WO2003085090A2 - A human ribonucleotide reductase m2 subunit - Google Patents
A human ribonucleotide reductase m2 subunit Download PDFInfo
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- WO2003085090A2 WO2003085090A2 PCT/US2003/009301 US0309301W WO03085090A2 WO 2003085090 A2 WO2003085090 A2 WO 2003085090A2 US 0309301 W US0309301 W US 0309301W WO 03085090 A2 WO03085090 A2 WO 03085090A2
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Definitions
- Ribonucleotide reductase is a highly regulated enzyme involved in the DNA synthesis pathway. It is responsible for the de novo conversion of ribonucleoside diphosphates to deoxyribonucleoside diphosphates that are essential for DNA synthesis and repair (Cory and Sato, (1983) Mol Cell Biochem 53, 257-266; and Thelander and Berg (1986) Mol Cell Biol 6, 3433-3442). Ribonucleotide reductase consists of two subunits, Ml and M2. Ml is a 170 kDa dimer required for enzyme regulation (Chang and Cheng (1979) Cancer Res 39, 5081-5086). M2 is an 88 kDa dimer containing a tyrosine free radical and a non-heme iron.
- the invention relates to a newly isolated genomic sequence of the human ribonucleotide reductase M2 subunit (hRRM2) gene.
- the 10.3 kb sequence (SEQ ID NO:l shown below) was deposited in GenBank (Accession Number AY032750).
- the hRRM2 gene was found to consist of 10 exons. Two transcription initiation sites were identified, corresponding to mRNA transcripts of 3.4 kb and 1.65 kb, respectively. Deletion analysis of the 5 '-flanking region showed that the promoter activity is consistent with the presence of two separate promoters driving expression of the two hRRM2 transcripts: one is located between bp 1908-2310 of SEQ ID NO:l and designated SEQ ID NO:2; the other is located between bp 2366-2567 of SEQ ID NO: 1 and designated SEQ LD NO:3.
- the composite promoter (bp 1908-2567 of SEQ ID NO:l) is designated SEQ ID NO:4.
- the promoter region of hRRM2 contains many binding sites for transcriptional factors.
- these promoters are useful for fine-tuned regulation of gene expression.
- the invention features an isolated nucleic acid containing a promoter sequence at least 60% (including any percentage between 60% and 100%, e.g., 70%, 80%, 90%, 95%, or 100%) identical to SEQ ID NO:2, 3, or 4 and a nucleotide sequence encoding a transcript.
- the promoter sequence is operably linked to the nucleotide sequence, i.e., transcription of the nucleotide sequence is under the control of the promoter.
- isolated nucleic acid is a nucleic acid the structure of which is not identical to that of any naturally occurring nucleic acid or to that of any fragment of a naturally occurring genomic nucleic acid.
- the term therefore covers, for example, (a) a DNA which has the sequence of part of a naturally occurring genomic DNA molecule but is not flanked by both of the coding sequences that flank that part of the molecule in the genome of the organism in which it naturally occurs; (b) a nucleic acid incorporated into a vector or into the genomic DNA of a prokaryote or eukaryote in a manner such that the resulting molecule is not identical to any naturally occurring vector or genomic DNA; (c) a separate molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), or a restriction fragment; and (d) a recombinant nucleotide sequence that is part of a hybrid gene, i.e., a gene encoding a
- the "percent identity" of two nucleic acid sequences is determined using the algorithm of Karlin and Altschul ((1990) Proc Natl Acad Sci USA 87, 2264-2268), modified as in Karlin and Altschul ((1993) Proc Natl Acad Sci USA 90, 5873-5877). Such an algorithm is incorporated into the XBLAST programs of Altschul et al. ((1990) J Mol Biol 215, 403-410). BLAST nucleic acid searches are performed with the XBLAST program. Where gaps exist between two sequences, Gapped BLAST is utilized as described in Altschul et al. ((1997) Nucleic Acids Res 25, 3389-3402). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST) are used. See the World Wide Wed address ncbi.nlm.nih.gov.
- the invention also features an isolated nucleic acid at least 86% (including any percentage between 86% and 100%, e.g., 90%, 95%, or 100%) identical to SEQ ID NO:l, which can be used, e.g., for detection of amplified RRM2 gene in a subject, or screening of therapeutic compounds for treating a cell proliferation-associated disorder.
- the invention features a method of determining whether a subject is suffering from or at risk for developing a cell proliferation-associated disorder (e.g., cancer).
- the method involves providing a test sample (e.g., a tissue sample or a body fluid sample) containing genomic DNA from a subject, and determining the level of RRM2 genomic DNA in the test sample, e.g., by Southern blot analysis. If the level of RRM2 genomic DNA in the test sample is higher than that in a normal sample, it indicates that the subject is suffering from or at risk for developing a cell proliferation-associated disorder.
- the invention further features a method of identifying a compound for treating a cell proliferation-associated disorder.
- the method involves contacting a compound with a cell containing amplified RRM2 genomic DNA, and determining the level of RRM2 genomic DNA. If the level of RRM2 genomic DNA in the presence of the compound is lower than that in the absence of the compound, it indicates that the compound is a candidate for treating a cell proliferation-associated disorder.
- the method involves contacting a compound with a system
- a cell system or a cell-free system containing a nucleic acid that includes a marker nucleotide sequence operably linked to a promoter sequence of SEQ ID NO:2, 3, or 4, and determining the expression level of the marker nucleotide sequence. If the expression level of the marker nucleotide sequence in the presence of the compound is different from that in the absence of the compound, it indicates that the compound is a candidate for treating a cell proliferation-associated disorder.
- the marker nucleotide sequence can be, e.g., the coding region of the l RRM2 gene, or any of other suitable genes.
- the system can include a nucleic acid encoding various transcriptional factors interacting with SEQ ID NO:2, 3, or 4, e.g., SP1, c-Ets, MZF1, E2F, Lyf-1, GATA-X, HSF2, AP-1, CdxA, IK-2, Sox-5, SRY, Brn-2, HNF-1, STATx, GATA-1, USF, Pbx-1, Oct-1, GATA-2, CRE- Bp, or Nkx-2.
- the system can contain one or more of these factors themselves.
- the method involves contacting a compound with a cell containing amplified RRM2 genomic DNA and expressing the ribonucleotide reductase Ml subunit (RRMl) or p53R2 gene, and determining the level of RRMl or p53R2 mRNA or protein in the cell. If the level of RRMl or p53R2 mRNA or protein in the presence of the compound is lower than that in the absence of the compound, it indicates that the compound is a candidate for treating a cell proliferation-associated disorder.
- RRMl ribonucleotide reductase Ml subunit
- the method involves contacting a compound with a cell containing amplified RRM2 genomic DNA and expressing RRMl and p53R2 genes, and determining the level of RRMl and RRM2 or RRMl and p53R2 interaction in the cell. If the level of RRMl and RRM2 or RRMl and p53R2 interaction in the presence of the compound is lower than that in the absence of the compound, it indicates that the compound is a candidate for treating a cell proliferation-associated disorder. Also within the scope of the invention is a method of treating a cell proliferation- associated disorder in a subject whose RRM2 genomic DNA is amplified.
- the method involves administering to the subject an effective amount of a compound that reduces the level of RRM2 genomic DNA, transcription from a promoter sequence of SEQ ID NO:2, 3, or 4, RRMl mRNA or protein, RRM2 mRNA or protein, p53R2 mRNA or protein, RRMl and RRM2 interaction, or RRMl and p53R2 interaction in the subject.
- a compound that reduces the level of RRM2 genomic DNA, transcription from a promoter sequence of SEQ ID NO:2, 3, or 4, RRMl mRNA or protein, RRM2 mRNA or protein, p53R2 mRNA or protein, RRMl and RRM2 interaction, or RRMl and p53R2 interaction in the subject can be reduced by using an antibody to RRMl, RRM2, or p53R2, respectively.
- RRMl and RRM2 can be reduced by using an antibody to RRMl or RRM2
- the interaction between RRMl and p53R2 can be reduced by using an antibody to RRMl or ⁇ 53R2.
- the interaction between RRMl and RRM2 or RRMl and p53R2 can also be inhibited by using a competitor peptide or polypeptide such as a 7-amino acid peptide FTLDADF (SEQ ID NO:5).
- a cell proliferation- associated disorder in a subject whose RRM2 genomic DNA is amplified can also be treated by administering to the subject an effective amount of an RRM2 inhibitor (e.g., hydroxyurea) followed by administering to the subject an effective amount of a nucleotide or nucleoside analog (e.g., gemcitabine).
- an RRM2 inhibitor e.g., hydroxyurea
- a nucleotide or nucleoside analog e.g., gemcitabine
- the invention features a method of developing a procedure for treating a cell proliferation-associated disorder in a subject, e.g., a subject who has amplified RRM2 genomic DNA.
- the method involves providing a plurality of subjects suffering from a cell proliferation-associated disorder; administering to each subject an effective amount of an RRM2 inhibitor; administering to each subject an effective amount of a nucleotide or nucleoside analog following administration of the RRM2 inhibitor, each at a unique time point; and selecting an optimal time point at which the cell proliferation-associated disorder is inhibited to the greatest extent.
- the invention is based on the identification of genomic sequence of the human ribonucleotide reductase M2 subunit gene and the unexpected discovery that this sequence is amplified in drug-resistant cancer cells. Further, it was found that low-dose hydroxyurea (HU, an S-phase specific inhibitor of ribonucleotide reductase with a broad spectrum of anti-tumor effects) enhanced the activity of gemcitabine (Gem, a deoxycytidine analogue whose active metabolite, dFdCTP, blocks DNA elongation and has a cytotoxic effect) in a time and sequence dependent manner. Unexpectedly, this enhancement is associated with a decrease in hRRM2 mRNA, protein, or activity. Moreover, a phase I trial demonstrated that hydroxyurea followed by gemcitabine can be safely administered to human patients and has cytotoxic effect on cancer cells.
- the invention features an isolated nucleic acid containing a promoter sequence at least 60% identical to SEQ ID NO:2, 3, or 4 and a nucleotide sequence operably linked to the promoter sequence and encoding a transcript. Expression of the nucleotide sequence is directed by the promoter sequence.
- the invention features an isolated nucleic acid at least 86% identical to SEQ ID NO:l.
- This nucleic acid is useful, e.g., for diagnosing a cell proliferation-associated disorder, or identifying a compound for treating such a disorder.
- a nucleic acid of the invention can be expressed in vitro by DNA transfer into a suitable host cell by methods known in the art.
- the nucleic acid can be inserted into a recombinant expression vector.
- a variety of host-expression vector systems can be utilized to express a nucleic acid of the invention.
- microorganisms such as bacteria transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors; yeast transformed with recombinant yeast expression vectors; and human cell lines infected with recombinant virus or plasmid expression vectors.
- Isolation and purification of recombinant polypeptides, or fragments thereof, can be carried out by conventional means including preparative cliromatography and immunological separations involving monoclonal or polyclonal antibodies.
- the invention also features methods for diagnosing and treating a cell proliferation-associated disorder, and identifying therapeutic compounds for treating such a disorder.
- a diagnostic method of the invention involves comparing the RRM2 genomic DNA level in a sample prepared from a subject (i.e., an animal or a human) with that in a sample prepared from a normal subject, i.e., a subject who does not suffer from or at risk for developing a cell proliferation-associated disorder.
- a higher RRM2 genomic DNA level indicates that the subject is suffering from or at risk for a cell proliferation-associated disorder.
- This method can be used on its own or in conjunction with other procedures to diagnose a cell proliferation-associated disorder in appropriate subjects.
- Amplification of a gene locus can be detected by a variety of methods known in the art. For example, the copy number of a gene locus can be determined and compared by PCR amplification of genomic DNA prepared from a test sample and a control sample. Amplification of a gene locus can also be identified by Southern blot analysis. Fluorescence in situ hybridization (FISH) of a DNA sequence to a metaphase chromosomal spread can further be used to provide a precise chromosomal location and an amount of the DNA sequence present in the chromosome.
- FISH Fluorescence in situ hybridization
- the invention also provides a method for identifying and manufacturing compounds (e.g., proteins, peptides, peptidomimetics, peptoids, antibodies, or small molecules) that decrease the RRM2 genomic DNA level, modulate expression of a marker nucleotide sequence under the control of a promoter sequence of SEQ ID NO:l, 2, or 3, decrease the level of RRMl or p53R2 mRNA or protein, decrease the level of RRMl and RRM2 interaction, or decrease the level of RRMl and p53R2 interaction in a system (a cell system or a cell-free system).
- Compounds thus identified can be used, e.g., for preventing and treating a cell proliferation-associated disorder.
- the candidate compounds of the invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art.
- libraries include: peptide libraries, peptoid libraries (libraries of molecules having the functionalities of peptides, but with a novel, non-peptide backbone that is resistant to enzymatic degradation); spatially addressable parallel solid phase or solution phase libraries; synthetic libraries obtained by deconvolution or affinity chromatography selection; and the "one-bead one-compound” libraries. See, e.g., Zuckermann et al. (1994) J Med Chem 37, 2678-85; and Lam (1997) Anticancer Drug Des 12, 145.
- the system is contacted with a candidate compound and the RRM2 genomic DNA level, the expression of the marker nucleotide sequence, the level of RRMl or p53R2 mRNA or protein, the level of RRMl and RRM2 interaction, or the level of RRMl and p53R2 interaction is evaluated relative to that in the absence of the candidate compound, h a cell system, the cell can be one that contains amplified RRM2 genomic DNA (e.g., a cancer cell), one naturally expresses RRMl, RRM2, or p53R2, or one that is modified to express a recombinant nucleic acid
- the system can contain a transcriptional factor that interacts with the promoter sequence of SEQ ID NO:2, 3, or 4, e.g., SP1, c-Ets, MZF1, E2F, Lyf-1, GATA-X, HSF2, AP-1, CdxA, IK-2, Sox-5, SRY, Brn-2, HNF-1, STATx, GATA-1, USF, Pbx-1, Oct-1, GATA-2, CRE-Bp, or Nkx-2, or a nucleic acid encoding such a transcriptional factor.
- a transcriptional factor that interacts with the promoter sequence of SEQ ID NO:2, 3, or 4, e.g., SP1, c-Ets, MZF1, E2F, Lyf-1, GATA-X, HSF2, AP-1, CdxA, IK-2, Sox-5, SRY, Brn-2, HNF-1, STATx, GATA-1, USF, Pbx-1, Oct-1, GATA-2, C
- the candidate compound is identified as being useful for preventing and treating a cell proliferation-associated disorder.
- the RRM2 genomic DNA level can be determined by methods described above and any other methods well known in the art.
- the expression of the marker nucleotide sequence, RRMl, or p53R2 can be determined at either the mRNA level or at the protein level.
- Methods of measuring mRNA levels in a tissue or a body fluid sample are known in the art.
- cells can be lysed and the levels of mRNA in the lysates or in RNA purified or semi-purified from the lysates can be determined by any of a variety of methods including, without limitation, hybridization assays using detectably labeled DNA or RNA probes and quantitative or semi-quantitative RT-PCR methodologies using appropriate oligonucleotide primers.
- RNA protection assay RPA
- SAGE SAGE.
- antibodies e.g., monoclonal or polyclonal antibodies
- the antibody itself or a secondary antibody that binds to it can be detectably labeled.
- the antibody can be conjugated with biotin, and detectably labeled avidin (a polypeptide that binds to biotin) can be used to detect the presence of the biotinylated antibody.
- Multi-layer sandwich assays can be used to enhance the sensitivity of the methodologies.
- Some of these protein-measuring assays e.g., ELISA or Western blot
- ELISA or Western blot can be applied to lysates of cells
- others e.g., immunohistological methods or fluorescence flow cytometry
- Methods of measuring the amount of label will be depend on the nature of the label and are well known in the art.
- Appropriate labels include, without limitation, radionuclides (e.g., 125 L 131 1, 35 S, 3 H, or 32 P), enzymes (e.g., alkaline phosphatase, horseradish peroxidase, luciferase, or ⁇ -glactosidase), fluorescent moieties or proteins (e.g., fluorescein, rhodamine, phycoerythrin, GFP, or BFP), or luminescent moieties (e.g., QdotTM nanoparticles supplied by the Quantum Dot Corporation, Palo Alto, CA).
- Other applicable assays include quantitative immunoprecipitation or complement fixation assays.
- the level of RRMl and RRM2 interaction, or the level of RRMl and p53R2 interaction can be determined by any method known in the art, e.g., by in vitro binding assay or by using a yeast hybrid system.
- the binding domains of RRMl, RRM2, and p53R2 can be identified, e.g., by mutagenesis, and used in the screening assays.
- This invention further provides a method for preventing and treating a cell proliferation- associated disorder.
- Subjects to be treated can be identified, for example, by determining the RRM2 genomic DNA level in a sample prepared from a subject by methods described above.
- the subject is a candidate for treatment with an effective amount of compound that decreases the RRM2 genomic DNA level, transcription from a promoter sequence of SEQ ID NO:l, 2, or 3, the level of RRMl, RRM2, or p53R2 mRNA or protein, or the level of RRMl and RRM2 or RRMl and p53R2 interaction in the subject.
- treating is defined as administration of a composition to a subject, who has a cell proliferation-associated disorder, with the purpose to cure, alleviate, relieve, remedy, prevent, or ameliorate the disorder, the symptom of the disorder, the disease state secondary to the disorder, or the predisposition toward the disorder.
- An "effective amount” is an amount of the composition that is capable of producing a medically desirable result, e.g., as described above, in a treated subject. This method can be performed alone or in conjunction with other drugs or therapy.
- a therapeutic composition e.g., a composition containing a compound identified as described above
- a therapeutic composition is administered to the subject.
- the compound will be suspended in a pharmaceutically-acceptable carrier (e.g., physiological saline) and administered orally or by intravenous infusion, or injected or implanted subcutaneously, intramuscularly, intrathecally, intraperitoneally, intrarectally, intravaginally, intranasally, intragastrically, intratracheally, or intrapulmonarily.
- a pharmaceutically-acceptable carrier e.g., physiological saline
- the compound can be delivered directly to the cancer tissue.
- the dosage required depends on the choice of the route of administration; the nature of the formulation; the nature of the subject's illness; the subject's size, weight, surface area, age, and sex; other drugs being administered; and the judgment of the attending physician. Suitable dosages are in the range of 0.01-100.0 mg/kg. Wide variations in the needed dosage are to be expected in view of the variety of compounds available and the different efficiencies of various routes of administration. For example, oral administration would be expected to require higher dosages than administration by intravenous injection. Variations in these dosage levels can be adjusted using standard empirical routines for optimization as is well understood in the art. Encapsulation of the compound in a suitable delivery vehicle (e.g., polymeric microparticles or implantable devices) may increase the efficiency of delivery, particularly for oral delivery.
- a suitable delivery vehicle e.g., polymeric microparticles or implantable devices
- a polynucleotide containing a nucleic acid sequence encoding an anti-sense RRMl, RRM2, or p53R2 RNA can be delivered to the subject, for example, by the use of polymeric, biodegradable microparticle or microcapsule delivery devices known in the art.
- liposomes prepared by standard methods.
- the vectors can be incorporated alone into these delivery vehicles or co-incorporated with tissue-specific antibodies.
- Poly-L-lysine binds to a ligand that can bind to a receptor on target cells (Cristiano et al. (1995) J Mol Med 73, 479).
- tissue specific targeting can be achieved by the use of tissue-specific transcriptional regulatory elements (TRE) which are known in the art.
- Delivery of "naked DNA" i.e., without a delivery vehicle) to an intramuscular, intradermal, or subcutaneous site is another means to achieve in vivo expression.
- the nucleic acid sequence encoding an anti-sense RRMl, RRM2, or p53R2 RNA is operatively linked to a promoter or enhancer-promoter combination.
- Enhancers provide expression specificity in terms of time, location, and level. Unlike a promoter, an enhancer can function when located at variable distances from the transcription initiation site, provided a promoter is present. An enhancer can also be located downstream of the transcription initiation site.
- Suitable expression vectors include plasmids and viral vectors such as herpes viruses, retroviruses, vaccinia viruses, attenuated vaccinia viruses, canary pox viruses, adenoviruses and adeno-associated viruses, among others.
- Polynucleotides can be administered in a pharmaceutically acceptable carrier.
- Pharmaceutically acceptable carriers are biologically compatible vehicles that are suitable for administration to an animal or a human, e.g., physiological saline or liposomes.
- a preferred dosage for administration of polynucleotide is from approximately 10 ⁇ to 10 12 copies of the polynucleotide molecule. This dose can be repeatedly administered, as needed. Routes of administration can be any of those listed above.
- Antibodies can be used to reduce the level of RRMl, RRM2, or p53R2 protein, or to decrease the level of RRMl and RRM2 or RRMl and p53R2 interaction in a subject.
- the term "antibody” includes intact molecules as well as fragments thereof, such as Fab, F(ab') 2 , and Fv which are capable of binding to an epitopic determinant present in the RRMl, RRM2, or p53R2 protein.
- Methods of making monoclonal and polyclonal antibodies and fragments thereof are known in the art. See, for example, Harlow and Lane, (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York.
- RRMl and RRM2 or RRMl and p53R2 interaction include a peptide or polypeptide containing an amino acid sequence at the C-terminus of RRM2.
- a 7-amino acid peptide (FTLDADF, SEQ ID NO: 5) at the C-terminus of RRM2 was found to dramatically suppress the human RR activity when added to the enzyme in vitro at a ratio of 10:1 (peptide:enzyme). It was further observed that a truncated form of the M2 subunit, lacking these 7 amino acids at the C-terminus, fails to form a complex with the corresponding large subunit Ml.
- Peptides or polypeptides that are functionally equivalent to SEQ ID NO:5 can be either longer or shorter than SEQ ID NO:5, or contain substitute amino acids within SEQ ID NO:5, as long as they are capable of inhibiting the interaction between RRMl and RRM2 or RRMl and p53R2.
- Such peptides or polypeptides can be generated and used according to the methods described above or any other methods well known in the art.
- the invention provides a method of developing a procedure for treating a cell proliferation-associated disorder by providing a plurality of subjects suffering from a cell proliferation-associated disorder (e.g., subjects having amplified RRM2 genomic DNA); administering to each subject an effective amount of an RRM2 inhibitor (e.g., hydroxyurea) followed by an effective amount of a nucleotide or nucleoside analog (e.g., gemcitabine), each at a unique time point; and selecting an optimal time point at which the cell proliferation-associated disorder is inhibited to the greatest extent. Once the optimal time point has been identified, the procedure can be used to treat a cell proliferation- associated disorder in appropriate subjects.
- a cell proliferation-associated disorder e.g., subjects having amplified RRM2 genomic DNA
- an RRM2 inhibitor e.g., hydroxyurea
- a nucleotide or nucleoside analog e.g., gemcitabine
- hRRM204 5' ATTTAGAAGTCAGCATCCAAG3'
- hRRM208 5' TAGACAAACCATCGGAGGAGAGAG3'.
- the cDNA clones representing the 5 '-termini of RRM2 mRNA were obtained by 5 'RACE following the manufacturer's protocol (BRL Lifesciences).
- the primers used to identify the transcription initiation sites corresponded to the hRRM2 cDNA sequence (GenBank Accessing Number x59618) (Pavloff et al. (1992) DNA Seq 2 (4), 227-234) at positions 358 to 338 (hRRM225: 5' GGCTCCTGGAAGATCCTCCT 3') and at positions 242 to 225 (hRRM231: 5' CTGCTGCGGGTCCGTGAT 3').
- a luciferase reporter plasmid containing the 5 '-flanking region of the hRRM2 gene from bp 1787 to bp 2581 of SEQ ID NO:l was constructed by inserting a designated DNA fragment from P6.2 subclone into the pGL3-basic vector (Promega) using the Erase A Base technique. (4) Cell Culture and Transient Transfections
- Human oropharyngeal carcinoma KB cells (American Type Culture Collection) were cultured on plastic tissue culture plates in RPMI 1640 medium supplemented with 10%) fetal calf serum and 100 units/ml of penicillin and streptomycin at 37°C in a humidified atmosphere containing 5% carbon dioxide.
- PC-3 human prostate cancer cells (American Type Culture Collection) were grown under the same conditions. Transient transfections were performed using lipofectin according to the manufacturer's suggested protocol (BRL Lifesciences). Briefly, cells were cultured in 60 mm cell culture dish at a density of 1 x 10 5 cells/dish and transfected with 5 mg/dish of the reporter plasmid.
- hRRM2 Expression After Treatment of Chemotherapeutic Agents and UV Irradiation PC-3 prostate cancer cells were treated with IC 5 Q dose of each chemotherapeutic agent for 48 hours before RNA was extracted from the cells, hi order to induce DNA damage, KB cells were exposed to 2000 J/min UV irradiation and then returned to normal culture conditions. RNA was then extracted at various times. Gel electrophoresis was performed and 20 mg of total RNA was loaded in each lane. The separated RNA was transferred to a membrane and probed with full-length hRRM2 cDNA as described above. Blots were also probed with GAPDH housekeeping gene as an internal control for RNA loading. Blots were quantified on Phosphorlmager. The intensities of the bands in each lane were normalized to the intensity of the GAPDH band. RESULTS (1) Cloning and Sequencing of Genomic hRRM2
- Two hRRM2 cDNAs of 3.4 kb and 1.65 kb have been identified. They include identical coding regions and differ only in the lengths of their untranslated regions. These cDNAs are shown to be aligned with the genomic structure.
- the human RRM2 gene was found to show significant structural similarity to the mouse RRM2 gene, which also consists of 10 exons.
- the transcription initiation site corresponding to the 1.65 kb mRNA is designated as +1 (corresponding to bp 2567 of SEQ ID NO: 1) while transcription of the 3.4 kb mRNA was found to initiate at position -187 (corresponding to bp 2380 of SEQ ID NO:l).
- the site that has been designated as +1 here corresponds to +151 in the RRM2 sequence published by Pavloff et al. ((1992) DNA Seq 2 (4), 227-234).
- a consensus TATA box was found upstream of the first transcription initiation site by manual scanning, but no such site was found upstream of the second transcription initiation site.
- putative transcription factor binding sites include c-Ets, MZF1, E2F, Lyf-1, GATA-X, HSF2, AP-1, CdxA, IK-2, Sox-5, SRY, Brn-2, HNF-1, STATx, GATA-1, USF (c-Myc), Pbx-1, Oct-1, GATA-2, CRE-Bp, and Nkx-2 sites.
- a deletion to -659 was found to express luciferase at a level comparable to the pGL3 promoter control in which luciferase expression is driven by the SV40 promoter lacking any enhancer sequence. Luciferase activity dropped only minimally for deletions between -659 and -257, and then a significant drop in activity occurred with deletion of the sequence between -257 and -236. As there are no identified binding sites for transcription factors within this region, it is unlikely that this change in activity reflects a change due to such a factor.
- the proximity of the upstream site of transcription initiation suggests that the drop in activity may reflect the presence of two separate RRM2 promoter regions, wherein the region between -659 and -257 acts as a promoter for the 3.4 kb RRM2 mRNA while the region between -204 and +1 acts as a promoter for the 1.65 kb RRM2 mRNA.
- This is supported by the observation that significant promoter activity is again observed for the deletion constructs with endpoints at -204, -184, and -156. Luciferase activity again dropped significantly with the deletion of the sequence between -156 and -106, suggesting that some important site was lost.
- There are two CCAAT boxes within this segment of DNA which are likely to be responsible for this drop in activity.
- hRRM2 mRNA Transcripts in Human Tissues
- hRRM2 transcripts As two hRRM2 transcripts have been identified and they appear to be expressed from separate promoters, whether they lead to different expression patterns was further examined, h order to determine whether they are differentially expressed in a tissue specific manner, Northern blot analysis was used to measure the level of each transcript in a variety of human tissues. All tissues examined expressed both forms of mRNA, although their relative levels varied significantly. In placenta, lung, and kidney, the 3.4 kb mRNA is the major form. Conversely, in thymus, testis, colon, and small intestine, the 1.65 kb form predominates.
- the gene is expressed at high levels in thymus, small intestine, colon, and testis, and at lower levels in lung and liver. Very low levels were detected in prostate, skeletal muscle, brain and leukocyte tissues. Interestingly, the size of the hRRM2 mRNA found in the heart tissue was slightly larger than the others, which may reflect differential splicing or alternative transcription. (5) Alteration of l RRM2 mRNA Expression Induced by Chemotherapeutic Agents or UV Treatment
- hRRM2 may also reflect a need to have multiple means of regulating expression in response to different cellular events.
- hRRM2 may require one set of regulatory factors to control expression levels throughout cell cycle and a different means for regulating expression in response to DNA damage.
- the effect of various chemotherapeutic drugs and of UV irradiation on the relative expression levels of the two forms of hRRM2 mRNA was examined.
- PC-3 prostate cancer cell was treated with the IC 50 dose of each agent for 48 hours, and RNA was extracted and examined by Northern blot. The expression of each form of hRRM2 mRNA in drug-treated cells was measured as a relative ratio to the level in untreated cells.
- hRRM2 In order to more specifically examine the effect of DNA damage on the level of hRRM2 expression, cells were exposed to UV irradiation, and then RNA was extracted from cells after 6, 24, and 48 hours. The amount of each hRRM2 mRNA after UV treatment is measured relative to the level in untreated cells. Again, both forms of hRRM2 mRNA are affected in a similar manner by UV treatment. Expression is significantly increased after 6 hours, after which the level dropped back to the level found in untreated cells by 24 hours, and then significantly reduced at the 48-hour time point. The level of induction after 6 hours suggests that the downstream promoter may be preferentially regulated.
- Manassas, VA were grown in plastic tissue culture plates in RPMI 1640 supplemented with 10% fetal bovine serum at 5% CO 2 at 37°C.
- a gemcitabine-resistant clone (KBGem) was selected by stepwise exposure to increasing concentrations of gemcitabine. This cloned cell line was selected and maintained in the presence of 8 mM gemcitabine.
- the ID 5 Q of KB wild-type (KBwt) cells is 0.3 mm, whereas the KBGem clone possesses an ID 50 of 32.3 mm (Goan et al. (1999) Cancer Res 59, 4204-4207).
- a hydroxyurea-resistant clone (KBHURs) was sequentially selected in a stepwise manner in the presence of HU.
- This cloned cell line was selected in the presence of ImM HU (Yen et al. (1994) Cancer Res 54, 3686-3691). To avoid contamination, these two clones were selected and maintained separately by different personnel and facilities.
- the genomic sequence of the human M2 subunit of RR has been published (GenBank Accession Number AY032750) (Zhou et al. (2001) Cytogent Cell Genet 95, 52- 59).
- the following PCR primers were designed to amplify the promoter region of hRRM2 based on the published genomic sequence:
- PAC 621 5' GATCGCTTGATACCAACCTGGG 3'
- PAC 624 5' GGCCACGCCGACATGACTCA 3'
- PAC 6210 5' TCCCTGGGAGATGGATGC 3'
- PAC 6211 5' AAGCCTGGAAAACGCTCC 3'
- PAC 6228 5' TCCCGTAGTTTGAAGGTTTACAA 3'
- HRM 228 5' ACTCCAGCAGCCTTTAAATCTC 3'.
- a hot start PCR technique was employed (Qiagen, CA). The reaction was initiated by heating for 15 min at 95°C, followed by 35 cycles of 40 sec at 94°C, 40 sec at 55°C and 2 min at 72°C, followed by a final incubation for 7 min at 72°C. (3) RRM2 mRNA and Northern Blot Analyses
- the blots were hybridized for 1 hr at 68°C with 32 P -labeled probes. After washing twice with 2 x SSC/0.1% SDS and twice with 0.1 x SSC/0.1% SDS, membranes were exposed for 18-48 hr at -70°C to Kodak XAR-5 film (Eastman Kodak, Rochester, NY) with intensifying screens or exposed to a Phosphohnager screen for 18 hr and then scanned by a Phospholmager.
- Kodak XAR-5 film Eastman Kodak, Rochester, NY
- Protein extracts from each clone were separated by 10% SDS-PAGE, loaded with equal amounts of proteins. Following electrophoresis, the proteins were transferred to a PVDF membrane (American Pharmacia Biotech). Goat anti-human RRM2 polyclonal antibody, purchased from Santa Cruz Biotechnology, Inc. (SC-10844:R2(N-18)), was used for Western blot analysis. The ⁇ -tubulin anti-serum was employed as an internal control.
- DNA sequences of in vitro synthesized double-stranded DNA fragments are as follows:
- NF- B 5' AGTTGAGGGGACTTTCCCAGG 3 '
- OCT1 5' TGTCGAATGCAAATCACTAGAA 3'
- Metaphase images were captured and analyzed on a SKY vision cytogenetic workstation (Applied Spectral Imaging) attached to a Zeiss Axioplan 2 microscope with a 150 W Xenon UV light source.
- Five fluorochrom.es were used in the SKY combinatorial labeling: Spectrum Orange, Texas Red, Cy5, Spectrum Green, and Cy5.5. Instrument adjustments were implemented to reduce artifact caused by weak fluorescence signals or misalignment of the optical system prior to initiation of the study (Garini et al. (1999) Cytometry 35, 214-226).
- 20 or more GTG-banded metaphase cells were analyzed.
- For spectral karyotyping validation 5 to 20 metaphase cells were analyzed.
- chromosome enumeration satellite DNA probes Vysis, Downers Grove, IL
- whole chromosome painting probes Oncor, Gaithersburg, MD
- region-specific probes including YAC (YA153A6) for BCL2 (provided by Dr. Gary A. Silverman, Harvard Medical School, Boston, MA), YAC (812fl 1) for AML1 (provided by Dr.
- chromosome arm 12p and 12q arm-specific paint probes AL Technologies, Rockville, MD
- chromosome arm 19q telomere probe Vysis
- translocation probe Oncor
- chromosome 22 BCR probe Oncor
- MYCN locus-specific probe Oncor
- the KBwt cell line was found to be near triploid ( ⁇ 3n) with hRRM2 hybridization signals on the short arm of tliree "A group” cliromosomes.
- DAPI staining identified the "A group” chromosomes as human chromosome 2.
- the spectrum green labeled hRRM2 PAC DNA probe exhibited signals on chromosome 2p 24-25 of mitotic cells.
- Partial G-banding analysis of the KBGem clone exhibited a modal range of near triploid.
- Hybridization signals were localized to two morphologically normal chromosome 2s, an isochromosome of the short arm of chromosome 2 or i(2)(pl0), and a highly amplified region on a marker chromosome.
- 24-color spectral karyotyping (SKY) was performed. SKY painting indicated the marker chromosome was a derivative of chromosome 13.
- the hsr-bearing derivative chromosome 13 maybe described as der(13)del(13)(ql2ql4)t(2;13)(p24;pll.2)hsr(2)(p24p25). Furthermore, partial G-banding analysis of the KBHURs clone exhibited near triploidy with a highly amplified marker chromosome apparently derived from an I(2p). SKY painting indicated that the marker chromosome also contained chromosome 5 material. FISH using a chromosome 5 painting probe confirmed the chromosome 5 material at the distal end of the der (2) marker chromosome. Suggested nomenclature for this marker is: der(2) i(2) (plO) inv dup hsr (2) (p24 p25)t (2;5) (p25; ?).
- the two forms of hRRM2 mRNA (3.4 kb and 1.65 kb) were detected by the M2 probe.
- KBHURs clones expressed the 1.65 kb form of hRRM2 mRNA at a level 30-fold higher than that of KBwt.
- the 3.4 kb form of hRRM2 mRNA expression in the KBGem clone and the KBHURs clone increased 25-fold relative to KBwt.
- the 1.65 kb mRNA corresponds to transcription from the downstream PI promoter and the 3.4 kb mRNA corresponds to expression initiated from the upstream P2 promoter (Zhou et al. (2001) Cytogenet Cell Genet 95, 52-59).
- hRRM2 protein To determine the amount of hRRM2 protein, equal amounts of total protein extracted from KBwt, KBGem and KBHURs were analyzed by Western blot. The molecular weight of M2 protein is 44 kD. After quantitation, normalized to a-tubulin, KBGem showed a 10.2-fold and KBHURs showed an 11.3-fold increase over KBwt.
- the KBGem and KBHURs clones Compared to KBwt cells, the KBGem and KBHURs clones expressed 5 times more l RRM2 mRNA at the 4-hour time point. After 8 hours, the resistant clones exhibited 10- fold higher expression than the wild-type control. At the 12-hour time point, the KBGem and KBHURs clones expressed l RRM2 at a level 20-25-fold higher than that of KBwt. In these clones, the 3.4 kb mRNA expression was equal to that of the 1.65 kb mRNA at the 4- and 8-hr points, but at 12-hr point, the 1.65 kb mRNA was significantly overexpressed in both resistant clones.
- KBGem sample shows an additional band on the NF- ⁇ B gel that was competed out.
- the KBGem clone did not demonstrate a different binding pattern for AP- 1, AP-2, CREB, and OCT1 when compared to KBwt.
- AP-1 KBGem clone showed less binding than KBwt.
- the binding of AP-2, CREB, and OCT1 was more prominent for the KBGem clone than KBwt.
- KBHURs clone different DNA-protein complexes were seen for almost all of the transcription factors examined when compared to KBwt or KBGem.
- Human oropharyngeal carcinoma KB cells (American Type Culture Collection) were cultured on plastic tissue culture plates in RPMI 1640 supplemented with 10% fetal bovine serum at 37°C in a humidified atmosphere containing 5% carbon dioxide. Growth media were dialyzed to remove purines and pyrimidines in all Gem studies. In all studies, HU was included in the growth medium where indicated at a concentration of 0.1 mM while Gem was provided at a concentration of 0.3 ⁇ M.
- 5xl0 5 cells were seeded in 35-mm dishes and then treated with 0.1 mM HU. At the indicated time points, 7 ⁇ l of 3 H-2'-Deoxycytidine or 4 ⁇ l of 3 H-Gem was added and the plates were incubated for 30 minutes at 37°C in a humidified atmosphere containing 5% carbon dioxide. The plates were then harvested, the DNA purified, its concentration measured, and 10 ⁇ g of DNA were suspended in 5 ml Ecoscint A and counted for radioactivity using a Beckman LS 5000 CE liquid scintillation counter.
- Protein extracts from each clone were separated by 14% SDS-PAGE and then transferred to PVDF membrane (Amersham Pharmacia Biotech). After transferred, the PVDF membranes were suspended in 1% I- blockTM (Applied Biosystems) blocking buffer until detection.
- Goat anti-human RRM2 polyclonal antibody Santa Cruz Biotechnology, Inc. (SC-10844:R2(N-18)
- ⁇ -tubulin anti-serum internal control
- PVDF membranes were incubated with above-mentioned buffer for 45 minutes at room temperature. After washed with 0.5% I-blockTM blocking buffer six times, the PVDF membranes were re-incubated with 1% I-blockTM blocking buffer including 1 :2000 diluted second antibody conjugated to alkaline phosphatase (bovine anti- goat antibody, Santa Cruz) for 30-60 minutes. After washed with 0.5% I-blockTM blocking buffer and assay buffer (200 mM Tris, 10 mM MgCl 2 ), the PVDF membranes were loaded a thin layer of CSPD ® Ready-to-Use substrate solution (Applied Biosystem) and incubated for 5 minutes, then exposed to X-ray film for 3 minutes.
- I-blockTM blocking buffer including 1 :2000 diluted second antibody conjugated to alkaline phosphatase (bovine anti- goat antibody, Santa Cruz) for 30-60 minutes.
- I-blockTM blocking buffer and assay buffer 200 mM Tris, 10 mM Mg
- lxlO 6 permeabilized cells were incubated at 37°C for 10 minutes in 300 ⁇ l of 50 mM HEPES, pH 7.4, 10 mM MgCl 2 , 8 mM dithiothreitol, 0.06 mM FeCl 3 , 7.5 mM potassium phosphate, pH 7.4, 0.75 mM CaCl 2 , 10 mM phosphoenolpyruvate, 0.2 mM 3 H-rCDP, 0.2 mM rGDP, 0.2 mM rADP, 0.2 mM dTDP.
- This assay which measures the amount of DNA synthesis generated by an unknown pool of dNTPs, was conducted according to the method of Sherman and Fyfe as previously described (Zhou et al. (1998) Biochem Pharmaco 55, 1657-1665; and Sherman and Fyfe (1989) Anal Biochem 180, 222-226).
- the reaction mixture contained 50 mM Tris-HCl (pH 7.5), 10 mM MgCl 2 , 5 mM DTT, 0.25 ⁇ M template/primer, 1.25 ⁇ M 3 H-dATP (for dCTP, dGTP, and dTTP assays) or 3 H-dTTP (for dATP assay), and 0.2 units of Sequenase (2.0) in a total volume of 50 ⁇ l. DNA synthesis was allowed to proceed for twenty minutes at room temperature and then the reaction was stopped and 40 ⁇ l of the reaction mixture was spotted onto Whatman DE81 ion exchange paper (2.4cm diameter).
- the cell cycle analysis was performed at flowcytometry core facility of City of Hope National Medical Center. The percent of cells in each phase of the cell cycle was analyzed by ModFit software. The protocol was summarized as below. Cells were trypsinized, washed twice with PBS, and resuspended in 1 ml PBS. 7 ml 0.5 M citric acid buffer (pH 2.35) was added and samples were stored overnight at room temperature in the dark. On the second day, the cells were placed at 4°C in a dark environment. On the third day, cells were spun down, resuspended in citric acid buffer (pH 4.5) and adjusted to a concentration of lxl 0 6 cells/ml.
- the cells were spun down and resuspended in 1 ml BSA-H (0.1 % BSA in PBS buffer). 150 ⁇ g RNase A (DNase free) was added to the solution and cells were incubated at 37°C for 30 minutes. The cells were washed once with BSA-H and the pellet was resuspended in 1 ml lOO ⁇ g/ml propidium iodine (PI) and stored overnight at room temperature in the dark. On the following day, cells were analyzed by flowcytometry (Gao et al. (1998) Biochem Pharmaco 56,105-112). RESULTS
- the colony-forming ability of KB cells treated with HU and/or Gem was assayed using the ID 50 concentrations for KB cells. KB cells were exposed to 0.1 mM HU. 0.3 ⁇ M Gem was added after 2 to 24 hours of HU treatment, and then cells were assayed for their colony forming ability. When cells were treated with HU or Gem alone, colony formation was reduced to approximately 50% of that seen for untreated controls. When HU and Gem were added together or 2 hours apart, colony-forming ability was similar to that seen for HU or Gem alone. When Gem was added to the growth medium 4 hours after initiation of HU treatment, a markedly enhanced inhibition was observed. It reached a nadir when Gem was added after 8 hours of exposure to HU. Under these conditions, colony formation was reduced by 86% when compared to untreated cells. The inhibition of colony forming ability gradually abated when treatments were separated by 12 to 24 hours but continued to show some enhancement over that seen for either drug alone.
- Northern blot analysis was used to measure the level of l ⁇ RRM2 mRNA after exposure to HU at various time points. KB cells growing in logarithmic phase were exposed to 0.1 mM HU and harvested after 0, 2, 4, 8, 12 16 and 24 hours. Total cellular RNA was extracted and analyzed by Northern blot (20 ⁇ g per lane). PCR products, consisting of full-length hRRM2 subunit cDNA sequences, were used as probes. The amount of RNA detected was measured by phosphorimager and results were normalized to the amount of GAPDH mRNA detected on the same blot. The amount of hRRM2 mRNA decreased by 20% relative to the untreated sample after 2 hours and then decreased to 60% of control levels at the 4-hour time point.
- each dNTP pool was measured as described in the Materials and Methods.
- dATP and dCTP were the most susceptible of the four dNTP pools to inhibition by HU.
- a decrease in these intracellular pools commenced after 1 hour of exposure to HU. The decline continued, with the dCTP pool reaching a nadir of approximately 15% of the control at 4 hours.
- the dATP pool reached a nadir after 8 hours.
- the dATP and dCTP pools slowly recovered to about 80% of their starting level.
- the dTTP and dGTP pools did not vary significantly over time.
- Hydroxyurea was administered orally at 500 mg every 6 h for four doses on days 1 and 8 of each cycle.
- Gemcitabine was administered as a 30-min infusion 6 h after the fourth dose of hydroxyurea (i.e., on days 2 and 9). Patients were required to have a platelet count
- G-CSF Granulocyte colony stimulating factor
- Hematologic dose-limiting toxicity was defined as a platelet count ⁇ 75,000/ ⁇ l on day 8, or a platelet count ⁇ 25,000/ ⁇ l lasting seven or more days, an ANC ⁇ 1,000/ ⁇ l on day 8 or an ANC ⁇ 500/ ⁇ l lasting seven or more days.
- Hepatic DLT was defined as bilirubin 4.5-6.0 mg/dl or transaminase 8.0-20.0 times normal (grade 3), and bilirubin > 6.0 mg/dl or transaminase more than 20.0 times normal (grade 4). Other toxicities were graded according to the NCI Common Toxicity Criteria Version 1.0.
- MTD maximum tolerated dose
- Plasma samples for determination of plasma gemcitabine were collected on days 2 and 9 of the first cycle. Samples were collected at the following times: before gemcitabine, just prior to the end of the 30 min infusion, and 5, 10, 15, 30, and 45 min after the end of the infusion. Blood sample were collected in heparinized tubes and kept on ice until plasma could be separated by centrifugation (within 30 min). Plasma was transferred to polypropylene tubes containing tetrahydrouridine to inhibit cytidine deaminase and stored at -70°C until analysis.
- Gemcitabine in plasma was measured by a novel reversed-phase HPLC assay that was developed and validated in the City of Hope Analytical Pharmacology Core Facility. Following addition of the internal standard (IS), 2'3'-dideoxycyditine (Sigma Chemical Company, St. Louis, MO.), gemcitabine was extracted from plasma by solid-phase extraction using a 1-ml aromatic sulfonic acid cartridge (JT Baker, Phillipsburg, N. J.).
- Plasma gemcitabine data were analyzed by compartmental methods using ADAPT II software (USC Biomedical Simulations Resource, Los Angeles, Calif). Data were fitted to a two-compartment model with first-order elimination. Secondary pharmacokinetic parameters (CL SVS V, and t ⁇ ) were determined and compared within and among patients.
- gemcitdbine one patient was replaced because the patient did not receive a complete cycle due to a pleural effusion, and became inevaluable for both toxicity and response. Another patient at this dose was not treated on days 8 and 9 of cycle 1 due to a grade 3 rash, but completed a second cycle. This patient was replaced in the evaluation of toxicity, but was included in the evaluation of response. At the dose of 875 mg/m 2 , one patient was not treated on days 8 and 9 of cycle 1 due to a urinary tract infection, but eventually completed five cycles of therapy. This patient was replaced in the evaluation of toxicity also, but was included in the evaluation of response.
Abstract
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KR10-2004-7015589A KR20050002917A (en) | 2002-03-29 | 2003-03-25 | Human ribonucleotide reductase m2 subunit |
JP2003582269A JP2005521418A (en) | 2002-03-29 | 2003-03-25 | Human ribonucleotide reductase M2 subunit |
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US9228186B2 (en) | 2002-11-14 | 2016-01-05 | Thermo Fisher Scientific Inc. | Methods and compositions for selecting siRNA of improved functionality |
US9839649B2 (en) | 2002-11-14 | 2017-12-12 | Thermo Fisher Scientific Inc. | Methods and compositions for selecting siRNA of improved functionality |
US9719094B2 (en) | 2002-11-14 | 2017-08-01 | Thermo Fisher Scientific Inc. | RNAi targeting SEC61G |
US20090227780A1 (en) * | 2002-11-14 | 2009-09-10 | Dharmacon, Inc. | siRNA targeting connexin 43 |
US7951935B2 (en) | 2002-11-14 | 2011-05-31 | Dharmacon, Inc. | siRNA targeting v-myc myelocytomatosis viral oncogene homolog (MYC) |
US7619081B2 (en) * | 2002-11-14 | 2009-11-17 | Dharmacon, Inc. | siRNA targeting coatomer protein complex, subunit beta 2 (COPB2) |
US9771586B2 (en) | 2002-11-14 | 2017-09-26 | Thermo Fisher Scientific Inc. | RNAi targeting ZNF205 |
EP2314691A3 (en) * | 2002-11-14 | 2012-01-18 | Dharmacon, Inc. | Fuctional and hyperfunctional siRNA |
US9879266B2 (en) | 2002-11-14 | 2018-01-30 | Thermo Fisher Scientific Inc. | Methods and compositions for selecting siRNA of improved functionality |
US7612196B2 (en) | 2002-11-14 | 2009-11-03 | Dharmacon, Inc. | siRNA targeting cyclin-dependent kinase inhibitor 1B (p27, Kip1) (CDKN1B) |
US10011836B2 (en) | 2002-11-14 | 2018-07-03 | Thermo Fisher Scientific Inc. | Methods and compositions for selecting siRNA of improved functionality |
US20080268457A1 (en) * | 2002-11-14 | 2008-10-30 | Dharmacon, Inc. | siRNA targeting forkhead box P3 (FOXP3) |
US9719092B2 (en) | 2002-11-14 | 2017-08-01 | Thermo Fisher Scientific Inc. | RNAi targeting CNTD2 |
US7592442B2 (en) * | 2002-11-14 | 2009-09-22 | Dharmacon, Inc. | siRNA targeting ribonucleotide reductase M2 polypeptide (RRM2 or RNR-R2) |
US7691998B2 (en) * | 2002-11-14 | 2010-04-06 | Dharmacon, Inc. | siRNA targeting nucleoporin 62kDa (Nup62) |
US7781575B2 (en) | 2002-11-14 | 2010-08-24 | Dharmacon, Inc. | siRNA targeting tumor protein 53 (p53) |
US8198427B1 (en) | 2002-11-14 | 2012-06-12 | Dharmacon, Inc. | SiRNA targeting catenin, beta-1 (CTNNB1) |
US7635770B2 (en) * | 2002-11-14 | 2009-12-22 | Dharmacon, Inc. | siRNA targeting protein kinase N-3 (PKN-3) |
US20100113307A1 (en) * | 2002-11-14 | 2010-05-06 | Dharmacon, Inc. | siRNA targeting vascular endothelial growth factor (VEGF) |
US7977471B2 (en) * | 2002-11-14 | 2011-07-12 | Dharmacon, Inc. | siRNA targeting TNFα |
US7605250B2 (en) * | 2004-05-12 | 2009-10-20 | Dharmacon, Inc. | siRNA targeting cAMP-specific phosphodiesterase 4D |
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US9799416B2 (en) * | 2009-11-06 | 2017-10-24 | Terrapower, Llc | Methods and systems for migrating fuel assemblies in a nuclear fission reactor |
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WO2011003194A1 (en) * | 2009-07-10 | 2011-01-13 | The Governors Of The University Of Alberta | Oncolytic viruses and methods for treating neoplastic disorders |
US8679509B2 (en) | 2009-07-10 | 2014-03-25 | The Governors Of The University Of Alberta | Oncolytic viruses and methods for treating neoplastic disorders |
US9370550B2 (en) | 2009-07-10 | 2016-06-21 | The Governors Of The University Of Alberta | Oncolytic viruses and methods for treating neoplastic disorders |
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