Influence of Mouse Major Histocompatibility Complex (H-2) …...The influence of the major histocompatibility complex (MHC) of the mouse (H-2) on carcinogen-induced tumorigenesis was

[CANCER RESEARCH 48, 6634-6641. December 1, 1988]

Influence of Mouse Major Histocompatibility Complex (H-2) on TV-Ethyl-TV-nitrosourea-induced Tumor Formation in Various Organs1

L. C. J. M. Oomen,2 M. A. van der Valk, A. A. M. Hart, P. Demant, and P. Emmelot

Divisions of Molecular Genetics [L. C. J. M. O., M. A. v. d. V., P. D.J, Chemical Carcinogenesis [P. E.], and Clinical Oncology {A. A. M. H.J, The Netherlands CancerInstitute, Plesmanlaan IH, 1066 CX Amsterdam, The Netherlands

ABSTRACT

The influence of the major histocompatibility complex (MHC) of themouse (H-2) on carcinogen-induced tumorigenesis was investigated. Miceof five 11-2 congenie strains on the CS7BL/10 background were treatedwith the direct-acting carcinogen /V-ethyl-A'-nitrosourea at the age of 15

days, and examined for tumors when moribund. Significant differencesbetween strains in susceptibility to jV-ethyl-W-nitrosourea-induced tumorsin lung, small intestine, and liver were found. For lung tumors the strainsB10.A and 2R were most susceptible, the strains 4R and BIO wererelatively resistant. The strain 5R was intermediate. Susceptibility tosmall intestine tumors was highest in the strain 2K, intermediate in thestrain B10.A, the strains 4R, 5R, and BIO were relatively resistant. Thelocation of the tumors in the intestine was also affected by 11-2. In the

strain 2R most tumors are located in the proximal part, in 4R in thedistal part. Tumorigenesis in the liver was highest in the strain 2R,intermediate in the strains B10.A, 4K. and BIO, and lowest in the strain5R.

We conclude that susceptibility to carcinogen-induced tumors in thelung, small intestine, and liver in congenie strains on the C57BL/10background is 11-2 haplotype dependent. Susceptibility to tumors in thelung and intestine has a similar strain distribution, but differs from thatfor liver tumors. Males were more susceptible than females in the strainBIO (lung tumors) and 4R (small intestine and liver tumors). Thisindicates haplotype- and organ-specific, sex-related influences on tumordevelopment.

The possible mechanism(s) of H-2 effects on chemically inducedtumorigenesis are discussed. Apart from the well-known immunologicalfunctions of the MHC, the involvement of hormonally related effects ofthe MHC is considered as well.

INTRODUCTION

The MHC3 of the mouse (H-2) influences the susceptibilityto spontaneous, virally and carcinogen-induced tumors. Tumorswith a viral etiology, i.e., leukemias or mammary tumors havebeen studied most extensively (for reviews see References 1 and2). In virally induced leukemogenesis the MHC control ofimmunological response against viral antigens on virions or oninfected cells accounts for the haplotype-linked resistance.These immunological effects of the MHC are based on the well-known functions of the Class I and Class II products in H-2-restricted antigen presentation to cytotoxic or helper T-lym-phocytes. In contrast to virally induced tumors, tumors inducedby chemical carcinogens often express antigens that are uniqueto a given tumor. This holds even for tumors of the samehistológica! type induced in mice of the same strain by the samecarcinogen (3). Therefore the mechanism(s) of the MHC-re-lated resistance to carcinogen-induced tumorigenesis might differ from those reported for virally induced leukemogenesis.Thus the study of the MHC-linked effects on carcinogen-

Received2/17/88;revised8/15/88;accepted8/24/88.Thecostsof publicationof thisarticleweredefrayedin part bythe payment

of pagecharges.Thisarticlemustthereforebe herebymarkedadvertisementinaccordancewith18U.S.C.Section1734solelyto indicatethisfact.

1This work was financially supported by the Scientific Advisory Board on

Smoking and Health, The Netherlands.2To whom requests for reprints should be addressed.3The abbreviations used are: MHC, major histocompatibility complex; ENU,

/V-ethyl-JV-nitrosourea; h.c., hepatocellular.

induced tumorigenesis may not only be relevant for understanding of factors influencing tumor resistance, but may also helpto elucidate some presently little known functions of the MHC.

An //-¿-related influence on carcinogen-induced tumorigenesis has been reported for tumors of lymphoid tissue and skin(4) and lung (for review see Reference 5). The development oflung tumors induced by several kinds of carcinogens in adults(6, 7), is under H-2 control. Also in mice treated prenatallywith ENU, lung tumor formation is affected by the H-2 complex. Different H-2 genes affect specifically the two majorhistológica! types of lung tumors (8). Thus in all these experimental systems the 11-2 complex influences susceptibility to

lung tumor formation. It must, however, be emphasized thatbesides the H-2 complex other genes also influence lung tumorformation (6, 7, 9-11).

To obtain further insight into the role of the MHC in resistance to carcinogen-induced tumors we tested in five H-2 con-genie strains on the C57BL/10 background whether the H-2complex affects ENU-induced carcinogenesis also in organsother than lung.

MATERIALS AND METHODS

Animals and Carcinogen Treatment

The H-2 congenie strains, their H-2 genotypes, and numbers of miceused are given in Table 1. All mice were reared and housed underspecific pathogen-free conditions (for further details on housing seeReference 8). Mice received a single i.p. injection of a freshly preparedsolution of ALethyl-ALnitrosourea (K & K Laboratories, NY) in trioc-tanoin (Fluka; A. G. Buchs, Switzerland; 0.5 mmol ENU/kg bodyweight) at the age of 15 days. They were weaned at 4-5 weeks of age

and separated according to sex and litter.

Tumor Examination

Mice were killed when visibly sick (moribund). Some mice diedduring the experiment. The very few cases in which no satisfactory(histological) examination was possible, were excluded from the experiment. At autopsy all macroscopically visible lesions were fixed in 4%neutral buffered formaldehyde solution and embedded in Paraplast formicroscopic examination, performed on 5 /mi sections stained withhematoxylin & eosin. From enlarged lymphoid organs also Giemsa-

stained dip preparations were prepared. For tumors of the small intestine in each mouse the number of tumors and their location wererecorded. To evaluate lung tumorigenesis in detail, the left lung lobefrom all mice, whether appearing macroscopically as lung tumor bearing or not, was histologically examined. All left lung lobes were uniformly sectioned som¡serially:5-/uin sections were taken at ISO-urnintervals. In each section (stained with hematoxylin & eosin) the diameter of tumors was measured; only tumors greater than ±200»<mwere taken into account. In this way detailed information as to thehistological type, number and size of lung tumors in individual micewas obtained.

Pathology

Lung Tumors. In this study three histological lung tumor types (12,13) were found: alveolar, papillary, and mixed. The papillary type oflung tumor is characterized by a papillary structure and growth into

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H-2 AND EMU-INDUCED TUMOR1GENESIS

Table 1 Strains, numbers of mice used, and survival data

StrainBlO.A/SgSnAB10.A(2R)/SgSfABIO.A(4R)/SgSfAB10.A(5R)/SgSnAC57BI/10ScSnAAbbreviatednameB

10.A2R4R5RBIOH-2

hap-lotypeatah4iSbGenotype"

(alíelesat particularregion)KkkkbbA„kkkbbA.kkkbbE,kk(k)b(b)E.kkokoSddbdbDdbbdbSex9è9<JSi$ÕΫNumber

ofmice*61444044SI5741475451Mean

agedaysc35737029030542138442940938840450%

survivaldays'*375400280315445430440405400440

" Strains are congenie for ¡1-2on the BIO background.* The number of female and male mice from each strain.' Mean age of death has been calculated from the age of death of each individual mouse.d The 50% survival age has been deduced from the survival curve of the strain.

alveoli, bronchioli, and possibly bronchi. The tumors of the alveolartype grow along the preexisting septal framework and are not clearlyseparated from the surrounding lung tissue. Mixed-type lung tumors,which contained areas characteristic of the alveolar and papillary type,were also found. Sometimes, mostly in the older mice, examples offusion of papillary and alveolar tumors were revealed by serial-stepsectioning. The constituting parts of such fused tumors were consideredas separate tumors. In most cases, however, the serial step-sectionsrevealed that the mixed-type lesions represent single tumors with oneor more alveolar and papillary areas.

Tumors of the Small Intestine. In this study macroscopically visibletumor (-like) lesions could be found all along the small intestine. Ineach strain approximately 30% of these lesions were taken proportionately from different parts of the intestine for histológica!investigation.As nearly 100% of these lesions were (papillary-) adenocarcinomas of

the epithelium (for detailed description of these tumors see Reference14), the results were further based on macroscopica! findings.

Liver Tumors. Microscopical examination of liver lesions with macroscópica!appearance of neoplasia revealed in several instances merehyperplasia. Therefore the results on liver tumors are based on microscopical classification. Liver parenchymal tumors usually are classifiedinto two categories: h.c. adenoma and hepatocellular carcinoma (IS).To classify h.c. tumors as carcinomas, we evaluated features (IS) usuallyassociated with malignancy: atypia of cells, markedly disturbed tissuearchitecture (and compression), invasion into surroundings, pleio-

morphism, high mitotic rate, atypical mitoses, and anaplasia. Métastases were found only in mice with h.c. carcinomas, and were neverencountered in animals with h.c. adenomas only.

Tumors in Other Organs. Published criteria were used to diagnoseHarderíangland tumors (16, 17) and ovarian tumors (18, 19).

Lymphoid neoplasms were diagnosed as "T-cell," when obviouslythymus-derived, "B-cell," when exhibiting B-cell-related characteristics(morphologically, both micro- and macroscopically), and as "non-B,non-T," when lacking all of these characteristics (20).

Tumors of the reticuloendothelial system were denoted "histiocytic"or "malignant histiocytic" in case of (erythro-) phagocytosis; endothelialtumors were subdivided only into "benign" (haemangioma, lymphan-gioma) and "malignant" (haemangioendothelio-sarcoma, lymphangio-

endothelio-sarcoma) using general criteria for malignancy.

Statistical Analysis

For each tumor type the results are given in a numerical form(percentage and mean age of tumor-bearing animals, mean number oftumors), and as actuarial, "at risk" curves. In the actuarial method of

statistical analysis (21) curves are constructed, showing the proportionof mice still free of the investigated tumor type at the end of each 50-day period. Mice that die of other causes are accounted for in thestatistical analysis. The analysis of a certain tumor type in differentstrains thus is not influenced by other tumors or other causes of deathas long as these are not related to the tumor under study and the lattercan be considered to be the cause of death. Inter-strain differences in

susceptibility to tumor formation in different organs are evaluated bycomparing the respective "at risk" curves using the logrank test, rather

than a single total value (e.g., tumor incidence).In many mice simultaneous occurrence of tumors in several organs

has been found in these experiments. Although this might complicatethe application of the actuarial method, there is no indication that thishas been the case here. The appearance of tumors in one organ did notaffect in any obvious way the occurrence of tumors in other organs.Not all tumors under study cause death shortly after being detectable,as the actuarial method and the logrank test require. To check the effectof this violation of assumptions, the data were also analyzed using theprevalence method. This method is valid for tumors not causing deathbut remaining in situ throughout the life of the animal. The use of theprevalence method did not materially change the conclusions reachedbased on the actuarial method and therefore the results are presentedaccording to the latter method of analysis.

RESULTS

Survival

Mice became moribund and were killed (or died) at agesranging from 100 to 800 days. Mean age of death, and 50%survival age are given in Table 1. For each strain there is onlya small difference between the mean age of death and the 50%survival age (see Table 1), indicating that the deaths were ratherevenly distributed in time. Between strains, however, differencesin survival are found. On the average mice from strains 4R, 5R,and BIO lived longest, those from strain 2R have the shortestmean survival time and strain BIO.A is intermediate.

Tumorigenesis

In some of the strains sex-related differences in developmentof tumors in the lung, small intestine, and liver were observed(see below). Therefore the results on tumorigenesis in theseorgans are reported separately for males and females.

Lung Tumors

The percentage of tumor-bearing animals, and the mean ageand mean number of tumors per tumor-bearing animal for eachlung tumor type are given in Table 2. The "at risk" curves for

the three lung tumor types (alveolar, papillary, and mixed) aregiven in Figs. 1-3.

Alveolar Lung Tumors. The "at risk" curves for females are

given in Fig. \A. Comparable curves were constructed for males(not shown). There are differences in susceptibility for alveolarlung tumors between strains (Fig. 1/Õ).Both in females and inmales the strain 2R is highly susceptible and different from thestrains 4R and BIO which are resistant. Strains BIO.A and 5Rare intermediate; in females they differ from strain 2R on the

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H-2 AND EMU-INDUCED TUMORIGENESIS

Table 2 Incidence, mean age, and number of tumors in mice from five H-2 congenie strains treated with ENV at the age of 15 days

LungtumorsAlveolarStrainB10.A2R4R5RBIOSexF

MFMF

MF

MFM%*

Mean age* Meannrc44

68597117

2151751329433

396337

355561

417479

411553

4761.5

±0.12.0±0.22.2

±0.22.2±0.21.0

±0.11.5±0.11.8

±0.22.4 ±0.21.0

±0.11.5 ±0.1Papillary%

Mean age Meannr60

6623

3839

4046

542340373

377373

322519

410467

415474

4171.9

+ 0.11.8±0.11.3

±0.11.4 +0.11.6

+ 0.11.2±0.11.6

+ 0.11.6±0.11.3

±0.11.2 ±0.1IntestinaltumorsMixed-type%

Mean age Meannr31

4910262

838522

15486

441458

446504

558567

4896055231.6

+ 0.11.4±0.11.8

±0.21.7±0.11.5

+0.11.5

+0.12.5±0.21.1

±0.1%

Mean age Meannr74

8793

95657561836169393

390304

314486

4404764204224263.8

±0.43.7 +0.35.5

±0.36.2 ±0.52.3

±0.23.5±0.32.3

+ 0.22.0 +0.21.9

+ 0.12.4 ±0.2Liver

tumorsAdenomasCarcinomas%

Mean age % Meanage20

3315

3232

5533

263943486

485511444545

477555

527543

4807

293

1414255

131114521

433484

409536

510542

558550

490" Percentage of mice with one or more tumors.* Mean age (days) of tumor-bearing mice.c Mean number of tumors per tumor-bearing mouse and SEM.

A °° ~

40-

20-

O-1 r nr rI ' I I ' I3456

\ ' I7 ex100 days

B

BIO A

B

B10A / \ ^

BIO

Fig. 1. Alveolar lung tumors in female and male mice from five 11-2congeniestrains on the C57BL/10 background treated postnatally with ENU. A, "at risk"

curves show the percentage of female mice still free of alveolar lung tumors(vertical axis) at the end of each successive 50 days interval (horizontal axis).Arrows, mean tumor age in each strain. Comparable curves were calculated formales (not shown). B, results of the statistical analysis of differences betweencurves are given both for females (F) and males (A/). Significant differences (P ë0.01) between strains are indicated by arrows (the arrow Head pointing towardsthe strain of highest susceptibility), whereas broken lines between strains indicatethat they do not differ significantly (P > 0.01).

B10.A

M

B10A

Fig. 2. Papillary lung tumors in female and male mice from five 112 congeniestrains on the C57BL/10 background treated postnatally with ENU. A, "at risk"

curves show the percentage of female mice still free of papillary lung tumors(vertical axis) at the end of each successive 50-day interval (horizontal axis).Arrows, mean tumor age in each strain. Comparable curves were calculated formales (not shown). B, results of the statistical analysis of differences betweencurves are given both for females (F) and males (A/). Significant differences (P ë0.01) between strains are indicated by arrows (the arrow head pointing towardsthe strain of highest susceptibility), whereas broken lines between strains indicatethat they do not differ significantly (P > 0.01).

one hand and from strains 4R and BIO on the other, in malesthe same strain differences are found, except that 2R and BIO.Ado not differ.

Papillary Lung Tumors. The "at risk" curves for females are

given in Fig. 2A. Comparable curves were constructed for males(not shown). Statistical analysis of these curves revealed inter-

strain differences (Fig. IB). For females, the strain BIO.A hasthe highest susceptibility and is significantly different from thestrains 4R, 5R, and BIO which are less susceptible and similarto each other. The strain 2R has an intermediate position. Formales, the strains BIO.A and 2R are relatively susceptible, thestrain 5R is intermediate, and the strains BIO and 4R areresistant.

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H-2 AND EMU-INDUCED TUMOR1GENESIS

Mixed (Alveolar/Papillary) Type of Lung Tumors. These tumors were found largely (and in some strains exclusively) latein life (see Fig. 3A and Table 2). The "at risk" curves for females

are given in Fig. 3A. Comparable curves were constructed formales (not shown). Statistical analysis showed strain differencesin susceptibility (Fig. 3B). Both for females and males strains2R, B10.A, and 5R are susceptible and different from strains4R and BIO, which are resistant.

It can be concluded that in female as well as in male micefrom H-2 congenie strains on the C57BL/10 background,treated postnatally with ENU, susceptibility to alveolar, papillary, and mixed-(alveolar/papillary) type lung tumor formationis haplotype dependent. For alveolar and mixed-type lung tumors the respective patterns of strain differences imply that the(putative) gene(s) involved can be allocated to particular regionsof the H-2 complex (see Discussion). In case of papillary tumorsmore than one H-2 gene is involved.

Tumors of the Small Intestine

Tumors of the small intestine were found in a high percentageof females and males in all strains (Table 2). They were foundat a very early age but also in old animals. The time of the firstdetection ranged from 104 days (2R) to 212 days (5R).

"At risk" curves for females are given in Fig. 4A. Comparable

curves were constructed for males (not shown). Significantstrain differences in susceptibility were found (Fig. 4B). Both

80-

40-

xlOOd.yl

B

Fig. 3. Mixed (alveolar/papillary)-type of lung tumors in female and malemice from live H-2 congenie strains on the CS7BL/10 background treatedpostnatally with ENU. A, "at risk" curves show the percentage of female micestill free of mixed (alveolar/papillary)-type of lung tumors (vertical axis) at theend of each successive 50-day interval (horizontal axis). Arrows, mean tumor agein each strain. Comparable curves were calculated for males (not shown). /;.results of the statistical analysis of differences between curves are given both forfemales (/•')and males (M). Significant differences (P £0.01) between strains are

indicated by arrows (the arrow head pointing towards the strain of highestsusceptibility), whereas broken lines between strains indicate that they do notdiffer significantly (P > 0.01).

- 100-i

80-

60-

20-

1 ' 1

1 211

1

31

I"1411*15*A1

'6»

â„¢¿�1

' 1

7 8«100days

B

B10A B10A

Fig. 4. Tumors of the small intestine in female and male mice from five H-2congenie strains on the C57BL/10 background treated postnatally with ENU. A,"at risk" curves show the percentage of female mice still free of tumors of thesmall intestine (vertical axis) at the end of each successive 50-day interval(horizontal axis). Arrows, mean tumor age in each strain. Comparable curves werecalculated for males (not shown), lì.results of the statistical analysis of differencesbetween curves are given both for females (F) and males (A/). Significant differences (P £0.01 ) between strains are indicated by arrows (the arrow head pointingtowards the strain of highest susceptibility), whereas broken lines between strainsindicate that they do not differ significantly (P > 0.01).

for females and males the strain 2R is most susceptible andsignificantly different from strains B10.A, 4R, 5R, and BIO.For females the strain B10.A is intermediate and significantlydifferent from the least susceptible strains 4R, 5R, and BIO,for males these four strains group together. The strain differences in mean number of tumors in the small intestine matchalmost completely the pattern deduced from the "at risk" curves

(Table 2). Thus, in female as well as in male mice, susceptibilityto tumor formation in the small intestine is haplotype dependent. The patterns of strain differences found indicate involvement of more than one //-2-associated gene (see Fig. 4 andTable 1).

Unexpectedly, strain differences with respect to the localization of tumors along the small intestine were found (Fig. 5). Asno sex differences in tumor localization exist, the data arecombined for both sexes. In mice from strain 2R most tumorsare found in the proximal part of the small intestine, althoughthe distal part exhibits also an appreciable number of tumors.In contrast, in mice from strain 4R most tumors are found inthe distal pan of the small intestine, while the proximal partcontains only a few. In the strains BIO.A, 5R, and BIO thetumors are distributed more evenly along the small intestine,although the strain B10.A is more 2R-like and the strain BIOis more similar to 4R.

Thus the differences between H-2 congenie strains on the

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H-2 AND ENU-INDUCED TUMORIGENESIS

o.o

Fig. 5. Distribution patterns of the tumors along the small intestine of micefrom five // _'congenie strains on the C57BL/10 background treated postnatallywith ENU. Per strain the mean number of tumors (vertical axis; combined forfemales and males) found in each S-cm segment of the small intestine (horizontalaxis) is given, from stomach to cecum.

80-

20-

C57BL/10 background in ENU-induced tumorigenesis in thesmall intestine are manifest in: (a) overall susceptibility (asbased on the "at risk" curves), (b) the mean number of tumors

per tumor-bearing animal, and (c) the localization of tumorsalong the small intestine.

Liver Tumors

The results on liver tumors (Table 2) are given separately forh.c. adenomas and h.c. carcinomas. The susceptibility to livertumors without distinction of tumor type was evaluated as well.No accurate data on h.c. adenoma numbers in individual micecan be given, as it is often impossible to identify these lesionsas single or (confluent) multiple tumors; the mean number ofh.c. carcinomas per tumor-bearing animal is close to one in allstrains. Therefore no data on the number of h.c. adenomas orh.c. carcinomas are presented in Table 2.

Hepatocellular Adenomas. H.c. adenomas were found in allstrains in both sexes. The age at first detection ranged from274 days (4R) to 377 days (5R), but for most h.c. adenoma-bearing animals the age of defection ranged between 400 and600 days.

"At risk" curves for males are given in Fig. 6/4. Such curves

were also constructed for females (not shown). Only for malessignificant strain differences in susceptibility for h.c. adenomaformation exist (Fig. 6Ä). Strain 2R is most susceptible anddiffers only from strain 5R, which is relatively resistant; thestrains 4R, B10.A, and BIO each have a different intermediateposition.

Hepatocellular Carcinomas. H.c. carcinomas occur (especiallyin females) less frequently than h.c. adenomas (Table 2). Mosth.c. carcinomas were detected at age 400-600 days. "At risk"

curves for males are given in Fig. 1A. Such curves were alsoconstructed for females (not shown). Significant differenceswere found between males of the strain 5R (relatively resistant)on one hand and B10.A and 2R (relatively susceptible) on theother hand, while the strains 4R and BIO are intermediate andnot significantly different from any strain. No interstrain differences have been seen in females. The tumor age, rather thantumor incidence, is an important underlying factor of the straindifferences in males. In males of the strain 5R h.c. carcinomasare seen much later than in 2R and B10.A while there is nodifference in incidence between 5R and 2R (Table 2). Comparing the mean age at detection of h.c. adenomas and h.c. carcinomas in males two patterns are found: In strains B10.A and2R h.c. carcinomas are found earlier than h.c. adenomas, in the

B

BW A T \ 4R

B10 B10- -5R

Fig. 6. Hepatocellular adenomas in female and male mice from five //--'congenie strains on the CS7BL/10 background treated postnatally with ENU.. I."at risk" curves show the percentage of male mice still free of hepatocellularadenomas (vertical axis) at the end of each successive SO-day interval (horizontalaxis). Arrows, mean tumor age in each strain. Comparable curves were calculatedfor females (not shown). II. results of the statistical analysis of differences betweencurves are given both for females (F) and males (A/). Significant differences (P S0.01) between strains are indicated by arrows (the arrow head pointing towardsthe strain of highest susceptibility), whereas broken lines between strains indicatethat they do not differ significantly (/' > 0.01).

other strains h.c. adenomas are found earlier or at the same ageas h.c. carcinomas.

Thus, susceptibility to h.c. adenoma and h.c. carcinoma formation in males is haplotype-dependent. No H-2 influence isseen in females. Both for h.c. adenoma and h.c. carcinomaformation the patterns of strain differences imply that at leasttwo genes of the H-2 complex are involved. When liver tumorsare considered as a whole, making no distinction betweenadenomas and carcinomas, the H-2 influence is seen only inmales and again the strain 2R is the most susceptible, strain5R the most resistant.

Tumors in Other Organs

Although the vast majority of tumors was found in lung,small intestine, and liver, appreciable numbers of tumors werealso found in other tissues. The percentage and mean age ofanimals with tumors of the lymphoid system, Harderian gland,and ovaries are given in Table 3. For lymphoid and Harderiangland tumors "at risk" curves for females and males revealed

no significant differences between sexes. For further analysisthe data of two sexes were combined. No differences betweenstrains in lymphoid, Harderian gland, and ovarian tumor formation were found.

Tumors were furthermore sporadically found in other organs,but never in more than 10% of mice. Tumors of the reticulo-endothelial system (both the histiocytic- and the endothelial

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H-2 AND EMU-INDUCED TUMOR1GENESIS

-100-,

80-

40-

20-

ir ir i*x100 days

BBIO A 2R B10A

B10

Fig. 7. Hepatocellular carcinoma in female and male mice from five H-2congenie strains on the C57BL/10 background treated postnatally with END. A,"at risk" curves show the percentage of male mice still free of hepatocellular

carcinomas (vertical axis) at the end of each successive 50 days interval (horizontalaxis). Arrows, mean tumor age in each strain. Comparable curves were calculatedfor females (not shown). B, results of the statistical analysis of differences betweencurves are given both for females (F) and males (A/). Significant differences (P £0.01) between strains are indicated by arrows (the arrow head pointing towardsthe strain of highest susceptibility), whereas broken lines between strains indicatethat they do not differ significantly (P > 0.01 ).

Table 3 Incidence oflymphoid, ovarian, and Harderíangland tumors in micefrom five H-2 congenie strains on the C57BL/IO background, treated with ENU

at age of 15 days

Lymphoid tumors" Harderian glandtumors" Ovarian tumors*

StrainPercentage'B

10.A2R4R5RBIO35

36302439Mean

age1*Percentage'268

18822830131312

6131816Mean

age1*Percentage' Meanage"1438

4604444554488

8222324476

504556559569

" The data on females and males are combined.* Females only.' Percentage of tumor-bearing animals.'' Mean age (days) of tumor-bearing animals.

type) were found in 2-10% of mice from all strains, usually atolder ages (more than 300 days). In the stomach, colon, kidney,skin, backbone, neurilemma, and fat tissue tumors were foundoccasionally in animals from two or three strains. Tumors ofthe vagina, gallbladder, urinary bladder, muscle, thyroid gland,and mammary gland were found only once.

Sex-related Differences in Tumorigenesis

In addition to the above-reported strain-related differences intumorigenesis in lung, small intestine, and liver, in two strainssex-related differences were also found. In all strains suscepti

bility to tumor formation tended to be higher in males than infemales (Table 2), but analysis of the "at risk" curves showed

significant differences between sexes only in strains 4R andBIO. In strain BIO susceptibility to tumorigenesis in the lung(all three tumor types) is significantly higher in males than infemales; in strain 4R this holds true for tumorigenesis in thesmall intestine and liver (both tumor types).

DISCUSSION

The incidence of spontaneous tumors in the strains used inthis study has been reported by Faraldo et al. ( 10), and Faraldo."

They observed low incidence and late appearance of lung tumors, only sporadic appearance of liver tumors, and no tumorsof small intestine, indicating that the tumors observed in thisstudy were induced by carcinogen treatment.

The differences in tumorigenesis observed in mice from H-2congenie strains on the C57BL/10 background treated postnatally with ENU, are summarized in Fig. 8. A marked H-2-related influence on carcinogen-induced tumor formation inlung, small intestine, and liver was demonstrated.

In lung, three different tumor types, i.e., alveolar, papillaryand mixed (alveolar/papillary), were found. The alveolar andpapillary tumors are presumed to originate from alveolar typeII cells (22), although origin of papillary' tumors from Claracells has been proposed (12). The papillary and mixed-typetumors are thought to be more malignant than the alveolartumors (for review see Reference 12). The alveolar and papillarytumors occur in variable proportions in different H-2 congenie

(8) and inbred strains (23, 24) after carcinogen treatment, hencetheir development is probably under different genetic control.Therefore, in the present study we have analyzed the resultsseparately for the different lung tumor types (see Table 3 andFigs. IA, 14, and 3A). The pattern of strain differences is notthe same for the three different lung tumor types (see Fig. 8),indicating that a particular H-2 haplotype can have differenteffects on the three lung tumor types. The regions of the H-2complex responsible for these effects are discussed below. Theoverall results on lung tumors are similar to those obtained inH-2 congenie strains on C57BL/10 background in untreated

RELATIVE SUSCEPTIBILITY

LUNG TUMORS

Alveolar

Papillary

Mixed-type

INTESTINAL TUMORS

LIVER TUMORS

Adenoma

Carcinoma

Fig. 8. Patterns of strain differences for lung-, intestinal-, and liver tumorsfound in mice from five H-2 congenie strains on the C57BL/10 backgroundtreated postnatally with ENU. The relative strain susceptibility is based onstatistical analysis of the "at risk" curves shown in Figs. 1-4, 6, and 7. For a

given tumor type those strains not contained within a box differ significantly (PS 0.01). For lung and intestinal tumors the data for females are given, whereasfor liver tumors those for males are presented. Comparable results are obtainedfor the opposite sex in case of lung- and intestinal tumors (see Figs. 1-4); forliver tumors no strain differences for females were found (see Figs. 6 and 7).

High IntermediateLow2R

IB10A.|R] |4R,B1O|IB10.A

|2R| 4R.5R.B10[SIDA,

2R.5R|2R

BW./2R

4R,B10Õ|2R.

BW. A 4R, B|

4R.Biolk

4R,5R.B10.

BWSR10

SR

4 Unpublished data.

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H-2 AND ENU-INDUCED TUMORIGENES1S

mice (10, 25), and carcinogen-treated mice (7, 8). In all thesedifferent experimental systems H-2°is always associated withsusceptibility and H-2b with resistance. Recently these two

haplotypes have also been shown to have the same distinctiveeffect in strains on the A/Sn background (6).

Among the tumor types found in this study those in the smallintestine were the most abundant. This is in accordance withour data on prenatal ENU treatment (14). We show for thefirst time that there is an //-2-related influence on the incidence

and time of appearance and number of tumors in the smallintestine. Surprisingly, haplotype-related effects on the localization along the small intestine are also found. Thus thishaplotype-related influence on tumorigenesis in the small intestine is demonstrable at different levels. The patterns of straindifferences suggest that the H-2 effects on tumor incidence,

number, and localization may be due to the same or similargenetic factors. However, these aspects of tumor developmentmay be selectively modified indirectly, possibly by H-2 effectson intestinal bacterial flora or on physiology of the intestine.

Tumors of the liver were found commonly in mice from moststrains tested. In males the H-2 haplotype influences the occurrence of both the hepatocellular adenoma and carcinoma. Thesetwo liver tumor types are under similar genetic control (Fig. 8).Information from the literature on the relation between the H-

2 complex and liver tumor formation is scanty and contradictory. Firstly, spontaneous (25) and carcinogen-induced (7) tumors in the liver of mice with C57BL/10 background werefound very infrequently. Secondly, the results from studies usingH-2 congenie strains on C3H background, which are moresusceptible to liver tumor formation, are contradictory: an H-2influence on liver tumor formation in mice from these strainshas been suggested (25), but this could not be confirmed in astudy from this Institute (7).

For all tumor types discussed above, the patterns of straindifferences are summarized in Fig. 8. Overall, 2R and B10.Aare the most susceptible strains. For lung (all three types) andintestinal tumors 4R and BIO are resistant whereas 5R occupiesvariable positions. For both liver tumor types, however, strain5R is the most resistant and strains 4R and BIO are intermediate. From the genotype of the strains studied (Table 1) it canbe concluded that tumorigenesis in lung, small intestine, andliver is influenced by more than one H-2 gene. Barring nonad-ditive gene interactions, the major effects on alveolar lungtumors map to the right of E¿and on mixed-type lung tumorsbetween Eg and D. For the other tumor types the putative genesinvolved cannot yet be assigned to particular regions. It has,however, to be noted that no significant differences betweenstrains 4R and BIO were found for any of the tumor typeslisted; this holds both for females and males (see also Figs. 1-4, 6, and 7). This consistent similarity of strains 4R and BIOwas not found for any other pair of strains. As the strains 4Rand BIO, as opposed to the other strains, have the regions Ea-S of H-20 origin (Table 1), it is possible that this segment of

the H-2 complex has an important effect on tumorigenesis,although other regions are involved as well.

The finding that more than one H-2 gene is involved in tumorformation has been reported in many studies on H-2 control oftumorigenesis (1, 2, 8,10, 26). This is hardly surprising. Firstly,the H-2 complex contains several groups of structurally andfunctionally related genes located in different regions, whichmay affect tumorigenesis through the same mechanism. Secondly, the different types of genes in the H-2 complex might

affect different steps in the neoplastic process. The evidence forvirally induced leukemias and mammary tumors indicates that

the MHC effects on tumorigenesis reside predominantly at thesystemic level (27), while non-MHC genes are known to affectthe susceptibility of target cells (28). Whether all H-2 effectson neoplastic development observed here are systemic orwhether some act at the target cell level remains to be seen.

Most studies on mechanisms of H-2 effects on tumorigenesisfocused on tumors with viral etiology, i.e., leukemias and mammary tumors (for review see References 1 and 2). In leukemias,the //-2-associated effects on tumorigenesis are mainly due to

immunological defense mechanisms against the antigens encoded by the inducing virus (1), which are very similar or thesame in all tumors induced by this virus. These effects arelargely explained in terms of Class I and II MHC restriction,which is dependent on expression of MHC products on tumorcells. Both loss or enhancement of Class I gene expression canaffect tumor growth and progression (i.e., metastasis). Tumor-specific expression of histocompatibility antigens alien to thestrain from which the tumor originates has been reported, usingtransplantable tumor lines (reviewed in References 29 and 30).However, anomalies in Class I expression are not apparent inprimary AKR leukemias (31). Therefore, the relevance of thesefindings in explaining tumor resistance in primary hosts studiedhere may be limited.

Alternatively, the MHC might influence the susceptibility ofthe immune system to the immunosuppressive effects of ENU(32). In different strains this could result in generalized differences in the immune response to ENU-induced tumors. Thisnotion is, however, not compatible with our finding that, contrary to the case for virally induced leukemias, carcinogen-induced leukemias were not influenced by the H-2 haplotype(Table 3). Finally, while virally induced leukemias express thetumor antigen of viral origin, chemically induced tumors oftenexpress antigens that are unique to a given tumor (3). Therefore,the H-2 effects observed here may be nonimmunological, although the possibility that //-2-regulated immunological responses are also involved cannot be ruled out.

Apart from its function in immune response, the mouse MHCinfluences other processes. Expression of certain Class I geneshas led to alteration of oncogene expression in tumor cells (33).The MHC affects also some hormone-related phenomena.Genes of the H-2 complex influence resistance to glucocorti-coid-induced cleft palate in embryos (34, 35), levels of gluco-corticoid receptor in lung (reviewed in Reference 36) and estrogen receptor in uterus (37). We have evidence5 that there is anH-2 haplotype-specific modifying effect of glucocorticoid hormone on ENU-induced tumorigenesis in lung and intestinewhen ENU and hormone are administered simultaneously tomouse embryos. The possible significance of hormonal mechanisms in H-2 effects on tumorigenesis is also indicated by theobservation of Mühlbockand Dux (2) of an H-2 influence onmammary tumor induction by prolactin (without involvementof the mammary tumor virus), which has recently been confirmed and extended in our laboratory (26). Haplotype-relatedsex differences in tumorigenesis of lung, small intestine, andliver, observed in this study, may be due to involvement of H-2in hormone-regulated processes in tumorigenesis. Alternatively,they might reflect the higher susceptibility of males to livertumors, which becomes evident only in susceptible strains. Thislatter possibility, however, is not supported by the fact thatthere is no correlation between strain susceptibility and presence of sex differences in our data.

"'Oomen, van der Valk, Hart, and Demant. Glucocorticoid hormone elicci on

transplacental carcinogenesis and lung differentiation: Influence of histocompat-ibility-2 (H-2) complex, submitted for publication.

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H-2 AND EMU-INDUCED TUMORIGENESIS

In this study the organs for which an //-2-related influenceon carcinogen-induced tumorigenesis was found, i.e.. lung,small intestine, and liver, are developmentally related. They alloriginate from the primitive gut and their development is, atleast partially, under similar glucocorticoid hormone-mediatedcontrol (38-40). Our observation that H-2 affects glucocorti-coid-regulated fetal lung development5 suggests that the effectsoíH-2 on hormonally regulated organogénesisand carcinogen-induced tumorigenesis may have a common basis. The elucidation of the cellular and molecular mechanisms involved inthe H-2 effects on chemical carcinogenesis described here requires further study. Such study may also throw new light onthe presently little understood nonimmunological functions ofthe MHC.

ACKNOWLEDGMENTS

The authors thank M. Butzelaar, L. Kuyper-Pietersma, E. van Muyl-wijk, J. Bulthuis for excellent technical assistance. Our thanks are dueto Dr. A. Dux and Dr. A Berns for critical reading of the manuscript.

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1988;48:6634-6641. Cancer Res   L. C. J. M. Oomen, M. A. van der Valk, A. A. M. Hart, et al.   Organs

-nitrosourea-induced Tumor Formation in VariousN-Ethyl-N) on H-2Influence of Mouse Major Histocompatibility Complex (

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