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Definitions

• This invention is generally in the field of the use of oligonucleotides/DNA fragments for biological and therapeutical applications, and more specifically in the field of nucleic acids that interfere with DNA damage signaling and repair pathways, in particular the non- homologous end joining (NHEJ) pathway of double-stranded break (DSB) repair.
• NHEJ non- homologous end joining pathway of double-stranded break
• the invention relates to nucleic acids useful as tools for triggering cell lethality of tumors submitted to anticancer therapies .
• Ionizing radiations cause directly or indirectly double-strand DNA breaks (DSBs) and trigger cell/tissue death (necrosis or apoptosis) .
• the cytotoxic effect of ionizing radiation forms the basis for radiation therapy, which is widely used in the treatment of human cancer.
• the efficacy of radiation therapy is currently limited by the radiation resistance of certain tumors (for example, glioblastomas) and by the side effects caused by irradiation of nearby normal tissues (for example, in treatment of breast and cervical cancer) .
• chemotherapeutic agents can cause DNA damages, including direct or indirect DSBs.
• chemotherapeutic agents include inhibitors of topoisomerases I or II (camptothecin/topotecan, epirubicin/etoposide) , DNA crosslinkers (cisplatin/carboplatin /oxaliplatin) , DNA alkylating agents (carmustine/dacarbazine) or anti-metabolic agents (5- fluorouracil/ge citabine/capecitabine) , as well as inhibitors of the mitotic spindles (paclitaxel/docetaxel/vinorelbine) .
• genes of particular interest to be targeted with ionizing radiations are those involved in the regulation of radiation-induced lethality mechanisms, such as apoptosis or DNA repair.
• Cell death induced by ionizing radiation depends mostly on the repair of DSBs .
• NHEJ non homologous end-joining
• HR homologous recombination
• DNA-PKsc proteins are important in the repair of radiation- or chemo-induced DNA damage. If damage cannot be repaired on time, cells die. Therefore, they are interesting molecular targets for sensitizing target cells and tissues to radiotherapy and chemotherapy. Many approaches have been conceived and carried out to inhibit these key proteins (Ku70/Ku80, DNA-PKsc, etc.) involved in the NHEJ pathway which is considered as predominant in mammalian cells: 1) Inhibitors of PI3K (phosphatidylinositol-3-kinase) (i.e. DNA-PKsc, PARP-1, ATM, ATR) (Boulton et al . , 2000; Durant & Karran, 2003 ; Willmore et al . ,2004; Vauger et al . , 2004)
• PI3K phosphatidylinositol-3-kinase
• the inventors have found that the tumor sensitivity to direct or indirect DNA damaging anticancer therapies can be enhanced by using chemically modified or not double-stranded nucleic acid molecules, acting as mimetics of broken DNA fragments and recognized as DSB sites induced by the DNA damaging treatments.
• the molecules may have a non replicative structure due said modifications.
• An object of the invention is then to provide such double- stranded nucleic acid fragments, also named "DNA repair induced lethality” (DRIL in short) molecules in the following, capable of enhancing the response of treatment- resistant tumors to radiotherapy and chemotherapy.
• DRIL DNA repair induced lethality
• the invention aims at providing new DRIL molecules to be used in combination with physical and chemical agent (s) which can cause directly or indirectly DSBs of DNA and a method for treating cancer combining the use of said DRIL molecules with anticancer therapies which cause direct or indirect DNA damage.
• Another object of the invention relates to the use of DRIL molecules for making anti-tumoral therapeutic adjuvant for enhancing efficiency of cancer treatment, particularly for highly resistant tumors to radio-and/or chemotherapies.
• the DRIL molecules of the invention are substrates for proteins involved in the NHEJ pathway (sequence -independent pathway)., particularly Ku proteins and comprise a sequence- independent backbone of at least 4-10000 base pairs (bp) , particularly 4-1000 bp.
• the double-stranded DRIL molecules are capable of being uptaken by cell / tissue body into the cell nucleus when used with pharmaceutically acceptable carriers; the free ends of the DRIL molecules are recognizable by the DNA binding proteins involved in double- strand breaks repair and damaging signalling, - the free ends of the DRIL molecules is amenable by said enzymes to be incorporated in the tumoral cell genomic DNA. According to the mechanism of action of DRIL molecules via
• NHEJ pathway their length are not a limitation per se, except for practical considerations, but must include at least 4 bp, more preferably at least 8 bp .
• the DRIL molecules of the invention then comprise
• Particularly preferred DRIL molecules comprise I ⁇ -lOObp, and more advantageously 24-100 bp.
• the DRIL molecules according to the invention have a native phosphodiester backbone or a chemically modified phosphodiester backbone, or another backbone with chemical groups or mixtures of chemical groups, provided the modified oligomers remain substrates for proteins Involved in the NHEJ pathway, particularly Ku proteins,, and DSB damage signalling pathway.
• the chemical modifications are intended to confer chemical stability to DRIL molecules and/or to prevent them for further replication (potential cause of mutagenic effect) upon their- genomic integration if it occurs. ⁇
• sugar mimetics such as 2 ' -O-alkylribose, 2 ' -0-alkyl-C4' branched ribose, cyclobutyls or other carbocyclics or hexitol in place of the pentofuranosyl group.
• They can be made linear or made of hairpin double-stranded nucleic acids in which the loop can be nucleic acids, or other chemical groups known by skilled person, preferably a linker such as hexaethyleneglycol or tetradeoxytliymidylate (T4) .
• a linker such as hexaethyleneglycol or tetradeoxytliymidylate (T4) .
• DRIL molecules of the invention can be made of at least one free dsDNA end; said free end may be blunt or 5'-/3' - protruding end and comprise modified nucleic acid backbones or other chemical groups or mixture of chemical groups known by skilled person.
• Preferred fragments comprise one or several chemical groups at the end of each strand.
• Preferred chemical .groups comprise phosphorothioates .
• preferred fragments have 3'- 3' nucleotide linkage.
• modified backbones of the invention comprise methylphosphonates, phosphoramidates, morpholino nucleic acid, 2'-0,4'-C methylene/ethylene bridged locked nucleic acid, peptide nucleic acid (PNA) , and short chain alkyl, or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intrasugar linkages of variable length, or any modified nucleotides known by skilled person.
• the phophodiester backbone of the oligonucleotide may be replaced with a polyamide backbone, the bases being bound directly or indirectly to the aza nitrogen atoms of the polyamide backbone.
• oligonucleotides may contain substituted sugar moieties comprising one of the following at the 2' position: OH, SH, 0CH 3 , SCH 3/ - F, OCN, OCH 2 CH 2 OCH 3 , 0(CH 2 ) n NH 2 or 0(CH 2 ) n CH 3 where n is from 1 to about 10 ; CI to CIO lower alkyl, substituted lower alkyl, alkaryl or aralkyl ; CI ; Br ; CN ; CF 3 ; OCF 3. ; 0- ; S- ; or N-alkyl ; 0-, S-, or
• N-alkenyl SOCH 3 ; S0 2 CH 3 ; 0N0 2 ; N0 2 ; N 3 ; NH 2 ; heterocycloalkyl ; heterocycloalkaryl ; aminoalkylamino ; polyalkylyamino ; substitued silyl ; or a group for improving the pharmacokinetic and/or pharmacodynamic properties of an oligonucleotide, and " other substituents having similar properties .
• the DRIL molecules are essentially based on natural nucleotides either 2 ' -deoxynucleotides or 2 ' -ribonucleotides and optionally comprise one or several modified nucleotides and/or nucleobases other than adenine, cytosine, guanine, thymine and uracil.
• nucleobases other than the usual bases are for example C5-methylcytosine, uracile, pseudoisocytosine, C5- propynyluracil, N7-deazaguanine, N7-glycosylated guanine, or alpha anomer, or other modified nucleobases or a basic residue .
• the chemically modified DRIL molecules which will be in the cell in the tissue or in the body when they are irradiated or treated by chemotherapies, will be either incorporated into genomic DNA at the DSB sites, or recognized as DSB sites induced by ionizing radiation by cellular DNA repair mechanism as NHEJ. Then, they will be bound by DSB repair proteins, either being integrated into the broken chromosomes or saturating the repair system.
• said DRIL molecules further comprise at least one embedded element which hampers DNA replication, DNA repair, or damage signalling process .
• Said non-replicable element can be incorporated at the internal position or at the end of the double-stranded fragment. It (they) may comprise: a) a unit which cannot be used as a template for DNA replication, such as a polyethylene ⁇ jlycol chain, preferably a hexaethyleneglycol chain, or any hydrocarbon chain, eventually interrupted and/or substituted by one or more heteroatoms e.g.
• T4 tetradeoxythymidylate
• Said strands are made by chemical synthesis, semi-biosynthesis or biosynthesis, any method of amplification, followed by any extraction and preparation methods and any chemical modification.
• the invention thus also relates to adjuvant compositions to be used in association with a DNA breaking treatment, said compositions comprising a DRIL molecule such as above defined, in combination with a pharmaceutically acceptable carrier, in an efficient amount to be introduced in the nucleus of tumoral cells .
• the invention also relates- to a method for promoting tumor sensibility to anticancer therapies which comprises, in association,
• a transfection agent is -used in said introduction step.
• the invention provides rational to establish clinical protocol of the use of DRIL molecules in combination with radiotherapy or chemotherapy.
• the rational underlying any protocol is that the DRIL molecules should be delivered in the nucleus of cells when DNA damaging event occurs. Therefore, DRIL molecules must to be administrated several hours prior radiotherapy, whereas they can be given along with chemotherapeutic agent (s) depending on the administration mode and the pharmacokinetics of each component.
• the protocol on mice comprises administration of DRIL molecules several hours -before irradiation, for example 5 hours, and 3 times a week, a total dose of irradiation corresponding to 30 Gy over 6 weeks of treatments.
• the use of a fractionated irradiation is particularly efficient.
• said method comprises coupling the treatment with DRIL molecules with a double chemotherapy.
• a double chemotherapy For example 5- FU and CPT 11 are injected together 3 times, 3 consecutive days, spaced by a full week of rest.
• the treatment with DRIL molecules is coupled with radiotherapy. It will be easily adapted for humans by the one skilled in the art, particularly depending on the weight of the patient.
• the DRIL molecules are chemically modified DRIL molecules such as above defined and other practice in human therapy.
• the DRIL molecules are not chemically modified and correspond to native nucleic acid fragments, but exhibit the characteristics of chemically modified fragments, particularly have the number of base pairs and properties defined with respect to said chemically modified DRIL molecules .
• DNA strand breakage is achieved by ionized radiation (radiotherapy) or chemical reaction (chemotherapy) .
• Such a method is a new therapeutic adjuvant in conjunction with DNA damaging therapies to tumors.
• the invention also relates to the use of said non-chemically modified DRIL molecules for making anti-tumoral drugs for treating tumors, particularly highly resistant tumors to radio-and/or chemotherapies, said drugs being used in association with a DNA breaking treatment, particularly radiotherapy or chemotherapy.
• the chemically modified or non-modified DRIL molecules are administrated by any appropriate route, with appropriate acceptable carrier, such as oral, or intravenous, or intratumoral administration, or sub-cutaneous inj ections, or others.
• appropriate acceptable carrier such as oral, or intravenous, or intratumoral administration, or sub-cutaneous inj ections, or others.
• Figure 1.1 Band-shift assays performed on different 32 P radio- labeled DRIL molecules in the presence of various amounts of nuclear extract from Hep2 cells;
• Figure 1.2 Identification of the presence of Ku proteins in the retarded bands of different 32 P radio-labelled DRIL molecules involving proteins in Hep2 cell nuclear extract;
• Figure 2.1 Clonogenic survival assay of Hela cells after irradiation performed with ⁇ -rays of DRIL molecules) ; •
• Figure 2.2 Inhibition of radiation-enhanced illegitimate integration of a linear plasmid fragment (2 ⁇ g) carrying the gene coding for neomycin resistance by DRIL32-PEG molecules;
• Figure 2.3 Hela cells transfected by fluorescent DRIL32-FITC molecules after irradiation
• Figure 3.1 FACS analyses of the untreated GMA32 cells, the cells transfected alone, or transfected with different DRIL molecules by lipofectamine, but without further irradiation or mitotic inhibitor treatment;
• Figure 3.2 Immunodetection of DNA repair foci by ⁇ -H2AX labeling in the untreated GMA32 cells, the cells transfected alone, or transfected with different DRIL molecules by lipofectamine ;
• Figure 3.3 Western blot analysis of the phosphorylation status of p53 serine 15 residue of the untreated GMA32 cells, the cells transfected alone, or transfected with different DRIL molecules by lipofectamine, but without further irradiation or mitotic inhibitor treatment.
• Figure 3.4 Clonogenical survival of untreated and treated GMA32 cells by irradiation or by different mitotic inhibitors
• Figure 4 Growth of the xenografted human larynx tumor on mice monitored as the ratio of the tumor volume at time t over the initial volume (V t /Vi) with or without treatments;
• Figure 5.1 Chemosensitization of the treatment of digestive tumors induced in K-Ras vl2G x Apc 1638N transgenic mice.
• Figure 5.2 Mean number of digestive tumors per animal by macroscopy or histology examination.
• FIG. 5.3 Panel A: Protocol schema (i.p.: intraperitoneal injection; o.: oral administration).
• Panel B Fluorescence of DRIL32-FITC (left) and of immunofluorescence labelled ⁇ —H2AX (right) on the 5 ⁇ m section from the tumor tissue of the treated animal according to the protocol given in panel A. Lower parts show the details (using 63x lens) of the indicated zone in the upper parts (using lOx lens, white box) . Colocalization of fluorescentDRIL32-FITC and labelled ⁇ —H2AX appears as bright dots over DAPI counterstained nuclei. Color pictures are available upon request.
• Example 1 Design, synthesis and preparation of DRIL molecules
• DRIL molecules Two types were designed: linear or hairpin dsDNA fragments.
• a hexaethyleneglycol linker (abbreviated as PEG) or a tetradeoxythymydylate (abbreviated as T4) was used as loop.
• PEG hexaethyleneglycol linker
• T4 tetradeoxythymydylate
• the end(s) of dsDNA stem can be protected against chemical degradation by 3' -exonucleases by incorporation of phosphorothioates, or 3' -3' nucleotide linkage.
• other chemical modifications can be used provided that they are compatible with Ku70/Ku80-DNA PKsc binding (Martensson & Hammarten, 2002).
• DRIL8-PEG 16 bp
• DRIL16-PEG 16 bp
• DRIX.24- PEG 24 bp
• DRIL32-PEG 32 bp
• DRIL32-PEG 32 bp
• DRIL32-2xPEG A dumbell dsDNA fragment where both ends were sealed by two PEG loops was also designed, as control.
• Some DRIL molecules were labelled v ⁇ a a T tagged with fluorescein (DRIL32-FITC) , cyanine 3 (DRIL32- Cy3) , or biotin (DRIL32-Bt) .
• Table 1.1 and 1.2 summarized the sequences and chemical structures of DRIL molecules used in this work.
• DRIL8-PEG 3 TGCGTGCC5
• D DRIL32ss 5 ⁇ CGCACGGGTGTTGGGTCGTTTGTTCGGATCT-3 '
• t fluorescein (FITC), cyanine 3 (Cy3) or biotin (Bt)-tagged T
• Table 1.1 Sequences and chemical structures of DRIL molecules.
• the Bold letters are nucleotides with phosphorothioate backbone. Solid line symbolizes liexaethyleneglycol linker (PEG).
• DRIL32-T4 contains T 4 as a linker instead of PEG linlcer.
• DRIL32-2xPEG is a dumbbell (closed) molecule.
• DRIL32s33-PEG has a shuffled sequence (same base composition but in different order) and a 3 '-3' linkage.
• DRIL molecules Sequences and chemical structures
• Table 1.2 Sequences and chemical structures of 64-bp DRIL molecules.
• the Bold letters are nucleotides with phosphorothioate backbone. Solid line symbolizes hexaethyleneglycol linker (PEG).
• DRIL molecules were made by automated solid phase oligonucleotide synthesis (Eurogentec, Belgium) . They were purified by denaturing reverse phase HPLC. Denaturing capillary gel electrophoresis and MALDI-TOF/LC-MS were used for quality control. More than 90% of oligonucleotides are full length. All samples were lyophilized before shipping.
• dumbell dsDNA fragment (DRIL32-2xPEG) was prepared by annealing and ligation by DNA T4 ligase (BioLabs) of two semi hairpins carrying PEG loop and with 3' protruding and complementary ends.
• DRIL32, DRIL64 and DRIL64-PEG bi-molecular DRIL molecules
• DRIL32, DRIL64 and DRIL64-PEG The mixture of 1:1 stock solution (highest concentration possible) of each strand in bi-distilled water has to be heated at 90 °C for 5 minutes for complete denaturation of each strand. The annealing was carried out by smooth return to room temperature (the samples are typically left in water bath) and the resulting duplex molecules were stored in aliquot at -20°C.
• a series of band-shift assays were carried out with different 32 P radio-labelled DRIL molecules in the presence of nuclear protein extracts from Hep2 cells according to standard protocol.
• 10 nM 32 P radio-labelled DRIL molecules were incubated in the presence of various concentrations of nuclear proteins (0, 10, 20, 40, 80, 160, and 320 ng/ ⁇ l) at 30 °C for 10 minutes in TBE buffer.
• the samples were loaded on a 5% acrylamide native gel.
• the electrophoresis was run at 95V for 2 hours at 4°C.
• the gel was dried and scanned by phosphorimager (Molecular Dynamics) .
• Figure 1.1 shows that, except the 8-bp DRIL8-PEG molecules (the shortest DRIL molecule) , up to 3 retarded bands were observed for longer DRIL molecules with a common band (band 1) . Other bands occurred in the presence of long DRIL molecules.
• the retarded band pattern of the titration of Hep2 nuclear protein extracts with 32-bp long DRIL molecules (DRIL32-PEG, DRIL32po-PEG and DRIL32) is more complex.
• the intensity of the retarded band 1 increases and then decreases as the concentration of protein increases.
• the intensity of the retarded bands 2 and 3 increases as a function of protein concentration until it reaches a plateau.
• Band 1 and 2 are super-shifted into band 1* and 2* upon anti- Ku70 antibody addition. It is likely that band 1 has one Ku70/80 complex bound to the DRIL and band 2 two Ku70/80 complexes bound to the DRIL. Control experiments performed with purified Ku proteins confirmed this interpretation. It is noted that the band 3 disappeared upon addition of anti-Ku70 antibody (clearly seen with DRIL24-PEG and DRIL32po-PEG) , showing that the band 3 also contains the Ku complex.
• Example 3 In vitro activity of DRIL molecules
• DRIL molecules The activity of DRIL molecules in cultured cells was studied by clonogenic survival assay in two radio-resistant human cancer cell lines derived from a female cervix carcinoma (HeLa) and from carcinoma of larynx (Hep2) in association with ionizing radiation.
• HeLa female cervix carcinoma
• Hep2 carcinoma of larynx
• the hairpin DRIL molecules (DRIL32-PEG, DRIL32-T4 and DRIL24-PEG) and the linear double-stranded DRIL molecules (DRIL64-PEG and DRIL64) significantly reduced clonogenic survival.
• the dumbell DRIL molecule (DRIL32-2xPEG) which lacks free dsDNA ends (capped by hexaethyleneglycol linker at both ends) did not exhibit any effect.
• the chemical nature of loop did not matter (for example, DRIL32-PEG versus DRIL32-T4) .
• Ionizing radiation is known to improve transfection of exogenous DNA, a process termed radiation-enhanced integration.
• Hela cell culture was used for this assay.
• Cells were transfected during 8 hours by 2 ⁇ g of a linear plasmid (carrying the gene coding for neomycin resistance) and three different ratio of DNA/superfect (1:2, 1:5, 1:10).
• the cells were exposed to different irradiation protocols: no irradiation, one single irradiation of 1 Gy and 2 Gy, as well as a 2 Gy irradiation delivered by split doses of 0.5 Gy every 2 hours (4 x 0.5 Gy) .
• Plasmid integration was significantly enhanced by the split irradiation protocol. When 2 ⁇ g of DRIL32-PEG molecules were added to the transfection mix, the radiation-enhanced integration was abolished (figure 2.2).
• DSB damages in nuclei can be immunodetected by using ⁇ -H2AX antibody which labels DNA breaks. Most of the H2AX foci appear rapidly after irradiation and disappeared as DSBs repair process progressed. Few H2AX foci were detected two hours after irradaiation in non transfected cells.
• Figure 2.3 gives the results obtained with Hela cells transfected by fluorescent DRIL32-FITC molecules at 2 hours after 2 Gy irradiation.
• Transfection and irradiation protocoles were similar to those described above.
• the cells were grown on surface coverslip in 5 cm diameter Petri dishes, transfected with 2 ⁇ g DRIL32-FITC molecules labeled with FITC with Superfect (Qiagene) according to the manufacturer's instruction.
• cells were irradiated (2 Gy) , then rest for 2 hours in the medium at 37 °C. After 3 washing cycles, the cells were fixated with 2% PFA for 10 ' minutes.
• D-H2AX was detected with rabbit anti-D-H2AX antibody (4411-PC, Trevigen) diluted 1/100 in lxPBS, 1% BSA.
• rabbit anti-D-H2AX antibody 4411-PC, Trevigen
• lxPBS 0.5% TritonX-100
• rhodamine-conjugated goat anti-rabbit antibodies diluted 1/100 in lxPBS, 1% BSA. Cells were visualized by epifluorescence microscopy.
• Example 4 Effects of DRIL molecules in GM ⁇ 32 cell line and their association with irradiation or mitotic inhibitors .
• the GMA32 Chinese hamster fibroblast cells permissive of DNA breaks were maintained in MEM medium (Gibco) supplemented with 1 mM sodium pyruvate, 2 mM glutamine, lx MEM non essential a ino acids, lx penicillin/streptomycin and 10% horse serum.
• MEM medium Gibco
• 2xl0 5 to 4xl0 5 cells were seeded in medium without antibiotics, in 5 cm diameter Petri dishes 24 hours before the transfection of different DRIL molecules (4.5 ⁇ g) with lipofectamine 2000 (LifeTechnologies) as transfection agent (in a 1:3 ratio), according to the manufacturer's instructions.
• the cells were either irradiated (4 Gy) or treated with mitotic inhibitors: nocodazole (200 nM) , navelbine (100 nM) or taxol (200 nM) . About 16 hour later ' the drug was removed and the cells were allowed to recover. Cell irradiation was performed with ⁇ -rays from a 137 Cesium source. After a 24 hour recovery, the cells were collected and used either for FACS, western blot analyses or to determine the clonogenicity (survival) and the effect of each treatment.
• Figure 3.1 shows FACS analyses of the untreated GMA32 cells, the cells transfected alone, or transfected with different DRIL molecules by lipofectamine, but without further irradiation or mitotic inhibitor treatment.
• the Ml phase represents the percentage of cells in sub-Gl stage indicative of cell death. Significant cell death was observed only in the presence of double-stranded DRIL32 and hairpin DRIL32-PEG molecules, whereas "hairpin DRIL16-PEG and single-strand
• DRIL32ss induced intermediate and moderate cell death, respectively.
• the shortest hairpin DRIL8-PEG failed to trigger cell death as compared to the control (cells transfected by lipofectamine alone) .
• the experiments were performed with a FACScalibur flow cytometer (Becton Dickinson) .
• Cells were collected, suspended in 1ml of cold GM buffer ( 6.5mM glucose, 137mM NaCl, 5.4mM KC1, 2mM Na 2 HP0 4 , ImM KHP0 4 , 0.5mM EDTA) , and stored at 4°C for at least 2 hours after addition of 3ml of cold 100% ethanol.
• cold GM buffer 6.5mM glucose, 137mM NaCl, 5.4mM KC1, 2mM Na 2 HP0 4 , ImM KHP0 4 , 0.5mM EDTA
• the cells were grown on coverslip in 5 cm diameter Petri dishes 24 hours before the transfection with different DRIL molecules. One day after the transfection,
• FITC-DiOC 6 (Molecular probes) was added in the medium 5 minutes at 37 °C (to counterstain the membranes). After 3 washing cycles, the cells were fixated with 4% PFA for 20 minutes. After additionnal washings, H2AX phosphorylated on serine 139 ( ⁇ -H2AX) was detected with, rabbit anti- ⁇ -H2AX antibody (4411-
• the protein p53 is a well known major protein in mediating DNA damage signaling and in coordinating appropriate responses (DNA repair, apoptosis, etc.) by changing its phosphorylation status.
• the phosphorylation of serine 15 residue is involved in the interaction with MDM2 protein which acts as a feed back control.
• the phosphorylation status of the serine 15 of p53 was assessed by Western blot.
• Figure 3.3 shows that the p53 serine 15 ' was , highly phosphorylated when cells were transfected by either double-stranded DRIL32 or hairpin DRIL32-PEG molecules, whereas the shorter hairpin DRIL16-PEG induced moderate phosphorylation. Neither the shortest DRIL8- PEG nor single strand DRIL32ss molecules were able to induced significant phosphorylation on the serine 15 of p53 protein. This experiment provides additional evidence that the presence of both double-stranded DRIL32 and hairpin DRIL32-PEG in GMA32 cells was detected as DNA damage and induced the signal to transducer responses such as p53 protein phosphorylation, likely through ATM activation pathway.
• DRIL32-PEG molecules when they were treated by mitotic inhibitors (200 nM nocodazole, 100 nM navelbine (vinorelbine) or 200 nM taxol (paclitaxel) ) . These drugs block either polymerization or depolymerization of microtubules, and can induce indirectly DNA breakage.
• clonogenicity assay serial dilutions were made after, counting the cells to seed 5 cm Petri dishes with different amounts of cells. The number of cells range from 100-200 (control cells) to 3000 (transfected or/and treated cells) . Ten days after, the cells (forming clone) were fixed with 4% paraformaldehyde (20 minutes) , then colored with methylene blue (15minutes), and the number of clone in each plate (in triplicates) was scored.
• DRIL molecules in association with radiotherapy were assessed by using nude mice xenografted with human tumors by subcutaneous injection of radio-resistant cell lines (Hep2 derived from carcinoma of larynx) or tumor fragments (previously obtained by subcutaneous injection of the U87 cell lines derived of glioblastoma) .
• radio-resistant cell lines Hep2 derived from carcinoma of larynx
• tumor fragments previously obtained by subcutaneous injection of the U87 cell lines derived of glioblastoma
• mice xenografted with radio-resistant human larynx tumors were mainly carried out on the mice xenografted with radio-resistant human larynx tumors in order to establish proof of concept in vivo. Irradiation was performed with ⁇ -rays from a 137 Cesium source with appropriate protection of mice in order to perform localized irradiation of tumors.
• Typical assay condition consists of intratumoral injection of an appropriate preparation of 1 nmole DRIL molecules with transfecting agents (cationic dendrimer (Superefct, Qiagene) , dioctadecylamidoglycyispermine (DOGS, Polyplus transfection) , polyethyleneimine (PEI, Polyplus Transfection) according to manufacturer's instruction, 5 hours prior irradiation.
• transfecting agents cationic dendrimer (Superefct, Qiagene) , dioctadecylamidoglycyispermine (DO
• a total dose of 30 Gy was delivered in 4-5 weeks: i) 3x2 Gy/week (about one every two days); ii) 5 Gy/week; iii) 15 Gy/2 weeks.
• the size of tumor was measured 2-3 times a week.
• Treatment by irradiation and intratumoral injection of MEM medium (the DRIL dilution buffer) , was used as a control 'of irradiation treatment without DRIL (MEM) .
• the ratio of volume measured at t .time over the initial volume (V t /Vi) was used as indicator of tumor progression.
• the mice were followed up to 100 days. At least 4 independent series of six animals were tested.
• MEM or DRIL32-PEG was delivered by intratumoral injection 5 hours prior irradiation.
• the split irradiation dose (2 Gy) was given one of every two days, three times a week.
• the treatment lasted 5 weeks totalling 30 Gy irradiation.
• the dots represent the time course of tumor volume of each mouse.
• the solid lines are the best polynomial fitting.
• Panel D shows a Kaplan-Meyer plot of all mice of which the increase in tumor volume (V t /Vi) ⁇ 5) .
• mice with xenografted human larynx tumors were subsequently carried out on mice with xenografted human larynx tumors in order to define molecular features of DRIL molecules and optimal protocol for in vivo activity.
• the data obtained from .the studied cohort were consistent with molecular features of DRIL molecules observed in biochemical and in vi tro studies (cf. examples 2, 3 and 4).
• mice xenografted with human glioblastome tumors were also observed in mice xenografted with human glioblastome tumors.
• the glioblastome is the highest grade of brain ' tumor, and is characterized by its extraordinary aggressive progression with fast fatal outcome and resistance to radio- and chemotherapies. 2-3 millions of U87 cells derived from human glioblastome was first injected subcutaneously in nude mouse. The grafted tumor was then took out and used to seed subsequently other nude mice by subcutaneous transplant of about 8 mm 3 glioblastome tumor. Table 2 shows data of a pilot series of xenografted human glioblastome tumors on nude mice.
• Table 2 Assay of radiosensitization of xenografted human glioblastome on nude mice by DRIL32-PEG (1 nmole/intratumoral injection). Two protocols of irradiation were used: 1x15 Gy/week, or 3x5 Gy/week, followed by one week rest, the second irradiation cycle. The total irradiation dose was 30 Gy. Control groups were the untreated or the groups received saline solution (PBS) injection.
• PBS saline solution
• Example 6 Chemosensitization of the treatment of digestive tumors induced in K-Ras yl2G x Apc 1638N transgenic mice
• An endogenous mouse tumor model was chosen to assess the ability of DRIL molecules to sensitize anticancer chemotherapy.
• transgenic mice carrying K-Ras vl2G and Apc 1638N mutations were used. They ' were obtained by breeding two transgenic mice: one carries K-Ras vl2G mutant under the control of the mouse villin promoter (pVill/ K-Ras vl G ) (Janssen et al., 2002), the other contains Apc 1638N mutation in one allele (Fodde et al . , 1994).
• Ras vl2G x Apc 1638N mutations developed spontaneous tumors in the digestive tract at the age of about 5 months and died rapidly.
• the protocol includes three treatment cycles. Each cycle consists of intraperitoneal injection of 0.6 mg 5FU and 0.6 mg CPT11, along with 0.1 mg DRIL32-PEG by oral administration, three times a week, followed by one week rest.
• 5FU 5 fluorouracile, Teva
• CPTll/Irinotecan (Campto, Aventis) was prepared in 0.9% NaCl solution at the concentration of 20 mg/ml. The health status and survival of the mice were monitored till the death. No clinical indication of additional toxic effect due to DRIL molecules was observed.
• Panel A Treatment protocol for three groups/arms of the K-Ras vl2G x Apc 1638N" transgenic mice at the mean age of 12 weeks: the control group (untreated) , the group treated by 5FU+CPT11, the group treated by 5FU+CPT11 and DRIL32-PEG. It was performed by three cycles of treatment. Each cycle consists of intraperitoneal injection of 0.6 mg 5FU and 0.6 mg CPT11, along with 0.1 mg DRIL32-PEG by oral administration, three times a week, followed by one week rest. The number of mice involved in each groups is indicated in parenthesis. The end point is the time of survival; Panel B: Kaplan-Meyer plot of survival curves of the three groups; Panel C: The median survival time of three groups as shown in panel B.
• mice A series of mice was sacrificed two weeks after the end of treatment (at the mean age of 18 weeks) in order to evaluate the mean number of tumors per animal.
• the intestine was examined by macroscopy and histology examination (standard staining by Hematoxyline-Eosine-Safran) .
• Figure 6.3 shows an additional assay where a 18 week-old K-Ras vl G x Apc 1638N transgenic mouse was consecutively treated by chemotherapy (5FU+CPT11) and DRIL32-FITC for three days and sacrificed two hours later after the last treatment as indicated in the panel A. The intestine was taken out and washed by PBS. Then the tumor tissues were sampled and frozen at -80°C. For the analysis, 5 ⁇ m histological samples were made from the frozen tumor tissues by cryostat.
• DNA repair foci were detected by immunofluorescence with polyclonal rabbit anti- ⁇ -H2AX antibody (Trevigen) diluted 1/500 in PBS, then with goat anti-rabbit antibody tagged by cyanine 3 (Jackson) diluted 1/200 in PBS. The samples were also counterstained by DAPI (Sigma) . Samples were visualized by epifluorescence microscopy. It was found that the fluorescence of DRIL32-FITC was heterogeneously disseminated in tumor tissues (epithelium and stroma between . glandular structures) and had preferential nucleus localization (figure 6.3, panel B, left). Similar pattern was found for ⁇ -H2AX sites (figure 6.3, panel B, right) . The colocalized DRIL32-FITC and ⁇ -H2AX signals were observed almostly.
• RNA antisense decreases the radioresistance of human fibroblasts. Cancer Gene Ther.
• a Ku80 fragment with dominant negative activity imparts a radiosensitive phenotype to CHO-K1 cells.
• RNA antisense decreases the radioresistance of human fibroblasts. Cancer Gene Ther.
• Radiation-induced recombination is dependent on KU80. Radiat Res. (1999), 151, 408-13.

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