[CANCER RESEARCH 28, 82ÃŽ-830,May 1968]

Karyotypes of Rats from Strains of Different Susceptibility toMammary Cancer Induction1

E. Douglas Rees, Amy Eversole Shuck, Joseph C. Christian, and Joe R. Pugh

Departments o¡Medicine and Pharmacology, University o¡Kentucky, Lexington, Kentucky 40506

SUMMARY

The female Sprague-Dawley rat is quite vulnerable to theinduction of mammary carcinomas by 3-methylcholanthreneand by 7,12-dimethylbenz(a)anthracene, a majority of the carcinomas induced are sex hormone dependent. The Long-Evansrat is relatively resistant to mammary carcinoma induction butis more susceptible to leukemia. Karyologic studies were performed on cells from rats of both strains. No difference wasnoted in the X or Y chromosome. In the Sprague-Dawley,both members of the #3 pair were subterminal, whereas themembers were generally heteromorphic (one subterminal andone terminal) in cells from Long-Evans rats. In inbred Long-Evans rats in particular, the #12 chromosome was interestingin that one of the members frequently had larger upper armsthan the other, and, not infrequently, one of the homologs hadlarge satellites on prominent upper arms. Study of diploid cellsof both strains indicated a tendency, especially after administration of 3-methylcholanthrene or 7,12-dimethylbenz(a)an-thracene, for a chromosome of subterminal morphology to bemissing and a terminal chromosome to be gained, apparentlythrough loss of upper arms of the former. Karyotypes of inbredFischer, Marshall, and Osborne-Mendel rats were also established. The present data do not indicate a correlation betweenkaryotype and susceptibility to mammary cancer induction.

INTRODUCTION

In a study of the karyotypes of three strains of laboratoryrats, Hungerford and Nowell (6) noted polymorphism of theX chromosome in the noninbred Lewis and Wistar (Shay)strains. In these two strains, the Y chromosome was the smallest terminal chromosome, whereas in the BN strain, the Ychromosome could not be distinguished from medium-sizedterminal autosomes. Fitzgerald (2) previously determined aWistar karyotype and found that the Y chromosome was thesmallest terminal chromosome and the X chromosome was oneof the largest terminal chromosomes (but less distinctive thanthe Y). The results were in accord with those obtained earlieron the laboratory rat at Lund by Tjio and Levan (14). In each

1Supported by a grant from the AMA-ERF. J. C. C. and J. R. P.were summer student fellows supported by institutional fundsprovided by the American Cancer Society and the National Cancer Institute.

Received September 7, 1967; accepted January 13, 1968.

of these rat strains, there were 5 subterminal, 8 terminal, and7 median autosomal pairs. More recently, Yosida and Amana(16) reported similar findings in their karyologic studies ofseveral strains of laboratory and wild rats; however, polymorphism of the #3 chromosome pair was noted in some strains.Bianchi and Molina have described polymorphism of the smallest subterminal chromosome in their strain of laboratory rat(1).

Sydnor et al. (13; personal communication) have demonstrated differences between rat strains in susceptibility to mammary cancer induction by oral administration of the polycyclicaromatic hydrocarbon carcinogens, o-methylcholanthrene and7,12-dimethylbenz(a)anthracene. In view of (a) the sex-hormone dependency of these induced mammary cancers (3), (b)the differential susceptibility of rats of different strains to formthese cancers (13), and (c) the X chromosome polymorphismnoted in at least one rat strain (6), a study of the karyotypesof rat strains of differing susceptibility to mammary cancerinduction was initiated. The susceptible Sprague-Dawley strainand the relatively resistant Long-Evans strain were primarilyemphasized in the present study, but the karyotypes of Fischer,Marshall, and Osborne-Mendel rats were also established.

MATERIALS AND METHODS

Sprague-Dawley rats were obtained from the Holtzman Company (Madison, Wisconsin). Long-Evans rats were providedby Dr. Katherine Sydnor and were all descended from a singlemating pair (obtained from Diablo Farms, Inc., Berkeley,California). The animals were maintained in stainless steelcages with wire bottoms in an air-conditioned room. Waterand chow pellets were available ad libitum. Liver was providedonce a week and lettuce twice a week. Most of the animalswere 50-100 days old at the time of the study, but youngerand older animals were also studied; however, no variation inkaryotype was noted with age, though older animals generallydid not provide as many good spreads. Chromosomes wereprepared from marrow of the femur and tibia by the methodof Tjio and Whang (15) and stained with aceto-orcein. Photomicrographs of 22 suitable spreads were obtained and enlargedto about X 5500 for chromosome measurements. At the timeof karyotype construction, each chromosome in a photographwas checked against the preparation by microscopic examination. Karyotypes (Figs. 1-5) were arranged according toHungerford and Nowell (6). The mean relative length ofchromosomes and the accompanying standard deviations andstandard errors were also calculated.

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E. Douglas Rees, Amy Eversole Shuck, Joseph C. Christian, and Joe R. Pugh

In the latter part of this study, chromosome spreads wereprepared using the fixation procedure of Moorhead et al. (9),since a greater number of good spreads were obtained. Theseanimals were injected intraperitoneally with 0.75 mg of col-chicine in 1 ml of isotonic saline a half hour before the animalwas decapitated, and the femoral and tibial marrows were removed for study. The slides were stained with aceto-orcein orwith Giemsa stain. In order to minimize statistical bias inevaluating the distribution of chromosome number per cell,counts of chromosome number were limited to no more than10 cells per rat (225 cells from 23 Sprague-Dawley rats and137 from 14 Long-Evans rats). For the Sprague-Dawley strain,90% of all cells were diploid; for Long-Evans, 95% were dip-loid. With few exceptions, deviations from diploidy werenumbers smaller than 42 that probably represented an artifactof the technic mainly. The study of both rat strains was concurrent.

Since a karyotypic difference between the Long-Evans andSprague-Dawley strains was found, additional strains of inbredrats of the Fischer, Marshall, and Osborne-Mendel strains wereobtained for study from Dr. Katherine Sydnor, who had determined their susceptibility to mammary cancer induction. Thekaryotypic studies of these three strains were not as extensiveas for the Long-Evans and Sprague-Dawley animals; but atleast 10 good preparations from each of at least 5 rats of eachstrain were carefully examined. Also, the influence of polycyclichydrocarbons on marrow cell chromosomes of 50-day-old female Sprague-Dawley rats was determined by studying preparations 1-16 days after intragastric administration of a singledose of either 3-methylcholanthrene (100 mg) or 7,12-dimethyl-

benz(a)anthracene (20 mg).

RESULTS

It is evident by microscopic examination, as well as by statistical analysis of karyologic measurements, that certain pairsof chromosomes are distinguishable and others are not. Although the largest median chromosomes are significantly largerthan the shortest terminal autosomes, the karyotype systemof Hungerford and Nowell (6) was followed for reasons ofsimplicity and in recognition of the limitation (10) of pairingby length (adjoining chromosomes in the 4-10 and 14-20 groupcould not be distinguished from one another). The two largestsubterminal (#1 and #3) pairs, the largest terminal (#2)pair, and generally the Y chromosome were readily distinguishable in both Sprague-Dawley and Long-Evans rats. The Xchromosome seemed to be the second largest terminal chromosome, but often could not be distinguished definitely. Polymorphism was not recognized in the X chromosome of eitherthe Sprague-Dawley or the Long-Evans rats. On plotting thearm ratios of the six smallest subterminal chromosomes againsttheir relative lengths in the manner of Patau (10), the #11and #13 chromosomes of both strains fell into completely distinct groups, but the group of #12 chromosomes overlappedsomewhat the margins of the #11 and #13 groups. Due toshorter upper arms, the #12 subterminal pairs had greaterlong arm/short arm ratios than did the #11 and #13 pairs.One of the #12 homologs not infrequently had larger upperarms than did the other, especially in cells from Long-Evans

rats. In good preparations, each subterminal chromosome couldgenerally be classified on careful microscopic examination. Itshould be mentioned that, according to the criteria and nomenclature of Levan et al. (8), the centromere of the #11 and#13 chromosomes is in a submedian rather than subterminalposition; more specificially, these authors suggest that whatwe specify for convenience as subterminal chromosomes in therat should be designated as smst (submedian-subterminal)chromosomes.

Most of the variability in gross chromosome morphologyobserved in both strains was in the subterminal chromosomes.In the Long-Evans rats, almost invariably one of the #3homologs was terminal and the other was either satellited orhad definite upper arms (Figs. 6, 7). Occasionally both members were terminal. Generally both members of the #3 pairin the Sprague-Dawley rat were subterminal ; but it was notunusual for satellites, rather than definite upper arms, to bepresent on one or both members (Fig. 8). Rarely one of thehomologs appeared to be terminal. Both members of the #11pair were subterminal in both strains, and, in some cells ofsome Long-Evans rats, at least one of the homologs possessedsatellites. One or both of the members of the #12 pair oftenhad satellites rather than definite upper arms in both Sprague-Dawley and Long-Evans rats (Fig. 6). In both strains theupper arms of the #12 chromosomes were shorter, less plump,and less spread apart than those of the #11 chromosomes(Figs. 6-9). Not infrequently a single morphologically unique#12 (Fig. 7) was seen in cells from the inbred Long-Evansrats but not Sprague-Dawley rats. The general appearance ofthis chromosome was one of large satellites extending fromprominent upper arms; sometimes a secondary constriction ofthe lower arms was suggested instead. We have not seen thissort of chromosome in cells from noninbred Long-Evans ratsobtained from several commercial sources. Occasionally satellites were seen on one or both of the #13 sub terminal chromosomes in the Sprague-Dawley rats (Fig. 9) ; this was notedquite often in the Long-Evans rats (Fig. 7). More recently,we have carried out karyologic studies on Sprague-Dawley ratsobtained from commercial sources other than Holtzman, andwe have detected no definite difference in Sprague-Dawleykaryotype in these rats.

Only minor differences were observed in the karyotypes ofthe Osborne-Mendel, Fischer, and Marshall strains. The Xchromosome(s) could frequently be distinguished in the Marshall and Fischer strain but very seldom in the Osborne-Mendelanimals. The Y chromosome also was quite distinctive in theMarshall and Fischer rats, though appearing somewhat moreglobular in the latter. In Osborne-Mendel males, the Y chromosome could only occasionally be definitely recognized. Inall three strains, both members of the #3 chromosome pairwere subterminal; they were frequently satellited in the Marshall and Fischer rats. Only occasionally was a satellited chromosome noted in the marrow cells of the Osborne-Mendel rats(generally on a #11 chromosome). Satelliting was also common on #13 chromosomes in the Fischer and Marshall rats.Differences in the size of the upper arm of the #12 chromosomewas not nearly so marked or frequent in these strains as in theLong-Evans animals.

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Karyotypes of Rats of Different Cancer Susceptibility

Table 1

RatstrainHoltzmanLong-EvansCellsanalyzed9788Subterminalchromosomes/cell1090.7«5.795271.684.121.6701.1t00

Distribution of number of subterminal chromosomes in diploidcells.

0 Percent of cells.

DISCUSSION

The present study and that of Yosida and Amano (16) haveonly two rat strains in common, the Long-Evans and Fischer.With respect to the Fischer, the observations on the #3 chromosome pair were the same: both members were subterminal.In the case of their inbred Long-Evans strain, both membersof the #3 pair were subterminal, whereas the strain we usedhad a heteromorphic pair. Yosida and Amano (16) felt thatthe X chromosome is intermediate in size between the fourthand fifth largest chromosome pairs; our observations are morein accord with those of Hungerford and Nowell (6), who placedthe X between the #3 and #4 pairs. It is difficult to be certainon this point, however, for in many instances the X chromosome cannot be distinguished definitely.

In most good chromosome preparations it is possible toidentify the morphology of each chromosome and to determinethe number of chromosomes in each morphologic group. Counting the number of chromosomes in each morphologic grouppermits a convenient analysis for gross chromosome alterations,since the procedure can be done directly under the microscopeand the results can be expressed quantitatively. Variation ofthe number of chromosomes in each morphologic group canbe due to actual karyologic differences, to an artifact of preparation, and to an error in assigning a morphologic classification. In any case, it is important to know for comparative purposes the magnitude of variation in normal cells. Approximately10 percent of the diploid cells in Sprague-Dawley rats showedan alteration in the morphologic grouping, and generally thisinvolved a missing subterminal chromosome (Table 1) with corresponding gain of a terminal chromosome. Intragastric administration of 3-methylcholanthrene or 7,12-dimethylbenz(a)an-thracene more than doubled the incidence of morphologicalterations in diploid cells (Table 2), and, again, the primaryalteration was loss of a chromosome of subterminal morphologyand gain of a terminal chromosome. Presumably this representsloss of the upper arms of a subterminal chromosome with con-

Table 2

Controls, untreated3-MC, i.g.DMBA, i.g.Cells

analyzed165

115107Cells

lackingsubterminal

chromosome(s)(%)8.5

27.023.3

Proportion of diploid cells lacking one or more chromosomes ofsubterminal morphology (Sprague-Dawley females). 3-MC, i.g.,3-methylcholanthrene given intragastrically ; DMBA, i.g., 7,12-di-methylbenz( a) anthracene given intragastrically.

version to terminal morphology. In the untreated rats, a #3chromosome was predominately involved in this change, whereaswith 3-methylcholanthrene or 7,12-dimethylbenz(a)anthracenetreatment, the other subterminal chromosomes were mainlyaffected. Although the technic of chromosome preparation forcultured cells differs from that for marrow cells, it is of interes't

that diploid cells cultured from induced mammary carcinomaswere found to have a diminished number of subterminal chromosomes (11; unpublished studies). A nonrandom representation of chromosome types in human tumor stemlines has beennoted by Levan (7) and Steenis (12).

The female Long-Evans rat is relatively resistant (13) toinduction of mammary cancer by intragastric instillation of3-methylcholanthrene or 7,12-dimethylbenz(a)anthracene ascompared to the marked susceptibility of the female Sprague-Dawley rat. On the other hand, the incidence of leukemia ismuch higher in Long-Evans rats than in Sprague-Dawley ratsafter intravenous administration of 7,12-dimethylbenz(a)an-thracene (4, 5). A relationship between heteromorphism of the#3 chromosome pair in the Long-Evans rat and its responseto the polycyclic hydrocarbon carcinogens was considered,though it did not seem likely. Any simple relationship is ruledout, however, by the study of the Fischer, Marshall, andOsborne-Mendel karyotypes and the observation that intragastric instillation of a single dose of 7,12-dimethylbenz(a)an-thracene (100 mg/kg body weight) induces mammary cancersin 90-100% of female Sprague-Dawley and Osborne-Mendelrats but in only about 10% of Long-Evans, Fischer, and Marshall rats (Katherine Sydnor, personal communication). Theonly other karyotypic difference noted between the strains wasthe presence of a morphologically unique #12 chromosomewhich was seen not infrequently in cells from Long-Evans rats.No apparent difference in the sex chromosomes of these tworat strains was noted. On the basis of present data there seemsto be no direct relationship between strain karyotype and tumor susceptibility.

ACKNOWLEDGMENTS

We express our thanks to Dr. Katherine Sydnor who generouslysupplied the inbred rats and provided data on the tumor susceptibilities of the different rat strains. Dr. Peter C. Nowell kindlyreviewed some of our slides and offered helpful advice.

REFERENCES

1. Bianchi, X. O., and Molina, 0. Autosomal Polymorphism in aLaboratory Strain of Rat. J. Heredity, 57: 231-232, 1966.

2. Fitzgerald, P. H. Cytological Identification of Sex in SomaticCells of the Rat, Rattus Norvegicus. Exptl Cell Res., 25: 191-193, 1961.

3. Huggins, C., Briziarelli, G., and Sutton, H. Rapid Induction ofMammary Carcinoma in the Rat and the Influence of Hormones on the Tumors. J. Exptl. Med., 109: 25-42, 1959.

4. Huggins, C. B., and Grand, L. Neoplasms Evoked in MaleSprague-Dawley Rat by Pulse Doses of 7,12-Dimethylbenz-(a)anthracene. Cancer Res., S6: 2255-2258, 1966.

5. Huggins, C. B., and Sugiyama, T. Induction of Leukemia inRat by Pulse Doses of 7,12-Dimethylbenz(a)anthracene. Proc.Nati. Acad. Sci., 55: 74-81, 1966.

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E. Douglas Rees, Amy Eversole Shuck, Joseph C. Christian, and Joe R. Pugh

6. Hungerford, D. A., and Nowell, P. C. Sex Chromosome Polymorphism and the Normal Karyotype in Three Strains of theLaboratory Rat. J. Morphol., 113: 275-286, 1963.

7. Levan, A. Non-random Representation of Chromosome Typesin Human Tumor Stemlines. Hereditas: Lund, 56: 28-38, 1966.

8. Levan, A., Fredga, K., and Sandberg, A. A. Nomenclature forCentromeric Position on Chromosomes. Hereditas: Lund, 52:201-220, 1964-65.

9. Moorhead, P. S., Nowell, P. C., Mellman, W. J, Battips, D. M.,and Hungerford, D. A. Chromosome Preparations of Leukocytes Cultured from Human Peripheral Blood. Exptl. Cell Res.,£0:613-616, 1960.

10. Patau, K. The Identification of Individual Chromosomes, Especially in Man. Am. J. Human Genet., 13: 250-276, 1960.

11. Rees, E. D., and Mukerjee, D. Vulnerability of the Subtermi

nal Chromosomes of Cultured Rat Mammary Cancer Cells toAlterations. Proc. Soc. Exptl. Biol. Mod., 117: 869-871, 1964.

12. Steenis, H. V. Chromosomes and Cancer. Nature, W9: 819-821,1966.

13. Sydnor, K. L.. Butenandt, O., Brillantes, F. P., and Huggins, C.Race-strain Factor Related to Hydrocarbon-induced MammaryCancer in Rats. J. Nati. Cancer Inst., 39: 805-814, 1962.

14. Tjio, J. H., and Levan, A. Comparative Idiogram Analysis ofthe Rat and the Yoshida Rat Sarcoma. Hereditas: Lund, iß:218-234, 1956.

15. Tjio, J. H., and Whang, J. Chromosome Preparations of BoneMarrow Cells Without Prior In Vitro Culture or In VivoColchicine Administration. Stain Technol., 37: 17-20, 1962.

16. Yosida, T. H., and Amano, K. Autosomal Polymorphism inLaboratory Bred and Wild Norway Rats, Rattus Norvegicus,Found in Misima. Chromosoma, Berlin, 16: 658-667, 1965.

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Karyotypes of Rats of Different Cancer Susceptibility

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Fig. 1. Karyotype of male Sprague-Dawley (Holtzman) rat.Fig. 2. Karyotype of male Long-Evans rat.Fig. 3. Karyotype of male Fischer rat.Fig. 4. Karyotype of male Marshall rat.Fig. 5. Karyotype of male Osborne-Mendel rat.

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E. Douglas Rees, Amy Eversole Shuck, Joseph C. Christian, and Joe R. Pugh

Fig. 6. Metaphase chromosome of female Long-Evans rats arrow indicates satellited #12 chromosome. Giemsa, X 2000.Fig. 7. Metaphase chromosomes of female Long-Evans rats upper arrow indicates a #12 chromosome with large satellites on promi

nent upper arms, and lower arrow indicates a satellited #13 chromosome. Giemsa, X 2400.Fig. 8. Metaphase chromosomes of female Sprague-Dawley rat; a satellited #3 chromosome is indicated by arrow. Giemsa, X 1700.Fig. 9. Metaphase chromosomes of female Sprague-Dawley rat; arrow points to satellited #13 chromosome. Giemsa, X 1800.

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1968;28:823-830. Cancer Res   E. Douglas Rees, Amy Eversole Shuck, Joseph C. Christian, et al.   Mammary Cancer InductionKaryotypes of Rats from Strains of Different Susceptibility to

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