Folate and Pterin Metabolism by Cancer Cells in Culture1

[CANCER RESEARCH 38, 2378-2384, August 1978) 0008-5472/78/0038-0000$02.00 Folate and Pterin Metabolism by Cancer Cells in Culture1

Baldassarre Stea, Peter S. Backlund, Jr., Phillip B. Berkey, Arthur K. Cho, Barbara C. Halpern, Richard M. Halpern, and Roberts A. Smith2

Departments of Chemistry (B. S., P. S. B., P. B. B., B. C. H.Â¡,Pharmacology Â¡A.K. C.], Medicine, Â¡Ft.M. H.]. and Chemistry [Ft. A. SJ and Molecular Biology Institute Â¡R.M. H., R. A. S.], University of California, Los Angeles, California 90024

ABSTRACT cells, a significant peak of radioactivity was observed in the blue-fluorescent region. This peak of radioactivity was ab Malignant cells grown in culture excrete into their sent in chromatograms of growth media of normal cells growth medium a folate catabolite that can be seen as a grown under the same conditions. blue-fluorescent region on paper chromatograms of such We tentatively identified this blue-fluorescent compound media. This folate catabolite has now been identified by as Pt-6-CHO,3 primarily on the basis of its inhibitory power paper chromatography, thin-layer chromatography, and toward xanthine oxidase. However, we later found that Pt- combined gas chromatography-mass spectrometry as 6- 6-CH2OH also is a potent inhibitor of the same enzyme hydroxymethylpterin and not as pterin-6-carboxaldehyde system. This prompted us to identify by unequivocal means as previously reported. the fluorescent folate catabolite characteristic of cultured Moreover, when pterin-6-carboxaldehyde was added to malignant cells. the growth medium of logarithmically growing malignant Here we report the definitive identification of this blue- cells, it was primarily reduced to 6-hydroxymethylpterin. fluorescent compound isolated from growth media of KB In contrast pterin-6-carboxylate was the principal product cells as Pt-6-CH2OH.We also show that Pt-6-CHO, a putative formed from added pterin-6-carboxaldehyde by normal catabolite in the cancer cell-mediated cleavage of folie established cell lines in culture. These results have been acid, is reduced to Pt-6-CH2OH. interpreted as indicative of a possible mechanism of We believe that the elucidation of pterin and folate catab folate catabolism in malignant cells. Folie acid or another olism in malignant cells, as opposed to normal cells grown folate derivative is oxidatively cleaved at the C-9-N-10 in tissue culture, may be of great value in determining a bond to yield pterin-6-carboxaldehyde as one of the prod metabolic difference that could be exploited to differentially ucts. This derivative is subsequently reduced to 6-hydrox and specifically inhibit malignant growths. Moreover, as we ymethylpterin, which is excreted into the growth medium. have already suggested (12), monitoring for the levels of the highly fluorescent products of folate catabolism in body INTRODUCTION fluids may have a wide application as a diagnostic tool in the detection of malignant diseases. Controversial views exist in the literature as to whether active folate catabolism takes place in humans and other mammals. When [2-14C]folic acid was administered p.o. to MATERIALS AND METHODS rats (3), radioactivity in the urine could be identified only as Tissue Culture. Cells were usually grown in McCoy's folate metabolites in which the C-9-N-10 bond had re Modified Medium 5A (Grand Island Biological Co., Grand mained intact. No radioactive pterins were detected in the Island, N. Y.) containing Bacto-peptone (600 mg/liter), 15% first 24-hr urine samples following administration of the [2- undialyzed PCS, and gentamicin (50 /^g/ml). In some exper 14C]folic acid. However, a more recent study (19), in which rats received tracer doses of [3',5',9-3H]folic acid i.m., has iments, modifications and additions were made to this basic medium as specified in each case. All cultures were shown shown that after a period of equilibration of the tracer with to be concurrently pneumonia-like organism-negative by the folate pool the principal urinary catabolite is acetami- culture for 3 weeks in Grand Island Biological Co. broth dobenzoylglutamate. This finding suggests that active fol and pour plates. Of the cell lines used, the BALB/c3T3 ate catabolism takes place in vivo via cleavage of the C-9- fibroblasts and the SV40-transformed 3T3 cells (SV3T3) N-10bond. were kindly supplied by Dr. J. M. Jordan, UCLA; the We have previously shown (12) that malignant cells cata- benzo(a)pyrene-transformed 3T3 cells were a gift from Julie bolize folie acid yielding a compound visible as a blue- Baldwin, The Salk Institute, La Jolla, Calif. fluorescent region on paper chromatograms of their growth Determination of Cell Growth. Growth rates were esti media. At the limit of detection of paper chromatography, mated either by cell counts or by the following procedure. we could not see the same blue-fluorescent region in The cells left in the flasks after the medium had been chromatograms of growth media of normal cells. When [2- removed were carefully rinsed 4 times with cold 0.9% NaCI 14C]folate was added to the growth media of malignant solution, and the amount of protein remaining in the flask was determined by the method of Lowry ef a/. (18) with 1 This investigation was supported by the USPHS Grant CA 17332 and the bovine serum albumin as the standard. Julius and Dorothy Fried Research Foundation, Department of Chemistry Contribution 3926. 3 The abbreviations used are: Pt-6-CHO, pterin-6-carboxaldehyde; Pt-6- 1 To whom requests for reprints should be addressed at the Department CHjOH, 6-hydroxymethylpterin; PCS, fetal calf serum; HPLC, high-pressure of Chemistry, University of California, Los Angeles, Calif. 90024. liquid chromatography; Pt-6-COOH, pterm-6-carboxylic acid; TLC, thin-layer Received December 27, 1977; accepted April 24, 1978. chromatography.

2378 CANCER RESEARCH VOL. 38

Analysis of Media. In all cases, growth media were Table 1 centrifugea in an unrefrigerated International Model V cen TLC of Pt-6-CHO and Pf-6-CHjOH trifuge at 2500 rpm for 3 min to remove floating cells. R,CompoundR-6-CHO Isopropyl alcohol was then added to the supernatant to a final concentration of 70% (v/v). This solution was stirred at 4Â°for at least 2 hr before the precipitated proteins were System1Â°0.32 System20.17 System30.43 System40.62 removed by centrifugaron. The isopropyl alcohol was then Pt-6-CHzOHSolvent 0.43Solvent 0.32Solvent 0.42Solvent 0.32 removed by 3 successive extractions with 6 volumes of " Solvent systems: 1, 3% NH4CI; 2, 5% citrate (adjusted to pH 9 toluene. The remaining aqueous phase was either filtered with concentrated NH4OH); 3, CH^Ishisopropyl alcohol:NH4OH:H2O through a Millipore filter and directly chromatographed by (7:1:1:1); 4, ethyl acetate :n-propyl alcohohHjO (4:1:2), upper HPLC or lyophilized and then redissolved in a small volume phase. of 0.1 N NH,OH as specified later in each case. HPLC was performed on a 25-cm x 4.6-mm Partisil 10- POPOP, 120 g naphthalene per liter of 1,4-dioxane). Count SCX column (Whatman, Inc., Clifton, N. J.) with an Altex ing was performed with a Beckman LS-250 or LS-100 Model 100 pump equipped with an Altex Model 904-42 sam scintillation counter. Gas chromatography-mass spectrom- ple injector (Altex Scientific, Inc., Berkeley, Calif.). Isocratic etry was performed with a Hewlett-Packard 5981A instru elution of the column was performed with 5 mM H3PO4 at a ment equipped with a 2-ft x 2-mm column of 3% OV-101 on flow rate of 3 ml/min. Under these conditions, Pt-6-COOH, 100 to 200 mesh Gas-chrom Q (Applied Science Labs, Inc., Pt-6-CHO, and Pt-6-CH2OH were separated as reported Inglewood, Calif.). Helium was used as the carrier gas (20 elsewhere.4 The eluant was detected with a Farrand A4 ml/min), and the eluting temperature was 200Â°. fluorometer equipped with a 7-60 excitation filter (band Chemicals. [2-'4C]Folate (specific activity, 58.2 mCi/ center at 360 nm) and a 3-72 emission filter with a cutoff at mmol) was purchased from Amersham/Searle Corp. (Ar 450 nm (Farrand Optical Co., Inc., Valhalla, N. Y.). Concen lington Heights, III.), and [3',5',9-3H]folic acid (specific trations of various pterins in the growth media were deter activity, 24 Ci/mmol) was purchased from Schwarz/Mann mined from calibration curves obtained by plotting peak (Orangeburg, N. Y.). [2-14C]Pt-6-CHO was prepared from [2- area values versus concentrations. All of the standards 14C]folate by the method of Thijssen (24) and used without used in constructing the calibration curves were incubated further purification. This preparation was found to be con in McCoy's Modified Medium 5A containing 15% FCS for 36 taminated by small amounts of [2-14C]Pt-6-COOH and [2- hr at 37Â°before being subjected to the extraction procedure 14C]Pt-6-CH2OH probably originating by dismutation reac and the Chromatographie separation. Incubation of the tion of the aldehyde. Pt-6-COOH and buttermilk xanthine standards for longer time periods did not have further effect oxidase were purchased from Sigma Chemical Co. (St. on recoveries or retention times. Louis, Mo.) and used without further purification. Pt-6- Miscellaneous Methods. When media supplemented CH2OH was synthesized from Pt-6-CHO also by the method with folate were analyzed, all of the operations were carried of Thijssen (24), modifed as follows. Pt-6-CHO was sus out in semidarkness. Paper chromatography and TLC were pended in 5% NaHCO3 (w/v) and dissolved by dropwise always carried out in the dark, and the tanks were flushed addition of 2 N NaOH under a nitrogen atmosphere. After with argon before chromatograms were introduced. TLC reduction with NaBH4 the solution was filtered by gravity, was carried out on precoated cellulose plates (EM Labora and the filtrate was acidified to pH 5 with glacial acetic acid. tories, Inc., Elmsford, N. Y.; Catalog No. 5757-9H) with The precipitated Pt-6-CH2OH was washed with water, either System 1,3% NH4CI, or System 2, 5% citrate (adjusted ethanol, and ether; after decolorization with charcoal, the to pH 9 with concentrated NH4OH). Silica gel Woelm plates crude product was recrystallized from hot dilute HCI (0.01 (ICN Pharmaceuticals, Inc., Cleveland, Ohio; Catalog No. N). The Pt-6-CH2OH thus purified appeared as a single 04609) were also used and developed with either System 3, fluorescent spot on TLC with the 4 solvent systems de CH3CN:isopropyl alcohol:NH4OH:H2O (7:1:1:1); or System 4, scribed previously and as a single peak when it was chro the upper phase of ethyl acetate:n-propyl alcohol:H20 matographed on HPLC with the same solvent system used (4:1:2). RF's of standard Pt-6-CHO and Pt-6-CH2OH in these to analyze the media. 4 solvent systems are reported in Table 1. Paper chromatog raphy was carried out on Whatman No. 3MM paper with the RESULTS following 4 systems: System A, ferf-butyl alcohol:0.1 M ammonium acetate (7:3); System B, 1% NH4OH; System C, Purification of the Blue-fluorescent Compound Present ethanohacetic acid:H2O (30:2:68); System D, n-propyl alco- in the Growth Media of KB Cells by Paper Chromatography hol:1% NH4OH (2:1). When media supplemented with radio and Identification by TLC. When KB cells are grown in the active compounds were analyzed, the paper chromato presence of [2-14C]folate and their growth media are chro grams were sectioned into 1-cm rectangles and counted in matographed on paper with System A, a considerable peak a toluene-based scintillation fluid containing 4.0 g PPO and of radioactivity appears in correspondence to a blue-flu 0.005 g POPOP per liter. TLC plates were counted by orescent region visible on the chromatogram under UV. scraping the adsorbent layer directly into scintillation vials; However, chromatograms of media from normal cells or 0.75 ml of 1 N NH4OH was then added to each vial, followed control media (results not shown) incubated without cells by 5 ml of modified Bray's solution (4.0 g PPO, 0.05 g in the presence of [2-14C]folate do not show the character istic blue-fluorescent region or any significant peak of 4 B. Stea, R. M. Halpern, and R. A. Smith, manuscript in preparation. radioactivity in that same region of the chromatogram.

AUGUST 1978 2379

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1978 American Association for Cancer Research. B. Stea et al. authentic p-aminobenzoylglutamate in System A and was present in proportion roughly equivalent to that that would be expected from biological cleavage of folate at the C-9- N-10 bond. Similar results were obtained with human HT-29 cells, also a malignant cell line (results not shown). To obtain the radioactive blue-fluorescent compound from the growth medium of KB cells in sufficient amount for further characterization, 2.5 x 105 KB cells were grown in a 150-sq cm Corning flask containing 25 ml of reconsti tuted McCoy's Modified Medium 5A without added Bacto- peptone and unlabeled folate but supplemented with 2 /Â¿Ci of [2-14C]folate (specific activity, 38.8 mCi/mmol) and 15% undialyzed FCS. When the cells reached confluency, the Origin CM medium was collected, deproteinized, lyophilized, and re- ~ 50 dissolved in 1 ml of 0.1 N NH,OH; 0.5 ml of this solution was IO -Folie Acid b streaked on a large piece of Whatman No. 3MM paper and X chromatographed with System A. The blue-fluorescent 30 i band, visible under UV (R, 0.40), was excised, and the > radioactivity associated with it was eluted with 0.5 N NH,OH, K Blue Fluorescent Region > 90% of the radioactivity was removed with the first ml of Rf=033| eluate. This solution was directly used for TLC analysis on cellulose and silica gel plates. We found that the radioactiv ity eluted from the paper Chromatogram of KB cell growth i 0.5- medium comigrated with authentic Pt-e-CHjOH on either f cellulose plates or silica gel plates with 4 different solvent 10 20 JO 40 50 systems. Chart 2 exemplifies 1 of these TLC plates; similar Origin CM Front results were obtained with the other 3 solvent systems. Chart 1. Chromatogram of medium of KB cells grown in presence of both [2-"C]folate and [3',5 .9-'H]folate KB cells (1.5 â€¢¿10')were plated in a 25-sq Furthermore, the radioactive compound clearly had a differ cm Falcon flask and grown on the basic McCoy's Modified Medium 5A ent Chromatographie mobility from Pt-6-CHO in at least 3 of supplemented with 15% undialyzed FCS. 0.25 MCi of [2-14C]folate. and 0.5 the 4 solvent systems used (Table 1). /iCi of [3' ,5' ,9-3H]folate The medium was collected after 8 days of incubation and treated as described in "Materials and Methods." The residue obtained Identification of the Blue-fluorescent Compound by Pa after lyophilization was dissolved in 300 *J of 0.1 NNH.OH, and 150 Ml of this per Chromatography and Combined Gas Chromatogra- solution were chromatographed on Whatman No. 3MM paper with Solvent phy-Mass Spectrometry. A relatively large amount of the A. After development the Chromatogram was observed under UV in a blue-fluorescent compound was obtained by growing cells ChromatoVue box (Ultra-Violet Products, Inc., San Gabriel, Calif.) for detec tion of fluorescent areas. Only 1 fluorescent band with an RFof 0.41 was in roller bottles. KB cells (2.5 x 107) were inoculated in observed on the Chromatogram of the medium from KB cells. The distribu each of 4 roller bottles containing 100 ml of McCoy's tion of radioactivity on the Chromatogram was determined as described in "Materials and Methods," and counting was performed with a Beckman LS- Modified Medium 5A supplemented with 15% FCS. After a 250 liquid scintillation counter with the external standard set at 0.737, the growth period of 7 days, the medium was collected, depro gain at 2.68, and the Automatic Quench Compensation at 5.1. The cpm were teinized, and lyophilized. The residue obtained after lyoph converted to dpm as described in the Beckman Technical Bulletin (20) with Beckman radioactive standards. Top, distribution of the radioactivity due to ilization was redissolved in 50 ml of 5 mw sodium phosphate "C, bottom, distribution of the radioactivity due to 'H. The characteristic buffer (pH 7) and applied to an anion-exchange column fluorescent band observed in the Chromatogram of the KB cell medium and the peak of radioactivity corresponding to it were absent from the Chromato gram of a control medium without cells incubated simultaneously with the experimental flask in the presence of the same amount of [2-14C]folate and [3',5',9-JH]folate.

When KB cells were grown in a medium supplemented with [2-14C]folate and [2',5',9-3H]folate, the fluorescent metabo lite with the characteristic RF of 0.41 in System A was labeled with both 3Hand 14C(Chart 1). Under the conditions of the experiment described in Chart 1, nearly 4.5% of the 14Cand 3.5% of the 3H present on the Chromatogram were found under the peak corresponding to the blue-fluorescent region. In addition the ratio of 14Cto 3Hassociated with the Chart 2. TLC of the radioactive blue-fluorescent compound isolated from KB growth medium. A silica gel plate was streaked with 50 Â¿Â¿Iofthe fluorescent region was found to be greater than that found radioactive solution derived from the blue-fluorescent band of a paper in the folie acid region. This finding indicated to us that the Chromatogram of KB cell growth medium developed with System A. In fluorescent folate catabolite originated from cleavage of the addition, standard Pt-6-CHO and Pt-6-CH.,OH were also streaked on each plate. After development with System 4, the plates were dried in a hood and molecule at the C-9-N-10 bond. Furthermore, although no examined under UV in a ChromatoVue box for detection of fluorescent attempts were made to identify other peaks of radioactivity bands corresponding to standard Pt-6-CHO and Pt-6-CH2OH (their relative present on the Chromatogram, the peak of 3H,with an RKof mobility with each solvent system had been determined in previous experi ments). Finally, the silica gel layer was sectioned and scraped directly into 0.33, had the same Chromatographie mobility as that of scintillation vials for determination of radioactivity.

2380 CANCER RESEARCH VOL. 38

(Dowex 1X8, 200 to 400 mesh, 2.5 x 20 cm) that had been by the Dowex 1 column was chromatographed on paper prepared by the method of Rembold (22). The column was with System A, which had also been used for our previous developed in the dark with deionized, degassed water, and work (12), its RFwas identical with that of the characteristic the elution was monitored with a Vari-Chrom UV-Vis detec blue-fluorescent region observed in paper chromatograms tor (Varian Associates, Palo Alto, Calif.) at 345 nm. The flow of the media of various malignant cell lines (12). was adjusted to about 1 ml/min, and 20-ml fractions were The remaining lyophilized residue was used for gas chro- collected. A blue-fluorescent material was eluted after ap matographic-mass spectrometric studies. Under the condi proximately 5 bed volumes of the eluant had passed tions of chromatography used, the sample originating from through the column. Elution was continued with another 10 the KB cells growth medium gave rise to 5 major peaks; 1 of bed volumes of water, but no other blue-fluorescent mate them had the same retention time (280 sec) as the peak rial was eluted from the column. When standard Pt-6- obtained from the trimethylsilyl derivative of authentic Pt-6- CH2OH was chromatographed under identical conditions, it CH2OH. When this peak was scanned, it gave a mass was eluted from the column with water and appeared to spectrum consistent with the fragmentation pattern of the have the same retention volume as the unknown blue- trimethylsilyl derivative of Pt-6-CH2OH and essentially iden fluorescent compound. However, standard Pt-6-CHO re tical with the mass spectrum produced by the derivative of quired dilute formic acid for elution. The blue-fluorescent authentic Pt-6-CH2OH (Table 3). The structure shown in fractions collected from the column were pooled and lyoph- Chart 3 illustrates the trimethylsilyl derivative of Pt-6- ilized, and the residue obtained was used for analysis by CHjOH. The parent Â¡onwith a mass of 409 corresponds to paper chromatography and combined gas chromatography- the molecular weight of such a derivative of Pt-6-CH.jOH, mass spectrometry. and the base peak at mass 394 represents loss of a methyl For paper Chromatographie analysis, 4 mg of the lyophi- group from the parent ion. lized residue were dissolved in 200 /*! of 0.5 N NH4OH and Inhibitory Power of the Blue-fluorescent Compound on chromatographed on Whatman No. 3MM paper with 4 Xanthine Oxidase. We have previously shown (12) that the different solvent systems. In addition to the unknown, fluorescent compound excreted by malignant cells is inhib authentic Pt-6-CH2OH and Pt-6-CHO, as well as their mix itory to the activity of buttermilk xanthine oxidase. In fact, tures, were applied to each chromatogram. We found that the ability to inhibit this enzyme was used in our preliminary the unknown comigrated with standard Pt-6-CH,OH in each report as a criterion to identify the cancer cell catabolite as of the 4 solvent systems and not with Pt-6-CHO, as previ Pt-6-CHO because this compound has been reported (14, ously reported (12). Table 2 shows the RK'sof the unknown, 17) as the most powerful inhibitor of xanthine oxidase Pt-6-CH2OH, and Pt-6-CHO in the 4 different solvent sys among all the pterins tested. However, when we tested tems. When the unknown fluorescent compound purified synthetic Pt-6-CH2OH for its ability to inhibit the same enzyme, we found that on a molar basis the inhibitory Table 2 power of the unknown fluorescent catabolite isolated from Paper chromatography of pterins and blue-fluorescent material the culture medium of KB cells was comparable with that of isolated from growth medium of KB cells Pt-6-CH2OH and not with that of Pt-6-CHO. The results in The blue-fluorescent material used in this study had been par tially purified by chromatography on Dowex 1 as described in the text. The composition of Solvent Systems A, B, C, and D is described in "Materials and Methods."

CompoundPt-6-CHzOH Si(CH3)3 Fluorescent material from 0.420.28B0.630.630.52C0.610.610.50D0.470.470.52 KB medium Chart 3. TrimethylsilylderivativÂ«ofPt-6~CH,OH.Thestructure shown is R-6-CHOA0.42 the expected reaction product of Pt-6-CH,OHwith /V,O-bis(trimethylsilyl)acetamide.

Table 3 Mass spectral fragmentation data of the trimethylsilyl derivatives of authentic and metabolically formed Pt-6-CHÂ£>H The lyophilized residue (5 mg) was mixed with 100 /Â¿Iof/V,O-bis(trimethylsilyl)acetamide (Pierce Chemical Co., Rockford, III.) and 200 Â¿ilof acetonitrile (silylation grade) in a 600-/Â¿lmicro-V-vial (Wheaton Scientific, Millville, N. J.). Authentic Pt-S-CHjOH (1 mg) was also reacted with the silylating reagent in acetonitrile under the same conditions. After incubation in an oven at 65Â°for 20 min, the unreacted material was removed by centrifugation, and 3 /Â¿Iofthe supernatant were chromatographed on a Hewlett-Packard 5981A gas chromatograph-mass spectrometer. Peaks were scanned with an electron impact energy of 20 eV. Relative % of abundance of selected common ions in the mass spectra

Compound m/e % mie % m/e % m/e % mie % m/e % Authentic Pt-6-CHjOH 26.7 12.6 10.6 20.0 100.0 88.0 232 247 306 322 394 409 KB cell metabolite 29.0 16.2 9.7 18.7 100.0 90.2

AUGUST 1978 2381

Table 4 Table 5 Inhibition of xanthine oxidase by pterin derivatives and the KB cell Products of /2-14C/Pf-6-CHO metabolism by KB cells and human folate catabolite breast fibroblasts Xanthine oxidase activity was measured by the method of Kalc- Two x 10s KB cells and human breast fibroblasts were plated and kar (13), which follows the production of uric acid at 290 nm. grown in 25-sq cm Falcon flasks containing 4 ml of McCoy's Modi Hypoxanthine was present at concentrations of 14 to 16 Â¿Â¿Min0.05 fied Medium 5A supplemented with 15% undialyzed FCS. [2-14C]Pt- M potassium phosphate buffer (pH 7.5). The blue-fluorescent com 6-CHO (3.1 and 1.54 nmol; approximate specific activity, 1.2 mCi/ pound was isolated from paper chromatograms of KB cell growth mmol) in Hanks' solution was added to each of the cultures on the medium developed with System A; its concentration was estimated second and fifth days of growth, respectively. Concomitant growth with published extinction coefficients for R-6-CHzOH (5). A control was measured on Days 1, 3, 5, and 6, and both cell lines were eluate from a Whatman No. 3MM paper developed with the same found to be in the logarithmic phase of growth up to Day 5. A system was not inhibitory. control medium without cells but supplemented with the same amount of [2-MC]Pt-6-CHO was incubated under the same condi Substrate Inhibitor (hypoxan- tions. After 6 days of incubation, the media were collected, depro- concentra- thine) con- teinized, and lyophilized. The residues were each redissolved in tion centration % inhibi- 200 /Â¿Iof 0.1 N NH4OH; 100 fi\ of this solution were chromatographed on Whatman No. 3MM paper with Solvent System A, Compound (^M) (/Â¿M) tion together with standard Pt-6-COOH, Pt-6-CHO, and Pt-6-CHzOH. (In R-6-CHOR-6-CH2Ã›HCompound Solvent System A, the standards migrated with RF's of 0.09, 0.29, and 0.41, respectively.) After development the chromatograms KBcell isolated from were examined under UV, and the position of the standards was medium0.0320.350.48141416853682 recorded. The distribution of radioactivity on the chromatogram was then determined as described in "Materials and Methods," and the fraction of radioactivity present under each of the beaks Table 4 show that Pt-6-CHO is at least 10 times more potent comigrating with the standards was expressed as percentage of an inhibitor of xanthine oxidase than is either Pt-6-CH2OH total radioactivity. The small amounts of radioactivity present as R-6-COOH and R-o-CHjOH in the control medium are contami or the cancer cell catabolite. nants already present in the [2-14C]R-6-CHO preparation used and Pt-6-CHO as Precursor of the Blue-fluorescent Com do not arise during incubation with the medium. They were not pound Found in the Medium of KB Cells. A possible removed as they served the purpose of Chromatographie stan mechanism of cleavage of folie acid would yield Pt-6-CHO dards. as a product (4); indeed, some investigators appear to have % total radioactivity present as isolated Pt-6-CHO as one of the products of folie acid cleav age by an HCI or heat-activated enzymatic system from hu R-6-COOHHumanCell type incubated man blood cells (1, 7). If Pt-6-CHO were a product of folie CHsOH10 acid cleavage, it may be expected to be reduced to Pt-6- breast fibroblasts 52 KB cells 17 4 CHjOH because of the intrinsic reactivity (and probable 68 Control medium without 16 58Pt-6- 6 toxicity) of aldehyde groups. Reduction of aldehyde groups cellsR-6-CHO17 to alcohols has long been a recognized pathway in drug detoxification (2). Evidence supporting the reductive metabolism of Pt-6- 6 reveals that the ability to reduce Pt-6-CHO to Pt-6-CH2OH CHO comes from an experiment in which [2-14C] Pt-6-CHO is lowest for the Walker 256 carcinosarcoma cells among was added to the growth medium of logarithmically growing all of the malignant cell lines. However, when those same cells in culture to a final concentration of 1.16 Â¿Â¿M.The Walker 256 cells were tested for their ability to cause tumors results from this experiment (Table 5) clearly indicate that in female Sprague-Dawley rats, only 1 of 10 rats developed the malignant KB cells convert virtually all of the added [2- a tumor when they were given s.c. injections of 3.6 x 106 14C]Pt-6-CHO to [2-14C]Pt-6-CH2OH. In contrast, the normal Walker 256 cells per rat. Clearly, the Walker 256 cell line human breast fibroblasts metabolize Pt-6-CHO mainly by tested is only weakly tumorigenic, and there are probably oxidation to the Pt-6-COOH. less than 103 malignant cells per 3.6 x 106 cells (11). This is The same experiment was repeated by adding unlabeled reflected in their ability to reduce added Pt-6-CHO that, Pt-6-CHO to the growth medium of several cell lines and although lower than any other malignant cell line tested, is analyzing the medium after 6 days of growth by HPLC. The nevertheless greater than any normal cell line. The BALB/ results of these experiments are shown in Tables 6 and 7 c3T3 fibroblasts as well as the SV40 and the benzo(a)py- and are expressed as the mol fractions of the total pterins rene-transformed 3T3 cells were also tested for their ability recovered that are present as Pt-6-COOH, Pt-6-CHO, and to metabolize Pt-6-CHO. The results in Table 7 show that the Pt-6-CH2OH. The data shown in Table 6 are in good agree transformed fibroblasts reduced R-6-CHO to Pt-6-CH.Â¡OHto ment with those of Table 5 and extend the observation a greater extent than did their normal counterpart, although made for KB and human breast fibroblasts to other malig they also showed an increased rate of oxidation to the Pt-6- nant and normal cell lines. The general trend is that malig COOH with respect to the normal 3T3 fibroblasts. The nant cell lines tend to reduce the added Pt-6-CHO to the Pt- possibility that these results could be attributed simply to a 6-CKOH, whereas normal cell lines tend to oxidize it to the difference in cell density between the normal and the Pt-6-COOH or not metabolize it at all. Although experiments transformed cells was eliminated by plating transformed with a tumor of varying growth rate, such as the Morris cells at a lower density than the normal cells, so that by the hepatoma, would be more indicative, the ability of a certain end of a 5-day growth period both cell lines reached cell line to reduce Pt-6-CHO to Pt-6-CH2OH can be corre approximately the same density. The results of this experi lated with the degree of oncogenicity of that cell line. Table ment were similar to those obtained when transformed and

2382 CANCER RESEARCH VOL. 38

Table 6 Mol fractions of recovered pterins present as Pt-6-CHÂ£>H, Pt-6-CHO, and Pt-6-COOH in media of various cell lines Cells (2 x 105)were grown in 25-sq cm Falcon flasks containing 4 ml of reconstructed McCoy's Modified Medium 5A without Bacto-peptone and folie acid but supplemented with 15% undialyzed PCS and gentamicin (for the L1210 6% PCS was used instead of 15%). After 24 hr of growth, 32 nmol of Pt-6-CHO in 0.1% NaHCO3were added to each flask. This concentration of aldehyde did not seem to affect growth rates of either KB or human breast fibroblasts in previous experiments. Cells were grown in this medium for either 5 or 6 days. Control flasks were incubated as the experimental flask except that no cells were added. Concomitant cell growth was measured on Days 2, 5, and 6, and by the end of the sixth day normal cell lines had doubled an average of 3.5 times and malignant cell lines had doubled an average of 4.25 times. All of the cell lines were approaching confluency by Day 5 with the exception of the L1210 line. Media from cultures grown for 5 and 6 days were analyzed by HPLC as described in "Materials and Methods," and the results were expressed as mol fractions of recovered pterins present as Pt-6-CHÃ¼OH,Pt-6- CHO, and Pt-6-COOH. No significant difference was found between results for the 5 days and those for the 6 days of growth. The data for the 6 days of growth are shown in the table below. Total recoveries of the pterins averaged 86 Â±10% of the control. Mol %

CelllineHuman KBHuman carci nomaUterine HeLaMouse cervical carci nomaLymphatic L1210Rat leukemiaBreast W-256Human carcinosarcomaNormal breast fibro fibroblastsThymusbreast blastsRat normalMouse fibroblastsKidney normalControl fibroblastsPt-6-COOH192472731251212Pt-6-CHO00456063718888Pt-6-CHjOH817649136400 mediumOriginNasopharyngeal

normal cells were plated at the same density and grown for the same length of time. We therefore concluded on the basis of these experiments that the ability to reduce Pt-6- CHO to Pt-6-CH,OH is a characteristic of the malignant Table 7 state of a cell and not a coincidental observation in many Mol fractions of recovered pterins present as Pt-6-COOH, Pt-6- malignant cell lines. CHO, and Pt-6-CHÂ£>H in media of normal and transformed 373 fibroblasts In this set of exerpiments, 5 x 104 BALB/c3T3 fibroblasts and DISCUSSION SV40-transformed 3T3 (SV3T3) and benzo(a)pyrene-transformed 3T3 cells (BP3T3) were plated in 100-mm i.d. Lux plastic Petri An increase in the catabolism of folie acid may be respon dishes containing 8 ml of the basic McCoy's medium supplemented sible for the folate deficiency observed to occur in many with 15% PCS and gentamicin; 64 nmol of R-6-CHO in 1% NaHCO3 forms of cancer and in other pathological conditions as were also added on Day 1. Controls consisted of simultaneously well (6, 27). We have recently (12) reported that malignant incubated media without cells. Cells were grown for 5 days without cells in culture degrade folie acid to a blue-fluorescent a change of media. Growth curves were also determined; they showed that all 3 cell lines were still in the logarithmic phase of pterin but that normal amniotic and embryonic cells do not, growth by the end of Day 5. By this time 3T3 fibroblasts had grown or at least not at the same elevated rate as do malignant for 4 generations; SV3T3 and BP3T3 both had grown for 5 genera cells. We also demonstrated a close correlation between tions. After 5 days the media were collected, deproteinized, and the presence in human urines of a blue-fluorescent com analyzed by HPLC as described in "Materials and Methods." pound, tentatively identified as Pt-6-CHO, and a pathologi Results are expressed as in Table 4. Total recoveries of pterins averaged 84 Â±2% of the control. Transformed as well as normal cal diagnosis of malignant disease (12). Additional support cells were also tested for oncogenicity in BALB/c mice by giving for our findings comes from literature reports describing i.p. injections of 2 x 10' cells/mouse. One hundred % incidence of the occurrence of as yet uncharacterized blue-fluorescent tumor was found for both SV3T3 and BP3T3; all of the mice with substances in urines from patients with corpus uteri carci the tumor died within 8 to 10 weeks from the time of cell inocula tion. However, when 2x10Â« 3T3 fibroblasts were injected i.p. into noma, myelomatosis (25), and bronchial carcinoma (26), as each of 9 BALB/c mice, only 1 tumor resulted. well as in urine of mice carrying the Ehrlich ascites tumor Mol % (10). Furthermore, higher levels of fluorescent substances thought to originate from the nonfluorescent tetrahydro- CelllineBP3T3 biopterin have been reported to occur in the blood of tumor-bearing rats as well as in the blood of patients with SV3T3 23 58 19 various forms of cancer (15). 3T3 13 82 5 In this report, we have unequivocally identified the blue- Control mediumPt-6-COOH475Pt-6-CHO28 95Pt-6-CHzOH260 fluorescent product of folate catabolism by malignant cells

AUGUST 1978 2383

in culture as Pt-6-CH2OH. We have also investigated the REFERENCES nature of a possible precursor of the Pt-6-CH2OH that is 1. Aravmdakshan. I., and Braganca, B. M Enzymic Cleavage of Folie Acid present in the media of malignant cells. We found that by Extracts from Human Blood Cells. II. Observations on the Liberation of the Active Form of the Enzyme and Its Specificity. Biochim. Biophys. added Pt-6-CHO can indeed be reduced to Pt-6-CH2OH by Acta, 27: 345-354, 1958. the action of growing cancer cells. This conversion did not 2. Bachur, N. R. Cytoplasmic Aldo-Keto Reductases: A Class of Drug take place in cell-free conditioned media from those same Metabolizing Enzymes. Science, 793. 595-597, 1976. 3. Barford, P. A., and Blair, J. A. In: W. Pfleiderer (ed.), Chemistry and cultures, thus indicating that the presence of growing Biology of Pteridines, pp. 413-426. Berlin: Walter De Gruyter & Co., malignant cells is necessary for the reduction of Pt-6-CHO 1975. 4. Blair, J. A. Isolation of Isoxanthopterin from Human Urine. Biochem. J., to occur. Although purified folie acid preparations are 68. 385-387, 1958. inevitably contaminated by traces of Pt-6-CHO arising by 5. Blakley, R. L. In A . Neubergerand E. L. Tatum (eds.). The Biochemistry photodecomposition of folie acid itself (16), the amount of of Folie Acid and Related Pteridines: Chemical and Physical Properties this contamination in our preparations of [2-14C]folic acid of Rerins and Folate Derivatives, Chap. 3, pp. 66-71. Amsterdam: North Holland Publishing Co., 1969. was very low and could not account for the quantities of Pt- 6. Blakley, R. L. In A. Neubergerand E. L. Tatum (eds.), The Biochemistry 6-CH2OHusually recovered from growth media of malignant of Folie Acid and Related Pteridines: Folate Deficiency, Chap. 12, pp. 425-426. Amsterdam: North Holland Publishing Co., 1969. cells, even if we assumed complete conversion of the 7. Braganca, B. M , Aravindakshan, I., and Ghanekar, D. S. Enzymic contaminant Pt-6-CHO into Pt-6-CH2OH.We believe that the Cleavage of Folie Acid by Extracts from Human Blood Cells. I. Prepara production of Pt-6-CH2OH by neoplastic cells is indicative tion and Co-factor Requirements of the Enzyme System. Biochim. Biophys. Acta, 25. 623-634, 1957. of active folie acid catabolism by these cells and implies the 8. Dinning, J. S., Sime, J. T., Work, P. S., Allen, B., and Day, P. L. The existence of possibly 2 enzymatic systems: one involved in Metabolic Conversion of Folie Acid and Citrovorum Factor to a Diazotiz able Amine. Arch. Biochem. Biophys.. 66. 114-119, 1957. the cleavage of folie acid (or one of its derivatives), which 9. Felsted, R. L., Richter, D. R., and Bachur, N. R. Rat Liver Aldehyde would give Pt-6-CHO as one of the products, and the other Reductase. Biochem. Pharmacol.,26.- 1117-1124, 1977. involved in the reduction of this product to Pt-6-CH2OH, 10. Gantner, G., Grassmayr, K., Hausen, A., and WÃ¤chter,H. Increased Excretion of a Fluorescing Substance in the Urine of Ehrlich Ascites which is excreted into the medium. Aldehyde reductases Tumor-bearing Mice. Mikrochim. Acta,2. 33-38, 1977. are widely distributed among animal tissues (2) and are 11. Halpern. R. M., Halpern, B. C., Conklin, K. A., and Smith, R. A. Inhibition of Neoplastic Cell Growth by Autogenous DNA. Proc Nati. Acad. Sci. thought to play key metabolic roles in aldehyde detoxifica U. S., 67: 207-214, 1968. tion and drug metabolism (2, 9, 28); of special importance 12. Halpern, R. M., Halpern, B. C., Stea, B., Dunlap. A., Conklin, K., Clark, to this discussion are 2 such reductases recently character B., Ashe, H., Sperling, L., Halpern, J. A., Hardy, D., and Smith, R. A. Pterin-6-Aldehyde, A Cancer Cell Catabolite: Identification and Applica ized from rat and human livers, which have been shown to tion in Diagnosis and Treatment of Human Cancer. Proc. Nati. Acad. prefer aromatic to aliphatic aldehydes (9, 28). The ubiqui Sei. U. S., 74: 587-591, 1977. tous and constitutive presence of an aldehyde reductase in 13. Kalckar, H. M. Differential Spectrophotometry of Purine Compounds by Means of Specific Enzymes. I. Determination of Hydroxypurine Com malignant cells, as suggested by our results, could be pounds. J. Biol. Chem., 767. 429-443,1947. justified easily in terms of a potential role in detoxifying the 14. Kalckar, H. M.. Kjeldgaard, N. 0., and Klenow, H. 2-Amino-6-Hydroxy-6- Pt-6-CHO produced by a folate cleavage enzyme. Formylpteridine, an Inhibitor of Purine and Pterine Oxidases. Biochim. Biophys. Acta. 5. 586-594, 1950. The enzymatic system responsible for the metabolic dis 15. Kokolis, N., and Ziegler, I., On the Levels of Phenylalanme, Tyrosineand Tetrahydrobiopterin in the Blood of Tumor-bearing Organisms. Cancer posal of folate cofactors has not been the object of many Biochem. Biophys.,2. 79-85, 1977. investigations during the 3 decades since the discovery of 16. Lowry, O. H., Bessey, O. A., and Crawford, E. J. Photolytic and folie acid; consequently, information about this enzyme Enzymatic Transformations of Pteroylglutamic Acid. J. Biol. Chem., 780: system is limited. Only sporadic reports exist in the litera 389-398, 1949. 17. Lowry, 0. H., Bessey, 0. A., and Crawford, E. J. Rerin Oxidase. J. Biol. ture concerning the in vitro catabolism of folie acid. It has Chem., 780. 399-410, 1949. been reported (8, 23) that liver extracts from a variety of 18. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. Protein species are capable of cleaving citrovorum factor (NMor- Determination With the Folin Phenol Reagent. J. Biol. Chem., 793: 265- 275,1951. myltetrahydrofolate) to a diazotizable amine, presumably p- 19. Murphy, M., Keating, M., Boyle, P., Weir, D. G., and Scott, J. M. The Elucidation of the Mechanism of Folate Catabolism in the Rat. Biochem. aminobenzoylglutamate. An enzymatic activity capable of Biophys. Res. Commun., 77: 1017-1024, 1976. degrading folie acid was also found to exist in human blood 20. Neary, M. P. Beckman Technical Bulletin 015-082-045-A, pp. 45-52. Ful- cells in an inert state but could be activated by heat lerton. Calif.: Beckman Instruments, Inc., 1971. 21. Poe, M. Dihydrofolate Reductase from a Methotrexate Resistant Strain treatment or acid extraction (1, 7). Finally, a recent study of of Escherichia coli: Dihydrofolate Monooxygenase Activity. Biochem. dihydrofolate reductase from Escherichia coli (21) has Biophys. Res. Commun.. 54: 1008-1014,1973. shown that this enzyme is also capable of catalyzing the 22. Rembold, H. Isolation of Reridines by Ion-Exchange Chromatography. Methods Enzymol.. 788: 652-660, 1971. oxidative cleavage of dihydrofolate and folate (although to 23. Silverman, M. Enzymatic Cleavage of the Citrovorum Factor. J. Am. a lower extent) at the C-9-N-10 bond. The products of Chem.Soc.,75. 1512, 1953. these reactions were shown to be 7,8-d ihydro-Pt-6-CHO 24. Thijssen, H. H. W. A Simple Method for Preparing 2-Amino-4-Hydroxy-6- Formylpteridine, a Precursor of the Reridine Substrate of Dihydropter- and Pt-6-CHO, respectively. The possibility exists that this oate Biosynthesis. Anal. Biochem., 54: 609-611, 1973. enzyme or some unregulated form of it present in malignant 25. WÃ¤chter,H., Grassmayr, K., Gutter, W., Hausen, A., and Sallaberger, G. Fluorometric Investigations in Situ of Low-molecular Urinary Constitu cells could be responsible for the cleavage of folie acid that ents Separated by High-voltage Electrophoresis. Wien. Klin. Wochschr., we believe takes place in cancer cells. 84: 586-590, 1972. 26. WÃ¤chter,H., Grassmayr, K., Gutter, W., Hausen, A., Sallaberger, G., and Gabi, F. Urinary Low Molecular Weight Products in Neoplasia. Brit. Med. J.,2,-322, 1971. ACKNOWLEDGMENTS 27. Weir, D. G. The Pathogenesis of Folie Acid Deficiency in Man Irish J. Med. Sci., 743. 3-20, 1974. The authors would like to thank Dorothy Hardy for her assistance in tissue 28. Wermuth, B., Munch, J. D. B., and von Wartburg, J. P. Purification and culture work and Emma DiStefano and Cam Pintauro for assistance in taking Properties of NADPH-dependent Aldehyde Reductase from Human Liver. mass spectra. J. Biol. Chem..252: 3821-3828, 1977.

2384 CANCER RESEARCH VOL. 38

Baldassarre Stea, Peter S. Backlund, Jr., Phillip B. Berkey, et al.

Cancer Res 1978;38:2378-2384.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/38/8/2378

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/38/8/2378. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.