Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
  • A61K31/4164—1,3-Diazoles
  • A61K31/4184—1,3-Diazoles condensed with carbocyclic rings, e.g. benzimidazoles
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7084—Compounds having two nucleosides or nucleotides, e.g. nicotinamide-adenine dinucleotide, flavine-adenine dinucleotide
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7088—Compounds having three or more nucleosides or nucleotides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

• the invention relates to compositions and methods for diagnosing, prognosing and treating cancer which generally utilize Rad51 inhibitors and Rad51 expression detectors
• BRCA1 and BRCA2 are responsible for more than 65% of familial forms of breast cancer whereas mutations in these genes are rare in sporadic cases (Feunteun, J , Mol Med Today 4 263-270 (1998)
• BRCA1 function is also lost in sporadic breast cancer due to down-regulation of BRCA1 protein levels in tumor cells (Yoshikawa, K , et al , din Cane Res 5 1249-1261 (1999), Wilson, C A , et al , Nat Genet 21 236-240 (1999), Dobrovic, A and Simpfendorfer, D , Cane Res 57 3347-3350 (1997, Mancini,
• BRCA1 is directly involved in pathways that respond to DNA damage (Zhang, H , et al , Ce// 92 433-436 (1998))
• the poly peptide is part of a multi protein complex, which also contains Rad51 (Scully, R , et al , Cell 88 265-275 (1997)), a key enzyme of homologous recombination and the repair of DNA double strand breaks (Feunteun, J , Mol Med Today 4 263-270 (1998), Baumann, P and West, S C , Trends Biochem Sci 23 247-251 (1998))
• homologous recombination is one of the mechanisms involved in the repair of DNA double strand breaks
• One of the key-factors catalyzing these processes is the product of the rad51 gene Induced disruption of the rad51 gene in chicken cells leads to cell death accompanied by the accumulation of DNA double-strand breaks (Sonoda, E , et al , EMBO J , 17 598- 608 (1998)) Elevated expression of Rad51 enhances radioresistance of human tumor cells
• Homologous recombination of DNA is one of the driving forces of genetic variety and evolution, but on the other hand, the same mechanism guarantees maintenance of genomic stability by participation in the repair of DNA double strand breaks
• the product of the recA gene is known as one of the key factors, catalyzing homologous recombination processes in prokaryotes like Esche ⁇ chia coll In eukaryotes, members of the Rad51 family of proteins share remarkable structural and functional homology with E coll RecA In bacteria and yeast, RecA/Rad51 deficiency leads to a drop in recombination rate and high sensitivity to y-irradiation without affecting overall cell survival
• mouse embryos lacking functional Rad51 die early in development just prior to gastrulation and efforts to establish Rad51 deficient mammalian cell lines have failed (Lim, D S & Hasty, P , Mol Cell Biol 16 7133-43 (1996), Tsuzuki, T , et al , Proc Natl Acad Sci USA
• Rad51 physically interacts with several tumor suppressors like the BRCA-1 (Scully, R , et al , Cell 88 265-75 (1997b)) and BRCA-2 (Sharan, S K , et al , Nature 386 804-10 (1997)), polypeptides defective in hereditary forms of breast and ovarian cancer
• BRCA-1 Stemcells
• BRCA-2 Stemcells
• Mouse embryos lacking functional BRCA1 or BRCA2 display a similar phenotype as Rad51 deficient mouse embryos (Hakem, R , et al , Cell 10 85 1009-23 (1996), Suzuki, A , et al , Genes Dev 11 1242-52 (1997), Chen, J J , et al , Mol Cell 2 317-
• the tumor suppressor p53 is the most prominent regulator of p21 Waf1 expression p53 maintains genomic stability by controlling for DNA integrity and, as appropriate, responds with halting the cell cycle or by inducing cell death by apoptosis (for review see, (Janus, F , et al , Cell Mol Life Sci 55 12-27 (1999)) p53 also forms protein complexes with Rad51 and suppresses biochemical activities of the bacterial homologue RecA in vitro (Sturzbecher, H W , et al , Embo J 15 1992-2002
• the present invention provides a number of methods of diagnosis and prognosis, predictive outcome methods and methods of treating cancer
• Methods of inhibiting and inducing apoptosis are also provided
• a method of diagnosing an individual for cancer comprises determining the level of Rad51 expression in a sample from an individual, and comparing said level to a control level wherein a change from said control indicates cancer
• the sample is preferably a tissue sample or cells which have been cultured in spheroids
• Various cancers can be diagnosed by said method, including but not limited to breast cancer, brain cancer, pancreatic cancer, prostate cancer, colon cancer, lymphoma, and skin cancer
• Rad51 expression can be determined by the level of Rad51 protein or nucleic acid, protein being preferred In one embodiment, the level is determined through the use of polyclonal antibodies Preferably, the level is determined through the use of monoclonal antibodies In one embodiment, said antibodies are raised against eukaryotic Rad51 , preferably, mammalian Rad51 Alternatively, the Rad51 expression is determined by the level of Rad51 nucleic acid
• a method of prognosing an individual for cancer comprises determining the level of Rad51 expression in a sample from an individual, and comparing said level to a control which indicates the severity of cancer so as to provide a prognosis Generally, the higher level of Rad51 expression in said individual the less time the patient has to live without treatment
• the method comprises determining the level of Rad51 in a primary tissue sample of interest and comparing said level of Rad51 to a non-cancer tissue sample, wherein a difference in said level indicates a cancer cell is in the tissue sample of interest
• a kit for detecting a normal or abnormal level of Rad51 expression in a tissue sample comprises a binding agent for detecting Rad51, a detectable label, and a control which indicates a normal level of Rad51 expression or Rad51 expression at various seventies of cancer
• a Rad51 inhibitor is administered to said individual in an amount effective to inhibit cancer in said individual
• Rad51 or a Rad51 inhibitor is administered to a cell which comprises dysfunctional p53 As shown herein, there is not a requirement that p53 be present for the methods provided herein Therefore, in one embodiment, p53 is excluded from administration in conjuction with a Rad51 inhibitor
• Also provided herein is a method for inducing sensitivity to radiation and DNA damaging chemotherapeutics in an individual with cancer comprising administering inhibiting Rad51 activity in said individual.
• a composition comprising a Rad51 inhibitor is administered to said individual in an amount effective to induce said sensitivity
• a method of inducing apoptosis in a cell which comprises administering a Rad51 inhibitor to said cell
• the cell is a cancer cell
• a method of determining a predictive outcome of a treatment for cancer Predictive outcome is a term which indicates whether or not a treatment will be effective for a certain condition
• the method comprises determining the level of Rad51 expression in a tissue sample of a patient and correlating said level with a control which indicates the resistance a patient will have to chemotherapy or radiation treatments The greater the level of Rad51 , generally the greater resistance the patient will have
• a method of inhibiting apoptosis in a cell comprising inducing overexpression of Rad51 in a cell Overexpression means more expression than would be found in a normal unaffected cell Inducing overexpression can be by a variety of ways including administering
• Rad51 protein Rad51 nucleic acid or by indirectly stimulating Rad51 expression
• inducing is by administration of a Rad51 nucleic acid
• a nuclear localization signal is joined to said nucleic acid
• Also provided herein is a method of enhancing survival of a cell comprising inducing overexpression of
• the method comprises cultunng cells in spheroids and adding a candidate agent to said spheroids and determining Rad51 expression levels before and after adding said candidate agent, wherein a change indicates said candidate agent modulates Rad51 expression
• the agent inhibits expression
• the spheroids can also be used to determine effective treatments
• the spheroids can be used to identify agents which modulate Rad51 activity BRIEF DESCRIPTION OF THE DRAWINGS
• Figures 1A- F show comparison by Rad51 and p53 expression levels in monolayer cell culture by immunohistochemistry (Figuers 1A-1 D) and Western-Blotting (Figures 1E-1 F) More particularly, in Figures 1A-1D, immunocytochemistry of Rad51 ( Figure 1B and 1 D) and p53 ( Figures 1A and 1C) in pancreatic cancer cell lines are shown Immunostaining of cell lines 818-4 ( Figures 1A-1 B) and BXPC-3 ( Figures 1C-1 D), respectively, was performed using monoclonal antibody 1G8 for Rad51 and monoclonal antibody PAb1801 for p53 Before staining, cells were fixed in 3,7% neutral buffered formalin and permeabi zed with 0,2% ⁇ ton X-100 Rad 51 and p53 proteins were visualized using diaminobenzidme tetrahydrochlonde as substrate for peroxidase, counterstainmg with hemalum
• Figures 2A-2H show accumulation of Rad51 protein in human pancreatic cancer cell lines grown as spheroids More particularly, Figures 2A-2F show PancTU-l and 818-4 cells which were grown as spheroids and analyzed by immunohistochemistry Spheroids were harvested, formalin fixed and paraffin embedded Rad51 was detected using monoclonal antibody 1G8 and visualized using diaminobenzidme tetrahydrochlonde as substrate for peroxidase, counterstainmg with hemalum The order of magnification as indicated Figures 2G-2H show Western blot analysis of Rad51 protein in 50 ⁇ g of total lysates from PancTU-l ( Figure 2G) and 818-4 ( Figure 2H) pancreatic cell lines grown as monolayer (lane a) or as spheroid (lane b) using monoclonals 1G8 for Rad51 detection
• FIG. 3A-3H shows comparison of Rad51 expression in pancreatic cancer cells, grown either as monolayer, as tumor in SCID mice after orthotopic transplantation or in different tumor specimens of pancreatic adenocarcinoma Rad51 expression was determined by immunohistochemistry in A-B) Panc-TUI monolayer cells, C-E) Panc-TUI cells growing as tumors in SCID mice after orthotopic transplantation or F-H) in different specimens of human pancreatic adenocarcinoma Specimens were harvested, formalin fixed and paraffin embedded Rad51 was detected using monoclonal antibody 1 G8 and visualized using diaminobenzidme tetrahydrochlonde as substrate for peroxidase, counterstainmg with hemalum Order of magnification as indicated
• Figure 4 shows specificity of Rad51 immunohistochemistry determined by peptide competition More particularly, Figure 4 shows a competition experiment using a peptide corresponding to the 1G8 epitope Rad51 was stained with the monoclonal
• Figure 5 shows mutation analysis-of the Rad51 coding sequence using the non-isotopic RNASE cleavage assay (NIRCA) Negative control NIRC-assay performed on hybridized sense and anti-sense wild-type Rad51 mRNA, positive control RNASE cleavage using wild-type sense and an in vitro generated point mutant (anti-sense) of Rad51 , Capan-1, HPAF NIR-assay performed on Rad51 mRNA isolated from the pancreatic tumor cell lines Capan-1 and HPAF, respectively, hybridized to genuine wild-type Rad51 mRNA, a) control without RNASE digestion, b) c) d) RNASE 1 , 2, 3 digestion with the enzymes provided by the supplier, wt wild-type Rad51 mRNA, mt mutant Rad51 mRNA, S sense mRNA, AS anti-sense mRNA RNA fragments were analyzed on 2% agarose gels and stained with ethidium bromide Shown is an inverted print of the gel using
• Figure 6 shows biological consequences of Rad51 over-expression
• Figure 6A shows that p53 levels are unaffected by over-expression of Rad51 U ⁇ Rad51 and UiLacZ cells were plated and allowed to sit for 24h At that time, induction of ectopic protein production was induced with 1 ⁇ M mu ⁇ sterone A, non-induced cells received 1%o ethanol, the solvent used for munsterone A UV irradiation was carried out after additional 24h and cells were harvested 24h thereafter Equal numbers of cells were subjected to Western blotting for p53 protein detection
• Figure 6B shows over-expression of Rad51 confers resistance to DNA double strand breaks U ⁇ Rad51 cells were plated at identical cell numbers and allowed to adhere for 24h
• Induction of ectopic protein production was induced with 1 ⁇ M munsterone A, non-induced cells received 1%o ethanol 24h after induction, cells were treated with calicheamicin ⁇ 1 at the concentrations indicated for 16h, washed three times in complete medium and allowed
• Figure 7 shows a map of the 5'-reg ⁇ on of the human rad51 gene Regulatory region fine hatched, exons black, mtrons coarsed, nucleotides 700 to 1560 are shown, putative factor binding sites are boxed
• Figures 8A-L show expression of RadG1, p53, K ⁇ 67-ant ⁇ gen and BRCA1 in relation to tumor grading
• a collection of specimens from invasive ductal carcinoma of different histological grading (G1 , G2, G3) were stained with monoclonal antibodies 1G8 (anti Rad51) Do-1 (anti p53), MIB-1 (anti K ⁇ 67-ant ⁇ gen), and AB-1 (anti BRCA1) Counterstainmg with Hemalum
• Figure 9 shows a graph showing the correlations between Rad51 , p53, K ⁇ 67-ant ⁇ gen and BRCA1 expression and established tumor parameters r s Spearman s rank correlation coefficient, */**/*** p ⁇ 0 05/0 01/0 001 , T tumor size, N nodal status, G histological grading, ES estrogen receptor status, PS progesterone receptor status, PCI positive stained cell index, IRS immunoreactive score (Remmele and Stegner, 1987), SH staining intensity index
• Figures 10A-10D show representative staining patterns of BRCA1 Various specimens of invasive ductal breast cancer were stained for BRCA1 expression using monoclonal antibody AB-1 (0) no BRCA1 specific staining (blue nuclei due to Mayer's Hemalum counter-staining), (1) more than 10% of tumor cells show weak BRCA1 staining (grey color), (2) clear BRCA1 staining with more than 10% of brown tumor cell nuclei, (3) intense brown staining of more than 10% of tumor cell nuclei
• Figures 11 A-11C show ectopic expression of Rad51 causes cell cycle arrest in U ⁇ Rad51 cells More particularly, Figure 11 A shows U ⁇ Rad51 cells mducibly over-express ectopic Rad51 protein Cells were grown in the presence or absence of munsterone A as indicated by .+ ' and . - " , respectively
• Figures 12A-12C shows ectopic expression of Rad51 transcnptionally induces expression of p2l Waf1 protein without p53 activation More particularly, Figure 12A shows Rad51 triggers p21Waf-l protein expression U ⁇ Rad51 cells were plated and allowed to sit for 24h At that time, cells shown in lanes 1 and 2 received 1 %o ethanol, the solvent used for munsterone A, while those represented in lanes 3 and 4 were supplemented with munsterone A UV irradiation was carried out after additional 24h (lanes 2 and 4) and cells were harvested 24h thereafter Equal numbers of cells were subjected to Western blotting for p21 Waf1 protein detection by using monoclonal antibody 6B6 (Pharmingen) Figure 12B shows Rad51 induces transcriptional activation of the waf-1 promoter Equal numbers of non-induced UiLacZ and U ⁇ Rad51 cells were transfected with reporter plasmid WWP-Luc ((el Deiry, W S ,
• Figures 13A-13C show Rad51 induced cell cycle arrest is lost after prolonged Rad51 over i expression
• Figure 13A shows Rad51 arrested U ⁇ Rad51 cells re-enter the cell cycle despite over-expression of Rad51 Cells were induced with munsterone A for the time indicated and the distribution of cell cycle phases determined by flow cytometry G, dark gray, S black, G 2 /M light gray
• Figure 13B shows p21 Wa 1 protein level decreases while cells re-enter proliferation
• Equal cell numbers derived from ( Figure 13A) were analyzed for p21 Waf1 expression by Western Blot using monoclonal antibody 6B6 (Pharm gen)
• Figure 13C shows Rad51 does not induce transcriptional activation of the waf-1 promoter after adaptation
• Equal numbers of non-induced and adapted U ⁇ Rad51 cells were transfected with reporter plasmid WWP-Luc (el Deny, W.S ⁇ _et al , Cell 75 817-25 (1993)) Cells were induced with
• Figures 14A-14B show adaptation to Rad51 over-expression does not affect UV triggered cell cycle arrest pathways Particularly, Figure 14A shows re-induction of Rad51 in adapted cells does not lead to cell cycle arrest Ponasterone A was removed from long-term induced (>28d) U ⁇ Rad51 cells for 14d (Panel 1) or for 11d followed by re-induction for 72h (Panel 2) and cells analyzed by flow cytometry
• Panels 3 and 4 Cells were treated as in panels 1 and 2, respectively, but in addition cells were UV-irradiated 24h prior to harvest
• Figure 14B shows that p53 accumulates after UV-irradiation of adapted cells
• Cells were treated as in (A) An aliquot each was lysed for Western blot analysis Lysates from equal cell numbers were applied to each lane and analyzed for p53 protein by using a polyclonal sheep anti p53 serum Lane numbers correspond to panel numbers in Figure 14A DETAILED DESCRIPTION OF THE INVENTION
• the present invention is directed to a series of discoveries relating to the pivotal role that Rad51 plays in a number of cellular functions, including those involved in disease states
• compositions and methods for inhibiting Rad51 and methods of treatment for disease states associated with Rad51 activity as further defined below using Rad51 inhibitors
• methods regarding the regulation of apoptosis are also provided.
• a method which comprises first determining the level of Rad51 expression in a first tissue type of a first individual, i e the sample tissue for which a diagnosis or prognosis is required
• the testing may be done on one or more cells cultured as spheroids or a primary tissue sample
• the first individual, or patient is suspected of being at risk for the disease state, and is generally a human subject, although as will be appreciated by those in the art, the patient may be animal as well, for example in the development or evaluation of animal models of human disease
• animals including mammals such as rodents (including mice, rats, hamsters and guinea pigs), cats, dogs, rabbits, farm animals including cows, horses, goats, sheep, pigs, etc , and primates (including monkeys, chimpanzees, orangutans and gorillas) are included within the definition of patient
• the tissue type tested will depend on the disease state under consideration
• potentially cancerous tissue may be tested, including breast tissue, skin cells, pancreas, prostate, colon, solid tumors, brain tissue, etc
• the disease state under consideration is cancer and the tissue sample is a potentially cancerous tissue type
• Rad51 expression as used herein means any form of expression, at the protein or nucleic acid level Preferably, expression level is determined at the protein level and the nucleic acid level is excluded from the determination
• the control may be another control experiment on an unaffected sample such as from another individual or another tissue of the same individual, or it may be a chart, graph or diagram which indicates the "normal" range of levels of Rad51 in an individual similar to the one being tested.
• the severity may be determined by having one or more controls and determining how different the test results are from the control. The greater the level of Rad51 in the test results over the control indicates a greater severity of cancer.
• a number of controls may be provided such that the results can be matched with a control which shows a predetermined severity of cancer. By determining the severity, a prognosis can also be provided.
• a change is generally from at least about 5% to about 500% or more, more preferably 20% to 100%, and sometimes more than a 200% increase, sometimes more than a 300% increase, sometimes more than a 400% increase, sometimes more than a 500% increase, and sometimes more than a 750% increase, Generally, to see this effect, at least about 100 cells should be evaluated, with at least about 500 cells being preferred, and at least about 1000 being particularly preferred.
• the level of Rad51 expression can be determined in a variety of ways.
• a labeled binding agent that binds to Rad51 is used.
• labeled herein is meant that a compound has at least one element, isotope or chemical compound attached to enable the detection of the compound.
• labels fall into three classes: a) isotopic labels, which may be radioactive or heavy isotopes; b) immune labels, which may be antibodies or antigens; and c) colored or fluorescent dyes.
• the labels may be incorporated into the compound at any position.
• Preferred labels are fluorescent or radioactive labels.
• the binding agent can either be labeled directly, or indirectly, through the use of a labeled secondary agent which will bind to the first binding agent.
• the spheroids or tissue sample are prepared as is known for cellular or in situ staining, using techniques well known in the art, as outlined in the Examples.
• the binding agent used to detect Rad51 protein is an antibody.
• the antibodies may be either polyclonal or monoclonal, with monoclonal antibodies being preferred.
• antibodies to the particular Rad51 under evaluation be used; that is, antibodies directed against human Rad51 are used in the evaluation of human patients.
• homology between different mammalian Rad51 molecules is quite high (73% identity as between human and chicken, for example), it is possible to use antibodies against Rad51 from one type of animal to evaluate a different animal (mouse antibodies to evaluate human tissue, etc.).
• antibodies raised against eukaryotic Rad51 are used, with antibodies raised against mammalian Rad51 being especially preferred
• antibodies raised against yeast, human, rodent, primate, and avian Rad51 proteins are particularly preferred
• the protein used to generate the antibodies need not be the full-length protein, fragments and derivatives may be used, as long as there is sufficient immunoreactivity against the sample Rad51 to allow detection Alternatively, other binding agents which will bind to Rad51 at sufficient affinity to allow visualization can be used
• expression levels are determined by determining mRNA levels of Rad51
• a method for inhibiting at least one Rad51 biological or biochemical activity comprises administering a Rad51 inhibitor to a composition comprising Rad51
• the composition can be an in vitro solution comprising Rad51 and Rad51 binders such as DNA and ATP under conditions which allow Rad51 activity
• the composition is a cell
• the Rad51 inhibitor is a small molecule
• Rad51 biological or biochemical activity as used herein can be selected from the group consisting of DNA dependent ATPase activity, formation of Rad51 foci, nucleic acid strand exchange, DNA binding, nucleoprotem filament formation, DNA pairing and DNA repair DNA repair and recombination are generally considered biological activities
• DNA repair can be double stranded break repair, single stranded annealing or post replication recombinational repair
• a Rad51 inhibitor inhibits cell proliferation
• a Rad51 inhibitor results in the cells containing it to be more sensitive to radiation and/or chemotherapeutic agents
• a Rad51 inhibitor induces apoptosis as further described below
• a Rad51 inhibitor or an agent or composition having Rad51 inhibitory activity is defined herein as an agent or composition inhibiting expression or translation of a Rad51 nucleic acid or the biological activity of a Rad51 peptide by at least 30%, more preferably 40%, more preferably 50%, more preferably 70%, more preferably 90%, and most preferably by at least 95%
• a Rad51 inhibitor inhibits expression or translation of a Rad51 nucleic acid or the activity of a Rad51 protein by 100%
• inhibition is defined as any detectable decrease in Rad51 activity compared to a control not comprising the Rad51 inhibitor
• Rad51 inhibitors can include inhibitors of Rad51 homologues such as RecA and/or inhibitors that sensitize cells to radiation and also affect aspects of recombination in vivo, which were not previously known to inhibit Rad51
• Rad51 as used herein refers to Rad51 and its homologues, preferably human homologues
• Rad51 excludes non-human homologues
• Rad51 inhibitors provided herein were not previously known to inhibit RecA or other Rad51 homologues and were not known to induce sensitizing of cells to radiation
• Rad51 as used herein excludes homologues thereof
• the Rad51 inhibitor can inhibit Rad51 directly or indirectly, preferably directly by interacting with at least a portion of the Rad51 nucleic acid or protein Additionally, the inhibitors herein can be utilized individually or in combination with each other
• the small molecule is preferably 4 kilodaltons (kd) or less In another embodiment, the small molecule is less than 3 kd, 2kd or 1 kd In another embodiment the small molecule is less than 800 daltons (D), 500 D, 300 D, 200 D or 100 D
• the Rad51 inhibitor is an inorganic or organic molecule
• the Rad51 inhibitor is a small organic molecule, comprising functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically will include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups
• the Rad51 inhibitor may comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more chemical functional groups
• Rad51 inhibitors can comprise nucleotides, nucleosides, and nucleotide and nucleoside analogues
• Nucleotides as used herein refer to XYP, wherein X can be U, T, G, C or A (base being uracil, thymme, guanme, cytosme or adenme, respectively), and Y can be M, D or T (mono, di or tri, respectively)
• nucleotides can include xathanine,
• the Rad51 inhibitor is a nucleotide diphosphate
• the Rad51 inhibitor is selected from the group consisting of ADP, GDP, CDP, UDP and
• ADP is excluded
• the Rad51 inhibitor is a nucleotide analogue
• the Rad51 inhibitor is a nucleotide diphosphate complexed with aluminum fluoride
• the Rad51 inhibitor is selected from the group consisting of ADP AIF4, GDP AIF4,
• CDP AIF4 UDP AIF4 and TDP AIF4
• the Rad51 inhibitor is a non-hydrolyzable nucleotide
• the Rad51 inhibitor is selected from the group consisting of ATPyS, GTPyS, UTPyS, CTPyS, TTPYS, ADPyS, GDPyS, UDPyS, CDPYS, TDPyS, AMPyS, GMPyS, UMPyS,
• ADPyS is excluded
• the Rad51 inhibitor is a DNA minor groove binding drug
• the Rad51 inhibitor is selected from the group consisting of distamycin, netropsm, bis- benzimidazole and actinomycm
• the Rad51 inhibitor is a peptide
• peptide herein is meant at least two covalently attached ammo acids, which includes proteins, poiypeptides, oligopeptides and peptides
• the protein may be made up of naturally occurring ammo acids and peptide bonds, or synthetic peptidomimetic structures
• “ammo acid”, or “peptide residue”, as used herein means both naturally occurring and synthetic ammo acids
• homo-phenylalanme, citrullme and noreleucine are considered ammo acids for the purposes of the invention
• “Ammo acid” also includes imino acid residues such as prolme and hydroxyprolme
• the side chains may be in either the (R) or the (S) configuration In the preferred embodiment, the ammo acids are in the (S) or L-configuration If non-naturally occurring side chains are used, non-ammo acid substituents may be used, for example to prevent or retard in vivo degradations
• the peptides can be naturally occurring or fragments of naturally occu ⁇ ng proteins
• cellular extracts containing proteins, or random or directed digests of protemaceous cellular extracts may be used
• procaryotic and eukaryotic proteins can
• the Rad51 inhibitors are peptides of from about 5 to about 30 ammo acids, with from about 5 to about 20 ammo acids being preferred, and from about 7 to about 15 being particularly preferred
• the peptides may be digests of naturally occu ⁇ ng proteins as is outlined above, random peptides, or "biased" random peptides
• ' randomized or grammatical equivalents herein is meant that each nucleic acid and peptide consists of essentially random nucleotides and ammo acids, respectively Since generally these random peptides (or nucleic acids, discussed below) are chemically synthesized, they may incorporate any nucleotide or ammo acid at any position
• the synthetic process can be designed to generate randomized proteins or nucleic acids, to allow the formation of all or most of the possible combinations over the length of the sequence
• Preferred peptides include p53 and Rad51 antibodies and include but are not limited to ammo acids 94-160 and 264-315 of p53 and fragments of Rad51 antibodies
• the Rad51 inhibitors are nucleic acids By “nucleic acid” or
• oligonucleotide or grammatical equivalents herein means at least two nucleotides covalently linked together
• a nucleic acid of the present invention will generally contain phosphodiester bonds, although in some cases, as outlined below, nucleic acid analogs are included that may have alternate backbones, comprising, for example, phosphoramide (Beaucage et al , Tetrahedron 49(10) 1925 (1993)) and references therein, Letsmger, J Org Chem 35 3800 (1970), Sprmzl et al , Eur J
• Rad51 inhibitors may bind to Rad51 , but exclude agents which generally activate Rad51 such as DNA on which Rad51 normally binds to in the process of recombmational activity, DNA repair, -etc
• nucleic acid Rad51 inhibitors may be naturally occurring nucleic acids, random nucleic acids, or "biased' random nucleic acids
• digests of procaryotic or eucaryotic genomes may be used as is outlined above for proteins
• Rad51 inhibitors are obtained from a wide variety of sources, as will be appreciated by those in the art, including libraries of synthetic or natural compounds Any number of techniques are available for the random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means Known pharmacological agents may be subjected to directed or random chemical modifications to produce structural analogs
• the methods include both in vitro and in vivo applications, preferably in vivo Accordingly, in a preferred embodiment, the methods comprise the steps of administering a
• Rad51 inhibitor to a sample comprising Rad51 under physiological conditions, preferably to a cell
• the cell that the Rad51 inhibitor is administered to may be a variety of cells
• the cell is mammalian, and preferably human
• the cell may be any cell in a site in need of Rad51 inhibition such as diseased cells including cancerous cells and cells infected with viruses such as HIV as further discussed below
• NLS nuclear localization signal
• SV40 monkey virus
• T Antigen Pro Lys Lys Lys Lys Arg Lys Val
• ARRRRP human retmoic acid receptor- ⁇ nuclear localization signal
• NFKB p50 EVQRKRQKL
• Ghosh et al Cell 62 1019 (1990)
• NFKB p65 EKRKRTYE, Nolan et al , Cell 64 961 (1991 )
• others see for example Bouhkas, J
• NLSs incorporated in synthetic peptides or grafted onto reporter proteins or other molecules not normally targeted to the cell nucleus cause these molecules to be concentrated in the nucleus See, for example, Dmgwall, and Laskey, Ann, Rev Cell Biol 2 367-390, 1986, Bonnerot, et al , Proc Natl Acad Sci USA 84 6795-6799, 1987, Galileo,
• the inhibitory agents may be administered in a variety of ways, orally, systemically, topically, parenterally e g , subcutaneously, intraperitoneally, intravascularly, etc in one embodiment, the inhibitors are applied to the site of a tumor (or a removed tumor) mtra-operatively during surgery Depending upon the manner of introduction, the compounds may be formulated in a variety of ways
• the concentration of therapeutically active compound in the formulation may vary from about 0 1-100 wt % Generally, a therapeutic amount for the need is used, for example, to achieve inhibition of cellular proliferation, radiation or chemotherapeutic sensitization or inducing apoptosis
• the Rad51 inhibitory molecules can be combined in admixture with a pharmaceutically or physiologically acceptable carrier vehicle
• Therapeutic formulations are prepared for storage by mixing the active ingredient having the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed.
• Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate and other organic acids, antioxidants including ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, proteins, such as serum albumin, gelatin or immunoglobulins, hydrophi c polymers such as polyvmylpyrrolidone, ammo acids such as glycme, glutamme, asparagine, arginme or lysme, monosaccha ⁇ des, disacchandes and other carbohydrates including glucose, mannose, or dextrms, chelating agents such as EDTA, sugar alcohols such as mannitol or sorbitol, salt-forming countenons such as sodium, and/or nonionic surfactants such as Tween, Pluronics or PEG
• compositions can be prepared in various forms, such as granules, aerosols, tablets, pills, suppositories, capsules, suspensions, salves, lotions and the like
• Pharmaceutical grade organic or inorganic carriers and/or diluents suitable for oral and topical use can be used to make up compositions containing the therapeutically-active compounds
• Diluents known to the art include aqueous media, vegetable and animal oils and fats Stabilizing agents, wetting and emulsifying agents, salts for varying the osmotic pressure or buffers for securing an adequate pH value, and skin penetration enhancers can be used as auxiliary agents
• an agent that targets the target cells such as an antibody specific for a cell surface membrane protein or the target cell, a ligand for a receptor on the target cell, etc
• proteins which bind to a cell surface membrane protein associated with endocytosis may be used for targeting and/or to facilitate uptake,
• Dosages and desired drug concentrations of pharmaceutical compositions of the present invention may vary depending on the particular use envisioned The determination of the appropriate dosage or route of administration is well within the skill of an ordinary physician Animal experiments provide reliable guidance for the determination of effective doses for human therapy lnterspecies scaling of effective doses can be performed following the principles laid down by Mordenti, J and Chappell, W "The use of mterspecies scaling in toxicokmetics" In Toxicok etics and New Drug Development,
• the methods comprise identifying the inhibitory effect of the Rad51 inhibitor For example, determining the effect on double strand break repair, homologous recombination, sensitivity to ionizing radiation, class switch recombination, cellular inhibition, induction of apoptosis, etc Assays are detailed in Park, J Biol Chem 270(26) 15467 (1995) and Li et al , PNAS USA 93 10222 (1996), Shmohara et al , supra, (1992), all of which are hereby incorporated by reference Further assays are discussed below in the examples
• the invention provides methods of treating disease states requiring inhibition of cellular proliferation
• the disease state requires inhibition of at least one of Rad51 expression, translation or the biological activity of Rad51 as described herein
• a disease state means either that an individual has the disease, or is at risk to develop the disease
• Disease states which can be treated by the methods and compositions provided herein include, but are not limited to hyperpro ferative disorders More particular, the methods can be used to treat, but are not limited to treating, cancer (further discussed below), premature aging, autoimmune disease, arthritis, graft rejection, inflammatory bowel disease, proliferation induced after medical procedures, including, but not limited to, surgery, angioplasty, and the like
• the invention herein includes application to cells or individuals afflicted or impending affliction with any one of these disorders
• compositions and methods provided herein are particularly deemed useful for the treatment of cancer including solid tumors such as skin, breast, brain, cervical carcinomas, pancreas, testicular carcinomas, etc More particularly, cancers that may be treated by the compositions and methods of the invention include, but are not limited to Cardiac sarcoma (angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma), myxoma, rhabdomyoma, fibroma, lipoma and teratoma, Lung bronchogenic carcinoma (squamous cell, undifferentiated small cell, undifferentiated large cell, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatous hamartoma, mesothelioma, Gastrointestinal esophagus (squamous cell carcinoma, adenocarcino
• the individual, or patient is generally a human subject, although as will be appreciated by those in the art, the patient may be animal as well Thus other animals, including mammals such as rodents (including mice, rats, hamsters and guinea pigs), cats, dogs, rabbits, farm animals including cows, horses, goats, sheep, pigs, etc , and primates (including monkeys, chimpanzees, orangutans and gorillas) are included within the definition of patient
• the individual requires inhibition of cell proliferation More preferably, the individual has cancer or a hyperpro ferative cell condition.
• compositions provided herein may be administered in a physiologically acceptable carrier to a host, as previously described Preferred methods of administration include systemic or direct administration to a tumor cavity or cerebrospmal fluid (CSF)
• CSF cerebrospmal fluid
• the Rad51 inhibitors herein induce sensitivity to alkylatmg agents, DNA cross-linkers, intra and inter strand, ⁇ splatin and related compounds and radiation Induced sensitivity (also called sensitization or hypersensitivity) is measured by the cells tolerance to radiation or alkylatmg agents
• sensitivity which is measured, i e , by toxicity, occurs if it is increased by at least 20%, more preferably at least 40%, more preferably at least 60%, more preferably at least 80%, and most preferably by 100% to 200% or more
• the methods comprising administering the Rad51 inhibitors provided herein further comprise administering an alkylatmg agent or radiation
• ionizing radiation shall mean all forms of radiation, including but not limited to alpha, beta and gamma radiation and ultra violet light, which are capable of directly or indirectly damaging the genetic material of a cell or virus
• irradiation shall mean the exposure of a sample of interest to ionizing radiation
• radiosensitive shall refer to cells or individuals which display unusual adverse consequences after receiving moderate, or medically acceptable (i e , nonlethal diagnostic or therapeutic doses)
• Alkylatmg agents include BCNU and CCNU
• radiation sensitizers e g , xanthine and xanthine derivatives including caffeine
• the Rad51 inhibitors provided herein are administered to prolong the survival time of an individual suffering from a disease state requiring the inhibition of the proliferation of cells
• the individual is further administered radiation or an alkylatmg agent
• a fragment of Rad51 wherein said fragment consists essentially of a binding site for a small molecule, wherein said small molecule regulates the biological or biochemical activity of Rad51
• the regulation is inhibitory
• the binding site is the binding site for p53
• the binding site is identified by combining the inhibitor with fragments of Rad51
• the fragments are from between ammo acids 125 and 220
• Rad51 125-220 is fragmented to fragments of 5-25 ammo acids and then tested separately or in random recombinations to determine the binding site by standard binding techniques
• Hyb ⁇ doma cell line 1G8 was grown as described previously (Buchhop, S , et al , Hybndoma 15 205- 210 (1996) Production of antibody was performed using a mimPERM Bioreactor (Heraeus, Osterode, Germany) according to the recommendations of the supplier
• the ecdysone analogues munsterone A and ponasterone A, respectively (Invitrogen) were dissolved at ImM in absolute ethanol and used at a final concentration of 1 ⁇ M to induce expression of ectopic Rad51 (U ⁇ Rad51) or ⁇ -galactosidase
• Non-induced controls were supplemented with the same amount of ethanol
• media were removed and cells irradiated for 1 second on a TFL-20M transillummator (Biometra, Gottmgen, Germany) equipped with 312nm bulbs According to biological calibration, this corresponds to approximately 270J/m 2
• Cells were then grown in fresh medium, as were non- irradiated controls
• Calicheamicin y (Wyeth-Ayers" research, Pearl River, NY, USA) was dissolved in absolute ethanol at 100 ⁇ M and stored at -80°C
• cells were treated with increasing concentrations of calicheamicin ⁇ 1 for 16h, allowed to recover for 72h at 37°C under standard conditions and stained with crystal violet
• Hybndoma cell line 1G8 was isolated as described previously (Buchhop, S , et al , Hybndoma 15 205- 210 (1996)) Monoclonal antibody 1G8 specifically recognizes Rad51 protein Prof J Gerdes, Klasteil Borstel, Germany kindly provided the monoclonal antibody MIB-1 directed against the proliferation marker protein KI-67 Monoclonal anti p53 antibodies PAbl801 and DO-1 were supplied by Dianova, Hamburg, Germany Roche Biochemicals, Mannheim, Germany, supplied the polyclonal sheep anti p53 serum HRP-conjugated goat anti-mouse IgG (Amersham Buchler KG, Braunschweig, Germany) and HRP-conjugated donkey anti-sheep IgG (Sigma-Ald ⁇ ch Chemie, Steinheim, Germany) were used as second antibody Immunohistochemistry
• tissue from 41 surgical resection specimens 38 Whipple specimens and 3 left pancreatectomies
• tissue from 41 surgical resection specimens 38 Whipple specimens and 3 left pancreatectomies
• the mean age of the patients was 59 7 years (45-75)
• PBS phosphate buffered saline
• Non-isotopic RNASE cleavage assay was performed following the manufacturer's guidelines provided with the Mismatch Detection Kit II (Ambion, Texas, USA) Briefly, total cell RNA was prepared from cultured cells using the RNeasy kit supplied by Quiagen (Hilden, Germany) Reverse Transcription and PCR was performed in a one-tube reaction (Life Technologies, Düsseldorf, Germany), using the "outer primer pairs" for the PCR reaction described below Subsequently, an aliquot of this reaction was subjected to a nested PCR using the primers listed, which include the SP6 or T7 promoter sequence PCR products were transcribed in vitro with T7 or SP6 RNA polymerase to produce sense and antisense RNA probes, respectively Sense RNA products were hybridized with antisense from wild-type control and vice versa The hybridized samples were treated in three different reactions with the RNases provided with the kit The cleaved products were analyzed on ethidium bromide-stained agarose gel
• a human genomic PAC library (RPC11 ,3-5 Human PAC Library No 704, Pieter de Jong, Rosewell Park Cancer Institute) was kindly provided by Resource Center/Primary Database of the German Human Genome Project, Berlin, Germany This library was screened with a random labeled PCR fragment amplified from human genomic DNA by using the rad51 specific primers 5'- ATGGCAATGCAGATGCAGCTTGAAGC-3' and
• Rad51 protein level During cell cycle progression only minor variations in Rad51 protein level occur (Yamamoto, A , et al ,
• FIG. 2A demonstrates that Rad51 accumulates to much higher levels in a sub- population of tumor cell nuclei compared to monolayer cultures. The proportion of cells over- expressing Rad51 varies between cell lines with about 5-10 percent of cells for PancTU-l and more than 30 percent for 818-4. There is no apparent correlation between Rad51 over-expression and the genetic status of p53 in the respective cell lines (data not shown). Western blot analysis of Rad51 in
• PancTU-l and 818-4 cells grown as monolayers or as spheroids confirmed the immunocytochemical data (Figure 2B).
• Expression of p53 was not affected by cell culture conditions (data not shown).
• Quantification of the Western blot reveals about a five-fold difference for Rad51 between monolayer and spheroid. Given that Rad51 is over-expressed only in 20 percent of 818-4 cells, this argues that the level of Rad51 in over-expressing spheroid cells is increased 25-fold compared to monolayers.
• PancTU-l cells were inoculated into the pancreas in SCID mice. Mice were sacrificed on day 21 after inoculation. PancTU-l tumors were fixed, paraffin embedded, and prepared for immunohistochemistry. To rule out that paraffin embedding would affect the outcome of the experiment, PancTU-l cells grown as monolayer were harvested by centrifugation after scraping and processed under identical technical conditions concerning fixation, paraffin embedding, and histochemical analysis.
• Rad51 expression was investigated in paraffin embedded specimens of human pancreatic adenocarcinoma. As shown in Figure 3, panel C, Rad51 protein accumulates to high levels in tumor cell nuclei. Rad51 over-expression is restricted to tumor cells and not found in nuclei of surrounding tissue. Under identical staining conditions, nuclear antigens like Ki- 67 and p53 were also easily detectable in the tumor cell population. There was apparent correlation between Rad51 and p53 expression in tumor specimens (data not shown).
• Intense staining was highly specific for Rad51 protein, since pre-incubation of the anti Rad51 monoclonal 1G8 with a peptide corresponding to the epitope recognized by the antibody (Buchhop, S., et al., Hybridoma. 15:205-210 (1996)) completely blocked the staining reaction (Figure 4).
• the percentage of Rad51 positive tumor cells ranged from 5% to nearly 50% between different specimens. Tumor specimens were scored positive when more than 5% of tumor cell nuclei were stained as intense as in spheroids or xeno-transplants. According to thee criteria, 27 (66%) out of 41 pancreatic adenocarcinoma specimens expressed Rad51 protein at high-levels.
• NIRCA non-isotopic RNase cleavage assay
• Rad51 confers resistance to DNA double strand breaks
• the human osteosarcoma cell line U-20S was used as parental cell line to establish clone U ⁇ Rad51 which inducibly expresses Rad51
• the mducibly E coli ⁇ -galactosidase producing clone UiLacZ was developed Treating these cells with munsterone A or ponasterone A, analogues of the insect steroid hormone ecdysone, induces expression of the respective ectopic proteins (Miska, et al , submitted for publication) All cell clones express wild-type p53, which accumulates and becomes activated to induce cell cycle arrest in response to DNA damage This system was used to elucidate the link between Rad51 over- expression and the cellular response to DNA double strand breaks To test whether over-expression of Rad51 can affect p53 levels, these were compared in non-induced versus induced U ⁇ Rad51
• the promoter region of human rad51 shows characteristics of a housekeeping gene Recent evidence suggests that expression of Rad5 1 is regulated at the transcriptional level (Xia, et al , Mol Cell Biol , 17 7151-7158 (1997), Ohnishi, et al , Biochem Biophys Res Commun 245 319- 324 (1998)) This prompted us to study the 5 -regulatory region of the human rad51 gene An 8 1kb DNA fragment of the 5'-reg ⁇ on of this gene was sequenced (GenBank accession number AF203691 ) The 5'-UTR involves the first exon and a 3 3kb nucleotide sequence encompasses the first intron
• Pancreatic adenocarcinoma is regarded a paradigm for a chemo- and radioresistant tumor entity
• These data confirm previous reports on Rad51 expression in monolayer cell systems (Yamamoto et al , Oral Oncol 34 524-528 (1996), Chen et al , Mutat Res 384 205-211b (1997), Xia et al , Mol Cell Biol 17 7151-7158 (1997))
• Different results emerge when tumor cells were grown as spheroids or as xeno-transplants in SCID mice Under these conditions, Rad51 protein accumulates to high levels in the nuclei of a sub-population of cells
• tumor cells in specimens of human pancreatic adenocarcinoma also showed Rad51 over-expression Since we did not detect any mutations in the coding sequence, we assert that the over-expressed protein represents wild-type Rad51 In summary, our data present Rad
• Rad51 protein While cancer cells predominantly express high levels of mutant rather than wild-type p53, over-expression of Rad51 protein is not associated with alterations in the coding region Up-regulation of rad51 expression has been reported on the transcriptional level during immortalization of primary human fibroblasts (Xia et al , Mol Cell Biol 17 7151-7158 (1997) From our analysis of the 5 -regulatory region, the rad51 gene appears to contain a TATA-less, GC- ⁇ ch promoter known from housekeeping genes. Nutritional deficits like shortage of oxygen, common to inner cell layers of spheroid cultures are not believed to trigger Rad51 over-expression (data not shown).
• EXAMPLE 2 BREAST CANCER IS ACCOMPANIED BY OVER-EXPRESSION OF RAD51
• over-expression of wild-type Rad51 protein in tumor specimens of invasive ductal mammary carcinoma.
• Statistical analysis of more than one hundred tumor specimens revealed that Rad51 over-expression significantly correlates with tumor grading. These data qualify Rad51 overexpression as a marker for diagnosis and prognosis of invasive ductal mammary carcinoma.
• over-expression of Rad51 is shown to contribute to the pathogenesis of sporadic breast cancers.
• the mouse monoclonal antibody 1G8 specifically recognizing Rad51 protein in paraffin embedded tissues was isolated as described previously (Buchhop, S., et al., Hybridoma 15:205-210 (1996); Maacke et al., submitted for publication)).
• Monoclonal antibody MIB-1 directed against the proliferation marker Ki67-antigen was kindly provided by Prof. J. Gerdes (Forschungstechnik Borstel, Germany).
• Monoclonal anti p53 antibody DO-1 was supplied by Dianova (Hamburg, Germany), and monoclonal antibody AB-1 (MS110 (Wilson, C.A., et al., Nat. Genet. 21 :236-240 (1999)) directed against BRCA1 was purchased from Calbiochem (Schwalbach, Germany).
• Tissue sections were fixed routinely in neutral buffered formalin (4%) and subsequently embedded in paraffin. Consecutive 4 ⁇ m thick sections were dewaxed in xylene, passed through alcohol and washed in phosphate buffered saline (PBS). In order to improve antigen retrieval, sections were immersed in citrate buffer (100 mM Na-citrate, pH 6.0) and boiled for 3 minutes in a pressure cooker.
• citrate buffer 100 mM Na-citrate, pH 6.0
• Endogenous peroxidase activity was blocked by incubation in 3,5% hydrogen peroxide in PBS for 5min. After permeabilizing the cells with Triton-X 100 for 5min, specimens were blocked in horse serum and subsequently with avidin and biotin (Vector Laboratories Inc., Burlingame, USA). Sections were incubated with the different antibodies for Ih at room temperature in a wet chamber. After washing in PBS, a biotinylated anti-mouse antibody (Vektor Laboratories Inc.,
• the antigen retrieval solution used for this modified procedure was purchased from DAKO, (Hamburg, Germany). Antibody incubation was performed overnight at 4°C.
• BRCA1 staining intensity was classified using the following criteria: (0): no BRCA1 specific staining (blue nuclei due to Mayer's Hemalum counter-staining); (1): more than 10% of tumor cells show weak BRCA1 staining (grey color); (2): clear BRCA1 staining with more than 10% of brown tumor cell nuclei; (3): intense brown staining of more than 10% of tumor cell nuclei.
• Non-isotopic RNase cleavage assay was performed following the manufacturer's guidelines provided with the Mismatch detection Kit II (Ambion, Texas, USA). Briefly: total cell RNA was prepared using the RNeasy kit supplied by Quiagen (Hilden, Germany). Reverse Transcription and PCR was performed in a one-tube reaction (GibcoBRL, Düsseldorf, Germany), using the "outer primer pairs" described below for PCR. An aliquot of this reaction was subjected to nested PCR using primers, which include SP6 or T7 promoter sequences. PCR products were transcribed in vitro with T7 or SP6
• RNA polymerase to produce sense and antisense RNA probes, respectively.
• Sense RNA products were hybridized with antisense from wild-type control and vice versa.
• the hybridized samples were treated in three different reactions with the RNases provided with the kit.
• the cleaved products were analyzed on ethidium bromide-stained agarose gels (2%) using the Fluor-S imaging system (Biorad, Munchen, Germany).
• Rad51 mutation analysis the following primers were used for amplification of the Rad51 coding region 4 (Rad51 sequences are underlined):
• 5'-primer GATAATACGACTCACTATAGGGAAGAAGAAAGCTTTG.
• 3'-primer TCATTTAGGTGACACTATAGGAAGACAGGGAGAGTC.
• S'-primer GATMTACGAGTCACTATAGGGCGCTTCCCGAGGC-
• recombinatio ⁇ al processes may act to maintain genetic stability, but if deregulated or increased, genomic instability and malignant transformation can result.
• genomic instability and malignant transformation can result.
• PCI positive cell index
• Figure 8 shows representative examples of Rad51, ⁇ 53 and 30 Ki87-antigen staining patterns in relation to tumor grading.
• RECT1FIED SHEET (RULE 81) ISA/EP summary, recombination factor Rad51 over-expression in invasive ductal breast cancer classified as PCI correlates with clinical tumor parameters like tumor grading and hormonal receptor status. Since Rad51 in the tumors represents the wild-type form of the protein, over-expression is the result of epigenetic changes in tumor cells.
• n number of cases
• n c number of cases in a particular class
• PCI positive stained cell index
• IRS immunoreactive score (Remmele and Stegner, 1987)
• Sll staining intensity index
• nn test not necessary
• ns not significant
• H-test according to Kruskal and Wallis
• U-test according to Mann and Whitney.
• cancer related genes may also be dysregulated by epigenetic mechanisms as demonstrated recently for the BRCA1 tumor suppressor gene in sporadic breast cancer (Wilson, C.A., et al., Nat. Genet. 21 :236-240 (1999)).
• BRCA1 The biological function of BRCA1 is not understood in detail, but it appears to be involved in DNA double strand break (DSB) repair (Feunteun, J , Mol Med Today 4 263-270 (1998)) via a pathway, which relies on homologous recombination (Hend ⁇ ckson, E A , Am J Hum Genet 61 795-800 (1997)) BRCA1 protein is found in complex with Rad51 , the key enzyme of homologous recombination (Scully, R , et al , Cell 88 265-275 (1997))
• Rad51 cDNA (St ⁇ rzbecher, H.W., et al., Embo J 15:1992-2002 (1996)) was inserted into plasmid plND and co-transfected with a modified pVgRxR (Invitrogen, de Schelp, the
• coli ⁇ - galactosidase reporter for p53 transcriptional activation pRGC ⁇ FosLacZ (Frevier, T., et al., Cancer Res 52:6976-8 (1992)) and a plasmid conferring hygromycin resistance (pDSP Hygro; (Pfarr, D.S., et al., Dna 4:461-7 (1985)).
• UiRad51-blue cells were selected with 80 ⁇ g/ml hygromycin B (Roche Molecular Biochemicals, Mannheim, Germany) and maintained in 40 ⁇ g/ml.
• Equal cell numbers were pelleted and lysed in 2x SDS sample buffer (5% SDS; 125mM Tris/CI, pH 6,8; 10% glycerol; 0,02 % bromophenol blue and 12,5% freshly added ⁇ -mercaptoethanol), boiled for 10 minutes and nucleic acids were removed by digestion with 260 U Benzonase for 30min at 37°C (Merck, Darmstadt, Germany). Transfer was essentially carried out as described (Towbin, H., et al., Proc Natl Acad Sci USA 76:4350-4 (1979)). Rad51 was detected with monoclonal antibody 1G8
• Cells were trypsinised, washed in PBS and lysed in cell lysis buffer (Promega, Mannheim, Germany). Cell lyeates were assayed for protein content using the BCA assay kit (Pierce) and diluted with lysis buffer to contain identical protein levels. Luciferase activity was measured in triplicate using the Steady-GloTM as say kit (Promega) in a Microlumate LB 96P luminom eter ( K G & G B erthold , Freiburg, Germany) according to the suppliers recommendations.
• the human osteosarcoma cell line U-2 OS was used as parental cell line to create clone UiRad51 which inducibly expresses Rad51.
• the inducibly E. coli ⁇ -galactosidase producing clone UiLacZ was established. Treating the cells with muristerone A or ponasterone A, analogues of the insect steroid hormone ecdysone, induces expression of the respective ectopic proteins. From experiments obtained in rats pharmacological effects of these metamorphosing insect hormones can be excluded (Masuoka, M., et al., Jap. J. Pharmac. 20:142-156 (1970)).
• Figure 11A shows the induction of ectopic Rad51 protein by muristerone A treatment. Equal cell numbers were applied to each lane in this analysis and in all other relevant experiments in need of quantitative evaluation. Due to the short exposure time in the experiment shown, only ectopic protein is visible. In order to test, whether high-level expression of Rad51 would affect cell proliferation, growth curves were recorded. Proliferation of U-2 OS parental cells was not affected by muristerone A treatment confirming that steroid treatment alone does not affect cell proliferation of this cell line, while growth of UiLacZ controls was slightly retarded, presumably due to the high synthesis rate of ectopically expressed protein.
• Rad51 induces p21 Waf1 in a p53 independent manner by activating the waf-1 promoter
• p21 Waf1 mediates cell cycle arrest (el Deiry, W S , et al , Cell 75 817-25 (1993)) Over-expression of p21 Waf1 is sufficient to trigger both, G 1 and G 2 /M arrest, in
• p53 and p53 dependent cell cycle arrest are at least partially mediated by p21 Waf (el Deiry, W.S., et al., Cell 75:817-25 (1993)).
• p21 Waf el Deiry, W.S., et al., Cell 75:817-25 (1993)
• Rad51 requires the transactivator activity of p53 to induce p21 WaM
• cell line UiRad51-bIue was established from UiRad51 by stable integration of the highly p53 specific reporter pRGC ⁇ fos-LacZ (Frebourg, T., et al., Cancer Res 52:6976-8 (1992)).
• substrate cleavage by ⁇ -galactosidase can be used as direct measure of p53 transactivator activity in this cell clone.
• Muristerone A dependent Rad51 over-expression in UiRad51-blue was verified by Western blot analysis as was Rad51 dependent cell cycle arrest (data not shown).
• UV irradiated UiRad51-blue cells exhibit elevated levels of ⁇ -galactosidase activity compared to non-irradiated controls, indicating that under these conditions p53 becomes competent to work as transactivator
• waf-1 promoter activity was analyzed after transient transfection of reporter construct WWP-Luc and assayed 48h thereafter.
• adapted UiRad51 i.e., cells that had been induced for 28 days.
• Rad51 induces a transient cell cycle arrest in non-adapted cells independent of p53
• the p53 dependent and p21 Waf1 mediated cell cycle arrest in response to UV-irradiation is intact p53 accumulates (not shown), is activated as transcription factor ( Figure 12C), p21 Waf1 accumulates (Figure 12A) and cells arrest in G, and G 2 /M ( Figure 11 B)
• Figure 14A an aliquot of the cells shown in Figure 14A were used for the determination of p53 levels
• Western blot analysis demonstrates that the ability of p53 to accumulate in response to UV-irradiation is still intact Consequently, the p53 dependent pathway is not affected by adaptation to high level expression of Rad51 Moreover, serum dependence is conserved since after serum withdrawal adapted U ⁇ Rad51 cells accumulate in G

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