Decreases in Histamine Forming Enzyme Activity ofNon-metastasizing Fibrosarcomas in Hamsters

with Progressive Tumor Growth

THOMAS C. MOORE, M.D., LOIS E. KOPPELMANN, PH.D.,* CARLOS A. E. LEMMI, PH.D.

A marked and progressive decrease in the activity of the his-tamine forming enzyme, histidine decarboxylase (HDC), oftumors was found to be associated with the progressive growthof SV-40 virus induced and transplanted syngeneic non-metastasizng Sbrosarcomas in inbred LSH Syrian hamsters. His-tamine forming enzyme activity was highest in the smallesttumors (p < .005) and in the tumors with the slowest growthrate (p < .005, r - 0.84). Tumor histamine forming enzymeactivity was highest for each interval of animal exposure toinoculated tumor cells in those animals which had limited theirtumor growth to the smallest tumor size. These findings sug-gested a local anti-infammatory effect of progressive tumorgrowth. Induced local inflammation by repeated intratumorinjections of bradykinin markedly elevated tumor hisamineforming enzyme activity above expected levels for tumors ofthe same size in a small group of individual animals whichwere sampled at random from a larger group of animals whichwere being studied for the tumor growth kinetics effects ofrepeated intralesional injections of bradykinin. Tumor his-tamine forming enzyme activity was highest in those animalswhich were managed by the frequency of injection and doseschedules which were found in the tumor growth kinetics studyto be most effective in limiting tumor growth. These findingssuggested that the observed anti-inflammatory effects of pro-gressive tumor growth may be reversed by locally inducedinflammation at the tumor site with beneficial effects on tumorgrowth.

V ASOACTIVE SUBSTANCES which act as mediators ofinflammation (histamine, bradykinin, prostaglan-

din E1, serotonin, etc.) bear close relationship to oneanother. They may release and be released by oneanother1'10'11'19'27 and may potentiate the actions of oneanother.2'4'26'3122 These substances, from their actionsas local hormones and as potent neurovascular regula-tors, are uniquely equipped to influence the anatomi-cally diffusely dispersed segments of the immune sys-

From the Research Laboratory of the Division ofPediatric Surgery, UCLA-Harbor General Hospital,

Torrance, California and the Department of Surgery,University of California, Los Angeles, California

tem and to coordinate these activities with target arearequirements.A marked increase in allograft levels of histidine de-

carboxylase (HDC) the histamine forming enzyme, hasbeen found following both skin20 and renal22 allograftingin the rat. This increase in HDC activity was not foundfollowing autografting or isografting. The peak eleva-tion in allograft HDC activity (29-fold with renal allo-grafting) occurred approximately ten days postgrafting.A peak eight-fold, four day elevation in splenic HDCenzyme activity was found following renal allografting.22An increase in the urinary excretion of histamine has

been found to occur after skin, kidney and heart allo-grafting in rats, dogs and man.16'17'24'25'29 Both chem-ical inhibition ofHDC enzyme and antihistamine activ-ity have been found by us to prolong the survival ofskin allografts in rats'13'1423 and renal allografts in dogsand man.15'30

Elevated levels of kallikrein.(bradykinin forming en-zyme) and prostaglandin E activity also have been en-countered after kidney and heart allografting in rats.5'9"1821The purpose of this report is to describe the marked

and progressive decrease in tumor levels of HDC en-zyme activity found to be associated with progressivegrowth from small to large size of transplanted anduntreated SV-40 virus induced non-metastasizing fibro-sarcomas in hamsters and to compare these findingswith those of bradykinin injected tumors.

* Recipient of an NIH post-doctoral research fellowship fromthe National Cancer Institute (F32-CA05950).

Supported by GRS (G-2761) and NIH (HL-18936) researchgrants.

Submitted for publication: May 10, 1977.

Material and Methods

Inbred male LSH Lakeview colony Syrian hamsterswere used. The tumor employed was a well-character-ized syngeneic SV-40 virus induced and non-metastasiz-

0003-4932-78-0800-0175-0075 © J. B. Lippincott Company

175

Ann. Surg. * August 1978MOORE, KOPPELMANN, AND LEMMI

ing fibrosarcoma (obtained from S. Tevethia of TuftsUniversity). Tumor cells were grown in Eagle's MEM(EMEM) culture medium with 10% fetal bovine serum

(FBS) in a Wedco incubator. Prior to use for inocula-tion, the cells were dissociated in 0.25% trypsin andcentrifuged at 200 x g in EMEM-FCS for ten minutes.Following this, they were washed in Hank's balancedsalt solution (HBSS) and resuspended in HBSS at con-

centrations of 1 x 105 or 1 x 106 cells per 0.5 ml (byhemocytometer count).Two studies were carried out relating to tumor con-

centrations of the histamine forming enzyme, histidinedecarboxylase (HDC). One involved a correlation oftumor HDC activity with tumor size, intervals sincetumor cell inoculation and tumor growth rates. In thisstudy, 1 x 106 tumor cells were inoculated at one siteover the pelvic girdle in 137 animals. The inoculationsite was inspected daily for the appearance ofa palpableand measurable tumor. Tumors developed in 55% oftheanimals. Palpable tumors generally developed betweentwo and two and one-half weeks after tumor cell in-oculation. Tumor diameters were measured daily witha flat-edged sliding caliper after wetting the fur withalcohol. The skin of these animals was quite loose andtumor growth was observed to be spherical well pastthe 10 mm in diameter size. Tumor volume calculationswere based on the observed spherical sizes of thesetumors. Fifteen animals which developed tumors were

selected at random for study at sacrifice when the tumorshad reached varying sizes. The tumors were removed,weighed and quick frozen in liquid nitrogen for laterassay for HDC activity.The second study involved an evaluation of the ef-

fects of repeated intralesional injections of bradykininon tumor HDC activity in animals with tumors of com-parable growth rate. TumorHDC activity was correlatedwith dose and frequency of injections of bradykinin(obtained from the Sigma Chemical Co., St. Louis,Mo.) and with the effects of bradykinin injections on

tumor HDC activity as compared with the HDC activityof control animals (saline injected and noninjected) whichwere studied at the same period after the tumors hadreached 10 millimeters in diameter. In this study, 1 x 105tumor cells were inoculated at two locations, over therump and between the shoulder blades in 82 animals.Fifty-four per cent of the animals developed one tumorand 46% developed two tumors. Tumor diameters were

measured as above. The tumors of the animals em-

poyed in this study had achieved diameters of 10 mmbetween 28-32 days after the inoculation of tumorcells. Fifty-one per cent ofthe tumors which developedhad comparable growth kinetics up to the tumor sizeof 10 mm in diameter. Repeated intratumor injectionsof bradykinin and saline (control) in equal volumes

were initiated when the tumors had reached 10 mm indiameter. The sandard injection schedule was one ofincreasing intervals between injections (daily x 4, everytwo days x2, every three days x 1 and every four daysx 1). Two doses of bradykinin were used. 50 ug and250 ,ug. Five bradykinin injected animals and two con-trol animals were sacrificed for study between five and14 days after the tumors had reached 10 mm in diameterand after the initiation of injections in animals whichreceived injections. The majority of animals were keptalive beyond the 14 day period for tumor growth kineticsstudies.HDC enzyme activity of tumors was assayed by the

14C radioisotopic method of Schayer.28 This methodinvolved the incubation of 14C histidine and pyridoxal-5-phosphate with a known weight of tissue for a threehour period at 370 in a Dubnoff metabolic incubator.The 14C histamine produced was converted to benzenesulfonyl histamine (BSH). The BSH was purified byrepeated recrystallizations over a period of five days.The HDC activity, corrected for tissue blank, back-ground activity and recovery of BSH, was calculatedand expressed as disintegrations per minute (dpm) per100mg BSH per gram oftissue or dpm/gm tissue. Statis-tical significance was determined by the Student's t test.

Results

When the effects of progressive tumor growth on thetumor levels ofthe histamine forming enzyme, histidinedecarboxylase (HDC) of the 15 animals studied wereevaluated with respect to tumor size, a marked and pro-gressive decrease in tumor HDC activity was foundwith progressive tumor growth. The mean tumor HDCactivity of three very small tumors measuring 0-1 mmin diameter was 48,702 ± 15,685 dmp/g. It fell sharplyto 10,595 ± 2,177, 7,076 ± 2,272, 3,125 ± 1,374 and781 ± 353 dpm/g as the tumors grew to two, seven,8-9 and over 12 mm in diameter (Fig. 1). The meanHDC activity of the 0-1 mm tumors was significantlyhigher than that oftwo and 7 mm tumors (p < .05) and of8-9 and 12-30 mm tumors (p < .025). The significanceof the difference between the mean HDC activity of 2mm tumors and the 12-30 mm tumors was greater(p < .005) than that found for 0-1 mm tumors as com-pared with 12-30 mm tumors despite the much highermean HDC activity level of the 0-1 mm tumors. Thegreater spread in HDC activity levels within the 0-1mm counted for this difference in significance. In aneffort to evaluate other factors which might influencetumor HDC activity, in addition to tumor size, tumorHDC activities of individual tumors were studied withrespect to the interval in weeks from the time of tumorcell inoculation to the time ofanimal sacrifice and tumor

176

ENZYME ACTIVITY OF FIBROSARCOMAS 177

LJJC/,

-I 50P00-

40,000-

E 30,000-

Z Ea cLF X 20,000-

I

CK10,000-0

I-

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FIG. 1. Bar graph showingeffects of progressive in-creases in tumor size on theactivity of the histamineforming enzyme, histidinedecarboxylase (HDC) oftumors. Numbers of ani-mals studied are indicatedat the top of each bar.2

2 7 8-9 12-30

TUMOR DIAMETER(mm)

assay for HDC activity. For the five intervals studiedtwo, three, four, five and seven weeks), it was foundthat for each interval that the smallest tumors in theinterval group had tumor HDC activities ofmuch higherlevels than the animals with larger tumors (Table 1).The findings of these two means of evaluating tumor

HDC activity suggested that both the duration of ex-posure to inoculated tumor cells and the size of the

TABLE 1. Influence ofTumor Size on Tumor Histidine DecarboxylaseEnzyme Activity with Respect to Interval of Exposure

ofAnimals to Inoculated Tumor Cells

Interval from Tumor Tumor HistidineCell Inoculation Tumor Diameter Decarboxylase

(wks) (mm) dpm/gm)

2 2 12,77212 569

3 0-1 28,0152 8,418

4 8 1,1409 2,47113 512

5 0-1 79,4697 9,4487 4,7038 5,76412 569

7 0-1 38,62118 22330 430

tumor influenced tumor HDC activity in the presence ofprogressive tumor growth. Within all of the intervalsstudied, the animals which were able to maintain theirtumors at the smallest size were found to have tumorswith the highest levels of HDC activity.These observations suggested the value of studying

tumor HDC activity with respect to tumor growth rate.When the growth rate of each of the 15 tumors wascalculated from the volume of the tumor divided bythe weeks of growth (interval since tumor cell inocula-tion), a unit of tumor growth rate was obtained andexpressed as the log mm3/wk for each tumor. The vol-ume of each tumor was obtained by the formula: vol-ume = 0.4 x d3, where d = diameter in mm (thetumors grew in a spherical shape). By plotting the HDCenzyme activity in dpm/gm of each tumor versus thegrowth rate (log mm3/wk), a statistically significant nega-tive correlation (the slower the growth rate, the greaterthe HDC) was found (p < .005). The coefficient of cor-relation (r) for the 15 tumors analyzed by this methodwas -0.84 (Fig. 2).

In the second study, which involved animals of com-parable tumor growth kinetics, five animals which hadreceived repeated intratumor injections of 50 or 250,ug of bradykinin and two control animals were with-drawn from the larger group for sacrifice for study oftumor HDC activity. The intervals of withdrawal rangedfrom five to 14 days after the tumors had reached thesize of 10 mm in diameter. The majority of animalswere kept alive for tumor growth kinetic studies rela-

Le- L"

VOl. 188.9 NO. 2

T

Ann. Surg. . August 1978MOORE, KOPPELMANN, AND LEMMI

tive to the effectiveness of the two doses of bradykininin slowing tumor growth in comparison with saline in-jection and no injection controls. One animal whichhad received daily intralesional injections of 50 ,ug ofbradykinin was sacrificed at five days after the initia-tion of injections. This tumor had grown 1.5 mm indiameter and had an HDC activity of 155,464 dpm/gm(Fig. 3). Another animal which had received 50 uginjections was sacrificed at ten days and at a time whenthe interval between injections was three days. Thistumor had grown 7.0 mm in diameter and had an HDCactivity of 35,733 dpm/g. Five animals were sacrificed14 days after the tumor had reached the size of 10 mmin diameter. Three animals were sacrificed 14 days afterthe initiation of bradykinin injections, at a time whenthe interval between injections was four days. Two ofthese animals had received tumor injections of 250 ,ugofbradykinin. Their tumors had grown 7.0 and 11.0mmin diameter and had HDC activities of 15,858 and 18,603dpm/gm. The tumor of the animal which had receivedtumor injections of 50 ,tg of bradykinin had grown11.0 mm in diameter and had an HDC activity of 9,955dpm/g. The saline injected tumor had grown 20.0 mmin diameter and had an HDC activity of 4,309 dpm/g.The noninjected tumor had grown 15.0 mm and had anHDC activity of 2,680 dpm/g.The mean tumor growth after reaching the 10 mm

size for tumor of comparable growth kinetics which

Wc)

-J

ct

C...)

W E

W EZ a

I

0

155,00C

50,OOC

40,00C

30,00C

20,000

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22 0

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p0< 1,000 0080,0

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TUMOR HISTIDINE DECARBOXYLASE(dpm/gm)

FIG. 2. Graph showing a statistically significant (p < 0.005) negativecorrelation of tumor growth rates (expressed as log mm3/wk) andtumor histidine decarboxylase (HDC) enzyme activity for the 15animals studied (the slower the growth rate, the higher the tumorHDC). The coefficient of correlation (r) for the 15 tumors analyzedby this method was -0.84.

50pg BK

50 pg BK

250jug BK25Ojug BK

50jug BK

SLINE NOT IN,

5 10 14DAYS AFTER TUMORS REACHED 10mm SIZE

FIG. 3. Bar graph showingthe levels of activity of thehistamine forming enzyme,histidine decarboxylase(HDC) in the tumors of an-imals which were managedby repeated intralesional in-jections of bradykinin or sa-line or by no injections. Ani-mals with tumors of com-parable growth kinetics wereused. Repeated tumor in-jections were initiated whenthe tumors reached 10 mmin diameter and were givenby a standard injectionschedule (daily x4, q2 daysx1, q3 days x1 and q4days x 1). These tumorswere from individual ani-mals sampled at randomfrom a larger group of ani-mals which were kept alivefor tumor growth kineticsstudies. Tumor HDC levelswere highest in those ani-mals shown in the growthkinetics study7 to havethe greatest growth inhibi-tion (more frequent injec-tions and higher doses ofbradykinin).

178

Vol. 188 . No. 2 ENZYME ACTIVITY OF FIBROSARCOMAS 179

had not received bradykinin injections (saline injectionsor no injections) was 4.5 mm at five days, 7.5 mm at tendays and 11.5 mm at 14 days. The significance of thegrowth inhibiting effects of the repeated 250 Ag intra-lesional injections of bradykinin for all animals, includingthose not sacrificed, in comparison with the growth ofnoninjected and saline injected tumors (which did notdiffer) was calculated by the Student's t test to bep < .001 at five days, p < .01 at ten days and p < .005at 14 days. For repeated 50 Ag injections, it was p< .0025 at five days and not statistically significant atten and 14 days, although the growth of those tumorswas somewhat less than that of controls.7The individual tumor levels ofHDC activity ofbrady-

kinin injected tumors reflected the findings ofthe tumorgrowth kinetics studies which showed statistically sig-nificantly greater tumor growth inhibition when the in-jections ofbradykinin were more frequent and when thedoses were higher. Individual tumor HDC activitieswere higher when the injections of bradykinin weremore frequent and when the doses were higher.

Furthermore, it may be of interest to note that allof the bradykinin injected tumors were 11.5 mm or moreat the time of study for HDC activity. The mean tumorHDC activity of the five tumors in the first study whichwere in the over 12 mm in diameter range (three meas-ured 12 or 13 mm in diameter) was 781 ± 353. TheHDC activities of all of the bradykinin injected tumorswere substantially above these levels. The incidence oftumor development after tumor cell injections in thefirst and second studies was comparable (55% versus54%) and the rates of tumor growth were similar.

Discussion

The mounting of an effective cellular immune re-sponse at a target area involves the escape ofantigen andthe access of cells. It also involves the mobilization,processing and delivery of essential cellular and non-cellular components of the immune response to thetarget area and their transport through the vascularendothelium to the sites ofimmunological assault. Theseprocesses require communications and interactions be-tween target areas and the diffusely scattered segmentsand centers of the lymphoid system.The studies ofMcGregor and Logie12 with the "hom-

ing" characteristics on transfer of isotope labelled syn-geneic "immunoblasts" to inflammatory foci led themto conclude that the "homing" was determined by ageneral property of the cells rather than by their im-munological commitment and that the tissue disposi-tion of sensitized lymphocytes in the body was de-termined by a complementary relationship between bloodimmunoblasts and the vascular endothelium of inflamed

tissue. More recent studies of Hall, Hopkins and Orians3also suggest that the "homing" of immunoblasts isnot primarily "antigen-driven."The findings of the study reported here provide ad-

ditional evidence of an anti-inflammatory effect of pro-gressive malignant tumor growth as reflected in a markedand progressive reduction in the histamine formingenzyme activity of these tumors in association withprogressive tumor growth. The extent of reduction intumor histamine forming enzyme activity was found tobe influenced significantly by tumor size and the rate oftumor growth. The highest levels of histamine formingenzyme activity in tumors was found in the tumors ofanimals which had been exposed to inoculated tumorcells for the longest periods of time and had still kepttheir tumors at relatively small sizes. The elevated levelsof histamine formation in these tumors may have con-tributed to limiting the speed of their growth.

In the second part of this study, repeated intratumorinjections of bradykinin were found to elevate markedlythe levels of tumor histamine forming enzyme activityin comparison with the levels which might have beenexpected for tumors of this size as observed in thefirst part of this study. These studies were carried outon a small number of individual animals which weresampled from a larger group ofanimals which were keptalive for tumor growth kinetics studies.7 In these in-dividual animals, the observed effects on tumor his-tamine forming enzyme activity reflected the tumorgrowth inhibition effects of the frequency and doses ofbradykinin. Tumor histamine forming enzyme activitywas highest in animals which received more frequentinjections and higher doses of bradykinin. It was inthese groups of animals that significantly greater tumorgrowth inhibition was found in the tumor growth kineticsstudy.7

In addition to its effects on the levels of histamineformation and release in injected tumors, bradykininalso may have had an influence in vivo on the levelsof sensitized lymphoid cell antitumor cell cytolytic ac-tivity. In a recent study, daily intratumor injectionsof 250 ,ug of bradykinin for 21 days halted tumor growth.8Splenic lymphoid cells from these animals, in the pres-ence of in vitro added bradykinin, were found tohave significantly enhanced antitumor cell cytolytic ac-tivity by the 51Cr release assay (p < .005).6

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of Kallikrein and Phospholipase A on Histamine Release.J. Clin. Lab. Invest., (suppl) 107:95, 1969.

2. Goldberg, M. R., Chapnick, B. M., Joiner, P. D., et al.: In-fluence of Inhibitors of Prostaglandin Synthesis on Vasocon-strictor Responses to Bradykinin. J. Pharmacol., Exp. Ther.,198:357, 1976.

180 MOORE, KOPPELMANN, AND LEMMI Ann. Surg. * August 1978

3. Hall, J. G., Hopkins, J. and Orians, E.: Studies on the Lympho-cytes of Sheep; III. Destination of Lymph-borne Immuno-blasts in Relation to their Tissue of Origin. Eur. J; Immunol.,7:30, 1977.

4. Ikeda, K., Tanaka, K. and Katori, M.: Potentiation of Brady-kinin-Induced Vascular Permeability by Prostaglandin E2 andArachiodonic Acid in Rabbit Skin. Prostaglandin, 10:747,1975.

5. Jaffe, B. M., Moore, T. C. and Vigran, T. S.: Tissue Levels ofProstaglandin E following Heterotopic Rat Heart Allografting.Surgery, 78:481, 1975.

6. Koppelmann, L. E. and Moore, T. C.: Bradykinin-Induced En-hancement In-Vitro of Sensitized Hamster Spleen Cell Anti-Tumor Cell Cytotoxicity. IRCS Med. Sci., 4:524, 1976.

7. Koppelmann, L. E., Moore, T. C., Lemmi, C. A. E. and Porter,D. D.: Accumulation of Mononuclear Cells in Tumors withGrowth Slowing and Elevation in Host Splenic Histidine De-carboxylase Activity following Repeated Tumor Injectionswith Bradykinin. Surgery, 78:181, 1975.

8. Koppelmann, L. E., Moore, T. C. and Porter, D. D. (Unpub-lished Data).

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10. Lewis, G. P. and Winsey, M. J. P.: The Action of Pharmacologi-cally Active Substances on the Flow and Composition of CatHind Limb Lymph. Br. J. Pharmacol., 40:446, 1970.

1 1. McGiff, J. C., Terragno, N. A., Malik, K. V. and Lonigro, A. J.:Release of Prostaglandin E-Like Substance from Canine Kid-ney by Bradykinin. Circ. Res., 31:36, 1972.

12. McGregor, D. D. and Logie, P. S.: The Mediator of CellularImmunity. VII. Localization of Sensitized lymphocytes inInflammatory Exudates. J. Exp. Med., 139:1415, 1974.

13. Moore, T. C.: Histidine Decarboxylase Inhibitors and the Sur-vival of Skin Homografts. Nature, 215:871, 1967.

14. Moore, T. C.: Histidine Decarboxylase Inhibitors and Second-Set Allograft Survival. Arch. Surg., 99:470, 1969.

15. Moore, T. C.: Effective use of Isoniazid and an Anti-Histaminein Clinical Renal Transplantation. Surg., Gynecol. Obstet.,133:75, 1971.

16. Moore, T. C., and Chang, J.: Urinary Histamine Excretion in theRat following Skin Homografting and Autografting. Ann.Surg., 167:232, 1968.

17. Moore, T. C., Cleveland, R. J., Thompson, D. P. and Nelson,R. J.: Vasoactive Amine Metabolism following Canine andHuman Heart Transplantation. Ann. Surg., 154:51, 1971.

18. Moore, T. C. and Jaffe, B. M.: Prostaglandin E Levels ofHeterotopic Rat Heart Allografts and of Host LymphoidTissues at Intervals Post-Grafting. Transplantation, 18:383,1974.

19. Moore, T. C., Normell, L. and Eiseman, B.: Effect of SerotoninLoading on Histamine Release and Blood Flow of IsolatedPerfused Liver and Lung. A.M.A. Arch. Surg., 84:42, 1963.

20. Moore, T. C., and Schayer, R. W.: Histidine DecarboxylaseActivity ofAutografted and Allografted Rat Skin. Transplanta-tion, 7:99, 1969.

21. Moore, T. C., Sinclair, M. C. and Lemmi, C. A. E.: Elevationin Rat Plasma Kallikrein Activity following Renal Allograft-ing. Am. J. Surg., 127:289, 1974.

22. Moore, T. C., Sinclair, M. C. and Lemmi, C. A. E.: Elevationin Host Splenic and Allograft Parenchymal and Capsu-lar Histidine Decarboxylase Activity and Weights followingRenal Allografting in Rats. Surgery, 75:203, 1964.

23. Moore, T. C. and Thompson, D. P.: Prolongation of RatSkin Allograft Survival with Isonicotinic Acid Hydrazideand Methapyrilene Hydrochloride. Surg. Gynecol. Obstet.,132:275, 1971.

24. Moore, T. C., Thompson, D. P. and Glassock, R. J.: Eleva-tion in Urinary and Blood Histamine following Clinical RenalTransplantation. Ann. Surg., 173:381, 1971.

25. Moore, T. C., Thompson, D. P. and Hayes, M.: Correlation ofRenal Blood Flow Indices and Histamine Metabolism Dataafter Canine Renal Allografting. Arch. Surg., 101:45, 1970.

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28. Schayer, R. W.: Formation and Binding of Histamine by RatTissues In Vitro. Am. J. Physiol., 187:63, 1956.

29. Sinclair, M. C., Lemmi, C. A. E. and Moore, T. C.: Elevationin Urinary Excretion of Histamine following Renal Allograft-ing in Rats. J. Surg. Res., 17:43, 1974.

30. Smellie, W. A. B. and Moore, T. C.: Effect of Semicarbazideand Pyridoxine Deficiency on Survival of Canine RenalHomografts. Surg. Gynecol. Obstet., 128:81, 1969.

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