had successfully mobilized PB out of the genome was unsuccessful. Unexpectedly, 293T HEK cells that had been transfected with ENEAS and selected for puromycin resistance were insensitive to GCV.