6639.Full-Text.Pdf

[CANCER RESEARCH 62, 6639–6644, November 15, 2002] Methylthioadenosine Phosphorylase, a Gene Frequently Codeleted with p16cdkN2a/ARF, Acts as a Tumor Suppressor in a Breast Cancer Cell Line1

Scott A. Christopher, Paula Diegelman, Carl W. Porter, and Warren D. Kruger2 Fox Chase Cancer Center, Division of Population Science, Philadelphia, Pennsylvania 19111 [S. A. C., W. D. K.], and Roswell Park Cancer Institute, Pharmacology and Therapeutics Department, Buffalo, New York 14263 [P. D., C. W. P.]

ABSTRACT leukemic cells also lack MTAP activity (15). These enzymatic studies also reported MTAP deficiency in some cell lines derived from solid The human methylthioadenosine phosphorylase (MTAP) gene is lo- tumors including melanoma and breast cancer (11, 16). These early cated on 9p21 and is frequently homozygously deleted, along with studies were hampered by the lack of commercial availability of the p16cdkN2a/ARF, in a wide variety of human tumors and human tumor- derived cell lines. The function of MTAP is to salvage methylthioad- radiochemical substrate. However, this problem was partially over- enosine, which is produced as a byproduct of polyamine metabolism. We come with the development of polyclonal MTAP antiserum. Using have reintroduced MTAP into MCF-7 breast adenocarcinoma cells and this antibody, it was shown that loss of MTAP was a very common have examined its effect on the tumorigenic properties of these cells. occurrence in glioblastomas and non-small cell lung cancer (17, 18). MTAP expression does not affect the growth rate of cells in standard Because loss of MTAP expression in tumors appears to be mostly the tissue culture conditions but severely inhibits their ability to form colonies result of homozygous deletions of the MTAP gene (see below), more in soft agar or collagen. In addition, MTAP-expressing cells are sup- recent studies have examined loss of MTAP DNA in various cancers. pressed for tumor formation when implanted into SCID mice. This sup- About one-third of all acute lymphoblastic leukemia patients have a pression of anchorage-independent growth appears to be because of the enzymatic activity of MTAP, as a protein with a missense mutation in the homozygous deletion for the gene encoding MTAP (19, 20). Genetic active site does not exhibit this phenotype. MTAP expression causes a studies show high rates of MTAP loss in non-small cell lung cancer, significant decrease in intracellular polyamine levels and alters the ratio of melanoma, bladder cancer, pancreatic, osteosarcoma, and endometrial putrescine to total polyamines. Consistent with this observation, the poly- cancer (21–24). amine biosynthesis inhibitor ␣-difluoromethylornithine inhibits the ability MTAP was initially mapped to human chromosome 9p, and later of MTAP-deficient cells to form colonies in soft agar, whereas addition of studies refined this region to 9p21–22 (see Fig. 1B; Refs. 25, 26). This the polyamine putrescine stimulates colony formation in MTAP-express- region of the human genome is especially interesting because it is ing cells. These results indicate that MTAP has tumor suppressor activity homozygously deleted in a variety of cancers, including gliomas, and suggest that its effects may be mediated by altering intracellular polyamine pools. melanomas, pancreatic, non-small cell lung cancers, mesothelioma, and acute leukemias (26–28). These deletions are unusual in that the INTRODUCTION majority of them are quite large, eliminating multiple genes including MTAP. A detailed study of Ͼ500 primary tumors by Cairns et al. (29) A quarter century ago, Toohey (1) first recognized that certain found that almost all of the deletions remove a 170 kb region, which murine malignant hematopoietic cell lines lacked MTAP3 activity. includes at least three transcripts: MTAP, CDKN2A, and p14ARF. MTAP is a key enzyme in the methionine salvage pathway (see Fig. CDKN2A (also known as p16ink4a/MTS1/INK4) encodes p16, an 1). This pathway functions to salvage MTA, which is formed as a inhibitor of the cyclin D-dependent kinase cdk4 (30). Germ line point by-product of polyamine metabolism. Phosphorylation of MTA by mutations in CDKN2A are associated with familial multiple mela- MTAP results in the conversion of MTA into adenine and MTR1P. A noma (31, 32), indicating that it is a tumor suppressor gene. The series of reactions then salvages the methyl-thio group from MTR1P P14ARF gene is an alternatively spliced transcript of CDKN2A (33). to form methionine. This pathway has been most extensively studied The first exon (1␤) is not shared with CDKN2A, but exons two and in Klebsiella pneumoniae (2–5), but has also been shown to exist in rat three are read in an alternative reading frame. Thus, the p14ARF liver (6–8) and in Saccharomyces cerevisiae (9, 10). MTAP, the first protein is entirely unrelated to the p16 protein. Molecular studies have enzyme in the pathway, is found in cells derived from a variety of established that p14ARF binds to and inactivates MDM2, preventing different tissues and appears to be expressed in all normal human the degradation of p53 (34). Thus in the absence of p14ARF, p53 is tissues (11, 12). These facts suggest that the entire salvage pathway is destabilized, and unable to function in growth arrest and apoptosis. present in all of the cells in the human body. The deletion of these two genes causes dysregulation of the two Early studies on human leukemia cell lines revealed that loss of pathways, Rb and p53, that are thought to be important in most ϳ MTAP activity was relatively common ( 30%; Refs. 13, 14). This cancers, and has therefore led to the assumption that loss of MTAP loss does not appear to be an artifact of cell culture as primary activity is incidental and not of pathogenic consequence (35). How- ever, there are several reasons to suspect that this is not the case. First, Received 5/29/02; accepted 9/11/02. homozygous deletion is an unusual mechanism for inactivation of a The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with tumor suppressor gene. Most tumor suppressor genes are inactivated 18 U.S.C. Section 1734 solely to indicate this fact. by point mutation of one allele followed by loss of the other allele 1 Supported by United States Army Grant DAMD17-97-1-7707, USPHS Grant CA- 22153, and Core Grant CA-06927 from the NIH, and by an appropriation from the (LOH). This is rarely observed for CDKN2A (29). Why is this the Commonwealth of Pennsylvania. case? A reasonable hypothesis is that homozygous deletion can re- 2 To whom requests for reprints should be addressed, at Fox Chase Cancer Center, move more than one gene from the region, whereas point mutation 7701 Burholme Avenue, Philadelphia, PA 19111. Phone: (215) 728-3030; Fax: (215) 214- 1623; E-mail: [email protected]. followed by LOH cannot. (It should be noted that a single point 3 The abbreviations used are: MTAP, methylthioadenosine phosphorylase; MTA, mutation can knock out both p16 and ARF because they share exons methylthioadenosine; MTR1P, methylthioribose-1-phosphate; ODC, ornithine decarboxylase; DFMO, ␣-difluoromethylornithine; LOH, loss of heterozygosity; BrdUrd, bro- 2 and 3). Second, in certain cancers, loss of MTAP has been observed modeoxyuridine. in cells that retain p16. Schmid et al. (21) found in a study of 6639

reaction was preincubated to remove trace contaminants of adenine. Adenine release rates were linear with protein concentrations up to 250 ␮g protein under these conditions. A unit of MTA phosphorylase activity is defined as the enzyme amount that catalyzes the formation of 1 ␮mol of adenine/min under the conditions of the assay described above. Cell Growth Assays. Growth rates in standard tissue culture conditions were measured by measuring BrdUrd incorporation into cellular DNA using an ELISA assay according to manufacturer’s instructions (Roche Biomedical). Cells were plated at a density of 1000 cells/well in 96-well plates and allowed to grow for 4 days before being labeled with BrdUrd for 18 h. Soft Agar and Collagen Growth. Soft agar was prepared by dissolving

1% Seaplaque agarose (BioWhittaker, Rockland, ME) in double-distilled H2O at 65°C. A base layer counting 5 ml of 0.5% agarose and 1ϫ medium per 60-mm plate was solidified at 4°C for 15 min. A cell layer containing 104 cells in 0.33% agarose in 3 ml of medium was solidified at 4°C for 15 min, then

incubated at 37°C under 5% CO2. Cells were fed at 7-day intervals with an overlay of 0.33% agarose by adding 1 ml, chilling at 4°C, and returning to the incubator. After 21 days the number of colonies was determined by counting Fig. 1. A. Polyamine biosynthetic pathway. Metabolites are in bold lettering and under a low-power microscope. enzymes are in italics. B, map of chromosome 9p21 region. Exons are indicated by Collagen gel was prepared as described by Russo et al. (39). In brief, a basal vertical lines. layer of 300 ␮l 80% vitrogen 100 (Cohesion tech) collagen gel was solidified at 37°C for 30 min. A second layer of 500 ␮l collagen gel was solidified for 1 h under the same conditions. Cells (2500) were overlaid on collagen gel in non-small cell lung cancer that homozygous deletion of MTAP oc- a 1.5-ml suspension of culture medium. curred in 38% (19 of 50) of samples compared with only 18% (9 of Tumor Formation in Mice. Cells were grown in culture, trypsinized using 50) for p16. In another report, it was found that 3 of 7 primary 0.04% trypsin, washed in medium and 2ϫ PBS, and resuspended to a con- 8 astrocytomas were deleted for MTAP, but only 2 of 7 were deleted for centration of 10 cells/ml. One-hundred ␮l of each suspension was inoculated p16 (36). The fact that MTAP is lost independently of p16 hints that in 16–18-gram athymic SCID mice implanted with 0.25 mg 21-day release s.c. ␤-estradiol pellets (Innovative Research of America, Sarasota, FL). Tumors loss of MTAP may have some functional basis in tumor biology. were detected by palpation and quantitated by measuring with calipers in three In this paper we examine the effect of reintroduction of MTAP to dimensions. a MTAP-deleted tumor cell line. Our results indicate that MTAP acts Polyamine Measurements. Intracellular polyamines were extracted from as a tumor suppressor and may exert its effect through altering cell pellets with 0.6 N perchloric acid, dansylated, and measured by reverse- polyamine metabolism. phase high-performance liquid chromatography as described (40).

MATERIALS AND METHODS RESULTS

Plasmids and Site-directed Mutagenesis. The plasmid pTre2:MTAP was It was shown previously by our group that MCF-7 cells lack MTAP created by cloning the MTAP containing BamHI/EcoRV fragment from pCR: activity and are deleted for markers in 9p21 (37). We transfected sMTAP (37) into pTre2 (Clontech, Palo Alto, CA). The MTAP D220A mutant MCF-7 cells with a construct that would express MTAP under a was created from pTre2:MTAP by site-directed mutagenesis using the Tet-repressible promoter and isolated several MTAP expressing QuikChange XL site-directed mutagenesis kit (Stratagene, La Jolla, CA). The clones (Fig. 2A; Table 1). These clones expressed MTAP at levels that primers used to engineer the mutation were: 5Ј-ATGGCGACAGCTTAT- were similar to a control fibroblast cell line. When the tetracycline GACTGC-3Ј and 5Ј-GCAGTCATAAGCTGTCGCCAT-3Ј. Cell Line and Stable Transfectants. MCF-7 Tet-off cells (Clontech) were analogue, doxycycline, was added, two of the lines showed a decrease cultured in Clontech-recommended medium using heat-inactivated fetal bo- vine serum and supplemented with 250 ␮g/ml G418. Transfections were performed using Fugene6 reagent (Roche, Indianapolis, IN), a nonliposomal transfection agent according to the manufacturer’s instructions. A total of 2 ␮g of plasmid DNA in a 10:1 ratio of pTK-Hyg to pTRE2-MTAP was used for each transfection. Clones were selected using 250 ␮g/ml Hygromycin from a 50 mg/ml stock solution in PBS (Sigma). After 3 weeks individual colonies were subcloned, expanded, and tested for MTAP expression by Western blot. Western Analysis and Enzyme Assays. Cell lysates were prepared by Ϫ three cycles of freeze-thawing at 80°Cin20mM KH2PO4 (pH 7.4) contain- ing1mM phenylmethylsulfonyl fluoride (Sigma) and 1 mM DTT (Sigma). Lysates was suspended in 1ϫ SDS sample buffer and separated by SDS- PAGE. Proteins were then transferred onto an Immobilon P membrane (Mil- lipore Corp., Bedford, MA). The primary antibody used in this study was MTAP antibody produced from chicken yolk (from the laboratory of Dennis Carson, University of San Diego, San Diego, CA). MTA phosphorylase activity was measured by a modification of a method described previously by Savarese et al. (38). In this assay the liberation of Fig. 2. A, SDS-PAGE/Western analysis of stable MTAP-expressing clones. The indicated clones were grown either in the presence or absence of doxycycline for 48 h, adenine by MTAP is monitored by its conversion to 8-dihydroxyadenine by cells were harvested, and extracts prepared. After SDS-PAGE, the extracts were trans- xanthine oxidase. This results in an increased absorbance at 305 nm. Assays ferred into membrane and probed with anti-MTAP serum, as described. WS1 is a were performed using an Agilent 8453 multidiode array spectrophotometer at nontransformed human fibroblast line. B, native-gel/Western analysis of MTAP-expressing and D220A-mutant lines. Ten ␮g of total cell extract from the indicated cell lines were 37°C, with cuvettes containing a total volume of 1 ml of 40 mM potassium loaded on a 14% native polyacrylamide gel. After electrophoresis, proteins were trans- phosphate (pH 7.4), 0.8 units of xanthine oxidase, 0.5 mM MTA, and crude cell ferred to membrane and probed with anti-MTAP antibody. The slight difference in extracts (100–250 ␮g protein). Before the addition of xanthine oxidase, the migration between D220A versus wild-type is expected because of the change in charge. 6640

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2002 American Association for Cancer Research. MTAP AS A TUMOR SUPPRESSOR IN BREAST CANCER CELLS in enzyme levels and activity, but MTAP was still expressed at Table 2 Suppression of colony formation by MTAP on soft agar detectable levels. We also transfected a construct expressing the The 10,000 cells were plated on 60-mm dishes in soft agar, and colony formation was assessed after 21 days by screening under a microscope with ϫ40 magnification. A grid luciferase gene as a negative control. pattern was placed in the back of each plate to aid in the counting process. The rows below We compared the growth rates in standard tissue culture conditions the dark line are from a separate experiment. (anchorage-dependent growth) of three MTAP-expressing and non- Cells # Plated # Colonies % Colony formation expressing subclones using BrdUrd labeling. We observed no signif- MCF-7 parent 10,000 2118 21.2 icant difference in BrdUrd uptake between MTAP-expressing and Luc1 10,000 1979 19.8 nonexpressing cells (Fig. 3). We next tested the ability of MTAP- Luc2 10,000 2214 22.1 MTAP1 10,000 15 0.15 expressing and nonexpressing cells to exhibit anchorage-independent MTAP2 10,000 13 0.13 growth by examining their ability to form colonies in soft agar (Table MTAP4 10,000 0 0 Ϫ ϩ 2). Approximately 20% of the cells from the three MTAP control MTAP4 Dox 10,000 0 0 MTAP8 10,000 2 0.2 lines formed colonies on soft agar, whereas we observed almost no MTAP8 ϩ Dox 10,000 0 0 colonies forming from three different MTAPϩ lines. The MTAP- WS1 (fibroblast) 10,000 0 0 expressing lines also failed to form colonies when doxycycline was MTAP4 3610 0 0 added to the medium, indicating that low levels of MTAP were MTAP8 3020 0 0 MTAP9 3660 0 0 sufficient to suppress anchorage-independent growth. These experi- D220A4 2410 214 8.9 ments show that MTAP suppresses anchorage-independent growth D220A5 4000 502 12.6 but does not affect anchorage-dependent growth. D220A6 1670 181 11.6 We next tested whether MTAP enzymatic activity was necessary for suppression of anchorage-independent growth or whether the MTAP protein might have some additional nonenzymatic function. The D220A mutant cell lines were next tested for their ability to On the basis of the crystal structure of human MTAP, aspartate 220 is form colonies on soft agar (Table 2) and on collagen (Fig. 4). All three hypothesized to play a key role in the catalytic mechanism (41). of the cell lines were able to form colonies in both soft agar and Therefore, we mutated this residue to alanine (D220A) and isolated collagen, whereas the MTAP-expressing cells were not. In addition, three stable lines that express the D220A. Western analysis using we tested the ability of three MTAP-expressing and two D220A- native PAGE shows that the mutants are expressed and form a expressing cell lines to form tumors in SCID mice (Fig. 5). After 30 multimer that migrates similar to wild-type MTAP (Fig. 2B). Also, days, measurable tumors appeared on the mice implanted with the extracts from these D220A-expressing cells had no detectable MTAP D220A-expressing cells, and these tumors grew rapidly until the mice activity (Table 1). These results show that we have created a stable had to be sacrificed. No measurable tumors appeared in animals MTAP protein that is catalytically inactive. injected with the MTAP-expressing cells. These results show that MTAP enzymatic activity is required for the suppression of anchor- Table 1 MTAP activity in stable transfectant age-dependent growth and that MTAP can suppress tumor formation MTAP activity was determined as described in “Materials and Methods.” in vivo. Cell line Dox Activity (units/mg extract) Cells lacking MTAP would be expected to build up the polyamine byproduct MTA (Fig. 1). MTA has been shown in vitro to be an MCF-7 ϪϽ0.05 MCF-7 ϩϽ0.05 inhibitor of spermine synthase and spermidine synthase, two key MTAP4 Ϫ 1.09 enzymes in polyamine biosynthesis. Inhibition of these enzymes MTAP4 ϩ 0.25 MTAP8 Ϫ 1.48 would be expected to cause an increase in putrescine inside MTAP- MTAP8 ϩ nta deficient cells. Therefore, we tested the hypothesis that MTAP ex- MTAP9 Ϫ 4.14 pression might affect polyamine levels and distribution. We measured MTAP9 ϩ 1.35 D220A4 ϪϽ0.05 intracellular putrescine, spermidine, and spermine levels in three D220A5 ϪϽ0.05 D220A and three MTAP-expressing cell lines (Table 3). As expected D220A6 ϪϽ0.05 putrescine levels were elevated in D220A cells relative to MTAP- WS1 0.86 expressing cells (3285 versus 571; P Ͻ 0.00001). However, unex- a nt, not tested. pectedly we found that spermine and spermidine levels were also significantly elevated (3225 versus 1126; P Ͻ 0.00001), although the ratio of putrescine to the total polyamine pools was still significantly different between the MTAPϩ and MTAPϪ cells (0.19 versus 0.32; P Ͻ 0.001). These findings show that cells lacking MTAP have increased polyamine pools and suggests that there may be feedback regulation of polyamine biosynthesis by a downstream product in the salvage pathway. We next tested the effects of putrescine (2 mM) and DFMO (50 ␮M) on anchorage-independent growth of three MTAPϩ and three MTAPϪ cell lines (Fig. 4; Table 4). DFMO is an inhibitor of ODC and has been shown to cause depletion of intracellular polyamines (42). At the concentrations used there was no effect on growth rate of either MTAPϩ or MTAPϪ cells as judged by BrdUrd uptake in standard tissue culture conditions (anchorage-dependent growth; data not shown). However, DFMO severely inhibited colony formation Fig. 3. BrdUrd incorporation of MTAPϩ and MTAPϪ cell lines. BrdUrd incorporation MTAPϪ cells on soft agar, and this inhibition could be reversed by the of three MTAP-expressing and nonexpressing cell lines was determined by ELISA assay. addition of putrescine. Putrescine alone did not appear to have any The plot shows the mean (dot) and SE (shaded box) of the three measurements; Ϫ bars, Ϯ SD. additional effect on colony formation in the MTAP cells. Addition 6641

Fig. 4. Representative example of colony formation on collagen gel. Shown above are portions of a collagen gel in which either MCF-7 cells are expressing D220A (above) or wild-type MTAP (below). Pictures were taken after 12 days under ϫ40 magnification. Along the middle is the indicated treatment: Ϫ, no treatment; DFMO, 50 ␮M DFMO; PUT, 2mM putrescine; PUTϩDFMO, both PUT and DFMO. Results are nearly identical for other MTAP-expressing and nonexpressing lines.

gous deletions are so common in this chromosomal region in tumors. Most tumor suppressor genes are inactivated by mechanisms involv- ing point mutation of one copy followed by LOH of the other allele. The 9p21–22 region is unusual because LOH is relatively rare, but homozygous deletion is not. To inactivate three genes by point mutation would be difficult, but all of the three genes could be inactivated by a large deletion event. Consistent with this idea, a large mapping study of 545 primary tumors shows that tumors containing homozygous deletions of 9p21 have a minimal 170-kb region deleted that includes both MTAP and p16 (29). Our data suggest that MTAP may suppress anchorage-independent growth via its effect on polyamine metabolism. We found that MCF-7 cell lines expressing MTAP had significantly lower levels of all three major polyamines and had a reduced ratio of putrescine to total polyamines. We also found that addition of putrescine to MTAP- expressing MCF-7 cells stimulated their ability to grow on soft agar. Fig. 5. Tumor growth in SCID mice. One ϫ 107 of the indicated cell line was injected Conversely, the ODC inhibitor, DFMO, inhibited anchorage-indepen- s.c. behind the shoulder blades into three female SCID mice implanted with slow release dent growth of MTAPϪ cells. These results suggest that elevated estrogen pellets. Mice were monitored for a maximum of 56 days, but mice injected with the D220A5 and D220A6 had to be euthanized early because of the large size of the levels of polyamines, especially putrescine, are correlated with an- tumors. The bars show the SD between the different mice. chorage-independent growth. Another way to elevate polyamine pools is to overexpress ODC.

of putrescine to MTAPϩ cells resulted in the stimulation of colony Ϫ ϩ formation and anchorage-independent growth. Taken together, these Table 3 Polyamine levels in MTAP and MTAP cells experiments demonstrate that elevated putrescine is required for an- Each of the cell lines was grown in triplicate in separate tissue culture dishes, and polyamine levels were determined as described in “Materials and Methods.” The number Ϫ ϩ chorage-independent growth and are consistent with the hypothesis shown is the average of the three experiments. The MTAR and MTAP averages are Ϫ that at least some of the tumorigenic effects of MTAP are because of from all three cell lines (nine different measurements). The difference between MTAP ϩ its influence on polyamine levels. and MTAP cells are all highly significant (P Ͻ 0.001). Putrescine Spermidine Spermine Putrescine/ (␮moles/ (␮moles/ (␮moles/ total DISCUSSION Cell line 106 cells) 106 cells) 106 cells) polyamins Ϫ D220A4 (MTAP ) 4.0 4.8 4.1 0.31 Ϫ In this report we demonstrate that when MTAP is expressed in D220A5 (MTAP ) 2.9 3.5 2.7 0.31 Ϫ MTAP-deficient MCF-7 cells, it results in a suppression of anchorage- D220A6 (MTAP ) 3.0 3.3 2.9 0.33 Ϫ independent growth in vitro and tumor formation in vivo. We saw no MTAP /Avg. 3.3 3.9 3.2 0.32 MTAP 4 0.7 1.7 0.9 0.21 effect of MTAP expression on growth rate under standard tissue MTAP 8 0.4 1.1 1.2 0.16 culture conditions, indicating that this effect is not a general effect on MTAP 9 0.6 1.6 1.3 0.21 ϩ cell-cycle, but rather appears to be specific to growth in an anchorage- MTAP Avg. 0.6 1.5 1.1 0.19 independent environment. This phenomenon is unusual, as most cell cycle-related tumor suppressor genes affect growth under both con- Table 4 Effect of putrescine and DFMO on colony formation ditions (43–45). However, our observation is not unprecedented, Cells (10,000) of the indicated lines were plated in soft agar with medium containing ␮ because certain oncogenes, such as Rho and Bcr-Abl, allow anchor- nothing, 50 M DFMO, or 2 mM putrescine. Colony formation was assessed after 21 days. age-independent growth but have limited effect on cell proliferation Cell line ϩ treatment Colonies formed % Colonies/total (46). MCF7 ϩ none 2198 22 MCF7 ϩ DFMO 221 2.2 On the basis of our data, MTAP is the third protein identified in the MCF7 ϩ PUT 2126 21 9p21–22 region with tumor suppressor activity. The two other con- MTAP4 ϩ none 91 0.9 firmed suppressor proteins are p16 and p14ARF. Each protein appears MTAP4 ϩ DFMO 0 0 MTAP4 ϩ PUT 397 4.0 to affect different cellular functions important in transformation: p16 MTAP8 ϩ none 77 0.77 affecting cell-cycle, p14ARF affecting apoptosis, and MTAP affecting MTAP8 ϩ DFMO 0 0 anchorage dependence. Our findings may help explain why homozy- MTAP8 ϩ PUT 458 4.58 6642

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Detection of methylthioadenosine phosphorylase Ϫ (MTAP) and p16 gene deletion in T cell acute lymphoblastic leukemia by real-time number of different cancers (53, 54). Our data suggest that MTAP quantitative PCR assay. Leukemia (Baltimore), 14: 935–940, 2000. tumors may be particularly sensitive to DFMO. We found that DFMO 21. Schmid, M., Malicki, D., Nobori, T., Rosenbach, M. D., Campbell, K., Carson, D. A., inhibited anchorage-independent growth at concentrations that had no and Carrera, C. J. Homozygous deletions of methylthioadenosine phosphorylase (MTAP) are more frequent than p16INK4A (CDKN2) homozygous deletions in effect on growth rates in standard tissue culture conditions. It may be primary non-small cell lung cancers (NSCLC). Oncogene, 17: 2669–2675, 1998. worthwhile stratifying the ongoing trials for MTAP status of the 22. Della Ragione, F., Russo, G., Oliva, A., Mastropietro, S., Mancini, A., Borrelli, A., primary tumor to determine whether DFMO treatment might be more Casero, R. A., Iolascon, A., and Zappia, V. 5Ј-Deoxy-5Ј-methylthioadenosine phosphorylase and p16INK4 deficiency in multiple tumor cell lines. Oncogene, 10: effective in MTAP-deleted tumors. 827–833, 1995. 23. Stadler, W. M., Sherman, J., Bohlander, S. K., Roulston, D., Dreyling, M., Rukstalis, D., and Olopade, O. I. Homozygous deletions within chromosomal bands 9p21–22 in bladder cancer. Cancer Res., 54: 2060–2063, 1994. ACKNOWLEDGMENTS 24. Garcia-Castellano, J. M., Villanueva, A., Healey, J. H., Sowers, R., Cordon-Cardo, C., Huvos, A., Bertino, J. R., Meyers, P., and Gorlick, R. Methylthioadenosine We thank Dr. Dennis Carson for the use of his anti-MTAP antiserum, phosphorylase gene deletions are common in osteosarcoma. Clin. 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Scott A. Christopher, Paula Diegelman, Carl W. Porter, et al.

Cancer Res 2002;62:6639-6644.

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