WO1995009926A1 - Genes codant la resistance aux agents d'alkylation d'adn - Google Patents

Genes codant la resistance aux agents d'alkylation d'adn Download PDF

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WO1995009926A1
WO1995009926A1 PCT/US1994/011279 US9411279W WO9509926A1 WO 1995009926 A1 WO1995009926 A1 WO 1995009926A1 US 9411279 W US9411279 W US 9411279W WO 9509926 A1 WO9509926 A1 WO 9509926A1
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dna
cell
mcra
dna sequence
resistance
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PCT/US1994/011279
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David H. Sherman
Paul R. August
Michael C. Flickinger
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Regents Of The University Of Minnesota
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Priority to US08/624,447 priority Critical patent/US6524812B1/en
Publication of WO1995009926A1 publication Critical patent/WO1995009926A1/fr

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    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004Oxidoreductases (1.)
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    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/36Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Actinomyces; from Streptomyces (G)
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    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/11DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52Genes encoding for enzymes or proenzymes
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    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813Hybridisation assays
    • C12Q1/6827Hybridisation assays for detection of mutation or polymorphism

Definitions

  • Agents that act to damage DNA by alkylation, DNA-DNA crosslinking, DNA-protein crosslinking, and/or DNA cleavage have been used as potent chemotherapeutic agents. Some of these agents are reductively activated by electron transfer to a moiety such as semi-quinone. Agents which are thought to be reductively activated include the naturally occurring and synthetic mitomycins and the enediynes such as neocarzinostatin. Kasai et al., SynLett. H):788 (1992) (mitomycins); and Nicolaou et al., An ⁇ ewandte Chemie. fisl387 (1991) (enediynes). Cellular resistance to these types of chemotherapeutic agents develop, especially in tumor cells. The mechanism of resistance of tumor cells and the organisms that produce the agents are not known.
  • Mitomycins are agents that are reductively activated and catalyze DNA alkylation and DNA-DNA crosslinks. Mitomycins are antitumor antibiotics produced by Strepto yces lavendulae and other Streptomyces species. Several mitomycins have been characterized including the naturally occurring mitomycin A, mitomycin B, mitomycin C (MMC), porfiro ycin, and mitiromycin. The structural properties and biological activity of mitomycins have motivated a large number of studies to determine cellular target sites and mechanism of action. Complete structural characterization of MMC has been followed by studies showing the basis of its remarkable activity against mammalian tumor cells. Iyer et al. , Science, 145 . :55 (1964); Schwartz et al. , Science, 142:1181
  • the molecule has three important functional groups, which include a quinone ring system, carbamate moiety, and a highly strained aziridine group that contribute together to determine MMC target site specificity and ability to alkylate DNA. Significantly, the precise regions in DNA that undergo mono- and bifunctional alkylation by MMC, leading to inhibition of replication and subsequent cell death have also been determined. Cera et al. , Biochemistry. 21:3908 (1989); Kumar et al. , Biochemistry. 11:1364 (1993); Teng et al. , Biochemistry, .28:3901 (1989); and Tomasz et al. , Science, 235:1204 (1987).
  • Mitomycins are DNA bioreductive alkylating agents which present a difficult and unique challenge for cellular resistance in producing microorganisms.
  • MDR multidrug resistance
  • the invention provides for an expression cassette and vectors including the expression cassette.
  • the expression cassette comprises a DNA sequence that provides resistance to a cell to a DNA bioreductive alkylating or cleaving agent operably linked to a promoter functional in the cell.
  • the preferred DNA bioreductive alkylating or cleaving agent is a mitomycin.
  • Preferred DNA sequences are those that substantially correspond to the mcr and mrd loci of S. lavendulae B619.
  • the promoter is preferably functional in Streptomyces and provides for a sufficient level of gene expression so that resistance of the cell to the DNA bioreductive alkylating or cleaving agent can be detected.
  • a DNA probe has sufficient complementarity to all or a portion of a known DNA or RNA sequence that provides resistance to the agent so that it can hybridize to the DNA or RNA sequence, preferably under low stringency conditions. Portions of a DNA or RNA sequence preferably are restriction endonuclease fragments of the mcr or mrd DNA sequence. The preferred probes are complementary to the 6.7 kb Bell fragment of pDHS3000 encoding mcr and the 4.2 kb Bell fragment of pDHS3001 encoding mrd.
  • mcr Two loci have been identified that provide mitomycin resistance to mitomycin sensitive host cells.
  • One locus found on a 6.2 kb Bell fragment from S. lavendulae has now been designated mcr and is the same as the locus designated mcrA in U.S. Application Serial No. 08/133,963.
  • mcrA which is the same as the DNA sequence identified as mcrAl
  • mcrB which is the same as the DNA sequence identified as mcrAl
  • mcrORF3 which is the same as the DNA sequence previously identified as mcrAORF3 in U.S. Application Serial No. 08/133,963.
  • the other locus is found on a 4.2 kb Bell fragment on plasmid pDHS3001 and is now referred to as mrd and is the same as the locus previously identified as mcrB in U.S. Application No. 08/133,963.
  • the identifiers of the gene loci and DNA sequences in this application have been changed from the parent application Ser. No. 08/133,963 as described above. Subject matter from the parent application that referred to the previous identifiers has been modified to the new identifiers, but the gene loci and DNA sequences remain the same as those disclosed in the parent application Ser. No. 08/133,963.
  • the invention also provides for polypeptides and antibodies specific for the polypeptides.
  • a polypeptide can be encoded by a DNA sequence that provides resistance to a cell to a DNA bioreductive alkylating or cleaving agent such as the mcr DNA sequence.
  • the preferred polypeptide is the MCRA polypeptide which is about a 56,000 dalton polypeptide encoded by mcrA.
  • the invention also provides transformed cells.
  • Transformed cells comprise an expression cassette comprising a DNA sequence that substantially corresponds to a DNA sequence that provides resistance to the cell to a DNA bioreductive alkylating or cleaving agent operably linked to a promoter.
  • the preferred cell is a cell sensitive to the DNA bioreductive alkylating or cleaving agent such as Streptomyces lividans.
  • the preferred DNA sequence substantially corresponds to the DNA sequence of the mcrA and mcrB genes.
  • the expression cassette preferably is expressed in an amount sufficient to confer resistance to the cell to the agent.
  • the invention also provides methods for identifying agents that inhibit the resistance of the cell to the DNA bioreductive alkylating or cleaving agent.
  • One method involves using transformed cells.
  • Transformed cells resistant to the agent comprise an expression cassette as described herein.
  • the transformed cells are incubated with an effective amount of an agent suspected to inhibit resistance of the cell to the DNA bioreductive alkylating or cleaving agent and an effective amount of the DNA bioreductive alkylating or cleaving agent. After incubation for a suitable amount of time, it can be determined if the suspected agent inhibited the resistance of the cell to the DNA bioreductive alkylating or cleaving agent.
  • an inhibitory agent can be identified by its ability to inhibit the function of a polypeptide encoded by the DNA sequence.
  • a substantially pure polypeptide such as MCRA is incubated with a DNA sample and the DNA bioreductive alkylating or cleaving agent and the inhibitory agent. After incubation, it can be determined whether the inhibitory agent inhibited the function of MCRA polypeptide by measuring the binding of the DNA bioreductive alkylating or cleaving agent to the DNA sample or by determining whether DNA alkylation has occurred. If the suspected inhibitory agent inhibits the function of MCRA, binding and/or activity of the DNA bioreductive alkylating or cleaving agent is increased in the presence of the suspected inhibitory agent, preferably 2 to 20-fold.
  • the invention also provides a method for identifying sequences homologous to the mcr or mrd sequences in other cell types and/or organisms.
  • the cells are multi-drug resistant or mitomycin C-resistant tumor cells.
  • the method involves generating a DNA library and amplifying selected sequences in the library using poiymerase chain reaction. Amplification of selected sequences is accomplished by selecting oligonucleotide primers that are complementary to a portion of the mcr or mrd loci. Once formed, the amplified products are isolated and screened for homology to mcr or mrd by hybridization to a DNA probe. Sequences that hybridize can be mapped using restriction enzymes and sequenced using standard methods.
  • FIG. 1 Model of the mechanism whereby the MCRA polypeptide confers resistance against MMC.
  • the left hand side of the figure demonstrates an activation cascade for MMC while the right side shows how MCRA can prevent MMC activation.
  • an FAD bound to MCRA would remove a single electron to form FADH•.
  • FADH- could again be reduced completely to FADH 2 or undergo reoxidation to FAD.
  • FIG. 1 Nucleotide sequence and deduced amino acid sequence of mcrA. mcrB, and mcrORF3 genes of S. lavendulae B619. Shaded circles with arrows represent the translational start point (TSP) for promoters Pl and P2 as determined by primer extension.
  • the thin arrowed line labeled PE primer indicates the location of the oligonucleotides used for TSP determination. Potential -35 and -10 regions are labeled for both TSPs.
  • the dark line labeled RBS indicates a potential RBS for a polypeptide translated from the shorter transcript originating from P2.
  • the shaded valine proximal to the RBS indicates a potential start codon for the shortened mcrA polypeptide. Facing arrows reveal two divergent repeats, which may have roles in transcription termination, with ⁇ G values lower
  • Figure 3 Alignment of the mcrA and 6-hydroxy-D-nicotine oxidase deduced amino acid sequences. Identical amino acids are shaded. The histidine responsible for attachment of FAD to 6- hydroxy-D-nicotine oxidase and conserved in the deduced mcrA amino acid sequence is marked by a dot (•).
  • Figure 4 Dot plot comparisons of the mcrA deduced protein sequence with 6-hydroxy-D-nicotine oxidase (6HDN0) and L-gulono- ⁇ -lactone oxidase.
  • FIG. 5A Streptomyces lavendulae strains which produce MMC, probed with a 6.7 kb Bell insert of pDHS3000 encoding mcr. Lanes: 1, 1 kb ladder; 2, Bell S. lividans 1326; 3, BamHI S. lividans 1326; 4, Bell S. lavendulae B619; 5, BamHI S.
  • Figure 5B S. lavendulae probed with a 4.2 kb Bell insert of pDHS3001 encoding mrd. Lanes: 1, 1 kb ladder; 2, Bell S. lividans 1326; 3, BamHI S. lividans 1326; 4, Bell S. lavendulae B619; 5, BamHI S. lavendulae B619; 6, Bell S. lavendulae PB1000; 7, BamHI S. lavendulae PB1000; 8, Bell S. lavendulae NRRL 2564; 9, BamHI S. lavendulae NRRL 2564; 10, Bell S. lavendulae KY681; 11, BamHI S. lavendulae KY681; 12, ⁇ Hindlll DNA ladder.
  • FIG. 6 Frame analysis of mcr, and restriction enzyme map of pDHS3000 illustrating subcloning performed to determine the DNA sequence necessary to confer MMC resistance to S. lividans.
  • a (+) in the mitomycin C resistance column indicates that the vector to the right was able to confer MMC resistance, whilst a (-) indicates no resistance. Shaded areas represent DNA cloned from S. lavendulae B619, whereas dark areas represent vector DNA.
  • FIG. 7 Restriction-enzyme map of the pPRA112 cosmid clone containing S. lavendulae DNA adjacent to the mcr locus.
  • Figure 8. Restriction enzyme maps of the cosmid clones containing S. lavendulae DNA adjacent to the mrd locus. The central shaded region denotes the boundaries of the mrd locus.
  • Figure 9 Strategy for gene-disruption by homologous recombination.
  • the thiostrepton resistance gene (tsr) was used for selection by cloning into the middle of mcr0RF3. Single-stranded DNA was then used to transform S. lavendulae, followed by selection with thiostrepton to obtain recombinant organisms.
  • FIG 10. SDS-PAGE of purified MCRA. MCRA was purified as described in Example 4.
  • Figure 11. DNA sequence of mrd locus derived from 4.2 kb Bell fragment from plasmid pDHS3001.
  • FIG. 12 Genetic map of mrd resistance locus from Streptomyces lavendulae. Shaded regions are defined as indicated. A putative MMC hydroxylase is located at the 3' end of mrd locus. Map of subclones of mrd locus and the ability of the subclones to confer resistance to MMC at 25 ⁇ g/ml to S. lividans.
  • FIG. 13 MMC induction of MCRA expression in S. lividans/pDHS3000.
  • Panel A shows Commassie blue stained SDS-PAGE gel of extracts from S.
  • Lane 1 purified MCRA control; Lane 2, _____ lividans/piJ702 grown with 1 ⁇ g/ml MMC; 3, S. lividans/pIJ702 grown with 0 ⁇ g/ml MMC; 4, S_-_ 1ividans/PDHS3000 grown with 0 ⁇ g/ml MMC; 5, S.
  • Panel B shows Western blot of SDS-PAGE using anti-MCRA antibodies
  • Panel C shows MCRA expression as a function of MMC concentration.
  • Figure 15 Induction of MCRA expression as measured by ELISA assay in S. lividans cells containing pDHS3000. Cells were treated with 76 mM neocarzinostatin (NCS), mephalan, ( ⁇ ) -1,2:3,4 diepoxybutane (DEB), daunomycin and mitomycin C.
  • NCS neocarzinostatin
  • DEB diepoxybutane
  • Panel B amplified spectrum from panel A between 300-500 nm.
  • Figure 17 15 L fermentation of S. lavendulae showing MMC production, dry mass and MCRA expression over the course of a 240 hr culture.
  • the invention provides for an expression cassette including a DNA sequence that provides resistance to a cell to a DNA bioreductive alkylating or cleaving agent as well as a polypeptide encoded by the DNA sequence.
  • the invention also provides transformed cell lines and methods for identifying agents that inhibit the resistance of a cell to a DNA bioreductive alkylating or cleaving agents.
  • DNA probes of the invention are substantially complementary to all or a portion of a DNA sequence that provides resistance to a cell to a DNA bioreductive alkylating or cleaving agent and are useful in a method for detecting homologous DNA sequences in other organisms.
  • DNA sequences providing for resistance to a cell to a DNA bioreductive alkylating or cleaving agent • have been identified.
  • a DNA sequence that provides resistance to a DNA bioreductive alkylating or cleaving agent can encode a polypeptide that imparts resistance to cells from these agents and/or protects DNA from these agents.
  • a bioreductive DNA alkylating or cleaving agent is an agent that has a moiety that is reductively activated by electron transfer and can catalyze cleavage or alkylation of DNA.
  • Agents that have a moiety such as a benza-quinone which is reduced to a semiquinone and/or hydroquinone species to become bioreductively activated include mitomycins. While not meant to be a limitation of the invention, it is believed that once the DNA bioreductive alkylating or cleaving agent is reductively activated it binds to DNA and can catalyze one or more of the following reactions: DNA alkylation, DNA-DNA crosslinking, DNA-protein crosslinking, or DNA cleavage. It is further believed that one way that a polypeptide imparts resistance to cells from these agents is by binding to and deactivating the reduced active form of the agent. It is believed that deactivation occurs by oxidation.
  • FIG. 1 A specific example of one cellular resistance mechanism to DNA bioreductive alkylating or cleaving agents is shown in Figure 1.
  • the DNA bioreductive alkylating or cleaving agent shown in Figure 1 is mitomycin C and a polypeptide that imparts resistance to mitomycin C (MMC) is designated MCRA.
  • MMC mitomycin C
  • MCRA polypeptide that imparts resistance to mitomycin C
  • the MCRA polypeptide includes a covalently bound cofactor, such as flavin adenine dinucleotide (FAD), that mediates the oxidation of MMC in the event that it undergoes sequential reduction through the semi- quinone radical.
  • FAD flavin adenine dinucleotide
  • One electron reduction of MMC can result in immediate oxidation by MCRA bound FAD to the FADH- species.
  • the FADH- species would be available to accommodate a second electron transfer from MMC to result in a non-reduced form of MMC that cannot bind to or catalyze alkylation or crosslinking of DNA.
  • Identification of DNA sequences that provide resistance to a cell to a DNA bioreductive alkylating or cleaving agent provides for polypeptides, DNA probes, and transformed cell lines. These DNA sequences can be used in methods to identify agents that inhibit resistance of cells to a DNA bioreductive alkylating or cleaving agent as well as to identify homologous DNA sequences that provide for resistance in other cells, such as tumor cells.
  • the invention also provides for antibodies to
  • MCRA and methods for detecting expression of MCRA or related polypeptides in cells resistant to DNA bioreductive alkylating and/or cleaving agents.
  • DNA-DNA crosslinking can be potent cancer chemotherapeutic agents .
  • These agents are characterized by the ability to bind to and alter DNA.
  • a DNA cleaving agent is one that binds DNA and creates single or double-stranded breaks.
  • a DNA alkylating agent is an agent that binds to DNA and forms covalent bonds to the DNA molecule.
  • DNA alkylation includes the formation of DNA-DNA and DNA-protein crosslinks.
  • a DNA bioreductive alkylating or cleaving agent is a compound that is reductively activated. Reductive activation can occur by electron transfer to a moiety in the agent such as a benza-quinone. Molecules containing moieties such as anthraquinone, aminoquinone, or enediyne can become reductively activated and then catalyze damage to DNA.
  • Compounds that can be reductively activated preferably include the following structural component:
  • Compounds that include both an aromatic ring as shown above and optionally include an aziridine nitrogen group at carbons 1 and 2 are also perferred DNA bioreductive alkylating or cleaving agents. While not meant to be a limitation of the invention, it is believed that once the agent is activated, it can bind to DNA and catalyze DNA alkylation, crosslinking or cleavage.
  • An aromatic group can function as a site for reductive activation and an aziridine group is involved in creating DNA damage.
  • the DNA bioreductive alkylating and/or cleaving agents are also preferably those compounds that can induce expression of a DNA sequence that codes for resistance to the agent.
  • a DNA bioreductive alkylating or cleaving agent can also be a compound that does not induce expression of a DNA sequence that encodes resistance but that is inactivated by a polypeptide encoded by a DNA sequence that provide resistance to a cell to the agent. While not meant to limit the invention, it is believed that inactivation of the DNA bioreductive alkylating or cleaving agent by a polypeptide occurs ' by binding of the agent to the polypeptide followed by deactivation by oxidation of the DNA bioreductive alkylating or cleaving agent.
  • bioreductive DNA alkylating or cleavage agents include the mitomycins and related compounds. The structure of mitomycins is as follows:
  • Mitomycins contain a benza-quinone moiety that can be reduced by electron transfer to form activated mitomycins. Activated mitomycins bind to DNA and catalyze DNA-DNA and DNA-protein crosslinks and DNA alkylation.
  • Mitomycins can be naturally occurring compounds produced by Streptomyces species and include mitomycin A, mitomycin B, mitomycin C, porfiromycin, and mitiromycin. Other mitomycins include mitomycins E, G, I, J, L, M, K, mitiromycin, albomitomycin A, isomitomycin A, KW 2149, KW-2149 metabolites such as M- 16 and M-18.
  • Naturally occurring mitomycins can be isolated from growth media of Streptomyces species by known methods, as described Herr et al. , Antimicrobial Agents Annual, at page 23, Plenum Press, NY (1960). Other Mitomycin derivatives have also been chemically synthesized as described Kasai et al. in SynLett. 10:777 (1992). Mitomycin analogs can also be obtained by directed biosynthesis as described by Claridge et al., J. Antibiotics, 31:437 (1986).
  • FR900482 Another specific example of DNA bioreductive alkylating or cleaving agents are substituted dihydrobenzoxazines such as FR900482, as described in J. Am. Chem. Soc.. 109:4106 (1987).
  • FR900482 The structure of FR900482 is as follows:
  • the agent FR900482 can be obtained from growth medium of Streptomyces sandaensis 6897, as described by Kiyoto et al., J. Antibiotics. 40 . :594 (1987).
  • DNA bioreductive alkylating or cleaving agents can be produced by Streptomyces species. Because of their toxic and damaging effects on DNA, the producing microorganisms have resistance mechanisms that protect their DNA from these agents. DNA sequences that impart resistance to DNA bioreductive alkylating or cleaving agents are isolated as described herein.
  • Expression cassettes including a DNA sequence that provides resistance to a cell to a DNA bioreductive alkylating or cleaving agent
  • the invention provides an expression cassette comprising a DNA sequence that provides resistance to a cell to a DNA bioreductive alkylating or cleaving agent operably linked to a promoter functional in the cell.
  • the DNA sequence can contain one or more genes and/or portions of the genes or gene locus.
  • the DNA sequence can encode a single polypeptide or more than one polypeptide.
  • the DNA sequence can provide resistance to a DNA bioreductive alkylating or cleaving agent by encoding a polypeptide that provides resistance to the cell or protects DNA from the action of the DNA bioreductive alkylating or cleaving agent.
  • the promoter is preferably a DNA sequence that provides for a sufficient level of expression of the gene or genes encoded on the DNA sequence in the cell so that resistance of the cell to the DNA bioreductive alkylating or cleavage agent can be detected.
  • the DNA sequence can encode a gene locus.
  • a DNA sequence can also include portions of a gene locus or a gene. Portions of a gene or gene locus, as used herein, refer to restriction enzyme fragments of the gene or gene locus.
  • a cell is resistant to a DNA bioreductive alkylating or cleaving agent if it can grow in the • presence of amounts of the agent that typically prevent growth of this type of cell. Growth preferably is about 2 to 1000-fold, and more preferably 2 to 100-fold increased over a sensitive cell. Those amounts can vary depending on the cell and the agent, but can be readily determined using standard dose response methods as described in Masuda et al., cited supra.
  • the amount of DNA bioreductive alkylating or cleavage agent is in the range of about 1-1000 ⁇ g/ml, more preferably about 10-500 ⁇ g/ml, and most preferably about
  • a cell is resistant to a DNA bioreductive alkylating or cleavage agent if the DNA from the cell does not develop interstrand DNA-DNA crosslinks or breaks in the DNA in the presence of the agent.
  • DNA-DNA crosslinks can be determined by standard methods, such as the alkaline elution method or DNA renaturation method as described by Masuda et al., cited supra.
  • Single strand DNA breaks in cells incubated in the presence of a DNA bioreductive alkylating or cleaving agent can be determined using a method such as the alkaline elution method described by Masuda et al. , cited supra.
  • resistant cells show a decrease in DNA-DNA crosslinks of about 2 to 20 fold compared to sensitive cells.
  • a DNA sequence that provides resistance to a DNA bioreductive alkylating or cleaving agent can encode a polypeptide that provides resistance to the cell or protects the DNA from the action of the agent. This polypeptide can act to prevent transport of the agent into the cell or nucleus or can act to inactivate the agent. While not in any way meant to be a limitation of the invention, a DNA sequence can also provide resistance to a cell by specifying a structural alteration of the agent, or modifying cellular target sites. Preferably the polypeptide inactivates the bioreductive DNA alkylating or cleaving agent by oxidation.
  • a DNA sequence that imparts resistance to a cell to a DNA bioreductive alkylating or cleaving agent can include one gene or more than one gene.
  • resistance is imparted by the mcrA and mcrB genes on the mcr locus providing for the expression of MCRA.
  • a DNA sequence that confers resistance to a cell to DNA bioreductive alkylating or cleavage agents can be isolated from the genome of the cell as follows .
  • the cell is a microorganism that produces a DNA bioreductive alkylating or cleaving agent.
  • a DNA library can be generated from the cell using standard methods as described in Sambrook et al. , cited supra. One such method includes digesting the total chromosomal DNA with the restriction enzyme and ligating the fragments into a vector, such as a plasmid. The vectors are then introduced into a host cell.
  • the host cell is preferably a cell that does not grow in the presence of the DNA bioreductive alkylating or cleaving agent (i.e., is sensitive) .
  • Transformed cells are then selected for the ability to grow in the presence of different amounts of the DNA bioreductive alkylating or cleaving agent.
  • An additional or alternative screening method that can be utilized optionally is to determine whether the transformed cells that can grow in the presence of the DNA bioreductive alkylating or cleaving agent develop DNA-DNA crosslinks or breaks as described herein.
  • Vectors from the transformed cells resistant to the agent are isolated and the DNA sequences conferring resistance are identified.
  • the ability of the DNA sequences to impart resistance to the cell is confirmed by subcloning of the sequence and transfer of the sequence into a sensitive host cell. Analysis of the sequence of DNA is conducted by standard methods .
  • the DNA sequence and/or the predicted amino acid sequence can then be compared to other known DNA sequences using a computer databank, such as the GenBank, and homologous sequences from other organisms identified.
  • Portions of a gene or gene locus can be obtained by digesting a DNA sequence encoding the gene or gene locus with one or more restriction enzymes such as shown in Figure 6 and Figure 12.
  • the restriction enzyme fragments of the DNA sequence are those fragments of the DNA sequence that can confer resistance to a DNA bioreductive alkylating or cleaving agent to a cell.
  • the fragments generated are ligated into a vector and the vector is introduced into a suitable sensitive host cell.
  • the transformed cells are selected for the growth in the presence of the DNA bioreductive alkylating or cleaving agent. Fragments of the gene or gene locus are identified by standard methods including restriction endonuclease mapping.
  • Methods of transformation and suitable host cells are known to those of skill in the art. Methods of transformation include calcium chloride or phosphate precipitation, electroporation, polybrene, liposomes, and protoplast fusion with polyethylene glycol and the like. Suitable host cells are those that exhibit sensitivity (i.e., do not grow in the presence of the DNA bioreductive alkylating or cleaving agent) . Specific examples of suitable host cells are sensitive Streptomyces species such as Streptomyces lividans, Streptomyces coelicoler. Streptomyces parvulus, and Streptomyces griseus.
  • DNA sequences that provide resistance to a DNA bioreductive alkylating or cleaving agent include DNA sequences that provide resistance to mitomycins such as the mcr gene locus and the mrd gene locus from S. lavendulae B619. These two gene loci provide for resistance of S. lividans to mitomycin C.
  • the mcr locus confers resistance to >100 ⁇ g/ml mitomycin C, and the mrd locus confers resistance to about 25 ⁇ g/ml.
  • the mcr locus includes three different coding sequences designated mcrA, mcrB, and mcrORF3 and has the DNA sequence as shown in Figure 2.
  • the mcr locus is contained on the 6.7 kb Bell fragment of pDHS3000.
  • the mcrA and mcrB sequences are found on the 2.2 kb BclII-Sphl fragment of pDHS3005.
  • the mrd locus is contained on the 4.2 kb Bell fragment of pDHS3001.
  • the mrd locus is contained on the 4.2 kb Bell fragment of pDHS3001 and has the sequence shown in Figure 11. Preferred examples of portions of the DNA sequence are shown in Figure 12.
  • Subclones of the mrd locus are generated as follows. A 3280 bp subclone of pDHS3001 is generated with Afllll/Ascl. A 3152 bp subclone of pDHS3001 was generated with Notl. These subclones confer resistance to a cell to 25ug/ml mitomycin C.
  • portions of DNA sequences include those shown in Figure 6. Portions of the mcr locus were generated by digesting a 6.7 kilobase Bell fragment encoding the mcr locus with Sphl, PuvII, BamHI, Bglll, PstI, Fspl, Ncol, Stul, and Clal. The fragments are subcloned into plasmids, as shown in Table I. The subclones are tested for the ability to confer resistance to mitomycin C, as described herein. The minimum DNA sequence of the mcr locus identified that provides resistance to mitomycin C corresponds to the DNA sequence of the 2.2 kilobase Bglll-Sphl subclone of the plasmid designated pDHS3005.
  • DNA sequences can also substantially correspond to DNA sequences encoding the mcr gene locus, mcrA, mcrB, and mcrORF3, respectively.
  • a DNA sequence that substantially corresponds is a DNA sequence that shares sufficient continuous DNA sequence identity to a DNA sequence encoding the mcr locus, mcrA gene, mcrB gene, or mcrORF3 gene or mrd locus so that the DNA sequence provides resistance to a cell to mitomycins, and can hybridize to a probe derived from mcr or mrd locus.
  • a DNA sequence that substantially corresponds preferably shares about 75-100% DNA sequence identity, and more preferably about 90-100% DNA sequence identity.
  • probes can readily be designed and synthesized using methods known two those of skill in the art.
  • the sequence preferably hybridizes to a probe derived from the mcr or mrd loci under conditions of high stringency.
  • a DNA sequence that substantially corresponds to the DNA sequence of mcrA is a DNA that includes a CAT codon for His 64 rather than CAC. Changes in the nucleotide sequence that do not result in a change in the amino acids encoded by the DNA sequence are sequences that are likely to also provide resistance to a cell to mitomycins.
  • DNA sequences that can encode polypeptides encoded by mcr and mrd loci such as MCRA. These DNA sequences can vary in the DNA sequence due to changes in a codon for a particular amino acid, but still encode a polypeptide with an amino acid sequence such as that of MCRA. These DNA sequences are also those that impart resistance to a cell to a DNA bioreductive alkylating and/or cleaving agent.
  • a DNA sequence that provides for resistance to a DNA bioreductive alkylating or cleaving agent to a cell functional in the cell in the expression cassette is operably linked to a promoter.
  • a promoter is an untranslated DNA sequence that provides for expression of the DNA sequence and is preferably located immediately upstream from the DNA sequence.
  • the promoter provides for a level of expression of the DNA sequence in an amount effective to render the cell resistant to the DNA bioreductive alkylating or cleaving agent.
  • the promoter can be a native promoter that is associated with and provides for expression of the DNA sequence in the source organism.
  • the promoter can be a constitutive or inducible promoter.
  • the promoter can also be a heterologous promoter obtained from a different gene and/or a different organism.
  • the promoter is functional in the host cell carrying the expression cassette.
  • the promoter can be derived from prokaryotic, viral, or eukaryotic sources.
  • prokaryotic promoters include the P Lac promoter, the P tac promoter, mel promoter on pIJ702, tipA of Streptomyces lividans and ermE of Saccharopolyspora erythrea.
  • viral promoters include the SV40 early promoter, the Herpes Simplex thymidine kinase promoter, the Rous sarcoma LTR promoter, the bacteriophage T7 promoter, the bacteriophage ⁇ P L promoter, and the like.
  • eukaryotic promoters include yeast promoters such as ADHI promoter, the TPI promoter, the GALI promoter, and the metalothionein promoter.
  • the preferred promoters for an expression cassette are the P tac promoter and the SV40 early promoter.
  • the especially preferred promoter is a promoter inducible by the DNA bioreductive alkylating or cleaving agent. Promoters are commercially available in vectors or can be obtained using known methods as described in Sambrook et al., cited supra., and Nielsen et al., Appl. Microbiol. Biotech.. 3_3:307 (1990).
  • the promoter sequence is combined with a DNA sequence that provides resistance to a DNA bioreductive alkylating or cleaving agent by standard methods to form an expression cassette.
  • One such method involves digesting a plasmid containing the DNA sequence and a plasmid containing the promoter with the same restriction enzymes, and then ligating the fragments into a third vector.
  • the third vector is then transformed into host cells and the host cells are selected for expression of the DNA sequence by selecting cells resistant to the DNA bioreductive alkylating or cleaving agent.
  • Other methods utilizing subcloning of the DNA sequence into established expression vectors can be conducted as described by Sambrook et al. , cited supra , for expression of cloned DNA sequences in mammalian or prokaryotic cells.
  • an expression cassette can also be subcloned into a vector so that efficient transmission into sensitive host cells can be achieved.
  • Vectors can include viral or plasmid vectors.
  • the vector is a known expression vector such as the SV40 vectors, the pSMG, the pSVT7, the pMT2, the p205, the pHeBo, the pBV-lMTHA, the pASl, the pET-3A, and the pKK177-3 as described by Sambrook et al., at pages 16.17 to 16.27.
  • the preferred expression vector is a high copy number plasmid from Streptomyces species such as the pIJ702. Many of the expression vectors are commercially available. pIJ702 can be obtained from the John Innes Institute, Norwich, England.
  • An expression cassette can also comprise a selectable marker gene.
  • Selectable marker genes are known to those of skill in the art and are present in the commercially available expression vectors. Specific examples of selectable marker genes include thymidine kinase, dihydrofolate reductase, aminoglycoside phosphotransferase, hygromycin B phosphotransferase, adenosine deaminase, arginine synthetase, and antibiotic resistance genes.
  • a preferred plasmid comprising an expression cassette in accordance with the invention is the plasmid pDHS3000 with a 6.7 kb Bell DNA sequence from S. lavendulae B619 containing the mcr locus.
  • This plasmid has been designated 1326/pDHS3000 and deposited in Streptomyces lividans with the American Type Culture Collection in Rockville, MD and given Accession No. 69448.
  • Another preferred plasmid is the pDHS3001 with a 4.2 kb Bell DNA sequence from S. lavendulae B619 that contains the mrd locus .
  • This plasmid has been designated 1326/pDHS3001 and deposited in Streptomyces lividans with the American Type Culture Collection in Rockville, MD and given Accession No. 69449.
  • an expression cassette Once an expression cassette is formed, it can be used to form transformed cells.
  • the transformed cells can provide polypeptides encoded by the DNA sequence and that provide resistance to DNA bioreductive alkylating or cleaving agents.
  • the transformed cells are also useful in methods for identifying agents that inhibit the resistance of cells to the DNA bioreductive alkylating or cleaving agents.
  • DNA probes can be useful in a method for identifying homologous DNA sequences that provide resistance to a DNA bioreductive alkylating or cleaving agent in other cells such as drug-resistant tumor cells.
  • a DNA probe in accordance with the invention comprises a DNA sequence that has sufficient DNA sequence complementarity to all or a portion of a known DNA or RNA sequence that provides for resistance to a DNA bioreductive alkylating or cleaving agent so that the probe can detect the DNA or RNA sequence by hybridization under low stringency conditions.
  • the probe preferably hybridizes to all or a portion of mcr or mrd locus under high stringency conditions.
  • the DNA sequence can be as small as about 17 nueleotides and can be single or double stranded.
  • the DNA probe has a size of about 17-2500 nueleotides and more preferably about 50-2200 nueleotides.
  • a preferred DNA probe includes a sequence complementary to nueleotides 310-330 of the mcrA sequence. This nucleotide sequence includes the codon for the histidine residue (His 64 ) believed to be important for binding of the FAD cofactor.
  • Other preferred probes include DNA sequences complementary to the DNA sequence found at nueleotides 142 to 166 for amino acids 171 to 177, and nueleotides 646 to 663 for amino acids 429 to 436 of the mcrA gene. As shown in Figure 3, these regions share amino acid homology with the 6-hydroxy-D-nicotine oxidase.
  • polypeptides that provide resistance to mitomycins and other DNA bioreductive alkylating or cleaving agents have a similar mechanism of action as 6-hydroxy-D- nicotine oxidase.
  • Region of the DNA sequence that encode amino acids that share homology with the DNA sequence for the 6-hydroxy-D-nicotine oxidase are selected to develop DNA probes.
  • Preferred DNA probes also include probes that are complementary to all or a portion of the DNA sequence for the mcr and mrd gene locus. These probes include sequences complementary to the DNA sequence of 6.7 kb Bell fragment of pDHS3000 encoding the mcr locus and the 4.2 kb Bell fragment of pDHS3001 encoding the mrd locus. These probes have been used to identify DNA sequences in the genome of resistant Streptomyces spp. by hybridization as shown in Figure 5. Portions of the DNA sequence can include restriction enzyme fragments, preferably those shown in Figure 6 or Figure 12.
  • DNA probes can be prepared by standard methods such as automated DNA synthesis or as restriction endonuclease fragments.
  • DNA probes are preferably labelled with a detectable agent such as a radioactively labelled nucleotide. Once a sequence is selected, a DNA probe labelled with the detectable agent can be prepared by automated DNA synthesis, poiymerase chain reaction, nick translation, and other methods as described in Sambrook et al. DNA probes are used to detect homologous sequences by hybridization.
  • a DNA probe according to the invention has sufficient complementarity to a DNA or RNA sequence so that it can hybridize to a DNA or RNA sequence under either low or high stringency conditions.
  • Sufficient complementarity depends on the length of the probe, whether any mismatches are present on the probe, and the stringency conditions and the effect of these factors on hybridization are known to those of skill in the art. Typically, in hybridizations conducted under lower stringency conditions, the probe can be smaller in length and have some mismatches in sequences. Methods of DNA-DNA and DNA-RNA hybridization as well as high and low conditions of stringency are known to those of skill in the art and are described in Sambrook et al.
  • Transformed cells carrying a expression cassette can be used in method to identify agents that inhibit resistance of the cell to the DNA bioreductive alkylating or cleaving agent.
  • Transformed cells include an expression cassette comprising a DNA sequence that provides resistance to a cell to a DNA bioreductive alkylating or cleaving agent operably linked to a promoter functional in the cell.
  • the preferred DNA sequence substantially corresponds to the DNA sequence for the mcrA and mcrB genes.
  • the expression cassette provides the cell with resistance to the DNA bioreductive alkylating or cleaving agent.
  • a transformed cell can be formed by standard methods using an expression cassette of the invention prepared as described herein. Briefly, a preferred expression cassette comprising a DNA sequence that substantially corresponds to a DNA sequence of mcrA and mcrB genes operably linked to a promoter is combined with a vector such as the plasmid pIJ702. A plasmid carrying the expression cassette is introduced into a suitable host by transformation methods such as calcium phosphate or calcium chloride precipitation, liposomes, electroporation, and the like. Transformants are selected for growth in the presence of the DNA bioreductive alkylating or cleaving agent. Transformed cells that are selected for growth in the presence of the agent are considered to be resistant to the agent.
  • Preferred host cells are those cells whose growth is inhibited in the presence of the DNA bioreductive alkylating or cleaving agent in the absence of the expression cassette (i.e., are sensitive).
  • sensitive cells include Streptomyces species such as Streptomyces lividans, tumor cells such as the L1210, the EMT6, HG-29, BE human carcinoma, and the L5178Y.
  • the preferred host cell is Streptomyces lividans.
  • Specific examples of a DNA bioreductive alkylating or cleaving agent include mitomycin A, mitomycin B, mitomycin C, profiromycin, mitiromycin, and FR900147.
  • the preferred DNA bioreductive alkylating or cleaving agents are mitomycin compounds.
  • the plasmid pDHS3005 is introduced into protoplasts of S. lividans by polyethylene glycol transformation.
  • Plasmid pDHS3005 comprises the 2.2 kb Bglll-Sphl DNA sequence including the mcrA and mcrB gdne sequences.
  • S. lividans transformants are selected for growth in the presence of about 10 to 100 ⁇ g/ml of mitomycin C.
  • Transformed cells that can grow in the presence of at least about 10 ⁇ g/ml of mitomycin C are considered resistant to mitomycin C.
  • a transformed cell can also be a transformed cell line.
  • a transformed cell line can be formed by amplifying the transformed cells selected for resistance to the agent. Methods of amplification and subculturing of cells to form homogeneous cell lines are known to those of skill in the art.
  • a preferred sensitive cell line is a tumor cell line sensitive to mitomycins such as the L1210 cell line.
  • a transformed cell line can exhibit transient expression of resistance of about 48 to 72 hours or can exhibit stable expression of resistance over several generations of growth (i.e., about 50-100 generations) in the presence of the DNA bioreductive alkylating or cleaving agent.
  • polypeptides that are Encoded by DNA Sequences that Provide Resistance to a Cell to a DNA Alkylating or Cleaving Agent and Antibodies Thereto The invention also provides polypeptides that are encoded by DNA sequences that provide resistance to a cell to a DNA bioreductive alkylating or cleaving agent. While not meant to be a limitation of the invention, polypeptides can provide resistance to the cell to a DNA bioreductive alkylating or cleaving agent by inhibiting transport of the agent, deactivating the agent, inhibiting binding of the agent to DNA, or inhibiting the crosslinking or cleaving of DNA by the agent.
  • a polypeptide encoded by the DNA sequence that provides resistance to a cell to the agent can be identified by its presence in cell lysates of transformed cells compared with its absence in non- transformed cells using standard methods.
  • a polypeptide can also be identified by determining whether the polypeptide can inhibit the binding to or activity of the DNA bioreductive alkylating or cleaving agent with a DNA sample by standard methods. Development of DNA-DNA crosslinks or breaks in the presence of the polypeptide and the agent can be assayed by the alkaline elution method or the DNA renaturation method as described by Masuda et al., cited supra.
  • polypeptides can be isolated from transformed cell lysates using standard methods.
  • Preferred polypeptides are encoded by the mcr locus can have a molecular weight within the range of about 10 to 60 kD as measured by SDS-PAGE.
  • MCRA polypeptide An especially preferred example of a polypeptide that is encoded by a DNA sequence that provides resistance to a DNA alkylating or cleaving agent is the MCRA polypeptide.
  • the MCRA polypeptide is about a 56,000 dalton molecular weight polypeptide as determined by SDS-PAGE that can be isolated from a transformed cell such as S. lividans carrying the plasmid pDHS3000.
  • a portion of the N-terminal sequence of the MCRA polypeptide isolated from transformed cell lysates is identical to that of the predicted amino acid sequence for the polypeptide encoded by the mcrA gene as shown below:
  • the predicted amino acid sequence of the MCRA polypeptide shares amino acid homology with the 6- hydroxy-D-nicotine oxidase and L-gulono-lactone oxidase. Both of these enzymes catalyze reactions using FAD cofactor-mediated oxidation.
  • a cofactor such as FAD binds to the His 64 residue of MCRA and mediates oxidative deactivation of a DNA bioreductive alkylating or cleaving agent such as mitomycin compounds.
  • the UV spectrum of purified MCRA and electrospray spectrometry indicates that FAD is covalently bound to MCRA as a cofactor.
  • MCRA and other polypeptides that are encoded by DNA sequences that impart resistance to DNA bioreductive alkylating or cleaving agents can be analyzed by inhibition of DNA or binding to alkylating or cleaving agents that are radiolabeled. This inhibition can be assessed by binding of the polypeptide to a radiolabeled agent or by inhibition of activation of the agent in the presence of a reducing agent.
  • the polypeptides preferably have oxidase activity and prevent reductive activation of the alkylating and/or cleaving agents.
  • the invention also includes antibodies to polypeptides encoded by DNA sequences that provide resistance to a cell to a DNA alkylating or cleaving agent. Once such polypeptides are identified, antibodies specific for the polypeptides can be prepared by standard methods known to those of skill in the art.
  • the antibodies can be used in assays to detect expression of polypeptides, to isolate and purify polypeptides and DNA sequences encoding them, and to identify and isolate related polypeptides in other resistant cells.
  • Polyclonal antibodies can be formed by injecting an animal such as a mouse several times intravenously with a polypeptide such as MCRA polypeptide. Typically about 0.5 to 2.0 mg are injected with Freund's incomplete adjuvant on at least three separate occasions. About one to two weeks after the last immunization, sera can be collected and the antibodies to a polypeptide such as MCRA can be quantitated using a standard ELISA test.
  • Monoclonal antibodies can be formed using the standard Kohler, Milstein technique. Pursuant to the Kohler, Milstein technique, immunization of the mammalian host is accomplished within the dose parameter by subcutaneous or intraperitoneal injection of the immunogen compound in adjuvant. Administration is repeated periodically and preferably for at least three injections. Three days before the spleen is removed, a priming injection of the immunogen compound is again administered. After their separation, the spleens are fused with the immortal mammalian cells such as mouse myeloma cells using the techniques outlined by Kohler and Milstein. Polyethylene glycol (PEG) or electrical stimulation will initiate the fusions. The fused cells are then cultured in cell wells according to culture techniques known in -the art. Cellular secretions in the culture medium are tested after an appropriate time for the presence of the desired monoclonal antibodies.
  • the immortal mammalian cells such as mouse myeloma cells using the techniques outlined by Kohler and Milstein. Polyethylene glycol (PEG
  • the selection technique for identifying the appropriate monoclonal antibodies is an important aspect for determining the immunospecificity of the monoclonal antibody.
  • the selection techniques call for determining the binding affinity of the hybridoma cellular products for polypeptides such as the MCRA and against cross- reactive controls.
  • hybridoma culture fluid is tested in screening assays for immunoreactivity with the polypeptide MCRA and lack of immunoreactivity with bovine serum albumin.
  • Screening assays can be performed by immunoenzymatic assay, immunofluorescence, fluorescence- activated cell sorter, radioimmunoassay, immuno- precipitative assay or inhibition of biological activity.
  • the hybridoma culture selected will exhibit strong binding characteristics to a polypeptide such as the MCRA polypeptide and exclude binding with a variety of controls including bovine serum albumin and a polypeptide encoded with the mcrB gene.
  • the invention also provides for methods of identifying agents that inhibit the resistance of cells to a DNA bioreductive alkylating or cleaving agent.
  • Applications of such methods include identification of drugs or agents that might be useful to combat development of tumor drug resistance and design of analogs of the DNA bioreductive alkylating or cleaving agents that resistant cells are sensitive to.
  • One method of identifying agents that inhibit resistance of cells to a DNA bioreductive alkylating or cleaving agent involves the use of a transformed cell.
  • a step of the method involves providing a transformed cell that is resistant to the DNA bioreductive alkylating or cleaving agent.
  • the transformed cell comprises an expression cassette having a DNA sequence that provides resistance to a cell to a DNA bioreductive alkylating or cleaving agent, preferably the DNA sequence that substantially corresponds to the DNA sequence for the mcrA and mcrB genes, operably linked to a promoter functional in the cell.
  • the transformed cell is incubated with an effective amount of an agent suspected to inhibit resistance of the cell to the DNA bioreductive alkylating or cleaving agent and an effective amount of the DNA bioreductive alkylating or cleaving agent. After suitable incubation, it can be determined whether the suspected agent inhibited the resistance of the cell to the DNA bioreductive alkylating or cleaving agent.
  • the transformed cell can be obtained as described herein.
  • a preferred transformed cell is S. lividans carrying the pDHS3005 plasmid encoding the mcrA and mcrB genes.
  • the DNA bioreductive alkylating or cleaving agent is preferably a mitomycin and the especially preferred agent is mitomycin C.
  • the agent suspected to inhibit resistance to mitomycin C can be agents that inhibit polypeptides such as the MCRA polypeptide or it can be analogs of the DNA alkylating or cleaving agents .
  • polypeptides such as the MCRA polypeptide
  • analogs of the DNA alkylating or cleaving agents Preferred examples of several analogs of mitomycins and methods for synthesizing such analogs are disclosed by Kasai et al., cited supra.
  • Effective amounts of the DNA bioreductive alkylating or cleavage agent or the agent suspected of inhibiting resistance to the DNA alkylating or cleaving agent are those amounts of the agent that would typically inhibit the growth of a sensitive cell.
  • Effective amounts of mitomycin are known to those of skill in the art and preferably are 1 to 1,000 ⁇ g/ml and more preferably about 25 to 100 ⁇ g/ml.
  • Effective amounts of an agent suspected to inhibit resistance are preferably within the same range and can be determined using standard dose response methodology.
  • Transformed cells are incubated in the presence of the DNA alkylating or cleaving agent and the agent suspected of inhibiting resistance of the cell to the DNA alkylating or cleaving agent.
  • the incubation period depends on the assay used to determine whether there has been a change in the resistance of the cell to the DNA alkylating or cleaving agent. If the change in resistance of the cell is measured by DNA-DNA crosslinking as described by Masuda et al., cited supra, incubation can be as short as one hour. If the change in resistance of the cell is determined by growth of the transformed cells, the incubation period is about 1 to 10 days.
  • An inability of the transformed cell to grow during the incubation period indicates that resistance of the cell to the DNA bioreductive alkylating or cleaving agent has been inhibited. Determining whether the suspected agent inhibits resistance of the cell to a DNA alkylating or cleaving agent can be accomplished by several methods. As described herein, if the transformed cell's resistance to the DNA alkylating or cleaving agent is inhibited, its growth is inhibited about 10 to 100-fold. If the transformed cell's resistance is inhibited, DNA- DNA alkylation or crosslinking is increased about 2 to 20-fold over that of resistant cells.
  • an agent suspected of inhibiting resistance of a cell to a DNA bioreductive alkylating or cleaving agent can be identified by determining if the suspected agent inhibits the function of a polypeptide encoded by a DNA sequence that provides resistance to the agent.
  • the steps of this method involve providing a substantially pure MCRA polypeptide and a sample of a DNA containing CpG residues.
  • the MCRA polypeptide and the sample of DNA are then incubated with an agent suspected to inhibit the function of the MCRA polypeptide and the DNA bioreductive alkylating and cleaving agent. After incubation, it is determined whether the suspected agent can inhibit the function of the MCRA polypeptide.
  • Substantially pure MCRA polypeptide can be obtained from a transformed cell line as described herein.
  • a substantially pure polypeptide is a polypeptide that does not contain other polypeptides as determined by SDS-PAGE. See Figure 10.
  • a sample of DNA containing CpG residues can be obtained commercially or synthesized by automated DNA synthesis or isolated from a microorganism such as S. lividans. The agent suspected of inhibiting the MCRA polypeptide can bind to and block the function of MCRA.
  • Such an agent could be an antibody to MCRA, an analog or derivative of mitomycin or a polypeptide designed to inhibit the binding or function of the FAD cofactor, e.g., a compound that covalently binds to or otherwise alters the His 64 residue of MCRA.
  • the function of the MCRA is predicted to include deactivation of the DNA alkylating or cleaving agent so that the DNA alkylating or cleaving agent can no longer bind to and/or cleave or alkylate DNA.
  • An inhibition of the function of the MCRA polypeptide can be determined by whether the DNA bioreductive or alkylating or cleaving agent can bind to DNA and/or catalyze DNA crosslinks or breaks. Binding of the DNA alkylating or cleaving agent to the DNA and crosslinking or cleaving of DNA can be determined by standard methods. If the suspected agent inhibits the MCRA polypeptide, binding of the DNA alkylating or cleaving agent to DNA will increase about 2 to 10 fold or the DNA-DNA crosslink formation will increase about 2 to 20 fold.
  • the invention also provides a method for identifying DNA sequences of other organisms that are homologous to the mcr and mrd gene loci that provide resistance to mitomycins.
  • Homologous sequences in other organisms or cells, especially tumor cells can be involved in the development of tumor drug resistance to DNA bioreductive alkylating or cleaving agents such as mitomycins.
  • a preferred method involves generating a DNA library from cells of the organism using standard methods, followed by amplifying DNA sequences of the library using poiymerase chain reaction.
  • the poiymerase chain reaction (PCR) includes the use of oligonucleotide primers that are complementary to portions of the mcr or mrd DNA sequences.
  • the amplified products are isolated and analyzed for homology to mcr and mrd DNA sequence by hybridization to a DNA probe complementary to all or a portion of the mcr or mrd DNA sequence.
  • a DNA library from the cells of the organism can be generated using standard methods.
  • the organism is preferably mammalian and the cells are preferably mitomycin C or multidrug-resistant tumor cells.
  • the DNA sequences in the DNA library are amplified by standard PCR techniques as described by Sambrook et al., cited supra.
  • Oligonucleotide primers are preferably about 20 to 30 nueleotides in length.
  • the sequence of the primers is selected to be complementary to the DNA coding sequence of the mcr gene locus or the mrd gene locus.
  • two different primers can be used in a single PCR reaction.
  • the preferred primers are complementary to DNA sequences of the mcrA gene that encode amino acids 171 to 177:
  • primers can be selected from other sequences by identifying regions of the DNA sequence of the mcr or mrd locus that encode amino acids that are shared by several related proteins such as 6-hydroxy-D-nicotine oxidase. Amino acids 171 to 177 and 429 to 436 of the MCRA polypeptide are very similar to the amino acids found in 6-hydroxy-D-nicotine oxidase. Primers can be prepared by standard methods such as automated DNA synthesis. Once primers are selected, they can be synthesized and combined with DNA sequences of the DNA library of the organism in a poiymerase chain reaction.
  • poiymerase chain reaction results in amplified products complementary to DNA sequences found in the source organism's genome.
  • amplified products are then isolated and it is determined whether the amplified products are substantially homologous to the mcr or mrd gene loci.
  • Homologous amplified products can be identified by hybridization under low stringency conditions to a DNA probe by standard methods. Hybridization conditions and DNA probes have been described herein.
  • An amplified product is substantially homologous if it hybridizes to a DNA probe complementary to a portion of the mcr or mrd gene locus under low stringency conditions and preferably shares about 75-100% DNA sequence identity and more preferably, shares about 90-100% DNA sequence identity.
  • the preferred probes are those that are complementary to all or a portion of the 2.2 kb Bglll- Sphl DNA sequence from pDHS3005 encoding the mcrA and mcrB genes and the 4.2 kb Bell fragment of the pDHS30001 encoding the mrd locus .
  • Hybridization can be detected under both low and high stringency conditions using standard methods. Identification of such homologous DNA sequences from other organisms or cells can be used to identify agents that can inhibit resistance to DNA bioreductive alkylating or cleaving agents as described herein. Sequences that hybridize to the probes can be analyzed by standard methods such as restriction endonuclease mapping and DNA sequencing.
  • Polypeptides that are homologous or related to MCRA can also be detected in other types of resistant cells using antibodies specific for MCRA.
  • antibodies specific for MCRA can be used to identify homologous or related polypeptides in mitomycin resistant tumor cell lines using ELISA assays.
  • the antibodies can also be used to isolate and purify related polypeptides using affinity chromatography.
  • the invention also provides methods for screening for novel DNA bioreductive alkylating or cleaving agents.
  • a method involves incubating cells that are resistant to a DNA bioreductive alkylating or cleaving agents and detecting induction of expression of a DNA sequence that imparts resistance to a DNA bioreductive alkylating or cleaving agent such as mitomycin C.
  • the cells can be naturally occurring resistant cells such a S. lavendulae or the cells can be transformed with an expression cassette including a DNA sequence that provides resistance to a cell to a DNA bioreductive alkylating or cleaving agent.
  • a cellular extract from a resistant Streptomyces spp is incubated with a transformed cell such S. lividans carrying plasmid pDHS3000 and the cells are grown in the presence of the cellular extract for 48 hours to stationary phase. After 48 hours induction of mcrA is monitered. Induction of mcrA can be detected by detecting the presence of mcrA using ELISA as discussed in Example 3. All extracts exhibiting the ability to induce expression of mcrA or mrd can be further fractionated to identify the compounds that act as inducers of expression of resistance. These compounds can include novel DNA bioreductive alkylating or cleaving agents which could be useful as anticancer agents.
  • EXAMPLE 1 Cloning of Mitomycin C Resistance Genes Genes encoding resistance to mitomycin C were identified and cloned from S. lavendulae DNA. Two gene loci were identified and designated mcr and mrd. The gene locus designated mcr encodes mcrA, mrd and 0RF3. The gene locus designated mrd was also identified and isolated.
  • the E. coli strain used was DH5 ⁇ F' .
  • the S.lividans strain used was 1326 (John Innes strain S.lividans 66).
  • Streptoverticillium spp. used in this study have been identified as Streptomyces lavendulae in accordance with the proposition that the genus Streptoverticillium be unified with the members of the genera Streptomyces, in the species lavendulae (Witt et al., System. Appl. Microbiol.. 13 . :361 (1990)).
  • S. lavendulae strains used in this study were B619, NRRL 2564, KY681, and PB1000. Strain B619 was a gift from Abbott Laboratories.
  • Strain PB1000 was derived from strain B619 as described below.
  • Strain NRRL 2564 was obtained from the American Type Culture Collection (ATCC 27422).
  • Strain KY681 was kindly provided by Kyowa Hakko Kogyo, Co., Ltd.
  • Strain PB1000 is a highly resistant MMC mutant. Mycelia of S. Lavendulae B619 was plated on MMC gradient plates. Mutants resistant to 250 ⁇ g/ml of mitomycin C were obtained. After several rounds of replating, selected mutants were identified with resistance to greater than 1000 ⁇ g/ml of MMC. One such mutant designated PB1000 was highly resistant to MMC and had a normal morphological phenotype.
  • pDHS3000 which contained a 6.7 kb insert (mcr) and conferred high level resistance (>100 ⁇ g/ml).
  • Clones possessing pDHS3001 contained a 4.2 kb insert (mrd) and conferred lower levels of MMC resistance (25 ⁇ g/ml) in liquid culture. See Table I.
  • pDHS3002 was constructed by the digestion of pDHS3000 with PvuII and Clal, Klenow treatment of the total digestion products, and ligation into the blunt- ended Bglll site of pIJ702.
  • pDHS3003 was constructed by the digestion of pDHS3000 with BamHI and Clal, Klenow treatment of the total digestion products, and ligation into the blunt-ended Bglll site of pIJ702.
  • pDHS3004 was constructed by the digestion of pDHS3003 with Bglll and Pstl, Klenow treatment of the total digestion products, gel purification of the 3.5 kb fragment, and ligation of the purified fragment into the blunt-ended Bglll site of pIJ702.
  • pDHS3005 was constructed by the digestion of pDHS3003 with Belli and Sphl, Klenow treatment of the total digestion products, gel purification of the 2.2 kb fragment, and ligation of the purified fragment into the blunt-ended Bglll site of pIJ702.
  • pDHS3006 was constructed by the digestion of pDHS3003 with Fspl and Stul - Klenow treatment of the total digestion products, gel purification of the 1.9 kb fragment, and ligation of the purified fragment into the blunt-ended Bglll site of pIJ702.
  • pDHS3007 was constructed by the digestion of pDHS3003 with Ncol and Sphl.
  • pDHS3008 was constructed by the digestion of pDHS3003 with Pstl and Stul, Klenow treatment of the total digestion products, gel purification of the 1.8 kb fragment, and ligation of the purified fragment into the blunt-ended Bglll site of pIJ702.
  • pDHS3009 was constructed by the digestion of PDHS3003 with Pstl and Ncol.
  • pDHS3010 was constructed by the digestion of pDHS3000 with Bell, Klenow treatment of the total digestion products, and ligation into the BamHI site pBR322. See Table I. TABLE I
  • Streptomyces spp. were grown in tryptic soy broth (TSB) for DNA isolation, or in yeast extract malt extract (YEME) broth supplemented with 5mM MgCl 2 and 5mM glycine for transformation. Streptomyces were grown on R2YE agar medium and E. coli was grown on LB agar medium.
  • TLB tryptic soy broth
  • YEME yeast extract malt extract
  • Growth media were supplemented with the appropriate antibiotics at the following concentrations: Agar plate; ampicillin, 50 ⁇ g/ml; mitomycin C, 20 ⁇ g/ml; thiostrepton, 20 ⁇ g/ml: Luria Broth; ampicillin, 50 ⁇ g/ml; mitomycin C., 5 ⁇ g/ml; thiostrepton, 5 ⁇ g/ml. Bacterial transformations.
  • Competent E. coli DH5 ⁇ F' were prepared and transformed according to Sambrook et al., Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY (1985).
  • the 3.2 kb BamHI/Clal fragment from pDHS3003 was cloned into pUC119.
  • the internal 2.5 kb Apal fragment was purified using the GeneClean II kit (BIO 101, La Jolla, CA) according to the manufacturer's instructions. Different allotments of this fragment were digested with Sail, BstEII, and Rsal. In addition, the fragment was double digested with Xhol and Ncol. The digestion products were blunt-ended using Klenow as described by Sambrook et al., cited supra. These products were cloned into the Smal site of pUC119.
  • Single-stranded DNA was isolated from clones containing inserts using the helper phage MK037.
  • Single-stranded DNA sequencing was performed using the dideoxy chain termination method. (Sequenase kit, version 2.0; deazaguanosine dinucleotides, United States Biochemical; and [ ⁇ - 35 S] dATP.)
  • DNA sequence analysis was performed using the Intelligenetics, and the Wisconsin Genetics Computer Group software programs version 7.0 (Madison, WI).
  • a 5.5 kb Bell fragment from plasmid pDHS3001 was isolated and subjected to shotgun cloning into pUC119. Briefly, the Bell generated fragments were ligated using T4 DNA ligase. The ligated product was disrupted using sonication to generate randomly sized fragments. Selected fragments ranging in size from 500 to 1000 bp were isolated via agarose gel electrophoresis. Fragments were cloned into Smal site in pUC119. Single stranded DNA was produced and sequenced via the dideoxy chain termination method of Sanger et al. Analysis of sequencing gel is being conducted with the aid of
  • the sequence for the mrd (formerly mcrB) locus of S. lavendulae is shown in Figure 11.
  • the sequence has two complete and two partial open reading frames (ORF) as follows: from the 5' end an incomplete reading frame at 1-320 nueleotides; an open reading from 1055-1519 nueleotides; an open reading frame from 1871-
  • the translation product of the ORF at the 3' end of the clone aligns with mycinamicin IV hydroxylase from Micromonospora griseorubida.
  • a polypeptide having the predicted amino acid sequence encoded by this ORF shows a strong similarity to mycinamicin hydroxylase.
  • a _____ lavendulae DNA library was constructed by isolating total chromosomal DNA, digesting completely with BcJ.1, and ligating into the Bglll site in the high copy Streptomyces vector pIJ702 as described by Hopwood et al., cited supra.
  • Protoplasts of S. lividans (______ lividans determined to be suitable hosts because of their high sensitivity to MMC in liquid culture, and on agar medium (total inhibition of growth was observed at 10 ⁇ g/ml)) were generated and transformed using standard methodology.
  • S. lividans 1326 The sensitivity of S. lividans 1326 was tested for its resistance to MMC by plating spores onto plates containing 1, 10, and 25 ⁇ g/ml MMC. Some growth was observed at 10 ⁇ g/ml MMC, however, growth was not observed at 25 ⁇ g/ml MMC. B619 genomic DNA was digested with Bell to completion and ligated into the Bglll site pIJ702. S. lividans protoplasts were transformed with the ligation mixture and plated onto R2YE agar. After 24 hours, regenerated protoplasts were overlaid with 25 ⁇ g/ml thiostrepton. Approximately 8000 mitomycin C resistant colonies appeared several days later.
  • Plasmid preparations of MMC resistant colonies revealed two distinct DNA fragments.
  • the clones that grew at a normal rate possessed pDHS3000, which contained a 6.7 kb fragment (mcr) and conferred high levels of resistance (>100 ⁇ g/ml) in liquid medium.
  • the clones that grew at a reduced rate possessed pDHS3001, which contained a 4.2 kb DNA insert (mrd) and conferred lower levels of MMC resistance (25 ⁇ g/ml) in liquid culture (Table I).
  • ORFs designated mcrA. mcrB, and ORF3.
  • the predicted direction of transcription for mcrA and mcrB is left to right, while ORF3 would be transcribed divergently.
  • the predicted start site for mcrA is an ATG codon at nucleotide position 131.
  • the predicted stop codon for mcrA is located at nucleotide position 1475 and a non- coding region of 49 bp separates it from mcrB.
  • the putative translational start site for mcrB is a GTG codon at nucleotide position 1527, preceded by a ribosome-binding site centered ⁇ 8 nueleotides upstream.
  • a characteristic stem loop structure suggests the presence of a rho-dependent terminator at the 3 '-end of mcrB.
  • the 3' end of 0RF3 is separated by a 112 bp non-coding region from the 3' end of mcrB.
  • the predicted start site of 0RF3 is an ATG codon at nucleotide position 2862.
  • the copy number of mcr showed variability in the MMC- producing strains of S. lavendulae. and corresponded to the level of MMC resistance exhibited by the specific MMC-producing strains. This is particularly evident in S. lavendulae strain PB1000, which expressed the highest level of resistance to MMC and has the highest copy number of mcr.
  • PB1000 was generated specifically as a highly resistant variant, which was isolated after a strain development protocol using high level exposure to MMC.
  • a series of subclones from mrd locis have been prepared. Briefly, the 4.2 kb Bell fragment from pDHS3001 has been subcloned into plJ702 (plasmid) as shown in Figure 12. The subclones have been introduced into S. lividans and resistance to MMC evaluated as described previously. A 3280 bp Afllll/Ascl fragment did confer resistance as well as a 3152 bp Not I fragment. A 1696 bp Pvul/Ascl fragment and 1032 bp Not 1/ Pvu I fragment are being evaluated. Resistance was conferred at 25 ⁇ g/ml.
  • RNA isolation and transcriptional start point determination were performed.
  • RNA was extracted from S. lividans 1326 containing pDHS3000 grown in YEME medium with the addition of glycine to 5mM and mitomycin C or thiostrepton to a concentration of 5 ⁇ g/ml. The cultures were allowed to grow for 72 hours. Additional drug was added to the mitomycin C induced culture at 5 ⁇ g/ml 35 minutes before RNA extraction.
  • RNA was isolated using the Kirby protocol (from Hopwood et al., cited supra. ) .
  • Primer extension was performed using the primer extension system of Promega Biotech (Madison, WI) according to the instructions of the manufacturer.
  • the oligonucleotide used for the extension reaction was 5 ' - CCACCTCCTGCTCGTCGGCC-3 ' , synthesized by Keystone Laboratories, Inc. (Menlo Park, CA) .
  • the primer extension protocol resulted in two primer extension products designated Pl and P2.
  • the primer extension products were sequenced using dideoxy chain termination method.
  • the results established the presence of two transcriptional start points, Pl and P2 with an expression ratio of ⁇ 5:1.
  • the transcriptional start point for Pl is also the first nucleotide (bp 131) of the translational start codon.
  • the mcrA transcript represents a leaderless mRNA, a phenomenon that has been described for several other Streptomyces resistance genes.
  • the second, weaker primer extension product (P2) was observed that represents an mRNA with a transcriptional start point at nucleotide 170. This transcriptional start point may ensure basal levels of mcrA and mcrB mRNA.
  • Hybridization of mcr to a 1.8 kb band in a Northern blot from S. lavendulae total RNA provides compelling evidence for a polycistronic mRNA including mcrA and mcrB.
  • mcrA Gene expression of mcrA is induced by low levels of MMC. Completion of the DNA sequence for mcr suggested strongly that the mcrA gene product represented the protein induced after addition of the drug. It was determined whether other compounds in the mitomycin class were capable of inducing expression of the mcrA-mcrB operon.
  • MCRA 1.0-0.5 ug/ml antigen
  • the plates were incubated for 1 hour at 37°C, the contents were decanted and the plates were shaken dry.
  • the wells were washed four times with PBS- T20 and were shaken dry.
  • the plates were incubated at 37°C for 1 hour.
  • the wells were washed four times with PBS-T20 and were shaken dry.
  • One hundred microliters of secondary antibody (goat anti-rabbit HRP), at a dilution of 1:3000 in PBS-T20, was added to the wells and incubated at 37°C for 1 hr or RT for 2 hrs.
  • the contents were decanted and the plates were shaken dry.
  • the wells were washed four times with PBS-T20 and were shaken dry.
  • One hundred microliters of OPD solution was added to each well. The plates were
  • MCRA MMC concentration dependent and maximum expression of MCRA occurred at concentrations of MMC greater than 10 ug per ml ( Figure 13). Concentrations of MMC above this level result in a decreased growth rate of the MMC producer S. lavendulae when it is grown in MMC, which indicates that the mechanism of resistance conferred by MCRA can be physiologically saturated (data not shown) . Significantly, MCRA is induced by concentrations of MMC as low as 0.01 ug per ml (30 nM) . The induction of MCRA may be induced at a much lower concentration physiologically in S. lavendulae.
  • NCS neocarzinostatin
  • mcr locus The ability of mcr locus to confer resistance to other mitomycins was examined. To determine whether resistance to other mitomycins and mitosanes is conferred by MCRA. we examined the ability of S. 1ividans/pDHS3000 to grow in the presence of MMC related molecules. As shown in Table II, resistance against all the compounds tested was conferred by pDHS3000. However, mitomycin D and H were not lethal to the control culture, S. lividans pIJ702. The non-lethality of mitomycins D and H does not reflect an inability of mitomycin D and H to penetrate cell barriers since these compounds were able to induce MCRA expression.
  • MCRA a novel resistance protein
  • the protein was purified to confirm its identity, and to perform a series of in vitro experiments concerning its precise biological function.
  • a purification scheme has been developed for MCRA.
  • S. lividans containing pDHS3000 was grown to stationary phase in the presence of 5 ⁇ g/ml MMC.
  • the mycelia was harvested after 54 hours of growth, centrifuged at 5,000 rpm for 10 minutes. The supernatant was decanted and the mycelial pellet resuspended in two volumes of protein extraction buffer (50 mM Tris -HC1 pH 8.0, 10% glycerol, 2 mM EDTA pH 8.0).
  • One half of the total mycelia was frozen at -80°C while the other half was processed to isolate MCRA. All of the subsequent steps were performed at 4°C.
  • the mycelia were disrupted by passing twice through a French press at 1500 psi. The homogenate was centrifuged at 10,000 rp for 1 hour.
  • the homogenate supernatant was removed and proteins precipitated from it by the gradual addition of ammonium sulfate until 100% saturation.
  • the saturated ammonium sulfate solution was allowed to stir overnight and then centrifuged at 10,000 rpm for 30 minutes. The supernatant was discarded and the pellet was resuspended in protein extraction buffer precooled to 4°C.
  • the solution was dialyzed against 4 liters of protein extraction buffer five times over two days.
  • the dialysis tubing was placed in a tray containing 200 g of PEG 6000 for 6 hours. Dialysis was performed against 4 liters of protein extraction buffer for 4 hours.
  • the protein solution was removed from the dialysis tubing and centrifuged at 5,000 rpm for 10 minutes.
  • the supernatant was removed and ammonium sulfate added to a concentration of 50%.
  • the solution was allowed to stir for one hour and then centrifuged at 7,000 rpm for 10 minutes.
  • the supernatant was removed and ammonium sulfate was added to bring the concentration up to 70%.
  • the solution was allowed to stir for one hour and then centrifuged at 7,000 rpm for 10 minutes.
  • the supernatant was removed and the pellet was resuspended in protein extraction buffer followed by dialysis against 4 L of buffer twice for 12 hours.
  • the dialysis tubing was placed in a tray containing 100 g of PEG 6000 for 6 hours.
  • the dialysis tubing was washed with distilled water and dialyzed overnight against 2 L of 50 mM phosphate buffer, pH 7.0. The dialysate was centrifuged at 5,000 rpm for 5 minutes. The supernatant was removed and sterile filtered.
  • This protein solution was loaded onto a DEAE column equilibrated with 50 mM phosphate buffer pH 7.0 using an Econosystem (BIORAD) . A distinct yellow band could be seen at the head of the column.
  • the column was washed with 1 L of the same buffer.
  • the column was eluted with 1 L of 0 to 0.3 mM KC1 gradient; 4 ml fractions were collected. Fractions were run on SDS- PAGE evaluated for a protein with the ability to co- migrate with the MC inducible protein from S. lividans 1326/pDHS3000 MMC induced cell extracts.
  • Fractions containing the MCRA protein were concentrated using Centriprep (Amicon, Beverly Mass.) with a MW cut off of 30,000 according to instructions from the manufacturer. The concentrated protein solution was a bright yellow color. Gel filtration (Sephadex HR200, Pharmacia) was performed to further purify MCRA. The column (size) was washed with 50 mM phosphate buffer, pH 7.0 and loaded with 2 mis of the protein solution. The column was run at a flow rate of 0.2 mls/min. and 4 ml fractions collected. Fractions were analyzed by SDS-PAGE and revealed that the protein co-migrating with the MC inducible protein was substantially pure.
  • N-terminal sequence analysis of the MCRA protein was conducted by standard methods using an Applied Biosystems, Inc. 476 Sequencer (pulse liquid mode) . Data analysis was done with the AB1 610 Sequence Analysis Software. The analysis shown below shows that the sequence of the isolated MCRA polypeptide agrees with the predicted amino acid sequence from the nucleotide sequence:
  • Monoclonal antibodies can be prepared by standard methods as described below.
  • Anti-MCRA polyclonal antibodies were produced as follows. 500 ug of purified MCRA was mixed in a ratio of 1:2 with complete Freunds adjuvant to homogeneity. The mixture formed an emulsion when passed back and forth through two connected syringes. Two New Zealand white rabbits were each inoculated with a suspension containing 500 ug of MCRA. After two weeks they were inoculated again and one month later they were reinoculated. After seven days, blood was removed from the rabbits and the serum was titered for antibodies against MCRA by ELISA assay. The polyclonal antibody titer was 1:8000. The ELISA assay was conducted as described in Example 3.
  • Monoclonal antibodies can be formed using the standard Kohler, Milstein technique. Pursuant to the Kohler, Milstein technique, immunization of the mammalian host is accomplished within this dose parameter by subcutaneous or intraperitoneal injection of the immunogen compound in adjuvant. Administration is repeated periodically and preferably for at least four injections. Three days before the spleen is removed, a priming injection of immunogen compound is again administered. After their separation, the spleen cells are fused with immortal mammal cells such as mouse myeloma cells using the techniques outlined by Kohler and Milstein. Polyethylene glycol (PEG) or electrical stimulation will initiate the fusions. The fused cells are then cultured in cell wells according to culture techniques known in the art. Cellular secretions in the culture medium are tested after an appropriate time for the presence of the desired cellular products.
  • PEG polyethylene glycol
  • the selection technique for identifying the appropriate monoclonal antibody is an important aspect for determining the immunospecificity of the monoclonal antibody.
  • the selection techniques call for determining the binding affinity of the hybridoma cellular products for the mitomycin C resistance polypeptide MCRA and against cross-reactive controls.
  • hybridoma culture fluid is tested in screening assays for reactivity with mitomycin C resistance polypeptide MCRA and lack of immunoreactivity with bovine serum albumin.
  • Screening assays can be performed by immunoenzymatic-assay, immunofluorescence, fluorescence- activated cell sorter, radioimmunoassay, immuno- precipitative assay or inhibition of biological activity.
  • the hybridoma cultures selected will exhibit strong binding characteristics to the MCRA polypeptide and exclude binding with a variety of controls, including BSA and mcrB.
  • subcloning to refine the selected culture can be performed. These techniques are known to those skilled in the art. See, for example, Goding, James Goding, Monoclonal Antibodies: Principles and Practice. 2nd Edition. Academic Press, San Diego, CA (1986), the disclosure of which is incorporated herein by reference. Briefly, the appropriately selected cell culture is separated into one-cell units which are then recultured. The subclone cultures are then again tested for specific immunoreactivity, lack of cross-reactivity, and the amount of monoclonal antibody secreted. Those subcultures exhibiting the highest amounts of secreted monoclonal antibody are chosen for subsequent pilot development.
  • the antibodies specific for MCRA can be used in a variety of assays including ELISA assays and Western blot assays.
  • the antibodies are useful for analyzing induction of MCRA in cells and identifying other related mitomycin resistance proteins and to analyze the functional domains of MCRA.
  • Western blots of cells expressing MCRA were conducted as follows. Completely dry filter with protein. Dip the filter for 2 sec into 100% Methanol and then soak in IX TBS (Tris buffered saline) (20 mM Tris -HC1 pH 7.5, 150 mM NaCl) for 2 minutes. Gently shake at room temperature in blocking solution (5% non ⁇ fat dry milk in TBS). Wash 3 x 10 minutes wash solution (0.1% non-fat dry milk in TBS). Add primary antibody to antibody incubation solution (1% non-fat dry milk, 0.05% Tween-20 in TBS) at 1:8000 and incubate for 2 hours at room temperature on orbital shaker. Wash 3 x 10 minutes wash solution.
  • IX TBS Tris buffered saline
  • TBS + .05% Tween-20 (1:2000 goat anti-rabbit HRP Zymed) . Incubate 2 hours on gentle shaker. Wash 3 x 10 minutes wash solution. Incubate with color development solution 25 mM Tris -HC1 pH 7.5, 4-Chloro-l-napthol (3 mg/ml MeOH) 30% H202). Rinse with distilled H 2 0 and blot with Whatman paper to dry and store.
  • DNA adjacent to one or both MMC resistance genes includes a cluster of biosynthetic genes for the metabolite
  • S. lavendulae genomic library using the Streptomyces- E. coli shuttle vector, pNJl (Tuan et. al. , 1990).
  • High molecular weight genomic DNA was subjected to partial digestion to generate fragments about 30-40 kb in length.
  • the library was subsequently screened with 32 P- labeled mcr or mrd.
  • a single cosmid clone (encompassing ⁇ 25 kb) was identified using mcr as a probe, and its map is shown in Fig. 7. For mrd.
  • clones Selection for thiostrepton resistance allowed convenient identification of several clones that are currently under analysis (Fig. 9). Initially these clones will be screened for the presence of tsr in the chromosome and integration of the construct used for gene disruption. Fermentation and analysis of MMC production will then be performed on clones of S. lavendulae in which the mcrORF3 gene is inactivated.
  • Another strategy for obtaining S. lavendulae mutants blocked in MMC production includes transposon mutagenesis method for Streptomyces spp.
  • This approach involves use of a hypertransposing element Tn5099-10 and a temperature sensitive delivery system from pGM160. Briefly, the vector containing Tn5099-10 will be transformed into the MMC-producing S. lavendulae wild- type strain. Incubation at elevated temperatures induces transposition randomly into the chromosome. Transposon mutants blocked in MMC production will be identified using a biological assay as described previously. Loss of MMC production will be confirmed by analysis of extracts by HPLC.
  • a method for identifying agents that inhibit resistance of a cell to a DNA alkylating or cleaving agent can be conducted as described below.
  • Transformed cultures of S. lividans carrying plasmid pDHS3005 can be cultured in the presence of 0,
  • the transformed cells can be incubated for about 7 days.
  • Cells that are resistant to mitomycin C will grow in the presence of at least about 10 ⁇ g/ml of mitomycin C.
  • a sensitive cell will preferably not show any growth at 25 ⁇ g/ml of mitomycin C. It is likely that an inhibitory agent will prevent or inhibit growth of the transformed S. lividans in the presence of concentrations of mitomycin of 10 ⁇ g/ml or greater, preferably growth is inhibited at least about 10-fold.
  • EXAMPLE 8 Determination of Mechanism of Action of MCRA An assay for catalytic activity and to ascertain the mechanism of resistance toward MMC was developed. A UV spectrophotometric assay has been developed using the absorbance of MMC at 363 nm to determine whether reduction of MMC is prevented by MCRA. In this assay, freshly prepared extracts of S.
  • MCRA MCRA in S. lavendulae was detected using an ELISA assay on cell lysates taken at various time points during growth of the cells in culture.
  • Samples were centrifuged at 5K for 10 minutes. For dry weight determinations the pellets were washed 4 times with 5 volumes of distilled water to remove salts and calcium carbonate. Mycelia for protein isolation was washed 4 times with phosphate buffered saline (PBS). Samples for dry weights were resuspended in 1 volume of distilled water and poured into pre- weighed aluminum cupcake tins which were dried at 80°C for 24 hrs after which they were re-weighed and the dry mass determined. Fermentation broth was sterile filtered at room temperature and the presence of MMC was assayed by HPLC immediately. Samples for protein isolation were resuspended in one volume of PBS.
  • PBS phosphate buffered saline
  • the mycelia were incubated on ice for 15 minutes and then sonicated as described.
  • the cell extract was centrifuged at 4°C at 10K for 30 minutes. The supernatant was removed and sterile filtered through a 0.2 uM filter. These extracts were frozen at -80°C.
  • the cell extracts were analyzed for the presence of MCRA.
  • S. lavendulae has the ability to produce MMC at high concentrations when it is grown in the proper fermentation medium. Given this ability it is expected that it would be resistant to MMC at high levels. Additionally, if MCRA confers MMC resistance then it should be detected in cells producing MMC. As shown in Figure 17, S. lavendulae was grown to stationary phase and produced MMC as determined by HPLC analysis. In addition, ELISA assays revealed that MCRA was expressed. MCRA was expressed at a low level prior to MMC production, and was strongly expressed just prior to MMC biosynthesis. Most likely the induction is due to the presence of MMC produced which is below the limit of our ability to detect MMC. Thus, MCRA is produced in the naturally resistant host cell that produces MMC and is indueibly expressed by MMC as has been observed in S_ ⁇ . Lividans pDHS3000.
  • Polyclonal and monoclonal antibodies can be used to identify and/or isolate polypeptides related to MCRA present in MMC resistant tumor cells.
  • MMC resistant tumor cell lines have been described by Pan et al. Dr. SuShu PAN, University of Columbia.
  • Tumor cells can be exposed to varying concentrations of MMC and then cell extracts containing cytoplasmic proteins can be obtained.
  • the cell extracts can be screened for polypeptides reactive with polyclonal and/or monoclonal antibodies specific for MCRA in an ELISA as described previously for S. lavendulae cell extracts in Example 3.
  • Cell lines exhibiting positive reactivity with antibodies to MCRA can be further analyzed using affinity chromatography and/or Western blots using standard methods. It is expected that the antibodies to MCRA would identify mammalian polypeptides related to MCRA. These antibodies can be used to isolate these polypeptides with affinity chromatography.
  • MOLECULE TYPE DNA (genomic)
  • ORGANISM Streptomyces lavendulae
  • GATCTTCCTC GTTTTGGGGA GGTGCTGACG AGCCGGCCTT CGCGCCGGGC TTCCCCGCGG 60
  • Trp lie Glu Ala Gly Ala Arg Trp Arg Lys Val Leu Glu His Thr Ala 95 100 105 CCG CAC GGG CTC GCG CCG CTG AAC GGC TCG AGC CCC AAC GTG GGC GCT 505 Pro His Gly Leu Ala Pro Leu Asn Gly Ser Ser Pro Asn Val Gly Ala 110 115 120 125
  • MOLECULE TYPE DNA (genomic)
  • ORGANISM Streptomyces lavendulae
  • MOLECULE TYPE DNA (genomic)
  • ORGANISM Streptomyces lavendulae
  • ORGANISM Streptomyces lavendulae
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • ORGANISM Streptomyces lavendulae

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Abstract

L'invention se rapporte à une cassette d'expression comprenant une séquence d'ADN qui confère à une cellule une résistance par rapport à un agent bioréducteur de clivage ou d'alkylation d'ADN. Les agents bioréducteurs de clivage ou d'alkylation d'ADN sont notamment les mitomycines et les énediynes. La cassette d'expression permet d'obtenir des cellules transformées résistantes, des polypeptides de résistance, ainsi que des amorces et des sondes d'ADN. L'invention se rapporte également à des procédés permettant d'identifier des agents qui inhibent la résistance cellulaire à des agents tels que les mitomycines. Des sondes d'ADN et des amorces d'amplification par polymérase peuvent être utilisées selon un procédé permettant d'identifier des séquences d'ADN homologues dans d'autres organismes et/ou types de cellules.
PCT/US1994/011279 1993-10-07 1994-10-06 Genes codant la resistance aux agents d'alkylation d'adn WO1995009926A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000053737A2 (fr) * 1999-03-12 2000-09-14 Regents Of The University Of Minnesota Groupe de genes biosynthetiques destines a la mitomycine

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* Cited by examiner, † Cited by third party
Title
ABSTRACTS OF THE GENERAL MEETING OF THE MICROBIOLOGY SOCIETY, issued 1992, AUGUST et al., "Cloning and Expression of the Streptomyces Lavendulae Mitomycin C Resistance Genes in Streptomyces Lividans", page 31, Abstract 0-12. *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6495348B1 (en) 1993-10-07 2002-12-17 Regents Of The University Of Minnesota Mitomycin biosynthetic gene cluster
WO2000053737A2 (fr) * 1999-03-12 2000-09-14 Regents Of The University Of Minnesota Groupe de genes biosynthetiques destines a la mitomycine
WO2000053737A3 (fr) * 1999-03-12 2000-12-21 Univ Minnesota Groupe de genes biosynthetiques destines a la mitomycine

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