Increase of Induced Skin Tumors in the Mouse by the Lethal Yellow Gene (A 11) 1

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1 Increase f Induced Skin Tumrs in the Muse by the Lethal Yellw Gene (A 11) 1 GEORGE VLAHAKIS and W. E. HESTON, Labratry f Bilgy, Natinal Cancer Institute,2 Bethesda, Maryland SUMMARY The lethal yellw gene (Au) increased r nt highly significant statistically. susceptibility t skin tumrs induced There was a shrter latent perid in the in the muse by painting with methyl yellw mice than in the aguti mice f chlanthrene. This was mst clearly bth the male and female grups and a shwn by the statistically significant shrter time between the appearance greater average number f papillmas f the first tumr and the time the and carcinmas f the skin in the yel animal was autpsied. All these: differlw (AuA) mice than in the aguti (Aa) ences were in the same directin, mice. The final incidence f papill althugh sme f the individual differmas and epidermid carcinmas was ences were nt highly significant sta greater in the yellw than in the aguti tistically. Females shwed a greater mice f bth the male and female tumr respnse t the painting f the grups. All these incidences, hwever, skin with methylchlanthrene than did apprached 100 percent and the indi males.-j Nat Cancer Inst 31: , vidual differences were nly brderline PREVIOUS REPORTS presented evidence fr a relatinship between the lethal yellw gene (All) f the aguti series and ccurrence f a number f neplasms in mice. Lethal yellw, which augments bdy weight, increases susceptibility t bth induced and spntaneus pulmnary tumrs (1, 2), an effect interpreted as due t the All gene itself. Lethal yellw als increases susceptibility t spntaneus hepatmas in males and t mammary gland tumrs as seen in the reduced age at which the latter arse in females (3). The average tumr age f the aguti (Aa) virgins was 15 mnths, while that f the yellw (All A) virgins was nly 8 mnths. In breeders this difference was eliminated, fr the average tumr age was apprximately 8 mnths fr bth AliA and Aa females. It seemed wrth while, therefre, t extend these studies t induced neplasms f the skin. Data presented herein shw that the gene als des increase susceptibility t induced skin neplasms. 1 Received March 5, Natinal Institutes f Health, Public Health Service, U.S. Department f Health, Educatin, and Welfare. 189

190 VLAHAKIS AND HESTON MATERIALS AND METHODS Because the Swiss muse is highly susceptible t induced skin tumrs, as riginally shwn by Cramer and Stwell (4), we chse this muse fr ur study but used the

2 190 VLAHAKIS AND HESTON MATERIALS AND METHODS Because the Swiss muse is highly susceptible t induced skin tumrs, as riginally shwn by Cramer and Stwell (4), we chse this muse fr ur study but used the highly inbred Swiss strain designated as SWR. The ther strain used was YBR that carries the lethal yellw gene (All). SWR females, which althugh albin in clr are hmzygus fr aguti (AA), were utcrssed t yellw strain YBR males, heterzygus fr lethal yellw (Alia), t prduce tw types f (SWR X YBR)F 1 animals: AliA (yellw) andaa (aguti). The SWR females, btained frm Dr. M. K. Deringer f this labratry, were f the F65 and F66 brther X sister inbred generatins. The YBR males frm ur wn clny were als highly inbred, f the F72 and F77 brther X sister inbred generatins. Since bth parental strains were highly inbred and, therefre, wuld be expected t be hmzygus at all lci except fr the aguti lcus where the YBR males wuld be Alia (AIIAII has a lethal effect), all the (SWR X YBR)Fl hybrids wuld be expected t be genetically alike except that half wuld be AliA (yellw) and half Aa (aguti). With all nngenetic factrs unifrm, any difference between the tw clr grups culd be attributed t this lethal yellw gene. Apprximately 50 (SWR X YBR)F/s f each gentype and sex were weaned when abut 1 mnth ld, placed in plastic cages measuring 7 X 11 X 5 inches, with 8 animals t a cage, and segregated as t sex but nt as t gentype. Each cage was equipped with a water bttle and feed hpper cntaining NCI pellets, the frmula fr which has been published (5). At apprximately 2 mnths f age each animal was weighed and then, with the use f a 1 cc tuberculin syringe, painted nce a week fr 12 weeks with 0.1 cc f 0.25 percent methylchlanthrene (Eastman Kdak) in thiphene-free benzene (Fisher). The methylchlanthrene, kept in a dark, glass-stppered cntainer, was applied t the interscapular area that had been clipped with an electric hair clipper. Once the painting was begun, each animal was examined fr skin tumrs at least nce a week until autpsy, and the number f thse bserved was recrded. The majrity f these early tumrs appeared as keratinized grwths and were classified as papillmas. They ccurred usually in the area painted with the carcingen. A few papillmas, hwever, develped in the ventral skin, a few in the psterir drsal skin, and a few in the skin f the head, arund r near the muth, and arund the eyes. The few early tumrs that appeared t be invading deeper tissues were classified tentatively as carcinmas. These were fund essentially in the painted area. The animals were autpsied when their skin tumrs were ulcerated and were large, measuring up t 21 mm in diameter, and induratin at the base indicated invlvement f the deeper tissues. Sme animals were autpsied when ther tumrs ccurred grssly, and sme when they were in pr cnditin frm ther causes. All the tumrs that appeared t be carcinmas r sarcmas, several papillmas frm each f mst animals, and all ther tumrs were taken JOURNAL OF THE NATIONAL CANCER INSTITUTE

INDUCED SKIN TUMORS AND THE A Y GENE 191 fr micrscpic examinatin, fixed in Fekete's mdificatin f Tellyesniczky's fixative (70% alchl, 20 parts; frmalin, 2 parts; glacial acetic acid, 1 part), munted

3 INDUCED SKIN TUMORS AND THE A Y GENE 191 fr micrscpic examinatin, fixed in Fekete's mdificatin f Tellyesniczky's fixative (70% alchl, 20 parts; frmalin, 2 parts; glacial acetic acid, 1 part), munted in paraffin, cut, and stained with hematxylin and esm. All tumrs were classified histlgically.s It was smetimes difficult t distinguish between papillmas and epidermid carcinmas, but thse classified as papillmas were fr the mst part smaller and mre elevated. Histlgically they shwed the thickened epithelial layer with rather nrmal rientatin f epidermis and intact basement membrane, althugh marked keratinizatin and crnificatin were usually present. Thse classified as epidermid carcinmas were usually larger and histlgically shwed anaplasia f the epithelial cells with invasin thrugh the basement membrane int the dermis. RESULTS AND DISCUSSION All the animals, except an Aa male in which the papillmas regressed althugh it had a subcutaneus fibrsarcma, had ne r mre f the tumr types mentined. As seen in table 1, the All gene increased susceptibility t these induced skin tumrs, particularly as shwn by the average number f tumrs. The AliA males had an average f 5.5 papillmas while the Aa males had an average f 4.3, a difference brderline in significance (t = 1.848; P is between 0.1 and 0.05). Fr the females the difference was highly significant, the AliA grup having an average f 9.3 papillmas, the Aa grup an average f nly 4.5 (t = 6.064; P< 0.001). When the sexes were cmbined, the AliA animals had an average f 7.1 papillmas and the Aa animals an average f 3.8. This difference is highly significant (t = 5.425; P<O.OOl). When cmpared with respect t carcinmas (bth epidermid and basal cell), the AliA males had an average f 1.8 while the Aa males had an average f 1.3, a difference that is highly significant (t = 3.008; P<O.005). In the females the AliA grup had an average f 2.5 carcinmas, the Aa grup an average f 1.7, and this difference was als highly significant (t = 3.794; P<O.OOl). When the sexes were cmbined, the AliA animals had an average f 2.1 while the Aa animals had an average f 1.4. This difference, f curse, is als highly significant (t = 4.640; P<O.OOl). If the papillmas and carcinmas are cmbined, the effect f the lethal yellw gene n skin tumr frmatin can be demnstrated in the fllwing averages btained fr the respective grups: All A males, 6.7 skin tumrs; Aa males, 4.8 skin tumrs (t = 2.429; P is between 0.02 and 0.01); AliA females, 11.6 skin tumrs; Aa females, 5.7 skin tumrs (t = 6.601; P <0.001). On the average there were mre skin tumrs in the AliA grup than in the Aa grup fr either sex, and the differences were significant. 8 We are indebted t Dr. Thelma Dunn, Pathlgist f the Natinal Cancer Institute, fr ensuitatin ensultatln n the classificatin f the neplasms. VOL. 31, NO.1, JULY 1963

4 ... q t< ":l t< (J :> (J....., UJ... cl TABLE I.-Respnse f (SWRXYBR)FI mice t 12 weekly paintings f the skin with 0.25 percent methylchlanthrene in benzene Average Average time time Mice with skln tumrs and subcutaneus sarcmas fund at autpsy between between first first Number Percent Average palntlng papillma Average with with Number Average number f Number and first and age at Number Percent Average epider- epider- with basal number f skln with sub- Number papillma autpsy autpsy with with number f mid mid cell carclnmas tumrs cutaneus Gentype Sex f mice (weeks) (weeks) (mnths) papillma papillma papillmas carcinma carclnma carclnma cmblned cmblned sarcmas A-A cj'cj' Aa cj'cj' A-A Aa A-A- cj'cj' and ci'ci' Aa' and Abve data cmbined.... t t>.:) tl :r:... rfl :> ti U1 z...

INDUCED SKIN TUMORS AND THE A]' GENE 193 Since nt all papillmas were taken frm each animal fr histlgic examinatin, it is pssible that sme may have been carcinmatus.

5 INDUCED SKIN TUMORS AND THE A]' GENE 193 Since nt all papillmas were taken frm each animal fr histlgic examinatin, it is pssible that sme may have been carcinmatus. Hwever, when incidence data were btained frm the histlgic classificatin, the yellw mice had a higher incidence f papillmas and epidermid carcinmas than the aguti animals in bth the male and the female grups. All these incidences apprached 100 percent, and differences within grups were nt statistically significant r were nly brderline in statistical significance. In an Aa male there was metastasis f an epidermid carcinma t the lung. In bth the male and female grups the average time between first painting and the appearance f the first papillma was less in the yellw than in the aguti animals, but in neither grup was the difference highly significant statistically. Furthermre, in the male and female grups the time between the appearance f the first papillma and autpsy was less in the yellw than in the aguti animals, but the difference was statistically significant nly fr the females (t = 2.498; 0.01 <P <0.02). Hwever, that all these differences were in the same directin has sme significance and suggests that the effect f this gene n increasing susceptibility is als expressed in decreasing latent perid and increasing grwth rate f the tumr. There was a significant sex difference in the papillma and carcinma respnse f the skin t the painting with methylchlanthrene, the females cnsistently shwing a greater respnse than the males. Andervnt and Edgcmb (6) in their study f the respnse f 7 inbred strains f mice t induced skin tumrs als fund a sex difference fr mst strains. The differences they bserved, hwever, were nt unifrm, fr greater susceptibility ccurred in either the males r the females f the respective strains. In the present study the yellw females had an average f 9.3 papillmas, which was significantly greater than the average f 5.5 fr the yellw males (t = 4.564; P <0.001). The aguti females had a slightly higher average than the aguti males but the difference was nt significant. The average number f carcinmas in the yellw females was 2.5, which was significantly greater than the average f 1.8 in the yellw males (t = 3.220; P <0.005). The average number f carcinmas in the aguti females was als significantly greater than that in the aguti males (t = 2.529; P <0.02). In bth clr grups the females had a higher incidence f papillmas and carcinmas but, as indicated earlier, all these incidences apprached 100 percent, and the differences were nt statistically significant. The latent perid was less in females than in males. The average time between the first painting and the appearance f the first papillma in the yellw females was 12.7 weeks, significantly less than the 15.5 weeks fr the yellw males (t = 4.073; P <0.001), and the average time f 13.5 weeks fr the aguti females was significantly less than the average time f 16.4 weeks fr the aguti males (t = 3.412; P <0.001). Furthermre, the tumrs apparently grew mre rapidly in the females than the males. The average time between the appearance f the first tumr and autpsy VOL. 31, NO.1, JULY 1963

194 VLAHAKIS AND HESTON in the yellw females was 6.4 weeks, significantly less than the 9.3 weeks in the yellw males (t = 3.657; P <0.001). The average time in the aguti females was 8.

6 194 VLAHAKIS AND HESTON in the yellw females was 6.4 weeks, significantly less than the 9.3 weeks in the yellw males (t = 3.657; P <0.001). The average time in the aguti females was 8.3 weeks, which was less than the 10.1 weeks in the aguti males, but this difference is nly brderline in significance. The subcutaneus sarcmas, ccurring in this grup f mice nly in the males (table 1), were fund in the area painted with the carcingen. In additin, there were 4 All A and 12 Aa females that develped carcinsarcmas f the mammary gland, a rather unusual finding since this is usually a rarely ccurring tumr. One AliA and 7 Aa females had the usual mammary adenca.rcinmas and in 2 AI/A and 3 Aa females there were adenacanthmas f the mammary gland. One All A male and 1 Aa male shwed an accumulatin f plasma cells, which may have been neplastic, adjacent t papillmas. Small areas f dark-pigmented cells appeared usually at the base f papillmas in 12 Aa males and 7 Aa females. These pigmented areas were similar t thse bserved by thers in the skin f mice painted with carcingenic hydrcarbns (7). Such pigmented areas were nt bserved in the All A animals. A lymphcytic leukemia was fund in a yellw female and a hepatma in an aguti male. All gentypic grups were susceptible t the develpment f pulmnary tumrs, but, since this study was nt designed t study pulmnary tumrs and the age at autpsy was nt unifrm, n attempt was made t cunt the multiple small tumrs ccurring with relatively high frequency in mst animals. The data presented in this paper thus add anther grup f neplasms, induced neplasms f the skin, t the list f thse whse ccurrence is increased by the intrductin f this remarkable lethal yellw gene. In additin t increasing susceptibility t skin tumrs, pulmnary tumrs, mammary tumrs, and hepatmas, this gene als increases nrmal grwth. Data in table 2 shw the effect f the gene n increasing the weight f the mice in this study. In earlier studies (3, 8) it was shwn that nt nly are the yellw mice fatter, but they als have an increase in grwth f skeletn and muscle tissue as cmpared with their nnyellw littermates. TABLE 2.-Bdy weights f (SWR X YBR)F 1 mice painted nce a week fr 12 weeks with 0.25 percent methylchlanthrene in benzene Average bdy A verage bdy Number weight at weight at f start f apprximate time Gentype Sex mice painting (g) f autpsy (g) AyA 0'0' Aa 0'0' AyA <? <? Aa <? <? '0' AyA* and <? <? 0'0' Aa* and <? <? Abve data cmbined. JOURNAL OF THE NATIONAL CANCER INSTITUTE

INDUCED SKIN TUMORS AND THE A Y GENE 195 There is evidence that this effect f the gene n nrmal grwth is related t its effect n susceptibility t the varius neplasms.

7 INDUCED SKIN TUMORS AND THE A Y GENE 195 There is evidence that this effect f the gene n nrmal grwth is related t its effect n susceptibility t the varius neplasms. The gene als causes the hair t be yellw, and when it is hmzygus it has a lethal effect, the animals dying III the early embrylgic stages f develpment. The effect f the gene n cat clr suggests that its primary actin invlves an enzyme r enzyme deficiency that causes the metablism f tyrsine t be shunted ver t the prductin f yellw pigment thrughut the grwth f the hair. Much wrk n the bichemical level must. be dne if we are t determine whether such a primary actin culd cause all these ther apparently divergent effects. The system prvided by the FI hybrids ffers an apprach t ne further step n a smewhat higher level in the analysis f the effect f the gene: that f determining whether its actin n increasing susceptibility t these induced neplasms f the skin is lcalized in the end rgan, the skin. It is hped that this questin can be answered thrugh the bservatin f the inductin f tumrs in bth All A and Aa skin transplanted int All A hsts and III skin f bth gentypes transplanted int Aa hsts. REFERENCES (1) HESTON, W. E.: Relatinship between the lethal yellw (Au) gene f the muse and susceptibility t induced pulmnary tumrs. J Nat Cancer Inst 3: , (2) HESTON, W. E., and DERINGER, M. K.: Relatinship between the lethal yellw (Au) gene f the muse and susceptibility t spntaneus pulmnary tumrs. J Nat Cancer Inst 7: , (3) HESTON, W. E., and VLAHAKIS, G.: Influence f the Au gene n mammary-gland tumrs, hepatmas, and nrmal grwth in mice. J Nat Cancer Inst 26: ,1961. (4) CRAMER, W., and STOWELL, R. E.: Skin carcingenesis by a single applicatin f 20-methylchlanthrene. Cancer Res 3: 36-42, (5) HESTON, W. E., VLAHAKIS, G., and DERINGER, M. K.: High incidence f hepatmas and the increase f this incidence with urethan in C3H, C3Hf, and C3He male mice. J Nat Cancer lnst 24: , (6) ANDERVONT, H. B., and EDGCOMB, J. H.: Respnses f seven inbred strains f mice t percutaneus applicatins f 3-methylchlanthrene. J Nat Cancer lnst 17: , (7) STEWART, H. L.: Experimental cutaneus carcinma. In The Physipathlgy f Cancer (Hmburger, F., ed.), 2d ed. New Yrk, Heber-Harper, 1959, chapt. 1, pp (8) HESTON, W. E., and VLAHAKIS, G.: Eliminatin f the effect f the Au gene n pulmnary tumrs in mice by alteratin f its effect n nrmal grwth. J Nat Cancer lnst 27: , VOL. 31, NO.1, JULY 1963

The estimator, X, is unbiased and, if one assumes that the variance of X7 is constant from week to week, then the variance of X7 is given by

The estimator, X, is unbiased and, if one assumes that the variance of X7 is constant from week to week, then the variance of X7 is given by ESTIMATION PROCEDURES USED TO PRODUCE WEEKLY FLU STATISTICS FROM THE HEALTH INTERVIEW SURVEY James T. Massey, Gail S. Pe, Walt R. Simmns Natinal Center fr Health Statistics. INTRODUCTION In April 97, the