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
  • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
  • C07K14/4721—Lipocortins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the present invention relates in general to multidrug resistance (MDR) in ceils.
• MDR multidrug resistance
• the present invention relates to the identification of a new member of the MDR gene family, P-40, as well as to the identification of P-40 related genes (homologs) as being further members of the MDR gene family.
• the present invention therefore relates to nucleic acid molecules encoding P-40 protein and P-40 proteins homologs, to multidrug resistant cells containing these nucleic acid molecules; to hybridomas containing antibodies to P-40 and P-40 homologs; to nucleic acid probes for the detection of these nucleic acid molecules; to a method of detection of such nucleic acid molecules or of the P-40 protein or P-40 homologs; to bioassays comprising the nucleic acid molecules encoding P-40 or P-40 homologs, P-40 protein or P-40 protein homologs, or antibodies of the present invention to diagnose, assess or prognose MDR in an animal; to therapeutic uses of the nucleic acid molecules of the present invention (i.e. antisense), protein or antibodies of the present invention; and to methods of preventing MDR in an animal.
• nucleic acid molecules of the present invention i.e. antisense
• P-gp P-glycoprotein
• MRP multidrug resistance associated protein
• P-gp and MRP belong to a large family of ATP trafficking proteins that are evolutionary conserved and mediate the transport of numerous ligand ranging from ions to large polypeptides (Higgins et al., 1992, Ann. Rev. Cell Biol. 8: 67-113).
• P-gp and MRP reduce the accumulation of drugs via an energy dependent drug efflux mechanism (Shapiro et al., 1994, J. Biol. Chem. 269(5.: 3745-3754; Doige et al., 1992, Biochim. Biophys. Acta 1109: 161-171).
• LRP the Lung Resistance Protein, a the component of human vaults
• alteration in functions or levels of proteins mediating apoptosis or programmed cell death (Lowe et al., 1993, Cell 74(6): 957-967; Lowe et al., 1994, Science 266(5186): 807-810).
• the present invention seeks to meet these and other needs.
• the invention concerns the demonstration that P-40 has a direct role in multidrug resistance.
• the invention relates to the identification of P-40 as a member of the MDR gene family and to the identification of P-40/Annexin I related genes as members of this broadened MDR gene family. In addition, the invention relates to the identification of
• Annexin II and IV as potential MDR determinants.
• the invention relates to the identifications of Annexins (I to XI, also referred thereto herein P-40 and P- 40 homolgs) as potential members of the MDR gene family.
• the present invention also relates to the identification of Annexin-based MDR in cells.
• the present invention further relates to the isolated nucleic acid molecules encoding P-40 or fragment thereof and to the identification of P-40 as Annexin I.
• the invention in addition relates to purified P-40 polypeptides, homologs thereof, or epitope binding portions thereof and the use thereof in multidrug resistance.
• the invention also provides a specific detection method for P-40 nucleic acids encoding P-40 proteins or homologs thereof, polypeptides or fragments thereof in a sample.
• the invention provides a recombinant nucleic acid molecule comprising P-40 (or homologs thereof) operationally linked to a promoter, efficient in initiating transcription thereof in a host cell as well as to such a host cell.
• the invention provides a non-human organism containing the nucleic acid molecule mentioned above. Further, the invention provides an antisense P-40 or P-40-related (Annexin l-related) nucleic acid molecule.
• the invention further provides an antibody having specific binding affinity towards P-40, P-40 homologs or an epitope-containing-region thereof.
• the antibody is a monoclonal.
• the invention also provides the hybridoma producing the monoclonal antibody.
• the invention also seeks to provide a method for the detection of P-40 or P-40 protein homologs or portions thereof in a sample. In one embodiment, such a method is quantitative.
• the invention seeks to provide a diagnostic kit comprising a first contained means containing the above-mentioned antibody, and second container means containing a conjugate comprising a binding partner of the monoclonal antibody and a label.
• the invention seeks to provide diagnostic methods for human disease and particularly for cancer and the multidrug resistance of cancer cells.
• a method for evaluating the predisposition of a cancer tumor to be and/or become multidrug resistance is provided herein.
• the invention further seeks to provide therapeutic methods involving the P-40 nucleic acid homologs, variants or parts thereof, antisense thereof, P-40 protein, P-40 protein homologs or P-40 antibodies.
• the present invention also relates to a kit comprising the oligonucleotide primers or agents or ligands of the present invention which are specific to annexins and more particularly to annexin I.
• a compartmentalized kit in accordance with the present invention includes any kit in which reagents are contained in separate containers.
• Such containers include small glass containers, plastic containers or strips of plastic or paper.
• Such containers allow the efficient transfer of reagents from one compartment to another compartment such that the samples and reagents are not cross-contaminated ant the agents or solutions of each container can be added in a quantitative fashion from one compartment to another.
• Such containers will include a container which will accept the test sample (DNA or cells), a container which contains the primers used in the assay (or antibodies or other ligands), containers which contain enzymes, containers which contain wash reagents, and containers which contain the reagents used to detect the extension products (or reagents to detect the antibodies or ligands).
• Annexin I is a member of a large family of Ca2+ phospholipid binding proteins [for review see (Raynal et al., 1994, Biochim. Biophys. Acta. 1197: 63-93), implicated in several cellular mechanisms including intracellular membrane vascular trafficking and exocytosis process (Creutz, 1992, Science. 258: 924-93; 1 Lin et al., 1992, Cell 70: 283-291 ; Creutz et al., 1992, J. Cell Science. 103: 1 177-1 192).
• Annexin I has not been previously implicated in drug resistance or suggested to be implicated hereinto.
• the inventors are then the first to show the role of Annexin I in expression of drug resistance to anticancer drugs.
• the present invention is the first to show an Annexin-based mutlidrug resistance pathway in cells.
• Nucleotide sequences are presented herein by single strand, in the 5' to 3' direction, from left to right, using the one letter nucleotide symbols as commonly used in the art and in accordance with the recommendations of the IUPAC-IUB Biochemical Nomenclature Commission.
• rDNA recombinant DNA
• isolated nucleic acid molecule refers to a polymer of nucleotides. Non-limiting examples thereof include DNA and RNA molecules purified from their natural environment.
• recombinant DNA refers to a DNA molecule resulting from the joining of DNA segments. This is often referred to as genetic engineering.
• DNA segment is used herein, to refer to a DNA molecule comprising a linear stretch or sequence of nucleotides. This sequence when read in accordance with the genetic code, can encode a linear stretch or sequence of amino acids which can be referred to as a polypeptide, protein, protein fragment and the like.
• amplification pair refers herein to a pair of oligonucleotides (oligos) of the present invention, which are selected to be used together in amplifying a selected nucleic acid sequence by one of a number of types of amplification processes, preferably a polymerase chain reaction.
• amplification processes include ligase chain reaction, strand displacement amplification, or nucleic acid sequence-based amplification, as explained in greater detail below.
• the oligos are designed to bind to a complementary sequence under selected conditions.
• the nucleic acid (i.e. DNA or RNA) for practising the present invention may be obtained according to well known methods.
• Oligonucleotide probes or primers of the present invention may be of any suitable length, depending on the particular assay format and the particular needs and targeted genomes employed. In general, the oligonucleotide probes or primers are at least 12 nucleotides in length, preferably between 15 and 24 molecules, and they may be adapted to be especially suited to a chosen nucleic acid amplification system.
• the oligonucleotide probes and primers can be designed by taking into consideration the melting point of hydrizidation thereof with its targeted sequence (see below and in Sambrook et al., 1989, Molecular Cloning - A Laboratory Manual, 2nd Edition, CSH Laboratories; Ausubel et al., 1989, in Current Protocols in Molecular Biology, John Wiley & Sons Inc., N.Y.).
• Nucleic acid hybridization refers generally to the hybridization of two single-stranded nucleic acid molecules having complementary base sequences, which under appropriate conditions will form a thermodynamically favored double-stranded structure. Examples of hybridization conditions can be found in the two laboratory manuals referred above (Sambrook et al., 1989, and Ausubel et al., 1989) and are commonly known in the art.
• a nitrocellulose filter can be incubated overnight at 65°C with a labeled probe in a solution containing 50% formamide, high salt ( 5 x SSC or 5 x SSPE), 5 x Denhardt's solution, 1% SDS, and 100 ⁇ g/ml denatured carried DNA ( i.e. salmon sperm DNA).
• the non-specifically binding probe can then be washed off the filter by several washes in 0.2 x SSC/0.1 % SDS at a temperature which is selected in view of the desired stringency: room temperature (low stringency), 42°C (moderate stringency) or 65°C (high stringency).
• the selected temperature is based on the melting temperature (Tm) of the DNA hybrid.
• Tm melting temperature
• RNA-DNA hybrids can also be formed and detected.
• the conditions of hybridization and washing can be adapted according to well known methods by the person of ordinary skill. Stringent conditions will be preferably used (Sambrook et al.,1989, supra).
• Probes of the invention can be utilized with naturally occurring sugar-phosphate backbones as well as modified backbones including phosphorothioates, dithionates, alkyl phosphonates and -nucleotides and the like. Modified sugar-phosphate backbones are generally taught by Miller, 1988, Ann. Reports Med. Chem. 23:295 and Moran et al., 1987, Nucleic acid molecule. Acids Res., 14:5019. Probes of the invention can be constructed of either ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), and preferably of DNA.
• RNA ribonucleic acid
• DNA deoxyribonucleic acid
• probes can be used include Southern blots (DNA detection), dot or slot blots (DNA, RNA), and Northern blots (RNA detection). Although less prepared, labeled proteins could also be used to detect a particular nucleic acid sequence to which it binds. Other detection methods include kits containing probes on a dipstick setup and the like.
• Probes can be labeled according to numerous well known methods (Sambrook et al., 1989, supra). Non-limiting examples of labels include 3 H, 14 C, 32 P, and 35 S. Non-limiting examples of detectable markers include ligands, fluorophores, chemiluminescent agents, enzymes, and antibodies. Other detectable markers for use with probes, which can enable an increase in sensitivity of the method of the invention, include biotin and radionucleotides. It will become evident to the person of ordinary skill that the choice of a particular label dictates the manner in which it is bound to the probe.
• radioactive nucleotides can be incorporated into probes of the invention by several methods.
• Non-limiting examples thereof include kinasing the 5' ends of the probes using gamma 32 P ATP and polynucleotide kinase, using the Klenow fragment of Pol I of E. coli in the presence of radioactive dNTP (i.e. uniformly labeled DNA probe using random oligonucleotide primers in low-melt gels), using the SP6/T7 system to transcribe a DNA segment in the presence of one or more radioactive NTP, and the like.
• radioactive dNTP i.e. uniformly labeled DNA probe using random oligonucleotide primers in low-melt gels
• oligonucleotides or “oligos” define a molecule having two or more nucleotides (ribo or deoxyribonucleotides). The size of the oligo will be dictated by the particular situation and ultimately on the particular use thereof and adapted accordingly by the person of ordinary skill.
• An oligonucleotide can be synthetised chemically or derived by cloning according to well known methods.
• a "primer” defines an oligonucleotide which is capable of annealing to a target sequence, thereby creating a double stranded region which can serve as an initiation point for DNA synthesis under suitable conditions.
• Amplification of a selected, or target, nucleic acid sequence may be carried out by a number of suitable methods. See generally Kwoh et al., 1990, Am. Biotechnol. Lab. 8:14-25. Numerous amplification techniques have been described and can be readily adapted to suit particular needs of a person of ordinary skill. Non-limiting examples of amplification techniques include polymerase chain reaction (PCR), ligase chain reaction (LCR), strand displacement amplification (SDA), transcription-based amplification, the Q ⁇ replicase system and NASBA (Kwoh et al., 1989, Proc. Natl. Acad. Sci.
• PCR polymerase chain reaction
• LCR ligase chain reaction
• SDA strand displacement amplification
• transcription-based amplification the Q ⁇ replicase system
• NASBA Kermuth et al., 1989, Proc. Natl. Acad. Sci.
• amplification will be carried out using PCR.
• PCR Polymerase chain reaction
• U.S. Pat. Nos. 4,683,195; 4,683,202; 4,800,159; and 4,965,188 the disclosures of all three U.S. Patent are incorporated herein by reference.
• PCR involves, a treatment of a nucleic acid sample (e.g., in the presence of a heat stable DNA polymerase) under hybridizing conditions, with one oligonucleotide primer for each strand of the specific sequence to be detected.
• An extension product of each primer which is synthesized is complementary to each of the two nucleic acid strands, with the primers sufficiently complementary to each strand of the specific sequence to hybridize therewith.
• the extension product synthesized from each primer can also serve as a template for further synthesis of extension products using the same primers.
• the sample is analysed to assess whether the sequence or sequences to be detected are present. Detection of the amplified sequence may be carried out by visualization following EtBr staining of the DNA following gel electrophores, or using a detectable label in accordance with known techniques, and the like.
• EtBr staining of the DNA following gel electrophores, or using a detectable label in accordance with known techniques, and the like.
• Ligase chain reaction LCR is carried out in accordance with known techniques (Weiss, 1991 , Science 254:1292).
• SDA Strand displacement amplification
• the term "gene” is well known in the art and relates to a nucleic acid sequence defining a single protein or polypeptide.
• a "structural gene” defines a DNA sequence which is transcribed into RNA and translated into a protein having a specific amino acid sequence thereby giving rise the a specific polypeptide or protein. It will readily recognized by the person of ordinary skill, that the nucleic acid sequence of the present invention can be incorporated into anyone of numerous established kit formats which are well known in the art.
• the term “vector” is commonly known in the art and defines a plasmid DNA, phage DNA, viral DNA and the like, which can serve as a DNA vehicle into which DNA of the present invention can be cloned.
• expression defines the process by which a structural gene is transcribed into mRNA (transcription), the mRNA is then being translated (translation) into one polypeptide (or protein) or more.
• expression vector defines a vector or vehicle as described above but designed to enable the expression of an inserted sequence following transformation into a host.
• the cloned gene (inserted sequence) is usually placed under the control of control element sequences such as promoter sequences.
• control element sequences such as promoter sequences.
• the placing of a cloned gene under such control sequences is often referred to as being operably linked to control elements or sequences.
• Expression control sequences will vary depending on whether the vector is designed to express the operably linked gene in a prokaryotic or eukaryotic host or both (shuttle vectors) and can additionally contain transcriptional elements such as enhancer elements, termination sequences, tissue-specificity elements, and/or translational initiation and termination sites.
• the designation "functional derivative” denotes, in the context of a functional derivative of a sequence whether an nucleic acid or amino acid sequence, a molecule that retains a biological activity
• This functional derivative or equivalent may be a natural derivatives or may be prepared synthetically. Such derivatives include amino acid sequences having substitutions, deletions, or additions of one or more amino acids, provided that the biological activity of the protein is conserved. The same applies to derivatives of nucleic acid sequences which can have substitutions, deletions, or additions of one or more nucleotides, provided that the biological activity of the sequence is generally maintained.
• the substituting amino acid as chemico-physical properties which are similar to that of the substituted amino acid.
• the similar chemico-physical properties include, similarities in charge, bulkiness * hydrophobicity, hydrophylic ⁇ ty and the like.
• the term "functional derivatives" is intended to include “fragments", “segments", “variants”, “analogs” or “chemical derivatives” of the subject matter of the present invention.
• variant refers herein to a protein or nucleic acid molecule which is substantially similar in structure and biological activity to the protein or nucleic acid of the present invention.
• the functional derivatives of the present invention can be synthesized chemically or produced through recombinant DNA technology, all these methods are well known in the art.
• chemical derivatives is meant to cover additional chemical moieties not normally part of the subject matter of the invention. Such moieties could affect the physico-chemical characteristic of the derivative (i.e. solubility, absorption, half life and the like, decrease of toxicity). Such moieties are exemplified in Remington's Pharmaceutical Sciences (1980). Methods of coupling these chemical-physical moieties to a polypeptide are well known in the art.
• allele defines an alternative form of a gene which occupies a given locus on a chromosome.
• a “mutation” is a detectable change in the genetic material which can be transmitted to a daughter cell.
• a mutation can be, for example, a detectable change in one or more deoxyribonucleotide.
• nucleotides can be added, deleted, substituted for, inverted, or transposed to a new position.
• Spontaneous mutations and experimentally induced mutations exist.
• the result of a mutations of nucleic acid molecule is a mutant nucleic acid molecule.
• a mutant polypeptide can be encoded from this mutant nucleic acid molecule.
• the term "purified” refers to a molecule having been separated from a cellular component.
• a “purified protein” has been purified to a level not found in nature.
• a “substantially pure” molecule is a molecule that is lacking in all other cellular components. While the property of a host cell to become MDR is demonstrated with human P-40/Annexin I other Annexins (ll-XI), non-human Annexins, other biologically functional genes/cDNA-related to Annexins can also be used in the context of the present invention. For example, Mouse Annexin I could be used in some embodiments of the present invention as will be recognized by a person of ordinary skill.
• Annexins are part of a gene family of multifunctional calcium - and phospholipid - binding protein (for a review see Raynald et al., 1994, Biochem., Biophys. Acta., 1197:45-62). They have been described in many organisms from mammals, to molds and even plants, and their similar functional properties in Ca 2+ and phospholipid are explainable by their common primary structure (Raynald et al., 1994, supra). Indeed, some of the Annexins are thought to have originated from a common ancestor (Raynald et al., 199, supra). Moreover, the family of Annexin genes shows very significant identity between human, rat, and mouse homologs (Raynald et al., 1994, supra). It will be clear to the person of ordinary skill that the present invention is not to be limited to human Annexins as homologs having the biological function of Annexin-based MDR can be used within the context of the present invention.
• Annexins are found in diverse evolutionary distant organisms such as plants, yeasts, and parasites (Raynald et al., 1994, supra), the present invention has very broad implications.
• the present invention opens the way to use Annexins form diverse organisms, such as yeast (i.e. Candida albicans) and parasites as therapeutic targets.
• Antifungal drugs for example, could be identified by using yeast Annexins as therapeutic targets.
• Annexins in plants could find utility in the development of specific crop resistance, by for example, increasing the expression level of at least one Annexin.
• a method of reducing Annexin-based MDR in a cell or animal comprising the step of administering a therapeutically effective amount of a pharmaceutical composition according to the instant invention.
• the said polypeptide may be incorporated into preparations in either liquid or solid forms using carriers and excipients conventionally employed in the pharmaceutical art, optionally in combination with further active ingredients.
• the preparation may, for example, be applied orally, parenterally, enterally or preferably topically.
• Preferred forms include, for example, solutions, emulsions, gels, sprays, lotions, ointments, creams or powders.
• Annexin-based MDR reducing agent to be administered. It is apparent that the dosage will be dependent on the particular treatment used. It should also be clear that the dosage should be chosen to display the biological activity without causing adverse effects. It will be understood that age, sex, type of disease, of formulation and other variables known to the person of ordinary skill will affect determination of the dosage to be used.
• compositions may be formulated as dosage units, each unit being adapted to supply a fixed dose of active ingredient.
• the total daily dose may, of course, be varied depending on the subject treated and the particular use of the composition. This can obviously be adapted by the treating professional.
• the present invention also provides polyclonal, monclonal antibodies, or humanized versions thereof, chimeric antibodies and the like which inhibit or neutralize their respective Annexin targetted antigens in vivo and/or specific thereto.
• Treatments comprise parenteral administration of multiple or single doses of the above listed antibodies and derivatives thereof.
• the dosage will be varied by the practitioning professional depending on the usual parameters such as pharmacodynamic characteristics the route of administration, recipient's characteristics, symptoms and/or disease thereof and the like.
• a daily dosage of active ingredient can be for example about 0.1 to 100 mg/kg of body weight, ordinarily 0.5 - 50 and preferably 1-10 mg/kg per day, i.e. divided doses ranging from 1-6 times per day or alternatively in sustained release form.
• the non-human animals of the present invention having a transgenic interruption or alteration of the Annexin endogenous gene(s) (knockout animal) and/or into the genome in which transgenes directing expression of Annexin(s) has been introduced include vertebrates such as rodents, non- human primates, amphibians, reptiles and the like. These animals are prepared in accordance with known methods. The same applies to transgenic plants. BRIEF DESCRIPTION OF THE DRAWINGS
• Figure 1 A shows a restriction map of the gene encoding P-40.
• FIG. 1 B shows the nucleic acid sequence of P-40/Annexin I.
• Figure 1 C shows the amino acid sequence of P-40/Annexin I.
• Figure 1 D shows the nucleic acid sequence of both strands of P-40/Annexin I and hence shows examples of antisense nucleic acid molecules which can be used in accordance with the present invention.
• Figure 2 shows the in vitro expression of P-40.
• FIG. 2a shows in vitro transcribed and translated mixes containing PCRII vector only immunoprecipitated with IPM96 mAb or PCRII plus 1.4 kb insert immunoprecipitated with IPM96 mAb or an irrelevant lgG2b (lanes 2 and 3, respectively).
• Lanes 1-4 of figure 2b show the same samples as in lanes 1 and 2 of Fig. 2a but transferred to nitrocellulose membrane and probed with a specific antibody to P-40 (IPM96 mAb ; Wang et al., 1997, Biochem. Biophys. Res. Com.
• FIG. 3 shows the analysis of protein and mRNA levels in drug sensitive and resistant cells.
• MCF-7, SKOV3 and H69 drug sensitive
• MCF-/Adr, SKOV/VLB 1 ° and H69/AR drug resistant human MDR cell lines
• RNA levels in the same cell lines total RNAs were resolved on agarose gel and transferred to nylon membrane and probed with 32 P-dATP labelled 1.4 kb fragment encoding for P-40 (or annexin I) and actin (Fig. 3b).
• Figure 3b shows that the expression of a 1.6 kb mRNA in drug sensitive and resistant MDR cells correlates with levels of P-40 (or annexin I) in the same cell lines.
• FIG. 4 shows the post-translational modification of P-40 or
• Annexin I in MCF-7/AR cells were metabolically labeled with 35 Smethionine or 32 P inorganic phosphate and radiolabeled protein were immunoprecipitated with an irrelevant lgG2b or IPM96 mAb (lanes 1 and 3 or 2 and 4, respectively).
• Figure 5 shows the expression of Annexins I, II, IV and VI in drug sensitive and resistant cells.
• H69, SKOV3 and AuxB1) and resistant (MCF-7/Adr, H69/AR, SKOV3 ⁇ /LB 1 0 , SKOV3/VLB0.06 and CH r C5) or revertant (H69/PR) were resolved on
• FIG. 6 shows the drug sensitivity assays for P-40 transient transfectants.
• the level of P-40 (Annexin I) expression in MCF-7 transiently transfected cells was determined by Western blotting (figure 6a).
• MCF-7 cells transfected with pCDNA3 vector without or with P-40 (Annexin I) gene were incubated in the absence and presence of increasing concentrations of Taxol
• FIG. 6b shows the P-40 (or annexin I) construct pCIN4P-40.
• the full length P-40 (or annexin I) cDNA was cloned into the Not-1 site of eukaryotic expression vector pCIN4 in both sense and antisense orientations.
• the neomycin phosphotransferase gene is also present on the construct with its expression being driven by the same promoter element located ahead of multi-cloning sites (indicated in the map), thus providing G418 resistance to cells containing a construct.
• the encyphalomyocarditis virus internal ribosome entry sites IRS
• IVS Intervening sequences
• Figure 8 shows the expression of P-40 (or annexin I) in MCF- 7 stable transfectants.
• the expression level of P-40 was determined by Western blot and indirect immunofluorescence.
• Figure 8a shows equal number of cells from MCF-7 transfected with vector alone (lane 1) or vector plus P- 40cDNA (P-40-MCF-7) (lane 2) were lysed and the total lysates were separated by 10% SDS PAGE and blotted onto PVDF membrane. The blot was probed with IPM96 monoclonal antibody.
• Figure 8b shows immunohistochemical staining of MCF-7 cells transfected with vector alone or vector plus P-40cDNA.
• P-40 (or annexin I) is stable transfectants (P-40-MCF-7) was determined using the cytospins probed with IPM96 mAb and FITC conjugated goat anti-mouse secondary antidoby as described in Materials and Methods.
• Figure 8b shows the expression of P-40/annexin I by immunofluorescence analysis using the IPM96 mAb.
• Figure 9 shows the effects of anticancer drugs on MCF-7 cells stably expressing P-40 (or annexin I).
• MCF-cells transfected with P-40 cDNA or vector only were incubated in the absence and presence of increasing concentration of adriamycin (doxorubicin), actinomycin D, Taxol and cisplatin.
• doxorubicin adriamycin
• actinomycin D actinomycin D
• Taxol Taxol
• cisplatin adriamycin
• the sensitivity of the cells to the drugs was measured by the tetrazolium salt based assay as described in Materials and Methods. Experiments were performed in triplicates.
• Drug sensitive ( MCF-7, H69, SKOV3 and HL60) and resistant (MCF-7/Adr, H69/Adr, SKOV3/VLB 1 ° and HL60/AR) cells were grown in the absence of antibiotics in ⁇ -MEM or RPMI-1640 media supplemented with 5% to 15% fetal calf serum (Hyclon. Inc.) as previously described (Mirski et al., 1987, Cancer Res. 47: 2594-2598; McGrath et al., 1987, Biochem. Biophys. Res. Com. 145(3): 1 171-1176; Bradley et al., 1989, Cancer Res. 49: 2790- 2796; Batist et al., 1986, J. Biol.
• Chem. 261(33): 15544-15549 Briefly, cells were grown at 37°C in humid atmosphere of 5% CO2 and 95% air. Cells were passaged when 70-80% confluent for adherent cells and 1X106 cell/ml for cells in suspension. Drug resistant cells were grown continuously with the appropriate concentrations of cytotoxic drugs 24 hours following subculturing. All cells were examined for Mycoplasma contamination using the Mycoplasma PCR method (Strategen Inc. San Diego, CA).
• MCF-7/Adr cells at 70-80% confluency were metabolically labeled with [ 35 S] methionine (100 ⁇ Ci/ml; 1000 Ci/mmol; Amersham Life Sciences, Inc.) or Carrier free 32 P inorganic phosphate (8 mCi/ml; Amersham Life Sciences, Inc.) for 3-4 hours at 37° C in methionine- or phosphate-free ⁇ -MEM.
• Cells were lysed and the cell lysates were immunoprecipitated with lgG 2b , or IPM96 mAb. Screening an expression library with IPM96 monoclonal antibody
• a 5' stretch cDNA expression library of HeLa cells constructed into ⁇ gtl 1 vector was obtained from Clontech (Palo Alto, CA). About
• 1X10 6 plaque forming units were plated using Escherichia coli Y1090 as host and screened with IPM96 monoclonal antibody. Briefly, plates containing phage plaques were incubated at 42°C for 4 hours and then overlayed with a dried nitrocellulose filter saturated in 10 mM X-gal. The plates were continuously incubated at 37°C for another 3 hours and the filters were rinsed with TBST buffer (50 mM Tris-HCL pH 7.9, 150 mM NaCI, 0.05% Tween-20) and incubated in TBST containing 5% of skin milk for 30 minutes with gentle agitation. The nitrocellulose disks were incubated with TBST buffer containing 2 ⁇ g/ml of
• Protein G purified IPM96 monoclonal antibody The reactive plaques were detected with a second goat anti-mouse IgG conjugated to horseradish peroxidase and visualized chemiluminescence using Amersham ECL kit. The immunoreactive plaques were verified by duplicate lifts and purified by subsequent rounds of screening using decreased plaque density.
• Immunoreactive plaques were eluted in ddH2O and utilized as template for PCR directed by the 3' and 5' insert screening amplimer sequence of ⁇ gt-11 and the fragment from PCR was cloned into PCR II vector (InVitrogen Inc.) following standard procedures (Sambrook et al., 1989, Molecular cloning: A laboratory
• the cDNA clones of the present invention were sequenced by the dideoxy chain termination method using M13 universal primer and sequence specific primers via the automated DNA sequencing service at the Sheldon Biotech Centre at McGill University. Both strands of two different pick clones were completely sequenced. Computer analysis of the DNA and protein sequence was done using the Blast DNA search programs. Northern blot and DNA slot blot analyses
• RNAs from drug sensitive and resistant cells were extracted with the Trazol solution (GibcoBRL, Gaithersburg,
• RNA from each cell line was run on a 1% formaldehyde-denatured agarose gel and transferred to HybondsTM-N nylon membrane (Amersham, Oakvill, Ont.) by pressure blotting with 20X SSC (1X
• RNA integrity and equal loading was assessed in all cases by hybridization with actin or rRNA probes.
• genomic DNA from MCF-7 and MCF-7/Adr cells was prepared as described (Sambrook et al., 1989, supra) and 10 ⁇ g DNA from each sample was denatured by 1 M NaOH. Following 10 minute boiling at 100°C. The denatured DNA was diluted serially loaded onto Nylon membrane by using Manifold Slot Blot apparatus (Pharmacia Inc.). The blot was then hybridized with a 32P-labeled probe at 42°C in 50% formamide, 2.5XDenhardt's solution, 25 mg/ml denatured summon sperm DNA, 1 % SDS and 1.25X SSPE overnight. The highest stringent wash of the blot was 0.5X SSC with 1% SDS at 65°C for 30 minutes.
• 35S-methionine (Dupont/NEW, Mississauga, Ont.) according to the manufacturer's protocol. Briefly, cDNA clone encoding full length P-40 was cloned into Notl and Xhol sites of a pCDNA3 expression vector containing T7 and SP6 promotor sequences (In Vitrogene, Inc.). The pCDNA3 with and without P-40 insert were added to a coupled reticulocyte lysate transcription and translation system in the presence of [35S]-methionine. Following a 2 hour incubation at 30°C, in vitro synthesized proteins were immunoprecipitated with IPM96 monoclonal antibody as previously described (Georges et al., 1991 , J. Cell. Physiol. 148: 479-484; Wang et al., 1997, supra). DNA Transfection
• MCF-7 cells were transfected with the pCDNA3 or pCIN4 vectors or vector containing P-40 coding sequence using NpofectAMINE (Gibco, Burlington, Ontario, Canada) according to the manufacturer's procedure. Briefly, 4 X 10 5 cells in a 60 mm plate with 5 ml of serum-free minimal essential medium were overlaid with 200 ⁇ l of serum-free essential medium containing 5 ⁇ g of supercoiled DNA mixed with 10 ⁇ l of lipofectAMINE. After 5 hour incubation, the medium was replaced with 5 ml of minimal essential medium (MEM) supplemented with 10% fatal calf serum (Hyclone Laboratories) and cells were further cultured at 37°C for 24 - 48 hours.
• MEM minimal essential medium
• Transient transfectants were harvested 48 hours later and aliquoted at 7.5X10 3 cells per well of a 96-well plate. Drugs were added to cells 24 hours later and incubated for another 48 hours at 37°C. The assay was developed by adding 50 ⁇ l (5 mg/ml) of an MTT dye and allowed to incubate for four hours at 37°C as previously described (Pouliot et al., 1997, Biochem. Pharmacol. 53(1): 17-25) .
• Total cell lysates cell cultures or from transcription/translation reaction mixture were mixed with equal volumes of sample buffer containing SDS and the denatured proteins were resolved on 10% polyacrylamide gels according to the method of Laemmeli (Laemmli et al., 1970, Nature 227(259): 680-685).
• acrylamide gels containing 35S-methionine labeled proteins gels were fixed in 50% methanol/water and soaked in Amplify TM (Amersham, Oakvill , Ont.) for 30 minutes prior to drying and exposing to Kodak film at -70°C.
• proteins were transferred to nitrocellulose membrane for 1 hour at 50 volts according to the method of Towbin et al., (Towbin et al., 1979, Proc. National Acad. of Sciences of the United States of America 76(9): 4350-4354).
• the nitrocellulose membrane was blocked with 5% skin milk/PBS and incubated overnight at 4°C with 1 ⁇ g/ml of IPM96 monoclonal antibody.
• the immunoreactive proteins were detected with horseradish peroxidase conjugated goat anti-mouse antibody and visualized by chemoluminence using Amersham ECL kit (Amersham, Oakvill , Ont.). Cytotoxicity assays
• chemosensitivity patterns of stably transfected MCF-7 cells with pCIN4 vector alone or full length P-40 cDNA were determined by a tetrazolium salt-based microplate assay as described previously (Pouliot et al., 1997). Briefly, 100 ⁇ l aliquots of cells were plated into 96-well plates at 5000 cells per well. The cells were then incubated at 37° C for 24 hours before the addition of increasing concentration of different cytotoxic agents.
• FIG. 2a shows the immuno- precipitation of proteins with IPM96 mAb from an in vitro expression reactions containing vector only (lane 1) or vector plus 1.4 kb insert (lane 2).
• an irrelevant lgG2b was used to immunoprecipitate proteins from a reaction mix containing vector plus the 1.4 kb insert (figure 2a, lane 3).
• FIG. 2a shows a 35 S-methionine labeled 40 kDa protein immunoprecipitated with IPM96 mAb but not with an irrelevant lgG2b (lanes 2 and 3, respectively).
• Figure 2b also shows Western blotting of the proteins identical to those in lanes 1 and 2 of figure 2a, but probed with IPM96 mAb (lanes 1 and 2) or an irrelevant lgG2b
• Annexin I cloned from HeLa cells is similar or different from that found in MCF-7/AR ceils, Annexin I was cloned from MCF-7/AR cells by reverse PCR using 5' and 3' primers encoding Annexin I. Sequence analysis of Annexin I clones from MCF-7/AR cells revealed no differences from that isolated from HeLa cells cDNA expression library (data not shown). Northern blot analyses In an earlier study (Wang et al., 1997, supra) , the levels of
• FIG. 3a shows a Western blot analysis of total cell lysates from drug sensitive (MCF-7, SKOV3 and H69) and their resistant (MCF-7/AR, SKOV3 ⁇ LB 1 ° and H69/AR) cells probed with IPM96 mAb.
• MCF-7, SKOV3 and H69 drug sensitive
• MCF-7/AR, SKOV3 ⁇ LB 1 ° and H69/AR resistant cells probed with IPM96 mAb.
• the results of the latter Western blot analysis shows clearly the increase in P-40 expression in resistant cells relative to the parental drug sensitive cells.
• the SKOV3 cells show lower levels of P-40 than the resistant SKOV3/VLB 1 °.
• the SKOV3 cell line was derived from a patient with ovarian tumor that was considered to be clinically resistant Cis-platinum and adriamycin [(ogh et al., 1975, Plenum Press, New York : 155-159).
• Cis-platinum and adriamycin (ogh et al., 1975, Plenum Press, New York : 155-159).
• Northern blot analysis were performed with total RNA extracted from drug sensitive (MCF-7, SKOV3 and H69) and resistant (MCF-7/AR, SKOV3/VLB 1 ° and H69/AR) cells and blotted membrane probed with a 32P labeled 1.4 kb fragment.
• FIG 3b shows the 1.8 kb mRNA band in resistant MCF-7/AR and H69/AR but not in the parental drug sensitive cell lines, MCF-7 and H69/AR.
• the level of 1.8 kb mRNA hybridizing band was 4-fold higher drug resistant SKOV/VLB 1 ° cells relative to drug sensitive SKOV-3 cells (figure 3b).
• the Northern blot demonstrate clearly that the observed increase in P-40 or Annexin I cells is due to an increase in mRNA of P-40.
• the Northern blot results are consistent with the Western blot data, especially those relating to the protein and mRNA levels of P40 or Annexin I in SKOV3 versus SKOV3/VLB 1 ° cells (figure 3).
• Annexin I is a phosphoprotein phosphorylated at serine and tyrosine amino acids (Wang et al., 1994, Biochem. , 33: 276-282; Varticovski et al., 1988, Biochem., 27: 3682-3690). Further, it has been shown that phosphorylation of Annexin I at its N-terminal domain decreases its affinity for negatively charged phospholipids or membrane vesicle aggregation (Wang et al., 1994, Biochem., 33: 276-282 ]. Given the above results, it was of interest to examine the post-translational modification of P-40 or Annexin I in MDR cells.
• FIG. 5 shows the results of the Western blots probed with anti-Annexin I, II, IV and VI monoclonal antibodies.
• the results of the Western blot probed with anti-Annexin I show similar results to the Western blot in figure 3a probed with IPM96, confirming the antigen specificity of IPM96 mAb towards to Annexin I.
• Annexin II and IV are also increased in MDR cells relative to the parental cell lines, however to a lesser extend as that of Annexin I.
• both annexin II and IV are expressed, at lower levels, in drug sensitive cells (figure 5).
• cDNA transfections (transient and stable) of Annexins II- XI, as described below will be carried out to verify the drug resistance of Annexin ll-XI-transfected cells.
• results in lane 3 of figure 6a show a 40 kDa protein in MCF-7 cells transfected with pCDNA3 vector plus P-40 or Annexin I.
• MCF-7 cells transfected with pCDNA3 plus P-40 or Annexin I full length cDNA were analyzed by indirect immunofluorescence with IPM96 mAb.
• the results in figure 6b showed the relative number ( ⁇ 5%) of MCF-7 cells that overexpress P-40 or Annexin I.
• the efficiency of the transient transfection to -5% of the cells was confirmed following transfection with pCDNA3 containing a beta-gal gene (data not shown). Having established the expression of P-40 or Annexin I in
• MCF-7 cells it was of interest to examine the effect of P-40 or Annexin I overexpression (albeit ⁇ 5%) on the sensitivity of MCF-7 cells to anticancer drugs.
• the results in figure 6b and 6c show MCF-7 cells transfected with vector only and with vector plus P-40 incubated with increasing concentrations of Taxol or adriamycin, respectively.
• overexpression of P-40 or Annexin I in MCF-7 cells decreases their sensitivity to Taxol and adriamycin.
• the analysis as to whether higher levels of P-40 or Annexin I expression will lead to a larger decrease in the sensitivity of transfectant cells to anticancer drugs has also been formerly tested using for stable cell lines expressing P-40-cDNA (see below). The transient transfected cells were incubated with a chelator
• EGTA EGTA
• Verapamil calcium channel blocker
• P-40 (or annexin I) is highly expressed in several MDR cell lines relative to their parental drug sensitive cells (Wang et al., 1997, supra).
• the MDR cell lines used in our study were previously shown to contain amplified copies of Pgp MDR-1 or MRP genes.
• the genes encoding Pgp, MRP and P-40 (or annexin I) are localized on chromosome 7, 16 and 9 respectively (Cole et al., 1993, supra; Trent and Witkowski, 1987, supra; Wallner et al., 1986, supra). Therefore, the observed increase in P-40 protein is not due to a co-amplification of P-gp or MRP.
• Annexins share a similar core domain with four or eight conserved 70 amino acid repeats and an amino terminal domain that varies in length and sequence between the different members of the annexin family Although the physiological function(s) of annexins is not clear, they have been implicated in calcium-regulated exocytosis (Drust et al., 1988, Nature 331 : 88- 91 ; Creutz et al., 1987, J. Biol. Chem. 262: 1860-1865). Annexin I has also been shown to mediate the calcium-dependent fusion of liposomes with isolated neutrophil plasma membranes (Meers et al., 1986, Nature 321: 81-84).
• Annexins are generally phosphorylated in response to varieties of stimuli.
• Annexin I is phosphorylated by EGF receptor-kinase at tyrosine residues found in the N-terminal head domain (Pepinsky et al., 1986, Nature 321 : 81-84) and by protein kinases C and A (Varticovski et al., 1988, Biochem. 27: 3682- 3690].
• phosphorylation of Annexin I at the amino terminal domain by protein kinase C inhibits its ability to aggregate chromaffin granules (Wang et al., 1986, J. Biol. Chem. 261: 6548-6553.).
• Annexin I was shown to be rapidly phosphorylated upon stimulation of cells to secrete normal cell metabolites Wang et al. 1992, Biochem, 31: 9934-9939) Taken together, our finding that Annexin I is not phosphorylated in MDR cells is consistent with its increased capacity to cause aggregation of membrane vesicles.
• P-40 cannot be labelled by photoactive analogues of cytotoxic agents (data not shown) and that its amino acid sequence does not encode an ATP binding domain suggest that P-40 (or annexin I) itself does not transport drugs directly to the extracellular environment.
• the intracellular distribution of P-40 in both in vitro selected P-40 expressing cells and in the transfectant cells suggests that the membrane localization of P-40 (annexin I) is essential for its function in drug resistance.
• Translocation of P-40 from the plasma membrane to cytoplasm after treatment with verapamil and EDTA has been observed in both P-40 expressing cell lines and P-40 cDNA transfectants (data not shown). This finding suggests that P-40 may participate in sequestering drugs from their targets.
• the intracellular action concentration (eg. Ca 2+ ) may be important for the functions of P-40 (annexin I) in drug resistance.
• P-40 annexin I
• MDR modulators Given the role of annexins in promoting aggregation of membrane vesicles through Ca 2+ dependent phospholipid binding, it is proposed that P-40 (annexin I) confers a drug resistance phenotype by aggregation of drug filled membrane vesicles or exocytosis of such drug filed vesicles (Warrt et al., Cancer Res. 46: 825- 830). The observed increase in membrane voculisation in many MDR cell lines (Beck, 1987, Biochem.
• Phamacol. _36(18): 2879-2887; Sehested et al., 1987 British J. of Cancer 56: 747-751) supports this proposition. Furthermore, P- glycoprotein an MRP have been detected in the endosomal membranes in in vitro selected cell lines (Abbaszadegan et al., 1997, Cancer Res. 56(23): 5435- 5442; Klohs et al., 1988, Mol. Pharmacol. 34: 180-185). Thus ' P-40 (or annexin I) or other members of the annexin family could function together with P- glycoprotein or MRP to cause the aggregation and possibly exocytosis of drug filled vesicles.
• the present invention strongly suggests their importance in the establishment of the MDR phenotype in cancer cells.
• the present invention presents means to further dissect the structure/function relationship of annexins in MDR and provides methods and assays to identify modulators of this MDR phenotype.
• FIG 9 shows the results of the chemosensitivity assays using P-40-MCF-7 cells in the presence of increasing concentration of anticancer drugs to that of MCF-7 cells transfected with vector alone.
• P-40-MCF-7 cells displayed low level of resistance to adriamycin, actinomycin D, Taxol and cisplatin ( Figure 9) relative to the cells transfected with vector alone.
• P-40-MCF-7 cells did not show cross-resistance to colchicine and vincristine (data not shown). These results suggest that the overexpression of P-40 (or annexin I) does confer a low level of drug resistance to a variety of anticancer drugs.
• the present invention provides convincing evidence that annexin and particularly annexin I are important in the development of the MDR phenotype in cancer cells.
• the herein presented findings are also important for providing a further understanding of the functions of the annexin family. Analysis of clinical tumour samples for P-40 (or annexin I) expression (primary or relapsed after chemotherapy) will provide further evidence for the diagnostic role of P-40 in clinical drug resistance.

Landscapes

• Chemical & Material Sciences (AREA)
• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Organic Chemistry (AREA)
• General Health & Medical Sciences (AREA)
• Gastroenterology & Hepatology (AREA)
• Biochemistry (AREA)
• Biophysics (AREA)
• Zoology (AREA)
• Genetics & Genomics (AREA)
• Medicinal Chemistry (AREA)
• Molecular Biology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Toxicology (AREA)
• Preparation Of Compounds By Using Micro-Organisms (AREA)
• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
• Peptides Or Proteins (AREA)

Applications Claiming Priority (3)

Publications (1)

Family

ID=4161689

Family Applications (1)

Country Status (4)

Families Citing this family (5)

Family Cites Families (1)

• 1997
  • 1997-10-24 CA CA 2219299 patent/CA2219299A1/en not_active Abandoned
• 1998
  • 1998-10-26 EP EP98949842A patent/EP1025225A1/de not_active Withdrawn
  • 1998-10-26 WO PCT/CA1998/000992 patent/WO1999021980A1/en not_active Application Discontinuation
  • 1998-10-26 AU AU96174/98A patent/AU9617498A/en not_active Abandoned

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events