774
Nucleic Acids. 11. Cytotoxicity Studies with Nucleotides and Dinucleoside Phosphates Containing ura-Cytidine
'Thirtx-one derivative3 and analogs of l - ~ - D - ~ ~ a b i l l ~ f l l r i t l i ~1 nix-cytidiiie), ~ ~ ~ ~ ~ ~ ! ~ ~ iiirliitliiig i ! l ~ ~ nirnple subituted nucleosides, niicleotides, and dinucleoside phosphates, were tested for cytotoxicity to Kl3 c*arciriorn:i vells in culture. The simple nucleotides showed good activity in this system which was preverited by deoxycytidine in the cases tested. The 3i-.5'-dinucleoside phosphates of am-cytidine aiid varioiis other iiiicleosideq showed significant cytotoxic activity os. KB cells and were iisually more active tha their c>iiuei.poiiding2'+5' XU cntic~lusions( ' : i l l analogs. The 5'-+5'-diniicleoside phosphates of urn-cytidine also ..hoR-ed good iti'tiv yet be drawn regarding the possible penetration and/or action of the intact diruicleo I)ilosphat es. r r Felt t of the compounds were tested for cytot,oxicitJ-1's. L5178Y t i i o i i + 1eitkemi:r cells iri rill : : r i l l 1 i t $ crrtr-cytidineted, iiicliidiiig t lit* simple niirleoresistant mutant ("kinaseless"). For the moit. part, the cornpoi tidrh, xere highly c.ytotiixic to the parent cell. , , I . I:.( i t & d > ,C ' , L I , ~ ( ~ l > c s . , 19, kl:: I l!I>!)J,
NUCLEIC ACIDS. II
September 1967
TABLE I1
TABLE I CYTOTOXICITY O F UT-@CYTIDINE DERIV.4TIVES T O KB CELLS Median IDKO .. m M X 104 ~
Conipd
d m l
CA CAP CAP PCA MepCA PhpCA C.4p.4 CADA CApdA CApdL4 CACA CA,CA CAI)U CA,,U CAI)T CA,,T AC.4 A,,CA UpCA IJ,CA dU,CA pCAdC pC.4Ch pCAC pCAdU
0.05 0,05 0.06 0.06 0.4 0.07 0.60 0.12 2.3 0.12 0.66 0.06 0.47 0.l i 7.7 0 . l!) I),0.5 0 . ox
0.13 0.1s 0.16 0.11 0.20 0.1 0.1
2 1.5 1.85 1.75 11 1.7 10 2 40 2.1 12 1 8 . :;
2.65 30 3 ,4 0 .!) 1.4 2.3 2.3 3 2.1 3.6 2 1.6
775
Range of IDse, m/ml
No. of
0.03-0.11 0.03-0.05 0.03-0.07 0.04-0.15 0.3-0..55 0.05-0.08 0.48-0.67 0.08-0.16 1 . 7 and 2 , 8 0.12-0.22 > 0 . 4 and 0 . 6 6 0.03 and 0.07 I),44 alld 0 . 5 0 0.0:)-1).24 1 .7-2. 0 0,1+1.9 0 , 0:1-0. OT 0.03-0. 1 0 . 12-0. 1.5 0.12-0.15
22
n.0 O . 1-0.14 0.07 and 0 , 1 2
EFFECTOF DEOXYCYTIDINE O N CYTOTOXICITY OF SELECTED COMPOUKDS IN KB CELLS IDso, pg, ml-----.igent - dC +dC (10 pg'inl) CA 0 06 >I PCA 0 1 >1 0 1 >1 pCAdC 0 09 >1 CApU 0 12 >1 CA,dX 0 13 >0 4 CApA CAPA 0 67 >2 0 55 >2 MepCh
expts
7 -
> )
10 6 1
6
6 2 3 2 2 2 :3 J
:1 J
lacking a kinase for phosphorylating this nucleoside. These cultures were obtained through the courtesy of Dr. Fischer and a variety of ma-C derivative4 arid diriucleoside phosphates were tested for cytotoxic, activity therein. The results of these investigations are shown in Table 111. I n some of these eabes, the
3 3 3 4 8 6 3 2
Z'-dinucleoside phosphate equalled that of ara-C per se on a niolar basis. 5'-G'-Dinucleoside phosphates were approximately as active against KB as was am-C. It is interesting that pCA4dCshowed striking activity. particularly when one considers that dC prevents the activity of am-C when added to the assay niediuni in excess. It should be noted that PhpCA is several times more cytotoxic t o IiB cells than is IlepCA, both on a weight and on a niolar basis. Under various test conditions, deoxycytidine prevents or delays the cytotoxic attion of am-C both in vitro 2's. mammalian cells and D K A viruses and in whole a n i n ~ a l s . ' ~The ~ - ~ ~cytotoxicity of selected dinucleoside phosphates was determined in the presenre and absence of 10 pg of deoxycytidine/ml to determine whether direct evidence could be obtained that these compounds were acting by a mechanism not involving cleavage t o am-C. The results of these experiments are presented in Table 11. These data show significant prevention of the cytotoxic action of pCA and the three dinucleoside phosphates tested in addition to the free nucleoside. Further studies in which the concentration of deoxycytidine was varied from 0.08 to 10 pg/nil eonfirmed the niarlied reduction of activity at the highest level of deoxyrytidine. Significant reversal was observed a t the lower roncentrations of deoxycytidine but not in a direct ratio to the amount of this nucleoside present. Thus, no eviderwe was obtained with the nwleosides investigated that their action at tlie ~nolec~ular level was signihcaiit 1.v different froni a/ a-C: per se by this test. Cytotoxicity Studies with L5178Y and Its Resistant Mutant.-In :L rrceiit In~l)lictLtioil,2°('hu :ud I ' 1 ~ 1 i e r reported the isolation of a mutant cell line of L.51781mouseleukemiawhichwas resistant to ara-C,presumably (11) J . >. E b a n s and G U .\Ien:.~l, B z a c l i ~ m .I ' l i u i m u ~ o l, 13,989 (1Yb.l).
TABLE I11 CI~OSS-~~ESIST.~KCS STUDIESIK Ll5i8T CELLLISSS .\gent
CA CAPA CA,A APCA ApCA pCAA ChpdA C.ipdA PCA pCA(NRL)
CAP CAP
TDC.4 CA,,T CL4pT
CA4,dU CApdU
Aledian IDsa, pg/ml Kinaseless Parent mutant"
0.02 0.09 0.07 0.05 0 . 03 0.05 0.06 0.05 0.03 O..i 0 . 03
0.03 0,03 0.03 0.07 0.04 0.02 0.04
so
of expts
35 3.2h 1.6D
7 6
1 .:3 1 . .i 1.7 8.i >,XI >10 > 10 >tX) >IO > 10 l!)
2 2
>;,o
>10 >10 2.i pCACA pCAdU 0.0'3 > 10 pCAC 0.04 30 PhpCA 0.03 >23 LIepCA 0.07 23 .4 0.3 0.4 0.5 0.44 PA 0.3 0.3 AD A,*& 0.6 0.3 11 >40 UDA ApU >4c >4 0.03 20 U,CA UpCA 0.04 20 Clone Ca55 izolated from a subline of L51iXS. ', /J 1-allies for three assays were 3.2, >4, and >4 pg/ml. fL
I
:3 3
:3 I 2 2 2 1 2 2 2 2 2 2 2 J
3 -r
1 1 1 3
:z
-> 2
< 0.01,
absolute end point of activity was not determined since clear-cut cross-resistance was demonstrated, which waq the primary purpose of the cxperiment. The unique cytotoxicditp of acieiioiiiie (A) and *Irontainirig diilucleo4ide pho+phates .hould be iiotetl. Although the p:+rti:il c*rosh-resiit>illcaeof nll the ronipounds w1iic.h inlii1)itetl tlic Ca55 liiic tw c~plninctl by hydrolysis of the dinucleoside phosphate t o liberate adenosine, reversal studies with deoxyc.ytidine :we not wholly corisisterit with this oxplanation. As shown c 4 : m
in Table IT', dC: reversed the cytotoxic activity of -1 i n the parent L51iSY cells to a much greater extent than was observed with the Ca55 mutant. With the ,tc.ontaining conipounds CX,,,l and CLipA, however. the reversal of activity in the mutant cell line with dC \v~L< greater than observed with either A or am-C alone. If the compounds which were only partially crossresistant in the Ca55 mutant are active by virtue of hydrolysis to liberate the cytotoxic coniporient I J J and X,U show a loner order of activity than anticipated. When coinbinations of X and U n'erc tested together, the cytotoxicity of was reversed by IT,as shown in Table IV. These data were confirmed on repeat experiments and suggest that the low order o f wtivity of dinucleoside phosphates containing +I : ~ n dU i b due to reversal of the cytotoxic activity of -1 b y U rather than by slo~verhydrolysis of tlieqe conipainid5 *
Discussion Bused on the studies reported l i e i ~ cwtaiii . structure artivity relationships can bo dedured. The simplc iiuvleotides of am-C show niarked cytotoxic. actir it>:$gainst both KB cells arid the mouse LT,1781' cell i i i c.ulturc. Converting the 3'-phoiphate of am-C t o t h ( L phenyl ester allom retention of coiisider:ible activity. n hereas conversion of this nucleotide t o the methyl ester results in a marked 10% of activity in I S . Ahlost all of the am-C-contaiuing (soinpounds examined were highly cytotoxic to LZliST cells in culture. In general, the 3'+5'-dinurleoside phosphate estei containing am-C are more active against KB cells than the (horresponding 2 ' 4 5 ' derivatives (APCA and UPCh arc notable exceptions). 011 the whole, the 5'+5'diriucleoside phosphates containing am-C showed good cytotoxicity equalling. in some cases, that of ara-C' per se o n a niolecular basis. In all cases investigated thus far, including a small nuniber of nucleotides and dinucleoside phosphates, cytotoxicity to KB or L5178T cells was decreased by the addition of deoxycytidine at zero time, as is the case for a ~ a - C . Although this observation suggests that the substituted compounds ultiniately act in the cell at the same locus as does ara-C, it is based on an indirect nicasure. This conclusion
