6-THIOGUANINE RESISTANCE IN A HUMAN COLON CARCINOMA CELL LINE WITH UNALTERED LEVELS OF HYPOXANTHINE GUANINE PHOSPHORIBOSYLTRANSFERASE ACTIVITY Valentina BEMI 1 , Gino TURCHI 2 , Elisa MARGOTTI 1 , Francesco GIORGELLI 1 , Rossana PESI 1 , Francesco SGARRELLA 3 , Maria Grazia TOZZI 3 and Marcella CAMICI 1 * 1 Dipartimento di Fisiologia e Biochimica, Pisa, Italy 2 Istituto di Mutagenesi e Differenziamento del CNR, Pisa, Italy 3 Dipartimento di Scienze del Farmaco, Sassari, Italy Cell populations resistant to high doses (30 M) of 6-thio- guanine (6-TG, 6-TG r cells) were selected from a human colon carcinoma cell line, LoVo. This cell line, which lacks hMSH2, a component of the human mismatch binding het- erodimer hMutS, is resistant to low doses of 6-T G. T he level of activity of hypoxanthine-guanine phosphoribosyltransfer- ase, the enzyme responsible for the phosphoribosylation of the thiopurine, was comparable to that expressed in the parental cells. N o significant difference was found in the levels of enzyme activities involved in the conversion of 6-TG or its derivatives into non-toxic compounds. In contrast, a signifi- cant difference wasfound in the uptake kineticsof 6-T G in the 2 cell types. Net uptake of 6-TG ceased after 100-sec incuba- tion in the 6-T G r cells, while it appeared to continue through- out the 10-min incubation in the wild-type cells. As a conse- quence, after 10-min incubation, the total amount of 6-TG taken up by the parental LoVo cells was approximately 3 times higher than that present in the 6-T G r cells. Int. J. Cancer 82:556–561, 1999.  1999 Wiley-Liss, Inc. The thiopurine 6-thioguanine (6-TG) has been used as a chemotherapeutic agent for the treatment of childhood and adult leukemias since the 1950s. The mechanism of 6-TG cytotoxicity in proliferating cells involves conversion of the thiopurine to the nucleotide by the purine salvage pathway, followed by incorpora- tion into DNA, instead of dGTP (Uribe-Luna et al., 1997). Incorporation of 6-TG into DNA appears to prevent its replication since modified DNA is a poor template for DNA polymerases and a poor substrate for several DNA ligases (Ling et al., 1992). Therefore, the cytotoxic effect of 6-TG appears to be strictly dependent on its conversion to the nucleotide by hypoxanthine- guanine phosphoribosyltransferase (HGPRT); absence of this en- zyme activity represents one of the best characterized mechanisms of resistance (Lennard, 1992). Additional mechanisms of cytotoxic- ity have been indicated for 6-TG. The growth arrest induced by 6-TG in 6-mercaptopurine-resistant (HGPRT - ) human leukemia cells has been related to an epigenetic mechanism, where an alteration in tRNA queuine modification has been suggested (Morgan et al., 1994). Post-replicative DNA mismatch repair has been shown to play a relevant role in the mechanism of cytotoxicity of 6-TG (Swann et al., 1996; Waters and Swann, 1997). The observation that certain eukaryotic cells resistant to N-methyl-N- nitrosourea are also resistant to 6-TG (Aquilina et al., 1990) led to the conclusion that a common alteration was responsible for both phenotypes. Indeed, after incorporation into DNA, 6-TG is chemi- cally methylated to S 6 -methylthioguanine by S-adenosyl methio- nine (SAM) (Swann et al., 1996). S 6 -Methylthioguanine, during DNA replication, directs incorporation of either thymine or cyto- sine with roughly equal probability, and the S 6 -methylthioguanine- thymine pairs are recognized and bound by hMutS, the human mismatch binding heterodimer. In this regard, LoVo cells, lacking hMSH2, a component of the heterodimer which regulates the repair mechanism, have been described as resistant to low doses of 6-TG. Since the exact mode of action of 6-TG is not fully understood, the use of LoVo cells in experiments where the toxicity of the thiopurine is tested rules out the mismatch repair pathway as a possible mechanism and enables us to look for alternative mecha- nisms of toxicity. We report here the selection obtained at high doses of the thiopurine (30 μM) of a large population of 6-TG- resistant (6-TG r ) cells from LoVo and compare the enzymatic pattern and kinetics of uptake of the analog shown by parental and resistant cells. MATERIAL AND METHODS Material [8- 14 C]Hyp (55 mCi/mmol), [ 14 C-methyl]SAM (59 mCi/mmol), [8- 14 C]Gua (53.3 mCi/mmol), allopurinol, dithiothreitol, p- nitrophenylphosphate (p-NPP), 6-TG, Ham’s F-12 medium and xanthine oxidase (XOD) (EC 1.1.3.22) from milk were from Sigma (St. Louis, MO). PEI-cellulose thin-layer plastic sheets (0.1 mm thick) (Merck, Darmstadt, Germany) were pre-washed once with 10% NaCl and 3 times with deionized water before use. Scintilla- tion liquid HiSafe III was purchased from Pharmacia LKB (Uppsala, Sweden). Human colon carcinoma cell lines LoVo and HT29 were provided by Dr. O. Sanfilippo (Milan, Italy). FCS was from GIBCO (Berlin, Germany). All other chemicals were of analytical grade. Cell culture and preparation of cell extracts Cells were grown as monolayers in Ham’s F-12 medium with 7% FCS and antibiotics (standard medium) at 37°C in a humidified 5% CO 2 /95% air atmosphere. In our conditions, LoVo and HT29 cells have a cloning efficiency of 60% to 65% and a doubling time of 31.4 and 38.6 hr, respectively. For the preparation of cell extracts, when cells were in the logarithmic phase of growth, monolayers were washed twice with PBS and cells were scraped off with a cell scraper, collected and centrifuged at 800 g for 3 min. Pellets were washed with PBS and stored at –75°C. Cell pellets were resuspended in approximately 3 vol of 50 mM Tris-HCl (pH 7.4) supplemented with 10 mM dithiothreitol. The suspension was subjected to ultrasonic treatment and centrifuged at 40,000 g for 1 hr at 4°C. The supernatant (cell extract) was kept at –75°C. Selection of 6-TG r variant LoVo cells The 6-TG r variant was selected as previously described (Turchi et al., 1992). Briefly, 5  10 5 cells were seeded in 4 dishes for selection in the presence of the selective agent, the purine analog 6-TG. The drug was added to the dishes immediately after seeding to give a final concentration of 30 μM. The concentration of the selected drug was determined by the inhibition curve of LoVo cell growth in the presence of increasing concentrations of 6-TG (5 to 100 μM). At the dose of choice, a substantial part of the population, Grant sponsors: MURST (Italy); CNR; Target Project ‘‘Biotechnologie.’’ *Correspondence to: Dipartimento di Fisiologia e Biochimica, Via S. Maria 55, 56100 Pisa, Italy. Fax: +39–050–502583. E-mail: camici@dfb.unipi.it Received 30 November 1998; Revised 24 March 1999 Int. J. Cancer: 82, 556–561 (1999)  1999 Wiley-Liss, Inc. Publication of the International Union Against Cancer Publication de l’Union Internationale Contre le Cancer