pancreatic cancer is the eighth most common cause of death from cancer in the world , with a very poor prognosis and a 5-year survival rate of about 3% [ 1 , 2 ] . due to late diagnosis because of the lack of marked symptoms , less than 20% of the patients are candidates for surgical resection  the only known possible treatment to cure this disease . most of the patients present unresectable tumors and , because of the natural aggressiveness of the disease , micro- and macro - metastases are frequent in patients . several clinical trials are being conducted combining different chemotherapies and radiotherapies , but none has yet shown more than a 10-month survival result [ 3 , 4 ] . given those facts , our group previously reported the use of dna - based therapy for cancer treatment which drives the expression of diphtheria toxin a chain by the regulatory sequence of the h19 gene only into cancer cells [ 5 , 6 ] . the human h19 gene is mapped at the short arm of chromosome 11 , p15.5 , homologous to a region of murine chromosome 7 . it is an imprinted gene , expressed from the maternal allele in several tissues during embryo development and repressed right after birth . h19 gene was shown to be re - expressed in several cancer tissues derived from those tissues which expressed the gene during embryonic development , such as bladder cancer , hepatocellular carcinoma , adrenocortical tumors , choriocarcinoma , colorectal cancer , ovarian carcinoma , and lung carcinoma . h19 rna shares all known characteristics of messenger rna but has no known protein product . h19 gene was also shown to play an important role in tumor growth ; its expression caused differential expression of other genes . it was demonstrated that oncogenes , tumor suppressor genes , and angiogenesis factors like tnf- and nfb are regulated by the h19 gene . the present work tested the feasibility of using the regulatory sequences of the h19 gene to express the diphtheria toxin a subunit for a dna - based treatment for pancreatic cancer . ish was performed as described by ariel et al . , using the digoxigenin labeled h19 rna probe or by a modification of this method using   h19-lna ( locked nucleic acid ) dig - labeled probe synthesized at exiqon ( see table sa in supplementary mateial available online at doi:10.1155/2007/69141 ) . total rna was extracted from cell lines or frozen tissue , using tri reagent solution , following manufacture 's instructions ( sigma - aldrich israel ltd . , cdna was synthesized starting from 2  g total rna using the m - mlv reverse transcriptase kit following manufacturerr 's instructions ( promega , madison wi , usa ) . pcr reactions were carried out using gotaq green master mix ( promega ) , starting from 2  l cdna in a final volume of 25  l , according to the manufacture instructions , in the presence of 0.1  g/l of each of the forward and the reverse primers . primers designation sequences , and pcr conditions are detailed in supplement table a. the integrity of the cdna was assayed by pcr analysis of the ubiquitous beta actin , which was used as internal control . as negative control for all pcr analysis , the reaction was carried out using water in the absence of cdna . for analysis of hamster h19 gene expression , rt - pcr was performed using 2 different sets of primers designed for mouse h19 gene . the resulting bands in the gel were purified using the wizard sv gel and pcr clean - up system kit , following the manufacture 's instruction . the purified pcr product was sent to the center for genomic technologies , the hebrew university of jerusalem . sequences were verified by blast analysis using two different databases , ensembl ( http://www.ensembl.org/ ) and ncbi ( http://www.ncbi.nlm.nih.gov/ ) , and by sequence alignment using mafft version 6 ( http://align.bmr.kyushu-u.ac.jp/mafft/online/server ) . the different cell lines used in this investigation were chosen because of their differences in the level of h19 gene expression and their response to transfection procedure . human pancreatic adenocarcinoma cell lines crl-1687 , crl-2119 , crl-1997 , and crl-2547 and ductal adenocarcinoma cell line crl-1469 were obtained from the american type culture collection ( atcc ; rockville , md , usa ) and cultured in 90% dmem - f12 medium and 10% fetal bovine serum ( fbs ) . the hamster pancreatic ductal carcinoma cell line pc.1 - 0 was kindly provided by dr . buscail l. ( institut national de la sant et de la recherche mdicale u531 , institut louis bugnard , institut federatif de recherche-31 , centre hospitalier universitaire rangueil , 31403 toulouse , france ) and was cultured in 90% rpmi-1640 medium supplemented with 10% fbs . antibiotic solution ( penicillin ( 180 units / ml ) , streptomycin ( 100  g / ml ) , and amphotericin - b ( 0.2  g / ml ) ) were added to all medium solution . the reporter plasmids and the dta - h19 construct were prepared as previously described [ 5 , 6 ] . a total of 79  10 cells / well were grown overnight in a twelve - well nunc multidish . cells were cotransfected with 2  g of the luc - sv40 reporter vector and increasing concentrations of the dta - h19 vector using the transfectant jetpei ( polyplus , illkirsh , france ) according to manufacturer 's instructions ( n / p = 5 ) . the luciferase activity in the cotransfected cells ( dta - h19 and luc - sv40 vectors ) was compared to that obtained in the cells transfected with luc - sv40 only , in order to determine the cytotoxic effect of the dta - h19 plasmid . a final volume of 100  l of cell suspension was injected subcutaneously into athymic nude mice ( 56 weeks old and 2030  grs ) purchased from harlan ( zeist , the netherlands ) . after tumor reached an approximated size of 3  mm  3  mm , 3 treatments were given , with a 2-day interval between each treatment , by direct injection into the tumor . treatment consisted of 25  g plasmid ( dta - h19 for treated group and luc - h19 for control group ) mixed with the nonviral transfectant polyethylenimine ( polyplus ) ( n / p = 6 ) diluted in 50  l of 5% w / v glucose ( prepared according to manufacturer 's instructions ) . animals were sacrificed 3 days after the third treatment ; tumors were excised for ex vivo measurements and for pathology and molecular studies . the following pancreatic cancer animal model was based on previous studies [ 18 , 19 ] . 56-week - old male syrian golden hamsters ( 7080  grs ) were purchased from harlan and used to generate the orthotopic model . after anesthetization by subcutaneous injection of ketamine and xylazine ( 85  mg / kg and 15  mg / kg , resp . ) , the pancreas was surgically exposed , and 25  l of cell suspension containing 250,000 pc.1 - 0 cells in pbs x1 were injected subcapsularly into the pancreas using a catheter with a 27-gauge needle . tumors reached a treatable size on day 7 , and metastases were visible on day 9 following cell injection . after tumors were generated , the animals bearing the pancreatic tumors were treated at days seven and nine following cell injection . on day eleven the treatments were given under anesthesia , consisting of an intratumoral injection of 50  g of dna vector ( luc - h19 for the control group and dta - h19 for the treated group ) resuspended in 25  l of saline solution . before each treatment and before sacrifice , tumor sizes were measured using a caliber , and abdominal cavity was searched for visible metastases . tumors were excised and their ex vivo dimensions were recorded following sacrifice and tumor measurement in vivo . the tumors were cut in the middle ; one half of each tumor was fixed in formalin , processed , and embedded in paraffin for pathology studies , and the other half was frozen in liquid nitrogen for further molecular analysis . results showed that 65% of the tested samples were positive for h19 expression ( figures 1(a ) and 1(b ) ) . three of the analyzed cell lines ( crl-2547 , crl-1687 , and crl-1469 ) showed high level of h19 expression ( figure 2(b ) , lanes 2 and 3 and figure 2(a ) , lane 2 , resp . ) , whereas the other two cell lines ( crl-2119 and crl-1997 ) showed low levels of h19 expression ( figure 2(b ) , lanes 4 and 5 , resp . ) . h19 expression was also analyzed on pc.1 - 0 cells , a hamster pancreatic carcinoma cell line . since there is no report of the hamster 's h19 gene sequence at the gene databases ensembl and ncbi , primers designed originally for human and murine known h19 sequence were tested to determine h19 rna level in hamster pc.1 - 0 cells . a faint band was visualized when primers designed originally for mouse h19 gene sequence were used ( figure 2(c ) , middle panel , lane 3 ) . previous studies showed that h19 gene expression may be induced by hypoxia and/or serum starvation conditions and that the h19 gene may play a role in tumorogenesis [ 17 , 20 ] . therefore , the level of h19 rna in pc.1 - 0 was determined by rt - pcr extracted from cells cultured under hypoxia conditions for four hours , and also in total rna extracted from tumors generated in athymic nude mice back by injecting 2 * 10 pc.1 - 0 cells ( cells injected were cultured under normal conditions ) . when amplifying total rna from cells cultured under hypoxia , a weak band was visualized using the set of primers h19_mouse ( figure 2(c ) , lane 4 , middle panel ; primers 454  bp supplement table a ) . on the other hand , when rna extracted from the pc.1 - 0 developed tumors was analyzed , strong bands were visualized using the same set of primers ( figure 2(c ) , lane 5 , upper and middle panel ) . blast analysis and sequences alignment showed high homology between the pcr products and the available sequences of mouse and human on gene databases ( data not shown ) . despite the high homology between the sequences , further analysis must be performed in order to assure that the sequence corresponds indeed to the hamster h19 rna . in order to test the in vitro therapeutic potential of dta - h19 plasmid , the human pancreatic carcinoma cells crl-1687 , crl-2119 , crl-1997 , crl-2547 , and crl-1469 and hamster pancreatic ductal carcinoma cell line pc.1 - 0 were cotransfected with 2  g of luc - sv40 at the indicated concentrations of dta - h19 plasmids ( figure 3 ) . luciferase activity was measured 48 hours after transfection , and data was normalized to total protein per well . the toxic effect was calculated as the relative luciferase activity in the cotransfected cells compared to the activity in cells transfected only with the luc - sv40 plasmid . the results showed that the dta - h19 vector was effective in reducing the luciferase activity in all the cell lines and that there is a dose - response effect ( figure 3 ) . exogenous h19 regulatory sequence activity was assayed by expressing the luciferase gene under the control of h19 regulatory sequences ( luc - h19 plasmid ) . results showed that the cytotoxic effect of h19-dta observed in the different cell lines is directly correlated to the luciferase activity expressed from the luc - h19 plasmid ( figure 3 ) . tumors were generated into athymic nude mice back by subcutaneous injection of crl-1469 cells and then treated with dta - h19 ( n = 7 ) or luc - h19 ( n = 8 , control group ) plasmid by direct injection into the tumor . results showed that treatment with dta - h19 resulted in an arrest of tumor growth , leading to a relative maintenance of tumor volume ( figure 4(a ) ) . a significant difference in tumor growth progression between the two groups was found , indicating that the growth of the dta - h19 treated group was arrested ( p - value =  .036 ) . this observation was confirmed by the final ex vivo tumor volume , which showed to be significantly smaller for the dta - h19 group as compared to the control group ( p - value =  .038 ) ( figure 4(b ) ) . similar results were obtained when the model was generated by injecting crl-2547 cells ( p - value <  .1 ) ( figure 4(c ) ) . a hamster model of pancreatic cancer was developed to evaluate the therapeutic efficacy of dta - h19 for this indication . seven days after cell implantation , all the animals presented visible and measurable tumors in the pancreas . the relevance of this orthotopic pancreatic carcinoma animal model was tested by studying the expression of the h19 gene which is a prerequisite to evaluate the therapeutic potential of the toxin expression vector in this animal model . all the analyzed samples from tumors generated in the pancreas showed high h19 expression when tested by rt - pcr ( figures 5(d ) and 5(e ) ) , signifying that our model was suitable for testing the dta - h19 efficacy in vivo . following h19 expression confirmation , histological studies were performed in order to determine if the tumors were compatible with pancreatic carcinoma . all the tumors presented necrotic areas which were detected on the center of each tumor , caused by poor oxygen and nutrients supply ( figure 5(b ) ) . seven days after cell injection , 100% ( n = 16 ) of the animals presented a single tumor in the pancreas . the average size of the tumor was 0.019  cm , and some animals presented possible metastases . the animals were divided into 2 groups , the first was treated with 50  g of dta - h19 plasmid and the second group with luc - h19 plasmid ( control group ) . the treatments were given at days 7 and 9 following tumor cells injection . as expected , dta - h19 treatment was effective in inhibiting the tumor growth compared with the luc - h19 treated tumors . while the control group showed a greater than 200% increment in tumor size , the dta - h19 group showed approximately 100% ( p <  .03 ) ( figure 6(a ) ) . at the end of the experiment , the tumors were excised and their ex vivo size was determined . a significant difference on tumor size was detected when comparing dta - h19 tumors to the control . the ex vivo tumor volume average of the dta - h19 treated group was almost half that of the control group ( p  <  .0075 ) ( figures 6(b ) and 6(c ) ) . the level of dta transcript was monitored by rt - pcr analyses ( figure 7 ) . all the samples showed the presence of dta transcript verifying that the intratumoral injection of the plasmid leads to transfection and expression of the dta gene . in order to test the systemic toxicity of dta - h19 , different organ samples were collected from animals treated with the vector at terminal sacrifice and subjected to histopathology examination . no gross or microscopic significant alterations were noted in the following organs : pancreas , liver , lung , bowel , spleen , heart , kidney , adrenal gland , lymph - node , salivary glands , gall - bladder , urinary - bladder , and brain ( data not shown ) . although treatment with naked dna proved to be sufficient for inhibiting tumor growth progression , we tested if the use of a transfection enhancer reagent such as pei could improve the efficacy results . according to the developed animal model described previously , pc.1 - 0 cells were injected into the pancreas . on day 7 , when tumors were visible , they were treated with 100  l of a complex containing 50  g of dta - h19 plasmid and in vivo pei ( prepared according to manufacturer 's instructions ) . this treatment was repeated on day 9 , and animals were sacrificed on day 11 . the results obtained showed that no significant improvement in the therapeutic effect was found when pei was used as a carrier for delivering the plasmid as compared to that obtained using naked dna ( data not shown ) . as mentioned above , metastases occurrence was also analyzed during the present experiment . metastases were visible mostly on day 9 , after the first treatment , while few animals showed metastases on day 7 . all the animals in the control group showed multiple visible metastases at the end of the experiment , whereas 70% of the animals treated with dta - h19 did not show visible metastases , and the number of metastases present in those animals was significantly lower ( table 1 ) . the present study proposes a successful approach for the differential killing of pancreatic cancer cells , without affecting the surrounding normal cells . this approach consists of a dna - based therapy expressing a toxin under the control of regulatory sequences of a differentially expressed gene , the h19 gene . it was previously demonstrated that the dta - h19 construct was able to kill tumor cells both in vitro and in vivo in animal models for bladder cancer and colorectal liver metastases [ 5 , 6 ] . this toxin vector was used in order to kill pancreatic cancer cells either in culture or in tumors developed in heterotopic and orthotopic animal models of pancreatic cancer . we successfully demonstrated by ish analysis that the h19 gene is expressed in 65% of the analyzed human pancreatic cancer samples ( n = 49 , figure 1 ) , indicating that treatment of human pancreatic cancer with the dta - h19 is very likely to be effective in this disease . promoter activity was analyzed in all human and hamster cell lines using a plasmid carrying the luciferase gene under the control of h19 regulatory sequence . a relatively high activity of the regulatory sequences was detected in all the tested cell lines , and this activity was correlated with the cytotoxic effect observed when using these sequences for driving the expression of the diphtheria toxin a chain ( figure 3 ) . the effect of the dta - h19 plasmid cotransfected into cells with a second plasmid which encodes the luciferase gene under the control of the sv40 regulatory sequence caused inhibition of the luciferase activity at a dose - response manner in all the studied cell lines ( figure 3 ) . the highest amount of the dta - h19 plasmid used in cotransfection with luc - sv40 construct was about 40 times lower than that of the luc - sv40 plasmid . therefore , the observed effect is more likely due to the cytotoxic effect of the dta - h19 plasmid expression and not the result of competition between the two plasmids for the entry into the cell . these results lead us to test the toxic effect of the plasmid in vivo . we tested the cytotoxic effect of the therapeutic vector both in a heterotopic and an orthotopic model for pancreatic cancer which are complementary and allow the study of tumors with distinctive histopathologies . the results obtained in the heterotopic model using crl-1469 cells showed a significant difference in tgp ( p  =  .036 ) between the dta - h19 treated group and the control group . these results were confirmed by the reduction in tumor size ex vivo , where the average tumor size of the dta - h19 group was almost 1/3 the size of the control group ( p =  .037 ) ( figure 4 ) . furthermore , when using a different cell line to generate the tumors , crl-2547 cells , similar results were obtained , corroborating that the treatment with dta - h19 was effective in arresting tumor growth . the fact that all the animals developed a similar size of tumors after pc.1 - 0 cell injection ensured its reproducibility . h19 expression was previously determined in the developed tumors by rt - pcr analyses in order to validate the use of this animal model in evaluating the potential therapeutic effect of the toxin vector . high levels of h19 gene expression were detected in the developed tumors even though no expression was detected in cultured cells ( figure 5 ) , which is in agreement with previous observations establishing that h19 gene expression may be induced by hypoxia and/or serum starvation conditions developed following injection of tumor cells into the animal tissue . animals presenting pancreatic tumors were then treated with either dta - h19 plasmid or the control plasmid ( luc - h19 ) administrated twice by direct injection into the tumor . we demonstrated that although the treatment consisted of an injection of naked dna into the tumor , the vector indeed entered the cells and the h19 sequence was effective in leading to the dt - a gene expression ( figure 7 ) . it was previously observed that no significant difference in efficacy was found when comparing the use of naked dna to dna - pei when administrated by direct injection into the tumor . the activity of the h19 promoter was demonstrated in the human and hamster pancreatic tumor cells tested , leading to dta expression ( figures 2 and 3 ) . therefore , inhibition of the growth of dta - h19 treated tumors in the hamster results from the action of the dta produced ( figure 6(a ) ) . intratumoral injection of the toxin vector under the control of the h19 regulatory sequences induced a 50% decrease in the median tumor volume as compared to that of the tumor treated with the luc - h19 vector ( figures 6(b ) and 6(c ) ) . although the tumors were not completely ablated , the treatment with the toxin vector significantly reduced the growth rate of the dta - h19 treated tumors as compared to the control group . we observed that after dta - h19 treatment , 67% of the treated animals did not present visible metastases . the number of metastases found in the rest of the treated group ( 33% ) was significantly lower and more localized than those of the control group . these results indicate that the lack of visible metastases in 2/3 of the animals is related to the reduction of tumor progression in the dta - h19 treated animals . metastases developed in the toxin - treated group may have been already present before the first treatment , as micrometastases , and therefore their growth was not completely prevented by the intratumoral treatment . the h19 promoter is known to be differentially activated in various tumor types and to show none or undetectable activity in normal tissue [ 5 , 6 ] . the presented results lead us to advocate a new dna - based treatment for pancreatic cancer , inducing cytotoxic effect in cancer cells by intratumoral injection of a naked dna vector containing the dt - a gene under the control of h19 regulatory sequences for human pancreatic cancer .
pancreatic cancer is the eighth most common cause of death from cancer in the world , for which palliative treatments are not effective and frequently accompanied by severe side effects . 
 we propose a dna - based therapy for pancreatic cancer using a nonviral vector , expressing the diphtheria toxin a chain under the control of the h19 gene regulatory sequences . 
 the h19 gene is an oncofetal rna expressed during embryo development and in several types of cancer . 
 we tested the expression of h19 gene in patients , and found that 65% of human pancreatic tumors analyzed showed moderated to strong expression of the gene . in vitro experiments showed that the vector was effective in reducing luciferase protein activity on pancreatic carcinoma cell lines . 
 in vivo experiment results revealed tumor growth arrest in different animal models for pancreatic cancer . 
 differences in tumor size between control and treated groups reached a 75% in the heterotopic model ( p = .037 ) and 50% in the orthotopic model ( p = .007 ) . 
 in addition , no visible metastases were found in the treated group of the orthotopic model . 
 these results indicate that the treatment with the vector dta - h19 might be a viable new therapeutic option for patients with unresectable pancreatic cancer .