WO2000062075A1 - Proteine a activite croisee avec l'ubiiquitine, utilisee comme marqueur de la chimiosensibilite de cellules tumorales - Google Patents

Proteine a activite croisee avec l'ubiiquitine, utilisee comme marqueur de la chimiosensibilite de cellules tumorales Download PDF

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WO2000062075A1
WO2000062075A1 PCT/US2000/009959 US0009959W WO0062075A1 WO 2000062075 A1 WO2000062075 A1 WO 2000062075A1 US 0009959 W US0009959 W US 0009959W WO 0062075 A1 WO0062075 A1 WO 0062075A1
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ubiquitin
reactive protein
cells
cross
ubiquitin cross
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Shyamal D. Desai
Leroy Fong Liu
Edmond J. Lavoie
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Rutgers, The State University Of New Jersey
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6842—Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • cancer remains one of the leading causes of death in the United States and the world. Many such treatments are described in CANCER: Principles & Practice of Oncology, 2nd Edition (1985), DeNita et al, eds., J.B. Lippincott Company, Philadelphia, Pa. Since many forms of cancer exhibit rapid progression and are not readily detectable until there is widespread dissemination of the disease, it is critically important to the survival of the patient that the disease be detected as early as possible and that the most effective treatment be found at an early stage.
  • chemotherapeutic agents have been developed for the treatment of various forms of cancer, most of which rely upon the most characteristic feature of tumor cells—rapid and relatively uncontrolled cell division.
  • D ⁇ A- damaging agents including alkylating agents such as melphalan, bisulfan and nitrosoureas, antimetabolites such as mercaptopurine, fiuorouracil and methotrexate, topoisomerase I "poisons” such as camptothecin, bi- and terbenzimidazoles, as well as certain benzo[c]phenanthridine and protoberberine alkaloids, and topoisomerase II poisons such as adriamycin, etoposide (NP-16), actinomycin D and daunomycin.
  • alkylating agents such as melphalan, bisulfan and nitrosoureas
  • antimetabolites such as mercaptopurine, fiuorouracil and methotrexate
  • these agents have shown promise in the treatment of various forms of cancer.
  • the challenge has become the identification of the appropriate agent for the treatment of a specific tumor type.
  • Treatment with an ineffective chemotherapeutic agent costs the patient valuable time that could have been used for effective treatment, and during that time the disease often progresses. Furthermore, the progress of the disease may actually be aided by ineffective treatment, since many of the chemotherapeutic agents also depress the immune system and allow further dissemination of tumor cells to seed remote sites.
  • a cancer patient can be subjected to serious side effects caused by many of the chemotherapeutic agents currently in use.
  • side effects are nausea and vomiting, hair loss, anemia, infection, diarrhea, and blood clotting disorders. Permanent damage to the heart, lung, kidney, reproductive, or other tissue may result.
  • Tumor cells can sometimes be detected by the presence of "markers”— detectable molecules characteristic of the abnormal metabolic processes in those cells. Many of the markers discovered to date have aided in the early detection of the disease. In some cases, trial and error, combined with epidemiological studies, has enabled physicians to associate a more effective treatment with a particular tumor type. In most cases, however, prediction of efficacy of a particular treatment regimen is difficult, since some tumor cell types may respond to treatment while others do not.
  • Tumor marker levels can be elevated in individuals with benign conditions, and are not specific to a particular type of cancer. The level of a tumor marker can be elevated in more than one type of cancer. Also, tumor marker levels are not elevated in every person with cancer and may be totally undetectable in the early stages of the disease. Tumor markers alone provide no prediction of success for a given treatment regimen. What is needed in cancer therapeutics is a method for quickly and accurately predicting the effectiveness of a particular chemotherapeutic agent against a patient's individual tumor type.
  • TOPI is multiubiquitinated and destroyed by 26S-proteasome in normal cells. Many tumor cells are defective in this process in response to camptothecin treatment in tumor cells, but that in those tumor cells in which the multiubiquitinated TOPI is not destroyed by proteo lysis, there is an associated and readily detectable accumulation of ubiquitin cross-reactive protein.
  • the correlation between ubiquitin cross-reactive protein levels and defective destruction of ubiquitin-tagged TOPI provides a prognostic marker for identification of cancers in which camptothecin and other antineoplastic agents known to act through a DNA-damage mechanism are more likely to provide effective treatment. Since malignant tumor cells exhibit more of the defects leading to uncontrolled cell division than do benign tumor cells, particularly those defects associated with proteolytic regulation in the cell, the present invention also provides a method for distinguishing between malignant tumor cells and benign tumor cells, using ubiquitin cross-reactive protein as a marker for the malignant state.
  • Camptothecin and other agents associated with DNA damage have previously been considered cytotoxic agents.
  • the defective proteolytic degradation pathway described in the invention is common to cancer cells, rather than all cells, and therefore indicates that these agents target tumor cells more specifically than rapidly proliferating cells.
  • Cellular ubiquitin cross- reactive protein therefore provides a marker for cells for which such anti-tumor agents have therapeutic value.
  • the invention therefore describes a method for identifying cells sensitive to DNA-damaging agents by assaying to determine the relative level of cellular ubiquitin cross-reactive protein.
  • An elevated level of cellular ubiquitin cross-reactive protein in some cancer cells relative to that found in normal cells is correlated with sensitivity to a DNA-damaging agent.
  • DNA-damaging agents include alkylating agents (e.g., BCNU, CCNU, chlorambucil, cis-platinum, melphalan, mitomycin C, cyclophosphamide, and semustine), antimetabolites (e.g., thioguanine, thiopurine, hydroxyurea, guanazole, cyclocytidine, ara-C, and 5-aza-2'-deoxycytidine), and topoisomerase II inhibitors (e.g., doxorubicin, daunorubicin, mitoxantrone, menogaril, ribidazine, and NP-16), and topoisomerase I inhibitors (e.g., topotecan, irinotecan, 9-aminocamptothecin, 9- nitrocamptothecin, homocamptothecin, and mo holinodoxorubicin).
  • alkylating agents e.g.
  • the relative level of cellular ubiquitin cross- reactive protein as determined by the method is a marker for sensitivity to a topoisomerase I inhibitor such as camptothecin.
  • the correlation between a higher relative level of cellular ubiquitin cross-reactive protein, as described in the invention, is also used to distinguish malignant tumor cells from benign tumor cells, malignancy being associated with an elevated level of ubiquitin cross-reactive protein as compared to the level of ubiquitin cross-reactive protein found in normal cells.
  • Cells with a defective ubiquitin/proteasome proteolytic pathway are also identified by the method wherein an elevated level of ubiquitin cross- reactive protein are found in those cells as compared to normal cells.
  • the invention provides a method for identifying cells having a defective ubiquitin/proteasome proteolytic processing pathway, comprising determining the presence of a cellular ubiquitin cross-reactive protein in the cells wherein the presence of the ubiquitin cross-reactive protein correlates with a defective ubiquitin/proteasome proteolytic processing pathway.
  • Figure 1A illustrates a western blot analysis of topoisomerase I (topi) trapped in the form of cleavable complexes in the presence of c camptothecin over a period of time.
  • Figure IB illustrates a western blot analysis of topoisomerase I- co ⁇ jugate to SUMO-1 (small ubiquitin modifiers- 1) in the presence of camptothecin over a period of time.
  • Figure IC illustrates an immunoblot analysis of levels of topoisomerase I (topi) remaining in cells treated with camptothecin over a period of time.
  • Figure ID illustrates a western blot analysis of levels of top2 ⁇ in cells treated with camptothecin over a period of time.
  • FIG. 1 A illustrates immunoblots of topoisomerase I (topi) levels in nine established human breast cancer cell lines treated with camptothecin for different periods of time.
  • FIG. 2B illustrates immunoblots of topoisomerase I (topi) levels in nine established human colon cancer cell lines treated with camptothecin for different periods of time.
  • Figure 2C illustrates a graphical analysis of the topoisomerase I (topi) levels remaining in human cancer cells of Fig. 2 A and Fig. 2B over a period of time after camptothecin treatment.
  • Figure 3 A illustrates western blot analysis of topoisomerase I (topi) levels in three cell lines transformed with different oncogenes and with their non transformed counterpart after camptothecin treatment.
  • Figure 3B illustrates a graphical analysis of the topoisomerase I
  • Figure 4A illustrates topoisomerase I (topi) trapped in DNA in the form of cleavable complexes in breast cancer and colon cancer lines after camptothecin treatment.
  • Figure 4B illustrates topoisomerase I (topi) SUMO-1 conjugates formed in breast cancer and colon cancer lines after camptothecin treatment.
  • FIG. 4C illustrates topoisomerase I (topi) levels in breast cancer and colon cancer lines after camptothecin treatment for various times.
  • Figure 5 A illustrates a graphical analysis of the effect of CPT on transcription arrest and restart in BSC cells.
  • Figure 5B illustrates a graphical analysis of the effect of CPT on transcription arrest and restart in V79 cells.
  • Figure 5C illustrates a graphical analysis of the effect of CPT on transcription arrest and restart in BSC cells.
  • Figure 6 A illustrates a graphical analysis of transcription recovery in breast cancer cell line.
  • Figure 6B illustrates a graphical analysis of topoisomerase I (topi) levels remained after camptothecin treatment in breast cancer cell lines after camptothecin treatment.
  • Figure 6C illustrates a graphical analysis of the CPT hypersensitivity of ZR-75-1 cells defective in topoisomerase I (topi) down- regulation.
  • Figure 6D illustrates a graphical analysis of transcription restart in N79 cells treated with MG132 (proteasome inhibitor).
  • Figure 6E illustrates a graphical analysis of a correlation of topoisomerase I (topi) down regulation and transcription restart in a panel of breast cancer cell lines.
  • Figure 6F illustrates an analysis of topoisomerase 1 degradation in N79 cells treated with CPT in the presence of MG132 (proteasome inhibitor).
  • Figure 7 illustrates the accumulation of ubiquitin cross-reactive protein in tumor cells sensitive to camptothecin.
  • Figure 8 illustrates accumulation of ubiquitin cross-reactive protein in transformed cells.
  • Figure 9 A illustrates western blot analysis of ubiquitin cross- reactive protein (UCRP) using anti-ubiquitin and anti ubiquitin cross-reactive protein (UCRP) antibodies.
  • UCRP ubiquitin cross- reactive protein
  • Figure 9B illustrates the western blot analysis of ubiquitin cross- reactive protein (UCRP) from different cell lines grown in the presence of IF ⁇ antibodies.
  • UCRP ubiquitin cross- reactive protein
  • Ubiquitin cross-reactive protein includes cellular proteins which are bound by an antibody that binds to ubiquitin.
  • ubiquitin cross-reactive proteins/ISG15 include cellular proteins which show cross- reactivity with ubiquitin antisera.
  • the ubiquitin cross-reactive proteins/ISG15 are about 15 to about 18 kilodaltons (kDa), preferably about 15 to about 17 kDa as measured from Western blot analysis.
  • Specific examples of ubiquitin cross-reactive proteins/ISG15 in the art include those described in Bebington et al. (Mol. Hum. Repro.. l ⁇ , 966 (1999)), Bebington et al.
  • Ubiquitin cross-reactive protein further includes diubiquitin, which consists of two ubiquitin units ligated together (e.g., through the formation of an isopeptide bond between lysine 48 and glycine 76).
  • the ubiquitin cross-reactive protein can be ubiquitin cross-reactive protein/ISGl 5, as disclosed in Haas et al. (J. Biol. Chem.. 262, 11315 (1987).
  • the ubiquitin cross-reactive protein used as a biomarker (i.e., prognastic marker) for tumor cell chemosensitivity can be ubiquitin cross- reactive protein/ISGl 5, as disclosed in Haas et al. (J. Biol. Chem., 262, 11315 (1987).
  • the ubiquitin cross-reactive protein has been characterized as an interferon inducible UCRP/ISG15 based on the following observations: (1) Topoisomerase 1 (topi) is ubiquinated and destroyed upon campothecin (CPT) treatment via 26S-proteasome pathway in normal cells; (2) this process is defective in many tumor cells; (3) tumor cells defective in topoisomerase 1 (topi) down regulation are hypersensitive to CPT; (4) there is an up-regulation (or elevation) of UCRP/ISG15 protein in many tumor cells and this upregulation was correlated to topi down-regulation (destruction); and (5) UCRP/ISG15 was not present in many normal cells or tumor cells resistant to campothecin (CPT).
  • CPT campothecin
  • a preferred agent to detect ubiquitin cross-reactive protein in the methods of the invention is a rabbit antiserum containing anti-ubiquitin antibody which was raised against bovine red blood cell ubiquitin conjugated to keyhole limpet hemocyanin (e.g., Sigma, product No. U-5379).
  • Other preferred agents include antibodies to ubiquitin cross-reactive protein/ISGl 5.
  • Ubiquitin cross-reactive protein is a marker for cells with abnormal proteolytic processing mechanisms and for cells which have an increased sensitivity to treatment with cytotoxic agents. Normal cells grow and divide in a controlled manner, primarily due to the cellular regulation mechanisms for proteins involved in cellular reproduction. Among these mechanisms is a proteolytic degradation process commonly known as the ubiquitin proteasome degradation pathway.
  • the ATP/ubiquitin-dependent non- lysosomal proteolytic pathway of the 26S proteasome utilizes the 8.6 kDa, 76 amino acid protein ubiquitin as a covalent signal to target cellular proteins to the 26S proteasome.
  • Ubiquitin binds the target protein through formation of a peptide bond between its C-terminal glycine residue and the epsilon amino group of a lysine residue in the target protein.
  • Tagging of cellular proteins for proteolytic destruction in the proteasome generally involves the addition of multiple ubiquitin units ligated together through the formation of isopeptide bonds between Lys 48 and Gly 76 of successive ubiquitins.
  • the target protein, recognized by the proteasome through its ubiquitin signal, is degraded, while the ubiquitin units are regenerated for use in subsequent proteolytic cycles. Hochstrasser, M. (1996) Annu. Rev. Genet. 30, 405-439.
  • a tumor can be either composed of non-cancerous cells (benign) or cancerous cells (malignant).
  • a cancerous cell is a potentially malignant neoplastic cell, characterized by more rapid than normal cellular proliferation, partial or complete lack of structural organization and functional coordination with the normal tissue, and continued unregulated growth after initial stimulation by cellular factors.
  • cancerous cells are characterized by relatively uncontrolled cell growth and DNA synthesis, most of the therapies for treating cancer have been targeted at damage to the DNA of the rapidly-dividing cell.
  • DNA-damaging agents are physical agents, such as radiation, and chemotherapeutics.
  • chemotherapeutics are alkylating agents, which bind to the DNA (often at a preferred guanine residue) and inhibit DNA synthesis.
  • Alkylating agents recognized by the National Cancer Institute include: Asaley, AZQ, BCNU, Busulfan, carboxyphthalatoplatinum, CBDCA, CCNU, CHIP, chlorambucil, chlorozotocin, cis-platinum, chlomesone, cyanomo ⁇ holinodoxorubicin, cyclodisone, dianhydrogalactitol, fluorodopan, hepsulfam, hycanthone, melphalan, methylCCNU, mitomycin C, mitozolamide, nitrogen mustard, PCNU, piperazine, piperazinedione, pipobroman, porfiromycin, spirohydantoin mustard, teroxirone, tetraplatin, thio-tepa, triethylenemelamine,uracil nitrogen mustard, and Yoshi-864.
  • Antimetabolites such as 5-fluorouracil (which interferes with DNA synthesis by inhibiting thymidylate synthetase) and cytosine arabinosine (ara-C, which inhibits DNA synthesis through the inhibition of DNA polymerase), are also used for cancer therapy.
  • 5-fluorouracil which interferes with DNA synthesis by inhibiting thymidylate synthetase
  • arabinosine cytosine arabinosine
  • Antimetabolites also listed with the National Cancer Institute include: 3-HP, 2'-deoxy-5-fluorouridine, 5-HP, alpha-TGDR, aphidicolin glycinate, 5-aza-2'deoxycytidine, beta-TGDR, cyclocytidine, guanazole, hydroxyurea, inosine glycodialdehyde, macbecin II, pyrazoloimidazole, thioguanine, and thiopurine.
  • topoisomerase II inhibitors such as mitoxantrone, which mediates DNA strand breakage by inhibiting the action of topoisomerase II.
  • Other topoisomerase II inhibitors include doxorubicin, amonafide, m-AMSA, anthrapyrazole derivative, pyrazoloacridine, bisantrene HCl, daunorubicin, deoxydoxorubicin, menogaril, N,N-dibenzyl daunomycin, oxanthrazole, ribidazine, VM-26, and NP-16.
  • topoisomerase I inhibitors exemplified by camptothecin, a number of camptothecin analogs, such as topotecan, irinotecan, 9-aminocamptothecin, 9- nitrocamptothecin, and mo ⁇ holinodoxorubicin.
  • camptothecin a number of camptothecin analogs, such as topotecan, irinotecan, 9-aminocamptothecin, 9- nitrocamptothecin, and mo ⁇ holinodoxorubicin.
  • camptothecin a number of camptothecin analogs, such as topotecan, irinotecan, 9-aminocamptothecin, 9- nitrocamptothecin, and mo ⁇ holinodoxorubicin.
  • the inventors have described new topoisomerase I poisons in U.S. application number 09/023,147 (filed February 12, 1998) and PCT application number PCT/
  • a D ⁇ A-damaging agent is any physical or chemical agent which interferes with the synthesis or repair of D ⁇ A, and can include any of the listed therapeutic agents. It is to be understood, however, that this list is not meant to be limiting, since methods of synthesizing and identifying new compounds with alkylating, antimetabolite, or topoisomerase I or Il-inhibiting properties are known to those of skill in the art and these new compounds are often reported in the literature. Ubiquitin Cross-Reactive Protein Levels Correlate with Sensitivity to a Topoisomerase I Poison
  • camptothecin treatment produces a complex between camptothecin (CPT), topoisomerase I (TOPI), and DNA in which the TOPI is ultimately polyubiquitinated and degraded.
  • topoisomerase I in the polyubiquitinated complex is not degraded.
  • Many breast and colon cancer cells are defective in TOPI down- regulation in response to CPT treatment as compared to non-transformed cells. These cells can be identified by the correlated increase in ubiquitin cross-reactive protein in those cells as compared to levels detected in normal cells.
  • the relative level of ubiquitin cross-reactive protein is, therefore, used in the present invention as a predictive marker for determining which tumor cell types will likely respond to treatment with agents known to induce DNA damage.
  • Ubiquitin has also been associated with proteolytic degradation and regulation of a number of cell cycle proteins.
  • the description of the present invention is not intended to be limited solely to detection of cells for which DNA damaging agents will be therapeutically effective, but includes other uses for the ubiquitin cross-reactive protein marker, including detection of cells with abnormal ubiquitin-associated proteolytic degradation processes, which can be useful in both clinical practice and laboratory research.
  • Deregulation of the ubiquitin/proteasome pathway in tumor cells has been documented previously. For example, MHC class I-restricted peptide presentation, which is carried out by proteasome-mediated degradation, is reduced in tumors (Restifo, N.P. et al., 122, 265-272 (1993)). Such a reduction may contribute to escape of tumor cells from immune surveillance (Spataro, V. et al., British J.
  • Cyclins Dl , E and B are known to be elevated in tumors due to decreased proteasome-dependent degradation.
  • IKBOC the NF- ⁇ B inhibitor
  • the CDK inhibitor p27 is also known to be down-regulated in many colon and breast cancers due to increased proteasome-dependent degradation (Catzavelos, C. et al., Nature Med.. 1, 227-230 (1997); Loda, M. et al., Nature Med. : 1, 231-234 (1997)). Therefore, deregulation of the ubiquitin/proteasome pathway in tumor cells appears to lead to either elevated or reduced degradation depending on the substrates.
  • Camptothecin is a quinoline-based alkaloid found in the bark of the Chinese camptotheca tree. It is a naturally-occurring DNA topoisomerase I (TOP 1) inhibitor.
  • Related compounds in the family with topoisomerase I- inhibiting activity include 9-aminocamptothecin, CPT-11, also known as irinotecan, DX-8951 f, and topotecan.
  • CPT-11 also known as irinotecan
  • DX-8951 f DX-8951 f
  • topotecan topotecan.
  • Many tumor cells have been demonstrated to be hypersensitive to CPT (Pantazis, P. et al., Cancer Res. ⁇ 51, 1577-1582 (1993); Pantazis, P. et al., Tnt. J. Cancer, 51, 863-871 (1993)), and the inventors have demonstrated a correlation between resistance to CPT and extent of TOPI down-regulation among a panel of breast and
  • Camptothecin has been demonstrated to stabilize TOPI cleavable complexes within an actively transcribed region, and to transiently arrest the elongating RNA polymerase complex.
  • the TOPI cleavable complex is rapidly multi-ubiquitinated by an E2 or E2/E3 enzyme.
  • Multi-ubiquitinated TOPI is either released from the DNA template, and destined for degradation by 26S proteasome, or degraded directly on the DNA template by 26S proteasome. Release of multi-ubiquitinated TOPI from the DNA template may explain the observed redistribution of TOPI (Danks, M.K. et al., Cancer Res., 5 ⁇ , 1664- 1673 (1996); Buckwalter, C.A.
  • camptothecin inhibits the rejoining step of the two-step breakage/rejoining process for relaxing superhelical DNA that is normally mediated by topoisomerase I.
  • the essential functions of topoisomerases in DNA replication, RNA transcription, chromosome condensation and segregation depend on their action in breaking/rejoining DNA strands (Wang, J.C., Ann. Rev. Biochem.. 61, 635-692 (1996)). Alteration of this breakage/religation reaction by xenobiotics (e.g. anticancer drugs and antibiotics) (Chen, AN. et al., Ann. Rev. Pharmacol. Toxicol..
  • Camptothecin is a specific inhibitor (poison) of eukaryotic topoisomerase I (TOPI) and a potent anticancer drug (Hsiang, Y.-H. et al., J. Biol. Chem , 26 ⁇ , 14873-14878 (1985)). Its antitumor activity has also been reflected in its preferential cytotoxicity against tumor cells in vitro (Pantazis, P. et al., Cancer Res., 54, 771-776 (1994)). However, the molecular mechanism(s) underlying the antitumor activity of CPT remains unclear.
  • CPT is known to inhibit TOPI by blocking the religation step, resulting in rapid accumulation of the cleavage intermediate, the reversible cleavable complex (Hsiang, Y.-H et al., supra; Porter, S.E. et al., Nucl. Acids Res.., 12, 8521-8532 (1989)).
  • the formation of ternary TOPI cleavable complexes in CPT-treated cells results in rapid arrest of both DNA replication and RNA transcription (Hsiang, Y.-H. et al., Cancer Res , 42, 5077-5082 (1989); D'A ⁇ a, P. et al., Cancer Res.. 5 ⁇ , 6919-6924 (1990)).
  • the topoisomerase I can be marked for degradation by the addition of multiple units of the ubiquitin protein—a process known as multi-ubiquitination.
  • Camptothecin (CPT)-induced TOPI -mediated DNA damage triggers a ubiquitin-proteasome pathway resulting in multi- ubiquitination and 26S proteasome-dependent destruction (down-regulation) of TOPI.
  • CPT Camptothecin
  • ATP/ubiquitin- dependent non-lysosomal proteolytic pathway of the 26S proteasome there is decreased sensitivity to the effects of camptothecin in terms of double-strand damage associated with the covalently-bound complex during DNA replication.
  • tumor cells are isolated by methods known to those of skill in the art. Generally, a biopsy is taken from an intact or resected tumor. For Western blot analysis, a single biopsy sample of approximately 2 x 10 5 cells is taken. Primary tumor tissue is stored in liquid nitrogen at -70 degree C. For extraction, the tumor cells are lysed by methods known to those of skill in the art. One such method of extraction from tumor cells has been described in U.S. Patent Number 5,723,302 (Diamandis, issued March 3, 1998). Cells may be lysed directly with SDS-PAGE sample buffer. Cell lysates are then sonicated and subjected to SDS-PAGE analysis using a 20% acrylamide gel, with diubiquitin as control marker. Following transfer of proteins onto nitrocellulose membranes, detection of the control marker and ubiquitin cross-reactive protein bands can be performed using a suitable anti- ubiquitin antibody (e.g., ubiquitin antisera).
  • a suitable anti- ubiquitin antibody e.g
  • Anti-ubiquitin antibodies are readily available from commercial vendors, such as Berkeley Antibody Company (Richmond, CA), Research
  • ubiquitin is a highly conserved protein, antibody against any known form of ubiquitin is cross-reactive between species.
  • An elevated relative level of ubiquitin cross-reactive protein indicates a tumor type that is most likely to benefit from anticancer treatment with a DNA-damaging agent (e.g., camptothecin, irinotecan, topotecan, 9-nitro-camptothecin, and related analogs).
  • a DNA-damaging agent e.g., camptothecin, irinotecan, topotecan, 9-nitro-camptothecin, and related analogs.
  • western blot analysis can be performed using antibodies specific to UCRP/ISG15.
  • ubiquitin cross-reactive proteins/ISG15 Background levels of ubiquitin cross-reactive proteins/ISG15 are expected to be found in many normal cells. For the average biopsy sample size, however,ubiquitin cross-reactive protein levels in normal cells are generally undetectable by Western blot analysis. Therefore, the increased amount of ubiquitin cross-reactive protein in certain cancer cells, relative to the background levels found in normal cells, is often defined as detectable presence of ubiquitin cross-reactive protein in cancer cells from an average biopsy as compared to the undetected lower levels of background ubiquitin cross-reactive protein in normal cells analyzed by Western blot.
  • Ubiquitin cross-reactive protein is a prognostic indicator insofar as ubiquitin cross-reactive protein levels in cancer cells sensitive to DNA- damaging agents such as camptothecin are elevated in relation to background levels of ubiquitin cross-reactive protein in normal cells.
  • An Elevated Relative Level of Ubiquitin Cross-Reactive Protein Indicates a Cellular Abnormality in the Ubiquitin/Proteasome Pathway
  • Topoisomerase I is targeted for pro teo lysis by addition of ubiquitin units in a "branched" fashion, most often utilizing either lysine residue 63 or lysine residue 48 in the ubiquitin protein as the attachment point for multiple ubiquitin units.
  • TOPI is ubiquitinated, but not proteolytically degraded.
  • the defective proteolytic degradation of TOPI in these cells is correlated with an increased relative level of cellular ubiquitin cross-reactive protein.
  • the marker is therefore used to detect a defective ubiquitin-associated proteolytic processing mechanism, such as that detected in cells with defective proteolytic degradation of TOPI.
  • the most preferred method for detection is immunoassay, especially Western blot analysis, utilizing anti-ubiquitin antibody (either monoclonal or polyclonal).
  • any suitable method for detection of the presence of a ubiquitin cross-reactive protein can be used in the methods of the invention.
  • a ubiquitin cross-reactive protein could be detected using an antibody that specifically binds to a ubiquitin cross-reactive protein without significantly binding to ubiquitin.
  • cells are lysed directly with 2x SDS-PAGE sample buffer, then sonicated and subjected to 20% acrylamide SDS-PAGE analysis.
  • the human fibroblast cell line WI38 and its SN40-transformed variant 2RA, colon cancer cell lines, HT29, KM12, Colo205, SW480 and HCT116, monkey kidney fibroblast cell lines, BSC, and the Chinese hamster lung cell line N79 were obtained from American Type Culture Collection (Rockville, MD).
  • N79, CB17, CB17/ER1, CB17/ER4, ⁇ IH/3T3, and KNIH/3T3 cells were grown in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum.
  • DMEM Dulbecco's Modified Eagle's Medium
  • Monkey kidney BSC cells were grown in Eagle's minimum essential medium supplemented with 10% fetal bovine serum. All media were supplemented with L-glutamine (2 mM), penicillin (100 units/ml), and streptomycin (100 ⁇ g/ml). All cultures were grown in a 37° C incubator with 5% CO 2 .
  • Uridine Tnco ⁇ oration Cells (5 x 10 5 ) were grown in 35 x 10 mm tissue culture plates and treated with 25 ⁇ M CPT for various times followed by pulse-labeling with 1 ⁇ Ci/ml of [5,6- 3 H] uridine (ICN, 47 Ci/mmole). Following uridine labeling, cells were lysed with 10% TCA and labeled RNA was collected on glass fiber filters. Alternatively, cells were lysed with 4 M isothioguanidine, 0.5% sarkosyl, 2 mM Na-citrate, and 0.1 M ⁇ -mercaptoethanol. Samples were directly spotted onto DE81 papers. Filters were washed as described (Desai, S.D. et al., J. Biol Chem.., 222, 24159-24164 (1997)). C. Immunoblotting of TOPI. TOP2 ⁇ and TOPI -SUMO- 1 conjugates
  • Cells (10 sample) were treated with CPT (25 ⁇ M, 1% DMSO) for various time at 37°C. Subsequently, cells were either lysed directly with 2 x SDS PAGE sample buffer or 0.2 N NaOH containing 2 mM EDTA (for monitoring trapped covalent TOP 1 -DNA complexes) or placed in fresh CPT-free tissue culture media for 30 minutes to reverse the cleavable complexes (for monitoring total cellular levels of topoisomerases) prior to lysis. To detect TOPl-Ub conjugates, cells were lysed in 0.2 N NaOH and 2 mM EDTA. Cell lysates were sonicated and then neutralized with 1/10 volume of 2 N HCl.
  • CPT induces multi-ubiquitination and degradation of TOPI in normal and certain cancer cells N79 cells were treated with 25 ⁇ M CPT for various times and were then lysed directly with SDS. Under these conditions, TOPI was found to be covalently trapped on the D ⁇ A (due to formation of covalent TOPI -D ⁇ A complexes) resulting in disappearance of the 100 kDa TOPI band in SDS- polyacrylamide gel (Fig. 1A) (the doublet labeled pTOPl and TOPI represent the phosphorylated and dephosphorylated forms of mouse TOPI, respectively) (D'A ⁇ a, P. et al, Rxp. Cell Res., 212, 125-131 (1995)). When the same gel shown in Fig.
  • CPT-induced TOPI degradation is deficient in many tumor cells
  • nine established breast cancer and five colon cancer cell lines were analyzed for their TOPI levels upon treatment with CPT by immunoblotting (Fig. 2 A).
  • These human tumor cells exhibited highly variable extent of TOPI down-regulation in response to CPT treatment (Fig. 2A).
  • many breast caucer cell lines such as ZR-75-1 and MCF7 were completely defective in TOPI down- regulation.
  • BT20, T47D, MDA-MB-468, MDA- MB-435, and MDA-MB-231 exhibited a modest level of degradation (less than 30%) of TOPI after 6 hours of continuous CPT treatment (Fig. 2A and 2C).
  • the BT474 cells like FM3A mouse mammary carcinoma cells described previously (Maniatis, T. et al, Tn Molecular Cloning: A Laboratory Manua Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., p.473 (1982)), exhibited the highest level of degradation (60%) of TOPI (Fig. 2A and 2C). Similarly, most colon cancer cell lines exhibited reduced extent of TOPI down- regulation (Fig. 2B and 2C).
  • Colo205 and HCTl 16 cells were found to be most deficient in degradation of TOPI (20% degraded in 6 hours) as compared to KM 12 cells which showed modest degradation of TOPI (40% in 6 hours) (Fig. 2B).
  • CPT-induced TOPI down-regulation was extensive in all non-transformed mouse and human fibroblasts (Fig. 3). Within two hours, more than 80% of the TOPI was destroyed in all these cells (Fig. 3).
  • Oncogenic transformation reduces the extent of TOPI deregulation in response to CPT Since the extent of TOPI degradation was found to be highly variable in tumor cells, studies were conducted to investigate whether this phenomenon was related to oncogenic transformation. For this pu ⁇ ose, pairs of cell lines transformed with different oncogenes were used. These cell lines were treated with CPT for different times and TOPI levels were monitored. As shown in Fig. 3 A, TOPI was efficiently degraded (>80% degraded in 4 hours) in normal human fibroblast WI38 cells, in immortalized monkey kidney fibroblast CV1 cells, in mouse fibroblast CB17 cells and in immortalized NIH/3T3 cells.
  • the 2RA cell line which is a T- antigen-transformed variant of WI38 cells, was completely defective in TOPI down-regulation in response to CPT treatment (Fig. 3A and B).
  • the ER1 and ER4 cell lines which are variants of the mouse fibroblast CB17 co-transformed with El A and Raf oncogenes, exhibited reduced extent of down-regulation of TOPI as compared with their untransformed CB17 cells (Fig. 3 A and B).
  • the KNIH/3T3 cell line, the K-Ras-transformed NIH/3T3 cells exhibited only modest reduction in extent of TOPI down-regulation (Fig. 3 A and B).
  • Down-regulation of TOPI could conceivably be an efficient mechanism to remove TOPI -mediated DNA damage and to confer tolerance of cells to CPT treatment.
  • CPT is known to cause transcription arrest due to blockage of the RNA polymerase elongation complexes by TOPI cleavable complexes (Zhang, H. et al, Proc. N tl Acad. Sci. USA, West, 1060-1064 (1988); Ljungman, M. et al, Carcinogenesis. 17, 31-35 (1996)).
  • Down-regulation of TOPI in response to CPT is therefore expected to reduce these transcription roadblocks and to allow transcription restart.
  • BSC cells treated with CPT resulted in rapid arrest of transcription.
  • the rate of transcription was reduced to less than 10% of the untreated control as monitored by pulse-labeling with 3 H-uridine (Fig. 5A).
  • Transcription arrest was also confirmed by nuclear run-on assays.
  • recovery of transcription was evident within 15 minutes and continued to increase for the next few hours.
  • the rate of transcription recovered to 22% of the untreated control.
  • NM-26 a TOP2 poison, inhibited uridine inco ⁇ oration as rapidly as CPT.
  • no significant recovery of transcription was observed in the next four hours as evidenced by pulse-labeling with 3 H-uridine.
  • Cells (10 6 /sample) were treated with CPT (25 mM, 1% DMSO) for various times at 37°C. Subsequently, the drug was removed and the cells were placed in fresh CPT-free tissue culture media for 30 minutes to reverse the cleavable complexes and enable monitoring of total cellular levels of topoisomerases prior to lysis with SDS sample buffer.
  • Immunoblotting analysis of cell lysates was carried out using TOPI antisera from scleroderma patients, using the ECL Western blot procedure (Pierce). The intensity of each band in the autoradiogram was quantitated by densitometric scanning.

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Abstract

Cette invention concerne des méthodes d'identification de cellules qui présentent une sensibilité accrue à un traitement aux agents cytotoxiques, ainsi qu'une méthode d'identification de cellules caractérisées par des mécanismes de transformation protéolytiques anormaux. Plus précisément, l'invention a trait à une méthode qui fait intervenir une protéine à activité croisée avec l'ubiquitine comme marqueur, à des fins de pronostic, de cellules tumorales qui présentent une sensibilité marquée pour un traitement avec des agents endommageant l'ADN, dont la camptothécine et ses analogues.
PCT/US2000/009959 1999-04-13 2000-04-13 Proteine a activite croisee avec l'ubiiquitine, utilisee comme marqueur de la chimiosensibilite de cellules tumorales WO2000062075A1 (fr)

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Cited By (8)

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US7135464B2 (en) 2002-06-05 2006-11-14 Supergen, Inc. Method of administering decitabine
US7250416B2 (en) 2005-03-11 2007-07-31 Supergen, Inc. Azacytosine analogs and derivatives
US7700567B2 (en) 2005-09-29 2010-04-20 Supergen, Inc. Oligonucleotide analogues incorporating 5-aza-cytosine therein
US20100111874A1 (en) * 2007-03-19 2010-05-06 University Of Medicine And Dentistry Of New Jresey Method of cancer detection and treatment
US7842513B2 (en) 2002-05-02 2010-11-30 Aspenbio Pharma, Inc. Pregnancy detection
US9381207B2 (en) 2011-08-30 2016-07-05 Astex Pharmaceuticals, Inc. Drug formulations
US10485764B2 (en) 2015-07-02 2019-11-26 Otsuka Pharmaceutical Co., Ltd. Lyophilized pharmaceutical compositions
US10519190B2 (en) 2017-08-03 2019-12-31 Otsuka Pharmaceutical Co., Ltd. Drug compound and purification methods thereof

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Title
DESAI S.D. ET AL.: "Roles of ubiquitin and ubiquitin-related proteins in camptothecin sensitivity/resistance", PROCEEDINGS OF THE AMERICAN ASSOCIATION FOR CANCER RESEARCH ANNUAL MEETING, March 2000 (2000-03-01), pages 819, XP000912155 *
DESAI S.D. ET AL.: "Ubiquitin-mediated destruction of topoisomerase I: a potential role of 26S proteasome in the repair of topoisomerase I-mediated DNA damage", PROCEEDINGS OF THE AMERICAN ASSOCIATION FOR CANCER RESEARCH ANNUAL MEETING, vol. 40, March 1999 (1999-03-01), pages 155, XP000912157 *
LIU L.F. ET AL.: "The roles of ubiquitin-dependent proteolysis in determining the sensitivity/resistance of tumor cells to topoisomerase inhibitors", PROCEEDINGS OF THE AMERICAN ASSOCIATION FOR CANCER RESEARCH ANNUAL MEETING, vol. 40, March 1999 (1999-03-01), pages 775, XP000912156 *

Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7842513B2 (en) 2002-05-02 2010-11-30 Aspenbio Pharma, Inc. Pregnancy detection
US7144873B2 (en) 2002-06-05 2006-12-05 Supergen, Inc. Kit for delivering decitabine in vivo
US7135464B2 (en) 2002-06-05 2006-11-14 Supergen, Inc. Method of administering decitabine
US7250416B2 (en) 2005-03-11 2007-07-31 Supergen, Inc. Azacytosine analogs and derivatives
US10456415B2 (en) 2005-09-29 2019-10-29 Astex Pharmaceuticals, Inc. Oligonucleotide analogues incorporating 5-aza-cytosine therein
US7700567B2 (en) 2005-09-29 2010-04-20 Supergen, Inc. Oligonucleotide analogues incorporating 5-aza-cytosine therein
US8461123B2 (en) 2005-09-29 2013-06-11 Astex Pharmaceuticals, Inc. Oligonucleotide analogues incorporating 5-aza-cytosine therein
US9358248B2 (en) 2005-09-29 2016-06-07 Astex Pharmaceuticals, Inc. Oligonucleotide analogues incorporating 5-aza-cytosine therein
US9480698B2 (en) 2005-09-29 2016-11-01 Astex Pharmaceuticals, Inc. Oligonucleotide analogues incorporating 5-aza-cytosine therein
US10933079B2 (en) 2005-09-29 2021-03-02 Astex Pharmaceuticals, Inc. Oligonucleotide analogues incorporating 5-aza-cytosine therein
US20100111874A1 (en) * 2007-03-19 2010-05-06 University Of Medicine And Dentistry Of New Jresey Method of cancer detection and treatment
US9381207B2 (en) 2011-08-30 2016-07-05 Astex Pharmaceuticals, Inc. Drug formulations
US10517886B2 (en) 2011-08-30 2019-12-31 Astex Pharmaceuticals, Inc. Drug formulations
US9913856B2 (en) 2011-08-30 2018-03-13 Astex Pharmaceuticals, Inc. Drug formulations
US10485764B2 (en) 2015-07-02 2019-11-26 Otsuka Pharmaceutical Co., Ltd. Lyophilized pharmaceutical compositions
US10519190B2 (en) 2017-08-03 2019-12-31 Otsuka Pharmaceutical Co., Ltd. Drug compound and purification methods thereof

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