Effects of Various Oncogenic Agents on Tumor-Producing Capabilities of Series D BALB/c Mammary Nodule Outgrowth Lines 1.2

Transcription

1 Effects f Varius Oncgenic Agents n Tumr-Prducing Capabilities f Series D Mammary Ndule Outgrwth Lines 1.2 DANIEL MEDINA 3 ancj K. B. DeOME, Deprtment 01 Zlgy ancj Its Cancer Research Genetics Labratry, University f Califrnia, Berkeley, Califrnia SUMMARY-In previus papers, we reprted the effects f viral, chemical, and physical carcingens n the tumr-prducing capabilities f the ndule utgrwth line D1. The present paper describes the tumr-prducing capabilities f a series f ndule utgrwth lines in BALBI c mice negative Fr mammary tumr virus (MTV) and in BALBlc f. C3H mice psitive Fr MTV. OF 7 utgrwth lines tested fr their respnsiveness t MTV, 6 shwed increased tumr-prducing capabilities. The respnsiveness f any utgrwth line t MTV was nt crrelated with its backgrund tumr ptential. Als described are the effects f MTV, ndule-inducing virus (NIV), 3-methylchlanthrene (MCA), and prlnged hrmne stimulatin, applied singly and cmbined, n the tumr-prducing capabilities f ndule utgrwth line D2. Single applicatin f prlnged hrmne stimulatin, MTV, r MCA increased the tumr-prducing capabilities f line D2; hwever, NIV had n measurable effect. Cmbined applicatins f MTV and MCA, MTV and hrmne stimulatin, and MCA and hrmne stimulatin had additive effects. The D series f ndule utgrwth lines is a gd system t study the effects f MTV's and chemical carcingens, applied individually r cmbined. The present studies and the previusly reprted nes indicate that different utgrwth lines respnd differently t varius ncgenic agents and that the respnse f an utgrwth line t a cmbinatin f several carcingens cannt be predicted by its respnse t a single applicatin f the agents.-j Nat Cancer Inst 45: ,1970. Dwnladed frm at Pennsylvania State University n May 10, 2016 THE ORIGIN and histlgical and bilgical characteristics f a series f mammary ndule utgrwth lines, derived frm hyperplastic alvelar ndules in mice, were described in (1). Fr cnvenience, these utgrwth lines were designated series D f ndule utgrwth lines and were numbered sequentially D 1-D8. Previus reprts described the effects f mammary tumr virus (MTV), ndule-inducing virus (NIV), 3- methylchlanthrene (MeA), and prlnged hrmne stimulatin, applied singly and cmbined, n the tumr-prducing capabilities f ndule utgrwth line Dl (2-4). The present reprt describes 1 Received February 27, 1970; accepted May 5, Supprted by Public Health Service grants CA 5045 and CA 5388 frm the Natinal Cancer Institute and Predctral Fellwship 4-FOI GM38, t Dr. Medina frm the Natinal Institute f General Medical Sciences. 3 Present address: Department f Anatmy, Baylr Cllege f Medicine, Hustn, Texas

2 354 MEDINA AND DEOME the tumr-prducing capabilities f ndule utgrwth lines DI-D8 in MTV-negative, NIVnegative mice and in MTV-psitive, NIVnegative J. C3H mice. In additin, it describes the effects f MTV, NIV, MCA, and prlnged hrmne stimulatin, applied singly and cmbined, n the tumr-prducing capabilities f ndule utgrwth line D2. A preliminary reprt f these experiments was presented in (5). MATERIALS AND METHODS Hsts.-Several different strains and hybrids f inbred mice were used. The cnstitutin f the mice with respect t MTV and its variant NIV is as fllws: mice are MTV negative; J. C3H mice are MTV psitive; and (C3Hf X )F t mice are MTV negative and NIV psitive. The rigin and mammary tumr incidences f the varius strains were dcumented in (6, 7). All mice were btained frm the Cancer Research Genetics Labratry Clny, hused per cage in temperature- and light-cycle-cntrlled rms, fed Purina Breeder Chw r Berkeley Diet,4 and given water ad libitum. Samples f the ndule utgrwths were transplanted int the inguinal mammary gland-free fat pads by the standard methd develped by DeOme et al. (8). All animals were palpated weekly fr tumrs. When first discvered, tumrs were remved immediately frm the animals. Samples f the tumr were embedded in paraffin, sectined at 7 1-', and stained with hematxylin and esin. Hrmne stimulatin.-fr hrmne stimulatin f the hst's mammary glands and f ndule utgrwths, the pituitary glands were taken frm 3- t 5-mnth-ld isgenic male mice and transplanted. Either 3 pituitary glands were transplanted int the left inguinal mammary fat pads f hst mice, r I r 2 pituitary glands were transplanted under the kidney capsule. Under bth cnditins, pituitary glands, free frm hypthalamic cntrl, secrete primarily lutetrpic hrmne (9). Pituitary isgrafts in intact mice induce recurrent pseudpregnancy and lbulalvelar develpment f the mammary glands (9-11). The pituitary glands remained permanently in place. Chemical carcingen.-mca was administered by the prcedure f Faulkin (12). When 8, 9, and 10 weeks ld, the mice were given nce each week 0.20 ml f a 0.25% slutin (w/v) f MCA in sesame il by gastric intubatin. MCA was administered t unanesthetized mice by means f a blunted 16-gauge needle ending in a silver-sldered bulb. Statistics.-Tw types f nnparametric statistical tests were used fr these data: the Mann- Whitney U and the Median. In bth tests, it was assumed that the nn-tumr-bearing mice, which were terminated at a time earlier than the 50% tumr endpint, wuld have shwn the same tumr distributin as the rest f the mice in the experimental grup. The 50% tumr endpint was defined as the time it tk 50% f the ndule transplants t prduce tumrs. With the 50% tumr endpint, different grups culd be cmpared directly, especially grups that had similar tumr incidences, but whse tumrs ccurred at different rates, r grups in which the appearance f hst tumrs prevented direct measurements f the final tumr incidence in the transplanted utgrwths. RESULTS Tumr-Prducing Capabilities f Series D f Ndule Outgrwths in Mice The tumr-prducing capabilities are shwn in table l. The results fr each utgrwth line (except D7) were cmbined frm several experiments. In all cases, the criteria used fr including the utgrwths in the data were the fllwing: 1) The utgrwths had filled at least 50% f the fat pads; and 2) the utgrwths had been transplanted fr at least 6 mnths fr lines Dl, D3, D4, D5, and D7 and fr at least 3 mnths fr lines D2, D2a, and D8. The tumr-prducing capabilities f utgrwth line D 1 have been the best dcumented. The results included its tumr-prducing capabilities ver 12 transplant generatins. Of 253 utgrwths, nly 10 tumrs (4%) appeared, at a mean latent perid f 404 days. This incidence remained fairly cn- 4 "Berkeley Diet" Muse Breeder Chw, Feedstuff Prcessing C., San Francisc, Calif Dwnladed frm at Pennsylvania State University n May 10, 2016 JOURNAL OF THE NATIONAL CANCER INSTITUTE

3 TUMOR POTENTIAL OF MAMMARY OUTGROWTH LINES 355 TABLE I.-Tumr-prducing capabilities f series D ndule utgrwths in and f. C3H mice Tumrs Mean age at death (days) Ndule N umber f tumrs Mice with Mice withut utgrwths Hst Percent TE50* tumrs in tumrs in Number f transplants transplants transplants Dl f. C3H 10/253 91/124 D2 41/92 f. C3H 43/53 D2a D3 D4 D5 f. C3H f. C3H f. C3H f. C3H 11/42 19/22 20/62 22/28 15/44 13/21 3/45 3/50 D7 2/11 D8 f. C3H 28/46 14/16 *The time (in days) it tk 50% f the transplants t prduce tumrs. stant ver 12 transplant generatins (table 2). In generatins 3, 6, 11, 13, and 14, where there were many transplants, the tumr ptential varied frm 2-9%. These results were nt significantly different at the 5% level (P>0.05). The earliest tumr appeared at 235 days and the latest at 620 days. The tumr-prducing capability f ndule utgrwth line D2 was high. Amng 92 transplants, 41 prduced tumrs (45%), at a mean latent perid f 280 days. The tumr incidence varied slightly with transplant generatin (table 3). Text-figure 1 shws the rate f tumr frmatin in D2 utgrwths by transplant generatin. The rate was calculated frm the slpes f the lines derived frm generatins 2, 3, and 8, which included enugh transplants fr cmparisn. It was similar in the 3 generatins (text-fig. 1). The significant difference was in the initial time f tumr appearance (fr cmparisn, taken at the 10% tumr endpint rather than at first appearance f tumr). Once tumrs started t appear, they frmed at a cnstant rate in the 3 generatins. Generatin 6A f utgrwth line D2 prduced few tumrs. By 259 days, generatin 3 had prduced 20% tumrs and generatin 8 30% tumrs, 70 GO genl!fclin 2 generatin 3 A generatin / // O,L!---- L L L , Time (days) TEXT-FIGURE 1.--Rate f tumr frmatin in D2 utgrwths by transplant generatin. whereas generatin 6A had prduced nly 4% tumrs. These tumr incidences differed significantly at the 5% level. The significant characteristic f generatin 6A was its unusual mrphlgy. The differences in the mrphlgical and secretry characteristics f this subline were presented in (1). The tumr-prducing capabilities f utgrwth line D2a were intermediate between thse f DI and D2. Amng 42 transplants, 11 prduced tumrs Dwnladed frm at Pennsylvania State University n May 10, 2016 VOL. 45, NO.2, AUGuST 1970

4 356 MEDINA AND DEOME TABLE 2.-Tumr-prducing capabilities f different transplant generatins f D1 ndule utgrwths in mice Generatin Tumrs Number f tumrs Number f transplants 3 2/23 4 2/13 6 2/36 9 0/7 10 0/4 11 1/ / / /72 Ttal 10/253 (26%), at a mean latent perid f 310 days. These results were cmbined frm 2 experiments n 2 different transplant generatins. The rate f tumr frmatin was lwer in utgrwth line D2a than in line D2 (text-fig. 2). Outgrwth line D3 prduced 32% mammary tumrs. Amng 62 transplants, 20 prduced tumrs, at a mean latent perid f 306 days. Althugh the initial time f tumr appearance (10% tumr endpint) was similar t that seen in utgrwth line D2a (275 days), utgrwth line D3 had a higher rate f tumr frmatin (text-fig. 2). The tumr-prducing capabilities f utgrwth line D4 were different frm thse f utgrwth lines D 1, D2, r D3. Althugh lines D4 and D3 had similar tumr incidences (36% and 32%, respectively), the rate f tumr frmatin was lwer in D4 utgrwths than in utgrwths D2 and D3 (text-fig. 2). Mean age at death (days) Mice with Mice withut Percent tumrs in tumrs in transplants transplants The tumr-prducing capabilities f utgrwth line D5 were similar t thse f line D 1. Amng 45 transplants, nly 3 prduced tumrs (7%), at a mean latent perid f 303 days. The tumr ptential f utgrwth line D5 was tested at the third and sixth transplant generatin. In the third generatin, utgrwths remained in mice fr ver 500 days and prduced nly 3 tumrs. In the sixth generatin, they remained in mice fr 330 days and prduced n tumrs. The tumr-prducing capability f utgrwth line D7 was nt well dcumented. Amng 11 transplants, nly 2 prduced tumrs (18%), at a mean latent perid f 315 days. The tumr-prducing capabilities f utgrwth line D8 were the highest f all lines tested. Amng 46 transplants, 28 prduced tumrs (61 %), at a mean latent perid f 243 days. TABLE 3.-Tumr ptential f different transplant generatins f D2 ndule utgrwths in mice Tumrs Mean age at death (days) Generatin Number f tumrs Mice with Mice withut Percent TE50* tumrs in tumrs in Number f transplants transplants transplants 2 5/ / / / A 2/ / Subttalt 41/ Ttalt 43/ *The time (In days) it tk 50% f the transplants t prduce mammary tumrs. tdatll d nt Include generatin 6A. :Data include all "eneratins. Dwnladed frm at Pennsylvania State University n May 10, 2016 JOURNAL OF THE NATIONAL CANCER INSTITUTE

5 TUMOR POTENTIAL OF MAMMARY OUTGROWTH LINES D2 D2a.. D3 l> D4 cds 40 '" E F 30 c 20 TEXT-FIGURE 2.-Turnr-prducing capabilities f ndule utgrwths D2, D2a, D3, D4, and DB in BALBfc mice. 10 0',!,,,, I Time (days) Respnse f Series 0 f Ndule Outgrwth Lines tmtv c:::j SALS/C czza SALS/cfC3H The effect f MTV n the tumr-prducing capabilities f series D ndule utgrwth lines was determined by transplanting utgrwths int the gland-free mammary fat pads f 3-week-ld, MTV-psitive j. C3H mice. The results are shwn in table 1. In 6 f the 7 utgrwth lines tested, MTV infectin increased the mammary tumr incidence and decreased the mean latent perid as cmpared t the same utgrwths transplanted int MTV-free mice. Amng the utgrwths frm DI, 73% prduced mammary tumrs; frm D2, 81 %; frm D2a, 86%; frm D3, 79%; frm D4, 62%; and frm D8, 88%. Each utgrwth line reached the 50% tumr endpint at days 308, 182, 161, 238, 364, and 168, respectively. MTV had n effect n the tumr-prducing capabilities f D5 utgrwths. Of 50 transplants, nly 3 prduced mammary tumrs (6%). There was little crrelatin between the mammary tumr incidence in utgrwths in mice and that in utgrwths in j. C3H mice (text-fig. 3). The figures were adjusted fr the mammary tumr incidence f the utgrwths at the end f 1 year. Outgrwth line Dl, which had a lw backgrund tumr incidence (2%), prduced 75% tumrs in j. C3H mice, whereas line D5 failed t shw an increased tumr incidence in j. C3H mice. Outgrwth line D2a, which prduced nly 26% tumrs in mice, prduced 86% tumrs in j. C3H mice and reached the 50% tumr endpint at 161 days. When transplanted int j. C3H Dwnladed frm at Pennsylvania State University n May 10, 2016 '" F.., c u TEXT-FIGURE 3.-Turnr-prducing capabilities f series D ndule utgrwths in BALBfc and BALBfcf. C3H mice. 10 Outgrwth Line VOL. 45, NO.2, AUGUST

6 358 MEDINA AND DEOME 4.-Tumr-prducing capabilities f ndule utgrwths Dl, D2, D2a, D3, and D4 in f. C3H mice. TEXT-FIGURE mice, the high tumr lines, D2 and D8 (45% in BALBjc and 61 % in BALBjc, respectively), prduced 81 and 88% tumrs, respectively. Thus the respnse f utgrwths t MTV was independent f their backgrund tumr incidence. Text-figure 4 plts the percentage f mammary tumrs against time. In all 5 utgrwth lines, the appearance f tumrs was linear with time. The prbability that a ndule cell ppulatin wuld develp a mammary tumr within a given perid was the same between the 10-70% tumr endpints. In utgrwth lines D2, D2a, and D3, the 50% tumr incidence was btained at different times; hwever, the slpes f the lines were almst identical. Fr these utgrwths, tumr incidence depended n the time f initial tumr frmatin s (266) _(357) (2.0) J' /./'" /;/. /ji " c 02a I b 350 4b 450 sb Time (days) The stability f the respnse f utgrwths t MTV can be inferred frm the data in tables 4 and 5. Table 4 shws the tumr-prducing capabilities f line Dl fr several different transplant generatins in BALBjc j. C3H mice. The respnse t MTV f generatin 14 (70% tumrs; TE50 = 287 days) was nt significantly different frm that t MTV f generatin 6 (85% tumrs; TE50 = 294 days). Similarly, the respnse f D2 utgrwths t MTV did nt change significantly in different generatins (table 5). Generatin 2 prduced 80% tumrs, with the 50% tumr endpint reached at 168 days. Thus the stable respnse f utgrwth lines Dl and D2 t MTV represented a prperty f each utgrwth line. TABLE 4.-Tumr-prducing capabilities f different transplant generatins f D1 ndule utgrwths in f. C3H mice Tumrs Mean age at death (days) Generatin N umber f tumrs Mice with Mice withut Percent TE50* tumrs in tumrs in Number f transplants transplants transplants 6 28/ / / / Ttal 91/ Dwnladed frm at Pennsylvania State University n May 10, 2016 "The time (In days) It tk 60% f the transplants t prduce mammary tumrs. JOURNAL OF THE NATIONAL CANCER INSTITUTE

7 TUMOR POTENTIAL OF MAMMARY OUTGROWTH LINES 359 TABLE 5.-Tumr ptential f different transplant generatins f D2 ndule utgrwths in J. C3H mice Tumrs Mean age at death (days) Generatin Number f tumrs Mice with Mice withut Percent TE50* tumrs in tumrs in Number f transplants transplants transplants 2 12/ / / Ttal 43/ "The time (In days) It tk 50% f the transplants t prduce mammary tumrs. Respnse f Ndule Outgrwth Line 02 t Prlnged Hrmne Stimulatin Table 6 summarizes the effects f hrmne stimulatin n the tumr-prducing capabilities f ndule utgrwth line D2 in virus-free and virus-psitive mice. Mice in grups I-V received utgrwths frm generatin 8 f line D2. In grup I, 19 f 40 utgrwths (48%) prduced tumrs in untreated female mice, at a mean latent perid f 246 days. In grups II and III, hrmne stimulatin f the mammary glands f mice was achieved by pituitary isgrafts under the kidney capsule. Grup II mice received a single pituitary isgraft, whereas grup III mice received 2 pituitary isgrafts. In grup II, 25 f 35 utgrwths (71%) prduced tumrs, with the 50% tumr endpint reached at 259 days. In grup III, 25 f 30 utgrwths (83%) prduced tumrs, with the 50% tumr endpint reached at 203 days. Bth 50% tumr endpints differed significantly frm each ther at the 5% level and als frm the tumr endpint in grup I. In grups IV and V, utgrwths were transplanted int the cleared fat pads f MTV-psitive f. C3H females. In grup IV, 23 f 26 utgrwths (88%) prduced tumrs, with the 50% tumr endpint reached at 182 days in bth grups. These results differed significantly frm thse btained in grup I at the 5% level. In grup V, 16 f 17 utgrwths (94%) prduced tumrs in hrmne-stimulated f. C3H mice, with the 50% tumr endpint reached at 147 days. These results indicate an additive effect between MTV and hrmne stimulatin rather than a synergistic effect between the 2 agents. Respnse f Outgrwth Lines 02 and 03 t NIV The effects f NIV n the tumr-prducing capabilities f utgrwth lines D2 and D3 were determined by transplanting samples f the utgrwths int MTV-negative, NIV-psitive (C3Hf X )F 1 mice (table 6). The dnr D2 utgrwths were taken frm 2 mice f generatin 5. Of 16 transplants taken frm 1 dnr, 9 prduced mammary tumrs (56%), with the 50% tumr endpint reached at 357 days (grup VI). Outgrwths frm the ther dnr prduced nly 2 tumrs in 14 transplants (grup VIII). The mrphlgy f 12 utgrwths in grup VII which did nt prduce tumrs was similar t that f the variants f D2 utgrwths described in (1). The alveli were extremely distended and filled with a white secretin resembling milk. The tumr incidences between the 2 grups bearing D2 utgrwths were significantly different (P<0.05). Outgrwth line D3 in (C3Hf X )F 1 mice (grup X) prduced a high incidence f mammary tumrs, with 19 tumrs ccurring in 31 transplants (61 %) and the 50% tumr endpint reached at 406 days. The tumr incidence f NIV-infected D3 utgrwths differed significantly frm that f NIV-infected Dl utgrwths (3). Tumr Ptential f Ndule Outgrwth Lines 02 and 03 in MeA-Treated Virus-Free and Virus. Psitive Mice The results f experiments n the effect f MCA n utgrwth lines D2 and D3 are shwn in table 7. Bth utgrwth lines respnded t MCA differently than did line D 1. D2 utgrwths expsed t 1.5 mg MCA pr- Dwnladed frm at Pennsylvania State University n May 10, 2016 VOL. 45, NO.2, AUGUST 1970

8 360 MEDINA AND DEOME III., 's III.::: rn i '> III > b() III % '>'".S Cf,) A C'I A III '" d ;::::: :S t.<j III ;3 d '0 s 0.. '" S I co P1 < '00' :S ex! III.<J ex! ex! :gl le.<j '" '" ij4.<j =!S 0... d.d",ex!.<j",_ '-.1 0 Srn ;::!._+=» J-4 :g.<j.dd!s.-i..s III S ;g,:::,ex! "",.<J",.<J * ll';) ril..,., III '" p., rn '" S... rn '" S III d.0 Z ex! S.!3 Z d III 0 s::. ex! ;3 S ij4 Z :g d 'OJ.!3 rn Ill.d '" ;3-; 0 0'" Z g 0.. c'5 C'lt-t-OC'lCf,)... ll';)c'lo C'lOO... ll';)... ll';)t-cf,)ooco Cf,)C'lC'lCf,)... Cf,)Cf,)Cf,)... Cf,) O.<J....<J 1lleX! '" d.0- COOCf,)C'lO-.t<O>COtt:>t- -.t<ll';)-.t<c':>c'lo-.t<cf,) C'lC'IC'I... C':>C':>C':>C'lC':> 1:15 I C'lC'I... C':> C'I-.t< Cf,)-.t<c-.t<C'I0>... -.t<t-ooo>ll';)... C':>t-c Ott:>O COt-CO-.t<C'I t<c':>c':>c'i... COC'lC':> O>ll';)ll';)C':>CO O>C'lOC'l 0>... C'lC'lC'I... C'lC'I ""' f- c.qq r;r; '0''0 '0 ij4ij4 ij4 C':>C':>...:l...:l C':>... 00«Ojil :...:....:..,.,.,.,.,xx.,.,x :l...:l...:l...:l...:l ij4ij4...:l...:lij4 ««<C':>C':>«C':> sss C'lC'IC'IC'IC'IC'IC'IC':>C':>C':> AAAAAAAAAA :::»».; U;"'3...; '"' fil '0 0", '" 3" 8 '" _.. ;.; ca:g a W '" ".!:l "", G:I a5.!! "O "go=",c :S p. 0'" '" s i g =.!!l '" 1!!3 >.... tee; 8 ';s s - s::::.- c.. U;:Ee "''''bi> c",.!!l... OC.a 8:1"'- :Q.9... O'l 0 til 'fii'.d.ate.5 -.d.d ctleq..el.el cp 8 CD 1l::;J r++.. duced 90% tumrs (18/20), with the 50% tumr endpint reached at 238 days (grup I). Thse expsed t 1.5 mg MCA and cntinuus hrmne stimulatin (1 pituitary under kidney capsule) prduced 100% tumrs (11/11), with the 50% tumr endpint reached at 196 days (grup II). Cmparing the tumr incidences in grups I and II with the tumr incidence f line D2 in pituitary-bearing mice (table 6) suggested that hrmne stimulatin and MCA had an additive effect. In grup III, D2 utgrwths expsed t 1.5 mg MCA in MTV-psitive j. C3H mice prduced 94% (30/32) mammary tumrs, with the 50% tumr endpint reached at 168 days. The 50% tumr endpint f D2 utgrwths in MCA-treated, MTV-psitive mice was similar t that f D2 utgrwths in untreated, MTV-psitive mice (table 1). The tumr-prducing capabilities f MCA-treated D2 utgrwths differed significantly (P<0.05) frm thse f untreated D2 utgrwths. D3 utgrwths expsed t 1.5 mg MCA In mice prduced 92% tumrs (24/26, grup IV), with the 50% tumr endpint reached at 252 days. Thse expsed t 1.5 mg MCA in hrmne-stimulated mice (1 pituitary under kidney capsule) prduced 94% tumrs (16/17, grup V), with the 50% tumr endpint reached at 238 days. The tumr-prducing capabilities f MCA-treated D3 utgrwths in hrmnestimulated and unstimulated mice did nt differ significantly frm each ther (P>0.05) nr did the tumr-prducing capabilities f MCA-treated D3 utgrwths differ significantly (P>0.05) frm thse f MCA-treated D2 utgrwths. DISCUSSION The mrphlgical, histlgical, and grwth characteristics f series D ndule utgrwth lines (reprted elsewhere) identified these ndule cell ppulatins as preneplastic rather than neplastic (1). The data reprted herein demnstrate that MTV-free, NIV-free ndule utgrwth lines had a wide variatin in tumr ptential. This wide variatin in tumr ptential als was demnstrated in C3H ndule utgrwth lines (13, 14). Outgrwth lines riginating frm BALB/- ccrgl hyperplastic alvelar ndules (Dl, D2, D2a, Dwnladed frm at Pennsylvania State University n May 10, 2016 JOURNAL OF THE NATIONAL CANCER INSTITUTE

9 and DB) had tumr-prducing capabilities f 4, 26, 45, and 61%, respectively. Outgrwth lines riginating frm AnDe hyperplastic alvelar ndules (D3, D4, and D5) had tumr-prducing capabilities f 32, 36, and 7%, respectively. Thus, the MTV-negative, NIV-negative ndule utgrwth lines had the same variatin in tumr ptential, regardless f whether they riginated in a lw-mammary tumr strain (OrgI) r in a higher mammary tumr strain (AnDe). Since the tumr-prducing capability f ndules is essentially a prbability statement, the incidence f a spntaneus tumr in a given strain may reflect the number f ndules induced and hw early ndules ccur in the lifetime f the muse. This hypthesis is supprted by the tempral pattern f nduligenesis in virgin and hrmne-stimulated f. 03H female mice (I5). Ndules ccurred earlier and in greater numbers in hrmnestimulated f. 03H mice than in virgin f. C3H mice; hwever, the tumrprducing capabilities f ndules were the same in bth virgin and hrmne-stimulated mice. This hypthesis emphasizes the imprtance f the ndule transfrmatin in determining mammary tumr incidence, in additin t the well-dcumented rle f MTV's and chemical carcingens in the ndule t tumr transfrmatin (2-5, 16). Previus results indicated that MTV increased significantly the tumr-prducing capabilities f utgrwth line DI (2, 3). Of 6 additinal utgrwth lines f series D tested fr their respnse t MTV, 5 shwed increased tumr-prducing capabilities. The tumr-prducing capabilities f utgrwths free f MTV did nt crrelate with their increased tumr-prducing capabilities fllwing infectin with MTV. Fr example, utgrwth lines DB and D2a respnded similarly t MTV; yet line DB prduced twice as many tumrs in MTV-free mice than did line D2a. Thus, the behavir f an utgrwth line t MTV culd nt be predicted n the basis f its backgrund tumrprducing capabilities. It was expected that the respnse f varius ndule utgrwth lines t MTV wuld be variable and, thus, upn examinatin f numerus utgrwth lines, ne line wuld be seen t respnd very prly t MTV. Tested at transplant generatins 3 and 6, line D5 failed t respnd t TUMOR POTENTIAL OF MAMMARY OUTGROWTH LINES <> S > ril '".:;: ;....:;:..., :l..;: :;g.s C!:) ; c<l '" : ;; '3 s::... OJ :0 al b A... I r..: r.1..:l < :l b.o :l ; ::g 8..., '"., il:: 8...,..., gs::.j:l'-...,,,,as s::... J.._ Sgj C>aS :@+>.J:lS::...,;l... =..$ 00-. S gj ",,:l ""+>..., * t:> 'il +> til <> p..... ", 0..., "'I 8'"... S:: 15 8 z..., f! Z s:: 0)0 S:: ::s S il::: '" -< :;g ::g s:: 00. 0)J1 '3 0 0'" z 6 0- ::s O':l c<l Ot:>t:> '1"""'4 c<lcr.lc<l t:>c<l 00-' cr.lcl c<l-'-'c<lc<l 0000c<l00 cr.lclt:>cr.l c<l-'... c<lc<l OOc<l ClOClClCl -' '" O... c<lt C"I'I""""4C'r.:lC"t'l"'""'i... 1"""41"""4 C'r.:l C'I, , il:: cr.l...: ii:fii:fii:fii:fii:f...:l...:l...:l...:l...:l -<-<-<-<-< jl:ljl:ljl:ljl:ljl:l c<lc<lc<lcr.lcr.l ::::::> i m:s.>4... :5 '"... '8... ",c 5l'ta ;j!:l...!:l " ; '5.8 :... ta :::a ".s 5l '" ",.c... ll'".!l ll... ". 8;:;1 361 Dwnladed frm at Pennsylvania State University n May 10, 2016 VOL. 45, NO.2, AuGUST 1970

10 362 MEDINA AND DEOME MTV with an increased tumr-prducing capability. The pssibility existed that the hst mice were nt infected with MTV. Assay f the hsts' mammary tissues, hwever, revealed cnsiderable MTV activity (1). In additin, mammary tumrs develped in the hsts' mammary glands in 5 f 50 mice by 1 year. Anther pssibility was that the ndule utgrwth line was refractry t MTV. Thin sectins f 6 samples f utgrwth line D5 were examined under the electrn micrscpe at 16, 32, and 36 weeks pst transplantatin; nne had B particles. Samples f utgrwth line D5 were examined lng after B particles were readily fund in utgrwth lines DI and D2 (17). Thus the unrespnsiveness f D5 t MTV might be due either t its refractriness t MTV infectin r replicatin r t a lw level f MTV infectin. Analysis f the tumr-prducing capabilities f the utgrwth lines in MTV-free mice and in MTV-psitive f. C3H mice indicated that 2 variables cntributed t the final tumr-prducing capabilities. If the percentage f tumrs was pltted against time (text-figs. 2 and 4), a straight line generally was btained. The slpe f the line indicated the rate f tumr prductin fr any given utgrwth. Outgrwth lines D2, D3, and D8 had similar rates f tumr frmatin in mice; hwever, they differed in the initial time f tumr frmatin (cnsidered the 10% tumr incidence pint). Thus, the final tumr-prducing capability differed in the 3 utgrwth lines. It appeared the ndule cell ppulatins differed with regard t the time required t exceed sme threshld; but nce the threshld was exceeded, the prcess f tumr frmatin ccurred at a similar rate. In sme cases, the initial time f tumr frmatin was similar; hwever, the rates f tumr frmatin were different (see lines D2A and D3 in, text-fig. 2; lines Dl, D3, and D4 in J. C3H, text-fig. 4). In almst all cases, the cnstant slpe suggested that the prbability f tumr frmatin was cnstant ver a lng perid; hwever, this prbability varied fr different lines. The basis fr this difference was tissue dependent, since all ther experimental cnditins were equal. Previus studies indicated that NIV was effective in the ndule t neplastic transfrmatin in DI utgrwths, althugh the respnse t NIV was less than that t MTV (3). The data reprted herein suggest that NIV affected the tumr ptential f line D3 but nt that f D2. They d nt suggest that NIV did nt infect utgrwth line D2, but merely suggest that the tumr ptential f line D2 was high enugh t mask the effect f NIV. These data als suggest a crrelatin between the mrphlgical characteristics f an utgrwth line and its tumrigenic respnse. All 12 utgrwths in grup VII (table 6) were cmpsed f cystic alveli, whereas utgrwths in grup VI (table 6) were cmpsed f nrmal lbulalvelar tissue. The failure f the D2 utgrwths in grup VII t prduce tumrs in NIVinfected (C3Hf X )F\ mice and in mice (generatin 6A-table 3) suggests an assciatin f the lw tumrigenic ptential with the cystic alveli f these utgrwths. This subline f ndule utgrwth D2 cmpsed f cystic alveli is being maintained, and further studies are in prgress t determine its tumrigenic ptential in MTV-negative and MTV-psitive mice and its grwth characteristics in nrmal and hrmnally manipulated mice. These data als indicate that MCA and prlnged hrmne stimulatin each culd increase the tumr-prducing capabilities f D2 and D3 ndule utgrwths. Previus results indicated that MCA, but nt prlnged hrmne stimulatin, increased the tumr-prducing capabilities f ndule utgrwth line Dl (4). In the present experiments, MCA increased the tumr ptential f utgrwth lines D2 and D3 frm 45 and 32%, respectively, t 90 and 92%, respectively. Hrmne stimulatin increased the tumr ptential f utgrwth line D2 frm 71 t 83%. Thus, these experiments, tgether with thse n DI utgrwths (2-4), demnstrate that the respnsiveness f the D series f ndule utgrwth lines t varius viral and chemical ncgenic agents cannt be predicted frm their backgrund tumr ptential. Furthermre, they indicate that a wide variety f respnses t viral and chemical agents-frm ttal refractriness t a high degree f susceptibility-can be expected when ne examines a series f cell ppulatins frm ne tissue type. It has been suggested that the actin f MCA with either MTV r NIV was additive rather than Dwnladed frm at Pennsylvania State University n May 10, 2016 JOURNAL OF THE NATIONAL CANCER INSTITUTE

11 TUMOR POTENTIAL OF MAMMARY OUTGROWTH LINES 363 synergistic (4). The present experiments further supprt this hypthesis. MTV cmbined with MeA increased the tumr ptential f ndule utgrwth line D2, equal t the summatin f the 2 agents alne. A similar situatin ccurred when the effects f MTV plus hrmne stimulatin and MeA plus hrmne stimulatin n the tumr ptential f D2 were examined. Thus, these experiments suggested: 1) MeA acted independendy and additively with MTV r prlnged hrmne stimulatin; and 2) hrmne stimulatin acted additively with MTV r MeA (D2 utgrwths) r synergistically with MTV r Me (Dl utgrwth), depending n the ndule utgrwth used. Sme experiments have demnstrated that the D series f ndule utgrwths are susceptible t MTV, NIV, and a variety f chemical and physical carcingens (2-5, 16). The system can be manipulated t study the individual, cmbined, and sequential effects f viral and chemical carcingens. Thus, series D ndule utgrwth lines is a gd mdel system in which t study the prblem f virus-chemical interactins resulting in neplasia. REFERENCES (1) MEDINA D, DEOME KB: Bilgical characteristics f the D series f BALBfc ndule utgrwth lines. In preparatin (2) ---: Influence f mammary tumr virus n the tumr-prducing capabilities f ndule utgrwths free f mammary tumr virus. J Nat Cancer Inst 40: , 1968 (3) ---: Respnse f hyperplastic alvelar ndule utgrwth-line Dl t mammary tumr virus, nduleinducing virus, and prlnged hrmnal stimulatin acting singly and in cmbinatin. J Nat Cancer Inst 42: , 1969 (4) MEDINA D, FAULKIN LJ JR, DEOME KB: Cmbined effects f 3-methylchlanthrene, mammary tumr virus, ndule-inducing virus, and prlnged hrmnal stimulatin n the tumr-prducing capabilities f the ndule utgrwth line DI. J Nat Cancer Inst 44: , 1970 (5) DEOME KB, MEDINA D: A new apprach t mammary tumrigenesis in rdents. Cancer 24: , 1969 (6) NANDI S: New methd fr detectin f muse mammary tumr virus. I. Influence f fster nursing n incidence f hyperplastic mammary ndules in Crgl mice. J Nat Cancer Inst 31 :57-73, 1963 (7) PI'I'ELKA DR, BERN HA, NANDI S, et al: On the significance f virus-like particles in mammary tissues f C3Hf mice. J Nat Cancer Inst 33 : , 1964 (8) DEOME KB, FAULKIN LJ JR, BERN HA, et al: Develpment f mammary tumrs frm hyperplastic alvelar ndules transplanted int gland-free mammary fatpads ffemale C3H mice. Cancer Res 19: , 1959 (9) Mfuu.BOCK 0, BOOT LM: Inductin f mammary cancer in mice withut the mammary tumr agent by isgrafts f hypphysis. Cancer Res 19: , 1959 (10) LOEB L, KIRTZ, MM: Effects f transplants f anterir lbes f the hypphysis n the grwth f the mammary gland and n the develpment f mammary gland carcinma in varius strains f mice. Amer J Cancer 36:56-82, 1939 (11) BARDIN CW, LIEBELT AG, LIEBELT RA: Mammary gland develpment after hypphysial isgrafts in intact mice f high and lw mammary cancer strains. J Nat Cancer Inst 36: , 1966 (12) FAULKIN LJ JR: Hyperplastic lesins f muse mammary glands after treatment with 3-methylchlanthrene. J Nat Cancer Inst 36: , 1966 (13) BLAIR PB, DEOME KB, NANDI S: The preneplastic stage in muse mammary carcingenesis. In Bilgical Interactins in Nrmal and Neplastic Grwth. Henry Frd Hspital Sympsium. Bstn, Little, Brwn & C., 1962, pp (14) INGRAHAM RL: Variability f muse mammary hyperplastic ndules maintained in rgantypic culture. J Nat Cancer Inst 40: , 1968 (15) MEDINA D, DEOME KB, YOUNG L: Tumr-prducing capabilities f hyperplastic alvelar ndules in virgin and hrmne-stimulated BALBfcf. C3H and C3Hf mice. J Nat Cancer Inst 44: , 1970 (16) MEDINA D, DEOME KB: Carcingen-induced mammary tumrs frm preneplastic ndule utgrwths in BALBfc mice. Cancer Res. In press (17) MEDINA D, DEOME KB, PlTELKA DR, et al: Appearance f virus particles in BALBfc ndule utgrwth lines transplanted int BALBfc f. C3H and (C3Hf X BALBfc)F t mice. In preparatin Dwnladed frm at Pennsylvania State University n May 10, 2016 VOL. 45, NO, 2, AUGUST 1970