<Original Article> Sister Chromatid Exchange (SCE) Frequency in Human and Simian Lymphoid Cell Lines Infected with Simian Retroviruses Closely Related to Human T-cell Leukemia Virus Type-1 利用統計を見る

Yamanashi Med. J. 6(l), Sl--87, l991

Sister Chromatid Exchange (SCE) Frequency in Human and Simian

Lymphoid Cell Lines Infected with Simian Retroviruses Closely Related

to Human T-Cell Leukemia Virus Type-1

Kumiko IuiMA, Atsumi Ts(.L}iMoToi), Hajime TsLEriMoTo2), Masanoyi HAyAMi3), Makoto HiGuRAsHi,

and Munehiro HiRAyAMA

DePartment ofMaternal and Child Health, School ofHealth Sciences, Facudy ofMedicine, The Universdy ofToltyo,

Bundyo-ku, Todyo 113, i)Division ofCarcinogenesis, National Cancer Center, Research Institute, Chuo-ku, Tokyo 104, 2)DePartment ofVeterinary InternalMedicine, Faculty ofAgricz`lture, The Universdy ofToltyo, Bundyo-ku, Todyo U3, and 3)l7zstitzLte Virzts Research, Klryoto UniwersiCv, Saflyo-kze, Klyoto 606,.IaPan

Abstract: Sister chromatid exchange (SCE) frequencies were examined on 4 simian lymphoid

cell lines producing simian retroviruses closely related to human T-cell leukemia virus type-1 and 6

human lymphoid cells immortalized by these simian viruses. The frequencies of SCE in both simian and human lymphoid cell lines were higher than those in normal cultured simian ancllor human lymphocytes.

We also observed the change of SCE frequencies in human cell lines immortalized by STLV at

several passage levels. At an early passage level the frequency of SCE in these human cell lines was

already higher than that in normal lymphocytes culture. This rise in SCE frequency did not change significantly during the observation period for 180 days.

From these findings we concluded that human lymphocytes were infected with STLV as simian lymphocytes, and that STLV caused same DNA damage in human lymphocytes as in simian lymphocytes, These phenomena induced by the retrovirus iRfections may correlate with leukemogenesis.

Key words: Simian T-cell leukemia virus (STLV), Adult T-cell leukemia (ATL), Sister chromatid

exchange (SCE), Human T-cell leukemia virus type-1 (HTLV-1), Lymphoid ceMine

INTRODUCTION

HumaR T-cell Ieukemia virus type-1

(HTLV-1) was isela￡ed from mature T-cell malignancies in man, and is known to be an etiological agent of adult T-cell leukemia (ATL)i-5). By comparison, the serurfl anti-bodies cross-reactive with HTLV-l were found in Old World monkeys and apes6rmiO). From these seropositive monkeys, simian retrovir-t}ses (STLVs) highly homologous to HTLV-1

Received Accepted September 7, December 12, 1990 1990

were isolated in culturesii). Nucleotide

sequ-eRce homology of STLV with HTLV-1 was

90-95%i2) and it was demonstrated that STLV

had the same leukemogenicity as

HTLV-li3･i4). To examine the biological function of

STLV, chromosomal damage induced by

STLV infection was investigated.

The role of viruses in causing chromosomal damage has received considerable attentioR, particularly in oncogenic viruses such as HSV, SV40 and Rauscher leukemia virusi5-i8). On the other hand, during the last decade, it has been firmly established that siste}' chromatid exchange (SCE) arises during replication on

damaged templates and SCE bioassay has now become popular as a very sensitive method for identifying potential DNA-damaging agents. It should be Roted, however, that although

con-ventional chromosome aberrations and SCE

are both cytogenetic rrianifestations of damage to the genome, they are seemingly unrelated and arise very different mechanismi9). This paper reports observations of the SCE frequeflcies and chromosomal instabilities in STLV-produciRg lymphoid cell lines and in the human cells immortalized by these viruses. It also estimates the potential ofchromosomal

damage by STLV.

MATERIALS AND METHODS

Cells

Four STLV-producing lymphoid cell Iines: JM86, PtM3, RfM26-l altd BM5 were used for

examination, which were established from

STLV-infected Japakese monkey (Macaca jus-cata), pig-tailed macaque (Macaca nemestrina), red-faced macaque (Macaca arctoides) and bon-net monkey (Macaca radiata), respectively were usedil).

Cells were cultivated for more than 12

months in RPMI 1640 medium supplemented

with IO-20% heat-inactivated fetal bovine ser"m (FBS) at 370C in a humidified atmos-phere of7% C02 in air. For cell growth, BM5 required exogeneous interleukin-2 (IL-2) pre-pared from simian spleen lymphocytes stimu-lated with concanavalin-A (Con-A) (Pharmacia Uppsala Sweden). All of these cell lines gave positive reactions with two monoclonal

anti-bodies coryespoRding to HTLV-1 p19 and

p24, respec￡ively.

(PHA-P) (Dilico Laboratories, Detroit, Mich, U.S.A.) iR RPMI 1640 medium supplemented with 20% FBS for 24 hrs. These cells were then co-cultivated with equal numbers of lethally-irradiated (l5,OOO rads) lymphoid cell line priducing STLV. The co-cultivated cells were

fed twice a week with the growth medium

containing 10% simiaR crude IL-2X). The

expression of STLV proteiRs was examined by the irRrfluRofiuorescence assay as describedii).

For comparison, HTLV-1 frorr} MT-1 and

MT-2 cell lines2'20) were also transmitted to

human PBMC using the same procedure.

Cytogenetic Studies

To examine chromosomal aberrations, the cultured cells were incubated with colcemid (2×IOr7 M) for the filtal 12 hrs. After cen￡ri-fugation, the cells were treated with hypotonic O.075 M KCI solution. Fo}lowing fur￡her cen-trifuga￡ion, the cell pellet was fixed with a mixture of methanol and acetic acid (8:I).

After the slides were stained with GierRsa, the

chromosoma} preparation was aRalysed by

examination of 30 to }OO mitoses in each cell line.

For sister chrorna￡id differential stainiRg, the cells were incubated iB medium containing bromodeoxyuridine (BrdUrd) at a concentra-tion of 40paM for the last 72 hr ofculture aRd were kept in a light free environment. After

treatment for chromosome preparatieR as

described abeve, a modification ofthe fiueresc-ence-plus-Giemsa (FPG) techRique2i) was used to obtain harlequilt chromosomes. TweRty to fifty fully difllerentiated and secondary

divid-ing metaphases were scored for examination ef SCE in each cell line.

Transmission of the Virus by Co--cultivation

For the virus recipient cells, human

peripheral blood mononuclear cells (PBMC)

obtained from two healthy womeR, testing

Regative for the HTLV-1 antibody, were

seeded at a density of 1× 106 cellslml aRd were stimulated with O.2% ofphytohemaggu}tinin-P

REsuLTs

The four original STLV-infected cell lines:

JM86, PtM3, RfM26-1 and BM5, were

analy-sed cytogeRetically. The frequeRcies of SCE in these STLV-producing cell lines were signi-ficantly higher than those in short-term

cul-STLV induced SCE 33

Table l. Cytogenetic features ofsimian lymphoid cell lines producing STLV

Cell Origin Virus Antigen

posMve

cells(%)

Chromosome number

-41 42-46

47-60

Cells Cells

"Jith MC with DMC

(%) (%)

SCE per chromosome

JM86

PtM3.

RfM

26-1

BM5

Japanese

Monkey

Pig-tailed macaque Red-faced macaque Bonnet monkey

STLV

-JA

STLV

-PT

STLV

-RF

STLV

-BO

1OO IOO 30 70 22 (51.2)

18

(94.7) 7 (}6.7)

46

(80.7)

14

(32.6) ( 1 (5.3) (

27

(64.3) (

7

(12,3) ( 2.2) (14.0)

o)(e)

4 95

9.5) (4 )

O)(7D)

75

o 9.5 e 1.9 o 26.2 3.5 e.54 O.22 O.27 O.39 Control Red-faced macaque Japanese monkey (-) (-) o o 65 (34.2) 8 (15A) I25 (65.8) (

44

(84,6) (

o) (o )

o)(o)

o o o o O.14

NT

Table 2. Cytogenetic features of human lymphoid cell lines proclucing HTLV

Cell Origin Virus Antigen posltlve cells(%)

Chromosome number

-45

46

47-60

Cells Cells

with MC with DMC

(%) (%)

SCE per chromosome

MT-1

MT-2

TAK

Human

Hurr}an

Human

HTLV

HTLV

HTLV

IOO 1oe 1OO 28 (84.8) ( 38 (67.9) ( 43 (89.5) (

5

4.2) (

6

11.5) (

2

4.2) ( 8.3) (2.7) 5.4) (l5.2) 2.1) ( 4.2) 2.8 5.8 2.1 2.8 7.6 2.1 O.25 O.27 O.24 Control

Human

(-) o e

(e )

lll (loo )(

o) (o )

o o O.I 7

tured lymphocytes from Srl-LV

antibody-nega￡ive healthy moRkeys

(O.14±O.051chromo-sorne) (Table 1). The SCE frequencies iR

MT-1, MT-2 and TAK cells producing

HTLV-1 were O,25!chromosome O.27!chromosome

and O.241chromosome, respectively, aRd signi-ficaittly higher than those in short-term cul-tured human lyrnphocytes from HTLV-1

anti-body-Regative healthy women (O.17±O.031

chromosome) (Table 2).

Normally, chromosome number (2n) iR

these macaque species is 42. In all cell lines, the

total chromosome number was distributed

over a wide range, and many hypo- and

hyper-diploid cells were observed. Minute

chromosomes (MC) and double minute

chromosomes (DMC) were also observed, and pulverizations were occasionally found. Human PBMCs were immortalized by

infec-tion with STLV from JM86, PtM3 and

RfM26--}. After 1 to 2 months from the

beginning of co-cultivation, several constantly

Table S. Cytogenetic features of newly establishecl cell lines by co-cultivation with cells producing STLV Cell Origin Virus Antigen

posmve

cells(%)

Chromosome number

-45 46 47-60

Cells Cells

with MC with DMC

(%) (%)

SCE per chromosome H ll

JM86

H21

JM86

H ll

PtM3

H ll Rf'M26-1

Human

Human

Human

Human

STLV

-.}A

STLV

-JA

STLV

-PT

s"rLv

-RF

IOO 100 70 70

10 5

(8.2) (65.3)

6 13

(22.2) (48.2)

18 25

(28.6) (39.7)

ll 29

(25.6) (67.4)

O 19

(O ) (26.5)

17

(3.7) (25.9)

3 l7

(4.7) (27.0)

os

(O ) ( 7.0)

4H

o 12.7 4.7 47.I 29.6 1.6 9.3 O.36

NT

N"r

NT

Table 4. Change of cytogenetic features in newly established cell lines by co-cultivation with JM86

Culture Antigen days positive cells(%)

-45

Chromosome number

46 47-60

Cells Cells

with MC with DMC

(%) (%)

SCE per chromosome H }1

JM86

60 se 115 l40 160 180 lOO 100 1OO lOO }oo 100 4 ( 8.2) 14 (2SA) l8 (28.1)

6

(l6.7) 11 (22.9)

10

(29A) 32 (65.3) 2S (38.3) 27 (42.2) 8 (22.2) 21 (48.8) 5 (l4.7) ( ( ( ( ( ( o

o)

2 3.3) 2 3.l) 3 8.3) 1 2.1) o

o)

13 (26.5) 21 (35.0) 17 (26.6) 19 (52.8) l5 (31.2) 19 (55.9) 8.2 o o ILI 8.3

4Ll

20.4 o o 2.8 2.1 47.1 O.34

NT

O.37 O.57 O.30 O.36 H21

JM86

40 60 80 70 1OO 1OO l2 (31.6) 7 (l2.7) 6 (22.2) 25 (65.8) 44 (80.0) IS (48.2) ( ( ( o

o)

o

o)

l 3.7) l ( 2.6) 4 ( 7.3) 7 (25.9) 2.6 S.6 o 2.6 1.8 3.7

NT

N[I'

NT

proliferating lymphoid cells were obtaiRed

(HIUM86, H2UM86, Hl/PtM8 and Hl/

RfM26-1, respectively). Also in these human cell lines immortalized by STLV, hypo- aRd hyper-diploid cells, MC and DMC were

fre-quen￡ly observed as shown in

STLV-producing simian cell lines. SCE frequency was

examined only iR HIUM86 cell line, and

elevatioR of SCE frquency was fouRd (Table

3).

IR HIUM86 and H2UM86 chromosome

abnormalities were determined at several pas-sage levels. At an early paspas-sage level SCE frequeRcy in HIGM86 was already higher than

STLV induced SCE 35

that in short-term cultured humaR lympho-cytes from healthy doRors (Table 4). The SCE

frequency showed the peak after 4 moRths

fyom the beginning of the co-cultivatioR. The number of total chromosomes was distributed over a wide raRge aRd the frequency of MC and DMC was high after 40-60 days from the initiation of the culture. These abBormalities

did not change significantly during the

observation period for i80 days, however, aneuploid cells had a tendency to increase.

DIscussloN

In this study, original STLV-infected simian cell lines were examined cytogeitetically. These cell lines showed higher SCE frequencies than those in short term lymphocyte cultures from normal controls. Furthermore, we observed that the total chromosome number was distri-buted over a wide range, and the frequency of MC aRd DMC was higher than that in normal short term cultured simian lymphocytes. We occasioRally found chromosomal pulveriza-tions. Human cell liltes immortalized by STLV showed chromosomal instabilities similar to those observed in the original STLV-infected cell lines after infection by the virus. The same

phenomena were observed in hvtman

iym-phoid cells immortalized by HTLV-1. A number of viruses have beeR observed to induce SCE. Wolff et al22). and Nichols et al2B). reported higher frequency ofSCE incidence in SV-40-transformed hurflan diploid cells com-pared with Rormal coRtrol cells. Similarly, increased incidence of SCE was demonstrated in a mouse cell line IRfected with Rauscher leukemia viyus24) and in peripheral blood lymphocytes infected with the herpes simplex virus25). Elevated levels of SCE have been reported iR lymphocytes from patients with various malignancies, such as leukemia26･27)

maligRant lymphoma28), and malignaRt

melanoma29). This SCE elevatioR has also been found in lymphoma cell lines 3e) and.in brleast tumor cell lines8i). IR our study, elevated SCE

frequencies were observed in cell lines immor-talized by both STLV and HTLV-1. To obtaiR further imformation about the relationship

between leukemogenicity and SCE, much

more data must be collected on ATL patients aRd on virus carrier.

Moreover, these cell lines immortalized by

STLV and HTLV-1 showed aReuploidy,

espe-cially the incidence of hypei--ploidy cells in-creased iR proportion to culture day. There are some reports that using flow cytometry

DNA aneuploidy were detected iR various

types of leukemia altd the incidence in ATL patients was significantly high32'33). on the othe}' hand, this increase was not parallel to the

increas of SCE frequency. The highest SCE frequency was observed only at an early pas-sage level.

We also found MC, DMC, and chromosomal

pulverizations in these cell lines. The

mechan-ism of the formation of MC aBd DMC is Rot

well understood. Since Spriggs34) first

observed DMC iR human lung cancer cells,

there have been some studies reporting the presence of DMC iB several kinds of cancer

cells35'36) By comparisoR, DMC aad

homogeneously staining chromosomah"egions (HSR) have bcen shown to be associated with

DNA amplification, and these phenomena

were originally discovered iR tumor cells37･38). In this study we did not ascertained by G-band,

so we Reed further information about the

appearance of MC and DMC.

Recently, a high proportion of macaques

that developed spontaneous lymphoma were

found to have antibodies cross-reacting with HTLV-1 antigens39). Furthermore, a typical ATL-like disease and its preleukemic state were found in African green monkeys

natural-ly infected with STLV. Our present data

indicates that STLV has similar Ieukemogenic-ity to HrrLv-l.

In this study it was found that STLV

induced chromosomal damage; 1) the increase

of aneuploid cells were observed both }n

hurflan cells immortalized by STLV and

HTLV-1, 2) in these cell lines the frequencies

of SCE and DMC were higher than those in

short term cultured lymphocytes from healthy donors, and 3) at an early passage Ievels from the beginning of the cocultivation SCE fre-queRcy was the highest.

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