SHORT COMMUNICATIONS 185

sc 93099

Conversion of 6-chloropurine to its ribonucleotide by oscites tumor cells

6-Chloropurine inhibits the growth of a variety of transplantable mouse neo- plasms 1-4 and can cause temporary remission in some cases of acute leukemia in h u m a n s 5. SARTORELLI and coworkers e3 demonstrated that in mouse ascites tumor cells 6-chloropurine reduced synthesis de novo of guanine nucleotides, but not that of adenine nucleotides, indicating that the analog affected some stage in the conversion of IMP to GMP. HAMPTON s,9 reported that the ribonucleoside 5'-phosphate of 6- chloropurine did not significantly inhibit the enzymatic conversion of IMP to AMP in cell-free systems but strongly inhibited conversion of IMP to XMP. 6-Chloro- purine did not inhibit XMP formation, and it was suggested 9 that 6-chloropurine might retard synthesis de novo of guanine nucleotides by prior anabolism to its ribonucleoside 5'-phosphate. We now present evidence that this ribonucleotide is synthesized by Sarcoma 18o ascites tumor cells in vivo and by cell-free extracts of these cells in the presence of Mg 2+ and 5-phosphoribosyl-I-pyrophosphate (PRPP).

Six mice bearing the ascitic form of Sarcoma 18o were used 8 days after transplantation and each was injected intraperitoneally with 5 t~moles of 6-chloro- [8-1*CJpurine dissolved in o.5 ml saline. After 3o min the tumor cells (16 g) were collected and extracted with 2 vol. of cold o.4 M HC104. The tissue residue was re-extracted with 1.5 vol. of cold o.2 M HC10, and the combined extracts were neu- tralized in the cold with 20 % KOH. After removal of KC104, the extract was chro- matographed on a I cm × I5 cm column of Dowex-z X8 resin (2oo-4oo mesh, formate form) using a linear gradient of formic acid which started at zero and reached I I . 7 M at 1ooo ml of eluant*. Two peaks of radioactivity were found: I (Fractions 12-24) which contained chloropurine, and I I (Fractions 71-83) which represented 15.4 o/ /0

of the radioactivity recovered. Peak I I fractions were pooled, freeze-dried, and the residue taken up in water for paper chromatography in three systems: 5 % disodium phosphate-isoamyl alcohol; n-butanol-acetic acid-water (25 : 15 :IO, v/v); isopro- panol - I 07 o ammonium sulfate (2 : I, v/v). In all systems 75-78 % of the radioactivity of the Peak I I sample co-chromatographed with authentic 6-chloropurine ribo- nucleoside 5'-phosphate z0. About 15 % of the radioactivity was found with the IMP markers. When subjected to paper electrophoresis in phosphate buffer (pH 7.I), 9 ° % of the Peak I I radioactivity had the same mobility (1I. 3 cm/h at 20 V/cm) as the chloropurine ribonucleotide.

A portion of the Peak I I concentrate was made i N in HCI and heated at IOO ° for I h. In the butanol and in the isopropanol systems, 94 % and 9 ° O//o of the applied radioactivity co-chromatographed with hypoxanthine. This observation and the known acid-lability of the glycosyl linkage of purine ribonucleosides and of the chloro-group of 6-chloropurine z° are consistent with the proposal that Peak I I represented 6-chloropurine ribonucleotide.

In a similar experiment in vivo, the synthesis of the analog nucleotide was measured in tumor cells which had been treated with azaserine* (Fig. I). Prior

" In this system, when 5 ml fractions of eluate were collected, 2/ ,mole samples of AMP, 6-chloropurine and IMP appeared in Fractions 13-19, 12-18 and 47-55, respectively.

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I86 SHORT COMMUNICATIONS

t r ea tmen t with azaserine enhances the conversion of other purine analogs to their nucleotide derivat ives in ascites tumor cellsn, 1~.

16iT I I, -lJ6

, .

: H lh Absorbonce II!/ / /

<0.4 4

Q2 2 ~

0 ~ 0 0 20 40 60 60 100

Froction number

Fig. I. Extract of [14C]chloropurine-treated ascites tumor cells chromatographed on anion- exchange resin. Eight days after transplantation, five mice bearing the Sarcoma 180 ascites tumor were each injected with 5 ° #g azaserine dissolved in o. i ml saline. Six hours later each mouse was injected with 4.64/,moles of 6-chloro[8-14C]purine (7.7' lO5 counts]min). After 30 rain the tumor cells (13.o ml) were collected and an extract prepared and chromatographed as described in the text. The A2e 0 of each eluate fraction (5 ml) is plotted (open area) as is the radioactivity (counts] min per ml) (black area). Peak II (black) contained 25 % of the radioactivity shown and represent- ed 0. 7/zmole of chloropurine nucleotide.

Washed Sarcoma 18o ascites t umor cells were suspended in 6 vol. of water and were t rea ted with a 2o kcycles probe-type sonic oscillator using the m i n i m u m time (about I min) necessary to disrupt the cells. The sonicate was centrifuged at IOO ooo ×g for I h and the clear supe rna tan t was stored at - -20 °. Pre l iminary experiments in

which 25/~1 portions of this enzyme preparat ion were incuba ted with o.15 t~mole 6-chloro[8-14C~purine, 1 .5~moles magnes ium sulfate, o.6¢~mole 5-phosphoribosyl- I -pyrophosphate and 20 ~moles Tris, pH 7.5, in a final vol. of 0.25 ml, showed tha t a radioactive product was formed which had the pale blue fluorescence under ultra- violet light (254 m#) tha t is characteristic of 6-chloropurine ribonucleotide. The product represented 8.2 % of the total radioactivity** and migrated at the same rate as au thent ic chloropurine nucleotide when subjected to electrophoresis in I M sodium borate, (pH 7.0) at a gradient of 22 V/cm. Mobilities in this system were: 6-chloropurine, 5.0 cm/h; 6-chloropurine ribonucleoside, 8.7 cm/h; and 6-chloropurine nucleoside 5 '-phosphate, 15 cm/h. Electrophoretograms of control incubat ions which lacked 5-phosphor ibosyl- i -pyrophosphate had only one radioactive area corre- sponding to the free base.

The tumor cell extract was concentra ted three-fold with Sephadex G-25 gel (0.22 g per ml of supernate) . This enzyme preparat ion (0. 4 ml) was incuba ted at 37 °

* Provided by the Cancer Chemotherapy National Service Center, Bethesda, Md. ** Reaction mixtures buffered at differel~t pH values had lower percentages of radioactivity

in the ribonucleotide region: pH 7 (7 .2 %), pH 8 (6. 9 °/o), pH 8. 5 (4.0 %), pH 9 (3.4 %).

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SHORT COMMUNICATIONS 187

for 3 h with 7.2/*moles 6-chloropurine, 7.2 #moles 5-phosphoribosyl-I-pyrophosphate, 24/,moles magnesium chloride and 12o/,moles Tris buffer (pH 7.5) in a final volume of I.O ml.

The solution was chromatographed on paper in n-butanol-acetic acid-water (5:3:2, v/v). In ultraviolet light, a blue fluorescent band (RF 0.39) was visible which corresponded in color and position to 6-chloropurine ribonucleoside 5'-phos- phate; a dark band (RF o.15) and a yellow fluorescent band (RF 0.72) were also present and corresponded to IMP and 6-chloropurine, respectively. 6-Chloropurine nucleoside (Re 0.64) was not detected but a dark band (RF 0.35, 2max 248 m/, at pH 5) corresponded to inosine. Elution of the 6-chloropurine (eM 9.1" 103 at 265 m/,) 13 and its nucleotide (eM 8.4" lO 8 at 263 m/*) into buffer of pH 4.5 and spectrophotometric assay showed that 2.4 % of the chloropurine had been converted to its ribonucleotide. The spectroscopic properties of the solution of 6-chloropurine nucleotide were: maximum at 263m/,, minimum at 23om/,, absorbance ratio 25om/,/26o m/*, 0.82 (reported 1° 263 m/*, 226 m/* and 0.78 ). Also present was a weak maximum at 305 m/, due to an unknown contaminant; the absorbance ratio 263 m/,/3o5 m# (approx. 7) increased upon more prolonged chromatography of the reaction mixture in the butanol-acetic acid system.

Sodium hydroxide (final concn, o.I N) was added to the above solution of 6-chloropurine nucleotide; after 16 h the absorption maximum at 263 m/* was replaced by new maxima at 254 and 29 ° m/,. A similar instability to alkali, accom- panied by the above spectral changes, has been reported 14 for 6-chloropurine ribo- nucleoside and is also shown by the synthetic 6-chloropurine ribonucleotide.

These findings demonstrate that cells of the ascitic form of Sarcoma 18o converted 6-chloropurine to a product identified as 6-chloropurine ribonucleotide. The finding that this substance was also formed when 6-chloropurine was incubated together with 5-phosphoribosyl-i-pyrophosphate and an extract prepared from the tumor cells suggests that its synthesis took place by the pyrophosphorylase route and indicates that the product is a nucleoside 5'-phosphate ester.

This investigation was supported by the National Cancer Institute of Canada and the Medical Research Council of Canada.

University o/ Alberta Cancer Research Unit ALEXANDER HAMPTON (McEachern Laboratory) and the Department o/ A . R . P . PATERSON Biochemistry, Edmonton, Alberta (Canada)

I D. A. CLARKE, V. S. PHILIPS, S. S. STERNBERG AND C. C. STOCK, Ann. N . Y . Acad. Sci., 60 (1954) 235.

2 D. A. CLARKE, H. C. REILLY AND C. C. STOCK, Antibiot. Chemotherap., 7 (1957) 653. 3 G. S. TARNOWSKY AND C. C. STOCK, Cancer Res., 17 (1957) lO33. 4 A. C. SARTORELLI ANn B. A. BOOTH, Cancer Res., 2o (196o) 198. 5 R. R. ELLISON, R. T. SILVER AND R. L. ENGLE, Ann. Internal Med., 51 (1959) 322. 6 A. C. SARTORELLI AND B. A. BOOTH, Arch. Biochem. Biophys., 89 (196o) 118. 7 A. C. SARTORELLI, J. R. AKERS AND B. A. BOOTH, Biochem. Pharmacol., 5 (196o) 238. 8 A. HAMPTON, Federation Proc., 2I (1962) 37 o. 9 A. HAMPTON, J. Biol. Chem., 238 (1963) 3o68.

IO A. HAMPTON AND M. H. MAGUIRE, J. Am. Chem. Soc., 83 (1961) 15o. I I A. C. SARTORELLI, G. A. LEPA~E AND E. C. MOORE, Cancer Res., 18 (1958) 1232. I2 A. R. P. PATERSON, Can. J. Biochem. Physiol., 37 (1959) IOl i . 13 S. F. MASON, J. Chem. Soc., (1954) 2o71. 14 M. P. GORDON, V. S. WELIKV AND G. B. BROWN, J. Am. Chem. Soc., 79 (1957) 3245-

Received July 26th, 1965

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