[CANCER RESEARCH 40, 2796-2801 , August 198010008-5472/80/0040-0000$02.00
Inhibition of Chemically Induced Morphological Transformation andReversion of the Transformed Phenotype by Ascorbic Acid inC3H/1OT% Cells1
William F. Benedict, William L. Wheatley, and Peter A. Jones
Division of Hematology-Oncology, Deportment of Medicine, Childrens Hospital of Los Angeles 1W. F. B., W. L. W., P. A. J.j, and Departments of Pediatrics (W. F.B., W. L. W.. P. A. J.j and Biochemistry (P. A. J.j, University of Southern California School of Medicine, Los Angeles, california 90027
diesterase inhibitors (4), and protease inhibitors (13). Increased serum concentration has also been reported to produce this effect (3). However, the inhibition has been completely reversible, and the nontransformed 10T½cells presentin the culture have been implicated as a factor in the growtharrest of the transformed cells produced by phosphodiesteraseinhibitors and increased serum (3, 4).
We wish to report that not only can ascorbic acid at bow,noncytotoxic concentrations completely prevent the expression of MCA2-produced transformation when added up to 3weeks after treatment with the carcinogen, but it can alsocause cells which are already morphobogically transformed toresume a normal morphological appearance. Moreover, thisreversion to a normal phenotype is often irreversible.
MATERIALS AND METHODS
Chemicals. Ascorbic acid and /1-aminopropionitrile fumaratewere obtained from Sigma GhemicabGo., St. Louis, Mo. Ascorbic acid was recrystallized from ethanol, dissoved in PBS at0.02 to 2.5 mg/mI, and stored in frozen aliquots at —20°.Bacterial colbagenase,type CLS, was obtained from Worthington Biochemicabs,Freehold, N. J., and Viokase was purchasedfrom the Viobin Corp., Montecelbo, Ill. Fetal calf serum wasfrom either Irvine Scientific, Irvine, Calif., or Flow Laboratories,Inc., Rockville, Md. The MCA was obtained from Gabbiochem,San Diego, Calif., and retinyb acetate was a gift from Dr. L.DeLuca, Bethesda, Md.
Cells and Transformation Studies. The 10T½cell line wasused between passages 9 and 13. GeII culture conditions forthis line have been previously described (2, 10, 20). Transformation assays were carried out in 60-mm plastic tissue culturedishes (Lux Scientific, Thousand Oaks, Calif.). The basic procedures for performing the transformation assay also havebeen previously described (2, 10, 20). In these experiments,2000 cells were seeded into the tissue culture dishes. Twentyfour hr later, 20 @tlof acetone containing MCA were added togive a final MCA concentration of either 1 or 5 @,tg/mI.Theexposure to MGA was for 24 hr, and control dishes receivedan equal volume of acetone for the same duration. The mediumwas then removed, the dishes washed once with PBS, andfresh medium was added. Ascorbic acid was then added to thedishes at a final concentration ranging from 1 to 25 pg/mb. Thehalf-life of the ascorbate in the medium is approximately 1 hr(5). In some experiments, the cultures were exposed to ascor
2 The abbreviations used are: 1 0T½, C3H/1 0T'/aCL8; MCA, 3-methylcholan
This work was supported by Grant CA-i 4226 from the National CancerInstitute.
Received February 15, 1980; accepted May 12, 1980.
CANCERRESEARCHVOL. 402796
ABSTRACT
C3H/10T½ mouse embryo cells were exposed to 3-methylchobanthrene (MCA) for 24 hr. The daily addition of ascorbicacid at the noncytotoxic concentration of 1 @ag/mlcompletelyprevented the expression of transformed foci at 42 or 56 daysafter the experiment was begun if the ascorbate was addedimmediately after MCA exposure and daily thereafter for a totalof 22 days. Ascorbic acid also could be added as late as 23days after MCA treatment and still completely inhibit morphological transformation if maintained in the cultures until thedishes were stained.
The expression of the transformed phenotype in approximately 80% of the cultures containing transformed foci wasalso completely blocked following subculture at 6 to 8 weeksafter MCA treatment if the transformed cells from these focisubsequently received ascorbic acid, 1 @tg/mI,daily. Only 40%of cultures containing transformed foci returned to a normalmorphological phenotype if the cultures were maintained for10 weeks after MCA exposure before passage. The reversionof transformed cells to a normal morphological pattern oftenappeared to be irreversible since, after four subcultures in thepresence of ascorbate, the ascorbic acid could subsequentlybe removed without reexpression of the morphological transformation. Transformed cells, however, became refractory tothe effect of ascorbic acid if they were initially subcubtured
several times without ascorbate and then were exposed to thevitamin. This variation in the response to ascorbic acid whencomparing early- and late-passaged transformed cells mayindicate a fundamental biological difference between thesetransformed cells although they have a similar morphologicalpattern.
The exact mechanism(s) responsible for inhibition and reversion of the transformed phenotype is presently unknown. However, differential cytotoxicity between transformed and nontransformed cells, the production of an extracellular matrix inascorbate-treated cultures, adipocyte formation, or increasesin adenosine cyclic 2':3'-monophosphoric acid were not foundto be implicated in producing these effects.
INTRODUCTION
Recently, it has been shown that several substances withdiverse structures can inhibit the chemical transformation ofC3H/i OT½CL8cells. These include retinoids (16), phospho
bate at various times after the MGA treatment as described inâ€â€˜Results.― The ascorbate was added daily, producing no
measurable change in pH, and the medium was changed twiceweekly. Six to 10 weeks following the initiation of each experiment, the cultures were fixed with methanol and stained withGiemsa. They were then scored for the presence of type Ill fociwhich are known to produce sarcomas when injected intoimmunosuppressed syngeneic mice (10, 20).
Growth Curves. 10T½cells or the well-characterized transformed cell clones MCA-TGL1 5 (1 1) or J2BB (1 1) derived from
the parental line were trypsinized and plated at a concentrationof 1O@cells/60-mm dish. Ascorbate (1 @tg/mI)was added toone-half of the dishes of each cell line beginning the day afterseeding, and fresh ascorbate was added daily thereafter. Cellnumbers were determined at various times after plating by firstwashing the dishes with PBS and subsequently adding PBScontaining 0.05% coblagenase and 0.25% Viokase. The cellswere then aspirated to form a single-cell suspension andcounted in a Coulter counter. Duplicate counts were taken,and 2 separate dishes were used for each point.
Insoluble Collagen Production. Monolayers of 10T½cellsgrowing in 60-mm dishes were treated with L-[3,4-3H]proline(New England Nuclear, Boston, Mass.; 1 sGi/ml; specific activity, 20 to 50 Gi/mmob) for 7 days with the daily addition ofascorbic acid at the indicated concentrations. The cells thenwere lysed by the addition of 0.25 M NH4OHfor 30 mm at roomtemperature, and the insoluble extracellular matrix which remained anchored to the bottom of the culture dishes waswashed with water and ethanol (12). The matrices were airdried and treated with trypsin (10 @eg/ml)for 3 hr at 37°toremove glycoproteins and other trypsin-sensitive proteins. Thetotal residual radioactivity which was sensitive to bacterialcoblagenase (Worthington type GLSPA) was then determinedusing previously published methods (12).
In some experiments, the unlabeled extraceblubarmatricesproduced by 10T½cells in the presence (1 peg/mI)or absenceof daily additions of ascorbic acid for 21 days were preparedas indicated above. These matrices were sterilized in 70%ethanol for I 0 mm, washed with PBS, and then used as asubstrate for freshly added 10T½cells.
cAMP DetermInations. 10T½cells were seeded into 100-mm dishes In the presence or absence of daily additions ofascorbic acid (1 @tg/mb/day).Medium was changed twice aweek; 10 days after seeding, the medium obtained from 48 hrincubation with the cells was harvested, and the cAMP levelwas determined (4) using a radioimmunoassay kit obtainedfrom New England Nuclear. The cells were also harvested, andthe intracellular levels of cAMP were determined using theprocedure supplied with the assay kit.
RESULTS
Inhibition of the Expression of Transformation by Ascorbate following MCA Treatment. In our initial studies, we foundthat ascorbic acid completely inhibited the morphological transformation of 10T½cells caused by MGA when the vitamin wasadded Immediately after the exposure if the cultures were fixed6 weeks after the MGA exposure (Table 1). Since the vitaminat a concentration of 1 @sg/mlproduced a total inhibition oftransformation, all subsequent studies were done at this concentratlon. It was observed that the ascorbate could be added
daily for the period from Day 2 to Day 23 following MGAtreatment and still completely block the transformation if theexperiment was terminated on Day 42 (Chart 1) or Day 56(Chart 2). Two transformed foci were observed under similarconditions if the experiment was continued for a total of 72days before fixation (Chart 2). Exposure to ascorbic acid fromDay 2 to Day 9 or Day 2 to Day 16 did not inhibit the expressionof transformed foci entirely (Chart 1).
We next determined how late after MCA treatment the ascor
Table 1Concentration-dependent inhibition of MCA-induced morphological
transformation by ascorbic acid
aMCA(1@ug/ml)wasaddedastheinitiatingconcentrationinExperimenti.b MCA (5 @g/ml) was added as the initiating concentration in Experiment 2.
NQDI$hSSw,1t@TypeID@NaI@shesTreated
0 0/23
I 32/68
0/27
0/28
0/29
0/25
7/29
4/30
0/30
DAYS AFTER MCA ADDIT1ON
Chart 1. Inhibition of MCA-initiated transformation by ascorbic acid. Cellswere treated with MCA (@)or acetone (0). Ascorbate (1 @g/ml)was subsequentlyadded daily as shown (@. Dishes were then stained at Day 42 and scored for thepresence of type Ill foci.
Time (wk) after initiating the experiment at which cultures containingfoci were passagedNo.
of original cultures in which thetransformed phenotype was cornpletely suppressed when subcultured and treated withascorbateTotal
no. of original cultures treatedwith ascorbate in subsequent pas
sage6
781015/20(75)a
5/6 (83)8/11(73)
6/15(40)a
Numbers in parentheses, percentage.
w.F.Benedictetal.bate could be added and still inhibit the expression of transformation. It was found that ascorbate could be added as late as23 days after MGA exposure and still completely block theappearance of transformed foci (Gharts 1 and 2).
Reversion of Transformed Cells to a Normal Phenotype.Since ascorbate was able to inhibit the formation of transformed foci following MCA treatment, it was also important todetermine whether the vitamin could block the reappearanceof type Ill foci when cultures containing such foci were passaged in the presence of ascorbate. Therefore, several culturescontaining visible type Ill foci obtained 6 to 10 weeks afterexposure to MCA were subcultured. One-half of the cultures ineach group were treated with ascorbate. The transformedphenotype in the majority of cultures was completely suppressed by ascorbic acid at the first subculture, and this effectwas seen independently of the time at which the cultures werepassaged (Table 2). Exceptions to this generality were seen incells from foci subcultured 10 weeks after the start of theexperiments. Such cells were more resistant to reversion to anormal morphological pattern than were cells from foci whichwere subcultured earlier in the experiments. In addition, unliketransformed cells passaged at 6 to 8 weeks, transformed cellssubcultured at 10 weeks which initially reverted to a normalmorphological phenotype following ascorbate treatment usually reexpressed their transformed phenotype at passage 2even when the ascorbic acid exposure was continued.
It has been shown previously by others that the weeklyaddition of retinybacetate at a concentration of 0. 1 @sg/mbcouldprevent MCA-induced transformation using the same C3H/10T½system (16). Consequently, we wished to determinewhether retinybacetate could also block the expression of thetransformed phenotype when the transformed cells were passaged in the presence of retinyl acetate. Therefore, cells fromseveral transformed foci which had previously been found tobe suppressed by ascorbate (Table 2) were exposed to 0. 1of retinyl acetate weekly. None of the particular transformedcells used in this experiment had ever been exposed to ascorbate. In contrast to the effect of ascorbic acid, none of thetransformed cells showed any suppression of the transformedmorphological phenotype when exposed to retinyl acetate(data not shown).
Irreversibility of the Response to Ascorbate. We wishedalso to examine whether the response to ascorbate was reversibbe, since all chemicals and conditions which previouslyhad been reported to inhibit transformation require continuoustreatment, and the transformed phenotype is expressed oncethe chemical is removed (3, 4, 13, 16). Three separate disheswhich contained transformed foci were subcultured 6 weeksafter exposure to MCA. One-half of each subculture receivedascorbic acid and one-half did not. The expression of thetransformed phenotype was suppressed in each culture receiving ascorbate (Chart 3, M-9-A through M-i 1-A). Numerous fociwere observed in the cultures which did not recieve ascorbate(Chart 3, M-9-N through M-i I -N). The cultures initially exposedto ascorbate were then maintained on the vitamin for 3 subsequent subcultures. No reexpression of morphological transformation was observed. The ascorbate was then removed fromone-half of the cultures which had previously been exposed tothe vitamin. No transformed foci subsequently appeared in anycultures whether they remained on ascorbate or not (Chart 3).Thus, the inhibition of the transformed phenotype by ascorbate
Table 2Suppression of the transformed phenotype by ascorbic acid upon initial
subcultureAt various times after the experiment was begun, numerouscultures containing
type Ill foci were each subcultured into 4 dishes. One-half of each subculturereceived daily ascorbate (1 tug/mI). Approximately 2 weeks later, one representative dish for each ascorbate-treated and non-treated subculture was stainedand examined for the presence of type Ill transformed foci. The data were thenexpressed as the number of original cultures in which the expression of thetransformed phenotype was completely inhibited by ascorbate on subsequentpassage compared to the total number of original cultures passaged and thentreated with ascorbate. All subcultures not treated with ascorbate containednumerous type III foci.
Chart 3. Irreversible suppression of the transformed phenotype by ascorbicacid. Three separate cultures containing type III foci (M-9 through M-i 1) weresubcultured as described in the text. One-haft of the dishes received ascorbate(A). and one-half did not(N). Representative dishes were then stained and scoredfor the presence fU@or absence (EDof type III foci. All cultures treated withascorbate contained no transformed foci (M-9-A through M-i 1-A), whereas allparallel cultures not exposed to ascorbic acid contained numerous foci (M-9-Nthrough M-i 1-N)' The cells from the remaining dishes which did not Initiallyreceive ascorbate were passaged an additional 3 times without ascorbate, andcells from the ascorbate-treated dishes were subcultured 3 more times in thepresence of ascorbate, Subsequently, at passage 5, one-half of the cultures notpreviously exposed to ascorbate were treated with ascorbic acid, whereasascorbate was removed from one-half of the cultures which had received ascorbicacid continuously from the first subculture. At each passage, dishes were stainedand scored for the presence or absence of type Ill foci.
was maintained over numerous cell divisions even after theascorbate was removed. In contrast, parallel transformed cubtures which received no ascorbic acid (M-9-N through M-1 1-N) were not suppressed by the vitamin if they were passagedseveral times before being treated with ascorbate (Chart 3).Therefore, it appears that there is a critical time at which themorphobogically transformed cells lose the capacity to be inhibited by ascorbate.
Effect of Ascorbate on Cell Growth. The effects of ascorbicacid on the expression of the transformed phenotype mighthave been due to cytotoxicity. However, daily additions ofascorbic acid (1 sg/ml/day) had no significant influence onthe growth rate either of nontransformed 1OT'/acells or of 2transformed clones derived from them, J2BB or MGA-TGL 15(Chart 4). Ascorbate also did not alter the plating efficiency at
Chart 4. Growth curves of 10T½cells and 2 transformed 1OT',4cell clones (MCA-TCL15 and J2BB) in the presence (0) or absence (•)of daily ascorbate.
this concentration (results not shown). Therefore, the effectsof ascorbate on the expression of the transformed phenotypecannot be related to any differential cytotoxicity between the10T½cells and their transformed counterparts.
Relationship between Extracellular Matrix Production andInhibition of Transformation by Ascorbic Acid. We haveshown in previous studies that the deposition of insolublecollagen into the extraceblular matrix produced by culturedcells is markedly modulated by the daily addition of ascorbicacid (7). We therefore tested the abilities of different concentrations of ascorbate to alter the composition of the extracellular matrix elaborated by 10T½cells (Chart 5). Little insolublecollagen was associated with the matrix produced in the absence of the vitamin, and almost all of the insoluble proteinpresent was trypsin sensitive. Considerable colbagenase-sensitive radioactivity, however, did appear in the insoluble matrixproduced by 10T½cells grown in the presence of ascorbate(1 @sg/ml/day).
Itwasthereforepossiblethatthe insolublecollagenproducedin the presence of ascorbic acid was responsible for the abilityof the vitamin to block the expression of the transformedphenotype. This possibility was discounted by 2 further experiments. (a) If the transformation assays were carried outwith cells seeded onto extraceblubarmatrices previously produced by 10T1I4cells in the presence or absence of ascorbicacid, no significant differences were observed in the transformation frequencies compared to cultures not containing apreformed matrix (Table 3). The presence of a preexistingextraceblularmatrix, in fact, appearedto stimulatethe transformation frequency when compared to cells grown on plastic. (b)The ability of ascorbate to inhibit the morphological transformation induced by MCA was not affected by simultaneousexposure to @-aminopropionitrilefumarate (Table 4), a compound which inhibits collagen cross-linking (12). We thereforeconclude that, although ascorbic acid is necessary for theproduction of insoluble collagen by 10T½cells, the presenceof this collagen is not responsible for the effects of the vitaminon the expression of the transformed phenotype.
Effect of Aecorbate on cAMP Levels. It has been shownpreviously that phosphodiesterase inhibitors can block the
500
400
300
200
Jig ascorbote/day
1025
Chart 5. Effect of ascorbic acid on the presence of insoluble collagen in theextracellular matrix produced by 10T½cells. 10T½cells were grown with theindicated concentration of ascorbic acid in the presence of (3Hjproline for 7 days.Subsequently, the cells were removed by NH40H lysis, and the total radioactivitywhich was sensitive to bacterial collagenase was determined.
expression of MCA-induced transformation (4) and thatchanges in levels of cyclic nucleotides may be involved in thisresponse (4). The ability of ascorbic acid to influence cAMPlevels in the culture medium and within 10T½cells was therefore examined (Table 5). The vitamin had little effect on theintracellular cAMP level and appeared to decrease the extracellular level of the cyclic nucleotide. The effects of ascorbateon the transformed phenotype were not, therefore, due tomarked increases in cAMP bevels.
Adipocyte Formation following Ascorbate Exposure. Aswith our early studies (25), it was noted that many adipocytesappeared in ascorbate-treated cultures. However, when cellsfrom transformed foci were subcubtured and exposed to ascorbic acid, adipocytes were observed with equal frequency inthose cultures where no suppression of the transformedphenotype occurred as in the cultures where the expression oftransformation was completely inhibited. Therefore, no apparent relationship was noted between adipocyte formation andthe inhibition of the transformed phenotype.
Effect of preexisting 10T½ matrix on MCA-induced transformation
10T½cells were grown in the presence or absence of ascorbic acid (1 g@g/ml)for 21 days. Subsequently, the cells were removed by lysis with 0.25 N NH4OHto leave the extracellular matrix anchored to the bottom of the culture dishes.These matrices were then used as substrates for 10T½cells which were addedsubsequently as in the standard transformation assay (outlined in the text) andtreated with MCA (5 @g/ml).The dishes were fixed and stained 6 weeks aftertreatment in the absence of further additions of ascorbic acid and scored for thepresence of type III foci. Results are cumulative data from 2 separate experiments.
appear to be directly relevant to our findings. These haveincluded studies on the inhibition of carcinogenic nitroso compounds (15), antiviral activity (17, 26) including the inhibitionof RNA tumor virus replication and infectivity (5), antimutagenicactivity (9, 21), cytotoxicity (19), differentiation (22), production of chromosomal aberrations (23, 24), increases in sisterchromatid exchanges (8), and the production of single- anddouble-strand DNA and RNA breaks (18). Several have norelationship to our studies because they concern modificationby ascorbate of known mutagenic or carcinogenic chemicalsin order to prevent their direct interaction with DNA or RNA.Since ascorbate was added after the exposure to MGA in ourexperiments, the vitamin could not modify the initial binding ofMCA or its various metabobitesto macromolecules.
The other main reason why the previous papers on ascorbateseem to have little relationship to our studies is that between20 and 2000 times higher concentrations of ascorbate wereused for those experiments compared to the concentrationsused in our own experiments. For example, a concentration of1 x 1O-@ M or more of ascorbate was required to produce asignificant increase in sister chromatid exchanges (8) or chromosomal aberrations (22, 23). This is over 200 times theconcentration used for our studies and, in fact, ascorbate (1@zg/ml)in cell culture produces no increases in sister chromatidexchanges or chromosomal aberrations.3 An exception to thislack of relevancy may be the preliminary observation by Leuchenberger and Leuchenberger (14) that transformed hamsterlung cultures seemed to revert to a more normal appearanceafter approximately 12 weeks of continuous exposure to vitamin C at a concentration of 20 pg/mI.
The exact mechanism(s) by which ascorbate inhibits MGAinduced transformation and causes morphobogicalby transformed cells to revert back to a normal morphological phenotype is yet undetermined. Differential cytotoxicity, productionof an extracellular matrix, adipocyte formation, or large increases in cAMP bevelswere not responsible for this inhibition.Its mechanism of inhibition also differs from that produced byretinyl acetate (16) since transformed cells which respond toascorbate did not revert to a normal morphological phenotypewhen retinybacetate was added to the cultures.
It has also been shown that cells from transformed foci areless responsive to ascorbate after 10 weeks in culture than arecells from foci maintained for shorter periods (see Table 2). Inaddition, transformed cells also become refractory to the effectof ascorbate after several passages in culture without ascorbate (see Chart 3). One could therefore suggest that there arefundamental biological differences between ascorbate-responsive and -nonresponsive transformed cells. It has been shownin Syrian hamster cells that neoplastic transformation in cell.culture is progressive in nature and that morphological transformation is an early change, whereas boss of anchorageindependent growth as measured by colony formation in semisolid agar occurs many population doublings after the carcinogen exposure (1). An obvious possibility, therefore, is thatmorphologically transformed cells which can have their transformed phenotype suppressed by ascorbic acid have not progressed to the stage where they can grow in semisolid mediumor be tumorigenic. In contrast, those transformed cells which
3 W. Benedict and A. Banerjee, unpublished observation.
Table 4Effect of simultaneous exposure to ascorbic acid and BAPN' on MCA-induced
transformation1OT/2cells (2000/dish) were treated 24 hr after seeding with MCA (5 @g/ml)
for a further 24 hr. Subsequently. the medium was replaced with mediumcontaining the indicated additions of BAPN (50 @g/ml)or ascorbate (1 @.tg/ml).BAPN was included in the medium which was changed twice a week, andascorbic acid was added daily. Cultures were fixed and stained 6 weeks afterseeding and scored for the presence of type Ill foci. Results given are thecumulative data from 2 separate experiments.
Table 5Effect of ascorbic acid on intra- and extracellular levels of cAMP
1OT'/2cells were seeded into 100-mm dishes and treated with ascorbic acid(1 .tg/ml) for 10 days. Subsequently, the intra- and extracellular levels of cAMPwere determined as detailed in the text.
2800 CANCERRESEARCHVOL. 40
DISCUSSION
Many papers have been written concerning ascorbic acidand cancer, much of which has been the subject of a recentreview (6). In our studies, we have shown that ascorbic acid atthe bow,noncytotoxic concentration of 1 zg/ml (6 x 10_6 M)can completely inhibit morphological transformation in 10T½cells when added daily, even when begun 23 days after exposure to the carcinogen, MCA. Moreover, ascorbate cansuppress the expression of the transformed phenotype in cellswhich are already transformed. It is of particular importancethat the suppression appears often to be irreversible (see Chart3), since all other chemicals and conditions which have beenreported to inhibit the expression of the transformed phenotypewere completely reversible (3, 4, 13, 16).
Although studies on ascorbate in vitro are numerous, none
Heidelberger, C. Oncogenic transformation of C3H/10T½clone 8 mouseembryo cells by halogenated pyrimidine nucleosides. Cancer Res., 36: 101-107, 1976.
11. Jones, P. A., Laug, W. E., Gardner, A., Nye, C. A., Fink, L. M., and Benedict,W. F. In vitro correlates of transformation of C3H/1 0T½clone 8 mousecells. Cancer Res., 36: 2863—2867,1976.
12. Jones, P. A., Scott-Burden, T., and Gevers, W. Glycoprotein, elastin, andcollagen secretion by rat smooth muscle cells. Proc. NatI. Acad. Sci. U. S.A. 76: 353-357, 1979.
13. Kuroki, T., and Drevon, C. Inhibition of chemical transformation in C3H/10T½cells by protease inhibitors. Cancer Res., 39: 2755—2761, 1979.
14. Leuchenberger, C., and Leuchenberger, R. Protection of hamster lungcultures by L-cysteine or vitamin-C against carcinogenic effects of freshsmoke from tobacco or manhuana cigarettes. Br. J. Exp. Pathol., 58: 625—634, 1977.
15. Lo, L. W., and Stith, H. F. The use of short term tests to measure thepreventive action of reducing agents on formation and activation of carcinogenic nitroso compounds. Mutat. Res., 57: 57—67,1978.
16. Merriman, R. I.., and Bertram, J. S. Reversible inhibition by retinoids of 3-methylcholar.threne-induced neoplastic transformation in C3H/ 1OT'i4clone8 cells. Cancer Res., 39: 1661—1666,1979.
17. Murata, A., Oyadomari, R., Ohoshi, T., and Kitagawa, K. Mechanism ofinactivahon on bacteriophage A containing single-stranded DNA by ascorbicacid. J. Nutr. Sci. Vitaminol., 21: 261—269,1975.
18. Omura, H., liyama, S., Tomita, V., Narazaki, V., Shinohara, K., and Murakami, H. Breaking action of ascorbic acid on nucleic acids. J. Nutr. Sci.Vitaminol., 21: 237—249,1975.
19. Peterkofsky, B., and Prather, W. Cytotoxicity of ascorbate and other reducing agents towards cultured fibroblasts as a result of hydrogen peroxideformation. J. Cell Physiol., 90: 61—70,1976.
20. Reznikoff, C. A., Bertram, J. S., Brankow, D. W., and Heidelberger, C.Quantitative and qualitative studies of chemical transformation of clonedC3H mouse embryo cells sensitive to postconfluence inhibition of celldivision. Cancer Res., 33: 3239-3249, 1973.
21. Rosin, M. P., and Stich, H. F. Assessment of the use of the Salmonellamutagenesisassay to determine the influence of antioxidants on carcinogeninduced mutagenesis. Int. J. Cancer, 23: 722—727,1979.
22. Schwarz, R. I., and Bissell, M. J. Dependence of the differentiated state onthe cellular environments: Modulation of collagen synthesis in tendon cells.Proc. Natl. Acad. Sct. U. S. A., 74: 4453—4457,1977.
23. Stich, H. F., Karim, J., Korapatnick, J., and Lo, L. Mutagenic action ofascorbic acid. Nature (Lond.), 260: 722—724,1976.
24. Stich, H. F., Wei, L., and Whiting, R. F. Enhancement of the chromosomedamaging action of ascorbate by transition metals. Cancer Res., 39: 4145—4151, 1979.
25. Taylor, S. M., and Jones, P. A. Multiple new phenotypes induced in 10T½and 3T3 cells treated with 5-azacytidine. Cell, 17: 771—779,1979.
26. Wong, K., Morgan, A. R., and Paranchych, W. Controlled cleavage of phageRi 7 RNA within the vinon by treatment with ascorbate and copper(Il). Can.J. Biochem., 52: 950-958, 1974.
2801
no longer are inhibited by ascorbate may have now acquiredthe capacity to proliferate in soft agar and produce tumors inviva. Studies to determine whether this hypothesis is correctare being done in our laboratory at present.
Theoretically, it could also be hypothesized that cells earlyin their neoplastic development in vivo might also be suppressed by continuous bowlevels of exposure to ascorbic acid.Unfortunately, there are no studies in vivo to date that addressthis hypothesis. The use of guinea pigs, which bikehumans donot synthesize ascorbate, should be chosen for such experiments so that the importance of exogenous daily ascorbateand the relevancy of our findings to the human situation can beadequately determined. We believe that such studies need tobe undertaken now.
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1980;40:2796-2801. Cancer Res William F. Benedict, William L. Wheatley and Peter A. Jones in C3H/10T½ Cellsand Reversion of the Transformed Phenotype by Ascorbic Acid Inhibition of Chemically Induced Morphological Transformation
