Patent Application: US-92091006-A

Abstract:
prostaglandin - endoperoxide h synthase converts arachidonic acid to prostaglandin h 2 . pghs - 2 is an inducible gene product undetectable in most normal human tissues , but abundant in cancer cells . the present invention exploits a previously undisclosed transcriptional function of pghs - 2 distinct from its well - established enzymatic role to identify potential therapeutic agents useful in treating cancer . the method comprises a dna binding assay in which pghs - 2 protein binding to the c / ebp , cre and nf - κb regions of the pghs - 2 promoter in the presence and absence of test compounds is evaluated to identify inhibitors of pghs - 2 transactivation activity .

Description:
previous studies of subcellular localization of pghs isoforms have shown that pghs - 1 and pghs - 2 are both found in the endoplasmic reticulum and at the nuclear envelope of fibroblasts and macrophages and that there is no substantial difference in the subcellular distribution of the two isozymes ( morita et al ., 1995 ; smith et al ., 1981 ; rollins et al ., 1980 ; kujubu et al ., 1993 ). in the current studies , we compared the distributions of pghs - 1 and pghs - 2 in human prostate ( dui45 ) and head and neck ( um - scc - 22b ) cancer cells . shown in fig1 are confocal photomicrographs of cancer cells stained by indirect immunofluorescence , using primary antibodies specific for either pghs - 1 or pghs - 2 . pghs - 1 immunoreactivity was evenly distributed in peripheral cytoplasm . pghs - 1 is known to be present in the endoplasmic reticulum of many cells and it is likely that the fluorescent staining of pghs - 1 we observed largely reflects localization of the enzyme in endoplasmic reticulum . in contrast to pghs - 1 , pghs - 2 in cancer cells was concentrated at the nuclear envelope and was present in abundant quantities within the cell nucleus ( fig1 ). results from the cancer cell lines were compared with those from endothelial cells ( ecy 304 ). only trace pghs - 2 immunoreactivity appeared in cytoplasm of ecy 304 cells and there was no pghs - 2 staining at the nuclear envelope or inside the nuclei of these cells . as shown in fig1 b , the fluorescence staining of pghs - 2 ( green ) overlaps with nuclear staining ( red ). on the other hand , the staining of pghs - 1 in um - scc - 22b cells did not overlap with nuclear staining . relationship of high levels of pghs - 2 protein and mrna in cancer cell lines to elevated nf - κb , mapk ( erk1i2 ) and p38 activity western blots of nucleoproteins obtained by fractionation of du145 and um - scc - 22b cells showed that pghs - 2 protein was recovered in the nuclear fractions of these cells , more so in the um - scc - 22b cells than in du145 cells ( fig2 a ). pghs - 2 mrna levels were also found to be readily detectable in du145 and um - scc - 22b cancer cells ( fig2 b ). pghs - 2 protein and mrna levels were undetectable in untreated ecy 304 cells . it should be noted that these findings were made in cells unexposed to cytokines or growth factors that can influence cellular pghs - 2 levels and cause nuclear translocation of the enzyme ( liou et al ., 2001 ). pghs - 1 protein was abundant in the head and neck cancer cells ( um - scc - 22b ), as well as in du145 prostate cancer and ecy 304 endothelial cells ( fig2 a ). phorbol - myristate acetate ( pma ) in short - term studies activates protein kinase c ( pkc ) and rapidly induces pghs - 2 gene transcription ( pang et al ., 2002 ). in the three cell lines studied here , abundance of pghs - 2 mrna and of pghs - 2 protein in nuclei was increased by treatment of cells with pma ( fig2 a and b ), with most marked changes seen in the um - scc - 22b cells . cytosolic pghs - 2 also increased with pma treatment ( fig2 a ). as expected , incubation of cancer cell lines with pma ( 100 nm ) for 12 h had no effect on abundance of pghs - 1 . evidence developed elsewhere indicates that mapk , p38 and nf - κb play important roles in up - regulation of pghs - 2 in response to cytokines and growth factors in normal cells ( goppelt - struebe et al ., 2000 ; wang and tai , 1998 ). we therefore studied whether mapk , p38 and nf - κb were implicated in the constitutive expression of pghs - 2 in cancer cells . when confluent um - scc - 22b cells were treated for 12 h with pd98059 ( 10 μm ), an inhibitor of the mapk cascade at mapk kinase ( mek ), pghs - 2 protein decreased to very low levels . sb23058 , a p38 kinase pathway inhibitor , and pyrrolidine dithiocarbamate ( pdtc ), an inhibitor of nf - κ b , minimally inhibited pghs - 2 expression in um - scc - 22b cells ( fig3 a ). although pkc is integral to constitutive expression of pghs - 2 in the tumor cell lines studied here , and pma is shown to increase pghs - 2 levels in this figure , these results indicate that there are multiple intracellular signaling pathways involved in constitutive expression of pghs - 2 in um scc - 22b cells . we transfected um - scc - 22b cells with dominant negative erk1 and / or erk2 expression plasmids . individually , dnerk1 or dnerk2 had little effect on pghs - 2 expression ( fig3 b ). as expected , dner1 and 2 together inhibited kinase activity of both isoforms leading to decreased pghs - 2 expression , although phosphorylation of the mutant erks was seen ( fig3 b ). this latter response has previously been described by robbins et al . ( 1992 ). this set of results suggests that constitutive expression of pghs - 2 in prostate cancer cells is in part dependent on the mapk and nf - kb signal transduction pathways . prostate and head and neck cancer cells produced levels of pge 2 ( 12 . 7 ± 1 . 3 ng / ml , and 14 . 3 ± 1 . 5 ng / ml [ mean ± sd ], respectively ) that were similar to those of noncancerous endothelial cells ( ecv 304 , 10 . 1 ± 1 . 2 ng / ml ) ( fig4 ). pma ( 100 nm ) has been shown elsewhere to enhance pge 2 production in a number of cell lines ( habib et al ., 1995 ; molina - holgado et al ., 2000 ). in the present studies , however , pma did not significantly increase pge 2 production in either du145 or um - scc - 22b cells ( fig4 ). pd98059 decreased pge 2 production in cancer cells to basal levels , as did indomethacin , a non - specific inhibitor of pghs - 1 and pghs - 2 , and ns398 , a selective inhibitor of pghs - 2 . although pma - induced increased expression of pghs - 2 did not result in significantly higher levels of pge 2 production in du145 and um - scc - 22b cells , pge 2 production was subject to down - regulation by conventional inhibitors of pghs - 2 enzyme activity . association of nuclear pghs - 2 with other transcriptional activators in cancer cell lines to investigate whether pghs - 2 associates with transcriptionally - active nucleoproteins , double antibody staining experiments were carried out in quiescent um - scc - 22b cells , and demonstrated that c - fos and pghs - 2 co - localized in cell nuclei ( fig5 ). co - immunoprecipitation studies also indicated that nuclear pghs - 2 associates with mdm2 and with the nuclear coactivator protein p300 . mdm2 is a determinant of the turnover of p53 and , when bound to p53 , facilitates nuclear export of the oncogene suppressor protein ( momand et al ., 1992 ). the treatment of um - scc - 22b cells with pma , 100 nm , caused a marked increase in nuclear association of pghs - 2 with c - fos ( fig5 ), but little increase in association with mdm2 or p300 . pghs - 2 binding to pghs - 2 promoter and to other genes that modulate cell division to assess pghs - 2 promoter activity in um - scc - 22b cells , the proximal − 831 /+ 123 base pairs of the human pghs - 2 promoter were fused to a luciferase reporter ( pghs - 2 luc ) ( cao et al ., 1999 ). this pghs - 2 reporter or sv40 luc ( pgl2 promoter ) were transfected into near confluent um - scc - 22b tumor cells . as fig6 indicates , pghs - 2 promoter activity was readily detected in quiescent um - scc - 22b cells and was similar to that obtained with transfection of the sv 40 promoter and luciferase reporter . this result is consistent with the constitutive expression of pghs - 2 found in um - scc - 22b cells . while pma ( 100 μm ) only negligibly increased pghs - 2 promoter activity , treatment of cells with pd98059 , pdtc , or sb203580 inhibited pghs - 2 promoter activity . these results implicate the mapk signal transduction pathway and nf - κb in constitutive expression of pghs - 2 . in chip experiments in prostate cancer cells , using monoclonal anti - pghs - 2 , we compared the amount of immunoprecipitable pghs - 2 promoter sequence with the amount of input dna for each sample ; a pghs - 2 orf primer served as a negative control . we found that pghs - 2 bound constitutively to the pghs - 2 promoter , and pma treatment of cells resulted in minimal increases in binding of pghs - 2 to the promoter ( fig7 a ). pghs - 2 also bound to the promoter region of mdm2 . binding of pghs - 2 to both promoters was inhibited by pdtc and pd98059 , suggesting dependence of these effects on nf - κb and mapk activation . we next defined which domains of the pghs - 2 promoter were capable of binding pghs - 2 protein . we used a dna - protein binding assay in which a biotinylated short pghs - 2 promoter oligonucleotide probe was incubated with streptavidin - coated beads previously interacted with nuclear extract proteins . as shown in fig7 b , pghs - 2 bound to c / ebp , cre , and two nf - κ b sites on the pghs - 2 promoter . there was only weak binding to the dna sequence between the two nf - κ b sites in the pghs - 2 promoter region . we also determined whether binding of pghs - 2 to its own promoter had any effect on transcriptional activity of the promoter . a pghs - 2 promoter - driven luciferase plasmid was co - transfected with pghs - 2 expression plasmids into 293t cells . these cells were used because of their low endogenous content of pghs - 2 , as alterations in transcriptional activity of pghs - 2 could not be assessed in cell lines in which the gene is constitutively expressed . when the amount of pghs - 2 protein was increased in 293t cells at 24 h after transfection of pghs - 2 expression plasmids , luciferase activity was increased ( fig8 ). thus , the binding of pghs - 2 to its own promoter provided positive feedback on pghs - 2 gene transcription . in contrast , transfection of the empty expression vector did not affect pghs - 2 promoter activity . much recent attention has been directed at pghs enzymes in cancer cells ( zimmermann et al ., 1999 ; yoshimura et al ., 2000 ; yu et al ., 2002 ; xiong et al ., 2003 ). located in the cytoplasm , particularly in the perinuclear region and in endoplasmic reticulum , these enzymes are involved in prostanoid production that by several mechanisms can support tumor growth ( gately , 2000 ; hendrickx et al ., 2003 ; pai et al ., 2002 ). constitutively - expressed pghs - 1 has been assumed to be particularly relevant in this regard , because pghs - 2 is seen as an inducible enzyme with important roles in inflammatory responses in which noncancerous cells participate ( cao et al ., 2003 ; cao and smith , 1999 ; han et al ., 2002 ). evidence from several laboratories , however , indicates that pghs - 2 may be constitutively expressed in cancer cells ( howe et al ., 2001 ; hanahan and weinberg , 2000 ). indeed , pghs - 2 was found in the current studies to be constitutively expressed in human prostate and head and neck cancer cell lines , but not in an immortalized noncancerous endothelial cell line ( ecv 304 ). the possible significance of such findings is suggested by observations that nonsteroidal anti - inflammatory drugs ( nsaids ) that inhibit the enzymatic activity of both pghs - 1 and pghs - 2 can serve a protective function against development of several tumors ( arber and dubois , 1999 ; moran em , 2002 ; oshima et al ., 1996 ; davies gl , 2003 ; piazuelo et al ., 2003 ; parfenova et al ., 2001 ). confocal microsocopy in the present studies revealed that pghs - 2 protein was not only found in a perinuclear location , but was abundant within the nucleus of these cancer cell lines . although pghs - 2 protein has been reported to be present in the nucleus of noncancerous cells , this translocation of pghs - 2 has been the result of cytokine treatment or other manipulations and has not been constitutive ( parfenova et al ., 2001 ; marvin et al ., 2000 ). our finding of nuclear pghs - 2 was confirmed by two techniques ( confocal microscopy , cell fractionation ) and caused us to investigate the possibility that pghs - 2 has a transcriptional function distinct from its well - described enzymatic role . chromatin immunoprecipitation studies revealed that pghs - 2 bound to the promoter region of pghs - 2 in human prostate and head and neck cancer cell lines . we identified the several specific domains of the pghs - 2 promoter region in which dna - pghs - 2 protein interactions occurred . in transcriptional studies carried out in 293t cells into which the relevant constructs were transfected , we showed that pghs - 2 protein was capable of directly regulating the transcription of its own gene . extrapolating these results to cancer cells , we propose that a mechanism of the ‘ constitutive ’ expression of the pghs - 2 gene in cancer cells may be the transactivator function of its own gene product . several lines of evidence have implicated nf - kb , mitogen - activated protein kinase , c / ebp and cre involvement in upregulation of pghs - 2 proteins ( zhu et al ., 2002 ). using a protein - dna binding assay , we found that pghs - 2 in fact bound strongly to two nf - kb sites and to c / ebp and cre sites of the pghs - 2 promoter region . pghs - 2 bound only weakly to control sequences between two nf - kb sites of the pghs - 2 promoter . we did not determine whether pghs - 2 directly binds to its own promoter or binds in association with other activators . an additional finding was that pghs - 2 protein bound to the mdm2 promoter . if pghs - 2 functions as a transactivator for mdm2 , then this becomes a mechanism by which the cancer cell that constitutively expresses pghs - 2 may regulate cellular levels of the oncogene suppressor protein , p53 . mdm2 binds to the transcriptional activation domain of p53 in the nucleus and causes translocation of the protein to the cytoplasm where p53 is rapidly degraded ( momand et al ., 1992 ). the present studies also revealed that pghs - 2 complexed with several nucleoproteins . these proteins include c - fos , which , when bound to c - jun , is a transactivator ( ap - 1 ) ( jang et al ., 1996 ). ap - 1 is involved in the induction of the pghs - 2 gene ( subbaramaiah et al ., 2002 ; guo et al ., 2001 ), but a direct interaction of ap - 1 and pghs - 2 has not previously been described . this interaction raises the possibility , as yet unstudied , that pghs - 2 may be transcriptionally active as a heterodimer with c - fos . the co - activator protein p300 was also found to complex with pghs - 2 . this was not surprising since p300 has intrinsic transacetylase activity that has been shown to be relevant to transcription of the pghs - 2 gene ( deng et al ., 2003 ). finally , nuclear pghs - 2 bound to mdm2 protein , as well as to the mdm2 promoter . while the significance of this finding is not yet clear , the possibility exists that , like p53 , pghs - 2 is subject to nuclear export and turnover that is directed by mdm2 . the human head and neck cancer cell line um - scc - 22b was maintained in dmem supplemented with 10 % fetal bovine serum ( fbs ). du145 human prostate cancer cells were cultured in eagle &# 39 ; s minimal essential medium supplemented with 10 % fbs . ecv 304 endothelial cells were grown in m199 medium that contained 10 % fbs . 293t cells were grown to 60 % confluence in dmem with 5 % fbs . all cell cultures were maintained in a 5 % c02 / 95 % 02 incubator at 37 ° c . signal transduction reagents used in cell treatments included pd98059 and sb203580 , obtained from calbiochem , la jolla , calif ., and pdtc and pma , obtained from sigma - aldrich , st . louis , mo . fractionation of cells in a microfuge and preparation of nucleoproteins was by a previously reported method ( lin et al ., 2002 ). nuclear extracts were prepared by resuspension of the crude nuclear fraction in high salt buffer ( 420 mn nacl , 20 % glycerol ) at 4 ° c . with rocking for 1 h . the supernatants were then collected after subsequent centrifugation at 4 ° c . and 13 , 000 rpm for 10 min . the techniques used were those previously described by our laboratory ( lin et al ., 1999 ). in brief , nucleoproteins were separated on discontinuous sds - page ( 9 % gels ) and then transferred by electroblotting to immobilon membranes ( millipore , bedford , mass .). after blocking with 5 % milk in tris - buffered saline containing 0 . 1 % tween , the membranes were incubated with various antibodies overnight . secondary antibodies were either goat anti - rabbit igg ( 1 : 1000 ) or rabbit anti - mouse igg ( 1 : 1000 ) ( dako , carpenteria , calif . ), depending upon the origin of the primary antibody . immunoreactive proteins were detected by chemiluminescence . 293t cells were transfected with dominant negative plasmids , or with vector alone , using lipofectamine plus ( invitrogen ) according to the supplier &# 39 ; s instructions . aliquots of cells from each treatment group were then exposed to the experimental variables and subsequently harvested for preparation of nuclear proteins . an internal control plasmid , p - galactosidase , was co - transfected in all experiments for normalization of results . ecv 304 , um - scc - 22b and du145 cells were cultured in slide chambers , were washed in phosphate - buffered saline ( pbs ), fixed by incubation for 10 min in 2 % formaldehyde in pbs and then washed twice with pbs . monoclonal antibodies to pghs - 1 , pghs - 2 ( cayman , ann arbor , mich .) or c - fos ( santa cruz , santa cruz , calif .) were diluted 1 : 200 in pbs , then added to the chamber and the samples incubated for 45 min . following washing in pbs , the samples were incubated for 30 min with a 1 : 100 dilution of fitc - conjugated rabbit anti - mouse igg in pbs . a zeiss scanning confocal microscope ( 510ls ) with an argon ion laser as the excitation source was used for microscopy . 25 × and 60 × objectives and a 50 % laser power source were used for subcellular localization of pghs - 1 and pghs - 2 . plasmids containing fragments of the human pghs - 2 promoter were generously provided by dr . s . m . prescott , university of utah , salt lake city , utah ( meade et al ., 1999 ). the plasmid − 1800pgl2 contained − 1840 /+ 123 , and plasmid + 800pgl2 contained the sequence − 831 /+ 123 . psv - f3 - galactosidase was used as a transfection efficiency control . dominant negative plasmids of mapk ( erk1 / 2 ) were a gift of dr . melanie cobb ( university of texas southwestern , dallas , tex .). using lipofectamine plus , 60 % confluent um - scc - 22b cells in 10 cm plates were transfected with approximately 6 ug of plasmids of dominant negative erk1 and erk2 . cultures were incubated at 37 ° c . for 5 h . the medium was replaced with 10 % fetal bovine serum containing medium for 24 h and then with serum - free medium for 24 h . transfected cells were incubated with 100 nm phorbol - 12 - myristate - 13 - acetate ( pma , sigma , st . louis , mo .) for 12 h and subsequently harvested for western blot analysis of pghs - 2 protein levels . chromatin immunoprecipitation was performed as described by luo et al . ( 1998 ) and braunstein et al . ( 1996 ). aliquots of cells ( 6 × 10 6 ) in monolayers were treated with 1 % formaldehyde for 15 min at room temperature to effect crosslinking . monolayers were then washed twice with pbs . extracts were prepared by scraping cells in 1 ml of buffer ( 150 mm nacl , 1 % np - 40 , 0 . 5 % doc , 0 . 1 % sds , 50 mm tris [ ph 8 . 0 ], 5 mm edta ) that contained the protease inhibitors leupeptin ( 10 μg / ml ) and pepstatin a ( 10 μg / ml ), phosphatase inhibitors ( 50 mm naf and 0 . 2 mm sodium orthovanadate ) and the deacetylase inhibitor , trichostatin a , 5 μm ( calbiochem , san diego , calif .). cell lysates were sonicated to yield chromatin fragments of approximately 600 bp , as assessed by agarose gel electrophoresis . immunoprecipitation was carried out with anti - p53 ( santa cruz , santa cruz , calif . ), anti - phosphoser392 - p53 ( cell signaling , beverly , mass . ), or anti - acetylated p53 ( upstate biotechnology , lake placid , n . y .). for studies of the pghs - 2 promoter , anti - pghs - 2 ( cayman ) was used . primers used in pcr were for promoters of mdm2 [ 5 ′- gga ttgggccggttcagtgg - 3 ′ ( forward ) and 5 ′- ggtct accctccaa - tcgccac - 3 ′ ( reverse )]; pghs - 2 [ 5 ′- ctgttgaaagcaacttagct - 3 ′ ( forward ) and 3 ′- agactgaaa accaagcccat - 3 ′ ( reverse )]. pcr results were resolved by 1 % agarose gel electrophoresis , and gels were stained with ethidium bromide . relative levels of dna were determined with quantifyone software ( bio - rad laboratories , inc ., hercules , calif .). aliquots ( 200 μg ) of nuclear extract proteins containing 6 μg biotinylated pghs - 2 - specific nf - κb , c / ebp , or cre sequences ( invitrogen ) and 60 μl of 4 % streptavidin - coated beads were incubated for 1 hour with shaking . the streptavidin - coated beads , with associated nuclear extract proteins bound to the biotinylated probe , were pelleted and washed three times with cold pbs . proteins bound to the beads were eluted by boiling and were separated on sds - page . western blot analysis was carried out with anti - human pghs - 2 antibody as described above . arber , n ., and dubois , r n . 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