ascites tumor development i. an analysis of the in vivo ... · 5.41; 1.8 mg. dry weight) on the...

Ascites Tumor Development

I. An Analysis of the in Vivo Effect of Nucleoproteinfrom Ehrlich Ascites Cells*

EDWARDC. HoRNf

WlTH THE TECHNICAL ASSISTANCE OF MART ELIZABETH HOUSE

(Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tenn.)

The intraperitoneal injection of nucleoprotein(NP) extracted from Ehrlich ascites tumor cellsreduces the mean survival time of mice when theyare subsequently challenged with living ascitescells (13). Identical injections with or after theintroduction of the tumor produced no significantchange in the average survival period. The purposeof this paper is to extend the original observationsand to discover, if possible, whether the accelerated death is caused by (a) a sensitivity reaction toNP, in view of the demonstrated antigenicity ofthis type of material (5, 18) ; (6) the neutralizationof a naturally occurring antitumor factor by NPresulting in more rapid tumor growth; (c) a directstimulating effect of NP on ascites cells; (d) thepossible presence of a toxin in the NP; or (e) theremoval by the NP of some substance, present intrace amounts, which is also removed by thetumor.

MATERIALS AND METHODSMice.â€”All mice used in these experiments

were male hybrids (C3H/R1 X 101/R1)1 supplied

through the generosity of Dr. William L. Russelland staff. Age variation in the experimental micedid not exceed 3 weeks; all animals were about 65months of age at the beginning of the experimentand averaged ca. 30 gm. in weight.

Ascites tumor cells.â€”The subline of Ehrlich

ascites tumor used was originally transferred fromMontefiore Hospital, New York City, to Duke

* This work was performed under Contract No. W-7405-eng-26 for the Atomic Energy Commission, and was supported in part by research grant (RG-4203) from the National Institutes of Health, Public Health Service.

â€¢fOn sabbatical leave from the Department of Zoology,Duke University, Durham, North Carolina.

1Designated after Standardized Nomenclature for InbredStrains of Mice, Committee on Standardized Nomenclaturefor Inbred Strains of Mice, Cancer Research, 12:602-13,1952.

Received for publication May 18, 1955.

University early in 1954 and in June of that yearbrought to Oak Ridge. Thereafter the subline wasmaintained in the hybrid stock and occasionally inCSH/Jax1 stock of both sexes. Early transfers

were made by injecting intraperitoneally 0.2 ml.of clear, undiluted ascites fluid from 7-12-day

growths. Because of inconsistent results, themajority of transfers to hybrids was made with asaline dilution of a suspension containing 16.0 XIO6 to 18.0 X IO6 cells in a 0.2-ml. aliquot. In the

smaller C3H/Jax mice more satisfactory resultshave been obtained by transferring 5.0 X IO6 to7.0 X IO6 cells as a 0.1-0.2-ml. injection diluted

with saline. Injections of these quantities of ascitescells consistently resulted in growth of the tumorand subsequent death of the mouse.

Nucleoprotein extraction procedure.â€”The nucleo

protein preparations used in these experimentswere extracted from nuclei of ascites tumor cellsisolated after the manner of Goldberg, Klein, andKlein (11). Pooled ascites fluids were obtained bydraining the peritoneal contents of ascitic miceinto a small quantity of neutralized Versene2 solu

tion to prevent clotting. The poolings were alwaysobtained from a single group of 10-30 mice whichhad received ascites tumor cells at the same time.All ascitic fluid used in these experiments wasfreshly obtained. The pooled fluid was cooled rapidly, and subsequent steps in extraction were carried out with chilled glassware and refrigeratedequipment kept at 4Â°C. Quick-frozen pooled fluidstored at â€”20Â°C. and later thawed for use was

found not to yield satisfactory extracts. Afterscreening through bolting silk, the fluid was cen-trifuged for 15 minutes at relatively low forces(230 X <7),3and the residue was resuspended in

2Disodium salt of ethylenediamine tetraacetic acid,Bersworth Chemical Company, Framingham, Massachusetts.

3The relative centrifugal forces given were calculated forthe bottom of the centrifuge tube.

663

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664 Cancer Research

0.14 M NaCl. Figure 1 illustrates a preparation atthis stage. In those cases where contaminationwith erythrocytes was heavy, the red blood corpuscles were hemolyzed by suspension in 0.07 MNaCl, and the ascites cells were separated from theghosts centrifugally. The ascites cells were not affected for extraction purposes by this treatment.Successive washes with 0.14 M NaCl were repeateduntil the residue was untinged and the supernateclear. The resulting packed cells were then suspended in 10 times their volume of 1 per cent citricacid and blended4 for 2 minutes at a reduced speed(70 volts).6 The dilute homogenate was then cen-

trifuged at an RCF of 600 X g for 15 minutes.Washing of the residue in 5 times its volume of 1per cent citric acid (centrifuged 10 min. at 450 Xg) was repeated until the supernate was clear; thisprocedure gave good yields of microscopicallyclean nuclei. A homogenate after one citric acidwash is shown in Figure 2. Examination of suspensions at this and subsequent stages of preparationreveals what appear to be isolated chromatic figures(indicated in Fig. 2). Work is in progress to determine the true nature of these configurations. Figure 3 shows the same homogenate after washingand suspending in 0.14 M NaCl. Since whole cellsand nuclei respond differently to changes in tonici-ty or ionic composition (1), they may be readilydistinguished (6). Observations on the effects ofvarious solutions on nuclei isolated by the methoddescribed here showed that these preparations wereremarkably free from whole cells or nuclei coatedwith a thin layer of cytoplasm.

To prepare nucleoprotein, the isolated nucleiwas suspended in 10 times their packed volume ofdistilled water adjusted to neutrality with a smallamount of NaHCOs. If, as indicated by the increased residue volume after centrifugation, thenuclei had begun to swell, the final suspension ofnuclei was made in a volume of neutralized distilled water determined by the concentration ofthe product desired, e.g., suspension in 3 times theoriginal packed nuclear volume yielded (afterfinal neutralization) an extremely viscous mass,the most concentrated employed in these experiments. If the nuclei had not swollen, they werewashed in 10 times their packed volume of neutralized distilled water; this usually sufficed toremoved most of the citric acid. The final suspension in distilled water was then neutralized with afew drops of 0.01 N NaOH to produce the viscousmaterial termed "nucleoprotein" in these experi

ments.

4Waring Blendor.

170-Volt setting on an autotransformer (Variac).

Various attempts to purify this product or tovary the extraction procedure resulted in extremely poor yields of products with low viscosity andlow solubility in distilled water. Therefore, theoutlined procedure was maintained although itwas realized that the yield was a complex productof extracted whole nuclei which, among otherthings, should contain a large measure of undis-sociated deoxyribonucleoprotein (3, 4) rather thana dissociated, poorly soluble, purified deoxyribo-nucleic acid-basic protein product.

Injections were made with freshly preparedproducts.

Chemical analyses.â€”Nitrogen-phosphorus ra

tios were obtained for all but the very earliestproducts used. For four preparations, these ratiosranged between 5.41 and 5.86. Biuret determinations for total protein nitrogen equivalents and theDische diphenylamine reaction for deoxyribonu-cleic acid (DNA) determinations were made togive a basis for determining relative concentrations for different nucleoprotein preparations. Afew DNA/RNA ratios were determined after thetechnic of Ogur and Rosen (21); these yieldedvalues of approximately 5:1.

RESULTSDifference in mean survival with time following a

single pretreatment with N P.â€”Thirty mice were

injected intraperitoneally with 0.4 ml. of a NPpreparation (N/P = 5.86; 10.3 mg. dry weight).Thirty control animals were set aside at the sametime and treated identically except for lack of pretreatment with NP. At weekly intervals beginningon the 14th day and continuing for 5 weeks thereafter, five experimental and five control mice werechallenged with an ascites cell suspension. Theirdeaths were recorded twice daily and the mean survival time for each group calculated. The resultsare shown graphically in Chart 1; the data aresummarized in Table 1. Since the difference between the mean survival time of the controls (Sc)and that of the expÃ©rimentais (Se) fluctuated withthe number of ascites cells used for challenge, aquotient obtained by dividing the difference inmean survival time of the two groups by the meansurvival of the controls

So-Se

s.allowed for comparison between different challenges. This quotient is plotted against time inall of the graphs which involve differences inmean survival time. The data in Chart 1 for thesimultaneous injection of NP and ascites cells(day 0) and that for the 7-day interval were



HORNâ€”AscitesTumor Development. I 665

taken from another experiment which was comparable in treatment but did not use the sameseries of animals.

The data showed some irregularity, but a maximum decrease in mean survival time (high quotient) was evident 3 weeks after the injection ofNP; thereafter this difference decreased somewhatmore gradually than the initial rise to the maximum. Similar results were reported earlier in muchless complete form and have been corroborated byadditional experiments.

Modification of the difference in mean survivaltime of pretreated mice by a second injection of N P.â€”Twenty mice which had been prepared identically to those used for the time data above weregiven a second injection (0.1 ml.) of NP (N/P =5.41; 1.8 mg. dry weight) on the 35th day of theexperiment. Ten of these animals received thematerial intravenously; ten, intraperitoneally.Five of each group were challenged with ascitescells 7 and 14 days after this second NP injection(or 42 and 49 days after the initial injection).Chart 1 is marked with a dotted arrow at 35 daysto indicate the time of second injection, and thetwo broken lines connect points which mark thedifferences in mean survival for these two groups.It is readily apparent that the mean survival timefor the mice receiving a second injection of NP wasdrastically shortened not only when comparedwith controls but also with those mice receivingbut one injection. Table 1 includes the data forthis experiment.

Modification of the difference in mean survivaltime of pretreated mice by an injection of killedascites cells or of ascites fluid.â€”In these experiments cell-free ascites fluid or washed ascites cellskilled with weak formalin solution were used fora second injection into NP-prepared animals. Thecell-free fluid was obtained by centrifuging clear,pooled ascites suspensions and by passing thesupernatant fluid through a sterilized, sinteredglass filter. The formalin-killed cells were obtainedby suspending saline-washed cells in 0.5 per centformalin-saline for 24 hours at 4Â°C. These cells

were then washed thoroughly in 0.14 M NaCl bysuccessive suspension and centrifugation. Finalsuspensions for injection were made in saline.

Twenty-one days after receiving the initial NPinjection, four mice were given a second injection(0.5 ml. intravenously) of a 50 per cent suspensionof killed cells. Five uninjected controls receivedthe same treatment. Twenty-one days later (day42 of the experiment) both groups were challengedwith the same ascites suspension used for the injection of mice already described for day 42 in thetime study. The mice which had received killed

cells alone showed the greatest decrease in meansurvival time yet observed, whereas the mice prepared first with NP and then with killed cellsshowed no difference in mean survival time fromthe control ascites-injected mice of day 42(Table 2).

Five NP-injected mice were each given intravenously a second injection of 0.4 ml. of undilutedcell-free ascites fluid (approximately 8 mg. of protein) 21 days after the initial NP injection; fourunprepared controls received identical treatment.Twenty-one days later (day 42 of the experiment)both of these groups were challenged with thesame ascites cell suspension used for all other 42-day challenges. The animals which received thefluid alone actually survived slightly better thanthe ascites control, whereas those that received

INTERVAL FROMNP INJECTION TO ASCITES CHALLENGE I doyÂ«)

CHART1.â€”Relativedifferences in survival between controlmice (Sc) and NP-injected mice (Se) when challenged withascites suspensions at different time intervals after the NP injection. The dotted arrow indicates a second NP injection at35 days. The broken lines connect points indicating relativesurvival for mice receiving the second injection either intraperitoneally (IP) or intravenously (IV).

NP and later the fluid injections showed a difference in mean survival from the controls identicalto that of the group which had been prepared withNP alone 42 days earlier (Table 2).

Modification of the difference in mean survivaltime of pretreated mice by cortisone.â€”A fourth

group of 20 mice which had been prepared withNP on day 0, identical in every respect to all previously mentioned NP-injected mice, were givenan additional 0.1-ml. injection intravenously ofNP on the 56th day of the experiment. Ten ofthese mice were administered 1 mg. each of cortisone6 intramuscularly on days 60, 61, and 63. Onthe 63d day all twenty of these animals were challenged, together with uninjected controls. As expected the "boosted" NP-injected animals suc

cumbed earlier than the controls, but the NP-prepared animals which received the cortisone treatment outlived the controls (Table 2). Although

6Cortone acetate, Merck, in saline suspension.



666 Cancer Research

suggestive, these results must be conditioned bythe fact that data from a separate experiment indicate that cortisone given in the manner described does extend slightly the mean survivaltime of untreated mice challenged with Ehrlichascites tumor (Table 2), contrary to the findings ofGottschalk and Grollman (12).

Difference in mean survival time as a function of

the amount of N P injected. â€”Each of sixteen mice re

ceived intraperitoneally 0.1 ml. of a NP preparation; each of sixteen more received 0.5 ml. of thesame preparation ; and twelve more received 2.0 ml.of the preparation. Each week thereafter for 4weeks untreated control animals of the same ageand four of each category which had received NP(except those receiving 2.0 ml.) were challenged

TABLE1RANGEOFSURVIVALANDSTANDARDERRORSFORDATAPRESENTEDINCHARTl

(Survival of Tumor-challenged Mice as a Function of Time after NP Treatment)

STANDARDERROB

Â©* (Ie)INTERVAL* S t

TREATMENT (days) (days)None (7-day control) 16.6NP 7 13.5 0.81 0.06None (14-day control) 16.0NP 14 12.0 0.75 0.06None (21-day control) 9.6NP 21 6.8 0.71 0.21None (28-day control) 13.0NP 28 10.4 0.80 0.16None (35-day control) 14.4NP 35 10.6 0.74 0.12None (42-day control) 11.0NP 42 8.6 0.78 0.16NP+35 days, then NP 42 7.0 0.64 0.14

(intraperitoneal)NP+S5 days, then NP 42 7.4 0.67 0.14

(intravenous)None (49-day control) 11.4NP 49 13.0 1.14 0.14NP+35 days, then NP 49 11.2 0.98 0.11

(intraperitoneal)NP+SS days, then NP 49 10.2 0.89 0.10

(intravenous)* Time in days from beginning of treatment to tumor challenge,

t Mean survival time in days.; So denotes mean survival time in days of the treated and tumor challenged mice ; Se the mean survival time

of the untreated challenged mice.{ The shortest and longest individual survival periods within the group in days after tumor challenge.

RANQEÂ§(days)16-189-15

16-1710-146-164-8

8-179-16

9-179-159-176-115-10

5-10

10-1612-1611-12

9-12

TABLE 2*

DATAFORCOMPARISONOFSURVIVALAFTERTREATMENTWITHNP, KILLEDCELLS,ASCITESFLUID,CORTISONE,ANDCOMBINATIONSOFTHESE

INTERVAL STREATMENT (days) (days)

None (42-day control) 11.0NP 42 8.6NP+21 days, then killed cells+21 days 42 11.0None+21 days, then killed cells+21 days 42 5.4NP+21 days, then asciticfluid+21 days 42 8.6None+21 days, then ascitic fluid+21 days 42 12.5None (63-day control) 7.4NP+56 days, then NP+7 days 63 6.2NP+56 days, then NP+7 days+cortisone 63 9.0None (cortisone control) 10.9Cortisone 0 18.4

* See Table 1 for explanation of headings.

0.781.000.490.781.14

0.841.22

1.23

STANDARDERROR

/Se\ /Se\W \sj

0.160.220.110.110.18

0.150.22

0.16

RANGE(days)

9-176-118-164-89-10

11-146-106-126-15

8-134-18



HORNâ€”AscitesTumor Development. I 667

with ascites tumor cells. The data from the differences in mean survival time are plotted in Chart 2;detailed data are presented in Table 3. Those receiving 0.1 ml. and 0.5 ml. showed the usual responsewithin the limits of the experiment, although themice with the lower dosage of NP responded later.The mice which received the highest dosage (2.0

0300-

Vs.0.200

7 14 21 28INTERVAL FROM NP INJECTION TO ASCITES

CHALLENGE (days)

CHART2.â€”Relative differences in survival when challengedwith ascites suspensions at different time intervals after different initial NP injections. (â€¢)0.1 ml.; (O) 0.5ml.; (A) 2.0ml.

TABLE 8*

RANGEOFSURVIVALANDSTANDARDERRORSFORDATA PRESENTED IN CHART 2

(Survival after Tumor Challenge as a Functionof Time and Dosage of NP Treatment)

STANDARD

INTERVALTREATMENT (days)

None(7-daycontrol)0.1

ml.NP0.5ml.NP2.0

ml.NPNone(14-daycontrol)0.1

ml.NP0.5ml.NP2.0ml.

NPNone(21-daycontrol)0.1

ml.NP0.5ml.NP2.0ml.

NP777141414212121S

(dayÂ»)16.615.512.816.014.313.79.712.716.712.712.216.5(1)0.930.770.960.960.680.890.760.690.93(1)0.040.100.050.120.120.120.080.090.02RANGÂ«

(dayÂ«)16-1816-179-1515-176-1712-177-1310-1517-1911-1710-1516-17

None (28-day 15.3control)

O.lml. NP 28 11.2 0.740.5ml. NP 28 15.5 1.01

* See Table 1 for explanation of headings.

0.060.07

11-18

11-1315-17

ml.) did not show the characteristic response during the first 3 weeks.

Specificity of the reaction.â€”A number of substances have been tested to determine the specificity of the response to the NP. Included amongthese have been protamine,7 histone extracted

'Protamine sulfate, General Biochemical, Inc., ChagrinFalls, Ohio.

from chicken erythrocyte nuclei, histone extractedfrom ascites cell nuclei, and NP extracted from ratlymphoma tissue.

Protamine and chicken erythrocyte histone produced no significant depression of the mean survival time. Since ascites cell histone, on the otherhand, produced irregular reductions in the meansurvival time, an account of its preparation isgiven here.

Isolated nuclei, prepared as for NP extraction,were treated with 0.2 N HC1 overnight at 4Â°C.

The suspension was centrifuged at 8000 X g for30 min., and the clear supernate was neutralizedby the gradual addition of dilute NaOH. Any precipitate which appeared during this process wasremoved by further high-speed centrifugation for30 minutes. At or near a pH of 6 a white precipi-

0.300-

Sc-Se 0200

0100

0 10 20 30 40INTERVAL FROM INJECTION TO ASCITES

CHALLENGE (doys)

CHART3.â€”Relative differences in survival when challengedwith ascites suspensions at different time intervals following aninitial injection of NP treated with deoxyribonuclease.

tate invariably appeared, probably an acid proteincomponent. When a clear supernate with a pH of7 was obtained, it was dialyzed against severalchanges of distilled water for 24 hours. The histonesolution was then centrifuged again at 8000 X gwhether or not it appeared clear, and the supernate was quick frozen and lyophilized. The purewhite, water-soluble product was administered asan aqueous solution.

Twenty mice were each given 4.64 mg. of ascitescell histone as a 1-ml. intraperitoneal injection.Twenty other mice of the same age served as untreated controls. Groups of five treated and fiveuntreated mice were challenged each week for 4weeks, and their survival was recorded. The resultsare shown in Chart 3. Although obvious differencesin mean survival time are exhibited, the pattern iserratic.

In a small series of experiments involving tenmice injected with a NP preparation derived froma rat lymphoma, no depression in the mean survival time was discovered.

In an attempt to elucidate the criticality of un-dissociated NP for the effect, twenty mice were pre-



668 Cancer Research

pared with an intraperitoneal injection of NPtreated with deoxyribonuclease;8 the injected ma

terial had lost all typical viscosity. These animalswere challenged, in groups of five each, 14, 21, 28,and 42 days after receiving the initial injection.The differences in mean survival time are shown inChart 4, a result which resembles that obtainedwith histone in its irregularity.

Effects on tumor cells.â€”A small group of ani

mals which had received a NP injection 35 daysprior to an ascites challenge was sacrificed alongwith untreated controls at days 2, 3, 4, 5, and 7after the tumor inoculation. The spleen, liver, fatbodies, and a kidney were fixed, stained, sectioned,and examined for tumor invasion. The results wereessentially those reported by Klein and RÃ©vÃ©sz(16), namely, initial invasion of the tissues was not

Sc-Se 0300-

02OO-

0100

7 (4 27 28INTERVAL FROM HISTONE INJECTION TO

ASCITES CHALLENGE (doys)

CHART4.â€”Relative differences in survival when challengedwith ascites suspensions at different time intervals following aninitial injection of histone extracted from ascites cell nuclei.

observed until the 4th or 5th day after tumorinoculation. No difference was seen between treated and untreated animals.

Cell counts and hematocrits were also made ofthe ascites cell suspensions from control and treated animals. Again no difference in T-cell counts/unit volume of ascites fluid was found betweencontrols and treated animals although the hematocrits were invariably higher in the ascites fromtreated mice, suggesting either larger T-cells ormore contaminating cells of other types. This finding is being investigated more fully.

Tests for NP-induced sensitivity.-â€”Several tests

were conducted in an effort to show more conclusively whether the NP injections produced a sensitivity in the mice demonstrable through anaphy-lactic responses. Twenty-one, 28, and 35 days afteran initial intraperitoneal injection of NP when,according to the mean survival data, a titer against

8Desoxyribonuclease, Worthington (D394) Biochemicals.

NP should be high, a number of animals weregiven a second injection of NP at different doselevels (0.1, 0.4, and 0.6 ml.) and by a variety ofroutes (intravenous, intraperitoneal, subcutaneousand intravenous, and intravenous and intraperitoneal). In no case could any anaphylactic manifestation be demonstrated either by examining theear vessels (17) or by noting more obvious symptoms. The Arthrus reaction, so clearly demonstrated by Benedict and Tips (2) with bovineplasma albumin, was attempted with no success.

It should be pointed out that many instancesoccurred during the experiments when animals received as a second injection various amounts ofdifferent types of material (killed ascites cells,ascitic fluid) which might be expected to giveanaphylaxis, yet none was noted. Furthermore,death never occurred in animals which had receivedmany times the amount of NP used routinely toproduce the reduction in survival time, nor hadany animals in any of the experiments died fromthe NP injections alone.

DISCUSSIONThe various hypotheses outlined in the intro

duction to this paper to explain the decreased meansurvival time of NP-treated animals may be dealtwith more surely in light of the available data. Theassumption that the ascites cells themselves mightbe affected appears less attractive after studyingthe observations made at successive intervals ontheir infiltration into organs and on their multiplication in NP-injected animals. Furthermore, thishypothesis would lead to the prediction that NPand cells injected simultaneously should decreasethe mean survival time; such is not the case. Noris there any uncomplicated explanation of thelatent period with this hypothesis.

If the NP were acting to combine with someessential factor in the physiology of the mouse,thus reducing a pool of this factor to a level wheremultiplying ascites cells might finish the task morequickly, multiple injections of NP might be expected to accomplish the same end. Such was notthe case. As a matter of fact, increase in theamount of NP injected seemed to lead to a lengthened latent periodâ€”quite the opposite effect from

that which would be predicted from the assumption that a vital material was being withdrawnfrom the system.

More difficult to dispose of are the hypothesesbased on immunological phenomena. The resemblance of the curve that results when differences inthe mean survival time are plotted against time(Chart 1) to the classical curve of titratable antibody production with time following an initial



HORNâ€”AscÃ¼esTumor Development. I 669

antigenic stimulus is striking. So, too, is the survival curve resulting from a second NP injection(Chart 1, broken lines) and the curve of secondaryresponse to a second homologous antigenic stimulus. As measured by titratable antibody, the typical secondary response to homologous antigen administered to an immunized animal is characterized by an immediate negative phase (drop intiter), followed by a rapid rise in titer to levels farabove those of the primary response, and then adecrease in titer which falls off very rapidly atfirst, slowly later (20). The pattern of decreasedsurvival of tumor-challenged mice which have received two NP injections spaced relatively widelyapart seems analogous to the secondary responsein two respects: (a) the greater decrease in survivalof these animals when compared with singly injected mice, and (6) the rapid decline in this effect1 week later. The protective effect of cortisone,which is known to decrease sensitivity to an antigen, is also suggestive of an immunological reaction.

With this evidence there still remains the choiceof sensitivity versus neutralization of a naturallyoccurring antitumor factor. The data may bebroadly interpreted to support either hypothesis,although the weight of evidence seems to lie withthe production of a sensitivity to NP. Once againthe quantitative aspects of the data are illuminating. If neutralization of an antitumor factor werethe mechanism producing the lowered survival oftreated mice, then an increase in the amount ofNP injected would be expected to produce both adecrease in the latent period before response and anincrease in the number of animals affected. Thesetwo factors operating together would then be expected to lower the survival time markedly. Actually, the data show quite the opposite response,for when a large amount of NP was used for asingle injection (2.0 ml.) the survival of these animals was not much poorer than that of the controls. On the other hand, interpretation of theseresults based on the assumption that NP anti-genicity is playing a major role follows readily.The injection of massive amounts of antigen isknown to produce little or no titratable antibody,and the resultant condition has been aptly termed"immunological paralysis" (10, 14). Presumably,

the antibody production sites are saturated underthese circumstances, and antibody production issuppressed. Applying these facts to the presentdata and assuming NP antigenicity, one could predict that the survival of heavily injected animalsshould resemble closely that of the controls.

Another aspect of the data can be interpretedlogically only when antigenicity of NP is assumed.

The injection of dead cells produced a remarkabledecrease in survival of challenged animals. If, however, the animals were first treated with NP andthen later given the same dead cell inoculation, thesurvival of the challenged animals was essentiallythe same as that of the controls. No reasonable explanation of these results based on neutralizationof an antitumor factor comes to mind. On the otherhand, if the tumor cells are simply considered tobe antigenic, then it logically follows that their introduction into an animal already producing antibodies would bind the available antibody and thusproduce the response that was obtained. Hereagain the quantity of cells inoculated is assumedto be a massive dosage of antigen.

Until more definitive contrary evidence is forthcoming, therefore, these data are interpreted asdemonstrating the production of a sensitivity toNP in the strain of mice used. Maculla (18,19) andBlix, Hand, and Stacey (5) have demonstrated theantigenicity of NP, thus removing one objectionwhich formerly might have been leveled at theseconclusions. If sensitivity is established, the premature death of a sensitized mouse must then beassumed to depend on the reintroduction of antigen in the form of living, multiplying ascites cellssupplied at the challenge.

The effects reported here have an obvious parallel in the numerous observations already describedthoroughly in the literature as the "XYZ," "enhancement," or "accelerant" factors. Unequivo

cally positive results of this nature have been obtained heretofore when lyophilized tumor (7, 8,24, 25), mitochondria and microsomes of tumorcells (22, 23), and supernatant fractions from tumor cells (9, 15) were used. The major differencebetween the reports cited and the present experiments seems to lie in the material utilized to produce the effect. The method of NP extraction usedhere should preclude the contamination of theproduct with cytoplasmic constituents. Carefulphase contrast microscopy during the isolationprocedure and the observed DNA/RNA ratios ofthe product of approximately 5:1 further suggestlittle if any mitochondrial or microsomal contamination. Moreover, a microsomally contaminatedsupernatant fraction from ascites cells did notproduce the effect in preliminary tests. The possibility exists, of course, that the material extractedfrom ascites cell nuclei might represent cytoplasmic RNA precursors.

SUMMARYMice pretreated with a single injection of nu-

cleoprotein extracted from Ehrlich ascites tumorcells were predisposed to a shorter survival time



670 Cancer Research

when challenged with ascites cells than mice notso pretreated. The decrease in mean survival timevaried with the amount of nucleoprotein injectedand with the time after the injection in such amanner as to suggest the production of a sensitivity to the nucleoprotein. The earlier death of pretreated mice is thought to be caused by a reactionbetween the sensitized mouse and the reintroducedantigen in the form of the ascites cell challenge.

ACKNOWLEDGMENTSThis work has been accomplished in the laboratory of Dr.

Norman G. Anderson, to whom the author is indebted for hisgenerosity with equipment but especially for his criticisms,suggestions, and advice during the course of the experiments.Indebtedness to Dr. T. Makinodan for his suggestions on andcriticisms of the serological data and their interpretation isgratefully acknowledged. Mr. William D. Gude prepared theslides used in the study of organ invasion by the tumor. Thedeterminations of standard errors were accomplished by Dr.A.|W. Kimball and G. J. Atta of the ORNL MathematicsPanel, to whom I am also grateful.

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lated Cell Components. IV. The Effect of Various Solutions on the Isolated Rat Liver Nucleus J. Gen. Physiol.,35:781-96, 1952.

2. BENEDICT.A. A., and TIPS, R. L. Active y and PassivelyInduced Arthrus Reactions in the Mouse. Proc. Soc.Exper. Biol. & Med., 87:618-22, 1954.

3. BEBNSTEIN,M. H., and MAZIA,D., The Desoxyribonucleo-protein of Sea Urchin Sperm. I. Isolation and Analysis.Biochim. et Biophys. Acta, 10:600-606, 1953.

4. . The Desoxyribonucleoprotein of Sea UrchinSperm. II. Properties. Ibid., 11:59-68, 1953.

5. lii.i \, U.; ILAND,C. N.; and STACET,M. The SerologicalActivity of Deoxypentosenucleic Acids. Brit. J. Exper.Path., 35:241-51, 1954.

6. BROWN,J. R. C. Contamination of Nuclear Fractions ofThymus Homogenates with Whole Cells. Science, 121:511-12, 1955.

7. CASEY,A. E.; DHYSDALE,G.; Ross, G. L.; and ELROD,B. A. Filtrates of Fresh Tumor Injected Prior to Transplantation of the Homologous Tumor. Proc. Soc. Exper.Biol. & Med., 77:221-24, 1951.

8. CASEY,A. E.; Ross, G. L.; and LANQSTON,R. R. SelectiveXYZ Factor in C57 Black Mammary Carcinoma Eo771.Proc. Soc. Exper. Biol. & Med., 72:83-89, 1949.

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All photomicrographs were made with American Opticaldark phase contrast. Magnification throughout 750X.

FIG. 1.â€”Ascitescells in 0.14 MNaCl suspension before extensive washing.

FIG. 2.â€”Suspensionof homogenate after blending and onewash in 1 per cent citric acid. Note what appear to be isolatedchromatic figures (see arrows).

FIG. 3.â€”Suspension of isolateli nuclei in 0.14 M NaCl following three 1 per cent citric acid washes.



1955;15:663-670. Cancer Res Edward C. Horn and Mary Elizabeth House of Nucleoprotein from Ehrlich Ascites CellsAscites Tumor Development: I. An Analysis of the in Vivo Effect

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