Trisomy of rat chromosome 1 associated with mesothelial cell transformation

1991;51:4059-4066. Published online August 1, 1991.Cancer Res Kenji Funaki, Jeffrey Everitt, Edilberto Bermudez, et al. TransformationTrisomy of Rat Chromosome 1 Associated with Mesothelial Cell  

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[CANCER RESEARCH 51, 4059-4066, August 1. 1991]

Trisomy of Rat Chromosome 1 Associated with Mesothelial Cell TransformationKenji Funaki1, Jeffrey Everitt, Edilberto Bermudez, and Cheryl Walker2

Chemical Industry Institute of Toxicology, Research Triangle Park, North Carolina 27709

ABSTRACT

Identification of specific chromosomal aberrations in transformedmesothelial cells is an important step in elucidating the mechanism oftransformation of these cells which are targets for occupational andenvironmental carcinogens, such as asbestos fibers. Cytogenetic analysisof normal rat mesothelial cell lines revealed that at late passage (p20-p34), trisomy of chromosome 1 was present in >80% of the cells in fourspontaneously immortalized lines examined, whereas at early passage(p8-plO), only 15-44% of the cells had trisomy 1. Trisomy of chromo

some 1 had increased in the population as a function of passage, suggesting that cells with trisomy 1 had a selective growth advantage underin vitro culture conditions and that this alteration was associated withtransformation. A commercially available rat mesothelial cell line (4/4RM4, ATCC), was also found to have a duplication of a portion of thelong arm of chromosome 1. To determine if chromosome I alterationshave relevance to the transformed phenotype in vivo, a neoplastic cellline was established from a spontaneous rat mesothelioma. At passage15, trisomy of chromosome 1 was observed in 26% of the metaphases inthis line. However, when these cells were injected into nude mice, 99%of the cells from the resulting tumor contained an additional copy ofchromosome 1. Therefore, trisomy 1 also conferred a selective growthadvantage in vivoand/or was associated with the malignant subpopulationin the tumor derived cell line. These studies suggest that chromosome 1contains a gene(s) involved in transformation of rat mesothelial cells.

INTRODUCTION

The mesothelium lines the pleural and peritoneal body cavities and is composed of mesoderm-deprived epithelial cells.Normal human mesothelial cells display many of the characteristics of both epithelial and mesenchymal cells, includingcoexpression of keratin and vimentin (1,2) and responsivenessto both platelet-derived growth factor and transforming-growthfactor ß(3). Mesothelioma is a neoplastic disease of this celltype, which in humans is associated with exposure to asbestosand other mineral fibers (4). In rats, mesothelioma can beinduced experimentally by instillation or inhalation of variousnatural and man-made mineral fibers (5-8), but can also occurspontaneously (9). The histogenesis of mesothelioma in rats isvery similar to that in humans (10), making them a usefulanimal model for the study of this disease.

The mechanism of mesothelioma development is at presentpoorly understood (7). Transformation in vivo may occurthrough a direct interaction of fibers with target mesothelialcells or may occur through an indirect interaction of fibers withother cell populations (such as inflammatory cells) that producecellular mediators, such as cytokines or oxygen radicals (7, 11).In vitro, asbestos fibers have been shown to interact with met-aphase chromosomes (12), and are clastogenic in vitro (13-23),leading to the hypothesis that aneuploidy or structural alterations of chromosomes may be directly induced as a result ofexposure to mineral fibers. A common finding in the mesothe-liomas of rats and humans is a complex set of numerical and

Received 1/4/91; accepted 5/23/91.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.

' Present address: Tolleri University School of Medicine, Yonago 683, Japan.2To whom correspondence should be addressed.

structural chromosomal changes. Chromosome aberrationshave been associated with many diverse types of cancer (24,25), and can be used as indicators for the presence of activatedoncogenes and/or loss of tumor suppressor gene function (26-28) that may be an important component of tumordevelopment.

Very little is known about specific cytogenetic alterationsassociated with transformation of rodent mesothelial cells. Ratmesothelial cells can be transformed in vitro by asbestos (29),and although it is clear that asbestos can induce aneuploidy inthese cells (12, 19, 23), no information about specific chromosomal alterations associated with in vitro transformation ofthese cells has been reported. Libbus and Craighead (30) reported, in a study of 15 rat cell lines derived from asbestos-induced mesotheliomas, that loss of chromosome 8, 16, 18, 20,and the X-chromosome occurred often in the cell lines and thattranslocations involving consistent breakpoints on chromosomes 5, 10, and 13 were frequently observed. To date, this isthe only report in which specific chromosome alterations associated with transformation of rodent mesothelial cells havebeen identified.

Normal rat mesothelial cells can be successfully cultured invitro (31, 32), and these cell strains spontaneously immortalize(acquire indefinite growth capacity) with a high frequency ( 19).3

In order to determine if specific chromosome aberrations areassociated with transformation of mesothelial cells, a panel ofspontaneously transformed rat mesothelial cells were subjectedto cytogenetic analysis. In the study reported here, bandinganalysis revealed that trisomy of chromosome 1 was a commonabnormality in late passage immortal rat mesothelial cell cultures. A subpopulation of cells with trisomy 1 could be detectedin early passages of the cell lines, suggesting a selective advantage for these cells which were to comprise the predominantpopulation in cultures with indefinite growth capacity. A lineof cells derived from a spontaneous rat mesothelioma was alsoestablished and contained trisomy 1 in a small percentage ofthe cells, but tumors which developed upon injection of thiscell line into nude mice exhibited trisomy 1 in greater than 99%of the cells. These results suggest that a gene(s) located onchromosome 1 is associated with the transformation of ratmesothelial cells.

MATERIALS AND METHODS

Normal Mesothelial Cell Lines. Normal mesothelial cells were isolated from the parietal pleura of rats as previously described (32).Briefly, cell lines (NRM1, NRM2, NRM3, and NRM4) were isolatedfrom individual epithelial-like colonies in primary mesothelial cellcultures from different animals (except NRM1 and NRM2 which werefrom the same animal), and maintained in Ham's F-12 medium(GIBCO, Grand Island, NY) supplemented with 10% FBS" (HyClone,

Logan, UT) as previously described (32). All cell lines analyzed havebeen in continuous culture for more than 100 population doublings.For the purpose of this study, cells were determined to be transformedwhen they had acquired indefinite growth capacity, i.e., immortalization. Line 4/4 RM4 (American Type Culture Collection, Rockville,

3E. Bermudez, unpublished observation.4The abbreviation used is: FBS. fetal bovine serum.

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RAT CHROMOSOME 1 TRISOMY IN MESOTHELIAL CELL TRANSFORMATION

MD) is a normal mesothelial cell line derived from the visceral pleuraof the rat. All the cell lines were passaged by brief treatment with amixture of 0.05% trypsin/0.05 HIM EDTA (GIBCO) in calcium- andmagnesium-free Hanks' balanced salt solution (GIBCO), followed by

replating in growth medium.Rat Mesothelioma-derived Cell Line. Cell line MEI was isolated from

a peritoneal metabolism that arose spontaneously in a 12-month-oldmale F344 rat. The macroscopic and histológica! appearance of thistumor was characteristic of mesotheliomas described for this speciesand strain. Briefly, nodular masses on the peritoneal surface werecollected into Dulbecco's phosphate-buffered saline (GIBCO). Excess

tissue was removed and the tissue was rinsed with fresh saline solution.The tissue was then finely minced with sterile scalpels and dispersed ina 150-mm tissue culture dish (Falcon, Lincoln Park, NJ) containing 30ml of Hams' F-12 medium supplemented with hydrocortisone (0.1 Mg/

ml; Sigma), insulin (10 ^g/ml; Sigma), transferrin (2.5 fig/ml; Sigma),selenium (2.5 ng/ml; Sigma), and 20% FBS. Once colonies appearedand grew to approximately 200 cells the tissue pieces were removedand the culture was refed with the same medium but containing only10% FBS (complete growth medium). A continuous culture was thenestablished from the expiant culture and maintained in complete growthmedium.

Cytogenetic Analysis. Cultures were exposed to colcemid (0.02 ^g/ml) (GIBCO) for 1.5-2 h before reaching confluency. Cultured cellswere detached by treatment with 0.05% trypsin/EDTA for 5 min at37°Cand centrifuged. Cell pellets were resuspended in hypotoniesolution of 0.075 M KC1 for 15 min at 37"C and then fixed with

methanol-acetic acid (3:1, v/v). After three changes of fresh fixative,cell suspensions were dropped on glass slides and air dried. Preparationsstained with a 2% Giemsa solution were used for chromosome counts.The chromosome number in 80 to 100 metaphases was counted toconstruct the chromosome number distribution. For chromosome banding analysis, destained preparations were stained by Q- or G-bandingmethod (33, 34). Between 13 and 35 banded metaphases from each cellline were used for determining the karyotypes of the cell lines. Thekaryotype that was most frequently observed in each cell line wasjudged to be modal. Chromosome rearrangements were classified intocommon and recurrent abnormalities; the former being an abnormalityobserved in all the cells examined from a cell line, and the latter anabnormality found in three or more cells. The incidence of rearrangements of chromosome 1 was estimated from partial karyotypesof all the cells used for chromosome counts.

RESULTS

Rat Mesothelial Cells. Normal rat mesothelial cell lines thathad spontaneously immortalized in tissue culture were kary-otyped by using G- and Q-banding techniques to identify non-random chromosome alterations that occurred in the cell populations. Table 1 summarizes the results from cell lines analyzed

during passage under in vitro culture conditions. The chromosome numbers tended to range widely with advanced passage,but modal chromosome numbers were exclusively near diploidin all the cell lines at all passages examined. At later passages,a small percentage of the population (1-13%) from 3 of thelines was comprised of cells with a tetraploid range ofchromosomes.

In the NRM1 cell line, 79% of the cell population countedat passage 13 had a diploid chromosome number (42 chromosomes) and the remaining cells were aneuploid with 43 chromosomes. All cells examined that had 42 chromosomes werepseudodiploid, having lost one normal copy of chromosome 16and containing an additional marker chromosome. The markerchromosome resulted from a translocation between an extrachromosome 1 (at breakpoint ql2) and chromosome 16 (atbreakpoint pi 1) (Fig. 1) (Table 2). Cells with 43 chromosomeswere found to contain an additional copy of chromosome 13 aswell as the -16, +der(16)t(l;16)(ql2;pl 1) alterations. At pas

sage 20, the dominant karyotype of the cells was still 42, XY,—16, +der( 16)t( 1; 16). In some pseudodiploid cells, other chromosome changes (-5, -12, -20), and + marker chromosomes)

were involved in addition to the common abnormalities, butthese missing chromosomes were not recurrent abnormalities.

In the NRM2 cell line, which was derived from the samedonor rat as NRM1, the chromosome distribution was withina diploid range at all passages. At passage 10, 93% of the cellpopulation had 42 chromosomes, and almost all of the diploidcells analyzed by banding techniques were karyotypically normal. However, an excess of chromosome 1 was found in 15%of the cells examined (Table 2). This abnormality existed aseither a duplication of chromosome Iql2—»qter,observed in asingle diploid metaphase, or complete or partial trisomy 1.Partial trisomy 1 in the aneuploid cells existed as either anextra chromosome with deletion of short arm or dup(l)(ql2—»qter). At passage 20, the modal chromosome number hadincreased to 43, with only 20% of the cells retaining a diploidor pseudodiploid chromosome number. Banded chromosomepreparations of cells with 43 chromosomes showed that theyhad an extra copy of chromosome 1, and more than one-halfof them had a deletion of chromosome 16 [del(16)(22)] inaddition to trisomy of 1 (Fig. 2) (Table 1). All of the metaphaseskaryotyped that contained 42 chromosomes also had trisomyof chromosome 1. Trisomy of 1 in these cells was accompaniedby chromosome loss, most frequently —16or del(16)(q22),although other, random, chromosome losses were also observed. Therefore the incidence of trisomy 1 had increased to

Table 1 Cytogenetic findings in rat pleural mesothelial cell lines

CelllineNRM1NRM2NRM3Passage13

20102047834Cells

counted/kary°tyPed100/35

100/26100/20

100/26100/30100/29

100/14Chromosome

range42-43,

40-44,42-43

41-4441-4641-43

38-45.84

8482-8642,42,42,

4.1.43,42,

43,XY,

XY.XY

XY,XY,Chromosome

abnormalitiesModal

karyotype-16.+der(16)t(l;

-16,+der(16)t(l;+

1, del(16)(q22)+ I.del(16)(q22)16)(ql2;pll)

16)(ql2;pll)Common-16,+der( 16)1(1:16)

-16, +der( 16)1(1;16)+

1+I.del(16)(q22)XYX.

-Y. +l,+Mar+3+

1.-16-9.+

1.Recurrentdup(l)(ql2-»qter)

, del(16)(q22)-Y,rec(l;3)t(l;3)

(pll;qll)-13, - Y

NRM4 31 100/23 40-46,82-88 43. XY.+1

RM4 15 80/13 40-45 43. XX, +20, dup(l) (q37-K)ter)

l,+del(l)(q34)dup( 1) (q33q36)

+20. dup( 1) (q37-K)ter) +1

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RAT CHROMOSOME 1 TRISOMY IN MESOTHELIAL CELL TRANSFORMATION

100% at passage 20 compared with 15% at the passage 10(Table 2). At passage 47, the modal karyotype was still 43, XY,+1, del(16)(q22). In addition to the common abnormalities,loss of chromosome 9 and a reciprocal translocation betweenchromosome 1 (break point q34) and chromosome 3, (breakpoint q42), and an identical marker chromosome were involvedin 20% (6 of 30) of the metaphases karyotyped.

In the NRM3 cell line, chromosome analysis was performedat passages 8 and 34. At passage 8, 91% of the cells countedhad a diploid chromosome number. Approximately 60% (19 of31) of metaphases with 42 chromosomes were karyotypicallynormal (Table 1), although pseudodiploid metaphases that hadlost one copy of chromosome 3 and acquired a marker chromosome derived from a translocation between an additionalchromosome 1 (at breakpoint pll) and chromosome 3 (atbreakpoint qll) comprised 40% of this population. Overall,cells with an additional chromosome 1 detected as a markerchromosome were observed in 44% of the cell population atpassage 8 (Table 2). At passage 34, the modal chromosomenumber had increased to 43. Banding analysis showed that thekaryotype of the NRM3 cells at the late passage were the mostvariable of all the cell strains examined. Of the 14 cells karyotyped, 93% had a duplication of chromosome 1; 11 of 14metaphases had an extra copy of chromosome 1, and 2 of 14had a large marker chromosome caused by translocation amongadditional chromosome 1 (at breakpoints pll and q36), chromosome 2 (at breakpoint qll), and unidentified material. Inaddition, cells at this passage contained an unidentified markerchromosome as a common abnormality, and had lost a copy ofthe Y-chromosome (Fig. 3) (Table 1).

A fourth cell line, NRM4, was also analyzed at late passage.At passage 31, 40 and 30% of the cells counted had 43 and 42chromosomes, respectively. Banding analysis showed that 88%

SHI*/* f\•£* B

«C•ï

8

Table 2 Incidence oftrisomy I in rat pleura! mesothelial cell lines

CelllineNRM1NRM2NRM3NRM4RM4Passage13

201020

478

343115Cells

examined100

100100

100100100

10010080Cells

with excessof chromosome

199

10015100

100449580100

(14 of 16) of cells with chromosomes had trisomy of chromosome 1 (Fig. 4) (Table 1). In addition, some aneuploid cells(11%) were observed to have a partial trisomy 1, as a result ofa deletion in the long arm region (breakpoint q34) of anadditional chromosome 1. In metaphases with 42 chromosomes, trisomy of chromosome 1 was found in 4 of the 7 bandedmetaphases (all of which also had additional random chromosome loss), and the remaining 3 metaphases were karyotypicallynormal. Despite the diversity in karyotype of the cells, thetrisomy of chromosome 1 was observed with high frequency inthese cells, being present in 80% of the metaphases examined(Table 2).

In a commercially available rat mesothelial cell line, RM4,an excess of chromosome 1 was observed in all the cells partiallykaryotyped with attention to rearrangements of chromosome 1(Table 2). In this line, 78% of the cells (62 of 80) contained 43chromosomes, including a large marker chromosome. Detailedbanding analysis showed that the marker chromosome was

ifn n11 MIt If

9 10

K5 6

II •»11 12

II H II I II II13

ft19

14

•ft20

15 16 17 18

IFig. I. G-banded karyotype showing a 45, XY, —16,+Mar in the NRM1 cell line at passage 20. Arrow indicates the marker chromosome from a translocation

between an extra chromosome I and chromosome 16, with breakpoints at ql2 and pll, respectively.

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RAT CHROMOSOME 1 TRISOMY IN MESOTHELIAL CELL TRANSFORMATION

»iKil U U MII IIII

13

II19

8

II14

II20

II II II M10 11 129

II15

i'16

II17

IIr.X Y

Fig. 2. G-banded karyotype showing a 43, XY, +l. del(l6)(q22) (arrow) in NRM2 at passage 47.

Fig. 3. Q-banded karyotype showing a 43, X, -Y, +1, +Mar in NRM3 at passage 34.

chromosome 1 with a duplication of the long arm region (q37—»qter) (Fig. 55). In addition, these metaphases also containedan extra chromosome 20. Thus, the modal karyotype was 43,XX, +20, dup(l)(q37-»qter) (Fig. 5A). Metaphases with 44chromosomes were also analyzed, and were found to containanother copy of chromosome 1 in addition to the dup(l)(q37—>qter) observed in the metaphases containing 43 chromosomes.

In all the spontaneously immortalized cell lines, similar alterations in cell growth were observed during passage in culture.At early passages, low density cells grew as discrete coloniescontaining tightly packed cells. At later passages, colonies ofcells appeared more disorganized and disperse. In addition, allthe cell lines exhibited a dramatic decrease in doubling time. Inthe NRM2 line for example, the population doubling time

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RAT CHROMOSOME I TRISOMY IN MESOTHELIAL CELL TRANSFORMATION

Fig. 4. Q-banded karyotype showing a complete trisomy I in NRM4 at passage 31.

decreased from 43 h at passage 5 to 26 h at passage 26.Therefore, the increase in trisomy 1 observed in the cell cultureswith increasing passage also correlated with altered growthpotential of these cells. The regions of chromosome 1 found tobe in excess in the cell lines examined are schematically represented in Fig. 6. Although the portion of duplication varied,the region of chromosome 1 from q37—Kjterwas duplicated in

all the cell lines.Mesothelioma Cell Line. A neoplastic cell line. MEI, derived

from a spontaneous rat mesothelioma, was also examined foralterations of chromosome 1. Cells from this line were examined at passage 15 and were found to be aneuploid with thenumber of chromosomes ranging from 42 to 46 (Table 3). Asshown in Table 3, an additional copy of chromosome 1 wasobserved in 26% (26 of 100) of the MEI metaphases partiallykaryotyped for estimating the incidence of chromosome alterations. The most frequent karyotype (observed in 36% of thecells) was 44,XY,+9,+12. When cells from this line were injected into nude mice, and the resulting tumor was explantedinto primary culture, chromosome numbers of the tumor cells,ME1-T (Table 3), were more widely distributed than the parental cell line, and the modal number was increased to 45. However, the incidence of trisomy 1 was remarkably increased to99% in the ME1-T cells, suggesting that trisomy 1 eitherconferred a selective growth advantage under in vivo conditions,or served as a marker for the neoplastic subpopulation in thiscell line.

DISCUSSION

In the spontaneously immortalized rat mesothelial cell linesexamined in this study, trisomy (including partial trisomy) ofchromosome 1 was the only common chromosome changeobserved in all the cell lines. Moreover, cells with an excess of

chromosome 1 were present in a higher proportion of the cellpopulation at later passages, suggesting that the cells withtrisomy 1 had a selective growth advantage under in vitro cultureconditions. In addition, a cell line derived from a spontaneousrat mesothelioma also exhibited trisomy of chromosome 1 whenreinjected in nude mice. This finding is significant as only 26%of the mesothelioma-derived Mel cells exhibited trisomy 1 inculture prior to injection, whereas >99% of the cells in thetumor had an additional chromosome 1. Therefore selection tohomogeneity of the trisomy 1 population had not occurred invitro prior to injection. In contrast, trisomy of chromosome 12which also occurred in the Mel cell line at passage 15, was notpresent in the tumor cell population (Table 3). Therefore trisomy 1 also correlated with malignant potential in vivo. Theseresults suggest that one or more genes located on chromosome1 are involved in acquisition and/or maintenance of the transformed phenotype in rat mesothelial cells. Whole or partial lossof chromosome 16 was also observed as a common abnormalityin two cell strains, NRM1 and NRM2. Because these cells linesoriginated from independent clones from the same donor rat,and loss of chromosome 16 was not observed in other cell lines,this change could be specific for cells from that particularanimal.

It is not possible at this time to determine when duringtransformation trisomy 1 first appeared in the cell cultures. Inthe NRM1 cell line, by passage 13, 99% of the cells containedan extra chromosome 1. In contrast, in the NRM2 cell line(derived from the same animal as NRM1) only 15% of the cellshad an additional chromosome 1 at passage 10, but by passage20, 100% of the cells exhibited trisomy 1. Therefore, althoughtrisomy of chromosome 1 was consistently observed in all thetransformed cells examined, the kinetics of the appearance ofthis cytogenetic abnormality remain to be investigated.

Structural alterations of rat chromosome 1 have been ob-

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RAT CHROMOSOME 1 TRISOMY IN MESOTHELIAL CELL TRANSFORMATION

Fig. 5. Q-banded karyotype showing a duplication of chromosome l in RM4. A, modal karyotype, 42, XX, +20, dup(q37—»ter)(arrow). B, partial karyotype ofhigh resolution band metaphase indicating the region of Iq37—»terduplicated (bracket).

ÕC

Fig. 6. A summary of the duplications on chromosome 1 in normal ratmesothelial cell lines. Solid bars indicate the regions duplicated in each cell line.In NRM2, the regions of duplication were different between early and late passage.The region from q37—»qterappears to be commonly duplicated in all the celllines examined.

served previously in several types of transformed cells. Rattrachéalepithelial cells transformed in vitro with TV-methyl-W-nitro-./V-nitrosoguanidine exhibited structural alterations in 4of 5 cell lines that resulted in a net gain of Iq (35), and thischange could be detected in the primary transformed coloniesfrom which the cell lines arose. In rat mammary cancer cellstransfected with H-ras, 7 of 9 transfectants had structuralabnormalities of chromosome 1 (36). Trisomy of chromosome1 has also been observed in rat carcinomas and sarcomastransformed with 7,12-dimethylbenz(a)anthracene (37, 38) andpolycyclic hydrocarbons (39, 40).

Whether trisomy of chromosome 1 observed in the spontaneously transformed mesothelial cells also occurs in asbestos-transformed rat cells remains to be determined. However, in apreliminary report, Palekar et al. (41) noted consistent alterations of chromosome 1 in cell lines derived from erionite andchrysotile asbestos-induced rat mesotheliomas. Although notobserved as frequently as other cytogenetic alterations, chromosome 1 alterations also occurred in asbestos-induced mesotheliomas in the study reported by Libbus and Craighead(30). This suggests that alteration of chromosome 1 occursduring both spontaneous and asbestos-induced transformation

of rat mesothelial cells. Therefore, it appears that the gene(s)4064

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Mel-T

RAT CHROMOSOME 1 TRISOMY IN MESOTHELIAL CELL TRANSFORMATION

Table 3 Cytogenetic findings in spontaneous rat mesothlioma

CelllineMEIPassage15Cellscounted/

karyotyped100/14Chromosomerange42-46Chromosome

abnormalitiesModal

karyolype44.

XY, +9, +12Common+12Recurrent+ l,-6, +9+MarCells

with excess ofchromosome I(%)26

Pri." 100/17 41-47, 86-90 45, XY, +1.+9, +Mar + 1, +Marl +9 991Pri.. primary cell culture from tumor produced in nude mouse following injection of MEI cells at passage 17.

located on rat chromosome 1 involved in transformation of ratmesothelial cells may play an important role in the transformation of this as well as many other cell types.

In humans, cytogenetic analysis of mesothelioma and meso-thelioma-derived cell lines has revealed a wide variety of chromosome alterations associated with these tumors. Deletionsand/or rearrangements involving chromosome 1 have beenreported in several studies of human mesothelial cells (20, 42-49), and have been reported to occur in one report with afrequency of >60% (45). A second commonly observed cyto-

genic alteration in human mesothelioma cells is deletion ormonosomy of chromosome 3 (45, 47, 48, 50). A tumor suppressor gene involved in several human cancers, including lung(51-53), renal cell carcinoma (54-56), and cervical carcinoma(57) is thought to be located on chromosome 3p. Althoughalterations of 3p are frequently observed in mesothelioma, inone study, mesothelial cells directly exposed to asbestos did notexhibit this alteration (20), suggesting that it may be a secondarychange occurring late in mesothelioma development. In a recentreport, Pelin-Enlund et al. (44) have reported an excess of the

short arm of chromosome 5 in 6 of 7 human mesothelioma celllines. Alterations of chromosome 5 have been observed inhuman mesothelioma only infrequently by other investigators(43, 48, 58, 59). Alterations of chromosome 11 (42, 43, 46,60), chromosome 22 (20, 42,43, 45,46,49, 50), and monosomyof chromosome 13 (44, 49), known to contain the humanretinoblastoma tumor suppressor gene (61), have also beenobserved in human mesothelioma cell lines.

Trisomy of chromosome 11 observed in human mesothelioma is especially interesting in light of the fact that a largelinkage group located on rat chromosome 1 is syntenic withhuman chromosome 11 (62). This would suggest that the samegene(s) involved in the transformation of rat mesothelial cellsmay also be involved in human mesothelial cell transformation.This linkage group also contains the H-ras protooncogene (62),but no significant increase in the expression of H-ras has beendetected in rat mesothelial cells trisomie for chromosome 1relative to normal cells,5 and it would not be expected that a50% increase in expression of the normal H-ras protooncogenewould be a transforming event. However, human chromosome11 does contain at least one tumor suppressor gene (63-65).Mutation in a tumor suppressor gene and subsequent duplication of that portion of the chromosome on which the gene islocated (observable cytogenetically as partial or complete tri-somy) might produce a dosage effect that could allow a mutantform of the tumor suppressor gene to exert a dominant negativeeffect on the product of the normal alíele.No cases of trisomyhave yet been shown at the protein level to have this effect,although tumor-specific trisomies, such as chromosome 7 associated with human melanoma (66), have been documented.

In summary, immortalized rat mesothelial cells and meso-thelioma-derived cells exhibit trisomy of chromosome 1. Aberrations in this chromosome have been observed in rat cells

from various tissues, transformed by a wide variety of agents,suggesting that alteration of gene(s) on this chromosome maybe an important event common to transformation of cells ofmany types, including asbestos-transformed mesothelial cells.Identification of the gene(s) involved will be a first step inunderstanding the molecular basis for the development ofmesothelioma.

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