(CANCER RESEARCH 50, 7920-7925, December 15, 1990]

Relationship of Polymorphisms near the Rat Prolactin, N-ras, and RetinoblastomaGenes with Susceptibility to Estrogen-induced Pituitary Tumors1

Laurie A. Shepel2 and Jack Gorski3

Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706

ABSTRACT

Chronic treatment of rats with the synthetic estrogen diethystilbestrolis known to induce the formation of pituitary tumors, and such tumorinduction is highly dependent on the strain of rat used. We examinedthree previously discovered restriction fragment length polymorphismsin rats to determine whether these correlated with susceptibility to tumorformation. The results indicate that the presence of particular alíelesofthe polymorphic N-ra.vand retinoblastoma (Rh) genes does not correlatewith tumor susceptibility. A polymorphism upstream of the rat prolactin(Prl) gene is due to the presence or absence of an /(/«-like sequence.

Results of this study indicate that animals bearing the alíelelacking this/</u-like insertion are more likely to develop larger pituitary tumors inresponse to diethylstilbestrol than are animals in which the I'rl alíelecontains the insertion. In addition, we show that the N-ras, Rb, and I'rl

genes are dispersed in the rat genome and that the polymorphic alíelesof the fri genes are segregating as classical Mendelian alíeles.Theseresults suggest that the difference in the fri gene itself or in some closelylinked gene is related to tumor resistance or susceptibility.

INTRODUCTION

Chronic treatment of rats with estrogens, such as 170-estra-diol or DES,4 results in the induction of pituitary tumors

(hyperprolactinomas). Susceptibility to such tumor inductiondiffers between rat strains. The inbred F344 rat strain is extremely sensitive to tumor formation by DES, while the outbredHoltzman strain is very resistant. The genetics of pituitarytumor susceptibility in the F344 and Holtzman strains has beenexamined (1), and a model was proposed in which the straindifference resided in three independently segregating loci.RFLPs in known genes can serve as genetic markers for tumorsusceptibility and may aid in the identification of the genesresponsible for resistance or susceptibility.

During the characterization of the upstream region of the ratPrl gene, a polymorphism was discovered by Schuler et al. (2).The alici io variation is caused by the insertion of a 112-basepair Alu-like repetitive DNA element located approximately7.6 kilobases upstream of the first exon. This dimorphism hasbeen observed in the DNA of Holtzman and Sprague-Dawley

rats but not in F344 rats, and DNA from the F344 rats did notcontain the Alu-\ike insertion. Additionally, Kumar et al. (3)reported a defective distal regulatory element at the 5' upstream

region of the rat Prl gene. The distal regulatory elements (bothpositive and negative) were shown to be nonfunctional, sincethe cells containing them were totally nonresponsive to steroidhormone (3). The distal element was localized to the general

Received 7/2/90; accepted 9/14/90.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1This work was supported by the College of Agricultural Life Sciences,University of Wisconsin, and by NIH Grant HD08192.

1Present address: The Jackson Laboratory, 600 Main Street, Bar Harbor, ME

04609.3To whom requests for reprints should be addressed.*The abbreviations used are: DES, diethystilbestrol; RFLP, restriction frag

ment length polymorphism; Rb, retinoblastoma; Prl, prolactin; ANOVA, analysisof variance; cDNA, complementary DNA.

area between 3.8 and 7.8 kilobases upstream of the transcriptioninitiation site of the Prl gene. Interestingly, this region overlapsthe area where the Alu-\\\x insertion was found in the DNA ofHoltzman and Sprague-Dawley rats. These observations areenticing with respect to the DBS-induced pituitary tumors inthe F344 rats, especially since the tumors are composed primarily of lactotrophs (/V/-producing cells). The presence of atotally defective regulatory region would not be entirely consistent with previous data showing that the Prl synthesis response to estrogen is similar in both Holtzman and F344 strains(4). Nevertheless, the Prl polymorphism may be involved in ormay be an important marker for susceptibility to pituitarytumor formation.

We previously examined differences in the structure of 15known protooncogenes and/or antioncogenes (5) and observedRFLPs in the N-ras protooncogene and the Rb antioncogenebetween DNA samples from F344, Holtzman, and four otherrat strains, and also within the Holtzman population. It wasobserved that unique 'N-ras restriction fragments were present

in the DNA of rat strains resistant to pituitary tumor inductionby estrogens. Similar results were observed for the Rb gene,although the correlation was not as strong as it was for 'N-ras.

Such observations are merely correlative in nature, and wewished to test this correlation with a direct comparison ofpituitary growth to the observed RFLPs.

In this paper we address the relationship of the N-ras, Rb,and Prl Alu-\\\x polymorphisms to estrogen-induced pituitarytumor susceptibility. The pattern of inheritance of the Prlpolymorphism and an analysis of linkage of the three genes arealso reported. The results indicate that the Prl polymorphicalíelesare inherited in an independent Mendelian fashion andthat the three genes are dispersed to separate chromosomes. Itappears that the N-rai and Rb polymorphic alíelesdo notcorrelate in any way with the degree of pituitary tumor growth.However, the Prl Alu-like polymorphic patterns are associatedwith different pituitary weights. Thus, the Prl RFLP may serveas a marker for pituitary tumor susceptibility.

MATERIALS AND METHODS

Animals Used. F344 and Holtzman rats were obtained from Harían/Sprague-Dawley, Madison, WI. In addition, various crosses betweenF344 and Holtzman rats were produced in our breeding colony asfollows. Female Holtzman rats were mated with male F344 rats toproduce the FI generation. These FI animals were used to produce theF2 generation and parental backcrosses. The F2 was produced by non-sibling matings of the I7, animals. F344 backcrosses were produced by

mating female F] rats with male F344 rats, and Holtzman backcrosseswere produced by mating male F, rats with female Holtzman rats.Reciprocal-type crosses were not performed, since it was previouslydetermined that female Holtzman rats are superior mothers to femaleF344 rats and that there are no apparent maternal effects on theresponse to DES (l).

Implants. DES-containing implants were prepared by cutting 1-cmpieces of Silastic tubing (inner diameter, 0.078 inches; outer diameter,0.125 inches), plugging one end with Silastic medical adhesive, andallowing to cure overnight. Implants were filled with 25 n\ of a solution

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GENETIC SUSCEPTIBILITY TO ESTROGEN-INDUCED PITUITARY TUMORS

of DES in absolute ethanol (200 mg of DES/ml); thus, each implantcontained 5 mg of DES. The ethanol was allowed to evaporate overnightin a vented hood. The other end of the tubing was then sealed withSilastic adhesive and cured overnight.

Induction of Pituitary Tumors. Weanling male F344 rats (21 days ofage) were given s.c. Silastic tubing implants containing 5 mg of DES.The implants were placed s.c. into the rats under light ether anesthesiathrough a small incision over the right hip, and the incisions wereclosed with one or two wound clips. Control animals received notreatment. After 12 wk, the animals were sacrificed by decapitation,and pituitarios were removed and weighed.

Genomic DNA Isolation and Southern Analysis. Portions of liver (SOto 200 mg) were removed from individual animals, and DNA wasisolated as described previously (5). Genomic DNA samples (5 to 20Mg)were cleaved with various restriction endonucleases and analyzedby gel electrophoresis and Southern transfer as described previously (5,6).

DNA Probes. The DNA probes used are as follows. The N-ras probeis p6al, a Sall/Nco\ 450-base pair cDNA fragment (designated as N450in this paper) containing exons 2, 3, and 4 of the human message (7).The Rb probe is a 4.5-kilobase mouse cDNA £coRIinsert isolated fromplasmid pMRB102. The probe (designated Prl-Alu) for the /'/•/upstreamAlu-liite polymorphism is a plasmid containing two Hindlll fragments:a 0.73-kilobase sequence that detects the ^/w-like RFLP and a 1.0-kilobase nonpolymorphic sequence upstream and adjoining the 0.73-kilobase fragment of the gene. The 0.73-kilobase fragment detects both0.73- and 0.84-kilobase (which contains the /i/u-like insertion) fragments of the I'rl upstream region (2).

In the case of the Rb probe, a cDNA for the gene was digested awayfrom the plasmid DNA, separated by electrophoresis in SeaPlaque(FMC BioProducts) low-melting-point agarose, and oligolabeled with"P in the gel slice (8). The A'-ras probe was a restriction fragment

digested away from the plasmid sequences and purified by preparativegel electrophoresis, whereas the Prl probe was not excised from theplasmid before labeling. DNA probes were labeled with [a-32P]dATPor [a-"P]dCTP by either nick-translation (9) to 1 to 4 x 10" cpm/Mg

or random hexanucleotide primer extension (oligolabeling) (8, 10) to0.3 to 1 x IO9cpm/Mg.

Statistical Analysis. Analysis of variance between means was performed to test for a correlation between the RFLP patterns and thedegree of tumor formation. The restriction patterns observed for eachgene were determined by Southern analysis of DNA samples from ratsthat had been treated for 7 wk with DES. The pituitary weight dataand the type of restriction pattern for each gene were entered into aMacintosh Plus Statview 512+ (Version 1.0) spreadsheet. A one-factorANOVA test was computed by the Statview program. The analysisprovided the mean pituitary weight for each restriction pattern, thestandard errors of the means, and a F test P value. A P value greaterthan O.OS indicates that the mean pituitary weights for the threerestriction patterns of a particular gene are not significantly differentas a group.

The mean pituitary weights and standard errors for each restrictionpattern of the three genes were entered into a Macintosh Cricket Graph(Version 1.2) spreadsheet. Bar graphs were then plotted by the programas they appear in Fig. 4.

RESULTS

Analysis of Vra.v, Kh, and Prl-Alu Polymorphisms in Individual Rats. In order to characterize the nature and distribution ofthe N-ras, Rb, and Prl-Alu RFLPs in the F344 and Holtzmanstrains, DNA was isolated from the liver of individual animalsand analyzed by Southern hybridization (Fig. 1). Liver was usedas an abundant tissue source because it is easy to homogenizeand yields a sufficient quantity of genomic DNA. Southernanalysis of the N-ras Pstl polymorphism was performed using21 individual F344 and 54 individual Holtzman liver DNAsamples. The data for 14 of the DNA samples tested are shown

N-rasF344 Holtzman

12345 6 7 8 9 10 11 12 13 14Kb

-11.5

I- 6.8

- 4.5

BRb

15

Prl-Alu19 20 21 22 23 24

Kb

20

-10.5- 7.4

- 3.8

Fig. 1. Southern analysis of N-ras, Rb, and Prl-Alu polymorphisms in DNAsamples from individual animals. In A, Pjrl-digested liver DNA samples (10 /igin Lanes I to 5, 5 ¿igin Lanes 6 to 14) from five individual F344 rats and nineindividual Holtzman rats were analyzed by Southern hybridization using the N-ras N450 probe. In B, ^¿si-digested liver DNA samples (10 Mg,Lanes 15 to 18)from four individual rats were analyzed by Southern hybridization to the Kl>4.5probe. The rats used were four unrelated offspring of genetic crosses discussed inthe text (Lane IS, Rat E5; Lane 16, Rat CCI; Lane 17, Rat NN3; Lane IS, RatT6). In C, ////»/Ill-digested liver DNA samples (20 >ig.Lanes 19 to 24) from sixindividual Holtzman rats were analyzed by Southern transfer and hybridizationusing the Prl-Alu probe. All filters were washed at medium stringency prior toexposure of the film. The examples shown are representative of all the polymorphic patterns observed for each of the three genes, kh. kilobases.

in Fig. \A. All 21 F344 samples tested displayed an identicalpattern, which has been designated Pattern 1, whereas theoutbred Holtzman strain contains a distribution of three patterns (Lanes 6, JO, and 12 display Pattern 1, identical to thatseen in the F344 samples; Lanes 8, 9, and 11 display Pattern 2;and Pattern 3 is represented by Lanes 7, 13, and 14). It wasshown previously that the distribution of the N-ras alíelesinthe Holtzman population follows the Hardy-Weinberg law ofgene equilibrium, and that inheritance and segregation of thealíelesoccur in a Mendelian fashion (5).

Sixty-four individual animals were also tested for Rb polymorphisms. As opposed to the three N-ros patterns, thereappear to be four distinct Xbal patterns observed for the Rbgene (Fig. 1B). There is a range of two to five variable bands,and the four observed Xbal patterns may be the net result oftwo separate but linked polymorphisms. For the first polymorphism, which contains the 20- and 10.5-kilobase fragments,Lanes 18, 16, and 17 are designated as Patterns 1, 2, and 3,respectively, by analogy with the N-ras patterns. Patterns for

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GENETIC SUSCEPTIBILITY TO ESTROGEN-INDUCED PITUITARY TUMORS

the second polymorphism, which includes the 6.4-, 7.4-, and8.0-kilobase fragments, are designated as 4, 5, and 6 for Lanes18,16, and / 7, respectively. The Hardy-Weinberg relation couldnot be applied here to test the distribution of genotypes, sincethe animals tested were not produced by random matings;however, it is probable that one alíeleis fixed in the F344population (5).

Individual DNA samples were also analyzed for the Prl Alu-like polymorphism (Prl-Alu) using a Hindlll digestion, and Fig.1C shows six representative examples of the patterns observed.The 1.0-kilobase fragment is common to all samples and isdetected because the probe used contains this fragment. Thepolymorphism reported by Schuler et al. (2) consists of an Alu-like insertion in the 0.73-kilobase Hindlll fragment to generatea 0.84-kilobase sequence. Similarly, we also see that someanimals are homozygous for the 0.73-kilobase fragment, someare heterozygous for the two fragments, and others are homozygous for the alíelethat contains the inserted DNA (Fig. 1C).The three Prl-Alu patterns are designated as 1, 2, and 3,corresponding to Lanes 19 and 20, 21 and 22, and 23 and 24,respectively, of Fig. 1C. Schuler et al. (2) stated that eight F344rats tested did not contain the Alu-like insert, indicating thatthe F344 animals have the 0.73-kilobase alíelefixed withintheir population. All 15 F344 samples tested in our studieswere also homozygous for the 0.73-kilobase alíele,whereas theHoltzman samples maintained a distribution of the three patterns (data not shown). If one combines the data from thisstudy and those of Schuler et al. (2) and applies the Hardy-Weinberg equilibrium test, x2 analysis yields a P value of 0.25for the 26 Holtzman rats tested (d.f. = 1). This P value is lowerthan that calculated for the N-ras genotypes (P = 0.65), but itis still significant at the 95% confidence level. The data indicatethat the alíelesof this polymorphism also follow the Mendelianlaw of segregation. Pedigree analysis (to follow) of the Prl-AluRFLP supports this interpretation.

Inheritance of the Prl-Alu Polymorphism. In order to determine the pattern of inheritance, genetic crosses of F344 andHoltzman rat strains were performed as described in "Materialsand Methods." Liver DNA samples from 113 offspring (of both

sexes) of 12 litters of the genetic crosses were examined bySouthern analysis for their Prl-Alu Hindlll restriction patterns.The autoradiograms from an analysis of four litters are shownin the center of Fig. 2 and are representative of the resultsobtained by Southern analysis of all 12 litters tested. The topof Fig. 2 shows a schematic representation of the alíeles(designated as a and b) present for each of the three patterns. Thetype of cross and the predicted and observed numbers of offspring with each of the three Hindlll patterns are indicated atthe bottom of the figure.

The results indicate that the polymorphism is inherited aspredicted. In addition, the alíelesdo not appear to be sex linked,since all three patterns are observed for both sexes, x2 analysis

of the cumulative results from all 12 litters, which includes alltypes of crosses except Pattern 3 x Pattern 3, yields a P valueof 0.92 (data not shown). Thus, the inheritance appears tofollow the proposed model, and when combined with the distribution according to the Hardy-Weinberg law, these datastrongly indicate that the polymorphic Prl-Alu alíelesare segregating in a Mendelian fashion.

Inheritance of the Rb RFLP(s) was not analyzed because ofthe large number of samples involved in determining inheritance for any of the genes. Given that the Prl-Alu and N-rasRFLPs are composed of alíelesthat follow Mendel's law of

KbAllelic 1-0 '

Distributions 0840.73-

Alíele b b

Predicted numberof offspring

Type ofcross

1 x 1

1 x 2

1 X 3

Observed numberof offspring

Type ofcross

1 x 1

1 x 2

1 x 3

Pattern1106.50Pattern206.59Pattern3000

Pattern1

Pattern2

Pattern3

10

0 0

Fig. 2. Representative Southern analyses of the Prl-Alu Hindlll polymorphismin offspring from known genetic crosses. The top of the figure indicates the alleilecomposition of each of the three patterns observed: the a alíelecontains the 0.73-kilobase fragment without the . l/»-likcinsertion, and the b alíelecontains the0.84-kilobase fragment containing the .-(/// like insertion. Both alíelescontain the1.0-kilobase Hindlll fragment. Liver DNA samples from individual animals infour representative litters of genetic crosses were digested with Hindlll andanalyzed by Southern hybridization using the Rb 4.5-kilobase probe. The middleof the figure shows autoradiograms from such an analysis of Litters A, D, C, andB. The Hindlll patterns of the parents of each litter are known, and the type ofcross for each litter (i.e.. Pattern I x Pattern 1) is indicated at the right of eachautoradiogram. The bottom of the figure lists the numbers of animals predicted(by our model) to display each pattern for the litters shown, as well as the numberof animals actually observed.

segregation, it is likely that the same situation exists for the RbRFLP.

Analysis of Linkage of the Three Polymorphic Genes. If theN-ras, Prl, and Rb genes have any effect on the degree of DES-induced pituitary tumor formation, each gene's effect may be

qualitatively and quantitatively different and may depend onthe number of copies of each alíele.Cooperative or additiveeffects of the genes are also possible. If the genes are linked(i.e., on the same chromosome), the alíeleswould be inheritedtogether. However, if the genes are not linked, many moreallelic combinations are possible, potentially allowing for morevariation in additive, cooperative, dominant, or suppressiveeffects of the genes. Therefore, it was of interest to determinewhether the N-ras, Prl, and Rb gene pairs are linked or assortingrandomly.

To determine whether the alíelesof two genes are segregatingindependently, a contingency test (or test for independence)was performed between different combinations of the genepatterns. The three gene patterns for N-ras, Rb, and Prl-Aluwere determined and tabulated for 60 or 36 individual rats fromthe genetic crosses described previously, x2 values were calculated for each of the three possible 3x3 tables (i.e., N-ras

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GENETIC SUSCEPTIBILITY TO ESTROGEN-INDUCED PITUITARY TUMORS

Patterns 1, 2, and 3 versus Rb Patterns 1, 2, and 3, etc.), and Pvalues were obtained. The results are shown in Fig. 3. Onewould expect that linked genes should yield a P value of <0.05by this analysis, and unlinked genes (i.e., independent) shouldyield a P value >0.05. The resulting P values for all threecombinations of the N-ros, Rb, and Prl genes are above 0.05(Fig. 3), indicating that the different gene pairs are independent.Both Rb polymorphisms (Patterns 1 to 3 and 4 to 6) were testedagainst Prl and N-ras and gave very similar results. This is asexpected, since both Rb polymorphisms are part of only onegene; therefore, only the data for Rb Patterns 1 to 3 are shownin Fig. 3. As a control for linkage, the two Rb polymorphismswere tested against each other, and the very low P value obtained

Totals:

N-ras

patterns

Totals:

Rbpatterns

Totals:

Rbpatterns

Totals:

Rbpatterns

Prolactin patterns

1 231

11401117205025

305Prolactin

patterns

1 23171

116901019

161N-ras

patterns

1 23061

127801117

172Rb

patterns

1 2320001460014

22

36 0.2264

Totals: 20

Totals:

20

Totals:

20

36

Totals:

20

36

0.8022

0.5804

0.0001

Fig. 3. Observed frequency tables for contingency tests: analysis of linkage ofthe three polymorphic genes. Sixty rat DNA samples from F,, F;, and Holtzmanbackcrosses were analyzed by Southern analysis for their restriction patterns ofthe I'rl-Alu and N-ras polymorphisms, and 36 samples were analyzed for the Rbpolymorphism. F344 backcrosses were not included in order to avoid linkagedisequilibrium as discussed in the text for Fig. 4. For each possible combinationof patterns for pairs of genes, the number of rats containing such a combinationwas tabulated in 3 x 3 tables as shown. For the Rb gene, Patterns 1 to 3 and 4 to6 refer to the RFLPs for fragments of 10.5/20 kilobases and 6.4/7.4/8.0 kilobases,respectively, as discussed for Fig. IB. Using a Macintosh Statview program,predicted values for each combination were calculated, and a total x2 value was

determined for each 3x3 table. The degrees of freedom were 4 for each table,and the /' values for independence of the two genes are listed at the right of each

table. The test indicates that genes are not linked (i.e., they are independent)when P> 0.05.

(<0.001) indicates that they are linked, as expected for a singlegene.

Relationship between the N-ras, Rb, and Prl-Alu Polymorphisms and Pituitary Tumor Susceptibility. In order to analyzethe relationship between the presence of the three RFLPs andthe susceptibility or resistance to induction of pituitary tumorsby DES, DNA was isolated from the livers of animals treatedwith DES and was subjected to Southern analysis of the threegenes. For such an analysis, a subset of animals was chosen sothat the range of pituitary weights was maximal. However,genotypes of two polymorphic genes require more than onegeneration to reach genetic equilibrium, and nonrandom matingcan result in a situation known as linkage disequilibrium. If twoor more genes (whether those being tested or genes that arerelevant but unidentified) are not segregating randomly becauseof nonrandom matings, one might incorrectly conclude that aparticular gene correlates with a particular phenotype. In orderto avoid bias due to such linkage disequilibrium, the F344backcross animals could not be used in this analysis because allthree genes appear to have one alíelefixed in the F344 strain.Since the Holtzman alíelesare not fixed for any of the threegenes, those backcrosses may be included. The F, and F2animals should contain equal contributions of alíelesfrom theparental strains (i.e., the Hardy-Weinberg equilibrium shouldbe maintained) and are therefore included in the analysis.

The presence of restriction Pattern 1, 2, or 3 of each individual gene was determined by Southern analysis of DNA fromindividual animals. (Since it was shown in the previous sectionthat the two Rb polymorphisms are linked, only the RFLPrepresented by Patterns 1,2, and 3 was used in this experiment.)These data were tabulated with the pituitary tumor weights forthe corresponding animals. The animals were grouped according to their restriction patterns for each gene, and the meanpituitary weight was calculated for all animals in each group.The mean pituitary weights and their standard errors are shownby the bar graphs in Fig. 4. A one-factor ANO VA test (95%confidence level) was performed between the mean pituitaryweights of the three patterns for each gene in order to determinewhether the means were significantly different. Such a testindicates that the means are significantly different from oneanother if P < 0.05. In such an analysis, comparing all threepatterns together as a group, the mean pituitary weight valuesare not significantly different (P » 0.05), indicating that thesegenotypes do not appear to have any relationship to the development of the pituitary tumors. However, a closer look at thePrl-Alu data, as presented in Fig. 4, reveals that the mean

pituitary weight for Pattern 3 is different from that for eitherPattern 1 or 2. Therefore, further statistical analysis was performed on the Prl-Alu data as described below.

By decreasing the confidence level at which the ANO VA testis performed, the mean pituitary weight of Prl-Alu Pattern 3 isdifferent from either Pattern 1 or 2 at a confidence level ofapproximately 89%. However, the mean pituitary weights ofthe N-ras and Rb patterns become significantly different onlyat confidence levels of 10 to 20%. The latter holds true for acomparison of Prl-Alu Patterns 1 and 2 against each other.Since the mean pituitary weights of Prl-Alu Patterns 1 and 2are essentially the same, the data for these two patterns werepooled into a single class, and the mean of this class (designatedas Class 4) was compared against the mean of Pattern 3.Analysis of variance between the means of Pattern 3 and Class4 yields a P value that approaches significance at the 95%confidence level (P = 0.0816) and may indicate some associa-

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GENETIC SUSCEPTIBILITY TO ESTROGEN-INDUCED PITUITARY TUMORS

Õ2o

oíOí

Prl-Alu

Patterns 1&2 vs. 3are significantlydifieren! at 91%confidence level.

N-ras

Rb

RestrictionPattern

Fig. 4. Tesi for a correlation between the Prl-Alu, N-ras, and Rb polymorphisms and degree of DES-induced pituitary tumor formation. The F344 back-cross animals could not be used for this analysis in order to avoid linkagedisequilibrium. !•',.I-'...and Holtzman backcross rats were treated with DES (asdescribed in "Materials and Methods"), pituitary weights were obtained, andsubsets of 60, 89, and 36 rats (for Prl-Alu, N-roj, and Rb, respectively) were usedfor Southern analysis. Animals were chosen such that the distribution of pituitaryweights was as large as possible. DNA was extracted from the liver of each of therats and analyzed for each of the polymorphisms by Southern analysis. ForPatterns 1, 2, and 3 of each gene, the mean pituitary weights of the correspondingrats were calculated using a Macintosh Statview program. These means arerepresented by the bars in each panel, along with their standard errors and thenumber of animals (\ >that fell into each category. The horizontal line across thecenter of the Prl-Alu indicates the average weight of control pituitaries. A one-factor ANOVA test was performed among the mean pituitary weights of the threepatterns for a given gene. The resulting 1' values were all greater than 0.05,

indicating that the means are not significantly different among the three groupsat the 95% confidence level (P < 0.05 indicates a significant difference).

tion of this polymorphism with tumor development. The meanpituitary weights of Pattern 3 and Class 4 become significantlydifferent when the confidence level is reduced to 91%.

A comparison of Prl-Alu mean pituitary weights from DES-

treated animals (represented by the bars in Fig. 4) with pituitaryweights from control, untreated animals makes the lower valuefor Pattern 3 even more striking. Pituitary weights from allcontrol rats of the F,, F2, and Holtzman backcrosses wereaveraged; this value (13.4 mg) is indicated in Fig. 4 by the solidline. Strictly speaking, the proper control would include aproportionate number of control animals for each pattern, butan average of all control animals used is sufficient here. Allthree restriction patterns are represented in the controls (determined in the inheritance studies), and the control pituitaryweights do not vary significantly between the types of crosses.Compared with the control value, the terminal pituitary weightupon DES treatment for Pattern 3 animals is 1.55-fold, whereasthe terminal weight is 2.1-fold for Pattern 1 and 2 animals. If

the control value is subtracted from the treatment values, theestrogen-induced increase in pituitary weights for Prl-Alu Pat

terns 1 and 2 is approximately 2 times higher than for Pattern3. Since animals displaying Patterns 1 and 2 have at least one

alíele("F344 type") with the /1/w-like insertion and the animals

displaying Pattern 3 do not have that alíele,the significantdifference in mean pituitary weights may indicate a dominanteffect of the F344-type alíele.Thus, either one or two copies ofthe F344-like alíelemay be important in rendering the animalsusceptible to tumor formation.

One of our initial hypotheses was that if any of the genesappeared to be correlated with tumor susceptibility, perhapsparticular combinations of the polymorphic genes would display a greater correlation with tumor susceptibility than for anysingle gene. The results discussed above indicate that only thePrl-Alu RFLP is correlated with the degree of tumor formation.Since the N-ras and Rb RFLPs do not appear to be involved,there is no reason to believe that any additive effects existbetween these two RFLPs and the Prl RFLP. However, we didtest the combinations of Prl-Alu with the other two RFLPs bythe same methods we used for the individual RFLPs andobserved significant differences between groups. When the datawere examined more closely, it was obvious that the differencesseen for the combinations were due entirely to the differencesalready noted for the Prl-Alu RFLP alone. Therefore, the Prlpolymorphism is the only one of the three tested that correlateswith the degree of tumor formation, and no additive effectsappear to exist for combinations of the genes.

DISCUSSION

We examined RFLPs in the N-ras and Rb genes and in theupstream region of the Prl gene. The results indicate that eachof the three genes is composed of alíelesthat appear to be fixedin the F344 strain, but are heterogeneous in the Holtzmanstrain. The polymorphic alíelesof each gene are segregating ina Mendelian fashion, and the three genes are not linked in thegenome. In the human, the N-ras, Rb, and Prl genes are dispersed to three separate chromosomes, i.e., 1, 13, and 6, respectively (11-13). In the rat, the Prl gene is located on chromosome 17 (14), but the locations of the other genes have notbeen determined (or even cloned in their entirety) for the rat.Our observation that the three genes appear to be dispersed inthe rat is consistent with data for the human, indicating thatthese three genes were probably dispersed to different chromosomes before the divergence of rats and humans. It appearsthat linkage homology between rodents is considerably higherthan that predicted by their differences in chromosomal bandingpatterns, implying that the evolution of genetic content andbanding patterns are independent events (15). The same maybe true between rats and humans.

We examined the N-ras, Rb, and Prl-Alu polymorphisms interms of their relationship to DES-induced pituitary tumors.The results indicate that the N-ras and Rb genes display nocorrelation with tumor susceptibility. In contrast, animals having different patterns of the Prl-Alu polymorphism exhibitsignificantly different degrees of tumor response to DES. Examination of the individual genotypes has shown that thehétérozygotesand homozygotes containing a F344-type alíele(no Alu-\iie insertion) display the largest pituitaries, whileanimals homozygous for the alíelecontaining the /4/u-like insertion (a Holtzman-specific alíele)have the smallest pituitariesof the three groups. Thus, there may be a dominant effect ofthe F344-type alíele(or a closely linked alíele)on tumor formation. These results suggest that the difference in either thePrl gene itself or some closely linked gene is important in thedetermination of tumor resistance or susceptibility.

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GENETIC SUSCEPTIBILITY TO ESTROGEN-INDUCED PITUITARY TUMORS

An interesting hypothesis is that the Prl alíelecontaining the/l/«-likeinsertion has a negative regulatory element, either forPrl or for a gene that exists upstream of the Prl gene. As statedearlier, Kumar et al. (3) identified the presence of defectivedistal regulatory elements (both positive and negative) upstreamof the Prl gene in the same region as the /4/w-like insertion. Inthose experiments, the DNA source was a pituitary tumor cellline (GH) derived from a Wistar/Furth rat. We analyzed thePrl-Alu pattern in Wistar rats (from which Wistar/Furths werederived) and observed the absence of the /1/w-like insertion (datanot shown). Both Wistar and Wistar/Furth rats are susceptibleto estrogen-induced hyperprolactinomas. Such results supportthe notion that negative regulatory elements exist in the .(///-like insertion.

Other investigators have reported the presence of a negativeregulatory element within an Alu sequence in monkey DNA(16). This "reducer" element seems to function as an enhancer;

i.e., it decreased transcription on both directions independentlyof orientation and position. Sequence homology between thisreducer and the Prl-Alu insertion is not significant, but thiscould be due to the divergence of primates and rodents. It ispossible that reducer elements exist in members of the rodentAlu family, perhaps accounting for the preferential, uncontrolled growth of the lactotrophs in the F344 rat pituitary. It isalso possible that the difference in strain susceptibility to tumorinduction may be related to a difference in the nuclear factorsavailable to bind to such regulatory elements.

Although the presence of one or two copies of the F344-likePrl-Alu alíelecorrelates with tumor formation, the Prl alíele(or

a linked alíele)does not appear to be sufficient for full tumordevelopment. Two points support this claim, (a) Although thepituitary weights for animals of the various genetic crossespossessing Patterns 1 and 2 are significantly larger than incontrol animals, the tumor induction is not as dramatic as thatobserved in the F344 strain (in which the pituitaries are 2 to 3times larger than they are in the crosses), (b) The frequency ofPrl-Alu hétérozygotesin the Holtzman population is 0.538, yetthe tumor incidence in this strain is extremely low (2 to 6%;Ref. l). The Prl polymorphism is apparently one indicator oftumor susceptibility, but it is likely that several genes areinvolved that may interact, cooperate, or counteract each otherin pituitary tumor development. Genes derived from the Holtzman strain may be suppressing full tumor formation in crossesthat contain the Prl-Alu alíele.

Although they do not appear to be directly involved in theDES-induced pituitary tumor system, it is possible that the N-ras and Rb (and possible Prl-Alu) gene polymorphisms observedin this study are related to other types of chemically inducedtumors to which the F344 rat is much more susceptible thanthe Holtzman. For example, the F344 rat is extremely susceptible to the induction of mammary carcinomas by dimethylben-zanthracene and by neutron or y radiation, or if the animalsare glucocorticoid deficient and contain high levels of Prl.Aflatoxin BI induces hepatocellular carcinomas in F344 rats,and methyl(methoxymethyl)nitrosamine induces renal mesen-chymal tumors. Perhaps the polymorphisms between strainsfor the N-ras, Rb, and Prl-Alu (or a gene linked to it) arecorrelated with the susceptibility to one or more of these othertumors.

In this study, we analyzed the structure of only a small subsetof the protooncogenes known to exist. It is possible that RFLPsexist for other protooncogenes or even for those that have beenanalyzed but which remained undetected because of the enzyme

sites or probes chosen for analysis. Small differences (such asthe Alu-type insertion) may go unnoticed, and point mutationsare not detectable by the method used unless the mutation is atthe particular enzyme site used to digest the DNA. In addition,protooncogenes or antioncogenes that have not yet been discovered may be responsible for the strain differences in tumorsusceptibility.

The Prl polymorphism appears to be a marker for one of thefactors involved in the genetic predisposition to pituitary tumorformation. It is known that the rat Prl gene resides on chromosome 17. Through further studies of linkage and gene mapping, and as more becomes known about the linkage map ofthe rat, the Prl-Alu RFLP may serve as a marker to define thechromosomal region containing one gene that appears to bepartially responsible for the development of pituitary tumors.

ACKNOWLEDGMENTS

We thank the following for providing the plasmids or bacteriacontaining the DNA probes: N-ras from Dr. Henry Pitot at the University of Wisconsin-Madison, who originally obtained the probe fromDr. Mitchell Goldfarb at Cold Spring Harbor Laboratories; Rb fromDr. Rene Bernards at the Massachusetts General Hospital CancerCenter; the probe for the Prl Alu-\ike insertion was purified by Dr.James Weber, previously of our laboratory. We also thank TamaraGreco for technical assistance; Dr. Linda Schuler for technical adviceand helpful discussions; Dr. James Crow, Dr. Margaret Dentine, andDr. Jack Rutledge for helpful criticisms and discussions; and KathrynHoltgraver for help in preparation of the manuscript.

REFERENCES

1. Wiklund. .1..Rutledge, J.. and Gorski, J. A genetic model for the inheritanceof pituitary tumor susceptibility. Endocrinology, 109: 1708-1714, 1981.

2. Schüler,L. A., Weber, J. L., and Gorski, J. Polymorphism near the ratprolactin gene caused by insertion of an /1/u-like element. Nature (Lond.),305: 159-160, 1983.

3. Kumar, V., Wong, D. T. W., Pasion, S. G., and Biswas, D. K. Defectivedistal regulatory1element at the 5' upstream of rat prolactin gene ofsteroid-nonresponsive GH-subclone. Hindi im. Biophys. Acta, 910: 213-223, 1987.

4. Wiklund, J., Wertz, N., and Gorski, J. A comparison of estrogen effects onuterine and pituitary growth and prolactin synthesis in F344 Holtzman rats.Endocrinology, 109: 1700-1707, 1981.

5. Shepel, L. A., and Gorski, J. Restriction fragment length polymorphisms inthe rat N-ras and retinoblastoma genes: their identification and characterization. Oncogene, in press, 1990.

6. Southern, E. M. Detection of specific sequences among DNA fragmentsseparated by gel electrophoresis. J. Mol. Biol., 98: 503-517, 1975.

7. Taparowsky, E., Shimizu, K., Goldfarb, M., and Wigler, M. Structure andactivation of the human N-ras gene. Cell, 34: 581-586, 1983.

8. Feinberg, A. P., and Vogelstein, B. Addendum: a technique for radiolabelingDNA restriction endonuclease fragments to high specific activity. Anal.Biochem., 137: 266-267, 1984.

9. Rigby, P. W. I.. Dieckman, M., Rhodes. C, and Berg. P. Labeling deoxyri-bonucleic acids to high specific activity in vitro by nick translation with DNApolymerase I. J. Mol. Biol., 133: 237-251, 1977.

10. Feinberg, A. P., and Vogelstein, B. A technique for radiolabeling DNArestriction endonuclease fragments to high specific activity. Anal. Biochem.,m-6-13, 1983.

11. Sparkes, R. S., Sparkes, M. C., Wilson, M. G., Towner, J. W.. Benedict, W.,Murphree, A. L., and Yunis. J. J. Regional assessment of genes for humanesterase D and retinoblastoma to chromosome band 13ql4. Science (Washington DC), 20«:1042-1044, 1980.

12. Dryja, T. P., Rapaport, J. M., Weichselbaum, R., and Bruns, G. A. Chromosome 13 restriction fragment length polymorphisms. Hum. Genet., 65:320-324, 1984.

13. Owerbach, D., Rutter, W. J., Cooke, N. E., Martial, J. A., and Shows, T. B.The prolactin gene is located on chromosome 6 in humans. Science (Washington DC), 212: 815-816, 1981.

14. Cooke, N. E., Szpirer, C., and Levan, G. The related genes encoding growthhormone and prolactin have been dispersed to chromosomes 10 and 17 inthe rat. Endocrinology, 119: 2451-2454, 1986.

15. Womack, J. F. The linkage map of the rat. Behav. Genet.. //: 437-444,1981.

16. Saffer, J. D.. and Thurston, S. J. A negative regulatory element with properties similar to those of enhancers is contained within an tin sequence. Mol.Cell. Biol., 9: 355-364, 1989.

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1990;50:7920-7925. Cancer Res   Laurie A. Shepel and Jack Gorski  Estrogen-induced Pituitary Tumorsand Retinoblastoma Genes with Susceptibility to

,rasRelationship of Polymorphisms near the Rat Prolactin, N-

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