[CANCER RESEARCH 46, 6333-6338, December 1986]

Presence of Chromosomal Abnormalities and Lack of AIDS Retrovirus DNASequences in AIDS-associated Kaposi's Sarcoma1

Pasquale Delli Bovi, Emilio Donti, Daniel M. Knowles II, Alvin Friedman-Kien, Paul A. Luciw, DiñoDina,Riccardo Dalia-Favoni, and Claudio Basilico2

Departments of Pathology and Dermatology, New York University School of Medicine, New York, New York 10016 [P. D. B., E. D., D. M. K., A. F-K., R. D-F., C. B.],and Chiron Research Laboratories, Chiron Corporation, Emeryville, California 94608 [P. A. L., D. D.]

ABSTRACT

The frequent occurrence of Kaposi's sarcoma (KS) in association with

the acquired immune deficiency syndrome (AIDS) could be due to thefact that the etiological agent of this tumor is the same retrovirus causingAIDS, to another oncogenic virus frequently found in AIDS patients, orto the unmasking of the tumorigenic potential of KS cells by immuno-suppression. We have therefore investigated the presence of DNA sequences homologous to the AIDS retrovirus, cytomegalovirus (CMV),and hepatitis B virus in 13 KS necropsies and biopsies from AIDSpatients. All KS DNA samples were negative for AIDS retrovirus orhepatitis B DNA sequences. Two DNAs from necropsies contained CMVDNA, but the data suggested the presence of replicating CMV DNA dueto generalized infection. We have also studied cell cultures derived fromKS skin biopsies of AIDS patients. These cultures had a short lifetimein vitro and expressed some markers of endothelial cells. The cells werenot tumorigenic in nude mice but contained a number of chromosomalrearrangements which were often monoclonal within the same culture.However, these abnormalities were different from culture to culture andeven in cultures from the same biopsy. The presence of these chromosomal abnormalities seemed to correlate with the cell positivity forendothelial markers. Taken together these results indicate that neitherthe AIDS retrovirus, CMV, or hepatitis B virus is directly responsiblefor the altered growth of KS cells, that KS may be polyclonal even withinthe same lesion, and that KS cells have a tendency to karyotypic rearrangements.

INTRODUCTION

The recently identified epidemic of AIDS3 is associated with

a significant increase in the occurrence of some tumors, namelyKS and B-cell lymphomas (1-4). Thus, AIDS and its associatedneoplasms may represent a unique model system to study theimmunological, virological, and genetic factors which influencethe development of malignancies.

KS is a multifocal neoplasm, generally considered an angio-sarcoma of endothelial origin (5), which presents itself as s.c.nodules or pigmented plaques of the skin or oral mucosa. Thistumor often has a rather benign clinical course, although aninvasive visceral form is described (5). KS was until recentlymost common in the United States and Europe in men ofEastern European or Mediterranean origin over 50 yr old (5),but patients with AIDS (particularly in the homosexual group)can incur KS at any age (1-3, 6). We report studies performedon KS skin biopsies and necropsies obtained from AIDS patients aimed at determining the presence or absence of specificviral DNA sequences in these lesions. We have also derived cellcultures from KS biopsies, in an attempt to study the nature of

Received 4/28/86; revised 8/21/86; accepted 9/3/86.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 investigation was supported by USPHS Grant CA37295 from the

National Cancer Institute.2To whom requests for reprints should be addressed.3The abbreviations used are: AIDS, acquired immune deficiency syndrome;

KS, Kaposi's sarcoma; ARV, acquired immune deficiency syndrome retrovirus;

cDNA, complementary DNA; CMV, cytomegalovirus; HBV, hepatitis B virus;III VI , human umbilical cord vein endothelia.

these tumor cells, whose cellular origin and degree of malignancy are not well known (5, 7).

MATERIALS AND METHODS

Tumor Biopsy Processing and Cell Culture Conditions. Cell cultureswere derived from KS biopsies using the technique described by Folk-man et al. (8), for the isolation of skin capillary endothelial cells withslight modifications. After the specimens were processed and treatedwith collagenase as described (8), the pellet of cells obtained wasresuspended in E199 medium containing 10% fetal calf serum, distributed in two 60-mm Falcon Primaria dishes, and incubated at 37"C.

One to 2 h later the medium and unattached cells were removed andreplaced with Medium E199 containing 20% fetal calf serum, heparin(Sigma, St. Louis, MO) (90 fig/ml), and endothelial cell growth supplement (Collaborative Research, Lexington, MA) (60 ¿¿g/ml)(9). Mediumwas replaced every 2 days. Microcolonies of nonfibroblastic-lookingcells were identified, removed, and replated in individual Falcon Primaria dishes or normal tissue culture dishes coated with a 1.5% gelatinsolution in Dulbecco's phosphate-buffered saline. The same medium

was used for all further passages.Immunofluorescence Staining. The cultures were tested for the pres

ence of specific cell markers by indirect immunofluorescent staining ofcells fixed on glass coverslips. For Factor Vlll-related antigen, Ulexeuropaeus Agglutinin I, and HLA-DR, fixation was in absolute niellianol for 20 min at -20°C. For vimentin and keratin, the fixation wasperformed in acetone:methanol (3:1) for 20 min at —20"C.For OKM5,fixation was in acetone for 20 min at —20°C.The Factor Vlll-related

antigen antibody was from Calbiochem (San Diego, ÇA);U. europaeusAgglutinin I was from Vector Laboratories (Burlingame, CA); themouse monoclonal antibodies to human HLA-DR and OKM5 werefrom Dr. C. Y. Wang (Sloan Kettering Institute, New York, NY) andfrom Ortho Pharmaceuticals, Inc. (Raritan, NJ), respectively; the antibodies against vimentin and keratins were kindly provided by Dr. R.Lyim (Department of Pharmacology, N.Y.U. Medical Center) and T.T. Sun (Department of Dermatology, N.Y.U. Medical Center), respectively. Goat anti-rabbit and sheep anti-mouse fluorescein isothiocya-nate-conjugated -y-globulins were from Cappel Laboratories (West

Chester. PA).Cytogenetic Analysis. As soon as a sufficient number of dividing cells

was available, chromosome preparations were obtained after treatmentof cell cultures with colchicine (0.04 /ig/ml) for 2 to 3 h. Wheneverpossible, a minimum of 20 metaphases was counted for each culture,and 5 to 10 karyotypes were prepared from well-spread GTG-bandedmetaphases (10).

DNA Preparation and Filter Hybridization. The DNAs from bothnecropsies and biopsies were extracted in the following manner: thespecimens were cleaned of all extraneous nontumorous tissues; washedin buffered saline; and then minced with a scalpel. The tissue wasresuspended in lysis buffer [10 mM Tris (pH 7.9): 10 mM NaCl:10 mMEDTA:0.5% sodium dodecyl sulfate:proteinase K (500 Mg/ml)]. Thelysate was incubated overnight at 50°C.The DNAs were extracted twice

with phenol, once with phenol:chloroform:isoamyl alcohol, and oncewith chloroform:isoamyl alcohol. Following addition of sodium acetateto a final concentration of 300 mM, the DNAs were alcohol precipitatedand washed several times in 75% alcohol, dried under vacuum, andresuspended in 1 mM Tris (pH 7.9):0.5 mM EDTA. The isolated DNAswere digested with the appropriate restriction enzyme, size fractionatedby agarose gel electrophoresis, and transferred to nitrocellulose paper.

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CHROMOSOME ABNORMALITIES AND LACK OF VIRAL SEQUENCES IN KS

For detecting AIDS-related retrovirus sequences, the filter was hybridized to an entire ARV-2 provirus probe [obtained by gel purification ofthe 9.5-kilobase EcoR.1 proviral insert from the recombinant plasmici.pARV-X la/2, representing the entire ARV-2 genome circularly permuted at the £coRIsite (11)] and nick translated to a specific activityof 3 x 10" cpm/ng of DNA. Hybridization conditions and washingwere as described (11). The filter was then exposed to Kodak XAR-2film in the presence of an intensifying screen at -70°C.The same filters

were generally rehybridized to a human N-ras cDNA probe (12). Theentire pN-ras plasmid was nick translated to a specific activity of 2 x10" cpm/jug of DNA. For detecting CMV sequences, the filters were

hybridized to two different human CMV (strain Towne) DNA probescloned in pBR322 plasmid, precisely pRL103 and pRL3. The recombinant plasmid, pRL103, contains a 21.5-kilobase Hindlll insert [corresponding to the "C" fragment of the physical map for Hindlll (13)cleavage of human CMV strain Towne], and pRL3 contains a 16-kilobase BamHl insert (corresponding to the "C" fragment of thephysical map for BamH\ cleavage4 of human CMV strain Towne).

They were kindly provided by Dr. G. S. Hayward. The two fragmentsrepresent different parts of the unique large sequences of the humanCMV strain Towne. The 21.5-kilobase Hindlll fragment represents animmediate early gene and has focus formation activity (Refs. 24 and25; Footnote 4). The 16-kilobase BamHl fragment represents latesequences (13, 14). The probes were labeled with 32Pby nick translationat a specific activity of 3.0 x 108cpm//¿g.The hybridization and washing

conditions were those described by Wahl et al. (15).

RESULTS AND DISCUSSION

Presence of Viral Sequences ¡nKS DNA. The frequent occurrence of KS in AIDS patients could be due to the fact that theetiological agent of KS is the same retrovirus causing AIDS(designated LAV, HTLV III, or ARV in different laboratories)(11, 16-19), to another infectious agent with oncogenic potential frequently found in AIDS patients or, in general, in im-munosuppressed individuals, or could be merely due to theimmunosuppression which unmasks the unregulated growthproperties of cells having genetic alterations that are normallykept in check by the host immune surveillance mechanisms.

We have therefore investigated the presence of ARV DNA( 11) in total DNA extracted from KS biopsies and necropsies,as well as in cell cultures derived from KS biopsies (see later).The results of Southern blots with an ARV DNA probe areshown in Fig. 1. All cellular DNAs tested (a total of 13 biopsiesand necropsies and 2 cell lines) were uniformly negative forARV DNA sequences at levels of sensitivity that should havedetected approximately 0.2 ARV genome equivalents/cell. Toensure that the various DNAs had been retained by the filtersand undergone proper enzyme digestion, the blots were alsohybridized to a human N-ras cDNA probe (12). The expectedhuman DNA restriction fragments hybridizing to the N-rasprobe were detected in all samples. It appears therefore that theAIDS retrovirus is unlikely to be directly involved in the etiology of AIDS-associated KS.

We next turned our attention to human CMV and hepatitisB virus, since both of these viruses have been implicated in KS(20-23). All KS DNA samples were negative for HBV DNAsequences (data not shown), but two DNA samples from KSnecropsies contained sequences which hybridized to CMVDNA probes. Fig. 2 shows the results obtained with one sample.CMV DNA sequences are present in relatively high abundance(approximately 5 copies per cell genome equivalent), and allexpected viral DNA fragments homologous to the probes usedappeared to be present (13, 14), including those hybridizing toanother probe (not shown) corresponding to the CMV repeated

Kb 123456 789 10 11 12 13

23.1 »

9.4 »

6.5 »

5.4

3.8

2.3»

2.0»

Kb 1 2 3 4 5 6 7 8 9 10 11 1213

23.1-

9.4»

4.3»

2.3«2.0»

JlS.

5.4

BFig. 1. Absence of ARV-2 (I l)-related sequences in the DNA extracted from

Kaposi's sarcoma biopsies and necropsies. The DNAs were digested with Sad

and electrophoresed in 0.9% agarose gel [an enzyme that produces two internalfragments of 3.8 and 5.7 kilobases from the DNA of the ARV-2 provirus (11)].A, hybridization with the ARV-2 probe (see "Materials and Methods"); B,hybridization with a human N-ros probe to visualize the DNAs on the filter (see"Materials and Methods"). Lanes I to 10, DNAs from 4 independent skin biopsiesand 6 necropsies of Kaposi's sarcomas from 10 AIDS victims (7 to 10 fig/lane);

Lane 11, DNA (7 ¿tg)from the human T24 cell line as negative control; Lane 12,50 pg of pARV-X 7a/2 plasmidic DNA as marker digested with Sad; Lane 13,50 pg of pARV-X 7a/2 DNA digested with EcoRl. The arrows on the right indicatethe molecular weight of the specific proviral fragments obtained by digestingpARV-\ 7a/2 DNA with the Sad enzyme. In Lanes 12 and 13 the bands visiblein B and not in . I represent plasmid sequences which were detected only with theprobe used in B, which contained pBR322 DNA. Analysis with three otherrestriction enzymes (£coRI,Hindlll, and Pst\) followed by hybridization to theARV-2 probe also failed to reveal the presence of DNA sequences homologousto this probe (data not shown).

' G. S. Hayward. personal communication.

sequences. The other DNA sample produced similar results,but the CMV DNA sequences appeared to be present in loweramounts (approximately 2 copies/cell, data not shown). Only2 of the 13 cases tested contained CMV DNA sequences. Inaddition, the DNA preparation containing the highest amountsof CMV sequences was derived from the skin necropsy of apatient who had died of CMV infection. These findings therefore are likely to reflect the occurrence of opportunistic CMVinfections which are frequently observed in AIDS patients andto be due to the presence of replicating CMV DNA in somecells of the original biopsy, rather than of specific CMV-

transforming genes (24, 25) in all tumor cells.Taken together these results suggest that neither the AIDS

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CHROMOSOME ABNORMALITIES AND LACK OF VIRAL SEQUENCES IN KS

Hind-Ill Bam HI Eco R I

Kb 1 2 1 2 m 1 2 m

23.1:

9.4 »-

ó.5 »_

4.3

2.3

2.0:

t ~

Hind-Ill Bam HI Eco RI

Kb l 2 m 12m 12m

23.1l

9.4 :

6.5

4.3

2.3

20

BFig. 2. Detection of CMV sequences by Southern blot analysis of DNA

extracted from a KS necropsy from an AIDS patient. The DNA digested with theenzymes shown was electrophoresed in 0.8% agarose gel and hybridized to twodifferent human CMV DNA probes cloned in pBR322 plasmids (see "Materialsand Methods"). In A, the DNA samples were hybridized with the purified 21.5-kilobase Hind\\l insert of the plasmid pRL103 (13), that had been 32P labeled bynick translation. In B, the DNA samples were hybridized with the gel-purifiedI6-kilobase BamHl insert of the plasmid pRL3 (see "Materials and Methods").

Lane I contained 5 ng of DNA from the KS necropsy; Lane 2 contained 5 jig ofnormal human T-lymphocyte DNA; Lane m contained 100 pg of DNA of theplasmid pRLI03 (A) or plasmid pRL3 (B), mixed with 5 jig of mouse DNA.

retrovirus, HBV, or CMV is required for the altered growthproperties of KS cells.

Studies of KS-derived Cell Cultures. Studies on the genetic

alterations of KS cells would be facilitated by the availability ofcell lines originating from these tumors. Experiments carriedout with DNA from biopsies and necropsies, although useful,suffer from limitations due to the fact that the proportion oftumor cells in the samples may be low, the supply of DNA islimited, and the properties of the tumor cells themselves cannotbe easily studied. For these reasons, we have attempted to obtaincell cultures derived from KS tumors. All patients from whomthese cultures were derived had not yet undergone any treatment. The techniques used to establish KS cell cultures weresimilar to those used for culturing endothelial cells and aredescribed in "Materials and Methods." The procedure involved

the isolation of microcolonies of cells that were morphologicallydistinct from the background of rapidly growing fibroblasts.Thus, our KS cultures are generally "clonal" in nature, or they

originate from a small number of cells.Cells cultured from KS biopsies were propagated in the

medium described, and their properties were studied in a varietyof ways to attempt to determine their relationship to the primary tumor cells and to assess their degree of transformationin vitro and in vivo. The cultures were compared to human skinfibroblasts, normal HUVE, and SV40-transformed HUVE cellcultures. The growth rate of cells in cultures was variable.Division time was on the order of 24 to 30 h; most culturesslowed their growth rate after six or seven passages and stoppedgrowing after about 2 mo. One culture (L-9) has been maintained for over 4 mo (approximately 70 cell generations). DNAextracted from L-9 cells did not contain any sequences homologous to ARV or CMV DNA probes (see above). The presenceof specific cell markers was tested by immunofluorescence orimmunoperoxidase reactions on fixed cells (Table 1). All cellswere negative for keratins but positive for vimentin, indicatingtheir mesenchymal origin (26). The presence of Factor VIII-related antigen, a marker of endothelial cells (27, 28), was notdetected in any of the cultures. /. europaeus type I agglutinin(29, 30) did not react with most cultures, but reacted with a fewcells in Bart 3 that, however, were no longer detectable at laterpassages.

The lack of Factor VIII positivity in the cells was consistentwith the observation that Factor VIII is not uniformly presentin KS sections (30-32), particularly in the so-called spindlecells; also SV40-transformed capillary (33) and HUVE cells(Ref. 34; Footnote 5) become Factor VIII negative after a fewpassages in culture. The KS cell cultures were assessed for thepresence of HLA-DR antigens (35, 36) and the antigen(s)recognized by OKM5, a monoclonal antibody which has beenshown to stain specifically the surface of capillary endothelialcells (37), as well as the spindle cell in a large number of KSsections tested (32). Similarly, HLA-DR antigens have beenshown to be frequently expressed by the spindle cells of KS inAIDS patients (32). Several cultures were positive for OKM-5,and two (including L-9, the line with the highest growth potential) for HLA-DR. Cultures that, although derived from KSbiopsies, were considered on the basis of morphology, growthpattern, and requirements to be mainly composed of fibroblasts(e.g., L-4) were all negative for OKM-5 and HLA-DR antigens.Since both OKM5 and HLA-DR antigens are also expressedby monocytes and macrophages, we tested the positive culturesfor the presence of eight markers characteristic of this celllineage (38). All were negative (data not shown). The positivityfor OKM-5 and HLA-DR markers by endothelial cells as wellas KS indicates that the cultures described here are likely to be

! Personal observation.

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CHROMOSOME ABNORMALITIES AND LACK OF VIRAL SEQUENCES IN KS

Table 1 Histochemical and karyotypic characteristics of cell cultures derived from KS

Cells Vimentin"Keratins"HUVE

XIIIe +

HUVE-SV +Factor

VIIIa-

relatedantigen+V.

europaeus"Agglutinin HLA-DR"

I (la) OKM5"Frequency

ofabnormal

karyotypedmetaphases0/7

(30)"

(15)Chromosome

analysis'Karyotype46,XY

Major karyotypic abnormalities

KSL*L-4L-9

KS19"19-119-2

19-3

KS26*26-MC26-1

KS B*B-lB-2B-3

0/5 (15)10/10(40)

46,XY46,X Y,- 14,+der( 14)t( 14; 17)(p 13;q 11)

NT*

NTNT

NT NT NT+

NT

20-30%

10/10(19) 46,XY,t(19;21)(ql3;qll)3/3 (21) 46,XY,6q-,8p+,inv(10)(q22.1q24.3),

12p+,13q-il4q+5/5 (22) 49,XY,+2,+7,+ 10,6q-,8p+,13q-

1/11(16) 46,X Y/46.X Y.del(8)(q21 )0/7 (25) 46,XY

2/5 (18) 46,XY/46,XY,8p-/46,XY,+ 17,-10/4 (23) 46,XY7/9 (46) 46,XY(60%)/46,XY,12p+(30%)/

91 .XXYY-12,-12,+der( 12),t(12;12)(pl3;pl3)(10%)

°Specific cell markers used in the characterization of the cell cultures.* The karyotype of most KS cultures was determined between the second and third passage in vitro. For HUVE cells, HUVE-SV, and FS4, the karyotype was

determined at the 13th passage in vitro or after the cells had been in culture for approximately 3 mo, respectively.' Normal human umbilical cord vein cells at the 13th passage in vitro.d Numbers in parentheses, total number of metaphases counted.1 Human umbilical cord vein cells transformed with a plasmid containing the SV40 early region.' Ihi man foreskin fibroblasts.'•'Individual biopsies from KS lesions (all from different patients) from which each culture indicated below was originated.* NT, not tested.

derived from KS spindle cells. It is interesting to note that,while normal endothelia are positive for both these markers,our KS cultures tested so far appeared to react with OKM5 oranti-HLA-DR antibodies, but not with both.

Karyotypic analysis of the KS cell cultures revealed variablechromosomal aberrations in different cultures (Fig. 3; Table 1).Metaphases from the L-9 cell cultures showed a partial trisomy17 due to a selective duplication of the segment 17ql 1 —>qter.This extra material was translocated to the short arm ofchromosome 14, resulting in a 46,XY,-14,+der(14)t(14;17)

(pl3;ql 1) karyotype. This karyotype was observed in all metaphases examined. The KS 19-1 cultures had a[t(19;21)(ql3;qll)] translocation, while the KS 19-2 and -3cultures had several numerical and/or structural rearrangements, but not the t(19;21) translocation. The 6q-,8p+ and13q—anomalies were present in both these latter cultures,suggesting a clonal evolution. Analysis of the KS B-3 culture

revealed three cytogenetic clones. The first (approximately 60%of the metaphases) had a normal 46,XY karyotype, the second(30%), a normal diploid chromosome number with a 12p+chromosome derived from an end-to-end duplication of thesegment 12p 11 —»p 13, while the third (about 10% of the cells)showed a near tetraploid karyotype in which the only commonanomaly was a t(12;12) translocation.

The karyotypic abnormalities of our KS cultures contrastedwith the situation in HUVE cells or skin fibroblast cultures,which showed no detectable chromosomal aberrations. Thesame was true of those cultures derived from KS samples (e.g.,L-4 and KS26-1) that had been considered to be mainly composed of normal fibroblasts on the basis of morphology andfailure to react with either OKM5 or anti-HLA-DR antibodies.

These findings suggested that KS cells are not characterizedby specific chromosomal abnormalities. However, the presenceof a different chromosomal rearrangement in all KS culturessuggests a tendency towards chromosomal rearrangements. Todetermine whether this situation reflected the development andclonal evolution of cells carrying chromosomal abnormalitiesduring propagation in culture, or whether those cells existed inthe tumor itself, we examined two KS primary cultures. In thecase of the KS26 mass culture, we found a 8q deletion in oneout of 16 metaphases examined. In KSB-mess culture we found

several chromosomal abnormalities, but unfortunately the poorquality of banding did not allow a satisfactory interpretation ofthe karyotypes. Two later analyses of both these mass culturesdid not reveal any clonal evolution of the cells with abnormalkaryotype. We could never detect any metaphase in fresh tumorcells.

The presence of chromosomal abnormalities in the KS cellcultures described here suggests that these cells may have someof the properties of tumor cells (39). However, the morphologyof these cultured cells and their growth patterns did not appeartransformed, and the cultures (with the possible exception ofL-9) did not have an indefinite growth potential but underwent

senescence rather rapidly. The cells did not exhibit any of theproperties commonly associated with transformation in vitro,such as growth in soft agar medium, and were not tumorigenicin nude mice (at IO6 cells/mouse) (not shown). The lack of

transformed growth properties may be due to suboptimalgrowth conditions. A variety of growth factors and hormonesare being tested to determine whether any of them wouldinfluence growth properties in these cultures.

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Fig. 3. G-banded karyotypes of two cellcultures derived from Kaposi's sarcoma biop

sies of AIDS patients. A shows the46,XY,t(19;21)(ql3,qll) karyotype of the KS19-1 cell culture. The arrows indicate thebreakpoints of the translocation. While thismetaphase shows only one No. 3 chromosome,this monosomy was not present in any othermetaphase. B shows the karyotype of the pol--,pliiiil cell population identified within the B-3 culture. The t(12;12)(pl3:pl3) translocationis clearly visible.

HA•~J

B »11 12

ft N17 18

15

21 x y

Sii «l2 3

l»l

¡i»

13

w14 15 18

19 2l 22 xx

Conclusions

The absence of DNA sequences homologous to those of theAIDS-associated retrovirus or of hepatitis B virus in all of theKS samples we tested argues against a direct viral etiology forKS. Although 2 samples of 13 contained CMV DNA, the factthat the whole CMV genome was present and the number ofcopies was relatively high suggested an opportunistic infection,rather than a direct causative role of this virus in KS. We cannotrule out the presence of minute fragments of ARV, HBV, orCMV DNA integrated into the DNA of KS cells, nor thepossibility that these viruses may have acted by a "hit and run"

(40,41 ). Our results, however, suggest that immunosuppressionmay be the main factor determining the frequent occurrence ofKS in AIDS patients. Subtle genetic alterations in KS cells orabnormal concentrations of growth factors may lead to prolif

eration and dedifferentiation of skin endothelial cells, a phenomenon that normally would be kept in check by the immunesystem. Accordingly, the phenotype of the cells in the KScultures is not typically "transformed," and these cells exhibit

only some markers characteristic of endothelial cells. Mostimportantly, they appear to have undergone chromosomal rearrangements which were not "specific" and varied from culture

to culture, including cultures derived from a single biopsy (e.g.,KS 19-1 and-2).

We could not assess whether the chromosomal abnormalitiesdiscussed above were already present in the original KS lesionsor whether they arose during propagation in culture. We found,however, several cultures (most notably L-9 and KS19-1) inwhich all cells contained the same characteristic chromosomalalteration, suggesting a tendency toward chromosomal rearrangement and clonal evolution in these cells. Since the small

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CHROMOSOME ABNORMALITIES AND LACK OF VIRAL SEQUENCES IN KS

colonies which originated them were picked a few days afterplating the tumor in culture, these rearrangements probablyexisted in the original cell population. In other cases, however,not all the cells in the culture carried detectable chromosomalrearrangements. This indicates a considerable heterogeneity inthe cell populations making up the KS lesion; chromosomalrearrangements could occur at different stages of growth, bothin the tumor and in tissue culture. Taken together these observations suggest that KS is a multifocal tumor that is polyclonaleven within the same skin lesion.

The presence of many different chromosomal aberrations inour KS cell cultures could be also due to a phemonenon ofgeneralized chromosomal instability in our patients. In thisrespect, it is interesting to note that recently nonspecific chromosomal aberrations have been reported in lymphocytes frompatients with sporadic KS, as well as in "fibroblast-like" cells

derived from KS lesions of the same patients (42). This couldsuggest that KS is associated with a yet unidentified virus whichcould cause chromosomal instability.

ACKNOWLEDGMENTS

We thank Dr. G. Teebor and Dr. M. Ittmann for providing us withsome KS specimens and Dr. D. Moscatelli for the helpful suggestionsand technical advice on endothelial cell cultures.

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2. Friedman-Kien, A. E. Disseminated Kaposi's sarcoma syndrome in younghomosexual men. J. Am. Acad. Dermatol.. 5: 468-471, 1981.

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14. Stinski, M. F., Thomsen. D. R., Stenberg. R. M.. and Goldstein, L. C.Organization and expression of the immediate early genes of human cytomegalovirus. J. Virol., 46: 1-14, 1983.

15. Wahl, G. M., Stern, M., and Stark, G. R. Efficient transfer of large DNAfragments from agarose gels to diazobenzyloxymethyl-paper and rapid hybridization by using dextran sulfate. Proc. Nati. Acad. Sci. USA, 76: 3683-3687, 1979.

16. Barre-Sinoussi, F., Chermann. J. C.. Rey, F., Nugeyre, M. T., Chamare!, S.,Grues!, J., Dauguet, C., Axler-Blin, C., Vezinet-Brun, F., Rouzioux, C.,Rozenbaum. W., and Montegnier, L. Isolation of a T-lymphotropic retrovirusfrom a patient at risk for acquired immune deficiency syndrome (AIDS).Science (Wash. DC). 220: 868-871. 1983.

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1986;46:6333-6338. Cancer Res   Pasquale Delli Bovi, Emilio Donti, Daniel M. Knowles II, et al.   SarcomaRetrovirus DNA Sequences in AIDS-associated Kaposi's Presence of Chromosomal Abnormalities and Lack of AIDS

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