WO1996024688A1

WO1996024688A1 - Diagnostics based on dna duplex repair

Info

Publication number: WO1996024688A1
Application number: PCT/US1996/000638
Authority: WO
Inventors: Christopher P. Adams; Lawrence Grossman
Original assignee: Mosaic Technologies Inc
Current assignee: Mosaic Technologies Inc
Priority date: 1995-02-07
Filing date: 1996-01-16
Publication date: 1996-08-15
Anticipated expiration: 1997-08-07
Also published as: AU4701396A

Abstract

Assay protocols are provided for determining the interrelationship between lesion formation and cellular repair mechanisms of such lesions. The assay provides for synthesis of duplex oligomers, which may then be subjected to agents, which result in lesion formation. The modified DNA oligomers are desirably bound to a solid support and contacted with cell lysates and the ability of the cell lysate to correct lesion determined. The assay may be employed in various diagnostic contexts, such as evaluating response of neoplastic cells to therapeutic treatments, the effect of environments on causing lesions, and the ability of disease cells to repair various lesions, particularly those associated with the disease.

Description

DIAGNOSTICS BASED ON DNA DUPLEX REPAIR

INTRODUCTION Technical Field

The field of this invention is diagnostics related to repair of DNA lesions. Back^ground DNA is a stable composition, since it is required to maintain its integrity over extended periods of time; As the transmitter of genetic information, which defines species, the information must be transmitted reliably from parent to progeny. In addition, during the lifetime of the host, changes in the genetic information can adversely affect the performance capability or viability of the host or even be lethal. In mammals, mutations can frequently lead to devastating diseases, such as cancer, lupus, muscular dystrophy, and cystic fibrosis.

For hyperproliferative diseases, treatments are employed, such as high energy irradiation or chemotherapeutic drugs, which are intended to introduce lesions into the

DNA, so as to reduce the viability of the diseased cells. In the complex world in which we live, there are many compounds which can act as mutagens. causing lesions in

DNA. Exposure to ultraviolet light can also induce lesions in the DNA.

Because hosts have been exposed to an environment which induces genetic modification, the hosts have developed repair mechanisms. These mechanisms are predicated on recognizing the lesion, excising the lesion, and replacing the lesion, if possible, with the original nucleosides. In many individuals, these mechanisms may be compromised, or the system may be more efficient as to one lesion, as compared to another. In the case of cancer, where one is using chemotherapy or irradiation, the ability for the cancer cells to repair the lesions introduced by the treatment, can seriously compromise a particular methodology. It is therefore of substantial interest in a wide variety of situations to relat materials causing lesions and the ability of a host to repair the lesion. This interest ca be applied to mutagens in the environment, cancer treatments, or other situation wher one is interested in the interrelationship between materials causing lesions and th ability of the cells to repair such lesions. Relevant I-iteratιιre

Simmons, British Medical Journal 306,29(1993); Harris, Science 262,53 (1993); Boerrigter, J. of Gerontology 48,B11-16 (1993); Tanhes, Science 262,29 (1993).

SUMMARY OF THE INVENTION Duplex DNA which has been exposed to one or more lesion induction agents is combined with a cellular lysate. After sufficient time for the cellular repair system to have acted on lesions present in the DNA, the DNA is assayed for any repairs which have occurred. The method finds application in evaluating therapeutic treatments based on the inducing of lesions in cells, mutagenic agents and the ability to repair lesions resulting from the mutagenic agent, and analysis of agents in enhancing or inhibiting the cellular repair system.

DESCRIPTION OF SPECIFIC EMBODIMENTS The subject invention is concerned with those situations where one is interested in the relationship between lesion formation in duplex DNA and its repair by a cellular repair system. This interest exists in a wide variety of contexts. In the therapeutic context, there is therapy, either irradiation or chemotherapy, associated with hyperproliferative diseases, such as cancer. In this situation, one is interested in the ability of the neoplastic cells to repair lesions which are induced by the therapeutic regimen. In exposure situations, such as exposure to radiation, e.g. ultraviolet, as from the sun, or high energy radiation, when working with radioactive materials or in nuclear power plants, exposure to mutagens, which may be in the water, soil, food, or the like, there is an interest in determining the ability for the environmental agent to induce lesions, as well as the ability of the host, particularly humans, exposed to the agent, to repair any induced lesions. There is also an interest in being able to develop drugs which may enhance or inhibit the cellular repair mechanism, depending upon the context in which the drug is to be used. Other situations may also employ the subject invention.

The invention employs duplex DNA which has been subjected to an agent which induces lesions. Depending upon the source of the DNA, the lesion may be as a result of environmental exposure, therapy, or the like. To evaluate a host's ability to repair lesions, one may synthesize an oligomeric duplex, of at least 30 bp, usually about 50 bp, and not more than about 500 bp, usually not more than about 250 bp. The synthetic duplex is then exposed to an agent of interest, as in the therapeutic treatment of cancer, or a sample composition, such as wastewater, soil, by employing a UV mimetic agent, or the like, to determine the mutagenic potential for components of the sample, as well as the ability for a repair system to remove the lesions.

Depending upon the particular agent which is employed for introducing a lesion, a wide variety of lesions may result. For example, by employing a mimetic nucleoside, such as 5-fluorodeoxyuridine, the incorporation of the 5-fluorodeoxyuridine results in replication errors in the complementary strand. Therefore, by preparing strands and replicating them using DNA polymerase, where 5-fluorodeoxyuridine triphosphate is one of the nucleosides present with the other four nucleoside triphosphates, one can prepare a large number of dimeric oligomers, which will have coding errors. The ability to excise the 5-fluorodeoxyuridine and replace it with a natural nucleoside would be indicative of repair.

Under ionizing radiation conditions and oxidative metabolism, the DNA adduct molecule 8'-hydroxyguanine is produced. Oligomers comprising the 8'-hydroxyguanine may be synthesized so as to have the 8'-hydroxyguanine one or more times in the chain. Synthesis of the sequence specific adduct has been described by Bodepudi et al., Chemical Research in Toxicology 5,608(1992).

UV radiation has been shown to produce photodimers, particularly pyrimidine dimers, as the major product and 6-4 adducts, generally not more than about 10% of the total adducts, as a minor product. Duplex oligomers may be produced which may then be subjected to irradiation so as to produce the different types of adducts. Irradiation may be employed, so that on the average one or a few adducts may be formed, usually not more than about 5 adducts per molecule, preferably not more than about 3 adducts, more preferably from about I to 2 adducts per molecule on the average. An ultraviolet light source of about 225- 275nm, usually about 240-260nm, generally at a power of about 10-20, usually about 14 joules/sec/m², may be employed for about 0.5 to 15, usually about 1 to 12 nm to provide the desired level of adducts. The adduct products may then be separated and purified using high pressure liquid chromatography, where substantial enrichment of each of the adducts may be achieved. To prepare molecules which have been modified using chemotherapeutic agents, the duplex DNA may be combined with the therapeutic agent under conventional conditions, where the chemotherapeutic agent reacts with the duplex DNA. Thus, one can combine the duplex DNA with cis-platin, sulfur or nitrogen mustards, enediene compounds, methylating compounds, etc. In this manner, lesions may be introduced into the DNA related to the reaction of chemotherapeutic agent.

The repair assay can also be used as a biosensor where one is interested in the mutagenic capacity of a composition, one may combine the DNA oligomers with the composition for sufficient time for lesions to form. With any particular composition, this may require an empirical determination or one may choose an arbitrary time period, by which if no lesions are formed, one may assume that the mutagenic potential of the composition is below a threshold level. Thus, one may introduce the oligomer into an aqueous medium, where the medium may be a source of unknown mutagen or carcinogen in an existing stream, e.g., obtained as an extract from soil, as a wash from gas, as an extract from clothing, or the like. The damaged DNA is incubated with the composition, where cellular components may be included, such as a cell lysate from a repair deficient host cell, to provide for an environment more closely associated with the environment in which mutagenesis occurs.

The oligomer may be prepared for reaction in solution or be bound to a solid surface. Since the oligomer will be synthesized, or will be formed by the polymerase chain reaction or analogous reaction, one can control the terminal functionality of one or both chains. Thus, one can provide for a wide variety of functionalities which, if desired, may be used for binding to a solid support, covalently or non-covalently. For example, for covalent bonding, one may provide for carboxy. oxo, e.g. aldehyde, hydroxy, thiol, amino, phosphate, etc. One can then prepare a surface with a complementary functionality, so that covalent bond formation can be achieved. For example, with an amino functionality on the oligomer, one can link to an activated carboxyl or carboxyl ester, to form an amide, or link to an oxo group under reductive amination conditions to form an alkylamine group, or the like. With thiol, one can prepare disuliϊdes, where a sulfenyl halide is present on the surface, or form a thioether, with an activated olefϊn, e.g. maleimide. With phosphorus, one can activate the phosphate, so as to be capable of forming phosphate esters or amides. For hydroxyl, one may form esters with carboxyl groups or ethers with active halo or epoxide. The literature has extensive procedures for reacting functionalized DNA oligomers with a wide variety of other functionalities to covalently bond the oligomer to another molecule. Alternatively, one may have a need for non-covalent bonding, by employing various ligands bonded to the oligomer, where it is the ligand receptor which is bonded to a solid substrate. Thus, one may use haptens, e.g. digoxin, with anti-digoxin on a surface, biotin with streptavidin, sugars with, lectins, and the like. By using a solid support, one can readily separate the DNA bound to the surface from other components in an assay solution. In addition, where one is interested in dimerization of duplex DNA, one can provide for denaturation of the DNA bound to the solid support, so as to remove any strands which are non-covalently bonded to the strand bound to the support, and if desired, one may degrade the single-stranded DNA, so as to leave only cross-linked duplex DNA. Depending upon the nature of the support, the support may be functionalized in a variety of ways. For example, with glass or silicate supports, the glass or silicate may be functionalized with carboxyalkyl or aminoalkyl silyl ester to provide carboxy or amino groups. Plastics such as polystyrene may be functionalized with chloromethyl groups to provide for active halogen, which may then react with amino groups or hydroxyl groups, may be nitrated, followed by reduction to provide for amino groups, or the like. Acrylates may have free carboxyl groups, hydroxyl groups, or the like, which may be used for esterification, amidification, or the like. If desired, linkers may be employed, so that the nucleic acid oligomer may be spaced away from the surface. Various linkers may be employed, such as alkylene groups, polyethylene oxy groups, or the like. Usually the linker will vary from about 6 to 100 A length in the chain, more usually from about 8 to 60 A length in the chain.

Various substrates or containers may be employed in carrying out the assays. Thus, the solid support may be a microtiter well plate, a petri dish, a microscope slide test tube, capillary tube, or the like. In some instances, one may wish to us microparticles, generally form 10 to 500 μ as a substrate, for ease of handling an transfer. The particular solid substrate or vessel which is employed will vary with th nature of the assay.

The sample DNA oligomer may be combined with an appropriate cellular lysat for carrying out the assay. Depending upon the nature of the assay, the cellular sourc may vary. For example, where one is interested in the host's capability to respond to lesion formation as a result of exposure to irradiation, environment, or the like, one may use normal cells as the source of the lysate. Thus, one may use peripheral blood lymphocytes, keratinocytes, hepatocytes, bone marrow, or the like, depending upon the nature of the exposure and the purpose of the assay.

The lysate may be prepared in accordance with conventional ways, using sonication, mild detergent, homogenization, protease inhibitors, etc., as described by Manley et al., Proc. Natl. Acad. Sci. USA 77,3855 (1980); Wood, et al., Cell 53,95(1988). Usually, the number of cells for an assay will be 10⁴ to 10⁶ with 46 x 10³ to 46,000 x 10³ number of DNA molecules. The volume of the lysate will generally be in the range of about 1 to 10, more usually about 4 to 6 ml with the lysate in a appropriately buffered medium, generally at a pH in the range of about 6.5 to 7.5. Where one is interested in chemotherapy in the case of cancer, one will normally use cells from the tumor obtained by biopsy for the lysate.

In each case, one may expand the cells in culture, usually not providing for more than about 50 generations, more usually not more than about 30 generations. The cells may be lysed as described above and used accordingly. Where the subject technique is being used as an environmental biosensor, one will normally employ two cellular sources, a DNA repair deficient cell line, and a normal cell population. In this way, one may compare the results of the DNA repair deficient cell line and the normal cell line. DNA repair deficient cell lines may be obtained from xeroderma pigmentosum cell line; ataxia telangctasia cell lines; Bloom's syndrome cell lines, etc. The xeroderma pigmentosum cell lines are associated with UV mimetic or large bulky carcinogenic polycyclic aromatic hydrocarbon induced deficiency; the ataxia telangctasia cells are associated with oxidomimetic and oxidative damage, or colorectal cancer cells mismatch repair; while the Bloom's syndrome cells are associated with ligase defective cells. In this assay, one would expose the DNA oligomer to the environmental composition in an appropriate medium for a sufficient time to induce lesions, if a lesion forming component is present. One would then divide the DNA composition into two aliquots, subjecting one aliquot to a DNA repair deficient cell lysate and the other aliquot to lysate from normal cells, e.g. peripheral blood lymphocytes. If desired, one may divide the DNA sample into a plurality of aliquots, subjecting the DNA sample to different repair deficient cell lines. One would then compare the results obtained with the DNA exposed to the one or more repair deficient cell line lysates and the result with the repair proficient cell lysate. The assay will be carried out under conditions, depending upon the nature of the DNA sample, the nature of the lysate, the information desired, and the like. Usually, incubations of the DNA with the lysate will be for at least 0.5 h, more usually at least I h, and may be for 24 h or more. The particular time will depend upon the sensitivity of the assay, the rate of repair, the nature of the lesions, the size of the sample, and the like.

The temperature will generally range from about 20 to 40°C, conveniently 25 °C or 37 °C. particular precautions may be sealing of the vessels to prevent evaporation, protection against light, and the like.

A wide variety of protocols may be employed for detecting the occurrence and degree of repair. The particular approach will depend on the nature of the lesion and the nature of the repair. For example, where the lesion will be substituted with naturally occurring dNTP, one may use labeled dNTP and determine the amount of labeled dNTP incorporated into the DNA oligomers. This can be achieved by adding ³²P radiolabeled dNTPs to the lysate. The oligomers can be bound to the surface of the assay vessel. After sufficient time for repair to occur, the supernatant may be washed away and the radioactivity of the residual DNA determined. One or more washings may be employed, to ensure the substantial absence of non- incorporated radioactive nuclectides. Alternatively, one may use biotin labeled dNTPs, so that biotin becomes incorporated into the oligomeric DNA. After carefully washing away any unincorporated biotin labeled nucleosides, one may then use labeled streptavidin, to detect the presence of biotin bound to the surface. Labels may include radioisotopes, enzymes, fluorescers, and the like. With enzymes, one would need to add a substrate which provided a detectable signal, such as light absorptio fluorescence, chemiluminescence, or the like.

Where the lesion allows for detection by specific binding with an antibody, on can determine the residual number of lesions, as compared to a control where no repa has occurred. Thus, one may use antibodies to the particular modification, such as 8' hydroxyguanine, 6-4 adduct, pyrimidine dimer, 6-4 adduct, the particul chemotherapeutic agent, such as cis-platin, the alkylating agent, intercalating agent, e.g psoralen, or the like. The antibody may be labeled or one may use labeled anti-antibod to detect the presence of the lesion. Depending upon the nature of the label, one may detect the presence of the label in accordance with conventional ways. Scintillatio counters may be used with radioisotopes, fluorimeters may be used with fluorescen molecules, photodetectors may be used with chemiluminescers, spectrophotometers ma be used with dyes, and the like. For the most part, determinations will be made with solid substrate bound oligomers.

Usually, a control may be employed, where one may compare the resul obtained with the sample, with the result obtained with oligomers having known lesion and/or with lysates which may be repair defective or repair deficient. One may use a plurality of controls to obtain a more accurate determination of the proficiency of the sample lysate in repairing a particular lesion or the ability of a sample component to cause lesions.

As indicated, the subject invention may be used to screen drugs for efficacy in cancer treatment. By using a lysate from neoplastic cells from a patient, one may determine those lesions which are most refractory to repair by the tumor cells. In this way, efficacy of chemotherapy can be enhanced by ensuring that the lesions produced by the particular therapeutic treatment are refractory to repair by the particular tumor. The subject invention can also be used to investigate drugs which may enhance or inhibit the repair system. Thus, one may add a component to the lysate with oligomer comprising a particular lesion and determine the effect of the drug on the capability of the lysate to repair the lesion and the rate at which the lesion may be repaired. Similarly, with a repair proficient lysate, one may add drugs to determine the ability for the drug to inhibit repair. Also, as indicated previously, one may investigate the ability of components of the sample to introduce lesions, as determined by the ability of normal cells to repair the induced lesion.

The following examples are offered by way of illustration and not by way of limitation.

EXPERIMENTAL

Example I

Oxidative metabolism and radiation result in production of the DNA adduct molecule 8 '-hydroxyguanine. To investigate the ability for cellular lysates to repair such lesion, 8 '-hydroxyguanine containing DNA molecules are synthesized as 80 mer oligonucleosides having a 5 '-amino functionality with a hexaethyleneoxy spacer group. The synthesis is performed in accordance with conventional ways. See Crea et al., Nucleic Acids Res. 8,2331 (1980); and Bodepudi et al., (1992) supra. Both strands are produced, while only one strand has the amino functionality. The two strands are hybridized to provide dimeric oligomers. A glass cover slip is functionalized with 3'- carboxypropylsilyl triethyl ester. The carboxy group is then activated with l-ethyl-3- (3'-dimethylaminopropyl) carbodiimide ("EDC"). The oligomer is added to the activated carboxy groups at a concentration of 8mg/^l in lOmM N-methyl imidazole to provide the oligomer at a density of about 10¹⁰ molecules/cm². (See Rasmussen, Anal. Biochem. 198, 138(1991)

A lysate of 10⁶ human peripheral blood lymphocytes ("PBL") is prepared by the method of Manley et al. (1980) supra. To 100 μl of the lysate is added 500 μM of ³²P radiolabeled deoxynucleoside triphosphates ("dNTPs"). The cover slip is covered with the lysate solution and sealed to prevent evaporation. The assay mixture is incubated at 37°C for 6 h.

After incubation, the assay mixture is washed from the cover slip, followed by washing to the cover slip twice with 0.4 M sodium hydroxide. The slip is then removed to a scintillation counter and the radioactivity determined. The assay is repeated with oligomer prepared in the same way as the sample oligomer, except that 8'- hydroxyguanine is excluded. By comparing the radioactivity of the sample oligomer in the absence of 8' -hydroxyguanine with the oligomer comprising 8 '-hydroxyguanine one can determine a human patient's capability to repair lesions resulting fro radiation.

Example 2 In the next study, pyrimidine dimers or 6-4 adducts are the lesions. These ar produced by ultraviolet radiation and may be the cause of skin cancer in humans. In thi study, one uses epidermal cells as the source of the lysate.

The sample is prepared by synthesizing 100 mer oligonucleosides, where one of the chains includes a 5' amino group, as described above. 5 μg/ml of the 100 mer duplex oligomer is subjected to UV radiation (254 nm) for 0. Ih in PBS. The irradiation results in lesions comprising a major proportion of pyrimidine dimer, but a small portion, 10%, of 6-4 adduct. The pyrimidine dimer containing oligomers are separated from the 6-4 adduct oligomers by HPLC.

Each of the two types of adducts is combined with a microtiter well plate functionalized by chloromethylating polystyrene, resulting in amino substitution of the activated chlorine.

The response of normal or DNA repair deficient patients can be determined. One prepares a lysate from patients suffering from xeroderma pigmentosum to evaluate the repair deficiency of the diseased patient as compared with normal individuals. In this assay, a 200 μl whole cell lysate is prepared as described above. The lysate is added to the wells comprising the pyrimidine dimer and incubated for 1 h at 37°C. After 1 h, the wells are washed twice with phosphate buffered saline-Tween 20 to remove the lysate. Mouse monoclonal antibodies specific for the pyrimidine dimer in 1 % BSA/PBS at a concentration of .Olppm are then added to each of the wells and the mixture incubated for 1 h at 37° C. Each of the wells is then washed thoroughly with PBS to remove any non-specific bound antibody, followed by the addition of fluorescein labeled goat anti-mouse antibody. The number of pyrimidine dimer molecules is then determined by determining the fluorescence in each well. This result is compared to a well in which no lysate was added, so that there could be no repair of the pyrimidine dimer. Example 3

In this example, the ability to repair lesions resulting from chemotherapeutic agents is investigated.

100 mer oligonucleosides are synthesized with sequence specific cis-platin containing adducts, and having on one chain, a 5'-alkylthiol group. The binding of the cis-platin is achieved by intrastrand cross-linking to the oligomer. The adduct oligomer is then combined with thiol activated polyacrylate under oxidative condition to provide the formation of disulfide groups between the ligonucleoside duplex and the polymer surface. Whole cell lysate of PBL (lOOμl) is incubated over the immobilized adduct oligomer for 2 h at 37°C. The plastic surface is washed 3X with PBS -T ween to remove any interfering components. Mouse monoclonal antibodies specific for cis-platin are added in 1 % BSA/2X PBS at a concentration of 1:10³ and incubated for 30 min. After thorough washing to remove any non-specifically bound mouse monoclonal antibody, sheep anti-mouse antibody conjugated A-HRP are added at a concentration of 1: 10⁴ in 1 % BSA/PBS and the mixture allowed to incubate for 30 min at 37° C. The surface is then thoroughly washed with PBS to ensure the substantial absence of non-specific bound conjugate. o-Phenylene diamine at 20 mg in 10 ml phosphate buffer and hydrogen peroxide (20 μl 3 % H₂O₂ are added and the rate of formation of color determined as indicative of the amount of HRP bound to the surface. A control is performed where the cis-platin adduct is exposed to a medium incapable of repair.

Alternatively, after washing the oligomer free from the lysate, the oligomer is released from the surface using dithiothreitol, the oligomer isolated and the amount of platinum remaining determined by atomic absorption.

Example 4

The subject process may be used as an environmental biosensor. In this application, standardized microtiter well plates or chips may be employed where a known amount of an oligomer is present on the surface. Aminoalkyl substrated polystyrene is employed as the surface. For this purpose, 100 mer duplex DNA molecules are employed having terminal phosphate, where the terminal phosphate is activated by polynucleoside kinase and excess ATP. The activated phosphate is then reacted with 0.2M l-ethyl-3-(3-dimethyl-aminopropyl) carbodiamide dissolved in lOmM N-methyl imidazole to provide a concentration of 10⁹ immobilized molecules per mm².

In carrying our the assay, oligonucleoside probes individually bound to solid substrates are immersed in an unknown solution taken from a stream located near a hazardous waste site and incubated for 12 h. The probes are DNA duplexes bound to the support through an amide linkage to only one chain. The solution is removed and the surfaces washed once with PBS. Probes A, B and C are then incubated 1 h with different PBL whole cell lysates from DNA repair deficient human cell lines: xeroderma pigmentosum, ataxia telangctasia and Bloom's syndrome cell lines. Probe D is incubated 1 h with PBL whole cell lysate from a repair proficient cell line. After 1 h, the probes are removed from the incubation chamber, and washed once with PBS. A probe is reserved as a control which is subjected to neither the repair deficient cell lines, nor the repair proficient cells. The probes are then subjected to denaturating conditions, 90°C for 5 min in PBS so that uncrosslinked DNA loses the strand unbound to the surface. If desired, the single-stranded DNA may be degraded with SI nuclease. To the probes is then added mouse monoclonal antibody specific for duplex DNA in 1 % BSA/2x PBS at a dilution of 1: 10- and the mixture incubated for 1.5h. The presence of the mouse monoclonal antibody bound to the surface is then determined by adding rabbit anti-mouse antibody conjugated with fluorescein.

The fluorescence of the samples combined with the repair deficient cell line lysates is then compared with the proficient cell line and the control. Where the control demonstrates the retention of double-stranded DNA, the sample may be considered to comprise components which induce crosslinking of duplex DNA, resulting in lesions in the DNA. The retention of the crosslinking in the presence of the repair deficient cell line lysates is indicative that those individuals suffering from the different diseases are particularly susceptible to such components. In addition, retention of the crosslinking in the presence of the repair proficient cell line lysate is indicative that the component is mutagenic and may have serious disease potential in its ability to cause mutagenesis of DNA, which is not readily repaired by the repair system of normal cells,

It is evident from the results, that the subject invention provides a convenient and rapid method for determining the interrelationship between mutagenesis and repair in a variety of contexts. Of particular importance is the use of the subject method to evaluate chemotherapeutic drugs as to their efficacy in creating lesions in neoplastic cells, where the repair system of the necplasti°c cells in inadequate to repair the injury to the DNA. In addition, one may screen various compounds as to their ability to enhance or inhibit the repair systems of normal cells or aberrant cells. The assay is simple to carry out, can be carried out fairly quickly and can provide for information which may then be used in evaluating therapeutic treatments, susceptibility to mutagenesis, and the like.

All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A method for evaluating the interrelationship between DNA lesions and cellular repair, said method comprising: combining duplex DNA suspected of comprising a lesion with a lysate from a candidate cell population for sufficient time for said lysate to act upon said duplex DNA; and determining any change in the composition of said duplex DNA as a result of said lysate acting on said duplex DNA and repairing any lesions.

2. A method according to Claim 1 , wherein said duplex DNA is bound to a solid surface, and said determining is with an antibody which binds to a specific lesion or a specific lesion forming agent.

3. A method according to Claim 1, wherein said duplex DNA is bound to a solid surface, and said determining is with radioisotope labeled deoxynucleoside triphosphates, whereby radioisotope incorporation into said duplex DNA is indicative of repair.

4. A method for determining the ability of neoplastic cells to repair lesions formed by therapeutic agents, said method comprising: combining duplex DNA subjected to said therapeutic agent, said duplex DNA bound to a solid surface, with a cellular lysate of said neoplastic cells for sufficient time for said lysate to act on lesions present in said duplex DNA; and determining the extent of repair of said lesions by said lysate.

5. A method according to Claim 4, wherein said lesions are as a result of radiation.

6. A method according to Claim 5, wherein said determining is by means of an antibody to said lesion.

7. A method according to Claim 4, wherein said lesions are as a result of a chemotherapeutic agent.

8. A method according to Claim 7, wherein said determining is by means of an antibody to said lesion or said chemotherapeutic agent.

9. A method according to Claim 7, wherein said determining is by means of a radioisostope labeled deoxynucleoside triphosphate.

10. A method for determining the mutagenicity of a sample and the ability of cells to repair the lesion resulting from said sample, said method comprising: combining duplex DNA with said sample for sufficient time for lesions to be formed in said duplex DNA in the presence of a mutagenic agent in said sample to provide specimen DNA; separating said specimen DNA from said sample and combining portions of said specimen DNA with at least one lysate from a repair deficient cell source and a repair proficient cell source; and determining any difference in said specimen DNAs between said specimen DNA combined with said repair deficient lysate and said repair proficient lysate, whereby a difference indicates that said sample had mutagenic capability and said lesion is capable of repair.