Nucleotide sequence preservation of human leukemic mitochondrial DNA

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Nucleotide Sequence Preservation of Human Leukemic Mitochondria! DMA1 .... Raymond J. Monnat, Jr.,2 Clare L. Maxwell, and Lawrence A. Loeb The Joseph Gottstein Memorial Cancer Research Laboratory, Department of Pathology, University of Washington, Seattle, Washington 98195

ABSTRACT Nucleotide sequence variation in mitochondria! DNA isolated from human leukemic cells has been analyzed by recombinant DNA techniques. Three hundred eighty-seven independent recombinant DNA clones, each containing one of three defined segments of mitochondrial DNA isolated from the neoplastic cells of four leukemic patients, were analyzed. Partial nucleotide se quence determination of the 387 clones yielded a total of 81.7 kilobases of nucleotide sequence information. The only evidence of within-individual nucleotide sequence divergence consisted of three clones containing deletions of one or two nucleotides in one mitochondrial DNA region. These clones were three of 113 independent clones isolated from a patient with acute lymphocytic leukemia. The low level of nucleotide sequence divergence in the mitochondrial DNA population of neoplastic cells from individual leukemic patients suggests that a mechanism or mech anisms exist that limit the development of nucleotide sequence divergence in mammalian mitochondrial DNA. The results further suggest that this mechanism does not appear to be abrogated by neoplastic transformation in leukemic patients.

INTRODUCTION Prior to the advent of recombinant DNA techniques, it was not possible to directly identify and characterize mutations occurring in the DNA of normal or neoplastic human cells. Now that individual cellular genes can be isolated, characterized, and reintroduced into mammalian cells, it has become possible to establish causal links between the presence of specific genetic alterations and either resistance to therapy or the development of métastases. We have applied recombinant DNA techniques to a specific question related to the phenomenon of neoplastic progression. Do mutations accumulate within human neoplastic cell popula tions? We have examined mtDNA3 isolated from neoplastic cells of patients with leukemia. We chose to study the mtDNA of human leukemic cells for 3 reasons: (a) mtDNA is well charac terized; its nucleotide sequence is known in entirety (3), and a great deal is known about between-individual nucleotide se quence differences; (b) mtDNA can be easily and reproducibly isolated from leukemic cells present in small amounts of periph eral blood; (c) Our previous work (29) demonstrated a low level of nucleotide sequence divergence within the mtDNA population of lymphocytes from individual normal donors. Thus, mtDNA mutations occurring in leukemic cells should be easily detected. 1This work was supported by grants from the NIH (CA-24845) and the United States Department of Energy (DE-AM06-76L02225). 1 Postdoctoral Fellow of the NIH (GM-07187). To whom requests for reprints should be addressed. 'The abbreviations used are: mtDNA, mitochondrial DNA; CO III, cytochrome oxidase subunit III; ALL, acute lymphocytic leukemia; CML/BC, chronic myelogenous leukemia in blast crisis; CLL, chronic lymphocytic leukemia. Received 9/5/84; revised, 12/11/84; accepted 1/10/85.

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In this paper, we report on the isolation and nucleotide sequence determination of 387 independently isolated recombinant clones containing mtDNA from the neoplastic cells of 4 patients with leukemia.

MATERIALS AND METHODS Materials. Mononuclear cells were isolated from peripheral blood samples from 4 patients with clinical diagnoses of leukemia. The mononuclear cell fractions were isolated and stored at -70°C until confirmation of the clinical diagnosis. The cloning vector, M13mp11, and host, Escherichia coli strain JM103, were gifts of Dr. Joachim Messing, University of Minnesota. Protocols for growth of vectors and host strains are given in Ref. 27. Restriction endonucleases, T4 DNA ligase, and M13 sequencing primer were obtained from New England Biolabs or Bethesda Research Labo ratories, Inc. Calf intestinal alkaline phosphatase was obtained from Boehringer-Mannheim. E. coli DNA polymerase I large fragment was prepared by digestion of homogeneous E. coli DNA polymerase I (22) with Bacillus subtilis subtilisin, followed by a separation on Sephadex G100 (23), or obtained from Bethesda Research Laboratories. FicolhHypaque was obtained from Pharmacia Fine Chemicals. Deoxy- and dideoxynucleoside triphosphates and ATP were obtained from Pharma cia P-L Biochemicals. [«-32P]dCTPand [«-32P]dTTPwere obtained from New England Nuclear. Methods. Peripheral blood mononuclear cells were isolated from 4 patients with leukemia by gradient centrifugation in FicolhHypaque (10). The resulting mononuclear cell fractions were washed twice in phos phate-buffered saline, pelleted, and frozen at -70°C. mtDNA was iso lated from the frozen cell pellets by a modification of the "no gradient" technique of Bogenhagen and Clayton (9) [Tapper ef al. (38)]. The resulting mtDNA was resuspended in 50 M!of 50 rriM Tris (pH 7.5):1 HIM EDTA. Prior to cloning, each mtDNA preparation was digested sequen tially with the restriction endonucleases Sacl and Xbal, extracted with phenol, and precipitated with ethanol. To test for complete digestion, 5 ti\ (1/10 volume) of each restriction digest containing approximately 10 ng of mtDNA were end-labeled with 2 MCiof [«-32P]dCTP(3000 Ci/mmol) (17). The labeled DNA was precipitated with ethanol at -70°C and washed with cold 70% ethanol to remove unincorporated dCTP. The labeled DNA fragments were separated on a 0.8% neutral agarose gel by electrophoresis in Trisiborate buffer [90 rtiM Tris-HCI:90 rriM boric acid:1 mM EDTA, pH 8.2 (26)]. The gel was transferred to Whatman No. 3MM chromatography paper and dried at 80°Cfor 30 min under vacuum. Autoradiography was performed with an enhancing temperature (22°C)for 12 to 24 h.

screen at room

The cloning vector, M13mp11, was prepared by cutting the viral replicative form sequentially with the restriction endonuclease Sacl and Xbal. The 2 resulting fragments were dephosphorylated with calf intes tinal alkaline phosphatase (14), extracted with phenol, and precipitated with ethanol. Ligation reactions were performed in a volume of 30 ^l of 50 ÕTIM Tris-HCI (pH 7.5):10 mM MgCI2:10 mw dithiothreitoM mM ATP containing 100 ng of dephosphorylated M13 DNA, approximately 50 ng of mtDNA, and 0.1 to 0.5 units of T4 DNA ligase. After ligation at 18°C for 24 h, aliquots of the mixture were used to transform the E. coli host strain JM103 (27). DNA Sequence Analysis. Single-stranded M13 DNA containing mi tochondrial inserts was prepared from individual plaque-purified colonies

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HUMAN LEUKEMIA by virus precipitation with polyethylene glycol and extraction with phenol (27). DMA sequence determination was by a modification of the dideoxy chain termination method of Sanger ef al. (36). Single-stranded recom binant DNA template (0.5 Mg) was hybridized wtih a 2-fold molar excess of a synthetic oligonucleotide sequencing primer (New England Biolabs; 17-mer) in 13 n\ of 12 mw Tris-HCI (pH 7.9):70 mM NaCI:7.5 HIM MgCI2; annealing was at 55°C for 1 h. Each base-specific chain termination reaction contained in 6 ^l of 20 mw Tris-HCI (pH 7.5): 67.5 mw NaCI: 7.5 mM MgCI2: 10 mw dithiothreitol: 1.67 ^M [a-^PJdTTP (150 Ci/mmol),

mtDNA

ments 4, 5, or 7 was used as a template for nucleotide sequence determination by the dideoxy chain termination method of Sanger ef a/. (36). The nucleotide sequences of the light (L)-strand of Fragment 4 and of the H-strand of Fragment 7 were determined from the Sacl site that divides the CO III gene into halves with a cut between L-strand nucleotides 9647 and 9648. The H-strand sequence of Fragment 5 was determined from the Sacl site that cuts between H-strand nucleotides 36 and 37 (Chart 1).

Patient information and a summary of the nucleotide sequence information obtained are given in Table 1. A total of 81.7 kiloside triphosphates were 4.2 MMfor dATP, dCTP, and dGTP and 1.67 /¿M bases of sequence information, an average of 211 nucleotides per clone, was determined. Single-base substitutions were found for dTTP in the respective chain termination reactions. The dideoxy: deoxynucleoside triphosphate ratios used in individual termination reac approximately once every 230 nucleotides when patient mtDNA tions were 60:1 for adenosine, 30:1 for cytidine, 22.5:1 for guanosine, sequences were compared with the published human mtDNA and 50:1 for thymidine. The concentrations of nonlimiting deoxynucleo sequence. The published sequence was derived largely from side triphosphates were 42 ^M in adenosine-, cytidine-, and guanosinemtDNA isolated from a single placenta (3). A small portion of the specific base termination reactions and 31 ^M in thymidine-specific base published sequence (45 cell population doublings required to convert the zygote into a fully formed organism. Most importantly, a progressive increase in base substitutions would be expected if tumor progression in leukemic cells proceeded by an error-prone DNA replication mechanism that was able to affect mtDNA (25). In this study, only 3 mitochondrial mutations were identified in 81,700 nucleotides of DNA sequence information obtained from the neoplastic cells of 4 leukemic patients. All 3 of the withinindividual differences consisted of deletion of one or 2 nucleo tides in the one patient with ALL. These clones were 3 of a total of 81 containing Fragment 7 that were isolated as independent clones from the patient with ALL. The single-base deletions of cytidine in 2 of the clones produce a -1 frame shift in the CO III gene and new chain termination codons 24, 48, and 70 down stream from the deletion site. The deletion of a cytidine and thymidine from the L-strand nucleotide region 9727-9732 of the third clone produces a -2 frame-shift mutation in the CO III gene and new chain termination codons 4,26, 70, and 84 downstream from the 2 nucleotide deletion sites. These within-individual mutations could have originated in at CANCER

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9732 (29). Thus, there does not appear to be preferential muta tion of these nucleotide positions when human mtDNA is cloned in M13 and the resulting clones used for nucleotide sequence determination by the dideoxy chain termination method. Concerted Preservation of Human Leukemic mtDNA. Our results strongly suggest that nucleotide sequence divergence is not present in the mtDNA population of neoplastic cells of leukemic patients at a level of greater than one nucleotide substitution per molecule. Two implications of this finding are: (a) that a mechanism (or mechanisms) exist to restrict the de velopment of mitochondrial nucleotide sequence divergence; and (b) that this mechanism does not appear to be abrogated by neoplastic transformation. With only 3 exceptions, this study revealed an invariant mtDNA population in neoplastic cells of individual leukemic patients. The low level of mitochondrial nucleotide sequence divergence in both normal (29) and neoplastic human cells suggests the exist ence of a mechanism to limit the accumulation of mutations in the mtDNA population derived from the zygote. How such a mechanism might work is not clear. The most direct mechanism, a passive loss of altered or damaged mtDNA molecules, fails to explain the absence of all nucleotide substitutions, regardless of their coding effects, from human mtDNA. A second mechanism of mitochondrial nucleotide sequence preservation could be more accurate mtDNA replication or mtDNA repair. For example, a hundredfold increase in the accuracy of mtDNA replication, pre dicted from measurements of the fidelity of the suspected mtDNA polymerase, 7, in vitro (24) would be sufficient to limit the development of mitochondrial nucleotide sequence divergence during development. However, neither more faithful mtDNA rep lication nor mtDNA repair appears adequate to limit mutation accumulation during mtDNA turnover in normal somatic cells following completion of development (35) or in an expanding neoplastic cell population. A third possible mechanism that could explain the absence of nucleotide substitutions in the mtDNA population of an individual is the repeated correction of all mtDNA molecules in a cell against a master copy. This mechanism in its simplest form is unlikely to be correct, as genetically distinct mtDNA molecules appear to be able to persist in single human cells (39). In this study, the nucleotide sequence of mtDNA isolated from the neoplastic cells of individual leukemic patients was found to be highly conserved. Thus, critical tests of a somatic mutational origin of tumor cell heterogeneity and tumor progression (19, 20, 25, 32, 34, 40) must focus on defined nuclear genes. The methods and approach developed in the course of this investi gation can be used to identify and characterize nucleotide se quence divergence in specific nuclear genes from individual tumor cell populations. Two families of nuclear genes that will be particularly important to analyze in this manner are cellular VOL. 45 APRIL 1985

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17. Drouin, J. Cloning of human mitochondrial DNA in Escherichia coli. J. Mol. Biol., 740: 15-34, 1980. 18. Fialkow, P. J. Cell lineages in hematopoietic neoplasia studied with glucose-6phosphate dehydrogenase cell markers. J. Cell. Physiol., (Suppl. 1): 37-43, 1982. 19. Fidler, I. J., and Hart, I. R. Biological diversity in metastatic neoplasms: origins and implications. Science (Wash. DC), 277: 998-1003, 1982. 20. Foulds, L. Tumour progression and neoplastic development. In: P. Emmelot and O. Muhlbock (eds.), Cellular Control Mechanism and Cancer, pp. 242258. New York: Elsevier Publishing Company, 1964. 21. Greenberg, B. D., Newbold, J. E., and Sugino, A. Intraspecific nucleotide sequence variability surrounding the origin of replication in human mitochondrial DNA. Gene, 27: 33-49, 1983. 22. Jovin, T. M., Englund, P. T., and Bertsch, L. L. Enzymatic synthesis of deoxyribonucleic acid. XXVI. Physical and chemical studies of a homogeneous deoxyribonucleic acid polymerase. J. Biol. Chem., 244: 2996-3008, 1969. 23. Klenow, H., and Henningsen, I. Selective elimination of the exonuclease activity of the deoxyribonucleic acid polymerase from Escherichia coli B by limited proteolysis. Proc. Nati. Acad. Sci. USA, 65: 168-175, 1970. 24. Kunkel, T. A., and Loeb, L. A. Fidelity of mammalian DNA polymerases. Science (Wash. DC), 273: 765-767,1981. 25. Loeb, L. A., Springgate, C. F., and Battula, N. Errors in DNA replication as a basis of malignant changes. Cancer Res., 34: 2311 -2321, 1974. 26. Maniatis, T., Fritsch, E. F., and Sambrook, J. Molecular Cloning. A Laboratory Manual, pp. 156-162. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1982. 27. Messing, J. New M13 vectors for cloning. Methods Enzymol., 707: 20-78, 1983. 28. Monnat, R. J., Jr., and Loeb, L. A. Mechanisms of neoplastic transformation. Cancer Invest., 7: 175-183, 1983. 29. Monnat, R. J., Jr., and Loeb, L. A. Nucleotide sequence preservation of human mitochondrial DNA. Proc. Nati. Acad. Sci. USA, in press, 1985. 30. Nichols, W. W. Viral interactions with the mammalian genome relevant to neoplasia. In: J. German (ed.). Chromosome Mutation and Neoplasia, pp. 317332. New York: Alan R. Liss, 1983. 31. Niranjan, B. G., Bhat, N. K., and Avadhani, N. G. Preferential attack of mitochondrial DNA by aflatoxin B, during hepatocarcinogenesis. Science (Wash. DC), 275: 73-75,1982. 32. Nowell, P. C. The clonal evolution of tumor cell populations. Science (Wash. DC), 794: 23-28, 1976. 33. Olivo, P. D., Van de Walle, M. J., Laipis, P. J., and Hauswirth, W. W. Nucleotide sequence evidence for rapid genotypic shifts in the bovine mitochondrial DNA D-loop. Nature (Lond.). 306: 400-402, 1983. 34. Poste, G., and Greig, R. The experimental and clinical implications of cellular heterogeneity in malignant tumors. J. Cancer Res. Clin. Oncol., 706:159-170, 1983. 35. Rabinowitz, M., and Swift, H. Mitochondrial nucleic acids and their relation to the biogenesis of mitochondria. Physiol. Rev., 50: 376-427, 1970. 36. Sanger, F., Nicklen, S., and Coulson, A. R. DNA sequencing with chainterminating inhibitors. Proc. Nati. Acad. Sci. USA, 74: 5463-5467, 1977. 37. Springgate, C. F., and Loeb, L. A. Mutagenic DNA polymerase in human leukemic cells. Proc. Nati. Acad. Sci. USA, 70: 245-249,1973. 38. Tapper, D. P., Van Etten, R. A., and Clayton, D. A. Isolation of mammalian mitochondrial DNA and RNA and cloning of the mitochondrial genome. Meth ods Enzymol., 97: 426-434, 1983. 39. Wallace, D. C. Assignment of the chloramphenicol resistance gene to mito chondrial deoxyribonucleic acid and analysis of its expression in cultured human cells. Mol. Cell. Biol., 7: 697-710, 1981. 40. Wheldon, T. E., and Kirk, J. An error cascade mechanism for tumor progres sion. J. Theor. Biol., 42: 107-111,1973.

protooncogenes and those genes whose products play essential roles in cell growth and division (8, 28). ACKNOWLEDGMENTS We thank Dr. Bernard Poiesz and Dr. Marshall Kadin for providing leukemic cells and R. Martene Koplitz for help with DNA sequence determinations.

REFERENCES 1. Allen, J. A., and Coombs, M. M. Covalent binding of polycyclic aromatic compounds to mitochondrial and nuclear DNA. Nature (Lond.), 287: 244-245, 1980. 2. Anderson, S. Shotgun DNA sequencing using cloned DNase l-generated fragments. Nucleic Acids Res., 9: 3015-3027,1981. 3. Anderson, S., Bankier, A. T., Barrell, B. G., de Bruijn, M. H. L., Coulson, A. R., Drouin, J., Eperon, I. C., Nierlich, D. P., Roe, B. A., Sanger, F., Schreier, P. H., Smith, A. J. H., Staden, R., and Young, I. G. Sequence and organization of the human mitochondrial genome. Nature (Lond.), 290: 457-465, 1981. 4. Anderson, S., de Bruijn, M. H. L., Coulson, A. R., Eperon, I. C., Sanger, F., and Young, I. G. Complete sequence of bovine mitochondrial DNA. Conserved features of the mammalian mitochondrial genome. J. Mol. Biol., 756:683-717, 1982. 5. Aquadro, C. F., and Greenberg, B. D. Human mitochondrial DNA variation and evolution: analysis of nucleotide sequences from seven individuals. Genetics, 703. 287-312, 1983. 6. Backer, J. M., and Weinstein, I. B. Mitochondrial DNA is a major cellular target for a dihydrodiol-epoxide derivative of benzo[a]pyrene. Science (Wash. DC), 209:297-299, 1980. 7. Bibb, M. J.. Van Etten, R. A., Wright, C. T., Walberg. M. W., and Clayton, D. A. Sequence and gene organization of mouse mitochondrial DNA. Cell, 26: 167-180,1981. 8. Bishop, J. M. Cellular oncogenes and retroviruses. Annu. Rev. Biochem., 52: 301-354, 1983. 9 Bogenhagen, D., and Clayton, D. A. The number of mitochondrial deoxyribo nucleic acid genomes in mouse L and human HeLa cells. J. Biol. Chem., 249: 7991-7995, 1974. 10. Boyum, A. Separation of leucocytes from blood and bone marrow. IV. Isolation of mononuclear cells and granulocytes from human blood. Scand. J. Clin. Lab. Invest., 27(Suppl. 97): 77-89, 1968. 11. Brown, W. M. Polymorphism in mitochondrial DNA of humans as revealed by restriction endonuclease analysis. Proc. Nati. Acad. Sci. USA, 77: 3605-3609, 1980. 12. Brown, W. M. Evolution of animal mitochondrial DNA. In: M. Nei and R. K. Koehn (eds.), Evolution of Genes and Proteins, pp. 62-88. Sunderiand, MA: Sinauer Associates, Inc., 1983. 13. Cann, R. L., Brown, W. M., and Wilson, A. C. Polymorphic sites and the mechanism of evolution of human mitochondrial DNA. Genetics, 706: 479499, 1984. 14. Chaconas, G., and van de Sande, J. H. 5'-MP labeling of RNA and DNA restriction fragments. Methods Enzymol., 65: 75-85, 1980. 15. Cifone, M. A., and Fidler, I. J. Increasing metastatic potential is associated with increasing genetic instability of clones isolated from murine neoplasms. Proc. Nati. Acad. Sci. USA, 78: 6949-6952, 1981. 16. Dimnik, L. S., and Hoar, D. I. Thymidine deprivation is mutagenic to the mitochondrial genome. Genetics, 97 (Suppl. 1).• s31,1981.

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NO. OF CLONES: DELETION(S): POSITIONS*:

78/81 — —

2/81

-C 9731 or 32

1/81 -C,T 9727-32

GTACGTACGT 9735

Fig. 1. Nucleotide deletions identified in mtDNA Fragment 7 of Patient ALL. The published L-strand sequence of nucleotide positions 9721 to 9738 of human mtDNA is given (left). Seventy-eight of 81 clones containing Fragment 7 from Patient ALL had a nucleotide sequence identical to that of the published sequence. The nucleotide sequencing gel autoradiogram of one of these 78 clones is shown (Lanes 1 to 4). Two of 81 clones contained a deleted cytidine at position 9731 or 9732. The nucleotide sequencing gel autoradiogram of one of these 2 clones is shown (Lanes 5 to 8). One of 81 clones contained a deletion of the thymidine at position 9730 and of a cytidine from positions 9727 to 9729 or 9731 to 9732. The nucleotide sequencing gel autoradiogram of this one double-deletion clone is shown (Lanes 9 to 72). •, it is not possible to assign the deleted cytidines unambiguously to positions 9727 to 9729 or 9731 to 9732.

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