1382 Blood. Vol 71, No 5 (May), 1988: pp 1382-1389

Reed-Sternberg Cells in Hodgkin’s Cell Lines HDLM, L-428, and KM-H2 Are notActively Replicating: Lack of Bromodeoxyuridine Uptake

by Multinuclear Cells in Culture

By Su-Ming Hsu, Xun Zhao, Subendu Chakraborty, Y-Fa Liu, Jacqueline Whang-Peng, Ming S. Lok, and Shiroh Fukuhara

We compared the proliferation of mononuclear and multi-

nuclear cells in four Hodgkin’s cell lines, HDLM-1 , HDLM-

1 d, 1-428, and KM-H2, by examining their capacity to

incorporate bromodeoxyuridine (BrdUrd) into nuclei.

Approximately 5% of all cells in HDLM-1 cultures had two

or more nuclei, a characteristic of Reed-Sternberg (RS)

cells. Unlike mononuclear Hodgkin’s (H) cells, these RS

cells exhibited no uptake, or only minimal uptake of

BrdUrd, suggesting that they did not replicate actively.

Cytogenetic study showed that 25% of the HDLM-1 cells

contained a tetraploid (4X) set of chromosomes with a

characteristic two-peak distribution. Following treatment

of HDLM-1 cells with phorbol ester. the percentages of 4X

cells and RS cells increased to 50% and 12%. respectively.

This increase in RS cells was not likely to be due to cell

fusion as shown by the absence of hybridization of BrdUrd-

positive and -negative nuclei. Phorbol ester has a short-

term effect of blocking the exit of cells from G1 into S

phase, but no effect on the transition from S phase to G2IM

phase. The block is more prominent in 2X cells than in 4X

cells, which may explain the increase in percentage of 4X

T HE MONONUCLEAR Hodgkin’s (H) cells and the

binuclear or multinuclear Reed-Sternberg (RS) cells

display similar, if not identical, markers.’5 These two types

of cells make up the neoplastic cells in Hodgkin’s disease

(HD). However, the mechanism involving the transforma-

tion of H cells to RS cells is not yet completely known. Kadin

and Asbury6 have attributed the formation of RS cells to

abnormal multipolar mitoses and disturbed cytokinesis

rather than to cell fusion.

In earlier investigations, RS cells were thought to be

end-stage cells that, although important for the diagnosis of

HD, had little or no capacity to replicate.78 This conclusion

was based on the fact that mitoses and tritiated-thymidine

uptake could be seen in H cells, but only rarely, if at all, in

RS cells. Later autoradiographic studies on long-term cul-

tunes of Hodgkin’s tissue indicated that RS cells are capable

of DNA synthesis.6’9 However, these studies, in which cul-

From the Department of Pathology. University of Texas Health

Science Center at Houston; Cytogenetic Oncology Section. Mcdi-

cine Branch, National Cancer Institute, Bethesda, MD; Hematolo-

gy-Oncology Service, Veterans Administration Hospital, Leaven-

worth, KS; and First Division of Internal Medicine, Faculty of

Medicine, Kyoto University. Japan.

Submitted July 20, 1987; accepted January 4. 1988.

Address reprint requests to Su-Ming Hsu. MD, Department of

Pathology. University of Texas Health Science Center at Houston.

P0 Box 20708, Houston, TX 77225.

The publication costs ofihis article were defrayed in part by page

charge payment. This article must therefore be hereby marked

“advertisement” in accordance with 18 U.S.C. §1 734 solely to

indicate this fact.

C I 988 by Grune & Stratton, Inc.

0006-4971/88/7105-0026$3.OO/O

cells in phorbol ester-treated cultures. In addition, phorbol

ester induced the differentiation of H-RS cells, which was

accompanied by loss of the marker HeFi-1 from the cell

surface. Approximately one third of the RS cells did notexpress HeFi-1 , or expressed only minimal amounts. The

findings led us to the following conclusions: (1 ) The 4X cellsprobably are formed from 2X H cells as a result of disturbed

cytokinesis, but not a cell fusion. (2) A considerable num-

ber of 4X cells were H cells, because the number of 4X cells

consistently exceeded that of RS cells. (3) Since mitotic

figures are extremely rare in RS cells and these cells did

not show active BrdUrd uptake, the increased number of

RS cells must also be a consequence of disturbed cytokine-

sis of H cells or a result of nuclear transformation (twisting,

convolution, or separation of the nucleus) in H cells. (4)

Most RS cells lose their proliferating capacity and some RS

cells may undergo further differentiation. Uptake of BrdUrd

and phorbol ester induction were also studied on the other

three H-RS cell lines, HDLM-1 d. 1-428, and KM-H2, with

results similar to those for HDLM-1.

0 1988 by Grune & Stratton, Inc.

tuned cells prepared from HD tissue were used, may have

suffered from serious artifacts. For example, fibroblasts and

Epstein-Barr virus-transformed B cells tend to overgrow in

culture.’#{176}Although the neoplastic nature ofthe cultured cells

in one study was clearly confirmed by the finding of cellular

aneuploidy and by the capacity of the cells to grow in the

brains of nude mice,9 contaminating cells may have been

present, some of which could display cytologic characteris-

tics resembling those of RS cells. Such artifacts may

preclude a precise evaluation of the number of true RS cells

in culture.

Recently, the establishment of H-RS cell lines and the

development of monoclonal antibodies (MoAbs) specific for

H-RS cells have made studies of the biologic nature of these

cells easier than before. The study of H-RS cells can yield

more meaningful and interpretable results than as obtainable

with heterogeneous cell cultures.

In a previous ‘ we observed that the percentage of

RS cells increased following the I 2-O-tetra-decanoyl phor-

bol- 1 3-acetate (TPA)-induced differentiation of H-RS cells.

Therefore, we became interested in investigating the mecha-

nism of RS cell formation. In the present study, we showed

that the increase in the percentage of RS cells following TPA

induction paralleled the decreased proliferation of the cul-

tuned H-RS cells. This observation raises the question

whether RS cells are capable of replication. We tested the

capacity of H cells and RS cells to take up bromodeoxyurid-

inc (BrdUrd), an analogue of thymidine that is incorporated

into the nuclei of replicating cells. Unlike H cells, RS cells

took up BrdUrd only minimally, indicating such cells usually

do not proliferate. Furthermore, we obtained evidence that

cell fusion is not responsible for the formation of RS cells.

Instead, RS cells could be generated as a result of disturbed

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LACK OF BRDURD UPTAKE IN RS CELLS 1383

cytokinesis, as has been suggested in previous studies,6 or by

twisting and convoluting of the nucleus.

MATERIALS AND METHODS

Tissue culture ofH-RS cells. HDLM is a series of four cell linesderived from a patient with HD. All ofthesecell lines (HDLM-l, -2,

-ld, -2d) have similar, if not identical, phenotypes and cytogeneticmarkers.’2’5 The HDLM-ld and -2d cells had been cultured in thepresence of phorbol ester (TPA) for more than 1 year; they were then

cultured in a TPA-free medium. We used HDLM-l and HDLM-ldcells in the present study because, in our laboratory, these cells grew

more rapidly than did HDLM-2 and -2d cells. In addition, we used

two H-RS cell lines, L-428 and KM-H2.’#{176}”6These cells show aphenotypic expression (ie, they react with MoAbs Ki-l, IRac, 2H9,

and HeFi-1) similar to that of HDLM cells. All cells were grown at 4

x l0� to 2 x 106 cells/mL in RPM! 1640 medium (GIBCO, GrandIsland, NY) supplemented with 10% fetal calf serum, 2 mmol/Lglutamine, 50 �mol/L 2-mencaptoethanol, and 50 j.tg/mL gentamy-

cm at 37#{176}Cin a humidified, 5% CO2 atmosphere. The medium was

changed every two to three days. The viability of these cells has

generally been maintained at 95%. Cell viability was determined by

the Trypan Blue dye exclusion test.All of these cells have a doubling time of approximately 60 to 84

hours. Smears were prepared by cytocentnifugation during thestationary phase of culture and were stained with Diff-Quik forcytologic evaluation. A total of 4,000 cells were counted, and thenumber of RS cells among them was identified as those cells

containing two or more nuclei that were either widely separated or inintimate contact. The result was expressed as the number of RS cells

per 100 cells in culture.Ultrastructural study of HDLM-1 cells. HDLM- 1 cells were

fixed in 3% glutanaldehyde, embedded in Epon, and processed forelectnon-microscopic examination of the nuclear structure.

MoAbs and immunoperoxidase reagents. To compare the phe-

notypes of mononuclear H cells and RS cells, we used the MoAbsHeFi-l , 2H9, and anti-IRac for immunostaining on cytospin smears.

The specificities of these MoAbs and the staining procedure havebeen described in detail previously.5’72#{176} The labeling reagents usedwere biotin-labeled horse anti-mouse Ig and avidin-biotin-penoxi-dase complex (ABC), both obtained from Vector Laboratories

(Burlingame, CA). In addition, we also examined the expression of anuclear antigen, Ki-67, in these cells. Ki-67 has been reported to bean antigen for proliferating cells.2’

TPA induction of cells. To study the effect of TPA on theformation of RS cells, we used a protocol for TPA induction ofHDLM-1 cells that has been described previously.”22 Briefly, TPA

dissolved in DMSO (14 �.tg/mL) was added at a final concentrationof 2 i�g/mL to cultures of H-RS cells. Every second day, two-thirdsof the medium was replaced with fresh medium containing TPA.The induction was carried out for three to four days. Cytospinsmears were prepared during the course of induction and wereevaluated for the percentage of RS cells. The increase in the numberof RS cells was also determined with night-angle light scatter, whichwe used as a detection parameter in flow cytometry.23

Effect of TPA on cell cycle. To determine the effects of TPA on

cell proliferation, we examined the DNA cycle of HDLM-l andHDLM-ld cells by using propidium-iodide staining and flow cytom-

etry.’5’22 Briefly, the cells were cultured in medium with or without

TPA for two days. The cells were washed in RPM! 1640 medium,

and then 1 mL aliquots of suspensions containing 2 x 106 cells wereincubated with 95% ethanol (2.5 mL) for 24 hours at 2 to 8#{176}C.After

being washed in phosphate buffered saline (PBS), the cells were

incubated with 100 ML RNase A (1,000 U/lOO �tL, WorthingtonDiagnostic, NJ) for one minute. The samples were then stained with

propidium iodide (50 �ig/mL) for two minutes and vortexed gently.Finally, the cells were analyzed by flow cytometny (Ontho Cytofluor-

ograf system 50H). The phase of TPA-tneated cells in the DNA

cycle was compared with that of control cells.BrdUrd uptake. HDLM-I, HDLM-ld, L-428, and KM-H2

cells were cultured in the presence of BndUrd (1 x 106 mol/L) forvarious periods (one to 72 hours). The cells were washed with Tnis

buffered saline (TBS) (0.1 mol/L, pH 7.6), prepared as cytospinsmears, and examined for uptake of BrdUrd by the nuclei. We used

the ABC immunoperoxidase method with an anti-BrdUrd MoAb(Becton Dickinson, Sunnyvale, CA) to detect BrdUrd uptake in the

nuclei.2426 Briefly, smears were treated with 1 mol/L HCI in normal

saline for 30 minutes and then washed with TBS for ten minutes.

Next, the cells were stained with anti-BndUnd MoAb ( 1:40), biotin-labeled horse anti-mouse IgG, and ABC as described previously)”26

Cells that take up BrdUnd exhibit dank granular staining in thenuclei. At various intervals, the percentage of BndUrd uptake by RScells was compared with that by mononuclear cells.

To test for possible fusion of mononuclear cells, we took cellspreviously treated with BrdUrd for 36 hours and cultured them withcontrol, untreated cells for one to two days. The RS cells were thenexamined for the presence of hybrid nuclei (BrdUrd-positive nucleusand Brd-Urd-negative nucleus in the same cell).

Cytogenetic study. We studied the chromosome distribution in

both TPA-treated and control HDLM-l cells. Both TPA-inducedand control cells were treated with colcemide (0. 1 �ig/mL, 90minutes) and hypotonic solution and were examined for the number

of chromosomes that they contained. The colcemide treatment did

not affect the number of RS cells in culture. A total of 2 15 cells wereincluded.

Sorting ofcells with high DNA content. We sorted HDLM-ld

cells according to their DNA content and determined the percent-

ages of RS cells in populations with high and in those with low DNAcontent. The sorting procedure was carried out with an OrthoCytofluorognaf 50H controlled by a 21 50 computer system that wasattached to the machine. Cells with high (highest 25%) and lowDNA content were deflected into separate containers, centrifuged

onto slides, and stained with Diff-Quik. The percentage of RS cellsin each sample was then determined as described in a previoussection (Tissue culture of H-RS cells).

Single-cell culture. The uptake of nucleic acid into cell nucleidoes not necessarily indicate that these cells can successfully com-plete the replication cycle. We used a limiting-dilution technique toculture single cells in 96-well plates to determine the replicationcapacity of the cells. We examined each well under a phase contrastmicroscopy to determine the number of cells and the size of cell.

Cells with multiple nuclei generally had a size three to four timesthat of mononuclear cells. Frequently, the number of nuclei in thelarge cell can be determined by the presence of two or more refractilenucleoli. Wells that contained more than one cell and those in whichthe number of nuclei per cell could not be ascertained were excludedfrom the study. The plates were cultured for ten to 14 days andexamined for the formation of colonies in each well.

RESULTS

Number of RS cells in culture and their morphologic

characteristics. The percentages of RS cells varied among

the four cell lines studied. From 7% to 1 1% of HDLM-ld and

L-428 cells were RS cells, followed by 4% to 6% for HDLM-

1, and 3% to 5% for KM-H2 cells, respectively (Fig 1). The

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EJD0

30

UCo

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Control TPA

Fig 1 . Percentages of RS cells in control and TPA-treatedH-RS cells in cultures. The result obtained with 1-428 is similar tothat of HDLM-ld.

percentage of RS cells remained constant in control cultures

throughout the 18-month study period.

Most of the RS cells contained two nuclei that were eitherwidely separated or in contact. Often, the two nuclei were

connected to one another by a thin, thread-like substance. A

highly twisted nucleus was frequently observed in H cells;

these cells presumably became RS cells after going through

several stages of transformation (Fig 2). The twisting and

deep convolution of nuclei were apparent on electron micros-

copy (Fig 3).

Phenotype ofRS cells v H cells. The phenotypic expres-

sion of RS cells was similar, if not identical, to that of H cells.

Both expressed HeFi-l, 2H9, and IRac. The staining inten-

sity of the H cells was fairly uniform; however, the expression

of HeFi-l in RS cells was quite variable. The intensity of

HeFi- 1 staining in 20% to 35% of the RS cells was very weak

or negative (Fig 4), but, in 10% to 20% of these cells, itappeared to be more intense than that in H cells. The

TPA-induced RS cells and H cells both had diminishedexpression of HeFi-l and 2H9, but remained IRac-positive.

The expression of Ki-67 was highly variable among cell

lines (Table 1 ). In L-428, most H as well as RS cells showed

� �

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A, W ,�. E”W’,� ‘�

Fig 2. Nuclei of HDLM-1 cells vary morphologically. (A-C) Thenuclei appear to be twisted, but remain connected. (D-F) the two

nuclei began to separate. but remain attached to each other by athin thread. (G) the connection has been broken. Original magnifl-

cation x 400.

Fig 3. Electron microscopy demonstrates the highly indented

or convoluted nuclei in HDLM-1 cells. The two nuclei in a cell may

appear separate or connected to one another depending on theplane of the section.

variable staining. In HDLM, intense staining was observed

in most H cells, but a considerable number of RS cells were

not stained on stained only weakly (Fig 5). The staining of

KM-H2 cells was uniformly weak. Treatment with TPA

reduced the staining intensity in all of the H-RS cell lines.

Effect of TPA on the formation of RS cells and on their

phase in the cell cycle. Induction with TPA had a dramatic

effect on the formation of RS cells. In all cultures, the

percentage of RS cells increased during a three-day induc-

tion period (Figs 1 and 6). For example, the percentage of RScells in HDLM-l cultures increased from 4% to 6% to I 1% to

15%. The cell viabilities were slightly lower in TPA-treated

cultures than in control cultures.

The TPA induction also affected the cell replication cycle.The precise percentage of cells in each phase is difficult to

determine because the aneuploidy of chromosome. However,

we estimated that, in control cultures, approximately 25% of

HDLM-l (2X) cells were in GO/GI phase (region 1, Fig 7)

and 20% in S phase (region 2). In TPA-treated cultures, 46%

,

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Fig 4. HeFi-1 staining of HDLM-1 cells. Most H cells expressedthe HeFi-1 antigen. However, approximately 30% of RS cells(arrows) did not express, or expressed only minimal amounts of,

HeFi-1. (A, HDLM-1; B, HDLM-ld; C, KM-H2).

1384 HSU El AL

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LACK OF BROURD UPTAKE IN RS CELLS 1385

Table 1 . Ki-67 Expression and BrdUrd Uptake in H-RS Cells

HDLM-1 L428 KM-H2

H RSH RS H RS

Ki-67

BrdUrd

>95% (3+)

>90% (3+)30-40% (2+13+) 60-70% (2+13+) 40-50% (2+)

<30% (±) >90% (3+) <30% (±)>90% (+)

>90% (3+)80% (±)

<25% (+)

The stainin 9 was performed on cells in stationary phase (Ki-67) or on cells incubated with BrdUrd for th rae days. 3 + . Strongl y positive; 2 +.

moderately positive; + , weakly positive; - negative.

(region 1) were in GO/GI phase and 6% in S phase. There was

no significant change in regions 4 and 5 (S and G2/M phase

of 4X cells, respectively). These data were interpreted as

showing that TPA enhanced entry of cells into , phase, but

also blocked their exit from late G, into S phase, and the

blocking is more prominent for 2X cells than 4X cells. A

similar GO/GI block was obtained for the other three cell

lines (L-428, HDLM-ld, and KM-H2).

BrdUrd uptake in H-RS cells. The uptake of BrdUrd by

the nuclei of H cells depended on the length of time of

exposure to BrdUrd. Most H cells (85% to 90%) had BrdUrd

in their nuclei after 18 to 24 hours of incubation (Fig 8).

However, the nucleic-acid incorporation in RS cells was

minimal. At 30 to 36 hours, only approximately 25% of RS

cells showed weak, granular or rare scattered staining in

their nuclei (Fig 9 and Table I). Intense BrdUrd uptake was

detected in <5% of RS cells (at 36 hours), and in 25% of the

RS cells after prolonged (>72 hours) incubation with

BrdUrd. The BrdUrd uptake by the cells appeared to be

affected by TPA treatment. Staining with anti-BrdUrd was

weaker in TPA-treated cells than in control cells (not

shown). The results were similar for all four cell lines tested.

In studying the possibility ofcell fusion, we did not observe

any RS cells that contained hybrid nuclei or nuclei with

intermediate staining intensity. Both nuclei in an RS cell

either were not stained or were stained weakly with anti-

BndUrd, indicating that the formation of RS cells was not a

result ofcell fusion.

Cytogenetic study. The numbers of chromosomes in

HDLM-l cells are illustrated in Table 2 and in the histogram

shown in Fig 10. The distribution remained similar through

the 18-month study period. Most HDLM-l cells had chro-

mosome numbers between 30 and 42 (hypodiploid) with

modal numbers at 2 peaks, at 37 to 38 and 74 to 76

chromosomes. Approximately 25% of the cells had 74 on

more chromosomes (tetraploid, 4X). The chromosome distri-

bution was similar in HDLM-l and HDLM-ld cells, even

Fig 5. Ki-67 staining of HDLM-1 cells. Thenuclei of most (>90%) H cells expressed Ki-67,compared with only 30% of the nuclei of RS cells(arrow heads). Few small binucleated RS cellswere positively stained by Ki-67 (arrows).

though the HDLM-ld cells had been treated continuously

with TPA for more than 1 year. TPA treatment apparently

increased the numbers of 4X cells (50%) over those in

untreated (control) cultures (25%).

Correlation ofDNA content with number ofnuclei. We

sorted the cells with the highest DNA content (highest 25%)

and compared the number of nuclei in these cells with that of

the remaining cells, which had a low DNA content. The

percentage of RS cells (7% to 10%) in the high-DNA-

content population was very similar to that for low DNA

content and that for presorted cell samples. This result

indicates that cells with high DNA content (4X cells ?) did

not necessarily contain multiple nuclei.

Capacity ofRS cells to divide. A total of 563 single-cell

cultures were evaluated for their capacity to proliferate.

Among 476 cultured small cells (H cells), 125 (25%) multi-

plied successfully. However, among 87 cultured large cells

(RS cells), only five cells multiplied. It appeared that most

large RS cells with widely separated nuclei did not prolifer-

ate in single-cell culture.

DISCUSSION

By using H-RS cell lines, we have shown that RS cells do

not participate actively in cell replication, and that these are

likely to be end-stage cells, as had been suggested by

Peckham and Cooper.7’8 This is confirmed by the lack of

BrdUrd uptake in the majority of RS cells during three days

of incubation, and by the lack of proliferation of large RS

cells in single-cell culture. Furthermore, we have provided

evidence that cell fusion is not responsible for the formation

of RS cells by showing that no cells containing hybrid

BrdUrd-positive and -negative nuclei were present. Dis-

turbed cytokinesis may contribute to the formation of 4X

cells, and probably of RS cells as well. Other possible

mechanisms of RS cell formation include convolution, twist-

ing, and separation of nuclei.

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#{163}Mononuclear Cell

0 RS Cell

18

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31-I 31-I

1386 HSU El AL

a

a

a0a.aa

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Hours of BUDR IncubatIon

Fig 8. BrdUrd uptake by HDLM-1 cells. The nuclei of most Hcells contained BrdUrd after 24 hours of incubation, comparedwith only 1 5% of the nuclei of RS cells. The numbers of RS cellscontaining BrdUrd increased as the incubation period increased.

Since RS cells are likely to be derived from H cells, the increase inthe number of BrdUrd-positive AS cells may result from a transfor-mation of BrdUrd-positive H cells into AS cells.

Fig 6. Increase in number of AS cells after TPA induction. Thiseffect of TPA was observed best with HDLM-1 d calls. Theincreased number of AS cells can be measured by the increase onright-angle scatter on flow cytometry (A. control HDLM-1 d cells;B. TPA-treated HDLM-ld cells). as indicated by the shift to theright in B. (C) Some of the TPA-induced HDLM-1 d cells that

contained two or more nuclei (arrows).

We observed BrdUrd uptake in virtually all H cells, but in

only 25% of RS cells, after 36 hours ofculture. To reach 50%

uptake, H cells had to be cultured with BrdUrd for more than

I 2 hours. The extent of nucleic-acid incorporation in H cells

in this study was similar to that in a previous study in which

36.5% of cells were labeled after incubation with 3H-thymid-

inc (1 zCi/mL) for 1 7 to 1 8 hours.9 However, Kadin and

Asbury6 reported a similar degree of uptake (24% to 43%) by

H cells when they used the same amount of 3H-thymidine,

but only one hour of incubation. In both studies,6’9 multinu-

clear (RS ?) cells made up between 1% and 2% of the total

number of cells, and 20.7% to 44% of these were labeled. The

difference in the source of cells and the conditions of culture

could account for the difference in results in their studies and

ours.

In our study, we used BrdUrd, a pyrimidine analogue of

thymidine, to examine the DNA synthesis of the cells,24’26 By

using a MoAb to BrdUrd, one can measure the incorporation

of this thymidine analogue into the DNA of H-RS cells

exposed in vitro, without the need for a radioactive isotope.

The method is very sensitive requiring only l0� or l06

mol/L BrdUrd.2�26 Thus, the lack of uptake by RS cells

cannot be attributed to the sensitivity of the method used.

The uptake of BrdUrd by RS cells, if it occurs, is far less

than that by H cells. The staining in RS cells usually consists

of a few granules and specks, unlike the intense, diffuse

Fig 7. Cell cycles of TPA-treated HDLM-1

cells (A. left) and control cells (B. right). Theprecise percentage of cells in each phase of cellcycle cannot be determined because the aneu-ploidy of cells. The cell cycle can be separatedinto five regions. Aegion 1 corresponds to theG0/G1 of 2X cells; region 2. S phase of 2X cells;region 3. G2 /M phase of 2X cells plus G0/G1phase of 4X cells; region 4, S phase of 4X cells;region 5, G2/M phase of 4X cells. Note thatthere is a significant decrease of percentage of

cells in region 2. but not in region 4 followingTPA treatment. Aegions 4 and 5 consisted ofapproximately 27% of total cells in bothtreated and control cultures.

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LACK OF BRDURD UPTAKE IN RS CELLS 1387

Fig 9. The extent of BrdUrd uptakein AS cells was minimal compared withthat in H cells. (A) A Diff-Quik-stainedsmear of HDLM-1 cells for cytologicevaluation. (B) Most H cells containabundant BrdUrd, whereas several AScells (arrows) without significant up-

take are seen. The same result wasobserved with KM-H2 cells (C). Thefaint nuclear staining is due to themethyl-green counterstain applied tocontrols (D).

staining seen in most H cells. In limiting-dilution cultures,

we rarely observed any proliferation of RS cells. It is

apparent that, even though we observed BrdUrd uptake in a

few (<25%) RS cells, the probability that these will complete

the cellular replication cycle is small because of their mini-

mal uptake of nucleic acid.

There was a considerable discrepancy between the number

of cells positive for BrdUrd and those positive for Ki-67.

Ki-67 is a nuclear antigen for proliferating cells. It has been

reported that H-RS cells in tissues express Ki-67,2’ but

whether the expression alone can be used to indicate the

replicating capacity of cells is not known. In this study, the

staining by Ki-67 in the H-RS cells used yielded some

confusing results. For example, most (>95%) mononuclear

HDLM-l cells were Ki-67-positive even though at least 25%

of them were in GO/GI phase. In KM-H2 cells, the staining

was uniformly weak and did not vary among cells. A

sucessful cell division is a result of multiple, integrated

biochemical processes, and one cannot deduce the entire

replicating function of cells by detecting a single protein,

especially when the cells are neoplastic and have biochemical

derangements.

Unlike most human neoplastic cell lines, HDLM-l cells

are characterized by a two-peak (2X and 4X) distribution of

the number of chromosomes; this is seen also for other H-RS

cell lines, including L-428, L-439, L-591, and possibly,

KM-H2.’#{176}”6 The precise percentage of 4X cells in culture,

however, cannot be determined. On cytogenetic study, one

can detect only those cells entering the mitotic cycle that are

arrested in metaphase on colcemide treatment. We do not

know whether 2X cells and 4X cells have the same rate of

mitosis. Nevertheless, judging by the DNA content distribu-

tion in our cell cycle analysis, we estimate that the percent-

age of 4X HDLM- 1 cells was in the vicinity of 25%.

Since the number of 4X cells exceeded the number of RS

cells, it is quite clear that a considerable number of 4X cells

have a single nucleus. This conclusion is also supported by

the following observations: (1) The cells with high DNA

content included a large number (90%) of H cells. (2) Most

RS cells had absent or minimal BrdUrd uptake and thus

were not likely to enter the mitotic cycle. It was clear that

most mitotic cells in prophase, which we examined, had only

one nucleus. Since RS cells are not likely to undergo cell

division, it is impossible to determine the number of chromo-

some in RS cells.

The formation of 4X cells is probably attributable to

disturbed cytokinesis of 2X cells, rather than to cell fusion.

The 4X sibling cells may contain two nuclei that are widely

separated (as in some RS cells). The RS cells lose the

capacity to replicate because the two nuclei are incapable of

synchronization in the cell cycle. A large number of 4X cells

may have a single nucleus because of incomplete separation

during mitosis. These cells can undergo mitosis and thus can

be detected on cytogenetic study. Some of the single-

nucleated cell may become RS cells as a result of convolu-

tion, twisting, and subsequent separation of nuclei. We

suspect that mitosis without cytokinesis is a property of some

2X cells, but generally not of 4X cells, because there were

only rare cells with four times (8X) the normal number of

chromosomes (Fig 1 1).

The increases in the percentage of 4X cells in metaphase

and in the percentage of RS cells following TPA induction

are also of interest. TPA can produce a G, block and delay

entry into S phase, whereas cells in S phase can proceed to

G2/M phase. The effect of TPA on the cell cycle of H-RS

cells is similar to that on human peripheral-blood lympho-

cytes, HeLa cells, and the cells of the mouse fibroblast line

C3H/lOTl/2.27’2’ Since the increased number of 4X cells

can be detected as early as two days after TPA induction,

and since the cell doubling time is approximately 60 hours, it

Table 2. Distribution of Chromosome Number in HDLM-1 Cells

<35 36 37 38 39 40 41 42 43-69 70 71 72 73 74 75 76 77 78 79-92 100-150 >200

Total No.of Ce8s

Examined

HDLM-1/TPA 2 3 7 10 1 3 0 0 5 2 1 7 0 8 2 11 3 7 6 5 2 85

HDIM-1 11 1 18 16 8 10 8 3 10 4 3 3 1 3 2 13 0 1 5 8 2 130

Total 13 4 25 26 9 13 8 3 15 6 4 10 1 11 4 24 3 8 11 13 4 215

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10

0

S

10

0

n �J,n nTPA-treated HDLM- 1 Cells

L1� nfl n n flrJ1�1fJ�l{�ln 1138

Number of chromosomes

Fig 1 1 . Hypothetical scheme of AS-cell formationdue to disturbed cytokinesis and nuclear twisting andseparation. Most AS cells are not likely to enter thecell division cycle. as shown by minimal nucleic-acidincorporation. M + C. mitosis with cytokinesis;M - C. mitosis without cytokinesis; 2X. diploid; 4X.tetraploid.

�i\

Ox

I II� -

1388 HSU El AL

HDLM-1 Cells

a.h� �� flmnJ-.. ..- - . . .. .. 111111#{149}811]

is difficult to attribute the increase to the inhibition of

cytokinesis by TPA, as such an effect would require two cell

cycles to become evident. It appears that the block exerted by

TPA is more prominent on 2X cells than on 4X cells, which

causes a relative increase in the percentage of 4X cells

detected on cytogenetic study.

It is highly unlikely that the 4X and 2X cells represent two

separate clones, because the chromosome distribution is not

much different in HDLM-l and HDLM-ld cells. (HDLM-

ld cells were derived from HDLM-1 cells after incubation of

the latter with TPA for more than 1 year.) Since TPA affects

the mitosis of 4X cells to a lesser degree, one would expect a

gradual replacement of 2X cells by 4X cells in HDLM-ld

culture, if two different clones exist. Phenotypic study by

multiple markers in HDLM-l and HDLM-ld cells has not

shown any significant heterogeneity of cells, or any other

evidence in support of the two-clone theory. Furthermore, a

considerable number of cells in other H-RS cell lines are also

4X. One cannot argue that all H-RS cell lines contain two

distinct populations. Taken together, some of the 4X cells

must be generated continuously from 2X cells as a result of

distributed cytokinesis.

It has been known that TPA induces the differentiation of

H-RS cells.” Most H-RS cells express markers such as

Fig 1 0. Chromosome distribution in untreatedand TPA-treated HDLM-1 cells. Note the character-istic two-peak distribution.

HeFi-l and Ki-l,’7’29 which are considered to be markers of

immature or neoplastic cells because they are absent from

normal lymphoreticular cells.’6 Thus, it is not surprising that,

after TPA induction, the H-RS cells become negative for

these markers.” The increased number of RS cells following

TPA induction may be consistent with the status of cellular

differentiation of H-RS cells, because a small portion of the

RS cells did not express HeFi-l , or expressed it only mini-

mally. However, cellular differentiation cannot totally

explain the mechanism of RS-cell formation, because most of

the RS cells still expressed amounts of HeFi-l antigen

comparable with those of H cells. The formation of RS cells

following TPA induction may result from a decreased rate of

cellular proliferation that allows the twisted nuclei in the

cells to become separated.

In conclusion, the mechanism of RS-cell formation is

probably a multifaceted rather than a simple process. The

disturbed cytokinesis of H cells and the twisting, convolution,

and separation of their nuclei lead to the formation of 4X

cells and RS cells. RS cells do not proliferate to any extent,

even though they take up a small amount of nucleic acid and

express Ki-67 antigen. The capacity of RS cells to complete

the cell division cycle is doubtful.

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LACK OF BROURD UPTAKE IN AS CELLS 1389

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1988 71: 1382-1389  

SM Hsu, X Zhao, S Chakraborty, YF Liu, J Whang-Peng, MS Lok and S Fukuhara cells in culturenot actively replicating: lack of bromodeoxyuridine uptake by multinuclear Reed-Sternberg cells in Hodgkin's cell lines HDLM, L-428, and KM-H2 are 

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