Patent Application: US-201314415064-A

Abstract:
the present invention relates to the construction of a new hypoxia inducible factor responsive reporter gene construct , the genetic constructs and vectors containing the same . further , the present invention relates to a stable cell line comprising the hif responsive reporter construct , as well as methods and uses of the inventive constructs and cell lines to identify modulators of hif activity .

Description:
establishment of this hif cell - based screening assay was achieved by an initial co - transfection of the 4xepo - hre - fluc plasmid and the pcdna3 plasmid ( invitrogen ) carrying an antibiotic resistant gene , into the saos - 2 osteosarcoma cell line ( atcc ). a subpopulation of cells stably transfected with the plasmids was later obtained by the g418 antibiotic selection . single cell clones were isolated from the mass population of stable transfectants using a limited dilution cloning strategy . a single clone with the highest reporter activity was used for a proof - of - concept study using known inhibitors of hif . these inhibitors are the commercially available drugs , bortezomib ( velcade ) and cisplatin . the 4xepo - hre - fluc plasmid construct contained four copies of the hre of the epo gene while the pcdna3 vector carries a neomycin - resistant gene . co - transfection of these plasmids into saos - 2 cells was performed at a 10 : 1 ratio ( 4xepo - hre - fluc : pcdna3 ) using the lipofectamine 2000 ( invitrogen ) fusogen . initially , 8 . 3 × 10 5 saos - 2 cells were seeded in an antibiotic - free dmem with 10 % fbs in a t - 25 flask for 24 hours . on the following day , a plasmid mixture was prepared by adding 11 . 4 μg of 4xepo - hre - fluc and 1 . 14 μg of pcdna3 in 625 μl serum - free and antibiotic - free dmem . at the same time a transfection reagent mixture was prepared by adding 18 . 75 μl of lipofectamine 2000 into 606 . 25 μl serum - free and antibiotic - free dmem . the reagent mixture was gently mixed and incubated at room temperature for 5 minutes . after the incubation , the plasmid mixture and the reagent mixture were combined , vortexed and incubated at room temperature for 20 minutes . while the complexes were incubating , cultured media from the overnight t - 25 flask saos - 2 cells was discarded , and the cells were washed once with 5 ml of serum - and antibiotic - free dmem . at the end of the 20 minutes incubation , the 1250 μl of plasmid - lipofectamine 2000 complexes was added into the cells . the flask was then swirled followed by an addition of 3750 □ l of serum - and antibiotic - free dmem . the cells were incubated for 6 hours in a humidified incubator supplied with 5 % co2 at 37 ° c . five ml of dmem with 10 % serum and antibiotic - free was added into the flask after 6 hours in order to make a final concentration of 5 % reduced - serum dmem . five ml of antibiotic - free dmem with 10 % fbs was then added into the in order to make final concentration of 5 % reduced - serum dmem . selection of a stably - transfected population of saos - 2 cells from the transfected cells above was performed using the g418 antibiotic . after 48 hours of incubation , the transfection media mixture was removed from the cells . they were then washed twice with 1 × pbs followed by the addition of dmem containing 1 mg / ml g418 and 10 % fbs . the cells were continued to be incubated in a humidified incubator supplied with 5 % co2 at 37 ° c . for 3 more days . after the incubation period , spent media was removed and replaced with fresh media containing 1 mg / ml g418 and 10 % fbs was added . this media replacement was done at every 3 days for a period of 3 weeks . after 3 weeks , the surviving cells , which constitute the mass population of stably - transfected , neomycin - resistant parental cells , were propagated further . this parental population was then tested for its ability to upregulate the luciferase reporter protein upon hypoxia induction . to confirm the hif reporter activity of neomycin - resistant parental population , a single luciferase assay was conducted . initially , 2 . 5 × 10 4 of the cells were plated in wells of a 48 - well plates in antibiotic - free dmem with 10 % fbs for 16 hours . one of the plates was then incubated for 24 hours in a normoxic ( 21 % 02 ) gaseous atmosphere in a humidified co2 incubator while the other was incubated in a hypoxic ( 0 . 5 % 02 ) gaseous environment in a hypoxic chamber ( biospherix , usa ) controlled by an oxygen regulator ( proox model 110 , biospherix , usa . after 24 hours of exposure to different oxygen concentrations , bright - glo ™ luciferase assay ( promega , usa ) was conducted to analyse the hif reporter activity . initially , growth media in each well was discarded and the cells were rinsed once with 300 μl of 1 × pbs . room temperature - equilibrated glo lysis buffer ( glb ; 60 μl ) was then added into the wells . plates were then gently rocked on a shaker for 5 min at room temperature . cell lysis was confirmed visually by observing the wells of the 48 - well plate under a light microscope . thirty microliters of the resulting cell lysates were then transferred into individual glass vials . bright - glo ™ assay reagent was then added into each tube at a 1 : 1 ( v / v ) ratio . luminescence intensity was immediately read and recorded by a luminometer ( sirius - 2 , titertek - berthold ). after confirmation of the hif reporter activity in the mass population of the stable parental neomycin - resistant cells , single cell cloning was performed using a limiting serial dilution cloning strategy . the parental cells were seeded in the first row wells ( a1 - a8 ) of a 96 - well plate at 1 . 5 × 10 4 cells for 24 hours . after an overnight incubation , spent media in the wells was pippeted out carefully and the cells were washed with 50 μl 1 × pbs . trypsinization of cells was done by adding 50 μl of 1 × trypsin - edta in each well for 1 minute at 37 ° c . after cells became detached , the content of each well was topped up to 100 μl with dmem supplemented with g 418 and 10 % fbs . one hundred microliters of the same media was pipetted into the remaining 9 rows of empty wells . cell suspension from the first well ( 100 μl ) was then transferred into the adjacent well and the mixture was pipetted up and down . the same volume of cell suspension was then transferred from this well to the next . this process was repeated to obtain a two - fold serial dilution of the cell suspension . all of the cell suspension from the last well ( 200 μl ) was diluted in 10 ml of conditioned dmem supplemented with g 418 and 10 % fbs . the mixture was then inverted up and down to ensure even distribution of cells . this cell suspension ( 100 μl ) was aliquoted into wells of a 96 - well plate . the plate was then incubated in a 45 ° slanted position for 5 hours at 37 ° c . this was done to ensure attachment of single cells at the edge of each respective well . after the incubation each well was examined for the presence of a single attached cell . wells containing single cells were properly labeled . the plate was subjected to further incubation at 37 ° c . for 7 to 14 days . individual cells in the wells were visualized only every 4 days to reduce disturbance of the cell culture media . during visualization , images of the cells were captured . single cells which survived and grew into a colony after the 7 to 14 days incubation were trypsinized and transferred into wells of a 48 - well plate . as the number of cells gradually increased the subculturing was performed into bigger tissue culture containers . parameters for the propagation and upscaling of the cells are summarized in table 1 . each cell clone population was then cryopreserved for long term storage . after pure stable cell clones were obtained , their reporter activity towards hif induction was re - tested using the single luciferase assay as described above . the cells were also confirmed to be free from mycoplasma contamination using a 4 ′- 6 - diamidino - 2 - phenylindole ( dapi ) staining . to determine the optimum cell density to be used in the hif activity assay , 2 . 7 × 10 4 , 3 . 3 × 10 4 and 4 . 0 × 10 4 cells were seeded into wells of a 48 - well plate for 24 hours . the seeded cells were then exposed to either normoxic or hypoxic conditions for 24 hours , as described above . following lysis and mixture with the bright - glo assay reagent , the bioluminescence intensity was analysed as described in the earlier methods . to verify the property of the stable single cell population as an assay system to measure hif activity , a proof of concept study was performed with commercially available and known hif inhibitors . these inhibitors are bortezomib ( velcade ) and cisplatin . treatment of these drugs in certain cancer cells leads to cell death . to confirm that the inhibition of bioluminescence in our cell assay system is due to hif inhibition rather than this cytotoxic effect , we simultaneously determined the viability of cells under various concentrations of each drug used for the inhibition assay . the 3 -( 4 , 5 - dimethylthiazol - 2 - yl )- 2 , 5 - dimethyltetrazolium bromide mtt assay was used . cells were seeded at 1 . 5 × 10 4 cells per well in 96 - well plates for 24 hours followed by treatment with selected concentrations of bortezomib and cisplatin . the cells were then incubated in either normoxic or hypoxic conditions for 24 hours . after the treatment , cell culture media was replaced with fresh serum - free dmem containing 0 . 5 mg / ml mtt . after 4 hours of incubation in a humidified co2 incubator at 37 ° c ., formazan precipitates were dissolved with 100 μl of dmso . the resulting reaction was read using a microplate reader at 570 nm absorbance and 630 nm as the reference wavelength . the half maximal inhibitory concentration ( ic50 ) of each drug towards the cells was determined by plotting graphs using the graphpad prism 5 software . hif inhibitory effects at the various concentrations of drugs tested were also measured using the single luciferase assay system . a correlation between the drugs &# 39 ; effects on cell viability and hif inhibition was then evaluated . saos - 2 osteosarcoma cells , that showed a typical epitheloid morphology ( fig1 ), were co - transfected with the recombinant pluc - mcs carrying the 4xepo - hre ( 4xepo - hre - fluc ) and pcdna3 plasmids , harboring the neomycin resistant gene ( fig2 ). the resulting transfectants were then treated with g418 antibiotics . throughout the treatment , changes in the cell culture were monitored under a light microscope . representative images of the observed morphological changes at selected times during the treatment are shown in fig3 . by day 3 , most of the cells in the culture were rounded up and floating suggesting their sensitivity to the g418 treatment . cells that appear to be resistant to the drug , on the other hand , continued to proliferate . after 20 days of treatment , this resistant population of cells became confluent in the tissue culture flasks . these cells , designated as the ‘ mass population ’, were propagated for further selection and stored for future use . the mass population of cells was evaluated for their ability to respond to hypoxia stimulation by measuring their luciferase expression level . the hypoxic cells were found to be able to produce 90 - fold higher luciferase signal compared to the cells in normoxia ( fig4 ). therefore , we conclude that this mass population retained their 4xepo - hre gene construct , and they are responsive to hypoxia . a limiting serial dilution cloning strategy was then performed on these cells . a number of single cells were obtained . four of them proliferated to form cell colonies . representative images of clone # 3 , at different times of the culture , are shown in fig5 . by day 21 each clone became a confluent population in the culture flask . to ensure that no mycoplasma contamination was introduced during the process , the cells were stained using dapi staining method . results obtained showed that they are free from contamination ( fig6 ). to test the level of hypoxia - responsive luciferase signal of each cell clone , they were subjected to hypoxia exposure . all comparisons were done against their normoxia culture controls . out of the four clones , clone 3 gave the highest luciferase signal , that was 517 - fold higher than the normoxia control ( fig7 ). to the best of our knowledge , this is the highest hypoxia - specific signal that has been obtained thus far . previously , using a vegf promoter construct , woldemichael et al ., ( 2006 ) obtained a 40 - fold signal intensity . using a similar promoter , bo et al ., ( 2008 ) however only managed to obtain a 14 - fold increase in signal . even using a novel hif reporter construct containing tandem repeats of minimum hif binding sequences , zhou et al ., ( 2011 ) obtained just a 100 - fold greater luciferase signal under hypoxia . our clone 1 gave a fold difference comparable to their findings . however , our clones 2 , 3 and 4 all gave a superior sensitivity . this increase in sensitivity will greatly improve the signal to noise ratio of various experimental conditions . microscopic observation of all of the four stable saos - 2 clones showed no morphological differences compared to their parental cells ( fig7 ). high cell density is known to be a factor in increased hif activity in certain cell lines ( kaluz et al ., 2006 ). to test whether cell density affects the signal sensitivity of our clone 3 , cells were cultured at different cell numbers and then exposed to hypoxia . results showed that at densities of 2 . 7 × 10 4 to 4 . 0 × 10 4 , no statistically significant variation was observed in the signals ( fig9 ). hence , we conclude that the hypoxia - responsive signal produced by this stable saos - 2 clone remain consistent at different cell densities . we have also tested signal stability of the clone 3 as the passage numbers increased . thus far , our findings showed that the signal intensity is still stable up to 30 passages ( data not shown ). we will monitor the signal further as the passage numbers increase over time . a proof of concept study was conducted using known inhibitors of hif which are bortezomib and cisplatin . bortezomib , a proteasomal inhibitor , was shown to inhibit transcriptional activity of hif - 1 via specific effect on hif1 - 1α c - terminal activation domain ( kaluz et al ., 2006 ). treatment of bortezomib , at concentrations of up to 500 nm , significantly reduced the hypoxia - induced luciferase signal in our clone 3 cells without affecting cell viability ( fig1 ). at higher concentrations , the inhibition was associated with cell death . in addition to bortezomib , cisplatin was also shown to inhibit hif . the mechanism of inhibition , however , was achieved via a repression of hif1 - 1α protein expression ( duyndam et al ., 2007 ). in the present study , addition of cisplatin at 25 μm concentration led to almost 60 % inhibition of luciferase signals in hypoxic clone 3 . viability of the cells was also reduced . previous studies have shown that cisplatin treatment resulted in hif inhibition and also apoptotic cell death ( tanaka et al ., 2005 ). in summary , our proof of concept using bortezomib and cisplatin showed that the hypoxia - inducible signal in our clone 3 population is responsive to the inhibitory effects of these drugs suggesting the reliability and sensitivity of the assay system . duyndam m c , van berkel m p , dorsman j c , rockx d a , pinedo h m , boven e . cisplatin and doxorubicin repress vascular endothelial growth factor expression and differentially down - regulate hypoxia - inducible factor i activity in human ovarian cancer cells . biochem pharmacol . 2007 74 : 191 - 201 . ek e t , dass c r , choong p f . commonly used mouse models of osteosarcoma . crit rev oncol hematol . 2006 60 : 1 - 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