MITOCHONDRIAL DNA SEQUENCE VARIATION IN HUMAN LEUKEMIC CELLS Rayna IVANOVA 1,2 *, Virginia LEPAGE 2 , Marie-Noe ¨lle LOSTE 2 , Franc ¸ois SCHA ¨ CHTER 3 , Eveline WIJNEN 4 , Mark BUSSON 1 , Jean-Michel CAYUELA 5 , Franc ¸ois SIGAUX 5 and Dominique CHARRON 1,2 1 INSERM U396, Ho ˆ pital St Louis, Paris, France 2 Laboratoire d’Immunologie et d’Histocompatibilite ´, Ho ˆ pital St Louis, Paris, France 3 CESTI-ISMCM Universite ´ Leonard de Vinci, Paris, France 4 Faculty of Technology and Science, Etten-Leur, The Netherlands 5 INSERM U462 and Laboratoire d’he ´matologie moleculaire, Ho ˆ pital St Louis, Paris, France The Long PCR followed by the RFLP technique has been used to search for abnormally structured mitochondrial DN A (mtDN A) and specific sequence differences implicated in the pathogenesis of acute lymphoblastic leukaemia (ALL). W e have studied 54 specific sites whose combinations define groups of mtDNA types, in 30 leukemic patients of French Caucasian origin. Results were compared with those in 100 French heathy individuals. N ucleotide substitutionshave been defined in 11 patients. This polymorphism is expressed by single base substitution at 6 sites which corresponds to 5 morphs, 2 of which were not found in the reference group. Combining the 11 observed morphs, we have identified 7 different mtDN A types, defined in 30 patients with ALL. T wo of the morphs (MspI-2 and AvaII-3) and 3 of the types (17-2, 55-2, N ew Fr 150) were not found in the group of healthy individuals. W e have observed significant statistical changes in type 28-2 in ALL patientscompared with the controls. Int. J. Cancer 76:495–498, 1998.  1998 Wiley-Liss, Inc. The human mtDNA genome contains 16569 bp and plays a limited but essential role in the biogenesis of the organelles that contain these. Since 1988, mtDNA variation resulting from base substitutions, insertions, or deletions have been postulated to play an important role in a broad spectrum of human diseases. Whereas only about 7% of the nuclear DNA is ever expressed at any particular differentiated stage, the expression of the whole mtDNA is essential for the normal functioning of the cells. Genetic mutation, whether spontaneous or induced by chemical mutagens, is a random process and occurs in the mtDNA as well as nDNA, but the mitochondrial rate of mutation is about 10 times higher than that of nDNA (Linnane et al., 1989). At the same time, mitochon- drial DNA is a privileged target for particular mutagens and carcinogens (Baggetto et al., 1993). Yamamoto et al., (1992) suggest that some mtDNA mutations could be one of the endog- enous factors that induce somatic mutations in the nuclear genome and contribute etiologically to human carcinogenesis. Brown et al., (1979) have expressed the opinion that mitochondrial mutations might play an important role in the initiation and progression of tumors as well as in their spontaneous regression. Several anoma- lies of mtDNA have been reported, i.e., deleted mtDNA in human colon adenocarcinoma cells (Savre-Train et al., 1992, Heerdt et al., 1994) breast cancer (Bianchi et al., 1995, Sharp et al., 1992) thyroid and renal oncocytic tumors (Tallini et al., 1994,; Kovacs et al., 1992) human gliomas (Liang and Hays, 1996) cirrhotic liver surrounding hepatic tumours (Yamamoto et al., 1992) abnormal length in leukaemia lymphoblasts (Gianni et al., 1980). However, to date, very few biological characteristics of mtDNA alterations in leukemic cells have been reported. We have studied the correlation between the existence of sequence variants (morphs and types) in mtDNA and predisposi- tion for the carcinogenesis process. To obtain and characterise a mtDNA sequence variability in our patients, we have screened the entire mtDNA molecules, using restriction mapping with 6 restric- tion enzymes. We have studied the basal level of mtDNA polymor- phism in normal and cancer cells and its biological significance in malignant transformation processes. MATERIAL AND METHODS Patients Thirty patients of French Caucasian origin, both adults and children classified as having ALL were studied. Of these, 15 were B cell proliferation (BCP) ALL and 15 T-ALL. Controls consisted of 100 unrelated adults, healthy individuals (CEPH families) from the French population (details not shown). DNA extraction Total DNA (mt and nuclear) was extracted from lymphoblastoid cell pellets and isolated by using the standard phenol/chloroform extraction procedure. Human mtDNA restriction endonuclease fragment patterns were analysed using total cell isolated DNA isolated from total cells. PCR All samples were screened by Long and Standard PCR. Approxi- mately 150 ng of genomic DNA were used as template. The nucleotide sequences of the primers used and the size of the amplified fragments are listed in Table I. Long PCR. In order to do avoid Southern blot analysis and to facilitate the rapid screening for mtDNA polymorphism, we used the Long PCR (Expand Long PCR System Boehringer Manheim, Germany). The primers (forward 571–598 and reverse 16220– 16193), the amplification conditions and the thermal cycling were carried out according to the method described by Paul et al., (1996). Standard PCR. All samples were segmentally amplified by PCR with 10 sets of primers in 10 overlapping fragments (Table I) using standard conditions (Passarino et al., 1993) with Taq DNA polymerase (Boehringer Manheim). Cycling conditions were: 1 min at 95°C, at 56°C and at 72°C for 30 cycles. The resulting 10 mtDNA segments were screened for sequence variations with the appropriate 6 endonucleases. Restriction endonuclease digestion For each PCR fragment, 200 ng of amplified mtDNA was digested for 12 hr at 37°C with the HpaI, BamHI, HaeII, MspI, AvaII, HincII restriction enzymes. The conditions and buffers used were those recommended by the manufacturer (Eurogentec, Sera- ing, Belgium). Electrophoresis of mtDNA The size and quantity of the PCR products were confirmed by electrophoresis of 3 μl of amplified DNA on 0.7% agarose gels. The resulting restriction fragments were resolved according to size by Grant sponsor: Association pour la Recherche sur le Cancer; Grant number: 95-1402. *Correspondance to: Laboratoire d’Immunologie et d’Histocompatibilite, Ho ˆpital St Louis, 1 Ave Cl Vellefaux, Paris Cedex 75475, France. Fax: (33)1-4249-4889. E-mail: reni@histo.chu-stlouis.fr Received 22 December 1997; Revised 16 January 1998 Int. J. Cancer: 76, 495–498 (1998)  1998 Wiley-Liss, Inc. Publication of the International Union Against Cancer Publication de l’Union Internationale Contre le Cancer